EDSTAC
Final
Report
August
1998
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC)

Final
Report
August
1998
EDSTAC
Final
Report
August
1998
Table
of
Contents
Executive
Summary
Chapter
One:
Introduction
I.
Overview
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1­
1
II.
The
EDSTAC's
Origin
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1­
1
III.
About
the
EDSTAC
Report
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1­
3
Chapter
Two:
Background
I.
The
Endocrine
System
as
it
Relates
to
the
Endocrine
Disruptor
Screening
and
Testing
Program
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2­
1
II.
Statutory
Basis
for
Endocrine
Disruptor
Screening
and
Testing
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2­
5
III.
Literature
Cited
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2­
12
Chapter
Three:
Conceptual
Framework
and
Principles
I.
Introduction
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3­
1
II.
Description
of
Endocrine
Disruptor
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3­
3
III.
Definition
of
Other
Key
Terms
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3­
4
IV.
Overview
of
the
EDSTAC
Conceptual
Framework
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3­
5
V.
Scope
of
the
EDSTAC
Conceptual
Framework
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3­
6
VI.
Where
Endocrine
Disruption
Fits
in
the
Broader
Context
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3­
6
VII.
General
Principles
to
Guide
the
Development
of
the
Endocrine
Disruptor
Screening
and
Testing
Program
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3­
7
VIII.
The
EDSTAC
Conceptual
Framework
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3­
9
IX.
Additional
Components
and
Clarifications
to
the
Original
EDSTAC
Conceptual
Framework
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3­
11
X.
Compilation
of
Chapter
Three
Recommendations
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3­
14
XI.
Literature
Cited
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3­
18
Chapter
Four:
Priority
Setting
I.
Introduction
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4­
1
II.
Overview
of
the
Sorting
and
Priority
Setting
Recommendations
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4­
4
III.
Exposure­
Related
Information
and
Criteria
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4­
15
IV.
Effects­
Related
Information
and
Criteria
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4­
23
V.
High
Throughput
Pre­
Screening
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4­
28
VI.
Recommendations
for
Handling
Polymers
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4­
36
EDSTAC
Final
Report
August
1998
VII.
Recommendations
for
Handling
Mixtures
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4­
45
VIII.
Recommendation
to
Screen
Naturally
Occurring
Non­
Steroidal
Estrogens
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4­
51
IX.
Recommendation
for
a
Nominations
Process
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4­
53
X.
The
Endocrine
Disruptor
Priority
Setting
Database
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4­
56
XI.
Recommended
Approach
to
Priority
Setting
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4­
65
XII.
Compilation
of
Chapter
Four
Recommendations
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4­
78
XIII.
Literature
Cited
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4­
87
Chapter
Five:
Screening
and
Testing
I.
Chapter
Overview
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5­
1
II.
Tier
1
Screening
Concepts
and
Design
Parameters
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5­
2
III.
Recommended
Tier
1
Screening
Battery
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5­
4
IV.
General
Principles
in
Evaluating
Tier
1
and
Tier
2
Results
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5­
32
V.
Tier
2
Testing
Concepts
and
Design
Parameters
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5­
40
VI.
Recommended
Tier
2
Testing
Battery
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5­
54
VII.
Validation,
Standardization,
Methods
Development,
and
Research
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5­
67
VIII.
Compilation
of
Chapter
Five
Recommendations
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5­
77
IX.
Literature
Cited
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5­
80
Chapter
Six:
Communications
and
Outreach
I.
Introduction
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6­
1
II.
Need
for
Communication
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6­
2
III.
Recommendations
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6­
3
IV.
Generalized
Schedule
for
Implementation
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6­
17
V.
Compilation
of
Chapter
Six
Recommendations
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6­
19
VI.
Literature
Cited
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6­
21
Chapter
Seven:
Compilation
of
EDSTAC
Recommendations
I.
Chapter
Overview
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7­
1
II.
Recommendations:
Chapter
Three
 
Conceptual
Framework
and
Principles
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7­
1
III.
Recommendations:
Chapter
Four
 
Priority
Setting
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7­
5
IV.
Recommendations:
Chapter
Five
 
Screening
and
Testing
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7­
14
V.
Recommendations:
Chapter
Six
 
Communications
and
Outreach
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7­
18
EDSTAC
Final
Report
August
1998
Acronyms
ACGIH
American
Conference
of
Governmental
Industrial
Hygienists
ADME
Absorption,
Distribution,
Metabolism,
and
Excretion
AGD
Anogenital
Distance
AMRT
Alternative
Mammalian
Reproduction
Test
AR
Androgen
Receptor
ASO
Accessory
Sex
Organ
ATSDR
Agency
for
Toxic
Substances
Disease
Registry
CAS
Chemical
Abstracts
Service
CASRN
Chemical
Abstracts
Service
Registry
Number
CAT
Chloramphenicol
Acetyl
Transferase
CBI
Confidential
Business
Information
CDFA
California
Department
of
Food
and
Agriculture
CDPR
California
Department
of
Pesticide
Regulation
CERCLA
Comprehensive
Environmental
Response,
Compensation,
and
Liability
Act
CFR
Code
of
Federal
Regulations
COWG
Communications
and
Outreach
Work
Group
CWA
Clean
Water
Act
DCI
Data
Call­
In
DDT
dichlorodiphenyltrichloroethane
DES
diethylstilbestrol
DSP
Daily
Sperm
Production
EAFUS
Everything
Added
to
Food
in
the
United
States
EAC
Endocrine
Active
Compounds
EAT
Estrogen,
Androgen,
and
Thyroid
ECA
Enforceable
Consent
Agreement
EDCs
Endocrine
Disrupting
Compound
EDPSD
Endocrine
Disruptor
Priority
Setting
Database
EDSTAC
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
EDSTP
Endocrine
Disruptor
Screening
and
Testing
Program
EFDB
Environmental
Fate
Databases
EMAP
Environmental
Monitoring
and
Assessment
Program
EPA
U.
S.
Envrionmental
Protection
Agency
ER
Estrogen
Receptor
EU
European
Union
FDA
U.
S.
Food
and
Drug
Administration
FIFRA
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
FQPA
Food
Quality
Protection
Act
FSIS
Food
Safety
Inspection
Service
GD
Gestational
Day
GLP
Good
Laboratory
Practice
GRAS
Generally
Regarded
As
Safe
GUI
Graphical
User
Interface
EDSTAC
Final
Report
August
1998
GUS
Groundwater
Ubiquitous
Source
HPG
Hypothalamic­
Pituitary­
Gonadal
HPT
Hypothalamic­
Pituitary­
Thyroid
HSDB
Hazardous
Substances
Databank
HSEES
Hazardous
Substances
Emergency
Event
Surveillance
HTPS
High
Throughput
Pre­
Screening
IARC
International
Agency
for
Research
on
Cancer
ITC
International
Trade
Commission
K
ow
Octanol:
Water
Partition
Coefficient
MARCIS
Microbiological
and
Residue
Computer
Information
Systems
MBP
Myelin
Basic
Protein
NAS
The
National
Academy
of
Sciences
NGO
Non­
Governmental
Organizations
NIOSH
National
Institute
for
Occupational
Safety
and
Health
NLM
National
Library
of
Medicine
NOAA
National
Oceanic
and
Atmospheric
Administration
NOEC
Naturally
Occurring
Estrogenic
Compounds
NONE
Naturally
Occurring
Nonsteroidal
Estrogenic
Compound
NPA
Not
Publicly
Available
NTIS
National
Technology
Information
Service
OECD
Organization
of
Economic
Cooperation
and
Development
OPP
Office
of
Pesticide
Programs
OPPTS
Office
of
Prevention,
Pesticides,
and
Toxic
Substances
OSHA
Occupational
Safety
and
Health
Administration
PAH
Polyaromatic
Hydrocarbon
PCBs
Polychlorinated
Biphenyls
PCNA
Proliferating
Cell
Nuclear
Antigen
PDM
Probabilistic
Dilution
Model
PDMS
Pesticide
Document
Management
System
PELs
Permissible
Exposure
Limit
PMN
Pre­
Manufacture
Notification
PND
Postnatal
Day
PSWG
Priority
Setting
Work
Group
PVC
Persistent
Vaginal
Cornification
QA/
QCQuality
Assurance/
Quality
Control
QSAR
Quantitative
Structure
Activity
Relationship
RBA
Relative
Binding
Affinity
RED
Registration
Eligibility
Decision
REL
Recommended
Exposure
Limits
RfD
Reference
Dose
RVIS
Residue
Violation
Information
System
SAB
Science
Advisory
Board
SAP
Science
Advisory
Panel
SAR
Structure
Activity
Relationship
SARA
Superfund
Amendments
and
Reauthorization
Act
EDSTAC
Final
Report
August
1998
SDWA
Safe
Drinking
Water
Act
SHC
Spermatid
Head
Counts
SI
Steroidogenesis
Inhibition
STWG
Screening
and
Testing
Work
Group
SuCCSES
Substructure­
based
Computerized
Chemical
Selection
Expert
System
SWDA
Safe
Drinking
Water
Act
T1S
Tier
1
Screening
T2T
Tier
2
Testing
TCDD
tetrachlorodibenzodioxin
TLV
Threshold
Limit
Value
TR
Thyroid
Receptor
TRH
Thyrotropin­
Releasing
Hormone
TRI
Toxic
Release
Inventory
TSCA
Toxic
Substances
Control
Act
TSCATS­
HE
Toxic
Substances
Control
Act
Test
Submissions
­
Health
Effects
VO
Vaginal
Opening
VOCs
Volatile
Organic
Compounds
VP
Vaginal
Patency
EDSTAC
Final
Report
August
1998
Appendices
Appendix
A:
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
Participant
List
Appendix
B:
Principles
Work
Group
Participant
List
Appendix
C:
Priority
Setting
Work
Group
Participant
List
Appendix
D:
Screening
and
Testing
Work
Group
Participant
List
Appendix
E:
Communications
and
Outreach
Work
Group
Participant
List
Appendix
F:
References
and
Sources
for
Chapter
Four,
Priority
Setting
Appendix
G:
Data
Source
Matrices
Appendix
H:
Fate
and
Transport
Tables
Appendix
I:
Feasibility
Demonstration
Project
for
HTPS
Appendix
J:
References
and
Sources
for
Chapter
Five,
Screening
and
Testing
Appendix
K:
Brief
Overview
of
Assays
Considered
for
Tier
1
Screening
Appendix
L:
Protocols
for
Tier
1
Screening
Assays
Appendix
M:
Assays
not
Included
in
Tier
1
Screening
Appendix
N:
Endocrine
Disruption
and
Invertebrates
Appendix
O:
Protocol
for
Possible
In
Utero
Developmental
Screening
Assay
Appendix
P:
Examples
of
"
Weight
of
Evidence"
Determinations
Appendix
Q:
Tier
2
Testing
Study
Designs
Appendix
R:
Preliminary
Categorization
of
Tier
1
Screens
and
Tier
2
Tests
by
the
Screening
and
Testing
Work
Group
Appendix
S:
Survey
of
Cost
Estimates
for
EDSTAC's
Proposed
Endocrine
Disruptor
Screening
and
Testing
Assays
Appendix
T:
Summary
of
EPA's
September,
1997
Outreach
Questionnaire
Appendix
U:
Compilation
of
Public
Comment
EDSTAC
Final
Report
Executive
Summary
August
1998
Executive
Summary
EDSTAC
Final
Report
Executive
Summary
August
1998
Table
of
Contents
I.
Introduction
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1
II.
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
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1
A.
EDSTAC
Formation
and
Structure
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1
B.
Scope
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2
III.
Chapter
Three
 
Conceptual
Framework
and
Principles
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3
A.
Conceptual
Framework
Overview
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3
B.
Recommended
Principles
and
Guidance
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6
IV.
Chapter
Four
 
Priority
Setting
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6
A.
Initial
Sorting
and
Phased
Approach
to
Screening
and
Testing
the
Universe
of
Chemicals
6
B.
Information
Useful
for
Prioritization
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7
C.
High
Throughput
Pre­
Screening
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8
D.
Endocrine
Disruptor
Priority
Setting
Database
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9
E.
Recommended
Approach
to
Priority
Setting
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9
1.
Compartment­
Based
Approach
to
Priority
Setting
for
T1S
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9
2.
Special
Compartments
of
Chemicals
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10
3.
Recommended
Approach
to
Priority
Setting
for
T2T
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11
V.
Chapter
Five
 
Screening
and
Testing
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11
A.
Tier
1
Screening
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11
1.
Recommended
Screening
Assays
.
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11
2.
Criteria
for
Evaluating
Tier
1
Results
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13
B.
Tier
2
Testing
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13
1.
Test
Selection
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13
2.
Low
Dose
Issues
in
T2T
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14
C.
Validation,
Standardization,
Methods
Development,
and
Research
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15
VI.
Chapter
Six
 
Communications
and
Outreach
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15
A.
Principles
to
Guide
a
Communications
and
Outreach
Strategy
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15
B.
Basic
Features
of
a
Communications
and
Outreach
Strategy
.
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16
VI.
Chapter
Seven
 
Compilation
of
EDSTAC
Recommendations
.
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17
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
1
I.
Introduction
Concerns
regarding
both
the
presence
of
endocrine
disruptors
in
food,
water,
or
other
environmental
media,
and
the
potential
risk
they
pose
to
humans
and
wildlife
have
been
growing
in
recent
years.
Passage,
in
1996,
of
the
Food
Quality
Protection
Act
(
FQPA)
and
Amendments
to
the
Safe
Drinking
Water
Act
(
SDWA)
reflected
these
concerns
and
required
EPA
to:

develop
a
screening
program,
using
appropriate
validated
test
systems
and
other
scientifically
relevant
information,
to
determine
whether
certain
substances
may
have
an
effect
in
humans
that
is
similar
to
an
effect
produced
by
a
naturally
occurring
estrogen,
or
other
such
endocrine
effect
as
the
Administrator
may
designate.

Specifically,
EPA
was
required
to
develop
a
screening
program
by
August
1998;
to
implement
the
program
by
August
1999;
and
to
report
to
Congress
on
the
program's
progress
by
August
2000.
In
1996,
EPA
formed
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC),
charging
the
Committee
to
provide
advice
on
how
to
design
a
screening
and
testing
program
for
endocrine
disrupting
chemicals.
This
report
contains
the
findings
of
the
Committee,
and
is
organized
into
sections
discussing
the
EDSTAC
recommendations
on:
a
Conceptual
Framework;
Priority
Setting;
Screening
and
Testing;
and
Communications
and
Outreach.
The
Final
Report
contains
many
references
to
scientific
papers
that
are
intended
to
provide
background
information
and/
or
justification
for
the
EDSTAC's
recommendations.
These
references
reflect
the
EDSTAC's
understanding
of
the
science
of
endocrine
disruption
as
of
the
final
plenary,
held
on
June
17
and
18,
1998
in
Washington,
DC.
The
Committee
recognizes
the
science
of
endocrine
disruption
is
rapidly
and
continually
evolving
and
EPA
will
need
to
incorporate
the
results
of
on­
going
research
and
recent
publications
when
implementing
the
Committee's
recommendations.

The
EDSTAC
describes
an
endocrine
disruptor
as
an
exogenous
chemical
substance
or
mixture
that
alters
the
structure
or
function(
s)
of
the
endocrine
system
and
causes
adverse
effects
at
the
level
of
the
organism,
its
progeny,
populations,
or
subpopulations
of
organisms,
based
on
scientific
principles,
data,
weight­
of­
evidence,
and
the
precautionary
principle.

II.
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
A.
EDSTAC
Formation
and
Structure
Chapter
One
provides
an
introduction
and
overview
of
both
the
EDSTAC
process
and
the
report
itself.
The
EDSTAC
was
composed
of
individuals
representing
various
stakeholder
groups
and
scientific
expertise.
The
members
included
scientists
and
other
representatives
from:
EPA,
other
federal
agencies,
state
agencies,
various
sectors
of
industry,
water
providers,
worker
protection
organizations,
national
environmental
groups,
environmental
justice
groups,
public
health
groups,
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
2
and
research
scientists.
The
Committee
began
their
deliberations
in
October
1996
and
completed
their
recommendations
in
July
1998.

The
Committee
organized
itself
into
the
following
working
groups,
each
with
a
specific
assignment:

·
Principles
Work
Group
to
develop
the
EDSTAC
conceptual
framework
(
which
is
contained
in
Chapter
Three);
·
Priority
Setting
Work
Group
to
develop
a
recommended
approach
to
setting
priorities
for
endocrine
disruptor
screening
and
testing
(
which
are
contained
in
Chapter
Four);
·
Screening
and
Testing
Work
Group
to
develop
recommendations
on
the
screening
assays
and
tests
to
include
in,
and
implementation
of,
a
screening
and
testing
program
(
which
are
contained
in
Chapter
Five);
and
·
Communications
and
Outreach
Work
Group
to
develop
recommendations
on
communication
issues
for
the
screening
and
testing
program
(
which
are
contained
in
Chapter
Six).

The
work
groups
were
comprised
of
Committee
members
as
well
as
other
individuals
who
were
not
Committee
members
but
were
asked
to
participate
in
the
EDSTAC
process
because
of
their
particular
expertise
and
perspective.
These
work
groups
met
periodically
to
accomplish
their
tasks.
The
full
Committee
held
nine
meetings,
all
open
to
the
public,
in
different
locations
around
the
country.

B.
Scope
Chapter
Two
describes
the
scope
of
the
EDSTAC's
deliberations.
In
addition,
the
chapter
contains
background
information
on
the
function
of
the
endocrine
system,
the
issue
of
endocrine
disruptors,
and
the
complex
statutory
and
chemical
universe
within
which
priority
setting
and
screening
and
testing
must
be
accomplished.
The
primary
scope
of
the
EDSTAC
was
to
develop
recommendations
for
a
screening
and
testing
program
for
endocrine
disrupting
chemicals.
The
EDSTAC
interpreted
this
scope
to
include
not
only
the
1996
FQPA
and
SDWA
provisions,
but
also
those
of
the
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA),
the
Toxic
Substances
Control
Act
(
TSCA),
and
the
Federal
Food,
Drug,
and
Cosmetic
Act
(
FFDCA).
Together
these
acts
provide
testing
authorities
for
a
variety
of
chemicals
for
both
human
health
as
well
as
ecological
effects.
As
a
result,
the
EDSTAC
recommended
that
EPA's
endocrine
disruptor
screening
and
testing
program
(
EDSTP)
should:

Address
both
human
and
ecological
(
wildlife)
effects:
The
EDSTAC
recommended
that
the
EDSTP
scope
should
include
screening
for
adverse
effects
to
wildlife,
as
well
as
humans,
recognizing
that
wildlife
are
an
inherently
valuable
element
of
ecosystems
and
their
well­
being
can
be
an
indication
of
the
overall
health
of
the
environment
in
which
humans
live.
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
3
Examine
effects
to
estrogen,
androgen,
and
thyroid
hormone­
related
processes:
The
Committee
recommended
that
three
primary
hormone
systems
be
included
in
the
EDSTP
-
estrogen,
androgen,
and
thyroid
-
because
they
are
important
hormones
in
both
humans
and
wildlife
with
a
relatively
large
body
of
available
relevant
data
and
screening
assays
and
tests
from
which
to
select.
While
the
EDSTAC
focused
on
these
three
hormones,
it
is
aware
that
the
science
regarding
the
endocrine
system
is
rapidly
developing.
As
more
data
become
available
on
other
hormones,
and
assays
are
developed
to
identify
effects
on
them,
it
is
essential
these
additional
hormones
be
incorporated
into
the
screening
and
testing
program.
For
example,
as
part
of
its
recommendations
for
a
phased
approach
to
implementation,
the
EDSTAC
calls
upon
EPA
to
periodically
evaluate
and,
where
appropriate,
incorporate
new
screens
and
tests,
as
well
as
other
scientific
developments,
into
the
program.
In
addition,
the
EDSTAC
recommends
a
performancebased
approach
to
the
use
of
screens
and
tests,
as
well
as
species
selection.
As
improved
screens,
tests,
and/
or
screens/
tests
utilizing
more
appropriate
species
are
developed
and
validated,
the
EDSTAC
strongly
encourages
their
use.

Evaluate
endocrine
disrupting
properties
of
both
chemical
substances
and
common
mixtures:
The
universe
of
chemicals
to
be
prioritized
for
endocrine
disruptor
screening
and
testing
numbers
more
than
87,000
and
includes
those
listed
in
the
TSCA
Inventory,
active
pesticide
ingredients,
and
ingredients
in
cosmetics
and
food
additives.
In
addition,
EDSTAC
recommends
that
EPA
should
determine
the
technical
feasibility
and,
where
feasible,
should
screen
and
test
representative
samples
of
mixtures
from
six
distinct
types
of
mixtures
(
i.
e.,
combinations
of
two
or
more
chemicals).
The
inclusion
of
these
six
types
of
mixtures
is
to
determine
whether
they
may
have
endocrine
effects,
different
from
those
of
the
individual
component
chemicals,
which
can
only
be
detected
when
tested
as
a
mixture.

III.
Chapter
Three
 
Conceptual
Framework
and
Principles
A.
Conceptual
Framework
Overview
The
conceptual
framework
provides
the
structure
for
the
EDSTAC's
recommendations
for
screening
and
testing
(
see
ES­
1).
The
Committee
determined
that
a
tiered
approach
would
be
most
effective
in
utilizing
reasonably
available
resources
to
detect
endocrine
disrupting
chemicals
and
quantify
their
effects.
The
core
elements
of
the
approach
include
initial
sorting,
priority
setting,
Tier
1
Screening
(
T1S),
and
Tier
2
Testing
(
T2T).
Chapters
Four
and
Five
describe
the
program
aspects
in
more
detail.

Initial
Sorting:
A
chemical
entering
the
framework
would
go
through
initial
sorting
based
on
existing
data.
An
evaluation
and
analysis
of
this
information
would
direct
the
chemical
to
one
of
four
categories.
The
first
would
lead
to
the
"
hold
box"
indicating
the
chemical
is
not
likely
to
interact
with
the
EAT
hormone
systems
and
no
further
analysis
is
required
at
this
time.
The
second
category
contains
chemicals
without
sufficient
data
to
make
a
determination
to
proceed
to
T2T
or
hazard
assessment.
These
chemicals
enter
the
priority
setting
and,
from
there,
the
T1S
portions
of
the
EDSTP.
The
EDSTAC
anticipates
most
chemical
substances
and
mixtures
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
4
entering
the
program
will
fall
into
this
category.
The
third
category
includes
chemicals
with
sufficient
existing
data
to
move
directly
to
T2T
(
i.
e.,
existing
data
meet
Tier
1
requirements).
The
fourth
category
includes
chemicals
with
sufficient
existing
data
to
move
directly
to
hazard
assessment
(
i.
e.,
existing
data
are
adequate
for
both
Tier
1
and
Tier
2
requirements).
In
addition,
the
EDSTAC
has
included
a
voluntary
bypass
scenario,
whereby
the
owner
of
a
chemical
could
voluntarily
go
to
T2T
without
having
completed
the
full
T1S
battery.
This
bypass
option
brings
with
it
a
set
of
requirements
discussed
in
Chapter
Five.

Priority
Setting:
The
term
priority
setting
refers
primarily
to
the
need
to
set
priorities
for
the
chemicals
that
move
into
T1S
(
the
second
category)
after
the
initial
sorting
stage.
Chemicals
will
be
prioritized
based
on
exposure­
related
information,
effects­
related
information,
and
statutory
criteria,
and
then
phased
into
the
program.
This
phasing
of
chemicals
is
recommended
for
practical
reasons
 
the
available
laboratories
and
resources
for
screening
and
testing
cannot
handle
the
large
number
of
chemicals
entering
this
category
simultaneously.

Tier
1
Screening:
T1S
is
designed
to
detect
chemical
substances
and
mixtures
capable
of
interacting
with
the
EAT
hormonal
systems.
Completion
of
this
tier
will
result
in
either
a
decision
to
move
the
chemical
into
T2T
and
serve
to
guide
test
selection
and
dosages,
or
an
indication
that
no
further
analysis
is
needed.
In
the
latter
case,
the
chemical
would
not
be
subjected
to
any
further
screening
or
testing,
at
that
time,
and
would
proceed
to
the
"
hold
box."

Tier
2
Testing:
T2T
is
intended
to
determine
whether
a
chemical
substance
or
mixture
exhibits
endocrine­
mediated
adverse
effects
and
to
identify,
characterize,
and
quantify
those
effects
for
EAT
hormones.
As
with
Tier
1,
there
are
two
possible
outcomes.
If
endocrine
mediated
adverse
effects
are
not
observed,
the
chemical
would
move
to
the
"
hold
box."
If
such
effects
are
observed,
the
information
collected
during
T2T
would
be
used
in
the
Hazard
Assessment
process.
As
results
are
obtained,
additional
data
may
be
required
which
would
require
additional
testing,
especially
to
determine
whether
the
identified
effects
are
endocrinemediated

"
Hold
Box:"
The
EDSTAC
uses
this
term
throughout
the
Report
to
mean
that
either
no,
or
no
further,
endocrine
disruptor
screening
and
testing
is
necessary
for
a
chemical
substance
or
mixture
at
the
time
the
decision
is
made
to
place
the
compound
in
the
"
hold
box."
A
chemical
substance
or
mixture
can
be
placed
in
the
"
hold
box"
at
a
variety
of
points
within
the
recommended
tiered
approach
to
the
EDSTP.
These
include
priority
setting,
T1S,
or
T2T.
As
part
of
the
EDSTAC's
recommendation
for
a
phased
approach
to
implementation,
the
EDSTAC
sets
forth
criteria
to
be
used
during
periodic
evaluations
of
chemical
substances
and
mixtures
placed
in
the
"
hold
box"
to
determine
whether
new
or
additional
screening
and
testing
may
be
necessary.
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
5
Figure
ES.
1.
EDSTAC
Conceptual
Framework
Providing
the
Structure
for
Screening
and
Testing
for
Endocrine
Disruptors
INITIAL
SORTING
Obtain
and
Analyze
Existing
Data
SUFFICIENT
DATA
or
VOLUNTARY
BYPASS
of
Tier
1
Screening
to
go
to
Tier
2
Testing
SET
PRIORITIES
For
Tier
1
Screening
TIER
1
SCREENING
Detect
Interaction
With
Endocrine
System
[
estrogen/
androgen/
thyroid]

TIER
2
TESTING
Determine
and
Characterize
Endocrine
Disrupting
Effects
HAZARD
ASSESSMENT
Yes
Yes
No
No
*
For
a
more
detailed
version
of
the
initial
sorting
and
priority
setting
components
of
this
framework,
please
see
Figure
4.1.
SUFFICIENT
DATA
to
go
to
Hazard
Assessment
HOLD
No
Further
Analysis
Required
at
This
Time
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
6
B.
Recommended
Principles
and
Guidance
To
guide
development
of
the
screening
and
testing
program,
the
EDSTAC
provided
several
sets
of
principles
for
general
development
of
the
conceptual
framework,
including:

Provisions
to
Bypass
Tiers:
The
ordered
sequence
described
above
should
not
exclude
the
possibility
that
a
chemical
substance
or
mixture
could
bypass
one
or
more
tiers
when
information
warrants
such
a
move.
In
addition,
the
EDSTAC
recommends
that
a
voluntary
bypass
of
T1S
should
be
incorporated
into
the
program;
however,
specific
information
requirements
are
recommended
for
those
chemicals.

Proactive
Effort
to
Generate
Adequate
Information:
In
cases
where
existing
information
is
inadequate
to
determine
whether
a
chemical
substance
or
mixture
should
proceed
to
the
next
tier,
there
should
be
an
active
process
for
generating
the
needed
information.
It
is
anticipated
that
the
process
would
be
specifically
tailored
to
the
chemical
under
review.

Moving
Through
the
Program:
Criteria
and
assumptions
for
deciding
whether
a
chemical
should
move
from
one
tier
to
the
next
should
be
developed
in
advance
of
the
initiation
of
screening
and
testing.

The
Committee
also
provided
additional
principles
to:

·
guide
overall
development
of
the
screening
and
testing
strategy;
·
more
specifically
guide
decisions
regarding
the
selection
of
screens
and
tests;
·
guide
how
the
T1S
battery
should
be
designed
and
used;
and,
·
guide
how
the
T2T
battery
should
be
designed
and
used.

IV.
Chapter
Four
 
Priority
Setting
A.
Initial
Sorting
and
Phased
Approach
to
Screening
and
Testing
the
Universe
of
Chemicals
The
EDSTAC
estimates
the
initial
universe
of
chemicals
that
needs
to
be
considered
for
prioritization
for
endocrine
disruptor
screening
and
testing
numbers
approximately
87,000
including:
pesticides,
commodity
chemicals,
naturally
occurring
non­
steroidal
estrogens,
food
additives,
cosmetics,
nutritional
supplements,
and
representative
mixtures.
Simultaneous
screening,
testing,
and
evaluation
of
this
universe
is
far
beyond
the
capabilities
of
available
facilities
and
resources.
Consequently,
the
EDSTAC
recommends
both
an
initial
sorting
of
this
universe
and
a
phased
approach
to
handle
the
chemicals.
This
approach
would
identify
high
priority
chemicals
and
permit
them
to
proceed
through
the
program
first,
followed
by
medium
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
7
priority
chemicals,
and
then
low
priority
ones.
The
phased
approach
also
provides
guidance
to
permit
the
bypassing
of
portions
of
the
screening
and
testing
process
speeding
review
and
evaluation.

Building
on
the
Conceptual
Framework,
the
EDSTAC
recommends
the
universe
of
chemicals
should
undergo
an
initial
sorting
into
the
following
four
categories
(
see
Figure
4.1):

1.
Chemicals
(
primarily
polymers,
as
described
below)
that
are
unlikely
to
have
endocrine
disrupting
effects
that
enter
the
"
hold
box;"
2.
Chemicals
with
insufficient
data
that
will
undergo
HTPS
and
T1S
and,
possibly,
T2T;
3.
Chemicals
with
sufficient
data
to
bypass
T1S
and
go
directly
to
T2T;
and
4.
Chemicals
with
sufficient
data
to
go
directly
to
hazard
assessment.

The
EDSTAC
concluded
that,
in
most
cases,
polymers
with
a
number
average
molecular
weight
(
NAMW)
greater
than
1,000
daltons
are
unlikely
to
be
able
to
cross
biological
membranes
and
barriers
and
would,
therefore,
not
be
biologically
available
to
cause
endocrine­
mediated
effects.
Consequently,
the
Committee
recommends
exempting
the
approximately
25,000
polymers
that
meet
this
NAMW
criterion
from
screening
and
testing
by
placing
them
in
the
"
hold
box,"
pending
review
of
their
components.
The
EDSTAC
further
estimates
that
the
number
of
chemical
substances
that
fall
into
categories
3
and
4
probably
number
no
more
than
1,000.

Thus,
the
EDSTAC
estimates
that
approximately
62,000
chemicals
will
remain
and
need
to
be
at
least
considered
for
screening
and,
if
necessary,
testing.
The
EDSTAC
recognizes,
however,
that
it
is
not
likely
to
be
possible
or
necessary
to
screen
and
test
62,000
chemicals.
Although
the
EDSTAC
incorporated
a
phased
approach
to
implementation
in
order
to
address
the
volume
of
chemicals
ultimately
needing
to
be
screened
and
tested,
the
EDSTAC
did
not
define
the
number
of
phases,
how
long
each
phase
should
be,
or
the
number
of
chemicals
that
should
be
screened
and/
or
tested
in
each
phase.

B.
Information
Useful
for
Prioritization
The
EDSTAC
recommends
that
the
following
types
of
exposure­
and
effects­
related
information
be
used
to
prioritize
chemicals
for
T1S.
Exposure­
related
information
includes:

·
Biological
sampling
data
(
e.
g.,
blood
or
tissue
analyses)
for
humans
and
other
biota;
·
Environmental,
occupational,
consumer
product,
and
food­
related
data;
·
Data
on
environmental
releases;
·
Production
volume;
and
·
Fate
and
transport
data
and
models.
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
8
Sources
of
data
on
effects­
related
information
are:

·
Toxicological
laboratory
studies
and
databases;
·
Epidemiological
and
field
studies
and
databases;
·
Predictive
biological
activity
or
effects
models
(
e.
g.,
SARs,
QSARs);
and
·
Results
of
high
throughput
pre­
screening.

The
EDSTAC
presents
a
detailed
discussion
of
the
strengths
and
limitations,
as
well
as
guiding
principles,
for
using
each
of
these
information
categories
for
the
purpose
of
priority
setting.

C.
High
Throughput
Pre­
Screening
The
EDSTAC
recognized
that
biological
effects
data
are
incomplete
or
lacking
for
most
chemicals,
a
condition
which
makes
priority
setting
difficult.
To
help
address
this
problem,
the
EDSTAC
recommends
that
some
of
the
T1S
assays
be
conducted
in
a
high­
speed,
automated
fashion
to
provide
preliminary
hormonal
or
biological
activity
information.
This
approach
is
called
"
high
throughput
pre­
screening"
(
HTPS)
where,
rather
than
following
traditional
manual
sample
preparation,
handling,
and
analysis
procedures,
automated
techniques
and
robotics
are
used
to
accelerate
the
assay
process.
Such
a
process
permits
a
large
volume
of
chemicals
to
be
tested
in
a
short
period
of
time.
The
EDSTAC
recommends
that
HTPS
be
conducted
on:
(
1)
all
chemicals
with
current
production
volumes
greater
than
10,000
pounds
per
year
(
estimated
to
be
approximately
15,000
chemicals);
(
2)
all
pesticide
active
ingredients
and
formulation
inerts
;
and
(
3)
all
chemicals
that
are
proposed
to
bypass
either
T1S
or
both
T1S
and
T2T
for
any
reason.

The
EDSTAC
recommends
that
the
T1S
in
vitro
transcriptional
activation
assays
be
modified
and
validated
for
use
in
the
high
throughput
mode.
It
further
recommends
that,
when
used
in
the
screening
and
testing
program,
the
HTPS
assays
should:

·
provide
information
about
the
ability
of
chemicals
to
bind
to
the
estrogen,
androgen,
or
thyroid
hormone
receptors;
·
be
used
with
other
exposure­
and
effects­
related
data
in
prioritizing
chemicals
for
T1S;
·
improve
QSAR
predictive
models;
and
·
provide
information
to
assist
in
the
design
of
the
tests
in
T2T
for
chemicals
bypassing
T1S.

The
EDSTAC
has
recommended,
and
EPA
has
already
initiated,
a
feasibility
demonstration
pilot
program
be
created
to
assess
the
proposed
use
of
HTPS.
The
EDSTAC's
recommendations
are,
therefore,
contingent
upon
technical
feasibility
of
the
HTPS
technology
and
successful
standardization
and
validation
of
the
HTPS
assays.
If
HTPS
is
technically
feasible
and
validation
is
successful,
the
EDSTAC
believes
that
HTPS
can
be
a
powerful,
cost
effective
tool
in
the
EDSTP.
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
9
D.
Endocrine
Disruptor
Priority
Setting
Database
The
EDSTAC
recommends
that
data
used
as
the
basis
for
sorting
and
priority
setting
be
organized
into
a
relational
database
called
the
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD).
Development
of
this
database
was
initiated
by
the
EDSTAC
but,
due
to
time
and
resource
limitations,
was
not
completed.
The
EDSTAC
recommends
that
EPA
complete
and
maintain
the
EDPSD.
The
EDPSD
can
be
an
invaluable
resource
for
initial
sorting
and
priority
setting
provided
it
contains
current
data
and
its
use
is
simple
and
open
to
review
by
all.

The
EDSTAC
recommends
that
the
EDPSD
development
proceed
in
three
stages:

1.
capture
data
from
existing
databases
such
as
use
data
on
fate,
transport,
and
toxicity;
2.
use
data
not
readily
available
in
existing
databases
such
as
chemicals
and
concentrations
from
the
National
Health
and
Nutrition
Examination
Survey
and
the
Hazardous
Substances
Emergency
Events
Surveillance;
and
3.
incorporate
HTPS
data
and
improved
Quantitative
Structure
Activity
Relationships
(
QSAR)
models.

E.
Recommended
Approach
to
Priority
Setting
1.
Compartment­
Based
Approach
to
Priority
Setting
for
T1S
In
general,
the
EDSTAC
recommends
that
whatever
priority
setting
approach
is
used
by
EPA
it
should
be
open
and
simple.
The
EDSTAC
further
recommends
that,
while
the
process
should
be
driven
by
empirical
data,
it
should
allow
for
chemicals
of
concern,
which
have
less
data,
especially
less
effects­
related
data,
to
be
included
in
the
higher
priority
rankings.

Based
on
these
principles,
the
EDSTAC
recommends
that
EPA
use
a
"
compartment­
based"
approach
to
priority
setting
that
builds
directly
upon
the
recommended
exposure­
and
effectsrelated
information
categories
described
above.
The
term
"
compartment­
based"
refers
to
an
approach
whereby
different
combinations
of
information,
and
criteria
that
flow
from
this
information,
are
used
to
generate
a
set
of
priorities
for
each
phase
of
the
program.
The
EDSTAC
recommends
four
broad
categories
of
compartments
should
be
developed,
including
those
that:
(
1)
would
be
based
on
the
integration
of
exposure
and
effects
information
and
criteria;
(
2)
rely
only
on
exposure­
related
information
and
criteria;
(
3)
rely
only
on
effects­
related
information
and
criteria;
and
(
4)
focus
on
special
compartments
of
chemicals.

While
the
EDSTAC
has
not
agreed
upon
specific
compartments,
nor
the
order
in
which
they
should
be
used
in
priority
setting,
it
did
provide
a
number
of
illustrations
to
show
how
the
compartment­
based
approach
might
be
used
in
practice.
The
EDSTAC
also
made
the
following
recommendations
for
the
development
of
the
compartment­
based
approach:
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
10
·
have
a
multi­
stakeholder
group
use
the
EDPSD
to
characterize
and
define
what
will
be
included
in
each
compartment;
·
determine
whether
and,
if
so,
how
the
compartments
should
be
weighted;
and
·
address
the
possibility
of
overlaps
between
compartments.
2.
Special
Compartments
of
Chemicals
The
EDSTAC
identified
a
number
of
specially
targeted
compartments
for
purposes
of
priority
setting
which
include:

Nominations:
The
priority
setting
process
recommended
by
the
EDSTAC
will
give
high
priority
to
chemicals
with
widespread
exposure
at
the
national
level.
However,
there
are
chemicals
that
result
in
disproportionately
high
exposure
to
identifiable
groups,
communities,
or
ecosystems.
The
EDSTAC
recommends
EPA
establish
a
parallel
but
separate
priority
setting
process
where
chemicals
with
regional
or
local
exposure
can
be
nominated
by
affected
citizens
to
receive
a
priority
for
T1S.
The
EDSTAC
recommends
that
a
goal
for
each
phase
of
the
EDSTP
is
for
no
less
than
5%
of
the
total
number
of
chemicals
be
drawn
from
those
that
are
nominated
but
not
otherwise
selected
in
the
core
process
as
a
high
priority
for
T1S.
The
EDSTAC
recognizes
that
the
total
number
of
nominations
or
their
quality
may
be
such
that
this
goal
cannot
be
met
in
specific
phases.
The
EDSTAC
further
recommends
the
use
of
a
specialized
set
of
criteria
for
prioritizing
nominated
chemicals
that
would
focus
primarily
on
exposure
and,
secondarily,
on
available
effects
data.
The
EDSTAC
also
sets
forth
recommendations
on
the
procedures
EPA
should
use
for
submitting
a
nomination
and,
where
necessary,
protecting
the
identity
of
the
person
submitting
the
nominations.

Mixtures:
The
EDSTAC
felt
that
mixtures,
defined
as
a
combination
of
two
or
more
chemicals,
needed
special
attention
during
the
initial
stages
of
sorting
and
prioritization.
Consequently,
the
EDSTAC
recommends
that
EPA
should
determine
the
technical
feasibility
and,
where
feasible,
should
screen
and
test
representative
samples
of
mixtures
from
six
distinct
types
of
mixtures,
including:

·
contaminants
in
human
breast
milk;
·
phytoestrogens
in
soy­
based
infant
formula;
·
mixtures
of
chemicals
commonly
found
at
hazardous
waste
sites;
·
pesticide/
fertilizers
mixtures;
·
disinfection
byproducts;
and
·
gasoline.

Some
of
the
technical
challenges
for
screening
and
testing
mixtures
mirror
those
of
single
compounds,
however,
the
EDSTAC
acknowledges
that
the
technical
feasibility
of
screening
and
testing
mixtures
is
by
no
means
certain.
Technical
feasibility
for
screening
and
testing
mixtures
will
include
an
evaluation
of
whether
it
is
possible
to
identify
a
reasonable
number
of
representative
samples
of
mixtures
from
each
of
the
recommended
six
types
of
mixtures,
as
well
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
11
as
the
ability
to
send
the
representative
samples
of
mixtures
through
HTPS,
T1S,
and
T2T
depending
on
their
physical
properties,
and
validation
and
standardization
of
the
results.

Naturally
Occurring
Non­
Steroidal
Estrogens
(
NONEs):
Another
special
class
of
chemicals
of
concern
to
the
EDSTAC
are
naturally
occurring
non­
steroidal
estrogens.
These
are
natural
products
derived
from
plants
(
phytoestrogens)
and
fungi
(
mycotoxins).
These
chemicals
occur
widely
in
foods
and
have
the
potential
to
act
in
an
additive,
synergistic,
or
antagonist
fashion
with
other
hormonally
active
chemicals.
EDSTAC
recommends
that
EPA
include
representatives
from
the
seven
chemical
classes
of
NONEs
in
the
EDSTP.
3.
Recommended
Approach
to
Priority
Setting
for
T2T
While
the
EDSTAC
recommended
a
compartment­
based
approach
to
setting
priorities
for
T1S,
it
also
recommended
that
priority
setting
for
T2T
for
chemicals
that
bypass
T1S
should
be
done
as
follows:

·
Food
use
pesticides
should
use
the
schedule
EPA
has
established
for
tolerance
reassessments
and
pesticide
re­
registration
under
the
FQPA.
·
For
all
other
chemicals
voluntarily
bypassing
T1S,
priorities
should
be
established
on
a
case­
specific
basis
using
all
available
information,
including
the
priority
ranking
for
T1S.
The
EDSTAC
recommends
that
priority
setting
for
these
chemicals
should
be
generally
driven
by
the
same
priorities
as
those
set
during
the
priority
setting
phase
of
the
EDSTP,
and
that
voluntary
action
on
the
part
of
owners/
producers
should
expedite,
but
not
delay,
testing.

The
EDSTAC
did
not
develop
an
explicit
set
of
recommendations
for
how
to
set
priorities
for
chemicals
that
produce
positive
results
in
T1S
and
must
move
forward
into
T2T.

V.
Chapter
Five
 
Screening
and
Testing
A.
Tier
1
Screening
1.
Recommended
Screening
Assays
The
screening
tier
of
the
Conceptual
Framework
detects
whether
a
chemical
substance
or
mixture
may
interact
with
the
endocrine
system
for
estrogen,
androgen,
and
thyroid
hormones.
The
EDSTAC
developed
several
criteria
to
guide
the
selection
of
T1S
assays:

·
maximize
sensitivity
which
serves
to
minimize
false
negatives;
·
include
a
range
of
organisms
representing
differences
in
metabolism;
·
detect
all
known
modes
of
action
for
the
endocrine
endpoints
of
concern;

·
include
a
sufficient
range
of
taxonomic
groups
among
the
test
organisms;
and
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
12
·
incorporate
sufficient
diversity
among
the
endpoints,
permitting
weight­
of­
evidence
conclusions.

The
EDSTAC
recommends
both
in
vitro
and
in
vivo
assays
be
included
in
T1S.
Each
type
of
assay
has
strengths
and
weaknesses
but
when
used
in
combination,
the
weaknesses
can
be
minimized.
Those
chemical
substances
and
mixtures
which
go
through
the
HTPS
program,
if
it
is
technically
feasible
and
validated,
would
not
be
required
to
do
the
first
two
in
vitro
assays
at
the
bench.
The
recommended
assays
include:

·
In
Vitro
Assays
·
Estrogen
Receptor
Binding/
Reporter
Gene
Assay
·
Androgen
Receptor
Binding/
Reporter
Gene
Assay
·
Steroidogenesis
Assay
with
minced
testis
·
In
Vivo
Assays
·
Rodent
3­
day
Uterotrophic
Assay
·
Rodent
20­
day
Pubertal
Female
with
thyroid
·
Rodent
5­
7­
day
Hershberger
Assay
·
Frog
Metamorphosis
Assay
·
Fish
Gonadal
Recrudescence
Assay
The
battery
of
T1S
assays
is
designed
to
work
as
a
whole.
The
EDSTAC
believes
that
data
from
all
the
assays
are
necessary
if
EPA
is
to
make
accurate
decisions
about
the
chemicals
that
are
screened.
The
Committee
also
believes
this
battery
of
assays
meet
all
the
established
criteria
for
T1S
but
the
assays
must
be
validated
and
standardized
before
final
inclusion
in
the
T1S
battery.
At
present
no
T1S
assay
is
fully
validated.

The
EDSTAC
believes
the
recommended
T1S
battery,
if
validated,
will
have
the
necessary
breadth
and
depth
to
detect
any
currently
known
endocrine
disruptors.
The
EDSTAC
recognizes
that
chemical
substances
or
mixtures
might
produce
effects
from
prenatal/
pre­
hatch
exposure
that
would
not
be
detected
from
pubertal
or
adult
exposures.
To
address
these
concerns,
the
EDSTAC
recommends
that
EPA
take
affirmative
steps,
in
collaboration
with
industry
and
other
interested
parties
to
attempt
to
develop
the
protocol
for
a
full
life
cycle
(
i.
e.,
with
embryonic
exposure
and
evaluation
of
the
adult
offspring)
developmental
exposure
screening
assay
that
can
be
subjected
to
validation
and
standardization.

The
EDSTAC
also
recommends
four
assays
be
examined
as
possible
alternatives
to
some
of
those
proposed
above:

·
In
Vitro
·
Placental
Aromatase
Assay
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
13
·
In
Vivo
·
Modified
Rodent
3­
day
Uterotrophic
Assay
(
intraperitoneal
dosing);
·
Rodent
14­
day
Intact
Adult
Male
Assay
with
thyroid;
and
·
Rodent
20­
day
Thyroid/
Pubertal
Male
Assay.

These
alternatives
should
also
be
included
in
the
validation
and
standardization
program.
If
they
are
at
least
as
sensitive
as
the
assays
proposed
for
the
T1S
battery,
they
might
replace
some
of
the
recommended
assays,
particularly
if
they
offer
reductions
in
time,
cost,
and
complexity
of
T1S.
2.
Criteria
for
Evaluating
Tier
1
Results
The
EDSTAC
recommends
that
a
weight­
of­
evidence
approach
be
used
to
evaluate
T1S
results
and
make
decisions
about
going
on
to
T2T.
This
approach
would
include:
(
1)
the
balance
of
positive
and
negative
responses
observed
in
both
the
in
vitro
and
in
vivo
assays;
(
2)
the
nature
and
range
of
the
biological
effects
observed;
(
3)
the
shape
of
the
dose­
response
curves;
(
4)
the
severity
and
magnitude
of
effects
induced;
and
(
5)
the
presence
or
absence
of
response
in
multiple
taxa.
Ten
principles
were
recommended
for
evaluating
the
T1S
results
under
a
weight­
ofevidence
approach.

The
evaluation
of
T1S
data,
and
other
available
information
(
e.
g.,
HTPS
or
literature
data),
will
result
in
a
decision
either
that
the
chemical
needs
no
further
STET
and
can
move
to
the
"
hold
box"
or
that
the
chemical
needs
to
be
tested
in
Tier
2.

B.
Tier
2
Testing
1.
Test
Selection
The
purpose
of
T2T
is
to
determine
whether
a
chemical
substance
or
mixture
exhibits
endocrine­
mediated
adverse
effects
and
to
identify,
characterize,
and
quantify
those
effects
for
EAT
hormones.
The
EDSTAC
identified
three
principles
to
guide
selection
of
tests
for
inclusion
in
T2T:

·
Tests
must
include
the
most
sensitive
developmental
lifestage.
·
Tests
must
identify
the
specific
hazard
caused
by
the
chemical
and
establish
a
doseresponse
relationship.
·
A
range
of
taxa
must
be
included
in
Tier
2
tests.
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
14
With
these
criteria
in
mind,
the
EDSTAC
recommends
that
the
following
tests
be
incorporated
into
Tier
2:

·
Two­
Generation
Mammalian
Reproductive
Toxicity
Study
or
a
less
comprehensive
test
(
e.
g.,
Alternative
Mammalian
Reproductive
Test)
·
Avian
Reproduction
Test
·
Fish
Life
Cycle
Test
·
Mysid
Life
Cycle
Test
·
Amphibian
Development
and
Reproduction
Test
As
with
T1S,
the
battery
of
Tier
2
tests
is
designed
to
work
as
a
whole.
The
outcome
of
T2T
is
designed
to
be
conclusive
in
relation
to
the
outcome
of
T1S,
and
any
other
prior
information,
in
the
sense
that
a
negative
outcome
in
T2T
will
supersede
a
positive
outcome
in
T1S.
Furthermore,
each
full
test
in
T2T
has
been
designed
to
include
those
endpoints
that
will
allow
one
to
reach
a
definitive
conclusion
as
to
whether
or
not
the
tested
chemical
substance
or
mixture
is
or
is
not
an
endocrine
disruptor
for
EAT
in
that
species/
taxa.
Conducting
all
five
tests
in
the
T2T
battery
would
provide
a
more
comprehensive
profile
of
the
effects
a
chemical
substance
or
mixture
could
induce
via
EAT
endocrine
disruption
mode(
s)/
mechanism(
s)
of
action
than
would
be
the
case
if
only
a
subset
of
tests
were
performed.
The
EDSTAC
recommends
that
the
"
default"
action,
in
the
absence
of
any
prior
information,
should
be
to
perform
all
tests
in
the
T2T
battery
with
all
endpoints.

However,
performance
of
the
entire
battery
with
multiple
generations
may
not
always
be
necessary.
Therefore,
the
EDSTAC
developed
guidance
in
the
selection
of
Tier
2
tests,
focusing
upon:
(
1)
the
determination
of
which
of
the
five
taxonomic
groups
should
be
included
in
the
Tier
2
testing
of
a
specific
chemical
substance
or
mixture;
(
2)
the
circumstances
under
which
it
may
be
appropriate
to
perform
an
alternative
test,
with
a
particular
focus
on
the
selection
of
alternative
mammalian
tests;
(
3)
the
selection
of
endpoints;
(
4)
the
special
case
of
chemicals
that
bypass
T1S
and
go
directly
to
T2T;
and
(
5)
the
potential
need
for
supplemental
information
to
complete
T2T.

The
Committee
believes
that
while
this
battery
of
assays
meets
all
the
established
T2T
criteria
and
objectives,
the
test
protocols
must
be
validated
and
standardized
before
final
incorporation
into
the
screening
and
testing
program.
At
present,
none
of
the
new
tests
or
enhancements
to
existing
test
guidelines
are
fully
validated
or
standardized.
2.
Low
Dose
Issues
in
T2T
The
EDSTAC
recognized
that
questions
have
been
raised
as
to
the
adequacy
of
conventional
toxicology
study
designs
for
assessment
of
endocrine
active
substances,
particularly
with
regard
to
low
dose
selection
and
the
identification
of
no­
observed­
adverse­
effect­
levels
(
NOAEL).
To
address
these
questions,
the
EDSTAC
recommends
that
a
project
be
performed
to
resolve
the
underlying
uncertainties
and
controversy
about
these
issues.
The
purpose
of
the
project
is
to
address
the
nature
of
the
dose­
response
curves
for
exogenous
estrogenic
substances
in
order
to
allow
more
informed
judgments
about
appropriate
toxicology
study
designs
for
substances
that
have
hormonal
activity.
In
addition,
the
EDSTAC
summarized
their
preliminary
discussions
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
15
regarding
the
exact
design
of
the
research
program
and
laid
out
implications
and
corresponding
actions
to
be
taken
based
upon
the
possible
results
of
the
research.

C.
Validation,
Standardization,
Methods
Development,
and
Research
The
EDSTAC
believes
validation
and
standardization
of
the
recommended
screens
and
tests
are
essential
for
implementation
of
the
EDSTP.
EDSTAC
also
believes
the
validation
and
standardization
program
is
of
highest
priority,
and
recommends
that
it
proceed
on
an
accelerated
schedule.
The
EDSTAC
recommends
that
the
validation
and
standardization
program
be
consistent
with
the
principles
articulated
by
the
national
(
ICCVAM,
1996;
Zeiger,
1998)
and
international
(
OECD,
1996)
alternative
methods
validation
groups.
As
mentioned
throughout
Chapter
Five,
each
assay
and
test
recommended
for
T1S
or
T2T
needs
some
level
of
validation,
standardization,
methods
development,
or
further
research
before
being
accepted
as
a
regulatory
toxicity
screen
or
test
for
inclusion
in
the
EDSTP.
The
level
of
effort
needed
to
fully
validate
and
standardize
may
be
different
for
each
individual
assay
or
test
(
including
all
recommended
endpoints)
for
each
individual
assay
or
test.
The
effort
required
for
each
assay
or
test
will
be
defined
by
a
variety
of
criteria
including:
period
of
time
in
use,
existing
level
of
general
acceptance
in
the
endocrine
toxicology
field,
and
existing
understanding
of
relevancy
and
reliability.
Regardless
of
the
effort
required,
the
EDSTAC
believes
all
of
the
assays
and
tests
recommended
for
T1S
and
T2T
must
be
fully
validated
and
standardized
before
being
included
in
the
EDSTP.
The
EDSTAC
recommends
that
as
individual
assays
and
tests
are
validated
and
standardized,
they
can
be
utilized
in
the
EDSTP
without
waiting
for
all
assays
and
tests
in
the
batteries
to
be
validated.
EDSTAC
further
recommends
that
a
multi­
stakeholder
process,
involving
government,
industry,
and
academia,
be
utilized
in
validating
and
standardizing
the
T1S
and
T2T
batteries.

VI.
Chapter
Six
 
Communications
and
Outreach
A.
Principles
to
Guide
a
Communications
and
Outreach
Strategy
Good
communication
is
essential
for
the
success
of
the
screening
and
testing
program.
Particular
care
is
needed
to
ensure
that,
to
the
extent
possible,
potential
misuse
of
information
generated
by
the
EDSTP
does
not
occur.
EPA
should
develop
a
strategy
for
clear
and
accurate
communication
to
all
the
stakeholders
during
the
development
and
implementation
of
the
screening
and
testing
program.
It
is
of
particular
importance
that
EPA
clearly
communicate
the
limitations
that
must
be
placed
on
the
results
of
the
screening
and
testing
as
well
as
the
meaning
and
implications
of
the
decisions
made
by
the
Agency
based
upon
these
results.
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
16
The
EDSTAC
recommends
that
five
principles
be
used
in
developing
a
communication
and
outreach
strategy
for
the
screening
and
testing
program:

·
the
process
and
results
of
the
program
should
be
clear
and
open;
·
the
results
should
be
communicated
within
the
context
set
forth
in
the
EDSTAC
final
report;
·
the
limitations
and
uncertainties
associated
with
the
available
data
and
the
screening
and
testing
results
should
be
clearly
articulated;
·
any
changes
in
the
program
should
be
promptly
and
clearly
communicated;
and
·
a
quality
assurance
program
should
be
developed
to
ensure
that
the
Endocrine
Disruptor
Priority
Setting
Database
is
current
and
accurate.

B.
Basic
Features
of
a
Communications
and
Outreach
Strategy
The
EPA
strategy
should
address:
(
1)
what
should
be
communicated;
(
2)
to
whom
it
should
be
communicated;
(
3)
how
it
should
be
communicated;
and
(
4)
when
it
should
be
communicated.
Specifically,
the
EDSTAC
recommends
that
EPA
communicate
information
on
the
screening
and
testing
approach,
the
status
and
results
for
chemicals
that
have
been
evaluated
in
the
program,
and
the
nominations
process.
This
information
should
be
communicated
to
all
stakeholders
who
have
expressed
an
interest
in
the
program,
including
the
general
public.
To
facilitate
communications,
it
is
recommended
that
EPA
tailor
the
information
to
specific
target
audiences.
Some
examples
of
target
audiences
include,
but
are
not
limited
to,
farm
workers,
environmental
justice
organizations,
or
industries
that
formulate
products
but
do
not
manufacture
the
component
chemicals,
often
referred
to
as
"
downstream"
industries.

The
Committee
recommends
information
be
communicated
in
a
variety
of
ways.
EPA
should
develop
a
tracking
system
for
chemicals
entering
the
EDSTP
that
is
compatible
and
fully
integrated
with
the
Endocrine
Disruptor
Priority
Setting
Database
described
in
Chapter
Four.
This
database
should
not
exist
in
isolation;
rather
it
should
be
integrated
into
those
being
developed
elsewhere
in
the
Agency.
The
tracking
system
should
be
designed
to
enable
the
public
to
have
quick
access
to
determine
the
status
of
a
chemical.
Access
to
information
about
the
EDSTP
should
be
available
via
Internet,
telephone,
fax,
mail,
and
the
Federal
Register.

Finally,
information
should
be
made
available
on
a
regular
basis
using
a
bulletin
or
newsletter
of
limited
length.
This
update
could
summarize
the
status
of
the
overall
program
and
individual
chemicals
that
have
entered
it
as
well
as
important
developments
or
changes
in
the
program.
It
could
also
be
a
vehicle
where
EPA
could
issue
a
call
for
nominations.
EPA
will
need
to
commit
adequate
resources
to
implement
a
newsletter
and
the
other
EDSTAC
communications
and
outreach
recommendations.
EDSTAC
Final
Report
Executive
Summary
August
1998
ES
­
17
VI.
Chapter
Seven
 
Compilation
of
EDSTAC
Recommendations
Chapter
Seven
includes
all
of
the
Committee's
recommendations
made
in
Chapters
Three,
Four,
Five,
and
Six.
Each
set
of
recommendations
can
also
be
found
at
the
end
of
their
respective
chapters.
EDSTAC
Final
Report
Chapter
One
August
1998
Chapter
One
Introduction
EDSTAC
Final
Report
Chapter
One
August
1998
Table
of
Contents
I.
Overview
................................................................................................................................
1
II.
The
EDSTAC's
Origin.........................................................................................................
1
III.
About
the
EDSTAC
Report
...............................................................................................
2
Appendices
Appendix
A:
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
Participant
List
Appendix
B:
Principles
Work
Group
Participant
List
Appendix
C:
Priority
Setting
Work
Group
Participant
List
Appendix
D:
Screening
and
Testing
Work
Group
Participant
List
Appendix
E:
Communications
and
Outreach
Work
Group
Participant
List
EDSTAC
Final
Report
Chapter
One
August
1998
1
­
1
I.
Overview
A
growing
body
of
scientific
research
indicates
that
man­
made
industrial
chemicals
and
pesticides
may
interfere
with
the
normal
functioning
of
human
and
wildlife
endocrine,
or
hormone,
systems.
These
endocrine
disruptors
may
cause
a
variety
of
problems
with
development,
behavior,
and
reproduction.

Although
many
pesticides,
and
some
industrial
chemicals,
have
undergone
extensive
toxicological
testing,
this
testing
may
have
been
inadequate
to
determine
whether
they
interact
with
the
endocrine
system
and
whether
additional
testing
is
needed
for
the
U.
S.
Environmental
Protection
Agency
(
EPA)
to
assess
and
characterize
both
human
health
and
ecological
risk.
Notwithstanding
recognition
that
the
scientific
knowledge
related
to
endocrine
disruptors
is
still
evolving,
there
is
appropriate
widespread
agreement
that
the
development
of
a
screening
and
testing
program
is
needed.

This
report
contains
the
consensus
recommendations
of
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC).
This
chapter
describes
the
origin
of
the
EDSTAC,
including
its
mission,
purpose,
composition,
and
outcome.
This
chapter
also
describes
the
work
groups
established
by
the
EDSTAC
and
the
other
chapters
of
the
report,
which
are
the
products
of
these
work
groups
and
the
Committee
as
a
whole.

II.
The
EDSTAC's
Origin
Reflecting
increasing
scientific
knowledge
about,
and
concern
for,
endocrine
disruption,
EPA
convened
a
workshop
in
April
1995
to
craft
a
strategy
for
assessing
the
risk
of
endocrine
disruption
and
to
define
research
needs
in
the
areas
of
human
and
ecological
effects.
A
second
workshop
was
convened
in
June
1995
to
further
define
the
research
needs
for
ecological
effects.

In
May
1996,
EPA
sponsored
a
stakeholder
meeting
to
further
develop
its
response
to
the
issue.
Attendees
urged
the
Agency
to
address
screening
and
testing
issues,
and
stressed
the
essential
need
for
broad
stakeholder
involvement
in
what
was
recognized
as
an
evolving
program.
Three
months
later,
in
August
1996,
Congress
passed
both
the
Food
Quality
Protection
Act
(
FQPA)
and
amendments
to
the
Safe
Drinking
Water
Act
(
SDWA).
Both
of
these
laws
contained
provisions
calling
for
the
screening
and
testing
of
chemicals
and
pesticides
for
possible
endocrine
disrupting
effects.
Specifically,
these
laws
require
EPA
to:

develop
a
screening
program,
using
appropriate
validated
test
systems
and
other
scientifically
relevant
information,
to
determine
whether
certain
substances
may
have
an
effect
in
humans
that
is
similar
to
an
effect
produced
by
a
naturally
occurring
estrogen,
or
other
such
endocrine
effect
as
the
Administrator
may
designate.
These
laws
required
EPA
to
develop
a
screening
program
by
August
1998,
to
implement
the
program
by
August
1999,
and
to
report
to
Congress
on
the
program's
progress
by
August
2000.
EDSTAC
Final
Report
Chapter
One
August
1998
1
­
2
As
a
result
of
the
May
1996
meeting
and
the
passage
of
the
FQPA
and
the
SDWA,
EPA
formed
the
EDSTAC.
EPA
charged
the
EDSTAC
with
providing
advice
to
the
Agency
on
how
to
design
a
screening
and
testing
program
for
endocrine
disrupting
chemicals.
In
part
because
deliberations
about
forming
the
EDSTAC
predated
enactment
of
the
FQPA
and
the
amendments
to
the
SDWA,
both
EPA
and
the
EDSTAC
itself
decided
not
to
limit
the
Committee's
deliberations
to
the
types
of
chemicals,
hormonal
systems,
or
effects
specifically
covered
under
these
statutes.
The
scope
of
the
EDSTAC's
effort
is
further
explained
in
Chapter
Three,
which
sets
forth
the
Conceptual
Framework
within
the
recommendations
of
the
following
chapter.

The
EDSTAC
was
composed
of
individuals
representing
various
stakeholder
groups
and
scientific
expertise.
The
members
included
scientists
and
other
representatives
from:
EPA,
other
federal
agencies,
state
agencies,
various
sectors
of
industry,
water
providers,
worker
protection,
national
environmental
groups,
environmental
justice
groups,
public
health
groups,
and
research
scientists.
Committee
members
were
asked
by
EPA
to
serve
as
members
of
the
EDSTAC,
following
a
fourmonth
convening
process
conducted
by
the
facilitation
team.
A
list
of
Committee
members
and
alternates
is
provided
in
Appendix
A.

As
a
federally
chartered
advisory
committee,
all
EDSTAC
plenary
meetings
were
open
to
the
public.
A
total
of
ten
Committee
meetings
were
held,
starting
with
an
organizational
meeting
in
October
1996
and
the
final
plenary
in
June
1998.
The
majority
of
these
plenary
meetings
were
held
in
different
locations
across
the
country,
including
San
Francisco,
Houston,
Baltimore
Chicago,
New
York,
Orlando
and
Washington,
D.
C.
Numerous
work
group
meetings
and
conference
calls
were
also
convened.
Public
comment
sessions
were
held
at
seven
of
the
ten
Committee
meetings
in
order
to
provide
members
of
the
public
an
opportunity
to
comment
to
Committee
members
about
the
EDSTAC
process
and
development
of
the
screening
and
testing
program.
A
wide
diversity
of
constituents
expressed
interest
in
the
actions
of
the
Committee
and
the
issue
of
endocrine
disruptors,
including:
advocacy
organizations,
disease­
impacted
groups,
environmental
groups,
environmental
justice
networks,
farmers
and
farm
workers,
governmental
organizations,
industry,
environmental
and
health
non­
governmental
organizations
(
NGOs),
trade
unions,
students,
affected
or
"
downstream"
industries,
and
concerned
citizens.

The
Committee
organized
itself
into
four
work
groups:
the
Principles
Work
Group,
the
Priority
Setting
Work
Group
(
PSWG),
the
Screening
and
Testing
Work
Group
(
STWG),
and
the
Communications
and
Outreach
Work
Group
(
COWG).
Work
groups
were
facilitated
by
members
of
the
facilitation
team
with
technical
assistance
from
EPA.
Each
work
group
consisted
of
Committee
members,
as
well
as
other
individuals
who
were
not
members
of
the
Committee
but
who
were
asked
to
participate
in
the
EDSTAC
process
because
of
their
particular
expertise
and
perspective.
A
list
of
the
members
for
each
of
these
work
groups
is
included
in
Appendices
B
(
Principles),
C
(
PSWG),
D
(
STWG),
and
E
(
COWG).

III.
About
the
EDSTAC
Report
EDSTAC
Final
Report
Chapter
One
August
1998
1
­
3
The
EDSTAC
Report
was
developed
through
a
deliberative
process
that
encouraged
the
development
of
consensus
solutions
to
complex
problems
and
issues
at
both
the
work
group
and
Committee
levels.
The
work
groups
were
the
primary
drafters
of
the
chapters
for
the
final
report.
Discussion
papers
and
drafts
of
these
chapters
were
presented
by
the
work
groups
to
the
Committee.
The
Committee
then
discussed
the
issues
raised
by
these
discussion
papers
and
drafts
and
developed
the
final
consensus,
which
is
reflected
in
this
report.

Chapter
Two
of
this
report
provides
the
reader
with
background
information
on
the
function
of
the
endocrine
system,
the
issue
of
endocrine
disruptors,
and
the
complex
statutory
and
chemical
universe
within
which
priority
setting
and
screening
and
testing
must
be
accomplished.
The
chapter
is
intended
to
provide
a
context
for
those
individuals
not
well­
versed
in
either
the
scientific
or
regulatory
basis
of
this
very
technical
issue.
It
is
hoped
that
this
chapter
will
provide
the
reader
with
an
understanding
of
the
basis
for
the
EDSTAC's
recommendations
that
follow.

The
EDSTAC
formed
the
Principles
Work
Group
to
further
develop
and
refine
a
set
of
principles
that
the
Committee
"
brainstormed"
at
its
first
plenary
meeting
in
San
Francisco.
The
Principles
Work
Group
helped
to
create
a
document
that
was
called
the
EDSTAC
Conceptual
Framework.
This
document,
which
was
made
public
in
May
1997,
has
been
revised
slightly
from
the
original
version
and
is
now
included
as
Chapter
Three
of
the
EDSTAC's
final
report.
Initially,
the
Conceptual
Framework
was
intended
to
inform,
focus,
facilitate,
and
expedite
the
work
of
the
EDSTAC
work
groups.
In
its
finalized
form,
the
goal
of
the
EDSTAC
Conceptual
Framework
is
to
provide
broad
guidance
to
EPA
regarding
the
development
and
implementation
of
its
endocrine
disruptor
screening
and
testing
strategy.

Chapter
Four
addresses
the
need
to
set
priorities
for
endocrine
disruptor
screening
and
testing,
and
builds
upon
the
information
contained
in
Chapter
Two
regarding
the
universe
of
chemicals
that
need
to
be
considered
for
endocrine
disruptor
screening
and
testing.
Chapter
Four
also
shows
how
various
complexities
are
addressed
in
the
recommendations
for
sorting
and
priority
setting.
The
PSWG
was
charged
by
the
EDSTAC
to
address
the
following
tasks:

·
specify
types
of
information
that
should
be
gathered
and
analyzed
to
sort
and
prioritize
chemical
substances
and
mixtures
for
screening
and
testing;
·
develop
criteria
for
evaluating
the
quality,
adequacy,
and
reliability
of
the
information
that
will
be
used
in
sorting
and
prioritizing
chemical
substances
and
mixtures
for
screening
and
testing;
·
develop
criteria
for
sorting
chemical
substances
and
mixtures
into
four
possible
next
steps,
including:
(
1)
hold
screening
and
testing;
(
2)
prioritize
for
Tier
1
Screening
(
T1S);
(
3)
go
to
Tier
2
Testing
(
T2T);
or
(
4)
go
to
hazard
assessment;
·
develop
criteria
for
setting
priorities
for
T1S.
These
criteria
will
address
the
relative
order
of
priority
in
which
chemical
substances
that
are
sorted
into
this
category
will
actually
proceed
to
T1S;
and
·
suggest
how
information
used
for
priority
setting
should
be
combined
with
screening
and
testing
results
to
generate
a
"
weight­
of­
evidence"
determination
for
proceeding
from
screening
to
testing
or
from
testing
to
hazard
assessment.

Chapter
Five
describes
the
EDSTAC
recommendations
regarding
development
of
a
screening
and
EDSTAC
Final
Report
Chapter
One
August
1998
1
­
4
testing
program
within
the
overarching
framework
set
forth
in
Chapter
Three.
The
work
of
the
STWG,
established
by
the
EDSTAC
to
assist
in
developing
guidance
regarding
development
of
the
screening
and
testing
program,
formed
the
basis
for
Chapter
Five.
The
EDSTAC
charged
the
STWG
with
developing
recommendations
on:

·
the
specific
assays
to
be
included
in
a
standardized
T1S
battery;
·
guidance
for
using
available
information
to
generate
a
"
weight­
of­
evidence"
determination
for
moving
a
specific
chemical
substance
or
mixture
from
screening
to
testing;
·
guidance
for
how
to
tailor
specific
T2T;
and
·
a
process
and
criteria
to
standardize
and
validate
screens
and
tests.

The
Communications
and
Outreach
Work
Group's
purpose
was
threefold:
(
1)
to
assist
in
the
coordination
and
input
on
overall
outreach
and
communication
efforts
surrounding
the
EDSTAC
plenary
meetings;
(
2)
to
develop
recommendations
for
the
EDSTAC
report
on
communication
issues
regarding
the
screening
and
testing
program;
and
(
3)
to
review
draft
recommendations
and
the
draft
report
of
the
EDSTAC
with
the
objective
of
ensuring
effective
communication
to
both
EPA
and
the
public.
The
recommendations
of
the
COWG
for
the
second
task
can
be
found
in
Chapter
Six
of
the
report,
along
with
a
description
of
the
efforts
undertaken
by
the
work
group
regarding
ongoing
communication
efforts
of
the
Committee
throughout
the
process,
as
well
as
ensuring
effective
communication
of
the
report
itself.

Chapter
Seven
includes
all
of
the
Committee's
recommendations,
made
in
Chapters
Three,
Four,
Five,
and
Six.
Each
set
of
recommendations
can
also
be
found
at
the
end
of
their
respective
chapters.
EDSTAC
Final
Report
Chapter
Two
August
1998
Chapter
Two
Background
EDSTAC
Final
Report
Chapter
Two
August
1998
Table
of
Contents
I.
The
Endocrine
System
as
it
Relates
to
the
Endocrine
Disruptor
Screening
and
Testing
Program.....................................................................................................................................
1
II.
Statutory
Basis
for
Endocrine
Disruptor
Screening
and
Testing.......................................
5
A.
FQPA
and
SDWA
Endocrine
Disruptor
Screening
and
Testing
Provisions
.........................
5
B.
Additional
Chemical
Screening
and
Testing
Authorities......................................................
6
C.
Scope
of
the
EDSTAC.......................................................................................................
6
1.
Other
Key
FQPA
Provisions...........................................................................................
7
2.
FIFRA
Testing
Provisions
and
Universe
of
Chemicals
....................................................
7
3.
TSCA
Testing
Provisions
and
Universe
of
Chemicals
.....................................................
9
4.
Relevance
of
the
FFDCA
and
Universe
of
Chemicals....................................................
11
III.
Literature
Cited................................................................................................................
12
Figures
Figure
2.1
Description
of
the
Biochemical
Events
of
a
Steroid
Hormone
Actions
................
3
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
1
I.
The
Endocrine
System
as
it
Relates
to
the
Endocrine
Disruptor
Screening
and
Testing
Program
The
purposes
of
this
section
are:
to
provide
a
brief
overview
of
the
issue
of
endocrine
disruption;
to
explain
the
scientific,
regulatory,
and
societal
concerns
related
to
this
issue;
and
to
explain
why
the
EPA
created
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC).
The
evidence
for
endocrine
disruption
as
an
ecological
and/
or
public
health
issue
was
reviewed
by
EPA
in
a
February
1997
report
(
U.
S.
EPA,
1997).
The
National
Academy
of
Sciences
is
preparing
a
more
definitive
report
on
the
scientific
evidence
for
endocrine
disruption
at
the
request
of
the
Agency,
and
should
be
available
in
late
1998.
This
section
reflects
the
EDSTAC
member's
views
on
the
science
of
the
endocrine
system
as
it
is
relevant
to
the
design
of
a
screening
and
testing
program.

The
endocrine
system
 
also
referred
to
as
the
hormone
system
 
is
made
up
of
glands
located
throughout
the
body,
hormones
which
are
synthesized
and
secreted
by
the
glands
into
the
bloodstream,
and
receptors
in
the
various
target
organs
and
tissues
which
recognize
and
respond
to
the
hormones.
The
function
of
the
system
is
to
regulate
a
wide
range
of
biological
processes,
including:
control
of
blood
sugar
(
through
the
hormone
insulin
from
the
pancreas);
growth
and
function
of
reproductive
systems
(
through
the
hormones
testosterone
and
estrogen
and
related
components
from
the
testes
and
ovaries);
regulation
of
metabolism
(
through
the
hormones
cortisol
from
the
adrenal
glands,
and
thyroxin
from
the
thyroid
gland);
development
of
the
brain
and
the
rest
of
the
nervous
system
(
estrogen
and
thyroid
hormones);
and
development
of
an
organism
from
conception
through
adulthood
and
old
age.
Normal
functioning
of
the
endocrine
system,
therefore,
contributes
to
homeostasis
(
the
body's
ability
to
maintain
itself
in
the
presence
of
external
and
internal
changes),
and
to
the
body's
ability
to
control
and
regulate
reproduction,
development,
and/
or
behavior.
An
endocrine
system
is
found
in
nearly
all
animals,
including
mammals,
non­
mammalian
vertebrates
(
e.
g.,
fish,
amphibians,
reptiles,
and
birds),
and
invertebrates
(
e.
g.,
snails,
lobsters,
insects,
and
other
species).
In
humans,
the
system
is
comprised
of
more
than
50
different
hormones,
and
the
complexity
in
other
species
would
appear
to
be
comparable.

There
are
four
chemical
classes
of
hormones:
(
1)
steroids
derived
from
cholesterol
(
e.
g.,
the
sex
hormones
estrogen
and
androgen);
(
2)
amines
synthesized
from
amino
acids
(
e.
g.,
tyrosine
and
histidine),
giving
rise
to
thyroid
hormones
and
catecholamines
(
e.
g.,
adrenalin
and
nonaldrenalin);
(
3)
peptides
and
proteins
consisting
of
chains
of
amino
acids
(
e.
g.,
growth
hormone);
and
(
4)
eicosanoids
which
are
derived
from
a
20­
carbon
fatty
acid
called
arachidonic
acid
(
e.
g.,
prostaglandins
and
leukotrienes).

There
also
are
three
major
classes
of
receptors
to
which
hormones
might
bind:
(
1)
receptors
found
on
the
surface
of
cells
(
to
which
the
peptide
hormones
bind);
(
2)
receptors
found
in
the
cytoplasm
of
cells
(
to
which
the
steroid
hormones
bind);
and
(
3)
receptors
found
in
the
nuclei
of
cells
(
to
which
the
thyroid
hormones
bind).
There
are
two
major
mechanisms
of
hormone
action:
activation
of
plasma
membrane
receptors
either
via
binding
or
catecholamines,
peptides,
or
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
2
protein
hormones;
and
activation
of
intracellular
receptors
as
illustrated
in
Figure
2.1.
Steroid
hormones
and
thyroid
hormones
operate
in
this
latter
manner.

A
vast
array
of
receptor
proteins
and
genes
are
associated
with
the
cells
of
the
body.
Cells
may
contain
as
many
as
10,000
protein
receptors
for
a
single
steroid
hormone,
and
as
many
as
50
to
100
genes
in
a
cell
may
be
controlled
by
the
binding
of
a
single
type
of
hormone
to
the
various
receptors
in
a
cell.
Also,
some
genes
are
affected
by
more
than
one
receptor­
hormone
complex.

The
focus
of
the
EDSTAC
is
on
identifying
disruptors
of
estrogen,
androgen,
and
thyroid
hormones
in
terms
of
interference
with
their
functioning
by
one
or
more
of
the
following
mechanisms
of
action:

·
synthesis;
·
release
into
the
blood
stream;
·
transport
and
serum
binding;
·
cell
receptors
(
at
cell
surface
to
allow
entry
into
cell);
·
nuclear
receptors
(
receptor
binding);
·
signal
transduction
(
which
causes
activation
of
a
gene);
·
transcription
(
to
generate
messenger
RNA);
·
translation
(
to
generate
proteins,
e.
g.,
enzymes,
regulatory
proteins,
structural
proteins,
other
receptors,
etc.);
and/
or
·
metabolism
(
in
general,
to
form
more
polar
metabolites
by
oxidation
for
urinary
excretion,
conjugation,
activation/
inactivation,
etc.).

Below
is
a
description
of
the
biochemical
events
of
a
steroid
(
estrogen/
androgen)
hormone
action,
as
depicted
in
Figure
2.1:

1.
Estrogens
and
androgens
(
EA)
are
synthesized
in
the
gonad
(
i.
e.,
ovary
or
testis).
2.
EA
are
secreted
into
the
blood
and
transported
in
free
form
or
bound
to
a
transport
protein
(
i.
e.,
steroid
hormone
binding
globulin
[
SHBG]).
(
As
an
aside,
after
EA
binds
to
the
SHBG,
it
cannot
diffuse
into
the
cell.)
3.
Free
EA
diffuses
passively,
through
the
cell
membrane,
into
the
cytoplasm
of
the
target
cell
and
then,
4.
through
the
nuclear
membrane,
into
the
nucleus
which
contains
the
genetic
machinery
and
the
EA
receptors.
5.
EA
hormones
bind
to
their
receptor
(
R).
6.
Two
receptors,
each
bound
to
an
EA
hormone
molecule,
bind
to
one
another
forming
a
dimer.
The
receptor
dimer
binds
to
a
protein
transcription
factor
(
TF).
This
entire
complex
then
binds
to
a
hormone
response
element
(
HRE)
on
a
gene
(
Gene
A).
7.
Gene
A
is
subsequently
activated
such
that
the
DNA
(
Gene
A)
is
transcribed
and
messenger
ribonucleic
acid
(
mRNA)
is
synthesized.
8.
mRNA
is
transported
out
of
the
nucleus
into
the
cytoplasm.
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
3
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
4
9.
The
mRNA
is
"
translated,"
by
the
ribosomes
(
r)
and
additional
translational
machinery,
into
protein
A
by
linking
together
the
amino
acids
(
aa)
specified
by
the
mRNA
code
(
which
reflects
the
DNA,
or
gene
A,
code).
10.
Protein
A
can
be
one
of
the
following
kinds
of
proteins,
including,
but
not
limited
to,
an
enzyme,
peptide
hormone,
hormone
receptor,
or
growth
factor.

Each
of
the
steps
listed
above
offers
an
opportunity
for
a
substance
to
alter
the
way
hormones
exert
control
over
the
essential
processes
in
an
animal.
There
also
are
feedback
systems
in
the
body
which
control
the
actions
of
the
hormones,
increasing
hormone
production
when
the
amount
in
the
body
is
too
low
and
decreasing
production
when
the
amount
is
too
large.
While
in
adult
organisms,
these
control
mechanisms
may
help
to
blunt
mild
to
moderate
fluctuations
in
hormone
or
hormone­
like
actions
or
stresses
produced
from
the
environment,
feedback
systems
are
less
well­
developed
in
developing
organisms,
making
these
organisms
potentially
more
vulnerable.
In
addition,
it
is
worth
noting
that
changes
in
the
endocrine
system
may
take
place
at
any
point
in
time
during
the
conception,
development,
birth,
growth,
and
eventual
reproduction
of
the
organism
or
its
parents.
Such
changes
may
appear
as
effects
in
the
individual
organism
and/
or
in
the
population.

The
following
text
box
contains
an
example
in
more
technical
language
that
certain
audiences
may
find
helpful,
describing
how
the
estrogen,
androgen,
and
thyroid
hormone
systems
function.

An
Example
of
How
Estrogen,
Androgen,
and
Thyroid
Hormone
Systems
Function
An
excellent
example
of
the
process
by
which
EAT
hormone
systems
act
is
the
neuroendocrine
control
of
the
development
of
the
male
reproductive
system
in
utero,
beginning
at
4­
6
weeks
in
humans
and
at
the
end
of
the
second
week
(
of
three)
of
gestation
in
rodents.
Initially
in
rodents
(
and
presumably
in
humans),
the
indifferent
gonads
begin
to
differentiate
into
testes
triggered
by
products
from
male­
determining
genes
on
the
y
chromosome
(
male
determining
chromosome
in
mammals).
Within
a
few
days
in
rodents,
the
hypothalamus
in
the
brain
begins
to
produce
and
secrete
gonadotropin­
releasing
hormone
(
GnRH)
which
travels
via
the
blood
to
the
anterior
lobe
of
the
pituitary
gland
(
just
under
the
brain).
In
a
receptor­
mediated
process,
GnRH
stimulates
the
production
and
secretion
of
two
gonadotropins
 
luteinizing
hormone
(
LH),
and
follicle
stimulating
hormone
(
FSH)
 
which
travel
via
the
blood
to
the
developing
testes.
In
the
testes,
in
receptor­
mediated
processes,
LH
stimulates
cells
(
Leydig
cells)
to
produce
testosterone,
some
of
which
is
converted
to
dihydrotestosterone
(
DHT),
in
the
fetal
testis
by
the
enzyme
5­
alpha­
reductase.
Testosterone
and
DHT
in
receptor­
mediated
processes,
within
and
outside
the
developing
testis,
induce
the
formation
of
male
reproductive
structures.
FSH
stimulates
other
cells
in
the
testis
(
Sertoli
cells)
which
act
as
"
nurse
cells"
to
the
developing
germ
cells
in
the
presence
of
high
concentrations
of
testosterone.
The
same
processes
at
puberty
in
males
release
FSH
and
LH
which
again
act
within
and
outside
the
testes,
this
time
to
initiate
spermatogenesis
(
formation
of
sperm)
and
to
trigger
development
of
male
secondary
sex
characteristics.
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
5
EPA
originally
charged
the
EDSTAC
with
designing
a
screening
and
testing
program
to
identify
substances
and
common
mixtures
capable
of
altering
the
way
estrogen,
androgen,
and
thyroid
hormones
exert
control
over
the
essential
processes
described
above.
As
discussed
in
Section
II
of
this
chapter,
the
decision
to
create
such
a
program
was
based
both
on
requirements
specified
by
the
Food
Quality
Protection
Act
and
the
Safe
Drinking
Water
Act,
and
on
the
EPA
review
of
scientific
evidence
for
the
presence
of
endocrine
disrupting
substances
in
the
environment.

Knowledge
of
the
endocrine
system
has
grown
sufficiently
for
scientists
to
believe
they
can
improve
their
methods
of
screening
and
testing
chemical
substances
and
mixtures
for
toxicity
to
the
endocrine
system.
The
EDSTAC
believes
it
is
important
to
acknowledge
the
rapidly
evolving
state­
of­
the­
science
surrounding
the
issue
of
endocrine
disruption
while,
at
the
same
time,
recognizing
there
are
still
many
unanswered
questions
yet
to
be
resolved.
(
For
additional
information
see
the
following
references:
Kavlock
et
al.,
1996;
Ankley
et
al.,
1998;
Colborn
and
Clement,
1992;
Rolland
et
al.,
1997;
Kendall
et
al.,
1998).
Regardless
of
whether
and
how
widespread
endocrine
disruption
is
at
present,
an
important
way
to
help
protect
human
health
and
the
environment
against
possible
endocrine
disruption,
is
to
screen
and
test
chemical
substances
and
mixtures
for
their
ability
to
interact
with
and
disrupt
the
endocrine
system.
For
this
reason,
the
EDSTAC
has
devised
the
screening
and
testing
program
for
endocrine
disruption
that
is
described
in
the
remainder
of
this
report.

II.
Statutory
Basis
for
Endocrine
Disruptor
Screening
and
Testing
A.
FQPA
and
SDWA
Endocrine
Disruptor
Screening
and
Testing
Provisions
As
noted
above,
the
1996
Food
Quality
Protection
Act
(
FQPA)
and
the
1996
Amendments
to
the
Safe
Drinking
Water
Act
(
SDWA)
require
the
U.
S.
Environmental
Protection
Agency
(
EPA)
to:

develop
a
screening
program,
using
appropriate
validated
test
systems
and
other
scientifically
relevant
information,
to
determine
whether
certain
substances
may
have
an
effect
in
humans
that
is
similar
to
an
effect
produced
by
a
naturally
occurring
estrogen,
or
other
such
endocrine
effect
as
the
Administrator
may
designate.

The
laws
require
EPA
to
develop
a
screening
program
by
August
1998,
to
implement
the
program
by
August
1999,
and
to
report
on
the
program's
progress
by
August
2000.

The
two
laws
target
different
sets
of
chemical
substances.
Section
304
of
the
FQPA
states
that
in
carrying
out
the
program,
the
Administrator
shall:

(
A)
provide
for
the
testing
of
all
pesticide
chemicals;
and
(
B)
may
provide
for
the
testing
of
any
other
substance
that
may
have
an
effect
that
is
cumulative
to
an
effect
of
a
pesticide
chemical
if
the
Administrator
determines
that
a
substantial
population
may
be
exposed
to
such
a
substance.
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
6
Section
136
of
the
SDWA
Amendments
states
that:

in
addition
to
the
substances
referred
to
in
(
FQPA),
the
Administrator
may
provide
for
testing
under
the
screening
program
authorized
by
(
FQPA)
for
any
other
substance
that
may
be
found
in
sources
of
drinking
water
if
the
Administrator
determines
that
a
substantial
population
may
be
exposed
to
such
substance.

B.
Additional
Chemical
Screening
and
Testing
Authorities
The
FQPA
and
the
SDWA
did
not
arise
in
a
vacuum.
Rather,
the
FQPA
and
SDWA
requirements
for
endocrine
disruptor
screening
and
testing
place
another
layer
of
screening
and
testing
activity
on
an
extensive
regulatory
system
to
which
new
and
existing
pesticide
and
industrial
chemicals
are
already
subjected.
These
include:

·
Federal
Food,
Drug
and
Cosmetic
Act
(
1938)
as
amended
(
1958)
 
As
it
applies
to
EPA,
FFDCA
regulates
the
use
of
pesticides
as
food
additives.
Pesticide
tolerances
for
food
are
established
under
the
Act.
A
tolerance
is
defined
as
the
maximum
amount
of
residue
allowed
to
remain
on
an
agricultural
commodity
at
the
time
of
harvest.
·
Federal
Insecticide,
Fungicide
and
Rodenticide
Act
(
1947)
as
amended
 
FIFRA
provides
a
regulatory
framework
for
the
registration
and
use
of
pesticides.
·
Clean
Water
Act
(
Federal
Water
Pollution
Control
Act,
1972,
as
amended)
 
The
CWA
regulates
toxic
water
pollutants.
·
Safe
Drinking
Water
Act
(
1974)
 
The
SDWA
sets
enforceable
standards
for
substances
in
drinking
water.
·
Toxic
Substances
Control
Act
(
1976)
 
TSCA
requires
notification
before
new
chemicals
can
be
placed
into
commerce
and
gives
authority
for
testing,
information
reporting,
and
for
controlling
new
and
existing
industrial
chemicals.

C.
Scope
of
the
EDSTAC
In
convening
the
EDSTAC,
EPA
did
not
limit
the
Committee
to
the
narrow
set
of
chemicals
and
the
single
hormonal
system
explicitly
mentioned
in
the
FQPA
and
SDWA
endocrine
disruptor
screening
and
testing
provisions.
Nor
did
the
EDSTAC
limit
its
recommendations
to
the
protection
of
human
health.
Rather,
as
described
more
fully
in
Chapter
Three,
the
EDSTAC
strongly
recommends
that
EPA's
endocrine
disruptor
screening
and
testing
program
should:

·
address
both
human
health
and
ecological
effects;
·
initially
emphasize
identifying
and
characterizing
effects
that
enhance,
mimic,
or
inhibit
estrogen,
androgen,
and
thyroid
hormone­
related
processes;
and
·
be
capable
of
evaluating
the
endocrine
disrupting
properties
of
both
chemical
substances
and
common
mixtures.
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
7
The
EDSTAC
believes
that
this
scope
properly
reflects
a
broad
concern
about
the
potential
human
health
and
ecological
effects
of
endocrine
disruption.
Given
the
recommended
scope
of
the
program,
the
EDSTAC
discussed
additional
testing
authorities.
These
included
FIFRA
and
FFDCA
(
as
amended
in
FQPA),
TSCA,
and
SDWA.
An
overview
of
FQPA
and
TSCA
is
provided
below.
A
very
brief
summary
of
other
key
components
of
the
FQPA
is
also
provided.
These
overviews
are
provided
for
informational
purposes
only.
They
do
not
represent
any
interpretation
of
statutory
authority
by
either
the
EDSTAC
or
EPA.

1.
Other
Key
FQPA
Provisions
The
FQPA
revised
the
Federal
Food,
Drug
and
Cosmetic
Act
(
FFDCA)
and
the
Federal
Insecticide,
Fungicide
and
Rodenticide
Act
(
FIFRA).
The
major
FQPA
amendments
to
the
FFDCA
include:
(
1)
health
based
safety
standards
for
pesticide
residues
in
food;
(
2)
special
provisions
for
infants
and
children;
(
3)
limits
on
"
benefits"
considerations;
(
4)
review
of
all
existing
pesticide
tolerances
by
the
year
2006;
(
5)
uniformity
of
tolerances;
and
(
6)
screening
and
testing
for
endocrine
disruptors.
Specific
FQPA
amendments
to
FIFRA
include:
(
1)
pesticide
reregistration
is
required
every
15
years;
(
2)
EPA
is
required
to
develop
procedures
for
expedited
review
of
safer
pesticides;
(
3)
provisions
to
facilitate
"
minor
use"
registrations;
and
(
4)
requires
EPA
to
expedite
the
review
and
registration
of
anti­
microbial
pesticides.

2.
FIFRA
Testing
Provisions
and
Universe
of
Chemicals
Under
FIFRA,
EPA
regulates
pesticides
 
a
group
of
chemicals
which
includes
insecticides,
herbicides,
fungicides,
rodenticides,
disinfectants,
plant
growth
regulators,
biological
agents,
and
other
pest
control
agents.
FIFRA
gives
EPA
the
authority
to
register
pesticides
to
ensure
no
unreasonable
adverse
effects
to
human
health
or
the
environment
exist,
taking
into
account
the
economic,
social,
and
environmental
costs
and
benefits
of
the
pesticide
use.
As
such,
FIFRA
is
a
cost­
benefit
statute.
In
other
words,
the
determination
of
what
constitutes
an
"
unreasonable
adverse
effect"
must
account
for
socioeconomic
factors
as
well
as
scientific
judgments.
The
primary
regulatory
vehicle
under
FIFRA
is
the
pesticide
label
("
the
label
is
the
law").
Every
registered
pesticide
product
must
bear
a
label
that
includes
the
producer
number,
product
registration
number,
active
ingredient
statement,
warning
or
precautionary
statements,
and
directions
for
use.

Registration
and
re­
registration
decisions
are
based
in
part
on
the
evaluation,
synthesis,
and
integration
of
pesticide
studies
conducted
by
registrants
and
others
and
submitted
to
the
Agency.
The
data
requirements,
and
the
Agency's
ability
to
require
special
studies
when
deemed
necessary,
are
substantial.
Studies
are
routinely
conducted
in
mammalian
toxicology,
occupational
and
residential
exposure,
residue
chemistry,
environmental
fate
and
transport,
and
ecological
effects.
Individual
studies
are
evaluated
by
EPA
scientists,
and
subsequently
used
in
human
health
and
ecological
risk
assessments.
The
risk
assessments
are
then
used
by
regulatory
decision­
makers
who
make
the
final
risk
management
decisions.
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
8
Until
FQPA
was
passed,
risk
assessments
for
pesticide
registration
characterized
estimates
of
risk
only
for
single
active
ingredients.
Dietary
risk
assessments
included
an
estimate
of
risk
from
all
use­
sites
(
e.
g.,
corn,
cotton,
wheat,
ornamental
plants,
etc.),
but
non­
dietary
(
e.
g.,
occupational
or
residential)
risk
assessments
addressed
each
exposure
scenario
separately.
Ecological
risk
assessments
continue
to
be
done
only
for
single
uses
of
single
chemicals.
However,
EPA's
Office
of
Pesticide
Programs
(
OPP)
is
evaluating
the
feasibility
and
appropriateness
of
conducting
more
complex
assessments.
The
scope
and
complexity
of
any
specific
pesticide
risk
assessment
varies
with
the
specific
chemical
and
use
pattern(
s),
but
a
tiered,
iterative
approach
is
common.
In
the
initial
tier
assessment
for
human
health,
reasonable
worst
case
assumptions
are
utilized
as
estimates
of
exposure
(
e.
g.,
residues
in
food
are
at
the
tolerance
level,
and
all
of
the
crop
is
treated).
If
the
risk
estimate
exceeds
the
level
of
concern,
additional
empirical
or
surrogate
data
are
used
to
refine
the
exposure
assessment,
until
such
time
as
it
can
be
shown
that
the
level
of
concern
is
not
really
exceeded,
or
the
decision
is
made
that
risk
reduction
measures
should
be
taken.
For
ecological
assessments,
the
tiers
progress
through
simple
risk
quotients
derived
from
laboratory
fate,
transport,
and
toxicity
data
in
early
tiers,
to
a
"
weight­
of­
evidence"
approach
in
later
tiers.

When
a
pesticide
undergoes
evaluation
for
registration,
re­
registration,
or
Special
Review
(
see
below),
the
scientific
disciplines
review
and
evaluate
registrant­
submitted
and
other
studies
in
a
comprehensive
manner
to
ensure
the
studies
meet
scientific
and
regulatory
policy
standards
established
for
carrying
out
risk
assessments.
The
studies
are
evaluated
and
integrated
in
such
a
manner
that
routes
of
dissipation,
significant
environmental
degradates,
residue
levels,
and
residence
time
of
persistent
degradates
in
the
various
environmental
compartments
are
elucidated.
This
information
along
with
the
hazard
profile
of
the
pesticide,
as
determined
in
the
required
studies
and
available
incident
data,
is
used
to
determine
risk
in
aquatic
and
terrestrial
environmental
compartments.
If
a
high
level
of
concern
is
identified,
risk
mitigation
options
are
identified
and
considered
for
inclusion
on
the
pesticide
label.
If
the
available
options
are
not
adequate
to
reduce
the
level
of
concern
to
an
acceptable
level,
the
use
of
the
pesticide
may
not
be
approved
or
may
be
rescinded.

OPP
currently
reviews
approximately
5,000
pesticide
registration
submissions
annually.
The
scope
of
the
submissions
ranges
from
simple
label
amendments
to
registration
of
new
active
ingredients.
Since
1947,
thousands
of
pesticide
products
have
been
registered.
Not
surprisingly,
perhaps,
standards
for
approval
and
test
data
requirements
reflect
changes
in
science
and
pesticide
regulatory
policy
over
time.
To
ensure
compliance
with
current
scientific
and
regulatory
standards,
FIFRA
now
requires
the
review
and
re­
registration
of
existing
pesticides
every
15
years.
At
any
time,
registrants
may
delete
pesticide
uses
or
voluntarily
withdraw
products
or
uses
that
are
not
economically
feasible
to
maintain.
Further,
EPA
has
the
authority
to
cancel
registrations
for
pesticide
products
that
do
not
meet
the
requirements
for
re­
registration
(
or
registration,
for
that
matter).
The
number
of
registered
products
subjected
to
re­
registration
in
response
to
the
1988
amendments
to
FIFRA
was
approximately
50,000.
The
total
number
of
products
remaining
on
the
market
is
now
approximately
20,000.

Presently,
there
are
approximately
900
registered
pesticide
active
ingredients
and
2500
inert
ingredients.
Inert
ingredients
used
in
pesticide
formulations
are
subjected
to
test
requirements
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
9
that
are
less
comprehensive
than
those
for
active
ingredients.
Under
the
FQPA
screening
and
testing
program,
both
active
and
inert
ingredients
are
to
be
included.
Many
of
the
pesticide
"
inerts"
are
also
listed
in
the
TSCA
Inventory,
which
is
described
below,
as
are
a
number
of
the
active
ingredients
(
because
they
also
have
non­
pesticidal
uses).

In
the
registration
or
re­
registration
process,
problems
that
arise
during
the
review
of
a
particular
pesticide
may
be
investigated
under
the
Special
Review
Process.
Special
Review
is
a
formal
scientific
and
legal
process
in
which
EPA
presents
its
case
that
the
use(
s)
of
a
currentlyregistered
pesticide
may
be
presenting
risks
of
concern
and,
thus,
risk
reduction
or
cancellation
of
the
use(
s)
may
be
warranted.
Special
Review
is
conducted
by
notice
and
comment
rulemaking.
The
science
issues
are
developed
and
must
be
presented
to
the
FIFRA
Scientific
Advisory
Panel
for
review.
Additionally,
the
U.
S.
Food
and
Drug
Administration,
the
U.
S.
Department
of
Agriculture,
and
congressional
committees
are
invited
to
provide
formal
comments.
Once
a
decision
is
made,
the
registrant
may
appeal
the
decision
through
administrative
procedure
or
judicial
review.

The
FQPA
amendments
to
FIFRA
require
EPA
to
reassess
all
existing
pesticide
tolerances
of
food
use
pesticides
by
the
end
of
the
year
2006.
The
data
requirements
for
pesticide
registration
are
substantial,
and
the
burden
of
proof
to
demonstrate
safety
lies
with
the
registrant.
As
such,
the
EPA
has
significant
authority
to
issue
a
"
data­
call­
in"
requiring
the
registrant
to
conduct
studies
to
rebut
a
presumption
of
risk
identified
by
EPA.
Nevertheless,
the
databases
for
any
given
pesticide
may
vary
substantially.
The
types
and
minimum
amounts
of
data
that
registrants
are
required
to
submit
or
cite
in
support
of
an
application
are
listed
in
40
Code
of
Federal
Regulations
(
CFR)
Part
158.
The
data
requirements
vary
according
to
use
patterns
(
e.
g.,
terrestrial
food
crop,
indoor
domestic,
etc.)
and
physicochemical
properties
(
e.
g.,
gas,
volatile
liquid,
dust,
chemical
class,
etc.).
As
such,
for
purposes
of
priority
setting,
it
is
important
that
each
pesticide
be
critically
examined
on
a
case­
specific
basis
with
respect
to
the
adequacy
of
existing
data
for
the
evaluation
of
endpoints
due
to
endocrine
disruption,
as
well
as
for
evaluation
of
exposure
potential.

3.
TSCA
Testing
Provisions
and
Universe
of
Chemicals
TSCA
was
signed
into
law
in
1976
and
most
of
its
provisions
became
effective
on
January
1,
1977.
TSCA
requires
EPA
to
"
compile,
keep
current,
and
publish
a
list
of
each
chemical
substance
which
is
manufactured
or
processed
in
the
United
States."
TSCA
exempts
chemicals
used
only
in
small
quantities
(
as
defined
by
EPA
by
rule)
for
research
purposes
from
this
listing.

Chemical
regulation
under
TSCA
is
quite
different
than
that
described
above
for
FIFRA.
Under
the
New
Chemical
Review
Program,
manufacturers
must
submit
Pre­
Manufacture
Notification
(
PMN)
for
new
chemicals.
By
statute,
EPA
must
review
the
submission
within
90
days.
Because
there
is
no
obligation
on
the
part
of
the
manufacturer
to
develop
toxicity
data
prior
to
notification,
the
main
tools
the
Agency
uses
in
this
review
are
Structure
Activity
Relationship
(
SAR)
models.
In
practice,
EPA
often
drops
review
and
gives
approval
for
most
chemicals.
Where
appropriate,
the
Agency
prohibits
or
limits
manufacture,
processing,
distribution,
use,
or
disposal
when
it
judges
the
chemical
may
present
an
unreasonable
risk
and
data
are
inadequate.
The
Agency
can
require
testing
for
chemicals
that
will
have
substantial
production,
significant
exposure,
or
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
10
substantial
release.
Testing
may
also
be
required
for
chemicals
that
pose
significant
risk.
Testing
is
tied
to
affordability.

Testing
of
existing
chemicals
under
TCSA
is
conducted
differently
than
for
new
chemicals.
Test
requirements
for
existing
chemicals
are
determined
by
a
rule­
making
or
through
a
negotiated
Enforceable
Consent
Agreement
(
ECA).
To
require
testing
of
existing
chemicals,
the
Agency
must
make
a
finding
that
the
chemical
may
present
an
unreasonable
risk
to
human
health
or
the
environment
or,
alternatively,
that
it
is
produced
in
substantial
quantities
and
there
is
substantial
or
significant
human
exposure
or
substantial
environmental
release.
These
findings
which
EPA
makes
under
TSCA
4(
a)(
1)(
A)
and
4(
a)(
1)(
B)
are
discussed
in
the
following
paragraph.
In
addition,
EPA
must
find
that
there
are
inadequate
data
to
reasonably
determine
or
predict
the
effects
of
the
chemical
on
human
health
or
the
environment
and
that
testing,
therefore,
is
necessary.
This
testing
may
include
health
effects,
environmental
effects,
chemical
fate
in
the
environment,
and
exposure.

Under
TSCA
Section
4(
a)(
1)(
A),
EPA
must
have
a
suggestion
of
hazard
and
there
must
be
an
exposure
to
the
chemical
for
EPA
to
require
testing
data.
Under
TSCA
Section
4(
a)(
1)(
B)
data
may
be
required
when
there
is
substantial
production
(
one
million
pounds
per
year
threshold
value)
and:
(
a)
substantial
release
(
the
lessor
of
one
million
pounds
per
year
or
10%
of
production);
(
b)
substantial
human
exposure
(
widespread
human
exposure
indicated
by
1,000
workers,
10,000
consumers,
or
100,000
members
of
the
general
population);
or
(
c)
significant
human
exposure
under
special
high
exposure
scenarios.

EPA's
initial
listing
of
chemicals
in
commerce,
commonly
called
the
"
Initial
Inventory"
or
the
"
1977
Inventory,"
consisted
of
those
chemicals
manufactured
in
the
U.
S.
or
imported
into
the
U.
S.
on
or
after
January
1,
1975
and
before
the
end
of
the
initial
reporting
period.
This
period
varied
depending
on
the
chemical/
company
circumstances
and
certain
allowances
were
made
for
later
additions
and
corrections.
The
Initial
Inventory
was
published
in
1979
and
contained
about
60,000
chemicals.
This
represented
the
initial
set
of
"
existing
chemicals"
and
the
basis
for
distinguishing
between
"
new"
and
"
existing"
chemicals
under
TSCA.
Chemicals
not
on
the
Inventory
are
considered
"
new"
and
are
subject
to
the
PMN
requirements
of
TSCA.
After
EPA
completes
the
pre­
manufacture
review
of
a
new
chemical,
and
when
the
manufacturer
or
importer
of
the
chemical
notifies
the
Agency
that
manufacture
or
importation
has
commenced,
EPA
adds
the
new
chemical
to
the
Inventory.

As
of
August
18,
1997,
based
on
a
search
performed
by
EPA
for
the
EDSTAC,
there
were
about
75,500
chemicals
in
the
TSCA
Inventory.
Of
the
75,500
chemicals,
2,643
are
inorganics,
24,160
are
polymers,
48,697
are
organics,
and
about
500
are
complex
substances
from
petroleum
refining
streams.
The
"
metals"
are
distributed
among
the
inorganics,
polymers,
and
organics.

At
the
time
the
Initial
Inventory
was
compiled,
production
data
were
also
collected
for
those
chemicals.
Production
data
have
been
updated
three
times
for
a
subset
of
Inventory
chemicals.
The
Inventory
Update
Rule
(
IUR)
has
required
reporting
of
the
quantities
of
subject
chemicals
produced
in
1985,
1989,
and
1993.
Categories
of
chemicals
exempted
from
IUR
reporting
are
polymers,
inorganics,
microorganisms,
and
naturally
occurring
substances.
Additionally,
the
IUR
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
11
has
a
reporting
threshold
of
10,000
pounds
per
site
for
each
chemical
(
i.
e.,
reporting
is
required
for
a
chemical
only
if
a
company
manufactured
or
imported
at
least
10,000
pounds
of
the
chemical
at
any
single
site
during
the
year
covered
by
the
rule).
Of
the
organics,
approximately
12,340
have
been
produced
or
imported
in
excess
of
10,000
pounds
in
1985,
1989,
or
1993.
Of
these,
approximately
11,037
are
organics
that
are
non­
petroleum
fractions.
Available
recent
production
or
importation
data
on
inorganics
or
polymers
are
not
easily
accessible.

EPA
estimates
that
a
total
of
approximately
15,000
non­
polymeric
chemicals
are
manufactured
or
imported
at
levels
above
10,000
pounds
per
year
(
the
12,000
IUR
chemicals
plus
an
estimated
3,000
chemicals
from
exempt
categories
(
primarily
inorganics)).
Within
this
set
of
15,000
nonpolymeric
chemicals,
there
are
approximately
3,000
chemicals
produced
in
amounts
greater
than
1
million
pounds
per
year.
Approximately
25,000
chemicals
potentially
subject
to
the
IUR
have
never
been
reported
on
the
IUR,
indicating
they
are
manufactured
or
imported
in
amounts
less
than
10,000
pounds
per
year
and,
in
some
cases,
may
no
longer
be
produced
at
all.

Although
EPA
has
authority
to
order
testing
of
chemicals
under
TSCA,
in
the
nearly
20
years
of
TSCA's
existence,
this
authority
has
been
used
for
only
121
chemicals.
This
is
not
an
indication
of
how
much
more
information
might
really
be
needed
but,
rather,
the
administrative
challenges
of
mounting
an
information
request.
Because
of
the
expense
in
justifying
and
preparing
test
rules,
and
due
to
concern
over
litigation,
EPA
tends
to
rely
on
negotiated
consent
orders
and
voluntary
testing
which
have
resulted
in
testing
of
an
additional
443
chemicals.

4.
Relevance
of
the
FFDCA
and
Universe
of
Chemicals
In
addition
to
the
chemicals
regulated
by
EPA
under
TSCA,
FIFRA,
and
FFDCA,
there
are
a
large
number
of
chemicals
that
are
regulated
under
FFDCA
and
other
statutes
by
other
agencies
that
may
present
significant
exposures
to
humans
and
for
which
there
are
essentially
no
data
on
the
potential
for
endocrine
disruption.
The
EDSTAC
is
recommending
that
ingredients
in
cosmetics,
food
additives
(
including
those
Generally
Regarded
As
Safe
(
GRAS),
under
the
FFDCA),
and
nutritional
supplements
also
receive
serious
consideration
for
priority
setting
within
the
endocrine
disruptor
screening
and
testing
program.
This
recommendation
is
made
even
though
it
is
understood
that
FQPA
and
SDWA
do
not
confer
on
any
other
agency
the
regulatory
authority
to
require
screening
and
testing
for
endocrine
disruption
potential.
EDSTAC
Final
Report
Chapter
Two
August
1998
2
­
12
III.
Literature
Cited
Ankley,
G.,
et
al.,
"
Overview
of
a
Workshop
on
Screening
Methods
for
Detecting
Potential
(
Anti­
)
Estrogenic/
Androgenic
Chemicals
in
Wildlife,"
Environmental
Toxicology
and
Chemistry,
17,
1998,
pp.
68­
87.
Colborn,
T.,
and
C.
Clement,
"
Chemically­
Induced
Alterations
in
Sexual
and
Functional
Development:
The
Wildlife/
Human
Connection,"
C.
Princeton
Scientific
Publishing
Co.,
Inc.,
Princeton,
NJ.,
1992.
Kavlock,
R.
J.,
et
al.,
"
Research
needs
for
risk
assessment
of
health
and
environmental
effects
of
endocrine
disruptors:
A
review
of
the
U.
S.
EPA­
sponsored
workshop,"
Environmental
Health
Perspectives,
1996.
Kendall,
R.,
R.
Dickerson,
J.
Geisy,
and
W.
Suk,
"
Principles
and
Processes
for
Evaluating
Endocrine
Disruption
in
Wildlife,"
SETAC
Press,
Pensacola,
FL,
1998,
p.
515.
Rolland,
R.,
M.
Gilbertson,
and
R.
E.
Peterson,
"
Chemically
Induced
Alterations
in
Functional
Development
and
Reproduction
of
Fishes,"
SETAC
Press,
Pensacola,
FL,
1997.
U.
S.
EPA,
"
Special
Report
on
Endocrine
Disruption:
An
Effects
Assessment
and
Analysis,"
Office
of
Research
and
Development,
EPA/
630/
R­
96/
012,
Washington,
D.
C.,
1997.
EDSTAC
Final
Report
Chapter
Three
August
1998
Chapter
Three
Conceptual
Framework
and
Principles
EDSTAC
Final
Report
Chapter
Three
August
1998
Table
of
Contents
I.
Introduction...........................................................................................................................
1
II.
Description
of
Endocrine
Disruptor
....................................................................................
3
III.
Definition
of
Other
Key
Terms
..........................................................................................
4
IV.
Overview
of
the
EDSTAC
Conceptual
Framework...........................................................
5
V.
Scope
of
the
EDSTAC
Conceptual
Framework
..................................................................
6
VI.
Where
Endocrine
Disruption
Fits
in
the
Broader
Context
...............................................
6
VII.
General
Principles
to
Guide
the
Development
of
the
Endocrine
Disruptor
Screening
and
Testing
Program
................................................................................................................
7
VIII.
The
EDSTAC
Conceptual
Framework
..........................................................................
9
A.
Obtain
and
Analyze
Existing
Information
to
Set
Priorities
..................................................
9
B.
Tier
1
Screening
to
Detect
Interactions
With
the
Endocrine
System....................................
9
C.
Tier
2
Testing
to
Determine
and
Characterize
Endocrine
Disruption.................................
10
IX.
Additional
Components
and
Clarifications
to
the
Original
EDSTAC
Conceptual
Framework
..............................................................................................................................
11
A.
High
Throughput
Pre­
Screening.......................................................................................
12
B.
Alternative
Means
of
Meeting
Versus
Bypassing
Tier
1
Screening....................................
13
1.
Alternative
Means
to
Meet
T1S
Information
Requirements
..........................................
13
2.
Bypassing
T1S
.............................................................................................................
13
X.
Compilation
of
Chapter
Three
Recommendations
...........................................................
14
XI.
Literature
Cited
................................................................................................................
18
Figures
Figure
3.1
EDSTAC
Conceputal
Framework
.........................................................................
2
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
1
I.
Introduction
In
this
chapter
the
EDSTAC
presents
the
conceptual
framework
and
principles
that
have
served
as
the
foundation
upon
which
all
the
other
recommendations
contained
in
this
report
have
been
built.
In
addition
to
this
introductory
section,
the
chapter
includes
sections
on:
the
description
of
"
endocrine
disruptor;"
definitions
for
several
other
key
terms;
the
purpose
and
context
for
endocrine
disruptor
screening
and
testing;
an
overview,
scope,
and
general
principles
for
the
framework;
the
conceptual
framework
itself;
and
a
discussion
of
other
important
conceptual
agreements
reached
after
the
framework
was
originally
developed.

The
initial
purpose
of
the
EDSTAC
Conceptual
Framework
was
to
lay
the
groundwork
for
future
EDSTAC
recommendations
and
to
inform,
facilitate,
and
expedite
the
work
of
the
EDSTAC
work
groups.
The
Principles
Work
Group
developed
the
original
version
of
the
framework
in
early
1997,
and
the
EDSTAC
reached
a
tentative
consensus
agreement
on
the
contents
of
this
chapter
in
May
1997.
Subsequently,
the
document
was
used
by
the
work
groups
and
the
EDSTAC
to
guide
their
deliberations.
At
the
time
the
EDSTAC
agreed
to
the
framework's
content,
members
recognized
the
evolutionary
nature
of
the
document
and
agreed
to
revisit
it,
as
appropriate,
throughout
their
deliberations.

During
the
EDSTAC's
deliberations,
another
concept
was
identified
to
be
considered
in
the
context
of
the
original
Conceptual
Framework,
and
other
concepts
that
were
already
contained
within
the
framework
were
clarified.
The
new
concept
incorporated
into
the
framework
was
the
use
of
"
high
throughput
pre­
screening."
The
concepts
further
clarified
relate
to
the
scenarios
under
which
chemicals
would
be
permitted
to
bypass
Tier
1
Screening
(
T1S)
and
the
interconnectedness
of
these
bypass
scenarios
with
other
elements
of
the
Conceptual
Framework.
These
concepts
are
introduced
at
the
end
of
the
chapter
and
further
elaborated
upon
in
subsequent
chapters.

In
its
final
version,
the
EDSTAC
Conceptual
Framework
is
intended
to
provide
guidance
to
EPA
regarding
development
and
implementation
of
its
endocrine
disruptor
screening
and
testing
program,
as
well
as
future
expansion,
as
appropriate,
of
the
program.
The
EDSTAC
believes
the
Conceptual
Framework
it
has
developed,
as
well
as
the
principles
underlying
the
recommendations
it
is
providing
to
EPA
for
priority
setting
and
the
screening
and
testing
program
itself,
are
applicable
to
the
consideration
of
other
hormone
systems
in
addition
to
those
involving
estrogen,
androgen,
and
thyroid
hormone.
All
of
the
recommendations
contained
herein
are
premised
on
the
principle
of
scientific
validity.

The
Conceptual
Framework
is
summarized
in
the
decision
flowchart
contained
in
Figure
3.1,
which
shows
how
screens
and
tests
are
used
to
evaluate
potential
endocrine
disruptors.
The
structure
of
the
Conceptual
Framework
was
placed
into
tiers
to
illustrate
how
chemical
substances
and
mixtures
can
be
sequentially
sorted
into
groups
that
are
increasingly
likely
to
be
classified
as
endocrine
disruptors,
thereby
warranting
additional
attention.
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
2
Figure
3.1
EDSTAC
Conceptual
Framework*

INITIAL
SORTING
Obtain
and
Analyze
Existing
Data
SUFFICIENT
DATA
or
VOLUNTARY
BYPASS
of
Tier
1
Screening
to
go
to
Tier
2
Testing
SET
PRIORITIES
For
Tier
1
Screening
TIER
1
SCREENING
Detect
Interaction
With
Endocrine
System
[
estrogen/
androgen/
thyroid]

TIER
2
TESTING
Determine
and
Characterize
Endocrine
Disrupting
Effects
HAZARD
ASSESSMENT
Yes
Yes
No
No
*
For
a
more
detailed
version
of
the
initial
sorting
and
priority
setting
components
of
this
framework,
please
see
Figure
4.1.
SUFFICIENT
DATA
to
go
to
Hazard
Assessment
HOLD
No
Further
Analysis
Required
at
This
Time
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
3
II.
Description
of
Endocrine
Disruptor
In
any
emerging
scientific
specialty
area,
numerous
terms
and
definitions
are
used.
As
examples,
environmental
estrogens,
environmental
hormones,
endocrine
disrupting
chemicals,
endocrine
modulators,
endocrine
disrupters,
and
endocrine
disruptors
may
all
be
found
in
recent
scientific
publications.
Several
definitions
of
endocrine
disruptor
have
also
been
published
(
e.
g.,
Kavlock
et
al.,
1996;
European
Commission,
1996;
U.
S.
EPA,
1997).
Tattersfield
et
al.,
1997
distinguished
between
endocrine
disruption
(
reduction
or
enhancement
of
hormone
levels
beyond
natural
bounds)
and
endocrine
modulation
(
adjustment
within
natural
bounds),
but
viewed
the
terms
as
interchangeable
for
their
purposes.
This
ambiguity
was
reflected
in
the
EDSTAC's
deliberations.

In
its
initial
discussions,
the
EDSTAC
acknowledged
the
Kavlock
et
al.,
1996
definition
of
an
endocrine
disruptor
as:

An
exogenous
agent
which
interferes
with
the
synthesis,
secretion,
transport,
binding,
action,
or
elimination
of
natural
hormones
in
the
body
which
are
responsible
for
the
maintenance
or
homeostasis,
reproduction,
development
or
behavior.

Certain
EDSTAC
members
were
concerned
that
the
Kavlock
et
al.,
1996
definition
was
formulated
for
research,
and
was
too
open
ended
for
regulatory
operations.
In
May
1997,
the
EDSTAC
developed
the
following
working
definition:

An
exogenous
substance
that
changes
endocrine
function
and
causes
adverse
effects
at
the
level
of
the
organism,
its
progeny,
and/
or
(
sub)
populations
of
organisms.

The
working
definition
served
a
useful
purpose
in
guiding
the
deliberations
of
the
EDSTAC
and
its
work
groups.
However,
over
time
it
became
clear
that
the
EDSTAC
was
divided
regarding
the
acceptability
of
this
working
definition.

Two
opposing
viewpoints
emerged.
One
view
held
that
the
definition
must
include
the
term
adverse,
whereas
the
second
view
held
that
adverse
was
inappropriate
and
should
be
excluded
from
the
definition.
Proponents
for
including
adverse
reasoned
that
a
definition
should
distinguish
disruption
from
the
wide
range
of
hormone
fluctuations
necessary
for
normal
physiological
adaptation.
Proponents
for
excluding
adverse
reasoned
that
hormone
function
is
so
sensitive
to
xenobiotic
challenge,
that
any
biochemical
alteration
during
key
developmental
stages
above
background
may
lead
to
serious,
but
subtle
pathology
later
in
life
or
in
subsequent
generations.
In
addition,
they
argued
that
effects
not
adverse
for
an
individual
may
be
adverse
at
the
population
level.
Both
sides
acknowledged
that
clear
delineation
of
adverse
is
at
times
subjective
and
may
be
open
to
differences
in
interpretation.
Toxicological
effects
occur
along
a
continuum
from
subtle
biochemical
events
to
gross
pathology.
The
point
at
which
an
observable
effect
becomes
truly
adverse
is
therefore
a
judgment
that
may
differ
among
individual
scientists.
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
4
The
EDSTAC
acknowledges
that,
at
this
time,
knowledge
and
experience
in
endocrine
disruptor
toxicology
do
not
permit
the
simple
categorization
of
all
endocrine
effects
into
adverse
and
nonadverse
The
capacity
to
make
this
distinction
will
improve
as
understanding
of
the
assay
systems
and
long­
term
consequences
of
endocrine
effects
increases.

In
order
to
achieve
consensus,
the
EDSTAC
agreed
to
the
following
general
description
to
express
the
range
of
members'
views
within
the
context
of
the
proposed
screening
and
testing
program:

The
EDSTAC
describes
an
endocrine
disruptor
as
an
exogenous
chemical
substance
or
mixture
that
alters
the
structure
or
function(
s)
of
the
endocrine
system
and
causes
adverse
effects
at
the
level
of
the
organism,
its
progeny,
populations,
or
subpopulations
of
organisms,
based
on
scientific
principles,
data,
weight­
of­
evidence,
and
the
precautionary
principle.

III.
Definition
of
Other
Key
Terms
The
EDSTAC
agreed
to
utilize
the
following
definitions
in
this
report:

"
Priority
setting"
is
defined
as
the
collection,
evaluation,
and
analysis
of
existing
relevant
information
to
determine
whether,
and
in
what
relative
order
of
priority,
chemical
substances
or
mixtures
will
be
subjected
to
screening,
testing,
or
hazard
assessment.

"
Screening"
is
defined
as
the
application
of
assays
to
determine
whether
a
chemical
substance
or
mixture
may
interact
with
the
endocrine
system.

"
Testing"
is
defined
as
a
customized
combination
of
tests
and
endpoints
designed
to
determine
whether
a
chemical
substance
or
mixture
causes
endocrine­
mediated
adverse
effects
and
to
identify,
characterize,
and
quantify
these
effects.

"
Hazard
assessment,"
as
used
in
this
document,
includes:
(
1)
identification
of
the
chemical
substances
and
mixtures
that
have
endocrine
disruption
effects,
which
is
often
referred
to
as
"
hazard
identification,"
and
(
2)
establishment
of
the
relationship
between
dose
and
effect,
which
is
often
referred
to
as
"
dose­
response
assessment."

"
Chemical
substances,"
as
used
in
this
document,
include
naturally
occurring
and
synthetic
chemicals
and
elements.
"
Mixtures"
refers
to
commonly
found
combinations
of
chemical
substances,
including
those
found
in
the
environment.

The
term
"
functional
equivalency"
is
used
at
several
critical
junctures
in
the
document.
The
EDSTAC
defines
an
assay,
test,
or
endpoint
as
being
"
functionally
equivalent"
to
a
T1S
or
T2T
assay,
test,
or
endpoint
when
it
provides
equivalent
information
for
each
endpoint
being
studied.
For
purposes
of
the
endocrine
disruptor
screening
and
testing
program,
EDSTAC­
recommended
assays,
tests,
and
endpoints
must
be
validated
and
standardized
prior
to
EPA's
use
of
functionally
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
5
equivalent
information.
As
discussed
elsewhere
in
this
document,
EPA
should
provide
clear
guidance
on
the
use
of
functionally
equivalent
assays,
tests,
and
endpoints
prior
to
the
implementation
of
the
screening
and
testing
program.

In
general,
the
term
"
weight­
of­
evidence"
is
typically
used
to
refer
to
a
process
by
which
trained
professionals
judge
the
strengths
and
weaknesses
of
a
collection
of
information
to
render
an
overall
conclusion
that
may
not
be
evident
from
consideration
of
the
individual
data.
Further
clarification
of
how
"
weight­
of­
evidence"
principles
will
be
applied
to
the
EDSTP
can
be
found
in
Chapter
Five,
Section
IV.

IV.
Overview
of
the
EDSTAC
Conceptual
Framework
The
EDSTAC
Conceptual
Framework
places
activities
in
an
ordered
sequence.
The
elements
of
this
sequence
include:
(
a)
priority
setting,
which
includes
the
sorting
and
prioritization
of
chemical
substances
and
mixtures
for
evaluation
in
screening
and/
or
testing
batteries;
(
b)
screening
to
detect
chemical
substances
and
mixtures
capable
of
acting
on
endocrine
systems;
and
(
c)
testing
to
determine,
characterize,
and
quantify
the
nature
of
the
endocrine
disrupting
properties
of
the
chemical
substances
and
mixtures
identified
by
prior
information
and/
or
T1S.

The
Conceptual
Framework
contains
a
series
of
decision
points.
At
each
of
these
points
in
the
process,
all
available
information
is
evaluated
to
determine
whether
and
how
to
proceed
to
the
next
step(
s).
A
"
weight­
of­
evidence"
approach
is
commonly
used
to
make
such
a
determination.

Three
guiding
principles
should
be
adopted
in
the
use
of
such
a
tiered
decision­
making
system:

·
This
ordered
sequence
should
not
exclude
the
possibility
that
a
chemical
substance
or
mixture
could
bypass
one
or
more
tiers
when
information
warrants
such
a
move
(
e.
g.,
sufficient
prioritization
data
on
endocrine
disrupting
properties
may
be
available
to
initiate
Tier
2
Testing
(
T2T)
or
hazard
assessment).
·
If
information
is
not
adequate
to
determine
whether
a
chemical
substance
or
mixture
should
move
to
the
next
tier,
there
should
be
an
active
process
for
generating
the
information
needed
to
make
such
a
decision.
·
The
criteria
and
default
assumptions
for
deciding
whether
chemical
substances
or
mixtures
move
from
one
tier
to
the
next,
to
the
degree
possible,
should
be
developed
in
advance
of
initiating
a
screening
and
testing
strategy.
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
6
V.
Scope
of
the
EDSTAC
Conceptual
Framework
The
Conceptual
Framework
is
consistent
with
several
central
issues
defined
in
the
scope
of
activity
for
EDSTAC:

·
Screening
and
testing
should
be
relevant
to
both
human
health
and
ecological
effects.
·
Screening
and
testing
should
initially
emphasize
identifying
and
characterizing
effects
that
enhance,
mimic,
or
inhibit
estrogen,
androgen,
and
thyroid
hormone­
related
processes.
EPA
should
consider
tests
that
detect
multiple
hormone
interactions,
address
endpoints
in
multiple
species,
and
predict
long­
term
or
delayed
effects.
EPA
should
periodically
revisit
the
scope
of
this
strategy
to
permit
inclusion
of
additional
hormone
systems,
animals
other
than
vertebrates,
other
hormone­
mediated
effects,
or
new
screens
and
tests
as
they
become
available.
·
Screening
and
testing
should
be
capable
of
evaluating
both
chemical
substances
and
common
mixtures.

VI.
Where
Endocrine
Disruption
Fits
in
the
Broader
Context
Many
of
the
effects
of
endocrine
disruption
are
manifested
as
disease
processes
that
are
already
recognized
and
addressed,
to
some
degree,
in
current
toxicological
assessments.
For
example,
endocrine
disruption
may
result
in
cancer,
neurotoxicity,
or
reproductive
or
developmental
toxicity
(
i.
e.,
infertility,
birth
defects,
etc.).
It
is
important
to
realize
that
these
issues
are
interconnected.
For
example,
some
cancers
have
their
origin
in
prenatal
life
as
do
some
neurological
problems.
In
addition,
there
may
be
some
endocrine
disruptive
effects
that
may
not
fit
clearly
into
any
of
the
three
more
well­
recognized
categories.

While
considering
the
potential
adverse
human
health
and
environmental
effects
due
to
endocrine
disruption,
it
should
also
be
noted
that
the
effects
of
human
exposures
to
endocrine
active
chemicals
are
not
necessarily
adverse.
Knowledge
of
the
functioning
of
human
endocrine
systems
has
led
to
the
development
of
numerous
important
medical
applications
of
therapies
that
operate
through
chemical
modulation
of
endocrine
systems.
The
applications
represent
the
positive
effects
of
human
exposures
to
endocrine
active
agents.
Examples
include
birth
control,
adjunct
therapies
for
prostate
and
breast
cancer,
prevention
of
postmenopausal
osteoporosis
and
heart
disease,
treatment
of
hypothyroidism,
and
prevention
or
reversal
of
hair
loss.
In
addition,
the
consumption
of
a
diet
high
in
soy
(
and
its
phytoestrogens)
is
thought
to
contribute
to
low
breast
cancer
rates
in
some
Asian
populations.
It
must
be
kept
in
mind
that
endocrine
active
chemicals
with
beneficial
effects
may
still
lead
to
adverse
effects
under
circumstances
of
unintended,
inappropriate,
or
environmental
exposures.

The
scope
of
the
program
recommended
by
the
EDSTAC
addresses
a
small
portion
of
all
possible
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
7
hormonal
effects.
The
scope
includes
disruption
of
estrogen,
androgen,
and
thyroid
hormonal
activities.
This
group
of
hormones
includes
those
for
which
there
exists
the
most
data,
and
for
which
standardized
assays
have
been
developed.
These
hormonal
systems
are
a
very
limited
part
of
the
potential
universe
of
endocrine
systems
in
all
animals
that
may
be
affected
by
chemical
exposures.
It
is
important,
as
the
science
evolves,
for
EPA
to
be
creative
in
developing
and
including
new
screens
for
additional
modes
of
action
or
to
use
different
modeling
systems
that
will
improve
the
ability
to
detect
endocrine­
mediated
environmental
hazards,
especially
for
nonmammalian
species.

While
estrogen,
androgen,
and
thyroid
hormones
are
extremely
important,
and
it
is
critical
to
focus
initial
development
of
screens
and
tests
to
look
for
effects
on
them,
the
Committee
wants
it
to
be
clear
that
the
scope
of
the
EDSTP
is
quite
limited.
The
EDSTAC
has
not
devised
a
way
to
test
for
all
possible
endocrine
disruptive
effects,
nor
is
the
Committee
addressing
the
many
nonhormone
mediated
causes
of
cancer,
neurological
toxicity,
and
toxicity
to
reproduction
or
to
early
life
stage
developmental
processes.
When
a
chemical
proceeds
to
the
"
hold
box"
of
the
Conceptual
Framework,
it
is
because
the
chemical
tested
negative
for
the
hormone
systems
assessed,
not
for
all
hormonal
effects
or
for
other
mechanisms
that
may
cause
these
effects.

EPA
has
already
developed
toxicological
screening
and
testing
protocols
to
evaluate
carcinogenicity,
developmental
and
reproductive
toxicity,
and
neurotoxicity.
Some
of
the
information
developed
from
these
screening
and
testing
activities
will
be
useful
in
evaluating
the
endocrine
disruption
potential
of
chemical
substances
and
mixtures.
For
example,
results
from
developmental
toxicity
testing
could
suggest
the
need
to
undertake
T1S
or
T2T.
Similarly,
results
from
screening
and
testing
related
to
endocrine
disruption
could
suggest
the
need
for
neurotoxicity,
developmental,
or
other
toxicity
screening
or
testing.
The
EDSTAC
recommends
that
EPA
examine
the
interrelationships
between
these
screening
and
testing
protocols
and
take
advantage
of
potential
opportunities
to
streamline
protocols
and
ensure
that
the
results
of
the
related
screens
and
tests
are
taken
advantage
of
in
assessing
the
risk
of
endocrine
disruption.

VII.
General
Principles
to
Guide
the
Development
of
the
Endocrine
Disruptor
Screening
and
Testing
Program
Several
principles
have
guided
the
development
of
the
EDSTAC
Conceptual
Framework,
and
should
guide
further
development
of
specific
processes
to
sort
and
prioritize,
screen,
and
test
chemical
substances
and
mixtures
for
endocrine
disruption.
These
principles
help
ensure
that
the
strategy
of
screening
and
testing
will
serve
the
general
purpose
stated
above,
while
recognizing
that
societal
resources
must
also
be
allocated
to
sources
of
environmental
risk
other
than
endocrine
disruption.
Thus,
the
screening
and
testing
strategy
should:

·
require
the
minimal
number
of
screens
and
tests
necessary
to
make
sound
decisions,
thereby
reducing
the
time
needed
to
make
these
decisions;
·
examine
existing
screens
and
tests
for
their
potential
to
predict,
detect,
and/
or
characterize
endocrine
disruptors,
ensuring
that
any
modification
to
existing
screens
and
tests
does
not
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
8
compromise
their
ability
to
predict
other
toxicity
endpoints;
1
·
systematically
examine
existing
screening
and
testing
data,
not
only
for
adverse
endpoints
in
high
dose
groups,
but
also
for
physiological
changes
in
low
dose
groups;
·
not
detract
from
current
and
new
efforts
to
assess
the
toxicity
of
chemical
substances
and
mixtures
through
mechanisms
other
than
endocrine
disruption;
·
provide
data
that
can
be
used
for
a
broad
range
of
management
and
regulatory
programs
in
a
form
that
supports
international
harmonization
of
the
data's
use;
·
include
periodic
review
of
new
scientific
information;
·
use
a
performance­
based
approach
to
the
selection
of
screens,
tests,
and
species,
including
the
use
of
more
appropriate
screens,
tests,
and
species
as
they
are
developed
and
validated;
·
be
dynamic
in
order
to
stay
current
with
the
rapidly
evolving
science
related
to
the
endocrine
system;
and
·
be
conducted
at
a
minimal
cost
necessary
to
make
the
decisions
within
the
EDSTAC
Conceptual
Framework.

In
addition
to
these
nine
broad
principles,
which
place
screening
and
testing
for
endocrine
disruption
into
a
larger
framework
of
environmental
risk,
there
are
several
principles
specific
to
the
screens
and
tests
themselves:

·
To
make
decisions
within
the
EDSTAC
Conceptual
Framework,
all
screens
and
tests
should
have
well­
defined
endpoints.
·
The
use
of
animals
should
be
reduced
to
the
minimal
level
needed
to
obtain
scientifically
valid
results
and
interpretations.
·
The
results
of
screens
and
tests
should
support
further
research
on
effects
of
endocrine
disruptors
on
populations,
communities,
and
ecosystems.
·
In
interpreting
screening
and
testing
results,
a
"
weight­
of­
evidence"
approach
should
be
used,
but
should
be
consistent
with
a
principle
of
prudence
in
protecting
human
health
and
the
environment.
In
the
case
of
T1S,
this
means
that
a
relatively
higher
value
is
placed
on
sensitivity
as
opposed
to
specificity.
The
goal
is
to
minimize
false
negative
results
while
also
ensuring
that
false
positive
results
do
not
become
so
frequent
that
chemical
substances
cannot
be
sorted
meaningfully
with
respect
to
T2T.
·
Screening
and
testing
results
should
be
reported
in
a
format
that
facilitates
database
development
and
analysis
by
a
broad
array
of
scientific,
regulatory,
and
management
organizations.
·
Decision
criteria,
such
as
those
for
statistical
significance
(
e.
g.,
necessary
confidence
intervals)
and
biological
plausibility,
should
be
clearly
defined.

1.
If
a
necessary
modification
does
compromise
the
existing
toxicity
assays,
separate
endocrine
disruptor
and
toxicity
screens
and/
or
tests
should
be
conducted.
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
9
VIII.
The
EDSTAC
Conceptual
Framework
A.
Obtain
and
Analyze
Existing
Information
to
Set
Priorities
An
ordered
screening
and
testing
strategy
should
begin
with
an
effort
to
obtain
and
analyze
available
information
on
new
and
existing
chemical
substances
or
mixtures.
Information
on
toxic
and
physiological
effects,
chemical
structure
activity
relationships
(
SARs),
use
information,
product
chemistry,
exposure
information,
and
legal
mandates
will
be
examined.
Given
limited
resources
and
capacity,
as
well
as
the
potential
magnitude
of
the
task,
it
will
be
necessary
to
develop
a
priority
setting
system
to
determine
the
relative
order
in
which
chemical
substances
and
mixtures
will
be
subjected
to
T1S.
An
evaluation
and
analysis
of
this
information
will
lead
to
one
of
four
possible
determinations:

·
polymers
which
will
be
placed
into
a
"
hold"
status
(
with
some
exceptions)
pending
a
review
of
their
monomers
and
oligomers;
·
chemicals
for
which
insufficient
data
exists
to
proceed
to
either
T2T
or
hazard
assessment
and
will,
therefore,
need
to
be
prioritized
for
T1S;
·
chemicals
for
which
sufficient
data
exists,
or
voluntary
bypass
of
T1S,
to
go
to
T2T;
and
·
chemicals
for
which
sufficient
data
exists
to
go
to
hazard
assessment.

B.
Tier
1
Screening
to
Detect
Interactions
With
the
Endocrine
System
The
purpose
of
T1S
is
to
obtain
a
minimum,
yet
sufficient,
set
of
valid
and
reliable
data
to
detect
whether
a
chemical
substance
or
mixture
may
interact
with
the
endocrine
system.
Included
in
T1S
is
a
battery
of
assays
designed
to
detect
effects
that
enhance,
mimic,
or
inhibit
estrogen,
androgen,
and
thyroid
hormone­
related
processes.
In
contrast
to
the
more
refined
and
detailed
tests
of
Tier
2,
the
T1S
assays
should:

·
be
inexpensive,
quick,
and
easy
to
perform;
·
be
validated
and
standardized
as
soon
as
possible,
defining
characteristics
such
as
sensitivity
and
specificity
against
a
clearly
defined
standard,
once
it
is
identified;
·
be
more
"
sensitive"
than
they
are
"
specific,"
meaning
they
should
have
as
their
primary
objective
the
minimization
of
false
negative
or
(
Type
II)
errors,
while
permitting
an
as­
of­
yet
undetermined,
but
acceptable,
level
of
false
positive
or
(
Type
I)
errors;
·
capture
multiple
endpoints
and
reflect
as
many
modes
of
endocrine
action
as
possible;
·
be
broadly
predictive
across
species,
gender,
and
age;
and
·
yield
data
capable
of
being
interpreted
as
either
positive
or
negative
for
the
purpose
of
determining
whether
and
how
to
conduct
T2T.
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
10
Information
gathered
during
T1S
should
be
used
to
make
initial
judgments
about
areas
of
concern
and
should
direct
and
focus
T2T.
The
interpretation
of
T1S
results
should
be
consistent
with
best
scientific
judgment,
formed
on
the
basis
of
considerations
such
as
"
weight­
of­
evidence,"
consistency
of
the
data
set,
and
methodological
strengths
and
limitations.
Based
on
the
evaluation
made
during
this
phase,
one
of
two
decisions
is
possible:

1.
Proceed
to
T2T
 
If
the
interpretation
of
results
from
the
full
battery
of
T1S
assays
is
determined
to
be
"
positive"
(
i.
e.,
the
screens
produced
evidence
of
interaction
with
the
endocrine
system,
within
the
scope
of
endocrine
functions
addressed
by
the
program),
the
chemical
substance
or
mixture
will
enter
T2T
to
characterize
the
nature
of
any
endocrine
disrupting
effects.

2.
Hold
Screening
and
Testing
 
If
the
interpretation
of
results
from
the
full
battery
of
T1S
assays
is
determined
to
be
"
negative"
(
i.
e.,
the
screens
have
not
produced
evidence
of
interactions
with
the
endocrine
system,
within
the
scope
of
endocrine
functions
addressed
by
the
program),
and
these
results
are
not
contravened
by
the
"
weight­
of­
evidence"
developed
during
the
prioritization
phase,
no
additional
screening
or
testing
is
necessary
unless:

a)
existing
statutes
require
periodic
review
(
e.
g.,
FIFRA
re­
registration);
b)
new
statutory
requirements
mandate
review;
c)
new
screens
for
endocrine
disruption
are
incorporated
into
the
strategy
and
it
is
determined
that
these
new
screens
may
either
generate
significant
new
information
or
they
invalidate
prior
screens
and
therefore
warrant
the
re­
screening
of
chemical
substances
and
mixtures
that
have
already
been
subjected
to
T1S;
and/
or
d)
new
information
on
the
endocrine
disrupting
potential
of
the
chemical
substance
or
mixture
becomes
available
which
warrants
the
re­
screening
of
the
chemical
substance
or
mixture.

C.
Tier
2
Testing
to
Determine
and
Characterize
Endocrine
Disruption
The
purpose
of
T2T
is
to
determine
and
characterize
the
nature,
likelihood,
and
dose­
response
relationship
of
estrogen­,
androgen­,
and
thyroid­
related
effects
in
humans
and
wildlife.
Selection
of
Tier
2
tests
should
be
based
upon
T1S
results
and
other
relevant
information.
An
underlying
principle
of
T2T
is
that
it
should
provide
information
useful
for
human/
ecological
hazard
assessment.
The
T2T
scheme
should
be
flexible
enough
to
allow
for
scientific
judgment
in
the
selection
of
the
most
appropriate
tests
and
endpoints,
and
costs
should
be
practical.
T2T
should
be
aimed
at
determining
whether
the
chemical
substance
or
mixture
is
an
endocrine
disruptor.
In
addition,
these
tests
should
be
designed
to
establish
the
relationship
between
different
exposure
levels,
timing
and
duration
of
exposure,
and
adverse
effects,
including
developmental
and
reproductive
effects
on
the
individual
and
its
progeny.
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
11
In
comparison
to
the
components
of
the
T1S
phase,
T2T
should
ideally
be
both
sensitive
and
specific.
In
other
words,
it
should
be
designed
to
minimize
both
false
positive
(
Type
I)
and
false
negative
(
Type
II)
errors.
Additionally,
this
battery
of
tests
should:

·
include
assessment
of
endpoints
identified
as
relevant
from
T1S;
·
include
parental/
offspring
developmental
endpoints
(
e.
g.,
two­
generation
studies)
in
order
to
adequately
evaluate
all
life
stages;
·
include
the
life
cycle
of
both
viviparous
(
live
birth)
and
oviparous
(
egg­
laying)
organisms;
·
be
conducted
at
a
range
of
doses
that
allows
full
characterization
of
the
adverse
effects
of
the
chemical
substance
or
mixture
being
tested;
·
be
conducted
in
accordance
with
Good
Laboratory
Practice
(
GLP)
regulations
to
the
degree
consistent
with
resources
and
the
goal
of
timely
decisions;
and
·
be
validated,
if
need
be,
as
soon
as
possible
against
a
clearly
defined
standard.

Interpretation
of
results
from
T2T
should
reflect
current
scientific
judgment,
including
considerations
such
as
"
weight­
of­
evidence"
and
consistency
of
the
data
set.

A
"
negative"
result
in
T2T
should
abrogate
any
additional
endocrine
screening
and
testing
for
that
particular
chemical
substance
or
mixture
within
the
scope
of
endocrine
functions
defined
by
the
program
unless:

a)
existing
statutes
require
periodic
review
(
e.
g.,
FIFRA
re­
registration);
b)
new
statutory
requirements
mandate
review;
c)
new
screens
or
tests
for
endocrine
disruption
are
incorporated
into
the
EDSTAC
strategy
which
will
generate
significant
new
information,
or
invalidate
prior
screens
or
tests
upon
which
decisions
have
been
made
to
stop
screening
and
testing;
d)
new
information
on
the
endocrine
disrupting
potential
of
the
chemical
substance
or
mixture
becomes
available
and
it
is
determined
that
this
new
information
warrants
additional
testing;
and/
or
e)
there
are
changes
in
the
use
and
expected
exposure
patterns
upon
which
the
initial
selection
of
tests
was
made.

In
the
event
of
a
"
positive"
outcome,
the
chemical
substance
or
mixture
will
proceed
to
the
hazard
assessment
phase
of
decision­
making,
whereupon
it
may
be
decided
that
additional
T2T
is
required
before
a
final
determination
of
hazard
can
be
made.

IX.
Additional
Components
and
Clarifications
to
the
Original
EDSTAC
Conceptual
Framework
During
the
course
of
its
deliberations,
the
EDSTAC
identified
an
additional
concept,
the
incorporation
of
"
high
throughput
pre­
screening,"
to
be
considered
in
the
context
of
the
original
Conceptual
Framework.
In
addition,
the
EDSTAC
clarified
conditions
under
which
a
chemical
substance
might
be
permitted
to
bypass
T1S
assays
and,
instead,
go
directly
to
T2T.
These
two
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
12
issues
are
closely
connected
to
other
issues
discussed
later
in
the
report,
including
testing
at
low
doses
and
the
definitiveness
of
T2T.

A.
High
Throughput
Pre­
Screening
During
its
deliberations
the
EDSTAC
concluded
that
biological
effects
data
were
incomplete
or
lacking
for
most
chemical
substances.
In
the
absence
of
biological
effects
data,
EPA
would
be
left
with
the
choice
of
either
raising
or
lowering
the
priority
of
a
chemical
based
on
the
lack
of
effects
information.

To
address
this
problem,
the
EDSTAC
recommends
that
a
subset
of
the
in
vitro
assays
recommended
for
inclusion
in
the
T1S
battery
should
be
conducted
with
the
assistance
of
automated
technology,
in
advance
of
the
priority
setting
step
of
the
overall
sorting
and
priority
setting
process.
The
EDSTAC
came
to
refer
to
this
technology,
which
uses
robotics
and
other
automated
processes,
and
the
role
that
it
will
play
in
the
overall
endocrine
disruptor
screening
and
testing
program,
as
"
high
throughput
pre­
screening"
(
HTPS).

HTPS
results,
by
themselves,
will
not
be
sufficient
to
make
a
determination
about
whether
a
chemical
may
interact
with
the
endocrine
system
of
an
intact
animal.
Such
determinations
will
require
additional
screening
and
testing.

The
primary
purpose
of
the
HTPS
is
to
provide
preliminary
biological
activity
information
for
a
large
number
of
chemicals
in
a
relatively
short
period
of
time.
This
information
will
simultaneously
be
of
value
for:
(
a)
detecting
hormonal
activity
(
as
a
component
of
T1S);
and
(
b)
providing
at
least
some
biological
effects­
related
information
for
the
estrogen,
androgen,
and
thyroid
hormonal
systems
to
assist
in
the
effort
to
set
priorities
for
T1S.
The
in
vitro
assays
that
would
be
performed
as
part
of
the
HTPS
include
transcriptional
activation
assays.
Performance
of
these
assays
would
still
be
required
as
part
of
T1S
for
all
chemicals
that
do
not
go
through
HTPS.

The
EDSTAC
recommends
that
all
chemicals
currently
produced
in
an
amount
equal
to
or
greater
than
10,000
pounds
per
year
(
estimated
to
be
about
15,000
chemicals)
be
subjected
to
HTPS.
Also,
it
is
expected
that
all
pesticides
(
i.
e.,
both
active
ingredients
and
formulation
inerts)
will
be
subjected
to
HTPS.
Any
chemicals
subjected
to
the
assays
conducted
in
the
HTPS
step
would
not
be
required
to
repeat
the
ER
binding/
transcriptional
activation
assay
and
the
AR
binding/
transcriptional
activation
assay
as
part
of
T1S.
On
the
other
hand,
for
any
chemicals
not
subjected
to
HTPS
(
e.
g.,
production
volumes
less
than
10,000
pounds
per
year),
the
assays
in
T1S
would
result
in
information
equivalent
to
that
which
would
have
been
provided
from
HTPS.
The
HTPS
concept
is
explained
in
more
detail
in
Chapter
Four,
Section
V,
and
referred
to
often
in
Chapter
Five.
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
13
B.
Alternative
Means
of
Meeting
Versus
Bypassing
Tier
1
Screening
The
EDSTAC
expects
the
vast
majority
of
chemicals
included
in
the
EDSTP
to
go
through
the
program
in
the
logical,
hierarchical
manner
in
which
the
program
was
designed.
Notwithstanding
this
expectation,
the
EDSTAC
recognizes
there
will
be
circumstances
where
it
may
be
inefficient
to
follow
all
steps
of
the
EDSTP.
For
example,
the
EDSTAC
Conceptual
Framework
allows
chemicals
that
have
already
been
subjected
to
tests
that
are
the
"
functional
equivalents"
of
the
T2T
two­
generation
reproductive
toxicity
tests,
endocrine
disruptor
endpoints,
taxa,
and
dosing
considerations
recommended
by
the
EDSTAC,
to
bypass
both
T1S
and
T2T
and
proceed
directly
to
hazard
assessment.

The
EDSTAC
has
identified
two
other
circumstances
where
a
chemical
substance
or
mixture
may
not
be
required
to
be
evaluated
in
the
assays
included
in
the
recommended
T1S
battery.
Both
of
these
scenarios
are
discussed
below
and
then
in
further
detail
in
Chapters
Four
and
Five.

1.
Alternative
Means
to
Meet
T1S
Information
Requirements
The
EDSTAC
recommends
that
it
should
be
permissible
to
complete
the
information
requirements
of
T1S
through
the
submission
of
data
that
are
"
functionally
equivalent"
to
the
data
that
would
be
generated
from
the
recommended
T1S
battery.
Further,
functionally
equivalent
information
could
be
submitted
for
one
or
more
of
the
recommended
T1S
assays
or
for
the
entire
battery.
The
EDSTAC
believes
it
is
helpful
to
distinguish
this
scenario,
which
is
in
essence
an
alternative
means
of
meeting
the
information
requirements
associated
with
T1S,
from
two
other
scenarios,
which
are
considered
bypassing
T1S.

2.
Bypassing
T1S
There
are
two
scenarios
in
which
the
EDSTAC
recommends
that
the
owner
of
a
chemical
should
be
permitted
to
bypass
T1S.
Each
of
these
two
scenarios
has
different
implications
for
the
information
requirements
associated
with
completing
T2T
and
hazard
assessment
following
T2T.

a)
Chemicals
That
Have
Previously
Been
Subjected
to
Two­
Generation
Reproductive
Toxicity
Tests
The
first
scenario
includes
those
chemicals
that
have
previously
been
subjected
to
mammalian
and
wildlife
developmental
toxicity
and/
or
reproductive
toxicity
testing,
but
such
testing
may
not
include
additional
EAT
endpoints
for
T2T,
as
specified
in
Chapter
Five,
Section
VI.
The
EDSTAC
expects
that
food­
use
pesticides
will
fall
into
this
category,
given
the
requirements
of
FIFRA
and
FQPA,
as
well
as
a
small
number
of
other
types
of
pesticides
and
industrial
chemicals.
The
EDSTAC
agrees
that
chemicals
which
meet
this
criterion
for
bypassing
T1S
would
still
be
subjected
to
the
assays
that
will
be
part
of
the
HTPS.
In
addition,
chemicals
which
meet
this
criterion
will
also
be
the
most
likely
candidates
for
the
alternative
approaches
for
completing
T2T,
as
discussed
in
Chapter
Five,
Section
V,
C,
2.
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
14
b)
Chemicals
for
Which
There
is
No
Prior
Toxicity
Testing
The
second
scenario
includes
those
chemicals
for
which
the
owner
of
the
chemical
has
decided
to
voluntarily
go
to
T2T
without
having
completed
the
full
T1S
battery
or
any
prior
two­
generation
reproductive
toxicity
testing.
These
chemicals
must
be
evaluated
in
the
HTPS
assays.
In
addition,
chemicals
that
bypass
T1S
under
this
second
scenario
must
be
evaluated
in
all
the
tests
of
the
T2T
battery
(
i.
e.,
the
mammalian
and
non­
mammalian
multi­
generation
tests
with
all
the
recommended
endpoints),
consistent
with
the
principles
governing
T2T,
which
are
set
forth
in
Chapter
Five,
Section
V,
C.

X.
Compilation
of
Chapter
Three
Recommendations
1.
The
EDSTAC
developed
a
tiered
Conceptual
Framework
that
formed
the
basis
for
its
screening
and
testing
strategy
and
all
subsequent
recommendations.
The
tiered
framework
consists
of
the
following
three
major
activities:

·
Priority
setting
includes
the
sorting
and
prioritization
of
chemical
substances
and
mixtures
based
on
existing
information.
The
existing
information
would
be
used
to
sort
chemicals
into
four
categories.
An
evaluation
and
analysis
of
this
information
will
lead
to
sorting
chemicals
into
one
of
four
categories:

·
Polymers,
which
are
placed
into
a
"
hold"
status
(
with
some
exceptions)
pending
a
review
of
their
monomers
and
oligomers.
·
Chemicals
for
which
there
is
insufficient
data
regarding
endocrine
disruption
and
will
therefore
need
to
be
prioritized
for
Tier
1
Screening.
·
Chemicals
for
which
sufficient
data
exists
to
proceed
to
Tier
2
Testing.
·
Chemicals
for
which
sufficient
data
exists
to
go
to
hazard
assessment.

·
Tier
1
Screening
(
T1S)
to
detect
chemical
substances
and
mixtures
capable
of
acting
on
endocrine
systems.

·
Tier
2
Testing
(
T2T)
to
determine,
characterize,
and
quantify
the
nature
of
the
endocrine
disrupting
properties
of
the
chemical
substances
and
mixtures
identified
by
prior
information
and/
or
T1S.

2.
The
EDSTAC
recommended
the
adoption
of
several
principles
to
guide
the
use
of
the
Conceptual
Framework.

·
A
chemical
may
bypass
one
or
more
tiers
when
warranted
by
appropriate
information
(
e.
g.,
sufficient
prioritization
data
on
endocrine
disrupting
properties
to
initiate
T2T
or
hazard
assessment).
·
If
information
is
inadequate
to
determine
if
a
chemical
should
move
to
the
next
tier,
an
active
process
should
be
developed
for
generating
the
needed
information
to
make
such
a
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
15
decision.
·
Criteria
and
default
assumptions
for
deciding
whether
chemicals
move
from
one
tier
to
the
next
should
be
developed
in
advance
of
initiating
screening
and
testing.

3.
Within
the
context
of
the
Conceptual
Framework,
the
EDSTAC
recommends
that
the
overall
scope
of
the
screening
and
testing
strategy
should:

·
be
relevant
to
both
human
health
and
ecological
effects;
·
initially
emphasize
identifying
and
characterizing
effects
that
enhance,
mimic,
or
inhibit
estrogenic,
androgenic,
and
thyroid
hormone­
related
processes;
·
consider
tests
that
detect
multiple
hormone
interactions,
address
endpoints
in
multiple
species,
and
predict
long­
term
or
delayed
effects;
·
be
periodically
revisited
to
permit
inclusion
of
additional
hormone­
mediated
effects
or
new
screens
and
tests
as
they
become
available;
·
be
capable
of
evaluating
the
endocrine
disrupting
properties
of
chemical
substances
and
common
mixtures;
and
·
allow
determination
of
possible
additive,
synergistic,
or
antagonistic
effects
caused
by
interactions
among
the
components
of
mixtures.

4.
The
EDSTAC
recommends
nine
broad
principles
to
guide
the
implementation
of
the
endocrine
disruptor
screening
and
testing
program.
The
screening
and
testing
program
should:

·
require
the
minimal
number
of
screens
and
tests
necessary
to
make
sound
decisions,
thereby
reducing
the
time
needed
to
make
these
decisions;
·
examine
existing
screens
and
tests
for
their
potential
to
predict,
detect,
and/
or
characterize
endocrine
disruptors,
ensuring
that
any
modification
to
existing
screens
and
tests
does
not
compromise
their
ability
to
predict
other
toxicity
endpoints;
·
systematically
examine
existing
screening
and
testing
data
not
only
for
adverse
endpoints
in
high
dose
groups,
but
also
for
physiological
changes
in
low
dose
groups;
·
not
detract
from
current
and
new
efforts
to
assess
the
toxicity
of
compounds
and
mixtures
through
mechanisms
other
than
endocrine
disruption;
·
provide
data
that
can
be
used
for
a
broad
range
of
management
and
regulatory
programs
in
a
form
that
supports
international
harmonization
of
their
use;
·
include
periodic
review
of
new
scientific
information;
·
use
a
performance­
based
approach
to
the
selection
of
screens,
tests,
and
species,
including
the
use
of
more
appropriate
screens,
tests,
and
species
as
they
are
developed
and
validated;
·
be
dynamic
in
order
to
stay
current
with
the
rapidly
evolving
science
related
to
the
endocrine
system;
and
·
be
conducted
at
the
minimum
cost
necessary
to
make
the
decisions
within
the
EDSTAC
Conceptual
Framework.

5.
The
EDSTAC
also
recommended
six
guiding
principles
specific
to
the
screens
and
tests
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
16
themselves.

·
To
facilitate
making
decisions
within
the
EDSTAC
Conceptual
Framework,
all
screens
and
tests
should
have
well­
defined
endpoints.
·
The
use
of
animals
should
be
reduced
to
the
minimum
level
needed
to
obtain
scientifically
valid
results
and
interpretations.
·
The
results
of
screens
and
tests
should
support
further
research
on
effects
of
endocrine
disruptors
on
populations,
communities,
and
ecosystems.
·
In
interpreting
screening
and
testing
results,
a
"
weight­
of­
evidence"
approach
should
be
used,
but
should
be
consistent
with
a
principle
of
prudence
in
protecting
human
health
and
the
environment.
In
the
case
of
T1S,
this
means
that
the
strategy
will
err
on
the
side
of
false
positive
identifications
rather
than
false
negatives.
·
Screening
and
testing
results
should
be
reported
in
a
format
that
facilitates
database
development
and
analysis
by
a
broad
array
of
scientific,
regulatory,
and
management
organizations.
·
Decision
criteria,
such
as
those
for
determining
statistical
significance
(
e.
g.,
necessary
confidence
intervals)
and
biological
plausibility,
should
be
clearly
defined.

6.
The
EDSTAC
recommends
that
T1S
provide
the
minimum,
yet
valid
and
reliable,
data
to
detect
interactions
with
the
endocrine
system.
In
contrast
to
Tier
2
tests,
T1S
assays
should:

·
be
inexpensive,
quick,
and
easy
to
perform;
·
be
validated
and
standardized
as
soon
as
possible,
defining
characteristics
such
as
sensitivity
and
specificity
against
a
"
gold
standard,"
once
it
is
identified;
·
be
more
"
sensitive"
than
they
are
"
specific,"
meaning
that
they
should
have
as
their
primary
objective
the
minimization
of
false
negative
or
(
Type
II)
errors,
while
permitting
an
as­
of­
yet
undetermined,
but
acceptable,
level
of
false
positive
or
(
Type
I)
errors;
·
capture
multiple
endpoints
and
reflect
as
many
modes
of
endocrine
action
as
possible;
·
be
broadly
predictive
across
species,
gender,
and
age;
and
·
yield
data
capable
of
being
interpreted
as
either
positive
or
negative
for
the
purpose
of
determining
whether
and
how
to
conduct
T2T.

7.
The
EDSTAC
recommends
that
T1S
be
used
to
make
initial
judgments
about
areas
of
concern
in
order
to
direct
the
focus
of
T2T.
The
interpretation
of
T1S
results
should
be
consistent
with
best
scientific
judgment,
formed
on
the
basis
of
considerations
such
as
"
weight­
of­
evidence,"
consistency
of
the
data
set,
and
methodological
strengths
and
limitations.

8.
The
EDSTAC
recommends
that
T2T
be
based
upon
T1S
results
and
other
relevant
information.
An
underlying
principle
of
T2T
is
that
it
should
provide
information
useful
for
human
and
ecological
hazard
assessment.
The
T2T
scheme
should
be
flexible
enough
to
allow
for
scientific
judgment
in
the
selection
of
the
most
appropriate
tests
and
endpoints,
and
costs
should
be
reasonable.
Tests
should
be
aimed
at
determining
whether
the
chemical
substance
or
mixture
is
an
endocrine
disruptor
and
whether
the
effects
are
a
result
of
primary
or
secondary
disturbances
of
endocrine
function.
In
addition,
these
tests
should
be
designed
to
establish
the
relationship
between
different
exposure
levels,
timing
and
duration
of
exposure,
and
adverse
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
17
effects,
including
developmental
and
reproductive
effects
on
the
individual
and
its
progeny.

9.
In
contrast
to
T1S,
the
EDSTAC
recommends
that
T2T
should
be
both
sensitive
and
specific,
and
designed
to
minimize
false
positive
(
Type
I)
and
false
negative
(
Type
II)
errors.
Additionally,
this
battery
of
tests
should:

·
include
assessment
of
endpoints
identified
as
relevant
from
Tier
1
screens;
·
include
parental/
offspring
developmental
endpoints
(
e.
g.,
two­
generation
studies)
in
order
to
adequately
evaluate
all
life
stages;
·
include
the
life
cycle
of
both
viviparous
(
live
birth)
and
oviparous
(
egg­
laying)
organisms;
·
be
conducted
at
a
range
of
doses
that
allow
full
characterization
of
the
adverse
effects
of
the
chemical
substance
or
mixture
being
tested;
·
be
conducted
in
accordance
with
Good
Laboratory
Practice
(
GLP)
regulations
to
the
degree
consistent
with
resources
and
the
goal
of
timely
decisions;
and
·
be
validated,
if
need
be,
as
soon
as
possible
against
a
clearly
defined
standard.

10.
The
EDSTAC
recommends
that
a
subset
of
the
T1S
in
vitro
assays
be
conducted
with
the
assistance
of
automated
technology
to
provide
biological
effects
information
to
assist
in
the
overall
sorting
and
priority
setting
process.
Because
of
the
role
this
technology
will
play
in
the
overall
EDSTP,
the
EDSTAC
refers
to
it
as
"
high
throughput
pre­
screening"
(
HTPS).
The
EDSTAC
recommends
that
all
chemicals
currently
produced
in
an
amount
equal
to
or
greater
than
10,000
pounds
per
year
(
estimated
to
be
about
15,000
chemicals)
be
subjected
to
HTPS.
Also,
it
is
expected
that
all
pesticides
(
i.
e.,
both
active
ingredients
and
formulation
inerts)
will
be
subjected
to
HTPS.
Any
chemicals
subjected
to
the
assays
conducted
in
the
HTPS
step
would
not
be
required
to
repeat
the
ER
binding/
transcriptional
activation
assay
and
the
AR
binding/
transcriptional
activation
assay
as
part
of
T1S.
On
the
other
hand,
for
any
chemicals
not
subjected
to
HTPS
(
e.
g.,
production
volumes
less
than
10,000
pounds
per
year),
the
assays
in
T1S
would
result
in
information
equivalent
to
that
which
would
have
been
provided
from
HTPS.

11.
The
EDSTAC
recommends
that
the
vast
majority
of
chemicals
go
through
priority
setting,
T1S,
and
T2T
in
a
sequential
manner.
However,
the
EDSTAC
also
recognizes
there
may
be
individual
cases
in
which
T1S
is
bypassed.
Three
situations
were
identified
where
a
chemical
may
bypass
T1S,
each
with
different
implications
for
information
requirements
in
T2T.

·
Alternative
means
to
meet
T1S
information
requirements
through
the
generation
of
data
which
are
"
functionally
equivalent"
to
data
derived
from
the
recommended
screening
battery.
·
Bypassing
T1S
for
chemicals
(
e.
g.,
food­
use
pesticides)
that
have
previously
been
subjected
to
two­
generation
reproductive
toxicity
tests.
Such
chemicals
should
still
be
subjected
to
high
throughput
pre­
screening
assays.
·
Bypassing
T1S
for
chemicals
for
which
there
is
no
prior
toxicology
testing
but
the
owner
has
voluntarily
decided
to
proceed
directly
to
testing.
Such
chemicals
must
be
evaluated
in
the
high
throughput
pre­
screening
assays,
and
all
of
the
tests
in
the
T2T
battery.
EDSTAC
Final
Report
Chapter
Three
August
1998
3
­
18
XI.
Literature
Cited
European
Commission,
"
European
Workshop
on
the
Impact
of
Endocrine
Disrupters
on
Human
Health
and
the
Environment,"
Environment
and
Climate
Research
Programme,
DG
XII,
European
Commission,
Report
EUR
17549,
1997.
Kavlock,
R.
J.
et
al.,
"
Research
needs
for
risk
assessment
of
health
and
environmental
effects
of
endocrine
disruptors:
A
review
of
the
U.
S.
EPA­
sponsored
workshop,"
Environmental
Health
Perspectives,
104,
1996,
pp.
715­
740.
Tattersfield,
L.
et
al.
(
Ed.).
"
SETAC­
Europe/
OECD/
EC
Expert
Workshop
on
Endocrine
Modulators
and
Wildlife:
Assessment
and
Testing,"
SETAC­
Europe,
Brussels,
1997.
U.
S.
EPA,
"
Special
Report
on
Environmental
Endocrine
Disruption:
An
Effects
Assessment
and
Analysis.
Office
of
Research
and
Development,"
EPA/
630/
R­
96/
012,
1997.
EDSTAC
Final
Report
Chapter
Four
August
1998
Chapter
Four
Priority
Setting
EDSTAC
Final
Report
Chapter
Four
August
1998
Table
of
Contents
I.
Introduction...........................................................................................................................
1
A.
Charge
to
the
PSWG
.........................................................................................................
1
B.
The
Need
for
Priority
Setting
.............................................................................................
1
II.
Overview
of
the
Sorting
and
Priority
Setting
Recommendations
......................................
4
A.
Initial
Sorting
Step.............................................................................................................
4
B.
Phased
Approach
...............................................................................................................
5
C.
Polymers............................................................................................................................
6
D.
Chemicals
With
Sufficient
Data
to
go
to
T2T
or
Voluntary
Bypass
of
T1S.........................
8
E.
Chemicals
With
Sufficient
Data
to
Proceed
to
Hazard
Assessment......................................
9
F.
Chemicals
with
Insufficient
Data
to
go
to
T2T
or
Hazard
Assessment.................................
9
G.
Priority
Setting
Information
Categories
and
Criteria
.........................................................
10
H.
Role
of
the
Statutory
Criteria
...........................................................................................
11
I.
High
Throughput
Pre­
Screening
(
HTPS)
Step
...................................................................
12
J.
Inclusion
of
Mixtures
and
Naturally
Occurring
Non­
Steroidal
Estrogens
and
Recommendation
for
a
Nominations
Process.........................................................................
14
K.
Introduction
of
the
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD)
...................
14
L.
Overview
of
the
Recommended
Approach
to
Priority
Setting
...........................................
15
III.
Exposure­
Related
Information
and
Criteria
...................................................................
15
A.
Biological
Sampling
Data.................................................................................................
16
B.
Environmental,
Occupational,
Consumer
Product,
and
Food­
Related
Data
.......................
18
C.
Environmental
Releases....................................................................................................
19
D.
Production
Volume
Data
.................................................................................................
20
E.
Fate
and
Transport
Data
and
Models................................................................................
21
IV.
Effects­
Related
Information
and
Criteria........................................................................
23
A.
Toxicological
Laboratory
Studies
and
Databases..............................................................
25
B.
Epidemiological
and
Field
Studies
and
Databases
.............................................................
26
C.
Predictive
Biological
Activity
or
Effects
Models...............................................................
27
V.
High
Throughput
Pre­
Screening
.......................................................................................
28
A.
Introduction.....................................................................................................................
28
B.
Purpose
of
HTPS
.............................................................................................................
29
C.
Which
Assays
Will
be
Conducted
in
HTPS?
.....................................................................
29
D.
Limitations
of
the
Assays
to
be
Conducted
During
HTPS.................................................
31
E.
Technical
and
Logistical
Issues.........................................................................................
31
F.
Which
Chemicals
Should
be
Subjected
to
HTPS?..............................................................
32
G.
How
Will
HTPS
Results
be
Used?
...................................................................................
33
H.
Practical
Considerations
and
Constraints
to
Be
Considered
in
HTPS
Implementation
.......
35
VI.
Recommendations
for
Handling
Polymers.......................................................................
36
A.
Introduction.....................................................................................................................
36
B.
Key
Issues
Associated
With
the
Prioritization
of
Polymers
...............................................
39
EDSTAC
Final
Report
Chapter
Four
August
1998
C.
Options
Considered
by
the
PSWG....................................................................................
42
D.
Recommendation
for
Handling
Polymers..........................................................................
44
VII.
Recommendations
for
Handling
Mixtures
.....................................................................
45
A.
Introduction.....................................................................................................................
45
B.
Definition.........................................................................................................................
45
C.
Categorization
Scheme
for
Mixtures
................................................................................
45
D.
Determining
the
Composition
of
Mixtures
to
be
Considered.............................................
47
E.
Criteria
for
Prioritizing
Mixtures
......................................................................................
47
F.
Recommendations
............................................................................................................
48
VIII.
Recommendation
to
Screen
Naturally
Occurring
Non­
Steroidal
Estrogens
...............
51
A.
Background
.....................................................................................................................
51
B.
Recommendation
.............................................................................................................
52
IX.
Recommendation
for
a
Nominations
Process
..................................................................
53
A.
Introduction.....................................................................................................................
53
B.
Description
of
the
Nominations
Process
...........................................................................
54
C.
Criteria
for
Evaluating
Nominated
Chemicals
...................................................................
54
D.
Submission
of
Nominations..............................................................................................
55
E.
Mixtures
in
the
Context
of
the
Nominations
Process.........................................................
56
F.
Ability
to
Track
Nominations............................................................................................
56
X.
The
Endocrine
Disruptor
Priority
Setting
Database
........................................................
56
A.
Introduction.....................................................................................................................
56
B.
Recommendation
and
Principles
to
Guide
the
Continued
Development,
Utilization,
and
Maintenance
of
the
Prototype
EDPSD..................................................................................
57
C.
Description
of
the
Prototype
EDPSD
...............................................................................
57
D.
Preliminary
Recommendation
for
Data
Fields
to
be
Included
in
the
EDPSD
.....................
59
E.
Special
Handling
of
Effects
Data
in
the
Context
of
the
EDPSD
........................................
59
F.
Continued
Development
of
the
EDPSD
............................................................................
63
G.
Use
by
Multi­
Stakeholder
Group
.....................................................................................
64
H.
Maintenance
....................................................................................................................
65
XI.
Recommended
Approach
to
Priority
Setting...................................................................
65
A.
Introduction.....................................................................................................................
65
B.
Obstacles
to
an
Ideal
Priority
Setting
System....................................................................
65
C.
Principles
for
Setting
Priorities
.........................................................................................
66
D.
Recommended
Strategy
for
Setting
Priorities
for
Tier
1
Screening
...................................
67
E.
Examples
of
Compartments
for
the
Recommended
Priority
Setting
Strategy.....................
68
F.
Numbers
of
Chemicals
Prioritized
and
Associated
Weightings
of
Compartments...............
72
G.
Next
Steps
(
Reaching
Closure)
on
Phase
I
Priorities
for
Screening
and
Testing
................
74
H.
Recommended
Approach
to
Setting
Priorities
for
Tier
2
Testing
During
Phase
I
of
the
EDSTP.................................................................................................................................
74
XII.
Compilation
of
Chapter
Four
Recommendations
..........................................................
78
A.
Summary
and
Scope
of
Effort
..........................................................................................
78
EDSTAC
Final
Report
Chapter
Four
August
1998
B.
The
Universe
of
Chemicals
and
Initial
Sorting
..................................................................
78
C.
Polymers..........................................................................................................................
79
D.
Priority
Setting
Information
Categories
and
Criteria
.........................................................
80
E.
High
Throughput
Pre­
Screening
.......................................................................................
80
F.
Mixtures...........................................................................................................................
81
G.
Naturally
Occurring
Non­
Steroidal
Estrogens
(
NONEs)...................................................
82
H.
Nominations
....................................................................................................................
83
I.
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD)...................................................
84
J.
Recommended
Approach
to
Priority
Setting......................................................................
85
XIII.
Literature
Cited
.............................................................................................................
87
Figures
Figure
4.1
EDSTAC
Conceptual
Framework
........................................................................
7
Figure
4.2
Example
of
Data
Fields
Arranged
Into
a
Form
as
They
Might
Appear
on
a
Computer
Screen
...............................................................................................
60
Tables
Table
4.1
Existing
Files
in
the
Endocrine
Disruptor
Priority
Setting
Database
....................
61
Table
4.2
Examples
of
File
Types
That
Could
be
Placed
in
the
Endocrine
Disruptor
Priority
Setting
Database....................................................................................
62
Appendices
Appendix
F:
References
and
Sources
for
Chapter
Four
 
Priority
Setting
Appendix
G:
Exposure
&
Effects
Data
Source
Matrices
Appendix
H:
Fate
and
Transport
Tables
Appendix
I:
Feasibility
Demonstration
Project
for
HTPS
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
1
I.
Introduction
A.
Charge
to
the
PSWG
This
chapter
of
the
EDSTAC
report
addresses
the
need
to
set
priorities
for
endocrine
disruptor
screening
and
testing.
It
was
developed
by
the
Priority
Setting
Work
Group
(
PSWG)
and
was
reviewed,
refined,
and
endorsed
by
the
EDSTAC.
The
PSWG
consisted
of
nineteen
individuals
representing
a
wide
diversity
of
perspectives
and
backgrounds
including
various
sectors
of
industry;
a
variety
of
state
and
federal
government
agencies;
national
environmental,
worker,
and
public
health­
oriented
organizations;
and
local
citizen
and
environmental
justice
groups.
A
complete
list
of
work
group
members
is
included
in
Appendix
C.
References
and
sources
for
this
chapter
can
be
found
in
the
Literature
Cited
section
at
the
end
of
the
chapter
and
in
Appendix
F.

The
charge
given
to
the
PSWG
was
to:

·
specify
types
of
information
that
should
be
gathered
and
analyzed
to
sort
and
prioritize
chemical
substances
and
mixtures
for
screening
and
testing;
·
develop
criteria
for
evaluating
the
quality,
adequacy,
and
reliability
of
the
information
that
will
be
used
in
sorting
and
prioritizing
chemical
substances
and
mixtures
for
screening
and
testing;
·
develop
criteria
for
sorting
chemical
substances
and
mixtures
into
four
possible
next
steps,
including:
(
1)
hold
screening
and
testing;
(
2)
prioritize
for
Tier
1
Screening
(
T1S);
(
3)
go
to
Tier
2
Testing
(
T2T);
or
(
4)
go
to
hazard
assessment;
·
develop
criteria
for
setting
priorities
for
T1S.
These
criteria
will
address
the
relative
order
of
priority
in
which
chemical
substances
that
are
sorted
into
this
category
will
actually
proceed
to
T1S;
and
·
suggest
how
information
used
for
priority
setting
should
be
combined
with
screening
and
testing
results
to
generate
a
"
weight­
of­
evidence"
determination
for
proceeding
from
screening
to
testing
or
from
testing
to
hazard
assessment.

B.
The
Need
for
Priority
Setting
Priority
setting
for
endocrine
disruptor
screening
and
testing
is
not
a
trivial
exercise.
Industrial
chemicals,
pesticides,
commercial
products,
and
environmental
contaminants
have
been
subjected
to
various
screening
and
testing
regimes
for
decades
(
Swanson
and
Socha,
1997).
However,
the
existing
regulatory
screening
and
testing
schemes
do
not
specifically
address
endocrine
disrupting
mechanisms.
The
chemicals
in
commerce
and
the
environment
exhibit
a
range
of
physical
and/
or
chemical
and
toxicological
properties,
as
well
as
varied
production
and
use
patterns.
Only
some
chemicals
are
likely
to
cause
endocrine
disruption,
and
only
some
of
these
chemicals
will
be
produced
or
used
in
such
a
fashion
that
humans
or
other
living
organisms
will
be
exposed
to
them.
Because
screening
and
testing
can
be
such
a
resource­
intensive
process
for
both
the
public
and
private
sectors,
priorities
must
be
set
carefully
to
ensure
that
the
chemicals
of
greatest
concern
are
given
priority
over
chemicals
of
little
or
no
concern.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
2
The
challenge
is
daunting.
Building
on
the
information
contained
in
Chapter
Two,
the
EDSTAC
recommends
the
universe
of
chemicals
to
at
least
be
considered
for
endocrine
disruptor
screening
and
testing
should
include:

·
all
of
the
approximately
75,500
chemicals
currently
listed
on
the
TSCA
Inventory
(
James
Darr,
U.
S.
EPA,
personal
communication);
·
all
of
the
approximately
900
active
ingredients
(
approximately
500
of
which
are
food­
use
pesticides
which
will
be
prioritized
for
screening
and
testing
according
to
the
schedule
and
requirements
of
the
FQPA
(
see
Chapter
Four,
Section
XI,
H)
and
approximately
2,500
inert
ingredients
that
are
used
to
formulate
over
20,000
pesticide
products
(
Penny
Fenner­
Crisp,
U.
S.
EPA,
personal
communication);
·
approximately
8,000
chemicals
regulated
by
the
Food
and
Drug
Administration
(
FDA)
including
5,000
ingredients
in
cosmetics
and
3,000
food
additives
(
Bern
Schwetz,
FDA,
personal
communication);
·
naturally
occurring
non­
steroidal
estrogens
(
NONEs)
and
other
naturally
occurring
or
environmentally
degraded
chemicals;
and
·
nutritional
supplements,
for
which
a
number
cannot
be
estimated
because
these
chemicals
are
not
currently
regulated
by
the
FDA
or
any
other
agency.

Thus,
the
number
of
individual
chemical
substances
that
should
be
considered
for
endocrine
disruptor
screening
and
testing
exceeds
87,000.
Later
in
this
chapter
the
EDSTAC
presents
recommendations
for
screening
and
testing
"
commonly
found
mixtures"
as
per
the
scope
of
the
recommended
program
set
forth
in
Chapter
Three.
The
EDSTAC
recognizes
that
the
evaluation
of
some
of
the
individual
chemical
substances,
if
they
are
determined
to
be
a
priority
for
screening
and
testing,
will
require
a
cooperative
effort
among
the
responsible
agencies.

In
responding
to
the
challenge,
the
PSWG
grappled
with
a
number
of
practical
considerations:

·
What
scientific
criteria
should
be
used
in
establishing
priorities?
·
What
information
is
available
with
respect
to
these
criteria
and
how
readily
can
the
information
be
analyzed?
·
What
are
the
major
gaps
in
information
needed
for
setting
priorities
and
how
can
these
gaps
be
filled?
·
How
should
the
priority
setting
system
be
designed
to
maximize
"
transparency"
(
i.
e.,
public
understanding
of
the
rationale
underlying
the
established
priorities)?
·
Should
priorities
be
governed
by
existing
statutory
authorities?
·
How
might
priorities
be
set,
without
regard
to
EPA's
statutory
authority,
to
encourage
voluntary
private
sector
testing
and
to
ensure
compounds
of
concern
are
addressed?

The
EDSTAC's
efforts
to
develop
a
coherent,
scientifically
sound
framework
for
setting
screening
and
testing
priorities
have
required
EDSTAC
members
to
carefully
review
the
way
in
which
EPA
gathers
information
about
new
and
existing
chemicals.
The
Committee
examined
the
authority
provided
to
EPA
by
Congress
which
guide
the
Agency's
data­
gathering
efforts,
and
reviewed
the
Agency's
management
of
the
data
available
to
it.
The
Committee
also
reached
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
3
beyond
EPA
in
its
quest
for
pertinent
information
sources
to
guide
priority
setting.
Data
on
chemical
hazards
in
the
environment
are
also
gathered
by
the
Occupational
Safety
and
Health
Administration,
the
U.
S.
Department
of
Agriculture,
the
Food
and
Drug
Administration,
the
U.
S.
Department
of
the
Interior,
and
other
federal
and
state
agencies,
as
well
as
parties
in
the
private
and
academic
sectors.

Despite
the
multitude
of
data­
gathering
authorities
and
databases
on
chemicals,
information
on
exposure
to,
and
the
health
and
environmental
effects
of,
most
chemicals
is
incomplete,
and
inadequate
with
respect
to
endocrine
disrupting
effects.
For
example,
much
more
information
is
available
on
the
effects
of
pesticides
regulated
under
FIFRA
than
is
available
on
the
effects
of
industrial
chemicals
addressed
under
TSCA.
The
EDSTAC's
priority
setting
scheme
attempts
to
address
these
information
disparities.

The
priority
scheme
recommended
in
this
chapter
reflects
an
integrated,
scientifically
driven
concern
for
chemical
exposures
and
effects
that
transcends
the
barriers
that
exist
under
current
federal
law.
The
priority
setting
scheme
described
in
this
chapter
is
noteworthy
in
several
respects,
including:

·
First,
even
though
the
immediate
impetus
for
endocrine
screening
and
testing
lies
in
provisions
contained
in
the
FQPA
and
the
amendments
to
the
SDWA,
as
described
further
below,
the
EDSTAC
has
not
limited
its
priority
setting
scheme
to
chemicals
addressed
only
under
the
endocrine
disruptor
screening
and
testing
provisions
contained
in
those
two
statutes.
·
Second,
as
described
in
the
Conceptual
Framework
contained
in
Chapter
Three,
the
EDSTAC
has
not
limited
its
attention
solely
to
the
estrogen
mimics
that
are
explicitly
mentioned
in
the
FQPA
and
the
SDWA,
but
is
recommending
that
the
initial
screening
and
testing
program
also
include
androgen­
and
thyroid­
related
hormones.
The
Committee
also
recommends
periodic
review
of
the
initial
program
to
evaluate
whether
the
inclusion
of
additional
hormonal
systems
is
warranted
in
the
future.
·
Third,
even
though
the
FQPA
and
the
SDWA
focus
on
human
health,
the
EDSTAC
decided
early
in
its
deliberations
that
the
Endocrine
Disruptor
Screening
and
Testing
Program
(
EDSTP)
should
address
environmental
impacts
as
well.
·
Fourth,
the
EDSTAC
recommends
that
the
EDSTP
should
address
chemical
mixtures
in
addition
to
single
chemicals.
·
Fifth,
the
priority
setting
scheme,
by
promoting
the
use
of
robotic
screening
technologies
(
referred
to
as
High
Throughput
Pre­
Screening,
or
HTPS),
is
designed
both
to
generate
new
information
about
chemicals
and
to
help
validate
chemical
modeling
techniques
that
are
used
to
judge
hazards
in
the
absence
of
empirical
data.
·
Sixth,
the
Committee
deliberately
included
so­
called
NONEs
(
e.
g.,
phytoestrogens,
mycotoxins)
 
substances
that
naturally
occur
in
the
environment
 
in
its
priority
setting
scheme.

It
is
important
to
note
that
the
following
discussion
of
the
EDSTAC's
recommended
priority
setting
scheme
does
not
reflect
any
interpretation
by
the
EDSTAC
of
EPA's
authority
to
implement
these
recommendations.
The
EDSTAC's
priority
setting
scheme
is
driven
by
an
overarching
concern
with
exposures
to
and
effects
from
chemicals.
The
Committee
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
4
acknowledges
that
EPA's
screening
and
testing
actions
will
be
both
heavily
driven
and
constrained
by
its
statutory
authority.

II.
Overview
of
the
Sorting
and
Priority
Setting
Recommendations
A.
Initial
Sorting
Step
As
described
in
Chapter
Three
and
graphically
depicted
in
Figure
4.1,
the
EDSTAC
Conceptual
Framework
consists
of
three
major
components:
(
1)
the
sorting
and
priority
setting
component;
(
2)
the
T1S
component;
and
(
3)
the
T2T
component.
Within
the
sorting
and
priority
setting
component,
the
EDSTAC
has
made
a
distinction
between
the
tasks
of
"
sorting"
and
of
"
priority
setting."

The
term
"
sorting"
is
used
to
refer
to
the
initial
effort
to
sort
the
universe
of
chemicals
that
will
be
considered
for
endocrine
disruptor
screening
and
testing
into
four
distinct
categories.
Coming
out
of
the
"
initial
sorting"
box,
the
four
possibilities
include:

1.
polymers
which
will
be
placed
into
a
"
hold"
status
(
with
some
exceptions)
pending
a
review
of
their
monomers,
oligomers,
and
other
components;
2.
chemicals
for
which
insufficient
data
exist
to
proceed
to
either
T2T
or
hazard
assessment
and
will,
therefore,
need
to
be
prioritized
for
T1S;
3.
chemicals
for
which
sufficient
data
exist
to
go
to
T2T;
and
4.
chemicals
for
which
sufficient
data
exist
to
go
to
hazard
assessment.

The
term
"
priority
setting"
refers
primarily
to
the
need
to
set
priorities
for
the
chemicals
that
fall
into
the
second
category
after
the
initial
sorting
stage
 
namely,
those
chemicals
for
which
insufficient
data
exist
to
proceed
to
either
T2T
or
hazard
assessment
and
will,
therefore,
need
to
be
prioritized
for
T1S.

The
remainder
of
this
section
provides
an
explanation
of
the
phased
approach
to
screening
and
testing,
a
brief
overview
of
each
of
the
four
categories
of
chemicals
that
flow
from
the
initial
sorting
step
(
referred
to
above),
and
some
of
the
other
key
features
of
the
priority
setting
system
recommended
by
the
EDSTAC.
The
rest
of
the
chapter
builds
upon
this
overview
section.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
5
B.
Phased
Approach
The
EDSTAC
agreed
that
the
EDSTP
should
be
implemented
in
a
phased
manner.
In
general,
this
means
the
chemicals
determined
to
be
a
high
priority
should
be
screened
and,
if
necessary,
tested
prior
to
those
determined
to
be
a
lower
priority.
Two
of
the
reasons
for
this
general
agreement
on
the
use
of
a
phased
approach
to
implementation
are:
(
1)
to
ensure
that
the
program
does
not
get
bogged
down
by
taking
on
too
much
too
fast,
both
in
terms
of
laboratory
capacity
and
the
administrative
challenges
with
implementing
a
program
of
this
magnitude;
and
(
2)
to
ensure
that
periodic
programmatic­
level
evaluations
occur
that
includes
the
incorporation
of
new
scientific
findings,
new
screens
and/
or
tests,
etc.
However,
the
EDSTAC
did
not
have
a
sufficient
amount
of
information
nor
the
time
to
develop
more
refined
recommendations
about
precisely
how
many
phases
there
should
be,
how
long
each
phase
should
be,
or
the
number
of
chemicals
that
should
be
screened
and/
or
tested
in
each
phase.
The
EDSTAC
understands
that
some
of
these
issues
will
be
addressed
by
EPA
when
it
issues
its
detailed
implementation
plan
after
the
conclusion
of
the
EDSTAC.

Given
the
elements
of
the
screening
and
testing
program
upon
which
EDSTAC
was
able
to
agree,
it
is
clear
there
are
a
number
of
activities
which
will
need
to
occur
immediately
following
the
conclusion
of
the
EDSTAC
process
and
prior
to
the
actual
screening
and
testing
of
compounds.
These
activities
include
the
validation
and
standardization
of
the
recommended
T1S
assays
and
Tier
2
tests,
the
completion
of
the
HTPS
assays
(
assuming
they
are
shown
to
be
technically
feasible
and
are
validated),
and
the
completion
of
the
T1S
priority
setting
process.
Therefore,
if
"
Phase
I"
of
the
program
is
defined
as
the
start
of
the
actual
screening
and
testing
of
chemicals,
its
start
date
is
dependent
upon
the
completion
of
these
preliminary
activities.
EPA
and
some
industry
representatives
have
indicated
that
they
may
wish
to
make
use
of
a
screening
assay
or
test
as
soon
as
it
is
validated,
rather
than
waiting
for
all
screens
and
tests
to
be
validated.
Thus,
the
start
of
Phase
I
may
be
staggered
depending
upon
the
results
and
timing
of
the
validation
process.

During
"
Phase
I"
of
the
program
(
as
defined
in
the
preceding
paragraph)
T1S
will
only
include
those
chemicals
determined
to
be
a
high
priority,
and
T2T
will
only
include
those
chemicals
that
bypass
T1S.
During
the
second
phase
of
the
program,
those
chemicals
that
were
determined
to
be
positive
in
T1S
will
move
into
T2T,
and
a
new
set
of
priority
chemicals
will
then
be
subjected
to
T1S.

Finally,
as
noted
above,
one
of
the
reasons
for
recommending
a
phased
approach
to
implementation
is
to
ensure
that
EPA
conducts
periodic
programmatic­
level
evaluations
of
the
EDSTP.
The
EDSTAC
has
stated
in
several
places
in
this
report
that
the
design
of
the
EDSTP
needs
to
be
flexible
to
account
for
the
newly
emerging
and
rapidly
evolving
scientific
investigation
of
endocrine
disruptors.
Although
the
EDSTAC's
recommendations
regarding
flexibility
are
meant
to
imply
that
new
scientific
findings
and
new
screens
and/
or
tests
should
be
incorporated
into
the
program
as
they
emerge,
the
EDSTAC
believes
it
is
critically
important
to
include
an
explicit
evaluation
step
into
the
program.
The
use
of
a
phased
approach
to
implementation
can
help
to
ensure
that
such
evaluations
occur.
Some
of
the
issues
that
should
be
evaluated
at
the
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
6
conclusion
of
one
phase
and
prior
to
the
start
of
another
relate
to
the
criteria
for
chemicals
coming
back
into
the
program
when
they
are
placed
in
the
"
hold
box."
These
include
an
evaluation
of
whether
new
screens
and
tests
have
been
developed
for
the
EAT
hormonal
systems,
including,
for
example,
in
utero/
developmental
assays
and
whether
new
screens
and
tests
have
been
developed
for
other
hormonal
systems.

C.
Polymers
In
an
effort
to
grapple
with
the
very
large
number
of
chemicals
that
the
PSWG
had
identified
as
candidates
for
endocrine
disruptor
screening
and
testing,
the
group
spent
considerable
time
addressing
the
question
of
which,
if
any,
chemicals
should
be
placed
in
the
"
hold
box"
as
part
of
the
initial
sorting
step.
It
was
thought
that
a
class,
or
classes,
of
chemicals
with
a
very
low
probability
of
being
endocrine
disruptors
for
the
hormonal
systems
addressed
by
the
screening
and
testing
program
could
be
set
aside
so
as
to
avoid
"
clogging
up"
the
system.

The
group
initially
identified
polymers
as
one
type
of
chemical
that
warranted
consideration
for
exclusion
because
of
their
molecular
size.
It
was
initially
thought
that
polymers
would
not
pose
a
threat
to
the
endocrine
systems
of
humans
and
other
biota.
Subsequently,
the
group
learned
there
are
instances
where
polymers
could
be
absorbed,
particularly
in
neonates.

Consequently,
the
EDSTAC
recommends
that:

1.
All
monomer
and
oligomer
components
of
polymers
should
be
prioritized
for
and
subjected
to
endocrine
disruptor
screening
and
testing.

2.
All
"
new"
polymers
(
i.
e.,
those
produced
after
the
Initial
TSCA
Inventory,
which
was
published
in
1979)
with
number
average
molecular
weight
(
NAMW)
less
than
1,000
daltons
should
also
be
prioritized
for
and
subjected
to
endocrine
disruptor
screening
and
testing.
Throughout
this
document,
the
term
"
number
average
molecular
weight,"
or
"
NAMW"
of
polymers
is
utilized.
This
term
indicates
a
numerical
mean,
with
the
actual
MW
of
the
polymers
ranging
about
this
mean.
The
EDSTAC
recommends
embracing
the
language
in
the
1995
Final
TSCA
Polymer
Rule
(
60
FR
16333)
which
uses
a
NAMW
cutoff
of
1,000
daltons,
provided
that
the
polymer
does
not
contain
other
than
certain
specified
reactive
functional
groups
and
that
the
polymer
contains
less
than
10%
oligomers
with
MW
less
than
500
daltons
and
less
than
25%
oligomers
with
a
MW
of
less
than
1,000
daltons.

3.
All
previously
manufactured
polymers
(
regardless
of
NAMW)
and
all
"
new"
polymers
with
a
NAMW
greater
than
1,000
daltons
should
be
set
aside
pending
the
outcome
of
the
screening
and
testing
of
their
monomer
and
oligomer
and
other
components.

4.
If
the
component
is
determined
to
have
endocrine
disrupting
properties,
the
component
should
proceed
to
hazard
assessment.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
7
1
This
flow
chart
represents
a
more
detailed
description
of
the
sorting
and
priority
setting
components,
and
how
they
relate
to
the
Tier
1
Screening
(
T1S),
Tier
2
Testing
(
T2T),
and
Hazard
Assessment
components.
2
See
Chapter
Four,
Section
I.
B.
3
See
Chapter
Four,
Section
VI.
A.
2.
4
See
Chapter
Four,
Section
II.
K.
Essentially,
this
number
results
from
subtracting
25,000
polymers
from
the
total
universe
of
86,000
chemicals.
5
See
Chapter
Four,
Section
VI.
A.
1.
6
See
Chapter
Four,
Section
V.
F.
Polymers
(
Est.
25,000)
3
Hold
Polymers
Pending
Screening
and
Testing
and
Exposure
Assessment
of
Their
Components
Sufficient
Data
to
go
to
Hazard
Assessment
Sufficient
Data
or
Voluntary
Bypass
of
T1S
to
go
to
T2T
Insufficient
Data
to
go
to
T2T
or
Hazard
Assess.
(
Est.
62,000)
4
Initial
Sorting
Total
Universe
of
Chemicals
(
Est.
87,000)
2
High
Throughput
Pre­
Screening
Set
Priorities
for
T1S
Using
Both
Exposure
and
Effects
Information
Tier
1
Screening
(
T1S)

Tier
2
Testing
(
T2T)

Hazard
Assessment
Figure
4.1
Detailed
Depiction
of
EDSTAC
Conceptual
Framework1
Phase
I
Currently
Produced
in
Quantities
>
10,000
lbs/
yr
(
Est.
15,000)
6
Yes
No
Is
It
a
"
New"
Polymer
w/
NAMW
<
1000
Daltons?
5
No
Yes
Phase
II
Phase
III
Hold
No
Further
Analysis
Required
at
This
Time
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
8
5.
As
with
any
chemical
shown
to
have
endocrine
disrupting
properties,
an
exposure
assessment
should
be
performed.
It
is
at
this
stage,
that
all
potential
exposure
routes
for
a
component
would
be
determined,
including
the
potential
for
the
component
to
be
available
from
the
polymer.
Implications
with
respect
to
the
polymer
itself
would
be
dependent
upon
the
results
of
such
an
exposure
assessment.

As
indicated
in
Figure
4.1,
if
this
approach
is
utilized
it
will
place
approximately
25,000
polymers
of
the
approximately
87,000
chemicals
being
considered
for
endocrine
disruptor
screening
and
testing
into
a
"
hold
box"
pending
a
review
of
their
monomers
and
oligomers
(
James
Darr,
U.
S.
EPA,
personal
communication).
The
rationale
for
these
recommendations,
as
well
as
the
recommendations
themselves,
are
elaborated
upon
in
Section
VI
of
this
chapter.

D.
Chemicals
With
Sufficient
Data
to
go
to
T2T
or
Voluntary
Bypass
of
T1S
As
noted
in
Chapter
Three,
there
are
two
scenarios
in
which
the
EDSTAC
recommends
the
owner
of
a
chemical
should
be
permitted
to
voluntarily
bypass
T1S.
Each
of
these
two
scenarios
has
different
implications
for
the
information
requirements
associated
with
completing
T2T.

