DRAFT
STUDY
PLAN
FOR
PREVALIDATION
OF
THE
SLICED
TESTIS
ASSAY
EPA
Contract
Number
68­
W­
01­
023
WA
3­
5,
Task
16
August
1,
2003
PREPARED
FOR
GARY
E.
TIMM
WORK
ASSIGNMENT
MANAGER
U.
S.
ENVIRONMENTAL
PROTECTION
AGENCY
ENDOCRINE
DISRUPTOR
SCREENING
PROGRAM
WASHINGTON,
D.
C.

BATTELLE
505
KING
AVENUE
COLUMBUS,
OH
43201
Battelle
Report
i
August,
2003
TABLE
OF
CONTENTS
Page
1.0
ENDPOINT
MEASUREMENTS/
BACKGROUND
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3
1.1
STEROIDOGENESIS
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4
1.1.1
Signal
Transduction
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4
1.1.2
Cholesterol
Synthesis
and
Transport
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5
1.1.3
Enzymatic
Conversions
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5
1.2
SLICED
TESTIS
STEROIDOGENESIS
ASSAY
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7
1.2.1
Basis
for
Selection
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7
1.2.2
Summary
of
Optimization
Studies
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9
1.2.3
Sliced
Testis
Steroidogenesis
Assay
Procedure
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10
2.0
REFERENCE
CHEMICAL
RESOLUTION
ISSUES
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11
2.1
Chemical
Characterization
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12
2.2
Formulation
Development
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12
2.3
Formulation
Analysis
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13
2.4
Stability
Determination
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13
3.0
STUDY
DESIGN
(
PREVALIDATION
OF
THE
SLICED
TESTIS
ASSAY)
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14
3.1
BASELINE
AND
CONTRALATERAL
TESTIS
FRAGMENT
STUDY
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14
3.2
EVALUATION
OF
AMINOGLUTETHIMIDE
(
AG)
AS
A
POSITIVE
CONTROL
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17
3.3
SELECTION
OF
A
CYTOTOXICANT
AS
A
POSITIVE
CONTROL
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19
3.4
TRAINING
AND
HANDS­
ON
EXPERIENCE
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21
3.5
BASELINE
EXPERIMENT
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22
3.6
POSITIVE
CONTROL
EXPERIMENT
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24
3.7
MULTICHEMICAL
EXPERIMENTS
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27
4.0
RECOMMENDED
REFERENCE
CHEMICALS
FOR
MULTICHEMICAL
TESTING
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29
5.0
STUDY
PROTOCOL
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30
6.0
DATA
FORMAT
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30
7.0
GLP
MONITORING­
QUALITY
ASSURANCE
UNIT
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31
8.0
TECHNICAL
MONITORING/
COORDINATION
­
ADMINISTRATIVE
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31
9.0
REFERENCES
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32
Battelle
Report
ii
August,
2003
List
of
Figures
Page
Figure
1.
Sliced
Testis
Assay
Experimental
Design
for
Optimization
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2
Figure
2.
Intracellular
Biochemical
Steroidogenic
Pathway
Following
Trophic
Hormone
Stimulation
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4
Figure
3.
Enzymatic
Conversions
of
Cholesterol
and
Intermediate/
End­
Product
Hormones
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6
Figure
4.
Technical
Flow
Illustration
of
the
Sliced
Testis
Steroidogenesis
Assay
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10
List
of
Tables
Table
1.
Prevalidation
Study
Plan
Experiments
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3
Table
2.
Representative
Studies
Using
the
Sliced
Testis
Assay
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8
APPENDIX
A
 
Draft
Protocol
for
the
Prevalidation
of
the
Sliced
Testis
Assay
APPENDIX
B
 
Data
Coordination
and
Distribution/
Report
Submission
Battelle
Report
1
August,
2003
Endocrine
Disruptor
Screening
Program
Contract
No.
68­
W­
01­
023
Work
Assignment
3­
5,
Task
16
Study
Plan
for
Prevalidation
of
the
Sliced
Testis
Assay
INTRODUCTION
In
1996,
the
Food
Quality
Protection
Act
(
FQPA)
amendments
were
enacted
by
Congress
to
authorize
the
EPA
to
implement
an
Endocrine
Disruptor
Screening
Program
(
EDSP)
on
pesticides
and
other
substances
found
in
food
or
water
sources
for
endocrine
effects
in
humans
(
FQPA,
1996).
In
this
program,
comprehensive
toxicological
and
ecotoxicological
screens
and
tests
are
being
developed
for
identifying
and
characterizing
the
endocrine
effects
of
various
environmental
contaminants,
industrial
substances,
and
pesticides.
A
two­
tiered
approach
will
be
utilized.
Tier
1
employs
a
combination
of
in
vivo
and
in
vitro
screens,
and
Tier
2
involves
in
vivo
testing
methods
using
two­
generation
reproductive
studies.
A
steroidogenesis
assay
is
proposed
as
one
of
the
Tier
1
screening
battery
assays.

A
detailed
review
paper
(
DRP)
about
steroidogenesis
was
prepared
(
EPA
2002).
The
DRP
(
1)
summarized
the
state
of
the
science
of
the
in
vivo,
ex
vivo,
and
in
vitro
methodologies
available
for
measuring
gonadal
steroidogenesis;
(
2)
for
each
methodology,
presented
a
review
of
the
individual
assays
and
representative
data
generated
by
investigators
who
used
the
assay
to
evaluate
a
substance
for
steroidogenic­
altering
activity;
(
3)
provided
an
evaluation
of
the
various
methodologies
and
the
assays
as
tools
for
screening
substances
with
suspected
steroidogenic
activity;
(
4)
recommended
a
particular
screening
method
and
assay
as
a
screening
tool;
and
(
5)
described
the
strengths,
weaknesses,
and
implications
for
further
research
associated
with
the
recommended
screening
assay.

Although
a
promising
tool,
the
sliced
testis
assay
remains
to
be
fully
tested
as
an
assay
that
can
meet
all
the
demands
of
an
endocrine
disruptor
screening
tool.
Concerns
raised
by
the
EPA
and
EDMVS
during
discussions
on
June
11,
2002,
and
thereafter
suggested
that
experiments
be
conducted
to
ensure
the
optimization
of
the
assay
prior
to
more
rigorous
prevalidation
and
validation
testing.
The
most
notable
concerns
were
associated
with
1)
various
incubation
variables,
2)
variables
that
affect
optimal
hCG
stimulation,
3)
characterization
of
the
parenchymal
post­
slicing
equilibration
time,
4)
parenchymal
viability,
and
5)
detection
of
Leydig
cell
toxicity.
In
addition
to
these
most
notable
concerns,
other
factors
that
could
potentially
affect
the
optimal
performance
of
this
assay
were
identified.
The
factors
tested
are
illustrated
in
Figure
1.
The
results
of
these
experiments
have
been
evaluated
to
a
large
extent
but
the
evaluation
is
not
completed.
Upon
completion,
sensitivity
analysis
of
the
optimization
study
results
will
also
be
performed
in
order
to
determine
whether
a
few
additional
tests
are
needed.
If
not,
then
the
optimization
study
results
will
be
used
to
proceed
with
assay
prevalidation.
Battelle
Report
2
August,
2003
Figure
1.
Sliced
Testis
Assay
Experimental
Design
for
Optimization
The
planned
purpose
for
the
sliced
testis
assay
is
to
develop
a
screening
tool
that
will
assess
the
steroidogenic
pathway
capacity
of
the
Leydig
cell
so
as
to
identify
substances
with
toxic
effects
on
hormone
production.
The
objective
of
this
study
plan
is
to
use
the
now
optimized
assay
to
obtain
baseline
(
media
only)
information,
obtain
intra­
and
inter­
laboratory
variability
estimates,
identify
a
chemical
and
corresponding
concentration
that
can
be
used
as
a
positive
control,
evaluate
cytotoxicants
for
assay
responsiveness
and
selection
of
a
cytotoxicant
positive
control,
and
test
several
chemicals
with
different
modes
of
action
in
order
to
test
assay
relevance.
The
study
plan
includes
using
multiple
laboratories
­
one
lead
laboratory
and
three
participating
laboratories.
Also,
the
study
plan
describes
the
training
procedures
to
be
used
to
transfer
the
assay
conduct
method
from
the
lead
laboratory
to
the
participating
laboratories.
The
study
plan
distributes
the
experiments
to
the
lead
laboratory
and/
or
participating
laboratories
based
on
the
level
of
experience
a
given
lab
has
conducting
the
assay
and
purpose
for
conducting
Battelle
Report
3
August,
2003
the
experiments.
If
information
is
needed
that
will
eventually
be
incorporated
into
the
design
of
the
experiment,
then
only
the
lead
laboratory
was
assigned
to
conduct
the
study.
Obviously,
if
inter­
laboratory
variability
estimates
were
a
goal,
then
the
participating
laboratories
were
involved.
Table
1
below
summarizes
the
prevalidation
experiments,
order
that
the
studies
will
be
conducted,
and
laboratory
assignments.

Table
1.
Prevalidation
Study
Plan
Experiments
Study
Number
Study
Type
Laboratory
Assignment
1
Baseline/
Contralateral
Testis
Fragment
Lead
Laboratory
2
Evaluation
of
AG
as
Positive
Control
Lead
Laboratory
3
Selection
of
Cytotoxicant
as
Positive
Control
Lead
Laboratory
4
Training/
Hands­
On
Experience
Lead
Laboratory
+
3
Participating
Laboratories
5
Baseline
Experiment
Lead
Laboratory
+
3
Participating
Laboratories
6
Positive
Control
Experiment
Lead
Laboratory
+
3
Participating
Laboratories
7
Multichemical
Experiments
Lead
Laboratory
Prevalidation
of
the
sliced
testis
assay
is
the
main
focus
of
this
study
plan.
This
study
plan
covers
the
following
topics:

°
Endpoint
measurements/
background
information
°
Protocol
issues
needing
resolution
°
Study
design
to
address
the
protocol
issues
°
Recommended
test
substances
°
The
detailed
study
protocol
°
Statistical
methods
for
comparing
the
performance
of
the
assay
°
Data
format.

1.0
ENDPOINT
MEASUREMENTS/
BACKGROUND
Steroidogenesis
is
a
specific
pathway
of
chemical
reactions
that
result
in
the
production
of
gonadal
intermediary
and
end­
product
hormones.
The
pathway
(
1)
begins
with
intracellular
signal
transduction,
(
2)
continues
with
cholesterol
production
in
the
cytoplasm
and
transport
to
the
mitochodrial
inner
membrane,
and
(
3)
ends
with
a
set
of
multi­
step
enzymatic
conversions
from
cholesterol
to
the
end­
product
hormones.
Battelle
Report
4
August,
2003
1.1
STEROIDOGENESIS
1.1.1
Signal
Transduction
Signal
transduction
describes
the
intracellular
biochemical
reactions
that
occur
after
stimulation
of
the
luteinizing
hormone
(
LH)
membrane
bound
receptor
and
up
to
initiation
of
cholesterol
transport
to
the
mitochondria.
The
LH
receptor
is
coupled
with
a
G­
protein
and,
when
stimulated
by
LH
or
human
chorionic
gonadotropin
(
hCG),
interacts
with
adenylate
cyclase
to
form
cyclic
adenosine
3',
5'­
cyclic
monophosphate
(
cAMP).
Increased
cAMP,
the
second
messenger,
stimulates
protein
kinase
A,
which
initiates
cholesterol
biosynthesis
and
cholesterol
transport
protein
synthesis
(
Cooke,
1996;
Stocco,
1999).
The
receptor­
mediated
biochemical
reactions
are
illustrated
in
Figure
2
(
Stocco,
1999).

Figure
2.
Intracellular
Biochemical
Steroidogenic
Pathway
Following
Trophic
Hormone
Stimulation
Calcium
(
Ca2+)
is
involved
in
the
signal
transduction
of
the
steroidogenic
pathway
(
Janszen
et
al.,
1976).
In
order
for
the
maximal
stimulation
of
steroidogenesis
to
occur,
intracellular
calcium
levels
must
increase
following
LH
binding.
The
calcium­
mediated
reactions
also
involve
calmodulin,
a
calcium
binding
protein
(
Hall
et
al.,
1981).
Chloride
(
Cl­)
and
arachidonic
acid
have
also
been
implicated
in
steroidogenic
signal
transduction
(
Choi
and
Cooke,
1990;
Naor,
1991;
Cooke,
1996).
Arachidonic
acid
appears
to
produce
a
direct
inhibitory
effect
and
an
indirect
stimulatory
effect
on
steroidogenesis.
Steroid
hormone
production
is
inhibited
when
arachidonic
acid
activates
protein
kinase
C.
However,
metabolism
of
arachidonic
acid
to
its
metabolites,
e.
g.,
leukotrienes,
stimulates
cholesterol
transport
into
the
mitochondria,
thereby
enhancing
steroid
hormone
production.
Other
intracellular
substances
shown
to
affect
steroidogenesis
include
free
radicals,
i.
e.,
superoxide
anion
and
hydroxyl
free
radical,
as
well
as
hydrogen
peroxide
and
nitric
oxide
(
Clark
et
al.,
1994;
Davidoff
et
al.,
1995).
Battelle
Report
5
August,
2003
1.1.2
Cholesterol
Synthesis
and
Transport
Cholesterol
is
the
common
precursor
to
the
formation
of
all
gonadal
steroid
hormones.
The
primary
source
of
cellular
cholesterol
is
the
serum.
Cholesterol
is
transported
to
the
cell
via
serum
protein
carriers,
e.
g.,
high­
or
low­
density
lipoprotein
(
HDL
or
LDL).
Once
inside
the
cell,
cholesterol
is
immediately
utilized,
or
it
can
be
stored,
e.
g.,
in
lipid
droplets.
A
second,
minor
source
of
cholesterol
is
de
novo
synthesis,
which
increases
following
hormone
stimulation.
Upon
LH­
induced
stimulation,
mobilization
of
newly
synthesized
and
stored
cholesterol
(
enzymatic
hydrolysis
of
cholesterol
esters)
in
lipid
droplets
occurs.
Cholesterol
is
transported
out
of
the
cytoplasm
and
into
the
mitochondria.
In
the
mitochondria,
cholesterol
is
transported
from
the
outer
to
the
inner
membrane,
which
is
the
rate­
limiting
step
in
steroidogenesis.
The
transport
of
cholesterol
from
the
outer
to
the
inner
mitochondrial
membrane
requires
a
transport
protein.
LH
stimulation
of
steroidogenic
cells
activates
de
novo
production
of
the
cholesterol
transport
protein.
This
protein
is
essential
for
steroidogenesis
and,
since
it
mediates
the
rate­
limiting
step
of
steroid
hormone
production,
it
is
referred
to
as
the
steroid
acute
regulatory
(
StAR)
protein.
In
the
mitochondria,
StAR
protein
transports
cholesterol
to
the
inner
mitochondrial
membrane,
where
the
side­
chain
cleavage
enzyme
(
P450SCC)
catalyzes
cholesterol
into
pregnenolone.

1.1.3
Enzymatic
Conversions
Enzymatic
conversion
of
cholesterol
to
pregnenolone
constitutes
the
initial
step
in
a
series
of
biochemical
reactions
that
culminate
in
end­
product
hormone
production.
Figure
3
illustrates
the
final
stage
of
the
steroidogenic
biosynthetic
pathway,
as
well
as
the
cell
types
for
males
and
females
and
the
intracellular
location
of
various
enzymatic
steps
of
the
steroidogenic
pathway.

The
first
enzyme
reaction
is
the
conversion
of
cholesterol
to
pregnenolone
by
the
cytochrome
P450
cholesterol
side­
chain
cleavage
enzyme
(
P450SCC).
P450SCC
activity
is
also
considered
to
be
a
rate­
limiting
step
in
the
production
of
gonadal
steroid
hormones
(
Kagawa
&
Waterman,
1995).

The
second
enzymatic
reaction
results
in
the
conversion
of
pregnenolone
to
progesterone
by
the
enzyme
3$­
hydroxysteroid
dehydrogenase/
ª
5
­
ª
4
isomerase
(
3$­
HSD).
At
this
point,
the
steroidogenic
pathway
bifurcates
into
a
ª
5­
hydroxysteroid
pathway
(
starting
with
pregnenolone)
and
a
ª
4­
ketosteroid
pathway
(
starting
with
progesterone)
and,
even
though
the
same
enzymes
use
different
substrates
along
the
parallel
pathways,
both
pathways
eventually
converge,
culminating
in
the
production
of
androstenedione.
Battelle
Report
6
August,
2003
'
'
Corpus
Luteum
Figure
3.
Enzymatic
Conversions
of
Cholesterol
and
Intermediate/
End­
Product
Hormones
'
Theca
Cells
Leydig
Cells
Granulosa
Cells
Testis
and
Peripheral
Tissues
Intracellular
Compartment
(
Females
Only)

(
Males
Only)
Mitochondria
Cytoplasm
Testis
Ovary
Battelle
Report
7
August,
2003
The
third
enzymatic
reaction
involves
cytochrome
P450
17"­
hydroxylase/
C17
­
20
lyase
(
P450c17).
For
the
ª
5­
hydroxysteroid
pathway,
P450c17
initially
catalyzes
the
conversion
of
pregnenolone
to
17"­
hydroxypregnenolone,
which
is
then
converted
to
DHEA.
As
mentioned
above,
DHEA
is
converted
to
androstenedione
by
3$­
HSD.
Likewise
for
the
ª
4­
ketosteroids,
P450c17
converts
progesterone
to
17"­
hydroxyprogesterone,
which
is
then
converted
to
androstenedione.

The
next
enzymatic
reaction
involves
the
conversion
of
androstenedione
to
testosterone
by
17­
ketosteroid
reductase
(
17KSR),
which
is
also
referred
to
as
17$­
hydroxysteroid
dehydrogenase
(
17$­
HSD).
The
production
of
testosterone
is
considered
an
end­
hormone
product.
A
second
possible
reaction
involving
androstenedione
occurs
in
the
female,
whereby
androstenedione
is
converted
to
estrone
by
aromatase.

In
the
male,
testosterone
is
converted
to
dihydrotestosterone
(
DHT)
by
5"­
reductase.
DHT
is
significantly
more
potent
as
an
androgen
than
testosterone
and
is
also
considered
an
endproduct
hormone.
DHT
is
produced
primarily
in
peripheral
tissues,
although
it
is
also
found
in
the
testis.

The
last
enzyme
in
the
steroidogenic
pathway
is
aromatase.
Aromatase
converts
testosterone
into
estradiol
and,
in
the
female,
androstenedione
into
estrone.
In
short,
aromatase
converts
androgenic
substances
into
estrogenic
substances.
As
mentioned
above
for
testosterone
and
DHT,
estradiol
and
estrone
are
considered
end­
product
hormones
of
the
steroidogenic
pathway.
Aromatase
is
found
in
many
different
peripheral
tissues,
as
well
as
male
and
female
gonadal
tissue.

In
summary,
cholesterol
is
the
common
precursor
for
production
of
steroid
hormones.
A
series
of
biochemical
reactions
involving
different
enzymes
results
in
conversion
of
cholesterol
to
end­
hormone
products:
testosterone,
DHT,
estradiol,
and
estrone.
The
steroidogenic
pathway
is
regulated
by
gonadotropins
and
end­
product
hormones.
An
alteration
of
the
regulatory
mechanisms,
as
well
as
direct
effects
on
the
substrates
and
enzymes
of
the
steroidogenic
pathway,
can
affect
end­
hormone
product
formation,
thereby
possibly
resulting
in
reproductive
system
toxicity.

1.2
SLICED
TESTIS
STEROIDOGENESIS
ASSAY
1.2.1
Basis
for
Selection
Steroid
hormones
produced
by
the
gonads
affect
most
of
the
organs
in
the
body
including
bone,
muscle,
brain,
and
reproductive
organs.
It
is
for
this
reason
that
the
EDSTAC
recommended
that
an
assay
that
measures
steroidogenic
function
be
considered
as
a
component
of
the
Tier
1
Screening
(
T1S)
battery.
An
evaluation
of
steroidogenic
assays
and
the
criteria
for
a
screen
as
presented
in
the
DRP
on
steroidogenesis
resulted
in
selection
of
the
sliced
testis
assay.
The
objective
of
this
assay
is
to
detect
disruption
of
the
steroidogenic
pathway.
It
may:
(
1)
be
used
as
one
of
the
protocols
recommended
by
EDSTAC
for
the
Tier
1
screening
battery,
Battelle
Report
8
August,
2003
(
2)
serve
as
a
follow­
up
test
for
certain
substances
for
which
additional
data
are
required
or
desired,
and/
or
(
3)
predict
the
likelihood
that
steroidogenesis
and
downstream
biologically
dependent
processes
would
be
affected
by
the
same
or
similar
substances
in
vivo.
The
endpoint,
testosterone,
was
selected
for
its
potential
to
detect
toxicant­
induced
alterations
of
steroidogenesis
in
testicular
tissue.

This
in
vitro
assay
has
been
used
with
fetal,
neonatal,
and
adult
testes,
and
is
not
limited
to
mammalian
species,
having
been
used
to
assess
steroidogenesis
in
fish,
reptile,
avian,
and
amphibian
systems
as
well.
Thus,
the
steroidogenesis
bioassay
as
a
component
in
the
T1S
phase
should
be
broadly
understood
to
screen
for
any
disruption
of
the
overall
steroid
biosynthetic
pathway.
The
sliced
testis
steroidogenesis
assay
has
the
capacity
to
evaluate
simultaneously
all
of
the
processes
involved
with
gonadal
synthesis
of
steroid
hormones
(
signal
transduction,
transcription,
translation,
synthesis,
and
cellular
secretion
of
the
steroids).

The
sliced
testis
assay
has
been
used
to
identify
substances
that
alter
steroidogenesis.
Examples
of
experimental
studies
from
the
literature
that
used
the
sliced
testis
assay
for
measuring
steroidogenesis
are
summarized
in
Table
2.

