*2.3. Evaluation of the Biosurfactant Cytotoxic Potential*

The biosurfactant cytotoxicity was assessed using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide) (MTT) method [16,17]. The L929 (mouse fibroblast) cells and the Vero (renal epithelial) cells from the African green monkey were maintained in Dulbecco's Modified Eagle Medium supplemented with 10% inactivated fetal bovine serum and a 1% antibiotic (penicillin and streptomycin) solution. The cells were kept in a chamber at 37 ◦C in an atmosphere enriched with 5% CO<sup>2</sup> and 95% humidity.

The cells were plated (10<sup>5</sup> cells/mL) in 96-well plates and incubated for 24 h. Next, 10 µL of the biosurfactant solutions was added to the wells at a final concentration of 3.12 to 400 µg/mL. After a further incubation for 72 h, 25 µL of the MTT solution (5 mg/mL) was added, followed by an incubation of 3 h. The culture with the MTT was then aspirated and 100 µL of dimethyl sulfoxide was added to each well. The absorbance was measured in a microplate reader at a wavelength of 560 nm. The experiments were conducted in triplicate and the percentage of inhibition was calculated with the aid of the demo version of GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA, USA).

An intensity scale was used for the determination of toxicity. Samples with 95 to 100% inhibitory activity were considered to be highly toxic, those with 70 to 90% inhibitory activity were considered to be moderately toxic and those with inhibitory activity less than 50% were considered to be non-toxic [18].
