United
States
Science
Advisory
EPA­
SAB­
EC­
99­
013
Environmental
Board
(
1400)
July
1999
Protection
Agency
Washington
DC
www.
epa.
gov/
sab
REVIEW
OF
THE
EPA'S
PROPOSED
ENVIRONMENTAL
ENDOCRINE
DISRUPTOR
SCREENING
PROGRAM
REVIEW
OF
THE
ENDOCRINE
DISRUPTOR
SCREENING
PROGRAM
BY
A
JOINT
SUBCOMMITTEE
OF
THE
SCIENCE
ADVISORY
BOARD
AND
SCIENTIFIC
ADVISORY
PANEL
July
28,
1999
EPA­
SAB­
EC­
99­
013
Honorable
Carol
M.
Browner
Administrator
U.
S.
Environmental
Protection
Agency
401
M
Street,
S.
W.
Washington,
DC
20460
Subject:
Review
of
EPA's
Proposed
Environmental
Endocrine
Disruptor
Screening
Program.

Dear
Ms.
Browner:

In
1996,
the
passage
of
the
Food
Quality
Protection
Act
(
FQPA)
and
amendments
to
the
Safe
Drinking
Water
Act
(
SDWA)
required
EPA
to
develop
a
screening
and
testing
strategy
for
environmental
endocrine
disruptors.
The
Agency
established
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC)
to
provide
advice
on
the
screening
and
testing
of
pesticides
and
other
chemicals
for
their
potential
to
disrupt
the
endocrine
system.
The
EPA
subsequently
asked
the
Science
Advisory
Board
(
SAB)
and
the
FIFRA
Scientific
Advisory
Panel
(
SAP)
to
form
a
Joint
Subcommittee
to
review
a
set
of
scientific
issues
being
considered
by
the
Agency
concerning
the
development
of
the
Agency's
endocrine
disruptor
screening
and
testing
program
as
required
by
the
legislation
noted
above.
A
Joint
Subcommittee
(
the
Joint
Environmental
Disruptor
Screening
Program
(
EDSP)
review
Subcommittee)
met
on
March
30­
April
1,
1999,
in
Arlington
VA,
and
produced
this
report.

The
Charge
was
broad
and
complex,
posing
18
major
questions
within
four
broad
areas:
a)
scope
of
the
program;
b)
priority­
setting;
c)
the
high
throughput
pre­
screening
approach;
and
d)
the
proposed
endocrine
disruptor
screening
program
(
the
complete
Charge
is
provided
in
section
2.2
of
this
report).

At
the
outset,
we
wish
to
note
that
although
our
review
identified
several
areas
of
concern,
and
the
EDSP
has
provided
recommendations
to
improve
EPA's
planned
program,
we
wish
to
congratulate
the
Agency
for
dealing
effectively
with
an
extraordinarily
complex
set
of
issues,
many
of
which
are
on
the
cutting
edge
of
the
relevant
science.
The
EDSP's
detailed
2
response
to
each
element
of
the
Charge
can
be
found
in
section
3
of
the
report,
and
our
major
issues
and
recommendations
are
summarized
below:

a)
Evaluating
the
Program:
We
find
no
provision
for
mid­
course
evaluation
or
optimization
of
the
process.
Although
an
approach
may
look
fine
on
paper
or
in
a
small
research
setting,
translating
it
into
a
volume­
screening
mode
may
be
quite
another
thing.
There
was
broad
support
among
the
Subcommittee
for
the
concept
that
the
Agency
should
convene
a
panel
of
independent
scientists
to
review
all
the
screening
data
for
50­
100
compounds,
with
an
eye
towards
revising
the
process
and
eliminating
those
methods
that
don't
work.

b)
Mixture
Issues:
The
Subcommittee
agreed
that
the
initial
focus
of
the
methods
development
effort
must
focus
necessarily
on
single
compounds
and
leave
the
question
of
testing
of
mixtures
until
accepted
single­
compound
methods
have
been
completed.

c)
Case
Studies:
The
Subcommittee
strongly
encourages
the
Agency
to
include
more
and
better­
detailed
case
studies
in
the
evolution
of
the
priority­
setting
scheme.
This
will
facilitate
a
realistic
test
of
the
plan,
checking
the
sensitivity
of
the
system
and
its
practicality
to
prioritize
properly
chemicals
for
further
testing.

d)
Sub­
population
Compartment:
The
question
of
the
need
for
a
separate
compartment
to
address
sub­
populations
(
e.
g.,
developing
children)
was
addressed
to
the
EDPS.
Our
conclusions
supported
the
use
of
sub­
populations
as
a
criterion
within
the
existing
compartments
already
identified,
but
not
as
a
separate
stand­
alone
compartment.

e)
Use
of
the
Integrated
Risk
Information
System
(
IRIS):
The
priority
testing
scheme
relies
on
the
use
of
several
databases
summarizing
the
environmental
fate
and
effects
of
chemicals.
Several
Members
of
the
Committee
expressed
concern
about
problems
with
the
validation
of
IRIS
and
other
databases.
Before
placing
heavy
reliance
on
these
computerized
systems,
users
need
to
be
aware
of
these
validation
problems
and
proceed
with
caution
before
incorporating
these
values
unilaterally.

f)
Exposure:
The
EDPS
believes
that
consideration
of
the
toxicological
implications
of
exposure
should
include
both
dose
and
timing
of
exposure,
particularly
with
respect
to
developmental
or
reproductive
events.
The
current
3
scheme
does
not
adequately
cover
the
time
aspect
of
exposure
and
this
needs
to
be
remedied
before
broad­
scale
application
of
the
approach.

g)
Use
of
Animals
and
Routes
of
Exposure:
We
are
concerned
about
the
large
number
of
animals
that
would
be
needed
by
the
EDSTAC
program.
The
Subcommittee
is
cognizant
of
the
essential
role
animals
play
in
tests
to
detect
endocrine
disruption,
and
aware
that
there
are
no
substitutes
for
tests
currently
available
for
the
Tier
2
tests.
This
fact
notwithstanding,
the
Agency
has
an
obligation
to
conserve
all
resources
in
developing
new
testing
protocols,
and
the
use
of
animals
in
such
tests
poses
both
ethical
and
practical
problems.
In
addition,
in
this
role
of
hazard
assessment
(
as
opposed
to
hazard
definition)
biologically
relevant
routes
of
exposure
are
indicated..
The
current
EPA
synthesis
of
the
EDSTAC
recommendations
is
inconsistent
on
the
matter
of
route
of
exposure,
but
animal
testing
should
be
restricted
to
biologically
relevant
routes
of
exposure.

h)
Need
for
an
Introductory
Statement:
The
previous
EDSTAC
meeting
suggested
that
the
final
document
needed,
as
a
introductory
section,
a
description
of
the
problem
or
the
scientific
or
health­
based
reason
for
the
EDSTAC
program.
The
EDPS
urges
the
EPA's
EDSTAC
team
to
include
a
description
of
both
the
health
and
ecological
problems
associated
with
exposure
to
the
endocrine
disruptors
and
to
show
how
the
these
findings
relate
to
the
program.

i)
Support
for
Decisions:
Decisions
about
which
assays
are
selected,
and
which
protocols
are
adopted
for
those
assays,
should
be
supported
with
data
that
are
generally
available.

j)
Exceptions:
Testing
strategies
will
always
have
exceptions.
Care
should
be
taken
to
be
aware
of
the
imperfect
nature
of
any
future
agreed
strategy.

k)
Negative
Control
Agents:
There
is
a
need
to
define
and
agree
on
some
negative
control
agents
for
ED
assay
validation.
Assay
specificity
will
not
be
capable
of
assessment
unless
such
agents
can
be
made
available
for
general
study.

l)
Expanding
the
Universe
of
Agents:
Developing
massive
amounts
of
screening
information
on
a
large
universe
of
chemicals
does
not
necessarily
expedite
the
development
of
the
appropriate
scientific
underpinning
that
the
Agency
needs
to
broaden
this
effort.
Consequently,
the
Subcommittee
recommends
that
EPA
4
should
not
expand
the
set
of
agents
until
the
Agency
develops
or
adopts
validated
systems
and
can
provide
clear
decision
criteria.

We
appreciate
the
opportunity
to
review
these
proposed
revisions,
and
look
forward
to
receiving
your
response
to
the
issues
raised.

/
Signed
/

Dr.
Joan
Daisey,
Chair
Science
Advisory
Board,
and
Co­
chair,
Endocrine
Disruptor
Screening
Program
Review
Subcommittee
/
Signed
/
Dr.
Gene
McConnell,
Co­
chair
Endocrine
Disruptor
Screening
Program
Review
Subcommittee
i
NOTICE
This
report
has
been
written
as
part
of
the
activities
of
the
Science
Advisory
Board,
a
public
advisory
group
providing
extramural
scientific
information
and
advice
to
the
Administrator
and
other
officials
of
the
Environmental
Protection
Agency.
The
Board
is
structured
to
provide
balanced,
expert
assessment
of
scientific
matters
related
to
problems
facing
the
Agency.
This
report
has
not
been
reviewed
for
approval
by
the
Agency
and,
hence,
the
contents
of
this
report
do
not
necessarily
represent
the
views
and
policies
of
the
Environmental
Protection
Agency,
nor
of
other
agencies
in
the
Executive
Branch
of
the
Federal
government,
nor
does
mention
of
trade
names
or
commercial
products
constitute
a
recommendation
for
use.

Distribution
and
Availability:
This
Science
Advisory
Board
report
is
provided
to
the
EPA
Administrator,
senior
Agency
management,
appropriate
program
staff,
interested
members
of
the
public,
and
is
posted
on
the
SAB
website
(
www.
epa.
gov/
sab).
Information
on
its
availability
is
also
provided
in
the
SAB's
monthly
newsletter
(
Happenings
at
the
Science
Advisory
Board).
Additional
copies
and
further
information
are
available
from
the
SAB
Staff.
ii
ABSTRACT
The
1996
passage
of
the
Food
Quality
Protection
Act
and
amendments
to
the
Safe
Drinking
Water
Act
(
SDWA)
required
EPA
to
develop
a
screening
and
testing
strategy
for
environmental
endocrine
disruptors.
The
EPA
subsequently
asked
the
Science
Advisory
Board
(
SAB)
and
the
FIFRA
Scientific
Advisory
Panel
(
SAP)
to
form
a
Joint
Subcommittee
to
review
a
set
of
scientific
issues
concerning
the
development
of
the
Agency's
endocrine
disruptor
screening
and
testing
program.
The
review
Subcommittee
met
on
March
30­
April
1,
1999,
in
Arlington
VA.

The
Charge
was
broad
and
complex,
posing
18
major
questions
within
four
broad
areas:
a)
scope
of
the
program;
b)
priority­
setting;
c)
the
high
throughput
pre­
screening
approach;
and
d)
the
proposed
endocrine
disruptor
screening
program
The
Subcommittee
recommended:
a
mid­
course
evaluation
or
optimization
of
the
screening;
an
initial
focus
on
the
methods
development
effort;
the
inclusion
of
more
and
betterdetailed
case
studies;
the
use
of
sub­
populations
as
a
criterion
within
the
existing
compartments
already
identified,
but
not
as
a
separate
stand­
alone
compartment;
making
users
aware
of
validation
problems
in
systems
like
IRIS;
the
inclusion
of
both
dose
and
timing
of
exposure,
particularly
with
respect
to
developmental
or
reproductive
events;
minimizing
the
number
of
animals
needed
for
testing;
inclusion
of
an
introductory
statement;
support
with
data
decisions
about
which
assays
are
selected,
and
which
protocols
are
adopted
for
those
assays,
should
be
with
data;
be
aware
of
the
imperfect
nature
of
any
future
agreed
strategy;
define
and
agree
on
some
negative
control
agents
for
environmental
disruption
assay
validation;
do
not
expand
the
set
of
agents
until
the
Agency
develops
or
adopts
validated
systems
and
can
provide
clear
decision
criteria.

Although
the
review
identified
several
areas
of
concern,
we
wish
to
congratulate
the
Agency
for
dealing
effectively
with
an
extraordinarily
complex
set
of
issues,
many
of
which
are
on
the
cutting
edge
of
the
relevant
science.

KEYWORDS:
endocrine;
hormone;
environmental
endocrine
disruptors;
screening;
assays;
environmental
mixtures.
iii
ROSTER
Joint
SAB/
SAP
Subcommittee
On
Endocrine
Disruptor
Screening
March
30­
April
1,
1999
CO­
CHAIRS
Dr.
Joan
Daisey,
Lawrence
Berkeley
Laboratory,
Indoor
Environmental
Program,
Berkeley
CA
Dr.
Ernest
McConnell,
ToxPath,
Raleigh
NC
MEMBERS
AND
CONSULTANTS
Dr.
John
Ashby,
Zeneca
Corporation,
Cheshire,
United
Kingdom
Dr.
Richard
Bull,
Molecular
Biosciences,
Battelle
Pacific
Northwest
Laboratories,
Richland,
WA
Dr.
Charles
Capen,
Department
of
Veterinary
Biosciences,
The
Ohio
State
University,
Columbus,
OH
Dr.
Kenneth
Davis,
Ecological
Research
Center,
University
of
Memphis,
Memphis,
TN
Dr.
John
Doull,
Department
of
Pharmacology,
Toxicology
and
Therapeutics,
The
University
of
Kansas
Medical
Center,
Kansas
City,
KS
Dr.
Paul
M.
D.
Foster,
Chemical
Industry
Institute
of
Toxicology,
Research
Triangle
Park,
NC
Dr.
James
Gibson,
Dow
AgroSciences,
Indianapolis,
IN
Dr.
Philippe
Grandjean,
Institute
of
Community
Health,
Odense
University,
Denmark
Dr.
Diane
Henshel,
School
of
Public
and
Environmental
Affairs,
Indiana
University,
Bloomington,
IN
Dr.
Alan
Maki,
Exxon
Company,
USA,
Houston,
TX
Dr.
Genevieve
Matanoski,
School
of
Hygiene
and
Public
Health,
Johns
Hopkins
University,
Baltimore,
MD
Dr.
Margaret
McCarthy,
Department
of
Physiology,
School
of
Medicine,
University
of
Maryland
at
Baltimore,
Baltimore,
MD
Dr.
Michael
McClain,
University
of
Medicine
and
Dentistry
of
New
Jersey,
R.
W.
Johnson
Medical
School,
Randolph,
NJ
Dr.
F.
M.
Anne
McNabb,
Department
of
Biology,
Virginia
Polytech.
Institute
&
State
University,
Blacksburg,
VA
Dr.
Mary
Anna
Thrall,
Department
of
Pathology,
College
of
Veterinary
Medicine
&
Biomedical
Sciences,
Colorado
State
University,
Fort
Collins,
CO
Dr.
John
G.
Vandenbergh,
Department
of
Zoology,
College
of
Agriculture
and
Life
Sciences,
North
Carolina
State
University,
Raleigh,
NC
Dr.
Tim
Zacharewski,
Department
of
Biochemistry,
Michigan
State
University,
East
Lansing,
MI
FEDERAL
EXPERTS
Dr.
Robert
Chapin,
National
Institute
of
Environmental
Health
Sciences,
Research
Triangle
Park,
NC
Dr.
James
Hanson,
National
Cancer
Center,
Bethesda,
MD
iv
CO­
DESIGNATED
FEDERAL
OFFICIAL
(
SAP)
Mr.
Larry
Dorsey,
FIFRA
Scientific
Advisory
Panel,
Office
of
Prevention,
Pesticides
and
Toxic
Substances,
Environmental
Protection
Agency,
Washington,
DC
CO­
DESIGNATED
FEDERAL
OFFICIAL
(
SAB)
Mr.
Samuel
Rondberg,
Science
Advisory
Board
(
1400),
Environmental
Protection
Agency,
Washington,
DC
v
TABLE
OF
CONTENTS
1
EXECUTIVE
SUMMARY
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1
2.
INTRODUCTION
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4
2.1
Background
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4
2.2
Charge
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4
3.
DETAILED
RESPONSE
TO
THE
CHARGE
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11
3.1
Scope
of
the
Program
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11
3.1.1
The
proper
Scope
for
the
Endocrine
Disruptor
Screening
Program
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11
3.1.2
Use
of
the
expanded
set
of
Agents
in
the
EDSP
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
13
3.1.3
Exemptions
from
the
EDSP
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
14
3.1.4
Mixtures
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
.
.
.
.
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.
.
.
15
3.2
Priority
Setting
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
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.
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.
.
.
.
.
.
.
.
.
16
3.2.1
The
Component­
based
Approach
to
Priority­
setting
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
16
3.2.2
The
Relational
Database
and
Priority
Setting
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
17
3.3
High
Throughput
Prescreening
Approach
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
19
3.3.1
High
Throughput
Technology
As
A
Tool
for
Priority
Setting
.
.
.
.
.
.
.
.
.
19
3.3.2
High
Throughput
Technology
As
A
Prescreening
Device
.
.
.
.
.
.
.
.
.
.
.
.
20
3.4
The
Proposed
Endocrine
Disruptor
Screening
Program
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
21
3.4.1
The
Two­
phase
Sorting
Strategy
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
21
3.4.2
Adequacy
of
the
Screening
Battery
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
22
3.4.3
Adequacy
of
Thyroid
Coverage
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
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.
.
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.
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.
.
.
.
24
3.4.4
In
utero
and
In
Ovo
Screens
and
Single
Dose
Screening
.
.
.
.
.
.
.
.
.
.
.
.
.
25
3.4.5
Rigor
of
The
Five
Compartment
Test
Protocol
Design
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
26
3.4.6
Adequacy
of
Detection
of
Critical
Endpoints
in
The
EAT
Systems
.
.
.
.
.
27
3.4.7
Additional
Screening
for
Agents
Initially
Found
to
Be
Negative
.
.
.
.
.
.
29
3.4.8
Endocrine
Disruptors
and
Hazard
Assessment
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
29
3.4.9
Validation
of
The
Proposed
Screens
and
Tests
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
29
3.4.10
Subcommittee
Recommendations
to
Help
EPA
Meet
its
Charge
.
.
.
.
.
.
31
4.
MAJOR
FINDINGS
AND
RECOMMENDATIONS
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
32
1
1
EXECUTIVE
SUMMARY
In
1996,
the
passage
of
the
Food
Quality
Protection
Act
(
FQPA)
and
amendments
to
the
Safe
Drinking
Water
Act
(
SDWA)
required
EPA
to
develop
a
screening
and
testing
strategy
for
endocrine
disruptors
within
two
years
and
implement
the
plan
by
August,
1999.
EPA
established
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC)
under
the
Federal
Advisory
Committee
Act
to
advise
the
Agency
on
the
screening
and
testing
of
pesticides
and
other
chemicals
for
their
potential
to
disrupt
the
endocrine
system.
Consequently,
the
EPA
asked
the
Science
Advisory
Board
(
SAB)
and
the
FIFRA
Scientific
Advisory
Panel
(
SAP)
to
form
a
Joint
Subcommittee
to
review
a
set
of
scientific
issues
being
considered
by
the
Agency
concerning
the
development
of
the
Agency's
endocrine
disruptor
screening
and
testing
program
as
required
by
the
legislation
noted
above.
This
Joint
Subcommittee
(
the
Joint
Environmental
Disruptor
Screening
Program
(
EDSP)
review
Subcommittee
met
on
March
30­
April
1,
1999,
and
produced
this
report.