1.
Chemicals
That
Have
Previously
Been
Subjected
to
Two­
Generation
Reproductive
Toxicity
Tests
The
first
scenario
includes
those
chemicals
that
have
previously
been
subjected
to
mammalian
and
wildlife
developmental
and/
or
reproductive
toxicity
testing,
but
where
such
testing
may
not
have
included
additional
endpoints
for
T2T,
as
specified
in
Chapter
Five,
Section
V,
C.
The
EDSTAC
expects
that
food­
use
pesticides
will
fall
into
this
category,
given
the
requirements
of
FIFRA,
as
will
a
small
number
of
other
types
of
pesticides
and
industrial
chemicals.
The
EDSTAC
agrees
that
chemicals
that
meet
this
criterion
for
bypassing
T1S
would
still
be
subjected
to
the
assays
that
will
be
part
of
the
HTPS,
for
the
reasons
outlined
in
Chapter
Four,
Section
V,
G,
2.

In
addition,
chemicals
that
meet
this
criterion
will
also
be
the
most
likely
candidates
for
the
alternative
approaches
for
completing
T2T,
as
discussed
in
Chapter
Five,
Section
VII,
C.
As
described
in
more
detail
in
Chapter
Four,
Section
XI,
H,
the
recommended
approach
for
setting
priorities
for
T2T
of
food­
use
pesticides
is
basically
to
follow
the
schedule
for
pesticide
reregistration
and
tolerance
reassessments
for
these
chemicals,
as
per
the
schedule
and
requirements
of
the
FQPA.
Also
in
Section
XI,
H,
the
EDSTAC
discusses
the
need
for
special
treatment
of
those
pesticides
that
are
likely
to
complete
their
tolerance
reassessments
prior
to
the
completion
of
the
validation
and
standardization
of
recommended
Tier
2
tests.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
9
2.
Chemicals
for
Which
There
is
no
Prior
Toxicity
Testing
The
second
scenario
includes
those
chemicals
for
which
the
owner
of
the
chemical
has
decided
to
voluntarily
complete
T2T
without
having
completed
the
full
T1S
battery
or
any
prior
twogeneration
reproductive
toxicity
testing.
The
EDSTAC
recommends
that
these
chemicals
must
also
be
evaluated
in
the
HTPS
assays.
In
addition,
chemicals
that
bypass
T1S
under
this
second
scenario
must
be
evaluated
in
all
the
tests
of
the
T2T
battery
(
i.
e.,
the
mammalian
and
nonmammalian
multi­
generation
tests
with
all
the
recommended
endpoints),
consistent
with
the
principles
governing
T2T,
which
are
set
forth
in
Chapter
Five,
Section
V,
C.
Finally,
as
discussed
in
Section
XI,
H
of
this
chapter,
these
chemicals
should
retain
their
T1S
priority
ranking
for
T2T
even
though
they
will
voluntarily
bypass
the
screening
tier.
In
other
words,
if
these
chemicals
are
deemed
to
be
a
high
priority
for
T1S
and
the
owner
of
the
chemical
decides
to
voluntarily
bypass
T1S,
they
should
also
be
a
high
priority
for
T2T.

E.
Chemicals
With
Sufficient
Data
to
Proceed
to
Hazard
Assessment
The
EDSTAC
recommends
that
chemicals
for
which
there
are
sufficient
data
to
conduct
a
hazard
assessment
should
be
permitted
to
bypass
both
T1S
and
T2T
and
proceed
directly
to
the
hazard
assessment
step
of
the
process.
This
option
should
be
available
for
chemicals
that
have
sufficient
data
to
make
either
a
definitive
positive
or
negative
determination
that
the
chemical
either
does
or
does
not
have
endocrine
disrupting
properties
for
the
estrogen,
androgen,
and
thyroid
hormonal
systems
addressed
by
the
program.

This
step
in
the
process
will
require
a
case­
specific
review
and
determination
that
the
same
type
and
quality
of
information
exist
for
the
chemical
as
would
be
necessary
to
move
from
T2T
to
hazard
assessment.
The
owner
of
such
a
chemical
(
i.
e.,
the
company
or
companies
that
produced
the
chemical)
or
EPA
in
the
case
of
an
"
orphan"
chemical
(
i.
e.,
one
that
has
no
owner)
would
need
to
show
that
the
screens
and
tests
conducted
yielded
data
that
are
the
"
functional
equivalent"
of
data
that
would
have
been
produced
from
T1S
and
T2T.
Such
functional
equivalency
will
certainly
include
sufficient
dose­
response
relationship
clarification
before
proceeding
to
the
hazard
assessment
phase.

The
EDSTAC
believes
that
only
a
small
number
of
chemicals
will
meet
this
criterion;
however,
it
did
not
attempt
to
identify
these
chemicals.
Rather,
the
Committee
has
appropriately
deferred
this
determination
to
EPA
as
part
of
the
implementation
of
the
EDSTP.
As
noted
above,
such
a
determination
will
need
to
be
made
on
a
case­
specific
basis.
When
EPA
formally
proposes
its
approach
to
implementing
the
EDSTP,
the
Agency
should
publish
more
detailed
decision­
making
criteria,
data
and
reporting
requirements,
and
procedures
that
should
be
followed
to
provide
the
degree
of
clarity
necessary
to
implement
this
recommendation.

F.
Chemicals
with
Insufficient
Data
to
go
to
T2T
or
Hazard
Assessment
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
10
A
very
large
number
of
chemicals
will
remain
after
the
initial
sorting
step
has
separated
out
polymers
(
with
some
exceptions),
food­
use
pesticides,
and
any
chemicals
that
have
functionally
equivalent
data
to
bypass
T1S,
and
the
small
number
of
chemicals
that
will
be
ready
for
the
hazard
assessment
step.
The
EDSTAC
estimates
the
number
of
chemicals
that
will
fall
into
this
category
to
be
approximately
62,000.
The
EDSTAC
has
developed
a
set
of
recommendations
to
guide
the
T1S
priority
setting
process
for
these
chemicals.

As
discussed
in
more
detail
below,
the
EDSTAC
recommends
an
approach
to
setting
priorities
for
T1S
without
specifying
the
precise
number
of
chemicals
that
should
ultimately
be
subjected
to
T1S.
Building
on
the
discussion
of
the
phased
approach
to
implementing
the
EDSTP,
it
should,
however,
be
noted
that
even
though
approximately
62,000
chemicals
will
remain
after
sorting
out
polymers
and
the
relatively
small
number
of
chemicals
that
will
meet
the
criteria
for
bypassing
T1S
and/
or
T2T,
the
EDSTAC
does
not
expect
that
62,000
chemicals
will
be
subjected
to
T1S.
(
See
Chapter
Four,
Section
XI,
F)

G.
Priority
Setting
Information
Categories
and
Criteria
When
the
PSWG
began
its
deliberations,
the
group
sought
to
address
the
following
questions:

·
What
information
is
relevant
to
the
task
of
priority
setting?
·
Is
this
information
readily
available?
·
If
so,
how
easily
can
the
information
be
accessed?
·
What
is
the
quality,
variability,
and
reliability
of
this
information?
·
Can
the
information
be
used
as
the
basis
for
criteria
to
determine
priorities
for
endocrine
disruptor
screening
and
testing?

In
grappling
with
these
questions,
the
PSWG
established
three
main
categories
for
organizing
information
and
criteria
related
to
priority
setting:
exposure­
related,
effects­
related,
and
statutory
criteria.
The
exposure
and
effects
categories
and
information
are
consistent
with
those
in
Swanson
and
Socha,
1997.
Under
each
of
these
main
headings,
the
group
identified
a
number
of
subheadings:

1.
Exposure­
Related
Information
and
Criteria
a)
Biological
sampling
data
i.
Human
ii.
Other
biota
b)
Environmental,
occupational,
consumer
product,
and
food­
related
data
i.
Air
ii.
Water
(
including
surface
water,
groundwater,
and
drinking
water)
iii.
Soil/
Sediments
iv.
Consumer
products
v.
Food
c)
Environmental
releases
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
11
d)
Production
volume
e)
Fate
and
transport
data
and
models
2.
Effects­
Related
Information
and
Criteria
a)
Toxicological
laboratory
studies
and
databases
b)
Epidemiological
and
field
studies
and
databases
c)
Predictive
biological
activity
or
effects
models
(
e.
g.,
SAR,
QSAR)
d)
Results
of
high
throughput
pre­
screening
3.
Statutory
Criteria
a)
Pesticides,
as
per
FQPA
b)
Chemicals
found
in
sources
of
drinking
water
affecting
significant
populations,
as
per
SDWA
c)
Chemicals
that
may
have
a
cumulative
effect
with
pesticides,
as
per
FQPA
For
the
exposure
and
effects
criteria,
the
PSWG
identified
a
significant
number
of
data
sources,
evaluated
the
quality
and
strengths
and
limitations
of
these
data
sources,
and
determined
how
to
best
utilize
these
data
sources
to
accomplish
the
task
of
priority
setting.
The
results
of
this
effort
are
set
forth
in
Section
III
for
the
exposure­
related
criteria,
and
in
Section
IV
for
the
effectsrelated
criteria.
Appendix
G
includes
a
series
of
detailed
matrices
containing
a
list
and
preliminary
evaluation
of
data
sources
organized
under
the
exposure
and
effects
subheadings.

H.
Role
of
the
Statutory
Criteria
The
PSWG
of
the
EDSTAC
discussed
the
proper
role
of
the
statutory
criteria
listed
above
in
relation
to
the
other
criteria.
The
EDSTAC
understands
that
the
screening
and
testing
requirement
for
pesticides
(
both
active
and
"
inert"
ingredients)
contained
in
the
FQPA
is
mandatory.
However,
the
EDSTAC
also
understands
the
screening
and
testing
of
chemicals
found
in
sources
of
drinking
water
affecting
significant
populations
under
the
SDWA
and
chemicals
that
may
have
a
cumulative
effect
with
pesticides
under
the
FQPA
to
be
discretionary.

While
recognizing
the
importance
of
the
statutory
criteria
in
relation
to
EPA's
implementation
authorities,
the
Committee
has
developed
its
priority
setting
recommendations
based
on
public
health
and
environmental
concerns
rather
than
on
existing
regulatory
requirements.
Thus,
the
Committee
recommends
that
the
statutory
criteria
should
not
be
used
as
a
sole
basis
for
establishing
priorities
for
endocrine
disruptor
screening
and
testing.
The
Committee
recognizes
that
this
recommendation
might
result
in
a
chemical
substance
or
mixture
being
identified
as
a
high
priority
for
endocrine
disruptor
screening
and
testing
for
which
EPA
does
not
have
authority
to
require
such
screening
and
testing
under
FQPA.
Nevertheless,
the
Committee
believes
it
is
important
to
have
priorities
driven
by
scientific
considerations
and
explicit
value
judgments,
rather
than
by
existing
regulatory
requirements.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
12
The
Committee
is
hopeful
that
when
a
chemical
is
identified
as
a
high
priority
for
T1S
that
falls
outside
the
scope
of
the
FQPA
and
the
SDWA,
the
owner
of
the
chemical
would
voluntarily
conduct
T1S
and,
if
necessary,
T2T.
The
Committee
acknowledges,
however,
that
reliance
on
authority
other
than
FQPA
may
affect
the
timing
of
actually
conducting
T1S,
notwithstanding
the
priority
ranking
of
the
chemical.

I.
High
Throughput
Pre­
Screening
(
HTPS)
Step
One
problem
the
PSWG
identified
early
on
in
its
deliberations
is
the
lack
of
endocrine
disruptor
effects­
related
data
on
the
vast
majority
of
chemicals
and
their
breakdown
products.
The
PSWG
considered
recommending
the
use
of
published
and
available
Quantitative
Structure
Activity
Relationship
(
QSAR)
models
to
obtain
predictions
for
the
endocrine
disrupting
potentials
of
untested
compounds.
Although
promising,
available
QSAR
models
are
generally
thought
to
be
insufficiently
validated
for
the
diversity
of
chemicals
that
will
be
included
in
endocrine
disruptor
screening
and
testing
(
Ankley
et
al.,
1997).
Therefore,
it
was
the
PSWG's
determination
that
QSARs
were
currently
incapable
of
providing
accurate
predictions
for
this
highly
diverse
universe
of
chemicals.
To
rectify
this
problem,
the
work
group
recommended,
and
the
plenary
endorsed
(
subject
to
a
demonstration
of
feasibility),
incorporating
into
the
EDSTAC
Conceptual
Framework
the
use
of
"
high
throughput
pre­
screening,"
or
the
use
of
automated
processes
(
robotic
and
specialized
instrumentation)
to
aid
in
the
screening
of
compounds
(
discussed
in
more
detail
in
Chapter
Four,
Section
V).
The
feasibility
demonstration
effort
for
HTPS
is
described
more
fully
both
in
Section
V
of
this
chapter
and
in
Appendix
I.

The
primary
purpose
of
HTPS
would
be
to
address
the
fact
that
there
is
very
little,
if
any,
biological
effects
information
for
humans,
and
even
less
for
other
species,
on
the
vast
majority
of
chemicals
to
be
considered
for
endocrine
disruption
screening
and
testing.
The
assays
that
will
be
conducted
during
the
HTPS
step
of
the
process
are
transcriptional
activation
assays
for
the
three
hormonal
systems
(
estrogen,
androgen,
and
thyroid
hormone­
related).
Two
of
the
HTPS
assays
(
ER
and
AR
binding/
transcriptional
activation)
are
part
of
the
T1S
battery.
Any
chemicals
subjected
to
the
assays
conducted
in
the
HTPS
step
would
not
be
required
to
repeat
the
ER
and
AR
binding/
transcriptional
activation
assays
as
part
of
T1S.
On
the
other
hand,
any
chemicals
which
are
subjected
to
T1S
but
not
to
HTPS
(
e.
g.,
production
volumes
less
than
10,000
pounds
per
year)
would
go
through
the
in
vitro
assays
on
the
bench
as
part
of
T1S,
thereby
resulting
in
information
equivalent
to
that
which
would
have
been
provided
from
HTPS.

However,
the
assays
in
the
HTPS
step
will
be
far
from
comprehensive
or
definitive.
The
HTPS
assays
will
certainly
provide
valuable
information
on
the
potential
of
a
chemical
to
bind
to
the
relevant
receptor
in
cell
culture
and
result
in
transcriptional
activation,
which
is
information
that
is
missing
for
a
large
number
of
chemicals.
However,
the
results
of
HTPS
will
not
be
sufficient
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
13
by
themselves
to
support
the
conclusion
that
a
chemical
is
or
is
not
an
endocrine­
mediated
toxicant.

Data
resulting
from
HTPS
will
be
combined
with
exposure­
related
information,
and
with
any
other
effects­
related
information
that
is
available,
for
each
chemical
for
the
purpose
of
setting
priorities
for
T1S.
In
other
words,
HTPS
data
will
not
be
used
in
isolation
of
other
relevant
data,
nor
will
it
become
the
de
facto
determinant
of
priorities
for
T1S.

Although
the
use
of
robotic
technology
will
greatly
expand
the
"
throughput"
of
chemicals
over
a
given
period
of
time
for
the
selected
assays,
the
EDSTAC
does
not
recommend
that
all
chemicals
be
subjected
to
HTPS.
Rather,
the
EDSTAC
recommends
that
the
estimated
15,000
chemicals
currently
produced
in
an
amount
equal
to
or
greater
than
10,000
pounds
per
year
and
all
pesticides
be
subjected
to
HTPS.
The
EDSTAC
makes
this
recommendation
to
help
EPA
avoid
a
task
that
might
never
be
completed
if
a
higher
number
of
chemicals
were
to
be
recommended
for
HTPS.
Also,
the
EDSTAC
believes
that
15,000
chemicals
is
not
an
insignificant
number
of
chemicals,
especially
given
the
history
of
TSCA.

The
EDSTAC
further
recommends
that
chemicals
permitted
to
bypass
T1S
and
go
directly
to
T2T,
as
well
as
those
permitted
to
bypass
both
T1S
and
T2T
and
go
directly
to
hazard
assessment
due
to
functional
equivalency
of
data,
also
be
subjected
to
HTPS.
There
are
several
generic
reasons
why
the
EDSTAC
recommends
conducting
HTPS
assays
on
these
chemicals
which
include:
(
1)
the
data
generated
from
the
HTPS
assays
will
be
valuable
in
and
of
themselves,
even
though
they
are
limited
to
receptor­
binding
mechanisms
and
cannot
be
used
by
themselves
to
determine
whether
a
chemical
is
or
is
not
an
endocrine
disruptor;
(
2)
as
an
ancillary
benefit,
the
data
can
be
used
to
improve
and
validate
QSARs;
and
(
3)
beyond
these
generic
benefits,
in
the
case
of
food­
use
pesticides
that
will
complete
tolerance
reassessments
prior
to
the
availability
of
validated
Tier
2
tests,
HTPS
data
can
be
used
along
with
other
relevant
information
to
help
prioritize
whether
and,
if
so,
when
these
chemicals
should
be
subjected
to
any
additional
endocrine
disruptor
testing.
The
rationale
for
recommending
that
food­
use
pesticides
complete
HTPS
assays
is
further
elaborated
upon
in
Chapter
Four,
Section
XI,
H.

The
EDSTAC
recommends
that
existing
QSAR
models
be
rederived
and
supplemented
with
data
from
the
HTPS
assays,
thereby
expanding
the
predictive
ability
of
these
models.
Existing
QSARs
are
derived
using
data
from
cell­
free
receptor
binding
and
cellular
proliferation
assays.
These
assays
are
part
of
the
T1S
battery,
as
specified
in
Chapter
Five,
Section
III.
New
QSARs
using
HTPS
data
and
transcriptional
activation
potencies
from
whole
cell
assays
will
need
to
be
developed.
These
new
models
will
likely
be
expansions
of
existing
QSARs
if
the
same
chemical
compounds
are
included
in
both.

Thus,
when
it
comes
time
to
set
priorities
for
the
first
phase
of
T1S,
HTPS
data
(
as
well
as
improved
QSARs)
should
be
used
along
with
other
relevant
exposure
and
effects
data.
Chemicals
not
subjected
to
HTPS
(
because
they
are
produced
in
amounts
less
than
10,000
pounds
per
year),
but
which
are
selected
for
T1S
during
the
first
phase
of
the
program,
would
still
have
to
complete
the
transcriptional
activation
assays
as
part
of
the
T1S
battery.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
14
It
is
envisioned
that
the
process
of
QSAR
model
expansion
and
improvement
will
continue
in
a
cyclical
feedback
manner,
thus
providing
the
opportunity
to
validate
the
QSAR
models
using
external
data
sets
for
screens
and
tests
of
compounds
not
subjected
to
HTPS.
Eventually,
predictions
of
endocrine
disruption
potentials
obtained
from
validated
QSAR
models
could
be
used
as
surrogates
for
HTPS
data
in
the
case
of
compounds
for
which
effects
data
are
not
available.

J.
Inclusion
of
Mixtures
and
Naturally
Occurring
Non­
Steroidal
Estrogens
and
Recommendation
for
a
Nominations
Process
The
EDSTAC
recommends
in
subsequent
sections
of
this
chapter
that
EPA
include
a
discrete
number
of
mixtures
(
Section
VII)
and
naturally
occurring
non­
steroidal
estrogens
(
Section
VIII)
in
the
EDSTP.
In
addition,
the
EDSTAC
recommends
that
a
process,
separate
and
distinct
from
the
core
priority
setting
process,
be
conducted
to
allow
affected
communities
and
members
of
the
public
to
nominate
chemicals
for
screening
and,
if
necessary,
testing
(
Section
IX).

K.
Introduction
of
the
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD)

The
PSWG
struggled
with
how
to
use
the
information
sources
and
criteria
it
identified
to
sort
and
prioritize
chemicals
for
endocrine
disruption
screening
and
testing.
The
EDSTAC,
in
response
to
work
group
information
and
queries,
directed
the
PSWG
to
consider
developing
a
computer
database
to
electronically
store
information
related
to
criteria
that
could
be
used
for
sorting
and
prioritizing.
The
EDSTAC
was
careful
to
instruct
the
PSWG
not
to
develop
a
list
of
what
were
then
referred
to
as
"
high
priority
chemicals
for
Phase
I
screening,"
but
rather
to
develop
a
tool
to
illustrate
different
scenarios
that
could
show
the
implications
of
alternative
choices
for
setting
priorities.

The
PSWG
asked
two
of
its
members
to
develop
a
relational
database
containing
information
sources
associated
with
various
criteria
to
facilitate
the
sorting
and
prioritizing
processes.
The
resulting
prototype
database
is
referred
to
as
the
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD).
A
preliminary
version
of
the
EDPSD
was
presented
to
the
EDSTAC
in
December
1997.

The
EDSTAC
was
impressed
by
the
speed
with
which
the
EDPSD
could
provide
different
scenarios,
and
gave
unanimous
support
for
continued
development
of
the
EDPSD.
However,
it
became
clear
that
sufficient
time
and
resources
were
not
available
to
adequately
develop
the
EDPSD
within
the
time
frame
of
EDSTAC's
deliberations.
Accordingly,
the
PSWG
was
told
that
EPA
would
complete
and
validate
the
EDPSD
as
a
post­
EDSTAC
exercise.
Section
X
of
this
chapter
provides
a
more
detailed
description
of
the
prototype
EDPSD,
including
the
data
fields
that
were
included
by
the
December
1997
plenary,
the
data
fields
the
EDSTAC
recommends
that
EPA
include,
and
a
process
for
using
the
EDPSD.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
15
L.
Overview
of
the
Recommended
Approach
to
Priority
Setting
In
Section
XI
of
this
chapter,
the
EDSTAC
presents
its
recommendations
for
how
to
set
priorities:
(
a)
for
chemicals
that
will
need
to
be
considered
for
T1S,
and
(
b)
for
chemicals
that
meet
the
criterion
for
bypassing
T1S
and
going
directly
to
T2T.
The
recommended
approach
for
setting
priorities
for
the
approximately
62,000
chemicals
that
will
need
to
be
considered
for
T1S
builds
upon
the
EDSTAC's
recommendations
to:
screen
mixtures
and
naturally
occurring
nonsteroidal
estrogens;
establish
a
separate
and
distinct
nominations
process;
separate
out
food­
use
pesticides
and
other
chemicals
that
have
sufficient
data
to
bypass
T1S;
and
utilize
a
database
tool
to
help
analyze
information
relevant
to
priority
setting.
The
recommended
approach
is
one
that
would
have
EPA,
with
continued
advice
and
assistance
from
a
multi­
stakeholder
group,
use
the
EDPSD
to
help
set
priorities
that
flow
from
a
simple
and
transparent
application
of
the
exposureand
effects­
related
information
categories
and
criteria.
The
EDSTAC
recommends
that
EPA
apply
the
information
categories
and
criteria
outlined
in
Sections
III
and
IV
in
a
manner
that
would
explicitly
state
the
percentage
of
the
total
number
of
chemicals
to
be
subjected
to
T1S
in
any
one
phase
of
the
program
to
be
drawn
from
the
data
sources
for
each
criterion,
or
from
the
explicit
combinations
of
criteria.
This
approach,
which
is
referred
to
as
a
"
compartment­
based"
approach
to
priority
setting,
is
described
in
more
detail
in
Section
XI.

The
recommended
approach
for
setting
priorities
for
chemicals
that
meet
the
criterion
for
bypassing
T1S
and
going
directly
to
T2T,
in
the
case
of
food­
use
pesticides,
is
to
use
the
schedule
EPA
has
established
for
tolerance
reassessments
and
pesticide
re­
registration
under
the
FQPA.
All
other
chemicals
that
meet
this
criterion
would
be
addressed
on
a
case­
specific
basis.

III.
Exposure­
Related
Information
and
Criteria
This
section
describes
in
more
detail
the
types
of
exposure­
related
information
and
criteria
that
the
EDSTAC
recommends
be
used
as
the
foundation
for
the
priority
setting
process
for
T1S.
Exposure­
related
information
and
criteria
consist
of
four
exposure
information
categories
and
one
fate
and
transport
information
category.

The
four
exposure­
related
information
categories
are:
(
a)
biological
sampling
data
for
humans
and
other
biota;
(
b)
environmental,
occupational,
consumer
product,
and
food­
related
data;
(
c)
data
on
environmental
releases;
and
(
d)
data
on
production
volume.
These
four
exposure­
related
information
categories
can
be
viewed
as
a
hierarchy
or
spectrum
in
an
exposure
chain.
At
one
end
of
the
exposure
spectrum
is
the
detection
of
chemicals
in
animal
or
human
tissues
and/
or
fluids
via
biomonitoring
studies.
Such
detection
indicates
that
systemic
exposure
has
actually
occurred.
Detection
of
a
chemical
in
an
environmental
medium,
or
knowledge
that
a
chemical
is
in
food
or
a
consumer
product,
indicates
it
is
probable
that
exposure
can
occur.
Knowledge
that
a
chemical
is
released
to
the
environment
indicates
that,
depending
upon
its
physical
and/
or
chemical
properties,
exposure
is
possible.
Production
volume
data
show
that
a
given
chemical
is
produced
and
could
be
released
to
the
environment
and
exposure
may
occur.
At
the
other
end
of
the
spectrum,
some
chemicals
are
entirely
consumed
in
making
a
subsequent
product
(
e.
g.,
in
a
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
16
closed
system)
and,
thus,
are
never
released
to
the
environment.
Regardless
of
which
data
are
used,
special
attention
should
be
paid
to
chemicals
for
which
there
is
evidence
of
embryonic,
post
partum
or
post
hatch,
early
life
stage,
or
pre­
maturation
exposures.

A
major
limitation
of
the
more
direct
measures
of
exposure
is
that
data
are
available
for
only
a
limited
number
of
chemicals.
Human
exposure
information
is
not
currently
collected
for
the
purpose
of
priority
setting
and/
or
risk
assessment.
Data
that
do
exist
have
been
collected
for
other
purposes.
U.
S.
population
exposure
data
that
exist
from
the
Centers
for
Disease
Control
and
Prevention
are
limited
to
some
heavy
metals,
volatile
organic
compounds,
persistent
organochlorines,
and
some
non­
persistent
pesticides.
The
ongoing
National
Health
and
Nutrition
Examination
Survey
(
NHANES)
family
of
surveys
(
see
Appendix
G)
does
provide
an
opportunity
to
sample
human
tissue
for
additional
chemicals.
However,
funding
for
analysis
of
the
NHANES
samples
has
not
been
secured.
In
contrast,
while
production
data
exist
for
a
large
number
of
chemicals,
the
link
between
production
data
and
exposure
is
tenuous.

The
fate
and
transport
information
category
includes
chemical
and/
or
physical
properties
that
may
be
used
to
predict
or
estimate
the
medium
or
media
where
a
chemical
is
likely
to
be
found
and
whether
or
not
a
chemical
is
likely
to
remain
in
the
environment
over
time.
This
information
can
be
used
in
several
ways.
Since
new
chemicals
will
not
have
any
data
in
the
four
exposure­
related
information
categories,
the
fate
and
transport
information,
along
with
estimates
on
production
volumes
or
environmental
releases
can
be
used
to
estimate
concentrations
in
environmental
media.
Fate
and
transport
information
can
also
be
combined
with
known
production
volumes
or
environmental
release
information
to
estimate
concentrations
in
environmental
media.
The
more
direct
the
measure
of
exposure
that
is
combined
with
fate
and
transport
information,
the
more
likely
one
would
anticipate
the
estimates
to
be
of
actual
conditions.
Unlike
the
other
exposurerelated
information
categories
which
contain
measurable
empirical
data,
fate
and
transport
information
consists
of
estimations
and
predicted
and/
or
calculated
data.

The
remainder
of
this
section
describes
in
more
detail
the
nature
of
the
information
included
in
each
exposure­
related
information
category,
the
strengths
and
limitations
of
the
type
of
information
in
each
category,
and
a
recommended
set
of
guiding
principles
for
how
to
use
the
information
contained
in
each
category
to
complete
the
task
of
setting
priorities
for
endocrine
disruptor
screening
and
testing.

A.
Biological
Sampling
Data
Biological
sampling
refers
to
the
monitoring
of
tissues
or
media
from
living
or
dead
organisms
for
chemicals
to
document
actual
human
or
animal
exposure.
The
biological
sampling
information
category
includes
data
that
falls
into
two
subcategories:
(
1)
human
biomonitoring,
and
(
2)
monitoring
of
other
biota.
Human
biomonitoring
refers
to
monitoring
of
human
tissues
and
media
(
e.
g.,
blood,
breast
milk,
adipose
tissue,
and
urine).
Monitoring
of
other
biota
encompasses
the
sampling
of
a
very
wide
range
of
species
(
invertebrates,
vertebrates
such
as
fish,
and
other
wildlife)
and
sample
matrices
(
e.
g.,
carcass,
liver,
kidney,
egg,
feathers,
etc.)
for
exposure
to
environmental
contaminants.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
17
Strengths
Human
·
Data
are
evidence
of
actual
human
exposure
·
Many
data
sets
are
representative
of
large
populations;
other
data
sets
are
representative
of
disproportionately
exposed
populations
·
Can
be
used
to
provide
data
to
address
mixtures
·
Generally
good
quality
data;
however,
this
must
be
determined
on
a
case­
specific
basis
·
May
be
used
to
identify
trends
·
For
those
substances
monitored,
can
evaluate
frequency
and
magnitude
of
exposure
detections
relative
to
each
other
to
help
prioritize
·
Addresses
multiple
routes
of
exposure
Other
Biota
·
Data
document
actual
exposure
·
Analytical
data
sets
are
generally
of
high
quality
·
Multiple
routes
of
exposure
are
addressed
·
Broad
coverage
of
phylogenetic
groups
(
e.
g.,
fish,
reptiles,
birds,
wild
mammals,
shellfish
and
other
invertebrates,
etc.),
habitats,
and
environmental
matrices
·
Information
on
various
animal
species
will
substantially
enhance
understanding
of
the
phenomenon
of
human
effects
·
Many
monitoring
programs
are
spatially
and
temporally
replicated
Limitations
Human
·
Limited
number
of
compounds
monitored;
limited
data
available
may
not
capture
any
short­
lived
compounds
or
peak
exposure
·
Biologic
half­
life,
metabolism,
and
tissue
distribution
vary
from
substance
to
substance
·
Limited
opportunities
to
collect
appropriate
specimens
·
May
not
be
representative
with
respect
to
time,
population,
or
exposure
distribution
·
Population
surveys
(
e.
g.,
NHANES)
may
not
characterize
particularly
susceptible
or
disproportionately
or
highly
exposed
subpopulations
(
e.
g.,
workers)
·
Identified
compounds
may
not
be
traceable
to
a
particular
producer
·
Need
to
separate
biomarkers
of
exposure
from
those
of
susceptibility
or
effect
·
Analyses
often
focus
on
the
"
usual
suspects"
and
additional
substances
need
to
be
measured
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
18
Other
Biota
·
Limited
number
of
compounds
monitored;
limited
data
available
may
not
capture
any
short­
lived
compounds
or
peak
exposure
·
Biologic
half­
life,
metabolism,
and
tissue
distribution
vary
from
substance
to
substance
·
Limited
opportunities
to
collect
appropriate
specimens
·
Population
surveys
may
not
characterize
particularly
susceptible
or
disproportionately
or
highly
exposed
subpopulations
·
Identified
compounds
may
not
be
traceable
to
a
particular
producer
Need
to
separate
biomarkers
of
exposure
from
those
of
susceptibility
or
effect
·
"
Exposure"
or
"
potential
exposure"
are
generally
monitored;
"
biological
effects"
are
not
Guiding
Principles
for
Using
These
Data
for
Priority
Setting
·
The
greater
the
relevance
of
the
data
set
to
large
populations,
disproportionately
exposed
subpopulations,
or
particularly
susceptible
subpopulations,
the
more
weight
the
data
set
should
be
given.
·
Data
sets
with
good
quality
assurance/
quality
control
(
QA/
QC)
data
should
be
given
greater
weight
than
those
data
sets
with
lower
QA/
QC
data.
·
The
lower
the
detection
limits
and
the
greater
the
efforts
to
test
organisms
that
are
likely
to
be
exposed,
the
greater
the
weight
"
non­
detect"
data
should
be
given.

B.
Environmental,
Occupational,
Consumer
Product,
and
Food­
Related
Data
Environmental,
occupational,
consumer
product,
and
food­
related
data
include:
(
1)
monitoring
data
for
chemical
contaminants
found
in
a
variety
of
environmental
media
to
which
humans
and
animals
are
exposed,
such
as
water
(
surface,
ground,
and
drinking),
air,
soil,
sediment,
and
food;
and
(
2)
use
information
for
chemicals,
when
it
is
available.

Strengths
·
Provides
data
on
likely
exposures
to
humans
and
other
biota
·
Databases
exist
for
air,
water,
soil,
and
food
·
May
be
used
to
identify
trends
·
Data
can
be
used
to
identify
relevant
media
for
exposures
(
e.
g.,
food,
air,
and/
or
water)

Limitations
·
Limited
number
of
compounds
monitored
·
Quantitative
exposure
levels
must
be
inferred
in
many
cases
·
"
Detect"
limits
may
vary
from
one
data
set
to
another
·
Use
data
sources
are
not
comprehensive,
are
frequently
secondary
sources,
and
may
not
be
independently
verified.
The
highest
quality,
most
comprehensive
data
sources
are
usually
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
19
maintained
by
fee­
for­
service
organizations.
Consequently,
no
use
information
databases
for
existing
chemicals
have
been
included
in
the
EDPSD.

Guiding
Principles
for
Using
These
Data
for
Priority
Setting
·
The
greater
the
relevance
of
the
data
set
to
large
populations,
disproportionately
exposed
subpopulations,
or
particularly
susceptible
subpopulations,
the
more
weight
the
data
set
should
be
given.
·
The
more
likely
a
chemical
is
to
be
internalized
by
an
organism
from
its
environment,
the
greater
weight
it
should
be
given.
·
Data
sets
with
good
QA/
QC
data
should
be
given
greater
weight
than
those
data
sets
with
lower
QA/
QC
data.
·
The
lower
the
detection
limits
and
the
greater
the
efforts
to
test
organisms
that
are
likely
to
be
exposed,
the
greater
the
weight
"
non­
detect"
data
should
be
given.

C.
Environmental
Releases
Environmental
release
information
includes
data
on
chemicals
released
to
the
environment
to
which
humans
and
animals
may
be
exposed,
such
as
permitted
industrial
discharges
to
air
or
water
and
accidental
release
or
spill
data.
An
example
of
the
industrial
discharge
data
is
the
Toxic
Release
Inventory
(
TRI)
reporting
required
by
EPA.
An
example
of
accidental
release
or
spill
data
is
the
Hazardous
Substance
Emergency
Surveillance
System
maintained
by
the
Agency
for
Toxic
Substances
Disease
Registry
(
ATSDR).

Strengths
·
Provides
data
on
potential
and
known
exposures
to
humans
and
other
biota
·
Databases
exist
for
air
and
water
·
May
be
used
to
identify
trends
·
Data
can
be
used
to
identify
relevant
media
for
exposures
(
e.
g.,
food,
air,
and/
or
water)
·
TRI
is
updated
annually
·
Databases
include
location­
specific
data
which
are
relevant
to
disproportionately
exposed
populations
Limitations
·
Data
exist
for
a
limited
number
of
industrial
chemicals
(
528
in
the
case
of
the
TRI)
·
Quantitative
exposure
levels
are
difficult
to
estimate
in
many
cases
·
No
data
are
available
in
the
TRI
for
releases
under
10,000
pounds
per
year
from
single
sources
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
20
Guiding
Principles
for
Using
These
Data
for
Priority
Setting
·
The
greater
the
relevance
of
the
data
set
to
large
populations,
disproportionately
exposed
subpopulations,
or
particularly
susceptible
subpopulations,
the
more
weight
the
data
set
should
be
given.
·
The
more
likely
the
environmental
releases
are
to
lead
to
organism
exposure,
the
greater
the
weight
the
release
data
should
be
given
(
e.
g.,
TRI
releases
to
air
and
water
should
be
given
more
weight
than
TRI
releases
to
disposal
such
as
permitted
landfills,
etc.).

D.
Production
Volume
Data
Production
volume
data
include
production
information,
primarily
volume,
for
chemical
substances
and
are
mainly
relevant
to
existing
chemical
substances.
Such
information
can
only
be
estimated
for
new
products
and
is
not
relevant
to
environmental
contaminants.
The
discussion
of
strengths
and
limitations
which
follows
distinguishes
among
existing
industrial
(
i.
e.,
TSCAregulated
chemicals,
existing
pesticides
(
i.
e.,
FIFRA­
regulated),
and
new
chemicals.

Strengths
Existing
Industrial
Chemicals
(
TSCA­
Regulated)
·
Quick,
easy
way
to
obtain
a
rough
estimate
of
exposure
potential
·
Readily
available
(
to
EPA)
for
chemicals
other
than
polymers
and
inorganics
produced
or
imported
in
amounts
greater
than
10,000
pounds
per
year
·
Reliable
and
comprehensive
·
Identifies
site­
limited
chemicals
·
Excludes
non­
isolated
intermediates
·
Includes
data
on
imported
chemicals
Existing
Pesticides
(
FIFRA­
Regulated)
·
Production
data
available
at
national
level
(
but
not
state
level)
for
all
covered
products
only
as
composite,
not
manufacturer­
specific
·
Available
to
EPA
and
the
public
New
Chemicals
·
Estimated
production
volume
data
available
to
EPA
for
all
new
chemicals
·
Comprehensive
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
21
Limitations
Existing
Industrial
Chemicals
(
TSCA­
Regulated)
·
TSCA
inventory
update
identifies
site­
limited
intermediates,
but
does
not
contain
information
on
uses
of
individual
chemicals
·
TSCA
inventory
data
may
be
protected
as
Confidential
Business
Information
(
CBI),
which
means
that
they
are
available
to
the
EPA
but
not
to
the
public
·
Does
not
contain
data
on
degradates,
mixtures
of
chemical
substances,
inorganics,
polymers,
or
chemicals
produced/
imported
in
amounts
less
than
10,000
pounds
per
year
Existing
Pesticides
(
FIFRA­
Regulated)
·
Often
lacking
information
on
number
of
potentially
exposed
workers,
"
fence­
line"
concentrations,
and
environmental
release
pathways
·
Currently
contains
information
on
currently
registered
and
used
products
only
New
Chemicals
·
TSCA
data
for
new
chemicals
may
be
protected
as
CBI,
which
means
that
they
are
available
to
the
EPA
but
not
to
the
public
·
Production
data
are
estimates
·
Many
Pre­
Manufacture
Notification
(
PMN)
chemicals
are
never
commercialized;
fewer
are
commercially
successful
Guiding
Principles
for
Using
These
Data
for
Priority
Setting
·
Production
volume
provides
only
a
very
rough
indication
of
potential
human
and
ecological
exposure.
Combining
production
data
with
other
data
(
e.
g.,
effects
data)
minimizes,
to
a
certain
extent,
some
of
the
inherent
weaknesses
of
using
production
data
as
a
surrogate
for
exposure.
Production
information
should
not
be
used
to
prioritize
between
existing
industrial
chemicals
and
pesticides
or
between
new
chemicals
and
pesticides
because
production
volume
ranges
are
too
divergent.
For
example,
production
volumes
for
high­
volume
industrial
chemicals
are
several
orders
of
magnitude
higher
than
those
for
either
new
chemicals
or
pesticides.

E.
Fate
and
Transport
Data
and
Models
Environmental
fate
and
transport
information
is
available
from
various
reference
sources,
including
databases,
textbooks,
and
monographs
(
e.
g.,
Swanson
and
Socha,
1997;
Cowan
et
al.,
1996).
Although
the
data
source
matrix
for
environmental
fate
and
transport
data
and
models
included
in
Appendix
G
highlights
a
number
of
specific
sources
of
information,
no
single
source
is
really
superior
to
another
in
that
each
is
a
collection
of
data.
Because
there
is
a
lot
of
environmental
fate
and
transport
data
from
which
to
choose,
the
challenge
is
to
identify
the
critical
fate
and
transport
data
useful
for
sorting
and
prioritization
purposes.

The
EDSTAC
recommends
that
EPA
focus
on
three
subcategories
of
environmental
fate
and
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
22
transport
information:
persistence,
mobility,
and
bioaccumulation.
Each
of
these
factors
can
affect
the
bioavailability
of
a
chemical
substance
because
each
is
directly
correlated
to
potential
exposure.
The
definitions
used
by
the
EDSTAC
for
these
terms
are
as
follows:

Persistence
is
the
tendency
of
a
chemical
substance
or
its
degradation
products
to
persist
(
survive)
in
the
environment
without
transformation
into
another
chemical
form.

Mobility
is
the
tendency
of
a
chemical
substance
to
move
within
environmental
media
(
e.
g.,
air
or
water)
or
between
media
(
e.
g.,
to
migrate
from
soil
to
groundwater).

Bioaccumulation
is
the
capacity
of
a
chemical
to
accumulate
(
be
stored
in
the
tissue)
in
an
organism
as
a
result
of
uptake
from
all
environmental
sources.

Strengths
·
Environmental
fate
and
transport
tests
pertaining
to
the
three
categories
are
already
in
place
and
have
a
long
history
of
use
for
many
chemicals
·
EPA
has
identified
thresholds
for
various
environmental
fate
and
transport
tests
that
trigger
regulatory
concern;
however,
at
this
time,
the
quantification
of
these
potential
thresholds
(
or
"
triggers")
and
their
application
to
determine
the
potential
for
endocrine
disruption
may
be
lacking
or
subjective
·
Modeling
can
also
be
used
to
estimate
environmental
fate
and
transport
characteristics
of
persistence,
bioaccumulation,
and
mobility
when
test
data
on
specific
substances
are
lacking
Limitations
·
No
single
source
of
information
on
fate
and
transport
includes
all
chemical
substances
·
There
are
gaps
in
the
data
sources,
making
direct
comparisons
between
chemical
substances
difficult
·
Most
fate
and
transport
estimating
procedures
have
not
been
validated
over
the
range
of
possible
chemical
substances
that
will
need
to
be
considered
for
endocrine
disruptor
screening
and
testing
·
Test
data
for
the
three
selected
parameters
may
not
be
available
for
all
chemical
substances
·
At
this
time,
there
are
no
generally
established
or
accepted
environmental
fate
or
transport
criteria
directly
related
to
endocrine
disruption
·
Fate
and
transport
of
chemical
substances
may
vary
widely
depending
on
environmental
conditions;
arbitrary
standard
conditions
are
established
for
regulatory
and
comparative
purposes
Guiding
Principles
for
Using
These
Data
for
Priority
Setting
·
For
each
of
the
three
environmental
fate
and
transport
characteristics
 
persistence,
mobility,
and
bioaccumulation
 
the
tables
contained
in
Appendix
H
specify
relevant
physicochemical
criteria
along
with
their
corresponding
threshold
(
or
"
trigger").
These
"
triggers"
are
those
which
EPA
generally
takes
into
consideration
when
evaluating
a
pesticide
or
chemical
for
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
23
registration.
However,
it
should
be
noted
that
EPA
does
not
rely
solely
on
"
trigger"
values,
but
considers
other
environmental
effects
(
e.
g.,
wildlife
toxicology)
before
granting
product
registrations.
·
The
use
of
fate
and
transport
data
to
help
set
priorities
for
T1S
should
take
into
account
all
three
environmental
compartments
 
air,
water,
and
soil.
·
Fate
and
transport
characteristics
should
be
based
on
laboratory
or
field
tests
when
good
quality
data
are
unavailable.
If
laboratory
or
field
data
are
lacking
for
a
chemical,
EPA
should
calculate
the
predicted
fate
and
transport
data
for
use
in
priority
setting
by
means
of
reliable
methodology
or
by
use
of
an
algorithm.
·
The
physicochemical
measures
recommended
for
each
of
the
three
environmental
fate
and
transport
characteristics
identified
above
 
persistence,
mobility,
and
bioaccumulation
 
are
listed
below:

·
Hydrolysis
half­
life
 
persistence;
·
Biodegradation
 
persistence;
·
Photooxidation
 
persistence;
·
Volatility
(
Henry's
Law)
 
mobility;
·
Absorption
Coefficient
(
Koc)
 
mobility;
and
·
Octanol:
Water
Partition
Coefficient
(
Kow/
LogP)
 
mobility
and
bioaccumulation.

·
Fate
and
transport
measures
that
provided
redundant
information
were
eliminated.
Some
measures,
such
as
photolysis,
must
be
determined
experimentally.
A
surrogate
measure,
photooxidation
in
this
case,
can
help
to
fill
gaps
in
the
data
(
photooxidation
is
one
estimate
of
the
atmospheric
half­
life
of
a
parent
compound
due
to
reaction
with
photochemicallyproduced
hydroxyl
radicals).

IV.
Effects­
Related
Information
and
Criteria
In
addition
to
HTPS,
which
is
described
separately
in
Chapter
Four,
Section
V,
the
effects­
related
information
categories
the
EDSTAC
recommends
as
the
foundation
for
the
priority
setting
process
include:
(
a)
toxicological
laboratory
studies
and
databases;
(
b)
epidemiological
and
field
studies
and
databases;
and
(
c)
predictive
biological
activity
or
effects
models,
commonly
referred
to
as
Structure
Activity
Relationship
(
SAR)
and/
or
Quantitative
Structure
Activity
Relationship
(
QSAR)
models.

Toxicological
laboratory
studies
and
databases
include
all
published,
publicly
available,
or
otherwise
useable
information
related
to
the
laboratory
study
of
toxic
effects
of
chemical
substances
and
mixtures
on
living
organisms
or
cell
systems,
including
humans,
wildlife,
and
ecological
systems.

Epidemiological
and
field
studies
and
databases
range
from
hypothesis­
generating
descriptive
studies,
such
as
case
reports
and
ecological
field
analyses,
to
prospective
cohort
studies
and
rigorously
controlled
hypothesis­
testing
clinical
trials
or
community
interventions.
The
most
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
24
common
studies
are
descriptive.

Empirical
toxicological
and
epidemiological
data
are
reported
in
a
large
number
of
peer
reviewed
scientific
journals.
Published
studies
are
conducted
with
varying
degrees
of
methodological
rigor
and
data
are
reported
in
widely
varying
detail.
Consequently,
information
obtained
from
the
general
literature
must
be
reviewed
in
detail
in
order
to
determine
its
applicability
and
adherence
to
generally
acceptable
investigatory
practices.
Some
positive
reproductive
effects
data
are
included
in
several
regularly
updated
databases
which
are
described
in
more
detail
in
Appendix
G.

Predictive
biological
activity
or
effects
models
attempt
to
identify
correlation
between
properties
that
can
be
derived
from
the
chemical
structure
or
properties
of
molecules
and
biological
activities,
including
those
that
can
be
identified
through
in
vitro
or
in
vivo
screens
and
tests.
SAR
and
QSAR
models
are
also
used
to
predict
physicochemical
properties
such
as
solubility,
volatility,
and
lipophilicity
(
LogP).
QSARs
are
useful
for
estimating
or
predicting
how
a
chemical
may
behave
when
empirical
toxicological
or
epidemiological
data
are
unavailable.

General
Guiding
Principles
for
Effects­
Related
Criteria
·
The
EDSTAC
believes
that
using
published
toxicological
laboratory,
epidemiological,
or
field
studies
for
priority
setting
without
first
narrowing
the
universe
of
chemicals
subject
to
detailed
review
would
be
virtually
impossible
in
the
appropriate
time
frame
and
with
available
resources.
Accordingly,
the
EDSTAC
recommends
that
data
from
the
general
scientific
literature,
which
is
not
organized
into
logical
databases,
be
used
to
help
set
priorities
after
an
initial
selection
is
made
based
on
effects­
related
data
organized
into
logical
databases.
This
issue
is
discussed
in
more
detail
in
Chapter
Four,
Section
X,
E.
·
QSAR
models
should
not
override
HTPS
information.
Rather,
HTPS
data
should
be
used
to
improve
the
QSAR
database
as
described
more
fully
in
Chapter
Four,
Section
V,
G,
3.
·
Positive
epidemiological
studies
should
be
considered
of
higher
value
for
priority
setting
purposes
even
in
the
presence
of
negative
toxicological
studies.
·
EPA
has
provided
considerable
guidance
on
how
to
interpret
the
results
of
toxicity,
epidemiology,
and
other
relevant
data.
This
guidance
should
be
relied
upon
in
interpreting
the
available
database
for
prioritizing
effects
information.
The
most
relevant
guidance
for
endocrine
disruptor
information
are
the
Guidelines
for
Developmental
Toxicity
Risk
Assessment
(
U.
S.
EPA,
1991),
the
Guidelines
for
Reproductive
Toxicity
Risk
Assessment
(
U.
S.
EPA,
1996),
and
the
Guidelines
for
Neurotoxicity
Risk
Assessment
1998.

The
remainder
of
this
section
describes
in
more
detail
the
nature
of
the
information
included
in
each
effects­
related
information
category,
the
strengths
and
limitations
of
the
type
of
information
in
each
category,
and
a
recommended
set
of
guiding
principles
for
how
to
use
the
information
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
25
contained
in
each
category
to
complete
the
task
of
setting
priorities
for
endocrine
disruptor
screening
and
testing.

A.
Toxicological
Laboratory
Studies
and
Databases
Strengths
·
For
a
few
chemicals,
particularly
those
regulated
under
FIFRA,
a
wide
variety
of
whole
animal
studies
have
been
completed
using
modern
protocols
with
some
endocrine
sensitive
endpoints
(
e.
g.,
developmental
toxicity,
reproductive
toxicity)
and
conducted
under
Good
Laboratory
Practices
(
GLPs)
·
Allows
for
testing
of
single
agents
and/
or
mixtures
to
establish
cause­
and­
effect
relationships
·
Studies
are
likely
to
provide
useful
dose­
response
data
for
the
endpoints
and
species
studied
·
Good
coverage
for
a
few
chemicals
and/
or
substances
(
e.
g.,
petroleum
crudes,
organochlorine
pesticides)
with
respect
to
aquatic
species,
birds,
and
wildlife
Limitations
·
Toxicological
database
for
industrial
chemicals
is
less
complete
than
that
for
pesticides
·
Ability
to
extrapolate
endocrine­
related
knowledge
in
test
species
to
other
species,
including
humans,
is
limited
by
the
lack
of
knowledge
about
interspecies
comparative
endocrinology
·
Effects
at
very
low
doses
and
the
presence
of
an
inverted
"
U­
shaped"
dose­
response
curve
or
the
"
inverted
J­
shaped"
curve
indicative
of
hormesis
have
generally
not
been
examined
in
toxicological
studies
·
Studies
may
not
be
designed
to
detect
the
relevant
endpoints
·
Relevance
of
in
vitro
data
to
organisms
and
populations
is
not
well
characterized
·
Very
little
data
on
TSCA­
related
chemicals
especially
for
effects
on
birds
and
fish
·
Little
is
known
of
endocrine
disruptor
effects
in
wild
mammals,
lower
vertebrates,
and
invertebrates
·
Relatively
few
studies
have
looked
at
subtle
and
multi­
generation
effects
Guiding
Principles
for
Using
These
Data
for
Priority
Setting
·
Whenever
possible,
in
vivo
studies
with
relevant
endpoints
and
with
wide
dose­
response
data
should
be
viewed
as
more
relevant
for
priority
setting
than
in
vitro
studies.
This
is
especially
true
when
considering
in
vitro
studies
featuring
receptor­
mediated
mechanisms,
which
typically
do
not
correlate
well
with
endocrine­
mediated
in
vivo
effects.
·
Studies
that
have
any
or
all
of
the
following
characteristics
should
be
valued
greater
than
those
that
do
not:
·
inclusion
of
relevant
endpoints
sensitive
to
endocrine
disruption
·
indication
of
a
dose
response
for
endocrine
disruptor
effects
·
receipt
of
peer
review
·
GLP
compliance
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
26
B.
Epidemiological
and
Field
Studies
and
Databases
Strengths
·
These
data
may
provide
actual
observation
of
impacts
upon
humans,
organisms,
or
ecological
communities,
removing
many
of
the
uncertainties
inherent
in
assessing
risk
based
on
laboratory
studies
·
When
these
data
include
biomarkers
of
exposure
or
effects,
they
can
serve
to
document
a
completed
exposure
pathway
·
Data
from
these
studies
can
provide
information
on
vulnerable
populations
or
high­
exposure
subgroups,
such
as
the
occupationally
exposed
Limitations
·
Human
disease
and
organ
system
dysfunction
is
multifactorial
in
causality,
making
it
difficult
to
identify
the
contribution
of
individual
factors
unless
they
are
dominant
causes
·
The
mechanisms
which
lead
to
specific
human
diseases
are
often
unknown,
and
the
specific
endocrine
disruption
mechanisms
which
cause
specific
diseases
are
poorly
understood
·
Small
excess
risks
for
common
health
outcomes
may
be
difficult
to
identify
without
appropriate
consideration
of
study
power.
·
Studies
of
highly
exposed
individuals
may
not
be
relevant
to
much
lower
population
exposures
or
to
more
vulnerable
subpopulations;
extrapolation
of
high­
exposure
effects
to
low­
exposure
circumstances
or
between
subpopulations
introduces
uncertainty
and
decreases
the
utility
of
the
data
·
Studies
often
address
only
one
route
of
exposure;
this
route,
however,
may
not
be
the
most
relevant
route
for
the
general
population
·
Human
and
ecological
communities
are
seldom
exposed
to
only
one
compound;
it
is
difficult
to
identify
and
examine
the
effects
of
multiple
exposures
and
their
possible
interactions
Guiding
Principles
for
Using
These
Data
for
Priority
Setting
·
Despite
the
many
limitations
inherent
in
epidemiological
and
field
study
data,
statistically
positive
studies
should
be
a
priority
indicator
for
additional
screening
and
testing.
·
When
multiple
studies
exist
and
there
is
a
consistently
positive
association
between
exposure
and
an
effect,
but
individually
the
studies
do
not
reach
statistical
significance,
this
finding
should
be
given
weight
when
determining
the
priority
for
screening
and
testing.
·
Weight
given
to
statistically
negative
studies
should
be
dependent
upon
the
study
design,
quality
of
the
data,
and
the
power
of
the
study
to
detect
an
effect.
Negative
human
epidemiological
studies
and
ecological
field
studies
should
be
considered,
but
should
not
necessarily
override
positive
toxicological
studies
when
determining
priority
for
screening
and
testing.
·
When
multiple
studies
exist,
weight
should
be
given
to
those
studies
that
have
received
peer
review
and
which
are
of
high
design
quality.
A
checklist
of
issues
important
to
evaluating
a
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
27
study
should
be
developed
to
assist
in
the
review.
Such
a
checklist
would
include
likelihood
of
misclassification
of
exposure
or
disease,
likelihood
of
introduction
of
bias,
and
utilization
of
standardized
tools
or
methods.
·
Descriptions
of
studies
should
include
a
characterization
of
their
design.
Study
design
is
important
in
determining
the
inferences
that
can
be
drawn
from
the
results.
Commonly
used
descriptors
include:
descriptive
(
case
reports,
case
series,
calculations
of
rates
of
prevalence,
incidence,
mortality);
observational
(
ecological,
cross­
sectional,
case­
control,
cohort,
proportionate
morbidity/
mortality
ratio);
and
experimental
(
clinical
trial,
community
trial).

C.
Predictive
Biological
Activity
or
Effects
Models
Strengths
·
SARs/
QSARs
can
be
used
to
rapidly
and
relatively
inexpensively
predict
biological
activities
of
large
numbers
of
compounds,
thereby
avoiding
the
need
to
prioritize
on
the
basis
of
"
no
data"
·
Current
SAR/
QSAR
models
developed
for
application
to
endocrine
disruption
analysis
predict
binding
affinity
and,
therefore,
have
the
same
advantages
and
disadvantages
as
the
in
vitro
models
upon
which
they
are
based
·
The
use
of
SARs/
QSARs
in
sorting
and
prioritizing
allows
for
transparency
and
comparative
consistency
and
avoids
the
problem
of
comparing
different
experimental
data
types
against
each
other
(
e.
g.,
two­
generation
reproduction
study
versus
in
vitro
binding)

Limitations
·
No
models
are
perfect,
and
the
current
receptor
binding
models
suffer
both
from
the
imperfections
of
receptor
binding
modeling
and
the
ability
of
receptor
binding
to
predict
in
vivo
activity
·
Not
all
mechanisms
of
endocrine
disruption
are
known
or
have
enough
data
to
model;
it
is,
therefore,
not
possible
to
generate
models
for
all
possible
ways
in
which
the
endocrine
system
can
be
disrupted
Guiding
Principles
for
Using
These
Data
for
Priority
Setting
·
Guiding
principles
applicable
to
the
biological
effects
data
used
as
the
basis
for
the
SAR,
as
well
as
to
the
QSAR
itself,
should
be
applied
to
the
results
of
the
SAR/
QSAR.
·
The
applicable
chemical
domain
of
the
SAR/
QSAR
should
be
as
diverse
as
possible.
·
SARs/
QSARs
should
be
developed
using
the
most
complete
and
accurate
data
sets
available.
·
SARs/
QSARs
should
be
validated
and
used
only
within
the
range
of
conditions
for
which
they
are
validated.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
28
V.
High
Throughput
Pre­
Screening
A.
Introduction
During
the
course
of
its
investigations
the
EDSTAC
realized
that,
with
the
exception
of
food­
use
and
consumer
pesticides
with
regulatory
mandates
requiring
developmental
and
two­
generation
reproductive
toxicity
testing,
substantial
endocrine
effects
data
were
lacking
for
most
chemical
substances.
Developmental
and
reproductive
toxicity
screening
and
testing
data
are
available
in
the
literature
for
an
estimated
5,000
chemicals,
a
large
fraction
of
which
are
pesticides
and
pharmaceuticals
(
John
D.
Walker,
U.
S.
EPA,
personal
communication).
In
addition,
existing
QSAR
methods
for
endocrine­
mediated
effects
are
presently
insufficiently
validated
to
be
universally
accepted
as
a
source
of
effects
data
(
Ankley
et
al.,
1997).

In
the
absence
of
biological
effects
data,
the
scientists
and
officials
within
EPA
charged
with
carrying
out
the
priority
setting
process
will
be
left
with
the
choice
of
either
raising
or
lowering
the
priority
of
a
chemical
based
on
a
lack
of
effects
information.
Raising
the
priority
seems
to
make
sense
from
a
public
health
protection
standpoint,
but
in
reality
it
will
accomplish
nothing
because
the
vast
majority
of
chemicals
being
evaluated
are
likely
to
be
in
the
"
no
data"
category
for
endocrine­
mediated
effects.
In
essence,
if
a
lack
of
data
became
a
rationale
for
making
a
chemical
a
high
priority
for
screening
and
testing,
it
could
render
the
biological
effects
portion
of
the
prioritization
process
meaningless.

To
address
the
problem
of
having
little
or
no
endocrine
disruptor
effects
data
on
the
majority
of
chemicals
that
will
need
to
be
screened
and
possibly
tested,
the
EDSTAC
recommends
that
EPA
use
"
high
throughput
pre­
screening"
(
HTPS).
As
the
term
is
used
throughout
this
document,
HTPS
refers
to
the
use
of
automated
processes
(
robotic
and
specialized
instrumentation)
to
aid
in
the
screening
of
compounds.
These
automated
processes
involve
a
number
of
preparatory
operations,
some
of
which
are
also
associated
with
traditional
screening
approaches,
such
as
sample
preparation
(
weighing
and
dissolving
in
the
appropriate
medium),
screening,
and
the
reading
of
screening
results.
However,
in
the
case
of
HTPS,
the
process
of
placing
the
samples
into
a
microliter
plate,
the
sampling
process
itself,
and
the
reading
of
sampling
results,
are
all
automated.
Since
all
processes
are
automated
and
can
be
programmed
to
run
continuously,
it
is
possible
for
large
numbers
of
samples
to
be
assayed
in
a
relatively
short
period
of
time
using
this
technology.

High
throughput
screening
technology
is
used
extensively
in
the
pharmaceutical
and
agrochemical
industries
to
identify
chemicals
that
have
commercial
potential
or
that
may
have
desirable
or
undesirable
biological
effects
(
Christopher
Waller,
OSI
Pharmaceuticals,
Inc.,
personal
communication).
The
EDSTAC
proposes
that
high
throughput
screening
technology
be
employed
as
a
prioritization
tool
 
hence
the
term
"
pre­
screening"
 
for
the
endocrine
disruptor
screening
and
testing
program.
HTPS
results,
although
limited
in
the
scope
of
information
they
generate,
will
be
useful
in
identifying
chemicals
that
have
an
affinity
for
the
estrogen,
androgen,
or
thyroid
hormone
receptor.
This
information
could
be
used
in
conjunction
with
other
exposureand
effects­
related
information
to
determine
the
priority
by
which
chemicals
should
be
advanced
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
29
to
the
screening
and
testing
tiers
of
the
program.
However,
HTPS
results
will
not
be
sufficient
to
make
a
definitive
determination
about
whether
a
chemical
does
or
does
not
have
endocrine
disrupting
properties.
This
is
the
function
of
T2T.

The
EDSTAC's
recommendations
regarding
HTPS
are
contingent
upon
the
demonstration
of
feasibility
of
this
process
in
the
context
of
the
EDSTP.
Appendix
I
contains
information
on
the
demonstration
of
feasibility
proposal.
The
remainder
of
this
section
explains
the
purposes
of
HTPS
and
how
it
will
be
used
to
improve
the
endocrine
disruptor
priority
setting,
screening,
and
testing
processes.
Chapter
Five
describes
some
of
the
HTPS
assays
in
more
detail
and
their
relation
to
the
other
assays
in
the
T1S
battery.

B.
Purpose
of
HTPS
First
and
foremost,
HTPS
will
provide
a
baseline
of
systematically
gathered
data
for
the
endocrine
hormones
that
are
currently
addressed
in
the
program
 
estrogen,
androgen,
and
thyroid.
This
is
especially
important
for
those
chemicals
for
which
such
data
on
endocrine
relevant
effects
are
otherwise
lacking,
namely
most
chemicals
on
the
TSCA
Inventory.
The
use
of
HTPS
data
should
make
screening
more
productive,
as
it
is
likely
that
a
higher
proportion
of
chemicals
sent
to
T1S
during
the
early
phases
of
the
program
will
have
some
evidence
of
biological
activity.

Second,
given
the
exploratory
nature
of
HTPS,
it
is
important
to
gain
some
perspective
on
the
effectiveness
of
this
methodology
compared
to
other
methodologies,
such
as
QSARs,
that
can
be
used
to
identify
compounds
for
screening.
There
is
some
concern
that
pre­
screening
chemical
substances
 
especially
some
pesticides,
for
which
substantial
reproduction
and
developmental
(
whole
animal)
testing
data
may
already
exist
 
is
a
redundant
exercise.

The
EDSTAC
recognizes
that
the
inappropriate
use
of
HTPS
data
could
result
in
a
certain
stigma
or
in
product
de­
selection.
This
potential
is
not
unique
to
HTPS,
but
is
a
broader
communication
issue
related
to
endocrine
disruptor
screens
and
tests
in
general.
This
issue
is
addressed
in
Chapter
Six.
EDSTAC
members
believe
that
if
communication
of
the
results
of
HTPS
is
handled
effectively,
inappropriate
use
of
the
data
and
potential
adverse
marketplace
reactions
to
such
inappropriate
use
will
be
minimized.

C.
Which
Assays
Will
be
Conducted
in
HTPS?

As
noted
in
Chapter
Two,
one
of
the
key
mechanisms
by
which
chemicals
affect
the
endocrine
system
is
by
interacting
with
receptors.
There
is
substantial
evidence
to
support
this
statement
for
estrogen
and
androgen
receptors.
Existing
data
suggest
that
receptor
binding
may
not
be
a
significant
mechanism
for
thyroid­
related
effects.
As
discussed
in
Chapter
Five,
both
the
transcriptional
activation
and
receptor
binding
assays
for
estrogen
alpha
and
androgen
hormones
are
recommended
for
inclusion
in
the
standardization
and
validation
program
for
T1S.
If
transcriptional
activation
assays
can
be
standardized,
validated,
and
shown
to
be
as
reliable
as
receptor
binding
assays,
the
EDSTAC
recommends
that
they
be
included
in
the
screening
battery
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
30
as
the
preferred
assay
to
detect
receptor
interactions.
The
receptor
binding
assay
is
an
acceptable
alternative
that
has
decades
of
use,
but
may
be
less
informative
than
the
transcriptional
activation
assays
in
terms
of
the
nature
of
the
interaction
(
agonism
or
antagonism).
The
potential
contributions
of
both
types
of
assays
in
the
context
of
priority
setting
are
discussed
below.

Receptor
binding
assays
are
cell­
free
biochemical
preparations
in
which
one
determines
the
amount
of
chemical
that
binds
to
the
hormone
receptor
as
a
function
of
the
concentration
of
the
chemical
in
solution,
thus
determining
the
affinity
of
the
chemical
for
the
receptor.
Because
receptor
binding
assays
only
measure
binding,
whether
a
substance
is
an
agonist
(
turns
on
or
turns
off
gene
expression
like
the
natural
ligand)
or
an
antagonist
(
which
has
the
ability
to
block
the
action
of
the
natural
hormone)
cannot
be
determined.

Transcriptional
activation
assays
are
conducted
with
intact
cells
that
have
been
genetically
modified
to
contain
a
hormone
receptor
and
a
reporter
gene.
The
reporter
gene
produces
a
protein
that
can
be
quantitatively
measured
to
reflect
the
ability
of
a
chemical
to
act
like
a
hormone,
or
to
block
the
action
of
a
hormone.
The
chemical
may
bind
to
the
receptor
and
the
resulting
receptor­
ligand
complex
binds
to
a
specific
place
on
the
reporter
gene
called
the
hormone
response
element.
Subsequent
steps
include
transcription
of
DNA
of
the
gene
to
form
RNA
and
translation
of
the
RNA
to
form
the
marker
protein.
There
are
several
different
kinds
of
marker
proteins
that
have
been
used
in
these
assays.
The
common
property
is
that
they
produce
detectable
signals
that
gene
expression
has
taken
place.
For
example,
one
marker
protein,
luciferase,
is
derived
from
fireflies
and
causes
the
emission
of
light
when
acting
on
luciferin,
which
is
introduced
into
the
culture
medium.
Thus,
the
activity
of
a
hormone
mimic
is
detected
by
the
amount
of
light
produced
by
the
cell.
In
practice,
the
amount
of
light
produced
can
be
compared
with
that
produced
when
the
natural
hormone
or
a
reference
substance
is
added
to
the
culture.

Transcriptional
activation
assays
incorporate
receptor
binding,
but
may
be
more
relevant
to
responses
in
whole
animals
because
they
use
intact
cells
and
measure
biological
processes
that
result
from
receptor
binding.
However,
relevance
must
be
balanced
with
the
fact
that,
because
of
the
added
complexity
inherent
in
these
processes,
it
is
possible
for
the
marker
protein
to
be
expressed
by
actions
of
the
chemical
unrelated
to
receptor
binding.
The
cells
used
may
have
some
ability
to
metabolize
tested
chemicals.
This
metabolic
competence
can
be
enhanced
by
genetically
incorporating
the
ability
to
make
one
or
more
of
the
enzymes
typically
involved
in
metabolism
of
exogenous
chemicals.
This
may
provide
the
assay
with
the
ability
to
detect
compounds
which
must
be
metabolically
altered
in
order
to
bind
to
the
receptor.
These
enzymes
can
also
be
added
to
the
receptor
binding
assays.

Both
the
transcriptional
activation
and
receptor
binding
assays
can
be
run
automatically
at
several
concentrations
to
determine
an
EC­
50
(
the
concentration
at
which
50%
response
is
obtained).
The
EC­
50
can
be
used
to
compare
potencies
of
chemicals
within
each
assay,
which
is
a
useful
index
for
setting
priorities
among
chemicals
for
additional
screening.
EPA
has
selected
the
transcriptional
activation
assay
utilizing
the
luciferase
reporter
gene
for
demonstration
purposes
and,
if
shown
to
be
technically
feasible
and
valid,
intends
to
use
it
for
the
HTPS.
In
this
assay
system
the
test
material
is
run
in
the
assays
listed
below
with
and
without
metabolic
activation
for
agonist
and
antagonist
potential.
Multiple
doses
(
probably
five
plus
a
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
31
control)
would
be
run
so
that
an
EC­
50
for
transcriptional
activation
can
be
determined
as
a
measure
of
potency
as
discussed
above.

1.
Estrogen
Alpha
Receptor
Transcriptional
Activation
Assay
(
no
metabolism)
2.
Estrogen
Alpha
Receptor
Transcriptional
Activation
Assay
(
metabolism)
3.
Estrogen
Beta
Receptor
Transcriptional
Activation
Assay
(
no
metabolism)
4.
Estrogen
Beta
Receptor
Transcriptional
Activation
Assay
(
metabolism)
5.
Androgen
Receptor
Transcriptional
Activation
Assay
(
no
metabolism)
6.
Androgen
Receptor
Transcriptional
Activation
Assay
(
metabolism)
7.
Thyroid
Receptor
Transcriptional
Activation
Assay
(
no
metabolism)
8.
Thyroid
Receptor
Transcriptional
Activation
Assay
(
metabolism)

D.
Limitations
of
the
Assays
to
be
Conducted
During
HTPS
There
are
two
noteworthy
limitations
to
the
types
of
assays
being
considered
for
the
HTPS
step.
First,
these
assays
cover
only
one
of
the
possible
mechanisms
of
action
for
endocrine­
mediated
toxic
effects.
At
present,
this
includes
biological
activity
resulting
directly
from
the
binding
of
a
chemical
to
the
hormone
receptor.
On
the
other
hand,
existing
data
on
thyroid­
active
substances
(
other
than
the
natural
ligand)
have
not
shown
that
thyroid
receptor
binding/
activation
is
a
key
component
of
the
mode(
s)
or
mechanism(
s)
of
action
by
which
that
substance
exerts
its
thyroidrelated
effects.
Nonetheless,
the
HTPS
will
include
the
thyroid
receptor
transcriptional
activation
assays.
This
will
be
done
for
two
reasons:
(
1)
to
do
so
will
constitute
only
a
minor
increase
in
cost
and
effort;
and,
perhaps
more
importantly,
(
2)
to
confirm
or
refute
the
current
hypotheses.
If
the
results
of
HTPS
show
that
thyroid
receptor
binding/
activation
is
a
key
component
of
the
mechanism(
s)
of
action,
then
thyroid
receptor
assays
would
be
included
in
the
basic
T1S
battery.

Assays
that
assess
the
activity
of
enzymes
involved
in
hormone
synthesis
are
technically
possible
to
conduct
using
high
throughput
technologies
but
are
not
being
recommended
for
inclusion
in
HTPS
by
the
Committee.
Despite
this
limitation,
there
are
good
scientific
reasons
to
believe
that
most
androgen­
and
estrogen­
mediated
toxicants
capable
of
eliciting
adverse
effects
at
low
doses
do
so
by
binding
to
a
receptor.
Therefore,
the
overarching
goal
of
protecting
human
and
ecological
health
is
likely
to
be
served
by
evaluating
this
mechanism
early
in
the
EDSTP.

The
second
significant
limitation
of
the
assays
being
considered
for
use
in
the
HTPS
step
of
the
process
is
that
they
are
unlikely
to
produce
the
same
spectrum
of
metabolites
that
an
intact
animal
produces.
That
is,
chemicals
that
need
to
be
metabolized
in
order
to
be
active
may
not
be
detected
by
HTPS.
Again,
this
limitation
will
be
addressed
in
the
screening
tier.
Both
of
these
limitations
will
also
need
to
be
considered
in
the
interpretation
and
utilization
of
the
results
of
HTPS
for
purposes
of
priority
setting.

E.
Technical
and
Logistical
Issues
Estimates
of
the
speed
of
using
high
throughput
technology
are
encouraging.
Once
the
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
32
preliminary
collection
and
handling
of
the
chemicals
are
completed,
it
is
not
out
of
the
question
for
several
thousand
assays
to
be
run
in
one
month,
depending
on
whether
confirmatory
assays
are
also
run
(
Christopher
Waller,
OSI
Pharmaceuticals,
Inc.,
personal
communication).
However,
there
are
technical
and
logistical
constraints,
as
well
as
policy
issues,
that
will
need
to
be
addressed
in
determining
the
number
of
chemicals
that
can
or
should
be
subjected
to
HTPS.

With
regard
to
the
technical
constraints,
some
compounds
have
physical
and/
or
chemical
characteristics
such
as
insolubility,
high
volatility,
and
high
reactivity
that
are
not
amenable
to
any
in
vitro
screening
system.
There
are,
however,
scientific
reasons
to
assume
that
highly
insoluble
and
highly
reactive
chemicals
are
unlikely
to
be
endocrine
disruptors.

There
are
also
some
significant,
but
not
insurmountable,
logistical
hurdles
to
be
overcome.
One
so­
called
hurdle
includes
validation
of
the
assays
for
the
significantly
diverse
kinds
of
chemicals
that
will
be
subjected
to
HTPS.
While
it
is
intended,
and
expected,
that
HTPS
will
provide
some
false
positives
while
minimizing
false
negatives,
there
is
currently
no
history
of
use
for
HTPS
methodology
to
evaluate
large
numbers
of
diverse
chemical
substances
for
potential
endocrinemediated
effects.
Until
now,
HTPS
endocrine
assays
have
been
used
mainly
as
a
tool
to
identify
new
leads
or
to
assess
biological
activity
of
an
existing
lead.
The
possibility
exists
that
HTPS
may
not
provide
sufficient
effects
data
to
warrant
continued
use,
or
that
it
may
result
in
an
unacceptable
number
of
false
negatives.
However,
all
screens,
whether
automated
or
not,
must
undergo
the
process
of
validation.

In
addition
to
validation,
the
problem
of
obtaining
the
chemicals
must
be
overcome
before
HTPS
can
be
implemented.
This
process
involves
not
only
collection
but
quality
assurance
of
the
collected
samples.
Some
chemicals
in
the
environment
(
e.
g.,
NONEs)
are
simply
not
commercially
available.
Moreover,
since
there
is
no
registrant
or
chemical
manufacturer
for
such
"
orphan"
chemical
substances,
the
ownership
and
responsibility
to
shepherd
them
through
the
screening
and
testing
processes
will
rest
with
EPA
or
other
government
agencies.
Obviously,
if
chemical
substances
cannot
be
procured
they
must
either
be
isolated
or
synthesized
in
order
to
be
screened
and,
if
necessary,
tested.
At
this
time,
the
EDSTAC
is
not
aware
of
how
many
compounds
could
fall
into
this
category.

EPA
has
launched
a
feasibility
demonstration
effort
designed
to
ensure
that
the
types
of
assays
being
considered
for
HTPS
can
be
used
on
the
wide
range
of
chemicals
that
will
need
to
be
subjected
to
this
step
in
the
process.
For
more
information
on
the
HTPS
feasibility
demonstration
project,
see
Appendix
I.

F.
Which
Chemicals
Should
be
Subjected
to
HTPS?

Although
the
use
of
robotic
technology
will
greatly
expand
the
throughput
of
chemicals
over
a
given
period
of
time
for
the
selected
assays,
the
EDSTAC
is
not
recommending
that
all
chemicals
needing
to
be
prioritized
for
T1S
be
subjected
to
HTPS.
Rather,
the
EDSTAC
recommends
that
the
set
of
chemicals
currently
produced
in
an
amount
equal
to
or
greater
than
10,000
pounds
per
year
(
estimated
to
be
about
15,000
chemicals)
should
be
subjected
to
HTPS.
Also,
it
is
expected
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
33
that
all
pesticides
(
i.
e.,
all
pesticide
active
ingredients
and
formulation
inerts)
will
be
subjected
to
HTPS.
As
indicated
earlier
the
EDSTAC
makes
this
recommendation
to
help
EPA
avoid
an
unachievable
task
that
might
never
be
completed.
Given
the
history
of
TSCA,
the
EDSTAC
believes
that
15,000
chemicals
is
not
an
insignificant
number.

The
EDSTAC
recommends
that
chemicals
determined
to
be
a
high
priority
for
T1S
that
do
not
undergo
the
HTPS
assays
because
they
are
produced
in
amounts
less
than
10,000
pounds
per
year
should
still
undergo
the
transcriptional
activation
assays
contained
in
HTPS.
However,
rather
than
using
the
HTPS
process,
these
lower
production
volume
chemicals
should
undergo
transcriptional
activation
assays
on
the
bench
as
part
of
T1S.

The
EDSTAC
also
recommends
that
chemicals
permitted
to
bypass
T1S
and
go
directly
to
T2T,
as
well
as
those
permitted
to
bypass
both
T1S
and
T2T
and
go
directly
to
hazard
assessment
(
due
to
functional
equivalency
of
data),
be
subjected
to
HTPS.
However,
as
described
more
fully
below,
the
results
of
HTPS
from
these
chemicals
would
not
be
used
to
set
priorities
for
T1S.

G.
How
Will
HTPS
Results
be
Used?

1.
For
Chemicals
That
Will
be
Prioritized
for
T1S
In
the
context
of
setting
priorities
for
T1S,
the
EDSTAC
recommends
that
EPA
use
the
results
of
HTPS
in
conjunction
with
other
exposure­
and
effects­
related
priority
setting
information.
In
other
words,
HTPS
results
should
be
considered
along
with
any
other
biological
effects
information
that
may
be
available,
as
well
as
information
on
exposure­
related
considerations
(
e.
g.,
biological
sampling;
environmental,
occupation,
consumer
product,
and
food­
related
data;
releases
to
the
environment;
production
volumes;
and
fate
and
transport
models
and
data).

The
HTPS
results,
by
themselves,
cannot
be
regarded
as
definitively
proving
or
disproving
endocrine­
mediated
toxicity
in
whole
animals.
Such
determinations
can
only
be
made
with
confidence
at
the
end
of
the
entire
screening
and
testing
process.
There
is
concern
that
the
results
of
HTPS
will
be
over­
interpreted
because
they
may
be
the
first
data
that
will
be
generated
in
the
endocrine
disruptor
evaluation
process.
Therefore,
it
is
important
to
stress
the
limitations
of
these
assays.
Most
importantly,
the
HTPS
assays
are
very
simple
in
vitro
assays.
Like
any
in
vitro
method,
the
simplicity
that
makes
the
assays
attractive
for
rapid
generation
of
data
also
limits
their
reliability
as
predictors
of
what
might
occur
in
the
intact
organism.
They
do
not
possess
all
of
the
complexities
of
pharmacokinetics,
pharmacodynamics,
metabolism,
and
multi­
system
interactions
that
are
inherent
in
the
whole
organism.
It
is
rare
for
an
in
vitro
assay
for
any
toxicity
to
have
better
than
an
80%
concordance
with
in
vivo
results.
For
this
reason,
most
in
vitro
assays
are
used
only
as
a
preliminary
step
of
a
more
comprehensive
assessment.

HTPS
is
primarily
useful
as
a
pre­
screen
to
indicate
the
need
for
further
evaluation,
but
will
not
always
predict
toxicity
in
whole
animals.
The
HTPS
results,
coupled
with
data
from
the
remainder
of
the
screening
and
testing
program
will
be
useful
in
interpreting
whether
a
chemical
evokes
endocrine­
mediated
responses.
For
these
reasons,
the
EDSTAC
strongly
recommends
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
34
that
a
negative
HTPS
result
not
be
used
as
a
basis
for
placing
a
chemical
into
the
"
hold
box."
Further,
the
Committee
recommends
that
a
negative
HTPS
result
not
be
used,
in
isolation,
to
decrease
the
priority
of
a
chemical
for
screening
and
testing;
nor
should
a
positive
HTPS
result
be
the
only
factor
considered
in
setting
priorities
for
T1S.

2.
For
Chemicals
That
Meet
the
Criteria
for
Going
Directly
to
T2T
or
Hazard
Assessment
Chemicals
that
meet
the
criteria
for
proceeding
directly
to
T2T
or
hazard
assessment
would
also
be
subjected
to
HTPS
according
to
the
EDSTP.
However,
unlike
the
large
number
of
chemicals
that
do
not
meet
these
criteria,
the
results
of
HTPS
from
this
set
of
chemicals
will
not
be
used
to
help
set
priorities
for
T1S.

There
are
several
generic
reasons
why
the
EDSTAC
recommends
conducting
HTPS
assays
on
food­
use
pesticides
and
other
chemicals
that
have
previously
been
subjected
to
two­
generation
reproductive
toxicity
tests.
These
generic
reasons
include:
(
1)
the
data
generated
from
the
HTPS
assays
will
be
valuable
in
and
of
themselves,
even
though
they
are
limited
to
a
single
mechanism
of
action
and
cannot
be
used
by
themselves
to
determine
whether
a
chemical
is
or
is
not
an
endocrine
disruptor;
(
2)
as
an
ancillary
benefit,
the
data
can
be
used
to
improve
and
validate
QSARs;
and
(
3)
beyond
these
generic
benefits,
in
the
case
of
food­
use
pesticides
that
will
complete
tolerance
reassessments
prior
to
the
availability
of
validated
Tier
2
tests,
HTPS
data
can
be
used,
along
with
other
relevant
information,
to
help
prioritize
whether
and,
if
so,
when
these
chemicals
should
be
subjected
to
any
additional
endocrine
disruptor
testing.
The
last
rationale
for
recommending
food­
use
pesticides
complete
HTPS
assays
is
further
elaborated
upon
in
Chapter
Four,
Section
XI,
H.

There
may
be
concern
that
it
is
redundant
to
subject
pesticides
and
other
chemicals
to
HTPS
for
which
substantial
two­
generation
reproductive
and
developmental
(
whole
animal)
toxicity
testing
data
already
exist.
However,
the
EDSTAC
believes
the
value
of
generating
HTPS
data
outweighs
the
relatively
low
cost
associated
with
subjecting
these
chemicals
to
HTPS.

3.
To
Improve
QSARs
The
EDSTAC
recommends
that
existing
QSAR
models
be
rederived
and
supplemented
with
data
from
the
HTPS
assays,
thereby
expanding
the
predictive
ability
of
these
models.
Thus,
when
it
comes
time
to
set
priorities
for
the
first
phase
of
T1S,
HTPS
data,
as
well
as
improved
QSARs,
should
be
used
along
with
other
relevant
exposure
and
effects
data.
It
is
envisioned
that
the
process
of
QSAR
model
expansion
and
improvement
will
then
continue
in
a
cyclical
feedback
manner,
thus
providing
the
opportunity
to
validate
evolving
QSAR
models
using
external
data
sets
for
screens
and
tests
of
compounds
not
subjected
to
HTPS.
Eventually,
predictions
of
endocrine
disruption
potential
obtained
from
validated
QSAR
models
could
be
used
as
surrogates
for
HTPS
data
in
the
case
of
compounds
for
which
effects
data
are
not
available.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
35
H.
Practical
Considerations
and
Constraints
to
Be
Considered
in
HTPS
Implementation
There
is
widespread
agreement
that
several
practical
considerations
will
need
to
be
addressed
for
HTPS
to
work
as
intended.
These
include:

·
Demonstrating
the
feasibility
of
HTPS
 
An
important
first
step
in
implementing
the
recommendation
of
the
EDSTAC
to
incorporate
the
use
of
HTPS
into
the
EDSTP
is
to
undertake
an
effort
to
demonstrate
the
feasibility
of
using
this
technology
for
the
wide
range
of
chemicals
that
will
need
to
be
considered
for
endocrine
disruptor
screening
and
testing.
A
detailed
discussion
of
the
feasibility
demonstration
effort
that
is
already
underway
is
contained
in
Appendix
I.
·
Collecting,
handling,
and
QA/
QC
of
the
chemicals
to
be
tested
 
The
procurement
of
sufficient
quantities
of
relevant
chemical
substances,
the
shipment
of
these
materials,
and
the
assurance
of
the
chemical
identity
and
purity
of
these
chemicals
will
be
the
most
time
consuming
phase
of
HTPS.
While
these
issues
are
inherent
in
any
of
the
assays
being
considered
for
screening
or
testing,
they
must
be
taken
into
consideration
when
planning
for
HTPS
as
they
are
likely
to
contribute
to
the
cost
and
time
for
this
step
of
the
program.
The
EDSTAC
recommends
that
EPA
explore
the
feasibility
of
creating
an
archive
of
a
subset
of
these
chemicals,
which
can
be
accessed
by
researchers
interested
in
studying
endocrinemediated
toxicity
or
in
validating
new
screens
for
endocrine
disruptors.
This
may
be
particularly
important
for
radio­
labeled
compounds
that
are
costly
to
synthesize.
There
is
precedence
for
such
activities,
including
the
EPA
Pesticide
Repository,
the
National
Institute
of
Standards
and
Technology
(
formerly
the
Bureau
of
Standards)
in
the
Department
of
Commerce,
and
the
NTP
chemical
repository
for
the
validation
of
in
vitro
developmental
toxicity.
·
Patent
issues
 
Many
or
all
of
the
HTPS
assays
under
consideration
are
patented
and
the
intellectual
property
issue
must
be
addressed
before
implementing
any
endocrine
disruptor
screening
and
testing
program.
This
is
unlikely
to
be
a
critical
issue
for
a
massive
screening
effort
because
it
is
almost
certain
that
such
work
would
be
done
under
contract
by
the
holder
of
the
patent.
It
may,
however,
be
a
significant
issue
for
individual
investigators
or
companies
who
wish
to
work
with
the
assays
on
an
investigative
basis
in
their
own
laboratories.
Licensing
agreements
should
be
worked
out
before
any
final
decisions
are
made.
·
Overall
costs
and
specific
cost
factors
 
As
with
all
screening
assays,
the
cost
of
performing
an
assay
needs
to
be
taken
into
account
in
selecting
which
HTPS
assays
to
recommend,
as
high
cost
may
limit
the
number
of
chemicals
that
can
be
evaluated.
·
Validation
of
the
HTPS
assays
for
the
wide
range
of
chemicals
that
are
intended
for
prescreening
 
High
throughput
screening
technology
has
been
used
in
the
pharmaceutical
and
agrochemical
industries
to
find
chemicals
with
novel
and
relevant
biological
activity
at
high
potency,
as
these
are
the
ones
that
are
likely
to
be
candidates
for
lead
optimization.
However,
environmental
chemicals
that
have
been
identified
so
far
as
having
endocrine­
mediated
effects
typically
have
low
potency.
Issues
such
as
how
good
the
assays
will
be
at
detecting
such
chemicals;
the
limit
of
detection;
and
how
easily
these
assays
will
accommodate
a
range
of
chemical
properties
(
such
as
solubility,
pH,
and
high
vapor
pressure)
can
be
addressed,
but
doing
so
may
require
some
research
involving
a
representative
group
of
chemicals
before
HTPS
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
36
can
be
implemented
on
a
large
scale.
Based
on
the
results
of
using
HTPS
as
a
tool
for
identifying
discovery
leads,
one
generally
should
expect
a
"
hit
rate"
of
1.8­
2.0%
for
a
very
weak
lead
(
activity
at
100
uM),
0.6%
for
a
weak
lead
(
activity
at
10
uM),
and
0.15%
for
an
average
lead
(
1
uM)
(
Christopher
Waller,
OSI
Pharmaceuticals,
Inc.,
personal
communication).

Other
implementation
issues,
such
as
who
will
be
responsible
for
conducting
various
parts
of
the
HTPS
process,
how
much
each
step
will
cost,
etc.,
are
not
addressed
in
this
document.
However,
it
is
envisioned
that
EPA
will
undertake
the
coordination
(
and,
perhaps,
expense)
of
conducting
the
HTPS
step
of
the
program.
It
is
also
assumed
that
implementing
the
HTPS
process
will
require
EPA
to
work
cooperatively
with
industry
to
collect
what
will
be
a
very
large
number
of
chemical
samples.
Moreover,
the
issue
of
"
orphan
chemicals"
 
those
for
which
there
is
no
current
manufacturer
or
registrant
 
is
an
issue
that
EPA
must
address.

VI.
Recommendations
for
Handling
Polymers
A.
Introduction
This
section
presents
some
key
issues
associated
with
the
prioritization
of
polymers
for
endocrine
disruptor
screening
and
testing
along
with
several
options
and
a
recommended
approach
for
how
polymers
should
be
treated.

1.
Chemical
Nature
of
Polymers
Polymers
are
defined
in
40
Code
of
Federal
Regulations
(
CFR)
Part
723
as
a
chemical
substance
consisting
of
one
or
more
types
of
monomer
units
and
comprising
a
simple
weight
majority
of
molecules
containing
at
least
three
monomer
units
which
are
covalently
bound
to
at
least
one
other
monomer
unit
or
other
reactant
and
which
consists
of
less
than
a
simple
weight
majority
of
molecules
of
the
same
molecular
weight.
Such
molecules
must
be
distributed
over
a
range
of
molecular
weights
wherein
differences
in
the
molecular
weight
are
primarily
attributable
to
differences
in
the
number
of
monomer
units.

Polymers
result
from
chemical
reactions
that
permit
varying
numbers
of
monomers
or
monomer
units
and
other
precursors
to
be
chemically
incorporated
into
the
products
of
the
reactions.
According
to
40
CFR
Part
723,
the
term
"
monomer
unit"
means
"
the
reacted
form
of
the
monomer
in
a
polymer."
That
is,
the
monomer
must
have
formed
at
least
one
covalent
bond
with
another
like
or
unlike
molecule
under
the
conditions
of
the
relevant
polymerforming
reaction.

Polymer
molecules
typically
vary
in
their
degree
of
polymerization,
or
the
extent
to
which
they
have
incorporated
varying
numbers
of
monomers,
oligomers,
and
other
precursors.
However,
polymer
products
might
be
composed
of
various
other
substances
that
usually
are
not
the
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
37
result
of
the
polymerization
reaction
including:

·
Residuals
 
unreacted
polymer
precursors,
monomers,
oligomers,
and
other
reactants
·
Byproducts
 
catalyst
residues,
free­
radical
initiator
byproducts,
etc.
·
Impurities
 
precursor
impurities,
oxidation
products,
etc.
·
Other
substances
 
those
that
are
mixed
into
the
product,
such
as
fire
retardants,
plasticizers,
solvents,
inhibitors,
fillers,
colorants,
antioxidants,
slip
agents,
etc.

2.
Present
Regulatory
Status
of
Polymers
The
initial
TSCA
Inventory
(
or
Initial
Inventory),
published
in
1979,
consisted
of
those
chemicals
that
were
manufactured
in
the
U.
S.
or
imported
into
the
U.
S.
on
or
after
January
1,
1975,
and
before
the
end
of
the
initial
reporting
period
(
which
varied
depending
on
the
chemical
and/
or
company
circumstances).
Certain
allowances
were
made
for
later
additions
and
corrections.
The
Initial
Inventory
contained
about
60,000
chemicals,
approximately
half
of
which
were
polymers.
Chemicals
on
the
Initial
Inventory
are
referred
to
as
"
existing
chemicals."
Chemicals
not
on
the
Initial
Inventory
are
considered
"
new"
and
are
subject
to
the
Pre­
Manufacture
Notification
(
PMN)
requirements
of
TSCA.
After
EPA
completes
the
pre­
manufacture
review
of
a
new
chemical
and
when
the
manufacturer
or
importer
of
the
chemical
notifies
the
Agency
that
manufacture
or
importation
has
commenced,
EPA
adds
the
new
chemical
to
the
Inventory.

The
existing
chemical
polymers
are
described
in
the
Initial
Inventory
using
a
simplified
procedure
for
naming
the
polymers.
Polymers
on
the
Initial
Inventory
are
named
as
"
Polymer
of
A,
B,
C,
D..."
where
A,
B,
C,
D...
are
the
monomers
which
are
reacted
to
form
the
polymer.
The
Inventory
chemical
name
does
not
include
any
description
of
the
chemical
identity
of
the
specific
polymer
or
polymers
that
are
made
from
these
monomers.
For
example,
there
is
no
information
about
whether
the
polymer
is
in
the
form
of
a
carbamate,
amide,
isocyanate,
or
some
combination
of
these
functional
groups;
nor
is
there
any
mention
of
the
presence
or
absence
of
reactive
functional
groups,
such
as
isocyanate
or
epoxy
groups.
In
addition,
the
number
average
molecular
weight
(
NAMW)
 
which
refers
to
the
arithmetic
average
(
mean)
of
the
molecular
weight
of
all
molecules
in
the
polymer
 
distribution
of
the
polymer
or
polymers
made
from
the
listed
monomers
is
not
reported.
The
Initial
Inventory,
however,
does
include
a
number
of
low
NAMW
oligomers
(
dimers,
trimers,
etc.)
which
are
purposefully
manufactured
as
such.

In
contrast
to
the
estimated
30,000
polymers
reported
on
the
Initial
Inventory
(
John
Walker,
U.
S.
EPA,
personal
communication),
new
chemical
polymers
that
are
reported
to
EPA
include
a
chemical
description
of
the
polymer
containing
information
on
the
NAMW
distribution,
the
presence
of
reactive
functional
groups,
etc.
In
addition,
EPA
receives
information
on
the
anticipated
uses,
exposures
(
occupational,
environmental,
consumer,
etc.),
and
environmental
releases
of
the
new
polymers.

The
EPA,
under
TSCA,
first
proposed
the
exemption
of
certain
polymers
(
NAMW
greater
than
20,000
daltons)
from
PMN
in
1982
(
47
Federal
Register
(
FR)).
The
Final
Rule
for
this
early
exemption
was
published
in
1984.
In
making
its
no­
risk
finding,
EPA
concluded
with
regard
to
polymers
that:
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
38
Molecular
weight
is
clearly
the
prime
determinant
of
risk.
For
a
chemical
to
elicit
a
toxic
response
within
an
organism,
it
must
come
into
direct
contact
with
the
biological
cells
from
which
it
elicits
the
response.
Because
all
organisms
are
encased
in
protective
membranes,
a
chemical
must
penetrate
these
membranes
and
be
translocated
to
various
parts
of
the
organism
to
gain
access
to
its
target
sites.
If
a
chemical
cannot
penetrate
the
protective
membranes
to
access
a
target
site,
and
it
cannot
elicit
a
toxic
response,
it
will
not
generally
present
a
risk.
(
49
FR
46081,
also
cited
in
60
FR
16328)

EPA
operated
with
this
exemption
for
almost
a
decade
until
a
proposal
to
expand
the
exemption
was
made
in
1993
(
58
FR).
That
proposal
was
published
as
a
final
rule
in
1995,
and
it
sets
out
the
exemption
policy
under
which
the
TSCA
program
now
operates
(
60
FR).
EPA
extensively
reviewed
over
10,000
polymers
from
1980
to
1995
and
concluded
that:

Of
these
10,000,
the
polymers
that
would
have
qualified
under
the
final
polymer
exemption
rule
[
1995]
have
consistently
been
characterized
as
posing
low
concern
for
both
adverse
health
and
environmental
risks
by
the
Agency
during
the
course
of
PMN
review.
The
characteristics
of
a
significant
number
of
polymers
(
i.
e.,
their
NAMW
and/
or
physical/
chemical
properties)
are
such
that
they
are
neither
absorbed
by
biological
systems
nor
do
they
interact
with
biological
systems,
as
described
above.
(
60
FR
16329)

As
required
by
section
5(
h)(
4)
of
TSCA,
the
current
polymer
exemption
is
based
wholly
on
a
finding
by
the
EPA
that
the:

manufacture,
processing,
distribution
in
commerce,
use,
and
disposal
of
new
chemical
substances
meeting
the
revised
polymer
exemption
criteria
will
not
present
an
unreasonable
risk
of
injury
to
human
health
or
the
environment
under
the
terms
of
the
exemption.
(
60
FR
16316)
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
39
The
present
regulation
(
60
FR
16333)
exempts:

·
polymers
with
average
NAMW
between
1,000
and
10,000
daltons
if
they
do
not
contain
other
than
certain
specified
reactive
functional
groups
(
as
well
as
containing
less
than
10%
oligomers
with
NAMW
less
than
500
daltons
and
less
than
25%
oligomers
with
NAMW
less
than
1,000
daltons);
·
polymers
with
average
NAMW
greater
than
10,000
daltons
(
and
less
than
2%
oligomers
with
NAMW
less
than
500
daltons
and
less
than
5%
oligomers
with
NAMW
less
than
1,000
daltons);
·
polyester
polymers
made
with
any
of
a
long
list
of
specified
reactants;
and
·
polymers
produced
in
quantities
less
than
10,000
kilograms
per
year.

Polymers
that
are
ineligible
for
the
exemption
include:

·
polymers
that
degrade,
decompose,
or
depolymerize;
·
polymers
that
are
prepared
from
monomers
or
other
reactants
that
are
not
on
the
TSCA
Inventory;
and
·
water­
absorbing
polymers
with
NAMW
greater
than
or
equal
to
10,000
daltons.

B.
Key
Issues
Associated
With
the
Prioritization
of
Polymers
The
PSWG
originally
considered
exempting
polymers
with
a
NAMW
greater
than
1,000
daltons
from
priority
setting,
similar
to
the
reporting
exemption
which
exists
for
new
chemical
polymers
under
TSCA.
All
polymers
with
a
NAMW
less
than
1,000
daltons
would
be
treated
like
all
other
chemicals
and
would
be
subjected
to
priority
setting.
The
exempt
polymers
would
be
put
into
a
"
hold
box"
pending
information
on
monomers
or
other
low
NAMW
polymers
of
potential
concern
and
screening
and
testing
of
the
monomers
themselves.
However,
concerns
surfaced
within
the
PSWG
upon
further
examination.

Bioaccumulation/
Potential
Exposure
The
original
proposal
to
exempt
polymers
was
based
on
an
assumption
that
molecules
larger
than
1,000
daltons
would
not
cross
biological
membranes
and
barriers.
If
a
neonate
is
orally
exposed
to
a
polymer
with
a
NAMW
greater
than
1,000
daltons,
some
of
the
polymer
could
enter
the
body
and
interact
with
cells.
Such
an
interaction
is
unlikely
to
occur
in
a
more
mature
animal
but
could
occur
in
neonates
due
to
delayed
intestinal
closure.
Gastrointestinal
absorption
is
dependent
on
factors
such
as
lipophilicity,
molecular
weight,
particle
size,
and
metabolism
of
chemicals
in
the
gastrointestinal
tract
(
Baintner,
1986;
Kleinman
and
Walker,
1984;
Lecce
and
Broughton,
1973;
Walker,
1978;
Westrom
and
Tagesson,
1989;
Westrom,
Svendsen,
and
Tagesson,
1984;
Weaver,
Laker,
et
al.,
1987;
Westrom,
Tagesson,
et
al.,
1989).

The
potential
for
gastrointestinal
absorption
of
high
molecular
weight
substances
was
taken
into
consideration
by
the
EPA
as
early
as
1982.
The
Agency
concluded
that:
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
40
·
Substances
with
NAMW
greater
than
1,000
daltons
are
generally
not
readily
absorbed
through
the
intact
gastrointestinal
tract.
(
49
FR
46081);
·
"
For
a
chemical
to
elicit
a
toxic
response
within
an
organism,
it
must
come
into
direct
contact
with
the
biological
cells
from
which
it
elicits
the
response.
Because
all
organisms
are
encased
in
protective
membranes,
a
chemical
must
penetrate
these
membranes
and
be
translocated
to
various
parts
of
the
organism
to
gain
access
to
its
target
sites.
If
a
chemical
cannot
penetrate
the
protective
membranes
to
access
a
target
site,
and
it
cannot
elicit
a
toxic
response,
it
will
not
generally
present
a
risk."
(
49
FR
46081,
also
cited
in
60
FR
16328)
·
Dermal
exposure,
rather
than
inhalation
or
ingestion,
is
the
major
route
of
exposure
for
most
polymers.
(
47
FR
33930)

Based
on
the
available
data,
EPA
was
able
to
proceed
in
making
its
no­
risk
finding
as
a
basis
for
the
polymer
exemption.
The
physical
properties
of
a
polymer
affect
not
only
its
functional
ability,
but
its
fate
and
transport
in
the
environment.
Generally,
as
the
molecular
weight
and
degree
of
polymerization
increase,
the
affinity
for
adsorption
to
solids
(
soil
and
sediment)
increases
and
the
potential
for
biodegradation
and
bioaccumulation
decreases.

Polymer
Complexity
Polymers
are
complex
substances
consisting
of
additives
such
as
fire
retardants,
antioxidants,
slip
agents,
colorants,
residual
monomers,
catalysts,
additive
reaction
products,
catalyst
residues
and
reaction
products,
byproducts,
low
molecular
weight
polymer
chains,
etc.
Although
additives,
monomers,
catalysts,
many
oligomers,
and
many
other
substances
will
be
included
in
the
priority
setting
scheme,
some
of
the
other
polymer
components
may
not
be.
Concern
about
their
toxicity
arose
on
the
part
of
the
PSWG
after
reviewing
some
work
done
in
the
early
1970'
s
on
a
complex
polymer
fluid
showing
that
the
fluid's
polysiloxane
dimers
and
trimers
were
more
toxic
components
of
the
mixture
than
were
the
monomers.
Although
the
issue
was
not
one
of
incomplete
breakdown
products,
but
rather
of
intentionally
made
dimers
and
trimers,
the
work
highlighted
the
fact
that
by
only
studying
the
monomer,
it
is
conceivable
that
one
might
miss
a
higher
order
of
toxicity
reached
in
the
dimers,
trimers,
etc.
The
toxicity
of
these
other
polymer
components
may
not
be
the
same
as
the
toxicity
of
the
monomers.
Thus,
testing
data
from
the
monomers
and
additives
may
not
provide
complete
guidance
as
to
the
toxicity
of
the
entire
polymer.

Composition
of
Copolymers
Most
polymers,
for
regulatory
purposes,
are
described
and
assigned
Chemical
Abstracts
Service
Registry
Numbers
(
CASRNs)
on
the
basis
of
the
monomers
used
in
their
manufacture.
For
polymers
having
multiple
monomers
(
or
copolymers,
as
opposed
to
homopolymers),
the
relative
concentrations
of
the
various
monomers
can
vary
widely,
but
the
polymers
can
still
be
assigned
the
same
CASRN.
For
example,
poly(
A/
B)
with
an
A/
B
ratio
of
95/
5
or
5/
95
is
still
described
by
the
same
CASRN.
Consequently,
for
purposes
of
prioritizing
polymers
for
testing,
the
CASRN
does
not
represent
a
unique
chemical
composition.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
41
In
addition,
for
many
condensation
polymers,
chemically
identical
polymers
can
be
made
from
slightly
different
monomers.
In
this
instance,
the
CASRN
would
be
different,
even
though
the
polymers
are
compositionally
indistinguishable.

The
variable
compositions
within
a
CASRN
listing,
and
identical
compositions
for
different
CASRN
listings,
are
problematic
for
both
new
polymers,
as
well
as
for
those
nominated
to
the
original
Inventory.

Testing
of
Polymers
Many
polymer
components
do
not
have
an
identity
apart
from
their
role
as
a
component
of
a
polymer.
Hence,
they
do
not
exist
independently
and,
in
general,
cannot
be
readily
synthesized
or
purified
for
screening
and
testing.
If
such
components
were
to
be
tested,
they
would
have
to
be
extracted
from
the
polymer
matrix
in
which
they
exist.
Such
an
extraction
would
be
a
highly
complex
undertaking,
requiring
the
identification
of
a
long
list
of
parameters
such
as:

·
solvent
for
the
extraction;
·
time
for
the
extraction;
·
temperature
for
the
extraction;
·
surface
area
of
the
polymer
to
be
extracted;
and
·
volume
of
the
extracting
solvent.

Varying
any
of
these
parameters
would
affect
which
polymer
components
are
extracted
and
how
much
of
any
component
is
extracted.
Variation
in
these
parameters
can
reflect
different
use
conditions
of
the
polymer,
different
potential
exposure
conditions,
different
properties
of
the
polymer,
and
different
components
which
one
desires
to
extract.
In
addition,
any
extraction
would
yield
an
extract
which
is
a
mixture
consisting
of
the
polymer
components,
primarily
the
smaller
monomer
and
additive
compounds.
Thus,
any
test
will
yield
a
result
which
does
not
describe
the
endocrine
behavior
of
the
polymer
components
by
themselves.
Further,
since
the
conditions
of
the
extraction
determine
the
composition
of
the
extract
and
the
concentration
of
the
components
in
the
extract,
the
test
may
not
necessarily
yield
useful
information
regarding
the
potential
toxicity
of
the
polymer.

Once
the
polymer
components
are
extracted,
the
extract
may
need
to
be
concentrated,
for
example,
to
obtain
an
appropriate
concentration
for
testing
or
to
remove
extracting
solvents.
This
"
concentration"
step
must
be
very
carefully
conducted
so
as
to
ensure
that
no
part
of
the
extract
is
lost
or
altered.
In
most
cases,
validation
of
this
step
would
be
very
difficult.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
42
Migration
of
Polymer
Components
Two
types
of
components
are
of
interest:
(
1)
the
lower
molecular
weight
monomers
and
oligomers
that
may
be
present
in
the
matrix
of
high
NAMW
polymers;
and
(
2)
the
additives,
catalysts,
etc.
Because
essentially
all
of
these
components
are
on
the
TSCA
Inventory,
they
will
be
considered
individually
along
with
other
chemicals
during
prioritization
and
will
receive
due
consideration
for
screening
and
testing.

Degradation
Products
of
Polymers
The
EDSTAC
considered
the
issue
of
the
potential
for
polymers
to
degrade
in
the
environment
and
therefore
pose
risk
of
organismic
exposure
to
substances
which
would
not
be
captured
under
the
priority
setting
scheme.
Most
polymers
are
chemically
designed
to
be
used
in
applications
where
stability
is
essential
to
their
functional
and
commercial
success.
Although
most
polymers
would
not
be
expected
to
degrade
in
the
environment,
data
are
not
complete
for
all
polymer
classes.
However,
concern
about
the
ability
of
chemical
degradates
to
enter
the
environment,
especially
water,
is
not
limited
to
the
potential
degradates
of
polymers
alone,
but
includes
essentially
all
chemicals
which
are
released
to
the
environment.
The
EDSTAC
does
not
consider
it
necessary
to
give
special
consideration
to
the
potential
degradates
of
polymers.
These
issues
will
be
considered
for
polymers
as
well
as
other
chemicals
in
the
priority
setting
scheme
in
the
context
of
the
exposure
criteria.

C.
Options
Considered
by
the
PSWG
1.
Include
all
Polymers
(
Regardless
of
NAMW)
From
Priority
Setting
This
option
would
ensure,
in
theory,
that
no
molecules
are
overlooked
in
priority
setting.
The
polymers
would
be
subject
to
the
same
exposure­
and
effects­
related
criteria
as
are
the
smaller
molecules.
From
a
practical
standpoint,
however,
exposure
data
would
be
the
primary
driver
in
this
application,
and
such
data
would
be
hard
to
obtain
for
most
of
the
polymers.

For
most
polymers,
the
likelihood
is
small
when
humans
and
other
animals
come
in
contact
with
polymers
that
a
significant
bioavailable
dose
would
be
received.
Therefore,
the
public
health
value
of
including
all
polymers
in
the
prioritization
exercise
would
be
negligible.
This
needs
to
be
balanced
in
light
of
the
significant
resources
that
would
be
required
to
actually
characterize
the
polymeric
substance,
obtain
and
evaluate
the
available
exposure
and
effects
data,
and
make
a
prioritization
decision
for
thousands
of
polymers.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
43
2.
Include
Polymers
With
NAMW
Greater
Than
1,000
Daltons
to
Which
Neonates
are
Likely
to
be
Exposed;
Put
the
Others
in
Hold
Criteria
would
need
to
be
developed
to
identify
those
polymers
which
are
used
in
materials
most
likely
to
come
in
contact
with
neonates.
It
must
be
acknowledged
that
such
criteria
are
most
workable
for
humans
and
less
readily
ascertainable
for
fish
and
wildlife.
Examples
of
the
kinds
of
polymers
that
would
need
to
be
considered
relevant
to
human
neonates
include
those
used
in
food
contact
materials,
infant
toys,
etc.
A
significant
advantage
of
such
an
effort
over
option
number
1
would
be
to
focus
the
priority
setting
on
those
molecules
most
likely
to
present
a
potential
exposure
to
the
sensitive
population.
The
technical
difficulties
associated
with
screening
and
testing
polymers,
which
are
described
above,
would
still
remain.

3.
Hold
Polymers
With
NAMW
Greater
Than
1,000
Daltons
From
Priority
Setting
No
priority
setting
of
the
polymer
would
occur
unless
data
indicate
leachable
monomers
or
oligomers
have
endocrine
disruption
potential.
This
option
focuses
resources
on
the
polymers
that
contain
or
might
release
monomers
or
oligomers
of
concern.
This
still
entails
a
significant
technical
investment
to
determine
the
nature
and
amount
of
leachable
"
other
components"
from
the
polymer.
Priority
setting
would
initially
take
place
on
the
monomers
under
the
same
criteria
as
other
single
chemicals.

4.
Exempt
all
Polymers
With
NAMW
Greater
Than
1,000
Daltons;
Concentrate
on
Monomers
This
option
obviates
the
resource­
intensive
step
of
considering
the
"
other"
chemicals
present
in
a
typical
polymer
mixture.
Priority
setting
would
take
place
on
the
monomers
and
the
appropriate
ones
would
be
screened
and
tested.
This
is
the
least
resource­
intensive
option
(
at
the
priority
setting
stage,
at
least)
and
focuses
on
identification
of
monomers
of
concern.
Concerns
about
a
monomer's
use
in
a
polymer
arise
not
during
priority
setting
but
after
screening
and
testing
is
completed.
At
this
point,
the
results
of
screens
and
tests
of
the
monomer,
along
with
the
proper
dose­
response
analysis,
would
be
considered
with
exposure
assessment
(
including
use
and
migration
from
polymers)
to
assess
risk.
This
option
would
still
require
detailed
consideration
of
polymers,
but
at
a
later
stage
in
the
program
and
only
for
polymers
where
screening
and
testing
of
the
monomer
and
other
components
indicate
a
concern.

5.
Modified
Option
4
 
Treat
Polymers
as
Mixtures
and
Consider
Them
Along
With
Other
Mixtures
The
issues
that
complicate
the
consideration
of
polymers
are
similar,
if
not
identical,
to
those
faced
by
mixtures
in
general.
These
include
often
broadly
defined
composition,
wide
range
of
chemicals
present
in
one
CASRN
(
chemical
nature
and
NAMWs),
etc.
By
considering
polymers
along
with
mixtures,
the
consideration
of
exposure­
and
effects­
related
criteria
would
be
similar
for
both.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
44
D.
Recommendation
for
Handling
Polymers
The
EDSTAC
prefers
option
number
4.
In
particular,
the
EDSTAC
recommends
that
existing
and
new
chemical
monomers
and
oligomers,
as
well
as
new
chemical
polymers
with
a
NAMW
of
less
than
1,000
daltons,
should
be
considered
within
the
broader
priority
setting
scheme
and
undergo
screening
and
testing
as
appropriate.
The
priority
setting
scheme
will
consider
the
potential
for
sensitive
populations
to
be
exposed
(
e.
g.,
the
exposure
of
neonates).
Existing
chemical
polymers,
regardless
of
NAMW,
are
viewed
as
presenting
a
lower
priority
for
initial
action
because
of
the
unavailability
of
critical
information
such
as
NAMW
and
explicit
information
about
the
chemical
nature
of
the
polymer.
In
addition,
many
of
the
existing
polymers
are
very
large
molecules
(
NAMW
greater
than
50,000
daltons).
As
such,
potential
exposure
to
residual
monomers
and
low
molecular
weight
oligomers
contained
in
existing
polymers
is
limited.

Thus,
the
EDSTAC
recommends:

1.
All
monomer
and
oligomer
components
of
polymers
should
be
prioritized
for
and
subjected
to
endocrine
disruptor
screening
and
testing.

2.
All
"
new"
polymers
(
i.
e.,
those
produced
after
the
Initial
TSCA
Inventory,
which
was
published
in
1979)
with
number
average
molecular
weight
(
NAMW)
less
than
1,000
daltons
should
also
be
prioritized
for
and
subjected
to
endocrine
disruptor
screening
and
testing.
Throughout
this
document,
the
term
"
number
average
molecular
weight,"
or
"
NAMW,"
of
polymers
is
utilized.
This
term
indicates
a
numerical
mean,
with
the
actual
MW
of
the
polymers
ranging
about
this
mean.
The
EDSTAC
recommends
embracing
the
language
in
the
1995
Final
TSCA
Polymer
Rule
(
60
FR
16333)
which
uses
a
NAMW
cutoff
of
1,000
daltons,
provided
that
the
polymer
does
not
contain
other
than
certain
specified
reactive
functional
groups
and
that
the
polymer
contains
less
than
10%
oligomers
with
MW
less
than
500
daltons
and
less
than
25%
oligomers
with
a
MW
of
less
than
1,000
daltons.

3.
All
previously
manufactured
polymers
(
regardless
of
NAMW)
and
all
"
new"
polymers
with
a
NAMW
greater
than
1,000
daltons
should
be
set
aside
pending
the
outcome
of
the
screening
and
testing
of
their
monomer
and
oligomer
components.

4.
If
the
component
is
determined
to
have
endocrine
disrupting
properties,
the
component
should
proceed
to
hazard
assessment.

5.
As
with
any
chemical
shown
to
have
endocrine
disrupting
properties,
an
exposure
assessment
should
be
performed.
At
this
stage,
all
potential
exposure
routes
for
a
component
would
be
determined,
including
the
potential
for
the
component
to
be
available
from
the
polymer.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
45
Finally,
the
EDSTAC
recommends
that
EPA
gain
experience
with
monomers,
oligomers,
and
new
polymers
with
NAMW
less
than
1,000
daltons
and
learn
how
to
apply
that
experience
toward
the
development
of
an
approach
to
address
existing
polymers.
This
would
focus
the
EDSTP
on
the
polymers
about
which
the
best
information
is
available
and
on
those
most
biologically
relevant
to
the
endpoints
of
concern.
To
the
extent
that
data
generated
during
implementation
of
the
EDSTP
on
new
chemical
polymers
indicate
a
problem,
EPA
should
obtain
information
on
molecular
weight,
production
volume,
chemical
identity,
and
any
other
appropriate
information
needed
to
identify
and
evaluate
existing
chemical
polymers
in
the
priority
setting
step
of
the
EDSTP.
This
could
be
done
through
the
TSCA
Inventory
Update
Rule.

VII.
Recommendations
for
Handling
Mixtures
A.
Introduction
The
EDSTAC
has
acknowledged
the
importance
of
considering
mixtures,
and
public
comment
at
plenary
meetings
reinforced
the
Committee's
concern
over
mixtures.
This
section
discusses
several
of
the
key
issues
relating
to
the
screening
and
testing
of
chemical
mixtures
for
endocrine
disruption
and
presents:
a
scheme
for
organizing
the
various
mixtures,
recommended
priority
setting
criteria,
and
recommendations
for
initial
action.

B.
Definition
Quite
simply,
a
mixture
is
any
combination
of
two
or
more
chemicals.
The
number
of
chemical
mixtures
present
in
the
environment
is
practically
infinite.
In
addition
to
the
approximately
87,000
chemicals
considered
for
priority
setting,
many
other
metabolites,
degradates,
and
combustion
products
may
also
occur
in
the
environment.
Given
this
huge
array
of
possible
mixtures,
the
EDSTAC
focused
on
grouping
mixtures
into
general
classes.

C.
Categorization
Scheme
for
Mixtures
Mixtures
can
be
sorted
on
where
they
are
found
in
the
environment,
their
source,
and
their
chemical
makeup.
The
EDSTAC
proposes
a
simple
categorization
approach
to
mixtures
focused
on
the
range
of
mixtures
found
in
products,
the
environment,
and
human
tissues
and
fluids.
The
proposed
scheme
for
organizing
mixtures,
along
with
examples
of
categories
of
data
that
fit,
is
outlined
below:

1.
Products
commonly
containing
mixtures
a.
Pesticide
formulations
b.
Cosmetics,
toiletries,
cleaners,
and
other
consumer
products
c.
Petroleum
derived
products
 
gasoline,
solvents,
metalworking
fluids
d.
Food
 
including
additives,
contaminants,
and
phytoestrogens
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
46
e.
Pharmaceuticals/
Over­
the­
counter
drugs
f.
Other
commercial,
formulated
products
For
products
commonly
containing
mixtures,
a
further
three­
part
distinction
can
be
made
between:

·
Formulated
products
 
These
are
products
mixed
to
contain
a
specified
proportion
of
chemicals
necessary
for
product
function.
Examples
include
pesticides,
cosmetics,
medicines,
etc.
·
Commercial
non­
formulated
products
 
These
are
products
which
are
blended
to
attain
certain
performance
criteria.
In
contrast
to
the
formulated
products,
the
proportion
of
ingredients
is
generally
not
fixed.
Although
knowledge
about
the
precise
identity
or
proportion
of
the
chemicals
contained
therein
is
limited,
some
information
about
the
chemical
nature
(
e.
g.,
aliphatic/
aromatic)
is
available.
Examples
include
fuels,
solvents,
and
lubricants.
·
Industrial
chemicals
 
For
the
purposes
of
priority
setting,
these
will
be
considered
as
single
chemicals.
However,
even
though
one
chemical
predominates,
other
chemical
impurities
may
be
present
as
well.
The
potential
activity
of
impurities
must
be
considered
in
the
screening
and
testing
of
these
"
single
chemicals."
Commodity
chemicals
such
as
styrene,
propylene,
and
toluene
are
examples
of
such
"
single
chemicals."

2.
Environmental
media
commonly
containing
mixtures
(
including,
but
not
limited
to,
TSCA
and
FIFRA
chemicals,
metabolites,
degradates,
and
combustion
products)

a.
Contaminated
media
at
Superfund
sites
b.
Toxic
chemicals
in
urban
air
c.
Contaminated
drinking
water
i.
Pesticides/
Fertilizers
ii.
Disinfection
byproducts
iii.
Chemicals
commonly
found
in
drinking
water
d.
Surface
water
and
groundwater
i.
Effluents
e.
Indoor
air
f.
Sediments/
Sludge
g.
Occupational
media
(
e.
g.,
welding
fumes,
coke
oven
emissions,
etc.)

3.
Tissues
and
media
from
humans
and
other
animals
(
including
animals
produced
for
food,
fish,
and
wildlife)
commonly
containing
mixtures
(
including,
but
not
limited
to,
TSCA
and
FIFRA
chemicals,
metabolites,
degradates,
and
combustion
products)
from:

a.
Blood
b.
Breast
milk
c.
Exhaled
breath
d.
Fat
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
47
e.
Urine
f.
Miscellaneous
tissues
(
e.
g.,
finfish,
shellfish,
meat,
poultry,
etc.)

D.
Determining
the
Composition
of
Mixtures
to
be
Considered
Determining
the
precise
composition
of
mixtures
to
be
considered
for
prioritization
is
challenging
given
the
large
number
of
possibilities.
This
task
is
somewhat
easier
for
mixtures
found
in
products
because
the
basic
formulations
are
usually
well­
defined
and
are
not
likely
to
drift
widely
over
time.
However,
the
composition
of
mixtures
found
in
environmental
and
biological
samples
is
highly
variable
with
respect
to
specific
components
present
and
their
relative
amounts.
In
such
cases,
higher
priority
should
be
given
to
mixture
combinations
typically
or
frequently
found
in
environmental
and
biological
media.

E.
Criteria
for
Prioritizing
Mixtures
The
following
are
some
recommended
criteria
for
prioritizing
mixtures
for
the
purpose
of
endocrine
disruptor
screening
and
testing:

1.
Exposure
data
on
mixtures
(
same
criteria
as
with
single
chemicals)
·
Biological
sampling
(
human
and
other
biota)
data
for
components
of
mixtures
·
Environmental,
occupational,
consumer
product,
and
food­
related
data
·
Environmental
releases
·
Production
volume
·
Fate
and
transport
data
and
models
2.
Toxic
effects
associated
with
the
mixture
in
question
(
same
criteria
as
with
single
chemicals)
·
Toxicological
laboratory
studies
and
databases
·
Epidemiological
and
field
studies
and
databases
(
populations
affected)
·
Predictive
biological
activity
or
effects
models
(
e.
g.,
SARs,
QSARs)

3.
Toxic
effects
data
on
major
components
·
Use
the
ranking
developed
for
individual
components
by
the
EDSTAC
to
rank
mixtures
based
on
the
relative
ranking
of
the
components
they
contain
·
This
approach
is
especially
useful
for:
·
mixtures
for
which
there
are
no
toxic
effects
data
on
the
mixture
itself.
If
toxic
effects
data
are
available
on
the
mixture,
those
data
should
be
given
primary
consideration
in
priority
setting
for
the
mixture.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
48
·
environmental
contaminants
and
complex
product
mixtures,
especially
if
the
mixture
contains
a
component
with
a
high
priority
for
screening
and
testing.

F.
Recommendations
The
EDSTAC
and
EPA
are,
in
many
ways,
entering
uncharted
territory.
The
evaluation
(
including
the
design,
implementation,
and
interpretation
of
screens
and
tests)
of
the
potential
for
endocrine
disruption
of
single
compounds
is
still
emerging
and
fraught
with
much
scientific
uncertainty.
Nonetheless,
the
Committee
urges
EPA
to
rigorously
address
the
science
of
mixture
toxicology
in
their
research
efforts,
and
recognizes
the
need,
under
the
auspices
of
the
EDSTP,
to
begin
to
confront
mixtures.

The
science
of
evaluating
mixtures
remains
complex
and
unclear
for
any
toxic
endpoint.
Given
the
potentially
overwhelming
task
of
establishing
priorities
for
endocrine
disruptor
screening
and
testing
of
mixtures,
the
Committee
recommends
a
well­
considered,
step­
wise
approach
to
the
inclusion
and
prioritization
of
mixtures
in
the
EDSTP.
The
EDSTAC
urges
EPA
to
identify
the
challenges
it
faces
early
in
this
endeavor,
and
to
address
these
issues
in
a
transparent
fashion.

The
recommendations
that
follow
are
based
on
the
assumption
that,
prior
to
undertaking
the
T1S
step
of
the
program,
the
following
will
occur:

·
Demonstration/
Validation
(
D/
V)
of
both
HTPS
and
the
T1S
battery
 
a
limited
number
of
chemicals
will
be
selected
and
evaluated
in
the
battery
of
screens
recommended
by
the
EDSTAC.
The
purpose
of
this
D/
V
phase
is
to
show
the
utility
and
validity
of
the
screens
to
be
used
in
both
HTPS
and
T1S.

·
HTPS
 
a
series
of
transcriptional
activation
assays
will
be
selected
for
utilization
in
the
high
throughput
mode.

Specific
recommendations
for
mixtures:

1.
D/
V
 
Include
a
limited
set
of
mixtures
in
the
D/
V
phase
of
screening,
including
those
to
be
included
in
HTPS.
For
the
purpose
of
this
phase,
a
set
of
mixtures
should
be
selected
that
spans
a
range
of
physical
and
chemical
properties.
The
goal
here
is
to
challenge
HTPS
and
T1S
with
a
variety
of
chemicals
to
ensure
feasibility
and
robustness
before
evaluating
other
mixtures.
Clearly,
the
mixtures
chosen
for
validation
may
be
drawn
from
mixtures
found
in
the
environment
and
may
include
"
known"
endocrine
disruptors,
but
the
primary
selection
criterion
should
be
chemical
diversity.
This
component
of
the
D/
V
phase
is
in
addition
to
any
D/
V
efforts
done
for
individual
chemicals,
as
described
above.

2.
HTPS
 
If
the
screens
are
shown
to
be
capable
of
handling
single
components
as
well
as
a
diverse
set
of
mixtures
in
the
D/
V
phase,
expert
judgment
(
e.
g.,
EDSTAC
consensus),
guided
by
a
set
of
prioritization
criteria,
should
be
used
to
evaluate
the
literature
and
to
decide
on
a
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
49
limited
set
of
mixtures
to
enter
HTPS.
Rather
than
focusing
on
chemical
diversity
as
in
the
initial
D/
V
phase,
these
mixtures
should
be
representative
of
those
found
in
environmental
media
or
biological
tissues.
For
each
mixture,
a
set
of
chemicals
should
be
identified
that
are
deemed
representative
of
the
chemicals
and
their
proportions
found
in
the
selected
mixture.
The
EDSTAC
recommended
that
the
PSWG
develop
prioritization
criteria
for
mixtures
and
identify
a
set
of
mixtures
to
enter
HTPS.
These
criteria
and
the
set
of
mixtures
are
described
in
Chapter
Four,
Section
VII,
E,
and
Section
VII,
F,
4,
respectively.

3.
Screening
and
Testing
 
The
battery
of
assays
validated
for
use
in
the
screening
program
should
be
used
to
evaluate
the
mixtures
examined
in
HTPS.
If
appropriate,
screening
should
be
followed
by
testing.
Additionally,
a
comprehensive
literature
evaluation
should
be
undertaken
to
identify
exposure
and
effects
data
on
mixtures
that
have
not
already
undergone
HTPS.
This
information
should
be
used
to
inform
the
prioritization
for
Phase
II
and
subsequent
phases.
During
the
time
it
would
take
to
accomplish
this,
data
could
be
gathered
from
the
screening
and
testing
of
single
compounds
during
Phase
I
and
from
a
limited
number
of
mixtures
to
help
inform
the
prioritization
of
other
candidate
mixtures.
The
prioritization
of
mixtures
for
Phase
II
and
subsequent
phases
would
use
the
same
prioritization
criteria
as
those
used
for
single
chemicals.

4.
Highest
Priority
Mixtures
for
Screening
and
Testing
 
The
EDSTAC
is
concerned
that
the
sheer
complexity
of
the
mixtures
issue
could
produce
"
paralysis
by
analysis"
and
result
in
no
meaningful
progress.
To
overcome
this
potential
inertia,
the
EDSTAC
urges
EPA
to
focus
initially
on
six
types
of
mixtures.
These
six
types
of
mixtures
have
been
identified
by
applying
the
exposure
and
effects
criteria
for
priority
setting
outlined
earlier
in
this
chapter.
In
suggesting
that
EPA
focus
its
initial
attention
on
these
six
types
of
mixtures,
by
no
means
does
the
EDSTAC
underestimate
the
enormous
challenge
of
addressing
just
these
six.
However,
the
EDSTAC
believes
a
systematic
approach
that
focuses
initially
on
these
six
types
of
mixtures
could
shed
light
on
a
wide
range
of
technical
challenges,
help
validate
screens
and
tests,
and
promote
development
of
decision­
making
protocols
for
screening
and
testing
other
types
of
mixtures.
Thus,
the
EDSTAC
recommends
that
EPA
focus
its
initial
efforts
on
identifying
a
relatively
small
number
of
representative
samples
of
mixtures
(
i.
e.,
more
than
one
and
fewer
than
ten)
from
the
following
six
types
of
mixtures;
and
second
whether
it
is
technically
feasible
to
run
these
representative
samples
through
the
HTPS,
T1S,
and
T2T.
If
such
steps
are
determined
to
be
technically
feasible,
the
EDSTAC
recommends
that
the
selected
representative
samples
of
mixtures
be
subjected
to
HTPS,
T1S,
and,
if
necessary,
T2T.
In
presenting
the
six
candidate
types
of
mixtures,
in
several
instances
the
EDSTAC
identifies
some
data
sources
that
EPA
might
use
to
initiate
the
first
step
of
this
activity
(
i.
e.,
to
identify
a
small
number
of
representative
samples):

a)
Contaminants
in
human
breast
milk
 
The
contaminants
in
human
breast
milk
are
recommended
for
immediate
attention
because
infants
are
directly
exposed
to
them.
Existing
literature
demonstrates
that
human
breast
milk
in
the
United
States
and
elsewhere
is
contaminated
with
a
sizable
number
of
chemicals
that
tend
to
exist
in
common
proportions
(
Jensen
and
Slorach,
1990).
Scientific
opinion
favors
breast
feeding
over
reliance
on
infant
formulas
and
cows'
milk
in
most
cases.
Therefore,
the
results
of
testing
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
50
contaminants
in
human
breast
milk
must
be
communicated
with
great
sensitivity.

The
EDSTAC
acknowledges
that
if
hazards
are
recognized
in
breast
milk,
no
techniques
exist
for
reducing
immediately
the
hazards
to
those
exposed.
But
women
have
a
right
to
know
the
extent
to
which
they
have
been
exposed
to
endocrine
disrupting
chemicals
and
are
entitled
to
know
the
hazards
to
which
they
are
subjecting
their
infants.
Over
the
longterm
the
evidence
from
analysis
of
contaminants
in
breast
milk
can
be
an
impetus
to
the
evaluation
of
policies
for
reducing
further
exposure
to
such
chemicals.

b)
Phytoestrogens
in
soy­
based
infant
formulas
 
Soy­
based
infant
formulas
contain
a
complex
mixture
of
plant­
derived
NONEs
 
often
referred
to
as
"
phytoestrogens."
In
particular,
the
formulas
contain
a
category
of
phytoestrogens
called
isoflavones,
specifically
genistein
and
daidzein.
But
the
formulas
also
contain
a
wide
array
of
other
isoflavones,
present
as
minor
components,
which
also
possess
estrogenic
characteristics
(
Chapter
Four,
Section
VIII).

c)
Mixtures
of
chemicals
most
commonly
found
at
hazardous
waste
sites
 
ATSDR
has
published
a
summary
of
the
combinations
of
chemicals
most
commonly
found
at
hazardous
waste
sites
(
Johnson
and
De
Rosa,
1995).
These
mixtures
pose
a
potential
hazard
to
the
communities
in
which
these
sites
are
located
and,
to
the
extent
that
such
sites
are
located
in
lower­
income
areas,
their
presence
raises
issues
of
environmental
justice.
Such
sites
are
distributed
broadly
across
the
United
States.

d)
Pesticide/
Fertilizer
mixtures
 
Pesticides
and
fertilizers
have
commonly
been
detected
in
surface
water
and
groundwater
across
the
United
States.
The
National
Toxicology
Program
(
NTP)
of
the
National
Institute
for
Environmental
Health
Sciences
(
NIEHS)
has
conducted
tests
for
traditional
reproductive
and
developmental
toxicological
endpoints
of
the
most
commonly
occurring
mixtures
in
California
and
Iowa,
two
heavily
agricultural
states
(
Heindel
et
al.,
1994).
Screening
and
testing
these
mixtures
will
provide
an
opportunity
to
compare
results
to
the
toxicological
data
already
available.

e)
Disinfection
byproducts
 
Some
of
the
chemicals
used
for
purifying
drinking
water
supplies
produce
byproducts
that,
ironically,
may
themselves
pose
a
hazard
to
human
health.
EPA
currently
is
reviewing
monitoring
data
on
disinfection
byproducts,
with
the
objective
of
setting
priorities
for
screening
and
testing.
EPA
is
whittling
down
a
list
of
several
hundred
such
byproducts
and
anticipates,
in
the
short
run,
NIEHS/
NTP
initiating
testing
on
approximately
ten
of
these
chemicals
for
carcinogenicity,
immunotoxicity,
and
reproductive
effects.
Based
on
whatever
results
are
available
from
this
review
and
testing,
and
subject
to
technical
feasibility,
the
EDSTAC
recommends
subjecting
representative
mixtures
of
the
most
commonly
occurring
disinfection
byproducts
to
screening
and
possible
testing
for
endocrine
disruption.

f)
Gasoline
 
Gasoline
is
a
complex
mixture
of
volatile
organic
compounds
to
which
large
numbers
of
the
population
are
exposed
by
inhalation.
Dermal
exposure
can
also
occur,
particularly
in
occupational
settings.
Subject
to
technical
feasibility,
the
EDSTAC
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
51
recommends
that
representative
samples
of
this
mixture
be
subjected
to
screening
and,
if
necessary,
testing.
The
EDSTAC
did
not
have
time
to
identify
potential
data
sources
for
determining
whether
it
is
possible
and,
if
so,
what
might
constitute,
representative
samples
of
gasoline.

VIII.
Recommendation
to
Screen
Naturally
Occurring
Non­
Steroidal
Estrogens
A.
Background
Naturally
occurring
non­
steroidal
estrogens
(
NONEs)
include
natural
products
derived
from
plants
(
phytoestrogens)
and
fungi
(
mycotoxins).
NONEs
are
less
active
than
estradiol
and
diethylstilbestrol
(
DES)
in
in
vitro
and
in
vivo
assays,
but
the
ubiquitous
presence
of
these
compounds
in
foods
indicate
that
NONEs
cannot
be
ignored
(
e.
g.,
Cassidy,
1996;
Clarke
et
al.,
1996;
Gavaler
et
al.,
1995;
Sheehan
and
Medlock,
1995).
Moreover,
the
potential
additive,
antagonist,
and
synergistic
effects
of
NONEs
with
other
endogenous
and
exogenous
hormonally
active
chemical
substances
are
issues
that
warrant
investigation.
Significant
research
on
NONEs
is
being
conducted
in
the
United
States
and
other
countries
to
better
characterize
the
benefits
and
potential
hazards
(
effects)
of,
as
well
as
the
levels
of
exposure
to,
these
estrogenic
compounds.

NONEs
are
commonly
perceived
as
safe,
generally
beneficial,
and
overall
innocuous
to
humans.
For
example,
the
low
incidence
of
breast
cancer
in
women
within
Asia
has
been
attributed
to
the
beneficial
effects
of
the
phytoestrogen
genistein.
Genistein
is
a
major
component
in
soybeans,
which
comprise
a
large
part
of
some
Asian
diets.
Moreover,
phytoestrogens
are
recommended
as
safer,
natural
alternatives
to
steroidal
estrogens
for
hormone
replacement
therapy.
However,
over
the
last
40
years,
adverse
effects
of
naturally
occurring
non­
steroidal
estrogenic
compounds
have
been
well­
documented
in
wildlife
(
range
livestock)
and
laboratory
animals.
In
humans,
there
are
reports
that
phytoestrogens
prolong
the
menstrual
cycle
and
cause
(
weak)
proliferation
of
reproductive
epithelial
cells.

Exposure
to
NONEs
through
food
sources
can
occur
throughout
one's
lifetime
(
i.
e.,
in
utero,
infancy,
childhood,
and
adulthood).
Significant
quantities
of
a
complex
mixture
of
isoflavone
phytoestrogens
(
predominately
genistein
and
daidzein)
are
present
in
various
soy­
based
foods.
Soybean
infant
formulas
are
widely
used
in
the
U.
S.
and
abroad,
and
there
is
research
under
way
to
determine
the
effects
of
these
compounds
on
male
infants.
Additionally,
the
exposure
and
uptake
of
NONEs
in
adults
is
evident
because
phytoestrogens
have
also
been
detected
in
human
breast
milk
and
urine.

The
potential
effects
of
NONEs,
beneficial
and
detrimental,
should
not
be
dismissed
or
assumed
to
be
non­
existent
because
organisms
have
the
ability
to
rapidly
metabolize
these
compounds.
Many
of
the
endocrine
disruption
issues
and
concerns
for
pesticides
and
industrial
chemicals
may
be
just
as
relevant
for
NONEs.
There
is
substantial
evidence
to
justify
a
designation
of
high
priority
for
screening
and
testing
of
these
compounds
based
on
the
exposure
to
and
potential
effects
of
NONEs
to
both
wildlife
and
human
populations.
While
there
is
an
abundant
amount
of
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
52
in
vitro
and
in
vivo
screening
data
(
mainly
uterotrophic
and
estrogen
receptor
binding
assays)
on
NONEs,
broad­
based
mechanistic
screening
and
two­
generation
reproductive/
developmental
toxicity
testing
(
according
to
current
guideline
standards
for
pesticides
and
chemicals)
is
lacking.
A
review
of
the
literature
indicates
that:

·
Estrogenic
plant
and
fungal
natural
products
are
ubiquitous
in
nature
and
occur
in
significant
quantities
in
at
least
20
fruits
and
vegetables
including
legumes,
coffee,
beer,
wine,
and
bourbon
whiskey
and
forage
clover.
(
Cassidy,
1996;
Clarke
et
al.,
1996;
Gavaler
et
al.,
1995;
Richard
and
Thompson,
1997;
Thomas,
1998;
Verdeal
and
Ryan,
1979;
Wiese
and
Kelce,
1997).
·
NONE
levels
vastly
exceed
pesticide
residues
in
food.
The
typical
daily
intake
of
isoflavones
by
humans,
estimated
to
be
0.6
mg/
kg/
day,
can
prolong
a
human
female's
menstrual
cycle.
The
daily
intake
of
a
vegetarian
who
consumes
very
large
quantities
of
soy­
derived
nutrients
could
be
much
higher.
(
Adams,
1996;
Cassidy,
1996;
Clarke
et
al.,
1996;
Wiese
and
Kelce,
1997).
·
Coumesterol
is
uterotrophic
in
female
rats
fed
over
a
90­
hour
period
at
dose
levels
within
the
range
reported
in
human
foods.
Exposure
of
newborn
rats
to
100
ug/
day
of
coumesterol
accelerates
the
onset
of
puberty,
increases
the
incidence
of
persistent
vaginal
keratinisation
and
induces
bloody
ovarian
follicles.
(
Clarke,
1996;
Sheehan
and
Medlock,
1995;
Verdeal
and
Ryan,
1979;
Wiese
and
Kelce,
1997).
·
The
deleterious
effects
of
clover
phytoestrogens
on
grazing
sheep
is
well­
documented.
Effects
range
from
temporary
and
permanent
infertility
to
permanent
abnormalities
in
their
reproductive
organs.
(
Adams,
1996;
Thomas,
1997;
Verdeal
and
Ryan,
1979).
·
At
doses
up
to
50
mg/
day
by
oral
administration,
zearalenone,
a
corn
mycotoxin,
produces
effects
on
the
vulva,
uterus,
ovary,
cervix,
and
mammary
glands
of
swine.
Sows
receiving
5
mg
of
purified
zearalenone
daily
throughout
the
last
month
of
pregnancy
produced
litters
with
stillborn
pigs
or
pigs
with
a
"
splayleg"
incoordination
of
hind
limbs.
(
Adams,
1996;
Wiese
and
Kelce,
1997;
Verdeal
and
Ryan,
1979).
·
Phytoestrogens
(
genistein)
can
be
both
tumor
promoters
and
inhibitors
depending
on
the
target
organ
and
the
dose.
Genistein
has
been
observed
to
inhibit
both
tyrosine
kinase
and
topoisomerase
II.
The
latter
is
the
target
site
of
action
for
taxol,
a
drug
currently
used
to
treat
breast
cancer.
(
Clarke
et
al.,
1996;
Lien
and
Lien,
1996;
Markovits,
1989).
·
NONEs
may
produce
various
biological
responses
in
vivo.
NONEs
may
act
as
estrogen
agonists
or
antagonists
(
anti­
estrogenic
effects).
These
effects
could
either
be
beneficial
or
deleterious
depending
on
the
target
tissue
and
dose.
Additionally,
NONEs
may
cause
other
responses
through
other
mechanisms
that
do
not
involve
the
estrogen
receptor,
for
example
genistein.
Additionally,
it
is
reported
that
some
phytoestrogens
may
alter
the
concentration
of
sex­
hormone­
bonding
globulin
which,
in
turn,
alters
the
bioavailability
of
endogenous
hormones.
(
Adams,
1996;
Cassidy,
1996;
Clarke
et
al.,
1996;
Safe
and
Gaido,
1998;
Sheehan
and
Medlock,
1995;
Wiese
and
Kelce,
1997;
Wiseman,
1996).

B.
Recommendation
The
EDSTAC
therefore
recommends
screening
and,
if
necessary,
testing:
(
1)
representative
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
53
NONEs
singularly;
and
(
2)
a
complex
mixture
of
NONEs
(
e.
g.,
soy­
based
infant
formulas
as
discussed
in
Section
VII
of
this
chapter).
Data
from
the
representative
compounds
should
be
compared
to
hormones
and
synthetic
chemicals.
The
representative
compounds
should
come
from
the
major
chemical
classes
of
estrogenic
natural
products.
Testing
soy­
based
infant
formulas
should
be
made
part
of
the
initial
investigation
to
evaluate
mixtures.

The
following
NONEs
were
chosen
from
the
literature
based
on
their
reported
effects
(
beneficial
and
deleterious)
to
wildlife
and/
or
humans
and
on
their
widespread
occurrence
in
nature.
These
NONEs
should
be
screened
and,
if
necessary,
tested.

Representative
NONEs:
1.
Isoflavones:
genistein,
daidzein,
miroestrol,
biochanin
A,
formononetin,
equol
2.
Flavones:
kaemferol,
naringenin
3.
Coumestans:
coumesterol
4.
Dihydrochalcones:
phoretin
5.
Triterpenes:
betulafolienetriol
(
ginseng)
6.
Lignans:
enterolactone
Representative
estrogenic
mycotoxin:
7.
Beta­
resorcyclic
lactones:
zearalenone,
zearalenol,
zearanol
IX.
Recommendation
for
a
Nominations
Process
A.
Introduction
The
EDSTAC
recommends
that
EPA
establish
a
process
that
would
allow
affected
citizens
to
nominate
chemical
substances
or
mixtures
for
endocrine
disruptor
screening
and
testing.
In
general,
the
nominations
process
recommended
by
the
EDSTAC
is
intended
to
focus
on
chemical
substances
or
mixtures
where
exposures
are
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems
rather
than
on
chemical
substances
or
mixtures
where
exposures
are
more
broadly
experienced
by
the
general
population
at
the
regional
and/
or
national
levels.
As
such,
the
nominations
process
is
intended
to
provide
a
mechanism
for
prioritizing
chemical
substances
or
mixtures
that
are
unlikely
to
be
considered
as
high
priority
through
the
core
priority
setting
process.
For
this
reason,
the
EDSTAC
recommends
that
the
nominations
process
should
run
parallel
to,
but
be
separate
and
distinct
from,
the
core
priority
setting
process
described
earlier
in
this
chapter.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
54
B.
Description
of
the
Nominations
Process
Consistent
with
the
overall
philosophy
of
the
core
priority
setting
process,
as
described
in
Section
XI
of
this
chapter,
chemical
substances
and
mixtures
that
are
nominated
will,
in
effect,
be
placed
in
one
of
the
"
compartments"
of
the
overall
compartment­
based
approach
to
priority
setting
(
Chapter
Four,
Section
XI).
The
EDSTAC
recommends
a
goal
for
each
phase
of
the
EDSTP
of
no
less
than
5%
of
the
total
number
of
chemical
substances
or
mixtures
subjected
to
T1S
drawn
from
substances
receiving
nominations
but
not
selected
through
the
main
priority
setting
process.
The
EDSTAC
recognizes
that
the
total
number
of
nominations
or
their
quality
may
be
such
that
this
goal
cannot
be
met
in
specific
phases.
For
each
phase
of
the
EDSTP,
the
nominated
chemicals
should
be
evaluated
against
the
specialized
criteria
described
below.
Priorities
for
the
nominated
chemicals
should
be
established
in
accordance
with
these
specialized
criteria
on
a
separate
track,
rather
than
attempting
to
integrate
the
prioritization
of
the
nominated
chemicals
with
the
chemicals
that
are
selected
for
T1S
through
the
core
priority
setting
process.
Any
nominated
chemical
substance
or
mixture
that
becomes
a
priority
for
T1S
through
the
core
priority
setting
process
should
be
removed
from
consideration
within
the
list
of
nominated
chemicals
in
order
to
ensure
that
the
priorities
drawn
from
the
nominations
process
will
compete
only
against
other
nominated
chemicals.

The
nominations
process
should
allow
for
an
early
opportunity
to
submit
nominations
during
each
phase
of
the
EDSTP.
A
call
for
nominations
should
be
made
via
a
public
notice
specifying
both
the
criteria
by
which
nominations
will
be
evaluated
and
the
deadline
for
submitting
nominations.
The
time
period
for
submitting
nominations
should
end
prior
to
the
expected
Federal
Register
(
FR)
notice
announcing
EPA's
formal
proposal
for
T1S
priorities.
As
a
part
of
the
public
comment
period
following
such
an
announcement,
members
of
the
public
should
be
given
an
opportunity
to
comment
on
all
chemicals
that
are
proposed
for
T1S.
Chemicals
not
included
in
the
priority
list
for
each
phase
of
the
EDSTP
could
be
nominated
at
the
start
of
the
next
phase.
However,
the
public
comment
period
following
the
FR
notice
should
not
be
considered
a
second
opportunity
to
nominate
chemicals
for
the
current
phase
of
the
program.

C.
Criteria
for
Evaluating
Nominated
Chemicals
As
noted
above,
the
EDSTAC
recommends
that
the
nominations
process
should
utilize
a
different
set
of
criteria
than
will
be
used
for
the
core
priority
setting
steps
of
the
EDSTP,
particularly
with
respect
to
exposure.
The
exposure­
related
criteria
for
the
nominations
process
should
be
designed
to
allow
for
chemical
substances
and
mixtures
for
which
there
may
not
be
widespread
exposures
on
a
national
scale,
but
for
which
there
are
exposures
on
a
smaller
scale,
to
be
eligible
to
receive
a
priority
status
for
T1S.
Thus,
the
nominations
process
should
be
designed
to
focus
on
exposures
that
are
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
55
After
exposure­
related
criteria
have
been
considered
in
the
evaluation
of
nominated
chemical
substances
and
mixtures,
it
is
likely
that
effects­
related
information
will
need
to
be
considered
to
help
further
set
priorities
among
nominations.
This
is
potentially
problematic
because
there
is
likely
to
be
a
lack
of
effects­
related
information.
In
fact,
the
lack
of
effects
data
may
be
the
very
reason
for
public
concern.
That
is,
communities
may
be
regularly
exposed
to
a
chemical
substance
or
mixture
that
has
not
undergone
meaningful
toxicological
evaluation.
Nevertheless,
if
there
are
effects
data,
or
if
the
chemical
substance
or
mixture
is
chemically
similar
to
another
chemical
substance
or
mixture
for
which
effects
data
are
available,
the
Committee
recommends
that
EPA
utilize
those
data
as
a
secondary
source
of
information
to
help
set
priorities
among
nominees.

In
summary,
when
evaluating
nominations,
EPA
should
consider
nominated
chemical
substances
and
mixtures
that
meet
the
following
criteria
to
be
a
higher
priority
than
those
that
do
not
meet
these
criteria:

·
chemical
substances
and
mixtures
for
which
there
is
a
likelihood
of
a
regularly
completed
exposure
pathway
as
compared
to
chemical
substances
and
mixtures
for
which
the
exposure
pathway
is
likely
to
be
completed
only
rarely
or
occasionally;
·
chemical
substances
and
mixtures
that
affect
a
high
proportion
of
people
within
a
given
community
or
workplace;
and
·
chemical
substances
and
mixtures
for
which
there
may
be
direct
or
indirect
(
i.
e.,
model
derived)
effects­
related
data
regarding
the
endocrine
disrupting
potential
of
the
nominated
chemical
substance
or
mixture.

D.
Submission
of
Nominations
Members
of
the
public
should
be
encouraged
to
submit
nominations
with
as
much
information
as
possible,
but
it
should
still
be
permissible
to
do
so
without
data
or
evidence
as
it
relates
to
the
specialized
criteria.
Lack
of
such
information
should
not
preclude
EPA
from
evaluating
a
nominated
chemical
on
par
with
nominated
chemicals
for
which
data
have
been
submitted.
EPA
should
make
use
of
all
information
available
to
the
Agency,
including
anecdotal
information
that
may
be
submitted,
as
well
as
information
gathered
as
part
of
the
core
priority
setting
process
(
e.
g.,
information
contained
within
the
EDPSD
recommended
in
Section
X
of
this
chapter).
Recognizing
that
the
nominations
process
may
be
vulnerable
to
misuse
for
various
reasons,
the
EDSTAC
recommends
that
the
Agency
design
a
nominations
process
that
protects
nominators,
workers,
communities,
registrants,
manufacturers,
etc.,
as
appropriate.
Recognizing
that
misuse
could
significantly
detract
from
its
intended
purpose,
the
EDSTAC
recommends
that
the
nominations
process
be
as
transparent
as
possible
and
that
EPA
provide
the
list
of
nominations
with
any
appropriate
supporting
information
in
appropriate
publications,
such
as
the
Federal
Register
Notice
and/
or
the
Internet.

The
identity
of
nominating
organizations
should
be
made
public.
The
identity
of
individual
nominators
should
be
kept
confidential
by
EPA
upon
written
request.
In
order
to
assist
EPA
in
its
evaluation
of
nominated
chemicals,
the
Committee
recommends
that
nominations
should
include
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
56
the
following
types
of
information:

·
how
exposure
to
the
nominated
chemical
substance
or
mixture
may
be
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems;
·
the
reasons
for
the
nomination
(
which
may
include
both
exposure­
and
effects­
related
concerns)
and
any
information
that
provides
a
basis
for
those
concerns;
and
·
the
degree
of
support
for
the
nomination
from
the
potentially
affected
communities
and/
or
workplaces.

E.
Mixtures
in
the
Context
of
the
Nominations
Process
The
EDSTAC
expects
nominations
of
chemical
mixtures
as
well
as
individual
chemical
substances.
However,
as
with
the
broader
discussion
of
mixtures
contained
in
Section
VII
of
this
chapter,
the
EDSTAC
recognizes
there
are
difficult
technical
and
policy
issues
surrounding
the
issue
of
screening
and
testing
mixtures.
The
EDSTAC
is
particularly
concerned
that
EPA
and
other
governmental
agencies,
in
anticipation
of
the
nominations
process
raising
expectations
for
action,
be
prepared
to
take
whatever
steps
may
be
appropriate
to
address
potential
public
health
and
environmental
impacts
that
are
identified
through
the
EDSTP.
Similarly,
the
EDSTAC
recommends
that
the
communication
and
outreach
effort
that
will
accompany
the
nominations
process
should
address
the
capabilities,
as
well
as
the
limitations,
which
EPA
and
other
governmental
agencies
are
likely
to
face
in
any
effort
subsequent
to
the
screening
and
testing
stage
of
the
process.

F.
Ability
to
Track
Nominations
As
recommended
in
Chapter
Six
of
this
report,
members
of
the
public
should
be
able
to
track
and
locate
the
progress
of
all
chemicals
in
the
EDSTP
through
a
centralized,
on­
line
database
run
by
EPA.
This
on­
line
database
will
provide
an
opportunity,
in
addition
to
the
FR
notice,
for
members
of
the
public
to
determine
the
status
of
chemicals
that
may
be
of
concern
to
them.

X.
The
Endocrine
Disruptor
Priority
Setting
Database
A.
Introduction
As
described
in
other
sections
of
this
chapter,
the
PSWG
began
its
work
by
describing
exposureand
effects­
related
information
categories
and
criteria
to
be
used
for
sorting
and
prioritizing
chemicals
for
endocrine
disruptor
screening
and
testing.
The
PSWG
also
identified
and
evaluated
data
sources
associated
with
these
categories
and
criteria.
These
data
sources
are
listed
in
matrices
contained
in
Appendix
G.

After
identifying
these
data
sources,
the
PSWG
grappled
with
how
to
use
them
to
sort
and
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
57
prioritize
chemicals
for
endocrine
disruptor
screening
and
testing.
Over
time
it
became
clear
that
there
was
much
value
and
utility
in
assembling
the
relevant
and
useful
data
sources
into
a
single
relational
database,
which
is
referred
to
as
the
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD).
The
PSWG
had
contemplated
developing
and
using
the
EDPSD
to
assist
in
the
EDSTAC's
deliberations
and,
in
particular,
to
help
the
work
group
and
the
Committee
understand
the
implications
of
alternative
approaches
to
priority
setting.
After
making
significant
progress
on
the
prototype
EDPSD,
the
PSWG
and
the
EDSTAC
came
to
realize
that
the
tool
could
not
be
completed
given
time
and
resource
constraints.

This
section
presents
recommendations
on
the
further
development,
utilization,
and
maintenance
of
the
prototype
EDPSD.
The
recommended
approach
to
priority
setting
contained
in
Section
XI
of
this
chapter
builds
upon
the
recommendations
contained
in
this
section.

B.
Recommendation
and
Principles
to
Guide
the
Continued
Development,
Utilization,
and
Maintenance
of
the
Prototype
EDPSD
The
EDSTAC
recommends
that
EPA
continue
to
develop
and
maintain
the
EDPSD
as
a
tool
that
can
be
used
to
expeditiously
sort
and
prioritize
chemicals
for
endocrine
disruptor
screening
and
testing.
The
EDSTAC
identified
several
principles
that
should
guide
EPA's
use
of
the
EDPSD,
along
with
the
process
EPA
should
follow
in
conjunction
with
use
of
the
EDPSD.

Most
importantly,
the
EDPSD
itself,
as
well
as
the
process
by
which
it
is
utilized,
should
be
open
and
transparent.
As
described
in
more
detail
below,
EPA
should
convene
a
multi­
stakeholder
group
prior
to
the
completion
of
the
tool.
This
group
would
serve
to
help
ensure
that
the
tool
was
developed
and
ultimately
used
according
to
the
guidelines
suggested
by
the
EDSTAC.
EPA
and
the
multi­
stakeholder
group
should
develop
ground
rules
to
prevent
the
use
of
the
EDPSD
to
confirm
a
priori
assumptions
regarding
the
priority
for
screening
specific
chemicals
or
as
a
means
to
hide
or
obfuscate
the
basis
for
priority
setting
decisions.
Furthermore,
EPA
should
provide
notice
and
opportunity
to
comment
on
the
proposed
database
tool
before
it
is
used
by
the
Agency.
This
will
allow
an
opportunity
for
additional
chemical­
specific
data
that
might
not
otherwise
be
included
in
the
identified
data
sources
to
be
incorporated
into
the
EDPSD.

C.
Description
of
the
Prototype
EDPSD
The
prototype
EDPSD
is
a
relational
database
that
(
as
of
December
1997)
contains
records
for
approximately
87,000
chemicals
with
Chemical
Abstracts
Service
Registry
Numbers
(
CASRNs)
from
data
sources
related
to
the
information
categories
and
criteria
described
in
Sections
III
and
IV
of
this
document.
It
was
created
using
Molecular
Design
Limited
Information
Systems'
Integrated
Scientific
Information
System.
The
CASRNs
of
discrete
organic
chemicals,
polymers,
and
inorganic
chemicals
from
each
data
source
were
entered
in
a
multi­
field
format.
The
number
of
chemical
records
in
the
EDPSD
is
determined
by
the
cumulative
number
of
chemical
records
contained
in
each
data
field.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
58
The
data
fields
included
in
the
prototype
EDPSD
were
used
to
develop
a
form
that
appears
on
the
computer
screen
during
operation
of
the
EDPSD
(
Figure
4.2).
When
queried
using
particular
scenarios
(
e.
g.,
how
many
TRI
chemicals
produced
between
10,000
and
one
million
pounds
appear
in
Great
Lakes
fish
(
GLC
Fish)
and
also
occur
in
the
ATSDR
database,
etc.),
the
EDPSD
provides
the
number
of
chemicals
meeting
the
criteria
used.
Any
number
of
scenarios
can
be
developed
depending
upon
user
interests.
The
prototype
EDPSD
showed
great
promise
in
providing
numbers
of
chemicals
that
displayed
certain
criteria,
and
also
has
potential
to
develop
algorithms
combining
different
criteria.
However,
early
queries
using
different
scenarios
occasionally
resulted
in
numbers
that
were
known
to
be
inaccurate.
It
was
assumed
that
the
inaccuracies
were
resolvable
by
adequately
cross­
referencing
the
different
data
fields
and
conducting
appropriate
QA/
QC
corrections
to
the
data
included
in
the
fields.
However,
the
QA/
QC
exercise
could
not
be
completed
in
the
tight
time
frame
of
the
EDSTAC
schedule.
As
such,
final
development,
demonstration,
and
validation
of
the
EDPSD
was
viewed
as
a
high
priority,
post­
EDSTAC
task
for
EPA
with
multi­
stakeholder
involvement.
A
more
detailed
description
of
the
EDPSD
follows.

All
data
fields
in
the
EDPSD
(
Figure
4.2,
Tables
4.1,
and
4.2)
are
linked
by
CASRNs,
and
there
are
no
duplicate
records
for
any
chemical.
For
most
discrete
organic
chemicals,
chemical
formulas,
molecular
weights,
and
Simplified
Molecular
Input
Line
Entry
System
(
SMILES)
notations
and
chemical
structures
were
entered
into
the
EDPSD.

There
are
two
types
of
fields
in
the
EDPSD
 
logical
and
numerical.
Logical
fields
are
binary
in
nature
(
i.
e.,
+/­,
yes/
no,
true/
false,
etc.).
For
example,
a
chemical
either
is
present
or
absent
in
a
particular
data
source
that
is
included
in
the
EDPSD.
Numerical
fields,
by
contrast,
are
quantitative.
They
provide
an
actual
measured
value
or,
alternatively,
an
estimated
number
associated
with
a
particular
data
source
or
environmental
fate
parameter
(
e.
g.,
an
estimated
hydrolysis
half­
life
of
two
hours).

As
a
relational
database,
the
EDPSD
may
be
queried
in
a
wide
variety
of
ways
to
answer
questions
in
minutes
that
would
otherwise
take
hours,
days,
or
weeks
to
answer.
As
noted
earlier,
the
EDPSD
can
be
used
to
rapidly
estimate
the
numbers
and
types
of
chemicals
in
different
data
sources
that
meet
different
criteria
(
e.
g.,
the
number
of
chemicals
with
annual
production/
importation
volumes
greater
than
one
million
pounds
per
year
and
log
octanol
water
partition
coefficients
(
LogP)
>
6
that
are
measured
in
Great
Lakes
fish
and
identified
by
California's
Proposition
65
as
reproductive
toxicants).

The
EDPSD
is
a
very
powerful
tool
for
exploring
alternative
approaches
to
the
application
of
the
criteria
described
in
Sections
III
and
IV
in
this
document.
As
described
more
fully
in
Chapter
Four,
Section
X,
G,
the
EDSTAC
recommends
that
EPA
and
the
multi­
stakeholder
group
make
full
use
of
the
EDPSD
in
an
effort
to
advise
the
Agency
on
its
final
decisions
for
priority
setting
for
T1S.
However,
the
EDSTAC
recommends
that
EPA
and
the
multi­
stakeholder
group
not
be
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
59
limited
to
data
that
can
easily
be
placed
into
a
database
format
such
as
the
EDPSD
when
providing
advice
and
making
final
decisions
on
priorities
for
T1S
(
Chapter
Four,
Section
X,
E).

D.
Preliminary
Recommendation
for
Data
Fields
to
be
Included
in
the
EDPSD
As
noted
above,
significant
progress
was
made
in
developing
the
EDPSD,
but
the
tool
was
not
completed
given
the
time
period
and
resources
available
to
the
EDSTAC.
During
the
course
of
its
work,
the
PSWG
spent
time
grappling
with
the
question
of
what
data
sources
should
be
considered
for
inclusion
in
the
EDPSD.
This
section
outlines
some
of
the
PSWG's
preliminary
conclusions,
which
should
be
a
starting
point
for
the
recommended
development
and
implementation
of
the
EDPSD
to
be
completed
by
EPA
and
the
proposed
multi­
stakeholder
group.
The
following
data
sources
should
be
included,
but
are
not
considered
to
comprise
a
final
comprehensive
list.
Rather,
they
illustrate
the
kinds
of
data
sources
that
might
be
included
in
the
final
version
of
the
EDPSD.
The
data
field
examples
(
Figure
4.2,
Tables
4.1,
and
4.2)
are
categorized
by
type,
and
each
data
field
example
is
further
described
in
Appendix
G.

E.
Special
Handling
of
Effects
Data
in
the
Context
of
the
EDPSD
The
proposed
EDPSD
is
a
relational
database
tool
that
the
EDSTAC
recommends
be
used
to
assist
in
prioritizing
chemicals
for
endocrine
disruptor
screening
and
testing.
The
prototype
EDPSD
has
purposely
been
designed
to
be
user­
friendly,
transparent,
and
flexible.
However,
these
very
qualities
make
it
difficult,
if
not
impossible,
to
include
information
from
the
general
scientific
literature
that
is
not
organized
into
accessible
numerical
or
logical
databases.
Though
this
represents
a
significant
shortcoming,
the
EDSTAC
believes
the
EDPSD
is
sufficiently
versatile
to
justify
its
use.
However,
the
EDPSD
should
not
be
used
in
isolation
from
other
"
tools,"
nor
should
it
be
used
to
perform
functions
that
do
not
lend
themselves
to
its
design.

There
are
numerous
data
sources
that
provide
toxicological,
epidemiological,
or
field
study
data
that
may
be
useful
in
prioritizing
chemicals
for
endocrine
disruptor
screening
and
testing.
Although
far
from
comprehensive,
published
studies
can
be
identified
through
widely
available
scientific
literature
databases
such
as
Medline,
Toxline,
and
NIOSHTIC.

Substance­
specific
reports
are
also
widely
available
that
include
summarized
data
reviewed
by
the
authors.
Such
reports
are
prepared
by
various
organizations
and
agencies
such
as
the
International
Agency
for
Research
on
Cancer
(
IARC),
NIOSH
(
Criteria
Documents),
ATSDR
(
Toxicologic
Profiles),
to
name
a
few.
Other
sources
of
compiled
data
exist
in
the
substancespecific
rules
and
rule­
making
dockets
of
regulatory
agencies
such
as
OSHA,
EPA,
CPSC,
or
online
data
summaries
such
as
the
EPA
IRIS
system.
Less
exhaustive
reviews
are
also
found
in
agency
investigative
reports
such
as
the
NIOSH
Health
Hazard
Evaluation
reports
or
ATSDR
hazardous
site
evaluations.
Research
grant
progress
and
final
reports
submitted
to
NIH,
EPA,
private
foundations,
etc.,
on
the
other
hand,
are
not
widely
available.
Lastly,
some
companies
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
60
Figure
4.2.
Example
of
data
fields
arranged
into
a
form
as
they
might
appear
on
a
computer
screen
CASRN
MW
Formula
Name
SMILES
Exposure­
Related
Criteria:

Biological
Sampling
Environmental
Sampling
Release
to
the
Environment
Invertebrates,
Fish,
and
Wildlife
GLC
Fish
ATSDR/
PL
Chemical
Production
or
Importation
<
10,000
lbs.
>
10,000
<
million
lbs.
>
million
<
billion
lbs.
>
billion
lbs.
Site­
limited
intermediates
Polymers
Inorganics
Fate
and
Transport
LogP
Hydrolysis
half­
life
(
d)
Atmospheric
(
OH
radical)
half­
life
(
d)
HLC
(
atm/
cum
/
mole)
VP
(
mm/
Hg)
Water
solubility
(
mg/
l)
KOC
BCF
Effects­
Related
Criteria:

Predicted
Biological
Activity/
Effect
Laboratory
Studies
Epidemiology/
Field
Studies
RTECS
TSCATS
8(
e)
HE
RTOX
TSCATS
8(
e)
EE
RTOX
Prop
65
Statutory­
Related
Criteria:

FQPA
SDWA
Active
Ingredients
Inert
Ingredients
NHATS
TRI
ER
QSAR
SDWA
CCL
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
61
Table
4.1.
Existing
files
(
and
field
type)
in
the
Endocrine
Disruptor
Priority
Setting
Database
More
information
on
the
existing
and
proposed
data
fields
recommended
for
inclusion
in
the
EDPSD
may
be
found
in
Appendix
G
of
this
report.

Generic
and
Notation
Files:

1.
Descriptive
(
all
textual)
 
CASRN,
Chemical
name,
Chemical
formula,
and
SMILES
2.
Quantitative
(
numerical)
 
Molecular
weight
Exposure­
Related
Criteria
Files:

1.
Biological
Sampling
Data
(
logical)
 
NHATS*

2.
Environmental,
Occupational,
Food,
and
Consumer
Product
Data
(
logical)
 
Great
Lakes
Fish,
Invertebrates,
Fish,
and
Wildlife,
ATSDR/
PL
3.
Environmental
Release
Data
(
logical)
 
Toxics
Release
Inventory*

4.
Production/
Importation
Volume
Data
(
logical)
 
Annual
production
volume
categories*
for
discrete
organic
chemicals
(
x
<
10,000
lbs.;
10,000
<
x
<
1,000,000
lbs.;
1,000,000
<
x
<
1,000,000,000
lbs.;
x
>
1,000,000,000);
Site­
limited
intermediates,
Polymers,
and
Inorganics
5.
Fate
and
Transport
Data
and
Models
(
all
numerical
)
 
Estimated
LogP
(
based
on
QSARs);
Hydrolysis
half­
life,
Atmospheric
half­
life,
Henry's
Law
Constant,
Vapor
pressure,
KOC,
Water
solubility,
and
Bioconcentration
factor
Effects­
Related
Criteria
Files:

1.
Toxicology
Laboratory
Studies
&
Epidemiology
and
Field
Studies
and
Databases
(
all
logical)
 
RTECS,
TSCATS
8(
e),
HE
RTOX,
EE
RTOX,
and
Proposition
65
2.
Predictive
Biological
Activity
or
Effects
Models
(
numerical)
 
Hologram
QSAR
for
estrogen
receptor
binding
Statutory­
Related
Criteria
Files:

1.
FQPA
(
logical)
 
Pesticide
active
ingredients,
Inerts*

2.
SDWA
(
logical)
 
Contaminant
Candidate
List*

(*)
Indicates
data
files
that
are
currently
logical,
but
could
be
changed
to
numerical
with
appropriate
quality
control
and
analysis.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
62
Table
4.2.
Examples
of
file
types
that
could
be
placed
in
the
Endocrine
Disruptor
Priority
Setting
Database
Exposure­
Related
Criteria
Files:

1.
Biological
Sampling
Data
 
NHANES,
TEAM,
NHEXAS
(
when
available)

2.
Environmental,
Occupational,
Consumer
Product,
and
Food­
Related
Data
 
Published
data
on
measured
concentrations
of
industrial
chemicals,
pesticide
active
ingredients
and
inerts
in
air,
drinking
water,
ground
water,
surface
water,
sediment,
and
soil
(
e.
g.,
ACGIH/
TLV,
FDA/
GRAS,
OSHA/
PEL,
FDA/
PAFA)

3.
Environmental
Release
Data
(
logical
or
numerical
)
 
ATSDR/
HSEES
(
logical
numerical),
USGS
Pesticide
Monitoring
Program
4.
Production/
Importation
Volume
Data
(
logical
or
numerical
)
 
Non­
CBI
individual
production
volumes
for
industrial
chemicals,
discrete
organic
chemicals,
polymers,
inorganics,
pesticide
active
ingredients,
and
inerts
5.
Fate
and
Transport
Data
and
Models
(
all
numerical
)
 
Measured
data
for
LogP,
Hydrolysis
half­
life,
Atmospheric
half­
life,
Henry's
Law
Constant,
Vapor
pressure,
KOC,
Water
solubility,
and
Bioconcentration
factor,
Estimated
and
measured
biodegradation
rate
data
Effects­
Related
Criteria
Files:

1.
Laboratory
Toxicology
Studies
&
Epidemiology
and
Field
Studies
and
Databases*
(
all
logical)
 
RTECS,
TSCATS
8(
e),
HE
RTOX,
EE
RTOX,
and
Proposition
65
2.
Predictive
Biological
Activity
or
Effects
Models
(
numerical)
 
Hologram
QSAR
for
estrogen
receptor
binding
Statutory­
Related
Criteria
Files:

1.
FQPA
(
logical)
 
Pesticide
active
ingredients
and
inerts*

2.
SDWA
(
logical)
 
Contaminant
Candidate
List*

(*)
Indicates
data
files
that
are
currently
logical,
but
could
be
changed
to
numerical
with
appropriate
quality
control
and
analysis.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
63
maintain
published
literature
databases
relevant
to
their
products
as
well
as
epidemiological
data
on
the
health
experience
of
their
work
force.
Unfortunately,
for
any
given
chemical
substance
or
mixture,
the
process
of
collecting
and
assessing
most
of
these
data
is
extraordinarily
time
consuming
and
resource
intensive.

For
these
reasons,
the
EDSTAC
recommends
that
EPA
make
use
of
the
potentially
valuable
information
contained
in
the
scientific
literature
in
an
efficient
and
cost­
effective
manner.
In
particular,
EPA
should
make
use
of
all
of
the
data
that
is
available
to
it
in
a
step­
wise
fashion,
starting
first
with
data
that
lends
itself
for
inclusion
in
the
EDPSD.
This
will
include
data
from
databases
such
as
RTECS
and
TSCATS,
which
are
limited
to
positive
findings
from
the
literature.
Other
databases
that
contain
abstracts
of
studies
but
are
not
limited
to
positive
findings
could
be
searched
next
for
those
chemicals
that
either
have
positive
findings
in
RTECS
or
TSCATS
or
that
warrant
further
review
due
to
the
application
of
other
effects­
related
information
or
criteria
(
e.
g.,
positive
HTPS
or
QSAR
results).
Finally,
if
necessary
and
helpful
to
the
process
of
either
making
or
justifying
the
basis
for
final
priority
setting
decisions,
EPA
could
review
the
literature
available
on
a
particular
chemical.

F.
Continued
Development
of
the
EDPSD
In
order
to
complete
data
collection
in
anticipation
of
the
use
of
the
EDPSD,
data
from
additional
files
need
to
be
included
in
the
database,
and
the
relevance
of
those
files
to
priority
setting
for
endocrine
disruptor
screening
and
testing
needs
to
be
provided
as
part
of
the
justification
for
their
addition.
All
new
chemicals
from
each
additional
file
must
include,
at
a
minimum,
CASRNs
and
molecular
weights.
All
new
discrete
organic
chemicals
from
each
additional
file
must
also
include
SMILES
notations
and
chemical
structures.

The
EDSTAC
recommends
that
EPA
provide
resources
to
complete
the
QA/
QC
investigations
of
files
that
are
currently
in
the
EDPSD.
The
EDSTAC
further
recommends
that
EPA
provide
resources
to
add
new
files
to
the
EDPSD
in
stages.
These
files
and
the
stages
for
their
addition
could
include:

1st
stage:
EPA
and
other
databases
that
provide
data
on
use
for
industrial
chemicals
and
pesticides;
information
from
pesticide
ecotoxicity,
fate,
and
toxicity
one­
liners;
chemicals
that
are
non­
food­
use
pesticide
active
ingredients
and
non­
food­
use
other
pesticide
ingredients;
chemicals
on
the
Generally
Regarded
As
Safe
(
GRAS)
list;
and
chemicals
in
the
Priority
Assessment
of
Food
Additives
(
PAFA)
database.
2nd
stage:
Data
on
chemical
use
that
were
not
readily
available
in
databases;
chemicals
and
concentrations
of
chemicals
in
National
Health
and
Nutrition
Examination
Survey
(
NHANES),
Total
Exposure
Assessment
Methodology
(
TEAM),
and
ATSDR's
Hazardous
Substances
Emergency
Events
Surveillance
(
HSEES)
files;
measured
chemical
fate
data;
and
additional
QSARs
for
endocrine
disruptors.
3rd
stage:
Inclusion
of
HTPS
data
and
improved
QSARs.

The
EDSTAC
recognizes
that
the
time
and
resources
required
to
add
new
files
will
depend
upon
a
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
64
number
of
factors,
including:
when
pending
files
are
received,
the
format
of
received
files,
the
determination
of
whether
to
use
files
as
sources
of
numerical
or
logical
data,
conversion
of
logical
files
to
numerical
files,
completion
of
QA/
QC
investigations
of
the
files
and
data,
and
expediency
of
the
input
process.

G.
Use
by
Multi­
Stakeholder
Group
The
EPA
should
convene
a
multi­
stakeholder
group
prior
to
completion
of
the
tool.
This
group
would
serve
to
ensure
that
the
tool
was
developed
and
ultimately
used
according
to
the
guidelines
provided
by
the
EDSTAC.
This
multi­
stakeholder
group
should
provide
input
and
assistance
to
EPA
in
completing
the
development
of
this
tool.
Once
the
tool
is
completed,
the
multistakeholder
group
should
be
provided
an
opportunity
to
make
use
of
the
tool
to
provide
input
on
the
priorities
for
T1S.
However,
EPA
would
ultimately
be
responsible
for
setting
priorities
for
T1S.
Presumably,
the
group
would
follow
the
approach
to
priority
setting
recommended
in
Section
XI
of
this
chapter.
Specifically,
the
group
should
make
use
of
the
EDPSD
to
understand
the
implications
of
its
recommendations
to
EPA
regarding
the
number
and
types
of
chemicals
that
should
be
included
on
the
list
of
priority
chemicals
for
T1S
in
Phase
I
of
the
program.

The
EDSTAC
recommends
that
the
multi­
stakeholder
group
convened
for
this
purpose
be
approximately
half
the
size
of
the
EDSTAC,
but
with
the
same
degree
of
balance
and
diversity
of
interests.
EPA
should
establish
ground
rules
for
the
multi­
stakeholder
group
that
encourage
the
group
to
stay
focused
on
the
development
of
a
fair
and
scientifically
sound
set
of
final
recommendations
of
priorities
for
T1S.
As
indicated
earlier,
the
ground
rules
should
encourage
the
assembled
group
not
to
use
the
EDPSD
as
a
tool
that
simply
confirms
or
justifies
a
set
of
a
priori
assumptions.

Finally,
the
EDSTAC
recommends
that
the
Agency
provide
an
opportunity
for
public
comment
on
the
content
and
structure
of
the
database
tool,
as
well
as
on
the
approach
or
way
in
which
the
Agency
intends
to
use
the
tool.
Among
other
things,
this
will
allow
an
opportunity
for
submission
of
additional
chemical­
specific
data
to
be
incorporated
into
the
database
tool.
The
EDSTAC
also
recommends
that,
after
receiving
comment
on
the
tool
itself,
EPA
propose
for
public
comment
its
T1S
priorities.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
65
H.
Maintenance
In
order
for
the
EDPSD
to
remain
a
timely
and
viable
tool,
the
EDSTAC
recommends
that
EPA
update
the
database
every
six
months
at
a
minimum,
and
more
frequently
if
time
and
resources
permit.
If
maintained
properly,
the
EDSTAC
believes
the
tool
will
not
only
provide
the
capability
to
understand
the
"
real­
world"
implications
of
alternative
approaches
to
priority
setting,
but
the
tool
will
also
have
broad
application
and
pertinence,
once
knowledge
of
the
existence
of
the
tool
spreads.

XI.
Recommended
Approach
to
Priority
Setting
A.
Introduction
The
EDSTAC's
recommended
approach
to
priority
setting
establishes
an
initial
sorting
step
to
separate
the
universe
of
chemicals
that
need
to
be
considered
for
endocrine
disruptor
screening
and
testing
into
four
distinct
categories:

1.
polymers
that
will
be
placed
into
a
"
hold"
status
(
with
some
exceptions)
pending
a
review
of
their
monomers
and
oligomers;
2.
chemicals
for
which
there
are
insufficient
data
to
proceed
to
either
T2T
or
hazard
assessment
and
will
therefore
need
to
be
prioritized
for
T1S;
3.
chemicals
for
which
sufficient
data
exists
to
go
to
T2T;
and
4.
chemicals
for
which
sufficient
data
exists
to
go
to
hazard
assessment.

In
this
concluding
section
of
the
Priority
Setting
chapter,
a
number
of
issues
are
presented
which
the
PSWG
considered
in
developing
its
recommendations,
followed
by
the
EDSTAC's
recommended
approach
to
setting
priorities
for
T1S.
Also
included
is
the
EDSTAC's
rationale
for
its
recommendation
to
rely
on
EPA's
schedule
for
tolerance
reassessments
under
the
FQPA
as
the
basis
for
setting
priorities
for
food­
use
pesticides
that
will
be
permitted
to
bypass
T1S
and
go
directly
to
T2T.

B.
Obstacles
to
an
Ideal
Priority
Setting
System
In
an
ideal
world,
EPA
would
have
sufficient
information
on
exposures
to
and
effects
from
candidate
chemicals
to
provide
a
basis
for
priority
setting.
In
reality,
existing
data
sets
are
uneven
in
quality
and
quantity.
The
EDSTAC's
review
of
available
data,
contained
in
Sections
III
and
IV
of
this
chapter
and
in
Appendix
G,
attests
to
these
problems.
Major
characteristics
of
this
unevenness
include
the
following:

·
Many
more
data
are
available
on
the
effects
of
the
relatively
small
number
of
active
ingredients
in
pesticides
(
approximately
900)
than
on
the
thousands
of
industrial
chemicals
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
66
produced
in
much
larger
quantities.
·
Biological
monitoring
data
for
humans
are
scarce.
A
relatively
small
number
of
chemicals
(
on
the
order
of
100
or
less)
have
been
routinely
sampled
in
human
blood
and
urine
in
the
United
States,
and
the
major
U.
S.
national
program
for
sampling
concentrations
in
human
tissues
was
discontinued
in
1990.
·
Monitoring
data
for
other
organisms,
while
more
numerous
than
human
data,
still
focus
on
a
relatively
small
number
of
chemicals.
·
Data
on
routine
chemical
releases
to
the
environment,
while
markedly
better
than
they
were
prior
to
the
creation
of
the
Toxic
Release
Inventory
about
10
years
ago,
still
encompass
only
528
industrial
chemicals
and
pesticides
and
frequently
rely
on
engineering
estimates
rather
than
on
actual
releases.

C.
Principles
for
Setting
Priorities
The
EDSTAC's
report
could
have
been
designed
primarily
to
assist
EPA
in
implementing
the
screening
program
provisions
of
the
FQPA
and
the
SDWA.
But,
as
noted
earlier,
the
EDSTAC
saw
its
charge
as
reaching
beyond
these
specific
statutes
and
EPA's
regulatory
authority.
The
EDSTAC
acknowledges
that
EPA's
implementation
of
these
priority
setting
recommendations
will
be
influenced
most
heavily
by
its
statutory
authorities.
Nevertheless,
the
EDSTAC
hopes
its
broad,
scientifically
derived
approach
will
encourage
voluntary
testing
behavior
within
the
private
sector
and
new
screening
and
testing
initiatives
by
other
agencies.

The
proposed
priority
setting
system
for
T1S
is
based
on
the
following
three
principles:

1.
The
system
should
be
"
transparent."

Environmental
health
concerns
in
the
United
States
are
usually
addressed
in
decisions
that
represent
a
mix
of
scientific
judgment
and
individual
and
shared
values.
Priority
setting
for
endocrine
active
chemicals
is
especially
value­
laden,
because
necessary
knowledge
of
effects
and
exposures
is
so
lacking.
There
are
many
different,
reasonable,
and
not
obviously
wrong
ways
of
deciding
how
to
apply
the
information
categories
and
criteria
identified
by
the
EDSTAC.
The
manner
in
which
these
are
used
should
identify
as
clearly
as
possible
the
weights
assigned
to
various
categories
and
the
rationales
underlying
those
weights.

2.
The
system
should
reflect
guiding
principles
derived
from
the
EDSTAC's
review
of
existing
data
on
effects
and
exposures.

Sections
III
and
IV
of
this
chapter
present
the
EDSTAC's
major
conclusions
about
the
strengths
and
limitations
of
the
information
included
in
each
exposure­
and
effects­
related
information
category,
as
well
as
on
a
set
of
guiding
principles
for
how
to
use
the
information
in
setting
priorities.
These
guiding
principles
are
principles
for
weighting
data.
A
nonexhaustive
list
includes,
for
example:
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
67
·
The
greater
the
relevance
of
a
biological
sampling
data
set
to
large
populations,
disproportionately
exposed
subpopulations,
or
particularly
susceptible
subpopulations,
the
more
weight
the
data
set
should
be
given.
·
The
more
likely
a
chemical
is
to
be
internalized
by
an
organism
from
its
environment,
the
greater
weight
it
should
be
given.
·
The
more
likely
environmental
releases
are
to
lead
to
organism
exposure,
the
greater
weight
the
release
data
should
be
given.
·
Production
volume
should
not
be
used
to
prioritize
between
existing
industrial
chemicals
and
pesticides,
because
production
volumes
for
high­
volume
industrial
chemicals
are
several
orders
of
magnitude
higher
than
those
for
pesticides.

3.
The
system
should
rely
heavily
on
empirical
data,
but
the
highest
priority
should
not
be
assigned
solely
to
those
chemicals
for
which
the
most
empirical
information
on
exposures
and
effects
has
been
gathered.

The
most
solid
evidence
of
exposures
comes
from
monitoring
of
organisms,
including
humans.
Chemicals
detected
in
organisms
should
be
weighted
heavily
in
the
priority
setting
system.
However,
the
number
of
chemicals
monitored
in
this
fashion
is
limited.
Therefore,
chemicals
that
may
not
be
widely
monitored
in
organisms
or
environmental
media,
yet
are
of
potential
concern,
should
not
be
excluded
completely
from
the
highest
priority
rankings.
Existing
empirical
data
on
selected
chemicals
can
and
should
be
used
to
improve
the
predictive
capacity
of
models
for
chemicals
lacking
empirical
data.

The
EDSTAC
also
prefers
weighting
heavily
empirical
evidence
of
effects,
at
least
until
it
is
learned
how
to
develop
better
models
for
use
in
the
assessment
process.
The
EDSTAC
recognizes
that
there
is
a
risk
that
heavily
weighting
those
chemicals
about
which
the
most
is
known
may
penalize
those
chemical
producers
who
have
evaluated
the
potential
effects
of
their
products.
The
Committee
acknowledges
this
possibility,
but
it
should
be
kept
in
perspective.
It
applies
mainly
to
active
ingredients
in
pesticides.
Since
the
food­
use
pesticides
(
approximately
500
of
almost
900
currently
registered
active
ingredients)
may
go
directly
to
T2T
anyway,
thereby
skipping
T1S,
the
availability
of
large
amounts
of
data
on
these
pesticides
will
not
raise
their
priority
for
T1S
higher.

D.
Recommended
Strategy
for
Setting
Priorities
for
Tier
1
Screening
The
EDSTAC
advocates
adoption
of
a
"
compartment­
based
priority
setting
strategy."
This
strategy
builds
directly
upon
the
several
distinct
exposure­
and
effects­
related
information
categories
and
criteria
found
in
Sections
III
and
IV,
respectively,
as
well
as
several
specially
targeted
priorities
identified
elsewhere
in
this
chapter,
including:
mixtures
(
Section
VII),
naturally
occurring
non­
steroidal
estrogens
(
Section
VIII),
and
nominations
(
Section
IX).
The
basic
premise
of
a
compartment­
based
priority
setting
strategy
is
to
establish
separate
priorities
for
a
limited
number
of
separate
compartments.
The
term
"
compartment"
simply
refers
to
the
particular
information
category
or
criterion
or
combinations
of
information
categories
or
criteria
that
define
each
set
of
priorities.
Such
compartments
can
be
defined
by
the
integration
of
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
68
exposure
and
effects
data,
the
consideration
of
exposure
data
on
their
own,
effects
data
on
their
own,
or
specially
targeted
priorities,
as
described
below.

A
compartment­
based
approach
can
be
contrasted
with
approaches
that
strive
to
develop
a
single
rank­
ordered
priority
list
that
integrates
all
exposure­
and
effects­
related
information
categories
and
criteria.
The
Committee
believes
the
proposed
compartmentalized
approach
best
accommodates
its
principles
for
priority
setting
and
the
real­
world
situation
of
uneven
data.

E.
Examples
of
Compartments
for
the
Recommended
Priority
Setting
Strategy
While
the
EDSTAC
endorses
the
general
framework
of
a
"
compartment­
based
priority
setting
strategy,"
the
specific
compartments
and
the
weights
and/
or
order
in
which
they
should
be
utilized
have
not
yet
been
agreed
upon.
Thus,
the
compartments
described
immediately
below
are
intended
solely
as
examples.

Where
the
EDSTAC
was
confident
of
the
data
that
are
pertinent
to
a
particular
compartment,
the
number
of
chemicals
estimated
to
fall
within
each
compartment
are
indicated
below.
For
some
of
the
example
compartments,
the
EDSTAC
did
not
have
sufficient
data
to
provide
estimates.
The
compartments
are
not
listed
in
order
of
agreed­
upon
priority.

As
noted
above,
the
following
examples
of
compartments
fall
within
four
major
categories:

·
Integrated
Exposure/
Effects
 
Each
of
these
compartments
draws
first
from
databases
containing
information
on
exposures.
Within
each
compartment,
priorities
are
set
on
the
basis
of
effects
data.
For
purposes
of
illustration
only,
these
data
on
effects
are
presumed
to
come
from
TSCATS,
RTECS,
HTPS,
and
QSAR
models.
These
are
the
databases
currently
projected
for
inclusion
in
the
EDPSD.
Elsewhere
in
this
chapter,
the
challenge
of
readily
assessing
effects
data,
and
the
desirability
of
taking
a
"
tiered
approach"
to
such
assessments
that
goes
more
deeply
into
or
beyond
databases
such
as
those
mentioned
specifically
above
are
described.
It
is
anticipated
that
most
of
the
chemicals
in
Phase
I
will
be
prioritized
based
on
integrated
exposure
and
effects
data.

·
Exposure
Only
 
Compartments
in
this
category
would
prioritize
chemicals
based
on
exposure
data
only,
without
using
effects
data.
It
is
anticipated
that
chemicals
in
these
compartments
would
be
relatively
few
compared
to
those
taken
from
integrated
compartments.
These
compartments
would
focus
on
identifying
chemicals
with
high
production
volumes.
Special
attention
should
be
paid
to
chemicals
for
which
there
is
evidence
of
embryonic,
post
partum
or
post
hatch,
early
life
stage,
and
pre­
maturation
exposures.

·
Effects
Only
 
Compartments
in
this
category
would
prioritize
chemicals
based
on
effects
data
only,
without
using
exposure
data.
It
is
anticipated
that
chemicals
prioritized
in
these
compartments
taken
for
screening
in
any
one
phase
would
be
relatively
few
compared
to
those
taken
from
integrated
compartments.
These
compartments
would
focus
on
identifying
chemicals
with
noteworthy
effects
data.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
69
·
Specially
Targeted
Categories
 
These
categories
 
which
presume
widespread
exposure
and
the
possibility
of
widespread
effects
 
include
mixtures,
nominations,
and
non­
steroidal
estrogens.
The
nominations
category
can
include
less
widespread,
yet
elevated
exposures
and
can
be
driven
by
reported
effects
that
might
be
associated
with
exposures
to
chemicals.

1.
Examples
of
Integrated
Exposure/
Effects
Compartments
a)
Chemicals
found
in
human
biological
samples
These
are
the
most
solid
indicators
of
human
exposure.
They
number
approximately
100
chemicals
and
include
chemicals
from
the
NHATS,
NHANES,
and
TEAM
studies
described
earlier
in
this
chapter
and
in
Appendix
G,
Table
1.
Some
of
these
substances
may
bypass
T1S
and
go
directly
to
T2T.
Priorities
for
screening
among
the
remaining
substances
can
be
established
based
on
effects
data,
with
the
highest
priority
given
to
chemicals
on
this
list
for
which
there
is
some
indication
of
possible
biological
effects.
The
EDSTAC
acknowledges
that
some
of
the
human
sampling
data
are
not
current,
but
believes
they
are
nevertheless
worthwhile
to
use.

b)
Chemicals
found
in
wildlife
samples
These
are
the
most
solid
indicators
of
wildlife
exposure.
U.
S.
Fish
and
Wildlife
Service's
Environmental
Contaminant
Data
Management
System
lists
625
compounds
and
the
Great
Lakes
Fish
Monitoring
Program
lists
over
550
compounds.
(
See
Appendix
G,
Table
1.)
Priorities
among
these
chemicals
can
be
set
based
on
effects.

c)
Highest
volume
chemical
releases
from
industrial
sites
This
component
draws
on
the
Toxic
Release
Inventory,
which
includes
528
chemicals.
Priorities
within
the
compartment
would
be
based
on
evaluation
of
effects
data.

d)
Commonly
occurring
chemicals
at
hazardous
waste
sites
ATSDR
has
published
a
list
of
the
most
commonly
occurring
chemicals
at
hazardous
waste
sites
(
Johnson
and
De
Rosa,
1995).
These
pose
a
potential
hazard
to
the
communities
in
which
these
sites
are
located
and,
to
the
extent
that
such
sites
are
located
in
lower­
income
areas,
the
presence
of
these
sites
raises
profound
issues
of
environmental
justice.
These
sites
are
distributed
broadly
across
the
United
States.
Priorities
within
this
compartment
would
be
based
on
evaluation
in
Environmental
Fate
and
Transport
models
and
assessment
of
pertinent
effects
data.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
70
e)
Cosmetics,
food
additives,
and
related
substances
within
FDA
jurisdiction
This
compartment
includes
substances
like
cosmetics
and
food
additives
which
are
eaten
or
are
intended
to
be
put
on
the
skin
of
humans.
Therefore,
exposure
is
widespread.
Priorities
for
screening
within
this
compartment
would
be
based
on
evaluation
of
data
on
effects,
to
the
extent
that
such
data
are
readily
available.

f)
Chemicals
to
which
there
is
significant
occupational
exposure
Occupational
exposures
can
be
orders
of
magnitude
higher
than
environmental
exposures.
This
compartment
includes
workplace
chemicals:
(
1)
to
which
large
numbers
of
workers
are
exposed,
or
(
2)
that
are
present
in
large
quantities/
high
concentrations
and
therefore
represent
a
disproportionately
high
risk
to
workers.
EPA
regulates
occupational
exposures
to
pesticides
and
pesticide
products.
OSHA
regulates
approximately
400
chemicals
in
the
workplace;
however,
the
majority
of
occupational
exposures
are
unregulated.
Integrating
chemicals
identified
in
this
category
with
effects
information
will
yield
a
group
of
chemicals
which
pose
a
high
risk
to
worker
populations.

g)
Chemicals
to
which
there
is
widespread
environmental
exposure
An
example,
because
of
the
potentially
large
number
of
people
exposed,
would
be
consumer
exposure.
The
EDSTAC
believes
that
chemicals
in
consumer
products
for
which
there
is
evidence
of
endocrine­
disrupting
effects
should
be
given
a
high
priority
for
screening
and
testing.
In
the
environmental
realm,
chemicals
for
which
there
is
evidence
of
their
presence
in
environmental
media
and
for
which
there
is
evidence
of
endocrine­
disrupting
effects
should,
likewise,
be
given
a
high
priority
for
screening
and
testing.

2.
Examples
of
Exposures
Only
Compartments
a)
High­
production
volume
chemicals
A
limited
number
of
chemicals
would
be
drawn
from
this
compartment.
These
chemicals
would
have
very
high
production
or
import
volumes
and
would
be
included
unless
there
were
clear
reasons
to
believe
that
exposures
would
not
be
likely
(
e.
g.,
a
chemical
is
site
limited
and
not
stable).
This
category
would
identify
chemicals
with
high
exposure
potential
that
are
unlikely
to
be
selected
in
an
exposure/
effects
integrated
approach
because
of
few
or
no
effects
data.

b)
Chemicals
to
which
there
is
widespread
or
significant
environmental,
occupational,
consumer,
or
food­
related
exposure
but
no
effects
data:

Evidence
of
widespread
or
significant
human
exposure
(
in
the
environment,
workplace,
consumer
products,
or
food)
should
be
sufficient
to
put
a
chemical
on
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
71
the
priority
list
for
screening
and
testing.
The
rationale
is
that
the
majority
of
commercial
chemicals
have
not
been
tested
for
endocrine
disrupting
effects,
and
therefore
a
proactive
approach
is
needed
for
chemicals
that
entail
significant
human
exposure.

