United States Prevention, Pesticides EPA712-C-96-258
Environmental Protection and Toxic Substances June 1996
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.8600
Developmental
Neurotoxicity Screen
'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 870.8600 Developmental neurotoxicity screen.
(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 the OPPT guideline under 40 CFR
795.250 Developmental Neurotoxicity Screen.
(b) Purpose. In the assessment and evaluation of the toxic character-
istics of a chemical, it is important to determine when acceptable expo-
sures in the adult may not be acceptable to a developing organism. This
test is designed to provide information on the potential functional and mor-
phologic 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 lactation, one
dose level being used per group. Offspring are randomly selected from
within litters for neurotoxicity evaluation. The evaluation includes observa-
tion to detect gross neurological and behavioral abnormalities, determina-
tion of motor activity, neuropathological evaluation, and brain weights.
Measurements are carried out periodically during both postnatal develop-
ment and adulthood.
(d) Test procedures—(1) Animal selection—(i) Species and strain.
Testing should be performed in the Sprague Dawley rat.
(ii) Age. Young adult animals (nulliparous females) should be used.
(iii) Sex. Pregnant females should be used at each dose level.
(iv) Number of animals. The objective is for a sufficient number
of pregnant rats to be exposed to ensure that an adequate number of off-
spring are produced for neurotoxicity evaluation. At least 20 litters are
recommended at each dose level. This number assumes a coefficient of
variation of 20 to 25 percent for most behavioral tests. If, based upon
experience with historical control data or data for positive controls in a
given laboratory, the coefficient of variation for a given task is higher
than 20 to 25 percent, calculation of appropriate sample sizes to detect
a 20 percent change from control values with 80 percent power would
need to be done. For most designs, calculations can be made according
to Dixon and Massey under paragraph (f)(5) of this guideline, Neter and
Wasserman under paragraph (f)(10) of this guideline, Sokal and Rohlf
under paragraph (f)(ll) of this guideline, or Jensen under paragraph (f)(8)
of this guideline.
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(A) On day-4 after birth, the size of each litter should be adjusted
by eliminating extra pups by random selection to yield, as nearly as pos-
sible, four males and four females per litter. Whenever the number of male
or female pups prevents having four of each sex per litter, partial adjust-
ment (for example, five males and three females) is permitted. Adjustments
are not appropriate for litters of less than eight pups. Elimination of runts
only is not appropriate. Individual pups should be identified uniquely after
standardization of litters. A method that may be used can be found under
paragraph (f)(l) of this guideline.
(B) After standardization of litters, males and females should be ran-
domly assigned to one of each of three behavioral tasks. Alternatively,
more than one of the behavioral tasks may be conducted in the same ani-
mal. In the latter case, a minimum of 1 to 2 days should separate the
tests when conducted at about the same age.
(C) One male and one female should be randomly selected from each
litter for sacrifice at weaning as specified in paragraph (d)(8) of this guide-
line.
(2) Control group. A concurrent control group should be used. This
group should be a sham treated group, or, if a vehicle is used in admin-
istering the test substance, a vehicle control group. Animals in the control
groups should be handled in an identical manner to test group animals.
The vehicle should neither be developmentally toxic nor have effects on
reproduction.
(3) Dose levels and dose selection, (i) At least three dose levels plus
a control (vehicle control, if a vehicle is used) should be used.
(ii) If the substance has been shown to be developmentally toxic ei-
ther in a standard developmental toxicity study or a pilot study, the highest
dose level should be the maximum dose which will not induce in utero
or neonatal deaths or malformations sufficient to preclude a meaningful
evaluation of neurotoxicity.
(iii) In the absence of standard developmental toxicity, unless limited
by the physicochemical nature or biologicial properties of the substance,
the highest dose level should induce some overt maternal toxicity but
should not result in a reduction in weight gain exceeding 20 percent during
gestation and lactation.
(iv) The lowest dose should not produce any grossly observable evi-
dence of either maternal or developmental neurotoxicity.
(v) The intermediate doses should be equally spaced between the
highest and lowest dose.
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(4) Dosing period. Day-0 in the test is the day on which a vaginal
plug and/or sperm are observed. The dose period should cover the period
from day-6 of gestation through weaning (21 days postnatally).
