United States Prevention, Pesticides EPA712-C-96-239
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.6300
Developmental
Neurotoxicity Study
"Public Draft'
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INTRODUCTION
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7U.S.C. I36,etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the
OPP Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0132 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 870.6300 Developmental neurotoxicity study.
(a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline is OPP 83-6 Postnatal Developmental Tox-
icity (Pesticide Assessment Guidelines, Subdivision F—Hazard Evalua-
tion; Human and Domestic Animals) EPA report 540/09-82-025, 1982.
(b) Purpose. In the assessment and evaluation of the toxic character-
istics 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 expo-
sure 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 observa-
tions to detect gross neurologic and behavioral abnormalities, determina-
tion 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 tox-
icity and/or adult neurotoxicity study, or as part of a two-generation repro-
duction study, with assessment of the offspring conducted on the second
(p2) generation.
(d) Test procedure—(1) Animal selection—(i) Species and strain.
Testing should be performed in the rat. Because of its differences in timing
of developmental events compared to strains that are more commonly test-
ed 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 justifica-
tion/reasoning for this selection must be provided.
(ii) Age. Young adult (nulliparous females) animals should be used.
(iii) Sex. Pregnant female animals should 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 ade-
quate number of offspring are produced for neurotoxicity evaluation. At
least 20 litters are recommended at each dose level. For behavioral tests,
one female and one male pup per litter should be randomly selected and
assigned to one of the tests.
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(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)(l) of this guideline.
(v) Assignment of animals for behavioral tests, brain weights, and
neuropathological evaluations. After standardization of litters, one male
and one female from each litter should be randomly assigned to one of
the following tests: Motor activity, auditory startle, and learning and mem-
ory, in weanling and adult animals. On postnatal-day-11, either 1 male
or 1 female pup from each litter (total of 10 males and 10 females per
dose group) should be sacrificed. Brain weights should be measured in
all of these pups and, of these pups, six per sex per dose should be selected
for neuropathological evaluation. At the termination of the study, either
1 male or 1 female from each litter (total of 10 males and 10 females
per dose group) should be sacrificed and brain weights should be meas-
ured. An additional group of six animals per sex per dose group (one male
or one female per litter) should be sacrificed at the termination of the
study for neuropathological evaluation.
(2) Control groups. Concurrent control groups are required. This
group should be a sham-treated group or, if a vehicle is used in administer-
ing the test substance, a vehicle control group. The vehicle should neither
be developmentally toxic nor have effects on reproduction. Animals in
the control groups should be handled in an identical manner to test group
animals.
(3) Dose levels and dose selection, (i) At least three dose levels of
the test substance plus a control group (vehicle control, if a vehicle is
used) should be used.
(ii) If the test substance has been shown to be developmentally toxic
either in a standard developmental toxicity study or in a pilot study, the
highest dose level should be the maximum dose which will not induce
in utero or neonatal death or malformations sufficient to preclude a mean-
ingful evaluation of neurotoxicity.
(iii) If a standard developmental toxicity study has not been con-
ducted, the highest dose level, unless limited by the physicochemical na-
ture or biological properties of the substance, should induce some overt
maternal toxicity, but should not result in a reduction in weight gain ex-
ceeding 20 percent during gestation and lactation.
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(iv) The lowest dose should not produce any grossly observable evi-
dence of either maternal or developmental neurotoxicity.
(v) The intermediate doses should be equally spaced between the
highest and lowest doses used.
(4) Dosing period. Day-0 of gestation is the day on which a vaginal
plug and/or sperm are observed. The dosing period should cover the period
from day-6 of gestation through day- 10 postnatally. Dosing should not
occur on the day of parturition in those animals who have not completely
delivered their offspring.
(5) Administration of the test substance. The test substance or vehi-
cle should be administered orally. Other routes of administration may be
acceptable, on a case-by case basis, with ample justification/reasoning for
this selection. The test substance or vehicle should be administered based
on the most recent weight determination.
(6) Observation of dams, (i) A gross examination of the dams should
be made at least once each day before daily treatment. The animals should
be observed by trained technicians who are unaware of the animals' treat-
ment, using standardized procedures to maximize interobserver 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 interobserver reliability is required.
(ii) During the treatment and observation periods, observations should
include:
(A) Assessment of signs of autonomic function, including but not lim-
ited to:
(7) Ranking of the degree of lacrimation and salivation, with a range
of severity scores from none to severe.
(2) Presence of absence of piloerection and exophthalmus.
