367
Environmental
Protection
Agency
§
799.9380
Pharmakologie
und
Experimentelle
Pathologie.
247:
367
(
1964).
(
20)
Spark,
C.
and
Dawson,
A.
B.
The
order
and
time
of
appearance
of
centers
of
ossification
in
the
fore
and
hind
limbs
of
the
albino
rat,
with
special
reference
to
the
possible
influence
of
the
sex
factor.
American
Journal
of
Anatomy.
41:
411
 
445
(
1928).
(
21)
Staples,
R.
E.
Detection
of
visceral
alterations
in
mammalian
fetuses.
Teratology.
9(
3):
A37
 
A38
(
1974).
(
22)
Staples,
R.
E.
and
Schnell,
V.
L.
Refinements
in
rapid
clearing
technique
in
the
KOH
 
alizarin
red
S
method
for
fetal
bone.
Stain
Technology.
39:
61
 
63
(
1964).
(
23)
Strong,
R.
M.
The
order
time
and
rate
of
ossification
of
the
albino
rat
(
mus
norvegicus
albinus)
skeleton.
American
Journal
of
Anatomy.
36:
313
 
355
(
1928).
(
24)
Stuckhardt,
J.
L.
and
Poppe,
S.
M.
Fresh
visceral
examination
of
rat
and
rabbit
fetuses
used
in
teratogenicity
testing.
Teratogenesis,
Carcinogenesis,
and
Mutagenesis.
4:
181
 
188
(
1984).
(
25)
Van
Julsingha,
E.
B.
and
Bennett,
C.
G.
Eds.
Neubert,
D.,
Merker,
H.
J.,
and
Kwasigroch,
T.
E.
A
dissecting
procedure
for
the
detection
of
anomalies
in
the
rabbit
foetal
head.
Methods
in
Prenatal
Toxicology
(
University
of
Chicago,
Chicago,
IL,
1977)
pp.
126
 
144.
(
26)
Whitaker,
J.
and
Dix,
D.
M.
Doublestaining
for
rat
foetus
skeletons
in
teratological
studies.
Laboratory
Animals.
13:
309
 
310
(
1979).
(
27)
Wilson,
J.
G.
Eds.
Wilson,
J.
G.
and
Warkany,
J.
Embryological
considerations
in
teratology.
Teratology:
Principles
and
Techniques
(
University
of
Chicago,
Chicago,
IL,
1965)
pp.
251
 
277.

§
799.9380
TSCA
reproduction
and
fertility
effects.

(
a)
Scope.
This
section
is
intended
to
meet
the
testing
requirements
under
section
4
of
the
TSCA.
This
section
is
for
two­
generation
reproduction
testing
and
is
designed
to
provide
general
information
concerning
the
effects
of
a
test
substance
on
the
integrity
and
performance
of
the
male
and
female
reproductive
systems,
including
gonadal
function,
the
estrous
cycle,
mating
behavior
conception,
gestation,
parturition
lactation,
and
weaning,
and
on
the
growth
and
development
of
the
offspring
The
study
may
also
provide
information
about
the
effects
of
the
test
substance
on
neonatal
morbidity,
mortality
target
organs
in
the
offspring,
and
preliminary
data
on
prenatal
and
postnatal
developmental
toxicity
and
serve
as
a
guide
for
subsequent
tests.
Additionally,
since
the
study
design
includes
in
utero
as
well
as
postnatal
exposure
this
study
provides
the
opportunity
to
examine
the
susceptibility
of
the
immature/
neonatal
animal.
(
b)
Source.
The
source
material
used
in
developing
this
TSCA
test
guideline
is
the
OPPTS
harmonized
test
guideline
870.3800
(
February
1996
Public
Draft).
This
source
is
available
at
the
address
in
paragraph
(
g)
of
this
section.
(
c)
Good
laboratory
practice
standards.
The
study
shall
be
conducted
in
compliance
with
40
CFR
part
792
 
