INFLUENCE OF ETHANOL ON THE ANTIBACTERIAL AND ANTI-PLAQUE EFFICACY OF AN AMINE FLUORIDE/STANNOUS FLUORIDE MOUTHRINSE SOLUTION

Introduction

Mouthrinses are common adjuncts to oral hygiene measures, especially for patients in risk for caries, gingivitis or periodontitis. Since side effects of high concentrated chlorhexidine rinses restrict their use to few weeks, there is a search for alternative antibacterial agents. One of those alternatives, triclosan, is effective when used in toothpastes (0.3 % concentration of the active) and when additional components (copolymer, zinc citrate, pyrophosphate) are used to increase its substantivity ^{(Gaffar et al., 1997} ; ^{Brecx et al., 2001)}. Triclosan-containing mouth rinses generally contain a lower concentration of the ingredient (0.03-0.05 %) and have been proven to be clinically effective. Another alternative, the amine fluoride/stannous fluoride system (ASF) was tested in short term trials ^{(Netuschil et al., 1995}) as well as in long term clinical investigations ^{(Banoczy et al., 1989} ; ^{Brecx et al., 1990} ; ^{Brecx et al., 1993}). It was found that, based on a strong antibacterial activity, the ASF combination reduced plaque and gingivitis, especially when used as a toothpaste together with mouth rinsing ^{(Banoczy et al., 1989}). However, all of these elder studies mentioned were conducted with the old formulation containing alcohol. Nowadays efforts are made to omit or at least reduce the alcohol content of mouthwash solutions. Side effects of ethanol, especially when in daily contact with oral mucosal surfaces, have been discussed recently ^{(Arweiler et al., 2001} ; ^{Netuschil and Brecx, 2001}) e.g. the smoothening effect on composite fillings ^{(Penugonda et al., 1994}), the possible potential for carcinogenic effects in the oropharynx ^{(Winn et al., 1991}) and a oral pain (^{Bolanowski et al., 1995}). Moreover, mouthwashes may be a source of acute intoxications ^{(Weller-Fahy et al., 1980}). For children with a high caries risk or with orthodontic appliances and also for patients with a sensitive mucosa one should provide alcohol-free mouthrinse solutions as an alternative to the commercially available alcohol-containing mouthrinse preparations. A new formulated alcohol-free ASF solution has shown significant antibacterial and clinical properties compared to a placebo solution ^{(Arweiler et al., 2001}). However, its efficacy in relation to the former (alcohol-containing) ASF solution and compared to the test standard 0.2 % chlorhexidine was still unknown. Anyway, any new formulated (mouthrinse) preparation has to be compared with conventional (mouthrinse) products in vivo to prove and to precisely compare the antiplaque and antibacterial effectiveness ^{(Addy et al., 1990} ; ^{Harper et al., 1995}).

The purpose of this study was to assess the plaque inhibitory properties and the effect on plaque vitality of an alcohol-free amine fluoride/stannous fluoride mouthrinse (ASF-) in comparison to the former alcohol-containing solution (ASF+), to 0.2 % chlorhexidine (CHX) as a positive and to a placebo solution as a negative control.

MATERIALS AND METHODS

Study population

After receiving the study information and written consent 12 dental students (8 females, 4 males) of the University of the Saarland, age varying from 20 to 31 years (mean age 24.2), volunteered for the study. Criteria for exclusion were the use of antibiotics during the last six months or other antibacterial medicaments that could have affected plaque growth, poor oral hygiene (papillary bleeding index (PBI) according to ^{Saxer and Mühlemann (1975)} > 40 %), less than 20 teeth available for evaluation, fixed or removable orthodontic appliances, partial dentures, known allergy against components of mouthrinses, pregnancy and an age < 18 years.

Study design

The 4 cell cross-over study was performed using the classical 4-day plaque regrowth design ^{(Addy et al., 1983} ; ^{Saxton et al., 1988} ; ^{Jenkins et al., 1994a} ; ^{Jenkins et al., 1994b} ; ^{Riep et al., 1999}) with an assessment of plaque index ^{(Silness and Löe, 1964)} and plaque vitality ^{(Netuschil et al., 1989} ; ^{Brecx et al., 1995}) on day 1 and day 4 (PlI 1, PlI 2, VF 1, VF 2) and plaque area on day 4 (PA, ^{Arweiler et al., 2000} ; ^{Arweiler et al., 2001}). As well the papillary bleeding index (PBI) according to ^{Saxer and Mühlemann (1975)} was used at the begin and the end of every test period to control the maintainance of healthy gingival conditions during the 8 weeks of the study. This was necessary because plaque formation and the influence of mouthrinses differ at healthy and inflamed gingival sites ^{(Quirynen et al., 1991} ; ^{Ramberg et al., 1994} ; ^{Ramberg et al., 1996}).

