In Vitro Toxicity Evaluation of Carrageenan on Cells and Tissues of the Oral Cavity.

Carrageenan is a highly potent anti-human papillomavirus (HPV) agent with the potential for formulation as a mouthwash against oral HPV infection. However, its toxic effect on tissues of the oral cavity is currently unknown. This study aims to evaluate the safety of carrageenan on human cells and tissues of the oral cavity. Human salivary gland cells and reconstructed human oral epithelium (RHOE) were used for this in vitro study. The cells were subjected to 0.005-100 µg/mL of carrageenan for 4, 12, and 24 h in quadruplicate. RHOE were exposed to 100 µg/mL of carrageenan for 24 h in triplicate and stained with hematoxylin/eosin for histological analyses. All experiments had saline and 1% sodium dodecyl sulphate (SDS) as negative and positive controls, respectively. Carrageenan tissue toxicity was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to quantify cell viability. Tissue toxicity was further evaluated histologically by an oral pathologist to assess morphological changes. Our data showed that carrageenan did not significantly decrease cell and tissue viability when compared to the positive control. The histological evaluation of the RHOE also showed no loss of viability of the carrageenan-treated sample compared to untreated tissue. In contrast, 1% SDS-treated RHOE showed extensive tissue destruction. Our experiments suggest that carrageenan is safe for use in the oral cavity.

Safety evaluation of MA-T after ingestion in mice.

MA-T (Matching Transformation System®) is a proprietary chemical mixture for on-demand production of aqueous chlorine dioxide that is used for the treatment of oral malodor. MA-T is also an effective disinfectant against at least 39 pathological microorganisms, including severe acute respiratory syndrome coronavirus 2, and therefore may be useful as a disinfectant mouthwash to prevent the spread of infection. Accidental ingestion is the putative worst hazard scenario associated with mouthwash use; therefore, here we investigated the safety of MA-T ingestion in mice. Mice were provided drinking water containing 0-3000 µg/ml MA-T for 7 days followed by non-spiked drinking water for an additional 14 days. At day 7, mice ingesting 1000 or 3000 µg/ml MA-T showed significantly decreased body weight and significantly increased liver, kidney, and heart tissue injury biomarkers compared with control. However, at 14 days after stopping MA-T ingestion, body weight and tissue injury biomarkers had returned to normal. Histological analysis revealed that MA-T-induced injuries in liver, kidney, spleen, stomach, duodenum, colon, and rectum had also recovered at 14 days after stopping MA-T ingestion; however, mild vascular endothelial injuries remained in heart, jejunum, and ileum in the worst-case scenario. Taken together, MA-T may be potentially safety for further development as a disinfectant mouthwash by risk management, such as placing a caution of the label and adding a distinctive flavor.

Effects of Na-DNA mouthwash solutions on oral soft tissues. A bioreactor-based reconstituted human oral epithelium model.

