LuSens: A keratinocyte based ARE reporter gene assay for use in integrated testing strategies for skin sensitization hazard identification

• Published in: Toxicology in vitro Vol. 28; no. 8; pp. 1482 - 1497
• Main Authors: Ramirez, Tzutzuy, Mehling, Annette, Kolle, Susanne N., Wruck, Christoph J., Teubner, Wera, Eltze, Tobias, Aumann, Alexandra, Urbisch, Daniel, van Ravenzwaay, Ben, Landsiedel, Robert
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.12.2014
• Subjects: Adverse outcome pathway (AOP); Animals; Antioxidant response element (ARE); Antioxidant Response Elements - genetics; Cell Line; Genes, Reporter; Humans; In vitro; Keratinocyte activation; Keratinocytes - drug effects; Rats; Skin Irritancy Tests - methods; Skin sensitization; Antioxidant response element (ARE); Adverse outcome pathway (AOP); Skin sensitization; Keratinocyte activation; In vitro
• ISSN: 0887-2333; 1879-3177
• DOI: 10.1016/j.tiv.2014.08.002

•The LuSens assay uses a human keratinocyte ARE reporter cell line.•The LuSens addresses a key step of the adverse outcome pathway of skin sensitization.•The LuSens provides the same readout as KeratinoSens™; both can be used equivalently.•The test battery comprised of LuSens, DPRA and h-CLAT attained a predictivity similar to LLNA. Allergic contact dermatitis can develop following repeated exposure to allergenic substances. To date, hazard identification is still based on animal studies as non-animal alternatives have not yet gained global regulatory acceptance. Several non-animal methods addressing key-steps of the adverse outcome pathway (OECD, 2012) will most likely be needed to fully address this effect. Among the initial cellular events is the activation of keratinocytes and currently only one method, the KeratinoSens™, has been formally validated to address this event. In this study, a further method, the LuSens assay, that uses a human keratinocyte cell line harbouring a reporter gene construct composed of the antioxidant response element (ARE) of the rat NADPH:quinone oxidoreductase 1 gene and the luciferase gene. The assay was validated in house using a selection of 74 substances which included the LLNA performance standards. The predictivity of the LuSens assay for skin sensitization hazard identification was comparable to other non-animal methods, in particular to the KeratinoSens™. When used as part of a testing battery based on the OECD adverse outcome pathway for skin sensitization, a combination of the LuSens assay, the DPRA and a dendritic cell line activation test attained predictivities similar to that of the LLNA.