The allocated formulations (10 ml) were used as 1 min rinses 2 times a day after meals (on the last test day only one rinse). Every test week was followed by a 10 days wash-out phase to avoid carry-over effects, during which the normal individual oral hygiene habits were conducted using a fluorided toothpaste (Aronal^®, Gaba, Switzerland).

After every test week volunteers were asked with a question sheet about quality of life during the use of the mouthrinses.

Test solutions

^{Table I} lists the test substances. Both ASF solutions contained the same ingredients (0.0125 % Olaflur and 0.0125 % stannous fluoride = 250 ppm fluoride), but ASF- was prepared without alcohol while ASF+ contained 5 % ethanol. The 0.2 % Corsodyl^® mouthrinse solution (SB, Bühl, Germany) with 7 % ethanol (CHX) served as a positive control, the placebo solution (CON) contained the same ingredients and flavours like the ASF solutions. All test products were supplied by Gaba International AG, Basel, Switzerland.

Statistical evaluation

After completing the final examination, the statistical evaluations were conducted by the computer program Statistical Package of Social Science/SPSS 7.5.2G. For each rinsing solution the mean values of the clinical parameters (PlI 1, PlI 2, PA) and the vitality of the supragingival plaque flora (VF 1, VF 2) were calculated. Analysis of variance (ANOVA) was performed to determine the significance of differences between the solutions. Since the analysis proved significant individual comparisons were made. Normal distribution was looked for by the Kolmogorov-Smirnov-test. The data were normally distributed, thus parametric tests (Students paired t-test) were used to compare between placebo and the other treatments. Differences of interest were those between both ASF solutions. According to ^{Perneger (1998)}, Bonferroni adjustments for multiple tests were not considered to be necessary ^{(Arweiler et al., 2001}).

Further analysis of variance (ANOVA) was used to examine significant differences in mean whole mouth PBI on day 0 of each test cycle. Significance was determined at p < 0.05.

RESULTS

All twelve subjects completed the 4 test periods of the study. Severe unwanted events were not observed. The active solutions showed slight side effects only in a few subjects. Five volunteers complained about staining, nine about mouth burning and taste disturbance during rinsing with the CHX. During the use of both ASF solutions two participants each showed aphthous ulcera. Subjectively, very soft alterations of the marginal gingiva were recorded by four dental students. All side effects disappeared during the wash-out periods.

The mean whole mouth papillary bleeding index (PBI) at the beginning of each of the 5 test cycles (day 0) is shown in ^{table II} . There were no significant differences between the groups. Thus, very similar and healthy gingival conditions existed during the entire course of the clinical investigation. The mean plaque indices (PlI 1 and PlI 2), plaque area (PA; in %) and vitality (VF 1 and VF 2; in %) combined with the statements of significance (compared to the placebo) are depicted in ^{table III} .

On the first test day, only the CHX reduced PlI significantly when compared to the placebo rinse. The ASF- and ASF+ showed a 5.7 % and a 12.9 % reduction, respectively, which both were not significant. On day 4 all active solutions (ASF-, ASF+, CHX) retarded the PlI as well as the PA values significantly as compared to placebo (reductions of 27.2 %, 32.4 % and 73.5 % for PlI 2 and 50.4 %, 60 % and 76.7 % for PA) (^{fig. 1}, ^1b, ^1c, ^1d and ²).

Compared to the placebo both biofilm vitality values (VF 1, VF 2) were significantly reduced with all active solutions. While the vitality when using CHX ranged between 44.3 % and 46.6 %, the ASF solutions led to values between 41.1 % and 63.0 %.

The statistical comparison of the two ASF solutions revealed no significant differences in any of the parameters (^{table IV}).

