2.3.1. Cell Viability (MTT Test)

Cell viability was evaluated using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide assay, as previously described [25]. Briefly, keratinocytes were homogeneously seeded in 96-well plates at a density of 1.5 × 10⁴ and incubated at 37 °C, with a 5% CO₂ humidified atmosphere. After 24 h, cells were treated with the cosmetic product, starting at 40 mg/mL, following serial dilution (1:2) in cell medium (tested range 0.313–40 mg/mL). Untreated cells were used as the control (Ctrl). The test was conducted in three replicates for each dilution. After 24 h of treatment, cells were incubated with 1 mg/mL MTT solution (Merck, Darmstadt, Germany) at 37 °C for 2 h. After removing the medium from each well, isopropanol was added to dissolve formazan crystals and the absorbance was read at a 570 nm wavelength using a microplate reader (MultiSkan, Thermo Scientific, Waltham, MA, USA). Cell survival was calculated measuring the difference in optical density (OD) of the tested cosmetic product with respect to the control (Equation (1)).

Reduction of cell viability by more than 30% is considered a cytotoxic effect.