Table
2.
Representative
Studies
Using
the
Sliced
Testis
Assay
Animal/
Type
of
Preparation
Treatment
&
Stimulant
Measured
Response
Reference
Crl:
CD
BR
rats/
Testes
Slices
(
50
mg)
Various
conditions,
factors,
and
chemicals
Various
changes
in
testosterone
production
Powlin
et
al.,
1998
Adult
male
Long­
Evans
rats/
Testes
Slices
(
1/
4)
Ethane
dimethanesulfonate
@
0,
3,
10,
32,
100,
320,
1000,
or
3200
µ
g/
mL
media/
ovine
LH
(
100
ng/
mL)
9
testosterone
production
Gray
et
al.,
1995
Male
Long­
Evans
Hooded
rats
(
3­
25
weeks
of
age)/
Testes
Slices
(
1/
4)
Vinclozolin
@
5
to
100
mg/
kg/
day,
gavage,
for
22
weeks/
hCG,
50
IU
8
basal
and
hCG
stimulated
testosterone
@
15
and
100
mg/
kg/
day
Fail
et
al.,
1995
Male
Long­
Evans
Hooded
rats
(
3­
14
weeks
of
age)/
Testes
Slices
(
1/
4)
Methoxychlor
@
50
or
200
mg/
kg/
day,
gavage,
for
11
weeks/
hCG,
50
IU
9
basal
testosterone
production
no
effect
on
HCG
stimulated
testosterone
production
Fail
et
al.,
1994
Adult
male
SD
rat/
Testes
Slices
(
1/
4)
Ethane
dimethanesulfonate
@
0,
500,
or
3000
µ
M/
100
mIU/
mL
hCG
9
testosterone
production
Laskey
et
al.,
1994
Adult
male
OFA
rat/
Testis
Slices
(~
1/
4)
14C­
pregnenolone
(
50
mCi/
mmol;
200
nCi
­
a
tracer
amount)
~
70
and
15
percent
of
the
14Cradioactivity
was
testosterone
and
androstenedione
Gurtler
and
Donatsch,
1979
In
summary,
the
most
salient
features
of
this
assay
and
the
basis
for
its
consideration
are
that
it
identifies
substances
that
alter
steroid
hormone
production
and
can
be
conducted
at
a
minimal
cost,
quickly,
and
simply
with
standard
laboratory
equipment
and
basic
laboratory
training,
which
are
all
important
characteristics
of
an
assay
to
be
used
as
a
screen
for
identifying
Battelle
Report
9
August,
2003
substances
with
steroidogenic­
altering
activity.
The
sliced
testis
assay
is
stable
and
the
parenchyma
remains
viable
over
a
sufficient
time
period
to
measure
changes
in
end­
product
hormone
production.
In
addition,
the
assay
is
relatively
sensitive
and
specific;
uses
parenchyma
that
maintains
the
cytoarchitecture
of
the
organ;
uses
a
reduced
number
of
animals;
will
be
relatively
easy
to
standardize
(
by
optimization);
and
has
a
well­
defined
endpoint
in
testosterone,
which
can
be
modified
to
include
additional
intermediate
hormonal
endpoints
if
so
desired.

1.2.2
Summary
of
Optimization
Studies
Although
the
advantages
and
strengths,
as
described
above,
provide
a
basis
for
further
consideration
of
this
assay
as
the
screening
tool
for
substances
that
alter
steroidogenesis,
the
sliced
testis
assay
also
has
some
limitations,
which
prompted
performing
experiments
to
optimize
the
assay.
Some
limitations
of
the
assay
are
not
able
to
be
resolved.
For
example,
substances
that
require
metabolic
activation
will
not
be
identified
as
substances
that
alter
steroid
hormone
production.
Also,
substances
that
are
insoluble
in
the
media
or
cannot
be
formulated
in
a
soluble
vehicle
are
unable
to
be
tested.
Aside
from
these
limitations,
there
are
a
number
of
variables
that
were
tested
in
order
to
determine
the
settings
that
optimized
the
assay.
An
important
objective
of
the
optimization
experiments
was
to
determine
the
level
of
a
given
factor
that
would
reduce
to
a
minimum
the
variability
of
the
assay.

The
experimental
design
factors
that
were
tested
in
an
attempt
to
optimize
the
sliced
testis
assay
are
illustrated
in
Figure
1.
Although
the
optimization
experiments
are
completed
and
the
results
analyzed,
a
complete
analysis
(
including
sensitivity
analysis)
is
in
progress.
However,
an
initial
evaluation
of
the
optimization
experimental
findings
suggest
the
following
as
preliminary
optimal
conditions:

°
incubation
temperature
­
34
to
37
degrees
°
vessel
type
­
scintillation
vial
°
shaker
speed
­
low
(~
135
rpm)
°
incubation
medium
volume
­
5
mL
°
hCG
concentration
­
0.1
IU/
mL
(
final
concentration)
°
parenchymal
fragment
wt
­
0.15
mg
(
range
0.130
­
0.170
mg)
°
testis
preparation
conditions
­
cold
buffered
saline
°
testis
preparation
time
­
less
than
1
hour
°
media
sampling
volume
­
less
than
0.5
mL
°
cell
damage
and
destruction
was
sensitive
to
LDH
analysis
The
present
prevalidation
study
plan
and
protocol
include
these
conditions
for
conducting
the
sliced
testis
assay
prevalidation
experiments.
However,
pending
complete
statistical
sensitivity
analysis,
these
conditions
are
subject
to
modification.
Battelle
Report
10
August,
2003
Shake
and
Incubate
at
34
°
C,
95%
O2,
5%
CO2
hCG
Challenge
(
stimulated)

Sliced
Rat
Testis
No
Challenge
(
unstimulated)

Time
Equilibration
Period
(
1
hr)
Add
Testis
Fragment
to
Media
Media
sampled;
then
add
media
and
hCG,
with
or
without
Test
Chemical
Media
Samples,
then
Add
Media
with
or
without
Test
Chemical
Discard
old
and
replenish
with
new
media
Media
Sample
Media
Sample
Media
Sample
Media
and
Tissue
Fragment
Sample
Equilibration
Period
(
1
hr)

T0
0
hr
Baseline
T1
+
1
hr
Post­
Challenge
T2
+
2
hr
Post­
Challenge
T3
+
3
hr
Post­
Challenge
T4
+
4
hr
Post­
Challenge
1.2.3
Sliced
Testis
Steroidogenesis
Assay
Procedure
As
can
be
deduced
by
the
previous
information,
the
sliced
testis
assay
has
been
used
by
many
researchers
over
the
past
couple
of
decades.
The
parameters
and
settings
that
are
used
can,
and
often
do,
vary
from
laboratory
to
laboratory
and
researcher
to
researcher.
However,
based
on
the
results
of
the
optimization
studies,
the
experimental
factors
and
their
levels
can
now
be
more
standardized.
The
optimal
assay
procedure
is
described
and
illustrated
below
(
Figure
4).

Figure
4.
Technical
Flow
Illustration
of
the
Sliced
Testis
Steroidogenesis
Assay
The
assay
procedure
will
begin
at
a
time
that
will
enable
the
technician
to
complete
all
incubations
and
sample
collections
before
approximately
noon
on
a
given
day
(
does
not
include
the
analyses).
An
11
to
15
week
old
male
Sprague­
Dawley
rat
will
be
euthanized.
The
testes
will
be
removed
[
develop
SOP
for
testis
removal
and
preparation].
The
testes
will
be
weighed
Battelle
Report
11
August,
2003
[
acceptance
criterion,
i.
e.,
>
1000
mg]
and
placed
in
cold
(
40
C)
Dulbeccos
Phosphate
Buffered
Saline
(
DPBS;
undiluted;
pH
7.2
­
7.4).
The
time
from
removal
of
the
testis
to
the
time
of
slicing
will
be
less
than
1
hour.
Each
testis
will
have
the
tunica
albicans
removed
and
will
be
sliced
longitudinally
to
yield
pieces
weighing
within
10%
of
0.150
grams
(
between
0.135
g
and
0.165
g).
Each
slice
will
be
placed
into
a
tightly
capped
scintillation
vial
containing
5
mL
of
95%
O2
/
5%
CO2
gassed
media.
[
develop
SOP
for
testis
slice
distribution
among
test
tubes].
The
media
will
be
medium­
199
without
phenol
red
(
Invitrogen),
which
will
have
been
adjusted
to
a
pH
of
7.4.

The
vials
containing
the
testicular
sections
and
media
will
be
incubated
at
340C
[
range
34
to
370C]
on
a
shaker
at
low
speed,
i.
e.,
135
rpm.
After
the
first
period
of
incubation,
1
hour,
the
contents
of
the
vial
will
be
gently
centrifuged.
The
equilibration
media
will
be
discarded
without
analysis.
New
media
(
5.0
mL)
will
be
placed
in
the
original
vial.
One
aliquot
(
0.5
mL)
of
media
[
need
further
evaluation
of
aliquot
volume]
will
be
collected
for
analysis
(
Time
0,
Baseline
Sample).
The
sample
aliquot
will
be
analyzed
for
testosterone.
[
develop
SOPs
for
testosterone
RIA]
To
one­
half
of
the
vials,
a
0.5
mL
volume
of
media
without
hCG
(
unstimulated)
will
be
added,
and
to
the
other
half
of
the
vials,
0.5
mL
of
media
with
hCG
(
stimulated)
will
be
added
to
the
vials
to
replace
the
volume
removed
for
analysis,
as
well
as
to
initiate
testosterone
production
(
hCG
stimulated
vials
only).
The
final
hCG
concentration
will
be
0.1
IU/
mL.
[
Note:
For
assays
using
a
test
chemical,
the
test
chemical
will
also
be
formulated
in
the
media
so
that
when
the
replacement
media
is
added,
at
time
0,
the
final
target
concentration
will
be
achieved.]

At
1,
2,
3
and
4
hours
post­
challenge,
one
0.5
mL
sample
(
or
smaller)
of
media
will
be
collected
from
each
of
the
hCG­
stimulated
and
non­
stimulated
vials.
After
each
sampling,
the
appropriate
media
with
or
without
hCG
will
be
added
to
the
vial
to
replace
the
volume
removed
for
analysis.
The
sample
aliquots
removed
at
each
time
point
will
be
analyzed
for
testosterone.
Also,
at
the
end
of
the
incubation
period,
e.
g.
4
hours,
remove
the
tissue
fragment
and
collect
a
sample
for
microscopic
examination.

Media
samples
collected
at
the
specified
time
points
will
be
frozen
and
stored
at
­
70
degrees
C
and
analyzed
within
one
month
after
collection.
Testosterone
will
be
analyzed
in
duplicate
for
each
sample
using
an
RIA
method
modified
for
rat
samples.
All
samples
for
a
given
day's
set
of
runs
will
be
analyzed
in
the
same
testosterone
RIA
,
if
possible.
Tissue
samples
will
be
thawed,
sectioned,
stained,
and
mounted
for
microscopic
examination.
The
staining
procedure
is
specific
for
beta­
HSD,
an
enzyme
specific
to
Leydig
cells.

2.0
REFERENCE
CHEMICAL
RESOLUTION
ISSUES
There
are
no
sliced
testis
assay
issues
that
require
resolution
by
experimentation
prior
to
initiation
of
the
protocol
included
in
this
study
plan.
The
protocol
is
believed
to
be
complete
and
ready
for
implementation.
However,
prior
to
implementation
of
the
multichemical
study,
there
are
chemistry
issues
that
must
be
resolved.
Reference
chemical
use
issues
include:
Battelle
Report
12
August,
2003
°
chemical
characterization
°
formulation
development
°
formulation
analysis
°
stability
determination
In
addition,
there
are
some
unanswered
questions
that
are
believed
necessary
to
address
during
prevalidation
in
order
to
design
the
validation
study
plan.
The
experiments
needed
to
obtain
this
additional
information
were
built
into
the
design
of
the
prevalidation
study
plan.
The
issues
yet
to
be
resolved
include:

°
contralateral
fragment
variability
°
evaluation
of
aminoglutethimide
as
a
positive
control
°
evaluation
of
cytotoxicants
The
experimental
design
for
these
studies
are
described
in
Section
4.0.

Prevalidation
will
use
a
variety
of
reference
chemicals
to
test
how
the
sliced
testis
assay
responds.
Some
chemicals
will
be
evaluated
for
their
use
as
a
positive
control
and
others
will
be
used
to
determine
the
relevance
of
the
assay.
These
latter
reference
chemicals
will
be
selected
to
have
different
modes
of
action.
All
such
chemical
usage
will
require
that
the
reference
chemicals
be
characterized
prior
to
use,
as
well
as
prepared
into
a
formulation
that
can
be
added
to
the
incubation
mixture
as
a
solution.
In
addition,
confirmation
of
the
formulation
concentration
requires
development
of
an
analysis
procedure.
Finally,
the
stability
of
each
formulation
needs
to
be
determined.
Each
of
these
issues
are
addressed
in
more
detail
in
the
following
subsections.

2.1
CHEMICAL
CHARACTERIZATION
A
single
lot
of
each
reference
chemical
will
be
procurred
so
that
all
prevalidation
experiments
are
performed
using
the
same
lot.
The
lot
obtained
will
have
a
purity
>
95
­
98
percent.
Upon
receipt
of
each
reference
chemical,
the
identity
will
be
confirmed
by
running
an
IR
spectrum
and
comparing
it
to
a
reference
spectrum
from
the
manufacturer
or
from
the
literature.
Within
30
days
of
using
a
reference
chemical
in
the
assay,
the
purity
will
be
determined
using
one
chromatographic
method
and
one
additional
or
complementary
method.

2.2
FORMULATION
DEVELOPMENT
Prior
to
testing,
the
solubility
of
the
reference
chemical
in
the
media
at
a
concentration
that
will
result
in
the
desired
final
concentration
in
the
incubation
mixture
will
need
to
be
determined.
Formulation
development
will
be
based
on
the
highest
concentration
needed
for
a
given
reference
chemical.
In
addition,
a
formulation
will
be
developed
using
a)
media
only
or
b)
a
vehicle
that
can
be
used
to
dissolve
the
reference
chemical
prior
to
mixing
it
with
the
media.
Battelle
Report
13
August,
2003
Only
select
solvents
will
be
used
as
vehicles.
The
vehicles
that
will
be
used
and
their
concentrations
were
determined
during
the
optimization
studies.
Based
on
these
results,
selected
concentrations
of
ethanol,
DMSO,
or
Tween
20
will
be
used
as
vehicles
if
the
chemical
is
not
soluble
in
the
media.

It
will
be
important
to
confirm
that
the
formulation
is
a
solution.
This
will
be
done
by
analyzing
the
formulation
before
and
after
filtering
since
the
results
of
the
two
analyses
will
be
similar
if
the
formulation
is
a
solution.
In
addition,
since
the
media
is
gassed
in
the
assay,
the
solubility
of
gassed
formulation
will
be
tested.

Formulation
development
will
seek
to
prepare
a
solution
of
the
reference
chemical
in
media,
with
and
without
hCG,
so
that
a
given
aliquot
of
the
formulation,
when
added
to
the
incubation
mixture,
will
result
in
the
desired
final
concentration
of
reference
chemical
to
be
tested,
as
well
as
the
final
total
incubation
volume.
Also,
if
a
vehicle
is
needed
to
make
the
formulation,
then
the
vehicle
concentration
would
be
formulated
at
an
acceptable
level.

When
formulations
for
multiple
laboratories
are
required,
a
formulation
will
be
prepared
in
a
sufficient
batch
size
that
will
allow
the
same
formulation
to
be
dispensed
to
all
laboratories.
If
different
concentrations
are
to
be
tested
for
a
given
reference
chemical,
then
the
central
laboratory
will
prepare
a
stock
solution,
which
will
be
shipped
to
the
laboratories.
Each
laboratory
will
then
use
the
stock
solution
to
prepare
the
various
concentrations
to
be
tested.

Formulations,
if
solutions,
will
be
prepared
to
within
5
percent
of
target.

2.3
FORMULATION
ANALYSIS
For
each
chemical
to
be
tested,
a
method
for
analysis
will
be
developed
for
the
reference
chemical
in
media/
vehicle
over
the
concentration
range
to
be
tested.

Prior
to
shipment
of
a
formulation
to
the
laboratory(
ies),
a
sample
of
the
stock
solution
formulation
to
be
shipped
will
be
taken
and
analyzed.
Samples
will
be
analyzed
in
duplicate.
If
the
determined
concentration
is
not
within
5
percent
of
the
target
concentration,
then
the
formulation
will
be
re­
prepared.

After
the
laboratory(
ies)
have
used
the
diluted
formulations,
a
sample
will
be
shipped
back
to
the
central
laboratory
and
archived
for
possible
analysis
at
a
later
date
if
there
is
some
question
about
the
results
of
the
assay.

2.4
STABILITY
DETERMINATION
Prior
to
being
used
in
the
assay,
the
formulations
will
be
tested
for
stability.
Stability
testing
will
be
performed
at
the
concentration
of
the
stock
solution
formulation.
Stability
will
be
determined
at
time
0
(
day
of
preparation)
and
at
weekly
intervals
for
a
period
of
at
least
6
weeks
and,
thereafter,
at
9
and
12
weeks.
Stability
will
be
tested
at
room
temperature
and
at
Battelle
Report
14
August,
2003
refrigerated
temperatures.
Stability
will
be
tested
using
amber
glass
bottles
with
Teflon­
lined
lids.

3.0
STUDY
DESIGN
(
PREVALIDATION
OF
THE
SLICED
TESTIS
ASSAY)

Each
of
the
studies
planned
for
prevalidation
is
summarized
in
the
table
below
and
are
described
in
more
detail
in
the
following
subsections.

Study
Number
Study
Type
Laboratory
Assignment
1
Baseline/
Contralateral
Testis
Fragment
Lead
Laboratory
2
Evaluation
of
AG
as
Positive
Control
Lead
Laboratory
3
Selection
of
Cytotoxicant
as
Positive
Control
Lead
Laboratory
4
Training/
Hands­
On
Experience
Lead
Laboratory
+
3
Participating
Laboratories
5
Baseline
Experiment
Lead
Laboratory
+
3
Participating
Laboratories
6
Positive
Control
Experiment
Lead
Laboratory
+
3
Participating
Laboratories
7
Multichemical
Experiments
Lead
Laboratory
3.1
BASELINE
AND
CONTRALATERAL
TESTIS
FRAGMENT
STUDY
The
lead
laboratory
will
conduct
this
experiment
using
the
optimized
assay
conditions.
The
objectives
of
this
experiment
are
two­
fold.
First,
to
have
an
experienced
laboratory
conduct
the
assay
under
conditions
considered
optimal
for
minimizing
the
assay
variability
in
testosterone
concentrations
measured
over
time.
Second,
to
evaluate
the
variability
in
contralateral
testes
fragments
for
the
purposes
of
deciding
whether
future
experimental
designs
can
use
randomized
fragment
assignments
or
if
a
blocking
design
is
essential
and,
if
the
latter,
what
blocking
design
would
be
most
favorable.

The
purposes
of
this
experiment
are:

°
to
demonstrate
competence
of
lead
lab
using
optimized
assay
conditions
°
to
estimate
response
trends
in
T
production
in
the
absence
of
inhibiting
chemicals
°
to
estimate
Leydig
cell
density
after
four
hours
of
incubation,
in
the
absence
of
inhibiting
chemicals
°
to
determine
the
variability
in
assay
response
associated
with
animals,
contralateral
testes
within
animals,
testis
fragments
within
testes
The
experimental
design
is
summarized
in
the
following
table:
Battelle
Report
15
August,
2003
Time
(
Hours
from
Equilibration)
hCG
Animal
Testis
Fragment
#
Incubation
(
Run)
#

0
no
1
­
3
A
1
1
­
3
yes
2
4
­
6
1
no
1
­
3
A
1
1
­
3
yes
2
4
­
6
2
no
1
­
3
A
1
1
­
3
yes
1
­
3
A
2
4
­
6
1
B
1,
2
7,
8
2
A
3,
4
9,
10
B
1,
2
11,
12
3
A
3,
4
13,
14
B
1,
2
15,
16
4
A
1,
2
17,
18
B
1,
2
19,
20
5
A
1,
2
21,
22
B
1,
2
23,
24
3
no
1
­
3
A
1
1
­
3
yes
2
4
­
6
4
no
1
­
3
A
1
1
­
3
yes
1
­
3
A
2
4
­
6
1
B
1,
2
7,
8
2
A
3,
4
9,
10
B
1,
2
11,
12
3
A
3,
4
13,
14
B
1,
2
15,
16
4
A
1,
2
17,
18
B
1,
2
19,
20
5
A
1,
2
21,
22
B
1,
2
23,
24
Battelle
Report
16
August,
2003
The
experiment
summarized
in
the
above
table
represents
one
replicate
study.
A
replicate
study
is
an
independently
conducted
experiment;
separated
by
day
and
using
freshly
prepared
reagents.
Two
full
replicates
will
be
conducted
for
runs
1
to
24
and
a
third
partial
replicate
will
be
carried
out
for
runs
1
to
6.
Runs
1
to
6
within
each
replicate
will
use
two
fragments
taken
from
a
single
testis
from
each
of
three
animals.
Runs
7
to
24
within
each
of
the
two
full
replicate
will
use
five
animals,
two
testes
within
each
animal,
and
two
fragments
from
each
testis.
Each
replicate
will
use
different
animals.
The
total
number
of
individual
fragments
and
incubations
used
to
conduct
this
experiment
is
24.

The
sampling
time
points
from
the
media
range
from
2
to
5
depending
on
the
sample
type.
If
two
samples,
then
the
time
points
are
at
2
and
4
hours
post­
equilbration.
If
five,
then
the
sampling
time
points
are
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
postequilibration
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
324
samples
[
Runs
1
to
6
has
5
time
points
with
duplicate
analyses
for
60
samples
and
runs
7
to
24
has
2
time
points
with
duplicate
analyses
for
72
samples,
which
gives
a
total
of
132
samples/
replicate.

The
principal
endpoint
is
testosterone
(
T)
concentration.
T
analyses
will
be
carried
out
in
duplicate.
Results
of
the
duplicate
T
analyses
will
be
averaged.
Statistical
analysis
will
be
carried
out
on
the
averages
the
duplicates.

The
statistical
analysis
will
be
carried
out
by
the
lead
laboratory.
Mixed
effects
repeated
measures
models
will
be
fitted
to
the
data
to
describe
trends
in
concentration
vs.
time
and
to
estimate
animal­
to­
animal,
testis­
to­
testis
within
animal,
and
fragment­
to­
fragment
within
testis
components
of
variation.
For
the
T
concentration
analyses
fixed
effects
terms
in
the
models
will
describe
linear
and
non­
linear
trends
in
T
for
hCG
stimulated
testis
fragments
and
for
nonstimulated
testis
fragments.
Random
effects
terms
in
the
model
will
estimate
variance
components
that
account
for
variation
among
fragments
within
testes,
testes
within
animals,
animals
within
replicates,
and
variation
among
replicates,
primarily
for
the
hCG
stimulated
testis
fragments.
A
correlation
structure
will
be
fitted
to
describe
the
relation
among
the
responses
based
on
the
same
vial
(
i.
e.,
the
same
fragment)
at
the
various
times.
The
testis­
to­
testis
variation
within
animals
and
the
fragment­
to­
fragment
variation
within
testes
will
be
determined
only
for
the
hCG
stimulated
testis
fragments.

The
results
of
the
model
fit
will
be
parametric
functions
that
describe
the
time
trends
and
variance
components
that
describe
the
within
and
among
replicate
variation
about
the
time
trends.

Residuals
from
the
model
fits
will
be
examined
to
determine
goodness­
of­
fit
to
the
model
assumptions,
to
assess
the
nature
of
the
random
variation
about
the
model,
and
to
search
for
outlying
observations.
Outliers
may
be
individual
responses
that
deviate
from
the
trend
within
their
vial,
individual
vials
that
deviate
from
the
average
across
vials
within
their
replicate,
and
individual
replicates
that
deviate
from
the
average
across
replicates.
Each
of
these
types
of
residuals
will
be
examined.
Battelle
Report
17
August,
2003
Based
on
the
model
fits
multiple
endpoints
will
be
reported.
Endpoints
include
°
T
concentrations
at
1,
2,
3,
4
hours
following
equilibration,
with
and
without
hCG
stimulation
°
Ratios
of
T
concentrations
with
and
without
hCG
stimulation,
at
1,
2,
3,
4
hours
following
equilibration
°
Response
trends,
with
and
without
hCG
stimulation
°
Ratios
of
trends
with
and
without
hCG
stimulation
°
Average
Leydig
cell
densities
after
two
hours
of
incubation
and
after
four
hours,
with
and
without
hCG
stimulation.

