APPENDIX
D
STUDY
PROTOCOLS
FOR
ETHYLENE
DICHLORIDE
This
Appendix
contains
detailed
study
protocols
for
the
toxicity
studies
to
be
conducted
for
ethylene
dichloride,
and
is
organized
into
the
following
sections:

D.
1
Combined
Acute
Toxicity
with
BAL
and
Histopathology
and
Acute
Neurotoxicity
(
Inhalation)

D.
2
Subchronic
Neurotoxicity
(
Oral)

D.
3
Reproductive
Toxicity
(
Oral)

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RECEIVED
OPPT
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2003
MAR11
5:
03PM
OPPT­
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0010­
0010
D.
1
Combined
Acute
Inhalation
Toxicity
with
BAL
and
Histopathology
and
Acute
Neurotoxicity
(
Inhalation)

Proposed
Test
Parameters
All
proposed
test
parameters
(
except
test
concentrations)
are
per
TSCA
guidelines
for
acute
inhalation
toxicity
requirements
(
40
CFR
799.9135,
provided
in
Appendix
B),
and
are
to
be
conducted
in
compliance
with
good
laboratory
practice
standards
(
40
CFR
792).
For
testing
and
reporting
schedule,
see
Appendix
A.

Parameter
Proposed
Test
species
Rat
Strain
F344
Age
Young
adult
Sex
Male
and
Female
Health
Status
Evaluate
initially
Number
ofanimals
5/
sex/
dose
for
acute
toxicity
(
with
BAL
and
histopathology)
plus
10/
sex/
dose
for
acute
neurotoxicity
testing
Control
Groups
Concurrent
sham
control
Positive
Controls
(
behavioral
and
neuropathology)
Acceptable
historical
control
data
from
testing
laboratory
Concentration
level
and
selection
Control
+
3
exposure
concentrations
Limit
dose
To
be
determined
from
range
finding
study
andlor
existing
studies
8­
hr
study
If
triggered
(
3
dose
groups)
Exposure
Whole
body
Environmental
conditions
c22
°
C;
40
­
60%
humidity,
12­
15
air
changes/
hr
Exposure
periodicity
4
hr
(
8
hr
if
triggered)
Physical
measurements
Environmental
conditions
monitored
Observation
period
24
hr
for
acute
toxicity;
14
days
for
acute
neurotoxicity
Gross
pathology
Full
necropsy,
organ
weights
Histopathology
Respiratory
tract,
liver,
and
kidney
of
controls
and
high
dose
group
Bronchoalveolar
lavage
Provide
indicators
oflung
damage
Equipment
and
test
methods
reporting
Adequately
defined
Results
reporting
Tabular
results,
per
animal,
statistics
Time
ofneurotoxicity
testing
0,
4
hr,
7d,
14d
Functional
observational
battery
Standard
evaluations
for
appearance,
behavior,
and
functional
integrity
List
of
neurotoxicity
measures
Autonomic
function,
abnormal
motor
movements,
response
to
general
and
sensory
stimuli,
alertness,
grip
strength,
landing
foot
splay,
body
weight,

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behavioral
changes
Motor
activity
Individually
assessed,
automated
Neuropathology
Neuropathological
examinations
(
CNS
and
peripheral)
Results
Tabular,
per
animal,
statistics
Evaluation
Adequately
described
Study
Design
and
Conduct:
Acute
Toxicity
with
BAL
and
Histopathology
The
test
species
will
consist
of
young
adult
male
and
female
(
nulliparous
and
nonpregnant)
F344
rats.
At
least
five
males
and
five
females
shall
be
used
in
each
concentrationlduration
and
control
group.
Animals
shall
be
randomly
assigned
to
treatment
and
control
groups.
The
control
group
shall
be
a
sham­
treated
group.
Except
for
treatment
with
the
test
substance,
animals
in
the
control
group
shall
be
handled
in
a
manner
identical
to
the
test­
group
animals.

Test
animals
will
be
exposed
to
the
substance
using
a
whole­
body
exposure
chamber.
The
laboratory
shall
measure
the
following:
chemical
purity,
rate
of
airflow,
actual
concentrations
of
the
test
substance
in
the
breathing
zone,
temperature
and
humidity.
Food
and
water
shall
be
withheld
during
exposure.

