Genotoxicity is commonly evaluated during the chemical safety assessment together with other toxicological endpoints. The micronucleus test is always included in many genotoxic test guidelines for long time in many classes of chemicals, e.g., pharmaceutical chemicals, agricultural chemicals, food additives. Although the trend of the safety assessment of chemicals faces to animal welfare and in vitro systems are more welcome than the in vivo systems, the in vivo test systems are paid more attention in the field of genotoxicity because of its weight of evidence. In this review, I will summarize the following points: 1) historical consideration of the test development, 2) characteristics of the test including advantages and limitations, 3) new approaches considering to the animal welfare.

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The micronucleus test-most widely used in vivo genotoxicity test.

The mouse has traditionally been used for the micronucleus test, with bone marrow the usual target organ. The aim of the 9th collaborative study by CSGMT was to evaluate the suitability of the rat for the micronucleus test, with bone marrow and peripheral blood as the target organ. Since the rat spleen eliminates circulating micronucleated erythrocytes, a rat peripheral blood micronucleus assay might not be feasible. Thirty-four Japanese laboratories and six overseas laboratories participated in this collaboration, and 40 chemicals were studied. As a rule, rat bone marrow and peripheral blood were analyzed using acridine orange staining. Among 36 mouse micronucleus-positive rat carcinogens, 34 of which had been evaluated by CSGMT, we observed 33 positive and three negative results with rat bone marrow and 30 positive, three equivocal, and three negative responses with rat peripheral blood. Of the two mouse micronucleus-negative rat carcinogens, acrylonitrile was positive in rat bone marrow and 4,4'-methylene bis(2-chloroaniline) was negative in both rat bone marrow and peripheral blood. Two chemicals reported to be mouse micronucleus-negative and rat-positive, azobenzene and Solvent Yellow 14, and one chemical reported to be mouse-positive and rat-negative, 1,2-dimethylhydrazine, gave positive responses in rat bone marrow and peripheral blood. The concordance between bone marrow and peripheral blood with rats was 92%. The concordance between rat and mouse erythrocytes was 88%. We concluded that the rat micronucleus assay, using either bone marrow or peripheral blood, can be used as an alternative to the mouse micronucleus assay.

Evaluation of the rat micronucleus test with bone marrow and peripheral blood: Summary of the 9th collaborative study by CSGMT/JEMS. MMS

During almost 40 years of use, the micronucleus assay (MN) has become one of the most popular methods to assess genotoxicity of different chemical and physical factors, including ionizing radiation-induced DNA damage. In this minireview, we focus on the position of MN among the other genotoxicity tests, its usefulness in different applications and visibility by international organizations, such as International Atomic Energy Agency, Organization for Economic Co-operation and Development and International Organization for Standardization. In addition, the mechanism of micronuclei formation is discussed. Finally, foreseen directions of the MN development are pointed, such as automation, buccal cells MN and chromothripsis phenomenon.

https://www.mdpi.com/1422-0067/21/4/1534/pdf

Micronucleus Assay: The State of Art, and Future Directions

To examine whether micronucleus tests can be incorporated into general toxicology assays, we performed micronucleus tests applying the treatment protocols typically used in such assays. In this 13th Collaborative Study of the CSGMT, both rats and mice were tested, although rats were used in the majority of the studies. Fifteen mutagens were tested in rats, mainly by oral (p.o.) administration. Micronucleus induction was evaluated 2, 3, and 4 days, and 1, 2, 3, and 28 days after the beginning of the treatment in the peripheral blood, and at 28 days in the bone marrow. Of the 15 chemicals that induced micronuclei in rats in short-term assays, two chemicals (1,2-dimethylhydrazine.2HCl and mitomycin C) were negative in all our experiments, possibly because of insufficient dose levels. The remaining 13 were positive within the estimated dose range of a general toxicology assay, suggesting the possibility of integrating the micronucleus assay into general toxicology assays. Three patterns were observed in micronucleus induction during the period of repeated treatment: (1) gradual increases in micronucleus frequency with sequential doses, (2) a peak at 3-5 days followed by gradual decreases in micronucleus frequency with sequential doses, and (3) a rapid increase in micronucleus frequency followed by a plateau. We evaluated factors that might have been involved in those patterns, such as the spleen function, target organ exposure, extramedullary hematopoiesis, hypothermia, and hypoxia. Another factor we considered was dosage. Because the dosages employed in a general toxicity assay are usually lower than those used in short-term micronucleus assays, this discrepancy was considered the greatest potential problem for integrating the micronucleus assay into general toxicology assays. Our results indicate that the integration of the micronucleus assay into a 28-day toxicological assay is feasible. To serve this purpose, blood samples collected 4 days after the beginning of treatment and blood and bone marrow samples collected at autopsy should be examined. Furthermore, although it is recognized that mice may be suitable for performing independent micronucleus assays, we propose that rats can provide biologically important and relevant information regarding potential chemical mutagens that can be evaluated under conditions used in the conduct of general toxicology studies.