3.
Examples
of
Effects
Only
Compartments
a)
Results
of
HTPS
A
relatively
small
number
of
chemicals
is
expected
in
this
category.
HTPS
is
designed
to
increase
available
knowledge
on
effects
of
chemicals,
especially
for
those
chemicals
about
which
little
is
known.
The
results
of
HTPS
can
assist
in
setting
priorities
within
other
compartments,
but
can
also
be
used
on
a
"
standalone
basis,
as
indicated
in
Section
V
of
this
chapter.
Alternatively,
this
compartment
could
contain
any
chemicals
that
have
a
positive
result
in
the
HTPS
assays,
but
are
not
otherwise
identified
as
a
priority
under
any
of
the
compartments
described
above.
Chemicals
in
this
compartment
could
be
ranked
based
on
HTPS
determinations
regarding
their
potency,
while
acknowledging
that
HTPS
does
not
address
the
full
range
of
endocrine
disrupting
mechanisms.

b)
Results
of
Epidemiology
Assessments
Epidemiological
analyses
may
or
may
not
provide
useful
information
about
human
exposures
to
chemicals.
Epidemiological
studies
can
provide
evidence
of
health
effects
related
to
chemicals.
The
strength
of
a
causal
association
between
exposure
and
health
effects
will
vary
depending
on
study
design
and
quality.
In
general,
the
weight­
of­
evidence
is
greatest
for
a
randomized
controlled
trial
and
weakest
for
simple
case­
reports.
Cohort
studies,
case­
control
studies,
ecological
analyses,
and
simple
demographic
or
temporal
analyses
of
disease
fall
in
between
randomized
control
trials
and
case­
reports,
providing
decreasing
weight­
ofevidence
for
a
causal
association
between
exposure
and
disease.

c)
Results
of
Laboratory
and
Field
Studies
This
compartment
would
include
chemicals
that
were
identified
by
laboratory
or
field
studies
as
having
the
potential
to
cause
effects
in
humans
or
wildlife.
However,
these
chemicals
would
have
either
no
or
inadequate
exposure
data.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
72
4.
Specially
Targeted
Compartments
a)
Mixtures
People
and
other
living
organisms
are
continually
exposed
to
mixtures.
The
EDSTAC
does
not
underestimate
the
difficulty
of
addressing
these
mixtures.
Nevertheless,
initial
steps
must
be
taken
to
understand
the
implications
of
these
exposures.
In
Section
VII,
the
EDSTAC
identified
six
types
of
mixtures
from
which
representative
samples
of
mixtures
should
be
selected
as
a
priority
for
T1S.
It
is
the
Committee's
belief
that
its
recommendations
represent
a
reasonable
and
prudent
approach.

b)
Naturally
occurring
non­
steroidal
estrogens
(
NONEs)

As
described
in
Section
VIII,
humans
and
other
living
organisms
are
broadly
exposed
to
a
wide
range
of
naturally
occurring
chemicals
that
affect
hormones.
These
substances
are
ubiquitous
in
food.
Individuals
exposed
to
them
should
be
made
aware
of
the
benefits
and
hazards
that
may
be
associated
with
their
consumption.
Based
on
such
information,
consumers
may
be
able
to
voluntarily
alter
their
diets.
As
indicated
in
Section
VIII,
twelve
such
substances
should
be
addressed
in
Phase
I.

c)
Nominations
The
EDSTAC
recommends
EPA
establish
a
process
to
allow
citizens
to
nominate
chemicals
for
endocrine
disruptor
screening
and
testing.
The
purpose,
criteria,
and
principles
that
should
guide
EPA
in
developing
and
implementing
the
recommended
process
are
described
in
Section
IX
of
this
chapter.

F.
Numbers
of
Chemicals
Prioritized
and
Associated
Weightings
of
Compartments
EPA
has
not
provided
the
EDSTAC
with
a
target
for
the
number
of
chemicals
the
Agency
believes
should
go
through
T1S
in
either
Phase
I,
subsequent
phases,
or
for
the
life
of
the
program.
The
PSWG
of
the
EDSTAC
exchanged
views
about
potential
targets
for
the
number
of
chemicals
for
each
phase
of
the
program
but
did
not
attempt
to
reach
consensus
on
this
matter
in
the
hopes
of
using
the
EDPSD
as
a
tool
that
could
be
used
to
explore
alternative
scenarios
and
targets.
The
PSWG
and
the
EDSTAC
had
hoped
to
use
the
tool
to
develop
precise
recommendations
on
how
to
structure
the
compartments
(
i.
e.,
how
many
compartments
there
should
be
and
how
many
chemicals
should
be
drawn
from
each
compartment).
However,
because
the
EDPSD
was
not
completed
before
the
drafting
of
the
EDSTAC's
final
report,
the
EDSTAC
(
and,
in
particular,
the
PSWG
that
conducted
this
work
on
the
EDSTAC's
behalf)
was
unable
to
conduct
a
"
reality
check"
on
how
the
illustrative
compartments
might
work
in
practice.

In
the
absence
of
having
a
tool
that
could
be
used
to
both
ground
its
recommendations
in
the
most
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
73
up­
to­
date
and
relevant
data
available
and
to
test
out
alternative
priority
setting
scenarios,
the
EDSTAC
was
reluctant
to
develop
recommendations
on
such
questions
as
how
many
compartments
there
should
be,
how
many
chemicals
should
be
drawn
from
each
compartment,
and
how
many
chemicals
should
be
screened
and/
or
tested
in
each
phase
of
the
program.
Additional
uncertainties
that
made
it
difficult
to
develop
such
recommendations
include
unknown
results
of
the
validation
and
standardization
process,
laboratory
capacity
to
conduct
screens
and
tests,
and
the
feasibility
of
conducting
screens
and
tests
on
chemical
substances
and/
or
mixtures
with
certain
physical
properties
(
e.
g.,
gases).

Thus,
the
number
of
chemicals
to
be
selected
for
T1S
is
a
major
unknown
in
achieving
greater
specificity
at
this
time
on
how
the
system
should
work
in
practice.
For
example,
if
only
a
small
number
of
chemicals
can
be
screened
in
Phase
I,
this
dramatically
reduces
the
number
of
chemicals
that
can
be
selected
from
each
compartment,
and
may
dictate
the
selection
of
a
smaller
number
of
compartments.
On
the
other
hand,
if
the
number
of
chemicals
to
be
screened
is
relatively
large,
this
provides
somewhat
greater
flexibility
in
selecting
chemicals
and
could
alter
the
weights
assigned
to
different
compartments.
Factors
such
as
laboratory
capacity,
private
sector
testing
response,
and
the
universe
of
eligible
chemicals
are
variables
that
may
be
considered
in
the
determination
of
the
number
of
chemicals
to
be
screened
in
Phase
I.

Whatever
number
is
chosen,
it
should
encompass
chemicals
most
widely
found
in
biological
samples,
produced
at
highest
volumes,
released
in
greatest
amounts,
and
most
likely
to
be
of
environmental
concern,
and
several
mixtures
to
which
there
is
widespread
exposure.
Moreover,
should
a
decision
be
made
to
raise
the
priority
for
screening
of
those
chemicals
that
rank
highest
in
multiple
compartments,
this
will
provide
increased
assurance
that
screening
resources
are
being
directed
where
they
can
be
most
helpful.
Beyond
the
chemicals
that
rise
to
the
top
because
of
their
high
rankings
in
multiple
compartments,
the
question
of
how
many
chemicals
should
be
selected
from
each
compartment
is
a
heavily
value­
driven
exercise.
For
example
and
for
illustrative
purposes
only,
one
could
take
all
or
almost
all
of
the
chemicals
from
a
compartment
(
e.
g.,
measured
concentrations
in
tissues
and
fluids
of
living
organisms)
that
is
deemed
highly
important
relative
to
other
compartments.

For
example
and
for
illustrative
purposes
only,
if
the
number
of
chemicals
chosen
is
relatively
large,
chemicals
could
be
prioritized
by:

·
Selecting
72%
from
the
integrated
exposure/
effects
compartments;
·
Selecting
10%
from
the
exposure­
only
compartment(
s);
·
Selecting
10%
from
the
effects­
only
compartment(
s);
and
·
Selecting
8%
from
the
specially
targeted
compartments.

Conversely,
and
again
for
example
and
for
illustrative
purposes
only,
if
the
number
of
chemicals
chosen
is
relatively
small,
chemicals
could
be
prioritized
by:

·
Selecting
60%
from
the
integrated
exposure/
effects
compartments;
·
Selecting
15%
from
the
exposure­
only
compartment(
s);
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
74
·
Selecting
15%
from
the
effects­
only
compartment(
s);
and
·
Selecting
10%
from
the
specially
targeted
compartments.

G.
Next
Steps
(
Reaching
Closure)
on
Phase
I
Priorities
for
Screening
and
Testing
The
EDSTAC
believes
it
has
created
a
strong,
logical,
transparent
basis
for
setting
priorities
for
T1S.
The
Committee
recommends
that
the
multi­
stakeholder
group,
described
in
Section
X,
G,
use
the
EDPSD
tool
to
experiment
with
the
above
categories
and
compartments
to
determine
more
finely
the
numbers
of
chemicals
that
emerge
for
T1S.
The
experiment
can
encompass
including
or
excluding
different
quantitative
thresholds
for
guiding
decisions
on
the
larger
categories
of
priority
chemicals,
including
parameters
related
to
environmental
fate
and
transport
and
parameters
related
to
reported
effects
data.

H.
Recommended
Approach
to
Setting
Priorities
for
Tier
2
Testing
During
Phase
I
of
the
EDSTP
As
described
in
Chapter
Three,
the
EDSTAC
is
recommending
that
the
owners/
producers
of
chemicals
should
be
permitted
to
bypass
T1S
under
two
alternative
scenarios.
"
Scenario
1"
covers
chemicals
for
which
two­
generation
reproductive
toxicity
studies
are
either
required
by
statute
(
i.
e.,
FIFRA),
or
where
such
studies
have
been
completed
in
the
past,
but
in
both
cases
the
studies
did
not
include
the
additional
T2T
endocrine
disruptor
endpoints
recommended
by
the
EDSTAC.
"
Scenario
2"
covers
chemicals
where
the
owner/
producer
has
decided
to
voluntarily
complete
T2T
without
having
completed
the
full
T1S
battery
or
any
prior
two­
generation
reproductive
toxicity
testing.

This
section
focuses
primarily
on
the
need
to
set
priorities
for
food­
use
pesticides
regulated
under
FQPA,
which
is
a
subset
of
chemicals
covered
under
Scenario
1,
during
the
first
phase
of
implementing
the
EDSTP.
As
discussed
below,
the
EDSTAC
recommends
that
priorities
for
conducting
T2T
on
food­
use
pesticides
should
be
based
on
the
FIFRA/
FQPA
re­
registration
and
tolerance
reassessment
processes.

Priority
setting
for
T2T
for
chemicals
other
than
food­
use
pesticides
for
which
two­
generation
reproductive
toxicity
tests
have
been
completed
in
the
past
but
where
the
chemical
is
not
regulated
under
FIFRA/
FFDCA,
as
well
as
chemicals
that
bypass
T1S
under
Scenario
2,
will
generally
be
driven
by
the
same
priorities
set
during
the
priority
setting
phase
for
T1S
unless
the
producer/
owner
of
the
chemical
wishes
to
voluntarily
expedite
testing.
In
other
words,
the
EDSTAC
recommends
that
a
chemical
which
receives
a
high
priority
ranking
for
T1S
should
retain
that
high
priority
ranking
for
T2T
even
when
the
owner
wishes
to
voluntarily
bypass
T1S.
Food­
use
pesticides
that
bypass
T1S
under
Scenario
1
are
likely
to
be
the
prime
candidates
for
the
alternative
approaches
to
completing
the
information
requirements
for
T2T
described
in
Chapter
Five,
Section
V.
It
is
also
assumed
that
it
may
be
necessary
to
assess
endocrine­
mediated
endpoints
that
had
not
been
adequately
assessed
in
past
two­
generation
reproductive
toxicity
tests
on
these
compounds.
The
determination
of
which
alternative
tests
and/
or
additional
endpoints
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
75
need
to
be
conducted
will
be
made
on
a
case­
specific
basis.

The
EDSTAC
recognizes
that
it
may
be
necessary
to
conduct
a
limited
number
of
assays
that
are
similar,
if
not
identical,
to
those
that
would
have
been
conducted
during
T1S
for
chemicals
which
are
permitted
to
bypass
the
T1S
battery.
The
purpose
of
conducting
these
assays
as
part
of
T2T
is
to
gain
knowledge
about
specific
mechanisms
of
action
necessary
to
complete
the
hazard
assessment
step
and/
or
to
determine
whether
any
adverse
effects
observed
in
T2T
are
in
fact
endocrine­
mediated.

The
decision
to
consider
pesticides
separately
for
priority
setting
was
based
on
practical
realities
associated
with
scheduling
in
EPA's
Office
of
Pesticide
Programs.
These
include
ongoing
reregistration
activities,
which
have
been
in
progress
for
more
than
a
decade,
and
new
requirements
for
tolerance
reassessment
and
registration
renewal
mandated
under
the
Food
Quality
Protection
Act.
These
represent
the
primary
scheduling
priorities
in
the
Pesticides
Program
for
the
foreseeable
future.

Under
the
re­
registration
program
mandated
in
1988,
EPA
reviews
older
pesticides
to
ensure
compliance
with
current
scientific
and
regulatory
policies.
Re­
registration
is
intended
to
update
test
data
requirements
and
standards
for
approval
which
change
over
time.
During
re­
registration
the
Agency
issues
Data
Call­
Ins
(
DCIs).
The
interval
between
issuance
of
the
DCI
and
receipt
of
data
is
dependent
upon
the
number
and
the
kind
of
studies
requested.
Presently,
re­
registration
is
being
conducted
on
compounds
for
which
DCIs
were
issued
en
masse
shortly
after
passage
of
the
1988
amendments
to
FIFRA
or
on
a
case­
specific
basis
thereafter.
For
the
most
part,
these
data
have
been
received
by
the
Agency.
Data
were
requested
for
436
active
ingredients,
and
Registration
Eligibility
Decisions
(
REDs)
have
been
issued
for
approximately
200
of
the
436
pesticide
ingredients
(
John
Housenger,
U.
S.
EPA,
personal
communication).
Generally,
neither
the
DCIs
nor
REDs
issued
to­
date
have
systematically
dealt
with
endpoints
acknowledged
to
be
endocrine­
mediated.

In
addition
to
the
re­
registration
process,
food­
use
pesticides
represent
a
category
of
pesticides
for
which
EPA
has
already
undertaken
a
hazard­
based
priority
setting
exercise.
The
food­
use
pesticides
are
being
reviewed
with
an
eye
to
tightening
regulatory
treatment
in
light
of
new
scientific
data
and
statutory
requirements.
This
priority
setting
exercise
was
mandated
by
Congress
under
Section
408(
q)(
3)
of
the
FQPA.
EPA
is
required
to
reassess
all
existing
tolerances
for
pesticide
residues
in
or
on
raw
and
processed
foods
for
both
active
and
inert
ingredients
by
August
2006.
EPA
is
directed
to
give
priority
review
to
pesticides
that
appear
to
present
risk
concerns
based
on
existing
data.
In
reassessing
tolerances,
EPA
must
consider:

·
aggregate
exposure
to
the
pesticide;
·
cumulative
effects
from
other
substances
with
a
common
mode
of
toxicity;
·
whether
there
is
an
increased
susceptibility
to
the
pesticide
for
infants
and
children;
and
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
76
·
whether
the
pesticide
produces
an
effect
in
humans
similar
to
an
effect
produced
by
a
naturally
occurring
estrogen
and
other
endocrine
effects.

The
FQPA
requires
EPA
to
review,
within
ten
years,
all
tolerances
and
exemptions
established
prior
to
FQPA's
enactment
on
August
3,
1996.
EPA
is
required
to
review
33%
of
applicable
tolerances
and
exemptions
by
August
1999,
66%
by
August
2002,
and
100%
by
August
2006.
FQPA
also
required
EPA
to
publish
its
review
schedule
within
one
year
of
the
law's
enactment,
which
EPA
did
on
August
4,
1997
(
62
FR
42019­
42030).
This
general
schedule
developed
by
EPA
for
tolerance
reassessment,
along
with
re­
registration
and
registration
renewal,
are
the
primary
driving
forces
in
scheduling
regulatory
actions
for
pesticides
and
their
formulations
and
inert
ingredients.
With
respect
to
tolerance
reassessment,
EPA
has
divided
the
pesticide
reevaluation
process
into
three
categories,
which
will
be
reviewed
in
chronological
order
over
ten
years:

·
Group
1,
the
highest
priority
class,
includes
organophosphate,
carbamate,
and
organochlorine
pesticides.
It
also
includes
pesticides
classified
by
EPA
as
probable
human
carcinogens
(
Groups
B1
and
B2
in
EPA's
carcinogen
ranking
system),
and
possible
human
carcinogens
for
which
EPA
has
quantified
a
cancer
potency
(
Group
CQ*
in
EPA's
carcinogen
ranking
system).
Group
1
also
includes
high­
hazard
inert
ingredients
and
any
pesticides
that
appear
to
exceed
their
reference
dose
(
RfD).
[
Note
that
RfD
is
defined
as
the
daily
exposure
level
of
a
pesticide
which,
during
the
entire
70­
year
human
lifetime,
appears
to
be
without
appreciable
risk
of
non­
cancer
effects
on
the
basis
of
all
of
the
facts
known
at
the
time.
It
is
expressed
in
milligrams
of
the
pesticides
as
it
appears
in
the
diet,
per
kilogram
of
body
weight
per
day
(
mg/
kg/
day).]
The
exposure
must
not
exceed
100%
of
the
RfD
to
meet
the
reasonablecertainty
of­
no­
harm
health­
based
standard
in
the
FQPA.
The
inclusion
of
certain
pesticides
in
Group
1
is
also
driven
by
EPA's
need
to
complete
their
re­
registration
by
2002,
even
though
their
tolerances
may
not
appear
to
pose
the
greatest
risk
to
public
health.
Also
in
Group
1
are
pesticides
for
which
tolerances
and
exemptions
are
in
the
process
of
being
proposed
for
revocation.
·
Group
2
includes
possible
human
carcinogens
not
included
in
Group
1.
Group
2
also
includes
remaining
pesticides
for
which
re­
registration
must
be
completed
by
2002,
and
other
pesticides
included
for
other
scheduling
reasons.
·
Group
3
includes
biological
pesticides,
those
inert
ingredients
not
identified
as
high
hazard,
and
selected
other
pesticides.
It
should
be
noted
that
biopesticides,
mainly
the
pathogenic
microorganisms,
are
probably
not
amenable
to
endocrine
disruption
screening
and
testing.

At
the
time
of
the
FQPA's
enactment,
there
were
9,728
tolerances
and
exemptions
for
active
and
formulation
inert
ingredients
subject
to
the
reassessment
requirement.
According
to
the
EPA,
8,190
of
these
are
tolerances
for
active
ingredients,
712
are
exemptions
for
active
ingredients,
and
826
are
exemptions
for
inert
ingredients
(
John
Housenger,
U.
S.
EPA,
personal
communication).
The
total
number
of
all
active
pesticide
ingredients
and
inerts
currently
registered
by
EPA
is
approximately
3,400
(
Penny
Fenner­
Crisp,
U.
S.
EPA,
personal
communication).
This
includes
approximately
900
active
ingredients
and
approximately
2,500
inerts.
(
Some
of
the
inerts
are
also
listed
in
the
TSCA
Inventory.)
Of
these
3,400,
469
active
ingredients
are
scheduled
to
be
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
77
addressed
through
the
tolerance
reassessment
process.
This
includes
228
in
Group
1
(
scheduled
for
review
by
August
1999),
93
in
Group
2
(
scheduled
for
review
by
August
2002),
and
148
in
Group
3
(
scheduled
for
review
by
August
2006).
There
are
an
additional
823
inert
ingredient
exemptions
that
will
be
dealt
with
as
part
of
Group
3.

There
are
both
advantages
and
limitations
to
using
the
re­
registration
and
tolerance
reassessment
processes
as
the
basis
for
setting
priorities
for
endocrine
disruption
screening
and
testing:
·
Re­
registration
and
tolerance
reassessment
priorities
were
not
established
specifically
with
endocrine
disruption
endpoints
in
mind.
On
the
other
hand,
registration
renewal,
which
has
yet
to
begin,
could
take
them
into
consideration.
The
current
database
on
reproduction
and
developmental
toxicity
for
most
food­
use
pesticides
reflects
the
application
of
the
1985
test
guidelines.
Non­
food­
use
pesticides
may
or
may
not
have
reproductive
or
complete
developmental
toxicity
data,
depending
on
their
specific
use
patterns.
·
The
priority
setting
process
for
food­
use
pesticides
is
driven
by
human
health
considerations,
so
the
entire
set
of
non­
human,
ecosystem­
protection
concerns
of
EDSTAC
is
not
explicitly
incorporated.
However,
most
of
the
food­
use
pesticides
with
ecological
concerns
to
nontarget
organisms
appear
on
the
Group
1
and
Group
2
lists.
·
Tolerance
reassessment
for
Group
1
pesticides
is
likely
to
be
completed
prior
to
the
completion
of
the
validation
and
standardization
of
all
EDSTAC
recommended
screens
and
tests.

Even
though
the
tolerance
reassessment
process
may
be
complete
for
Group
1
chemicals
before
the
screening
and
testing
program
is
fully
operational,
other
opportunities
will
arise
during
which
the
human
health
risk
assessments
done
for
the
ten­
year
tolerance
reassessment
exercise
will
be
revisited.
One
of
those
opportunities
will
be
during
the
fifteen­
year
registration
renewal
cycle.
Other
opportunities
may
arise
sooner
than
that,
for
example
during
the
course
of
periodic
modifications
to
the
registration
status
of
a
specific
active
and/
or
other
product
ingredient(
s).
For
instance,
requests
may
be
submitted
for
emergency
exemptions
(
Section
18'
s),
new
uses,
and/
or
modifications
to
existing
uses.
Each
of
these
actions
requires
an
updating
of
the
previous
risk
assessment.
If
the
pesticide
under
evaluation
has
been
shown
to
share
a
common
mechanism
of
action
with
other
pesticides,
the
other
pesticides
will
have
to
be
revisited,
as
well.
Also,
when
test
guidelines
are
updated,
the
program
will
assess
whether
or
not
additional
data
might
be
needed
to
upgrade
the
database
on
all
pesticides
for
which
that
test
guideline
is
appropriate.
(
Such
an
assessment
will
need
to
be
done
soon
in
light
of
the
recent
upgrading
of
the
multigeneration
reproductive
toxicity
and
developmental
toxicity
test
guidelines.)
In
light
of
these
possibilities,
the
EDSTAC
recommends
that
HTPS
data
be
used,
along
with
other
relevant
information,
to
help
prioritize
whether
and,
if
so,
when
these
pesticides
should
be
subjected
to
any
additional
endocrine
disruptor
testing
prior
to
the
next
mandated
fifteen­
year
registration
renewal
cycle.

Notwithstanding
these
disadvantages,
the
EDSTAC
recommends
that
the
priorities
EPA
has
established
for
the
re­
registration
and
tolerance
reassessment
processes
be
used
as
the
basis
for
the
priorities
for
subjecting
food­
use
pesticides
to
T2T.
When
planning
for
the
registration
renewal
process
begins,
the
FQPA
requirement
for
endocrine
disruptor
screening
and
testing
should
be
designated
as
a
criterion
for
priority
setting.
EPA's
priority
scheme
for
tolerance
reassessment
and
exemption
reviews
encompasses
many
pesticides
of
potential
concern
for
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
78
endocrine
disruption.
However,
it
leaves
out
several
hundred
non­
food­
use
active
and
inert
ingredients.
These
will
be
addressed
using
the
recommended
process
for
setting
priorities
for
T1S
as
described
above.

XII.
Compilation
of
Chapter
Four
Recommendations
A.
Summary
and
Scope
of
Effort
The
Priority
Setting
Work
Group
based
its
deliberations
on
the
original
Conceptual
Framework
described
in
Chapter
Three.
The
work
of
the
group
revolved
around
adapting
the
Conceptual
Framework
and
included
the
operational
elements
necessary
for
sorting
and
prioritizing
chemicals.
The
core
priority
setting
process
that
emerged
contained
several
elements:

·
the
use
of
all
available
existing
information;
·
the
development
of
a
relational
database
to
efficiently
access
and
utilize
information;
·
an
initial
sorting
of
the
universe
of
chemicals
into
categories
based
on
an
operationalized
Conceptual
Framework;
·
the
development
of
high
throughput
pre­
screening
data
and
its
incorporation
into
the
database;
·
the
use
of
the
database
to
summarize
empirical
data
and
estimate
fate
and
effect
parameters
where
possible;
·
the
use
of
the
database
to
establish
criteria
for
sorting
chemicals
where
appropriate;
and
·
the
use
of
a
compartment­
based
concept
to
accommodate
subjective
weighting
where
appropriate.

The
EDSTAC
viewed
its
role
within
EPA's
broader
mandate
to
protect
human
health
and
the
environment
and
the
broader
testing
authorities
available
to
EPA.
As
such,
the
EDSTAC
did
not
limit
itself
solely
to
requirements
of
the
Food
Quality
Protection
Act
and
the
Safe
Drinking
Water
Act
Amendments
of
1996.
The
Committee
believes
it
is
important
to
have
priorities
driven
by
scientific
considerations
and
explicit
value
judgments,
rather
than
by
existing
regulatory
requirements.

B.
The
Universe
of
Chemicals
and
Initial
Sorting
1.
The
EDSTAC
recommends
that
pesticides,
commodity
chemicals,
environmental
contaminants,
naturally
occurring
non­
steroidal
estrogens
(
e.
g.,
phytoestrogens,
mycotoxins),
food
additives,
cosmetics,
nutritional
supplements,
and
a
set
of
representative
mixtures
be
prioritized
for
endocrine
disruptor
screening
and
testing.

2.
The
EDSTAC
recommends
that
scientific
considerations
be
used
as
the
primary
basis
for
prioritizing
chemicals
for
endocrine
disruptor
screening
and
testing.
Statutory
authority
to
protect
human
health
and
the
environment
is
embedded
in
long­
standing
federal
legislation,
as
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
79
well
as
the
Food
Quality
Protection
Act
and
the
Safe
Drinking
Water
Act.

3.
The
EDSTAC
recommends
that
the
chemicals
under
consideration
(
approximately
87,000
compounds)
be
sorted
into
the
following
four
primary
categories
based
on
the
operationalized
Conceptual
Framework:

·
Polymers
are
to
be
placed
on
hold
(
with
some
exceptions)
pending
review
of
their
monomers,
oligomers,
other
components,
additives,
and
degradation
products
(
approximately
20,000
to
25,000
compounds).
·
Chemicals
to
be
considered
for
endocrine
disruptor
screening
(
approximately
62,000
compounds)
which
lack
sufficient
data
to
be
placed
on
hold
or
to
proceed
to
definitive
testing
or
hazard
assessment
will
be
subjected
to
the
priority
setting
process
for
T1S.
·
Chemicals
with
sufficient
data
are
to
bypass
screening
and
proceed
directly
to
testing
or
hazard
assessment
(
approximately
500
to
600
compounds).
·
Chemicals
with
sufficient
data
are
to
go
to
hazard
assessment
(
expected
to
number
approximately
50
to
100
compounds)

C.
Polymers
4.
With
some
exceptions,
the
EDSTAC
concluded
that,
due
to
molecular
weight,
polymers
are
less
cause
for
concern
than
other
classes
of
chemicals
with
regard
to
endocrine
disruption.
However,
there
is
some
concern
regarding
the
intestinal
absorption
capacity
of
neonates.
Because
of
the
lack
of
information
on
polymers
produced
prior
to
1979
(
the
date
of
the
initial
TSCA
Inventory),
coupled
with
the
low
likelihood
that
polymers
themselves
are
a
concern
for
endocrine
disruption,
the
EDSTAC
offers
the
following
recommendations.

·
All
new
polymers
with
a
number
average
molecular
weight
(
NAMW)
greater
than
1,000
daltons
and
all
previously
manufactured
(
or
"
existing")
polymers
(
regardless
of
NAMW)
are
to
be
held
from
priority
setting
for
endocrine
disruptor
screening
and
testing
pending
the
outcome
of
the
screening
and
testing
of
their
monomer,
oligomer,
and
other
components.
·
The
monomers,
oligomers,
and
other
components
of
polymers,
as
well
as
"
new"
polymers
(
i.
e.,
those
that
went
into
production
after
1979)
with
a
NAMW
less
than
1,000
daltons
are
to
undergo
priority
setting,
screening,
and
testing
as
appropriate.
·
Chemicals
on
the
EPA
SDWA
Contaminant
Candidate
List
(
CCL)
should
be
used
to
identify
the
potential
degradates
of
polymers
which
are
most
likely
to
present
environmental
exposure
and
which
should,
therefore,
be
subjected
to
priority
setting,
screening,
and
testing,
as
appropriate.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
80
·
If
monomers,
oligomers,
or
other
components
of
a
polymer
are
determined
to
have
endocrine
disrupting
properties,
an
exposure
assessment
should
be
performed.
At
this
stage,
all
potential
exposure
routes
for
a
component
would
be
determined,
including
the
potential
for
the
component
to
be
available
from
the
polymer.
·
As
the
Agency
gains
experience
with
endocrine
disruptor
screening
and
testing
of
monomers,
oligomers,
and
"
new"
polymers
(
i.
e.,
those
that
went
into
production
after
1979)
with
NAMW
less
than
1,000
daltons,
it
should
apply
that
experience
toward
development
of
an
approach
to
address
"
existing"
polymers
(
i.
e.,
those
that
went
into
production
before
1979).

D.
Priority
Setting
Information
Categories
and
Criteria
5.
The
EDSTAC
recommends
using
existing
exposure­
related
and
effects­
related
data
and
information
to
establish
criteria
for
accomplishing
initial
sorting.
The
Committee
identified
the
following
subcategories
of
information
that
could
be
used
as
the
basis
for
sorting
and
priority
setting
and
developed
principles
regarding
their
use.

Exposure­
Related
Information
and
Criteria
a)
Biological
sampling
data
b)
Environmental,
occupational,
consumer
product,
and
food­
related
data
c)
Environmental
releases
d)
Production
volume
e)
Fate
and
transport
data
and
models
Effects­
Related
Information
and
Criteria
a)
Toxicological
laboratory
studies
and
databases
b)
Epidemiological
and
field
studies
and
databases
c)
Predictive
biological
activity
or
effects
models
(
e.
g.,
SARs,
QSARs)
d)
Results
of
high
throughput
pre­
screening
E.
High
Throughput
Pre­
Screening
6.
The
EDSTAC
found
there
was
a
general
lack
of
endocrine
effects
data
for
the
vast
majority
of
chemicals.
To
address
this
problem,
the
EDSTAC
recommends
that,
if
demonstrated
to
be
feasible,
eight
in
vitro
transcriptional
activation
assays
should
be
conducted
in
a
high
throughput
pre­
screening
mode
(
i.
e.,
with
the
use
of
robotics
and
other
automated
processes).
The
objectives
for
conducting
these
assays
in
a
high
throughput
mode
is
to:

·
provide
some
information
about
the
affinity
of
chemicals
to
bind
to
the
estrogen,
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
81
androgen,
and/
or
thyroid
hormone
receptors;
·
use
this
information
in
conjunction
with
other
exposure­
and
effects­
related
information
to
determine
the
priority
by
which
chemicals
should
be
advanced
to
T1S;
·
improve
QSAR
models;
·
provide
a
source
of
information
to
help
focus
the
selection
of
Tier
2
tests
for
those
chemicals
that
bypass
T1S;
and
·
generate
data
that
can
be
used
to
identify
chemicals
that
may
be
of
concern
at
low
doses.

7.
The
EDSTAC
recommends
that
the
high
throughput
pre­
screening
(
HTPS)
transcriptional
activation
assays
be
conducted
on:

·
the
estimated
15,000
chemicals
that
are
currently
produced
in
an
amount
equal
to
or
greater
than
10,000
pounds
per
year;
·
chemicals
that
are
permitted
to
bypass
T1S
and
go
directly
to
T2T;
·
chemicals
that
are
permitted
to
bypass
both
T1S
and
T2T
and
go
directly
to
hazard
assessment;
and
·
all
pesticides
(
both
active
ingredients
and
formulation
inerts).

8.
The
EDSTAC
recommends
that
HTPS
results
for
the
"
bypass"
chemicals
not
be
used
to
set
priorities
for
T1S,
but
to
improve
QSARs
and
inform
dosing
considerations,
particularly
during
the
interim
period
when
research
on
low
dose
is
being
conducted,
and
to
inform
decisions
regarding
the
types
of
tests
that
would
need
to
be
conducted
in
T2T.

9.
The
EDSTAC
recommends
that
existing
QSAR
models
be
derived
and
supplemented
with
data
from
the
HTPS
assays,
thereby
expanding
the
predictive
ability
of
these
models.

10.
The
EDSTAC
recommends
that
EPA
explore
the
feasibility
of
creating
an
archive
of
a
subset
of
HTPS
project
chemicals
which
can
be
accessed
by
researchers
interested
in
studying
endocrine
mediated
toxicity
or
in
validating
new
screens
for
endocrine
disruptors.

F.
Mixtures
11.
The
EDSTAC
recommends
that
EPA
include
a
limited
set
of
mixtures
that
span
a
range
of
physical
and
chemical
properties
in
both
the
feasibility
demonstration
project
for
the
HTPS
assays,
as
well
as
the
validation
effort
for
the
T1S
assays.

12.
If
the
screens
are
shown
to
be
capable
of
handling
a
diverse
set
of
mixtures
in
the
HTPS
feasibility
demonstration
project
and
the
T1S
validation
steps,
EPA
should
use
expert
judgment,
guided
by
a
set
of
prioritization
criteria,
to
evaluate
the
literature
and
to
decide
on
a
limited
set
of
mixtures
to
enter
HTPS.

13.
The
battery
of
screens
validated
for
use
in
the
screening
program
should
be
used
to
evaluate
the
mixtures
examined
in
HTPS.
If
appropriate,
screening
should
be
followed
by
testing.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
82
14.
The
EDSTAC
recommends
that
a
comprehensive
literature
evaluation
be
undertaken
to
identify
exposure
and
effects
data
on
mixtures
that
do
not
undergo
HTPS.
This
information
would
be
used
to
inform
the
prioritization
for
Phase
II
and
subsequent
phases
of
the
screening
and
testing
program
which
would
use
the
same
prioritization
criteria
as
those
used
for
single
chemicals.

15.
The
EDSTAC
recommends
that
representative
sample
mixtures
be
selected
from
the
following
categories
and
be
subjected
to
HTPS
(
if
feasible)
and
to
T1S:

·
contaminants
in
human
breast
milk;
·
phytoestrogens
in
soy­
based
infant
formulas;
·
mixtures
of
chemicals
most
commonly
found
at
hazardous
waste
sites;
·
pesticide/
Fertilizer
mixtures;
·
disinfection
byproducts;
and
·
gasoline.

G.
Naturally
Occurring
Non­
Steroidal
Estrogens
(
NONEs)

16.
Naturally
occurring
non­
steroidal
estrogens
include
natural
products
derived
by
plants
(
phytoestrogens)
and
fungi
(
mycotoxins).
Due
to
the
ubiquitous
presence
of
these
compounds
in
foods,
and
due
to
the
potential
additive
and
antagonist
effects
of
NONEs
with
other
endogenous
and
exogenous
hormonally
active
chemical
substances,
the
EDSTAC
recommends
that:

·
NONEs
be
included
in
the
endocrine
disruptor
screening
and
testing
program
singly
and
in
complex
mixtures;
and
·
the
following
NONEs
be
screened
and,
if
necessary,
tested.

Representative
NONEs:

·
Isoflavones:
genistein,
daidzein,
miroestrol,
biochanin
A,
formononetin,
equol
·
Flavones:
kaemferol,
naringenin
·
Coumestans:
coumesterol
·
Dihydrochalcones:
phoretin
·
Triterpenes:
betulafolienetriol
(
ginseng)
·
Lignans:
enterolactone
Representative
estrogenic
mycotoxins:

·
Beta­
resorcyclic
lactones:
zearalenone,
zearalenol,
zearanol
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
83
H.
Nominations
17.
The
core
priority
setting
process
recommended
by
the
EDSTAC
focuses
on
giving
high
priority
to
chemicals
with
widespread
exposure
at
the
national
level.
The
EDSTAC
recognizes
such
a
process
could
result
in
a
low
priority
for
chemicals
where
exposures
are
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems.
Therefore,
the
EDSTAC
recommends
that
EPA
establish
a
nominations
process
that:

·
runs
parallel
to,
but
is
separate
and
distinct
from,
the
core
priority
setting
process;
·
is
designed
to
allow
chemical
substances
and
mixtures
for
which
there
may
not
be
widespread
exposures
on
a
national
scale,
but
for
which
there
are
exposures
on
a
smaller
scale,
to
be
eligible
to
receive
a
priority
for
T1S;
·
allows
for
an
early
opportunity
to
submit
nominations
during
each
phase
of
the
Endocrine
Disruptor
Screening
and
Testing
Program;
and
·
draws
no
less
than
5%
of
the
total
number
of
chemical
substances
or
mixtures
subjected
to
T1S
from
substances
receiving
nominations
but
not
selected
through
the
main
priority
setting
process.

18.
The
EDSTAC
recommends
that
any
nominated
chemical
substances
and/
or
mixtures
that
becomes
a
priority
for
T1S
through
the
core
priority
setting
process
be
removed
from
consideration
within
the
list
of
nominated
chemicals
in
order
to
ensure
that
the
priorities
drawn
from
the
nominations
process
will
compete
only
against
other
nominated
chemicals.

19.
In
keeping
with
the
overall
purpose
of
the
nominations
process,
the
EDSTAC
recommends
that
a
different
set
of
exposure­
related
criteria
be
used
to
evaluate
the
priority
for
nominated
chemicals
compared
to
the
exposure­
related
criteria
that
will
be
used
for
the
core
priority
setting
process.
Specifically,
the
nominations
process
should
focus
on
exposures
that
are
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems
rather
than
focusing
on
chemicals
for
which
there
is
widespread
exposure
in
the
aggregate.

20.
The
EDSTAC
recommends
that
if
there
are
effects
data
for
the
nominated
chemical,
or
if
the
chemical
is
similar
to
another
chemical
substance
or
mixture
for
which
effects
data
are
available,
EPA
should
utilize
those
data
as
a
secondary
source
of
information
to
help
set
priorities
among
nominees.

21.
The
EDSTAC
recommends
that
when
the
relative
priorities
of
nominated
chemical
substances
or
mixtures
are
evaluated,
EPA
should
consider
those
that
meet
the
following
criteria
to
be
a
higher
priority
than
those
that
do
not:

·
chemical
substances
or
mixtures
where
there
is
a
likelihood
of
regular
exposure,
in
contrast
to
those
for
which
exposure
occurs
only
rarely
or
occasionally;
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
84
·
chemical
substances
or
mixtures
that
affect
a
high
proportion
of
people
within
a
given
community
or
workplace;
and
·
chemical
substances
or
mixtures
for
which
there
may
be
empirical
or
estimated
(
i.
e.,
model
derived)
effects­
related
data
regarding
endocrine
disrupting
potential.

22.
The
EDSTAC
recommends
that
EPA
make
use
of
all
available
information
when
evaluating
nominations,
including
anecdotes,
and
other
information
gathered
as
part
of
the
core
priority
setting
process
(
e.
g.,
information
contained
within
the
Endocrine
Disruptor
Priority
Setting
Database).

23.
To
assist
EPA
in
evaluating
nominated
chemicals,
the
EDSTAC
recommends
that
EPA
request
the
following
types
of
information
from
the
public
regarding
nominations:

·
how
exposure
to
the
nominated
chemical
substances
or
mixtures
may
be
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems;
·
the
reasons
for
the
nomination
(
which
may
include
both
exposure­
and
effects­
related
concerns)
and
any
information
that
provides
a
basis
for
those
concerns;
and
·
the
degree
of
support
for
the
nomination
from
the
potentially
affected
communities
and/
or
workplaces.

I.
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD)

24.
The
EDSTAC
identified
and
evaluated
numerous
data
sources
associated
with
the
exposure
and
effects
information
categories
and
criteria
(
Appendix
G).
The
Committee
endorsed
the
integration
of
relevant
and
useful
data
sources
into
a
prototype
relational
database,
referred
to
as
the
Endocrine
Disruptor
Priority
Setting
Database.
Although
promising,
the
EDPSD
could
not
be
completed
within
the
EDSTAC's
time
and
resource
constraints.
Consequently,
EDSTAC
made
a
number
of
recommendations
regarding
continued
development
and
use
of
the
EDPSD.

·
EPA
should
continue
to
develop
and
maintain
the
EDPSD
as
a
tool
that
can
be
used
to
expeditiously
sort
and
prioritize
chemicals
for
endocrine
disruption
screening
and
testing.
·
The
process
used
by
EPA
in
developing
the
EDPSD,
as
well
as
the
process
by
which
it
is
used,
should
be
open
and
transparent.
·
EPA
should
convene
a
multi­
stakeholder
group
prior
to
the
completion
of
the
EDPSD
tool
to
ensure
effectiveness,
openness,
and
transparency.
·
After
completion
of
the
HTPS
assays,
this
group
should
make
use
of
the
tool,
along
with
the
"
compartment­
based"
approach
to
priority
setting
described
below,
in
assisting
EPA
as
it
develops
the
final
priorities
for
T1S.
·
The
EDPSD
should
not
be
limited
to
effects
data
that
can
be
easily
placed
into
a
database
format,
but
should
also
include
data
from
peer
reviewed
literature.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
85
·
EPA
should
update
the
EDPSD
at
least
every
six
months,
and
more
frequently
if
time
and
resources
permit.

25.
The
EDSTAC
recommends
that
EPA
provide
resources
to
complete
the
Quality
Assurance/
Quality
Control
investigations
of
files
that
are
currently
in
the
EDPSD.
The
EDSTAC
further
recommends
that
EPA
provide
resources
to
add
new
files
to
the
EDPSD
in
stages.
These
files
and
stages
for
their
addition
could
include:

1st
stage:
EPA's
and
others'
databases
that
provide
data
on
use
for
industrial
chemicals
and
pesticides;
information
from
pesticide
ecotoxicity,
fate,
and
toxicity
one­
liners;
chemicals
that
are
non­
food­
use
pesticide
active
ingredients
and
non­
food­
use
other
pesticide
ingredients;
chemicals
on
the
Generally
Regarded
As
Safe
(
GRAS)
list;
and
chemicals
in
the
FDA
Priority
Assessment
of
Food
Additives
(
PAFA)
database.
2nd
stage:
Data
on
chemical
use
that
were
not
readily
available
in
databases;
chemicals
and
concentrations
of
chemicals
in
National
Health
and
Nutrition
Examination
Survey
(
NHANES),
Total
Exposure
Assessment
Methodology
(
TEAM),
and
Agency
for
Toxic
Substances
Disease
Registry's
(
ATSDR)
Hazardous
Substances
Emergency
Events
Surveillance
(
HSEES)
files;
measured
chemical
fate
data;
and
additional
QSARs
for
endocrine
disruptors.
3rd
stage:
Inclusion
of
HTPS
data
and
improved
QSARs.

The
EDSTAC
recognizes
that
the
time
and
resources
required
to
add
new
files
will
depend
upon
a
number
of
factors,
including:
when
pending
files
are
received,
the
format
of
received
files,
the
determination
of
whether
to
use
files
as
sources
of
numerical
or
logical
data,
conversion
of
logical
files
to
numerical
files,
completion
of
QA/
QC
investigations
of
the
files
and
data,
and
expediency
of
the
input
process.

J.
Recommended
Approach
to
Priority
Setting
26.
The
EDSTAC
identified
a
number
of
obstacles
to
the
development
of
an
"
ideal"
priority
setting
system,
including
the
uneven
quality
and
quantity
of
both
exposure­
and,
even
more
so,
effects­
related
data
sources.
Major
characteristics
of
this
unevenness
include:

·
Many
more
data
are
available
on
the
effects
of
the
relatively
small
number
of
currently
registered
active
ingredients
in
pesticides
(
approximately
900)
than
on
the
thousands
of
industrial
chemicals
produced
in
much
larger
quantities.
·
Biological
monitoring
data
for
humans
are
scarce.
A
relatively
small
number
of
chemicals
(
on
the
order
of
100
or
less)
have
been
routinely
sampled
in
human
blood
and
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
86
urine
in
the
United
States,
and
the
major
U.
S.
national
program
for
sampling
concentrations
in
human
tissues
was
discontinued
in
1990.
·
Monitoring
data
for
other
organisms,
while
more
numerous
than
human
data,
still
focus
on
a
relatively
small
number
of
chemicals.
·
Data
on
routine
chemical
releases
to
the
environment,
while
markedly
better
than
they
were
prior
to
the
creation
of
the
Toxic
Release
Inventory
about
10
years
ago,
still
encompass
only
528
industrial
chemicals
and
pesticides
and
frequently
rely
on
engineering
estimates
rather
than
actual
releases.

27.
The
EDSTAC
recommended
several
principles
to
guide
the
development
of
a
strategy
for
setting
priorities
for
the
large
number
of
chemicals
for
which
there
are
insufficient
data
to
go
to
T2T
or
hazard
assessment.
The
selected
system
should
be
transparent,
should
make
use
of
the
guiding
principles
for
exposure­
and
effects­
related
data
sources,
and
should
be
driven
by
empirical
data,
but
not
be
held
captive
by
them.

28.
The
EDSTAC
recommends
a
"
compartment­
based
priority
setting
strategy"
for
prioritizing
chemicals
for
T1S.

·
The
strategy
builds
upon
the
identification
and
evaluation
of
the
different
exposure­
and
effects­
related
information
categories
and
criteria.
·
The
term
"
compartment"
refers
to
the
consideration
of
these
information
categories
either
singly
or
in
combination.
·
Illustrative
examples
of
the
four
different
categories
of
compartments
include:
·
the
integration
of
exposure
and
effects
information;
·
the
consideration
of
exposure
information;
·
the
consideration
of
effects
information;
and
·
specially
targeted
priorities
(
mixtures,
nominations,
and
naturally
occurring
nonsteroidal
estrogens).

The
specific
compartments
and
the
weights
and/
or
order
in
which
they
should
be
utilized
have
not
yet
been
agreed
upon.
A
target
number
of
chemicals
to
go
through
T1S
in
the
first
phase
of
the
program
or
during
the
life
of
the
program
has
not
been
determined.
Possible
targets
and
how
these
targets
might
be
affected
by
the
compartmentalized
approach
to
priority
setting
have
not
been
agreed
upon.

29.
The
EDSTAC
recommends
a
number
of
next
steps
to
further
develop
and
refine
the
compartment­
based
approach
to
priority
setting,
including:

·
use
of
the
EDPSD
by
a
multi­
stakeholder
group
to
further
characterize
and
define
what
will
be
contained
in
each
compartment;
·
whether,
and
if
so,
how
to
prioritize
the
compartments;
and
·
how
to
address
the
possibility
of
overlaps
between
compartments.
EDSTAC
Final
Report
Chapter
Four
August
1998
4
­
87
30.
The
EDSTAC
recommends
using
the
schedule
EPA
has
established
for
tolerance
reassessments
and
pesticide
re­
registration
under
the
FQPA
for
setting
priorities
for
those
food­
use
pesticides
that
meet
the
criteria
for
bypassing
T1S
and
going
directly
to
T2T.
When
planning
for
the
registration
renewal
process
begins,
the
FQPA
requirement
for
endocrine
disruptor
screening
and
testing
should
be
designated
as
a
criterion
for
priority
setting.

31.
The
EDSTAC
recommends
that
priorities
for
T2T
for
all
other
chemicals
(
i.
e.,
non­
food­
use
pesticides
and
other
chemicals
where
the
owner
either
wishes
to
voluntarily
bypass
T1S,
or
where
the
owner
has
met
the
criteria
for
completing
the
alternative,
functionally
equivalent,
T1S
assays)
should
be
established
on
a
case­
specific
basis.
However,
the
EDSTAC
recommends
that
chemicals
which
receive
a
high
priority
ranking
for
T1S
should
retain
that
high
priority
ranking
for
T2T,
even
when
the
owner
wishes
to
voluntarily
bypass
T1S.

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pp.
520­
525.
Westrom,
B.
R.,
C.
Tagesson,
et
al.,
"
Decrease
In
Intestinal
Permeability
to
Polyethylene
Glycol
1000
During
Development
in
the
Pig,"
J
Dev
Physiol.,
11(
2),
1989,
pp.
83­
87.
Wiseman,
H.,
"
Role
of
Dietary
Phyto­
Oestrogen
in
the
Protections
Against
Cancer
and
Heart
Disease,"
Bioactive
Components
of
Food,
658th
Meeting
of
the
Biochemical
Society,
Liverpool
England
(
UK)
April
16­
19,
1996
Biochemical
Society
Transactions
24
(
3),
1996,
pp.
795­
800.
EDSTAC
Final
Report
Chapter
Five
August
1998
Chapter
Five
Screening
and
Testing
EDSTAC
Final
Report
Chapter
Five
August
1998
Table
of
Contents
I.
Chapter
Overview
.................................................................................................................
1
II.
Tier
1
Screening
Concepts
and
Design
Parameters
............................................................
2
A.
Introduction
to
T1S
...........................................................................................................
3
B.
Criteria
for
T1S..................................................................................................................
3
III.
Recommended
Tier
1
Screening
Battery
...........................................................................
4
A.
Outline
of
Recommended
T1S
Battery
and
Possible
Alternatives........................................
4
1.
Recommended
T1S
Battery............................................................................................
4
2.
Alternative
Assays
for
Possible
Inclusion........................................................................
5
3.
Validation
of
the
Battery
................................................................................................
8
4.
Assays
not
Included
in
T1S
............................................................................................
8
5.
Developmental
Exposure
Screening
Assay
.....................................................................
9
6.
Methods
to
Select
the
Appropriate
Dose
Level(
s)
for
In
Vivo
Assays
...........................
10
7.
Routes
of
Administration
.............................................................................................
11
B.
Scientific
Basis
for
In
Vitro
Screening
for
Estrogen,
Androgen,
and
Thyroid
Activities.....
11
C.
In
Vitro
Assay
Overviews.................................................................................................
13
1.
Estrogen
Receptor
Assays............................................................................................
14
2.
Androgen
Receptor
Assays
..........................................................................................
17
3.
Steroidogenesis............................................................................................................
18
D.
Scientific
Basis
for
In
Vivo
Screening
for
Estrogen,
Androgen,
and
Thyroid
Activities
.....
19
1.
Unique
Thyroid
Action
Properties
to
be
Considered
in
Design
and
Interpretation
of
T1S
...................................................................................................
20
2.
In
Vivo
Assays
Using
Other
Vertebrates.......................................................................
23
E.
In
Vivo
Assay
Overviews..................................................................................................
24
1.
Rodent
3­
Day
Uterotrophic
Assay
(
Subcutaneous).......................................................
25
2.
Rodent
20­
Day
Pubertal
Female
Assay
With
Thyroid
...................................................
26
3.
Rodent
5­
7
Day
Hershberger
Assay..............................................................................
27
4.
Frog
Metamorphosis
Assay
..........................................................................................
28
5.
Fish
Gonadal
Recrudescence
Assay
..............................................................................
28
F.
Alternative
Assays
for
Possible
Inclusion
..........................................................................
29
1.
Placental
Aromatase
Assay...........................................................................................
29
2.
Modified
Rodent
3­
Day
Uterotrophic
Assay
(
Intraperitoneal).......................................
29
3.
14­
Day
Intact
Adult
Male
Assay
..................................................................................
29
4.
Rodent
20­
Day
Thyroid/
Pubertal
Male
Assay...............................................................
30
IV.
General
Principles
in
Evaluating
Tier
1
and
Tier
2
Results............................................
31
A.
Introduction.....................................................................................................................
31
B.
False
Negatives
and
False
Positives
Within
the
Context
of
T1S
and
T2T
..........................
34
C.
Specific
Principles
for
Evaluating
T1S..............................................................................
35
V.
Tier
2
Testing
Concepts
and
Design
Parameters
..............................................................
40
A.
Introduction
to
T2T
.........................................................................................................
40
B.
Outline
of
Recommended
T2T
Battery
.............................................................................
41
EDSTAC
Final
Report
Chapter
Five
August
1998
C.
Guidance
for
Selecting
Tier
2
Tests..................................................................................
42
1.
Determining
Which
Taxa
Should
be
Included
in
Tier
2
Testing
for
a
Specific
Chemical.........................................................................................................
43
2.
Determining
When
to
Perform
Alternative
Tests...........................................................
45
3.
Determining
Selection
of
Endpoints
.............................................................................
47
4.
Chemicals
That
Bypass
T1S
and
go
Directly
to
T2T.....................................................
47
5.
Potential
Need
for
Supplemental
Information
to
Complete
T2T....................................
48
D.
Low
Dose
Considerations
for
T2T...................................................................................
48
1.
Introduction
to
the
Issue
..............................................................................................
48
2.
Recommended
Project
to
Address
Low
Dose
Issues.....................................................
50
E.
Methods
to
Select
the
Target
Doses
for
T2T....................................................................
52
F.
Testing
Antithyroid
Activities
in
T2T................................................................................
53
VI.
Recommended
Tier
2
Testing
Battery
.............................................................................
54
A.
Outline
of
Recommended
T2T
Battery.............................................................................
54
B.
Two­
Generation
Mammalian
Reproductive
Toxicity
Study...............................................
54
C.
Alternative
Approaches
to
Mammalian
T2T
.....................................................................
55
1.
Alternative
Mammalian
Reproduction
Test...................................................................
58
2.
One­
Generation
Test....................................................................................................
59
D.
Description
of
the
Tests
for
Other
Animal
Taxa
...............................................................
60
1.
Avian
Reproduction
Test
.............................................................................................
61
2.
Fish
Life
Cycle
Test
.....................................................................................................
62
3.
Mysid
Life
Cycle
Test
..................................................................................................
64
4.
Amphibian
Development
and
Reproduction..................................................................
66
VII.
Validation,
Standardization,
Methods
Development,
and
Research.............................
66
A.
Concept
of
Assay
Validation
and
Standardization.............................................................
67
B.
Statutory
Need
for
Validation
..........................................................................................
67
C.
Addressing
the
Validation
Issue........................................................................................
67
D.
Validation
and
Standardization
Process............................................................................
68
E.
Levels
of
Effort
Necessary
to
Validate
the
Recommended
Screens
and
Tests....................
70
F.
Screens
and
Tests
Recommended
for
Further
Research.....................................................
71
G.
Stakeholder
Involvement
in
the
Validation
Program
.........................................................
73
H.
Preliminary
Cost
Estimates
for
the
T1S
and
T2T
Batteries
...............................................
74
VIII.
Compilation
of
Chapter
Five
Recommendations..........................................................
77
A.
Tier
1
Screening...............................................................................................................
77
B.
Principles
for
Evaluating
Tier
1
and
Tier
2
Results
...........................................................
79
C.
Tier
2
Testing
..................................................................................................................
79
D.
Validation
of
the
Screening
and
Testing
Batteries.............................................................
80
IX.
Literature
Cited
................................................................................................................
80
Figures
EDSTAC
Final
Report
Chapter
Five
August
1998
Figure
5.1
Potential
Sources
of
False
Results
in
Screening
and
Testing................................
36
Figure
5.2
False
Positives
Possibility
Graph.........................................................................
37
Figure
5.3
False
Negative
Possibility
Graph.........................................................................
38
Tables
Table
5.1
Assays
Included
in
Recommended
T1S
Battery
and
Possible
Alternatives.............
6
Table
5.2
T1S
Assays
Related
to
Biological
Activities
Detected...........................................
7
Table
5.3
Mammalian
Tier
2
Test
Endpoints......................................................................
56
Table
5.4
Avian
Reproduction
Test
Endpoints
...................................................................
63
Table
5.5
Fish
Life
Cycle
Test
Endpoints
...........................................................................
65
Table
5.6
Estimated
Costs
per
Chemical
for
Tier
1
Screening
............................................
75
Table
5.7
Estimated
Costs
per
Chemical
for
Tier
2
Testing
................................................
76
Appendices
Appendix
J:
References
and
Sources
for
Chapter
Five
­
Screening
and
Testing
Appendix
K:
Brief
Overview
of
Assays
Considered
for
Tier
1
Screening
Appendix
L:
Protocols
for
Tier
1
Screening
Assays
Appendix
M:
Assays
not
Included
in
Tier
1
Screening
Appendix
N
Endocrine
Disruption
and
Invertebrates
Appendix
O:
Protocol
for
Possible
In
Utero
Developmental
Screening
Assay
Appendix
P:
Examples
of
"
Weight­
of­
Evidence"
Determinations
Appendix
Q:
Tier
2
Testing
Study
Designs
Appendix
R:
STWG
Preliminary
Categorization
of
Tier
1
Screens
and
Tier
2
Tests
Appendix
S:
Survey
of
Cost
Estimates
for
the
EDSTAC's
Proposed
Endocrine
Disruptor
Screening
and
Testing
Assays
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
1
I.
Chapter
Overview
This
chapter
describes
the
EDSTAC
recommendations
regarding
development
of
a
screening
and
testing
program
for
assessing
the
potential
of
pesticides
and
other
chemicals
to
disrupt
endocrine
function
in
humans
and
wildlife.
Where
appropriate,
strengths
and
limitations
of
options
are
discussed
and
possible
future
research
projects
are
identified
to
develop
needed
procedures.
The
EDSTAC
established
the
Screening
and
Testing
Work
Group
(
STWG)
(
see
Appendix
D
for
a
list
of
work
group
members)
to
assist
in
their
efforts
to
provide
guidance
to
EPA
regarding
the
development
and
implementation
of
its
endocrine
disruptor
screening
and
testing
program.
The
STWG
work
formed
the
basis
for
this
chapter
and
recommendations.
Literature
cited
for
all
sections
are
found
at
the
end
of
the
chapter.
Additional
sources
can
be
found
in
Appendix
J.

After
this
introduction,
the
chapter
is
comprised
of
seven
main
sections:
(
1)
the
concepts
and
design
parameters
involved
with
Tier
1
Screening
(
T1S);
(
2)
the
Recommended
T1S
Battery;
(
3)
the
general
principles
in
evaluating
Tier
1
and
Tier
2
results;
(
4)
the
concepts
and
design
parameters
involved
with
Tier
2
Testing
(
T2T);
(
5)
the
recommended
T2T
Battery;
(
6)
a
discussion
of
validation,
standardization,
methods
development,
and
research;
and
(
7)
a
summary
of
the
recommendations
made
throughout
the
chapter.

The
T1S
sections
begin
with
an
explanation
of
the
purpose
of
screening
and
identification
of
five
criteria
used
to
design
the
screening
battery.
An
outline
of
the
recommended
T1S
battery
follows
with
brief
overviews
of
each
recommended
assay
and
discussions
of
the
value
of
including
both
in
vitro
and
in
vivo
assays.
Four
alternative
assays
for
consideration
are
also
discussed.
Finally,
a
section
on
evaluating
the
battery
includes
a
discussion
of
a
"
weight­
of­
evidence"
approach
to
evaluating
T1S
results.

In
developing
the
T1S
battery,
the
EDSTAC
considered
screening
endpoints
for
their
utility
in
screening
chemical
substances
or
mixtures
for
their
potential
to
interact
with
the
endocrine
system.
The
goal
of
T1S
is
to
detect
chemical
substances
or
mixtures
capable
of
interacting
with
estrogen,
androgen,
or
thyroid
(
EAT)
hormone
systems.
Assessing
these
activities
is
relevant
as
changes
in
them
may
adversely
affect
the
development,
reproductive
function,
or
chronic
health
status
of
humans
or
animals.
The
objective
of
T1S
is
not
to
determine
dose­
response
relationships,
confirm
the
mechanism
of
action,
or
determine
the
adversity
of
the
chemicals'
effect
on
reproduction
and/
or
development;
however,
screening
assays
must
be
sensitive
enough
to
detect
all
known
xenobiotics
that
act
via
the
mechanism
of
action
each
assay
is
designed
to
detect.

The
screening
battery
presented
here
has
been
designed
to
ensure
that
interaction
with
hormone
systems
will
be
detected.
There
are
instances
in
which
a
choice
had
to
be
made
between
an
assay
that
was
highly
specific
for
a
hormonal
activity
and
one
that
may
be
less
specific
but
more
sensitive
and
apical
(
i.
e.,
a
more
comprehensive
assessment
of
functions
that
are
relevant
to
reproduction,
development,
or
chronic
health).
In
those
instances,
the
EDSTAC
opted
for
the
latter
since
it
better
fulfills
the
first
criterion
for
screens
(
that
they
be
sensitive),
and
is
better
aligned
with
the
overall
mission
of
detecting
effects
regardless
of
mechanism
of
action.
These
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
2
assays
require
varying
levels
of
additional
development,
standardization,
and
validation
before
they
can
be
reliably
and
routinely
implemented
as
part
of
T1S.

The
T2T
sections
begin
with
discussions
of
the
purpose
of
testing,
guidance
for
selecting
Tier
2
tests,
and
the
issues
of
low
dose
considerations
and
selecting
target
doses
for
components
of
T2T.
An
outline
of
the
recommended
T2T
battery
is
followed
by
overviews
of
the
mammalian
twogeneration
reproductive
toxicity
study,
alternative
mammalian
tests,
and
tests
using
other
vertebrate
and
invertebrate
taxa.
Finally,
recommendations
regarding
implementation
of
the
standardization,
validation,
and
research
program
are
presented.

The
goal
of
T2T
is
to
determine
whether
a
chemical
substance
or
mixture
causes
endocrinemediated
adverse
effects
for
EAT
and
to
determine
the
consequences
to
the
organism
of
the
activities
observed
in
T1S
and
their
dose
response
relationships.
This
is
done
in
the
larger
context
of
testing
for
reproductive
and
developmental
toxicity
potential
by
any
mechanism
(
including
EAT)
using
study
designs
that
provide
a
comprehensive
assessment
of
relevant
functions.

II.
Tier
1
Screening
Concepts
and
Design
Parameters
Chemical
substances
or
mixtures
can
alter
endocrine
function
by
affecting
the
availability
of
a
hormone
to
the
target
tissue,
and/
or
affecting
the
cellular
response
to
the
hormone.
Mechanisms
regulating
hormone
availability
to
a
responsive
cell
are
complex
and
include
hormone
synthesis,
serum
binding,
metabolism,
cellular
uptake
(
e.
g.,
thyroid),
and
neuroendocrine
control
of
the
overall
function
of
an
endocrine
axis.
Mechanisms
regulating
cellular
response
to
hormones
are
likewise
complex
and
are
tissue
specific.
Because
the
role
of
receptors
is
often
crucial
to
cellular
responsiveness,
specific
nuclear
receptor
binding
assays
are
included.
In
addition,
tissue
responses
that
are
particularly
sensitive
and
specific
to
a
hormone
are
included
as
endpoints
for
Tier
1
screens.

The
following
definitions
are
utilized
in
this
chapter.
Estrogenic
refers
to
compounds
whose
effects
are
mediated
through
the
estrogen
receptor
(
ER),
initiating
a
cascade
of
cell/
tissue
specific
effects
similar
to
those
initiated
by
estradiol,
as
opposed
to
estrogen­
like
for
those
chemicals
resembling
estrogen
which
are
not
or
have
not
been
shown
to
be
mediated
through
the
ER.
Similarly,
androgenic
effects
are
androgen
receptor
(
AR)
mediated,
as
opposed
to
androgen­
like
effects,
which
may
not
be
mediated
via
the
AR.
In
contrast,
the
terms
antiandrogenic
and
antiestrogenic
are
not
specifically
limited
to
AR­
and
ER­
mediated
interactions.
In
this
context,
agonists
bind
to
the
receptor
and
act
like
the
endogenous
hormone;
antagonists
bind
to
the
receptor
and
appear
to
act
opposite
to
the
endogenous
hormone.
Antihormones
can
act
via:
(
1)
the
steroid
hormone
receptor;
(
2)
steroid
hormone
synthesis
inhibition;
(
3)
reduction
of
bioavailability
by
reducing
the
amount
of
free
hormone
in
the
serum;
(
4)
increased
hormone
metabolism
leading
to
reduced
serum
hormone
levels;
and
(
5)
other
mechanisms.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
3
A.
Introduction
to
T1S
The
number
of
chemicals
needing
evaluation
is
huge.
T1S
is
intended
to
make
the
evaluation
process
more
efficient
by
distinguishing
those
chemical
substances
and
mixtures
which
may
interact
with
the
endocrine
system
from
those
that
may
not.
The
EDSTAC
considered
all
known
endocrine
disruptors
of
EAT
in
developing
the
T1S
battery
and
believes
that
the
recommended
battery,
if
validated,
will
have
the
necessary
breadth,
and
depth
to
detect
all
currently
known
disruptors
of
EAT.
Therefore,
following
application
of
the
T1S
battery,
a
chemical
substances
or
mixture
will
be
designated
as
having
either:
(
1)
the
potential
for
EAT
activity,
which
will
require
further
analysis
in
T2T
to
verify
and
evaluate
that
potential;
or
(
2)
low
or
no
potential
for
EAT
activity,
which
will
allow
assignment
of
chemical
substances
or
mixtures
to
the
"
hold
box"
(
see
Section
IV
in
this
chapter,
on
general
principles
in
evaluating
Tier
1
and
Tier
2
results
for
further
discussion
of
how
this
decision
is
made).

In
developing
the
recommended
T1S
battery,
many
existing
and
potential
assays
were
evaluated
for
their
relative
strengths
and
weaknesses
(
overviews
of
evaluated
assays
can
be
found
in
Appendix
K).
The
recommended
T1S
battery
contains
mammalian
in
vitro
and
in
vivo
assays
and
in
vivo
nonmammalian
assays.
The
T1S
battery
is
designed
to
be
a
sufficiently
sensitive
screening
mechanism
so
that
chemical
substances
and
mixtures
which
ultimately
prove
to
be
endocrine
active
for
EAT
in
humans
and
wildlife
are
not
missed.
In
this
regard,
sensitivity
of
the
battery
has
been
prioritized
above
specificity.
In
addition,
T1S
results
should
inform
T2T,
in
terms
of
providing
guidance
on
which
tests
to
perform,
which
endpoints
to
include,
and
to
assist
in
determining
the
range
of
doses
to
be
used.
These
goals
include
identifying
those
doses
(
doseresponse
life
stages
(
most
sensitive),
and
organisms
(
most
appropriate,
sensitive,
and
at
risk)
in
which
adverse
effects
are
likely
to
occur.

B.
Criteria
for
T1S
The
T1S
battery
recommended
by
the
EDSTAC
has
been
developed
such
that,
at
the
completion
of
the
selected
assays,
the
EPA
and
other
stakeholders
will
accept,
both
scientifically
and
as
a
matter
of
policy,
the
assignment
of
chemical
substances
or
mixtures
as
either
having:
(
1)
low
or
no
potential
for
estrogen,
androgen,
or
thyroid
endocrine
activity;
or
(
2)
as
having
such
potential.
The
ability
to
accept
either
outcome
requires
that
the
chosen
T1S
battery
meets
the
five
criteria
identified
below.

1.
The
T1S
battery
should
maximize
sensitivity
to
minimize
false
negatives
while
permitting
an
as
of
yet
undetermined,
but
acceptable,
level
of
false
positives.
This
criterion
expresses
the
need
to
"
cast
the
screening
net
widely"
in
order
not
to
miss
potential
EAT
active
materials.

2.
The
T1S
battery
should
include
a
range
of
organisms
representing
known
or
anticipated
differences
in
metabolic
activity.
The
battery
should
include
assays
from
representative
vertebrate
classes
to
reduce
the
likelihood
that
important
pathways
for
metabolic
activation
or
detoxification
of
parent
chemical
substances
or
mixtures
are
not
overlooked.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
4
3.
The
T1S
battery
should
be
designed
to
detect
all
known
modes
of
action
for
the
endocrine
endpoints
of
concern.
All
chemicals
known
to
affect
the
action
of
EAT
hormones
should
be
detected.

4.
The
T1S
battery
should
include
a
sufficient
range
of
taxonomic
groups
among
the
test
organisms.
There
are
known
differences
in
endogenous
ligands,
receptors,
and
response
elements
among
taxa
that
may
affect
endocrine
activity
of
chemical
substances
or
mixtures.

5.
The
T1S
battery
should
incorporate
sufficient
diversity
among
the
endpoints
and
assays
to
reach
conclusions
based
on
"
weight­
of­
evidence"
considerations.
Decisions
based
on
the
battery
results
will
require
weighing
the
data
from
several
assays.

The
T1S
must
be
relatively
fast
and
efficient
while
meeting
the
criteria
described
above.
The
EDSTAC
recommends
that
if
changes
are
made
to
the
recommended
T1S
battery,
based
upon
development
of
new,
validated
assays,
the
"
amended"
battery
also
needs
to
meet
these
criteria.

III.
Recommended
Tier
1
Screening
Battery
A.
Outline
of
Recommended
T1S
Battery
and
Possible
Alternatives1
1.
Recommended
T1S
Battery
The
T1S
battery
recommended
by
the
EDSTAC
includes
three
in
vitro
assays,
three
in
vivo
mammalian
assays,
and
two
in
vivo
nonmammalian
assays.
Those
chemicals
which
go
through
the
HTPS
program,
if
it
is
technically
feasible
and
validated,
would
not
be
required
to
do
the
first
two
in
vitro
assays
at
the
bench.
Based
on
existing
data,
the
EDSTAC
believes
this
battery
will
detect
EAT
activity,
provided
all
of
the
component
assays
can
be
properly
developed,
standardized,
and
validated.

In
Vitro
1.
Estrogen
Receptor
(
ER)
Binding/
Transcriptional
Activation
Assay;
2.
Androgen
Receptor
(
AR)
Binding/
Transcriptional
Activation
Assay;
and
3.
Steroidogenesis
Assay
with
Minced
Testis.

In
Vivo
1.
Rodent
3­
Day
Uterotrophic
Assay
(
Subcutaneous);
2.
Rodent
20­
Day
Pubertal
Female
Assay
with
Thyroid;
3.
Rodent
5­
7­
Day
Hershberger
Assay;
4.
Frog
Metamorphosis
Assay;
and
1
Protocols
for
all
these
assays
can
be
found
in
Appendix
L.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
5
5.
Fish
Gonadal
Recrudescence
Assay.

2.
Alternative
Assays
for
Possible
Inclusion
In
addition,
the
EDSTAC
has
identified
one
in
vitro
assay
and
three
in
vivo
assays
as
possible
substitutes,
if
properly
developed,
standardized,
and
validated,
for
some
of
the
component
assays
in
the
recommended
battery.
These
assays
are:

In
Vitro
1.
Placental
Aromatase
Assay.

In
Vivo
1.
Modified
Rodent
3­
Day
Uterotrophic
Assay
(
Intraperitoneal);
2.
Rodent
14­
Day
Intact
Adult
Male
Assay
With
Thyroid;
and
3.
Rodent
20­
Day
Thyroid/
Pubertal
Male
Assay.

Combinations
of
the
alternative
assays,
if
validated
and
found
to
be
functionally
equivalent,
could
potentially
replace
three
of
the
component
assays
in
the
recommended
T1S
battery
(
in
vitro
steroidogenesis
assay
with
testis,
20­
day
pubertal
female
assay,
and
5­
7­
day
Hershberger
assay),
thereby
possibly
reducing
the
overall
time,
cost,
and
complexity
while
maintaining
equivalent
performance
of
the
overall
T1S
battery.
Table
5.1
shows
the
assays
included
in
the
proposed
battery
as
well
as
two
possible
batteries
that
would
include
the
alternative
assays.
In
addition,
Table
5.2
shows
the
assays
in
relation
to
which
of
the
biological
activities
they
are
expected
to
detect,
that
may
be
affected
by
exogenous
agents
and
lead
to
EAT­
related
toxicity.

One
alternative
battery
would
include
the
ER
binding
or
transcriptional
activation
assay,
the
AR
binding
or
transcriptional
activation
assay,
the
modified
rodent
3­
day
uterotrophic
assay
(
administered
by
intraperitoneal
injection),
the
rodent
14­
day
intact
adult
male
assay
with
thyroid,
the
frog
metamorphosis
assay,
the
fish
gonadal
recrudescence
assay,
and,
possibly,
the
placental
aromatase
assay.

The
other
alternative
battery
would
include
the
ER
binding
or
transcriptional
activation
assay,
the
AR
binding
or
transcriptional
activation
assay,
placental
aromatase
assay,
the
rodent
3­
day
uterotrophic
assay
(
administered
by
subcutaneous
injection),
the
rodent
20­
day
thyroid/
pubertal
male
assay,
the
frog
metamorphosis
assay,
and
the
fish
gonadal
recrudescence
assay.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
6
Table
5.1
Assays
Included
in
Recommended
T1S
Battery
and
Possible
Alternatives
Assays
Recommended
T1S
Possible
Possible
Battery
Alternative
1
Alternative
2
In
Vitro
Estrogen
receptor
binding
x
x
x
Androgen
receptor
binding
x
x
x
Steroidogenesis
x
Placental
aromatase
?
1
x
In
Vivo
3­
day
uterotrophic
x
(
sc)
2
x
(
ip)
3
x
20­
day
pubertal
female
x
Hershberger
x
14­
day
intact
male
x
20­
day
pubertal
male
x
Frog
metamorphosis
x
x
x
Fish
gonadal
recrudescence
x
x
x
1
=
may
be
needed
in
battery
to
meet
criteria
2
=
subcutaneous
3
=
intraperitoneal
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
7
Table
5.2
T1S
Assays
Related
to
Biological
Activities
Detected
Anticipated
to
Detect:

Assay
in
Option
Estrogen
Agonism
Estrogen
Antagonism
Androgen
Agonism
Androgen
Antagonism
Thyroid­
Related
Effects
Steroid
Synthesis
Aromatase
Inhibition
5­
a
­
Reductase
Inhibition
HPG1
In
Vitro
Estrogen
receptor
binding
1,2,3
X
X
Androgen
receptor
binding
1,2,3
X
X
Steroidogenesis
1
X
Placental
aromatase
3,
2?
X
In
vivo
3­
day
uterotrophic
1,2,3
X
(
X)
2
20
day
pubertal
female
1
X
X
X
X
X
X
Hershberger
1
X
LH3
Hershberger
+
T
1
(
X)
4
X
X
LH3
14­
day
intact
male
2
X
X
X
X
(
X)
5
X
X
20­
day
pubertal
male
3
X
X
X
X
X
X
X
Frog
metamorphosis
1,2,3
X
?
6
?
6
?
6
X
X
?
6
?
6
X
Fish
gonadal
recrudescence
1,2,3
X
X
X
X
?
6
X
X
?
6
X
Notes:
1
HPG
 
indicates
that
the
model
has
an
intact
hypothalamic­
pituitary­
gonadal
axis
(
except
for
the
Hershberger
assay
which
does
not
have
an
intact
HPG
axis),
and
that
effects
on
hypothalamic­
pituitary
control
of
gonadal
endocrine
function
would
be
evaluated.
2
It
is
likely
that
aromatizable
androgens
would
be
detected
in
this
assay;
however,
given
that
there
are
no
examples
of
environmental
androgens,
this
point
cannot
be
empirically
demonstrated.
3
Agents
that
affect
LH
level
would
be
detected
in
the
assay.
4
Empirical
demonstration
that
the
assay
detects
estrogens
is
limited.
The
biology
of
the
system
suggests
that
they
will
be
detected.
5
Empirical
demonstration
that
aromatase
inhibitors
are
detected
is
limited.
If
sensitivity
to
aromatase
inhibitors
is
lacking,
a
placental
aromatase
assay
would
be
added
to
this
option.
6
The
biology
of
these
organisms
suggests
that
these
effects
may
be
detectable.
However,
there
are
no
empirical
data
to
support
the
sensitivity
of
the
assay
for
these
endpoints.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
8
3.
Validation
of
the
Battery
In
order
to
provide
sufficient
data
to
allow
informed
decisions
about
the
relative
merits
of
the
recommended
T1S
battery
component
assays
and
alternative
assays
(
based
on
sensitivity,
specificity,
technical
complexity,
inter­
and
intra­
laboratory
variability,
time,
and
cost),
EDSTAC
recommends
that
validation
studies
be
initiated
on
all
of
the
assays
in
the
recommended
battery
as
well
as
the
alternatives.

If
the
assays
comprising
either
of
these
alternative
combinations
(
see
Table
5.1)
are
validated,
the
EDSTAC
recommends
performance
of
the
alternative
battery
containing
these
assays
using
the
same
standard
test
substances
recommended
for
validation
of
the
initial
battery,
which
were
selected
on
the
basis
of
predetermined
criteria
(
see
Chapter
five,
Section
VII,
G).
Sufficient
information
could
then
be
available
to
allow
an
informed
choice
between
the
recommended
battery
or
a
variation,
including
the
alternative
assays,
as
the
preferred
T1S
battery.
This
approach
would
be
most
expedient
in
the
event
that
one
or
more
of
the
recommended
battery
assays
cannot
be
properly
standardized
and
validated,
since
information
would
be
immediately
available
on
the
alternative
assays.
The
EDSTAC
believes
this
process
provides
a
model
for
validation
and
incorporation
of
new
assays,
as
they
may
be
developed
and
proposed,
into
the
T1S
battery.

The
EDSTAC
believes
it
is
critical
to
acknowledge
that
the
state­
of­
the­
science,
with
respect
to
assay
development
and
species
selection,
is
rapidly
evolving,
and
bioassays
are
currently
being
developed
that
may
offer
distinct
advantages
over
those
assays
and
species
presently
recommended
for
use.
This
is
particularly
the
case
for
selection
of
non­
mammalian
species
currently
recommended
for
use
in
in
vivo
assays.
Specific
bioassays
and
species
should
be
selected
on
a
performance­
based
approach.
As
improved
bioassays
and/
or
those
utilizing
more
appropriate
species
are
developed
and
validated,
EDSTAC
strongly
encourages
their
use
as
assays
for
screening
and/
or
testing.
Selected
assays
identified
as
research
priorities
by
the
EDSTAC
are
discussed
in
Section
VII,
F
of
this
chapter.
The
EDSTAC
recommends
that
EPA
set
up
a
specific
mechanism
for
evaluating
and
incorporating
these
and
other
new
developments,
as
appropriate,
into
the
program.

Given
the
wide
range
of
species
that
may
be
adversely
affected
by
endocrine
disruptors,
continued
development
of
screens
and
tests
is
particularly
important
to
ensure
that
a
representative
range
of
species
and
potential
endocrine­
related
effects
can
be
evaluated.

4.
Assays
not
Included
in
T1S
Currently,
there
are
no
data
available
to
suggest
that
thyroid
effects
of
chemical
substances
or
mixtures
are
mediated
through
the
receptor.
Therefore,
the
recommended
T1S
battery
does
not
currently
include
a
thyroid
receptor
(
TR)
binding
and/
or
transcriptional
activation
assay.
Nevertheless,
the
EDSTAC
is
recommending
that
the
HTPS
program
include
evaluation
of
the
TR.
The
Committee
believes
including
the
thyroid
assays
in
the
HTPS
program
will
enable
EPA,
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
9
and
others,
to
obtain
a
better
understanding,
at
relatively
low
costs,
of
whether
effects
could
be
mediated
through
the
TR.

Brief
overviews
of
all
assays
considered
by
the
STWG
can
be
found
in
Appendix
K.
In
addition,
Appendix
M
includes
more
thorough
discussions
of
assays
that
the
work
group
considered
in
detail,
but
decided
not
to
include
in
the
recommended
T1S
battery.
The
EDSTAC's
thoughts
on
the
role
of
invertebrates
in
T1S
can
be
found
in
Appendix
N.

5.
Developmental
Exposure
Screening
Assay
The
EDSTAC
considered
all
known
endocrine
disruptors
of
EAT
in
developing
the
T1S
battery
and
believes,
to
the
best
of
its
knowledge,
that
the
recommended
battery,
if
validated,
will
have
the
necessary
breadth
and
depth
to
detect
any
currently
known
disruptors
of
EAT.
There
is
a
concern,
however,
that
chemical
substances
or
mixtures
could
produce
effects
from
prenatal/
prehatch
exposure
that
would
not
be
detected
from
pubertal
or
adult
exposure.
Furthermore,
there
are
differing
views
within
the
EDSTAC
about
whether
there
is
scientific
evidence
of
known
endocrine
disruptors
or
reproductive
toxicants
that
can
affect
the
prenatal
stage
of
development
without
affecting
the
adult
or
prematuration
stages,
and
whether
effective
doses
and
affected
endpoints
may
differ
among
the
three
life
stages.

Notwithstanding
these
differing
views,
the
EDSTAC
recommends
that
EPA
take
affirmative
steps,
in
collaboration
with
industry
and
other
interested
parties,
to
attempt
to
develop
a
protocol
for
a
full
life
cycle
(
i.
e.,
with
embryonic
exposure
and
evaluation
of
the
adult
offspring)
developmental
exposure
screening
assay
that
can
be
subjected
to
validation
and
standardization.
In
addition
to
the
general
principles
and
criteria,
set
forth
in
Chapter
Three,
that
guide
selection
of
all
T1S
assays,
the
EDSTAC
believes
such
an
assay
or
assays
must
involve
prenatal
or
prehatch
exposure
and
retention
of
offspring
through
puberty
to
adulthood
and
structural,
functional,
and
reproductive
assessment.

The
EDSTAC
recognizes
it
may
be
difficult
to
develop
a
developmental
exposure
screening
assay
that
meets
both
the
criteria
specified
above,
and
the
more
general
criteria
for
selecting
T1S
assays
set
forth
in
Chapter
Three.
However,
the
EDSTAC
believes
it
is
worth
the
effort.
Furthermore,
in
Section
VII,
F,
the
EDSTAC
has
summarized
protocols
for
in
utero
and
in
ovo
developmental
screening
assays
that
could
be
further
evaluated
for
this
purpose
(
an
expanded
discussion
of
an
in
utero
protocol
is
included
in
Appendix
O).
Inclusion
of
these
protocols
is
not
intended
to
limit
the
creative
effort
that
will
be
necessary
to
achieve
the
EDSTAC's
recommendation.

Finally,
the
EDSTAC
recommends
that
if
such
an
assay
were
identified,
validated,
and
standardized,
the
decision
on
whether
it
should
be
included
in
the
T1S
battery
should
include
an
evaluation
of
its
potential
to
replace
one
or
more
of
the
recommended
T1S
assays
and
its
overall
impact
on
the
cost
effectiveness
of
the
T1S
battery.
It
should
be
noted,
however,
full
life
cycle
assessments
are
included
in
the
recommended
T2T
battery
for
mammals,
other
vertebrates,
and
invertebrates.
These
tests
will
employ
a
full
range
of
doses,
embryonic
exposures,
rearing
offspring
to
adulthood,
and
a
full
complement
of
reproductive
and
developmental
endpoints.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
10
6.
Methods
to
Select
the
Appropriate
Dose
Level(
s)
for
In
Vivo
Assays
All
T1S
in
vitro
assays
(
including
the
steroidogenesis
assay)
will
involve
multiple
dose
levels,
whether
performed
by
HTPS
or
bench
level
methods,
so
a
dose­
response
curve
and
assessment
of
relative
potencies
can
be
developed.
Results
from
the
HTPS
(
or
its
equivalent)
will
provide
potency
information
(
i.
e.,
EC
50)
relative
to
a
positive
control
such
as
17
beta
estradiol
(
E2),
diethylstilbestrol
(
DES),
testosterone,
or
T4
for
those
chemical
substances
or
mixtures
which
bind
to
the
E,
A,
or
T
receptor.
Information
on
the
in
vitro
effective
doses
of
E2,
DES,
testosterone,
or
T4
can
be
used
to
set
the
dose
level(
s),
based
on
the
validation
process,
for
the
remaining
T1S
assays
for
these
chemical
substances
or
mixtures.
There
are
no
current
data
which
indicate
that
thyroid
toxicants
act
via
binding
to
the
thyroid
hormone
receptor(
s).
Thus,
the
recommended
in
vitro
receptor
binding/
transcriptional
activation
assay
may
not
inform
dose
selection
for
in
vivo
T1S
assays
for
thyroid
endpoints.
For
these
chemical
substances
or
mixtures,
prior
information
and
range­
finding
studies
will
be
critical.

Subject
to
the
results
of
the
validation
process,
the
EDSTAC
recommends
using
one
or
more
dose
levels
in
the
performance
of
the
in
vivo
assays.
Information
to
assist
in
selecting
the
dose
level(
s)
includes:

1.
prior
information,
such
as
that
available
during
the
priority
setting
phase;
2.
results
from
the
HTPS
(
or
its
equivalent
bench­
level
assays);
and
3.
results
from
range­
finding
studies,
utilized
for
T1S
dose
selection
(
see
below).

A
range­
finding
study
can
be
performed
at
multiple
dose
levels
(
at
least
five)
with
a
few
animals
per
dose
level
and
a
limited
number
of
relevant
endpoints.
The
range­
finding
studies
specifically
performed
for
each
in
vivo
TIS
assay
will
include
the
following:

·
use
of
the
same
species
strain,
sex(
es),
and
age
as
in
the
T1S
assay;
·
use
of
the
same
route
of
administration,
vehicle,
and
duration
of
dosing
as
in
the
T1S
assay;
·
use
of
multiple
dose
levels
(
the
number
of
dose
levels
will
depend
on
the
availability
and
extent
of
prior
information);
·
use
of
multiple
animals
per
dose
level
which
may
be
fewer
than
the
number
used
per
group
in
the
T1S
assay;
·
use
of
relevant
endpoints,
which
may
be
more
limited
than
those
in
the
T1S
assay
(
for
example,
the
range­
finding
study
for
the
T1S
uterotrophic
assay
may
employ
only
body
weights
and
uterine
wet
weight,
while
the
assay
may
also
evaluate
uterine
gland
height,
serum
hormone
levels,
and/
or
vaginal
cornification,
etc.);
·
use
of
comparable
animals
(
e.
g.,
ovariectomized
females
for
the
uterotrophic
range­
finding
study
or
castrated
males
for
the
Hershberger
range­
finding
assay).
However,
there
may
be
circumstances
under
which
exceptions
occur
(
e.
g.,
use
of
intact
males
in
the
range­
finding
study
for
the
Hershberger
assay
to
define
doses
producing
systemic
toxicity
and
any
effects
on
the
reproductive
system
as
a
first
pass
approximation);
and
·
use
of
more
than
one
range­
finding
study
if
the
initial
version
does
not
identify
the
dose
level(
s)
to
be
used
in
the
specific
T1S
assay
if
necessary
by
extrapolation
or
interpolation.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
11
The
dose
level(
s)
to
be
selected
for
the
in
vivo
assays
should
not
result
in
excessive
systemic
toxicity,
but
should
result
in
effects
useful
for
detection
of
potential
EAT
disruption.
However,
no
dose
level
higher
than
one
gram/
kilogram
body
weight/
day
(
i.
e.,
a
"
limit"
dose)
should
be
utilized.
The
rationale
for
selection
of
dose
levels
for
each
range­
finding
study,
all
of
the
results
for
such
studies,
and
the
logic
employed
to
select
the
dose
level(
s)
for
the
T1S
assay
should
be
included
in
the
submission
of
T1S
results
for
evaluation
by
the
Agency
as
to
the
appropriateness
of
the
study
design,
conduct,
and
conclusions.

7.
Routes
of
Administration
The
route
of
administration
for
the
recommended
uterotrophic
assay
is
subcutaneous
(
sc)
injection
while
the
route
for
the
modified
uterotrophic
assay
and
14­
day
intact
adult
male
assay
with
thyroid
is
intraperitoneal
(
ip)
injection.
The
route
for
all
other
mammalian
in
vivo
assays
is
gavage
(
orogastric
intubation).
The
parenteral
(
non­
oral)
routes
avoid
the
first­
pass
metabolic
effect
of
the
liver
and
will
permit
detection
of
potential
EDCs
that
are
active
as
parent
compounds
and
which
undergo
significant
first­
pass
metabolism.
Hepatic
xenobiotic
metabolism
does
occur
eventually
after
parenteral
administration
(
substantially
with
ip),
so
the
potential
effects
of
metabolites
will
be
evaluated
as
well
by
these
routes.
Compounds
are
occasionally
metabolized
by
the
gut
microflora;
this
type
of
metabolism
has
been
shown
to
be
important
for
some
plantderived
estrogens.
The
oral
route
of
exposure
will
allow
for
this
type
of
metabolism.

The
EDSTAC
believes
EPA
should
propose
a
policy
for
route
of
administration
for
each
assay
and
test.
Since
T1S
seeks
activity,
an
ip
route
or
other
in
vivo
approach
seems
most
realistic
for
detecting
potential
endocrine
activity.
Conversely,
T2T
should
focus
on
developing
a
policy
for
route
of
administration
based
on
exposure
route(
s)
which
approximates
the
ecologically
relevant
exposure
pathway,
dependent
on
the
test
species
and
fate
of
the
chemical
in
the
environment.

B.
Scientific
Basis
for
In
Vitro
Screening
for
Estrogen,
Androgen,
and
Thyroid
Activities
General
agreement
has
been
reached
on
the
strengths
and
limitations
of
most
currently
available
in
vitro,
in
vivo,
and
ex
vivo
methods
for
detection
of
toxicants
that
act
via
ER,
AR,
steroid
hormone
synthesis
inhibition,
and/
or
altered
hypothalamic­
pituitary­
gonadal
(
HPG)
mechanisms.
With
this
in
mind,
several
short­
term
in
vitro
assays
for
AR
and
ER
receptor
binding
or
transcriptional
activation
and
minced
testis
steroidogenesis
inhibition
(
SI)
activity
were
identified
as
quite
useful
in
screening.
In
vitro
methods
also
include
steroidogenic
enzyme/
hormone
synthesis,
biochemical
assays,
and
in
vitro
and
testis
steroid
hormone
synthesis.

Advantages
of
in
vitro
assays
include:

a)
sensitivity
to
low
concentrations
increases
detectability;
b)
high
specificity
of
response;
c)
low
cost;
d)
small
amount
of
chemical
substance
or
mixture
required;
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
12
e)
in
vitro
assays
can
be
automated,
including
use
of
robotics;
f)
high
throughput
assays
(
thousands/
month)
can
be
developed;
g)
results
can
be
coupled
with
QSAR
models
and
for
database
screening;
h)
can
be
used
for
complex
mixtures
(
sludge,
water
contaminants);
and
i)
reduces
or
replaces
animal
use.

The
EDSTAC
recognizes
two
categories
of
in
vitro
assays
that
may
be
used
in
T1S
to
assess
the
binding
of
test
substances
to
receptors,
i.
e.,
cell­
free
assays
for
receptor
binding
and
transfected
cells
designed
to
detect
transcriptional
activation.
The
specific
assays
chosen,
whether
done
"
at
the
bench"
or
through
the
high
throughput
pre­
screening
process
(
discussed
in
detail
in
Chapter
Four,
Section
V),
should
have
the
following
characteristics:

a)
evaluate
binding
to
estrogen,
androgen,
and
perhaps
thyroid
nuclear
receptors;
b)
evaluate
binding
to
the
receptor
in
the
presence
and
absence
of
metabolic
capability
(
e.
g.,
one
or
more
of
the
P450
isozymes,
CYP1A1,
CYP3A4,
etc.);
c)
distinguish
between
agonist
and
antagonist
in
functional
assays;
and
d)
yield
dose
responses
for
relative
potency
of
chemical
substances
or
mixtures
with
endocrine
activity.

If
high
throughput
procedures
are
used,
receptor
binding
assays
should
be
performed
for
EAT
receptors.
If
the
assays
are
done
at
the
bench
level,
only
estrogen
and
androgen
receptor
assays
are
recommended
and/
or
functional
assays
should
be
performed
for
estrogen,
androgen,
and
perhaps
thyroid
receptors
(
specifically
recommended
is
a
stably
transfected
cell
line
like
the
MVLN
cell
line,
if
available,
to
assess
transcriptional
activation).
If
stably
transfected
cell
lines
are
not
available,
then
transiently
transfected
reporter
gene
assays
should
be
used.
MCF­
7
proliferation
assays
are
also
acceptable;
however,
yeast­
based
assays
are
not
recommended
at
this
time.
These
assays
can
be
performed
either
high
throughput
or
at
the
bench
level.

Receptor
binding
assays
can
use
rat,
mouse,
or
human
ER
or
AR.
These
assays
evaluate
the
ability
of
the
xenobiotic
chemical
substances
or
mixtures
to
displace
the
radio­
labeled
endogenous
ligand
from
the
binding
site,
in
a
cell­
free
or
whole
cell
system.
Relative
potency
can
be
determined
for
positive
chemical
substances
or
mixtures.
Assay
limitations
are
solubility
in
the
culture
medium,
inability
to
distinguish
agonists
from
antagonists,
lack
of
metabolic
capability,
and
risk
of
degradation
of
the
receptor.

The
functional
assay,
specifically
transcriptional
activation,
requires,
for
agonist
or
antagonist
activity,
that
the
chemical
substance
or
mixture
bind
to
the
receptor.
In
addition,
there
is
a
consequence
to
the
binding,
i.
e.,
transcription
(
synthesis
of
mRNA)
of
a
reporter
gene
and
translation
of
the
mRNA
to
an
identifiable
detectable
protein
such
as
firefly
luciferase
or
betagalactosidase
In
the
case
of
the
firefly
luciferase,
with
substrate
and
cofactors
present
in
the
culture,
there
is
a
light
flash
detected
from
formation
of
the
product
when
the
enzyme
is
synthesized
in
response
to
transcriptional
activation
and
acts
on
the
provided
substrate.
In
the
case
of
the
beta­
galactosidase,
with
substrate
and
cofactors
present
in
culture,
the
product
is
detected
colorimetrically
when
the
enzyme
is
synthesized
in
response
to
transcriptional
activation
and
acts
on
the
provided
substrate.
The
assay
uses
intact
cells
and
may
use
different
cell
lines
for
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
13
assessment
of
effects
on
EAT
binding
domains
with
transfected
(
transiently
or
permanently)
receptors
and
reporter
gene
constructs.
This
assay
can
distinguish
between
agonists
and
antagonists.
Assay
limitations
are
solubility,
toxicity,
permeability
of
the
cell
membrane,
and
lack
of
or
limited
metabolic
capability.
If
a
chemical
substance
or
mixture
must
be
metabolized
to
an
active
moiety,
it
will
not
be
detected
unless
the
limited
residual
metabolic
capacity
of
the
cultured
cells
is
sufficient
to
transform
the
chemical
to
its
active
form.
Metabolic
activity
might
be
provided
by
either
preincubating
the
chemical
substance
or
mixture
with
an
S9
fraction
(
supernatant
from
9000g
x
centrifugation
of
homogenized
liver
from
a
metabolically
induced
rat)
or
incorporating
the
S9
fraction
into
the
treatment
mixture.
In
addition,
cell
lines
are
being
genetically
engineered
to
incorporate
genes
for
P450
enzymes
as
a
method
for
extending
their
metabolic
capacity
and,
perhaps,
obviate
the
need
for
use
of
the
S9
fraction.

For
assessing
receptor
binding
in
vitro,
EDSTAC
recommends
both
the
receptor
binding
assays
and
the
transcriptional
activation
assays
be
incorporated
into
the
T1S
battery,
and
subjected
to
validation
and
standardization.
There
is
agreement
that
the
transcriptional
activation
assays
can
provide
more
information
than
the
receptor
binding
assays,
since
they
measure
not
just
binding
capacity
but
also
the
physiological
and
biochemical
consequences
of
that
binding.
However,
the
limited
database
on
the
relative
utilities
of
receptor
binding
and
transcriptional
activation
assays
do
not
allow
the
EDSTAC
to
recommend
one
category
of
assay
over
the
other
at
this
time.
Including
the
receptor
binding
and
transcriptional
activation
assays
in
the
standardization
and
validation
program
is
expected
to
provide
the
data
needed
to
reach
a
decision
on
whether
both
assays
should
be
required
or,
if
not,
whether
the
receptor
binding
or
transcriptional
activation
is
preferred.
It
is
important
to
keep
in
mind
that
these
assays
evaluate
just
one
of
the
possible
mechanisms
of
endocrine
disruption;
if
a
chemical
substance
or
mixture
acts
via
another
mechanism
than
the
receptor,
it
will
not
be
detected
in
these
assays.

Large­
scale
high
throughput
pre­
screening
(
HTPS)
programs
for
chemicals
have
been
employed,
using
standardized
in
vitro
functional
assays
(
i.
e.,
transcriptional
activation
of
a
reporter
gene),
in
the
pharmaceutical
industry.
Several
companies
involved
in
drug
design
routinely
screen
chemicals
for
hormonal
activity
on
a
large
scale
(
thousands
per
month).

In
vitro
evaluations
can
provide
both
false
positive
and
false
negative
results.
In
vitro
false
positives
(
i.
e.,
active
in
vitro
but
not
in
vivo)
arise
when
a
chemical
is
not
absorbed
or
distributed
to
the
target
tissue,
is
rapidly
metabolically
inactivated
and
excreted,
and/
or
when
some
other
form
of
toxicity
predominates
in
vivo.
False
negatives
are
considered
to
be
of
greater
concern
if
in
vitro
tests
were
used
to
the
exclusion
of
in
vivo
methods.
In
vitro
evaluations
can
result
in
false
negatives
due
to
their
inability,
or
unknown
capacity,
to
metabolically
activate
toxicants.
As
a
result,
the
EDSTAC's
recommended
battery
includes
in
vivo
methods
in
conjunction
with
in
vitro
techniques.
Nevertheless,
some
in
vitro
assays
may
offer
distinct
advantages
over
in
vivo
assays
when
investigating
the
activity
of
specific
metabolites.

C.
In
Vitro
Assay
Overviews
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
14
The
EDSTAC
recommends
a
specific
assay
for
each
of
the
ER
receptor
binding,
ER
transcriptional
activation,
AR
receptor
binding,
AR
transcriptional
activation,
and
steroidogenesis
categories
in
order
for
standardization
and
validation
to
occur
efficiently.
The
receptor
binding
and
transcriptional
activation
assays
would
be
performed
only
on
those
chemical
substances
or
mixtures
not
going
through
HTPS,
while
the
steroidogenesis
assay
would
be
performed
on
all
chemical
substances
or
mixtures
going
through
T1S.
Equivalent
assays
could
replace
these
if
they
meet
specific
performance
criteria
and
were
similarly
validated.
Even
if
HTPS
is
implemented,
standardization
and
validation
of
these
additional
in
vitro
assays
would
allow
them
to
be
conducted
in
individual
labs
on
a
more
limited
basis.
The
following
assays
are
the
specific
ones
recommended
for
inclusion
in
the
standardization
and
validation
program.

1.
Estrogen
Receptor
Assays
a)
ER
Binding:
Cell­
Free
ER
Alpha
Binding
b)
ER
Transcriptional
Activation:
MVLN
2.
AR
Assays
a)
AR
Binding:
Cell­
Free
AR
Binding
b)
AR
Transcriptional
Activation:
AR
Transcriptional
Activation
3.
Steroidogenesis
a)
Minced
testis
1.
Estrogen
Receptor
Assays
In
vitro
rat
ER
binding
assays
provide
a
rapid
and
fairly
inexpensive
method
for
quantifying
the
ability
of
chemicals
to
compete
with
DES
or
estradiol
for
ER.
The
assay
can
be
used
for
measuring
ER
in
cell­
free
extracts
obtained
from
various
tissue
homogenates
following
in
vivo
exposure
to
an
environmental
chemical.
In
addition,
the
assay
may
be
used
to
determine
the
ability
of
a
given
compound
to
compete
with
radio­
labeled
estradiol
for
binding
to
the
ER.
The
technical
aspects
of
the
ER
binding
assay
are
well
documented
for
receptors
obtained
from
cytosolic
or
nuclear
extracts
of
various
mammalian
and
other
vertebrate
tissues
(
Anderson
et
al.,
l972;
Korach
et
al.,
l979).
In
brief,
cytosolic
or
nuclear
extracts
containing
ER
are
incubated
with
[
3H]
estradiol
for
18
hours
at
4o
C
in
the
presence
or
absence
of
increasing
concentrations
of
radio­
inert
DES
or
test
chemicals.
Nonspecific
binding
is
assessed
by
the
addition
of
100
molar
excesses
of
radio­
inert
DES.
Bound
[
3H]­
and
free
ligands
are
separated
using
hydroxyapatite
extraction,
or
charcoal­
dextran
adsorption,
and
are
quantified
by
scintillation
counting.

The
ER
binding
assays
are
less
sensitive
than
the
functional
assays,
of
short­
term
duration,
and
can
be
standardized
between
laboratories.
The
assay
is
useful
for
evaluating
effects
of
a
test
compound
on
ER
distribution
and
number
following
in
vivo
exposure.
In
addition,
the
assay
can
be
used
to
rapidly
evaluate
test
compounds
for
their
ability
to
bind
to
the
ER
in
the
absence
of
any
of
their
metabolites.
Comparison
of
IC50
and
Ki
values
for
the
chemicals
tested
in
vitro
with
that
of
endogenous
and
synthetic
estrogens
provide
an
indication
of
the
potential
of
a
given
chemical
to
disrupt
ER
function
in
vivo.
However,
this
assay
does
not
distinguish
between
ER
agonist
and
antagonists.
The
cytosolic
rat
ER
binding
assay
may
also
yield
false
negative
results
if
metabolic
activation
is
required
prior
to
binding
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
15
to
the
ER
or
if
the
test
chemical
is
not
completely
solubilized
in
the
assay
buffer.
In
addition,
the
results
may
be
artifactual
if
ER
is
altered
by
detergent/
denaturation
effects
of
the
test
chemical,
particularly
if
concentrations
greater
than
10
micromolar
are
used.
At
present,
ER
binding
data
are
not
entirely
comparable
from
lab
to
lab
because
of
methodological
differences
between
labs
in
the
conduct
of
this
assay.
However
the
rat
cytosolic
ER
binding
assay
has
been
used
for
about
20
years;
it
is
less
complex
than
whole
cell
binding
assays,
and
competent
laboratories
should
be
able
to
obtain
similar
results
with
minimal
effort.

Cell­
free
and
whole­
cell
binding
assays
using
human
ER
(
hER)
are
rapidly
being
developed
and
offer
both
advantages
and
disadvantages
over
the
above
assay,
one
advantage
being
the
use
of
the
human
rather
than
the
rat
ER.
However,
being
relative
new,
they
have
not
been
standardized
in
their
examination
of
xenoestrogens.
Assays
for
ER
beta
binding
and/
or
transcriptional
activation
should
be
considered
as
they
become
more
widely
available,
and
included
in
screening
if
warranted
(
i.
e.,
if
it
is
determined
that
some
xenobiotics
bind
only
to,
or
more
avidly
to,
ER
beta
and
would
be
missed
in
current
assays
with
ER
alpha).

a.
ER
Binding
The
cell­
free
estrogen
receptor
alpha
binding
assay,
a
long­
standing
and
relatively
simple
in
vitro
assay
that
detects
specific
mechanisms
of
endocrine
activity,
is
recommended.
This
is
important
because
several
xenobiotics
display
affinity
for
the
estrogen
and/
or
androgen
receptors.
Binding
assays
identify,
but
do
not
discriminate
between,
agonists
and
antagonists.
The
apical
nature
of
these
assays
is
an
advantage
rather
than
a
limitation
because
either
activity
can
produce
adverse
reproductive
effects.
These
assays
typically
lack
metabolic
activity,
which
is
an
advantage
if
one
wishes
to
identify
the
specific
compound
with
endocrine
activity.
However,
the
lack
of
metabolic
activation
is
also
a
limitation
because
some
xenobiotics
require
metabolic
activation.

b.
ER
Transcriptional
Activation
Binding
of
estrogen
to
ER
alpha
in
target
cells
results
in
the
initiation
of
specific
transcription
activation
events.
Various
estrogen­
regulated
genes
have
been
identified
in
MCF­
7
cells
(
pS2,
Cath
D,
PgR,
TPA),
and
their
corresponding
gene
products
can
be
measured
as
an
endpoint
for
estrogen
action
(
VanderKuur
et
al.,
1993a;
Pilat
et
al.,
1993;
Davis
et
al.,
1995).
However,
such
endogenous
genes
are
additionally
regulated
by
other
cellular
mechanisms
(
Nunez
et
al.,
1989;
Cavailles
et
al.,
1989;
Zacharewski
et
al.,
1994),
and
the
quantification
of
gene
products
(
mRNA)
may
be
relatively
laborious
and
difficult.
Therefore,
the
introduction
of
artificial,
ER­
regulated
reporter
gene
constructs
into
MCF­
7
cells
has
become
a
routine
method
of
measuring
ER
transcriptional
activation
(
VanderKuur
et
al.,
1993b;
Meyer
et
al.,
1994).
These
reporter
assays
utilize
the
human
ER
of
MCF­
7
cells
for
transcriptional
regulation
of
a
reporter
gene
that
codes
for
an
exogenous
enzyme
that
can
be
easily
measured
in
a
cell
lysate.
Of
the
typical
reporter
gene
products
of
chloramphenicol
acetyl
transferase
(
CAT)
and
luciferase
(
Luc),
the
more
sensitive
assays
utilize
luciferase.
Reporter
genes
can
be
introduced
into
cells
for
the
duration
of
the
experiment
only
(
transient
transfection)
or
permanently,
creating
a
genetically
altered
subline
(
stable
transfection).
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
16
Transcriptional
activation
assays
are
a
direct
manifestation
of
receptor­
mediated
responses
on
gene
expression
(
i.
e.,
the
presence
of
a
functional
estrogen
receptor
and
a
reporter
gene
are
sufficient
to
express
estrogen­
mediated
induction).
The
MVLN
assay
(
stably
transfected
MCF­
7
cell
line
with
an
artificial
gene
including
ER
alpha,
a
controller
segment
of
vitellogenin,
and
promotor
regulating
expression
of
luciferase),
which
detects
transcriptional
activation
after
receptor
binding
using
a
luciferase
reporter
gene,
is
recommended.
This
rapid
and
sensitive
assay
(
IC50=
20
pM
range)
confirms
ER
binding
and
appropriate
controls
can
distinguish
agonists
from
antagonists.
These
assays
should
be
conducted
in
a
manner
that
allows
them
to
detect
receptor
antagonists
as
well
as
agonists.
Although
these
assays
often
provide
information
similar
to
the
above
binding
assays,
this
is
not
always
the
case,
and
there
are
well­
founded
biological
reasons
for
a
chemical
to
be
positive
in
either
the
binding
or
the
transcriptional
activation
assay
but
not
both.
However,
due
to
a
higher
degree
of
difficulty,
concern
exists
that
proper
execution
of
whole­
cell
assays
requires
a
level
of
skill
and
training
that
may
not
currently
exist
in
the
toxicology
community.
If
so,
these
assays
might
be
much
more
difficult
to
implement
than
the
binding
assays,
some
of
which
have
been
used
for
decades
and
are
less
complex.

In
spite
of
the
difficulty
of
establishing
stably
transfected
cell
lines,
various
MCF­
7
cell
derivatives
have
been
created.
As
mentioned
above,
the
MVLN
cell
line
is
an
MCF­
7
cell
derivative
containing
an
artificial
gene
consisting
of
the
ER­
controlled
segment
of
the
vitellogenin
promoter,
regulating
the
expression
of
luciferase
(
Pons
et
al.,
1990;
Gagne
et
al.,
1994).
These
cells
also
contain
a
neomycin
resistance
gene
that
was
used
in
the
stable
transfectant
selection
process.
Therefore,
since
all
MVLN
cells
contain
the
reporter
gene,
estrogen­
regulated
transcription
can
be
measured
with
a
high
sensitivity.
However,
the
metabolic
capability
of
the
MVLN
assay
has
not
been
studied
in
detail;
it
is
assumed
to
be
similar
to
that
of
MCF­
7
cells
from
which
they
are
derived.
In
principle,
there
are
several
advantages
of
this
assay
over
other
in
vitro
assays
that
assess
estrogen
action.
The
MVLN
cell
assay
is
easy
to
use
because
it
is
permanently
transfected
and
it
is
a
short­
term
assay.
In
addition,
the
MVLN
cell
assay
has
been
standardized
to
the
degree
that
is
has
been
employed
in
high
throughput
transcription
assays
involving
robotic
manipulation
of
large
numbers
of
sample
wells
containing
relatively
few
cells
(
e.
g.,
96­
well
plates).
A
procedure
that
has
been
used
to
characterize
estrogen
agonists
as
well
as
antagonists
can
be
characterized
with
the
MVLN
assay
(
Gagne
et
al.,
1994).
In
addition,
a
systematic
comparison
of
more
than
25
chemicals,
including
phthalates,
alkylphenols,
chlorinated
pesticides,
and
steroids
in
the
MVLN
and
the
MCF­
7
proliferation
assay
found
that
these
assays
were
of
equivalent
sensitivity
and
responsiveness.
Assays
like
the
MVLN
are
deemed
desirable
because
they
are
stably
transfected
and
hence
relatively
easy
to
use
and
standardize,
have
high
throughput
potential,
and
are
typically
run
to
detect
both
agonists
and
antagonists.

The
MVLN
assay
has
been
reported
to
have
a
disadvantage
though,
namely,
that
when
the
cells
are
briefly
exposed
to
hydroxytamoxifen,
their
reporter
gene
cannot
respond
to
estrogens.
The
mechanism
underlying
this
effect
is
presently
unknown.
In
principle,
avoiding
exposure
to
hydroxytamoxifen
should
prevent
this
from
happening;
however,
this
raises
the
issue
of
instability
due
to
inadvertent
exposure
to
chemicals
during
maintenance
or
propagation
of
the
cells
(
this
requires
a
serumsupplemented
medium).
The
MVLN
cells,
like
all
other
cell
culture
models,
requires
monitoring
in
order
to
ascertain
that
the
initial
response
is
preserved
through
extensive
propagation
(
Badia
et
al.,
1994).
In
addition
to
the
MVLN,
other
stably
transfected
cell
lines
have
been
or
are
being
used
to
detect
for
ER
and
AR
action.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
17
2.
Androgen
Receptor
Assays
a.
AR
Binding
The
cell­
free
AR
binding
assay,
used
to
determine
the
ability
of
environmental
chemicals
to
compete
with
endogenous
ligand
for
binding
to
AR,
is
recommended.
This
is
an
easy,
time­
honored
task,
with
decades
of
use,
and
relatively
simple
to
standardize
and
execute.
Equilibrium
binding
assays
require
overnight
incubation
at
4oC
with
AR
isolated
from
castrated
rat
reproductive
tissues
(
e.
g.,
epididymis,
ventral
prostate,
seminal
vesicle)
with
increasing
concentrations
of
radio­
labeled
ligand
at
different
fixed
concentrations
of
inhibitor
or
a
fixed
concentration
of
labeled
androgen
with
increasing
concentrations
of
unlabeled
competitors.
Following
the
incubation,
hydroxyapatite
or
dextran­
coated
charcoal
is
used
to
separate
protein­
bound
ligand
from
free
ligand
and
specific
binding
is
plotted
in
double
reciprocal
plots
(
i.
e.,
Lineweaver­
Burke)
and
as
Scatchard
plots
as
a
function
of
competing
inhibitor
concentrations.
Data
analysis
yields
apparent
equilibrium
binding
affinity
constants
for
the
inhibitor
(
Ki),
which
reflects
the
affinity
of
the
chemical
for
the
AR.
Ki
values
can
be
used
to
rank
chemicals
for
their
ability
to
bind
AR
and
therefore
for
their
potential
to
be
endocrine
active.
IC50
values
can
be
used
to
calculate
Ki
values
and
the
relative
binding
affinity
(
RBA)
of
the
toxicant
for
AR,
as
compared
to
DHT
or
T,
but
this
method
is
less
accurate
than
experimental
determination
of
the
Ki.
Within
the
last
few
years,
a
surprising
number
of
chemicals
in
the
environment
of
anthropogenic
origin
have
been
shown
to
act
as
AR
ligands,
including
pesticides
(
e.
g.,
vinclozolin,
procymidone),
pesticide
metabolites
(
p,
p'
DDE
and
other
DDT
metabolites,
methoxychlor
metabolites),
hydroxylated
PCBs,
and
steroidal
and
non­
steroidal
natural
and
synthetic
estrogens
(
Waller
et
al.,
1996).

Advantages
of
the
cell­
free
binding
assay
include
ease
of
use,
low
cost,
the
potential
to
standardize
receptor
preparations
for
distribution
to
many
labs,
and
metabolism
(
but
not
spontaneous
degradation)
of
chemicals
in
the
assay
is
minimized.
The
absence
of
metabolism
is
an
important
consideration
as
parent
chemicals
and/
or
metabolites
can
be
individually
examined
to
determine
which
structure
is
responsible
for
AR
binding,
information
that
is
critical
if
the
data
are
to
be
used
in
a
QSAR
model.
Disadvantages
include
the
need
for
radio­
labeled
ligands
and
that
data
are
restricted
only
to
ligand
binding
affinity
with
no
information
on
agonist
or
antagonist
activity,
AR
stabilization,
or
degradation
or
rates
of
association
and
dissociation
from
the
AR.

b.
AR
Transcriptional
Activation
For
AR­
mediated
activity,
stably
transfected
cell
lines
are
under
development,
but
not
yet
widely
available.
The
AR
transcriptional
activation
(
Cis­
Trans)
assay,
using
monkey
kidney
CV­
1
cells,
is
recommended.
A
MCF­
7
cell
stably
transfected
with
wild
type
androgen
receptor
has
recently
become
available;
however,
only
a
few
androgen
agonists
and
antagonists
have
been
tested
using
this
cell
proliferation
assay
(
Szelei
et
al.,
1997).
Hence,
like
the
CV­
1,
cell
lines
transiently
cotransfected
with
hAR
and
a
promoter
construct
with
a
Luc
reporter
are
recommended
at
this
time.
It
is
noteworthy
that
as
compared
to
MCF­
7
cells,
the
CV­
1
has
some
metabolic
capability.
Here
again,
the
YAS
is
not
acceptable
as
it
is
unable
to
detect
the
AR­
mediated
activity
of
chlorinated
pesticides.

Cells
transiently
transfected
with
hAR
and
reporter
construct
to
detect
transcriptional
activation
after
receptor
binding
distinguish
agonist/
antagonist.
Such
assays
have
been
used
extensively
and
can
be
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
18
employed
in
a
HTS
mode
for
rapid
screening.
Transcriptional
activation
assays
are
used
to
determine
whether
chemicals
which
bind
AR
act
as
AR
agonists
or
antagonists
(
Zhou
et
al.,
1994;
Simental
et
al.,
1991).
CV­
1
cells
are
transiently
transfected
with
the
hAR
expression
vector
together
with
a
reporter
construct
(
e.
g.,
chloramphenicol
acetyl
transferase
(
CAT),
beta­
galactosidase,
or
firefly
luciferase)
containing
an
AR­
dependent
promoter
such
as
the
mouse
mammary
tumor
virus
promoter.
Transfected
cells
are
cultured
in
the
presence
(
for
antagonist
activity)
or
absence
(
for
agonist
activity)
of
a
single
concentration
of
androgen
(
0.1
nM
DHT)
together
with
increasing
concentrations
of
inhibitor.
Following
a
48
hour
culture
period,
cells
are
harvested
and
luciferase
activity
is
measured
in
the
resultant
solubilized
cell
extract
as
an
estimate
of
AR­
induced
transcriptional
activity.

Advantages
of
these
types
of
assays
are
that
they
use
human
AR,
they
display
some
metabolic
activity,
and
they
establish
whether
a
chemical
that
binds
hAR
acts
as
an
agonist
or
antagonist.
This
information
is
critical
in
understanding
the
mechanism
responsible
for
the
induction
of
adverse
endocrine­
mediated
effects.
Disadvantages
of
these
assays
are
that
they
require
the
AR
expression
vector,
reporter
vectors,
and
transient
cotransfections,
which
can
be
difficult.
The
assay
requires
close
adherence
to
the
standard
operating
procedure
for
reproducibility,
and
a
48
hour
incubation
during
which
time
metabolism
of
the
treatment
chemicals
may
confound
the
data.
In
this
regard,
media
from
this
assay,
and
other
in
vitro
assays,
should
be
analyzed
before
and
after
the
incubation
period
to
account
for
potential
degradation
and
metabolism
of
the
exogenous
test
chemicals
and
hormones.

3.
Steroidogenesis
Antiandrogens
and
antiestrogens
act
via
a
number
of
direct
mechanisms
in
addition
to
those
that
directly
involve
the
steroid
hormone
receptors.
One
prominent
mechanism
of
antihormonal
activity
is
inhibition
of
hormone
synthesis
by
inhibiting
the
activity
of
P450
enzymes
in
the
steroid
(
and
fungal)
pathway.
Such
activity
can
be
detected
in
vitro
with
a
fairly
simple
in
vitro
procedure
with
minced
testicular
tissue
obtained
from
adult
male
rats,
because
for
many
of
the
pesticides
known
to
alter
this
pathway
the
parent
material
is
active.
Although
aromatase,
another
P450
enzyme
is
present
only
at
very
low
levels
in
the
testis
and
male
reproductive
tract,
it
was
proposed
that
inhibition
of
aromatase
need
not
be
included
in
vitro
because
it
will
be
assessed
in
the
in
vivo
pubertal
female
assay
that
follows.
However,
aromatase
activity
cannot
be
assessed
in
the
recommended
testis
culture
assay
or
in
any
of
the
in
vivo
assays
using
male
rats.

The
testis
culture
in
vitro
assay
using
minced
(
50
mg)
pieces
of
single
testis,
which
can
be
used
to
evaluate
hormone
synthesis
with
and
without
stimulation
with
cAMP,
hCG,
or
substrates,
is
recommended.
This
assay
assesses
non­
receptor
mediated
effects
on
P450
steroidogenic
enzymes.
Incomplete
metabolism
in
vitro
is
of
concern,
except
for
those
classes
of
chemicals
where
the
parent
material
is
active
(
e.
g.,
certain
classes
of
fungicides,
drugs,
and
agricultural
products).
This
assay
has
been
used
with
fetal,
neonatal,
and
adult
testis,
and
is
not
limited
to
mammalian
species,
having
been
used
to
assess
steroidogenesis
in
fish,
reptile,
avian,
and
amphibian
systems
as
well.

It
is
also
possible
to
use
cultures
of
Leydig
cells
isolated
from
testicular
tissue
to
perform
steroidogenesis
assays.
Leydig
cells
are
the
cells,
within
the
testis,
responsible
for
steroid
synthesis.
The
advantage
of
using
these
isolates
is
that
they
are
enriched
for
the
cells
that
synthesize
testosterone.
The
disadvantage
is
that
there
are
extra
steps
in
the
preparation
of
the
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
19
cells.
Both
approaches
are
expected
to
be
comparable
in
their
ability
to
detect
steroidogenesis
inhibitors.
The
utility
of
the
minced
testis
culture
is
primarily
based
on
data
generated
using
Leydig
cell
cultures
(
Klinefelter
and
Kelce,
1996).

Substances
that
interfere
with
steroidogenesis
act
primarily
by
inhibiting
cytochrome
P450
enzymes
in
the
steroid
pathway.
For
example,
the
mechanism
of
action
of
two
major
classes
of
herbicides,
the
imidazoles
and
the
triazoles,
involves
inhibition
of
P450
enzymes
in
the
sterol
synthesis
pathway
for
lanosterol,
a
vital
component
of
fungal
membranes
(
Taton
et
al.,
1988).
Cytochrome
P450
inhibitors
tend
to
be
non­
specific,
and
these
fungicides
can
also
inhibit
other
P450
enzymes
such
as
those
required
for
mammalian
steroid
hormone
synthesis
(
Murray
and
Reidy,
1990).
Inhibition
of
mammalian
steroid
synthesis
can
potentially
result
in
a
broad
spectrum
of
adverse
effects
in
vivo,
including
abnormal
serum
hormone
levels,
pregnancy
loss,
delayed
parturition,
demasculinization
of
male
pups,
lack
of
normal
male
and
female
mating
behavior,
altered
estrous
cyclicity,
and
altered
reproductive
organ
weights.

D.
Scientific
Basis
for
In
Vivo
Screening
for
Estrogen,
Androgen,
and
Thyroid
Activities
The
EDSTAC
believes
inclusion
of
in
vivo
methods
in
T1S
can
help
reduce
false
negatives
in
the
absence
of
knowledge
of
absorption,
distribution,
metabolism,
and
excretion.
In
vivo
assays
are
often
apical
(
that
is,
while
they
incorporate
endocrine­
specific
endpoints,
disruption
of
a
number
of
hormone
regulation/
delivery
mechanisms
can
be
evaluated
in
the
same
assay).
Therefore,
they
are
less
specific,
but
more
comprehensive,
than
in
vitro
assays.
In
vivo
assays
can
be
made
more
specific
if
accompanied
by
target
organ/
cell
dosimetry
of
biologically
active
metabolites.
In
vitro
data
are
enhanced
if
the
actual
concentration
of
the
chemicals
in
the
media
is
determined,
to
account
for
metabolism,
stability,
and
solubility,
and
to
determine
whether
these
concentrations
compare
to
those
that
can
be
achieved
in
vivo.
Cellular
assays
should
determine
viability,
and
the
specificity
and
limitations
of
each
assay
should
be
defined.
It
is
clear
a
combination
of
in
vivo
and
in
vitro
assays
is
necessary
in
order
to
detect
EAT
alterations
that
act
via
the
ER,
AR,
TR,
inhibition
of
steroid
hormone
synthesis,
and/
or
alterations
of
the
hypothalamic­
pituitary­
gonadal
(
HPG)
and
HPT
(
thyroid)
axis.

More
than
50
assays,
and
related
endpoints,
were
considered
by
the
STWG,
including
in
vitro,
in
vivo,
and
ex
vivo
(
in
vivo
dosing
followed
by
in
vitro
assessment
of
function)
techniques.
In
vivo
endpoints
considered
include
reproductive
organ
weights
and
histology,
serum
hormone
levels,
in
vivo
gene
activation,
protein
synthesis,
behavior,
growth,
development,
pregnancy
maintenance,
and
anatomy/
morphology.
For
each
endpoint,
the
sensitivity
(
defined
here
as
the
response
of
the
assay
to
low
concentrations
or
dosage
levels),
specificity
(
pathognomonic
for
a
mechanism
of
action,
since
the
lack
of
specificity
leads
to
false
positives),
relative
simplicity,
difficulties
encountered
running
the
assay,
confounding
factors,
and
limitations,
test
duration,
and
costs
were
discussed.
In
addition,
items
such
as
degree
of
acceptance
of
the
method,
how
many
chemicals
had
been
screened,
and
the
relative
"
newness"
of
the
assay
(
state­
of­
the­
art)
were
considered.

Advantages
of
in
vivo
assays
include:
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
20
a)
account
for
absorption,
distribution,
metabolism,
and
excretion;
b)
well­
defined,
acceptable
methods
used
for
decades;
c)
general
acceptability
in
toxicity
testing;
d)
some
endpoints
are
toxicologically
relevant
and
have
been
used
in
risk
assessment;
e)
evaluate
a
broader
range
of
mechanisms;
f)
provide
a
comprehensive
evaluation
of
the
whole
endocrine
system
as
a
unit;
and
g)
give
comparative
perspective
to
other
endpoints
of
toxicity.

It
is
important
to
reiterate
that
the
screening
battery
is
being
designed
to
minimize
false
negatives,
based
on
an
assessment
of
the
ability
of
the
battery
to
detect
known
EDCs
that
act
via
EAT.
In
this
regard,
the
value
of
each
individual
assay
cannot
be
considered
in
isolation
from
the
other
assays
in
the
battery,
as
they
have
been
combined
in
a
manner
such
that
limitations
of
one
assay
are
complemented
by
strengths
of
another.

The
EDSTAC
believes
the
recommended
screening
battery,
once
validated,
will
detect
all
of
the
EDCs
mediated
by
EAT
including
xeno(
anti)
estrogens
(
that
act
via
the
ER
or
inhibition
of
aromatase
by
oral
or
parenteral
administration),
xeno(
anti)
androgens
(
via
AR
or
hormone
synthesis),
altered
HPG
axis,
and
antithyroid
action
(
via
synthesis,
metabolism,
and
transport,
and
the
TR).
However,
results
of
even
the
most
specific
in
vivo
assays
can
be
affected
by
endocrine
mechanisms
other
than
those
directly
related
to
ER,
AR,
and
TR
action.
For
example,
uterine
weight
in
the
ovariectomized
female
rat
is
affected
in
an
estrogen­
like
manner
by
high
doses
of
aromatizable
and
nonaromatizable
androgens
and
growth
factors
like
EGF.
The
age
at
puberty
(
vaginal
opening
in
the
female
or
preputial
separation
in
the
male
rat)
can
be
affected
by
chemicals
that
act
on
the
hypothalamus,
pituitary,
or
thyroid
or
alter
growth
hormone
secretion.
If
gonadally
intact
females
are
used,
uterine
weight
can
also
be
affected
by
toxicants
that
stimulate
hypothalamic­
pituitary
or
gonadal
endocrine
secretions.
Clearly,
castration
of
the
treated
male
or
female
markedly
affects
the
specificity
of
the
test.
The
lack
of
specificity
of
in
vivo
assays
is
a
limitation
if
the
goal
is
to
only
identify
ER,
AR,
and
TR
alterations.
In
contrast,
this
lack
of
specificity
could
be
considered
an
advantage
if
a
broader,
more
apical
screening
strategy
is
desired.

1.
Unique
Thyroid
Action
Properties
to
be
Considered
in
Design
and
Interpretation
of
T1S
Thyroid
dysfunction
leads
to
abnormal
development,
altered
growth
patterns,
and
a
variety
of
physiological
perturbations
in
mammals
(
Dussault
and
Ruel,
1987;
Myant,
1971;
Porterfield
and
Hendrich,
1993;
Porterfield
and
Stein,
1994;
Timiras
and
Nzekwe,
1989),
as
well
as
in
birds
(
Tsai
and
Tsai,
1997),
reptiles
(
Schrebier
and
Richardson,
1997),
amphibians
(
Brown
et
al.,
1995;
Tata,
1994),
and
fish
(
Leatherland,
1994).
Considering
the
consequences
to
wildlife
populations
and
human
health
of
the
presence
in
the
environment
of
synthetic
compounds
with
thyroid
disrupting
activities,
the
EDSTAC
has
recommended
a
series
of
assays
that
will
detect
whether
substances
may
interact
with
the
thyroid.

The
chemistry
of
thyroid
hormone,
the
endocrine
mechanisms
governing
its
regulation,
and
the
mechanisms
by
which
thyroid
hormone
exerts
its
effects
are
surprisingly
similar
among
vertebrates
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
21
(
Gorbman
et
al.,
1983).
The
EDSTAC
deliberations
have,
therefore,
been
guided
by
research
focused
on
a
variety
of
vertebrates
to
develop
this
series
of
screens.
Despite
the
volume
of
literature
reviewed,
the
rapid
pace
of
research
into
thyroid
hormone
action
makes
it
predictable
that
the
present
screens
will
become
obsolete,
both
because
more
effective
assays
will
likely
be
developed
and
because
new
information
about
thyroid
hormone
action
may
reveal
mechanisms
of
thyroid
disruption
not
identified
by
the
recommended
T1S
battery.
The
following
background
information,
about
functioning
within
the
thyroid
axis
and
methods
used
to
evaluate
anti­
thyroid
activities,
is
intended
to
provide
a
rationale
for
the
recommended
Tier
1
thyroid­
specific
assays.

Endocrinology
of
the
Vertebrate
Thyroid:

Cells
of
the
thyroid
gland
are
arranged
in
follicles;
the
epithelial
cells
surround
a
fluid­
filled
core
containing
proteinaceous
material
 
the
colloid
(
Fawcett,
1986).
Individual
follicular
cells
respond
to
a
pituitary
hormone,
thyrotropin
(
TSH),
by
increasing
the
synthesis
and
release
of
thyroid
hormones
(
Wondisford
et
al.,
1996).
In
mammals,
TSH
release
from
the
pituitary
is
stimulated,
in
turn,
by
a
neuroendocrine
peptide,
thyrotropin­
releasing
hormone
(
TRH)
(
Greer
et
al.,
1993;
Morley,
1981;
Taylor
et
al.,
1990),
and
inhibited
by
the
negative
feedback
effects
of
thyroid
hormone
itself
(
Franklin
et
al.,
1987;
Mirell
et
al.,
1987;
Shupnik
and
Ridgway,
1987).
In
a
redundant
negative­
feedback
loop,
thyroid
hormone
also
exerts
an
inhibitory
effect
on
brain
cells
that
manufacture
TRH
(
Koller
et
al.,
1987;
Zoeller
et
al.,
1993).
The
functional
relationships
among
levels
of
this
endocrine
axis
are
so
tightly
linked
that
perturbations
within
one
level
produce
compensatory
changes
in
the
other
levels.
The
details
of
the
relationship
within
this
axis
have
not
been
explored
fully
for
all
non­
mammalian
vertebrates.

Thyroid
Hormone
Actions:

The
majority
of
biological
actions
of
thyroid
hormones,
including
the
regulation
of
brain
development,
are
believed
to
be
mediated
by
nuclear
receptors
for
triiodothyronine
(
T3)
(
Lazar,
1993).
Although
the
responsiveness
to
thyroid
hormone
requires
the
presence
of
nuclear
TRs,
the
specific
effects
of
thyroid
hormone
vary
from
tissue
to
tissue
(
Schwartz,
1983).
Pleiotropic
effects
of
thyroid
hormone
may
be
in
part
attributable
to
different
levels
and
combinations
of
TR
isoform
expression
(
Lazar,
1993;
Lazar,
1994).
However,
an
important
mechanism
by
which
thyroid
hormone
effects
can
be
regulated
within
cells,
tissue,
and
across
developmental
stages
is
the
interaction
between
receptors
for
thyroid
hormone
and
those
for
retinoids
(
Forman
and
Samuels,
1990;
Kliewer
et
al.,
1992;
Mano
et
al.,
1994;
Yu
et
al.,
1991;
Zhang
et
al.,
1992).
The
implication
of
these
observations
is
that
thyroid
hormone
action
can
be
modified,
even
disrupted,
by
agents
which
interfere
with
retinoid
metabolism.

Despite
the
recognition
that
thyroid
hormone
exerts
its
effects
through
nuclear
receptors,
there
are
very
clearly
defined
endpoints
of
thyroid
hormone
action
during
development.
There
are
a
few
genes
expressed
in
mammals
whose
expression
has
been
rigorously
defined
as
directly
regulated
by
thyroid
hormone
in
the
mammal.
These
include
myelin
basic
protein
(
MBP)
(
Mitsubashi
et
al.,
1988)
neuroganin/
RC3
(
Iniguez
et
al.,
1993),
TRH
(
Hollenberg
et
al.,
1995),
malic
enzyme
(
Song
et
al.,
1986),
thyrotropin
(
Carr
et
al.,
1993),
and
some
neuron­
specific
genes
(
Thompson,
1996).
In
amphibians,
a
number
of
genes
have
been
identified
in
frogs
(
Xenopus
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
22
laevis),
which
are
shown
to
mediate
effects
of
thyroid
hormone
on
metamorphosis
(
Brown
et
al.,
1996;
Brown
et
al.,
1995;
Denver
et
al.,
1997;
Furlow
et
al.,
1997;
Kanamori
and
Brown,
1996),
as
well
as
more
apical
endpoints
such
as
tail
resorption.

Mechanisms
of
Antithyroid
Activity:

A
variety
of
environmental
compounds
are
known
to
affect
thyroid
function
or
thyroid
hormone
action
(
Gaitan
and
Cooksey,
1989;
Green,
1996).
Processes
known
to
be
affected
and
a
brief
description
of
the
effects
are
included
below:

a)
Active
transport
of
iodide
into
the
thyroid
gland.
Inhibitors
include
complex
anions
(
e.
g.,
ClO4,
TcO4,
thiocyanate).
Reduces
thyroid
iodide
uptake
and
can
reduce
thyroid
hormone
synthesis
and
circulating
levels.
Elevated
TSH
can
overcome
modest
inhibition
in
the
absence
of
other
thyroid
pathologies.
b)
Iodination
of
thyroglobulin
(
by
thyroid
peroxidase
(
TPO)).
Inhibitors
include
thionamides
(
e.
g.,
propylthiouracil,
methiazole,
carbimazole),
thiocyanate,
aniline
derivatives
such
as
sulfonamides,
substituted
phenols
(
resorcinol),
flavonoids,
and
iodide.
Reduces
thyroid
hormone
synthesis
and
circulating
levels,
but
can
be
overcome
by
elevated
TSH.
c)
Coupling
reaction.
Iodinated
tyrosine
residues
of
thyroglobulin
must
be
coupled
by
an
ether
linkage
to
form
iodothyronines,
which
are
released
from
the
thyroid
gland.
Inhibition
of
this
coupling
reaction
reduces
thyroid
hormone
synthesis.
This
reaction
may
be
controlled
by
TPO
itself.
Inhibitors
include
thionamides
and
other
inhibitors
of
iodination,
minocycline,
and
lithium
salts.
d)
Hormone
release.
This
is
a
cAMP­
dependent
process
stimulated
by
TSH.
Inhibitors
include
iodide
and
lithium
salts.
e)
Iodotyrosine
deiodination.
This
process
is
important
for
recovery
of
iodide
within
the
thyroid
gland.
Inhibition
causes
the
reduction
in
thyroid
iodide
content
and
thus,
inhibition
of
thyroid
hormone
synthesis.
Inhibitors
include
nitrotyrosines.
f)
Iodothyronine
deiodination.
This
reaction
is
important
for
conversion
of
thyroxine
to
the
hormonally
active
tri­
iodothyronine,
and
for
the
conversion
of
T3
to
the
hormonally
inactive
T2.
Inhibitors
include
thiouracil
derivatives,
oral
cholecystographic
agents,
and
amiodarone.
g)
Hormone
excretion
or
inactivation.
This
process
is
affected
by
inducers
of
hepatic
drugmetabolizing
enzymes.
Inhibitors
include
phenobarbital,
phenytoin,
carbamazepine,
rifampicin,
and
organochlorines.
h)
Hormone
action.
Thyroid
hormone
action
is
largely
mediated
by
binding
to
specific
nuclear
receptors.
There
is
limited
evidence
that
compounds
such
as
phenytoin
(
dilantin)
and
amiodarone
can
displace
T3
from
nuclear
binding
sites
in
vitro.
However,
there
is
little
in
vivo
evidence
that
this
interaction
may
compromise
T3
action.
In
addition,
there
are
predictions
that
specific
PCBs
may
interfere
with
T3
binding
to
the
nuclear
receptor
because
of
similarities
in
structure.
However,
these
predictions
have
not
yet
been
experimentally
verified.

Recommended
T1S
Assays
for
Anti­
Thyroid
Activity:
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
23
To
the
EDSTAC's
knowledge,
all
known
antithyroid
compounds
so
far
reported
in
vertebrates
affect
circulating
levels
of
thyroxine
(
T4).
The
physiological
consequences
of
these
effects
are
variable
and
may
require
considerable
time
to
develop
in
a
screening
paradigm.
In
addition,
they
may
represent
endpoint
measures
that
are
not
solely
responsive
to
thyroid
disruption.
Therefore,
the
recommended
T1S
battery
includes
in
vivo
measures
of
circulating
levels
of
thyroxine
and
TSH,
and
changes
in
the
histopathology
of
the
thyroid
gland,
as
initial
endpoints
for
antithyroid
screening.
These
measures
can
be
made
in
animals
that
are
treated
in
the
commission
of
other
in
vivo
screens
(
e.
g.,
uterotrophic
assay).
Human
serum­
based
commercial
kits
are
available
to
measure
T4
and
TSH
by
radio­
immunoassay,
and
these
assays
have
been
validated
and
standardized
for
some
other
species.
It
is
important
to
recognize
that
a
significant
change
in
circulating
thyroxine
or
TSH
should
be
considered
a
positive
finding.
This
determination
is
based
upon
the
fact
that
some
compounds,
such
as
PCBs,
have
been
reported
to
reduce
circulating
levels
of
T4,
but
leave
TSH
unaffected
(
Goldey
et
al.,
1995).
In
addition,
weak
antithyroid
agents,
especially
those
affecting
some
aspects
of
iodide
metabolism
in
the
thyroid
(
Gaitan
and
Cooksey,
1989;
Gaitan
et
al.,
1989;
Green,
1996),
may
be
compensated
for
by
elevated
TSH.
Thus,
T4
may
appear
normal,
but
TSH
would
be
elevated.
Finally,
the
absence
of
an
effect
on
circulating
levels
of
T4
or
TSH
does
not
preclude
the
possibility
that
an
agent
is
antithyroid.
It
is
well
known
that
goitrogens
can
affect
thyroid
function
over
long
periods
and
not
be
manifested
by
significant
changes
in
circulating
levels
of
T4
or
TSH
measured
by
radio­
immunoassay
(
Gaitan
et
al.,
1989).
These
compounds
would
produce
a
measurable
effect
on
the
thyroid
gland.
For
these
reasons,
the
EDSTAC
recommends
thyroid
histopathologic
evaluations.

During
their
deliberations,
the
STWG
extensively
discussed
the
timing
of
exposure
to
a
chemical
substance
or
mixture.
EDSTAC
recommends
evaluation
of
antithyroid
effects
in
animals
prepared
for
testing
other
actions
(
either
14­
day
or
20­
day
exposure).
Although
no
cases
are
known
in
which
exposure
to
xenobiotics
of
greater
than
14
days
are
required
to
significantly
affect
circulating
levels
of
T4,
TSH,
or
thyroid
histopathology,
EDSTAC
believes
longer
periods
may
be
required
(
DeVito
et
al.,
1998).
The
effects
of
duration
of
chemical
exposure
must
be
quickly
evaluated
in
the
validation
phase.

These
measures
in
mammals
represent
evaluation
of
thyroid
function;
there
are
no
clear
markers
of
thyroid
hormone
action
that
could
be
used
within
the
context
of
a
T1S
assay.
In
contrast,
tail
resorption
in
amphibian
metamorphosis
represents
an
assay
which
utilizes
specific
thyroid
hormone­
dependent
effects
as
an
endpoint
for
a
T1S
assay.

2.
In
Vivo
Assays
Using
Other
Vertebrates
The
T1S
battery
includes
an
amphibian
and
a
fish
assay,
which
fill
important
needs
in
the
battery
and
complements
the
information
from
assays
using
mammals.
These
assays
help
the
battery
meet
design
criteria
2
and
4,
which
express
the
need
for
a
sufficient
range
of
taxonomic
subjects
and
range
of
metabolic
functions
be
evaluated
in
the
battery.
While
the
basic
biochemical
processes
of
receptor
binding
and
cellular
activation
by
hormones
are
known
to
be
similar
among
many
organisms,
detailed
comparative
data
do
not
exist
to
assess
the
extent
of
the
homology
across
vertebrate
classes.
In
particular,
fish
ER
differs
from
mammalian
ER
more
than
the
ER
of
other
classes,
and
fish
have
some
unique
androgens.
Hence,
including
fish
as
subjects
makes
sense
as
it
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
24
is
the
class
most
likely
to
show
differences
from
mammals
in
EAT
activity.
In
addition,
there
are
known
differences
in
the
ability
of
organisms
to
metabolize
xenobiotics,
due
partially
to
the
route
of
exposure.
Fish
and
amphibians
receive
more
dermal
exposure
to
chemical
substances
or
mixtures
than
other
vertebrate
classes,
and
thus
chemical
substances
or
mixtures
avoid
immediate
metabolism
in
the
liver.
These
assays
also
meet
the
need
for
the
battery
to
have
a
clear
cut
response
to
measure
the
effects
of
thyroid
hormone,
which
frog
metamorphosis
does,
and
hence
complements
the
data
on
serum
concentration
of
thyroid
hormones
and
thyroid
gland
histology
derived
from
the
pubertal
female
rodent
assay.

Unlike
the
mammalian
assays,
the
assays
recommended
for
fish
and
frogs
have
not
been
used
in
regulatory
testing,
and
hence
they
need
more
work
before
being
implemented
for
that
purpose.
In
fact,
however,
both
procedures
using
these
species
and
similar
endpoints
have
been
used
to
investigate
endocrine
activity
of
chemical
substances
or
mixtures
in
the
research
literature.
The
work
needed
for
standardization
of
protocols
and
validation
of
the
assay
with
known
endocrine
disruptors
should
proceed
as
soon
as
possible
as
these
assays
play
a
crucial
role
in
the
T1S
battery.
In
addition,
EDSTAC
encourages
development
of
other
assays
in
the
event
that
either
of
these
two
fail
to
be
adequately
standardized
and
validated,
so
that
a
complete
screening
battery
can
be
implemented.

E.
In
Vivo
Assay
Overviews
Several
measures
of
estrogenicity
(
reviewed
by
Gray
et
al.,
1997a;
Reel
et
al.,
1996;
Parker,
1966,
Chapter
30)
have
been
used
for
over
70
years,
including
uterine
size,
vaginal
cornification,
female
sexual
receptivity,
and
age
at
puberty/
vaginal
opening
(
see
Parker,
1966,
for
a
thorough
review).
For
example,
Dodds
et
al.,
1938,
found
that
DES
produced
full
estrus
in
ovariectomized
rats,
so
far
as
vaginal,
uterine,
and
mating
reactions
were
concerned.
These
remain
some
of
the
most
useful
shortterm
in
vivo
methods
for
screening
for
estrogenicity.
Studies
of
xenoestrogens
typically
indicate
that
the
sensitivity
of
these
endpoints
is
as
follows:
uterine
weight
measured
5
hours
after
the
last
treatment,
with
fluid,
is
generally
more
sensitive
than
the
age
at
vaginal
opening
or
vaginal
cornification;
however,
this
is
not
always
the
case.
Uterine
histology
and
biochemical
measures
appear
to
be
at
least
as
sensitive
to
estrogens
as
uterine
weight,
but
these
endpoints
are
slightly
more
difficult
to
evaluate
as
they
require
specialized
skills
and
equipment
and
are
more
expensive.

The
sensitivity
of
the
age
at
vaginal
opening
to
methoxychlor
appears
to
be
about
twofold
greater
than
the
onset
of
persistent
vaginal
cornification
(
PVC)
and
at
least
equivalent
to
the
sensitivity
of
the
uterotrophic
assay
(
Gray
et
al.,
1989;
Gray
and
Ostby,
1998).
However,
in
another
study
hydroxylated
PCBs
induced
vaginal
cornification
at
dosage
levels
that
failed
to
induce
an
increase
in
uterine
weight
(
Gillesby
and
Zacharewski,
1996).
PVC
was
not
detected
in
a
long
term
study
of
the
estrogenicity
of
octylphenol
that
doubled
uterine
weight
in
long­
term
ovariectomized
rats
after
10
weeks
of
oral
administration
and
after
three
days
of
administration
in
juvenile
rats
(
Gray
and
Ostby,
1998).
Hence,
some
of
the
original
measures
of
estrogenicity,
in
use
now
for
nearly
three­
quarters
of
a
century,
are
still
regarded
as
the
most
useful
indicators
of
estrogenic
activity
in
vivo.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
25
Recent
studies
to
evaluate
methoxychlor,
4­
tert­
octylphenol,
nonylphenol,
bisphenol
A,
DES
(
Reel
et
al.,
1985),
estrogens,
and
antiestrogens
(
Conner
et
al.,
1996)
have
demonstrated
the
utility
of
these
biological
assays,
since:
(
1)
pro­
estrogens/
metabolites
may
be
detected
following
in
vivo
exposure;
(
2)
agonistic/
antagonistic
properties
may
be
addressed;
(
3)
bioaccumulation
and/
or
the
development
of
tolerance
to
exposure
may
be
evaluated;
(
4)
multiple
routes
and
lengths
of
exposure
may
be
easily
compared;
and
(
5)
acute
exposure
regimes
may
be
used.
However,
care
should
be
taken
when
interpreting
results
from
these
biological
assays
since:
(
1)
some
environmental
chemicals
do
not
test
positive
for
all
measures;
(
2)
exposure
route
and
time
of
assessment
following
exposure
affect
the
results;
and
(
3)
the
observed
biological
response
may
result
from
other
mechanisms
of
action.
For
example,
in
the
ovariectomized
female,
increased
uterine
weight
can
be
induced
by
aromatizable
and
nonaromatizable
androgens
(
Salamon,
1938)
and
EGF
(
Nelson
et
al.,
1991).

In
the
intact
(
ovaries
present
versus
castrate
or
ovariectomized)
juvenile
female
rat,
the
age
at
which
treatment
is
initiated
(
typically
19­
21
days
of
age)
and
the
duration
of
treatment
are
critical
variables
that
affect
uterine
weight.
Exposure
duration
longer
than
3
or
4
days
or
the
use
of
juvenile
females
24­
25
days
of
age
at
the
start
of
the
study
are
not
recommended
because
of
the
potential
confounding
of
the
treatment
effect
with
the
onset
of
natural
estrous
cyclicity
and
its
concurrent
fluctuations
in
uterine
weight
and
histology.
As
long
as
the
uterine
weight
bioassay
has
been
used,
it
still
has
not
been
completely
standardized,
a
fact
that
leads
to
some
variation
in
results
from
lab
to
lab.
For
example,
there
are
differences
with
respect
to
how
thoroughly
the
mesenteric
fat
along
the
uterine
curvature
is
removed,
and
some
labs
weigh
the
uterus
with
its
contents,
while
others
remove
the
fluid
before
weighing.
Uterine
weight,
serum
hormone
concentrations,
and
other
evaluations
in
intact
female
rats
are
difficult
to
interpret
unless
great
care
is
taken
to
assure
that
females
are
necropsied
at
the
same
stage
of
the
estrous
cycle.
With
regard
to
the
measurement
of
serum
hormones
in
the
cycling
female
rat,
the
time
of
day
is
also
critical,
in
addition
to
the
day
of
the
cycle.
Effects
on
estrous
cyclicity
are
not
limited
to
ER­
mediated
alterations;
several
other
reproductive
(
hypothalamic­
pituitary)
and
nonreproductive
(
hypothyroidism)
endocrine­
related
alterations
can
alter
estrous
cyclicity
in
the
female
rodent.
The
detection
of
vaginal
cornification
in
juvenile,
and
ovariectomized
adult
rodents
is
one
of
the
original
assays
used
to
detect
estrogenicity
and,
as
indicated
above,
this
assay
appears
to
be
relatively
sensitive
to
weak
estrogens.
However,
higher
levels
of
xenoestrogens
are
required
to
disrupt
estrous
cyclicity
and
induce
constant
estrus
PVC
in
intact
female
rats
(
Gray
et
al.,
1989).

1.
Rodent
3­
Day
Uterotrophic
Assay
(
Subcutaneous)

Assay
for
estrogenicity
An
increase
in
uterine
weight
is
generally
considered
to
be
one
of
the
best
indicators
of
estrogenicity
when
measured
in
the
ovariectomized
(
ovx)
or
immature
female
rat
or
mouse
after
1­
3
days
of
treatment.
The
recommended
3­
day
uterotrophic
assay
(
sc
injection)
uses
the
ovariectomized
adult
female
rat
(
the
duration
can
be
extended
if
so
desired)
with
n
=
10/
group.
Subcutaneous
treatment
is
recommended
at
this
time
because
most
of
the
historical
data
are
collected
in
this
manner
and
there
is
relatively
few
data
concerning
the
effects
of
other
routes
of
administration
at
this
time.
At
necropsy
one
should
carefully
trim
the
uterus
of
fat
and
weigh
with
and
without
fluid
and
save
uterus
and
vaginal
tissues
for
histopathology.
Most
xenoestrogens
have
been
examined
in
this
assay.
It
also
should
be
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
26
executed
in
a
manner
to
detect
antiestrogens.
In
this
regard,
a
control
and
xenobiotic­
treated
group
are
coadministered
with
estradiol
sc
and
necropsied.

2.
Rodent
20­
Day
Pubertal
Female
Assay
With
Thyroid
Assay
for
thyroid,
HPG
axis,
aromatase,
and
estrogens
that
are
only
effective
orally
or
after
longer
dosing
than
the
uterotrophic
assay.

The
determination
of
the
ages
at
"
puberty"
in
the
female
rat
is
an
endpoint
that
has
already
gained
acceptance
in
the
toxicology
community.
Vaginal
opening
(
VO)
in
the
female
is
a
required
endpoint
measured
in
the
new
EPA
two­
generation
reproductive
toxicity
test
guideline.
In
this
regard,
this
assay
would
be
easy
to
implement
because
these
endpoints
have
been
standardized
and
validated
and
VO
data
are
currently
being
collected
under
GLP
conditions
in
most
toxicology
laboratories.
In
addition,
VO
data
are
reported
in
many
recently
published
developmental
and
reproductive
toxicity
studies
(
i.
e.,
see
studies
from
R.
E.
Peterson's,
R.
Chapin's
and
L.
E.
Gray's
laboratories
on
dioxins,
PCBs,
antiandrogens,
and
xenoestrogens).

In
the
pubertal
female
assay,
oral
dosing
is
initiated
in
weanling
rats
at
21
days
of
age
(
10
per
group,
selected
for
uniform
body
weights
at
weaning
to
reduce
variance).
The
animals
are
dosed
daily,
7
days
a
week,
and
examined
daily
for
vaginal
opening
(
one
could
also
check
for
age
at
first
estrus
and
onset
of
estrous
cyclicity).
Dosing
continues
until
VO
is
attained
in
all
females
(
typically
two
weeks
after
weaning,
unless
delayed).
Age
at
VO
is
also
determined
in
the
female
rat.
Rats
are
dosed
by
gavage
with
xenobiotic
and
examined
daily
for
VO.
The
advantage
over
the
uterotrophic
assay
is
that
one
test
detects
both
agonists
and
antagonists,
it
detects
xenoestrogens
like
methoxychlor
that
are
almost
inactive
via
sc
injection,
it
detects
aromatase
inhibitors,
altered
HPG
function,
and
unusual
chemicals
like
betasitosterol.
In
addition,
at
necropsy
one
should
weigh
the
ovary
(
increased
in
size
with
aromatase
inhibitors,
but
reduced
with
betasitosterol),
save
the
thyroid
for
histopathology,
take
serum
for
T4,
and
measure
TSH.

Exposure
of
weanling
female
rats
to
environmental
estrogens
can
result
in
alterations
of
pubertal
development
(
Ramirez
and
Sawyer,
1964).
Exposure
to
a
weakly
estrogenic
pesticide
after
weaning
and
through
puberty
induces
pseudoprecocious
puberty
(
accelerated
vaginal
opening
without
an
effect
on
the
onset
of
estrous
cyclicity)
after
only
a
few
days
of
exposure
(
Gray
et
al.,
1989).
Pubertal
alterations
also
result
in
girls
exposed
to
estrogen­
containing
creams
or
drugs,
which
induce
pseudoprecocious
puberty
and
alterations
of
bone
development
(
Hannon
et
al.,
1987).

Several
examples
of
estrogenic
chemicals
affecting
vaginal
opening
in
rodents
are
known
and
include
methoxychlor
(
Gray
et
al.,
1989),
nonylphenol,
and
octylphenol
(
Gray
and
Ostby,
1998).
This
endpoint
appears
to
be
almost
as
sensitive
as
the
uterine
weight
bioassay,
but
the
evaluation
is
easier
to
conduct
and
does
not
require
that
the
animals
be
euthanized,
so
they
can
be
used
for
additional
evaluations.
For
example,
treatment
with
methoxychlor
at
weaning
(
6
mg/
kg/
day
or
higher)
caused
pseudoprecocious
puberty
in
female
rats.
Vaginal
opening
occurs
from
two
to
seven
days
earlier
in
treated
animals
than
controls,
in
a
dose­
related
fashion,
but
methoxychlor
did
not
alter
estrous
cyclicity
at
the
low
dosage
levels,
indicating
a
direct
estrogenic
effect
of
methoxychlor
on
vaginal
epithelial
cell
function
without
an
effect
on
hypothalamic­
pituitary
maturation.
Similar
effects
have
been
achieved
EDSTAC
Final
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Chapter
Five
August
1998
5
­
27
with
chlordecone,
another
weakly
estrogenic
pesticide,
and
octylphenol.
Chlordecone
also
induces
neurotoxic
effects
(
hyperactivity
to
handling
and
tremors).
In
addition
to
estrogens,
the
age
at
vaginal
opening
and
uterine
growth
can
be
affected
by
alteration
of
several
other
endocrine
mechanisms,
including
alterations
of
the
hypothalamic­
pituitary­
gonadal
axis
(
Shaban
and
Terranova,
1986;
and
Gonzalez
et
al.,
1983).
In
rats,
this
event
can
also
be
induced
by
androgens
(
Salamon,
1938)
and
EGF
(
Nelson
et
al.,
1991).
In
the
last
20
years
there
have
been
over
200
publications
which
demonstrate
the
broad
utility
of
this
assay
to
identify
altered
estrogen
synthesis,
ER
action,
growth
hormone,
prolactin,
FSH
or
LH
secretion,
or
CNS
lesions.

3.
Rodent
5­
7
Day
Hershberger
Assay
Assay
for
Antiandrogens
In
the
castrated
male
rat,
the
gonads
have
been
removed
and
effects
on
androgen­
dependent
accessory
sex
organs
and
tissues
are
likely
to
be
direct
and
not
a
result
of
pituitary
or
gonadal
secretion.
The
assay
(
Hershberger,
1953)
requires
two
stages
as
below:

·
castrated
male
rat
+
T
+
Xenobiotic
(
to
detect
antagonist)
·
castrated
male
+
X
(
to
detect
agonist)

In
this
in
vivo
test,
sex
accessory
gland
weights
(
ventral
prostate
and
seminal
vesicle
separately)
are
measured
in
castrated,
testosterone­
treated
adult
male
rats
after
4­
7
days
of
treatment
by
gavage
with
the
test
compound.
The
advantage
of
this
assay
is
that
it
is
fairly
simple,
short
term,
and
relatively
specific
compared
to
other
in
vivo
procedures.
Although
the
androgens,
testosterone,
and
dihydrotestosterone
(
DHT),
play
a
predominant
role
in
the
growth
and
maintenance
of
the
size
of
these
structures,
several
other
hormones
and
growth
factors
can
influence
sex
organ
weights
including
the
thyroid
and
growth
hormones,
prolactin,
and
epidermal
growth
factor
(
EGF)
(
Luke
and
Coffey,
1994).
Exposure
to
estrogenic
pesticides
can
also
reduce
sex
accessory
gland
size;
however,
it
is
unclear
to
what
degree
these
reductions
result
from
direct
versus
indirect
action
of
the
chemical.
Other
useful
endpoints
that
help
reveal
the
mechanism
of
action
include
serum
hormone
levels
of
T,
DHT,
LH,
AR
distribution,
TRPM2/
C3
gene
activation,
ODC,
and
5­
alpha­
reductase
activity
in
the
prostate.
The
prostate
and
seminal
vesicles
should
be
weighed
separately
because
these
organs
differ
with
respect
to
the
androgen
that
controls
their
growth
and
differentiation.
The
prostate
is
dependent
upon
enzymatic
activation
of
T
to
DHT,
whereas
the
seminal
vesicle
is
less
dependent
upon
this
conversion.
Hence,
effects
on
5­
alpha­
reductase
can
be
distinguished
from
AR­
mediated
mechanisms
by
determining
whether
the
prostate
is
preferentially
affected.
Growth
of
the
levator
ani
muscle
is
T
dependent,
having
little
capacity
to
convert
T
to
the
more
potent
androgen
DHT.
Weight
of
this
muscle
is
useful
in
identifying
anabolic
androgens
and
antiandrogens,
and
for
this
reason
has
been
used
extensively
in
the
pharmaceutical
industry.
In
order
to
detect
androgenic
rather
than
antiandrogenic
action
one
would
simply
delete
the
hormone
administration
from
the
protocol.

Data
from
this
assay
(
often
with
slight
modifications),
using
drugs
and
xenoantiandrogens,
are
widely
available
in
the
literature.
For
a
non­
in
utero
assay,
this
assay
robustly
detects
androgens
and
antiandrogens
with
a
dynamic
response
that
typically
exceeds
that
of
the
intact
adult
male
(
Raynaud,
1984).
Most
of
the
studies
are
able
to
detect
significant
effects
with
only
five
animals
per
group.
In
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
28
fact,
in
one
study
which
used
10­
15
drugs,
the
Hershberger
assay
was
more
responsive
than
was
the
intact
male
for
every
chemical
examined
(
Wakeling
et
al.,
1981).
The
power
of
the
castrate­
male
assay
arises
from
the
fact
that
castration
creates
a
"
fetal­
like"
endocrine
system
with
regard
to
the
regulation
of
androgen
secretion,
as
the
HPG
axis
can
no
longer
compensate
for
the
effect
of
the
chemical
on
the
AR.
For
example,
p,
p'
DDE
reduces
sex
accessory
gland
weights
in
this
assay,
but
not
when
administered
to
intact
male
rats
(
Kelce
et
al.,
1997).

4.
Frog
Metamorphosis
Assay
This
assay
employs
intact
larval
(
tadpole)
stages
of
the
African
clawed
frog
(
Xenopus
laevis)
exposed
over
a
14­
day
time
period,
50­
64
days
of
age,
to
observe
the
rate
of
tail
resorption
(
Fort
and
Stover,
1997).
Tail
resorption
can
be
easily
quantified
with
computer­
aided
video
image
processing
(
Fort
and
Stover,
1997).
The
molecular
mechanisms
involved
in
tail
resorption
are
well
characterized
(
Brown
et
al.,
1995;
Hayes,
1997a)
and
this
assay
is,
therefore,
considered
to
be
a
simple
and
specific
assay
for
thyroid
action.
It
will
detect
thyroid
(
increase
in
tail
resorption
rate)
and
antithyroid
(
decrease
in
tail
resorption
rate)
effects.
Because
evidence
also
suggests
that
thyroid
action
on
tail
resorption
is
regulated
by
corticoids,
estrogens,
and
prolactin
(
Hayes,
1997b),
this
assay
will
address
distinctive
modulating
pathways
and,
in
tandem
with
the
14­
day
mammalian
pubertal
assay,
a
comprehensive
screen
for
thyroid
hormone
activity
is
achieved.

5.
Fish
Gonadal
Recrudescence
Assay
Intact
mature
fish
maintained
under
simulated
"
winter"
conditions
(
short
day
length,
cool
temperatures)
exhibit
regressed
secondary
sex
characteristics
and
gonad
maturation.
In
this
assay,
intact
fish
of
both
sexes
(
fathead
minnow,
Pimephales
promelas,
or
other
appropriate
species)
are
simultaneously
subjected
to
an
increasing
photoperiod/
temperature
regime
and
test
substance
to
determine
potential
effects
on
maturation
from
the
regressed
position
(
recrudescence).
The
primary
endpoints
examined
in
the
assay
include
morphological
development
of
secondary
sexual
characteristics,
ovary
and
testis
development
(
weight
increases),
gonadosomatic
index
(
ratio
of
gonadal
weight
to
body
weight),
final
gamete
maturation
(
ovulation,
spermiation),
and
induction
of
vitellogenin.
This
assay
is
sensitive
to
HPG
axis
effects
in
addition
to
androgen­
and
estrogenrelated
activity.

Fish
differ
in
steroid
profiles
from
mammals
(
e.
g.,
11­
ketotestosterone
as
opposed
to
testosterone
is
the
most
important
androgen
in
fish).
The
estrogen
receptor
in
fish
appears
to
differ
structurally
and
functionally
from
the
mammalian
estrogen
receptor
(
Petit
et
al.,
1995;
Gustafsson,
1996).
Also,
steroid
receptors
in
eggs
and
hepatic
vitellogenin
production
have
no
known
analogous
receptors
in
mammals,
which
would
suggest
sites
of
endocrine
disruption
unique
to
oviparous
animals.
Therefore,
this
assay
is
essential
to
address
these
known
endocrine
differences.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
29
F.
Alternative
Assays
for
Possible
Inclusion
1.
Placental
Aromatase
Assay
One
critical
enzyme
present
at
very
low
levels
in
the
testis,
and
at
higher
levels
in
the
ovary,
uterus,
and
placenta,
is
aromatase,
which
converts
testosterone
to
estradiol
and
is
another
P450
isozyme.
Human
placental
aromatase
is
commercially
available
and
could
be
used
in
vitro
to
assess
the
effects
of
toxicants
on
this
enzyme
fairly
easily.

2.
Modified
Rodent
3­
Day
Uterotrophic
Assay
(
Intraperitoneal)

This
is
an
in
vivo
assay
(
O'Conner
et
al.,
1996)
for
estrogenic
activity
in
ovariectomized
female
rats.
It
can
detect
certain
antiestrogens
with
mixed
activity,
i.
e.,
those
with
some
agonistic
activity
(
e.
g.,
tamoxifen).
The
rats
are
injected
intraperitoneally
with
the
test
agent
daily
for
three
days.
The
ip
injection
method
may
enhance
the
sensitivity
of
the
assay
and
is
capable
of
detecting
the
estrogenic
potential
of
methoxychlor,
which
has
been
cited
as
an
example
of
a
compound
not
detectable
by
the
sc
route.
The
females
are
necropsied
either
6
hours
or
24
hours
after
the
final
treatment,
depending
on
the
protocol
employed
by
the
laboratory.
Vaginal
cytology
is
evaluated
by
vaginal
lavage
to
determine
whether
the
epithelium
has
become
cornified,
indicative
of
estrus.
Presence
of
fluid
in
the
uterine
lumen
is
noted
and
recorded,
and
the
number
of
animals
that
have
fluid
in
the
uterus
is
reported.
Fluid
imbibition
(
uptake)
is
indicative
of
estrogenic
potential.
The
uterus
is
excised
and
weighed.
It
is
then
preserved
in
an
appropriate
fixative
for
subsequent
histological
evaluation,
if
needed.

Subsequent
histological
evaluation
will
be
triggered
by
an
equivocal
uterine
weight
or
uterine
fluid
response
(
i.
e.,
an
increase
that
is
not
statistically
significant).
This
evaluation
will
consist
of
a
characterization
of
the
appearance
of
the
uterine
epithelium,
a
measurement
of
uterine
epithelial
cell
height,
and
epithelial
mitotic
index
or
proliferating
cell
nuclear
antigen
(
PCNA)
immunohistochemistry.
Uterine
cell
height
and
cell
proliferation
are
sensitive
indicators
of
estrogenic
potential.

3.
14­
Day
Intact
Adult
Male
Assay
This
in
vivo
assay
is
intended
to
detect
effects
on
male
reproductive
organs
that
are
sensitive
to
antiandrogens
and
agents
that
inhibit
testosterone
synthesis
or
inhibit
5­
alpha­
reductase
(
Cook
et
al.,
1997).
The
duration
of
the
assay
is
anticipated
to
be
sufficient
to
detect
effects
on
thyroid
gland
activity.
The
rats
are
anatomically
intact
and
mature;
therefore,
they
have
an
intact
HPG
axis,
allowing
an
assessment
of
the
higher
order
neuroendocrine
control
of
male
reproductive
function
and
the
thyroid.

Young
adult
male
rats
(
70­
90
days
of
age)
are
used
in
this
assay.
They
are
dosed
daily
with
the
test
agent
for
14
days.
The
recommended
route
of
administration
is
ip,
which
may,
in
some
cases,
maximize
the
sensitivity
of
the
assay.
They
are
necropsied
24
hours
after
the
final
dose.
Immediately
after
sacrifice
one
cauda
epididymis
is
weighed
and
processed
for
evaluation
of
sperm
motility
and
concentration.
The
following
organs
are
weighed:
testes,
epididymides,
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
30
seminal
vesicles,
and
prostate.
The
following
are
fixed
and
evaluated
histologically:
one
testis
and
epididymis,
and
the
thyroid.
The
following
hormones
are
measured
in
blood
plasma:
T4,
TSH,
LH,
testosterone,
DHT,
and
estradiol.

Empirical
assessment
of
this
assay
has
shown
it
to
be
sensitive
to
agents
that
are
directly
antiandrogenic,
inhibit
5­
alpha­
reductase,
inhibit
testosterone
synthesis,
or
affect
thyroid
function.
The
sensitivity
of
this
assay,
as
defined
as
the
ability
to
detect
a
hazard,
may
be
comparable
to
other
assays
that
have
been
recommended.

4.
Rodent
20­
Day
Thyroid/
Pubertal
Male
Assay
This
assay
detects
androgens
and
antiandrogens
in
vivo
in
a
single
stage
apical
test.
"
Puberty"
is
measured
in
male
rats
by
determining
age
at
PPS
(
preputial
separation).
Animals
are
dosed
by
gavage
beginning
one
week
before
puberty
(
which
occurs
at
about
40
days
of
age)
and
PPS
is
measured.
Androgens
will
accelerate
and
antiandrogens
and
estrogens
will
delay
PPS.
The
assay
takes
about
3
weeks,
and
allows
for
comprehensive
assessment
of
the
entire
endocrine
system
in
one
study
(
10
per
group,
selected
for
uniform
body
weights
to
reduce
variance).
The
animals
are
dosed
daily,
seven
days
a
week,
and
examined
daily
for
PPS.
Dosing
continues
until
53
days
of
age;
the
males
are
then
necropsied.
The
body,
heart
(
thyroid),
adrenal,
testis,
seminal
vesicle
plus
coagulating
glands
(
with
fluid),
ventral
prostate,
and
levator
ani
plus
bulbocavernosus
muscles
(
as
a
unit)
are
weighed.
The
thyroid
is
retained
for
histopathology
and
serum
is
taken
for
T4,
T3,
and
TSH.
Testosterone,
LH,
prolactin,
and
dihydrotestosterone
analyses
are
optional.
These
endpoints
take
several
weeks
to
evaluate
and
are
affected
not
only
by
estrogens
but
by
environmental
antiandrogens,
drugs
that
affect
the
hypothalamic­
pituitary
axis
(
Hostetter
and
Piacsek,
1977;
Ramaley
and
Phares,
1983),
and
by
prenatal
exposure
to
TCDD
(
Gray
et
al.,
1995a;
Bjerke
and
Peterson,
1994)
or
dioxin­
like
PCBs
(
Gray
et
al.,
1995b).
In
contrast
to
these
other
mechanisms,
only
peripubertal
estrogen
administration
accelerates
this
process
in
the
female
and
delays
it
in
the
male.
Preputial
separation
in
the
male
rodent
is
easy
to
measure
and
this
is
not
a
terminal
measure
(
Korenbrot
et
al.,
1977).

Age
and
weight
at
puberty,
reproductive
organ
weights,
and
serum
hormone
levels
can
also
be
measured.
Delays
in
male
puberty
result
from
exposure
to
both
estrogenic
and
antiandrogenic
chemicals
including
methoxychlor
(
Gray
et
al.,
1989),
vinclozolin
(
Anderson
et
al.,
1995b)
and
p,
p'
DDE
(
Kelce
et
al.,
1995).
Exposing
weanling
male
rats
to
the
antiandrogenic
pesticides
p,
p'
DDE
or
vinclozolin
delays
pubertal
development
in
weanling
male
rats
as
indicated
by
delayed
preputial
separation
and
increased
body
weight
(
because
they
are
older
and
larger)
at
puberty.
In
contrast
to
the
delays
associated
with
exposure
to
estrogenic
substances,
antiandrogens
do
not
inhibit
food
consumption
or
retard
growth
(
Anderson
et
al.,
1995).
Antiandrogens
cause
a
delay
in
preputial
separation
and
affect
a
number
of
endocrine
and
morphological
parameters
including
reduced
seminal
vesicle,
ventral
prostate,
and
epididymal
weights.
It
is
apparent
that
PPS
is
more
sensitive
than
are
organ
weights
in
this
assays.
In
addition,
responses
of
the
HPG
are
variable.
In
studies
of
vinclozolin,
increases
in
serum
LH
were
a
sensitive
response
to
this
antiandrogen,
whereas
serum
LH
is
not
increased
in
males
exposed
to
p,
p'
DDE
during
puberty
(
Kelce
et
al.,
1997).
Furthermore,
a
systematic
review
of
the
literature
indicates
that
the
sex
accessory
glands
of
the
immature
intact
male
rat
are
consistently
more
affected
than
in
the
adult
intact
male
rat.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
31
In
summary,
preputial
separation
and
sex
accessory
gland
weights
are
sensitive
endpoints.
However,
a
delay
in
preputial
separation
is
not
pathognomonic
for
antiandrogens.
Pubertal
alterations
result
from
chemicals
that
disrupt
hypothalamic­
pituitary
function
(
Huhtaniemi
et
al.,
1986),
and,
for
this
reason,
additional
in
vivo
and
in
vitro
tests
are
needed
to
identify
the
mechanism
of
action
responsible
for
the
pubertal
alterations.
For
example,
alterations
of
prolactin,
growth
hormone,
gonadotrophin
(
LH
and
FSH)
secretion,
or
hypothalamic
lesions
alter
the
rate
of
pubertal
maturation
in
weanling
rats.

As
indicated
above,
the
determination
of
the
age
at
"
puberty"
in
the
male
rat
are
endpoints
that
already
have
gained
acceptance
in
the
toxicology
community.
Preputial
separation
in
the
male
is
a
required
endpoint
in
the
new
EPA
two­
generation
reproductive
toxicity
test
guideline.
In
this
regard,
this
assay
would
be
easy
to
implement
because
these
endpoints
have
been
standardized
and
validated
and
PPS
data
are
currently
being
collected
under
GLP
conditions
in
most
toxicology
laboratories.
In
addition,
PPS
data
are
reported
in
many
recently
published
developmental
and
reproductive
toxicity
studies
(
i.
e.,
see
studies
from
R.
E.
Peterson's,
J.
Ashby's,
R.
Chapin's
and
L.
E.
Gray's
laboratories
on
dioxins,
PCBs,
antiandrogens,
and
xenoestrogens).

Sex
accessory
gland
weights
in
intact
adult
male
rats
also
can
be
affected
directly
or
indirectly
by
toxicant
exposure.
The
HPG
axis
in
an
intact
animal
is
able
to
compensate
for
the
action
of
antiandrogens
by
increasing
hormone
production,
which
counteracts
the
effect
of
the
antiandrogen
on
the
tract
(
Raynoud
et
al.,
1984;
Edgren,
1994;
Hershberger,
1953).

IV.
General
Principles
in
Evaluating
Tier
1
and
Tier
2
Results
A.
Introduction
Apart
from
substances
yielding
negative
results
in
all
assays,
it
is
likely
that
most
substances
will
produce
a
unique
array
of
results
requiring
a
judgment
as
to
whether
the
weight
of
evidence
indicates
the
substance
should
or
should
not
be
judged
a
candidate
for
T2T
(
after
completing
T1S),
and,
designated
as
an
endocrine
disruptor
for
EAT
(
after
completing
T2T).
A
table
consisting
of
18
chemical
types
along
with
known
or
expected
T1S
results
can
be
found
in
Appendix
P,
Examples
of
"
Weight­
of­
Evidence"
Determinations.

There
are
two
senses
in
which
a
"
weight­
of­
evidence"
determination
will
need
to
be
made.
The
first
is
with
respect
to
the
question
of
whether
consistent
results
are
being
obtained
across
multiple
assays.
If
the
results
are
not
consistent,
it
will
be
necessary
to
"
weight"
the
conflicting
results,
allowing
some
to
carry
more
weight
than
others.
The
second
sense
is
with
respect
to
the
question
of
whether
a
particular
body
of
evidence,
even
if
it
is
fully
consistent,
is
sufficient
to
justify
a
decision.
In
this
sense,
it
is
the
"
weight"
of
the
entire
body
of
evidence,
relative
to
some
minimal
level
established
as
being
required
for
sound
decisions,
that
is
being
judged.

Assessing
the
"
weight­
of­
evidence,"
and
using
that
assessment
in
forming
judgments
about
a
substance,
can
be
done
in
a
variety
of
ways.
On
one
extreme
are
approaches
based
solely
on
expert
judgment
in
which
an
individual
reflects
on
the
data
and
offers
an
informed,
yet
personal,
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
32
opinion.
On
the
other
extreme
are
more
formal
and
mathematical
procedures
such
as
Bayesian
analysis
in
which
data
are
viewed
sequentially
and
used
to
formulate
a
priori
and
a
posteriori
judgments.
An
intermediate
approach
is
one
in
which
a
group
debates
the
available
data,
presents
alternative
arguments,
and
collectively
reaches
a
judgment.

All
three
of
these
possibilities
are
forms
of
"
weight­
of­
evidence"
assessments.
The
EDSTAC
has
agreed
not
to
prescribe
a
particular
"
weight­
of­
evidence"
approach,
as
these
are
controversial
and
a
matter
of
science
policy
to
be
established
by
the
Agency.
Instead,
the
EDSTAC
offers
general
guidelines
for
reasoning
from
the
data
produced
in
the
two
tiers,
which
conform
to
the
outline
provided
in
the
NAS/
NRC
report
Science
and
Judgment
in
Risk
Assessment
(
National
Academy
Press,
1994).
These
guidelines
provide
a
framework
within
which
one
may
take
into
account
multiple
features
of
relevant
screening
and
testing
data
in
determining
whether
the
substance
should
be
a
priority
for
T2T
(
after
T1S)
and/
or
is
determined
to
have
endocrine
disrupting
effects
(
after
T2T).

"
Weight­
of­
evidence"
considerations
will
arise
at
two
places
within
the
EDSTP.
It
first
will
arise
in
considering
whether
the
evidence
collected
solely
within
a
given
tier
(
T1S)
warrants
a
particular
conclusion
(
e.
g.,
that
the
substance
may
have
endocrine
activity
for
EAT).
The
second
place
where
it
will
arise
is
in
considering
whether
results
from
the
previous
tier
(
T1S)
should
affect
the
conclusions
drawn
from
the
subsequent
tier
(
T2T).
By
this,
the
EDSTAC
is
not
referring
to
the
fact
that
T1S
results
may
guide
selection
and/
or
design
of
Tier
2
tests
(
with
the
results
of
the
Tier
2
tests
then
being
interpreted
without
further
reference
to
the
T1S
results).
The
EDSTAC
is,
instead,
referring
to
the
possibility
that
the
T1S
assay
results
may
be
"
weighted
into"
the
determination
of
whether
a
substance
has
passed
or
failed
the
Tier
2
tests.

A
broad
range
of
results
may
need
to
be
weighted
into
a
final
judgment
at
either
tier.
Information
routinely
taken
into
consideration
in
determining
the
"
weight­
of­
evidence"
will
include:

·
the
balance
of
assays/
tests
that
gave
positive
and
negative
results;
·
results
of
in
vitro
versus
in
vivo
assays/
tests;
·
the
nature
of
the
biological
effects
induced;
·
the
range
of
effects
observed;
·
the
slope
and
shape
of
the
dose­
response
curves;
·
the
level,
magnitude,
or
severity
of
the
effects
induced;
and
·
the
presence
or
absence
of
response
in
multiple
taxa.

The
"
weight­
of­
evidence"
approach
makes
explicit
the
assumption
that
results
of
some
assays/
tests,
in
some
taxa,
at
some
level
of
severity,
are
intrinsically
"
worth"
more
than
others
and
should,
therefore,
carry
more
weight
in
decisions
following
T1S
and
T2T.
For
example,
positive
results
showing
reproducible,
high
levels
of
effects
at
low
doses
(
near
the
doses
produced
by
environmental
or
human
exposures)
are
likely
of
greater
weight
than
weak
effects
observed
only
at
very
high,
perhaps
excessively
toxic,
levels
of
exposure.

The
EDSTAC
has
taken
the
approach
here
of
providing
guidance
on
the
use
of
"
weight­
ofevidence
Any
approach
used
must
satisfy
several
broad
criteria
which
are
essential.
The
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
33
weighting
system
should
be
transparent,
allowing
individuals
to
review
the
"
weight­
of­
evidence"
determination.
It
should
be
possible
to
understand
the
procedure
before
viewing
the
data,
so
individuals
have
a
reasonable
expectation
of
the
final
decision
at
the
time
when
the
data
are
presented.
This
does
not
mean
the
decision
is
fully
determined
by
the
data,
removing
the
need
for
scientific
judgment,
but
it
does
mean
that
any
deviations
from
the
expected
decision
should
be
supported
by
an
explanation
detailing
the
"
weight­
of­
evidence"
assigned.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
34
B.
False
Negatives
and
False
Positives
Within
the
Context
of
T1S
and
T2T
The
guiding
principle
for
the
treatment
of
false
positives
and
negatives
should
be
one
of
valuing
sensitivity
more
than
specificity
at
the
screening
level
unless
this
compromises
the
ability
to
sort
chemical
substances
or
mixtures
into
a
subset
most
likely
to
be
of
concern.
False
positives
and
negatives
can
arise
in
at
least
three
different
ways
in
the
screening
and
testing
batteries
(
see
Figure
5.1):

·
The
false
result
may
be
due
to
the
stochastic
nature
of
screens
and
tests.
A
false
result
leading
to
an
incorrect
claim
that
the
screen/
test
is
positive
is
a
Type
I
(
false
positive)
error.
A
false
result
leading
to
an
incorrect
claim
that
the
screen/
test
is
negative
is
a
Type
II
(
false
negative)
error.
The
frequency
of
these
types
of
errors
is
expressed
by
the
p
value2
for
an
assay,
so
the
selection
of
a
required
p
value
to
classify
a
result
as
positive
will
determine
the
frequency
of
Type
I
and
Type
II
errors.
The
guiding
principle
above
suggests
that
required
p
values
should
be
chosen
so
Type
II
errors
are
minimized,
while
also
ensuring
that
Type
I
errors
do
not
become
so
frequent
that
chemical
substances
or
mixtures
can
no
longer
be
sorted
meaningfully.

·
False
positives
and
negatives
may
arise
due
to
unknown
or
unexpected
limitations
of
the
test
or
assays,
such
as
anomalous
activity
of
chemicals
or
classes
in
a
particular
assay
or
interference
from
assay
procedures.

·
The
third
source
of
error
arises
from
a
potential
lack
of
predictivity
of
results
in
T1S
for
endocrine
disruptive
responses
in
T2T.
This
source
of
error
is
shown
in
Figure
5.1
by
the
bold
arrow
going
from
positive
results
in
T1S
to
T2T.
A
negative
result
in
T1S
may
simply
mean
the
assay
battery
misses
a
mechanism
of
action
that
would
have
been
active
in
a
Tier
2
Test.
This
will
result
in
a
false
classification
of
the
substance
as
not
having
endocrine
activity,
an
error
that
would
have
been
caught
in
T2T
had
the
chemical
substance
or
mixture
proceeded
to
that
stage.
For
this
reason,
the
T1S
battery
was
designed
to
capture
all
known
endocrine
mechanisms
for
EAT
and
to
minimize
false
negative
results
specifically
as
opposed
to
false
positives.
A
positive
result
in
T1S
could
be
followed
by
negative
results
in
T2T
because
the
endpoints
measured
in
T1S
may
not
accurately
predict
adverse
effects
in
longterm
whole
animal
tests.
This
will
result
in
unnecessary
testing
of
some
chemicals
in
T2T,
a
possibility
considered
more
acceptable
than
missing
potential
endocrine
disruptors
for
EAT.

In
treating
the
frequency
of
Type
I
and
Type
II
errors,
it
is
important
to
consider
both
the
frequency
of
these
errors
in
each
particular
assay/
test
and
the
number
of
assays/
tests
in
a
battery.
As
the
number
of
assays/
tests
in
a
battery
increases,
the
probability
that
at
least
one
of
the
assays/
tests
will
show
a
false
positive
increases.
This
is
shown
in
Figure
5.2,
which
displays
the
2
"
P
value"
is
the
statistical
probability
that
two
groups,
e.
g.,
control
and
treated
animals,
come
from
the
same
population
(
i.
e.,
p=
0.05
means
that
the
probability
is
5
out
of
100
that
the
two
groups,
based
on
values
for
a
given
parameter,
belong
to
the
same
population
and
conversely,
that
95
out
of
100
do
not
belong
to
the
same
population
and
therefore
they
are
significantly
different).
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
35
relationship
between
the
probability
of
any
one
assay/
test
showing
a
false
positive
(
the
X
axis),
the
number
of
assays/
tests
in
the
battery
(
the
Y
axis),
and
the
probability
that
the
battery
shows
at
least
one
false
positive
result.
In
this
figure,
it
is
assumed
that
the
chemical
substance
or
mixture
tested
actually
has
no
endocrine
activity,
but
might
yield
a
false
positive
result
due
primarily
to
stochastic
variation.
The
goal
of
the
Tier
1
or
Tier
2
stages
should
be
to
minimize
the
probability
of
a
battery
producing
a
Type
II
error
for
a
chemical
substance
or
mixture,
while
not
causing
the
probability
of
a
Type
I
error
from
getting
so
large
that
the
battery
becomes
ineffective
at
sorting
chemical
substances
or
mixtures.
Figure
5.3
displays
the
analogue
of
Figure
5.2
(
i.
e.,
the
probability
that
a
battery
produces
a
false
negative
result
if
each
assay/
test
has
a
given
false
negative
frequency,
there
are
N
assays/
tests
in
the
battery,
and
the
chemical
substance
or
mixture
truly
is
an
endocrine
disruptor
for
EAT).

The
EDSTAC
cautions
that
the
statistical
properties
of
actual
assays/
tests
in
a
battery
will
not
be
identical,
so
Figures
5.2
and
5.3
are
simply
illustrative
and
must
be
modified
for
any
particular
battery
developed.
What
the
figures
indicate
is
that
weighting
a
single
positive
result
from
a
battery
into
the
"
weight­
of­
evidence"
judgment
should
reflect
a
concern
for
both
Type
I
and
Type
II
errors.
From
these
figures,
it
can
be
seen
that
a
large
battery
(
e.
g.,
with
10
assays/
tests),
each
with
a
false
positive
frequency
of
only
10%,
can
result
in
a
very
high
probability
of
producing
at
least
one
assay/
test
showing
a
false
positive
when
applied
to
a
substance
that
in
reality
has
no
endocrine­
disrupting
properties.
Such
a
battery
would
be
essentially
useless
in
sorting
chemical
substances
or
mixtures
and
focusing
society's
resources.
The
final
advice
here
is
that
an
effort
should
be
made
to
characterize
statistically
the
frequency
of
Type
I
and
Type
II
errors
associated
with
any
selected
battery,
and
to
use
this
characterization
in
deciding
the
weight
assigned
to
a
single
positive
result
from
that
battery.

C.
Specific
Principles
for
Evaluating
T1S
There
are
several
specific
criteria
to
be
met
by
the
decision
process
assuming
appropriate
dose
and
route
of
exposure
as
discussed
previously
in
this
chapter:

1.
If
functionally
equivalent
information
is
available
(
e.
g.,
from
the
sorting
and
prioritization
phase),
it
may
be
appropriate
that
only
those
T1S
assays
which
evaluate
the
endocrine
activity
of
concern
(
based
on
prior
information)
of
a
chemical
substance
or
mixture
would
be
performed
(
i.
e.,
only
a
subset
of
assays
would
be
run).
Similarly,
the
results
of
the
T1S
assays
may
require
that
only
a
subset
of
the
Tier
2
tests
be
conducted.

2.
If
all
assays
are
performed,
and
all
assays
are
negative,
then
the
chemical
substance
or
mixture
does
not
have
endocrine
activity
for
estrogen,
androgen,
or
thyroid
hormone
at
this
time.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
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36
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
37
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
38
EDSTAC
Final
Report
Chapter
Five
August
1998
5
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39
3.
In
vitro
assays
cannot
and
will
not
be
"
gatekeepers;"
they
cannot
constitute
a
"
decision
node;"
they
are
useful
as
information
for
possible
mechanisms
(
or
site
of
action)
but
not
as
"
yes/
no"
determinants
to
proceed
to
the
in
vivo
screens
or
T2T
because:
a)
in
vitro
assays
mediated
by
receptor
binding
evaluate
only
one
of
many
possible
sites
and
modes
of
action;
b)
negative
results
may
mean
relatively
little
due
to
limitations
of
the
assays,
e.
g.,
lack
of
metabolic
capability,
solubility,
etc.
(
i.
e.,
false
negatives);
and
c)
positive
results
may
be
false
positives.

4.
Results
from
in
vivo
assays
have
more
weight
than
results
from
in
vitro
assays
since:
a)
in
vitro
assays
will
generate
false
negatives
as
well
as
false
positives,
based
on
differences
in
access
to
the
target
tissue,
metabolism,
etc.
relative
to
in
vivo
assays;
and
b)
in
vivo
results
are
considered
to
be
more
relevant.

5.
Results
from
in
vitro
assays
that
assess
endocrine
activity
with
and
without
metabolic
activation
are
worth
more
than
results
from
in
vitro
assays
without
metabolic
activation
(
since
the
former
can
assess
the
activity
of
metabolites
generated
within
the
culture
if
the
correct
metabolic
activation
is
used
(
e.
g.,
rat
liver
S9)
and
the
latter
can
only
assay
the
parent
compound).

6.
Results
from
apical
in
vivo
assays
are
worth
more
than
results
from
specific
in
vivo
assays
(
since
they
indirectly
assay
many
more
sites
of
action
to
get
to
the
same
endpoint;
e.
g.,
uterotrophic
assay
in
ovariectomized
adult
females
[
specific
assay;
chemical
substance
or
mixture
must
act
at
level
of
uterus]
versus
in
intact
immature
females
[
apical
assay;
chemical
substance
or
mixture
can
act
at
level
of
hypothalamus,
pituitary,
gonad,
thyroid,
and/
or
uterus]).
A
positive
specific
assay
provides
mechanistic
information
but
other
mechanisms
of
action
may
also
be
present
and
go
undetected;
a
negative
specific
assay
is
less
informative.

7.
Biologically
plausible
results
are
worth
more
than
biologically
implausible
results
(
obviously
dependent
on
the
state
of
current
scientific
knowledge).

8.
Statistical
significance
is
a
useful
tool,
but
must
be
interpreted
within
the
context
of
biological
significance.
For
example,
an
observed
association
which
does
not
achieve
statistical
significance,
but
which
is
consistent
with
results
from
related
assays
suggesting
a
common
mechanism
of
action,
might
be
interpreted
as
biologically
significant.
This
means
the
use
of
any
particular
criterion
such
as
p
equal
to
0.05
should
be
carefully
considered,
and
there
may
be
no
hard
and
fast
rule
for
weighting
by
statistical
significance.

9.
A
consistent
pattern
of
positive
(
or
negative)
results
in
various
related
assays
is
worth
more
than
a
single
isolated
positive
(
or
negative)
result
(
e.
g.,
positive
results
for
binding
to
ER
and
transcriptional
activation
in
vitro
and
positive
results
in
an
apical
or
specific
uterotrophic
assay
in
vivo
are
worth
more
than
a
positive
result
for
receptor
binding
and
transcriptional
activation,
but
no
uterotrophic
response)
(
see
additional
comments
in
discussion
of
false
negatives
and
false
positives
above).
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
40
10.
The
decision
which
will
emerge
from
T1S
is:
a)
The
chemical
substance
or
mixture
does
not
require
further
testing
for
EAT
activity
at
this
time
(
the
chemical
substance
or
mixture
goes
to
the
"
hold
box");
or
b)
The
chemical
substance
or
mixture
should
be
tested
further
for
EAT
activity
at
this
time,
and
i)
proceed
to
T2T;
or
ii)
proceed
to
Hazard
Assessment/
Regulatory
Action.

V.
Tier
2
Testing
Concepts
and
Design
Parameters
A.
Introduction
to
T2T
The
purpose
of
T2T
is
to
characterize
the
nature,
likelihood,
and
dose­
response
relationship
of
endocrine
disruption
of
estrogen,
androgen,
and
thyroid
in
humans
and
wildlife.
T2T
is
a
complement
to
T1S
and
T2T
results
supersede
T1S
results.
As
already
discussed,
T1S
is
composed
of
a
battery
of
in
vitro
and
in
vivo
assays
designed
to
detect
whether
a
substance
may
have
EAT
activity.
The
in
vitro
screening
assays
are
highly
sensitive
and
quite
selective
for
a
particular
mode
of
action.
They
are,
however,
quite
far
removed
from
the
biological
complexity
of
an
intact
animal
and
may
give
false
positive
readings
because,
for
instance,
not
all
substances
which
bind
to
a
receptor
will
cause
an
adverse
biological
effect;
false
negative
readings
may
also
result
from
the
in
vitro
receptor
binding
or
transcriptional
assays
because
not
all
endocrine
disruptors
act
via
a
receptor.
In
vivo
assays
encompass
the
metabolic
and
response
capability
of
a
whole
organism
but
focus
on
such
a
short
time
frame
that
the
full
effects
of
exposure
to
a
chemical
substance
may
not
be
identified
and
characterized.
Since
there
is
considerable
biological
conservation
in
the
endocrine
system,
it
is
not
necessary
to
screen
in
every
major
taxonomic
group.
Screens
based
on
mammalian
cell
lines
or
intact
animals
will
determine
whether
a
chemical
substance
or
mixture
may
interact
with
the
endocrine
system,
and
if
so,
the
effects
of
the
chemical
substance
or
mixture
must
be
characterized
in
longer­
term
studies
in
species
representing
a
variety
of
taxa.

T2T
is
the
final
phase
of
the
screening
and
testing
program
and
is
intended
to
provide
more
detailed
information
regarding
endocrine
disruption
activity
of
a
tested
chemical
substance
or
mixture.
This
tier
should
assess
the
concentrations
which
elicit
endocrine
disruption
and
the
consequences
of
such
disruption
to
inform
hazard
assessments.
To
fulfill
this
purpose,
tests
are
longer­
term
studies
designed
to
encompass
critical
life
stages
and
processes,
a
broad
range
of
doses,
and
administration
by
a
relevant
route
of
exposure,
so
a
more
comprehensive
profile
of
biological
consequences
of
chemical
exposure
can
be
identified
and
related
to
the
dose
or
exposure
which
caused
them.
Effects
associated
with
endocrine
disruption
may
be
latent
and
not
manifested
until
later
in
life
or
may
not
appear
until
the
reproductive
period
is
reached.
Tests
for
endocrine
disruption
will
usually
encompass
two
generations
including
effects
on
fertility
and
mating,
embryonic
development,
sensitive
neonatal
growth
and
development,
and
transformation
from
the
juvenile
life
stage
to
sexual
maturity
unless
a
rationale
exists
to
limit
the
mammalian
test
to
one
generation.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
41
The
outcome
of
T2T
is
designed
to
be
conclusive
in
relation
to
the
outcome
of
T1S,
and
any
other
prior
information,
in
the
sense
that
a
negative
outcome
in
T2T
will
supersede
a
positive
outcome
in
T1S.
Furthermore,
each
full
test
in
T2T
has
been
designed
to
include
those
endpoints
that
will
allow
one
to
reach
a
definitive
conclusion
as
to
whether
or
not
the
tested
chemical
substance
or
mixture
is
or
is
not
an
endocrine
disruptor
for
EAT
in
that
species/
taxa.
Conducting
all
five
tests
in
the
T2T
battery
would
provide
a
more
comprehensive
profile
of
the
effects
a
chemical
substance
or
mixture
could
induce
via
EAT
endocrine
disruption
mode(
s)/
mechanism(
s)
of
action
than
would
be
the
case
if
only
a
subset
of
tests
were
performed.
The
EDSTAC
recommends
that
the
"
default"
action,
in
the
absence
of
any
prior
information,
be
to
perform
all
tests
in
the
T2T
battery
with
all
endpoints.
However,
performance
of
the
entire
battery
with
multiple
generations
may
not
always
be
necessary,
as
is
discussed
below.

The
next
section
of
this
report
provides
guidance
on
the
selection
of
the
taxa
to
be
subjected
to
T2T,
and
the
use
of
alternative
tests,
focusing
particularly
on
alternative
mammalian
tests.
Conducting
Tier
2
tests
on
less
than
the
five
recommended
taxa
and/
or
the
use
of
an
alternative
mammalian
test
will
result
in
T2T
outcomes
that
are,
by
definition
and
design,
less
comprehensive
in
comparison
to
the
outcomes
from
T2T
using
the
recommended
two­
generation
mammalian
test
and/
or
the
full
set
of
recommended
taxa.
However,
the
EDSTAC
believes
the
guidance
set
forth
below
will
ensure
that
the
information
generated
from
tests
conducted
with
less
than
the
full
set
of
recommended
taxa
and/
or
using
alternative
Tier
2
tests
will
still
be
sufficient,
along
with
prior
information,
for
EPA
to
make
a
decision
about
whether
the
chemical
substance
or
mixture
should
be
placed
in
the
"
hold
box"
or
forwarded
to
hazard
assessment.

While
two­
generation
tests
are
designed
to
fully
characterize
potential
effects
of
concern,
there
may
be
instances
(
depending
on
available
prior
information)
when
a
less
comprehensive
study
design
would
provide
adequate
information
on
which
to
make
decisions.
In
addition,
there
may
be
reasons
why
all
of
the
non­
mammalian
tests
need
not
be
conducted
in
the
same
time
frame,
or
at
all.
Below
is
a
discussion
of
how
the
decisions
of
which
tests
to
perform
should
be
informed.

In
determining
which
mammalian
and
non­
mammalian
tests
to
include
in
T2T,
the
EDSTAC
chose
tests
which
would
require
the
least
modification
from
existing
standardized
methods
in
order
to
minimize
the
time
necessary
to
standardize
and
validate
those
tests.
Thus,
the
Tier
2
tests
for
mammals
(
rats),
fish,
birds,
and
invertebrates
are
based
on
existing
test
guidelines
(
the
recommended
study
design
for
amphibians
has
never
been
utilized
in
a
testing
scheme).
The
existing
test
guidelines
were
considered
adequate
for
evaluating
the
most
obvious
consequences
of
EAT­
related
endocrine
disruption,
but
not
adequate
to
pick
up
some
of
the
more
subtle
or
insidious
effects.
However,
more
modification
of
the
non­
mammalian
guidelines
was
required
than
for
their
mammalian
counterparts
and
the
elements
described
in
the
existing
non­
mammalian
guidelines,
in
general,
have
not
been
widely
and
routinely
run,
as
have
the
elements
described
in
the
mammalian
guidelines.

B.
Outline
of
Recommended
T2T
Battery
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
42
The
EDSTAC
recommends
that
the
T2T
battery
include
a
mammalian
two­
generation
reproductive
toxicity
study,
or
a
less
comprehensive
mammalian
test
in
accordance
with
guidelines
outlined
below,
and
tests
addressing
four
additional
taxonomic
groups,
including
birds,
amphibians,
fish,
and
invertebrates
as
follows.

Mammalian
Tests
1.
Two­
Generation
Mammalian
Reproductive
Toxicity
Study;
or
2.
A
Less
Comprehensive
Test:
a)
Alternative
Mammalian
Reproductive
Test;
or
b)
One­
Generation
Test.

