FIRST PHASE INTER-LABORATORY VALIDATION OF THE IN VITRO EYE IRRITATION TESTS FOR COSMETIC INGREDIENTS: (9) EVALUATION OF CYTOTOXICITY TESTS ON HeLa CELLS AND CHL/IU

HAJIME KOJIMA1,2, JUNKO OHUCHI1,3 JOSHIN OKADA1,3, HIROSHI KAKSHIMA1,4, ERIKO MIYAI1,5, JUNICHI AKIYAMA1,5, YUUKO OKAMOTO1,6, MAYUMI KOTANI1,7, KAORI INOUE1,8, MICHIO SHIBATA1,8, HIDENOBU OKUMURA1,9, MASAKI ARASHIMA1,9, TAKAMASA ATSUMI1,9, IKUYO MAKINO1,10, KATSUYOSHI CHIBA1,10, YASUO OHNO11 and AKIRA TAKANAKA11
1Japan Cosmetic Industry Assoc. (JCIA), 4th floor Hatsumei Bldg., 9-14, Tranomon, 2-chome, Minato-ku, Tokyo 105, Japan; 2Biochemical Research Institute, Nippon Menard Cosmetic Co Ltd., 4-66, Asakusa, Ohgaki-shi, Gifu 503, Japan; 3KAO Corp. Tochigi Biological Sciences, 2606 Akabane, Ichikai, Haga, Tochigi 321-34, Japan; 4Kanebo Cosmetic Laboratory, 3-28, 5-chome, Kotobuki-cho, Odawara-shi, Kanagawa 256, Japan; 5Kaminomoto Co., Ltd., 3-3-35, Kumochibashidori, Chuo-ku, Kobe 658, Japan; 6Div. Fundamental Research, KOSE Corporation, 1-18-4 Azusawa, Itabushi-ku, Tokyo 174, Japan; 7SUNSTAR Inc., 3-1 Asahi-machi, Takatsuki-shi, Osaka 569, Japan; 8Shiseido Safety & Analytical Research Center, 1050 Nippa-cho, Kohoku-ku. Yokohama 223, Japan; 9Shiga Central Research Laboratory, NOEVIR Co. Ltd., 112-1, Okada-cho, Yokaichi-shi, Shiga 527, Japan; 10Safety Research Center, Yakult Central Institute for Microbiological Research, 1796 Yuo, Kunitachi-shi, Tokyo 186, Japan; 11Div. of Phamacology, Biological Safety Resource Center (BSRC), Mitamura Building, 26-1 Kaitaicho, Shinjuku-ku, Tokyo 112-0012 Japan 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan

Regular Article :AATEX 3(4):191-198

Abstract
The first phase inter-laboratory validation of cytotoxicity tests using established cell lines was conducted by the collaboration of 7-8 laboratories for the purpose to evaluate the test for their relevance as alternative methods to the Draize rabbit eye irritation test (Draize test). The methods studied were the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) assay in HeLa cells (HeLa-MTT assay) and crystal violet staining assay in CHL/IU cells (CHL/IU-CVS assay). We tested nine surfactants and isotonic sodium chloride solution (phosphate saline) under the common standard operating procedure (SOP) and determined the concentrations of test chemicals that showed 50% absorbance reduction as compared to control (the median effective concentration: EC50).
Inter-laboratory coefficients of variation of the EC50 values obtained for the surfactants obtained were below 0.50 except for polyoxyethylene octylphenylether (10 E.O.: Triton X-100). The correlation coefficients of these EC50 values with maximal average Draize total score (MAS) were -0.902 and -0.817 for the HeLa-MTT and CHL/IC-CVS assays respectively. The cytotoxicity potentials (EC50) of test substances were similar between these two assay methods.
We conclude that the cytotoxicity tests in HeLa-MTT and CHL/IU-CVS assays are promising as alternative methods to the Draize eye test for surfactants. Because surfactants constitute only a minor part of total cosmetic ingredients, these tests should be evaluated with a wider range of chemicals used as cosmetic ingredients.

Key words: validation study, Draize eye irritation test, cytotoxicity test, MTT, crystal violet, HeLa cells, CHL/IU cells, alternatives, in vitro, surfactant.


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