Validation Study on Five Cytotoxicity Assays by JSAAE -- IV. Details of The Colony Formation Assay

Noriho Tanaka1, Masumi Asakura2, Chiharu Hattori3, Akira Hayasaka4, Makoto Hayashi5, Takashi Yahashi6, Hiroshi Hori7, Hiroyoshi Hoshi8, Ikuo Imazeki4, Takuya Ishibashi7, Hiroshi Itagaki9, Miyako Kakuma10, Shinya Kaneda11, Mayako Kato1, Akio Kawakami12, Akihiko Kido13, Michiyo Kitazawa14, Hajime Kojima15, Daijyo Maki16, Chihomi Mitsuoka16, Satoru Miyazaki17, Fumio Mizuno13, Matsuko Moriysu16, Madoka Nakajima14, Nahoko Nakano16, Shinobu Nakamura16, Tamotsu Nishitomi14, Tadao Ohno18, Masato Omori19, Hiroshi Ono1, Makoto Ono10, Yuko Osanai10, Kaoru Saijo18, Tetsuji Sasaki18, Hidetaka Sato20, Shinichiro Sato21, Hiroyasu Shimada3, Kazuyuki Shimono11, Hideaki Sugawara17, Yasuko Sugiki22, Shigeo Sugimoto20, Shigeru Sugimoto20, Junko Suzuki20, Ken Takahashi23, Minoru Takizawa24, Jirou Taniya24, Noriko Teramoto18, Toshiyuki Tsuchiya26, Motoo Uejima11, Hayao Ueno27, Yuji Ugai14, Shinobu Wakuri1, Xinahi Wang18, Masanori Yoshida27, Isao Yoshimura19, Kouichi Yuhki25

1Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257;
2Japan Bioassay Laboratory, 2445 Hirasawa, Hadano-shi, Kanagawa 257;
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;
6Ina Research Inc., 8047 Nishiminowa, Ina-shi, Nagano 399-45;
7Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga-shi, Futui 914;
8Research Institute for the Functional Peptides, 11-26 Minamisanban-cho, Yamagata-shi 990;
9Shiseido Safety & Analytical Research Center,1050 Nippa-cho, Kohoku-Ku, Yokohama-shi 223;
10Beauty Care Products Laboratory, Nagase, Co., Ltd., 5-1 Kobuna-cho, Nihonbashi, Chuo-ku, Tokyo 103;
11Naruto Research Institute, Otsuka Pharmaceutical Factory, Inc., 115 Kuguhara, Tateiwa, Muya-cho, Naruto-shi, Tokushima 772;
12Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 1-1-8 Azusawa, Itabashi-ku, Tokyo 174;
13Kashima Laboratory, Mitsubishi-kagaku Institute of Environmental and Toxicological Sciences, 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki 314-02;
14Biosafety Research Center, Foods, Drugs and Pesticides, 582-2 Arahama, Shioshinden, Fukude-cho, lwata-gun, Shizuaka 437-12;
15Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd., 2-7 Torimi-cho, Nishi-ku, Nagoya-shi 451;
16Panapharm Laboratories Co., Ltd. 1285 Kurisaki-cho, Uto-shi, Kumamoto 869-04;
17Life Science Research Information Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01;
18RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukaba Science City, Ibaraki 305;
19Department of Management Science, Faculty of Engineering, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162;
20Japan Food Research Laboratories, 6-11-1O Nagayama, Tama-shi, Tokyo 206;
21Division of Radiation Medicine, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi 263;
22Menicon Co., Ltd., 5-1-10 Takamoridai, Kasugai-shi, Aichi 487;
23The Institute of Environmental Toxicology, 4321 Uchimoriya-cho, Mitsukaido-shi, Ibaraki 303;
24Hakko Electric Machine Works Co. Ltd., 3055 Togura, Toguramachi, Hanishina-gun, Nagano 389-08;
25Koken Bioscience Institute, Koken Co., Ltd., 2-11-12 Nakane, Meguro-ku, Tokyo 152;
26Safety Evaluation Center, Central Research Laboratory, Showa Denko K K, 1-1-1 Ohnodai, Midori-ku, Chiba-shi 267;
27Takeda Analytical Research Laboratories, Ltd., 2-17-85 Juso-honmachi, Yodogawa-ku, Osaka 532

Original paper :AATEX 5(1-2):74-86,1998
Abstract
The inter-laboratory validation study on 5 cytotoxicity assays conducted by JSAAE has been described in the preceding articles in this issue. Presented here are precise data and the protocols on the colony formation (CF) assay with two cell lines, HeLa S3 (SC) that is common to the other 4 assays and BALB/3T3 A31-1-1 that is frequently used in this assay. The CF assay showed high performance rates with both HeLa S3 (SC) and BALB/3T3 A31-1-1 cell lines. Ninety-five percent of submitted data files were acceptable before ED50 calculation in this assay. Variations on negative controls in the assay revealed technical differences among laboratories and the characteristics of cells applied. The CF assay with BALB/3T3 A31-1-1 cells resulted in the lowest median values of log(ED50) for Tween 20, Tween 80 and cetylpyrimidinium chloride monohydrate. For propylene glycol assayed with HeLa S3 (SC) cells, the lowest median value of log(ED50) was observed. Thus the CF assay was able to detect with the highest sensitivity the least toxic chemical (propylene glycol) and the most severely toxic chemical (cetylpyrimidinium chloride monohydrate). CF assay with HeLa S3 (SC) cells gave the largest power for distinction when observing the most and the least toxic chemicals. However, the toxicities of moderately toxic chemicals were not sharply distinguishable from each other. Comparing the mean hinge-spreads, CF assay with BALB/3T3 A31-1-1 cells showed the largest variation of the log(ED50) values among all assay systems examined in this study. These results suggest that the CF assay is a highly sensitive assay but is influenced by many factors such as culture conditions and techniques of cell handling.

Key words: alternatives, colony formation assay, cytotoxicity assay, inter-laboratory validation


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