For
each
response
the
average
value
across
runs,
the
standard
error
of
estimate,
and
confidence
intervals
about
the
average
will
be
reported.
Estimates
of
run­
to­
run
variation
within
replicates
and
replicate­
to­
replicate
variation
will
be
reported.
Separate
variance
components
will
be
estimated
for
responses
with
hCG
stimulation
and
for
responses
without
hCG
stimulation.

Random
effects
terms
in
the
model
will
describe
variance
components
that
account
for
animal­
to­
animal
variation,
testis­
to­
testis
variation
within
animal,
and
fragment­
to­
fragment
variation
within
testis.
These
components
of
variation
will
be
estimated
for
hCG
stimulated
testis
fragments
at
two
hours
and
at
four
hours
following
equilibration.
A
correlation
structure
will
be
fitted
to
describe
the
relation
among
the
responses
at
the
various
times
based
on
the
same
vial
(
i.
e.
the
same
fragment).
The
results
of
the
model
fit
will
be
parametric
functions
that
describe
the
time
trends
and
estimates
of
the
variance
components.

For
each
response
estimates
of
variance
components
will
be
reported.

The
results
of
this
experiment
would
require
technical
review
and
approval
to
determine
whether
the
assay
is
performing
satisfactorily
before
proceeding
to
the
next
study.

3.2
EVALUATION
OF
AMINOGLUTETHIMIDE
(
AG)
AS
A
POSITIVE
CONTROL
The
lead
laboratory
will
conduct
this
positive
control
experiment
with
AG
using
the
optimized
assay
conditions.
Although
AG
is
anticipated
to
be
the
positive
control
for
the
assay,
verification
is
warranted.
In
order
to
evaluate
the
responsiveness
of
the
assay
on
a
day­
to­
day
basis,
this
study
plan
proposes
to
use
a
positive
control
with
every
set
of
samples.
Along
with
the
testis
fragments
used
for
the
media­
vehicle
control
and
test
chemicals
(
multichemical
testing),
the
tissue
fragment
response
to
a
known
gonadal
steroidogenic
inhibitor
is
proposed
for
use
as
a
positive
control.
One
concentration
of
the
inhibitor
would
be
used
once
the
inhibitor
and
concentration
are
finalized.
Now
that
the
optimized
assay
is
available,
the
use
of
aminoglutethimide
(
AG)
as
a
potential
positive
control
substance
can
be
evaluated.
In
addition,
this
experiment
attempts
to
determine
a
single
concentration
of
AG
that
could
be
used
when
included
in
the
multichemical
tests.
Furthermore,
once
the
positive
control
substance
and
the
concentration
are
determined,
then
the
response
from
this
treatment
will
serve
to
build
historical
control
data.
Battelle
Report
18
August,
2003
The
Lead
Laboratory
alone
will
evaluate
AG
for
its
potential
use
as
a
positive
control.
This
experiment
will
be
conducted
immediately
following
an
evaluation
of
the
results
from
the
contralateral
fragment
experiment
and
approval
has
been
given
to
proceed.
The
experimental
design
for
this
experiment
is
described
as
follows:

Sample
Type
hCG
Number
of
Incubations
(
Runs)
Testis
Fragment
ID
Number
Media­
Vehicle
control
no
3
1
­
3
Media­
Vehicle
control
yes
3
4
­
6
Media
+
AG
(
low)
yes
3
7
­
9
Media
+
AG
(
mid)
yes
3
10
­
12
Media
+
AG
(
high)
yes
3
13
­
15
The
experiment
summarized
in
the
above
table
represents
one
replicate
study.
A
replicate
study
is
an
independently
conducted
experiment;
separated
by
day
and
using
freshly
prepared
reagents.
Two
replicates
would
be
conducted.
The
overall
experiment
would
use
three
rats/
replicate
study;
three
testes
total/
replicate
study
(
based
on
using
5
fragments/
testis).
Within
each
replicate,
the
five
fragments
from
a
single
testis
would
be
distributed
among
the
five
test
conditions.
Thus,
each
repetition
of
the
test
within
a
replicate
will
involve
testes
fragments
from
the
same
testes.
The
overall
total
number
of
individual
fragments
and
incubations
used
to
conduct
this
experiment
is
30.

The
sampling
time
points
(
5)
from
the
media
are
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
In
addition,
at
the
end
of
the
incubation
period,
a
piece
of
the
tissue
fragment
will
be
collected
and
snap­
frozen
for
beta­
HSD
staining
and
microscopic
analysis.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
300
samples
[
30
runs
x
5
sampling
time
points
x
2
(
duplicate)
analyses]
and
the
overall
total
number
of
tissue
samples
is
30
[
30
runs
x
1
time
point].

The
data
analysis
and
statistical
evaluation
will
be
similar
to
the
intra­
laboratory
analysis
that
described
later
in
this
study
plan
when
this
positive
control
experiment
is
conducted
by
the
Lead
Laboratory
and
the
three
participating
laboratories.
While
the
purpose
of
this
initial
experiment
is
to
determine
whether
AG
will
be
useful
as
a
positive
control
and,
if
so,
determine
a
single
concentration
to
use
in
subsequent
studies,
the
second
conduct
of
this
experiment
using
multiple
laboratories
has
as
its
primary
purpose
to
begin
to
estimate
inter­
and
intra­
laboratory
variability.

The
results
of
this
experiment
would
require
technical
review
and
approval
to
determine
whether
the
assay
is
performing
satisfactorily,
AG
is
a
satisfactory
positive
control,
and
a
single
concentration
is
selected
before
proceeding
to
the
next
study.
Battelle
Report
19
August,
2003
3.3
SELECTION
OF
A
CYTOTOXICANT
AS
A
POSITIVE
CONTROL
The
lead
laboratory
will
conduct
this
positive
control
experiment
with
various
cytoxicant
substances
to
be
tested
using
the
optimized
assay
conditions.
It
is
of
interest
to
determine
whether
a
given
test
chemical
is
cytotoxic
to
the
cellular
components
of
the
testes.
Chemicals
with
cytotoxic
activity
are
classified
as
(
for
the
purposes
of
the
present
study
plan)
a)
general
cellular
toxicants,
which
refers
to
those
chemicals
that
are
non­
specific
cytotoxicants,
or
b)
specific
cytoxicants,
which
refers
to
chemicals
that
are
specifically
toxic
to
the
Leydig
cell.
The
purpose
of
this
set
of
experiments
is
to
test
different
known
general
cytoxicants
for
their
effect
on
testosterone
production
(
RIA
analysis)
and
on
the
Leydig
cell
(
beta­
HSD
staining).
From
these
results,
a
chemical
that
can
be
used
as
a
cytotoxicant
positive
control
will
be
selected
in
order
to
provide
information
about
the
tissue
fragment
response
when
cellular
destruction
is
the
cause
of
the
decrease
in
testosterone
concentrations.
As
was
described
above
for
AG,
the
cytotoxicant
positive
control
will
be
used
at
one
concentration
along
with
the
media­
vehicle
control
when
performing
multichemical
testing.

The
four
general
chemical
cytotoxicants
presently
being
considered
for
testing
are
SDS;
2,
4­
dinitrophenol;
methylnitrosourea;
sodium
azide.
Three
concentrations
of
each
will
be
tested.
Prior
to
being
tested
in
the
sliced
testes
assay,
it
is
necessary
to
determine
whether
these
agents
interfere
with
the
testosterone
RIA.
Consequently,
a
mid
concentration
testosterone
standard
will
be
spiked
with
the
cytotoxicant
and
compared
to
the
results
obtained
from
a
vehicle
spiked
control
(
same
final
volumes).
If
there
is
no
interference,
then
the
cytotoxicant
will
be
tested
using
the
sliced
testis
assay.

A
Leydig
cell
specific
staining
method
and
microscopic
evaluation
will
be
used
to
evaluate
the
viability
of
these
cells.
At
the
end
of
the
incubation
period,
a
piece
will
be
taken
from
each
fragment
for
staining.
The
media­
vehicle
control
fragment
will
be
used
to
evaluate
the
result
of
incubation
with
the
test
chemical
treatment.
The
staining
procedure
is
specific
for
cells
containing
3 ­
HSD
(
see
Section
1.0),
a
steroidogenic
enzyme
specific
to
the
Leydig
cells
when
examining
testis
tissue.
The
general
procedure
is
described
as
follows:

°
after
the
last
sample
collection,
snap
freeze
the
tissue,
section
it
(~
15
um),
and
mount
it
on
slides.
Stain
the
slide
using
the
following
solutions
and
procedure:
a)
etiocholanolone
stock
solution
(
1
mg/
ml
in
DMSO),
b)
2
mg
Nitroblue
Tetrazolium
in
0.6
ml
Etiocholanolone
stock,
c)
10
mg
NAD+
dissolved
in
9.5
ml
warm
Dulbeccos
Phosphate
buffered
saline
(
DPBS),
and
d)
10
mg
NAD+
dissolved
in
9.5
ml
warm
Dulbeccos
Phosphate
buffered
saline
(
DPBS).
Mix
solutions
b
and
c.
Cover
section
tissue
on
slide
with
staining
solution
for
1
­
2
hours.
Rinse
in
deionized
water.
Fix
in
10%
formalin
in
DPBS
with
5%
sucrose.
Coverslip
with
glycerol:
DPBS
(
1:
1)
and
seal
with
nail
polish.
[
Ref.:
Payne
et
al.,
(
1980).
Endocrinology
106:
1424;
Klinefelter
et
al.,
(
1993).
In:
Methods
in
Toxicology,
Vol.
3,
pp.
166­
181.
Battelle
Report
20
August,
2003
In
order
to
increase
efficiency
and
conserve
animal
usage,
only
the
stimulated
(
hCGwith
incubations
will
be
performed.
This
seems
reasonable
since
the
cytotoxicant
response
will
decrease
the
testosterone
concentration
and
it
will
be
difficult
to
assess
a
chemical­
induced
decrease
in
testosterone
concentration
in
a
non­
stimulated
tissue
fragment.
The
experimental
design
for
this
experiment
is
described
as
follows:

Sample
Type
hCG
Number
of
Incubations
(
Runs)
Testis
Fragment(
s)

Media­
Vehicle
control
no
3
1
­
3
Media­
Vehicle
control
yes
3
4
­
6
Positive
control
yes
3
7
­
9
Media
+
Cytotoxicant
A
(
low)
yes
3
10
­
12
Media
+
Cytotoxicant
A
(
mid)
yes
3
13
­
15
Media
+
Cytotoxicant
A
(
high)
yes
3
16
­
18
Media
+
Cytotoxicant
B
(
low)
yes
3
19
­
21
Media
+
Cytotoxicant
B
(
mid)
yes
3
22
­
24
Media
+
Cytotoxicant
B
(
high)
yes
3
25
­
27
The
experiments
listed
in
the
above
table
represent
one
replicate
study.
Note
that
two
of
the
four
cytotoxicants
are
tested
per
replicate
study.
A
replicate
study
is
an
independently
conducted
experiment;
separated
by
day
and
using
freshly
prepared
reagents.
Two
replicates
would
be
conducted.
The
overall
experiment
would
use
three
rats/
replicate
study;
three
testes
total/
replicate
study
(
based
on
obtaining
8
to10
fragments/
testis).
Testes
will
be
at
least
1000
mg
so
that
at
least
nine
fragments
can
be
obtained
from
a
testis.
These
nine
fragments
will
be
divided
among
the
nine
test
conditions.
The
overall
total
number
of
individual
fragments
and
incubations
used
to
conduct
this
experiment
is
54.
The
two
replicate
experimental
designs
will
be
repeated
with
the
other
two
cytotoxicants.

The
sampling
time
points
(
5)
from
the
media
are
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
In
addition,
at
the
end
of
the
incubation
period,
a
piece
of
the
tissue
fragment
will
be
collected
and
snap­
frozen
for
beta­
HSD
staining
and
microscopic
analysis.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
540
samples
[
54
runs
x
5
sampling
time
points
x
2
(
duplicate)
analyses]
and
the
overall
total
number
of
tissue
samples
is
54
[
54
runs
x
1
time
point].

Regarding
data
analysis
and
the
statistical
evaluation,
the
principal
endpoints
are
testosterone
(
T)
concentration
and
Leydig
cell
counts.
T
analyses
will
be
carried
out
in
duplicate.
Results
of
the
duplicate
testosterone
analyses
will
be
averaged.
Statistical
analysis
will
be
carried
out
on
the
averages
of
the
duplicates.
Mixed
effects
repeated
measures
models
Battelle
Report
21
August,
2003
will
be
fitted
to
the
data
to
describe
trends
in
T
concentration
vs.
time
and
trends
in
the
cytotoxicant
concentration.
Fixed
effects
terms
in
the
models
will
describe
linear
and
non­
linear
T
time
trends
and
cytotoxicant
concentration
trends
for
hCG
stimulated
testis
and
differences
relative
to
the
hCG
stimulated
media­
vehicle
control.
Random
effects
terms
in
the
model
will
describe
variance
components
that
account
for
variation
among
the
repeat
vials
within
replicates
and
variation
among
replicates.
A
correlation
structure
will
be
fitted
to
describe
the
relation
among
the
responses
at
the
various
times
based
on
the
same
vial
(
i.
e.
the
same
fragment)
and
at
the
various
cytotoxicant
concentrations,
based
on
fragments
taken
from
the
same
testis.
The
results
of
the
model
fit
will
be
parametric
functions
that
describe
the
time
trends
and
concentration
trends
and
variance
components
that
describe
the
within
and
among
replicate
variation
about
the
time
trends
and
concentration
trends.

Residuals
from
the
model
fits
will
be
examined
to
determine
goodness­
of­
fit
to
the
model
assumptions,
to
assess
the
nature
of
the
random
variation
about
the
model,
and
to
search
for
outlying
observations.
Outliers
may
be
individual
responses
that
deviate
from
the
time
trend
within
their
vial
or
from
the
concentration
trend
across
vials,
individual
vials
that
deviate
from
the
average
across
vials
with
the
same
conditions
within
their
replicate,
and
individual
replicates
that
deviate
from
the
average
across
replicates.
Each
of
these
types
of
residuals
will
be
examined.

Based
on
the
model
fits
multiple
endpoints
will
be
reported.
Possible
endpoints
include
T
concentrations
at
1,
2,
3,
4
hours
following
equilibration
for
the
media­
vehicle
control
and
graded
cytotoxicant
concentrations,
differences
or
ratios
of
T
concentrations
from
the
same
groups
with
hCG
stimulation
to
the
media­
vehicle
control
with
hCG
stimulation
at
1,
2,
3,
4
hours
following
equilibration,
response
trends
in
time
or
in
cytotoxicant
concentration
with
hCG
stimulation,
and
differences
or
ratios
of
trends
to
the
media­
vehicle
control
with
hCG
stimulation.
For
each
response
the
average
value
across
runs,
the
standard
error
of
estimate,
and
confidence
intervals
about
the
average
will
be
reported.
Estimates
of
run­
to­
run
variation
within
replicates
and
replicate­
to­
replicate
variation
will
be
reported.
Differences
between
the
T
concentrations
in
the
presence
of
the
cytotoxicant
and
the
T
concentrations
in
the
media­
vehicle
control
group
will
also
be
analyzed.

The
results
of
this
experiment
would
require
technical
review
and
approval
to
determine
whether
the
assay
is
performing
satisfactorily,
a
single
chemical
is
selected
as
a
positive
control,
and
a
single
concentration
is
selected
before
proceeding
to
the
next
study.

3.4
TRAINING
AND
HANDS­
ON
EXPERIENCE
In
addition
to
the
lead
laboratory,
additional
laboratories
will
be
required
to
participate
in
order
for
inter­
laboratory
assay
variability
to
be
estimated.
These
participating
laboratories
were
selected
following
a
review
of
their
responses
to
a
sliced
testis
assay
prevalidation
proposal.
Three
laboratories
were
selected
from
those
solicited
for
participation.
It
is
believed
important
to
provide
training
and
give
the
participating
laboratories
experience
prior
to
using
their
results
from
conducting
the
assay
for
estimating
inter­
laboratory
variability.
In
this
way,
the
assay
Battelle
Report
22
August,
2003
variability
that
is
generated
by
laboratories
judged
to
be
competent
in
the
conduct
of
the
assay
can
be
evaluated.
The
procedures
planned
for
training
the
participating
laboratories
and
providing
hands­
on
experience
are
described
in
the
following
paragraphs.

Standardized
study
documents
will
be
produced
by
the
lead
laboratory.
These
documents
will
include
a
study
protocol
and
standard
operating
procedures
(
SOPs).
While
the
standard
protocol
and
study
specific
SOPs
may
be
customized
according
to
each
laboratory
format,
the
content
of
the
documents
will
be
the
same.
The
study
specific
SOPs
to
be
developed
by
the
lead
laboratory
will
focus
on
aspects
of
the
assay
that,
if
varied,
will
impact
on
the
assay
variability
and
performance.
The
procedures
to
be
addressed
by
study
specific
SOPs
include:

°
Testis
removal/
Fragment
production
°
Reagent
preparation
°
Fragment
assignment,
incubation
and
media
sampling
°
Testosterone
RIA
°
Fragment
processing
for
staining
and
slide
preparation
In
addition,
forms
for
data
collection
will
be
standardized.
In
this
way,
the
data
deemed
necessary
for
collection
is
more
likely
to
occur.
In
addition,
if
collected
on
the
same
form,
then
the
data
will
be
readily
amenable
to
efficient
review
by
outside
laboratory
monitors,
e.
g.
lead
laboratory,
Data
Coordination
Center
(
DCC).

Training
of
the
participating
laboratories'
technical
staff
will
take
place
at
the
lead
laboratory.
The
senior
investigator/
head
technician
for
each
participating
laboratory
will
be
trained
in
the
conduct
of
the
assay
at
the
lead
laboratory.
The
training
will
include
a
review
of
the
relevant
study
documents
described
above
and
any
necessary
demonstrations
regarding
various
aspects
of
the
assay
(
starting
at
testis
removal
through
to
data
analysis).
Furthermore,
the
lead
laboratory
will
be
set­
up
to
allow
the
participating
laboratory
staff
to
demonstrate
proficiency
in
conducting
the
assay
by
supervising
them
as
they
conduct
a
baseline
(
media
only)
experiment
as
described
in
subsection
3.5
and
a
positive
control
experiment
as
described
in
subsection
3.6,
except
only
one
replicate
per
laboratory
would
be
conducted.

The
results
of
these
proficiency
experiments
will
be
reviewed
by
the
lead
laboratory
and
the
EPA
prior
to
proceeding
to
the
next
studies.

3.5
BASELINE
EXPERIMENT
This
study
will
be
conducted
by
the
lead
laboratory
and
three
participating
laboratories.
The
purposes
of
this
study
are:

°
to
estimate
baseline
inter­
and
intra­
laboratory
variability
(
which
will
also
provide
information
to
design
the
validation
experimental
design)
°
to
evaluate
the
knowledge
transfer
of
assay
procedures
to
participating
laboratories
°
to
begin
collection
of
historical
control
and
variability
data.
Battelle
Report
23
August,
2003
The
experimental
design
of
this
study
is
summarized
in
the
following
table.

Sample
Type
hCG
Number
of
Incubations
(
Runs)
Testis
Fragment
ID
Number
Media
no
3
1
­
3
Media
yes
3
4
­
6
The
information
presented
in
the
table
is
one
replicate
of
the
experiment.
A
total
of
three
independent
replicates
would
be
conducted
by
each
laboratory.
The
overall
study
would
use
one
rat/
replicate
study
and
one
testis
total/
replicate
study.
The
overall
total
number
of
individual
fragments
or
incubations
used
is
18
for
each
laboratory
[(
3
runs
with
+
3
runs
without
hCG)
x
3
replicate
studies].

The
sampling
time
points
(
5)
from
the
media
are
at
time
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
No
fragments
will
be
collected
for
3$­
HSD
staining
and
microscopic
examination.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
180
samples/
laboratory
[
18
runs
x
5
time
points
x
2
(
duplicate)
analyses].

Regarding
the
data
analysis
and
statistical
evaluation,
each
laboratory
will
carry
out
three
complete
study
replicates.
The
results
for
each
analysis
will
be
reported
individually,
with
sufficient
identifying
information
to
determine
which
results
correspond
to
duplicate
analyses,
to
different
time
points
within
one
vial,
to
different
vials
within
the
same
replicate,
and
to
different
replicates.
Each
laboratory
will
maintain
databases
to
include
all
data
generated
during
the
study.
The
databases
will
have
uniform
structure,
formatting,
and
variable
naming
across
laboratories.
Data
will
be
reported
for
each
individual
sample
generated.
Test
conditions,
background
environmental
conditions,
and
results
for
each
analysis
for
each
sample
at
each
time
point
will
be
reported.
Analysis
results
to
be
reported
are
testosterone
(
T)
concentration
(
ng/
mg
testis/
hr).
Detection
limits
and
indications
of
inability
to
detect
will
be
reported,
as
will
confirmation
of
the
acceptability
or
non­
acceptability
of
each
individual
value.

The
statistical
analysis
will
be
divided
into
intra­
laboratory
and
inter­
laboratory
components.
The
intra­
laboratory
analyses
will
be
carried
out
by
each
laboratory
individually,
based
on
a
common
analysis
plan.
Mixed
effects
repeated
measures
models
will
be
fitted
to
the
data
to
describe
trends
in
concentration
vs.
time.
Fixed
effects
terms
in
the
models
will
describe
linear
and
non­
linear
trends
in
T
time
trends
for
hCG
stimulated
testis
fragments
and
for
nonstimulated
testis
fragments.
Random
effects
terms
in
the
model
will
describe
variance
components
that
account
for
variation
among
the
repeat
vials
within
replicates
and
variation
among
replicates.
A
correlation
structure
will
be
fitted
to
describe
the
relation
among
the
responses
at
the
various
times
based
on
the
same
vial
(
i.
e.
the
same
fragment
and
among
fragments
from
the
same
testis).
The
results
of
the
model
fit
will
be
parametric
functions
that
Battelle
Report
24
August,
2003
describe
the
time
trends
and
variance
components
that
describe
the
within
and
among
replicate
variation
about
the
time
trends.

Residuals
from
the
model
fits
will
be
examined
to
determine
goodness­
of­
fit
to
the
model
assumptions,
to
assess
the
nature
of
the
random
variation
about
the
model,
and
to
search
for
outlying
observations.
Outliers
may
be
individual
responses
that
deviate
from
the
trend
within
their
vial,
individual
vials
that
deviate
from
the
average
across
vials
within
their
replicate,
and
individual
replicates
that
deviate
from
the
average
across
replicates.
Each
of
these
types
of
residuals
will
be
examined.

Based
on
the
model
fits,
multiple
endpoints
will
be
reported.
Possible
endpoints
include
T
concentrations
at
1,
2,
3,
4
hours
following
equilibration
with
and
without
hCG
stimulation,
ratios
of
T
concentrations
with
and
without
hCG
stimulation
at
1,
2,
3,
4
hours
following
equilibration,
response
trends
with
and
without
hCG
stimulation,
and
ratios
of
trends.
For
each
response
the
average
value
across
runs,
the
standard
error
of
estimate,
and
confidence
intervals
about
the
average
will
be
reported.
Estimates
of
run­
to­
run
variation
within
replicates
and
replicate­
to­
replicate
variation
will
be
reported.