Multigeneration
Tests
in
Other
Taxa
1.
Avian
Reproduction
(
with
bobwhite
quail
and
mallard)
2.
Fish
Life
Cycle
(
fathead
minnow)
3.
Mysid
Life
Cycle
(
Americamysis)
4.
Amphibian
Development
and
Reproduction
(
Xenopus)

C.
Guidance
for
Selecting
Tier
2
Tests
The
Conceptual
Framework,
found
in
Chapter
Three,
states
that
existing
information
on
biological
effects
and
exposure
and
the
results
of
T1S
should
be
used
to
inform
decisions
regarding
the
selection
and
design
of
Tier
2
tests.
The
EDSTAC
believes
that
T1S
information
may
be
of
use
in
determining
whether
to
evaluate
thyroid
effects,
for
example,
but
may
be
of
limited
value
in
determining
whether
all
five,
or
some
subset
of
the
recommended
Tier
2
tests
should
be
conducted
since
only
a
limited
number
of
taxa
are
recommended
for
the
in
vivo
assays
and
mammalian
cell
lines
are
used
for
in
vitro
assays
in
T1S.
Therefore,
the
EDSTAC
recommends
that
the
choice
of
whether
Tier
2
tests
will
be
conducted
on
all
five
of
the
recommend
taxa,
or
a
more
limited
subset
of
the
five
taxa,
should
be
based
on
the
physicochemical
characteristics
and
environmental
release
and
exposure
information
of
the
chemical
substance
or
mixture
to
be
tested,
together
with
biological
data
from
T1S.

This
section
addresses
the
need
for
guidance
in
the
selection
of
Tier
2
tests,
focusing
upon:
(
1)
the
determination
of
which
of
the
five
taxonomic
groups
should
be
included
in
the
Tier
2
testing
of
a
specific
chemical
substance
or
mixture;
(
2)
the
circumstances
under
which
it
may
be
appropriate
to
perform
an
alternative
test,
with
a
particular
focus
on
the
selection
of
alternative
mammalian
tests;
(
3)
the
selection
of
endpoints;
(
4)
the
special
case
of
chemicals
that
bypass
T1S
and
go
directly
to
T2T;
and
(
5)
the
potential
need
for
supplemental
information
to
complete
T2T.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
43
1.
Determining
Which
Taxa
Should
be
Included
in
Tier
2
Testing
for
a
Specific
Chemical
In
general,
the
number
of
studies
underway
at
any
one
time
is
limited,
in
part,
by
laboratory
capacity.
In
addition,
the
pace
at
which
each
study
can
be
completed
is
controlled
by
the
nature
of
the
protocol
for
the
study.
Priority
should
be
given
to
conducting
tests
for
those
species/
taxa
to
which
exposure
is
known
or
expected
to
occur.
The
EDSTAC
recommends
that
in
the
absence
of
information,
as
described
below,
the
default
assumption
should
be
that
chemical
substances
or
mixtures
should
be
subjected
to
the
full
set
of
five
recommended
tests
with
all
endpoints,
once
they
are
fully
standardized
and
validated.
Building
on
these
overarching
principle,
the
EDSTAC
recommends
that
EPA
should
use
the
following
guidelines
in
determining
which
taxa/
tests
should
be
included
T2T
for
a
specific
chemical
substance
or
mixture:

a)
Where
use,
exposure,
and
release
of
a
substance
are
well
characterized,
it
may
be
possible
to
tailor
T2T
for
particular
exposure
scenarios.
Conversely,
if
they
are
poorly
characterized,
positive
results
in
T1S
would
trigger
the
entire
battery
of
Tier
2
tests
unless
other
data
clearly
indicated
that
certain
taxa
would
not
be
affected.
As
noted
above,
the
EDSTAC
recommends
the
default
assumption,
in
the
absence
of
information
that
certain
taxa
would
not
be
affected,
be
to
conduct
all
five
of
the
recommended
tests
with
all
endpoints.

b)
If
sufficient
exposure
data
are
available,
the
owner
of
the
chemical
may
conduct
an
exposure
assessment
to
provide
information
that
will
help
inform
the
selection
of
Tier
2
tests.
Exposure
assessments
vary
in
scope
and
complexity,
but,
for
this
purpose,
"
sufficient
data"
at
a
minimum
includes
chemical
identity,
basic
chemical
properties
(
water
solubility,
octanol:
water
partition
coefficient,
vapor
pressure,
Henry's
Law
constant),
rates
of
significant
transformation
processes
such
as
biodegradation,
and
use
and
release
profiles,
including
accidental
releases.
Measured
values
are
preferable
for
chemical
properties
and
environmental
fate,
but
estimation
methods
are
often
satisfactory
for
supplying
missing
data.
Ideally,
the
chemical
use
and
release
profile
should
provide
information
on
the
distribution
of
releases
(
if
any)
to
air,
water,
soil,
etc.,
and
the
amounts
and
frequency
of
such
releases.

For
example,
freshwater
aquatic
exposure
modeling
often
can
be
conducted
using
models
which
incorporate
environmental
degradation
processes.
One
such
model,
the
Probablistic
Dilution
Model,
yields
the
frequency
of
exceedance
of
an
ecological
concern
level
preset
by
the
modeler,
and
is
useful
not
only
when
releases
are
from
known
point
sources
but
also
when
only
the
category
of
use
(
via
Standard
Industrialization
Classification
Codes)
is
known.
At
a
higher
level
of
complexity,
even
site­
specific
models
such
as
EXAMS­
II
may
be
appropriate
under
some
circumstances,
for
example,
when
there
are
only
a
few
point
sources
and
the
site
of
release
and
downstream
environments
are
well
characterized.

Soil
fate
models
such
as
SESOIL
and
PRZM
exist
but
also
have
extensive
parameterization
requirements.
Short
of
this,
rough
estimates
of
mobility
in
soil
and
thus
likelihood
of
reaching
groundwater
or
surface
waters
that
are
in
hydrological
contact
with
ground
water
can
be
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
44
made
using
certain
screening­
level
tools
routinely
applied
by
EPA
in,
for
example,
PMN
review.

For
air
releases
numerous
fate
models
such
as
ISCLT
are
available,
and
like
the
water
models,
they
vary
in
complexity.
They
calculate
concentrations
of
chemicals
in
air
at
assumed
locations
of
human
receptors
(
e.
g.,
at
certain
distances
from
stack
releases),
but
such
exposure
data
may
also
be
used
as
input
for
aquatic
or
terrestrial
ecological
exposure
assessments.

Models
for
calculating
environmental
concentrations
of
chemicals
released
to
estuaries
are
less
well
developed
in
general,
but
the
model
ESTUARY
contains
extensive
hydrologic
data
for
several
major
estuaries
in
the
U.
S.
and
is
potentially
useful.
It
was
designed
for
high­
volume
consumer
products
that
are
widely
used
and
dispersed
in
the
environment
(
i.
e.,
surfactants).

c)
If
a
chemical
is
released
to
or
can
be
predicted
to
reach
streams,
rivers,
or
lakes,
a
fish
life
cycle
test
with
freshwater
species
and
invertebrate
life
cycle
test
should
be
conducted.
If
release
is
to
an
estuary,
marine
species
should
be
substituted
for
freshwater
species
in
toxicity
testing.
If
release
is
to
both
types
of
environment,
freshwater
species
are
preferred.
In
a
broad
sense,
freshwater
fish
species
are
often
more
sensitive
than
saltwater
fish
species
in
laboratory
toxicity
tests.
The
greater
sensitivity
is
due,
in
part,
to
greater
bioavailability
in
freshwater
which
has
fewer
dissolved
solids
than
brackish
or
saltwater.
Moreover,
currently
freshwater
species
are
more
widely
available
and
somewhat
easier
to
accommodate
in
laboratory
settings
than
saltwater
species.
Species
selection,
independent
of
taxa,
should
be
performance­
based,
and
as
more
appropriate
species
are
developed
for
use
and
validated,
the
EDSTAC
strongly
encourages
their
use.

d)
Pesticides
with
agricultural
or
other
outdoor
uses,
and
chemicals
that
would
be
expected
to
bioaccumulate
and
biomagnify
through
the
food
chain
or
that
present
a
potential
exposure
to
birds
and
aquatic
organisms,
should
be
tested
in
the
appropriate
tests.
Alternatively,
if
the
pesticide
or
other
chemical
is
not
likely
to
be
used
in
a
manner
that
results
in
exposure
to
birds
and
aquatic
organisms,
it
does
not
need
to
be
tested
in
either
the
avian
or
aquatic
tests.
For
example,
as
per
current
regulatory
requirements
under
FIFRA
(
40
CFR
158),
this
would
be
the
case
if
a
pesticide
registrant
is
requesting
the
first
use
of
a
pesticide
only
for
application
to
flowers
grown
only
in
greenhouses.
Since
a
greenhouse
is
considered
a
confined­
use
situation,
the
studies
to
assess
the
impact
on
birds
or
aquatic
organisms
would
not
be
required.
If,
at
a
later
time,
the
registrant
requests
additional
uses,
then
additional
studies
might
be
required.
In
this
example,
assume
the
second
use
is
for
application
to
turf.
Then,
impacts
on
birds
and
aquatic
organisms
would
be
of
concern,
and
therefore
studies
on
those
organisms
would
be
necessary.

A
similar
situation
exists
for
commercial
chemicals
under
TSCA.
A
manufacturer
may
supply
information
to
EPA
that
a
substance
is
a
site­
limited
intermediate
with
no
significant
releases
to
the
environment.
In
this
case,
the
exposure
of
concern
would
be
exposure
to
workers
and
the
appropriate
test
would
be
the
mammalian
reproduction
test.
If
new
uses
were
developed
for
the
chemical
which
could
result
in
environmental
exposure,
the
ecological
tests
could
be
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
45
required.
The
rationale
for
testing
should
not
be
limited
solely
to
use
considerations
but
should
also
consider
potential
releases
from
manufacturing,
storage,
transportation,
and
disposal.

e)
Finally,
as
noted
in
Chapter
Three,
if
a
chemical
is
placed
in
the
"
hold
box"
based
on
the
results
of
Tier
2
tests
that
are
conducted
on
less
than
the
full
set
of
the
five
recommended
taxa/
tests,
and
the
use
and
exposure
patterns
for
the
chemical
substance
subsequently
change,
EPA
should
determine
whether
the
new
use
or
uses
warrant
testing
in
one
or
more
of
the
taxa
that
had
previously
not
been
included
in
Tier
2
testing
of
the
chemical.

2.
Determining
When
to
Perform
Alternative
Tests
In
Chapter
Three
the
EDSTAC
sets
forth
a
definition
of
"
function
equivalency."
In
presenting
the
guidelines
for
determining
when
it
may
be
appropriate
to
perform
alternative
Tier
2
tests,
the
EDSTAC
wishes
to
emphasize
the
difference
between
alternative
tests
that
meet
the
definition
of
functional
equivalency
from
those
that
do
not.
As
stated
in
Chapter
Three,
an
assay,
test,
or
endpoint
can
be
considered
to
be
"
functionally
equivalent"
to
a
T1S
or
T2T
assay,
test,
or
endpoint
when
it
provides
equivalent
information
for
each
endpoint
being
studied.
Furthermore,
the
EDSTAC­
recommended
assays,
tests,
and
endpoints
must
be
validated
and
standardized
prior
to
EPA's
use
of
functionally
equivalent
information.

The
EDSTAC
recommends
that,
as
new
tests
are
developed
and
proposed
for
inclusion
in
T2T,
EPA
should
determine
whether
such
tests
are
functionally
equivalent
to
the
recommended
Tier
2
tests.
If
they
are
determined
not
to
be
functionally
equivalent,
the
decision
about
whether
and,
if
so,
under
what
condition,
any
newly
developed
alternative
tests
should
be
used,
should
be
subjected
to
the
criteria
listed
below.

This
section
sets
forth
guidelines
for
determining
when
to
conduct
alternative
tests,
which
are
less
comprehensive
than
the
recommended
tests,
and
therefore
do
not
meet
the
definition
of
"
functional
equivalency."

The
EDSTAC
believes
there
are
instances
when
a
less
comprehensive
test
for
any
of
the
five
recommended
taxa,
when
considered
along
with
existing
information,
would
provide
sufficient
information
upon
which
to
make
a
decision
that
meets
the
overall
purpose
of
T2T
(
i.
e.,
whether
to
go
to
the
hold
box
or
hazard
assessment).
The
EDSTAC
identified
two
alternative
study
designs
for
mammalian
species.
The
EDSTAC
believes
such
alternative
designs
could
be
developed
for
all
species
currently
included
in
T2T.
When
and
if
such
alternative
tests
are
developed
for
the
other
taxa,
in
making
the
decision
to
perform
a
less
comprehensive
test
EPA
should
consider
the
same
issues,
and
apply
the
same
principles
that
are
described
below
for
the
alternative
mammalian
tests.
The
EDSTAC
recommends
alternative
study
designs
be
developed
for
other
species/
taxa
only
after
their
respective
multi­
generation
study
protocols
are
validated
and
standardized.

The
primary
considerations
for
determining
whether
an
alternative,
less
comprehensive
test
should
be
conducted
include
an
understanding
of
the
toxicity
profile
of
the
chemical
substance
or
mixture
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
46
under
study;
its
mechanisms
of
action;
exposure
scenarios;
use
patterns;
populations
at
risk;
and
other
prior
information.
The
criteria
for
determining
when
an
alternative
test
might
be
run
include:
(
1)
a
full
two­
generation
reproductive
toxicity
study
has
been
run
in
the
past,
but
it
either
was
conducted
in
accordance
with
the
"
old"
guideline
and/
or
the
results
in
the
previous
study
require
additional
follow­
up
that
may
be
accomplished
using
one
of
the
alternative
protocols;
(
2)
production
volume
and
potential
for
exposure
is
low;
or
(
3)
there
is
low
probability
of
at­
risk
populations
being
exposed.
In
applying
criteria
2
and
3,
the
alternative
test
may
be
used
more
as
a
preliminary
evaluation
and,
therefore,
not
necessarily
as
the
last
evaluation
of
the
potential
for
EAT
effects
in
a
reproductive
toxicity
study.
Furthermore,
the
EDSTAC
wishes
to
note
that
chemical
substances
determined
to
be
a
high
priority
for
the
early
phases
of
T1S
based
primarily
on
exposure
considerations
are
not
likely
to
meet
these
criteria;
whereas
the
chemicals
permitted
to
bypass
T1S
due
to
existing
statutory
requirements
(
i.
e.,
food­
use
pesticides)
will
more
often
than
not
meet
the
first
of
these
three
criteria.

Below
are
potential
scenarios
for
a
specific
chemical
substance
or
mixture
that
requires
a
decision
on
whether
to
perform
the
two­
generation
mammalian
test
or
one
of
the
alternative
tests.
These
examples
pertain
only
to
the
mammalian
reproduction
tests
as
they
are
the
only
ones
currently
discussed
in
the
report.

a)
A
chemical
substance
or
mixture
has
not
been
through
T1S,
and
thus,
a
weight­
of­
evidence
evaluation
cannot
be
performed.
One
may
or
may
not
have
prior
information
which
offers
an
understanding
of
the
chemical
substance
or
mixture's
potential
to
show
endocrine
activity
for
the
EAT
hormone
systems.
In
this
case,
the
EDSTAC­
recommended
two­
generation
reproduction
study
would
be
conducted
for
each
taxa/
species
which
has
been
identified
as
being
exposed
or
likely
to
be
exposed.
Whether
or
not
all
four
of
the
non­
mammalian
tests
would
be
performed
should
be
determined
in
accordance
with
the
principles
described
in
Section
V,
C,
1
of
this
chapter.

b)
A
chemical
substance
or
mixture
has
been
through
T1S
and
the
weight­
of­
evidence
evaluation
concludes
it
does
not
have
endocrine
activity
for
EAT.
The
chemical
substance
or
mixture
has
use
patterns
suggesting
no
or
low
exposure
potential
to
populations
of
concern.
In
this
case,
no
further
screening
or
testing
for
EAT
would
be
needed.
However,
a
decision
might
be
made
to
ask
for
a
reproductive
toxicity
study
to
satisfy
other
regulatory
requirements.
One
might
argue
that
an
alternative
test
protocol
would
provide
sufficient
information
at
this
time
since
it
is
important
to
evaluate
the
potential
for
inducing
reproductive
effects
of
concern
that
may
have
other
underlying
mode(
s)/
mechanism(
s)
of
action.
Obviously,
depending
upon
the
outcome
of
the
study
and
later
discovery
of
additional
relevant
information,
additional,
follow­
up
studies
could
be
required.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
47
3.
Determining
Selection
of
Endpoints
In
addition
to
guidance
regarding
selection
of
tests,
the
EDSTAC
believes
it
is
necessary
to
offer
some
guidance
related
to
selection
of
which
endpoints
to
include
in
performance
of
the
tests
for
each
chemical
substance
or
mixture.
Below
are
some
potential
scenarios
regarding
selection
of
which
particular
endpoints
should
be
evaluated.

a)
A
chemical
substance
or
mixture
has
been
through
T1S
and
the
"
weight­
of­
evidence"
evaluation
concludes
it
may
have
endocrine
activity
in
all
three
hormone
systems
(
E,
A,
and
T).
The
chemical
substance
or
mixture
has
use
patterns
suggesting
exposure
could
possibly
occur
to
populations
of
concern.
In
this
case,
the
two­
generation
test,
including
the
thyroid
related
enhancements
(
recommended
later
in
the
report),
should
be
conducted.

b)
A
chemical
substance
or
mixture
has
been
through
T1S
and
the
"
weight­
of­
evidence"
evaluation
concludes
it
may
have
endocrine
activity
for
E
and
A,
but
not
for
T.
The
chemical
substance
or
mixture
has
use
patterns
suggesting
exposure
could
possibly
occur
to
populations
of
concern.
In
this
case,
it
would
be
appropriate
to
require
the
EDSTACrecommended
mammalian
two­
generation
test,
without
the
recommended
enhancements
for
evaluating
T.
In
other
words,
the
study
design
would
be
tailored
to
provide
results
only
for
the
endpoints
of
concern
identified
in
T1S.

4.
Chemicals
That
Bypass
T1S
and
go
Directly
to
T2T
In
Chapter
Three,
the
EDSTAC
makes
a
distinction
between
two
different
scenarios
under
which
a
chemical
would
be
permitted
to
bypass
T1S
and
go
directly
to
T2T.
The
first
includes
chemicals
that
have
previously
been
subjected
to
two­
generation
mammalian
and
wildlife
developmental
and
reproductive
toxicity
testing.
The
second
includes
chemicals
for
which
the
owner
of
the
chemical
has
decided
to
voluntarily
go
to
T2T
without
having
completed
the
full
T1S
battery
or
any
prior
two­
generation
reproductive
toxicity
testing.
The
EDSTAC
recommends
elsewhere
that
both
categories
of
"
bypass
chemicals"
should
be
required
to
complete
the
HTPS
assays.

T1S
assays,
in
the
aggregate,
provide
preliminary
information
on
the
presence
of
endocrine
activity
for
EAT,
the
mechanism
of
action,
and
the
species
and
sex
at
risk.
The
EDSTAC
believes
the
absence
of
such
information
in
the
case
of
chemicals
that
voluntarily
bypass
T1S
without
having
completed
the
full
T1S
battery
or
any
prior
two­
generation
reproductive
toxicity
testing
provides
sufficient
justification
for
requiring
such
chemicals
to
complete
all
five
tests
in
the
T2T
battery
(
i.
e.,
the
two­
generation
mammalian
and
non­
mammalian
tests
with
all
the
recommended
endpoints).
However,
the
determination
of
whether
all
of
the
non­
mammalian
tests
would
need
to
be
conducted
should
be
made
consistent
with
the
principles
governing
their
selection,
which
were
set
forth
in
Section
V,
C,
1
of
this
chapter.

For
chemicals
that
bypass
T1S
because
they
have
previously
been
subjected
to
two­
generation
mammalian
and
wildlife
developmental
and
reproductive
toxicity
testing,
as
noted
above,
the
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
48
EDSTAC
believes
these
chemicals
are
the
primary
candidates
for
meeting
the
criteria
specified
for
possible
use
of
alternative
Tier
2
tests.
Once
again,
the
determination
of
whether
all
of
the
nonmammalian
tests
would
need
to
be
conducted
should
be
made
consistent
with
the
principles
governing
their
selection,
which
were
set
forth
in
Section
V,
C,
1
of
this
chapter.

The
EDSTAC
recognizes
it
may
be
necessary,
after
completion
of
T2T,
to
conduct
a
limited
number
of
assays
that
are
similar,
if
not
identical,
to
those
that
would
have
been
conducted
during
T1S
for
chemicals
which
are
permitted
to
bypass
the
T1S
battery.
The
purpose
of
conducting
these
assays
as
part
of
T2T
is
to
gain
knowledge
about
specific
mechanisms
of
action
that
are
necessary
to
complete
the
hazard
assessment
step
and/
or
to
determine
whether
any
adverse
effects
observed
in
T2T
are
in
fact
endocrine
mediated.

5.
Potential
Need
for
Supplemental
Information
to
Complete
T2T
The
EDSTP
focuses
on
identifying
agents
that
act
as
reproductive
or
developmental
toxicants
through
the
EAT
endocrine­
mediated
mechanisms.
Properly
conducted,
Tier
2
tests
are
intended
to
be
the
final
arbiter
of
whether
or
not
a
substance
is
an
endocrine
disruptor
for
EAT.
In
other
words,
when
the
results
of
T2T
are
unambiguous,
they
provide
a
conclusive
answer.
Given
the
current
definition
of
testing,
both
the
expression
of
"
endocrine­
mediated"
and
"
adverse
effects"
are
necessary
conditions
for
designation
as
an
endocrine
disruptor
for
EAT.
In
some
cases,
particularly
for
those
chemicals
that
have
received
a
positive
in
T1S,
it
will
be
apparent
that
the
type
of
effect
seen
in
T2T
is
endocrine­
mediated.
In
other
cases,
particularly
for
those
chemicals
that
bypass
T1S,
the
results
of
T2T
may
not
allow
a
judgment
to
be
reached
that
the
adverse
effect
is
endocrine­
mediated.
The
EDSTAC
recommends
that
such
information,
if
determined
to
be
necessary
to
achieve
a
"
weight­
of­
evidence"
judgment,
should
be
generated
through
further
study
using
more
focused
assays,
some
of
which
may
be
drawn
from
the
T1S
battery.

Furthermore,
it
is
inevitable
with
toxicological
testing
that
equivocal
results
will
sometimes
be
obtained
in
T2T.
When
this
occurs
one
must
look
at
the
possible
reasons
for
the
ambiguity
and
see
if
there
are
ways
to
resolve
it.
Conducting
other
tests,
or
running
assays
to
investigate
the
mechanism
of
action
(
if
T1S
was
bypassed),
may
resolve
the
ambiguity
and
allow
a
more
informed
"
weight­
of­
evidence"
determination
of
the
hazard
potential
of
the
chemical
substance
or
mixture.
Alternatively,
repeating
the
study,
perhaps
at
different
exposure
levels,
may
resolve
the
ambiguity.

D.
Low
Dose
Considerations
for
T2T
1.
Introduction
to
the
Issue
Issues
have
been
raised
regarding
approaches
to
regulatory
toxicity
testing
that
employ
dosing
regimens
up
to
maximally
tolerated
dosages
in
order
to
identify
a
hazard
and
extrapolate
from
these
doses
to
estimate
risk
or
safe
levels
in
the
range
of
environmentally
relevant
exposures.
There
are
two
principal
issues:
(
1)
whether
or
not
a
threshold
dose
exists
for
receptor
mediated
toxicity;
and
(
2)
whether
the
dose
response
curve
is
monotonic
or
non­
monotonic
in
nature.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
49
With
respect
to
the
first
issue,
some
believe
that
there
is
no
threshold
for
effects
of
exogenous
endocrine
disruptors
since
there
exist
background
levels
of
endogenous
hormones
that
are
already
biologically
active.
Thus,
any
additional
exposure
constitutes
an
exceedance
of
the
threshold
(
e.
g.,
McLachlan
et
al.,
1982;
Wibbels
et
al.,
1991;
Russo,
1996;
Gray
et
al.,
1997b).

The
second
issue
relates
to
the
shape
of
the
dose­
response
curve.
Although
monotonic
curves
may
vary
in
slope,
the
slope
of
such
curves
is
always
in
the
same
direction
(
either
positive
or
negative
including
zero)
and
therefore
there
are
no
local
maximum
or
minimum
points
along
the
curve.
With
non­
monotonic
dose­
response
curves,
local
minima
can
exist
such
that
an
effect
may
be
pronounced
at
low
doses,
then
becomes
statistically
insignificant
or
disappears
at
intermediate
doses,
and
finally
reappears
(
or
a
different
effect
appears)
at
higher
doses.
The
issue
raised
by
non­
monotonic
curves
is
that
"
high
dose"
testing
may
fail
to
detect
toxicity
that
occurs
in
the
"
low
dose"
region
of
the
dose­
response
curve.
This
is
because
the
classical
approach
to
finding
the
NOAEL
by
progressive
reduction
of
the
dose
beginning
at
high
doses
will
locate
the
nadir
(
i.
e.,
a
local
minimum
at
some
intermediate
dose)
but
will
not
locate
the
second
region
of
increased
effect
at
doses
below
the
apparent
NOAEL.
This
issue
is
further
complicated
by
the
possibility
of
different
effects
at
low
doses
as
compared
to
high
doses.
There
are
examples
of
this
phenomenon
for
endogenous
hormones.
For
example,
it
is
well
known
that
testosterone
stimulates
sperm
production
up
to
a
point,
but
at
excess
levels
inhibits
it.
There
is
evidence
that
the
developing
mouse
prostate
responds
in
a
non­
monotonic
manner
to
estrogens,
in
that
prostate
weight
is
increased
initially,
then
decreased
by
higher
maternal
dosages
of
potent
estrogens
like
DES
and
ethinyl
estradiol.
There
is
one
report
in
the
literature
indicating
that
the
weak
estrogen
bisphenol
A
has
effects
similar
to
these
potent
estrogens
at
low
doses
(
Nagel
et
al.,
1997).
There
is
intense
scientific
debate
surrounding
these
issues
that
centers
on
two
principal
questions:
first,
are
data
implicating
xenobiotics
in
such
phenomena
reproducible
and
broadly
generalizable
to
endocrine
endpoints
and
endocrine
active
chemicals?
and
second,
is
the
low
dose
phenomenon
indicative
of
adverse
effects
at
the
individual
or
population
level?
If
low
dose
phenomena
are
reproducible,
generalizable,
and
related
to
adverse
effects,
the
implications
for
regulatory
toxicity
testing
and
risk
assessment
are
profound.
The
EDSTAC
recognizes
there
are
divergent
scientific
opinions
on
the
"
low
dose"
issue
at
the
present
time
and
that
more
research
is
necessary
to
answer
these
questions.

The
EDSTAC
notes
that,
historically,
testing
has
sometimes
missed
critical
endpoints
either
by:
(
1)
failing
to
dose
during
the
most
sensitive
life
stage
(
Morrissey
et
al.,
1987);
(
2)
failing
to
test
in
a
susceptible
organism
(
Chamberlin,
1979;
Fraser,
1988);
or
(
3)
failing
to
examine
subtle
(
yet
biologically
important)
endpoints.
For
example,
early
studies
on
the
developmental
effects
of
PCBs
in
rodents
identified
fetotoxicity
as
a
critical
endpoint,
yet
these
studies
failed
to
test
at
low
enough
doses
or
to
measure
subtle
enough
endpoints.
As
a
result
these
early
studies
missed
the
neurotoxic
effects
of
PCBs
which
occur
at
much
lower
doses
(
Tilson
et
al.,
1990).

These
omissions
in
testing
may
lead
either
to
missing
a
critical
effect
completely,
or
to
identifying
a
NOAEL
which
is
erroneously
high.
The
EDSTAC
has
attempted
to
minimize
the
likelihood
of
these
types
of
errors
by
requiring
testing
in
a
variety
of
organisms
during
sensitive
life
stages.
A
variety
of
endpoints
which
appear
to
be
low­
dose
sensitive
have
also
been
added
to
the
EDSTP
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
50
testing
protocols.
In
addition,
a
number
of
endocrine
disruptor­
sensitive
endpoints
have
already
been
added
to
EPA's
reproductive
toxicity
testing
guidelines
(
OPPTS
Public
Draft,
1996;
U.
S.
EPA
TSCA
finalized
testing
guidelines,
1997;
U.
S.
EPA
FIFRA
finalized
testing
guidelines,
1998).

The
Committee
agrees,
however,
that
dose
selection
in
T2T
must
include
special
attention
to
setting
the
low
dose.
In
particular,
the
low
dose
should
not
be
selected
by
identifying
the
high
dose
and
then
dropping
the
dose
by
a
fixed
formula
of
a
couple
of
orders
of
magnitude.
Instead,
a
number
of
considerations
need
to
go
into
selecting
the
low
dose,
including
the
results
of
prior
information
(
e.
g.,
HTPS,
toxicity
testing,
pharmacokinetic,
and
epidemiology
data).
Information
about
environmental
exposure
levels
might
also
be
used
where
appropriate.
Finally,
range­
finding
studies
are
currently
constructed
so
as
to
inform
both
the
high
dose
and
the
low
dose
for
the
definitive
testing,
with
inclusion
of
low­
dose
sensitive
endpoints
in
the
range­
finding
study.
Range­
finding
studies,
with
fewer
animals
per
group
and
a
more
limited
set
of
endpoints
than
in
definitive
tests,
will
not
necessarily
identify
the
appropriate
low
dose
or
a
NOAEL
but
they
will
indicate
whether
effects
are
observed
for
low
dose
sensitive
endpoints
and
therefore
whether
low
doses
should
be
included
in
the
definitive
tests.
These
precautions
will
minimize
the
likelihood
that
critical
effects
will
be
missed
or
that
erroneously
high
NOAELs
will
be
identified
in
T2T.

2.
Recommended
Project
to
Address
Low
Dose
Issues
As
previously
discussed,
questions
have
been
raised
as
to
the
adequacy
of
conventional
toxicology
study
designs
for
assessment
of
endocrine
active
substances,
particularly
with
regard
to
low
dose
selection
and
the
identification
of
no­
observed­
adverse­
effect­
levels
(
NOAELs).
The
EDSTAC
believes
a
project
is
required
to
resolve
the
underlying
uncertainties
and
controversy
about
these
issues.
The
purpose
of
the
project
is
to
address
the
nature
of
the
dose­
response
curves
for
exogenous
estrogenic
substances
in
order
to
allow
more
informed
judgments
about
appropriate
toxicology
study
designs
for
substances
that
have
hormonal
activity.
The
recommended
project
focuses
only
on
substances
with
estrogenic
activity,
since
it
has
been
reported
that
some
estrogens
can
produce
nonmonotonic
dose
response
curves
at
environmentally
relevant
dosage
levels.
However,
the
results
of
the
study
could
be
more
broadly
applicable
to
substances
with
other
types
of
hormonal
activity
(
e.
g.,
those
that
have
androgenic
or
antiandrogenic
activity).

Several
very
important
studies
related
to
low
dose
issues
are
currently
in
progress.
The
exact
design
of
the
project
(
e.
g.,
chemicals
to
be
tested,
strains
of
animals,
endpoints
to
be
evaluated)
will
be
based
on
a
thorough
evaluation
of
all
relevant
data
that
can
be
made
available.
The
EDSTAC
believes
this
evaluation
should
be
completed
within
a
reasonable
timeframe,
such
as
four
to
six
months.
Preliminary
discussions
to
date
have
focused
on
a
two­
phased
project.
Phase
I
(
Replication
Studies)
is
intended
to
replicate
previously
reported
low
dose
results
in
male
and
female
mice
with
one
test
substance
and
a
positive
control
substance
(
e.
g.,
DES).
The
replication
studies
would
be
done
in
at
least
3
different
laboratories
using
an
identical,
mutually
agreed
upon
protocol.
The
Phase
I
studies
will
allow
a
systematic
evaluation
of
inter­
laboratory
variability
in
the
assessment
of
various
estrogen­
responsive
endpoints
in
male
mice.
Depending
on
the
timing
for
completing
studies
currently
in
progress,
or
results
of
studies,
if
they
have
been
completed,
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
51
Phase
I
may
not
be
required.
If
Phase
I
is
required,
it
should
be
conducted
simultaneously
with
Phase
II
(
described
below).

Phase
II
(
Exploratory
Studies)
is
intended
to
evaluate
potential
new
endpoints
and
compare
the
effects
in
male
and
female
rats
and
mice
with
approximately
four
to
seven
different
substances,
with
varying
degrees
of
estrogenic
potency
(
including
estradiol
and
at
least
one
natural
estrogenic
substance)
using
an
identical
protocol
for
each
substance.
The
number
of
substances,
as
well
as
the
number
of
laboratories,
will
depend
on
available
resources.
The
Phase
II
studies
will
allow
a
systematic
determination
of
any
species
or
sex
differences
in
sensitivity,
as
well
as
elucidation
of
any
qualitative
differences
in
the
responses
to
the
various
test
substances.
Phase
II
will
allow
assessment
of
the
complete
time
course
for
any
observed
effects,
and
a
determination
of
whether
or
not
observations
early
in
life
are
predictive
of
any
specific
adverse
outcomes
during
later
life
stages.

If
the
results
of
the
project
confirm
there
is
a
need
to
include
additional
low
doses
for
Tier
2
testing
of
substances
with
estrogen,
androgen,
or
thyroid
activity,
there
are
implications
for
additional
testing
of
any
substances
that
may
already
have
been
subjected
to
dosing
in
the
EDSTAC­
recommended
Tier
2
tests.
For
any
substance
that
has
previously
been
tested
and
found
to
be
positive
in
T2T
(
i.
e.,
produces
adverse
effects
on
EAT
sensitive
endpoints),
it
will
be
necessary
to
retest
that
substance
with
additional
low
doses.
This
additional
low
dose
testing
would
be
done
for
the
purpose
of
conclusively
identifying
the
NOAEL.

If
the
results
of
the
project
demonstrate
the
need
to
include
any
additional
EAT
endpoints,
there
would
be
a
need
for
retesting
to
assess
those
new
endpoints
for
any
substances
previously
tested
using
the
EDSTAC­
recommended
T2T
protocols.
Additional
endpoints
would
be
those
that
are
plausibly
linked
to
adverse
effects
and
not
covered
in
current
tests,
or
those
that
would
be
more
sensitive
than
those
in
current
tests.

Given
the
current
state
of
the
science,
the
EDSTAC
believes
its
recommended
T2T
protocols
(
once
standardized
and
validated)
will
be
adequate
to
determine
whether
chemicals
exhibit
EATmediated
adverse
effects.
If
the
results
of
the
project
confirm
there
is
a
need
to
include
additional
low
doses,
but
there
is
not
a
need
to
include
additional
EAT
endpoints,
then
it
will
be
necessary
to
retest
chemicals
that
have
been
found
positive
in
the
EDSTAC
recommended
T2T
protocols
to
conclusively
identify
the
NOAEL,
if
one
exists.
However,
it
will
not
be
necessary
to
retest
chemicals
that
have
already
been
found
negative,
and
placed
in
the
"
hold
box,"
using
the
EDSTAC
recommended
T2T
protocols.
As
indicated
in
Chapter
Three,
all
chemicals
placed
in
the
"
hold
box,"
can
be
reconsidered
for
additional
testing
if
"
new
information
on
the
endocrine
disrupting
potential
of
the
chemical
substance
or
mixture
becomes
available
and
it
is
determined
that
this
new
information
warrants
additional
testing."

If
retesting
is
required,
a
test
concentration
that
produced
adverse
effects
in
the
EDSTACrecommended
T2T
protocol
(
if
any)
would
be
required
as
the
high
dose
of
the
repeat
study
to
demonstrate
reproducibility
of
the
initial
finding.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
52
If
the
project
demonstrates
that
conventional
protocols
are
adequate
for
identifying
the
NOAEL
of
endocrine
active
substances,
then
the
NOAEL
derived
from
conventional
T2T
should
not
be
contested.

The
EDSTAC
recommends
that
a
collaborative
group,
involving
government,
industry,
and
appropriate
individuals
in
academia,
design
the
study
protocols,
be
kept
abreast
of
the
conduct
of
the
studies,
evaluate
results,
and
develop
overall
conclusions
and
recommendations.

E.
Methods
to
Select
the
Target
Doses
for
T2T
For
T2T
in
mammals,
other
vertebrates,
and
invertebrates,
the
EDSTAC
recommends
that
the
information
to
select
doses
used
in
the
performance
of
these
tests
include:

1.
existing
information
such
as
that
available
during
the
priority
setting
phase
including
results
from
the
HTPS
(
or
its
equivalent
by
bench­
level
assays);
2.
results
from
T1S
(
including
the
range­
finding
study
results);
3.
results
from
other
assays
or
tests
for
pesticide
registration,
etc.;
and
4.
results
from
range­
finding
studies.

Range­
finding
studies
specifically
for
T2T
should
be
performed
at
multiple
doses
(
at
least
five)
with
a
limited
number
of
animals
per
dose,
an
abbreviated
duration
(
which
must
include
exposures
during
gestation
or
egg
development
and
lactation),
and
a
limited
number
of
relevant
endpoints
including
low
dose
sensitive
ones.
If
further
research
validates
the
low
dose
concern,
the
EDSTAC
would
recommend
using
the
low
dose
sensitive
endpoints
in
the
range­
finding
study
to
determine
the
need
for
inclusion
of
low
doses
in
the
definitive
T2T.
Range­
finding
studies
are
already
conducted
for
toxicity
studies,
and
have
the
principal
purpose
of
defining
both
the
maximal
level
that
can
be
dosed
and,
at
the
other
end
of
the
dose
range,
informing
a
dose
that
will
have
no
observable
effect
on
the
most
sensitive
endpoint
in
the
full­
scale
study.
Endpoints,
identified
in
recent
publications,
which,
at
present,
appear
to
be
low
dose
sensitive
include:
prostate
weight
(
for
mammals),
epididymal
sperm
concentration
(
for
mammals),
other
accessory
sex
organ
weights
(
all
vertebrates),
thyroid
weight
(
all
vertebrates),
reproductive
capability
(
all
T2T),
and
vaginal
threads
(
for
mammals).
All
of
these,
except
for
vaginal
threads,
are
included
in
the
1996
guidelines;
however,
vaginal
threads
would
be
identified
during
the
examination
of
offspring
females
for
vaginal
patency.
New
and/
or
different
low
dose
sensitive
endpoints
may
be
identified
as
new
data
are
generated.

Current
toxicological
test
guidelines
generally
require
testing
at
a
minimum
of
three
dose
levels
plus
a
control.
These
guidelines
specify
that
the
top
dose
level
should
be
a
maximally
tolerated
dose
(
MTD),
that
is,
a
dose
which
by
definition
is
toxic
but
which
does
not
result
in
excessive
mortality
(
not
to
exceed
10%).
In
reproductive
and
developmental
toxicity
studies,
the
MTD
is
usually
based
on
parental
or
maternal
toxicity,
which
is
expressed
as
depressed
body
weight
gain,
actual
weight
loss,
reduced
feed
and/
or
water
consumption,
treatment­
related
clinical
signs
of
toxicity,
etc.
The
MTD
is
based
on
available
toxicity
information
such
as
data
from
a
range
finding
study.
The
next
lower
dose
is
ideally
set
at
an
intermediate
toxic
dose
and
the
lowest
dose
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
53
at
a
level
at
which
no
toxic
effects
are
observed.
If
additional
lower
doses
are
included
in
the
study
because
of
identified
concerns
at
low
doses,
the
additional
doses
should
be
widely
spaced
(
perhaps
in
orders
of
magnitude)
to
identify
the
nature
of
the
dose­
response
curve
in
the
low
dose
region.
The
toxicity
upon
which
the
MTD
is
based
may
or
may
not
be
related
to
the
endpoints
that
are
the
object
of
the
investigation
(
e.
g.,
cancer,
neurotoxicity,
reproductive,
or
developmental
effects).

The
rationale
and
results
of
range­
finding
studies
should
be
included
in
the
submission
of
the
T2T
results
for
evaluation
by
the
Agency.

F.
Testing
Antithyroid
Activities
in
T2T
Thyroid
hormones
are
well
known
to
play
essential
roles
in
vertebrate
development
(
Dussault
and
Ruel,
1987;
Myant,
1971;
Porterfield
and
Hendrich,
1993;
Porterfield
and
Stein,
1994;
Timiras
and
Nzekwe,
1989).
Experimental
work
focused
on
the
effects
of
thyroid
hormone
on
brain
development
in
the
neonatal
rat
supports
the
concept
of
a
"
critical
period,"
during
which
thyroid
hormone
must
be
present
to
avoid
irreversible
damage
(
Timiras
and
Nzekwe,
1989).
Though
the
duration
of
this
critical
period
may
be
different
for
different
thyroid
hormone
effects,
the
general
view
has
developed
that
this
is
the
period
of
maximal
developmental
sensitivity
to
thyroid
hormone,
and
it
occurs
during
the
lactational
period
in
the
rat
(
Oppenheimer
et
al.,
1994;
Timiras
and
Nzekwe,
1989).
Although
thyroid
hormone
receptors
are
expressed
in
fetal
rat
brains
(
Bradley
et
al.,
1989;
Strait
et
al.,
1990)
and
thyroid
hormone
can
exert
effects
on
the
fetal
brain
(
Escobar
et
al.,
1990;
Escobar
et
al.,
1987;
Escobar
et
al.,
1988;
Porterfield,
1994;
Porterfield
and
Hendrich,
1992;
Porterfield
and
Hendrich,
1993;
Porterfield
and
Stein,
1994),
the
lactational
period
represents
a
stage
of
rapid
expansion
of
the
central
nervous
system
that
coincides
with
a
large
increase
in
the
expression
of
thyroid
hormone
receptors
(
Perez­
Castillo
et
al.,
1985)
and
an
increase
in
the
number
of
demonstrated
effects
of
thyroid
hormone
on
brain
development.

In
conducting
thyroid­
related
tests
in
Tier
2,
the
EDSTAC
recommends
using
an
approach
in
which
dosing
occurs
during
the
fetal
and
lactational
period.
In
addition,
there
are
a
variety
of
endpoints
that
would
provide
reliable
markers
of
thyroid
disruption.
Brain
weight
offers
a
simple
measure,
though
it
is
not
thyroid
specific.
Characteristics
of
myelination,
or
of
myelin
basic
protein
expression
(
either
mRNA
or
protein),
would
provide
a
more
selective
measure
(
Bhat
et
al.,
1981;
Bhat
et
al.,
1979;
Farsetti
et
al.,
1991;
Figueiredo
et
al.,
1993;
Rodriguez­
Pena
et
al.,
1993;
Shanker
et
al.,
1987).
In
this
regard,
the
expression
of
myelin
basic
protein
and/
or
neurogranin/
RC3
may
offer
the
simplest
and
most
specific
endpoints
of
thyroid
disruption
during
the
perinatal
period
(
Farsetti
et
al.,
1991;
Iniguez
et
al.,
1993).
These
mRNAs
are
both
enormously
abundant
and
robustly
affected
by
thyroid
hormone.
However,
their
sensitivity
to
xenobiotics
has
not
been
studied.
A
list
of
existing
endpoints
for
thyroid
hormone
function,
and
additional
ones
recommended
by
the
EDSTAC
for
validation
and
inclusion,
are
found
in
Table
5.3.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
54
VI.
Recommended
Tier
2
Testing
Battery
A.
Outline
of
Recommended
T2T
Battery
The
EDSTAC
recommends
that
the
T2T
battery
include
a
mammalian
two­
generation
reproductive
toxicity
study,
or
a
less
comprehensive
test
in
accordance
with
the
guidelines
outlined
above,
and
tests
addressing
four
additional
taxonomic
groups,
including
birds,
amphibians,
fish,
and
invertebrates
as
follows:

Mammalian
Tests
1.
Two­
Generation
Mammalian
Reproductive
Toxicity
Study;
or
2.
A
Less
Comprehensive
Test:
a)
Alternative
Mammalian
Reproductive
Test;
or
b)
One­
Generation
Test.

Multigeneration
Tests
in
Other
Taxa
1.
Avian
Reproduction
(
with
bobwhite
quail
and
mallard)
2.
Fish
Life
Cycle
(
fathead
minnow)
3.
Mysid
Life
Cycle
(
Americamysis)
4.
Amphibian
Development
and
Reproduction
(
Xenopus)

B.
Two­
Generation
Mammalian
Reproductive
Toxicity
Study
The
two­
generation
reproductive
toxicity
study
in
rats
(
TSCA
799.9380,
August
15,
1997;
OPPTS
870.3800,
Public
Draft,
February,
1996;
OECD
no.
416,
1983;
FIFRA,
Subdivision
F,
Guidelines
83­
4)
is
designed
to
comprehensively
evaluate
the
effects
of
a
chemical
on
gonadal
function,
estrous
cycles,
mating
behavior,
fertilization,
implantation,
pregnancy,
parturition,
lactation,
weaning,
and
the
offspring'
ability
to
achieve
adulthood
and
successfully
reproduce,
through
two
generations,
one
litter
per
generation.
Administration
is
usually
oral
(
dosed
feed,
dosed
water,
or
gavage)
but
other
routes
are
acceptable
with
justification
(
e.
g.,
inhalation).
In
addition,
the
study
also
provides
information
about
neonatal
survival,
growth,
development,
and
preliminary
data
on
possible
teratogenesis.
The
experimental
design
for
a
two­
generation
reproductive
toxicity
study
is
presented
in
Figure
Q.
1,
which
is
found
in
Appendix
Q,
Tier
2
Test
Study
Designs.

In
the
existing
two­
generation
reproductive
toxicity
test,
a
minimum
of
three
treatment
levels
and
a
concurrent
control
group
are
required.
At
least
20
males
and
sufficient
females
to
produce
20
pregnant
females
must
be
used
in
each
group
as
prescribed
in
this
current
guideline.
The
highest
dose
must
induce
toxicity
but
not
exceed
10%
mortality.
In
this
study,
potential
hormonal
effects
can
be
detected
through
behavioral
changes,
ability
to
become
pregnant,
duration
of
gestation,
signs
of
difficult
or
prolonged
parturition,
apparent
sex
ratio
(
as
ascertained
by
anogenital
distances)
of
the
offspring,
feminization
or
masculinization
of
offspring,
number
of
pups,
stillbirths,
gross
pathology
and
histopathology
of
the
vagina,
uterus,
ovaries,
testis,
epididymis,
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
55
seminal
vesicles,
prostate,
and
any
other
identified
target
organs,
and
gynecological
assesments
(
evaluation
of
parental
estrous
cyclicity,
onset
of
puberty,
acquisition
of
VO).

Table
5.3
provides
a
summary
of
the
endpoints
evaluated
within
the
framework
of
the
experimental
design
of
the
updated
two­
generation
reproductive
toxicity
test
(
and
some
recommended
additional
endpoints
for
validation
and
inclusion
to
cover
EAT
concerns).
These
endpoints
are
comprehensive
and
cover
every
phase
of
reproduction
and
development.
Tests
that
measure
only
a
single
dimension
or
component
of
hormonal
activity,
(
e.
g.,
in
vitro
or
short­
term
assays)
provide
supplementary
and/
or
mechanistic
information,
but
cannot
provide
the
breadth
of
information
listed
in
Table
5.3,
which
is
critical
for
risk
assessment.

Additionally,
in
this
study
type,
hormonally
induced
effects
such
as
abortion,
resorption,
or
premature
delivery
as
well
as
abnormalities
and
anomalies
such
as
masculinization
of
the
female
offspring
or
feminization
of
male
offspring,
can
be
detected.
Substances
such
as
the
phytoestrogen,
coumesterol,
and
the
antiandrogen,
cyproterone
acetate,
which
possess
the
potential
to
alter
normal
sexual
differentiation,
were
similarly
detected
in
this
study
test
system
(
i.
e.,
1982
Guideline).
The
initial
prebreed
exposure
period
(
10
weeks)
of
the
two­
generation
reproductive
toxicity
test
also
provides
information
on
subchronic
exposures
which
can
be
used
for
other
regulatory
purposes.

C.
Alternative
Approaches
to
Mammalian
T2T
The
EDSTAC
acknowledges
that
the
developing
organism
may
be
uniquely
sensitive
to
the
effects
of
endocrine­
active
agents.
Therefore,
any
mammalian
Tier
2
test
should
include
a
careful
assessment
of
the
consequences
of
in
utero
and
lactational
exposure
on
subsequent
growth
and
development.
Below
are
two
alternative
mammalian
tests
the
EDSTAC
recommends
be
included,
once
validated
and
standardized,
as
part
of
T2T.
The
determination
on
whether
to
perform
the
two­
generation
test
or
one
of
the
alternative
tests
should
be
made
consistent
with
the
criteria
described
in
Chapter
Five,
Section
V,
C,
2.

Although
EAT­
relevant
endpoints
are
not
optimally
detected
by
developmental
toxicity
study
designs,
both
of
these
alternative
tests
can
be
modified
to
allow
assessment
of
term
fetuses
by
adding
additional
F0
females
to
each
dose
group
(
i.
e.,
create
satellite
groups)
and
terminating
these
satellite
females
just
prior
to
anticipated
parturition
(
i.
e.,
on
gd
20­
21)
and
performing
gestational
and
fetal
structural
evaluations
(
i.
e.,
ovarian
corpora
lutea,
uterine
implantation
sites,
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
56
Table
5.3
Mammalian
Tier
2
Test
Endpoints
Below
are
two
types
of
lists.
First,
those
endpoints
required
in
current
EPA
test
guidelines
1996.
Second,
additional
endpoints
recommended
by
EDSTAC
for
validation
and
inclusion
in
both
the
recommended
two­
generation
test,
as
well
as
the
alternative
mammalian
tests
discussed
below.
These
additional
endpoints
will
detect
estrogen,
androgen,
and
thyroid
hormone
perturbations.

As
discussed
above,
in
Chapter
Five,
Section
V,
C,
the
default
assumption
is
that
all
of
these
endpoints
would
be
evaluated
unless
the
conditions
which
are
set
forth
in
the
guidelines
for
determining
the
selection
of
endpoints
are
met.

Current
Guideline
Endpoints
Sensitive
to
Estrogens/
Antiestrogens
·
sexual
differentiation
·
gonad
development
(
size,
morphology,
weight)
>
accessory
sex
organ
(
ASO)
development
·
ASO
weight
±
fluid;
histology
·
sexual
development
and
maturation:
acquisition
of
vaginal
patency
(
VP),
preputial
separation
(
PPS)
·
fertility
·
fecundity
·
time
to
mating
·
mating
and
sexual
behavior
·
ovulation
·
estrous
cyclicity
·
gestation
length
·
abortion
·
premature
delivery
·
dystocia
·
spermatogenesis
·
epididymal
sperm
numbers
and
morphology;
testicular
spermatid
head
counts;
daily
sperm
production
(
DSP);
efficiency
of
DSP
·
gross
and
histopathology
of
reproductive
tissues
·
anomalies
of
the
genital
tract
·
viability
of
the
conceptus
in
utero
(
prenatal
demise)
·
survival
and
growth
of
offspring
·
maternal
lactational
behaviors
(
e.
g.,
nursing,
pup
retrieval,
etc.)

Recommended
Additional
Endpoints
for
Validation
and
Inclusion
·
accessory
sex
organ
function
(
secretory
products)
·
sexual
development
and
maturation
(
nipple
development
and
retention)
·
androgen
and
estrogen
levels
·
LH
and
FSH
levels
·
testis
descent
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
57
Current
Guideline
Endpoints
Sensitive
to
Androgens/
Antiandrogens
·
altered
apparent
sex
ratio
(
based
on
AGD)
·
malformations
of
the
urogenital
system
·
altered
sexual
behavior
·
changes
in
testis
and
accessory
sex
organ
weights
·
effects
on
sperm
numbers,
morphology,
etc.
·
retained
nipples
in
male
offspring
·
altered
AGD
(
now
triggered
from
PPS/
VP)
·
reproductive
development;
PPS/
VP
(
puberty)
·
male
fertility
·
agenesis
of
prostate
·
changes
in
androgen­
dependent
tissues
in
pups
and
adults
(
not
limited
to
sex
accessory
glands)

Current
Guideline
Endpoints
Sensitive
to
Thyroid
Hormone
Agonists/
Antagonists
(
general)
·
growth,
body
weight
·
food
consumption,
food
efficiency
·
developmental
abnormalities
·
perinatal
mortality
·
testis
size
and
DSP
·
VP;
PPS
Recommended
Additional
Endpoints
for
Validation
and
Inclusion
·
neurobehavioral
deficits
(
see
developmental
landmarks
below)
·
TSH,
T4,
thyroid
weight
and
histology
(
e.
g.,
goiter)
·
developmental
landmarks:
·
prewean
includes
pinna
detachment,
surface
righting
reflex,
eye
opening,
acquisition
of
auditory
startle,
negative
geotaxis,
mid­
air
righting
reflex,
motor
activity
on
PND
13,
21,
etc.
·
postwean
includes
motor
activity
PND
21
and
postpuberty
ages
(
sex
difference);
learning
and
memory
PND
60
­
active
avoidance/
water
maze
·
brain
weight
(
absolute),
whole
and
cerebellum
·
brain
histology
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
58
total,
resorbed,
dead
and
live
fetuses,
live
fetal
number,
sex,
weight,
external,
visceral,
and
skeletal
alterations),
i.
e.,
a
"
standard"
developmental
toxicity
evaluation
by
OPPTS
1996
draft
guidelines;
USEPA
TSCA
guidelines
870.3700,
1997;
FDA
guidelines,
1993.
The
remaining
females
in
each
group
would
continue
on
study
as
described
below.

1.
Alternative
Mammalian
Reproduction
Test
A
graphical
representation
of
the
study
design
(
Figure
Q.
2)
and
additional
descriptive
text
for
the
Alternative
Mammalian
Reproduction
Test
(
AMRT)
are
provided
in
Appendix
Q,
Tier
2
Test
Study
Designs.
The
objectives
of
this
test
are
to
describe
the
consequences
of
in
utero
and/
or
lactational
exposure
on
reproduction
and
development
from
compounds
that
displayed
EAT
activity
in
the
T1S.
If
validated,
this
test
may
be
used,
under
certain
defined
circumstances,
instead
of
the
recommended
two­
generation
reproductive
toxicity
test
(
TSCA
guidelines,
1997)
in
T2T.
In
this
regard
it
will
be
conducted
with
at
least
three
treatment
groups
plus
a
control
and
include
endpoints
sensitive
to
chemicals
that
alter
development
via
EAT
activities.
As
with
the
two­
generation
mammalian
reproductive
toxicity
study,
the
default
assumption
is
that
all
of
the
endpoints
in
Table
5.3
would
be
evaluated
in
the
AMRT,
unless
the
conditions
set
forth
in
the
guidelines
for
determining
the
selection
of
endpoints
are
met.

The
AMRT
involves
exposure
of
maternal
rats
(
designated
F0
generation)
from
gestational
day
6
(
time
of
implantation),
through
parturition
(
birth),
and
through
the
lactation
period
until
weaning
of
offspring
(
designated
F1
generation)
on
postnatal
day
21.
F1
offspring
(
both
sexes)
are
retained
after
weaning
with
no
exposures
for
10
weeks
and
then
mated
within
groups.
F1
males
are
necropsied
after
the
mating.
F1
females
and
their
litters
(
designated
the
F2
generation)
are
retained
until
the
F2
generation
is
weaned.
F0
females
(
and
a
subset
of
F1
weanlings)
are
necropsied
with
organ
weights
and
possible
histopathology.
F1
animals
are
evaluated
for
reproductive
development
(
VP,
PPS),
estrous
cyclicity,
and,
at
necropsy,
for
organ
weights,
possible
histopathology,
andrological
assessments,
and
T3/
T4
(
with
TSH
triggered).
F2
weanlings
are
counted,
sexed,
weighed,
examined
externally,
and
discarded.

The
AMRT
differs
from
the
"
standard"
two­
generation
study
design
in
that
it:

a)
does
not
include
exposures
prior
to
mating,
during
mating,
or
during
the
early
preimplantation
stage
of
pregnancy
in
the
dams;
b)
does
not
include
exposures
to
parental
males;
and
c)
does
not
include
direct
exposure
to
the
postweanling
offspring;
potential
exposure
is
limited
to
in
utero
transplacental
and/
or
lactational
routes.

The
AMRT
differs
from
the
one­
generation
test
(
see
below)
in
that
its
study
design
provides
for:

a)
exposure
to
the
F0
dam
only
from
gd6,
through
weaning
of
the
F1
offspring
on
pnd
21;
b)
no
exposure
to
parental
males;
c)
mating
of
the
F1
animals
(
who
have
not
been
directly
exposed)
to
produce
F2
offspring;
and
d)
following
the
F2
offspring
to
weaning
on
pnd
21.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
59
2.
One­
Generation
Test
A
second
alternative
to
the
standard
two­
generation
reproductive
toxicity
test
is
a
one­
generation
reproductive
toxicity
test.
A
graphical
representation
of
the
one­
generation
test
(
Figure
Q.
3),
and
additional
text,
is
provided
in
Appendix
Q,
Tier
2
Test
Study
Designs.
It
has
been
used
in
rats
and
mice.
It
has
been
used
as
a
range­
finding
study
prior
to
performance
of
a
guideline
twogeneration
(
or
more)
study
for
the
last
10
years
under
EPA
(
TSCA/
FIFRA)
GLPs;
the
design
is
similar
to
that
used
by
Sharpe
et
al.,
1996.
This
is
a
shortened,
scaled­
down
version
of
the
new
draft
OPPTS
and
Final
TSCA
guidelines
for
reproductive
toxicity
testing.
As
with
the
twogeneration
mammalian
reproductive
toxicity
study,
the
default
assumption
is
that
all
of
the
endpoints
in
Table
5.3
would
be
evaluated
in
the
one­
generation
test,
unless
the
conditions
set
forth
in
the
guidelines
for
determining
the
selection
of
endpoints
are
met.

The
one­
generation
test
is
a
less
comprehensive
evaluation
of
functional
reproductive
development
than
the
AMRT
(
since
it
does
not
follow
F1
animals
through
production
of
F2
offspring),
but
it
has
the
advantage
of
assessing
postnatal
development
and
adult
reproductive
capacity
after
in
utero
lactational
and
post­
lactational
exposure.
In
the
presence
of
continued
exposure,
the
postnatal
component
of
the
test
is
extended
to
evaluate
acquisition
of
vaginal
patency,
preputial
separation,
estrous
cyclicity,
and
andrological
assessments
in
the
F1
offspring.
Inappropriate
retention
of
Mullerian
duct
derivations
(
e.
g.,
oviducts)
in
males
and
of
Wolffian
duct
derivatives
(
e.
g.,
seminal
vesicles,
epididymides)
in
females
can
be
identified
in
all
three
proposed
tests
(
with
or
without
satellite
F0
females
and
examination
of
term
fetuses).

The
one­
generation
test
involves
a
short
prebreed
exposure
period
for
male
and
female
rats
of
the
initial
parental
generation
(
designated
F0),
and
exposure
continues
through
mating,
gestation,
and
lactation
of
F1
litters.
F0
males
are
necropsied
after
F1
deliveries;
F0
females
are
necropsied
after
F1
weaning.
Postweanling
F1
animals
are
directly
exposed
for
a
10­
week
postwean
period
and
are
then
necropsied.
F1
animals
are
evaluated
for
reproductive
development
(
VP,
PPS),
estrous
cyclicity
and
at
necropsy
for
organ
weights,
possible
histopathology,
andrological
assessments,
and
T3/
T4
(
TSH
triggered).
F0
animals
will
undergo
the
same
necropsy
assessments.

The
one­
generation
test
differs
from
the
"
standard"
two­
generation
study
design
in
that
it:

a)
is
shorter
(
basic
design
calls
for
two
weeks,
can
be
prolonged)
than
in
the
standard
twogeneration
study
(
10
weeks
to
encompass
one
full
spermatogenic
cycle
in
rats),
though
it
does
include
a
prebreed
exposure
period;
and
b)
does
not
evaluate
effects
of
in
utero
and/
or
lactational
exposure
(
and
beyond)
on
generation
of
F2
offspring
though
it
does
include
direct
exposure
of
F1
offspring
after
weaning,
including
exposure
through
puberty
and
sexual
maturation.
F1
male
and
female
reproductive
organs
(
weight/
histology),
estrous
cyclicity,
and
andrological
endpoints
are
assessed
at
scheduled
necropsy
on
PND
90
±
2.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
60
The
one­
generation
test
differs
from
the
AMRT
in
that
its
study
design
provides
for:

a)
exposure
to
both
male
and
female
F0
parental
animals
prior
to
mating,
during
mating,
and
during
gestation
and
lactation
of
F1
offspring
(
F0
males
are
necropsied
after
F1
deliveries,
F0
females
are
necropsied
after
F1
weaning);
b)
direct
exposure
of
postweanling
F1
offspring
after
lactation
until
termination;
and
c)
no
mating
of
F1
animals
to
produce
F2
offspring.

D.
Description
of
the
Tests
for
Other
Animal
Taxa
The
EDSTAC
agrees
T2T
should
address
at
least
four
other
animal
taxonomic
groups,
including
birds,
amphibians,
fish,
and
invertebrates.
Each
of
the
four
basic
non­
mammalian
tests
should
be
multigenerational,
as
is
the
basic
mammalian
reproduction
study.
It
is
recommended
that
the
following
standardized
tests
be
used
as
a
basis
for
a
non­
mammalian
battery:

1.
Avian
Reproduction
(
with
bobwhite
quail
and
mallard)
2.
Fish
Life
Cycle
(
fathead
minnow)
3.
Mysid
Life
Cycle
(
Americamysis)
4.
Amphibian
Development
and
Reproduction
(
Xenopus)

Except
for
the
amphibian
study,
these
tests
are
routinely
performed
for
chemicals
with
widespread
outdoor
exposures
that
are
expected
to
affect
reproduction.
Modifications
to
each
may
be
warranted
to
enhance
the
ability
to
detect
endocrine­
related
effects.
The
amphibian
test,
though
not
standardized,
is
considered
warranted
because
of
the
extensive
fundamental
knowledge
base
on
amphibian
development
and
reproduction.

Just
as
for
mammalian
testing,
there
may
be
instances
when
less
comprehensive
study
designs
would
be
adequate.
Considerations
for
determining
whether
the
full
battery
of
comprehensive
non­
mammalian
tests
should
be
implemented
include
an
understanding
of
mechanisms
of
action,
environmental
fate
and
transport,
persistence,
potential
for
bioaccumulation,
and
potential
ecosystems
exposed.

Production
volume
is
also
a
consideration
for
less
comprehensive
approaches.
Comprehensive
assessments
of
environmental
toxicity,
including
chronic
toxicity
assays
in
a
variety
of
species,
are
already
generated
for
pesticides
and
very
high
production
volume
chemicals
(>
1,000
tons
per
annum)
in
Europe.
The
European
Union
explicitly
requires
less
comprehensive
assessments
for
lower
production
volume
chemicals,
with
additional
testing
required
as
production
increases.
As
with
mammalian
assessments,
these
moving
triggers
recognize
that
the
potential
for
exposure
is
correlated
with
production
volume.
While
there
are
no
explicitly
required
data
sets
in
the
U.
S.
under
TSCA,
similar
decisions
are
made
on
data
adequacy
based
on
other
information,
including
production
volume.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
61
There
are
a
number
of
alternative,
less
comprehensive
assays
that
may
be
appropriate
to
consider
for
environmental
toxicity
assessments.
These
might
include
shorter­
term
avian
development
tests,
and
the
fish
early
life
cycle
test
and
Daphnia
reproduction
test,
both
of
which
are
already
established
protocols.

1.
Avian
Reproduction
Test
While
birds
are
not
included
as
subjects
in
the
T1S
battery,
it
is
important
to
evaluate
the
effects
of
exposure
of
birds
to
chemical
substances
or
mixtures
with
endocrine
activity.
Furthermore,
birds
are
fundamentally
different
from
mammals
in
the
control
of
sexual
differentiation
(
males
are
the
homogametic
sex)
so
results
using
mammalian
subjects
will
not
provide
complete
information
relevant
to
birds.

Use
of
the
EPA's
Avian
Reproduction
Test
guidelines
(
OPPTS
850.2300)
is
recommended,
modified
to
include
the
additional
endpoints
presented
below
to
make
the
test
more
sensitive
to
chemical
substances
or
mixtures
with
endocrine
activity.
Table
5.4
provides
a
summary
of
the
endpoints
evaluated
within
the
framework
of
the
Avian
Reproduction
Test
(
and
recommended
additional
endpoints
for
validation
and
inclusion
to
cover
EAT
concerns).
Two
important
extensions
of
this
guideline
are
recommended:
(
1)
modification
and
standardization
of
the
husbandry
and
dosing
of
the
offspring
from
EPA's
Avian
Reproduction
Test
guidelines
(
OPPTS
850.2300)
to
create
a
two­
generation
avian
reproduction
test;
and
(
2)
using
the
procedures
of
the
modified
Avian
Reproduction
Test
protocol,
evaluate
an
additional
exposure
pathway
(
i.
e.,
direct
topical
exposure,
which
is
common
in
the
wild,
by
dipping
eggs).
The
recommended
extensions
to
the
guideline
are
outlined
in
Appendix
Q.

In
the
current
Avian
Reproduction
Test
guidelines,
two
species
are
commonly
used,
mallards
and
northern
bobwhite.
Exposure
of
adults
begins
prior
to
the
onset
of
maturation
and
egg
laying
and
continues
through
the
egg­
laying
period;
their
offspring
are
exposed,
in
early
development,
by
material
deposited
into
the
egg
yolk
by
the
females.
These
offspring
can
be
used
efficiently
to
test
for
the
effects
of
chemical
substances
or
mixtures
on
avian
development.
There
are
several
endpoints
currently
required
(
see
OPPTS
850.2300,
c,
2)
that
are
particularly
relevant
to
disruption
of
endocrine
activity,
including:
eggs
laid,
cracked
eggs,
eggshell
thickness,
viable
embryos,
and
chicks
surviving
to
14
days.
The
guidelines
should
be
extended
with
additional
observations
made
for
circulating
steroid
titers,
thyroid
hormones,
major
organ
(
including
brain)
weights,
gland
weights,
bone
development,
leg
and
wing
bone
lengths,
ratios
of
organ
weights
to
bone
measurements,
skeletal
x­
ray,
histopathology,
functional
tests,
and
reproductive
capability
of
offspring
(
Baxter
et
al.,
1969;
Bellabarba
et
al.,
1988;
Dahlgren
and
Linder,
1971;
Emlen,
1963;
Cruickhank
and
Sim,
1986;
Fleming
et
al.,
1985a;
Fleming
et
al.,
1985b;
Fox,
1976;
Fox
et
al.,
1978;
Freeman
and
Vince,
1974;
Hoffman
and
Eastin,
1981;
Hoffman
and
Albers,
1984;
Hoffman,
1990;
Hoffman
et
al.,
1993;
Hoffman
et
al.,
1996;
Jefferies
and
Parslow,
1976;
Kubiak
et
al.,
1989;
Maguire
and
Williams,
1987;
Martin,
1990;
Martin
and
Solomon,
1991;
McArthur
et
al.,
1983;
McNabb,
1988;
Moccia
et
al.,
1986;
Rattner
et
al.,
1982;
Rattner
et
al.,
1987;
Summer
et
al.,
1996;
Tori
and
Mayer,
1981).
Other
avian
assays
were
considered
including
the
Japanese
quail
androgenic
assay
(
proctodeal
gland),
egg
injection,
draft
OECD
Japanese
quail
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
62
reproduction,
and
two
generation
avian
reproduction
tests,
but
were
not
selected
because
the
endpoints
addressed
were
limited
or
there
was
a
lack
of
accepted
and
standardized
methods.

2.
Fish
Life
Cycle
Test
The
freshwater
fathead
minnow
Pimephales
promelas
is
the
recommended
species
to
be
used
and
is
continuously
exposed
from
fertilization
through
development,
maturation,
reproduction,
and
early
development
of
offspring
with
a
test
duration
of
up
to
300
days.
The
fathead
minnow
is
also
the
recommended
species
for
use
in
the
screening
battery
for
the
fish
gonadal
recrudescence
assay,
and
as
such,
the
relevance
of
any
activity
detected
in
the
screening
assay
would
be
evaluated.
However,
EDSTAC
recommends
a
performance­
based
approach
to
species
selection
and,
as
more
appropriate
species
are
developed
and
validated,
EDSTAC
strongly
encourages
their
use.
For
example,
if
exposure
to
a
particular
chemical
substance
or
mixture
is
predominantly
estuarine
or
marine,
the
estuarine
sheepshead
minnow
Cyprinodon
variegatus
may
be
substituted
since
experience
and
an
established
method
exist
for
this
species.

Likewise,
other
species
may
offer
more
specificity
for
certain
endpoints
or
geographic
relevance
and
should
be
considered
in
a
performance­
based
approach.
For
example,
the
sex­
linked
color
gene
in
a
medaka
strain
may
afford
early,
non­
intrusive
specificity
for
determining
the
genetic
sex
of
test
animals
following
estrogenic
and
androgenic
screens
or
tests
and
anadromous
salmon
may
have
better
value
to
examine
thyroid
function
in
a
sea­
water
challenge
(
smoltification)
assay.

Fish
are
the
most
diverse
and
least
homologous
to
mammals
of
all
vertebrates.
Reproductive
strategies
extend
from
oviparity,
to
ovoviviparity,
to
true
viviparity.
The
consequences
of
an
endocrine
disruptor
may
be
quite
different
across
the
many
families
of
fishes.
As
a
first
step
though,
only
a
fathead
minnow,
or
in
special
cases
the
sheepshead
minnow,
life
cycle
test
is
suggested
to
confirm
and
quantify
any
effects
detected
by
the
Tier
1
battery.
Subsequent
tests
with
other
species
will
then
be
a
function
of
the
risk
assessment
and
nature
of
the
hormones
involved
and
effects
expected/
obtained.

The
fish
life
cycle
test
(
OPPTS
850.1500)
follows
procedures
outlined
in
Benoit,
1981,
for
the
fathead
minnow,
and
Hansen
et
al.,
1978,
for
the
sheepshead
minnow.
In
general,
the
test
begins
with
200
embryos
distributed
among
eight
incubation
cups
in
each
treatment
group.
When
hatching
is
completed,
the
number
of
larvae
are
reduced
to
25
individuals,
if
available,
which
are
released
to
each
of
four
replicate
larval
growth
chambers.
Four
weeks
following
their
release
into
the
larval
growth
chambers,
the
number
of
juvenile
fish
are
reduced
again
and
25
individuals,
if
available,
are
distributed
to
each
of
two
replicate
adult
test
chambers.
When
fish
reach
sexual
maturity,
fish
are
separated
into
spawning
groups
(
pairs
or
one
male/
two
females)
with
a
minimum
of
eight
breeding
females.
Remaining
adults
will
be
maintained
in
the
tank
but
will
be
segregated
from
the
spawning
groups.
Adults
will
be
allowed
to
reproduce,
at
will,
until
the
300th
day
of
exposure.
Alternatively,
the
test
may
be
continued
past
300
days
until
one
week
passes
in
which
no
eggs
from
any
group
have
been
laid.
The
embryos
and
fish
are
exposed
to
a
geometric
series
of
at
least
five
test
concentrations,
a
negative
(
dilution
water)
control,
and,
if
necessary,
a
solvent
control.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
63
Table
5.4
Avian
Reproduction
Test
Endpoints
Current
Guideline
Endpoints
Sensitive
to
Estrogens/
Antiestrogens,
Androgens/
Antiandrogens,
and/
or
Hypothalamic­
Pituitary­
Gonadal
Axis
·
egg
production
·
eggs
cracked
·
viable
embryos
(
fertility)
·
eggshell
thickness
·
fertilization
success
·
live
18­
day
embryos
·
hatchability
·
14­
day­
old
survivors
Recommended
Additional
Endpoints
for
Validation
and
Inclusion
·
sex
ratio
·
major
organ
(
including
brain)
weights
·
gland
weights
·
histopathology
·
plasma
steroid
concentrations
·
neurobehavioral
test
(
e.
g.,
nest
attentiveness)

Current
Guideline
Endpoints
Sensitive
to
Thyroid
Hormone
Agonists/
Antagonists
·
body
weight
of
adults
·
food
consumption
of
adults
·
body
weight
of
14­
day­
old
survivors
·
developmental
abnormalities
Recommended
Additional
Endpoints
for
Validation
and
Inclusion
·
plasma
T3/
T4
·
thyroid
histology
·
bone
development
(
skeletal
x­
ray)
·
ratio
of
organ
weights
to
bone
measurements
·
neurobehavioral
test
(
cliff
test)
·
cold
stress
test
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
64
Assessment
of
effects
on
offspring
of
the
parental
group
(
first
filial
or
F1
generation)
will
be
made
by
collecting
two
groups
of
50
embryos
from
each
experimental
group
and
incubating
those
embryos.
When
embryos
hatch,
the
number
of
larvae
hatched
from
each
group
will
be
impartially
reduced
to
25,
if
available,
and
released
into
the
larval
growth
chambers.
After
four
weeks
of
exposure,
lengths
and
weights
of
surviving
individuals
will
be
made.

Observations
are
made
of
the
effects
of
the
test
substance
on
embryo
hatching
success,
larvaejuvenile
adult
survival,
growth
of
parental
and
F1
generation,
and
reproduction
of
the
adults.
Table
5.5
provides
a
summary
of
the
endpoints
evaluated
within
the
framework
of
the
Fish
Life
Cycle
Test
(
and
recommended
additional
endpoints
for
validation
and
inclusion
to
cover
EAT
concerns).

3.
Mysid
Life
Cycle
Test
Invertebrates
(
especially
arthropods
such
as
insects
and
crustaceans)
constitute
the
vast
majority
of
animal
species
on
earth.
Yet,
relatively
few
invertebrate
toxicity
test
protocols
are
routinely
used
in
regulatory
toxicity
testing,
and
none
have
been
designed
with
endocrine
endpoints
in
mind.
Nevertheless,
invertebrate
growth,
reproduction,
and
development
are
under
endocrine
control.
However,
invertebrate
endocrine
systems
and
hormones
are
not
directly
analogous
to
those
of
vertebrates.

Two
invertebrate
life
cycle
toxicity
tests
are
commonly
used
in
chemical
and
pesticide
testing,
both
using
crustaceans.
The
opossum
or
mysid
(
Order
Mysidacea)
shrimp
is
an
estuarine
species,
whereas
water
fleas
or
daphnids
(
Daphnia
magna
or
Daphnia
pulex)
are
freshwater
species.
The
former
is
sexually
dimorphic
with
males
and
females,
whereas
the
latter
undergoes
parthenogenetic
reproduction
for
the
majority
of
its
life
cycle.
Although
sexual
reproduction
occurs
in
Daphnia,
the
standard
test
protocol
(
U.
S.
EPA
Public
Draft
OPPTS
850.1300
and
OECD
202)
is
designed
solely
for
the
parthenogenetic
reproductive
phase.
The
chronic
tests
for
both
species
are
designed
to
provide
No
Observed
Effect
Levels
(
NOEL)
using
apical
effect
endpoints
for
fecundity
and
growth.

Although
neither
chronic
test
was
designed
to
examine
endocrine
specific
endpoints,
both
species
are
crustaceans
and
therefore
share
common
physiology.
Ecdysone
is
a
steroid
hormone
that
regulates
growth
and
molting
in
arthropods,
and
exhibits
some
functional
and
structural
similarities
to
estrogen.
The
central
role
of
ecdysone
makes
it
an
attractive
candidate
for
examining
endocrine
effects
in
invertebrates;
however,
other
possibilities
also
exist.
Morphogenetic
and
reproductive
development
of
arthropods
is
controlled
in
part
by
juvenile
hormone
(
JH).
Methyl
farnesoate
is
a
JH
like
compound
that
may
play
a
role
in
reproduction
and
development
(
Borstet
et
al.,
1987;
Laufer
et
al.,
1987a,
b).

Invertebrate
hormones
are
beyond
the
immediate
scope
of
the
EDSTAC
which
has
focused
on
the
vertebrate
estrogen,
androgen,
and
thyroid
hormones.
Nevertheless,
invertebrate
hormones
that
are
functionally
equivalent
to
estrogen,
androgen,
and
thyroid
need
to
be
examined
in
more
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
65
Table
5.5
Fish
Life
Cycle
Test
Endpoints
Current
Guideline
Endpoints
Sensitive
to
Estrogens/
Antiestrogens,
Androgens/
Antiandrogens,
and/
or
Hypothalamic­
Pituitary­
Gonadal
Axis
·
viability
of
embryos
·
time
to
hatch
·
spawning
frequency
·
egg
production
·
fertilization
success
Recommended
Additional
Endpoints
for
Validation
and
Inclusion
·
sexual
differentiation
(
tubercle
formation,
gonadal
histology)

·
sex
ratio
·
gonadosomatic
index
·
gamete
maturation
(
production,
final
oocyte
maturation,
sperm
motility
test,
etc.)

·
vitellogenin
·
plasma
steroid
concentrations
·
in
vitro
gonadal
steroidogenesis
Current
Guideline
Endpoints
Sensitive
to
Thyroid
Hormone
Agonists/
Antagonists
·
growth,
length,
and
body
weight
·
developmental
abnormalities
Recommended
Additional
Endpoints
for
Validation
and
Inclusion
·
plasma
T3/
T4
·
neurobehavioral
tests
(
e.
g.,
activity
level
and
swimming
performance,
nesting
(
spawning)
behavioral
endpoints
such
as
territory
defense,
courtship,
and
egg
protection
and
care,
or
whether
test
fish
retain
the
ability
to
avoid
known
deterrent
chemicals
after
exposure
to
a
test
chemical)

·
thyroid
histology
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
66
depth.
More
importantly,
chemicals
that
affect
these
vertebrate
hormones
may
also
affect
invertebrate
hormones
resulting
in
altered
reproduction,
development,
and
growth.

Chemicals
with
estrogenic
properties
are
reported
to
have
altered
normal
function
of
ecdysone
systems
(
Mortimer,
1993;
1994;
1995a;
1995b;
Chu
et
al.,
1997).
(
Satyanarayana
et
al.,
1994)
showed
stimulation
of
vitellogenin
in
insect
prepupae
and
pupae
by
methoprene,
a
JH
mimic
with
retinoid
properties.
Whether
vitellogenin
production
is
controlled
through
an
estrogen
receptor
or
an
alternative
mechanism
is
not
crucial
for
obtaining
test
results
that
show
alteration
occurs.

Therefore,
the
mysid
shrimp
chronic
life
cycle
test
(
OPPTS
850.1350)
may
be
adapted
to
determine
whether
chemicals
that
affect
hormonal
activity
in
vertebrates
also
affect
arthropods.
Once
adapted
to
include
reproductive
and
developmental
endpoints
relevant
to
the
EDSTP,
the
test
could
be
a
useful
component
in
screening
and
testing.
Although
mysids
would
provide
some
indication
of
endocrine
effects
for
arthropods,
it
is
unlikely
to
identify
vertebrate
effects.

The
other
common
invertebrate
bioassay,
one
using
the
water
flea,
Daphnia,
is
used
internationally
(
OECD
202).
It
incorporates
life
cycle
assessment
and
reproductive
and
developmental
endpoints,
albeit
applied
quite
differently
in
this
group
of
animals.
Reproduction
is
usually
parthenogenic
in
the
laboratory
in
these
animals,
limiting
the
applicability
to
endpoints
identified
in
this
report.
The
particular
aspect
of
this
system
is
that
the
Daphnia
is
sensitive
to
estrogenic
compounds
(
Baldwin
et
al.,
1995;
Baldwin
et
al.,
1997;
Shurin
and
Dodson,
1997),
and
possesses
receptors
for
testosterone,
making
the
system
sensitive
to
another
vertebrate
hormone.
Again,
this
bioassay
would
have
to
be
adapted
for
the
endpoints
and
process
of
interest
in
the
EDSTP
as
a
protocol
for
including
invertebrate
species
in
the
endpoints
addressed
by
the
EDSTP
screening
and
testing
battery.
Other
invertebrates,
such
as
molluscs,
crayfishes,
and
echinoderms,
do
have
EA
systems,
but
again
relevant
standardized
tests
for
evaluating
the
consequences
of
interfering
with
these
systems
are
not
currently
available.
We
simply
do
not
know
whether
one
(
mysid)
or
two
(
mysid
and
daphnia)
Tier
2
tests
will
provide
sufficiently
valid
information
for
other
invertebrate
groups
not
tested.
This
is
a
source
of
uncertainty,
potentially
leading
to
Type
II
errors
of
unknown
magnitude.

4.
Amphibian
Development
and
Reproduction
A
definitive
amphibian
test,
which
exposes
larvae
through
metamorphosis
and
reproduction,
is
important
to
evaluate
the
consequences
of
endocrine
disruption
in
a
poikilothermic
oviparous
vertebrate
distinct
from
fishes.
A
rich
literature
on
metamorphosis,
growth,
and
reproduction
exists
for
frogs
and
promising
methods
are
being
developed.
No
established
method
has
been
identified
which
is
suitably
comprehensive
to
stand
as
a
Tier
2
test
at
this
time.
The
EDSTAC
feels
a
test
to
address
this
taxonomic
group
and
set
of
endpoints
is
needed
in
T2T
and
should
be
given
a
high
priority
for
development
and
standardization.

VII.
Validation,
Standardization,
Methods
Development,
and
Research
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
67
A.
Concept
of
Assay
Validation
and
Standardization
As
stated
earlier,
the
role
of
standardization
and
validation
is
to
provide
sufficient
data
to
allow
informed
decisions
about
the
relative
merits
of
the
recommended
T1S
battery
component
assays
and
alternative
assays
(
based
on
sensitivity,
specificity,
technical
complexity,
inter­
and
intralaboratory
variability,
time,
and
cost).

Validation
is
the
scientific
process
by
which
the
reliability
and
relevance
of
an
assay
method
are
evaluated
for
the
purpose
of
supporting
a
specific
use
(
ICCVAM,
1996).
Relevance
refers
to
the
ability
of
the
assay
to
measure
the
biological
effect
of
interest.
Measures
of
relevance
can
include
sensitivity
(
the
ability
to
detect
positive
effects),
specificity
(
the
ability
to
give
negative
results
for
chemicals
that
do
not
cause
the
effect
of
interest),
statistically
derived
correlation
coefficients,
and
determination
of
the
mechanism
of
the
assay
response
with
the
toxic
effects
of
interest.
Reliability
is
an
objective
measure
of
a
method's
intra­
and
inter­
laboratory
reproducibility.
The
process
of
validation
includes
standardization,
that
is,
definition
of
conditions
under
which
the
assay
is
run
(
species,
strain,
culture
medium,
dosing
regimen,
etc.).
Standardization
is
critical
to
ensure
reliability,
that
is,
valid
results
from
time
to
time
and
between
laboratories.
Even
in
those
instances
where
there
is
currently
some
degree
of
de
facto
acceptance
of
a
given
screening
method
as
valid,
there
is
a
need
for
such
standardization.

B.
Statutory
Need
for
Validation
The
Food
Quality
Protection
Act
(
FQPA)
requires
EPA
"
to
develop
a
screening
program,
using
appropriate
validated
test
systems
and
other
scientifically
relevant
information,
to
determine
whether
certain
substances
may
have
an
effect
in
humans
that
is
similar
to
an
effect
produced
by
a
naturally
occurring
estrogen,
or
other
such
endocrine
effect
as
the
Administrator
shall
designate"
by
August
1998
with
implementation
of
the
peer
reviewed
program
by
August
1999.
In
requiring
the
use
of
validated
test
systems
the
FQPA
is
merely
mandating
good
science.
There
are
numerous
reasons
for
using
only
validated
assays.
These
include:
having
confidence
that
the
assay
is
detecting
the
effect
it
purports
to
be
detecting,
that
the
results
of
the
assay
are
reproducible
and
comparable
from
laboratory
to
laboratory,
and
that
the
results
permit
a
comparison
of
the
toxicity
of
various
chemicals.
These
factors
are
important
in
being
able
to
interpret
results
to
establish
a
relative
priority
among
chemicals
for
progressing
from
screening
to
testing
and
ultimately
to
perform
a
hazard
and
risk
assessment.

C.
Addressing
the
Validation
Issue
The
assays
and
tests
recommended
by
the
EDSTAC
vary
considerably
in
terms
of
their
degree
of
development
and
validation.
EPA
(
Dr.
Lynn
Goldman,
April
24,
1997,
letter
to
EDSTAC)
recognized
that
few
screening
assays
have
actually
met
the
"
gold
standard"
of
validation
and
that
other
assays
have
been
accepted
on
the
basis
of
peer
review
and
general
use
without
formal
validation.
Because
the
number
of
validated
off­
the­
shelf
assays
is
so
limited,
EDSTAC
was
asked
by
EPA
to
extend
its
consideration
to
all
existing
assays.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
68
Thus,
although
formal
validation
was
not
a
prerequisite
for
assay
selection
by
the
EDSTAC,
the
ability
or
potential
of
an
assay
to
be
validated
must
be
considered
because
assays
must
be
capable
of
passing
the
"
validation
test"
before
the
screening
program
is
fully
implemented.
The
following
are
a
list
of
factors
that
EPA
might
consider
in
estimating
the
likelihood
that
a
candidate
assay
will
actually
survive
the
validation
process.
If
possible,
it
would
be
useful
to
devise
a
quantitative
or
semi­
quantitative
scale
for
expressing
these
characteristics
so
that
test
methods
reviewed
by
various
people
could
more
easily
be
compared.

1.
Number
of
independent,
peer­
reviewed
publications
reporting
results
from
the
assay.
2.
Similarity
of
results
from
independent
publications
performing
the
assay.
3.
Number
of
independent
laboratories
publishing
results
from
the
assay.
4.
Consistency
of
the
methods
used
across
laboratories.
5.
Consistency
of
results
of
the
assay
between
labs
(
to
the
extent
results
are
available
for
the
same
chemicals).
6.
Known
variability
of
the
assay
within
single
laboratories
(
may
not
preclude
use
as
a
valuable
research
tool,
but
may
have
important
implications
for
use
as
a
widespread
screening
tool).
7.
Age
of
the
assay
­
is
it
an
"
old
standby"
or
a
"
new
kid
on
the
block."
8.
The
extent
to
which
the
assay
relies
upon
calibrated
equipment
and
calibrated
standards.
9.
The
extent
to
which
the
assay
depends
upon
the
skill
of
the
technician
(
a
professional
opinion
from
those
who
know
about
the
assay).
10.
The
extent
to
which
the
assay
utilizes
internal
controls
or
standards.
11.
Use
of
the
assay
to
develop
clinically
effective
drugs
(
although
not
of
itself
proof
of
validity,
success
speaks
well
for
itself).
12.
Number
of
examples
of
false
positives/
false
negatives
from
the
assay
(
although
we
have
a
clear
charge
to
eliminate
false
negatives
at
the
screening
stage,
it
is
nonetheless
important
to
consider
the
overall
performance
of
an
assay
in
order
to
estimate
its
likelihood
of
being
validatable).
13.
Any
known
species­
or
strain­
specific
sensitivities
of
the
assay.
For
in
vitro
assays,
any
knowledge
of
critical
sensitivity
to
cell
characteristics
such
as
passage
number,
plating
density,
doubling
time,
etc.
or
other
specific
sensitivities
of
the
assay,
such
as
receptor
number,
transfection
technique,
serum
requirements,
media
composition,
etc.
(
trying
to
get
another
angle
on
how
finicky
the
assay
is).

Information
related
to
validation
status
of
the
assays
is
summarized
in
Chapter
Five,
Section
VII,
E,
and
was
among
the
factors
considered
in
deciding
among
assays.
These
same
considerations
regarding
validation
and
standardization
apply
to
T2T.

D.
Validation
and
Standardization
Process
While
not
all
assays
would
necessarily
need
to
be
validated
since
they
may
have
de
facto
acceptance
as
being
valid
in
the
scientific
community
due
to
their
long
history
of
use
and
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
69
performance,
others
have
little
or
no
data
that
would
allow
judgments
to
be
made
regarding
their
validity.

The
following
is
a
description
of
elements
of
a
such
a
validation
process
for
endocrine
disruptor
screening
assays.

1.
Characterize
Reference
Substances/
Vehicles.
·
A
reference
substance
for
each
hormone
endpoint
(
minimum)
·
Reference
substances
for
each
hormone
endpoint:
·
positive
control
substances
·
negative
control
substances
·
Natural
and
man­
made
substances
·
Composition
and
purity
defined
(
e.
g.,
GC/
MS)
·
Stability
verified
·
Coded
·
Centralized
distribution
(
using
same
batch
number,
lot
number,
etc.)

2.
Develop
a
Standard
Protocol
for
Each
Assay
Method.
·
Assay
system
to
be
used
(
species,
strain,
sex,
age,
cell
line,
clone,
gene
construct,
etc.)
·
Dose
levels/
exposure
concentrations
·
General
criteria
for
selection
·
MTD
(
whole
animal
systems;
e.
g.,
mortality,
decreased
body
weight,
etc.)
·
Cell
viability
(
in
vitro
systems):
2
methods
·
Solubility
limitations
·
Specific
for
each
reference
substance
·
Dose/
exposure
regimen
·
Number
of
doses
·
Duration
·
Guidance
of
mixtures
·
Concentrations
of
reference
substances
·
Interpretation
of
data
·
Route
of
exposure
(
whole
animal
systems)
·
Description
of
endpoint(
s)
to
be
measured
·
Materials
(
equipment,
media,
vehicles,
etc.)
·
Time(
s)
of
measurements
·
Criteria
for
positive/
negative
response
·
Statistical
methods
to
used
·
Number
of
replications
required
(
depending
on
the
study
design)

3.
Define
Specialized
Skills
and
Equipment
Required
for
Each
Assay
Method
4.
Conduct
in
a
Variety
of
Laboratories.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
70
5.
Compile
and
Evaluate
Data
·
Expert
scientific
oversight
group
·
Inter­
laboratory/
intra­
laboratory
variability
·
Positive
versus
negative
response
·
Relative
potency
(
in
comparison
to
reference
substance)
·
Viability/
maintenance
of
assay
system
(
including
passage
number
and
growth
curves)
·
Sensitivity
of
assay
system
(
e.
g.,
minimal
effective
dose/
concentration)
·
Specificity
of
assay
system
(
positive
versus
negative
controls)

E.
Levels
of
Effort
Necessary
to
Validate
the
Recommended
Screens
and
Tests
The
EDSTAC
believes
validation
and
standardization
of
the
recommended
screens
and
tests
are
essential
for
implementation
of
the
EDSTP.
The
EDSTAC
also
believes
the
validation
and
standardization
program
is
of
highest
priority,
and
recommends
that
it
proceed
on
an
accelerated
schedule.
As
indicated
earlier,
the
EDSTAC
recommends
that
the
validation
and
standardization
program
be
consistent
with
the
principles
articulated
by
the
national
(
ICCVAM,
1996;
Zeiger,
1998)
and
international
(
OECD,
1996)
alternative
methods
validation
groups.
As
mentioned
throughout
the
chapter,
each
screen
and
test
recommended
for
T1S
or
T2T
needs
some
level
of
validation,
standardization,
methods
development,
or
further
research
before
being
accepted
as
a
regulatory
toxicity
screen
or
test
for
inclusion
in
the
EDSTP.
The
level
of
effort
needed
to
fully
standardize
and
validate
may
be
different
for
each
individual
screen
or
test
(
including
all
recommended
endpoints).
The
effort
required
for
each
screen
or
test
will
be
defined
by
a
variety
of
criteria
including:
period
of
time
in
use,
existing
level
of
general
acceptance
in
the
endocrine
toxicology
field,
and
existing
understanding
of
relevancy
and
reliability.
Regardless
of
the
effort
required,
EDSTAC
believes
all
the
screens
and
tests
recommended
for
T1S
and
T2T
must
be
fully
validated
and
standardized
before
being
included
in
the
EDSTP.
The
EDSTAC
recommends
that
as
individual
screens
and
tests
are
validated
and
standardized,
they
can
be
utilized
in
the
EDSTP
without
waiting
for
all
screens
and
tests
in
the
batteries
to
be
validated.
The
EDSTAC
further
recommends
that
a
multi­
stakeholder
process,
involving
government,
industry,
and
academics,
be
utilized
in
validating
and
standardizing
the
T1S
and
T2T
batteries
(
see
Section
VII,
G,
of
this
chapter,
for
more
explanation).

As
mentioned
earlier,
the
screens
and
tests
being
recommended
by
the
EDSTAC
vary
considerably
in
terms
of
the
effort
necessary
to
be
fully
validated
and
standardized.
Each
screen
or
test
lies
somewhere
along
a
spectrum
of
validation
from
already
fully
validated
and
standardized
to
needing
further
research
to
determine
their
utility
in
the
EDSTP.
Outlined
in
the
following
few
paragraphs,
in
some
detail,
is
the
spectrum
of
possible
effort
needed
to
validate
and
standardize
the
screens
and
tests.
In
addition,
the
Screening
and
Testing
Work
Group
attempted
to
categorize
the
levels
of
validation
needed
of
the
recommended
screens
and
tests
and
their
preliminary
efforts
are
found
in
Appendix
R.
It
is
possible
that
the
status
of
these
screens
and
tests
may
have
changed
as
work
has
progressed
in
the
interim.
The
EDSTAC
recommends
that
EPA
update
this
categorization
scheme
as
part
of
their
validation
and
standardization
program.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
71
The
recommended
screens
and
tests
(
including
all
endpoints)
will
have
to
meet
all
the
criteria
of
relevance
and
reliability
for
use
in
regulatory
toxicity
screening
or
testing
for
EAT
in
order
to
be
considered
fully
validated
and
standardized
(
ICCVAM,
1996;
Zeiger,
1998).
As
screens
and
tests
become
fully
validated
and
standardized,
they
will
warrant
inclusion
in
the
EDSTP
according
to
their
specific
and
appropriate
use.
None
of
the
screens,
new
tests,
or
enhancements
to
existing
test
guidelines
included
in
T1S
or
T2T
completely
fulfill
these
criteria
to
date.

Some
of
the
recommended
screens
and
tests
have
been
in
use
for
a
sufficient
period
of
time
and
have
therefore
gained
sufficient
general
acceptance
within
the
field
of
endocrine
toxicology
to
be
considered
de
facto
validated
(
reliable
and
relevant).
These
assays
measure
relevant
endpoints,
are
responsive
to
endocrine
active
compounds
with
a
high
degree
of
specificity,
are
sufficiently
sensitive
to
identify
all
known
active
agents,
and
can
reasonably
be
expected
to
give
reproducible
results
from
laboratory
to
laboratory,
assuming
a
general
level
of
competence
and
expertise.
Nonetheless,
variations
in
protocols
for
these
screens
and
tests
can
produce
disparate
results.
Therefore,
before
the
recommended
screens
and
tests
are
utilized
in
the
EDSTP,
a
standard
performance
guideline
should
be
developed
that
produces
an
acceptable
level
of
consistency
in
results
for
each
one.

Some
of
the
recommended
screens
and
tests
have
sufficiently
broad
use
to
be
generally
considered
relevant
OR
reliable
to
either
screening
for
endocrine
activity
(
Tier
1)
or
to
testing
for
adverse
endocrine­
mediated
effects
(
Tier
2).
These
screens
and
tests
cannot,
however,
be
generally
considered
to
be
both
relevant
and
reliable.
The
level
of
performance
that
can
be
expected
of
these
screens
and
tests
with
respect
to
identifying
endocrine
active
agents
or
endocrine
disruptive
effects
of
chemicals
must
be
clarified.
Therefore,
these
assays
should
undergo
further
but
focused
validation
and
standardization
to
define
their
relevance
and
reliability
for
the
task
of
endocrine
disruptor
screening
or
testing.
The
validation
required
may
be
focused
to
answer
specific
questions
about
relevance
and
to
provide
information
regarding
specificity
and
sensitivity.

Some
of
the
recommended
screens
and
tests
may
have
relevance
to
the
task
of
either
screening
for
endocrine
activity
or
testing
for
endocrine
disruptive
effects,
but
their
performance
in
identifying
endocrine
active
agents
or
endocrine
disruptive
effects
has
seen
only
limited
testing.
Questions
as
to
whether
these
assays
measure
endpoints
that
are
relevant
to
endocrine
activity
or
endocrine
disruptive
effects,
whether
these
assays
respond
with
specificity
and
sensitivity
to
known
endocrine
active
agents,
or
whether
they
identify
endocrine
disruptive
effects
cannot
be
addressed
with
information
currently
available.
In
addition,
questions
regarding
the
specific
protocols
and
conditions
under
which
the
assays
should
be
conducted
must
be
answered
before
relevance
and
reliability
can
be
assessed.
Nonetheless,
the
EDSTAC
feels
these
assays
would
have
sufficient
utility,
if
further
developed
and
validated,
to
enhance
or
augment
the
screening
and
testing
program.
Therefore,
the
EDSTAC
recommends
that
resources
be
made
available
to
pursue
methods
development
and
validation
and
standardization
of
these
assays.

F.
Screens
and
Tests
Recommended
for
Further
Research
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
72
As
discussed
in
Section
III,
A,
5,
the
EDSTAC
recognizes
the
importance
of
evaluating
postnatal
consequences
of
in
utero
and
in
ovo
exposures
to
chemicals
with
EAT
activities.
Such
an
assay
has
not
been
incorporated
into
T1S
due,
in
part,
to
the
lack
of
an
appropriate,
short­
term,
costeffective
assay.
The
EDSTAC,
however,
does
recommend
that
EPA
take
the
lead
in
designing,
standardizing,
and
validating
such
an
assay.
The
EDSTAC
also
identified
other
screens
and
tests
which,
if
available,
could
have
an
important
utility
in
the
screening
and
testing
program,
and
recommends
that
research
be
conducted
to
determine
whether
such
assays
can
be
developed,
and
if
so,
what
purpose
the
assays
could
fulfill
within
the
endocrine
disruptor
screening
and
testing
program.
The
rationale
for
including
each
of
the
following
assays
and
tests
is
found
below.

·
in
utero
developmental
screening
assay
·
in
ovo
developmental
screening
assay
·
avian
androgenicity
screening
assay
·
invertebrate
screening
assays
·
amphibian
development
and
reproduction
test
(
Xenopus)
·
reptilian
reproduction
test
In
Utero
Developmental
Screening
Assay:
The
rationale
for
developing
an
in
utero
developmental
screening
assay
was
discussed
earlier;
however,
the
EDSTAC
did
consider
one
possible
study
design
which
is
summarized
here
(
and
further
elaborated
upon
in
Appendix
O).
In
the
study
design,
pregnant
rats
would
be
exposed
to
the
tested
chemical
from
the
start
of
the
embryonic
period
through
weaning
of
the
offspring
three
weeks
after
birth.
Types
of
EATsensitive
endpoints
that
could
be
measured
in
this
assay
might
include
apparent
sex
ratio
(
based
on
anogenital
distance),
numbers
of
offspring
per
litter,
anogenital
distance,
retention
of
nipples
in
males,
precocious
puberty
in
females,
uterine
and
ovarian
weight
and
various
uterine
histological
and
biochemical
parameters
in
females,
and
reproductive
tract
anomalies
in
both
sexes.
Blood
samples
could
be
analyzed
for
estradiol,
testosterone,
T4,
and
TSH.
Myelin
basic
protein
in
brains
could
be
measured
as
an
indicator
of
thyroid
hormone
activity.

How
much
effort
such
an
assay
would
entail
or
what
its
cost
might
be
is
not
currently
known.
There
may
be
additional
protocols
that
could
be
predictive
of
EAT
in
developing
systems
and
which
are
more
amenable
to
screening
applications
than
the
protocol
included
and
therefore
the
EDSTAC
encourages
development
of
other
assays
to
address
this
issue.

In
Ovo
Developmental
Screening
Assay:
A
major
route
of
excretion
of
lipophilic
contaminants
for
female
birds
is
into
the
yolk
of
their
eggs;
therefore,
their
embryos
can
have
high
levels
of
exposure
from
the
earliest
stages
of
development.
In
addition,
the
endocrine
control
of
sexual
and
reproductive
development
is
fundamentally
different
in
birds
than
in
mammals.
Hence,
a
shortterm
screening
assay
for
chemical
substances
or
mixtures
that
alter
avian
development
is
highly
desirable.
There
is
a
moderate
amount
of
research
on
the
effects
of
environmental
contaminants
injected
into
bird
eggs
that
could
be
the
basis
for
developing
such
an
assay.
The
more
general
rationale
for
developing
an
in
ovo
developmental
screening
assay,
as
with
an
in
utero
one,
is
found
earlier
in
the
chapter.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
73
Avian
Androgenicity
Screening
Assay:
This
assay
would
be
useful
in
the
T1S
battery
to
improve
and
extend
our
assessment
of
chemical
substances
or
mixtures
for
androgenic
and
antiandrogenic
activity
in
birds.
Development
of
this
screen
will
become
important
if
data
from
T2T
point
to
differences
in
the
actions
of
chemical
substances
or
mixtures
in
birds
versus
mammals.

Reptilian
Reproduction
Test:
Several
distinctive
features
of
reptilian
reproduction
(
e.
g.,
ovoviviparity,
temperature­
dependent
sex
determination),
and
a
generally
long
life
span
that
allows
high
body
burdens
of
environmental
contaminants
to
accumulate
in
reptiles,
underscore
the
importance
of
developing
a
practical
reproductive
test
in
this
class
of
ecologically
important
vertebrates.

G.
Stakeholder
Involvement
in
the
Validation
Program
The
EDSTAC
recommends
that
a
multi­
stakeholder
process
involving
government,
industry,
and
academics
be
utilized
in
standardizing
and
validating
the
T1S
and
T2T
batteries.
One
key
step
in
instituting
a
validation
program
for
T1S
assays
is
the
identification
of
a
set
of
"
standard
test
substances"
for
the
individual
assays
as
well
as
for
the
overall
T1S
battery.
To
the
extent
possible,
the
standard
test
substances
will
be
chosen
according
to
the
following
criteria:

1.
known
EAT
positives
which
act
via
receptor
binding;
2.
known
EAT
positives
that
do
not
appear
to
act
via
receptor
binding
(
i.
e.,
via
some
other
mechanism
such
as
alterations
of
hormone
synthesis,
degradation,
transport,
etc.);
3.
known
EAT
negatives
(
i.
e.,
substances
known
not
to
have
hormonal
activity);
4.
known
EAT
positives
which
are
active
as
the
parent
compound;
5.
known
EAT
positives
which
require
metabolic
activation;
6.
substances
that
cover
a
wide
range
of
EAT
potencies;
7.
substances
with
a
wide
range
of
physical
properties
(
e.
g.,
pH,
reactivity,
volatility,
etc.);
and
8.
substances
with
extensive
in
vivo
databases
with
in
vivo
effects
that
have
been
well
documented.

It
may
not
be
possible
to
satisfy
every
one
of
the
above
criteria
(
e.
g.,
there
are
currently
no
known
examples
of
environmental
thyroid
or
androgen
receptor
agonists),
but
every
standard
test
substance
selected
should
meet
at
least
one
of
the
criteria.

In
addition,
careful
definition
of
the
expected
use
of
the
set
of
chemicals
is
necessary
to
avoid
inappropriate
use.
Such
a
set
of
chemicals,
developed
with
the
already
mentioned
criteria
in
mind,
would
be
used
in
the
validation
program
to
assist
in
defining
their
relevance
and
reliability
for
the
task
of
endocrine
disruptor
screening,
(
i.
e.,
to
identify
whether
a
specific
chemical
substance
or
mixture
has
endocrine
activity,
or
can
be
placed
in
the
"
hold
box").

Further,
as
was
also
stated
earlier,
it
is
critical
to
acknowledge
state
of
the
science
in
this
area
is
evolving
rapidly,
and
assays
currently
being
developed,
or
ones
developed
in
the
future,
may
offer
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
74
distinct
advantages
over
some
included
in
the
current
options.
As
they
are
developed,
validated,
and
standardized,
the
use
of
these
new
assays
for
screening
is
strongly
encouraged.

H.
Preliminary
Cost
Estimates
for
the
T1S
and
T2T
Batteries
An
EDSTAC
member
completed
a
survey
of
contract
labs
in
an
effort
to
estimate
the
cost
of
the
recommended
T1S
and
T2T
batteries.
The
results
of
this
survey
are
summarized
in
Tables
5.6
and
5.7
and
Appendix
S.
A
detailed
description
of
the
methodology
used
to
conduct
the
survey
is
included
in
the
EDSTAC
Docket
(#
OPPTS­
42189,
TSCA
Public
Docket
Office,
U.
S.
EPA).
The
EDSTAC
includes
these
estimates
in
its
final
report,
but
acknowledges
they
are
both
preliminary
and
uncertain
given
the
inherent
uncertainties
regarding
the
outcome
of
the
validation
and
standardization
process.
The
EDSTAC
also
notes
that
these
cost
estimates
should
be
viewed
in
the
context
of
the
near­
and
long­
term
public
health
and
environmental
protection
benefits
to
society.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
75
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
76
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
77
VIII.
Compilation
of
Chapter
Five
Recommendations
A.
Tier
1
Screening
1.
The
EDSTAC
recommends
that
any
T1S
battery
designed
to
detect
endocrine
disruptors
should
meet
five
criteria.
The
battery
should:

·
maximize
sensitivity
to
minimize
false
negatives
while
permitting
an
acceptable
level
of
false
positives;
·
include
a
range
of
organisms
representing
known
or
anticipated
differences
in
metabolic
activity;
·
detect
all
known
modes
of
action
for
endocrine
endpoints
of
concern;
·
include
a
sufficient
range
of
taxonomic
groups
among
the
test
organisms;
and
·
incorporate
sufficient
diversity
and
complementarity
among
the
endpoints
and
assays
to
reach
conclusions
based
on
weight
of
evidence
considerations.

2.
The
EDSTAC
recommends
the
following
assays
for
inclusion
in
the
T1S
battery:

In
Vitro
1.
Estrogen
Receptor
(
ER)
Binding/
Transcriptional
Activation
Assay;
2.
Androgen
Receptor
(
AR)
Binding/
Transcriptional
Activation
Assay;
and
3.
Steroidogenesis
Assay
With
Minced
Testis.
In
Vivo
1.
Rodent
3­
day
Uterotrophic
Assay
(
subcutaneous);
2.
Rodent
20­
day
Pubertal
Female
Assay
With
Thyroid;
3.
Rodent
5­
7
day
Hershberger
Assay;
4.
Frog
Metamorphosis
Assay;
and
5.
Fish
Gonadal
Recrudescence
Assay.

3.
The
EDSTAC
identified
the
following
four
assays
as
possible
alternatives
to
some
components
of
the
proposed
battery
and
recommends
that
they
also
be
standardized
and
validated:

In
Vitro
1.
Placental
Aromatase
Assay
In
Vivo
1.
Modified
Rodent
3­
day
Uterotrophic
Assay
(
intraperitoneal);
2.
Rodent
14­
day
Intact
Adult
Male
Assay
With
Thyroid;
and
3.
Rodent
20­
day
Thyroid/
Pubertal
Male
Assay.

Combinations
of
the
alternative
assays,
if
validated
and
found
to
be
functionally
equivalent,
could
potentially
replace
three
of
the
component
assays
in
the
proposed
T1S
battery
(
in
vitro
steroidogenesis
assay
with
testis,
20­
day
pubertal
female
assay,
and
5­
7
day
Hershberger
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
78
assay)
thereby
possibly
reducing
the
overall
time,
cost,
and
complexity
while
maintaining
equivalent
performances
of
the
overall
T1S
battery.
The
EDSTAC
recognizes
that
the
stateof
the­
science
in
this
area
is
evolving
quickly
and
strongly
encourages
the
use
of
new
or
improved
assays
for
screening
as
they
become
available.

4.
The
EDSTAC
recommends
that
validation/
standardization
studies
be
conducted
on
all
assays
in
the
proposed
battery
as
well
as
the
alternatives.

5.
The
EDSTAC
agrees
that
EPA
should
take
affirmative
steps,
in
collaboration
with
industry
and
other
interested
parties,
to
attempt
to
develop
the
protocol
for
a
full
life
cycle
(
i.
e.,
with
embryonic
exposure
and
evaluation
of
the
adult
offspring)
developmental
exposure
screening
assay
that
can
be
subjected
to
validation
and
standardization.
The
EDSTAC
further
recommends
that,
if
such
an
assay
were
identified,
validated,
and
standardized,
the
decision
on
whether
it
should
be
included
in
the
T1S
battery
should
include
an
evaluation
of
its
potential
to
replace
one
or
more
of
the
recommended
T1S
assays
and
its
overall
impact
on
the
cost
effectiveness
of
the
T1S
battery.

6.
The
EDSTAC
recommends
that
all
T1S
in
vitro
assays
involve
multiple
dose
levels,
whether
performed
by
HTPS
or
bench
level
methods,
so
a
dose­
response
curve
and
assessment
of
relative
potencies
can
be
developed.
Subject
to
the
results
of
the
validation
process,
the
EDSTAC
recommends
using
one
or
more
dose
levels
in
the
performance
of
the
in
vivo
assays.

7.
For
assessing
receptor
binding
in
vitro,
the
EDSTAC
is
recommending
that
both
the
cell­
free
receptor
binding
assays
and
the
transcriptional
activation
assays
for
ER
and
AR
be
incorporated
into
the
T1S
battery,
and
be
subjected
to
validation
and
standardization.

8.
As
noted
in
Chapter
Four,
the
EDSTAC
recommends
the
use
of
a
high
throughput
pre­
screen
(
HTPS)
for
toxicants
operating
though
the
ER,
AR,
and
TR
using
stably
transfected
cell
lines
with
and
without
metabolic
activation,
if
available.
Substances
which
have
not
been
assessed
in
the
HTPS
should
be
subject
to
assays
for
detection
of
ER
and
AR
activity
performed
at
the
bench.
Two
types
of
assays
are
considered
acceptable:
cell
free
receptor
binding
and
transcriptional
activation
in
transfected
cells.
The
latter
is
preferred.
Assays
must
meet
the
following
characteristics:

·
evaluate
binding
to
EAT
receptors;
·
evaluate
binding
with
and
without
metabolic
capability;
·
distinguish
between
agonist
and
antagonist
potential;
and
·
yield
dose
responses
to
establish
relative
potency.

9.
The
EDSTAC
is
recommending
evaluation
of
antithyroid
effects
in
animals
in
the
longer
term
rodent
screen
(
either
14­
day
or
20­
day
exposure).
Although
it
is
not
known
whether
exposure
to
xenobiotics
for
greater
than
14
days
is
required
to
significantly
affect
circulating
levels
of
T4,
TSH,
or
thyroid
histopathology,
the
EDSTAC
believes
these
longer
periods
may
be
required.
The
effects
of
duration
of
chemical
substance
and
mixture
exposure
must
be
quickly
evaluated
in
the
validation
phase.
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
79
B.
Principles
for
Evaluating
Tier
1
and
Tier
2
Results
10.
The
EDSTAC
recommends
that
a
"
weight­
of­
evidence"
approach
be
used
in
evaluating
T1S
and
T2T
results
and
has
developed
general
criteria
for
applying
"
weight­
of­
evidence"
to
ensure
that
decisions
are
transparent
and
predictable.

C.
Tier
2
Testing
11.
The
EDSTAC
recommends
that
the
following
tests
be
included
in
the
Tier
2
battery:

Mammalian
tests
·
Two­
generation
reproductive
toxicity
study,
or
·
An
alternative
less
comprehensive
test:
1.
Alternative
mammalian
reproductive;
and
2.
One­
generation
test.

Non­
mammalian
multigeneraton
tests
·
Avian
reproduction;
·
Fish
life
cycle;
·
Mysid
life
cycle;
and
·
Amphibian
development
and
reproduction.

12.
The
EDSTAC
recommends
that
the
"
default"
action,
in
absence
of
any
prior
information,
be
to
perform
all
tests
in
the
T2T
battery
with
all
endpoints.
Further,
the
EDSTAC
recommends
that
the
choice
of
whether
Tier
2
tests
will
be
conducted
on
all
five
of
the
recommend
taxa,
or
a
more
limited
subset
of
the
five
taxa,
should
be
based
on
the
physicochemical
characteristics
and
environmental
release
and
exposure
information
of
the
chemical
substance
or
mixture
to
be
tested,
together
with
biological
data
from
T1S.
The
results
of
T1S
or
other
information
may
also
allow
tailoring
of
T2T
such
as
the
inclusion
or
deletion
of
certain
endpoints
(
e.
g.,
thyroid
effects)
or
use
of
alternative
tests.

13.
The
EDSTAC
believes
that
a
project
is
required
to
resolve
the
underlying
uncertainties
and
controversy
about
issues
related
to
low
dose
selection
and
the
identification
of
no­
observedadverse
effect­
levels
(
NOAEL).
Further,
the
EDSTAC
recommends
that
a
collaborative
group
involving
government,
industry,
and
appropriate
individuals
in
academia
design
the
study
protocols,
be
kept
abreast
of
the
conduct
of
the
studies,
evaluate
results,
and
develop
overall
conclusions
and
recommendations.

14.
The
EDSTAC
recommends
that
information
used
to
select
doses
in
the
performance
of
Tier
2
tests
include:
EDSTAC
Final
Report
Chapter
Five
August
1998
5
­
80
·
existing
information
such
as
that
available
during
priority
setting
including
the
results
or
the
HTPS;
·
results
from
T1S;
·
results
from
other
assays
or
tests;
and
·
results
from
range
finding
studies.

15.
The
EDSTAC
recommends
including
thyroid­
sensitive
endpoints
in
T2T
and
that
dosing
in
mammalian
tests
include
fetal
and
lactational
exposure.

D.
Validation
of
the
Screening
and
Testing
Batteries
16.
The
EDSTAC
believes
the
validation
and
standardization
program
is
of
highest
priority,
and
recommends
that
it
proceed
on
an
accelerated
schedule.
The
EDSTAC
further
recommends
that
the
validation
and
standardization
program
be
consistent
with
the
principles
articulated
by
the
national
(
ICCVAM,
1996;
Zeiger,
1998)
and
international
(
OECD,
1996)
alternative
methods
validation
groups.

17.
The
EDSTAC
recommends
that,
as
individual
screens
and
tests
are
validated
and
standardized,
they
can
be
utilized
in
the
EDSTP
without
waiting
for
all
screens
and
tests
in
the
batteries
to
be
validated.

18.
The
EDSTAC
recommends
that
a
multi­
stakeholder
process,
involving
government,
industry,
and
academics,
be
utilized
in
validating
and
standardizing
the
T1S
and
T2T
batteries.

19.
The
EDSTAC
identified
other
screens
and
tests
which,
if
available,
could
have
an
important
utility
in
the
screening
and
testing
program,
and
recommends
that
research
be
conducted
to
determine
whether
such
assays
can
be
developed,
and
if
so,
what
purpose
the
assays
could
fulfill
within
the
endocrine
disruptor
screening
and
testing
program.

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utero
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"
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Methoprene
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1998
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C.,
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"
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Zacharewski,
"
Failure
of
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derived
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L.
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"
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315­
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M.
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W.
B.
Butler,
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C.
Brooks
"
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by
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52,
1995,
pp.
421­
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Denver,
R.
J.,
S.
Pavgi,
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Y.
B.
Shi,
"
Thyroid
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dependent
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272,
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M.,
K.
Crofton,
and
S.
McMaster,
"
Screening
Methods
for
Chemicals
That
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Synergism
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1993,
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244­
254.
EDSTAC
Final
Report
Chapter
Six
August
1998
Chapter
Six
Communications
and
Outreach
EDSTAC
Final
Report
Chapter
Six
August
1998
Table
of
Contents
I.
Introduction...........................................................................................................................
1
II.
Need
for
Communication.....................................................................................................
2
III.
Recommendations...............................................................................................................
3
A.
Principles
to
Guide
Implementation
of
a
Communications
and
Outreach
Strategy...............
3
B.
Basic
Features
of
the
Communications
and
Outreach
Strategy............................................
3
1.
What
Should
be
Communicated?....................................................................................
4
2.
To
Whom
Should
Information
be
Communicated?
.......................................................
11
3.
How
Should
Information
be
Communicated?
...............................................................
12
4.
When
Should
Information
be
Communicated?
..............................................................
14
C.
Adequacy
of
Resources
Devoted
to
Communication
and
Outreach...................................
16
IV.
Generalized
Schedule
for
Implementation.......................................................................
16
V.
Compilation
of
Chapter
Six
Recommendations................................................................
19
A.
Need
for
Communication
.................................................................................................
19
B.
Principles
to
Guide
Implementation
of
a
Communications
Strategy...................................
19
C.
Basic
Features
of
a
Communications
and
Outreach
Strategy.............................................
19
VI.
Literature
Cited
................................................................................................................
21
Figures
Figure
6.1
Generalized
Schedule
for
Implementation
of
the
Endocrine
Disruptor
Screening
and
Testing
Program
........................................................................................
18
Appendices
Appendix
T:
Summary
of
EPA's
September
1997
Outreach
Questionnaire
Results
Appendix
U:
Summary
of
Public
Comments
to
the
EDSTAC
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
1
I.
Introduction
The
EDSTAC
recognized
early
on,
that
effective
communication
about
the
endocrine
disruptor
screening
and
testing
program
and
its
results,
would
be
critical
to
the
success
of
the
Endocrine
Disruptor
Screening
and
Testing
Program
(
EDSTP)
(
Heckler,
1985;
Banquet,
1985;
Chilton,
1989;
NRC,
1989;
NRC,
1994;
NRC,
1996;
NRC,
1983).
To
address
this
need,
the
EDSTAC
created
the
Communications
and
Outreach
Work
Group
(
COWG),
which
was
charged
with
three
principal
tasks:

1.
providing
advice
on
the
coordination
of
the
overall
communications
and
outreach
efforts
surrounding
the
EDSTAC
process;
2.
developing
recommendations
to
be
incorporated
into
the
EDSTAC
final
report
on
communication
issues
regarding
key
decision
points
of
the
Conceptual
Framework
and
implementation
of
the
EDSTP;
and
3.
improving
the
understandability
of
the
final
report
and
any
other
materials
distributed
by
the
Committee.

A
significant
portion
of
the
work
completed
by
the
COWG
during
the
EDSTAC
process
fell
under
the
first
task
 
coordination
and
input
on
overall
communications
and
outreach
efforts
surrounding
the
EDSTAC
plenary
meetings.
Activities
of
the
work
group
included:
providing
feedback
to
EPA
on
the
public
comment
period
session;
developing
the
Description
of
the
EDSTAC
Charge,
which
was
used
by
EPA
to
describe
the
process
to
the
public;
recommending
to
EPA
that
the
Agency
coordinate
an
outreach
mailing
to
interested
and
potentially
interested
parties;
assisting
in
the
subsequent
assembly
of
materials
for
the
mailing;
and
discussing
additional
outreach
efforts
for
EPA
and
the
Committee.
Included
in
the
EPA
outreach
mailing
was
a
questionnaire
developed
by
the
COWG
and
disseminated
to
over
1,500
addressees.
This
questionnaire
was
created
in
an
effort
to
obtain
information
as
to
the
public's
interest
in
the
EDSTAC
and
its
activities
during
the
Committee's
existence,
as
well
as
to
help
in
future
outreach
efforts
by
the
Agency.
The
information
received
in
response
to
this
questionnaire
will
assist
the
Agency
in
determining
the
most
effective
way(
s)
to
communicate
with
those
individuals
and
organizations
interested
in
the
EDSTAC
process.
A
summary
of
the
results
of
the
survey
can
be
found
in
Appendix
T.

The
recommendations
provided
in
this
chapter
focus
primarily
on
the
second
of
these
three
tasks
 
developing
recommendations
to
be
incorporated
into
the
EDSTAC
final
report
on
communication
issues
regarding
key
decision
points
of
the
Conceptual
Framework
and
implementation
of
the
EDSTP.
In
some
instances,
however,
communication
recommendations
regarding
key
decisions
of
the
Conceptual
Framework
and
the
EDSTP
are
included
elsewhere
in
the
report
where
they
are
more
appropriate.

The
work
group's
efforts
surrounding
the
third
task
above
 
improving
the
understandability
of
the
final
report
and
any
other
materials
distributed
by
the
Committee
 
included:
review
of
the
Priority
Setting
and
Screening
and
Testing
Work
Group
chapters
to
ensure
communication
issues
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
2
are
addressed
where
appropriate;
development
of
language
appropriate
for
distribution
to
the
public
describing
various
aspects
of
the
screening
and
testing
program;
and
development
of
background
materials
describing
the
basic
science
of
the
endocrine
system,
as
well
as
reasons
why
the
EDSTAC
was
created
by
EPA,
for
use
in
Chapter
One
of
this
Report.

II.
Need
for
Communication
The
EDSTAC
Conceptual
Framework,
which
has
been
described
elsewhere
in
this
document,
is
premised
on
a
phased
or
tiered
approach
to
decision­
making
regarding
screening
and
testing
chemical
substances
or
mixtures
for
endocrine
disruption.
Under
this
approach,
increasingly
more
specific
and
precise
information
produced
in
each
tier
is
used
to
reach
key
decisions,
which
begin
with
judgments
as
to
how
chemicals
and
mixtures
should
be
selected
for
movement
into
the
screening
and
testing
stages
(
i.
e.,
priority
setting).
This
will
lead
eventually
to
judgments
about
whether
a
chemical
or
mixture
may
or
may
not
interact
with
the
endocrine
system
(
i.
e.,
T1S)
or
produce
endocrine­
mediated
adverse
effects
(
i.
e.,
T2T)
for
the
hormone
systems
currently
addressed
by
the
program
(
i.
e.,
estrogen,
androgen,
and
thyroid
systems
(
EAT)).

The
first
steps
of
the
program
utilize
broad
criteria
relating
to
exposure­
and
effects­
related
information
for
the
purposes
of
sorting
and
prioritizing
chemicals
for
T1S.
Criteria
for
moving
a
chemical
into
screening
are
less
restrictive
than
criteria
used
later
in
the
program
to
move
chemicals
from
screening
into
testing
or,
similarly,
from
testing
into
hazard
assessment.
The
purpose
of
using
less
restrictive
criteria
initially
is
to
ensure
that
chemicals
which
may
cause
endocrine
activity
are
not
missed
in
early
steps
of
the
program,
when
information
less
specific
for
evaluating
interaction
with
the
endocrine
system
is
used
to
make
decisions.
Thus,
because
the
information
gathered
becomes
more
specific
as
a
chemical
moves
through
the
EDSTP,
the
criteria
for
progressing
through
the
program
need
to
be
more
restrictive.
Such
increasing
rigor
will
focus
attention
and
resources
on
those
chemicals
and
mixtures
most
likely
to
cause
endocrine­
mediated
adverse
effects.

When
little
or
no
effects
data
on
a
chemical
are
available,
additional
information
to
guide
sorting
and
priority
setting
decision
making
will
come
from
the
results
of
High
Throughput
Pre­
Screening
(
HTPS),
as
described
in
Chapter
Four.
The
T1S
battery
is
intended
to
identify,
through
the
application
of
various
assays,
whether
a
chemical
substance
or
mixture
may
interact
with
the
EAT
components
of
the
endocrine
system
and,
if
so,
to
forward
such
compounds
to
the
testing
phase
of
the
program.
T2T
is
intended
to
determine
whether
a
particular
compound
does
or
does
not
produce
endocrine­
mediated
adverse
effects
and
whether
it
should,
therefore,
be
subjected
to
the
hazard
assessment
phase
of
decision­
making.

Communication
is
most
important
when
decisions
are
made
to
move
chemicals
from
one
step
in
the
process
to
the
next
(
i.
e.,
from
initial
sorting
to
priority
setting,
to
screening,
to
testing).
The
tiered
approach
is
constructed
so
the
Agency
will
have
increased
certainty,
with
each
progressing
tier,
that
a
chemical
does
or
does
not
disrupt
the
endocrine
system
for
the
hormone
systems
currently
addressed
by
the
program.
It
is
important
for
EPA
to
clearly
communicate
the
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
3
limitations
that
must
be
placed
on
the
interpretation
of
information
from
the
EDSTP,
as
well
as
the
meaning
and
implication
of
its
decisions.

One
significant
concern
identified
by
the
Committee
is
that
information
could
be
misused
to
label
chemicals
as
"
endocrine
disruptors"
prior
to
the
existence
of
evidence
to
support
such
a
claim.
Such
potential
misuse
of
information
could
lead
to
unnecessary
and
undue
concern,
along
with
a
failure
to
focus
society's
attention
on
those
substances
that
are
most
likely
to
be
endocrine
disruptors.
Such
a
result
could,
in
the
end,
create
problems
serving
the
interest
of
no
one.
This,
therefore,
necessitates
that
the
public
and
other
interested
stakeholders
be
provided
with
accurate
information
about
the
meaning
of
the
EDSTP
results.
The
recommendations
provided
in
the
remainder
of
this
chapter
seek
to
emphasize
the
importance
of
communication
as
EPA
moves
forward
with
implementation
of
the
EDSTP.

III.
Recommendations
A.
Principles
to
Guide
Implementation
of
a
Communications
and
Outreach
Strategy
EPA
should
develop
and
implement
an
effective
communications
and
outreach
strategy
for
the
EDSTP,
as
this
is
an
element
vital
to
the
program's
success.
EPA
should
follow
a
set
of
principles
regarding
implementation
of
the
communications
and
outreach
strategy,
which
include:

·
Both
the
process
and
results
of
the
EDSTP
should
be
open
and
transparent.
·
The
results
of
the
EDSTP
should
be
interpreted
and
communicated
within
the
context
set
forth
in
the
EDSTAC
final
report.
·
The
limitations
and
uncertainties
of
the
available
data
and
the
results
of
EDSTP
should
be
articulated
clearly
when
the
screening
and
testing
program
is
discussed.
·
As
new
scientific
evidence
emerges,
the
uncertainties
and
limitations
of
the
data
may
also
change.
These
changes
should
be
communicated
clearly.
·
EPA
should
develop
quality
assurance
processes
to
assure
that
any
database
maintained
for
the
public
relative
to
the
EDSTP
is
accurate
and
current.

B.
Basic
Features
of
the
Communications
and
Outreach
Strategy
It
is
anticipated
that
the
EDSTP
will
produce
an
abundance
of
information
shortly
after
its
initiation,
some
of
which
may
be
preliminary
and
difficult
to
interpret.
As
results
of
the
program
are
generated,
it
will
be
imperative
for
EPA
to
make
them
available
to
the
public
in
a
timely
manner
and
to
provide
guidance
on
their
interpretation
(
while
recognizing
there
may
be
legitimate
disagreement
as
to
the
appropriate
interpretation).
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
4
This
program
of
communication
and
outreach
should
consider
the
following
four
issues:

1.
What
should
be
communicated?
2.
To
whom
should
information
be
communicated?
3.
How
should
information
be
communicated?
4.
When
should
information
be
communicated?

The
following
discussion
further
addresses
these
four
issues
in
detail.

1.
What
Should
be
Communicated?

The
EDSTAC
has
focused
on
several
aspects
of
the
program,
described
below,
about
which
EPA
should
be
prepared
to
provide
information.
Where
appropriate,
the
Committee
has
provided
suggested
language
that
could
be
used
to
communicate
such
information
to
those
interested
in
the
issues
and
the
outcomes
of
the
EDSTP.

a)
Description
of
the
Endocrine
Disruptor
Screening
and
Testing
Program
The
EDSTAC
determined
that
one
of
the
central
issues
for
EPA's
communication
and
outreach
efforts
is
the
explanation
of
the
screening
and
testing
program
itself.
Committee
members
recognized
the
importance
of
explicitly
describing
what
information
generated
by
the
EDSTP
means
and
does
not
mean,
so
as
to
avoid
misinterpretation
and
misperception
of
the
information.
This
potential
problem
was
identified
as
a
major
area
of
concern
by
Committee
members.

To
address
this
concern,
an
explanation
of
the
various
components
of
the
EDSTP
is
provided
below
in
less
technical
terms
than
is
found
throughout
the
report.
This,
it
is
hoped,
will
help
minimize
miscommunication
about
the
EDSTP
and/
or
its
results.
Each
phase
of
the
EDSTP,
(
i.
e.,
priority
setting,
screening,
and
testing)
is
briefly
described.
The
Committee
envisions
such
language
filling
a
variety
of
needs
for
EPA
and
others.
For
example,
it
could
be
used
in
the
following
ways:
to
fill
requests
of
the
Agency
for
information
about
the
EDSTP
and,
more
specifically,
what
it
means
when
chemicals
reach
certain
steps
in
the
process;
as
background
information
in
the
development
of
booklets
or
brochures
about
the
screening
and
testing
program;
in
EPA
outreach
mailings;
and
as
information
placed
on
EPA's
Web
site.
The
language
below
could
also
be
used
more
widely,
as
Committee
members
and
others
seek
to
explain
the
EDSTP
to
their
constituencies.
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
5
Steps
of
the
EDSTP
The
following
examples
were
developed
by
the
Committee
to
explain
each
phase
of
the
EDSTP.

PRIORITY
SETTING
PROCESS
During
the
priority
setting
process,
existing
information
is
gathered
and
evaluated
on
new
and
existing
chemical
substances
and
mixtures
to
determine
their
priority
to
be
set
aside,
screened,
tested,
or
forwarded
to
hazard
assessment.
Information
on
exposure
and
human
health
and
ecological
effects,
as
well
as
statutory
requirements
about
chemicals,
will
be
used
by
EPA
to
set
priorities.
The
exposure­
related
information
and
criteria
will
include:
biological
sampling
data;
environmental,
occupational,
consumer
product,
and
food­
related
data;
data
on
environmental
releases;
production
volume;
and
fate
and
transport
data
and
models.
The
effects­
related
information
and
criteria
will
include:
toxicological
laboratory
studies
and
databases;
epidemiological
and
field
studies
and
databases;
predictive
biological
activity
or
effects
models;
and
results
of
high
throughput
pre­
screening.

Since
most
chemicals
lack
adequate
data
on
human
health
and
ecological
effects
for
purposes
of
priority
setting
for
endocrine
screening
and
testing,
the
EDSTAC
recommends
that
chemicals
produced
in
amounts
equal
to
or
greater
than
10,000
pounds
per
year,
as
well
as
pesticides,
be
subjected
to
High
Throughput
Pre­
Screening
(
HTPS)
assays.
These
assays
are
intended
both
to
provide
a
cursory
assessment
of
the
chemicals'
potential
to
interact
with
estrogen,
androgen,
and
thyroid
receptor
systems
and
to
assist
in
the
effort
to
set
priorities
for
Tier
1
Screening
(
T1S).
Using
a
limited
number
of
assays
that
are
appropriate
for
automated
processing
and
that
rely
on
robotics
technology,
HTPS
is
designed
to
generate
results
quickly
and
inexpensively.
HTPS
results,
by
themselves,
will
not
be
sufficient
to
make
a
determination
about
whether
a
chemical
may
interact
with
the
endocrine
system
of
an
intact
animal.
Such
determinations
will
require
additional
screening
and
testing.

In
addition,
a
nominations
program
which
allows
citizens
and
communities
to
nominate
chemicals
for
EPA's
EDSTP
will
constitute
another
criterion
for
consideration
by
EPA
in
the
priority
setting
process.

For
information
on
the
possible
decisions
resulting
from
the
priority
setting
process,
see
the
accompanying
information
on
"
Priority
Setting
Decisions."
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
6
PRIORITY
SETTING
DECISIONS
The
priority
setting
process
will
result
in
one
of
four
possible
decisions:

(
1)
Hold.
No
further
analysis
required
(
at
this
time).
(
2)
Set
priorities
for
Tier
1
Screening
(
T1S).
(
3)
Sufficient
data,
or
voluntary
bypass
of
T1S,
to
go
to
Tier
2
Testing
(
T2T).
(
4)
Sufficient
data
to
go
to
hazard
assessment.

The
first
category
is
for
those
chemical
substances
and
mixtures
that
have
a
low
probability
of
interacting
with
the
endocrine
system
or
exhibiting
endocrine­
mediated
adverse
effects.
The
Committee
identified
one
class
of
chemicals
 
polymers
 
that,
with
some
exceptions,
falls
into
this
category.
A
polymer
is
a
chemical
compound
or
mixture
of
compounds
composed
of
many
small
units
bound
together
to
form
a
larger
compound.
Because
of
their
molecular
size,
most
polymers
are
of
low
concern,
and
therefore
should
be
placed
into
a
"
hold"
status
pending
a
review
of
their
components.
For
information
on
how
these
chemicals
can
be
recalled
into
the
priority
setting
process,
see
the
accompanying
information
describing
the
"
hold
box."

The
second
category
is
for
those
chemicals
with
insufficient
data
to
proceed
to
T2T,
which
therefore
will
need
to
be
prioritized
for
T1S.
A
combination
of
exposure
and
effects
data
will
be
used
to
set
these
priorities.

The
third
category
is
for
those
chemicals
for
which
sufficient
data
exist
to
permit
them
to
go
directly
to
T2T,
or
for
which
the
owner
of
the
chemical
has
decided
to
voluntarily
bypass
T1S
and
go
directly
to
T2T,
according
to
the
specific
recommendations
found
in
Chapter
Four
of
the
EDSTAC
report.
As
with
T1S,
it
is
important
to
note
that
prioritizing
a
chemical
for
T2T
does
not
mean
the
chemical
is
an
endocrine
disruptor.
It
means,
simply,
that
sufficient
data
exist
to
indicate
the
chemical
substance
or
mixture
has
shown
the
potential
to
interact
with
the
specific
parts
of
the
endocrine
system
examined
in
the
EDSTP
and
should
therefore
be
evaluated
in
T2T
in
accordance
with
the
priority
it
receives.

The
fourth
category
is
for
those
chemicals
for
which
existing
data
provide
equivalent
information
to
the
T1S
and/
or
T2T
batteries.
Such
data
demonstrate
that
a
chemical
is
an
endocrine
disruptor
for
the
hormone
systems
addressed
by
the
EDSTP
(
i.
e.,
estrogen,
androgen,
and
thyroid).
These
chemicals
will
proceed
directly
to
hazard
assessment.
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
7
TIER
1
SCREENING
Tier
1
Screening
(
T1S)
is
defined
as
the
application
of
assays
to
determine
whether
a
chemical
substance
or
mixture
may
interact
with
the
endocrine
system
for
the
estrogen,
androgen,
or
thyroid
(
EAT)
hormone
systems.
It
is
intended
to
provide
a
fast,
cost­
efficient,
and
sensitive
means
of
determining
which
chemicals
should
be
subject
to
the
more
comprehensive
and
specific
Tier
2
Testing
(
T2T).
Screening
consists
of
in
vitro
and
in
vivo
assays
designed
to
screen
for
activity
in
EAT
hormone
systems.
However,
it
is
not
designed
to
quantify
such
activity
(
i.
e.,
to
determine
doseresponse
The
screening
process
can
result
in
one
of
two
decisions:

(
1)
No
further
screening
or
testing
required
(
at
this
time).
(
2)
Further
analysis
requiring
T2T.

T1S
is
designed
to
provide
sensitivity
sufficient
to
minimize
the
chance
that
a
chemical
substance
or
mixture
that
may
interact
with
the
endocrine
system
will
pass
through
T1S
undetected
(
i.
e.,
to
minimize
false
negative
results).
This,
however,
is
likely
to
result
in
an
increased
number
of
false
positive
results
(
i.
e.,
chemicals
which
screen
positively
in
Tier
1
screens
without
ultimately
demonstrating
adverse
effects
on
the
endocrine
system).
For
this
reason,
a
positive
result
in
screening
warrants
further
investigation
in
T2T.

Chemicals
judged
to
be
positive
in
T1S
will
proceed
to
T2T.
Chemicals
judged
to
be
negative
in
T1S,
are
considered,
unlikely
to
interact
with
the
EAT
hormone
systems,
because
of
the
emphasis
on
sensitivity.
These
chemicals
will
not
go
on
to
T2T,
but
instead
will
be
placed
in
the
"
hold
box."
For
information
on
how
chemicals
can
be
recalled
into
the
screening
and
testing
process,
see
the
accompanying
information
describing
the
hold
box.
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
8
TIER
2
TESTING
Chemical
substances
or
mixtures
enter
Tier
2
Testing
(
T2T)
if:
(
1)
existing
laboratory,
field,
or
epidemiological
data
suggest
the
chemical
substance
or
mixture
has
shown
the
potential
to
interact
with
the
estrogen,
androgen,
or
thyroid
hormone
systems;
(
2)
Tier
1
Screening
(
T1S)
results
are
positive;
or
(
3)
statutory
or
regulatory
mandates
require
testing.
The
Tier
2
battery
includes
both
mammalian
and
non­
mammalian
tests
designed
to
evaluate
a
variety
of
adverse
reproductive
and
developmental
effects.

The
purposes
of
T2T
are:
to
determine
whether
chemical
substances
or
mixtures
may
produce
changes
in
endocrine
activity
that
will
likely
result
in
adverse
effects;
to
characterize
the
nature
of
the
effects;
and
to
evaluate
dose­
response
relationships.
What
constitutes
an
adverse
effect
may
differ
with
taxonomic
groups
and
is
a
matter
of
scientific
judgment
that
may
evolve
with
new
scientific
information.
The
T2T
process
can
result
in
one
of
two
decisions
relating
to
the
hormone
systems
addressed
by
the
Endocrine
Disruptor
Screening
and
Testing
Program
(
i.
e.,
estrogen,
androgen,
and
thyroid):

(
1)
No
evidence
of
endocrine­
mediated
adverse
effects
for
estrogen,
androgen,
or
thyroid
hormone
systems
(
at
this
time);
and
(
2)
Evidence
of
endocrine­
mediated
adverse
effects
for
estrogen,
androgen,
and/
or
thyroid
hormone
systems
in:
·
mammals;
·
birds;
·
fish;
·
reptiles;
·
amphibians;
and/
or
·
invertebrates.

Positive
T2T
results
may
trigger
additional
testing
and/
or
a
hazard
assessment.

Chemicals
that
test
negative
in
T2T
are
generally
considered
to
possess
low
or
no
potential
to
affect
the
endocrine
system,
within
the
scope
of
endocrine
functions
addressed
by
the
program.
Such
chemicals
can,
however,
be
recalled
into
the
testing
process,
even
if
it
has
previously
received
a
negative
testing
result.
For
information
on
the
criteria
used
to
determine
whether
a
chemical
is
reentered
into
the
testing
process,
see
the
accompanying
"
hold
box"
description.
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
9
"
HOLD
BOX"

At
three
different
points
in
the
EDSTP,
chemical
substances
or
mixtures
are
evaluated
and
may
potentially
be
placed
in
what
is
referred
to
as
the
"
hold
box."
The
first
example
of
this
is
illustrated
in
the
priority
setting
phase,
where
polymers
with
a
number
average
molecular
weight
of
greater
than
1,000
daltons
are
placed
in
a
"
hold"
status,
pending
screening
and
testing
and,
if
necessary,
exposure
assessment
of
their
components.
Those
polymers
that
are
equal
to
or
less
than
a
number
average
molecular
weight
of
1,000
daltons
will
also
be
prioritized
for
and
subjected
to
endocrine
disruptor
screening
and
testing.

The
second
situation
where
chemicals
are
placed
in
a
"
hold
box"
takes
place
in
the
context
of
Tier
1
Screening
(
T1S).
If
results
of
a
screening
battery
are
deemed
negative,
the
chemical
substance
or
mixture
is
placed
into
a
"
hold
box"
and
no
further
activity
occurs
unless
certain
criteria
are
met.
Specifically,
the
possibility
exists
for
a
chemical
substance
or
mixture
to
re­
enter
the
screening
and
testing
program
if:

(
1)
existing
statutes
require
periodic
review
(
e.
g.,
FIFRA
re­
registration);
(
2)
new
statutory
requirements
mandate
review;
(
3)
new
screens
for
endocrine
disruption
are
incorporated
into
the
strategy
and
it
is
determined
that
these
new
screens
may
either
generate
significant
new
information
or
they
invalidate
prior
screens
and
therefore
warrant
the
re­
screening
of
chemical
substances
and
mixtures
that
have
already
been
subjected
to
T1S;
and/
or
(
4)
new
information
on
the
endocrine
disrupting
potential
of
the
chemical
substance
or
mixture
becomes
available
which
warrants
re­
screening.

The
third
situation
where
chemicals
are
placed
in
the
"
hold
box"
takes
place
in
the
context
of
Tier
2
Testing
(
T2T).
If
results
of
the
T2T
battery
are
deemed
negative,
the
chemical
substance
or
mixture
is
placed
in
the
"
hold
box"
and
no
further
testing
is
performed
unless
certain
criteria
are
met.
In
addition
to
(
1)
through
(
4)
above,
a
fifth
possibility
exists
for
re­
entry
into
the
screening
and
testing
program.
Specifically
a
chemical
substance
or
mixture
could
re­
enter
the
screening
and
testing
program
if:

(
5)
there
is
a
change
in
the
use
and
expected
exposure
patterns
upon
which
the
selection
of
tests
were
made.
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
10
A
potential
outcome
of
each
phase
of
the
screening
and
testing
program,
is
the
development
of
lists
of
chemicals.
The
Committee
developed
a
series
of
questions
EPA
should
be
prepared
to
address
when
EDSTP
decisions
result
in
the
creation
of
a
list
of
chemicals
demonstrating
a
common
decision
having
been
made
regarding
a
chemical's
status.
The
Committee
developed
these
questions
as
a
way
to
alert
EPA
to
concerns
that
may
arise
regarding
the
results
of
screening
and
testing.
It
will
be
important
for
the
Agency
to
clearly
communicate
about
the
issues
that
have
been
raised
to
ensure
results
of
the
EDSTP
are
accurately
reflected.
The
questions
include:

·
What
does
this
list
mean?
·
For
what
purpose
will
the
list
be
used?
·
What
are
the
chemicals
on
the
list?
·
How
was
this
list
derived?
·
What
are
the
selection
criteria
for
inclusion
on
the
list?
·
What
are
the
limitations
and
uncertainties
of
knowledge
associated
with
the
list?
·
Who
compiled
the
list?
·
Are
there
other
ways
to
get
a
chemical
on
the
list
or
considered
for
inclusion?
·
How
can
a
chemical
be
removed
from
the
list?

b)
Screening
and
Testing
Results
Regular
EDSTP
status
reports
should
be
produced
and
distributed.
These
documents
should
include:

·
the
status
of
all
chemicals
and
mixtures
within
the
EDSTP;
·
a
list
of
all
chemicals
and
mixtures
whose
status
within
the
EDSTP
has
changed
since
the
last
update;
and
·
important
EDSTP
decisions
and
developments
at
decisive
points
in
the
program,
such
as
calls
for
nominations
of
compounds
to
be
considered
in
priority
setting;
lists
of
chemicals
that
have
been
prioritized
for
T1S;
lists
of
chemicals
that
have
been
identified
for
T2T;
lists
of
chemicals
that
have
produced
endocrine­
mediated
adverse
effects
in
T2T
and
are
now
subject
to
hazard
assessment;
significant
scientific
advances
in
the
field;
the
incorporation
of
new
assays
into
the
EDSTP;
and
expansion
of
the
scope
of
work
(
e.
g.,
looking
at
additional
hormones).

c)
Nominations
Process
As
described
in
Chapter
Four,
Section
IX,
of
this
report,
the
EDSTAC
recommends
EPA
establish
a
process
that
would
allow
stakeholders,
including
members
of
the
general
public,
to
nominate
chemical
substances
or
mixtures
for
endocrine
disruptor
screening
and
testing.
In
general,
the
nominations
process
is
intended
to
focus
on
chemical
substances
or
mixtures
where
exposures
are
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems
rather
than
those
where
exposures
are
more
broadly
experienced
by
the
general
population
at
the
regional
and/
or
national
levels.
The
process
should
provide
a
mechanism
for
prioritizing
chemicals
unlikely
to
be
considered
as
having
a
high
priority
through
the
core
priority
setting
process.
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
11
It
is
important
for
EPA
to
alert
the
public
about
the
opportunity
to
nominate
chemicals,
as
well
as
to
provide
accurate
and
up­
to­
date
information
about
the
status
of
all
chemicals
considered
for
prioritization.
Members
of
the
public
should
be
encouraged
to
provide
comments
during
the
formal
public
comment
period,
which
is
expected
to
take
place
after
EPA
has
publicized
its
proposed
list
of
chemical
substances
or
mixtures
prioritized
for
screening
and
testing.
An
opportunity
to
nominate
chemicals
will
occur
at
the
start
of
each
phase
of
the
EDSTP.

d)
Background
Information
on
the
EDSTAC
Process
EPA
should
communicate
information
to
the
public
about
the
EDSTAC,
including
its
purpose,
goals,
and
process,
as
needed.
The
language
contained
in
Chapter
Two
of
this
report
could
be
used
by
the
Agency
for
this
purpose.

2.
To
Whom
Should
Information
be
Communicated?

a)
Members
of
the
Public
and
Other
Stakeholders
Throughout
the
EDSTAC
process,
an
interest
in
the
issue
of
endocrine
disruption
and
the
development
of
a
screening
and
testing
program
was
evident.
This
was
demonstrated
via
the
public
comment
sessions
held
at
seven
of
the
nine
plenary
meetings,
where
members
of
the
public
representing
 
industry,
environmental
groups,
advocacy
organizations,
farmers
and
farm
workers,
governmental
organizations,
environmental
and
health­
oriented
non­
governmental
organizations
(
NGOs),
trade
unions,
disease­
impacted
groups,
environmental
justice
networks,
students,
industries
that
formulate
products
but
do
not
manufacture
the
component
chemicals
(
i.
e.,
"
downstream"
industries),
and
concerned
citizens,
among
others
 
were
given
the
opportunity
to
present
their
comments
to
the
Committee
regarding
the
deliberations
of
the
EDSTAC
and
its
work
groups.
A
compilation
of
the
statements
made
by
members
of
the
public
at
each
of
the
EDSTAC
meetings
can
be
found
in
Appendix
U.
Furthermore,
each
of
these
stakeholders
was
also
represented
either
in
one
of
the
work
groups
or
on
the
Committee
itself,
further
demonstrating
the
variety
of
interests
contributing
to
this
effort.

It
is
recommended
that
EPA
proactively
communicate
with
groups,
such
as
those
listed
above,
which
have
clearly
demonstrated
an
interest
in
the
issue,
particularly
those
organizations
and
individuals
who
have
requested
to
receive
program
information
directly
from
EPA.
The
database
of
names
and
organizations
already
collected
by
the
EDSTAC
could
be
used
as
a
base
of
contacts
for
proactive
communication
to
stakeholders.
In
fact,
much
of
the
data
entry
has
already
been
done.
Other
stakeholders
to
include
can
be
found
in
the
list
of
organizations
that
received
EPA's
September
1997
mailing,
as
well
as
The
Keystone
Center's
list
of
interested
parties
accumulated
over
the
duration
of
the
EDSTAC
process.

b)
Specific
Audiences
For
some
stakeholders,
EPA
will
find
it
necessary
to
go
beyond
the
generic
EDSTP
status
reports.
A
tailored
set
of
messages
about
the
program
targeted
to
specific
audiences
will
be
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
12
needed.
It
is
clear
that
the
"
public"
consists
of
a
variety
of
people
and
organizations,
each
with
varying
levels
of
knowledge
and
interest
in
endocrine
disruptor­
related
issues.
In
addition,
many
communities
face
other
challenges
such
as
language
barriers
and
differences
in
culture
or
economic
viability.
Such
differences
create
a
need
for
informational
materials
to
be
tailored
to
such
audiences.
In
particular,
EPA
should
consider
this
type
of
communication
with
environmental
justice
organizations,
"
downstream"
industries,
farm
workers,
and
patient­
specific
groups.
To
find
out
more
about
communicating
with
such
constituencies,
the
Committee
recommends
EPA
conduct
a
follow­
up
survey,
building
on
the
information
gathered
from
the
September
1997
survey
described
in
Section
I
of
this
chapter.

3.
How
Should
Information
be
Communicated?

As
EPA
carries
the
important
new
responsibility
of
screening
and
testing
chemical
substances
and
mixtures
for
endocrine
disruption,
it
will
be
necessary
to
develop
a
capacity
to
quickly
respond
to
requests
for
information,
both
about
specific
chemicals
and
about
the
EDSTP
in
general.

a)
Electronic
Communication
The
EDSTAC
recommends
that
EPA
create
a
tracking
database
with
the
ability
to
handle
inquiries
about
the
status
of
specific
chemicals
and
classes
of
chemicals,
as
well
as
summaries
for
defined
sets
of
chemicals
(
e.
g.,
organophosphates).
The
goal
for
the
creation
of
such
a
database
is
that
any
member
of
the
public
should
be
able
to
query
and
quickly
determine
the
status
of
a
chemical
or
mixture
in
the
EDSTP.
Inquiries
might
come
from
within
the
Agency,
from
the
public,
or
from
industry.
As
a
result,
it
is
important
that
the
database
be
organized
so
people
can
submit
inquiries
in
many
different
ways
and
with
varying
levels
of
expertise.
For
example,
the
inquiry
might
begin
with
a
chemical
name
or
Chemical
Abstracts
Service
(
CAS)
registry
number,
a
chemical
structure,
or
a
stage
in
the
EDSTP
process.

The
tracking
database
should
be
compatible
with,
and
fully
integrated
into,
the
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD),
described
in
Chapter
Four.
As
proposed
in
Chapter
Four,
Section
X,
G,
a
multi­
stakeholder
group
should
be
created
by
the
Agency
to
continue
development
of
the
EDPSD
as
a
tool
for
priority
setting
purposes.
In
addition,
the
Committee
recommends
that
the
same
group
assist
in
development
of
the
tracking
database,
in
order
to
promote
consistency
and
ensure
it
meets
the
needs
of
the
diverse
groups
likely
to
use
the
database
in
the
future.
The
database
should
also
be
compatible
with,
and
integrated
into,
those
being
developed
elsewhere
in
the
Agency
(
e.
g.,
for
carcinogens
or
reproductive
toxicants).
The
EDPSD
should
not
exist
in
isolation.

In
creating
this
database,
several
characteristics
are
desirable
if
it
is
to
address
the
needs
of
a
wide
range
of
potential
users:

·
The
database
should
be
useful
over
the
Internet,
and
a
Web
site
should
be
established
for
this
purpose.
Since
it
will
be
integrated
with
other
databases
at
the
Agency,
the
Web
site
should
be
reached
through
links
that
begin
at
several
locations
(
e.
g.,
the
main
Agency
site,
a
page
dedicated
to
inquiries
about
toxic
substances
in
general,
a
page
dedicated
to
searching
for
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
13
information
on
endocrine
disruptors,
and
pages
dedicated
to
searching
for
other
effects
such
as
developmental,
reproductive,
or
carcinogenic).

·
The
database
should
include
the
ability
to
search
by
specific
chemical
names,
by
classes
of
chemicals,
and,
where
appropriate,
by
chemical
structure.
Searching
should
be
by
chemical,
not
by
product
name.

·
The
database
should
include
the
ability
to
search
for
the
place
of
each
chemical
in
the
EDSTP
process
and
subsequent
regulatory
decisions.
This
includes
the
ability
to
obtain
a
listing
of
all
chemicals
that
presently
are:
(
i)
in
HTPS;
(
ii)
undergoing
review
for
priority
setting;
(
iii)
in
T1S;
(
iv)
in
T2T;
(
v)
undergoing
hazard
assessment;
or
(
vi)
have
had
a
regulatory
decision
made.

·
Whenever
the
location
of
a
chemical
in
the
system
is
provided
to
an
inquirer,
or
a
listing
of
chemicals
at
particular
points
in
the
EDSTP
is
provided,
it
is
essential
that
this
information
have
appended
to
it
a
brief
description
of
what
it
means
for
a
chemical
to
be
at
that
location.
This
description
should
be
consistent
with
guidance
provided
elsewhere
in
this
chapter.

·
The
database
should
include
the
ability
to
obtain
the
decision
results
of
each
step
a
chemical
has
completed
to
date.
The
designations
for
these
results
should
be
consistent
with
those
detailed
elsewhere
in
the
EDSTAC
Report.
This
should
include
information
describing:
(
i)
the
result
of
a
chemical's
priority
setting;
(
ii)
the
results
of
T1S;
(
iii)
the
results
of
T2T.

·
The
database
should
not
attempt
to
summarize
the
rationale
for
the
Agency's
decisions
discussed
in
the
previous
item
and
based
on
specific
positives/
negatives
for
particular
screens/
tests.
Instead,
the
database
should
direct
the
inquirer
to
the
appropriate
documentation,
explaining
how
that
documentation
can
be
viewed
and/
or
obtained.

·
To
facilitate
the
utility
of
the
database
as
a
research
tool,
it
would
be
useful
if
the
database
contained
information
on
whether
the
chemical
was
positive
or
negative
for
each
individual
screening
assay
and/
or
test,
including
the
results
of
HTPS.
In
stating
whether
the
result
of
a
screen
or
test
was
positive
or
negative,
it
is
important
that
the
database
also
provide
information
about
the
criteria
by
which
a
result
is
considered
to
be
a
pass
or
fail
for
that
assay.

In
considering
the
range
of
questions
users
might
have
in
directing
inquiries
to
the
database,
several
kinds
of
information
should
be
available
through
the
database.
These
include:

·
chemical
name
and
CAS
registry
number;
·
common
synonyms
(
but
not
product
names);
·
chemical
structure;
·
information
on
the
stage
in
which
a
chemical
currently
is
found
(
priority
setting;
T1S;
T2T;
HTPS;
hazard
assessment;
"
hold
box");
·
one­
sentence
descriptions
of
the
purpose
and
possible
outcomes
of
each
of
the
stages;
this
description
would
be
provided
whenever
the
inquiry
indicates
a
particular
stage
has
been
reached;
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
14
·
summary
of
the
Agency
decision
on
a
chemical
at
each
of
the
stages
through
which
it
has
passed
(
priority
setting;
T1S;
T2T;
HTPS;
hazard
assessment;
"
hold
box");
·
summary
of
the
result
obtained
from
each
HTPS
screen;
·
one
sentence
descriptions
of
reasonable
interpretation(
s)
of
a
chemical's
having
positive
or
negative
results
in
a
particular
HTPS
screen;
·
summary
of
the
result
obtained
from
each
T1S
assay;
·
one
sentence
descriptions
of
reasonable
interpretation(
s)
of
a
chemical's
having
positive
or
negative
results
for
a
particular
T1S
assay;
·
summary
of
the
result
obtained
from
each
T2T
test;
·
one
sentence
descriptions
of
reasonable
interpretation(
s)
of
a
chemical's
having
positive
or
negative
results
of
a
particular
T2T
test.

The
availability
of
a
tracking
system
will
be
a
particularly
important
tool
as
it
relates
to
the
nominations
program.
Members
of
the
public
should
be
able
to
rely
on
this
database
to
provide
timely
and
accurate
information
about
chemicals
that
have
been
prioritized
for
T1S,
either
through
the
nominations
process
or
other
means.
The
availability
of
such
information
will
be
imperative
as
affected
communities,
in
particular,
review
the
list
to
determine
if
chemicals
of
concern
to
them
have
been
selected
for
T1S.

b)
Telephone,
Fax,
Mail,
and
Other
Communication
For
those
who
do
not
have
access
to
the
Internet,
the
contents
of
the
EPA
Web
site
should
be
available
by
other
media
through
EPA
staff
support.
A
centralized,
automated
telephone
system
should
be
developed.
In
addition,
regular
EDSTP
status
reports
and
important
program
developments
should
be
posted
in:
the
Federal
Register;
pesticide
registration
notices;
press
releases;
and
Web
announcements.
In
addition,
where
appropriate,
EPA
should
provide
information
about
the
EDSTP
through
a
variety
of
media,
such
as
general
fact
sheets,
questionand
answer
documents,
information
booklets,
EPA
newsletters,
brochures,
pamphlets,
trade
journals,
videotapes,
slide
presentations,
and
other
publications
as
appropriate.

EPA
should
initiate
contact
with
stakeholders,
providing
them
with
the
address
of
the
Web
site
and
the
number
of
the
centralized
telephone
site.
The
Agency
should
maintain
proactive
communication
with
these
groups
until
the
groups
indicate
they
plan
to
receive
information
electronically
or
are
no
longer
interested.

To
be
successful,
EPA
should
invest
resources
into
how
to
effectively
manage
professional
communication
efforts.

4.
When
Should
Information
be
Communicated?

Communication
should
occur
regularly
and
frequently
given
the
rapid
developments
in
the
science
of
endocrine
disruption
and
the
increasing
public
interest
in
the
issue.
There
are
two
kinds
of
information
that
EPA
should
be
prepared
to
communicate
at
specific
points
in
time.
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
15
a)
Public
Updates
About
the
EDSTP
The
COWG
discussed
the
means
and
mechanisms
available
for
disseminating
information
to
the
public
regarding
the
progress
and
results
of
the
EDSTP.
One
option
EPA
has
used
in
other
programs
is
a
regular
bulletin
or
newsletter
that
identifies
specific
actions,
events,
and
program
directions
taken
by
EPA
staff.
The
Committee
recommends
that
EPA
explore
this
option
for
disseminating
information
to
members
of
the
public
for
whom
e­
mail
is
either
not
available
or
is
not
an
effective
means
of
receiving
such
information.

The
Committee
recognizes
that
this
type
of
informing
effort
needs
to
be
goal­
oriented
and
have
some
specific
parameters
in
order
to
provide
the
best
use
of
Agency
funds.
Therefore,
the
following
operating
conditions
should
be
taken
into
account
in
creating
an
updating
bulletin:

·
The
output
should
be
in
the
form
of
a
newsletter
or
bulletin
for
public
review
with
the
purpose
of
informing
the
public
of
the
program
and
its
progress.
·
The
publication
should
be
of
a
limited
length
and
in
a
desk
top
format.
·
Publication
should
start
shortly
after
EPA
initiated
the
program
in
late
1998
or
mid­
1999,
and
continue
for
a
defined
period
of
time.
·
The
publication
should
be
produced
for
the
duration
of
the
screening
phase
and
into
the
testing
phase,
with
some
predetermined
ending
time.
·
The
content
of
the
publication
could
be
chemical­
specific,
but
more
likely
would
direct
interested
readers
to
sources
where
more
detailed
information
could
be
found,
rather
than
list
volumes
of
scientific
technical
information.
·
The
publication
should
draw
heavily
on
the
Web
site
information,
if
not
duplicate
much
of
what
is
on
the
Web
site.

The
survey
conducted
by
EPA
with
advice
from
the
COWG
indicated
that
there
are
members
of
the
public,
including
individual
citizens,
organizations,
and
small
businesses,
for
whom
electronic
access
is
not
an
effective
mode
of
communicating,
or
is
not
available.
For
these
constituents,
EPA
should
provide
information
in
an
accessible
and
easy
to
understand
form.

b)
Whenever
Important
EDSTP
Developments
Warrant
Communication
Important
developments
in
the
EDSTP
of
a
definitive,
non­
preliminary
nature
should
be
communicated
as
soon
as
that
information
is
available,
rather
than
waiting
for
the
generation
of
regular
public
updates.
Examples
might
include:
calls
for
nominations
of
compounds
at
the
outset
of
each
phase
of
the
EDSTP
that
are
to
be
considered
in
priority
setting;
lists
of
chemicals
that
have
been
prioritized
for
T1S;
lists
of
chemicals
that
have
identified
for
T2T;
lists
of
chemicals
that
have
been
identified
as
exhibiting
endocrine­
mediated
adverse
effects
in
T2T
and
are
subject
to
hazard
assessment;
significant
scientific
advances
in
the
field;
the
incorporation
of
new
assays
into
the
EDSTP;
expansion
of
the
scope
of
the
EDSTP
(
e.
g.,
looking
at
additional
hormone
systems);
and
other
key
decisions
or
developments
within
the
EDSTP.
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
16
C.
Adequacy
of
Resources
Devoted
to
Communication
and
Outreach
Management
of
the
EDSTP
will
be
a
significant
new
responsibility
for
EPA,
and
providing
public
information
on
the
program
will
be
essential
for
the
full
cooperation
of
affected
and
interested
parties.
EPA
should
allocate
sufficient
resources
with
high­
level
responsibility
to
manage
its
communications
and
outreach
strategy.

It
is
important
that
all
information
be
available
through
a
small
number
of
centralized
sites.
It
is
vital
that
the
public
and
other
interested
parties
be
able
to
obtain
information
through
such
a
centralized
site
rather
than
having
to
track
the
material
to
a
specific
office
in
the
Agency.

The
Committee
identified
the
following
tasks
that
must
be
provided
resources
on
a
continuing
basis:

·
creation
and
maintenance
of
a
centralized
tracking
system
in
the
form
of
a
database,
which
may
be
queried
for
the
status
of
particular
chemicals
and
for
summaries
of
status
across
classes
of
compounds;
·
creation
and
maintenance
of
a
component
of
a
Web
site
with
an
appropriate
graphical
user
interface
allowing
individuals
to
make
these
inquiries;
·
creation
and
maintenance
of
a
component
of
the
same
Web
site
allowing
individuals
to
obtain
background
documents
and
regular
EDSTP
status
reports;
·
creation
and
maintenance
of
a
centralized,
automated
telephone
system
allowing
individuals
to
access
the
tracking
system
database
and
to
order
specific
program
documents;
and
·
assignment
of
staff
to
monitor
the
above
four
items,
and
to
disseminate
materials
that
are
requested
through
the
automated
telephone
system
or
other
ways.
In
addition,
this
staff
resource
should
proactively
send
regular
EDSTP
status
reports,
as
well
as
important
program
updates,
to
stakeholders
who
have
requested
such.

Management
of
the
EDSTP
should
continue
to
be
the
responsibility
of
the
EPA
Assistant
Administrator
of
the
Office
of
Prevention,
Pesticides,
and
Toxic
Substances.
Concurrently,
coordination
across
the
entire
Agency
should
enable
all
EPA
staff
to
locate
and
supply
requested
information.

IV.
Generalized
Schedule
for
Implementation
During
the
EDSTAC
process,
the
importance
of
communicating
EPA's
schedule
for
implementing
the
EDSTP
became
evident.
To
help
inform
the
public
of
the
estimated
schedule
for
implementation,
including
opportunities
to
provide
public
comment
on
the
EDSTP,
a
generalized
schedule
was
developed.
This
schedule,
while
not
precise
in
dates,
provides
the
reader
with
a
sense
of
the
direction
that
EPA
will
be
taking
as
they
seek
to
fully
implement
the
EDSTP.
It
should
also
be
noted
that
the
schedule
was
developed
assuming
adequate
resources
to
carry
out
each
activity
as
scheduled.
Lower
levels
of
funding
will
cause
a
stretching
of
various
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
17
activities
and
the
other
activities
that
depend
on
their
completion.
Specific
resources
have
not
yet
been
approved
for
EDSTP
implementation.

Several
fundamental
requirements
that
must
be
met
as
set
forth
in
the
FQPA
will
serve
as
the
basis
for
EPA's
implementation
plan.
These
requirements
include:

·
Using
validated
assays,
EPA
must
propose
a
screening
program
by
August
1998.
·
EPA
must
implement
the
proposed
screening
program
by
August
1999.
·
EPA
must
report
to
Congress,
the
progress
of
the
screening
and
testing
program
to
date
by
August
2000.

Other
key
elements
of
the
EDSTP
will
be
implemented
according
to
the
attached
generalized
schedule
(
Figure
6.1).
The
schedule
describes
the
key
processes
and
their
relationship
to
each
other.
The
public
will
have
an
opportunity
to
comment
on
the
report
once
it
is
made
final
and
released
in
the
FR
Notice.

The
key
elements
of
the
schedule
include:

·
EDSTAC
and
SAB/
SAP
peer
review
processes;
·
High
Throughput
Pre­
Screening
feasibility
demonstration
and
utilization;
·
Final
development,
utilization,
and
maintenance
of
the
EDPSD
and
completion
of
the
priority
setting
process;
·
Standardization,
validation,
and
utilization
of
the
T1S
battery
and
newly
developed
Tier
2
Tests;
and
·
EPA
regulatory
and
administrative
processes
(
e.
g.,
FQPA
Orders,
TSCA
consent
agreements
and/
or
rulemaking)
related
to
the
EDSTP.
Final
EDSTAC
Report
Chapter
Six
August
1998
6
­
18
Figure
6.1
Generalized
Schedule
for
Implementation
of
the
Endocrine
Disruptor
Screening
&
Testing
Program
(
EDSTP)
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
19
V.
Compilation
of
Chapter
Six
Recommendations
A.
Need
for
Communication
As
described
in
Chapter
Two,
Section
II,
the
Communications
and
Outreach
Work
Group
(
COWG),
and
then
later
the
full
EDSTAC,
recognized
the
importance
of
communication
about
the
EDSTP
to,
among
other
things,
prevent
misuse
of
information.
Because
the
EDSTP
applies
a
tiered
approach,
results
become
increasingly
definitive
as
chemicals
progress
through
each
step
of
the
screening
and
testing
program.
This
type
of
system
leaves
room
for
interpretation
of
results,
particularly
in
the
early
stages
of
the
EDSTP
(
i.
e.,
during
priority
setting
or
screening),
that
may
or
may
not
be
accurate.
Therefore,
the
Committee
emphasizes
the
need
for
clear
and
accurate
communication
to
interested
stakeholders
throughout
the
development
and
implementation
of
the
EDSTP.
In
particular,
it
is
important
that
EPA
clearly
communicate
about
the
limitations
that
must
be
placed
on
the
interpretation
of
information
and
results
from
the
EDSTP,
as
well
as
the
meaning
and
implications
of
its
decisions.
The
recommendations
identified
in
Chapter
Six
seek
to
emphasize
this
point,
while
providing
guidance
to
EPA
as
it
further
develops
its
communications
strategy
for
the
EDSTP.

B.
Principles
to
Guide
Implementation
of
a
Communications
Strategy
1.
The
EDSTAC
recommends
that
EPA
develop
and
implement
an
effective
communications
and
outreach
strategy
for
the
EDSTP
based
on
the
following
set
of
principles
intended
to
help
ensure
accurate
and
open
communication
to
stakeholders:

·
Both
the
process
and
results
of
the
EDSTP
should
be
open
and
transparent.
·
The
results
of
the
EDSTP
should
be
interpreted
and
communicated
within
the
context
set
forth
in
the
final
EDSTAC
Report.
·
The
limitations
and
uncertainties
of
the
available
data
and
the
results
of
EDSTP
should
be
articulated
clearly
when
the
screening
and
testing
program
is
discussed.
·
As
new
scientific
evidence
emerges,
the
uncertainties
and
limitations
of
the
data
may
also
change.
These
changes
should
be
communicated
clearly.
·
EPA
should
develop
quality
assurance
processes
to
assure
that
any
database
maintained
for
the
public
relative
to
the
EDSTP
is
accurate
and
current.

C.
Basic
Features
of
a
Communications
and
Outreach
Strategy
2.
The
Committee
recommends
that
EPA
base
their
communications
and
outreach
strategy
on
the
following
four
questions:

·
What
should
be
communicated?
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
20
·
To
whom
should
information
be
communicated?
·
How
should
information
be
communicated?
·
When
should
information
be
communicated?

Details
of
the
recommendations
for
each
of
the
four
questions
are
located
in
Chapter
Six,
Section
III,
B.
The
basic
recommendations,
however,
follow.

3.
Under
"
What
should
be
communicated?,"
the
Committee
recommends
that
EPA
be
prepared
to
provide
information
to
interested
stakeholders
on
the
EDSTP
itself,
on
screening
and
testing
results,
the
nominations
process,
and
background
information
about
the
EDSTAC
process.
Suggested
language
explaining
the
various
components
of
the
EDSTP
in
less
technical
terms
than
is
found
throughout
the
report,
is
included
in
the
chapter.

4.
Under
"
To
whom
should
information
be
communicated?,"
the
Committee
recommends
that
EPA
actively
communicate
with
members
of
the
public
and
other
stakeholders,
such
as
those
who
have
demonstrated
interest
in
the
process
through
their
attendance
of
the
public
EDSTAC
meetings
and
public
comment
periods.

5.
The
Committee
recognizes
the
need
for,
and
recommends
EPA
develop,
tailored
information
to
be
relayed
through
a
variety
of
mechanisms.
This
would
help
to
ensure
that
specific
audiences
 
such
as
environmental
justice
organizations,
"
downstream"
industries,
farm
workers,
and
patient
groups
 
who
may
not
have
the
ability
to
access
information
via
traditional
means
and
who
have
varying
levels
of
knowledge
and
interest
in
endocrine
disruptor­
related
issues,
have
the
opportunity
to
learn
about
the
EDSTP
and
its
results.

6.
The
Committee
recommends
that
EPA
conduct
a
follow­
up
to
their
September
1997
outreach
questionnaire
in
order
to
find
out
more
information
about
how
best
to
communicate
with
certain
groups,
such
as
those
listed
above
in
recommendation
number
five.

7.
Under
"
How
should
information
be
communicated?,"
the
Committee
recommends
that
EPA
develop
a
tracking
system
as
part
of
the
priority
setting
database
described
in
Chapter
Four.
They
recommend
that,
if
possible,
such
a
database
be
incorporated
into
existing
EPA
systems
to
promote
efficiency
and
cost­
effectiveness.
Several
characteristics
of
a
desirable
database
intended
to
address
the
needs
of
a
wide
range
of
potential
users
have
been
included.
The
EDSTAC
believes
it
is
important
for
members
of
the
public
to
have
access
to
information
about
the
screening
and
testing
program
as
it
progresses,
including
the
ability
to
query
and
quickly
determine
the
status
of
a
chemical
or
mixture
in
the
EDSTP,
as
well
as
to
access
and
download
relevant
EDSTP
documents.

8.
For
those
without
Internet
access,
information
should
be
available
through
a
variety
of
sources,
including
telephone,
fax,
mail,
Federal
Register
notices,
and
other
forms
of
communication,
as
necessary.

9.
Under
"
When
should
information
be
communicated?,"
the
Committee
recommends
that
EPA
develop
a
newsletter
or
bulletin,
as
has
been
done
in
other
EPA
programs,
that
would
be
made
EDSTAC
Final
Report
Chapter
Six
August
1998
6
­
21
available
on
a
regular
basis.
The
report
should
be
of
a
limited
length
and
should
be
available
for
a
limited
duration.

10.
The
Committee
also
recommends
that
information
be
communicated
when
warranted
by
important
EDSTP
developments,
such
as
a
call
for
nominations,
when
lists
of
chemicals
have
been
prioritized
for
T1S,
identified
for
T2T,
or
identified
as
being
subjected
to
hazard
assessment
after
exhibiting
endocrine­
mediated
adverse
effects
in
T2T,
as
well
as
regarding
other
key
decisions
relating
directly
to
the
program.

11.
As
described
in
Chapter
Six,
Section
III,
C,
the
Committee
strongly
recommends
that
EPA
commit
adequate
resources
to
the
communication
aspects
of
this
program.
Several
tasks
requiring
such
support
are
identified
in
the
report,
such
as
the
creation
and
maintenance
of
a
tracking
database,
maintenance
of
a
Web
site
with
an
appropriate
graphical
user
interface,
creation
and
maintenance
of
a
centralized,
automated
telephone
system,
and
assignment
of
staff
to
monitor
such
items.

VI.
Literature
Cited
Banquet,
C.
and
K.
Ringen,
"
Cancer
Control
in
Blacks;
Epidemiology
and
NCI
Program
Plans,"
Progress
in
Clinical
and
Biological
Research,
216,
1986,
pp.
215­
27.
Chilton,
J.
A.,
(
ed.),
"
The
Cancer
Information
Service
in
Jones,
LA,"
Minorities
and
Cancer,
New
York:
Springer­
Verlag,
1989,
pp.
59­
65.
Heckler,
M.,
"
Report
of
the
Secretary's
Task
Force
on
Black
&
Minority
Health,"
Vol.
I:
Executive
Summary
­
U.
S.
Department
of
Health
and
Human
Services,
1985.
National
Research
Council,
"
Improving
Risk
Communication,"
National
Academy
Press,
1989,
332
pp.
National
Research
Council,
"
Risk
Assessment
in
the
Federal
Government,"
National
Academy
Press,
1983,
191
pp.
National
Research
Council,
"
Science
and
Judgment
in
Risk
Assessment,"
National
Academy
Press,
1994,
651
pp.
National
Research
Council,
"
Understanding
Risk,"
National
Academy
Press,
1996,
249
pp.
EDSTAC
Final
Report
Chapter
Seven
August
1998
Chapter
Seven
Compilation
of
EDSTAC
Recommendations
EDSTAC
Final
Report
Chapter
Seven
August
1998
Table
of
Contents
I.
Chapter
Overview
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
II.
Recommendations:
Chapter
Three
-
Conceptual
Framework
and
Principles
.
.
.
.
.
.
.
.
1
III.
Recommendations:
Chapter
Four
-
Priority
Setting
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
5
A.
Summary
and
Scope
of
Effort
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
5
B.
The
Universe
of
Chemicals
and
Initial
Sorting
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
5
C.
Polymers
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6
D.
Priority
Setting
Information
Categories
and
Criteria
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
7
E.
High
Throughput
Pre­
Screening
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
7
F.
Mixtures
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
8
G.
Naturally
Occurring
Non­
Steroidal
Estrogens
(
NONEs)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
9
H.
Nominations
.
.
.
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.
10
I.
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD)
.
.
.
.
.
.
.
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.
11
J.
Recommended
Approach
to
Priority
Setting
.
.
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.
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.
12
IV.
Recommendations:
Chapter
Five
-
Screening
and
Testing
.
.
.
.
.
.
.
.
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.
14
A.
Tier
1
Screening
.
.
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.
.
14
B.
Principles
for
Evaluating
Tier
1
and
Tier
2
Results
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
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.
.
.
.
16
C.
Tier
2
Testing
.
.
.
.
.
.
.
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.
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.
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.
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.
.
16
D.
Validation
of
the
Screening
and
Testing
Batteries
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
17
V.
Recommendations:
Chapter
Six
-
Communications
and
Outreach
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
18
A.
Need
for
Communication
.
.
.
.
.
.
.
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.
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.
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.
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.
18
B.
Principles
to
Guide
Implementation
of
a
Communications
Strategy
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
18
C.
Basic
Features
of
a
Communications
and
Outreach
Strategy
.
.
.
.
.
.
.
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.
.
18
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
1
7
­
I.
Chapter
Overview
The
following
chapter
provides
a
summary
of
the
recommendations
agreed
to,
in
Chapters
Three,
Four,
Five,
and
Six,
by
the
EDSTAC
regarding
establishment
of
an
endocrine
disruptor
screening
and
testing
program.
For
more
information
regarding
these
recommendations,
please
see
the
respective
chapters
within
this
report.

II.
Recommendations:
Chapter
Three
-
Conceptual
Framework
and
Principles
1.
The
EDSTAC
developed
a
tiered
Conceptual
Framework
that
formed
the
basis
for
its
screening
and
testing
strategy
and
all
subsequent
recommendations.
The
tiered
framework
consists
of
the
following
three
major
activities:

·
Priority
setting
includes
the
sorting
and
prioritization
of
chemical
substances
and
mixtures
based
on
existing
information.
The
existing
information
would
be
used
to
sort
chemicals
into
four
categories.
An
evaluation
and
analysis
of
this
information
will
lead
to
sorting
chemicals
into
one
of
four
categories:

·
Polymers,
which
are
placed
into
a
"
hold"
status
(
with
some
exceptions)
pending
a
review
of
their
monomers
and
oligomers.
·
Chemicals
for
which
there
is
insufficient
data
regarding
endocrine
disruption
and
will
therefore
need
to
be
prioritized
for
Tier
1
Screening.
·
Chemicals
for
which
sufficient
data
exists
to
proceed
to
Tier
2
Testing.
·
Chemicals
for
which
sufficient
data
exists
to
go
to
hazard
assessment.

·
Tier
1
Screening
(
T1S)
to
detect
chemical
substances
and
mixtures
capable
of
acting
on
endocrine
systems.

·
Tier
2
Testing
(
T2T)
to
determine,
characterize,
and
quantify
the
nature
of
the
endocrine
disrupting
properties
of
the
chemical
substances
and
mixtures
identified
by
prior
information
and/
or
T1S.

2.
The
EDSTAC
recommended
the
adoption
of
several
principles
to
guide
the
use
of
the
Conceptual
Framework.

·
A
chemical
may
bypass
one
or
more
tiers
when
warranted
by
appropriate
information
(
e.
g.,
sufficient
prioritization
data
on
endocrine
disrupting
properties
to
initiate
T2T
or
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
2
7
­
hazard
assessment).
·
If
information
is
inadequate
to
determine
if
a
chemical
should
move
to
the
next
tier,
an
active
process
should
be
developed
for
generating
the
needed
information
to
make
such
a
decision.
·
Criteria
and
default
assumptions
for
deciding
whether
chemicals
move
from
one
tier
to
the
next
should
be
developed
in
advance
of
initiating
screening
and
testing.

3.
Within
the
context
of
the
Conceptual
Framework,
the
EDSTAC
recommends
that
the
overall
scope
of
the
screening
and
testing
strategy
should:

·
be
relevant
to
both
human
health
and
ecological
effects;
·
initially
emphasize
identifying
and
characterizing
effects
that
enhance,
mimic,
or
inhibit
estrogenic,
androgenic,
and
thyroid
hormone­
related
processes;
·
consider
tests
that
detect
multiple
hormone
interactions,
address
endpoints
in
multiple
species,
and
predict
long­
term
or
delayed
effects;
·
be
periodically
revisited
to
permit
inclusion
of
additional
hormone­
mediated
effects
or
new
screens
and
tests
as
they
become
available;
·
be
capable
of
evaluating
the
endocrine
disrupting
properties
of
chemical
substances
and
common
mixtures;
and
·
allow
determination
of
possible
additive,
synergistic,
or
antagonistic
effects
caused
by
interactions
among
the
components
of
mixtures.

4.
The
EDSTAC
recommends
nine
broad
principles
to
guide
the
implementation
of
the
endocrine
disruptor
screening
and
testing
program.
The
screening
and
testing
program
should:

·
require
the
minimal
number
of
screens
and
tests
necessary
to
make
sound
decisions,
thereby
reducing
the
time
needed
to
make
these
decisions;
·
examine
existing
screens
and
tests
for
their
potential
to
predict,
detect,
and/
or
characterize
endocrine
disruptors,
ensuring
that
any
modification
to
existing
screens
and
tests
does
not
compromise
their
ability
to
predict
other
toxicity
endpoints;
·
systematically
examine
existing
screening
and
testing
data
not
only
for
adverse
endpoints
in
high
dose
groups,
but
also
for
physiological
changes
in
low
dose
groups;
·
not
detract
from
current
and
new
efforts
to
assess
the
toxicity
of
compounds
and
mixtures
through
mechanisms
other
than
endocrine
disruption;
·
provide
data
that
can
be
used
for
a
broad
range
of
management
and
regulatory
programs
in
a
form
that
supports
international
harmonization
of
their
use;
·
include
periodic
review
of
new
scientific
information;
·
use
a
performance­
based
approach
to
the
selection
of
screens,
tests,
and
species,
including
the
use
of
more
appropriate
screens,
tests,
and
species
as
they
are
developed
and
validated;
·
be
dynamic
in
order
to
stay
current
with
the
rapidly
evolving
science
related
to
the
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
3
7
­
endocrine
system;
and
·
be
conducted
at
the
minimum
cost
necessary
to
make
the
decisions
within
the
EDSTAC
Conceptual
Framework.

5.
The
EDSTAC
also
recommended
six
guiding
principles
specific
to
the
screens
and
tests
themselves.

·
To
facilitate
making
decisions
within
the
EDSTAC
Conceptual
Framework,
all
screens
and
tests
should
have
well­
defined
endpoints.
·
The
use
of
animals
should
be
reduced
to
the
minimum
level
needed
to
obtain
scientifically
valid
results
and
interpretations.
·
The
results
of
screens
and
tests
should
support
further
research
on
effects
of
endocrine
disruptors
on
populations,
communities,
and
ecosystems.
·
In
interpreting
screening
and
testing
results,
a
"
weight­
of­
evidence"
approach
should
be
used,
but
should
be
consistent
with
a
principle
of
prudence
in
protecting
human
health
and
the
environment.
In
the
case
of
T1S,
this
means
that
the
strategy
will
err
on
the
side
of
false
positive
identifications
rather
than
false
negatives.
·
Screening
and
testing
results
should
be
reported
in
a
format
that
facilitates
database
development
and
analysis
by
a
broad
array
of
scientific,
regulatory,
and
management
organizations.
·
Decision
criteria,
such
as
those
for
determining
statistical
significance
(
e.
g.,
necessary
confidence
intervals)
and
biological
plausibility,
should
be
clearly
defined.

6.
The
EDSTAC
recommends
that
T1S
provide
the
minimum,
yet
valid
and
reliable,
data
to
detect
interactions
with
the
endocrine
system.
In
contrast
to
Tier
2
tests,
T1S
assays
should:

·
be
inexpensive,
quick,
and
easy
to
perform;
·
be
validated
and
standardized
as
soon
as
possible,
defining
characteristics
such
as
sensitivity
and
specificity
against
a
"
gold
standard,"
once
it
is
identified;
·
be
more
"
sensitive"
than
they
are
"
specific,"
meaning
that
they
should
have
as
their
primary
objective
the
minimization
of
false
negative
or
(
Type
II)
errors,
while
permitting
an
as­
of­
yet
undetermined,
but
acceptable,
level
of
false
positive
or
(
Type
I)
errors;
·
capture
multiple
endpoints
and
reflect
as
many
modes
of
endocrine
action
as
possible;
·
be
broadly
predictive
across
species,
gender,
and
age;
and
·
yield
data
capable
of
being
interpreted
as
either
positive
or
negative
for
the
purpose
of
determining
whether
and
how
to
conduct
T2T.

7.
The
EDSTAC
recommends
that
T1S
be
used
to
make
initial
judgments
about
areas
of
concern
in
order
to
direct
the
focus
of
T2T.
The
interpretation
of
T1S
results
should
be
consistent
with
best
scientific
judgment,
formed
on
the
basis
of
considerations
such
as
"
weight­
of­
evidence,"
consistency
of
the
data
set,
and
methodological
strengths
and
limitations.
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
4
7
­
8.
The
EDSTAC
recommends
that
T2T
be
based
upon
T1S
results
and
other
relevant
information.
An
underlying
principle
of
T2T
is
that
it
should
provide
information
useful
for
human
and
ecological
hazard
assessment.
The
T2T
scheme
should
be
flexible
enough
to
allow
for
scientific
judgment
in
the
selection
of
the
most
appropriate
tests
and
endpoints,
and
costs
should
be
reasonable.
Tests
should
be
aimed
at
determining
whether
the
chemical
substance
or
mixture
is
an
endocrine
disruptor
and
whether
the
effects
are
a
result
of
primary
or
secondary
disturbances
of
endocrine
function.
In
addition,
these
tests
should
be
designed
to
establish
the
relationship
between
different
exposure
levels,
timing
and
duration
of
exposure,
and
adverse
effects,
including
developmental
and
reproductive
effects
on
the
individual
and
its
progeny.

9.
In
contrast
to
T1S,
the
EDSTAC
recommends
that
T2T
should
be
both
sensitive
and
specific,
and
designed
to
minimize
false
positive
(
Type
I)
and
false
negative
(
Type
II)
errors.
Additionally,
this
battery
of
tests
should:

·
include
assessment
of
endpoints
identified
as
relevant
from
Tier
1
screens;
·
include
parental/
offspring
developmental
endpoints
(
e.
g.,
two­
generation
studies)
in
order
to
adequately
evaluate
all
life
stages;
·
include
the
life
cycle
of
both
viviparous
(
live
birth)
and
oviparous
(
egg­
laying)
organisms;
·
be
conducted
at
a
range
of
doses
that
allow
full
characterization
of
the
adverse
effects
of
the
chemical
substance
or
mixture
being
tested;
·
be
conducted
in
accordance
with
Good
Laboratory
Practice
(
GLP)
regulations
to
the
degree
consistent
with
resources
and
the
goal
of
timely
decisions;
and
·
be
validated,
if
need
be,
as
soon
as
possible
against
a
clearly
defined
standard.

10.
The
EDSTAC
recommends
that
a
subset
of
the
T1S
in
vitro
assays
be
conducted
with
the
assistance
of
automated
technology
to
provide
biological
effects
information
to
assist
in
the
overall
sorting
and
priority
setting
process.
Because
of
the
role
this
technology
will
play
in
the
overall
EDSTP,
the
EDSTAC
refers
to
it
as
"
high
throughput
pre­
screening"
(
HTPS).
The
EDSTAC
recommends
that
all
chemicals
currently
produced
in
an
amount
equal
to
or
greater
than
10,000
pounds
per
year
(
estimated
to
be
about
15,000
chemicals)
be
subjected
to
HTPS.
Also,
it
is
expected
that
all
pesticides
(
i.
e.,
both
active
ingredients
and
formulation
inerts)
will
be
subjected
to
HTPS.
Any
chemicals
subjected
to
the
assays
conducted
in
the
HTPS
step
would
not
be
required
to
repeat
the
ER
binding/
transcriptional
activation
assay
and
the
AR
binding/
transcriptional
activation
assay
as
part
of
T1S.
On
the
other
hand,
for
any
chemicals
not
subjected
to
HTPS
(
e.
g.,
production
volumes
less
than
10,000
pounds
per
year),
the
assays
in
T1S
would
result
in
information
equivalent
to
that
which
would
have
been
provided
from
HTPS.

11.
The
EDSTAC
recommends
that
the
vast
majority
of
chemicals
go
through
priority
setting,
T1S,
and
T2T
in
a
sequential
manner.
However,
the
EDSTAC
also
recognizes
there
may
be
individual
cases
in
which
T1S
is
bypassed.
Three
situations
were
identified
where
a
chemical
may
bypass
T1S,
each
with
different
implications
for
information
requirements
in
T2T.
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
5
7
­
·
Alternative
means
to
meet
T1S
information
requirements
through
the
generation
of
data
which
are
"
functionally
equivalent"
to
data
derived
from
the
recommended
screening
battery.
·
Bypassing
T1S
for
chemicals
(
e.
g.,
food­
use
pesticides)
that
have
previously
been
subjected
to
two­
generation
reproductive
toxicity
tests.
Such
chemicals
should
still
be
subjected
to
high
throughput
pre­
screening
assays.
·
Bypassing
T1S
for
chemicals
for
which
there
is
no
prior
toxicology
testing
but
the
owner
has
voluntarily
decided
to
proceed
directly
to
testing.
Such
chemicals
must
be
evaluated
in
the
high
throughput
pre­
screening
assays,
and
all
of
the
tests
in
the
T2T
battery.

III.
Recommendations:
Chapter
Four
-
Priority
Setting
A.
Summary
and
Scope
of
Effort
The
Priority
Setting
Work
Group
based
its
deliberations
on
the
original
Conceptual
Framework
described
in
Chapter
Three.
The
work
of
the
group
revolved
around
adapting
the
Conceptual
Framework
and
included
the
operational
elements
necessary
for
sorting
and
prioritizing
chemicals.
The
core
priority
setting
process
that
emerged
contained
several
elements:

·
the
use
of
all
available
existing
information;
·
the
development
of
a
relational
database
to
efficiently
access
and
utilize
information;
·
an
initial
sorting
of
the
universe
of
chemicals
into
categories
based
on
an
operationalized
Conceptual
Framework;
·
the
development
of
high
throughput
pre­
screening
data
and
its
incorporation
into
the
database;
·
the
use
of
the
database
to
summarize
empirical
data
and
estimate
fate
and
effect
parameters
where
possible;
·
the
use
of
the
database
to
establish
criteria
for
sorting
chemicals
where
appropriate;
and
·
the
use
of
a
compartment­
based
concept
to
accommodate
subjective
weighting
where
appropriate.

The
EDSTAC
viewed
its
role
within
EPA's
broader
mandate
to
protect
human
health
and
the
environment
and
the
broader
testing
authorities
available
to
EPA.
As
such,
the
EDSTAC
did
not
limit
itself
solely
to
requirements
of
the
Food
Quality
Protection
Act
and
the
Safe
Drinking
Water
Act
Amendments
of
1996.
The
Committee
believes
it
is
important
to
have
priorities
driven
by
scientific
considerations
and
explicit
value
judgments,
rather
than
by
existing
regulatory
requirements.

B.
The
Universe
of
Chemicals
and
Initial
Sorting
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
6
7
­
1.
The
EDSTAC
recommends
that
pesticides,
commodity
chemicals,
environmental
contaminants,
naturally
occurring
non­
steroidal
estrogens
(
e.
g.,
phytoestrogens,
mycotoxins),
food
additives,
cosmetics,
nutritional
supplements,
and
a
set
of
representative
mixtures
be
prioritized
for
endocrine
disruptor
screening
and
testing.

2.
The
EDSTAC
recommends
that
scientific
considerations
be
used
as
the
primary
basis
for
prioritizing
chemicals
for
endocrine
disruptor
screening
and
testing.
Statutory
authority
to
protect
human
health
and
the
environment
is
embedded
in
long­
standing
federal
legislation,
as
well
as
the
Food
Quality
Protection
Act
and
the
Safe
Drinking
Water
Act.

3.
The
EDSTAC
recommends
that
the
chemicals
under
consideration
(
approximately
87,000
compounds)
be
sorted
into
the
following
four
primary
categories
based
on
the
operationalized
Conceptual
Framework:

·
Polymers
are
to
be
placed
on
hold
(
with
some
exceptions)
pending
review
of
their
monomers,
oligomers,
other
components,
additives,
and
degradation
products
(
approximately
20,000
to
25,000
compounds).
·
Chemicals
to
be
considered
for
endocrine
disruptor
screening
(
approximately
62,000
compounds)
which
lack
sufficient
data
to
be
placed
on
hold
or
to
proceed
to
definitive
testing
or
hazard
assessment
will
be
subjected
to
the
priority
setting
process
for
T1S.
·
Chemicals
with
sufficient
data
are
to
bypass
screening
and
proceed
directly
to
testing
or
hazard
assessment
(
approximately
500
to
600
compounds).
·
Chemicals
with
sufficient
data
are
to
go
to
hazard
assessment
(
expected
to
number
approximately
50
to
100
compounds)

C.
Polymers
4.
With
some
exceptions,
the
EDSTAC
concluded
that,
due
to
molecular
weight,
polymers
are
less
cause
for
concern
than
other
classes
of
chemicals
with
regard
to
endocrine
disruption.
However,
there
is
some
concern
regarding
the
intestinal
absorption
capacity
of
neonates.
Because
of
the
lack
of
information
on
polymers
produced
prior
to
1979
(
the
date
of
the
initial
TSCA
Inventory),
coupled
with
the
low
likelihood
that
polymers
themselves
are
a
concern
for
endocrine
disruption,
the
EDSTAC
offers
the
following
recommendations.

·
All
new
polymers
with
a
number
average
molecular
weight
(
NAMW)
greater
than
1,000
daltons
and
all
previously
manufactured
(
or
"
existing")
polymers
(
regardless
of
NAMW)
are
to
be
held
from
priority
setting
for
endocrine
disruptor
screening
and
testing
pending
the
outcome
of
the
screening
and
testing
of
their
monomer,
oligomer,
and
other
components.
·
The
monomers,
oligomers,
and
other
components
of
polymers,
as
well
as
"
new"
polymers
(
i.
e.,
those
that
went
into
production
after
1979)
with
a
NAMW
less
than
1,000
daltons
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
7
7
­
are
to
undergo
priority
setting,
screening,
and
testing
as
appropriate.
·
Chemicals
on
the
EPA
SDWA
Contaminant
Candidate
List
(
CCL)
should
be
used
to
identify
the
potential
degradates
of
polymers
which
are
most
likely
to
present
environmental
exposure
and
which
should,
therefore,
be
subjected
to
priority
setting,
screening,
and
testing,
as
appropriate.
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
8
7
­
·
If
monomers,
oligomers,
or
other
components
of
a
polymer
are
determined
to
have
endocrine
disrupting
properties,
an
exposure
assessment
should
be
performed.
At
this
stage,
all
potential
exposure
routes
for
a
component
would
be
determined,
including
the
potential
for
the
component
to
be
available
from
the
polymer.
·
As
the
Agency
gains
experience
with
endocrine
disruptor
screening
and
testing
of
monomers,
oligomers,
and
"
new"
polymers
(
i.
e.,
those
that
went
into
production
after
1979)
with
NAMW
less
than
1,000
daltons,
it
should
apply
that
experience
toward
development
of
an
approach
to
address
"
existing"
polymers
(
i.
e.,
those
that
went
into
production
before
1979).

D.
Priority
Setting
Information
Categories
and
Criteria
5.
The
EDSTAC
recommends
using
existing
exposure­
related
and
effects­
related
data
and
information
to
establish
criteria
for
accomplishing
initial
sorting.
The
Committee
identified
the
following
subcategories
of
information
that
could
be
used
as
the
basis
for
sorting
and
priority
setting
and
developed
principles
regarding
their
use.