The
Charge
was
broad
and
complex,
posing
18
major
questions
within
four
broad
areas:
a)
scope
of
the
program;
b)
priority­
setting;
c)
the
high
throughput
prescreening
approach;
and
d)
the
proposed
endocrine
disruptor
screening
program
(
the
complete
Charge
is
provided
in
section
2.2
of
this
report).

The
EDSP's
detailed
response
to
each
element
of
the
Charge
is
found
in
section
3
of
the
report.
The
major
issues
and
recommendations
are:

a)
Evaluating
the
Program:
We
find
no
provision
for
mid­
course
evaluation
or
optimization
of
the
process.
The
Agency
is
mandated
to
assemble
and
evaluate
this
proposed
panel
of
tests
and
then
to
implement
them,
but
a
correlate
responsibility
is
to
make
sure
that
what's
being
done
is
the
best
that
can
be.
Although
something
looks
fine
on
paper
or
in
a
small
research
setting,
translating
it
into
volume­
screening
mode
may
be
quite
another
thing.
There
was
broad
support
among
the
Subcommittee
for
the
concept
that
the
Agency
should
convene
a
panel
of
independent
scientists
to
review
all
the
screening
data
for
50­
100
compounds,
with
an
eye
towards
revising
the
process
and
eliminating
those
methods
that
don't
work.

b)
Mixtures
Issues:
The
Subcommittee
agreed
that
the
initial
focus
of
the
methods
development
effort
must
focus
necessarily
on
single
compounds
and
leave
the
question
of
testing
of
mixtures
until
accepted
single­
compound
methods
have
been
completed.
The
Subcommittee
concluded
that
very
promising
methods
already
exist
in
the
field
of
ecotoxicology.
These
include
the
Whole
Effluent
Testing
(
WET)
and
Toxicity
Identification
Evaluation
(
TIE)
procedures
developed
by
the
Agency
in
concert
with
the
Society
for
Environmental
Toxicology
and
Chemistry.
Those
methods
have
been
developed
to
test
effects
of
effluents
and
should
have
direct
application
to
the
Endocrine
Disruptor
Screening
Program.
2
c)
Case
Studies:
The
Subcommittee
strongly
encourages
the
Agency
to
include
more
and
better­
detailed
case
studies
in
the
evolution
of
the
priority­
setting
scheme.
Case
studies
will
enable
a
realistic
test
of
the
scheme,
checking
sensitivity
of
the
system
and
its
practicality
to
prioritize
chemicals
for
further
testing.

d)
Sub­
population
Compartment:
The
question
of
the
need
for
a
separate
compartment
to
address
sub­
populations
(
e.
g.,
developing
children)
was
addressed
to
the
EDPS.
Our
conclusions
supported
the
use
of
sub­
populations
as
a
criterion
within
the
existing
compartments
already
identified,
but
not
as
a
separate
stand­
alone
compartment.

e)
Use
of
the
Integrated
Risk
Information
System
(
IRIS):
The
priority
testing
scheme
relies
on
the
use
of
several
databases
summarizing
the
environmental
fate
and
effects
of
chemicals.
Several
Members
of
the
Committee
expressed
concern
that
there
are
numerous
problems
with
the
validation
of
IRIS
and
other
databases.
Before
placing
heavy
reliance
on
these
computerized
systems,
users
need
to
be
aware
of
these
validation
problems
and
proceed
with
caution
before
incorporating
these
values
unilaterally.

f)
Exposure:
The
EDPS
expressed
concern
that
consideration
of
the
toxicological
implications
of
exposure
should
include
both
dose
and
timing
of
exposure,
particularly
with
respect
to
developmental
or
reproductive
events.
The
current
scheme
does
not
adequately
cover
the
time
aspect
of
exposure
and
this
needs
to
be
remedied
before
broad­
scale
application
of
the
approach.

g)
Animal
Tests
and
Routes
of
Exposure:
We
are
concerned
about
the
large
number
of
animals
that
would
be
needed
by
the
EDSTAC
program,
and
there
is
significant
international
concern
on
the
proposed
use
of
animals
for
such
screening.
The
Subcommittee
is
cognizant
of
the
essential
role
animals
play
in
tests
to
detect
endocrine
disruption.
There
are
no
substitutes
for
tests
currently
available
for
the
Tier
1
and
Tier
2
tests.
This
notwithstanding,
the
Agency
has
an
obligation
to
conserve
all
resources
in
developing
new
testing
protocols,
and
the
use
of
animals
in
such
tests
poses
both
ethical
and
practical
problems.
In
this
role
of
hazard
assessment
(
as
opposed
to
hazard
definition)
biologically
relevant
routes
of
exposure
are
indicated
(
oral
gavage,
diet,
water,
inhalation,
skin
painting).
At
present,
use
of
the
subcutaneous
injection
or
intraperitoneal
injection
routes
are
recommended
in
thequest
of
increasing
assay
sensitivity.
In
fact,
irrespective
of
the
outcome
of
this
suggestion
it
should
be
noted
that
the
current
EPA
synthesis
of
the
EDSTAC
recommendations
is
inconsistent
on
the
matter
of
route
of
exposure,
and
that
al
animal
testing
should
use
only
biologically
relevant
routes..
3
h)
Need
for
an
Introductory
Statement:
The
previous
EDSTAC
meeting
suggested
that
the
final
document
needed,
as
a
introductory
section,
a
description
of
the
problem
or
the
scientific
or
health­
based
reason
for
the
EDSTAC
program.
The
EDPS
urges
the
EPA's
EDSTAC
team
to
include
a
description
of
both
the
health
and
ecological
problems
associated
with
exposure
to
the
endocrine
disruptors
and
to
show
how
the
program
relates
to
these
findings.

i)
Support
for
Decisions:
Decisions
about
which
assays
are
selected,
and
which
protocols
are
adopted
for
those
assays,
should
be
supported
with
data
that
are
generally
available.

j)
Exceptions:
Testing
strategies
will
always
have
exceptions.
Care
should
be
taken
to
be
aware
of
the
imperfect
nature
of
any
future
agreed
strategy.

k)
Negative
Control
Agents:
There
is
a
need
to
define
and
agree
on
some
negative
control
agents
for
ED
assay
validation.
Assay
specificity
will
not
be
capable
of
assessment
unless
reliable
agents
can
be
made
available
for
general
study.

l)
Expanding
the
Universe
of
Agents:
Developing
massive
amounts
of
screening
information
on
a
large
universe
of
chemicals
does
not
necessarily
expedite
the
development
of
the
appropriate
scientific
underpinning
that
the
Agency
needs
to
broaden
this
effort.
Consequently,
the
Subcommittee
recommends
that
EPA
should
not
expand
the
set
of
agents
until
the
Agency
develops
or
adopts
validated
systems
and
can
provide
clear
decision
criteria.

Although
the
review
identified
several
areas
of
concern,
and
the
EDSP
has
provided
recommendations
to
improve
EPA's
planned
program,
we
wish
to
congratulate
the
Agency
for
dealing
effectively
with
an
extraordinarily
complex
set
of
issues,
many
of
which
are
on
the
cutting
edge
of
the
relevant
science.
4
2.
INTRODUCTION
2.1
Background
Chemicals
which
may
interfere
with
endocrine
system
functioning
(
endocrine
disruptors)
have
concerned
the
U.
S.
Environmental
Protection
Agency
(
EPA)
for
some
time.
Such
chemicals
have
the
potential
to
impact
human
and
wildlife
populations.
A
variety
of
human
health
and
ecological
effects
have
been
attributed
to
endocrine
disruptors.

In
1996,
the
passage
of
the
Food
Quality
Protection
Act
(
FQPA)
and
amendments
to
the
Safe
Drinking
Water
Act
(
SDWA)
required
EPA
to
develop
a
screening
and
testing
strategy
for
endocrine
disruptors
within
two
years
and
implement
the
plan
by
August,
1999.
The
legislation
cites
the
Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA)
and
the
Toxic
Substances
Control
Act
(
TSCA)
as
the
two
statutes
under
which
EPA
should
implement
an
endocrine
screening
and
testing
strategy.
EPA
established
the
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC)
under
the
Federal
Advisory
Committee
Act
to
advise
the
Agency
on
the
screening
and
testing
of
pesticides
and
chemicals
for
their
potential
to
disrupt
the
endocrine
system.

Consequently,
the
Science
Advisory
Board
(
SAB)
and
the
FIFRA
Scientific
Advisory
Panel
(
SAP)
were
asked
to
form
a
Joint
Subcommittee
to
review
a
set
of
scientific
issues
being
considered
by
the
Agency
concerning
the
development
of
the
Agency's
endocrine
disruptor
screening
and
testing
program
as
required
by
the
legislation
noted
above.

2.2
Charge
The
specific
issues
to
be
addressed
by
the
Joint
Subcommittee
are:

a)
Scope
of
the
Program
1)
The
amendments
to
the
Food
Quality
Protection
Act
(
FQPA)
and
the
Safe
Drinking
Water
Act
(
SDWA)
mandate
or
support
the
development
of
a
screening
program
that
will
determine
whether
pesticides
and
certain
drinking
water
source
contaminants
"
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."
Very
early
in
its
deliberations,
EPA's
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC)
determined
that
there
was
both
a
strong
scientific
basis
and
feasibility,
considering
time
and
resource
constraints,
to
expand
the
scope
of
the
screening
program
to
include
the
androgen­
and
thyroid­
hormone
systems,
and
to
include
evaluation
of
the
potential
impact
on
wildlife
as
well
as
on
human
health.
EPA
agrees
and
is
developing
a
screening
program
which
incorporates
these
modifications.
Does
the
Joint
Subcommittee
agree
that
this
expanded
scope
is
appropriate
to
serve
as
the
starting
point
for
the
Endocrine
Disruptor
Screening
Program
5
(
EDSP),
given
the
understanding
that
the
framework
for
the
Program
can
support
for
further
expansion
at
a
later
date?

2)
The
FQPA
and
SDWA
identify
a
universe
of
substances
that
should
be
evaluated
in
a
an
EDSP.
EDSTAC
noted
that
there
exist
many
other
substances
in
addition
to
pesticides
and
certain
drinking
water
source
contaminants
that
may
exhibit
endocrine­
disrupting
potential.
They
recommended
that
the
"
candidate
pool"
for
the
EDSP
include
substances
on
the
Toxic
Substances
Control
Act
(
TSCA)
Inventory,
certain
complex
environmental
mixtures
as
well
as
non­
pesticide
food
additives,
cosmetics
and
nutritional
supplements.
EPA
agrees
that
there
are
substances
in
addition
to
pesticides
and
certain
drinking
water
source
contaminants
that
warrant
consideration
for
inclusion
in
the
EDSP..
Does
the
Joint
Committee
agree
that
this
expanded
universe
of
substances
should
be
included
in
the
EDSP
process,
at
a
minimum
in
the
priority­
setting
phase,
and
continuing
on
if
a
potential
for
concern
is
identified?

3)
FQPA
contains
a
provision
which
would
exempt
from
the
EDSP
"
any
biological
substance
or
other
substance
if
the
Administrator
determines
that
the
substance
is
anticipated
not
to
produce
any
effect
in
humans
similar
to
an
effect
produced
by
a
naturally­
occurring
estrogen"
or,
presumably,
"
such
other
endocrine
effect
as
the
Administrator
may
designate."
EPA
has
identified
some
chemical
categories
that
may
be
candidates
for
exemption.
Examples
include
certain
polymers
with
a
number
average
molecular
weight
(
NAMW)
greater
than
1000
daltons,
certain
List
4
pesticide
inerts
such
as
cookie
crumbs,
strong
mineral
acids
and
bases,
which
are
most
likely
to
interact
with
tissue
at
the
portal
of
entry
giving
rise
to
localized
lesions
rather
than
systemic
effects,
certain
biopesticides
such
as
plant
pesticides
or
microbials
or
non­
chemical
pesticides
such
as
parasitic
wasps.
Does
the
Joint
Committee
agree
that
there
are
categories
of
pesticides
and
other
substances
that
should
be
exempt
from
the
EDSP?
In
addition
to
the
examples
noted
here,
are
there
additional
categories
that
should
be
considered
for
exemption?

