427
Environmental
Protection
Agency
§
799.9620
Erythrocytes
Using
Hoechst
33258
and
Pyronin
Y.
Mutation
Research.
120,
269
 
275
(
1983).
(
12)
Romagna,
F.
and
Staniforth,
C.
D.
The
automated
bone
marrow
micronucleus
test.
Mutation
Research.
213,
91
 
104
(
1989).
(
13)
Gollapudi,
B.
and
McFadden,
L.
G.
Sample
size
for
the
estimation
of
polychromatic
to
normochromatic
eruthrocyte
ratio
in
the
bone
marrow
micronucleus
test.
Mutation
Research.
347,
97
 
99
(
1995).
(
14)
Richold,
M.,
Ashby,
J.,
Bootman,
J.,
Chandley,
A.,
Gatehouse,
D.
G.,
and
Henderson
L.
Ed.
Kirkland,
D.
J.
In
Vivo
Cytogenetics
Assays.
Basic
Mutagenicity
Tests,
UKEMS
Recommended
Procedures.
UKEMS
Subcommittee
on
Guidelines
for
Mutagenicity
Testing.
Report.
Part
I
revised
(
Cambridge
University
Press,
Cambridge,
New
York,
Port
Chester,
Melbourne,
Sydney,
1990)
pp.
115
 
141.
(
15)
Lovell,
D.
P.,
Anderson,
D.,
Albanese,
R.,
Amphlett,
G.
E.,
Clare,
G.,
Ferguson,
R.,
Richold,
M.,
Papworth,
D.
G.,
and
Savage,
J.
R.
K.
Ed.
D.
J.
Kirkland.
Statistical
Analysis
of
In
Vivo
Cytogenetic
Assays.
Statistical
Evaluation
of
Mutagenicity
Test
Data.
UKEMS
Sub­
Committee
on
Guidelines
for
Mutagenicity
Testing,
Report,
Part
III.
(
Cambridge
University
Press,
Cambridge,
New
York,
Port
Chester,
Melbourne,
Sydney,
1989)
pp.
184
 
232.
(
16)
Heddle,
J.
A.,
Salamone,
M.
F.,
Hite,
M.,
Kirkhart,
B.,
Mavournin,
K.,
MacGregor,
J.
G.,
and
Newell,
G.
W.
The
Induction
of
Micronuclei
as
a
Measure
of
Genotoxicity.
Mutation
Research.
123:
61
 
118
(
1983).
(
17)
MacGregor,
J.
T.,
Heddle,
J.
A.,
Hite,
M.,
Margolin,
G.
H.,
Ramel
C.,
Salamone,
M.
F.,
Tice,
R.
R.,
and
Wild,
D.
Guidelines
for
the
Conduct
of
Micronucleus
Assays
in
Mammalian
Bone
Marrow
Erythrocytes.
Mutation
Research.
189:
103
 
112
(
1987).
(
18)
MacGregor,
J.
T.,
Wehr,
C.
M.,
Henika,
P.
R.,
and
Shelby,
M.
E.
(
1990).
The
In
Vivo
Erythrocyte
Micronucleus
Test:
Measurement
at
Steady
State
Increases
Assay
Efficiency
and
Permits
Integration
with
Toxicity
Studies.
Fundamental
Applied
Toxicology
14:
513
 
522.
(
19)
MacGregor,
J.
T.,
Schlegel,
R.
Choy,
W.
N.,
and
Wehr,
C.
M.
Eds.
Hayes,
A.
W.,
Schnell,
R.
C.,
and
Miya,
T.
S.
Micronuclei
in
Circulating
Erythrocytes:
A
Rapid
Screen
for
Chromosomal
Damage
During
Routine
Toxicity
Testing
in
Mice.
Developments
in
Science
and
Practice
of
Toxicology
(
Elsevier,
Amsterdam,
1983)
pp.
555
 
558.

[
62
FR
43824,
Aug.
15,
1997,
as
amended
at
64
FR
35079,
June
30,
1999]

§
799.9620
TSCA
neurotoxicity
screening
battery.

