FIRST PHASE INTER-LABORATORY VALIDATION OF THE IN VITRO EYE IRRITATION TESTS FOR COSMETIC INGREDIENTS: (3) EVALUATION OF HEMOLYSIS TEST

YUUKO OKAMOTO1,2, KENJI OHKOSHI1,2, HIROSHI ITAGAKI1,3, KAORI INOUE1,3, MICHIO SHIBATA13, HIROSHI KAKASHIMA1,4, TOMOYASU OGAWA1,4, KIMIYO KONISHI1,5, KAZUTAMI SAKAMOTO16, YOSHINOBU TAKINO1,6, MINA KANARI1,6, KIYOJI MATSUKAWA1,7, KUNIO MASUDA1,7, HAJIME KOJIMA1,8, KATASUYOSHI CHIBA1,9, IKUYO MAKINO1,9, TOYOZO KANEKO10, AKIHIKO HIROSE10, YASUO OHNO11 and AKIRA TAKANAKA11
1Japan Cosmetic Industry Assoc. (JCIA), 4th floor Hatsumei Bldg., 9-14, Tranomon, 2-chome, Minato-ku, Tokyo 105, Japan; 2Div. Fundamental Research, KOSE Corporation, 1-18-4 Azusawa, Itabushi-ku, Tokyo 174, Japan; 3Shiseido Safety & Analytical Research Center, 1050 Nippa-cho, Kohoku-ku, Yokohama 223, Japan; 4Kanebo Cosmetic Laboratory, 3-28, 5-chome, Kotobuki-cho, Odawara-shi, Kanagawa 256, Japan; 5SUNSTAR Inc., 3-1 Asahi-machi, Takatsuki-shi, Osaka 569, Japan; 6Applied Research Laboratories, Central Research Laboratories, Azinomoto Co. Inc., 1-1 Suzuki-cho, Kawasuki-ku, Kawasaki 210, Japan; 7OPPEN Cosmetic Co. Ltd., 2-28-2 Shinaike, Setsu-shi, Osaka 569, Japan; 8Biochemical Research Institute, Nippon Menard Cosmetic Co Ltd., 4-66, Asakusa, Ohgaki-shi, Gifu 503, Japan; 9Safety Research Center, Yakult Central Institute for Microbiological Research, 1796 Yuo, Kunitachi-shi, Tokyo 186, Japan; 10Div. of Toxicology, Mitamura Building, 26-1 Kaitaicho, Shinjuku-ku, Tokyo 112-0012 Japan 1-18-1, Kamiyoga, Setagaya-ku, Tokyo 158, Japan; 11Div. of Phamacology, 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):146-153

Abstract
A hemolysis test using sheep red blood cell (RBC) as an alternative method to Draize rabbit eye irritation test (Draize test) was evaluated by nine laboratories using 9 surfactants and physiological saline as test substances. The test procedure was controlled under a common standard operating procedure (SOP) among laboratories. The concentrations of test substances that showed 50%, hemolysis (HC50) were determined, and was compared in vivo Draize test scores. Interlaboratory coefficients of variation among the nine laboratories were calculated for each test substance and the average of those was 23.7%. In this study, only one test substance, Tween20, had relatively large variation of HC50 values among laboratories. This seemed to be because Tween20 results were affected more sensitively by the slight changes in experimental conditions than the other test substances in this study, because the reproducibility of Tween 20 results was markedly improved in a supplemental test conducted after a detailed review of the SOP. Rank order correlation coefficients between hemolysing potencies and Draize scores were relatively good. When 1/HC50 was compared to maximal average Draize total score (MAS), the correlation coefficient was 0.738. From these results and its ability to measure direct effects of chemicals on cell membrane we considered that the hemolysis test is a promising alternative method to the Draize test.

Key words: validation study, Draize eye irritation test, alternatives, in vitro, red blood cells, hemolysis, surfactant.


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