EN 2000-32-EC pdf free download

EN 2000-32-EC pdf free download. MUTAGENICITY – IN VITRO MAMMALIAN CHROMOSOME ABERRATION TEST.
The purpose of the in vitro chromosomal aberration test is to identify agents that cause structural chromosome aberrations in cultured mammalian cells (1) (2) (3). Structural aberrations may be of two types, chromome or chromatid. With the majority of chemical mutagens, induced aberrations are of the chromatid type, but chromosome-type aberrations also occur. An increase in polyploidy may indicate that a chemical has the potential to induce numerical aberrations. However, this method is not designed to measure numerical aberrations and is not routinely used for that purpose. Chromosome mutations and related events are the cause of many human genetic diseases and there is substantial evidence that chromosome mutations and related events causing alterations in oncogenes and tumour suppressor genes of somatic cells are involved in cancer induction in humans and experimental animals. The in vitro chromosome aberration test may employ cultures of established cells lines, cell strains or primary cell cultures. The cells used are selected on the basis of growth ability in culture, stability of the karyotype, chromosome number, chromosome diversity and spontaneous frequency of chromosome aberrations. Tests conducted in vitro generally require the use of an exogenous source of metabolic activation. This metabolic activation system cannot mimic entirely the mammalian in vivo conditions. Care should be taken to avoid conditions which would lead to positive results which do not reflect intrinsic mutagenicity and may arise from changes in pH, osmolality or high levels of cytotoxicity (4) (5).This test is used to screen for possible mammalian mutagens and carcinogens. Many compounds that are positive in this test are mammalian carcinogens; however, there is not a perfect correlation between this test and carcinogenicity. Correlation is dependent on chemical class and there is increasing evidence that there are carcinogens that are not detected by this test because they appear to act through mechanisms other than direct DNA damage. See also General Introduction Part B.Exposure concentrations Among the criteria to be considered when determining the highest concentration are cytotoxicity, solubility in the test system and changes in pH or osmolality. Cytotoxicity should be determined with and without metabolic activation in the main experiment using an appropriate indication of cell integrity and growth, such as degree of confluency, viable cell counts, or mitotic index. It may be useful to determine cytotoxicity and solubility in a preliminary experiment. At least three analysable concentrations should be used. Where cytotoxicity occurs, these concentrations should cover a range from the maximum to little or no toxicity; this will usually mean the concentrations should be separated by no more than a factor between 2 and Ó10. At the time of harvesting, the highest concentration should show a significant reduction in degree of confluency, cell count or mitotic index (all greater than 50%). The mitotic index is only an indirect measure of cytotoxic/cytostatic effects and depends on the time after treatment. However, the mitotic index is acceptable for suspension cultures in which other toxicity measurements may be cumbersome and impractical. Information on cell cycle kinetics, such as average generation time (AGT), could be used as supplementary information AGT, however, is an overall average that does not always reveal the existence of delayed subpopulations, and even slight increases in average generation time can be associated with very substantial delay in the time of optimal yield of aberrations. For relatively non-cytotoxic substances, the maximum test concentration should be 5µl/ml, 5 mg/ml or 0,01 M, whichever is the lowest. EN 2000-32-EC  pdf download.