United States

Environmental Protection

Agency

Prevention, Pesticides

and Toxic Substances

(7101)

EPA712–C–96–239

June 1996

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 published 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. Environmental Protection Agency under the Toxic Substances Control Act (15

U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act

(7 U.S.C. 136, et seq.).

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 ‘‘Environmental 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 comments. 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, Arlington, 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 Bulletin 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 testing 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 harmonized OPPTS test guideline is OPP 83–6 Postnatal Developmental Toxicity (Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation; Human and Domestic Animals) EPA report 540/09–82–025, 1982.

(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 should 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 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 adequate 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)(1) 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 memory, 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 measured. 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 administering 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 meaningful evaluation of neurotoxicity.

(iii) If a standard developmental toxicity study has not been conducted, the highest dose level, unless limited by the physicochemical nature or biological properties of the substance, should induce some overt

maternal toxicity, but should not result in a reduction in weight gain exceeding 20 percent during gestation and lactation.
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(iv) The lowest dose should not produce any grossly observable evidence 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 vehicle 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’ treatment, 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 limited to:

(1) 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.

(3) 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 facilitate interpretation of the data.

(iii) Signs of toxicity should be recorded as they are observed, including 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 technicians, who are unaware of the treatment being used, 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. At a minimum, the end points outlined in paragraph

(d)(6)(ii) of this guideline should be monitored as appropriate for the developmental 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 separation should be determined. General procedures for these determinations

may be found in paragraphs (f)(1) and (f)(11) 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 increases 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 asymptotic 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 variables 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 conduct of this testing may be obtained under paragraph (f)(1) of this guideline. 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 requirement, 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 separate 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 (acquisition), 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, motivational, 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 absence 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 described 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 paragraphs (f)(20) and (f)(12) of this guideline, and for adult rats under paragraph (f)(16) of this guideline.

(vi) Neuropathology. Neuropathological evaluation should be conducted 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 fixative. The remaining animals will be sacrificed in a similar manner and

immediately thereafter their brains removed and weighed. At the termination 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 animals 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 histological 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 appropriately 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 evidence of neuropathological alterations, to identify types of neuropathological alterations resulting from exposure to the test substance, and to determine the range of severity of the neuropathological alterations. Representative 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 recommended 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 additional methods such as Bodian’s or Bielchowsky’s silver methods and/

or immunohistochemistry for glial fibrillary acid protein be used in conjunction with more standard stains to determine the lowest dose level at

which neuropathological alterations are observed. Evaluations of postnatalday–11 pups is described in paragraphs (d)(7)(vi)(B)(1) 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.

(1) 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 cerebellum) 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 paragraphs (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:

(i) Gross changes in the size or shape of brain regions such as alterations in the size of the cerebral hemispheres or the normal pattern of

foliation of the cerebellum.

(ii) The death of neuronal precursors, abnormal proliferation, or abnormal 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).

(iii) Abnormal differentiation, while more apparent with special

stains, may also be indicated by shrunken and malformed cell bodies.
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(iv) Evidence of hydrocephalus, in particular enlargement of the ventricles, stenosis of the cerebral aqueduct and general thinning of the cerebral hemispheres.

(C) Subjective diagnosis. If any evidence of neuropathological alterations is found in the qualitative examination, then a subjective diagnosis

will be performed for the purpose of evaluating dose-response relationships. 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 severity 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 relationships. 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 developmental processes is sometimes more clearly reflected in the rate or extent 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 minimum, 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 specific 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 include:

(i) A detailed description of the procedures used to standardize observations and procedures as well as 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 evaluation effects in neonatal animals perinatally exposed to chemicals and establish test norms for the appropriate age group.

(iii) Procedures for calibrating and ensuring the equivalence of devices and the balancing of treatment groups in testing procedures.
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(iv) A short justification explaining any decisions involving professional 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 acquisition 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 recommended.

(B) Any diagnoses derived from neurological signs and lesions, including 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 observation 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 animal 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 additional 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 embedded in plastic for light microscopy, with special reference to glycol methacrylate, 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, Pesticides 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 of Histologic 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 measures. In: Neurobehavioral Toxicology, Z. Annau (ed). Johns Hopkins University Press, Baltimore. pp. 94–149 (1986).

(17) Pender, M. P. Asimple method for high resolution light microscopy 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 interference 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 system. In: Experimental and Clinical Neurotoxicology, P. S. Spencer and

H. H. Schaumburg (eds.) Williams and Wilkins, Baltimore. pp. 48–61

(1980).