Causes and countermeasure for blank absorbance increase in the ROS assay

A reactive oxygen species (ROS) assay is an in chemico photoreactivity test listed in ICH S10 guideline and OECD Test Guideline No. 495. We currently utilize the ROS assay to assess the photosafety of cosmetic ingredients. We have recently confronted a problem that there was an absorbance increase of blank assessing superoxide anion generation after irradiation, whereas this did not occur in the negative control (sulisobenzone), leading to a dissatisfaction of the acceptance criteria. Therefore, we aimed to investigate the causes and find countermeasures. No significant effects of impurities and manufacturer differences of sodium phosphate and DMSO on blank absorbance increases were observed. In contrast, when Cu²⁺ was added to the buffer, the increase of blank absorbance after irradiation did not occur. We then confirmed the dose-response relationship and found that adding 0.1 μM of Cu²⁺ (corresponding to 6 ppb of Cu²⁺) was sufficient in suppressing the blank absorbance increase, suggesting the need of Cu²⁺ supplementation to the buffer. Finally, we confirmed that the ROS assay using the buffer supplemented with 0.1 μM of Cu²⁺ obtained stable test results by using 17 proficiency chemicals listed in TG 495. Our results suggest that the modified ROS assay protocol would be useful for obtaining stable test results.