The
inter­
laboratory
analysis
will
be
carried
out
by
the
DCC.
The
objective
of
the
interlaboratory
analysis
is
to
assess
the
extent
of
variation
across
laboratories
with
respect
to
average
response
and
variability
of
response.
For
each
endpoint
the
average
response
and
associated
standard
error,
the
variation
among
runs
within
replicates,
and
the
variation
among
replicates
will
be
determined
for
each
laboratory.
Comparisons
of
within
laboratory
variance
components
will
be
made
among
the
participating
laboratories
based
on
control
charts
to
assess
homogeneity
of
variance.
The
reference
for
comparisons
will
be
either
the
lead
laboratory
results
or
the
average
results
across
all
laboratories.
If
there
is
no
evidence
of
lab­
to­
lab
variation
the
within
laboratory
variance
components
will
be
pooled
among
laboratories.
Otherwise
separate
within
laboratory
variance
components
will
be
specified
for
each
laboratory.

The
average
responses
will
be
compared
across
laboratories
based
on
random
effects
one­
way
analysis
of
variance.
The
among
laboratories
variance
in
average
response
will
be
added
to
the
within
laboratory
variance,
resulting
in
the
total
assay
variability.
Estimates
and
confidence
intervals
will
be
reported
for
each
laboratory
based
on
the
total
assay
variability.
Estimates
and
confidence
intervals
will
be
reported
on
the
differences
between
the
participating
laboratory
results
and
the
lead
laboratory
results
and
the
differences
between
each
laboratory's
results
and
the
average
of
the
laboratory
results
based
on
the
total
assay
variability.

3.6
POSITIVE
CONTROL
EXPERIMENT
This
study
will
be
conducted
by
the
lead
laboratory
and
three
participating
laboratories.
The
purposes
of
this
study
are:

°
to
evaluate
aminoglutethimide
(
AG)
as
a
positive
control
for
the
assay
°
to
determine
AG
(
positive
control)
inter­
and
intra­
laboratory
variability
Battelle
Report
25
August,
2003
°
to
determine
time
effect
on
the
assay
by
comparing
Baseline
to
Pilot
experiments
using
the
media
control
data
°
to
begin
collection
of
historical
positive
control
and
variability
data.

The
experimental
design
of
this
study
is
summarized
in
the
following
table.

Sample
Type
hCG
Number
of
Incubations
(
Runs)
Testis
Fragment(
s)

Media­
Vehicle
control
no
3
1
­
3
Media­
Vehicle
control
yes
3
4
­
6
Cytotoxicant
control
yes
3
7
­
9
Media
+
AG
(
low)
yes
3
10
­
12
Media
+
AG
(
mid)
yes
3
13
­
15
Media
+
AG
(
high)
yes
3
16
­
18
The
information
presented
in
the
table
is
one
replicate
of
the
experiment.
Three
replicates
of
the
study
will
be
run.
The
non­
stimulated
incubations
(
non
hCG)
were
excluded
from
the
study
since
the
affect
of
AG
on
the
non­
stimulated
tissue
fragments
would
not
be
expected
to
reduce
the
baseline
levels
to
any
great
extent.
The
overall
study
would
use
three
rats/
replicate
study
and
three
testes
total/
replicate
study.
Six
fragments
will
be
taken
from
one
testis
from
each
rat
and
allocated
to
the
six
test
conditions.
The
overall
total
number
of
individual
fragments
or
incubations
used
is
54
for
each
laboratory.

The
sampling
time
points
(
5)
from
the
media
are
at
time
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
In
addition,
at
the
end
of
the
incubation
period,
a
piece
of
the
tissue
fragment
will
be
collected
and
snap­
frozen
for
3$­
HSD
staining
and
microscopic
examination.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
540
samples/
laboratory
[
54
runs
x
5
time
points
x
2
(
duplicate)
analyses].
The
overall
total
number
of
tissue
samples
for
microscopic
examination
are
54/
laboratory
[
54
runs
x
1
time
point].

Regarding
data
analysis
and
statistical
evaluation,
the
statistical
analysis
will
be
divided
into
intra­
laboratory
and
inter­
laboratory
components.
The
intra­
laboratory
analyses
will
be
carried
out
by
each
laboratory
individually,
based
on
a
common
analysis
plan.
Mixed
effects
repeated
measures
models
will
be
fitted
to
the
data
to
describe
trends
in
T
concentration
vs.
time
and
trends
in
AG
concentration.
Fixed
effects
terms
in
the
models
will
describe
linear
and
nonlinear
T
time
trends
and
AG
concentration
trends
for
hCG
stimulated
testis
and
differences
relative
to
the
media­
vehicle
control.
Random
effects
terms
in
the
model
will
describe
variance
components
that
account
for
variation
among
the
repeat
vials
within
replicates
and
variation
among
replicates.
A
correlation
structure
will
be
fitted
to
describe
the
relation
among
the
Battelle
Report
26
August,
2003
responses
at
the
various
times
based
on
the
same
vial
(
i.
e.
the
same
fragment
and
fragments
taken
from
the
same
testis)
and
at
the
various
AG
concentrations.
The
results
of
the
model
fit
will
be
parametric
functions
that
describe
the
time
trends
and
concentration
trends
and
variance
components
that
describe
the
within
and
among
replicate
variation
about
the
time
trends
and
concentration
trends.

Residuals
from
the
model
fits
will
be
examined
to
determine
goodness­
of­
fit
to
the
model
assumptions,
to
assess
the
nature
of
the
random
variation
about
the
model,
and
to
search
for
outlying
observations.
Outliers
may
be
individual
responses
that
deviate
from
the
time
trend
within
their
vial
or
from
the
concentration
trend
across
vials,
individual
vials
that
deviate
from
the
average
across
vials
with
the
same
conditions
within
their
replicate,
and
individual
replicates
that
deviate
from
the
average
across
replicates.
Each
of
these
types
of
residuals
will
be
examined.

Based
on
the
model
fits
multiple
endpoints
will
be
reported.
Possible
endpoints
will
be
include
T
concentrations
at
1,
2,
3,
4
hours
following
equilibration
for
the
media­
vehicle
control
and
graded
AG
concentrations,
with
hCG
stimulation,
or
in
AG
concentration
with
hCG
stimulation.
For
each
response
the
average
value
across
runs,
the
standard
error
of
estimate,
and
confidence
intervals
about
the
average
will
be
reported.
Estimates
of
run­
to­
run
variation
within
replicates
and
replicate­
to­
replicate
variation
will
be
reported.
Differences
or
ratio
between
the
T
concentrations
in
the
presence
of
AG
and
the
T
concentrations
in
the
media­
vehicle
control
group
will
also
be
analyzed.

The
inter­
laboratory
analysis
will
be
carried
out
by
the
DCC.
The
objective
of
the
interlaboratory
analysis
is
to
assess
the
extent
of
variation
across
laboratories
with
respect
to
average
response
and
variability
of
response.
For
each
endpoint
developed
in
the
intra­
laboratory
analysis
the
average
response
and
associated
standard
error,
the
variation
among
repetitions
within
replicates,
and
the
variation
among
replicates
will
be
calculated
for
each
laboratory.
Comparisons
of
within
laboratory
variance
components
will
be
made
among
the
participating
laboratories
based
on
control
charts
to
assess
homogeneity
of
variance.
The
reference
for
comparisons
will
be
either
the
lead
laboratory
results
or
the
average
results
across
all
laboratories.
If
there
is
no
evidence
of
lab­
to­
lab
variation
the
within
laboratory
variance
components
will
be
pooled
among
laboratories.
Otherwise
separate
within
laboratory
variance
components
will
be
specified
for
each
laboratory.

The
average
responses
will
be
compared
across
laboratories
based
on
random
effects
one­
way
analysis
of
variance.
The
among
laboratories
variance
in
average
response
will
be
added
to
the
within
laboratory
variance,
resulting
in
the
total
assay
variability.
Estimates
and
confidence
intervals
will
be
reported
for
each
laboratory
based
on
the
total
assay
variability.
Estimates
and
confidence
intervals
will
be
reported
on
the
differences
between
the
participating
laboratory
results
and
the
lead
laboratory
results
and
between
each
laboratory's
results
and
the
average
of
the
laboratory
results
based
on
the
total
assay
variability.
Battelle
Report
27
August,
2003
The
responses
included
in
the
intra­
laboratory
and
inter­
laboratory
analyses
will
be
categorized
as
primary
or
secondary
endpoints.
The
primary
endpoints
will
be
restricted
to
a
small
number
(
two
to
five)
of
the
most
important
responses
for
comparisons.
Power
calculations
to
assess
inference
sensitivity
versus
sample
size
will
be
based
on
the
primary
endpoints.
The
studies
will
be
powered
to
attain
desired
sensitivity
for
comparisons
of
the
primary
endpoints
within
or
among
laboratories.
The
secondary
endpoints
are
the
(
relatively
large
number
of)
remaining
responses
that
are
included
in
the
statistical
analyses.
The
studies
will
not
be
powered
for
comparisons
among
the
secondary
endpoints.

For
purposes
of
assessing
sample
size
versus
inference
sensitivity
for
the
validation
tests,
two
responses
have
been
selected
as
primary
endpoints.
These
are:

°
differences
between
T
concentrations
associated
with
the
high
aminoglutethimide
concentration
and
the
media­
vehicle
control
at
three
hours
past
equilibration,
with
hCG
stimulation.

°
Sum
of
absolute
differences
between
T
concentrations
associated
with
the
high
aminoglutethimide
concentration
and
the
media­
vehicle
control
at
1,
2,
3,
and
4
hours
past
equilibration,
with
hCG
stimulation.

The
second
response
approximates
the
area
between
the
T
concentration
versus
time
curves
for
these
two
conditions.

Sensitivity
analyses
will
be
carried
out
based
on
response
variability
estimates
obtained
from
the
analyses.
Numbers
of
replicates
per
laboratory
necessary
to
detect
heterogeneity
of
replicate­
to­
replicate
variability
within
laboratories
will
be
assessed.
Numbers
of
laboratories
and
numbers
of
replicates
per
laboratory
necessary
to
detect
various
levels
of
coefficient
of
variation
across
laboratories
with
high
power
or
to
detect
various
levels
of
ratio
of
between
laboratory
standard
deviation
to
within
laboratory
standard
deviation
with
high
power
will
be
determined.

3.7
MULTICHEMICAL
EXPERIMENTS
This
study
will
be
conducted
by
the
lead
laboratory
only.
The
purposes
of
this
study
are:

°
to
determine
the
response
of
the
assay
when
challenged
with
putative
positive
test
chemicals.
The
test
chemicals
will
differ
in
their
mode(
s)
of
action,
which
will
include
altering
signal
transduction,
cholesterol
transport
into
the
mitochondria,
and
the
series
of
enzymatic
reactions
that
lead
to
the
production
of
testosterone
in
the
gonadal
steroidogenic
pathway.
°
to
determine
the
response
of
the
assay
when
challenged
with
putative
negative
test
chemicals.
°
to
select
test
chemicals
and
concentrations
that
would
be
used
for
validation
of
the
assay.
Battelle
Report
28
August,
2003
°
to
estimate
inter­
laboratory
variability
based
on
the
results
from
the
assay
using
the
selected
test
chemicals.

The
experimental
design
of
this
study
for
any
two
test
chemicals
is
summarized
in
the
following
table:

Sample
Type
hCG
Number
of
Incubations
(
Runs)
Testis
Fragment(
s)

Media­
Vehicle
control
no
3
1
­
3
Media­
Vehicle
control
yes
3
4
­
6
Positive
control
­
Aminogluthethimide
@
1
conc
yes
3
7
­
9
Cytotoxicant
control
­
TBD
@
1
conc
yes
3
10
­
12
Media
+
Test
Chemical
A
(
low)
yes
3
13
­
15
Media
+
Test
Chemical
A
(
mid)
yes
3
16
­
18
Media
+
Test
Chemical
A
(
high)
yes
3
19
­
21
Media
+
Test
Chemical
A
(
high)
no
3
22
­
24
Media
+
Test
Chemical
B
(
low)
yes
3
25
­
27
Media
+
Test
Chemical
B
(
mid)
yes
3
28
­
30
Media
+
Test
Chemical
B
(
high)
yes
3
31
­
33
Media
+
Test
Chemical
B
(
high)
no
3
34
­
36
The
information
presented
in
the
table
is
one
replicate
of
the
experiment.
The
nonstimulated
incubations
(
non
hCG)
were
excluded
from
the
study
since
the
affect
of
a
test
chemical
on
the
non­
stimulated
tissue
fragments
would
not
be
expected
to
reduce
the
baseline
levels
to
any
great
extent.
However,
a
high
concentration
test
chemical
with
non­
stimulated
tissue
fragment
was
retained
in
the
experimental
design
to
confirm
this
response.
In
addition,
in
the
case
when
the
assay
would
be
used
to
evaluate
a
chemical
with
an
unknown
effect
on
the
testosterone
concentration,
it
would
be
important
to
test
the
non­
stimulated
fragment
at
the
high
concentration
to
determine
whether
the
test
chemical
had
a
stimulatory
effect
on
the
testosterone
concentration.
A
total
of
two
independent
replicates
would
be
conducted.
The
overall
study
would
use
two
rats/
replicate
study
and
four
testes
total/
replicate
study.
The
overall
total
number
of
individual
fragments
or
incubations
used
is
72.

The
sampling
time
points
(
5)
from
the
media
are
at
time
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
In
addition,
at
the
end
of
the
incubation
period,
a
piece
of
the
tissue
fragment
will
be
collected
and
snap­
frozen
for
3$­
HSD
staining
and
microscopic
examination.
Thus,
the
overall
Battelle
Report
29
August,
2003
total
number
of
testosterone
samples
for
analysis
is
720
samples
[
72
runs
x
5
time
points
x
2
(
duplicate)
analyses].
The
overall
total
number
of
tissue
samples
for
microscopic
examination
are
72
[
72
runs
x
1
time
point].

Separate
statistical
analyses
will
be
carried
out
for
each
test
chemical.
Consideration
will
be
given
to
pooling
the
media­
vehicle
control
groups
across
those
chemical
tests
that
use
the
same
vehicle.
The
statistical
analysis
considerations
are
the
same
as
those
described
above
in
the
previous
section,
except
that
there
are
two
replicates
per
chemical
rather
than
three.
Extent
of
replicate­
to­
replicate
variation
can
be
determined
from
these
tests
only
in
a
qualitative
manner.

4.0
RECOMMENDED
REFERENCE
CHEMICALS
FOR
MULTICHEMICAL
TESTING
The
purpose
of
this
study
plan
is
to
design
experiments
that
will
provide
information
to
evaluate
the
relevance
of
the
sliced
testis
assay.
For
this
reason,
reference
chemicals
are
recommended
for
testing
in
this
study
plan.
In
this
prevalidation
study
plan,
the
optimized
sliced
testis
assay
will
be
evaluated
for
its
capacity
to
detect
an
effect
of
a
substance
on
gonadal
steroidogenesis.
It
is
predicted
that
the
assay
will
detect
interference
with
key
steps
in
the
steroidogenic
pathway
because
interference
with
any
of
these
steps
will
result
in
a
statistically
significant
change
in
measured
testosterone
production
relative
to
the
controls.
The
key
processes
include
a)
decreases
in
signal
transduction,
b)
interference
with
the
transport
of
cholesterol
from
the
cytoplasm
to
the
mitochondria
by
the
steroid
acute
regulatory
protein
and
c)
inhibition
of
various
enzymes
in
the
steroidogenic
pathway
(
see
Section
1.0).

Selection
of
reference
chemicals
were
primarily
based
on
their
reported
mechanism
of
action
as
it
relates
to
the
gonadal
steroidogenic
pathway.
The
most
desired
reference
chemicals
for
testing
are
those
with
a
single
site
of
action
(
high
specificity)
regardless
of
the
exposure
concentration.
However,
most
reference
chemicals
are
inhibitory
at
more
than
one
site
of
the
steroidogenic
pathway.
In
addition,
in
those
instances
when
a
reference
chemical
is
active
at
one
site,
the
effect
is
usually
only
at
the
low
concentration
and,
as
the
concentration
increases,
the
effect
may
include
more
than
a
single
site
(
decreased
specificity).
While
mostly
positive
reference
chemicals
were
selected,
there
was
also
a
negative
reference
chemical
included
in
order
to
evaluate
whether
a
false
positive
effect
would
be
seen.

The
proposed
reference
chemicals
for
testing
are
summarized
below:

Test
Chemical
Mode
of
Actiona
Test
Concentrationsb
Reference
Aminoglutethimide
(
positive
control)
P450SCC,
aromatase
10,
100,
and
1000
µ
M
Powlin
et
al.,
1998;
Johnston,
1997;
Uzgiris
et
al.,
1977
Diethylumbelliferyl
phosphate
cAMP­
stimulated
accumulation
of
StAR
TBD
Choi
et
al.,
1995
Dimethoate
StAR
protein
TBD
Walsh
et
al.,
2000
Test
Chemical
Mode
of
Actiona
Test
Concentrationsb
Reference
Battelle
Report
30
August,
2003
Ketoconazole
P450SCC,
P450c17
0.1,
1,
and
10
µ
M
Powlin
et
al.,
1998;
Kan
et
al.,
1985;
Albertson
et
al.,
1988;
DeCoster
et
al.,
1989;
Chaudhary/
Stocco,
1989;
Malozowski
et
al.,
1985,
1986
Trilostanec
3$­
HSD
TBD
Takahashi
et
al.,
1990
Genistein
or
epostane
3$­
HSD
TBD
Ohno
et
al.,
2002;
Tanaka
et
al.,
1992
Flutamide
P450c17
10,
100,
and
1000
µ
M
Powlin
et
al.,
1998;
Ayub
and
Levelll,
1987
Finasterided
or
MK­
434
5"­
reductase
10,
100,
and
1000
µ
M
(
MK­
434
­
TBD)
Morris,
1996
Fenarimol
aromatase
TBD
Vinggaard
et
al.,
2000
Vinclozolin
(
negative
chemical)
antiandrogen
TBD
­­

Bisphenol
A
c
AMP
TBD
­­

Lindane
c
AMP
TBD
­­

a.
Site
of
action
that
leads
to
a
decrease
in
Testosterone
concentration
for
those
chemicals
with
inhibitory
activity.
Negative
reference
chemicals
would
have
no
effect
on
the
testosterone
concentration.
b.
Final
concentrations
in
the
incubation
mixture.
c.
Requires
synthesis
by
commercial
laboratory.
Availability
may
be
difficult
due
to
proprietary
claim.
d.
Commercial
source
may
limit
availability.

5.0
STUDY
PROTOCOL
The
study
protocol
is
included
with
this
study
plan
as
an
attachment.

6.0
DATA
FORMAT
A
uniform
data
format
will
be
developed
for
the
laboratories
to
record
and
present
data
generated
by
conducting
the
sliced
testis
assay.
Data
forms
will
be
developed
for
the
testosterone
concentrations
measured
at
each
time
point
for
a
given
fragment
of
testis
and
the
fragments
used
for
staining
in
order
to
determine
the
number
of
viable
Leydig
cells.
Regarding
the
data
forms
for
collecting
data,
the
results
for
each
analysis
will
be
reported
individually,
with
sufficient
identifying
information
to
determine
which
results
correspond
to
duplicate
analyses,
to
different
time
points
within
one
vial,
to
different
vials
within
the
same
replicate,
and
to
different
replicates.
These
raw
data
collection
forms
will
be
used
to
process
the
data
and
report
it
in
a
summarized
format
as
ng
T/
mg
testis/
hr
for
the
testosterone
analysis
and
cell
number/
cm2
for
the
Battelle
Report
31
August,
2003
Leydig
cell
analysis.
The
forms
and
format
to
be
used
are
under
development.

The
lead
laboratory
will
maintain
a
database
to
include
all
data
generated
during
the
study.
The
databases
will
have
uniform
structure,
formatting,
and
variable
naming
across
laboratories.
Test
conditions,
background
environmental
conditions,
and
results
for
each
analysis
for
each
sample
at
each
time
point
will
be
reported.
These
data
bases
are
under
development.

Additional
details
regarding
data
review
and
distribution,
as
well
as
reporting
format
and
submission
are
included
in
Appendix
B.

7.0
GLP
MONITORING­
QUALITY
ASSURANCE
UNIT
Prevalidation
will
be
conducted
in
accordance
with
Good
Laboratory
Practice
(
GLP)
regulations.
A
GLP­
compliant
protocol
will
direct
the
work
of
the
laboratory(
ies).
The
overall
prevalidation
work
will
be
monitored
by
Battelle's
Quality
Assurance
Officer
and
the
laboratory
work
at
each
laboratory
will
be
monitored
by
the
individual
laboratories'
Quality
Assurance
Unit.
A
QAPP
for
prevalidation
will
be
prepared
by
the
lead
laboratory
and
this
QAPP
will
apply
to
all
participating
laboratories
as
well.
Battelle's
QA
Officer
will
visit
the
participating
laboratories
prior
to
initiation
of
testing
to
ensure
they
are
GLP
compliant.

8.0
TECHINICAL
MONITORING/
COORDINATION
­
ADMINISTRATIVE
SUPPORT
Administrative
support,
in
the
way
of
technical
monitoring
and
coordination
of
activities,
will
be
performed
by
an
individual
from
Battelle
or
the
lead­
laboratory
with
relevant
experience
with
the
overall
program,
purposes
for
conducting
individual
experiments
in
the
prevalidation,
set­
up
and
conduct
of
assay,
interpretation
of
results,
and
timely
submission
of
deliverables.

This
individual
will
make
visits
to
the
participating
labs.
Visits
to
each
laboratory
will
occur
prior
to
initiating
laboratory
work
and
during
the
conduct
of
the
baseline
experiment.
In
addition,
the
focus
of
this
individual
will
be
to:

°
to
establish
milestones
for
lead/
participating
laboratories
based
on
EPA
schedule
°
to
coordination
of
knowledge
transfer
between
lead
laboratory
and
participating
laboratories.
Includes
working
with
lead
laboratory
to
develop/
transfer
protocol
and
study
specific
SOPs
to
participating
laboratories
and
the
implementation
of
such
SOPs
at
the
participating
laboratories
°
to
coordinate
the
development
and
production
of
a
particular
report
format
with
the
lead
laboratory
and
EPA,
which
can
then
be
transferred
and
used
by
the
participating
laboratories
in
order
to
report
results
in
a
consistent
format
°
to
coordinate
the
schedule
for
chemical
procurement,
formulation
preparation
and
analysis,
and
stability
determinations
with
the
Chemical
Repository
and
lead/
participating
laboratories
based
on
EPA
milestone
schedule.
Battelle
Report
32
August,
2003
9.0
REFERENCES
Albertson,
B.
D.,
Frederick,
K.
L.,
Maronian,
N.
C.,
Feuillan,
P.,
Schorer,
S.,
Dunn,
J.
F.,
Lariaux,
D.
L.
(
1988).
The
effect
of
ketoconazole
on
steroidogenesis:
I.
Leydig
cell
enzyme
activity
in
vitro.
Res.
Commun.
Chem.
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Pharmacol.,
Jul;
61:
17­
26.