(5) Administration of test substance. The test substance or vehicle
should be administered orally by intubation. The test substance should be
administered at the same time each day. The animals should be weighed
periodically and the dosage 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 blind with respect to the ani-
mal's treatment, using standardized procedures to maximize inter-observer
reliability. Where possible, it is advisable that the same observer be used
to evaluate the animals in a given study. If this is not possible, some dem-
onstration of interobserver reliability is required.
(ii) During the treatment and observation periods, cage-side observa-
tions should include:
(A) Any responses with respect to body position, activity level, co-
ordination of movement, and gait.
(B) Any unusual or bizarre behavior including, but not limited to,
headflicking, head searching, compulsive biting or licking, self-mutilation,
circling, and walking backwards.
(C) The presence of convulsions, tremors, increased levels of
lacrimation and/or red-colored tears, increased levels of salivation,
piloerection, pupillary dilation or constriction, unusual respiration
(shouldow, labored, dyspneic, gasping, and retching) and/or mouth breath-
ing, diarrhea, excessive or diminished urination, or vocalization.
(iii) Signs of toxicity should be recorded as they are observed, includ-
ing the time of onset, the degree and duration.
(iv) Animals should be weighed at least weekly.
(v) The day of delivery of litters should be recorded.
(7) Study conduct—(i) Observation of offspring. (A) All offspring
should be examined cage-side daily for gross signs of mortality and mor-
bidity.
(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 animal's treatment, using standardized pro-
cedures to maximize interobserver reliability. Where possible, it is advis-
able that the same observer be used to evaluate the animals in a given
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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, the degree, and duration.
(ii) Developmental landmarks. Live pups should be counted and lit-
ters weighed by weighing each individual pup at birth, or soon thereafter,
and on days 4, 7, 13, 17, and 21, and biweekly thereafter. The age of
the pups at the time of the appearance of the following developmental
landmarks should be determined:
(A) Vaginal opening. General procedure for this determination may
be found under paragraph (f)(l) of this guideline.
(B) Testes descent. General procedure for this determination may be
found under paragraph (f)(l) of this guideline.
(iii) Motor activity. (A) Motor activity should be monitored specifi-
cally on days 13, 17, 21, 45 (±2 days), and 60 (±2 days). Motor activity
should be monitored by an automated activity recording apparatus. The
device used should be capable of detecting both increases and decreases
in activity, i.e. baseline activity as measured by the device should not be
so low as to preclude decreases nor so high as to preclude increases. Each
device should be tested by standard procedures to ensure, to the extent
possible, reliability of operation across devices and testing of animals with-
in dose groups should be balanced across devices.
(B) Each animal should be tested individually. The test session should
be long enough to demonstrate habituation of motor activity in control
animals, i.e. to approach asymptotic levels by the last 20 percent of the
session. Animals' activity counts should be collected in equal time periods
of no greater than 10 min duration. All sessions should have the same
duration. Treatment groups should be counterbalanced across test times.
(C) Efforts should be made to ensure that variations in the test condi-
tions are minimal and are not systematically related to treatment. Among
the variables which can affect motor activity are sound level, size, and
shape of the test cage, temperature, relative humidity, lighting conditions,
odors, use of home cage or novel test cage, and environmental distractions.
(D) Additional information on the conduct of a motor activity study
may be obtained in the TSCA motor activity guideline, in OPPTS
870.6200.
(iv) Auditory startle test. An auditory startle habituation test should
be performed on the offspring on day-22 and day-60. Details on the con-
duct of this testing may be obtained under paragraph (f)(l) of this guide-
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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 may be used at the discretion of the investigator. Details
on the conduct of this testing may be obtained under paragraph (f)(7) of
this guideline.
(v) Active avoidance test. Active avoidance testing should be con-
ducted beginning at 60 to 61 days of age. Details on the apparatus may
be obtained under paragraph (f)(4) of this guideline and on the conduct
of testing under paragraph (f)(2) of this guideline, respectively; reviews
on active avoidance conditioning can be found under paragraphs (f)(3) and
(f)(9) of this guideline. In performing the active avoidance task, the follow-
ing measures should be made:
(A) Mean number of shuttles during the adaptation period preceding
each daily session.
(B) Mean number and latency of avoidances per session, presented
in blocks of 10 trials (2 blocks of 10 trials per session across five sessions).
