Application of the improved BALB/c 3T3 cell transformation assay to the examination of the initiating and promoting activities of chemicals: the second interlaboratory collaborative study by the non-genotoxic carcinogen study group of Japan.

The Non-genotoxic Carcinogen Study Group in the Environmental Mutagen Society of Japan organised the second step of the inter-laboratory collaborative study on one-stage and two-stage cell transformation assays employing BALB/c 3T3 cells, with the objective of confirming whether the respective laboratories could independently produce results relevant to initiation or promotion. The method was modified to use a medium consisting of DMEM/F12 supplemented with 2% fetal bovine serum and a mixture of insulin, transferrin, ethanolamine and sodium selenite, at the stationary phase of cell growth. Seventeen laboratories collaborated in this study, and each chemical was tested by three to five laboratories. Comparison between the one-stage and two-stage assays revealed that the latter method would be beneficial in the screening of chemicals. In the test for initiating activity with the two-stage assay (post-treated with 0.1microg/ml 12-O-tetradecanoylphorbol-13-acetate), the relevant test laboratories all obtained positive results for benzo[a]pyrene and methylmethane sulphonate, and negative results for phenanthrene. Of those laboratories assigned phenacetin for the initiation phase, two returned positive results and two returned negative results, where the latter laboratories tested up to one dose lower than the maximum dose used by the former laboratories. In the exploration of promoting activity with the twostage assay (pretreated with 0.2microg/ml 3-methylcholanthrene), the relevant test laboratories obtained positive results for mezerein, sodium orthovanadate and TGF-beta1, and negative results for anthralin, phenacetin and phorbol. Two results returned for phorbol 12,13-didecanoate were positive, but one result was negative - again, the maximum dose to achieve the latter result was lower than that which produced the former results. These results suggest that this modified assay method is relevant, reproducible and transferable, provided that dosing issues, such as the determination of the maximum dose, are adequately considered. The application of this two-stage assay for screening the initiating and promoting potential of chemicals is recommended for consideration by other research groups and regulatory authorities.

Verification of the BALB/c 3T3 cell transformation assay after improvement by using an ITES-medium.

We carried out the first-step verification study on our ITES-medium-improved BALB/c 3T3 cell transformation assay. In order to estimate its potential use as a short-term screening method for putative carcinogens, 31 chemicals were tested in the improved transformation assay. The test chemicals consisted of 18 carcinogens and 13 noncarcinogens. The present improved transformation assay did not use an exogenous metabolizing system. Data analysis was carried out on 34 chemicals, including assay data for three chemicals reported previously by the authors. As a result, the improved transformation assay showed a concordance of 73.5% with a rodent bioassay, a sensitivity for carcinogens of 71.4%, and a specificity for detection of noncarcinogens of 76.9%. The improved transformation assay detected all of the genotoxic carcinogens, and five of 11 nongenotoxic carcinogens as positive. It can be expected that the improved transformation assay will be able to detect not only genotoxic carcinogens with high probability but also approximately 50% of nongenotoxic carcinogens within about 3 weeks. Hence, these preliminary findings suggest that our improved transformation assay will be a reliable and useful short-term test procedure of screening for potential carcinogens, and it encourages us to conduct further experiments on many carcinogens and noncarcinogens.