Utility of Rat Liver S9 Fractions to Study Skin-Sensitizing Prohaptens in a Modified KeratinoSens Assay

Prohaptens are chemicals, which may cause skin sensitization after being converted into electrophilic molecules by skin enzymes. Aroclor-induced rat liver S9 fractions represent the metabolic activation system most commonly used in in vitro toxicology. This system contains much higher enzyme activities compared with those reported in skin, but it may still serve as a surrogate system to study the potential of chemicals to act as prohaptens. To test this concept, the luciferase induction in KeratinoSens reporter cells treated with chemicals in presence and absence of S9 fractions was measured. Suspected prohaptens such as methyl isoeugenol, eugenol, or trans-anethole gave no, or only weak, ge ne induction in absence of S9 fractions, and a significantly enhanced luciferase induction in presence of S9, proving their prohapten status. Direct-acting haptens like 2,4-dinitrochlorobenzene or cinnamic aldehyde gave a reduced response in presence of S9. We evaluated whether this metabolic activation assay might be implemented in a tiered screening strategy to counter-screen negatives in the KeratinoSens assay to enhance sensitivity. To this aim, all chemicals classified negative were retested with this activation step. Among the 77 chemicals found as correct-negatives, 73 were also negative in presence of metabolic activation, thus this counterscreen would reduce specificity only slightly. However, this comprehensive screening showed that only a small fraction of the known skin sensitizers need activation by the S9 system. Therefore, the KeratinoSens-S9 assay appears useful for the in vitro evaluation of specific classes of potential prohaptens and to mechanistically rationalize their prohapten status.