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In Vivo Micronucleus Assay. Chronic Toxicity Study, Pursuant to the (A) Purpose

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In Vivo Micronucleus Assay. Chronic Toxicity Study, Pursuant to the (A) Purpose Environmental Protection Agency § 79.64 Program 13-week Studies.’’ Fundam. period and femoral bone marrow is ex- Appl. Toxicol. 11:343–358. tracted. The bone marrow is then (7) Russell, L.D., Ettlin, R.A., smeared onto glass slides, stained, and Sinhattikim, A.P., and Clegg, E.D PCEs are scored for micronuclei. Re- (1990) Histological and searchers may need to run a trial at Histopathological Evaluation of the the highest tolerated concentration of Testes, Cache River Press, Clearwater, the test atmosphere to optimize the FL. sample collection time for [59 FR 33093, June 27, 1994, as amended at 61 micronucleated cells. FR 36513, July 11, 1996] (ii) This assay may be done sepa- rately or in combination with the sub- § 79.64 In vivo micronucleus assay. chronic toxicity study, pursuant to the (a) Purpose. The micronucleus assay provisions in § 79.62. is an in vivo cytogenetic test which (2) Species and strain. (i) The rat is uses erythrocytes in the bone marrow the recommended test animal. Other of rodents to detect chemical damage rodent species may be used in this to the chromosomes or mitotic appa- assay, but use of that species will be ratus of mammalian cells. As the justified by the tester. erythroblast develops into an eryth- (ii) If a strain of mouse is used in this rocyte (red blood cell), its main nu- assay, the tester shall sample periph- cleus is extruded and may leave a eral blood from an appropriate site on micronucleus in the cell body; a few the test animal, e.g., the tail vein, as a micronuclei form under normal condi- source of normochromatic tions in blood elements. This assay is erythrocytes. Results shall be reported based on an increase in the frequency as outlined later in this guideline with of micronucleated erythrocytes found ‘‘normochromatic’’ interchanged for in bone marrow from treated animals ‘‘polychromatic’’, where specified. compared to that of control animals. (3) Animal number and sex. At least The visualization of micronuclei is fa- five female and five male animals per cilitated in these cells because they experimental/sample and control group lack a main nucleus. (b) Definitions. For the purposes of shall be used. The use of a single sex or this section the following definitions a smaller number of animals shall be apply: justified. Micronuclei mean small particles con- (4) Positive control group. A single con- sisting of acentric fragments of chro- centration of a compound known to mosomes or entire chromosomes, produce micronuclei in vivo is adequate which lag behind at anaphase of cell di- as a positive control if it shows a sig- vision. After telophase, these frag- nificant response at any one time ments may not be included in the point; additional concentration levels nuclei of daughter cells and form single may be used. To select an appropriate or multiple micronuclei in the concentration level, a pilot or trial cytoplasm. study may be advisable. Initially, one Polychromatic erythrocyte (PCE) concentration of the test substance means an immature red blood cell that, may be used, the maximum tolerated because it contains RNA, can be dif- dose or that producing some indication ferentiated by appropriate staining of toxicity, e.g., a drop in the ratio of techniques from a normochromatic polychromatic to normochromatic erythrocyte (NCE), which lacks RNA. erythrocytes. Intraperitoneal injection In one to two days, a PCE matures into of 1,2-dimethyl-benz-anthracene or ben- a NCE. zene are examples of positive control (c) Test method—(1) Principle of the test exposures. A concentration of 50–80 per- method. (i) Groups of rodents are ex- cent of an LD50 may be a suitable posed by the inhalation route for a guide. minimum of 6 hours/day over a period (d) Test performance—(1) Inhalation ex- of not less than 28 days to three or posure. (i) All data developed within more concentrations of a test sub- this study shall be in accordance with stance in air. Groups of animals are good laboratory practice provisions sacrificed at the end of the exposure under § 79.60. 587 VerDate Mar<15>2010 10:54 Sep 22, 2011 Jkt 223159 PO 00000 Frm 00597 Fmt 8010 Sfmt 8010 Y:\SGML\223159.XXX 223159 erowe on DSK2VPTVN1PROD with CFR § 79.64 40 CFR Ch. I (7–1–11 Edition) (ii) The general conduct of this study at any one of the test points is consid- shall be in accordance with the vehicle ered nonmutagenic in this system. emissions inhalation exposure guide- (3) Test evaluation. (i) Positive results line in § 79.61. in the micronucleus test provide infor- (2) Preparation of slides and sampling mation on the ability of a chemical to times. Within twenty-four hours of the induce micronuclei in erythrocytes of last exposure, test animals will be sac- the test species under the conditions of rificed. One femur from each test ani- the test. This damage may have