The results in this research showed the toothpastes with different whitening ingredients have no genotoxic effect (statistically significant difference in number of micronuclei, binucleated cells or nuclear buds were found neither between evaluation periods within the same group nor between the groups for the same evaluation periods), while for some of them – Colgate Max Expert (CMEW), Rembrandt Daily White + Peroxide (RDWP) and Splat Extreme White (SEW), cytotoxic effect in terms of statistically significant difference in the number of karyorrhexis and condensed chromatin was identified between the sampling periods. Based on this fact the null hypothesis was partially rejected.
Statistically significant increase in the number of karyorrhexis and condensed chromatin cells was detected after 30 days (T1) and 60 days (T2) of using the toothpaste compared to baseline sampling at T0. Furthermore, the results point to statistically significant increase in the number condensed chromatin cells even at T2 compared to T1 for RDWP and SEW. Both cytotoxic parameters were decreased in the follow up period (T3) for the three whitening toothpastes. Interestingly, no statistically significant difference for the three whitening toothpastes was observed against control non-whitening toothpaste (Kalodont Multi Repair) as for no other tested whitening toothpaste. Based on these results, it can be judged that a slight cytotoxic effect may be present for all tested whitening toothpastes, while only for some of them (CMEW, RDWP, SEW) this effect is statistically significant. The differences between the evaluation periods of the three whitening toothpastes further indicate direct correlation of toothpaste application period with the observed alterations. Finally, it is important to highlight that all the three whitening toothpastes revealing statistically significant difference for the two cytotoxic parameters (karyorrhexis and condensed chromatin) contain peroxides in its composition (hydrogen peroxide and urea peroxide) as an active whitening ingredient. This implies peroxides to dominantly influence the revealed cytotoxic effect of the three whitening toothpastes.
Due to a limited number of studies (3, 4, 10, 28–30) in the available literature that analyze the cytotoxicity and genotoxicity performance of toothpastes classified as whitening toothpastes, this study was compared to studies that evaluated similar effects of other over the counter products and bleaching agents for professional usage.
Complementary to the results of this study, an in vitro study of Rode et al. (10) applying the micronucleus test (MNT) suggest no genotoxic potential of the whitening toothpastes while the elements of cytotoxic potential exist. The cytotoxic potential was presented by fluoride whitening toothpastes for which they showed cytotoxicity in gingival fibroblasts was related to concentration of the toothpaste (higher concentration caused cytotoxicity). Based to their research, they claim the cytotoxicity of the toothpastes is mainly caused due to fluoride in its composition. Opposed to this, results of this study conclude that the increased cytotoxicity alterations (in terms of karyorrhexis and condensed chromatin) are dominantly caused by peroxide in the whitening toothpaste composition, despite the concentration of peroxides in toothpaste is usually low − 1% hydrogen peroxide or 0.5–0.7% calcium peroxide (31, 32). However, it is important to highlight here that all tests in this study were conducted with constant same concentration of the toothpaste throughout all sampling time. Following, based on the results in this study solely, the influence of other ingredients, such as fluoride, cannot be entirely neglected. Similar results for cytotoxic effect of toothpastes to those of Rode et al (10) were shown by Camargo et al. (3) that were using in vitro MNT test on Chinese hamster fibroblasts and reported higher concentration of the toothpaste caused cytotoxicity in V79 cells. In their work, cytotoxicity of whitening toothpastes was presented through cell survival. The most cytotoxic toothpaste they showed to be Colgate Whitening, which had viable cells lower than 5%. Based on this fact, they concluded that the whitening toothpastes promote the highest cytotoxicity amongst toothpastes. Unlike the work of Rode et al. (10) and this study, Camargo et al. (3) report also genotoxic effect of the whitening toothpastes. In their in vitro study, using the methyl tetrazolium test (MTT) on human gingival fibroblasts (HGF-1), they reported Oral-B whitening toothpaste, having fluoride and abrasives as the main whitening ingredients, as the most genotoxic one. Although in this study no test was conducted on Oral-B whitening toothpaste, other fluoride and abrasives based toothpastes were examined. Irrespective of the whitening ingredients of the toothpastes, the results of this study point to a conclusion of no genotoxic effect of the whitening toothpastes. In their study, Bruno et al.(29) used the MTT assay in in vitro conditions and reported that all tested toothpastes, including whitening toothpaste, Colgate Luminous White, were classified as highly cytotoxic - cell viabilities were lower than 50%. They emphasize that decrease in cell viability, implying cytotoxicity, can be attributed to the various components in toothpastes, either alone or combined. This study points to nearly same conclusion yet implying peroxides to dominantly attribute to cytotoxicity of the whitening toothpastes. In their research, Tadin et al. (4) compared the toxicity of whitening toothpastes and non-whitening toothpastes, and only one of these tested whitening toothpastes showed significant increase in number of micronucleated cells after 60 days of usage - Colgate Whitening toothpaste. They reported that whitening toothpastes can express genotoxic effects on buccal epithelial cells, but the obtained results were classified as limited and biologically insignificant. The results of this research suggest no genotoxic potential of the observed whitening toothpastes at all.