4)
EDSTAC
concluded,
and
EPA
agrees,
that
there
are
important
complex
environmental
mixtures
that
deserve
inclusion
in
the
EDSP.
EDSTAC
recommended
that
EPA
include
in
the
EDSP
representative
mixtures
to
which
large
or
identifiable
key
segments
of
the
population
(
e.
g.
children)
are
exposed.
They
suggested
that
high­
priority
mixture
categories
include:
Chemicals
in
breast
milk,
phytoestrogens
in
soy­
based
infant
formulas,
mixtures
commonly
found
at
Superfund
sites,
common
pesticide/
fertilizer
mixtures
found
in
ground
and
surface
water,
disinfection
byproducts,
and
gasoline.
EPA
proposes
to
screen
and
test
(
if
appropriate)
one
representative
mixture
from
each
category,
after
it
confirms
that
the
screening
and
testing
components
of
the
EDSP
are
satisfactory
for
the
handling
of
single
substances.
6
A)
Is
the
proposal
a
reasonable
way
to
address
the
practicality
of
screening
and
testing
mixtures?
B)
Are
the
six
categories
of
mixtures
the
most
appropriate
to
address
first?
C)
Are
there
other
mixture
categories
that
should
be
included
in
addition
to,
or
instead
of
those
identified
(
Note:
During
the
May
Consultation,
it
was
suggested
that
mixtures
found
in
fish
tissue,
benthic
sites
and
eggs
of
Great
Lakes
birds
should
replace
gasoline
as
a
priority
mixture).
D)
Can/
should
standardized
representative
mixtures
be
developed?
If
so,
how
should
the
chemical
combinations,
ratios,
and
doses
be
selected
for
mixtures?
E)
If
a
mixture
is
positive
in
the
screening
tier,
should
the
whole
mixture
be
tested
in
the
testing
tier
or
should
only
the
active
component(
s)
in
the
screen(
s)
be
tested
in
the
second
tier?

b)
Priority­
setting
1)
EDSTAC
recommended
a
component­
based
approach
to
priority­
setting.
EPA
agrees
that
this
is
the
appropriate
framework.
Under
this
approach,
EPA
will
group
chemicals
into
sets,
based
on
the
existence
of
factual
information
in
a
given
area.
Thus,
priority
ranking
can
be
made
fairly
among
substances,
i.
e.,
chemicals
will
compete
for
priority
with
others
on
the
basis
of
comparable
data
and
will
not
be
assigned
lower
priority
for
lack
of
information.
Are
these
principles
and
the
component­
based
approach
to
priority
setting
reasonable?
Are
there
other
approaches
that
would
be
more
useful?

2)
EPA
is
developing
a
relational
database
to
assist
in
developing
priorities
for
screening.
The
relational
database
is
intended
to
import
existing
data
and
allow
its
synthesis,
as
well
as
the
estimation
of
certain
parameters
through
modeling.
The
relational
database
was
considered
to
have
great
value
in
helping
to
identify
the
specific
compartments
under
the
EDSTAC's
component­
based
priority
setting
approach.
The
database
will
also
be
helpful
in
selecting
chemicals
for
the
first
and
subsequent
rounds
of
screening.
Would
the
Joint
Subcommittee
comment
on
the
approach
and
provide
additional
insights
to
improve
the
content
of
the
relational
database
or
its
implementation?

c)
High
Throughput
Prescreening
Approach
1)
EDSTAC
recommended,
and
EPA
proposes
to
implement
a
priority
setting
strategy
that
includes
initial
sorting
based
on
an
examination
of
existing
information.
This
initial
sorting
strategy
leads
to
four
possible
outcomes:
i)
polymers;
ii)
chemicals
with
sufficient
data
to
proceed
to
testing;
iii)
chemicals
with
sufficient
data
to
proceed
to
hazard
assessment;
and
iv)
chemicals
with
7
insufficient
data,
which
presumably,
would
go
into
the
screening
phase.
EPA
anticipates
that
a
large
number
of
substances
will
end
up
in
category
iv­
chemicals
with
insufficient
data.
To
provide
at
least
a
minimum
number
of
biological
data
to
assist
in
the
sorting
process,
EPA
proposes
to
conduct
High
Throughput
Prescreening
on
a
significant
number
of
substances
(
perhaps,
as
many
as
15,000),
using
in
vitro
assay
systems
incorporating
transcriptional
activation
or
reporter
gene
systems
for
the
estrogen­,
androgen­
and
thyroid­
hormone
systems.

A)
On
the
assumption
that
the
technology
can
be
shown
to
be
applicable
to
the
large
number
and
wide
range
of
chemical
substances
under
consideration,
and
the
limited
relevant
test
data
which
are
available
for
many
industrial
chemicals,
is
this
a
reasonable
approach
and
sorting
strategy
to
support
priority
setting?

B)
EPA
has
been
funding
a
pilot
study,
using
about
80
chemicals,
to
determine
the
applicability
of
the
high
throughput
technology
in
a
prescreening
component
of
the
EDSP.
Based
upon
your
review
of
the
data
developed
to
date,
does
the
Joint
Subcommittee
believe
that
this
technique
can
be
used
as
a
prescreening
device?
If
not,
what
modifications/
improvements
must
be
made
in
order
to
assure
its
usefulness?

d)
The
Proposed
Endocrine
Disruptor
Screening
Program
1)
EPA
is
proposing
to
develop
and
implement
an
Endocrine
Disruption
Screening
Program
that
consists
of
two
phases.
The
first
phase
is
screening,
currently
consisting
of
eight
components.
The
second
phase
is
testing,
currently
envisioned
to
have
one
to
five
components,
depending
upon
the
need
for
identifying
effects
in
various
sectors
of
the
animal
kingdom.
Is
it
reasonable
and
appropriate
to
develop
and
implement
a
two­
phase
program,
the
first
phase
focused
on
identifying
a
substance's
potential
to
interact
with
one
or
more
of
the
three
hormone
systems,
the
second
phase
to
characterize
the
effects
of
concern
that
interaction
with
these
hormone
systems
might
elicit?

2)
EDSTAC
recommended,
and
EPA
proposes
to
implement,
a
screening
battery
consisting
of
eight
assays,
three
in
vitro
and
five
in
vivo,
to
address
estrogen­,
androgen­
and
thyroid­
hormone
system
effects.
At
the
time
(
a
year
ago
or
so)
and
continuing
to
today,
based
upon
our
knowledge
of
the
state­
of­
the­
science,
the
Agency
believed
that
these
eight
assays,
once
validated
and
standardized,
would
detect
all
substances
currently
known
to
interact
with
the
three
hormone
systems
to
be
covered
in
the
Program.
Does
the
Joint
Subcommittee
agree
with
this
assessment?
If
not,
what
changes
should
be
made
in
the
battery
to
assure
the
identification
of
substances
of
potential
concern?
8
3)
Interaction
with
a
receptor
is
the
principal
or
key
mechanism
by
which
substances
exert
their
effects
on
the
estrogen­
and
androgen­
hormone
systems.
This
appears
NOT
to
be
the
case
for
the
thyroid­
hormone
system.
In
light
of
this,
does
the
Joint
Subcommittee
believe
that
there
is
adequate
coverage
of
the
thyroid
provided
in
the
proposed
screening
battery?
If
not,
what
modifications
should/
could
be
made?

4)
EPA
would
prefer
to
have
a
screening
battery
which
included
assays
containing
an
in
utero
or
in
ovo
exposure
component,
given
its
great
and
continuing
concern
about
the
potential
for
effects
on
the
developing
vertebrate
organism.
At
the
time
the
proposed
screening
battery
was
being
assembled,
EPA
was
not
aware
of
the
existence
of
any
such
screens.
Is
the
Joint
Subcommittee
aware
of
any
such
assays
that
may
exist
or
are
under
development
that
could
supplant
or
complement
one
or
more
components
of
the
proposed
screening
battery?

5)
EDSTAC
recommended,
and
EPA
would
prefer,
for
efficiency
and
cost
reasons
given
the
numbers
of
substances
that
may
be
involved
in
the
EDSP,
to
conduct
each
in
vivo
screening
assay
using
only
one
dose,
with
the
appropriate
use
of
range
finding
studies
and
other
information
to
inform
dose
selection.
Does
the
Joint
Subcommittee
agree
with
this
approach,
and
if
not,
what
suggestions
would
it
have
to
modify
the
approach,
keeping
pace,
volume,
cost
and
efficiency
in
mind?
What
would
be
the
public
health
consequences
of
these
false
negatives?
(
Note:
At
the
May
consultation,
some
members
raised
concern
about
relying
on
a
single
dose
and
suggested
that
a
minimum
of
two
doses,
and
perhaps
even
three,
be
used
to
ensure
that
the
screens
do
not
yield
false
negative
results.
It
has
also
been
suggested,
elsewhere,
that
this
issue
could/
should
be
solved
during
validation/
standardization.).

6)
EDSTAC
recommended,
and
EPA
is
proposing,
a
testing
phase
in
the
EDSP
which
could
have
as
many
as
five
components
(
i.
e.
covering
mammals,
birds,
fish,
invertebrates
and
amphibians).
Each
test
would
be
designed
to
delineate
the
dose­
response
relationships
of
effects
of
concern
for
chemicals
which
yielded
positive
results
during
the
screening
phase.
The
testing
protocols
to
be
used
are
either
upgrades
or
modifications
of
existing
guidelines,
except
for
the
amphibian.
In
this
case,
a
protocol
is
being
developed
de
novo.
Does
the
Joint
Subcommittee
believe
that
these
test
protocol
designs
will
provide
sufficient
rigor
to
identify
effects
of
concern
and
establish
their
dose
responses
for
disruption
in
the
estrogen­,
androgen­
and/
or
thyroid­
hormone
systems?

7)
There
could
be
circumstances
in
which
substances
bypass
the
screening
phase,
and
go
directly
into
the
testing
phase.
EPA
is
proposing
for
those
cases
that
the
chemical
under
evaluation
be
tested
in
all
five
tests.
Does
the
Joint
9
Subcommittee
believe
that
the
tests
in
the
testing
phase
will
be
adequate
to
detect
all
known
critical
endpoints
in
the
estrogen­,
androgen­,
and
thyroidhormone
systems?
If
not,
what
modifications
should
be
made?

8)
If
the
results
of
any
of
the
testing
phase
tests
are
negative,
what,
if
any,
additional
screening
or
testing
should
be
conducted
to
assure
that
the
chemical
is
not
an
endocrine
disruptor
in
the
estrogen­,
androgen­
or
thyroid
hormone
systems
of
that
sector
of
the
animal
kingdom?

9)
Testing
phase
tests
will
identify
effects
of
concern
that
are
the
consequence
of
endocrine
disruption.
They
may
also
identify
effects
of
concern
that
are
not
the
consequence
of
endocrine
disruption.
Thus,
it
may
not
be
possible
to
determine
if
a
substance
is
an
endocrine
disruptor
if
it
has
not
been
subjected
to
some
or
all
components
of
the
screening
battery.
Is
it
important
to
be
able
to
identify
substances
as
endocrine
disruptors
from
the
standpoint
of
conducting
a
hazard
assessment.
If
so,
why?
If
not,
why
not?

10)
EPA
is
proposing
a
validation
program
in
which
the
maximum
validation
effort
will
consist
of
conducting
each
assay
in
three
laboratories.
EPA
believes
that
there
currently
is
a
wide
variation
in
the
state
of
validation
of
each
of
the
proposed
screens
and
tests,
and
that
the
validation
efforts
should
be
tailored
for
each
assay/
test
accordingly.
EPA
plans
to
focus
first
on
the
validation
of
the
mammalian
assays
as
they
are
both
better
developed
than
the
non­
mammalian
assays
and
are
more
directly
relevant
to
meeting
the
FQPA
and
SDWA
mandates
for
a
screening
program
for
potential
human
health
impacts.
EPA's
preliminary
assessment
of
the
work
needed
is
as
follows:

The
uterotrophic
assay
requires
the
development
of
a
standardized
protocol
but
may
need
little
or
no
additional
laboratory/
protocol
development
effort
since
the
assay
has
been
in
extensive
use
for
many
years.

The
Hershberger
assay
may
require
some,
but
not
much,
additional
laboratory/
protocol
development
in
addition
to
standardization.

The
pubertal
male
and
pubertal
female
assays
need
some
additional
developmental
work
and
will
require
the
full
regime
of
interlaboratory
validation.

The
mammalian
two­
generation
reproduction
test
will
require
limited
testing
in
one
laboratory
to
validate
the
new
endpoints
since
the
basic
protocol
is
already
considered
to
be
valid.
10
Both
of
the
non­
mammalian
screens
and
some
of
the
non­
mammalian
tests
will
require
the
full
validation
regime;
some
will
require
further
prevalidation
development
(
e.
g.
amphibian
test).

The
mammalian
two­
generation
test
will
require
limited
testing
in
one
laboratory
to
validate
the
new
endpoints
since
the
basic
protocol
is
already
considered
to
be
valid.
All
of
the
non­
mammalian
assays
will
require
the
full
validation
regime
and
some
will
require
further
pre­
validation
development.
Does
the
Joint
Committee
agree
with
the
Agency's
assessment
of
the
current
status
of
the
screens
and
tests?
If
not,
what
is
the
Joint
Committee's
own
assessment
of
any
screen
or
test
which
differs
from
EPA's,
and
what
is
the
basis
for
your
opinion?

11)
Does
the
Joint
Subcommittee
have
any
other
suggestions
or
recommendations
that
would
help
EPA
meet
its
charge?
11
3.
DETAILED
RESPONSE
TO
THE
CHARGE
3.1
Scope
of
the
Program
The
amendments
(
1996)
to
the
Food
Quality
Protection
Act
(
FQPA)
and
the
Safe
Drinking
Water
Act
(
SDWA)
mandate
or
support
the
development
of
a
screening
program
that
will
determine
whether
pesticides
and
certain
drinking
water
source
contaminants
"
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."
Very
early
in
its
deliberations,
EPA's
Endocrine
Disruptor
Screening
and
Testing
Advisory
Committee
(
EDSTAC)
determined
that
there
was
scientific
basis
for
expanding
the
scope
of
the
screening
program
to
include
the
androgen­
and
thyroid­
hormone
systems,
and
to
include
evaluation
of
the
potential
impact
on
wildlife
as
well
as
on
human
health
(
EDSTAC,
1999).
EPA
agrees
and
is
developing
a
screening
program
which
incorporates
these
modifications.
Sections
3.4.1
through
3.4.4
address
significant
issues
in
designing
the
screening
program
3.1.1
The
proper
Scope
for
the
Endocrine
Disruptor
Screening
Program
(
EDSP)

The
initial
element
of
the
Charge
(
a)
(
1)
for
this
review
asks
if
the
Joint
Subcommittee
agrees
that
this
expanded
scope
is
appropriate
to
serve
as
the
starting
point
for
the
Endocrine
Disruptor
Screening
Program
(
EDSP),
given
the
understanding
that
the
framework
for
the
Program
can
support
further
expansion
at
a
later
date.

Expansion
of
the
scope
of
the
program
under
the
FQPA
and
the
SDWA
from
simply
estrogen­
like
effects
on
human
health
to
androgen
and
thyroid
active
compounds
is
reasonable.
This
expansion
raises
the
sights
above
that
demanded
by
the
authorization
language,
but
is
clearly
within
the
guidance
provided.

The
EDSTAC
review
of
endocrine
effects
recognized
that
issues
related
to
endocrine
disruption
are
even
broader
than
the
three
categories
identified
(
EDSTAC,
1998).
However,
there
will
be
significant
technical
difficulties
in
addressing
estrogen,
androgen
and
thyroid
active
compounds.
Further
expansion
at
this
stage
in
the
development
of
the
program
would
have
created
an
unmanageable
task.
Nevertheless,
it
should
be
recognized
that
modification
of
the
activity
of
other
hormonal
systems
can
be
as
important,
or
perhaps
more
important,
than
the
systems
identified.
Moreover,
the
technology
to
begin
integrating
these
systems
is
now
becoming
available.
Overall,
the
proposed
framework
should
enable
the
agency
to
integrate
knowledge
of
these
systems
and
assay
techniques
as
they
mature.

The
expansion
of
the
concerns
to
the
broader
environmental
concerns
over
endocrine
disruption
is
not
only
appropriate,
but
crucial.
Modifications
in
reproductive
and
developmental
processes
in
the
environment
have
been
related
to
endocrine
disruption.
Experience
has
shown
that
effects
on
wildlife
and
ecosystems
are
seen
before
there
is
any
significant
impact
in
humans,
particularly
for
chemicals
that
bioaccumulate.
It
is
unfortunate
that
effects
on
populations
in
actual
ecosystems
cannot
be
practically
included
in
the
program
because
in
some
cases
these
12
have
been
most
sensitive
measures
for
agents
of
this
type.
Consequently,
there
are
substantive
reasons
for
expanding
the
scope
to
non­
human
species.
As
with
the
expansion
to
the
androgen
and
thyroid
hormone
systems,
however,
it
is
important
to
recognize
that
this
further
complicates
implementation.

