Optimisation of Photo-KeratinoSens™ for <i>in vitro</i> photoallergenicity assessment

The aim of this work was to develop and validate a cell-based in vitro assay for predicting photoallergenicity by expanding the scope of our previously reported in vitro method, photo-KeratinoSens™, which is a luciferase-based assay dependent on activation of the Keap1-Nrf2-ARE pathway. First, we increased the maximum starting test concentration from a fixed 2000 µM in the original KeratinoSens™ to either 5000 μg/mL or 4 times the concentration providing a cell viability of 75% (under UV irradiation), depending on the cytotoxicity. Then, we established that 0.5% ethanol, 0.5% acetone and 0.1% tetrahydrofuran are available as solvents in addition to 1% DMSO, which is used in the standard KeratinoSens™ method (OECD TG442D). We confirmed that a representative photoallergen, 6-methylcoumarin, gave reproducible results. To validate the developed assay, we used it to evaluate a library of 90 chemicals consisting of 60 known photoallergens and 30 non-photoallergens. The accuracy, sensitivity, specificity and balanced accuracy of photo-KeratinoSens™ were 76.7% (69/90), 66.7% (40/60), 96.7% (29/30) and 81.7%, respectively. When we excluded chemicals with no UVA absorption, the accuracy, sensitivity, specificity and balanced accuracy were improved to 81.8% (45/55), 80.0% (36/45), 90.0% (9/10) and 85%, respectively. Our results suggest that photo-KeratinoSens™, which combines the OECD TG442D in vitro skin sensitization test detecting key event 2 in the adverse outcome pathway (AOP) of skin sensitization with exposure to UV iradiation, may be useful as a contributory input in a weight-of-evidence approach for evaluating photoallergenicity potential without animal testing.