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Toxicol In Vitro. 2014 Jun;28(4):515-23. doi: 10.1016/j.tiv.2013.11.014. Epub 2013 Dec 30.

Intra-/inter-laboratory validation study on reactive oxygen species assay for chemical photosafety evaluation using two different solar simulators.

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Department of Pharmacokinetics and Pharmacodynamics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Electronic address:
Non-Clinical Research Group, Ophthalmic Research and Development Center, Santen Pharmaceutical Co., Ltd., 8916-16 Takayama-cho, Ikoma, Nara 630-0101, Japan.
Drug Developmental Research Laboratories, Shionogi & Co., Ltd., 3-1-1 Futaba-cho, Toyonaka, Osaka 561-0825, Japan.
Research Center, Taisho Pharmaceutical Co., Ltd., 1-403, Yoshino-cho, Kita-ku, Saitama 331-9530, Japan.
Safety Research Department, ASKA Pharmaceutical Co., Ltd., 5-36-1, Shimosakunobe, Takatsu-ku, Kawasaki, Kanagawa 213-8522, Japan.
Pharmaceuticals Research Center, Asahi Kasei Pharma Corporation, 632-1 Mifuku Izunokuni-shi, Shizuoka 410-2321, Japan.
Laboratory of Cell Toxicology, Hatano Research Institute, Food and Drug Safety Center, 729-5 Ochiai, Hadano, Kanagawa 257-8523, Japan.
Safety Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 1-1-1, Kazusakamatari, Kisarazu, Chiba 292-0818, Japan.
Safety Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 2-2-50, Kawaguchi, Toda-shi, Saitama 335-8505, Japan.
Product Development Regulatory Affairs Department, Shionogi & Co., Ltd., 2-17-5 Shibuya, Shibuya-ku, Tokyo 150-8673, Japan.
National Institute of Health Sciences (NIHS), 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.
Japanese Center for the Validation of Alternative Methods (JaCVAM), National Institute of Health Sciences (NIHS), 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.


A previous multi-center validation study demonstrated high transferability and reliability of reactive oxygen species (ROS) assay for photosafety evaluation. The present validation study was undertaken to verify further the applicability of different solar simulators and assay performance. In 7 participating laboratories, 2 standards and 42 coded chemicals, including 23 phototoxins and 19 non-phototoxic drugs/chemicals, were assessed by the ROS assay using two different solar simulators (Atlas Suntest CPS series, 3 labs; and Seric SXL-2500V2, 4 labs). Irradiation conditions could be optimized using quinine and sulisobenzone as positive and negative standards to offer consistent assay outcomes. In both solar simulators, the intra- and inter-day precisions (coefficient of variation; CV) for quinine were found to be below 10%. The inter-laboratory CV for quinine averaged 15.4% (Atlas Suntest CPS) and 13.2% (Seric SXL-2500V2) for singlet oxygen and 17.0% (Atlas Suntest CPS) and 7.1% (Seric SXL-2500V2) for superoxide, suggesting high inter-laboratory reproducibility even though different solar simulators were employed for the ROS assay. In the ROS assay on 42 coded chemicals, some chemicals (ca. 19-29%) were unevaluable because of limited solubility and spectral interference. Although several false positives appeared with positive predictivity of ca. 76-92% (Atlas Suntest CPS) and ca. 75-84% (Seric SXL-2500V2), there were no false negative predictions in both solar simulators. A multi-center validation study on the ROS assay demonstrated satisfactory transferability, accuracy, precision, and predictivity, as well as the availability of other solar simulators.


Inter-laboratory precision; Photoreactivity; Phototoxicity; Reactive oxygen species; Validation

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