An interlaboratory validation study of the improved transformation assay employing Balb/c 3T3 cells: Results of a collaborative study on the two-stage cell transformation assay by the Non-genotoxic Carcinogen Study Group

The Non-genotoxic Carcinogen Study Group of the Environmental Mutagen Socie ty of Japan organised the first step of an interlaboratory validation study on an improved cell transformation assay employing Balb/c 3T3 A31-1-1 cell s. Nineteen laboratories participated in this study. The modified transform ation assay was evaluated for its responsiveness, its interlaboratory repro ducibility and its transferability. In this study, a mixture of Dulbecco's modified Eagle's medium and nutrient mixture F12, supplemented with insulin -transferrin-ethanolamine-sodium selenite and 2% fetal bovine serum (FBS) w as used during the period of expression of transformed foci, intead of the usual minimum essential medium with 10% FBS. 20-Methylcholanthrene (MCA) an d 12-O-tetradecanoylphorbol-13-acetate (TPA) were selected as a prototype i nitiator and a tumour promoter, respectively. Two series of experiments wer e conducted. In the first series, the transformation activity of MCA was ex amined at various concentrations. In the absence of the promoting treatment with TPA, exposure to MCA only weakly induced transformed foci. In the pre sence of 0.1 mu g/ml TPA, all laboratories observed significant dose-depend ent increases in the number of transformed foci with increasing MCA concent rations. In the second series of experiments, various concentrations of TPA were tested. In the absence of initiating treatment with MCA, exposure to TPA weakly induced transformed foci in about half of the laboratories. In t he presence of 0.2g/ml MCA, all the laboratories observed significant dose- dependent increases in the number of transformed foci with increasing TPA c oncentrations. The results from this study support the usefulness of this m odified two-stage transformation assay with Balb/c 3T3 cells.