Validation Study on Five Cytotoxicity Assays by JSSSE — I. Overview of The Study And Analyses of Variations of ED50 Values

Tadao Ohno¹, Masumi Asakura², Takumi Awago³, Yoshihiro Futamura⁴, Akihiro Harihara⁵, Masato Hatao⁶, Chiharu Hattori⁷, Akira Hayasaka⁸, Makoto Hayashi⁹, Takashi Yahashi¹⁰, Zenzou Hirata⁴, Hiroshi Hori¹¹, Hiroyoshi Hoshi¹², Koichi Imai¹³, Ikuo Imazeki⁸, Takuya Ishibashi¹¹, Hiroshi Itagaki⁶, Tatsuo Iwata¹⁴, Miyako Kakuma¹⁵, Shinya Kaneda¹⁶, Ikuo Kato⁵, Mayako Kato¹, Tooru Kawahatsu¹⁷, Akio Kawakami¹⁸, akemi Kazama¹⁹, Akihiko Kido²⁰, Shigemi Kimura²¹, Michiyo Kitazawa²², Shigefumi Kogiso²³, Hajime Kojima²⁴, Mayumi Kotani²⁵, Masahiro Kuramochi²¹, Daijyo Maki²⁶, Masako Matsuda²⁷, Chihomi Mitsuoka²⁶, Satoru Miyazaki²⁸, Fumio Mizuno²⁰, Minako Mori²⁹, Katsunori Morimoto³⁰, Matsuko Moriysu²⁶, Keiya Nakajima²⁹, Madoka Nakajima²², Masaaki Nakamura¹³, Masaki Nakamura⁴, Nahoko Nakano²⁶, Shinobu Nakamura²⁶, Akira Inegami³¹, Masato Nishino¹⁹, Tamotsu Nishitomi²⁰, Kenji Ohkoshi³², Yuuko Okamoto³², Takashi Omori³⁵, Hiroshi Ono³⁴, Makoto Ono¹⁵, Yuko Osanai¹⁵, Kaoru Saijo¹, Yuka Sano³⁵, Kyoko Saotome³⁶, Kazunori Sasaki⁵, Tetsuji Sasaki¹, Hidetaka Sato³⁷, Shinichiro Sato³⁸, Hiroyasu Shimada⁷, Shoichiro Shimogo⁶, Kazuyuki Shimono¹⁶, Hiroshi Shionoya¹⁴, Hideaki Sugawara²⁸, Yasuko Sugiki³⁹, Shigeo Sugimoto⁴⁰, Shigeru Sugimoto³⁷, Junko Suzuki³⁷, Kinya Takagaki⁴, Ken Takahashi⁴¹, Minoru Takizawa⁴², Chieko Tamaki²³, Noriho Tanaka³⁴, Jirou Taniya³⁴, Noriko Teramoto¹, Hisashi Torishima⁴⁴, Toshiyuki Tsuchiya⁴⁵, Motoo Uejima¹⁶, Hayao Ueno⁴⁶, Yuji Ugai²², Shogo Wada⁴⁷, Shinobu Wakuri³⁴, Xinhai Wang¹, Ikuo Watanabe²⁹, Masami Watanabe⁴⁸, Satoshi Yajima⁴⁹, Yoshiki Yamagata¹⁹, Yuuzou Yamaguchi⁴⁹, Osamu yamakita⁴⁰, Ryohei Yamamoto⁴⁴, Masanori Yoshida⁴⁶, Isao Yoshimura³³, Kouichi Yuhki⁴³, Shiro Yukiyama⁴⁰