PURPOSE: To investigate whether the addition of sodium-DNA (Na-DNA) to chlorhexidine (CHX)-containing mouthwash influenced morphology and viability of a reconstituted human oral epithelium (ROE), and protects ROE against oxidative stress. METHODS: Multi-layered 0.5 cm² ROE specimens were positioned inside a continuous flow bioreactor and grown air-lifted for 24 hours. They were treated with phosphate-buffered saline (PBS) (n= 16) or 1 vol% H2O2 for 1 minute (n= 16). Then, they were treated for 5 (n= 8) or 30 minutes (n= 8) with the experimental mouthwash solutions containing: 0.2 wt% CHX, 0.2 wt% CHX + 0.2 wt% Na-DNA, 0.2 wt% Na-DNA, PBS. After 60 minutes washout specimens were subjected to tetrazolium-based viability assay (MTT) confocal laser-scanning microscopy (CLSM), and histological evaluation using optical microscopy and transmission electron microscopy (TEM). RESULTS: ROE treated with Na-DNA for 30 minutes revealed significantly higher viability than PBS, and CHX + Na-DNA showed higher viability after 30-minute treatment than after 5 minutes, suggesting a significant protective activity of Na-DNA. Moreover, the protective effect of Na-DNA on cell viability was higher after the induction of oxidative stress. After treatment with CHX, CLSM revealed cell stress, leading to cell death in the outer layer. On the contrary, specimens treated with Na-DNA showed a much lower number of dead cells compared to PBS, both in the absence or presence of oxidative stress. Histological examination showed that the protective action of Na-DNA formulations reached more in-depth into the epithelium exposed to oxidative stress, due to intercellular spaces opening in the outer epithelium layers, giving way to Na-DNA to the inner parts of the epithelium. It can be concluded that Na-DNA had a topical protective activity when applied for 30 minutes unless the epithelium barrier is damaged, allowing it to act more in-depth. CLINICAL SIGNIFICANCE: Na-DNA showed a clear and protective action against cellular degeneration due to oxidative stress and, partly, to the exposure to CHX. Its addition to chlorhexidine mouthwash or gels could be clinically helpful in contrasting the detrimental activity of CHX on oral tissues, and in the preservation of cell viability, control of inflammation and wound healing.

A 30-s exposure to ethanol 20% is cytotoxic to human keratinocytes: possible mechanistic link between alcohol-containing mouthwashes and oral cancer.

OBJECTIVES: To provide mechanistic evidence for the epidemiological link between long-term use of alcohol-containing mouthwashes and oral cancer. MATERIAL AND METHODS: Human epithelial keratinocytes were exposed for 30 s to concentrations of ethanol commonly present in mouthwashes. After a recovery period, cell viability was assessed with the MTT assay. RESULTS: A marked cytotoxic effect was observed for ethanol concentrations of 20% and above. CONCLUSIONS: The cytotoxicity of ethanol may explain the epidemiological association between mouthwash use and oral cancer. Evidence suggests that the risk of developing cancer in a tissue is strongly determined by the number of stem cell divisions accumulated by the tissue during a person's lifetime; cell division is a major source of mutations and other cancer-promoting errors. Since cell death activates the division of stem cells, the possible cytotoxicity of ethanol on the cells lining the oral mucosa will promote the division of the stem cells located in deeper layers to produce new cells to regenerate the damaged epithelium. If we regularly use mouthwashes containing cytotoxic concentrations of ethanol, the stem cells of the oral cavity may need to divide more often than usual and our risk of developing oral cancer may increase. CLINICAL RELEVANCE: Many mouthwashes contain percentages of ethanol above 20%. Because ethanol is not crucial to prevent and reduce gingivitis and plaque, members of the dental team should consider the potential risk of oral cancer associated with frequent use of alcohol-containing mouthwashes when advising their patients.

Effects of octenidine mouth rinse on apoptosis and necrosis of human fibroblasts and epithelial cells - an in vitro study.

This study aimed at comparing the cytotoxicity of a new octenidine mouth rinse (MR) on gingival fibroblasts and epithelial cells using different established MRs. Octenidol (OCT), Chlorhexidine 0.2% (CHX), Meridol (MER), Oral B (OB), and control (PBS only) were used. Human primary gingival fibroblasts (HGFIBs) and human primary nasal epithelial cells (HNEPCs) were cultivated in cell-specific media (2 × 105 cells/well) and treated with a MR or PBS for 1, 5, and 15 min. All tests were performed in duplicate and repeated 12 times. The apoptosis and necrosis were determined using a Caspase-3/7 assay and LDH assay, respectively. The data were analyzed using two-way analysis of variance with subsequent Mann-Whitney U-test. No significant differences could be found between the incubation times of the MR, neither for apoptosis nor necrosis (p > 0.05). Regarding apoptosis of HGFIBs, MRs had no influence at all. In HNEPCs, OCT induced relevantly lower apoptosis than CHX (p = 0.01). Considering necrosis, MER showed the lowest numbers of necrotic HGFIBs and HNEPCs, whereas OB induced the highest number of necrotic cells. The differences between both MR were statistically relevant (p < 0.01). OCT did neither differ from the other MRs nor from the control (PBS) in induction of necrosis in both cell types. In conclusion, the slightly negative effect of OCT considering apoptosis and necrosis of HGFIBs and HNEPCs is nearly the same or even lower compared to the established MRs included in this study. The results confirm that OCT is a potential alternative to CHX.

