Bacterial plaque plays an essential

Transcription

1 Influence of Additional Active Ingredients on the Effectiveness of Non-Alcoholic Chlorhexidine Mouthwashes: A Randomized Controlled Trial Antonio Bascones,* Sergio Morante,* Leopoldo Mateos,* Montserrat Mata, and Jorge Poblet Background: Non-alcoholic chlorhexidine mouthwashes are equally effective and have fewer potential risks than hydroalcoholic solutions. Nowadays, other active ingredients are added to these mouthwashes in an attempt to improve their effectiveness and reduce side effects. Following an experimental gingivitis model, this study examined three non-alcoholic commercial mouthwashes having 0.12% chlorhexidine digluconate (CHX) in common. Methods: Using a double masked, cross-over design, 30 subjects underwent three consecutive experimental phases with three mouthwashes: CHX, CHX % sodium fluoride (CHX- NaF), and CHX % cetylpyridinium chloride (CHX-CPC). In each one of these 21-day phases, the subjects discontinued all oral hygiene measures and were treated exclusively with the experimental mouthwash randomly assigned (an oral rinse twice a day). Each experimental phase was preceded by a 14-day washout period. Levels of gingivitis, dental plaque, supragingival calculus, and dental staining were assessed at baseline and end (day 21) of experimental phases. Results: The evolution of gingival and dental staining indices did not show statistically significant differences between the treatments. Differences were noticed in the plaque index (P = ), with CHX-NaF the treatment with the greatest increase. Differences were also observed in the supragingival calculus index (P = ), with CHX-CPC showing a smaller increase. Tongue staining was more frequent with CHX-CPC (P = ). Conclusion: In non-alcoholic 0.12% chlorhexidine mouthwashes, the addition of other active ingredients does not produce beneficial effects, but may even reduce the antiplaque effectiveness or increase tongue staining. J Periodontol 2005;76: KEY WORDS Adjunctive therapy; chlorhexidine/therapeutic use; comparison studies; cross-over studies; double-blind method; mouthrinses/ therapeutic use. * Department of Medicine and Buccofacial Surgery, Complutense University, Madrid, Spain. Lácer Personal Health, Lácer, S.A., Barcelona, Spain. Bacterial plaque plays an essential role in the development of periodontal disease since its accumulation inevitably leads to gingivitis. 1 The physiopathological mechanisms by which gingivitis progresses to periodontitis are not all well known, which is why preventing periodontal disease is based on an adequate control of plaque. The main measure for controlling bacterial plaque is mechanical hygiene procedures (toothbrushing and dental floss). However, both the absence of hygiene habits as well as the inability to perform a correct toothbrushing technique can make mechanical plaque control insufficient. Therefore, chemical control of plaque is becoming widespread as a coadjuvant treatment of mechanical control. Chlorhexidine is the most effective antibacterial agent for chemical plaque control. It is extensively used, reducing plaque and gingivitis by 60%. 2 Chemically, chlorhexidine is made up of two 4-chlorophenyl rings and two biguanide groups linked by a central hexamethylene chain, which confers a strong basicity and a bicationic charge. Nevertheless, its bicationic nature makes it highly interactive with any anionic element, thus making its formulation much more difficult. The mechanism of action of chlorhexidine begins with a strong binding to the bacterial cellular membrane. At low concentrations, it increases the membrane s 1469

2 Is Chlorhexidine Improved by Adding Other Active Ingredients? Volume 76 Number 9 permeability and thus also the potassium intracellular concentration, resulting in a bacteriostatic effect. At high concentrations, chlorhexidine produces a bactericidal effect due to the bacterial cytoplasm precipitation and subsequent cellular death. 2 In vitro, it is effective against Gram-negative and Gram-positive bacteria, including aerobes and anaerobes, as well as against fungi and yeasts. No significant bacterial resistances have been described, even in cases of long-lasting use over a two-year period. 3 Chlorhexidine is adsorbed rapidly on dental surfaces, salivary proteins, and mucous membranes, where it is released gradually over 8 to 12 hours. 