Ayub,
M.,
and
Levell,
M.
J.
(
1987).
Inhibition
of
rat
testicular
17$­
hydroxylase
and
17,20­
lyase
activities
by
anti­
androgens
(
flutamide,
hydroxyflutamide,
RU23908,
cytoproterone
acetate
(
in
vitro).
J.
Steroid
Biochem.,
28:
43­
47.

Chaudhary,
L.
R.
and
Stocco,
D.
M.
(
1989).
Inhibition
of
hCG­
and
cAMP­
stimulated
progesterone
production
in
MA­
10
mouse
Leydig
tumor
cells
by
ketoconazole.
Biochem.
Inter.
18,
251­
262.

Choi,
M.
S.
K.
and
Cooke,
B.
A.
(
1990).
Evidence
for
two
independent
pathways
in
the
stimulation
of
steroidogenesis
by
luteinizing
hormone
involving
chloride
channels
and
cyclic
AMP.
FEBS
Letters,
261:
402­
404.

Choi,
Y.
S.,
Stocco,
D.
M.,
and
Freeman,
D.
A.
(
1995).
Diethylumbelliferyl
phosphate
inhibits
steroidogenesis
by
interfering
with
a
long­
lived
factor
acting
between
protein
kinase
A
activation
and
induction
of
the
steroidogenic
acute
regulatory
protein
(
StAR).
Eur.
J.
Biochem.,
234(
2):
680­
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Clark,
B.
J.,
Wells,
J.,
King,
S.
R.,
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Stocco,
D.
M.
(
1994).
The
purification,
cloning,
and
expression
of
a
novel
luteinizing
hormone­
induced
mitochondrial
protein
in
MA­
10
mouse
Leydig
tumor
cells:
Characterization
of
a
steroidogenic
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protein
(
STAR).
J.
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269(
45),
28314­
28322.

Cooke,
B.
A.
(
1996).
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cell
structure
and
function
during
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In:
The
Leydig
Cell
(
eds.
Payne,
A.
H.,
Hardy,
M.
P.,
and
Russel,
L.
D.),
Cache
River
Press,
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IL.

Davidoff,
M.
S.,
Middendorff,
R.,
Mayer,
B.,
and
Holstein,
A.
F.
(
1995).
Nitric
oxide
synthase
(
NOS­
i)
in
Leydig
cells
of
the
human
testis.
Arch.
Histol.
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58:
17­
30.

Fail,
P.
A.,
Pearce,
S.
W.,
Anderson,
S.
A.,
Tyl,
R.
W.,
and
Gray,
L.
E.,
Jr.
(
1995).
Endocrine
and
reproductive
toxicity
of
vinclozolin
(
VIN)
in
male
Long­
Evans
hooded
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The
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15,
293
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1570).

Fail,
P.
A.,
Pearce,
S.
W.,
Anderson,
S.
A.,
and
Gray,
L.
E.,
Jr.
(
1994).
Methoxychlor
alters
testosterone
and
LH
response
to
human
chorionic
gonadotropin
(
hCG)
or
gonadotropin­
releasing
hormone
(
GNRH)
in
male
Long­
Evans
hooded
rats.
Biology
of
Reproduction
50(
1),
106
(
Abstract
206).

Gray,
L.
E.,
Klinefelter,
G.,
Kelce,
W.,
Laskey,
J.,
Ostby,
J.,
and
Ewing,
L.
(
1995).
Hamster
Battelle
Report
33
August,
2003
Leydig
cells
are
less
sensitive
to
ethane
dimethanesulfonate
when
compared
to
rat
Leydig
cells
both
in
vivo
and
in
vitro.
Toxicol.
Appl.
Pharmacol.
130,
248­
256.

Gürtler,
J.
and
Donatsch,
P.
(
1979).
Effects
of
two
structurally
different
antispermatogenic
compounds
on
the
synthesis
of
steroids
in
rat
testes.
Arch.
Toxicol.
41(
2),
381­
385.

Janszen,
F.
H.
A.,
Cooke,
B.
A.,
van
Driel,
M.
J.
A.,
and
Van
der
Molen,
H.
J.
(
1976).
The
effect
of
calcium
ions
on
testosterone
production
in
Leydig
cells
from
rat
testis.
Biochem
J.,
160:
433­
437.

Johnston,
J.
O.
(
1997).
"
Aromatase
Inhibitors."
In:
Biochemistry
and
Function
of
Sterols.
Eds.
Parish,
E.
J.
and
Nes,
W.
C.
CRC
Press,
pp
23­
53.
Battelle
Report
A­
1
July,
2003
APPENDIX
A
Draft
Protocol
for
the
Prevalidation
of
the
Sliced
Testis
Assay
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
1
of
37
EPA
Contract
No.:
68­
W­
01­
023
(
Battelle
Prime
Contractor)
Contract
No.:
Study
Code:
Master
Protocol
No.:

TITLE:
Draft
Prevalidation
of
the
Sliced
Testis
Assay
SPONSOR:
Battelle
Memorial
Institute
505
King
Avenue
Columbus,
OH
43201­
2693
TESTING
FACILITY:
[
Name
and
address
of
lab]

PROPOSED
EXPERIMENTAL
START
DATE:

PROPOSED
EXPERIMENTAL
TERMINATION
DATE:

AMENDMENTS:

Number
Date
Section(
s)
Page(
s)
1
2
3
4
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
2
of
37
APPROVED
BY:

___________________________________
[
Name,
title,
lab
affiliation]
Date
Study
Director
___________________________________
Gary
E.
Timm
Date
Work
Assignment
Manager
Endocrine
Disruptor
Screening
Program
U.
S.
EPA
___________________________________
L.
Greg
Schweer
Date
Project
Officer
Endocrine
Disruptor
Screening
Program
U.
S.
EPA
___________________________________
[
Name,
title,
lab
affiliation]
Date
Management
___________________________________
David
P.
Houchens,
Ph.
D.
Date
Program
Manager
Endocrine
Disruptor
Screening
Program
Battelle
Memorial
Institute
REVIEWED
BY:

___________________________________
J.
Thomas
McClintock,
Ph.
D.
Date
EPA
Quality
Assurance
Manager
___________________________________
Terri
L.
Pollock,
B.
A.
Date
Quality
Assurance
Manager
Battelle
Memorial
Institute
____________________________________
[
Name,
title,
and
lab
affiliation]
Date
Quality
Assurance
Specialist
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
3
of
37
TABLE
OF
CONTENTS
Page
1.0
BACKGROUND
AND
OBJECTIVES
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1.1
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1.2
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2.0
CHEMICALS
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7
2.1
TEST
CHEMICALS,
REAGENTS
AND
SOLUTIONS
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2.1.1
Aminoglutethimide
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7
2.1.2
Dimethoate
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7
2.1.3
Fenarimol
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8
2.1.4
Flutamide
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8
2.1.5
Genistein
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8
2.1.6
Ketoconazole
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9
2.1.7
Diethylumbelliferyl
phosphate
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9
2.1.8
MK­
434
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10
2.1.9
Trilostane
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10
2.1.10
Vinclozolin
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10
2.1.11
Bisphenol
A
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11
2.1.12
Lindane
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11
2.2
STANDARDS
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12
2.2.1
Testosterone
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12
2.2.2
Human
Chorionic
Gonadotropin
(
hCG)
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12
2.3
CHEMICAL
SAFETY
AND
HANDLING
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12
3.0
ANIMALS
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13
3.1
TEST
ANIMALS
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13
3.1.1
Species
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13
3.1.2
Supplier
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13
3.1.3
Rationale
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13
3.1.4
Number
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Sex
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13
3.1.5
Age
and
Weight
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14
3.1.6
Body
Weight
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14
3.1.7
Identification
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14
3.1.8
Limitation
of
Discomfort
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14
3.1.9
Culled
Animals
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14
3.1.10
Quality
Control
and
Sentinels
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14
3.2
HUSBANDRY
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15
3.2.1
Conditions
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15
3.2.2
Diet
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15
3.2.3
Water
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15
4.0
SLICED
TESTIS
ASSAY
PROCEDURE
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16
5.0
PREVALIDATION
STUDY
DESIGN
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18
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
4
of
37
5.1
BASELINE/
CONTRALATERAL
TESIS
FRAGMENT
EXPERIMENT
.
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.
18
5.2
EVALUATION
OF
AMINOGLUTETHIMIDE
(
AG)
AS
A
POSITIVE
CONTROL
.
.
.
.
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.
20
5.3
CYTOTOXICITY
.
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21
5.4
TRAINING/
HANDS­
ON
EXPERIENCE
.
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22
5.5
BASELINE
EXPERIMENT
.
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22
5.6
POSITIVE
CONTROL
EXPERIMENT
.
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23
5.7
MULTICHEMICAL
EXPERIMENTS
.
.
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24
6.0
CHEMISTRY
.
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.
27
6.1
CHEMICAL
PROCUREMENT
AND
CHARACTERIZATION
.
.
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.
27
6.2
FORMULATION
PREPARATIONS
.
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27
6.3
FORMULATION
ANALYSIS
.
.
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28
6.4
FORMULATION
STABILITY
.
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28
7.0
ENDPOINT
MEASUREMENTS
.
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28
7.1
TESTOSTERONE
RIA
PROCEDURE
.
.
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29
7.2
3$­
HSD
LEYDIG
CELL
STAINING
PROCEDURE
.
.
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.
30
8.0
DATA
ANALYSIS
.
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31
9.0
STATISTICAL
ANALYSIS
.
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.
31
9.1
CONTRALATERAL
TESTIS
FRAGMENT
EXPERIMENT
.
.
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.
31
9.2
BASELINE
EXPERIMENT
.
.
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.
32
9.3
POSITIVE
CONTROL
EXPERIMENT
.
.
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.
34
9.4
MULTICHEMICAL
EXPERIMENT
.
.
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.
36
10.0
RETENTION
OF
SPECIMENS
AND
RECORDS
.
.
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.
36
11.0
QUALITY
CONTROL/
QUALITY
ASSURANCE
PROCEDURES
.
.
.
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.
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.
36
12.0
REPORTING
.
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.
36
13.0
PERSONNEL
.
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.
37
14.0
STUDY
RECORDS
TO
BE
MAINTAINED
.
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37
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
5
of
37
1.0
BACKGROUND
AND
OBJECTIVES
1.1
BACKGROUND
In
1996,
the
Food
Quality
Protection
Act
(
FQPA)
amendments
were
enacted
by
Congress
to
authorize
the
EPA
to
implement
an
Endocrine
Disruptor
Screening
Program
(
EDSP)
on
pesticides
and
other
substances
found
in
food
or
water
sources
for
endocrine
effects
in
humans
(
FQPA,
1996).
In
this
program,
comprehensive
toxicological
and
ecotoxicological
screens
and
tests
are
being
developed
for
identifying
and
characterizing
the
endocrine
effects
of
various
environmental
contaminants,
industrial
substances,
and
pesticides.
A
two­
tiered
approach
will
be
utilized.
Tier
1
employs
a
combination
of
in
vivo
and
in
vitro
screens,
and
Tier
2
involves
in
vivo
testing
methods
using
two­
generation
reproductive
studies.
A
steroidogenesis
assay
is
proposed
as
one
of
the
Tier
1
screening
battery
assays.

A
detailed
review
paper
(
DRP)
about
steroidogenesis
was
prepared.
The
DRP
(
1)
summarized
the
state
of
the
science
of
the
in
vivo,
ex
vivo,
and
in
vitro
methodologies
available
for
measuring
gonadal
steroidogenesis;
(
2)
for
each
methodology,
presented
a
review
of
the
individual
assays
and
representative
data
generated
by
investigators
who
used
the
assay
to
evaluate
a
substance
for
steroidogenic­
altering
activity;
(
3)
provided
an
evaluation
of
the
various
methodologies
and
the
assays
as
tools
for
screening
substances
with
suspected
steroidogenic
activity;
(
4)
recommended
a
particular
screening
method
and
assay
as
a
screening
tool;
and
(
5)
described
the
strengths,
weaknesses,
and
implications
for
further
research
associated
with
the
recommended
screening
assay.

The
in
vitro
sliced
testis
steroidogenesis
assay
was
selected
as
the
most
promising
screening
tool
for
identifying
substances
with
steroidogenic­
altering
activity.
The
sliced
testis
assay
was
recommended
because
it
can
be
conducted
at
a
minimal
cost,
quickly,
and
simply
with
standard
laboratory
equipment
and
basic
laboratory
training;
the
preparation
is
stable
and
the
parenchyma
remains
viable
over
a
sufficient
time
period
to
measure
changes
in
end­
product
hormone
production;
the
assay
is
relatively
sensitive
and
specific;
the
assay
uses
parenchyma
that
maintains
the
cytoarchitecture
of
the
organ;
the
assay
uses
a
reduced
number
of
animals
(
up
to
quartered
testis
slices);
the
assay
will
be
relatively
easy
to
standardize
(
by
optimization);
and
the
assay
has
a
well­
defined
endpoint
in
testosterone
and,
if
desired,
can
be
modified
to
include
additional
intermediate
hormonal
endpoints.
The
experiments
necessary
to
identify
the
factors
and
conditions
that
optimize
the
assay
have
been
completed
and
much
of
the
data
analysis
has
been
finished.
While
there
may
be
some
minor
changes
to
the
procedure,
the
assay
as
described
in
the
present
protocol
describes
what
is
presently
believed
to
be
the
optimized
assay
procedure.
As
such,
this
procedure
will
be
used
to
proceed
with
the
experiments
believed
necessary
to
prevalidate
the
assay.
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
6
of
37
1.2
OBJECTIVES
The
objectives
of
the
prevalidation
studies
will
be
to
assess
the
relevance
of
the
sliced
testes
assay
for
detecting
compounds
that
inhibit
steroidogenesis.
Relevance
will
be
assessed
by
demonstrating
that
the
assay
can
detect
inhibition
of
steroid
hormone
synthesis
by
determining
the
change
in
the
production
of
testosterone.

These
prevalidation
studies
will
be
performed
by
a
lead
laboratory
and
three
additional
participating
laboratories.
The
table
below
summarizes
the
different
studies
to
be
conducted
and
the
laboratories
that
will
be
involved
in
the
conduct
of
each
study.

Study
Phase
Study
Type
Laboratory
Assignment
1
Baseline/
Contralateral
Testis
Fragment
Lead
Laboratory
2
Evaluation
of
AG
as
Positive
Control
Lead
Laboratory
3
Selection
of
Cytotoxicant
as
Positive
Control
Lead
Laboratory
4
Training/
Hands­
On
Experience
Lead
Laboratory
+
3
Participating
Laboratories
5
Baseline
Experiment
Lead
Laboratory
+
3
Participating
Laboratories
6
Positive
Control
Experiment
Lead
Laboratory
+
3
Participating
Laboratories
7
Multichemical
Experiments
Lead
Laboratory
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
7
of
37
2.0
CHEMICALS
Below
are
a
list
of
reagents
to
be
used
and
test
chemicals
to
be
tested
using
the
assay.
Information
provided
for
each
test
chemical
includes
identification
and
physical­
chemical
properties.

2.1
TEST
CHEMICALS
2.1.1
Aminoglutethimide
(
Positive
Control)

CAS
Number:
55511­
44­
9
Synonyms:
Lot
Number:
Purity:
Appearance:
Solid
Molecular
Formula:
C13H16N2O2
Molecular
Weight:
232.3
Storage,
Bulk
Chemical:
Storage,
Test
Solution:

2.1.2
Dimethoate
CAS
Number:
60­
51­
5
Synonyms:
MDL#
MFCD00053676
Lot
Number:
Purity:
Supplier:
Appearance:
Molecular
Formula:
C6H12NO3PS2
Molecular
Weight:
229.3
Storage,
Bulk:
2­
80C
Storage,
Test
Solution:
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
8
of
37
2.1.3
Fenarimol
CAS
Number:
60168­
88­
9
Synonyms:
MDL#
MFCD00055325
Lot
Number:
Purity:
Supplier:
Appearance:
Molecular
Formula:
C17H12Cl2NO
Molecular
Weight:
331.2
Storage,
Bulk:
Storage,
Test
Solution:

2.1.4
Flutamide
CAS
Number:
13311­
84­
7
Synonyms:
2
Methyl­
 
(
4
nitro­
3­[
trifluoromethyl]
phenyl
propanamide,
MDL#
MFCD00072009
Lot
Number:
Purity:
Supplier:
Molecular
Formula:
C11H11F3N2O3
Molecular
Weight:
276.2
Storage,
Bulk:
Room
Temp.
Storage,
Test
Solution:

2.1.5
Genistein
CAS
Number:
446­
72­
0
Synonyms:
5,7­
Dihydroxy­
3­(
4­
hydroxyphenyl)­
4H­
1­
benzopyran­
4­
one
4 ,
5,7­
Trihydroxyisoflavone,
MLD
#
MFCD00016952
Lot
Number:
Purity:
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
9
of
37
Supplier:
Appearance:
off­
white
solid
Molecular
Formula:
C15H10O5
Molecular
Weight:
270.2
Storage,
Bulk:
­
00C
Storage,
Test
Solution:

2.1.6
Ketoconazole
CAS
Number:
65277­
42­
1
Synonyms:(
±
)
­
cis­
1­
Acetyl­
4­(
4­[(
2­[
2,4­
dichlorophenyl]­
2­[
1H­
imidazol­
1­
ylmethyl]­
1,3­
dioxolan­
4­
yl)­
methoxy]
phenyl)
piperazine,
MLD
#
MFCD00058579
Lot
Number:
Purity:
Supplier:
Appearance:
Molecular
Formula:
C26H28Cl2N4O4
Molecular
Weight:
531.4
Storage,
Bulk:
2­
80C
Storage,
Test
Solution:

2.1.7
Diethylumbelliferyl
Phosphate
CAS
Number:
65277­
42­
1
Synonyms:(
±
)
­
cis­
1­
Acetyl­
4­(
4­[(
2­[
2,4­
dichlorophenyl]­
2­[
1H­
imidazol­
1­
ylmethyl]­
1,3­
dioxolan­
4­
yl)­
methoxy]
phenyl)
piperazine,
MLD
#
MFCD00058579
Lot
Number:
Purity:
Supplier:
Appearance:
Molecular
Formula:
C26H28Cl2N4O4
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
10
of
37
Molecular
Weight:
531.4
Storage,
Bulk:
2­
80C
Storage,
Test
Solution:

2.1.8
MK­
434
CAS
Number:
65277­
42­
1
Synonyms:(
±
)
­
cis­
1­
Acetyl­
4­(
4­[(
2­[
2,4­
dichlorophenyl]­
2­[
1H­
imidazol­
1­
ylmethyl]­
1,3­
dioxolan­
4­
yl)­
methoxy]
phenyl)
piperazine,
MLD
#
MFCD00058579
Lot
Number:
Purity:
Supplier:
Appearance:
Molecular
Formula:
C26H28Cl2N4O4
Molecular
Weight:
531.4
Storage,
Bulk:
2­
80C
Storage,
Test
Solution:

2.1.9
Trilostane
CAS
Number:
65277­
42­
1
Synonyms:(
±
)
­
cis­
1­
Acetyl­
4­(
4­[(
2­[
2,4­
dichlorophenyl]­
2­[
1H­
imidazol­
1­
ylmethyl]­
1,3­
dioxolan­
4­
yl)­
methoxy]
phenyl)
piperazine,
MLD
#
MFCD00058579
Lot
Number:
Purity:
Supplier:
Appearance:
Molecular
Formula:
C26H28Cl2N4O4
Molecular
Weight:
531.4
Storage,
Bulk:
2­
80C
Storage,
Test
Solution:

2.1.10
Vinclozolin
(
Negative
Test
Chemical)

Chemical
Name:
3­(
3,5­
Dichlorophenyl)­
5­
ethenyl­
5­
methyl­
2,4­
oxazolidinedione
CAS
Number:
5­
0471­
44­
8
Supplier:
BASF
Manufacturer's
Batch
No.:
To
be
added
to
the
protocol
by
amendment
Appearance:
To
be
added
to
the
protocol
by
amendment
Molecular
Formula:
C12H9Cl2NO3
Molecular
Weight:
286.114
Storage
Conditions
of
Bulk
Chemical:
To
be
added
to
the
protocol
by
amendment
DRAFT
PROTOCOL
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11
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37
Storage
Conditions
of
Dosing
Suspensions:
To
be
added
to
the
protocol
by
amendment
2.1.11
Bisphenol
A
CAS
Number:
1565­
94­
2
Synonyms:
MDL
#
MFCD01317012
Lot
Number:
Purity:
Appearance:
liquid
Molecular
Formula:
C29H36O8
Molecular
Weight:
512.6
Storage,
Bulk:
Storage,
Test
Solution:

2.1.12
Lindane
CAS
Number:
58­
89­
9
Synonyms:
1 ,
2 ,
3 ,
4 ,
5 ,
6 ­
Hexachlorocyclohexane,
 ­
1,2,3,4,5,6­
Hexachlorocyclohexane,
MDL
number:
MFCD00135947
Lot
Number:
Purity:
Supplier:
Appearance:
Molecular
Formula:
C6H6Cl6
Molecular
Weight:
290.8
Storage,
Bulk:
Room
Temp
Storage,
Test
Solution:
DRAFT
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2.2
STANDARDS
Standardization
of
the
analytical
assays
will
require
testosterone
for
the
RIA
method.
In
addition,
hCG
will
be
used
as
a
stimulant
of
the
sliced
testis
bioassay.
These
substances
will
be
considered
standards
and
will
be
handled
and
documented
according
to
the
laboratory's
Standard
Operating
Procedures
for
standards.

2.2.1
Testosterone
Chemical
Name:
Testosterone
CAS
No.:
58220
Molecular
Formula/
Weight:
288.4
Solubility:
Clear
colorless
to
very
faint
yellow
solution
at
100
mg/
mL
in
chloroform
Supplier:
Sigma­
Aldrich
Chemical
Company
Lot
No.:
TBD
Purity:
NLT
98%
Storage
Conditions:
2
Year
shelf
life
A
safety
protocol
exists
for
the
use
of
the
radioactive
form
of
testosterone.

2.2.2
Human
Chorionic
Gonadotropin
(
hCG)

Chemical
Name:
hCG
CAS
No.:
9002­
61­
3
Molecular
Formula/
Weight:
36,700
Solubility:
H2O
Supplier:
Calbiochem
Lot
No.:
TBD
Purity:
TBD
Storage
Conditions:
Freezer
(­
20
degrees
C).
Following
reconstitution,
aliquot
and
freeze
(­
20
degrees
C).
Stable
for
2
years
as
supplied.

2.3
CHEMICAL
SAFETY
AND
HANDLING
Once
the
chemicals
to
be
tested
and
used
according
to
the
final
protocol
are
determined,
then
an
MSDS
for
each
chemical
will
be
included
as
an
attachment
to
the
protocol.
DRAFT
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Page
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37
3.0
ANIMALS
3.1
TEST
ANIMALS
An
IACUC
protocol
will
be
submitted
and
approved
before
the
beginning
of
the
prevalidation
experiments.
Animals
will
be
sequentially
ordered
on
an
as
needed
basis
for
each
of
the
experiments.

