Optimization of the IL-8 Luc assay as an in vitro test for skin sensitization

- We created another data set of 122 chemicals of the IL-8 Luc assay.
- We analyzed the reason for poor performance of the original protocol.
- The solubilizing chemicals with X-VIVO 15 significantly improved its performance.
- Its performance was significantly improved, 90% of accuracy and 90% of specificity.

We previously reported a dataset of the IL-8 Luc assay covering reference chemicals published by ECVAM, in which the effects of chemicals on IL-8 promoter activity were evaluated by an IL-8 reporter cell line, THP-G8 cells. To clarify its performance, we created another dataset of 88 sensitizers and 34 non-sensitizers. Simultaneously, to improve its performance, we changed the incubation time from 5 h to 16 h, deleted the criterion regarding the effects of N-acetylcysteine, and set an exclusion criterion for detergents. These modifications significantly improved its performance. In addition, we examined the following three criteria to judge chemicals as sensitizers: Criterion 1: Fold induction of SLO luciferase activity (FlnSLO-LA) ⩾ 1.4, Criterion 2: the lower limit of the 95% confidence interval of FInSLO-LA ⩾ 1.0, Criterion 3: the intersection of criteria 1 and 2. Among them, Criterion 1 produced the best performance, demonstrating that the accuracy, sensitivity and specificity were 81%, 79%, and 90%, respectively. In addition, we found that the IL-8 Luc assay solubilizing chemicals with X-VIVO substantially improved its performance. Finally, the IL-8 Luc assay combined with DPRA and DEREK could improve substantially its performance. These data suggest that the IL-8 Luc assay is a promising test method to screen skin sensitizers.