The SkinEthic Skin Irritation Test assay (42 minutes application + 42 hours postincubation)is designed for the prediction of acute skin irritation of chemicals by measurementof its cytotoxic effect. The relative viability of the treated tissues was measured at the end of the treatment exposure(42 minutes) followed by a post-exposure period (42 hours) using the vital dye MTT. A cutoffvalue of 50% viability of the negative control value was considered and used to classifytest substances as irritant (I) or non irritant (NI). The culture environment might allow thedetection of very small quantities of cytokines secreted by the epidermis in response to topicalapplication of test substances. Cell viability determination is based on cellular mitochondrial dehydrogenase activity,measured by MTT reduction and conversion into blue formazan salt that is quantified afterextraction from tissues. The reduction of cell viability in treated tissuesis compared to negative controls and expressed as a percentage. The percentage reduction inviability is used to predict the irritation potential.
Each test substance (test material, negative and positive controls) is topically appliedconcurrently on three tissues replicates for 42 minutes at room temperature (RT, comprisedbetween 18°C to 24°C). Exposure to the test substance was followed by rinsing withphosphate buffer saline (PBS) and mechanically dried. Epidermis were then transferred tofresh medium and incubated at 37°C for 42 additional hours. Cell viability is assessed byincubating the tissues for 3 hours with 0.3 mL MTT solution (1 mg/mL). The formazancrystals are extracted using 1.5 mL isopropanol for 2 hours at RT and quantified byspectrophotometry at 570 nm wavelength. Sodium Dodecyl Sulphate (SDS 5%), and PBStreated epidermis are used as positive and negative controls, respectively. For each treatedtissue, the cell viability is expressed as the percentage of the mean negative control tissues.Values under 50% is qualified the test substance as irritant.