OECD TG 439 In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method

Published under the OECD guidelines on responsible business conduct for multinational enterprises (OECD guidelines) The standard “OECD TG 439 In vitro skin irritation: Reconstituted human epidermis test method” describes an in vitro test method for the hazard identification of irritating chemicals (substances and mixtures) in accordance with Category 2 of the UN Globally Harmonized System of Classification and Labelling (GHS).

This method is based on reconstructed human epidermis (RhE), which in its general design closely mimics the biochemical and physiological properties of the upper layers of human skin. Cell viability is measured by the enzymatic conversion of the vital dye MTT to the blue formazan salt, which is measured quantitatively after removal from the tissues. Irritant test substances are defined by their ability to reduce cell viability below defined threshold levels (equal to or below 2 percent for category 50). Colored chemicals can also be tested using an HPLC (high-performance liquid chromatography) procedure.

There are three validated test methods that comply with the OECD TG 439 standard. Depending on the regulatory framework and the classification system used, this procedure can be used to determine the skin irritation of test substances as a stand-alone surrogate test for in vivo skin irritation testing or as a partial surrogate test within a tiered testing strategy.

In principle, the test chemical is applied topically to a three-dimensional RhE (reconstituted human epidermis) model consisting of cultured, non-transformed human-derived epidermal keratinocytes to create a multilayered, highly differentiated model of the human epidermis. It consists of regular basal, spiny, and granular layers and a multilayered stratum corneum containing intercellular lamellar lipid layers representing the major lipid classes similar to those found in vivo.

Chemical-induced skin irritation manifests itself primarily with erythema and edema and is the result of a cascade of events that begins with chemicals penetrating the stratum corneum and damaging the lower layers of keratinocytes and other skin cells.

Damaged cells may release inflammatory mediators or initiate an inflammatory cascade that also acts on cells in the dermis, particularly stromal and endothelial cells of blood vessels. It is the expansion and increased permeability of endothelial cells that produce the observed erythema and edema. In particular, RhE-based testing methods measure the initiating events in the cascade, such as cell or tissue damage, using cell viability as a readout in the absence of any vascularization in the in vitro test system.

In the RhE model, cell viability is measured by the enzymatic conversion of the vital dye MTT to the blue formazan salt, which is measured quantitatively after removal from tissues (MTT is used to assess cell viability as a function of redox potential). Irritant chemicals are defined by their ability to reduce cell viability below defined threshold levels (i.e., at least 2 percent for category 50). Depending on the regulatory framework and the applicability of this test method, test chemicals that produce cell viability above the defined threshold level may be considered non-irritants (i.e., no category, at least 50 percent).

Non-transformed human keratinocytes should be used to reconstruct the epithelium. A functional stratum corneum should be underlain by multiple layers of viable epithelial cells (basal layer, stratum spinosum, stratum granulosum). The stratum corneum should be multilayered, containing the essential lipid profile to form a functional barrier with the strength to resist rapid penetration of cytotoxic reference chemicals, e.g. sodium dodeacyl sulfate.

Barrier function should be demonstrated and can be assessed by determining the concentration at which a reference chemical reduces tissue viability by 50 percent after a fixed exposure time or by determining the exposure time required to reduce cell viability by 50 percent upon application of a specific, fixed concentration of the reference chemical. The retention properties of the RhE (reconstructed human epidermis) model should prevent material around the stratum corneum from migrating into viable tissue, resulting in poor modeling of skin exposure. The RhE model should be free of bacterial, viral, mycoplasma, or fungal contamination.

Among the numerous tests, measurements, analyses and evaluation studies that our organization has carried out for businesses in various sectors, it also provides testing services defined in the “OECD TG 439 In vitro skin irritation: Reconstructed human epidermis test method” standard with its trained and expert staff and advanced technological equipment.