Experimental Study for the Development of an in vitro Test for Contact Allergens

Abstract

We have previously reported an in vitro hapten-specific sensitization method using Pam-212 cells (in vitro sensitization test) to identify the potential effectiveness of contact allergens. In the present study, we conducted comparison studies of 11 allergens and 2 irritants in order to evaluate the method as an alternative predictive test. The guinea pig maximization test (GPMT) was developed based on the test described by Magnusson and Kligman. Our assay was carried out as follows: we treated Pam-212 cells with 13 test chemical solutions, while T cells and macrophages of BALB/c mice were cultured with hapten-conjugated Pam-212 cells for 5 days. After incubation, 10⁵ T cells were stimulated with mitomycin-C-treated spleen cells conjugated with chemicals. Three days later, the [³H]methyl thymidine incorporation was counted. The results of the GPMT were in agreement with those reported in previous studies except for benzocaine. In our GPMT experiments, benzocaine was negative, but it had been classified as a moderate sensitizer in previous studies. Our assay detected extreme, strong and moderate sensitizers as previously classified by the GPMT. They could be summarized as follows: three of five chemicals classified as moderate sensitizers, and 100% of strong or extreme sensitizers were detected by both the GPMT and the in vitro sensitization test. No irritants showed a positive reaction in our assay. These results support the view that the sensitivity of our in vitro test may be equivalent to that of the GPMT and may be useful as a rapid and objective allergen screening test.