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Eye and Skin Irritation in 3-D Tissue Constructs Using MTT and ATP Endpoints
Published: March 30th, 2009
by H. Raabe
SUMMARY
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.
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