Implementation of an In Vitro Methodology for Phototoxicity Evaluation in 3T3 Cell Line

ROCO, JULIETA; LENZE, MARIELA; BENEDETTI, MARTINA; GUTIÉRREZ, MARÍA LAURA

Montevideo

Congreso; SETAC Latin America 15th Biennial Meeting; 2023

SETAC

Phototoxicity is an acute response induced by light, which occurs when photoreactive chemicals are activated by sunlight and transformed into cytotoxic products against cells of the skin and/or eye. In vivo methods using pigmented guinea pigs, mice or rats are used to simulate real human phototoxicity scenarios. Animals are exposed to chemicals topically or systemically and irradiated with an appropriate dose of ultraviolet A (UVA) light. However, the animal sacrifices, expense, and time required to perform the test pose many problems, especially in the era of widespread awareness of animal welfare and ethics. Nonanimal phototoxicity tests are gaining popularity these days to overcome these problems. The 3T3 in vitro phototoxicity assay can replace the use of experimental animals in the study of substances with phototoxic potential.The purpose of this work was to implement an in vitro test following the OECD guidelines No. 432 for the evaluation of the safety of multiple substances and products such as cosmetics, phytosanitary products, medical devices, drugs, effluents and industrial waste, among others, with in order to predict the potential effect of acute phototoxicity.Methodology: 3T3 cells were used and the cytotoxicity of different test substances was evaluated both in the presence and in the absence of UVA. Concentration-response curves (CRC) were performed using chlorpromazine (CPZ) as a positive control. To evaluate the response of the system, substances with a positive and negative response were evaluated, such as Norfloxacin and Sodium Lauryl Sulfate, respectively. Finally, the relative reduction in cell viability was evaluated and the inhibitory concentration 50(IC50) was calculated, which allows calculating two indices: the photoirritation factor (PIF) and thephotomedian effect (MPE).Results: The CRC for CPZ gave an IC50 of 23.04 ± 0.09 μg/mL (mean ± standard deviation) for thecondition without UVA radiation, meeting the acceptance criteria of the methodology (7.0 to 90.0 μg/ mL). In turn, under UVA irradiation the acceptance criteria were also met (0.1 to 2.0 μg/mL) since the IC50 was 0.76 ± 0.01 μg/mL. On the other hand, the indices calculated for CPZ were PIF > 5 and MPE > 0.15, revealing its positive phototoxic response.It is essential to have an effective in vitro test method to detect substances and products with phototoxic potential after application to skin, eyes and other external epithelia exposed to light.