The
Joint
Subcommittee
expressed
considerable
concern
with
respect
to
whether
the
program
is
focused
on
the
critical
endpoints.
The
focus
on
the
endocrine
system
is
very
much
a
focus
on
mechanism(
s)
that
do
not
necessarily
relate
in
a
holistic
manner
to
the
adverse
health
and
environmental
effects
that
are
of
ultimate
concern.
In
general,
the
major
concern
for
endocrine
disruptors
are
effects
on
normal
reproduction
and
development
that
also
extend
to
the
induction
of
certain
kinds
of
cancer.
Deviations
in
endocrine
levels
in
either
direction
can
have
biological
consequences,
although
the
consequences
can
be
beneficial
as
well
as
detrimental
in
some
cases.
Each
alteration
needs
to
viewed
in
terms
of
the
totality
of
the
data."
The
endocrine
disruptions,
as
characterized
by
interactions
with
these
three
hormone
systems,
are
not
the
only
way
in
which
such
effects
can
be
produced.
As
a
consequence,
there
is
some
inversion
of
the
normal
decision
logic
that
makes
it
crucial
that
a
concise
working
definition
of
endocrine
disruption
be
developed.
The
EDSP
is
being
developed
on
the
apparent
assumption
that
most
compounds
that
affect
the
three
endocrine
systems
identified
are
likely
to
be
of
toxicological
concern.
Secondary
testing
will
be
triggered
based
on
broad­
based
screening.
However,
significantly
more
thought
must
be
given
to
identifying
quantitative
"
trigger
points"
as
well
as
the
question
of
whether
the
response
is
positive
or
not.
These
might
include
an
exclusion
based
on
excessive
doses
required
to
induce
the
effect
or
a
requirement
for
a
minimum
level
of
response
in
the
test
system.
The
most
important
issue
is
to
identify
the
magnitude
and
perhaps
the
duration
of
a
response.
Most
of
this
difficulty
could
be
taken
care
of
by
crafting
a
more
concise
definition
of
what
constitutes
endocrine
disruption.
This
could
possibly
be
done
by
adding
a
phrase
to
the
endocrine
disruption
definition
that
states
"...
reproducible
effects
on
any
endocrine
sensitive
system
impairing
successful
reproduction
and
development
..."
"
Broader
definitions
could
also
raise
issues
relating
to
carcinogenesis
or
the
ability
to
maintain
homoeostatic
balance
in
response
to
biotic,
chemical
and
physical
stresses.
Extension
of
such
definitions
inevitably
leads
to
complications
in
the
application
of
the
test
scheme.

The
Joint
Subcommittee
also
identified
a
need
to
establish
an
on­
going
review
of
progress
of
the
EDSP.
There
are
both
methodological
and
interpretative
issues
involved.
The
methodological
issues
are
more
straightforward,
involving
a
process
for
reviewing
new
screening
and
testing
methods
for
incorporation
or
substitution
for
current
methods.
The
interpretative
problems
have
more
to
do
with
how
the
data
are
going
to
be
applied
to
improving
environmental
protection.
EPA
put
in
a
heroic
effort
in
reviewing
the
available
methodology
and
putting
together
a
framework
based
on
that
methodology.
They
have
addressed
many
of
the
interpretative
problems
that
evolve
from
the
current
structure
in
the
EDSTAC
report
(
EDSTAC,
1998;
Federal
Register,
1998).
It
is
not
as
clear
how
these
processes
are
to
be
moved
forward
in
a
rational
way.
A
regular
plan
to
revisit
both
the
methodological
and
interpretative
issues
can
be
used
as
a
vehicle
to
stimulate
that
progress.
13
3.1.2
Use
of
the
expanded
set
of
Agents
in
the
EDSP
The
FQPA
and
SDWA
identify
a
universe
of
substances
that
should
be
evaluated
in
an
EDSP.
The
EDSTAC
report
(
1998)
noted
that
there
are
many
other
substances
in
addition
to
pesticides
and
certain
drinking
water
source
contaminants
that
may
exhibit
endocrine­
disrupting
potential.
They
recommended
that
the
"
candidate
pool"
for
the
EDSP
include
substances
on
the
Toxic
Substances
Control
Act
(
TSCA)
Inventory,
certain
complex
environmental
mixtures
as
well
as
non­
pesticide
food
additives,
cosmetics
and
nutritional
supplements.
EPA
agrees
that
there
are
substances
in
addition
to
pesticides
and
certain
drinking
water
source
contaminants
that
warrant
consideration
for
inclusion
in
the
EDSP.
Charge
element
(
a)
(
2)
asked
the
Joint
Committee
to
comment
on
the
use
of
an
expanded
universe
of
substances
in
the
EDSP
process,
both
in
the
priority­
setting
phase,
and
continuing
on
to
later
phases
if
a
potential
for
concern
is
identified.

Expansion
of
the
scope
of
the
mandated
efforts
under
the
FQPA
and
the
SDWA
makes
sense
only
if
EPA
has
developed
or
adopted
validated
systems
and
can
apply
clear
decision
criteria
for
expanding
(
or
not
expanding)
the
effort.
Under
such
circumstances
there
would
little
reason
to
exclude
additional
chemicals
from
consideration.
At
the
present
time,
however,
this
particular
expansion
seems
to
add
a
level
of
complexity
that
may
be
counterproductive.
The
Subcommittee's
concerns
arise
from
considering
what
the
underlying
objective(
s)
of
a
screening
and
testing
program
are
in
the
environmental
programs
administered
by
the
EPA.
The
ultimate
goal
is
to
protect
health
and
the
environment
from
adverse
effects.
In
one
sense,
the
Agency
is
to
be
congratulated
for
attempting
to
focus
on
the
endocrine
disruptor
issue,
because
it
does
move
them
in
the
direction
of
identifying
and
perhaps
understanding
more
subtle
environmental
hazards.
However,
if
the
activity
loses
its
connectivity
to
recognized
or
newly
described
forms
of
compromised
health
and
ecological
effects,
it
will
difficult
for
the
program
to
establish
a
solid
rational
basis.
Expanded
consideration
of
diverse
types
of
candidate
"
endocrine
disrupting"
chemicals
has
the
potential
of
getting
ahead
of
our
knowledge
of
the
risks
actually
represented
by
screening
and
testing
of
large
numbers
of
chemicals.
The
interest
in
how
modified
cellular
function
leads
to
adverse
effects
is
a
necessary
step
involved
in
improving
hazard
identification
and
risk
assessment.
These
advancements
must
be
built
on
careful
development
of
the
science
that
establishes
clear
causal
associations
between
new
testing
tools
and
adverse
impacts.
The
precedent
that
could
be
set
by
pursuing
mechanisms
without
regard
to
effect
can
not
only
greatly
increase
the
expense
of
testing
of
products,
it
can
compromise
confidence
in
the
screening
program
that
will
eventually
have
to
include
all
aspects
of
endocrine
functions
represented.
The
application
of
these
tests
to
a
wide
variety
of
chemicals
(
as
many
as
15,000)
has
the
potential
for
building
up
a
data
base,
but
not
necessarily
increasing
our
knowledge
about
the
significance
of
any
effects
that
are
observed.
Consequently,
we
believe
that
the
most
important
area
on
which
to
focus
resources
is
that
intended
to
improve
our
ability
to
establish
clear
causal
connections
between
exposure
and
effects
in
target
organisms,
rather
then
expand
the
number
of
agents
in
the
screening
program.

This
point
may
be
illustrated
by
considering
some
multifaceted
problems
that
could
evolve
from
the
EDSP
in
the
form
of
questions
some
of
which
are
actually
discussed
in
the
14
report.
Is
the
intent
to
focus
entirely
on
direct
effects
mediated
through
recognized
estrogen,
androgen
and
thyroid
receptors?
How
will
indirect
effects
on
the
endocrine
receptor
be
identified?
Are
indirectly
mediated
effects
on
the
estrogenic,
androgenic
and
thyroid
systems
to
be
handled
differently
from
a
risk
assessment
standpoint?
The
High
Throughput
Prescreening
System
(
HTPS)
is
unlikely
to
detect
indirect
effects.
However,
this
may
provide
the
only
data
that
is
available
on
most
of
those
chemicals.
Are
the
ones
that
are
"
negative"
then
neglected?
Their
ability
to
harm
health
and
the
environment
has
not
been
evaluated.
Reporter
systems
can
only
dependably
detect
those
interactions
that
are
mediated
at
the
level
of
the
receptor.
Many
results
from
higher
level
tests
are
likely
to
arise
from
these
indirect
effects.
Does
the
inability
of
seeing
the
effect
on
a
cellular
system
containing
a
reporter
system
linked
to
the
hormone
response
element
provide
a
rationale
for
dismissing
endocrine
disruption
as
a
mechanism
that
is
likely
to
be
active
at
low
doses?
What
if
the
steroid
hormone
response
is
mimicked
by
a
membrane
hormone
effect,
as
has
been
demonstrated
with
Epidermal
Growth
Factor?
Where
does
mechanism
of
action
fit
into
this
process
and
how
does
it
modify
perceived
risks
at
low
dose?

On
the
other
hand,
there
is
the
clear
long­
term
advantage
of
beginning
to
focus
on
the
association
of
adverse
health
impacts
with
modifications
in
hormonal
control
mechanisms.
If
an
adverse
effect
of
a
chemical
can
be
clearly
associated
with
an
endocrine
effect
(
or
any
other
biochemical/
molecular
response
that
can
be
clearly
related
to
adverse
effects),
the
dose
response
relationships
can
be
explored
across
test
systems
and
into
the
impacted
species.
Thus,
the
impacts
of
low
dose
exposure
to
environmental
agents
can
be
explored
in
greater
detail
and
with
greater
understanding.
This
is
true,
however,
only
if
the
health
impacts
that
are
associated
with
changes
are
understood
in
fairly
explicit
ways.
Developing
massive
amounts
of
screening
information
on
a
large
universe
of
chemicals
does
not
necessarily
expedite
the
development
of
the
appropriate
scientific
underpinning
that
the
Agency
needs
to
broaden
this
effort.
Consequently,
the
Subcommittee
recommends
that
EPA
should
not
expand
the
set
of
agents
until
it
has
validated
systems
and
can
apply
clear
decision
criteria.

3.1.3
Exemptions
from
the
EDSP
The
1996
Food
Quality
Protection
Act
(
FQPA)
contains
a
provision
which
would
exempt
from
the
EDSP
"...
any
biological
substance
or
other
substance
if
the
Administrator
determines
that
the
substance
is
anticipated
not
to
produce
any
effect
in
humans
similar
to
an
effect
produced
by
a
naturally
occurring
estrogen"
or,
presumably,
"...
such
other
endocrine
effect
as
the
Administrator
may
designate."
EPA
has
identified
some
chemical
categories
that
may
be
candidates
for
exemption.
Examples
include
certain
polymers
with
a
number
average
molecular
weight
(
NAMW)
greater
than
1000
daltons,
certain
List
4
pesticide
inert
substances
(
such
as
cookie
crumbs,
strong
mineral
acids
and
bases),
which
are
most
likely
to
interact
with
tissue
at
the
portal
of
entry
giving
rise
to
localized
lesions
rather
than
systemic
effects,
certain
biopesticides
such
as
plant
pesticides
or
microbials
or
non­
chemical
pesticides
such
as
parasitic
wasps.
In
Charge
element
(
a)
(
3),
EPA
asked
if
the
Joint
Subcommittee
agreed
with
the
Agency's
position
that
there
are
categories
of
pesticides
and
other
substances
that
should
be
15
exempt
from
the
EDSP.
The
Subcommittee
was
also
asked
to
identify
any
additional
categories
that
should
be
considered
for
exemption.

The
Subcommittee
believes
that
there
are
clearly
categories
of
chemicals
that
should
be
exempt.
However,
the
boundaries
between
those
compounds
that
would
be
exempted
and
those
that
would
not
must
be
carefully
considered.
The
selection
of
1000
daltons
as
a
cutoff
for
polymers
based
on
nominal
molecular
weight
appears
to
have
precedent
under
TSCA,
but
the
scientific
justification
for
this
limit
was
not
provided.
Many
compounds
with
molecular
weight
approaching
500
are
known
to
have
biological
activity.
Presumably
this
precedent
will
allow
polymers
with
as
much
as
10%
of
their
total
mass
to
be
500
daltons
or
less.
A
more
concise
statement
of
the
scientific
reason
for
taking
the
specific
action
on
polymers
would
have
been
useful.
Clearly,
there
would
be
rationales
for
dismissing
other
types
of
chemicals
(
e.
g.
amino
acids,
fatty
acids,
sugars
that
are
part
of
normal
diets)
from
the
EDSP.

The
Joint
Subcommittee
did
not
respond
to
the
second
issue
of
this
Charge
element.
We
did
not
think
it
appropriate
for
the
Subcommittee
to
identify
additional
classes
of
chemicals
for
exemption.
The
Subcommittee
suggests
that
the
Agency
consider
handling
of
exemptions
through
a
rule
making
process
that
is
transparent
and
open
to
public
comment.

3.1.4
Mixtures
EPA
recognizes
that
there
are
important
complex
environmental
mixtures
that
deserve
inclusion
in
the
EDSP.
Consequently,
EPA
plans
to
include
in
the
EDSP
representative
mixtures
to
which
large
or
identifiable
key
segments
of
the
population
are
exposed.
Initial
choices
for
these
high­
priority
mixture
categories
include:
chemicals
in
breast
milk;
phytoestrogens
in
soybased
infant
formulas;
mixtures
commonly
found
at
Superfund
sites;
common
pesticide/
fertilizer
mixtures
found
in
ground
and
surface
water;
disinfection
byproducts;
and
gasoline.
EPA
proposes
to
screen
and
test
(
if
appropriate)
one
representative
mixture
from
each
category,
after
it
confirms
that
the
screening
and
testing
components
of
the
EDSP
are
satisfactory
for
the
handling
of
single
substances.

For
this
review,
EPA
asked
(
in
Charge
elements
(
a)
(
4)
(
A­
E))
if
the
proposal
is
a
reasonable
way
to
address
the
practicality
of
screening
and
testing
mixtures;
if
the
chosen
six
categories
are
the
right
ones
to
address
first;
if
there
other
mixture
categories
that
should
be
included
in
addition
to,
or
instead
of
those
identified;
and
if
standardized
representative
mixtures
be
developed.
The
Agency
also
sought
advice
on
dealing
with
those
mixtures
found
to
be
positive
in
the
screening
tier,
asking
if
the
whole
mixture
should
be
tested
in
the
testing
tier
or
only
the
active
component(
s)
identified
in
the
screens(
s).

The
recommendations
for
handling
mixtures
are
outlined
in
Section
VII
of
Chapter
4
of
the
EDSTAC
Final
Report
(
EDSTAC,
1998)
and
were
discussed
by
EPA
staff
and
were
the
subject
of
several
comments
from
the
public
during
the
Subcommittee's
public
meeting.
Although
there
was
general
recognition
of
the
key
importance
of
mixtures
as
a
part
of
the
overall
EDSP,
there
were
concerns
about
the
selection
process,
the
experimental
design
for
testing
the
1This
approach
first
assigns
environmental
toxicants
into
four
categories
(
based
on
available
data):
a)
Specially
targeted
priorities;
b)
Exposure­
related
information;
c)
Effects­
related
information;
and
d)
Integrated
Effects
and
Exposure.
Components
(
or
sets)
are
defined
within
each
category,
into
which
agents
are
assigned
on
the
basis
of
exposure
and/
or
effects
information.
The
individual
agents
are
then
ranked
within
each
component
from
highest
concern
to
lowest
concern
to
set
priorities
for
eventual
Tier
one
screening.