4 This property, called substantivity, affords a convenient dosage schedule for patients every 12 hours. Mouthwashes are the most widespread way to use chlorhexidine. 5 Chlorhexidine mouthwashes have shown anti-gingivitis activity, providing greater reductions in gingivitis occurrence and severity than placebo, but without significant differences between the 0.12% and 0.2% concentrations. 6 Lower concentrations have also been investigated, but with contradictory antiplaque and antimicrobial results. 7-9 Until recently, the majority of chlorhexidine mouthwashes contained alcohol, either as a preservative (without any therapeutic activity) or in order to stabilize the galenic formulation by dissolving other ingredients. High ethanol concentration together with a low ph make mouthwashes a potentially irritating element and, consequently, contraindicated for patients with mucositis, those who have undergone head and neck irradiation, have immunocompromised systems, or are chronic alcoholics. The presence of alcohol could also represent a supposed increase in the risk of suffering oral cancer, 10,11 although this has not been entirely verified. 12 If we add the other harmful effects of alcohol, such as epithelium desquamation 13 or white lesions induction as well as type I and type IV hypersensitivity reactions, there is an evident need for nonalcoholic chlorhexidine formulations having the same anti-plaque and anti-gingivitis effectiveness. Studies have shown the same effectiveness of nonalcoholic chlorhexidine mouthwashes compared with the same formulations in an alcoholic solution. 14,15 In view of the foregoing, it may be concluded that there is no justification for using chlorhexidine formulations containing alcohol; the same effectiveness with fewer potential risks can be obtained with a non-alcoholic solution. Furthermore, the objective is directed at obtaining chlorhexidine associations, with the purpose of reducing its side effects (soft and hard tissue staining and taste disturbances) without reducing, or even increasing, its effectiveness. The possibility exists that adding other chemical agents to chlorhexidine solutions in order to improve their effects may result in the inactivation of the antiseptic due to the high cationic value of its molecule. The aim of this study was to assess if adding active ingredients (sodium fluoride or cetylpyridinium) to nonalcoholic mouthwashes containing 0.12% chlorhexidine improved their effectiveness. In an attempt to reduce the customary side effects of chlorhexidine, an improvement of effectiveness would support future investigations with associations containing lower concentrations of chlorhexidine. Following an experimental gingivitis model, we assessed, with a double masked, cross-over design, the effectiveness of three non-alcoholic 0.12% chlorhexidine-containing mouthwashes available in Spain, one of them incorporating sodium fluoride and another cetylpyridinium chloride, in the prevention of gingivitis development and supragingival plaque formation. MATERIALS AND METHODS Study Population Thirty healthy volunteers of both genders and aged over 18 years were included in the study after giving their informed written consent. Volunteers were mostly dental students of the Complutense University Faculty of Dentistry. The following exclusion criteria were applied: active periodontal disease (pockets >4 mm); less than 20 natural teeth; periodontal surgery during the previous 3 months in the area of the teeth under study; treatment during the previous 6 months with antibiotics for a dental pathology or during the previous 30 days for any other reason; treatment during the previous 30 days with the mouthwashes under study or with any other medication which might affect the periodontal condition; history of hypersensitivity or specific oral allergy to any of the ingredients of the experimental mouthwashes; pregnancy or breast-feeding; and systemic diseases, especially chronic ones, which might interfere with the recording of representative data on gingivitis, dental plaque, supragingival calculus, and dental staining. The study was authorized by the Clinical Research Ethics Committee of Hospital Clínico San Carlos, Madrid, Spain. Study Design The study was a double-masked, cross-over design with random distribution of the experimental mouthwashes. The subjects underwent three consecutive 21-day experimental phases with the mouthwashes to be assessed. In each one of these phases the subjects discontinued all oral hygiene measures and were treated exclusively with the randomly assigned mouthwash. Mouthwashes were used as an oral rinse with 10 ml for about 1 minute twice a day, in the morning (after breakfast) and in the evening (after dinner). In addition, the subjects avoided rinsing with water, eating, and drinking for 30 minutes following the use of the mouthwash. 1470

