[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: 40CFR798.5385]

[Page 197-199]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 798_HEALTH EFFECTS TESTING GUIDELINES--Table of Contents
 
                       Subpart F_Genetic Toxicity
 
Sec. 798.5385  In vivo mammalian bone marrow cytogenetics tests: 
Chromosomal analysis.

    (a) Purpose. The in vivo bone marrow cytogenetic test is a 
mutagenicity test for the detection of structural chromosomal 
aberrations. Chromosomal aberrations are generally evaluated in first 
post-treatment mitoses. With the majority of chemical mutagens, induced 
aberrations are of the chromatid type but chromosome type aberrations 
also occur.
    (b) Definitions. (1) Chromosome-type aberrations are changes which 
result from damage expressed in both sister chromatids at the same time.
    (2) Chromatid-type aberrations are damage expressed as breakage of 
single chromatids or breakage and/or reunion between chromatids.
    (c) Reference substances. Not applicable.
    (d) Test method--(1) Principle. Animals are exposed to test 
chemicals by appropriate routes and are sacrificed at sequential 
intervals. Chromosome preparations are made from bone marrow cells. The 
stained preparations are examined and metaphase cells are scored for 
chromosomal aberrations.
    (2) Description. The method employs bone marrow of laboratory 
rodents which have been exposed to test chemicals. Prior to sacrifice, 
animals are further treated with a spindle inhibitor, (e.g., colchicine 
or Colcemid[reg]) to arrest the cells in c-metaphase. Chromosome 
preparations from the cells are stained and scored for chromosomal 
aberrations.

[[Page 198]]

    (3) Animal selection--(i) Species and strain. Any appropriate 
mammalian species may be used. Examples of commonly used rodent species 
are rats, mice, and hamsters.
    (ii) Age. Healthy young adult animals shall be used.
    (iii) Number and sex. At least five female and five male animals per 
experimental and control group shall be used. Thus, 10 animals would be 
sacrificed per time per group treated with the test compound if several 
test times after treatment are included in the experimental schedule. 
The use of a single sex or smaller number of animals should be 
justified.
    (iv) Assignment to groups. Animals shall be randomized and assigned 
to treatment and control groups.
    (4) Control groups--(1) Concurrent controls. (i) Concurrent positive 
and negative (vehicle) controls shall be included in the assay.
    (ii) Positive controls. A single dose positive control showing a 
significant response at any one time point is adequate. A compound known 
to produce chromosomal aberrations in vivo shall be employed as the 
positive control.
    (5) Test chemicals--(i) Vehicle. When possible, test chemicals shall 
be dissolved in isotonic saline or distilled water. Water insoluble 
chemicals may be dissolved or suspended in appropriate vehicles. The 
vehicles used shall neither interfere with the test chemical nor produce 
toxic effects. Fresh preparations of the test compound should be 
employed.
    (ii) Dose levels. For an initial assessment, one dose of the test 
substance may be used, the dose being the maximum tolerated dose (to a 
maximum of 5,000 mg/kg) or that producing some indication of 
cytotoxicity (e.g., partial inhibition of mitosis) or shall be the 
highest dose attainable (to a maximum of 5,000 mg/kg). Additional dose 
levels may be used. For determination of dose-response, at least three 
dose levels should be used.
    (iii) Route of administration. The usual routes are oral or by 
intraperitoneal injection. Other routes may be appropriate.
    (iv) Treatment schedule. In general, test substances should be 
administered once only. However, based on toxicological information a 
repeated treatment schedule may be employed.
    (e) Test performance--(1) Generally the test may be performed in two 
assays. (i) Animals should be treated with the test substance once at 
the selected dose(s). Samples should be taken at three times after 
treatment. For rodents, the central sampling interval is 24 hours. Since 
cell cycle kinetics can be influenced by the test substance, one earlier 
and one later sampling interval adequately spaced within the range of 6 
to 48 hours shall be applied. Where the additional dose levels are 
tested in a subsequent experiment, samples shall be taken at the 
predetermined most sensitive interval or, if this is not established, at 
the central sampling time. If the most sensitive interval is known and 
documented with data, only this one time point shall be sampled.
    (ii) If a repeated treatment schedule is used at the selected 
dose(s), samples shall be taken 6 and 24 hours after the last treatment; 
other sampling times may be used if justified. Where the additional dose 
levels are tested in a subsequent experiment, samples shall be taken at 
the predetermined most sensitive interval or, if this is not 
established, at 6 hours after the last treatment.
    (2) Administration of spindle inhibitor. Prior to sacrifice, animals 
shall be injected IP with an appropriate dose of a spindle inhibitor 
(e.g., colchicine or Colcemid[reg]) to arrest cells in c-metaphase.
    (3) Preparation of slides. Immediately after sacrifice, the bone 
marrow shall be obtained, exposed to hypotonic solution, and fixed. The 
cells shall then be spread on slides and stained. Chromosome 
preparations shall be made following standard procedures.
    (4) Analysis. The number of cells to be analyzed per animal should 
be based upon the number of animals used, the negative control 
frequency, the predetermined sensitivity, and the power chosen for the 
test. Slides shall be coded before microscopic analysis.
    (f) Data and report--(1) Treatment of results. Data should be 
presented in tabular form for both cells and animals. Different types of 
structural chromosomal aberrations should be

