Clin Oral Invest (2001) 5:8995 DOI 10.

1007/s007840100114

O R I G I N A L A RT I C L E

T. Hoffmann G. Bruhn S. Richter L. Netuschil M. Brecx

Clinical controlled study on plaque and gingivitis reduction under long-term use of low-dose chlorhexidine solutions in a population exhibiting good oral hygiene
Received: 12 January 2001 / Accepted: 25 April 2001 / Published online: 2 June 2001 Springer-Verlag 2001

Abstract The aim of this randomized, parallel-group, placebo-controlled, blind clinical study was to examine the dental plaque and the gingivitis inhibitory effects of two low-dose 0.06% chlorhexidine (CHX) preparations in comparison to a commercially available 0.1% CHX solution, an amine fluoride/stannous fluoride (ASF) solution and a water control as an adjunct to the daily mechanical oral-hygiene measures. After prophylaxis, 81 medical students performed their tooth cleaning and were asked to rinse for 30 s, according to the instructions given by the manufacturers with the assigned amount of the following solutions: once daily 10 ml ASF 250 ppm or twice daily each 10 ml CHX 0.06% with fluoride, 10 ml CHX 0.06% without fluoride, 15 ml CHX 0.1% or 100 ml tap water. The plaque index (PlI), the gingival index (GI) and the discoloration index (DI) were taken at day 14 and months (M) 0, 1, 2, 3 and 6. In this study group with good oral hygiene, all test products led to a reduction of the PlI scores when used as supplements to the usual mechanical hygiene measures. At M3 subjects using both the 0.1% as well as the 0.06% CHX showed significantly less plaque accumulation than those using ASF. In the GI a significant difference between the control and 0.1% CHX was found at M3 and at M6. If a further decrease in the GI is wanted, a minimum concentration of 0.1% CHX for long-term use in association with habitual cleaning is needed, which, however, should be limited to special patients. If maintaining clinical health is the goal, the other solutions are a good alternative to 0.1% CHX because they exhibit less staining. Keywords Plaque Index Gingival Index Discoloration Index Low-dose chlorhexidine Amine fluoride/stannous fluoride

Introduction
Gingivitis is caused by the bacteria present in the dental plaque biofilm [6, 33]. The frequency of gingivitis in the young population is nearly 100% [4, 41]. This shows that individual oral hygiene procedures do not result in overall gingivitis-free dentitions [24]. While the combination of individual and professional oral hygiene can lead to stable oral health in well-controlled subjects [3], additional preventive measurements are necessary for specific population groups. The use of mouthwashes or rinsing solutions is credited to Chinese medicine and goes back to 2700 B.C. More detailed information on inherited recipes arose from the time of Hippocrates (480 B.C.) [16]. Scientific research on antiseptic mouthwashes has a history of 50 years, starting with the investigations of Slanetz and Brown [45] and continuing through the work of Stralfors [46]. Chlorhexidine (CHX) digluconate has a 30-year history in dental medicine [35, 36] and is by far the most thoroughly studied and the most effective anti-plaque and anti-gingivitis agent known today (for review, see [2, 14, 20, 29]) Rinsing twice daily with 10 ml of a 0.2% CHX solution inhibited dental plaque formation [21, 36]. Furthermore, its anti-gingivitis efficacy was also well documented [1, 25, 29, 32]. Unfortunately, these positive effects are accompanied by side effects, the most disturbing being extrinsic tooth staining [15, 18, 31, 32, 42]. In a few cases, the occurrence of gingival desquamation and painful mucosa were reported [18, 29]. Different studies have shown that the dental plaqueand gingivitis-inhibitory qualities, as well as the side effects, of CHX are dose-dependent [11, 12, 23]. Therefore, starting in the late 1970s [5], studies were performed to investigate the plaque- and gingivitis-reducing efficacy of low-concentration CHX solutions or dentifrices [17, 19, 23, 26, 39]. The gold standard remained the 0.2% CHX. The combination of mechanical and chemical plaque control [8, 9, 37, 47], however, seemed to be even more

