Establishment and intra-/inter-laboratory validation of a standard protocol of reactive oxygen species assay for chemical photosafety evaluation

ABSTRACT A reactive oxygen species (ROS) assay was previously developed for photosafety evaluation of pharmaceuticals, and the present multi‐center study aimed to establish and validate a standard protocol for ROS assay. In three participating laboratories, two standards and 42 coded chemicals, including 23 phototoxins and 19 nonphototoxic drugs/chemicals, were assessed by the ROS assay according to the standardized protocol. Most phototoxins tended to generate singlet oxygen and/or superoxide under UV–vis exposure, but nonphototoxic chemicals were less photoreactive. In the ROS assay on quinine (200 µm), a typical phototoxic drug, the intra‐ and inter‐day precisions (coefficient of variation; CV) were found to be 1.5–7.4% and 1.7–9.3%, respectively. The inter‐laboratory CV for quinine averaged 15.4% for singlet oxygen and 17.0% for superoxide. The ROS assay on 42 coded chemicals (200 µm) provided no false negative predictions upon previously defined criteria as compared with the in vitro/in vivo phototoxicity, although several false positives appeared. Outcomes from the validation study were indicative of satisfactory transferability, intra‐ and inter‐laboratory variability, and predictive capacity of the ROS assay. Copyright © 2012 John Wiley & Sons, Ltd. In three participating laboratories, photochemical reactivities of 42 coded chemicals and two standard controls were assessed by reactive oxygen species (ROS) assay for photosafety evaluation. Outcomes from the validation study were indicative of satisfactory transferability, intra‐ and inter‐laboratory variability, and predictive capacity of the ROS assay. Thus, results from the present validation study provided sufficient support for the ROS assay as an alternative method for photosafety assessment.