[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2005]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR795.250]
[Page 75-80]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 795_PROVISIONAL TEST GUIDELINES--Table of Contents
Subpart D_Provisional Health Effects Guidelines
Sec. 795.250 Developmental neuro tox icity screen.
(a) Purpose. In the assessment and evaluation of the toxic
characteristics of a chemical, it is important to determine when
acceptable exposures in the adult may not be acceptable to a developing
organism. This test is designed to provide information on the potential
functional and morphologic hazards to the nervous system which may arise
in the offspring from exposure of the mother during pregnancy and
lactation.
(b) Principle of the test method. The test substance is administered
to several groups of pregnant animals during gestation and lactation,
one dose level being used per group. Offspring are randomly selected
from within litters for neurotoxicity evaluation. The evaluation
includes observation to detect gross neurological and behavioral
abnormalities, determination of motor activity, neuropathological
evaluation, and brain weights. Measurements are carried out periodically
during both postnatal development and adulthood.
(c) Test procedures--(1) Animal selection--(i) Species and strain.
Testing should be performed in the Sprague Dawley rat.
(ii) Age. Young adult animals (nulliparous females) shall be used.
(iii) Sex. Pregnant females shall be used at each dose level.
(iv) Number of animals. The objective is for a sufficient number of
pregnant rats to be exposed to ensure that an adequate number of
offspring are produced for neurotoxicity evaluation. At least 20 litters
are recommended at each dose level. This number assumes a coefficient of
variation of 20 to 25 percent for most behavioral tests. If, based upon
experience with historical control data or data for positive controls in
a given laboratory, the coefficient of variation for a given task is
higher than 20 to 25 percent, then calculation of appropriate sample
sizes to detect a 20 percent change from control values with 80 percent
power would need to be done. For most designs, calculations can be made
according to Dixon and Massey (1957) under paragraph (e)(5) of this
section, Neter and Wasserman (1974) under paragraph (e)(10) of this
section, Sokal and Rohlf (1969) under paragraph (e)(11) of this section,
or Jensen (1972) under paragraph (e)(8) of this section.
(A) On day 4 after birth, the size of each litter should be adjusted
by eliminating extra pups by random selection to yield, as nearly as
possible, 4 males and 4 females per litter. Whenever the number of male
or female pups prevents having 4 of each sex per litter, partial
adjustment (for example, 5 males and 3 females) is permitted.
Adjustments are not appropriate for litters of less than 8 pups.
Elimination of runts only is not appropriate. Individual pups should be
identified uniquely after standardization of litters. A method that may
be used can be found in Adams et al. (1985) under paragraph (e)(1) of
this section.
(B) After standardization of litters, males and females shall be
randomly assigned to one of each of three behavioral tasks.
Alternatively, more than one of the behavioral tasks may be conducted in
the same animal. In the
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latter case, a minimum of 1 to 2 days should separate the tests when
conducted at about the same age.
(C) One male and one female shall be randomly selected from each
litter for sacrifice at weaning as specified in paragraph (c)(8) of this
section.
(2) Control group. A concurrent control group shall be used. This
group shall be a sham treated group, or, if a vehicle is used in
administering the test substance, a vehicle control group. Animals in
the control groups shall be handled in an identical manner to test group
animals. The vehicle shall neither be developmentally toxic nor have
effects on reproduction.
(3) Dose levels and dose selection. (i) At least 3 dose levels plus
a control (vehicle control, if a vehicle is used) shall be used.
(ii) If the substance has been shown to be developmentally toxic
either in a standard developmental toxicity study or a pilot study, the
highest dose level shall be the maximum dose which will not induce in
utero or neonatal deaths or malformations sufficient to preclude a
meaningful evaluation of neu ro toxicity.
(iii) In the absence of standard developmental toxicity, unless
limited by the physicochemical nature or biologicial properties of the
substance, the highest dose level shall induce some overt maternal
toxicity but shall not result in a reduction in weight gain exceeding 20
percent during gestation and lactation.
(iv) The lowest dose should not produce any grossly observable
evidence of either maternal or developmental neurotoxicity.
(v) The intermediate dose(s) shall be equally spaced between the
highest and lowest dose.
(4) Dosing period. Day 0 in the test is the day on which a vaginal
plug and/or sperm are observed. The dose period shall cover the period
from day 6 of gestation through weaning (21 days postnatally).
(5) Administration of test substance. The test substance or vehicle
should be administered orally by intubation. The test substance shall be
administered at the same time each day. The animals shall be weighed
periodically and the dosage based on the most recent weight
determination.