16
mixtures,
and
the
ability
of
the
Agency
to
evaluate
and
interpret
the
results
of
the
studies
and
to
effectively
communicate
this
information
to
the
risk
managers
and
the
public.
There
was
a
consensus
by
the
Subcommittee
that
the
mixtures
section
of
the
EDSTAC
document
needed
to
be
re­
worked
and
there
were
several
public
comments
recommending
that
mixtures
not
be
included
in
the
program.

The
Subcommittee
suggests
a
compromise
proposal:
delay
starting
the
mixtures
testing
program
until
most
of
the
single
agent
testing
was
completed.
This
would
have
two
advantages;
first
it
would
provide
a
more
extensive
data
base
to
use
in
selecting
mixtures
for
testing;
and
second
it
would
enable
the
Agency
to
benefit
from
some
of
the
current
efforts
underway
with
pesticides
(
within
EPA
and
by
outside
research
groups)
to
improve
our
ability
to
define
and
test
mixtures.
The
Subcommittee
recognizes
that
the
Agency
is
currently
testing
some
mixtures
(
wastewater,
cholinesterase
inhibiting
pesticides
etc.)
and
that
relatively
little
effort
would
be
required
to
incorporate
these
into
EDSP.
We
are
also
aware
of
studies
that
compare
the
effects
of
mixtures
having
independent
actions
with
those
having
identical
actions
as
a
way
to
characterize
the
risk
assessment
of
mixtures
(
Yang
et
al.,
1998;
Feron
et.
al.,
1995;
NAS/
NRC/
COT,
1989).
Similar
approaches
could
be
used
to
standardize
or
characterize
mixtures
for
testing
in
the
EDSTAC
program
and
would
provide
more
interpretable
results
than
those
proposed
in
this
report.

The
EPA's
final
question
in
the
mixtures
section
addressed
phase
2
testing
of
mixtures
and/
or
the
components.
The
Subcommittee
believes
that
would
be
prudent
to
test
both
the
mixture
and
its
components.

3.2
Priority
Setting
3.2.1
The
Component­
based
Approach
to
Priority­
setting
The
EDSTAC
report
(
1998)
recommended
a
component­
based
approach
to
prioritysetting
EPA
agrees
that
this
is
the
appropriate
framework,
and
plans
to
group
chemicals
into
sets,
based
on
the
existence
of
factual
information
in
a
given
area.
Thus,
comparisons
can
be
made
between
like
substances
(
i.
e.,
chemicals
will
compete
for
priority
with
others
on
the
basis
of
comparable
data
and
will
not
be
assigned
lower
priority
for
lack
of
information).
In
Charge
element
(
b)
(
1),
EPA
asked
the
Joint
Subcommittee
to
comment
on
the
principles
of
the
component­
based
approach
to
priority
setting,
and
to
suggest
any
other
approaches
that
would
be
more
useful.

The
Subcommittee
finds
that
this
approach1
is
supportable
when
ranking
is
based
on
both
effect
and
exposure
data
following
guidance
in
NRC
and
EPA
risk
assessment
literature
(
NRC,
2
Such
a
database
links
(
or
relates)
all
data
elements
to
each
other,
allowing
a
broad
range
of
questions
to
be
answered.
In
this
case,
it
would
provide
a
means
of
relating
environmental
exposure
data
with
toxicological
effects
information.

17
1994,
1983;
EPA
1997,
1992,
1986).
The
greatest
weight
should
be
given
to
chemicals
for
which
we
have
data
that
indicates
actual
human
or
environmental
exposure
and
effects.
Lower
weight
should
be
given
to
agents
for
which
the
data
are
indicative
of
probable
exposure
(
in
food
or
drinking
water)
or
probable
effects
(
from
well
conducted
animal
studies).
The
lowest
weight
and
priority
ranking
should
be
given
to
chemicals
for
which
the
data
are
indicative
of
possible
exposure
(
based
on
Toxics
Release
Inventory
data
or
known
high
production
volume)
or
possible
effects
from
(
in
vitro
research
or
from
Structure
Activity
Research).
The
Subcommittee
supports
the
nomination
concept
(
i.
e.,
the
process
of
identifying
("
nominating")
probable/
possible
exposure
or
probable/
possible
effects
as
noted
above
by
citizens
who
are
disproportionately
exposed
because
of
the
group
or
community
to
which
they
belong,
or
because
an
ecosystem
is
disproportionately
exposed
(
EPA,
1999))
but
advises
the
Agency
that
the
process
needs
further
definition
and
that
no
unsubstantiated
claims
be
allowed.

The
Subcommittee
is
concerned
that
the
prioritization
process
is
not
as
"
transparent"
as
it
needs
to
be
for
public
understanding.
Also,
concern
was
expressed
at
the
public
meeting
that
health
care
professionals
(
both
personal
health
clinicians
and
public
health)
may
not
understand
EPA's
process,
intent,
and
implementation
methods,
especially
in
the
context
of
their
own
work.
Communication
at
this
level
needs
to
improve,
or
the
results
will
be
valueless.
Additional
concern
was
expressed
that
the
process
appears
to
have
undue
emphasis
on
chemistry
and
toxicology,
with
less
clear
emphasis
on
health
effects.
In
summary,
prioritization
should
be
based
on
a
sound
scientific
basis.

Other
than
the
comments
provided
above,
the
Subcommittee
has
no
suggestions
for
alternative
approaches.

3.2.2
The
Relational
Database
and
Priority
Setting
EPA
is
developing
a
relational
database2
to
assist
in
developing
priorities
for
screening.
This
database
is
intended
to
import
and
synthesize
existing
data,
allowing
EPA
to
estimate
certain
parameters
through
modeling.
It
is
expected
to
have
great
value
in
helping
to
identify
the
specific
components
under
the
EDSTAC's
component
­
based
priority
setting
approach.
The
database
will
also
be
helpful
in
selecting
chemicals
for
the
first
and
subsequent
rounds
of
screening.
The
EPA
asked
the
Joint
Subcommittee
to
comment
on
this
approach,
and
to
provide
advice
to
improve
the
content
of
the
relational
database
or
its
implementation
(
Charge
element
(
b)
(
2)).

The
Subcommittee
believes
that
the
proposed
relational
database
shows
strong
promise
of
being
a
useful
tool,
as
long
as
it
does
prove
to
be
truly
relational
This
step
is
the
very
core
of
18
the
risk
assessment
process
and
will
ultimately
provide
the
most
valuable
guidance
in
the
priority
setting
approach.

When
designing
a
relational
database,
it
is
important
to
consider
relationships
that
accomplish
more
than
simple
indexing
of
component
data,
so
that
the
database
is
not
just
a
resource
to
access
information
on
a
specific
agent.
Although
this
capability
is
valuable
in
itself,
one
needs
to
consider
biologic
relationships
in
modeling
the
database,
so
that
it
can
become
a
more
active
investigational
tool.
For
instance,
the
data
need
to
be
collected
and
organized
in
a
way
that
can
respond
to
our
growing
knowledge
in
gene
sequence
at
specific
loci
and
implications
for
health
and
disease.
Likewise,
the
data
need
to
be
organized
so
that
developmental
gene
networks
and
other
biological
hierarchies
can
be
reflected
appropriately
in
the
database.
We
are
moving
beyond
single
major
risk
factors
for
particular
outcomes
and
into
the
complex
gene­
gene
and
gene­
environment
relationships
which
characterize
common
diseases
(
cancer,
heart
disease,
behavioral
disorders,
aging
etc.)
This
is
a
very
difficult
challenge
for
genetic
epidemiologists
and
has
enormous
design
and
analytical
ramifications.
Since
the
type
of
information
provided
by
the
database
will
affect
greatly
the
interpretation
of
available
data,
its
design
will
inevitably
affect
prioritization.

The
database
needs
to
reflect
knowledge
throughout
the
specified
organism's
life
cycle,
and
should
be
able
both
to
examine
longitudinal
developmental
changes
within
a
system,
and
to
make
cross­
sectional
comparisons
across
the
organism.
The
goal
is
to
facilitate
creation
of
a
biologically
plausible
chain
of
causal
inference.
The
database
also
needs
to
be
prepared
to
deal
with
a
rapidly
growing
genetic
database
on
variation
in
endocrine
system­
related
genes.

The
database
should
be
designed
so
it
can
be
readily
interfaced
with
human
health
surveillance
data
on
disorders
such
as
birth
defects
and
cancer.
The
National
Institutes
of
Health,
the
Center
for
Disease
Control,
and
other
agencies
are
working
with
states
to
strengthen
these
surveillance
systems,
and
in
some
states
(
e.
g.
Iowa)
these
systems
have
been
constructed
together
with
environmental
quality
databases.
These
have
been
used
for
aggregate
(
ecologic)
studies
of
health
outcome
risk
factors.
It
is
expected
that
such
capabilities
will
be
substantially
expanded
in
the
next
few
years
with
corresponding
implications
for
priority
setting.

Priority
setting
should
also
address
those
persons
or
organisms
found
to
be
"
most
susceptible",
but
not
be
limited
to
this
subpopulation
alone.
There
needs
also
to
be
focus
on
population
disease
burden.
Individual
rare
genes
may
be
major
risk
factors
for
a
few
persons,
but
may
contribute
less
to
the
burden
of
a
disease
in
a
population
than
do
"
minor
risk
factor
genes"
which
are
common
in
the
population.

Finally,
there
are
two
important
problem
which
must
be
considered
in
using
the
relational
database
as
proposed.
The
Subcommittee
expressed
concern
that
the
lack
of
effects
data
on
the
universe
of
chemicals
currently
in
commercial
use
will
lead
to
a
relational
data
base
that
only
identifies
known
problem
chemicals
that
are
already
well
studied.
The
Subcommittee
encouraged
the
development
and
use
of
new
techniques
including
quantitative
structural
activity
relationships,
molecular
modeling,
and
androgen
binding,
in
addition
to
solubility
(
Kow)
and
19
other
measures
to
help
identify
the
bio­
available,
potentially
active
compounds
for
further
testing
in
the
EDSP).
In
addition,
we
are
concerned
by
potential
problems
caused
by
uninformed
use
of
data
from
EPA's
Integrated
Risk
Information
System
(
IRIS).
The
priority
testing
scheme
relies
on
the
use
of
several
databases
(
including
the
IRIS)
summarizing
the
environmental
fate
and
effects
of
chemicals.
Caution
was
expressed
by
several
members
of
the
Committee
that
there
are
numerous
problems
with
the
validation
of
IRIS
(
and
other
databases
as
well).
Before
placing
heavy
reliance
on
these
computerized
systems,
users
need
to
be
aware
of
these
validation
problems
and
proceed
with
caution
before
incorporating
these
values
on
their
face
value.

3.3
High
Throughput
Prescreening
Approach
Based
on
recommendations
from
the
EDSTAC
(
1998),
EPA
proposes
to
implement
a
priority
setting
strategy
that
includes
an
initial
sorting,
based
on
an
examination
of
existing
information.
This
initial
sorting
strategy
leads
to
four
possible
classifications:
i)
polymers;
ii)
chemicals
with
sufficient
data
to
proceed
to
testing;
iii)
chemicals
with
sufficient
data
to
proceed
to
hazard
assessment;
and
iv)
chemicals
with
insufficient
data,
which
presumably,
would
go
into
the
screening
phase.
EPA
anticipates
that
a
large
number
of
substances
will
end
up
in
category
iv.
To
provide
biological
data
to
assist
in
the
sorting
process,
EPA
proposes
to
conduct
High
Throughput
Prescreening
(
HTPS)
on
a
significant
number
of
substances
(
perhaps
as
many
as
15,000),
using
in
vitro
assay
systems
incorporating
transcriptional
activation
or
reporter
gene
systems
for
the
estrogen­,
androgen­
and
thyroid­
hormone
systems.

In
Charge
element
(
c)
(
1)
EPA
asks
two
questions
about
this
approach:
first,
is
this
a
reasonable
approach
and
sorting
strategy
to
support
priority
setting?;
and
second,
based
upon
the
data
developed
through
the
pilot
study
to
date,
can
this
technique
can
be
used
as
a
prescreening
device,
and
what
modifications/
improvements
must
be
made?
(
The
latter
two
questions
are
addressed
in
section
3.3.2,
below.)

3.3.1
High
Throughput
Technology
As
A
Tool
for
Priority
Setting
EDSTAC
has
recommended
the
use
of
HTPS
in
order
to
address
the
problem
that
most
chemical
substances
on
the
TSCA
Inventory
have
little
or
no
data
regarding
their
potential
to
interact/
modulate/
disrupt
the
endocrine
system.
HTPS
is
designed
to:
a)
provide
priority
setting
information
for
chemicals
to
be
examined
in
Tier
one
Screening
(
T1S);
b)
provide
a
prospective
on
the
effectiveness
of
HTPS
relative
to
other
methodologies
such
as
QSARs;
and
c)
satisfy
the
receptor
binding/
in
vitro
gene
expression
T1S
requirement
for
those
chemicals
that
go
through
HTPS.

EPA
does
not
intend
to
use
HTPS
data
to
establish
the
endocrine
disrupting
status
of
a
chemical.
Nevertheless,
there
is
considerable
concern
that
results
from
HTPS
will
be
the
first
available
data,
and
will
thus
be
(
inappropriately)
used,
resulting
in
a
certain
stigma
or
in
product
de­
selection.
This
is
a
concern
and
appropriate
measures
should
be
spelled
out
and
taken
in
order
to
ensure
that
the
data
from
HTPS
is
not
misused.
20
Unfortunately,
the
EPA­
funded
demonstration
project
with
OSI
Pharmaceuticals
failed
to
sufficiently
demonstrate
the
utility
of
their
HTPS
system
for
the
purpose
of
chemical
sorting
and
priority
setting
of
estrogen,
androgen
and
thyroid
active
chemicals.
The
Joint
Subcommittee
raised
several
concerns
regarding
the
responsiveness
and
selectivity
of
the
assays
developed
to
date.
We
believe
that
the
currently
available
data
obtained
from
the
OSI
assays
would
not
be
of
assistance
in
chemical
sorting
and
priority
setting.
However,
it
was
acknowledged
that
this
was
a
work
in
progress
and
that,
in
general,
the
HTPS
approach
had
merit
but
required
further
development
prior
to
implementation.
Therefore,
the
Joint
Subcommittee
agrees
that
in
conjunction
with
other
priority
setting
data,
results
from
estrogen
and
androgen
receptor
HTPS
assays
could
contribute
to
chemical
priority
setting
provided
the
assays
are
validated
and
standardized.
The
Subcommittee
also
questioned
the
utility
of
the
thyroid
receptor
HTPS
assay,
since
there
are
no
known
examples
of
endocrine
disruption
that
occur
as
a
result
of
chemical
interaction
with
this
receptor.

The
Subcommittee
had
one
additional
concern.
EPA's
plan
for
increasing
the
quality
of
the
assay
is
appropriate,
but
there
appears
to
be
no
contingency
plan
in
the
event
that
it
is
eventually
discovered
that
the
assay
is
not
working.
Also,
the
plan
says
nothing
about
a
time
frame
for
making
adjustments
to
the
assay,
nor
at
what
point
it
would
be
prudent
to
discontinue
it
and
seek
other
approaches.

3.3.2
High
Throughput
Technology
As
A
Prescreening
Device
Eight
transcriptional
activation
assays
have
been
recommended
by
EPA.
These
assays
include
the
estrogen
receptor
(
ER)
alpha
and
beta,
the
androgen
receptor
(
AR),
and
the
thyroid
receptor
(
TR)
in
the
absence
and
presence
of
metabolic
activation/
detoxification
system.
The
OSI
Pharmaceuticals
Corporation
(
under
an
EPA
contract
as
noted
above)
initiated
a
study
to
determine
the
feasibility
of
using
AR
and
TR
transcriptional
activation
assays
to
pre­
screen
chemicals
in
the
presence
and
absence
of
a
metabolic
system.
Sixty­
one
chemicals
were
examined
including
known
ER
or
AR
agonists
and
antagonists.
The
known
ER
and
AR
active
chemicals
were
selected
in
order
to
span
a
wide
range
of
potencies.
As
of
March
5,
1999,
stably
transfected
ER
and
TR
transcriptional
activation
assays
in
the
absence
of
metabolic
systems
have
been
used
to
assess
the
61
selected
chemicals.
A
stable
AR
cell
line
has
been
selected
and
was
used
in
an
initial
pilot
screen
of
16
chemicals.