Ranking or count of urination and defecation, including polyuria
and diarrhea.
(4) Pupillary function such as constriction of the pupil in response
to light or a measure of pupil size.
(5) Degree of palpebral closure, e.g., ptosis.
(B) Description, incidence, and severity of any convulsions, tremors,
or abnormal movements.
(C) Description and incidence of posture and gait abnormalities.
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(D) 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 fa-
cilitate interpretation of the data.
(iii) Signs of toxicity should be recorded as they are observed, includ-
ing the time of onset, degree, and duration.
(iv) Animals should be weighed at least weekly and on the day of
delivery and postnatal-day-11 and -21 (weaning) and such weights should
be recorded.
(v) The day of delivery of litters should be recorded and considered
as postnatal-day-0.
(7) Study conduct—(i) Observation of offspring. (A) All offspring
should be examined cage-side at least daily for gross signs of mortality
or morbidity.
(B) All offspring should be examined outside the cage for gross signs
of toxicity whenever they are weighed or removed from their cages for
behavioral testing. The offspring should be observed by trained techni-
cians, who are unaware of the treatment being used, using standardized
procedures to maximize interobserver reliability. Where possible, it is ad-
visable that the same observer be used to evaluate the animals in a given
study. If this is not possible, some demonstration of interobserver reliabil-
ity is required. At a minimum, the end points outlined in paragraph
(d)(6)(ii) of this guideline should be monitored as appropriate for the de-
velopmental stage being observed.
(C) Any gross signs of toxicity in the offspring should be recorded
as they are observed, including the time of onset, degree, and duration.
(ii) Developmental landmarks. Live pups should be counted and
each pup within a litter should be weighed individually at birth or soon
thereafter, and on postnatal-day-4, -11, -17, and -21 and at least once
every 2 weeks thereafter. The age of vaginal opening and preputial separa-
tion should be determined. General procedures for these determinations
may be found in paragraphs (f)(l) and (f)(ll) of this guideline.
(iii) Motor activity. Motor activity should be monitored specifically
on postnatal-day-13, -17, -21, and -60 (±2 days). Motor activity must
be monitored by an automated activity recording apparatus. The device
must be capable of detecting both increases and decreases in activity, (i.e.,
baseline activity as measured by the device must be capable of detecting
both increases and decreases nor so high as to preclude detection of in-
creases in activity). Each device should be tested by standard procedures
to ensure, to the extent possible, reliability of operation across devices
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and across days for any one device. In addition, treatment groups must
be balanced across devices. Each animal should be tested individually. The
test session should be long enough for motor activity to approach asymp-
totic levels by the last 20 percent of the session for nontreated control
animals. All sessions should have the same duration. Treatment groups
should be counter-balanced across test times. Activity counts should be
collected in equal time periods of no greater than 10 minutes duration.
Efforts should be made to ensure that variations in the test conditions are
minimal and are not systematically related to treatment. Among the vari-
ables that can affect motor activity are sound level, size and shape of the
test cage, temperature, relative humidity, light conditions, odors, use of
home cage or novel test cage, and environmental distractions. Additional
information on the conduct of a motor activity study may be obtained
in OPPTS 870.6200.
(iv) Auditory startle test. An auditory startle habituation test should
be performed on the offspring on day-22 and -60 + 2. Details on the con-
duct of this testing may be obtained under paragraph (f)(l) of this guide-
line. 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) should be made. While use of prepulse inhibition is not a re-
quirement, it is highly recommended. Details on the conduct of this test
may be obtained in paragraph (f)(10) of this guideline
(v) Learning and memory tests. A test of associative learning and
memory should be conducted around the time of weaning (postnatal-day-
21 to day-24) and at adulthood (postnatal-day-60 + 2). The same or sepa-
rate tests may be used at these two stages of development. Some flexibility
is allowed in the choice of tests for learning and memory in weanling
and adult rats. However, the tests must be designed so as to fulfill two
criteria. First, learning must be assessed either as a change across several
repeated learning trials or sessions, or, in tests involving a single trial,
with reference to a condition that controls for nonassociative effects of
the training experience. Second, the tests should include some measure
of memory (short-term or long-term) in addition to original learning (ac-
quisition), but note that this measure of memory cannot be reported in
the absence of a measure of acquisition obtained from the same test. If
the tests of learning and memory reveals an effect of the test compound,
it may be in the best interest of the sponsor to conduct additional tests
to rule out alternative interpretations based on alterations in sensory, moti-
vational, and/or motor capacities. In addition to the above two criteria,
it is recommended that the test of learning and memory be chosen on
the basis of its demonstrated sensitivity to the class of compound under
investigation, if such information is available in the literature. In the ab-
sence of such information, examples of tests that could be made to meet
the above criteria include: Delayed-matching-to-position, as described for
the adult rat (see paragraph (f)(3) of this guideline) and for the infant
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rat (see paragraph (f)(9) of this guideline); olfactory conditioning, as de-
scribed in paragraph (f)(13) of this guideline; and acquisition and retention
of schedule-controlled behavior (see paragraphs (f)(4) and (f)(5) of this
guideline). Additional tests for weanling rats are described under para-
graphs (f)(20) and (f)(12) of this guideline, and for adult rats under para-
graph (f)(16) of this guideline.
