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Special Feature: REACH and In Vitro Alternatives—Corrosive Potential Testing

Posted: November 28, 2007

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Currently there are commercially available skin tissue models that meet the OECD guidelines and have been validated by ECVAM for the in vitro testing of chemical corrosive potential.a,b The basis for this method is the observation that corrosive chemicals are cytotoxic to the viable layers of the epidermis after a short term exposure. In fact, the basic protocol for one of the available tissue modelsa uses just two exposure times. The materials to be screened for corrosive potential are topically applied to the tissues for three minutes and to a second set of tissues for one hour, followed by immediate determination of the cytotoxic effect.

Cytotoxicity is determined through the use of the vital dye MTT. In the tissues, viable keratinocytes will take up MTT, and the reduction of MTT by mitochondrial enzymes results in the formation of insoluble purple formazan crystals. The purple formazan crystals can be extracted with isopropanol and quantified spectrophotometrically. The intensity of the purple color is directly proportional to the number of viable keratinocytes in the tissue, and inversely proportional to the cytotoxicity of the test material. Thus, corrosive materials will reduce the number of viable cells, which in turn will result in less of the purple formazan crystal being formed in the MTT assay. This will be shown quantitatively by a low absorbance measurement of the extracted formazan solution for tissues treated with corrosive materials.4

The absorbance data from the MTT assay can easily be converted into a measurement of tissue viability. Since a negative control of distilled water being applied to tissues is included in the experiment, the average absorbance measurement for the MTT extracted from the negative control tissues can be used to represent 100% tissue viability, meaning all of the keratinocytes are alive and unharmed. Tissue viability can be calculated using the following formula:

Percent Viability = MTT absorbance of treated tissues/mean MTT absorbance of untreated tissues x 100

By dividing the absorbance measurement of the MTT extract from tissues treated with the materials being tested for corrosive potential by the average absorbance value of the MTT extract from negative control tissues, the result will give an index of the viability of the test material treated tissues. If the absorbance measurement of the MTT extract from the tissues treated with the test material is half the absorbance of the negative control extracts, then the tissue viability was reduced to 50%. This means that the test material had a cytotoxic effect and reduced the number of viable keratinocytes in the skin tissue model by half.