The
bronchoalveolar
lavage
and
respiratory
pathology
shall
be
conducted
24
hrs
following
exposure
to
allow
expression
of
signs
of
toxicity.
Animals
will
be
subjected
to
a
full
gross
necropsy.
Lungs,
liver,
kidneys,
adrenals,
brain,
and
gonads
shall
be
weighed
wet,
as
soon
as
possible
after
dissection.
The
following
organs
and
tissues,
or
representative
samples
thereof,
shall
be
preserved
in
a
suitable
medium
for
possible
future
histopathological
examination:
All
gross
lesions;
brain
including
sections
of
medullalpons;
cerebellar
cortex
and
cerebral
cortex;
pituitary;
thyroidlparathyroid;
thymus;
heart;
sternum
with
bone
marrow;
salivary
glands;
liver;
spleen;
kidneys;
adrenals;
pancreas;
gonads;
accessory
genital
organs;
aorta;
skin;
gall
bladder;
esophagus;
stomach;
duodenum;
jejunum;
ileum;
cecum;
colon;
rectum;
urinary
bladder;
representative
lymph
nodes;
thigh
musculature;
peripheral
nerve;
spinal
cord
at
three
levels
cervical,
midthoracic,
and
lumbar;
and
eyes.
Respiratory
tract
tissues
shall
also
be
preserved
in
a
suitable
medium.

Full
histopathology
shall
be
performed
on
the
respiratory
tract,
liver
and
kidney
of
all
animals
in
the
control
and
high
concentration
groups,
on
all
gross
lesions
which
differ
from
controls
in
frequency,
distribution,
type,
or
severity
in
all
concentration
groups
and
on
target
organs
in
all
animals,
as
indicated
by
the
observations
in
the
high
concentration
group
in
this
study.
Archived
organs
identified
as
targets
of
toxicity
from
results
of
the
90­
day
study.
The
lungs
will
be
lavaged
using
physiological
saline,
consisting
oftwo
washes
of
approximately
80%
of
the
total
lung
volume.
The
following
parameters
shall
be
determined
in
the
lavage
fluid
as
indicators
of
cellular
damage
in
the
lungs:
total
protein,
cell
count,
and
percent
leukocytes.
In
addition,
a
phagocytosis
assay
shall
be
performed
to
determine
macrophage
activity.

If
no
adverse
effects
are
seen
in
the
4­
hr
study
as
compared
with
controls,
no
further
testing
is
necessary.
If
the
4­
hr
study
shows
positive
effects
in
histopathology
or
the
bronchoalveolar
lavage,
an
8­
hr
study
shall
be
conducted.
This
will
be
determined
by
the
study
director
following
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consultation
with
the
HAP
Task
Force
and
USEPA.
Only
those
tissues
showing
positive
results
in
the
4­
hr
study
must
be
pursued
in
the
follow­
up
8­
hr
study.

The
final
test
report
shall
include
the
following
information:
a
description
of
equipment
and
test
methods,
a
description
of
exposure
apparatus,
and
a
description
of
the
measuring/
monitoring
equipment.
Exposure
data
shall
be
tabulated
and
presented
with
mean
values
and
measure
of
variability
(
e.
g.,
standard
deviation)
and
should
include:
chemical
purity,
airflow
rates,
temperature
and
humidity,
nominal
concentration,
actual
concentration.

The
following
information
shall
be
tabulated
by
test
group
exposure
level
to
indicate:
number
of
animals
exposed,
number
of
animals
dying,
number
of
animals
showing
overt
signs
of
toxicity,
pre­
and
post­
exposure
body
weight
change
in
animals,
counts
and
incidence
of
gross
and
histopathological
alterations,
amount
of
administered
lavage
fluid
and
recovered
lavage
fluid
for
each
test
animal,
magnitude
ofchange
ofbiochemical
and
cytologic
indices
in
lavage
fluids.

Study
Design
and
Conduct:
Acute
Neurotoxicity
An
additional
10
animals/
sex/
dose
group
shall
be
exposed
as
described
above
for
the
Acute
Toxicity
study.
At
least
five
males
and
five
females
should
be
used
in
each
dose
and
control
group
for
terminal
neuropathology.

