FIRST PHASE INTER-LABORATORY VALIDATION OF THE IN VITRO EYE IRRITATION TESTS FOR COSMETIC INGREDIENTS: (7) EVALUATION OF CYTOTOXICITY TESTS ON PRIMARY RABBIT CORNEAL EPITHELIAL CELLS (CORNEPACK)

HAJIME KOJIMA1,2, YOSHINOBU TAKINO1,3, MINA KANARI1,3, KAZUTAMI SAKAMOTO1,3, ERIKO MIYAI1,4, JUNICHI AKIYAMA1,4, MICHIO SHIBATA1,5, HISASHI TORISHIMA6, RYOHEI YAMAMOTO6, ATSUKO MIYAJIMA7, YASUO OHNO7 and AKIRA TAKANAKA7
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; 3Applied Research Laboratories, Central Research Laboratories, Ajinomoto Co. Inc., 1-1 Suzuki-cho, Kawasuki-ku, Kawasaki 210, Japan; 4Kaminomoto Co., Ltd., 3-3-35, Kumochibashidori, Chuo-ku, Kobe 658, Japan; 5Shiseido Safety & Analytical Research Center, 1050 Nippa-cho, Kohoku-ku. Yokohama 223, Japan; 6Technical Research Laboratory, Kurabo Industries Ltd., 14-5 Shimokida-cho, Neyagawa-shi, Osaka 572, Japan; 7Div. 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):175-181

Abstract
The cytotoxicity test of neutral red (NR) uptake in primary rabbit corneal epithelial ceils (CornePack) was evaluated as an alternative method to the Draize rabbit eye irritation test (Draize test). We tested 9 surfactants and isotonic sodium chloride solution (physiological saline) and controlled the test procedures under a common standard operating procedure (SOP) among laboratories. The concentrations of test chemicals that showed 50% reduction in NR uptake relative as compared to control (the median NR uptake concentration: NR50) were determined, and compared with in vivo scores.
Six laboratories participated in this study. Each laboratory exist a few quitter and inter-laboratory coefficients of variation (CV) of this assay were above 0.500 on 2 test substances. The correlation and rank correlation coefficients between maximal average Draize total score (MAS) and NR50 of surfactants were -0.619 and 0.846 respectively.
We concluded that this cytotoxicity test on CornePack assay is not reproducible and correlates poorly with Draize scores. However, we improved the protocol by omitting the pre-culture step and changing the NR assay. As a result, the uptake of NR of negative control samples became more stable and higher than the former data.
Because of its higher sensitivity than the other methods validated under the Ministry of Health and Welfare (MHW) project, we consider this assay may be evaluated with a wider range of chemicals used as cosmetic ingredients addition to improvement of methods.

Key words: validation study, Draize eye irritation test, cytotoxicity test, neutral red, primary normal rabbit corneal epithelial cells, alternatives, in vitro, surfactant.


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