3.1.1
Species
Sprague
Dawley
Derived
Outbred
Albino
Rat
[
Crl:
CD
®
(
SD)
IGSBR],
known
as
the
Charles
River
CD
®
Rat.

3.1.2
Supplier
Charles
River
Laboratories,
Inc.,
Raleigh,
NC.

3.1.3
Rationale
The
use
of
live
animals
is
necessary
to
provide
fresh
testicular
tissue
for
the
assay
optimization.
Alternative
test
systems
are
not
available
for
the
assessment
of
chemical
effects
on
the
cultured
testicular
cells.
The
albino
rat
is
the
species
of
choice
and
the
Sprague­
Dawley
rat
has
been
employed
in
previous
reproductive
toxicity
studies.

3.1.4
Number
and
Sex
Male
rats
will
be
used
to
conduct
the
experiments
in
this
protocol.
Regarding
the
number
of
animals
that
will
be
used,
an
approximation
can
be
made
based
on
the
design
of
each
experiment.
Approximately
8
­
10
fragments
can
be
obtained
from
each
testis.
The
numbers
below
are
nominal
numbers
and
actual
numbers
used
may
vary
based
individual
laboratory
procedures,
e.
g.
sentinel
animals
for
health
monitoring.

Study
Phase
Study
Type
Number
of
Male
Rats
1
Baseline/
Contralateral
Testis
Fragment
10
2
Evaluation
of
AG
as
Positive
Control
2
3
Selection
of
Cytotoxicant
as
Positive
Control
4
4
Training/
Hands­
On
Experience
8
5
Baseline
Experiment
12
DRAFT
PROTOCOL
[
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37
Study
Phase
Study
Type
Number
of
Male
Rats
6
Positive
Control
Experiment
36
7
Multichemical
Experiments
~
40
3.1.5
Age
and
Weight
The
animals
will
be
approximately
10
weeks
of
age
on
the
scheduled
animal
receipt
date
for
the
experiments.
Testes
removal
will
be
when
the
animals
are
between
11
and
15
weeks
of
age.
Males
should
be
approximately
275
to
400
grams.
Testis
that
weight
1000
±
200
mg
will
be
used
for
testing.

3.1.6
Body
Weight
All
animals
will
be
weighed
at
least
once
during
acclimation
and
quarantine.

3.1.7
Identification
Animals
shall
be
uniquely
identified
by
eartag
within
three
days
of
arrival.
The
method
of
identification
and
animal
numbers
will
be
documented
in
the
study
records.

3.1.8
Limitation
of
Discomfort
There
should
be
no
discomfort
or
injury
to
animals;
if
any
animal
becomes
severely
debilitated
or
moribund,
it
will
be
humanely
terminated
by
CO2
asphyxiation.
All
necropsies
will
be
performed
after
terminal
anesthesia
with
CO2.
Animals
will
not
be
subjected
to
undue
pain
or
distress.

3.1.9
Culled
Animals
Animals
received
with
the
shipment,
but
not
used
in
the
study,
will
be
removed
from
the
study
room.
Records
will
be
kept
documenting
the
fate
of
all
animals
received
for
the
study.

3.1.10
Quality
Control
and
Sentinels
Sentinel
Animals
will
not
be
necessary
since
the
animals
will
not
be
in
house
for
more
than
1
month.
The
animals
will
be
ordered
as
close
as
possible
to
the
time
of
use.
If
any
quality
control
is
necessary,
it
will
be
conducted
on
the
test
animals
during
the
quarantine
period.
DRAFT
PROTOCOL
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Page
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37
3.2
HUSBANDRY
3.2.1
Conditions
The
animal
portions
of
this
protocol
will
be
carried
out
under
standard
laboratory
conditions.
The
animals
will
be
housed
1
per
cage
upon
arrival,
during
the
acclimation
period,
and
until
used
for
testing
in
solid­
bottom
polycarbonate
cages
with
stainless
steel
wire
lids
(
Laboratory
Products,
Rochelle
Park,
NJ),
with
Sani­
Chip
®
cage
litter
(
P.
J.
Murphy
Forest
Products
Corp.,
Montville,
NJ).
The
cage
dimensions
will
be
8"
x19"
x10.5"
(
height)
for
all
phases
of
this
study.

All
animals
will
be
housed
in
approved
Animal
Facilities
following
arrival
and
until
needed.
The
animal
rooms
should
be
air­
conditioned,
and
temperature
and
relative
humidity
are
continuously
monitored,
controlled,
and
recorded
using
an
automatic
system.
The
target
environmental
ranges
will
be
64
to
79/
F
(
18­
26/
C)
for
temperature
and
30
to
70%
relative
humidity
with
a
12
hours
light:
12
hours
dark
cycle
per
day
(
NRC
Guide,
1996;
see
below).
Temperature
and/
or
relative
humidity
excursions
outside
the
target
ranges
will
be
documented
in
the
study
records
and
in
the
final
report.
The
specific
animal
rooms
used
will
be
documented
in
the
study
records
and
in
the
final
report.
At
all
times,
the
animals
will
be
handled,
cared
for,
and
used
in
compliance
with
the
NRC
Guide
for
the
Care
and
Use
of
Laboratory
Animals
(
1996).

3.2.2
Diet
Purina
Certified
pelleted
Rodent
Diet
®
(
No.
5002,
PMI
Feeds,
Inc.,
St.
Louis,
MO)
will
be
available
ad
libitum.
The
analysis
of
each
feed
batch
for
nutrient
levels
and
possible
contaminants
will
be
performed
by
the
supplier,
examined
by
the
Study
Director,
and
maintained
in
the
study
records.
It
is
anticipated
that
contaminant
levels
will
be
below
certified
levels
and
will
not
affect
the
design,
conduct,
or
conclusions
of
this
study.
The
feed
will
be
stored
at
approximately
60­
70/
F,
and
the
period
of
use
will
not
exceed
six
months
from
the
milling
date.

3.2.3
Water
Animals
will
receive
tap
water
(
at
RTI
this
will
be
the
city
municipal
supply.
Water
will
be
available
ad
libitum
by
plastic
water
bottles
with
butyl
rubber
stoppers
and
stainless
steel
sipper
tubes.
The
supplier
per
EPA
specifications
should
measure
contaminant
levels
of
the
city
water
at
regular
intervals.
Documentation
of
these
analyses
will
be
inspected
by
the
Study
Director
and
maintained
in
the
study
records.
It
is
anticipated
that
contaminant
levels
will
be
below
the
maximal
levels
established
for
potable
water
and
will
not
affect
the
design,
conduct,
or
conclusions
of
these
studies.
DRAFT
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4.0
SLICED
TESTIS
ASSAY
PROCEDURE
[
Note:
The
procedure
described
below
is
the
preliminary
optimized
assay
pending
completion
of
the
sensitivity
analysis
of
the
optimization
study
results.]

The
assay
procedure
will
begin
at
a
time
that
will
enable
the
technician
to
complete
all
incubations
and
sample
collections
before
approximately
noon
on
a
given
day
(
does
not
include
the
analyses).
An
11­
week
old
male
Sprague­
Dawley
rat
will
be
euthanized.
The
testes
will
be
removed
[
develop
SOP
for
testis
removal
and
preparation].
The
testes
will
be
weighed
[
acceptance
criterion]
and
placed
in
cold
(
40
C)
Dulbeccos
Phosphate
Buffered
Saline
(
DPBS;
undiluted;
pH
7.2
­
7.4).
The
time
from
removal
of
the
testis
to
the
time
of
slicing
will
be
less
than
1
hour.
Each
testis
will
have
the
tunica
albicans
removed
and
will
be
sliced
longitudinally
to
yield
pieces
weighing
within
10%
of
0.175
grams
(
between
0.157
g
and
0.192
g).
[
actual
slice
weight
to
be
determined
based
on
optimization
results].
Each
slice
will
be
placed
into
a
tightly
capped
scintillation
vial
containing
5
mL
of
95%
O2
/
5%
CO2
gassed
media.
[
develop
SOP
for
testis
slice
distribution
among
test
tubes].
The
media
will
be
medium­
199
without
phenol
red
(
Invitrogen),
which
will
have
been
adjusted
to
a
pH
of
7.4.
[
check
on
media
components].

The
vials
containing
the
testicular
sections
and
media
will
be
incubated
at
360C
[
temperature
to
be
determined
based
on
optimization
results]
on
a
shaker
at
135
rpm
[
actual
RPMs
will
be
based
on
optimization
study
results].
After
the
first
period
of
incubation,
1
hour
[
actual
equilibration
period
will
be
based
on
optimization
study
results],
the
contents
of
the
vial
will
be
filtered
using
filter
paper
[
check
on
use
of
gentle
centrifugation].
The
equilibration
media
will
be
discarded
without
analysis.
If
microscopic
examination
of
the
fragment
is
to
be
performed,
then
remove
a
piece
of
the
fragment,
transfer
it
to
a
screw­
cap
cryovial,
snap
freeze
the
sample
in
liquid
nitrogen,
and
store
it
in
a
­
70
degrees
freezer
until
removed
for
further
processing.
New
media
(
5.0
mL)
will
be
placed
in
the
original
vial
and
the
testis
tissue
will
be
returned
to
the
vial.
One
aliquot
(
0.5
mL)
of
media
[
need
further
evaluation
of
aliquot
volume]
will
be
collected
for
analysis
(
Time
0,
Baseline
Sample).
The
sample
aliquot
will
be
analyzed
for
testosterone.
[
develop
SOPs
for
testosterone
RIA]
To
one­
half
of
the
vials,
a
0.5
mL
volume
of
media
without
hCG
(
unstimulated)
will
be
added,
and
to
the
other
half
of
the
vials,
0.5
mL
of
media
with
hCG
(
stimulated;
final
concentration
to
be
determined
based
on
optimization
study
results)
will
be
added
to
the
vials
to
replace
the
volume
removed
for
analysis,
as
well
as
to
initiate
testosterone
production
(
hCG
stimulated
vials
only).
[
Note:
For
assays
using
a
test
chemical,
the
test
chemical
will
also
be
formulated
in
the
media
so
that
when
the
replacement
media
is
added,
at
time
0,
the
final
target
concentration
will
be
achieved.]

At
1,
2,
3
and
4
hours
post­
challenge,
one
0.5
mL
sample
of
media
will
be
collected
from
each
of
the
hCG­
stimulated
and
non­
stimulated
vials.
After
each
sampling,
the
appropriate
media
with
or
without
hCG
will
be
added
to
the
vial
to
replace
the
volume
removed
for
analysis.
The
sample
aliquots
removed
at
each
time
point
will
be
analyzed
for
testosterone.
Also,
at
the
end
of
the
incubation
period,
e.
g.
4
hours,
remove
the
tissue
fragment
and
collect
a
sample
for
DRAFT
PROTOCOL
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Page
17
of
37
Shake
and
Incubate
at
34
°
C,
95%
O2,
5%
CO2
hCG
Challenge
(
stimulated)

Sliced
Rat
Testis
No
Challenge
(
unstimulated)

Time
Equilibration
Period
(
1
hr)
Add
Testis
Fragment
to
Media
Media
sampled;
then
add
media
and
hCG,
with
or
without
Test
Chemical
Media
Samples,
then
Add
Media
with
or
without
Test
Chemical
Discard
old
and
replenish
with
new
media
Media
Sample
Media
Sample
Media
Sample
Media
and
Tissue
Fragment
Sample
Equilibration
Period
(
1
hr)

T0
0
hr
Baseline
T1
+
1
hr
Post­
Challenge
T2
+
2
hr
Post­
Challenge
T3
+
3
hr
Post­
Challenge
T4
+
4
hr
Post­
Challenge
microscopic
examination.
Transfer
the
tissue
piece
to
a
cryovial,
snap
freeze
it
in
liquid
nitrogen,
and
store
at
­
70
degrees
C
until
removed
for
processing.

Media
samples
collected
at
the
specified
time
points
will
be
frozen
and
stored
at
­
70
degrees
C
and
analyzed
within
one
month
after
collection.
Testosterone
will
be
analyzed
in
duplicate
for
each
sample
using
an
RIA
method
modified
for
rat
samples.
All
samples
for
a
given
day's
set
of
runs
will
be
analyzed
in
the
same
testosterone
RIA
,
if
possible.
Tissue
samples
will
be
thawed,
sectioned,
stained,
and
mounted
for
microscopic
examination.
The
staining
procedure
is
specific
for
beta­
HSD,
an
enzyme
specific
to
Leydig
cells.

Figure
1.
Technical
Flow
Illustration
of
the
Sliced
Testis
Assay
DRAFT
PROTOCOL
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37
5.0
Prevalidation
STUDY
DESIGN
5.1
BASELINE/
CONTRALATERAL
TESTIS
FRAGMENT
EXPERIMENT
This
experiment
will
be
conducted
by
the
lead
laboratory
only.

The
purposes
of
this
experiment
are:

°
to
demonstrate
competence
of
lead
lab
using
optimized
assay
conditions
°
to
estimate
response
trends
in
T
production
in
the
absence
of
inhibiting
chemicals
°
to
estimate
Leydig
cell
density
after
four
hours
of
incubation,
in
the
absence
of
inhibiting
chemicals
°
to
determine
the
variability
in
assay
response
associated
with
animals,
contralateral
testes
within
animals,
testis
fragments
within
testes
The
experimental
design
is
summarized
in
the
following
table:

Time
(
Hours
from
Equilibration)
hCG
Animal
Testis
Fragment
#
Incubation
(
Run)
#

0
no
1
­
3
A
1
1
­
3
yes
2
4
­
6
1
no
1
­
3
A
1
1
­
3
yes
2
4
­
6
2
no
1
­
3
A
1
1
­
3
yes
1
­
3
A
2
4
­
6
1
B
1,
2
7,
8
2
A
3,
4
9,
10
B
1,
2
11,
12
3
A
3,
4
13,
14
B
1,
2
15,
16
4
A
1,
2
17,
18
B
1,
2
19,
20
5
A
1,
2
21,
22
B
1,
2
23,
24
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
19
of
37
3
no
1
­
3
A
1
1
­
3
yes
2
4
­
6
4
no
1
­
3
A
1
1
­
3
yes
1
­
3
A
2
4
­
6
1
B
1,
2
7,
8
2
A
3,
4
9,
10
B
1,
2
11,
12
3
A
3,
4
13,
14
B
1,
2
15,
16
4
A
1,
2
17,
18
B
1,
2
19,
20
5
A
1,
2
21,
22
B
1,
2
23,
24
The
experiment
summarized
in
the
above
table
represents
one
replicate
study.
A
replicate
study
is
an
independently
conducted
experiment;
separated
by
day
and
using
freshly
prepared
reagents.
Two
full
replicates
will
be
conducted
for
runs
1
­
24
and
a
third
partial
replicate
will
be
carried
out
for
runs
1
­
6.
Runs
1­
6
within
each
replicate
will
use
two
fragments
taken
from
a
single
testis
from
each
of
three
animals.
Runs
7­
24
within
each
of
the
two
full
replicate
will
use
five
animals,
two
testes
within
each
animal,
and
two
fragments
from
each
testis.
Each
replicate
will
use
different
animals.
The
total
number
of
individual
fragments
and
incubations
used
to
conduct
this
experiment
is
24.

The
sampling
time
points
from
the
media
range
from
2
to
5
depending
on
the
sample
type.
If
two
samples,
then
the
time
points
are
at
2
and
4
hours
post­
equilbration.
If
five,
then
the
sampling
time
points
are
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
postequilibration
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
324
samples
[
Runs
1
­
6
has
5
time
points
with
duplicate
analyses
for
60
samples
and
runs
7
­
24
has
2
time
points
with
duplicate
analyses
for
72
samples,
which
gives
a
total
of
132
samples/
replicate.
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
20
of
37
5.2
EVALUATION
OF
AMINOGLUTETHIMIDE
(
AG)
AS
A
POSITIVE
CONTROL
This
experiment
will
be
conducted
by
the
lead
laboratory
only.

The
purpose
of
this
experiment
is
to
determine
the
optimized
assay
response
to
AG
when
tested
at
three
concentrations.
From
this
information,
AG
may
be
selected
as
a
positive
control
when
the
sliced
testis
assay
is
used
to
evaluate
the
effect
of
a
test
chemical.
The
experimental
design
is
summarized
in
the
following
table:

Sample
Type
hCG
Number
of
Incubations
(
Runs)
Testis
Fragment
ID
Number
Media­
Vehicle
control
no
3
1
­
3
Media­
Vehicle
control
yes
3
4
­
6
Media
+
AG
(
low)
yes
3
7
­
9
Media
+
AG
(
mid)
yes
3
10
­
12
Media
+
AG
(
high)
yes
3
13
­
15
The
experiment
summarized
in
the
above
table
represents
one
replicate
study.
A
replicate
study
is
an
independently
conducted
experiment;
separated
by
day
and
using
freshly
prepared
reagents.
Two
replicates
will
be
conducted.
The
overall
experiment
will
use
three
rats/
replicate
study;
three
testes
total/
replicate
study
(
based
on
using
5
fragments/
testis).
Within
each
replicate,
the
5
fragments
from
a
single
testis
will
be
distributed
among
the
5
test
conditions.
The
overall
total
number
of
individual
fragments
and
incubations
used
to
conduct
this
experiment
is
30.

The
sampling
time
points
(
5)
from
the
media
are
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
In
addition,
at
the
end
of
the
incubation
period,
a
piece
of
the
tissue
fragment
will
be
collected
and
snap­
frozen
for
beta­
HSD
staining
and
microscopic
analysis.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
300
samples
[
30
runs
x
5
sampling
time
points
x
2
(
duplicate)
analyses]
and
the
overall
total
number
of
tissue
samples
is
30
[
30
runs
x
1
time
point].
DRAFT
PROTOCOL
[
Name
and
address
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Page
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37
5.3
CYTOTOXICITY
STUDY
This
experiment
will
be
conducted
by
the
lead
laboratory
only.

The
purpose
of
this
experiment
is
to
determine
the
optimized
assay
response
to
various
cytotoxicants.
Each
cytotoxicant
will
be
tested
at
three
concentrations.
From
this
information,
one
cytotoxicant
may
be
selected
as
a
positive
control
and
included
in
the
experimental
design
when
the
sliced
testis
assay
is
used
to
evaluate
the
effect
of
a
test
chemical.

The
cytotoxicants
to
be
tested
are:
SDS;
2,4­
dinitrophenol;
methylnitrosourea;
sodium
azide.
Three
concentrations
of
each
cytotoxicant
will
be
tested.

Prior
to
using
a
given
cytotoxicant
in
the
sliced
testis
assay,
the
highest
concentration
of
the
cytotoxicant
to
be
tested
will
be
evaluated
for
interference
with
the
testosterone
RIA.
Briefly,
a
mid
concentration
testosterone
standard
will
be
spiked
with
the
cytotoxicant
and
compared
to
the
results
obtained
from
a
vehicle
spiked
control
(
same
final
volumes).
If
there
is
no
interference,
then
the
cytotoxicant
will
be
tested
using
the
sliced
testis
assay.

The
experimental
design
is
summarized
in
the
following
table:

Sample
Type
hCG
Number
of
Incubations
(
Runs)
Testis
Fragment(
s)

Media­
Vehicle
control
no
3
1
­
3
Media­
Vehicle
control
yes
3
4
­
6
Positive
control
yes
3
7
­
9
Media
+
Cytotoxicant
A
(
low)
yes
3
10
­
12
Media
+
Cytotoxicant
A
(
mid)
yes
3
13
­
15
Media
+
Cytotoxicant
A
(
high)
yes
3
16
­
18
Media
+
Cytotoxicant
B
(
low)
yes
3
19
­
21
Media
+
Cytotoxicant
B
(
mid)
yes
3
22
­
24
Media
+
Cytotoxicant
B
(
high)
yes
3
25
­
27
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
22
of
37
The
experiment
summarized
in
the
above
table
represents
one
replicate
study.
Note
that
two
cytotoxicants
are
tested
per
replicate
study.
A
replicate
study
is
an
independently
conducted
experiment;
separated
by
day
and
using
freshly
prepared
reagents.
Two
replicates
will
be
conducted.
The
overall
experiment
will
use
three
rats/
replicate
study;
three
testes
total/
replicate
study
(
based
on
obtaining
8
­
10
fragments/
testis).
Testis
will
be
at
least
1000
mg
so
that
at
least
9
fragments
are
obtained
per
testis.
The
overall
total
number
of
individual
fragments
and
incubations
used
to
conduct
this
experiment
is
54.

The
sampling
time
points
(
5)
from
the
media
are
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
In
addition,
at
the
end
of
the
incubation
period,
a
piece
of
the
tissue
fragment
will
be
collected
and
snap­
frozen
for
beta­
HSD
staining
and
microscopic
analysis.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
540
samples
[
54
runs
x
5
sampling
time
points
x
2
(
duplicate)
analyses]
and
the
overall
total
number
of
tissue
samples
is
54
[
54
runs
x
1
time
point].

5.4
TRAINING/
HANDS­
ON
EXPERIENCE
The
lead
and
participating
(
3)
laboratories
will
be
involved
in
the
conduct
of
this
phase
of
prevalidation.

Training
of
the
participating
laboratories'
technical
staff
will
take
place
at
the
lead
laboratory.
The
senior
investigator/
head
technician
for
each
participating
laboratory
will
be
trained
in
the
conduct
of
the
assay
at
the
lead
laboratory.
The
training
will
include
a
review
of
the
relevant
study
documents
and
any
necessary
demonstrations
regarding
various
aspects
of
the
assay
(
starting
at
testis
removal
through
to
data
analysis).
Furthermore,
the
lead
laboratory
will
be
set­
up
to
allow
the
participating
laboratory
staff
to
demonstrate
proficiency
in
conducting
the
assay
by
supervising
them
as
they
conduct
a
baseline
(
media
only)
experiment
and
a
positive
control
experiment
­
both
of
which
are
described
in
further
detail
in
the
following
subsections.
However,
only
one
replicate
of
each
experiment
will
be
conducted
by
each
participating
laboratory.

5.5
BASELINE
EXPERIMENT
The
lead
and
participating
(
3)
laboratories
will
be
involved
in
the
conduct
of
this
phase
of
prevalidation.

The
purposes
of
this
study
are:

°
to
estimate
baseline
inter­
and
intra­
laboratory
variability
(
which
will
also
provide
information
to
design
the
validation
experimental
design)
°
to
evaluate
the
knowledge
transfer
of
assay
procedures
to
participating
laboratories.
°
to
begin
collection
of
historical
control
and
variability
data.
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
23
of
37
The
experimental
design
of
this
study
is
summarized
in
the
following
table:

Sample
Type
hCG
Number
of
Incubations
(
Runs)
Testis
Fragment
ID
Number
Media
no
3
1
­
3
Media
yes
3
4
­
6
The
information
summarized
in
the
table
is
one
replicate
of
the
experiment.
A
total
of
three
independent
replicates
will
be
conducted
by
each
laboratory.
The
overall
study
will
use
one
rat/
replicate
study
and
one
testis
total/
replicate
study.
The
overall
total
number
of
individual
fragments
or
incubations
used
is
18
for
each
laboratory
[(
3
runs
with
+
3
runs
without
hCG)
x
3
replicate
studies].

The
sampling
time
points
(
5)
from
the
media
are
at
time
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
In
addition,
at
the
end
of
the
incubation
period,
a
piece
of
the
tissue
fragment
will
be
collected
and
snap­
frozen
for
3$­
HSD
staining
and
microscopic
examination.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
180
samples/
laboratory
[
18
runs
x
5
time
points
x
2
(
duplicate)
analyses].
The
overall
total
number
of
tissue
samples
for
microscopic
examination
are
18/
laboratory
[
18
runs
x
1
time
point].