3 Bascones, Morante, Mateos, Mata, Poblet Each one of the three experimental phases was preceded by a 14-day washout period. At the beginning of these periods, the subjects had their teeth professionally cleaned (removal of tartar, plaque, and stains as well as polishing with a rubber cup and prophylaxis paste). They also received instructions for accurate oral hygiene during the next 14 days by brushing their teeth for at least 2 minutes, three times a day. The objective of each washout period was to assure the least possible presence of plaque, as well as a practically non-existent level of gingivitis, before starting the experimental phase that followed. Experimental Treatments The subjects meeting the selection criteria were treated on a randomized, double-masked, cross-over basis, with three non-alcoholic experimental mouthwashes: 0.12% chlorhexidine digluconate (CHX), 0.12% chlorhexidine digluconate % sodium fluoride (CHX-NaF), or 0.12% chlorhexidine digluconate % cetylpyridinium chloride (CHX-CPC). At the beginning of each experimental phase, the subjects received the assigned mouthwash. The use of less than 60% of the planned amount of mouthwash was considered as an inadequate compliance. Since the original colors of the mouthwashes were not similar, they were homogeneously colored to ensure the blinding of the experimental treatments. Given that one of the indices assessed in the study was dental staining, a previous in vitro study was carried out to rule out the possibility that the change in coloring of original mouthwashes could affect this index. In order to do so, we used a modification in the method of Mendieta et al., 16 originally described by Addy et al. 17 to investigate in vitro the role of dietary factors in the etiology of tooth staining associated with chlorhexidine. Six formulations were assessed: the three original mouthwashes and their respective solutions specifically colored for the present study. Acrylic blocks measuring 30 mm 10 mm 6 mm were prepared. Groups of three blocks were placed in 60 ml of each solution for 2 minutes. After being rinsed for 30 seconds in 60 ml of distilled water, the blocks were dried and placed for 60 minutes in 60 ml of a standard tea solution. Once removed from the tea solution, the blocks returned to the first step; that is, they were placed into the assigned solution. After completing six cycles, the optical density of each block was then measured twice with a spectrophotometer at a fixed lambda of 395 nm. The data were analyzed by means of the Student t test. It concluded that the coloring of each experimental mouthwash did not cause, in relation to the respective original mouthwash, a significant change in the capacity to induce staining in vitro. To assure the homogeneity of dental hygiene measures, for each one of the three washout periods, all the subjects received a toothbrush # (bristles inches thick) and a tube of toothpaste** (1.89% sodium monofluorophosphate and 0.20% aldioxa). The subjects were carefully instructed not to use any product (toothpaste, mouthwash, etc.), other than those provided by the investigators. Clinical Assessment The levels of gingivitis, 18 dental plaque, 19 supragingival calculus, 20 and dental staining 21 were assessed at baseline and at the end (day 21) of each experimental phase. The assessment of gingival and dental plaque indices was made on the mesial, distal, vestibular, and lingual sides of the six Ramfjord teeth suggested for evaluating periodontal condition. 