The irritation potential of formulations and ingredients for industrial screening and product development is often conducted using in vitro 3-D human ocular and epidermal tissue constructs. To predict irritation potential after chemical exposure, tissue viability is typically determined by the ability of live cells to reduce MTT. Toxic exposures result in decreases in relative MTT reduction. However, two issues may contribute to inaccurate viability assessment: subtoxic exposures that induce higher metabolic rates typically greater than controls (i.e., hormesis) and chemicals that directly reduce MTT causing an overestimation of tissue viability (e.g., NaOH, α-tocopherol (α-t), ascorbic acid). For such chemicals, residues left on the tissues may increase the total MTT signal, so freeze-killed tissues are used to estimate chemical-mediated reduction of MTT. However, alternative methods of measuring tissue viability, such as amount of adenosine triphosphate (ATP) may be used. We compared these two methods by testing a series of model mild skin care formulations in 3-D human eye and skin constructs. The formulations were spiked with various concentrations of Triton® to induce a range of toxic effects, and were prepared with and without α-t, a MTT reducer. For formulations with α-t, freeze-killed tissues were tested in parallel in both the MTT and ATP assays. The results showed the same irritancy predictions for the 4 formulations containing α-t as for the 4 control formulations without α-t (e.g., formula with highest Triton conc.: ET50 eye = 172 and 157 min, ET50 skin = 778 and 772 min, with and w/o α-t). The ATP assay provided the same rank order of irritancy as did the MTT assay although the relative viability values from the ATP assay at each exposure were overall lower (e.g., formula with highest Triton conc.: ET50 eye = 14.5 and 12.9 min, ET50 skin = 202 and 231 min, with and w/o α-t). In summary, the MTT assay of formulas capable of MTT reduction should include freeze-killed tissues, and the ATP assay can confirm the relative rank order of the irritancy predictions.
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 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.