5.6
POSITIVE
CONTROL
EXPERIMENT
The
lead
and
participating
(
3)
laboratories
will
be
involved
in
the
conduct
of
this
phase
of
prevalidation.

The
purposes
of
this
study
are:

°
to
evaluate
aminoglutethimide
(
AG)
as
a
positive
control
for
the
assay
°
to
determine
AG
(
positive
control)
inter­
and
intra­
laboratory
variability
°
to
determine
time
effect
on
the
assay
by
comparing
Baseline
to
Pilot
experiments
using
the
media
control
data
°
to
begin
collection
of
historical
positive
control
and
variability
data
The
experimental
design
of
this
study
is
summarized
in
the
following
table.
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
24
of
37
Sample
Type
hCG
Number
of
Incubations
(
Runs)
Testis
Fragment(
s)

Media­
Vehicle
control
no
3
1
­
3
Media­
Vehicle
control
yes
3
4
­
6
Cytotoxicant
control
yes
3
7
­
9
Media
+
AG
(
low)
yes
3
10
­
12
Media
+
AG
(
mid)
yes
3
13
­
15
Media
+
AG
(
high)
yes
3
16
­
18
The
information
summarized
in
the
table
is
one
replicate
of
the
experiment.
The
nonstimulated
incubations
(
non
hCG)
were
excluded
from
the
study
since
the
affect
of
AG
on
the
non­
stimulated
tissue
fragments
would
not
be
expected
to
reduce
the
baseline
levels
to
any
great
extent.
A
total
of
three
independent
replicates
will
be
conducted
by
each
laboratory.
The
overall
study
will
use
three
rats/
replicate
study
and
three
testes
total/
replicate
study.
Six
fragments
will
be
taken
from
one
testis
from
each
rat
and
allocated
to
the
six
test
conditions.
The
overall
total
number
of
individual
fragments
or
incubations
used
is
54
for
each
laboratory.

The
sampling
time
points
(
5)
from
the
media
are
at
time
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
In
addition,
at
the
end
of
the
incubation
period,
a
piece
of
the
tissue
fragment
will
be
collected
and
snap­
frozen
for
3$­
HSD
staining
and
microscopic
examination.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
540
samples/
laboratory
[
54
runs
x
5
time
points
x
2
(
duplicate)
analyses].
The
overall
total
number
of
tissue
samples
for
microscopic
examination
are
54/
laboratory
[
54
runs
x
1
time
point].

5.7
MULTICHEMICAL
EXPERIMENTS
This
experiment
will
be
conducted
by
the
lead
laboratory
only.

The
purposes
of
this
study
are:

°
to
determine
the
response
of
the
assay
when
challenged
with
putative
positive
test
chemicals.
The
test
chemicals
will
differ
in
their
mode(
s)
of
action,
which
will
include
altering
signal
transduction,
cholesterol
transport
into
the
mitochondria,
and
the
series
of
enzymatic
reactions
that
lead
to
the
production
of
testosterone
in
the
gonadal
steroidogenic
pathway.
°
to
determine
the
response
of
the
assay
when
challenged
with
putative
negative
test
chemicals.
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
25
of
37
°
to
select
test
chemicals
and
concentrations
that
would
be
used
for
validation
of
the
assay.
°
to
estimate
interlaboratory
variability
based
on
the
results
from
the
assay
using
the
selected
test
chemicals.

The
test
chemicals
and
their
concentrations
that
will
be
tested
are
summarized
in
the
following
table:

Test
Chemical
Mode
of
Actiona
Test
Concentrationsb
Reference
Aminoglutethimide
(
positive
control)
P450SCC,
aromatase
10,
100,
and
1000
µ
M
Powlin
et
al.,
1998;
Johnston,
1997;
Uzgiris
et
al.,
1977
Diethylumbelliferyl
phosphate
cAMP­
stimulated
accumulation
of
StAR
TBD
Choi
et
al.,
1995
Dimethoate
StAR
protein
TBD
Walsh
et
al.,
2000
Ketoconazole
P450SCC,
P450c17
0.1,
1,
and
10
µ
M
Powlin
et
al.,
1998;
Kan
et
al.,
1985;
Albertson
et
al.,
1988;
DeCoster
et
al.,
1989;
Chaudhary/
Stocco,
1989;
Malozowski
et
al.,
1985,
1986
Trilostanec
3$­
HSD
TBD
Takahashi
et
al.,
1990
Genistein
or
epostane
3$­
HSD
TBD
Ohno
et
al.,
2002;
Tanaka
et
al.,
1992
Flutamide
P450c17
10,
100,
and
1000
µ
M
Powlin
et
al.,
1998;
Ayub
and
Levelll,
1987
Finasterided
or
MK­
434
5"­
reductase
10,
100,
and
1000
µ
M
(
MK­
434
­
TBD)
Morris,
1996
Fenarimol
aromatase
TBD
Vinggaard
et
al.,
2000
Vinclozolin
(
negative
chemical)
antiandrogen
TBD
­­

Bisphenol
A
cAMP
TBD
­­

Lindane
cAMP
TBD
­­
a.
Site
of
action
that
leads
to
a
decrease
in
Testosterone
concentration
for
those
chemicals
with
inhibitory
activity.
Negative
test
chemicals
would
have
no
effect
on
the
testosterone
concentration.
b.
Final
concentrations
in
the
incubation
mixture.
c.
Requires
synthesis
by
commercial
laboratory.
Availability
may
be
difficult
due
to
proprietary
claim.
d.
Commercial
source
may
limit
availability.
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
26
of
37
The
experimental
design
of
this
study
for
any
two
test
chemicals
is
summarized
in
the
following
table:

Sample
Type
hCG
Number
of
Incubation
s
(
Runs)
Testis
Fragment(
s)

Media­
Vehicle
control
no
3
1
­
3
Media­
Vehicle
control
yes
3
4
­
6
Positive
control
­
Aminogluthethimide
@
1
conc
yes
3
7
­
9
Cytotoxicant
control
­
TBD
@
1
conc
yes
3
10
­
12
Media
+
Test
Chemical
A
(
low)
yes
3
13
­
15
Media
+
Test
Chemical
A
(
mid)
yes
3
16
­
18
Media
+
Test
Chemical
A
(
high)
yes
3
19
­
21
Media
+
Test
Chemical
A
(
high)
no
3
22
­
24
Media
+
Test
Chemical
B
(
low)
yes
3
25
­
27
Media
+
Test
Chemical
B
(
mid)
yes
3
28
­
30
Media
+
Test
Chemical
B
(
high)
yes
3
31
­
33
Media
+
Test
Chemical
B
(
high)
no
3
34
­
36
The
information
summarized
in
the
table
is
one
replicate
of
the
experiment.
The
nonstimulated
incubations
(
non
hCG)
were
excluded
from
the
study
since
the
affect
of
AG
on
the
non­
stimulated
tissue
fragments
would
not
be
expected
to
reduce
the
baseline
levels
to
any
great
extent.
However,
a
high
concentration
test
chemical
with
non­
stimulated
tissue
fragment
was
retained
in
the
experimental
design
to
confirm
this
response.
In
addition,
in
the
case
when
the
assay
would
be
used
to
evaluate
a
chemical
with
an
unknown
effect
on
the
testosterone
concentration,
it
would
be
important
to
test
the
non­
stimulated
fragment
at
the
high
concentration
to
determine
whether
the
test
chemical
had
a
stimulatory
effect
on
the
testosterone
concentration.
A
total
of
three
independent
replicates
will
be
conducted.
The
overall
study
will
use
two
rats/
replicate
study
and
four
testes
total/
replicate
study.
The
overall
total
number
of
individual
fragments
or
incubations
used
is
72.

The
sampling
time
points
(
5)
from
the
media
are
at
time
0
(
after
a
1
hour
equilibration)
and
1,
2,
3,
and
4
hours
post­
equilibration.
Each
sample
will
be
analyzed
for
testosterone
in
duplicate.
In
addition,
at
the
end
of
the
incubation
period,
a
piece
of
the
tissue
fragment
will
be
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
27
of
37
collected
and
snap­
frozen
for
3$­
HSD
staining
and
microscopic
examination.
Thus,
the
overall
total
number
of
testosterone
samples
for
analysis
is
720
samples
[
72
runs
x
5
time
points
x
2
(
duplicate)
analyses].
The
overall
total
number
of
tissue
samples
for
microscopic
examination
are
72
[
72
runs
x
1
time
point].

6.0
CHEMISTRY
The
test
chemicals
and
AG
(
positive
control)
will
be
procurred,
characterized,
formulated,
and
analyzed
as
described
below.
A
central
chemistry
laboratory
will
perform
these
activities.
However,
the
formulations
prepared
by
the
central
chemistry
laboratory
will
be
limited
to
the
stock
solution,
which
will
be
shipped
to
the
assay
testing
laboratories,
where
the
appropriate
dilutions
will
be
made.

6.1
CHEMICAL
PROCUREMENT
AND
CHARACTERIZATION
A
single
lot
of
each
test
chemical
will
be
procurred
so
that
all
prevalidation
experiments
are
performed
using
the
same
lot.
The
lot
obtained
will
have
a
purity
>
95
­
98
percent.
Upon
receipt
of
each
test
chemical,
the
identity
will
be
confirmed
by
running
an
IR
spectrum
and
comparing
it
to
a
reference
spectrum
from
the
manufacturer
or
from
the
literature.
Within
30
days
of
using
a
test
chemical
in
the
assay,
the
purity
will
be
determined
using
one
chromatographic
method
and
one
additional
or
complementary
method.

6.2
FORMULATION
PREPARATIONS
Prior
to
testing,
the
solubility
of
the
test
chemical
in
the
media
at
a
concentration
that
will
result
in
the
desired
final
concentration
in
the
incubation
mixture
will
need
to
be
determined.
Formulation
development
will
be
based
on
the
highest
concentration
needed
for
a
given
test
chemical.
In
addition,
a
formulation
will
be
developed
using
a)
media
only
or
b)
a
vehicle
that
can
be
used
to
dissolve
the
test
chemical
prior
to
mixing
it
with
the
media.

Only
select
solvents
will
be
used
as
vehicles.
The
vehicles
that
will
be
used
and
their
concentrations
were
determined
during
the
optimization
studies.
Based
on
these
results,
selected
concentrations
of
ethanol,
DMSO,
or
Tween
20
will
be
used
as
vehicles
if
the
chemical
is
not
soluble
in
the
media.

It
will
be
important
to
confirm
that
the
formulation
is
a
solution.
This
will
be
done
by
analyzing
the
formulation
before
and
after
filtering
since
the
results
of
the
two
analyses
will
be
similar
if
the
formulation
is
a
solution.
In
addition,
since
the
media
is
gassed
in
the
assay,
the
solubility
of
gassed
formulation
will
be
tested.
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
28
of
37
Formulation
development
will
seek
to
prepare
a
solution
of
the
test
chemical
in
media,
with
and
without
hCG,
so
that
a
given
aliquot
of
the
formulation,
when
added
to
the
incubation
mixture,
will
result
in
the
desired
final
concentration
of
test
chemical
to
be
tested,
as
well
as
the
final
total
incubation
volume.
Also,
if
a
vehicle
is
needed
to
make
the
formulation,
then
the
vehicle
concentration
would
be
formulated
at
an
acceptable
level.

When
formulations
for
multiple
laboratories
are
required,
a
formulation
will
be
prepared
in
a
sufficient
batch
size
that
will
allow
the
same
formulation
to
be
dispensed
to
all
laboratories.
If
different
concentrations
are
to
be
tested
for
a
given
test
chemical,
then
the
central
laboratory
will
prepare
a
stock
solution,
which
will
be
shipped
to
the
laboratories.
Each
laboratory
will
then
use
the
stock
solution
to
prepare
the
various
concentrations
to
be
tested.

Formulations,
if
solutions,
will
be
prepared
to
within
5
percent
of
target.

6.3
FORMULATION
ANALYSIS
For
each
chemical
to
be
tested,
a
method
for
analysis
will
be
developed
for
the
test
chemical
in
media/
vehicle
over
the
concentration
range
to
be
tested.

Prior
to
shipment
of
a
formulation
to
the
laboratory(
ies),
a
sample
of
the
stock
solution
formulation
to
be
shipped
will
be
taken
and
analyzed.
Samples
will
be
analyzed
in
duplicate.
If
the
determined
concentration
is
not
within
5
percent
of
the
target
concentration,
then
the
formulation
will
be
re­
prepared.

After
the
laboratory(
ies)
have
used
the
diluted
formulations,
a
sample
will
be
shipped
back
to
the
central
laboratory
and
archived
for
possible
analysis
at
a
later
date
if
there
is
some
question
about
the
results
of
the
assay.

6.4
FORMULATION
STABILITY
Prior
to
being
used
in
the
assay,
the
formulations
will
be
tested
for
stability.
Stability
testing
will
be
performed
at
the
concentration
of
the
stock
solution
formulation.
Stability
will
be
determined
at
time
0
(
day
of
preparation)
and
at
weekly
intervals
for
a
period
of
at
least
6
weeks
and,
thereafter,
at
9
and
12
weeks.
Stability
will
be
tested
at
room
temperature
and
at
refrigerated
temperatures.
Stability
will
be
tested
using
amber
glass
bottles
with
Teflon­
lined
lids.

7.0
ENDPOINT
MEASUREMENTS
The
sliced
testis
assay
incubation
media
will
be
sampled
at
selected
time
points
and
the
media
analyzed
for
testosterone
concentration
using
an
RIA
method.
In
addition,
at
the
conclusion
of
the
incubation
period,
the
testis
fragment
will
be
sampled
and
processed
for
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
29
of
37
Leydig
cell
identification
using
a
staining
procedure
specific
for
3$­
HSD.

7.1
TESTOSTERONE
RIA
PROCEDURE
A
radioimmunoassay
(
RIA)
commercial
kit
(
Diagnostic
Products
Corporation,
Los
Angeles,
CA),
that
utilizes
125I­
testosterone
and
a
testosterone­
specific
antibody
affixed
to
polypropylene
culture
tubes,
will
be
used
to
measure
testosterone.

Testosterone
(
Sigma,
St.
Louis,
MO;
T­
1500)
to
be
used
for
preparing
the
standard
curve
will
be
stored
desiccated
at
room
temperature.
A
standard
will
be
prepared
in
ethanol
(
0.1
mg/
ml).
Up
to
an
8­
point
standard
curve
but
not
less
than
a
4­
point
standard
curve
will
be
prepared
using
standards
with
concentrations
of
0.07,
0.16,
0.41,
1.02
,
2.56,
6.4,
16,
and
40
ng/
ml
in
PBS­
Gel
Buffer
(
0.1
M
phosphate
buffered
saline
with
0.1%
(
w/
v)
sodium
azide
and
0.1%
(
w/
v)
gelatin,
pH
7.4).
In
addition,
procedural
controls
will
be
included
in
each
run.
These
procedural
controls
will
include
low
or
high
serum
testosterone
samples
and
two
reagent
blanks.
The
standard
curve
points
and
the
procedural
controls
will
be
prepared
in
quadruplicate;
the
bioassay
unknowns
and
the
internal
standards
(
see
below)
will
be
prepared
in
duplicate.
The
volume
of
all
standards
and
controls
(
including
bioassay
unknowns)
will
be
adjusted
to
50
:
L
by
adding
the
PBS­
Gel
Buffer.
Next,
1
ml
125I­
testosterone
will
be
added
to
each
antibody­
coated
tube
and
mixed
(
Vortex).
The
tubes
will
be
incubated
in
a
37/
C
water
bath
for
three
hours,
during
which
time
testosterone,
whether
labeled
or
unlabeled,
will
compete
for
testosterone
specific
antibody
binding
sites.
At
the
end
of
the
incubation
period,
the
free
(
unbound)
testosterone,
in
the
supernatant
fluid
of
all
tubes
will
be
aspirated
and
wiped
clean
of
fluid.
The
bound
testosterone
will
be
counted
in
a
gamma
counter
for
1
minute.
The
concentration
of
testosterone
will
be
estimated
against
the
standard
curve.
Values
will
be
reported
as
a
mean
concentration
(
ng/
mL)
of
duplicate
analyses.

Verification
of
the
testosterone
assay
will
involve
preparation
of
internal
standards
(
no
less
than
three)
using
spiked
media
with
concentrations
ranging
from
12.5
to
500
ng/
mL.
Each
concentration
will
be
run
at
each
of
three
volumes
­
10,
25,
and
50
µ
L,
to
check
for
parallellism,
and
each
sample
will
be
adjusted
to
50
µ
L
by
adding
the
PBS­
Gel
Buffer.
The
low
and
high
standards
will
be
analyzed
in
at
least
duplicate.
Verification
will
be
based
on
results
determined
for
accuracy,
precision,
specificity,
and
linearity.
Accuracy
will
be
expressed
as
the
relative
error,
which
will
be
determined
by
comparing
the
measured
to
the
target
concentration.
Relative
errors
within
10
percent
will
be
acceptable.
Precision
will
be
expressed
as
the
relative
standard
deviate
(
RSD)
or
coefficient
of
variance
(
CV),
which
will
be
determined
by
calculating
the
mean
and
standard
deviation
(
sd)
of
the
low
and
high
standards.
A
RSD
or
CV
within
10
percent
will
be
acceptable.
The
sensitivity
will
be
acceptable
if
the
means
of
the
blanks
and
low
standards
are
significantly
different
at
the
5
percent
significance
level.
Linear
determinations
of
the
standard
curve
line
will
be
made
and
a
correlation
coefficient
(
r)
calculated.
An
r
of
0.90
or
greater
will
be
considered
acceptable.
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
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of
37
Inter­
and
intra­
assay
variability
will
be
determined.
The
intra­
assay
variability
will
be
determined
from
the
precision
results
calculated
from
the
results
obtained
by
measuring
the
low
and
high
standards
in
triplicate.
The
inter­
assay
variability
will
be
determined
by
repeating
analysis
of
the
standards
by
generating
a
standard
curve
on
three
different
days.

7.2
3$­
HSD
LEYDIG
CELL
STAINING
PROCEDURE
Analysis
results
will
be
reported
as
Leydig
cell
density
(
cells/
cm2).

A
Leydig
cell
specific
staining
method
and
microscopic
evaluation
will
be
used
to
evaluate
the
viability
of
these
cells.
At
the
end
of
the
incubation
period,
a
piece
will
be
taken
from
each
fragment
for
staining.
The
media­
vehicle
control
fragment
will
be
used
to
evaluate
the
result
of
incubation
with
the
test
chemical
treatment.
The
staining
procedure
is
specific
for
cells
containing
3 ­
HSD,
a
steroidogenic
enzyme
specific
to
the
Leydig
cells
when
examining
testis
tissue.
The
general
staining
procedure
[
Ref.:
Payne
et
al.,
(
1980).
Endocrinology
106:
1424;
Klinefelter
et
al.,
(
1993).
In:
Methods
in
Toxicology,
Vol.
3,
pp.
166­
181.]
is
described
as
follows:

°
after
the
last
sample
collection,
snap
freeze
the
tissue,
section
it
(~
15
um),
and
mount
it
on
slides.
°
stain
the
slide
using
the
following
solutions
and
procedure:

a)
etiocholanolone
stock
solution
(
1
mg/
ml
in
DMSO),
b)
2
mg
Nitroblue
Tetrazolium
in
0.6
ml
Etiocholanolone
stock,
c)
10
mg
NAD+
dissolved
in
9.5
ml
warm
Dulbeccos
Phosphate
buffered
saline
(
DPBS),
and
d)
10
mg
NAD+
dissolved
in
9.5
ml
warm
Dulbeccos
Phosphate
buffered
saline
(
DPBS).
°
Mix
solutions
b
and
c.
°
Cover
section
tissue
on
slide
with
staining
solution
for
1
­
2
hours.
°
Rinse
in
deionized
water.
°
Fix
in
10%
formalin
in
DPBS
with
5%
sucrose.
°
Coverslip
with
glycerol:
DPBS
(
1:
1)
and
seal
with
nail
polish.

8.0
DATA
ANALYSIS
The
results
for
each
analysis
will
be
reported
individually,
with
sufficient
identifying
information
to
determine
which
results
correspond
to
duplicate
analyses,
to
different
time
points
within
one
vial,
to
different
vials
within
the
same
replicate,
and
to
different
replicates.
The
lead
and
participating
laboratories
will
maintain
a
database
to
include
all
data
generated
during
the
study.
The
databases
will
have
uniform
structure,
formatting,
and
variable
naming
across
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
31
of
37
laboratories.
Test
conditions,
background
environmental
conditions,
and
results
for
each
analysis
for
each
sample
at
each
time
point
will
be
reported.

Analysis
results
to
be
reported
are
testosterone
(
T)
concentration
(
ng
T/
mg
tissue/
hour)
and
Leydig
cell
density
(
cells/
cm2).
Detection
limits
and
indications
of
inability
to
detect
will
be
reported,
as
will
confirmation
of
the
acceptability
or
non­
acceptability
of
each
individual
value.

9.0
STATISTICAL
ANALYSIS
9.1
CONTRALATERAL
TESTIS
FRAGMENT
EXPERIMENT
The
principal
endpoint
is
testosterone
(
T)
concentration.
T
analyses
will
be
carried
out
in
duplicate.
Results
of
the
duplicate
T
analyses
will
be
averaged.
Statistical
analysis
will
be
carried
out
on
the
averages
the
duplicates.

The
statistical
analysis
will
be
carried
out
by
the
lead
laboratory.
Mixed
effects
repeated
measures
models
will
be
fitted
to
the
data
to
describe
trends
in
concentration
vs.
time
and
to
estimate
animal­
to­
animal,
testis­
to­
testis
within
animal,
and
fragment­
to­
fragment
within
testis
components
of
variation.
For
the
T
concentration
analyses
fixed
effects
terms
in
the
models
will
describe
linear
and
non­
linear
trends
in
T
for
hCG
stimulated
testis
fragments
and
for
nonstimulated
testis
fragments.
Random
effects
terms
in
the
model
will
estimate
variance
components
that
account
for
variation
among
fragments
within
testes,
testes
within
animals,
animals
within
replicates,
and
variation
among
replicates,
primarily
for
the
hCG
stimulated
testis
fragments.
A
correlation
structure
will
be
fitted
to
describe
the
relation
among
the
responses
based
on
the
same
vial
(
i.
e.
the
same
fragment)
at
the
various
times.
The
testis­
to­
testis
variation
within
animals
and
the
fragment­
to­
fragment
variation
within
testes
will
be
determined
only
for
the
hCG
stimulated
testis
fragments.

The
results
of
the
model
fit
will
be
parametric
functions
that
describe
the
time
trends
and
variance
components
that
describe
the
within
and
among
replicate
variation
about
the
time
trends.

Residuals
from
the
model
fits
will
be
examined
to
determine
goodness­
of­
fit
to
the
model
assumptions,
to
assess
the
nature
of
the
random
variation
about
the
model,
and
to
search
for
outlying
observations.
Outliers
may
be
individual
responses
that
deviate
from
the
trend
within
their
vial,
individual
vials
that
deviate
from
the
average
across
vials
within
their
replicate,
and
individual
replicates
that
deviate
from
the
average
across
replicates.
Each
of
these
types
of
residuals
will
be
examined.

