Troubles and countermeasure for blank absorbance increase in the micellar Reactive Oxygen species (ROS) assay

<p>A reactive oxygen species (ROS) assay is an in chemico photoreactivity test listed in ICH S10 Guideline, OECD Test Guideline (TG) No. 495 and Guidance on photosafety evaluation of Quasi-Drugs and Cosmetics. We currently utilize the ROS assay to assess photosafety of cosmetic ingredients, and we confronted a problem that there was an increase of blank absorbance assessing superoxide anion after irradiation, leading to a dissatisfaction of the acceptance criteria. We found that the recommended solvent DMSO was the main cause and contamination of Cu²⁺ (superoxide anion scavenger) in the phosphate buffer (NaPB) was determined as being able to suppress blank absorbance increases ¹⁾. We previously confirmed that the ROS assay using NaPB supplemented with 0.1 μM of Cu^{2+ 1)} and the ROS assay using acetonitrile as alternative vehicle instead of DMSO ²⁾ could be useful for obtaining stable test results by using 17 proficiency chemicals listed in TG495. A significant blank absorbance increase observed in the micellar ROS (mROS) assay ³⁾ using a micellar solution of 0.5% (v/v) Tween 20 for solubility enhancement, and thus in this study, we report results of micellar ROS assay for poorly water-soluble chemicals using acetonitrile instead of DMSO.</p><p></p><p>1) Ohtake and Hirota, 2022. J. Toxicol. Sci. 47, 109-116. </p><p>2) Ohtake and Hirota, 49^th JSOT Annual Meeting, P-91. </p><p>3) Seto et al., 2013. Toxicol. In Vitro 27, 1838-1846.</p>