Amending the U-SENS™ skin sensitization test method for interfering auto-fluorescent chemicals

Limitations of the applicability domain of new approach methodologies (NAM) present a major challenge for the testing of cosmetic ingredients in Europe, as the regulation does not allow to resort to in vivo test method. Therefore, research focused on overcoming such limitations of established in vitro test methods is frequently conducted. Here, we address a limitation of the U-SENS™, an in vitro skin sensitization test method that addresses the key event 3 on activation of dendritic cells of the adverse outcome pathway (AOP) for skin sensitization. The applicability domain of the U-SENS™ excludes autofluorescent substances that can interfere with the measurement of the expression of CD86, i.e., the primary readout. An evaluation of several fluorochromes identified APC as most suitable for testing auto-fluorescent chemicals. Acceptance criteria were reproducibly met when using the APC-labelled antibody. Equivalent performance in terms of reproducibility and skin sensitisation hazard assessment of the standard FITC-labelled antibodies and the APC-labelled antibodies was demonstrated by testing 40 substances. Finally, the value of the expanded technical applicability domain was highlighted with a case study using sulfuretin. In conclusion, we successfully demonstrated the expansion of the U-SENS™ applicability domain to interfering auto-fluorescent chemicals by using APC-labelled antibodies.