22 The assessment of supragingival calculus index was made on the lingual surfaces (mesial, medial, and distal position) of the four lower incisors. Extrinsic dental staining was assessed on the vestibular surfaces of the upper incisors, as well as on the vestibular and lingual surfaces of the lower incisors. Each dental surface was divided into two regions (coronal and gingival) where the area and intensity of staining were recorded. The staining index of each region was calculated by multiplying its area score by its intensity score. At the end of each experimental phase, the oral cavity was examined and all the subjects were systematically asked for the possible appearance of adverse events. The cases of tongue staining were described apart from the rest of adverse events. Statistical Analysis Non-parametric Kruskal-Wallis test was used to compare the treatment groups at each time period (baseline and final) with respect to gingivitis, dental plaque, supragingival calculus, and dental staining indices. Where a statistically significant difference was found, pair-wise treatment comparisons were undertaken. To analyze the change in the indices, the difference between the final and baseline score of the index at issue, for each subject and in each experimental phase, was used. In this way, the smaller the difference, the greater was the preventive effect of the mouthwash analyzed. A mixed model for fixed and random effects was used to assess change in indices as well as sequence and period effects. Chi square test was used to compare the frequency of tongue staining and the rest of adverse events. The significance level of statistical tests was established at α =0.05. The results are presented as mean ± standard deviation. Lácer Chlorhexidine Mouthwash, Lácer, S.A., Barcelona, Spain. Cariax Gingival Mouthwash, Laboratorios Kin, S.A., Barcelona, Spain. Perio-Aid without alcohol, Dentaid, Cerdanyola, Barcelona, Spain. Perspex, ICI, Macclesfield, Cheshire, U.K. # Lácer Medium Toothbrush, Lácer, S.A. ** Lácer Toothpaste, Lácer, S.A. 1471

4 Is Chlorhexidine Improved by Adding Other Active Ingredients? Volume 76 Number 9 RESULTS Study Population Thirty subjects (17 men and 13 women) with a mean age of ± 2.53 years (range: 19 to 30 years) were included. All completed the three experimental phases of the study and used the treatments correctly. The treatment groups were homogeneous at baseline for all the indices assessed (gingivitis, dental plaque, supragingival calculus, and dental staining) (Table 1). Gingivitis At the end of the experimental phases, there were no statistically significant differences between the three mouthwashes (Table 1). Even though it was close to significance, the evolution of the gingival index did not show statistically significant differences between the treatments (P = ) (Fig. 1). greater increase in the score than CHX (P = ) and CHX-CPC (P = ) (Fig. 2). Supragingival Calculus There were statistically significant differences between the mouthwashes (P = ) with regard to final calculus scores, with CHX-NaF having a significantly higher score than CHX-CPC (P = ). The change in the supragingival calculus index confirmed statistically significant differences between the treatments Dental Plaque After 21 days of use, there were statistically significant differences between the mouthwashes (P = ), with the CHX-NaF solution having a dental plaque score significantly higher both than CHX (P = ) and CHX-CPC (P = ) (Table 1). There were also statistically significant differences between the treatments with regard to the change in dental plaque index (P = ), with CHX-NaF showing a Table 1. Indices Scores (mean ± standard deviation) Figure 1. Box diagram of the change in gingival index.the central line of the box represents the median and the sides of the box, the first and second quartiles.the mean is detailed with the plus (+) symbol.the lateral lines of the box represent the minimum and maximum values. Index Formulation Baseline Final Evolution* Gingival CHX 0.26 ± ± ± 0.33 CHX-NaF 0.24 ± ± ± 0.36 CHX-CPC 0.23 ± ± ± 0.35 P = P = P = Dental CHX 0.13 ± ± ± 0.55 plaque CHX-NaF 0.13 ± ± ± 0.67 CHX-CPC 0.11 ± ± ± 0.59 P = P = P = Supragingival CHX 0.02 ± ± ± 0.27 calculus CHX-NaF 0.03 ± ± ± 0.19 CHX-CPC 0.02 ± ± ± 0.09 P = P = P = Dental CHX 0.08 ± ± ± 0.77 staining CHX-NaF 0.05 ± ± ± 0.49 CHX-CPC 0.06 ± ± ± 0.56 P = P = P = * Difference between the final and baseline values. P = (CHX-NaF versus CHX); P = (CHX-NaF versus CHX-CPC). P = (CHX-NaF versus CHX); P = (CHX-NaF versus CHX-CPC). P = (CHX-NaF versus CHX-CPC). P = (CHX-CPC versus CHX-NaF); P = (CHX-CPC versus CHX). Figure 2. Box diagram of the change in dental plaque index (P = , CHX- NaF shows a greater increase). For a description of the box diagram, see the legend to Figure 1.The outliers (anomalous values) are identified outside the lateral lines. 1472