(vi) Neuropathology. Neuropathological evaluation should be con-
ducted on animals on postnatal-day-11 and at the termination of the study.
At 11 days of age, one male or female pup should be removed from each
litter such that equal numbers of male and female offspring are removed
from all litters combined. Of these, six male and six female pups will
be sacrificed for neuropathological analysis. The pups will be killed by
exposure to carbon dioxide and immediately thereafter the brains should
be removed, weighed, and immersion-fixed in an appropriate aldehyde fix-
ative. The remaining animals will be sacrificed in a similar manner and
immediately thereafter their brains removed and weighed. At the termi-
nation of the study, one male or one female from each litter will be killed
by exposure to carbon dioxide and immediately thereafter the brain should
be removed and weighed. In addition, six animals sex per dose group (one
male or female per litter) should be sacrificed at the termination of the
study for neuropathological evaluation. Neuropathological analysis of ani-
mals sacrificed at the termination of the study should be performed in
accordance with the OPPTS 870.6200. Neuropathological evaluation of
animals sacrificed on postnatal-day-11 and at termination of the study
should include a qualitative analysis and semiquantitative analysis as well
as simple morphometrics.
(A) Fixation and processing of tissue samples for postnatal-day-
11 animals. Immediately following removal, the brain should be weighed
and immersion fixed in an appropriate aldehyde fixative. The brains should
be postfixed and processed according to standardized published histo-
logical protocols under paragraphs (f)(6), (f)(14), (f)(17), and (f)(21) of
this guideline. Paraffin embedding is acceptable but plastic embedding is
preferred and recommended. Tissue blocks and slides should be appro-
priately identified when stored. Histological sections should be stained for
hematoxylin and eosin, or a similar stain according to standard published
protocols under paragraphs (f)(2), (f)(14), and (f)(18) of this guideline.
For animals sacrificed at the termination of the study, methods for fixation
and processing of tissue samples are provided in paragraph (e)(4)(iv)(A)
of OPPTS 870.6200.
(B) Qualitative analysis. The purposes of the qualitative examination
are threefold—to identify regions within the nervous system exhibiting evi-
dence of neuropathological alterations, to identify types of neuropatholo-
gical alterations resulting from exposure to the test substance, and to deter-
mine the range of severity of the neuropathological alterations. Representa-
tive histological sections from the tissue samples should be examined mi-
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croscopically by an appropriately trained pathologist for evidence of
neuropathological alterations. The following stepwise procedure is rec-
ommended for the qualitative analysis. First, sections from the high dose
group are compared with those of the control group. If no evidence of
neuropathological alterations is found in animals of the high dose group,
no further analysis is required. If evidence of neuropathological alterations
are found in the high dose group, then animals from the intermediate and
low dose group are examined. Subject to professional judgment and the
kind of neuropathological alterations observed, it is recommended that ad-
ditional methods such as Bodian's or Bielchowsky's silver methods and/
or immunohistochemistry for glial fibrillary acid protein be used in con-
junction with more standard stains to determine the lowest dose level at
which neuropathological alterations are observed. Evaluations of postnatal-
day-11 pups is described in paragraphs (d)(7)(vi)(B)(7) and
(d)(7)(vi)(B)(2) of this guideline. For animals sacrificed at the termination
of the study, the regions to be examined and the types of alterations that
should be assessed are identified in paragraph (e)(4)(iv)(B) of OPPTS
870.6200.
(7) Regions to be examined. The brains should be examined for any
evidence of treatment-related neuropathological alterations and adequate
samples should be taken from all major brain regions (e.g., olfactory bulbs,
cerebral cortex, hippocampus, basal ganglia, thalamus, hypothalamus,
midbrain (tectum, tegmentum, and cerebral peduncles), brainstem and cer-
ebellum) to ensure a thorough examination.
