United States       Prevention, Pesticides      EPA712-C-98-240
          Environmental Protection    and Toxic Substances      August 1998
          Agency        (7101)
&EPA   Health Effects Test
          OPPTS 870.6500
          Operant Behavior

     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.).

     Final  Guideline Release: This guideline  is available from the U.S.
Government Printing Office,  Washington, DC 20402 on disks or paper
copies: call (202) 512-0132. This  guideline is also available electronically
in PDF (portable document format) from EPA's  World Wide Web  site
(http://www.epa.gov/epahome/research.htm) under the heading "Research-
ers and Scientists/Test Methods and Guidelines/OPPTS  Harmonized Test

OPPTS 870.6500  Schedule-controlled operant behavior.
     (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 798.6500 Schedule-Controlled
Operant Behavior  and OPP 85-5  Schedule-Controlled Operant Behavior
(Pesticide  Assessment  Guidelines,  Subdivision  F—Hazard  Evaluation;
Human and Domestic Animals, Addendum 10, EPA report  540/09-91-
123, March  1991).

     (b) Purpose. In the assessment and evaluation of the potential human
health effects of substances, it may be necessary to test for functional neu-
rotoxic effects.  Substances that have been observed to produce neurotoxic
signs in other toxicity studies (e.g. central nervous  system (CNS) depres-
sion or stimulation), as well  as substances with a structural similarity to
neurotoxicants affecting performance, learning,  or memory may be appro-
priate to evaluate with this test. This guideline defines procedures for con-
ducting studies  of schedule-controlled operant behavior, one way of evalu-
ating the rate and pattern of a class of learned behavior (under paragraphs
(g)(l),  (g)(4), (g)(5), and (g)(6) of this guideline. The purpose of the guide-
line  is  to evaluate  the effects of acute and repeated exposures on the rate
and pattern of responding under schedules of reinforcement. Any observed
effects should be evaluated in the context of both the concordance between
functional neurological and neuropathological effects and with respect to
any  other  toxicological  effects  seen. Operant behavior tests may be also
used to evaluate many other  aspects of behavior (under paragraph (g)(3)
of this guideline). Additional tests may be necessary to  completely assess
the effects of any substance on learning, memory,  or behavioral perform-

     (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.

     Behavioral toxicity is  any adverse change in the functioning of the
organism with respect to its environment in relation to exposure to a chem-
ical substance.

     ED is effective dose.

     Neurotoxicity  is  any adverse effect on the structure or function  of
the nervous  system related to exposure to a chemical substance.

     Operant, operant behavior,  operant conditioning:  An operant is  a
class of behavioral responses which changes or operates on the environ-

ment in the same way. Operant behavior is further distinguished as behav-
ior which  is modified by  its consequences.  Operant conditioning  is the
experimental procedure used to modify some class  of behavior by rein-
forcement or punishment.

     Schedule of reinforcement specifies the relation between behavioral
responses and the delivery of reinforcers, such as  food or water (under
paragraph (g)(2) of this guideline). For example, a fixed ratio (FR) sched-
ule requires a fixed number of responses to produce a reinforcer (e.g. FR
30). Under a fixed interval (FI) schedule, the first response after a fixed
period of time is reinforced (e.g. FI 5 min).

     (d) Principle of the test method. Experimental animals are  trained
to perform under a schedule of reinforcement and measurements of their
operant behavior are made. Several doses of the test substance are adminis-
tered according to the experimental design (between groups or within sub-
jects) and the duration of exposure (acute or repeated). Measurements of
the operant behavior are repeated. For  use of this test to study learning,
animals may be trained following exposure.  A descriptive and statistical
evaluation  of the data is made to evaluate the nature and extent  of any
changes in behavior in relation to exposures to the  test  substance. Com-
parisons are made between any exposures that influence  the behavior and
exposures that have neuropathological effects or effects on other targets
of the chemical.

     (e) Test procedures—(1) Experimental  design. These test proce-
dures may be used to evaluate the behavior of experimental animals receiv-
ing either acute  or repeated exposures.  For acute exposure studies, either
within-subject or between-groups experimental designs may be used. For
repeated exposure studies, between-groups  designs  should be used, but
within-subject  comparisons  (preexposure and  postexposure)  are rec-
ommended and encouraged.

     (2) Animal  selection—(i) Species. For most studies  the laboratory
mouse or rat is recommended.  Standard strains should be used. Under
some circumstances other species may be recommended.

     (ii) Age. Experimental animals should be young adults. Rats or mice
should be  at least  14  and 6 weeks old, respectively, prior to exposure.

     (iii) Sex. Because  of  the labor-intensive nature of this testing, only
one sex may be used. If data on the test chemical indicate that one sex
is more sensitive to the test  substance,  or if it receives greater exposure,
that sex should be used. If females are used, they should be virgins.

     (iv) Experimental history.  Animals  should be experimentally and
chemically naive.

     (3) Number of animals. Eight to ten animals should be exposed to
each level of the test substance and/or control procedure.

     (4) Control groups, (i) A concurrent control group or control ses-
sions (according to the design of the  study)  are  required. For control
groups, subjects should be  treated in the same way  as  for an exposure
group except that administration of the test substance is omitted.

     (ii) Positive control data from the laboratory performing the testing
should provide evidence that the experimental procedures are sensitive to
substances known to affect operant behavior. Both increases and decreases
in response  rate should be demonstrated.  Data based  on acute exposures
will be adequate. Permanently injurious substances need not be used. His-
torical data may be used if the essential aspects of the experimental proce-
dure remain the same.  Periodic updating  of positive control data is rec-
ommended. New positive control data should also be collected when per-
sonnel or some other critical element in the testing laboratory has changed.

