ATLA::Alternatives to Laboratory Animals

Volume 27, Number 4

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.

ATLA 27, 685-702, July/August 1999

Toshiyuki Tsuchiya,¹ Makoto Umeda,² Hiroshi Nishiyama,³ Isao Yoshimura,³ Shozo Ajimi,⁴ Masumi Asakura,⁵ Hiroshi Baba,⁶ Yasuaki Dewa,⁷ Youji Ebe,⁸ Yuici Fushiwaki,⁹ Shuichi Hamada,¹⁰ Tetsuo Hamamura,⁹ Makoto Hayashi,¹¹ Yumiko Iwase,⁶ Yoshitsugu Kajiwara,⁴ Yasushi Kasahra,⁷ Masayoshi Kawabata,¹² Emiko Kitada,¹³ Kinya Kubo,¹³ Kaori Mashiko,⁷ Daisaku Mirua,¹⁴ Fukutaro Mizuhashi,¹⁵ Fumio Mizuno,¹⁶ Madokanakajima,¹⁴ Yoshiyuki Nakamura,¹⁷ Naoko Nobe,¹⁰ Hidetoshi Oishi,¹⁸ Erina Ota,¹⁶ Ayako Sakai,¹¹ Miho Sato,¹⁷ Sawako Shimada,¹⁴ Toshie Sugiyama,⁵ Chitose Takahashi,¹² Yuko Takeda,⁶, Noriho Tanaka,² Chikako Toyoizumi,¹⁹ Takeki Tsutsui,²⁰ Shinobu Wakuri,² Satoshi Yajima²¹ And Nobuhiro Yajima¹²

¹Safety Evaluation Center, Central Research Laboratory, Showa Denko K.K., 1-1-1 Ohnodai, Midori-ku, Chiba 267-0056, Japan; ²Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257-0025, Japan; ³Faculty of Engineering, Science University of Tokyo, Kagurazaka, Tokyo 162-8601, Japan; ⁴Hita Research Laboratories Chemical Biotesting Center, Chemicals Inspection and Testing Institute, 822, 3-chome, Ishii-machi, Hita, Oita 877-0061, Japan; ⁵Japan Bioassay Research Center, 2445 Hirasawa, Hadano, Kanagawa 257-0015, Japan; ⁶Toxicology Laboratory, Yokohama Research Center, Mitsubishi Chemical Corporation, 1000 Kamoshida-cho, Aoba-ku, Yokohama 227-8502, Japan; ⁷Research Center, Kyorin Pharmaceutical Co. Ltd. 1848 Nogi, Nogi-machi, Shimotsuga-gun, Tochigi 329-0114, Japan; ⁸Life Sciences Laboratory, Performance Materials R&D Center, Mitsui Chemicals Inc., 1144 Togo, Mobara-shi, Chiba 297-0017, Japan; ⁹Water Section, Kanagawa Environmental Research Center, 842 Nakaharashimojuku, Hiratsuka, Kanagawa 254-0072, Japan; ¹⁰Central Research Laboratories, SSP Co. Ltd. 1143 Nanpeidai, Narita, Chiba 286-8511, Japan; ¹¹National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan; ¹²Snow Brand Milk Products Co. Ltd. 519 Shimoishibashi, Ishibashi-machi, Shimotsuga-gun, Tochigi 329-0512, Japan; ¹³Pharmaceutical Development Laboratory, Kirin Brewery Co. Ltd. 2-2 Souja-machi 1-chome, Maebashi-shi Gunma 371-0853, Japan; ¹⁴Experimental Toxicology Department, Biosafety Research Center, Foods, Drugs and Pesticides, 582-2 Shioshinden, Fukude-cho, Iwatagun, Shizuoka 437-1213, Japan; ¹⁵Kumiai Chemical Industry Co. Ltd. 3360 Kamo, Kikugawacho, Ogasa-gun, Shizuoka 439-0031, Japan; ¹⁶Kashima Laboratory, Mitsubishi Chemical Safety Institute Ltd. 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki 314-0255, Japan; ¹⁷School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Shizuoka-si Shizuoka 422-8002, Japan; ¹⁸Developmental Research Laboratories, Dainippon Pharmaceutical Co. Ltd. Enoki 3394, Suita, Osaka 564-0053, Japan; ¹⁹Chemical Products Quality Assurance Center, Canon Inc., 30-2 Shimomarubo 3-chome, Ohta-ku, Tokyo 146-8501, Japan; ²⁰School of Dentistry at Tokyo, The Nippon Dental University, 1-9-20 Fujimi, Chiyoda-ku, Tokyo 102-8159, Japan; ²¹Central Research Laboratory, Takasago International Corporation, 4-11, 1-chome Nishiyawata, Hiratsuka, Kanagawa 254-0073, Japan.

SUMMARY

The Non-genotoxic Carcinogen Study Group of the Ennronmental 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, instead 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 0.1 µg/ml TPA, all laboratories observed significant dosedependent increases in the number of transformed foci with increasing MCA concentrations. In the second series of experiments, venous 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 0.2 µg/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.

Keywords: improved cell transformation assay, Balb/c 3T3 cells, MCA, TPA, interlaboratory ualidation study