Good
Laboratory
Practice
Standards.
(
d)
Principle
of
the
test
method.
The
test
substance
is
administered
to
parental
(
P)
animals
prior
to
and
during
their
mating,
during
the
resultant
pregnancies,
and
through
the
weaning
of
their
F1
offspring.
The
substance
is
then
administered
to
selected
F1
offspring
during
their
growth
into
adulthood
mating,
and
production
of
an
F2
generation,
until
the
F2
generation
is
weaned.
(
e)
Test
procedures
 
(
1)
Animal
selection
 
(
i)
Species
and
strain.
The
rat
is
the
most
commonly
used
species
for
testing.
If
another
mammalian
species
is
used,
the
tester
shall
provide
justification
reasoning
for
its
selection,
and
appropriate
modifications
will
be
necessary.
Healthy
parental
animals,
which
have
been
acclimated
to
laboratory
conditions
for
at
least
5
days
and
have
not
been
subjected
to
previous
experimental
procedures,
should
be
used.
Strains
of
low
fecundity
shall
not
be
used.
(
ii)
Age.
Parental
(
P)
animals
shall
be
5
to
9
weeks
old
at
the
start
of
dosing.
The
animals
of
all
test
groups
should
be
of
uniform
weight,
age,
and
parity
as
nearly
as
practicable,
and
should
be
representative
of
the
species
and
strain
under
study.
(
iii)
Sex.
(
A)
For
an
adequate
assessment
of
fertility,
both
males
and
females
shall
be
studied.
(
B)
The
females
shall
be
nulliparous
and
nonpregnant.
(
iv)
Number
of
animals.
Each
control
group
shall
contain
a
sufficient
number
of
mating
pairs
to
yield
approximately
20
pregnant
females.
Each
test
group
shall
contain
a
similar
number
of
mating
pairs.

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368
40
CFR
Ch.
I
(
7
 
1
 
02
Edition)
§
799.9380
(
v)
Identification
of
animals.
Each
animal
shall
be
assigned
a
unique
identification
number.
For
the
P
generation,
this
should
be
done
before
dosing
starts.
For
the
F1
generation,
this
should
be
done
for
animals
selected
for
mating;
in
addition,
records
indicating
the
litter
of
origin
shall
be
maintained
for
all
selected
F1
animals.
(
2)
Administration
of
test
and
control
substances
 
