United States       Prevention, Pesticides     EPA712-C-96-258
          Environmental Protection    and Toxic Substances     June 1996
          Agency        (7101)
&EPA   Health Effects Test
          OPPTS 870.8600
          Neurotoxicity Screen
                'Public Draft"

     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

     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:

     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,  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

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.

     (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-

     (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

     (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.

     (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-

     (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

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

     (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-

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

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

     (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.

     (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

     (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.

     (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-

     (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).


    (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.

    (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).