United States Prevention, Pesticides EPA712-C-96-238
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.6200
Neurotoxicity Screening
Battery
'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 870.6200 Neurotoxicity screening battery.
(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 are 40 CFR 798.6050 Functional Observa-
tional Battery, 798.6200 Motor Activity, and 798.6400 Neuropathology;
and OPP 81-8 Acute Neurotoxicity—Rat, 82-7 90-Day Neurotoxicity—
Rat, and 83-1 Chronic Feeding—Two Species, Rodent and Nonrodent
(Pesticide Assessment Guidelines, Subdivision F—Hazard Evaluation;
Human and Domestic Animals) EPA report 540/09-82-025, 1982.
(b) Purpose. This neurotoxicity screening battery consists of a func-
tional observational battery, motor activity, and neuropathology. The func-
tional observational battery consists of noninvasive procedures designed
to detect gross functional deficits in animals and to better quantify behav-
ioral 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 pro-
vide data to detect and characterize histopathological changes in the central
and peripheral nervous system. This battery is designed to be used in con-
junction 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 chemi-
cal.
(c) Definitions. The definitions in section 3 of the Toxic Substances
Control Act (TSCA) and the definitions in 40 CFR Part 792—Good Lab-
oratory Practice Standards apply to this test guideline. The following defi-
nitions also apply to this test guideline.
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 suf-
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ficient 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 during each observation period.
Measurements of motor activity of individual animals are made in an auto-
mated device. The animals are perfused and tissue samples from the nerv-
ous 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 spe-
cies, 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) should be used.
(iii) Sex. Both males and females should be used. Females should
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 sched-
uled to be perfused before the end of the study. Animals should be ran-
domly assigned to treatment and control groups.
(3) Control groups, (i) A concurrent (vehicle) control group is re-
quired. Subjects should 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
should provide evidence of the ability of the observational methods used
to detect major neurotoxic endpoints including limb weakness or paralysis
(e.g. repeated exposure to acrylamide), tremor (e.g. p-p'-DDT), and auto-
nomic signs (e.g. carbaryl). Positive control data are also required to dem-
onstrate 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).
Positive control data should be collected at the time of the test study unless
the laboratory can demonstrate the adequacy of historical data for this pur-
pose, i.e. by the approach outlined in this guideline.
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(4) Dose level and dose selection. At least three doses should 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 maxi-
mally 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 indicence of fatalities
that would preclude a meaningful evaluation of the data. This dose may
be estimated by a BD procedure as described above, with the middle and
low dose levels chosen as fractions of the BD dose. The lowest dose
should produce minimal effect, e.g. an ED 10, 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 signifi-
cant neurotoxic 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 ED 10, 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 recog-
nized that more than one route of exposure may be important and that
these criteria may conflict with one another. In order to save resources,
initially only one route is being 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 should 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 should be made before the initiation of exposure, at the
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estimated time of peak effect within 8 hours 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 observa-
tions of gait and arousal.
(B) Subchronic and chronic studies. In a subchronic study, at a min-
imum, observations and activity measurements should 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
should be made before the initiation of exposure and before the daily expo-
sure, 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 should 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 should 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. Ex-
plicit, operationally defined scales for each measure of the battery are to
be used. The development of objective quantitative measures of the obser-
vational end-points specified is encouraged. Examples of observational
procedures using defined protocols may be found in paragraphs (g)(6),
(g)(8), and (g)(ll) of this guideline. The functional observational battery
should include a thorough description of the subject's appearance, behav-
ior, and functional integrity. This should 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 should
include the following list of measures:
(7) Assessment of signs of autonomic function, including but not lim-
ited to:
(/) Ranking of the degree of lacrimation and salivation, with a range
of severity scores from none to severe.
(//) Presence or absence of piloerection and exophthalmus.
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(///) Ranking or count of urination and defecation, including polyuria
and diarrhea. This is most easily conducted during the open field assess-
ment.
(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 re-
moval from the cage or handling, with a range of severity scores from
no reaction to hyperreactivity.
