Subject(s)
Hazardous Substances/toxicity , Animals , Benz(a)Anthracenes/toxicity , Bleomycin/toxicity , CHO Cells , Cell Count , Cell Survival/drug effects , Cricetinae , Cricetulus , Cyclophosphamide/toxicity , Dactinomycin/toxicity , Dose-Response Relationship, Drug , Etoposide/toxicity , Female , Griseofulvin/toxicity , Hydrogen Peroxide/toxicity , Hydroxyurea/toxicity , Methyl Methanesulfonate/toxicity , Mitomycin/toxicity , Phenol/toxicity , Vinblastine/toxicityABSTRACT
The in vitro micronucleus test is currently used as a screening assay during the early stages of drug development by pharmaceutical companies to identify chemicals likely to produce positive outcomes in the in vitro chromosome aberration assay. For several reasons the assay is being considered as an alternative to the aberration assay-it requires less laboratory time, less material and less training. However, the current screening protocols are not rigorous enough to fully satisfy concerns about genotoxic safety. Using a protocol previously developed by testing 16 chemicals, this manuscript contributes to the validation of the protocol using 10 additional chemicals. Furthermore, conclusions drawn from the developmental effort regarding the need for an extended exposure in the absence of metabolic activation, the number of cells to be counted, and the preferred statistical procedure for the assay are re-examined. The recommended, validated protocol utilizes cytochalasin B and 4h exposures in the presence and in the absence of metabolic activation, specifies the need to test to a relative survival rate of approximately 50%, requires the counting of 2,000 binucleated cells per treatment concentration, and employs a trend test for statistical analysis of the data.
Subject(s)
Micronucleus Tests/methods , Micronucleus Tests/standards , Animals , CHO Cells , Cell Division/drug effects , Cell Division/genetics , Cricetinae , Cricetulus , Cytochalasin B/toxicity , Data Interpretation, Statistical , Dose-Response Relationship, Drug , Mutagens/toxicityABSTRACT
The in vitro micronucleus (IVM) test is currently used as a screen during the early stages of pharmaceutical development to identify chemicals likely to produce positive outcomes in the in vitro chromosome aberration assay. For several reasons, the assay is being considered as an alternative to the aberration assay, but the current screening protocols are not rigorous enough to fully satisfy concerns about genotoxic safety. This manuscript describes the investigation of several protocol parameters to assist with the development of a regulatory guideline for the IVM test. The parameters investigated are: the effect of cytochalasin B on the outcome of the assay when conducted with continually growing cell lines; the need for an extended exposure in the absence of metabolic activation; and the number of cells to be counted for a valid assay. In addition, two statistical procedures for the analysis of data from the test are described. The results of the investigation indicate that cytochalasin B does not effect the outcome of the test, that the extended exposure treatment is not necessary, that counting 2000 cells is preferable to counting 1000, and that the data can be appropriately analyzed using a trend test.
