Arachidonic acid release: An in vitro alternative for dermal irritancy testing

Validated in vitro alternatives are being utilized extensively for mutagenicity and ocular irritancy testing. However, validation of alternative assays for dermal irritancy is progressing more slowly. As the irritant response in human skin is mediated, at least in part, by eicosanoids derived from arachidonic acid, the effect of relatively pure anionic surfactants (AS, n = 8) and surfactant-containing finished products (FP, n = 25) on the release of [3H]arachidonic acid from a prelabelled murine fibroblast cell line (C3H-10T12 cells) in vitro was examined. Test substances were administered at various non-lethal concentrations, in triplicate, to 12- and 24-well plates containing preconfluent monolayers (80–90% confluence) of C3 H-10T12 cells. Because it is impossible to test all concentrations of each test substance in a single assay, statistical techniques were developed to ‘standardize’ in vitro assay results. In each assay, radiolabel release due to a positive control was also measured, using 0.04, 0.05 and 0.06 mM concentrations of sodium dodecyl sulfate (SDS). Test substance releases were then transformed into ‘SDS equivalent’ responses, significantly reducing both inter- and intra-assay variability. A straight line was fitted to the test substance responses and compared with that for SDS to calculate the relative potency in vitro for individual AS and FP. Relative potencies correlated with in vivo responses, that is primary dermal irritation indices obtained in rabbits, with Spearman p = 0.408 (P< 0.03) for 32 tested agents, and p = 0.976 (P < 0.001) for the eight AS. Exclusion of extremely alkaline or acidic FP (pH > 1 1 or < 2, n = 4) and those which were insoluble in the aqueous cell culture media at the 1% stock dilution (n = 5), improved the overall in vivo-in vitro correlation significantly (p = 0.683, P < 0.001, n = 23) and produced a significant correlation for FP alone (p = 0.539, P < 0.05, n = 15). These results suggest that release of [3H]arachidonic acid from cultured skin cells represents a novel, mechanistically based in vitro screen for dermal irritancy testing.