This OECD 439 (July, 2015) protocol may be used to determine the skin irritancy of chemicals either as a stand-alone replacement test for in vivo skin irritation testing or as a partial replacement test within a testing strategy.

It is based on the in vitro test system of reconstructed human epidermis (RhE), which closely mimics the biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis. The RhE test system uses human derived non-transformed keratinocytes as cell source to reconstruct an epidermal model with representative histology and cytoarchitecture.

The in vitro 3T3 NRU phototoxicity test is used to identify the phototoxic potential of a test substance induced by the excited chemical after exposure to light. The test evaluates photo-cytotoxicity by the relative reduction in viability of cells exposed to the chemical in the presence versus absence of light.

Substances identified by this test are likely to be phototoxic in vivo following systemic application and distribution to the skin, or after topical application.

This protocol OECD 431 provides an in vitro procedure allowing the identification of non-corrosive and corrosive substances and mixtures in accordance with UN GHS. It also allows a partial sub-categorisation of corrosives.

This test makes use of reconstructed human epidermis (RHE), obtained from human derived non-transformed epidermal keratinocytes  which closely mimics the histological, morphological, biochemical and physiological properties of the upper parts of the human skin, i.e. the epidermis.

This protocol OECD 492 provides an in vitro procedure allowing the identification of chemicals (substances and mixtures) not requiring classification and labelling for eye irritation or serious eye damage in accordance with UN GHS.

It makes use of reconstructed human cornea–like epithelium (RhCE) which closely mimics the histological, morphological, biochemical and physiological properties of the human corneal epithelium.

This guideline presents general principles for measuring dermal absorption and delivery of a test substance using excised skin. Skin from many mammalian species, including 3D reconstructed skin, humans explants, or pigskin, can be used. The permeability properties of skin are maintained after excision from the body because the principal diffusion barrier is the stratum corneum.

The test substance, which may be radiolabelled, is applied to the surface of a skin sample separating the two chambers of a diffusion cell. The chemical remains on the skin for a specified time under specified conditions, before removal by an appropriate cleansing procedure. The receptor fluid is sampled at time points throughout the experiment and analysed for the test chemical and/or metabolites using HPLC-MS/MS, ELISA, etc.


The bacterial reverse mutation test uses amino-acid requiring strains of Salmonella typhimurium and Escherichia coli to detect point mutations, which involve substitution, addition or deletion of one or a few DNA base pairs. The principle of this bacterial reverse mutation test is that it detects mutations which revert mutations present in the test strains and restore the functional capability of the bacteria to synthesize an essential amino acid. The revertant bacteria are detected by their ability to grow in the absence of the amino acid required by the parent test strain.

Suspensions of bacterial cells are exposed to the test substance in the presence and in the absence of an exogenous metabolic activation system. In the plate incorporation method, these suspensions are mixed with an overlay agar and plated immediately onto minimal medium. In the preincubation method, the treatment mixture is incubated and then mixed with an overlay agar before plating onto minimal medium. For both techniques, after two or three days of incubation, revertant colonies are counted and compared to the number of spontaneous revertant colonies on solvent control plates.