(6) Observation of dams. (i) A gross examination of the dams shall
be made at least once each day, before daily treatment. The animals
shall be observed by trained technicians who are blind with respect to
the animal's treatment, using standardized procedures to maximize inter-
observer reliability. 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 inter-observer reliability is
required.
(ii) During the treatment and observation periods, cage-side
observations shall include:
(A) Any responses with respect to body position, activity level,
coordination of movement, and gait.
(B) Any unusual or bizarre behavior including, but not limited to
headflicking, head searching, compulsive biting or licking, self-
mutilation, circling, and walking backwards.
(C) The presence of:
(1) Convulsions.
(2) Tremors.
(3) Increased levels of lacrimation and/or red-colored tears.
(4) Increased levels of salivation.
(5) Piloerection.
(6) Pupillary dilation or constriction.
(7) Unusual respiration (shallow, labored, dyspneic, gasping, and
retching) and/or mouth breathing.
(8) Diarrhea.
(9) Excessive or diminished urination.
(10) Vocalization.
(iii) Signs of toxicity shall be recorded as they are observed,
including the time of onset, the degree and duration.
(iv) Animals shall be weighed at least weekly.
(v) The day of delivery of litters shall be recorded.
(7) Study conduct--(i) Observation of offspring. (A) All offspring
shall be examined cage-side daily for gross signs of mortality and
morbidity.
(B) All offspring shall be examined outside the cage for gross signs
of toxicity whenever they are weighed or removed from their cages for
behavioral testing. The offspring shall be observed by trained
technicians, who are blind with respect to the animal's treatment
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using standardized procedures to maximize inter-observer reliability.
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 inter-observer reliability is required. At a minimum,
the end points outlined in paragraph (c)(6)(ii) of this section shall be
monitored as appropriate for the developmental stage being observed.
(C) Any gross signs of toxicity in the offspring shall be recorded
as they are observed, including the time of onset, the degree, and
duration.
(ii) Developmental landmarks. Live pups should be counted and
litters weighed by weighing each individual pup at birth, or soon
thereafter, and on days 4, 7, 13, 17, and 21, and biweekly thereafter.
The age of the pups at the time of the appearance of the following
developmental landmarks shall be determined:
(A) Vaginal opening. General procedure for this determination may be
found in Adams et al. (1985) under paragraph (e)(1) of this section.
(B) Testes descent. General procedure for this determination may be
found in Adams et al. (1985) under paragraph (e)(1) of this section.
(iii) Motor activity. (A) Motor activity shall be monitored
specifically on days 13, 17, 21, 45 (2 days), and
60 (2 days). Motor activity shall be monitored by
an automated activity recording apparatus. The device used shall be
capable of detecting both increases and decreases in activity, i.e.,
baseline activity as measured by the device shall not be so low as to
preclude decreases nor so high as to preclude increases. Each device
shall be tested by standard procedures to ensure, to the extent
possible, reliability of operation across devices and testing of animals
within dose groups shall be balanced across devices.
(B) Each animal shall be tested individually. The test session shall
be long enough to demonstrate habituation of motor activity in control
animals, i.e., to approach asymptotic levels by the last 20 percent of
the session. Animals' activity counts shall be collected in equal time
periods of no greater than 10 minutes duration. All sessions shall have
the same duration. Treatment groups shall be counter-balanced across
test times.
(C) Efforts shall 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 home cage or novel test cage, and
environmental distractions.
(D) Additional information on the conduct of a motor activity study
may be obtained in the TSCA motor activity guideline, in Sec. 798.6200
of this chapter.
(iv) Auditory startle test. An auditory startle habituation test
shall be performed on the offspring on days 22 and 60. Details on the
conduct of this testing may be obtained in Adams et al. (1985) under
paragraph (e)(1) of this section. In performing the auditory startle
task, the mean response amplitude on each block of 10 trials (5 blocks
of 10 trials per session on each day of testing) shall be made. While
use of pre-pulse inhibition is not a requirement, it may be used at the
discretion of the investigator. Details on the conduct of this testing
may be obtained from Ison (1984) under paragraph (e)(7) of this section.
(v) Active avoidance test. Active avoidance testing shall be
conducted beginning at 60 to 61 days of age. Details on the apparatus
may be obtained in Brush and Knaff (1959) and on the conduct of testing
from Brush (1962), under paragraphs (e)(2) and (e)(4) of this section,
respectively; reviews on active avoidance conditioning by Brush (1971)
and McAllister and McAllister (1971) can be found under paragraphs
(e)(3) and (e)(9) of this section, respectively. In performing the
active avoidance task, the following measures should be made:
(A) Mean number of shuttles during the adaptation period preceding
each daily session.