5 Bascones, Morante, Mateos, Mata, Poblet (P = ), with CHX-CPC showing a smaller increase in the score than both CHX-NaF (P = ) and CHX (P = ) (Table 1). Dental Staining At the end of the experimental phases, no statistically significant differences between the three mouthwashes were found. The change in the dental staining index showed no statistically significant differences between the treatments (Table 1). Period and Sequence Effects Statistical analysis revealed no significant sequence effect for the change in any of the indices assessed (gingivitis, dental plaque, supragingival calculus, and dental staining) as well as no significant period effect for the evolution of supragingival calculus and dental staining indices. Change in gingivitis and dental plaque indices revealed a significant period effect (P = and P = , respectively) with greater increases of the scores over time (first to third experimental period). Tongue Staining/Adverse Events Examination of the oral cavity detected statistically significant differences between the treatments with regard to tongue staining (P = ). During the treatment with CHX-CPC, the frequency of tongue staining (12 cases) was greater than that observed with CHX-NaF (three cases, P = ) and CHX (five cases, P = ). Twenty-one adverse events were reported during the experimental phases: eleven with the use of CHX- CPC (four cases of dysgeusia, four burning pains, one aphthous ulcer, one dental erosion, and one recurrent chronic parotiditis); six with CHX (dysgeusia, dental hypersensitivity, aphthous ulcer, labial herpes, tongue ulcer, and cracked lip); and four with CHX-NaF (three aphthous ulcers and one burning pain). There was no statistically significant difference between the treatments. DISCUSSION The latest investigations are aimed at obtaining nonalcoholic chlorhexidine formulations with the same or even more effectiveness than alcoholic solutions, but reducing the side effects, which include oral surface staining, and taste and sensitivity disturbances as well as oral pain. The main purpose of our study was to assess if adding active ingredients (sodium fluoride or cetylpyridinium chloride) to non-alcoholic mouthwashes containing 0.12% chlorhexidine improved their effectiveness. For this purpose, we compared three non-alcoholic 0.12% chlorhexidine containing mouthwashes available in Spain, assessing their capacity to prevent the development of gingivitis and the formation of supragingival plaque. Moreover, we assessed the accumulation of supragingival calculus, as well as the incidence of side effects associated with chlorhexidine, such as staining of oral surfaces, and other adverse events associated with the mouthwashes. To obtain the best evidence, we designed a randomized, double-masked, cross-over study. We found no previously published randomized controlled trial exclusively assessing non-alcoholic chlorhexidine mouthwashes. The experimental gingivitis periods of the present study lasted 21 days. Unlike other studies, such as that of Mendieta et al. 16 which used a 7-day experimental period or Quirynen et al. 14 with 11 days, ours is based on the experimental gingivitis model described by Löe et al. in 1965, 1 where it was shown that, in the absence of mechanical plaque control, all the subjects developed a clinically detectable gingivitis after a 21-day period. Moreover, the prior 2-week washout period is essential for the recovery of the mechanical plaque control measures that assure the least possible presence of plaque and a practically non-existent level of gingivitis. After 21 days of use, there were no statistically significant differences between the experimental treatments with regard to the mean gingival index; however, its evolution (difference between the baseline and final scores) was close to significant (P = ). The change in gingival control tended to be better with the mouthwash containing chlorhexidine (without other active ingredients) than with the CHX-NaF