(2) Types of alterations. Guidance for neuropathological examination
for indications of developmental insult to the brain can be found in para-
graphs (f)(7) and (f)(22) of this guideline. In addition to more typical kinds
of cellular alterations (e.g., neuronal vacuolation, degeneration, necrosis)
and tissue changes (e.g., astrocytic proliferation, leukocytic infiltration, and
cystic formation) particular emphasis should be paid to structural changes
indicative of developmental insult including but not restricted to:
(/) Gross changes in the size or shape of brain regions such as alter-
ations in the size of the cerebral hemispheres or the normal pattern of
foliation of the cerebellum.
(if) The death of neuronal precursors, abnormal proliferation, or ab-
normal migration, as indicated by pyknotic cells or ectopic neurons, or
gross alterations in regions with active proliferative and migratory zones,
alterations in transient developmental structures (e.g., the external germinal
zone of the cerebellum, see paragraph (f)(15) of this guideline).
(///) Abnormal differentiation, while more apparent with special
stains, may also be indicated by shrunken and malformed cell bodies.
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(/v) Evidence of hydrocephalus, in particular enlargement of the ven-
tricles, stenosis of the cerebral aqueduct and general thinning of the cere-
bral hemispheres.
(C) Subjective diagnosis. If any evidence of neuropathological alter-
ations is found in the qualitative examination, then a subjective diagnosis
will be performed for the purpose of evaluating dose-response relation-
ships. All regions of the brain exhibiting any evidence of
neuropathological changes should be included in this analysis. Sections
of each region from all dose groups will be coded as to treatment and
examined in randomized order. The frequency of each type and the sever-
ity of each lesion will be recorded. After all sections from all sections
from all dose groups including all regions have been rated, the code will
be broken and statistical analyses performed to evaluate dose-response re-
lationships. For each type of dose related lesion observed, examples of
different ranges of severity should be described. The examples will serve
to illustrate a rating scale, such as 1+, 2+, and 3+ for the degree of severity
ranging from very slight to very extensive.
(D) Simple morphometric analysis. Since the disruption of devel-
opmental processes is sometimes more clearly reflected in the rate or ex-
tent of growth of particular brain regions, some form of morphometric
analysis should be performed on postnatal-day-11 and at the termination
of the study to assess the structural development of the brain. At a mini-
mum, this would consist of a reliable estimate of the thickness of major
layers at representative locations within the neocortex, hippocampus, and
cerebellum. For guidance on such measurements see Rodier and Gramann
under paragraph (f)(19) of this guideline.
(e) Data collection, reporting, and evaluation. The following spe-
cific information should be reported:
(1) Description of test system and test methods. A description of
the general design of the experiment should be provided. This should in-
clude:
(i) A detailed description of the procedures used to standardize obser-
vations and procedures as well as operational definitions for scoring obser-
vations.
(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 labora-
tory must demonstrate competence in evaluation effects in neonatal ani-
mals perinatally exposed to chemicals and establish test norms for the ap-
propriate age group.
(iii) Procedures for calibrating and ensuring the equivalence of de-
vices and the balancing of treatment groups in testing procedures.
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(iv) A short justification explaining any decisions involving profes-
sional judgement.
(2) Results. The following information must be arranged by each
treatment and control group:
(i) In tabular form, data for each animal must be provided showing:
(A) Its identification number and the litter from which it came.
(B) Its body weight and score on each developmental landmark at
each observation time.
(C) Total session activity counts and intrasession subtotals on each
day measured.
(D) Auditory startle response amplitude per session and intrasession
amplitudes on each day measured.
(E) Appropriate data for each repeated trial (or session) showing ac-
quisition and retention scores on the tests of learning and memory on each
day measured.
(F) Time and cause of death (if appropriate); any neurological signs
observed; a list of structures examined as well as the locations, nature,
frequency, and extent of lesions; and brain weights.
(ii) The following data should also be provided, as appropriate:
(A) Inclusion of photomicrographs demonstrating typical examples of
the type and extent of the neuropathological alterations observed is rec-
ommended.
(B) Any diagnoses derived from neurological signs and lesions, in-
cluding naturally-occurring diseases or conditions, should also be recorded.
(iii) Summary data for each treatment and control group must include:
(A) The number of animals at the start of the test.
(B) The body weight of the dams during gestation and lactation.
(C) Litter size and mean weight at birth.
(D) The number of animals showing each abnormal sign at each ob-
servation time.
