ORIGINAL ARTICLE
Effects of pre-culture conditions on the human Cell Line Activation Test (h-CLAT) results: Results of the 4th Japanese inter-laboratory study

Makoto Mizuno1, Mayumi Yoshida2, Tatsuji Kodama3, Nanae Kosaka4, Kenji Okamoto5, Sakiko Sono6, Takaaki Yamada7, Seiji Hasegawa7, Takao Ashikaga6, Hirofumi Kuwahara5, Hitoshi Sakaguchi4, Jun Sato3, Naoko Ota2, Yuko Okamoto1 and Yasuo Ohno8

1KOSÉ Corporation, 2POLA CHEMICAL INDUSTORIES, INC., 3LION CORPORATION, 4Kao Corporation, 5Kanebo Cosmetic Inc., 6Shiseido Co., Ltd., 7Nippon Menard Cosmetic Co., Ltd., 8National Institute of Health Science, Japan

AATEX 13(2):70-82, 2008

Abstract
The human Cell Line Activation Test (h-CLAT) is an in-vitro skin sensitization method based on the enhancement of CD86 and/or CD54 in THP-1 cells. Experimental conditions for h-CLAT were optimized inour previous study. This protocol defines that THP-1 cells are seeded between 0.1x106 and 0.2x106cells/mL, and pre-cultured for 48h or 72h before treated with a test chemical. In this study we evaluatedeffects of pre-culture conditions on the h-CLAT results minutely. We cultivated the cells on ninepre-culture conditions before exposure to allergens (dinitrochlorobenzene (DNCB) and nickel sulfate (Ni))or non-allergen (sodium lauryl sulfate (SLS)), and then measuredCD86 and CD54 expressions on thesecells after the exposure. All laboratories almost correctly evaluated the skin sensitization potential of thesethree chemicals on any pre-culture conditions. However only low CD86 and CD54 RFI values induced byDNCB tend to be obtained as the final cell concentration on pre-culture became higher. For maintainingthe response of THP-1 cells to allergens and distinguishing allergens and non-allergens more clearly,THP-1 cells should be avoided being in over-growth conditions during pre-culture. Therefore a supplementary experimental condition about pre-culture for h-CLAT that final cell concentration in pre-culturemust not exceed 1.0x106 cells/mL was defined.

Key words: h-CLAT, skin sensitization, alternatives, THP-1, culture

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(AATEX: Altern. Animal Test. EXperiment.: Alternatives to Animal Testing and EXperimentation)