Validation Study on Five Cytotoxicity Assays by JSAAE -- V. Details of Crystal Violet Staining Assay

Hiroshi Itagaki1, Tadao Ohno2, Masato Hatao3, Makoto Hayashi4, Ikuo Imazeki3, Miyako Kakuma5, Mayako Kato2, Akio Kawakami6, Shigemi Kimura7, Hajime Kojima8, Mayumi Kotani9, Masahiro Kuramochi7, Daijyo Maki10, Chihomi Mitsuoka10, Satoru Miyazaki11, Matsuko Moriysu10, Nahoko Nakano10, Shinobu Nakamura10, Takashi Omori12, Hiroshi Ono13, Makoto Ono5, Yuko Osanai5, Kaoru Saijo2, Kyoko Saotome14, Tetsuji Sasaki2, Hidetaka Sato15, Shinichiro Sato16, Shoichiro Shimogo1, Hideaki Sugawara11, Shigeru Sugimoto15, Junko Suzuki15, Minoru Takizawa17, Noriho Tanaka13, Noriko Teramoto2, Shinobu Wakuri13, Xinhai Wang2, Satoshi Yajima18, Yuuzou Yamaguchi18, Isao Yoshimura12

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 Koyodai, Tsukaba Science City, Ibaraki 305;
3Chugai Pharmaceutical Co., Ltd., 3-41-8 Takada, Toshima-ku, Tokyo 171;
4Division of Genetics and Mutagenesis, Mitamura Building, 26-1 Kaitaicho, Shinjuku-ku, Tokyo 112-0012 Japan 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158;
5Beauty Care Products Laboratory, Nagase, Co., Ltd., 5-1 Kobuna-cho, Nihonbashi, Chuo-ku, Tokyo 103;
6Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 1-1-8 Azasawa, Itabashi-ku, Tokyo 174;
7BML Inc., 1361-1 Matoba, Kawagoe-shi, Saitama 350;
8Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd., 2-7 Torimi-cho, Nishi-ku, Nagoya-shi 451;
9R&D Planning HQ, Sunstar Inc., 3-1 Asahi-machi, Takatsuki-shi, Osaka 569;
10Panapharm Laboratories Co., Ltd. 1285 Kurisaki-cho, Uto-shi, Kumamoto 869-04;
11Life Science Research Information Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01;
12Department of ManaBement Science, Faculty of Engineering, Science University of Tokyo, 1-3 Kagurazaka, Shinjutu-ku, Tokyo 162;
13Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257;
14Yokohama City Institute of Health, 1-2-17 Takigashira, Isogo-ku, Yokohama-shi 235;
15Japan Food Research Laboratories, 6-11-10 Nagayama, Tama-shi, Tokyo 206;
16Division of Radiation Medicine, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi 263;
17Hakko Electric Machine Works Co. Ltd., 3055 Togura, Togura-machi, Hanishina-gun, Nagano 389-08;
18Central Research Laboratory, Takasago International Co., 1-4-11 Nishiyawata, Hiratsuka-shi, Kanagawa 254, Japan

Original paper :AATEX 5(1-2):87-98,1998

Abstract
The inter-laboratory validation study on 5 cytotoxicity assays conducted by JSAAE has been described in the preceding articles. Presented here are precise data and the protocols for the crystal violet staining (CV) assay with two cell lines, namely, HeLa S3 (SC) which is common to the other 4 assays, and CHL which has been utilized widely in this assay. Hand-plotted dose-response curves revealed information that enables us to easily detect abnormal data files. Characteristics of data files from 14 laboratories were visualized in a Figure together with all the log(ED50) values. Very low OD590 values were found in negative controls of submitted data, suggesting the need to carefully examine whether the results of the assay fell within the linear dose-response range or not. High interlaboratory reproducibility, therefore low inter-laboratory variation, was observed with both cell lines in the CV assay. Any influence of cell lines was not apparent in the tested chemicals except for cetylpyridinium chloride monohydrate. Results will supplement our understanding on the CV assay carried out in the large-scale interlaboratory validation.

Key words: alternatives, crystal-violet staining assay, cytotoxicity assay, inter-laboratory validation, JSAAE Project.


return