(
i)
Dose
levels
and
dose
selection
(
A)
At
least
three­
dose
levels
and
a
concurrent
control
shall
be
used.
Healthy
animals
should
be
randomly
assigned
to
the
control
and
treatment
groups,
in
a
manner
which
results
in
comparable
mean
body
weight
values
among
all
groups.
The
dose
levels
should
be
spaced
to
produce
a
gradation
of
toxic
effects.
Unless
limited
by
the
physical/
chemical
nature
or
biological
properties
of
the
test
substance,
the
highest
dose
should
be
chosen
with
the
aim
to
induce
some
reproductive
and/
or
systemic
toxicity
but
not
death
or
severe
suffering.
In
the
case
of
parental
mortality,
this
should
not
be
more
than
approximately
10%.
The
intermediate
dose
levels
should
produce
minimal
observable
toxic
effects.
The
lowest
dose
level
should
not
produce
any
evidence
of
either
systemic
or
reproductive
toxicity
(
i.
e.,
the
no­
observed
adverse­
effect
level,
NOAEL)
or
should
be
at
or
near
the
limit
of
detection
for
the
most
sensitive
endpoint.
Two­
or
four­
fold
intervals
are
frequently
optimal
for
spacing
the
dose
levels,
and
the
addition
of
a
fourth
test
group
is
often
preferable
to
using
very
large
intervals
(
e.
g.,
more
than
a
factor
of
10)
between
dosages.
(
B)
It
is
desirable
that
additional
information
on
metabolism
and
pharmacokinetics
of
the
test
substance
be
available
to
demonstrate
the
adequacy
of
the
dosing
regimen.
This
information
should
be
available
prior
to
testing
(
C)
The
highest
dose
tested
should
not
exceed
1,000
mg/
kg/
day
(
or
20,000
ppm
in
the
diet),
unless
potential
human
exposure
data
indicate
the
need
for
higher
doses.
If
a
test
performed
at
the
limit
dose
level,
using
the
procedures
described
for
this
study,
produces
no
observable
toxicity
and
if
an
effect
would
not
be
expected
based
upon
data
from
structurally
related
compounds,
then
a
full
study
using
three
dose
levels
may
not
be
considered
necessary.
(
ii)
Control
group.
(
A)
A
concurrent
control
group
shall
be
used.
This
group
shall
be
an
untreated
or
sham
treated
group
or
a
vehicle­
control
group
if
a
vehicle
is
used
in
administering
the
test
substance.
(
B)
If
a
vehicle
is
used
in
administering
the
test
substance,
the
control
group
shall
receive
the
vehicle
in
the
highest
volume
used.
(
C)
If
a
vehicle
or
other
additive
is
used
to
facilitate
dosing,
consideration
should
be
given
to
the
following
characteristics
Effects
on
the
absorption,
distribution,
metabolism,
or
retention
of
the
test
substance;
effects
on
the
chemical
properties
of
the
test
substance
which
may
alter
its
toxic
characteristics
and
effects
on
the
food
or
water
consumption
or
the
nutritional
status
of
the
animals.
(
D)
If
a
test
substance
is
administered
in
the
diet
and
causes
reduced
dietary
intake
or
utilization,
the
use
of
a
pair­
fed
control
group
may
be
considered
necessary.
(
iii)
Route
of
administration.
(
A)
The
test
substance
is
usually
administered
by
the
oral
route
(
diet,
drinking
water,
or
gavage).
(
B)
If
administered
by
gavage
or
dermal
application,
the
dosage
administered
to
each
animal
prior
to
mating
and
during
gestation
and
lactation
shall
be
based
on
the
individual
animal
body
weight
and
adjusted
weekly
at
a
minimum.
(
C)
If
another
route
of
administration
is
used,
for
example,
when
the
route
of
administration
is
based
upon
the
principal
route
of
potential
human
exposure
the
tester
should
provide
justification
and
reasoning
for
its
selection
and
appropriate
modifications
may
be
necessary.
Care
should
be
taken
to
minimize
stress
on
the
maternal
animals
and
their
litters
during
gestation
and
lactation.
(
D)
All
animals
should
be
dosed
by
the
same
method
during
the
appropriate
experimental
period.
(
iv)
Dosing
schedule.
(
A)
The
animals
should
be
dosed
with
the
test
substance
on
a
7­
days­
a­
week
basis.
(
B)
Daily
dosing
of
the
parental
(
P)
males
and
females
shall
begin
when
they
are
5
to
9
weeks
old.
Daily
dosing
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369
Environmental
Protection
Agency
§
799.9380
of
the
F1
males
and
females
shall
begin
at
weaning.
For
both
sexes
(
P
and
F1),
dosing
shall
be
continued
for
at
least
10
weeks
before
the
mating
period.
(
C)
Daily
dosing
of
the
P
and
F1
males
and
females
shall
continue
until
termination.
(
3)
Mating
procedure
 
(
i)
Parental.
(
A)
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.
Animals
should
be
separated
as
soon
as
possible
after
evidence
of
copulation
is
observed.
If
mating
has
not
occurred
after
2
weeks
or
3
estrous
periods,
the
animals
should
be
separated
without
further
opportunity
for
mating.
Mating
pairs
should
be
clearly
identified
in
the
data.
(
B)
Vaginal
smears
shall
be
collected
daily
and
examined
for
all
females
during
mating,
until
evidence
of
copulation
is
observed.
(
C)
Each
day,
the
females
shall
be
examined
for
presence
of
sperm
or
vaginal
plugs.
Day
0
of
pregnancy
is
defined
as
the
day
a
vaginal
plug
or
sperm
are
found.
(
ii)
F1
mating.
For
mating
the
F1
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.
(
iii)
Second
mating.
In
certain
instances
such
as
poor
reproductive
performance
in
the
controls,
or
in
the
event
of
treatment­
related
alterations
in
litter
size,
the
adults
may
be
remated
to
produce
an
F1b
or
F2b
litter
If
production
of
a
second
litter
is
deemed
necessary
in
either
generation,
the
dams
should
be
remated
approximately
1
 