Genotoxicity of non-alcoholic mouth rinses: A micronucleus and nuclear abnormalities study with fluorescent microscopy.

AIM: The aim of the present study was to evaluate the genotoxicity of non-alcoholic mouth rinses on buccal epithelial cells using a micronucleus test. METHODS: A total of 105 patients were selected and randomly divided into five groups. Four different mouth rinses and normal saline were given for 2 weeks' duration, and cytological smears were collected before and after exposure. These smears were subjected to micronucleus (MN) and other nuclear abnormalities (ONA) tests using acridine orange stain, and their frequencies were obtained in 500 buccal epithelial cells. The statistical analysis included mean, χ2 -test, analysis of variance, and post-hoc analysis by Bonferroni test. RESULTS: Micronucleated cells (P < .00) and MN (P < .00) were higher in individuals exposed to chlorhexidine (CHX), followed by chlorine dioxide (ClO2 ), potassium nitrate (KNO3 ), and sodium fluoride (NaF), amine fluoride (AmF), and normal saline. ONA were greater (P < .00) in individuals exposed to CHX, followed by ClO2 , AmF, KNO3 , and NaF and normal saline. Overall, the results showed that genotoxic damage was greater in the case of CHX, followed by ClO2 , KNO3 , and NaF, AmF, and normal saline. CONCLUSION: Chronic exposure to mouth rinses can cause genotoxic damage to buccal epithelial cells. Long-term injudicious and inadvertent use of mouth rinses should be discouraged.

Cytotoxicity of three commercial mouthrinses on extracellular matrix metabolism and human gingival cell behaviour.

This study evaluated the effects of commercially available antiseptic mouthrinses on human gingival fibroblast and keratinocyte behaviour and metabolism. Three mouthrinses containing essential oil (EO), chlorhexidine (CHX) and amine fluoride/stannous fluoride (AFSF), were tested in an in vitro study. Human gingival fibroblasts and keratinocytes were washed with 10% or 30% concentration of the commercial mouthrinses and their effects on cell adhesion and proliferation were investigated as well as the specific gene expression of markers involved in oral mucosa metabolism. As markers of cell metabolism, type I and IV collagens, laminin, fibronectin, fibromodulin and integrins were studied with real-time PCR. Moreover, interleukin-1 secretion, one of the major pro-inflammatory cytokines, was evaluated. The results showed that CHX significantly reduced fibroblast and keratinocyte substrate adhesion capacities and CHX and EO inhibited cell proliferation better than AFSF rinse. The gene expression of several matrix components and cell adhesion receptors was downregulated in cells washed with CHX and EO compared with those washed with AFSF rinse. In conclusion, the AFSF mouthrinse does not induce or induces to a lesser extent the onset of irritation and/or cytotoxicity than CHX or EO. These findings and those of future studies will enable us to gain further insight into the clinical significance and effects of commercial mouthrinses. Pending further investigations, clinicians should be aware of the potentially adverse effects of mouthrinses and warn their patients against making improper use of these products.

Cytotoxic effects of octenidine mouth rinse on human fibroblasts and epithelial cells - an in vitro study.

