Chromosome damage tests

Mutagenicity Before human exposure, a bacterial mutation test (or in vitro mammalian cell mutation test), and a chromosomal damage test. A full battery of mutagenicity tests before Phase 111 clinical trials. 

An in-vitro test with mammalian cells (chromosomal damage or mouse lymphoma tk assay)... 

An in-vivo test in rodents (chromosomal damage in haematopoietic cells)... 

Monomethylhydrazine-induced mutagenesis was not observed in Ames Salmonella/ microsome with activation (Matheson et al. 1978). In vivo tests in mice (dominant lethal, revertants in host-mediated assay), and dogs (micronuclei) were negative (reviewed in Trochimowicz 1994). However, in vitro chromosomal damage in human and rat tissue has been demonstrated, although in vivo liver DNA damage (as determined by DNA alkaline elution) was equivocal (reviewed in Trochimowicz 1994). 

In vivo test for chromosomal damage using rodent hematopoietic cells Gene Mammalian In vivo... 

The in vitro cytogenetic assay is a short-term mutagenicity test for detecting chromosomal damage in cultured mammalian cells. 

Metaphase Analysis. Metaphase analysis can be performed in any tissue with actively dividing cells, but bone marrow is the tissue most often examined. Cells are treated with a test compound and are arrested in metaphase by the administration of colcemid or colchicine at various sampling times after treatment. Preparations are examined for structural chromosome damage. Because the bone marrow has a good blood supply, the cells should be exposed to the test compound or its metabolites in the peripheral blood supply. Additionally, these cells are sensitive to S-dependent and S-independent mutagens (Topham et al., 1983). 

Dean, B.J. and Danford, N. (1984). Assays for the detection of chemically induced chromosome damage in cultures mammalian cells. In Mutagenicity Testing. A Practical Approach, (Venitt, S. and Parry, J.M., Eds.). IRL Press, Oxford, pp. 187-232. 

Under Guideline S2B, the following standard test battery is recommended (1) a test for gene mutation in bacteria, (2) an in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells or an in vitro mouse lymphoma thymidine kinase (TK) assay, and (3) an in vivo test for chromosomal damage using rodent hematopoietic cells. 

The in vivo micronucleus test is used for the detection of damage to chromosomes as well as the mitotic apparatus in bone marrow or peripheral blood cells of rodents. The assay system has been well standardized.14-17 The basic features of the test system are (1) the effect of the test chemical is observed in anucleated polychromatic erythrocytes (PCEs) (2) PCEs have a relatively short lifespan, so that any micronuclei they contain must have been generated as a result of recently induced chromosome damage (3) micronuclei are readily identifiable and their distribution is well defined and (4) the frequency of induced micronuclei in PCEs is dependent on sampling times. 

Detection of chromosomal damage using in vitro method (mouse lymphoma tk test, a test to evaluate the potential of a drug to cause mutations to thymidine kinase [tk])... 

Animals, usually rodents, are exposed to the test substance by an appropriate route, usually by gavage or by intraperitoneal injection. Each treated and control group must include at least five animals per sex. Positive controls should produce micronuclei in vivo at exposure levels expected to give a detectable increase over background. No standard treatment schedule (i.e., 1, 2, or more treatments at 24-h intervals) has been recommended. Three dose levels are generally used these should cover a range from the maximum to little or no toxicity. The erythrocytes are sampled from the bone marrow and/or peripheral blood of the animals. If bone marrow is used, the animals are sacrificed at appropriate times after treatment, the bone marrow extracted, and preparations made and stained. When peripheral blood is used, the blood is collected at appropriate times after treatment and smear preparations are made and stained. Preparations are analyzed for the presence of micronuclei. An increase in the frequency of micronucleated polychromatic erythrocytes in treated animals is an indication of induced chromosome damage. 

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. 

Positive results from the in vitro micronucleus test indicate that the test substance induces chromosome damage and/or damage to the cell division apparams, in cultured mammahan somatic cells. Immunochemical labehng (FISH fluorescence in sim hybridization) of kinetochores, or hybridization with general or chromosome specific centromeric/telomeric probes can provide useful information on the mechanism of micronucleus formation. Use of cytokinesis block facilitates the acquisition of the additional mechanistic information (e.g., chromosome nondisjunction) that can be obtained by FISH techniques. The micronucleus assay has a number of advantages over metaphase analysis performed to measure chromosome aberrations (see OECD TG 487 draft). 

Mutagenicity - in vitro tests for mutagenicity and chromosome damage. Adapted from Scales ... 

An in vitro test with cytogenetic evaluation of chromosomal damage with mammalian cells OR an in vitro mouse lymphoma thymidine kinase assay... 

Before any human studies, results from two separate in vitro tests for mutation and chromosomal damage must be provided. The full test battery must be completed before initiation of Phase II trials. 

Mackay JM, Fox V, Griffiths K, et al Trichloroacetic acid Investigation into the mechanism of chromosomal damage in the in vitro human lymphocyte cytogenetic assay and the mouse bone marrow micronucleus test. Carcinogenesis 16(5) 1127-1133, 1995... 

CS has been tested for its capacity to induce mutations in bacteria and in the fruit fly Drosophila melanogaster and for its capacity to cause chromosomal damage, as measured by a micronucleus test, in mice. Its capacity to bind to DNA in mammalian liver and kidney has also been studied. The results have been predominantly negative. 

CS has been tested for its ability to cause chromosomal damage in a micronucleus test in mice. Micronucleus tests detect small nuclei that arise from chromosomal fragments or chromosomes that fail to be incorporated into normal daughter nuclei when cells divide. An increased frequency of micronuclei is evidence of chromosomal break-... 

Bis(bromomethyl)propane-l,3-diol was mutagenic in only one of several bacterial strains tested, and only with metabolic activation. In cultured mammalian cells, it was only weakly active in tests for chromosomal aberrations and sister chromatid exchanges. Micronucleus formation, indicative of chromosomal damage, was induced in cells from mice exposed to 2,2-bis(bromomethyl)propane-l,3-diol in vivo. 

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