Alternatives to Animal Testing and Experimentation
Online ISSN : 2185-4726
Print ISSN : 1344-0411
ISSN-L : 1344-0411
Original Articles
Development of a Novel in Vitro Skin Sensitization Test Method using a Collagen Vitrigel Membrane Chamber
Hiroshi MiyazakiKunihiko YamashitaTadashi UchinoToshiaki TakezawaHajime Kojima
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2017 Volume 22 Issue 2 Pages 141-154


Several in vitro cell-based methods for predicting the skin sensitizing potential have been reported; however, such methods are limited by the poor water solubility of many organic molecules. Therefore, we developed a novel test method using a collagen vitrigel membrane (CVM) chamber, termed the Vitrigel Skin Sensitization Test (Vitrigel-SST). We first determined the optimal concentration of dimethyl sulfoxide (DMSO) for test chemical solutions subjected to the Vitrigel-SST. When medium containing 10% DMSO was subjected to the Vitrigel-SST using THP-1 cells, cytotoxicity was not observed until the 3 h time point. Thus, we decided to use 10% DMSO for Vitrigel-SST. Test chemicals were dispersed or dissolved in medium containing 10% DMSO. THP-1 cells were exposed to the test solutions for 30 min through the CVM, and the test solutions were replaced to fresh medium. After 48 h incubation, interleukin 8 (IL-8) productions was measured. Twenty-four test chemicals were evaluated to demonstrate the capacity of the Vitrigel-SST to predict chemical-induced skin sensitization. Cells exposed fifteen chemicals, including five chemicals with poor water solubility, increased maximum IL-8 production (IL-8MAX) more than 2-fold (≥2) that of cells exposed to the control treatment. IL-8MAX of five non-skin sensitizers was less than 2. The results of 20 of 24 chemicals were matched with that of LLNA when the IL-8MAX of 2 was adopted as cut off value. These results demonstrate that the Vitrigel-SST is a promising new skin sensitization test and is suitable to evaluate the skin sensitization potential of chemicals with poor water solubility.

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© 2017 Japanese Society for Alternative to Animal Experiments
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