Exposure­
Related
Information
and
Criteria
a)
Biological
sampling
data
b)
Environmental,
occupational,
consumer
product,
and
food­
related
data
c)
Environmental
releases
d)
Production
volume
e)
Fate
and
transport
data
and
models
Effects­
Related
Information
and
Criteria
a)
Toxicological
laboratory
studies
and
databases
b)
Epidemiological
and
field
studies
and
databases
c)
Predictive
biological
activity
or
effects
models
(
e.
g.,
SARs,
QSARs)
d)
Results
of
high
throughput
pre­
screening
E.
High
Throughput
Pre­
Screening
6.
The
EDSTAC
found
there
was
a
general
lack
of
endocrine
effects
data
for
the
vast
majority
of
chemicals.
To
address
this
problem,
the
EDSTAC
recommends
that,
if
demonstrated
to
be
feasible,
eight
in
vitro
transcriptional
activation
assays
should
be
conducted
in
a
high
throughput
pre­
screening
mode
(
i.
e.,
with
the
use
of
robotics
and
other
automated
processes).
The
objectives
for
conducting
these
assays
in
a
high
throughput
mode
is
to:
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
9
7
­
·
provide
some
information
about
the
affinity
of
chemicals
to
bind
to
the
estrogen,
androgen,
and/
or
thyroid
hormone
receptors;
·
use
this
information
in
conjunction
with
other
exposure­
and
effects­
related
information
to
determine
the
priority
by
which
chemicals
should
be
advanced
to
T1S;
·
improve
QSAR
models;
·
provide
a
source
of
information
to
help
focus
the
selection
of
Tier
2
tests
for
those
chemicals
that
bypass
T1S;
and
·
generate
data
that
can
be
used
to
identify
chemicals
that
may
be
of
concern
at
low
doses.

7.
The
EDSTAC
recommends
that
the
high
throughput
pre­
screening
(
HTPS)
transcriptional
activation
assays
be
conducted
on:

·
the
estimated
15,000
chemicals
that
are
currently
produced
in
an
amount
equal
to
or
greater
than
10,000
pounds
per
year;
·
chemicals
that
are
permitted
to
bypass
T1S
and
go
directly
to
T2T;
·
chemicals
that
are
permitted
to
bypass
both
T1S
and
T2T
and
go
directly
to
hazard
assessment;
and
·
all
pesticides
(
both
active
ingredients
and
formulation
inerts).

8.
The
EDSTAC
recommends
that
HTPS
results
for
the
"
bypass"
chemicals
not
be
used
to
set
priorities
for
T1S,
but
to
improve
QSARs
and
inform
dosing
considerations,
particularly
during
the
interim
period
when
research
on
low
dose
is
being
conducted,
and
to
inform
decisions
regarding
the
types
of
tests
that
would
need
to
be
conducted
in
T2T.

9.
The
EDSTAC
recommends
that
existing
QSAR
models
be
derived
and
supplemented
with
data
from
the
HTPS
assays,
thereby
expanding
the
predictive
ability
of
these
models.

10.
The
EDSTAC
recommends
that
EPA
explore
the
feasibility
of
creating
an
archive
of
a
subset
of
HTPS
project
chemicals
which
can
be
accessed
by
researchers
interested
in
studying
endocrine
mediated
toxicity
or
in
validating
new
screens
for
endocrine
disruptors.

F.
Mixtures
11.
The
EDSTAC
recommends
that
EPA
include
a
limited
set
of
mixtures
that
span
a
range
of
physical
and
chemical
properties
in
both
the
feasibility
demonstration
project
for
the
HTPS
assays,
as
well
as
the
validation
effort
for
the
T1S
assays.

12.
If
the
screens
are
shown
to
be
capable
of
handling
a
diverse
set
of
mixtures
in
the
HTPS
feasibility
demonstration
project
and
the
T1S
validation
steps,
EPA
should
use
expert
judgment,
guided
by
a
set
of
prioritization
criteria,
to
evaluate
the
literature
and
to
decide
on
a
limited
set
of
mixtures
to
enter
HTPS.
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
10
7
­
13.
The
battery
of
screens
validated
for
use
in
the
screening
program
should
be
used
to
evaluate
the
mixtures
examined
in
HTPS.
If
appropriate,
screening
should
be
followed
by
testing.

14.
The
EDSTAC
recommends
that
a
comprehensive
literature
evaluation
be
undertaken
to
identify
exposure
and
effects
data
on
mixtures
that
do
not
undergo
HTPS.
This
information
would
be
used
to
inform
the
prioritization
for
Phase
II
and
subsequent
phases
of
the
screening
and
testing
program
which
would
use
the
same
prioritization
criteria
as
those
used
for
single
chemicals.

15.
The
EDSTAC
recommends
that
representative
sample
mixtures
be
selected
from
the
following
categories
and
be
subjected
to
HTPS
(
if
feasible)
and
to
T1S:

·
contaminants
in
human
breast
milk;
·
phytoestrogens
in
soy­
based
infant
formulas;
·
mixtures
of
chemicals
most
commonly
found
at
hazardous
waste
sites;
·
pesticide/
Fertilizer
mixtures;
·
disinfection
byproducts;
and
·
gasoline.

G.
Naturally
Occurring
Non­
Steroidal
Estrogens
(
NONEs)

16.
Naturally
occurring
non­
steroidal
estrogens
include
natural
products
derived
by
plants
(
phytoestrogens)
and
fungi
(
mycotoxins).
Due
to
the
ubiquitous
presence
of
these
compounds
in
foods,
and
due
to
the
potential
additive
and
antagonist
effects
of
NONEs
with
other
endogenous
and
exogenous
hormonally
active
chemical
substances,
the
EDSTAC
recommends
that:

·
NONEs
be
included
in
the
endocrine
disruptor
screening
and
testing
program
singly
and
in
complex
mixtures;
and
·
the
following
NONEs
be
screened
and,
if
necessary,
tested.

Representative
NONEs:

·
Isoflavones:
genistein,
daidzein,
miroestrol,
biochanin
A,
formononetin,
equol
·
Flavones:
kaemferol,
naringenin
·
Coumestans:
coumesterol
·
Dihydrochalcones:
phoretin
·
Triterpenes:
betulafolienetriol
(
ginseng)
·
Lignans:
enterolactone
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
11
7
­
Representative
estrogenic
mycotoxins:

·
Beta­
resorcyclic
lactones:
zearalenone,
zearalenol,
zearanol
H.
Nominations
17.
The
core
priority
setting
process
recommended
by
the
EDSTAC
focuses
on
giving
high
priority
to
chemicals
with
widespread
exposure
at
the
national
level.
The
EDSTAC
recognizes
such
a
process
could
result
in
a
low
priority
for
chemicals
where
exposures
are
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems.
Therefore,
the
EDSTAC
recommends
that
EPA
establish
a
nominations
process
that:

·
runs
parallel
to,
but
is
separate
and
distinct
from,
the
core
priority
setting
process;
·
is
designed
to
allow
chemical
substances
and
mixtures
for
which
there
may
not
be
widespread
exposures
on
a
national
scale,
but
for
which
there
are
exposures
on
a
smaller
scale,
to
be
eligible
to
receive
a
priority
for
T1S;
·
allows
for
an
early
opportunity
to
submit
nominations
during
each
phase
of
the
Endocrine
Disruptor
Screening
and
Testing
Program;
and
·
draws
no
less
than
5%
of
the
total
number
of
chemical
substances
or
mixtures
subjected
to
T1S
from
substances
receiving
nominations
but
not
selected
through
the
main
priority
setting
process.

18.
The
EDSTAC
recommends
that
any
nominated
chemical
substances
and/
or
mixtures
that
becomes
a
priority
for
T1S
through
the
core
priority
setting
process
be
removed
from
consideration
within
the
list
of
nominated
chemicals
in
order
to
ensure
that
the
priorities
drawn
from
the
nominations
process
will
compete
only
against
other
nominated
chemicals.

19.
In
keeping
with
the
overall
purpose
of
the
nominations
process,
the
EDSTAC
recommends
that
a
different
set
of
exposure­
related
criteria
be
used
to
evaluate
the
priority
for
nominated
chemicals
compared
to
the
exposure­
related
criteria
that
will
be
used
for
the
core
priority
setting
process.
Specifically,
the
nominations
process
should
focus
on
exposures
that
are
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems
rather
than
focusing
on
chemicals
for
which
there
is
widespread
exposure
in
the
aggregate.

20.
The
EDSTAC
recommends
that
if
there
are
effects
data
for
the
nominated
chemical,
or
if
the
chemical
is
similar
to
another
chemical
substance
or
mixture
for
which
effects
data
are
available,
EPA
should
utilize
those
data
as
a
secondary
source
of
information
to
help
set
priorities
among
nominees.

21.
The
EDSTAC
recommends
that
when
the
relative
priorities
of
nominated
chemical
substances
or
mixtures
are
evaluated,
EPA
should
consider
those
that
meet
the
following
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
12
7
­
criteria
to
be
a
higher
priority
than
those
that
do
not:

·
chemical
substances
or
mixtures
where
there
is
a
likelihood
of
regular
exposure,
in
contrast
to
those
for
which
exposure
occurs
only
rarely
or
occasionally;
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
13
7
­
·
chemical
substances
or
mixtures
that
affect
a
high
proportion
of
people
within
a
given
community
or
workplace;
and
·
chemical
substances
or
mixtures
for
which
there
may
be
empirical
or
estimated
(
i.
e.,
model
derived)
effects­
related
data
regarding
endocrine
disrupting
potential.

22.
The
EDSTAC
recommends
that
EPA
make
use
of
all
available
information
when
evaluating
nominations,
including
anecdotes,
and
other
information
gathered
as
part
of
the
core
priority
setting
process
(
e.
g.,
information
contained
within
the
Endocrine
Disruptor
Priority
Setting
Database).

23.
To
assist
EPA
in
evaluating
nominated
chemicals,
the
EDSTAC
recommends
that
EPA
request
the
following
types
of
information
from
the
public
regarding
nominations:

·
how
exposure
to
the
nominated
chemical
substances
or
mixtures
may
be
disproportionately
experienced
by
identifiable
groups,
communities,
or
ecosystems;
·
the
reasons
for
the
nomination
(
which
may
include
both
exposure­
and
effects­
related
concerns)
and
any
information
that
provides
a
basis
for
those
concerns;
and
·
the
degree
of
support
for
the
nomination
from
the
potentially
affected
communities
and/
or
workplaces.

I.
Endocrine
Disruptor
Priority
Setting
Database
(
EDPSD)

24.
The
EDSTAC
identified
and
evaluated
numerous
data
sources
associated
with
the
exposure
and
effects
information
categories
and
criteria
(
Appendix
G).
The
Committee
endorsed
the
integration
of
relevant
and
useful
data
sources
into
a
prototype
relational
database,
referred
to
as
the
Endocrine
Disruptor
Priority
Setting
Database.
Although
promising,
the
EDPSD
could
not
be
completed
within
the
EDSTAC's
time
and
resource
constraints.
Consequently,
EDSTAC
made
a
number
of
recommendations
regarding
continued
development
and
use
of
the
EDPSD.

·
EPA
should
continue
to
develop
and
maintain
the
EDPSD
as
a
tool
that
can
be
used
to
expeditiously
sort
and
prioritize
chemicals
for
endocrine
disruption
screening
and
testing.
·
The
process
used
by
EPA
in
developing
the
EDPSD,
as
well
as
the
process
by
which
it
is
used,
should
be
open
and
transparent.
·
EPA
should
convene
a
multi­
stakeholder
group
prior
to
the
completion
of
the
EDPSD
tool
to
ensure
effectiveness,
openness,
and
transparency.
·
After
completion
of
the
HTPS
assays,
this
group
should
make
use
of
the
tool,
along
with
the
"
compartment­
based"
approach
to
priority
setting
described
below,
in
assisting
EPA
as
it
develops
the
final
priorities
for
T1S.
·
The
EDPSD
should
not
be
limited
to
effects
data
that
can
be
easily
placed
into
a
database
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
14
7
­
format,
but
should
also
include
data
from
peer
reviewed
literature.
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
15
7
­
·
EPA
should
update
the
EDPSD
at
least
every
six
months,
and
more
frequently
if
time
and
resources
permit.

25.
The
EDSTAC
recommends
that
EPA
provide
resources
to
complete
the
Quality
Assurance/
Quality
Control
investigations
of
files
that
are
currently
in
the
EDPSD.
The
EDSTAC
further
recommends
that
EPA
provide
resources
to
add
new
files
to
the
EDPSD
in
stages.
These
files
and
stages
for
their
addition
could
include:

1st
stage:
EPA's
and
others'
databases
that
provide
data
on
use
for
industrial
chemicals
and
pesticides;
information
from
pesticide
ecotoxicity,
fate,
and
toxicity
one­
liners;
chemicals
that
are
non­
food­
use
pesticide
active
ingredients
and
non­
food­
use
other
pesticide
ingredients;
chemicals
on
the
Generally
Regarded
As
Safe
(
GRAS)
list;
and
chemicals
in
the
FDA
Priority
Assessment
of
Food
Additives
(
PAFA)
database.

2nd
stage:
Data
on
chemical
use
that
were
not
readily
available
in
databases;
chemicals
and
concentrations
of
chemicals
in
National
Health
and
Nutrition
Examination
Survey
(
NHANES),
Total
Exposure
Assessment
Methodology
(
TEAM),
and
Agency
for
Toxic
Substances
Disease
Registry's
(
ATSDR)
Hazardous
Substances
Emergency
Events
Surveillance
(
HSEES)
files;
measured
chemical
fate
data;
and
additional
QSARs
for
endocrine
disruptors.

3rd
stage:
Inclusion
of
HTPS
data
and
improved
QSARs.

The
EDSTAC
recognizes
that
the
time
and
resources
required
to
add
new
files
will
depend
upon
a
number
of
factors,
including:
when
pending
files
are
received,
the
format
of
received
files,
the
determination
of
whether
to
use
files
as
sources
of
numerical
or
logical
data,
conversion
of
logical
files
to
numerical
files,
completion
of
QA/
QC
investigations
of
the
files
and
data,
and
expediency
of
the
input
process.

J.
Recommended
Approach
to
Priority
Setting
26.
The
EDSTAC
identified
a
number
of
obstacles
to
the
development
of
an
"
ideal"
priority
setting
system,
including
the
uneven
quality
and
quantity
of
both
exposure­
and,
even
more
so,
effects­
related
data
sources.
Major
characteristics
of
this
unevenness
include:

·
Many
more
data
are
available
on
the
effects
of
the
relatively
small
number
of
currently
registered
active
ingredients
in
pesticides
(
approximately
900)
than
on
the
thousands
of
industrial
chemicals
produced
in
much
larger
quantities.
·
Biological
monitoring
data
for
humans
are
scarce.
A
relatively
small
number
of
chemicals
(
on
the
order
of
100
or
less)
have
been
routinely
sampled
in
human
blood
and
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
16
7
­
urine
in
the
United
States,
and
the
major
U.
S.
national
program
for
sampling
concentrations
in
human
tissues
was
discontinued
in
1990.
·
Monitoring
data
for
other
organisms,
while
more
numerous
than
human
data,
still
focus
on
a
relatively
small
number
of
chemicals.
·
Data
on
routine
chemical
releases
to
the
environment,
while
markedly
better
than
they
were
prior
to
the
creation
of
the
Toxic
Release
Inventory
about
10
years
ago,
still
encompass
only
528
industrial
chemicals
and
pesticides
and
frequently
rely
on
engineering
estimates
rather
than
actual
releases.

27.
The
EDSTAC
recommended
several
principles
to
guide
the
development
of
a
strategy
for
setting
priorities
for
the
large
number
of
chemicals
for
which
there
are
insufficient
data
to
go
to
T2T
or
hazard
assessment.
The
selected
system
should
be
transparent,
should
make
use
of
the
guiding
principles
for
exposure­
and
effects­
related
data
sources,
and
should
be
driven
by
empirical
data,
but
not
be
held
captive
by
them.

28.
The
EDSTAC
recommends
a
"
compartment­
based
priority
setting
strategy"
for
prioritizing
chemicals
for
T1S.

·
The
strategy
builds
upon
the
identification
and
evaluation
of
the
different
exposure­
and
effects­
related
information
categories
and
criteria.
·
The
term
"
compartment"
refers
to
the
consideration
of
these
information
categories
either
singly
or
in
combination.
·
Illustrative
examples
of
the
four
different
categories
of
compartments
include:
·
the
integration
of
exposure
and
effects
information;
·
the
consideration
of
exposure
information;
·
the
consideration
of
effects
information;
and
·
specially
targeted
priorities
(
mixtures,
nominations,
and
naturally
occurring
nonsteroidal
estrogens).

The
specific
compartments
and
the
weights
and/
or
order
in
which
they
should
be
utilized
have
not
yet
been
agreed
upon.
A
target
number
of
chemicals
to
go
through
T1S
in
the
first
phase
of
the
program
or
during
the
life
of
the
program
has
not
been
determined.
Possible
targets
and
how
these
targets
might
be
affected
by
the
compartmentalized
approach
to
priority
setting
have
not
been
agreed
upon.

29.
The
EDSTAC
recommends
a
number
of
next
steps
to
further
develop
and
refine
the
compartment­
based
approach
to
priority
setting,
including:

·
use
of
the
EDPSD
by
a
multi­
stakeholder
group
to
further
characterize
and
define
what
will
be
contained
in
each
compartment;
·
whether,
and
if
so,
how
to
prioritize
the
compartments;
and
·
how
to
address
the
possibility
of
overlaps
between
compartments.
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
17
7
­
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
18
7
­
30.
The
EDSTAC
recommends
using
the
schedule
EPA
has
established
for
tolerance
reassessments
and
pesticide
re­
registration
under
the
FQPA
for
setting
priorities
for
those
food­
use
pesticides
that
meet
the
criteria
for
bypassing
T1S
and
going
directly
to
T2T.
When
planning
for
the
registration
renewal
process
begins,
the
FQPA
requirement
for
endocrine
disruptor
screening
and
testing
should
be
designated
as
a
criterion
for
priority
setting.

31.
The
EDSTAC
recommends
that
priorities
for
T2T
for
all
other
chemicals
(
i.
e.,
non­
food­
use
pesticides
and
other
chemicals
where
the
owner
either
wishes
to
voluntarily
bypass
T1S,
or
where
the
owner
has
met
the
criteria
for
completing
the
alternative,
functionally
equivalent,
T1S
assays)
should
be
established
on
a
case­
specific
basis.
However,
the
EDSTAC
recommends
that
chemicals
which
receive
a
high
priority
ranking
for
T1S
should
retain
that
high
priority
ranking
for
T2T,
even
when
the
owner
wishes
to
voluntarily
bypass
T1S.

IV.
Recommendations:
Chapter
Five
-
Screening
and
Testing
A.
Tier
1
Screening
1.
The
EDSTAC
recommends
that
any
T1S
battery
designed
to
detect
endocrine
disruptors
should
meet
five
criteria.
The
battery
should:

·
maximize
sensitivity
to
minimize
false
negatives
while
permitting
an
acceptable
level
of
false
positives;
·
include
a
range
of
organisms
representing
known
or
anticipated
differences
in
metabolic
activity;
·
detect
all
known
modes
of
action
for
endocrine
endpoints
of
concern;
·
include
a
sufficient
range
of
taxonomic
groups
among
the
test
organisms;
and
·
incorporate
sufficient
diversity
and
complementarity
among
the
endpoints
and
assays
to
reach
conclusions
based
on
weight
of
evidence
considerations.

2.
The
EDSTAC
recommends
the
following
assays
for
inclusion
in
the
T1S
battery:

In
Vitro
1.
Estrogen
Receptor
(
ER)
Binding/
Transcriptional
Activation
Assay;
2.
Androgen
Receptor
(
AR)
Binding/
Transcriptional
Activation
Assay;
and
3.
Steroidogenesis
Assay
With
Minced
Testis.
In
Vivo
1.
Rodent
3­
day
Uterotrophic
Assay
(
subcutaneous);
2.
Rodent
20­
day
Pubertal
Female
Assay
With
Thyroid;
3.
Rodent
5­
7
day
Hershberger
Assay;
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
19
7
­
4.
Frog
Metamorphosis
Assay;
and
5.
Fish
Gonadal
Recrudescence
Assay.

3.
The
EDSTAC
identified
the
following
four
assays
as
possible
alternatives
to
some
components
of
the
proposed
battery
and
recommends
that
they
also
be
standardized
and
validated:

In
Vitro
1.
Placental
Aromatase
Assay
In
Vivo
1.
Modified
Rodent
3­
day
Uterotrophic
Assay
(
intraperitoneal);
2.
Rodent
14­
day
Intact
Adult
Male
Assay
With
Thyroid;
and
3.
Rodent
20­
day
Thyroid/
Pubertal
Male
Assay.

Combinations
of
the
alternative
assays,
if
validated
and
found
to
be
functionally
equivalent,
could
potentially
replace
three
of
the
component
assays
in
the
proposed
T1S
battery
(
in
vitro
steroidogenesis
assay
with
testis,
20­
day
pubertal
female
assay,
and
5­
7
day
Hershberger
assay)
thereby
possibly
reducing
the
overall
time,
cost,
and
complexity
while
maintaining
equivalent
performances
of
the
overall
T1S
battery.
The
EDSTAC
recognizes
that
the
stateof
the­
science
in
this
area
is
evolving
quickly
and
strongly
encourages
the
use
of
new
or
improved
assays
for
screening
as
they
become
available.

4.
The
EDSTAC
recommends
that
validation/
standardization
studies
be
conducted
on
all
assays
in
the
proposed
battery
as
well
as
the
alternatives.

5.
The
EDSTAC
agrees
that
EPA
should
take
affirmative
steps,
in
collaboration
with
industry
and
other
interested
parties,
to
attempt
to
develop
the
protocol
for
a
full
life
cycle
(
i.
e.,
with
embryonic
exposure
and
evaluation
of
the
adult
offspring)
developmental
exposure
screening
assay
that
can
be
subjected
to
validation
and
standardization.
The
EDSTAC
further
recommends
that,
if
such
an
assay
were
identified,
validated,
and
standardized,
the
decision
on
whether
it
should
be
included
in
the
T1S
battery
should
include
an
evaluation
of
its
potential
to
replace
one
or
more
of
the
recommended
T1S
assays
and
its
overall
impact
on
the
cost
effectiveness
of
the
T1S
battery.

6.
The
EDSTAC
recommends
that
all
T1S
in
vitro
assays
involve
multiple
dose
levels,
whether
performed
by
HTPS
or
bench
level
methods,
so
a
dose­
response
curve
and
assessment
of
relative
potencies
can
be
developed.
Subject
to
the
results
of
the
validation
process,
the
EDSTAC
recommends
using
one
or
more
dose
levels
in
the
performance
of
the
in
vivo
assays.

7.
For
assessing
receptor
binding
in
vitro,
the
EDSTAC
is
recommending
that
both
the
cell­
free
receptor
binding
assays
and
the
transcriptional
activation
assays
for
ER
and
AR
be
incorporated
into
the
T1S
battery,
and
be
subjected
to
validation
and
standardization.
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
20
7
­
8.
As
noted
in
Chapter
Four,
the
EDSTAC
recommends
the
use
of
a
high
throughput
pre­
screen
(
HTPS)
for
toxicants
operating
though
the
ER,
AR,
and
TR
using
stably
transfected
cell
lines
with
and
without
metabolic
activation,
if
available.
Substances
which
have
not
been
assessed
in
the
HTPS
should
be
subject
to
assays
for
detection
of
ER
and
AR
activity
performed
at
the
bench.
Two
types
of
assays
are
considered
acceptable:
cell
free
receptor
binding
and
transcriptional
activation
in
transfected
cells.
The
latter
is
preferred.
Assays
must
meet
the
following
characteristics:

·
evaluate
binding
to
EAT
receptors;
·
evaluate
binding
with
and
without
metabolic
capability;
·
distinguish
between
agonist
and
antagonist
potential;
and
·
yield
dose
responses
to
establish
relative
potency.

9.
The
EDSTAC
is
recommending
evaluation
of
antithyroid
effects
in
animals
in
the
longer
term
rodent
screen
(
either
14­
day
or
20­
day
exposure).
Although
it
is
not
known
whether
exposure
to
xenobiotics
for
greater
than
14
days
is
required
to
significantly
affect
circulating
levels
of
T4,
TSH,
or
thyroid
histopathology,
the
EDSTAC
believes
these
longer
periods
may
be
required.
The
effects
of
duration
of
chemical
substance
and
mixture
exposure
must
be
quickly
evaluated
in
the
validation
phase.

B.
Principles
for
Evaluating
Tier
1
and
Tier
2
Results
10.
The
EDSTAC
recommends
that
a
"
weight­
of­
evidence"
approach
be
used
in
evaluating
T1S
and
T2T
results
and
has
developed
general
criteria
for
applying
"
weight­
ofevidence
to
ensure
that
decisions
are
transparent
and
predictable.

C.
Tier
2
Testing
11.
The
EDSTAC
recommends
that
the
following
tests
be
included
in
the
Tier
2
battery:

Mammalian
tests
·
Two­
generation
reproductive
toxicity
study,
or
·
An
alternative
less
comprehensive
test:
1.
Alternative
mammalian
reproductive;
and
2.
One­
generation
test.

Non­
mammalian
multigeneraton
tests
·
Avian
reproduction;
·
Fish
life
cycle;
·
Mysid
life
cycle;
and
·
Amphibian
development
and
reproduction.
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
21
7
­
12.
The
EDSTAC
recommends
that
the
"
default"
action,
in
absence
of
any
prior
information,
be
to
perform
all
tests
in
the
T2T
battery
with
all
endpoints.
Further,
the
EDSTAC
recommends
that
the
choice
of
whether
Tier
2
tests
will
be
conducted
on
all
five
of
the
recommend
taxa,
or
a
more
limited
subset
of
the
five
taxa,
should
be
based
on
the
physico­
chemical
characteristics
and
environmental
release
and
exposure
information
of
the
chemical
substance
or
mixture
to
be
tested,
together
with
biological
data
from
T1S.
The
results
of
T1S
or
other
information
may
also
allow
tailoring
of
T2T
such
as
the
inclusion
or
deletion
of
certain
endpoints
(
e.
g.,
thyroid
effects)
or
use
of
alternative
tests.

13.
The
EDSTAC
believes
that
a
project
is
required
to
resolve
the
underlying
uncertainties
and
controversy
about
issues
related
to
low
dose
selection
and
the
identification
of
noobserved
adverse­
effect­
levels
(
NOAEL).
Further,
the
EDSTAC
recommends
that
a
collaborative
group
involving
government,
industry,
and
appropriate
individuals
in
academia
design
the
study
protocols,
be
kept
abreast
of
the
conduct
of
the
studies,
evaluate
results,
and
develop
overall
conclusions
and
recommendations.

14.
The
EDSTAC
recommends
that
information
used
to
select
doses
in
the
performance
of
Tier
2
tests
include:

·
existing
information
such
as
that
available
during
priority
setting
including
the
results
or
the
HTPS;
·
results
from
T1S;
·
results
from
other
assays
or
tests;
and
·
results
from
range
finding
studies.

15.
The
EDSTAC
recommends
including
thyroid­
sensitive
endpoints
in
T2T
and
that
dosing
in
mammalian
tests
include
fetal
and
lactational
exposure.

D.
Validation
of
the
Screening
and
Testing
Batteries
16.
The
EDSTAC
believes
the
validation
and
standardization
program
is
of
highest
priority,
and
recommends
that
it
proceed
on
an
accelerated
schedule.
The
EDSTAC
further
recommends
that
the
validation
and
standardization
program
be
consistent
with
the
principles
articulated
by
the
national
(
ICCVAM,
1996;
Zeiger,
1998)
and
international
(
OECD,
1996)
alternative
methods
validation
groups.

17.
The
EDSTAC
recommends
that,
as
individual
screens
and
tests
are
validated
and
standardized,
they
can
be
utilized
in
the
EDSTP
without
waiting
for
all
screens
and
tests
in
the
batteries
to
be
validated.
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
22
7
­
18.
The
EDSTAC
recommends
that
a
multi­
stakeholder
process,
involving
government,
industry,
and
academics,
be
utilized
in
validating
and
standardizing
the
T1S
and
T2T
batteries.

19.
The
EDSTAC
identified
other
screens
and
tests
which,
if
available,
could
have
an
important
utility
in
the
screening
and
testing
program,
and
recommends
that
research
be
conducted
to
determine
whether
such
assays
can
be
developed,
and
if
so,
what
purpose
the
assays
could
fulfill
within
the
endocrine
disruptor
screening
and
testing
program.

V.
Recommendations:
Chapter
Six
-
Communications
and
Outreach
A.
Need
for
Communication
As
described
in
Chapter
Two,
Section
II,
the
Communications
and
Outreach
Work
Group
(
COWG),
and
then
later
the
full
EDSTAC,
recognized
the
importance
of
communication
about
the
EDSTP
to,
among
other
things,
prevent
misuse
of
information.
Because
the
EDSTP
applies
a
tiered
approach,
results
become
increasingly
definitive
as
chemicals
progress
through
each
step
of
the
screening
and
testing
program.
This
type
of
system
leaves
room
for
interpretation
of
results,
particularly
in
the
early
stages
of
the
EDSTP
(
i.
e.,
during
priority
setting
or
screening),
that
may
or
may
not
be
accurate.
Therefore,
the
Committee
emphasizes
the
need
for
clear
and
accurate
communication
to
interested
stakeholders
throughout
the
development
and
implementation
of
the
EDSTP.
In
particular,
it
is
important
that
EPA
clearly
communicate
about
the
limitations
that
must
be
placed
on
the
interpretation
of
information
and
results
from
the
EDSTP,
as
well
as
the
meaning
and
implications
of
its
decisions.
The
recommendations
identified
in
Chapter
Six
seek
to
emphasize
this
point,
while
providing
guidance
to
EPA
as
it
further
develops
its
communications
strategy
for
the
EDSTP.

B.
Principles
to
Guide
Implementation
of
a
Communications
Strategy
1.
The
EDSTAC
recommends
that
EPA
develop
and
implement
an
effective
communications
and
outreach
strategy
for
the
EDSTP
based
on
the
following
set
of
principles
intended
to
help
ensure
accurate
and
open
communication
to
stakeholders:

·
Both
the
process
and
results
of
the
EDSTP
should
be
open
and
transparent.
·
The
results
of
the
EDSTP
should
be
interpreted
and
communicated
within
the
context
set
forth
in
the
final
EDSTAC
Report.
·
The
limitations
and
uncertainties
of
the
available
data
and
the
results
of
EDSTP
should
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
23
7
­
be
articulated
clearly
when
the
screening
and
testing
program
is
discussed.
·
As
new
scientific
evidence
emerges,
the
uncertainties
and
limitations
of
the
data
may
also
change.
These
changes
should
be
communicated
clearly.
·
EPA
should
develop
quality
assurance
processes
to
assure
that
any
database
maintained
for
the
public
relative
to
the
EDSTP
is
accurate
and
current.

C.
Basic
Features
of
a
Communications
and
Outreach
Strategy
2.
The
Committee
recommends
that
EPA
base
their
communications
and
outreach
strategy
on
the
following
four
questions:

·
What
should
be
communicated?
·
To
whom
should
information
be
communicated?
·
How
should
information
be
communicated?
·
When
should
information
be
communicated?

Details
of
the
recommendations
for
each
of
the
four
questions
are
located
in
Chapter
Six,
Section
III,
B.
The
basic
recommendations,
however,
follow.

3.
Under
"
What
should
be
communicated?,"
the
Committee
recommends
that
EPA
be
prepared
to
provide
information
to
interested
stakeholders
on
the
EDSTP
itself,
on
screening
and
testing
results,
the
nominations
process,
and
background
information
about
the
EDSTAC
process.
Suggested
language
explaining
the
various
components
of
the
EDSTP
in
less
technical
terms
than
is
found
throughout
the
report,
is
included
in
the
chapter.

4.
Under
"
To
whom
should
information
be
communicated?,"
the
Committee
recommends
that
EPA
actively
communicate
with
members
of
the
public
and
other
stakeholders,
such
as
those
who
have
demonstrated
interest
in
the
process
through
their
attendance
of
the
public
EDSTAC
meetings
and
public
comment
periods.

5.
The
Committee
recognizes
the
need
for,
and
recommends
EPA
develop,
tailored
information
to
be
relayed
through
a
variety
of
mechanisms.
This
would
help
to
ensure
that
specific
audiences
 
such
as
environmental
justice
organizations,
"
downstream"
industries,
farm
workers,
and
patient
groups
 
who
may
not
have
the
ability
to
access
information
via
traditional
means
and
who
have
varying
levels
of
knowledge
and
interest
in
endocrine
disruptor­
related
issues,
have
the
opportunity
to
learn
about
the
EDSTP
and
its
results.

6.
The
Committee
recommends
that
EPA
conduct
a
follow­
up
to
their
September
1997
outreach
questionnaire
in
order
to
find
out
more
information
about
how
best
to
EDSTAC
Final
Report
Chapter
Seven
August
1998
2­
24
7
­
communicate
with
certain
groups,
such
as
those
listed
above
in
recommendation
number
five.

7.
Under
"
How
should
information
be
communicated?,"
the
Committee
recommends
that
EPA
develop
a
tracking
system
as
part
of
the
priority
setting
database
described
in
Chapter
Four.
They
recommend
that,
if
possible,
such
a
database
be
incorporated
into
existing
EPA
systems
to
promote
efficiency
and
cost­
effectiveness.
Several
characteristics
of
a
desirable
database
intended
to
address
the
needs
of
a
wide
range
of
potential
users
have
been
included.
The
EDSTAC
believes
it
is
important
for
members
of
the
public
to
have
access
to
information
about
the
screening
and
testing
program
as
it
progresses,
including
the
ability
to
query
and
quickly
determine
the
status
of
a
chemical
or
mixture
in
the
EDSTP,
as
well
as
to
access
and
download
relevant
EDSTP
documents.

8.
For
those
without
Internet
access,
information
should
be
available
through
a
variety
of
sources,
including
telephone,
fax,
mail,
Federal
Register
notices,
and
other
forms
of
communication,
as
necessary.

9.
Under
"
When
should
information
be
communicated?,"
the
Committee
recommends
that
EPA
develop
a
newsletter
or
bulletin,
as
has
been
done
in
other
EPA
programs,
that
would
be
made
available
on
a
regular
basis.
The
report
should
be
of
a
limited
length
and
should
be
available
for
a
limited
duration.

10.
The
Committee
also
recommends
that
information
be
communicated
when
warranted
by
important
EDSTP
developments,
such
as
a
call
for
nominations,
when
lists
of
chemicals
have
been
prioritized
for
T1S,
identified
for
T2T,
or
identified
as
being
subjected
to
hazard
assessment
after
exhibiting
endocrine­
mediated
adverse
effects
in
T2T,
as
well
as
regarding
other
key
decisions
relating
directly
to
the
program.

11.
As
described
in
Chapter
Six,
Section
III,
C,
the
Committee
strongly
recommends
that
EPA
commit
adequate
resources
to
the
communication
aspects
of
this
program.
Several
tasks
requiring
such
support
are
identified
in
the
report,
such
as
the
creation
and
maintenance
of
a
tracking
database,
maintenance
of
a
Web
site
with
an
appropriate
graphical
user
interface,
creation
and
maintenance
of
a
centralized,
automated
telephone
system,
and
assignment
of
staff
to
monitor
such
items.
EDSTAC
Final
Report
August
1998
1
A
­
Appendix
A
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
Participant
List
EDSTAC
Chair
Lynn
Goldman
EDSTAC
Chair
Assistant
Administrator
Office
of
Prevention,
Pesticides,
and
Toxic
Substances
U.
S.
Environmental
Protection
Agency
401
M
Street
SW,
MC
7101
Washington,
DC
20460
202­
260­
2902
Fax
202­
260­
1847
goldman.
lynn@
epamail.
epa.
gov
U.
S.
EPA
Jim
Aidala
Associate
Assistant
Administrator
Office
of
Prevention,
Pesticides,
and
Toxic
Substances
U.
S.
Environmental
Protection
Agency
401
M
Street
SW,
MC
7101
Washington,
DC
20460
202­
260­
2897
Fax
202­
260­
1847
aidala.
jim@
epamail.
epa.
gov
Tudor
Davies
Director,
Office
of
Science
and
Technology
Office
of
Water
U.
S.
Environmental
Protection
Agency
401
M
Street
SW,
MC
7501C
Washington,
DC
20460
202­
260­
5400
Fax
202­
260­
5394
davies.
tudor@
epamail.
epa.
gov
EDSTAC
Final
Report
August
1998
2
A
­
Alternate
to
T.
Davies
Jeanette
Wiltse
Director,
Health
and
Ecological
Criteria
Division
Office
of
Water
U.
S.
Environmental
Protection
Agency
401
M
Street
SW,
MC
4304
Washington,
DC
20460
202­
260­
7317
Fax
202­
260­
1036
wiltse.
jeanette@
epamail.
epa.
gov
Penny
Fenner­
Crisp
Office
of
Prevention,
Pesticides,
and
Toxic
Substances,
7101
U.
S.
Environmental
Protection
Agency
401
M
Street
SW
Washington,
DC
20460
202­
260­
0947
Fax
202­
260­
1847
fenner­
crisp.
penelope@
epamail.
epa.
gov
Lawrence
Reiter
Director
National
Health
and
Environmental
Effects
Research
Laboratory
U.
S.
Environmental
Protection
Agency
2525
Highway
54
Research
Triangle
Park,
NC
27711
919­
541­
2281
Fax
919­
541­
4324
reiter@
herl45.
herl.
epa.
gov
EDSTAC
Final
Report
August
1998
3
A
­
Alternate
to
Lawrence
Reiter
Suzanne
McMaster
Assistant
Director
National
Health
and
Environmental
Effect
Research
Laboratory
(
MD1A)
U.
S.
Environmental
Protection
Agency
2525
Highway
54
Research
Triangle
Park,
NC
27711
919­
541­
3844
Fax
919­
541­
1440
mcmaster.
suzanne@
epamail.
epa.
gov
Other
Federal
Agencies
Pat
Basu
Director,
Chemistry
and
Toxicology
Division
Office
of
Public
Health
&
Science
Food
Safety
and
Inspection
Service
U.
S.
Department
of
Agriculture
Room
6912
Franklin
Court
Suite
1400
Independence
Avenue
S.
W.
Washington,
DC
20250
202­
501­
7319
Fax
202­
501­
7639
pat.
basu@
usda.
gov
Bryan
Hardin
Senior
Scientist
Office
of
the
Director
National
Institute
for
Occupational
Safety
and
Health
Centers
for
Disease
Control
and
Prevention
200
Independence
Avenue
SW
HHH
Building,
Room
715H
Washington,
DC
20201
202­
205­
8556
Fax
202­
260­
4464
bdh1@
cdc.
gov
George
Lucier
Director
of
the
Environmental
Toxicology
Program
National
Institute
for
Environmental
Health
Sciences
PO
Box
12233,
MD
A3­
02
111
TW
Alexander
Drive
Research
Triangle
Park,
NC
27709
919­
541­
3802
Fax
919­
541­
3647
Lucier@
niehs.
nih.
gov
Alternate
for
G.
Lucier
Michael
Shelby
National
Institute
for
Environmental
Health
Sciences
111
Alexander
Drive,
MD:
B3­
09
Research
Triangle
Park,
NC
27709
919­
541­
3455
Fax
919­
541­
4634
shelby@
niehs.
nih.
gov
Teri
Rowles
Coordinator
of
the
Marine
Mammal
Health
and
Stranding
Response
Program
National
Oceanic
and
Atmospheric
Administration
1315
East
West
Hwy
Silver
Spring,
MD
20910
301­
713­
2322
Fax
301­
713­
0376
teri.
rowles@
noaa.
gov
EDSTAC
Final
Report
August
1998
4
A
­
Alternate
to
T.
Rowles
Mary
Matta
Technical
Support
Team
Leader
Office
of
Ocean
Resources
Conservation
and
Assessment
Coastal
Resources
Coordination
Branch
National
Oceanic
and
Atmospheric
Administration
7600
Sand
Point
Way
NE
Bin
C15700
Seattle,
WA
98155
206­
526­
6315
Fax
206­
526­
6865
matta@
hazmat.
noaa.
gov
Mark
Schaefer
Deputy
Assistant
Secretary
for
Water
and
Science
U.
S.
Department
of
the
Interior
1849
C
Street
NW
Washington,
DC
20240
202­
208­
4811
Fax
202­
371­
2815
mark_
schaefer@
ios.
doi.
gov
Alternate
to
M.
Schaefer
Timothy
Kubiak
National
Water
Quality
Coordinator
U.
S.
Fish
and
Wildlife
Service
4401
N.
Fairfax
Drive
(
ARLSQ
330)
Arlington,
VA
22203
703­
358­
2148
Fax
703­
358­
1800
tim_
kubiak@
mail.
fws.
gov
Alternate
to
M.
Schaefer
Michael
Mac
Program
Manager
Biological
Resources
Division
U.
S.
Geological
Survey
12201
Sunrise
Valley
Drive
Reston,
VA
20192
703­
648­
4073
Fax
703­
648­
4238
michael_
mac@
nbs.
gov
Bernard
Schwetz
Director
National
Center
for
Toxicological
Research
U.
S.
Food
and
Drug
Administration
3900
N.
C.
T.
R.
Road
Jefferson,
AR
72079­
9502
501­
543­
7517
Fax
501­
543­
7576
bschwetz@
nctr.
fda.
gov
Alternate
to
B.
Schwetz
Barry
Delclos
Research
Pharmacologist
National
Center
for
Toxicology
Research
Division
of
Biochemical
Toxicology
(
HFT­
110)
U.
S.
Food
and
Drug
Administration
3900
NCTR
Road
Jefferson,
AR
72079
501­
543­
7372
Fax
501­
543­
7136
bdelclos@
nctr.
fda.
gov
EDSTAC
Final
Report
August
1998
5
A
­
Susan
Sieber
Deputy
Director
Division
of
Cancer
Epidemiology
and
Genetics
National
Cancer
Institute
Executive
Plaza
North,
Suite
540
6130
Executive
Blvd.
Rockville,
MD
20852
301­
496­
5946
Fax
301­
402­
3256
siebers@
epndce.
nci.
nih.
gov
Tom
Sinks
Associate
Director
for
Science
National
Center
for
Environmental
Health
Centers
for
Disease
Control
and
Prevention
4770
Buford
Highway
NE,
MS
F­
29
Atlanta,
GA
30341­
3724
770­
488­
7001
Fax
770­
488­
7015
ths2@
cdc.
gov
Alternate
to
T.
Sinks
Richard
Jackson
Director
National
Center
for
Environmental
Health
Centers
for
Disease
Control
and
Prevention
4770
Buford
Highway
NE,
MS
F­
29
Atlanta,
GA
30341
770­
488­
7000
Fax
770­
488­
7015
rxj4@
cdc.
gov
State
Agencies
Henry
Anderson
Chief
Medical
Officer
Wisconsin
Bureau
of
Public
Health
1414
E.
Washington
Avenue,
Room
96
Madison,
WI
53703
608­
266­
1253
Fax
608­
267­
4853
anderha@
DHFS.
state.
wi.
us
Anna
Fan
Chief
Pesticide
and
Environmental
Toxicology
Section/
OEHHA
California
Environmental
Protection
Agency
2151
Berkeley
Way,
Annex
11,
7th
Floor
Berkeley,
CA
94704
510­
540­
3066
Fax
510­
540­
3674
afan@
berkeley.
cahwnet.
gov
Nancy
Kim
Director
Division
of
Environmental
Health
Assessment
New
York
State
Department
of
Health
2
University
Place,
Room
350
Albany,
NY
12203
518­
458­
6438
Fax
518­
458­
6436
nkk01@
health.
state.
ny.
us
Industry
Christopher
Borgert
Pharmacologist/
Toxicologist
Consultant
to
Aquatrols,
Inc.
238
Turkey
Creek
10514
Palmetto
Blvd.
Alachua,
FL
32615
904­
462­
1266
Fax
904­
462­
1267
cjborgert@
APT­
PHARMATOX.
COM
George
Daston
Principal
Research
Scientist
Miami
Valley
Laboratories
Procter
&
Gamble
PO
Box
538707
Cincinnati,
OH
45253
513­
627­
2886
Fax
513­
627­
1908
daston.
gp@
pg.
com
EDSTAC
Final
Report
August
1998
6
A
­
Angelina
Duggan
Registrations
Manager
Development
and
Regulatory
Affairs
Business
Management
Department
FMC
Agricultural
Products
Group
1735
Market
Street
Philadelphia,
PA
19103
215­
299­
6670
Fax
215­
299­
6468
angelina_
duggan@
fmc.
com
Donald
Lamb
Vice
President,
Product
Safety
and
Regulatory
Affairs
Bayer
Corporation
100
Bayer
Road
Pittsburgh,
PA
15205­
9741
412­
777­
7431
Fax
412­
777­
7484
don.
lamb.
b@
bayer.
com
Joseph
LeBeau
Retired
Vice
President,
Product
Responsibility
Dow
Chemical
Company
4701
Butternut
Place
Midland,
MI
48640
517­
835­
6244
Fax
517­
835­
6244
melraccoon@
aol.
com
Thomas
Osimitz
Vice
President
Global
Safety
Assessment
and
Regulatory
Affairs
SC
Johnson
&
Son,
Inc.
1525
Howe
Street
Racine,
WI
53403
414­
260­
2669
Fax
414­
260­
0186
tgosimit@
scj.
com
James
Quance
Environmental
Issues
Manager
Exxon
Chemical
Company
13501
Katy
Freeway
Houston,
TX
77079
281­
870­
6281
Fax
281­
588­
4664
jim.
f.
quance@
chemical.
exxon.
sprint.
com
Abraham
Tobia
Manager,
Toxicology
and
Risk
Assessment
Senior
Registration
Scientist
BASF
Corporation
26
Davis
Drive
PO
Box
13528
Research
Triangle
Park,
NC
27709­
3578
919­
547­
2972
Fax
919­
547­
2880
tobiaa@
basf.
com
Water
Providers
Douglas
Crawford­
Brown
Director
Institute
for
Environmental
Studies
University
of
North
Carolina
Environmental
Sciences
and
Engineering
Bldg.
Chapel
Hill,
NC
27599­
1105
919­
966­
6026
Fax
919­
966­
9920
douglas_
crawford­
brown@
unc.
edu
Worker
Protection
and
Labor
Organization
Marion
Moses
President
Pesticide
Education
Center
PO
Box
420870
San
Francisco,
CA
94142­
0870
415­
391­
8511
Fax
415­
391­
9159
pec@
pesticides.
org
EDSTAC
Final
Report
August
1998
7
A
­
National
Environmental
/
Environmental
Justice
/
Public
Health
Groups
Theo
Colborn
Senior
Scientist
Wildlife
and
Contaminants
Program
World
Wildlife
Fund,
Inc.
1250
24th
Street,
NW
Washington,
DC
20037
202­
778­
9643
Fax
202­
530­
0743
colborn@
wwfus.
org
Alternate
to
T.
Colborn
Richard
Liroff
Senior
Program
Officer
World
Wildlife
Fund,
Inc.
1250
24th
Street,
NW
Suite
500
Washington,
DC
20037
202­
778­
9644
Fax
202­
530­
0743
rich.
liroff@
wwfus.
org
Peter
de
Fur
Affiliate
Associate
Professor
Center
for
Environmental
Studies
Virginia
Commonwealth
University
Science
and
Environmental
Health
Network
PO
Box
843050
816
Park
Avenue,
Room
111
Richmond,
VA
23284
804­
828­
1760
Fax
804­
225­
3559
pldefur@
saturn.
vcu.
edu
Lovell
Allan
Jones
Professor
and
Director
Experimental
Gynecology/
Endocrinology
University
of
Texas
Department
of
Gynecologic
Oncology
M.
D.
Anderson
Cancer
Center
Box
304
1515
Holcombe
Blvd.
Houston,
TX
77030
713­
792­
3316
Fax
713­
794­
5553
lovell_
jones@
gynonc.
mdacc.
tmc.
edu
Ted
Schettler
Co­
Chair
Committee
on
Human
Health
&
the
Environment
Physicians
for
Social
Responsibility
124
Peterborough
Street,
Apt
12
Boston,
MA
02215
617­
536­
7033
Fax
617­
536­
7033
tschettler@
igc.
apc.
org
Gina
Solomon
Senior
Scientist
Public
Health
Program
Natural
Resources
Defense
Council
71
Stevenson
Street,
Suite
1825
San
Francisco,
CA
94105
415­
777­
0220
Fax
415­
495­
5996
gsolomon@
nrdc.
org
EDSTAC
Final
Report
August
1998
8
A
­
Research
Scientists
William
Benson
Director
Environmental
and
Community
Health
Research
School
of
Pharmacy
University
of
Mississippi
347
Faser
Hall
University,
MS
38677
601­
232­
7077
Fax
601­
232­
1285
whbenson@
olemiss.
edu
Ron
Kendall
Director
and
Professor
The
Institute
of
Environmental
and
Human
Health
Texas
Tech
University
Texas
Tech
University
Health
Sciences
Center
Reese
Center,
1207
Gilbert
Drive
Lubbock,
TX
79416
806­
885­
4567
Fax
806­
885­
2132
rkendall@
ttu.
edu
Rochelle
Tyl
Research
Director
for
the
Center
for
Life
Sciences
and
Toxicology
Senior
Program
Director,
Reproductive
and
Developmental
Toxicology
Research
Triangle
Institute
3040
Cornwallis
Road
Herman
Laboratory
Bldg.
,
Room
245
PO
Box
12194
Research
Triangle
Park,
NC
27709
919­
541­
5972
Fax
919­
541­
5956
rwt@
rti.
org
Judith
Weis
Professor
of
Marine
Biology/
Aquatic
Toxicology
Department
of
Biological
Sciences
Rutgers
University
101
Warren
Street
Newark,
NJ
07102
973­
353­
5387
Fax
973­
648­
5518
jweis@
andromeda.
rutgers.
edu
EPA
Staff
Anthony
Maciorowski
Senior
Technical
Advisor
Office
of
Prevention,
Pesticides
and
Toxic
Substances
U.
S.
Environmental
Protection
Agency
401
M
Street
SW,
MC
7405
403
East
Tower
Washington,
DC
20460
202­
260­
3048
Fax
202­
260­
8168
maciorowski.
anthony@
epamail.
epa.
gov
Gary
Timm
Senior
Technical
Advisor
Office
of
Prevention,
Pesticides
and
Toxic
Substances
Chemical
Control
Division
U.
S.
Environmental
Protection
Agency
401
M
Street
SW,
MC
7405
Washington,
DC
20460
202­
260­
1859
Fax
202­
260­
8168
timm.
gary@
epamail.
epa.
gov
EDSTAC
Final
Report
August
1998
9
A
­
Facilitation
Staff
Jocelyn
Adkins
Program
Assistant
The
Keystone
Center
1030
Fifteenth
Street,
NW,
Suite
300
Washington,
DC
20005
202­
783­
0248
Fax
202­
783­
0328
jadkins@
keystone.
org
Paul
De
Morgan
Associate
The
Keystone
Center
1030
Fifteenth
Street,
NW,
Suite
300
Washington,
DC
20005
202­
783­
0248
Fax
202­
783­
0328
pdemorgan@
keystone.
org
Liz
Finn
Project
Support
Coordinator
The
Keystone
Center
1628
Sts.
John
Road
Keystone,
CO
80435
970­
513­
5816
Fax
970­
262­
0152
lfinn@
keystone.
org
Timothy
Mealey
Senior
Partner
Meridian
Institute
P.
O.
Box
4005
Dillon,
CO
80435
970­
513­
8340
ext.
208
Fax
970­
513­
8348
tmealey@
merid.
org
Judy
O'Brien
Associate
The
Keystone
Center
1030
Fifteenth
Street,
NW,
Suite
300
Washington,
DC
20005
202­
783­
0248
Fax
202­
783­
0328
jobrien@
keystone.
org
Chris
Spaulding
Program
Assistant
The
Keystone
Center
1628
Sts.
John
Road
Keystone,
CO
80435
970­
513­
5831
Fax
970­
262­
0152
cspaulding@
keystone.
org
EDSTAC
Final
Report
August
1998
1
B
­
Appendix
B
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
Principles
Work
Group
Participant
List
Douglas
Crawford­
Brown
Director
Institute
for
Environmental
Studies
University
of
North
Carolina
Environmental
Sciences
and
Engineering
Bldg.
Chapel
Hill,
NC
27599­
1105
919­
966­
6026
Fax
919­
966­
9920
douglas_
crawford­
brown@
unc.
edu
George
Daston
Principal
Research
Scientist
Miami
Valley
Laboratories
Procter
&
Gamble
P.
O.
Box
538707
Cincinnati,
OH
45253
513­
627­
2886
Fax
513­
627­
1908
daston.
gp@
pg.
com
Donald
Lamb
Vice
President,
Product
Safety
and
Regulatory
Affairs
Bayer
Corporation
100
Bayer
Road
Pittsburgh,
PA
15205­
9741
412­
777­
7431
Fax
412­
777­
7484
don.
lamb.
b@
bayer.
com
Joseph
LeBeau
Retired
Vice
President,
Product
Responsibility
Dow
Chemical
Company
4701
Butternut
Place
Midland,
MI
48640
517­
835­
6244
Fax
517­
835­
6244
melraccoon@
aol.
com
George
Lucier
Director
of
the
Environmental
Toxicology
Program
National
Institute
for
Environmental
Health
Sciences
P.
O.
Box
12233,
MD
A3­
02
111
TW
Alexander
Drive
Research
Triangle
Park,
NC
27709
919­
541­
3802
Fax
919­
541­
3647
Lucier@
niehs.
nih.
gov
John
McLachlan
Professor
of
Pharmacology
Director,
Tulane\
Xavier
Center
for
Bioenvironmental
Research
Tulane
University
1430
Tulane
Avenue,
SL­
3
New
Orleans,
LA
70112
504­
585­
6910
Fax
504­
585­
6428
jmclach@
mailhost.
tcs.
tulane.
edu
EDSTAC
Final
Report
August
1998
2
B
­
Lawrence
Reiter
Director
National
Health
and
Environmental
Effects
Research
Laboratory
U.
S.
Environmental
Protection
Agency
2525
Highway
54
Research
Triangle
Park,
NC
27711
919­
541­
2281
Fax
919­
541­
4324
reiter@
herl45.
herl.
epa.
gov
Gina
Solomon
Senior
Scientist
Public
Health
Program
Natural
Resources
Defense
Council
71
Stevenson
Street,
Suite
1825
San
Francisco,
CA
94105
415­
777­
0220
Fax
415­
495­
5996
gsolomon@
nrdc.
org
Rochelle
Tyl
Research
Director
for
the
Center
for
Life
Sciences
and
Toxicology
Senior
Program
Director,
Reproductive
and
Developmental
Toxicology
Research
Triangle
Institute
Herman
Laboratory
P.
O.
Box
12194
Research
Triangle
Park,
NC
27709
919­
541­
5972
Fax
919­
541­
5956
rwt@
rti.
org
Judith
Weis
Professor
of
Marine
Biology/
Aquatic
Toxicology
Department
of
Biological
Sciences
Rutgers
University
101
Warren
Street
Newark,
NJ
07102
973­
353­
5387
Fax
973­
353­
5518
jweis@
andromeda.
rutgers.
edu
Facilitators
Paul
De
Morgan
Associate
The
Keystone
Center
1030
Fifteenth
Street,
NW,
Suite
300
Washington,
DC
20005
202­
783­
0248
Fax
202­
783­
0328
pdemorgan@
keystone.
org
Timothy
Mealey
Senior
Partner
Meridian
Institute
P.
O.
Box
4005
Dillon,
CO
80435
970­
513­
8340
ext.
208
Fax
970­
513­
8348
tmealey@
merid.
org
EDSTAC
Final
Report
August
1998
1
C
­
Appendix
C
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
Priority
Setting
Work
Group
Participant
List
Lori
Abrams
Project
Industrial
Hygienist
Formerly
with
United
Auto
Workers
Health
and
Safety
2635
Brookwood
Road
614­
236­
2405
Fax
614­
236­
2405
loriabrams@
aol.
com
Henry
Anderson
Chief
Medical
Officer
Wisconsin
Bureau
of
Public
Health
1414
E.
Washington
Avenue,
Room
96
Madison,
WI
53703
608­
266­
1253
Fax
608­
267­
4853
anderha@
DHFS.
state.
wi.
us
Pat
Basu
Director,
Chemistry
and
Toxicology
Division
Office
of
Public
Health
&
Science
Food
Safety
and
Inspection
Service
U.
S.
Department
of
Agriculture
Room
6912
Franklin
Court
Suite
1400
Independence
Avenue
S.
W.
Washington,
DC
20250
202­
501­
7319
Fax
202­
501­
7639
pat.
basu@
usda.
gov
Chris
DeRosa
Director,
Division
of
Toxicology
Agency
for
Toxic
Substances
and
Disease
Registry
1600
Clifton
Road
MS­
29
Atlanta,
GA
30101
404­
639­
6300
Fax
404­
639­
6315
cyd0@
atsod3.
em.
cdc.
gov
Angelina
Duggan
Registrations
Manager
Development
and
Regulatory
Affairs
Business
Management
Department
FMC
Agricultural
Products
Group
1735
Market
Street
Philadelphia,
PA
19103
215­
299­
6670
Fax
215­
299­
6468
angelina_
duggan@
fmc.
com
Jack
Housenger
Associate
Director
Special
Review
and
Registration
Division
Office
of
Pesticide
Programs
U.
S.
Environmental
Protection
Agency
401
M
Street
SW
Washington,
DC
20460
703­
308­
8163
Fax
703­
308­
8005
housenger.
jack@
epamail.
epa.
gov
EDSTAC
Final
Report
August
1998
2
C
­
Nancy
Kim
Director
Division
of
Environmental
Health
Assessment
New
York
State
Department
of
Health
2
University
Place,
Room
350
Albany,
NY
12203
518­
458­
6438
Fax
518­
458­
6436
nkk01@
health.
state.
ny.
us
Richard
Liroff
Senior
Program
Officer
World
Wildlife
Fund,
Inc.
1250
24th
Street,
NW
Suite
500
Washington,
DC
20037
202­
778­
9644
Fax
202­
530­
0743
rich.
liroff@
wwfus.
org
Anthony
Maciorowski
Senior
Technical
Advisor
Office
of
Prevention,
Pesticides
and
Toxic
Substances
U.
S.
Environmental
Protection
Agency
401
M
Street
SW,
MC
7405
403
East
Tower
Washington,
DC
20460
202­
260­
3048
Fax
202­
260­
8168
maciorowski.
anthony@
epamail.
epa.
gov
Stephanie
Miles­
Richardson
Environmental
Health
Scientist
Division
of
Toxicology
Agency
for
Toxic
Substances
and
Disease
Registry
Executive
Park,
Building
4
1600
Clifton
Road,
NE,
E­
29
Atlanta,
GA
30333
404­
639­
5282
Fax
404­
639­
5084
srm7@
cdc.
gov
EDSTAC
Final
Report
August
1998
3
C
­
Marion
Moses
President
Pesticide
Education
Center
P.
O.
Box
420870
San
Francisco,
CA
94142­
0870
415­
391­
8511
Fax
415­
391­
9159
pec@
pesticides.
org
Thomas
Osimitz
Vice
President
Global
Safety
Assessment
and
Regulatory
Affairs
SC
Johnson
&
Son,
Inc.
1525
Howe
Street
Racine,
WI
53403
414­
260­
2669
Fax
414­
260­
0186
tgosimit@
scj.
com
Richard
Purdy
Senior
Ecotoxicologist
3M
Corporation
935
Bush
Avenue,
Bldg.
2­
3­
09
P.
O.
Box
33331
St.
Paul,
MN
55133
612­
778­
5379
Fax
612­
778­
6176
repurdy@
mmm.
com
James
Quance
Environmental
Issues
Manager
Exxon
Chemical
Company
13501
Katy
Freeway
Houston,
TX
77079
281­
870­
6281
Fax
281­
588­
4664
jim.
f.
quance@
chemical.
exxon.
sprint.
com
EDSTAC
Final
Report
August
1998
4
C
­
Barnett
Rattner
Deputy
Division
Chief
Patuxent
Wildlife
Research
Center
U.
S.
Geological
Survey
12011
Beech
Forest
Road
Laurel,
MD
20708­
4041
301­
497­
5671
Fax
301­
497­
5675
barnett_
rattner@
nbs.
gov
Florence
Robinson
North
Baton
Rouge
Environmental
Association
421
Springfield
Road
Baton
Rouge,
LA
70807
504­
775­
0341
Fax
504­
774­
2928
Ted
Schettler
Co­
Chair
Committee
on
Human
Health
&
the
Environment
Physicians
for
Social
Responsibility
124
Peterborough
Street,
Apt
12
Boston,
MA
02215
617­
536­
7033
Fax
617­
536­
7033
tschettler@
igc.
apc.
org
Tom
Sinks
Associate
Director
for
Science
National
Center
for
Environmental
Health
Centers
for
Disease
Control
and
Prevention
4770
Buford
Highway
NE,
MS
F­
29
Atlanta,
GA
30341­
3724
770­
488­
7001
Fax
770­
488­
7015
ths2@
cdc.
gov
John
Walker
Director
TSCA
Interagency
Testing
Committee
U.
S.
Environmental
Protection
Agency
401
M
Street
(
MC
7401)
E.
Tower,
Room
737C
Washington,
DC
20460
202­
260­
1820
Fax
202­
260­
7895
walker.
johnd@
epamail.
epa.
gov
Chris
Waller
Research
Manager
MYCOsearch
Natural
Products
Discovery
OSI
Pharmaceuticals,
Inc.
4727
University
Drive,
Suite
400
Durham,
NC
27707
919­
489­
4700
Fax
919­
490­
3745
cwaller@
osip.
com
Facilitation
Staff
Timothy
Mealey
Senior
Partner
Meridian
Institute
P.
O.
Box
4005
Dillon,
CO
80435
970­
513­
8340
ext.
208
Fax
970­
513­
8348
tmealey@
merid.
org
Chris
Spaulding
Program
Assistant
The
Keystone
Center
1628
Sts.
John
Road
Keystone,
CO
80435
970­
513­
5831
Fax
970­
262­
0152
cspaulding@
keystone.
org
EDSTAC
Final
Report
August
1998
1
D
­
Appendix
D
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
Screening
and
Testing
Work
Group
Participant
List
Christopher
Borgert
Pharmacologist/
Toxicologist
Consultant
to
Aquatrols,
Inc.
238
Turkey
Creek
10514
Palmetto
Blvd.
Alachua,
FL
32615
904­
462­
1266
Fax
904­
462­
1267
cjborgert@
APT­
PHARMATOX.
COM
George
Daston
Principal
Research
Scientist
Procter
&
Gamble
Miami
Valley
Laboratories
P.
O.
Box
538707
Cincinnati,
OH
45253
513­
627­
2886
Fax
513­
627­
1908
daston.
gp@
pg.
com
Penny
Fenner­
Crisp
U.
S.
Environmental
Protection
Agency
Office
of
Prevention,
Pesticides,
and
Toxic
Substances,
7101
401
M
Street
SW
WSM
East
Tower
633
Washington,
DC
20460
202­
260­
0947
Fax
202­
260­
1847
fenner­
crisp.
penelope@
epamail.
epa.
gov
John
French
Research
Biologist
U.
S.
Geological
Survey
Patuxent
Wildlife
Research
Center
12011
Beech
Forest
Road
Laurel,
MD
20708
301­
497­
5702
Fax
301­
497­
5744
john_
b_
french@
usgs.
gov
Earl
Gray
Section
Chief
U.
S.
Environmental
Protection
Agency
Developmental
and
Reproductive
Toxicology
Health
Effects
Research
Laboratory
MD­
72
Research
Triangle
Park,
NC
27711
919­
541­
7750
Fax
919­
541­
5138
gray.
earl@
epamail.
epa.
gov
Heraline
Hicks
Senior
Environmental
Health
Scientist
ATSDR
1600
Clifton
Road,
NE
MS
E29
Atlanta,
GA
30333
404­
639­
6306
Fax
404­
639­
6315
heh2@
atsod3.
em.
cdc.
gov
EDSTAC
Final
Report
August
1998
2
D
­
Lovell
Jones
Professor
and
Director
M.
D.
Anderson
Cancer
Center
University
of
Texas
Department
of
Gynecologic
Oncology
Box
304
1515
Holcombe
Blvd.
Houston,
TX
77030
713­
792­
3316
Fax
713­
794­
5553
lovell_
jones@
gynonc.
mdacc.
tmc.
edu
Ron
Kendall
Director
and
Professor
The
Institute
of
Environmental
and
Human
Health
Texas
Tech
University
Health
Sciences
Center
Reese
Center,
1207
Gilbert
Drive
Lubbock,
TX
79416
806­
885­
4567
Fax
806­
885­
2132
rkendall@
ttu.
edu
Ron
Miller
Project
Manager
Dow
Chemical
Company
Chemical
Environmental
Affairs
2020
Dow
Center
Midland,
MI
48674
517­
636­
3093
Fax
517­
636­
9899
rrmiller@
dow.
com
Teri
Rowles
Coordinator
of
the
Marine
Mammal
Health
and
Stranding
Response
Program
National
Oceanic
and
Atmospheric
Administration
1315
East
West
Hwy
Silver
Spring,
MD
20910
301­
713­
2322
Fax
301­
713­
0376
teri.
rowles@
noaa.
gov
Daniel
Sheehan
Research
Biologist
U.
S.
Food
and
Drug
Administration
NTCR/
Division
of
Reproductive
and
Developmental
Toxicology
3900
NCTR
Road
Jefferson,
AR
72079
501­
543­
7561
Fax
501­
543­
7682
dsheehan@
nctr.
fda.
gov
Michael
Shelby
National
Institute
for
Environmental
Health
Sciences
111
Alexander
Drive
MD:
B3­
09
Research
Triangle
Park,
NC
27709
919­
541­
3455
Fax
919­
541­
4634
shelby@
niehs.
nih.
gov
Ana
Soto
Associate
Professor
Tufts
University
Department
of
Cellular
Biology
School
of
Medicine
136
Harrison
Avenue
Boston,
MA
02111
617­
636­
6954
Fax
617­
636­
6536
asoto@
infonet.
tufts.
edu
Peter
Thomas
Professor
University
of
Texas,
Marine
Science
Institute
750
Channelview
Drive
Port
Aransas,
TX
78373
512­
749­
6768
Fax
512­
749­
6777
thomas@
utmsi.
zo.
utexas.
edu
Gary
Timm
Senior
Technical
Advisor
EDSTAC
Final
Report
August
1998
3
D
­
U.
S.
Environmental
Protection
Agency
Office
of
Prevention,
Pesticides
and
Toxic
Substances
Chemical
Control
Division
401
M
Street
SW,
MC
7405
Washington,
DC
20460
202­
260­
1859
Fax
202­
260­
8168
timm.
gary@
epamail.
epa.
gov
Abraham
Tobia
Manager,
Toxicology
and
Risk
Assessment
BASF
Corporation
26
Davis
Drive
P.
O.
Box
13528
Research
Triangle
Park,
NC
27709­
3578
919­
547­
2972
Fax
919­
547­
2880
tobiaa@
basf.
com
Leslie
(
Les)
Touart
Supervisory
Biologist
U.
S.
Environmental
Protection
Agency
Environmental
Fate
and
Effects
Division
Office
of
Pesticide
Program
401
M
Street
(
MC
7507C)
Washington,
DC
20460
703­
305­
6134
Fax
703­
308­
6181
touart.
les@
epamail.
epa.
gov
Rochelle
Tyl
Research
Director
for
the
Center
for
Life
Sciences
and
Toxicology
Research
Triangle
Institute
Senior
Program
Director,
Reproductive
and
Developmental
Toxicology
3040
Cornwallis
Road,
Herman
Laboratory
Bldg.
P.
O.
Box
12194
Research
Triangle
Park,
NC
27709
919­
541­
5972
Fax
919­
541­
5956
rwt@
rti.
org
Thomas
Zoeller
Associate
Professor
University
of
Massachusetts,
Amherst
Biology
Department
221
Morrill
Science
Center
Amherst,
MA
01003
413­
545­
2088
Fax
413­
545­
3243
tzoeller@
bio.
umass.
edu
Facilitator
Paul
De
Morgan
Associate
The
Keystone
Center
1030
Fifteenth
Street,
NW,
Suite
300
Washington,
DC
20005
202­
783­
0248
Fax
202­
783­
0328
pdemorgan@
keystone.
org
EDSTAC
Final
Report
August
1998
1
E
­
Appendix
E
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
Communications
and
Outreach
Work
Group
Participant
List
Davis
Baltz
Environmental
Education
Training
Specialist
Commonweal,
Inc.
451
Mesa
Road
P.
O.
Box
316
Bolinas,
CA
94924
Phone
415­
868­
0970
Fax
415­
868­
2230
dbaltz@
igc.
apc.
org
Christopher
Borgert
Pharmacologist/
Toxicologist
Consultant
to
Aquatrols,
Inc.
238
Turkey
Creek
10514
Palmetto
Blvd.
Alachua,
FL
32615
Phone
904­
462­
1266
Fax
904­
462­
1267
cjborgert@
APT­
PHARMATOX.
COM
Sarah
Brozena
Managing
Counsel
Chemical
Manufacturers
Association
1300
Wilson
Blvd.
Arlington,
VA
22209
703­
741­
5159
Fax
703­
741­
6092
sarah_
brozena@
mail.
cmahq.
com
Douglas
Crawford­
Brown
Director
University
of
North
Carolina
Institute
for
Environmental
Studies
Environmental
Sciences
and
Engineering
Bldg.
Chapel
Hill,
NC
27599­
1105
Phone
919­
966­
6026
Fax
919­
966­
9920
douglas_
crawford­
brown@
unc.
edu
EDSTAC
Final
Report
August
1998
2
E
­
Peter
de
Fur
Affiliate
Associate
Professor
Science
and
Environmental
Health
Network
Center
for
Environmental
Studies
P.
O.
Box
843050
816
Park
Avenue,
Room
111
Richmond,
VA
23284
Phone
804­
828­
1760
Fax
804­
225­
3559
pldefur@
saturn.
vcu.
edu
Anna
Fan
Chief
California
Environmental
Protection
Agency
Pesticide
and
Environmental
Toxicology
Section/
OEHHA
2151
Berkeley
Way,
Annex
11,
7th
Floor
Berkeley,
CA
94704
Phone
510­
540­
3066
Fax
510­
540­
3674
afan@
berkeley.
cahwnet.
gov
Lovell
Jones
Professor
and
Director
M.
D.
Anderson
Cancer
Center
University
of
Texas
Box
304
1515
Holcombe
Blvd.
Houston,
TX
77030
Phone
713­
792­
3316
Fax
713­
794­
5553
lovell_
jones@
gynonc.
mdacc.
tmc.
edu
EDSTAC
Final
Report
August
1998
3
E
­
Max
Lum
Director,
Health
Communications
National
Institute
for
Occupational
Safety
&
Health
200
Independence
Avenue
SW
Room
715H
Washington,
DC
20201
Phone
202­
260­
9723
Fax
202­
260­
1898
mrl1@
oddc1.
em.
cdc.
gov
John
McCarthy
Vice
President
American
Crop
Protection
Association
Science
and
Regulatory
Affairs
1156
15th
Street
NW
Washington,
DC
20005
Phone
202­
872­
3876
Fax
202­
463­
0474
john@
acpa.
org
Linda
Murray
Office
of
Pesticide
Programs
U.
S.
Environmental
Protection
Agency
401
M
Street
SW
(
MC
7501C)
Washington,
DC
20460
703­
305­
5401
Fax
703­
305­
5558
murray.
linda@
epamail.
epa.
gov
Timothy
O'Leary
Director,
Environmental
Health
Association
of
State
and
Territorial
Public
Health
Officials
1275
K
Street
NW
Suite
800
Washington,
DC
20005
Phone
202­
371­
9090
Fax
202­
371­
9797
toleary@
astho.
org
James
Quance
Environmental
Issues
Manager
Exxon
Chemical
Company
13501
Katy
Freeway
Houston,
TX
77079
Phone
281­
870­
6281
Fax
281­
588­
4664
jim.
f.
quance@
chemical.
exxon.
sprint.
com
Florence
Robinson
North
Baton
Rouge
Environmental
Association
421
Springfield
Road
Baton
Rouge,
LA
70807
Phone
504­
775­
0341
Fax
504­
774­
2928
robinsof@
rtk.
net
Tim
Tinker
Chief,
Communications
and
Research
ATSDR
1600
Clifton
Road
E­
60
Executive
Park
Drive,
Bldg.
35,
Suite
3530
Atlanta,
GA
30333
Phone
404­
639­
5013
Fax
404­
639­
0560
txt2@
cdc.
gov
Judith
Weis
Professor
of
Marine
Biology/
Aquatic
Toxicology
Rutgers
University
Department
of
Biological
Sciences
101
Warren
Street
Newark,
NJ
07102
Phone
973­
353­
5387
Fax
973­
353­
5518
jweis@
andromeda.
rutgers.
edu
EDSTAC
Final
Report
August
1998
4
E
­
Facilitator
Judy
O'Brien
Associate
The
Keystone
Center
1030
Fifteenth
Street,
NW,
Suite
300
Washington,
DC
20005
202­
783­
0248
Fax
202­
783­
0328
jobrien@
keystone.
org
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
1
F
­
Appendix
F
References
and
Sources
for
Chapter
Four
 
Priority
Setting
Adlercreutz
H.,
K.
Hockerstedt,
C.
Bannwart,
et
al.,
"
Effect
of
dietary
components,
including
lignans
and
phytoestrogens,
on
enterohepatic
circulation
and
liver
metabolism
of
estrogens
and
on
sex
hormone
binding
globulin
(
SHBG),"
J.
Steroid
Biochem,
27,
1987,
(
4­
6):
1135­
1144.
Constantinou,
A.,
and
E.
Huberman,
"
Genistein
as
an
Inducer
of
Tumor
Cell
Differentiation:
Possible
Mechanisms
of
Action,"
Proceedings
of
the
Society
for
Experimental
Biology
and
Medicine,
CCVIII,
1995.
Essex,
C.,
"
Phytoestrogens
and
Soy­
Based
Infant
Formula,"
British
Medical
Journal,
CCCXIII,
1996,
pp.
507­
508.
Hendrix,
S.
L.,
"
Nonestrogen
Management
of
Symposium
Symptoms,"
Endocrine
and
Metabolism
Clinics
of
North
America,
XXVI,
1997,
(
2):
379­
390.
Naftolin,
F.,
and
P.
L.
Whitten,
"
Dietary
Estrogens:
A
Biologically
Active
Background
for
Estrogenic
Action,"
Proceedings
from
the
New
Biology
of
Steroid
Hormones,
Budapest,
Hungary,
1990.
Okura,
A.,
et
al.,
"
Effect
of
Genistein
on
Topoisomerase
Activity
and
on
the
Growth
of
(
Val
12)
Ha
­
ras
­
Transformed
NIH
3T3
Cells,"
Biochemical
and
Biophysical
Research
Communications,
CLVII,
1988,
pp.
183­
189.
Richard,
S.
E.,
and
L.
U.
Thompson,
"
Phytoestrogens
and
Lignans:
Effect
on
Reproduction
and
Chronic
Disease,"
Antinutrients
and
Phytochemicals
in
Food.
American
Chemical
Society,
16,
1997,
pp.
273­
293.
Slama,
K.,
"
Animal
Hormones
and
Antihormones
in
Plants,"
Biochem
Physiol.
Pflanzen,
CVXXV,
1980,
pp.
177­
193.
Slavin,
J.,
"
More
Information
on
Phytoestrogens
in
Breast
Milk,"
Clinical
Chemistry,
XLIII,
1997,
pp.
548­
549.
"
Toxicants
Occurring
Naturally
In
Foods,"
National
Academy
of
Sciences,
1973.
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
1
G
­
Appendix
G
EXPOSURE
&
EFFECTS
DATA
SOURCE
MATRICES
TABLE
1.
BIOLOGICAL
SAMPLING
DATA
Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Bay
Delta
Estuary
Monitoring
Program
Description:
Fish,
surface
water,
various
contaminants
(
San
Francisco
Bay)
Citation:
Availability:
STORET
(
Phone:
800­
424­
9067).
?

Columbia
River
Basin
Fish
and
Wildlife
Program
Description:
Contaminants,
media
(
Columbia
River
Basin)
Citation:
Northwest
Power
Planning
Council.
Availability:
?

Environmental
Contaminant
Data
Management
System
Description:
Compilation
of
analytical
data
of
approximately
100,000
samples
of
invertebrates,
fish
and
wildlife
in
the
Environmental
Contaminant
Data
Management
System
of
the
Patuxent
Analytical
Control
Facility,
U.
S.
Fish
and
Wildlife
Service.
The
presence
and
concentration
of
about
625
compounds
(
pesticides
and
organochlorines,
contaminants,
metals,
and
petroleum
hydrocarbons)
are
recorded
from
a
wide
variety
of
environmental
biomonitoring
and
research
activities.
This
database
contains
many
unique
datasets
that
are
not
replicated
in
time
or
space.
The
analytical
procedures
do
not
favor
amines,
sulfides,
and
volatile
compounds.

Citation:
Patuxent
Analytical
Control
Facility,
U.
S.
Fish
and
Wildlife
Service.
Availability:
Y
Great
Lakes
Fish
Monitoring
Program
Description:
Fish
tissues,
pesticides.
Compilation
of
data
from
a
30­
year
biomonitoring
effort
conducted
by
the
Great
Lakes
Science
Center
of
the
U.
S.
Geological
Survey.
Composite
samples
of
whole­
fish
collected
from
the
Great
Lakes
and
nearby
rivers
were
analyzed
by
fullscan
gas
chromatography/
mass
spectral
analysis
for
over
550
compounds.
The
presence
and
concentration
of
a
wide
range
of
pesticides,
organochlorines,
ketones,
aldehydes,
alcohols,
phenols
and
other
oxygenates
contaminants
are
measured
in
fish
of
a
known
species,
size
and
age.
The
analytical
procedures
do
not
favor
amines,
sulfides,
and
volatile
compounds.
Polychlorinated
dibenzodioxins
and
dibenzofurans
are
not
quantified.
Citation:
Hesselberg,
R.
J.,
J.
P.
Hickey,
D.
A.
Nortrup,
and
W.
A.
Willford,
"
Contaminant
Residues
in
the
Bloater
(
Coregonus
hoyi)
of
Lake
Michigan,"
J.
Great
Lakes
Res.,
16(
1),
1990,
pp.
121­
129.
Great
Lakes
Science
Center,
U.
S.
Geological
Survey.
Availability:
EPA
Great
Lakes
Program
Office,
Chicago,
IL
Y
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
2
G
­
TABLE
1.
BIOLOGICAL
SAMPLING
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

National
Health
and
Nutrition
Examination
Survey
(
NHANES)
Description:
National
survey
of
U.
S.
population
includes
physical
examination
and
collection
of
blood
and
urine
samples.
Biological
measurements
have
included
specific
environmental
contaminants
(
e.
g.,
lead,
VOCs,
pesticides).
Four
surveys
have
been
completed:
(
National
Health
Service­
dates,
NHANES
II­
dates,
Hispanic
NHANES­
dates,
and
NHANES
III­
dates).
NHANES
III
was
conducted
in
two
phases
and
represents
two
samplings
of
the
U.
S.
population.
NHANES
III
and
IV
include
approximately
40,000
people.
Stored
serum
remains
available
for
NHANES
III
and
possibly
Hispanic
NHANES.
Representativeness
is
maintained
for
demographic
strata
including
age,
gender,
race,
and
region.
All
data
are
quality
controlled.
CLIA
regulations
for
analyses.
Assured
Quality
Assurance/
Quality
Control
(
QA/
QC)
with
analytic
comparability
over
time.
This
is
the
only
ongoing
survey
that
is
representative
of
the
U.
S.
population
with
weights
to
estimate
population
distributions
according
to
age,
race,
gender,
and
region.
Provides
unique
data
on
human
adipose
levels
of
contaminations
to
toxic
substances.
Analyses
by
standard
protocol.
Tissues
may
not
be
representative
of
the
US
population.
Citations:
·
Plan
and
Operation
of
the
Third
National
Health
and
Nutrition
Examination
Survey,
National
Center
for
Health
Statistics,
1988­
94.
·
Vital
Health
Statistics,
1994.
·
Exposure
of
the
U.
S.
Population
to
Environmental
Tobacco
Smoke:
NHANES
III,
1988­
91.
·
Pirkle,
J.
L.,
K.
M.
Flegal,
J.
T.
Bernert,
et
al.,
JAMA,
275,
1996,
pp.
1233­
1240.
·
VOC's
Ashley.
Availability:
Public
use
computer
tapes.
Stored
samples
for
NHANES
III;
authorization
must
be
sought.
Y
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
3
G
­
TABLE
1.
BIOLOGICAL
SAMPLING
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

National
Human
Adipose
Tissue
Survey
(
NHATS)

Office
of
Toxic
Substances,
U.
S.
EPA
Description:
National
survey
of
human
adipose
tissue
for
PCBs,
dioxins/
furans,
volatile
and
semivolatile
organics.
Analyses
of
adipose
tissue
from
autopsied
cadavers
and
surgical
patients.
Analyses
for
a
variety
of
toxic
compounds
using
standardized
protocols
on
composites
that
represent
nine
regions
and
three
age
groups.
Citations:
·
Human
Body
Burden
Database,
EPA
Field
Studies
Branch.
·
U.
S.
EPA,
"
Broad
scan
analysis
of
the
FY
82
National
Human
Adipose
Tissue
Survey
specimens,"
Vol.
1­
Executive
Summary,
U.
S.
EPA
Document
No.
EPA­
560/
5­
86­
035,
Washington,
D.
C.
·
Phillips
and
Birchard,
Arch.
Environ.
Contam.
Toxicol.,
21,
1991,
pp.
159­
168.
Availability:
EPA
Field
Studies
Branch,
Washington
DC
(
Phone:
202­
382­
3853),
Nationwide,
discontinued
1990
Y
National
Human
Exposure
Assessment
Survey
(
NHEXAS)
Description:
Surveys
designed
to
assess
human
exposure
via
multiple
pathways
(
food,
water,
air,
dust).
Limited
biomonitoring
(
urine,
blood).
Could
provide
unique
data
on
individual
(
nonoccupational
human
ambient
exposures
as
well
as
resulting
levels
of
pollutants
in
human
blood
and
urine.
Analysis
by
standard
protocol.
Could
provide
important
information
for
preventions
targeted
to
eliminating
specific
exposure
pathways.
May
also
be
focused
on
sensitive
populations.
Citation:
Office
of
Research
and
Development,
U.
S.
EPA.
Availability:
Not
yet
available.
Full
scale
funding
has
not
been
secured.
?

National
Neonatal
Bloodspot
Exposure
and
Effects
Survey
(
NNBEES)
­
proposed
Description:
NCEH
provides
national
quality
assurance
for
state
neonatal
blood
spot
programs.
All
babies
born
in
the
U.
S.
are
bled
and
tested
for
a
variety
of
metabolic
and
hereditary
conditions.
Could
provide
data
on
exposure
levels
to
endocrine
disrupting
substances
of
newborn
babies.
Measurements
of
newborn
hormone
levels
could
be
taken
from
same
blood
spot.
Contamination
is
an
issue
so
care
in
handling
must
be
emphasized,
unless
substance
to
be
measured
is
not
found
in
the
environment.
Citation:
National
Center
for
Environmental
Health
(
NCEH),
Centers
for
Disease
Control
and
Prevention.
Availability:
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
4
G
­
TABLE
1.
BIOLOGICAL
SAMPLING
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

National
Status
and
Trends:
Mussel
Watch,
Benthis
Surveillance
Description:
Mussels,
other
bivalves,
sediments,
bottom
fish,
major
and
trace
elements,
PAHs,
PCBs.
chlorinated
compounds,
coastal
areas
Citation:
National
Oceanic
and
Atmospheric
Administration
(
NOAA).
Availability:
National
status
and
trends
database,
Tom
O'Connor,
NOAA­
OAD
(
Phone:
301­
443­
8698).
?

Puget
Sound
Monitoring
Program
Description:
Fish,
surface
water,
sediments,
PAHs,
PCBs
(
Puget
Sound)
Citation:
Puget
Sound
Water
Quality
Authority.
Availability:
?

Total
Exposure
Assessment
Methodology
(
TEAM)
Study
­
1979­
1985
Description:
Breath
samples
analyzed
for
20
target
chemicals.
Included
nursing
study,
dry
cleaners
study,
swimming
pool
exposures
study,
and
indoor
air
study.
Assured
QA/
QC
with
analytic
comparability
over
time.
Citation:
Office
of
Acid
Deposition,
Environmental
Monitoring,
and
QA.
Availability:
Y
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
5
G
­
TABLE
2.
ENVIRONMENTAL,
OCCUPATIONAL,
FOOD,
AND
CONSUMER
PRODUCT
DATA
Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Agency
for
Toxic
Substances
Disease
Registry
(
ATSDR)
Priority
List
(
PL)
Description:
Biannual
list
of
hazardous
substances,
in
order
of
priority,
which
are
most
commonly
found
at
facilities
on
the
Comprehensive
Environmental
Response,
Compensation,
and
Liability
Act
(
CERCLA)
National
Priorities
List
and
that
pose
the
most
significant
potential
threat
to
human
health.
Identifies
hazardous
substances
commonly
found
at
certain
sites
that
pose
a
threat
to
human
health.
Sites
at
which
the
chemicals
are
found
and
the
frequency
of
finding
them
are
not
disclosed.
Citation:
Availability:
Publicly
available.
Y
American
Conference
of
Governmental
Industrial
Hygienists
(
ACGIH)
Threshold
Limit
Value
(
TLV)
List
Description:
The
ACGIH
is
a
non­
governmental
organization
that
issues
recommended
acceptable
workplace
exposure
limits
(
TLVs)
for
several
hundred
chemicals.
Citation:
Availability:
Publicly
available.
The
ACGIH
TLV
booklet
is
available
from
ACGIH
in
Cincinnati,
OH,
and
may
be
available
via
Internet.
Y
Ashford's
Dictionary
of
Industrial
Chemicals
Description:
Use
information.
Secondary
source.

Citation:
Ashford,
R.
D.
,
Ashford's
Dictionary
of
Industrial
Chemicals:
Properties,
Production,
Uses,
London,
England:
Wavelength
Publ,
Ltd.,
1994.

Availability:
Published
information.
Book.
N
California
Department
of
Food
and
Agriculture
(
CDFA)/
California
Department
of
Pesticide
Regulation
(
CDPR)
Description:
Subcategories:
Foreign,
domestic,
water,
worker
exposure,
retail,
pesticide
use,
fields
targeted
by
county
agriculture
commissioners.
Raw
data
and
summaries.
Very
close
to
GLPs.
Citation:
Availability:
Paul
Gosselin,
Division
of
Enforcement
and
Data
Management.
Contact
California
Department
of
Pesticide
Regulation/
Cal
EPA
for
fruits
and
vegetables;
Worker
Health
and
Safety
Group/
Cal
EPA
for
worker
exposure;
Environmental
Monitoring/
Cal
EPA
for
environmental
samples.
CDFA
analyzes
crops
under
contract
to
Cal
EPA.
?

Certified
and
Commercial
Pesticide
Applicator
Survey
Description:
Non­
agricultural
use
data
for
pesticides.
Citation:
Availability:
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
6
G
­
TABLE
2.
ENVIRONMENTAL,
OCCUPATIONAL,
FOOD,
AND
CONSUMER
PRODUCT
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Chem
Use
Database
Description:
Use
information.
Summary
of
information
presented
on
PMNs
and
results
of
patent
searches.
Citation:
Chem
Use
Data
Base.
Availability:
TSCA
Not
publicly
available.
Available
on
an
as­
needed
basis
with
TSCA
NPA
clearance.
?

Chesapeake
Bay
Monitoring
Program
Description:
Surface
water,
sediments,
air,
various
contaminants
(
Chesapeake
Bay)
Citation:
Availability:
?

chemicals
found
in
cosmetics
Description:
Citation:
FDA
Availability:.
?

Consumer
Product
Indoor
Air
Source
Ranking
Database
Description:
Data
on
approximately
1,400
chemicals
used
in
consumer
products.
Citation:
U.
S.
EPA,
Office
of
Pollution
Prevention
and
Toxic
Substances
(
OPPT)
Availability:.
Y?

Eastern
Fine
Particle
Visibility
Network
Description:
Air,
carbon,
volatile
organics,
NO3,
HNO3,
particulate
sulfate
Citation:
East
regional,
Acid
Deposition
System,
Batelle
PNL.
Availability:
Tom
Thomas,
(
Phone:
509­
375­
2783).
?

Environmental
Contaminant
Reference
Databook
Description:
Use
information.
Secondary
source.
Citation:
Prager,
Jan
C.
New
York,
NY:
Van
Nostrand
Reinhold.
Availability:
Published
information.
2
volume
book
set.
N
Environmental
Monitoring
and
Assessment
Program
(
EMAP)
Description:
Air,
groundwater,
surface
water,
biota,
soil
by
ecosystem
group,
pollutants
by
ecosystem
(
nationwide)
Citation:
EMAP
Research
&
Assessment
Center,
ORD,
U.
S.
EPA,
Research
Triangle
Availability:
Y?

EPA
Doane
3
Profile
Data
Base
Description:
Production
and
use
data
for
pesticides.
Produced
annually.
Computerized.
Citation:
Availability:
Not
publicly
available.
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
7
G
­
TABLE
2.
ENVIRONMENTAL,
OCCUPATIONAL,
FOOD,
AND
CONSUMER
PRODUCT
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Farmers
Pesticide
Use/
Satisfaction
Study
Description:
Product/
use
data
for
pesticides.
Survey
information
produced
annually.
Computerized.
Citation:
Availability:
Not
publicly
available.
?

FDA
­
Surveillance
and
Compliance
Description:
Subcategories:
Domestic,
import,
surveillance,
compliance.
Summary,
raw
data.
Most
QA
measures
observed;
not
strictly
GLP.
Citation:
FDA
Availability:
On
diskette,
web
site
in
future,
Mike
Bolger,
FDA,
(
Phone:
202­
205­
8705)
?

Florida
Department
of
Agriculture
and
Consumer
Services
Description:
Subcategories:
Domestic,
foreign,
ground
water,
surface
water.
Raw
data.
Violations
summarized.
Not
strictly
GLP.
Citation:
Availability:
George
Fong
(
Phone:
904­
488­
9670).
?

Food
Quality
Protection
Act
(
FQPA)
"
Cumulative
to
Pesticides"
List
Description:
List
of
chemicals
satisfying
statutory
requirements
of
being
cumulative
to
pesticides.
Citation:
Availability:.
Y?

Generally
Regarded
As
Safe
(
GRAS)
Substances
Description:
Citation:
FDA
Availability:.
Y
Handbook
of
Environmental
Data
on
Organic
Chemicals,
3rd
edition
Description:
Use
information.
Secondary
source.

Citation:
Verschueren,
Karel.
New
York,
NY:
Van
Nostrand
Reinhold.

Availability:
Published
information.
Book.
N
Hawley's
Condensed
Chemical
Dictionary
Description:
Use
information.
Secondary
source.

Citation:
Lewis,
R.
J.,
Sr.
(
ed.),
Hawley's
Condensed
Chemical
Dictionary.
12th
ed.
New
York,
NY:
Van
Nostrand
Reinhold,
1993.

Availability:
Published
information.
Book.
N
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
8
G
­
TABLE
2.
ENVIRONMENTAL,
OCCUPATIONAL,
FOOD,
AND
CONSUMER
PRODUCT
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Industry
Databases
Description:
Occupational
exposure
air
monitoring
data,
generally
for
chemicals
with
OSHA
Permissible
Exposure
Limits
(
PELs)
or
ACGIH
TLVs.
Relatively
few
chemicals.
Variable
quality.
Citation:
Availability:
?

International
Agency
for
Research
on
Cancer
(
IARC)
Monographs
Description:
Use
information
and,
in
some
cases,
producers
and
production
methods.
Review
of
existing
data.

Citation:
World
Health
Organization,
IARC.
IARC
Monographs
on
the
Evaluation
of
the
Carcinogenic
Risk
of
Chemicals
to
Humans.

Availability:
Published
information.
Multi­
volume
book
set.
N
International
Program
On
Chemical
Safety,
Environmental
Health
Criteria
Description:
Use
information,
as
well
as
some
production
information.
Chemical
review
based
on
the
collective
views
of
an
international
group
of
experts.

Citation:
International
Program
On
Chemical
Safety,
Environmental
Health
Criteria.
World
Health
Organization.

Availability:
Published
information.
Publication
of
the
World
Health
Organization.
?

Kirk­
Othmer
Encyclopedia
of
Chemical
Technology
Description:
Use
information
as
well
as
production
methods
and,
sometimes,
production
volumes.
Secondary
source.

Citation:
Kirk­
Othmer
Encyclopedia
of
Chemical
Technology,
4th
ed.,
Volumes
1­
present.
New
York,
NY:
John
Wiley
and
Sons,
1991­
present.

Availability:
Published
information.
Multi­
volume
encyclopedia
set.
N
Kline
&
Co.
Survey
Description:
Agricultural
and
non­
agricultural
use
data
for
pesticides
by
product/
use.
Produced
annually.
Citation:
Availability:
Publicly
available.
?

Market
Basket
Survey
Residue
Data
Description:
Pesticide
intake;
summary
data.
Not
strictly
GLP.
Citation:
FDA
Availability:
Mike
Bolger,
FDA,
(
Phone:
202­
205­
8705).
Y
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
9
G
­
TABLE
2.
ENVIRONMENTAL,
OCCUPATIONAL,
FOOD,
AND
CONSUMER
PRODUCT
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Microbiological
and
Residue
Computer
Information
Systems
(
MARCIS)
Description:
Analyzes
for
residues
of
chlorinated
hydrocarbons
and
chlorinated
organophosphates
in
eggs/
meat
and
poultry.
Residue
Violation
Information
System
(
RVIS)
reports
only
violative
findings
and
the
"
Red
Book"
published
annually
reports
the
same.
QA
measures
observed.
Not
strictly
GLP.
Citation:
Food
Safety
and
Inspection
Service/
USDA.
Availability:
Published
summaries
available
to
public.
Raw
data
available
to
other
government
agencies
through
FSIS.
Joanne
Hicks,
FSIS,
(
Phone:
202­
501­
6354).
?

Ministry
of
Agriculture,
Fisheries,
and
Food,
Food
Contaminants
Division
Description:
Monitoring
data
on
food
contaminants
including
phthalates.
Samples
analyzed
at
CSL
food
science
laboratory
(
Norwich).
Citation:
Availability:
Contact
Mr.
Steven
Wearne,
Ergon
House,
c/
o
Nobel
House,
17
Smith
Square,
London,
England
SW1P
31R.
?

National
Estuary
Program
Description:
Sediments,
surface
water,
fish,
PCBs,
heavy
metals,
fecal
coliform,
nutrients,
coastal/
estuarine
areas
in
the
U.
S.
Citation:
U.
S.
EPA
Availability:
Contact
regional
EPA
office.
?

National
Institute
for
Occupational
Safety
and
Health
(
NIOSH)
Health
Hazard
Evaluations
Description:
Occupational
exposure
air
monitoring
data
from
NIOSH
Health
Hazard
Evaluations.
Worst
case
rather
than
representative.
Citation:
Availability:
NIOSH,
Cincinnati,
OH
(
Phone:
800­
35­
NIOSH).
?

National
Institute
for
Occupational
Safety
and
Health
(
NIOSH)
Pocket
Guide
to
Chemical
Hazards
Description:
A
list
of
677
chemicals
or
substances
found
in
the
work
environment.
The
list
contains
all
chemicals
with
OSHA
PELs
(
as
of
the
last
printing
­
1994)
as
well
as
chemicals
for
which
NIOSH
has
Recommended
Exposure
Limits
(
RELs).
Citation:
Availability:
Publicly
available.
This
list
can
be
obtained
from
NIOSH
in
hard
copy
or
electronic
form
(
Phone:
800­
35­
NIOSH).
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
10
G
­
TABLE
2.
ENVIRONMENTAL,
OCCUPATIONAL,
FOOD,
AND
CONSUMER
PRODUCT
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

National
Occupational
Hazard
Survey
Description:
National
survey
of
U.
S.
workplaces.
Identified
chemicals
used
in
different
industries,
attempts
to
estimate
number
of
workers
exposed.
Outdated.
Numbers
are
estimates.
Perhaps
useful
for
identifying
endocrine
disruptors
to
which
a
large
number
of
workers
are
exposed.
Citation:
National
Institute
for
Occupational
Safety
and
Health.
Availability:
?

National
Toxics
Inventory
Description:
Reports
air
emissions
on
a
county
basis
but
are
moving
to
a
facility­
specific
basis
in
the
next
2
years.
Not
externally
peer
reviewed.
Updated
every
3
years.
Citation:
Office
of
Air,
U.
S.
EPA.
Availability:
Available
on
disk
as
ASCII
file.
?

National
Water
Information
System
(
NWIS)
Description:
Contains
4
sub­
data
sets:
Water
Quality
System,
Automated
Data
Processing
System
(
surface
water),
Ground­
Water
Site
Inventory
System,
and
the
Water
Use
Data
System.
Uses
multiresidue
methods.
GLP.
Citation:
U.
S.
Geological
Survey
Availability:
Subsets
through
STORET;
CD­
ROM.
Currently
undergoing
change
to
UNIXbased
workstations.
?

National
Water
Quality
Assessment
Program
(
NAWQA)
Description:
Surface
water,
ground
water,
trace
organics,
trace
elements.
Citation:
U.
S.
Geological
Survey.
Availability:
NAWDEX/
WATSTORE
USGS,
Water
Resources
Division,
Reston,
VA
(
Phone:
703­
860­
6031).
?

OSHA
Compliance
Database
Description:
OSHA
inspection
air
monitoring
data
for
occupational
exposures.
Raw
data.
Only
includes
chemicals
with
OSHA
Permissible
Exposure
Limits
PELs
(
relatively
few
chemicals).
Worst
case
rather
than
representative.
Useful
for
identifying
high­
end
exposures.
Citation:
Availability:
Occupational
Safety
and
Health
Administration.
?

OSHA
Consultation
Programs
for
small
businesses
Description:
Contains
air
monitoring
data
for
occupational
exposures
obtained
during
consultation.
Citation:
Availability:
Regional/
area
OSHA
offices.
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
11
G
­
TABLE
2.
ENVIRONMENTAL,
OCCUPATIONAL,
FOOD,
AND
CONSUMER
PRODUCT
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

OSHA
Permissible
Exposure
Limit
(
PEL)
List
Description:
The
PEL
list
is
a
list
of
chemicals
regulated
by
OSHA
(
approx.
400­
500
chemicals),
and
for
which
OSHA
has
recommended
acceptable
exposure
limits.
Citation:
Availability:
This
is
public
information
that
can
be
obtained
from
OSHA
or
the
29
Code
of
Federal
Regulations
(
CFR).
May
be
available
via
Internet,
as
well.
Y
Pesticide
Data
Program
Description:
Analyzes
for
pesticide
residues
on
food.
Raw
data.
Relational
database.
GLP
­
multiresidue
methods.
Citation:
AMS/
USDA.
Availability:
On
diskette,
hardcopy.
Bob
Epstein,
AMS,
(
Phone:
202­
720­
2158).
?

Pesticides
in
Ground­
Water
Database
Description:
Multiresidue
methods.
GLP.
Citation:
Office
of
Pesticide
Programs/
U.
S.
EPA.
Availability:
Hard
copy
(
1992),
future
Internet
access.
?

Permit
Compliance
System
Description:
Information
on
municipal
and
industrial
wastewater
discharge.
GLP.
Citation:
U.
S.
EPA
Availability:
?

Pre­
Manufacture
Notification
Database
(
PMN)
Description:
Use
information.
Summary
of
PMN
information.
Citation:
PMN
Database.
Availability:
TSCA.
Not
publicly
available.
Available
on
an
as­
needed
basis
with
TSCA
NPA
clearance.
?

Priority­
based
Assessment
of
Food
Additives
(
PAFA)
database,
U.
S.
FDA
Description:
Contains
administrative,
chemical,
and
toxicological
information
on
over
2,000
substances
directly
added
to
food,
including
substances
regulated
by
the
U.
S.
FDA
as
direct,
"
secondary"
direct,
and
color
additives,
and
Generally
Regarded
as
Safe
(
GRAS)
and
priorsanctioned
substances.
The
database
also
contains
only
administrative
and
chemical
information
on
less
than
1,000
such
substances.
The
more
than
3,000
total
substances
together
comprise
an
inventory
often
referred
to
as
"
Everything
Added
to
Food
in
the
United
States"
(
EAFUS).
While
this
is
a
comprehensive
list
of
substances
added
directly
to
food
that
FDA
has
either
approved
as
food
additives
or
listed
or
affirmed
as
GRAS,
it
contains
only
a
partial
list
of
all
food
ingredients
added
to
food
because,
under
federal
law,
some
ingredients
may
be
added
to
food
under
a
GRAS
determination
made
independently
from
the
FDA.
Citation:
U.
S.
Food
and
Drug
Administration
(
FDA).
Availability:
Publicly
available
on
the
Internet,
<
http://
vm.
cfscan.
fda.
gov/~
dms/
eafus.
html>.
Y
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
12
G
­
TABLE
2.
ENVIRONMENTAL,
OCCUPATIONAL,
FOOD,
AND
CONSUMER
PRODUCT
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Public
Health
Departments
Description:
Some
state
health
departments
collect
surveillance
data
on
specific
occupational
exposures
and
health
effects.
Citation:
Availability:
?

Safe
Drinking
Water
Act
(
SDWA)
Contaminant
Candidate
List
Description:
Citations:
·
U.
S.
EPA,
Announcement
of
the
Draft
Drinking
Water
Contaminant
Candidate
List;
Notice,
62
FR
#
193,
Oct.
6,
1997,
pp.
52194­
52219.
·
U.
S.
EPA,
Announcement
of
the
Drinking
Water
Contaminant
Candidate
List;
Notice,
63
FR
#
40,
Mar.
2,
1998,
pp.
10274­
10287.
Availability:
Y
Safe
Drinking
Water
Information
System
(
SDWIS)
Description:
Coliform,
lead,
copper,
radionuclides,
pesticides.
GLP.
Citation:
Office
of
Water,
U.
S.
EPA
Availability:
On
Internet
(
account
required),
but
not
user
friendly.
In
development,
but
will
contain
approximately
58,000
community
water
systems.
?

State
OSHA
databases
Description:
Approximately
one­
half
of
states
administer
state
OSHA
programs
that
collect
air
monitoring
data
during
inspections.
Worst
case
rather
than
representative.
Useful
for
identifying
high­
end
exposures.
Citation:
Availability:
Available
through
Freedom
of
Information
Act?
?

State
Water
Quality
Databases
Description:
Many
states
maintain
an
ambient
water
quality
database,
some
also
a
compliance
database.
Generally
GLP;
multi­
residue
methods.
Citation:
Availability:
Contact
individual
State
Departments
of
Environmental
Protection.
?

STOrage
and
RETrieval
System
(
STORET)
Description:
Point
and
non
point
source
ambient
water
monitoring
and
biological
monitoring
(
pesticides
are
one
parameter).
Generally
not
GLP.
Citation:
Office
of
Water,
U.
S.
EPA
Availability:
Currently
PC
based.
By
late
1997
will
use
UNIX/
Oracle
server
and
PC
based
Windows
95
configuration.
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
13
G
­
TABLE
2.
ENVIRONMENTAL,
OCCUPATIONAL,
FOOD,
AND
CONSUMER
PRODUCT
DATA
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Ullmann's
Encyclopedia
of
Industrial
Chemistry
Description:
Use
information
as
well
as
production
methods
and,
sometimes,
production
volumes.
Secondary
source.

Citation:
Gerhartz,
W.,
Ullmann's
Encyclopedia
of
Industrial
Chemistry,
5th
ed.,
Deerfield
Beach,
FL:
VCH
publishers,
1985.

Availability:
Published
information.
Multi­
volume
encyclopedia
set.
N
USDA
National
Agricultural
Statistics
Survey
Description:
Use
data
for
pesticides
by
production/
crop.
Updated
annually.
Citation:
Availability:
Published
information.
?

USDA
Management
Center
for
Food
and
Agricultural
Police
Survey
Description:
Use
data
for
pesticides
by
crop/
product/
site.
Citation:
Availability:
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
14
G
­
TABLE
3.
ENVIRONMENTAL
RELEASE
DATA
Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?
Comprehensive
Environmental
Response,
Compensation
and
Liability
Information
System
Description:
Contaminant
data
from
Superfund
sites.
Citation:
U.
S.
EPA
Availability:
Internet
access.
?

Hazardous
Substances
Emergency
Event
Surveillance
(
HSEES)
Description:
Citation:
Agency
for
Toxic
Substances
Disease
Registry
(
ATSDR).
Availability:
Y
Toxics
Release
Inventory
(
TRI)
Description:
Chemicals
released
from
manufacturing
facilities
by
pounds
per
year.
Provides
valuable
information
to
citizens
about
types
of
chemicals
released
from
manufacturing
facilities
in
communities
across
the
U.
S.
Information
may
be
useful
for
environmental
justice
concerns
to
identify
those
communities
to
which
potential
endocrine­
disrupting
chemicals
are
being
released.
Information
is
limited
to
chemicals
listed
in
Title
III
of
the
1986
Superfund
Amendments
and
Reauthorization
Act
(
SARA)
and
chemicals
added
to
that
list.
Current
TRI
contains
581
discrete
chemicals
and
28
chemical
groups.
Citation:
OPPT,
U.
S.
EPA
Availability:
Hard
copy
and
Internet
access.
Y
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
15
G
­
TABLE
4.
PRODUCTION
VOLUME
DATA
Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

FIFRA
Section
7
Data
Base
Description:
Production
and
use
volumes
for
pesticides
by
product/
company.
Produced
annually.
2­
3
year
lag
in
information.
Citation:
Availability:
Published
information.
?

Inorganic
chemicals
Description:
Citation:
Availability:
Y
Organic
chemicals
that
are
site­
limited
intermediates
Description:
Mostly
discrete
organic
chemicals
that
are
site­
limited
intermediates.
Industry
submitted
data
in
compliance
with
TSCA
section
8(
b).
Citation:
Availability:
Y
Organic
chemicals
with
annual
production/
importation
volumes
less
than
10,000
pounds
per
year
Description:
Mostly
discrete
organic
chemicals
with
annual
production/
importation
volumes
<
10,000
pounds.
Industry
submitted
data
in
compliance
with
TSCA
section
8(
b).
May
not
include
new
chemicals
that
meet
production
volume
threshold.
Citation:
Availability:
Y
Organic
chemicals
with
annual
production/
importation
volumes
between
10,000
pounds
and
1
million
pounds
per
year
Description:
Mostly
discrete
organic
chemicals
with
annual
production/
importation
volumes
between
10,000
pounds
and
1
million
pounds
per
year.
Industry
submitted
data
in
compliance
with
TSCA
section
8(
b).
Does
not
yet
include
about
5,600
chemicals
for
which
production
or
importation
volumes
need
to
be
cleared
for
Confidential
Business
Information
(
CBI).
May
not
include
new
chemicals
that
meet
production
volume
threshold.
Citation:
Availability:
Y
Organic
chemicals
with
annual
production/
importation
volumes
between
1
million
pounds
and
1
billion
pounds
per
year
Description:
Mostly
discrete
organic
chemicals
with
annual
production/
importation
volumes
between
1
million
and
1
billion
pounds.
Industry
submitted
data
in
compliance
with
TSCA
section
8(
b).
Does
not
yet
include
about
2,000
chemicals
for
which
production
or
importation
volumes
need
to
be
cleared
for
CBI.
May
not
include
new
chemicals
that
meet
production
volume
threshold.
Contains
many
petroleum
feedstock
chemicals
and
mixtures.
Citation:
Availability:
Y
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
16
G
­
TABLE
4.
PRODUCTION
VOLUME
DATA
Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Organic
chemicals
with
annual
production/
importation
volumes
greater
than
1
billion
pounds
per
year
Description:
Mostly
discrete
organic
chemicals
with
annual
production/
importation
volumes
greater
than
1
billion
pounds.
Industry
submitted
data
in
compliance
with
TSCA
section
8(
b).
Does
not
yet
include
about
60
chemicals
for
which
production
or
importation
volumes
need
to
be
cleared
for
CBI.
May
not
include
new
chemicals
that
meet
production
volume
threshold.
Contains
many
petroleum
feedstock
chemicals
and
mixtures.
Citation:
Availability:
Y
Other
pesticide
ingredients
Description:
Food­
use
other
pesticide
ingredients.
Does
not
yet
include
non­
food
use
other
pesticide
ingredients.
Citation:
Availability:
Y
Pesticide
active
ingredients
Description:
Food­
use
pesticide
active
ingredients.
Does
not
yet
include
non­
food
use
pesticide
active
ingredients.
Citation:
Availability:
Y
Polymers
Description:
Citation:
Availability:
Y
SRI
International
Directory
of
Chemical
Producers
Description:
Summary
of
producer
information:
producers;
production
sites;
and,
in
some
cases,
production
volumes.
Citation:
Directory
of
Chemical
Producers.
United
States
of
America.
Menlo
Park,
CA:
SRI
International,
1996.
Availability:
Published
information.
Supplied
on
an
annual
subscription
basis.
?

Toxic
Substances
Control
Act
(
TSCA)
Inventory
and
Updates
Description:
Producers,
production
sites,
and
production
volumes
by
Chemical
Abstracts
Service
Registry
Number
(
CASRN).
Updated
every
4
years.
Exemptions
<
10k/
lb.
Primary
source.
Some
errors,
but
difficult
to
check.
Not
much
use
information
included.

Citation:
TSCA
Inventory
and
Inventory
Update
Rule
information
(
Chemical
Update
System).

Availability:
TSCA
Confidential
Business
Information
(
Not
publicly
available.)
Available
on
an
as­
needed
basis
with
TSCA
NPA
clearance.
?

TABLE
4.
PRODUCTION
VOLUME
DATA
(
cont.)
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
17
G
­
Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

U.
S.
International
Trade
Commission
(
U.
S.
ITC)
Synthetic
Organic
Chemicals
Description:
Producers,
production
sites,
and,
in
some
cases,
production
volumes.
Updated
annually.
Data
supplied
by
producers,
summarized
by
U.
S.
ITC.

Citation:
"
U.
S.
ITC
Synthetic
Organic
Chemicals
 
U.
S.
Production
and
Sales,
1992,"
U.
S.
ITC
Publ
2720,
February
1994,
Washington,
DC.:
United
States
Trade
Commission,
1994.

Availability:
U.
S.
ITC
publication
which
may
be
ordered
from
The
Office
of
the
Secretary
Publications
Section,
Unites
States
U.
S.
ITC.;
1­
2
year
lag
in
information.
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
18
G
­
TABLE
5.
FATE
AND
TRANSPORT
DATA
AND
MODELS
Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Absorption
Coefficient
(
Koc)
Description:
Indicator
of
mobility.
Citation:
Availability:
Y
Biodegradation
Description:
Indicator
of
persistence.
Citation:
Availability:
Y
Environmental
Contaminant
Reference
Databook
Description:
In
some
cases,
biodegradation
data
and
bioconcentration
information
is
provided.
Secondary
source.

Citation:
Prager,
J.
C.,
Van
Nostrand
Reinhold:
New
York,
NY,
1996.

Availability:
Published
information.
2
volume
book
set.
N*

Environmental
Fate
Databases
(
EFDB)
Description:
Source
of
available
references
on
chemical
fate
and
transport.
Database
of
primary
sources
and
some
secondary
sources.

Citation:
Syracuse
Research
Corporation's
EFDB.

Availability:
EFDB
may
be
purchased
from
Syracuse
Research
Corporation.
On­
line
commercial
database
with
proven
usefulness.
N*

Handbook
of
Chemical
Property
Estimation
Methods
Description:
Chemical
and
physical
properties,
media­
specific
environmental
fate
properties.
Secondary
source.

Citation:
Lyman,
W.
J.,
W.
F.
Reehl,
and
D.
H.
Rosenblatt,
American
Chemical
Society:
Washington
D.
C.,
1990.

Availability:
Published
information.
Book.
N*

Handbook
of
Chemistry
and
Physics
(
57th
Ed.)
Description:
Chemical
and
physical
properties.
Secondary
source.

Citation:
Weast,
R.,
(
Ed.),
CRC
Press:
Cleveland,
OH,
1976.

Availability:
Published
information.
Book.
N*
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
19
G
­
TABLE
5.
FATE
AND
TRANSPORT
DATA
AND
MODELS
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Handbook
of
Environmental
Data
on
Organic
Chemicals
Description:
Chemical
and
physical
properties,
natural
and
anthropogenic
sources,
mediaspecific
fate
information.
Secondary
source.

Citation:
Verschueren,
K.,
Van
Nostrand
Reinhold
Co:
New
York,
NY,
1983.

Availability:
Published
information.
Book.
N*

Handbook
of
Environmental
Data
on
Organic
Chemicals,
3rd
edition
Description:
Biodegradation
rates
and
mechanisms,
atmospheric
reactions.
Secondary
source.

Citation:
Verschueren,
K.,
Van
Nostrand,
Reinhold:
New
York,
NY,
1996.

Availability:
Published
information.
Book.
N*

Handbook
of
Environmental
Degradation
Rates
Description:
Chemical­
specific
empirical
and
theoretical
environmental
degradation
rates.
Secondary
source.

Citation:
Howard,
P.
H.,
R.
S.
Boethling,
W.
F.
Jarvis,
W.
M.
Meylan,
and
E.
M.
Michalenko,
Lewis
Publishers:
Chelsea,
MI,
1991.

Availability:
Published
information.
Book.
N*

Handbook
of
Environmental
Fate
and
Exposure
Data
for
Organic
Chemicals:
Volumes
I­
III
Description:
Large
production
and
priority
pollutants,
solvents,
pesticides:
physical
and
chemical
properties,
environmental
fate
data,
natural
and
anthropogenic
sources,
media­
specific
background
concentrations.
Secondary
source.

Citation:
Howard,
P.
H.,
(
Ed.),
Lewis
Publishers:
Chelsea,
MI,
1991.

Availability:
Published
information.
Books
(
3
volumes).
N*

"
Health
Assessment
Guidance
Manual"
(
draft)
Description:
Guidance
for
performing
ATSDR
"
Health
Assessments"
to
sites
on
the
National
Priorities
List.
Secondary
source.

Citation:
U.
S.
Department
of
Health
and
Human
Services
for
the
Agency
for
Toxic
Substances
Disease
Registry,
1990.

Availability:
Available
from
US
Department
of
Health
and
Human
Services.
Atlanta,
GA.
Guidance
document.
N*
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
20
G
­
TABLE
5.
FATE
AND
TRANSPORT
DATA
AND
MODELS
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Hydrolysis
Half­
Life
Description:
Indicator
of
persistence.
Citation:
Availability:
Y
Illustrated
Handbook
of
Physical
and
Chemical
Properties
and
Environmental
Fate
for
Organic
Chemicals
(
Volume
I
&
II)
Description:
Physical
and
chemical
properties,
environmental
fate
data.
Secondary
source.

Citation:
Mackay,
D.,
W.
Y.
Shiu,
and
K.
C.
Ma,
Lewis
Publishers:
Chelsea,
MI,
1992.

Availability:
Published
information.
Books
(
2
volumes).
N*

Octanol:
Water
Partition
Coefficient
(
Kow)
Description:
Indicator
of
mobility
and
bioaccumulation.
Citation:
Availability:
Y
Pesticide
Document
Management
System
(
PDMS)
Description:
Bibliographic
information
on
unpublished
fate
and
transport
studies
on
pesticide
chemicals.
Information
is
variable
but
recent
information
is
of
good
quality.

Citation:
PDMS
available
from
Office
of
Pesticide
Programs
U.
S.
EPA.

Availability:
Available
from
U.
S.
EPA
(
contact
John
Jamula
at
703­
305­
6426).
Data
base
with
proven
usefulness.
N*

The
Pesticide
Manual,
Incorporating
The
Agrochemicals
Handbook,
10th
edition
Description:
Source
of
degradation
and
environmental
data
on
pesticides.
Reference
source.
Also
contains
some
information
on
use.

Citation:
Tomlin,
C.
(
Ed.),
The
Bath
Press:
Bath,
UK,
1994.

Availability:
Published
information.
Book.
N*

Photooxidation
Description:
Indicator
of
persistence.
Citation:
Availability:
Y
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
21
G
­
TABLE
5.
FATE
AND
TRANSPORT
DATA
AND
MODELS
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

"
Risk
Assessment
Guidance
for
Superfund
Volume
1.
Human
Health
Evaluation
Manual
Parts
A
&
B"
Description:
Federal
guidance
for
performing
risk
assessments
for
Superfund
sites.
Secondary
source.

Citation:
U.
S.
EPA,
Office
of
Emergency
and
Remedial
Response:
Washington
DC.
EPA/
540/
1­
89/
002,
1989.

Availability:
Available
from
U.
S.
EPA
and/
or
NTIS
(
703­
487­
4650).
Guidance
document.
N*

The
Soil
Chemistry
of
Hazardous
Materials
Description:
Interaction
and
fate
of
inorganic
and
organic
chemicals
in
soil.
Secondary
source.

Citation:
Dragun,
J.,
The
Hazardous
Materials
Control
Research
Institute:
Silver
Spring,
MD,
1988.

Availability:
Published
information.
Book.
N*

"
Superfund
Exposure
Assessment
Manual"
Description:
Federal
guidance
to
estimate
exposure
to
contaminants
migrating
from
uncontrolled
hazardous
waste
sites.
Secondary
source.

Citation:
U.
S.
EPA,
Office
of
Emergency
and
Remedial
Response:
Washington
DC.
EPA/
540/
1­
88/
001,
1988.

Availability:
Available
from
U.
S.
EPA
and/
or
NTIS
(
703­
487­
4650).
Guidance
document.
N*

Volatility
(
Henry's
Law)
Description:
Indicator
of
mobility.
Citation:
Availability:
Y
Syracuse
Research
Corporation's
EPI
Suite
Description:
Source
of
estimated
environmental
fate
data
including
adsorption
to
soil,
atmospheric
photooxidation,
volatilization
half­
lives
from
water,
biodegradability,
and,
sometimes,
hydrolysis
half­
lives.
Estimation
programs
for
which
the
methodology
has
been
peer
reviewed.

Citation:
Syracuse
Research
Corporation's
EPI
Suite.

Availability:
The
EPI
Suite
may
be
purchased
from
Syracuse
Research
Corporation.
Methodology
for
the
estimation
programs
in
EPI
has
been
published.
Computer
software
may
be
purchased
from
Syracuse
Research
Corporation.
N*
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
22
G
­
*
While
none
of
these
data
sources
is
being
recommended
for
inclusion
in
the
EDPSD,
specific
physiochemical
properties
related
to
environmental
fate
and
transport
are
recommended
for
inclusion.
These
properties
are
included
in
this
matrix
as
data
sources,
and
are
denoted
with
a
"
Y"
in
the
"
Recommended
for
Inclusion
in
EDPSD?"
column.
For
further
information,
and
for
a
specific
listing
of
these
properties,
please
refer
to
Chapter
Four,
Section
III,
E.
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
23
G
­
TABLE
6/
7.
TOXICOLOGY
LABORATORY
STUDIES
&
EPIDEMIOLOGY
AND
FIELD
STUDIES
±
±
For
text
explaining
the
combination
of
Toxicology
Laboratory
Studies
and
Epidemiology
and
Field
Studies
for
use
in
the
EDPSD,
please
refer
to
Chapter
Four,
Section
X,
E.

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Amphibian/
reptile­
related
toxicological
peer
reviewed
published
scientific
literature.
Description:
Morphology
of
gonads
and
vitellogenin
induction
for
amphibians/
reptiles.
Chemicals
studied
include
metabolites
of
DDT,
PCBs,
and
mercury.
Summary
data
principally
addressing
single
compounds.
Peer
reviewed;
not
GLP.
Citations:
·
Bergeron,
Crews,
and
McLachlan,
Environ.
Health
Perspectives,
102,
1994,
pp.
780­
781.
·
Crews,
Bergeron,
and
McLachlan.
Environ.
Health
Perspectives,
103,
1995,
pp.
73­
77.

·
Kanamadi
and
Daidapur,
J.
Herpetology,
25,
1991,
p.
497.
·
Punzo,
Bull.
Environ.
Contamin.
Toxicol.,
50,
1993,
pp.
385­
391.
Availability:
Publicly
available.
N
*
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
24
G
­
TABLE
6/
7.
TOXICOLOGY
LABORATORY
STUDIES
&
EPIDEMIOLOGY
AND
FIELD
STUDIES
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Avian­
related
toxicological
peer
reviewed
published
scientific
literature.
Description:
Responses
include
morphology
of
gonads
and
thyroid,
and
plasma
concentrations
of
steroids,
thyronines,
hypophyseal
hormones,
and
some
receptor
binding
data.
Studies
report
exposure
to
organochlorine
pesticides
and
metabolites,
PCBs,
TCDD,
and
metals.
Summary
data
principally
addressing
single
compounds.
Peer
reviewed;
not
GLP.
Citations:
·
Rattner,
Eroschenko,
Fox,
Fry,
and
Gorsline,
"
Organochlorine
Insecticides:
Persistent
Organic
Pollutants,"
J.
Exp.
Zool.,
232,
1975,
pp.
683­
689.
·
Rattner
et
al.,
"
Reviews
in
Environmental
Toxicology
I,"
Comp.
Biochem.
Physiol.,
83C,
1984,
pp.
451­
453.
·
Mayer
et
al.,
Biomarkers:
Biochemical,
Physiological,
and
Histological
Markers
of
Anthropogenic
Stress.
Huggett,
Kimerle,
Merhle
and
Bergman
(
Eds.),
1992.
·
Review
Article
 
Fox,
"
Chemically­
induced
alterations
in
sexual
and
functional
development:
The
wildlife/
human
connection,"
1992.
·
Guilette
et
al.,
Environ.
Health
Perspectives.,
103,
1995,
pp.
157­
164.
·
Fry,
Environ.
Health
Perspect.,
103,
1995,
pp.
165­
171.
·
Chen
et
al.,
Environ.
Toxicol.
Chem.,
13,
1994,
pp.
789­
796.
·
MacLellan
et
al.,
Arch.
Environ.
Toxicol.
Chem.,
30,
1996,
pp.
364­
372.
·
Janz
and
Bellward,
Toxicol.
Appl.
Pharmacol.,
139:
281­
291
and
292­
300.
·
Janz
and
Bellward,
Environ.
Toxicol.
Chem.,
16:
985­
989.
Availability:
Publicly
available.
N
*

California
Proposition
65
List
Description:
Citation:
Availability:
Y
ECOTOX
(
AQUIRE,
PHYTOTOX,
and
TERRETOX)
Description:
Bibliographic
information
and
data
on
published
ecotoxicity
studies
on
industrial
chemicals
and
pesticides
related
to
fish
and
aquatic
organisms.
Summary
data.
Citation:
ECOTOXicology
Data
Base
System
­
on­
line
U.
S.
EPA
Database.

Availability:
Not
publicly
available.
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
25
G
­
TABLE
6/
7.
TOXICOLOGY
LABORATORY
STUDIES
&
EPIDEMIOLOGY
AND
FIELD
STUDIES
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Fish­
and
aquatic
organism­
related
toxicological
peer
reviewed
published
scientific
literature.
Description:
In
vitro
responses
using
fish
and
wildlife
tissues.
Summary
data.
Peer
reviewed.
Citations:
·
Review
article
 
Nimrod
and
Benson.
"
Environmental
estrogenic
effects
of
alkylphenol
ethoxylates,"
Critical
Reviews
in
Toxicology,
26,
1996,
pp.
335­
364.
·
Review
article
­
Toppari
et
al.,
Male
reproductive
health
and
environmental
xenoestrogens.
Environ.
Health
Perspectives,
104,
1996,
pp.
741­
830.
·
Jobling
and
Sumpter,
Aquatic
Toxicology,
27,
pp.
361­
372.
Availability:
Publicly
available.
N
*

Fish­
and
aquatic
organism­
related
toxicological
peer
reviewed
published
scientific
literature.
Description:
Morphology
of
gonads,
steroidogenesis,
plasma
steroid
concentrations,
and
vitellogenin
concentration
in
fish
and
aquatic
organisms.
Chemicals
include
organochlorine
pesticides,
PCBs,
petroleum
hydrocarbons,
alkylphenols,
and
ethoxylates.
Summary
data
principally
addressing
single
compounds.
Peer
reviewed;
not
GLP.
Citations:
·
Review
article
 
Mayer
et
al.,
Biomarkers:
Biochemical,
Physiological,
and
Histological
Makers
of
Anthropogenic
Stress,
Huggett,
Kimerle,
Merhle
and
Bergman
(
Eds.),
Lewis
Publishers,
1992.
·
Review
article
 
Reijnders
and
Brasseur,
Chemically­
induced
alternations
in
sexual
and
functional
development:
The
wildlife/
human
connection,
Colburn
and
Clement
(
Eds.),
1992.
·
Jobling
et
al.,
Aquatic
Toxicology,
27,
1993,
pp.
361­
372.
·
Jobling
et
al.,
Environ.
Toxicol.
Chem.,
15,
1996,
pp.
194­
202.
·
Guillette
et
al.,
Environ.
Health
Perspectives,
103,
1995,
pp.
157­
164.
·
Sumpter
and
Jobling,
Environ.
Health
Perspectives,
103,
1995,
pp.
173­
178.
Availability:
Publicly
available.
N
*

Hazardous
Substances
Databank
(
HSDB)
Description:
Toxicity
and
bibliographic
information
on
many
chemicals.
Summary
information
of
studies.
Usually
peer
reviewed.
Not
GLP.
Citation:
National
Library
of
Medicine.
Availability:
Publicly
available.
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
26
G
­
TABLE
6/
7.
TOXICOLOGY
LABORATORY
STUDIES
&
EPIDEMIOLOGY
AND
FIELD
STUDIES
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?
Interagency
Testing
Committee
Screening
Information
Retrieval
Exchange
Network
(
ITCSIREN)
Description:
TSCA
Interagency
Testing
Committee
(
ITC)
testing
decisions
for
about
40,000
existing
and
new
chemicals
and
mixtures
and
rationales
for
decisions.
Discussion
of
endpoints
of
concern
related
to
humans.
Federal
Register
citations
of
ITC
Reports
to
the
U.
S.
EPA
Administrator
are
provided
for
chemicals
and
mixtures
added
to
the
Priority
Testing
List.
High
quality,
peer­
reviewed.
(
Some
not
publicly
available;
being
sanitized.)
Citation:
Walker,
ITCSIREN,
1997.
Availability:
?

Medline
Description:
Citation:
Availability:
N*

Pesticide
Document
Management
System
(
PDMS)
Description:
Bibliographic
information
on
unpublished
mammalian
and
ecotoxicity
studies
on
pesticide
chemicals.
Recent
data
are
of
high
quality.
Often
GLP.
Citation:
PDMS
available
from
Office
of
Pesticide
Programs
U.
S.
EPA.
Availability:
Publicly
available.
John
Jamula,
U.
S.
EPA,
(
Phone:
703­
305­
6426)
?

Registry
of
Toxic
Effects
of
Chemical
Substances
(
RTECS)
Reproductive
Effects
Data
Description:
Citation:
Availability:
Y
Toxline
Description:
Citation:
Availability:
N*

Toxic
Substance
Control
Act
Test
Submissions
­
Health
Effects
(
TSCATS­
HE)
Description:
Bibliographic
information
(
with
some
abstracts)
on
unpublished
human
health
effects,
chemical
fate,
ecological
effects,
and
industrial
hygiene
studies
on
industrial
chemicals.
Information
is
highly
variable,
from
well­
conducted
GLP
studies
to
very
brief
data
sheets.
Citation:
TSCATS
Data
Base
available
in
TOXLINE.
Availability:
Publicly
available
from
NLM,
CIS,
or
Syracuse
Research
Corporation.
On­
line
charges
apply.
Full
text
studies
available
from
NTIS
and
CIS.
Y
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
27
G
­
TABLE
6/
7.
TOXICOLOGY
LABORATORY
STUDIES
&
EPIDEMIOLOGY
AND
FIELD
STUDIES
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Toxic
Substance
Control
Act
Test
Submissions
­
Ecological
Effects
(
TSCATS­
EE)
Description:
Bibliographic
information
(
with
some
abstracts)
on
unpublished
human
health
effects,
chemical
fate,
ecological
effects,
and
industrial
hygiene
studies
on
industrial
chemicals.
Information
is
highly
variable,
from
well­
conducted
GLP
studies
to
very
brief
data
sheets.
Citation:
TSCATS
Data
Base
available
in
TOXLINE.
Availability:
Publicly
available
from
NLM,
CIS,
or
Syracuse
Research
Corporation.
On­
line
charges
apply.
Full
text
studies
available
from
NTIS
and
CIS.
Y
Web­
based
searches;
e.
g.,
http://
chemfinder.
camsoft.
com/
and
http://
ntp­
server.
niehs.
nih.
gov/
Description:
Toxicity
and
bibliographic
information
on
many
chemicals.
Information
is
highly
variable
from
well­
conducted
GLP
studies
to
very
brief
data
sheets.
Citation:
Various
Availability:
Publicly
available.
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
28
G
­
TABLE
6/
7.
TOXICOLOGY
LABORATORY
STUDIES
&
EPIDEMIOLOGY
AND
FIELD
STUDIES
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Wild
mammal­
related
toxicological
peer
reviewed
published
scientific
literature.
Description:
Morphology
of
gonads,
oviduct,
and
thyroid,
and
plasma
concentrations
of
steroids,
luteinizing
hormone,
and
thyronines,
and
some
steroid
receptor
binding
data
for
wild
mammals.
Responses
measured
following
exposure
to
organochlorine
pesticides
and
metabolites,
PCBs,
TCDD
petroleum
hydrocarbons,
mercury
and
lead.
Summary
data
principally
addressing
single
compounds.
Peer
reviewed;
not
GLP.
Citations:
·
Mayer
et
al.,
Biomarkers:
Biochemical,
Physiological,
and
Histological
Markers
of
Anthropogenic
Stress,
Huggett,
Kimerle,
Merhle
and
Bergman
(
Eds.),
Lewis
Publishers,
1992.
·
Wren,
J.,
Toxicol.
Environ.
Health,
33,
1991,
pp.
549­
585.
·
Sanders
and
Kirkpatrick,
Environ.
Res.,
13,
1977,
pp.
358­
363.
(
1977).
·
Rattner
and
Michael,
Toxicol.
Letters,
24,
1985,
pp.
65­
69.
·
Giesy,
J.
P.,
D.
A.
Verbrugge,
R.
A.
Othout,
et
al.,
Archives
of
Environmental
Contamination
and
Toxicology,
27,
1994,
pp.
21­
223.
·
Kihlstrom,
J.
E.,
M.
Olsson,
S.
Jensen,
A.
Johansson,
and
J.
Ahlbom,
"
Effects
of
PCB
and
different
fractions
of
PCBs
on
the
reproduction
of
the
mink
(
mustela
vison),"
Ambio.,
21,
1992,
pp.
563­
569.
·
Patnode,
K.
A.,
and
L.
R.
Curtis,
Toxicology
and
Applied
Pharmacol.,
127,
1994,
pp.
9­
18.
Availability:
Publicly
available.
N*

*
Peer
reviewed
scientific
literature
is
not
being
recommended
for
inclusion
in
the
EDPSD
due
to
the
fact
that
the
nature
of
the
data
does
not
lend
itself
to
such
use.
However,
the
Committee
recognizes
the
value
of
such
literature
and
recommends
that
it
be
used
outside
the
EDPSD
to
gather
further
information
on
chemical
substances
or
mixtures
of
concern.
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
29
G
­
TABLE
8.
PREDICTIVE
BIOLOGICAL
ACTIVITY
OR
EFFECTS
MODELS
Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

Androgen
Receptor
Model
(
AR)
Description:
Model
developed
that
predicts
chemicals
similar
to
the
chemicals
used
to
train
the
model.
Predicts
binding
affinity
like
in
vitro
models,
and
therefore
has
the
same
advantages
and
disadvantages.
Expert
opinions,
not
peer
reviewed.
Data
is
course,
but
fast
and
inexpensive
to
generate.
The
model
does
not
appear
to
be
as
robust
as
the
estrogen
receptor
model,
but
it
does
appear
to
be
predictive
and
useful.
Overall
data
quality
is
not
as
good
as
the
estrogen
receptor
model,
but
it
does
appear
to
be
fairly
accurate
at
predicting
in
vitro
activity.
Citation:
Waller,
C.
L.,
B.
W.
Juma,
W.
R.
Kelce,
and
L.
E.
Gray,
Jr.,
"
Three­
Dimensional
Quantitative
Structure­
Activity
Relationship
Models
for
Androgen
Receptor
Ligands,"
Toxicol.
Appl.
Pharmacol.,
137,
1996,
pp.
219­
227.
Availability:
Model
is
available
free
to
anyone.
One
would
need
the
CoMFA
software
and
proper
computer
to
run
the
model
(
which
EPA
has).
?

Aromatase
Inhibition
Model
Description:
There
is
currently
a
published
model
by
Oprea
and
Garcia
that
indicates
that
this
activity
can
be
modeled.
However,
not
many
nonsteroidal
chemicals
have
been
tested
and
included.
This
problem
has,
to
a
small
extent,
been
addressed
by
the
senior
author
who
has
an
unpublished
model
and
ideas
on
how
to
merge
it
with
another.
Citation:
Availability:
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
30
G
­
TABLE
8.
PREDICTIVE
BIOLOGICAL
ACTIVITY
OR
EFFECTS
MODELS
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

CoMFA
model
(
Estrogen
Receptor
Model)
Description:
Model
developed
that
predicts
chemicals
similar
to
the
chemicals
used
to
train
the
model.
Predicts
binding
affinity
like
in
vitro
models,
and
therefore
has
the
same
advantages
and
disadvantages.
Even
though
the
data
is
course,
but
fast
and
inexpensive
to
generate.
The
chemicals
used
to
train
the
model
are
not
very
diverse.
Overall
data
quality
is
almost
as
good
as
in
vitro
tests.
Citations:
·
Waller,
C.
L.,
T.
I.
Oprea,
K.
Chae,
H­
K.
Rhee­
Park,
K.
S.
Korach,
S.
Laws,
T.
E.
Wiese,
W.
R.
Kelce,
and
L.
E.
Gray,
Jr.,
"
Ligand­
Based
Identification
of
Environmental
Estrogens,"
Chem.
Res.
Toxicol.,
9,
1996,
pp.
1240­
1248.
·
Waller,
C.
L.,
and
J.
D.
McKinney,
"
Examination
of
the
Estrogen
Receptor
Binding
Affinities
of
Polychlorinated
Hydroxybiphenyls
Using
Three­
Dimensional
Quantitative
Structure­
Activity
Relationships,"
Environ.
Health
Perspectives,
103,
1995,
pp.
702­
707.
Availability:
Model
is
available
free
to
anyone.
One
would
need
the
CoMFA
software
and
proper
computer
to
run
the
model
(
which
U.
S.
EPA
has).
?

ECOSAR
Description:
An
automated
collection
of
over
300
SAR
equations
for
estimating
toxicity
to
aquatic
organisms
mainly
from
log
Kow
and
functional
groups.
Equations
for
predictive
use.
The
quality
of
the
value
calculated
by
the
program
varies
with
the
number
and
type
of
chemicals
used
to
derive
the
equation.
Separate
equations
are
used
for
different
functional
groups.
For
multi­
functional
compounds,
lowest
value
is
used.
Citation:
ECOSAR
Program;
Environmental
Effects
Branch,
OPPT;
U.
S.
EPA
­
7403;
401
M
Street,
S.
W.
Washington,
DC
20460.

Availability:
Publicly
available.
The
program
is
readily
available
from
U.
S.
EPA
or
National
Technical
Information
Service
NTIS
($
97).
NTIS,
Technology
Administration,
U.
S.
Department
of
Commerce,
Springfield,
VA
22161,
(
Phone:
703­
487­
4650).
Order
number
PB94­
500485INC
?
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
31
G
­
TABLE
8.
PREDICTIVE
BIOLOGICAL
ACTIVITY
OR
EFFECTS
MODELS
(
cont.)

Name
of
Data
Source
Description
/
Characteristics
of
Data
Source
Recommended
for
Inclusion
in
EDPSD?

QSAR
for
Androgen
Receptor
Binding
Description:
Would
be
developed
or
improved
upon
using
data
obtained
from
HTPS
assays.
Citation:
Availability:
Y
QSAR
for
Estrogen
Receptor
Binding
Description:
Would
be
developed
or
improved
upon
using
data
obtained
from
HTPS
assays.
Citation:
Availability:
Y
QSAR
for
Thyroid
Receptor
Binding
Description:
Would
be
developed
or
improved
upon
using
data
obtained
from
HTPS
assays.
Citation:
Availability:
Y
Substructure­
based
Computerized
Chemical
Selection
Expert
System
(
SuCCSES)
Description:
Chemical
substructures
(
121)
representing
over
13,000
chemicals
for
which
experts
have
reported
that
chemicals
containing
one
of
these
substructures
have
potential
to
cause
specific
health
or
ecological
effects.
Expert
opinions,
not
peer­
reviewed.
Citations:
·
Walker
and
Brink,
ASTM
STP,
1007,
1989,
pp.
507­
536.
·
Walker,
Sci.
Total
Environ.,
109/
110,
1991,
pp.
691­
700.
·
Walker,
Toxicol.
Model.,
1,
1995,
pp.
123­
141.
Availability:
Published
information.
Available
in
journals.
N
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
1
H
­
Appendix
H
1
2
Fate
and
Transport
Tables
3
4
5
Table
H.
1
6
7
Environmental
Fate
and
Transport
Criteria
8
9
PERSISTENCE:
The
tendency
of
a
chemical
substance
to
persist
(
survive)
in
the
10
environment
without
transformation
into
another
chemical
form.
11
12
13
PERSISTENCE
Measure
(
Potential
14
Utility)
15
Comment
Regulatory
Endpoint
Hydrolysis
Half­
16
Life
17
Degradation
in
water.
Measured
at
pH
5,
7,
and
9
(
acidic,
neutral,
and
alkaline)
at
25o
C
using
14C
material.
Half­
life
>
25
weeks
Aerobic
and
18
anaerobic
soil
metabolism
19
Degradation
due
to
the
biological
and
physical/
chemical
properties
of
the
soil.
Uses
radiolabeled
material.
The
specific
metabolites
are
identified,
and
persistent
ones
could
require
additional
toxicology,
ecotoxicity,
and
E­
fate
safety
evaluations.
Half­
life
>
2­
3
weeks
Photolysis
20
Degradation
due
to
sunlight.
Done
in
either
soil
or
aqueous
environment
with
radiolabeled
chemical
substance.
Half­
life
>
1
week
(
but
this
criterion
is
only
important
while
the
pesticide
is
on
the
surface
21
22
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
2
H
­
Table
H.
2
1
2
Environmental
Fate
and
Transport
Criteria
3
4
MOBILITY:
Ability
to
move
in
air
and/
or
potentially
leach
into
ground
water.
This
5
potential
is
altered
by
the
compound's
persistence.
6
7
8
MOBILITY
Measure
(
Potential
9
Utility)
10
Comment
Regulatory
Endpoint
Volatility,
Henry's
11
Law
Constant
12
Calculated
by
the
ratio
of
the
chemical's
vapor
pressure
to
its
solubility
in
water.
Indicator
of
volatilization
potential
when
pesticide
is
dissolved
in
water.
<
10­
2
atm­
m­
3/
mol
K
d,
K
oc
13
K
d
is
soil­
specific.
14
15
K
oc
is
normalized
to
16
%
organic
carbon
(
oc)
in
17
soil,
the
component
most
18
responsible
for
sorption.
19
Tendency
of
a
chemical
to
be
sorbed
to
soil.
K
d
<
5
and
usually
less
than
1
to
2.
Can
vary
widely
depending
on
the
soil
type.
K
oc
<
300
to
500
Ground
Water
20
Ubiquitous
Score
or
(
GUS)
21
Empirical
evaluation
GUS
=
log
soil
1/
2
life
x
(
4­
log
K
oc).
(
Log
soil
half
life)
<
1.8
is
improbable
leacher,
1.8
­
2.8
is
transitional
zone,
and
>
2.8
is
a
probable
leacher.

Aged
Soil
Column
22
Leaching
23
Lab
experiment
to
estimate
the
leaching
potential
of
parent
and
significant
soil
metabolite(
s)
in
various
soil
types.
No
quantitative
trigger.
Presence
of
parent
and/
or
metabolites
in
the
column
leachate
indicates
potential
to
contaminate
ground
water.

Terrestrial
Field
24
Dissipation
Studies
25
The
rate
of
dissipation
of
the
pesticide
after
application.
Measures
soil
degradation
in
the
environment
(
various
soils).
Expensive,
long­
term
and
involved.
Half
Life
of
2
to
3
weeks
is
considered
persistent,
and
detection
at
90
cm
(
30
inch)
indicates
leaching
26
27
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
3
H
­
Table
H.
3
1
2
Environmental
Fate
and
Transport
Criteria
34
BIOACCUMULATION:
The
capacity
of
a
chemical
to
accumulate
(
be
stored
in
the
5
tissue)
in
an
organism
as
a
result
of
uptake
from
all
environmental
sources.
6
7
8
BIOACCUMULATION
Measure
(
Potential
9
Utility)
10
Comment
Regulatory
Endpoint
Octanol
Water
11
Partition
Coefficient
12
(
K
OW)
13
Ability
of
a
chemical
substance
to
partition
between
an
aqueous
and
lipid
phase.
Classic
and
easy
measure
which
is
used
as
an
indication
of
a
chemical's
potential
for
bioconcentration
by
aquatic
organisms.
Log
K
OW
>
3
indicates
that
the
substance
has
the
propensity
to
accumulate
in
fat.

Bioaccumulation
14
Factor
(
BCF)
15
Used
to
help
assess
risks
to
fish
and
to
non­
target
organisms
(
including
humans)
above
them
in
the
food
chain.
During
an
accumulation
test,
at
any
time
during
the
uptake
phase,
the
concentration
of
test
substance
(
in
ppm)
in/
on
fish,
or
specified
tissues
thereof,
divided
by
the
concentration
of
the
chemical
in
the
surrounding
medium
=
BCF.
BCF
tests
are
required
for
chemicals
that
have
log
K
OW
values
>
3.0.
Remediation
required
if
EPA
water
branch
finds
pesticide
or
chemical
at
certain
levels
in
fish
during
random
sampling.
BCF
>
1000
Animal
16
Metabolism
17
This
is
part
of
mammalian
toxicology,
but
information
on
metabolism
and
excretion
can
be
useful
to
flag
potential
for
bioaccumulation.
Uses
radiolabeled
material.
Excretion
of
90%+
of
all
compound
in
the
first
24
hours
is
desirable.
18
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
4
H
­
1.
The
bioaccumulation
potential
is
considered
significant
if
the
substance
has
a
log
Kow
of
3
and
triggers
a
fish
1
bioaccumulation
test.

23
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
5
H
­
Literature
Cited
1
2
3
Hoppin,
P.
J.,
R.
A.
Liroff,
and
M.
M.
Miller,
"
WWF
Report:
Reducing
Reliance
4
on
Pesticides
in
Great
Lakes
Basin,"
Agriculture,
July
1996,
pp.
99­
101.
5
U.
S.
EPA
Draft
Waste
Min.,
"
Where
to
Begin?,"
Recommendations
of
the
Waste
6
Minimization
Prioritization
Team
on
Risk­
based
Tools
for
Identifying
Priority
7
Chemicals
8
and
Wastes,
Appendix
C,
July
1996.
9
Veith
et
al.,
J.
Fish
Res.
Board
Canada,
36,
1979,
pp.
1040­
1048.
10
Wiley,
J.,
D.
H.
Hutson,
T.
R.
Roberts
et
al.,
"
Progress
in
Pesticide
Biochemistry
11
and
Toxicology,"
Environmental
Fate
of
Pesticides,
7,
1990,
pp.
13­
25.
12
13
14
15
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
1
I
­
Appendix
I
1
2
Feasibility
Demonstration
Project
for
HTPS
3
4
[
Note
to
the
reader:
The
following
text
and
chart
were
supplied
by
OSI
Pharmaceuticals,
Inc.,
5
in
response
to
EPA's
RFP
on
the
demonstration
feasibility
study
for
high
throughput
pre­
6
screening
(
HTPS).
OSI
Pharmaceuticals,
Inc.,
was
the
only
company
to
submit
a
response
to
the
7
RFP
and
was
awarded
the
contract
for
the
demonstration
project.]
8
9
OSI
PHARMACEUTICALS,
INC.
10
HIGH
THROUGHPUT
PRE­
SCREENING
RESEARCH
PLAN
11
12
Executive
Summary
13
14
Profiling
of
chemical
entities
has
undergone
a
revolution
in
recent
years
with
emerging
rapid
15
technologies
measuring
endocrine
modulation.
Endocrine
modulation
by
chemicals
dispersed
into
the
16
environment
can
have
serious
consequences
for
wildlife
and
humans
and
has
resulted
in
legislation
17
included
in
the
Safe
Drinking
Water
and
Food
Quality
Protection
Acts
requiring
safety
assessments
of
18
manufactured
chemicals.
19
20
OSI
Pharmaceuticals,
Inc.,
has
recognized
the
need
for
rapid
chemical
assessment
tools
and
is
21
developing
a
core,
modular
chemical
profiling
technology
platform
which
builds
from
the
existing
22
strength
of
the
organization
and
provides
solutions
to
many
of
these
issues.
23
24
The
technology
has
focused
on
the
generation
of
panels
of
reporter
gene
constructs
which
will
provide
25
profiling
information
regarding
the
potential
for
chemical
compounds
to
activate
the
estrogen
receptor
26
(
a
and
b
)
,
androgen
receptor,
and/
or
thyroid
hormone
receptor.
A
panel
of
highly
sensitive,
stably
27
transfected
human
and/
or
rodent
cell
lines
are
available
which,
together
with
customized
automation
28
and
an
informatics/
quantitative
structure­
activity
relationship
(
QSAR)
package,
will
enable
the
rapid
29
generation
of
high
quality
and
quantitative
data
sets.
The
approach
provides
for
a
near
turn
key
system
30
enabling
the
routine
testing
of
active
molecules
from
a
variety
of
sources
(
pharmaceutical,
chemical,
31
agrochemical).
32
33
The
attached
research
plan
covers
the
core
features
of
the
program
and
a
proposed
initial
panel
of
gene
34
constructs
and
compound
libraries
which
could
be
employed
in
validating
the
approach.
Options
to
the
35
core
program
include
choices
in
cell
lines,
the
scale
and
throughput
capabilities
of
the
automation,
and
36
the
nature
and
make­
up
of
the
molecular
markers
used
in
profiling.
37
38
The
key
advantage
of
this
initiative
stems
from
its
harnessing
of
the
collective
skills,
experience,
know­
39
how
and
intellectual
property
(
US
5,665,543;
US
08/
267,834;
US
08/
458,691;
and
WO
94/
17208)
of
40
OSI
Pharmaceuticals,
Inc.,
and
that
cross­
licensed
from
Xenometrix,
Inc.,
to
deliver
a
technology
41
platform
which
we
believe
will
be
of
great
value
to
the
pharmaceutical,
chemical,
and
agrochemical
42
industries.
Implementation
time
of
a
staged
program
is
short:
(
1)
approximately
6
months
for
initial
43
validation/
demonstration
studies
on
50
­
100
compounds
(
a
workplan
is
attached
as
page
2;
(
2)
3
44
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
2
I
­
months
for
compound
acquisition
and
formatting
for
the
full
screening
phase
(
proposed);
and
(
3)
12
1
months
for
profiling
of
20,000
compounds
in
ten
assays
(
proposed).
It
should
be
emphasized
that
OSI
2
Pharmaceuticals,
Inc.,
will
work
extensively
with
the
Environmental
Protection
Agency
(
EPA)
to
tailor
3
the
screening
cell
panels,
robotics,
and
informatics
programs
to
suit
the
regulating
agency's
needs.
4
5
6
Staged
chemical
profiling
proposal
7
8
Stage
1:
6
month
validation/
demonstration
study
employing
50
B
100
chemical
compounds
(
specified
9
by
EPA)
designed
to
test
the
overall
performance
and
sensitivity
of
the
cell
based
assay
systems.
Cost
10
$
70K,
including
compound
acquisition
and
plating
up
to
$
5K.
We
have
sourced
and
priced
the
first
45
11
of
an
estimated
70
B
100
compounds
to
be
tested
and
anticipate
that
the
$
70K
project
budget
will
be
12
sufficient
to
procure
the
test
compounds.
Specialty
compounds
requiring
contract
synthesis
are
not
13
anticipated.
14
15
1)
Endocrine
modulator
assays
(
10
assays
with
controls)
16
·
Estrogen
receptor
assay
measuring
both
a
and
b
isoforms
in
MCF­
7
human
breast
cells
measuring
17
activation
and
inhibition,
with
and
without
endogenous
cyp3A4
metabolism.
18
·
Androgen
receptor
assay
in
MDA453
human
breast
cells
measuring
activation
and
inhibition,
with
19
and
without
endogenous
cyp3A4
metabolism.
20
·
Thyroid
hormone
receptor
assay
in
MCF7
human
breast
and/
or
HeLa
cervical
carcinoma
cells
21
measuring
activation
and
inhibition,
with
endogenous
cyp3A4
metabolism.
22
23
(
Proposed)
Stage
2:
3
month
compound
acquisition
phase
in
which
~
1mg
(
or
flick)
of
compound
is
24
dispensed
into
OSI
Pharmaceuticals,
Inc.
supplied
bar­
coded
vials
and
returned
to
OSI
25
Pharmaceuticals,
Inc.,
for
inventory
and
formatting
of
master
compound
screening
plates.
Compounds
26
can
be
supplied
with
structures
(
in
electronic
format)
or
anonymously.
Alternatively
OSI
27
Pharmaceuticals
has
the
capacity
to
source
compound
through
standard
suppliers
and
format
the
28
necessary
screening
plates
directly.
29
30
(
Proposed)
Stage
3:
12
month
high­
throughput
screening
campaign
of
a
maximum
of
50,000
31
compounds
at
a
rate
of
38,000
assays/
day
or
300
compounds/
day
in
10
assays
using
a
5
point
dose­
32
response
in
duplicate.
Data
reduction
and
QC
are
handled
on­
line.
QSAR
modeling
can
be
provided
33
given
structural
information.
Cost
$
3.9M.
34
35
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
3
I
­
Molecular
markers
1
2
Steroid
response
element
Cell
line
Steroid
target
3
ERE
­
MCF­
7
(
breast)
estrogen
(
a
 
and
b
receptors)
4
ARE
­
MDA­
453
(
breast)
androgen
5
TRE
­
MCF­
7
(
breast)
thyroid
hormone
6
HeLa
(
cervical)
7
8
ERE,
ARE,
and
TRE
are
the
DNA
response
elements
which
bind
the
corresponding
activated
receptor
9
and
modulate
gene
transcription.
The
fusion
constructs
contain
4
copies
of
the
ERE,
ARE
or
TRE
10
fused
to
the
HSV
TK
promoter
(
containing
mutations
to
reduce
basal
activity)
and
luciferase
reporter
11
gene.
Reporter
constructs
also
contain
selectable
marker
(
SV­
puro)
which
confers
resistance
of
12
transfected
cells
to
the
antibiotic
puromycin.
13
14
15
Cell
lines
and
characteristics
16
17
MCF­
7
18
The
cell
line
MCF­
7
was
derived
from
a
human
breast
carcinoma
and
will
be
used
to
evaluate
potential
19
compound
modulation
of
the
estrogen
receptor
(
ER).
The
assay
measures
binding
of
the
ER
to
the
20
ERE
DNA
sequence,
resulting
in
increased
reporter
gene
expression
(
luciferase).
It
can
detect
agonists
21
and
antagonists
of
estrogen.
The
ERE
DNA
sequence
does
not
bind
significantly
either
the
22
progesterone
receptor,
glucocorticoid
receptor,
androgen
receptor,
thyroid
receptor,
vitamin
D
23
receptor
or
retinoid
receptors.
MCF­
7
contains
both
a
and
b
ER
isoforms.
24
25
MDA­
MB­
453
26
Human
breast
cancer
cell
line
MDA­
MB­
453
will
be
used
for
measurement
of
androgen
receptor
27
modulation
through
the
ARE
construct.
This
line
has
high
androgen
receptor
number
and
responds
28
well
to
androgen
stimulation.
Androgen
receptor,
progesterone
receptor,
mineralocorticoid
receptors
29
and
glucocorticoid
receptors
all
can
bind
the
ARE
DNA
sequence.
However
the
MDA­
MB­
453
cell
30
line
exhibits
very
low
level
responses
to
estrogen,
progesterone
and
glucocorticoid
in
comparison
to
31
the
androgen
receptor
response,
making
this
line
ideal
for
these
studies.
32
33
MCF­
7/
HeLa
34
Both
the
breast
cell
line
MCF­
7
and
the
HeLa
cervical
carcinoma
cell
line
will
be
evaluated
for
thyroid
35
hormone
receptor
modulation
through
the
TRE
construct.
Both
lines
are
commonly
used
to
measure
36
TRE
activation
and
inhibition
and
the
line
showing
the
best
response
will
be
used
for
compound
37
screening..
It
should
be
noted
that
both
thyroid
hormone
receptors
(
TR
a
,
b
)
and
retinoic
acid
38
receptors
(
RARs
and
RXRs)
interact
with
the
TRE
construct,
either
as
homodimers
or
as
39
heterodimers.
Both
TR
and
RAR/
RXRs
serve
as
targets
for
compound
modulation
of
endocrine
and
40
development
functions.
The
proposed
cell
lines
are
suitable
for
detecting
TR/
RXR
interaction.
41
42
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
4
I
­
Cyp3A4
metabolic
activation
1
In
situations
where
it
is
desirable
to
screen
drug
candidates
in
the
presence
of
metabolic
activation
by
2
the
cytochrome
P450
isozyme,
CYP3A4,
we
propose
using
engineered
MCF­
7,
MDA453
and
HeLa
3
cell
lines
constitutively
expressing
3A4.
This
isozyme
is
responsible
for
metabolizing
70%
of
known
4
drugs.
The
cyp3A4
cDNA
is
licensed
through
the
National
Cancer
Institute
(
Dr.
F.
Gonzales).
5
6
Assay
systems
7
8
Promoter
sequences
will
be
cloned
upstream
of
the
luciferase
reporter
gene
from
P.
pyralis.
The
9
sensitive
and
quantitative
reporter
vector,
pUV120
includes
a
bacterial
origin
of
replication
for
10
propagation
in
E.
coli.
These
vectors
carry
splicing
and
polyadenylation
sequences
for
correct
11
processing
of
luciferase
mRNA
transcribed
by
the
target
promoter,
a
polylinker
sequence
for
insertion
12
of
foreign
promoters
5'
of
the
luciferase
reporter,
a
termination
signal
5'
of
the
inserted
promoter
13
sequence
that
serves
to
prevent
transcriptional
read­
through
from
upstream
transcription
units
14
following
integration
in
stable
cell
lines,
and,
finally,
eucaryotic
selectable
markers
(
SV­
puro)
for
15
positive
selection
of
recipient
cells
following
transfection.
These
vectors
have
been
constructed
and
16
optimized
by
OSI
Pharmaceuticals,
Inc.,
and
allow
for
sensitive
detection
of
even
weak
promoter
17
expression.
18
19
Electroporation
is
the
preferred
method
for
the
generation
of
stable
cell
lines
harboring
linearly
20
integrated
reporter
gene
constructs.
Briefly,
reporter
construct
DNAs
are
linearized
by
restiction
21
endonuclease
digestion,
the
linear
DNA
is
transferred
into
the
recipient
cells
by
electroporation
and
22
antibiotic
(
puromycin
or
neomycin)
is
added
to
select
for
those
cells
which
stably
integrate
the
23
construct
DNA.
Resistant
cells
clones
are
picked
and
analyzed
for
correct
regulation
of
the
integrated
24
luciferase
reporter
construct
and
luciferase
expression,
where
detergent
extracts
from
transfected
cells
25
are
incubated
with
luciferin,
ATP,
Mg2+
and
DTT
under
standard
conditions.
Luciferase
expression
26
from
the
target
promoter
is
further
characterized
for:
(
i)
faithful
integration
of
linear
construct
DNA
27
by
isolation
of
genomic
DNA
and
Southern
blot
hybridization
analysis;
and
(
ii)
correct
inducible
28
regulation
of
the
integrated
reporter
construct.
29
30
31
Molecular
and
Cellular
Biology
32
33
OSI
Pharmaceuticals,
Inc.,
has
considerable
strength
in
state­
of­
the­
art
molecular
and
cellular
biology.
34
This
includes
a
highly
trained
team
of
molecular
biologists
and
chemists,
who
are
capable
of
rapidly
35
identifying
and
isolating
regulatory
regions
involved
in
the
control
of
transcription
of
target
genes,
as
36
well
as
constructing
defined
reporter
vectors
used
to
monitor
even
minimal
changes
in
gene
expression.
37
These
vector
systems
are
stably
integrated
in
the
appropriate
cellular
background,
to
produce
cell
lines
38
for
high
throughput
screening.
Expertise
in
transcription
allows
the
multidisciplinary
team
to
perform
39
rapid
mechanism
of
action
studies
to
identify
mechanism(
s)
of
action
the
molecular
target
of
a
given
40
compound
on
a
molecular
target
rapidly
and
comprehensively.
OSI
Pharmaceuticals,
Inc.,
has
41
unparalleled
cell
culture
facilities
to
address
the
importance
of
cell
based
assay
systems
which
include
42
30
stand­
alone
tissue
culture
incubators,
hot
room
facilities
for
large
scale
suspension
culture,
and
20
43
custom
designed
and
developed
tissue
culture
incubators
incorporated
into
our
robotic
screening
44
systems.
45
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
5
I
­
1
Screening
technology
2
3
In
order
to
facilitate
the
screening
of
large
numbers
of
test
samples,
OSI
Pharmaceuticals,
Inc.
has
4
invested
extensively
in
the
development
of
proprietary
robotics
systems.
This
screening
approach
5
combines
live
genetically
engineered
cells
and
unique
robotics,
enabling
the
screening
of
greater
than
6
five
million
compounds
annually.
7
8
The
robotics
systems
handle
every
step
in
the
assay
procedure.
The
systems
consist
of
fully
automated
9
tissue
culture
incubators,
liquid
handling
/
dispensing
systems
for
the
dilution
and
addition
of
test
10
samples,
and
an
array
of
robotics
units
for
manipulation
of
each
step
of
the
assay
loop.
Robotic
arm
11
assemblies
are
employed
to
shuttle
microplates
through
the
assay
cycle.
Profiling
assays
culminate
with
12
a
read­
out
from
a
96­
well
luminometer.
Data
are
captured
automatically
into
a
processing
network
13
that
performs
quality
control
(
QC)
analysis
on
each
individual
microplate
and
carries
out
a
rapid
data
14
reduction
to
identify
active
compounds.
Automation
on
this
scale
has
proven
to
be
essential
for
15
producing
high
quality
data
from
cell
based
screens.
In
addition,
it
has
provided
a
number
or
major
16
advantages
over
other
screening
approaches:
17
18
(
i)
High­
Throughput:
The
robotic
system
designed
for
cell
panel
profiling
can
comfortably
evaluate
19
300
compounds
per
day,
in
a
five
point
dose
response
against
10
assay
targets
(
3
endocrine
targets
up
20
and
down;
2
targets
with
and
without
cyp3A4
metabolism).
Duplicate
determinations
are
averaged
to
21
yield
a
mean
inhibition
or
stimulation
of
receptor
activity.
Additional
positive
and
negative
controls
are
22
included
on
each
microtiter
plate
to
provide
clear
functional
plate
pass/
fail
criteria.
Potential
compound
23
cytotoxicity
is
minimized
by
using
a
short
incubation
time
of
between
12
and
24
hours.
Compounds
24
which
do
inhibit
basal
promoter
activity
by
greater
than
2
standard
deviations
will
be
further
studied
25
using
a
standard
cytotoxicity
assay
measuring
mitochondrial
respiration
(
MTT)
to
derive
a
cytotoxicity
26
IC50
value
for
that
compound.
27
28
(
ii)
Accuracy:
Automation
has
proven
to
be
highly
effective
in
removing
protocol
variation
and
in
more
29
accurately
controlling
and
synchronizing
procedures.
With
the
current
technology,
cell­
based
high­
30
throughput
screens
can
be
run
with
coefficients
of
variation
of
less
than
15%.
31
32
(
iii)
Cost
Effectiveness:
Support
for
each
robotics
system
requires
only
a
three­
person
team.
This
33
includes
the
staff
for
all
tissue
culture,
robotics
maintenance,
robotics
operation,
and
rudimentary
initial
34
data
analysis.
35
36
(
iv)
Compound
Handling:
Over
the
last
several
years,
OSI
has
been
successful
in
compiling
an
37
extensive
library
of
screening
samples.
We
have
developed
robotic
systems
which
enable
the
rapid
38
preparation
of
large
compound
libraries
into
a
96­
well
format
suitable
for
compound
screening.
This
39
approach
offers
the
possibility
of
archiving
sets
of
master
compound
plates
for
future
screening
needs.
40
This
robotic
system
is
capable
of
supporting
the
compound
needs
of
our
four
screening
systems
and
41
handles
our
growing
collection
(
including
collaborators
libraries)
of
more
than
1.5
million
synthetic
42
organic
compounds.
43
44
EDSTAC
Final
Report
Chapter
Four
Appendices
August
1998
6
I
­
(
v)
Automation
and
Information
Handling:
The
Informatics
Technology
Development
group
at
OSI
1
possesses
expertise
in
the
areas
of
database
administration,
application
development,
and
data
analysis.
2
Currently,
the
staff
consists
of
an
Oracle
certified
database
administrator,
an
Oracle
certified
master
3
developer,
an
ISIS
certified
database
administrator,
a
computational
chemist
experienced
in
data
4
analysis,
and
personnel
with
expertise
in
the
development
of
Internet/
Web­
based
tools.
The
primary
5
responsibility
of
the
team
is
to
provide
services
of
information
management
and
interpretation.
6
Currently,
we
have
developed
an
in­
house
version
of
Xenometrix's
molecular
toxicology
database.
7
8
(
vi)
Quality
Control
Criteria
and
Validation
of
the
Gene
Profiling
Panel:
The
proper
construction
of
the
9
profiling
panel
will
be
verified
by
several
criteria:
(
1)
the
DNA
constructs
will
be
properly
integrated
10
within
the
cell
line
(
unrearranged);
(
2)
the
reporter
signal
strength
will
be
sufficient
for
robotic
high­
11
throughput
screening
for
both
agonist
and
antagonist
activity;
(
3)
the
marker
genes
will
respond
12
appropriately
using
a
series
of
compounds
containing
chemical
classes
which
selectively
induce
13
expression
of
the
individual
target
genes.
14
15
(
vii)
Report
Format:
Reports
will
contain
data
tables
with
compound
ID
number,
compound
structure
16
(
if
available),
raw
data
(
luciferase
units),
EC
2X
(
agonist
activity),
IC
50
(
antagonist
activity).
Footnotes
17
can
include
specifics
with
regards
to
assay
conditions,
limitations
and/
or
difficulties
associated
with
a
18
given
data
point.
19
20
(
viii)
Success
in
applying
high­
throughput
screening
to
the
discovery
of
therapeutically
useful
21
compounds:
OSI
Pharmaceuticals,
Inc.,
has
successfully
utilized
with
HTS
robotic
screening
22
technology
in
the
area
of
cancer
with
compound
in
Phase
I
trials
(
in
collaboration
with
Pfizer),
and
23
with
compounds
active
in
animals
identified
in
the
therapeutic
areas
of
inflammation,
cardiovascular
24
disease,
diabetes,
anti­
viral,
prescription
cosmetics
and
anemia.
Last
year,
over
7
million
compound
25
equivalents
were
screened
in
multiple
therapeutic
programs.
26
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
Appendix
J
1
2
References
and
Sources
for
Chapter
Five
 
Screening
and
Testin
g
3
4
Table
of
Content
s
5
6
I.
Mammalian
In
Vitro
and
In
Vivo
Assays
(
Recommended
and
Considered)
.
.
.
.
.
.
.
.
.
.
.
1
II.
Fish
Gonadal
Recrudescence
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10
III.
Alternative
Mammalian
Reproduction
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11
IV.
Avian
Reproduction
(
EPA
OPPTS
850.2300;
OECD
206)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11
V.
Nest
Attentiveness/
Incubation
Behavior
Test
References
to
be
Used
for
Protocol
Development
and
Standardization
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
12
VI.
Visual
Cliff
Test
References
to
be
Used
for
Protocol
Development
and
Standardization
12
VII.
Cold
Stress
References
to
be
Used
for
Protocol
Development
and
Standardization
.
12
VIII.
Fish
Life
Cycle
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13
IX.
Methods
to
Select
the
Target
Doses
for
T2T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13
X.
Low
Dose
Consideration
for
T2T
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13
XI.
Documents
Distributed
to
Screening
and
Testing
Work
Group
Members
.
.
.
.
.
.
.
.
.
.
14
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
2
J
­
I.
Mammalian
In
Vitro
and
In
Vivo
Assays
(
Recommended
and
Considered)

Aakvaag
A.,
E.
Utaaker,
T.
Thorsen,
O.
A.
Lea,
and
H.
Lahooti,
"
Growth
control
of
human
mammary
cancer
cells
(
MCF­
7
cells)
in
culture:
effect
of
estradiol
and
growth
factors
in
serum­
containing
medium,"
Cancer
Research,
50,
1990,
pp.
7806­
10.
Aitken
S.
C.,
M.
E.
Lippman
,
A.
Kasid,
and
D.
R.
Schoenberg,
"
Relationship
between
the
expression
of
estrogen­
regulated
genes
and
estrogen­
stimulated
proliferation
of
MCF­
7
mammary
tumor
cells,"
Cancer
Research,
45,
1985,
pp.
2608­
15.
Allegretto,
E.,
and
R.
Heyman,
"
Intracellular
Receptor
Characterization
And
Ligand
Screening
By
Transactivation
And
Hormone­
Binding
Assays,"
Methods
in
Molecular
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EDSTAC
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August
1998
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­
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"
Retinoic
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binding
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ovarian
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T.
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"
A
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of
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oestrogen
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progesterone
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their
subcellular
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B.
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H.
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"
A
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resistant
MCF­
7
human
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cell
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Effects
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Saal,
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ICI
182,780,
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7
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dependent
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7
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1998
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7
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Optimization
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7
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L.
A.
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Horwitz,
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C.
Brooks,
S.
C.
"
Induction
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the
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Specific
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7
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Selected
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17J:
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QSAR
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MI,
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William,
J.,
J.
Odum,
R.
W.
Lewis,
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A.
M.
Brady,
"
The
Oral
Administration
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Polysorbate
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Wilson,
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M.,
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"
Binding
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"
Androgen
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Workshop:
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Simental,
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J.
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M.
Sar,
D.
B.
Lubahn,
K.
L.
Olsen,
F.
S.
French,
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E.
M.
Wilson,
"
A
single
base
mutation
in
the
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receptor
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"
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ovarian
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Zacharewski,
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vitro
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B.
K.
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"
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the
presence
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estrogen
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and
Ah
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black
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29,
1995,
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2146.

II.
Fish
Gonadal
Recrudescence
Assay
Arcand­
Hoy,
L.
D.,
and
W.
H.
Benson.
"
Fish
reproduction:
An
ecologically
relevant
indicator
of
endocrine
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Toxicol.
Chem.,
17(
1),
1998,
pp.
57.
Gustafsson,
J.
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Characteristics
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function
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29,
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Final
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Five
Appendices
August
1998
12
J
­
Nimrod,
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and
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"
Estrogenic
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Nimrod,
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Reproduction
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Petit,
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Y.
Valotaire,
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"
Differential
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1995,
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584­
592.

III.
Alternative
Mammalian
Reproduction
Test
Gray,
L.
E.
Jr.,
J.
Ostby,
R.
Simong,
J.
Ferrell,
G.
Rehnberg,
R.
Linder,
R.
Cooper,
J.
Goldman,
and
J.
Laskey,
"
The
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2,
1988,
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281­
287.
Zenick,
H.,
E.
D.
Clegg,
S.
D.
Perreault,
G.
R.
Klinefelter,
and
L.
E.
Gray,
"
Assessment
of
Male
Reproductive
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and
Methods
of
Toxicology,
3,
1994,
pp.
937­
988.

IV.
Avian
Reproduction
(
EPA
OPPTS
850.2300;
OECD
206)

Dvorak,
J.,
J.
L.
Halvorsen,
P.
Gulick,
K.
A.
Rauen,
U.
K.
Abbott,
B.
J.
Kelly,
and
F.
T.
Schultz,
"
DNA
cloning
of
a
Z­
and
W­
linked
gene
in
gallinaceous
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J.
Heredity,
83,
1992,
pp.
22­
55.
Halvorsen,
J.
L.,
"
Avian
sex
identification
by
recombinant
DNA
technology,"
Proceedings
of
the
Annual
Meeting
of
the
Association
of
Avian
Veterinarians,
Phoenix,
Arizona,
1990,
pp.
84­
90.
Ottinger,
M.
A.,
and
M.
Bakst,
"
Peripheral
androgen
concentrations
and
testicular
morphology
in
embryonic
and
young
Japanese
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and
Compare.
Endocrinol,
43,
1981,
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170­
177.
Ottinger,
M.
A.,
and
H.
J.
Brinkley,
"
Testosterone
and
sex­
related
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and
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Relationship
during
maturation
in
the
adult
Japanese
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Hormones
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Behavior,
11,
1978,
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182.
Panzica,
G.
C.,
N.
Aste,
C.
Viglietti­
Panzica,
and
M.
A.
Ottinger,
"
Structural
sex
differences
in
the
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Sharp,
P.
J.,
"
A
conparison
of
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of
plasma
luteinizing
hormone
concentrations
in
male
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female
domestic
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J.
Endocrinol.,
67,
1975,
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223.
Somers,
J.
D.,
E.
T.
Moran
Jr.,
B.
S.
and
Reinhart,
"
Effect
of
External
Application
of
Pesticides
to
the
Fertile
Egg
on
Hatching
Success
and
Early
Chick
Performance
3.
Consequences
of
Combining
2,4­
D
with
Picloram
and
Extremes
in
Contamination,"
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Tori,
G.
M.,
and
L.
P.
Mayer,
"
Effects
of
Polychlorinated
Biphenyls
on
the
Metabolic
Rates
of
Mourning
Doves
Exposed
to
Low
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682.

V.
Nest
Attentiveness/
Incubation
Behavior
Test
References
to
be
Used
for
Protocol
Development
and
Standardization
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
13
J
­
Fox,
G.
A.,
A.
P.
Gilman,
D.
B.
Peakall,
and
F.
W.
Anderka,
"
Behavioural
Abnormalities
of
Nesting
Lake
Ontario
Herring
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J.
Wildl.
Manage.,
42,
1978,
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477­
483.
McArthur,
M.
L.
B.,
G.
A.
Fox,
D.
A.
Peakall,
and
B.
J.
R.
Philogene,
B.
J.
R.,
"
Ecological
Significance
Of
Behavioral
and
Hormonal
Abnormalities
In
Breeding
Ring
Doves
Fed
An
Organochlorine
Chemical
Mixture,"
Arch.
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Contam.
Toxicol.,
12,
1983,
pp.
343­
353.

VI.
Visual
Cliff
Test
References
to
be
Used
for
Protocol
Development
and
Standardization
Baxter,
W.
L.,
R.
L.
Linder,
and
R.
B.
Dahlgren,
Dieldrin
Effects
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Dahlgren,
R
B.
and
R.
L.
Linder,
Effects
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J
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35,
2,
1971,
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319.
Emlen,
Jr.,
J.
T.
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Cliff
Edge
and
Escape
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Behaviour,
22,
1963,
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1­
15.
Fleming,
W.
J.,
G.
H.
Heinz,
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C.
A.
Schuler,
C.
A.
"
Lethal
and
Behavioral
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of
Chlordimeform
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36,
1985,
pp.
37­
47.
Fox,
G.
A.
"
Eggshell
Quality:
It's
Ecological
and
Physiological
Significance
In
a
DDE­
Contaminanted
Common
Tern
Colony,"
Wilson
Bull,
88,
3,
1976,
pp.
459­
477.

VII.
Cold
Stress
References
to
be
Used
for
Protocol
Development
and
Standardization
Fleming,
W.
J.,
G.
H.
Heinz,
J.
C.
Franson,
and
B.
A.
Rattner,
"
Toxicity
of
Abate
4E
(
Temephos)
in
Mallard
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the
Influence
of
Cold,"
Environmental
Toxicology
and
Chemistry,
4,
1985,
pp.
193­
199.
Maguire,
C.
C.
and
B.
A.
Williams,
"
Response
of
Thermal
Stressed
Bobwhite
to
Organophosphorous
Exposure,"
Environmental
Pollution,
47,
1987,
pp.
25­
39.
Martin,
P.
A.
and
K.
R.
Solomon,
"
Acute
Carbofuran
Exposure
and
Cold
Stress:
Interactive
effects
in
Mallard
Ducklings,"
Pesticide
Biochemistry
and
Physiology,
40,
1991,
pp.
117­
127.
Rattner,
B.
A.,
L.
Sileo,
and
C.
G.
Scanes,
"
Hormonal
Responses
and
Tolerance
to
Cold
of
Female
Quail
following
Parathion
Ingestion,"
Pesticide
Biochemistry
and
Physiology,
18,
1982,
pp.
132­
138.

VIII.
Fish
Life
Cycle
Test
Arcand­
Hoy,
L.
D.,
and
W.
H.
Benson,
"
Fish
reproduction:
An
ecologically
relevant
indicator
of
endocrine
disruption.
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Toxicol.
Chem.,
17,
1,
1998,
pp.
49­
57.
Benson,
W.
H.,
G.
van
der
Kraak,
C.
Tyler,
K.
E.
Brugger,
G.
Daston,
M.
Fry,
S.
Gimeno,
F.
Hunger,
M.
Kolossa,
R.
Länge,
and
P.
Matthiessen,
P.
"
Strategies
and
approaches
to
in
vivo
screening
and
testing
in
identifying
the
hazards
of
endocrine
modulating
chemicals
to
wildlife,"
SETAC­
Europe/
OECD/
EC
Expert
Workshop
on
Endocrine
Modulators
and
Wildlife:
Assessment
and
Testing,
1997,
pp.
59­
78.
Cyr,
D.
G.
and
J.
G.
Eales,
"
Interrealtionships
between
thyroidal
and
reproductive
endocrine
systems
in
fish,"
Rev.
Fish
Biol.,
6,
1996,
pp.
165­
200.
Ghosh,
S.
and
P.
Thomas,
"
Antagonistic
effects
of
xenobiotics
on
steroid­
induced
final
maturation
of
Atlantic
croaker
oocytes
in
vitro,"
Mar.
Environm.
Res.,
39,
1995,
pp.
159­
163.
Nimrod,
A.
C.
and
W.
H.
Benson,
"
Assessment
of
estrogenic
activity
in
fish,"
Chemically­
Induced
Alterations
in
the
Functional
Development
and
Reproduction
of
Fishes,
1997,
pp.
87­
100.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
14
J
­
Tyler,
C.
R.,
B.
van
der
Eerden,
S.
Jobling,
G.
H.
Panter,
and
J.
P.
Sumpter,
"
Measurement
of
vitellogenin,
a
biomarker
for
exposrue
to
estrogenic
chemicals,
in
a
wide
variety
of
cyprinid
fish,"
J.
Comp.
Physiol.,
B,
166,
1996,
pp.
418­
426.

IX.
Methods
to
Select
the
Target
Doses
for
T2T
Flaws,
J.
A.,
R.
J.
Sommer,
E.
K.
Silbergeld,
R.
E.
Peterson,
and
A.
N.
Hirshfield,
"
In
Utero
and
Lactational
Exposure
to
2,3,7,8
­
Tetrachlorodibenzo­
p­
dioxin
(
TCDD)
Induces
Gnital
Dysmorphogenesis
in
the
Female
Rat,"
Toxicol.
Appl.
Pharmacol.
147,
1997,
pp.
351­
362.
Gray,
L.
E.
Jr.,
C.
Wolf,
P.
Mann,
and
J.
S.
Ostby,
In
Utero
Exposure
to
Low
Doses
of
2,3,7,8
­
Tetrachlorodibenzo­
p­
dioxin
Alters
Reproductive
Development
of
Female
Long
Evans
Hooded
Rat
Offspring.
Toxicol.
Appl.
Pharmacol.,
146,
1997,
pp.
237­
244.

X.
Low
Dose
Consideration
for
T2T
Nagel,
S.
C.,
F.
S.
vom
Saal,
K.
A.
Thayer,
M.
G.
Dhar,
M.
Boechler,
and
W.
V.
Welshons,
"
Relative
Binding
Affinity­
Serum
Modified
Access
(
RBA­
SMA)
Assay
Predicts
the
Relative
in
Vivo
Bioactivity
of
the
Xenoestrogens
Bisphenol
A
and
Octylphenol."
Environmental
Health
Perspectives,
105,
1,
1997,
pp.
70­
76.
Reel,
J.
R.,
R.
W.
Tyl,
A.
D.
Lawton,
and
J.
C.
Lamb,
"
Bisphenol
A:
reproduction
and
fertility
assessment
in
CD­
1
mice
when
administered
via
subcutaneous
silastic
implants,"
NTIS
PB84­
155308,
1984.
Reel,
J.
R.,
J.
D.
George,
A.
D.
Lawton,
C.
B.
Myers,
and
J.
C.
Lamb,
"
Bisphenol
A:
reproduction
and
fertility
assessment
in
CD­
1
mice
when
administered
in
the
feed,"
NTIS
PB86­
103207,
1985.
vom
Saal,
F.
S.,
P.
S.
Cooke,
D.
L.
Buchanan,
P.
Palanza,
K.
A.
Thayer,
S.
C.
Nagel,
S.
Parmigiani,
and
W.
V.
Welshons,
"
A
physiologically
based
approach
to
the
study
of
bisphenol
A
and
other
estrogenic
chemicals
on
the
size
of
the
reproductive
organs,
daily
sperm
production
and
behavior,"
Toxicol.
Indus.
Health,
14,1­
2,
1998,
pp.
239­
260.

XI.
Documents
Distributed
to
Screening
and
Testing
Work
Group
Members
During
the
course
of
their
deliberations
to
evaluate
all
potentially
relevant
screening
and
test
methods,
the
STWG
utilized
an
extensive
set
of
resource
materials
including
peerreviewed
publications,
workshop
reports,
and
independent
assessments
by
various
international
scientific
groups
and
regulatory
bodies.
The
following
documents
were
of
particular
importance:

1.
Endocrine
Screening
Methods
Workshop:
Meeting
Report,
July,
1996.
Duke
University
Meeting.
2.
Workshop
on
Screening
Methods
for
Endocrine
Disruptors
in
Wildlife:
Draft
Workshop
Report,
March,
1997.
Kansas
City
Meeting.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
15
J
­
3.
OECD
Appraisal
of
Test
Methods
for
Sex­
Hormone
Disrupting
Chemicals,
1st
Draft,
October,
1996.
Prepared
by
the
MRC
Institute
for
Environmental
and
Health.
4.
Validation
and
Regulatory
Acceptance
of
Toxicological
Test
Methods.
A
Report
of
the
ad
hoc
Interagency
Coordinating
Committee
on
the
Validation
of
Alternative
Methods.
NIEHS,
Draft
Version,
October
16,
1995.

In
addition,
the
following
documents
were
disseminated
to
the
STWG
members:

Ankley,
G.
T.,
R.
D.
Johnson,
G.
Toth,
L.
C.
Folmar,
N.
E.
Detenbeck,
and
S.
P.
Bradbury,
"
Development
of
a
Research
Strategy
for
Assessing
the
Ecological
Risk
of
Endocrine
Disrupters,"
Rev.
Toxicol.,
In
Press.
Ashby,
J.,
J.
Oden,
and
J.
R.
Foster,
"
Activity
of
Raloxifene
in
Immature
and
Ovariectomized
Rat
Uterotrophic
Assays,"
Zenaca
Central
Toxicological
Laboratory,
Alderly
Park,
Macclesfield,
Cheshire,
UK,
1997.
Ashby,
J.,
J.
Odum,
H.
Tinwell,
and
P.
A.
Lefevre,
"
Assessing
the
Risks
of
Adverse
Endocrinemediated
Effects:
Where
to
From
Here?,"
(
submitted
to
Reg.
Tox
Pharmacol.
as
meeting
overview),
1997.
Cook,
Jon.
C.,
A.
M.
Kaplan,
L.
G.
Davis,
and
J.
C.
O'Connor,
"
Development
of
a
Tier
1
Screening
Battery
for
Detecting
Endocrine
Active
Compounds
(
EACs)."
Davis,
Paul
J.,
and
Faith
B.
Davis,
"
Nongenomic
Actions
of
Thyroid
Hormone,"
Thyroid,
6,
5,
1996,
497.
DeGroot,
M.
D.,
Leslie
J
"
Novel
Actions
of
Thyroid
Hormone,"
Thyroid,
6,
5,
1996,
"
NTP
Workshop
on
Validation
and
Regulatory
Acceptance
of
Alternative
Toxicological
Test
Methods,"
Final
Report,
1995.
Fingerman,
Milton,
"
Crustacean
Endocrinology:
A
Retrospective,
Prospective,
and
Introspective
Analysis,"
Physiological
Zoology.,
70,
3,
1997,
pp.
257­
269.
Hajek,
R.
A.,
A.
D.
Robertson,
D.
A.
Johnston,
N.
T.
Van,
R.
K.
Tcholakian,
L.
A.
Wagner,
C.
J.
Conti,
M.
L.
Meistrich,
N.
Contreras,
C.
L.
Edwards,
and
L.
A.
Jones,
"
During
Development,
17I­
Estradiol
Is
a
Potent
Estrogen
and
Carcinogen,"
Environmental
Health
Perspectives,
105,
2,
1997.
Committee
on
Environment
and
Natural
Resources
(
CENR)
of
the
National
Science
and
Technology
Council
"
The
Health
and
Ecological
Effects
of
Endocrine
Disrupting
Chemicals:
A
Framework
for
Planning,"
1996.
National
Research
Council,
Commission
on
Life
Sciences
Hormone­
Related
Toxicants
in
the
Environment;
Proposal
No.
95­
CLS­
056­
01
of
the
National
Academy
of
Science,
1994
Horwitz,
A.
F.
"
Integrins
and
Health,"
Scientific
American,
1997,
pp.
68­
75.
Katzenellenbogen,
B.
S.,
M.
M.
Montano,
P.
le
Goff,
D.
J.
Schodin,
W.
L.
Kraus,
B.
Bhardway,
and
N.
Fujimoto,
Antiestrogens:
Mechanisms
and
Actions
in
Target
Cells.
J.
Steroid
Biochem.
Molec.
Biol.,
53,
1­
6,
1995,
pp.
387­
393.
Kavlock,
et
al.
"
Research
Needs
for
the
Risk
Assessment
of
Health
and
Environmental
Effects
of
Endocrine
Disrupters:
A
Report
from
the
U.
S.
EPA­
Sponsored
Workshop,"
Environmental
Health
Perspectives,
104,
4,
1996.
Kohn,
M.
C.,
C.
H.
Sewall,
G.
W.
Lucier,
and
C.
J.
Portier,
"
A
Mechanistic
Model
of
Effects
of
Dioxin
on
Thyroid
Hormones
in
the
Rat,"
Toxicology
and
Applied
Pharmacology,
165,
1996,
pp.
29­
48.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
16
J
­
Leonard,
J.
L.,
and
A.
P.
Farwell,
"
Thyroid
Hormone­
Regulated
Actin
Polymerization
in
Brain,"
Thyroid,
7,
1,
1997,
pp.
147­
151.
Nagel,
S.
C.,
F.
S.
vom
Saal,
K.
A.
Thayer,
M.
G.
Dhar,
M.
Boechler,
and
W.
V.
Welshons,
"
Relative
Binding
Affinity­
Serum
Modified
Access
(
RBA­
SMA)
Assay
Predicts
the
Relative
in
Vivo
Bioactivity
of
the
Xenoestrogens
Bisphenol
A
and
Octylphenol,"
Environmental
Health
Perspectives,
105,
1,
1997,
pp.
70­
76.
Nagel,
S.
C.,
F.
S.
vom
Saal,
and
W.
V.
Welshons,
"
The
Effective
Free
Fraction
of
Estradiol
and
Xenoestrogens
in
Human
Serum
Measured
By
the
Whole
Cell
Uptake
Assays:
Physiology
of
Delivery
Modifies
Estrogenic
Activity,"
Proc.
Soc.
Exp.
Bio.
Med.,
In
Press.
O'Connor,
J.
C.,
J.
C.
Cook,
S.
C.
Craven,
C.
S.
Van
Pelt,
and
J.
D.
Obourn,
An
in
Vivo
Battery
for
Identifying
Endocrine
Modulators
that
are
Estrogenic
or
Dopamine
Regulators.
Fundamental
and
Applied
Toxicology.
3,
1996,
pp.
182­
195.
Odum,
J.,
P.
A.
Lefevre,
S.
Tittensor,
D.
Paton,
E.
J.
Routledge,
N.
A.
Beresford,
J.
P.
Sumpter,
J.
P.
and
J.
Ashby,
"
The
Rodent
Uterotrophic
Assay:
Critical
Protocol
Features,
Studies
With
Nonylphenols,
Comparison
With
a
Yeast
Estrogenicity
Assay,"
In
Press,
Reg.
Tox.
Pharmacol.
Rudel,
R.
"
Predicting
Health
Effects
of
Exposure
to
Compounds
With
Estrogenic
Activity.
Silent
Spring
Institute,"
Newton,
MA.
Environmental
Health
Perspectives,
105,
3,
1997.
Special
Report
on
Environmental
Endocrine
Disruption:
An
Effects
Assessment
and
Analysis
Document
(
Draft
Version);
EPA
Risk
Forum
White
Paper,
November
28,
1996.
Szelei,
J.,
J.
Jimenez,
A.
M.
Soto,
M.
F.
Luizzi,
and
C.
Sonnenschein,
C.
"
Androgen­
Induced
Inhibition
of
Proliferation
in
Human
Breast
Cancer
MCF7
Cells
Transfected
with
Androgen
Receptor,"
Endocrinology,
138,
4,
1997,
pp.
1406­
1412.
Tong,
W.,
L.
Xing,
W.
J.
Welsh,
and
D.
M.
Sheehan,
In
Press.
"
QSAR
Models
for
Binding
of
Estrogenic
Compounds
to
Estrogen
Receptor
a
and
b
Subtypes,"
R.
O.
W.
Sciences,
Jefferson,
Arkansas.
Tong,
W.,
R.
Perkins,
R.
Strelitz,
E.
R.
Collantes,
S.
Keenan,
W.
J.
Welsh,
W.
S.
Branham,
and
D.
M.
Sheehan,
In
Press.
"
Quantitative
Structure­
Activity
Relationships
(
QSARs)
for
Estrogen
Binding
to
the
Estrogen
Receptor:
Predictions
Across
Species,"
R.
O.
W.
Sciences,
Jefferson,
Arkansas.
Validation
and
Regulatory
Acceptance
of
Toxicological
Test
Methods:
A
Report
of
the
Ad
Hoc
Interagency
Coordinating
Committee
on
the
Validation
of
Alternative
Test
Methods.
NIEHS.
Final
Report,
March,
1997.
vom
Saal,
F.
S.,
B.
G.
Timms,
M.
M.
Montano,
P.
Palanza,
K.
A.
Thayer,
S.
C.
Nagel,
M.
D.
Dhar,
V.
K.
Ganjam,
S.
Parmigiani,
and
W.
V.
Welshons,
"
Prostate
Enlargement
in
Mice
Due
to
Fetal
Exposure
to
Low
Doses
of
Estradiol
rr
Diethystilbestrol
and
Opposite
Effects
at
High
Doses,"
Proc.
Natl.
Acad.
Sci.,
94,
1997,
pp.
2056­
2061.
vom
Saal,
F.
S.,
P.
S.
Cooke,
D.
L.
Buchanan,
P.
Palanza,
K.
A.
Thayer,
S.
C.
Nagel,
S.
Parmigiani,
and
W.
V.
Welshons,
"
A
Physiologically
Based
Approach
to
the
Study
of
Bisphenol
A
and
Other
Estrogenic
Chemicals
on
the
Size
of
Reproductive
Organs,
Daily
Sperm
Production
and
Behavior,"
Journal
of
Toxicology
and
Industrial
Health,
1997.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
Appendix
K
Brief
Overviews
of
Assays
Considered
for
Tier
1
Screening
Table
of
Contents
I.
Estrogen
and
Anti­
estrogen
­
Intrinsic
Activity
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
A.
Rat
(
and
other
non­
human
mammalian
and
avian)
ER
Binding
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
B.
hER
Binding
From
MCF­
7
Cell
Lysate
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
2
C.
Estrogen
Competitor
(
Binding)
Screening
Assay
(
A
Receptor/
Ligand
Assay,
PanVera)
.
.
2
II.
Estrogen
and
Anti­
estrogen
­
In
Vitro
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
3
A.
MCF­
7
Proliferation
Assay
(
ESCREEN)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
3
B.
YES­
Yeast
Estrogen
Screen
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
4
C.
MVLN
Assay.
Stably
Transfected
Reporter
Gene
Assay
in
Mammalian
Cells
.
.
.
.
.
.
.
.
.
5
D.
Cotransfected
Reporter
Gene
Assay
in
Mammalian
Cells
(
e.
g.,
CV­
1
or
COS
Cells)
.
.
.
.
6
E.
Stably
Transfected
Reporter
Gene
Assay
in
Mammalian
Cells
(
e.
g.,
MCF­
7
Cells)
.
.
.
.
.
.
6
III.
Estrogen
and
Anti­
estrogen
­
In
Vivo
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
7
A.
Uterine
Peroxidase
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
7
B.
Developmental
Uterotrophic
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
8
C.
Uterine
Weight
Bioassay
in
Juvenile
or
Adult
Ovariectomized
Female
Rats
.
.
.
.
.
.
.
.
.
.
10
D.
Vaginal
Smears
(
Mucification
and
Cornification)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11
E.
Puberty.
Age
at
Vaginal
Opening
(
First
Estrus,
Onset
of
Cyclicity)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
12
F.
Induction
of
Female
Sex
Behavior
(
Proceptive
and
Receptive
Behaviors)
.
.
.
.
.
.
.
.
.
.
.
13
G.
Feeding
Behavior.
Food
Consumption
and
Growth
Rate
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
14
H.
Estrous
Cyclicity
.
.
.
.
.
.
.
.
.
.
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.
.
15
I.
Super
Apical
Developmental
Toxicity
Test
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
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.
.
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.
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.
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.
.
.
.
.
16
IV.
Anti­
Estrogen
­
Synthesis
Metabolism
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
.
.
.
.
.
17
A.
Testis/
Ovary
Culture
In
Vitro
or
Ex
Vivo
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
17
V.
Androgen
and
Anti­
androgen
­
Intrinsic
Activity
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
18
A.
Rat
AR
Equilibrium
Binding
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
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.
.
.
.
.
.
.
.
18
B.
hAR
Whole
Cell
Binding
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
.
.
.
.
.
.
.
.
.
.
19
VI.
Androgen
and
Anti­
androgen
­
In
Vitro
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
20
A.
YAS­
Yeast
Androgen
Screen
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
20
B.
CV­
1
Cell
Assay.
A
hAR
Transcriptional
Activation
Assay
in
Mammalian
Cell
.
.
.
.
.
.
.
21
C.
hAR
Transactivation
Assays
Using
Stable
Cell
Lines
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
21
D.
Leydig
Cell
Culture
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
22
VII.
Androgen
and
Anti­
androgen
­
In
Vivo
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
23
A.
Endocrine
Challenge
Test
(
Fail
et
al.,
1995)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
23
B.
Super
Apical
Developmental
Toxicity
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
24
C.
Pubertal
Development
in
Male
Rodent
(
Preputial
Separation)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
25
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
D.
Hershberger
Assay
(
1953)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
25
VIII.
Anti­
androgen
­
Synthesis
Metabolism
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
26
A.
Testis/
Ovary
Culture
In
Vitro
or
Ex
Vivo
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
26
IX.
Thyroid
­
Intrinsic
Activity
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
27
A.
TR
Binding
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
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.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
27
X.
Thyroid
­
In
Vitro
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
28
A.
Whole
Cell
Binding
Assays.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
28
B.
Stably
Transfected
Cell
Lines.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
28
C.
Thyroid
Hormone­
Responsive
Cells.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
28
D.
Specialized
Cells.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
29
XI.
Thyroid
In­
vivo
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
29
A.
Short­
Term
Serum
T
4
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
29
B.
Long­
Term
Serum
T
4
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
30
C.
Thyroid
Peroxidase
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
30
D.
Malic
Enzyme
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
31
E.
Mammal
Development
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
32
XII.
Three
Alternative
Overviews
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
33
XIII.
Overview
of
Non­
Mammalian
Screens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
37
A.
Procedures
Using
Birds
and
Reptiles
to
Determine
Endocrine
Disruptive
Action
.
.
.
.
.
.
37
B.
Overview
of
Endocrine
Disruptor
Relevant
Screens
to
the
Lower
Vertebrates
and
Invertebrates
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
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.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
38
XIV.
Amphibian
Screens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
39
A.
Vitellogenin
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
39
B.
Frog
Embryo
Teratogenesis
Assay
Xenopus
(
FETAX)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
39
C.
Amphibian
Metamorphosis
Assay
(
Conceptual)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
40
D.
Frog
In
Vivo
Screening
Assay
(
Conceptual)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
40
E.
Metamorphosis
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
40
XV.
Bird
Screens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
41
A.
Avian
Egg­
Injection
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
41
B.
Japanese
Quail
Early
Life
Stage
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
42
C.
Japanese
Quail
Androgenicity
Screen
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
43
D.
Vitellogenin
Production
in
Female
Japanese
Quail
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
43
E.
Vitellogenin
Production
in
Adult
Male
Birds:
Japanese
Quail,
Chickens.
.
.
.
.
.
.
.
.
.
.
.
.
44
F.
Avian
"
Plaque
Assay"
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
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.
.
.
.
.
.
.
.
44
G.
Avian
Cell
Culture
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
45
H.
Chicken
Early
Life
Stage
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
45
I.
Cartilage
Growth
in
Chick
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
.
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.
.
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.
.
.
.
.
.
.
.
.
.
.
46
XVI.
Fish
Screens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
47
A.
Vitellogenin
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
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.
.
.
.
.
.
.
47
B.
In
Vitro
Vitellogenin
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
47
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
C.
In
Vivo
Screening
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
.
.
48
D.
Early
Life
Stage
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
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.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
48
E.
Embryo
and
Sac
Fry
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
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.
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.
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.
.
.
.
.
.
.
49
F.
Partial
Life
Cycle
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
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.
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.
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.
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.
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.
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.
.
.
.
.
49
G.
Full
Life
Cycle
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
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.
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.
.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
.
50
H.
Flounder
Metamorphosis
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
50
I.
In
Vitro
Steroid
Receptor
Competition
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
51
J.
In
Vitro
Steroid
Production
Bioassay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
.
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.
.
.
.
.
.
.
.
.
.
.
51
K.
In
Vitro
Germinal
Vesicle
Breakdown
(
GVBD)
Bioassay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
51
XVII.
Invertebrate
Screens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
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.
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.
.
.
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.
.
.
.
.
.
.
.
.
.
.
.
51
A.
Daphnia
Reproduction
(
Life
Cycle)
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
51
B.
Mysid
Life
Cycle
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
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.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
52
XVIII.
Reptilian
Screens
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
52
A.
Vitellogenin
Production
In
Adult
Male
Turtles
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
52
B.
Sex
Determination
in
Turtles
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
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.
.
.
.
.
.
.
.
53
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
1
K
­
This
Appendix
contains
judgments
about
the
utility
and
level
of
"
validation"
of
specific
assays
that
were
discussed
in
varying
levels
of
detail
by
the
Screening
and
Testing
Work
Group
(
STWG)
members.
The
summaries
should
not
be
considered
"
consensus"
materials,
but
rather
submissions,
from
various
members
of
the
STWG,
developed
to
assist
the
full
group
in
arriving
at
their
final
set
of
recommendations,
which
were
subsequently
forwarded
to
the
full
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
for
further
discussion.