2
weeks
following
weaning
of
the
last
F1a
or
F2a
litter.
(
iv)
Special
housing.
After
evidence
of
copulation,
animals
that
are
presumed
to
be
pregnant
shall
be
caged
separately
in
delivery
or
maternity
cages.
Pregnant
animals
shall
be
provided
with
nesting
materials
when
parturition
is
near.
(
v)
Standardization
of
litter
sizes.
(
A)
Animals
should
be
allowed
to
litter
normally
and
rear
their
offspring
to
weaning.
Standardization
of
litter
sizes
is
optional.
(
B)
If
standardization
is
performed,
the
following
procedure
should
be
used.
On
day
4
after
birth,
the
size
of
each
litter
may
be
adjusted
by
eliminating
extra
pups
by
random
selection
to
yield,
as
nearly
as
possible,
four
males
and
four
females
per
litter
or
five
males
and
five
females
per
litter.
Selective
elimination
of
pups,
i.
e.
based
upon
body
weight,
is
not
appropriate.
Whenever
the
number
of
male
or
female
pups
prevents
having
four
(
or
five)
of
each
sex
per
litter,
partial
adjustment
(
for
example,
five
males
and
three
females,
or
four
males
and
six
females
is
acceptable.
Adjustments
are
not
appropriate
for
litters
of
eight
pups
or
less.
(
4)
Observation
of
animals
 
(
i)
Parental
(
A)
Throughout
the
test
period,
each
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.
(
B)
Parental
animals
(
P
and
F1)
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.
(
C)
During
the
premating
and
gestation
periods,
food
consumption
shall
be
measured
weekly
at
a
minimum.
Water
consumption
should
be
measured
weekly
at
a
minimum
if
the
test
substance
is
administered
in
the
water.
(
D)
Estrous
cycle
length
and
pattern
should
be
evaluated
by
vaginal
smears
for
all
P
and
F1
females
during
a
minimum
of
3
weeks
prior
to
mating
and
throughout
cohabitation;
care
should
be
taken
to
prevent
the
induction
of
pseudopregnancy.
(
E)
For
all
P
and
F1
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
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40
CFR
Ch.
I
(
7
 