Following
a
review
of
the
data
and
an
up
to
date
presentation
by
OSI
at
the
public
meeting,
the
Joint
Subcommittee
believes
that
the
OSI
HTPS
assays
were
not
ready
to
be
used
as
a
pre­
screen
device.
The
following
modifications/
improvements
are
suggested
in
order
to
ensure
its
usefulness:

a)
improve
responsiveness
and
selectivity
of
assays
b)
conduct
a
thorough
statistical
analysis
of
the
results
to
identify
significant
chemical
effects
on
gene
expression
21
c)
validate
and
standardize
all
HTPS
assays
using
a
training
set
of
agents
known
to
be
either
positive
or
negative
with
regard
to
endocrine
disruption.
Use
of
this
set
should
identify
the
error
rate,
i.
e.,
the
percentage
of
false
positive
and
false
negative
findings.

d)
verify
the
results
by
comparing
to
other
bench
gene
expression
assays
e)
develop
assays
that
would
be
capable
of
distinguishing
interactions
between
estrogen
receptors
alpha
and
beta.

f)
establish/
define
criteria
for
positive,
negative
and
equivocal
results
The
Joint
Subcommittee
also
made
the
following
suggestions
regarding
the
use
of
HTPS
assays
for
the
purposes
of
priority
setting:

a)
re­
open
the
bidding
process
to
include
other
assays
(
e.
g.,
receptor
binding)
and
to
identify
additional
analytical
resources
b)
consult
with
intramural
EPA
scientists
and
extramural
scientists
with
expertise
in
receptor
binding/
gene
expression
assays
to
evaluate
responses
to
any
Agency
request
for
proposals
c)
investigate
the
development
and
utility
of
other
HTPS
assays
such
as
gene
chip/
cDNA
array
assays
and
computer
modeling
of
receptor
ligand
binding
domain­
chemical
interactions
3.4
The
Proposed
Endocrine
Disruptor
Screening
Program.

EPA
is
proposing
to
develop
and
implement
an
Endocrine
Disruption
Screening
Program
(
EDSP)
that
consists
of
two
phases:
screening,
currently
consisting
of
eight
components,
and
testing,
currently
envisioned
to
have
one
to
five
components,
depending
upon
the
need
for
identifying
effects
in
various
sectors
of
the
animal
kingdom.
EPA
posed
a
number
of
questions
to
the
Subcommittee
concerning
the
proposed
EDSP,
comprising
Charge
elements
(
d)(
1­
10).
The
response
to
these
questions
are
provided
in
the
following
report
sections
(
3.4.1­
3.4.10).

3.4.1
The
Two­
phase
Sorting
Strategy
In
Charge
element
(
d)
(
1),
the
Agency
asked
if
it
is
reasonable
and
appropriate
to
develop
and
implement
a
two­
phase
program,
the
first
phase
focused
on
identifying
a
substance's
potential
to
interact
with
one
or
more
of
the
three
hormone
systems,
the
second
phase
to
characterize
the
effects
of
concern
that
interaction
with
these
hormone
systems
might
elicit.
22
The
Subcommittee
supports
the
proposal
to
develop
a
two­
phase
program
for
endocrine
disruptor
screening
and
testing
(
EDST).
Further,
a
formal
reevaluation
of
the
screening
and
testing
process
at
regular
intervals
should
be
part
of
the
program.
The
purposes
of
this
reevaluation
process
would
be
to
evaluate
the
effectiveness
of
the
protocols
initially
adopted
for
screening
and
testing
and
to
adopt
new
protocols
in
cases
where
none
currently
exist
for
identifying
endocrine
alterations
or
the
effects
of
those
alterations.
Adoption
of
new
screens
and
tests
should
also
mean
the
elimination
of
previous,
less
useful
ones.

The
suggestion
was
made
that
non­
mammalian
systems
might
serve
as
developmental
screening
tests.
Amphibians,
birds
and
fish
have
all
been
used
for
developmental
screening
to
provide
an
integrative
assessment
system.
The
fish
and
the
bird
assays
seem
to
be
the
most
sensitive.
Of
the
three,
the
basic
mechanisms
underlying
development
are
best
understood
in
the
bird
to
date,
but
some
fish
(
especially
zebra
fish
and
medaka
species)
are
rapidly
catching
up.
Of
the
amphibians,
the
frog
embryo
teratogenesis
assay
Xenopus(
FETAX
)
may
be
adaptable
to
a
fully
integrative
screening
assay
(
Fort,
1995,
1996).

As
multiple
laboratories
are
likely
to
be
running
the
prescribed
assays,
it
is
important
to
establish
procedures
for
standardization
among
laboratories
and
for
training
of
the
technicians
and
scientists
who
will
run
the
screens
and
tests.
Significant
consideration
and
planning
needs
to
be
conducted
on
how
to
ensure
inter­
laboratory
standardization.

The
task
of
testing
for
endocrine­
disrupting
activity
and
related
potential
adverse
effects
is
at
the
cutting
edge
of
current
science
(
and
some
feel
that
it
is
ahead
of
the
state­
of­
the­
art),
and
that
it
is
therefore
particularly
important
to:

a)
be
as
explicit
as
possible
about
the
type
and
significance
of
the
effects
that
the
tests
are
attempting
to
assess.

b)
incorporate
updates
of
the
screening
and
testing
protocols
early
and
often.

c)
focus
resources
on
the
weakest
scientific
links
in
the
screening
and
testing
process
(
such
as
the
ability
to
clearly
link
the
tests
to
risks
of
adverse
effects).

Finally,
the
absence
of
an
anticipated
report
from
the
National
Research
Council
(
NRC)
study
committee
on
endocrine
disruptors
made
the
task
of
this
review
Subcommittee
more
difficult
from
a
procedural
perspective.
We
may
need
to
reevaluate
our
recommendations
on
the
underlying
science
for
screening
and
testing
once
conclusions
drawn
by
the
NRC
study
committee
are
available.

3.4.2
Adequacy
of
the
Screening
Battery
The
EDSTAC
(
1998)
recommended,
and
EPA
proposes
to
implement,
a
screening
battery
consisting
of
eight
assays,
three
in
vitro
and
five
in
vivo,
to
address
estrogen,
androgen,
and
thyroid
systems
(
EAT)
effects.
The
Agency
believes
that
these
eight
assays
would
detect
all
23
substances
currently
known
to
interact
with
the
EAT,
and
asked
the
Subcommittee
(
in
Charge
element
(
d)(
2))
if
it
agrees
with
this
position.
Also,
EPA
sought
advice
on
making
changes
in
the
battery
to
assure
the
identification
of
substances
of
potential
concern.

It
is
difficult
to
evaluate
the
proposals
provided
to
the
Subcommittee
without
a
clear
determination
of
the
scope
and
nature
of
the
problem
for
both
humans
and
wildlife.
A
concise
description
of
the
scope
and
nature
of
the
problem
for
both
humans
and
wildlife
would
be
useful
to
demonstrate
the
relationship
of
the
proposed
screens
to
the
effects
of
concern.
The
Subcommittee
agreed
that,
as
a
minimum,
the
Agency
must
develop
an
acceptable
methods
and
standardization
and
validation
program
for
all
proposed
testing
methods.
The
program
proposed
is
clearly
screening
more
for
mechanism
than
adverse
responses.
There
are
other
potential
mechanisms
in
the
EAT
systems
of
which
we
are
not
fully
aware.
Thus,
the
information
on
di­
nbutyl
phthalate
(
Gray,
1999a)
presented
by
EPA
staff
at
the
public
meeting
clearly
show
major
adverse
effects
produced
by
alterations
in
the
androgen
system
during
development.
These
effects
might
not
be
found
using
the
screens
currently
employed
in
Tier
1.
This
indicates
that
there
are
potential
critical
events
that
would
not
be
detected
with
the
current
screening
battery.
An
in
utero
(
or
in
ovo)
screen
that
is
recognized
as
the
most
sensitive
exposure
window
for
endocrine
disruptor
event
(
see
section
3.4.)
should
be
utilized.
The
Joint
Subcommittee
firmly
agreed
that
an
in
utero
assay
should
be
developed
by
the
EPA
and
that
it
should
be
considered
as
a
substitution,
not
an
addition
to
the
proposed
battery.
However,
it
is
imperative
that
it
be
validated
before
becoming
a
required
assay.

Thus,
the
screens
in
whole
animals
would
provide
access
to
more
potential
mechanisms
than
receptor
based
screens
since
these
animals
would
have
intact
hypothalamic/
pituitary/
gonadal
or
thyroid
axes
and
also
have
multiple
end
points
in
the
same
animals
related
to
endocrine
disturbances.
Moreover,
such
screens
would
also
provide
positive
information
on
reproductive
(
and
developmental)
toxicants
that
act,
for
example,
directly
on
the
gonads
via
non­
endocrine
primary
mechanisms,
such
as
methoxyethanol
(
Foster
et
al.,
1986),
but
would
also
affect
endocrine
end
points
subsequent
to
gonadal
damage
over
several
days.

The
Subcommittee
also
suggests
that
developmental
nervous
system
endpoints
should
be
incorporated
into
the
screening
assays.
This
could
be
done
at
the
level
of
a
"
to
be
developed"
integrative
screening
assay.
In
addition,
believe
that
due
regard
should
be
given
to
the
dose
route
employed
in
the
in
vivo
screens.
Some
flexibility
should
be
employed
but
it
was
considered
that
the
most
appropriate
route
of
exposure
(
that
which
mimics
the
typical
route
of
exposure
in
humans
and/
or
wildlife)
be
chosen
with
the
oral
route
being
the
default
exposure
route.
The
use
of
the
intra
peritoneal
route,
especially
for
the
uterotrophic
assay,
was
considered
to
be
inappropriate.

Further,
addressing
technique,
it
was
not
clear
why
the
fish
protocol
presently
being
refined
and
tested
by
EPA's
Duluth
laboratory
specifically
says
not
to
use
the
organ
weight
corrected
for
body
weight
(
organ/
somatic
index).
This
practice
is
generically
used
and
recommended
to
account
for
any
changes
in
overall
body
weight
induced
by
the
chemical
treatment.
The
protocols
should
be
consistent
with
each
other.
Since
exposure
to
the
chemicals
3
T3
and
T4
are
two
forms
of
thyroid
hormone,
the
digits
"
3"
and
"
4"
indicating
the
number
of
iodine
molecules
in
its
atomic
structure.

24
may
well
induce
changes
in
body
weight
as
a
separate
phenomenon
from
any
gonad
or
other
endocrine
effects,
it
would
seem
that
the
protocol
should
be
to
calculate
and
report
both
the
raw
organ
weights
and
the
body­
weight­
corrected
somatic
indices.

The
Subcommittee
was
also
aware
that
there
is
a
huge
gulf
in
terms
of
effort,
complexity
and
cost
between
Tier
1
screening
and
Tier
2
testing.
The
EPA
may
wish
to
consider
if
an
intermediate
tier
would
be
warranted
that
would
provide
valuable
information
without
the
expense
of
multi­
hundred
thousand­
dollar
efforts.

3.4.3
Adequacy
of
Thyroid
Coverage
Interaction
with
a
receptor
is
the
principal
or
key
mechanism
by
which
substances
exert
their
effects
on
the
EAT.
There
is
an
exception,
however,
in
that
this
appears
not
to
be
the
case
for
the
thyroid­
hormone
system.
Consequently,
the
Agency
asked
the
Joint
Subcommittee
to
comment
on
the
adequacy
of
coverage
of
the
thyroid
provided
in
the
proposed
screening
battery,
and
suggest
modifications
if
needed
(
Charge
element
(
d)(
3)).

The
Subcommittee
believes
that
the
proposed
screening
battery
should
detect
alterations
in
thyroid
function.
However,
the
screens
proposed
are
more
general
and
less
robust
than
those
designed
to
detect
alterations
in
estrogens
and
androgens.
It
would
be
prudent
to
have
thyroidhormone
sensitive
tests
in
the
screen.
Most
known
thyrotoxicants
produce
changes
in
thyroidrelated
hormones
and/
or
clearance
and/
or
thyroid
histology.
The
proposed
EDSTAC
screening
process
for
thyroid
hormone
appears
to
address
these
requirements.
Measuring
hormone
levels
and
thyroid
histopathology
in
rats,
and
amphibian
tail
resorption,
should
effectively
capture
the
strongest
thyrotoxicants.
The
Subcommittee
supports
the
inclusion
of
Thyroid
Stimulating
Hormone
(
TSH)
and
T3
in
addition
to
the
measurement
of
T4,
histopathology
and
amphibian
tail
resorption.
3
Only
the
proposed
amphibian
tail
resorption
test
specifically
evaluates
an
effect
of
thyroid
hormones
on
target
tissues.

No
data
were
offered
by
EPA
to
support
the
inclusion
of
additional
tests,
other
than
the
fact
that
T3
is
the
biologically
active
form
of
the
hormone,
and
that
an
elevation
in
TSH
would
confirm
a
physiologically
relevant
reduction
in
T4
or
T3
levels.
The
data
from
Cook
and
O'Connor
(
in
press)
showed
that
for
every
thyrotoxicant
that
reduced
T3,
there
were
also
changes
in
T4,
which
offers
direct
support
for
the
EPA
proposal.
If
the
EPA
wishes
to
have
the
extra
confirmation
of
a
thyroid
effect
(
or
lack
thereof),
the
Subcommittee
would
support
the
inclusion
of
TSH
and
T3
in
addition
to
the
measurement
of
T4,
histopathology
and
amphibian
tail
resorption.

Because
few
chemicals
that
alter
thyroid
function
do
so
by
binding
to
thyroid
receptors,
binding
assays
and
gene
reporter
screens
for
thyrotoxicants
have
been
omitted
from
Tier
1.
This
25
omission
is
appropriate
because
it
would
have
generated
false
negatives
for
thyroid
effects.
However,
if
these
receptor
assays
are
to
play
a
large
part
in
priority
setting
for
testing
in
Tier
1,
it
needs
to
be
recognized
that
less
information
about
potential
thyroid
alterations
will
be
available
from
the
Tier
1
screens
than
is
the
case
for
E
and
A.

There
is
broad
agreement
that
most
known
thyrotoxicants
produce
changes
in
thyroidrelated
hormones
and/
or
clearance
and/
or
thyroid
histology.
And
the
Subcommittee
agrees
that
it
is
prudent
to
have
some
degree
of
overlap
and
complementarity
in
the
screening
tests.
Consequently,
the
same
function
should
be
evaluated
by
more
than
one
test.
Finally,
because
hormone
signals
are
both
amplitude
and
frequency
modulated
signals,
and
a
single
time­
point
measurement
may
not
capture
or
identify
an
exposure­
related
change
when
one
is
primarily
measuring
a
hormone,
it
is
desirable
to
also
measure
some
downstream
functional
result
of
that
hormone.
The
proposed
EDSTAC
screening
process
for
thyroid
appears
to
address
these
requirements
by
measuring
hormone
levels
and
thyroid
histopathology
in
rats,
and
tail
resorption
in
amphibians.
These
measures
should
effectively
capture
the
strongest
thyrotoxicants.

3.4.4
In
utero
and
In
Ovo
Screens
and
Single
Dose
Screening
EPA
would
prefer
to
have
a
screening
battery
which
included
assays
containing
an
in
utero
or
in
ovo
exposure
component,
given
the
concern
about
the
potential
for
effects
on
the
developing
vertebrate
organism.
At
the
time
the
proposed
screening
battery
was
being
assembled,
EPA
was
not
aware
of
the
existence
of
any
such
screens.
The
question
posed
to
the
Subcommittee
in
Charge
element
(
d)
(
4)
asks
for
comment
on
any
such
assays
that
may
exist
or
are
under
development,
and
that
could
supplant
or
complement
one
or
more
components
of
the
proposed
screening
battery?
Charge
element
(
d)
(
5)
addressed
EPA
preference
to
conduct
each
in
vivo
screening
assay
using
only
one
dose,
selected
through
the
use
of
range
finding
studies
and
other
information.
The
Agency
asked
if
the
Subcommittee
agreed
with
the
single
dose
approach,
and
what
suggestions
it
had
to
modify
it.
The
EPA
also
sought
advice
on
the
possible
public
health
consequences
of
these
false
negatives.
The
Subcommittee
decided
that,
since
the
issues
were
inter­
related,
it
would
be
best
to
address
both
elements
of
the
Charge
in
a
single
response,
which
follows
below.

