Validation Study on Five Cytotoxicity Assays by JSAAE -- VIII. Details of The Neutral Red Uptake Assay

Tadao Ohno1, Yoshihiro Futamura2, Akihiro Harihara3, Masato Hatao4, Akira Hayasaka5, Makoto Hayashi6, Zenzou Hirata2, Ikuo Imazeki5, Hiroshi Itagaki4, Tatsuo Iwata7, Miyako Kakuma8, Ikuo Kato3, Mayako Kato1, Aakio Kawakami9, Akemi Kazama10, Akihiko Kido11, Daijyo Maki12, Masako Matsuda13, Chihomi Mitsuoka12, Satoru Miyazaki14, Fumio Mizuno11, Minako Mori15, Katsunori Morimoto16, Matsuko Moriysu12, Keiya Nakajima15, Masaki Nakamura2, Nahoko Nakano10, Tamotsu Nishitomi11, Kenji Ohkoshi17, Yuuko Okamoto17, Takashi Omori18, Hiroshi Ono19, Makoto Ono8, Yuko Osanai8, Kaoru Saijo1, Kazunori Sasaki3, Tetsuji Sasaki1, Shinichiro Sato20, Shoichiro Shimogo4, Hiroshi Shionoya7, Hideaki Sugawara14, Shigeo Sugimoto21, Kinya Takagaki2, Minoru Takizawa23, Noriho Tanaka19, Noriko Teramoto1, Hisashi Torishima23, Toshiyuki Tsuchiya25, Shogo Wada26, Shinobu Wakuri19, Xinhai Wang1, Ikuo Watanabe15, Yoshiki Ymagata10, Osamu Yamakita21, Ryohei Yamamoto24, Isao Yoshimura18, Shiro Yukiyama21

1RIKEN Cell Bank, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukaba Science City, Ibaraki 305;
2Toxicology Laboratory, Mochida Pharmaceutical Co., Ltd., 342 Gensuke, Fujieda-shi, Shizuoka 426;
3Shionogi Research Laboratories, Shionogi & Co., Ltd., 3-1-1 Futaba-cho, Toyonaka-shi, Osaka 561;
4Shiseido Safety & Analytical Research Center,1050 Nippa-cho, Kohoku-Ku, Yokohama-shi 223;
5Chugai Pharmaceutical Co., Ltd., 3-41-8 Takada, Toshima-ku, Tokyo 171;
6Division of Genetics and Mutagenesis, Mitamura Building, 26-1 Kaitaicho, Shinjuku-ku, Tokyo 112-0012 Japan 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158;
7Department of Drug Safety Research, R & D Division, Eisai Co., Ltd., Takehaya, Kawashima-cho, Hashima-gun, Gifu 501-61;
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;
10Biochemical Development & Production, Oriental Yeast Co., Ltd., 4-4-1 Minamisuita, Suita-shi, Osaka 564;
11Kashima Laboratory, Mitsubishi-kagaku Institute of Environmental and Toxicological Sciences, 14 Sunayama, Hasaki-machi, Kashima-gun, Ibaraki 314-02;
12Panapharm Laboratories Co., Ltd. 1285 Kurisaki-cho, Uto-shi, Kumamoto 869-04;
13Research and Development Center, Duskin Co., Ltd., 4-19-8 Minamisuita, Suita-shi, Osaka 564;
14Life Science Research Information Laboratory, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako-shi, Saitama 351-01;
15The Center of Japan Biological Chemistry, 52 Fukue, Kaizu-cho, Kaizu-gun, Gifu 503-06;
16Takara Belmont Co., Ltd., 5-1 Takamatsu, Kouzai-cho, Koga-gun, Shiga 520-32;
17Division of Fundamental Research, KOSE Co., 1-18-4 Azusawa, Itabashi-ku, Tokyo 174;
18Department of Management Science, Faculty of Engineering, Science University of Tokyo, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162;
19Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano-shi, Kanagawa 257;
20Division of Radiation Medicine, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-shi 263;
21Drug Safety Laboratory, Taiho Pharmaceutical Co., Ltd., 224-2 Ebisuno, Hiraishi, Kawauchi-Cho, Tokushima-shi 771-01;
22The Institute of Environmental Toxicology, 4321 Uchimoriya-cho, Mitsukaido-shi, Ibaraki 303;
23Hakko Electric Machine Works Co. Ltd., 3055 Togura, Toguramachi, Hanishina-gun, Nagano 389-08;
24Kurabo Industries Ltd., 14-41 Shimokida-cho, Neyagawa-shi, Osaka 572;
25Safety Evaluation Center, Central Research Laboratory, Showa Denko K K, 1-1-1 Ohnodai, Midori-ku, Chiba-shi 267;
26Wako Pure Chemical Industries, Ltd., 6-1 Takadacho, Amagasaki-shi, Hyogo 661

Original paper :AATEX 5(1-2):131-145,1998

Abstract
The inter-laboratory validation study on 5 cytotoxicity assays by JSAAE has been described in the preceding articles in this issue. Presented here are precise data and the protocols on the neutral red uptake (NR) assay with two cell lines, namely, HeLa S3 (SC) cells which are employed also in the other 4 assays, and the serum-free cultured NRCE cells. 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 24 laboratories were examined together with all the log(ED50) values. Variations in the negative controls in the assays revealed the level of technical competence of each laboratory. These results will supplement our understanding on the NR assay carried out in the large-scale inter-laboratory validation.

Key words: alternatives, cytotoxicity assay, inter-laboratory validation, neutral red uptake assay.


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