LLNA variability: An essential ingredient for a comprehensive assessment of non-animal skin sensitization test methods and strategies.

The development of non-animal skin sensitization test methods and strategies is quickly progressing. Either individually or in combination, the predictive capacity is usually described in comparison to local lymph node assay (LLNA) results. In this process the important lesson from other endpoints, such as skin or eye irritation, to account for variability reference test results - here the LLNA - has not yet been fully acknowledged. In order to provide assessors as well as method and strategy developers with appropriate estimates, we investigated the variability of EC3 values from repeated substance testing using the publicly available NICEATM (NTP Interagency Center for the Evaluation of Alternative Toxicological Methods) LLNA database. Repeat experiments for more than 60 substances were analyzed - once taking the vehicle into account and once combining data over all vehicles. In general, variability was higher when different vehicles were used. In terms of skin sensitization potential, i.e., discriminating sensitizer from non-sensitizers, the false positive rate ranged from 14-20%, while the false negative rate was 4-5%. In terms of skin sensitization potency, the rate to assign a substance to the next higher or next lower potency class was approx.10-15%. In addition, general estimates for EC3 variability are provided that can be used for modelling purposes. With our analysis we stress the importance of considering the LLNA variability in the assessment of skin sensitization test methods and strategies and provide estimates thereof.