[Code of Federal Regulations]
[Title 40, Volume 31]
[Revised as of July 1, 2007]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.6500]

[Page 221-224]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
 
                         Subpart G_Neurotoxicity
 
Sec.  798.6500  Schedule-controlled operant behavior.

    (a) Purpose. (1) In the assessment and evaluation of the potential 
human health effects of substances, it may be necessary to test for 
functional neurotoxic effects. Substances that have been observed to 
produce neurotoxic signs in other toxicity studies (e.g. CNS depression 
or stimulation), as well as substances with a structural similarity to 
known neurotoxicants should be evaluated for these effects.
    (2) This guideline defines procedures for conducting studies of 
schedule-controlled operant behavior, one way of evaluating functional 
neurotoxic effects (Dews, 1972 under paragraph (f)(1) of this section; 
NAS 1975, 1977, 1982 under paragraphs (f)(4), (5) and (6) of this 
section). Our purpose is to evaluate the effects of acute and repeated 
exposures on the rate and pattern of responding under schedules of 
reinforcement. Operant behavior tests may be used to evaluate many other 
aspects of behavior (Laties, 1978 under paragraph (f)(3) of this 
section). Additional tests may be necessary to completely assess the 
behavioral effects of any substance. Behavioral evaluation should be 
used in conjunction with neuropathologic evaluation and the evaluation 
of other toxic effects.
    (b) Definitions--(1) Neurotoxicity. Neurotoxicity or a neurotoxic 
effect is an adverse change in the structure or function of the nervous 
system following exposure to a chemical agent. Behavioral toxicity is an 
adverse change in the functioning of the organism with respect to its 
environment following exposure to a chemical agent.
    (2) Operant, operant behavior, operant conditioning. An operant is a 
class of behavioral responses which change or operates on the 
environment in the same way. Operant behavior is further distinguished 
as behavior which is modified by its consequences. Operant conditioning 
is the experimental procedure used to modify some class of behavior by 
reinforcement or punishment.
    (3) Schedule of reinforcement. A schedule of reinforcement specifies 
the relation between behavioral responses and the delivery of 
reinforcers, such as food or water (Ferster and Skinner, 1957 under 
paragraph (f)(2) of this section). For example, a fixed ratio (FR) 
schedule requires a fixed number of responses to produce a reinforcer 
(e.g. FR 30). On a fixed interval (FI) schedule, the first response 
after a fixed period of time is reinforced (e.g. FI 5 minutes).
    (c) 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 then 
administered according to the experimental design (between groups or 
within subjects) and the duration of exposure (acute or repeated). 
Measurements of the operant

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behavior are repeated. 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. Comparisons are 
made between any exposures that influence the behavior and exposures 
that have neuropathological effects or effects on other targets of the 
chemical.
    (d) Test procedures--(1) Experimental design. These test procedures 
may be used to evaluate the behavior of experimental animals receiving 
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 (pre-exposure and post-exposure) are 
recommended and encouraged.
    (2) Animal selection--(i) Species. (A) For most studies, the 
laboratory mouse or rat is recommended. Standard strains should be used.
    (B) 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. (A) Approximately equal numbers of male and female 
animals are required for each dose level and control group.
    (B) Virgin females should be used.
    (iv) Experimental history. Animals should be experimentally and 
chemically naive.
    (3) Number of animals. Six to twelve animals should be exposed to 
each level of the test substance and/or control procedure. If post 
exposure effects are examined, a separate group, 6 to 12 additional 
animals not sacrificed for pathology, will required in subchronic 
studies.
    (4) Control groups--(i) Untreated controls. A concurrent ``sham'' 
exposure or vehicle control group or session (according to the design of 
the study) is required. The subjects should be treated similarly except 
that administration of the test substance is omitted.
    (ii) Positive controls. Positive control data is required to 
demonstrate that the experimental procedures, under the specific 
conditions in the testing laboratory, 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. Data should be collected according to the same experimental 
design as that proposed for the test substance. Historical data on the 
procedure collected in the same species and under the same conditions in 
the testing laboratory may be acceptable, but the presentation of 
concurrent control data is strongly encouraged since it provides 
evidence that the test has remained sensitive.
    (5) Dose levels and dose selection. At least 3 doses, equally spaced 
over a log scale (e.g., 10, 30, 100), over a range of at least 1 log 
unit shall be used in addition to a zero dose or vehicle administration. 
The data should be sufficient to produce a dose-effect curve.
    (i) The highest dose shall produce: (A) Clear behavioral effects; or 
(B) life-threatening toxicity.
    (ii) The data from the lower doses must show either: (A) Graded 
dose-dependent effects at 2 dose levels; or (B) no effects at 2 dose 
levels, respectively.
    (6) Duration of exposure. The duration and frequency of exposure 
will be specified in the test rule.
    (7) Route of Administration. The route of administration will also 
be specified in the test rule and will usually be identical to one of 
the anticipated or actual routes of human exposure. For some chemicals, 
another route (e.g. parenteral) may be justified. The exposure protocol 
should conform to that outlined in the appropriate acute or subchronic 
toxicity study guideline under subpart B or subpart C of this part.
    (8) Study conduct--(i) Apparatus. Behavioral responses and the 
delivery of reinforcers shall 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 
periodically be calibrated.