OBJECTIVES: This study compared the cytotoxicity of a new octenidine mouth rinse (MR) against gingival fibroblasts and epithelial cells with different established MRs. METHODS: The following MRs were used: Octenidol (OCT), Chlorhexidine 0.2% (CHX), Listerine (LIS), Meridol (MER), Betaisodona (BET); and control (medium only). Human primary gingiva fibroblasts and human primary nasal epithelial cells were cultivated in cell-specific media (2 × 10(5) cells/ml) and treated with MR for 1, 5, and 15 min. Each test was performed 12 times. Metabolism activity was measured using a cytotoxicity assay. A cellometer analyzed cell viability, cell number, and cell diameter. The data were analyzed by two-way analysis of variance with subsequent Dunnett's test and additional t-tests. RESULTS: The cytotoxic effects of all MRs on fibroblasts and epithelial cells compared to the control depended on the contact time (p < 0.001). OCT and BET showed less influence on cell metabolism in fibroblasts than other MRs. OCT also demonstrated comparable but not significant results in epithelial cells (p > 0.005). Cell numbers of both cell types at all contact times revealed that OCT showed a less negative effect (p > 0.005), especially for epithelial cells compared to CHX after 15 min (p < 0.005). OCT and BET showed the best results for viability in fibroblasts (p > 0.005), but MER showed less influence than OCT in epithelial cells (p < 0.005). CONCLUSIONS: OCT is a potential alternative to CHX regarding cytotoxicity because of its lower cell-toxic effect against fibroblasts and epithelial cells.

Chitosan mouthwash: toxicity and in vivo validation.

A previous study showed that a chitosan mouthwash would be a valid alternative to current mouthwashes as it demonstrated, in vitro, significantly higher antibiofilm activity than two commercial mouthwashes. As such, the aim of this work was to verify the safety of the developed product and to validate, in vivo, the biological activity ascertained in vitro. Chitosan mouthwash safety was evaluated through Ames, MTT and V79 chromosomal aberration assay while antimicrobial activity was evaluated through in vivo assays. The results showed that the chitosan mouthwash was safe, presenting lower cytotoxicity than a commercial mouthwash, and that it effectively reduced viable counts of Streptococcus spp. and Enterococcus spp. by ca. 5.5 log of CFU. Furthermore, in direct comparison with a commercial mouthwash the chitosan mouthwash possessed significantly higher antimicrobial activity. The conjunction of these results proves that the chitosan mouthwash is a safe, effective, natural alternative to the existent chemical mouthwashes.

Increased number of micronuclei and nuclear anomalies in buccal mucosa cells from people exposed to alcohol-containing mouthwash.

The aim of this study was to evaluate the effects of alcohol-containing mouthwash on the induction of micronuclei and nuclear anomalies in exfoliated buccal cells, including binucleated cells, cells with nuclear buds, and karyolitic, karyorrhectic, condensed chromatin, and pyknotic cells. Buccal mucosa cells were collected from 107 healthy participants who were divided into three groups: control subjects who did not use mouthwash (n = 33), subjects who were exposed for 30 days and two times rinsing with 30 seconds each time to alcohol-containing mouthwash (n = 38; 26% ethanol concentration); and subjects exposed to a non-alcohol-containing mouthwash (n = 36). A slide was used to collect cells from the oral mucosa from the inner lining of both cheeks. Samples were spread directly onto two separate, precleaned and precoded slides. Smears were air-dried, fixed, stained, and analyzed by microscopy for micronuclei and nuclear anomalies. Frequency of micronuclei, nuclear buds, and karyolitic, karyorrhectic, and condensed chromatin cells increased significantly (P < 0.05) in the alcohol-containing mouthwash group after mouthwash exposition, compared with both the control and the non-alcohol-containing mouthwash groups. Our results suggest that subjects exposed to alcohol-containing mouthwash exhibited an increase in frequency of micronuclei and nuclear anomalies in oral mucosal cells, which is directly related to DNA damage.

Mouthwash and oral cancer risk quantitative meta-analysis of epidemiologic studies.