All
animals
shall
be
weighed
on
each
test
day
and
at
least
weekly
during
the
observation
period.
At
a
minimum,
observations
and
activity
testing
shall
be
made
before
the
initiation
ofexposure,
at
the
estimated
time
ofpeak
effect
at
the
end
ofthe
exposure,
and
at
7
and
14
days
after
dosing.

The
functional
observational
battery
shall
include
the
following:
ranking
of
the
degree
of
lacrimation
and
salivation,
with
a
range
of
severity
scores
from
none
to
severe,
presence
or
absence
of
piloerection
and
exophthalmus,
ranking
or
count
of
urination
and
defecation,
including
polyuria
and
diarrhea,
pupillary
function
such
as
constriction
of
the
pupil
in
response
to
light
or
a
measure
of
pupil
size,
degree
ofpalpebral
closure,
description
of
any
convulsions,
tremors,
or
abnormal
motor
movements,
ranking
of
the
subject's
reactivity
to
general
stimuli
such
as
removal
from
the
cage
or
handling,
ranking
of
the
subject's
general
level
of
activity
during
observations
of
the
unperturbed
subject
in
the
open
field,
descriptions
ofposture
and
gait
abnormalities,
forelimb
and
hindlimb
gripstrength,
a
quantitative
measure
of
landing
foot
splay,
sensorimotor
responses
to
stimuli
of
different
modalities,
body
weight,
description
of
any
unusual
or
abnormal
behaviors
and
apearances.
Further
information
on
the
neurobehavioral
integrity
ofthe
subject
may
be
provided
by:
count
of
rearing
activity
on
the
open
field,
ranking
of
righting
ability,
body
temperature,
excessive
or
spontaneous
vocalizations,
alterations
in
rate
and
ease
of
respiration,
sensorimotor
responses
to
visual
or
proprioceptive
stimuli.
Motor
activity
shall
be
monitored
by
an
automated
activity
recording
apparatus.
Each
animal
shall
be
tested
individually.
Positive
historical
control
data
for
functional
tests
will
be
provided
by
the
testing
laboratory.

Any
gross
abnormalities
should
be
noted.
Tissue
samples
taken
should
adequately
represent
all
major
regions
of
the
nervous
system.
Representative
histological
sections
from
the
tissue
samples
should
be
examined
microscopically
by
an
appropriately
trained
pathologist
for
evidence
of
neuropathological
alterations.
If
no
neuropathological
alterations
are
observed
in
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samples
from
the
high
dose
group,
subsequent
analysis
is
not
required.
If
any
evidence
of
neuropathological
alterations
is
found
in
the
qualitative
examination,
then
a
subjective
diagnosis
shall
be
performed
for
the
purpose
ofevaluating
dose­
response
relationships.
All
regions
ofthe
nervous
system
exhibiting
any
evidence
of
neuropathological
changes
should
be
included
in
this
analysis.
For
each
type
ofdose­
related
lesion
observed,
examples
ofdifferent
degrees
of
severity
should
be
described.
Photomicrographs
of
typical
examples
of
treatment­
related
regions
are
recommended
to
augment
these
descriptions.
Positive
historical
control
data
for
neuropathological
testing
methodology
will
be
provided
by
the
testing
laboratory.

The
final
test
report
shall
include
the
following
information:
description
of
equipment
and
test
methods,
a
short
justification
explaining
any
decisions
involving
professional
judgment,
a
detailed
description
of
the
procedures
used
to
standardize
observations,
positive
control
data
(
historical
data
preferred).
The
following
information
shall
be
arranged
by
test
group
dose
level:
identification
number,
body
weight,
score
on
each
sign
at
each
observation
time,
the
time
and
cause
of
death
(
if
appropriate),
total
session
activity
counts,
and
intrasession
subtotals
for
each
day
measured,
the
number
of
animals
at
the
start
ofthe
test,
the
number
ofanimals
showing
each
observation
score
at
each
observation
time,
mean
and
standard
deviation
for
each
continuous
endpoint
at
each
observation,
results
of
statistical
analyses
for
each
measure
(
where
appropriate).
Neuropathological
observations
may
be
presented
in
the
following
recommended
format:
description
of
lesions
for
each
animal,
a
list
of
structures
examined
as
well
as
the
locations,
nature,
frequency,
and
severity
oflesions.