I.
Estrogen
and
Anti­
estrogen
­
Intrinsic
Activity
A.
Rat
(
and
other
non­
human
mammalian
and
avian)
ER
Binding
Assay
DESCRIPTION
In
vitro
affinity
of
toxicants
for
rER.
DEGREE
OF
USE
Extensive,
for
20
years,
largest
in
vitro
database,
fairly
easy
in
vitro
assay.
DURATION
24
hours.
ASSAY
STABILITY
Good
at
low
temperatures,
receptor
degradation
at
higher
temperatures.
DOES
IT
METABOLIZE
TOXICANTS
No
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
can
be
trained
in
short
time.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
It
could
easily
be
standardized,
most
labs
run
it
at
4o
C
for
18
hours.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Very
sensitive
for
high
to
moderate
affinity
ligands,
can
be
run
over
a
wide
range
of
log
doses.
ARE
THERE
KNOWN
FALSE
POSITIVES
To
the
degree
that
it
and
all
other
in
vitro
assays
cannot
account
for
ADME,
things
that
bind
ER
may
not
be
active
in
vivo.
Other
than
this,
no
false
positives.
OR
FALSE
NEGATIVES
Yes,
if
assay
is
conducted
at
low
temp,
which
is
generally
true,
some
things
fail
to
bind
ER
under
these
conditions,
but
work
in
vivo
and
activate
ER
in
proliferation
assays
and
transfected
cells
(
and
effect
can
be
blocked
with
anti­
estrogen).
In
other
cases,
solubility
at
low
temperature
of
lipophilic
toxicants
may
preclude
detection.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
No
COMMENTS
Both
agonists
and
antagonists
bind
ER,
good
for
screening
but
additional
information
is
required
to
determine
if
it
is
estrogenic
or
anti­
estrogenic
in
vitro.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
2
K
­
Requires
radioactivity.
Toxicant
solubility
and
degradation
are
a
problem
in
this
and
all
other
in
vitro
assays.
GLP
requirements
should
be
established
to
verify
culture
conditions
to
the
same
degree
that
we
verify
dosing
solutions
for
in
vivo
tests.

B.
hER
Binding
From
MCF­
7
Cell
Lysate
DESCRIPTION
Competitive
binding
of
toxicants
to
hER
in
human
cell
lysate.
DEGREE
OF
USE
Widespread
since
1973.
DURATION
A
few
days.
COMMENTS
Problems
similar
to
rat
or
other
mammalian
RBA
assays
for
ER.

C.
Estrogen
Competitor
(
Binding)
Screening
Assay
(
A
Receptor/
Ligand
Assay,
PanVera)
DESCRIPTION
The
assay
utilizes
recombinant,
human
estrogen
receptor
and
an
autofluorescent,
high
affinity
estrogen
ligand.
Competitors
are
identified
by
their
ability
to
disrupt
binding
of
the
ER
and
fluorescent
estrogen.
Binding
is
quantified
by
fluorescence
polarization.
This
technique
allows
for
the
direct
measurement
of
the
bound
to
free
ratio
of
the
ligand
at
equilibrium,
in
solution,
with
no
precipitation,
dialysis,
extraction,
or
any
other
separation
of
bound
and
free
ligand
required.
DEGREE
OF
USE
This
assay
is
currently
in
use
in
drug
discovery
as
both
a
primary
screen
to
search
for
new
estrogen
ligands
and
as
a
secondary
screen
to
characterize
lead
compounds.
DURATION
Receptor/
ligand
equilibrium
is
the
rate
limiting
step.
Including
a
one­
hour
room
temperature
equilibration,
it
should
take
about
two
hours
from
sample
prep
to
measurement
of
polarization.
ASSAY
STABILITY
Recombinant
ER
is
stable
at
room
temperature
for
at
least
six
hours.
DOES
IT
METABOLIZE
TOXICANTS
No
ARE
SPECIAL
EQUIPMENT
REQUIREMENTS
MANDATED
A
fluorescence
polarimeter
is
required
($
18.5K
for
single
tube
instrument,
$
30K
for
96­
to
384­
well
instrument).
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
No,
only
basic
lab
skills
and
GLP
training
is
needed.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes,
typically
use
estradiol
as
a
standard
for
IC50
values
comparison.
Assay
has
been
used
to
determine
IC50
values
and
Ki
values
for
tamoxifen,
estradiol,
estrone,
estriol,
estrone,
chlordane,
and
others.
Because
assay
is
at
true
equilibrium,
Ki
values
can
be
calculated.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
3
K
­
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Similar
to
other
receptor/
ligand
binding
assays.
Sensitivity
dependent
not
on
assay,
but
on
solubility
of
the
test
compound.
For
example,
to
determine
an
IC50
value
of
50
mM,
the
test
compound
would
have
to
be
soluble
in
the
test
matrix
at
least
50mM.
ARE
THERE
KNOWN
FALSE
POSITIVES
As
with
other
receptor
binding
assays,
non­
competitive
inhibition
is
possible,
but
rare.
OR
FALSE
NEGATIVES
Probably
less
than
receptor/
ligand
assays
performed
at
lower
temperatures.
This
assay
can
be
performed
at
4
°
to
37
°
C.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
No
COMMENTS
Purified
receptor
is
well
characterized
and
therefore
activity
is
more
reproducible
than
lysates.
No
radioactivity.
Assay
is
performed
at
true
equilibrium.
Technique
is
non­
destructive.
Reactions
in
disposable
tubes
can
be
remeasured
under
various
conditions.
For
example,
competition
curve
can
be
measured
at
4
°
C,
incubated
at
20
°
C
and
then
measured
at
20
°
C.
Assay
can
be
performed
in
a
multi­
well
format
and
automated.
Technique
should
be
applicable
to
multiple
species
ER
to
determine
relative
potency
in
those
species.
Assay
is
very
simple:
(
1)
add
ER
and
fluorescent
estrogen
to
test
compound,
(
2)
incubate,
and
(
3)
measure
polarization.

II.
Estrogen
and
Anti­
estrogen
­
In
Vitro
A.
MCF­
7
Proliferation
Assay
(
ESCREEN)
DESCRIPTION
Measures
growth
of
cells
in
vitro
in
response
to
ER
modulators.
DEGREE
OF
USE
Thousands
of
chemicals
studied,
several
toxicants.
DURATION
One
rep
takes
a
week
ASSAY
STABILITY
Varies
with
serum
batch,
MCF­
7
cell
subclone,
passage
number,
etc.
DOES
IT
METABOLIZE
TOXICANTS
Relatively
unknown,
has
aromatase
activity,
but
much
less
than
kidney
or
liver
cells.
Reports
about
metabolic
activation
of
methoxychlor
have
not
been
verified.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
cell
culture
and
cell
counting
equipment.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
cells
must
be
maintained
the
same,
week
after
week.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Has
not
been
standardized.
Some
factors
could
be
(
subclone
type),
while
others
(
serum
variability)
would
be
difficult.
Competent
labs
appear
to
get
similar
qualitative,
if
not
quantitative
results.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
4
K
­
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Very
sensitive,
one
of
the
most
sensitive
assays.
ARE
THERE
KNOWN
FALSE
POSITIVES
Yes,
some
growth
factors,
other
steroids
may
TCDD,
etc.,
can
influence
proliferation
assay,
or
kill
cells
or
make
them
"
sick"
(
would
appear
anti­
estrogenic).
OR
FALSE
NEGATIVES
Things
that
require
metabolic
activation
may
be
missed
in
this
and
all
other
in
vitro
assays.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
As
above,
this
is
a
complex
response
of
an
unknown
number
of
genes
that
can
be
influenced
by
other
mechanisms.
Could
also
be
false
negatives
if
something
binds
ER
and
activates
different
genes.
COMMENTS.
Best
assay
of
the
`
70s
and
`
80s.
Controls
needed
for
cell
viability,
health
and
run
with
antiestrogen
to
reverse
effect
in
order
to
confirm
that
proliferation
was
mediated
via
ER.

B.
YES­
Yeast
Estrogen
Screen
DESCRIPTION
In
vitro
assay
using
yeast
cells
transformed
with
hER
(
whole
or
fragment),
VIT
promoter
and
reporter
(
luc
or
CAT)
construct.
DEGREE
OF
USE
Widespread
in
industry
for
drugs,
a
handful
of
papers
on
use
with
toxicants.
Results
quite
mixed.
DURATION
Short­
term,
24
hours.
ASSAY
STABILITY
Response
varies
greatly
from
subclone
to
subclone
for
certain
types
of
xenoestrogens.
Yeast
have
a
cell
wall
and
some
strains
have
transport
systems
that
render
them
drug
resistant
(
i.
e.
dexamethasone).
Other
strains
even
appear
to
transport
estradiol
out
of
the
cell.
Response
varies
greatly,
depending
upon
the
type
of
hER
gene
construct
(
whole
versus
fragment).
DOES
IT
METABOLIZE
TOXICANTS
Unknown
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Cell
culture
equipment
and
techniques.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Permanently
transformed
so
may
be
easier
than
transient
transfections,
but
in
vitro
training
required.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Has
not
been
standardized
at
present.
Standardization
could
be
achieved
if
the
"
best"
subclone
and
gene/
reporter
construct
could
be
determined.
May
be
premature
to
standardize
without
further
development
as
a
research
tool.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Good
for
some
(
alkylphenols),
some
strains
are
very
insensitive
to
chlorinated
pesticides.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
5
K
­
ARE
THERE
KNOWN
FALSE
POSITIVES
OR
FALSE
NEGATIVE
Depends
upon
specificity
of
reporter
construct,
but
as
good
as
any
ER­
binding
assay.
Several
false
negatives
noted
for
some
strains.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Depends
on
gene
construct
employed.
COMMENTS
As
employed
by
some,
is
more
like
a
binding
assay
because
it
fails
to
discriminate
between
agonist
and
antagonists.
Given
limitations
of
transport,
is
a
curious
choice
given
the
minimal
information
provided
and
high
potential
false
negatives.

C.
MVLN
Assay.
Stably
Transfected
Reporter
Gene
Assay
in
Mammalian
Cells
DESCRIPTION
The
assay
utilizes
a
mammalian
cell
line
(
MCF­
7
with
endogenous
human
ER)
that
has
been
stably
transfected
with
an
ER
specific
reporter
gene
(
Vit­
Luc).
DEGREE
OF
USE
Used
in
various
labs
for
pharmaceutical
and
environmental
research.
DURATION
From
plating
cells
to
harvesting
lysate
and
Luc
activity
takes
two
to
three
days.
ASSAY
STABILITY
Stably
transfected
cells
maintain
same
level
of
activity
for
at
least
30
passages.
Activity
is
maintained
after
typical
cell
culture
freezing
storage
cycles.
DOSE
IT
METABOLIZE
TOXICANTS
May
have
aromatase
and
other
limited
metabolic
capacities
of
other
MCF­
7
subclones.
ARE
SPECIAL
EQUIPMENT
REQUIREMENTS
MANDATED
Typical
cell
culture
equipment
as
well
as
a
luminometer
to
measure
Luc
activity.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Tissue
culture,
basic
lab
skills
GLP
training
is
needed.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes,
activity
for
various
steroids
and
nonsteroids
are
comparable
to
other
transient
reporter
assays.
Stably
transfected
cells
can
be
easily
distributed.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
COMPOUNDS
Highly
sensitive.
EC50
for
E2
in
the
pM
range.
ARE
THERE
KNOWN
FALSE
POSITIVES
No,
hypothetically
ER
could
be
activated
by
phosphorylation
pathways.
OR
FALSE
NEGATIVES
No,
would
expect
only
very
specific
activation
of
ER.
SPECIFICALLY
­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THE
EFFECT
(
FALSE
POSITIVES)
Hypothetically
ER
could
be
activated
by
phosphorylation
pathways.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
6
K
­
COMMENTS
Specific
for
ER
transcription
activation.
Done
in
mammalian
cells.
Utilizes
human
ER.
Easy
assay,
no
transactions.
High
assay
signal
due
to
all
cells
expressing
reporter.
Can
be
done
in
dishes,
12,
24
or
96
well
plates.
Can
be
automated.

D.
Cotransfected
Reporter
Gene
Assay
in
Mammalian
Cells
(
e.
g.,
CV­
1
or
COS
Cells)
DESCRIPTION
The
assay
utilizes
a
mammalian
cell
line
(
CV­
1,
COS)
that
has
been
transiently
transfected
with
ER
as
well
as
an
ER
specific
reporter
gene
(
Vit­
Luc).
DEGREE
OF
USE
Used
widely
in
various
labs
for
pharmaceutical
and
environmental
research.
DURATION
From
plating
cells
to
harvesting
lysate
and
Luc
activity
determination
takes
two
to
three
days.
ASSAY
STABILITY
Stability
depends
on
transfection
efficiency
between
experiments.
DOSE
IT
METABOLIZE
TOXICANTS
May
have
limited
metabolic
capacities.
ARE
SPECIAL
EQUIPMENT
REQUIREMENTS
MANDATED
Typical
cell
culture
equipment
as
well
as
a
luminometer
to
measure
Luc
activity.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Tissue
culture,
transfection,
basic
lab
skills
and
GLP
training
is
needed.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes,
when
standardized
relative
to
%
activity
of
E2,
results
are
comparable
from
assay
to
assay.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
COMPOUNDS
Sensitive.
EC50
for
E@
in
the
10pM
range.
ARE
THERE
KNOWN
FALSE
POSITIVES
No,
hypothetically
ER
could
be
activated
by
phosphorlyation
pathways.
OR
FALSE
NEGATIVES
No,
would
expect
only
very
specific
activation
of
ER.
SPECIFICALLY
­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THE
EFFECT
(
FALSE
POSITIVES)
Hypothetically
ER
could
be
activated
by
phosphorylation
pathways.
COMMENTS
Specific
for
ER
transcription
activation.
Done
in
mammalian
cells.
Can
utilize
ER
from
any
cloned
source
(
any
species).
Requires
transfections.
Can
be
done
in
dishes
or
12
well
plates.
Can
transfect
in
different
reporter
constructs
to
compare
ligand
selective
gene
regulation.

E.
Stably
Transfected
Reporter
Gene
Assay
in
Mammalian
Cells
(
e.
g.,
MCF­
7
Cells)
DESCRIPTION
The
assay
utilizes
a
mammalian
cell
line
(
MCF­
7
with
endogenous
human
ER)
that
has
been
stably
transfected
with
an
ER
specific
reporter
gene
(
Vit­
Luc).
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
7
K
­
DEGREE
OF
USE
Used
in
various
labs
for
pharmaceutical
and
environmental
research.
DURATION
From
plating
cells
to
harvesting
lysate
and
Luc
activity
determination
takes
two
to
three
days.
ASSAY
STABILITY
Stably
transfected
cells
maintain
same
level
of
activity
for
at
least
30
passages.
Activity
is
maintained
after
typical
cell
culture
freezing
storage
cycles.
DOSE
IT
METABOLIZE
TOXICANTS
May
have
aromatase
and
other
limited
metabolic
capacities
of
other
MCF­
7
subclones.
ARE
SPECIAL
EQUIPMENT
REQUIREMENTS
MANDATED
Typical
cell
culture
equipment
as
well
as
a
luminometer
to
measure
Luc
activity.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Tissue
culture,
basic
lab
skills
and
GLP
training
is
needed.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes,
activity
for
various
steroids
and
nonsteroids
are
comparable
to
other
transient
reporter
assays.
Stably
transfected
cells
can
be
easily
distributed.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
COMPOUNDS
Highly
sensitive.
EC50
for
E2
in
the
pM
range.
ARE
THERE
KNOWN
FALSE
POSITIVES
No,
hypothetically
ER
could
be
activated
by
phosphorylation
pathways.
OR
FALSE
NEGATIVES
No,
would
expect
only
very
specific
activation
of
ER.
SPECIFICALLY
­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THE
EFFECT
(
FALSE
POSITIVES)
Hypothetically
ER
could
be
activated
by
phosphorylation
pathways.
COMMENTS
Specific
for
ER
transcription
activation.
Done
in
mammalian
cells.
Utilizes
human
ER.
Easy
assay,
no
transaction.
High
assay
signal
due
to
all
cells
expressing
reporter.
Can
be
done
in
dishes,
12,
24
or
96
well
plates.
Can
be
automated.

III.
Estrogen
and
Anti­
estrogen
­
In
Vivo
A.
Uterine
Peroxidase
Assay
COMMENTS
Uterine
peroxidase
activity
is
thought
to
be
estrogen
regulated,
therefore
monitoring
its
levels
may
serve
as
a
means
to
determine
the
estrogenicity
of
chemicals.
Johri
et
al.
used
this
method
to
determine
estrogenic/
anti­
estrogenic
potential
of
anti­
fertility
substances.
In
this
assay
Charles
Foster
rats
where
given
either
oral
doses
ranging
from
1.5­
10.0
mg/
kg
or
subcutaneous
injections
(
e.
g.
estradiol)
of
0.1­
1.0
mg/
rat/
day.
After
3
days
(
or
longer)
of
dosing
the
animals
were
sacrificed
24
hours
after
the
last
dose.
Uteri
were
excised
and
homogenized
in
sodium
acetate.
Peroxidase
activity
was
determined
(
by
the
Alexander
Method)
and
reaction
rates
were
monitored
and
enzyme
activity
expressed
as
D
A353/
mg
protein/
min.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
8
K
­
B.
Developmental
Uterotrophic
Assay
DESCRIPTION
Rat;
treatment
on
postnatal
days
(
pnd)
10­
14
with
a
variety
of
estrogens/
antiestrogens
inhibits
uterine
gland
appearance
and
increases
uterine
weight
and
luminal
epithelial
height
measured
on
pnd
14
or
in
adults.
D­
R
curves;
replicated
in
mice.
ARE
SPECIAL
EQUIPMENT
REQUIREMENTS
MANDATED
Balance;
ocular
micrometer;
standard
histology
equipment
for
H&
E
staining
of
sections.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Animal
handling
and
dosing;
tissue
removal
and
weighing;
slide
preparation
including
vertical
placement
of
uteri
in
paraffin;
simple
microscopy.
AVAILABILITY
Available
in
open
literature.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Standardization;
use
of
positive
controls;
stable
values
for
control
and
max
responses
over
ten
years
and
~
ten
estrogens.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Sensitivity:
DES,
EE2~
1ug/
Kg/
day
ED50
for
luminal
epithelial
height
(
3x
increase
max)
or
for
doubling
uterine
weight,
~
0.3
ug/
Kg/
day
ED50
for
gland
inhibition
(~
4
glands/
section
inhibited
down
to
0
glands/
section.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Estrogens
and
antiestrogens;
need
to
examine
other
hormones
to
establish
specificity.
COMMENTS
General
and
specific
suggestions
today
for
the
estrogens/
antiestrogens.
General:
(
1)
Use
a
pure
antiestrogen
such
as
ICI
182,780
to
confirm
specificity
of
agonist/
antagonist
activity
in
vitro/
in
vivo?;
(
2)
All
tasks­
should
we
eventually
consider
standardizing
assays
so
data
can
be
compared/
modeled
and
quality
control
checked?,
include
positive/
negative
controls?;
(
3)
Are
we
assuming
multipoint
assays
or
(
hopefully
not)
single
point
assays?;
(
4)
Where
there
are
choices
sensitivity
should
be
considered;
all
other
things
being
equal
a
chemical
with,
for
example,
low
solubility,
would
be
more
likely
to
be
detected
in
the
more
sensitive
assay,
what
should
the
lower
limits
be
on
the
hormonal
activity
relative
to
a
standard
such
as
E2?;
(
5)
Do
cell
assays
need
cytotoxicity
measures
to
help
interpretation
of
data?
Specific:
For
in
vivo
detection
of
estrogenicity/
antiestrogenicity/
steroidogenesis
effects,
consider
the
following.
On
postnatal
days
(
PND)
10­
14,
the
rat
ovary
actively
makes
estrogens
which
increase
uterine
weight
(
normalized
to
body
weight)
by
30%
on
PND14.
After
about
PND
16,
estrogen
levels
drop
to
the
lower
but
still
detectable
values
found
in
immature
rats.
Ovariectomy
or
ICI
182,780
reduces
uterine
weight
to
control
or
lower
values
on
PND
14
or
beyond.
Treatment
with
agonists
on
PND
10­
14
increases
uterine
weight
several
fold,
increases
the
height
of
the
luminal
epithelium
(
LEH)
three­
fold
and
inhibits
the
appearance
of
uterine
glands.
Glands
begin
to
appear
on
PND
9
by
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
9
K
­
invagination
from
the
luminal
epithelium
and
the
process
is
over
by
PND
16­
17.
The
later
two
measures
are
done
on
cross­
sections
of
paraffin
embedded
uteri
by
ocular
micrometer
or
image
analysis
for
LEH
and
gland
counts
per
section.
Gland
numbers
are
4­
5
in
controls
and
decrease
to
almost
zero
from
agonist
treatment.
So
agonists
increase
uterine
weight
and
LEH
and
decrease
gland
numbers.
The
triphenylethylene
partial
agonists/
partial
antagonists
such
as
tamoxifen,
clomiphene,
toremifine,
etc.
show
marginal
uterine
weight
gain,
increase
LEH
three­
fold
and
inhibit
gland
genesis.
This
pattern
contrasts
with
the
complete
agonists.
ICI
182,780,
a
complete
antagonist,
reduces
uterine
weight
but
has
no
effect
on
gland
genesis
or
LEH,
a
different
pattern
than
those
above.
Important
here
is
that
the
patterns
of
responses
in
a
single
experiment
distinguishes
these
pharmacological
classes
rather
than
depending
on
sequential
experiments
each
with
a
different
design.
Additionally,
one
endpoint,
gland
genesis,
is
a
classical
developmental
toxicity
endpoint;
there
is
a
defined
ontogenic
pattern,
a
sensitive
period,
and
an
adverse
outcome
(
gland
numbers
are
reduced
or
absent
in
adults
following
PND10­
14
treatment).
We
don't
have
experience
with
other
mixed
agonists/
antagonists
with
the
possible
exception
of
some
phytoestrogens
for
which
the
verdict
is
not
yet
in.
An
additional,
but
untested
feature,
is
that
chemicals
which
interfere
with
steroidogenesis
should
be
active
in
this
system;
chemicals
which
increase
steroidogenesis
will
act
like
an
agonist
but
fail
to
bind
to
the
ER,
while
those
that
decrease
steroidogenesis
will
act
like
a
pure
antagonist
(
or
ovariectomy)
but
fail
to
bind
to
the
ER.
Any
nominations
for
chemicals
in
this
category
for
us
to
look
at?
Virtues
of
the
assay
are
that
there
are
an
average
of
six
female
pups
per
dam
which
can
be
randomized
to
different
dams
versus
waiting
for
animals
to
mature
or
the
need
for
ovariectomy
and
waiting
for
ten
days;
the
pups
stay
with
dams
in
a
single
cage
throughout
the
experiment.
Arrival
of
dams
on
GD
2
and
completion
of
the
study
on
PND
14
results
in
an
average
of
five
to
six
cage
days
per
animal,
while
providing
multiple
endpoints
and
the
ability
to
distinguish
the
pharmacological
activity,
and
includes
an
endpoint
for
developmental
toxicity.
These
features
should
be
compared
to
the
animal
numbers,
cage
costs,
personnel
time
and
length
of
time
to
conduct
the
sequential
experiments
as
suggested
in
the
outline.
Comments
on
metabolism:
Because
only
the
in
vivo
assays
have
the
potential
to
detect
prohormone
metabolism
to
an
active
hormone,
this
property
should
be
included
in
the
rationale
for
the
Tier
1
battery
including
an
in
vivo
component.
Comments
on
use
of
cell
constructs/
cultures:
These,
along
with
the
ER
binding
should,
if
positive,
be
seen
as
providing
high
priority
for
moving
into
Tier
1
and
Tier
2
in
vivo
tests.
Likewise,
chemicals
in
the
prescreens
that
are
"
positive"
should
be
moved
immediately
into
the
tiers.
This
sequence
is
differentiated
from
one
that
would
move
a
group
of
chemicals
lockstep
through
the
prescreen
and
Tier
1steps
with
no
priority
(
urgency)
to
move
to
higher
tiers.
With
lockstep
testing,
market
and
regulatory
decisions
on
chemicals
that
are
a
problem
would
ultimately
be
delayed
to
some
unknown
extent
while
exposure
continues.
In
particular,
the
in
vivo
assays
could
be
seen
as
inducing
significant
delays.
REFERENCES
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
10
K
­
Branham,
W.
S.,
D.
M.
Sheehan,
D.
R.
Zehr,
E.
Ridlon
and
C.
J.
Nelson,
"
The
postnatal
ontogeny
of
rat
uterine
glands
and
age­
related
effects
of
17ss­
estradiol,"
Endocrinology,
CXVII(
V),
1985,
pp.
2229­
2237.
Branham,
W.
S.,
D.
M.
Sheehan,
D.
R.
Zehr,
K.
L.
Medlock,
C.
J.
Nelson
and
E.
Ridlon,
"
Inhibition
of
uterine
gland
genesis
by
tamoxifen,"
Endocrinology,
CXVII(
V),
1985,
pp.
2238­
2248.
Branham,
W.
S.,
D.
R.
Zehr,
J.
J.
Chen,
and
D.
M.
Sheehan,
"
Postnatal
uterine
development
in
the
rat:
Estrogen
and
antiestrogen
effects
on
luminal
epithelium,"
Teratology,
XXXVIII,
1988,
pp.
29­
36.
Branham,
W.
S.,
D.
R.
Zehr,
J.
J.
Chen,
and
D.
M.
Sheehan,
"
Alterations
in
developing
rat
uterine
cell
populations
after
neonatal
exposure
to
estrogens
and
antiestrogens,"
Teratology,
XXXVIII,
1988,
pp.
271­
279.
Branham,
W.
S.,
D.
R.
Zehr,
and
D.
M.
Sheehan,
"
Differential
sensitivity
of
rat
uterine
growth
and
epithelium
hypertrophy
to
estrogens
and
antiestrogen,"
Proc.
Soc.
Exp.
Biol.
Med.,
CCIII,
1993,
pp.
297­
303.
Branham,
W.
S.
and
D.
M.
Sheehan,
"
Ovarian
and
adrenal
contributions
to
postnatal
growth
and
differentiation
of
the
rat
uterus,"
Biol.
Reprod.,
LIII,
1995,
pp.
862­
872.
Branham,
W.
S.,
R.
Fishman,
R.
S.
Streck,
K.
L.
Medlock,
J.
J.
DeGeorge,
and
D.
M.
Sheehan,
"
ICI
182,780
inhibits
endogenous
estrogen­
dependent
rat
uterine
growth
and
tamoxifen­
induced
developmental
toxicity,"
Biol.
Reprod.,
LIV,
1996,
pp.
160­
167.
Medlock,
K.
L.,
W.
S.
Branham,
and
D.
M.
Sheehan,
"
The
effects
of
phytoestrogens
on
neonatal
rat
uterine
growth
and
development,"
Proc.
Soc.
Exp.
Biol.
Med.,
CCVIII,
1995,
pp.
307­
313,
1995.
Medlock,
K.
L.,
W.
S.
Branham,
and
D.
M.
Sheehan,
"
Effects
of
coumestrol
and
equol
on
the
developing
reproductive
tract
of
the
rat,"
Proc.
Soc.
Exp.
Biol.
Med.,
CCVIII,
1995,
pp.
67­
71.
Medlock,
K.
L.,
W.
S.
Branham,
and
D.
M.
Sheehan,
"
Effects
of
toremifene
on
neonatal
rat
uterine
growth
and
differention,"
Biol.
Reprod.,
LVI,
1997,
pp.
1239­
1244.

C.
Uterine
Weight
Bioassay
in
Juvenile
or
Adult
Ovariectomized
Female
Rats
DESCRIPTION
One
to
three
oral
or
injected
doses
of
toxicant
to
immature
female
rat
(
18­
21
d),
longer
in
adult
ovx'ed
female.
DEGREE
OF
USE
One
of
original
"
gold"
standards
for
screening
for
estrogenicity,
used
for
about
80
years.
Extensive
database
on
toxicants.
DURATION
One
to
three
days,
or
weeks
in
adult.
ASSAY
STABILITY
Very
stable
unless
juvenile
females
older
than
24
days
at
necropsy,
or
sloppily
designed
at
weaning.
DOES
IT
METABOLIZE
TOXICANTS
Yes
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
11
K
­
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Good
balance
and
scissors.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Necropsy
skills.

HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Standardized
to
some
degree.
Could
be
improved
(
trim
fat,
weigh
with
and
without
fluid).
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
One
of
the
most
sensitive
in
vivo
short­
term
assays
using
immature
or
adult
females.
(
Rank
#
1).
Nonylphenol,
octylphenol,
bisphenol
A,
methoxychlor,
estradiol
17
I,
kepone,
etc.
are
positive.
ARE
THERE
KNOWN
FALSE
POSITIVES
Yes,
intact
female
is
used,
hence,
effects
on
hypothalamic­
pit
axis,
and
GH
or
prolactin
alterations
could
affect
this
process.
In
addition,
aromatizable
and
nonaromatizable
(
via
AR)
can
affect
weight.
Even
a
few
false
positives
in
ovariectomized
adult
females.
OR
FALSE­
NEGATIVES
Some
potential,
as
some
chemicals
are
poorly
absorbed
interaperitoneal
and
are
more
effective
when
given
orally,
while
many
others
are
less
effective
after
oral
administration.
As
with
any
in
vivo
assay,
chemicals
that
are
estrogenic
in
binding
or
cell
assays
may
be
negative,
albeit
not
falsely
so,
if
they
are
not
absorbed,
are
metabolically
inactivated
or
excreted
such
that
the
active
material
never
reaches
the
targets.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Yes,
as
indicated
above.
COMMENTS
Necropsy
of
animals
six
hours
after
last
dose,
of
three,
is
more
effective
than
24
hours
later
in
detection
of
weak
estrogens.
In
adult
female,
this
endpoint
can
be
coupled
with
several
others.

D.
Vaginal
Smears
(
Mucification
and
Cornification)
DESCRIPTION
Noninvasive
measurement
of
estrogenicity
in
intact
or
ovariectomized
female
rat.
Vaginal
lavages
are
examined
for
cell
types.
DEGREE
OF
USE
Extensive
use
for
over
80
years
(
Allen­
Doisy
Assay).
DURATION
Moderate
to
long
duration
screen
requires
daily
examination
of
vaginal
cells
by
microscopy.
At
least
one
week
in
duration
can
dose
for
months
at
low
dosage
levels.
ASSAY
STABILITY
Very
stable
across
and
within
labs.
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Microscope
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
12
K
­
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Not
difficult,
but
requires
more
expertise
than
determination
of
vaginal
opening
or
uterine
weight.
Data
analysis
can
also
be
relatively
complicated.

HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Not
standardized
with
regard
to
a
number
of
factors
(
staining
versus
no
staining,
read
and
discard
wet
sample
or
save
dry
and
read
later,
data
recording
and
classification,
method
of
data
analysis).
In
spite
of
the
lack
of
standardization,
this
is
a
robust
measure
as
I
am
not
aware
of
a
case
where
different
labs,
using
different
methods,
did
not
get
the
same
results.
Could
be
easily
standardized.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Xenoestrogen
toxicant
and
pesticide
data
indicate
clearly
that
vaginal
cornification
is
less
sensitive
that
a
number
of
other
screens
for
estrogenicity
(
Cluster
Rank
#
4)
below
uterine
weight,
uterine
histology,
uterine
biochemical
measures,
vaginal
histology,
vaginal
mucification,
vaginal
opening,
induction
of
mating
behavior,
etc.
However,
one
data
set
with
estrogenic
PCBs
reported
vaginal
cornification
in
the
absence
of
an
increase
in
uterine
weight.
Mucification
of
smears
occurs
before
or
at
lower
dosage
levels
than
cornification.
ARE
THERE
KNOWN
FALSE
POSITIVES
As
is
the
case
with
most
of
these
screens,
when
intact
animals
are
utilized,
there
are
many
treatments
that
alter
vaginal
cornification
via
nonestrogenic
mechanisms.
Conducting
the
test
in
an
ovariectomized
female
enhances
the
specificity
of
the
screen.
Still,
there
are
false
positives,
but
the
likelihood
is
greatly
reduced.
OR
FALSE
NEGATIVES
Yes.
Toxicants
that
accelerate
or
delay
hypothalamic
pituitary
development,
affect
the
reproductive
tract
directly,
alter
GH,
or
prolactin
can
alter
VO
in
the
intact
juvenile
female.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Yes,
especially
in
intact
animals.
COMMENTS
Good
assay
that
can
be
used
in
conjunction
with
several
other
endpoints.
For
example,
dose
ovx'ed
(
long­
term)
female
for
three
weeks,
take
vaginal
lavage
daily,
observe
lordosis
behavior,
necropsy
female
and
weight
uterus,
vaginal
and
do
histology
on
tract.
Could
also
add
biochemical
measures.

E.
Puberty.
Age
at
Vaginal
Opening
(
First
Estrus,
Onset
of
Cyclicity)
DESCRIPTION
In
vivo
test
of
estrogenicity
in
intact
juvenile
female
rats
or
mice.
One
major
advantage
over
other
assays
is
that
this
one
can
detect
estrogens
and
antiestrogens
(
delayed
VO).
Acceleration
and
delay
of
VO,
age
at
first
estrus
and
onset
of
estrous
cycles
can
occur
after
in
utero
exposure
to
xenoestrogens
and
other
toxicants
(
TCDD­
which
also
produces
a
permanent
anomaly
of
VO).
DEGREE
OF
USE
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
13
K
­
One
of
original
indices
used
to
screen
for
estrogens
80
years
ago.
Included
in
most
new
multigenerational
tests.
Fair
amount
of
xenoestrogen
data.
Methoxychlor,
octylphenol,
nonlyphenol
are
all
positive.
DURATION
Acceleration
of
VO
takes
from
2­
3
days
to
about
a
week,
after
which
the
process
begins
to
occur
normally
at
puberty
in
controls.
ASSAY
STABILITY
Strains
vary
slightly
(
except
Fischer
rat).
Age
at
VO
has
changed
considerably
since
the
1930'
s
due
to
improvements
in
diet.
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
None
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Ability
to
follow
a
simple
protocol.
Absolutely
the
easiest
assay
and
the
animals
do
not
need
to
be
killed.
Response
is
relatively
uniform,
such
that
fairly
small
differences
can
be
detected
with
modest
sample
sizes.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Falls
into
Cluster
#
1
or
2
for
sensitivity
below
uterine
weight,
etc.,
but
more
sensitive
than
is
vaginal
cornification
for
most
pesticides
and
toxic
substances.
Rivals
uterine
weight
in
juvenile
rat
for
some
chemicals
if
oral
dosing
is
employed
in
uterotropic
assay.
ARE
THERE
KNOWN
FALSE
POSITIVES
An
apical
test,
several
mechanisms
can
lead
to
accelerated
or
delays
in
VO.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Yes.
COMMENTS
Can
be
coupled
with
several
other
assays
if
dosing
is
continued
like
vaginal
cornification,
and
for
Ah­
receptor,
and
thyroid
hormone
effects.
If
VO
is
delayed,
ovary
can
be
studied
ex
vivo
for
inhibition
of
steroid
hormone
synthesis.
However,
cannot
be
conducted
along
with
uterotropic
assay
as
some
have
tried.

F.
Induction
of
Female
Sex
Behavior
(
Proceptive
and
Receptive
Behaviors)
DESCRIPTION
In
vivo
behavioral
test
of
lordosis
induction
(
lordosis
quotient)
provides
a
quantitative
screen
for
estrogenicity.
DEGREE
OF
USE
One
of
original,
80
year­
old,
tests
for
estrogenicity,
used
less
extensively
at
present,
as
compared
to
uterine
weight
or
vaginal
cornification.
Several
xenoestrogens
have
been
tested.
Methoxychlor,
nonylphenol,
octylphenol,
bisphenol
A
and
o,
p'
DDT
are
positive,
while
chlordecone
is
negative.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
14
K
­
DURATION
Three
days
ASSAY
STABILITY
Stable,
little
variability
in
data
from
lab
to
lab,
or
block
to
block.
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
need
reverse
photoperiod
(
or
willingness
to
work
nights)
with
dim
lights.
Minimal
equipment
requirements.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Some
training
required,
but
rather
simple
noninvasive
observation.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes,
most
protocols
are
quite
similar
and
could
be
easily
put
in
standard
SOP.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
One
of
the
most
sensitive
assays,
equivalent
to,
or
better
than
(
due
to
zero
variance
in
controls)
uterine
weight
and
histology.
ARE
THERE
KNOWN
FALSE
POSITIVES
None,
clearly
the
most
specific
test
for
an
estrogenic
response
In
Vivo.
OR
FALSE
NEGATIVES
Yes,
one
is
known,
chlordecone
blocks
rather
than
induces
lordosis
(
likely
through
the
effect
of
the
progesterone
receptor,
or
as
a
result
of
the
general
neurotoxicity).
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
None
known
COMMENTS
Can
be
coupled
with
other
assays.
For
example,
dose
animal,
observe
sex
behavior,
necropsy
female,
weigh
uterus
and
vagina
and
use
tissue
for
histology
and/
or
biochemical
measures
(
i.
e.,
ODC).

G.
Feeding
Behavior.
Food
Consumption
and
Growth
Rate
DESCRIPTION
Simple
in
vivo
assay
which
estrogens
specifically
retard
via
CNS
action.
DEGREE
OF
USE
Everyone
measures
it,
and
widely
recognized
as
a
sensitive
effect
in
the
toxicology
of
estrogens
in
the
male
rat.
Methoxychlor,
nonlyphenol,
octylphenol
are
positive.
Bisphenol
A
is
likely
positive.
DURATION
Appearance
of
estrogen­
inhibited
food
consumption
and
growth
are
dose­
related.
At
high
dosage
levels
the
effects
are
immediate,
while
at
lower
dosage
levels
the
effects
take
months
to
be
manifest.
ASSAY
STABILITY
Stable
DOES
IT
METABOLIZE
TOXICANTS
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
15
K
­
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Balances
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
No
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Likely
the
most
sensitive
response
in
weanling/
adult
male
rat
but
much
less
sensitive
in
intact
female
rat,
and
very
good
in
ovariectomized
female.
ARE
THERE
KNOWN
FALSE
POSITIVES
Obviously,
this
endpoint
can
be
affected
by
a
multiplicity
of
mechanisms,
so
although
sensitive
it
is
very
nonspecific.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Yes
COMMENTS
Always
collected,
but
rarely
recognized
in
a
multigen
study
as
an
estrogenic
effect.
Lack
of
recognition
of
body
weight
as
sensitive
endpoint
leads
to
serious
misinterpretation
of
multigen
data.

H.
Estrous
Cyclicity
DESCRIPTION
Repeated
daily
observation
of
vaginal
smears
allows
for
determination
of
alterations
of
estrous
cyclicity
in
rat.
Can
be
done
in
other
rodents,
but
mouse
is
more
variable.
Hamster
is
more
regular,
but
rarely
used
and
technique
is
quite
different
from
rat
or
mouse.
DEGREE
OF
USE
Very
widespread
in
reproductive
physiology,
required
in
most
new
multigen
studies.
Current
database
for
xenobiotics
modest,
but
growing.
DURATION
An
absolute
minimum
of
10
days
is
needed.
Guidelines
typically
require
15
days
or
longer.
More
useful
if
animals
are
dosed
for
some
time
period
prior
to
treatment
rather
than
initiating
treatment
with
onset
of
observation.
ASSAY
STABILITY
Some
variability
between
females
(
4,
5
versus
4/
5
d
cyclers
normal.
Not
unusual
to
see
6­
7
day
cycles
right
after
puberty).
Some
strain
variability,
possibly
seasonal
(
hypothesized,
but
not
proven),
and
social
influences
(
especially
in
mouse).
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Microscope
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Collecting
and
evaluating
smears
takes
a
little
practice,
data
analysis
is
more
difficult.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
16
K
­
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Methods
of
smear
collection,
staining
(
if
any),
preservation
of
sample
(
not
required
by
EPA,
if
any)
and
methods
of
recording
data
and
analyzing
data
vary
from
investigator
to
investigator.
However,
competent
labs
usually
get
similar
results
despite
variable
methodologies.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
With
exposure
to
adult
or
juvenile
female,
this
assay
is
less
sensitive
to
xenoestrogens
than:
(
1)
uterine
weight
and
histological
behavior;
(
2)
vaginal
opening;
and
(
3)
vaginal
mucification
and
cornification.
Less
sensitive
than
ovarian
measures
to
alterations
of
steroid
hormone
synthesis
and
ovarian
morphology,
due
to
compensation
within
ovary.
Sensitive
to
disruption
by
hypothalamic­
pituitary
endocrine
alterations
(
i.
e.,
atrazine)
of
LH,
FSH,
GnRH,
or
prolactin.

In
developmental
studies,
loss
of
estrous
cyclicity
is
a
sensitive
response
to
perinatal
xenoestrogen
exposure
via
CNS
defeminization.
Appearance
of
anovulation
is
dose­
related,
can
take
six
to
nine
months
to
appear.
Too
long
for
"
screening",
but
all
xenoestrogens
that
have
been
studied
produce
this
affect.
ARE
THERE
KNOWN
FALSE
POSITIVES
As
indicated
above,
this
is
an
apical
measure
that
responds
to
many
mechanisms
of
action
besides
estrogenicity.
OR
FALSE
NEGATIVES
Weak
estrogens
may
produce
many
other
effects
without
altering
estrous
cyclicity.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Estrogens,
androgens,
steroid
hormone
synthesis
inhibitors
and
toxicants
that
alter
LH,
FSH,
GnRH,
or
prolactin.
COMMENTS
Good
apical
test,
but
unfortunately
less
sensitive
by
about
ten­
fold
to
xenoestrogens
(
i.
e.
methoxychlor).

I.
Super
Apical
Developmental
Toxicity
Test
DESCRIPTION
Expose
pregnant/
lactating
dam
and
examine
hormone
(
AR,
ER,
SIS,
Ah,
and
T3)
sensitive
endpoints
in
progeny
up
to
puberty.
DEGREE
OF
USE
Such
a
protocol
has
been
used
at
EPA,
CIIT,
NIEHS
for
xenoestrogens,
environmental
antiandrogens,
Ah
receptor
agonists,
phthalates
and
antithyroidal
toxicants
(
PCBs
and
PTU).
DURATION
Relatively
long
(
two
to
three
months)
as
compared
to
other
"
screens,"
would
need
to
determine
if
it
is
quicker
and
cheaper
to
run
this
as
opposed
to
several
more
focused,
shorterterm
assays.
ASSAY
STABILITY
Unknown,
but
should
be
as
good
as
any
developmental
test.
DOES
IT
METABOLIZE
TOXICANTS
Yes
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
17
K
­
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
similar
to
needs
for
new
multigen
tests.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Multiplicity
of
in
vivo
techniques
are
required
that
are
currently
not
used
in
toxicology
labs,
but
they
should
be
coming
up
to
speed
to
implement
new
test
guidelines.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Not
standardized
as
used
in
different
labs
with
respect
to
dosing
or
some
of
the
assessments.
Some
standardization
is
now
taking
place
between
different
labs.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
18
K
­
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Antiandrogencity
via
AR,
or
SIS.
In
male
progeny,
AGD,
areolas,
nipples,
reduced
weight
of
prostate.
Higher
doses
of
AR­
mediated,
but
not
SIS,
produce
agenesis
of
prostate,
undescended
testis
and
agenesis
of
epididymis.
Altered
T
production
by
testis,
ex
vivo.

Estrogenicity.
Neonatal
uterine
weight
and
gland
development
(?),
vaginal
opening,
age
at
first
estrus,
vaginal
cornification.
Prostate
size
in
male
(?),
and
sperm
production
(
at
high
dosage
levels).
SIS
or
Antiestrogenicity.
Pregnancy
loss,
delay
in
deliver
by
dam,
delay
in
VO
and
possible
altered
ovarian
hormone
production
ex
vivo.

Antithyroid.
Reduced
perinatal
growth
and
brain
size.
Lower
serum
T4
and
possibly
T3,
elevated
TSH.
Also
detects
functional
developmental
alterations
induced
by
phthalates,
TCDD,
etc.
The
following
endpoints
are
considered
to
be
insensitive
because
they
take
too
long
to
assess
in
a
screen
and/
or
have
never
been
detected
with
a
pesticide
or
toxic
substance
following
developmental
exposure
(
i.
e.
for
ER­
mediated:
cancer,
reduced
AGD,
hypospadias
in
male
of
female,
undescended
testes
or
any
malformations).
ARE
THERE
KNOWN
FALSE
POSITIVES.
Very
Apical
Test
used
to
screen
for
several
mechanisms
at
once.
OR
FALSE
NEGATIVES.
Not
likely,
if
designed
properly.

IV.
Anti­
Estrogen
­
Synthesis
Metabolism
A.
Testis/
Ovary
Culture
In
Vitro
or
Ex
Vivo
DESCRIPTION
Determination
of
testosterone
production
from
testicular
tissue
from
animals
treated
in
vivo
(
ex
vivo)
or
using
in
vitro
dosing.
Used
for
EDS,
estrogens,
antiandrogens,
several
other
testicular
toxicants,
substances
that
inhibit
steroidogenesis.
In
female,
minced
ovary
culture
can
be
used
ex
vivo
from
pregnant
(
i.
e.,
GD
14­
16)
or
cycling
females
(
proestrus
for
estradiol
production).
DEGREE
OF
USE
A
few
hundred
publications
over
the
last
25
years
since
the
advent
of
RIAs
for
testosterone.
Used
in
other
vertebrates,
as
well
as
mammals.
Used
by
several
toxicology
laboratories
in
addition
to
NHEERL­
EPA.
DURATION
One
day
for
in
vitro,
longer
for
ex
vivo
(
duration
depends
upon
dosing).
ASSAY
STABILITY
Depends
on
methodology.
DOES
IT
METABOLIZE
TOXICANTS
Yes,
for
Ex
Vivo,
little
or
no
metabolism
for
in
vitro.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
19
K
­
Incubator,
freezer,
necropsy
equipment.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Some
practice,
but
a
rather
simple
assay
that
can
be
learned
by
competent
technicians
in
a
week
or
less.
Need
to
be
able
to
conduct
RIAs
for
T,
estradiol,
and
progesterone.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Has
not
been
standardized
between
labs,
but
could
be
without
too
much
difficulty.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Fairly
sensitive.
ARE
THERE
KNOWN
FALSE
POSITIVES
Cell
toxicants.
OR
FALSE
NEGATIVES
If
metabolic
activation
is
required
then
in
vitro
may
not
work.
COMMENTS.
These
assays
could
be
used
rapidly
to
screen
chemicals
for
ability
to
inhibit
steroidogenesis.

V.
Androgen
and
Anti­
androgen
­
Intrinsic
Activity
A.
Rat
AR
Equilibrium
Binding
Assay
DESCRIPTION
In
vitro
affinity
of
toxicants
for
rAR.
DEGREE
OF
USE
Extensive,
for
20
years,
large
in
vitro
data
base,
with
about
20
xenoantiandrogens
identified
to
date,
easiest
in
vitro
assay.
DURATION
24
hours
ASSAY
STABILITY
Acceptable
at
low
temperatures,
receptor
degradation
at
higher
temps.
DOES
IT
METABOLIZE
TOXICANTS
No
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
can
be
trained
in
short
time.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
It
could
easily
be
standardized.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Sensitive,
can
be
run
over
a
wide
range
of
log
doses.
ARE
THERE
KNOWN
FALSE
POSITIVES
To
the
degree
that
it
and
all
other
in
vitro
assays
cannot
account
for
ADME,
things
that
bind
AR
may
not
be
active
in
vivo.
Other
than
this,
no
false
positives.
OR
FALSE
NEGATIVES
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
20
K
­
Yes,
assay
is
conducted
at
low
temp
and
some
things
fail
to
bind
AR
under
these
conditions,
due
to
low
solubility
at
low
temperatures.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
No
COMMENTS
Both
agonists
and
antagonists
bind
AR,
good
for
screening
but
additional
information
is
required
to
determine
if
it
is
androgenic
or
anti­
androgenic
in
vitro.
Requires
radioactivity.
Toxicant
solubility
and
degradation
are
a
problem
in
this
and
all
other
in
vitro
assays.
GLP
requirements
should
be
established
to
verify
incubation
conditions
to
the
same
degree
that
we
verify
dosing
solutions
for
in
vivo
tests.
Should
be
simple,
as
labs
are
already
set
up
to
do
the
chemistry
for
in
vivo
studies.

B.
hAR
Whole
Cell
Binding
Assay
DESCRIPTION
In
vitro
whole
cell
binding
assay
with
human
AR
transiently
transfected
in
a
monkey
kidney
cell
line
(
COS).
DEGREE
OF
USE
Extensive
use
in
reproductive
medical
field.
Several
publications
now
with
fungicides
and
pesticides.
DURATION
A
few
days
for
entire
assay,
including
cell
culture
preparation
.
ASSAY
STABILITY
Quite
stable.
DOES
IT
METABOLIZE
TOXICANTS
These
cells
can
activate
some
antiandrogenic
fungicides.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
cell
culture
equipment
and
luminometer,
robot
optional.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Need
to
transfect
cells.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Can
be
standardized
fairly
easily,
especially
with
stable
cell
lines
expressing
hAR.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Sensitive,
or
more
sensitive,
than
rat
AR
cytosolic
binding
assays.
ARE
THERE
KNOWN
FALSE
POSITIVES
Theoretically,
chemical
cytotoxicity
may
reduce
radioligand
binding
to
hAR,
this
could
be
interpreted
as
a
false
positive
in
the
absence
of
appropriate
controls.
OR
FALSE
NEGATIVES
Some
toxicants
requiring
activation
may
not
be
detected.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Cell
death.
COMMENTS
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
21
K
­
Need
controls
in
this
and
other
similar
assays
for
cell
viability,
and
health.
Must
verify
toxicant
stability
in
media.

VI.
Androgen
and
Anti­
androgen
­
In
Vitro
A.
YAS­
Yeast
Androgen
Screen
DESCRIPTION
In
vitro
assay
using
yeast
cells
transformed
with
AR
(
whole
or
fragment),
and
a
reporter
(
luc,
J­
gal,
or
etc.)
construct.
DEGREE
OF
USE
Little
use,
one
paper
on
use
with
toxicants.
Results
for
sole
xenoantiandrogen
yielded
a
false
negative.
DURATION
Short­
term,
24
hours.
ASSAY
STABILITY
Response
likely
varies
greatly
from
subclone
to
subclone
for
certain
types
of
xenoandrogens.
Reservations
same
as
for
YES.
DOES
IT
METABOLIZE
TOXICANTS
Unknown
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Cell
culture
equipment
and
techniques.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Permanently
transformed
so
may
be
easier
than
transient
transfections,
but
in
vitro
training
required.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Has
not
been
standardized.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Insensitive
to
p,
p'
DDE.
ARE
THERE
KNOWN
FALSE
POSITIVES
OR
FALSE
NEGATIVE
Detects
antiandrogens
as
agonists,
not
as
good
as
an
AR­
binding
assay
due
to
false
negatives.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Unknown
COMMENTS
As
employed,
is
more
like
a
binding
assay
than
other
transfected
cell
assays
because
it
fails
to
discriminate
between
agonist
and
antagonists.
Given
limitations
of
transport
(
i.
e.,
cellular
mechanisms
accounting
for
multidrug
resistance),
is
a
curious
choice
given
the
minimal
information
provided
and
high
potential
false
negatives.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
22
K
­
B.
CV­
1
Cell
Assay.
A
hAR
Transcriptional
Activation
Assay
in
Mammalian
Cell
DESCRIPTION
Transiently
transfected
assay
measuring
transcriptional
activation
using
luciferase
reporter
and
an
hAR
construct
in
a
primate
kidney
cell
line
(
CV­
1).
DEGREE
OF
USE
Extensive
use
in
reproductive
medical
field.
Several
publications
now
with
fungicides
and
pesticides.
DURATION
A
few
days
for
entire
assay,
including
cell
culture
preparation.
ASSAY
STABILITY
Stable,
with
considerable
experience.
DOES
IT
METABOLIZE
TOXICANTS
These
cells
can
activate
some
antiandrogenic
fungicides.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
freezer,
incubator,
and
luminometer,
robot
optional.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Reported
to
be
a
difficult
assay
to
initially
establish.
Need
to
transfect
cells.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Can
be
standardized
fairly
easily,
especially
when
stable
cell
lines
have
been
established
which
will
eliminate
the
need
to
transfect
cells
for
each
experiment.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Very
sensitive.
ARE
THERE
KNOWN
FALSE
POSITIVES
Theoretically,
cytotoxicity
may
induce
apparent
inhibition
of
DHT
(
this
is
true
for
many
transfected
cell
reporter
assays
of
antiandrogenicity,)
hence,
use
constituitively
active
mutant
receptor
as
a
cytotoxicity
control
(
decline
in
luc
indicates
cytotoxicity).
OR
FALSE
NEGATIVES
Some
toxicants
requiring
activation
may
not
be
detected.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Cell
death
COMMENTS
Need
controls
in
this
and
other
similar
assays
for
cell
viability
and
health.
Must
verify
toxicant
stability
in
media.

C.
hAR
Transactivation
Assays
Using
Stable
Cell
Lines
DESCRIPTION
Transcriptional
activation
assay
using
cells
stably
expressing
a
MMTV­
luciferase
reporter
together
with
the
hAR
in
a
mammalian
cell
line
such
as
a
CV­
1.
DEGREE
OF
USE
Under
development,
likely
available
in
6
months,
could
be
automated
for
high
throughput.
DURATION
24
hours
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
23
K
­
ASSAY
STABILITY
Unknown
to
date,
should
be
stable.
DOES
IT
METABOLIZE
TOXICANTS
These
cells
can
activate
some
antiandrogenic
fungicides.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
freezer,
incubator,
and
luminometer,
robot
optional.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Do
not
need
to
transfect
cells,
but
training
required
and
cell
techniques
needed.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Not
yet,
but
can
be
easily
standardized.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Should
be
as
sensitive,
or
more
sensitive,
than
transient
transfection
assays
because
all
the
cells
are
responsive.
ARE
THERE
KNOWN
FALSE
POSITIVES
Theoretically,
cytotoxicity
may
induce
apparent
inhibition
of
DHT­
induced
effects
(
this
is
true
for
many
cell
reporter
assays
of
antiandrogenicity),
hence,
use
constituitively
active
mutant
receptor
as
a
cytotoxicity
control
(
decline
in
luc
indicates
cytotoxicity)
.
OR
FALSE
NEGATIVES
Toxicants
requiring
activation
may
not
be
detected.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Cell
death.
COMMENTS
Need
controls
in
this
and
other
similar
assays
for
cell
viability
and
health.
Must
verify
toxicant
stability
in
media.

D.
Leydig
Cell
Culture
DESCRIPTION
Determination
of
testosterone
production
in
purified,
isolated
Leydig
cells.
DEGREE
OF
USE
Limited
to
a
several/
few
research
laboratories.
DURATION
A
few
days
ASSAY
STABILITY
Variable
from
lab
to
lab
on
degree
of
purification
of
Leydig
cells
and
T
production
per
cell.
DOES
IT
METABOLIZE
TOXICANTS
No
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
quite
a
bit.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Requires
skills
that
are
not
widely
available.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
No
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
24
K
­
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Very
sensitive
to
SIS,
less
sensitive,
and
to
insensitive
to
estrogens.
ARE
THERE
KNOWN
FALSE
POSITIVES
Cell
toxicants.
OR
FALSE
NEGATIVES
Toxicants
that
require
metabolic
activation.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Cell
membrane
and
second
messenger
effects.

VII.
Androgen
and
Anti­
androgen
­
In
Vivo
A.
Endocrine
Challenge
Test
(
Fail
et
al.,
1995)
DESCRIPTION
Repeated
observation
of
serum
T,
LH
and
other
hormones
(
with
and
without
LH
or
GnRH
challenge)
in
catheterized
animal.
Clearly
the
best
way
to
detect
altered
hormone
secretion
in
vivo.
DEGREE
OF
USE
Limited
DURATION
Few
weeks.
ASSAY
STABILITY
Stable,
once
the
animal
is
catheterized.
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
quite
difficult.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Has
not
been
standardized.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
More
sensitive
than
single
determination
of
serum
hormones
levels.
ARE
THERE
KNOWN
FALSE
POSITIVES
Stress
reduces
serum
T,
increases
prolactin
and
corticosterone,
effect
that
is
rarely
accounted
for
in
most
studies.
Hormone
levels
are
also
subject
to
circadian
effects.
COMMENTS.
Excellent,
but
specialized.
Tail
bleeds
can
also
be
used
for
repeated
sampling
from
the
same
animal
if
only
a
small
amount
of
serum
is
needed.
This
method
also
is
not
standardized
and
requires
a
great
deal
of
practice.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
25
K
­
B.
Super
Apical
Developmental
Toxicity
Test
DESCRIPTION
Expose
pregnant/
lactating
dam
and
examine
hormone
(
AR,
ER,
SIS,
Ah,
and
T3)
sensitive
endpoints
in
progeny
up
to
puberty.
DEGREE
OF
USE
Such
a
protocol
has
been
used
at
EPA,
CIIT,
NIEHS
for
xenoestrogens,
environmental
antiandrogens,
Ah
receptor
agonists,
phthalates
and
antithyroidal
toxicants
(
PCBs
and
PTU).
DURATION
Relatively
long
(
two
to
three
months)
as
compared
to
other
"
screens,"
would
need
to
determine
if
it
is
quicker
and
cheaper
to
run
this
as
opposed
to
several
more
focused,
shorterterm
assays.
ASSAY
STABILITY
Unknown,
but
should
be
as
good
as
any
developmental
test.
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
similar
to
needs
for
new
multigen
tests.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Multiplicity
of
in
vivo
techniques
are
required
that
are
currently
not
used
in
toxicology
labs,
but
they
should
be
coming
up
to
speed
to
implement
new
test
guidelines.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Not
standardized
as
used
in
different
labs
with
respect
to
dosing
or
some
of
the
assessments.
Some
standardization
is
now
taking
place
between
different
labs.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Antiandrogencity
via
AR,
or
SIS.
In
male
progeny,
AGD,
areolas,
nipples,
reduced
weight
of
prostate.
Higher
doses
of
AR­
mediated,
but
not
SIS,
produce
agenesis
of
prostate,
undescended
testis
and
agenesis
of
epididymis.
Altered
T
production
by
testis,
ex
vivo.

Estrogenicity.
Neonatal
Uterine
weight
and
gland
development
(?),
vaginal
opening,
age
at
first
estrus,
vaginal
cornification.
Prostate
size
in
male
(?),
and
sperm
production
(
at
high
dosage
levels).
SIS
or
Antiestrogenicity.
Pregnancy
loss,
delay
in
deliver
by
dam,
delay
in
VO
and
possible
altered
ovarian
hormone
production
ex
vivo.

Antithyroid.
Reduced
perinatal
growth
and
brain
size.
Lower
serum
T4
and
possibly
T3,
elevated
TSH.
Also
detects
functional
developmental
alterations
induced
by
phthalates,
TCDD
and
etc.
The
following
endpoints
are
considered
to
be
insensitive
because
they
take
too
long
to
assess
in
a
screen
and/
or
have
never
been
detected
with
a
pesticide
or
toxic
substance
following
developmental
exposure
(
i.
e.,
for
ER­
mediated:
cancer,
reduced
AGD,
hypospadias
in
male
of
female,
undescended
testes
or
any
malformations).
ARE
THERE
KNOWN
FALSE
POSITIVES.
Very
Apical
Test
used
to
screen
for
several
mechanisms
at
once.
OR
FALSE
NEGATIVES.
Not
likely,
if
designed
properly.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
26
K
­
C.
Pubertal
Development
in
Male
Rodent
(
Preputial
Separation)
DESCRIPTION
In
vivo
determination
of
age
at
puberty
in
male
rat.
DEGREE
OF
USE
Data
base
includes
drugs
and
antiandrogens
and
estrogenic
pesticides
and
toxic
substance
(
vinclozolin,
pp
DDE,
methoxychlor,
phthalates,
TCDD,
PCBs).
Required
endpoint
in
new
multigenerational
test
guidelines.
DURATION
20­
30
days
ASSAY
STABILITY
Varies
from
block
to
block
and
from
LE
to
SD
by
less
than
two
days
if
designed
carefully.
Delays
of
two
to
three
days
are
generally
significant
with
sample
sizes
of
10­
12/
group.
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
No
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Some
practice
is
required.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Has
been,
is
almost
as
easy
as
puberty
in
female
rat.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Slightly
less
sensitive
than
are
some
developmental
AR­
mediated
alterations.
Also
moderately
sensitive
to
xenoestrogens
(
for
ER,
PPS
is
less
sensitive
than
growth,
but
much
better
than
spermatogenesis
and
dosing
duration
is
shorter).
ARE
THERE
KNOWN
FALSE
POSITIVES
Yes,
this
is
an
apical
assay
so
anti­
AR,
ER,
SIS
inhibition,
altered
hypothalamic­
pituitary
function
and
Leydig
cell
toxicants
will
all
delay
this
developmental
landmark.
OR
FALSE
NEGATIVES
Unknown
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Nonspecific,
apical
test.
COMMENTS
Noninvasive
test
that
could
be
coupled
with
both
Hershberger
test
and
biochemical
assays.

D.
Hershberger
Assay
(
1953)
DESCRIPTION
In
vivo
measurement
of
effects
of
antiandrogenic/
androgenic
toxicants
of
androgen­
dependent
tissues
in
peripubertal/
adult
male
rat.
Weigh
sex
accessory
glands
and
levator
ani/
bulbocavernosus
muscle
in
T­
implanted,
castrate
adult
or
intact
juvenile
male
rat
four
to
seven
days
after
start
of
study.
Can
also
measure
serum
T
and
DHT
(
to
discriminate
ARmediated
from
SIS
mechanisms
and
liver
effects
on
metabolism),
and
biochemical
indices
(
ODC)
and
tissue
gene
expression
(
TRPM2,
C3).
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
27
K
­
DEGREE
OF
USE
Extensive
in
drug
development
and
recently
in
toxicology
of
antiandrogens.
DURATION
One
week.
ASSAY
STABILITY
Stable
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Balance,
scissors.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Good
necropsy
and
surgical
skills.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Has
been
standardized
and
validated.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Responds
as
expected
to
moderate
dosage
levels
of
antiandrogenic
pesticides.
ARE
THERE
KNOWN
FALSE
POSITIVES
Yes,
especially
if
conducted
in
intact
peripubertal
male.
An
apical
test
in
intact
animal.
More
specific
in
castrate
but
some
endpoints
are
still
affected
by
other
mechanisms.
OR
FALSE
NEGATIVES
None
known.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)
Yes,
prolactin,
thyroid
hormone,
and
estrogens
affect
some
organ
weights.
COMMENTS
One
of
the
best
short­
term
assays
for
antiandrogens,
along
with
pubertal
development.

VIII.
Anti­
androgen
­
Synthesis
Metabolism
A.
Testis/
Ovary
Culture
In
Vitro
or
Ex
Vivo
DESCRIPTION
Determination
of
testosterone
production
from
testicular
tissue
from
animals
treated
in
vivo
(
ex
vivo)
or
using
in
vitro
dosing.
Used
for
EDS,
estrogens,
antiandrogens,
several
other
testicular
toxicants,
and
substances
that
inhibit
steroidogenesis.
In
female,
minced
ovary
culture
can
be
used
ex
vivo
from
pregnant
(
i.
e.,
GD
14­
16)
or
cycling
females
(
proestrus
for
estradiol
production).
DEGREE
OF
USE
A
few
hundred
publications
over
the
last
25
years
since
the
advent
of
RIAs
for
testosterone.
Used
in
other
vertebrates,
as
well
as
mammals.
Used
by
several
toxicology
laboratories
in
addition
to
NHEERL­
EPA.
DURATION
One
day
for
in
vitro,
longer
for
ex
vivo
(
duration
depends
upon
dosing).
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
28
K
­
ASSAY
STABILITY
Depends
on
methodology.

DOES
IT
METABOLIZE
TOXICANTS
Yes,
for
ex
vivo,
little
or
no
metabolism
for
in
vitro.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Incubator,
freezer,
necropsy
equipment.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Some
practice,
but
a
rather
simple
assay
that
can
be
learned
by
competent
technicians
in
a
week
or
less.
Need
to
be
able
to
conduct
RIAs
for
T,
estradiol,
and
progesterone.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Has
not
been
standardized
between
labs,
but
could
be
without
too
much
difficulty.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Fairly
sensitive.
ARE
THERE
KNOWN
FALSE
POSITIVES
Cell
toxicants.
OR
FALSE
NEGATIVES
If
metabolic
activation
is
required
then
in
vitro
may
not
work.
COMMENTS.
These
assays
could
be
used
rapidly
to
screen
chemicals
for
ability
to
inhibit
steroidogenesis.

IX.
Thyroid
­
Intrinsic
Activity
A.
TR
Binding
Assay
DESCRIPTION
Determines
whether
a
chemical
can
alter
T3
binding
to
its
nuclear
receptor.
Assay
is
performed
on
isolated
nuclei.
Nuclei
can
be
isolated
from
liver
of
any
species.
In
principal,
these
results
would
reveal
competition
for
the
binding
site
or
an
allosteric
effect.

DEGREE
OF
USE
Extensive
DURATION
Four
hours.
ASSAY
STABILITY
Prepared
nuclei
are
stable
at
­
80
¡
C
for
long
periods.
Assay
is
performed
at
RT.
DOES
IT
METABOLIZE
TOXICANTS
No
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
training
period
is
not
extensive.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
29
K
­
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Very
sensitive;
can
be
run
over
broad
dose
range.
ARE
THERE
KNOWN
FALSE
POSITIVES
No,
though
chemicals
that
bind
to
TR
in
vitro
may
not
affect
TR
signaling
in
vivo.
OR
FALSE
NEGATIVES
Conditions
of
the
assay
must
be
standardize
because
a
variety
of
factors
(
e.
g.,
oxidation)
blocks
T3
binding
to
its
receptor.
In
addition,
solubility
of
lipophilic
compounds
may
preclude
binding
in
this
in
vitro
system.
Finally,
chemicals
that
do
not
bind
to
the
TR
may
still
disrupt
thyroid
function
or
thyroid
hormone
action.
SPECIFICITY
High
COMMENTS
This
assay
should
be
practical
to
do
with
tissue
(
liver)
from
any
vertebrate.

X.
Thyroid
­
In
Vitro
A.
Whole
Cell
Binding
Assays.
Cell
lines
that
express
the
thyroid
hormone
receptor
can
be
used
as
a
whole­
cell
binding
assay.
Generally,
125I­
T
3
is
added
to
the
media
and
after
a
short
incubation
period,
nuclei
are
isolated
and
counted.
The
difference
between
a
whole­
cell
assay
and
other
binding
assays
is
that
the
ligand
must
be
taken
up
into
the
cell
by
stereospecific
uptake
sites
that
can
be
blocked
by
xenobiotics.
Thus,
this
type
of
screen
could
potentially
detect
a
broader
array
of
chemicals
that
affect
thyroid
hormone
action
(
i.
e.,
those
that
bind
to
the
TR
and/
or
those
that
affect
cellular
uptake).

Cell
lines
from
a
number
of
vertebrates
have
been
described
to
express
the
thyroid
hormone
receptor
and,
therefore,
may
be
suitable.
However,
cell
lines
often
exhibit
a
number
of
growth/
maintenance
characteristics
that
would
make
them
more
or
less
suitable
for
large
screens,
and
this
type
of
information
would
require
further
research
to
obtain.

B.
Stably
Transfected
Cell
Lines.
Cells
that
express
the
TR
can
also
be
stably
transfected
to
provide
a
number
of
reporters
that
would
respond
to
different
kinds
of
thyroid
responsive
elements
(
TREs).
An
example
would
be
GH
3
cells
which
have
been
used
to
study
the
role
of
thyroid
hormone
on
the
regulation
of
growth
hormone
gene
expression.
Because
there
are
a
number
of
TRE
motifs,
one
goal
would
be
to
establish
a
cell
line
that
would
allow
an
easy
screen
of
compounds
that
might
affect
the
ability
of
TR
to
influence
the
expression
of
several
types
of
regulatory
elements.
In
principle,
this
type
of
assay
could
be
established
in
cell
lines
from
a
variety
of
vertebrates
with
the
same
caveats
listed
above.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
30
K
­
C.
Thyroid
Hormone­
Responsive
Cells.
Various
cell
lines
change
phenotype
in
response
to
thyroid
hormone.
PC12
cells
are
an
example.
This
is
a
rat
pheochromocytoma
cell
line
which
can
be
induced
to
become
neuronlike
in
response
to
NGF.
Thyroid
hormone
can
block
this
change,
but
it
requires
transient
transfection
of
the
thyroid
hormone
receptor.

Another
example
is
that
of
the
XLT­
15
cell
line
of
Yaoita
and
Nakajima.
These
cells
can
be
induced
to
undergo
apoptosis
by
thyroid
hormone.
Thus,
a
screen
may
be
developed
to
determine
whether
a
compound
can
influence
this
induction.

D.
Specialized
Cells.
Various
cell
lines
exhibit
unique
features
that
can
be
recruited
for
development
of
a
screen.
For
example,
FRTL­
5
cells
are
derived
from
a
rat
thyrocarcinoma.
Marinovich
et
al.
[
Marinovich
1995;
1153]
have
reported
a
clonal
line
that
is
stably
transfected
with
the
human
thyroid
peroxidase
which
can
be
inhibited
by
a
number
of
chemicals
(
e.
g.,
ethylenethiourea,
a
metabolite
of
dithiocarbamate
pesticides).

XI.
Thyroid
In­
vivo
A.
Short­
Term
Serum
T4
DESCRIPTION
Determines
whether
a
chemical
can
alter
circulating
levels
of
thyroxine.
Several
mechanisms
for
this,
including
decreased
half­
life
(
displacing
from
serum
carrier
proteins,
activating
liver
enzymes),
or
decreasing
synthesis
by
effects
on
the
thyroid
itself.
T
4
is
more
sensitive
than
T
3
or
TSH.
T
3
and
TSH
can
be
measured
if
T
4
is
affected
but
there
are
a
number
of
compounds
that
affect
T
4
without
affecting
T
3
or
TSH.
Other
indices
would
be
required.
DEGREE
OF
USE
Extensive
DURATION
24
hours
or
more
ASSAY
STABILITY
N/
A
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
but
T
4
kits
are
widely
used
clinically.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
training
period
is
not
extensive.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
31
K
­
T
4
is
not
very
sensitive
in
a
short­
term
assay,
though
others
may
have
more
extensive
knowledge
on
this.
ARE
THERE
KNOWN
FALSE
POSITIVES
No
OR
FALSE
NEGATIVES
Because
the
half­
life
of
thyroid
hormone
in
blood
is
long
(~
120h)
and
because
some
compounds
may
interfere
with
thyroid
hormone
synthesis
(
e.
g.,
iodide
uptake),
short­
term
exposure
may
not
be
indicative
of
thyroid
affects.
SPECIFICITY
High
COMMENTS
This
could
be
coupled
to
a
screen
for
reproductive
effects.

B.
Long­
Term
Serum
T4
DESCRIPTION
Determines
whether
a
chemical
can
alter
circulating
levels
of
thyroxine
within
a
longer
time­
course.
This
could
be
performed
on
animals
being
evaluated
for
reproductive
effects
(
see
RTP
proposal).
DEGREE
OF
USE
Extensive
DURATION
48
hours
or
more.
ASSAY
STABILITY
N/
A
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Yes,
but
T
4
kits
are
widely
used
clinically.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
training
period
is
not
extensive.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Unclear
ARE
THERE
KNOWN
FALSE
POSITIVES
No
OR
FALSE
NEGATIVES
None
that
are
known.
SPECIFICITY
Unknown
COMMENTS
This
assay
can
be
included
in
an
experiment
for
reproductive
effects.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
32
K
­
C.
Thyroid
Peroxidase
DESCRIPTION
This
description
was
contributed
by
Dan
Sheehan
(
excerpted
by
TZ).
The
thyroid
peroxidase
has
a
broad
substate
specificity,
for
all
phenolic
chemicals
essentially.
One
group
of
prototypical
ED's
is
the
flavones
and
isoflavones;
they
inhibit
thyroid
peroxidase
with
IC0'
s
in
the
low
micromolar
range
and
can
act
as
either
competitive
inhibitors
or
as
suicide
substrate
inhibitors
which
inactivate
the
peroxidase.
Either
or
both
of
these
actions
could
lower
T3/
T4
production,
increase
TSH
and
lead
to
goiter/
carcinoma.
A
population
of
human
infants
consuming
soy
infant
formula,
which
has
a
high
isoflavone
content,
has
been
identified
with
Graves
disease
or
Hashimotos
thyroiditis,
both
autoimmune
thyroid
diseases
diagnosed
by
goiter.
The
prevalence
of
soy
formula
consumption
was
twice
as
high
in
these
patients
as
in
controls
who
had
consumed
cows
milk
formula.
The
assay
for
inhibition
is
simpler,
quicker,
and
cheaper
than
a
receptor
assay.
It
is
a
colorimetric
assay
of
peroxidase
activity
in
the
presence
of
various
concentrations
of
a
chemical
followed
by
graphical
or
computer
solutions
for
IC50'
s.
It
seems
likely
to
me
that
other
chemicals
may
act
via
the
same
mechanism.
Also
note
that
the
phenolic
"
A"
ring
(
or
its
equivalent)
is
crucial
for
estrogen
receptor
binding.
Chemicals
with
a
low
ER
RBA
(?)
(
i.
e.,
1/
1000
tha
of
E2)
and
a
phenolic
group
would
be
active
estrogens
in
the
micromolar
range
AND
would
also
possibly
be
active
on
the
peroxidase.
Given
the
co­
occurence
of
thyroid
and
reproductive
problems
in
some
geographical
areas,
this
may
be
due
to
estrogenic
activity
of
low
affinity
ER
ligands
which
also
have
peroxidase
inhibiting
activity.
While
this
assay
does
not
account
for
all
mechanisms
leading
to
thyroid
toxicity,
just
as
an
ER
RBA
assay
does
not,
it
is
cheap,
simple,
and
quick;
and
,
I
think,
should
be
in
the
in
vitro
battery
for
the
thyroid.
DEGREE
OF
USE
Extensive
DURATION
?
ASSAY
STABILITY
N/
A?
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Modest
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
training
period
is
not
extensive.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Unclear
ARE
THERE
KNOWN
FALSE
POSITIVES
No
OR
FALSE
NEGATIVES
Compounds
may
interfere
with
thyroid
hormone
action
without
affecting
TPO.
SPECIFICITY
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
33
K
­
Unknown
D.
Malic
Enzyme
DESCRIPTION
DEGREE
OF
USE
Extensive
DURATION
?
ASSAY
STABILITY
N/
A?
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Modest
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
training
period
is
not
extensive.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Unclear
ARE
THERE
KNOWN
FALSE
POSITIVES
No
OR
FALSE
NEGATIVES
Compounds
may
interfere
with
thyroid
hormone
action
without
affecting
TPO.
SPECIFICITY
Unknown
COMMENTS
See
above
E.
Mammal
Development
DESCRIPTION
This
assay
can
be
performed
on
animals
in
which
screens
for
reproductive
effects
are
being
evaluated
as
described
in
the
RTP
proposal.
Simplest
measures
would
be
circulating
T
4,
and
brain
weight.
DEGREE
OF
USE
Brain
weight
is
not
used
as
an
index
of
thyroid
hormone
action
during
development,
though
brain
weight
is
clearly
affected
by
thyroid
hormone
action.
DURATION
?
ASSAY
STABILITY
N/
A?
DOES
IT
METABOLIZE
TOXICANTS
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
34
K
­
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Modest
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
training
period
is
not
extensive.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Unclear
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Unclear
ARE
THERE
KNOWN
FALSE
POSITIVES
Yes,
compounds
that
affect
nutritional
status
or
eating.
However,
these
may
also
influence
thyroid
hormone
levels
which
are
linked
to
nutritional
status.
OR
FALSE
NEGATIVES
No?
SPECIFICITY
Unknown
COMMENTS
See
above.

XII.
Three
Alternative
Overviews
The
following
three
overviews
were
submitted
as
alternatives
to
existing
overviews
and
are
included
here
as
an
addendum.
(
submitted
by
Dr.
Soto)

A.
MCF7
Proliferation
Assay
(
E­
SCREEN)
DESCRIPTION
Measures
proliferation
of
cells
in
culture
in
response
to
estrogens.
DEGREE
OF
USE
Used
in
various
laboratories
in
the
U.
S.
A.,
Europe
and
Japan.
Many
chemicals
studied;
several
toxicants
were
discovered
to
have
estrogenic
properties
using
this
method.
Extensive
published
data
on
toxicants
with
estrogenic
activity.
DURATION
From
seeding
the
cells
to
harvesting
cells=
5
to
7
days.
It
requires
4
manipulations
only:
(
1)
seeding,
(
2)
changing
to
test
medium
24
hours
later,
(
3)
single­
step
harvesting
(
or
staining
in
situ)
and
(
4)
counting
(
or
reading
in
an
ELISA
plate
reader).
ASSAY
STABILITY
Very
stable
in
the
labs
that
started
with
a
line/
subline
of
proven
sensitivity.
Clones
reported
to
have
stable
activity
for
over
ten
years.
DOES
IT
METABOLIZE
TOXICANTS?
Not
thoroughly
characterized.
However,
some
proestrogens
(
methoxychlor,
bisphenol­
A
dimethacrylate,
alkylphenol­
monoethoxylates,
non­
hydroxylated
PCBs)
were
reported
to
have
activity
in
this
assay.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
35
K
­
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED?
Like
all
assays
performed
with
vertebrate
cell
cultures,
it
requires
laminar
flow
hood,
CO
2
incubator,
cell
freezing
storage,
inverted
microscope,
cell
counting
devise,
and
a
detector
for
the
specific
end
point
measured
(
it
may
use
an
electronic
cell
counter
or
an
ELISA
plate
reader
if
using
sulforhodamine­
B
assay
or
other
colorimetric
assay).
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED?
Like
all
assays
using
cell
culture,
it
requires
the
ability
of
maintaining
the
culture
stocks,
periodic
freezing
after
several
passages
to
maintain
the
cultures
in
case
of
infections
or
other
mishaps.
The
assay
itself
is
very
easy
to
perform.

HAS
IT
BEEN
STANDARDIZED
EASILY?
It
is
highly
reproducible
from
assay
to
assay.
It
is
used
in
several
labs
in
the
USA
and
Europe.
By
using
responsive
cells
to
begin
with,
it
has
provided
comparable
results
when
a
battery
of
20
coded
chemicals
were
tested
by
several
labs
(
manuscript
in
preparation;
Project
Coordinator:
Philip
Grandjean,
Odense
University,
Denmark).
Competent
labs
appear
to
get
similar
results.
Cell
of
appropriate
phenotype
can
be
easily
distributed.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Very
sensitive;
one
of
the
most
sensitive
assays
(
E2
EC50=
10­
15
pM
range;
maximal
activity
100
pM).
ARE
THERE
KNOWN
FALSE
POSITIVES?
In
competent
labs
no
false
positives
were
found
among
growth
factors.
Testosterone
is
the
only
steroid
that
shows
activity
at
TM
concentrations.
When
assaying
for
antagonists,
endocrinologists
have
used
a
two­
step
method
to
assess
whether
or
not
the
effect
is
truly
antiestrogenic
regardless
of
whether
the
end
point
is
inhibition
of
estrogen­
induced
cell
proliferation
or
inhibition
of
estrogen
induction
of
a
gene
product:
1)
assess
the
effect
of
a
range
of
doses
of
the
test
compound
together
with
the
minimal
estrogen
dose
needed
for
maximal
induction
of
the
gene
product
(
inhibition),
and
2)
assess
the
effect
of
increasing
doses
of
estradiol
administered
together
with
the
dose
of
toxicant
found
to
induce
maximal
inhibition
(
estradiol
rescue).
OR
FALSE
NEGATIVES?
False
negatives
due
to
toxicity
were
not
found
using
GLP
(
it
only
takes
looking
at
the
cells
with
the
inverted
microscope
to
detect
unspecific
toxicity).
TCDD
does
have
toxic
effects
both
in
the
presence
and
absence
of
estrogens.
Various
substances
that
require
metabolic
activation
like
alkylphenols
polyethoxylates
may
be
missed.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
OF
ACTION
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)?
So
far,
all
substances
found
to
be
estrogenic
with
the
E­
SCREEN
assay
that
were
also
tested
using
gene
activation
assays
were
found
to
be
consistently
estrogenic.
COMMENTS
As
with
all
bioassays,
it
requires
obvious
good
laboratory
practices.
In
the
case
of
rodent
bioassays
it
is
important
to
work
with
healthy
animals,
appropriate
light
cycles,
using
feeds
that
do
not
contain
estrogens,
etc.
When
working
with
cells
in
culture,
one
has
to
start
with
a
subline
that
expresses
the
appropriate
phenotype.
To
maintain
the
phenotype,
serum
used
for
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
36
K
­
cell
propagation
has
to
be
checked
before
using
it
for
propagation
(
therefore,
laboratories
stock
"
good
serum"
to
last
for
one
year).
Similarly,
stocks
have
to
be
frozen
periodically,
and
the
charcoal­
dextran
stripped
serum
(
which
can
be
stored
frozen
in
aliquots
for
up
to
one
year,
should
be
checked
once
before
use).
Recently,
it
was
shown
that
recombinant
serum
albumin
may
be
used
instead
of
charcoal­
dextran
stripped
serum.
The
assay
can
be
automated,
and
different
labs
use
12­,
24­
or
96­
well
plates,
depending
on
whether
one
uses
cell
numbers,
sulforhodamine­
B
staining
or
MTT
reaction
as
the
end
point).
Also,
cells
can
be
fixed
in
situ
at
the
end
of
the
experiment
and
stained
days
or
weeks
later.

B.
MVLN
Assay.
Stably
Transfected
Reporter
Gene
Assay
in
Mammalian
Cells
(
submitted
by
Dr.
Soto)
DESCRIPTION
The
assay
utilizes
a
mammalian
cell
line
(
MCF7
with
endogenous
ER)
that
has
been
stably
transfected
with
an
ER­
specific
reporter
gene
(
Vit­
luc).
DEGREE
OF
USE
Used
in
various
laboratories
for
pharmaceutical
and
environmental
research.
No
publications
are
listed
in
Medline
as
yet
attesting
its
use
for
estrogenic
toxicants.
DURATION
From
seeding
the
cells
to
harvesting
lysate
and
Luc
activity
takes
two
to
three
days.
The
following
manipulations
are
required:
(
1)
seeding
and
exposure
to
charcoal­
dextran
stripped
serum
containing
tamoxifen
for
24­
48
hours;
(
2)
changing
the
medium
to
tamoxifen­
free
for
12
hours
(
or,
treat
for
1­
2
weeks
with
charcoal­
dextran
stripped
serum,);
(
3)
incubate
with
test
substance
for
24
or
more
hours;
(
4)
finally
harvest
cells;
(
5)
homogenize
them;
(
6)
read
assay;
and
(
7)
run
a
protein
assay.
ASSAY
STABILITY
Like
the
parent
cell
line
MCF7,
it
should
be
stable
when
good
laboratory
practices
are
followed.
It
has
been
stable
for
30
passages.
Exposure
to
tamoxifen
renders
these
cells
unable
to
express
estrogen
induction
of
the
reported
gene.
DOES
IT
METABOLIZE
TOXICANTS?
Not
thoroughly
characterized.
It
may
maintain
same
capabilities
as
those
of
the
parental
cell
line.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED?
Like
all
assays
performed
with
vertebrate
cell
cultures,
it
requires
laminar
flow
hood,
CO
2
incubator,
cell
freezing
storage,
inverted
microscope,
cell
counting
devise,
and
a
detector
for
the
specific
end
point
measured
(
luminometer
to
measure
Luc
activity).
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED?
Like
all
assays
using
cell
culture,
it
requires
the
ability
of
maintaining
the
culture
stocks,
periodic
freezing
after
several
passages
to
maintain
the
cultures
in
case
of
infections
or
other
mishaps.
The
assay
itself
is
easy
to
perform.
HAS
IT
BEEN
STANDARDIZED
EASILY?
No
published
results
are
available
on
the
performance
of
this
assay
to
detect
estrogenic
toxicants.
Stably
transfected
cells
can
be
easily
distributed.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
37
K
­
Very
sensitive;
EC50
for
E2
in
the
20
pM
range;
maximal
activity
of
E2
was
reported
at
1
nM.
ARE
THERE
KNOWN
FALSE
POSITIVES?
Hypothetically,
ER
may
be
activated
by
non­
estrogenic
agents
through
phosphorylation
pathways.
When
assaying
for
antagonists,
endocrinologists
have
used
a
two­
step
method
to
assess
whether
or
not
the
effect
is
truly
antiestrogenic
regardless
of
whether
the
end
point
is
cell
proliferation
or
inhibition
of
estrogen
induction
of
a
gene
product:
(
1)
assess
the
effect
of
a
range
of
doses
of
the
test
compound
together
with
the
minimal
estrogen
dose
needed
for
maximal
induction
of
the
gene
product
(
inhibition);
and
(
2)
assess
the
effect
of
increasing
doses
of
estradiol
administered
together
with
the
dose
of
toxicant
found
to
induce
maximal
inhibition
(
estradiol
rescue).
OR
FALSE
NEGATIVES?
Similarly
to
the
parental
cell
line,
false
negatives
due
to
toxicity
should
be
excluded
by
GLP
(
it
only
takes
looking
at
the
cells
with
the
inverted
microscope
to
detect
unspecific
toxicity).
It
is
suspected
that
like
in
the
parent
cell
line
TCDD
may
have
toxic
effects
both
in
the
presence
and
absence
of
estrogens.
Various
substances
that
require
metabolic
activation
like
alkylphenols
polyethoxylates
may
be
missed.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
OF
ACTION
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)?
Hypothetically,
ER
may
be
activated
by
non­
estrogenic
agents
through
phosphorylation
pathways.
COMMENTS
Specific
ER
activation.
As
with
all
bioassays,
it
requires
obvious
good
laboratory
practices.
In
the
case
of
rodent
bioassays
it
is
important
to
work
with
healthy
animals,
appropriate
light
cycles,
using
feeds
that
do
not
contain
estrogens,
etc.
When
working
with
cells
in
culture,
one
has
to
start
with
a
subline
that
expresses
the
appropriate
phenotype.
To
maintain
the
phenotype,
serum
used
for
cell
propagation
has
to
be
checked
before
use
(
therefore,
laboratories
stock
"
good
serum"
to
last
for
at
least
one
year).
Similarly,
cell
stocks
have
to
be
frozen
periodically.
The
assay
can
be
automated;
can
be
done
in
12­,
24­
or
96­
well
plates.

C.
MCF7­
AR1
Assay.
MCF7
Cells
Stably
Transfected
With
"
Wild
Type"
Androgen
Receptor.
(
submitted
by
Dr.
Soto)
DESCRIPTION
The
assay
utilizes
a
mammalian
cell
line
(
MCF7)
stably
transfected
AR.
These
cells
proliferate
maximally
in
serumless
medium
supplemented
with
insulin
and
transferrin.
Androgens
inhibit
their
proliferation;
antiandrogens
abolish
the
inhibitory
effect
of
androgens.
DEGREE
OF
USE
This
method
was
just
published.
DURATION
From
seeding
cells
to
harvesting
them,
it
takes
5
days.
ASSAY
STABILITY
Like
the
parent
cell
line
MCF7,
it
should
be
stable
when
good
laboratory
practices
are
followed.
It
has
been
stable
for
4
years.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
38
K
­
DOES
IT
METABOLIZE
TOXICANTS?
Not
thoroughly
characterized.
It
should
keep
the
same
capabilities
as
those
of
the
parental
cell
line.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED?
Like
all
assays
performed
with
vertebrate
cell
cultures,
it
requires
a
laminar
flow
hood,
CO
2
incubator,
cell
freezing
storage,
inverted
microscope,
cell
counting
devise,
and
a
detector
for
the
specific
end
point
measured
(
it
may
use
an
electronic
cell
counter
or
an
ELISA
plate
reader
when
using
sulforhodamine­
B
assay
or
other
colorimetric
assay).

ARE
SPECIAL
SKILLS/
TRAINING
NEEDED?
Like
all
assays
using
cell
culture,
it
requires
the
ability
of
maintaining
culture
stocks,
periodic
freezing
after
several
passages
to
maintain
the
cultures
in
case
of
infections
or
other
mishaps.
The
assay
itself
is
easy
to
perform.
HAS
IT
BEEN
STANDARDIZED
EASILY?
It
can
be
standardized
easily,
since
cells
express
AR
constitutively.
No
published
results
are
available
on
the
performance
of
this
assay
to
detect
toxicants
that
are
androgen
agonists
or
antagonists.
Stably
transfected
cells
can
be
easily
distributed.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Very
sensitive;
EC50
for
DHT
in
the
pM
range.
ARE
THERE
KNOWN
FALSE
POSITIVES?
In
theory,
hyperphysiological
doses
of
glucocorticoids
may
bind
to
the
AR
and
this
may
activate
AREs.
OR
FALSE
NEGATIVES?
As
with
the
parental
cell
line,
false
negatives
due
to
toxicity
should
be
excluded
by
GLP
(
it
only
takes
looking
at
the
cells
with
the
inverted
microscope
to
detect
unspecific
toxicity).
In
addition,
since
androgens
inhibit
cell
proliferation,
"
rescue"
from
inhibition
by
an
antiandrogen
may
help
differentiating
toxicity
from
androgenicity.
Substances
that
require
metabolic
activation
may
be
missed.
SPECIFICITY­
ARE
THERE
OTHER
MECHANISMS
OF
ACTION
TO
PRODUCE
THIS
EFFECT
(
FALSE
POSITIVES)?
Probably
hyperphysiological
doses
of
glucocorticoids.
COMMENTS
Like
all
bioassays,
it
require
good
laboratory
practices.
In
the
case
of
rodent
bioassays
it
is
important
to
work
with
healthy
animals,
appropriate
light
cycles,
using
feeds
that
do
not
contain
estrogens,
etc.
When
working
with
cells
in
culture,
the
cell
line
should
express
the
appropriate
phenotype.
To
maintain
the
phenotype,
serum
used
for
cell
propagation
has
to
be
checked
before
use
(
therefore,
laboratories
stock
"
good
serum"
lots
for
at
least
one
year).
Similarly,
cell
stocks
have
to
be
frozen
periodically.
The
assay
can
be
automated;
can
be
done
in
12­,
24­
or
96­
well
plates.

XIII.
Overview
of
Non­
Mammalian
Screens
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
39
K
­
A.
Procedures
Using
Birds
and
Reptiles
to
Determine
Endocrine
Disruptive
Action
The
number
of
procedures
available
are
limited
for
taxa
other
than
mammals.
In
part,
this
is
because
many
procedures
used
in
mammals
have
just
not
been
tried
in
other
taxa,
as
well
as
because
they
are
not
appropriate
for
egg­
producing
vertebrates
or
any
phyla
other
than
Vertebrata.
Hence,
the
procedures
below
have
been
divided
into
two
matrices
with
identical
structure
as
the
mammalian
matrix,
but
including
both
birds
and
reptiles
as
subjects.
The
first
matrix
contains
procedures
that
have
already
been
used
to
determine
endocrine
action
of
contaminants,
and
the
second
contains
procedures
that
could
be
used,
but
have
not.
The
procedures
in
the
second
matrix
need
varying
amounts
of
development
to
be
ready
for
inclusion
in
a
screening
and
testing
program.
The
amount
of
development
needed
is
estimated
in
the
"
comments"
section.

Also,
the
number
and
variety
of
in
vitro
procedures
that
have
been
applied
to
wildlife
is
orders
of
magnitude
smaller
than
used
with
mammalian
material.
This
may
be
a
particular
problem
for
the
design
of
a
comprehensive
set
of
screening
procedures,
because
the
screening
battery
is
intended
to
cull
out
from
a
list
of
chemicals
to
be
tested,
all
those
chemical
that
have
no
intrinsic
endocrine
activity.
Hence,
the
screening
battery
should
be
comprehensive
for
all
types
of
endocrine
action
(
or
as
many
as
possible).
Thus
the
availability
of
short
duration
procedures
using
non­
mammalian
material
that
could
be
used
as
screens
would
seem
crucial
as
the
screening
and
testing
program
is
intended
to
be
protective
of
all
taxa,
not
just
humans
and
other
mammals.
The
issue
of
the
homology
of
steroid
hormone
and
receptor
structure
across
vertebrates
classes
needs
to
be
explored
more
fully
to
help
design
an
adequately
comprehensive
program,
especially
for
the
screening
phase.
At
this
date,
there
is
no
real
choice
to
be
made
as
there
are
very
few
if
any
procedures
using
non­
mammalian
material
that
could
be
used
as
a
screening
tool.
This
area
needs
thoughtful
development.
In
addition
to
development
of
non­
mammalian
vertebrate
in
vitro
procedures,
invertebrates
in
whole
animal
"
biological
activity"
tests
could
be
exploited,
which
by
dint
of
the
very
short
life
cycle,
of
Daphnia
for
instance,
could
be
economically
used
in
a
screening
battery.

B.
Overview
of
Endocrine
Disruptor
Relevant
Screens
to
the
Lower
Vertebrates
and
Invertebrates
Unlike
the
many
and
wide
array
of
screens
available
for
mammalians,
few
exist
for
the
animals
outside
that
taxonomic
group.
Some
assays
do
exist
which
can
be
employed
with
differing
degrees
of
specificity
and
sensitivity.
Predominantly,
the
assays
available
for
these
animals
will
be
in
vivo
and
somewhat
apical.
Although
this
means
these
assays
will
not
provide
detailed
mode
of
action
information,
the
endpoints
will
be
relevant
for
interpreting
"
adverse"
effects.

For
the
non­
mammalian
vertebrates,
vitellogenin
assays
are
available
and
will
generally
provide
suggestive
evidence
of
estrogen­
related
disruptions.
However,
there
is
some
evidence
of
thyroid
hormone
involvement
with
vitellogenin
production
which
may
compromise
any
conclusive
evidence
of
an
estrogen
specific
action.
Nonetheless,
compounds
which
affect
a
change
in
normal
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
40
K
­
vitellogenin
levels
should
be
captured
in
a
screening
program
for
more
definitive
investigation
of
the
effect
and
its
relevance.

Existing
standardized
tests
for
evaluating
conventional
toxicities
are
also
included
here
because
of
the
availability
of
such
information
for
certain
compounds
(
e.
g.,
pesticides).
Information
from
these
tests
can
be
used
to
screen
for
suggestive
endocrine
mediated
effects
to
be
flagged
for
further
investigation.

XIV.
Amphibian
Screens
A.
Vitellogenin
Assay
DESCRIPTION
An
assay
which
measures
the
amount
of
an
egg
yolk
protein
precursor
in
males
as
an
indicator
of
estrogenic
activity.
REFERENCES
Selcer,
"
Vitellogenin
induction
in
frogs
by
immersion
in
xenobiotic
estrogens,"
Am.
Zool.,
36,
1996,
p.
5.
Peterson,
G.
L.,
Determination
of
total
protein:
Methods
of
Enzymology,
91,
1993,
pp.
95­
121.
DURATION
>
72
hours
EQUIPMENT
Wet
lab,
antibody,
immunology
lab
STAFF
SKILLS
Can
be
trained
COST
Modest
AVAILABILITY
Limited
by
specificity
of
antibody,
but
Heppell
et
al.
efforts
at
developing
a
"
universal"
antibody
appear
promising
SENSITIVITY
Good
SPECIFICITY
Good
for
estrogen
activity,
but
thyroid
hormones
may
also
be
involved
STANDARDIZATION
Not
yet,
but
could
be
made
without
undue
difficulties
RELATEDNESS
Primarily
estrogen
box,
more
research
is
needed
to
ascertain
whether
thyroid
or
androgen
activity
is
or
is
not
connected
B.
Frog
Embryo
Teratogenesis
Assay
Xenopus
(
FETAX)
DESCRIPTION
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
41
K
­
A
96­
hour
whole
embryo
teratogenesis
screening
assay.
Because
the
exposure
is
through
primary
organogenesis
all
developmentally
important
processes
are
taken
into
account.
REFERENCES
Dumont
et
al.,
"
Frog
Embryo
Teratogenesis
Assay
­
Xenopus
(
FETAX),"
Shortterm
Bioassays
in
the
Analysis
of
Complex
Environmental
Mixtures
III,
1983.
Plenum,
ASTM,
Standard
guide
for
conducting
the
Frog
Embryo
Teratogenesis
Assay
­
Xenopus
(
FETAX),
E,
1991,
pp.
1439­
91.
DURATION
96
hours
EQUIPMENT
General
wet
lab
and
microscopy
lab
STAFF
SKILLS
Specialized
knowledge
of
amphibian
embryology
and
histology
COST
Modest
AVAILABILITY
Fair
SENSITIVITY
Fair,
not
fully
comprehensive
for
all
estrogenic,
androgenic,
or
thyroid
related
effects
SPECIFICITY
Does
not
distinguish
hormonal
from
non­
hormonal
developmental
effects
STANDARDIZATION
Yes
through
ASTM
RELATEDNESS
Apical
for
some
estrogenic,
androgenic,
and
thyroid
related
effects
other
C.
Amphibian
Metamorphosis
Assay
(
Conceptual)
COMMENTS
Similar
to
a
FETAX
like
assay
but
focused
on
tadpole
metamorphosis,
perhaps
specifically
on
tail
resorption.
Apical
for
thyroid
related
effects.

D.
Frog
In
Vivo
Screening
Assay
(
Conceptual)
COMMENTS
Similar
in
concept
to
what
is
proposed
for
fishes
evaluating
endpoints
such
as
gonadosomatic
index,
secondary
sex
characteristics,
oocyte
maturation,
plasma
steroids,
and
plasma
vitellogenin.
Apical
for
estrogen,
androgen,
and
thyroid
related
effects.

E.
Metamorphosis
DESCRIPTION
Determines
whether
a
chemical
can
affect
the
thyroid
hormone­
dependent
process
of
metamorphosis.
DEGREE
OF
USE
Extensive
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
42
K
­
DURATION
Depends
on
species
available.
Xenopus
or
Rana
would
require
about
a
week
of
treatment.
Scaphiopus
would
require
less
time
(
24
hours),
but
may
not
be
widely
available.
ASSAY
STABILITY
N/
A?
DOES
IT
METABOLIZE
TOXICANTS
Yes
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Modest
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
training
period
is
not
extensive
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
yes
ARE
THERE
KNOWN
FALSE
POSITIVES
No
OR
FALSE
NEGATIVES
Unclear
SPECIFICITY
Unknown
COMMENTS
The
ease
of
compound
administration
may
make
this
attractive.
The
use
of
±
T3/
T4
paradigm
would
allow
for
identification
of
thyroid
action
disruption.
Endpoints
(
e.
g.,
hind­
limb
growth
rate)
are
ÒintegratedÓ
measures;
thus,
the
screen
would
detect
compounds
that
affect
thyroid
hormone
action
along
the
entire
pathway.
There
are
clearly
a
number
of
endpoint
which
may
be
more
or
less
sensitive
or
reliable.
Structure
of
internal
organs
(
liver,
pronephros),
and
production
of
urea
are
two.

XV.
Bird
Screens
A.
Avian
Egg­
Injection
Assay
TEST
AND
FUNCTION:
The
effects
of
steroids
(
and
EDCs)
on
development
of
the
reproductive
tract
and
steroid
concentrations.
Subject
are
various
species
of
wildlife.
Exposure
is
by
egg­
injection
before
organogenesis;
responses
are
measured
at
hatching
and
include
the
morphology
histology
of
the
reproductive
tract
of
males
(
females
may
be
useful
also),
and
plasma
steroid
concentrations.
It
can
be
extended
into
the
breeding
age
of
the
affected
animal
to
look
check
for
functional
impairment.
REFERENCES:
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
43
K
­
Fry,
D.
M.,
C.
K.
Toone,
S.
M.
Speich,
and
R.
J.
Peard,
"
Sex
ratio
skew
and
breeding
patterns
of
gulls:
demographic
and
toxicological
considerations,"
Studies
in
Avian
Biology
10,
1987,
pp.
26­
43.
Nisbet
et
al.,
1996.
Several
other
studies
now
in
progress
using
similar
methodology
with
other
species.
DURATION:
Depends
on
the
length
of
incubation
(~
three
weeks
minimum),
and
availability
of
eggs:
wild
birds
usually
not
easily
available
unless
a
breeding
colony
exists.
EQUIPMENT:
Incubator,
hatchabator,
general
lab
facilities
and
facilities
for
RIAs.

STAFF
SKILLS:
Animal
husbandry
skills,
general
dissection
and
microscope
skills,
tissue
handling
and
preparation
for
sectioning,
biochemical
expertise
for
RIA
work.
AVAILABILITY:
Wide.
SENSITIVITY:
Good
for
estrogens.
SPECIFICITY:
Estrogenic
effects
in
males:
possibly
non­
aromatizable
androgen
effects
in
females.
STANDARDIZATION:
Needs
work,
especially
in
preparation
and
analysis
of
histological
responses.
OTHER:
The
choice
of
subject
species
is
crucial
for
operational
ease,
practicality
and
cost
control.
Embryos
must
be
large
enough
at
hatching
to
get
a
gonad
that
is
easily
handled
and
that
has
enough
blood
to
collect
for
RIA
on
the
plasma.
Very
similar
responses
to
procedure
R2.
Compare
to
A2.

B.
Japanese
Quail
Early
Life
Stage
TEST
AND
FUNCTION:
The
effects
of
steroids
(
and
EDCs)
on
development
of
sexual
maturity
in
Japanese
quail,
and,
if
desired,
of
the
reproductive
tract
and
plasma
steroid
concentrations
in
siblings.
This
tests
the
time
to
onset
of
maturity
as
measured
by
secondary
sex
characters
(
cloacal
or
foam
gland
size)
and
behavior
(
crowing)
in
males;
all
are
testosterone
dependent.
Exposure
varies,
see
below.
REFERENCES:
Ottinger,
M.
A.
and
H.
J.
Brinkley,
"
Testosterone
and
sex
related
behavior
and
morphology:
Relationship
during
maturation
in
the
adult
Japanese
quail,"
Hormones
and
Behavior
11,
1978,
pp.
175­
182;
and
other
references.
DURATION:
~
18
days
incubation
+
60
days
to
maturity.
EQUIPMENT:
Avian
husbandry,
general
lab,
tape
recorder.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
44
K
­
STAFF
SKILLS:
Husbandry,
general
lab.
AVAILABILITY:
Quail
are
widely
available;
strains
can
be
different,
recommend
out
bred
line.
SPECIFICITY:
Depending
on
the
timing
of
exposure
it
can
be
made
specific
to
estrogen
OR
androgens:
·
if
chicks
are
implanted
or
injected
with
EDC,
male
time
to
maturity
is
androgen
sensitive;
·
if
eggs
are
injected,
the
gonadal
development
of
male
offspring
is
impaired
by
estrogens
(
and
possibly
non­
aromatizable
androgens
in
female
offspring,
although
this
has
not
been
demonstrated
[
matrix
2]);
this
is
similar
to
procedure
A1.
Estrogenic
effects
on
time
to
maturity
in
males
is
not
known.
STANDARDIZATION:
This
test
has
been
used
widely
and
can
be
easily
standardized
further
.
OTHER:
Excellent
background
information
on
the
endocrinology
of
Japanese
quail;
this
procedure
might
be
developed
into
an
Androgen
antagonist
assay
in
males
with
co­
administration
of
EDC
and
androgen,
and
androgen
alone
as
control
[
matrix
2].

C.
Japanese
Quail
Androgenicity
Screen
TEST
AND
FUNCTION:
Foam
gland
size
and
crowing
behavior
in
photo­
regressed
adult
males
to
look
at
the
Androgenic
effect
of
EDCs
on
peripheral
targets,
using
Japanese
quail.
This
procedure
uses
individuals
that
are
not
secreting
GNRH,
and
thus
the
HPG
axis
is
shut
down.
REFERENCES:
[
Matrix
2]
DURATION:
Two
weeks
to
regress;
expose
for
two
days,
responses
in
five
to
seven
days.
EQUIPMENT:
Animal
husbandry,
photoperiod
control,
general
lab.
SPECIFICITY:
Measures
androgenic
effects
only.
STANDARDIZATION:
Good
potential.
RELATEDNESS:
Could
be
made
into
an
Androgen
antagonist
procedure,
with
the
co­
administration
of
testosterone
+
EDC,
and
using
testosterone
treatment
alone
as
a
control;
may
not
be
very
sensitive,
and
there
is
likely
to
be
a
threshold
effect
of
the
administered
testosterone,
making
the
test
for
an
antagonist
less
sensitive.
OTHER:
Almost
ready
to
go;
needs
testing
with
EDCs.

D.
Vitellogenin
Production
in
Female
Japanese
Quail
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
45
K
­
TEST
AND
FUNCTION:
Vitellogenenin
production
in
photo­
regressed
adult
females
to
look
at
the
estrogenic
effect
of
EDCs
on
peripheral
targets,
using
Japanese
quail.
REFERENCES:
[
Matrix
2]
DURATION:
Two
weeks
to
regress;
expose
for
two
days,
vitellogenin
response
in
24
hours.
EQUIPMENT:
Animal
husbandry,
photoperiod
control,
electrophoresis
lab.
STAFF
SKILLS:
As
above.

SPECIFICITY:
Specific
for
estrogen
binding
peripherally;
could
be
made
into
a
test
for
the
E­
antagonist
with
co­
administration
of
E2
and
the
potential
EDC,
along
with
E2
to
the
controls.
STANDARDIZATION:
Good
potential:
husbandry
can
be
made
standard,
but
a
common
source
of
antibody
is
needed;
ring
testing
probably
needed
for
electrophoresis
OTHER:
Needs
more
development
than
A3.
Could
be
used
as
a
procedure
for
estrogen
antagonists
with
co­
administration
of
estrogen
and
using
estrogen
alone
as
a
control.

E.
Vitellogenin
Production
in
Adult
Male
Birds:
Japanese
Quail,
Chickens.
REFERENCES:
Robinson,
G.
A.
and
A.
M.
V.
Gibbins,
"
Induction
of
vitellogenesis
in
Japanese
quail
as
a
sensitive
indicator
of
the
estrogen
mimetic
effect
of
a
variety
of
environmental
contaminants,"
Poultry
Science,
63,
1984,
pp.
1529­
1536.
(
and
other
references
)
Heppell,
S.
A.,
M.
D.
Denslow,
L.
C.
Folmar
and
C.
V.
Sullivan,
"
Universal
assay
of
vitellogenin
as
a
biomarker
of
environmental
estrogens,"
Environ.
Health
Perpsectives,
103
(
Suppl
7),
1996,
pp.
9­
15.
[
see
also:
Matter,
J.
M.,
A.
B.
Anthony,
K.
Alonso,
and
R.
L.
Dickerson,
"
TCDD
suppression
of
vitellogenin
synthesis
in
female
hens,"
Abstract.
17th
Annual
meeting
of
SETAC,
1996.]
DURATION:
Eight
to
ten
days.
EQUIPMENT:
Animal
facilities,
electrophoresis
equipment.
STAFF
SKILLS:
Animal
husbandry,
electrophoresis,
general
lab.
AVAILABILITY:
Depends
on
antibody:
birds
widely
available.
SENSITIVITY:
"
High",
but
o,
p­
DDT
did
not
induce
vitellogenesis.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
46
K
­
SPECIFICITY:
High.
OTHER:
Looks
pretty
good;
a
little
shorter
duration
than
A5
(
photo­
regressed
females)
but
relative
sensitivity
is
unknown.
Lots
of
current
work
on
different
species
of
wildlife
that
is
not
published
F.
Avian
"
Plaque
Assay"
TEST
AND
FUNCTION:
Avian
"
Plaque
assay";
slices
of
brain
are
prepared
in
vitro
and
infused
with
steroids,
peptides
or
EDCs
and
GNRH
production
is
measured.
Exploits
the
negative
feedback
control
of
steroids
on
GNRH
production.

REFERENCES:
Personal
communication;
Tom
Porter's
lab
at
Texas
A&
M;
reprints
in
the
mail
for
more
details.
AVAILABILITY:
Only
this
lab
uses
the
technique,
as
far
as
can
be
told.
OTHER:
This
assay
should
be
explored
as
it
is
one
of
the
only
in
vitro
assays
for
birds.
Drawbacks
include
killing
birds
for
brains,
independence
of
different
slices
from
the
same
brain
is
questionable,
and
the
expertise
rather
local,
I
believe.
Needs
development.

G.
Avian
Cell
Culture
TEST
AND
FUNCTION:
To
test
the
action
of
steroids
and
EDCs
on
the
handful
of
immortalized
avian
cell
lines.
In
general,
steroidogenic
endpoints
have
not
been
identified,
much
less
evaluated
for
sensitivity.
Further
only
one
avian
cell
line
(
fibroblasts)
is
spontaneously
immortal
and
not
chemically
or
virally
transformed,
hence
is
probably
the
best
for
results
relevant
to
an
intact
animal.
Again,
steroidogenic
responses
are
have
not
been
identified
in
these
fibroblasts
(
are
any
expected
in
a
fibroblast?).
REFERENCES:
Personal
communication,
Doug
Foster,
U
Minn.
AVAILABILITY:
Patented,
but
is
willing
to
license
for
this
use.
OTHER:
Needs
development,
but
promising.

H.
Chicken
Early
Life
Stage
TEST
AND
FUNCTION:
Coxcomb
size,
vent
sex
and
gonadal
morphology
and
histology
of
chickens;
EDCs
or
steroid
administered
by
injecting
the
eggs
before
organogenesis;
similar
to
B1
and
B2.
REFERENCES:
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
47
K
­
Elbrecht,
A.,
R.
G.
and
Smith,
"
Aromatase
enzyme
activity
and
sex
determination
in
chickens,"
Science,
225,
1992,
pp.
467­
470.
Snedecor,
J.
G.,
"
A
study
of
some
effects
of
sex
hormones
on
the
embryonic
reproductive
system
and
comb
of
the
white
leghorn
chick,"
J.
Exptl.
Zool.,
110,
1949,
pp.
205­
246.
­
and
others..
DURATION:
Long:
23
weeks
to
maturity,
but
for
morphology
of
the
reproductive
tract,
can
collect
material
from
hatchlings
after
21
day
incubation.
EQUIPMENT:
Animal
facilities,
RIA,
tissue
handling.
STAFF
SKILLS:
Husbandry,
RIA
techniques,
histological
imbedding
and
slicing.
AVAILABILITY:
No
problem.
SPECIFICITY:
For
Estrogenic
activity
in
males
chicks,
and
for
aromatase
inhibition
in
females;
aromatizable
androgen
has
no
effect
(
but
DHT,
and
hence
other
non­
aromatizable
androgens
?).
Needs
development
for
possible
A+
procedure.
STANDARDIZATION:
Easy
for
coxcomb,
subjective
assessments
of
gonadal
morphology
can
be
made
more
quantitative.
OTHER:
Coxcomb
assay
is
a
classic
and
has
been
widely
used
in
the
past,
but
it
is
very
time
consuming
as
maturity
is
reached
not
until
five
months;
however,
differences
can
be
seen
in
the
incompletely
mature
cock
and
may
be
useful.
A
nice
twist
exploited
by
Elbrecht
and
Smith
is
to
use
a
cross
that
produced
sexual
dimorphism
in
feather
color
which
was
independent
of
steroid
dependent
characters,
thus
revealing
genetic
sex.
In
some
studies
the
EDC
has
been
topically
applied
to
the
comb
directly.
Needs
development
for
possible
A+
procedure.

I.
Cartilage
Growth
in
Chick
DESCRIPTION
Determines
whether
a
chemical
can
alter
the
response
of
chick
cartilage
to
thyroid
hormone.
DEGREE
OF
USE
Extensive.
ASSAY
STABILITY
N/
A?
DOES
IT
METABOLIZE
TOXICANTS
Yes.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Modest.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
48
K
­
Yes,
but
training
period
is
not
extensive.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Unclear.
ARE
THERE
KNOWN
FALSE
POSITIVES
No.
OR
FALSE
NEGATIVES
Compounds
may
interfere
with
thyroid
hormone
action
without
affecting
cartilage.
SPECIFICITY
Unknown.

XVI.
Fish
Screens
A.
Vitellogenin
Assay
DESCRIPTION
An
assay
which
measures
the
amount
of
an
egg
yolk
protein
precursor
in
males
as
an
indicator
of
estrogenic
activity.
REFERENCES
Sumpter,
J.
P.,
"
The
purification,
radioimmunoassay
and
plasma
levels
of
vitellogenin
from
the
rainbow
trout.
Salmo
gairdneri.,"
Proceedings
of
the
Ninth
International
Symposium
on
Comparative
Endocrinology,
Hong
Kong
University
Press,
Hong
Kong,
1985,
pp.
335­
357.
Peterson,
G.
L.,
Determination
of
total
protein:
Methods
of
Enzymology,
99,
1993,
pp.
95­
121.
DURATION
>
72
hours.
EQUIPMENT
Wet
lab,
antibody,
immunology
lab.
STAFF
SKILLS
Can
be
trained.
COST
Modest.
AVAILABILITY
limited
by
specificity
of
antibody
although
Heppell
et
al.
are
developing
a
"
universal"
antibody.
SENSITIVITY
Good.
SPECIFICITY
Good
for
estrogen
activity,
but
thyroid
hormones
may
also
be
involved.
STANDARDIZATION
Not
yet,
but
could
be
made
without
undue
difficulties.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
49
K
­
RELATEDNESS
Primarily
estrogen
box,
more
research
is
needed
to
ascertain
whether
thyroid
or
androgen
activity
is
or
is
not
connected.

B.
In
Vitro
Vitellogenin
Assay
DESCRIPTION
A
procedure
isolating
trout
hepatocytes,
treating
them
with
a
xenobiotic,
and
then
measuring
the
amount
of
vitellogenin
secreted
into
the
culture
medium.
REFERENCES
Pelissero
et
al.,
"
Vitellogenin
synthesis
in
cultured
hepatocytes:
an
in
vitro
test
for
the
estrogenic
potency
of
chemicals,"
J.
Steroid
Biochemistry
and
Molecular
Biology
44,
1993,
pp.
263­
272.
EQUIPMENT
Immunology
lab.
STAFF
SKILLS
Moderate,
can
be
trained.
AVAILABILITY
Fair.
SENSITIVITY
Good.
SPECIFICITY
Good
for
estrogen
activity.
STANDARDIZATION
Practical.
RELATEDNESS
Applicable
to
estrogen
boxes
.

C.
In
Vivo
Screening
Assay
Refer
to
Peter
Thomas'
write
up.

D.
Early
Life
Stage
Test
DESCRIPTION
Newly
fertilized
eggs
are
exposed
to
a
test
chemical
through
hatching
and
early
development
and
growth
of
the
juvenile
fish.
Endpoints
measured
are
hatching
success,
survival,
and
growth.
REFERENCES
OECD
210,
EPA
850.1400,
ASTM
E
1241­
92.
DURATION
31
­
72
days.
EQUIPMENT
Wet
lab
sufficient
for
flow­
through
studies.
STAFF
SKILLS
General.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
50
K
­
AVAILABILITY
Commercially
available.
SENSITIVITY
Good.
SPECIFICITY
Poor.
STANDARDIZATION
Yes.
RELATEDNESS
Apical
for
all
boxes,
but
does
not
differentiate
hormonal
from
non­
hormonal
driven
effects
and
is
not
fully
comprehensive.

E.
Embryo
and
Sac
Fry
Test
DESCRIPTION
This
is
a
short­
term
test
in
which
the
life
stages
from
the
newly
fertilized
egg
to
the
end
of
the
sac­
fry
stage
are
exposed.
OECD
212.
DURATION
8
to
55
days.
EQUIPMENT
Wet
lab.
STAFF
SKILLS
General.
AVAILABILITY
Commercially
available.
SENSITIVITY
Unknown.
SPECIFICITY
None.
STANDARDIZATION
Yes.
RELATEDNESS
Apical
for
general
fish
health.

F.
Partial
Life
Cycle
Test
REFERENCES
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
APHA,
AWWA,
and
WPCF
810B,
1985,
p.
2.
DURATION
Long
term,
>
250
days.
EQUIPMENT
Wet
lab.
STAFF
SKILLS
Experienced.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
51
K
­
COST
High.
AVAILABILITY
Commercially
available.
SENSITIVITY
Good.
SPECIFICITY
Apical,
but
does
not
address
transgenerational
effects.
STANDARDIZATION
Fair.
RELATEDNESS
Applicable
to
all
boxes,
but
more
appropriate
in
definitive
testing
as
a
screen
it
should
only
be
used
if
the
test
has
already
been
completed.

G.
Full
Life
Cycle
Test
REFERENCE
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,
APHA,
AWWA,
and
WPCF,
810B,
1985,
p.
3.
DURATION
Long
term,
>
250
days.
EQUIPMENT
Wet
lab.
STAFF
SKILLS
Experienced.
COST
High.
AVAILABILITY
Commercially
available.
SENSITIVITY
Good.
SPECIFICITY
Apical,
but
does
not
address
transgenerational
effects.
STANDARDIZATION
Fair.
RELATEDNESS
Applicable
to
all
boxes,
but
more
appropriate
in
definitive
testing
as
a
screen
it
should
only
be
used
if
the
test
has
already
been
completed.

H.
Flounder
Metamorphosis
DESCRIPTION
Determines
whether
a
chemical
can
affect
the
thyroid
hormone­
dependent
process
of
metamorphosis.
DEGREE
OF
USE
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
52
K
­
Extensive.
DURATION
Seven
days.
ASSAY
STABILITY
N/
A?
DOES
IT
METABOLIZE
TOXICANTS
Yes.
ARE
SPECIAL
EQUIPMENT/
REQUIREMENTS
MANDATED
Modest.
ARE
SPECIAL
SKILLS/
TRAINING
NEEDED
Yes,
but
training
period
is
not
extensive.
HAS
IT
BEEN
OR
CAN
IT
BE
STANDARDIZED
EASILY
Yes.
SENSITIVITY
TO
LOW
DOSES
OR
WEAKLY
ACTIVE
CHEMICALS
Yes.
ARE
THERE
KNOWN
FALSE
POSITIVES
No.
OR
FALSE
NEGATIVES
Unclear.
SPECIFICITY
Unknown.
COMMENTS
The
ease
of
compound
administration
may
make
this
attractive.
The
use
of
±
T3/
T4
paradigm
would
allow
for
identification
of
thyroid
action
disruption.
Endpoints
(
e.
g.,
eye
migration)
are
ÒintegratedÓ
measures;
thus,
the
screen
would
detect
compounds
that
affect
thyroid
hormone
action
along
the
entire
pathway.

I.
In
Vitro
Steroid
Receptor
Competition
Assay
No
information
submitted.

J.
In
Vitro
Steroid
Production
Bioassay
No
information
submitted.

K.
In
Vitro
Germinal
Vesicle
Breakdown
(
GVBD)
Bioassay
No
information
submitted.

XVII.
Invertebrate
Screens
A.
Daphnia
Reproduction
(
Life
Cycle)
Test
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
53
K
­
DESCRIPTION
The
objective
of
this
test
is
to
assess
the
effect
of
a
chemical
on
the
reproductive
output
of
Daphnia
magna.
Information
on
growth
is
also
obtained.
REFERENCES
EPA
850.1300,
OECD
211,
ASTM
E
1193­
93.
DURATION
21
days.
EQUIPMENT
Wet
lab.
STAFF
SKILLS
Basic.
COST
Moderate.
AVAILABILITY
Commercially
available.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
54
K
­
SENSITIVITY
Unknown.
SPECIFICITY
Apical
for
invertebrate
endocrine
related
effects,
unknown
as
far
as
vertebrates
are
concerned.
STANDARDIZATION
Yes.
RELATEDNESS
B.
Mysid
Life
Cycle
Test
DESCRIPTION
The
objective
of
this
test
is
to
assess
the
survival,
growth,
and
reproduction
of
mysids
through
a
complete
life
cycle.
REFERENCES
EPA
850.1350,
ASTM
E
1191­
90
DURATION
28
days.
EQUIPMENT
Wet
lab,
saltwater
capability.
STAFF
SKILLS
Basic.
COST
Moderate.
AVAILABILITY
Commercially
available.
SENSITIVITY
Unknown.
SPECIFICITY
Apical
for
invertebrate
endocrine
related
effects,
unknown
as
far
as
vertebrates
are
concerned.
STANDARDIZATION
Yes.
RELATEDNESS
N/
A
XVIII.
Reptilian
Screens
A.
Vitellogenin
Production
In
Adult
Male
Turtles
TEST
AND
FUNCTION:
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
55
K
­
Vitellogenin
production
in
adult
male
turtles
(
Red­
eared
slider,
Trachemys
scripta)
and
male
frogs
(
Xenopus
laevis);
indicates
estrogen
receptor
binding
in
the
liver
and/
or
estrogen
receptor
production.
REFERENCES:
Palmer,
B.
D.
and
S.
K.
Palmer,
"
Vitellogenin
induction
by
xenobiotic
estrogens
in
the
Redeared
turtle
and
African
clawed
frog,"
Environ.
Health
Perspectives,
103
(
suppl
4),
1995,
pp.
19­
25.
(
and
others
in
the
bibliography)
DURATION:
Injection
daily
for
seven
days,
plasma
collected
on
day
14.
EQUIPMENT:
General
lab
space
for
animals,
equipment
for
ELISA.
STAFF
SKILLS:
ELISA.
COST:
Not
high;
turtles
and
frogs
purchased.
AVAILABILITY:
Animals
easily
obtainable.
SENSITIVITY:
Sliders
more
sensitive
than
frogs
when
comparing
DDT­
exposed
to
estradiol
controls,
but
frogs
equally
as
sensitive
as
sliders
when
comparing
the
absolute
of
vitellogenin
produced
in
response
to
DDT.
SPECIFICITY:
Estrogen­
dependant
response.
STANDARDIZATION:
Good
potential,
depends
on
common
source
of
antibody
.
OTHER:
Exact
same
protocol
used
for
amphibians;
possibly
useful
as
E­
procedure
with
coadministration
of
estrogen.

B.
Sex
Determination
in
Turtles
TEST
AND
FUNCTION:
Sex
determination
in
turtles;
the
red­
eared
slider
(
Trachemys
scripta)
and
snapping
turtles
(
Chelydra
serpentina)
have
be
used;
responses
measured
are
the
morphology
of
the
gonads
and
genital
ducts
and
the
histology
of
the
gonads.
REFERENCES:
Bergeron,
J.
M.,
D.
Crews,
and
J.
A.
McLaughlin,
"
PCBs
as
environmental
estrogens:
Turtle
sex
determination
as
biomarker
of
environmental
contamination,"
Environ
Health
Perspectives,
102,
1994,
pp.
780­
781.
Crews,
D.,
J.
M.
Bergeron,
and
J.
A.
McLaughlin,
"
The
role
of
estrogen
in
turtle
sex
determination
and
the
effects
of
PCBs,"
Environ
Health
Perpectives
103
(
suppl
7),
1994,
pp.
73­
77.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
56
K
­
Wibbles,
T.
and
D.
Crews,
"
Steroid­
induced
sex
determination
at
incubation
temperatures
producing
mixed
sex
rations
in
a
turtle
with
TSD,"
General
and
Comparative
Endocrinology
100,
1995,
pp.
53­
60.
DURATION:
Rather
long
~
12
weeks
to
hatch
and
collection
of
samples.
EQUIPMENT:
General
lab
facilities.
STAFF
SKILLS:
Tissue
handling.
AVAILABILITY:
Eggs
of
T.
scripta
are
commercially
available.
SENSITIVITY:
Incubation
temperature
is
crucial
and
needs
some
discussion
for
development
of
the
best
conditions
for
testing;
there
is
greater
physiological
sensitivity
to
steroids
at
incubation
temperatures
that
produce
mixed
sexes
from
the
clutch,
but
greater
statistical
sensitivity
when
clutch
is
incubated
at
male
producing
temperatures.
SPECIFICITY:
An
estrogen
dependant
alteration
of
male
gonads.
STANDARDIZATION:
?
Some
subjectivity
in
the
morphological
and
histological
analysis.
OTHER:
The
red­
eared
slider
(
Trachemys
scripta)
has
been
studied
most
extensively;
are
other
species
more
appropriate
or
available?
Again,
possibly
useful
as
a
E
antagonist
procedure
with
co­
administered
estrogen
at
male
temperatures,
OR
with
EDC
alone
at
female
temperatures.
This
needs
development,
but
would
be
worth
the
effort
as
so
much
of
the
basics
are
known
in
this
system.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
Appendix
L
Protocols
for
Tier
1
Screening
Assays
Many
of
the
following
draft
protocols
are
undergoing
internal
validation
within
NHEERL
laboratories
and
until
completion
of
this
process
they
should
be
considered
preliminary.
The
final
protocols
will
likely
differ
from
what
is
currently
presented.

Table
of
Contents
In
Vitro
Assays
(
presented
as
examples)
.
.
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1
I.
Rat
Estrogen
Receptor
Equilibrium
Exchange
Assay
.
.
.
.
.
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.
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.
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.
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.
.
.
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.
.
.
1
A.
Purpose
and
Applicability
.
.
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.
.
.
1
B.
Safety
and
Operating
Precautions
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
1
C.
Equipment
and
Materials
.
.
.
.
.
.
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.
.
1
D.
Methods
.
.
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.
2
II.
Protocol
to
Determine
Affinity
of
Compounds
for
the
Rat
AR
Androgen
Receptor
by
Equilibrium
Exchange
Assay
.
.
.
.
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.
5
A.
Purpose
and
Applicability
.
.
.
.
.
.
.
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.
.
.
5
B.
Safety
and
Operating
Precautions
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
.
.
5
C.
Equipment
and
Materials
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
5
D.
Methods
.
.
.
.
.
.
.
.
.
.
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.
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.
.
.
6
E.
Data
Processing
.
.
.
.
.
.
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.
10
F.
References
.
.
.
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.
11
III.
MVLN
Assay
.
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.
11
A.
Introduction
.
.
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.
11
B.
Maintenance
of
Cell
Stocks
.
.
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.
12
C.
MVLN
Assay
Setup
and
Time
Sequence
.
.
.
.
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.
.
.
.
13
D.
MVLN
Assay
Design
.
.
.
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.
.
14
E.
Example
Experiment
Setup
.
.
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.
14
F.
Example
Stock
and
Experiment
Schedule
.
.
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.
.
14
G.
Regarding
an
Estrogen
Free
Laboratory
Environment
.
.
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.
15
H.
Media
.
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.
15
I.
Sera
.
.
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.
15
J.
Buffers
.
.
.
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.
15
K.
References
.
.
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.
16
IV.
CV1
Cell
Transfections
.
.
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.
.
.
.
16
V.
Procedures
to
Assess
Individual
Steroidogenic
Enzyme
Activities
in
the
Rat
.
.
.
.
.
.
.
.
20
A.
Purpose
and
Applicability
.
.
.
.
.
.
.
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.
.
20
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
B.
Safety
and
Operating
Precautions
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
21
C.
Equipment
and
Materials
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
21
D.
Methods
.
.
.
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.
22
E.
Data
Processing
.
.
.
.
.
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.
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.
24
F.
References
.
.
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.
.
24
VI.
Research
Protocol
for
Assessment
of
Steroidogenesis
Using
In
Vitro
Testes
Culture
.
24
A.
50
Mg
Pieces
(
Rat
or
Other
Species)
.
.
.
.
.
.
.
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.
.
24
B.
Pieces
and
1/
4
Testes
(
Rat
And
Other
Species)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
25
C.
Stock
Solutions
and
Media
Preparation
.
.
.
.
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.
.
.
.
.
25
D.
Additional
Language
to
Consider
In
Developing
Steroidogenesis
Assay
Procedure
.
.
.
.
28
In
Vivo
Assays
.
.
.
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.
29
VII.
Research
Protocol
for
Assessment
of
Uterotropic
Activity
in
the
Adult
Ovariectomized
and
Juvenile
(
21
Day
Old)
Female
Rat
.
.
.
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.
29
A.
General
Conditions
.
.
.
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.
29
B.
Subjects
.
.
.
.
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.
.
29
VIII.
Research
Protocol
for
Assessment
of
Pubertal
Development
and
Thyroid
Function
in
Juvenile
(
21
Day
Old)
Female
Rats
.
.
.
.
.
.
.
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.
31
A.
General
Conditions
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
.
.
32
B.
Subjects
­
Juvenile
Female
Rats
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
32
C.
Experimental
Design
.
.
.
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33
D.
Treatment
.
.
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33
E.
Vaginal
Opening
.
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33
F.
Necropsy
.
.
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.
33
G.
Statistical
Analysis
.
.
.
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.
34
H.
Data
Summarization
.
.
.
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.
.
34
IX.
Research
Protocol
for
Assessment
of
(
Anti­)
Androgenic
Activity
in
the
Immature
Male
Rat:
The
"
Hershberger"
Assay
.
.
.
.
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.
34
A.
General
Conditions
.
.
.
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.
35
B.
Subjects
­
Castrated
Immature
Male
Rats
.
.
.
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.
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.
35
C.
Experimental
Design
.
.
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.
35
D.
Treatment
.
.
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36
E.
Necropsy
.
.
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.
36
F.
Statistical
Analysis
.
.
.
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.
36
G.
Data
Summarization
.
.
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.
36
X.
Fish
Gonadal
Recrudescence
Assay
.
.
.
.
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.
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.
37
A.
Scope
.
.
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.
37
B.
Introduction
.
.
.
.
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.
37
C.
Definitions
.
.
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.
37
D.
Principle
of
the
Test
.
.
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.
37
E.
Information
on
the
Test
Substance
.
.
.
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.
37
F.
Validity
of
the
Test
.
.
.
.
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.
.
.
38
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
G.
Description
of
the
Method
.
.
.
.
.
.
.
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.
38
H.
Procedure
.
.
.
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.
38
I.
References
.
.
.
.
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.
42
Alternative
In
Vivo
Assays
.
.
.
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.
43
XI.
Development
of
an
In
Vivo
Battery
for
Identifying
Endocrine
Modulators
in
Male
Crl:
Cd
Ò
Br
Rats
Protocol
.
.
.
.
.
.
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.
43
A.
Introduction
.
.
.
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.
43
B.
Study
Design
.
.
.
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.
43
C.
Materials
and
Methods
.
.
.
.
.
.
.
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.
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.
.
44
D.
Statistical
Analyses
.
.
.
.
.
.
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.
.
.
46
E.
Safety
And
Housekeeping
.
.
.
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.
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.
46
F.
References
.
.
.
.
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.
.
.
.
.
.
.
.
.
46
XII.
Development
of
an
In
Vivo
Battery
for
Identifying
Endocrine
Modulators
in
Female
Crl:
Cd
Ò
Br
Rats
Protocol
.
.
.
.
.
.
.
.
.
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.
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.
47
A.
Introduction
.
.
.
.
.
.
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.
47
C.
Materials
And
Methods
.
.
.
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.
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.
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.
48
D.
Statistical
Analyses
.
.
.
.
.
.
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.
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.
50
E.
Safety
And
Housekeeping
.
.
.
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.
.
50
XIII.
Research
Protocol
for
Assessment
of
Pubertal
Development
and
Thyroid
Function
in
Immature
(
33­
53
Day
Old)
Male
Rats
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
51
A.
General
Conditions
.
.
.
.
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.
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.
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.
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.
.
.
51
B.
Subjects
­
Peripubertal
Male
Rats
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
51
C.
Experimental
Design
.
.
.
.
.
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.
52
D.
Treatment
.
.
.
.
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.
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.
52
E.
Preputial
Separation
(
PPS)
.
.
.
.
.
.
.
.
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.
.
52
F.
Necropsy
.
.
.
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.
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.
.
52
G.
Statistical
Analysis
.
.
.
.
.
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.
.
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.
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.
.
53
H.
Data
Summarization
.
.
.
.
.
.
.
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.
.
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.
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.
.
53
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
1
L
­
In
Vitro
Assays
(
presented
as
examples)

I.
Rat
Estrogen
Receptor
Equilibrium
Exchange
Assay
A.
Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
a
procedure
for
the
quantitation
of
estrogen
receptor
number
and
binding
affinity
in
various
adult
female
rat
reproductive
tissues.
As
tissue
receptor
number
is
finite,
the
binding
of
ligand
to
the
receptor
(
i.
e.,
specific
binding)
is
a
saturable
process.
Unsaturable
binding
of
ligand
is
called
nonspecific
binding
and
is
due
to
ligand
binding
to
non­
receptor
proteins
etc.
Total
binding
is
saturable
binding
+
unsaturable
binding.
Total
and
nonspecific
binding
are
determined
empirically,
while
specific
binding
is
calculated
as
their
difference
(
i.
e.,
total­
nonspecific).
The
assay
described
below
measures
the
binding
of
radiolabelled
synthetic
ligand
(
i.
e.,
[
3H]­
E2)
by
cytosolic
and/
or
nuclear
receptor
extracts.
Total
[
3H]­
E2
binding
is
determined
by
incubating
the
extracts
with
increasing
concentrations
of
[
3H]­
E2
during
which
time
the
labelled
ligand
binds
to
the
unoccupied
receptors
in
the
cytosol
extract
or
exchanges
with
endogenous
hormone
bound
to
the
nuclear
receptors.
The
total
bound
ligand
(
i.
e.,
saturable
+
nonsaturable
binding)
is
separated
from
free
ligand
via
hydroxylapatite
extraction,
eluted
from
the
receptor
with
ethanol
and
quantified
using
liquid
scintillation
counting.
Nonspecific
binding
is
determined
exactly
as
above
except
that
a
100­
fold
molar
excess
of
radioinert
E2
is
included
in
each
incubation
together
with
the
increasing
concentrations
of
[
3H]­
E2
(
i.
e.,
binding
of
[
3H]­
E2
in
the
presence
of
a
100­
fold
molar
excess
of
radioinert
E2
represents
unsaturable
binding).
Specific
binding
is
calculated
as
total
­
nonspecific
binding
and
is
analyzed
graphically
via
Scatchard
analysis.

B.
Safety
and
Operating
Precautions
All
procedures
with
radioisotopes
should
follow
the
regulations
and
procedures
as
described
in
the
Hazardous
Agent
Protocol
(
HAP)
and
in
the
Radiation
Safety
Manual
and
Protocols.

C.
Equipment
and
Materials
1.
Equipment
·
Corning
Stir/
hot
Plates
·
Digital
Pipets
·
Balance
·
Polytron
PT
35/
10
Tissue
Homogenizer
·
Vacuum
Concentrator
·
Refrigerated
General
Laboratory
Centrifuge
·
High­
Speed
Refrigerated
Centrifuge
(
up
to
105,000
x
g)
·
pH
Meter
with
Tris
Compatible
Electrode
·
Scintillation
Counter
2.
Chemicals
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
2
L
­
·
Tris
HCL
&
Tris
Base
·
Glycerol
99%+
·
Ethylenediaminetetraacetic
acid
(
EDTA);
Disodium
salt
·
Dithiothreitol
(
DTT)
·
Potassium
Chloride
·
Hydroxylapatite
(
BIO­
RAD)
·
Scintillation
Cocktail
(
Flow
Scint
III)
·
Ethyl
Alcohol,
anhydrous
·
[
3H]­
Estradiol
&
Radioinert
Estradiol
(
NEN)
·
Triamcinolone
Acetonide
·
Steroids
(
Steraloids
­
recrystallized)
3.
Supplies
·
20
ml
Polypropylene
Scintillation
Vials
·
12
x
75
mm
Borosilicate
Glass
Test
Tubes
·
1000
ml
graduated
cylinders
·
500
ml
Erlenmeyer
flasks
·
Yellow
(
0­
200
ml)
pipet
tips
D.
Methods
1.
Preparation
of
TEDG
buffer
·
To
make
250ml:
·
Add
to
150ml
distilled
water:
·
.303g
Tris
base
·
.140g
EDTA
·
25ml
glycerol
·
Bring
volume
to
250
ml
and
stir.
·
pH
to
7.6
·
Add
.04g
DTT
before
use
2.
Preparation
of
50
mM
TRIS
Buffer
Add
50.0
ml
1.0
M
TRIS
to
950
ml
ddH
2
O.
Store
at
4o
C.
Check
pH
of
the
final
solution
to
make
sure
it
is
7.4
at
4o
C.

3.
Preparation
of
60%
Hydroxylapatite
(
HAP)
Slurry
Shake
BIO­
RAD
HT­
GEL
until
all
the
HAP
is
in
suspension
(
i.
e.,
looks
like
milk).
The
evening
before
the
receptor
extraction,
pour
100
mls
(
or
an
appropriate
volume)
into
a
100
ml
graduated
cylinder,
parafilm
seal
the
top
and
place
in
the
refrigerator
for
at
least
2hours.
Pour
off
the
phosphate
buffer
supernatant,
and
bring
the
volume
to
100
mls
with
50
mM
TRIS.
Suspend
the
HAP
by
parafilm
sealing
the
top
of
the
graduated
cylinder
and
inverting
the
cylinder
several
times.
Place
in
the
refrigerator
overnight.
The
next
morning,
repeat
the
washing
steps
two
times
with
fresh
50
mM
TRIS
buffer.
After
the
last
wash,
add
enough
50
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
3
L
­
mM
TRIS
to
make
the
final
solution
a
60%
slurry
(
i.
e.,
if
the
volume
of
the
settled
HAP
is
60
ml
bring
the
final
volume
of
the
slurry
to
100
mls
with
50
mM
TRIS).
Store
at
4o
C
until
ready
for
use
in
the
extraction.

4.
Standard
Curve
Construction
for
Saturation
and
Scatchard
Analysis
The
first
step
is
to
pipet
the
radioactive
ligand
(
i.
e.,
[
3H]­
E2)
with
and
without
a
100­
fold
excess
of
radioinert
E2
into
each
tube
so
that
the
final
concentrations
of
[
3H]­
E2
are
6,3,1,.
6,.
3,.
1,.
03,.
01
nM
in
a
300
ml
total
volume.
To
accomplish
this,
label
tubes
and
pipet
the
following
into
separate
12
x
75
mm
glass
test
tubes.

Total
Binding
Tubes
Nonspecific
Binding
Tubes
(+)

6
nM=
21
ml
of
10­
7
[
3H]­
E2
±
21
ml
of
10­
5
radioinert
E2
3
nM=
10.5
ml
of
10­
7
[
3H]­
E2
±
10.5
ml
of
10­
5
radioinert
E2
1
nM=
35
ml
of
10­
8
[
3H]­
E2
±
35
ml
of
10­
6
radioinert
E2
.6
nM=
21
ml
of
10­
8
mM
[
3H]­
E2
±
21
ml
of
10­
6
radioinert
E2
.3
nM=
10.5
ml
of
10­
8
[
3H]­
E2
±
10.5
ml
of
10­
6
radioinert
E2
.1
nM=
35
ml
of
10­
9
[
3H]­
E2
±
35
ml
of
10­
7
radioinert
E2
.03
nM=
10.5
ml
of
10­
9
[
3H]­
E2
±
10.5
ml
of
10­
7
radioinert
E2
.01
nM
=
3.5
ml
of
10­
9
[
3H]­
E2
±
3.5
ml
of
10­
7
radioinert
E2
After
the
tubes
have
been
pipetted
as
above,
place
them
in
the
speed­
vac
and
dry
the
tubes
according
to
the
instructions
in
the
speed­
vac
SOP.
When
tubes
are
dry
place
them
in
an
icewater
bath
and
cover
with
aluminum
foil
until
ready
to
pipet
the
nuclear
or
cytosolic
extract
into
each
tube
for
incubation.
(
Total
tubes
have
hot
only
and
NSB
tubes
have
hot
+
cold.)

5.
Estrogen
Receptor
Assay
Procedure
·
Make
TEDG
buffer
and
place
in
an
ice­
water
bucket.
·
Kill
the
rat
and
excise
uterus.
Tissues
should
be
trimmed
of
fat,
weighed
and
the
weights
recorded.
Place
on
dry
ice.
Before
assay,
place
tissues
into
a
homogenizing
tube
in
an
icewater
bath
and
add
TEDG
buffer
at
1.0
ml/
50mg
tissue.
It
is
extremely
important
to
keep
the
tissues/
extracts
at
4o
C
at
all
times.
·
Mince
tissues
with
Metzenbaum
scissors
until
all
pieces
are
small
1­
2mm
cubes.
Then
homogenize
the
tissues
at
4o
C
with
a
Polytron
homogenizer
using
five
second
bursts
of
the
Polytron.
[
Note:
place
probe
of
the
Polytron
in
an
ice­
water
bath
with
TEDG
buffer
to
cool
it
down
prior
to
its
use
for
homogenization]
·
Transfer
homogenates
to
pre­
cooled
centrifuge
tubes,
balance,
and
centrifuge
at
105,000
x
g
for
60
minutes
(
i.
e.,
35,000
rpm
using
90
Ti
Beckman
rotor).
The
supernatant
is
the
low­
salt
unoccupied
cytosolic
receptors.
·
While
these
tubes
are
spinning,
the
tubes
containing
[
3H]­
E2
±
radioinert
E2
for
the
saturation
experiment
can
be
pipetted
as
described
in
section
4.4
above.
Dry
these
tubes
in
the
speed­
vac,
and
place
in
a
rack
in
an
ice­
water
bath
as
indicated.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
4
L
­
·
Pipet
300
ml
of
the
cytosolic
extract
into
each
of
the
total
and
nonspecific
binding
tubes
containing
the
[
3H]­
E2
and
[
3H]­
E2
+
radioinert
E2,
respectively.
Gently
vortex
the
samples
and
place
them
in
the
refrigerator
overnight
in
rotor
covered
with
foil
for
20
hours.
·
Before
leaving
for
the
day,
prepare
the
first
wash
of
the
HAP
slurry
as
described
in
section
4.3
above.
Also,
label
the
HAP
tubes
and
the
scintillation
vials
to
be
used
the
following
day
­
see
underlines
below.
·
The
following
morning,
wash
the
HAP
as
described
above,
dilute
with
50
mM
TRIS
to
yield
a
60%
slurry,
and
transfer
contents
to
a
100
ml
Erlenmeyer
flask.
Place
a
stir
bar
in
the
flask
and
place
the
flask
into
a
beaker
containing
ice­
water;
stir
the
HAP
slurry
by
placing
the
beaker
on
a
magnetic
stir
plate.
·
While
the
HAP
slurry
is
constantly
being
stirred,
pipet
500
ml
of
the
HAP
slurry
into
clean
pre­
labelled
12
x
75
mm
glass
test
tubes.
These
tubes
should
be
prepared
in
duplicate
(
i.
e.,
two
tubes
for
every
incubation
tube).
Place
these
tubes
in
a
rack
in
an
ice­
water
bath
prior
to
pipetting
the
HAP
slurry
and
keep
them
in
the
ice­
water
bath
for
the
remainder
of
the
assay.
·
Take
the
incubation
tubes
from
the
refrigerator
and
place
them
in
an
ice­
water
bath
with
the
HAP
tubes.
Pipet
100
ml
in
duplicate
from
each
of
the
incubation
tubes
into
the
appropriate
pre­
labelled
tubes
containing
HAP.
Repeat
for
all
tubes.
Quickly
take
each
rack
from
the
ice­
water
bath
and
vortex
each
rack
of
tubes
using
the
whole­
rack
vortex
unit.
Place
racks
back
into
the
ice­
water
bath
and
vortex
as
above
every
five
minutes
for
20
minutes.
·
During
the
above
20
minute
HAP
extraction,
pipet
30
ml
in
duplicate
from
the
remaining
100
ml
in
each
incubation
tube
into
pre­
labelled
20
ml
scintillation
vials.
These
tubes
will
be
used
to
estimate
the
concentration
of
total
[
3H]­
E2
used
in
the
equilibrium
assay
and
will
be
called
the
total
counts
tubes.
The
extraction
tubes
containing
the
remaining
40
ml
of
incubate
can
be
discarded
into
the
plastic
radiation
safety
barrel
together
with
some
sawdust
to
adsorb
the
excess
liquid.
·
Centrifuge
the
HAP
tubes
for
two
to
three
minutes
at
4o
C
and
600
x
g
(
1780
rpm
in
a
Beckman
GLC
refrigerated
centrifuge).
Place
the
tubes
back
into
the
rack
and
into
the
icewater
bath.
·
While
the
tubes
remain
in
the
ice­
water
bath,
aspirate
the
supernatant
from
each
tube
using
a
9­
inch
pipet
connected
to
an
aspiration
apparatus
as
per
the
radiation
safety
protocol.
·
Add
2
ml
of
50
mM
TRIS
to
each
tube,
vortex
and
centrifuge
at
600
x
g
as
above.
Place
the
tubes
into
decanting
racks
in
an
ice­
water
bath
and
decant
the
supernatant
TRIS
wash
into
the
radiation
safety
container.
Gently
tap
the
tube
openings
on
a
clean
adsorbent
diaper,
place
the
rack
back
in
the
ice­
water
bath
and
add
2
mls
of
50
mM
TRIS.
Repeat
the
TRIS
washing
procedure
three
or
four
times
(
to
be
determined
empirically)
keeping
the
tubes
at
4oC
at
all
times.
·
Following
the
last
wash
and
decanting,
add
2
mls
of
ethanol
to
each
tube,
vortex
three
times
at
five
minute
intervals
and
centrifuge
the
tubes
at
600
x
g
for
ten
minutes.
Decant
the
supernatants
into
pre­
labelled
20
ml
scintillation
vials.
Add
14
ml
of
Optifluor
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
5
L
­
scintillation
cocktail
and
count
samples
using
the
single
label
DPM
program
with
quench
correction.

II.
Protocol
to
Determine
Affinity
of
Compounds
for
the
Rat
AR
Androgen
Receptor
by
Equilibrium
Exchange
Assay
A.
Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
a
procedure
for
the
quantitation
of
androgen
receptor
number
and
binding
affinity
in
various
adult
male
rat
reproductive
tissues.
As
tissue
receptor
number
is
finite,
the
binding
of
ligand
to
the
receptor
(
i.
e.,
specific
binding)
is
a
saturable
process.
Unsaturable
binding
of
ligand
is
called
nonspecific
binding
and
is
due
to
ligand
binding
to
non­
receptor
proteins
etc.
Total
binding
is
saturable
binding
+
unsaturable
binding.
Total
and
nonspecific
binding
are
determined
empirically,
while
specific
binding
is
calculated
as
their
difference
(
i.
e.,
total­
nonspecific).
The
assay
described
below
measures
the
binding
of
radiolabelled
synthetic
ligand
(
i.
e.,
[
3H]­
R1881)
by
cytosolic
and/
or
nuclear
receptor
extracts.
Total
[
3H]­
R1881
binding
is
determined
by
incubating
the
extracts
with
increasing
concentrations
of
[
3H]­
R1881
during
which
time
the
labelled
ligand
binds
to
the
unoccupied
receptors
in
the
cytosol
extract
or
exchanges
with
endogenous
hormone
bound
to
the
nuclear
receptors.
The
total
bound
ligand
(
i.
e.,
saturable
+
nonsaturable
binding)
is
separated
from
free
ligand
via
hydroxylapatite
extraction,
eluted
from
the
receptor
with
ethanol
and
quantified
using
liquid
scintillation
counting.
Nonspecific
binding
is
determined
exactly
as
above
except
that
a
100­
fold
molar
excess
of
radioinert
R1881
is
included
in
each
incubation
together
with
the
increasing
concentrations
of
[
3H]­
R1881
(
i.
e.,
binding
of
[
3H]­
R1881
in
the
presence
of
a
100­
fold
molar
excess
of
radioinert
R1881
represents
unsaturable
binding).
Specific
binding
is
calculated
as
total
­
nonspecific
binding
and
is
analyzed
graphically
via
Scatchard
analysis.

B.
Safety
and
Operating
Precautions
All
procedures
with
radioisotopes
should
follow
the
regulations
and
procedures
as
described
in
the
Hazardous
Agent
Protocol
(
HAP)
and
in
the
Radiation
Safety
Manual
and
Protocols.

C.
Equipment
and
Materials
1.
Equipment
·
Corning
Stir/
hot
Plates
·
Digital
Pipets
·
Balance
·
Polytron
PT
35/
10
Tissue
Homogenizer
·
Hewlett­
Packard
HPLC
with
on­
line
Radiomatic
Radiochromatograph
·
Vacuum
Concentrator
·
Hamilton
Syringes
(
50
ml)
·
Refrigerated
General
Laboratory
Centrifuge
·
High­
Speed
Refrigerated
Centrifuge
(
up
to
30,000
x
g)
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
6
L
­
·
pH
Meter
with
Tris
Compatible
Electrode
·
Scintillation
Counter
2.
Chemicals
·
Tris
HCL
&
Tris
Base
·
Phenylmethylsulfonyl
Fluoride
(
PMSF)
·
Glycerol
99%+
·
Sodium
Molybdate
·
Ethylenediaminetetraacetic
acid
(
EDTA);
Disodium
salt
·
Dithiothreitol
(
DTT)
·
Potassium
Chloride
·
Hydroxylapatite
(
BIO­
RAD)
·
Scintillation
Cocktail
(
Flow
Scint
III)
·
Ethyl
Alcohol,
anhydrous
·
[
3H]­
R1881
&
Radioinert
R1881
(
NEN)
·
Triamcinolone
Acetonide
·
Steroids
(
Steraloids
­
recrystallized)
3.
Supplies
·
20
ml
Polypropylene
Scintillation
Vials
·
12
x
75
mm
Borosilicate
Glass
Test
Tubes
·
1000
ml
graduated
cylinders
·
500
ml
Erlenmeyer
flasks
·
Yellow
(
0­
200
ml)
pipet
tips
D.
Methods
1.
Preparation
of
TEDG
Stock
Solutions
·
Add
7.444g
disodium
EDTA
to
100
ml
ddH
2
O
=
200mM.
Store
at
4o
C.
Use
750
ml/
100ml
TEDG
buffer
=
1.5
mM.
·
Add
1.742
g
PMSF
to
100
ml
ethanol
=
100
mM.
Store
at
4o
C.
Use
1.00
ml/
100ml
TEDG
buffer
=
1.0
mM.
·
Add
2.419
g
sodium
molybdate
to
8.0
ml
ddH
2
O
in
a
10
ml
volumetric
flask;
bring
the
total
volume
to
10
mls
=
1.0
M.
Store
at
4o
C.
Use
100ml/
100ml
TEDG
buffer
=
1.0
mM.
·
Add
15.4
mg
DTT
directly
to
100
ml
TEDG
buffer
the
morning
of
the
receptor
isolation
=
1.0
mM.
Add
147.24
g
Tris­
HCL
+
8.0
g
Tris
base
to
800mls
ddH
2
O
in
a
volumetric
flask;
bring
the
final
volume
to
1.0
liter.
Refrigerate
to
4o
C
and
pH
(
using
4oC
pH
standardizing
solutions)
the
cooled
solution
to
7.4.
Store
at
4o
C.
Use
1.0
ml/
100
ml
TEDG
buffer
=
10mM.
·
Add
298.2
g
KCL
to
600
ml
ddH
2
O
in
a
1000
ml
volumetric
flask;
bring
the
total
volume
to
1000
ml
=
4.0
M.
Store
at
room
temperature.
Use
10.0
ml
per
100
ml
high­
salt
TEDG
buffer
=
0.4M.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
7
L
­
2.
Preparation
of
Low­
Salt
TEDG
Buffer
(
pH
7.4)
To
make
100
mls
of
low­
salt
TEDG
buffer
add
the
following
together
in
this
order:
·
87.15
ml
ddH
2
O
·
1.0
ml
1M
TRIS
·
10.0
ml
glycerol
·
100
ml
1M
sodium
molybdate
·
750
ml
200mM
EDTA
·
1.0
ml
100mM
PMSF
·
15.4
mg
DTT
Check
pH
of
the
final
solution
to
make
sure
it
is
7.4
at
4o
C.

3.
Preparation
of
High­
Salt
TEDG
Buffer
(
pH
7.4)
To
make
100
mls
of
high­
salt
TEDG
buffer
add
the
following
together
in
this
order:
·
77.15
ml
ddH
2
O
·
10
ml
4.0
M
KCL
·
1.0
ml
1M
TRIS
·
10.0
ml
glycerol
·
100
ml
1M
sodium
molybdate
·
750
ml
200mM
EDTA
·
1.0
ml
100mM
PMSF
·
15.4
mg
DTT
Check
pH
of
the
final
solution
to
make
sure
it
is
7.4
at
4o
C.

4.
Preparation
of
50
mM
TRIS
Buffer
Add
50.0
ml
1.0
M
TRIS
to
950
ml
ddH
2
O.
Store
at
4o
C.
Check
pH
of
the
final
solution
to
make
sure
it
is
7.4
at
4o
C.

5.
Preparation
of
60%
Hydroxylapatite
(
HAP)
Slurry
Shake
BIO­
RAD
HT­
GEL
until
all
the
HAP
is
in
suspension
(
i.
e.,
looks
like
milk).
The
evening
before
the
receptor
extraction,
pour
100
mls
(
or
an
appropriate
volume)
into
a
100
ml
graduated
cylinder,
parafilm
seal
the
top
and
place
in
the
refrigerator
for
at
least
two
hours.
Pour
off
the
phosphate
buffer
supernatant,
and
bring
the
volume
to
100mls
with
50
mM
TRIS.
Suspend
the
HAP
by
parafilm
sealing
the
top
of
the
graduated
cylinder
and
inverting
the
cylinder
several
times.
Place
in
the
refrigerator
overnight.
The
next
morning,
repeat
the
washing
steps
two
times
with
fresh
50
mM
TRIS
buffer.
After
the
last
wash,
add
enough
50
mM
TRIS
to
make
the
final
solution
a
60%
slurry
(
i.
e.,
if
the
volume
of
the
settled
HAP
is
60
ml
bring
the
final
volume
of
the
slurry
to
100
mls
with
50
mM
TRIS).
Store
at
4o
C
until
ready
for
use
in
the
extraction.

6.
Preparation
of
[
3H­
17a­
Methyl]­
R1881
Stock
Solutions
Dilute
the
original
1.0
mCi/
ml
stock
of
[
3H­
17a­
methyl]­
R1881
to
0.1
mM
(
i.
e.,
1
x
10­
7
M).
This
is
most
easily
accomplished
by
pipeting
1
ml
of
the
stock
solution
for
every
specific
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
8
L
­
activity
unit
(
Ci/
mmol)
and
diluting
this
to
10.0
mls
with
ethanol.
Thus,
if
the
specific
activity
of
the
stock
vial
is
86
Ci/
mmol,
then
pipet
86.0
ml
into
an
amber
colored
vial
(
i.
e.,
R1881
is
photosensitive)
and
add
10.0
mls
ethanol
to
the
vial;
this
solution
is
1
x
10­
7M.

Calculation
Check
86
ml
x
1.0
mCi/
1000ml
=
86
x
10­
3
mCi
R1881
=
86
x
10­
6
Ci
R1881
86
x
10­
6
Ci
¸
86.0
Ci/
mmol
=
1
x
10­
6
mmol
R1881
=
1
x
10­
9
moles
R1881
1
x
10­
9
moles
R1881
¸
.010
liters
=
1
x
10­
7
moles/
liter
=
0.1
mM
To
prepare
the
1
x
10­
8M
stock
simply
make
a
10­
fold
dilution
of
the
1
x
10­
7M
stock
(
i.
e.,
pipet
1.0
ml
of
the
1
x
10­
7
M
stock
into
a
clean
amber
colored
vial
and
add
9
mls
ethanol
=
0.01
mM).

7.
Preparation
of
100X
Radioinert
R1881
Solutions
The
R1881
comes
as
a
5.00
mg
quantity.
Dilute
the
original
stock
to
5.0
ml
with
ethanol
=
3.52
mM.
Take
56.82
ml
and
dilute
to
20
ml
in
an
amber
vial
with
ethanol
=
1
x
10­
5
M
R1881.
This
is
the
10
mM
radioinert
R1881
stock.
To
make
the
1.0
mM
radioinert
R1881
stock,
pipet
2
ml
of
the
10
mM
stock
into
an
amber
vial
and
dilute
to
20
ml
with
ethanol
=
1
x
10­
6M
=
1.0
mM
radioinert
R1881
stock.

8.
Standard
Curve
Construction
for
Saturation
and
Scatchard
Analysis
The
first
step
is
to
pipet
the
radioactive
ligand
(
i.
e.,
[
3H]­
R1881)
with
and
without
a
100­
fold
excess
of
radioinert
R1881
into
each
tube
so
that
the
final
concentrations
of
[
3H]­
R1881
are
20,
16,
12,
8,
4,
2,
1,
0.5
nM
in
a
300
ml
total
volume.
To
accomplish
this,
label
tubes
and
pipet
the
following
into
separate
12
x
75
mm
glass
test
tubes.

Total
Binding
Tubes
Nonspecific
Binding
Tubes
a)
20nM=
60
ml
of
0.1
mM
[
3H]­
R1881
±
60
ml
of
10
mM
radioinert
R1881
b)
16nM=
48
ml
of
0.1
mM
[
3H]­
R1881
±
48
ml
of
10
mM
radioinert
R1881
c)
12nM=
36
ml
of
0.1
mM
[
3H]­
R1881
±
36
ml
of
10
mM
radioinert
R1881
d)
8nM=
24
ml
of
0.1
mM
[
3H]­
R1881
±
24
ml
of
10
mM
radioinert
R1881
e)
4nM=
12
ml
of
0.1
mM
[
3H]­
R1881
±
12
ml
of
10
mM
radioinert
R1881
f)
2
nM=
60
ml
of
0.01
mM
[
3H]­
R1881
±
60
ml
of
1.0
mM
radioinert
R1881
g)
1
nM=
30
ml
of
0.01
mM
[
3H]­
R1881
±
30
ml
of
1.0
mM
radioinert
R1881
h)
.5
nM=
15
ml
of
0.01
mM
[
3H]­
R1881
±
15
ml
of
1.0
mM
radioinert
R1881
After
the
tubes
have
been
pipetted
as
above,
place
them
in
the
speed­
vac
and
dry
the
tubes
according
to
the
instructions
in
the
speed­
vac
SOP.
When
tubes
are
dry
place
then
in
an
icewater
bath
and
cover
with
aluminum
foil
until
ready
to
pipet
the
nuclear
or
cytosolic
extract
into
each
tube
for
incubation.

9.
Androgen
Receptor
Assay
Procedure
·
Make
low­
and
high­
salt
TEDG
buffer
and
place
in
an
ice­
water
bucket.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
9
L
­
·
Kill
the
rat
and
excise
epididymis,
ventral
prostate,
and/
or
seminal
vesicles.
Tissues
should
be
trimmed
of
fat,
weighed
and
the
weights
recorded.
As
quickly
as
possible,
place
the
tissues
into
a
homogenizing
tube
in
an
ice­
water
bath
and
add
low­
salt
TEDG
buffer
at
10
ml/
gm
tissue.
It
is
extremely
important
to
keep
the
tissues/
extracts
at
4o
C
at
all
times.
·
Mince
tissues
with
Metzenbaum
scissors
until
all
pieces
are
small
1­
2mm
cubes.
Then
homogenize
the
tissues
at
4o
C
with
a
Polytron
homogenizer
using
five
second
bursts
of
the
Polytron.
[
Note:
place
probe
of
the
Polytron
in
an
ice­
water
bath
with
TEDG
buffer
to
cool
it
down
prior
to
its
use
for
homogenization]
·
Transfer
homogenates
to
pre­
cooled
centrifuge
tubes,
balance,
and
centrifuge
at
30,000
x
g
for
30
minutes
(
i.
e.,
15,262
rpm
using
JA­
17/
JA­
21
Beckman
rotors).
·
The
supernatant
is
the
low­
salt
unoccupied
cytosolic
receptors
and
this
material
can
be
assayed
directly
or
frozen
at
­
70o
C
for
future
analysis.
·
Add
high­
salt
TEDG
at
10
ml/
gm
original
tissue
weight
and
homogenize
the
pellet
thoroughly
at
15
minute
intervals
for
one
hour
(
i.
e.,
four
times).
This
procedure
extracts
the
occupied
androgen
receptor
from
the
nucleus.
·
Balance
the
tubes
and
centrifuge
at
30,000
x
g
for
30
minutes
at
4o
C.
While
these
tubes
are
spinning,
the
tubes
containing
[
3H]­
R1881
±
radioinert
R1881
for
the
saturation
experiment
can
be
pipetted
as
described
in
section
4.8
above.
Dry
these
tubes
in
the
speed­
vac,
and
place
in
a
rack
in
an
ice­
water
bath
as
indicated.
·
Transfer
the
supernatant
from
the
nuclear
extract
to
a
16
x
100
mm
polypropylene
tube
and
place
in
an
ice­
water
bath.
Pipet
300
ml
of
the
cytosolic
or
nuclear
extract
into
each
of
the
total
and
nonspecific
binding
tubes
containing
the
[
3H]­
R1881
and
[
3H]­
R1881
+
radioinert
R1881,
respectively.
Gently
vortex
the
samples
and
place
them
in
the
refrigerator
overnight
for
20
hours.
·
Before
leaving
for
the
day,
prepare
the
first
wash
of
the
HAP
slurry
as
described
in
section
4.5
above.
Also,
label
the
HAP
tubes
and
the
scintillation
vials
to
be
used
the
following
day
­
see
underlines
below.
·
The
following
morning,
wash
the
HAP
as
described
in
section
4.5
above,
dilute
with
50
mM
TRIS
to
yield
a
60%
slurry,
and
transfer
contents
to
a
100
ml
Erlenmeyer
flask.
Place
a
stir
bar
in
the
flask
and
place
the
flask
into
a
beaker
containing
ice­
water;
stir
the
HAP
slurry
by
placing
the
beaker
on
a
magnetic
stir
plate.
·
While
the
HAP
slurry
is
constantly
being
stirred,
pipet
500
ml
of
the
HAP
slurry
into
clean
pre­
labelled
12
x
75
mm
glass
test
tubes.
These
tubes
should
be
prepared
in
duplicate
­
i.
e.,
two
tubes
for
every
incubation
tube.
Place
these
tubes
in
a
rack
in
an
ice­
water
bath
prior
to
pipetting
the
HAP
slurry
and
keep
them
in
the
ice­
water
bath
for
the
remainder
of
the
assay.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
10
L
­
·
Take
the
incubation
tubes
from
the
refrigerator
and
place
them
in
an
ice­
water
bath
with
the
HAP
tubes.
Pipet
100
ml
in
duplicate
from
each
of
the
incubation
tubes
into
the
appropriate
pre­
labelled
tubes
containing
HAP.
Repeat
for
all
tubes.
Quickly
take
each
rack
from
the
ice­
water
bath
and
vortex
each
rack
of
tubes
using
the
whole­
rack
vortex
unit.
Place
racks
back
into
the
ice­
water
bath
and
vortex
as
above
every
five
minutes
for
20
minutes.
·
During
the
above
20
minute
HAP
extraction,
pipet
30
ml
in
duplicate
from
the
remaining
100
ml
in
each
incubation
tube
into
pre­
labelled
20
ml
scintillation
vials.
These
tubes
will
be
used
to
estimate
the
concentration
of
total
[
3H]­
R1881
used
in
the
equilibrium
assay
and
will
be
called
the
total
counts
tubes.
The
extraction
tubes
containing
the
remaining
40
ml
of
incubate
can
be
discarded
into
the
plastic
radiation
safety
barrel
together
with
some
sawdust
to
adsorb
the
excess
liquid.
·
Centrifuge
the
HAP
tubes
for
two
to
three
minutes
at
4o
C
and
600
x
g
(
1780
rpm
in
a
Beckman
GLC
refrigerated
centrifuge).
Place
the
tubes
back
into
the
rack
and
into
the
ice­
water
bath.
·
While
the
tubes
remain
in
the
ice­
water
bath,
aspirate
the
supernatant
from
each
tube
using
a
nine
inch
pipet
connected
to
an
aspiration
apparatus
as
per
the
radiation
safety
protocol.
·
Add
2
ml
of
50
mM
TRIS
to
each
tube,
vortex
and
centrifuge
at
600
x
g
as
above.
Place
the
tubes
into
decanting
racks
in
an
ice­
water
bath
and
decant
the
supernatant
TRIS
wash
into
the
radiation
safety
container.
Gently
tap
the
tube
openings
on
a
clean
adsorbent
diaper,
place
the
rack
back
in
the
ice­
water
bath
and
add
2
mls
of
50
mM
TRIS.
Repeat
the
TRIS
washing
procedure
three
or
four
times
(
to
be
determined
empirically)
keeping
the
tubes
at
4o
C
at
all
times.
·
Following
the
last
wash
and
decanting,
add
2
mls
of
ethanol
to
each
tube,
vortex
three
times
at
five
minute
intervals
and
centrifuge
the
tubes
at
600
x
g
for
ten
minutes.
Decant
the
supernatants
into
pre­
labelled
20
ml
scintillation
vials.
Add
14
ml
of
Optifluor
scintillation
cocktail
and
count
samples
using
the
single
label
DPM
program
with
quench
correction.

E.
Data
Processing
1.
Free
Concentration
of
[
3H]­
R1881
Multiply
the
DPM
in
the
total
counts
tubes
by
1.8047
x
10­
5.
This
value
will
yield
the
free
concentration
(
i.
e.,
nM)
of
[
3H]­
R1881
initially
present
in
each
incubation
tube.

Calculation
Check
X
DPM
=
4.5045
x
10­
13
Ci
=
5.4141
x
10­
15
mmole
=
5.4141
x
10­
18
moles
2.22
x
1012
dpm/
Ci
83.2
Ci/
mmole
1000
mmole/
mole
0.0003
liters
=
1.8047
x
10­
14
moles/
liter
=
X
(
1.8047
x
10­
5)
nM
1
x
10­
9
moles/
nmole
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
11
L
­
2.
Calculation
of
Total,
Nonspecific
and
Specific
[
3H]­
R1881
Binding
·
Total
binding
is
calculated
by
multiplying
the
DPM
from
the
tubes
that
contained
only
radiolabelled
R1881
x
(
1.6242
x
10­
2).
This
value
will
be
total
binding
in
fmoles.
·
Nonspecific
binding
is
calculated
by
multiplying
the
DPM
from
the
tubes
containing
radiolabelled
R1881
+
100­
fold
molar
excess
of
radioinert
R1881
x
(
1.6242
x
10­
2).
This
value
will
be
nonspecific
binding
in
fmoles.
·
Specific
binding
is
calculated
by
subtracting
nonspecific
binding
from
total
binding
i.
e.,
fmoles
total
binding
­
fmoles
nonspecific
binding
=
specific
binding
in
fmoles.

Calculation
Check
To
get
fmoles
multiply
the
DPM
values
by
1.6242
x
10­
2.
This
is
simply
nM
x
300,
i.
e.,

1.8047
x
10­
5
nM
x
0.0003
liter
=
1.6242
x
10­
2
fmoles
1
x
10­
6
nmoles/
fmole
3.
Graphical
Presentation
of
the
Data
An
example
of
a
typical
saturation
curve
and
the
associated
Scatchard
analysis
is
illustrated
in
Figure
1.
Maximal
binding
capacity
(
Bmax)
and
association/
dissociation
constants
(
Ka
/
Kd)
can
be
estimated
using
a
number
of
commercially
available
iterative
nonlinear
regression
analysis
programs.
One
of
the
better
programs
was
developed
by
Munson
and
Rodbard
and
is
called
LIGAND
(
Munson
and
Rodbard,
1980).

F.
References
Munson,
P.
J.,
and
D.
Rodbard,
Anal.
Biochem.,
107,
1980,
pp.
220­
239.
Nonneman,
D..
J.,
V.
K.
Ganjam,
W.
V.
Welshons,
and
F.
S.
vom
Saal,
Biol.
Reprod.,
47,
1992,
pp.
723­
729.
Segel,
I.
H.,
Enzyme
Kinetics:
Behavior
and
Analysis
of
Rapid
Equilibrium
and
Steady­
State
Enzyme
Systems.,
1st
ed.,
John
Wiley
and
Sons,
Inc.,
New
York,
NY,
1975.
Tekpetey,
F.
R.,
and
R.
P.
Amann,
Biol.
Reprod.,
38,
1988,
pp.
1051­
1060.

III.
MVLN
Assay
A.
Introduction
The
MVLN
estrogen
specific
transcription
assay
can
be
used
to
characterize
estrogenic
chemicals
(
Pons
et
al.
1990).
The
assay
utilizes
an
MCF­
7
(
Soule
et
al.
1973)
derivative
that
has
been
stably
transfected
with
the
Vit­
Luc
reporter
gene
(
Pons
et
al.
1990).
Thus,
the
MVLN
cell
line
expresses
the
endogenous
estrogen
receptor
of
MCF­
7
and
at
the
same
time,
contains
an
exogenous
estrogen
responsive
reporter
(
luciferase).
Therefore,
the
estrogen
specific
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
12
L
­
transcription
activity
of
a
test
chemical
is
directly
related
to
the
activity
of
luciferase
measured
in
the
lysate
of
treated
MVLN
cells.
The
MVLN
assay
procedure
presented
here
is
a
modified
version
of
published
methods
(
Gagne
et
al.
1994;
Pons
et
al.
1990).
While
the
protocol
herein
does
utilize
the
MVLN
subclone
of
MCF­
7,
all
tissue
culture
materials,
such
as
media
and
sera,
are
commercially
available.
In
brief,
MVLN
cells
are
seeded
into
12
well
plates,
fed
media
containing
treatment
compounds
and
then
two
days
later,
cell
lysates
are
harvested
and
measured
for
luciferase
activity.
When
cell
counting
is
called
for
in
this
procedure,
consult
the
method
"
Monolayer
Cell
Counting
with
a
Coulter
Counter"
from
this
laboratory.

B.
Maintenance
of
Cell
Stocks
The
MVLN
cell
line
must
be
obtained
from
its
source
(
Pons
et
al.
1990).
The
MVLN
clone
has
been
shown
to
maintain
a
stable,
estrogen
responsive
phenotype
in
this
laboratory
over
many
passages
(
at
least
15).
Stock
cultures
should
be
maintained
in
10%
fetal
bovine
sera
(
FBS)
media
under
5%
CO
2
in
a
37
°
C
incubator.
Such
culture
conditions
will
be
"
estrogen
rich"
and
tend
to
favor
cells
that
require
estrogen
for
growth
(
MCF­
7,
MVLN).
A
regular
schedule
of
passing
stocks
weekly
(
Monday
into
8,
T­
25
flasks
at
a
density
of
5
x
105
to
1
x
106
cells/
flask
should
provide
enough
cells
for
seeding
stocks
as
well
as
experimentals
(
8,
12
well
plates)
seven
days
later.
MVLN
cells
may
grow
slower
than
other
MCF­
7
derivitives.
In
addition,
MVLN
cells
are
very
sensitive
to
seeding
density.
If
seeded
too
light,
MVLN
cells
will
grow
exceedingly
slow
and
may
not
thrive.
The
common
pH
indicator
phenol
red
has
been
shown
to
be
estrogenic
and
therefore
should
not
be
used
in
cell
cultures
utilized
for
MVLN
assays.

For
routine
passage,
the
MVLN
cell
monolayer
is
removed
with
trypsin/
EDTA
treatment,
diluted
to
10
ml
with
whole
media
and
then
dispersed
into
a
suspension
(
mostly
single
cell)
through
the
use
of
a
10
ml
syringe
and
bent
canula.
This
canula
is
a
14
gauge,
blunt
tip,
Luer
lock
needle
(
Thomas
Scientific)
in
which
1
cm
at
the
tip
is
bent
30
°
to
45
°
.
The
10
cm
length
can
be
used
with
T­
25
flasks
while
the
15
cm
will
work
with
T­
25
or
T­
75
flasks.
First,
count
one
duplicate
flask.
Second,
remove
media
from
flask(
s)
to
be
passed
(
monolayer
of
cells
stays
attached).
Then,
wash
each
flask
three
times
with
Ca++
free
HBSS,
remove
and
then
add
1­
2
ml
trypsin
for
three
to
five
minutes
@
37
°
C.
Be
sure
to
disperse
the
trypsin
evenly
over
the
monlayer.
After
incubation,
dilute
trypsin
to
10
ml
with
whole
media.
With
the
sterile
canula­
syringe,
draw
the
10
ml
of
media
up
into
the
syringe.
Expel
the
media,
with
moderate
force,
through
the
bent
canula,
towards
the
cell
monolayer
(
almost,
but
not
touching
the
monolayer
surface)
with
a
circular
motion
covering
the
cell
surface
of
the
flask.
Repeat
for
a
total
of
three
cycles
making
sure
that
all
the
monolayer
has
been
removed
from
the
flask
(
keep
air
bubbles
to
a
minimum).
After
last
cycle,
leave
the
cell
suspension
in
the
flask.
Then,
with
a
10
ml
pipette,
rinse
down
the
inside
of
the
flask
three
times
with
the
cell
solution.
The
cells
will
not
be
harmed
by
this
procedure.
This
method
disperses
cells
better
than
trypsin
treatment
alone.
Note
that
since
all
MVLN
cells
can
express
the
reporter
gene
in
response
to
estrogen,
it
is
essential
that
cells
are
dispersed
evenly
before
seeding
experimental
plates.

An
aliquot
of
this
concentrated
media­
cell
solution
should
then
be
diluted
with
media
in
a
sterile
vessel,
mixed
and
used
to
seed
flasks.
For
precise
seeding,
it
is
recommended
that
the
entire
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
13
L
­
volume
of
cells
and
media
to
seed
all
the
flasks/
plates
desired
is
mixed
in
a
single
vessel.
For
example,
to
seed
eight
flasks
with
7
x
105
cells
each,
make
50
ml
of
a
seeding
solution
that
is
1.16
x
105
cells/
ml,
mix
well
and
then
add
6
ml
to
each
T­
25
flask.
Recall
that
you
know
how
many
cells
are
in
each
flask
since
a
duplicate
flask
was
counted
previously.
The
goal
of
this
method
is
to
seed
all
flasks/
plates
the
same
(
high
precision).
Since
all
MVLN
cells
can
express
the
reporter
gene
in
response
to
estrogen,
precision
in
MVLN
assays
is
completely
dependent
on
uniform
seeding
of
plates.
It
may
be
a
good
idea
to
practice
seeding
flasks
and
then
count
them
the
next
day.

C.
MVLN
Assay
Setup
and
Time
Sequence
With
the
following
exceptions,
passing
MVLN
cells
for
assays
should
be
done
as
described
above.
First,
it
is
essential
that
cells
used
to
seed
experimental
plates
were
withdrawn
from
estrogen
six
days
prior
to
passage.
A
suggested
way
to
withdraw
cells
from
estrogen
involves
seeding
stocks
(
eight
flasks)
in
10%
FBS
media
one
week
before
you
plan
on
seeding
experimentals.
The
day
after
seeding
the
stocks,
label
two
flasks
as
"
stock"
and
replace
the
media
in
these
with
10%
FBS
media
These
stock
flasks
should
be
fed
10%
FBS
media
every
other
day
until
used
to
seed
eight
more
stock
flasks
the
following
week.
The
remaining
6
flasks
are
then
withdrawn
from
estrogen
by
rinsing
three
times
with
sterile
PBS
(
all
flask
surfaces)
and
fed
10%
dextran
coated
charcoal
(
DCC)
FBS
media.
The
10%
DCC
FBS
media
is
almost
devoid
of
estrogens.
These
"
withdrawn"
flasks
should
be
rinsed
3X
and
fed
DCC
media
every
other
day
until
used
for
seeding
a
MVLN
experiment.
When
seeding
MVLN
stocks
or
experiments,
one
of
the
duplicate
flasks
is
always
counted
to
determine
the
cells
per
flask
count
for
that
series
(
one
of
two
stocks
is
counted,
one
of
six
withdrawn
is
counted).
Keep
in
mind
that
it
takes
a
week
to
get
cells
ready
for
a
MVLN
experiment.

MVLN
experiment
12
well
plates
are
seeded
on
day
­
1
(
Monday)
with
1
x
105
cells/
well
in
10%
DCC
FBS
media.
To
ensure
constant
seeding,
mix
the
required
cells
and
media
in
a
sterile
bottle
and
then
seed
aliquots
(
2
ml)
into
each
well.
The
seeding
mixture
bottle
should
be
mixed
often
during
this
procedure.
The
following
day
(
day
0,
Tuesday),
the
seeded
cells
are
left
alone
and
allowed
to
firmly
attach
to
the
plates.

Two
days
after
seeding
(
day
1,
Wednesday),
cells
are
fed
treatment
media
(
2
ml/
well).
Treatment
media
is
5%
DCC
FBS
into
which
treatments
in
ethanol
carrier
have
been
added.
Treatments
may
be
made
up
in
50
ml
polyethylene
tubes
(
do
not
use
polycarbonate
or
polystyrene
tubes!
(
Soto
et
al.
1991)
and
should
be
no
more
than
0.05%
v/
v
ethanol
carrier
solvent.
Higher
levels
of
ethanol
may
have
confounding
effects
on
MVLN
studies.
Treatment
carrier
solvents
such
as
DMSO
and
methanol
should
be
avoided
since
they
are
toxic
to
cells
and
will
have
confounding
effects
on
MVLN
studies.
Experimental
cells
should
have
new
treatment
media
applied
the
next
day
(
day
2,
Thursday).

On
day
3
(
Friday),
treated
cell
lysate
should
be
harvested
for
luciferase
assay.
First
remove
treatment
media
from
each
well.
Then,
wash
each
well
one
time
with
2
ml
PBS.
It
is
essential
that
all
PBS
is
removed
from
each
well
at
this
step.
Removal
of
the
PBS
by
aspiration
followed
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
14
L
­
by
a
one
minute
bench
top
incubation
with
the
plate
tipped
45
°
and
final
aspiration
of
drained
residue
is
recommended.
To
lyse
cells,
add
500
ml
lysis
buffer
to
each
well
and
then
incubate
at
room
temperature
on
a
rocker
for
at
least
30
minutes.
Transfer
lysate
from
wells
with
micro­
pipet
to
micro
tubes.
Make
sure
that
all
cell
debris
is
mixed
and
removed
uniformly
between
wells.
Lysate
can
be
left
at
room
temperature
for
up
to
six
hours,
kept
at
4
°
C
over
night
or
stored
indefinitely
at
­
70
°
C
without
loss
of
luciferase
activity.
Sample
tubes
should
be
equilibrated
at
room
temperature,
vortexed
and
then
spun
down
prior
to
sampling
(
20
to
50
ml)
for
luciferase
assay.

D.
MVLN
Assay
Design
Properly
designed
MVLN
assays
can
be
utilized
to
answer
only
the
following
four
questions.
First,
does
the
test
compound
stimulate
estrogen
receptor
mediated
transcription
(
what
is
the
shape
of
the
corresponding
dose
response
curve)?
Second,
if
the
test
compound
stimulates
transcription,
is
this
response
an
estrogen
receptor
mediated
mechanism
(
is
the
compound
an
estrogen
receptor
agonist)?
Third,
can
the
test
compound
block
the
agonist
effects
of
E
2
(
is
the
compound
an
antiestrogen)?
Last,
is
the
test
compound
toxic
to
MVLN
cells?
Attempts
to
obtain
additional
information
from
the
MVLN
assay
may
be
misleading.
Keep
in
mind
that
this
assay
does
not
necessarily
determine
if
the
test
compound
binds
to
the
ER.

Each
data
point
of
the
MVLN
assay
should
be
run
in
duplicate
(
minimum)
or
triplicate
during
a
trial.
Then,
that
same
trial
should
be
repeated
at
least
two
more
times.

E.
Example
Experiment
Setup
Blank
(
no
additions
to
DCC
FBS
media)
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
2
wells
E
2
(
positive
controls)
10­
12,
10­
11,
10­
10,
10­
9,
10­
8,
10­
7
M
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
12
wells
ICI­
182,780
10­
6
M
(
check
of
estrogen
free
conditions)
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
2
wells
E2
10­
10
M
+
ICI­
182,780
10­
6
M
(
Check
of
ICI)
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
2
wells
Test
compound
A:
10­
8,
10­
7,
10­
6,
10­
5
M
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
8
wells
Test
compound
B:
etc.

ICI
10­
6
M
+
TC
A
10­
5
M
(
ER
mechanism
test)
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
2
wells
ICI
10­
6
M
+
TC
B
10­
5
M
etc.

E2
10­
10
M
+
TC
A
10­
5
M
(
antiestrogen
test)
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
·
2
wells
E2
10­
10
M
+
TC
B
10­
5
M
etc.

to
a
Total
of
96
wells
Note:
Test
compound
toxicity
is
determined
by
comparing
Luc
activity
of
test
compound
treatments
with
blank
activity
and/
or
test
compound
with
ICI
to
ICI
alone.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
15
L
­
F.
Example
Stock
and
Experiment
Schedule
Day
­
1
(
Monday)
Pass
stocks,
experimentals
plated.
Day
0
(
Tuesday)
Cells
left
undisturbed
to
attach
to
flasks/
plates.

Day
1
(
Wednesday)
Feed
stocks
(
2
flasks
FBS,
6
flasks
withdrawn
in
DCC
FBS).

Feed
experimentals
treatments.
Day
2
(
Thursday)
Feed
experimentals
treatments.

Day
3
(
Friday)
Feed/
withdraw
stocks.

Experimentals
harvested
(
morning).

G.
Regarding
an
Estrogen
Free
Laboratory
Environment
All
glassware,
caps,
hoses,
etc.
that
may
contact
media
must
be
free
of
estrogens.
Soap
wash
and
3X
hot
rinse
followed
by
rinsing
three
times
with
ddH
2
O,
air
dry,
rinse
with
95%
ethanol
(
from
glass
bottle;
cans
and
plastic
may
have
estrogenic
contamination!)
followed
by
rinse
three
times
with
ddH
2
O
water.
Baking
of
foil
covered
glassware
for
12
hours
in
a
250
°
C
oven
will
sterilize
and
remove
more
organic
compounds.
Wash
and
rinse
bottle
caps
as
above,
dry
in
low
temp
oven
and
autoclave.
Alternatively,
bottles
and
caps
may
be
autoclaved
together
with
the
risk
of
contamination
by
estrogenic
condensates.
Your
cell
culture
environment
should
be
characterized
for
estrogen
contamination
by
proliferation
assay
tests
with
and
without
added
ICI­
182,
780
(
Wakeling
and
Bowler
1992).
If
the
"
estrogen
free"
cells
grow
faster
(>
10%)
than
the
ICI
treated
cells,
you
have
estrogen
contamination.
All
experiments
conducted
in
the
presence
of
such
contamination
are
useless
since
regardless
of
how
they
are
set
up,
you
are
testing
combinations
of
chemicals.
We
have
found
plastic
vessels
and
implements
to
be
the
major
source
of
estrogen
contamination.
Polystyrene
and
polycarbonate
seem
to
be
the
big
problems.
Do
not
use
culture
flasks
with
"
phenolic"
caps.
Filter
units
may
add
estrogenic
substances
to
media.
The
Corning
bottle
top
units
(
orange)
are
suspect.
Zap
Caps
seem
to
add
some
kind
of
nonestrogenic
mitogen
which
induces
MCF­
7
cells
to
grow
at
maximum
rate,
even
in
the
presence
of
ICI.
It
is
unclear
what
effect
Zap
Cap
contamination
has
on
MVLN
assays.
Also,
it
appears
to
be
relatively
easy
to
extract
estrogens
from
gloves
and/
or
hands
when
rinsing
items
with
ethanol.
Lastly,
ethanol
may
confound
MVLN
assays.
Ethanol
rinsed
vessels
and
implements
must
be
thoroughly
dry
before
use.

H.
Media
1.
DMEM
powder
for
10L
(
Gibco
13000­
096).
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
16
L
­
2.
gm
HEPES
(
Gibco
11344­
033),
media
will
be
20
mM.
3.
ml
non­
essential
amino
acids
(
Gibco
11140­
019),
media
will
be
0.1
mM.
4.
ml
sodium
pyruvate
(
Gibco
11360­
070),
media
will
be
1
mM.
5.
gm
NaHCO3.
6.
ml/
L
media
of
Antibiotic­
Antimycotic
solution
(
Gibco
15240­
062).

In
3
L
tissue
culture
grade
water,
add
1,
2,
3
&
4
above.
Mix
45
minutes
in
4
L
beaker.
Add
5,
mix
15
minutes.
pH
to
7.3.
Dilute
to
10
L
and
mix
15
minutes.
Check
pH.
Filter
into
500
ml
sterile
bottles
(
Gelman
VacuCap
4622
or
Gelman
Micro
Culture
Capsule
12158).
Store
media
at
4
°
C.
Add
Antibiotic­
Antimycotic
solution
to
media
bottle
when
used
(
made
up
with
sera).
To
make
one
bottle
of
10%
sera
media,
add
50
ml
sera
to
500
ml
of
media.

I.
Sera
FBS
Hyclone
Characterized
Fetal
Bovine
Sera
(
A­
1115­
L)
DCC
FBS
Hyclone
Charcoal/
Dextran
Fetal
Bovine
Sera
(
A­
1120­
L)

J.
Buffers
Ca++
Free
HBSS
Gibco
14185­
052
PBS
Gibco
14080­
055
Lysis
Buffer
Promega
E153A
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
17
L
­
K.
References
Gagne,
D.,
P.
Balaguer,
E.
Demirpence,
C
Chabret,
F.
Trousse,
J.
C.
Nicolas,
and
M.
Pons,
"
Stable
luciferase
transfected
cells
for
studying
steroid
receptor
biological
activity,"
Journal
of
Bioluminescence
&
Chemiluminescence,
9,
1994,
pp.
201­
209.
Pons,
M.,
D.
Gagne,
J.
C.
Nicolas,
and
M.
Mehtali,
"
A
new
cellular
model
of
response
to
estrogens:
a
bioluminescent
test
to
characterize
(
anti)
estrogen
molecules,"
Biotechniques,
9,
1990,
pp.
450­
459.
Soto,
A.
M.,
H.
Justicia,
J.
W.
Wray,
and
C.
Sonnenschein,
"
p­
Nonyl­
phenol:
an
estrogenic
xenobiotic
released
from
`
modified'
polystyrene,"
Environmental
Health
Perspectives,
92,
1991,
pp.
167­
173.
Soule,
H.
D.,
J.
Vazquez,
A.
Long,
S.
Albert,
and
M.
Brennan,
"
A
human
cell
line
from
a
pleural
effusion
derived
from
a
breast
carcinoma,"
Journal
of
the
National
Cancer
Institute,
51,
1973,
pp.
1409­
1416.
Wakeling,
A.
E.,
and
J.
Bowler,
"
ICI
182,780,
a
new
antioestrogen
with
clinical
potential
[
Review],"
Journal
of
Steroid
Biochemistry
&
Molecular
Biology,
43,
1992,
pp.
173­
177.

IV.
CV1
Cell
Transfections
Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
a
procedure
for
the
quantitation
of
AR­
mediated
alterations
of
transcriptional
activation
in
CV1
cells.

MONDAY
·
plate
0.2
x
106
CV1
cells
/
6cm
Corning
TC
dish
in
the
following
media
DMEM
­
(
high
glucose
+
L­
glutamine
+
25
mM
Hepes
+
NaHCO
3)
­
Take
a
0.5
liter
bottle
and
add
·
5
ml
of
100X
antibiotic/
antimycotic
(
frozen
aliquots)
·
50
ml
fetal
bovine
serum
(
frozen
50
ml
aliquots)
NOTES:
·
Grow
cells
up
in
T­
150
culture
flasks
in
10%
FCS
/
DMEM.
·
When
there
are
enough
cells
for
the
experiment,
decant
media
from
the
flasks
into
funnel
waste
beaker
layered
with
sterile
gauze
to
prevent
backsplash.
·
Wash
cells
twice
with
20
ml
of
1X
HBSS
and
decant
into
waste
funnel
(
this
gets
rid
of
the
serum
which
inactivates
trypsin).
·
Add
1­
2
ml
of
1X
Trypsin
(
from
1.0X
frozen
stock)
­
roll
the
trypsin
over
the
cells
and
place
cells
in
the
incubator
for
five
to
ten
minutes.
·
After
the
cells
detach
(
i.
e.,
round
up
and
float),
thump
the
side
of
the
flask
with
the
palm
of
your
hand
and
add
10
ml
of
10%
FCS
/
DMEM
(
the
serum
in
the
media
inactivates
the
trypsin).
·
Transfer
the
cells
to
a
50
ml
centrifuge
tube
(
be
sure
to
get
all
the
cells
off
the
plates)
and
spin
the
cells
down
at
1000
rpm
(
250
x
g).
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
18
L
­
·
Aspirate
or
decant
the
media
and
thump
pellet
loose
from
the
tube
­
add
10
ml
of
10%
FCS
/
DMEM
and
resuspend
the
cells
by
pipeting
up
and
down
with
a
10
ml
pipet.
·
Count
cells
on
a
hemocytometer
as
follows:
·
Total
cell
#
in
all
center
squares
x
0.01
=
#
cells
(
in
millions)
/
ml
·
Dilute
the
cell
suspension
out
to
50,000
cells/
ml
and
add
4ml
of
this
cell
stock
solution
to
each
6
cm
plate
using
a
10
ml
pipet.
·
In
general,
the
following
numbers
of
CV1
cells
are
obtained:
·
T­
150
culture
flask
of
almost
confluent
CV1
cells
will
contain
8­
10
x
106
cells
·
T­
75
culture
flask
of
almost
confluent
CV1
cells
will
contain
4­
5
x
106
cells
**
HANDS
ARE
WASHED
WELL
WITH
SOAP
AND
WATER
(
ESPECIALLY
UNDER
NAILS)
AND
BEFORE
PLACING
HANDS
IN
THE
CULTURE
HOOD,
HANDS
SHOULD
BE
SPRAYED
WITH
70%
ETOH.
·
THE
HOOD
SURFACES
ARE
SPRAYED
WITH
70%
ETOH
AND
WIPED
CLEAN
BEFORE
USE.
·
ANYTHING
THAT
GOES
IN
THE
HOOD
THAT
IS
NOT
STERILE
IS
SPRAYED
WITH
70%
ETOH
INCLUDING
MEDIA
BOTTLES,
TUBE
RACKS,
PIPETMEN,
ETC.
CAN
ALSO
FLAME
THE
OUTSIDE
OF
GLASS
CULTURE
MEDIA
BOTTLES
TUESDAY
·
Prepare
2X
HBS
(
100
ml)
as
follows:
·
90
ml
ddH
2
O
·
5.6
ml
5M
NaCl
(
from
sterile
stock)
·
2.5
ml
2M
HEPES
(
Na+
salt)
·
1.5
ml
0.1M
Na
2
HPO
4
****
pH
to
7.12­
7.13****
,
then
filter,
sterilize
and
store
in
the
refrigerator.

