[Code of Federal Regulations]
[Title 40, Volume 14]
[Revised as of July 1, 2003]
From the U.S. Government Printing Office via GPO Access
[CITE: 40CFR79.64]

[Page 560-562]
 
                   TITLE 40--PROTECTION OF ENVIRONMENT
 
         CHAPTER I--ENVIRONMENTAL PROTECTION AGENCY (CONTINUED)
 
PART 79--REGISTRATION OF FUELS AND FUEL ADDITIVES--Table of Contents
 
            Subpart F--Testing Requirements for Registration
 
Sec. 79.64  In vivo micronucleus assay.

    (a) Purpose. The micronucleus assay is an in vivo cytogenetic test 
which uses erythrocytes in the bone marrow of rodents to detect chemical 
damage to the chromosomes or mitotic apparatus of mammalian cells. As 
the erythroblast develops into an erythrocyte (red blood cell), its main 
nucleus is extruded and may leave a micronucleus in the cell body; a few 
micronuclei form under normal conditions in blood elements. This assay 
is based on an increase in the frequency of micronucleated erythrocytes 
found in bone marrow from treated animals compared to that of control 
animals. The visualization of micronuclei is facilitated in these cells 
because they lack a main nucleus.
    (b) Definitions. For the purposes of this section the following 
definitions apply:
    Micronuclei mean small particles consisting of acentric fragments of 
chromosomes or entire chromosomes, which lag behind at anaphase of cell 
division. After telophase, these fragments may not be included in the 
nuclei of daughter cells and form single or multiple micronuclei in the 
cytoplasm.
    Polychromatic erythrocyte (PCE) means an immature red blood cell 
that, because it contains RNA, can be differentiated by appropriate 
staining techniques from a normochromatic erythrocyte (NCE), which lacks 
RNA. In one to two days, a PCE matures into a NCE.
    (c) Test method--(1) Principle of the test method. (i) Groups of 
rodents are exposed by the inhalation route for a minimum of 6 hours/day 
over a period of not less than 28 days to three or more concentrations 
of a test substance in air. Groups of animals are sacrificed at the end 
of the exposure period and femoral bone marrow is extracted. The bone 
marrow is then smeared onto glass slides, stained, and PCEs are scored 
for micronuclei. Researchers may need to run a trial at the highest 
tolerated concentration of the test atmosphere to optimize the sample 
collection time for micronucleated cells.
    (ii) This assay may be done separately or in combination with the 
subchronic toxicity study, pursuant to the provisions in Sec. 79.62.
    (2) Species and strain. (i) The rat is the recommended test animal. 
Other rodent species may be used in this assay, but use of that species 
will be justified by the tester.
    (ii) If a strain of mouse is used in this assay, the tester shall 
sample peripheral blood from an appropriate site on the test animal, 
e.g., the tail vein, as a source of normochromatic erythrocytes. Results 
shall be reported as outlined later in this guideline with 
``normochromatic'' interchanged for ``polychromatic'', where specified.
    (3) Animal number and sex. At least five female and five male 
animals per experimental/sample and control group shall be used. The use 
of a single sex or a smaller number of animals shall be justified.
    (4) Positive control group. A single concentration of a compound 
known to produce micronuclei in vivo is adequate as a positive control 
if it shows a significant response at any one time point; additional 
concentration levels may be used. To select an appropriate concentration 
level, a pilot or trial study may be advisable. Initially, one 
concentration of the test substance may be used, the maximum tolerated 
dose or that producing some indication of toxicity, e.g., a drop in the 
ratio of polychromatic to normochromatic erythrocytes. Intraperitoneal 
injection of 1,2-dimethyl-benz-anthracene or benzene are examples of 
positive control exposures. A concentration of 50-80 percent of an LD50 
may be a suitable guide.
    (d) Test performance--(1) Inhalation exposure. (i) All data 
developed within this study shall be in accordance with good laboratory 
practice provisions under Sec. 79.60.
    (ii) The general conduct of this study shall be in accordance with 
the vehicle emissions inhalation exposure guideline in Sec. 79.61.
    (2) Preparation of slides and sampling times. Within twenty-four 
hours of the