T. Hoffmann () G. Bruhn S. Richter L. Netuschil M. Brecx Department of Periodontology, Dental School TU Dresden, Fetscherstrae 74, 01307 Dresden, Germany e-mail: th.hoffm@rcs.urz.tu-dresden.de Tel.: +49-351-4582712, Fax: +49-351-4585341

90

beneficial than only brushing, on the one hand, or only using mouthwash, on the other hand. When the main volume of the dental plaque is removed by mechanical actions, the oral chemopreventive agents only have to combat sparse biofilm remnants. Consequently, mouth rinses are recommended as adjuncts to mechanical oral hygiene [2, 6, 8, 9, 38]. The aim of this blind, parallel-group, placebo-controlled clinical study was to examine the plaque- and the gingivitis-inhibitory effects of two low-dose CHX preparations in comparison to a commercially available CHX solution, an amine fluoride/stannous fluoride solution (ASF) and a negative control on patients with good oral hygiene. The subjects used these preparations in addition to their habitual mechanical oral-hygiene measures over a 6-month period.

Substance

Manufactured brand

Amount Once daily 10 ml Twice daily 10 ml Twice daily 10 ml Twice daily 15 ml Twice daily 100 ml

ASF (250 ppm fluoride) Meridol (Wybert, Lrrach, Germany) CHX digluconate Corsodyl Zahnfleisch Fluid (0.06%) without (SmithKline Beecham, fluoride Bhl, Germany) CHX digluconate Odol med3 Depot (0.06%) with 250 ppm (SmithKline Beecham, sodium fluoride Bhl, Germany) (CHX/F) CHX digluconate 0.1% Chlorhexamed (Procter & Gamble, Mainz, Germany) Control: Water

Material and methods

Participant selection and distribution Eighty-one of 85 medical students (53 females and 28 males) from the Medical Faculty Carl Gustav Carus, University of Technology, Dresden ranging in age from 18 to 36 and exhibiting good oral hygiene were included in this study. Inclusion criteria were at least 20 teeth, gingival index (GI) <1.5, and no periodontitis. Exclusion criteria were allergic reactions to any of the components of any mouth rinse, antibiotic/antiphlogistic medication in the previous 8 weeks, dental-treatment needs (e.g. due to acute carious lesions), pregnancy or any kind of systemic diseases. All participants received complete information about the aim of the study, its duration, and the possible side effects of the rinsing solutions, and all signed a consent form. The study design fulfilled the regulations of clinical controlled trials [13, 27]. The ethics committee of the Medical Faculty of the University of Technology, Dresden, agreed to the study protocol (EK 890999). Investigation parameters Plaque accumulation was assessed by the criteria of the plaque-index system (PlI: [44]). The gingival status was scored according to the criteria of the gingival-index system (GI: [34]). Both parameters were recorded at four sites (mesio-vestibular, mid-vestibular, disto-vestibular and lingual) of each tooth. Tooth staining was evaluated by the discoloration index (DI: [9]) on the vestibular as well as on the lingual aspects of each tooth. One investigator assessed PlI and GI, while another estimated DI during the study. Test substances and controls The substances, tested according to the manufacturers instructions, included:

The different test solutions were presented in similar bottles. However, each participant got his/her specific information regarding the use of mouthwashes from an independent person not involved in the examinations. Investigation schedule At the time of selection (day 14) all participants received oral-hygiene instructions (Fig. 1). The three clinical parameters were evaluated, and professional oral hygiene was performed. The panelists received a new toothbrush and followed their habitual tooth-cleaning procedure, using their usual toothpaste for 2 weeks. At month 0 (M0) their teeth were polished once again. They

Fig. 1 Study design

Table 1 Number of panelists at the start (M0) and the end (M6) of the clinical investigation. F female, M male, ASF amine fluoride/stannous fluoride, CHX chlorhexidine Group Start (M0) F Control ASF CHX 0.06%/F CHX 0.06% CHX 0.10% M 15 16 17 17 16 81 End (M6) F M 11 15 17 16 14 73 Dropouts F 2 1 2 5 M 2 1 3 4 1 1 2 8

