Valencian Institute of Microbiology
(I V A M I)

        Skin irritation indicates irreversible skin damage after application of a chemical for up to 4 hours. The evaluation of skin irritation has been evaluated with experimental animals. However, to protect animal welfare, its use must be limited, and it has been prohibited since a few years to evaluate cosmetic products. This standard provides an in vitro procedure that can be used to evaluate the skin irritant capacity of isolated substances or mixtures of them, using an experimental model of reconstructed human epidermis (RhE: Reconstructed Human Epidermis). This model simulates the biochemical and physiological properties of the upper layers of human skin, that is, the epidermis, using non-transformed human epidermal keratinocytes as cells, which adapt in vitro to form a tissue equivalent in its cytoarchitecture to human skin. The RhE model uses human keratinocytes that represent the target organ in vitro. Furthermore, it involves the initial step of the inflammatory cascade/mechanism of action (cell damage and tissue damage resulting from localized trauma), which occurs during in vivo irritation in experimental animals. The test can be applied to solid, liquid, semi-solid substances and waxes. Liquids can be aqueous or non-aqueous; solids may be soluble or insoluble in water. Wherever possible solids should be ground to a fine powder prior to application and no further pretreatment of the sample prior to application is required. This method has the limitation that highly colored chemicals can interfere with cell viability measurements, in which a chromogenic substance is also used. For the evaluation, three replicates may be sufficient when the test results allow an unequivocal classification, but nevertheless, in cases of borderline situations, such as when there is no concordance between the replicates and/or viability percentages equal to 50 ± 5%, a second repetition should be performed, or even a third, in case discordant results have been obtained between the first two series.

            In the test, the test substance is applied directly topically to the three-dimensional model of RhE. This model consists of non-transformed human epidermal keratinocytes, which have been cultured to form a highly differentiated multilayer model of human epidermis. The RhE model is composed from bottom to top of the basal layer, the spinous layer (stratum spinosum), the granular layer (stratum granulosum), and the stratum corneum (stratum corneum), which contains intercellular lipids analogous to lipids found in vivo.

         Chemical-induced skin irritation is clinically manifested by erythema and edema resulting from a cascade of events beginning with penetration of the stratum corneum, followed by damage to the underlying layers of keratinocytes. Dying keratinocytes release mediators that initiate the inflammatory cascade, releasing cytokines that act on dermal cells, particularly stromal and endothelial cells. The increased permeability of endothelial cells causes the clinically observed erythema and edema. In vitro methods based on RhE models measure inducing changes in the inflammatory cascade that is initiated when chemicals disrupt the epidermis. Cell viability of RhE models is measured by the enzymatic conversion of the vital dye MTT (dimethyl-diphenyltetrazolium bromide) to formazan blue which can be quantitatively measured after extraction from tissue. Irritating substances cause a decrease in cell viability below a defined cut-off point (e.g. ≤ 50% for UN GHS category 2 products). Products that induce cell viability above the cut-off level can be considered non-irritant (e.g. > 50%, no irritant category).