(B) Mean number and latency of avoidances per session, presented in
blocks of 10 trials (2 blocks of 10 trials per session across 5
sessions).
(C) Mean number and latency of escapes per session, presented in
blocks of 10 trials as above.
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(D) Mean duration of shocks per session, presented in blocks of 10
trials as above.
(E) Mean number of shuttles during the inter-trial intervals.
(8) Post-mortem evaluation--(i) Age of animals. One male and one
female per litter shall be sacrificed at weaning and the remainder
following the last behavioral measures. Neuropathology and brain weight
determinations shall be made on animals sacrificed at weaning and after
the last behavioral measures.
(ii) Neuropathology. Details for the conduct of neuropathology
evaluation may be obtained in the TSCA neuropathology guideline, in
Sec. 798.6400 of this chapter. At least 6 offspring per dose group
shall be randomly selected from each sacrificed group (weaning and
adulthood) for neuropathologic evaluation. These animals shall be
balanced across litters, and equal numbers of males and females shall be
used. The remaining sacrificed animals shall be used to determine brain
weight. Animals shall be perfused in situ by a generally recognized
technique. After perfusion, the brain and spinal cord shall be removed
and gross abnormalities noted. Cross-sections of the following areas
shall be examined: The forebrain, the center of the cerebrum and
midbrain, the cerebellum and pons, and the medulla oblongata; the spinal
cord at cervical and lumbar swelling; Gasserian ganglia, dorsal root
ganglia, dorsal and ventral root fibers, proximal sciatic nerve (mid-
thigh and sciatic notch), sural nerve (at knee), and tibial nerve (at
knee). Tissue samples from both the central and peripheral nervous
system shall be further immersion-fixed and stored in appropriate
fixative for further examination. After dehydration, tissue specimens
shall be cleared with xylene and embedded in paraffin or paraplast
except for the sural nerve which should be embedded in plastic. A method
for plastic embedding is described by Spencer et al. under paragraph
(e)(12) of this section. Tissue sections shall be prepared from the
tissue blocks. The following general testing sequence is recommended for
gathering histopathological data:
(A) General staining. A general staining procedure shall be
performed on all tissue specimens in the highest treatment group.
Hematoxylin and eosin (H&E) shall be used for this purpose. The staining
shall be differentiated properly to achieve bluish nuclei with pinkish
background.
(B) Special stains. Based on the results of the general staining,
selected sites and cellular components shall be further evaluated by use
of specific techniques. If H&E screening does not provide such
information, a battery of stains shall be used to assess the following
components in all appropriate required samples: Neuronal body (e.g.,
Einarson's gallocyanin), axon (e.g., Kluver's Luxol Fast Blue), and
neurofibrils (e.g., Bielchosky). In addition, nerve fiber teasing shall
be used. A section of normal tissue shall be included in each staining
to assure that adequate staining has occurred. Any changes shall be
noted and representative photographs shall be taken. If lesions are
observed, the special techniques shall be repeated in the next lower
treatment group until no further lesions are detectable.
(C) Alternative technique. If the anatomical locus of expected neu
ro path ology is well-defined, epoxy-embedded sections stained with
toluidine blue may be used for small sized tissue samples. This
technique obviates the need for special stains.
(iii) Brain weight. At least 10 animals that are not sacrificed for
histo pathology shall be used to determine brain weight. The animals
shall be decapitated and the brains carefully removed, blotted, chilled,
and weighed. The following dissection shall be performed on an ice-
cooled glass plate: First, the rhombencephalon is separated by a
transverse section from the rest of the brain and dissected into the
cerebellum and the medulla oblongata/pons. A transverse section is made
at the level of the ``optic chiasma'' which delimits the anterior part
of the hypothalamus and passes through the anterior commissure. The
cortex is peeled from the posterior section and added to the anterior
section. This divides the brain into four sections, the telencephalon,
the diencephalon/mid-brain, the medulla oblongata/pons, and the
cerebellum. Sections shall be
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weighed as soon as possible after dissection to avoid drying. Detailed
methodology is available in Glowinski and Iversen (1966) under paragraph
(e)(6) of this section.
(d) Data reporting and evaluation. In addition to the reporting
requirements specified in part 792, subpart J of this chapter, the final
test report shall include the following information.
(1) Description of system and test methods. (i) A detailed
description of the procedures used to standardize observation and
operational definitions for scoring observations.
(ii) Positive control data from the laboratory performing the test
that demonstrate the sensitivity of the procedures being used. These
data do not have to be from studies using prenatal exposures. However,
the laboratory must demonstrate competence in testing neonatal animals
perinatally exposed to chemicals and establish test norms for the
appropriate age group.
