[Code of Federal Regulations]

[Title 40, Volume 31]

[Revised as of July 1, 2006]

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).