been mal will be removed and placed in fetal the result of chromosomal damage or bovine serum. The bone marrow is re- damage to the mitotic apparatus. moved, cells processed, and two bone (ii) Negative results indicate that marrow smears are made for each ani- under the test conditions the test sub- mal on glass microscope slides. The stance does not produce micronuclei in slides are stained with acridine- orange the bone marrow of the test species. (AO) or another appropriate stain (f) Test report. In addition to the re- (Giemsa + Wright’s, etc.) and examined porting recommendations as specified under a microscope. under § 79.60, the following specific in- (3) Analysis. Slides shall be coded for formation shall be reported: study before microscopic analysis. At (1) Test atmosphere concentration(s) least 1,000 first-division erythrocytes used and rationale for concentration per animal shall be scored for the inci- selection. dence of micronuclei. Sexes will be (2) Rationale for and description of analyzed separately. treatment and sampling schedules, tox- (e) Data and report—(1) Treatment of icity data, negative and positive con- results. In addition to the reporting re- trols. quirements specified under §§ 79.60 and (3) Historical control data (negative 79.61, the final test report must include and positive), if available. the criteria for scoring micronuclei. In- (4) Details of the protocol used for dividual data shall be presented in a slide preparation. tabular form including both positive (5) Criteria for identifying and negative controls and experimental micronucleated erythrocytes. groups. The number of polychromatic (6) Micronucleus analysis by animal erythrocytes scored, the number of and by group for each concentration micronucleated erythrocytes, the per- (sexes analyzed separately). centage of micronucleated cells, and, (i) Ratio of polychromatic to where applicable, the percentage of normochromatic erythrocytes. micronucleated erythrocytes shall be (ii) Number of polychromatic listed separately for each experimental erythrocytes with micronuclei. and control animal. Absolute numbers (iii) Number of polychromatic shall be included if percentages are re- erythrocytes scored. ported. (7) Statistical methodology chosen (2) Interpretation of data. (i) There are for test analysis. several criteria for determining a posi- (g) References. For additional back- tive response, one of which is a statis- ground information on this test guide- tically significant dose-related in- line, the following references should be crease in the number of consulted. micronucleated polychromatic (1) 40 CFR 798.5395, In Vivo, Mamma- erythrocytes. Another criterion may be lian Bone Marrow Cytogenetics Tests: based upon detection of a reproducible Micronucleus Assay. and statistically significant positive (2) Cihak, R. ‘‘Evaluation of Benzi- response for at least one of the test dine by the Micronucleus Test.’’ Muta- substance concentrations. tion Research, 67: 383–384 (1979). (ii) A test substance which does not (3) Evans, H.J. ‘‘Cytological Methods produce either a statistically signifi- for Detecting Chemical Mutagens.’’ cant dose-related increase in the num- Chemical Mutagens: Principles and ber of micronucleated polychromatic Methods for Their Detection, Vol. 4. erythrocytes or a statistically signifi- Ed. A. Hollaender (New York and Lon- cant and reproducible positive response don: Plenum Press, 1976) pp. 1–29. 588 VerDate Mar<15>2010 10:54 Sep 22, 2011 Jkt 223159 PO 00000 Frm 00598 Fmt 8010 Sfmt 8010 Y:\SGML\223159.XXX 223159 erowe on DSK2VPTVN1PROD with CFR Environmental Protection Agency § 79.65 (4) Heddle, J.A., et al. ‘‘The Induction and cells are harvested, fixed and of Micronuclei as a Measure of stained before scoring for SCEs. Re- Genotoxicity. A Report of the U.S. En- searchers may need to run a trial at vironmental Protection Agency Gene- the highest tolerated concentration of Tox Program.’’ Mutation Research, the test atmosphere to optimize the 123:61–118 (1983). sample collection time for second divi- (5) Preston, J.R. et al. ‘‘Mammalian sion metaphase cells. In Vivo and In Vitro Cytogenetics As- (ii) This assay may be done sepa- says: Report of the Gene-Tox Pro- rately or in combination with the sub- gram.’’ Mutation Research, 87:143–188 chronic toxicity study, pursuant to the (1981). provisions in § 79.62. (6) Schmid, W. ‘‘The micronucleus (2) Description. (i) The method de- test for cytogenetic analysis’’, Chem- scribed here employs peripheral blood ical Mutagens, Principles and Methods lymphocytes (PBL) of laboratory ro- for their Detection. Vol. 4 Hollaender dents exposed to the test atmosphere. A, (Ed. A ed. (New York and London: (ii) Within twenty-four hours of the Plenum Press, (1976) pp. 31–53. last exposure, test animal lymphocytes (7) Tice, R.E., and Al Pellom ‘‘User’s are obtained by heart puncture and du- guide: Micronucleus assay data man- plicate cell cultures are started for agement and analysis system’’, NTIS each animal. Cultures are grown in Order no.
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