(
a)
Scope.
This
section
is
intended
to
meet
the
testing
requirements
under
section
4
of
TSCA.
This
neurotoxicity
screening
battery
consists
of
a
functional
observational
battery,
motor
activity
and
neuropathology.
The
functional
observational
battery
consists
of
noninvasive
procedures
designed
to
detect
gross
functional
deficits
in
animals
and
to
better
quantify
behavioral
or
neurological
effects
detected
in
other
studies.
The
motor
activity
test
uses
an
automated
device
that
measures
the
level
of
activity
of
an
individual
animal.
The
neuropathological
techniques
are
designed
to
provide
data
to
detect
and
characterize
histopathological
changes
in
the
central
and
peripheral
nervous
system.
This
battery
is
designed
to
be
used
in
conjunction
with
general
toxicity
studies
and
changes
should
be
evaluated
in
the
context
of
both
the
concordance
between
functional
neurological
and
neuropatholgical
effects,
and
with
respect
to
any
other
toxicological
effects
seen.
This
test
battery
is
not
intended
to
provide
a
complete
evaluation
of
neurotoxicity,
and
additional
functional
and
morphological
evaluation
may
be
necessary
to
assess
completely
the
neurotoxic
potential
of
a
chemical.
(
b)
Source.
The
source
material
used
in
developing
this
TSCA
test
guideline
is
the
OPPTS
harmonized
test
guideline
870.6200
(
June
1996
Public
Draft).
This
source
is
available
at
the
address
in
paragraph
(
g)
of
this
section.
(
c)
Definitions.
The
following
definitions
apply
to
this
section.
ED
is
effective
dose.
Motor
activity
is
any
movement
of
the
experimental
animal.
Neurotoxicity
is
any
adverse
effect
on
the
structure
or
function
of
the
nervous
system
related
to
exposure
to
a
chemical
substance.
Toxic
effect
is
an
adverse
change
in
the
structure
or
function
of
an
experimental
animal
as
a
result
of
exposure
to
a
chemical
substance.
(
d)
Principle
of
the
test
method.
The
test
substance
is
administered
to
several
groups
of
experimental
animals,
one
dose
being
used
per
group.
The
animals
are
observed
under
carefully
standardized
conditions
with
sufficient
frequency
to
ensure
the
detection
and
quantification
of
behavioral
and/
or
neurologic
abnormalities,
if
present.
Various
functions
that
could
be
affected
by
neurotoxicants
are
assessed
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OPPT­
2003­
0010­
0007
428
40
CFR
Ch.
I
(
7
 
1
 
02
Edition)
§
799.9620
during
each
observation
period.
Measurements
of
motor
activity
of
individual
animals
are
made
in
an
automated
device.
The
animals
are
perfused
and
tissue
samples
from
the
nervous
system
are
prepared
for
microscopic
examination.
The
exposure
levels
at
which
significant
neurotoxic
effects
are
produced
are
compared
to
one
another
and
to
those
levels
that
produce
other
toxic
effects.
(
e)
Test
procedures
 