[[Page 561]]

last exposure, test animals will be sacrificed. One femur from each test 
animal will be removed and placed in fetal bovine serum. The bone marrow 
is removed, cells processed, and two bone marrow smears are made for 
each animal on glass microscope slides. The slides are stained with 
acridine- orange (AO) or another appropriate stain (Giemsa + Wright's, 
etc.) and examined under a microscope.
    (3) Analysis. Slides shall be coded for study before microscopic 
analysis. At least 1,000 first-division erythrocytes per animal shall be 
scored for the incidence of micronuclei. Sexes will be analyzed 
separately.
    (e) Data and report--(1) Treatment of results. In addition to the 
reporting requirements specified under Secs. 79.60 and 79.61, the final 
test report must include the criteria for scoring micronuclei. 
Individual data shall be presented in a tabular form including both 
positive and negative controls and experimental groups. The number of 
polychromatic erythrocytes scored, the number of micronucleated 
erythrocytes, the percentage of micronucleated cells, and, where 
applicable, the percentage of micronucleated erythrocytes shall be 
listed separately for each experimental and control animal. Absolute 
numbers shall be included if percentages are reported.
    (2) Interpretation of data. (i) There are several criteria for 
determining a positive response, one of which is a statistically 
significant dose-related increase in the number of micronucleated 
polychromatic erythrocytes. Another criterion may be based upon 
detection of a reproducible and statistically significant positive 
response for at least one of the test substance concentrations.
    (ii) A test substance which does not produce either a statistically 
significant dose-related increase in the number of micronucleated 
polychromatic erythrocytes or a statistically significant and 
reproducible positive response at any one of the test points is 
considered nonmutagenic in this system.
    (3) Test evaluation. (i) Positive results in the micronucleus test 
provide information on the ability of a chemical to induce micronuclei 
in erythrocytes of the test species under the conditions of the test. 
This damage may have been the result of chromosomal damage or damage to 
the mitotic apparatus.
    (ii) Negative results indicate that under the test conditions the 
test substance does not produce micronuclei in the bone marrow of the 
test species.
    (f) Test report. In addition to the reporting recommendations as 
specified under Sec. 79.60, the following specific information shall be 
reported:
    (1) Test atmosphere concentration(s) used and rationale for 
concentration selection.
    (2) Rationale for and description of treatment and sampling 
schedules, toxicity data, negative and positive controls.
    (3) Historical control data (negative and positive), if available.
    (4) Details of the protocol used for slide preparation.
    (5) Criteria for identifying micronucleated erythrocytes.
    (6) Micronucleus analysis by animal and by group for each 
concentration (sexes analyzed separately).
    (i) Ratio of polychromatic to normochromatic erythrocytes.
    (ii) Number of polychromatic erythrocytes with micronuclei.
    (iii) Number of polychromatic erythrocytes scored.
    (7) Statistical methodology chosen for test analysis.
    (g) References. For additional background information on this test 
guideline, the following references should be consulted.
    (1) 40 CFR 798.5395, In Vivo, Mammalian Bone Marrow Cytogenetics 
Tests: Micronucleus Assay.
    (2) Cihak, R. ``Evaluation of Benzidine by the Micronucleus Test.'' 
Mutation Research, 67: 383-384 (1979).
    (3) 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.
    (4) Heddle, J.A., et al. ``The Induction of Micronuclei as a Measure 
of Genotoxicity. A Report of the U.S. Environmental Protection Agency 
Gene-Tox Program.'' Mutation Research, 123:61-118 (1983).

[[Page 562]]

    (5) Preston, J.R. et al. ``Mammalian In Vivo and In Vitro 
Cytogenetics Assays: Report of the Gene-Tox Program.'' Mutation 
Research, 87:143-188 (1981).
    (6) Schmid, W. ``The micronucleus test for cytogenetic analysis'', 
Chemical Mutagens, Principles and Methods for their Detection. Vol. 4 
Hollaender A, (Ed. A ed. (New York and London: Plenum Press, (1976) pp. 
31-53.
    (7) Tice, R.E., and Al Pellom ``User's guide: Micronucleus assay 
data management and analysis system'', NTIS Order no. PB-90-212-598AS.