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D.
2
Subchronic
Neurotoxicity
(
Oral
Route)

Proposed
Test
Parameters
All
proposed
test
parameters
(
except
dose
selections)
are
per
TSCA
requirements
(
40
CFR
799.9620,
provided
in
Appendix
B),
and
in
compliance
with
good
laboratory
practice
standards
(
40
CFR
792).
For
testing
and
reporting
schedule,
see
Appendix
A.

Parameter
Proposed
Test
species
Rat
Strain
F344
Age
Young
adult
(>
42
d)
Sex
Male
and
Female
Number
ofanimals
10/
sex/
dose
Control
Groups
Concurrent
vehicle
control
Positive
controls
for
behavioral
and
neuropathology
Acceptable
historical
control
data
from
testing
laboratory
Concentration
level
and
selection
Control
+
3
doses
in
drinking
water
Dose
selection
Concentration
to
be
determined
based
on
acute
inhalation
findings
and
PBPK
modeling
Administration
ofthe
substance
In
drinking
water
for
subchronic
testing
Time
of
testing
Subchronic
(
0,
4
wk,
8
wk,
13
wk)
Functional
observational
battery
Standard
evaluations
for
appearance,
behavior,
and
functional
integrity
List
of
measures
Autonomic
function,
abnormal
motor
movements,
response
to
general
and
sensory
stimuli,
alertness,
grip
strength,
landing
foot
splay,
body
weight,
behavioral
changes
Motor
activity
Individually
assessed,
automated
Neuropathology
Neuropathological
examinations
Results
Tabular,
per
animal,
statistics
Evaluation
Adequately
described
Dose
Selection
The
preliminary
PBPK
model
and
results
ofthe
acute
studies
will
be
used
to
select
doses
for
the
oral
studies
based
on
expected
parent
blood
concentrations
and
amount
metabolized
below,
slightly
above,
and
well
above
saturation
of
the
P­
450
enzymes.
The
dose
levels
for
the
subchronic
study
may
be
modified
pending
the
results
of
the
acute
study.
Study
Design
and
Conduct
For
the
subchronic
neurotoxicity
studies,
the
test
species
will
consist
of
young
adult
male
and
female
(
nulliparous
and
nonpregnant)
F344
rats.
At
least
10
males
and
10
females
shall
be
used
in
each
concentrationlduration
and
control
group.
At
least
five
males
and
five
females
should
be
used
in
each
dose
and
control
group
for
terminal
neuropathology.

All
animals
shall
be
weighed
on
each
test
day
and
at
least
weekly
during
the
exposure
period.
For
the
subchronic
study,
observations
and
activity
measurements
shall
be
made
before
the
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initiation
of
exposure
and
before
the
daily
exposure,
and
during
the
4th,
8th,
and
13
th
weeks
of
exposure.

The
functional
observational
battery
shall
include
the
same
observations
as
noted
for
the
acute
neurotoxicity
studies.
Notation
of
any
gross
abnormalities,
tissue
sampling,
and
histopathology
shall
also
be
as
described
for
acute
neurotoxicity
testing
and
final
report
requirements
will
also
be
the
same
as
for
acute
neurotoxicity
testing.

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D.
3
Reproductive
Toxicity
(
Oral
Route)

Proposed
Test
Parameters
All
proposed
test
parameters
(
except
dose
selections)
are
per
TSCA
requirements
for
reproductive
toxicity
testing
(
40
CFR
799.9380,
provided
in
Appendix
B),
and
in
compliance
with
good
laboratory
practice
standards
(
40
CFR
792).
For
testing
and
reporting
schedule,
see
Appendix
A.

