In this randomized clinical trial, two mouthwashes were compared; one with ClO2 and one without ClO2, to investigate the malodor, salivary bacteria and reducing effects of ClO2. The results of this study demonstrate that rinsing with a mouthwash containing ClO2, used over a 7-day period, was effective in reducing morning oral malodor, plaque, tongue coating accumulation and the counts of Fusobacterium nucleatum in saliva in healthy subjects.
Chlorine dioxide (ClO2) is a stable free radical. It is readily soluble in water forming a light clear yellow-colored solution in which it can remain intact for considerable periods of time. Oral rinses containing ClO2 are now utilized in dental practices as a topical antiseptic for the oral cavity or for dentures [21, 35, 36]. Previous studies have suggested that ClO2 and ClO2
- are chemically reactive oxidants with powerful reducing capacity on VSCs. Lynch et al. reported that reaction of L-cystein, a thiol compound which contribute substantially towards oral malodor , with ClO2
. and/or ClO2
.-, which contained 0.10% (w/v) ClO2 (the same as the experimental mouthwash used in this study), yielded the disulfide cystine as a major product. The processes for the oxidation of thiols through the consecutive, two-step reaction sequence involving ClO2
. and/or ClO2
.- are shown as the following equations: (1) RSH (e.g. CH3SH) + ClO2
. → RS. + ClO2
.-+ H+; (2) 2RS. → RSSR (e.g. CH3SSCH3); (3) 4RSH + ClO2
.- → 2RSSR + Cl- + 2H2O . Grootveld et al. reported that the oral rinse containing ClO2 suppressed saliva numbers of Streptococcus mutans and lactobacilli in vivo, observed reflecting the bacterocidal action of oxohalogen oxidants present . Though a few subjects reported a problem of a chloric smell, chlorite anion is powerfully bactericidal to malodorous microorganisms . Chlorine dioxide penetrates the bacterial cells and reacts with vital amino acids in the cytoplasm to kill the organism [21, 35]. It is reported to exert its bactericidal effects by fixing cellular membrane proteins as a result of its oxidizing potential in a similar manner to oxidizing agents .
Several methods have been developed to identify these microorganisms, many of which are polymerase chain reaction (PCR)-based bacterial detection systems . Most of the reported PCR-based diagnostic systems are qualitative analysis methods and are therefore unsuitable for the accurate evaluation of bacteria causing oral malodor . A real-time PCR assay has been developed for the quantitative detection of DNA copy numbers . In this study we used a newly developed Invader PLUS technology. This is a sensitive, rapid method for detection and quantification of nucleic acid. While the original technology is based on the amplification by PCR of the target sequence followed by its detection using the Invader technology, the current modification allows simultaneous PCR amplification and Invader reaction. This allows simpler design and faster results. This technology has been applied for the quantification of periodontitis-related bacteria; F.n.; P.g.; T.f., and T.d. .
VSCs have been shown to result from the bacterial putrefaction of proteins with sulfur-containing amino acids . These proteins are derived from tongue epithelial cells and white blood cell debris [6, 7]. Bacteria such as P g., F.n., T.f., T.d., and several species of other oral bacteria associated with gingivitis and/or periodontitis are known to produce large amounts of VSCs, which are malodorous. Periodontal disease causes high concentrations of VSCs in mouth air. The concentrations of CH3SH are significantly higher in patients with periodontal disease than those in orally healthy individuals . Although the current study was conducted with orally healthy subjects, the results suggest that a mouthwash containing ClO2 might reduce bacterial load (as seen in F.n. reduction) and lower oral malodor in patients with periodontal disease.
F.n. produces both H2S and CH3SH from the saliva, dorsum of the tongue and sub-gingival plaque . F.n. is considered a 'bridge-organism' that facilitates colonization of other periodontal malodorous bacteria especially T.f. by coaggregation-mediated mechanisms [40, 41]. Moreover it was reported that F.n was an important bacterium in the development of complex dental plaque biofilms . Therefore the results of this study suggested that the reducing effects on morning oral malodor, plaque and tongue coating accumulation, was partially caused by reducing the counts of F. n. In this study, however, we were unable to find a statistically significant reduction of P.g., T.f. and T.d. bacterial load using the ClO2 mouthwash. Though we found a significant effect on plaque accumulation using the ClO2 mouthwash over a 7-day period, this was not translated into a significant inhibitory effect on gingivitis. Previous reports showed that the rinsing by CIO2 mouthwash reduced salivary bacteria such as Streptococcus mutans and lactobacilli . However there is no report about periodontitis-related bacteria. In this study, we demonstrated that 7-day rinsing by CIO2 mouthwash reduced oral malodor and only F.n. counts in saliva. The prevalence of F.n. was highest in saliva and it had shown a high capability of producing VSCs . On the other hand, the prevalence of the periodontal disease-associated bacteria such as P.g. and T.d. were lower than F.n. in saliva of these healthy subjects. Kurata et al. reported that the prevalence of P.g., T.f. and T.d. in saliva was related to periodontal health status and VSC levels in mouth air in patients with periodontal disease, however the prevalence of these bacteria was low in patients without periodontal disease . We need therefore to examine the long-term effects of ClO2, on periodontal diseases and microbiological activities, and use a larger sample size in future research.