(4) Ranking of the subject's arousal level or state of alertness during
observations of the unperturbed subject in the open field, with a range
of severity scores from coma to hyperalertness.
(5) Descriptions and incidence of posture and gait abnormalities ob-
served 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, e.g. that described by Meyer et al. under paragraph (g)(10) of
this guideline
Quantitative measure of landing foot splay; the procedure de-
scribed in paragraph (g)(4) of this guideline is recommended.
(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 tail-pinch, 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 fa-
cilitate interpretation of the data.
(C) Additional measures. Other measures may also be included and
the development and validation of new tests is encouraged. Further infor-
mation on the neurobehavioral integrity of the subject may be provided
by:
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(7) 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 should be monitored by an auto-
mated 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 should be tested by standard procedures to ensure, to the ex-
tent possible, reliability of operation across devices 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
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 hu-
midity, 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 pres-
ervation of cellular integrity for the detection of neuropathological alter-
ations, tissue should be prepared for histological analysis using in situ per-
fusion and paraffin and/or plastic embedding procedures. Paraffin embed-
ding is acceptable for tissue samples from the central nervous system. Plas-
tic embedding of tissue samples from the central nervous system is encour-
aged, when feasible. Plastic embedding is required for tissue samples from
the peripheral nervous system. Subject to professional judgment and the
type of neuropathological alterations observed, it is recommended that ad-
ditional methods, such as Bodian's or Bielchowsky's silver methods, and/
or glial fibrillary acidic protein (GFAP) immunohistochemistry be used
in conjunction with more standard stains to determine the lowest dose level
at which neuropathological alterations are observed. When such special
stains indicate evidence of structural alterations it is recommended that
the GFAP radioimmunoassay also be performed, particularly when addi-
tional animals are available for use in the radioimmunoassay. A description
of this technique can be found in paragraph (g)(12) of this guideline.
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(A) Fixation and processing of tissue. The nervous system should
be fixed by in situ perfusion with an appropriate aldehyde fixative. De-
tailed descriptions of vascular perfusions may be found in paragraphs
(g)(7), (g)(13), (g)(19), and (g)(21) of this guideline. Any gross abnormali-
ties should be noted. Tissue samples taken should adequately represent
all major regions of the nervous system. Detailed dissection procedures
are described in paragraphs (g)(19)(chapter 50), and (g)(13) of this guide-
line. The tissue samples should be postfixed and processed according to
standardized published histological protocols under paragraph (g)(l),
(g)(2), (g)(3), (g)(14), (g)(19), or (g)(20) of this guideline. Tissue blocks
and slides should be appropriately identified when stored. Histological sec-
tions should be stained for hematoxylin and eosin (H&E), or a comparable
stain according to standard published protocols under paragraphs (g)(l),
(g)(2), and (g)(14) of this guideline.
(B) Qualitative examination. Representative histological sections
from the tissue samples should be examined microscopically by an appro-
priately trained pathologist for evidence of neuropathological alterations.
The nervous system should be thoroughly examined for evidence of any
treatment-related 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 distin-
guished from artifacts resulting from influences other than exposure to the
test substance. Guidance for both regions to be examined and the types
of neuropathological alterations that typically result from toxicant exposure
can be found in paragraph (g)(20) of this guideline. A stepwise examina-
tion 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 exam-
ined sequentially.
(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 nervous system exhibiting any evidenceof
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 rela-
tionships. For each type of dose-related lesion observed, examples of dif-
ferent degrees of severity should be described. Photomicrographs of typical
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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 must include
the following information:
(1) Description of equipment and test methods. A description of
the general design of the experiment and any equipment used should be
provided. This should include a short justification explaining any decisions
involving professional judgment.
(i) A detailed description of the procedures used to standardize obser-
vations, including the arena and scoring criteria. Procedures for calibrating
and assuring the equivalence of activity devices and balancing treatment
groups should also be described.
(ii) Positive control data from the laboratory performing the test that
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 must be arranged by test
group dose level.
(i) In tabular form, data for each animal must 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 appro-
priate.
