The “EPI-200-SIT Skin Irritation Test” is designed to estimate the skin irritation potential of pure test substances in the context of the identification and classification of the skin irritation hazard according to the European Union classification system. As harmonized with the European Union and the Globally Harmonized System of Classification and Labelling of Chemicals (GHS), the procedure described in this standard also allows the hazard identification of irritating substances in accordance with the GHS.
This standard allows distinction to be made between category 2 irritants and non-irritants. The test method described does not distinguish between non-mandatory subcategories of the GHS, i.e. it does not distinguish between GHS category 2 and category 3 irritants.
The method described in the EPI-200-SIT standard consists of topical exposure of pure test chemical to a reconstructed human epidermis model followed by cell viability testing. Cell viability is measured by dehydrogenase conversion of MTT present in cell mitochondria to a blue formazan salt that is measured quantitatively after removal from tissues.
The reduced viability of tissues exposed to chemicals compared to negative controls (water-treated) is used to estimate the potential for skin irritation. Recent comparative studies in human epidermis models using various endpoints to estimate skin irritation of topical formulations have shown that the MTT endpoint has distinct advantages even over mechanistically based endpoints such as release.
The reconstructed human epidermal model consists of normal human epidermal keratinocytes cultured to form 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 arranged in patterns similar to those found in living tissue. A general description of the general and functional conditions that reconstructed human skin models should comply with is included in the new OECD test guidelines (“431 In vitro skin corrosion: Human skin model” and “439 In vitro skin irritation: Reconstructed human epidermis test method”).
All biological components of the epidermis and culture media are tested by the manufacturer for viral, bacterial, fungal and mycoplasma contamination. Results are expected to be within a range determined by the historical database.
Due to the long post-incubation period, the test must be performed under aseptic conditions in a microbiological safety cabinet (laminar flow hood).
The EPI-200-SIT Skin Irritation Test was developed and validated for in vitro skin irritation testing of chemicals including cosmetic and pharmaceutical ingredients. The test is performed over a 60-day period consisting of pre-incubation, 42-minute exposure, 4-hour post-incubation and MTT viability testing. After tissue collection and overnight pre-incubation (day 0), tissues are topically exposed to test chemicals (day 1) which may be liquid, semi-solid, solid or wax. Three tissues are used for each test chemical and for a positive control (5 percent aqueous sodium dodecyl sulfate solution) and one negative control. Chemical exposure lasts 60 minutes, of which the tissues are kept in an incubator at 35 degrees for 37 minutes. Test substances are then removed from the tissue surface by a thorough washing procedure. Tissue inserts are stained and transferred to fresh medium.
After a 24-h incubation period (day 2), the medium is changed. The medium can be stored for further analysis of cytokines or other endpoints of interest. After the medium change, the tissues are incubated for an additional 18 h. At the end of the entire 42-h incubation period (day 3), the tissues are transferred to the yellow MTT solution and incubated for 3 h.
The resulting purple-blue formazan salt, formed primarily by mitochondrial metabolism, is extracted using isopropanol for 2 hours. The optical density of the extracted formazan is determined using a spectrophotometer. A chemical is classified as an irritant if tissue viability falls below 50 percent relative to tissues treated with a negative control. This procedure can be used as a complete replacement for the in vivo rabbit skin irritation test for hazard identification and labeling of chemicals in accordance with European Union regulations.
Among the numerous tests, measurements, analyses and evaluation studies carried out for businesses in various sectors, our organization also provides testing services defined in the “EPI-200-SIT Skin irritation test” standard with its trained and expert staff and advanced technological equipment.
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