     (5) Dose levels and dose  selection.  At least three  doses should  be
used in addition to the vehicle control group (or sessions for within subject
studies).  Ideally, 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 maximally  support detection
of dose-effect relations.

     (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 incidence of fatalities
that would preclude 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.

     (ii) Subchronic (and chronic) studies.  The high dose need not  be
greater than 1 g/kg. Otherwise, the high  dose  should result in significant
neurotoxic effects  or other clearly toxic effects, but not  produce an inci-
dence 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

     (6) 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.  It should be recognized that for  many
materials more than one route of exposure may be important and that these
criteria may conflict with one  another. The route  that best meets these
criteria should be selected. Dietary feeding will be generally be acceptable
for repeated exposure studies.

     (7) 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.

     (8) Study conduct—(i) Apparatus. Behavioral responses and the de-
livery  of reinforcers  should be controlled and  monitored by automated
equipment located so that its operation does not provide unintended cues
or otherwise interfere with the ongoing  behavior.  Individual chambers
should be sound attenuated to  prevent disruptions of behavior by external
noise. The response manipulanda, feeders, and any stimulus devices should
be tested before each session;  these devices should be calibrated periodi-

     (ii) Chamber assignment. Concurrent treatment groups should  be
balanced across  chambers.  Each subject should be tested in the chamber
to which it is initially assigned.

     (iii) Schedule of food availability.  (A) If a  nonpreferred positive
reinforcer is used,  all subjects should be placed on a schedule of food
availability until they reach a  fixed percentage,  e.g.  80 to 90 percent, of
their ad libitum body weight, or kept at a  fixed weight and fed after each

     (B) Subjects must be trained until they display demonstrable stability
in performance across days prior to exposure. One simple and useful cri-
terion is a minimum number of sessions on the schedule and no systematic
trend during the 5 days before exposure.

     (iv) Time, frequency, and duration of testing—(A) Time of testing.
All experimental animals should be tested at the same time  of day and
with respect to the time of exposure. For  acute studies, testing should be
performed when  effects are estimated to  peak,  which may be estimated
from data on the functional observational  battery, motor  activity, or from
pilot studies. For subchronic  studies, subjects should be tested prior to
daily exposure in order to assess cumulative effects.

     (B) Frequency of testing. The maintenance of stable operant behav-
ior normally will require regular and frequent (e.g. 5 days a week) testing
sessions. Animals should be weighed on each test day.

     (C)  Duration of  testing. Experimental  sessions  should  be  long
enough to reasonably see the effects of exposure, but brief enough to  be
practical. Under most circumstances, a session length of 30-40 min should
be adequate.

     (v) Schedule selection. The schedule  of reinforcement chosen should
generate response rates that may increase or decrease as a function of ex-
posure. Many schedules of reinforcement  can do this: A single  schedule
maintaining  a moderate response rate, FI schedules, which engender a vari-

ety of response rates in each interval, or multiple  schedules, where  dif-
ferent components may maintain high and low response rates.

     (f) Data reporting and evaluation. The final test report must include
the following information:

     (1) Description  of equipment and test methods, (i) A description
of the experimental chambers, programming equipment, data collection de-
vices, and environmental test conditions should be provided. Procedures
for calibrating devices should also be described.

     (ii) A description of the experimental design including procedures for
balancing treatment groups  and the stability criterion should be provided.

     (iii) Positive control data from the laboratory performing the test that
demonstrates the sensitivity of the schedule used should be provided. His-
torical data may be used if all essential aspects of the experimental proto-
col are the same. Historical control data can be critical in the interpretation
of study findings. The Agency encourages submission of such data to fa-
cilitate the rapid and  complete review of the  significance  of effects seen.

     (2) Results. Data for each animal should be arranged in tabular form
by  test group including the animal identification  number, body weight,
preexposure rate and  patterns of responding, changes in response rate  and
patterns produced by  the chemical,  and group data for the  same variables,
including standard measures of central tendency and variability, e.g. means
and standard deviations, and results  of statistical analyses.

     (3) Evaluation of data, (i)  The findings 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 incidence
and magnitude of any observed effects, i.e. dose-effect curves for any ef-
fects seen.

     (ii)  The  evaluation should  include appropriate  statistical  analyses.
Choice  of analyses should consider tests appropriate to the experimental
design, including repeated measures. There may be many acceptable ways
to analyze the data.

     (iii)  Citations  from the literature related to the interpretation of the
neurotoxicity of the test material should also be included.

     (g) References. The following references should be consulted for ad-
ditional background material on this test guideline.

     (1)  Dews,  P.B.  Assessing  the  Effects of  Drugs,  In  Methods  in
Psychobiology,  Vol.  2,  (Ed.)  R.D. Myers. Academic,  NY.  pp. 83-124

     (2) Ferster, C.B. and Skinner, B.F. Schedules of Reinforcement. Ap-
pleton-Century-Crofts, NY (1957).

     (3) Laties, V.G. How Operant Conditioning Can Contribute to Behav-
ioral Toxicology, Environmental Health Perspectives 28:29-35 (1978).

     (4) National Academy of Sciences. Principles for Evaluating Chemi-
cals in the Environment. National Academy of Sciences, Washington, DC

     (5) National Academy of Sciences. Principles and Procedures for
Evaluating the Toxicity of Household Substances. National Academy of
Sciences, Washington, DC (1977).

     (6) National Academy of Sciences.  Strategies to determine needs and
priorities for toxicity testing. Appendix 3B. Reference Protocol Guidelines
for Neurobehavioral Toxicity Tests 2:123-129 (1982).