Discussion

The effectiveness of an alcohol-free ASF and an alcohol-free 0.1 % CHX solution in reducing plaque and plaque vitality compared to a placebo solution was previously demonstrated ^{(Arweiler et al., 2001}). However, the relation of the new ASF preparation (Meridol^® without alcohol) to the gold standard 0.2 % CHX as well as to the former alcohol containing ASF solution (Meridol^® with alcohol) remained unknown. As already discussed every "active" ingredient has to be tested as complete, formulated product ^{(Addy et al., 1990} ; ^{Harper et al., 1995}). So, good antibacterial and clinical properties of a preparation with alcohol will not guarantee a similar beneficial clinical efficacy of a corresponding alcohol-free solution. Therefore, the intention of this double blind cross-over study was to compare the new formulated alcohol-free ASF solution with the former preparation containing alcohol (5 % ethanol), including the standard CHX (0.2 %), for which excellent antibacterial and antiplaque properties are so far only proven for alcohol-containing solutions.

The classical 4-day plaque regrowth design ^{(Addy et al., 1983} ; ^{Saxton et al., 1988} ; ^{Jenkins et al., 1994a} ; ^{Jenkins et al., 1994b} ; ^{Riep et al., 1999} ; ^{Moran et al., 2000}) is the typical study design to test any anti-plaque efficacy of mouthrinse solutions (and other chemoprophylactic preparations) with acceptable expenditure. Both ASF solutions reduced the clinical parameters as well as the dental biofilm vitality significantly in comparison to the placebo. All clinical (PlI and PA) and bacteriological (VF) values were found to range between the positive (0.2 % CHX) and the negative control (placebo), the exceptions being the VF values at day 1 of the ASFs. Both, the alcohol-free and the alcohol containing ASF solution, did not inhibit plaque regrowth to the same extent than 0.2 % CHX. However, concerning the biofilm vitality, the ASF solutions exhibited similar results than the CHX. These results are in line with previous findings, showing that ASF solutions reveal a more pronounced antibacterial effect when directly compared to their plaque inhibitory efficacy in an experimental gingivitis design ^{(Brecx et al., 1990}). A strong and clinically relevant reduction of plaque accumulation by the ASF was documented, on the other hand, in a 3-day plaque regrowth study ^{(Netuschil et al., 1995}). Moreover, when the ASF was used in addition to habitual mechanical plaque control over a long period significant clinical benefits were established ^{(Banoczy et al., 1989} ; ^{Brecx et al., 1993}).

Conclusion

Strong antibacterial and antiplaque properties of the alcohol-free ASF mouthrinse solution were demonstrated in the present study. A comparison of both ASF solutions with and without alcohol showed a marginal advantage for the former alcohol containing ASF preparation, the differences, however, were not significant in any case. For persons who want to omit as much as possible any contact to alcohol, the newly formulated alcohol-free ASF solution provides a clinically acceptable alternative. This study was supported by a grant from the Gaba International AG, Basel, Switzerland.

This study was supported by a grant from the Gaba International AG, Basel, Switzerland