Based
on
the
model
fits
multiple
endpoints
will
be
reported.
Endpoints
include
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°
T
concentrations
at
1,
2,
3,
4
hours
following
equilibration,
with
and
without
hCG
stimulation
°
Ratios
of
T
concentrations
with
and
without
hCG
stimulation,
at
1,
2,
3,
4
hours
following
equilibration
°
Response
trends,
with
and
without
hCG
stimulation
°
Ratios
of
trends
with
and
without
hCG
stimulation.
°
Average
Leydig
cell
densities
after
two
hours
of
incubation
and
after
four
hours,
with
and
without
hCG
stimulation
For
each
response
the
average
value
across
runs,
the
standard
error
of
estimate,
and
confidence
intervals
about
the
average
will
be
reported.
Estimates
of
run­
to­
run
variation
within
replicates
and
replicate­
to­
replicate
variation
will
be
reported.
Separate
variance
components
will
be
estimated
for
responses
with
hCG
stimulation
and
for
responses
without
hCG
stimulation.

Random
effects
terms
in
the
model
will
describe
variance
components
that
account
for
animal­
to­
animal
variation,
testis­
to­
testis
variation
within
animal,
and
fragment­
to­
fragment
variation
within
testis.
These
components
of
variation
will
be
estimated
for
hCG
stimulated
testis
fragments
at
two
hours
and
at
four
hours
following
equilibration.
A
correlation
structure
will
be
fitted
to
describe
the
relation
among
the
responses
at
the
various
times
based
on
the
same
vial
(
i.
e.
the
same
fragment).
The
results
of
the
model
fit
will
be
parametric
functions
that
describe
the
time
trends
and
estimates
of
the
variance
components.

For
each
response
estimates
of
variance
components
will
be
reported.

9.2
BASELINE
EXPERIMENT
The
statistical
analysis
will
be
divided
into
intra­
laboratory
and
inter­
laboratory
components.
The
intra­
laboratory
analyses
will
be
carried
out
by
each
laboratory
individually,
based
on
a
common
analysis
plan.
Mixed
effects
repeated
measures
models
will
be
fitted
to
the
data
to
describe
trends
in
concentration
vs.
time.
Fixed
effects
terms
in
the
models
will
describe
linear
and
non­
linear
trends
in
T
time
trends
for
hCG
stimulated
testis
fragments
and
for
nonstimulated
testis
fragments.
Random
effects
terms
in
the
model
will
describe
variance
components
that
account
for
variation
among
the
repeat
vials
within
replicates
and
variation
among
replicates.
A
correlation
structure
will
be
fitted
to
describe
the
relation
among
the
responses
at
the
various
times
based
on
the
same
vial
(
i.
e.
the
same
fragment).
The
results
of
the
model
fit
will
be
parametric
functions
that
describe
the
time
trends
and
variance
components
that
describe
the
within
and
among
replicate
variation
about
the
time
trends.

Residuals
from
the
model
fits
will
be
examined
to
determine
goodness­
of­
fit
to
the
model
assumptions,
to
assess
the
nature
of
the
random
variation
about
the
model,
and
to
search
for
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outlying
observations.
Outliers
may
be
individual
responses
that
deviate
from
the
trend
within
their
vial,
individual
vials
that
deviate
from
the
average
across
vials
within
their
replicate,
and
individual
replicates
that
deviate
from
the
average
across
replicates.
Each
of
these
types
of
residuals
will
be
examined.

Based
on
the
model
fits,
multiple
endpoints
will
be
reported.
Possible
endpoints
include
T
concentrations
at
1,
2,
3,
4
hours
following
equilibration
with
and
without
hCG
stimulation,
ratios
of
T
concentrations
with
and
without
hCG
stimulation
at
1,
2,
3,
4
hours
following
equilibration,
response
trends
with
and
without
hCG
stimulation,
and
ratios
of
trends.
For
each
response
the
average
value
across
runs,
the
standard
error
of
estimate,
and
confidence
intervals
about
the
average
will
be
reported.
Estimates
of
run­
to­
run
variation
within
replicates
and
replicate­
to­
replicate
variation
will
be
reported.

The
inter­
laboratory
analysis
will
be
carried
out
by
the
DCC.
The
objective
of
the
interlaboratory
analysis
is
to
assess
the
extent
of
variation
across
laboratories
with
respect
to
average
response
and
variability
of
response.
For
each
endpoint
the
average
response
and
associated
standard
error,
the
variation
among
runs
within
replicates,
and
the
variation
among
replicates
will
be
determined
for
each
laboratory.
Comparisons
of
within
laboratory
variance
components
will
be
made
among
the
participating
laboratories
based
on
control
charts
to
assess
homogeneity
of
variance.
The
reference
for
comparisons
will
be
either
the
lead
laboratory
results
or
the
average
results
across
all
laboratories.
If
there
is
no
evidence
of
lab­
to­
lab
variation
the
within
laboratory
variance
components
will
be
pooled
among
laboratories.
Otherwise
separate
within
laboratory
variance
components
will
be
specified
for
each
laboratory.

The
average
responses
will
be
compared
across
laboratories
based
on
random
effects
one­
way
analysis
of
variance.
The
among
laboratories
variance
in
average
response
will
be
added
to
the
within
laboratory
variance,
resulting
in
the
total
assay
variability.
Estimates
and
confidence
intervals
will
be
reported
for
each
laboratory
based
on
the
total
assay
variability.
Estimates
and
confidence
intervals
will
be
reported
on
the
differences
between
the
participating
laboratory
results
and
the
lead
laboratory
results
and
the
differences
between
each
laboratory's
results
and
the
average
of
the
laboratory
results
based
on
the
total
assay
variability.

9.3
POSITIVE
CONTROL
EXPERIMENT
Regarding
data
analysis
and
statistical
evaluation,
the
statistical
analysis
will
be
divided
into
intra­
laboratory
and
inter­
laboratory
components.
The
intra­
laboratory
analyses
will
be
carried
out
by
each
laboratory
individually,
based
on
a
common
analysis
plan.
Mixed
effects
repeated
measures
models
will
be
fitted
to
the
data
to
describe
trends
in
T
concentration
vs.
time
and
trends
in
AG
concentration.
Fixed
effects
terms
in
the
models
will
describe
linear
and
nonlinear
T
time
trends
and
AG
concentration
trends
for
hCG
stimulated
testis
and
differences
relative
to
the
media­
vehicle
control.
Random
effects
terms
in
the
model
will
describe
variance
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components
that
account
for
variation
among
the
repeat
vials
within
replicates
and
variation
among
replicates.
A
correlation
structure
will
be
fitted
to
describe
the
relation
among
the
responses
at
the
various
times
based
on
the
same
vial
(
i.
e.
the
same
fragment)
and
at
the
various
AG
concentrations,
based
on
fragments
taken
from
the
same
testis.
The
results
of
the
model
fit
will
be
parametric
functions
that
describe
the
time
trends
and
concentration
trends
and
variance
components
that
describe
the
within
and
among
replicate
variation
about
the
time
trends
and
concentration
trends.

Residuals
from
the
model
fits
will
be
examined
to
determine
goodness­
of­
fit
to
the
model
assumptions,
to
assess
the
nature
of
the
random
variation
about
the
model,
and
to
search
for
outlying
observations.
Outliers
may
be
individual
responses
that
deviate
from
the
time
trend
within
their
vial
or
from
the
concentration
trend
across
vials,
individual
vials
that
deviate
from
the
average
across
vials
with
the
same
conditions
within
their
replicate,
and
individual
replicates
that
deviate
from
the
average
across
replicates.
Each
of
these
types
of
residuals
will
be
examined.

Based
on
the
model
fits
multiple
endpoints
will
be
reported.
Possible
endpoints
will
be
include
T
concentrations
at
1,
2,
3,
4
hours
following
equilibration
for
the
media­
vehicle
control
and
graded
AG
concentrations,
with
hCG
stimulation,
ratios
of
T
concentrations
from
the
same
groups
with
hCG
stimulation
at
1,
2,
3,
4
hours
following
equilibration,
response
trends
in
time
or
in
AG
concentration
with
hCG
stimulation,
and
ratios
of
trends.
For
each
response
the
average
value
across
runs,
the
standard
error
of
estimate,
and
confidence
intervals
about
the
average
will
be
reported.
Estimates
of
run­
to­
run
variation
within
replicates
and
replicate­
toreplicate
variation
will
be
reported.
Differences
between
the
T
concentrations
in
the
presence
of
AG
and
the
T
concentrations
in
the
media­
vehicle
control
group
will
also
be
analyzed.

The
inter­
laboratory
analysis
will
be
carried
out
by
the
DCC.
The
objective
of
the
interlaboratory
analysis
is
to
assess
the
extent
of
variation
across
laboratories
with
respect
to
average
response
and
variability
of
response.
For
each
endpoint
developed
in
the
intra­
laboratory
analysis
the
average
response
and
associated
standard
error,
the
variation
among
repetitions
within
replicates,
and
the
variation
among
replicates
will
be
calculated
for
each
laboratory.
Comparisons
of
within
laboratory
variance
components
will
be
made
among
the
participating
laboratories
based
on
control
charts
to
assess
homogeneity
of
variance.
The
reference
for
comparisons
will
be
either
the
lead
laboratory
results
or
the
average
results
across
all
laboratories.
If
there
is
no
evidence
of
lab­
to­
lab
variation
the
within
laboratory
variance
components
will
be
pooled
among
laboratories.
Otherwise
separate
within
laboratory
variance
components
will
be
specified
for
each
laboratory.

The
average
responses
will
be
compared
across
laboratories
based
on
random
effects
one­
way
analysis
of
variance.
The
among
laboratories
variance
in
average
response
will
be
added
to
the
within
laboratory
variance,
resulting
in
the
total
assay
variability.
Estimates
and
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confidence
intervals
will
be
reported
for
each
laboratory
based
on
the
total
assay
variability.
Estimates
and
confidence
intervals
will
be
reported
on
the
differences
between
the
participating
laboratory
results
and
the
lead
laboratory
results
and
between
each
laboratory's
results
and
the
average
of
the
laboratory
results
based
on
the
total
assay
variability.

The
responses
included
in
the
intra­
laboratory
and
inter­
laboratory
analyses
will
be
categorized
as
primary
or
secondary
endpoints.
The
primary
endpoints
will
be
restricted
to
a
small
number
(
two
to
five)
of
the
most
important
responses
for
comparisons.
Power
calculations
to
assess
inference
sensitivity
versus
sample
size
will
be
based
on
the
primary
endpoints.
The
studies
will
be
powered
to
attain
desired
sensitivity
for
comparisons
of
the
primary
endpoints
within
or
among
laboratories.
The
secondary
endpoints
are
the
(
relatively
large
number
of)
remaining
responses
that
are
included
in
the
statistical
analyses.
The
studies
will
not
be
powered
for
comparisons
among
the
secondary
endpoints.

For
purposes
of
assessing
sample
size
versus
inference
sensitivity
for
the
validation
tests,
two
responses
have
been
selected
as
primary
endpoints.
These
are:

°
differences
between
T
concentrations
associated
with
the
high
aminoglutethimide
concentration
and
the
media­
vehicle
control
at
three
hours
past
equilibration,
with
hCG
stimulation.

°
Sum
of
absolute
differences
between
T
concentrations
associated
with
the
high
aminoglutethimide
concentration
and
the
media­
vehicle
control
at
1,
2,
3,
and
4
hours
past
equilibration,
with
hCG
stimulation.

The
second
response
approximates
the
area
between
the
T
concentration
versus
time
curves
for
these
two
conditions.

Sensitivity
analyses
will
be
carried
out
based
on
response
variability
estimates
obtained
from
the
analyses.
Numbers
of
replicates
per
laboratory
necessary
to
detect
heterogeneity
of
replicate­
to­
replicate
variability
within
laboratories
will
be
assessed.
Numbers
of
laboratories
and
numbers
of
replicates
per
laboratory
necessary
to
detect
various
levels
of
coefficient
of
variation
across
laboratories
with
high
power
or
to
detect
various
levels
of
ratio
of
between
laboratory
standard
deviation
to
within
laboratory
standard
deviation
with
high
power
will
be
determined.

9.4
MULTICHEMICAL
EXPERIMENT
Separate
statistical
analyses
will
be
carried
out
for
each
test
chemical.
Consideration
will
be
given
to
pooling
the
media­
vehicle
control
groups
across
those
chemical
tests
that
use
the
same
vehicle.
The
statistical
analysis
considerations
are
the
same
as
those
described
above
in
the
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previous
section,
except
that
there
are
two
replicates
per
chemical
rather
than
three.
Extent
of
replicate­
to­
replicate
variation
can
be
determined
from
these
tests
only
in
a
qualitative
manner.

10.0
RETENTION
OF
SPECIMENS
AND
RECORDS
All
specimens
and
records
that
remain
the
responsibility
of
the
testing
laboratory
will
be
retained
in
the
testing
laboratory's
archives
for
the
life
of
the
contract.

11.0
QUALITY
CONTROL/
QUALITY
ASSURANCE
PROCEDURES
This
study
will
be
conducted
according
to
Good
Laboratory
Practice
Guidelines.
Quality
control
(
QC)
and
quality
assurance
(
QA)
procedures
will
follow
those
outlined
in
the
Quality
Assurance
Project
Plan
(
QAPP)
that
will
be
prepared
for
this
study.

12.0
REPORTING
Interim
data
sets
will
be
submitted
from
the
lead
laboratory
to
the
EPA
and
Battelle
upon
summarization
of
the
raw
data
and
completion
of
a
technical
review
for
the
experiments
listed
below:

°
baseline/
contralateral
tissue
fragment
and
positive
control
°
cytotoxicity
°
training
and
hands­
on
results
of
participating
laboratories
This
interim
data
submission
will
not
be
audited
by
the
Quality
Assurance
Unit
and
will
be
stamped
as
"
Preliminary
Data".
The
data
sets
will
be
reviewed
promptly
by
the
EPA
so
that
a
decision
can
be
reached
to
determine
whether
to
proceed
to
the
next
experiment.

Each
participating
laboratory
will
prepare
a
draft
final
report
upon
completion
of
the
baseline
and
positive
control
experiments.
A
single
draft
final
report
that
includes
both
experiments
will
be
prepared.
The
draft
final
reports
will
be
submitted
to
the
lead
laboratory,
Battelle,
and
the
EPA.
Review
comments
from
the
lead
laboratory
and
the
EPA
will
be
collected
by
Battelle
and
submitted
to
the
respective
participating
laboratory
for
inclusion
of
the
changes
prior
to
their
submission
of
a
final
report.

The
lead
laboratory
will
prepare
a
draft
final
report
that
will
include
the
results
of
all
experiments,
including
the
final
reports
submitted
by
the
participating
laboratories.
This
overall
draft
final
report
will
include
the
statistical
analyses
that
evaluates
inter­
and
intra­
laboratory
variability.
Upon
review
of
the
draft
final
report
by
the
EPA,
the
lead
laboratory
will
submit
a
final
report
that
incorporates
any
comments
from
the
EPA's
review
of
the
draft
final
report.
DRAFT
PROTOCOL
[
Name
and
address
of
lab]
Page
37
of
37
The
report
format
used
by
the
laboratories
will
have
a
standardized
format.
This
format
will
be
provided
to
the
laboratories
prior
to
initiating
report
preparation.
All
reports
will
include
introduction,
methods
and
materials,
results,
discussion,
and
conclusion
sections.
Any
difficulties
in
executing
the
studies
should
be
noted
and
there
should
be
data
summaries
as
well
as
the
raw
data
tables.

13.0
PERSONNEL
Personnel:

Study
Director:
Principal
Investigator:
Animal
Research
Facility
Vet.:
Animal
Research
Facility
Manager:
LORET
Laboratory
Supervisor:
Research
Toxicologist:
Research
Biologists:
Biologists:
Biological
Laboratory
Assistants:
Statistical
Analyses:
Quality
Assurance
Unit
(
QAU):

14.0
STUDY
RECORDS
TO
BE
MAINTAINED
°
Chemical
Receipt,
Storage
and
Use
Records
°
Animal
Receipt
Records
°
Quarantine
Animal
Health
Surveillance
Records
°
Balance
Check
Sheets
°
Temperature
and
Relative
Humidity
Records
°
Room
Log
Sheets
°
Feed
and
Water
Analyses
°
All
records
that
document
the
conduct
of
the
laboratory
experiments
and
results
obtained,
as
well
as
the
equipment
and
chemicals
used
°
Protocol
and
any
Amendments
°
List
of
any
Protocol
Deviations
°
List
of
Standard
Operating
Procedures
°
Computer
records:
all
records
of
data
sets
and
statistical
analyses
Battelle
Report
B­
1
August,
2003
APPENDIX
B
Data
Coordination
and
Distribution/
Report
Submission
Battelle
Report
B­
2
August,
2003
DATA
COORDINATION
AND
DISTRIBUTION/
REPORT
SUBMISSION
The
Data
Coordination
Center
(
DCC)
is
the
central
coordinator
of
all
information
being
generated
under
the
EDSP.
For
the
sliced
testis
assay
prevalidation,
the
DCC
will
foster
movement
of
data
from
the
laboratories
to
the
EPA.
Critical
data
coordination
and
distribution
points
are
anticipated
to
occur
in
between
the
completion
of
various
experiments.
To
ensure
accurate
and
timely
coordination
and
analysis
of
interim
data,
as
well
as
compilation
of
data
for
final
report
submission,
the
following
procedures
will
be
followed
and
serve
as
guidelines
to
the
laboratories.

In
general,
there
are
two
levels
of
deliverables.
The
first
level
consists
of
data
that
are
generated
upon
completion
of
a
given
individual
experiment
and
are
used
to
make
a
decision
to
proceed
to
the
next
experiment.
Such
data
sets
will
be
referred
to
as
interim
data.
The
second
level
consists
of
all
the
data
from
all
the
prevalidation
experiments.
This
data
set
is
designated
as
final
data
and
will
be
used
for
submission
of
the
final
report.
Further
descriptions
of
how
each
of
these
data
sets
will
be
handled
and
distributed
are
described
in
the
following
subsections.

INTERIM
DATA
REVIEW
There
are
several
different
decision
points
that
will
require
submission
of
interim
data
for
review.
The
interim
data
sets
to
be
submitted
for
review
will
be
generated
after
completion
of
the
following
experiments:

°
Lead
Laboratory's
conduct
of
the
baseline/
contralateral
tissue
fragment
and
positive
control
experiments
°
Lead
Laboratory's
conduct
of
the
cytotoxicity
experiments
°
Training
and
Hands­
on
experience
results
from
the
participating
laboratories
conduct
of
the
baseline
and
positive
control
experiments
using
the
assay
The
Lead
laboratory
will
generate
data
upon
completion
of
these
experiments
that
will
be
used
to
make
decisions
to
proceed
to
the
next
phase
of
prevalidation.
In
order
for
the
decision
to
proceed
to
be
made
promptly,
the
lead
laboratory
will
need
to
process
its
data
rapidly.
It
is
proposed
that
the
laboratory
will
provide
only
a
technical
review
of
the
data
for
accuracy
before
making
the
data
available
for
outside
review.
This
interim
data
review
would
proceed
without
the
data
going
through
a
formal
quality
assurance
audit.
As
such,
the
interim
data
sets
would
be
stamped
as
unaudited
interim
data.
In
this
way,
the
data
generated
after
each
phase
could
be
more
quickly
transferred
to
the
DCC
for
collation
and
statistical
analysis
of
all
data
sets,
thereby
providing
the
EPA
and
Lead
Laboratory
with
complete
data
sets
and
summarized
data
analysis
for
them
to
make
decisions
to
proceed.
The
flow
of
interim
data
that
is
critical
for
making
a
decision
to
proceed
with
the
next
phase
is
illustrated
in
Figure
B­
1.
Battelle
Report
B­
3
August,
2003
Lead
Laboratory
Participating
Laboratories
DCC
EPA
Lead
Laboratory
Decision
to
Proceed
to
Next
Phase
Technically­
Reviewed
Data
Cholate
Data,

Statistical
Analysis,
and
Inter­
laboratory
Analysis
Figure
B­
1.
Interim
Data
Flow
Diagram
FINAL
REPORT
SUBMISSION
Final
Reports
will
be
submitted
by
all
laboratories.
The
participating
laboratories
will
submit
a
final
report
for
the
baseline
and
positive
control
experimental
results.
Their
reports
will
be
written
after
the
positive
control
experiment
is
completed
since
the
participating
laboratories
are
not
involved
in
the
remaining
prevalidation
experiment
(
multi­
chemical
testing).
By
contrast,
the
lead
laboratory
is
assigned
the
task
of
putting
together
the
overall
study
report.
It
is
expected
that
the
lead
laboratory
will
need
the
participating
laboratories'
reports,
as
well
as
the
DCC's
summary
of
the
inter­
laboratory
analysis
to
complete
the
report.
These
reports
will
need
to
be
prepared
and
sent
to
the
lead
laboratory,
such
that
the
overall
study
report
can
be
completed
immediately
upon
the
completion
of
the
multi­
chemical
test
experiment.
In
general,
the
flow
of
information
during
the
course
of
this
study
should
proceed
as
illustrated
in
Figure
B­
2.

A
unified
format
for
data
and
report
submission
will
be
implemented.
Prior
to
the
start
the
training
phase,
the
DCC
staff
will
meet
with
the
lead
laboratory
staff
to
discuss
the
format
of
the
results
and
statistical
analysis
files
for
the
data.
Based
on
this
discussion,
the
DCC
will
put
together
a
draft
template
and
provide
it
to
the
lead
laboratory
and
the
EPA
for
review.
Comments
will
be
collected
via
a
conference
call
with
the
EPA
and
the
lead
laboratory
staff.
Following
the
conference
call,
a
final
template
will
be
developed.
The
lead
laboratory
will
distribute
this
information
to
the
participating
laboratories
at
the
training
session.
These
steps
are
recommended
to
ensure
that
data
is
presented
in
the
same
format
from
all
laboratories
in
order
to
foster
the
collating
process
and
the
inter­
laboratory
analysis.
By
being
proactive
at
the
beginning
of
the
study,
this
should
minimize
time
for
completing
the
inter­
laboratory
analysis,
as
well
as
problems
with
interpretation
of
data
from
each
laboratory.
Battelle
Report
B­
4
August,
2003
Participating
Laboratories
Lead
Laboratory
Statistical
Analysis
DCC
Lead
Laboratory
Inter­
laboratory
Analysis
Overall
Study
Report
Participating
Lab
Reports
Inter­
laboratory
analysis
report
Statistical
analysis
data
Participating
Laboratory
Final
Report
Figure
B­
2.
Data
Flow
for
Final
Report
Submission
It
is
anticipated
that
the
participating
laboratories
and
the
lead
laboratory
need
to
provide
data
to
the
DCC
at
interim
points
for
two
reasons:

1)
The
results
of
the
inter­
laboratory
analysis
at
the
end
of
the
baseline
and
positive
control
experiments
may
delay
submission
of
the
final
report.

2)
EPA
desires
to
receive
data
soon
after
generation
for
purposes
of
reviewing
the
data
for
additional
information
based
on
their
experience
and
the
goals
of
the
EDSP.
