Division 1.4 PRINCIPLE OF THE TEST

The test substance is applied topically to a three-dimensional reconstructed human epidermis model, comprised of normal, human-derived epidermal keratinocytes, which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organised basal, spinous and granular layers, and a multilayered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo.

The principle of the reconstructed human epidermis model test is based on the premise that irritant substances are able to penetrate the stratum corneum by diffusion and are cytotoxic to the cells in the underlying layers. Cell viability is measured by dehydrogenase conversion of the vital dye MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; EINECS number 206-069-5, CAS number 298-93-1)], into a blue formazan salt that is quantitatively measured after extraction from tissues (20). Irritant substances are identified by their ability to decrease cell viability below defined threshold levels (i.e. ≤ 50 %, for UN GHS category 2 irritants). Substances that produce cell viabilities above the defined threshold level, will not be classified (i.e. > 50 %, no category).

The reconstructed human epidermis model systems may be used to test solids, liquids, semi-solids and waxes. The liquids may be aqueous or non-aqueous; solids may be soluble or insoluble in water. Whenever possible, solids should be tested as a fine powder. Since 58 carefully selected substances, representing a wide spectrum of chemical classes, were included in the validation of the reconstructed human epidermis model test systems, the methods are expected to be generally applicable across chemical classes (16). The validation includes 13 GHS Cat. 2 irritants. It should be noted that non-corrosive acids, bases, salts and other inorganic substances were not included in the validation and some known classes of organic irritants such as hydroperoxides, phenols and surfactants were not included or were only included to a limited extent.