1
 
02
Edition)
§
799.9380
evaluation
of
sperm
motility
and
sperm
morphology.
(
1)
The
total
number
of
homogenization
resistant
testicular
sperm
and
cauda
epididymal
sperm
should
be
enumerated
The
method
described
in
the
reference
under
paragraph
(
g)(
8)
of
this
section
may
be
used.
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
F1
males
may
be
performed
unless
treatment­
related
effects
are
observed;
in
that
case,
the
lower
dose
groups
should
also
be
evaluated.
(
2)
An
evaluation
of
epididymal
(
or
vas
deferens)
sperm
motility
should
be
performed.
Sperm
should
be
recovered
while
minimizing
damage
(
the
evaluation
techniques
as
described
in
the
reference
under
paragraph
(
g)(
8)
of
this
section
may
be
used),
and
the
percentage
of
progressively
motile
sperm
should
be
determined
either
subjectively
or
objectively.
For
objective
evaluations,
an
acceptable
counting
chamber
of
sufficient
depth
can
be
used
to
effectively
combine
the
assessment
of
motility
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
F1
males
may
be
performed
unless
treatment
related
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.
(
3)
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
(
the
techniques
for
such
examinations
is
described
in
the
references
under
paragraphs
(
g)(
4)
and
(
g)(
8)
of
this
section
may
be
used)
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
and/
or
tails.
Evaluation
of
only
control
and
high­
dose
P
and
F1
males
may
be
performed
unless
treatment­
related
effects
are
observed;
in
that
case,
the
lower
dose
groups
should
also
be
evaluated.
(
ii)
Offspring.
(
A)
Each
litter
should
be
examined
as
soon
as
possible
after
delivery
(
lactation
day
0)
to
establish
the
number
and
sex
of
pups,
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.
(
B)
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
(
C)
The
age
of
vaginal
opening
and
preputial
separation
should
be
determined
for
F1
weanlings
selected
for
mating.
If
there
is
a
treatment­
related
effect
in
F1
sex
ratio
or
sexual
maturation
anogenital
distance
should
be
measured
on
day
0
for
all
F2
pups.
(
5)
Termination
schedule.
(
i)
All
P
and
F1
adult
males
and
females
should
be
terminated
when
they
are
no
longer
needed
for
assessment
of
reproductive
effects.
(
ii)
F1
offspring
not
selected
for
mating
and
all
F2
offspring
should
be
terminated
at
comparable
ages
after
weaning.
(
6)
Gross
necropsy.
(
i)
At
the
time
of
termination
or
death
during
the
study,
all
parental
animals
(
P
and
F1)
and
when
litter
size
permits
at
least
three
pups
per
sex
per
litter
from
the
unselected
F1
weanlings
and
the
F2
weanlings
shall
be
examined
macroscopically
for
any
structural
abnormalities
or
pathological
changes.
Special
attention
shall
be
paid
to
the
organs
of
the
reproductive
system.
(
ii)
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.

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371
Environmental
Protection
Agency
§
799.9380
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.
(
7)
Organ
weights.
(
i)
At
the
time
of
termination,
the
following
organs
of
all
P
and
F1
parental
animals
shall
be
weighed:
(
A)
Uterus
(
with
oviducts
and
cervix),
ovaries.
(
B)
Testes,
epididymides
(
total
weights
for
both
and
cauda
weight
for
either
one
or
both),
seminal
vesicles
(
with
coagulating
glands
and
their
fluids),
and
prostate.
(
C)
Brain,
pituitary,
liver,
kidneys,
adrenal
glands,
spleen,
and
known
target
organs.
(
ii)
For
F1
and
F2
weanlings
that
are
examined
macroscopically,
the
following
organs
shall
be
weighed
for
one
randomly
selected
pup
per
sex
per
litter
(
A)
Brain.
(
B)
Spleen
and
thymus.
(
8)
Tissue
preservation.
The
following
organs
and
tissues,
or
representative
samples
thereof,
shall
be
fixed
and
stored
in
a
suitable
medium
for
histopathological
examination.
(
i)
For
the
parental
(
P
and
F1)
animals
(
A)
Vagina,
uterus
with
oviducts,
cervix
and
ovaries.
(
B)
One
testis
(
preserved
in
Bouins
fixative
or
comparable
preservative),
one
epididymis,
seminal
vesicles,
prostate
and
coagulating
gland.
(
C)
Pituitary
and
adrenal
glands.
(
D)
Target
organs,
when
previously
identified,
from
all
P
and
F1
animals
selected
for
mating.
(
E)
Grossly
abnormal
tissue.
(
ii)
For
F1
and
F2
weanlings
selected
for
macroscopic
examination:
Grossly
abnormal
tissue
and
target
organs,
when
known.
(
9)
Histopathology
 
