Erythrocyte micronucleus test

Purpose. The purpose of the in vivo micronucleus test is to evaluate the potential of the test substance to cause chromosomal damage (clastogenicity) or damage to the mitotic apparatus (aneugenicity) by the analysis of micronuclei in erythrocytes sampled in bone marrow and/or in peripheral blood of experimental animals (usually rodents) (Schmid 1975 Heddle 1973 Heddle and Salamone 1981). 

Regulatory Acceptance. The rodent erythrocyte micronucleus assay is relatively easy to conduct and is considered able to detect the vast majority of clastogens and rodent carcinogens (when combined with the Ames test) and all human genotoxic carcinogens (Shelby and Zeiger 1990 Rosenkranz and Cunningham 

This test is well-validated and widely accepted by regulatory agencies as part of the standard battery of genetic toxicity assays, in addition to the two in vitro assays (BrambiUa and Martelh 2004 Cimino 2006). Because of the mechanism of micronuclei formation, the micronucleus test does not in principle detect gene mutations. It should therefore be considered complementary to in vitro gene mutation assays, and not as a follow-up assay to confirm in vitro gene mutations. 

The experimental conditions and data interpretation are described in OECD guideline 474. It is the most commonly used in vivo assay. Many laboratories are highly experienced, and a large database has been generated for comparison with carcinogenicity and other effects. 

Typically, micronucleus induction is measured in bone marrow PCEs after acute treatment. After repeated administration, it is also advisable to evaluate the induction of micronuclei in mature or normochromatic erythrocytes (NCEs). As erythrocytes persist for about one month in peripheral blood (named reticulocytes), the measurement of micronucleated reticulocytes in peripheral blood is considered equally acceptable in any species, provided that the spleen does not remove the micronucleated erythrocytes from blood and that both aneugens and clastogens are efficiently detected (see more in assays limitations and confounding factors section). Micronuclei are generally analyzed in the youngest (i.e., immature) reticulocytes in peripheral blood. 

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Disulfoton was negative in a dominant lethal test in male mice treated orally with a single dose ot S mg/kg (Herbold 1980) and in an erythrocyte micronucleus test in mice dosed with 6 or 12 mg/k /day for 2 days (Herbold 1981). Disulfoton also did not induce micronuclei in erythrocytes of mice dosed with 2, 4, or 8 mg/kg disulfoton (EPA 1984a Sandhu et al. ]985), but whether disulfoton was administered by the oral or intraperitoneal route was not clear. Other genotoxicity studies are discussed in Section 2.4. 

Mammalian Erythrocyte Micronucleus Test (Updated Guideline, adopted 21 July 1997)... 

B.ll Mutagenicity - In vivo mammalian bone-marrow chromosome aberration test B.12 Mutagenicity mammalian erythrocyte micronucleus test B.20 Sex-Unked recessive lethal test in Drosophila melanogaster B.22 Rodent dominant lethal test... 

The mammalian in vivo erythrocyte micronucleus test OECD TG 474 US-EPA OPPTS 870.5395 EU Annex V B.12... 

Positive results in the mammalian in vivo erythrocyte micronucleus test indicate that a substance produces micronuclei in the immature erythrocytes of the test species, which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species. 

Twenty-seven out of 44 FDA-approved biopharmaceuticals have been tested in a battery of genotoxicity assays. Eighty-five different assays performed yielded negative results. The most commonly performed assays were the Ames test, the chromosomal aberration assay in human lymphocytes, the mouse lymphoma gene mutation assay, and the mammalian in vivo erythrocyte micronucleus test. Examples of the range of biopharmaceutical products tested include, domase alfa (deoxyribonuclease I-DNAse), trastuzumab (mAb to human epidermal growth factor receptor 2), alteplase (tissue plasminogen activator), infliximab (mAb to the human tumor necrosis factor a). 

Developmental rats, rabbits Prenatal and postnatal development none Genetic toxicology Ames test, in vitro mammalian chromosome aberration test in V79 Chinese hamster cells, and in vivo mammalian erythrocyte micronucleus test in rats... 

Mouse erythrocytes Micronucleus test - Tsuchimoto and Matter 1981... 

OECD (1997c). Mammalian erythrocyte micronucleus test. OECD Guideline for Testing of Chemicals 474, 1-10. 

Mammalian erythrocyte micronucleus test Mammalian erythrocyte Structural and numerical chromosome aherrations in somatic cells B12/TG474... 

OECD, 1997c, Test Guideline 474, Mammalian Erythrocyte Micronucleus Test. 

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