Replacing HaCaT keratinocytes with 3D reconstructed human epidermis in a THP-1 coculture dendritic cell activation assay: quantitative skin sensitization potency assessment and structure-potency relationships.

Reliable assessment of skin sensitization hazard and potency remains challenging for fragrance and other substances. Although current in chemico and in vitro assays perform well within their respective applicability domains, challenges remain for highly lipophilic, poorly water-soluble, cytotoxic chemicals and substances requiring metabolic or abiotic activation. To address these challenges, the established 2D Cocultured Activation Test (COCAT), combining HaCaT keratinocytes with THP-1 cells as surrogate dendritic cells, was modified by incorporating a reconstructed human epidermis (RHE). The resulting 3D RHE/THP-1 coculture model was evaluated using chemicals that presented challenges for quantitative assessment in the 2D COCAT method. Results were compared with previously published 2D COCAT data and human and animal weight-of-evidence (WoE)-derived skin sensitization potency values (PVs). Incorporation of the RHE substantially expanded the applicability domain by enabling quantitative potency assessment of substances for which robust concentration-response relationships could not previously be established. The 3D RHE/THP-1 model differentiated all sixteen substances over more than three orders of magnitude in skin sensitization potency and enabled systematic comparison of structurally related substances. Alcohol-aldehyde potency relationships were highly structure dependent, ranging from minor differences between benzyl alcohol and benzaldehyde to approximately 15-fold differences between hydroxycitronellol and hydroxycitronellal. Potency estimates showed good overall agreement with published PVs. These findings demonstrate that incorporation of RHE extends the applicability domain of the COCAT method, provides a more physiologically relevant in vitro approach for quantitative skin sensitization potency assessment, and supports the further development of human-relevant, animal-free next-generation risk assessment strategies.