BACKGROUND: Use of mouthwash and an increased risk of oral cancer has been a source of controversy for decades. A meta-analysis of epidemiological studies of mouthwash and oral cancer and, specifically, mouthwash containing >25% alcohol, was undertaken. METHODS: Summary estimates were obtained with maximum likelihood estimates from random effects models. Sensitivity analyses were conducted to evaluate the influence of various inclusion. RESULTS: Eighteen studies were included in the meta-analysis. There was no statistically significant associations found between regular use of mouthwash and risk of oral cancer (RR=1.13; 95% CI (0.95-1.35)). There was no significant trend in risk of oral cancer associated with increased daily usage of mouthwash (p=0.11). There was no association between reported use of mouthwash specifically containing alcohol and risk of oral cancer (RR=1.16; 95% CI (0.44, 3.08)). CONCLUSIONS: This quantitative analysis of mouthwash use and oral malignancy revealed no statistically significant associations between mouthwash use and risk of oral cancer, nor any significant trend in risk with increasing daily use; and no association between use of mouthwash containing alcohol and oral cancer risk.

Cytogenetic damage induced by mouthrinses formulations in vivo and in vitro.

The aim of the present study was to comparatively evaluate DNA damage and cellular death in cells exposed to various commercially available mouthrinses: Listerine Cepacol, Plax alcohol free, Periogard, and Plax Whitening. A total of 75 volunteers were included in the search distributed into five groups containing 15 people each for in vivo study. Exfoliated buccal mucosa cells were collected immediately before mouthrinse exposure and after 2 weeks. Furthermore, blood samples were obtained from three healthy donors for in vitro study. The micronucleus test was used to evaluate mutagenicity and cytotoxicity in vivo. The single-cell gel (comet) assay was used to determine DNA damage in vitro. After 2 weeks exposure, Periogard showed 1.8% of micronucleated cells with significant statistical differences (p < 0.05) compared to before exposure (0.27%). Plax Whitening presented high tail moment value (4.5) when compared to negative control (0.6). The addition of all mouthrinses to cells incubated with methyl methanesulfonate did not alter the number of strand breaks in the genetic material. Listerine was able to reduce genetic damage induced by hydrogen peroxide because a decrease of tail moment was noticed. The results of the present study suggest that Periogard and Plax Whitening can induce genetic damage, whereas Listerine is an antioxidant agent. Since DNA damage is considered to be prime mechanism during chemical carcinogenesis, these data may be relevant in risk assessment for protecting human health and preventing carcinogenesis.

Efficacy of chlorhexidine digluconate-containing formulations and other mouthrinses against periodontopathogenic microorganisms.

OBJECTIVE: To determine in vitro the action of chlorhexidine digluconate and different commercially available mouthrinses on oral microorganisms. METHOD AND MATERIALS: Minimal inhibitory concentrations and possible induction of resistance by chlorhexidine digluconate, an essential oil-containing mouthwash and an amine fluoride/stannous fluoride solution, were determined against microorganisms normally found in the oral cavity (10 streptococci, 2 enterobacteria, 1 Candida albicans, 8 Porphyromonas gingivalis, 6 Aggregatibacter actinomycetemcomitans, and 1 Fusobacterium nucleatum). Further, the effect of a 1-minute exposure on cell and bacterial viability was studied. RESULTS: The susceptibility of the oral microorganisms to chlorhexidine digluconate ranged from 0.01% to 0.50%. Passages on agar plates containing subinhibitory concentrations of chlorhexidine digluconate resulted in a transitory moderate increase in the tolerance to chlorhexidine digluconate in five of the 24 isolates. After 1 minute of exposure, chlorhexidine digluconate solutions as well as the essential oil and the amine/stannous fluoride-containing solutions showed a high activity against the tested microorganisms. Commercially available chlorhexidine digluconate formulations (ie, those with antidiscoloration systems) were partly less efficient than the corresponding manually prepared chlorhexidine digluconate preparation. The determination of MTT resulted in a strong cytotoxicity of all tested preparations to gingival fibroblasts. CONCLUSION: The results indicate that most of the chlorhexidine digluconate formulations as well as essential oil and the amine fluoride/stannous fluoride solutions are active against oral microbes. Long-term use of these agents would not result in emergent antimicrobial resistance.

Properties of a new mouthrinse for patients receiving radiation therapy.

