REPORT FROM WORKING GROUP ON IN-VITRO TESTS FOR CHROMOSOMAL-ABERRATIONS

The following summary represents a consensus of the working group exce pt where noted. The items discussed are listed in the order in which t hey appear in the OECD guideline (473) for easy reference. Metabolic a ctivation. S9 from animals induced either with Aroclor 1254 or with th e combination of phenobarbital with beta-naphthoflavone is acceptable, and other systems could be used with suitable justification. Exposure concentrations. The upper limit of testing should be 10 mM (or 5 mg/m l where molecular weight is not known or mixtures are being tested), w hichever is lower. Where this limit is inappropriate the investigator should give detailed justification of the choice of top concentration. Cytotoxicity should be measured not only in range-finding tests but a lso concurrently with the assay for chromosomal aberrations. Cytotoxic ity. should be assessed by measurements of cell growth such as cell co unts or confluence estimation. Mitotic index data alone are not a suff icient measure of cytotoxicity, except in the case of blood cultures f or which other methods are impractical. Cytotoxicity at the top dose s hould be greater than 50% of concurrent negative/solvent controls, if this can be achieved without exceeding a concentration limit of 10 mM or 5 mg/ml. There should be at least three concentrations scored for a berrations (each with and without S9), covering a toxicity range down to a concentration giving little or no cytotoxicity. This will usually mean that the concentrations scored will be quite closely spaced. It was not possible to reach a consensus on the issue of solubility limit s. The group did not agree on whether (a) solubility rather than cytot oxicity should be the limiting factor, such that only one top dose wit h evident precipitate should be scored even if toxicity is not observe d, or (b) several concentrations with evident precipitate should be sc ored far aberrations if this were necessary to obtain cytotoxicity. It was agreed that evidence of precipitation should be determined in the final culture medium. Controls. Concurrent positive controls are requ ired but the working group thought it inappropriate to specify the con trol chemicals or the degree of response that should be obtained, leav ing it up to the test laboratory to demonstrate that the system was wo rking adequately based on historical data within the laboratory. It is not necessary to include both negative and solvent controls concurren tly with the aberration test; solvent controls alone are acceptable pr ovided that the laboratory has data to demonstrate that there is no ef fect of the solvent on baseline values. Preparation of cultures. A maj ority agreed that there need be no requirement for replicate cultures for each treatment in a test. Treatment length with test substance and culture harvest time. Treatment length both with and without S9 shoul d be for 3-6 h, followed by sampling at a time equivalent to about 1.5 normal cell cycle lengths from the beginning of treatment. If this pr otocol gives negative results both with and without S9, an additional test without S9 should be done, with continuous treatment for about 1. 5 normal cell cycle lengths. Certain chemicals may be more readily det ected by treatment/sampling times longer than 1.5 cycle lengths, e.g., some nucleoside/tide analogs may give stronger responses with longer treatments, and some nitrosamides give higher aberration yields at lat e sampling times in Chinese hamster cells.