[[Page 199]]

listed with their numbers and a mean frequency per cell for each animal 
in all treated and control groups. Gaps (achromatic lesions) should be 
recorded separately and not included in the total abberration frequency. 
Differences among animals within each group should be considered before 
making comparisons between treated and control groups.
    (2) Statistical evaluation. Data should be evaluated by appropriate 
statistical methods.
    (3) Interpretation of results. (i) There are several criteria for 
determining a positive result, one of which is a statistically 
significant dose-related increase in the number of structual chromosomal 
aberrations or abnormal metaphase figures. Another criterion may be 
based upon detection of a reproducible and statistically significant 
positive response for a least one of the test points.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of chromosomal 
aberrations or abnormal metaphase figures or a statistically significant 
and reproducible positive response at any one of the test points is 
considered nonmutagenic in this system.
    (iii) Both biological and statistical significance should be 
considered together in the evaluation.
    (4) Test evaluation. (i) Positive results in the in vivo bone marrow 
cytogenetics assay indicate that under the test conditions the test 
substance induces chromosomal aberrations in the bone marrow of the test 
species.
    (ii) Negative results indicate that under the test conditions, the 
test substance does not induce chromosomal aberrations in the bone 
marrow of the test species.
    (5) Test report. In addition to the reporting recommendations as 
specified under 40 CFR part 792, subpart J the following specific 
information shall be reported:
    (i) Species, strain, age, weight, number and sex of animals in each 
treatment and control group.
    (ii) Test chemical vehicle, dose levels used, rationale for dose 
selection.
    (iii) Route of administration, treatment and sampling schedules, 
toxicity data, negative and positive controls.
    (iv) Identity of spindle-inhibitor, its concentration and duration 
of treatment.
    (v) Details of the protocol used for chromosome preparation, number 
of cells scored per animal, type and number of aberrations given 
separately for each treated and control animal.
    (vi) Mitotic index, where applicable.
    (vii) Criteria for scoring aberrations.
    (viii) Number and frequency of aberrant cells per animal in each 
treatment and control groups.
    (ix) Total number of aberrations per group.
    (x) Number of cells with aberrations per group.
    (xi) Dose-response relationship, if applicable.
    (g) References. For additional background information on this test 
guideline the following references should be consulted:
    (1) Adler, I.D., Ramarao, G., Epstein, S.S. ``In vivo cytogenetic 
effects of trimethyl-phosphate and of TEPA on bone marrow cells of male 
rats,'' Mutation Research, 13:263-273 (1971).
    (2) Evans, H.J. ``Cytological methods for detecting chemical 
mutagens,'' Chemical Mutagens: Principles and Methods for Their 
Detection, Vol. 4. Ed. A. Hollaender (New York and London: Plenum Press, 
1976) pp. 1-29.
    (3) Kilian, J.D., Moreland, F.E. Benge, M.C., Legator, M.S., 
Whorton, E.B. Jr. ``A collaborative study to measure intralaboratory 
variation with the in vivo bone morrow metaphase procedure,'' Handbook 
of mutagenicity test procedures. Eds. Kilby, B.J., Legator, M. Nichols, 
C., Ramel, D., (Amsterdam: Elsevier/North Holland Biomedical Press, 
1977) 243-260.
    (4) Preston, J.R., Au, W., Bender, M.A., Brewen, J.G., Carrano, A.V. 
Heddle, J.A., McFee, A.F., Wolff, S., Wassom, J. ``Mammalian in vivo and 
vitro cytogenetics assays: Report of the Gene-Tox Program,'' Mutation 
Research, 87:143-188 (1981).

[50 FR 39397, Sept. 27, 1985, as amended at 52 FR 19080, May 20, 1987]

[[Page 200]]