¹ RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukaba Science City, Ibaraki 305;
² Japan Bioassay Laboratory, 2445 Hirasawa, Hadano-shi, Kanagawa 257;
³ Drug Safety Research Center, Tokushima Research Institute, Otsuka Pharmaceutical Co., Ltd., 463-10 Kagasuno, Kawauchi-cho, Tokushima-shi, 771-01;
⁴ Toxicology Laboratory, Mochida Pharmaceutical Co., Ltd., 342 Gensuke, Fujieda-shi, Shizuoka 426;
⁵ Shionogi Research Laboratories, Shionogi & Co., Ltd., 3-1-1 Futaba-cho, Toyonaka-shi, Osaka 561;
⁶ Shiseido Safety & Analytical Research Center,1050 Nippa-cho, Kohoku-Ku, Yokohama-shi 223;
⁷ Developmental Research Laboratory, Daiich Pharmaceutical Co., Ltd., 1-16-13 Kita-Kasai, Edogawa-ku, Tokyo 134;
⁸ Chugai Pharmaceutical Co., Ltd., 3-41-8 Takada, Toshima-ku, Tokyo 171;
⁹ Division of Genetics and Mutagenesis, Mitamura Building, 26-1 Kaitaicho, Shinjuku-ku, Tokyo 112-0012 Japan 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158;
¹⁰ Ina Research Inc., 8047 Nishiminowa, Ina-shi, Nagano 399-45;
¹¹ Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga-shi, Futui 914;
¹² Research Institute for the Functional Peptides, 11-26 Minamisanban-cho, Yamagata-shi 990;
¹³ Department of Biomaterials, Osaka Dental University, 1-5-31 Otemae, Chuo-ku, Osaka-shi 540;
¹⁴ Department of Drug Safety Research, R & D Division, Eisai Co., Ltd., Takehaya, Kawashima-cho, Hashima-gun, Gifu 501-61;
¹⁵ Beauty Care Products Laboratory, Nagase, Co., Ltd., 5-1 Kobuna-cho, Nihonbashi, Chuo-ku, Tokyo 103;
¹⁶ Naruto Research Institute, Otsuka Pharmaceutical Factory, Inc., 115 Kuguhara, Tateiwa, Muya-cho, Naruto-shi, Tokushima 772;
¹⁷ Pharmaceutical Research Center, Meiji Seika Kaisha, Ltd., 760 Morooka-cho, Kohoku-ku, Yokohama-shi 222;
¹⁸ Safety Research Laboratories, Yamanouchi Pharmaceutical Co., Ltd., 1-1-8 Azusawa, Itabashi-ku, Tokyo 174;
¹⁹ Biochemical Development & Production, Oriental Yeast Co., Ltd., 4-4-1 Minamisuita, Suita-shi, Osaka 564;
²⁰ Kashima Laboratory, Mitsubishi-kagaku Institute of Environmental and Toxicological Sciences, 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki 314-02;
²¹ BML Inc., 1361-1 Matoba, Kawagoe-shi, Saitama 350;
²² Biosafety Research Center, Foods, Drugs and Pesticides, 582-2 Arahama, Shioshinden, Fukude-cho, lwata-gun, Shizuaka 437-12;
²³ Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., 3-1-98 Kasugadanaka, Konohana-ku, Osaka-shi 554;
²⁴ Biochemical Research Institute, Nippon Menard Cosmetic Co., Ltd., 2-7 Torimi-cho, Nishi-ku, Nagoya-shi 451;
²⁵ R&D Planning HQ, Sunstar Inc., 3-1 Asahi-machi, Takatsuki-shi, Osaka 569;
²⁶ Panapharm Laboratories Co., Ltd. 1285 Kurisaki-cho, Uto-shi, Kumamoto 869-04;
²⁷ Research and Development Center, Duskin Co., Ltd., 4-19-8 Minamisuita, Suita-shi, Osaka 564;
²⁸ Life Science Research Information Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01;
²⁹ The Center of Japan Biological Chemistry, 52 Fukue, Kaizu-cho, Kaizu-gun, Gifu 503-06;
³⁰ Takara Belmont Co., Ltd., 5-1 Takamatsu, Kouzai-cho, Koga-gun, Shiga 520-32;
³¹ Deparment of Obstetrics and Gynecology, Fukui Medical School, 23-3 Shimoaizuki, Matsuoka-cho, Yoshida-gun, Futui 910-11;
³² Division of Fundamental Research, KOSE Co., 1-18-4 Azusawa, Itabashi-ku, Tokyo 174;
³³ Department of Management Science, Faculty of Engineering, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162;
³⁴ Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257;
³⁵ Biotechnology Research Laboratories, TAKARA SHUZO Co., Ltd., 3-4-1 Seta, Ohtsu-shi, Shiga 520-21;
³⁶ Yokohama City Institute of Health, 1-2-17 Takigashira, Isogo-ku, Yokohama-shi 235;
³⁷ Japan Food Research Laboratories, 6-11-1O Nagayama, Tama-shi, Tokyo 206;
³⁸ Division of Radiation Medicine, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi 263;
³⁹ Menicon Co., Ltd., 5-1-10 Takamoridai, Kasugai-shi, Aichi 487;
⁴⁰ Drug Safety Laboratory, Taiho Pharmaceutical Co., Ltd., 224-2 Ebisuno, Hiraishi, Kawauchi-Cho, Tokushima-shi 771-01;
⁴¹ The Institute of Environmental Toxicology, 4321 Uchimoriya-cho, Mitsukaido-shi, Ibaraki 303;
⁴² Hakko Electric Machine Works Co. Ltd., 3055 Togura, Toguramachi, Hanishina-gun, Nagano 389-08;
⁴³ Koken Bioscience Institute, Koken Co., Ltd., 2-11-12 Nakane, Meguro-ku, Tokyo 152;
⁴⁴ Kurabo Industries Ltd., 14-41 Shimokida-cho, Neyagawa-shi, Osaka 572;
⁴⁵ Safety Evaluation Center, Central Research Laboratory, Showa Denko K K, 1-1-1 Ohnodai, Midori-ku, Chiba-shi 267;
⁴⁶ Takeda Analytical Research Laboratories, Ltd., 2-17-85 Juso-honmachi, Yodogawa-ku, Osaka 532;
⁴⁷ Wako Pure Chemical Industries, Ltd., 6-1 Takadacho, Amagasaki-shi, Hyogo 661;
⁴⁸ Division of Radiation Biology, Department of Health Science, Faculty of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki-shi 852;
⁴⁹ Central Research Laboratory, Takasago International Co., 1-4-11 Nishiyawata, Hiratsuka-shi, Kanagawa 254, Japan
(pp. 1-38)