(
i)
Parental
animals
Full
histopathology
of
the
organs
listed
in
paragraph
(
e)(
8)(
i)
of
this
section
shall
be
performed
for
ten
randomly
chosen
high
dose
and
control
P
and
F1
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
high­
dose
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,
e.
g.,
those
that
failed
to
mate,
conceive,
sire,
or
deliver
healthy
offspring,
or
for
which
estrous
cyclicity
or
sperm
number,
motility,
or
morphology
were
affected,
shall
be
subjected
to
histopathological
evaluation.
Besides
gross
lesions
such
as
atrophy
or
tumors,
testicular
histopathological
examination
should
be
conducted
in
order
to
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
of
a
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.
Histopathological
examination
should
detect
qualitative
depletion
of
the
primordial
follicle
population
A
quantitative
evaluation
of
primordial
follicles
should
be
conducted
for
all
F1
females;
the
number
of
animals,
ovarian
section
selection,
and
section
sample
size
should
be
statistically
appropriate
for
the
evaluation
procedure
used.
Examination
should
include
enumeration
of
the
number
of
primordial
follicles,
which
can
be
combined
with
small
growing
follicles
(
see
paragraphs
(
g)(
1)
and
(
g)(
2)
of
this
section),
for
comparison
of
treated
and
control
ovaries.
(
ii)
Weanling.
For
F1
and
F2
weanlings,
histopathological
examination
of
treatment­
related
abnormalities
noted
in
macroscopic
examination
should
be
considered,
if
such
evaluation
were
deemed
appropriate
and
would
contribute
to
the
interpretation
of
the
study
data.
(
f)
Data
and
reporting
 
(
1)
Treatment
of
results.
Data
shall
be
reported
individually
and
summarized
in
tabular
form,
showing
for
each
test
group
the
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372
40
CFR
Ch.
I
(
7
 
1
 
02
Edition)
§
799.9380
types
of
change
and
the
number
of
animals
displaying
each
type
of
change.
(
2)
Evaluation
of
study
results.
(
i)
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
of
all
abnormalities.
(
ii)
When
appropriate,
historical
control
data
should
be
used
to
enhance
interpretation
of
study
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
of
study
conduct,
the
strain
and
source
of
the
animals,
and
the
vehicle
and
route
of
administration
should
be
included.
(
iii)
Statistical
analysis
of
the
study
findings
should
include
sufficient
information
on
the
method
of
analysis,
so
that
an
independent
reviewer/
statistician
can
reevaluate
and
reconstruct
the
analysis.
(
iv)
In
any
study
which
demonstrates
an
absence
of
toxic
effects,
further
investigation
to
establish
absorption
and
bioavailability
of
the
test
substance
should
be
considered.
(
3)
Test
report.
In
addition
to
the
reporting
requirements
as
specified
under
40
CFR
part
792,
subpart
J,
the
following
specific
information
shall
be
reported
Both
individual
and
summary
data
should
be
presented.
(
i)
Species
and
strain.
(
ii)
Toxic
response
data
by
sex
and
dose,
including
indices
of
mating,
fertility
gestation,
birth,
viability,
and
lactation;
offspring
sex
ratio;
precoital
interval,
including
the
number
of
days
until
mating
and
the
number
of
estrous
periods
until
mating;
and
duration
of
gestation
calculated
from
day
0
of
pregnancy.
The
report
should
provide
the
numbers
used
in
calculating
all
indices
(
iii)
Day
(
week)
of
death
during
the
study
or
whether
animals
survived
to
termination;
date
(
age)
of
litter
termination
(
iv)
Toxic
or
other
effects
on
reproduction
offspring,
or
postnatal
growth.
(
v)
Developmental
milestone
data
(
mean
age
of
vaginal
opening
and
preputial
separation,
and
mean
anogenital
distance,
when
measured).
(
vi)
Number
of
P
and
F1
females
cycling
pattern
and
mean
estrous
cycle
length.
(
vii)
Day
(
week)
of
observation
of
each
abnormal
sign
and
its
subsequent
course.
(
viii)
Body
weight
and
body
weight
change
data
by
sex
for
P,
F1,
and
F2
animals.
(
ix)
Food
(
and
water,
if
applicable)
consumption,
food
efficiency
(
body
weight
gain
per
gram
of
food
consumed
and
test
material
consumption
for
P
and
F1
animals,
except
for
the
period
of
cohabitation.
(
x)
Total
cauda
epididymal
sperm
number,
homogenization­
resistant
testis
spermatid
number,
number
and
percent
of
progressively
motile
sperm,
number
and
percent
of
morphologically
normal
sperm,
and
number
and
percent
of
sperm
with
each
identified
anomaly.
(
xi)
Stage
of
the
estrous
cycle
at
the
time
of
termination
for
P
and
F1
parental
females.
(
xii)
Necropsy
findings.
(
xiii)
Implantation
data
and
postimplantation
loss
calculations
for
P
and
F1
parental
females.
(
xiv)
Absolute
and
adjusted
organ
weight
data.
(
xv)
Detailed
description
of
all
histopathological
findings.
(
xvi)
Adequate
statistical
treatment
of
results.
(
xvii)
A
copy
of
the
study
protocol
and
any
amendments
should
be
included
(
g)
References.
For
additional
backgound
information
on
this
test
guideline,
the
following
references
should
be
consulted.
These
references
are
available
for
inspection
at
the
TSCA
Nonconfidential
Information
Center,
Rm.
NE
 
