82-86 JCD 18.3 Thrane:01-04 JCD 17.1 Harpenau

Objective: Volatile sulfur compounds (VSC), mainly derived from bacteria located in deep crypts at the back of the tongue and from periodontal pockets, are responsible for approximately 90% of halitosis (bad breath, malodor). The objective of this double blind clinical study was to assess the clinical efficacy of a new formulation for halitosis containing a combination of zinc (0.3% Zn) and chlorhexidine (0.025% CHX) in low concentrations. The new formulation was compared to some widely used and commercially available formulations containing various enzymes and antibacterial agents in a clinical setting under controlled conditions. • Methodology: Ten healthy volunteers participated in this study (5 female, 5 male, mean age: 46.6, range: 26–79). Each partici pant served as their own control, and neither the investigator nor the ten test subjects knew which formulation they were testing at any given time (double-blind design). Baseline H2S data were obtained by cysteine rinsing for 30 seconds, 90 seconds mouth closure, and gas chromatographic (GC) analysis of mouth air. On separate days, each participant then rinsed for 60 seconds with 10 ml of each of the eight various formulations. Cysteine rinses were repeated at 1 hour, 2 hours, and 3 hours, and GC measurements of oral H2S levels were again recorded. • Results: The test rinse (0.3% Zn + 0.025% CHX) reduced the intraoral H2S levels to 0.16% of control (range: 0.01–0.54%) after 1 hour, 0.4% after 2 hours, and 0.75% after 3 hours, providing superior efficacy in reducing H2S compared to the other formulations tested (p < 0.05). • Conclusion: A combination of Zn and CHX in low concentrations seems to be the most efficient way to remove the VSC that causes bad breath at present. Studies are underway to further explore the extraordinary efficacy of this combination (close to 100%), suggesting a specific mode of action and a synergistic effect of these two components. (J Clin Dent 18:82–86, 2007) A New Mouthrinse Combining Zinc and Chlorhexidine in Low Concentrations Provides Superior Efficacy Against Halitosis Compared to Existing Formulations: A Double-Blind Clinical Study Per Stanley Thrane, MD, DDS, PhD Alix Young, DDS, PhD Grazyna Jonski Gunnar Rölla, DDS, PhD Dental Faculty University of Oslo Norway 82 Introduction Offensive odor emanating from the oral cavity, often termed halitosis, is responsible for approximately 90% of bad breath cases.1-3 Halitosis is mainly caused by volatile sulfur compounds (VSC) derived from Gram negative anaerobic bacteria, mostly found in periodontal pockets and in the crypts at the back of the tongue.4,5 Hydrogen sulfide (H2S), methyl mercaptan (CH3SH), and, to a lesser extent, dimethyl sulfide (CH3SCH3) are the major components of the VSC that originate from the degradation of the sulfur-containing amino acids, cysteine, and methionine.6 They have an unpleasant odor, even in extremely low concentrations.7 In addition to causing halitosis, VSC may play an important role in the etiology of periodontal disease.8,9 In particular, methyl mercaptan has been shown to penetrate the various tissues in periodontal pockets,10 and increase the degradation of collagen, as well as inhibiting the protein synthesis of gin gival fibroblasts,11 thus adversely affecting critical events in the develop ment of periodontitis.8,9 The authors of this paper, and other researchers, have shown that certain metal ions, zinc (Zn) in particular, can be used to inhibit the formation of VSC12-14 and subsequently reduce or eradicate halitosis. Moreover, it has been shown that certain anti bacterial agents such as chlorhexidine (CHX) or cetylpyridinium chloride (CPC) may also inhibit VSC formation and thus reduce halitosis.15-17 If zinc ions and antibacterial agents operate by different mechanisms with regard to oral VSC inhibition, it is conceivable that combinations of two or more of these agents may provide an enhanced or synergistic anti-VSC effect.18 However, the opposite might also be the case; one or two components might reduce or block the effect of the other. In order to examine this further it was decided to: a) evaluate the clinical effec tiveness of a new anti-halitosis formulation (SB12®, Antula AB, Stockholm, Sweden) combining low concentrations of Zn (0.3%) and CHX (0.025%); b) use a double-blind clinical protocol to allow an unbiased comparison with other anti-halitosis formulations containing various enzymes and antibacterial agents, as shown in Table I; c) use a specially modified gas chromatograph particularly suited for measurements of low concentrations of VSC and considered the “gold standard” of halitosis measurements;6,19 and d) use cysteine rinsing according to Kleinberg and Codipilli20 to introduce bad breath in healthy volunteers in order to avoid some of the problems with including “patients,” as well as enabling each participant to serve as his or her own control. The aim of the present study was to examine the effectiveness of a new anti-halitosis formulation combining low levels of Zn and CHX, and to compare it with other widely used formulations Vol. XVIII, No. 3 The Journal of Clinical Dentistry 83 On test days, the subjects refrained from their normal oral hygiene and presented at the laboratory at 9:00 a.m. The par ticipants rinsed for 30 seconds with 5 ml of a 6 mM solution of L-cysteine (Sigma Chemical Co., St Louis, MO, USA) at pH 7.2. Subsequently, they kept their mouths closed for 90 seconds, after which mouth air samples were aspirated into a 3 ml sample loop connected to the auto injector of a gas chromatograph (Shimadzu, Kyoto, Japan), modified for this purpose as previously described.19 The obtained mouth air samples were thereafter analyzed directly by separation in the gas chromatograph using a Teflon column (3.66-mx 0.32 cm, temperature 70°C, nitrogen gas flow 32 ml min-1, hydrogen gas flow rate 125 ml min–1 and airflow rate 43 ml min-1) packed with polyphenol ether (5%)— phosphoric