In in vitro study on mouse fibroblasts cells L929, reported by Torrado et al. (28), the authors highlighted (based on MTT assay) that the commercially available Crest Extra Whitening toothpaste caused an inhibition percentage not greater than 50% and they concluded that cytotoxicity was not increased with the duration of process. Based on the results of this study, the cytotoxicity of the three tested whitening toothpastes (CMEW, RDWP and SEW) was directly related to the time of usage. Ghapanchi et al. (30) tested cytotoxicity in in vitro study of 16 commercial toothpaste in primary epithelial cells of the oral cavity and HeLa cell line for exposure from 1 to 5 minutes, and cytotoxic effects on buccal mucosa cells were evaluated for different duration, thus an increase in cytotoxicity was positively correlated with duration of exposure. All tested toothpastes showed cytotoxic potential, but to a different extent. Nearly the same results, but in various concentrations of hydrogen peroxide, was reported by Farukawa et al (33) for different duration exposure on cultured human gingival fibroblasts (HGFs). They results showed that the low concentration of hydrogen peroxide (0.0015%) had no effect on the survival of the cells after 30 minutes of exposure, but a reduced survival rate was obtained after 60 minutes. Lower concentration of hydrogen peroxide (0.00015%) didn't affect cell survivor even after 60 minutes. Concentration of ≥ 0.15% hydrogen peroxide affected cell survival after 90 seconds of exposure. Both studies (30, 33) showed correlation between the level of cytotoxicity and the time of exposure. Moreover, some chemical ingredients such as hydrogen peroxide, significantly increased cytotoxicity even in a short exposure time. Although the exact concentrations of peroxides and other active ingredients in tested whitening toothpastes was not compared in this study, the results suggest nearly same conclusion to those in (30, 33), but for different time intervals of exposure (1–5 minutes vs. 2 months of usage). It is important to note that in this study the toothpaste was always in contact with buccal mucosa for the same duration (3 minutes for each application of the toothpaste) during the experiment, so the magnitude of daily application was always the same during the 2 months treatment.