(C) Mean number and latency of escapes per session, presented in
blocks of 10 trials as above.
(D) Mean duration of shocks per session, presented in blocks of 10
trials as above.
(E) Mean number of shuttles during the intertrial intervals.
(8) Postmortem evaluation—(i) Age of animals. One male and one
female per litter should be sacrificed at weaning and the remainder follow-
ing the last behavioral measures. Neuropathology and brain weight deter-
minations should be made on animals sacrificed at weaning and after the
last behavioral measures.
(ii) Neuropathology. Details for the conduct of neuropathology eval-
uation may be obtained in the TSCA neuropathology guideline in OPPTS
870.6200 of this chapter. At least six offspring per dose group should be
randomly selected from each sacrificed group (weaning and adulthood)
for neuropathologic evaluation. These animals should be balanced across
litters, and equal numbers of males and females should be used. The re-
maining sacrificed animals should be used to determine brain weight. Ani-
mals should be perfused in situ by a generally recognized technique. After
perfusion, the brain and spinal cord should be removed and gross abnor-
malities noted. Cross-sections of the following areas should be examined:
The forebrain, the center of the cerebrum and midbrain, the cerebellum
and pons, and the medulla oblongata; the spinal cord at cervical and lum-
bar swelling; Gasserian ganglia, dorsal root ganglia, dorsal and ventral root
fibers, proximal sciatic nerve (midthigh and sciatic notch), sural nerve (at
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knee), and tibial nerve (at knee). Tissue samples from both the central
and peripheral nervous system should be further immersion-fixed and
stored in appropriate fixative for further examination. After dehydration,
tissue specimens should be cleared with xylene and embedded in paraffin
or paraplast except for the sural nerve which should be embedded in plas-
tic. A method for plastic embedding is described under paragraph (f)(12)
of this guideline. Tissue sections should be prepared from the tissue
blocks. The following general testing sequence is recommended for gather-
ing histopathological data:
(A) General staining. A general staining procedure should be per-
formed on all tissue specimens in the highest treatment group. Hema-
toxylin and eosin (H&E) should be used for this purpose. The staining
should be differentiated properly to achieve bluish nuclei with pinkish
background.
(B) Special stains. Based on the results of the general staining, se-
lected sites and cellular components should be further evaluated by use
of specific techniques. If H&E screening does not provide such informa-
tion, a battery of stains should be used to assess the following components
in all appropriate required samples: Neuronal body (e.g., Einarson's
gallocyanin), axon (e.g., Kluver's Luxol Fast Blue), and neurofibrils (e.g.,
Bielchosky). In addition, nerve fiber teasing should be used. A section
of normal tissue should be included in each staining to assure that adequate
staining has occurred. Any changes should be noted and representative
photographs should be taken. If lesions are observed, the special tech-
niques should be repeated in the next lower treatment group until no fur-
ther lesions are detectable.
(C) Alternative technique. If the anatomical locus of expected
neuropathology is well-defined, epoxy-embedded sections stained with to-
luidine blue may be used for small sized tissue samples. This technique
obviates the need for special stains.
(iii) Brain weight. At least 10 animals that are not sacrificed for
histopathology should be used to determine brain weight. The animals
should be decapitated and the brains carefully removed, blotted, chilled,
and weighed. The following dissection should be performed on an ice-
cooled glass plate: First, the rhombencephalon is separated by a transverse
section from the rest of the brain and dissected into the cerebellum and
the medulla oblongata/pons. A transverse section is made at the level of
the "optic chiasma" which delimits the anterior part of the hypothalamus
and passes through the anterior commissure. The cortex is peeled from
the posterior section and added to the anterior section. This divides the
brain into four sections, the telencephalon, the diencephalon/mid-brain, the
medulla oblongata/pons, and the cerebellum. Sections should be weighed
as soon as possible after dissection to avoid drying. Detailed methodology
is available under paragraph (f)(6) of this guideline.
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(e) Data reporting and evaluation. In addition to the reporting re-
quirements specified in 40 CFR part 792, subpart J, the final test report
should include the following information.
(1) Description of system and test methods, (i) A detailed descrip-
tion of the procedures used to standardize observation and operational defi-
nitions 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 labora-
tory must demonstrate competence in testing neonatal animals perinatally
exposed to chemicals and establish test norms for the appropriate age
group.