Validation Study on Five Cytotoxicity Assays by JSAAE — II. Statistical Analysis

Takashi Omori¹, Kaoru Saijo², Mayako Kato², Hiroshi Itagaki³, Makoto Hayashi⁴, Satoru Miyazaki⁵, Tadao Ohno², Hideaki Sugawara⁵, Noriko Teramoto², Noriho Tanaka⁶, Shinobu Wakuri⁶ and Isao Yoshimura<sup>1

1</sup> Department of Management Science, Faculty of Engineering, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162;
² RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukaba Science City, Ibaraki 305;
³ Shiseido Safety & Analytical Research Center,1050 Nippa-cho, Kohoku-Ku, Yokohama-shi 223; ⁴ Division of Genetics and Mutagenesis, Mitamura Building, 26-1 Kaitaicho, Shinjuku-ku, Tokyo 112-0012 Japan 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158;
⁵ Life Science Research Information Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01;
⁶ Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257
(pp. 39-58)

Validation Study on Five Cytotoxicity Assays by JSAAE — III. Quality of Collected Data Files

Takashi Omori¹, Kaoru Saijo², Mayako Kato², Hiroshi Itagaki³, Makoto Hayashi⁴, Satoru Miyazaki⁵, Tadao Ohno², Hideaki Sugawara⁵, Noriko Teramoto², Noriho Tanaka⁶, Shinobu Wakuri⁶, and Isao Yoshimura¹

¹Department of Management Science, Faculty of Engineering, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162;
²RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyodai Tsukuba Science City, Ibaraki 305;
³Shiseido Safety & Analytical Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama-shi 223;
⁴Division of Genetics and Mutagenesis, Mitamura Building, 26-1 Kaitaicho, Shinjuku-ku, Tokyo 112-0012 Japan 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158;
⁵Life Science Research Information Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01;
⁶Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257, Japan
(pp. 59-73)

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