The finding that there was no difference in the level of tongue coating between the experimental and placebo mouthwash may relate to the mechanism of using both mouthwashes in a 'gargling' fashion. For example, Hakuta et al. reported that 'gargling' with water everyday reduced the tongue coating of the elderly subjects in her study of oral function .
Frascella tested the effectiveness of a ClO2-containing mouthwash at different points of time for a total of 96 hours after rinsing . The results showed a significant improvement in OM scores and VSC levels measured by a portable sulfide monitor when the tested mouthwash was compared to a water control. The mean VSC concentration in the test group maintained its effective level at 8 hours after rinsing. In the present study, the interval from the last rinsing (before sleeping) with the experimental mouthwash to the assessment of subjects oral malodor was an average 8.80 (range 6 to 11) hours. We found that rinsing with ClO2 dramatically reduced the concentrations of all three kinds of VSCs, on the morning of the assessment day. However after the one week washout period, the VSCs level returned to those at the baseline. It is suggested that residual ClO2 remaining in the saliva or oral cavity may have reduced VSC level for at least about 9 hours. Further research should define the maximum effective time on VSC reduction and that trials should be conducted over longer time periods, 2-4 weeks or longer.
Recently, many over-the-counter mouthwashes have been used in the treatment of oral malodor. Some of these products merely mask malodor. The optimal mouthwash to treat oral malodor would be an antiseptic agent with proven long-lasting efficacy for reduction of OM and VSC concentrations, with no or few side effects. Chlorhexidine-containing mouthwashes inhibit formation of VSCs and are effective oral antiseptics with antiplaque and antigingivitis effects . Although CHX is considered the most effective oral antiseptic agent, Gürgan et al reported using 0.2% alcohol-free CHX mouthrinse for 1 week caused more irritation to oral mucosa, greater burning sensation, and increased altered taste perception compared to the placebo rinse . Listerine® (Johnson and Johnson, New Jersey, USA), a mouthwash containing essential oils, may also have antiplaque and antigingivitis activity . However, its high alcohol concentration reduces taste sensation and can cause oral pain . Zinc ions inhibit oral malodor but again had a taste problem . Triclosan and cetylpyridium chloride (CPC) are antimicrobial agents widely used as antiseptic agents . However, their clinical reduction of VSCs is questionable .
ClO2 is used widely in various fields for its safe and high antibacterial action . Sodium chlorite (NaClO2), equivalent to ClO2, the traditional ingredient in almost all oxygen supplementation today, is a non-toxic substance approved by the U.S. Food and Drug Administration (FDA) as an antimicrobial agent . We found ClO2 not only to be effective at reducing oral malodor, but also none of the volunteers complained about tongue stimulation or discoloring with the 0.10% ClO2 (0.16% NaClO2) mouthwash. For some subjects, the taste and smell of this mouthwash were disagreeable. This may be resolved in new formulations which masks these problems.
Kimoto et al investigated the antibacterial effects of a mouthwash containing ClO2 and it's cytotoxicity on human oral cells, for the purpose of using ClO2 as a bactericidal agent for natural teeth, dental implants and generally within the oral cavity. Their results suggest that the mouthwash containing ClO2 is harmless for human cells and possible to use as a bactericidal agent for dental implants . A proliferation of oral bacteria during sleep is responsible for the release of offending gases, most of which are VSCs. This is often described as "morning bad breath" and occurs even in healthy people . A substantial proportion of healthy people complain of this form of oral malodor. Healthy individuals who suffer from bad breath are likely to use mouthwashes containing several masking or antimicrobial agents. Therefore, recent papers have pointed out the relevance of comparative studies to verify the efficacy of the mouthwashes on "morning bad breath" in healthy subjects [8, 55, 56]. Most former mouthwash studies used healthy subjects with no complaints about oral malodor, often lacked an adequate control and were evaluated only over a short-term effect of the agent [8, 20, 55]. Our study also investigated only the short-term effects of the mouthwash in healthy subjects. It is not known therefore whether the same results would be obtained from patients presenting with halitosis as a clinical problem. Future research is needed to examine long-term effects, as well as effects on periodontal diseases and plaque accumulation in a well-defined sample of halitosis patients. It is also recognized that comparative efficacy studies need to be performed against the known effective mouthwashes containing CHX [12, 13]. Additional research should also be conducted in broader population samples, including females. Nonetheless, in this explorative study, the OM score was improved and VSCs concentrations were significantly reduced using the ClO2 mouthwash. Therefore, the mouthwash clearly demonstrated an anti-malodor effect on morning breath, potentially without any measurable side effects in healthy subjects.