(iii) Procedures for calibrating and assuring the equivalence of devices
and balancing treatment groups.
(iv) A short justification explaining any decisions where professional
judgement is involved such as fixation technique and choice of stains.
(2) Results. The following information should be arranged by test
group dose level.
(i) Data for each animal should be provided in tabular form showing:
(A) Its identification number and litter from which it came.
(B) Its body weight and score on each developmental landmark at
each observation time; total session activity counts and intrasession sub-
totals on each day measured; auditory startle response magnitude session
counts and intrasession subtotals on each day measured; avoidance session
counts and intrasession counts on each day measured; time and cause of
death (if appropriate); locations, nature or frequency, and severity of the
lesions; total brain weight; absolute weight of each of the four sections;
and weight of each section as a percentage of total brain weight. A com-
monly used scale such as 1+, 2+, 3+, and 4+ for degree of severity of
lesions ranging from very slight to extensive may be used for morphologic
evaluation. Any diagnoses derived from neurologic signs and lesions, in-
cluding naturally occurring diseases or conditions, should also be recorded.
(ii) Summary data for each group should include:
(A) The number of animals at the start of the test.
(B) Body weights of the dams during gestation and lactation.
(C) Litter size and mean weight at birth.
(D) The number of animals showing each observation score at each
observation time.
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(E) The percentage of animals showing each abnormal sign at each
observation time.
(F) The mean and standard deviation for each continuous end point
at each observation time. These will include body weight, motor activity
counts, acoustic startle responses, performance in active avoidance tests,
and brain weights (both absolute and relative).
(G) The number of animals in which any lesion was found.
(H) The number of animals affected by each different type of lesion,
the average grade of each type of lesion, and the frequency of each dif-
ferent type and/or location of lesions.
(3) Evaluation of data. An evaluation of the test results should be
made. The evaluation should include the relationship between the doses
of the test substance and the presence or absence, incidence, and severity
of any neurotoxic effect. The evaluation should include appropriate statis-
tical analyses. The choice of analyses should consider tests appropriate
to the experimental design and needed adjustments for multiple compari-
sons.
(f) References. The following references should be consulted for ad-
ditional background information on this test guideline.
(1) Adams, J. et al. Collaborative behavioral teratology study:Protocol
design and testing procedure. Neurobehavioral Toxicology and Teratology
7:579-586 (1985).
(2) Brush, F.R. The effects of inter-trial interval on avoidance learn-
ing in the rat. Journal of Comparative Physiology and Psychology 55:888-
892 (1962).
(3) Brush, F.R. Retention of aversively motivated behavior.
In-Adverse Conditioning and Learning. Brush, F.R., ed., Academic Press,
NY. (1971).
(4) Brush, F.R. and Knaff, P.R. A device for detecting and controlling
automatic programming of avoidance-conditioning in a shuttle-box. Amer-
ican Journal of Psychology 72:275-278 (1959).
(5) Dixon, W. J. and Massey, E.J. Introduction to Statistical Analysis.
2nd ed. McGraw-Hill, NY. (1957).
(6) Glowinski, J. and Iversen, L.L. Regional studies of catecholamines
in the rat brain-I. Journal of Neurochemistry 13:655-669 (1966).
(7) Ison, J.R. Reflex modification as an objective test for sensory
processing following toxicant exposure. Neurobehavioral Toxicology and
Teratology 6:437-445 (1984).
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(8) Jensen, D.R. Some simultaneous multivariate procedures using
Hotelling's T2 Statistics. Biometrics 28:39-53 (1972).
(9) McAllister, W.R. and McAllister, D.E. Behavioral measurement
of conditioned fear. In-Adverse Conditioning and Learning. Brush, F.R.,
ed., Academic Press, NY (1971).
(10) Neter, J. and Wasserman, W. Applied Linear Statistical Models.
Irwin, Homewood. (1974).
(11) Sokal, R.P. and Rohlf, E.J. Biometry. Freeman, San Francisco.
(1969).
(12) Spencer, P.S., et al. Neuropathological methods for the detection
of neurotoxic disease. ^'.Experimental and Clinical Neurotoxicology.
Spencer, P.S. and Schaumburg, H.H., eds., Williams & Wilkins Baltimore,
MD pp. 743-757 (1980).
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