Validation Study on Five Cytotoxicity Assays by JSAAE -- VII. Details of The MTT Assay

Hiroshi Itagaki1, Tadao Ohno2, Masato Hatao1, Chiharu Hattori3, Akira Hayasaka4, Makoto Hayashi5, Hiroshi Hori6, Koichi Imai7, Ikuo Imazeki4, Takuya Ishibashi6, Miyako Kakuma8, Mayako Kato2, Akio Kawakami9, Michiyo Kitazawa10, Shigefumi Kogiso11, Satoru Miyazaki12, Minako Mori13, Keiya Nakajima13, Madoka Nakajima10, Masaaki Nakamura7, Takashi Omori14, Hiroshi Ono15, Makoto Ono8, Yuko Osanai8, Kaoru Saijo2, Yuka Sano16, Tetsuji Sasaki2, Shinichiro Sato17, Hiroyasu Shimada3, Shoichiro Shimogo1, Hideaki Sugawara12, Shigeo Sugimoto18, Minoru Takizawa19, Chieko Tamaki11, Noriho Tanaka15, Noriko Teramoto2, Toshiyuki Tsuchiya20, Hayao Ueno21, Yuji Ugai10, Shogo Wada22, Shinobu Wakuri15, Xinhai Wang2, Ikuo Watanabe13, Osamu Yamakita18, Masanori Yoshida21, Isao Yoshimura14, Shiro Yukiyama18

1Shiseido Safety & Analytical Research Center,1050 Nippa-cho, Kohoku-Ku, Yokohama-shi 223;
2RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukaba Science City, Ibaraki 305;
3Developmental Research Laboratory, Daiich Pharmaceutical Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134;
4Chugai Pharmaceutical Co., Ltd., 3-41-8 Takada, Toshima-ku, Tokyo 171;
5Division of Genetics and Mutagenesis, Mitamura Building, 26-1 Kaitaicho, Shinjuku-ku, Tokyo 112-0012 Japan 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158;
6Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga-shi, Futui 914;
7Department of Biomaterials, Osaka Dental University, 1-5-31 Otemae, Chuo-ku, Osaka-shi 540;
8Beauty Care Products Laboratory, Nagase, Co., Ltd., 5-1 Kobuna-cho, Nihonbashi, Chuo-ku, Tokyo 103;
9Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 1-1-8 Azusawa, Itabashi-ku, Tokyo 174;
10Biosafety Research Center, Foods, Drugs and Pesticides, 582-2 Arahama, Shioshinden, Fukude-cho, lwata-gun, Shizuaka 437-12;
11Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugadanaka, Konohana-ku, Osaka-shi 554;
12Life Science Research Information Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01;
13The Center of Japan Biological Chemistry, 52 Fukue, Kaizu-cho, Kaizu-gun, Gifu 503-06;
14Department of Management Science, Faculty of Engineering, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162;
15Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257;
16Biotechnology Research Laboratories, TAKARA SHUZO Co., Ltd., 3-4-1 Seta, Ohtsu-shi, Shiga 520-21;
17Division of Radiation Medicine, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi 263;
18Drug Safety Laboratory, Taiho Pharmaceutical Co., Ltd., 224-2 Ebisuno, Hiraishi, Kawauchi-Cho, Tokushima-shi 771-01;
19Hakko Electric Machine Works Co. Ltd., 3055 Togura, Toguramachi, Hanishina-gun, Nagano 389-08;
20Safety Evaluation Center, Central Research Laboratory, Showa Denko K K, 1-1-1 Ohnodai, Midori-ku, Chiba-shi 267;
21Takeda Analytical Research Laboratories, Ltd., 2-17-85 Juso-honmachi, Yodogawa-ku, Osaka 532;
22Wako Pure Chemical Industries, Ltd., 6-1 Takadacho, Amagasaki-shi, Hyogo 661

Original paper :AATEX 5(1-2):119-130,1998

Abstract
The inter-laboratory validation study on 5 cytotoxicity assays conducted by JSAAE has been described in the preceding articles (see Validation Articles I-VI in this issue). Presented here are precise data and the protocols on the MTT assay with two cell lines, HeLa S3 (SC) and SQ-5. Hand-plotted dose-response curves provided unexpected information otherwise difficult to assume from calculated ED50 values and accompanying outputs from the logistic analysis program, LAP-JSAAE. The characteristics of data files from 18 laboratories were visualized together with all the log(ED50) values. Variations on negative controls in the assay revealed technical competence characteristics of each laboratory. Generally low OD590 values have been obtained in the present MTT assay but high interlaboratory reproducibility was observed with both cell lines. These results will supplement our understanding on the MTT assay carried out in the large-scale inter-laboratory validation.

Key words: Key words: alternatives, cytotoxicity assay, inter-laboratory validation, JSAAE project, MTT assay.


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