acid (0.05%) on 40/60 mesh Chromosorb T and a flame photometric detector.6 The standardized H2S formation in the mouth that was obtained after the cysteine rinsing constituted the baseline as a control for each tested subject. Immediately following, each subject rinsed for 30 seconds with one of the eight test solutions (A-H). Thereafter, cysteine rinses followed by mouth air analyses were repeated at 1, 2, and 3 hours. The H2S levels were subsequently compared with the baseline levels for each subject. At least one non-test day between uses of the different test solutions was introduced to avoid a putative cross-over effect between the different test solutions. Statistical Analyses Concentration of H2S in breath samples from the control measure ment, and from measurements taken 1, 2, and 3 hours after treatment were obtained from gas chromatograph software (EZStrat 7.2) as AUC (area under the curve) for the chromatogram peak. Those raw data were furthermore calculated as a % of control for each of the test subjects. Differences between the examined mouthrinses were statis tically tested by one-way ANOVA and LSD multiple comparisons. These tests were performed on both AUC (presented in Table II) and % of control (Figures 1, 2, and 3). The outcomes of the statistical analyses were similar in both cases. It was further investigated whether different active ingredients have or do not have an inhibitory effect on oral H2S formation; results greater than 100% were considered as “not having” inhibitory effect. The reason for those results greater than 100% needs closer investigation. Results A significant inhibition of H2S production was observed in mouth air samples taken 1, 2, and 3 hours after the rinse with a combination of Zn and CHX in low concentration (H) compared to the H2S baseline in all the 10 subjects tested. A great inter-individual variation in H2S levels was observed between the different test subjects. The results are summarized in Table II. A great variation in effectiveness among the various formulations was observed, ranging from virtually no observed effect (A, F) to almost 0% of control (H) over the whole testing period (3 hours). The results of the rinsing experiment (AUC) comparing the eight different anti-halitosis formulations are summarized in Table II and illustrated as % of control in Figures 1–3. against halitosis in a double-blind clinical design. The hypothesis to be tested was that Zn combined with CHX in low concen trations effectively inhibits H2S production induced in healthy individuals, and moreover, is comparatively more effective than other currently used antibacterial agents and/or enzymes. Materials and Methods Oral Rinses Eight different oral rinses were included in the study. All the oral rinses were commercially available at the time of the study except SB12® which was provided free-of-charge by the manufacturer (Antula AB, Stockholm Sweden). This study was performed at the Clinical Research Laboratory, Dental Faculty, University of Oslo, Norway. The following oral rinses were included in the experiment: A. Zendium® Munnskölj med Zink (Opus Healthcare, Malmö, Sweden) B. Listerine® Natural Citrus (Pfizer Consumer Healthcare, Morris Plains, NJ, USA) C. Listerine® Cool Mint (Pfizer Consumer Healthcare, Morris Plains, NJ, USA) D. Halita® Dentaid (S.L. Parc Tecnològic del Vallès, Cerdanyola, Spain ) E. retarDEX® (Periproducts Ltd, Middlesex, UK ) F. Dentyl® Refreshing Clove (Fresh Breath Ltd, London, UK) G. Dentyl® Smooth Mint (Fresh Breath Ltd, London, UK) H. SB12® (Antula Healthcare AB, Stockholm, Sweden) A summary of the active ingredients of the various rinses, as listed on the bottles, is shown in Table I. Test Subjects and Protocol Ten healthy volunteers participated in this study. They were recruited from the research staff at the Dental Faculty, comprising five females and five males, mean age: 46.6, range: 26–79. All test subjects took part in the experiment with informed consent, after having received an explanation of the protocol. They did not have any medical history that in any way could relate to halitosis. The trial followed a crossover, double-blind design. Table I A Summary of the Active Ingredients Listed on the Bottles of the Rinses Used in the Experiment Kode Mouthrinse Active Ingredients A Zendium® Zinc gluconate and various enzymes: amyloglycosidase, glycoxidase, and lactoperoxidase Listerine® Antibacterial agents: eucalyptol 0.092%, menthol B Citrus and 0.042%, menthyl salicylate 0.060%, and thymol 0.064% C Cool Mint D Halita® Chlorhexidine digluconate 0.05%, cetylpyridinium chloride (CPC) 0.05% and zinc lactate 0.14% E retarDEX® Antibacterial agent (cloSYSII®) Dentyl® Antibacterial agents: cetylpyridinium chloride, F Refreshing triclosan Clove and G Smooth Mint H SB12® Zn acetate 0.3% and chlorhexidine diacetate 0.025% test rinse 84 The Journal of Clinical Dentistry Vol. XVIII, No. 3 Discussion Given the design of this study with each test subject serving as their own control, the great inter-individual variation in H2S levels that was observed did not adversely influence the overall quality of the results. Moreover, by inducing halitosis in healthy volunteers, the difficulty with putative interference with various diseases and medication (drugs) that might influence H2S production was avoided. Halitosis is a symptom and not a disease, that often occurs in otherwise healthy individuals mainly due to local conditions in the mouth; i.e., putrefaction of anaerobic bacteria in crypts at the back of the tongue and in periodontal pockets.1,4-6,8 The choice of test subjects thus seemed appropriate. The subjective nature of bad breath per se, as well as rather subjective (organoleptic, nasopalatinal index)23 and less sensitive and specific measurement methods (i.e., portable sulfide monitor, e.g., Halimeter®),24 further complicate this picture making it more difficult to perform reliable comparative studies, as well as % o f C on tr ol 1 h r A fte r T re at m en t 100–