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    (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) Deprivation and training. (A) If a nonpreferred positive 
reinforcer is used, all subjects should be deprived of food until they 
reach a fixed percentage (e.g. 80 to 90 percent, commonly) of their ad 
libitum body weight or for a fixed period (e.g., 18 hours) prior to 
training. Deprivation should be kept constant throughout the study.
    (B) Subjects must be trained until they display demonstrable 
stability in performance across days prior to exposure. One simple and 
useful criterion is a minimum number of sessions on the schedule and no 
systematic trend during the 5 days before exposure.
    (C) Cumulative records of cumulative responding over time for each 
animal should be presented to demonstrate that the pattern of responding 
is representative of that generated by the schedule of reinforcement.
    (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, usually shortly after 
exposure. 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 behavior 
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. (1) 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 minutes 
should be adequate.
    (2) If the nature or duration of effects following cessation of 
repeated exposure are a concern, animals from the high dose group should 
be tested following exposure for a suitable period of time.
    (v) Schedule selection. The schedule of reinforcement chosen should 
generate response rates that may increase or decrease as a function of 
exposure. Many schedules of reinforcement can do this: a single schedule 
maintaining a moderate response rate; fixed-interval schedules, which 
engender a variety of response rates in each interval; or multiple 
schedules, where different components may maintain high and low response 
rates.
    (e) Data reporting and evaluation. In addition to the reporting 
requirements specified under 40 CFR part 792, subpart J the final test 
report should contain the following information:
    (1) Description of system, test methods, experimental design, and 
control data. (i) A description of the experimental chamber, programming 
equipment, data collection devices, and environmental conditions.
    (ii) A description of the experimental design including 
counterbalancing procedures, and the stability criterion.
    (iii) A description and statistical evaluation of positive control 
and other control data, including standard measures of central tendency, 
variability, coefficient of variation of response rates, and the slope 
of the dose-effect curve.
    (2) Results. (i) Data for each animal should be arranged by test 
group in tabular form including the animal identification number, body 
weight, pre-exposure rate of responding, changes in response rate 
produced by the chemical, and group data for the same variables, 
including standard measures of central tendency, variability and 
coefficient of variation.
    (ii) A description and statistical evaluation of the test results: 
With particular reference to the overall statistical procedures (e.g., 
parametric or nonparametric) dose-effect curve, and calculation of 
slope. Presentation of calculations is encouraged.
    (f) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Dews, P.B. ``Assessing the Effects of Drugs,'' Methods in 
Psychobiology, Vol. 2, Ed., R.D. Myers (New York: Academic Press, 1972) 
83-124.
    (2) Ferster, C.B. Skinner, B.F. Schedules of Reinforcement. (New 
York: Appleton-Century-Crofts, 1957).

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    (3) Laties, V.G. ``How Operant Conditioning can Contribute to 
Behavioral Toxicology,'' Environmental Health Perspectives, 28: 29-35 
(1978).
    (4) National Academy of Science. Principles for Evaluating Chemicals 
in the Environment. (Washington, DC: National Academy of Sciences, 
1975).
    (5) National Academy of Science. Principles and Procedures for 
Evaluating the Toxicity of Household Substances. (Washington, DC: 
National Academy of Sciences, 1977).
    (6) National Academy of Science. ``Strategies to determine needs and 
priorities for toxicity testing,'' Appendix 3B. Reference Protocol 
Guidelines For Neurobehavioral Toxicity Tests. 2: 123-129 (1982).