Allergic and irritant skin reactions caused by topical exposure to permanent hair dyes are a common problem. For regulatory and ethnical purposes, it is required to perform chemical safety assessment following the replacement, reduction, and refinement of animal testing (3Rs). Permanent hair dyes are formed by a mixture of ingredients that vary from low to extreme skin sensitizing potency and that inter-react to form unknown by-products. Because of the complex reaction, this cytotoxic mechanism has not yet been elucidated and is the subject of this study. Here, we topically exposed p-phenylenediamine (PPD), Resorcinol (RES), Hydrogen Peroxide (H2O2) alone or as a mixture to RhE and evaluated parameters related to skin irritation such as epidermal viability, keratinocytes damage, barrier loss and IL-1 α. Our data indicates that ingredients tested alone did not lead to an increase of cytotoxic parameters related to skin irritation. However, when the mixture of PPD/H2O2/RES and PPD/H2O2was applied to the RhE, some of the parameters such as morphological changes including the presence of apoptotic cells, barrier loss and increased IL- 1 α release were observed. The results indicate that the mixture of ingredients used in permanent hair dyes have an irritant effect in RhE while the ingredients alone not.