The
Subcommittee
prefers
those
tests
which
bundle
several
endpoints
into
a
single
"
test
unit."
The
Subcommittee
consequently
supports
the
use
of
gene
reporter
and
binding
assays
as
part
of
Tier
1.
Problems
may
be
encountered
because
of
differences
in
the
specificity
of
different
cell
systems
and
because
of
patent
control
of
some
assay
components.
A
screen,
using
animals
exposed
in
utero
and
possibly
during
lactation,
is
appealing.
The
Subcommittee
strongly
encourages
the
continued
development
and
evaluation
of
such
a
protocol.
It
could
replace
several
individual
assays.
No
protocol
for
such
a
test
has
been
evaluated
or
validated
as
a
screen
to
date.
However,
such
a
test
is
easily
developed
by
taking
pieces
of
existing
protocols
(
see
the
discussion
in
section
3.4.1).
The
development
of
such
a
protocol
would
significantly
improve
screening
effectiveness,
reduce
the
numbers
of
animals
used,
and
could
improve
overall
efficiency
of
screening.
26
The
consensus
of
the
Subcommittee
regarding
dose
levels
for
in
vivo
screens
was
focused
around
two
issues.
First,
for
those
relatively
non­
toxic
agents,
the
employment
of
a
single
limit
dose
(
as
specified
in
the
Federal
Register
(
1998)
document,
e.
g.,
1
g/
kg/
d
oral)
was
considered
to
be
appropriate.
Second,
in
other
cases
where
non­
specific
toxicity
could
be
possible,
the
highest
dose
level
tested
should
elicit
some,
but
not
overt,
systemic
toxicity
in
line
with
the
establishment
of
a
maximum
tolerated
dose
(
MTD).
A
second
dose
level
should
then
be
employed
at
one
quarter
of
the
MTD.
The
Subcommittee
felt
that
the
application
of
a
multiple
of
exposure
as
the
highest
dose
level
tested
would
not
be
appropriate
since
exposure
data
would
only
be
available
infrequently.
The
Subcommittee
also
found
that
false
negatives
from
high
dose
exposure
were
unlikely,
since
even
where
multiple
mechanisms
may
be
operating
at
different
parts
of
the
dose
response
curve,
one
would
not
expect
to
see
effects
only
at
low
dose
levels.
A
second
lower
dose
level
would
also
resolve
some
of
these
questions.

The
potential
for
identifying
effects
at
low
doses
of
putative
endocrine
disruptors
was
discussed.
In
view
of
the
preliminary
nature
of
these
potentially
important
findings
(
see
Nagel,
et
al.,
1997),
the
Subcommittee
recommends
that
EPA
continue
to
remain
alert
to
new
information
on
low
dose
effects.
The
Subcommittee
was
pleased
to
learn
that
EPA
is
sponsoring
a
workshop
on
this
issue
in
May,
1999.

It
should
be
noted
that
the
current
EPA
synthesis
of
the
EDSTAC
recommendations
is
inconsistent
on
the
matter
of
route
of
exposure.
The
uterotrophic
assay
uses
subcutaneous
or
intra
peritoneal
injection,
the
Hershberger
assay
oral
gavage,
the
multi­
generation
assay
uses
diet/
oral/
inhalation,
and
no
route
is
identified
for
the
pubertal
male
and
female
assays.
Consistency
is
preferred
unless
evidence
requires
otherwise.

An
integrative
developmental
assay
using
the
chicken
was
proposed
(
Henshel,
1998,
1996;
Henshel
et
al.,
1997).
The
assay
integrates
both
a
rapid
five­
day
screening
component
with
a
more
complete
developmental
assessment.
Many
chemicals
that
are
developmental
toxicants
interact
with
the
embryo
during
organogenesis.
Therefore,
using
modifications
of
established
procedures,
and
modeling
the
system
after
the
mammalian
embryo
culture
systems,
the
avian
embryo
may
provide
a
useful
assay.

3.4.5
Rigor
of
The
Five
Compartment
Test
Protocol
Design
EDSTAC
recommended,
and
EPA
is
proposing,
a
testing
phase
in
the
EDSP
which
could
have
as
many
as
five
subject
compartments(
i.
e.,
mammals,
birds,
fish,
invertebrates
and
amphibians).
Tests
for
each
compartment
would
be
designed
to
delineate
the
dose
response
relationships
of
effects
of
concern
for
chemicals
which
yielded
positive
results
during
the
screening
phase.
The
testing
protocols
to
be
used
are
either
upgrades
or
modifications
of
existing
guidelines,
except
for
the
amphibian.
In
this
case,
a
protocol
is
being
developed
de
novo.
The
Subcommittee
was
asked
if
the
planned
test
protocol
designs
would
provide
sufficient
rigor
to
identify
the
effects
of
concern
and
establish
their
dose
response
relationship
for
disruption
in
the
EAT
systems
(
Charge
element
(
d)
(
6)).
27
The
Subcommittee
believes
that
the
five
specified
compartments
should
be
adequate
for
detecting
endocrine
mediated
events
in
most
animal
species.
However,
the
Agency
needs
to
be
cognizant
that
there
may
be
exceptions
in
some
cases
(
e.
g.,
reptiles,
for
which
there
are
no
EPA
guidelines).
The
Subcommittee
also
concluded
that
many
of
the
proposed
tests
were
valid
assays
of
endocrine
disruptors.
They
also
concluded
that
methods
must
be
standardized
and
validated,
based
on
accepted
criteria
for
validation
and
regulatory
acceptance
of
toxicological
test
methods.
Other
tests,
however,
met
with
considerable
criticism:

a)
A
more
comprehensive,
in­
utero
test
battery
should
be
assembled
to
replace
several
tests
in
Tier
1
(
see
sections
3.4.1
and
3.4.4)

b)
The
Daphnia
developmental
assay
should
be
considered
as
a
replacement
for
the
mysid
assay
because
there
is
a
better
understanding
of
the
endocrine
mechanisms
in
Daphnia
(
Baldwin
et
al.,
1998;
Baldwin
and
LeBlanc,
1994).

c)
The
fish
assay
for
endocrine
disruption
should
include
the
measurement
of
vitellogenin
in
male
fishes.
Vitellogenin
is
a
yolk
precursor
protein
made
by
the
liver
in
response
to
estrogen
in
female
but
normally
not
in
male
oviparous
animals.
Its
detection
in
male
fish
is
a
highly
sensitive
assay
for
estrogenic
activity.
Many
laboratories
have
the
ability
to
detect
vitellogenin
by
radio
immunoassay
in
a
variety
of
species.
There
are
no
known
barriers
to
the
development
of
such
a
vitellogenin
test,
although
it
would
still
have
to
be
standardized
and
validated.

d)
The
fish
reproduction
assay
should
include
some
measure
of
the
reproductive
fecundity
of
the
selected
compounds.
Egg
production
and
developmental
success
will
detect
effects
which
may
not
be
obviously
toxic
to
the
organism
but
might
have
detrimental
effects
at
the
population
level.
None
of
the
proposed
tests
with
wildlife
detect
breeding
success.
Further,
exposure
to
a
variety
of
compounds
can
alter
the
sex
ratios
to
favor
one
sex
or
the
other.
The
effect
of
the
test
chemicals
should
include
an
evaluation
of
the
sex
ratios
of
eggs
(
or
other
stages
of
development)
treated
with
the
chemicals.

e)
The
Subcommittee
recommends
that
the
EPA
examine
the
use
of
the
Japanese
quail
to
substitute
for
the
proposed
avian
tests
on
Bob­
white
quail
and
mallard
ducks.
Japanese
quail
have
the
advantage
of
short
generation
time
and
provide
a
model
with
a
great
deal
of
background
information.

f)
Although
the
Tier
2
tests
designed
to
indicate
thyroid
alterations
should
"
identify
effects
of
concern"
they
will
not
effectively
determine
whether
those
hormone
alterations
have
adverse
effects
on
the
development
or
function
of
the
target
tissues
for
thyroid
hormones.
Thus,
the
proposed
tests
may
be
adequate
for
detecting
hormone
perturbations
but
they
don't
give
information
about
the
effects
of
those
perturbations
(
see
additional
information
in
section
3.4.6).
28
3.4.6
Adequacy
of
Detection
of
Critical
Endpoints
in
The
EAT
Systems
There
could
be
circumstances
in
which
EPA
decides
to
bypass
the
screening
phase
for
a
particular
agent
and
go
directly
into
the
testing
phase.
EPA
is
proposing
for
those
cases
that
the
chemical
under
evaluation
be
tested
in
all
five
tests.
The
Agency
is
asking
the
Subcommittee
if
the
tests
in
the
testing
phase
will
be
adequate
to
detect
all
known
critical
endpoints
in
the
EAT
hormone
systems,
and
what
modifications
the
Subcommittee
might
recommend
(
Charge
element
(
d)
(
7)).

A
central
point
in
the
Subcommittee's
discussion
was
that
the
tests
employed
in
Tier
2
will
be
the
ones
used
in
risk
assessment.
The
number
of
tests
employed
would
be
dependent
on
the
use
and
potential
exposure
for
each
chemical.
Clearly
no
single
test,
or
group
of
tests,
has
the
ability
to
cover
all
critical
endpoints
for
the
EAT
systems;
they
should,
however,
cover
most
endpoints.
Many
research
techniques,
especially
with
regard
to
the
thyroid
system,
are
not
at
a
stage
where
they
can
be
ready
for
application
in
a
regulatory
testing
scenario.
The
Subcommittee
recommends
that
the
EPA
should
remain
alert
for
new
techniques
and
end
points
to
improve
testing
protocols
when
these
become
robust
and
applicable
for
routine
testing.
The
Subcommittee
also
suggests
that
specific
consideration
be
given
for
the
use
of
Japanese
quail
in
the
avian
reproduction
study
and
the
use
of
Daphnia
spp.
as
a
useful
alternate
species
for
invertebrates.
It
was
also
unclear
during
the
discussions
if
the
proposed
Mysid
species
did
indeed
have
a
functioning
EAT
system.
Since
these
were
the
specific
endocrine
systems
laid
out
in
the
EDSP,
it
would
be
inappropriate
to
propose
a
species
in
which
estrogen,
androgen
and
thyroid
hormones
did
not
have
a
physiological
role.

The
immediate
focus
of
many
of
the
proposed
tests
is
on
mechanisms.
The
ultimate
goal,
however,
is
the
capability
to
detect
adverse
effects
on
reproduction
and
development
in
a
variety
of
species.
Thus,
all
chemicals
interfering
with
reproduction
and
development
should
be
detected
in
these
test
systems,
including
those
whose
primary
mechanism
is
not
via
a
disturbance
in
the
endocrine
or
EAT
systems.
Although
the
risk
assessment
for
any
adverse
effect
and
the
dose
response
data
for
that
adverse
effect
will
be
provided
by
these
tests,
it
will
be
unfortunate
if
all
reproductive
and
developmental
toxicants
are
labeled
as
"
endocrine
disruptors."
This
issue
further
raises
the
need
for
a
clear
definition
of
an
endocrine
disruptor
­­
if
it
is
to
receive
special
consideration
­­
as
opposed
to
being
treated
as
any
normal
reproductive
or
developmental
toxicant.

The
advent
of
new
test
end
points
(
especially
for
incorporation
into
the
mammalian
twogeneration
reproduction
study)
has
raised
questions
about
the
adversity
of
specific
responses
and
the
normal
range
for
these
end
points
(
e.
g.
anogenital
distance,
preputial
separation,
vaginal
opening).
Guidance
from
the
EPA
would
be
especially
welcome
in
these
specific
areas
of
testing.

The
Tier
2
tests
include
few
endpoints
that
will
detect
critical
target
tissue
effects
of
thyroid
hormone
alterations.
Such
tests
are
needed
to
provide
suitable
information
about
whether
alterations
in
thyroid
hormones
(
which
should
be
detected
by
the
proposed
screening
29
and
testing)
will
affect
other
developmental,
morphological
or
physiological
endpoints
in
target
tissues.
Currently
such
tests
are
not
available
for
quick
adoption.
However,
the
research
information
about
these
effects
is
available
and
could
be
used
as
the
basis
for
development
of
such
tests
at
later
stages
of
this
program.
With
respect
to
the
evaluation
of
thyroid
function,
the
proposed
Tier
2
tests
seem
marginally
adequate
for
providing
information
to
the
final
program
stages
of
hazard
evaluation
and
risk
assessment.
Thus,
although
the
addition
of
more
tests
to
Tier
2
should
not
be
done
lightly,
there
are
serious
questions
about
the
adequacy
of
the
thyroid
tests
for
assessing
whether
there
are
adverse
effects
of
thyroid
alterations.

The
proposed
Tier
2
tests
include
some
endpoints
affected
by
alterations
in
thyroid
function
(
e.
g.
growth).
However,
the
proposed
measurements
are
not
very
sensitive
and
most
are
ones
that
involve
the
interactions
of
several
hormone
systems.

3.4.7
Additional
Screening
for
Agents
Initially
Found
to
Be
Negative
EPA
wished
to
know
what,
if
any,
additional
screening
or
testing
would
be
required
to
assure
that
an
agent
is
not
an
EAT
disruptor,
if
the
results
of
any
of
the
testing
phase
tests
are
negative
(
Charge
element
(
d)
(
8))
.

The
Subcommittee
agreed
that,
if
an
agent
is
found
to
be
inactive
in
the
Tier
2
tests,
it
would
be
regarded
as
being
inactive
as
an
endocrine
disruptor.
This
is
axiomatic,
as
the
Tier
2
tests
were
selected
to
define
the
endocrine
toxicity
of
agents
found
to
be
potentially
active
in
the
Tier
1
tests.
So
the
answer
to
the
question
posed
is
that
no
further
testing
would
be
required.

The
Members
also
noted
that
an
agent
found
to
be
active
in
Tier
1
tests,
but
inactive
in
Tier
2
tests,
should
be
considered
to
be
inactive
as
an
endocrine
disruptor.
In
particular,
the
positive
Tier
1
data
should
not
assume"
a
life
of
its
own"
after
the
Tier
2
tests
are
found
to
be
negative.

3.4.8
Endocrine
Disruptors
and
Hazard
Assessment
Testing
(
as
opposed
to
screening)
phase
tests
will
identify
effects
of
concern
that
are
the
consequence
of
endocrine
disruption.
They
may
also
identify
effects
of
concern
that
are
not
the
consequence
of
endocrine
disruption.
Thus,
it
may
not
be
possible
to
determine
if
a
substance
is
an
endocrine
disruptor
if
it
has
not
been
subjected
to
some
or
all
components
of
the
screening
battery.
Because
of
this,
EPA
has
asked
the
Subcommittee
if
it
is
important
to
be
able
to
identify
substances
as
endocrine
disruptors
from
the
standpoint
of
conducting
a
hazard
assessment,
and
if
so,
why
(
Charge
element
(
d)
(
9).

It
is
important
to
be
able
to
identify
substances
as
endocrine
disruptors
from
the
standpoint
of
conducting
a
hazard
assessment.
If
a
compound
causes
toxicity,
it
should
be
treated
like
all
other
toxicants.
On
the
other
hand,
knowing
that
a
compound
is
more
toxic
to
developing
hormonal
systems
means
that
the
particularly
vulnerable
populations
are
more
likely
30
to
be
protected.
The
perception
among
the
Subcommittee
Members
is
that
hazard
assessment
will
not
likely
be
impacted,
but
that
risk
assessment
will
be
improved.