Parameter
Proposed
Species
Rat
Strain
F344
Age
Young
adult
Number
of
animals
20
pregnant
females/
dose
Doses
Control
+
3
concentrations
in
drinking
water.
Highest
dose
to
be
determined
from
acute
inhalation
study
and
PBPKmodeling
Route
Drinking
water
Exposure
Frequency
Daily
Design
Multigenerational;
exposure
>
10
wk
prior
to
mating
Mating
Random
Control
Vehicle
control
Observations
(
adult)
Daily/
weekly
Observations
(
litter)
PND
0,
4,
7,
14,
21
Endpoints
Fertility
index,
gestation
index,
sperm
morphology,
age
at
vaginal
opening
or
preputial
separation,
gross
necropsy,
organ
weight,
histopathology
Data
reporting
Tabular
Evaluation
Statistics
described
Study
Design
and
Conduct
Reproductive
toxicity
testing
shall
be
conducted
using
young,
healthy
F344
rats.
Parental
animals
shall
be
5
to
9
weeks
old
at
the
start
of
dosing.
The
females
shall
be
nulliparous
and
nonpregnant.
Each
group
shall
colltain
a
sufficient
number
of
mating
pairs
to
yield
approximately
20
pregnant
females.
The
animals
should
be
dosed
with
the
test
substance
on
a
7­
days­
a­
week
basis.
For
both
sexes
(
P
and
F1),
dosing
shall
be
continued
for
at
least
10
weeks
before
the
mating
period,
and
shall
continue
until
termination.
For
each
mating,
each
female
shall
be
placed
with
a
single
randomly
selected
male
from
the
same
dose
level
(
1:
1
mating)
until
evidence
of
copulation
is
observed
or
either
3
estrous
periods
or
2
weeks
has
elapsed.
For
mating
the
Fl
offspring,
at
least
one
male
and
one
female
should
be
randomly
selected
from
each
litter
for
mating
with
another
pup
of
the
same
dose
level
but
different
litter,
to
produce
the
F2
generation.
Throughout
the
test
period,
each
parental
animal
shall
be
observed
at
least
once
daily,
considering
the
peak
period
of
anticipated
effects
after
dosing.
Mortality,
moribundity,
pertinent
behavioral
changes,
signs
of
difficult
or
prolonged
parturition,
and
all
signs
of
overt
toxicity
shall
be
recorded
at
this
cageside
examination.
In
addition,
thorough
physical
examinations
should
be
conducted
weekly
on
each
animal.
Parental
animals
(
P
and
Fl)
shall
be
weighed
on
the
first
day
of
dosing
and
weekly
thereafter.
Parental
females
(
P
and
F1)
should
be
weighed
at
a
minimum
on
approximately
gestation
days
0,
7,
14,
and
21,
and
during
lactation
on
the
same
days
as
the
weighing
of
litters.
During
the
premating
and
gestation
periods,
food
and
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water
consumption
shall
be
measured
weekly
at
a
minimum.
Estrous
cycle
length
and
normality
should
be
evaluated
by
vaginal
smears
for
all
P
and
Fl
females
during
a
minimum
of
3
weeks
prior
to
mating
and
throughout
cohabitation;
care
should
be
taken
to
prevent
the
induction
of
pseudopregnancy.

