2.2. In Vitro Skin Irritation Test (OECD 439)

The 3D reconstructed human epidermis tissue model: SkinEthic™ RHE (SkinEthics, Lyon, France) was used to evaluate whether CPP_AIF cause skin irritation [8]. Testing procedure involved topical application of testing article (CPP_AIF) to surface of epidermis and subsequent assessment of effect on cell viability. All 3D-skin tissues were incubated with growth medium for 2 h and then CPP_AIF was add to final 1 mM for 42 min treatment. After treatment, testing substance was washed out by 25 times with 1 mL phosphate buffered saline (PBS) and tissues were further incubated in growth medium for 42 h. After incubation, growth medium was substituted by maintenance medium with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) agent for 3 h incubation. Next, insert (with tissue) were washed with PBS and air dried. Formazan in tissues was extracted with isopropanol and measured by determining the OD at 570 nm using a microplate spectrophotometer (iMark Microplate Absorbance Reader, Bio-Rad, Hercules, CA, USA). Cell viability of treated models was normalized to the negative control, which was set to 100%.