(
1)
Animal
selection
 
(
i)
Species.
In
general,
the
laboratory
rat
should
be
used.
Under
some
circumstances,
other
species,
such
as
the
mouse
or
the
dog,
may
be
more
appropriate
although
not
all
of
the
battery
may
be
adaptable
to
other
species.
(
ii)
Age.
Young
adults
(
at
least
42
days
old
for
rats)
shall
be
used.
(
iii)
Sex.
Both
males
and
females
shall
be
used.
Females
shall
be
nulliparous
and
nonpregnant.
(
2)
Number
of
animals.
At
least
10
males
and
10
females
should
be
used
in
each
dose
and
control
group
for
behavioral
testing.
At
least
five
males
and
five
females
should
be
used
in
each
dose
and
control
group
for
terminal
neuropathology.
If
interim
neuropathological
evaluations
are
planned,
the
number
should
be
increased
by
the
number
of
animals
scheduled
to
be
perfused
before
the
end
of
the
study.
Animals
shall
be
randomly
assigned
to
treatment
and
control
groups.
(
3)
Control
groups.
(
i)
A
concurrent
(
vehicle)
control
group
is
required.
Subjects
shall
be
treated
in
the
same
way
as
for
an
exposure
group
except
that
administration
of
the
test
substance
is
omitted.
If
the
vehicle
used
has
known
or
potential
toxic
properties
both
untreated
or
saline
treated
and
vehicle
control
groups
are
required
(
ii)
Positive
control
data
from
the
laboratory
performing
the
testing
shall
provide
evidence
of
the
ability
of
the
observational
methods
used
to
detect
major
neurotoxic
endpoints
including
limb
weakness
or
paralysis,
tremor,
and
autonomic
signs.
Positive
control
data
are
also
required
to
demonstrate
the
sensitivity
and
reliability
of
the
activity­
measuring
device
and
testing
procedures.
These
data
should
demonstrate
the
ability
to
detect
chemically
induced
increases
and
decreases
in
activity.
Positive
control
groups
exhibiting
central
nervous
system
pathology
and
peripheral
nervous
system
pathology
are
also
required.
Separate
groups
for
peripheral
and
central
neuropathology
are
acceptable
(
e.
g.
acrylamide
and
trimethyl
tin).
Permanently
injurious
substances
need
not
be
used
for
the
behavioral
tests.
Historical
data
may
be
used
if
the
essential
aspects
of
the
experimental
procedure
remain
the
same.
Periodic
updating
of
positive
control
data
is
recommended.
New
positive
control
data
should
also
be
collected
when
personnel
or
some
other
critical
element
in
the
testing
laboratory
has
changed.
(
4)
Dose
level
and
dose
selection.
At
least
three
doses
shall
be
used
in
addition
to
the
vehicle
control
group.
The
data
should
be
sufficient
to
produce
a
dose­
effect
curve.
The
Agency
strongly
encourage
the
use
of
equally
spaced
doses
and
a
rationale
for
dose
selection
that
will
maximally
support
detection
of
dose­
effect
relations.
For
acute
studies
dose
selection
may
be
made
relative
to
the
establishment
of
a
benchmark
dose
(
BD).
That
is,
doses
may
be
specified
as
successive
fractions,
e.
g.
0.5,
0.25,
...
n
of
the
BD.
The
BD
itself
may
be
estimated
as
the
highest
nonlethal
dose
as
determined
in
a
preliminary
range­
finding
lethality
study.
A
variety
of
test
methodologies
may
be
used
for
this
purpose,
and
the
method
chosen
may
influence
subsequent
dose
selection.
The
goal
is
to
use
a
dose
level
that
is
sufficient
to
be
judged
a
limit
dose,
or
clearly
toxic.
(
i)
Acute
studies.
The
high
dose
need
not
be
greater
than
2
g/
kg.
Otherwise,
the
high
dose
should
result
in
significant
neurotoxic
effects
or
other
clearly
toxic
effects,
but
not
result
in
an
incidence
of
fatalities
that
would
preclude
a
meaningful
evaluation
of
the
data.
This
dose
may
be
estimated
by
a
BD
procedure
as
described
under
paragraph
(
e)(
4)
of
this
section,
with
the
middle
and
low
dose
levels
chosen
as
fractions
of
the
BD
dose.
The
lowest
dose
should
produce
minimal
effect,
e.
g.
an
ED10,
or
alternatively,
no
effects.
(
ii)
Subchronic
and
chronic
studies.
The
high
dose
need
not
be
greater
than
1
g/
kg.
Otherwise,
the
high
dose
level
should
result
in
significant
neurotoxic
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Environmental
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§
799.9620
effects
or
other
clearly
toxic
effects,
but
not
produce
an
incidence
of
fatalities
that
would
prevent
a
meaningful
evaluation
of
the
data.
The
middle
and
low
doses
should
be
fractions
of
the
high
dose.
The
lowest
dose
should
produce
minimal
effects,
e.
g.
an
ED10,
or
alternatively,
no
effects.
(
5)
Route
of
exposure.
Selection
of
route
may
be
based
on
several
criteria
including,
the
most
likely
route
of
human
exposure,
bioavailability,
the
likelihood
of
observing
effects,
practical
difficulties,
and
the
likelihood
of
producing
nonspecific
effects.
For
many
materials,
it
should
be
recognized
that
more
than
one
route
of
exposure
may
be
important
and
that
these
criteria
may
conflict
with
one
another.
Initially
only
one
route
is
required
for
screening
for
neurotoxicity.
The
route
that
best
meets
these
criteria
should
be
selected.
Dietary
feeding
will
generally
be
acceptable
for
repeated
exposures
studies.
(
6)
Combined
protocol.
The
tests
described
in
this
screening
battery
may
be
combined
with
any
other
toxicity
study,
as
long
as
none
of
the
requirements
of
either
are
violated
by
the
combination.
(
7)
Study
conduct
 
(
i)
Time
of
testing.
All
animals
shall
be
weighed
on
each
test
day
and
at
least
weekly
during
the
exposure
period.
(
A)
Acute
studies.
At
a
minimum,
for
acute
studies
observations
and
activity
testing
shall
be
made
before
the
initiation
of
exposure,
at
the
estimated
time
of
peak
effect
within
8
hrs
of
dosing
and
at
7
and
14
days
after
dosing.
Estimation
of
times
of
peak
effect
may
be
made
by
dosing
pairs
of
rats
across
a
range
of
doses
and
making
regular
observations
of
gait
and
arousal.
(
B)
Subchronic
and
chronic
studies.
In
a
subchronic
study,
at
a
minimum,
observations
and
activity
measurements
shall
be
made
before
the
initiation
of
exposure
and
before
the
daily
exposure,
or
for
feeding
studies
at
the
same
time
of
day,
during
the
4th,
8th,
and
13th
weeks
of
exposure.
In
chronic
studies,
at
a
minimum,
observations
and
activity
measurements
shall
be
made
before
the
initiation
of
exposure
and
before
the
daily
exposure,
or
for
feeding
studies
at
the
same
time
of
day,
every
3
months.
(
ii)
Functional
observational
battery
 