·
Prepare
CaPO
4
precipitates
(
listed
below
are
the
amounts
for
14
dish
batches)
after
all
components
have
reached
room
temperature.
Add
the
following
to
a
sterile
5.0
ml
tube
in
this
order
·
1.53
ml
ddH
2
O
·
700
ng
pCMVhAR
(
P
9;
50
ng/
dish)
·
70
ug
MMTV­
LUC
(
5
ug/
dish)
**
vortex
to
mix**
·
219
ul
2M
CaCl
2
(
from
frozen
stock
made
by
adding
22.197g
CaCl
2
to
a
100
ml
volumetric
flask
and
bring
the
volume
to
100
mls
with
ddH2O,
sterile
filter).

**
vortex
to
mix**

·
While
vortexing
(
setting
2.5)
a
1.75
ml
aliquot
of
the
2X
HBS
solution
in
a
15
ml
screw
top
conical
c­
fuge
tube
add
the
contents
of
the
5.0
ml
tube
containing
CaCl
2­
DNA
with
a
5.0
ml
plastic
pipet.

**
DROPWISE
AND
SLOWLY**.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
19
L
­
·
Repeat
for
the
remaining
tubes
then
wait
10
minutes
for
ppt's
to
form.
·
At
ten
minutes
add
3.5
ml
of
media
to
each
tube
to
stop
the
precipitation
(
the
ppt
will
be
stable
for
at
least
30
minutes)....
repeat
for
the
other
tubes
at
their
ten
minute
time
periods.
After
all
ppts
have
been
stabilized,
check
the
ppts
to
be
sure
they
are
small
with
individual
grains.
If
particles
are
linked
into
chains
or
lumpy
looking
do
not
use
it.
Dilute
the
contents
of
each
tube
(
now
containing
7.0
mls)
with
49
mls
media
and
combine
all
in
a
sterile
flask
(
i.
e.,
for
multiple
14
dish
batches).
·
Aspirate
the
media
from
the
CV1
cell
dishes
and
immediately
add
4
ml
of
gently
mixed
DNA­
ppt
media
to
each
of
the
dishes
in
the
experiment
using
a
10
ml
glass
pipet.
·
Return
dishes
to
37
°
C
incubator
for
four
to
six
hours.
·
Aspirate
the
precipitate
from
each
dish
and
wash
each
dish
two
times
with
4
ml
DPBS
and
add
4
ml
5%
DCC­
FBS
DMEM
media.
·
Place
all
dishes
in
37
°
C
incubator
overnight
WEDNESDAY
·
Make
up
a
1000X
concentrated
stock
solution
for
each
treatment
in
ETOH
(
i.
e.,
so
won't
have
to
sterilize)
as
follows:
10.0
mM,
1
mM,
0.5
mM,
0.2
mM,
0.05
mM.
·
Make
up
10X
DHT
working
solution
by
adding
24
ul
of
10
uM
DHT
(
made
fresh
from
frozen
10
mM
stock)
to
a
5.0
ml
sterile
tube
and
combine
with
2.4
mls
of
5%
DCC­
media
(
this
is
enough
for
60
dishes).
·
Take
plates
out
of
incubator
and
add
4
ul
of
each
treatment
to
appropriately
labelled
dishes
(
in
duplicate).

**
Add
treatments
to
the
side
of
each
dish
and
let
run
down
into
the
media­
rock
plates
after
addition.**

·
Immediately
add
40
ul
of
10X
DHT
(
to
the
side
of
each
dish
to
receive
DHT)
using
the
positive
displacement
pipetting
technique.
·
Move
plates
back
and
forth
to
distribute
the
treatments
evenly
in
the
media.
·
Return
the
dishes
to
the
37
°
C
incubator
overnight.

**
Notes:
Final
[
]'
s
are
1nM
DHT
and
10
uM,
2
uM,
0.5
uM,
0.2
uM,
0.05
uM
treatments.

­­
2X
TBS
Buffer
(
500
ml)­­­­­­­­­­­­­­­­­­­­­
For
4
liters
­
8.18
g
NaCl
­
65.44
g
­
0.23
g
KCl
­
1.84
g
­
0.147
g
CaCl
2
­
2H
2
O
­
1.18
g
­
0.1
g
MgCl
2
­
6
H
2
O
­
0.80
g
­
0.128
g
Na
2
HPO
4
­
H
2
O
­
1.02
g
­
3.03
g
Tris
­
HCL
­
24.24
g
pH
the
buffer
to
7.4
and
filter
sterilize
into
a
sterile
bottle
before
use.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
20
L
­
THURSDAY
MORNING
­
Aspirate
media
from
all
dishes
and
replce
with
4.0
mls
of
fresh
5%
DCCmedia
using
a
10
ml
pipette.
Repeat
the
above
dosing
protocol.

AFTERNOON
­
Harvest
cells
·
Aspirate
media
from
all
dishes;
wash
cells
twice
with
2
ml
of
1X
HBSS
and
aspirate
the
HBSS
from
all
dishes.
·
add
0.5
ml
cell
culture
lysis
buffer
(
at
room
temperature)
to
each
plate
and
let
rock
at
room
temperature
for
20­
30
minutes.
·
using
a
rubber
scraper
(
if
necessary),
scrape
the
cells
loose
from
the
dish
and
then
slowly
pipette
the
lysis
buffer
and
cells
up
and
down.
­­
Need
to
go
slow
because
one
does
not
want
excessive
bubbles.

**
Can
use
the
same
rubber
scraper
for
all
plates,
if
the
scraper
is
rinsed
off
with
ddH
2
0
between
dishesand
go
from
high
treatment
dose
to
low
treatment
dose
(
i.
e.,
low
luciferase
to
high
luciferase).

**
Place
all
of
the
cell
lysate
into
prelabelled
1.0
ml
microcentrifuge
tubes
and
store
at
­
70
°
C
until
assay
(
i.
e.,
Friday
morning).

FRIDAY
­
LUCIFERASE
ASSAY
·
Prepare
20
mls
of
rxn
buffer
in
a
50
ml
centrifuge
tube
as
follows
(
20
mls
is
enough
for
50
samples):
·
Add
18
mL
ddH
2
O
to
a
50
ml
centrifuge
tube
·
Add
500
uL
of
1
M
Glycylglycine
(
make
a
50
ml
stock
by
adding
6.605g
gly­
gly
(
MW
132.1)
to
50
ml
ddH
2
O,
pH
to
7.8,
sterile
filter
and
store
at
4o
C).
·
Add
300
uL
1M
MgCl
2
(
from
Sigma
also
stored
at
4o
C)
·
Add
1.0
mL
of
100
mM
ATP
(
make
a
50
ml
stock
by
adding
2.755g
of
ATP
(
MW
551.1
g/
mole)
to
50
ml
ddH
2
O,
pH
the
soln
to
7.8,
aliquot
to
1.0
mL
and
store
at
­
80
°
C).
·
Add
200
uL
of
50
mg/
ml
BSA
(
make
a
10
ml
stock
by
adding
500
mg
BSA
to
10
ml
ddH
2
O,
aliquot
to
200
uL
and
store
at
­
20
°
C).
·
Place
the
tube
at
room
temperature
**
NOTE:
IT
IS
CRITICAL
THE
THAT
THE
pH
OF
THE
RXN
BUFFER
IS
7.8
AT
ROOM
TEMPERATURE.
IF
THE
GLY­
GLY
AND
ATP
SOLUTIONS
ARE
CAREFULLY
pH'ED,
THEN
THE
FINAL
MIX
WILL
BE
pH
7.8
­
CHECK
TO
BE
SURE.
**

·
Take
cell
lysates
out
of
the
freezer
and
allow
to
thaw
at
room
temperature.
·
Fill
brown
luminometer
bottle
with
ddH
2
O
and
connect
to
automatic
injector
in
the
front
panel
of
the
luminometer.
Make
sure
a
cuvette
is
in
the
chamber
and
flush
the
luminometer
lines
with
water
(
Service
14).
Empty
the
cuvette
and
replace
in
the
chamber.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
21
L
­
**
NOTE:
NEVER
PLACE
WET
CUVETTES
IN
THE
INSTRUMENT
AND
ALWAYS
KEEP
A
CUVETTE
IN
THE
INSTRUMENT
WHEN
NOT
IN
USE.
**

·
Flush
the
lines
with
air
(
i.
e.
disconnect
the
brown
bottle)
again
using
Service
14.
·
Add
the
luciferin
to
brown
bottle
#
1
and
fill
the
bottle
approximately
to
the
shoulder
(
should
be
enough
for
about
100
samples).
Add
rxn
buffer
to
the
brown
bottle
#
2
and
fill
the
bottle
approximately
to
the
shoulder
(
should
be
enough
for
about
100
samples).
Place
a
new
cuvette
in
the
chamber
and
flush
the
lines
with
the
luciferin/
rxn
solution.
·
The
1mM
D­
luciferin
stock
solution
is
made
and
stored
as
follows:
·
Add
159.21
mg
D­
luciferin
(
K+
salt;
MW
318.41
g/
mole)
to
500
ml
ddH
2
O.
Make
10
ml
aliquots
in
15
ml
centrifuge
tubes.
Wrap
tubes
with
aluminum
foil
(
i.
e.,
luciferin
is
light
sensitive)
and
store
at
­
20
°
C.

NOTE:
Can
also
use
the
Na+
salt
of
D­
luciferin
but
it
sometimes
turns
yellow
­
its
probably
still
ok
but
better
to
use
the
K+
salt
to
avoid
any
potential
problems.

·
Set­
up
luminometer
(
20
sec
read,
continuous
measurements,
raw
data,
no
duplicates)
so
will
get
raw
data
(
i.
e.,
relative
light
units)
for
each
sample.
·
Vortex
the
cell
lysate
and
spin
tubes
for
30
seconds
to
isolate
cell
debris.
·
Add
50
ul
of
vortexed
cell
lysate
to
a
new
cuvette,
place
in
the
luminometer
and
press
start.
While
the
sample
is
being
read
get
another
tube
ready
(
this
usually
works
out
so
that
by
the
time
the
machine
is
ready
the
next
sample
is
also
ready
to
go).
·
Use
a
new
cuvette
for
each
sample,
do
not
reuse
cuvettes.
·
When
finished,
exit
and
press
service
13
to
back
the
contents
of
each
line
back
into
the
respective
brown
bottles,
decant
to
stock
solution
and
place
the
rxn
buffer
in
the
refrigerator
and
the
luciferin
in
­
70C
freezer.
·
Wash
the
brown
bottles
out
with
ddH
2
O
and
place
ddH
2
O
in
each
bottle
and
place
back
in­
line.
Place
an
empty
cuvette
in
the
machine
and
flush
the
lines
(
service
14)
first
with
ddH
2
O
then
with
air.
Leave
the
lines
dry,
shut
off
the
luminometer
and
always
keep
an
empty
cuvette
in
the
sample
chamber.

V.
Procedures
to
Assess
Individual
Steroidogenic
Enzyme
Activities
in
the
Rat
A.
Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
a
procedure
for
the
radiometric
quantitation
of
steroidogenic
enzyme
activity.
The
principle
of
this
procedure
is
to
quantitate
the
conversion
of
radiolabelled
substrate
to
radiolabelled
product;
data
is
usually
expressed
as
pmoles
product
formed/(
unit
time
x
mg
protein).
To
this
end,
a
tissue
homogenate
or
cell
preparation
is
incubated
under
carefully
controlled
conditions
(
temperature,
pH,
time,
substrate
concentration,
cofactor
concentration,
and
osmolality)
with
radiolabelled
substrate.
Following
the
incubation
the
reaction
is
terminated
and
the
steroids
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
22
L
­
(
androgens,
progestins,
etc.)
are
extracted
from
the
biological
matrix
by
solid
phase
extraction
(
SPE;
see
SPE
SOP
for
this
procedure).
The
extracted
steroids
are
dried
in
vacuo
using
a
vacuum
concentrator
(
see
vacuum
concentrator
SOP
for
correct
operation
of
this
equipment)
and
injected
into
the
HPLC
with
on­
line
radiometric
quantitation
(
see
HPLC
SOP's).
The
HPLC
system
chromatographically
separates
the
substrate
from
product(
s),
mixes
the
HPLC
effluent
with
scintillation
fluid
and
quantitates
the
amount
of
radioactivity
in
each
substrate/
product(
s)
peaks.
See
the
references
listed
below
for
additional
clarification.
Please
note
the
specifics
for
the
radiometric
quantitation
of
the
following
enzyme
activities:

·
5a­
Reductase:
The
substrate
is
testosterone
(
5.0
mM)
and
the
products
of
the
reaction
are
5adihydrotestosterone
+
5a­
androstane­
3a/
ß,
17ß­
diols.
ß­
NADPH
(
0.5
mM)
is
the
cofactor.
·
17a­
Hydroxylase:
The
substrate
is
progesterone
(
5.0
mM)
and
the
products
of
the
reaction
are
17a­
hydroxyprogesterone
+
androstenedione
+
testosterone.
ß­
NADPH
(
0.5
mM)
is
the
cofactor.
·
C
17­
20­
Lyase:
The
substrate
is
17a­
hydroxyprogesterone
(
5.0
mM)
and
the
products
of
the
reaction
are
androstenedione
and
testosterone.
ß­
NADPH
(
0.5
mM)
is
the
cofactor.
·
17­
Ketosteroid
Reductase:
The
substrate
for
the
reaction
is
androstenedione
(
5.0
mM)
and
the
product
is
testosterone.
ß­
NADPH
(
0.5
mM)
is
the
cofactor.
·
5­
Ene­
3ß­
hydroxysteroid
Dehydrogenase/
Isomerase:
The
substrate
for
the
reaction
is
pregnenolone
(
5.0
mM)
and
the
product
is
progesterone.
ß­
NAD+
(
0.5
mM)
is
the
cofactor.

B.
Safety
and
Operating
Precautions
All
procedures
with
radioisotopes
should
follow
the
regulations
and
procedures
as
described
in
the
Hazardous
Agent
Protocol
(
HAP)
and
in
the
Radiation
Safety
Manual
and
Protocols.

C.
Equipment
and
Materials
1.
Equipment
·
Corning
Stir/
hot
Plate
·
Digital
Pipets
·
Balance
·
Polytron
PT
35/
10
Tissue
Homogenizer
·
Hewlett­
Packard
HPLC
with
on­
line
Radiomatic
Radiochromatograph
·
Heated
Shaking
Water
Bath
·
Vacuum
Concentrator
·
Hamilton
Syringes
(
50
ml)
2.
Chemicals
·
Sodium
Phosphate,
Monobasic
and
Dibasic
·
Sodium
Chloride
·
Magnesium
Chloride
·
Calcium
Chloride
·
Potassium
chloride
·
double­
distilled
de­
ionized
water
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
23
L
­
·
Recrystallized
Pregnenolone,
Progesterone,
17a­
Hydroxyprogesterone,
Androstenedione
or
Testosterone
·
3H­[
X,
X,
X]­
Pregnenolone,
Progesterone,
17a­
Hydroxyprogesterone,
Androstenedione
or
Testosterone
(
New
England
Nuclear)
·
Scintillation
Cocktail
(
Flow
Scint
III)
·
ß­
NAD+
or
ß­
NADPH
cofactor
·
Ethyl
Alcohol,
anhydrous
3.
Supplies
·
20
ml
Glass
Scintillation
Vials
·
12
x
75
mm
Glass
Test
Tubes
·
100
ml,
1000
ml
volumetric
flasks
·
100
ml
Erlenmeyer
flasks
D.
Methods
1.
Preparation
of
Krebs­
Ringer
Phosphate
Buffer
(
KRPB)
pH
6.9
Add
each
of
the
following
to
individual
100
ml
volumetric
flasks
and
fill
flasks
with
sodium
phosphate
buffer
(
prepared
as
described
below)
to
100
ml:
·
6.779
g
Sodium
Chloride
·
0.264
g
Magnesium
Chloride
·
0.336
g
Potassium
Chloride
·
0.386
g
Calcium
Chloride
Store
the
above
glass­
stoppered
solutions
in
the
refrigerator.
To
prepare
the
sodium
phosphate
buffer,
place
2.5811
g
of
sodium
phosphate
in
a
1000
ml
and
add
distilled
water
to
the
volume
line
and
then
add
a
stir
bar.
Put
the
beaker
and
contents
on
a
Corning
stir
plate
and
stir
the
solution
until
dissolved
on
a
stir
setting
of
3.5.
To
prepare
the
KRPB
add
10
mls
of
each
of
the
above
four
salt
solutions
to
a
100
ml
Erlenmeyer
flask.
Add
55
ml
of
phosphate
buffer
and
5.0
ml
of
glycerol
to
the
salt
solutions
in
the
Erlenmeyer
flask.
The
final
volume
of
KRPB
should
be
100
ml.
Adjust
the
pH
of
the
solution
with
5N
HCL
to
pH
6.9.
Store
the
KRPB
buffer
in
the
refrigerator.
This
buffer
is
stable
for
approximately
one
week.

2.
Preparation
of
Radioinert
Steroid
Standards
Prepare
1.0
mM
stock
solutions
in
ethanol.
Weigh
out
the
following
amounts
of
steroids
and
place
in
100
ml
ethanol.
Pregnenolone
=
31.65
mg/
100
ml
Progesterone
=
31.45
mg/
100
ml
17a­
Hydroxyprogesterone
=
33.05
mg/
100
ml
Androstenedione
=
28.64
mg/
100
ml
Testosterone
=
28.84
mg/
100
ml
For
5
mM
concentrations
in
1.0
ml
incubations
add
5.0
ml
to
each
incubation
vial
as
directed
below.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
24
L
­
3.
Preparation
of
3H­
Steroid
Solutions
Dilute
a
10
ml
aliquot
of
the
3H­
steroid
stock
with
ethanol
so
that
100
ml
yields
approximately
200,000
dpm.
***
Remember
to
count
the
sample
in
15
ml
of
scintillation
fluid
on
channel
1
(
i.
e.,
0­
400)
of
the
scintillation
counter.
***

4.
Preparation
of
Nicotinamide
Cofactor
Solutions
Prepare
5
mM
stock
solution
immediately
prior
to
the
initiation
of
the
reaction.
***
Note
that
the
addition
of
the
cofactor
should
be
the
final
step
in
the
reaction
commencement
­
see
specific
instructions
below.
***
Take
the
appropriate
cofactor
out
of
the
freezer
and
immediately
weigh
as
follows:
NAD+
=
3.32
mg/
ml
KRPB
NADPH
=
4.17
mg/
ml
KRPB
For
0.5
mM
concentrations
use
100
ml/
ml
incubation
volume
as
described
below.

5.
Steroidogenic
Enzyme
Assay
Procedure
The
first
step
is
to
pipet
the
radioinert
and
radioactive
substrate
into
each
vial.
Assays
of
steroidogenic
enzyme
activity
always
are
completed
in
duplicate;
thus,
label
two
20
ml
glass
scintillation
vials
for
each
tissue
sample,
plus
four
control
vials.
Pipet
5
ml
of
the
appropriate
radioinert
steroid
into
the
bottom
of
each
scintillation
vial.
Next
pipet
100
ml
of
the
radioactive
steroid
into
each
tube.
For
example,
if
the
objective
of
the
experiment
was
to
quantitate
17a­
hydroxylase
activity
then
add
5
ml
of
1
mM
progesterone
and
100
ml
of
3H­
progesterone
(
200,000
dpm)
to
each
vial.
Take
the
rotor
out
of
the
vacuum
concentrator
and
place
the
vials
inside
the
vacuum
concentrator.
Evaporate
the
ethanol
vehicle
in
vacuo
by
turning
on
the
vacuum
switch
of
the
vacuum
concentrator.
Allow
ethanol
to
evaporate
while
the
tissues
are
being
prepared
as
described
below.

Harvest
and
weigh
the
appropriate
tissues
from
the
control
and/
or
treated
animals.
Record
the
weights
and
place
each
separate
tissue
in
a
50
ml
polypropylene
homogenization
tube.
Place
the
tubes
in
an
ice­
water
bucket
and
add
10
ml
KRPB/
gm
tissue
wet
weight.
Homogenize
the
tissues
with
15
second
bursts
with
the
polytron
separated
in
time
by
at
least
30
seconds.
This
procedure
is
to
keep
the
tissues
at
0­
4o
C
at
all
times.
Filter
the
homogenates
through
100
mm
nylon
mesh
filters
into
clean
12
x
75
mm
test
tubes
and
place
each
tube
on
ice.

Turn
off
the
vacuum
switch
and
allow
the
pressure
on
the
inside
of
the
vacuum
concentrator
to
equilibrate
to
atmospheric
pressure.
Remove
the
scintillation
vials,
label
them
and
place
them
in
a
34o
C
water
bath
(
Dubanoff
­
with
metal
inserts
to
hold
each
vial).
Add
700
ml
of
KRPB
to
each
tube
and
allow
the
tubes
to
shake
for
ten
minutes
at
120
cycles/
min
in
order
to
resuspend
the
steroid
substrate.
Next,
add
200
ml
of
tissue
homogenate
(
20
mg
tissue)
to
each
labelled
tube.
Allow
the
tissues
to
shake
at
120
cycles/
min
for
five
minutes
then
add
100
ml
of
the
appropriate
cofactor
solution
to
begin
the
reaction.
***
Note
that
control
vials
will
contain
800
ml
of
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
25
L
­
KRPB
and
200
ml
of
tissue
homogenate
without
NADPH.
***
Time
the
incubation
duration
from
the
time
the
cofactor
was
added.
At
the
end
of
the
appropriate
incubation
time
(
need
to
ditermine
the
appropriate
incubation
time
for
the
tissue
being
examined)
stop
the
reaction
and
extract
the
steroids
from
the
biological
matrix
in
each
incubation
vial
as
described
in
the
solid­
phase
extraction
SOP.
Submit
the
samples
to
Dr.
Kelce
for
subsequent
HPLC
analysis.

E.
Data
Processing
Data
will
be
processed
by
the
software
analysis
package
of
the
HPLC.
The
detection
of
eluted
radioactive
substrate
and
product
peaks
is
reported
in
dpm
and
%
of
total
radioactivity
in
each
analysis.
Data
is
presented
as
enzyme
specific
activity
as
described
in
the
references
listed
below.

F.
References
Kelce,
W.
R.,
W.
J.
Krause,
and
V.
K.
Ganjam,
"
Unique
Regional
Distribution
of
Delta­
4­
3­
Ketosteroid
5a­
Oxidoreductase
and
Associated
Epididymal
Morphology
in
the
Marsupial
Didelphis
virginiana,"
Biology
of
Reproduction,
37,
1987,
pp.
403­
420.
Kelce,
W.
R.,
and
V.
K.
Ganjam,
"
Radiometric
Quantitation
of
Delta­
4­
3­
Ketosteroid
5a­
Oxidoreductase
Utilizing
High
Performance
Liquid
Chromatography,"
Steroids,
52,
1988,
pp.
217­
235.
Kelce,
W.
R.,
P.
K.
Rudeen,
and
V.
K.
Ganjam,
"
Prenatal
Ethanol
Exposure
Alters
Testicular
Steroidogenic
Enzyme
Activity
in
the
Newborn
Rat
Testis,"
Alcoholism:
Clinical
and
Experimental
Research,
13,
1989,
pp.
617­
621.
Kelce,
W.
R.,
M.
F.
Raisbeck,
and
V.
K.
Ganjam,
"
Gonadotoxic
Effects
of
2­
Hexanone
and
1,2­
Dibromo­
3­
chloropropane
on
the
Enzymatic
Activity
of
Rat
Testicular
17a­
Hydroxylase/
C17,20­
Lyase,"
Toxicology
Letters,
52,
1990,
pp.
331­
338.
Kelce,
W.
R.,
V.
K.
Ganjam,
and
P.
K.
Rudeen,
"
Inhibition
of
Testicular
Steroidogenesis
in
the
Neonatal
Rat
Following
Acute
Ethanol
Exposure,"
Alcohol,
1990,
pp.
75­
80.
Kelce,
W.
R.,
V.
K.
Ganjam,
and
P.
K.
Rudeen,
"
Effects
of
Fetal
Alcohol
Exposure
on
Brain
5a­
Reductase/
Aromatase
Activity,"
J.
Steroid
Biochem.,
35,
1990,
pp.
103­
106.
Kelce,
W.
R.,
A.
M.
Lubis,
W.
F.
Braun,
R.
S.
Youngquist,
and
V.
K.
Ganjam,
"
Influence
of
Rete
Testis
Fluid
Deprivation
on
the
Kinetic
Parameters
of
Goat
Epididymal
5a­
Reductase,"
Steroids,
55,
1990,
pp.
27­
31.

VI.
Research
Protocol
for
Assessment
of
Steroidogenesis
Using
In
Vitro
Testes
Culture
Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
a
procedure
for
the
quantitation
of
steroidogenic
hormone
production
from
minced
rat
testicular
tissue,
as
altered
hormone
production
is
indicative
of
altered
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
26
L
­
gonadal
enzyme
activity.

A.
50
Mg
Pieces
(
Rat
or
Other
Species)

1.
Each
testis
is
cut
into
pieces
that
weigh
approximately
50
mg,
placed
in
a
1.5
ml
microcentrifuge
tube
on
ice
and
media
is
added
to
all
of
them
at
the
same
time.
They
are
incubated
in
1.0
ml
of
media
and
are
assigned
to
various
treatment
as
dictated
by
the
protocol.

2.
The
tubes
are
capped
and
placed
into
a
microcentrifuge
tube
water
bath
rack
(
USA/
Scientific
Products).
The
cover
of
the
rack
is
fastened
down
in
order
to
hold
the
caps
of
the
tubes
closed.
These
racks
are
then
placed
in
a
reciprocating
shaker
set
at
72
cycles
per
minute.
The
shaker
is
set
in
an
incubator
at
36o
C
with
5%
filtered
carbon
dioxide.

3.
At
the
end
of
the
appropriate
amount
of
time
the
tubes
removed
from
the
incubator
and
are
spun
in
a
refrigerated
centrifuge
at
1500
RPM
(
430
x
g)
for
three
minutes.
The
supernatant
is
poured
into
a
1.5
ml
storage
tube
and
frozen
at
­
50o
C
until
assayed.

B.
Pieces
and
1/
4
Testes
(
Rat
And
Other
Species)

1.
The
testes
are
removed
from
the
animal
and
put
into
cold
Dulbecco's
phosphate
buffered
saline.

2.
The
testes
are
decapsulated
and
the
main
blood
vessels
are
removed.

3.
For
the
1/
4
testes
culture
each
testis
is
cut
in
half
through
the
long
axis
and
then
in
half
through
the
short
axis.
Each
piece
is
weighed
(
approximately
400
mg),
put
into
a
glass
20
ml
scintillation
vial
and
incubated
in
5
ml
of
media
199
(
Gibco,
catalog
#
400­
1100EB).
Each
piece
of
the
same
testis
is
assigned
to
+
or
­
hCG
treatment.

4.
The
scintillation
vials
are
capped
and
placed
in
racks
that
will
hold
them
securely.
They
are
placed
in
the
shaker
in
the
incubator
at
34o
C
and
5%
filtered
carbon
dioxide
and
shaken
gently
(
72
cycles/
minute)
for
one
hour.

5.
At
the
end
of
this
time
the
vials
are
removed
from
the
incubator.
A
200­
500
ml
sample
is
taken
from
each
one
and
placed
in
a
storage
vial
to
be
frozen
for
later
assays.

6.
hCG
is
added
to
the
appropriate
vial
and
placed
back
into
the
incubator.
The
process
is
repeated
every
hour
for
the
next
three
hours.

C.
Stock
Solutions
and
Media
Preparation
1.
PREGNENOLONE:
5­
Pregnen­
3b­
ol­
20­
one
M.
W.=
316.5
Sigma
#
P
9129
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
27
L
­
STORE
STOCK
IN
REFRIGERATOR­­
STABLE
FOR
ONE
MONTH
STOCK:
0.03165
grams
+
10
ml
ETOH=
10,000
µ
M/
ml
STOCK
1:
For
10
µ
M
Pregnenolone:
Make
a
1:
10
dilution
of
the
original
stock
=
1,000
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture
=
10
µ
M
Pregnenolone
STOCK
2:
For
2
µ
M
Pregnenolone:
Make
a
1:
10
dilution
of
STOCK
1
=
100
µ
M/
ml
ADD
20
µ
l
to
1
ml
of
M­
199
culture
=
2
µ
M
Pregnenolone
STOCK
3:
For
0.1
µ
M
Pregnenolone:
Make
a
1:
10
dilution
of
STOCK
2
=
10
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture
=
0.1
µ
M
Pregnenolone
2.
PROGESTERONE:
4­
Pregnene­
3,20­
Dione
M.
W.=
314.5
Sigma
#
P
0103
STORE
STOCK
IN
REFRIGERATOR­­
STABLE
FOR
TWO
MONTHS
STOCK:
0.03145
grams
+
10
ml
ETOH
=
10,000
µ
M/
ml
STOCK
1:
For
10
µ
M
Progesterone:
Make
a
1:
10
dilution
of
the
original
stock
=
1,000
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture
=
10
µ
M
Progesterone
STOCK
2:
For
2
µ
M
Progesterone:
Make
a
1:
10
dilution
of
STOCK
1
=
100
µ
M/
ml
ADD
20
µ
l
to
1
ml
of
M­
199
culture
=
2
µ
M
Progesterone
STOCK
3:
For
0.1
µ
M
Progesterone
Make
a
1:
10
dilution
of
STOCK
2
=
10
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture
=
0.1
µ
M
Progesterone
3.
CHOLESTEROL:
20
a­
Hydroxycholesterol
M.
W.=
402.5
Sigma
#
H
6378
STORE
STOCK
IN
REFRIGERATOR­­
STABLE
FOR
TWO
MONTHS
STOCK:
0.020
grams
+
5ml
ETOH
=
10,000
µ
M/
ml
STOCK
1:
For
10
µ
M
Cholesterol:
Make
a
1:
10
dilution
of
the
original
stock
=
1,000
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture
=
10
µ
M
Cholesterol
STOCK
2:
For
1
µ
M
Cholesterol:
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
28
L
­
Make
a
1:
10
dilution
of
STOCK
1
=
100
µ
M/
ml
ADD
20
µ
l
to
1
ml
of
M­
199
culture
=
1
µ
M
Cholesterol
STOCK
3:
For
0.1
µ
M
Cholesterol:
Make
a
1:
10
dilution
of
STOCK
2
=
10
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture
=
0.1
µ
M
Cholesterol
4.
ANDROSTENEDIONE:
4­
Androstene­
3,17­
Dione
M.
W.=
286.4
Sigma
#
A
9630
STORE
STOCK
IN
REFRIGERATOR­­
STABLE
FOR
TWO
MONTHS
STOCK:
0.02864
grams
+
10ml
ETOH
=
10,000
µ
M/
ml
STOCK
1:
For
10
µ
M
Androstenedione:
Make
a
1:
10
dilution
of
the
original
stock
=
1,000
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture
=
10
µ
M
Androstenedione
STOCK
2:
For
2
µ
M
Androstenedione:
Make
a
1:
10
dilution
of
STOCK
1
=
100
µ
M/
ml
ADD
20
µ
l
to
1
ml
of
M­
199
culture
=
2
µ
M
Androstenedione
STOCK
3:
For
0.1
µ
M
Androstenedione:
Make
a
1:
10
dilution
of
STOCK
2
=
10
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture=
0.1
µ
M
Androstenedione
5.
hCG:
Sigma
#
CG
10
(
p.
258)
=
10,000
IU/
ml
STOCK:
10,000
IU/
vial
in
1
ml
of
distilled
water
=
10,000
mIU,
aliquot
into
100
µ
l
aliquots
and
freeze.

WORKING
STOCK:
10
µ
l
of
original
stock
into
10
ml
of
M­
199
This
will
=
10,000
mIU/
ml.
ADD
10
µ
l
of
working
stock
to
each
ml
of
culture
media.

6.
db
cAMP:
N6,
O21
Dibutyl
Adenosine
3':
5'
Cyclic
Monophosphate
Sigma
#
D
0627,
M.
W.
491.4
PREPARE
FRESH
DAILY
STOCK:
0.04914
GRAMS
+
1
ml
M­
199
=
100
mM/
ml
10
µ
l
STOCK
+
1ml
M­
199
=
1
mM
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
29
L
­
ADD
10
µ
l
to
1ml
M­
199
20
µ
l
STOCK
+
1ml
M­
199
=
2
mM
ADD
20
µ
l
to
1ml
M­
199
40
µ
l
STOCK
+
1ml
M­
199
=
4
mM
ADD
40
µ
l
to
1ml
M­
199
7.
17
a
OH
PROGESTERONE
4­
PREGNEN­
17a,
20b­
DIOL­
3­
ONE
Sigma
#
P
6285,
M.
W.
332.5
STORE
STOCK
IN
REFRIGERATOR
STABLE
TWO
MONTHS
STOCK
=
0.3325
grams
+
10
ml
ETOH
STOCK
1:
For
10
µ
M
17
a
OH
Progesterone:
Make
a
1:
10
dilution
of
the
original
stock
=
1,000
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture
=
10
µ
M
Progesterone
STOCK
2:
For
a
2
µ
M
17
a
OH
Progesterone:
Make
a
1:
10
dilution
of
STOCK
1
=
100
µ
M/
ml
ADD
20
µ
l
to
1
ml
of
M­
199
culture
=
2
µ
M
Progesterone
STOCK
3:
For
a
0.1
µ
M
17
a
OH
Progesterone
Make
a
1:
10
dilution
of
STOCK
2
=
10
µ
M/
ml
ADD
10
µ
l
to
1
ml
of
M­
199
culture
=
0.1
µ
M
Progesterone
Medium
199
powder
is
purchased
from
Gibco
Laboratories
(
Catalog
#
400­
1122EB).
One
liter
of
liquid
medium
is
prepared
as
follows:

a.
Add
powder
to
a
1000
ml
graduated
cylinder.
Rinse
the
packet
three
times
with
distilled
water
and
bring
the
volume
up
to
1000
ml.
b.
Put
a
magnetic
stir
bar
into
the
cylinder
and
place
on
a
stirrer.
Stir
until
all
powder
is
off
the
bottom
of
cylinder
and
incorporated
into
liquid.
Pour
into
1000
ml
beaker
(
for
ease
in
mixing
and
determining
pH).
Position
pH
electrode
in
beaker.
c.
Add
while
stirring:
0.71
g
Sodium
Bicarbonate
(
Sigma,
S­
8875)
2.1
g
HEPES
(
Sigma,
H­
3375)
1.0
g
BSA
(
Swartz­
Mann,
#
802247)
0.025
g
Trypsin
Inhibitor
(
Sigma,
T­
9003)
d.
Adjust
pH
of
medium,
while
stirring,
to
pH
7.1­
7.3
(
0.2
to
0.3
pH
units
below
desired
final
pH
7.4)
using
1
N
NaOH
(
Sigma,
S­
5881)
or
1
N
HCL
(
Sigma,
920­
1).
e.
Sterilize
immediately
by
membrane
filtration
(
Krackler
Scientific,
cat.#
161­
0026,
pore
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
30
L
­
size,
0.2
microns)
using
negative
pressure.

Preparation
of
Media
containing
hCG
(
100
mIU/
ml)

One
vial
of
hCG
(
Sigma,
CG­
10)
containing
10,000
IU
is
diluted
in
1
ml
of
distilled
water.
This
is
separated
into
100ml
aliquots
and
stored
at
­
50o
C.
When
ready
for
use
a
vial
is
thawed
and
10
µ
l
are
added
to
10
ml
of
media
199.
This
is
then
added
to
the
culture
at
10
µ
l
per
ml
of
media
to
give
a
concentration
of
100
mIU's
per
ml.

D.
Additional
Language
to
Consider
In
Developing
Steroidogenesis
Assay
Procedure
An
in
vitro
testis
culture
can
be
used
as
a
screening
assay
to
evaluate
effects
on
the
mammalian
steroid
hormone
synthesis
pathway.
The
procedure
outlined
below
is
a
modification
of
that
used
by
Lasky
et
al.
1994.
Testes
are
removed
from
ten
week­
old
untreated
rats,
and
decapsulated.
Approximately
50
mg
of
testicular
parenchyma
is
then
placed
into
a
20
ml
scintillation
vial
containing
5
ml
of
culture
media
(
RMPI­
1640
media,
10%
fetal
calf
serum,
50
ug/
ml
soybean
trypsin
inhibitor).
The
test
materials
are
dissolved
in
DMSO
and
then
added
at
an
appropriate
concentration
to
each
scintillation
vial;
the
final
concentration
of
the
solvent
(
DMSO)
in
the
culture
media
should
not
exceed
0.2%
(
v/
v).
(
Guidance
on
selection
of
"
appropriate
concentrations"
needs
to
be
added.)
Finally,
either
10
ul
of
hCG
(
500
IU/
ml)
stock
solution,
or
10
ul
of
distilled
water
(
for
non­
hCG
stimulated
controls)
is
added.
The
vials
are
then
capped,
vortexed,
and
incubated
vertically
for
three
hours
at
34
°
C
under
vigorous
shaking
(
175
rpm).
At
the
end
of
the
incubation
period,
1.4
ml
of
the
culture
media
is
removed
and
centrifuged
at
14,000
x
g
for
five
minutes
(
4
°
C)
to
pellet
all
remaining
testicular
parenchyma.
The
resulting
supernatant
is
then
assayed
for
testosterone
or
estradiol
concentration
by
RIA.

In
Vivo
Assays
VII.
Research
Protocol
for
Assessment
of
Uterotropic
Activity
in
the
Adult
Ovariectomized
and
Juvenile
(
21
Day
Old)
Female
Rat
Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
procedures
for
the
quantitation
of
the
effects
of
potentially
estrogenic
compounds
on
the
uterus
of
the
adult
ovariectomized
female
rat.

Required
endpoints:
Uterine
Weight
Uterine
Histology
Optional
endpoints:
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
31
L
­
Bodyweight
change
Vaginal
cytology
and
histology
Serum
hormones
(
T4,
T3,
TSH,
LH,
E2,
and
Prolactin)
Liver
Weight
and
Histology
A.
General
Conditions
Typically,
prior
to
treatment,
female
rats
are
housed
in
groups
of
2
or
3
per
cage.
The
following
describes
the
housing
conditions
under
which
our
animals
are
housed.
Reasonable
variations
of
this
portion
of
the
protocol
should
be
tolerated.
Rats
are
housed
in
clear
plastic
cages
(
20x25x47
cm)
with
heat
treated
(
to
eliminate
resins
that
induce
liver
enzymes)
laboratory
grade
pine
shavings
(
Northeastern
Products,
Warrensburg,
NY)
as
bedding.
Animals
are
maintained
on
Purina
Rat
Chow
(
5001)
and
tap
water
ad
libitum,
in
a
room
with
a
14:
10
hour
photoperiod
(
L/
D,
lights
off
at
11:
00
EST)
and
temperature
of
20­
24o
C
with
a
relative
humidity
of
40­
50
%.

B.
Subjects
The
STWG
Selected
Option
2a
as
the
preferred
method.

·
OVARIECTOMIZED
ADULT
FEMALE
RATS.
Adult
SD
(
or
LE)
female
rat
can
be
ovariectomized
in
the
laboratory
under
appropriate
conditions
of
anesthesia,
and
sterility
or
purchased
from
the
supplier
(
about
$
6
per
rat).
Upon
receipt
animals
are
examined
and
weighed,
and
allowed
to
acclimate
from
surgery
for
at
least
three
weeks.
This
should
provide
the
animals
sufficient
time
to
recover
from
the
surgery
and
allows
for
regression
of
the
reproductive
tract.
For
example,
upon
examination,
the
vaginal
opening
should
be
small
and
difficult
to
lavage.
Vaginal
smears,
if
collected
would
lack
cellularity,
especially
cornified
or
nucleated
epithelial
cells.
Animals
that
displayed
any
characteristics
of
continued
ovarian
hormonal
exposure,
as
indicated
above,
should
be
deleted
from
the
study.
Forty
females
of
uniform
weight
are
selected
for
the
study
and
randomly
assigned
to
treatment
in
a
manner
that
provides
similar
means
and
variances
in
body
weight.

·
JUVENILE
FEMALE
RATS.
At
21
days
of
age
female
SD
(
or
LE)
rats
will
be
weaned
from
their
litters.
These
litters
are
derived
from
pregnant
females
that
were
generated
in
house
by
matings
or
purchased
from
a
supplier
as
"
timed
pregnant"
on
days
seven
to
ten
of
gestation.
Upon
birth,
the
litters
are
culled
to
eight
to
ten
pups
in
order
to
assure
normal
growth
rates
in
all
pups.
Growth
is
monitored
on
at
least
a
weekly
basis
and
any
unthrifty
litters
or
runted
pups
should
be
discarded
from
the
study.
Enough
litters
should
be
used
to
assure
that
about
55
pups
are
available
at
weaning.
Pups
are
weaned
at
21
days
of
age
and
weighed,
weight
ranked.
A
population
of
forty
female
rats
that
is
as
homogeneous
as
possible
is
selected
for
the
study
by
eliminating
the
"
outliers"
(
i.
e.,
the
largest
and
smallest
of
the
pups).
In
this
regard,
one
nuisance
variable,
i.
e.
body
weight
at
weaning,
is
experimentally
controlled.
In
addition,
body
weight
at
weaning
is
also
statistically
controlled,
by
assigning
the
forty
females
to
four
treatment
groups
in
a
manner
that
provides
each
group
with
similar
means
and
variances
in
weaning
weight.
This
source
of
variance
is
included
in
the
data
analysis
as
a
blocking
factor
or
by
using
weaning
weight
as
a
covariate.
In
addition,
it
is
imperative
that
treatments
should
be
initiated
no
later
than
22
days
of
age,
as
waiting
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
32
L
­
just
a
few
days
longer
can
result
in
failure
of
the
study
as
control/
untreated
female
rats
will
begin
to
show
dramatic
fluctuations
in
uterine
size
as
they
approach
puberty.

·
EXPERIMENTAL
DESIGN.
The
design
is
a
randomized
complete
block
design
with
ten
female
rats
in
each
of
four
treatment
groups.
The
treatment
conditions
are:
(
1)
Vehicle­
injected
control,
(
2)
Xenobiotic­
injected,
(
3)
Estradiol
treated,
and
(
4)
Estradiol
plus
xenobiotic
injected.
If
necessary,
the
study
can
be
conducted
in
blocks
rather
than
at
one
time.
In
this
case,
the
blocks
should
be
contain
all
treatment
conditions
and
balanced
with
respect
to
numbers
of
animals
in
each
block
(
i.
e.,
two
blocks
with
four
treatment
conditions,
with
five
females/
treatment/
block).

·
TREATMENT.
Treatments
are
administered
by
sc
injections
on
the
dorsal
surface,
caudal
to
the
nape
of
the
neck,
but
anterior
to
the
base
of
the
tail,
with
a
21
gauge
1.0
inch
needle,
using
a
1
cc
glass
tuberculin
syringe
for
each
treatment
condition,
in
corn
oil
(
1­
2.5
ml/
kg)
at
0700­
1000
for
three
consecutive
days.
The
estradiol­
treated
groups
should
be
dosed
with
1.0
m
g/
rat
of
free
estradiol
per
day
simultaneously
with
the
xenobiotic
treatment.
The
xenobiotic
should
be
administered
on
a
mg/
kg
body
weight
basis,
adjusted
daily
for
weight
changes
and
body
weight
and
volume
of
the
dose
administered
should
be
recorded
each
day.

·
NECROPSY.
On
the
third
day,
approximately
6
hours
after
the
final
treatment
the
females
are
anesthetized
in
CO
2
and
body
weight
is
recorded
to
the
nearest
0.1
g.
The
rat
is
subsequently
euthanized
by
decapitation,
and
serum
collected
for
optional
hormonal
analysis.
During
necropsy
care
must
be
taken
to
remove
mesenteric
fat
with
small
surgical
iris
scissors
from
the
uterine
horns
such
that
the
uterine
fluid
is
retained.
Once
free
from
the
fat
and
adnexa
the
uterus
and
cervices
are
separated
from
the
vagina
and
the
weight
of
the
uterus
with
fluid
is
recorded
to
the
nearest
mg.
Following
this
observation,
the
uterus
is
placed
on
a
paper
towel,
slit
to
allow
the
fluid
contents
to
leak
out,
gently
blotted
dry
and
reweighed.
The
uterus
and
vagina
also
can
be
examined
histologically
for
"
estrogen­
like"
alterations.
The
tissues
should
be
placed
in
Bouins
for
24
hours,
after
which
they
are
rinsed
and
stored
in
70
%
alcohol,
until
being
embedded
in
paraffin,
stained
with
H
and
E,
and
examined
for
histological
alterations
(
increased
endometrial
epithelial
cell
height,
increased
glandularity,
increased
vaginal
cornification).
In
addition,
as
estrogens
reduce
food
consumption,
induce
vaginal
cornification,
reduce
serum
LH
and
increase
prolactin,
these
endpoints
could
be
examined
at
this
time.
·
STATISTICAL
ANALYSIS.
Uterine
weight
data
are
analyzed
as
one­
way
ANOVAs
(
Treatment),
using
PROC
GLM
the
SAS
version
6.08
on
the
USEPA
IBM
mainframe.
The
regression
model
should
include
bodyweight
at
weaning
as
a
covariate.
If
the
study
was
conducted
in
blocks,
then
the
analysis
is
a
two­
way
ANOVA
with
Block
and
Treatment
as
main
effects,
again,
bodyweight
at
weaning
is
used
as
a
covariate.
Statistically
significant
effects
(
p
<
0.05,
F
statistic)
should
be
examined
using
the
LSMEANS
procedure
on
SAS
(
two­
tailed
t­
test)
to
compare
group
(
1)
vehicle­
treated
to
group
(
2)
xenobiotic­
treated,
and
group
(
3)
estradiol­
treated
to
group
(
4)
xenobiotic­
plus
estradiol­
treatments.

·
DATA
SUMMARIZATION.
Data
should
be
summarized
in
tablular
form
containing
the
mean,
standard
error
of
the
mean
and
sample
size
for
each
group
in
the
table.
Individual
data
tables
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
33
L
­
should
also
be
included.
The
mean,
SE
and
CV
values
for
the
control
data
should
be
examined
to
determine
if
they
meet
acceptable
QA
criteria
based
upon
normal
control
values.
Data
presented
should
include
at
least,
uterine
weight
with
and
without
fluid,
and
body
weight
at
necropsy
and
body
weight
change
from
day
21
to
24.
Data
may
be
also
be
presented
after
covariance
adjustment
for
body
weight,
but
this
should
not
replace
presentation
of
the
unadjusted
data.

VIII.
Research
Protocol
for
Assessment
of
Pubertal
Development
and
Thyroid
Function
in
Juvenile
(
21
Day
Old)
Female
Rats
Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
procedures
for
the
quantitation
of
the
effects
of
compounds
on
pubertal
development
and
thyroid
function
in
the
intact
juvenile
female
rat.
This
assay
detects
compounds
that
display
antithryoid,
estrogenic,
antiestrogenic
(
ER
or
steroid
enzyme
mediated)
activity,
or
alter
FSH,
LH,
prolactin,
Growth
Hormone
or
hypothalamic
function.

Required
Endpoints:
Growth
Age
and
Weight
at
Vaginal
Opening
Serum
T4
and
TSH
Thyroid
Histology
Uterine
and
Ovarian
Weights
and
Histology
Optional
Endpoints:
Serum
T3,
E2
and
prolactin
Thyroid
Weight
Vaginal
cytology
and
histology
Liver,
Kidney,
pituitary,
and
adrenal
Weights
and
Histology
Ex
Vivo
ovarian
and
pituitary
hormone
production
Hypothalamic
neurotransmitter
levels
Onset
of
estrous
cyclicity
and
cycle
length
(
requires
extension
of
dosing)

A.
General
Conditions
Typically,
prior
to
the
onset
of
the
study,
pregnant
female
rats
are
housed
individually.
After
assignment
to
treatments,
they
should
be
housed
in
pairs
of
similarly
treated
females.
The
following
describes
the
housing
conditions
under
which
our
animals
are
housed.
Reasonable
variations
of
this
portion
of
the
protocol
should
be
tolerated.
Rats
are
housed
in
clear
plastic
cages
(
20x25x47
cm)
with
heat
treated
(
to
eliminate
resins
that
induce
liver
enzymes)
laboratory
grade
pine
shavings
(
Northeastern
Products,
Warrensburg,
NY)
as
bedding.
Animals
are
maintained
on
Purina
Rat
Chow
(
5001)
and
tap
water
ad
libitum,
in
a
room
with
a
14:
10
hour
photoperiod
(
L/
D,
lights
off
at
11:
00
EST)
and
temperature
of
20­
24o
C
with
a
relative
humidity
of
40­
50
%.

B.
Subjects
­
Juvenile
Female
Rats
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
34
L
­
At
21
days
of
age
female
SD
(
or
LE
rats
will
be
weaned
from
their
litters.
These
litters
are
derived
from
individually
housed
pregnant
females
that
were
generated
in
house
by
matings
or
purchased
from
a
supplier
as
"
timed
pregnant"
on
days
seven
to
ten
of
gestation.
Upon
birth,
the
litters
are
culled
to
eight
to
ten
pups
in
order
to
assure
normal
growth
rates
in
all
pups.
Growth
is
monitored
on
at
least
a
weekly
basis
and
any
unthrifty
litters
or
runted
pups
should
be
discarded
from
the
study.
Enough
litters
should
be
used
to
assure
that
about
45
pups
are
available
at
weaning.
Pups
are
weaned
at
21
days
of
age
and
weighed
to
the
nearest
0.1
g,
weight
ranked.
A
population
of
thirty
female
rats
that
is
as
homogeneous
as
possible
is
selected
for
the
study
by
eliminating
the
"
outliers"
(
i.
e.,
the
largest
and
smallest
of
the
pups).
In
this
regard,
one
nuisance
variable,
i.
e.
body
weight
at
weaning,
is
experimentally
controlled.
In
addition,
body
weight
at
weaning
is
also
statistically
controlled,
by
assigning
the
thirty
females
to
two
treatment
groups
in
a
manner
that
provides
each
group
with
similar
means
and
variances
in
weaning
weight.
This
source
of
variance
is
included
in
the
data
analysis
as
a
blocking
factor
or
by
using
weaning
weight
as
a
covariate.
In
addition,
it
is
imperative
that
treatments
should
be
initiated
no
later
than
22
days
of
age,
as
waiting
just
a
few
days
longer
can
result
in
failure
of
the
study
as
control/
untreated
female
rats
will
begin
to
display
"
puberty"
(
i.
e.
vaginal
opening)
within
a
few
days.

C.
Experimental
Design
The
design
is
a
randomized
complete
block
(
bodyweight
at
weaning
is
the
blocking
factor)
design
with
fifteen
female
rats
in
each
of
two
treatment
groups.
The
treatment
conditions
are
(
1)
Vehicle­
treated
and
(
2)
Xenobiotic­
treated.
If
necessary,
the
study
can
be
conducted
in
blocks
rather
than
at
one
time.
In
this
case,
the
blocks
should
be
contain
all
treatment
conditions
and
balanced
with
respect
to
numbers
of
animals
in
each
block
(
i.
e.,
two
blocks
with
two
treatment
conditions,
with
eight
females/
treatment/
block).
Varying
dosage
levels
of
the
xenobiotic
can
be
employed,
although
only
one
high
dosage
level
(
at
or
just
below
the
MTD
or
limit
dose)
is
requried.

D.
Treatment
Treatments
are
administered
daily
by
oral
gavage
in
2.5
to
5.0
ml/
kg
bodyweight
corn
oil
from
22
days
of
age
for
20
days.
This
duration
of
treatment
is
unnecessary
to
detect
estrogenic
chemicals,
but
is
required
for
the
detection
of
pubertal
delays
and
antithyroid
effects.
Using
a
#
18
gavage
needle
and
a
1
cc
glass
tuberculin
syringe
for
each
treatment.
Xenobiotics
are
administered
in
corn
oil
at
2.5
ml/
kg
body
weight
at
0700­
1000
daily.
The
treatments
should
be
administered
on
a
mg/
kg
body
weight
basis,
adjusted
daily
for
weight
changes
and
body
weight
and
volume
of
the
dose
administered
should
be
recorded
each
day.

E.
Vaginal
Opening
Females
are
examined
daily
for
vaginal
opening.
The
appearance
of
complete
vaginal
opening,
a
small
"
pin
hole"
or
a
vaginal
thread
should
all
be
noted
if
and
when
they
occur.
In
addition,
the
weight
at
complete
vaginal
opening
should
be
noted.
Additional,
optional
measures
could
be
taken
that
would
facilitate
interpretation
of
changes
in
vaginal
opening
could
be
taken
on
these
females
prior
to
necropsy.
The
collection
of
daily
vaginal
lavages
to
identify
the
age
at
onset
of
estrous
cyclicity,
and
the
age
at
first
estrous
would
enable
one
to
distinguish
pseudoprecocious
puberty
from
true
precocious
puberty.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
35
L
­
F.
Necropsy
On
the
last
day
of
treatment,
the
females
are
anesthetized
in
CO
2
and
body
weight
is
recorded.
The
rat
is
subsequently
euthanized
by
decapitation,
and
serum
collected
for
optional
hormonal
analysis.
At
necropsy,
the
paired
ovarian,
uterine,
liver,
adrenal
and
body
weights
should
be
recorded.
During
necropsy
care
must
be
taken
to
remove
mesenteric
fat
with
small
surgical
iris
scissors
from
the
uterine
horns
such
that
the
uterine
fluid
is
retained
(
organ
weights
to
the
nearest
mg).
Once
free
from
the
fat
and
adnexa
the
uterus
and
cervices
are
separated
from
the
vagina
and
the
weight
of
the
uterus
with
fluid
is
recorded.
Following
this
observation,
the
uterus
is
placed
on
a
paper
towel,
slit
to
allow
the
fluid
contents
to
leak
out,
gently
blotted
dry
and
reweighed.
For
the
ovaries,
attached
fat
and
the
oviducts
should
be
carefully
removed
prior
to
weighing.
The
thyroid,
ovaries,
uterus
and
vagina
also
can
be
examined
histologically.
These
tissues
should
be
placed
in
Bouins
for
24
hours,
after
which
they
are
rinsed
and
stored
in
70
%
alcohol,
until
being
embedded
in
paraffin,
/
stained
with
H
and
E,
and
examined
for
histological
alterations.
In
addition,
serum
thyroxine
(
T4)
and
TSH
should
be
measured.

G.
Statistical
Analysis
All
data
(
age
at
vaginal
opening,
weight
at
vaginal
opening,
body
and
organ
weights
at
necropsy,
and
serum
hormones)
are
analyzed
as
one­
way
ANOVAs
(
Control
versus
Treatment),
using
PROC
GLM
the
SAS
version
6.08
on
the
USEPA
IBM
mainframe.
The
regression
model
should
include
bodyweight
at
weaning
as
a
covariate.
If
the
study
was
conducted
in
blocks,
then
the
analysis
is
a
twoway
ANOVA
with
Block
and
Treatment
as
main
effects,
and
again,
bodyweight
at
weaning
is
used
as
a
covariate.
Statistically
significant
effects
(
p
<
0.05,
F/
t
statistic)
should
be
examined
using
the
LSMEANS
procedure
on
SAS
(
two­
tailed
t­
test)
to
compare
group
(
1)
vehicle­
treated
to
the
(
2)
xenobiotic­
treated
group.
For
organ
weight
data,
bodyweight
at
necropsy
could
be
used
as
a
covariate
in
the
model,
although
this
is
rarely
useful
for
endocrine­
related
endpoints.
If
treatment
reduces
growth
and
delays
vaginal
opening,
the
mechanism
responsible
for
the
delay
is
always
in
question.
In
this
regard,
body
weight
change
from
day
22
until
the
average
age
of
vaginal
opening
in
the
control
group
could
be
used
as
a
covariate
in
the
regression
model,
however,
this
is
not
the
best
use
of
ANOCOVA.
If
serum
hormone
levels,
or
any
other
data,
display
heterogeneity
of
variance,
then
appropriate
data
transformations
should
be
employed.
Often
log
transformation
of
serum
hormone
data
is
required
because
the
variance
is
proportional
to
the
mean.

H.
Data
Summarization
Data
should
be
summarized
in
tablular
form
containing
the
mean,
standard
error
of
the
mean
and
sample
size
for
each
group
in
the
table.
Individual
data
tables
should
also
be
included.
The
mean,
SE
and
CV
values
for
the
control
data
should
be
examined
to
determine
if
they
meet
acceptable
QA
criteria
for
consistency
with
normal
values.
Data
presented
should
include
at
least,
age
and
weight
at
vaginal
opening,
ovarian,
uterine
(
with
and
without
fluid),
adrenal,
liver
and
body
weights
at
necropsy,
body
weight
change
from
day
21
to
necropsy
and
serum
T4
and
TSH.
Data
may
be
also
be
presented
after
covariance
adjustment
for
body
weight,
but
this
should
not
replace
presentation
of
the
unadjusted
data.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
36
L
­
IX.
Research
Protocol
for
Assessment
of
(
Anti­)
Androgenic
Activity
in
the
Immature
Male
Rat:
The
"
Hershberger"
Assay
Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
procedures
for
the
quantitation
of
the
effects
of
potentially
antiandrogenic
and
androgenic
compounds
on
the
hormone
dependent
tissues
in
the
immature
male
rat.
Adapted
from
Hershberger
et
al.
1953.
(
Proc
Soc
Exp
Biol
Med.,
83:
175.)

Required
Endpoints
:
Growth
Seminal
vesicle
plus
coagulating
gland
weight
(
with
and
without
fluid)
Ventral
Prostate
Weight
Levator
ani
plus
bulbocavernosus
weight
Optional
Measures:
Serum
testosterone,
estradiol,
LH,
prolactin,
T4,
TSH,
and
T3
Liver,
kidney,
thyroid,
adrenal
and
pituitary
weights
and
histology
Hypothalamic
neurotransmitter
levels
A.
General
Conditions
Typically,
prior
to
and
during
treatment,
male
rats
are
housed
in
groups
of
two
per
cage.
The
following
describes
the
housing
conditions
under
which
our
animals
are
housed.
Reasonable
variations
of
this
portion
of
the
protocol
should
be
tolerated.
Rats
are
housed
in
clear
plastic
cages
(
20x25x47
cm)
with
heat
treated
(
to
eliminate
resins
that
induce
liver
enzymes)
laboratory
grade
pine
shavings
(
Northeastern
Products,
Warrensburg,
NY)
as
bedding.
Animals
are
maintained
on
Purina
Rat
Chow
(
5001)
and
tap
water
ad
libitum,
in
a
room
with
a
14:
10
hour
photoperiod
(
L/
D,
lights
off
at
11:
00
EST)
and
temperature
of
20­
24o
C
with
a
relative
humidity
of
40­
50
%.

B.
Subjects
­
Castrated
Immature
Male
Rats
Castrated
immature
animals
can
be
purchased
from
a
supplier
or
produced
in
house.
For
in
house
efforts,
21
days
of
age
male
SD
(
or
LE)
rats
will
be
weaned
from
their
litters.
These
litters
are
derived
from
individually
housed
pregnant
females
that
were
generated
in
house
by
matings
or
purchased
from
a
supplier
as
"
timed
pregnant"
on
days
seven
to
ten
of
gestation.
Upon
birth,
the
litters
are
culled
to
eight
to
ten
pups
in
order
to
assure
normal
growth
rates
in
all
pups.
Growth
is
monitored
on
at
least
a
weekly
basis
and
any
unthrifty
litters
or
runted
pups
should
be
discarded
from
the
study.
Enough
litters
should
be
used
to
assure
that
about
55
pups
are
available
at
weaning.
At
21
days
of
age
males
are
weaned
and
castrated
under
appropriate
conditions
of
anesthesia
and
sterility
and
allowed
to
recover
for
one
week
being
house
in
cages
with
three
to
four
males
per
group.
Alternatively,
castrated
21
day
old
male
rats
can
be
purchased
from
a
supplier.
At
27
days
of
age
pups
are
weighed
to
the
nearest
0.1
g,
weight
ranked
and
a
homogeneous
population
of
forty
male
rats
is
selected
for
the
study
by
eliminating
the
"
outliers"
(
i.
e.,
the
largest
and
smallest
of
the
pups).
In
this
regard,
one
nuisance
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
37
L
­
variable,
i.
e.
body
weight
at
the
start
of
the
study,
is
experimentally
controlled.
In
addition,
body
weight
is
also
statistically
controlled,
by
assigning
the
forty
males
to
one
of
four
treatment
groups
in
a
manner
that
provides
each
group
with
similar
means
and
variances
in
weaning
weight.
This
source
of
variance
is
included
in
the
data
analysis
as
a
blocking
factor
or
by
using
the
initial
body
weight
as
a
covariate.

C.
Experimental
Design
The
design
is
a
randomized
complete
block
(
initial
body
weight
is
the
blocking
factor)
design
with
ten
28
day
old
male
rats
in
each
of
four
treatment
groups.
The
treatment
conditions
to
detect
androgenicity
are
(
1)
Oral
Vehicle­
treated
and
(
2)
Oral
Xenobiotic­
treated,
while
antiandrogens
are
detection
by
comparing
group
(
3)
sc
Testosterone
propionate
(
50
mg/
d)
plus
oral
vehicle
treated
versus
(
4)
sc
TP
and
oral
xenobiotic­
treatment.
If
necessary,
the
study
can
be
conducted
in
blocks
rather
than
at
one
time.
In
this
case,
the
blocks
should
be
contain
all
treatment
conditions
and
balanced
with
respect
to
numbers
of
animals
in
each
block
(
i.
e.,
two
blocks
with
two
treatment
conditions,
with
five
males/
treatment/
block).

D.
Treatment
Vehicle
(
groups
1
and
3)
and
xenobiotic
(
groups
2
and
4)
oral
treatments
are
administered
daily
for
seven
(
to
ten)
days
by
gavage
from
28
days
of
age
to
37
days
of
age.
Treatments
are
administered
using
a
separate
#
18
gavage
needle
and
a
1
cc
glass
tuberculin
syringe
for
each
treatment.
Xenobiotics
are
administered
in
corn
oil
at
2.5
ml/
kg
body
weight
at
0700­
1000
daily.
The
oral
treatments
should
be
administered
on
a
mg/
kg
body
weight
basis,
adjusted
daily
for
weight
changes
and
body
weight
and
volume
of
the
dose
administered
should
be
recorded
each
day.
Sc
injections
of
TP
(
50
m
g/
d
in
0.2
ml
oil
to
groups
3
and
4)
are
administered
at
the
same
time
of
day
on
the
dorsal
surface,
caudal
to
the
nape
of
the
neck,
but
anterior
to
the
base
of
the
tail,
with
a
21
gauge
1.0
inch
needle,
using
a
1
cc
glass
tuberculin
syringe
for
each
treatment
condition.

E.
Necropsy
On
the
day
after
the
last
treatment,
males
are
anesthetized
in
CO
2
and
body
weight
is
recorded.
The
rat
is
subsequently
euthanized
by
decapitation,
and
serum
collected
for
optional
hormonal
analysis.
At
necropsy,
the
paired
testicular,
paired
epididymal,
liver,
ventral
prostate,
seminal
vesicle
(
with
coagulating
glands
and
fluid),
levator
ani
plus
bulbocavernosus
muscles,
to
the
nearest
mg
and
body
weights
(
nearest
0.1
g)
should
be
recorded.
During
necropsy
care
must
be
taken
to
remove
mesenteric
fat
with
small
surgical
iris
scissors
from
these
tissues
such
that
the
fluid
in
the
sex
accessory
glands
is
retained.
Once
free
from
the
fat
and
adnexa
the
weight
with
fluid
is
recorded
and
these
tissues,
and
the
thyroid,
also
can
be
examined
histologically.
Tissues
should
be
placed
in
Bouins
for
24
hours,
after
which
they
are
rinsed
and
stored
in
70
%
alcohol,
until
being
embedded
in
paraffin,
stained
with
H
and
E,
and
examined
for
histological
alterations.
In
addition,
serum
thyroxine
(
T4)
and
TSH
should
be
measured.
Serum
LH
and
androgen
levels
are
optional.

F.
Statistical
Analysis
All
data
(
body
and
organ
weights
at
necropsy,
and
serum
hormones)
are
analyzed
as
one­
way
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
38
L
­
ANOVAs
for
two
orthogonal
contrasts.
The
first
is
between
groups
1
and
2
identifies
androgens,
while
the
comparison
between
groups
3
and
4
detects
antiandrogenicity.
Data
are
analyzed
on
PROC
GLM
the
SAS
version
6.08
on
the
USEPA
IBM
mainframe.
The
regression
model
should
include
initial
bodyweight
at
weaning
as
a
covariate.
If
the
study
was
conducted
in
blocks,
then
the
analysis
is
a
two­
way
ANOVA
with
Block
and
Treatment
as
main
effects,
again,
initial
bodyweight
is
used
as
a
covariate.
Statistically
significant
effects
(
p
<
0.05,
F/
t
statistic)
should
be
examined
using
the
LSMEANS
procedure
on
SAS
(
two­
tailed
t­
test)
to
compare
group
1)
vehicle­
treated
to
the
2)
xenobiotic­
treated
group.
For
organ
weight
data,
bodyweight
at
necropsy
could
be
used
as
a
covariate
in
the
model,
although
this
is
rarely
useful
for
endocrine­
related
endpoints.
If
serum
hormone
levels,
or
any
other
data,
display
heterogeneity
of
variance,
then
appropriate
data
transformations
should
be
employed.
Often
log
transformation
of
serum
hormone
data
is
required
because
the
variance
is
proportional
to
the
mean.

G.
Data
Summarization
Data
should
be
summarized
in
tablular
form
containing
the
mean,
standard
error
of
the
mean
and
sample
size
for
each
group
in
the
table.
Individual
data
tables
should
also
be
included.
The
mean,
SE
and
CV
values
for
the
control
data
should
be
examined
to
determine
if
they
meet
acceptable
QA
criteria
for
consistency
with
normal
values.
Data
presented
should
include
at
least,
levator
ani,
testicular,
epididymal,
ventral
prostate,
seminal
vesicle
(
with
coagulating
glands
and
fluid),
liver
and
body
weights
at
necropsy,
body
weight
change
from
day
28
to
necropsy
and
serum
T4
and
TSH.
Data
may
be
also
be
presented
after
covariance
adjustment
for
body
weight,
but
this
should
not
replace
presentation
of
the
unadjusted
data.

X.
Fish
Gonadal
Recrudescence
Assay
A.
Scope
·
Applicability.
This
guideline
is
intended
to
describe
a
method
to
screen
for
endocrine
disrupting
effects
by
exposing
intact
fish
to
a
test
substance
and
observing
gonadal
maturation
from
the
regressed
position
(
recrudescence)
and
other
endocrine
related
endpoints.
·
Background.
This
assay
is
based
on
recommendations
from
the
Workshop
on
Screening
Methods
for
Endocrine
Disruptors
in
Wildlife
held
in
Kansas
City,
MO
March
17­
19,
1997
(
Ankley
et
al.,
1998).

B.
Introduction
(
1)
Fish
are
the
most
phylogenetically
distant
class
of
vertebrates
from
mammals
so
the
degree
of
homology
with
this
latter
group
is
uncertain.
Assays
with
fish
to
screen
for
potential
endocrine
disruptive
activity
are
important
to
adequately
assess
this
group
of
important
vertebrates.

C.
Definitions
LOEC
(
Lowest­
observed­
effect­
concentration)
is
the
lowest
tested
concentration
of
a
test
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
39
L
­
substance
at
which
the
substance
is
observed
to
have
a
significant
effect
(
at
p
<
0.05)
when
compared
with
the
control.

NOEC
(
No­
observed­
effect­
concentration)
is
the
test
concentration
immediately
below
the
LOEC.

D.
Principle
of
the
Test
Fish
are
exposed
to
a
range
of
concentrations
of
the
test
substance
dissolved
in
water,
preferably
under
flow­
through
conditions,
or
where
appropriate,
semistatic
conditions.
Effects
are
assessed
and
compared
with
control
values
to
determine
the
LOEC
and
the
NOEC
for
the
endpoints
observed.
For
poorly
soluble
materials,
intraperitoneal
injection
may
be
considered.

E.
Information
on
the
Test
Substance
·
Results
of
an
acute
toxicity
test,
preferably
performed
with
the
species
chosen
for
this
test,
should
be
available.
This
implies
that
the
water
solubility
and
the
vapor
pressure
of
the
test
substance
are
known
and
a
reliable
analytical
method
for
the
quantification
of
the
substance
in
the
test
solutions
with
known
and
reported
accuracy
and
limit
of
detection
is
available.
·
Useful
information
includes
the
structural
formula,
purity
of
the
substance,
stability
in
water
and
light,
pKa,
Pow,
and
results
of
a
test
for
ready
biodegradability.

F.
Validity
of
the
Test
For
a
test
to
be
valid
the
following
conditions
apply:

·
The
dissolved
oxygen
concentration
must
be
between
60
and
100
percent
of
the
air
saturation
value
throughout
the
test.
·
The
water
temperature
must
not
differ
by
more
than
1.0o
C
between
test
chambers
and
should
be
within
the
temperature
prescribed
regime
specified
for
the
test
species.
·
Evidence
must
be
available
to
demonstrate
that
the
concentrations
of
the
test
substance
in
solution
have
been
satisfactorily
maintained
within
+
20
percent
of
the
mean
measured
values.
·
When
a
solubilizing
agent
is
used
it
must
have
no
significant
effect
on
survival
nor
produce
any
other
adverse
effects
as
revealed
by
a
solvent­
only
control.

G.
Description
of
the
Method
·
Test
chambers.
Any
glass,
stainless
steel,
or
other
chemically
inert
vessels
can
be
used.
The
dimensions
of
the
vessels
should
be
large
enough
to
allow
compliance
with
loading
rate
criteria
given
below.
It
is
desirable
that
test
chambers
be
randomly
positioned
in
the
test
area.
A
randomized
block
design
with
each
treatment
being
present
in
each
block
is
preferable
to
a
completely
randomized
design.
The
test
chambers
should
be
shielded
from
unwanted
disturbance.
·
Selection
of
species.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
40
L
­
a)
Recommended
fish
species
­­
fathead
minnow,
Pimephales
promelas.
b)
Feeding
and
handling
requirements
of
test
animals,
test
conditions,
duration,
and
survival
criteria
·
Holding
of
the
brood
fish.
c)
Details
on
holding
the
brood
stock
under
satisfactory
conditions
may
be
found
in
the
references
cited
under
paragraphs
(
j)(
1),
(
j)(
2),
and
(
j)(
3)
of
this
guideline.
d)
Test
fish
should
be
in
"
winter"
condition,
brought
about
by
holding
under
an
8
hour
light:
16
hour
dark
photoperiod
at
15
+
1o
C
for
a
minimum
of
30
days
prior
to
the
start
of
the
test.
·
Water.
Any
water
in
which
the
test
species
shows
control
survival
and
good
reproductive
viability.
It
should
be
of
constant
quality
during
the
period
of
the
test.
In
order
to
ensure
that
the
dilution
water
will
not
unduly
influence
the
test
result
(
for
example,
by
complexation
of
test
substance)
or
adversely
affect
the
performance
of
the
fish,
samples
should
be
taken
at
intervals
for
analysis.
Measurements
of
heavy
metals
(
e.
g.
Cu,
Pb,
Zn,
Hg,
Cd,
Ni),
major
anions
and
cations
(
e.
g.
Ca,
Mg,
Na,
K,
Cl,
sulfate),
pesticides,
total
organic
carbon,
and
suspended
solids
should
be
made,
for
example,
every
3
months
where
a
dilution
water
is
known
to
be
relatively
constant
in
quality.
Some
chemical
characteristics
of
an
acceptable
dilution
water
are
listed
in
the
following
Table
1.

H.
Procedure
·
Conditions
of
exposure
 
a)
Duration.
The
test
should
start
as
soon
as
possible
after
appropriately
conditioned
fish
are
placed
into
exposure
chambers.
Test
duration
will
be
21
days
from
the
start.
b)
Loading.
The
loading
rate
(
biomass
per
volume
of
test
solution)
should
be
low
enough
in
order
that
a
dissolved
oxygen
concentration
of
at
least
60
percent
of
the
air
saturation
value
(
ASV)
can
be
maintained
without
aeration.
For
flow­
through
tests,
a
loading
rate
not
exceeding
0.5
g/
L/
24
h
and
not
exceeding
5
g/
L
of
solution
at
any
time
has
been
recommended.

Table
1.­­
Some
Chemical
Characteristics
of
an
Acceptable
Dilution
Water
Substance
Maximum
Concentration
Particulate
matter
<
20
mg/
L
Total
organic
carbon
<
2
mg/
L
Un­
ionized
ammonia
<
1
g/
L
Residual
chlorine
<
10
g/
L
Total
organophosphorus
pesticides
<
50
ng/
L
Total
organochlorine
pesticides
plus
polychlorinated
biphenyls
<
50
ng/
L
Total
organic
chlorine
<
25
ng/
L
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
41
L
­
·
Test
solutions.
a)
For
flow­
through
tests,
a
system
which
continually
dispenses
and
dilutes
a
stock
solution
of
the
test
substance
(
e.
g.
metering
pump,
proportional
diluter,
saturator
system)
is
required
to
deliver
a
series
of
concentrations
to
the
test
chambers.
The
flow
rates
of
stock
solutions
and
dilution
water
should
be
checked
at
intervals
during
the
test
and
should
not
vary
by
more
than
10
percent
throughout
the
test.
A
flow
rate
equivalent
to
at
least
five
test
chamber
volumes
per
24
h
has
been
found
suitable.
b)
The
use
of
solvents
or
dispersants
(
solubilizing
agents)
may
be
required
in
some
cases
in
order
to
produce
a
suitably
concentrated
stock
solution.
c)
For
the
semistatic
technique,
two
different
renewal
procedures
may
be
followed.
Either
new
test
solutions
are
prepared
in
clean
vessels
and
surviving
eggs
and
larvae
gently
transferred
into
the
new
vessels,
or
the
test
organisms
are
retained
in
the
test
vessels
while
a
proportion
(
at
least
two­
thirds)
of
the
test
water
is
changed.
d)
Light
and
temperature.
The
test
begins
with
a
12
hour
light:
12
hour
dark
photoperiod
and
a
gradual
temperature
adjustment
from
15o
C
at
the
start
to
20o
C
at
24
hours.
On
day
seven
the
photoperiod
is
changed
to
16
hour
light:
8
hour
dark
and
the
temperature
adjusted
gradually
from
20o
C
to
25o
C
by
day
eight.
Day
eight
through
day
21
will
be
maintained
at
a
photoperiod
of
16
hour
light:
8
hour
dark
and
a
temperature
of
25o
C.
e)
Feeding.
Fish
should
be
fed
brine
shrimp
(
Artemia)
larvae.
Feeding
should
be
ad
libitum
while
minimizing
the
surplus.
Surplus
food
and
feces
should
be
removed
as
necessary
to
avoid
accumulation
of
waste.
f)
Test
concentrations.
1.
A
single
limit
concentration
identified
from
an
appropriate
rangefinding
test
or
five
concentrations
of
the
test
substance
spaced
by
a
constant
factor
not
exceeding
3.2
are
required.
The
curve
relating
LC50
to
period
of
exposure
in
the
acute
study
should
be
considered
when
selecting
the
test
concentration
or
range.
The
use
of
fewer
than
five
concentrations
and
a
narrower
concentration
interval
may
be
appropriate
in
some
circumstances.
Concentrations
of
the
substance
higher
than
the
96­
h
LC50
not
be
tested.
2.
Where
a
solubilizing
agent
is
used,
its
concentration
should
not
be
greater
than
0.1
mL/
L
and
should
be
the
same
in
all
test
vessels.
However,
every
effort
should
be
made
to
avoid
the
use
of
such
materials.
g)
Controls.
One
dilution­
water
control
and
also,
if
relevant,
one
control
containing
the
solubilizing
agent
should
be
run
in
addition
to
the
test
series.
a)
Fish.
Fish
which
are
mature
and
have
been
through
one
reproductive
cycle
are
used.
A
minimum
of
10
male
and
10
female
fish,
physically
separated
and
divided
into
2
replicates
are
used
per
test
level.
·
Frequency
of
analytical
determinations
and
measurements.
b)
During
the
assay,
the
concentrations
of
the
test
substance
are
determined
at
regular
intervals
to
check
compliance
with
the
validity
criteria.
A
minimum
of
five
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
42
L
­
determinations
is
necessary.
Samples
may
need
to
be
filtered
(
e.
g.,
using
a
0.45m
pore
size)
or
centrifuged
to
ensure
that
the
determinations
are
made
on
the
substance
in
true
solution.
c)
During
the
test,
dissolved
oxygen,
pH,
total
hardness
and
salinity
(
if
relevant),
and
temperature
should
be
measured
in
all
test
vessels.
Temperature
should
preferably
be
monitored
continuously
in
at
least
one
test
vessel.
·
Observations
 
a)
Gonadosomatic
index
(
GSI).
Defined
as
the
blotted
wet
weight
of
the
gonad
divided
by
the
blotted
wet
weight
of
the
intact
fish.
b)
Secondary
sex
characteristics.
Presence
and
extent
of
tubercles
on
male
fish
is
quantified.
c)
Final
oocyte
maturation
(
FOM)/
ovulation/
spermiation.
Maturity
and
production
of
gametes
is
quantified.
d)
Plasma
sex
steroids
and
vitellogenin.
Estradiol,
testosterone/
11­
ketotestosterone,
and
vitellogenin
may
be
quantified
by
appropriate
ELISA
or
RIA.
e)
Abnormal
appearance.
The
number
of
fish
showing
abnormality
of
body
form
should
be
recorded
at
adequate
intervals
depending
on
the
duration
of
the
test
and
the
nature
of
the
abnormality
described.
It
should
be
noted
that
abnormal
embryos
and
larvae
occur
naturally
and
can
be
of
the
order
of
several
percent
in
the
controls
in
some
species.
Abnormal
animals
should
only
be
removed
from
the
test
vessels
on
death.
f)
Abnormal
behavior.
Abnormalities,
e.
g.
hyperventilation,
uncoordinated
swimming,
atypical
quiescence,
and
atypical
feeding
behavior
should
be
recorded
at
adequate
intervals
depending
on
the
duration
of
the
test.
These
effects,
although
difficult
to
quantify,
can,
when
observed,
aid
in
the
interpretation
of
mortality
data
and
influence
a
decision
to
extend
the
exposure
period
beyond
the
recommended
duration.
g)
Weight.
At
the
end
of
the
test
all
surviving
fish
must
be
weighed
individually
as
wet
weights
(
blotted
dry).
h)
Length.
At
the
end
of
the
test,
measurement
of
individual
lengths
is
recommended:
Standard,
fork,
or
total
length
may
be
used.
If
however,
caudal
fin
rot
or
fin
erosion
occurs,
standard
lengths
should
be
used.
i)
Data
for
statistical
analysis.
These
observations
will
result
in
some
or
all
of
the
following
data
being
available
for
statistical
analysis:
i.
Cumulative
mortality.
ii.
Numbers
of
healthy
fish
at
end
of
test.
iii.
GSI
of
males
and
of
females.
iv.
Extent
of
tubercles
on
males.
v.
Length
and
weight
of
surviving
animals.
vi.
Gamete
production
and
maturity.
vii.
Numbers
of
fish
exhibiting
abnormal
behavior.
viii.
Plasma
titers
of
sex
steroids
and
vitellogenin
(
optional).
j)
Data
and
reporting
 
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
43
L
­
·
Treatment
of
results
a)
It
is
recommended
that
a
statistician
be
involved
in
both
the
design
and
analysis
of
the
test
results
since
this
test
guideline
allows
for
considerable
variation
in
experimental
design
as,
for
example,
in
the
number
of
test
chambers,
number
of
test
concentrations,
starting
number
of
fertilized
eggs,
and
number
of
parameters
measured.
b)
In
view
of
the
options
available
in
test
design,
specific
guidance
on
statistical
procedures
is
not
given
here.
However,
it
will
be
necessary
for
variations
to
be
analyzed
within
each
set
of
replicates
using
analysis
of
variance
or
contingency
table
procedures.
To
make
a
multiple
comparison
between
the
results
at
the
individual
concentrations
and
those
for
the
controls,
Dunnett's
method
might
be
found
useful.
However,
care
must
be
taken
where
applying
such
a
method
to
ensure
that
chamber­
to­
chamber
variability
is
estimated
and
is
acceptably
low.
Other
useful
methods
are
also
available.
·
Interpretation
of
results.
The
results
should
be
interpreted
with
caution
where
measured
toxicant
concentrations
in
test
solutions
occur
at
levels
near
the
detection
limit
of
the
analytical
method.
·
Test
report.
The
test
report
must
include
the
following
information:
a)
Test
substance.
i.
Physical
nature
and,
where
relevant,
physicochemical
properties.
ii.
Chemical
identification
data.
b)
Test
species.
Scientific
name,
strain,
source
and
method
of
collection
of
the
fertilized
eggs,
and
subsequent
handling.
c)
Test
conditions.
i.
Test
procedure
used
(
e.
g.,
semistatic
or
flow­
through
design).
ii.
Photoperiods.
iii.
Test
design
(
e.
g.
number
of
test
chambers
and
replicates,
number
of
embryos
per
replicate).
iv.
Method
of
preparation
of
stock
solutions
and
frequency
of
renewal
(
the
solubilizing
agent
and
its
concentration
must
be
given,
when
used).
v.
Nominal
test
concentrations,
means
of
the
measured
values,
their
standard
deviations
in
the
test
vessels,
and
the
method
by
which
these
were
attained,
and
evidence
that
measurements
refer
to
concentrations
of
the
test
substance
in
true
solution.
vi.
Dilution
water
characteristics:
pH,
hardness,
temperature,
dissolved
oxygen
concentration,
residual
chlorine
levels
(
if
measured),
total
organic
carbon,
suspended
solids,
salinity
of
the
test
medium
(
if
measured),
and
any
other
measurements
made.
vii.
Water
quality
within
test
vessels:
pH,
hardness,
temperature,
and
dissolved
oxygen
concentration.
viii.
Detailed
information
on
feeding
(
e.
g.,
type
of
feed,
source,
amount
given,
and
frequency).
d)
Results.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
44
L
­
i.
Evidence
that
controls
met
the
overall
survival
acceptability.
ii.
Data
on
mortality/
survival.
iii.
Data
on
the
observational
endpoints.
iv.
Data
for
length
and
weight.
v.
Incidence
and
description
of
morphological
abnormalities,
if
any.
vi.
Incidence
and
description
of
behavioral
effects,
if
any.
vii.
Statistical
analysis
and
treatment
of
data.
viii.
NOEC
for
each
response
assessed.
ix.
LOEC
(
at
p
=
0.05)
for
each
response
assessed.
x.
Any
concentration­
response
data
and
curves
available.
e)
Discussion
of
the
results.[
Reserved]

I.
References
The
following
references
should
be
consulted
for
additional
background
material
on
this
test
guideline.

4th
Aquatic
Toxicology
Symposium,
ASTM,
Philadelphia,
PA,
1980.
American
Society
for
Testing
and
Materials
(
ASTM),
"
Standard
Guide
for
Conducting
Early
Life­
Stage
Toxicity
Tests
with
Fishes,"
ASTM
E
1241­
92,
1992,
pp.
180­
207,
Philadelphia,
PA.
Brauhn,
J.
L.
and
R.
A.
Schoettger,
"
Acquisition
and
Culture
of
Research
Fish:
Rainbow
trout,
Fathead
minnows,
Channel
catfish
and
Bluegills,"
Ecological
Research
Series,
EPA­
660/
3­
75­
011,
1975,
pp.
54,
Duluth,
MN.
Brungs,
W.
A.,
and
B.
R.
Jones,
"
Temperature
Criteria
for
Freshwater
Fish:
Protocol
and
Procedures,"
Ecological
Research
Series,
EPA­
600/
3­
77­
061,
1977,
pp.
128,
Duluth,
MN.
Dunnett,
C.
W.,
"
A
multiple
comparisons
procedure
for
comparing
several
treatments
with
a
control,"
Journal
of
the
American
Statistical
Association,
50,
1955,
pp.
1096­
1121.
Dunnett,
C.
W.,
"
New
tables
for
multiple
comparisons
with
a
control,"
Biometrics
20,
1964,
pp.
482­
491.
McClave,
J.
T.
et
al.,
"
Statistical
Analysis
of
Fish
Chronic
Toxicity
Test
Data,"
Proceedings
of
McKim,
J.
M.
et
al.,
"
Metal
toxicity
to
embryos
and
larvae
of
eight
species
of
freshwater
fish­
II:
Copper,"
Bulletin
of
Environmental
and
Contamination
Toxicology,
19,
1978,
pp.
608­
616.
Rand,
G.
M.,
and
S.
R.
Petrocelli,
"
Fundamentals
of
Aquatic
Toxicology,"
Hemisphere
Publication
Corporation,
NY,
1985.
U.
S.
EPA,
Recommended
Bioassay
Procedure
for
Fathead
Minnows,
Pimephales
promelas
(
Rafinesque),
Chronic
Tests:
13,
National
Water
Quality
Laboratory,
Duluth,
MN.

Alternative
In
Vivo
Assays
XI.
Development
of
an
In
Vivo
Battery
for
Identifying
Endocrine
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
45
L
­
Modulators
in
Male
Crl:
Cd
Ò
Br
Rats
Protocol
A.
Introduction
The
primary
purpose
of
the
male
In
Vivo
battery
is
to
identify
compounds
that
have
the
potential
to
act
as
agonists
or
antagonists
to
the
estrogen,
androgen,
progesterone,
or
dopamine
receptor,
5
a
­
reductase
inhibitors,
steroid
biosynthesis
inhibitors
(
aromatase
and
testosterone
biosynthesis),
or
compounds
that
alter
thyroid
function.
This
approach
is
based
on
our
previous
work
(
Cook
et
al.,
1992;
Cook
et
al.,
1993;
Biegel
et
al.,
1995;
O'Connor
et
al,.
1996)
as
well
as
our
experiences
with
two
other
proprietary
compounds.

B.
Study
Design
All
experiments
will
use
sexually
mature
male
rats
approximately
ten
weeks
of
age.
Each
experiment
will
test
a
single
compound.
The
daily
dosage
for
each
compound
will
be
administered
at
approximately
8:
00
a.
m.
daily.

Dosing
by
intraperitoneal
injection
will
be
performed
for
15
consecutive
days
following
release
from
quarantine.
Rats
will
be
sacrificed
on
the
morning
of
test
day
+
15.
All
animals
(
15/
group)
will
be
evaluated
for
gross
observations
of
toxicity,
organ
weights
(
testes,
prostate,
seminal
vesicles,
epididymides,
accessory
sex
gland
unit),
and
serum
hormone
concentrations
(
testosterone,
estradiol,
dihydrotestosterone
(
DHT),
luteinizing
hormone
(
LH),
thyroid
stimulating
hormone
(
TSH),
thyroxine
(
T4)).
Epididymal
sperm
concentration
and
motility
will
be
evaluated.
Histology
of
one
testis
and
epididymis
and
the
thyroid
gland
will
be
performed.

C.
Materials
and
Methods
1.
Test
Species
Adult
male
Crl:
CD
Ò
BR
rats,
approximately
ten
weeks
of
age
and
weighing
between
260
and
300
grams,
will
be
acquired
from
Charles
River
Laboratories,
Raleigh,
North
Carolina.
The
Crl:
CD
Ò
BR
rat
has
been
selected
on
the
basis
of
extensive
experience
with
this
strain
and
its
suitability
with
respect
to
sensitivity
to
endocrine
modulators.

2.
Animal
Husbandry
All
rats
will
be
housed
in
stainless
steel,
wire­
mesh
cages
suspended
above
cage
boards.
Animal
rooms
will
be
targeted
at
a
temperature
of
23+
1
°
C
and
a
relative
humidity
of
50+
10%.
Animal
rooms
will
be
artificially
illuminated
(
fluorescent
light)
on
a
12­
hour
light/
dark
cycle.
All
rats
will
be
provided
tap
water
and
Purina
Ò
Certified
Rodent
Chow
Ò
#
5002
ad
libitum.
The
feed
is
guaranteed
by
the
manufacturer
to
meet
specified
nutritional
requirements
and
to
be
free
of
a
list
of
specified
contaminants.

3.
Pretest
Period
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
46
L
­
Upon
arrival,
all
rats
will
be
removed
from
shipping
cartons
and
housed
one
per
cage
in
a
quarantine
room.
The
rats
will
be:

·
quarantined
for
approximately
one
week,
·
identified
with
cage
card
identification,
·
weighed
three
times,
and
·
observed
with
respect
to
weight
gain
and
any
gross
signs
of
disease
or
injury.

The
rats
will
be
released
from
quarantine
by
the
laboratory
veterinarian
on
the
bases
of
body
weights
and
freedom
from
clinical
signs.

All
rats
accidentally
killed
during
the
pretest
period
will
be
discarded
without
necropsy.
All
rats
found
dead
or
sacrificed
in
extremis
during
the
pretest
will
be
necropsied
but
tissues
will
not
be
examined
microscopically.

4.
Assignment
to
Groups
During
the
pretest
period,
male
rats
will
be
divided
by
computerized,
stratified
randomization
into
groups
as
specified
in
a
protocol
amendment
so
that
there
are
no
statistically
significant
differences
among
group
body
weight
means.
For
each
experiment,
a
single
compound
will
be
tested.
Each
rat
will
be
housed
individually.

5.
Dosage
Preparation
and
Administration
All
dosing
solutions
will
be
made
within
three
days
of
study
start
and
will
be
prepared
weekly
for
the
duration
of
the
study.
Solutions
will
be
stored
in
the
refrigerator
when
not
in
use.
The
dose
volume
will
ideally
be
2
ml/
kg,
but
can
be
up
to
10
ml/
kg.
The
route
of
administration
will
be
intraperitoneal
injection.
This
route
was
selected
to
enhance
the
sensitivity
of
the
assay
and
to
facilitate
potency
comparisons.
Intraperitoneal
administration
reduces
variability
associated
with
uptake
which
would
occur
with
other
routes
of
administration
(
i.
e.,
gavage,
subcutaneous).
The
dosages
will
be
specified
in
a
protocol
amendment.
The
same
volume
of
vehicle
will
be
given
to
the
control
group.
Individual
rat
dose
volumes
will
be
based
on
the
daily
body
weight
except
on
test
day
+
15
which
will
use
the
previous
day's
weight.

6.
Body
Weights
All
rats
will
be
weighed
daily.

7.
Food
Consumption
and
Food
Efficiency
Individual
food
consumption
data
will
be
collected
weekly.
The
amount
of
food
consumed
by
each
group
will
be
determined.
From
these
determinations,
as
well
as
body
weight
data,
mean
daily
food
consumption
and
mean
food
efficiency
will
be
calculated
for
each
group.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
47
L
­
8.
Clinical
Observations
and
Mortality
Cage­
site
examinations
to
detect
moribund
or
dead
rats
and
abnormal
behavior
and
appearance
among
rats
will
be
conducted
at
least
once
daily
throughout
the
study.
Moribund
rats
will
be
sacrificed.
Moribund
and
dead
rats
will
be
given
a
gross
pathological
evaluation.
At
every
weighing,
each
rat
will
be
individually
handled
and
examined
for
abnormal
behavior
and
appearance.

9.
Pathological
Evaluation
All
rats
accidentally
killed
during
the
pretest
period
will
be
discarded
without
necropsy.
All
rats
found
dead
or
sacrificed
in
extremis
during
the
pretest
will
be
necropsied
but
tissues
will
not
be
examined
microscopically.
After
study
start,
all
rats
found
dead,
accidentally
killed,
sacrificed
in
extremis,
or
sacrificed
by
design
will
be
necropsied.
Rats
sacrificed
in
extremis
or
sacrificed
by
design
will
be
euthanized
by
carbon
dioxide
(
CO2)
anesthesia
and
exsanguination.
Blood
will
be
collected
from
the
inferior
vena
cava
for
preparation
of
serum
(
Section
K).
Time
of
death
will
be
recorded
for
all
animals.

Final
body,
testes,
accessory
sex
gland
unit,
prostate,
epididymides,
and
seminal
vesicles
(
with
fluid)
weights
will
be
measured.
Relative
organ
weights
(%
of
final
body
weight)
will
be
calculated.
Blood
will
be
collected
in
a
serum
separator
tube
and
placed
on
ice
until
serum
is
prepared.
One
epididymis
will
be
processed
immediately
for
evaluation
of
sperm
concentration
and
motility
according
to
procedures
recommended
in
the
EPA
reproductive
toxicity
testing
guidelines
(
870.3800).
The
other
epididymis
and
thyroid
from
each
rat
will
be
placed
in
formalin
fixative
and
the
testes
will
be
placed
in
Bouin's
fixative.
The
testes,
epididymides,
and
thyroid
will
be
evaluated
microscopically.
Microscopic
evaluations
will
be
performed
on
control
and
high
dose
animals
for
all
compounds.
Only
compounds
which
show
effects
in
the
high
dose
group
will
have
the
remaining
groups
evaluated.

10.
Hormonal
Evaluation
Blood
will
be
collected
at
the
time
of
sacrifice
from
all
animals.
The
blood
will
be
placed
in
a
serum
separator
tube
on
ice
until
the
serum
is
prepared.
Serum
will
be
stored
between
­
65
°
C
and
­
85
°
C
until
analyzed.
Serum
testosterone,
estradiol,
DHT,
LH,
TSH,
T4
levels
will
be
measured
by
commercially
available
radioimmunoassays
(
RIAs).
If
serum
is
limiting,
priority
of
analysis
will
be
determined
by
the
study
director.
Any
remaining
serum
will
be
discarded
after
the
report
is
issued.

D.
Statistical
Analyses
Mean
final
body
weights
and
organ
weights
will
be
analyzed
by
a
one­
way
analysis
of
variance
(
ANOVA).
When
the
corresponding
F
test
for
differences
among
test
group
means
is
significant,
pairwise
comparisons
between
test
and
control
groups
will
be
made
with
Dunnett's
test.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
48
L
­
Bartlett's
test
for
homogeneity
of
variances
will
be
performed
and,
when
significant
(
p
£
0.005),
will
be
followed
by
nonparametric
procedures
(
Dunn's
test).
Serum
hormone
levels
will
be
analyzed
using
Jonckheere's
test
for
trend.
If
a
significant
dose­
response
trend
is
detected,
data
from
the
top
dose
group
will
be
excluded
and
the
test
repeated
until
no
significant
trend
is
detected.
Except
for
Bartlett's
test,
all
other
significance
will
be
judged
at
p
£
0.05.

E.
Safety
And
Housekeeping
Appropriate
handling
precautions
will
be
used
for
each
compound.
Good
housekeeping
procedures
will
be
practiced
to
avoid
contamination
of
the
dose
preparation
facilities
and
potential
health
hazards.
To
avoid
skin
contact,
gloves
will
be
worn
when
handling
the
test
material.
In
addition,
the
neat
test
material
will
be
handled
in
a
chemical
hood.
Animal
carcasses,
feces,
and
unused
dosing
solutions
will
be
incinerated.

F.
References
Biegel,
L.
B.,
J.
C.
Cook,
J.
C.
O'Connor,
M.
Aschiero,
A.
J.
Arduengo
A.
J.,
and
T.
W.
Slone,
"
Subchronic
toxicity
study
in
rats
with
1­
methyl­
3­
propylimidazole­
2­
thione
(
PTI):
effects
on
the
thyroid,"
Fund.
Appl.
Toxicol.
27,
1995,
pp.
185­
194.
Cook,
J.
C.,
L.
S.
Mullin,
S.
R.
Frame,
and
L.
B.
Biegel,
"
Investigation
of
a
mechanism
for
Leydig
cell
tumorigenesis
by
linuron
in
rats,"
Toxicol.
Appl.
Pharmacol.
119,
1993,
pp.
195­
204.
Cook,
J.
C.,
S.
M.
Murray,
S.
R.
Frame,
and
M.
E.
Hurtt,
M.
E.,
"
Induction
of
Leydig
cell
adenomas
by
ammonium
perfluorooctanoate:
A
possible
endocrine­
related
mechanism,"
Toxicol.
Appl.
Pharmacol.
113,
1992,
pp.
209­
217.
Laskey,
J.
W.,
G.
R.
Klinefelter,
W.
R.
Kelce,
and
L.
L.
Ewing,
L.
L.,
"
Effects
of
ethane
dimethanesulfonate
(
EDS)
on
adult
and
immature
rabbit
Leydig
cells:
comparison
with
EDS­
treated
rat
Leydig
cells,"
Biol.
Reproduction,
50,
1994,
pp.
1151­
1160.
O'Connor,
J.
C.,
J.
C.
Cook,
S.
C.
Craven,
C.
S.
Van
Pelt,
and
J.
D.
Obourn,
J.
D.,
"
An
In
Vivo
battery
for
identifying
endocrine
modulators
that
are
estrogenic
or
dopamine
regulators,"
Fund.
Appl.
Toxicol,
1996,
in
press.
EPA/
FIFRA
Good
Laboratory
Practice
Standards,
1989,
40CFR
Part
160.
OECD
Principles
of
Good
Laboratory
Practice
,
1987,
C(
81)
30(
Final),
Annex
2.
MAFF
Japan
Good
Laboratory
Standards,
1985,
(
59
NohSan
No.
3850).

XII.
Development
of
an
In
Vivo
Battery
for
Identifying
Endocrine
Modulators
in
Female
Crl:
Cd
Ò
Br
Rats
Protocol
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
49
L
­
A.
Introduction
The
primary
purpose
of
the
female
in
vivo
battery
is
to
identify
compounds
that
have
the
potential
to
be
agonists
or
antagonists
to
the
estrogen
receptor.
This
approach
is
based
on
our
previous
work
(
O'Connor
et
al.,
1996).

B.
Study
Design
All
experiments
will
use
ovariectomized
female
rats
approximately
seven
weeks
of
age.
Each
experiment
will
test
a
single
compound.
The
groups
and
dosages
for
each
compound
will
be
described
in
protocol
amendments.
The
daily
dosage
for
each
compound
will
be
administered
at
approximately
8:
00
a.
m.
daily.

Rats
will
be
implanted
with
osmotic
minipumps
on
test
day­
one
for
evaluation
of
cell
proliferation.
Dosing
by
intraperitoneal
injection
will
be
performed
for
four
consecutive
days
following
release
from
quarantine.
Rats
will
be
sacrificed
on
the
morning
of
test
day­
five.

There
will
be
ten
animals
per
group.

The
rats
will
be
evaluated
for
vaginal
cytology,
organ
weights
(
uterus
and
liver),
gross
observations.
The
uterus
will
be
saved
in
formalin
fixative
for
possible
future
epithelial
cell
height
and
cell
proliferation
analysis.
Analyses
will
be
performed
at
the
discretion
of
the
study
director.

C.
Materials
And
Methods
1.
Test
Substances
Test
substances
will
be
prepared
in
appropriate
concentrations
and
solvents
to
deliver
no
more
than
10
ml/
kg
dose
volume.

2.
Test
Species
Adult
ovariectomized
female
Crl:
CD
Ò
BR
rats,
approximately
42
days
of
age
and
weighing
between
115
and
160
grams,
will
be
acquired
from
Charles
River.
The
female
rats
will
be
ovariectomized
on
the
day
of
shipment.
The
Crl:
CD
Ò
BR
rat
has
been
selected
on
the
basis
of
extensive
experience
with
this
strain
and
its
suitability
with
respect
to
sensitivity
to
endocrine
modulators.

3.
Animal
Husbandry
All
rats
will
be
housed
in
stainless
steel,
wire­
mesh
cages
suspended
above
cage
boards.
Animal
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
50
L
­
rooms
will
be
targeted
at
a
temperature
of
23+
1
°
C
and
a
relative
humidity
of
50+
10%.
Animal
rooms
will
be
artificially
illuminated
(
fluorescent
light)
on
a
12­
hour
light/
dark
cycle.
All
rats
will
be
provided
tap
water
and
Purina
Ò
Certified
Rodent
Chow
Ò
#
5002
ad
libitum.
The
feed
is
guaranteed
by
the
manufacturer
to
meet
specified
nutritional
requirements
and
to
be
free
of
a
list
of
specified
contaminants.

4.
Pretest
Period
Upon
arrival,
all
rats
will
be
removed
from
shipping
cartons
and
housed
one
per
cage
in
a
quarantine
room.
The
rats
will
be:

·
quarantined
for
approximately
one
week,
·
identified
with
cage
card
identification,
·
weighed
three
times,
and
·
observed
with
respect
to
weight
gain
and
any
gross
signs
of
disease
or
injury.

The
rats
will
be
released
from
quarantine
by
the
laboratory
veterinarian
on
the
bases
of
body
weights
and
freedom
from
clinical
signs.

All
rats
accidentally
killed
during
the
pretest
period
will
be
discarded
without
necropsy.
All
rats
found
dead
or
sacrificed
in
extremis
during
the
pretest
will
be
necropsied
but
tissues
will
not
be
examined
microscopically.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
51
L
­
5.
Assignment
to
Groups
During
the
pretest
period,
female
rats
will
be
divided
by
computerized,
stratified
randomization
into
treatment
groups
so
that
there
are
no
statistically
significant
differences
among
group
body
weight
means.
For
each
experiment,
a
single
compound
will
be
tested.
Each
rat
will
be
housed
individually.

6.
Dosage
Preparation
and
Administration
All
dosing
solutions
will
be
made
within
three
days
of
study
start.
Solutions
will
be
stored
in
the
refrigerator
when
not
in
use.
The
dose
volume
will
ideally
be
2
ml/
kg,
but
can
be
as
high
as
10
ml/
kg.
Route
of
administration
will
be
intraperitoneal
injection.
This
route
was
selected
to
enhance
the
sensitivity
of
the
assay
and
to
facilitate
potency
comparisons.
Intraperitoneal
administration
reduces
variability
associated
with
uptake
which
would
occur
with
other
routes
of
administration
(
i.
e.,
gavage,
subcutaneous).
The
dosages
will
be
specified
in
a
protocol
amendment.
The
same
volume
of
vehicle
will
be
given
to
the
control
group.
Individual
rat
dose
volumes
will
be
based
on
the
daily
body
weight.

7.
Body
Weights
All
rats
will
be
weighed
daily.

8.
Food
Consumption
and
Food
Efficiency
Individual
food
consumption
data
will
be
collected
for
the
treatment
period.
The
amount
of
food
consumed
by
each
group
will
be
determined.
From
these
determinations,
as
well
as
body
weight
data,
mean
daily
food
consumption
and
mean
food
efficiency
will
be
calculated
for
each
group.

9.
Clinical
Observations
and
Mortality
Cage­
site
examinations
to
detect
moribund
or
dead
rats
and
abnormal
behavior
and
appearance
among
rats
will
be
conducted
at
least
once
daily
throughout
the
study.
Moribund
rats
will
be
sacrificed.
Moribund
and
dead
rats
will
be
given
a
gross
pathological
evaluation.
At
every
weighing,
each
rat
will
be
individually
handled
and
examined
for
abnormal
behavior
and
appearance.

10.
Estrous
Cycle
Evaluation
Rats
assigned
to
the
biochemical
subset
will
be
evaluated
for
vaginal
cytology
on
test
days
one
through
four.
Vaginal
washes
will
be
collected
and
evaluated
using
established
cytological
markers
for
evidence
of
conversion
out
of
diestrus.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
52
L
­
11.
Pathological
Evaluation
All
rats
accidentally
killed
during
the
pretest
period
will
be
discarded
without
necropsy.
All
rats
found
dead
or
sacrificed
in
extremis
during
the
pretest
will
be
necropsied
but
tissues
will
not
be
examined
microscopically.
After
study
start,
all
rats
found
dead,
accidentally
killed,
sacrificed
in
extremis,
or
sacrificed
by
design
will
be
necropsied.
Rats
sacrificed
in
extremis
or
sacrificed
by
design
will
be
euthanized
by
carbon
dioxide
(
CO
2)
anesthesia
and
exsanguination.

Rats
assigned
for
necropsy
will
be
sacrificed
using
CO
2.
Time
of
death
will
be
recorded
for
all
animals.
Final
body,
liver,
and
uterine
weights
will
be
measured.
The
entire
uterus
from
ovarian
stump
to
cervix
will
be
removed,
quickly
dissected
free
of
fat
and
connective
tissue,
nicked,
blotted
to
express
luminal
fluid,
and
weighed.
The
presence
of
fluid
in
the
uterine
horns
will
be
recorded
as
a
gross
observation.
Ovarian
stumps
will
be
collected
from
all
animals,
placed
in
10%
neutral­
buffered
formalin
and
processed
to
confirm
the
absence
of
ovarian
tissue
at
the
discretion
of
the
pathologist.

The
uterus
will
be
collected
and
preserved
in
Bouin's
fixative
for
cell
proliferation
evaluation
and
morphometry.
Analyses
will
be
performed
at
the
discretion
of
the
study
director.

12.
Cell
Proliferation
Evaluation
The
uterus
will
be
collected
and
placed
in
Bouin's
fixative.
The
uterus
will
be
cut
into
sections
and
mitotic
index
will
be
counted
or
PCNA
in
the
nuclei
will
be
visualized
immunohistochemically
using
an
avidin­
biotin­
peroxidase
complex
method
with
a
monoclonal
antibody
against
PCNA.
Cell
proliferation
analysis
will
be
performed
at
the
discretion
of
the
study
director.

D.
Statistical
Analyses
Mean
final
body
weights
and
organ
weights
will
be
analyzed
by
a
one­
way
analysis
of
variance
(
ANOVA).
When
the
corresponding
F
test
for
differences
among
test
group
means
is
significant,
pairwise
comparisons
between
test
and
control
groups
will
be
made
with
Dunnett's
test.
Bartlett's
test
for
homogeneity
of
variances
will
be
performed
and,
when
significant
(
p
£
0.005),
will
be
followed
by
nonparametric
procedures
(
Dunn's
test).
Cell
proliferation
indices,
and
uterine
morphometry
measurements
will
be
analyzed
using
Jonckheere's
test
for
trend.
If
a
significant
dose­
response
trend
is
detected,
data
from
the
top
dose
group
will
be
excluded
and
the
test
repeated
until
no
significant
trend
is
detected.
Uterine
fluid
imbibition
and
estrus
conversion
data
will
be
analyzed
by
Fisher's
test.
Except
for
Bartlett's
test,
all
other
significance
will
be
judged
at
p
£
0.05.

E.
Safety
And
Housekeeping
Appropriate
handling
precautions
will
be
used
for
each
compound.
Good
housekeeping
procedures
will
be
practiced
to
avoid
contamination
of
the
dose
preparation
facilities
and
potential
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
53
L
­
health
hazards.
To
avoid
skin
contact,
gloves
will
be
worn
when
handling
the
test
material.
In
addition,
the
neat
test
material
will
be
handled
in
a
chemical
hood.
Animal
carcasses,
feces,
and
unused
dosing
solutions
will
be
incinerated.

XIII.
Research
Protocol
for
Assessment
of
Pubertal
Development
and
Thyroid
Function
in
Immature
(
33­
53
Day
Old)
Male
Rats
Purpose
and
Applicability
The
purpose
of
this
protocol
is
to
outline
procedures
for
the
quantitation
of
the
effects
of
compounds
on
pubertal
development
and
thyroid
function
in
the
intact
juvenile/
peripubertal
male
rat.
This
assay
is
detects
compounds
that
display
antithryoid,
estrogenic,
androgenic,
antiandrogenic
(
AR
or
steroid
enzyme
mediated)
activity,
or
alter
FSH,
LH,
prolactin,
Growth
Hormone
or
hypothalamic
function.

Required
Endpoints
Growth
Age
and
Weight
at
Preputial
Separation
Serum
T4
and
TSH
Thyroid
Histology
Seminal
vesicle
plus
coagulating
gland
weight
(
with
and
without
fluid)
Ventral
Prostate
Weight
Levator
ani
plus
bulbocavernosus
weight
Epididymal
and
Testis
Weights
and
Histology
Optional
Measures
Serum
testosterone,
estradiol,
LH,
prolactin
and
T3
Liver,
kidney,
adrenal
and
pituitary
weights
and
histology
Ex
Vivo
testis
and
pituitary
hormone
production
Hypothalamic
neurotransmitter
levels
A.
General
Conditions
Typically,
prior
to
the
onset
of
the
study,
pregnant
female
rats
are
housed
individually.
After
assignment
to
treatments,
subjects
are
housed
in
pairs
of
similarly
treated
males.
The
following
describes
the
housing
conditions
under
which
our
animals
are
housed.
Reasonable
variations
of
this
portion
of
the
protocol
should
be
tolerated.
Rats
are
housed
in
clear
plastic
cages
(
20x25x47
cm)
with
heat
treated
(
to
eliminate
resins
that
induce
liver
enzymes)
laboratory
grade
pine
shavings
(
Northeastern
Products,
Warrensburg,
NY)
as
bedding.
Animals
are
maintained
on
Purina
Rat
Chow
(
5001)
and
tap
water
ad
libitum,
in
a
room
with
a
14:
10
hours
to
period
(
L/
D,
lights
off
at
11:
00
EST)
and
temperature
of
20­
24o
C
with
a
relative
humidity
of
40­
50%.

B.
Subjects
­
Peripubertal
Male
Rats
At
21
days
of
age
male
SD
or
LE
rats
will
be
weaned
from
their
litters.
These
litters
are
derived
from
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
54
L
­
individually
housed
pregnant
females
that
were
generated
in
house
by
matings
or
purchased
from
a
supplier
as
"
timed
pregnant"
on
days
seven
to
ten
of
gestation.
Upon
birth,
the
litters
are
culled
to
eight
to
ten
pups
in
order
to
assure
normal
growth
rates
in
all
pups.
Growth
is
monitored
on
at
least
a
weekly
basis
and
any
unthrifty
litters
or
runted
pups
should
be
discarded
from
the
study.
Enough
litters
should
be
used
to
assure
that
about
45
pups
are
available
at
weaning.
Pups
are
weaned
at
21
days
of
age
and
weighed
to
the
nearest
0.1
g,
weight
ranked.
A
population
of
thirty
male
weanling
rats
that
is
as
homogeneous
as
possible
is
selected
for
the
study
by
eliminating
the
"
outliers"
(
i.
e.,
the
largest
and
smallest
of
the
pups).
In
this
regard,
one
nuisance
variable,
i.
e.
body
weight
at
weaning,
is
experimentally
controlled.
In
addition,
body
weight
at
weaning
is
also
statistically
controlled,
by
assigning
the
thirty
males
to
two
treatment
groups
in
a
manner
that
provides
each
group
with
similar
means
and
variances
in
weaning
weight.
This
source
of
variance
is
included
in
the
data
analysis
as
a
blocking
factor
or
by
using
weaning
weight
as
a
covariate.
In
addition,
it
is
imperative
that
treatments
should
be
initiated
no
later
than
33
days
of
age,
as
waiting
just
a
few
days
longer
can
result
in
failure
of
the
study
as
control/
untreated
male
rats
will
begin
to
display
"
puberty"
(
i.
e.,
preputial
separation)
within
five
to
seven
days.

C.
Experimental
Design
The
design
is
a
randomized
complete
block
(
bodyweight
at
weaning
is
the
blocking
factor)
design
with
fifteen
weanling
male
rats
in
each
of
two
treatment
groups.
The
treatment
conditions
are:
(
1)
Vehicletreated
and
(
2)
Xenobiotic­
treated.
If
necessary,
the
study
can
be
conducted
in
blocks
rather
than
at
one
time.
In
this
case,
the
blocks
should
be
contain
all
treatment
conditions
and
balanced
with
respect
to
numbers
of
animals
in
each
block
(
i.
e.,
two
blocks
with
two
treatment
conditions,
with
eight
males/
treatment/
block).

D.
Treatment
Treatments
are
administered
daily
by
oral
gavage
from
33
days
of
age
for
20
days.
This
duration
of
treatment
is
unnecessary
to
detect
androgenic
chemicals,
but
is
required
for
the
detection
of
pubertal
delays
and
antithyroid
effects.
Using
a
#
18
gavage
needle
and
a
1
cc
glass
tuberculin
syringe
for
each
treatment.
Xenobiotics
are
administered
in
corn
oil
at
2.5
ml/
kg
body
weight
at
0700­
1000
daily.
The
treatments
should
be
administered
on
a
mg/
kg
body
weight
basis,
adjusted
daily
for
weight
changes
and
body
weight
and
volume
of
the
dose
administered
should
be
recorded
each
day.

E.
Preputial
Separation
(
PPS)

Males
are
examined
daily
for
PPS.
The
appearance
of
partial
and
complete
PPS,
or
a
persistent
thread
of
tissue
between
the
glans
and
prepuce
should
all
be
noted
if
and
when
they
occur.
In
addition,
the
weight
at
complete
PPS
should
be
noted.

F.
Necropsy
On
the
last
day
of
treatment,
males
are
anesthetized
in
CO
2
and
body
weight
is
recorded.
The
rat
is
subsequently
euthanized
by
decapitation,
and
serum
collected
for
optional
hormonal
analysis.
At
necropsy,
the
paired
testicular,
paired
epididymal,
liver,
ventral
prostate,
seminal
vesicle
(
with
coagulating
glands
and
fluid),
levator
ani
plus
bulbocavernosus
muscles,
to
the
nearest
mg
and
body
weights
(
nearest
0.1
g)
should
be
recorded.
During
necropsy
care
must
be
taken
to
remove
mesenteric
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
55
L
­
fat
with
small
surgical
iris
scissors
from
these
tissues
such
that
the
fluid
in
the
sex
accessory
glands
is
retained.
Once
free
from
the
fat
and
adnexa
the
weight
with
fluid
is
recorded
and
these
tissues,
and
the
thyroid,
also
can
be
examined
histologically.
Tissues
should
be
placed
in
Bouins
for
24
hours,
after
which
they
are
rinsed
and
stored
in
70
%
alcohol,
until
being
embedded
in
paraffin,
stained
with
H
and
E,
and
examined
for
histological
alterations.
In
addition,
serum
thyroxine
(
T4)
and
TSH
should
be
measured.
Serum
LH
and
androgen
levels
are
optional.

G.
Statistical
Analysis
All
data
(
age
at
PPS,
weight
at
PPS,
body
and
organ
weights
at
necropsy,
and
serum
hormones)
are
analyzed
as
one­
way
ANOVAs
(
Control
versus
Treatment),
using
PROC
GLM
the
SAS
version
6.08
on
the
USEPA
IBM
mainframe.
The
regression
model
should
include
bodyweight
at
weaning
as
a
covariate.
If
the
study
was
conducted
in
blocks,
then
the
analysis
is
a
two­
way
ANOVA
with
Block
and
Treatment
as
main
effects,
again,
bodyweight
at
weaning
is
used
as
a
covariate.
Statistically
significant
effects
(
p
<
0.05,
F/
t
statistic)
should
be
examined
using
the
LSMEANS
procedure
on
SAS
(
two­
tailed
t­
test)
to
compare
group
(
1)
vehicle
to
the
(
2)
xenobiotic
treated
group.
For
organ
weight
data,
bodyweight
at
necropsy
could
be
used
as
a
covariate
in
the
model,
although
this
is
rarely
useful
for
endocrine­
related
endpoints.
If
treatment
reduces
growth
and
delays
PPS,
the
mechanism
responsible
for
the
delay
is
always
in
question.
In
this
regard,
body
weight
change
from
day
22
until
the
average
age
of
PPS
in
the
control
group
could
be
used
as
a
covariate
in
the
regression
model,
however,
this
is
not
the
best
use
of
ANOCOVA.
If
serum
hormone
levels,
or
any
other
data,
display
heterogeneity
of
variance,
then
appropriate
data
transformations
should
be
employed.
Often
log
transformation
of
serum
hormone
data
is
required
because
the
variance
is
proportional
to
the
mean.

H.
Data
Summarization
Data
should
be
summarized
in
tabular
form
containing
the
mean,
standard
error
of
the
mean
and
sample
size
for
each
group
in
the
table.
Individual
data
tables
should
also
be
included.
The
mean,
SE
and
CV
values
for
the
control
data
should
be
examined
to
determine
if
they
meet
acceptable
QA
criteria
for
consistency
with
normal
values.
Data
presented
should
include
at
least,
age
and
weight
at
PPS,
testicular,
epididymal,
ventral
prostate,
seminal
vesicle
(
with
coagulating
glands
and
fluid),
levator
ani,
liver
and
body
weights
at
necropsy,
body
weight
change
from
day
21
to
necropsy
and
serum
T4
and
TSH.
Data
may
be
also
be
presented
after
covariance
adjustment
for
body
weight,
but
this
should
not
replace
presentation
of
the
unadjusted
data.

Munson,
P.
J.,
and
D.
Rodbard
1980
Anal.
Biochem.
107,
220­
239.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
Appendix
M
Assays
not
Included
in
Tier
1
Screening
The
following
assays
were
considered
by
the
Screening
and
Testing
Work
Group
but
were
not
recommended
for
inclusion
in
the
T1S
battery
at
this
time.

Table
of
Contents
A.
Whole
Cell
ER
Binding
Assays
Using
MCF­
7
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
B.
Transiently
Transfected
Mammalian
Cell
With
hER,
Like
MCF­
7
With
Luciferase
or
CAT
Reporter
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
C.
AR
Whole
Cell
Binding
Assays:
Monkey
Kidney
COS
Cells
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
D.
MCF­
7
Proliferation
Assay
(
E­
Screen)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
2
E.
Yeast
Estrogen
Receptor
Assay
(
YES:
Yeast
Estrogen
Screen)
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
4
F.
Yeast­
Based
Androgen
Receptor
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
5
G.
Developmental
Uterotrophic
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6
H.
Temperature­
dependent
Sex
Determination
Assay
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6
I.
Avian
Assays
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
6
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
1
M
­
A.
Whole
Cell
ER
Binding
Assays
Using
MCF­
7
1
2
Cultures
of
MCF­
7
cells
can
be
utilized
for
whole
cell
ER
binding
assays
or
as
a
source
of
cell
3
homogenate
for
cell
free
binding
assays
(
Taylor
et
al.,
1984;
Kurebayashi
et
al.,
1987;
Shafie
et
al.,
4
1979).
Both
methods
involve
the
competition
of
a
given
compound
with
radiolabeled
estradiol
for
5
specific
binding
to
the
ER.
In
the
whole
cell
method,
cells
are
incubated
with
test
compounds
and
6
estradiol
(
Taylor
et
al.,
1984;
Kurebayashi
et
al.,
1987).
Bioavailability
as
well
as
the
metabolic
7
activation
of
a
test
compound
or
hormone
can
be
evaluated
with
whole
cell
ER
binding
studies.
In
8
contrast
with
homogenates,
whole
cell
assays
are
performed
at
physiological
temperature
(
37
°
C
for
9
mammalian
cells).
On
the
other
hand,
ER
in
MCF­
7
cell
lysate
can
be
used
to
measure
ER
binding
10
affinity
under
cell
free
conditions
(
Shafie
et
al.,
1979).
This
latter
method
is
analogous
to
ER
binding
11
assays
utilizing
receptor
obtained
from
rat
uterine
tissue.
ER
binding
assays
are
essential
for
the
12
characterization
of
a
compound
as
a
ligand
for
the
ER.
However,
ER
binding
determinations
do
not
13
define
the
ligand
as
agonist
or
antagonist.
Whole
cell
assays
may
able
to
metabolically
activate
some
14
chemicals,
which
could
be
an
important
advantage
over
cell
free
assays
that
do
not
display
this
15
capacity
(
MacIndoe
et
al.,
1981;
MacIndoe
et
al.,
1990),
but
in
general
the
metabolic
capacity
of
these
16
cell
lines,
if
any,
remains
uncharacterized
.
17
18
B.
Transiently
Transfected
Mammalian
Cell
With
hER,
Like
MCF­
7
With
Luciferase
19
or
CAT
Reporter
20
21
(
Unless
HTPS
is
implemented
in
combination
with
such
assays
they
are
somewhat
less
desirable
than
22
the
cell­
free
receptor
binding
assay
because
of
the
need
to
transfect
cells.)
23
24
The
transient
transfection
of
MCF­
7
cells
with
an
ER­
regulated
luciferase
reporter
gene
is
a
routine
25
procedure
which
yields
a
sensitive
evaluation
of
a
compound's
ability
to
induce
estrogen­
regulated
26
transcription
in
a
approximately
three
days
(
Meyer
et
al.,
1994).
Numerous
chemicals
and
mixtures
27
have
been
evaluated
for
estrogenicity
in
these
assays
(
organochlorines,
PCBs,
polycyclic
aromatic
28
hydrocarbons,
phytoestrogens,
alkylphenols,
phthalate
esters,
pulp
and
paper
mill
effluent,
urban
air
29
particulate
matter
and
sediment
extracts;
(
Ruh
et
al.,
1995;
Zacharewski
et
al.,
1995;
Connor
et
al.,
30
1996;
Moore
et
al.,
1996).
These
assays
can
distinguish
between
ER
agonist
and
antagonist
ligands
31
and
have
relatively
high
sensitivity
(
Zacharewski,
1996).
32
33
C.
AR
Whole
Cell
Binding
Assays:
Monkey
Kidney
COS
Cells
34
35
Whole­
cell
binding
assays
are
used
to
determine
the
relative
ability
of
environmental
chemicals
to
36
compete
with
endogenous
ligand
for
binding
to
AR
which
is
expressed
in
COS
cells
following
37
transient
transfection
with
a
cDNA
encoding
the
human
AR.
COS
cells
in
culture
are
transfected
with
38
the
pCMVhAR
expression
vector
which
promotes
high­
level
expression
of
hAR.
Cells
are
incubated
39
in
the
presence
of
a
single
saturating
concentration
of
radiolabeled
ligand
and
increasing
40
concentrations
of
the
toxicant/
competing
ligand.
Following
the
incubation,
the
cells
are
washed
to
41
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
2
M
­
separate
bound
from
free
ligand,
harvested
and
bound
radiolabeled
ligand
is
assessed
by
scintillation
1
counting.
These
dose­
response
data
frequently
are
presented
as
IC
50
values
or
the
concentration
of
2
inhibitor
necessary
to
reduce
specific
ligand
binding
by
50
percent.
3
4
Advantages
of
whole­
cell
binding
assays
include
the
fact
that
no
laboratory
animals
are
required,
the
5
AR
remains
intact,
the
assay
is
easy
to
perform
and
is
reproducible
from
lab
to
lab,
the
separation
of
6
bound
and
free
ligand
is
rapid,
and
incubations
are
performed
at
physiological
temperatures
which
can
7
aid
in
chemical
solubilization.
In
addition,
the
assay
may
be
homologous
to
humans
because
8
metabolism
of
the
parent
chemical
by
monkey
COS
cells
may
be
similar
to
human
cells
and
the
assay
9
uses
the
human
AR.
In
this
regard,
these
cell
assays
have
been
shown
to
metabolically
activate
10
proantiandrogenic
fungicides,
a
fact
that
may
render
moot
one
of
the
major
criticisms
of
in
vitro
11
screening.
Disadvantages
are
that
the
assay
requires
the
AR
expression
vector,
transient
cell
12
transfections,
tissue
cultures
which
are
expensive
to
maintain,
and
four
days
are
required
for
the
assay,
13
including
the
initial
seeding
of
the
cells
into
multiwell
plates.
14
15
D.
MCF­
7
Proliferation
Assay
(
E­
Screen)
16
17
The
MCF­
7
cell
proliferation
assay
is
not
specifically
recommended
in
the
view
of
the
majority
of
the
18
STWG
because
the
proliferative
response
is
indirect
(
i.
e.,
the
presence
of
functional
estrogen
receptor
19
is
necessary
but
not
sufficient
to
evoke
estrogen­
mediated
cell
proliferation).
Instead,
the
reporter
gene
20
assays
are
a
direct
manifestation
of
receptor­
mediated
responses
on
gene
expression
(
i.
e.,
the
presence
21
of
functional
estrogen
receptor
and
of
a
reporter
gene
are
sufficient
to
express
estrogen­
mediated
22
induction).
However,
if
one
wishes
to
use
this
assay,
the
data
are
acceptable
and
would
obviate
the
23
necessity
of
running
one
of
the
assays
mentioned
above.
Although
this
assay
is
typically
used
to
detect
24
estrogen
antagonists,
if
used
for
screening,
it
can
and
should
be
used
to
detect
antagonists
as
well.
25
26
The
MCF­
7
cell
line,
which
was
developed
at
the
Michigan
Cancer
Foundation
in
the
early
1970'
s,
27
derives
from
the
pleural
effusion
of
a
69
year
old
human
female
in
the
late
stages
of
metastatic,
28
mammary
carcinoma
(
Soule
et
al.,
1973).
In
addition
to
characterizing
MCF­
7
to
be
of
epithelial
29
origin,
early
investigations
found
it
to
express
the
estrogen,
androgen,
progesterone,
glucocorticoid,
30
vitamin
D
and
retinoic
acid
receptors
(
ER,
AR,
PR,
GR,
VDR,
RAR,
AhR;
Brooks
et
al.,
1973;
31
Horwitz
et
al.,
1975;
Eisman
et
al.,
1980;
Takenawa
et
al.,
1980).
The
MCF­
7
human
cell
line
has
32
been
widely
utilized
throughout
the
last
23
years
(
2800
citations
in
MedLine
through
May
1996)
in
the
33
study
of
cancer
biology,
steroid
hormone
biochemistry
and,
more
recently,
toxicology.
One
of
the
34
most
common
applications
of
MCF­
7
is
for
the
characterization
of
estrogenic
compounds.
Indeed,
35
most
of
the
data
on
newly
identified
estrogen
agonists
was
gathered
using
MCF­
7
proliferation
assays
36
(
Soto
et
al.,
1991,
1992,
1994,
1995,
1997;
Brotons
et
al.,
1995;
Olea
et
al.,
1996;
White
et
al.,
1994;
37
Jobling
et
al.,
1995;
Olea
et
al.,
1998).
The
estrogen­
specific
cell
growth
of
MCF­
7
was
first
identified
38
by
Lippman
et
al.,
1976.
In
recent
years,
any
assay
utilizing
this
effect
as
an
end
point
is
often
called
an
39
E­
Screen
(
Soto
et
al.,
1995).
The
MCF­
7
cell
proliferation
assay
is
one
of
the
most
sensitive
assays
40
for
assessing
estrogenicity
(
Welshons
et
al.,
1990;
Soto
et
al.,
1991;
Mayr
et
al.,
1992;
Soto
et
al.,
41
1995).
Estrogen
agonists
and
antagonists
can
be
differentiated
using
this
method
(
Wakeling
et
al.,
42
1988;
Wakeling
et
al.,
1989;
Jain
et
al.,
1992;
Wakeling
et
al.,
1992).
It
is
unclear
to
what
degree
43
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
3
M
­
proestrogens
may,
or
may
not,
be
activated
to
their
estrogenic
form
in
MCF­
7
cell
growth
studies
1
(
MacIndoe
et
al.,
1981;
MacIndoe
et
al.,
1990;
Soto
et
al.,
1995).
Utilizing
typical
cell
culture
2
equipment,
a
six
day
cell
growth
experiment
can
identify
if
a
chemical
has
estrogenic
activity
(
Wiese
et
3
al.,
1992;
Soto
et
al.,
1995).
While
numerous
reports
have
characterized
many
of
the
cell
culture
4
conditions
required
for
this
response,
the
estrogen­
mediated
mechanism
for
MCF­
7
cell
proliferation
is
5
still
being
debated.
Some
reports
define
a
serum­
born
inhibitor
of
cell
growth
that
is
specifically
6
inactivated
by
estrogens
(
Dell'Aquila
et
al.,
1984;
Soto
et
al.,
1984;
Soto
et
al.,
1992;
Briand
et
al.,
7
1986;
Lykkesfeldt
et
al.,
1986;
Soto
et
al.,
1992;
Sonnenschein
et
al.,
1996).
In
these
reports,
8
estrogenless
serum
is
required
for
expression
of
estrogen­
mediated
cell
proliferation.
In
the
absence
of
9
serum
(
serumless
medium),
proliferation
of
MCF7
cells
is
maximal
(
Butler
et
al.,
1983;
Briand
et
al.,
10
1986;
Soto
et
al.,
1984).
Others
have
failed
to
observe
such
a
role
for
serum
and
have
concluded
that
11
estrogen­
mediated
proliferation
is
the
result
of
a
complex
cascade
mechanism
initiated
by
a
small
12
number
of
ER
mediated
events
(
Wiese
et
al.,
1992).
Finally,
some
studies
suggest
that
a
combination
13
of
both
mechanisms
can
take
place
(
Aakvaag
et
al.,
1990).
The
inhibitory
effect
of
serum
is
mediated
14
by
serum
albumin
(
Laursen
et
al.,
1990;
Sonnenschein
et
al.,
1996).
15
16
Maintenance
of
the
MCF­
7
cell
proliferative
response
depends
on
three
primary
factors:
MCF­
7
cell
17
subclone,
culture
conditions
and
proper
experimental
design.
Wild
type
MCF­
7
cell
cultures
have
been
18
shown
to
preserve
their
phenotype
for
more
than
20
years
when
propagated
in
the
presence
of
19
estrogen
(
Welshons
and
Jordan,
1987;
Soto
et
al.,
1997).
They
become
estrogen
independent
after
20
multiple
passages
in
culture
under
stringent
conditions
designed
to
obtain
estrogen­
autonomous
21
phenotypes,
such
as
in
the
presence
of
charcoal­
dextran
stripped
serum
(
Katzenellenbogen
et
al.,
1987;
22
Welshons
et
al.,
1987;
Sonnenschein
et
al.,
1994).
Therefore,
MCF­
7
cell
subclones
to
be
used
for
23
measuring
estrogen­
mediated
proliferation
should
be
those
shown
to
maintain
the
estrogen
24
proliferative
response
over
long
term
passage
are
preferable.
The
proliferative
response
of
some
MCF­
25
7
cell
strains
are
more
variable
or
lower
(
wild
type,
ATCC,
BB,
BB104)
than
others
(
BUS
or
E3;
26
Nawata
et
al.,
1981;
Vickers
et
al.,
1988;
Wiese
et
al.,
1992;
Sonnenschein
et
al.,
1994;
Villalobos
et
27
al.,
1995;
Masamura
et
al.,
1995;
Klotz
et
al.,
1995).
Like
all
cell
lines,
preservation
of
the
initial
28
phenotype
requires
glp.
serum
effects
are
observed
in
practically
all
cell
lines;
hence,
testing
serum
29
batches
is
an
essential
standard
practice
for
maintenance
of
the
phenotype
(
Devleeschouwer
et
al.,
30
1987;
Jain
et
al.,
1991;
Welshons
et
al.,
1992;
Wiese
et
al.,
1992).
While
maintenance
of
the
estrogen­
31
responsive
phenotype
in
serum
containing
medium
requires
estrogens,
dextran­
coated
charcoal
32
treatment
of
serum
is
required
for
performing
the
proliferative
assay
(
Soto
et
al.,
1985;
Welshons
et
al.,
33
1992;
Wiese
et
al.,
1992;
Soto
et
al.,
1995).
Even
when
using
optimal
clones,
the
culture
conditions
34
required
for
maximal
cell
proliferation
growth
may
be
difficult
to
standardize
because
they
may
change
35
as
the
cells
evolve
through
time
(
e.
g.,
changes
in
doubling
times,
concentration
of
serum
required;
36
Katzenellenbogen
et
al.,
1987;
Wiese
et
al.,
1992).
Adaptation
periods
of
up
to
three
months
may
be
37
required
before
the
cells
regain
their
full
estrogen
responsiveness
after
freezing/
thawing
or
shipping.
In
38
addition,
partial
proliferation
may
be
obtained
with
chemicals
that
are
inactive
in
vivo
and
fail
to
39
activate
chimeric
ER
transfection
assays
in
MCF­
7
cells
(
Desaulniers
et
al.,
in
prep;
section
2.5
below).
40
Interpretation
of
the
results
of
MCF­
7
cell
proliferation
experiments
is
enhanced
if
one
includes
41
controls
that
can
identify
antiestrogens
as
well
as
estrogen­
independent
proliferation
effects
of
the
test
42
chemicals.
Trials
where
the
test
chemical
is
added
to
cultures
in
combination
with
antiestrogen
(
test
for
43
agonist)
or
estradiol­
17J
(
test
for
antagonist)
will
more
completely
characterize
a
compounds
effect
on
44
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
4
M
­
cell
growth
as
well
as
determine
if
such
a
response
is
ER­
mediated
(
Wiese
et
al.,
1992).
Potential
1
antagonists
are
to
be
confirmed
by
running
"
rescue"
experiments,
regardless
of
whether
the
end
point
is
2
proliferation
or
a
gene
product.
the
first
step
(
antagonist
activity)
evaluates
the
effect
of
a
range
of
3
doses
of
the
test
compound
together
with
the
minimal
estradiol
dose
needed
for
maximal
induction
of
4
response.
In
the
second
step,
inhibition
by
the
putative
antagonist
is
challenged
by
administering
5
increasing
doses
of
estradiol
together
with
the
dose
of
toxicant
found
to
induce
maximal
inhibition
6
(
estradiol
rescue)
(
Osborne
et
al.,
1984).
7
8
Due
to
the
fact
that
the
MCF­
7
cell
proliferation
assay
requires
optimization
of
various
laboratory
and
9
culture
conditions,
as
indicated
above,
it
may
be
difficult
to
standardize
for
large
scale
testing.
In
10
addition,
although
the
assay
is
sensitive
and
reproducible,
with
some
effort,
it
takes
longer
(
six
days)
11
than
other
in
vitro
assays.
In
the
case
of
the
MCF­
7
cell
proliferation
assay,
replication
of
an
initial
six
12
day
trial
lengthens
the
duration
of
the
assay
to
nearly
three
weeks.
Finally,
even
with
proper
controls
13
(
i.
e.
blocking
the
effect
with
antiestrogens),
there
is
potential
for
this
assay
to
identify
false
positives
14
(
general
cell
mitogens)
and
false
negatives
(
cytotoxic,
general
growth
inhibitors).
For
these
reasons,
15
opinions
were
quite
wide
ranging
on
the
future
utility
of
the
MCF­
7
cell
proliferation
assay
for
16
screening.
However,
competent
labs
should
be
able
to
obtain
comparable
results
(
in
fact,
an
17
interlaboratory
comparative
study
was
performed
in
Europe
and
the
U.
S.
to
be
published
in
1998).
18
Furthermore,
as
with
any
in
vitro
test,
the
results
should
be
replicated
(
three
times
appears
to
be
19
standard
in
the
in
vitro
field).
20
21
E.
Yeast
Estrogen
Receptor
Assay
(
YES:
Yeast
Estrogen
Screen)
22
23
Easy
to
run,
but
major
reservations
due
to
lack
of
standardization,
strain
differences
between
yeast
and
24
unique
physiology
of
yeast
as
compared
to
mammalian
cells.
Mammalian
steroid
receptors
introduced
25
into
the
yeast
strain
Saccharomyces
cerevisae
can
function
as
steroid­
dependent
transcriptional
26
activators
(
Metzger
et
al.,
1988;
Schena
and
Yamamoto,
1988;
McDonnell
et
al.,
1989).
Several
27
laboratories
have
begun
to
screen
chemicals
for
estrogenicity
in
yeast­
based
estrogen
receptor
assay,
28
commonly
referred
to
as
the
YES
(
yeast
estrogen
screen)
assay
(
Arnold
et
al.,
1996;
Gaido
et
al.,
29
submitted).
Yeast
cells
are
transformed
with
a
whole
or
a
fragment
of
the
human
ER
and
a
reporter
30
gene
containing
one
or
more
tandemly
linked
ER
response
elements
upstream
to
the
J­
galactosidase
31
reporter
(
lacZ)
or
some
other
reporter
construct
(
Conner
et
al.,
1996).
Yeast
cells
remain
32
"
permanently"
transformed
if
grown
under
the
appropriate
conditions.
Chemicals
that
bind
ER
and
33
activate
transcription
induce
J­
galactosidase
activity
during
an
incubation
period
ranging
in
duration
34
from
four
hours
to
overnight.
This
assay
has
been
utilized
to
examine
the
estrogenic
potency
of
several
35
xenoestrogens
including,
o,
p'­
DDT,
octylphenol,
nonylphenol,
and
bisphenol
A.
In
the
yeast
assay,
36
steroid
receptor
antagonists,
such
as
ICI
164,384,
have
positive
rather
than
negative
activity
(
Kohno
et
37
al.,
1994).
In
fact,
as
employed
above
some
YES
assays
detect
all
chemicals
as
agonists,
whether
they
38
are
agonists
or
antagonists
in
other
systems.
Major
advantages
of
the
YES
assay
include
ease
of
use
39
(
because
cells
do
not
have
to
be
continuously
transformed),
the
short­
term
duration,
and
the
ability
to
40
quantify
results
without
using
radioactive
materials.
This
assay
is
being
adapted
for
other
receptors
41
such
as
the
androgen
and
progesterone
receptors
and
it
has
been
automated
by
companies
in
the
42
pharmaceutical
industry
for
high
throughput
screening.
The
yeast­
based
assay
has
been
adapted
to
43
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
5
M
­
determine
the
effect
of
serum
binding
proteins
on
bioavailability
(
Arnold
et
al.,
1996)
and
to
assess
the
1
activity
of
chemical
mixtures
(
Arnold
et
al.,
1996).
In
addition,
the
YES
assay
has
been
successfully
2
used
to
identify
the
estrogenic
contaminants
in
sewage
treatment
water
effluents
(
estradiol,
estrone
and
3
ethinyl
estradiol
in
several
effluents
and
alkylphenols
in
the
river
Aire
in
the
UK)
which
were
4
responsible
for
induction
of
estrogenic
responses
in
the
fish
(
Desbrow
et
al.,
1996).
5
6
Although
the
YES
assay
is
an
extremely
useful
research
tool,
there
was
a
clear
consensus
by
the
7
STWG
that
the
YES
assay
should
not
be
recommended
for
screening
at
present.
In
spite
of
the
8
aforementioned
benefits,
several
reservations
regarding
the
YES
assay
also
were
expressed.
For
9
example,
significant
phylogenetic
differences
in
metabolism
of
steroids
and
toxic
substances
may
exist
10
and,
in
contrast
to
mammalian
cells,
yeast
cells
have
a
cell
wall
and
chemical
transport
systems
that
11
selectively
decrease
the
intracellular
levels
of
particular
steroid
hormones
and,
consequently,
the
12
potency
of
particular
chemicals
(
Krall
and
Yamamoto,
1996).
Of
major
concern
in
some
publications,
13
the
rate
of
false
negatives
is
high
for
ER.
The
YES
assay
is
not
considered
acceptable
at
this
time
14
because
of
it
inability
to
consistently
detect
estrogenic
activity
of
several
classes
of
xenobiotics
(
Gaido
15
et
al.,
1997;
Coldmann
et
al.,
1997).
For
this
reason,
positive
YES
data
are
acceptable,
but
negative
16
data
are
not.
In
addition,
as
the
YES
assay,
when
it
works,
does
not
distinguish
between
agonists
and
17
antagonists,
the
results
are
more
equivalent
to
a
binding
assay
than
other
transcriptional
activation
18
assays.
19
20
F.
Yeast­
Based
Androgen
Receptor
Assay
21
22
The
reservations
include
those
for
YES
and
the
fact
that
it
fails
to
detect
known
environmental
23
antiandrogens
(
Gaido
et
al.,
1997)
renders
it
a
dubious
choice
for
screening.
The
yeast­
based
estrogen
24
receptor
assay
can
also
be
adapted
for
screening
for
chemicals
that
interact
with
the
androgen
receptor
25
(
Purvis
et
al.,
1991;
Gaido
et
al.,
submitted).
In
these
assays,
yeast
are
permanently
transformed
with
26
the
human
AR,
or
AR
obtained
from
another
species,
and
a
reporter
gene
containing
one
or
several
27
androgen
response
elements
upstream
to
the
reporter
gene
(
e.
g.,
J­
galactosidase
­
lacZ).
Chemicals
28
that
bind
AR
and
activate
transcription
induce
reporter
gene
activity
during
an
incubation
that
can
last
29
from
four
hours
to
overnight.
Like
the
yeast­
based
estrogen
receptor
assay,
known
steroid
receptor
30
antagonists,
such
as
hydroxyflutamide,
have
positive
activity.
As
a
result,
the
yeast­
based
androgen
31
receptor
assay
detects
both
agonistic
and
antagonistic
chemicals
as
agonists.
The
assay
is
simple
to
32
perform,
large
numbers
of
samples
can
be
processed
quickly
and
the
results
quantified
without
using
33
radioactive
materials.
It
is
sensitive
to
steroidal
androgens
and
can
be
used
to
assess
chemicals
over
a
34
wide
dose
range.
Current
reservations
regarding
yeast­
based
androgen
receptor
assay
are
similar
to
35
those
expressed
for
the
YES
assay
and,
additionally,
p,
p
DDE,
which
binds
to
rat
AR
and
human
AR
36
in
COS
and
CV1
cells
with
high
affinity
is
poorly
detected
in
yeast­
based
androgen
receptor
assay.
37
38
G.
Developmental
Uterotrophic
Assay
39
40
The
developmental
uterotrophic
assay
provides
information
from
several
estrogen­
sensitive
41
endpoints,
and
can
be
combined
with
the
in
vivo
thyroid
assay.
Starting
on
day
ten
after
birth,
rats
42
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
6
M
­
are
dosed
daily
and
sacrificed
on
day
22.
After
uterine
weight
is
taken,
uteri
are
cross­
sectioned
1
and
the
number
of
uterine
glands
and
the
height
of
the
luminal
and
glandular
epithelia
are
2
measured.
Uterine
weight
may
suggest
complete
agonist
activity
(
large
weight
gain)
or
partial
3
agonist/
antagonist
activity
(
small
weight
gain).
Inhibition
of
gland
appearance
is
an
irreversible
4
developmental
toxicity
(
i.
e.,
not
dependent
on
the
continued
presence
of
the
estrogen)
and
unlike
5
uterine
weight,
is
completely
responsive
to
chemicals
from
either
pharmacological
class.
Epithelial
6
hypertrophy,
measured
as
cell
height,
occurs
at
all
ages
and
is
reversible,
as
is
uterine
weight
gain,
7
but
like
inhibition
of
gland
appearance,
is
completely
responsive
to
chemicals
in
both
8
pharmacological
categories.
Glandular
epithelial
hypertrophy
only
responds
to
mixed
9
agonists/
antagonists.
Furthermore,
the
ovary
makes
estrogens
starting
on
day
10
and
treatment
10
with
a
pure
antiestrogen
or
removal
of
the
ovary
lowers
uterine
weight.
Thus
lowered
uterine
11
weight
should
indicate
action
as
a
steroidogenesis
inhibitor
or
as
an
inhibitor
of
estrogen
12
production
via
the
hypothalamic­
pituitary­
ovarian
axis.
Finally,
increased
estrogen
production
via
13
the
same
route
would
increase
uterine
weight.
This
developmental
assay
define
patterns
of
14
activity
for
chemicals
that
act
via
the
receptor
and
additionally
detects
those
acting
via
the
15
hypothalamic­
pituitary­
ovarian
axis
or
other
mechanisms
that
cannot
be
detected
in
an
16
ovariectomized
animal.
17
18
H.
Temperature­
dependent
Sex
Determination
Assay
19
20
The
assay
involving
effects
on
temperature­
dependent
sex
determination
in
reptiles
(
turtles)
by
21
"
painting"
the
chemical
substance
and
mixture
on
the
eggs
was
not
selected.
This
assay
is
22
comparably
sensitive
to
E/
A
+
anti
E/
A
chemical
substances
and
mixtures
as
the
in
vitro
23
mammalian
assays,
can
only
be
performed
when
eggs
are
laid
(
four
months
out
of
the
year)
and
24
takes
a
long
time
in­
life
(
approximately
four
months).
It
is
a
sensitive,
specific
assay
for
E/
A
+
25
anti
E/
A
in
Reptilia,
involving
exposure
during
in
ovo
development.
26
27
I.
Avian
Assays
28
29
Assays
in
Avian
species,
for
example,
development
of
primary
and
secondary
sex
characteristics,
30
including
reproductive
structures,
after
exposure
during
in
ovo
development
(
egg
injection)
were
31
not
selected.
These
assays
are
comparably
sensitive
to
E/
anti­
E,
A/
anti­
A
as
the
mammalian
32
assays
and
take
a
long
time
in­
life
(
one­
two
months).
33
34
35
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
1
N
­
Appendix
N
1
2
Endocrine
Disruption
and
Invertebrates
3
4
5
Considerations
by
the
EDSTAC
Screening
and
Testing
Work
Group
(
STWG)
have
predominantly
6
dealt
with
vertebrate
animals
for
several
reasons.
The
first,
and
perhaps
overriding
one,
is
that
the
7
charge
given
to
the
work
group
of
focusing
on
estrogen,
androgen,
and
thyroid
hormone
actions
8
is
not
especially
relevant
to
important
and
well­
studied
hormones
of
invertebrates.
The
purported
9
endocrine
disruption
effects
of
public
concern
are
almost
exclusively
human
health
or
vertebrate
10
wildlife
related.
The
expertise
in
the
work
group
is,
also,
predominantly
with
the
vertebrate
11
classes.
However,
invertebrates
represent
over
95%
of
all
animals,
are
ubiquitous,
and
are
12
tremendously
important
ecologically
and
economically.
Commercial
fisheries
of
shrimp,
crab,
and
13
oyster
and
agriculturally
important
insect
pollination
are
but
a
few
key
examples.
Because
14
invertebrates
are
ubiquitous
and
are
easily
adapted
for
laboratory
testing,
they
can
serve
as
15
sentinels
and
surrogates
for
investigating
environmental
stress.
For
these
reasons,
invertebrates
16
should
not
be
ignored
from
consideration.
17
18
Endocrine
disruption
has
been
well
studied
and
well
exploited
for
certain
invertebrates,
especially
19
the
insects.
The
endocrine
systems
of
insects
have
been
intentionally
targeted
for
insecticidal
20
activity
and
several
insecticides
have
been
developed
and
used
to
suppress
insect
populations
by
21
disrupting
their
normal
endocrine
functions.
Juvenile
hormone
mimics
(
e.
g.,
methoprene),
22
antijuvenile
hormone
analogs
(
e.
g.,
precocene),
chitin
synthesis
inhibitors
(
e.
g.,
diflubenzuron),
23
ecdysone
analogs
(
e.
g.,
tebufeno­
zide),
and
molting
disruptants
(
e.
g.,
fenoxycarb)
are
some
24
examples.
These
insect
growth
regulating
compounds
have
also
been
observed
to
have
adverse
25
effects
in
related
arthropods
such
as
crustaceans,
including
disrupting
normal
molting
processes,
26
limb
regeneration,
and
reproduction
(
Christiansen
et
al.,
1977a,
b;
1979;
Cunningham,
1976;
27
Forward
and
Costlow,
1978;
Landau
and
Rao,
1980;
Nimmo
et
al.,
1980;
Touart
and
Rao,
1987).
28
Other
substances
like
the
organotin
TBT
have
caused
imposex
and
intersex
conditions
in
29
gastropods
(
Gibbs
and
Bryan,
1986;
Reijnders
and
Brasseur,
1992)
and
sewage
outfalls
have
30
caused
intersex
conditions
in
harpacticoid
copepods
(
Moore
and
Stevenson,
1994),
conditions
31
indicative
of
endocrine
disruption.
32
33
Although
the
relevance
of
estrogen
and
androgen
hormones
to
invertebrates
is
unclear,
34
invertebrates
may
be
useful
as
surrogates
for
investigating
phenomena
relevant
to
these
hormones
35
in
vertebrates.
Estrogens
have
been
reported
to
play
a
meaningful
role
in
development
and
36
reproduction
in
echinoderms
and
molluscs
(
Takeda,
1979;
Brueggemeier
et
al.,
1988;
Shirai
and
37
Walker,
1988).
Daphnids
have
been
used
to
investigate
the
effects
of
xenoestrogens
on
steroid
38
metabolism
(
Baldwin
et
al.,
1995;
Baldwin
et
al.,
1997)
and
sex
reversal
(
Shurin
and
Dodson,
39
1997).
Because
of
their
generally
shorter
life
cycles
and
relative
ease
of
handling
many
species
in
40
the
laboratory,
invertebrates
could
be
useful
for
evaluating
endocrine
disrupting
phenomena.
41
However,
additional
research
is
needed
before
this
promise
is
realized.
42
43
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
2
N
­
There
are,
therefore,
two
aspects
to
considering
endocrine
disruption
for
invertebrates,
one
is
1
relevance
to
the
health
of
invertebrate
organisms
themselves
and
the
other
is
relevance
of
2
invertebrates
as
surrogates
for
investigating
vertebrate­
related
phenomena.
Conventional
risk
3
assessment
of
toxic
chemicals
such
as
outdoor
use
pesticides
and
high
volume
industrial
chemicals
4
generally
include
a
crustacean
reproduction
or
life
cycle
test
in
the
data
set
used
in
the
assessment.
5
Although
specific
endocrine
system
endpoints
are
not
considered,
the
apical
nature
of
these
tests
6
may
be
adequate
to
detect
the
adverse
consequences
of
an
endocrine
disrupting
chemical
in
7
crustacean
arthropods.
Additional
information
is
needed
to
determine
what
is
most
useful
beyond
8
these
conventional
tests
for
the
wider
invertebrate
taxa.
As
surrogates,
more
information
on
the
9
correlation
of
endocrine
phenomena
between
invertebrates
and
vertebrates
would
be
helpful.
For
10
instance,
to
what
degree
does
a
substance
which
disrupts
ecdysteroid
metabolism
in
crustacea
11
disrupt
sex
steroid
metabolism
in
vertebrates?
Perhaps
good
correlations
may
be
found,
but
more
12
comparative
information
is
needed
before
recommendations
of
specific
invertebrate
tests
useful
for
13
evaluating
potential
endocrine
disrupting
activity
relevant
to
vertebrates
can
be
made.
14
15
No
invertebrate
assays,
therefore,
have
been
evaluated
for
use
in
T1S
for
detecting
estrogen,
16
androgen,
or
thyroid
hormone
disruption.
Invertebrate
tests
have
been
proposed
for
T2T.
It
is
17
recommended
that
a
workshop
of
invertebrate
endocrinologists
and
toxicologists
be
convened
to
18
address
first,
the
suitability
of
invertebrate
assays
for
estrogen
and
androgen
(
not
thyroid)
for
use
19
in
a
screening
battery,
and
second,
future
improvements
to
the
broader
consideration
of
endocrine
20
disruption
in
the
environment
and
the
utility
of
invertebrates
as
surrogate
test
organisms.
21
22
References:
23
24
Baldwin,
W.
S.,
D.
L.
Milam,
and
G.
A.
LeBlanc,
"
Physiological
and
biochemical
perturbations
in
25
Daphnia
magna
following
exposure
to
the
model
environmental
estrogen
diethylstilbestrol,"
26
Environ.
Toxicol.
Chem.,
14,
1995,
pp.
945­
952.
27
Baldwin,
W.
S.,
S.
E.
Graham,
D.
Shea,
and
G.
A.
LeBlanc,
"
Metabolic
androgenization
of
female
28
Daphnia
magna
by
the
xenoestrogen
4­
nonylphenol,"
Environ.
Toxicol.
Chem.,
16,
1997,
29
pp.
1905­
1911.
30
Brueggemeier,
R.
W.,
G.
D.
Yocum,
and
D.
L.
Denlinger,
"
Estranes,
androstanes,
and
pregnanes
in
31
insects
and
other
invertebrates,"
Physiological
Insect
Ecology,
1988,
pp.
885­
898.
32
Christiansen,
M.
E.,
J.
D.
Costlow,
Jr.,
and
R.
J.
Monroe,
"
Effects
of
the
juvenile
hormone
mimic
33
ZR­
515
(
Altosid)
on
larval
development
of
the
mud­
crab,
Rhithropanopeus
harrisii
in
34
various
salinities
and
cyclic
temperatures,"
Marine
Biol.,
39,
1977,
pp.
269­
279.
35
Christiansen,
M.
E.,
J.
D.
Costlow,
Jr.,
and
R.
J.
Monroe,
"
Effects
of
the
juvenile
hormone
mimic
36
ZR­
512
(
Altozar)
on
larval
development
of
the
mud­
crab
Rhithropanopeus
harrisii
at
37
various
cyclic
temperatures,"
Marine
Biol.,
39,
1977,
pp.
281­
288.
38
Christiansen,
M.
E.,
J.
D.
Costlow,
Jr.,
and
R.
J.
Monroe,
"
Effects
of
the
insect
growth
regulator
39
Dimilin
(
TH­
6040)
on
the
larval
development
of
two
estuarine
crabs,"
Marine
Biol.,
50,
40
1979,
pp.
29­
36.
41
Cunningham,
P.
A.,
"
Effects
of
Dimilin
(
TH­
6040)
on
reproduction
in
the
brine
shrimp
Artemia
42
salina,"
Environm.
Entomol.,
5,
1976,
pp.
701­
706.
43
Forward,
R.
B.,
Jr.
and
J.
D.
Costlow,
Jr.).
"
Sublethal
effects
of
insect
growth
regulators
upon
44
crab
larval
behavior,"
Water,
Air,
Soil
Pollution,
9,
1978,
pp.
227­
238.
45
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
3
N
­
Gibbs,
P.
E.,
and
G.
W.
Bryan,
"
Reproductive
failure
in
populations
of
the
dog­
whelk,
Nucella
1
lapillus,
caused
by
imposex
induced
by
tributyltin
from
antifouling
paints,"
J.
Mar.
Biol.
2
Assoc.,
UK
66,
1986,
pp.
767­
777.
3
Landau,
M.
and
K.
R.
Rao,
"
Toxic
and
sublethal
effects
of
precocene
II
on
the
early
4
developmental
stages
of
the
brine
shrimp
Artemia
salina
(
L.)
and
the
barnacle
Balanus
5
eburneus
Gould,"
Crustaceana,
39,
1980,
pp.
218­
221.
6
Moore,
C.
G.
and
J.
M.
Stevenson,
"
Intersexuality
in
benthic
harpacticoid
copepods
in
the
Firth
of
7
Forth,
Scotland,".
J.
Nat.
History,
28,
1994,
pp.
1213­
1230.
8
Nimmo,
D.
R.,
T.
L.
Hamaker,
J.
C.
Moore,
and
R.
A.
Wood,
"
Acute
and
chronic
effects
of
9
Dimilin
on
survival
and
reproduction
of
Mysidopsis
bahia,"
Aquatic
Toxicology,
ASTM,
10
1980,
pp.
366­
376.
11
Reijnders,
P.
J.
H.
and
S.
M.
J.
M.
Brasseur,
"
Xenobiotic
induced
hormonal
and
associated
12
disorders
in
marine
organisms
and
related
effects
in
humans;
an
overview,"
Chemically
13
Induced
Alterations
in
Sexual
and
Functional
Development:
The
Wildlife/
Human
14
Connection,
1992,
pp.
159­
174.
15
Shirai,
H.
and
C.
W.
Walker,
"
Chemical
control
of
asexual
and
sexual
reproduction
in
16
echinoderms,"
Endocrinology
of
Selected
Invertebrate
Types,
1988,
pp.
453­
476.
17
Shurin,
J.
B.
and
S.
I.
Dodson,
"
Sublethal
toxic
effects
of
cyanobacteria
on
nonylphenol
on
18
environmental
sex
determination
and
development
in
Daphnia,"
Environ.
Toxicol.
Chem.,
19
16,
1997,
pp.
1269­
1276.
20
Touart,
L.
W.
and
K.
R.
Rao,
"
The
influence
of
diflubenzuron
on
survival,
molting
and
limb
21
regeneration
in
the
grass
shrimp,
Palaemonetes
pugio,"
Pollution
Physiology
of
Estuarine
22
Organisms,
1987,
pp.
333­
349.
23
24
25
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
1
O
­
Appendix
O
1
2
Protocol
for
Possible
In
Utero
Developmental
Screening
Assay
3
4
5
As
discussed
in
Chapter
Five,
Sections
III,
A,
5,
and
VII,
F,
the
EDSTAC
recommends
that
EPA
take
6
affirmative
steps,
in
collaboration
with
industry
and
other
interested
parties,
to
attempt
to
develop
a
7
protocol
for
a
full
life
cycle
developmental
exposure
screening
assay
that
can
be
subjected
to
validation
8
and
standardization.
An
in
utero
protocol,
which
may
be
useful
in
trying
to
develop
such
an
assay,
is
9
described
below.
Inclusion
of
this
protocol
is
not
intended
to
limit
the
creative
effort
that
will
be
10
necessary
to
achieve
the
EDSTAC's
recommendation.
11
12
Possible
Protocol
13
14
In
order
to
assess
the
postnatal
developmental
and
reproductive
consequences
of
in
utero
and
15
lactational
exposures
to
chemical
substances
or
mixtures
with
possible
Estrogen,
Androgen,
and
16
Thyroid
activities,
the
screening
assay
should
include
the
following
design
parameters:
17
18
·
At
least
10
presumed
pregnant
(
sperm
positive)
females
per
group
19
·
Administration
of
chemical
substances
or
mixtures
in
vehicle
or
vehicle
alone
(
control
group)
20
by
gavage
once
daily
on
gestational
day
(
gd)
6
(
day
of
vaginal
sperm
detection
=
gd0)
through
21
at
least
postnatal
day
(
pnd)
10
(
preferably
pnd
20)
22
·
Collection
of
maternal
body
weights
and
feed
consumption
on
gd
0,
6,
9,
12,
15,
18,
20,
pnd
23
0,
4,
7,
14,
20,
and
21;
clinical
observations
once
daily
gd
0
through
5,
twice
daily
gd
6
24
through
pnd
21
25
·
On
day
of
parturition
(
pnd
0)
and
on
pnd
4,
7,
14,
and
21,
F1
pups
are
counted,
sexed,
26
weighed,
and
examined
grossly
27
·
Maternal
animals
are
sacrificed
when
pups
are
weaned
on
pnd
21
28
·
F1
offspring
are
necropsied
on:
pnd
0
(
one
per
sex
per
litter);
pnd
4
(
culled
pups
when
litters
29
are
culled
to
eight
with
as
equal
a
sex
ratio
as
possible);
pnd
14
(
one
female
per
litter);
pnd
21
30
(
all
remaining
pups)
or
pnd
21
(
one/
sex/
litter
with
remaining
pups
retained
until
pnd
50).
31
32
Endpoints
to
be
Evaluated
33
34
Maternal
35
36
·
In­
life:
body
weights,
feed
consumption,
clinical
observations.
37
·
Necropsy:
body
weight,
liver
weight,
thyroid
weight,
uterine
implantation
sites
counted
(
for
38
post­
implantation
prenatal
loss),
blood
samples
for
T4/
TSH;
thyroid
retained
in
fixation
for
39
possible
subsequent
histopathology.
40
41
42
43
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
2
O
­
Offspring
1
2
·
Apparent
sex
ratio
(
by
anogenital
distance)
with
body
weight
on
pnd
0
(
at
birth),
4,
7,
14,
and
3
21.
4
·
Postnatal
survival
and
development.
5
·
At
necropsy
on
pnd
0
and
4:
reproductive
tract
anomalies,
e.
g.,
hypospadias;
missing,
small,
6
or
ectopic
testes
or
ovaries;
missing
or
small
epididymides;
missing
seminal
vesicles
or
7
oviducts;
presence
of
Wolffian
ducts
or
their
derivatives
(
epididymides
and
seminal
vesicles)
8
in
females,
presence
of
Mullerian
ducts
or
their
derivatives
(
oviducts)
in
males;
weigh
uterus.
9
·
On
pnd
10­
12,
examine
males
for
retained
nipples.
10
·
On
pnd
14,
necropsy
females
(
one/
litter)
weigh
uterus
(
possible
histopathology
to
measure
11
uterine
gland
number
and
luminal
epithelial
cell
height);
examine
reproductive
system
for
12
anomalies.
13
·
On
pnd
21,
necropsy
one/
sex/
litter
or
all
remaining
pups;
examine
males
for
reproductive
tract
14
anomalies;
weigh
testes
and
epididymides;
examine
females
for
reproductive
tract
anomalies;
15
weigh
uterus
and
ovaries;
examine
for
precocious
puberty
(
acquisition
of
vaginal
patency)
and
16
vaginal
threads;
take
blood
samples
for
T4/
TSH
(
E2
in
females?
T
in
males?).
17
·
If
pups
are
retained
post
wean,
weigh
weekly;
clinical
observations
daily;
evaluate
for
18
acquisition
of
vaginal
potency
(
vp)
(
and
vaginal
threads)
for
females
starting
on
pnd
22;
19
evaluate
for
acquisition
of
preputial
separation
(
pps)
for
males
starting
on
pnd
30.
20
·
On
day
of
acquisition
of
VP
and
PPS,
weigh
animals
and
necropsy;
examine
as
on
pnd
21;
21
also
weigh
and
retain
thyroid,
testes,
epididymides,
ovaries,
and
uterus;
take
blood
samples
for
22
T4/
TSH,
E2,
and
T.
23
24
Interpretation
of
Endpoint
Changes
25
26
·
anogenital
distance
(
covary
by
body
weight
for
statistical
analysis)
27
28
·
increased
in
females
from
androgen
29
·
decreased
in
males
from
estrogen
or
anti­
androgen
30
·
uterine
weight
31
·
increased
precociously
by
estrogen
32
·
male
reproductive
tract
anomalies
from
anti­
androgens
or
possibly
estrogens
(
feminization)
33
·
female
reproductive
anomalies
from
androgens,
estrogens,
or
possibly
anti­
estrogens
34
·
T4/
TSH,
thyroid
weight
(
histopathology)
from
thyroid
or
anti­
thyroid
activity
35
·
accelerated
VP
from
estrogens;
delayed
VP
from
anti­
estrogens/
androgens;
accelerated
PPS
36
from
androgen;
delayed
PPS
from
anti­
androgen
(
covary
age
at
VP
or
PPS
by
body
weight
at
37
acquisition
for
statistical
analysis)
38
39
Notes
40
41
1.
If
run
one
dose
plus
control
(
T1S)
and
terminate
study
at
weaning
on
pnd
21,
approximate
42
duration
6.5
weeks
(
plus
quarantine).
43
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
3
O
­
This
type
of
assay
could
replace
intact
mammalian
pubertal
assays
(
all
apical
with
intact
HPG
1
axis)
2
3
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
1
P
­
Appendix
P
Examples
of
"
Weight­
of­
Evidence"
Determinations
The
following
charts
were
developed,
at
the
request
of
the
EDSTAC,
by
one
EDSTAC
member
and
one
STWG
member
(
not
an
EDSTAC
member).
The
Appendix
is
intended
to
give
the
reader
a
sense
of
how
results
could
be
interpreted,
however,
it
does
not
include
all
possible
examples.
For
instance,
the
document
does
not
include
an
example
of
how
one
might
handle
positive
in
vitro
results
coupled
with
negative
in
vivo
results
in
Tier
1,
which
might
go
into
the
"
hold
box."

T1S
RESPONSE/
"
Weight­
Of­
Evidence"
TABLE.
These
three
tables
(
parts
1,
2,
and
3)
include
known
or
expected
responses
o
the
assays
included
in
the
proposed
EDSTP
T1S
batttery.
The
list
of
endpoints
include
only
those
required.
After
th
follows:
+
is
a
positive;
­
is
negative;
+­?
is
a
possible
positive
response;
and
shaded
responses
are
more
certain
than
u
evidence"
determinations
are
made
to
determine
the
next
steps
for
the
chemical
in
question.
The
three
possible
de
or
(
3)
Other.
Most
chemicals
truly
exist,
and
in
vivo
dosage
levels
are
provided,
while
a
few
are
fictitious,
generate
outcomes
in
T1S.
The
substances
are
identified
by
brief
descriptions.

Part
1.

T1S
ASSAY
ENDPOINT
Antiandrogenic
Fungicide
Estrogenic
Toxic
Substance
Estrogenic
Pesticide
Antiandrogen
ic
Drug
PCBMixture
Anti­
A
pestici
de
IN
VITRO
HTPS
OR
BENCH
ASSAYS
HTPS
ER/
MVLN
ER
AGONIST
­
+
­+
­
­
­

ABOVE
PLUS
E2
ER
ANTAGONIST
­
­
­
­
­
­

HTPS
ER
PLUS
METABOLISM
METAB
IS
ER
AGONIST
­
­
++
­
­
­

ABOVE
PLUS
E2
METAB
IS
ER
ANTAGONIST
­
­
­
­
­
­

HTPS
AR/
CV­
1
AR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
DHT
AR
ANTAGONIST
+
+?
­+
­
­
+

HTPS
AR
PLUS
METABOLISM
METAB
IS
AR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
DHT
METAB
IS
AR
ANTAGONIST
++
­
++
­
­
­

HTPS
TR
TR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
T3
TR
ANTAGONIST
­
­
­
­
­
­

HTPS
TR
PLUS
METABOLISM
METAB
IS
TR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
T3
METAB
IS
TR
ANTAGONIST
­
­
­
­
­
­

IN
VITRO
TESTIS
CULTURE
INHIBITION
OF
P4
OR
T
SYNTHESIS
­
­
­
­
­
­

IN
VIVO
ASSAYS
3
DAY
UTEROTROPHIC
IN
ADULT
OVX'D
RAT
UTERINE
WEIGHT
WITH
AND
WITHOUT
FLUID
­
+
+?
­
­
­

UTERINE
HISTOLOGY
­
+
+?
­
­
­

BODY
WEIGHT
­
­
­
­
­
­
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
2
P
­
VAGINAL
CORNIFICATION
­
+?
­
­
­
­

3DAYUTEROTROPHICINADULTOVX'D
RAT
PLUS
E2
UTERINE
WEIGHT
WITH
AND
WITHOUT
FLUID
­
­
­
­
­
­

UTERINE
HISTOLOGY
­
­
­
­
­
­

BODY
WEIGHT
­
­
­
­
­
­

VAGINAL
CORNIFICATION
­
­
­
­
­
­

PUBERTAL/
THYROID
ASSAY
IN
WEANLING
FEMALE
RAT
GROWTH
­
­
­
­
­
­

AGE
AT
VAGINAL
OPENING
­
++
++
­
­
­

WEIGHT
AT
VAGINAL
OPENING
­
++
++
­
­
­

SERUM
T4
­
­
­
­
++
­

SERUM
TSH
­
­
­
­
+?
­

UTERINE
WEIGHT
WITH
AND
WITHOUT
FLUID
­
­
+?
¯
­
­

OVARIAN
WEIGHT
­
­
+?
¯
­
­
­

THYROID
HISTOLOGY
­
­
­
­
+­
­

NONREPRODUCTIVE
ORGAN
WEIGHTS
LIVER
­
­
­
­
LIVER
­
LIV
­
7­
10DAYHERSHBERGER­
TYPEASSAYIN
CASTRATED
MALE
RAT
SEMINALVESICLEWEIGHT,
WITH
AND
WITHOUT
FLUID
­
­
­
­
­
­

VENTRAL
PROSTATE
WEIGHT
­
­
­
­
­
­

LEVATOR
ANI
PLUS
BULBOCAVERNOSUS
WEIGHT
­
­
­
­
­
­

GROWTH
­
­
­
­
+­?
­

NONREPRODUCTIVE
ORGAN
WEIGHTS
LIVER
­
­
­
­
LIVER
­
LIV
­
7­
10DAYHERSHBERGER­
TYPEASSAYIN
CASTRATED
MALE
RAT:
PLUS
T
SEMINALVESICLEWEIGHT,
WITH
AND
WITHOUT
FLUID
+
¯
­
?
+?
¯
­
+­?
+

VENTRAL
PROSTATE
WEIGHT
+
¯
­
?
+?
¯
+
+
+­?
+

LEVATOR
ANI
PLUS
BULBOCAVERNOSUS
WEIGHT
+
¯
­
?
+?
¯
­
+­?
+

GROWTH
­
­
­+
­
+­?
­

NONREPRODUCTIVE
ORGAN
WEIGHTS
LIVER
­
­
­
­
LIVER
­
LIV
­
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
3
P
­
"
Weight­
of­
Evidence"
Determination:
Hold,
Go
to
T2T,
or
Other
T2T
T2T
T2T
Repeat
T1S
T2T
T2T
T2T
PART
2.
Known
or
expected
responses
of
well
characterized
toxicants
in
the
EDSTP
T1S
batttery.
The
list
of
endpoin
All
of
the
described
examples
would
have
sufficient
positive
responses
to
be
triggered
for
T2T.

T1S
ASSAY
ENDPOINT
IN
VITRO
HTPS
OR
BENCH
ASSAYS
Steroidoge
ne­
sis
Fungicide
Thyroid
Hormone
Analogue
Fungicide
Woodderived
Estrogen
Estrogenic
Pesticide
Antithyroi
dDrug
HTPS
ER/
MVLN
ER
AGONIST
­
­
­
+­
+
­

ABOVE
PLUS
E2
ER
ANTAGONIST
­
­
­
­
­
­

HTPS
ER
PLUS
METABOLISM
METAB
IS
ER
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
E2
METAB
IS
ER
ANTAGONIST
­
­
­
­
­
­

HTPS
AR/
CV­
1
AR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
DHT
AR
ANTAGONIST
­
­
­
­
­
­

HTPS
AR
PLUS
METABOLISM
METAB
IS
AR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
DHT
METAB
IS
AR
ANTAGONIST
­
­
­
­
­

HTPS
TR
TR
AGONIST
­
+
­
­
­
­

ABOVE
PLUS
T3
TR
ANTAGONIST
­
­
­
­
­
­

HTPS
TR
PLUS
METABOLISM
METAB
IS
TR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
T3
METAB
IS
TR
ANTAGONIST
­
­
­
­
­
­

IN
VITRO
TESTIS
CULTURE
INHIBITION
OF
P4
OR
T
SYNTHESIS
++
­
­?
+??
­
­

IN
VIVO
ASSAYS
3
DAY
UTEROTROPHIC
IN
ADULT
OVX'D
RAT
UTERINE
WEIGHT
WITH
AND
WITHOUT
FLUID
­
­
­
­
+
­

UTERINE
HISTOLOGY
­
­
­
­
+
­

BODY
WEIGHT
­
­
­
­
­
­

VAGINAL
SMEAR
­
­
­
­
­

3
DAY
UTEROTROPHIC
IN
ADULT
OVX'D
RAT
PLUS
E2
UTERINE
WEIGHT
WITH
AND
WITHOUT
FLUID
­
­
­
­
­
­

UTERINE
HISTOLOGY
­
­
­
­
­
­

BODY
WEIGHT
­
­
­
­
­
­

VAGINAL
SMEAR
­
­
­
­
­
­
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
4
P
­
PUBERTAL/
THYROID
ASSAY
IN
WEANLING
FEMALE
RAT
GROWTH
­
­?
­
­
­
+­?

AGE
AT
VAGINAL
OPENING
+
­
­
?
+
­
+
?
­
+
+­?

WEIGHT
AT
VAGINAL
OPENING
+
­
­
?
+
­
+
?
­
+
­

SERUM
T4
­
+
¯
?
­
­
­
++

SERUM
TSH
­
+
¯
?
­
­
­
++

UTERINE
WEIGHT
WITH
AND
WITHOUT
FLUID
+?
¯
­
?
+?
¯
­
­
+­?

OVARIAN
WEIGHT
+
­
­
?
+
­
­
­
+?

THYROID
HISTOLOGY
­
+?
¯
­
­
­
­

NONREPRODUCTIVE
ORGAN
WEIGHTS
LIVER
?
­
­
?
LIVER
­
LIVER
­
?
LIVER
­
THYROID
­
7­
10DAYHERSHBERGER­
TYPEASSAYIN
CASTRATED
MALE
RAT
SEMINALVESICLEWEIGHT,
WITH
AND
WITHOUT
FLUID
­
­
­
­
­
­

VENTRAL
PROSTATE
WEIGHT
­
­
­
­
­
­

LEVATOR
ANI
PLUS
BULBOCAVERNOSUS
WEIGHT
­
­
­
­
­
­

GROWTH
­
­?
­
­
­
+­?

NONREPRODUCTIVE
ORGAN
WEIGHTS
LIVER
?
­
­
+?
­
­
LIVER
­
+
­?

7­
10DAYHERSHBERGER­
TYPEASSAYIN
CASTRATED
MALE
RAT:
PLUS
T
SEMINALVESICLEWEIGHT,
WITH
AND
WITHOUT
FLUID
­
­
­
­
+
+­?

VENTRAL
PROSTATE
WEIGHT
­
­
­
­
+
+­?

LEVATOR
ANI
PLUS
BULBOCAVERNOSUS
WEIGHT
­
­
­
­
+?
+­?

GROWTH
­
­?
­
­
­
+­?

NONREPRODUCTIVE
ORGAN
WEIGHTS
LIVER
?
­
­
LIVER
­
­
LIVER
­
+
­?

"
Weight
­
of­
Evidence"
Determination:
Hold,
Go
to
T2T,
or
Other
T2T
REPEAT
T1S,
T2T
T2T
MAY
REPEAT
T1S,
T2T
T2T
T2T
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
5
P
­
PART
3.
Known
or
expected
responses
of
well
characterized
toxicants
in
the
EDSTP
T1S
batttery.
The
list
of
endpoin
T1S
ASSAY
ENDPOINT
IN
VITRO
HTPS
OR
BENCH
ASSAYS
Neurotoxic
Pesticide
Plasticizer
Antiandrogeni
cFungicide
Antiestrogenic
Drug
Herbicide
Xenoestroge
n
HTPS
ER/
MVLN
ER
AGONIST
+­
+­
­
­
­
++

ABOVE
PLUS
E2
ER
ANTAGONIST
­
­
­
­+
­
­

HTPS
ER
PLUS
METABOLISM
METAB
IS
ER
AGONIST
­
­
­
+
­
­

ABOVE
PLUS
E2
METAB
IS
ER
ANTAGONIST
­
­
­
+++
­
­

HTPS
AR/
CV­
1
AR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
DHT
AR
ANTAGONIST
­
­
­
­
­
­?

HTPS
AR
PLUS
METABOLISM
METAB
IS
AR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
DHT
METAB
IS
AR
ANTAGONIST
­
­
++
­?
­
­

HTPS
TR
TR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
T3
TR
ANTAGONIST
­
­
­
­
­
­

HTPS
TR
PLUS
METABOLISM
METAB
IS
TR
AGONIST
­
­
­
­
­
­

ABOVE
PLUS
T3
METAB
IS
TR
ANTAGONIST
­
­
­
­
­
­

IN
VITRO
TESTIS
CULTURE
INHIBITION
OF
P4
OR
T
SYNTHESIS
­
­?
­
­
­
­

IN
VIVO
ASSAYS
3
DAY
UTEROTROPHIC
IN
ADULT
OVX'D
RAT
UTERINE
WEIGHT
WITH
AND
WITHOUT
FLUID
­
­
­
+­
­
++

UTERINE
HISTOLOGY
­
­
­
­+
­
++

BODY
WEIGHT
+
­
­
­
­
­

VAGINAL
CORNIFICATION
­
­
­
+­
­
+

3
DAY
UTEROTROPHIC
IN
ADULT
OVX'D
RAT
PLUS
E2
UTERINE
WEIGHT
WITH
AND
WITHOUT
FLUID
­
­
­
++
­
­

UTERINE
HISTOLOGY
­
­
­
++
­
­

BODY
WEIGHT
+
­
­
­
­
­

VAGINAL
CORNIFICATION
­
­
­
++
­
­
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
6
P
­
PUBERTAL/
THYROID
ASSAY
IN
WEANLING
FEMALE
RAT
GROWTH
+
­
­
­
­
­

AGE
AT
VAGINAL
OPENING
­
­
­
+?
+
¯
?
­

WEIGHT
AT
VAGINAL
OPENING
­
­
­
+?
+
¯
?
­

SERUM
T4
­
­
­
­
­
­

SERUM
TSH
­
­
­
­
­
­

UTERINE
WEIGHT
WITH
AND
WITHOUT
FLUID
­
­
­
+
­
­

OVARIAN
WEIGHT
­
­
­
+
­
­

THYROID
HISTOLOGY
­
­
­
­
­
­

NONREPRODUCTIVE
ORGAN
WEIGHTS
+
­
­
­
­
­

­

7­
10DAYHERSHBERGER­
TYPEASSAYIN
CASTRATED
MALE
RAT
SEMINALVESICLEWEIGHT,
WITH
AND
WITHOUT
FLUID
­
­
­
­
­
­

VENTRAL
PROSTATE
WEIGHT
­
­
­
­
­
­

LEVATOR
ANI
PLUS
BULBOCAVERNOSUS
WEIGHT
­
­
­
­
­
­

GROWTH
+
­
­
­
­
­

NONREPRODUCTIVE
ORGAN
WEIGHTS
+
­
­
­
­
­

7­
10DAYHERSHBERGER­
TYPEASSAYIN
CASTRATED
MALE
RAT:
PLUS
T
SEMINALVESICLEWEIGHT,
WITH
AND
WITHOUT
FLUID
­
+?
++
­
­
­

VENTRAL
PROSTATE
WEIGHT
­
+?
++
­
­
­

LEVATOR
ANI
PLUS
BULBOCAVERNOSUS
WEIGHT
­
+?
+
­
­
­

GROWTH
+
­
­
­
­
­

NONREPRODUCTIVE
ORGAN
WEIGHTS
+
­
­
­
­
­

"
Weight­
of­
Evidence"
Determination:
Hold,
Go
to
T2T,
or
Other
REPEAT
T1S,
Hold1
T2T
T2T
T2T
REPEAT
T1S,
T2T
Other
2
[
Footnote:
(
1)
Ofthedescribedexamples,
theneurotoxicpesticidewouldgotothe"
holdbox"
orbestudiedfurtherbeforeitwouldgotoT2T.
Footnote
(
2)
Thexenoestrogenisonlyestrogenic
invitro
andbyinjection,
butwasnotestrogenicbyoraltreatment.
Theuterotrophicassayshouldberepeated
with
oral
dosing
to
determine
if
the
xenoestrogen
was
estrogenic
by
the
oral
route
before
it
went
to
T2T.]
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
Appendix
Q
Study
Designs
and
Protocols
for
Recommended
Tests
in
Tier
2
Testing
Battery
Table
of
Contents
I.
Two­
Generation
Mammalian
Reproductive
Toxicity
Study
Design
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
1
II.
Study
Design
for
the
Alternative
Mammalian
Reproduction
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
3
III.
Study
Design
for
the
One­
Generation
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
4
IV.
Recommended
Extensions
to
the
Avian
Reproduction
Guideline
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
8
V.
Fish
Life
Cycle
Test
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10
A.
Introduction
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
10
B.
Materials,
Methods,
and
Reporting
Requirements
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
11
C.
Literature
Cited
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
17
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
1
Q
­
I.
Two­
Generation
Mammalian
Reproductive
Toxicity
Study
Design
1
2
The
two­
generation
reproductive
toxicity
study
in
rats
(
TSCA
799.9380,
August
15,
1997;
3
OPPTS
870.3800;
Public
Draft,
February
1996;
OECD
no.
416
1983;
FIFRA
Subdivision
F
4
Guidelines
­
83­
4)
is
designed
to
comprehensively
evaluate
the
effects
of
a
chemical
on
gonadal
5
function,
estrous
cycles,
mating
behavior,
fertilization,
implantation,
pregnancy,
parturition,
6
lactation,
weaning,
and
the
offsprings'
ability
to
achieve
adulthood
and
successfully
reproduce,
7
through
two
generations,
one
litter
per
generation.
Administration
is
usually
oral
(
dosed
feed,
8
dosed
water,
or
gavage)
but
other
routes
are
acceptable
with
justification
(
e.
g.,
inhalation).
In
9
addition,
the
study
also
provides
information
about
neonatal
survival,
growth,
development,
and
10
preliminary
data
on
possible
teratogenesis.
The
experimental
design
for
a
two­
generation
11
reproductive
toxicity
study
is
presented
in
Figure
Q­
1.
12
13
In
the
existing
two­
generation
reproductive
toxicity
test,
a
minimum
of
three
treatment
levels
and
14
a
concurrent
control
group
are
required.
At
least
20
males
and
sufficient
females
to
produce
20
15
pregnant
females
must
be
used
in
each
group
as
prescribed
in
this
current
guideline.
The
highest
16
dose
must
induce
toxicity
but
not
to
exceed
10%
mortality.
In
this
study,
potential
hormonal
17
effects
can
be
detected
through
behavioral
changes,
ability
to
become
pregnant,
duration
of
18
gestation,
signs
of
difficult
or
prolonged
parturition,
apparent
sex
ratio
(
as
ascertained
by
19
anogenital
distances)
of
the
offspring,
feminization
or
masculinization
of
offspring,
number
of
20
pups,
stillbirths,
gross
pathology
and
histopathology
of
the
vagina,
uterus,
ovaries,
testis,
21
epididymis,
seminal
vesicles,
prostate,
and
any
other
identified
target
organs.
Table
5.3
(
Chapter
22
Five,
Section
VI,
B,
provides
a
summary
of
the
endpoints
that
are
evaluated
within
the
framework
23
of
the
experimental
design
of
the
updated
two­
generation
reproductive
toxicity
test
(
and
some
24
additions,
still
under
consideration,
to
cover
Estrogen,
Androgen,
and
Thyroid
concerns).
25
26
These
observations
are
comprehensive
and
cover
every
phase
of
reproduction
and
development.
27
Tests
that
measure
only
a
single
dimension
or
component
of
hormonal
activity,
(
e.
g.,
in
vitro
or
28
short­
term
assays)
provide
supplementary
and/
or
mechanistic
information,
but
cannot
provide
the
29
breadth
of
information
listed
in
Table
5.3,
which
is
critical
for
risk
assessment.
30
31
Additionally,
in
this
study
type,
hormonally­
induced
effects
such
as
abortion,
resorption,
or
32
premature
delivery
as
well
as
abnormalities
and
anomalies
such
as
masculinization
of
the
female
33
offspring
or
feminization
of
male
offspring
can
be
detected.
Substances
such
as
the
34
phytoestrogen,
coumesterol,
and
the
anti­
androgen,
cyproterone
acetate,
which
possess
the
35
potential
to
alter
normal
sexual
differentiation,
were
similarly
detected
in
this
study
test
system
36
(
i.
e.,
1982
Guideline).
The
initial
prebreed
exposure
period
(
ten
weeks)
of
the
two­
generation
37
reproductive
toxicity
test
also
provides
information
on
subchronic
exposures
which
can
be
used
38
for
other
regulatory
purposes.
39
40
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
2
Q
­
Figure
Q.
1
1
2
Two­
Generation
Reproductive
Toxicity
Study
Design
3
4
5
PBE
M
G
L
FO
6
Q
N2
7
N1
8
ECE
9
M
G
L
W
PBE
M
G
L
10
F1
N2
11
N3
|
N1
12
C
VP
EC
E
13
PPS
14
M
G
L
W
15
F2
N3
16
17
C
KEY
18
Q
=
Quarantine
(
one
week)
19
PBE
=
Pre­
Breed
Exposure
(
ten
weeks)
20
M
=
Mating
(
two
weeks)
21
G
=
Gestation
(
three
weeks)
22
L
=
Lactation
(
three
weeks)
23
VP
=
Vaginal
patency
(
evaluated
in
F1
females
on
postnatal
day
22
to
acquisition)
24
PPS
=
Preputial
separation
(
evaluated
in
F1
males
on
postnatal
day
35
to
acquisition)
25
W
=
Weaning
(
postnatal
day
21)
26
N1
=
Necropsy
of
all
paternal
animals
27
N2
=
Necropsy
of
all
maternal
animals
28
N3
=
Necropsy
of
selected
weanlings,
three/
sex/
litter,
if
possible
29
ECE
=
Estrous
Cyclicity
Evaluations
(
three
weeks)
30
C
=
Cull
litters
to
ten
pups
(
with
equal
sex
ratio)
on
postnatal
day
four
31
32
33
Direct
exposure
via
diet,
drinking
water,
inhalation,
etc.

34
Possible
indirect
exposure
from
transplacental
and/
or
translactational
exposure
35
Both
direct
and
possible
indirect
exposure
if
in
feed
or
water
(
nursing
pups
also
self­
feeding
and
drinking)
36
37
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
3
Q
­
II.
Study
Design
for
the
Alternative
Mammalian
Reproduction
Test
1
2
Female
P0
rats
are
quarantined
for
one
week
prior
to
mating.
Sufficient
numbers
of
animals
are
3
used
in
order
to
attain
20
pregnant
dams
per
dosage
group.
During
a
two­
week
mating
period
4
females
are
removed
from
breeding
cages
when
sperm
positive
smears
are
detected
and
they
are
5
randomly
assigned
to
treatment
groups.
Chemicals
are
administered
to
pregnant
female
rats
by
6
oral
gavage
beginning
on
day
seven
of
gestation
(
day
one
being
the
day
sperm
positive)
and
7
continued
throughout
gestation
and
lactation,
up
to
weaning
of
the
F1
generation.
F1
pups
are
8
never
exposed
directly,
but
receive
the
chemical
via
transplacental
and
lactational
exposures.
9
10
On
the
day
of
birth
(
postnatal
day
(
PND)
0),
anogenital
distance
(
AGD)
is
measured
in
both
male
11
and
female
F1
pups.
Animals
with
ambiguous
gender
should
be
uniquely
identified
such
that
sex
12
can
be
identified
at
a
later
age.
At
about
four
days
of
age,
litters
should
be
standardized
to
eight
13
pups
(
four
males
and
four
females
where
possible)
by
randomly
removing
excess
pups
(
necropsy
14
to
confirm
sex).
At
about
twelve
days
of
age,
the
pups
are
examined
for
the
presence
of
15
nipples/
aerolas,
which
should
be
clearly
visible
in
female
but
not
male
pups.
(
These
endpoints
are
16
sensitive
to
androgens
and
antiandrogens.)
17
18
After
weaning
at
21
days
of
age,
pups
(
at
least
20/
sex/
dose)
are
housed
in
unisexual
pairs
until
19
mating
ten
weeks
later
to
produce
the
F2.
All
surplus
weanlings
are
necropsied
and
examined
20
externally
and
internally
for
reproductive
tract
malformations.
P0
parental
females
will
be
21
necropsied
at
the
weaning
of
their
F1
litters
on
PND
21.
These
dams
will
be
subjected
to
a
gross
22
necropsy
with
organ
weights:
thyroid,
ovaries,
pituitary,
uterus
(
count
implants),
kidneys,
and
23
liver.
24
25
After
weaning,
female
F1
retained
rats
are
examined
daily,
beginning
on
PND
22,
for
vaginal
26
opening
until
complete.
Both
age
and
weight
at
vaginal
opening
are
determined.
For
retained
F1
27
male
pups,
the
age
of
preputial
separation
is
determined
from
PND
35
on,
with
age
and
weight
at
28
preputial
separation
recorded.
29
30
Three
weeks
prior
to
mating
of
the
F1,
estrous
cyclicity
is
monitored
for
three
weeks
in
female
31
rats.
Following
this,
F1
non
littermate
pairs
of
males
and
females
from
the
same
dose
group
are
32
established
for
a
two
week
period
in
order
to
produce
the
F2
generation.
Upon
delivery,
F2
pups
33
are
counted
by
sex
and
weighed,
being
terminated
at
weaning
after
individual
body
weights
are
34
taken.
The
F2
litters
are
not
standardized
to
eight
pups
per
litter.
35
36
After
mating
the
F1
males
are
necropsied
and
reproductive
organs
(
SV,
VP,
LA,
testis,
37
epididymides,
pituitary,
etc.)
are
weighed
and
preserved
for
histological
examinations.
In
38
addition,
one
testis
and
caput/
corpus
and
cauda
epididymis
is
used
for
enumeration
of
sperm
39
numbers.
The
thyroid
gland
also
is
weighed
and
saved
for
histological
examination
and
serum
is
40
taken
for
determination
of
serum
T4,
T3
and
TSH
(
especially
if
testing
was
triggered
by
thyroid
41
endpoints
in
screening).
Liver,
kidney,
and
brain
weights
also
should
be
determined.
42
43
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
4
Q
­
F1
females
should
be
necropsied
immediately
after
weaning
of
the
F2
pups
and
reproductive
1
organs
(
ovary,
uterus,
pituitary)
weighed
and
saved
for
histopathological
examination.
Liver,
2
kidney
and
brain
weights
also
should
be
taken.
The
thyroid
gland
also
should
be
weighed
and
3
saved.
4
5
III.
Study
Design
for
the
One­
Generation
Test
6
7
A.
Rats
of
both
sexes
seven­
nine
weeks
of
age,
quarantined
for
one
week,
distributed
into
8
treatment
groups
(
four
is
usual,
can
use
any
number
depending
on
previous
information)
9
ten/
sex/
group,
then
exposed
separately
(
housed
individually)
to
chemical
substance
or
mixture
10
by
whatever
route
(
we
have
used
gavage,
IV
injection,
inhalation,
dosed
feed,
dosed
water)
11
for
a
prebreed
exposure
period
(
suggest
two
weeks
but
would
depend
on
any
prior
12
information
on
chemical
substance
or
mixture
in
terms
of
bioaccumulation,
cumulative
13
toxicity,
etc.;
could
delete
to
shorten
duration)
14
B.
Animals
mated
one:
one
within
groups
for
two
weeks
(
can
use
one
week
to
shorten
15
duration);
vaginal
sperm/
copulation
plug
indicative
of
insemination
with
date
designated
16
gestational
day
(
gd)
0.
On
gd
0,
females
("
dams")
separated
from
males
and
housed
17
individually.
18
C.
Dams
deliver
on
gd
22
±
one
(
for
rats),
date
of
delivery
designated
postnatal
day
(
pnd)
0.
19
Anogenital
distance
measured
in
newborn
pups
(
pnd
0
or
one).
Litters
(
designated
F1)
culled
20
to
standard
size
on
pnd
four
(
usually
eight­
ten/
litter,
with
as
even
a
sex
ratio
as
possible).
21
Culled
pups
could
be
examined,
or
discarded.
F0
males
necropsied
after
all
dams
deliver,
with
22
organ
weights,
blood
samples
(?),
andrological
assessments
(?).
23
D.
At
weaning
on
pnd
21,
F0
dams
necropsied,
with
organ
weights,
blood
samples
(?),
etc.,
F1
24
one­
three
pups/
sex/
litter
necropsied
with
organ
weights,
blood
sampling
(?).
25
E.
Also
at
weaning,
pups
selected
from
all
litters,
ten/
sex/
group,
to
undergo
postwean
exposure.
26
F.
During
ten­
week
post­
wean
exposure
period,
F1
females
assessed
for
vaginal
patency
(
VP;
27
starting
on
pnd
22),
F1
males
assessed
for
preputial
separation
(
preputial
separation;
starting
28
on
pnd
35).
For
the
last
three
weeks,
F1
females
assessed
for
vaginal
cyclicity
by
daily
vaginal
29
smears
(
can
terminate
after
VP
and
preputial
separation).
30
G.
On
pnd
91,
F1
males
and
females
necropsied
with
organ
weights,
blood
sampling,
31
andrological
assessments,
etc.
32
33
Final
ESDTAC
Report
Chapter
Five
Appendices
August
1998
Q
­
5
Figure
Q.
2:
Alternative
Mammalian
Reproductive
Test
(
AMRT)
1
2
0
31
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
4
5
G
67
P
F1
10
week
holding
period
8M
L
20/
sex/
group
M
G
L
Q
9
10
11
W
gd
0
pnd
0
pnd
21
12
gd
0
gd
6
pnd
C
VP
EC
13
21
PPS
14
15
pnd
0
pnd
12
16
AGD
nipples
N
F1
males
N
F1
Females
17
Organ
weights
Organ
weights
18
N
F0
Females
Histology
Histology
19
Gross
necropsy
Sperm
T3/
T4
®
TSH
20
Organ
weights
T3/
T4
®
TSH
21
(
thyroid,
ovaries,
pituitary,
uterus,
liver,
kidneys,
brain)
F2
Weanlings
Count,
sex,
weigh,

22
discard
23
N
Subset
F1
Weanlings
24
Gross
necropsy
(
reproductive
tract
malformations)
25
KEY:
26
Dose
F0
females
Q
=
Quarantine
M
=
Mating
(
2
weeks)
G
=
Gestation
P
=
Parturition
L
=
Lactation
VP
=
Vaginal
Patency
PPS
=
Preputial
Separation
EC
=
Estrous
Cyclicity
gd
=
Gestational
Day
pnd
=
Postnatal
Day
AGD=
Anogenital
Distance
Final
ESDTAC
Report
Chapter
Five
Appendices
August
1998
Q
­
5
N
=
Necropsy
C
=
Cull
F1
pups
on
pnd
4
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
6
Q
­
H.
Information
on:
1
·
General
toxicity
(
body
weights,
feed
and/
or
water
consumption,
chemical
substance
or
mixture
2
intake,
clinical
observations,
etc.)
3
·
Mating
4
·
Fecundity
5
·
Fertility
6
·
Parturition
(
number
total,
dead,
live,
sex,
weight,
AGD,
gross
exam)
7
·
Prenatal
survival
and
growth
8
·
Postnatal
survival
and
growth
9
·
Reproductive
development
(
VP,
preputial
separation)
10
·
Vaginal
cyclicity
in
F1
females
11
·
Male
epididymal
sperm
number,
motility,
morphology;
testicular
homogenization­
resistant
12
spermatid
head
counts
(
SHC),
daily
sperm
production
(
DSP),
efficiency
of
DSP
13
·
Blood
samples
for
E,
A,
T
(
T4,
TSH)
14
·
Organ
weights:
reproductive
organs,
thyroid,
liver
(
EROD?)
15
·
Histology
of
reproductive
organs,
thyroid,
brain,
etc.,
optional
16
I.
Advantages
include:
17
·
Involves
in
utero
exposure
18
·
Assesses
reproductive
development,
structures
and
functions.
19
·
Can
be
modified
as
needed,
e.
g.,
shorten/
lengthen/
delete
prebred
exposure,
shorten
mating,
shorten
20
postwean
exposure
(
to
only
through
reproductive
development
or
to
~
pnd
50),
after
postwean
21
exposure,
mate
F1
to
produce
F2
generation
(
terminate
F2
on
pnd
four,
etc.),
add
other
assessments
22
(
e.
g.,
more
blood
samples,
histopathology,
etc.),
add
F1
developmental
landmarks
such
as
surface
23
righting
reflex,
pinna
detachment,
eye
opening,
acquisition
of
startle
reflex,
midair
righting
reflex,
24
motor
activity,
learning
and
memory,
etc.
25
·
Covers
the
mammalian
"
waterfront"
for
E,
A,
and
T.
26
·
Any
contract
lab
currently
performing
multi­
generation
studies
has
the
necessary
capabilities
(
staff,
27
facilities,
SOPs,
experience).
This
is
relatively
"
low
tech"
except
for
the
blood
assays
and
28
andrological
assessments.
29
J.
Sensitivity:
30
The
sensitivity
is
dependent
on
the
number
of
animals/
group
(
for
statistical
power),
the
type
of
31
assessments
included,
the
number
of
treatment
groups
and
the
range
of
doses.
It
is
a
powerful,
apical
32
assay.
33
34
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
7
Q
­
Figure
Q.
3
1
One­
Generation
Mammalian
Reproductive
Toxicity
Test
2
3
4
PBE
M
G
L
FO
5
Q
6
2
wks
2
wks
3
wks
3
wks
N,
B,
O
7
F0
females
89
N,
B,
A,
O
10
F0
males
11
12
G
P
L
W
10
week
PWE 

13
91
d
14
15
16
Cull
to
N
VP
VC
17
8­
10/
Litter
1­
3/
sex/
Litter
PPS
18
KEY:
19
N,
B,
A,
O
Q20
=
Quarantine
AGD
F1
males
PBE21
=
Prebred
exposure
and
females
M22
=
Mating
G23
=
Gestation
L24
=
Lactation
N25
=
Necropsy
B26
=
Blood
samples
A27
=
Andrological
assessments
O28
=
Organ
weights
P29
=
Parturition
(
delivery,
pnd
0)

AGD30
=
Anogenital
distance
VP31
=
Vaginal
patency
PPS32
=
Preputial
separation
VC33
=
Vaginal
cyclicity
PWE34
=
Postwean
exposure
35
Direct
exposure
36
Possible
indirect
transplacental
and/
or
translactational
exposure
37
Direct
and
possible
indirect
exposure
if
route
is
dosed
feed
or
water
38
39
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
1
Q
­
IV.
Recommended
Extensions
to
the
Avian
Reproduction
Guideline
A.
The
following
modifications
of
OPPTS
850.2300
are
recommended
to
assess
development
in
the
offspring
of
exposed
adults.