3.4.9
Validation
of
The
Proposed
Screens
and
Tests
EPA
is
proposing
a
validation
program
in
which
the
maximum
validation
effort
will
consist
of
conducting
each
assay
in
three
laboratories.
EPA
believes
that
there
currently
is
a
wide
variation
in
the
state
of
validation
of
each
of
the
proposed
screens
and
tests,
and
that
the
validation
efforts
should
be
tailored
for
each
assay/
test
accordingly.
EPA
plans
to
focus
first
on
the
validation
of
the
mammalian
assays,
as
they
are
both
better
developed
than
the
nonmammalian
assays
and
are
more
directly
relevant
to
meeting
the
FQPA
and
SDWA
mandates
for
a
screening
program
for
potential
human
health
impacts.
EPA's
preliminary
determination
of
the
areas
needing
development
are:
a)
the
uterotrophic
assay;
b)
the
Hershberger
assay;
c)
the
pubertal
male
and
pubertal
female
assays;
d)
the
mammalian
two­
generation
reproduction
test;
and
e)
the
non­
mammalian
screens
and
some
of
the
non­
mammalian
tests.

EPA
asked
if
the
Joint
Subcommittee
agrees
with
the
Agency's
assessment
of
the
current
status
of
the
screens
and
tests,
and,
if
it
reached
differing
conclusions,
to
provide
the
background
and
rationale
for
its
findings
(
Charge
element
(
d)
(
10)).

It
was
agreed
that
the
new
mammalian
multi­
generation
assay
protocol
would
require
validation
of
its
practicality.
It
cannot
be
validated
per
se
because
it
is
an
apical
Tier
2
test.
The
Subcommittee
recommended
that
the
validation
should
proceed
sequentially.
One
laboratory
should
establish
practicality,
and
that
result
should
then
be
confirmed
in
one
or
two
additional
laboratories.
An
objective
appraisal
of
the
result
of
the
first
run
could
well
indicate
that
the
protocol
is
practical,
and
the
second
phase
of
validation
may
be
canceled.
This
point
is
important,
given
the
time
taken
to
conduct
the
assay,
and
the
present
need
for
the
assay
as
the
most
informative
(
Tier
2)
test.

The
Subcommittee
also
agreed
that
the
non
mammalian
Tier
2
tests,
as
well
as
the
mammalian
tests,
would
require
formal
validation
as
to
their
practicality
and
sensitivity/
specificity.

The
purpose
of
the
Hershberger
assay
is
to
quantify
the
effects
of
potential
anti­
androgenic
and
androgenic
compounds
on
the
hormone­
dependant
tissues
in
the
immature
male
rat
(
Hershberger,
et
al.
1953).
Castrated
immature
male
rats,
reared
under
standardized
housing
conditions,
are
treated
with
a
potential
xenobiotic
or
the
vehicle
via
oral
gavage.
The
animals
are
then
euthanized
and
the
relevant
target
tissues
are
fixed
and
stained
and
examined
for
histopathology.
Serum
thyroxin
(
T4)
and
TSH
is
measured,.
as
is
serum
Luteinizing
Hormone
(
androgen
measurements
are
optional).
The
data
are
then
analyzed
for
statistical
significance
of
any
differences
found
between
the
treated
animals
and
the
controls.

The
Subcommittee
was
concerned
that
the
existing
animal
assays
in
Tier
I
may
not
be
sensitive
to
events
occurring
uniquely
in
the
fetus
or
in
the
developing
neonate/
weanling.
The
31
development
of
a
limited
in
utero
assay
is
currently
under
study,
and
several
laboratories
are
evaluating
the
effects
of
a
range
of
endocrine
active
chemicals
on
sexual
development
of
perinatal
rats
and
mice
(
Gray
et
al.,,
1999a;
1999b).
Uterotrophic
effects
in
the
female
weanling
rat
are
already
incorporated
as
an
alternative
assay
in
the
EDSTAC
proposals,
and
work
is
being
done
on
the
male
weanling
at
present
(
Ashby,
and
Lefevre,
1997a;
Gray
et
al.,
1997).

We
recommend
that
these
assays
be
kept
under
close
review,
with
attention
focused
on
the
results
obtained
when
testing
the
activity
of
the
same
agents
with
the
different
types
of
assay.
It
may
be
that
data
will
eventually
indicate
that
one
or
other
of
these
classes
of
assay
can
replace
the
existing
rodent
assays
in
Tier
I,
but
a
well
constructed,
robust,
database
will
be
needed
before
such
a
decision
can
be
made.
The
Subcommittee
endorsed
strongly
the
continuing
evaluation
of
endocrine
disruption
assays
that
cover
the
periods
of
gestation
and
sexual
development.

3.4.10
Subcommittee
Recommendations
to
Help
EPA
Meet
its
Charge
The
final
element
of
the
Charge
(
d)
(
11)
asked
the
Joint
Subcommittee
for
any
other
suggestions
or
recommendations
that
would
help
EPA
meet
its
charge.

The
body
of
this
report
provides
specific
recommendations
concerning
the
screening
and
testing
of
endocrine
disruptors,
as
posed
by
the
Charge.
The
following
section
of
this
report
contains
a
summary
of
our
major
findings
and
recommendations
including
some
issues
not
included
in
the
Charge
which
arose
during
the
Subcommittee's
public
meeting
and/
or
the
development
of
this
report..
32
4.
MAJOR
FINDINGS
AND
RECOMMENDATIONS
This
section
highlights
a
variety
of
recommendations
and
concerns
discussed
at
the
public
meeting,
or
generated
during
the
preparation
of
this
report.
These
findings
are:

a)
Evaluating
the
Program:
We
wish
to
reinforce
the
comments
concerning
the
lack
of
on­
going
program
evaluation
noted
in
section
3.1.1
of
this
report.
We
find
no
provision
for
mid­
course
evaluation
or
optimization
of
the
process.
The
Agency
is
mandated
to
assemble
and
evaluate
this
proposed
panel
of
tests
and
then
to
implement
them,
but
a
correlate
responsibility
is
to
make
sure
that
what's
being
done
is
the
best
that
can
be.
Edmund
Burke's
"
You
can
never
plan
the
future
by
the
past,"
and
Robert
Burns'
"
The
best­
laid
plans
of
mice
and
men
oft
gang
agley."
both
apply
here.
For
example,
evaluation
of
minced
testis
and
minced­
ovary
assays
finds
them
to
be
only
50%
effective
in
identifying
compounds
that
inhibited
steroid
biosynthesis
(
Powlin
et
al.,
1998).
Although
something
looks
fine
on
paper
or
in
a
small
research
setting,
translating
it
into
volume­
screening
mode
may
be
quite
another
thing.
There
was
broad
support
among
the
Subcommittee
for
the
concept
that
the
Agency
should
convene
a
panel
of
independent
scientists
to
review
all
the
screening
data
for
50­
100
compounds,
with
an
eye
towards
revising
the
process
and
eliminating
those
methods
that
don't
work.
The
dictum
that,
the
more
removed
a
screen
is
from
a
whole
model,
the
more
wrong
the
answers
are
likely
to
be,
also
supports
the
need
for
such
a
review.

Finally,
we
believe
that
the
regulated
community
and
the
public
interest
groups
would
be
more
willing
to
participate
if
they
knew
that
the
system
was
going
to
be
optimized
as
it
proceeded.
The
Agency
should
have
one
or
more
evaluations
of
the
process
as
we
proceed
with
this,
and
the
Subcommittee
strongly
encourages
this.

b)
Mixture
Issues:
Discussions
at
the
public
meeting
focused
on
whether
to
include
mixtures
in
the
listing
of
materials
to
be
screened
and
tested.
The
Subcommittee
agreed
that
the
initial
focus
of
the
methods
development
effort
must
necessarily
focus
on
single
compounds
and
leave
the
question
of
testing
of
mixtures
until
accepted
single­
compound
methods
have
been
completed.
However,
Agency
representatives
underscored
the
need
to
apply
the
methods
to
testing
of
effluents
and
source
waters
which
are
obviously
complex
mixtures.
The
Subcommittee
concluded
that
very
promising
methods
already
exist
in
the
field
of
ecotoxicology.
These
include
the
Whole
Effluent
Testing
(
WET)
and
Toxicity
Identification
Evaluation
(
TIE)
procedures
developed
by
the
Agency
in
concert
with
the
Society
for
Environmental
Toxicology
and
Chemistry.
Those
methods
have
been
developed
to
test
effects
of
effluents
and
would
have
direct
application
to
the
Endocrine
Disruptor
Screening
Program.
33
c)
Case
Studies:
The
Subcommittee
strongly
encourages
the
Agency
to
include
more
and
better­
detailed
case
studies
in
the
evolution
of
the
priority­
setting
scheme.
Case
studies
will
enable
a
realistic
test
of
the
scheme,
checking
sensitivity
of
the
system
and
its
working
practicality
to
actually
prioritize
chemicals
for
further
testing.

d)
Sub­
population
Compartment:
The
question
of
the
need
for
a
separate
defined
compartment
to
address
sub­
populations
(
i.
e.,
human
infants)
was
addressed
to
the
Subcommittee.
Our
conclusions
supported
the
use
of
sub­
populations
as
a
criterion
within
the
existing
compartments
already
identified,
but
not
as
a
separate
stand­
alone
compartment.

e)
Use
of
IRIS:
The
priority
testing
scheme
relies
on
the
use
of
several
databases
summarizing
the
environmental
fate
and
effects
of
chemicals.
Caution
was
expressed
by
several
members
of
the
Committee
that
there
are
numerous
problems
with
the
validation
of
IRIS
and
other
databases
as
well.
Before
placing
heavy
reliance
on
these
computerized
information
systems,
users
need
to
be
aware
of
these
validation
problems
and
proceed
with
caution
before
incorporating
these
data
at
their
face
value.

f)
Exposure:
Although
the
issue
was
not
explicitly
stated
in
the
Charge,
the
Subcommittee
expressed
concern
that
consideration
of
the
toxicological
implications
of
exposure
should
include
both
dose
and
timing
of
exposure,
particularly
with
respect
to
developmental
or
reproductive
events.
The
current
scheme
does
not
adequately
cover
the
time
aspect
of
exposure
and
this
needs
to
be
remedied
before
broad­
scale
application
of
the
approach.
e
of
exposure.
The
current
scheme
does
not
adequately
cover
the
time
aspect
of
exposure
and
this
needs
to
be
remedied
before
broad­
scale
application
of
the
approach.

g)
Use
of
Animals:
During
the
public
meeting,
concern
was
expressed
about
the
large
number
of
animals
that
would
be
needed
in
the
EDSTAC
program.
The
Subcommittee
was
asked
whether
alternatives
and
approaches
to
minimize
animal
use
had
been
appropriately
considered
in
developing
the
protocols.
The
Subcommittee
pointed
out
the
essential
role
animals
play
in
tests
to
detect
endocrine
disruption
to
reveal
adverse
effects
on
humans.
There
are
no
substitutes
for
tests
currently
available
for
the
Tier
1
or
Tier
2
tests
using
animals.
Because
of
the
complexity
of
the
biological
systems
involved
in
endocrine
disruptor
detection,
animals
will
remain
a
necessary
model
for
the
foreseeable
future.
Additional
comments
by
Subcommittee
Members
and
others
described
protocol
or
method
modifications
which
would
be
less
expensive,
faster
and
use
fewer
animals.
The
Agency
has
an
obligation
to
conserve
all
resources
in
developing
new
testing
protocols,
and
the
use
of
animals
in
such
tests
poses
both
ethical
and
practical
problems
34
h)
Need
for
an
Introductory
Statement:
The
previous
EDSTAC
meeting
suggested
that
the
final
document
needed,
as
a
introductory
section,
a
description
of
the
problem
or
the
scientific
or
health­
based
reason
for
the
EDSTAC
program
(
1998).
Although
the
anticipated
NAS/
NRC
report
is
expected
to
address
this
issue,
the
Joint
Subcommittee
now
urges
the
EDSTAC
team
to
include
a
description
of
both
the
health
and
ecological
problems
associated
with
exposure
to
the
endocrine
disruptors
and
to
show
how
the
program
relates
to
these
findings.

i)
Support
for
Decisions:
Decisions
about
which
assays
are
selected,
and
which
protocols
are
adopted
for
those
assays,
should
be
supported
with
data
that
are
generally
available.

j)
Exceptions:
Testing
strategies
will
always
have
exceptions.
Care
should
be
taken
to
be
aware
of
the
imperfect
nature
of
any
future
agreed
strategy.
In
particular,
there
is
the
present
danger
that
the
two
chemicals
dibutyl
phthalate
(
DBP)
and
methoxychlor
(
MC)
will
have
an
undue
influence
on
the
future.
The
status
of
DBP
as
an
anti­
androgen
(
Gray
et
al.,
1999c;
Bulger
et
al.,
1978)
has
yet
to
be
fuly
documented..
MC
is
sometimes
reported
as
being
inactive
for
in
vitro
estrogenicity
assays,
thereby
providing
a
precedent
for
using
animals
in
a
screening
mode.
However,
this
chemical
has
been
published
as
being
active
as
an
estrogen
in
vitro
(
Ashby,
1997b).

k)
Negative
Control
Agents:
There
is
a
need
to
define
and
agree
on
some
negative
control
agents
for
ED
assay
validation.
It
has
been
suggested
that
the
only
valid
one
at
present
is
diethyl
phthalate.
(
Foster,
1980).
This
position
is
supported
by
the
fact
that
it
gave
negative
results
in
a
full
and
updated
rodent
multi­
generation
study
at
the
National
Institute
of
Environmental
Health
Sciences
(
Chapin,
1997).
However,
this
view
is
questioned
by
work
in
progress
by
Ashby
and
by
Gray
indicating
positive
activity.
Assay
specificity
will
not
be
capable
of
assessment
unless
additional
reliable
negative
agents
can
be
found
and
made
available
for
general
study.

l)
Animal
Tests
and
Routes
of
Exposure:
As
noted
above,
MC
stands
alone
as
the
only
precedent
for
why
animals
should
be
used
in
the
screening
mode
(
Tier
1).
There
is
significant
international
concern
on
the
proposed
use
of
animals
for
screening.
Ashby
and
Lefevre
(
1997a)
proposed
that
short
term
animal
studies
should
be
recognized
as
an
intermediate
Tier,
much
as
happens
now
with
the
anticipation
of
animal
carcinogens
[
screen
in
vitro,
assess
in
short
term
in
vivo
assays,
and
then
define
in
lifetime
bioassays].
In
this
role
of
hazard
assessment
(
as
opposed
to
hazard
definition)
biologically
relevant
routes
of
exposure
would
be
indicated
(
oral
gavage,
diet,
water,
inhalation,
skin
painting).
At
present,
use
of
the
subcutaneous
injection
or
intra
peritoneal
injection
routes
are
recommended
in
the
frail
quest
of
increasing
assay
sensitivity.
In
fact,
irrespective
of
the
outcome
35
of
this
suggestion
it
should
be
noted
that
the
current
EPA
synthesis
of
the
EDSTAC
recommendations
is
inconsistent
on
the
matter
of
route
of
exposure
­­
the
uterotrophic
assay
uses
subcutaneous
or
intraperitoneal
injection,
the
Hershberger
assay
oral
gavage,
the
multi
generation
assay
uses
diet/
oral/
inhalation,
and
no
route
is
identified
for
the
pubertal
male
and
female
assays.

The
main
contributors
to
differences
in
test
outcome
between
assays
conducted
in
vitro
and
assays
in
rodents
will
be
delivered
dose,
pharmacodynamics,
and
pharmacokinetics.
Route
of
exposure
will
dominate
these
factors.
The
ultimate
role
to
be
adopted
for
animal
studies,
and
the
route
of
animal
exposures,
will
have
the
greatest
impact
on
the
successful
implementation
of
the
EPA
initiatives
in
the
area
of
endocrine
disruption.

m)
Expanding
the
Universe
of
Agents:
Developing
massive
amounts
of
screening
information
on
a
large
universe
of
chemicals
does
not
necessarily
expedite
the
development
of
the
appropriate
scientific
underpinning
that
the
Agency
needs
to
broaden
this
effort.
Consequently,
the
Subcommittee
recommends
that
EPA
should
not
expand
the
set
of
agents
until
the
Agency
develops
or
adopts
validated
systems
and
can
provide
clear
decision
criteria.
R­
1
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