(
A)
General
conduct.
All
animals
in
a
given
study
shall
be
observed
carefully
by
trained
observers
who
are
unaware
of
the
animals'
treatment,
using
standardized
procedures
to
minimize
observer
variability.
Where
possible,
it
is
advisable
that
the
same
observer
be
used
to
evaluate
the
animals
in
a
given
study.
If
this
is
not
possible,
some
demonstration
of
interobserver
reliability
is
required.
The
animals
shall
be
removed
from
the
home
cage
to
a
standard
arena
for
observation.
Effort
should
be
made
to
ensure
that
variations
in
the
test
conditions
are
minimal
and
are
not
systematically
related
to
treatment.
Among
the
variables
that
can
affect
behavior
are
sound
level,
temperature,
humidity,
lighting,
odors,
time
of
day,
and
environmental
distractions.
Explicit,
operationally
defined
scales
for
each
measure
of
the
battery
are
to
be
used.
The
development
of
objective
quantitative
measures
of
the
observational
end­
points
specified
is
encouraged.
Examples
of
observational
procedures
using
defined
protocols
may
be
found
in
the
references
under
paragraphs
(
g)(
5),
(
g)(
6),
and
(
g)(
9)
of
this
section.
The
functional
observational
battery
shall
include
a
thorough
description
of
the
subject's
appearance,
behavior,
and
functional
integrity.
This
shall
be
assessed
through
observations
in
the
home
cage
and
while
the
rat
is
moving
freely
in
an
open
field,
and
through
manipulative
tests.
Testing
should
proceed
from
the
least
to
the
most
interactive
with
the
subject.
Scoring
criteria
or
explicitly
defined
scales,
should
be
developed
for
those
measures
which
involve
subjective
ranking.
(
B)
List
of
measures.
The
functional
observational
battery
shall
include
the
following
list
of
measures:
(
1)
Assessment
of
signs
of
autonomic
function,
including
but
not
limited
to:
(
i)
Ranking
of
the
degree
of
lacrimation
and
salivation,
with
a
range
of
severity
scores
from
none
to
severe.
(
ii)
Presence
or
absence
of
piloerection
and
exophthalmus.
(
iii)
Ranking
or
count
of
urination
and
defecation,
including
polyuria
and
diarrhea.
This
is
most
easily
conducted
during
the
open
field
assessment.

