[Code of Federal Regulations]
[Title 40, Volume 30]
[Revised as of July 1, 2004]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR798.6200]
[Page 214-217]
TITLE 40--PROTECTION OF ENVIRONMENT
CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
Subpart G_Neurotoxicity
Sec. 798.6200 Motor activity.
(a) Purpose--(1) General. In the assessment and evaluation of the
toxic characteristics of a substance, determination of the effects of
administration of the substance on motor activity is useful when
neurotoxicity is suspected.
(2) Acute Motor Activity Test. The purpose of the acute motor
activity test is to examine changes in motor activity
[[Page 215]]
occurring over a range of acute exposure levels. These changes may then
be evaluated in the context of changes occurring in other organ systems.
This test is an initial step in determining the potential of a substance
to produce acute neurotoxicity and may be used to screen members of a
class of substances for known neurotoxicity, and/or to establish a
dosage regimen prior to the initiation of subchronic neurotoxicity
testing.
(3) Subchronic Motor Activity Test. The purpose of the subchronic
motor activity test is to determine whether the repeated administration
of a suspected neurotoxicant results in changes in motor activity. These
changes may be evaluated in the context of changes occurring in other
organ systems. This test is an initial step in determining the potential
of a substance to produce subchronic neurotoxicity.
(b) Definitions. (1) Neurotoxicity is the adverse effect on the
structure or function of the central and/or peripheral nervous system
related to exposure to a chemical substance.
(2) Motor activity is any movement of the experimental animal.
(3) A toxic effect is an adverse change in the structure or function
of an experimental animal as a result of exposure to a chemical
substance.
(c) Principle of the test method. The test substance is administered
to several groups of experimental animals, one dose being used per
group. Measurements of motor activity are made. The exposure levels at
which significant changes in motor activity are produced are compared to
those levels which produce toxic effects not originating in the central
and/or peripheral nervous system.
(d) Test procedures--(1) Animal selection--(i) Species and strain.
Testing shall be performed in a laboratory rat or mouse. The choice of
species should take into consideration such factors as the comparative
metabolism of the chemical and species sensitivity to the toxic effects
of the test substance, as evidenced by the results of other studies, the
potential for combined studies, and the availability of other toxicity
data for the species.
(ii) Age. Young adult animals (at least 42 days old for rat or
mouse) should be used.
(iii) Sex. (A) Equal numbers of animals of each sex are required for
each dose level for the motor activity test.
(B) The females shall be nulliparous and nonpregnant.
(2) Number of animals. Animals shall be randomly assigned to test
and control groups. Each test or control group must be designed to
contain a sufficient number of animals at the completion of the study to
detect a 40 percent change in activity of the test groups relative to
the control group with 90 percent power at the 5 percent level. For most
designs, calculations can be made according to Dixon and Massey (1957)
under paragraph (f)(1) of this section, Neter and Wasserman (1974) under
paragraph (f)(5) of this section, Sokal and Rohlf (1969) under paragraph
(f)(9) of this section, or Jensen (1972) under paragraph (f)(3) of this
section.
(3) Control groups. (i) A concurrent control group is required. This
group must be an untreated group, or, if a vehicle is used in
administering the test substance, a vehicle control group. If the toxic
properties of the vehicle are not known or cannot be made available,
both untreated and vehicle control group are required.
(ii) Positive control data are required to demonstrate the
sensitivity and reliability of the activity measuring device and testing
procedure. These data should demonstrate the ability to detect increases
or decreases in activity and to generate a dose-effect curve or its
equivalent using three values of the dose or equivalent independent
variable. A single administration of the dose (or equivalent) is
sufficient. It is recommended that chemical exposure be used to collect
positive control data. Positive control data shall be collected at the
time of the test study unless the laboratory can demonstrate the
adequacy of historical data for this purpose.
(iii) A satellite group may be treated with the high dose level for
90 days and observed for reversibility, persistence or delayed
occurrence of toxic effects for a post-treatment period of appropriate
length, normally not less than 28 days.
[[Page 216]]
(4) Dose levels and dose selection. At least 3 doses, equally spaced
on a log scale (e.g., \1/2\ log units) 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 effects on motor
activity 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.
