WHY CONDUCT A SKIN CORROSION TEST?
This in vitro skin corrosion test meets the requirements for hazard identification and labelling for compliance with a range of legislation including REACH and the CLP Regulation. The output is a binary classification of Corrosive or Non-Corrosive. An adaptation is available to distinguish between GHS sub-categories 1A and 1B/C for skin corrosives.
Skin corrosion is defined as the production of irreversible tissue damage, manifested as visible necrosis of the skin, according to the definition of the Globally Harmonized System for Classification and Labelling of Chemicals.
The test described here is fully accepted at a regulatory level for the hazard identification of corrosive and non-corrosive chemicals in accordance with the UN Globally Harmonized System (GHS) of Classification and Labelling. It can be used for single substances and for mixtures including finished products. It is appropriate for compliance with a range of legislation including REACH (Registration, Evaluation, Authorisation and restriction of CHemicals) and the CLP Regulation 1272/2008 (Classification, Packaging and Labelling of substances and mixtures).
The test method is based on reconstructed human epidermis (RhE), which in its overall design mimics the biochemical and physiological properties of the upper parts of the human skin. The procedure is based on the principle that corrosive chemicals are able to penetrate the skin barrier (stratum corneum) by diffusion or erosion, and are cytotoxic to the underlying cell layers. The test item is applied directly to the skin surface, providing a good model of “real life” exposure. Cell viability is measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that is quantitatively measured after extraction from the skin tissues. Corrosive test items are identified by their ability to decrease cell viability below defined threshold levels, resulting in classification as Corrosive (UN GHS Category 1) or Non-Corrosive (“no-label”). Sub-categorisation of corrosive test items into Category 1A or 1B/1C is possible.
TEST SYSTEM: RECONSTRUCTED HUMAN EPIDERMIS
Reconstructed human epidermis is a skin model composed of living human keratinocytes which have been cultured to form a multi-layered, highly differentiated epidermis. The levels of differentiation obtained are at the cutting edge of in vitro skin technology. The model consists of highly organised basal cells which progressively flatten out as the apical surface of the tissue is approached, analogous to the normal human in vivo epidermis. The model includes a functional skin barrier with an in vivo-like lipid profile. The profiles of key differentiation markers also mirror those seen in vivo.
The cells are both metabolically and mitotically active, and release many of the pro-inflammatory agents (cytokines) known to be important in skin irritation and inflammation. Reconstructed human epidermis is grown on special platforms at the air-liquid interface, allowing for direct application of test items in a way that accurately models “real life” skin exposure.
SUMMARY OF THE TEST METHOD
- Skin models are pre-warmed in a cell culture incubator (37°C / 5% CO2) for 60 minutes.
- The test item is applied to the surface of the skin models: triplicate models are dosed at the apical surface with 50μl (liquid) or 25mg (solid).
- Controls consist of ultrapure water (negative control) and potassium hydroxide (positive control).
- The dosed skin models are placed into a cell culture incubator for 3 minutes and 60 minutes (triplicate models for each time point).
- Test items and control substances are removed from the skin models’ surface by washing.
- The viability of the skin models is assessed by MTT conversion. MTT solution is applied to the surface of the skin models and placed into a cell culture incubator for 3 hours. The blue formazan metabolite produced by viable cells is then extracted into isopropanol by incubation at room temperature for 2 hours or overnight.
- Triplicate samples of the extracted formazan solution are transferred to a microplate and the formazan product is quantified by absorbance spectrophotometry (wavelength 570nm).
- Absorbance readings of the formazan product from skin models incubated with test items are compared with those of negative controls to calculate percentage viability.
- A range of acceptance criteria must be satisfied in accordance with OECD TG 431.
- If the viability is less than 50% after 3 minutes, the test item is classified as corrosive. If viability is 50% or greater after 3 minutes but less than 15% after 60 minutes, the test item is classified as corrosive. If viability is greater than 50% after 3 minutes and equal to or greater than 15% at 60 minutes, the test item is classified as non-corrosive.
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Skin corrosion test
At a glance
|What it measures||Viability of skin cells|
|Compliance||OECD TG 431|
|Turnaround time||6-8 weeks|
|Method||Reconstructed human epidermis|
|Sample required||10ml (liquids) / 10g (solids)|
Always animal and animal product-free