Evaluation of EndoSens: A reliable skin sensitization assessment model.

Abstract

The transition toward non-animal safety assessment has driven the development of in vitro models for skin sensitization, particularly those targeting key event 2 (KE2) within the adverse outcome pathway (AOP). While conventional KE2 assays often rely on randomly integrated reporter systems, the EndoSens model utilizes CRISPR/Cas9-mediated knock-in to precisely insert a luciferase reporter into the endogenous HMOX1 locus in HaCaT keratinocytes, providing a genomically anchored and physiologically relevant system. To enable reproducible adoption across laboratories, we established a standardized protocol with defined quality control (QC) criteria, incorporating cinnamyl alcohol as a quantitative positive control and a cytotoxicity pre-screen to identify non-cytotoxic test concentrations. Evaluation using ten OECD TG 442D reference chemicals and ten additional compounds demonstrated the assay's robust performance in discriminating skin sensitizers with high inter-laboratory consistency, supporting its integration as a reliable in vitro component within defined approaches (DA) for animal-free skin sensitization assessment.