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430
40
CFR
Ch.
I
(
7
 
1
 
02
Edition)
§
799.9620
(
iv)
Pupillary
function
such
as
constriction
of
the
pupil
in
response
to
light
or
a
measure
of
pupil
size.
(
v)
Degree
of
palpebral
closure,
e.
g.,
ptosis.
(
2)
Description,
incidence,
and
severity
of
any
convulsions,
tremors,
or
abnormal
motor
movements,
both
in
the
home
cage
and
the
open
field.
(
3)
Ranking
of
the
subject's
reactivity
to
general
stimuli
such
as
removal
from
the
cage
or
handling,
with
a
range
of
severity
scores
from
no
reaction
to
hyperreactivity.
(
4)
Ranking
of
the
subject's
general
level
of
activity
during
observations
of
the
unperturbed
subject
in
the
open
field,
with
a
range
of
severity
scores
from
unresponsive
to
hyperactive.
(
5)
Descriptions
and
incidence
of
posture
and
gait
abnormalities
observed
in
the
home
cage
and
open
field.
(
6)
Ranking
of
any
gait
abnormalities
with
a
range
of
severity
scores
from
none
to
severe.
(
7)
Forelimb
and
hindlimb
grip
strength
measured
using
an
objective
procedure
(
the
procedure
described
in
the
reference
under
paragraph
(
g)(
8)
of
this
section
may
be
used).
(
8)
Quantitative
measure
of
landing
foot
splay
(
the
procedure
described
in
the
reference
under
paragraph
(
g)(
3)
of
this
section
may
be
used).
(
9)
Sensorimotor
responses
to
stimuli
of
different
modalities
will
be
used
to
detect
gross
sensory
deficits.
Pain
perception
may
be
assessed
by
a
ranking
or
measure
of
the
reaction
to
a
tailpinch
tail­
flick,
or
hot­
plate.
The
response
to
a
sudden
sound,
e.
g.,
click
or
snap,
may
be
used
to
assess
audition.
(
10)
Body
weight.
(
11)
Description
and
incidence
of
any
unusual
or
abnormal
behaviors,
excessive
or
repetitive
actions
(
stereotypies),
emaciation,
dehydration
hypotonia
or
hypertonia,
altered
fur
appearance,
red
or
crusty
deposits
around
the
eyes,
nose,
or
mouth,
and
any
other
observations
that
may
facilitate
interpretation
of
the
data.
(
C)
Additional
measures.
Other
measures
may
also
be
included
and
the
development
and
validation
of
new
tests
is
encouraged.
Further
information
on
the
neurobehavioral
integrity
of
the
subject
may
be
provided
by:
(
1)
Count
of
rearing
activity
on
the
open
field.
(
2)
Ranking
of
righting
ability.
(
3)
Body
temperature.
(
4)
Excessive
or
spontaneous
vocalizations
(
5)
Alterations
in
rate
and
ease
of
respiration,
e.
g.,
rales
or
dyspnea.
(
6)
Sensorimotor
responses
to
visual
or
proprioceptive
stimuli.
(
iii)
Motor
activity.
Motor
activity
shall
be
monitored
by
an
automated
activity
recording
apparatus.
The
device
used
must
be
capable
of
detecting
both
increases
and
decreases
in
activity
i.
e.,
baseline
activity
as
measured
by
the
device
must
not
be
so
low
as
to
preclude
detection
of
decreases
nor
so
high
as
to
preclude
detection
of
increases
in
activity.
Each
device
shall
be
tested
by
standard
procedures
to
ensure
to
the
extent
possible,
reliability
of
operation
across
devices
and
across
days
for
any
one
device.
In
addition,
treatment
groups
must
be
balanced
across
devices.
Each
animal
shall
be
tested
individually.
The
test
session
shall
be
long
enough
for
motor
activity
to
approach
asymptotic
levels
by
the
last
20%
of
the
session
for
nontreated
control
animals.
All
sessions
shall
have
the
same
duration.
Treatment
groups
shall
be
counterbalanced
across
test
times.
Effort
should
be
made
to
ensure
that
variations
in
the
test
conditions
are
minimal
and
are
not
systematically
related
to
treatment.
Among
the
variables
which
can
affect
motor
activity
are
sound
level,
size
and
shape
of
the
test
cage,
temperature,
relative
humidity
lighting
conditions,
odors,
use
of
the
home
cage
or
a
novel
test
cage,
and
environmental
distractions.
(
iv)
Neuropathology:
Collection,
processing
and
examination
of
tissue
samples.
To
provide
for
adequate
sampling
as
well
as
optimal
preservation
of
cellular
integrity
for
the
detection
of
neuropathological
alterations,
tissue
shall
be
prepared
for
histological
analysis
using
in
situ
perfusion
and
paraffin
and/
or
plastic
embedding
procedures
Paraffin
embedding
is
acceptable
for
tissue
samples
from
the
central
nervous
system.
Plastic
embedding
of
tissue
samples
from
the
central
nervous
system
is
encouraged,
when
feasible.
Plastic
embedding
is
required
for
tissue
samples
from
the
peripheral
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Environmental