·
modify
(
d)
Test
procedures
(
2)
Definitive
test,
(
viii)
Chick
...,
to
require
that
all
offspring
be
maintained
separately
by
parental
(
dose)
group;
and,

·
modify
(
d)
Test
procedures
(
2)
Definitive
test,
(
ix)
Observations
of
record
on
chicks...
to
include:

1.
the
genetic
sex
ratio
at
hatching;
genetic
sex
can
be
determined
routinely
by
the
identification
of
restriction
fragments
from
sex
chromosomes
derived
from
red
blood
cells
in
a
small
blood
sample
(~
0.3ml)
(
Halvorsen
1990;
Dvorak
et
al.
1992);
identification
of
the
genetic
sex
of
offspring
allows
closer
control
of
subsequent
operations,
as
well
as
providing
an
endpoint
of
interest.

2.
at
14
days
of
age,
for
12
genetic
males
at
each
dose
level:

·
external
characteristics,
the
size
of
cloacal
protuberance
in
ducks
and
cloacal
gland
size
in
quail
(
Ottinger,
M.
A.
and
H.
J.
Brinkley.,
other
refs);
and,

·
internal
characteristics,
the
size
and
dimorphism
of
gonads,
the
presence
of
oviducts,
especially
on
the
left
side,
and
the
histology
of
the
gonad,
looking
especially
for
the
relative
amount
of
cortex
and
the
presence
of
primary
germ
cells
located
there,
(
Fry
et
al.,
1987;
Ottinger
and
Bakst,

1981);
also,
serum
sex
steroid
(
estrogens,
androgens)
should
be
determined
(
more
refs
needed)

3.
at
14
days
of
age,
for
12
genetic
females
at
each
dose
level:

·
internal
measurements
only,
looking
especially
for
presence
of
structures
on
the
right
side,
and
histologically
the
relative
amount
of
cortex
and
medulla
and
the
development
of
oocytes;
also,

serum
sex
steroid
(
estrogens,
androgens)
should
be
determined.

4.
at
14
days
of
age,
both
sexes
at
each
dose
level
will
be
assessed
for
general
growth
parameters
indicative
of
adequate
thyroid
function
major
organ
weights,
including
brain,
body
weights,
wing
and
bone
length,
thyroid
weight,
and
skeletal
x­
ray.
If
there
are
significant
differences
among
exposure
groups
in
these
responses,
thyroid
histopathology
will
be
performed
on
all
groups.
Otherwise,
only
the
high
dose
and
control
groups
will
be
so
assessed.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
2
Q
­
5.
at
14
days
of
age
all
surviving
chicks
will
be
subjected
to
the
visual
cliff
test
(
Baxter
et
al.,
1969;

Dahlgren
and
Linder,
1971;
Emlen,
1963;
Fleming
et
al.,
1985;
Fox,
1976).
The
F1
chicks
will
be
challenged
with
a
cold
stress
test
(
Fleming
et
al.,
1985a;
Fleming
et
al.,
1985b;
Maguire
and
Williams,
1987;
Martin
and
Solomon,
1991;
Rattner
et
al.,
1982;
Rattner
et
al.,
1987;
Tori
and
Mayer,
1981)
and
the
nest
attentiveness
test
(
Fox
et
al.,
1978;
McArthur
et
al.,
1983;
Kubiak
et
al.,

1989).
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
3
Q
­
·
Breeding
the
Second
Generation:

Complete
assessment
of
the
potential
endocrine
disruption
of
chemical
substances
or
mixtures
requires
a
test
to
the
reproductive
capability
of
birds
exposed
as
embryos.
This
is
distinct
from
data
gathered
in
part
A
that
evaluates
the
progress
of
sexual
differentiation
and
reproductive
development
in
birds
exposed
as
embryos.
There
are
no
codified
guidelines
for
a
two­
generation
avian
reproduction
test,
however,
an
OECD
work
group
is
currently
designing
protocols
for
such
a
test.

These
guidelines
should
be
used
if
they
will
be
available
soon
enough
to
implement
in
Tier
2
Testing.

If
not,
development
of
a
protocol
by
the
EPA
should
be
a
very
high
priority.

A
two­
generation
avian
reproduction
test
should
have
the
following
features:

·
uses
subjects
that
mature
and
breed
in
a
short
time;
Japanese
quail
are
an
obvious
choice,
and
in
addition
there
is
substantial
information
on
the
husbandry,
development,
endocrinology
and
reproduction
of
this
species,

·
exposure
of
the
parental
generation
should
be
exposed
through
the
feed
before
and
continue
through
egg­
laying;
chicks
(
F1
generation)
should
be
fed
from
hatching
diets
at
the
same
dose
levels
as
P1,

·
breeding
of
F1
should
begin
as
soon
as
possible,
consistent
with
the
biology
of
the
species
used;

Japanese
quail
mature
in
about
eight
weeks,
and
should
be
kept
on
long
day
photoperiod
from
hatching,

·
responses
measured
should
follow
the
guidelines
for
the
Avian
Reproduction
test
(
OPPTS
850.2300)
with
the
addition
of
measurement
of
the
age
at
reproduction
in
both
sexes.

·
From
the
12
hens
per
treatment
group,
60
eggs
will
be
needed
with
30
eggs
or
survivors
thereof
sacrificed
at
14
days
and
30
eggs/
chicks
maintained
for
F1
testing.

An
efficient
method
for
conducting
a
two­
generation
test
would
be
to
use
the
Japanese
quail
in
a
modified
Avian
Reproduction
test
as
described
in
part
A,
and
produce
extra
offspring
that
would
the
F1
breeders
in
a
two­
generation
test.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
4
Q
­
B.
In
the
expanded
two
generation
testing
protocol
described
in
section
A
above,
exposure
of
F1
embryos
occurs
only
through
maternal
depositions
of
chemical
substance
or
mixtures
to
eggs.
Often
avian
egg
exposure
occurs
directly,
such
as
during
spraying
of
pesticides
and
pesticide­
fertilizer
mixtures
or
crude
oils
and
derivatives
during
spills,
or
with
certain
air
pollutants.
Direct
exposure
differs
from
maternal
deposition
because
the
parent
compound
is
not
being
introduced
to
the
egg
as
adults
metabolize
the
parent
compounds.
These
facts
argue
for
an
alternative
exposure
to
embryos
via
the
direct
pathway.
For
chemicals
that
have
pathways
directly
to
eggs,
(
certain
pesticides,
fertilizers,
oils,

and
some
air
pollutants),
a
direct
exposure
protocol
needs
to
further
amend
existing
EPA
method
(
OPPTS
850.2300).
Experience
with
this
assessment
protocol
is
well
documented
(
Hoffman
and
Eastin,
1981;
Hoffman
and
Albers,
1984;
Hoffman,
1990;
Somers
et
al.,
1974).
It
is
recommended
that
eggs
be
dipped
in
appropriate
solutions,
including
registered
carrier
solvents
for
pesticides
for
ten
seconds
at
84
hrs
of
incubation
for
bobwhite
quail
and
mallards
and
63
hours
for
Japanese
quail.
This
assures
embryonic
development
is
proceeding
uniformly
prior
to
exposure
(
eggs
can
be
candled)
and
during
the
period
of
organogenesis.
Eggs
will
be
air
dried
for
five
minutes
prior
to
return
to
the
incubator.
All
eggs
will
be
clean
eggs
with
no
previous
adult
exposures.
Surplus
control
group
eggs
or
purchased
eggs
may
be
used.
Testing
of
these
eggs
and
offspring
will
be
identical
to
those
prescribed
in
Section
A,
1,
5
above.

·
Functional
Endpoint
Tests
for
the
Two­
Generation
Avian
Reproduction
Test:

i.
Nest
Attentiveness/
Incubation
Behavior:
Measuring
nest
attentiveness
assessment
of
adult
birds
during
incubation
would
complement
other
proposed
toxicity
endpoints.
It
is
a
functional
test
of
adult
reproduction
behavior
following
embryonic
and
subsequent
dietary
exposure
and
can
provide
a
functional
endpoint
of
high
ecological
relevance.

ii.
Visual
Cliff
Test:
A
short­
term
behavioral
assessment
of
hatchling
chicks
to
a
visual
challenge.
It
evaluates
depth
perception
and
motivation
needed
for
escape
responses
and
predatory
performance
to
capture
live
prey.
A
functional
deficit
in
these
behavior
patterns
early
in
life
can
result
in
reduced
growth
and
survival,
both
of
which
are
ecologically
important.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
5
Q
­
iii.
Cold
Stress
Test:
The
ability
of
chicks
and
adult
birds
to
regulate
body
temperature
is
a
vital
physiological
response
and
largely
controlled
by
thyroid
hormones.
Delay
or
impairment
of
thermoregulatory
ability
might
significantly
affect
survival
during
inclement
weather.

Likewise,
adult
birds
are
exposed
to
winter
cold
stress
and
inclement
weather
during
migration
periods.
This
test
provides
a
functional
evaluation
of
the
effect
of
cold
stress
on
body
temperature
and
the
ability
to
regulate
body
temperature
within
normal
ranges.

V.
Fish
Life
Cycle
Test
A.
Introduction
1.
Purpose
The
fish
life
cycle
test
is
designed
to
evaluate
growth,
maturation,
and
reproduction
of
fish
exposed
to
a
chemical,
substance,
or
mixture
from
a
continuous
exposure
through
a
complete
reproductive
life
cycle.

2.
Test
Material
The
test
material
must
be
soluble
or
dispersable
to
allow
distribution
to
test
aquaria.

3.
Acceptable
Protocols
Test
protocols
which
provide
guidance
for
performing
a
fish
life
cycle
test
can
be
found
in
the
following
references.

Benoit,
D.
A.,
"
User's
Guide
for
Conducting
Life­
Cycle
Chronic
Toxicity
Tests
with
Fathead
Minnows
(
Pimephales
promelas),"
Environ.
Res.
Lab.
­
Duluth,
MN.
EPA
600/
8­
81­
011,
1981
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
6
Q
­
Hansen,
D.
J.,
P.
R.
Parrish,
S.
C.
Schimmel,
and
L.
R.
Goodman,
"
Toxicity
Test
Using
Sheepshead
Minnows
(
Cyprinodon
variegatus),"
Bioassay
Procedures
for
the
Ocean
Disposal
Permit
Program,
EPA­
600/
9:
78­
010,
1978.

American
Public
Health
Association,
American
Water
Works
Association
and
Water
Pollution
Control
Federation,
"
Standard
Methods
for
the
Examination
of
Water
and
Wastewater,"
16:
854,

1985.

B.
Materials,
Methods,
and
Reporting
Requirements
[
Note:
numbers
in
parentheses
refer
to
the
numbered
references
at
the
end
of
this
section.]

1.
Biological
System
a)
Preferred
Species
The
preferred
test
species
are
fathead
minnow
(
Pimephales
promelas)
and
sheepshead
minnow
(
Cyprinodon
variegatus).

b)
Source
and
Acclimation
of
Fish
Adult
fish
are
obtained
from
either
wild
populations
or
suitable
culture
laboratories.
Fathead
minnows
should
be
maintained
at
25o
C.
Sheepshead
minnows
are
to
be
held
in
flowing
30o
C
seawater
of
>
15
o/
oo
salinity.
Source
fish
should
be
held
for
at
least
two
weeks
prior
to
breeding
and
should
not
exhibit
mortalities
>
5
%.

c)
Eggs
from
Adult
Fish
Artificial
inducement
and
natural
spawning
are
the
two
methods
for
obtaining
a
sufficient
number
of
eggs
for
a
chronic
exposure.
Artificial
inducement
entails
the
stimulation
of
egg
production
by
injection
of
human
gonadotrophic
hormone.
Sheepshead
minnow
females
can
be
injected
intraperitoneally
with
five
IU
HCG
on
two
consecutive
days.
Two
days
following
the
second
injection,
ova
from
females
are
stripped
and
mixed
with
sperm
derived
from
excised
macerated
testes.
Usually
ten
females
and
five
males
should
be
used.(
l)
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
7
Q
­
Natural
spawning
is
possible
with
a
few
considerations
for
each
fish
species
and
is
preferred.

Fathead
minnows
require
paired
spawning
in
order
to
eliminate
fighting
and
competition.

Culture
units
for
this
fish
can
consist
of
one
tank
measuring
30.5
x
30.5
x
61
cm
with
a
water
depth
of
18
cm
and
four
individual
spawning
chambers
(
15.2
x
30.5
cm)
formed
by
stainless
steel
screen
dividers
(
5
mesh,
0.89
mm
wire).(
2)
Sheepshead
minnow
embryos
are
obtained
by
combining
five
or
more
females
and
three
males
in
spawning
chambers
measuring
20
x
35
x
22
cm.
Mature
adults
should
attain
a
minimum
standard
length
of
2
>
mm
and
display
courtship
characteristics
(
sexual
dimorphism,
territoriality,
and
aggressive
behavior
by
the
male).
Fish
from
each
spawning
group
are
left
in
chambers
for
a
minimum
of
14
days.(
2)

Adult
deaths
during
spawning
should
be
noted;
dead
animals
are
removed,
but
not
replaced.
At
termination
of
each
spawning
group,
lengths
and
weights
of
individual
fish
are
measured.

d)
Feeding
Fry
of
both
fish
species
should
be
fed
equal
portions
of
live
brine
shrimp
nauplii
at
least
two
times
daily
about
six
hours
apart
for
three
weeks
(
frozen
nauplii
are
not
to
be
used).
juveniles
(
four
weeks
posthatch)
and
adults
can
be
fed
twice
daily
on
equal
portions
of
dry
food
(
e.
g.,

Tetramin
or
Biorell)
supplemented
with
frozen
adult
brine
shrimp.
Each
batch
of
food
should
be
checked
for
pesticides
and
metals.

e)
Embryo
Removal
A
record
of
numbers
and
egg
fertility
must
be
maintained
daily.
All
embryos
are
examined
daily
with
a
dissecting
scope
or
magnifying
viewer
to
remove
empty
shells
and
opaque,
or
abnormal
appearing
embryos.
If
less
than
50
percent
of
the
embryos
from
a
spawn
appear
to
be
healthy
and
fertile,
all
embryos
from
that
spawn
should
be
discarded.
(
2)
Embryos
should
be
removed
at
a
fixed
time
each
day
so
spawning
activity
is
not
disturbed
unnecessarily.

f)
Embryo
Exposure
(
Four­
Five
Days)

The
life­
cycle
chronic
toxicity
test
must
begin
with
embryos
from
at
least
three
separate
spawnings
that
are
<
24
hours
old
and
have
soaked
in
dilution
water
for
at
least
t­
waugh
hours.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
8
Q
­
(
2)
Testing
begins
by
randomly
distributing
50
embryos
to
each
of
the
four
replicate
larval
growth
chambers.
(
2)
Ten
embryos
are
transferred
with
a
large
bore
eye
dropper
to
successive
incubation
cups
which
are
standing
in
dilution
water.
This
is
repeated
until
50
embryos
are
in
each
cup.
The
incubation
cups
are
then
distributed
to
each
replicate
larval
chamber.

Survival
of
embryos,
time
required
to
hatch,
hatching
success,
and
survival
of
fry
for
four
weeks
are
determined
and
recorded.
Dead
embryos
usually
turn
opaque
and
must
be
counted
and
removed
each
day
until
hatching
is
complete.
Live
fungused
embryos
must
be
removed
daily
and
counted
as
dead.(
2)

g)
Larval­
Juvenile
Exposure
(
Eight
Weeks)

After
hatching,
each
group
of
larvae
is
randomly
reduced
to
25,
and
released
in
replicate
larval
growth
chambers.
(
2)
This
random
selection
must
include
any
fish
that
are
lethargic
or
deformed.

Survival
should
be
determined
in
each
replicate
growth
chamber
at
least
once
a
week.
Survival
during
this
period
is
determined
by
counting
the
number
of
live
fish,
since
dead
larvae
deteriorate
rapidly.

At
four
and
eight
weeks
after
hatching,
total
lengths
(
mm)
of
all
fish
must
be
recorded.
(
2)

Techniques
suggested
for
measuring
fish
include
direct
measurement
and
a
photographic
method
outlined
by
McKim
and
Benoit
1971.
(
3)
In
order
to
treat
growth
as
a
valid
endpoint,
the
amount
of
food
given
to
the
control
and
treated
fish
must
be
kept
constant
between
exposures.

h)
Juvenile­
Adult
Exposure
(
32­
40
Weeks)

All
fish
are
transferred
to
the
adult
spawning
tank
(
same
concentration)
eight
weeks
after
hatching.
(
2)
Each
tank
should
have
25
randomly
selected
fish
(
deformed
individuals
included).

When
secondary
sexual
characteristics
are
well­
developed,
fathead
minnow
(
20­
24
week
post
hatch)
males
will
exhibit
tubercles,
pads
and
body
color,
while
females
will
exhibit
extended
transparent
and
canals
(
urogenital
papilla).
At
this
time,
mature
fish
should
be
placed
in
spawning
tank,
separate
from
undeveloped
fish.
(
2)
The
spawning
tank
will
be
divided
into
four
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
9
Q
­
individual
spawning
chambers
with
appropriate
spawning
substrates.
Four
males
and
four
females
are
randomly
chosen
and
assigned
to
spawning
chambers.
Substrates
are
examined
daily
and
embryos
removed,
counted,
and
recorded
separately
for
each
pair.

The
adult
exposure
(
fathead
minnow)
should
be
terminated
when,
during
the
decreasing
daylength
photoperiod,
a
one­
week
period
passes
in
which
no
spawning
occurs.
(
2)
Testing
using
sheepshead
minnows
should
terminate
after
spawning
is
observed
for
two
weeks
because
this
fish
spawns
readily
and
almost
daily
unless
immature
or
affected
by
a
pollutant.
(
l)

i)
Second
Generation
Embryo
Exposure
(
Four­
Five
Days)

Fifty
embryos
from
each
concentration
level
are
randomly
selected
and
transferred
to
incubation
cups
for
hatch.
Those
embryos
not
selected
are
discarded.
Test
procedures
used
during
embryo
removal
and
embryo
exposure
(
sections
A,
5
and
A,
6,
respectively)
are
repeated
with
second
generation
embryo
reexposure.
(
l,
2)

j)
Second
Generation
Larval­
juvenile
Exposure
(
Four­
Eight
Weeks)

Eight
week
exposure
begins
with
the
release
of
two
groups
of
25
larvae
in
replicate
growth
chambers.
These
larvae
should
have
been
produced
from
different
breeding
pairs
in
each
spawning
tank.
Selection
of
each
group
should
be
from
early
spawnings.
(
1,
2)
Testing
procedures
are
the
same
as
those
described
in
section
A,
7.

Each
group
of
second
generation
fish
is
terminated
eight
weeks
after
hatching.
Fish
are
blotted,

weighed,
and
measured
before
being
discarded.
(
l,
2)

2.
Physical
System
a)
Test
Water
·
Fathead
Minnow
i.
Test
water
can
be
supplied
from
a
well
or
spring
provided
that
the
source
is
not
polluted;
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
10
Q
­
ii.
Water
should
be
sterilized
with
ultra
violet
irradiation
and
tested
for
pesticides,
heavy
metals,
and
other
possible
contaminants;

iii.
Hardness
of
40
to
48
mg/
L
as
CaCO3
and
pH
of
7.2
to
7.6
is
recommended;

iv.
Reconstituted
water
can
be
used.
Detailed
descriptions
of
acceptable
procedures
for
preparing
diluent
are
found
in
the
protocols
by
the
American
Society
of
Testing
Materials
1980.
(
4)

·
Sheepshead
Minnow
i.
Test
water
may
be
natural
(
sterilized
and
filtered
to
remove
particles
15
microns
and
larger)
or
a
commercial
mixture
(
provided
that
there
are
no
adverse
affects
to
test
organisms
or
alterations
in
test
material
toxicity);

ii.
Natural
seawater
is
considered
to
be
of
constant
quality
if
the
weekly
range
of
salinity
is
less
than
six
percent,
and
if
monthly
pH
range
is
less
than
0.8
of
a
pH
unit;

iii.
Salinity
should
be
>
15
parts
per
thousand;

iv.
Water
must
be
sterilized
and
free
of
Pollutants.
(
l)
Irradiation
with
ultraviolet
light
is
recommended
to
Sterilize
test
water.

b)
Temperature
A
continuous
record
of
temperature
of
test
water
must
be
kept.

·
Fathead
Minnow
Temperature
should
be
maintained
at
25
°
C
and
should
not
remain
outside
the
range
of
24
to
26
°
C
for
more
than
48
hours.
(
2)

·
Sheepshead
Minnow
Temperature
should
be
maintained
at
30
°
C.
(
l)

c)
Photoperiod
Lighting
above
each
replicate
must
be
balanced
and
must
simulate
the
wavelength
spectra
of
sunlight.
Light
intensities
at
the
water
surface
should
range
from
10
to
100
lumens.
One
lumen
per
square
meter
is
equal
to
one
lux.

·
Fathead
Minnow
A
graduated
photoperiod
as
described
in
Benoit
(
1981)
is
used.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
11
Q
­
·
Sheepshead
Minnow
A
16­
hour
light/
8­
hour
dark
cycle
is
maintained
throughout
the
test.
(
l)

d)
Dosing
Apparatus
Intermittent­
flow
proportional
diluters
as
described
by
Mount
and
Brungs
(
5)
or
continuous­
flow
serial
diluters,
as
described
by
Garton
(
6)
should
be
employed.
A
minimum
of
five
toxicant
concentrations
with
a
dilution
factor
not
greater
than
0.50
and
one
control
should
be
used.

e)
Toxicant
Mixing
A
mixing
chamber
is
recommended
to
assure
adequate
mixing
of
test
material.
Aeration
should
not
be
used
for
mixing.
Separate
flow
splitter
delivery
tubes
should
run
from
this
container
to
each
replicate
larval
and
adult
tank.
(
2)
Depending
upon
the
apparatus
used
a
mixing
chamber
may
not
be
required.
T­
t
must,
however,
be
demonstrated
that
the
test
solution
is
completely
mixed
before
introduction
into
the
test
system.
Flow
splitting
accuracy
must
be
within
10
percent
and
should
be
checked
Periodically
for
accurate
distribution
of
test
water
to
each
tank.

(
2)

f)
Test
Tanks
All
test
tanks
should
be
of
either
all
glass
or
glass
with
a
plastic
or
stainless
steel
frame.

·
Fathead
Minnow
Adult
spawning
tanks
should
measure
30.5
x
30.5
x
91.4
cm
or
30.5
x
30.5
x
61
cm
long
with
a
screened­
off
or
separate
larval
tank.
(
2)
Each
larval
section
is
divided
in
half
allowing
for
two
larval
growth
chambers
for
each
adult
spawning
tank.
Larval
chambers
should
be
designed
with
glass
bottoms
and
drains
that
allow
water
to
be
drawn
down
to
3
cm.
(
2)
Test
water
must
be
delivered
separately
to
each
adult
tank
and
larval
section,
with
one­
third
of
the
water
volume
going
to
the
latter.
Larval
tanks
can
also
be
conveniently
located
directly
above
spawning
tanks
containing
test
solutions
of
the
same
concentrations
so
they
can
be
drained
directly
into
the
spawning
tank.
Test
water
depth
in
adult
tanks
and
larval
chambers
should
be
a
minimum
of
15
cm.
(
2)

·
Sheepshead
Minnow
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
12
Q
­
Tanks
45
x
90
x
26
cm
with
a
water
depth
of
19
cm
have
been
successful.
Larval
chamber
design
and
test
water
divided
are
the
same
as
described
for
fathead
minnow.
(
l)

g)
Embryo
and
Fry
Chambers
Embryo
incubation
chambers
should
be
made
from
120
ml
glass
jars
with
the
bottoms
replaced
with
40
mesh
stainless
steel
or
nylon
screen.
Chambers
can
be
oscillated
vertically
(
2.5
to
4.0
cm)
in
the
test
water
(
rocker
arm
apparatus,
2
rpm
motor)
or
placed
in
separate
chambers
with
self­
starting
siphons.
Both
methods
should
insure
adequate
exchange
of
water
and
test
material.

(
1,
2)

h)
Flow
Rate
Flow
rates
to
adult
tanks
or
larval
chambers
should
provide
90
percent
replacement
in
8
to
12
hours.
(
2)
Flow
rate
must
be
capable
of
maintaining
dissolved
oxygen
at
above
75
percent
of
saturation
and
maintain
the
toxicant
level
(
concentration
cannot
drop
below
20%
with
fish
in
the
tank).

i)
Aeration
Dilution
water
should
be
aerated
vigorously
insuring
that
dissolved
oxygen
concentration
will
be
at
or
near
90
to
100
percent
saturation.
Test
tanks
and
embryo
chambers
should
not
be
aerated.

(
l,
2)

3.
Chemical
System
a)
Concentrations
A
minimum
of
five
concentrations
of
toxicant
and
a
control
(
all
duplicated)
are
used
in
this
chronic
test.
A
solvent
control
is
added
if
a
solvent
is
utilized.
As
a
minimum,
the
concentration
of
toxicant
must
be
measured
in
one
tank
at
each
toxicant
level
every
week.
Water
samples
should
be
taken
about
midway
between
top
and
bottom
and
the
sides
of
the
tank.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
13
Q
­
One
concentration
selected
must
adversely
affect
a
life­
stage
and
one
concentration
must
not
affect
any
life­
stage.
(
1)

b)
Measurement
of
other
Variables
Dissolved
oxygen
must
be
measured
at
each
concentration
at
least
once
a
week.
Freshwater
parameters
in
a
control
and
one
concentration
must
be
analyzed
once
a
week.
These
parameters
should
include
pH,
alkalinity,
hardness,
and
conductance.
Natural
seawater
must
maintain
a
constant
salinity
and
not
fluctuate
more
than
six
percent
weekly
or
a
monthly
pH
range
of
less
than
0.8
of
a
pH
unit.
(
2)

c)
Solvents
If
solvents
other
than
water
are
necessary,
they
should
be
used
sparingly
and
not
to
exceed
0.1
mL/
L
in
a
flow­
through
system.
The
following
solvents
are
acceptable:
(
4)

·
dimethylformamide
·
triethylene
glycol
·
methanol
·
acetone
·
ethanol
The
development
of
chemical
saturators
for
use
with
hydrophobic
chemicals
may
be
used
with
most
test
chemicals.
(
7,
8,
9)

d)
Calculations
Data
from
these
toxicity
studies
are
of
two
types,
continuous
(
i.
e.,
length,
weight)
and
discrete
(
i.
e.,
number
of
fish
hatching
or
surviving).
In
general,
continuous
data
should
be
analyzed
with
the
appropriate
analysis
of
variance
(
ANOVA)
technique
followed
by
an
appropriate
multiple
comparison
test.
Dichotomous
data
should
be
analyzed
using
some
form
of
a
2
x
2
contingency
table.
Final
EDSTAC
Report
Chapter
Five
Appendices
August
1998
14
Q
­
As
a
part
of
the
ANOVA,
it
is
desirable
to
plot
the
residuals
versus
concentration
and
determine
whether
there
have
been
any
obvious
violations
of
homoscedasticity
on
the
assumption
of
normality.
All
test
results
must
be
accompanied
by
the
original
(
raw)
data
for
the
reviewer's
evaluation.

C.
Literature
Cited
Hansen,
D.
J.,
P.
R.
Parrish,
S.
C.
Schimmel,
and
L.
R.
Goodman,
"
Toxicity
Test
Using
Sheepshead
Minnows
(
Cyprinodon
variegatus),"
Bioassay
Procedures
for
the
Ocean
Disposal
Permit
Program,
EPA­

600/
9­
78­
010,
1978.

Benoit,
D.
A.,
"
User's
Guide
for
Conducting
Life
Cycle
Chronic
Toxicity
Tests
with
Fathead
Minnows
(
Pimephales
promelas),"
Environ.
Res.
Lab.,
Duluth,
MN.
EPA­
600/
8:
81­
011,
1981.

McKim,
I.
M.,
D.
A.
Benoit,
"
Effect
of
long­
term
exposures
to
copper
and
survival,
reproduction
and
growth
of
rainbow
trout
(
Salvelinus
fontinalis),"
J.
Fish.
Res.
Board
Can.,
28:
655­
662,
1971.

ASTM
Standard
E
729­
80,
"
Practice
for
Conducting
Acute
Toxicity
Tests
with
Fishes,

Macroinvertebrates,
and
Amphibians,"
American
Society
for
Testing
and
Materials,
1916
Race
street,
Philadelphia,
PA
19103,
1971.

Mount,
D.
I.,
W.
A.
Brungs,
"
A
simplified
dosinq
apparatus
for
fish
toxicology
studies,"
Water
Res.,

1:
21­
29,
1967.

Garton,
R.
R.,
"
A
simple
continuous­
flow
toxicant
delivery
system,"
Water
Res.,
14:
227­
230
1980.

Chadwick,
G.
C.,
and
U.
Kiigemagi,
"
Toxicity
evaluation
of
a
technique
for
introducing
dieldrin
into
water,"
J.
Fish.
Res.
Board
Can.,
40:
76­
82,
1968.

Gingerich,
W.
H.,
W.
K.
Seim,
and
R.
D.
Schonbrod,
"
An
apparatus
for
the
continuous
generation
of
stock
solutions
of
hydrophobic
chemicals,"
Bull.
Environ.
Contam.
Toxicol.,
23:
685­
689,
1979.

Veith,
G.
D.,
and
V.
M.
Comstock,
"
Apparatus
for
continuously
saturating
water
with
hydrophobic
organic
chemicals,"
J.
Fish.
Res.
Board
Can.,
32:
1849­
1851,
1975.

U.
S.
EPA
Pesticide
Reregistration
Rejection
Rate
Analysis
(
EPA
738­
R­
94­
035),
United
States
Environmental
Protection
Agency.
Washington,
DC.,
1994.
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
1
R
­
Appendix
R
1
2
Preliminary
Categorization
of
Tier
1
Screens
and
Tier
2
Tests
3
by
the
Screening
and
Testing
Work
Group
4
5
As
mentioned
in
Chapter
Five,
the
screens
and
tests
being
recommended
by
the
EDSTAC
vary
6
considerably
in
terms
of
the
effort
necessary
to
be
fully
validated
and
standardized.
During
their
7
deliberations,
the
Screening
and
Testing
Work
Group
(
STWG)
attempted
to
categorize
the
levels
8
of
validation
required
of
the
recommended
screens
and
tests
and
their
preliminary
efforts
are
9
found
in
this
Appendix.
This
information
is
included
to
give
the
reader
a
sense
of
where
the
10
recommended
screens
and
tests
may
exist
across
the
spectrum
of
validation;
however,
the
11
EDSTAC
agreed
not
to
try
to
reach
consensus
agreement
on
where
all
the
screens
and
tests
lie,
as
12
they
will
all
need
to
validated
and
standardized
before
being
included
in
the
Endocrine
Disruptors
13
Screening
and
Testing
Program
(
EDSTP).
The
EDSTAC
recommends
that
EPA
update
this
14
categorization
scheme
as
part
of
their
validation
and
standardization
program.
15
16
The
recommended
screens
and
tests
(
including
all
endpoints)
will
have
to
meet
all
the
criteria
of
17
relevance
and
reliability
for
use
in
regulatory
toxicity
screening
or
testing
for
Estrogen,
Androgen,
18
and
Thyroid
(
EAT)
in
order
to
be
considered
fully
validated
and
standardized
(
ICCVAM,
1996;
19
Zeiger,
1998).
As
screens
and
tests
become
fully
validated
and
standardized,
they
will
warrant
20
inclusion
in
the
EDSTP
according
to
their
specific
and
appropriate
use.
None
of
the
screens,
new
21
tests,
or
enhancements
to
existing
test
guidelines
included
in
Tier
1
Screening
(
T1S)
or
Tier
2
22
Testing
(
T2T)
completely
fulfill
these
criteria
to
date.
As
mentioned
throughout
Chapter
Five,
23
each
assay
and
test
under
consideration
in
T1S
or
T2T
needs
some
level
of
standardization,
24
validation,
methods
development,
or
further
research
before
being
accepted
as
a
regulatory
25
toxicity
screen
or
test.
The
level
of
standardization
and
validation
varies
according
to
a
variety
of
26
criteria
applied
to
each
of
the
assays,
including:
period
of
time
in
use,
existing
level
of
general
27
acceptance
in
the
endocrine
toxicology
field,
and
existing
understanding
of
relevancy
and
28
reliability.
29
30
The
STWG
placed
the
proposed
screens
and
tests
in
one
of
four
general
categories
with
regard
to
31
the
level
of
validation,
standardization,
or
methods
development
required.
A
fifth
category,
32
discussed
in
Chapter
Five,
also
identified
assays
requiring
further
research.
33
34
Category
I:
35
36
Screens
and
tests
which
have
been
fully
validated
and
standardized
are
placed
in
Category
I.
37
These
procedures
meet
all
the
criteria
of
relevance
and
reliability
for
use
in
regulatory
toxicity
38
screening
or
testing
for
estrogen,
androgen,
and
thyroid.
As
other
procedures
become
sufficiently
39
standardized
and
validated
to
warrant
inclusion
in
Category
I,
such
screens
and
tests
should
be
40
incorporated
into
the
EDSTP
according
to
their
specific
and
appropriate
use.
Only
the
following
41
tests
are
included
in
Category
I:
42
43
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
2
R
­
·
Two­
Generation
Mammalian
Reproductive
Toxicity
Study
(
1996
Public
Draft
1
Guidelines
and
1997
TSCA
Final
Guidelines)
2
·
One­
Generation
Test
3
4
Category
II:
5
6
Screens
and
tests
which
have
been
in
use
for
a
sufficient
period
of
time
and
which
have
gained
7
sufficient
general
acceptance
within
the
field
of
endocrine
toxicology
to
be
considered
de
facto
8
validated
(
reliable
and
relevant)
are
included
in
Category
II.
These
assays
measure
relevant
9
endpoints,
are
responsive
to
endocrine
active
compounds
with
a
high
degree
of
specificity,
are
10
sufficiently
sensitive
to
identify
all
known
active
agents,
and
can
reasonably
be
expected
to
give
11
reproducible
results
from
laboratory
to
laboratory,
assuming
a
general
level
of
competence
and
12
expertise.
Nonetheless,
variations
in
protocols
for
these
screens
and
tests
can
produce
disparate
13
results.
Therefore,
standardization
of
the
protocol
to
be
recommended
for
these
screens
and
tests
14
should
be
accomplished
by
EPA
before
these
assays
are
implemented
as
screening
requirements
15
for
endocrine
activity
or
disruption.
The
following
screens
and
tests
are
included
in
Category
II:
16
17
·
ER
Binding
Assay
18
·
AR
Binding
Assay
19
·
Rodent
3­
Day
Uterotrophic
Assay
(
Subcutaneous)
20
·
Rodent
5­
7
Day
Hershberger
Assay
21
·
Rodent
3­
Day
Uterotrophic
Assay
(
Intraperitoneal);
22
·
Avian
Reproduction
Test
(
with
Bobwhite
Quail
and
Mallard)
(
as
currently
performed)
23
·
Fish
Life
Cycle
Test
(
Fathead
Minnow)
Test
(
as
currently
performed)
24
·
Mysid
Life
Cycle
Test
(
Americamysis)
25
26
Category
III:
27
28
Screens
and
tests
which
have
sufficiently
broad
use
to
be
generally
considered
relevant
OR
29
reliable
to
either
screening
for
endocrine
activity
(
Tier
1)
or
to
testing
for
adverse
endocrine­
30
mediated
effects
(
Tier
2)
are
included
in
Category
III.
These
assays
cannot,
however,
be
31
generally
considered
to
be
both
relevant
and
reliable.
The
level
of
performance
that
can
be
32
expected
of
these
assays
with
respect
to
identifying
endocrine
active
agents
or
endocrine
33
disruptive
effects
of
chemicals
must
be
clarified.
Therefore,
these
assays
should
undergo
further
34
but
focused
validation
and
standardization
to
define
their
relevance
and
reliability
for
the
task
of
35
endocrine
disruptor
screening
or
testing.
The
validation
required
may
be
focused
to
answer
36
specific
questions
about
relevance
and
to
provide
information
regarding
specificity
and
sensitivity.
37
The
following
screens
and
tests
are
included
in
Category
III:
38
39
·
ER
Transcriptional
Activation
Assay
40
·
AR
Transcriptional
Activation
Assay
41
·
Steroidogenesis
Assay
with
Minced
Testis
42
·
Rodent
20­
Day
Pubertal
Female
Assay
With
Thyroid
43
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
3
R
­
·
Placental
Aromatase
Assay
1
·
Rodent
14­
day
Intact
Adult
Male
Assay
with
Thyroid
2
·
Rodent
20­
day
Thyroid/
Pubertal
Male
Assay
3
·
Alternative
Mammalian
Reproduction
Test
4
·
Avian
Reproduction
Test
­
when
performed
in
multiple
generations
5
·
Turtle
Egg
Assay
6
7
Category
IV:
8
9
Screens
and
tests
which
may
have
relevance
to
the
task
of
either
screening
for
endocrine
activity
10
or
testing
for
endocrine
disruptive
effects,
but
whose
performance
in
identifying
endocrine
active
11
agents
or
endocrine
disruptive
effects
has
seen
only
limiting
testing
are
included
in
Category
IV.
12
Questions
as
to
whether
these
assays
measure
endpoints
that
are
relevant
to
endocrine
activity
or
13
endocrine
disruptive
effects,
whether
these
assays
respond
with
specificity
and
sensitivity
to
14
known
endocrine
active
agents,
or
whether
they
identify
endocrine
disruptive
effects
cannot
be
15
addressed
with
information
currently
available.
In
addition,
questions
regarding
the
specific
16
protocols
and
conditions
under
which
the
assays
should
be
conducted
must
be
answered
before
17
relevance
and
reliability
can
be
assessed.
Nonetheless,
these
assays
would
have
sufficient
utility,
if
18
further
developed
and
validated,
to
enhance
or
augment
the
screening
and
testing
program.
The
19
following
screens
and
tests
are
included
in
Category
IV:
20
21
·
Frog
Metamorphosis
Assay
22
·
Fish
Gonadal
Recrudescence
Assay
23
·
14­
Day
(
PND
9­
22)
Developmental/
Thyroid
Assay
24
25
26
EDSTAC
Final
Report
Chapter
Five
Appendices
August
1998
S
­
1
Appendix
S
1
2
Survey
of
Cost
Estimates
for
the
EDSTAC's
Proposed
Endocrine
Disruptor
3
Screening
and
Testing
Assays
4
5
6
This
Appendix
contains
a
detailed
summary
of
a
Cost
Estimate
Survey
for
Endocrine
Disruptor
7
Screening
and
Testing
Batteries
conducted
in
May
1998
by
Applied
Pharmacology
and
8
Toxicology,
Inc.
(
APT).
The
purpose
of
the
survey
was
to
project
costs
for
conducting
the
9
screening
and
testing
batteries
recommended
by
the
EDSTAC.
10
11
Based
upon
Chapter
Five
 
Screening
and
Testing
of
the
April
3,
1998
EDSTAC
Draft
Report,
12
and
Appendices
J,
K,
L,
and
what
was
P
(
and
is
now
Q),
APT
developed
detailed
protocols
for
13
thirteen
screening
assays
and
seven
tests
according
to
a
standardized
format.
These
protocols
14
were
sent
to
18
toxicology
laboratories
competent
to
conduct
the
types
of
assays
and
tests
15
recommended
by
the
EDSTAC.
Fourteen
laboratories
responded
by
providing
cost
estimates
for
16
one
or
more
of
the
assays
and
tests.
The
results
of
this
cost
estimate
survey
were
summarized
in
17
Tables
5.6
and
5.7
in
Chapter
Five
of
the
final
EDSTAC
report.
A
complete
copy
of
the
cost
18
estimate
survey,
including
protocols
for
the
thirteen
screening
assays
and
seven
tests,
is
included
19
in
the
EDSTAC
docket.
20
21
All
materials
related
to
APT's
cost
estimate
survey
except
the
twenty
detailed
protocols
are
22
contained
in
this
Appendix,
including:
an
example
of
the
cover
letter
that
accompanied
the
23
survey;
the
cost
estimate
form;
and
the
final
report.
The
final
report
describes
the
survey
design,
24
summarizes
the
results
in
Tables
1
­
3,
and
provides
the
individual
cost
estimates
in
its
own
25
Appendices
A
and
B.
26
27
28
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
1
T
­
Appendix
T
1
2
Summary
of
EPA's
September
1997
Outreach
Questionnaire
3
4
Following
the
establishment
of
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
5
Committee
(
EDSTAC),
the
Communications
and
Outreach
Work
Group
(
COWG)
developed
a
6
mailing
list
and
an
Outreach
Questionnaire
which
was
disseminated
to
over
1,500
addressees.
7
This
Questionnaire
was
created
in
an
effort
to
obtain
information
regarding
the
public's
interest
in
8
the
EDSTAC
and
its
activities
during
the
Committee's
tenure,
as
well
as
to
assist
in
future
9
outreach
efforts
conducted
by
the
U.
S.
Environmental
Protection
Agency
(
EPA/
the
Agency).
10
The
information
received
in
response
to
this
questionnaire
will
assist
the
Agency
in
determining
11
the
most
effective
way(
s)
to
communicate
with
those
individuals
and
organizations
interested
in
12
the
Endocrine
Disruptor
Screening
and
Testing
Program
that
EPA
will
implement
following
the
13
EDSTAC
process.
14
15
EPA
received
a
total
of
142
responses
to
the
Questionnaire
from
the
targeted
recipients
(
a
16
response
equal
to
9.4%).
A
break­
down
of
respondents
by
their
organizations
(
using
categories
17
as
described
in
the
Questionnaire
with
additions
in
some
cases)
is
as
follows:
18
21
with
Advocacy
Organizations
12
with
Professional
Organizations
2
with
Environmental
Justice
Organizations
18
with
Educational
Organizations
15
with
Government
Organizations
6
with
For­
Profit
Organizations
10
with
Non­
Profit
Organizations
11
with
Chemical/
Pesticide
Producer
Organizations
3
with
Chemical/
Pesticide
User
Organizations
4
with
Small
Businesses
4
with
News
Organizations
4
with
Law
Firms
5
with
Trade
Associations
3
with
Consulting
Organizations
2
Medical
Doctors
1
Student
1
Zoological
Organization
(
Zoo)
1
"
General
Public"
Individual
20
with
Multiple
Designations
of:
Advocacy/
Environmental
Justice/
Educational/
Non­
Profit
Organizations
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
2
T
­
Summary
of
Responses:

1.
All
respondents
were
interested
in
receiving
future
information
regarding
the
EDSTAC
and
its
activities.

2.
Respondents
were
asked
to
select
their
top
three
choices
indicating
what
types
of
materials
they
were
interested
in
receiving
regarding
EDSTAC
activities,
and
the
best
vehicles
for
receiving
those
materials.
[
Note:
Some
respondents
made
three
choices;
some
made
more
than
three;
and
some
made
less
than
three
choices.]

The
types
of
materials
most
useful
for
respondents
to
receive
regarding
the
EDSTAC
were
as
follows:

·
118
for
updates
on
the
progress
of
the
EDSTAC
as
a
whole
·
72
for
updates
on
the
progress
of
the
EDSTAC
work
groups
·
103
for
fact
sheets
on
endocrine
disruptors
·
42
for
resources
to
seek
out
additional
information
on
endocrine
disruptors
·
78
for
summary
memos
from
the
public
EDSTAC
meetings
·
Other:
1
wanted
scientific
summaries
1
wanted
minutes
of
meetings
with
copies
of
documents
that
were
distributed
1
wanted
on­
going
and
planned
wildlife
and
human
studies
According
to
the
responses
received,
the
types
of
vehicles
that
would
be
best
for
respondents
to
receive
information
regarding
EDSTAC's
activities
were
as
follows:

·
52
wanted
to
receive
information
via
the
EDSTAC
Web
Site
·
3
wanted
to
receive
information
via
the
EPA
Public
Docket
·
91
wanted
to
receive
information
via
e­
mail
transmissions
·
3
wanted
to
receive
information
via
an
electronic
bulletin
board
·
3
wanted
to
receive
information
via
speakers
·
58
wanted
to
receive
information
via
facsimile
(
fax)
transmissions
·
115
wanted
to
receive
information
via
the
U.
S.
mail
·
10
wanted
to
receive
information
via
attendance
at
public
plenary
meetings
·
9
wanted
to
receive
information
via
existing
EPA
communication
sources
·
21
wanted
to
receive
information
via
public
conferences/
workshops
·
Other:
­
1
wanted
to
receive
information
at
local
meetings
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
Appendix
U
Compilation
of
Public
Comment
to
the
EDSTAC
Table
of
Contents
I.
October
31
­
November
1,
1996
 
Washington,
D.
C.
.
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1
II.
December
12­
13,
1996
 
San
Francisco,
California
.
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.
4
Response
to
Public
Comment
.
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.
6
III.
February
5­
6,
1997
 
Houston,
Texas
.
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.
7
Response
to
Public
Comment
.
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.
10
IV.
April
29
­
May
1,
1997
 
Baltimore,
Maryland
.
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.
.
11
Response
to
Public
Comment
.
.
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.
.
18
V.
July
15­
16,
1997
 
Chicago,
Illinois
.
.
.
.
.
.
.
.
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.
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.
.
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.
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.
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.
.
.
.
.
.
.
19
Response
to
Public
Comment
.
.
.
.
.
.
.
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.
.
.
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.
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.
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.
.
.
.
26
VI.
October
7­
8,
1997
 
New
York,
New
York
.
.
.
.
.
.
.
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.
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.
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.
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.
.
.
.
.
27
Response
to
Public
Comment
.
.
.
.
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.
.
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.
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.
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.
32
VII.
December
2­
3,
1997
 
Orlando,
Florida
.
.
.
.
.
.
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.
.
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.
.
.
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.
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.
.
.
.
.
.
.
.
.
.
.
34
Response
to
Public
Comment
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
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.
.
.
.
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
1
U
­
I.
October
31
­
November
1,
1996
 
Washington,
D.
C.
1
2
3
Two
opportunities
were
given
at
this
meeting
for
the
members
of
the
public
observing
the
4
proceedings
to
comment
on
all
aspects
of
the
formation
of
the
EDSTAC.
5
6
October
31,
1996
7
8
Lynn
Bradley,
Director,
Environmental
Health,
Association
of
State
and
Territorial
Public
Health
9
Laboratory
Directors,
indicated
that
her
organization
has
members
interested
in
human
monitoring
10
and
the
related
issues
of
how
you
deal
with
exposure.
She
recommended
that
EPA
start
thinking
11
about
addressing
the
human
exposure
concerns
concurrently
with
the
efforts
to
design
a
screening
12
and
testing
program.
13
14
Chris
Wilkinson
of
the
Technology
Sciences
Group,
Inc.
related
that,
as
a
scientist,
he
is
appalled
15
with
this
process
because
law
is
mandating
science
and
therefore
science
is
under
the
gun.
He
also
16
expressed
concern
about
the
perceived
confusion
surrounding
the
scope
of
the
Committee.
His
17
advice
was
that
since
the
law
says
EPA
has
to
do
one
thing,
develop
a
screen
for
estrogenicity,
the
18
Committee
should
deal
with
the
law
and
then
do
the
other
tasks
as
time
permits.
The
Committee's
19
work
could
be
utilized
as
a
model
for
future
efforts.
He
also
suggested
a
set
of
steps
the
20
Committee
should
take:
first,
identify
substances
that
have
to
go
into
a
screen;
second,
complete
21
the
screens
(
a
simple
process
that
will
raise
flags);
third,
complete
actual
testing
(
dose­
response
22
issues);
and
fourth,
bring
in
exposure
because
EPA
will
be
doing
a
risk
assessment.
23
24
Elizabeth
Onon,
who
was
not
present
at
the
meeting,
asked
that
her
comments
be
read
into
the
25
record.
Gary
Timm
of
EPA
read
the
letter.
She
asked
the
Committee
to
address
the
potential
link
26
between
endocrine
disrupters
and
the
growing
body
of
scientific
knowledge
regarding
porphyria
as
27
relate
to
chemical
disabilities.
She
further
indicated
that
the
nominees
did
not
represent
the
victims
28
of
endocrine
disrupters.
29
30
Stuart
Cagen,
Shell
Chemical
Company,
indicated
his
support
for
the
concept
of
what
the
31
Committee
should
produce.
He
suggested
that
the
process
has
to
include
validation,
32
interpretation,
and
an
understanding
of
what
EPA
does
with
the
results.
33
34
Steve
Schraeder,
National
Institute
for
Occupational
Safety
and
Health
(
NIOSH),
expressed
35
concern
about
the
Committee
only
considering
estrogens,
especially
as
we
now
recognize
that
36
many
of
the
effects
we
originally
thought
to
be
estrogens
are
actually
anti­
estrogens
and
anti­
37
androgens.
He
suggested
that
the
time
is
right
for
the
Committee
to
go
broader.
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
2
U
­
Mary
Dadden,
President,
Long
Island
Breast
Cancer
Study,
relayed
her
disappointment
in
how
the
1
information
regarding
the
meeting
was
communicated.
She
requested
that,
in
the
future,
notice
be
2
given
at
least
four
to
six
weeks
in
advance.
In
addition,
she
indicated
her
desire
to
see
the
3
discussions
recognize
the
truly
dangerous
nature
of
chemicals
and
pesticides
in
the
environment
4
and
their
effects
on
humans.
She
suggested
that
the
Committee
consider
opening
at
least
two
seats
5
to
consumer
advocates
who
are
not
involved
from
a
scientific
perspective
but
can
bring
the
6
concerns
of
the
general
public.
7
8
In
response,
the
facilitator/
convenor
indicated
that
efforts
to
provide
more
notice
will
be
made.
He
9
also
indicated
that
the
National
Breast
Cancer
Coalition
had
offered
to
assist
in
coordinating
10
efforts
to
involve
disease­
specific
consumer
interests
in
addition
to
breast
cancer
groups.
One
11
nominee
added
that
the
addition
of
consumer
advocates
to
the
Navy
Breast
Cancer
Study
had
been
12
very
helpful
and
that
this
Committee
should
consider
their
inclusion.
The
idea
of
a
13
communications
work
group,
where
concerns
around
the
possibility
of
the
public
misunderstanding
14
the
results
of
screens
and
tests
could
be
addressed,
was
raised.
Another
nominee
added
that
the
15
Committee
seemed
to
be
missing
a
representative
of
the
people
affected
most
by
these
chemicals
16
and
pesticides.
The
idea
of
moving
the
EDSTAC
meetings
around
the
country
was
raised
as
one
17
way
to
include
a
wide
range
of
perspectives
and
interests
including
consumer
advocates.
In
18
addition,
using
the
Internet
to
disseminate
information
was
proposed,
though
its
ability
to
19
disseminate
information
to
a
wide
range
of
individuals
was
also
recognized.
20
21
Alan
Robeson
raised
the
issue
of
testing
mixtures.
He
indicated
that
a
lot
of
people
are
concerned
22
about
pesticide
runoff
from
agricultural
lands
and
the
combinations
of
pesticides.
He
23
recommended
that,
if
the
Committee
moves
its
meetings
around
the
country,
they
should
go
to
a
24
midwest
city
such
as
Chicago,
Kansas
City,
or
Des
Moines
in
the
spring.
25
26
John
Berlau
of
Consumer
Alert
asked
that
the
Committee
consider
consumer's
interest,
but
added
27
that
there
are
different
definitions
of
what
constitute
"
consumer
interests."
He
asked
that
the
28
Committee
look
at
the
benefits
of
these
substances
as
well
as
the
negatives
and
said
that
consumers
29
do
not
benefit
from
regulations
developed
without
good
science.
30
31
Miranda
Henning,
ChemRisk,
suggested
that,
given
the
exposure
issues
as
well
as
the
fact
that
the
32
information
might
be
used
in
risk
assessments,
the
Committee
should
involve
a
risk
assessor.
33
34
Eric
Juzenas,
Health
Policy
Analyst,
American
Public
Health
Association,
suggested
that
the
35
Committee's
environmental
justice
representation
should
be
enhanced
by
adding
some
additional
36
participants.
37
38
Rick
Hind,
Greenpeace,
stressed
that
the
Committee
should
keep
in
mind
the
issue
of
public
39
disclosure
and
participants
should
volunteer
information
regarding
their
financial
backing
and/
or
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
3
U
­
interests
as
they
relate
to
endocrine
disrupter
screening
and
testing
if
people
want
the
process
to
be
1
truly
transparent.
Relating
to
scope
he
said
that
the
Committee
should
focus
on
looking
at
this
2
through
the
lens
of
existing
FIFRA
and
TSCA
regulations.
In
addition,
he
felt
that
the
Committee
3
should
not
get
caught
looking
at
chemicals
one­
by­
one.
4
5
Charlie
Cray,
Greenpeace,
wanted
the
Committee
to
look
at
persistent
organic
pollutants.
He
also
6
added
that
the
Committee
should
incorporate,
from
an
environmental
justice
perspective,
some
of
7
the
folks
who
represent
the
people
who
suffer
as
a
result
of
the
migration
of
these
substances
to
8
the
colder
regions
of
the
world
(
e.
g.,
Alaska).
He
indicated
that
the
public
wants
a
list
of
products
9
they
should
not
buy
and
labeling
that
explains
what
is
in
a
product.
Other
countries
that
have
10
approached
these
issues,
(
e.
g.,
Denmark)
may
be
a
useful
source
of
information.
If
EPA
is
not
11
going
to
develop
these
lists,
he
asked,
who
will?
12
13
Nelsa
Ford
asked
whether
all
future
meetings
were
going
to
be
public
now?
EPA
indicated
that
all
14
EDSTAC
meetings
will
be
public,
however,
whether
all
work
group
meetings
will
be
public
15
remains
to
be
determined.
16
17
November
1,
1996
18
19
Maurice
Zeeman,
OPPTS,
EPA,
raised
the
issue
of
the
Committee
balancing
ecological
and
human
20
health
issues
because
so
much
uncertainty
exists
regarding
human
health
effects
whereas
we
are
21
more
certain
about
ecological
effects.
22
23
John
McCarthy,
American
Crop
Protection
Association,
commended
the
Committee
nominees,
24
EPA,
and
Keystone
on
their
efforts.
He
raised
three
points:
first,
focus
equally
on
human
and
25
ecological
effects;
second,
look
at
all
possible
hormonal
effect
endpoints
raised
during
the
26
deliberations
in
parallel,
while
recognizing
the
statutory
importance
of
specific
ones;
and
third,
the
27
Committee
should
get
on
with
the
task
at
hand
without
getting
too
bureaucratic
and
use
the
28
Science
Integration
and
Coordination
work
group
in
the
future
if
necessary.
29
30
Layla
Patarsi,
Center
for
Food
Quality,
Food
and
Drug
Administration,
explained
that
food
31
additives
comprise
a
set
of
chemicals
not
currently
regulated
under
TSCA
or
FIFRA,
and
asked
32
that
the
Committee
consider
them
in
their
deliberations.
33
34
Robert
Fisher,
National
Council
of
the
Paper
Industry
for
Air
and
Stream
Improvement,
talked
35
about
the
structure
of
the
Committee
and
recommended
that
the
group
consider
looking
at
how
36
the
EPA
structured
a
process
dealing
with
the
Gulf
of
Mexico
where
many
similar
issues
were
37
addressed.
They
used
a
tiered
process
and
included
issue
committees,
a
technical
steering
38
committee,
and
a
management
committee/
policy
review
board.
He
also
added
that
the
meetings
of
39
the
working
groups
should
be
open
to
the
public
for
observation
for
two
reasons,
first,
the
private
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
4
U
­
sector
is
often
involved
in
research
relevant
to
the
issues
being
discussed,
and
second,
the
private
1
sector
is
often
involved
in
the
decision
making
process
and
needs
to
know
about
the
findings
2
coming
out
of
these
efforts.
3
4
Bill
Kelley,
the
Institute
for
Regulatory
Policy,
recommended
the
nominees
develop
a
list
of
what
5
can
be
done
between
the
organizational
meeting
and
the
December
meeting
before
leaving.
He
6
also
raised
serious
misgivings
regarding
the
name
endocrine
"
disrupters"
and
suggested
changing
it
7
to
"
disruption"
or
"
effects"
as
the
Committee
is
talking
about
a
hazard
screening
process
rather
8
than
risk
assessment.
9
10
Elizabeth
Reeves,
requested
the
Committee
consider
using
cell
culture
testing
rather
than
animal
11
tests.
She
added
that
the
Committee
should
remember
the
ultimate
stakeholder
is
the
American
12
citizen
and
the
ecological
issues
should
not
be
overlooked.
13
14
Mary
Ann
Dodden,
expressed
support
for
the
evolution
toward
a
broader
definition
of
endocrine
15
disrupters
than
just
hormone
disruption.
If
the
definition
says
"
only
affect
the
hormone
system,"
16
she
felt,
the
Committee
will
not
examine
the
immune,
reproductive,
growth
and
neuro
systems.
17
She
also
stated
that
the
Committee
needs
to
recognize
that
other
issues
may
be
added
to
their
18
discussions
as
their
understanding
of
science
evolves.
19
20
Elsa
Ford,
asked
the
Committee
to
commit
to
follow­
up
testing
of
past
use
and
new
use
chemicals
21
as
pertains
to
endocrine
disruption.
Coordinated
documentation,
she
added,
is
important
in
these
22
types
of
efforts.
Finally,
she
said
that
the
public
needs
to
know
about
the
cumulative
effects
of
23
chemicals
and
pesticides
in
the
real
world
and
asked
the
Committee
not
to
focus
only
on
single
24
ones.
25
26
Patricia
Fail,
Research
Triangle
Institute,
told
the
Committee
nominees
she
had
been
working
27
mainly
on
FIFRA
issues
for
the
past
10­
15
years.
After
listening
to
the
need
for
various
screens
28
and
tests
during
the
past
two
days,
she
indicated
that
the
Committee
needs
to
take
already
existing
29
data
(
from
FIFRA
companies)
and
validate
the
tests
that
exist
and
see
if
the
chemicals
that
test
30
positive
are
actually
endocrine
disrupters.
31
32
John
Hines,
an
independent
consultant,
told
the
nominees
that
they
need
to
address
what
33
product(
s)
they
expect
as
a
result
of
this
effort.
He
also
indicated
that
the
discussions
on
34
prioritization
of
what
issues
to
address
were
good.
Regarding
membership,
he
added,
the
35
Committee
nominations
seem
to
be
carefully
structured
and
will
result
in
a
fairly
large
Committee,
36
so
do
not
add
too
many
more
people.
37
38
II.
December
12­
13,
1996
 
San
Francisco,
California
39
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
5
U
­
1
2
Lewell
D.
Brenneman,
MD,
Ph.
D.,
expressed
his
point
of
view
that
few
toxins
in
the
environment
3
are
tested
for
cancer
and
hormone
disruption.
He
outlined
two
approaches
for
dealing
with
4
manufactured
chemicals
such
as
carcinogens­­
acceptable
risk
and
public
health,
and
expressed
5
concern
about
the
fact
that
it
is
usually
the
consumer,
rather
than
the
producer,
that
has
the
burden
6
of
proving
chemical
toxicity.
Dr.
Brenneman
urged
the
EDSTAC
to
develop
long­
term
and
7
generational
testing
approaches
to
endocrine
disruption.
8
9
Joan
Reiss,
Project
Coordinator
of
the
Bay
Area
Breast
Cancer
Study,
urged
the
Committee
to
10
develop
outreach
activities
beyond
their
present
scope,
using
several
and
varied
means
of
11
communication.
Ms.
Reiss
encouraged
the
Committee
to
be
bold,
swift,
and
innovative
with
its
12
activities.
She
also
stressed
that
small
doses
of
chemicals
can
have
an
additive
effect,
and
she
13
urged
the
Committee
to
consider
this
when
looking
at
screening
and
testing.
Ms.
Reiss
went
on
to
14
request
that
the
Committee
identify
key
geographical
areas
and
criteria
to
start
studying
endocrine
15
disruption;
that
new
chemicals
should
be
kept
off
the
market
until
proven
not
harmful
to
humans
16
and
animals;
that
acceptable
dose
standards
be
shifted
from
levels
for
adult
males
to
those
17
acceptable
for
a
newborn
infant;
and
that
synergistic
effects
of
chemicals
be
analyzed.
18
19
Cindy
Dyer
of
the
U.
S.
Department
of
Agriculture,
Food
Safety
and
Inspection
Service,
20
commented
that
brominated
flame
retardants,
which
bioaccumulate,
and
are
widely
used
in
21
shipping
cartons,
are
a
prevalent
class
of
chemical
which
needs
to
be
scrutinized.
Dr.
Dyer
stated
22
her
view
that
there
is
currently
too
much
concentration
on
dioxin
and
PCBs
in
the
scientific
23
community,
and
that
brominated
compounds,
as
opposed
to
chlorinated
compounds,
deserve
24
closer
investigation.
Dr.
Dyer
also
urged
the
Committee
to
utilize
exposure
hazard
database
25
information.
26
27
Judy
Brady,
a
member
of
the
Cancer
Prevention
Coalition
and
the
Women's
Cancer
Resource
28
Center,
urged
the
Committee
to
look
at
the
safety
of
breast
milk
as
it
relates
to
endocrine
29
disruption.
30
31
Keith
Bowers,
of
the
IFS
Kaiser
Consulting
Group,
Global
Environmental
Issues
Unit
for
Eastern
32
Europe
and
the
former
Soviet
Union,
made
the
suggestion
that
the
EDSTAC
consider
identifying
33
and
reviewing
data
developed
in
other
countries
that
might
help
or
provide
insight
and
fill
data
34
gaps
in
its
own
and
other
domestic
efforts
related
to
endocrine
disruption.
Mr.
Bowers
stated
that
35
issues
such
as
endocrine
disruption
are
transborder
issues,
and
that
societies
all
across
the
globe
36
are
concerned
and
working
on
the
same
matters.
37
38
Sandra
Marquart,
of
the
West
Coast
office
of
Mothers
and
Others
for
a
Livable
Planet,
asked
the
39
Committee
to
consider
the
following
priorities
for
testing:
pesticides,
plastics,
solvents,
and
other
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
6
U
­
products
of
major
daily
use.
Ms.
Marquart
stressed
her
belief
that
this
is
an
international
issue,
1
heavily
influenced
by
trade
patterns.
She
also
encouraged
the
Committee
to
adjust
its
risk
2
considerations
from
levels
acceptable
for
adult
males
to
finding
the
levels
for
the
most
susceptible
3
organisms
(
e.
g.,
infants),
and
to
address
issues
of
synergism
and
threshold
levels.
Ms.
Marquart
4
asked
the
EDSTAC
to
develop
the
most
practical
tests
to
find
the
greatest
number
of
chemicals
5
given
its
budgetary
constraints.
She
urged
EPA
not
to
wait
for
final
endpoints
to
be
determined,
6
but
rather
to
take
action
and
to
extend
public
outreach
to
consumers
at
the
first
sign
of
possible
7
problems
with
chemicals.
Ms.
Marquart
further
urged
the
Committee
to
hold
future
meetings
as
8
close
to
mass
transit
systems
as
possible.
9
10
Leslie
Meister,
of
the
Silicon
Valley
Toxics
Coalition
in
San
Jose,
stressed
the
synergistic
effects
of
11
chemicals
in
Silicon
Valley
laborers'
and
consumers'
bloodstreams.
Ms.
Meister
urged
the
12
Committee
to
use
the
"
precautionary
principle"
in
its
decision
making
processes.
She
stated
that
13
issues
of
importance
in
the
screening
process
include
the
determination
of
exposure
thresholds
for
14
different
populations
(
e.
g.,
workers
or
children
or
in
utero
infants),
and
the
identification
of
15
endpoints.
16
17
Davis
Baltz,
of
Commonweal,
asked
the
EDSTAC
to
look
at
the
synergistic
effects
of
chemicals.
18
He
suggested
that,
in
order
to
streamline
the
EDSTAC's
process,
the
Committee
consider
classes
19
of
chemicals
as
a
whole
when
it
is
determined
that
several
individual
chemical
members
of
that
20
class
pose
problems.
Mr.
Baltz
also
stated
that
the
validation
of
screens
should
provide
21
information
that
is
both
useful
and
predictive,
allowing
EPA
to
enact
policies
and
draft
legislation
22
that
reduce
exposure
to
hazardous
chemicals.
He,
too,
encouraged
the
Committee
to
adopt
the
23
use
of
a
"
precautionary
principle."
24
25
Robert
Gould,
Associate
Pathologist
at
the
Santa
Theresa
Community
Hospital
in
San
Jose
and
a
26
member
of
Physicians
for
Social
Responsibility,
urged
the
Committee
to
act
promptly
and
to
create
27
a
timetable
for
developing
appropriate
methodologies.
Dr.
Gould
stated
that
screening
and
testing
28
must
be
a
part
of
an
ongoing
process
of
communication,
and
that
the
EDSTAC
process
should
be
29
completely
open
and
transparent,
including
activities
of
the
work
groups.
He
asked
the
Committee
30
to
address
estrogenic
compounds;
thyroid,
androgenic,
anti­
estrogenic,
and
anti­
androgenic
31
effects;
and
additive
and
synergistic
effects.
Dr.
Gould
suggested
EPA
regulate
whole
classes
of
32
chemicals,
and
to
use
a
"
precautionary
principle"
as
its
guiding
mechanism.
He
recommended
that
33
measures
of
high
sensitivity
and
low
specificity
be
used;
that
both
wildlife
and
human
health
34
effects
be
considered;
that
background
levels
in
humans
should
be
taken
into
account;
and
that
the
35
EDSTAC
study
entire
populations
and
communities.
36
37
Ritchie
Fraley,
a
scientist
at
SRI
International,
stated
that
while
receptivity
measurement
is
cost
38
effective
and
efficient,
adherence
to
receptor­
mediated
tests
could
miss
particular
mechanistic
39
indications.
Dr.
Fraley
indicated
that
technologies
such
as
accelerator
mass
spectrometry
might
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
7
U
­
prove
helpful
in
the
Committee's
work.
She
also
asked
how
testing
would
be
funded,
and
who
1
would
conduct
the
tests.
2
3
Jane
Williams,
of
California
Communities
Against
Toxics,
stated
that
California
is
an
industrialized
4
state,
subject
to
a
wide
range
of
impacts
including
dioxin
contamination
and
effects
from
military
5
facilities.
She
requested
that
the
Committee
address
human
health
and
ecological
effects,
as
well
6
as
synergistic
impacts.
Ms.
William
expressed
concern
that
a
lack
of
connection
to
the
policy
7
making
arena
would
render
the
process
ineffective.
She
asked
the
Committee
to
consider
the
task
8
of
implementation
in
the
course
of
developing
its
recommendations
and
develop
recommendations
9
in
such
a
way
that
all
producers
and
communities
could
understand
and
use
them.
10
11
Please
refer
to
Attachment
F
for
textual
statements
provided
by
Dr.
Brenneman,
Ms.
Reiss,
Dr.
12
Dyer,
Mr.
Baltz,
and
Dr.
Gould.
13
14
Response
to
Public
Comment
15
16
The
EDSTAC
chair
thanked
the
public
for
its
comments
and
perspectives,
adding
that
the
degree
17
of
the
quality
of
the
comments
at
this
meeting
were
exceptionally
intelligent
and
helpful.
Dr.
18
Goldman
explained
that
there
would
be
four
more
meetings
during
this
fiscal
year
in
different
parts
19
of
the
country,
and
that
EPA
hopes
to
improve
its
outreach
efforts
and
will
coordinate
a
20
communications
and
outreach
work
group
to
assist
in
this
effort.
21
22
Dr.
Goldman
stated
that
the
endocrine
disruption
screening
and
testing
process
came
about
as
part
23
of
legislation
on
drinking
water
standards
and
pesticides.
She
emphasized
that
the
matter
of
real
24
importance
in
the
process
is
to
do
the
necessary
work
using
the
best
available
science,
and
to
do
so
25
in
a
framework
that
will
help
make
policy
decisions
to
help
public
health
and
the
environment.
Dr.
26
Goldman
clarified
that
the
language
in
the
standard
in
the
new
pesticide
legislation
is
"
a
reasonable
27
certainty
of
no
harm."
She
stated
that
the
EDSTAC
will
take
into
account
a
multiplicity
of
28
exposures,
and
that
other
processes
are
also
under
way
to
advise
and
inform
the
agency
in
this
29
matter.
30
31
Regarding
the
process
of
the
advisory
Committee,
Dr.
Goldman
stated
that
all
decisions
made
by
32
the
Committee
will
be
made
in
the
full
Committee
in
open
session,
and
that
much
will
be
brought
33
to
the
Committee
by
work
groups
and
individual
scientists.
34
35
Dr.
Goldman
recognized
that
Toxics
Release
Inventory
(
TRI)
matters
are
not
of
peripheral
36
concern
for
her.
She
stated
her
belief
in
the
need
to
link
domestic
efforts
with
international
37
activities,
and
highlighted
the
fact
that
the
Office
of
Economic
Cooperation
and
Development
38
(
OECD),
which
has
been
working
on
issues
related
to
the
testing
of
chemicals,
recently
decided
to
39
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
8
U
­
address
endocrine
disruption
issues.
She
also
offered
other
examples
of
international
efforts
1
related
to
chemical
pollutants,
endocrine
disruption,
and
persistent
organic
pollutants,
and
that
the
2
United
States
is
presently
working
with
Europe
and
Canada
on
long­
range
transport,
global
policy
3
on
populations,
and
the
planning
of
a
meeting
at
the
United
Nations
Environmental
Programme
4
(
UNEP)
in
January
to
discuss
coordination
of
research.
5
6
III.
February
5­
6,
1997
 
Houston,
Texas
7
8
9
Due
to
changes
in
the
schedule,
some
members
of
the
public
were
able
to
provide
comments
prior
10
to
Dr.
Goldman's
departure
during
the
afternoon
of
the
first
day
of
the
Houston
plenary
meeting.
11
A
period
of
two
hours
was
also
devoted
to
public
comments
later
in
the
evening
of
the
first
day,
12
during
which
nearly
the
entire
Committee
was
present.
A
total
of
nineteen
members
of
the
public
13
offered
comments,
twelve
of
whom
also
submitted
written
comments
which
can
be
found
in
the
14
EPA
Docket.
This
meeting
summary
attempts
to
capture
the
essence
of
the
comments
made
by
15
the
public
to
the
EDSTAC
during
both
of
those
sessions.
In
addition,
some
members
of
the
public
16
who
were
not
present
at
the
meeting
sent
written
comments
to
The
Keystone
Center.
While
these
17
are
not
summarized
below,
they
are
also
available
through
the
Docket.
18
19
Sue
Pope,
a
member
of
Downwinders
At
Risk
and
an
advisory
board
member
of
the
American
20
Lung
Association
of
Texas,
expressed
great
concern
over
human
health
and
ecological
problems
in
21
her
community
seemingly
attributable
to
endocrine
disruption.
In
particular,
Ms.
Pope
chronicled
22
health
problems
of
her
family
and
livestock
alleged
to
stem
from
hazardous
waste
incineration
at
a
23
cement
kiln
in
her
area.
Ms.
Pope
entered
into
the
Docket
a
video
which
included
a
television
24
show
about
hazardous
waste­
related
problems
in
her
town,
Winona,
Texas.
This
segment
is
25
entitled
"
Winona
Residents
Demand
Justice
for
their
Children"
and
is
available
through
the
EPA
26
Docket.
27
28
LaNell
Anderson,
a
citizen
from
Channelview,
Texas,
detailed
health
problems
in
her
community
29
and
family
as
a
result
of
the
effects
of
dioxin
releases
along
the
Houston
Ship
Channel.
Ms.
30
Anderson
asked
that
the
Committee
make
explicit
its
goals
and
expected
steps
for
the
31
implementation
of
its
recommendations,
and
that
State
authorities
be
involved
in
the
EDSTAC
as
32
appropriate.
She
asserted
that
the
EDSTAC
process
merited
close
public
scrutiny
and
monitoring,
33
as
its
effects
will
have
national
impacts.
Ms.
Anderson
encouraged
full,
impartial,
firm,
and
34
informed
participation
by
each
of
the
Committee
members
in
the
EDSTAC
process.
35
36
Brandt
Mannchen
commented
on
the
composition
of
the
Committee
and
encouraged
awareness
37
that
no
specific
interest
in
the
Committee
have
the
ability
to
unduly
influence
the
process.
He
said
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
9
U
­
that
he
felt
the
Committee
was
not
broad
enough
in
its
composition,
especially
with
respect
to
1
community­
based
group
representation,
specific
health
problem/
developmental
effects
group
2
representation,
and
minority
representation.
Mr.
Mannchen
encouraged
the
EDSTAC
to
develop
a
3
screening
and
testing
program
that
would
be
flexible
enough
to
accommodate
chemical
synergies,
4
additive
effects,
and
new
information
as
it
becomes
available.
5
6
Chavel
Lopez,
a
member
of
the
Southwest
Public
Workers
Union
in
San
Antonio,
Texas,
7
commented
on
the
health
problems
surrounding
areas
adjacent
to
Kelly
Air
Force
Base
due
to
fuel
8
and
toxic
chemical
storage
and
toxic
chemical
dumping.
Mr.
Lopez,
a
member
of
a
group
of
9
approximately
500
concerned
citizens
in
Bear
and
Medina
counties,
raised
concerns
regarding
10
contamination
of
the
Edwards
Aquifer.
He
also
urged
that
the
Committee
include
more
11
representation
of
people­
of­
color.
12
13
LaNell
Anderson
read
a
statement
written
by
Ms.
Phyllis
Glazer,
President
of
Mothers
Organized
14
to
Stop
Environmental
Sins
(
MOSES),
which
raised
concerns
about
the
effects
of
a
hazardous
15
waste
incineration
facility
near
the
town
of
Winona,
and
expressed
dissatisfaction
with
the
16
requirements
of
State
agencies
in
requiring
testing
for
dioxins.
In
addition,
Ms.
Glazer's
statement
17
offered
concern
about
the
effects
of
other
hazardous
waste
sites
shipping
waste
to
the
incinerator
18
for
disposal
and/
or
storage.
19
20
Jane
Elioseff,
of
the
Galveston­
Houston
Association
for
Smog
Prevention
(
GHASP),
an
21
organization
devoted
to
improving
the
air
quality
of
the
Galveston­
Houston
area,
noted
that
the
22
Galveston­
Houston
area
is
a
severe
ozone
non­
attainment
area
with
high
levels
of
nitrogen
oxide.
23
She
stated
that
this
represented
a
large
air
quality
problem
for
a
suburban
area.
Ms.
Elioseff
said
24
that
GHASP,
which
receives
funding
from
the
W.
Alton
Jones
Foundation
to
pursue
its
work,
has
25
determined
that
endocrine
disruption
is
connected
to
air
pollution
and
has
just
published
a
report
26
on
air
quality
in
Houston,
called
"
Danger
in
the
Air,"
which
she
made
available
for
the
Docket.
27
28
Yolanda
Johnson,
a
member
of
the
Southwest
Public
Workers
Union
in
San
Antonio,
Texas,
29
expressed
great
concern
with
the
generational
effects
of
environmental
contamination
and
30
endocrine
disruption.
Both
she
and
her
husband
have
experienced
health
problems,
as
have
most
31
members
of
their
family
raised
near
the
fuel
and
chemical
storage
and
disposal
sites
at
Kelly
Air
32
Force
Base.
Ms.
Johnson
thanked
the
Committee
for
its
work,
and
urged
the
EDSTAC
to
33
encourage
greater
communication
and
disclosure
with
respect
to
endocrine
disrupting
hazards.
34
35
Susan
Pitman,
Network
Coordinator
of
The
Chemical
Connection,
A
Public
Health
Network
of
36
Texans
Sensitive
to
Chemicals,
commented
that
the
Committee
ought
to
consider
chemically­
37
sensitive
persons
and
vulnerable
populations
in
screening
and
testing
for
endocrine
disruptors,
38
rather
than
using
healthy
adult
males
as
its
benchmark.
39
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
10
U
­
Dominga
Adams,
a
member
of
the
Southwest
Public
Workers
Union
in
San
Antonio,
Texas,
1
detailed
numerous
ailments
experienced
by
both
herself
and
her
husband,
as
well
as
by
their
2
children
and
grandchildren.
Ms.
Adams
expressed
doubt
that
her
family's
health
problems
were
3
genetic,
and
indicated
she
felt
they
were
caused
by
chemicals
from
Kelly
Air
Force
Base.
She
also
4
encouraged
thorough
governmental
communication
with
the
public
in
instances
where
health
risks
5
may
or
do
exist.
6
7
Suzanne
Martine
Rohrer,
of
The
Endometriosis
Association,
explained
that
she
was
diagnosed
8
with
endometriosis,
discussed
the
effects
of
the
disease,
and
outlined
the
consequent
challenges
in
9
her
life
due
to
the
disease.
She
urged
the
Committee
to
work
diligently
and
asked
that
dioxin
10
receive
particular
scrutiny,
since
high
level
exposure
to
dioxin
has
been
associated
with
increased
11
incidents
of
endometriosis.
12
13
Kim
Phillips,
former
environmental
chairman
for
the
Texas
PTA,
said
that
she
was
grateful
for
and
14
supportive
of
the
Committee's
work,
and
asked
that
children,
rather
than
adult
males,
be
used
as
15
guides
for
exposure
effects.
Ms.
Phillips
cautioned
the
EDSTAC
that
consensus
processes
can
16
prove
difficult,
but
that
they
are
worth
the
effort.
She
outlined
some
of
her
organization's
17
principal
concerns
with
respect
to
endocrine
disruptors,
and
urged
the
Committee
to
consider
the
18
synergistic
effects
of
chemicals
and
to
err
on
the
side
of
caution.
19
20
Raul
Villar,
a
retired
welder
from
Kelly
Air
Force
Base,
commented
on
his
family's
health
21
problems
since
living
near
Kelly
Air
Force
Base,
and
expressed
anger
that
the
contamination
of
the
22
area
around
his
home
had
not
been
made
publicly
known
prior
to
his
purchase
of
the
lot.
23
24
David
Casen,
of
Save
Whitewright
and
Tri­
Counties
(
SWAT),
said
he
was
concerned
about
25
dioxins
in
his
community,
and
deeply
cynical
about
the
consensus
process
of
the
EDSTAC.
Mr.
26
Casen
stated
that
he
feared
the
Committee's
decisions
would
be
made
based
on
political
27
considerations.
He
also
expressed
his
concern
caused
by
having
representatives
from
chemical
28
production
companies
on
the
Committee.
Mr.
Casen
asked
the
EDSTAC
to
use
mechanistic
29
findings
as
suggestive
but
not
limiting
factors
in
screening
and
testing,
and
that
the
Committee
30
adopt
the
"
precautionary
principle."
31
32
Virginia
Castillo,
a
citizen
residing
near
Kelly
Air
Force
Base,
asked
that
someone
look
into
the
33
causes
of
numerous
health
problems
in
the
community
adjacent
to
the
Base.
34
35
Davis
Baltz,
a
Research
Associate
with
Commonweal,
outlined
eleven
points.
These
points
36
covered:
using
caution
to
determine
when
an
effect
was
"
adverse;"
the
importance
of
the
37
EDSTAC's
Principles
Work
Group,
its
charge,
and
its
composition;
deferral
of
chemicals
in
the
38
screening
and
testing
process;
the
importance
of
recognizing
new
information
on
endocrine
39
disruption
and
chemical
effects;
the
need
for
"
a
new
paradigm"
with
respect
to
timing
and
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
11
U
­
exposure
information;
a
re­
evaluation
of
quantitative
risk
assessment;
the
encouragement
of
the
1
use
of
the
"
precautionary
principle;"
the
need
for
EPA
to
identify
and
allocate
material
resources
2
to
permit
full
participation
and
expression
of
all
points
of
view
within
work
groups;
his
desire
to
3
have
a
defined
schedule
of
meeting
dates
for
the
duration
of
the
EDSTAC
process;
the
need
for
4
encouraging
public
education
and
comment
on
matters
related
to
endocrine
disruption
from
other
5
countries;
the
importance
of
studying
sex
steroid­
and
thyroid­
related
effects;
and,
the
need
for
6
screening,
testing,
and
evaluating
endocrine
disruptors
to
become
an
ongoing
responsibility
of
7
EPA.
8
9
Neil
Carman,
Ph.
D.,
of
the
Sierra
Club
and
formerly
of
the
Texas
Air
Quality
Board,
presented
10
comments
from
people
near
a
site
in
Jacksonville,
Arkansas,
which
expressed
doubts
about
the
11
fairness
of
health
studies
by
the
EPA
and
the
Agency
for
Toxic
Substances
and
Disease
Registry.
12
Mr.
Carman
also
said
he
had
little
faith
in
the
regulatory
process.
He
urged
the
EDSTAC
to
look
13
at
synergistic
effects
of
chemicals
and
to
use
the
"
precautionary
principle."
14
15
Winifred
Hamilton,
Ph.
D.,
of
GHASP,
stated
that
she
felt
most
affected
communities
are
not
16
represented
adequately
in
decisionmaking
processes
related
to
endocrine
disruption.
She
said
it
17
was
difficult
to
buy
a
non­
contaminated
house
in
Houston
due
to
the
prevalence
of
chlordane.
Dr.
18
Hamilton
urged
the
Committee
to
remain
focused
on
health
concerns,
and
offered
caution
that
19
consensus
processes
not
be
misused.
She
continued
by
outlining
principles
she
felt
were
20
important,
including
shifting
the
focus
of
acceptable
levels
of
endocrine
disruptors
to
protecting
21
the
most
vulnerable
populations
(
e.
g.,
children
and
conceptuses
in
utero).
22
23
Judy
Starns,
a
citizen
from
Channelview,
Texas,
outlined
the
impacts
of
contamination
in
her
24
community,
and
expressed
concern
that
her
community
has
not
been
able
to
have
a
fair
health
25
study
conducted.
26
27
Karla
Lamb,
a
citizen
from
Channelview,
Texas,
outlined
personal
health
problems
since
moving
to
28
Houston
in
1975.
She
expressed
anger
and
frustration
at
the
deleterious
effects
chemical
exposure
29
has
had
on
her
health,
and
urged
the
Committee
to
help
others
in
similar
situations.
30
31
Response
to
Public
Comment
32
33
Dr.
Fenner­
Crisp
thanked
the
members
of
the
public
for
their
comments.
She
stated
that
due
to
34
the
mandate
under
which
the
EDSTAC
was
operating,
the
scope
of
the
Committee's
deliberations
35
would
likely
have
to
start
by
focusing
on
the
sex
steroids,
but
that
such
a
focus
was
not
indicative
36
of
the
EPA's
research
priorities.
37
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
12
U
­
Regarding
the
public
request
for
a
document
detailing
the
state
of
the
science
related
to
endocrine
1
disruption,
Dr.
Fenner­
Crisp
stated
that
the
EPA
has
been
working
on
such
a
document
for
one
2
and
one­
half
years.
The
document
is
currently
undergoing
review
by
EPA's
Science
Policy
3
Council,
and
should
be
available
on
the
World
Wide
Web
and
in
hard
copy
through
conventional
4
avenues
shortly.
5
6
Referring
to
public
comments
on
the
EDSTAC
process
and
concerns
with
respect
to
consensus
7
processes
in
general,
the
facilitator
stated
that
comments
on
the
Committee's
process,
even
critical
8
ones,
are
very
welcome.
With
respect
to
the
availability
of
information,
he
said
that
he
hoped
the
9
use
of
the
World
Wide
Web
site
would
help
meet
the
needs
of
the
public,
and
added
that
the
pace
10
of
the
EDSTAC
would
hopefully
decrease,
thereby
allowing
members
of
the
public
an
easier
11
opportunity
to
track
its
deliberations.
12
13
Members
of
the
Committee
spoke
to
the
concerns
citizens
raised
about
having
representatives
14
from
chemical
producing
industries
sitting
on
the
EDSTAC.
Some
members
firmly
stated
that
15
there
was,
in
no
manner,
a
conflict
of
interest.
In
fact,
Committee
members
pointed
out,
it
is
only
16
in
places
like
the
EDSTAC
that
representatives
of
all
interested
parties
can
speak
and
share
17
commonalties
as
humans,
and
that
a
level
of
trust
in
the
commonality
of
commitment
would
have
18
to
evolve.
19
20
Committee
members
also
cautioned
members
of
the
public
against
putting
all
their
hope
in
the
21
EDSTAC
as
the
solution
to
endocrine
disruption
or,
more
generally,
to
environmental
22
contamination.
They
stated
that
the
EDSTAC
is
only
one
part
of
a
process
to
improve
the
23
situation,
and
that
the
limitations
of
science
alone
would
likely
present
very
real
constraints
to
the
24
effectiveness
of
the
Committee.
25
26
IV.
April
29
­
May
1,
1997
 
Baltimore,
Maryland
27
28
29
Following
a
brief
public
reception
hosted
by
the
EDSTAC
and
attended
by
several
members
of
the
30
Committee
on
the
first
day
of
the
plenary
meeting,
members
of
the
public
were
invited
to
offer
31
comments
related
to
the
scope
and
charge
of
the
EDSTAC.
The
public
comment
session
lasted
32
longer
than
two
hours,
and
allowed
time
for
more
than
forty
individuals
to
provide
comment
to
33
Committee
members,
nearly
all
of
whom
were
present.
Due
to
the
number
of
individuals
seeking
34
to
provide
public
comment,
all
those
who
signed
up
to
speak
to
the
EDSTAC
were
requested
to
35
keep
their
comments
to
no
more
than
four
minutes
in
length.
While
this
request
may
have
caused
36
frustration
on
behalf
of
some
individuals,
the
intended
and
effected
result
was
to
allow
all
persons
37
wishing
to
provide
comment
the
opportunity
to
do
so.
Written
comments
submitted
by
members
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
13
U
­
of
the
public
may
be
found
in
the
public
docket
(
Docket
Number­­
OPPTS­
42189;
phone
202­
260­
1
7099).
This
meeting
summary
attempts
to
capture
the
essence
of
the
comments
made
by
members
2
of
the
public
to
the
EDSTAC
during
the
public
comment
session.
3
4
Thomas
Hobbins,
MD,
from
Physicians
for
Social
Responsibility,
commented
that
one
of
the
most
5
important
recommendations
offered
by
his
organization
is
that
Congress
needs
to
provide
a
higher
6
level
of
funding
for
endocrine
disruption
research.
Dr.
Hobbins
related
anecdotally
the
potential
7
for
paradigmatic
shifts
in
thinking
related
to
perception
and
actual
awareness
of
health
problems
8
and
risks.
He
urged
the
Committee
to
look
at
zero
tolerance
as
a
guide
post
rather
than
acceptable
9
thresholds
for
risk.
10
11
Keith
Bowers,
of
ICF,
Inc.,
offered
comments
concerning
international
institutions
working
on
12
issues
of
endocrine
disruption.
Mr.
Bowers
commented
that
the
endocrine
disruptor­
related
work
13
being
done
by
agencies
and
organizations
such
as
the
United
Nations
Environmental
Programme
14
and
the
OECD
is
being
conducted
in
conjunction
with
their
work
on
persistent
organic
pollutants.
15
He
stated
that
it
was
important
for
the
EDSTAC
to
know
how
its
work
differs
conceptually
from
16
the
OECD's
work,
and
that
the
Committee
should
look
at
non­
persistent
organic
pollutant
types
of
17
chemicals.
18
19
Gail
LeMaire,
Ph.
D.,
of
the
Endometriosis
Association,
stated
that
there
are
currently
millions
of
20
American
women
and
girls
with
endometriosis,
and
expressed
frustration
at
the
lack
of
support
for
21
women
with
the
disease,
as
well
as
the
lack
of
information
and
research
on
the
disease
to
date.
Dr.
22
LeMaire
requested
that
more
research
be
conducted
on
the
relationship
of
chemicals
such
as
23
dioxin,
PCBs,
and
TCCD
and
their
effects
on
the
body.
She
added
that
studies
should
not
simply
24
examine
instances
of
adverse
effects,
but
should
be
proactive
before
such
effects
are
manifested
in
25
the
body.
Dr.
LeMaire
concluded
by
stating
that
progesterone
is
noticeably
missing
from
the
26
EDSTAC's
scope.
27
28
Anneke
Davis,
from
the
Maryland
Conservation
Council
commented
on
the
public
information
29
process
of
the
EDSTAC.
Recognizing
that
the
Committee
is
under
tremendous
pressure
to
30
produce
their
deliverables,
she
stated
that
testing
is
going
to
take
a
lot
of
time
and
money,
as
well
31
as
public
support.
In
order
to
accomplish
this
and
to
garner
public
support
for
adequate
funding
of
32
the
implementation
of
the
EDSTAC's
recommendations,
Ms.
Davis
encouraged
the
Committee
to
33
keep
groups
of
citizens
concerned
about
endocrine
disruption
issues,
such
as
the
Maryland
34
Conservation
Council,
informed.
Furthermore,
Ms.
Davis
encouraged
the
EDSTAC
to
draft
its
35
final
report
in
a
manner
that
will
be
intelligible
to
and
usable
by
the
general
public.
Finally,
she
36
urged
that
the
Committee
not
forget
that
the
effects
of
endocrine
disruptors
on
ecological
systems
37
can
have
a
tremendous
impact
on
humans
as
well.
38
39
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
14
U
­
John
Holtzman,
of
the
Chemical
Manufacturers
Association
noted
his
appreciation
of
the
1
Committee's
emphasis
on
insisting
upon
a
scientific
approach
to
its
deliberations
and
deliverables.
2
He
noted
that
the
EDSTAC's
work
will
benefit
the
large
number
of
studies
currently
being
3
conducted
in
the
field
of
endocrine
disruption.
4
5
Carolyn
Burridge,
of
the
Maryland
Chemical
Industry
Council
complimented
the
members
of
the
6
EDSTAC
on
their
progress,
and
noted
the
timeliness
of
the
Committee's
work
given
the
attention
7
endocrine
disruption
is
getting
in
state
and
national
legislatures.
However,
she
expressed
concern
8
that
the
state
of
the
science
on
endocrine
disruption
is
not
yet
sufficiently
advanced
to
allow
the
9
formulation
of
sound
public
policy
related
to
the
subject.
She
encouraged
the
EDSTAC
to
10
emphasize
the
need
for
solid
science
and
facts
in
its
work,
and
to
appropriately
apply
its
limited
11
resources
toward
this
end.
12
13
Susan
Gilson,
from
FMC
Corporation,
expressed
support
for
the
work
of
the
EDSTAC,
and
14
encouraged
the
Committee
to
develop
recommendations
useful
to
professionals
in
fields
affected
15
by
endocrine
disruption,
referencing
her
position
as
an
industrial
hygienist
as
an
example
of
such
16
fields.
In
addition,
she
noted
interest
in
the
work
of
the
STWG
in
gauging
dose
responses,
as
well
17
as
other
tools
the
work
group
employs.
18
19
John
Casana,
an
environmental
engineer,
stressed
his
desire
for
the
EDSTAC
to
use
a
scientific
20
approach
to
quantifying
risk
and
decision
making
in
its
work;
to
make
a
conscious
decision
as
to
21
what
degree
of
uncertainty
is
acceptable
in
its
recommendations;
and,
if
there
is
greater
uncertainty
22
than
appropriate,
to
devise
a
way
to
get
to
an
acceptable
level
of
uncertainty
for
a
defensible
23
position
for
future
generations.
24
25
Grace
Ziem,
MD,
Ph.
D.,
requested
that
the
EDSTAC
seek
testimony
from
physicians
who
are
26
treating
humans
suffering
from
problems
related
to
endocrine
disruption
when
making
27
recommendations
for
screening
and
testing.
Dr.
Ziem
encouraged
the
Committee
members
to
be
28
sensitive
to
frustrations
that
can
occur
in
the
interface
of
academics
and
physicians,
and
asked
that
29
the
Committee
allow
as
much
flexibility
as
possible
in
its
recommendations
for
physicians
to
treat
30
their
patients
according
to
their
own
judgment.
31
32
Linda
Roberts,
an
employee
of
Chevron,
requested
that,
whenever
possible,
the
public
have
access
33
to
documents
discussed
at
the
plenary
meetings
in
advance
of
such
meetings
to
allow
for
34
appropriate
comment
and
understanding
of
the
issues.
She
expressed
appreciation
for
35
Dr.
Goldman's
comments
with
respect
to
validation
and
encouraged
the
Committee
to
develop
36
clear
criteria
for
interpreting
the
results
from
screens
and
tests.
In
addition,
she
noted
that,
as
data
37
from
screens
and
tests
are
collected,
it
would
be
useful
to
check
this
data
against
known
endocrine
38
disruptors
to
see
how
predictive
the
testing
was.
Ms.
Roberts
also
requested
the
Committee
to
be
39
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
15
U
­
sensitive
to
the
issues
of
dose
selection,
and
to
hold
more
discussion
with
respect
to
the
issue
of
1
zero
tolerance.
2
3
Ajax
Eastman,
a
former
member
of
the
Maryland
Conservation
Council,
expressed
concern
about
4
the
development,
production,
and
release
of
potential
endocrine
disrupting
chemicals
during
the
5
time
it
takes
for
the
EDSTAC
to
reach
consensus
on
a
screening
and
testing
program
for
EPA.
He
6
questioned
whether
a
science
or
policy
measure
existed
to
slow
or
stop
the
introduction
of
such
7
new
chemicals
until
the
Committee's
recommendations
are
developed.
Mr.
Eastman
also
8
expressed
concern
that
the
CF
would
allow
issues
such
as
risk
assessment
and
cost/
benefit
analysis
9
to
put
greater
priority
on
economic
matters
than
human
and
ecological
health.
10
11
Pat
Lane
noted
great
concern
about
the
health
consequences
of
the
incineration
of
medical
and
12
municipal
wastes.
Ms.
Lane
expressed
appreciation
for
the
publicly
open
and
flexible
process
13
being
used
by
the
EDSTAC
in
developing
its
recommendations.
In
addition,
she
commented
that
14
terms
such
as
"
risk
assessment"
and
"
parts­
per­
trillion"
are
perhaps
not
clearly
or
fully
understood
15
by
the
general
public,
and
that
such
terminology
takes
on
international
implications
when
used
in
16
reference
to
chemicals
used
around
the
world.
Furthermore,
Ms.
Lane
noted
that
the
public's
17
perception
of
risk
is
a
critical
element
in
effective
communication
and
protection,
and
asked
the
18
Committee
to
use
any
influence
it
had
to
encourage
medical
facilities
to
sort
their
waste
and
reduce
19
the
incineration
of
PCBs.
20
21
Lisa
Carlson
and
Kirstin
Smith,
both
medical
students
at
Johns
Hopkins
University
and
members
of
22
the
student
chapter
of
Physicians
for
Social
Responsibility,
presented
a
joint
statement
and
23
expressed
both
concern
that
only
two
systems­­
the
gonadol
and
thyroidal
systems­­
were
24
mentioned
in
the
Committee's
scope,
as
well
as
hope
that
the
Committee
would
expand
its
scope
25
beyond
estrogen­,
anti­
estrogen­,
androgen­,
anti­
androgen­,
and
thyroid­
related
effects.
Ms.
26
Smith
read
a
statement
from
the
student
chapter
of
Physicians
for
Social
Responsibility
which
27
called
for:
(
1)
larger
studies
on
humans
and
animals
looking
at
chemical
effects
and
synergies;
(
2)
28
the
burden
of
proof
to
be
placed
on
manufacturers
to
prove
the
safety
of
chemicals
before
29
marketing
them;
(
3)
the
use
of
small
doses
in
testing
for
endocrine
disruption;
(
4)
restrictions
on
30
medical
waste
incinerators
and
annual
monitoring
by
EPA
of
waste
from
every
incinerator
for
31
levels
of
mercury
and
dioxin;
and
(
5)
aggressive
education
of
the
public
by
EPA
on
endocrine
32
disruption.
33
34
Phyllis
Glazer
and
Sherre
Holmer,
of
Mothers
Organized
to
Stop
Environmental
Sins
(
MOSES),
35
detailed
the
story
of
Winona,
Texas,
where
a
well
was
driven
through
the
local
aquifer
for
the
36
injection
of
hazardous
waste
into
the
ground,
which
resulted
in
contamination
of
the
aquifer.
They
37
commented
that,
in
most
cases,
those
affected
by
endocrine
disruption
lack
political
power,
and
38
urged
that
any
screening
and
testing
program
should
make
use
of
anecdotal
information
from
39
affected
persons
who
live
or
have
lived
within
two
miles
of
facilities
which
store,
produce,
or
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
16
U
­
dispose
of
hazardous
waste.
Furthermore,
Ms.
Glazer
and
Ms.
Holmer
requested
that
the
1
screening
and
testing
program
developed
by
the
Committee
take
into
account
the
synergistic
2
effects
of
multiple
chemicals,
due
to
their
view
that
the
regulation
of
exposure
to
single
chemicals
3
is
a
matter
of
regulatory
convenience.
They
recommended
that
the
screening
and
testing
program
4
focus
on:
the
most
vulnerable
life
stages;
both
human
health
and
the
environment;
and
the
5
transference
of
chemicals
through
bioaccumulation
and
human
relationships
(
e.
g.,
breast
feeding).
6
In
addition,
they
recommended
that:
information
about
the
potential
harm
of
any
substance
be
7
released
from
the
Committee
to
the
public
at
the
earliest
opportunity;
the
Committee
place
8
emphasis
on
the
certainty
of
safety.
They
noted
that
the
endocrinological
effects
do
not
necessarily
9
follow
a
linear
relationship,
and
that
current
levels
of
acceptable
risk
burden
populations
of
10
minorities.
Finally,
they
urged
the
EDSTAC
to
practice
good
science
without
allowing
an
uneven
11
balance
of
representation
on
the
Committee.
In
accord
with
this,
they
recommended
that
the
12
Committee
membership
be
restructured
to
decrease
the
number
of
agency
and
industry
13
representatives.
14
15
Albert
Donnay,
executive
director
of
MCS
Referral
and
Resources,
addressed
the
definition
of
16
"
endocrine
disruptor"
as
discussed
by
the
Committee.
Mr.
Donnay
commented
that
the
inclusion
17
of
the
notion
of
"
adverse"
in
the
definition
overlooks
the
fact
that
there
are
many
endocrine
18
disruptors
that
may
produce
beneficial
or
therapeutic
effects,
and
that
effects
of
endocrine
19
disruptors
may
vary
by
individual.
He
encouraged
the
Committee
to
remind
the
public
that
such
20
beneficial
endocrine
disruptors
do
exist.
In
addition,
he
asked
the
Committee
to
not
only
include
21
the
young
and
developing
in
recommended
field
studies,
but
to
also
include
elderly
animals.
Mr.
22
Donnay
also
encouraged
the
Committee
to
consider
its
definition
reference
to
exogenous
23
substance,
stating
that
light
can
have
an
effect
on
hormones
(
e.
g.,
diurnal
cycles).
Finally,
he
asked
24
that
the
EDSTAC
clarify
their
definition
inclusion
of
"(
sub)
populations"
to
identify
what
the
25
smallest
subpopulation
that
needed
to
be
considered
was.
26
27
Gwen
Dubois,
MD,
of
Physicians
for
Social
Responsibility,
stated
that
the
consideration
of
groups
28
and
classes
of
chemicals
rather
than
of
individual
chemicals
in
the
screening
and
testing
program
29
was
very
sensible.
She
urged
the
Committee
to
not
be
reticent
to
recommend
action
on
30
information
of
harmful
endocrine
disruption
to
ban
certain
chemicals.
In
addition,
Dr.
Dubois
31
expressed
hope
that
the
Committee's
recommendations
would
address
exposure
to
multiple
32
chemicals,
and
asked
the
Committee
to
consider
the
hazards
of
medical
waste.
33
34
Raymond
Yang,
a
professor
of
Toxicology
at
Colorado
State
University,
applauded
the
endurance
35
of
the
EDSTAC
members
through
the
course
of
difficult
deliberations.
He
expressed
his
view
that
36
the
figure
regarding
the
number
of
chemicals
released
into
the
environment
used
by
the
Committee
37
was
low,
and
encouraged
the
Committee
to
give
serious
consideration
to
the
issue
of
mixtures
in
38
all
of
its
deliberations,
products,
and
recommendations.
39
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
17
U
­
Bruce
Baird
Struminger,
a
student
at
Johns
Hopkins
University
Medical
School,
expressed
concern
1
regarding
the
way
that
EPA
formed
the
EDSTAC.
He
stated
his
view
that
the
Committee
lacked
a
2
clear
sense
of
consensus
on
the
definition
of
"
endocrine
disruptor"
and
noted
that,
while
simplicity
3
is
good
where
appropriate,
the
emphasis
in
the
definition
should
be
on
accuracy.
Mr.
Struminger
4
stated
that
the
issue
of
endocrine
disruption
is
a
complex
one,
and
that
a
simple
definition
would
5
not
help
protect
the
public
health.
He
urged
that
scope
be
enlarged
to
include
additional
6
hormones,
and
expressed
his
feeling
that
policy
on
endocrine
disruption
is
getting
ahead
of
the
7
clinical
science
on
the
issue.
He
expressed
dissatisfaction
with
the
level
of
education
on
8
endocrinology
in
medical
schools,
as
well
as
the
lack
of
communication
about
the
issue
of
9
endocrine
disruption
in
endocrine
journals.
He
asked
the
Committee
to
take
a
precautionary
10
stance
in
its
recommendations
and,
given
the
growing
and
irreversible
bioaccumulation
levels
of
11
dioxins
and
PCBs,
to
place
the
onus
on
industries
to
prove
their
products
do
not
compromise
12
human
health.
13
14
Audrey
R.
McMahon,
from
the
Learning
Disabilities
Association,
stated
that
maternal
thyroid
15
dysfunction
is
a
very
serious
endocrine
disruption
issue.
She
further
stated
that
EPA
research
has
16
clearly
related
PCBs
to
diminished
thyroid
function
and
subsequent
effects
on
humans,
including
17
embryonic
development.
Ms.
McMahon
expressed
her
view
that
developmental
disabilities
are
18
epidemic
in
the
United
States,
and
added
that
she
hoped
the
Committee's
recommendations
would
19
help
this
situation.
20
21
Mimi
Cooper,
of
the
League
of
Women
Voters
in
Baltimore,
expressed
her
hope
that
chlorines
and
22
the
burning
of
dioxins
would
be
affected
by
the
Committee's
work
and
would
eventually
be
23
brought
under
control.
24
25
Yataka
Aoki,
from
Johns
Hopkins
University
Public
Health
School,
stated
that
it
is
difficult
for
26
members
of
the
general
public
to
understand
what
Types
I
and
II
errors
are,
and
asked
the
27
Committee
to
use
commonly
familiar
language
in
its
recommendations
and
final
report.
28
29
Erica
Wexler,
a
science
teacher
from
South
Baltimore
and
a
member
of
the
Community
Advisory
30
Panel,
commented
that
it
was
important
for
the
EDSTAC
to
follow
a
scientific
process
and
take
all
31
the
advice
and
views
into
account
as
it
proceeds
with
its
tasks.
She
added
that
the
Committee
32
should
identify
the
best
screens
and
tests,
validate
them,
and
implement
their
use.
33
34
Greg
Merrill,
from
the
Chlorine
Chemistry
Council,
noted
a
threefold
increase
in
state
legislation
35
that
make
reference
to
endocrine
disruption,
and
that
those
who
were
responsible
for
creating
36
many
of
the
regulations
and
laws,
particularly
at
the
state
level,
are
looking
to
the
EDSTAC
for
37
guidance.
He
stated
that
it
was
important
that
the
foundations
EDSTAC
establishes
and
the
38
principles
adopted
be
based
on
sound
science
to
ensure
confidence
in
their
implementation.
39
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
18
U
­
Davis
Baltz,
of
Commonweal,
described
journal
articles
about
gender
maturation
and
exposure
to
1
certain
chemicals.
He
stated
that,
as
reflected
by
research
in
the
popular
press;
evidence
of
2
endocrine
disruption
is
increasing.
Mr.
Baltz
expressed
hope
that
the
Committee
would
draw
the
3
conclusion
to
increase
research
and
prevent
exposure.
He
stated
that
members
of
the
public
have
a
4
right
to
know
about
the
EDSTAC's
activities,
and
that
plenaries
should
continue
to
be
conducted
5
across
the
country
to
allow
input
from
different
geographical
areas.
6
7
Ann
Mulligan,
National
Coordinator
for
The
DES
Cancer
Network,
stated
that
DES­
affected
8
persons
represent
one
of
the
few
populations
known
to
be
affected
by
endocrine
disruption.
She
9
urged
the
Committee
to
construct
a
new
paradigm
for
endocrine
disruption,
and
to
adopt
zero
risk
10
and
precautionary
principles.
Ms.
Mulligan
described
generational
effects
of
DES,
adding
that
11
DES
is
still
being
given
to
women
in
other
countries
and
the
affected
population,
therefore,
is
a
12
global
one.
She
commented
that
traditional
risk
assessment
failed
those
exposed
and
needs
to
be
13
revisited,
noting
that
another
chemical,
AZT,
currently
being
prescribed
to
pregnant
women
14
shows
DES­
like
effects.
15
16
Alyce
Ortizar,
of
the
Well
Mind
Association
of
Greater
Washington,
detailed
cancer
rates
and
17
breast
cancer
incidence
stating
that
she
was
troubled
by
the
fact
that
companies
that
have
caused
18
endocrine
disruption
seem
to
be
benefiting
from
treating
endocrine
disruption.
She
urged
the
19
adoption
of
zero
tolerance
beliefs,
and
the
shifting
of
the
burden
of
proof
onto
industry.
20
21
Carolyn
S.
Van
Pelt,
of
DuPont
and
the
American
Crop
Protection
Association,
applauded
the
22
Committee
for
its
process
and
for
the
work
performed
thus
far.
23
24
Daniel
Pontious,
from
the
Maryland
Public
Interest
Research
Group,
stated
that
the
information
25
shared
with
the
public
is
only
as
good
as
the
testing
that
is
done
to
generate
that
information.
He
26
expressed
his
appreciation
that
the
Committee
would
be
looking
at
multiple
chemical
exposures,
27
and
encouraged
the
use
of
caution
and
scrutiny
in
looking
at
low­
dose
toxicity
of
some
chemicals.
28
29
Keary
Cope,
a
student
at
Johns
Hopkins
University,
suggested
the
consideration
of
certain
30
metabolites
in
the
Committee's
work.
31
32
Alan
Noe,
of
the
American
Crop
Protection
Association,
noted
that
the
large
number
of
public
33
commenters
indicated
a
high
level
of
interest
in
issues
related
to
endocrine
disruption.
He
34
commented
on
the
role
and
benefits
of
pesticides
in
the
American
agricultural
sector,
and
35
discouraged
the
use
of
the
precautionary
principle.
He
also
urged
that
science
"
drive"
the
36
EDSTAC
process.
37
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
19
U
­
Lara
Shane,
of
Responsible
Industry
for
a
Sound
Environment,
expressed
her
belief
that
the
1
EDSTAC
process
is
a
great
one
to
bring
people
from
the
different
sides
of
the
spectrum
to
come
2
together
in
productive
discussion
and
action,
and
offered
the
Committee
her
wholehearted
support.
3
4
Valerie
Jewitt,
from
the
American
Crop
Protection
Association,
commented
that
her
family
had
5
been
impacted
by
the
incidence
of
breast
cancer,
and
said
she
supported
getting
all
affected
parties
6
involved
in
the
EDSTAC
process.
She
encouraged
the
use
of
sound
science
in
effecting
the
7
parties'
mutual
goal
of
making
the
environment
safer
for
all
life,
and
added
that
she
looked
8
forward
to
when
this
type
of
process
is
commonplace
in
decision
making.
9
10
Charles
Cangerni,
of
Reliance
Fire
Protection,
discussed
the
role
of
small
business
in
the
United
11
States,
noting
that
they
do
not
generally
have
the
same
employment
and
financial
resources
as
12
large
companies.
Mr.
Cangerni
urged
the
Committee
to
keep
small
businesses
in
mind
when
13
recommending
screening
and
testing
techniques.
14
15
Melissa
Sheridan,
a
member
of
the
Endometriosis
Association,
stated
that
she
suffers
from
the
16
disease,
and
told
the
Committee
that
the
EDSTAC's
work
is
vitally
important
and
very
17
appreciated.
18
19
Gail
McPherson,
an
American
Agri­
woman
and
farmer,
expressed
great
concern
regarding
her
20
feeling
that
the
general
public
was
characterizing
the
agricultural
sector
as
polluting
and
causing
21
disease.
She
urged
the
public
and
the
EDSTAC
to
remember
the
role
of
the
agricultural
sector
in
22
the
development
of
the
United
States.
23
24
David
Fisk,
a
medical
student
at
Johns
Hopkins
University
and
member
of
the
student
chapter
of
25
Physicians
for
Social
Responsibility,
stated
that
endocrine
disruption
was
a
major
topic
of
26
discussion
at
a
recent
national
conference
in
Chicago.
He
urged
that
the
burden
of
proof
rest
on
27
chemical
manufacturers
and
incinerator
operators.
28
29
Polly
Walker,
MD,
encouraged
the
adoption
of
zero
exposure
and
precautionary
principles
and
a
30
new
type
of
risk
assessment.
She
stated
that
the
long
lasting
and
irreversible
effects
of
endocrine
31
disruption
are
unconscionable.
Dr.
Walker
further
stated
that
unless
the
real
levels
of
potential
32
harm
are
put
into
the
equation
of
risk
assessment,
new
technologies
will
not
be
developed
to
33
address
them.
She
encouraged
the
Committee
to
consider
the
synergistic
and
additive
effects
of
34
chemicals
in
endocrine
disruption.
35
36
Victor
Perez,
a
medical
student
at
Johns
Hopkins
University
and
a
member
of
a
south
Baltimore
37
community,
stated
that
his
community
receives
disproportionate
effects
from
hazardous
waste.
He
38
noted
that
amphibians
tend
to
be
the
first
type
of
organisms
to
be
noticeably
affected
by
39
environmental
changes
due
to
their
breeding
patterns
and
aquatic
life.
Similarly,
he
stated
that
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
20
U
­
inner­
city
and
urban
populations
have
emerged
as
somewhat
of
an
indicator
species
for
humankind,
1
especially
in
reference
to
endocrine
disruption.
He
encouraged
the
Committee
to
seriously
2
consider
such
populations
in
its
deliberations.
3
4
Response
to
Public
Comment
5
6
Judy
O'Brien,
Associate
of
The
Keystone
Center,
thanked
the
members
of
the
public
for
their
7
comments
and
asked
Committee
members
if
they
had
any
comments,
clarifications,
or
questions
8
related
to
the
public
comment.
9
10
With
respect
to
Dr.
Ziem's
comments,
one
Committee
member
noted
that
the
continued
need
to
11
integrate
the
work
of
the
EDSTAC
with
practitioners
is
one
that
has
been
reiterated
by
Committee
12
members
themselves
throughout
the
EDSTAC
process.
In
addition,
it
was
noted
that
the
13
EDSTAC
is
not
charged
with
developing
clinical
tests
to
assess
endocrinological
wellness,
but
14
rather
experimental
toxicology
related
to
endocrine
disruption.
15
16
Regarding
the
interface
between
science
and
public
policy,
one
Committee
member
noted
the
17
difficulties
attendant
in
banning
or
regulating
chemicals
already
on
the
market.
With
respect
to
the
18
Alar
scare,
another
Committee
member
noted
that
the
scientist
presented
the
Alar
information
19
correctly,
but
the
media
blew
the
information
out
of
proportion.
Due
to
this
unpredictability,
the
20
Committee
member
urged
the
EDSTAC,
and
the
COWG
in
particular,
to
try
to
anticipate
potential
21
public
relations/
perception
problems
to
the
greatest
extent
possible.
22
23
With
respect
to
the
EDSTAC's
scope,
Dr.
Goldman
commented
that
the
Committee
is
not
24
responsible
for
setting
standards
or
for
advising
EPA
on
how
to
set
them.
Nor,
she
added,
is
the
25
Committee
responsible
for
medical
waste
incineration
issues.
Dr.
Goldman
noted,
in
the
convening
26
phase,
many
Committee
members
advocated
for
taking
as
comprehensive
a
strategy
as
possible
to
27
address
the
widest
possible
scope
of
hormones.
However,
given
the
time
pressures
of
28
accomplishing
the
tasks
and
mandated
deliverables,
certain
hormones
were
necessarily
selected
as
29
a
starting
point
for
the
Committee's
scope.
The
EDSTAC's
recommendations
will,
however,
30
provide
a
general
framework
for
EPA
to
use
as
new
information
comes
forward
in
the
application
31
of
screening
and
testing
programs
for
a
variety
of
hormonal
endpoints
and
mechanisms.
32
33
Regarding
public
comments
about
research,
Dr.
Goldman
noted
that
the
EDSTAC
can
make
34
recommendations
concerning
research
and
studies
to
be
done,
but
that
the
Committee
would
not
35
be
performing
any
research
or
studies
itself.
In
response
to
comments
regarding
regulated
agency
36
membership
on
the
Committee,
Dr.
Goldman
noted
that
the
Agencies
that
generate
waste
are
not
37
part
of
the
EDSTAC,
but
that
many
agencies
represented
on
the
Committee
are
indeed
regulators.
38
39
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
21
U
­
One
Committee
member
replied
to
comments
regarding
the
EDSTAC's
scope
and
industry
onus
1
and
burden
of
proof,
noting
that
the
industry
representatives
on
the
Committee
are
sincerely
2
concerned
that
everything
that
is
produced
in
their
industry
is
safe
for
humans
and
the
3
environment.
He
added
that,
with
respect
to
scope,
the
hormonal
areas
identified
as
foci
for
the
4
Committee's
work
are
those
where
the
Committee
members
believe
the
EDSTAC
has
the
greatest
5
chance
of
making
solid
decisions
and
recommendations
given
the
deadlines
and
the
current
state
of
6
the
science.
7
8
Another
Committee
member
addressed
Dr.
Yang's
comments
regarding
the
consideration
of
9
mixtures
stating
that,
while
there
may
not
yet
seem
to
be
a
great
deal
of
discussion
on
the
subject
10
at
the
plenary
level,
much
work
on
mixtures
was
taking
place
at
the
work
group
level,
and
would
11
eventually
be
brought
before
the
full
Committee.
12
13
Another
Committee
member
pointed
out
that
there
is,
indeed,
representation
on
the
EDSTAC
by
14
physicians
who
are
treating
patients
with
endocrinological
disorders.
15
16
Dr.
Goldman
thanked
the
public
for
their
thoughts,
and
expressed
her
appreciation
of
the
public's
17
attention
and
patience
in
observation
of
the
involved,
and
sometimes
intricate,
discussions
of
the
18
EDSTAC.
One
Committee
member
urged
members
of
the
public
to
look
to
their
own
19
communities,
not
the
Committee,
to
find
the
solutions
to
community
problems.
Dr.
Goldman
20
echoed
these
sentiments,
adding
that
she
recognized
the
value
of
and
need
for
inter­
community
21
communication
and
encouraged
communities
affected
by
endocrine
disruption
and
hazardous
22
waste
to
begin
to
engage
each
other
in
dialogue.
23
24
After
the
public
comment
period,
several
EDSTAC
members
expressed
their
recognition
of
the
25
troubling
health
and
environmental
consequences
of
endocrine
disruptors,
but
urged
the
public
to
26
recognize
that
the
charge
of
the
EDSTAC
is
to
develop
a
screening
and
testing
program
for
27
endocrine
disruptors.
Committee
members
encouraged
future
public
comment
on
thoughts
and
28
ideas
specifically
related
to
the
development
of
an
effective,
strategic,
and
efficient
screening
and
29
testing
program,
rather
than
testimony
related
to
the
effects
and
perils
of
diseases
and
endocrine
30
disruption.
Public
comment
opportunities
at
the
Chicago
plenary
and
future
meetings
may,
31
therefore,
be
designed
to
facilitate
this
type
of
discourse.
32
33
V.
July
15­
16,
1997
 
Chicago,
Illinois
34
35
36
Following
the
Committee's
deliberations
on
the
first
day
of
the
plenary
meeting,
members
of
the
37
public
were
invited
to
offer
comments
related
to
the
scope
and
charge
of
the
EDSTAC.
The
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
22
U
­
public
comment
session
lasted
over
two
hours,
and
allowed
time
for
more
than
forty
individuals
to
1
provide
comment
to
Committee
members,
nearly
all
of
whom
were
present.
Due
to
the
number
of
2
individuals
seeking
to
provide
public
comment,
all
those
who
signed
up
to
speak
to
the
EDSTAC
3
were
requested
to
keep
their
comments
to
no
more
than
four
minutes
in
length.
While
this
request
4
may
have
caused
frustration
on
behalf
of
some
individuals,
the
intended
and
effected
result
was
to
5
allow
all
persons
wishing
to
provide
comment
the
opportunity
to
do
so.
Written
comments
6
submitted
by
members
of
the
public
may
be
found
in
the
public
docket
(
Docket
Number­­
OPPTS­
7
42189;
phone
202­
260­
7099).
This
meeting
summary
attempts
to
capture
the
essence
of
the
8
comments
made
by
members
of
the
public
to
the
EDSTAC
during
the
public
comment
session.
9
10
Patricia
Speth,
a
concerned
citizen
who
comes
from
a
family
of
farmers,
expressed
concern
that
11
the
agricultural
industry
may
be
adversely
impacted
by
endocrine
disruptors,
prior
to
gaining
12
additional
information
about
their
effects.
She
expressed
her
support
for
the
EDSTAC
process
13
and
suggested
that
the
approach
this
problem
should
be
deliberate
and
practical
and
urged
that
14
solutions
not
be
based
on
emotionalism
or
"
junk
science."
15
16
Joe
DiGangi,
of
Greenpeace
in
Chicago
and
formerly
a
corporate
scientist,
spoke
to
the
issue
of
17
risk
assessment.
He
urged
the
Committee
to
reopen
the
risk
assessment
discussion
within
the
18
Committee's
deliberations
because
he
feels
as
though
the
Committee
has
an
obligation
to
show
to
19
the
public
there
is
a
disparity
of
views
on
this
issue.
He
also
would
like
to
see
industry
take
on
the
20
burden
of
proof
to
demonstrate
the
safety
of
new
chemicals.
21
22
Brett
Hulsey,
of
the
Sierra
Club,
mentioned
his
role
on
the
FACA
for
water
monitoring
and
23
expressed
concern
about
the
fish
people
consume
today
and
the
lack
of
knowledge
about
fishing
24
advisories
that
exists
and
the
potential
for
harm
because
of
that
lack
of
knowledge.
He
supports
25
due
diligence
for
companies,
particularly
to
handle
the
high
costs
associated
with
cleaning
up
26
polluted
areas.
Finally,
he
urged
the
Committee
to
be
conservative
and
make
screens
as
extensive
27
as
possible
that
go
beyond
adverse
affect.
28
29
Mark
Richie,
of
the
Institute
for
Agriculture
and
Trade
Policy,
an
organization
that
is
concerned
30
about
pollution
and
health
concerns,
particularly
due
to
airborne
chemicals,
which
may
render
31
products
produced
in
an
organic
manner
polluted,
suggested
that
multiple
layers
of
impacts
are
of
32
great
concern,
as
is
the
eroding
market
niche
for
organic
products
due
to
this
pollution.
He
33
cautioned
the
Committee
to
take
the
precaution
that
is
necessary
to
protect
public
health,
but
to
34
also
remember
that
decisions
are
being
made
to
protect
other
industries
as
well.
35
36
Ellen
Kauffman,
of
the
Endometriosis
Association
in
Chicago,
stated
that
she
was
there
37
representing
women
with
endometriosis
and
related
information
about
the
disease
and
some
38
concerns
of
those
women
who
have
it.
She
indicated
that
studies
have
shown
that
a
transfer
of
39
toxins
can
occur
in­
utero
and
during
breast
feeding,
and
the
hormonal
effects
in
children
of
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
23
U
­
mothers
with
the
disease
are
increasingly
being
realized.
Ms.
Kauffman
related
that
there
are
5
1/
2
1
million
cases
of
endometriosis
today
and
is
estimated
to
occur
in
10%
of
women
of
reproductive
2
age.
Endometriosis
is
affecting
women
at
younger
and
younger
ages
with
greater
severity­­
the
3
costs
of
this
disease
are
high.
4
5
Davis
Baltz,
of
Commonweal,
Inc.,
spoke
directly
about
the
screening
and
testing
program,
6
suggesting
that
chemicals
in
the
Stop
Box
should
undergo
a
random
sampling
process
in
order
to
7
ensure
they
have
not
been
placed
there
erroneously.
He
also
suggested
breast
milk
should
be
8
tested.
Regarding
Production
and
Use
data,
he
supported
breaking
these
out
into
two
separate
9
categories
because
use
information
is
quite
different
that
production
information.
Mr.
Baltz
10
expressed
his
support
for
the
public's
right
to
know
about
these
issues
every
step
of
the
way,
11
which
includes
a
comprehensive
educational
effort.
12
13
Rob
Christie,
Director
of
Public
Affairs
for
FMC
Corporation,
expressed
concern
both
personally
14
and
professionally
about
the
work
of
the
EDSTAC.
He
related
his
opinion
that
any
direction
the
15
Committee
takes
with
public
policy
in
this
area
should
be
based
on
sound
science
and
he
urged
the
16
Committee
to
remain
focused
on
their
tasks,
in
order
to
reach
the
deadlines
ahead
of
them.
17
18
Jackie
Hunt
Christensen,
of
Health
Care
Without
Harm,
asked
the
Committee
to
do
the
following:
19
1)
recommend
to
EPA
that
chemicals
such
as
dioxins
be
phased
out
immediately,
rather
than
go
to
20
tier
2
testing;
2)
testing
of
chemicals
used
in
medical
products
should
be
reviewed,
as
well
as
21
products
produced
by
health
care
facilities;
and
3)
make
it
a
priority
that
chemicals
that
cross
the
22
placenta
or
are
present
in
breast
milk
should
be
tested.
23
24
Lynn
Fahey
McGrath,
manager
of
risk
assessment
for
Hoechst
Corporation,
a
multinational
25
chemical
company,
said
there
is
a
clear
need
for
chemical
products
to
pose
a
minimal
risk
to
26
consumers
and
the
environment.
She
urged
against
the
publication
of
premature
results
and
their
27
effects
(
e.
g.,
poor
deselection
choices),
noting
that
in
Europe,
positive
results
from
a
screen
can
28
result
in
deselection.
Her
experience
working
in
Europe
has
broadened
her
perspective
on
these
29
issues,
and
she
encourages
the
Committee
to
incorporate
greater
coordination
there.
She
also
30
encouraged
the
Screening
and
Testing
Work
Group
to
reevaluate
baseline
data
and,
at
a
minimum,
31
allow
flexibility
for
data
required
in
other
countries.
32
33
Jack
Weinberg
of
Greenpeace
discussed
some
of
the
policy
implications
of
the
decisions
EDSTAC
34
has
already
made.
He
noted
that
the
concept
of
endocrine
disruption
used
at
the
Wingspread
35
Conference
was
broader
than
that
used
by
the
EDSTAC
and
very
useful
because
it
captured
a
36
range
of
mechanisms
that
operated
at
very
minute
concentrations.
Mr.
Weinberg
recognized
that,
37
for
pragmatic
reasons,
the
EDSTAC
limited
the
scope
of
its
efforts
to
three
hormonal
systems,
but
38
asked
the
Committee
to
make
clear
to
the
public
that
the
extent
of
endocrine
disruption
was
not
39
circumscribed
nor
fully
addressed
by
consideration
of
the
three
hormones
in
the
EDSTAC's
scope.
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
24
U
­
1
Linda
Roberts,
of
Chevron,
directed
her
comments
to
the
Priority
Setting
Work
Group.
She
2
indicated
that
Chevron
has
had
some
experience
with
SAR
models
and,
in
terms
of
their
utility
and
3
use,
she
suggested
that
the
accuracy
of
SAR
output
is
a
function
of
the
degrees
of
structural
4
similarity
between
the
untested
chemicals
and
the
tested
chemicals.
Regarding
Option
3
of
the
5
PSWG's
presentation,
she
urged
the
work
group
to
clarify
what
is
meant
by
High,
Low,
and
No
6
Data,
especially
in
terms
of
production
and
use.
For
the
No
Data
option,
she
supported
using
the
7
term
"
inadequate
information".
Regarding
the
Stop
Box,
Ms.
Roberts
suggested
that
there
is
not
8
much
of
a
distinction
between
20,000
and
40,000
chemicals,
because
many
will
not
be
tested
in
9
our
lifetime.
She
urged
the
Committee
to
evaluate
the
chemicals
they
do
want
to
test
and
design
10
the
program
to
incorporate
them.
Regarding
the
STWG
activities,
Ms.
Roberts
encouraged
the
11
use
of
validation
in
the
biological
variability
of
the
process.
She
suggested
it
would
be
helpful,
for
12
example,
to
know
how
reproducible
the
endpoints
are,
how
many
animals
are
needed,
and
whether
13
the
results
are
reproducible
in
good
lab.
Finally,
regarding
dose
response,
she
asked
for
guidance
14
from
the
STWG
on
the
sizes
of
doses
it
thinks
is
appropriate,
particularly
for
the
high
and
low
15
doses.
16
17
Joanna
Hoelscher,
Director
of
the
Illinois
office
of
Citizens
for
a
Better
Environment,
thanked
the
18
Committee
for
holding
a
meeting
in
Chicago
and
for
the
opportunity
to
comment.
She
urged
the
19
EDSTAC
to
not
lose
sight
of
the
broader
mission
of
protecting
public
health
and
the
environment
20
in
both
the
short
term
and
the
long
term,
and
therefore
encouraged
the
use
of
the
"
precautionary
21
principle".
Ms.
Hoelscher
then
made
specific
comments
directed
toward
PSWG
and
COWG.
To
22
the
PSWG,
she
said
the
CF
must
take
into
account
the
fact
that
there
are
already
significant
23
amounts
of
information
in
the
literature
on
chemicals
already
known
to
be
endocrine
disruptors,
24
and
there
should
be
a
mechanism
to
fast
track
these
in
order
to
eliminate
exposure.
She
also
stated
25
that
traditional
risk
assessment
methodologies
are
inadequate
to
protect
public
health,
which
26
should
be
included
in
the
CF.
Finally,
she
suggested
prioritization
be
given
to
certain
chemicals
27
based
on
bioavailability
and
persistence.
To
the
COWG,
she
requested
that
scientific
uncertainty
28
not
be
considered
tantamount
to
an
assumption
of
no
danger.
29
30
Michael
Murry
of
the
National
Wildlife
Federation
directed
his
comments
to
the
PSWG
as
follows.
31
First,
he
suggested
the
uncertainty
that
exists
with
SARs
has
to
be
an
issue
when
looking
at
32
toxicological
effects.
Second,
regarding
the
log
K
ow,
he
suggests
that
increasing
the
K
ow
means
33
increasing
the
bioaccumulation
of
chemicals
and
there
is
no
reason
why
there
should
be
an
upper
34
limit
on
K
ow
s.
Third,
Mr.
Murray
suggested
that
there
are
two
potential
sources
of
breakdown
35
products
from
plants­­
monomers
and
dymers­­
and
the
only
way
to
control
discharge
of
them
is
to
36
control
discharge
of
their
parent
compounds.
Finally,
regarding
exposure
data,
he
encouraged
37
evaluation
in
a
precautionary
way
because
2000
new
chemicals
are
introduced
each
year
and
some
38
processes
are
still
behind
and
testing
old
chemicals
that
were
banned
20
years
ago.
39
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
25
U
­
Sarah
Janssen,
a
medical
student
who
is
a
member
of
Physicians
for
Social
Responsibility,
raised
1
several
concerns
regarding
potential
endocrine
disruptors.
Specifically,
she
is
concerned
about
2
toxic
releases
from
a
local
toxic
waste
incinerator
as
well
as
about
endocrine
disruptors
in
the
air,
3
water,
and
ground.
A
main
concern
lies
with
her
future
potential
patients
and
the
ramifications
of
4
these
chemicals
being
emitted
into
the
environment.
Ms.
Janssen
commended
the
Committee
for
5
its
intention
to
screen
and
test
mixtures
and
encouraged
them
to
look
at
low
doses.
She
also
urged
6
for
an
increase
in
federal
funding
for
the
development
of
more
sensitive
assays.
7
8
Mark
Holmer,
of
the
Chemical
Industry
Council
of
Illinois,
provided
some
general
comments
to
9
the
Committee.
He
stressed
the
importance
of
remaining
objective
when
examining
such
issues
10
and
urged
that
sound
science
be
applied.
He
praised
EPA
for
reaching
outside
of
its
own
11
employees
to
create
the
Committee
and
for
assembling
such
a
diverse
group.
Finally,
he
urged
that
12
science
lead
EPA
to
whatever
regulatory
path
they
take.
13
14
Ed
Gunderson,
chairman
of
regulatory
affairs
for
the
Chemical
Industry
Council
of
Illinois,
15
suggested
any
policies
developed
be
based
on
sound
science,
be
technically
realistic,
and
serve
to
16
protect
the
public
health
and
the
environment.
He
urged
that
protocols
for
the
Screening
and
17
Testing
program
provide
conclusive
data
demonstrating
beyond
any
doubt
that
an
adverse
effect
18
could
result
from
a
realistic
exposure
route.
Mr.
Gunderson
also
encouraged
the
use
of
a
19
scientific,
dosage­
risk­
based
approach
to
developing
the
program.
20
21
Lynn
Lawson,
with
the
Multiple
Chemical
Sensitivities:
Health
and
Environment,
described
health
22
effects
she
presently
experiences
due
to
chemical
sensitivity
and
lifestyle
changes
she
has
made
to
23
limit
exposures
to
such
chemicals.
Many
of
the
chemicals
indicated
in
personal
blood
tests,
she
24
asserted,
appear
on
lists
of
suspected
endocrine
disruptors.
Ms.
Lawson
asked
the
EDSTAC
to:
25
1)
address
the
endocrine
disrupting
potential
of
mixtures
first
in
their
screening
and
testing
26
program;
2)
consider
new
chemicals
guilty
until
proven
innocent;
3)
"
sunset"
chemicals
known,
or
27
likely,
to
be
endocrine
disruptors,
noting
her
feeling
that
any
threshold
for
such
chemicals
is
too
28
high.
29
30
Marjorie
Fischer,
of
the
League
of
Women
Voters,
stated
that
chemicals
in
the
environment
should
31
not
impinge
public
health,
especially
the
health
of
children.
She
added
that
the
current
position
of
32
the
League
of
Women
Voters
on
pesticides
is
that
exposures
to
people
should
be
reduced
and
that
33
nontoxic
alternatives
to
these
pesticides
should
be
used.
Ms.
Fischer
noted
that
many
suspected
34
endocrine
disruptors
would
appear
to
be
pesticides
and
would,
therefore,
seem
to
be
of
interest
to
35
the
League
due
to
its
interests,
stances,
and
publications.
She
expressed
appreciation
of
the
36
inclusive
definition
of
endocrine
disruptors
tentatively
being
used
by
the
Committee,
and
urged
that
37
zero
tolerance
parameters
be
imposed
on
endocrine
disrupting
chemicals,
especially
in
light
of
38
effects
caused
by
fetal
exposure
to
such
chemicals.
39
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
26
U
­
Cecile
Sastre,
from
the
Endometriosis
Association,
asked
the
EDSTAC
to
remember
the
real
1
effects
diseases
attributed
to
endocrine
disruptors
are
having
on
humans.
She
described
symptoms
2
and
illnesses
related
to
the
disease
of
endometriosis,
and
noted
that
evidence
indicates
certain
3
chemicals
are
responsible
for
endometriosis
and
a
myriad
of
other
diseases.
Ms.
Sastre
told
the
4
Committee
she
is
personally
afflicted
by
endometriosis,
and
described
the
effects
the
disease
has
5
had
on
her
life,
including
severe
chemical
sensitivities,
difficulties
finding
employment
due
to
the
6
hazards
of
workplaces,
as
well
as
other
lifestyle
complications.
She
urged
the
EDSTAC
to
set
7
zero
tolerance
standards,
and
to
consider
the
synergistic
actions
of
chemicals.
8
9
Dr.
George
Raab,
former
chair
of
the
Species
Survival
Unit
of
the
Chicago
Zoological
Society,
10
commented
on
the
synergies
of
chemical
compounds
affecting
species.
In
particular,
he
sought
to
11
report
recent
information
on
the
phenomenon
of
declining
amphibian
populations
in
several
parts
12
of
the
world.
Although,
he
admitted,
the
ultimate
causal
effects
and
factors
to
which
these
13
declines
may
be
attributed
have
not
yet
been
satisfactorily
identified,
he
noted
that
some
declines
14
indicate
there
might
be
endocrine
disruption
or
distortion
effects
at
work,
especially
in
parts
of
15
Central
America.
Dr.
Raab
urged
the
financing
of
studies
on
these
declines
and
effects
on
the
16
broadest
scale
possible.
17
18
Jim
Houston,
with
the
International
Joint
Commission's
Canadian
Section
in
Ottawa,
reflected
on
19
the
interface
between
efforts
directed
toward
endocrine
disruptors
in
his
country
and
in
the
United
20
States.
He
noted
that
the
earlier
Canadians
are
involved
in
similar
activities
and
are
informed
by
21
the
United
States
on
its
efforts,
the
more
efficiently
the
problem
may
be
addressed.
22
23
Charlie
Cray,
of
Greenpeace,
stated
that
the
fact
that
the
Committee
is
focusing
on
developing
a
24
screening
and
testing
program
should
not
delay
current
federal
or
state
actions
to
phase
out
25
products
proven
to
cause
endocrine
disrupting
effects.
He
noted
there
was
a
need
to
support
26
policies
on
endocrine
disruptors
even
when
uncertainties
exist.
Mr.
Cray
described
current
27
European
studies
indicating
the
timeliness
of
phasing
out
the
use
and
production
of
certain
28
chemicals.
Particularly
addressing
the
endocrine
disrupting
potential
of
chemicals
in
medical
29
products,
Mr.
Cray
noted
that
allegedly
safe
alternatives
are
available
and
urged
their
use.
He
30
encouraged
the
Committee
to
talk
about
releases
and
exposures
of
potentially
endocrine
disrupting
31
chemicals
from
common
products
into
waste
water
and
other
media.
32
33
Chuck
Elkins,
an
environmental
consultant,
commented
that
the
Committee's
T1S
design
sounded
34
fairly
sophisticated
and
seemed
consistent
with
the
current
state
of
the
science
and
with
similar
35
efforts.
However,
he
expressed
caution
that
a
decision
to
use
either
one
or
a
very
small
number
of
36
decision
makers
to
decide
what
testing
would
be
done
in
a
tailored
approach
could
prove
to
be
a
37
bottleneck
in
the
screening
and
testing
process.
Mr.
Elkins
asked
that
the
Committee
pay
38
particular
attention
to
communications
issues
in
the
sorting
and
prioritization
process,
as
these
39
stages
of
the
program
could
be
misunderstood
by
both
the
public
and
decision
makers.
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
27
U
­
Mary
Beth
Doyle,
from
the
Ecology
Center
of
Ann
Arbor,
thanked
the
Committee
for
conducting
1
a
meeting
in
the
Great
Lakes
region,
and
for
the
Committee's
hard
work.
She
encouraged
the
2
Committee
to
adopt
the
"
precautionary
principle"
in
its
program,
and
to
insure
chemicals
are
tested
3
thoroughly
before
being
released
into
the
environment.
With
respect
to
hormone
disruptors,
she
4
noted
the
inclusion
of
the
word
"
adverse"
in
the
working
definition
concerned
her,
and
urged
the
5
EDSTAC
to
develop
a
broader
definition.
In
addition,
Ms.
Doyle
expressed
concern
with
the
Stop
6
Box,
noting
her
feeling
that
the
scientific
tools
currently
available
to
screen
and
test
for
endocrine
7
disruptors
are
too
crude
to
send
a
chemical
substances
or
mixtures
definitively
to
the
Stop
Box.
8
Rather,
she
requested
that
such
chemical
substances
or
mixtures
be
assigned
to
a
low
priority
9
group.
Furthermore,
Ms.
Doyle
suggested
that
the
EDSTAC
"
groundtruth"
chemicals
sent
to
the
10
Stop
Box
to
ensure
the
accuracy
of
the
screening
and
testing
program.
She
also
said
that
11
community
concern
over
a
chemical
substances
or
mixtures
should
be
sufficient
to
pull
a
chemical
12
out
of
the
Stop
Box
for
re­
entry
into
the
screening
and
testing
program.
13
14
Penny
Richards,
the
Learning
Disabilities
Association
of
Illinois,
Inc.,
expressed
concern
about
the
15
impact
of
agricultural
chemicals
on
the
lifestyle
and
health
of
humans,
and
especially
on
her
16
developmentally
disabled
child.
Specifically,
Ms.
Richards
noted
concern
regarding
hormonal
17
disruption
due
to
fetal
exposure,
and
urged
the
EDSTAC
to
promote
and
protect
children's
health.
18
She
urged
the
Committee
to
adopt
the
"
precautionary
principle,"
adding
that
chemicals
should
not
19
have
a
"
civil
right"
of
being
considered
innocent
or
harmless
until
proven
guilty.
She
stated
that
no
20
permission
had
been
given
by
any
human
for
a
chemical
body
burden.
21
22
David
De
Rosa,
from
Greenpeace
in
Chicago,
expressed
his
concern
caused
by
the
recognition
23
that,
due
to
realistic
constraints,
all
chemicals
cannot
be
tested
for
endocrine
disruption.
24
Therefore,
he
noted,
he
hoped
that
scientists
would
be
able
to
learn
from
the
EDSTAC's
screening
25
and
testing
program
how
to
make
educated
guesses
about
classes
of
chemicals,
and
that
scientists
26
would
apply
that
knowledge
in
anticipating
any
effect,
not
just
adverse
effects.
Mr.
De
Rosa
27
stated
that
the
screening
and
testing
program
should
look
at
chemical
substances
or
mixtures,
as
28
well
as
their
breakdown
products,
with
which
people
come
into
contact
on
a
daily
basis.
He
29
encouraged
the
prioritization
process
to
use
production
volume
figures,
and
requested
that
any
30
chemical
proven
to
be
an
endocrine
disruptor
be
banned.
31
32
Bill
Holland,
of
Illinois
Public
Interest
Research
Group,
stated
his
concern
about
the
potential
33
hazards
of
minute
doses
of
endocrine
disrupting
chemicals,
noting
that
damage
from
exposure
may
34
not
be
apparent
until
much
later
in
an
organism's
development
process.
He
stated
that
his
35
organization
urged
the
adoption
of
both
the
"
precautionary
principle"
and
zero
exposure
36
tolerances.
Mr.
Holland
added
that
industry
should
be
responsible
for
showing
that
their
chemicals
37
are
safe
before
approval,
use,
and
release
into
the
environment.
Furthermore,
he
requested
EPA
38
make
significant
and
easily
understandable
efforts
to
educate
the
public
on
endocrine
disruption
39
issues,
chemicals
being
tested,
and
the
endocrine
disruptor
screening
and
testing
process.
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
28
U
­
Angel
Cohoon,
also
of
Greenpeace,
offered
great
concern
regarding
the
effects
of
endocrine
1
disruptors
on
early
stages
of
development,
especially
due
to
fetal
exposure
from
chemical
2
substances
or
mixtures
present
in
pregnant
mothers.
She
urged
the
Committee
to
consider
using
3
the
"
precautionary
principle"
and
to
immediately
phase
out
any
chemical
proven
to
be
an
endocrine
4
disruptor.
Despite
economic
and
practical
difficulties,
Ms.
Cohoon
expressed
confidence
that
5
scientific
ability
existed
to
eliminate
proven
endocrine
disruptors.
6
7
Barbara
Alexander
Malarky,
of
the
Waste
Handlers
Network,
praised
Illinois
EPA's
efforts
on
8
hormone
disruption
publications
to
date.
She
urged
that
the
Committee's
prioritization
process
9
place
significant
emphasis
on
the
cumulative
and
synergistic
effects
of
endocrine
disruptors,
and
10
that
it
not
look
at
chemicals
in
isolation.
Ms.
Malarky
expressed
concern
regarding
the
effects
of
11
waste
incinerators,
especially
from
the
disposal
of
dioxin
and
cows,
due
to
the
potential
for
12
interaction
of
airborne
endocrine
disrupting
chemicals.
13
14
Tracy
Easthope,
from
the
Ecology
Center
of
Ann
Arbor,
recommended
to
the
Committee
that
all
15
proceedings
of
the
International
Joint
Commission
and
their
science
advisory
board
be
included
as
16
part
of
the
official
proceedings
of
the
Committee.
She
noted
that
persistence
is
an
important
17
criteria
to
consider
in
the
screening
and
testing
program,
and
that
testing
should
be
sensitive
18
sentinel
species
as
an
"
early
warning
system"
in
order
to
avoid
economic
and
human
19
health/
environmental
costs.
In
addition,
she
questioned
the
scientific
ability
to
send
a
chemical
20
definitively
to
the
Stop
Box
as
indicated
in
the
Conceptual
Framework.
Ms.
Easthope
encouraged
21
the
Committee
to
look
at
research
indicating
that
the
timing
of
exposure
has
significant
22
implications
for
endocrine
disrupting
effects,
including
synergies
and
seasonal
variations,
due
to
23
the
fact
that
toxicity
differs
depending
on
stage
of
development
and
health
of
the
organism
at
the
24
time
of
exposure
(
e.
g.,
fetal
exposure
from
mother's
body
burden).
She
expressed
concern
about:
25
limitations
on
exposure
detection
due
to
the
state
of
the
science;
the
Committee's
working
26
definition
of
an
"
endocrine
disruptor";
and
the
potential
for
a
lack
of
clarity
about
screening
and
27
testing
results
(
e.
g.,
whether
or
not
a
chemical
substances
or
mixtures
is
an
endocrine
disruptor
if
28
results
of
T1S
or
T2T
are
mixed).
Finally,
Ms.
Easthope
emphasized
the
importance
of
risk
29
assessment
considerations
in
the
Committee's
development
of
a
screening
and
testing
program,
as
30
well
as
the
need
to
constantly
reevaluate
the
effectiveness
of
whatever
tools
are
designed
to
31
address
the
endocrine
disruptor
issue.
32
33
Response
to
Public
Comment
34
35
Dr.
Goldman
noted
that
while
the
Committee
is
not
yet,
as
a
whole,
in
agreement
on
the
role
of
36
risk
assessment
and
its
use
in
regulating
chemicals,
discussion
on
the
subject
had
not
at
any
point
37
been
constrained
or
inhibited.
She
asked
members
of
the
public
to
remember
that
this,
in
addition
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
29
U
­
to
other
issues,
are
still
at
a
level
of
tentative
agreement,
and
not
to
characterize
all
of
the
1
EDSTAC's
deliberations
as
firmly
agreed
upon.
2
3
In
response
to
one
commenter's
request
that
the
EDSTAC
look
at
the
international
context
and
4
regulatory
practices
in
the
European
Union
as
it
proceeds
in
its
deliberations,
Dr.
Goldman
5
indicated
this
was
an
issue
for
the
STWG
to
consider.
However,
she
cautioned
that
some
testing
6
data
used
by
European
authorities
might
not
be
available
for
the
EDSTAC's
screening
and
testing
7
program
due
to
confidential
business
information
constraints
and
to
differences
in
evaluation
8
methods.
9
10
With
respect
to
international
harmonization
and
coordination
issues,
Dr.
Goldman
reminded
the
11
Committee
and
members
of
the
public,
that
EPA
and
the
EDSTAC
have
begun
to
cooperate
with
12
other
international
organizations
on
endocrine
disruption
issues.
Specifically,
interaction
is
13
currently
underway
with
the
OECD,
with
one
of
EPA's
specific
goals
being
the
structuring
of
the
14
Committee's
work
to
make
its
efforts
available
to
the
OECD,
and
vice
versa.
15
16
She
also
highlighted
recent
and
ongoing
international
coordination
efforts
with
the
Summit
of
17
Eight,
where
an
environmental
agenda
was
put
together
for
protecting
children
on
an
international
18
level,
including
the
consideration
of
endocrine
disruption.
In
addition,
the
United
States
is
19
involved
in
the
International
Program
for
Chemical
Safety,
an
international
effort
that
will
involve
20
the
coordination
and
collaboration
of
research
on
matters
of
chemical
safety
and
endocrine
21
disruption.
22
23
With
respect
to
comments
regarding
the
Committee's
tentative
definition
of
an
"
endocrine
24
disruptor"
and
its
decision
to
focus
its
efforts
on
a
defined
set
of
endpoints
related
to
estrogen­,
25
androgen­,
and
thyroid­
related
effects,
Dr.
Goldman
agreed
with
one
commenter
that
the
26
Committee
should
take
care
to
recognize
that
these
hormones
did
not
constitute
the
full
extent
of
27
potential
endocrine
disruption.
In
addition,
she
noted
that,
while
the
EDSTAC
will
focus
on
the
28
three
specified
hormonal
systems,
the
Committee
plans
to
develop
broader
guiding
principles
for
a
29
broader
application
of
the
screening
and
testing
program
to
consider
endocrine
disruptors
in
30
general.
31
32
With
respect
to
the
issue
of
screening
and
testing
mixtures,
one
EDSTAC
member
noted
that
the
33
issue
is
being
taken
very
seriously,
and
is
presently
being
addressed
by
the
PSWG.
Although
a
34
specific
formula
for
addressing
mixtures
in
the
sorting,
priority
setting,
screening,
and
testing
35
processes
has
not
yet
been
developed,
it
was
indicated
that
more
information
would
likely
be
36
available
on
the
issue
in
upcoming
plenary
meetings.
37
38
Several
Committee
members
expressed
their
appreciation
of
the
public
comments,
noting
in
39
particular
the
scientific
and
policy­
related
sophistication
of
the
remarks.
Committee
members,
in
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
30
U
­
response
to
some
of
the
public
remarks,
noted
their
recognition
of
the
multiple
and
sensitive
issues
1
tied
to
communication,
and
reiterated
their
intention
to
give
this
aspect
of
the
screening
and
testing
2
program
great
attention.
3
4
VI.
October
7­
8,
1997
 
New
York,
New
York
5
6
7
Following
the
Committee's
deliberations
on
the
first
day
of
the
plenary
meeting,
members
of
the
8
public
were
invited
to
offer
comments
related
to
the
scope
and
charge
of
the
EDSTAC.
The
9
public
comment
session
lasted
over
two
hours,
and
allowed
time
for
approximately
twenty­
five
10
individuals
to
provide
comment
to
Committee
members,
nearly
all
of
whom
were
present.
Written
11
comments
submitted
by
members
of
the
public
may
be
found
in
the
public
docket
(
Docket
12
Number­­
OPPTS­
42189;
phone
202­
260­
7099).
This
meeting
summary
attempts
to
capture
the
13
essence
of
the
comments
made
by
members
of
the
public
to
the
EDSTAC
during
the
public
14
comment
session.
15
16
Frank
Stoppenbach,
a
resident
of
the
Hudson
Valley,
noted
that
he
was
concerned
about
the
17
presence
of
PCBs
in
his
community
and
about
the
impacts
on
small
communities
of
chemical
18
exposures.
He
encouraged
the
consideration
of
fetal
and
developmental
exposures
in
the
19
EDSTAC
screening
and
testing
program.
Furthermore,
Mr.
Stoppenbach
encouraged
that
data
20
regarding
endocrine
disruptors
generated
by
the
screening
and
testing
program
be
made
as
publicly
21
accessible
as
possible.
He
expressed
concern,
as
well,
about
the
use
of
SLAP
suits
in
his
22
community.
23
24
Greg
Koontz,
director
of
regulatory
affairs
for
the
Chemical
Producers
and
Distributors
25
Association,
described
benefits
of
the
products
supported
by
his
organization.
He
encouraged
26
support
for
efficient,
cost
effective
responses
to
environmental
problems
commensurate
with
the
27
problems
they
are
meant
to
address.
He
noted
concern
about
the
impact
of
regulatory
actions
on
28
small
businesses,
as
well
as
cost
impacts
from
such
regulation.
Mr.
Koontz
questioned
the
scope
29
of
the
endocrine
disruption
issue,
as
well
as
the
accuracy
and
ability
of
assays
to
detect
designated
30
endpoints.
He
encouraged
the
Committee
to
clarify
the
definitional
and
utilization
issues
of
false
31
positives
and
negatives,
and
urged
a
balanced
approach
to
risk
assessment.
32
33
Ralph
Magin,
a
research
and
development
scientist
with
the
Albermarle
Corporation,
expressed
his
34
support
for
toxicity
testing
requirements
that
afford
increased
public
health
protection.
He
noted
35
the
broad
array
of
assays
under
consideration,
and
encouraged
the
Committee
to
be
pragmatic
in
36
its
selection
process.
He
also
noted
that
toxicity
screening
and
testing
is
a
decision­
oriented
37
process
and
should
be
characterized
by
relevant
and
reliable
endpoints
and
methods,
upon
which
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
31
U
­
clear
interpretations
of
results
can
be
made.
Mr.
Magin
stated
that
those
who
review
and
interpret
1
the
testing
results
will
not
likely
be
research
scientists,
and
that
opportunities
to
clarify
ambiguities
2
will
not
be
forthcoming
once
the
EDSTAC
process
is
concluded.
He
questioned
the
present
3
potential
scope
of
screens
under
consideration
by
the
STWG,
and
encouraged
the
group
to
be
4
pragmatic
and
to
use
validated
assays
in
the
screening
and
testing
tiers
to
ensure,
among
other
5
things,
that
resources
are
not
expended
to
study
chemicals
of
little
concern.
6
7
Laura
Whatley,
with
American
Cyanamid
Company,
recognized
the
need
to
know
about
endocrine
8
disrupting
chemicals,
and
recommended
the
development
of
a
deliberate,
practical
approach
to
do
9
so,
in
order
to
save
time
and
resources.
She
furthermore
encouraged
the
Committee
to
base
its
10
recommendations
on
sound,
verifiable
science
so
that
future
policy
would
be
based
on
that
sound
11
science.
12
13
Geri
Barish,
president
of
the
Long
Island
Breast
Cancer
Coalition
and
a
member
of
One
in
Nine,
a
14
breast
cancer
support
and
advocacy
group,
noted
concern
about
the
growing
chemical
use
of
15
developing
countries
and
the
breast
cancer
rates
in
these
countries.
She
offered
DDT
use
in
India
16
as
an
example
of
this,
and
noted
the
lack
of
public
access
to
politicians,
scientists,
and
education
17
regardless
of
culture.
18
19
Ed
Sabo,
New
York
coordinator
for
the
Chlorine
Chemistry
Council,
stated
his
support
for
the
20
EDSTAC
process,
noting
that
the
endocrine
disruptor
issue
is
a
complex
subject
with
limited
21
scientific
information
on
which
to
base
public
policy.
He
commented
that
the
goal
of
achieving
22
consensus
on
a
screening
and
testing
program
requires
staying
focused,
and
urged
the
Committee
23
not
to
lose
focus
by
wanting
to
develop
a
list
of
endocrine
disruptors.
He
indicated
that
such
24
development
was
not
a
part
of
the
EDSTAC's
mission.
Mr.
Sabo
expressed
his
feeling
that
25
chemicals
entering
the
screening
and
testing
program
through
the
nominations
process
should
26
undergo
screening
through
the
same
criteria
as
all
other
chemicals.
Finally
he
stated
that
diligence
27
will
ensure
that
laws
and
regulations
will
be
based
on
sound
science.
28
29
Linda
Roberts,
of
Chevron
Research
and
Technology
Company,
commented
that
in
utero
tests
30
should
be
included
in
the
screening
and
testing
program
as
definitive
testing.
In
addition,
she
31
noted
that
definitive
testing
should
more
thoroughly
characterize
toxicity,
and
that
in
utero
assays
32
would
be
a
benefit
in
doing
this.
Ms.
Roberts
indicated
her
preference
that
laboratories
expend
33
resources
on
such
testing
rather
than
on
screens.
34
35
Mary
Dauden,
with
the
Coalition
Organized
to
Protect
the
Environment,
stated
that
the
first
and
36
foremost
role
of
government
is
the
protection
and
quality
of
human
health.
She
expressed
support
37
for
the
use
of
in
utero
developmental
assays
examine
responses
in
populations
at
greatest
risk,
38
from
pre­
natal
fetuses
to
infants
to
the
elderly.
She
said
that
fetuses
are
exposed
to
many
39
chemicals
which
can
cause
cancer
and
mental
disabilities.
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
32
U
­
Walter
Schroeder,
of
the
Nassau/
Suffolk
Landscape
Gardeners
Association,
Inc.,
on
Long
Island,
1
said
that
the
EDSTAC
was
doing
a
great
job
for
the
most
part.
He
noted
awareness
of
breast
2
cancer
incidences
in
his
community,
but
indicated
that
the
determination
of
pesticides
as
causative
3
factors
in
breast
cancer
is
not
clear
cut.
Mr.
Schroeder
cited
the
retraction
of
the
Tulane
study
and
4
Steve
Safe's
article
as
examples
of
the
continuing
debate
over
the
effects
of
endocrine
disruptors,
5
and
encouraged
the
Committee
to
act
prudently.
6
7
Diana
Hinchcliff,
executive
director
of
the
New
York
State
Chemical
Alliance,
indicated
her
8
support
of
the
EDSTAC
process
and
her
hope
that
the
Committee's
goal
will
be
accomplished
in
9
an
objective
manner.
Ms.
Hinchcliff
cited
the
public's
likely
misinterpretation
of
lists
and
past
10
examples
of
misuse
or
misunderstanding
of
lists
as
reasons
to
avoid
developing
lists
of
chemicals
11
or
to
deliberate
at
length
on
the
use
of
chemicals.
Rather,
she
suggested
the
Committee
create
an
12
endocrine
disruptor
screening
and
testing
program
based
on
the
best
available
science
to
determine
13
which
chemicals
have
endocrine
disruption
potential,
and
to
do
so
carefully,
expeditiously,
and
14
judiciously,
bearing
in
mind
issues
related
to
implementation.
15
16
Hamdy
Balba,
with
Uniroyal
Chemical
Company,
referenced
the
adverse
effects
of
many
endocrine
17
disruptors
in
urging
the
Committee
to
use
an
analytical
and
methodological
approach
in
creating
its
18
screening
and
testing
program.
He
suggested
the
establishment
of
a
database
for
all
documented
19
and
suspected
industrial
and
natural
chemicals
with
endocrinological
properties.
Mr.
Balba
20
encouraged
the
development
of
a
reliable
assay
for
confirming
endocrinological
activities
to
assist
21
the
regulatory
agencies
in
prioritizing
chemicals
for
screening
and
testing
and
protecting
public
22
health
and
the
environment.
Finally,
he
advised
the
Committee
to
be
aware
of
possible
23
antagonistic,
synergistic
effects
of
chemicals.
24
25
Joe
Gregella,
executive
director
of
the
Long
Island
Farm
Bureau,
noted
that
farmers
are
extremely
26
concerned,
as
are
commercial
applicators,
of
any
potential
impacts
of
pesticides.
He
stated
that
he
27
hoped
the
EDSTAC
could
help
instill
public
confidence
in
the
government
by
reintroducing
sound
28
science
to
policy.
Mr.
Gregella
urged
that
science
dictate
the
decisions
made
by
the
EDSTAC.
He
29
acknowledged
the
emotional
aspects
of
the
debate
over
the
endocrine
disruption
issue
and
urged
30
for
the
development
of
studies
that
can
be
verified
and
validated
by
peer
reviews
and
31
reproducibility.
Furthermore,
he
noted
that
public
policy
needs
to
balance
many
variables,
32
including
economic
and
social
concerns,
but
he
hoped
it
would
do
so
guided
by
sound
science.
33
34
Jim
Lamb,
a
toxicologist
with
Jelnecks,
Schwartz,
and
Connelly,
commented
that
the
design
of
the
35
entire
system
needs
to
be
built
with
primary
objective
of
being
able
to
do
a
risk
assessment
on
36
adverse
effects
on
the
endocrine
system,
noting
his
belief
that
the
"
adverse"
qualifier
was
critical.
37
Mr.
Lamb
also
stated
that
in
utero
testing
is
critical
to
the
endocrine
disruption
argument,
but
38
urged
that
it
be
conducted
in
the
testing
tier,
not
in
the
screening
tier.
He
stated
his
support
for
a
39
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
33
U
­
bypass
option
for
chemicals
to
go
straight
from
prioritization
to
testing,
contingent
upon
the
design
1
of
a
comprehensive
testing
tier.
2
3
Davis
Baltz,
with
Commonweal,
asked
whether
degradation
products
and
metabolites
be
captured
4
by
the
assay
battery
of
the
screening
tier
of
the
screening
and
testing
program,
and
expressed
5
concern
regarding
the
perceived
difficulty
of
moving
a
chemical
out
of
the
"
Hold"
box.
He
6
questioned
the
ability
of
the
Committee
to
focus
testing
on
the
hypothalamus­
pituitary­
gonadol
7
(
HPG)
axis,
given
the
rapidly
emerging
and
developing
state
of
the
science
related
to
endocrine
8
disruption.
Regarding
the
screening
battery,
Mr.
Baltz
commented
that
the
battery
needed
to
9
maintain
flexibility
to
address
issues
not
anticipated
by
the
Committee.
He
also
noted
his
belief
10
that
the
addition
of
an
invertebrate
assay
to
the
screening
and
testing
program
was
critical
to
11
support
EPA's
goal
of
protecting
the
environment.
Mr.
Baltz
noted
that
the
issue
of
how
to
12
address
commonly
found
mixtures
had
not
yet
been
agreed
upon
by
the
EDSTAC,
and
indicated
13
his
concern
about
the
chemical
body
burden
issue.
He
asked
how
new
chemicals
would
be
handled
14
by
the
screening
and
testing
program,
urging
that
they
be
handled
more
stringently
and
that
they
be
15
screened
and
tested
right
away.
Commenting
on
the
nominations
process,
Mr.
Baltz
indicated
his
16
support
for
the
concept
of
giving
the
public
a
process
to
offer
input
into
the
screening
and
testing
17
program,
and
noted
his
belief
that
a
separate
set
of
criteria
should
be
established
to
gauge
the
18
merits
of
the
chemical
nominations.
19
20
Marian
Feinberg,
of
the
South
Bronx
Clean
Air
Coalition,
referenced
concern
regarding
childhood
21
cancer
incidences
and
prenatal
sensitivity
in
requesting
that
screening
and
testing
tiers
agreed
upon
22
by
the
Committee
capture
the
broadest
possible
mechanisms.
Ms.
Feinberg
asserted
that
inner­
city
23
communities
are
being
heavily
impacted
by
endocrine
disruptor­
related
illnesses,
and
urged
that
the
24
roots
of
transmission
(
e.
g.,
ingestion,
inhalation)
of
such
diseases
be
examined.
She
also
25
commented
on
an
alleged
lack
of
union
representation
on
the
Committee
and
in
the
audience,
as
26
well
as
a
lack
of
representation
by
impacted
community
representatives.
She
encouraged
the
27
Committee
and
the
COWG
to
develop
publicly
accessible
and
intelligible
language
in
presenting
28
the
EDSTAC's
deliberations
and
recommendations.
29
30
Jim
Moore,
executive
director
of
the
New
York
Coalition
for
Alternatives
to
Pesticides,
31
encouraged
the
EDSTAC
to
adopt
the
precautionary
principle.
He
indicated
his
desire
for
the
32
onus
to
be
on
manufacturers
to
prove
the
safety
of
chemicals,
or
to
offer
compelling
reasons
for
33
bringing
products
to
market
that
transcend
doubt.
He
cited
DES,
DDT,
and
thalidomide
as
34
examples
of
science
policy
failures
that
did
not
determine
safety
prior
to
release,
and
noted
that
35
science
should
not
dominate
public
policy
but
that
it
should
be
a
part
of
policy
making.
He
36
commented
that
the
Committee's
definition
of
37
"
adverse"
effects
is
too
narrow
given
the
lack
of
knowledge
in
the
field,
and
stated
that
the
38
EDSTAC's
top
priority
should
be
a
true
understanding
of
the
true
mechanisms
of
endocrine
39
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
34
U
­
disruption.
Mr.
Moore
stated
his
desire
for
a
greater
emphasis
on
research
into
endocrine
1
disruption
issues.
2
3
Christian
Klossner,
with
the
New
York
Public
Interest
Research
Group,
noted
his
concern
4
regarding
the
increasing
use
of
pesticides
in
this
country
and
their
effects
on
endocrine
systems.
5
With
respect
to
priority
setting,
he
stated
his
belief
that
the
prioritization
criteria
should
be:
6
chemicals
that
are
implicated
by
current
toxicological
or
endocrinological
data;
chemicals
that
7
bioaccumulate
or
are
persistent;
likelihood
of
exposure;
consumer
products;
and
child
exposure.
8
He
indicated
that
he
felt
pesticides
fit
all
of
these
categories
and
should,
therefore,
be
of
priority
9
for
the
endocrine
disruptor
program.
Regarding
screening
and
testing,
he
requested
that
the
10
Committee
include
assays
and
tests
designed
to
encompass
developmental
and
fetal
life
stages.
11
Mr.
Klossner
indicated
his
support
for
the
EDSTAC's
careful
consideration
of
conventional
risk
12
assessment,
and
encouraged
the
Committee
to
examine
chemical
synergies.
He
asked
that
the
13
onus
of
proof
of
chemical
safety
lie
with
the
chemicals'
producers.
14
15
Wanda
Cohen
asked
the
Committee
about
timeframes,
required
resources,
and
risk
management
16
issues
related
to
the
screening
and
testing
program.
In
addition,
she
questioned
how
new
17
information
would
be
integrated
into
the
process,
and
whether
endocrine
effects
beyond
estrogen,
18
androgen,
and
thyroid
would
be
addressed.
She
also
asked
about
the
development
of
the
chemical
19
nominations
process
and
public
accessibility
of
information
coming
out
of
the
screening
and
testing
20
program.
Ms.
Cohen
commented
that
she
was
unsure
how
multisystem
effects
such
as
21
multineurological
effects,
degradation
products,
intermediates,
and
metabolites
would
be
22
accounted
for
in
the
priority
setting
criteria.
She
asked
about
additional
legislative
authorities
that
23
might
be
applicable
to
the
EDSTAC's
work,
such
as
the
Clean
Air
Act.
Ms.
Cohen
questioned
24
how
the
process
of
updating
databases
of
information
would
take
place
and
how
the
databases
25
would
be
kept
useful
and
accessible
to
the
public.
She
also
commented
that
exposure
is
not
26
necessarily
equivalent
to
effect.
27
28
Bill
Sanock,
with
the
Cornell
Cooperative
Extension,
commented
that
he
works
closely
with
breast
29
cancer
groups,
as
well
as
pesticide
users,
and
noted
that
those
who
use
pesticides
have
great
30
concern
about
their
safety
and
support
the
Committee's
work
to
provide
more
information.
He
31
stated
the
need
to
use
good,
practical
science
in
the
process,
and
asked
how
the
EDSTAC
would
32
communicate
results
and
recommendations
of
the
screening
and
testing
program
to
the
public.
He
33
encouraged
the
Committee
to
approach
risk
assessment
and
exposure
issues
using
common
sense
34
and
practical
research.
35
36
Judith
Helfund,
with
the
DES
Cancer
Network,
stated
that
Long
Island
has
the
highest
rate
of
37
DES
exposure
in
the
country,
and
indicated
the
region's
required
attention
on
related
issues.
She
38
offered
a
very
detailed
account
of
personal
physical
problems
due
to
DES
exposure
as
a
youth,
and
39
urged
the
Committee
to
adopt
the
precautionary
principle
to
avoid
future
incidences
similar
to
her
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
35
U
­
own.
In
addition,
Ms.
Helfund
asserted
that
transgenerational
impact
of
DES
exposure
and
1
endocrine
disruption
was
a
reality,
and
should
be
considered
as
the
effects
would
be
manifested
2
long
after
exposure.
She
stated
that
information
is
needed
to
make
informed
decisions
and
to
give
3
consent
to
exposure.
4
5
Minna
Barrett,
professor
of
environmental
psychology
at
the
State
University
of
New
York,
stated
6
that
her
mother
had
died
of
thyroid
and
breast
cancer.
She
encouraged
the
Committee
to
include
7
lifespan
scrutiny
and
mechanisms
(
especially
with
respect
to
fetal
exposure),
synergistic
chemical
8
reactions
(
including
low
dose
exposure
and
inert
synergies)
health
implications
for
endocrine
9
sensitive
cancers
and
other
diseases,
and
synchronicity,
bioaccumulative,
and
persistent
10
considerations
in
the
endocrine
disruptor
screening
and
testing
program.
She
encouraged
the
11
EDSTAC
to
include
an
analysis
of
developmental
endpoints
in
the
screening
and
testing
tiers
to
12
catch
effects
of
chemicals
to
which
neonatal
children
are
often
exposed.
Ms.
Barrett
stated
her
13
perception
that
many
of
the
endocrine
disruptor
issues
had
been
perpetuated
by
the
economy,
and
14
indicated
her
sense
that
industry
is
suspect.
She
commented
that
low
risk
allowances
are
not
15
acceptable,
and
that
only
no
risk
situations
would
be
acceptable.
16
17
Response
to
Public
Comment
18
19
Lynn
Goldman
thanked
the
public
for
their
comments,
noting
that
they
reflected
the
serious
20
challenge
to
the
Committee
and
EPA
to
protect
human
health
and
the
environment,
and
to
do
so
in
21
ways
that
are
effective,
cost
effective,
and
practical.
She
stated
that
she
expected
a
mutually
22
beneficial
outcome
for
both
economic
development
and
environmental
protection
if
the
EDSTAC
23
was
successful
in
reaching
its
goals.
With
respect
to
public
access
to
information,
Dr.
Goldman
24
commented
that
efforts
have
been
made
to
develop
an
endocrine
disruptor
Web
site
and
to
keep
it
25
updated.
26
27
With
respect
to
the
retraction
of
the
Tulane
study,
Dr.
Goldman
indicated
her
belief
that
the
28
portrayal
of
the
situation
surrounding
the
retraction
had
been
inaccurate.
She
noted
that,
while
29
that
particular
study
had
been
withdrawn
because
it
cannot
be
reproduced,
other
studies
exist
in
30
the
literature
supporting
the
synergy
of
endocrine
disrupting
chemicals.
Furthermore,
she
31
commented
that
the
legislation
that
started
the
EDSTAC
process
was
introduced
several
months
32
prior
to
the
publication
of
the
Tulane
study.
33
34
Dr.
Goldman
commented
on
the
obvious
thoughtful
preparation
by
commenters,
and
recognized
35
their
efforts.
She
reiterated
that
the
EDSTAC
process
has
been
a
science­
based
process
from
the
36
very
outset,
and
that
it
would
continue
to
be
so.
In
response
to
some
comments,
she
noted
that
37
EPA
is
engaged
in
activities
to
address
the
rising
rates
of
childhood
cancer.
38
39
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
36
U
­
One
Committee
member
responded
to
Mr.
Baltz'
concern
with
respect
to
the
handling
of
mixtures
1
by
noting
that
the
subject
was
being
addressed
by
the
PSWG
and
that
the
issues
of
identification,
2
prioritization,
and
technical
aspects
of
how
to
screen
and
test
mixtures
still
needed
to
be
discussed.
3
4
Dr.
Goldman,
in
response
to
public
comments
about
the
inclusion
of
an
in
utero
assay
in
the
5
screening
and
testing
program,
commented
that
there
is
no
debate
in
the
Committee
about
whether
6
it
should
be
part
of
the
process,
but
rather
where
in
the
process
it
should
be
included
(
i.
e.,
T1S
or
7
T2T).
With
respect
to
prenatal
detection
of
childhood
cancer,
she
commented
that
while
the
8
concern
is
valid,
it
is
not
within
the
scope
of
the
EDSTAC
but
that
other
EPA
offices
are
9
addressing
the
issue.
Regarding
public
comments
about
missing
or
inappropriate
Committee
10
representation,
she
stated
that
not
all
EDSTAC
members
were
present
at
the
New
York
meeting,
11
but
that
the
EDSTAC
did
have
union
representation
and
environmental
justice
representation,
and
12
that
membership
on
the
Committee
from
industry
was
important
and
vital
for
consensus.
Dr.
13
Goldman
noted
that
environmental
protection
has
often
been
done
in
an
adversarial
manner
with
14
industry,
but
that
the
EDSTAC
represents
a
coming
together
around
a
common
interest
and
in
15
good
will.
16
17
Some
members
of
the
public
commented
critically
about
the
location
of
the
New
York
plenary
as
18
being
inconvenient
and
inaccessible
by
mass
transportation.
Dr.
Goldman
acknowledged
these
19
criticisms
and
offered
an
explanation
of
why
the
meeting
had
to
be
held
in
the
determined
location.
20
However,
she
indicated
that
future
meetings
would
try
to
address
the
public's
concerns.
21
22
Committee
members
summarized
for
the
public
some
of
the
COWG's
efforts,
and
the
work
23
group's
goals
of
making
all
Committee
communication
palatable,
understandable,
and
clear
for
the
24
public.
It
was
also
noted
that
a
survey
had
been
widely
distributed
by
EPA
on
communications
25
issues
related
to
the
EDSTAC,
and
that
copies
of
the
survey
were
available
for
those
who
desired
26
to
complete
the
forms.
Members
of
the
COWG
expressed
interest
in
hearing
from
the
public
how
27
to
efficiently
and
effectively
communicate
with
and
to
the
public,
and
encourage
members
of
the
28
public
to
assist
COWG
by
offering
suggestions.
29
30
David
Parkinson,
a
Committee
member,
referenced
his
years
of
working
with
steelworkers
and
31
other
union
groups,
noting
that
he,
indeed,
did
lend
a
union
perspective
to
the
EDSTAC,
counter
32
to
public
statements
to
the
contrary.
He
noted
his
current
affiliation
with
occupational
and
33
environmental
health
clinics
in
the
New
York
area,
funded
partly
by
state,
and
his
desire
to
work
34
with
environmental
groups
in
the
area
to
design
something
they
can
do
for
themselves
to
test
and
35
monitor
health
concerns.
In
addition,
Mr.
Parkinson
indicated
the
willingness
of
the
clinics
to
36
assist
patients
who
are
sick
and
need
evaluation
given
an
environmental
problem
even
without
37
health
insurance.
38
39
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
37
U
­
One
Committee
member
thanked
the
public
for
their
comments,
and
acknowledged
her
1
appreciation
as
a
scientist
for
the
calls
from
observers
for
policy
based
on
sound
science.
In
2
addition,
she
acknowledged
the
need
to
balance
science
with
other
factors
to
create
sound
public
3
policy.
She
noted
that
repeatability
and
peer
review
are
of
great
importance
to
the
EDSTAC,
and
4
her
appreciation
for
the
public's
encouragement
of
the
adoption
of
the
precautionary
principle.
5
6
EDSTAC
members
indicated
their
support
for
a
public
nominations
process,
in
recognition
of
the
7
importance
that
the
public
have
a
continuing
role
in
the
endocrine
disruptor
screening
and
testing
8
process.
Members
of
the
Committee
noted
their
intention
to
scrutinize
similar
programs
already
in
9
place
at
other
organizations
or
agencies
in
the
design
of
the
EDSTAC's
process.
10
11
Industry
representatives
on
the
EDSTAC
encouraged
the
public
to
overcome
their
mistrust
of
the
12
sector
by
focusing
on
the
Committee's
product,
and
by
remembering
that
many
scientists
working
13
with
industry
have
spent
a
great
deal
of
time
training
to
learn
about
the
issues
without
malintent.
It
14
was
stated
that
public
input
is
very
important
to
the
EDSTAC
process,
and
that
industry
15
representation
is
present
because
industry
has
a
responsibility,
as
a
stakeholder,
to
be
involved
in
16
the
consensus­
building
process.
17
18
19
20
VII.
December
2­
3,
1997
 
Orlando,
Florida
21
22
23
Ellen
Bregg,
of
the
Coalition
to
Stop
Children's
Exposures
to
Pesticides,
urged
the
EPA
to
allow
24
independent
laboratories
to
perform
testing
in
addition
to
that
done
by
the
EPA.
She
expressed
25
concern
about
the
use
of
pesticides
in
urban
areas
and
the
effects
of
low
dose
exposures
to
26
malathion
and
pesticides.
Ms.
Bregg
said
she
would
like
to
see
more
educational
information
27
about
pesticides.
She
stated
she
wants
to
know
which
chemicals
will
be
addressed
by
the
28
screening
and
testing
program,
whether
chemical
effects
will
be
looked
at
individually
or
29
cumulatively,
and
whether
there
will
be
a
focus
on
the
effects
at
the
developmental
stage.
Ms.
30
Bregg
added
she
would
like
to
see
press
releases
providing
updates
on
the
Committee's
progress.
31
32
Greg
Koontz,
Regulatory
Affairs
Manager
for
the
Chemical
Producers
and
Distributors
33
Association,
emphasized
the
burden
of
the
screening
and
testing
program
needs
to
be
34
commensurate
with
the
gravity
of
the
adverse
effects
caused
by
endocrine
mediated
toxicity.
He
35
indicated
chemicals
with
health,
safety,
or
other
benefits
should
not
be
sacrificed
through
36
regulatory
overkill,
and
the
demands
of
an
endocrine
screening
and
testing
program
should
be
37
flexible
to
reflect
the
seriousness
of
the
threat
as
demonstrated
by
science.
Such
a
program,
Mr.
38
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
38
U
­
Koontz
stressed,
should
be
efficient
and
cost­
effective.
He
expressed
support
for
further
research
1
into
potential
endocrine
disruptor
effects
from
low
doses
but
opposes
a
test
program
including
low
2
dose
testing
unless
the
need
for
it
is
substantiated
by
good
science,
and
then
only
if
the
hypothesis
3
applies
generally
to
chemicals
and
not
just
exceptional
cases.
He
expressed
concern
with
the
4
testing
program
possibly
increasing
overall
risks
to
public
health
by
reducing
the
availability
of
5
certain
chemicals
or
by
scaring
the
public
into
not
using
particular
products
(
for
example,
6
mosquito­
control
chemicals,
the
use
of
which
aids
in
preventing
cases
of
viral
equine
encephalitis.)
7
Mr.
Koontz
suggested
using
a
nomination
approach,
whereby
chemicals
would
proceed
through
8
the
normal
priority
setting
process.
Mr.
Koontz
finished
by
saying
the
EPA,
if
needed,
should
ask
9
Congress
for
more
time
to
implement
a
sensible
approach.
10
11
Chuck
Elkins,
an
environmental
consultant
with
Jellinek,
Schwartz,
&
Connolly,
Inc.,
presented
12
five
ideas.
First,
the
success
of
EDSTAC
will
not
be
based
upon
how
many
chemicals
are
13
screened,
rather
on
whether
the
information
coming
out
of
the
program
is
useful.
Second,
while
14
chemicals
are
in
the
program
they
are
highly
vulnerable
as
a
full
data
set
is
not
available
until
the
15
chemical
in
question
gets
to
the
end
of
the
program.
Third,
each
stage
of
the
program
needs
to
be
16
clearly
defined
(
e.
g.,
what
is
screening).
Fourth,
only
EDSTAC
has
the
credibility
to
make
17
recommendations
and,
in
so
doing,
they
need
to
write
their
words
carefully,
work
on
principles,
18
and
avoid
using
lists.
Fifth,
too
many
chemicals
should
not
be
forced
into
the
program
at
the
19
beginning
as
the
system
will
break
down
if
chemicals
do
not
get
through
the
program
quickly.
20
21
John
Brennan,
a
high
school
biology
teacher
from
Orlando,
Florida,
discussed
his
concerns
about
22
the
influence
of
the
media
regarding
the
level
of
attention
the
issue
of
endocrine
disruption
23
receives.
He
commented
that
although
some
sensationalism
pushes
the
issue,
which
can
be
24
beneficial,
it
is
important
to
take
a
reasoned
approach
and
consider
all
sides.
As
an
example,
Mr.
25
Brennan
pointed
out
both
the
benefits
(
e.
g.
possible
safeguard
against
lime
disease)
and
detriments
26
(
e.
g.
health
risks
from
exposure)
of
applying
insecticide
to
a
child.
He
encouraged
the
Committee
27
to
keep
an
open,
scientific
mind
about
the
issue
of
endocrine
disruption
and
not
let
the
press
28
influence
its
decisions.
29
30
Nancy
Stephens,
Executive
Director
of
the
Florida
Manufacturing
and
Chemical
Council,
31
emphasized
the
need
for
the
Committee
to
balance
the
importance
of
chemical
use
with
the
safety
32
issues
associated
with
such
use.
She
requested
the
Committee
not
be
persuaded
by
political
and
33
emotional
agendas,
but
rather
address
the
issue
of
endocrine
disruption
on
a
scientific
basis.
Ms.
34
Stephens
suggested
the
Committee
provide
EPA
with
a
"
tool
box"
with
which
the
Agency
can
35
evaluate
chemicals.
36
37
Suzanne
Spencer,
representing
the
Northeast
Florida
Chapter
of
the
Sierra
Club,
stated
there
is
an
38
urgent
need
to
develop
a
screening
and
testing
program
to
detect
endocrine
disruption
in
humans,
39
fish,
and
wildlife.
She
brought
a
letter
from
Maurice
Coman,
Chair
to
the
Northeast
Florida
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
39
U
­
Group,
Sierra
Club,
discussing
the
link
between
dioxin
and
related
chemicals
to
cancer
and
1
reproductive,
immunological,
and
developmental
dysfunction.
Ms.
Spencer
also
brought
2
correspondence
from
the
Food
and
Drug
Administration
involving
consumer
exposure
to
dioxin
3
through
food
sources.
She
noted
the
need
to
do
significantly
more
research
concerning
the
health
4
effects
of
environmental
hazards
on
children,
and
added
the
threat
to
children
will
persist
until
this
5
happens.
6
7
Davis
Baltz,
of
Commonweal
and
member
of
the
EDSTAC
Communications
and
Outreach
Work
8
Group,
reminded
the
Committee
that
over
the
past
year
it
has
heard
from
a
broad
cross­
section
of
9
Americans
concerned
about
the
implications
of
endocrine
disruption,
including
the
following:
10
Sierra
Club;
Endometriosis
Association;
cancer
survivors;
learning
disability
networks;
consumers;
11
environmental
justice
representatives;
mothers;
pregnant
women;
farmers;
ranchers;
gardeners;
12
unions;
small
business;
environmental
consultants;
veterinarians;
the
League
of
Women
Voters;
13
and,
individual
citizens.
Mr.
Baltz
noted
a
common
theme
to
be
concern
for
children
and
added,
it
14
should
be
remembered,
that
as
adults,
we
impose
risks
on
our
children
without
their
consent.
He
15
reminded
the
Committee
of
its
agreement
regarding
the
definition
of
`
screening'
as
"
the
application
16
of
assays
to
detect
the
potential
for
endocrine
disrupting
properties
of
chemical
substances
and
17
mixtures."
Mr.
Baltz
emphasized
that,
for
the
promotion
of
children's
healthy
development,
the
18
EDSTAC
must
include
a
means
for
establishing
developmental
endpoints
for
low
doses
at
the
19
`
screening'
phase.
20
21
Betty
Mekdeci,
Director
of
the
Association
of
Birth
Defect
Children
Inc.
(
ABDC),
indicated
the
22
greatest
examples
of
how
exposure
to
herbicides
increases
the
likelihood
that
a
child
will
suffer
23
from
a
disability
or
birth
defect,
are
the
children
of
Vietnam
veterans.
She
spoke
of
an
analysis,
24
performed
by
ABDC
and
the
New
Jersey
Agent
Orange
Commission,
which
identified
disabilities
25
in
800
children
of
Vietnam
veterans
compared
to
400
children
of
non­
Vietnam
veterans
and
noted
26
further
evidence
suggested
the
effects
could
be
transgenerational.
Ms.
Mekdeci
said
the
types
of
27
problems
included;
chronic
skin
disorders,
benign
tumors/
cysts,
cancers,
emotional/
behavioral
28
problems,
chronic
fatigue,
tooth
problems,
and
others.
Ms.
Mekdeci
expressed
concern
with
the
29
lack
of
accurate
exposure
data
on
herbicides.
30
31
Andy
LaVigne,
Executive
Director
of
the
Florida
Fertilizer
and
Agrichemical
Association,
stated
32
extensive
testing
is
performed
on
agricultural
chemicals
and
pesticides
before
these
products
are
33
used.
He
said
no
scientist
will
give
a
determination
of
zero
risk
regarding
the
use
of
a
specific
34
chemical
because
there
are
so
many
variables
potentially
impacting
the
effects
related
to
use.
Mr.
35
LaVigne
said
claims
of
pesticides
not
being
well
tested
before
registration
and
release
for
public
36
use,
and
claims
they
are
not
specifically
tested
for
estrogenic
activity,
are
not
true.
He
noted
EPA
37
requires
each
pesticide
be
thoroughly
tested
for
its
potential
to
cause
adverse
effects,
including
38
hormone
disruption.
Mr.
LaVigne
requested
the
Committee
not
use
a
"
paparazzi
science"
39
approach
but
rather
develop
a
screening
program
based
on
the
best
available
scientific
data.
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
40
U
­
Pam
Williams,
of
the
Endo
Association,
indicated
that
dioxins
are
one
of
the
most
toxic
1
compounds
known
to
man
and
are
linked
to
endometriosis
and
other
health
problems.
Ms.
2
Williams
said
ninety
percent
of
human
dioxin
exposure
is
from
meat
and
dairy
products
because
3
the
animals
from
which
these
products
originate,
are
exposed
to
dioxins
via
pesticides,
herbicides,
4
plastics
incineration
(
PVC),
and
pulp
and
paper
production
processes.
Ms.
Williams
urged
the
5
Committee
to
consider
the
following
questions:
how
much
is
too
much;
can
the
current
levels
of
6
dioxins
already
present
be
reduced;
are
there
other
ways
to
do
what
we
are
doing;
and,
what
effect
7
will
these
changes
have
on
the
future
of
humanity?
8
9
Keith
Branly,
Vice
President
of
Research
and
Development
of
Micro
Flo,
a
small
pesticide
10
formulating
business,
stated
the
need
for
the
burdens
to
equal
the
benefits
which
result
from
11
pesticide
regulation.
He
noted
many
small
businesses
are
impacted
by
toxicity
testing
and
the
12
demands
of
a
screening
and
testing
program
need
to
be
viable
and
cost
effective.
Mr.
Branly
13
added
testing
and
screening
requirements
should
not
deprive
the
public
of
the
use
of
products
with
14
clear
health
benefits.
Regarding
the
issue
of
low­
dose
testing
raised
in
T2T,
he
said
he
feels
it
is
15
not
cost­
effective
and
is
premature
until
a
low­
dose
testing
hypothesis
is
accepted
by
the
scientific
16
community.
17
18
Katie
Holmes,
an
aquatic
toxicologist,
requested
the
Committee
verify
the
practicality,
not
just
the
19
theory
behind,
the
studies
it
recommends
be
used
to
analyze
potential
endocrine
disruptors.
Ms.
20
Holmes
encouraged
the
EDSTAC
to
consult
with
individuals
who
run
relevant
studies
dial,
and
are
21
aware
of
the
scientific
and
practical
issues
associated
with
the
tests.
She
expressed
particular
22
concern
regarding
the
mysid
life­
cycle,
fish
life­
cycle,
and
avian
reproduction
studies
being
23
considered
by
the
Committee.
24
25
Jeannie
Economos,
of
the
Farmworkers
of
Florida,
expressed
concern
about
farmworker
exposure
26
to
pesticides
and
related
health
effects.
She
stated
chemicals
are
being
screened
on
an
individual
27
basis
when,
in
fact,
people
are
exposed
to
all
kinds
of
chemicals
over
time
and
therefore,
the
28
cumulative
effects
associated
with
chemical
exposure
should
be
considered.
Ms.
Economos
29
commented
on
the
high
incidence
of
miscarriage
among
farmworkers
and
said
she
wants
to
see
30
studies
performed
on
this
particular
sector
in
order
to
evaluate
the
long­
term
effects
of
chemical
31
exposure.
32
33
Lukner
Millen,
speaking
on
behalf
of
farmworkers,
indicated
there
are
still
many
instances
where
34
pesticides
are
being
used
in
the
vicinity
of
workers.
He
told
a
story
of
a
female
farmworker
who
35
was
working
in
the
fields
when
she
broke
out
in
a
rash
all
over
her
body
and
was
unable
to
identify
36
the
pesticides
to
which
she
had
been
exposed.
Mr.
Millen
commented
that
exposure
problems
37
persist
and
he
wants
the
persons
responsible
for
exposing
farmworkers
to
pesticides
(
e.
g.,
spraying
38
the
fields
while
workers
are
present)
to
be
held
accountable
for
their
actions.
He
closed
by
saying
39
he
is
counting
on
the
Committee
to
act
to
change
this
situation.
40
EDSTAC
Final
Report
Chapter
Six
Appendices
August
1998
41
U
­
1
Joy
Cummings,
a
Florida
rancher
and
farmer,
explained
she
was
a
chemicals
sales
representative
2
until
she
realized
the
harmful
effects
associated
with
chemical
exposure.
After
moving
to
her
farm
3
she
found
her
community
was
contaminated.
Ms.
Cummings
remarked
that
people
are
eating
fish
4
contaminated
with
dioxins,
female
fish
are
taking
on
male
sexual
characteristics
(
as
are
some
5
female
humans
in
the
community),
and
there
is
a
high
rate
of
children
with
learning
disabilities
as
6
well
as
a
high
rate
of
leukemia.
She
stated
she
worked
on
a
dioxin
FACA
and
is
still
worried
7
about
the
dioxins
which
she
believes
are
probably
caused
from
chlorine.
Her
message
to
industry
8
was
to
stop
dumping
poisons
and
to
stop
using
chlorine.
9
10
Linda
Young,
Southeast
coordinator
of
the
Clean
Water
Network,
stated
she
was
also
speaking
on
11
behalf
of
Margaret
Williams
of
the
Citizens
for
Toxic
Exposure,
a
group
located
in
southern
12
Florida
which
evolved
due
to
concern
about
citizens'
health.
She
emphasized
the
public
health
13
must
not
go
unprotected
because
of
scientific
uncertainties
and
encouraged
the
application
of
the
14
precautionary
principle.
Ms.
Young
also
noted
the
need
to
assess
the
effects
of
chemicals
on
the
15
most
vulnerable
populations,
including
the
fetus.
16
17
Lisa
Doig,
of
the
Farmworkers
Association
of
Florida,
Inc.,
stated
she
strongly
suspects
a
direct
18
relationship
between
exposure
to
pesticides
and
worker
outbreaks
of
rashes,
welts,
bumps
on
the
19
body,
birth
defects,
and
miscarriages.
She
commented
on
cases
where
women
farmworkers
20
experienced
rashes,
believed
to
be
caused
by
chemical
exposure,
and
then
subsequently
had
21
miscarriages.
Ms.
Doig
suggested
an
in­
depth
study
into
the
cause
and
effect
relationship
of
22
pesticides
and
public
health
threats,
and
making
available
to
the
public
the
results.
23
24
Estaban
Caro
Jr.,
a
farmworker,
stated
that
as
a
common
worker
he
is
exposed
to
a
number
of
25
different
pesticides
and
suffers
from
itching
and
swelling
skin.
He
said
the
farmworkers
are
aware
26
of
their
exposure
to
such
chemicals
on
a
daily
basis.
With
the
help
of
organizations
like
the
27
Farmworkers
Association,
Mr.
Caro
explained,
workers
have
been
better
able
to
get
needed
28
information
about
how
to
protect
themselves
from
pesticide
exposure
and
how
to
get
treatment
29
once
exposure
occurs.
He
expressed
concern
about
how
the
exposure
will
affect
him
and
other
30
workers
in
the
long
run
and
asked
the
Committee
to
do
whatever
possible
to
make
the
31
farmworkers
lives
a
little
better.
32
Response
to
Public
Comment
33
34
Dr.
Lynn
Goldman
thanked
the
public
for
their
comments
and
reminded
them
of
the
Committee's
35
scope.
She
expressed
concern
about
the
fern
industry
and
indicated
more
attention
needs
to
be
36
focused
in
this
area.
Dr.
Goldman
noted
the
need
for
the
Committee
to
understand
the
extent
of
37
exposure
possible
and
to
understand
that
laws
and
regulations
do
not
always
work
as
they
should.
38
39