formula, the latter tending to be better than the CHX-CPC association. These results are slightly different from those found by Quirynen et al., 14 where the sodium fluoride rinse gave the worst gingival control, but without statistical significance either, of four alcoholic and non-alcoholic formulations containing 0.12 or 0.2% chlorhexidine. Statistically significant differences were shown for dental plaque index, with the CHX-NaF association having a higher final score and a worse change than the other two mouthwashes. These results coincide with those found by Quirynen et al., 14 which showed a lower capacity to retard the formation of new dental plaque with CHX-NaF, compared with CHX-CPC, both without alcohol, as well as with two alcoholic formulations containing 0.12 and 0.2% chlorhexidine. CHX-CPC formula showed a significantly better control of supragingival calculus accumulation (a commonly acknowledged potential side effect of chlorhexidine), with the CHX-NaF rinse showing both a significantly higher final score and a significantly worse change than CHX-CPC. However, in our opinion, supragingival calculus is more susceptible to bias factors, namely, autoclasis or diet, which could not be repeated in an identical manner throughout the experimental phases in the same subject. 1473

6 Is Chlorhexidine Improved by Adding Other Active Ingredients? Volume 76 Number 9 In the same way, staining of teeth and other oral surfaces can also be affected by factors such as diet and smoking habits, which in spite of the fact that it was a cross-over study, might not have been reproduced accurately during the different experimental phases. Nevertheless, dental staining showed no statistically significant differences between the three mouthwashes. The mouthwash containing sodium fluoride was that with a slightly lower dental staining at the end of the experimental phase, just like the results found by Quirynen et al. 14 The absence of significant differences for the dental staining potential of the experimental mouthwashes did not correlate with their capacity for tongue staining, where the mouthwash containing cetylpyridinium chloride caused a significant greater number of cases. These latter data coincide with what was observed in the previous in vitro study we carried out. Apart from concluding that in no case did the coloring of mouthwash cause, in relation to the respective original mouthwash, a significant change in the capacity to induce staining in vitro, the mouthwash containing cetylpyridinium chloride, both the original and the colored for this study, produced a significantly greater in vitro staining than the others (data not presented). With respect to adverse events, it should be repeated that the CHX-CPC formulation resulted in a greater number of cases of dysgeusia. Our study is in line with what has been indicated by authors such as Addy et al and Harper et al., 26 in the sense that the addition of other ingredients can reinforce or inhibit the activity of chlorhexidine. Thus, as Herrera et al. stated, 27 it is not possible to ensure the effectiveness of a formulation only by containing an active ingredient of proven effectiveness, since the variability between galenic formulations, as well as the addition of other active ingredients, may modify the properties of said formulation. Furthermore, as observed in our study for supragingival dental plaque, the partial reduction of chlorhexidine availability, reflected in some loss of clinical efficacy, had already been shown for mouthwashes containing sodium fluoride. 14,16 This inactivation also appears in the study of Herrera et al., 27 when a smaller bacterial reduction was found, both in aerobes and anaerobes, with the non-alcoholic mouthwash containing 0.12% chlorhexidine and sodium fluoride, as compared with the also non-alcoholic one containing 0.12% chlorhexidine and cetylpyridinium chloride. From the results obtained in the present study, it can be concluded that in non-alcoholic 0.12% chlorhexidine mouthwashes, the addition of other active ingredients does not produce beneficial effects, but may even reduce its antiplaque effectiveness or increase tongue staining. ACKNOWLEDGMENTS This study was supported by a grant from Lácer, S.A., Barcelona, Spain. Montserrat Mata is a technician in Research and Development, and