BIBLIOGRAPHY

• ADDY M, WILLIS L, MORAN J. Effect of toothpaste rinses compared with chlorhexidine on plaque formation during a 4-day period. J Clin Periodontol 1983;10:89-99.
• ADDY M, JENKINS S, NEWCOMBE R. The effect of triclosan, stannous fluoride and chlorhexidine product on : (I) Plaque regrowth over a 4-day period. J Clin Periodontol 1990;17:693-697.
• ARWEILER N B, DONOS N, NETUSCHIL L, REICH E, SCULEAN A. Clinical and antibacterial effect of tea tree oil - A pilot study. Clin Oral Invest 2000;4:70-73.
• ARWEILER N B, NETUSCHIL L, REICH E. Alcohol-free mouthrinse solutions to reduce supragingival plaque regrowth and vitality. J Clin Periodontol 2001;28:168-174.
• BANOCZY J, SZÖKE J, KERTESZ P, TOTH ZS, ZIMMERMANN P, GINTNER Z. Effect of amine fluoride/stannous fluoride-containing toothpaste and mouthrinsings on dental plaque, gingivitis, plaque and enamel F accumulation. Caries Res 1989;23:284-288.
• BOLANOWSKI SJ, GESCHEIDER GA, SUTTON SVW. Relationship between oral pain and ethanol concentration in mouthrinses. J Periodont Res 1995;30:192-197.
• BRECX M, NETUSCHIL L, REICHERT B, SCHREIL G. Efficacy of Listerine, Meridol and chlorhexidine mouthrinses on plaque, gingivitis and plaque bacteria vitality. J Clin Periodontol 1990;17:292-297.
• BRECX M, MACDONALD LL, LEGARY K, CHEANG M, FORGAY M GE. Long-term effects of Meridolâ and chlorehxidine mouthrinses on plaque, gingivitis, staining, and bacterial vitality. J Dent Res 1993;72:1194-1197.
• BRECX M, WEIGER R, NETUSCHIL L. Etude de la plaque dentaire humaine sus-gingivale par microscopie à fluorescence. J Parodontol 1995;14:249-258.
• BRECX M, NETUSCHIL L, PETERSSON LG. Mouthrinses and periodontal disease. Int Dent J, in press (2001).
• GAFFAR A, AFFLITO J, NABI N, HERLES S, KRUGER I, OLSEN S. Recent advances in plaque, gingivitis, tartar and caries prevention technology. Int Dent J 1994;44:63-71.
• GAFFAR A, AFFLITO J, NABI N. Chemical agents for the control of plaque and plaque microflora : an overview. Eur J Oral Sci 1997;105:502-507.
• HARPER PR, MILSOM S, WADE W, ADDY M, MORAN J, NEWCOMBE RG. An approach to efficacy screening of mouthrinses : studies on a group of French products. (II) Inhibition of salivary bacteria and plaque in vivo. J Clin Periodontol 1995;22:723-727.
• JENKINS S, ADDY M, NEWCOMBE R. Dose response of chlorhexidine against plaque and comparison with triclosan. J Clin Periodontol 1994a;21:250-255.
• JENKINS S, ADDY M, NEWCOMBE R. Comparison of cetylperidiniumchloride, triclosan and chlorhexidine mouthrinse for effects on plaque regrowth. J Clin Periodontol 1994b;21:441-444.
• LLEWELYN J. Oral squamous cell carcinoma. Mouthwashes may increase risk. Br Med J 1994;308:1508.
• MORAN J, ADDY M, JACKSON R, NEWCOMBE R G. Comparative effects of quaternary ammonium mouthrinses on 4-day plaque regrowth. J Clin Periodontol 2000;27:37-40.
• NETUSCHIL L, REICH E, BRECX M. Direct measurement of the bactericidal effect of chlorhexidine on human dental plaque. J Clin Periodontol 1989;16:484-488.
• NETUSCHIL L, WEIGER R, PREISLER R, BRECX M. Plaque bacteria counts and vitality during chlorhexidine, Meridol and Listerine mouthrinses. Eur J Oral Sciences 1995;103:355-361.
• NETUSCHIL L, BRECX M. How to select the right drug ? Int Dent J, in press (2001).
• PENUGONDA B, SETTEMBRINI L, SCHERER W, HITTELMANN E, STRASSLER H. Alcohol-containing mouthwashes : effect on composite hardness. J Clin Dent 1994;5:60-62.
• PERNEGER TV. Whats wrong with Bonferroni adjustments. Br Med J 1998;316:1236-1238.
• QUIRYNEN M, DEKEYSER C, VAN STEENBERGHE D. The influence of gingival inflammation, tooth type and timing on the rate of plaque formation. J Periodontol 1991;62:219-222.
• RAMBERG P, LINDHE J, DAHLEN G, VOLPE AR. The influence of gingival inflammation on de novo plaque formation. J Clin Periodontol 1994;21:51-56.
• RAMBERG P, FURUICHI Y, VOLPE A, GAFFAR A, LINDHE J. The effects of antimicrobial mouthrinses on de novo plaque formation at sites with healthy and inflamed gingivae. J Clin Periodontol 1996;23:7-11.
• RIEP BG, BERNIMOULIN JP, BARNETT ML. Comparative antiplaque effectiveness of an essential oil and an amine fluoride/stannous fluoride mouthrinse. J Clin Periodontol 1999;26:164-168.
• SAXER UP, MÜHLEMANN H. Motivation und Aufklärung. Schweiz Monatsschr Zahnheilk 1975;85:905-919.
• SAXTON CA, SVATUN B, LLYOD AA. Antiplaque effects and mode of action of a combination of zinc citrate and a nonionic antimicrobial agent. Scand J Dent Res 1988;96:212-217.
• SILNESS J, LÖE H. Periodontal disease in pregnancy II. Correlation between oral hygiene and periodontal condition. Acta Odontol Scand 1964;22:121-135.
• WINN DM, BLOT WJ, MCLAUGHLIN JK, AUSTIN DF, GREENBERG RS, PRESTON-MARTIN S, SCHOENBERG JB, FRAUMENI JF. Mouthwash use and oral condition in the risk of oral and pharygeal cancer. Cancer Res 1991;51:3044-3047.
• WELLER-FAHY E, BERGER LR, TROUTMAN W. Mouthwash : a source of acute ethanol intoxication. Pediatrics 1980;66:302-304.