[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: 40CFR798.5385]



[Page 197-200]

 

                   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.



[[Page 198]]



    (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.

    (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



[[Page 199]]



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 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



[[Page 200]]



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]