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EYE AND SKIN IRRITATION IN 3-D HUMAN TISSUE CONSTRUCTS USING MTT AND ATP ENDPOINTS
Published: October 5th, 2009
by Raabe, Hans; Burdick, Joel; Hanlon, Elizabeth; Hilberer, Allison; Hyder, Matthew; Inglis, Heather; Kong, Amanda; Majewski, Sh
SUMMARY
In vitro eye and skin model assays are typically used to assure safety prior to consumer use by employing them in product development to support the creation of products with minimal irritation potential. As part of a high quality program, the test systems are constantly monitored for applicability, investigated for potential limitations, and continuously improved to ensure accurate and reliable data and information to support safety assessments. The applied research presented here evaluates an additional endpoint (ATP assay) of consideration in special cases where potential confounders may skew interpretation of results in core standard model systems (MTT assay). Viability assessments in 3-D in vitro eye and skin constructs have historically been assessed using the MTT assay. The MTT conversion assay measures the NAD(P)H-dependent microsomal enzyme and succinate dehydrogenase reduction of MTT to a blue formazan precipitate in viable cells (Berridge, 1996). Two factors can affect the accuracy of the MTT assay. First, since the MTT assay measured the mean metabolic rate of a cell population, subtoxic exposures may induce hormesis, where increased metabolism in response to cell damage incorrectly suggests high viability. Second, chemicals that directly reduct MTT (e.g., α-tocopherol) may overestimate tissue viability if these chemicals persist in the tissue model after rinsing. Freeze-killed tissue controls (KC) are tested in parallel to the viable tissues to determine the extent, if any, of the direct reduction by the test chemical. The ATP endpoint is an alternative to MTT reduction which would not be affected by hormesis since the endpoint measures cellular ATP content, rather than metabolic rate. The ATP endpoint may also be more appropriate for chemicals that are strong reducers of MTT, including many that are commonly used in personal care products. The ViaLightŪ Plus ATP assay kit utilizes the bioluminescent measurement of ATP (Crouch, et al., 1993). Since cytotoxicity is expressed as a reduction in the bioluminescent measurement of ATP, the assay provides a direct measure of the number of viable cells present. Upon cell stress or cell death, the amount of ATP is rapidly depleted or hydrolyzed.
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