(iii) Procedures for calibrating and assuring the equivalence of
devices and balancing treatment groups.
(iv) A short justification explaining any decisions where
professional judgement is involved such as fixation technique and choice
of stains.
(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 and litter from which it came.
(B) Its body weight and score on each developmental landmark at each
observation time; total session activity counts and intrasession
subtotals on each day measured; auditory startle response magnitude
session counts and intrasession subtotals on each day measured;
avoidance session counts and intrasession counts on each day measured;
time and cause of death (if appropriate); locations, nature or
frequency, and severity of the lesions; total brain weight; absolute
weight of each of the four sections; and weight of each section as a
percentage of total brain weight. A commonly used scale such as 1+, 2+,
3+, and 4+ for degree of severity of lesions ranging from very slight to
extensive may be used for morphologic evaluation. Any diagnoses derived
from neurologic signs and lesions, including naturally occurring
diseases or conditions, shall also be recorded.
(ii) Summary data for each group shall include:
(A) The number of animals at the start of the test.
(B) Body weights of the dams during gestation and lactation.
(C) Litter size and mean weight at birth.
(D) The number of animals showing each observation score at each
observation time.
(E) The percentage of animals showing each abnormal sign at each
observation time.
(F) The mean and standard deviation for each continuous end point at
each observation time. These will include body weight, motor activity
counts, acoustic startle responses, performance in active avoidance
tests, and brain weights (both absolute and relative).
(G) The number of animals in which any lesion was found.
(H) The number of animals affected by each different type of lesion,
the average grade of each type of lesion, and the frequency of each
different type and/or location of lesions.
(3) Evaluation of data. An evaluation of the test results shall be
made. The evaluation shall include the relationship between the doses of
the test substance and the presence or absence, incidence, and severity
of any neurotoxic effect. The evaluation shall include appropriate
statistical analyses. The choice of analyses shall consider tests
appropriate to the experimental design and needed adjustments for
multiple comparisons.
(e) References. For additional background information on this test
guideline, the following references should be consulted:
(1) Adams, J., Buelke-Sam, J., Kimmel, C.A., Nelson, C.J., Reiter,
L.W., Sobotka, T.J., Tilson, H.A., and Nelson, B.K. ``Collaborative
behavioral teratology study: Protocol design and testing procedure.''
Neurobehavioral Toxicology and Teratology. 7: 579-586. (1985).
(2) Brush, F.R. ``The effects of inter-trial interval on avoidance
learning in the rat.'' Journal of Comparative Physiology and Psychology.
55: 888-892. (1962).
[[Page 80]]
(3) Brush, F.R. ``Retention of aversively motivated behavior.'' In:
``Adverse Conditioning and Learning.'' Brush, F.R., ed., New York:
Academic Press. (1971).
(4) Brush, F.R. and Knaff, P.R. ``A device for detecting and
controlling automatic programming of avoidance-conditioning in a
shuttle-box.'' American Journal of Psychology. 72: 275-278 (1959).
(5) Dixon, W.J. and Massey, E.J. ``Introduction to Statistical
Analysis.'' 2nd ed. New York: McGraw-Hill. (1957).
(6) Glowinski, J. and Iversen, L.L. ``Regional studies of
catecholamines in the rat brain-I.'' Journal of Neurochemistry. 13: 655-
669. (1966).
(7) Ison, J.R. ``Reflex modification as an objective test for
sensory processing following toxicant exposure.'' Neu ro be havioral
Toxicology and Teratology. 6: 437-445. (1984).
(8) Jensen, D.R. ``Some simultaneous multivariate procedures using
Hotel ling's T2 Statistics.'' Biometrics. 28: 39-53. (1972).
(9) McAllister, W.R. and McAllister, D.E. ``Behavioral measurement
of conditioned fear.'' In: ``Adverse Conditioning and Learning.'' Brush,
F.R., ed., New York: Academic Press (1971).
(10) Neter, J. and Wasserman, W. ``Applied Linear Statistical
Models.'' Homewood: Richard D. Irwin, Inc. (1974).
(11) Sokal, R.P. and Rohlf, E.J. ``Biometry.'' San Francisco: W.H.
Freeman and Co. (1969).
(12) Spencer, P.S., Bischoff, M.C., and Schaumburg, H.H.,
``Neuropathological methods for the detection of neurotoxic disease.''
In: ``Experimental and Clinical Neurotoxicology.'' Spencer, P.S. and
Schaumburg, H.H., eds., Baltimore, MD: Williams & Wilkins, pp. 743-757.
(1980).
[53 FR 5957, Feb. 26, 1988]