Protection
Agency
§
799.9620
nervous
system.
Subject
to
professional
judgment
and
the
type
of
neuropathological
alterations
observed
it
is
recommended
that
additional
methods,
such
as
glial
fibrillary
acidic
protein
(
GFAP)
immunohistochemistry
and/
or
methods
known
as
Bodian's
or
Bielchowsky's
silver
methods
be
used
in
conjunction
with
more
standard
stains
to
determine
the
lowest
dose
level
at
which
neuropathological
alterations
are
observed
When
new
or
existing
data
provide
evidence
of
structural
alterations
it
is
recommended
that
the
GFAP
immunoassay
also
be
considered.
A
description
of
this
technique
can
be
found
in
the
reference
under
paragraph
(
g)(
10)
of
this
section.
(
A)
Fixation
and
processing
of
tissue.
The
nervous
system
shall
be
fixed
by
in
situ
perfusion
with
an
appropriate
aldehyde
fixative.
Any
gross
abnormalities
should
be
noted.
Tissue
samples
taken
should
adequately
represent
all
major
regions
of
the
nervous
system
The
tissue
samples
should
be
postfixed
and
processed
according
to
standardized
published
histological
protocols
(
protocols
described
in
the
references
under
paragraphs
(
g)(
1),
(
g)(
2),
or
(
g)(
11)
of
this
section
may
be
used).
Tissue
blocks
and
slides
should
be
appropriately
identified
when
stored.
Histological
sections
should
be
stained
for
hematoxylin
and
eosin
(
H&
E),
or
a
comparable
stain
according
to
standard
published
protocols
(
some
of
these
protocols
are
described
in
the
references
under
paragraphs
(
g)(
1)
and
(
g)(
11)
of
this
section).
(
B)
Qualitative
examination.
Representative
histological
sections
from
the
tissue
samples
should
be
examined
microscopically
by
an
appropriately
trained
pathologist
for
evidence
of
neuropathological
alterations.
The
nervous
system
shall
be
thoroughly
examined
for
evidence
of
any
treatmentrelated
neuropathological
alterations.
Particular
attention
should
be
paid
to
regions
known
to
be
sensitive
to
neurotoxic
insult
or
those
regions
likely
to
be
affected
based
on
the
results
of
functional
tests.
Such
treatment­
related
neuropathological
alterations
should
be
clearly
distinguished
from
artifacts
resulting
from
influences
other
than
exposure
to
the
test
substance.
A
stepwise
examination
of
tissue
samples
is
recommended.
In
such
a
stepwise
examination
sections
from
the
high
dose
group
are
first
compared
with
those
of
the
control
group.
If
no
neuropathological
alterations
are
observed
in
samples
from
the
high
dose
group,
subsequent
analysis
is
not
required
If
neuropathological
alterations
are
observed
in
samples
from
the
high
dose
group,
samples
from
the
intermediate
and
low
dose
groups
are
then
examined
sequentially.
(
C)
Subjective
diagnosis.
If
any
evidence
of
neuropathological
alterations
is
found
in
the
qualitative
examination
then
a
subjective
diagnosis
shall
be
performed
for
the
purpose
of
evaluating
dose­
response
relationships.
All
regions
of
the
nervous
system
exhibiting
any
evidence
of
neuropathological
changes
should
be
included
in
this
analysis.
Sections
from
all
dose
groups
from
each
region
will
be
coded
and
examined
in
randomized
order
without
knowledge
of
the
code.
The
frequency
of
each
type
and
severity
of
each
lesion
will
be
recorded.
After
all
samples
from
all
dose
groups
including
all
regions
have
been
rated,
the
code
will
be
broken
and
statistical
analysis
performed
to
evaluate
dose­
response
relationships.
For
each
type
of
dose­
related
lesion
observed,
examples
of
different
degrees
of
severity
should
be
described.
Photomicrographs
of
typical
examples
of
treatment­
related
regions
are
recommended
to
augment
these
descriptions.
These
examples
will
also
serve
to
illustrate
a
rating
scale,
such
as
1=,
2=,
and
3=
for
the
degree
of
severity
ranging
from
very
slight
to
very
extensive.
(
f)
Data
reporting
and
evaluation.
The
final
test
report
shall
include
the
following
information:
(
1)
Description
of
equipment
and
test
methods.
A
description
of
the
general
design
of
the
experiment
and
any
equipment
used
shall
be
provided.
This
shall
include
a
short
justification
explaining
any
decisions
involving
professional
judgment.
(
i)
A
detailed
description
of
the
procedures
used
to
standardize
observations
including
the
arena
and
scoring
criteria.
(
ii)
Positive
control
data
from
the
laboratory
performing
the
test
that
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432
40
CFR
Ch.
I
(
7
 