B607,
Environmental
Protection
Agency,
401
M
St.,
SW.,
Washington,
DC,
12
noon
to
4
p.
m.,
Monday
through
Friday,
except
legal
holidays.
(
1)
Gray,
L.
E.
et
al.
A
dose­
response
analysis
of
methoxychlor­
induced
alterations
of
reproductive
development
and
function
in
the
rat.
Fundamental
and
Applied
Toxicology.
12:
92
 
108
(
1989).
(
2)
Heindel,
J.
J.
et
al.
Ed.
Hirshfield,
A.
N.
Histological
assessment
of
ovarian
follicle
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373
Environmental
Protection
Agency
§
799.9410
number
in
mice
as
a
screen
of
ovarian
toxicity
Growth
Factors
and
the
Ovary
(
Plenum,
NY,
1989)
pp.
421
 
426.
(
3)
Korenbrot,
C.
C.
et
al.
Preputial
separation
as
an
external
sign
of
pubertal
development
in
the
male
rat.
Biology
of
Reproduction
17:
298
 
303
(
1977).
(
4)
Linder,
R.
E.
et
al.
Endpoints
of
spermatoxicity
in
the
rat
after
short
duration
exposures
to
fourteen
reproductive
toxicants
Reproductive
Toxicology.
6:
491
 
505
(
1992).
(
5)
Manson,
J.
M.
and
Kang,
Y.
J.
Ed.
Hayes,
A.
W.
Test
methods
for
assessing
female
reproductive
and
developmental
toxicology.
Principles
and
Methods
of
Toxicology
(
Raven,
NY,
1989).
(
6)
Organisation
for
Economic
Co­
operation
and
Development,
No.
416:
Two
Generation
Reproduction
Toxicity
Study,
Guidelines
for
Testing
of
Chemicals.
[
C(
83)
44
(
Final)]
(
1983).
(
7)
Pederson,
T.
and
Peters,
H.
Proposal
for
classification
of
oocytes
and
follicles
in
the
mouse
ovary.
Journal
of
Reproduction
and
Fertility.
17:
555
 
557
(
1988).
(
8)
Seed,
J.,
Chapin,
R.
E.
E.
D.
Clegg,
L.
A.
Dostal,
R.
H.
Foote,
M.
E.
Hurtt,
G.
R.
Klinefelter,
S.
L.
Makris,
S.
D.
Perreault,
S.
Schrader,
D.
Seyler,
R.
Sprando,
K.
A.
Treinen,
D.
N.
R.
Veeramachaneni,
and
Wise,
L.
D.
Methods
for
assessing
sperm
motility,
morphology,
and
counts
in
the
rat,
rabbit,
and
dog:
a
consensus
report.
Reproductive
Toxicology.
10(
3):
237
 
244
(
1996).
(
9)
Smith,
B.
J.
et
al.
Comparison
of
random
and
serial
sections
in
assessment
of
ovarian
toxicity.
Reproductive
Toxicology.
5:
379
 
