Methyl green-pyronine staining of porcine organotypic skin explant cultures: An alternative model for screening for skin irritants

We describe a new alternative method for screening for skin irritants by us ing fresh intact porcine skin biopsies. Test chemicals were applied to the epidermis of the biopsies, which were then incubated for different times in tissue culture medium at 37 degrees C and with 5% carbon dioxide. A decrea se in epidermal keratinocyte RNA staining, visualised in frozen sections by using a modified methyl-green pyronine (MGP) staining procedure, was emplo yed as a marker of irritancy. If a decrease in epidermal RNA was observed a fter incubation for 4 hours (strong irritant), the chemical had an MGP scor e of 3; if after incubation for 24 hours (moderate irritant), the MGP score was 2; and if after incubation for 48 hours (weak irritant), the MGP score was 1. If no keratinocyte cytotoxicity was observed after incubation for 4 8 hours, the chemical was classified as non-irritant (MGP score = 0). At le ast three ears were used per chemical. The average MGP score was used to cl assify the chemical. Based on the MGP score for 20% sodium dodecyl sulphate , chemicals classified as strong or moderate irritants by using the MGP tes t were grouped together as category R38 chemicals. Weak irritants or non-ir ritants were not classified (NC). The MGP staining correctly identified 23 of 25 skin irritants for which reference data were available.