1
 
02
Edition)
§
799.9620
demonstrate
the
sensitivity
of
the
procedures
being
used.
Historical
data
may
be
used
if
all
essential
aspects
of
the
experimental
protocol
are
the
same.
Historical
control
data
can
be
critical
in
the
interpretation
of
study
findings.
The
Agency
encourages
submission
of
such
data
to
facilitate
the
rapid
and
complete
review
of
the
significance
of
effects
seen.
(
2)
Results.
The
following
information
shall
be
arranged
by
test
group
dose
level.
(
i)
In
tabular
form,
data
for
each
animal
shall
be
provided
showing:
(
A)
Its
identification
number.
(
B)
Its
body
weight
and
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.
(
ii)
Summary
data
for
each
group
must
include:
(
A)
The
number
of
animals
at
the
start
of
the
test.
(
B)
The
number
of
animals
showing
each
observation
score
at
each
observation
time.
(
C)
The
mean
and
standard
deviation
for
each
continuous
endpoint
at
each
observation
time.
(
D)
Results
of
statistical
analyses
for
each
measure,
where
appropriate.
(
iii)
All
neuropathological
observations
shall
be
recorded
and
arranged
by
test
groups.
This
data
may
be
presented
in
the
following
recommended
format:
(
A)
Description
of
lesions
for
each
animal
For
each
animal,
data
must
be
submitted
showing
its
identification
(
animal
number,
sex,
treatment,
dose,
and
duration),
a
list
of
structures
examined
as
well
as
the
locations,
nature
frequency,
and
severity
of
lesions.
Inclusion
of
photomicrographs
is
strongly
recommended
for
demonstrating
typical
examples
of
the
type
and
severity
of
the
neuropathological
alterations
observed.
Any
diagnoses
derived
from
neurological
signs
and
lesions
including
naturally
occurring
diseases
or
conditions,
should
be
recorded.
(
B)
Counts
and
incidence
of
neuropathological
alterations
by
test
group.
Data
should
be
tabulated
to
show:
(
1)
The
number
of
animals
used
in
each
group
and
the
number
of
animals
in
which
any
lesion
was
found.
(
2)
The
number
of
animals
affected
by
each
different
type
of
lesion,
the
locations
frequency,
and
average
grade
of
each
type
of
lesion.
(
3)
Evaluation
of
data.
The
findings
from
the
screening
battery
should
be
evaluated
in
the
context
of
preceding
and/
or
concurrent
toxicity
studies
and
any
correlated
functional
and
histopathological
findings.
The
evaluation
shall
include
the
relationship
between
the
doses
of
the
test
substance
and
the
presence
or
absence,
incidence
and
severity,
of
any
neurotoxic
effects.
The
evaluation
shall
include
appropriate
statistical
analyses,
for
example
parametric
tests
for
continuous
data
and
nonparametric
tests
for
the
remainder.
Choice
of
analyses
should
consider
tests
appropriate
to
the
experimental
design,
including
repeated
measures.
There
may
be
many
acceptable
ways
to
analyze
data.
(
g)
References.
For
additional
background
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)
Bennet,
H.
S.
et
al.
Science
and
art
in
the
preparing
tissues
embedded
in
plastic
for
light
microscopy,
with
special
reference
to
glycol
methacrylate,
glass
knives
and
simple
stains.
Stain
Technology.
51:
71
 
97
(
1976).
(
2)
Di
Sant
Agnese,
P.
A.
and
De
Mesy
Jensen
K.
Dibasic
staining
of
large
epoxy
sections
and
application
to
surgical
pathology.
American
Journal
of
Clinical
Pathology.
81:
25
 
29
(
1984).
(
3)
Edwards,
P.
M.
and
Parker,
V.
H.
A
simple
sensitive
and
objective
method
for
early
assessment
of
acrylamide
neuropathy
in
rats.
Toxicology
and
Applied
Pharmacology.
40:
589
 
591
(
1977).
(
4)
Finger,
F.
W.
Ed.
Myers,
R.
D.
Measuring
Behavioral
Activity.
Vol.
2.
Methods
in
Psychobiology
(
Academic,
NY,
1972)
pp.
1
 
19.
(
5)
Gad,
S.
A
neuromuscular
screen
for
use
in
industrial
toxicology.
Journal
of
Toxicology
and
Environmental
Health.
9:
691
 
704
(
1982).
(
6)
Irwin,
S.
Comprehensive
observational
assessment:
Ia.
A
systematic
quantitative
procedure
for
assessing
the
behavioral
physiological
state
of
the
mouse.
Psychopharmacologia.
13:
222
 
257
(
1968).

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433
Environmental
Protection
Agency
§
799.9630
(
7)
Kinnard,
E.
J.
and
Watzman,
N.
Techniques
utilized
in
the
evaluation
of
psychotropic
drugs
on
animals
activity.
Journal
of
Pharmaceutical
Sciences.
55:
995
 
1012
(
1966).
(
8)
Meyer,
O.
A.
et
al.
A
method
for
the
routine
assessment
of
fore­
and
hindlimb
grip
strength
of
rats
and
mice.
Neurobehavioral
Toxicology.
1:
233
 
236
(
1979).
(
9)
Moser
V.
C.
et
al.
Comparison
of
chlordimeform
and
carbaryl
using
a
functional
observational
battery.
Fundamental
and
Applied
Toxicology.
11:
189
 
206
(
1988).
(
10)
O'Callaghan,
J.
P.
Quantification
of
glial
fibrillary
acidic
protein:
Comparison
of
slot­
immunobinding
assays
with
a
novel
sandwich
ELISA.
Neurotoxicology
and
Teratology
13:
275
 
281
(
1991).
(
11)
Pender,
M.
P.
A
simple
method
for
high
resolution
light
microscopy
of
nervous
tissue
Journal
of
Neuroscience
Methods.
15:
213
 