INTRODUCTION: Patients receiving radiation therapy due to oral cancer develop complications such as hyposalivation, mucositis, oral infections, dental hypersensitivity and caries. Mouthrinses can alleviate some of these problems. AIMS AND OBJECTIVES: To investigate the in vitro antimicrobial properties and cytotoxicity of an experimental mouthrinse. METHODS: The mouthrinse contained 30% hexylene glycol (glycerine), 7% potassium nitrate and 0.025% sodium fluoride. The minimal inhibitory concentration (MIC) of these ingredients and the mixture was determined for C. albicans, S. aureus and S. mutans over 24 hours at different concentrations. The MICs of two commercial mouthrinses, Corsodyl and Plax, were also determined using the same organisms. All mouthrinses were then tested to determine the percentage kill over 1, 2, and 3 minutes. RESULTS: The MICs for hexylene glycol were 10%, 30% and 10% for C. albicans, S. aureus and S. mutons respectively. Potassium nitrate and sodium fluoride had no antimicrobial effects. The MIC of Corsodyl was 0.016 mg/ml for all the test organisms. The MIC for Plax varied from 0.0002 mg/ml to 0.001 mg/ml. The kill rates for all mouthrinses were acceptable, with no statistical differences between them. The experimental mouthrinse was not toxic to human oesophageal SCC cells after 1 minute exposure. At the time of the experiment, the costs of a similar quantity of the experimental mouthrinse, Corsodyl and Plax were R5.24, R30.00 and R10.00 respectively. CONCLUSIONS: The experimental mouthrinse was cost-effective and proved to have an antimicrobial effect and could be used safely to alleviate oral infections, desensitize teeth, improve oral hygiene and control dental caries in cancer patients after radiation therapy.

Genotoxicity of two mouthwash products in the Drosophila Wing-Spot Test.

In this study, genotoxicity of two mouthwash products (chlorexidin, benzidamine-HCl) were investigated in the Drosophila Wing-Spot Test which makes use of the wing cell markers multiple wing hairs (mwh) and flare (flr) and detects both mitotic recombination and various types of mutational events. Induced mutations are detected as single mosaic spots on the wing blade of surviving adults that show either the multiple wing hairs or flare phenotype. Induced recombination leads to mwh and flr twin spots and also, to some extent, to mwh single spots. Recording of the frequency and the size of different spots is allowed for a quantitative determination of the mutagenic and recombinogenic effects. Trans-heterozygous third-instar larvae were treated at different concentrations of the mouthwash products. Chlorexidin exposure concentrations were 0.5, 1 and 2mg/ml. Benzidamine-HCl exposure concentrations were 0.38, 0.75 and 1.5mg/ml. In addition, the observed mutations were classified according to size and type of mutation per wing. Both chlorexidin and benzidamine-HCl were genotoxic in terms of total mutations per wing at the highest doses. Survival rates of flies used in the experiments were significantly lower than those of the control group, with both mouthwash products showing toxic effects on Drosophila melanogaster larvae.

Safety assessment of ProBiora3, a probiotic mouthwash: subchronic toxicity study in rats.

Streptococcus viridans are commensal bacteria that constitute a significant portion of the resident oral microflora. The objective of the present study is to investigate adverse effects, if any, of a blend of 3 natural strains, Streptococcus uberis KJ2, Streptococcus oralis KJ3, and Streptococcus rattus JH145 (probiotic mouthwash, ProBiora(3)). The blend is administered to rats orally once daily (5 days per week) at doses of 0, 10(6), or 10(9) colony-forming units of each strain for 14 weeks. No treatment-related adverse effects are observed in the physiological parameters during the study or in the evaluation of blood and tissue samples taken from the animals at the end. Results of an in vitro antibiotic susceptibility study demonstrate that all 3 ProBiora(3) strains are susceptible to commonly used therapeutic antibiotics. The results of these investigations reveal that the no-observed-adverse-effect level of the probiotic mouthwash is 2.16 x 10(9) colony-forming units per strain per kilogram of body weight per day, the highest dose used.

