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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


Toshiyuki Tsuchiya, Makoto Umeda, Hiroshi Nishiyama, Isao Yoshimura, Shozo Ajimi, Masumi Asakura, Hiroshi Baba, Yasuaki Dewa, Youji Ebe, Yuichi Fushiwaki, Shuichi Hamada, Tetsuo Hamamura, Makoto Hayashi, Yumiko Iwase, Yoshitsugu Kajiwara, Yasushi Kasahara, Masayoshi Kawabata, Emiko Kitada, Kinya Kubo, Kaori Mashiko, Daisaku Miura, Fukutaro Mizuhashi, Fumio Mizuno, Madoka Nakajima, Yoshiyuki Nakamura, Naoko Nobe, Hidetoshi Oishi, Erina Ota, Ayako Sakai, Miho Sato, Sawako Shimada,1Toshie Sugiyama, Chitose Takahashi, Yuko Takeda, Noriho Tanaka, Chikako Toyoizumi, Takeki Tsutsui, Shinobu Wakuri, Satoshi Yajima and Nobuhiro Yajima

The Non-genotoxic Carcinogen Study Group of the Environmental Mutagen Society of Japan organised the first step of an interlaboratory validation study on an improved cell transformation assay employing Balb/c 3T3 A31-1-1 cells. Nineteen laboratories participated in this study. The modified transformation assay was evaluated for its responsiveness, its interlaboratory reproducibility 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) was used during the period of expression of transformed foci, intead of the usual minimum essential medium with 10% FBS. 20-Methylcholanthrene (MCA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) were selected as a prototype initiator and a tumour promoter, respectively. Two series of experiments were conducted. In the first series, the transformation activity of MCA was examined at various concentrations. In the absence of the promoting treatment with TPA, exposure to MCA only weakly induced transformed foci. In the presence of 0.1μg/ml TPA, all laboratories observed significant dose-dependent increases in the number of transformed foci with increasing MCA concentrations. 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 the presence of 0.2μg/ml MCA, all the laboratories observed significant dose-dependent increases in the number of transformed foci with increasing TPA concentrations. The results from this study support the usefulness of this modified two-stage transformation assay with Balb/c 3T3 cells.