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

Original paper :AATEX 5(1-2):1-38,1998

Abstract
In October 1992, the Japanese Society of Alternatives to Animal Experiments (JSAAE) organized an interlaboratory validation study on cytotoxicity assays for development of an alternative(s) to the in vivo Draize eye irritation test. The main purpose is to evaluate practicability of five proposed cytotoxicity assays with two cell lines each through a large scale inter-laboratory assessment. The five assays were colony formation (CF) assay, crystal-violet staining (CV) assay, lactate dehydrogenase release (LDH) assay, neutral red uptake (NR) assay, and MTT assay. They were selected because of their popularity in Japan. We chose six detergents as model chemicals for this first step validation. One of the six chemicals, which was revealed to be Tween 20 after breaking the code, was additionally included in the doubly-mask-coded chemical set for simultaneous evaluation of intra-laboratory variation. Technology transfer for all the assays was made cumulatively 118 times to less-experienced laboratories. Concomitantly, we performed the Draize test to confirm toxicity of the coded chemicals in vivo.
A total of 3,810 final data files including preliminary test results were submitted from 42 laboratories. Of 1,535 raw data files with final definitive assay results, 292 files were rejected because of not only apparent misunderstanding of the protocols provided by the Working Group but also for violating pre-set common rules. Acceptability of data files was also examined by a computer-assisted logistic analysis program (LAP-JSAAE) with a six step-wise check code to detect abnormality in the data file. After generating ED50 values through the program, 5 data file sets of 7 tested chemicals were judged unreliable since the large differences in ED50 values were found for the same but differently coded test chemical, Tween 20. This clearly indicates considerable intra-laboratory variation. After excluding these data files, analyses of inter-laboratory variation were made on 969 data files with the box-whiskand-er plot analysis.
The important results of our study are as follows:(1)CF, CV, MTT, and NR assays are recommendable from the view point of performance of these assays. Performance rate of each assay was calculated on the number of finally accepted assay data files divided by the expected number of data files. The highest rate was for the CF assay with BALB/3T3 AS 1-1-1 cells followed by the CV assay with two cell lines. Lower performance rates were observed in the sub-divided LDH assays. The performance rate was considered to reflect simplicity of the method and labor needed for the assay;(2)From the view point of the intralaboratory variation of the same but differently coded chemical Tween 20, medians of the log(ED50) values of each assay were satisfactorily close;(3)After eliminating the sub-divided LDH assays which gave a small number of acceptable data files per assay and therefore resulted in unstable hinge-spread of log(ED50) values, the CV assay with CHL cells and the MTT assay with SQ-5 cells were found to have given the smallest mean hinge-spread of log(ED50) followed closely by the CF assay with HeLa S3 (SC) cells and the CV assay with HeLa S3 (SC) cells. These assays were therefore considered to give small interlaboratory variation;(4)The CF assay with HeLa S3 (SC) cells resulted in the largest "power for distinction (PFD)" of toxicities between the least and the most toxic chemicals defined as the ratio of difference in medians of log(ED50) values of two chemicals to the mean hinge-spread, followed by the CV assay with HeLa S3 (SC) cells. However, the CF assay is not necessarily advantageous as far as distinguishing moderately irritating chemicals from non-irritating chemicals;(5)Medians of log(ED50) values enabled us to classify tested chemicals into at least three categories, namely, non-, moderately-, and highly-cytotoxic chemicals. Non- and highly-cytotoxic chemicals corresponded to non- and severe-irritants in the in vivo Draize test.
(6)Considering performance rate, inter-laboratory variation of data reflected on the mean hinge-spread, power for distinction of chemical cytotoxicity, time needed for an assay (i.e. the CF assay requires longer incubation time than others), and the data on the common cell line HeLa S3 (SC), we concluded that the CV assay is the most practical and recommendable as a part of alternatives to the in vivo Draize test.
Many problems that were revealed during the present validation study including human factors were discussed. The assay results will be further described in the ensuing articles in this issue, i. e., the calculation of ED50 values by LAP-JSAAE, problems on the CF, CV, LDH, MTT, and NR assays. A fact data base was constructed on the data files of this validation study which will be available on request.

Key words: alternatives, cytotoxicity assay, colony formation assay, crystal-violet staining assay, LDH assay, neutral red uptake assay, MTT assay, inter-laboratory validation, Draize eye imtation test.


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