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.

SkinEthic HCE Time-to-Toxicity on solids: A test method for distinguishing chemicals inducing serious eye damage, eye irritation and not requiring classification and labelling.

This study describes the development of a Time-to-Toxicity approach for solids (TTS) based on the SkinEthic™ HCE tissue construct, capable to distinguish chemicals that do not require classification for serious eye damage/eye irritation (No Cat.) from chemicals that require classification for eye irritation (Cat. 2), and serious eye damage (Cat. 1). Briefly, the time-to-toxicity of 69 solids was evaluated by exposing SkinEthic™ HCE tissue constructs to the test chemical for two different time periods (30-min, and 120-min). Based on the viability observed for the different exposure periods, a classification was assigned. The within laboratory reproducibility in terms of concordance in classifications (3 UN GHS categories), based on a set of 48 solids, was 93.7%. Furthermore, 73.6% Cat. 1 (N = 24), 55.6% Cat. 2 (N = 15) and 72.2% No Cat. (N = 30) were correctly identified with the SkinEthic™ HCE TTS test method. This study provides evidence that the SkinEthic™ HCE Time-to-Toxicity method (multiple exposure times) can distinguish Cat. 2 solids from Cat. 1 solids. This is an added value compared to the SkinEthic™ HCE EITS method (single exposure time) that can distinguish No Cat. chemicals from chemicals that do require classification and labelling for eye irritation/serious eye damage (Cat. 2/Cat. 1).