383
(
1991).
(
10)
Thomas,
J.
A.
Eds.
M.
O.
Amdur,
J.
Doull,
and
C.
D.
Klaassen.
Toxic
responses
of
the
reproductive
system.
Casarett
and
Doull's
Toxicology
(
Pergamon,
NY,
1991).
(
11)
Working,
P.
K.
and
Hurtt,
M.
Computerized
videomicrographic
analysis
of
rat
sperm
motility.
Journal
of
Andrology.
8:
330
 
337
(
1987).
(
12)
Zenick,
H.
et
al.
Ed.
Hayes,
A.
W.
Assessment
of
male
reproductive
toxicity:
a
risk
assessment
approach.
Principles
and
Methods
of
Toxicology
(
Raven,
NY,
1994).

[
62
FR
43824,
Aug.
15,
1997,
as
amended
at
64
FR
35078,
June
30,
1999]

§
799.9410
TSCA
chronic
toxicity.

(
a)
Scope
 
(
1)
Applicability.
This
section
is
intended
to
meet
the
testing
requirement
of
the
Toxic
Substances
Control
Act
(
TSCA)
(
15
U.
S.
C.
2601).
(
2)
Source.
The
source
material
used
in
developing
this
TSCA
test
guideline
is
the
Office
of
Prevention,
Pesticides
and
Toxic
Substances
(
OPPTS)
harmonized
test
guideline
870.4100
(
August
1998,
final
guidelines).
This
source
is
available
at
the
address
in
paragraph
(
h)
of
this
section
(
b)
Purpose.
The
objective
of
a
chronic
toxicity
study
is
to
determine
the
effects
of
a
substance
in
a
mammalian
species
following
prolonged
and
repeated
exposure.
A
chronic
toxicity
study
should
generate
data
from
which
to
identify
the
majority
of
chronic
effects
and
to
define
long­
term
dose­
response
relationships.
The
design
and
conduct
of
chronic
toxicity
tests
should
allow
for
the
detection
of
general
toxic
effects,
including
neurological
physiological,
biochemical,
and
hematological
effects
and
exposure­
related
morphological
(
pathological)
effects
(
c)
Definitions.
The
definitions
in
section
3
of
TSCA
and
in
40
CFR
Part
792
 
Good
Laboratory
Practice
Standards
apply
to
this
section.
The
following
definitions
also
apply
to
this
section.
Chronic
toxicity
is
the
adverse
effects
occurring
as
a
result
of
the
repeated
daily
exposure
of
experimental
animals
to
a
chemical
by
the
oral,
dermal,
or
inhalation
routes
of
exposure.
Cumulative
toxicity
is
the
adverse
effects
of
repeated
doses
occurring
as
a
result
of
prolonged
action
on,
or
increased
concentration
of,
the
administered
test
substance
or
its
metabolites
in
susceptible
tissue.
Dose
in
a
chronic
toxicity
study
is
the
amount
of
test
substance
administered
daily
via
the
oral,
dermal
or
inhalation
routes
for
a
period
of
at
least
12
months.
Dose
is
expressed
as
weight
of
the
test
substance
(
grams,
milligrams)
per
unit
body
weight
of
test
animal
(
milligram
per
kilogram),
or
as
weight
of
the
test
substance
in
parts
per
million
(
ppm)
in
food
or
drinking
water
per
day.
For
inhalation
exposure,
dose
is
expressed
as
weight
of
the
test
substance
per
unit
volume
of
air
(
milligrams
per
liter)
or
as
parts
per
million
per
day.
For
dermal
exposure,
dose
is
expressed
as
weight
of
the
test
substance
(
grams,
milligrams)
per
unit
body
weight
of
the
test
animal
(
milligrams
per
kilogram)
or
as
weight
of
the
substance
per
unit
of
surface
area
(
milligrams
per
square
centimeter)
per
day.
No­
observed­
effects
level
(
NOEL)
is
the
maximum
dose
used
in
a
study
which
produces
no
adverse
effects.
The
NOEL
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