Cytotoxic effect of Cuban propolis extracts on normal cells and in-vitro basal toxicity assay to estimate acute oral toxicity

In-vitro toxicity tests are recognized as alternative methods to animal acute toxicity testing. The aim of this study was to assess toxicity of 16 Cuban propolis extracts of different chemical types: brown (1, 4, 5, 16, and 17 BCP), red (9, 29, 35, 37, 45 and 72 RCP) and yellow (18, 39, 41, 50 and 60 YCP) against Balb/c 3T3 and Vero cells. Cells were treated at different concentrations (12.5; 25; 50 and 100 μg/mL) of propolis for 72 h and the IC50 value was determined. Furthermore we employed in-vitro cytotoxicity test described by Spielmann et al., red uptake (NRU) assay to estimate acute oral toxicity. Propolis showed differential cytotoxicity toward normal cells in a dose and tissue-dependence. RCP was the most interesting group because they did not affect normal cells evaluated. In Balb/c 3T3-A31 NRU cytotoxicity assay, after incubation for 48 h, IC50 values to RCP-45 and RCP-37 were 86.8 ± 1.14 and 96.1 ± 1.02 μg/mL, respectively. The LD50 values of these extracts were 558 and 579 mg/kg, respectively. For both samples the start dose for acute oral toxicity studies is 550 mg/kg in the case of using the Up-and-Down Procedure (UDP) and 300 mg/kg when Fixed Dose Procedure (FDP) and Acute Toxic Class Methods (ATC) were used. Results presented in this work can contribute to understand the toxicological profile of Cuban propolis and reduce the number of animals required in subsequent pharmacological / toxicological studies.