For
all
P
and
Fl
males
at
termination,
sperm
from
one
testis
and
one
epididymis
shall
be
collected
for
enumeration
of
homogenization­
resistant
spermatids
and
cauda
epididymal
sperm
reserves,
respectively.
In
addition,
sperm
from
the
cauda
epididymis
(
or
vas
deferens)
should
be
collected
for
evaluation
of
sperm
motility
and
sperm
morphology.
The
total
number
of
homogenization­
resistant
testicular
sperm
and
cauda
epididymal
sperm
should
be
enumerated.
Cauda
sperm
reserves
can
be
derived
from
the
concentration
and
volume
of
sperm
in
the
suspension
used
to
complete
the
qualitative
evaluations,
and
the
number
of
sperm
recovered
by
subsequent
mincing
and/
or
homogenizing
of
the
remaining
cauda
tissue.
Enumeration
in
only
control
and
high­
dose
P
and
Fl
males
may
be
performed
unless
treatment­
related
effects
are
observed;
in
that
case,
the
lower
dose
groups
should
also
be
evaluated.
An
evaluation
of
epididymal
(
or
vas
deferens)
sperm
motility
should
be
performed.
Sperm
should
be
recovered
while
minimizing
damage,
and
the
percentage
of
progressively
motile
sperm
should
be
determined.
For
objective
evaluations,
an
acceptable
counting
chamber
of
sufficient
depth
can
be
used
to
effectively
combine
the
assessment
ofmotility
with
sperm
count
and
sperm
morphology.
When
computer­
assisted
motion
analysis
is
performed,
the
derivation
of
progressive
motility
relies
on
user­
defined
thresholds
for
average
path
velocity
and
straightness
or
linear
index.
If
samples
are
videotaped,
or
images
otherwise
recorded,
at
the
time
of
necropsy,
subsequent
analysis
of
only
control
and
high­
dose
P
and
Fl
males
may
be
performed
unless
treatmentrelated
effects
are
observed;
in
that
case,
the
lower
dose
groups
should
also
be
evaluated.
In
the
absence
of
a
video
or
digital
image,
all
samples
in
all
treatment
groups
should
be
analyzed
at
necropsy.
A
morphological
evaluation
of
an
epididymal
(
or
vas
deferens)
sperm
sample
shall
be
performed.
Sperm
(
at
least
200
per
sample)
should
be
examined
as
fixed,
wet
preparations
and
classified
as
either
normal
(
both
head
and
midpiece/
tail
appear
normal)
or
abnormal.
Examples
of
morphologic
sperm
abnormalities
would
include
fusion,
isolated
heads,
and
misshapen
heads
andlor
tails.
Evaluation
of
only
control
and
high­
dose
P
and
Fl
males
may
be
performed
unless
treatment­
related
effects
are
observed;
in
that
case,
the
lower
dose
groups
should
also
be
evaluated.

