Raabe2, H., Burdick1, J., Hanlon2, E., Hilberer2, A., Hyder2, M., Inglis2, H., Kong2, A., Majewski1, S., McNamara1, M., Mun2, G., Nash2, J., Wilt2, N. 1Beauty Avenues, Reynoldsburg, OH, USA. 2Institute for In Vitro Sciences, Inc., Gaithersburg, MD, USA.

This study by scientists at Beauty Avenues and the Institute for In Vitro Sciences (IIVS) demonstrated that MatTek’s EpiDerm in vitro 3-D human skin tissue equivalent and EpiOcular in vitro 3-D human corneal tissue equivalent can be used, if/when necessary, with the ATP tissue viability assay in addition to the industry-standard MTT tissue viability assay. 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 reduce MTT (e.g., alpha-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 ViaLight® 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. 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. A series of model skin care formulations containing a range of concentrations of Triton® (to induce a range of cytotoxic effects) were tested, with and without the MTT reducer alpha-tocopherol (alpha-t), in the EpiOcular™ and EpiDerm™ 3-D in vitro eye and skin constructs. The MTT reducer, alpha-t, was not expected to add or reduce any cytotoxic effects in the formulation (see Table 1). Conclusions: • Freeze-killed control tissues demonstrated that test chemical (alpha-tocopherol) residues on treated tissues could directly reduce MTT in addition to the MTT reduction from viable cells. • Relative viability values from the MTT endpoint can be corrected for the amount of MTT reduced directly by test chemical residues in freeze-killed tissue controls. In some cases, the correction for direct MTT reducers gave a more conservative irritancy prediction. • Test chemical (alpha-tocopherol) residues on killed control tissues had no impact upon the ATP endpoint. • Both the MTT and ATP endpoint assays provided the same rank order of irritancies for the model formulations. • Since the relative viability values from the ATP assay were lower than those from the MTT assay, the ET50 values from the ATP endpoint may not be directly substituted for those from the MTT endpoint. • No evidence of hormesis was observed in the ATP endpoint data.


ATP assay, EpiDerm, EpiOcular, Freeze-killed control tissue, Hormesis, MTT reducers, ViaLight® Plus ATP assay

Materials Tested

Triton® X-100, alpha-tocopherol

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