Jorge Poblet is the medical manager at Lácer Personal Health, a Division of Lácer, S.A., Barcelona, Spain. REFERENCES 1. Löe H, Theilade E, Jensen SB. Experimental gingivitis in man. J Periodontol 1965;36: Ciancio SG, Nisengard RJ. Control and prevention of periodontal disease. In: Nisengard RJ, Newman MG, eds. Oral Microbiology and Immunology. Philadelphia: WB Saunders Company; 1994: Schiött CR, Briner WW, Kirkland JJ, Löe H. Two years oral use of chlorhexidine in man. III. Changes in sensitivity of the salivary flora. J Periodontal Res 1976;11: Rölla G, Melsen B. On the mechanism of the plaque inhibition by chlorhexidine. J Dent Res 1975;54(Spec. Issue B): Fardal O, Turnbull RS. A review of the literature on use of chlorhexidine in dentistry. J Am Dent Assoc 1986;112: Segreto VA, Collins EM, Beiswanger BB, et al. A comparison of mouthrinses containing two concentrations of chlorhexidine. J Periodontal Res 1986;21(Suppl.): Addy M, Wade WG, Jenkins S, Goodfield S. Comparison of two commercially available chlorhexidine mouthrinses: I. Staining and antimicrobial effects in vitro. Clin Prev Dent 1989;11(5): Jenkins S, Addy M, Newcombe R. Comparison of two commercially available chlorhexidine mouthrinses: II. Effects on plaque reformation, gingivitis, and tooth staining. Clin Prev Dent 1989;11(6): Yévenes I, Reyes J, Campos N, Saragoni V. Inhibitory effect in microbial plaque and antibacterial properties of chlorhexidine mouthwashes (in Spanish). Av Periodon Implantol 2003;15: Weaver A, Fleming SM, Smith DB. Mouthwash and oral cancer: Carcinogen or coincidence? J Oral Surg 1979; 37: Blot WJ, Winn DM, Fraumeni JF. Oral cancer and mouthwash. J Natl Cancer Inst 1983;70: Smith RG, Moran J, Addy M, Doherty F, Newcombe RG. Comparative staining in vitro and plaque inhibitory properties in vivo of 0.12% and 0.2% chlorhexidine mouthrinses. J Clin Periodontol 1995;22: Flötra L. Different modes of chlorhexidine application and related local side effects. J Periodontal Res 1973; 12(Suppl.): Quirynen M, Avontroodt P, Peeters W, Pauwels M, Coucke W, van Steenberghe D. Effect of different chlorhexidine formulations in mouthrinses on de novo plaque formation. J Clin Periodontol 2001;28: Leyes Borrajo JL, Garcia Varela L, Lopez Castro G, Rodriguez-Nuñez I, Garcia Figueroa M, Gallas Torreira M. Efficacy of chlorhexidine mouthrinses with and without alcohol: A clinical study. J Periodontol 2002;73: Mendieta C, Vallcorba N, Binney A, Addy M. Comparison of 2 chlorhexidine mouthwashes on plaque regrowth in vivo and dietary staining in vitro. J Clin Periodontol 1994;21: Addy M, Prayitno S, Taylor L, Cadogan S. An in vitro study of the role of dietary factors in the aetiology of 1474

7 Bascones, Morante, Mateos, Mata, Poblet tooth staining associated with the use of chlorhexidine. J Periodontal Res 1979;14: Löe H, Silness J. Periodontal disease in pregnancy. I. Prevalence and severity. Acta Odontol Scand 1963;21: Turesky S, Gilmore ND, Glickman I. Reduced plaque formation by the chloromethyl analogue of victamine C. J Periodontol 1970;41: Volpe AR, Manhold JH, Hazen SP. In vivo calculus assessment: A method and its examiner reproducibility. J Periodontol 1965;36: Lobene RR. Effect of dentifrices on tooth stains with controlled brushing. J Am Dent Assoc 1968;77: Ramfjord SP. Indices for prevalence and incidence of periodontal disease. J Periodontol 1959;30: Addy M, Jenkins S, Newcombe R. The effect of some chlorhexidine-containing mouthrinses on salivary bacterial counts. J Clin Periodontol 1991;18: Addy M, Moran J, Newcombe R, Warren P. The comparative tea staining potential of phenolic, chlorhexidine and anti-adhesive mouthrinses. J Clin Periodontol 1995; 22: Addy M, Wade W. An approach to efficacy screening of mouthrinses: Studies on a group of french products. (I). Staining and antimicrobial properties in vitro. J Clin Periodontol 1995;22: 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: Herrera D, Roldán S, Santacruz I, O Connor A, Sanz M. Antimicrobial activity in the saliva of four chlorhexidine mouthwashes (in Spanish). Periodoncia 2001;11: Correspondence: Prof. Antonio Bascones, Department of Medicine and Buccofacial Surgery, Faculty of Dentistry, Complutense University, Plaza Ramón y Cajal, s/n, Madrid, Spain. Fax: ; antbasco@arrakis.es. Accepted for publication January 26,