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(iii) All neuropathological observations should be recorded and ar-
ranged by test groups. This data may be presented in the following rec-
ommended format:
(A) Description of lesions for each animal. For each animal, data must
be submitted showing its identification (animal number, sex, treatment,
dose, duration), a list of structures examined as well as the locations, na-
ture, 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 de-
rived from neurological signs and lesions including naturally occurring dis-
eases or conditions, should be recorded.
(B) Counts and incidence of neuropathological alterations by test
group. Data should be tabulated to show:
(7) 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 should include the relationship between the doses of the test
substance and the presence or absence, incidence and severity, of any neu-
rotoxic effects. The evaluation should include appropriate statistical analy-
ses, for example, parametric tests for continuous data and nonparametric
tests for the remainder. Choice of analyses should consider tests appro-
priate to the experimental design, including repeated measures. There may
be many acceptable ways to analyze data. Statistical analysis comparing
total activity counts of treatment vs control animals at each measured time
must be made and supplied. The report must include dose-effect curves
for observations, motor activity expressed as actvity counts, and any gross
necropsy findings and lesions observed.
(g) References. The following references should be consulted for ad-
ditional background material on this test guideline.
(1) Armed Forces Institute of Pathology. Manual ofHistologic Stain-
ing Methods. McGraw-Hill, NY (1968).
(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).
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(3) 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).
(4) 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).
(5) Finger, F.W. Measuring behavioral activity. In: Methods in
Psychobiology Vol. 2, Ed. R.D. Myers. Academic, NY. pp. 1-19 (1972).
(6) Gad, S. A neuromuscular screen for use in industrial toxicology.
Journal of Toxicology and Environmental Health 9:691-704 (1982).
(7) Hayat, M.A. Volume 1. Biological applications. In: Principles and
Techniques of Electron Microscopy. Van Nostrand Reinhold, NY (1970).
(8) Irwin, S. Comprehensive observational assessment: la. A system-
atic quantitative procedure for assessing the behavioral physiological state
of the mouse. Psychopharmacologia 13:222-257 (1968).
(9) Kinnard, E.J. and Watzman, N. Techniques utilized in the evalua-
tion of psychotropic drugs on animals activity. Journal of Pharmaceutical
Sciences 55:995-1012 (1966).
(10) 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).
(11) Moser V.C. et al. Comparison of chlordimeform and carbaryl
using a functional observational battery. Fundamental and Applied Toxi-
cology 11:189-206 (1988).
(12) 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).
(13) Palay, S.L. and Chan Palay, V. Cerebellar Cortex: Cytology and
Organizatio. Springer Verlag, NY (1974).
(14) Pender, M.P. A simple method for high resolution light micros-
copy of nervous tissue. Journal of Neuroscience Methods 15:213-218
(1985).
(15) Ralis, H.M. et al. Techniques in Neurohistology, Butterworths,
London (1973).
(16) Reiter, L.W. Use of activity measures in behavioral toxicology.
Environmental Health Perspectives 26:9-20 (1978).
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(17) Reiter, L.W. and MacPhail, R.C. Motor Activity: A survey of
methods with potential use in toxicity testing. Neurobehavorial Toxicology
1—Suppl. 1:53-66(1979).
(18) Robbins, T.W. A critique of the methods available for the meas-
urement of spontaneous motor activity. Handbook of Psychopharmacology
Vol 7. Eds. Iversen, L.L., Iverson, D.S., Snyder, S.H. Plenum, NY. pp.
37-82 (1977).
(19) Spencer, P.S., Schaumburg, H.H. Eds., Experimental and Clini-
cal Neurotoxicology, Williams and Wilkins, Baltimore (1980).
(20) World Health Organization. Principles and Methods for the As-
sessment of Neurotoxicity Associated with Exposure to Chemicals (Envi-
ronmental Health Criteria 60), World Health Organizations Publications
Center USA, Albany, NY (1986).
(21) Zeman, W. and Innes, J.R. Craigie's Neuroanatomy of the Rat,
Academic, NY (1963).
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