In vitro analysis of various mouthwashes: cytotoxic, apoptotic, genotoxic and antibacterial effects.

Objective: This study aims to investigate the cytotoxic, apoptotic, and genotoxic effects of eleven mouthwashes, which are hypothesized to pose potential toxic risks to oral mucosal tissues, through in-vitro experiments using human gingival fibroblast(hGF) cell cultures and to compare the antibacterial efficacy of these mouthwashes.

Materials and methods: Healthy hGF cell lines were derived from individuals under ethical standards. The cytotoxic effects of the mouthwashes (Colgate 2in1,Colgate Optic White, Colgate Plax, Curasept, Elmex, Kloroben, Listerine Cool Mint, Listerine Zero, Meridol, Oral-B Pro Expert, Sensodyne Pronamel) were assessed in real-time using the xCELLigence system, which monitored cellular activity at 5-minute intervals over 72 h. Apoptotic effects of the IC50 concentrations of the mouthwashes on hGF cells were evaluated using Annexin V and Caspase-3 assays. Genotoxic effects at IC50 concentrations were analyzed through the Alexa Fluor^® 488 Mouse anti-H2AX assay. The antibacterial effect of mouthwashes on Streptococcus mutans and Lactobacillus rhamnosus was evaluated by modified microdilution method.

Results: According to the analysis of the IC50 values, Meridol was found to be the most cytotoxic mouthwash, while Listerine Zero was the least cytotoxic. The mouthwashes can be ranked in order of their cytotoxicity as follows: Meridol(0.011) > Elmex(0.029) > Colgate 2in1(0.187) > Colgate Plax(0.478) > Colgate Optic White (0.534) > Sensodyne Pronamel(0.577) > Oral-B Pro Expert(0.638) > Kloroben(0.766) > Curasept(1.872) > Listerine Cool Mint(2.334) > Listerine Zero(7.267)(p < 0.05). The Annexin V assay showed no major apoptotic impacts on human gingival fibroblast cell line at the IC50 values of the mouthwashes, except for Colgate Plax, Kloroben, and Oral B Pro Expert. The IC50 values of the evaluated mouthwashes did not show a significant apoptotic effect on the hGF cell line as evaluated by Caspase-3 assay and no significant genotoxic effect was observed as evaluated by H2AX assay(p > 0.05). Colgate Plax showed the most effective antibacterial effect on Streptococcus mutans and Lactobacillus rhamnosus(p < 0.05).

Conclusion: Mouthwash formulations showed varying cytotoxic effects on hGF and different antibacterial effectiveness. Essential oil-containing mouthwashes may be preferable to those with chlorhexidine or cetylpyridinium chloride, as they demonstrate less cytotoxicity, are more biocompatible, and have antibacterial efficacy.

Clinical relevance: The low cytotoxicity and potent antibacterial properties of essential oils render them a more safer choice for inclusion in mouthwash compositions.