Evaluation of the rodent micronucleus assay by a 28-day treatment protocol: Summary of the 13th Collaborative Study by the Collaborative Study Group for the Micronucleus Test (CSGMT)/Environmental Mutagen Society of Japan (JEMS)-Mammalian Mutagenicity Study Group (MMS).

The incidence of cancer increases with age in humans and in laboratory animals alike. There are different patterns of age-related distribution of tumors in different organs and tissues. Aging may increase or decrease the susceptibility of various tissues to initiation of carcinogenesis and usually facilitates promotion and progression of carcinogenesis. Aging may predispose to cancer by several mechanisms: (1) tissue accumulation of cells in late stages of carcinogenesis; (2) alterations in homeostasis, in particular, alterations in immune and endocrine system and (3) telomere instability linking aging and increased cancer risk. Increased susceptibility to the effects of tumor promoters is found both in aged animals and aged humans, as predicted by the multistage model of carcinogenesis. Available evidence supporting the relevance of replicative senescence of human cells and telomere biology to human cancer seems quite strong, however, the evidence linking cellular senescence to human aging is controversial and required additional studies. Data on the acceleration of aging by carcinogenic agents as well as on increased cancer risk in patients with premature aging are critically discussed. In genetically modified mouse models (transgenic, knockout or mutant) characterized by the aging delay, the incidence of tumors usually similar to those in controls, whereas the latent period of tumor development is increased. Practically all models of accelerated of aging in genetically modified animals show the increase in the incidence and the reduction in the latency of tumors. Strategies for cancer prevention must include not only measures to minimize exposure to exogenous carcinogenic agents, but also measures to normalize the age-related alterations in internal milieu. Life-span prolonging drugs (geroprotectors) may either postpone population aging and increase of tumor latency or decrease the mortality in long-living individuals in populations and inhibit carcinogenesis. At least some geroprotectors may increase the survival of a short-living individuals in populations but increase the incidence of malignancy.

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The relationship between aging and carcinogenesis: a critical appraisal.

The spontaneous frequencies of micronucleated reticulocytes (MNRETs) were examined monthly over the life spans of animals belonging to nine mouse strains for the 7th collaborative study organized by the CSGMT/JEMS.MMS. Both sexes of the BDF1 strain and females of the A/J strain showed a statistically significant increase in mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-1, B6C3F1, SAMR1, and MS/Ae, did not show significant age-related differences in mean of MNRET frequencies. More extensive statistical analyses are underway, and the outcomes will be reported separately.

Effect of aging on spontaneous micronucleus frequencies in peripheral blood of nine mouse strains: the results of the 7th collaborative study organized by CSGMT/JEMS · MMS*

The spontaneous frequencies of micronucleated reticulocytes (MNRETs) were examined monthly over the life spans of animals beloning to nine mouse strains for the 7th collaborative study organized by the CSGMT/JEMS · MMS. Both sexes of the BDF1 strain and females of the A/J strain showed a statistically significant increase ni mean spontaneous MNRET frequency in their last month of life, suggesting the possibility of strain-specific, age-dependent chromosomal instability. SAMP6/Tan, an accelerated senescence-prone strain, showed the same tendency, although it was not statistically significant. The other strains studied, ddY, CD-1, B6C3F1, SAMR1, and MS/Ae, did not show significant age-related differences in mean of MNRET frequencies. More extensive statistical analyses are underway, and the outcomes will be reported separately.

Effect of aging on spontaneous micronucleus frequencies in peripheral blood of nine mouse strains: the results of the 7th collaborative study organized by CSGMT/JEMS · MMS: Mutation Res. 338 (1995) 51–57

Takafumi Higashiguchi

Papers

Effect of aging on spontaneous micronucleus frequencies in peripheral blood of nine mouse strains: the results of the 7th collaborative study organized by CSGMT/JEMS · MMS*

Effect of aging on spontaneous micronucleus frequencies in peripheral blood of nine mouse strains: the results of the 7th collaborative study organized by CSGMT/JEMS · MMS: Mutation Res. 338 (1995) 51–57