Abstract
Phototoxicity assessment is a critical component of preclinical safety assessment of pharmaceuticals with potential photosensitivity. 3T3 Neutral Red Uptake (NRU) Phototoxicity test, OECD Test Guideline 432, has been widely used as a reference method but concerns about oversensitivity to UVA, insufficient cell attachment and resultant difficulties in achieving the acceptance criteria is raised, which is mainly attributable to aged and over-sensitized BALB/c 3T3 cell line. Here, we compared BALB/c 3T3, NIH 3T3, and HaCaT cell lines for in vitro NRU phototoxicity test using 3 phototoxic pharmaceuticals and non-phototoxic chemicals with well-established photo-reactivity profiles. BALB/c 3T3 cells showed oversensitivity to UVA, which often showed decreased viability below 80% of non-irradiated control. Cell counts were increased to 2 × 10(4) cells to meet the viability criteria. With established experimental condition, in vitro phototoxicity tests were done with three cell lines. While all three cell lines succeeded in distinguishing phototoxic pharmaceuticals, significant differences were noted in sensitivity metrics: BALB/c 3T3 displayed the highest responsiveness, whereas NIH 3T3 exhibited moderate sensitivity followed by HaCaT. In addition, NIH 3T3 and HaCaT showed higher tolerance to UV-induced damages, uniform adherence to culture surface, and minimal well-to-well variability, thereby improving assay reproducibility. Collectively, these findings demonstrated that NIH 3T3 and HaCaT cells can be used as better alternatives to BALB/c 3T3 in the in vitro phototoxicity test.