218
(
1985).
(
12)
Reiter,
L.
W.
Use
of
activity
measures
in
behavioral
toxicology.
Environmental
Health
Perspectives.
26:
9
 
20
(
1978).
(
13)
Reiter,
L.
W.
and
MacPhail,
R.
C.
Motor
activity:
A
survey
of
methods
with
potential
use
in
toxicity
testing.
Neurobehavorial
Toxicology
1
 
Supplement.
1:
53
 
66
(
1979).
(
14)
Robbins,
T.
W.
Eds.
Iversen,
L.
L.,
Iverson,
D.
S.,
and
Snyder,
S.
H.
A
critique
of
the
methods
available
for
the
measurement
of
spontaneous
motor
activity.
Vol
7.
Handbook
of
Psychopharmacology
(
Plenum,
NY,
1977)
pp.
37
 
82.

[
62
FR
43824,
Aug.
15,
1997,
as
amended
at
64
FR
35080,
June
30,
1999]

§
799.9630
TSCA
developmental
neurotoxicity.

(
a)
Scope
 
(
1)
Applicability.
This
section
is
intended
to
meet
the
testing
requirements
under
section
4
of
the
Toxic
Substances
Control
Act
(
TSCA).
(
2)
Source.
The
source
material
used
in
developing
this
TSCA
test
guideline
is
the
OPPTS
harmonized
test
guideline
870.6300
(
August
1998).
(
b)
Purpose.
In
the
assessment
and
evaluation
of
the
toxic
characteristics
of
a
chemical
substance
or
mixture
(
test
substance),
determination
of
the
potential
for
developmental
neurotoxicity
is
important.
This
study
is
designed
to
develop
data
on
the
potential
functional
and
morphological
hazards
to
the
nervous
system
which
may
arise
in
the
offspring
from
exposure
of
the
mother
during
pregnancy
and
lactation.
(
c)
Principle
of
the
test
method.
The
test
substance
is
administered
to
several
groups
of
pregnant
animals
during
gestation
and
early
lactation,
one
dose
level
being
used
per
group.
Offspring
are
randomly
selected
from
within
litters
for
neurotoxicity
evaluation.
The
evaluation
includes
observations
to
detect
gross
neurologic
and
behavioral
abnormalities,
determination
of
motor
activity,
response
to
auditory
startle,
assessment
of
learning,
neuropathological
evaluation,
and
brain
weights.
This
protocol
may
be
used
as
a
separate
study,
as
a
followup
to
a
standard
developmental
toxicity
and/
or
adult
neurotoxicity
study,
or
as
part
of
a
two­
generation
reproduction
study,
with
assessment
of
the
offspring
conducted
on
the
second
(
F2)
generation
(
d)
Test
procedure
 
(
1)
Animal
selection
 
(
i)
Species
and
strain.
Testing
must
be
performed
in
the
rat.
Because
of
its
differences
in
timing
of
developmental
events
compared
to
strains
that
are
more
commonly
tested
in
other
developmental
and
reproductive
toxicity
studies,
it
is
preferred
that
the
Fischer
344
strain
not
be
used.
If
a
sponsor
wishes
to
use
the
Fischer
344
rat
or
a
mammalian
species
other
than
the
rat,
ample
justification/
reasoning
for
this
selection
must
be
provided.
(
ii)
Age.
Young
adult
(
nulliparous
females
animals
must
be
used.
(
iii)
Sex.
Pregnant
female
animals
must
be
used
at
each
dose
level.
(
iv)
Number
of
animals.
(
A)
The
objective
is
for
a
sufficient
number
of
pregnant
rats
to
be
exposed
to
the
test
substance
to
ensure
that
an
adequate
number
of
offspring
are
produced
for
neurotoxicity
evaluation.
At
least
20
litters
are
recommended
at
each
dose
level.
(
B)
On
postnatal
day
4,
the
size
of
each
litter
should
be
adjusted
by
eliminating
extra
pups
by
random
selection
to
yield,
as
nearly
as
possible,
four
male
and
four
females
per
litter.
Whenever
the
number
of
pups
of
either
sex
prevents
having
four
of
each
sex
per
litter,
partial
adjustment
(
for
example,
five
males
and
three
females)
is
permitted
Testing
is
not
appropriate
for
litters
of
less
than
seven
pups.
Elimination
of
runts
only
is
not
appropriate.
Individual
pups
should
be
identified
uniquely
after
standardization
of
litters
A
method
that
may
be
used
for
identification
can
be
found
under
paragraph
(
f)(
1)
of
this
section.

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