Intoxication with mouthwash presenting as psychosis and delirium in a combat theater.

Alcohol is prohibited in combat zones, but it still can be obtained via covert sources. This can cause complex issues for the military physician. We present a case of a U.S. Marine in Iraq who was noted to have intermittent and unexplained behavioral changes. The patient had repeatedly denied alcohol use, but during a second medical evacuation from theater, a blood alcohol level was drawn. Results did not come back fast enough to prevent the evacuation, but did eventually confirm that he was intoxicated. Mouthwash was the source. This case illustrates the importance of screening for alcohol abuse and intoxication even in situations in which conventional use of alcohol is not anticipated.

Cytotoxicity evaluation of Persica mouthwash on cultured human and mouse cell lines in the presence and absence of fetal calf serum.

BACKGROUND AND AIMS: The effectiveness of an ideal antimicrobial agent depends on its ability to kill microbes while causing minimal toxicity to host cells. Several studies have been reported on the antimicrobial effects of chewing sticks (Salvadora persica) on oral bacteria. The purpose of this study was to evaluate the cytotoxic effects of Persica and chlorhexidine (CHX) mouthwashes on cultured human and mouse cell lines. MATERIALS AND METHODS: This was an experimental study. The toxic effects of four dilutions of Persica and CHX mouthwashes on KB, Saos-2, J744 A1, and gingival fibroblast cells were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. The effect of fetal calf serum (FCS) components on the cytotoxicity of these mouthwashes was also investigated. STATISTICAL ANALYSIS: Analysis of variance and the Kruskal-Wallis test were used to evaluate the results. RESULTS: The results indicated that Persica , at concentrations higher than 0.1%, exerted a very significant cytotoxic effect on all the cell lines (P < or = 0.01). CHX, at a concentration of 0.001%, exerted toxic effects only on gingival fibroblasts; concentrations higher than 0.001% were required to produce significant cell death in the other cell lines. At all the concentrations under study, both Persica and CHX exerted significantly greater cytotoxic effects in the absence of FCS than in its presence (i.e., in control culture medium). The toxicities of both mouthwashes were attenuated in the presence of FCS (10%). CONCLUSION: Our results indicate that both Persica and CHX mouthwashes are toxic to macrophage, epithelial, fibroblast, and osteoblast cells in a concentration-dependent manner.

Biologic assessment of antiseptic mouthwashes using a three-dimensional human oral mucosal model.

BACKGROUND: The biologic safety profile of oral health care products is often assumed on the basis of simplistic test models such as monolayer cell culture systems. We developed and characterized a tissue-engineered human oral mucosal model, which was proven to represent a potentially more informative and more clinically relevant alternative for the biologic assessment of mouthwashes. The aim of this study was to evaluate the biologic effects of alcohol-containing mouthwashes on an engineered human oral mucosal model. METHODS: Three-dimensional (3D) models were engineered by the air/liquid interface culture technique using human oral fibroblasts and keratinocytes. The models were exposed to phosphate buffered saline (negative control), triethylene glycol dimethacrylate (positive control), cola, and three types of alcohol-containing mouthwashes. The biologic response was recorded using basic histology; a cell proliferation assay; 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide tissue-viability assay; transmission electron microscopy (TEM) analysis; and the measurement of release of interleukin (IL)-1beta by enzyme-linked immunosorbent assay. RESULTS: Statistical analysis showed that there was no significant difference in tissue viability among the mouthwashes, cola, and negative control groups. However, exposure to the positive control significantly reduced the tissue viability and caused severe cytotoxic epithelial damage as confirmed by histology and TEM analysis. A significant increase of IL-1beta release was observed with the positive control and, to a lesser extent, with two of the tested mouthrinses. CONCLUSIONS: The 3D human oral mucosal model can be a suitable model for the biologic testing of mouthwashes. The alcohol-containing mouthwashes tested in this study do not cause significant cytotoxic damage and may slightly stimulate IL-1beta release.