(E) The percentage of animals showing each abnormal sign at each
observation time.
(F) The mean and standard deviation for each continuous endpoint
at each observation time. These will include body weight, motor activity
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counts, auditory startle responses, performance in learning and memory
tests, regional brain weights and whole 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 location, frequency and average grade of each type of lesion for each
animal.
(I) The values of all morphometric measurements made for each ani-
mal listed by treatment group.
(3) Evaluation of data. An evaluation of test results must be made.
The evaluation should include the relationship between the doses of the
test substance and the presence or absence, incidence, and extent of any
neurotoxic effect. The evaluation should include appropriate statistical
analyses. The choice of analyses should consider tests appropriate to the
experimental design and needed adjustments for multiple comparisons. The
evaluation should include the relationship, if any, between observed
neuropathological and behavioral alterations.
(f) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Adams, J. et al. Collaborative behavioral teratolgy study: Protocol
design and testing procedure. Neurobehavioral Toxicology and Teratology
7:579-586 (1985).
(2) Bennet, H. S. et al. Science and art in the preparing tissues embed-
ded in plastic for light microscopy, with special reference to glycol meth-
acrylate, glass knives and simple stains. Stain Technology 51:71-97
(1976).
(3) Bushnell, P.J. Effects of delay, intertrial interval, delay behavior
and trimethyltin on spatial delayed response in rats. Neurotoxicology and
Teratology 10:237-244 (1988).
(4) Campbell, B.A. and Harountunian, V. Effects of age on long-term
memory: Retention of fixed interval responding. Journal of Gerontology
36:338-341 (1981).
(5) Cory-Slechta, D.A. et al. Delayed behavioral toxicity of lead with
increasing exposure concentration. Toxicology and Applied Pharmacology
71:342-352(1983).
(6) 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).
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(7) Environmental Protection Agency. Office of Prevention, Pes-
ticides and Toxic Substances Neurotoxicity Screening Battery (1990).
(8) Fride, R. L. Developmental Neuropathology. New York: Springer
Verlag (1975).
(9) Green, R.J. and Stanton, M.E. Differential ontogeny of working
memory and reference memory in the rat. Behavioral Neuroscience
103:98-105 (1989).
(10) Ison, J.R. Reflex modification as an objective test for sensory
processing following toxicant exposure. Neurobehavioral Toxicology and
Teratology 6:437-445 (1984).
(11) Korenbrot, C.C. et al. Preputial separation as an external sigh
of pubertal development in the male rat. Biology of Reproduction 17:298-
303 (1977).
(12) Krasnegor, N.A. et al. (eds.) Perinatal Development: A
Psychobiological Perspective. Orlando: Academic Press (1986).
(13) Kucharski, D. and Spear, N. E. Conditioning of aversion to an
odor paired with peripheral shock in the developing rat. Developmental
Psychobiology 17;465-479 (1984).
(14) Luna, L. G. Manual ofHistologic Staining Methods of the Armed
Forces Institute of Pathology. (Third Edition) New York: McGraw Hill
(1968).
(15) Miale, I. E. and Sidman, R. An autoradiographic analysis of
histogenesis in the mouse cerebellum. Experimental Neurology 4:277-296
(1961).
(16) Miller, D. B. and Eckerman, D. A. Learning and memory meas-
ures. In: Neurobehavioral Toxicology, Z. Annau (ed). Johns Hopkins Uni-
versity Press, Baltimore, pp. 94-149 (1986).
(17) Pender, M. P. Asimple method for high resolution light micros-
copy of nervous tissue. Journal of Neuroscience Methods 15:213-218
(1985).
(18) Ralis, H. M. et al. Techniques in Neurohistology. Butterworths,
London (1973).
(19) Rodier, P. M. and Gramann, W. J. Morphologic effects of inter-
ference with cell proliferation in the early fetal period. Neurobehavioral
Toxicology 1:128-135 (1971).
(20) Spear, N. E. and Campbell, B. A. (eds.) Ontogeny of Learning
and Memory. Erlbaum, New Jersey (1979).
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(21) Spencer, P. S. and Schaumburg, H. H. (eds.) Experimental and
Clinical Neurotoxicology. Williams and Wilkins, Baltimore (1980)
(22) Suzuki, K. Special vulnerabilities of the developing nervous sys-
tem. In: Experimental and Clinical Neurotoxicology, P. S. Spencer and
H. H. Schaumburg (eds.) Williams and Wilkins, Baltimore, pp. 48-61
(1980).
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