(5) Duration of testing. The duration of exposure will be specified
in the test rule.
(6) Route of administration. The test substance shall be
administered by the method specified in the test rule. This will usually
be the route most closely approximating the route of human exposure. The
exposure protocol shall conform to that outlined in the appropriate
acute or subchronic toxicity study guideline.
(7) Combined protocol. The tests described herein 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) General. Motor activity must be monitored by
an automated activity recording apparatus. The device used must be
capable of detecting both increases and decreases in activity, i.e.
baseline activity as measured by the device must not be so low as to
preclude decreases nor so high as to preclude increases. Each device
shall be tested by standard procedure to ensure, to the extent possible,
reliability of operation across devices and across days for any one
device. In addition, treatment groups must be balanced across devices.
Each animal shall be tested individually. The test session shall be long
enough for motor activity to approach asymptotic levels by the last 20
percent of the session for most treatments and animals. All sessions
should have the same duration. Treatment groups shall be counter-
balanced across test times. Effort should be made to ensure that
variations in the test conditions 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. Tests shall be executed by an
appropriately trained individual.
(ii) Acute. Testing shall be timed to include the time of peak
signs.
(iii) Subchronic. All animals shall be tested prior to initiation of
exposure and at 30 2, 60 2
and 90 2 days during the exposure period. Testing
shall occur prior to the daily exposure. Animals shall be weighed on
each test day and at least once weekly during the exposure period.
(e) Data reporting and evaluation. In addition to the reporting
requirements specified under 40 CFR part 792, subpart J the final test
report must include the following information:
(1) Description of system and test methods. (i) Positive control
data from the laboratory performing the test which demonstrate the
sensitivity of the procedure being used.
(ii) Procedures for calibrating and assuring the equivalence of
devices and balancing treatment groups.
(2) Results. The following information must be arranged by test
group (dose level).
(i) In tabular form, data must be provided showing for each animal:
(A) Its identification number.
(B) Body weight, total session activity counts, and intrasession
subtotals for each date measured.
(ii) Group summary data should also be reported.
(3) Evaluation of data. An evaluation of the test results (including
statistical analysis comparing total activity counts at the end of
exposure of treatment vs control animals must be made and supplied. This
submission must include dose-effect curves for motor activity expressed
as activity counts.
(f) References. For additional background information on this test
guideline the following references should be consulted:
(1) Dixon, W.J., Massey, E.J. Introduction to Statistical Analysis
2nd Ed. (New York: McGraw-Hill, 1957).
(2) Finger, F.W. ``Measuring behavioral activity,'' Methods in Psy
ch o biology Vol. 2. Ed. R.D. Myers (New York: Academic, 1972) pp. 1-19.
[[Page 217]]
(3) Jensen, D.R. ``Some simultaneous multivariate procedures using
Hotelling's T2 Statistics,'' Biometrics, 28:39-53 (1972).
(4) Kinnard, E.J. and Watzman, N. ``Techniques utilized in the
evaluation of psychotropic drugs on animals activity,'' Journal of Phar
ma ceu ti cal Sciences, 55:995-1012 (1966).
(5) Neter, J. and Wasserman, W. Applied Linear Statistical Models.
Home wood, Richard D. Irwin, Inc., 1974.
(6) Reiter, L.E. ``Use of activity measures in behavioral
toxicology,'' Environmental Health Perspectives, 26:9-20 (1978).
(7) Reiter, L.W. and MacPhail, R.C. ``Motor Activity: A survey of
methods with potential use in toxicity testing,'' Neurobehavioral
Toxicology, 1: Suppl. 1, 53-66 (1979).
(8) Robbins, T.W. ``A critique of the methods available for the
measurement of spontaneous motor activity,'' Handbook of
Psychopharmacology. Vol. 7. Eds. Iversen, L.L., Iversen, D.S., Snyder,
S.H. (New York: Plenum, 1977) pp. 37-82.
(9) Sokal, R.P. and Rohlf, E.J. Biometry. (San Francisco: W.H.
Freeman and Co., 1969).
[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19082, May 20, 1987]