United States Prevention, Pesticides EPA712-C-98-240
Environmental Protection and Toxic Substances August 1998
Agency (7101)
&EPA Health Effects Test
Guidelines
OPPTS 870.6500
Schedule-Controlled
Operant Behavior
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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.).
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
Guidelines."
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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-
ance.
(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-
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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.
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(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
effects.
(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.
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(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-
cally.
(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
session.
(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-
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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
(1972).
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(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
(1975).
(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).
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