Development of a modified 3T3 Neutral Red Uptake Phototoxicity Test protocol for evaluation of poorly water-soluble...

The 3T3 neutral red uptake phototoxicity test (OECD TG432) is an alternative phototoxicity test method that is relatively easy and rapid to implement, with results obtainable in a short time, and is reported to have high reproducibility compared with in vivo assay methods. However, this method has been shown to be unsuitable for testing poorly water-soluble substances, which tend to separate out when mixed with the assay buffer solution. This causes difficulties in determining the dose dependency of substances and subsequent determination of the photoirritation factor because the ratio of cell viability, expressed as the half-maximal inhibitory concentration (IC₅₀) in the presence or absence of light, is not calculable. In this study, we investigated the optimum conditions for the evaluation of poorly water-soluble substances. In the conventional method, the final solvent concentration was 1% and the pre-incubation time was 60 min, but in the modified method, 10% and 5 min were used, respectively. Next, the results from the conventional method were compared with those of our modified method, which was found to be viable and comparable with the conventional method. Moreover, the false positive results frequently obtained with poorly water-soluble substances in the conventional method were not evident with the modified method, thus confirming its usefulness for the evaluation of such substances. We therefore propose that the modified method can be used for the in vitro testing of poorly water-soluble substances in phototoxicity evaluations.