Abstract
Many in vitro methods to screen skin irritants use keratinocyte cytotoxicity, assessed by reduced MTT metabolism. Since the three-dimensional structure of the skin may affect penetration, we preferred to use fresh intact porcine skin. In contrast to rodent skin, porcine skin has been shown to be very similar to human skin, morphologically, biochemically and immunologically. Porcine skin is derived from pig ears, a waste product of pig slaughter. After cleaning and decontaminating, biopsies were cut for porcine organotypic skin explant culture (pOSEC). We introduced a methyl-green pyronine (MGP) staining method to assess keratinocyte cell death. The MGP staining detects intracellular RNA. Viable cells express RNA to maintain viability, and the unstable RNAs are rapidly broken down by RNAses in dead cells. Pilot experiments have shown that the most interesting time-points at which to check for loss of skin viability in the pOSEC are after 4 hours, 24 hours and 48 hours of culture. We classified skin irritants based on the time-point of keratinocyte death in more than half of the skin cultures screened. Cytotoxicity within 4 hours was considered to be due to a strong irritant, within 24 hours to be due to a medium irritant, within 48 hours to be due to a weak irritant, and no chemicals inducing cell death after 48 hours was considered to be non-irritant. We used this method to classify approximately 40 chemicals with different skin irritation potentials. The MGP staining of pOSEC correctly identified the skin irritation hazard of most chemicals, including those used in a pilot ECVAM study.