Assessment of an in vitro model of human cells to evaluate the potential toxic and irritating of chemical compound.

To identify non-animal tests for predicting human skin irritation, the European Centre for the Validation of Alternative Methods, ECVAM, promotes prevalidation studies focused on the evaluation of suitable in vitro assays. Epidermal differentiation requires the sequential regulation of specific genes, with their simultaneous activation and inactivation, while the cells move upwards from the basal layer (BL) through the spinous layer (SL) and the granular layer (GL), ending up in the cornified envelope (CE). A pattern of markers, such as transglutaminases (TG), keratins, involucrin (INV), and loricrin (LOR), permits the discrimination between differentiating and terminally differentiated keratinocytes. We attempted to induce differentiation in undifferentiated NCTC2544 human keratinocyte line, by exposure to ZnSO4 and CaCl2. Analysis of specific markers, transglutaminase 1, involucrin and loricrin, show that basal NCTC2544 (BL) reached spinous- (SL) and granular-like (GL) phenotypes. BL-, Sl- and GL- NCTC were exposed to SDS, as irritant stimulus and Neutral red uptake (NRU) and MTT cytoxicity tests evidenced a relatively higher toxicity in SL-and GL cells on lysosomes respect to mitochondria. IL1α cytokine was monitored as early inflammation marker. The complex of data provides evidence for the suitability of our in vitro model to the analysis of cytotoxic /biological effects of topically applied exogenous compounds.