Concentrations of hydrogen peroxides in other bleaching agents for professional and non-professional usage are various (3–38%). The toxic potential of various bleaching substances has been evaluated in the literature. Del Real Garcia et al. (34) in their study assessed the impact of 10% hydrogen peroxide whitening strip exposure on the genotoxicity and oxidative damage. They concluded that strips with 10% hydrogen peroxide exhibit increased in NAs in oral epithelial cells and 8-OHdG levels in saliva which produce oxidative DNA damage. According to these results, they recommend careful and rational handling of self-application bleaching agents. Contrary to these results, Monteiro et al.(16) evaluated the genotoxic potential of 10% hydrogen peroxide at-home bleaching gels and concluded that there was no sign of genotoxic effect during the application of this bleaching agent for 30 min/day for 14 days. Klarić et al.(35) investigated the genotoxic effect of two hydrogen peroxide bleaching agents on oral mucosal cells and both demonstrated potentially genotoxic effect. It is important to mention that the concentration of hydrogen peroxide bleaching agent which they used was higher than in whitening toothpastes (25% and 38%). High concentration of peroxide is only allowed for professional usage, which is not the case with products with significantly lower concentrations of peroxide such as tooth whitening pastes. However, it is important to know that the hydrogen peroxide can interact with DNA and increase the concentration of reactive oxygen species and free radicals leading to consequent oxidative DNA damage (36, 37). The EU Scientific Committee on Consumer Safety (SCCP 2007) concluded that products with 0.1% hydrogen peroxide or the release up to 0.1% hydrogen peroxide are safe for human usage (38). In our study, none of the tested whitening toothpastes containing chemical agents (peroxides and/or enzymes; CMEW, HSWH, RDWP, SEW and SWP), abrasives (SDW and SWS) and charcoal (BDW and DBS) showed statistically significant increase of genotoxic parameters. Comparing our results to afore mentioned studies for the bleaching agents, we can conclude that the concentration of peroxides in the whitening toothpastes is low enough not to cause any genotoxic effects.
Generally, the side effects on the cells of the oral mucosa can be directly related to the presence of fluoride and fluoride concentration in fluoride containing products for oral hygiene, SLS, triclosan, sodium monofluorophosphate, silicon dioxide, sodium benzoate, preservatives, colors and flavors (2, 8, 30, 39). During the cytotoxic and genotoxic analysis of whitening toothpastes, it is important to note that components such as environmental, biological, demographic factors, as well as professional exposure to some toxins can be predilection factors for the appearance of increased toxic parameters in in vivo conditions. In our research the results of multiple regression showed significant dependence of genotoxicity parameters with demographic and lifestyle factors as possible predictors (numbers of cells with micronuclei with gender, alcohol consumption and number of amalgam and composite fillings; binucleated cells with alcohol consumption and number of composite fillings; nuclear buds with gender, coffee consumption and number of composite filling). Also, the cytotoxic parameters showed the significant dependence with demographic and lifestyle factors as possible predictors (number of cells with condensed chromatin with age, alcohol consumption and number of amalgam fillings; number with karyorrhexis with gender, coffee consumption and number of composite fillings; karyolysis with meat, coffee and alcohol consumption; pyknosis with gender and alcohol and coffee consumption.
The results in this study indicate a connection between the increase of cytotoxicity in tested toothpastes and the peroxide content in them. Namely, whitening toothpastes, that showed an increase in cytotoxicity, contained peroxide (urea or hydrogen peroxide) in their composition. As expected, with the cessation of the use of toothpastes with peroxides, a decrease in cytotoxic parameters was recorded. Our results suggest that the cytotoxic behavior of toothpastes might be due to peroxides in their compositions, although the influence of other components/ingredients in whitening toothpastes cannot be ruled out, especially in combination with peroxides in their composition. In clinical condition, the obtained results of tested whitening toothpastes, indicate a limited, biologically insignificant cytotoxic effect on buccal mucosal cells.
This study had some limitations. It did not test individual components/ingredients of toothpastes and their exact concentrations in them, so it is suggested for the future studies to consider testing individual components of whitening toothpastes and test the influence on cell survivor in in vitro conditions, using, for example MTT on human gingival fibroblasts (HGF). In addition, it would be desirable to analyze the cells of the buccal mucosa and samples from the gingiva or oropharynx to get a better insight into the toxicity of whitening toothpastes. It would be desirable to test the toxicity of different concentrations of individual components during the various exposure times – for example, for a duration of 1 to 5 minutes. Therefore, more in vitro/in vivo studies are required to investigate biocompatibility of individual ingredients of whitening toothpastes. Ultimately, for consumers, it is important to pay attention to the ingredients in whitening toothpastes when choosing toothpastes.