Silver nanoparticles (AgNPs) were successfully synthesized via the biological route using a 1 M silver nitrate (AgNO3) aqueous solution and an ethanolic peel extract of Punica granatum (Pg), at 60 °C. The physicochemical analysis revealed the formation of green synthesized Pg-AgNPs with a semi-spherical shape, non-uniformly distributed, and a particle size distribution between 5 and 100 nm. As regards the preliminary in vitro toxicological screening, the green synthesized Pg-AgNPs did not significantly affect the neonatal BALB/c epidermal cells’ viability (JB6 Cl 41-5a) at lower concentrations and did not produce visible changes in the morphology of the JB6 Cl 41-5a cells. In contrast, at higher concentrations (>50 μg/mL), the green Pg-AgNPs exhibited an important decrease in cell viability and confluency. In addition, the impact of Pg-AgNPs on cell membrane integrity suggests a potential cytotoxic effect. Contrary to the in vitro assays, after the evaluation of the anti-irritant effect in ovo, the lower concentration of Pg-AgNPs (10 μg/mL) produced hemorrhage and lysis when applied to the chorioallantoic membrane, while at 50 μg/mL, only slight coagulation was observed. Therefore, regarding the in ovo toxicological screening, the higher concentration of the Pg-AgNPs exhibited a better safety profile compared to the lower concentration, as indicated by the irritation score.