Each
litter
should
be
examined
as
soon
as
possible
after
delivery
(
lactation
day
0)
to
establish
the
number
and
sex
ofpups,
stillbirths,
live
births,
and
the
presence
of
gross
anomalies.
Pups
found
dead
on
day
0
should
be
examined
for
possible
defects
and
cause
of
death.
Live
pups
should
be
counted,
sexed,
and
weighed
individually
at
birth,
or
soon
thereafter,
at
least
on
days
4,
7,
14,
and
21
of
lactation,
at
the
time
of
vaginal
patency
or
balanopreputial
separation,
and
at
termination.
The
age
of
vaginal
opening
and
preputial
separation
should
be
determined
for
Fl
weanlings
selected
for
mating.
If
there
is
a
treatment­
related
effect
in
Fl
sex
ratio
or
sexual
maturation,
anogenital
distance
should
be
measured
on
day
0
for
all
F2
pups.
All
P
and
Fl
adult
males
and
females
should
be
terminated
when
they
are
no
longer
needed
for
assessment
of
reproductive
effects.
Fl
offspring
not
selected
for
mating
and
all
F2
offspring
should
be
terminated
at
comparable
ages
after
weaning.
At
the
time
of
termination
or
death
during
the
study,
all
parental
animals
(
P
and
Fl)
and
when
litter
size
permits
at
least
three
pups
per
sex
per
litter
from
the
unselected
Fl
weanlings
and
the
F2
weanlings
shall
be
examined
macroscopically
for
any
structural
abnormalities
or
pathological
changes.
Special
attention
shall
be
paid
to
the
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organs
of
the
reproductive
system.
Dead
pups
or
pups
that
are
terminated
in
a
moribund
condition
should
be
examined
for
possible
defects
and/
or
cause
of
death.
(
iii)
At
the
time
of
necropsy,
a
vaginal
smear
should
be
examined
to
determine
the
stage
of
the
estrous
cycle.
The
uteri
of
all
cohabited
females
should
be
examined,
in
a
manner
which
does
not
compromise
histopathological
evaluation,
for
the
presence
and
number
of
implantation
sites.
At
the
time
of
termination,
the
following
organs
of
all
P
and
Fl
parental
animals
shall
be
weighed:
uterus
(
with
oviducts
and
cervix),
ovaries,
testes,
epididymides
(
total
weights
for
both
and
cauda
weight
for
either
one
or
both),
seminal
vesicles
(
with
coagulating
glands
and
their
fluids),
prostate,
brain,
pituitary,
liver,
kidneys,
adrenal
glands,
spleen,
and
known
target
organs.
For
Fl
and
F2
weanlings
that
are
examined
macroscopically,
the
following
organs
shall
be
weighed
for
one
randomly
selected
pup
per
sex
per
litter:
brain,
spleen
and
thymus.
The
following
organs
and
tissues,
or
representative
samples
thereof,
shall
be
fixed
and
stored
in
a
suitable
medium
for
histopathological
examination.
For
the
parental
(
P
and
Fl)
animals:
vagina,
uterus
with
oviducts,
cervix,
and
ovaries,
one
testis
(
preserved
in
Bouins
fixative
or
comparable
preservative),
one
epididymis,
seminal
vesicles,
prostate,
coagulating
gland,
pituitary,
adrenal
glands,
any
target
organs
identified
upon
gross
necropsy.
For
Fl
and
P2
weanlings
selected
for
macroscopic
examination:
grossly
abnormal
tissue
and
target
organs
(
if
known).
Full
histopathology
of
the
organs
listed
above
shall
be
performed
for
ten
randomly
chosen
high
dose
and
control
P
and
Fl
animals
per
sex,
for
those
animals
that
were
selected
for
mating.
Organs
demonstrating
treatment­
related
changes
shall
also
be
examined
for
the
remainder
of
the
highdose
and
control
animals
and
for
all
parental
animals
in
the
low­
and
mid­
dose
groups.
Additionally,
reproductive
organs
of
the
low­
and
mid­
dose
animals
suspected
of
reduced
fertility.
Besides
gross
lesions
such
as
atrophy
or
tumors,
testicular
histopathological
examination
should
be
conducted
in
order
to
identify
treatment­
related
effects
such
as
retained
spermatids,
missing
germ
cell
layers
or
types,
multinucleated
giant
cells,
or
sloughing
of
spermatogenic
cells
into
the
lumen.
Examination
of
the
intact
epididymis
should
include
the
caput,
corpus,
and
cauda,
which
can
be
accomplished
by
evaluation
ofa
longitudinal
section,
and
should
be
conducted
in
order
to
identify
such
lesions
as
sperm
granulomas,
leukocytic
infiltration
(
inflammation),
aberrant
cell
types
within
the
lumen,
or
the
absence
of
clear
cells
in
the
cauda
epididymal
epithelium.
The
postlactational
ovary
should
contain
primordial
and
growing
follicles
as
well
as
the
large
corpora
lutea
of
lactation.
A
quantitative
evaluation
ofprimordial
follicles
should
be
conducted
for
all
Fl
females
if
any
of
the
following
treatment­
related
findings
were
observed:
reductions
in
ovarian
weight
and
abnormal
ovarian
histopathology
findings,
abnormal
estrous
cyclicity
and
female
infertility,
depletion
of
testicular
spermatid
counts
in
Fl
males
and
evidence
of
germ
cell
depletion
in
testicular
histopathology
evaluations.

Data
shall
be
reported
individually
and
summarized
in
tabular
form,
showing
for
each
test
group
the
types
ofchange
and
the
number
ofanimals
displaying
each
type
ofchange.
An
evaluation
of
test
results,
including
the
statistical
analysis,
shall
be
provided.
This
should
include
an
evaluation
of
the
relationship,
or
lack
thereof,
between
the
exposure
of
the
animals
to
the
test
substance
and
the
incidence
and
severity
ofall
abnormalities.
When
appropriate,
historical
control
data
should
be
used
to
enhance
interpretation
ofstudy
results.
Historical
data,
when
used,
should
be
compiled,
presented,
and
analyzed
in
an
appropriate­
and
relevant
manner.
In
order
to
justify
its
use
as
an
analytical
tool,
information
such
as
the
dates
ofstudy
conduct,
the
strain
and
source
of
the
animals,
and
the
vehicle
and
route
ofadministration
should
be
included.
Statistical
analysis
of
the
study
findings
should
include
sufficient
information
on
the
method
ofanalysis,
so
that
an
independent
reviewer/
statistician
can
reevaluate
and
reconstruct
the
analysis.

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