Genotoxicity test

Genotoxicity was evaluated with the micronuclei test (FluoroShield with DAPI) to detect some forms of chromosome mutations. For this detection, 3 × 10⁵ human pulp fibroblasts (FP6) were cultivated with 1 mL of DMEM supplemented with 10% FBS in 24-well plates (Prolab, São Paulo, SP, Brazil) for 24 h at 37 °C in an atmosphere of 5% CO₂. The cells were exposed to 1:8 and 1:16 dilutions of Brix 3000 and Papacarie Duo for 24 h. Additionally, there was a negative control (cells only) and a positive control with 5 mM EMS (Sigma-Aldrich, Brazil). Then, the supernatants were discarded, and two washes were performed with buffered saline solution (free from calcium and magnesium, CMF-PBS) to remove nonviable cells. Later, the cells were fixed with 4% formaldehyde for 10 minutes. After additional washing, 200 µL of PBS and a drop of FluoroShield with DAPI were added. The plate was agitated on an orbital table (Solab, Piracicaba, SP, Brazil) for 5 minutes under light protection. The plates were analyzed under a fluorescence microscope (Axiovert 200, Zeiss, Jena, Germany), and the micronuclei were counted every 2,000 cells.