9 6 9 7 12 5 12 5 11 5 53 28

7 4 9 6 12 5 11 5 9 5 48 25

91 Table 2 Comparison of the test products with the water control at month 3 (M3). n.s. not significant, CHX chlorhexidine, CHX/F chlorhexidine with sodium fluoride, ASF amine fluoride/stannous fluoride. * Significant, i.e. P0.05 in case without Bonferroni correction, P0.0051620 in case with Bonferroni correction With Bonferroni correction Without Bonferroni correction

Significance levels

P-values

Medians

0.50 0.66 0.83 0.68 0.96

DI

0.0109 0.0001 0.0642 0.0000

* * n.s. *

n.s. * n.s. *

Significance levels

With Bonferroni correction

Fig. 2 Box-plot diagrams of the plaque indices (PlI) of the groups using the different rinses during the investigation n.s. n.s. n.s. n.s. n.s. * * * 0.28 0.15 0.21 0.16 0.14 0.287 0.151 0.183 0.045 n.s. n.s. n.s. *

GI

Medians Significance levels With Bonferroni correction

P-values

Without Bonferroni correction

Without Bonferroni correction

Fig. 3 Box-plot diagrams of the gingival indices (GI) of the groups using the different rinses during the investigation

received a new toothbrush and were asked to rinse for 30 s after brushing, according to the special instructions for the assigned amount of the rinsing solution. During each visit the motivation to perform the study as meticulously as possible was reinforced for each individual without any attempt to influence their other cleaning habits. The clinical parameters were reevaluated after 1, 2 and 3 months (M1, M2, M3) as well as at the end of the study (M6), whereafter each subject received a new professional tooth cleaning. Randomization and statistics The patient was used as the statistical unit. According to their clinical parameters at day-14 and their sex, the 85 panelists were distributed at random and stratified to the

P-values

Medians

Control ASF 0.06% CHX/F 0.06% CHX 0.10% CHX

Products

0.55 0.23 0.20 0.14 0.15

PlI

0.0456 0.0022 0.0007 0.0013

* * * *

92 Table 3 Comparison of the products at month 3 (M3) a. n.s. not significant, CHX chlorhexidine, CHX/F chlorhexidine with sodium fluoride, ASF amine fluoride/stannous fluoride. *: significant, Parameters Compared products Median values i.e. P0.05 in case without Bonferroni correction, P0.0051620 in case with Bonferroni correction P-values Significance levels Without Bonferroni correction PlI DI ASF/0.06% CHX ASF/0.10% CHX ASF/0.06% CHX/F ASF/0.10% CHX 0.06%/0.10% CHX 0.23/0.14 0.23/0.15 0.66/0.83 0.66/0.96 0.68/0.96 0.048 0.038 0.021 0.003 0.038 * * * * * With Bonferroni correction n.s. n.s. n.s. * n.s.

Only significant differences without Bonferroni correction are listed.

five test groups (variables for stratification: 1: PlI0.7, 2: PlI>0.7; 1: GI0.6, 2: GI>0.6; 1: DI0.4, 2: DI>0.4; 1: male, 2: female). The Kolmogoroff-Smirnoff test was used, showing the data sets not being normally distributed. To compare all mouth rinses at M3 and M6, a non-parametric test (Kruskal-Wallis one-way ANOVA) was used. Specific differences at M3 and M6 were proven with the MannWhitney U test, using the statistical package SPSS for Windows 9.0. All three clinical parameters (PlI, GI, DI) were of equal interest, as were the investigation times M3 and M6. Moreover, it was of equal interest to evaluate whether each of the four test products (a) was different from the control and (b) differed from the other products. Therefore, a number of 10 inter-product comparisons were checked for each clinical parameter on M3 and M6. While Perneger [40] claims that Bonferroni adjustments do not apply in the clinical situation, these corrections are necessary from a mathematical point of view. As a consequence, the tables in the results section indicate both significance levels, without (P=0.05) and with (P=0.0051620) the Bonferroni adjustments.

Fig. 4 Box-plot diagrams of the discoloration indices (DI) of the groups using the different rinses during the investigation

in all groups except the 0.1% CHX group. Due to professional tooth cleaning, the DI was lower at M0 as compared to day 14. Thereafter, the DI increased with time in all test groups (Fig. 4). Results after 3 months (M3) Compared to the control, all test products revealed significantly lower PlI values. These differences were stated as clinically relevant. Concerning the GI, a significant difference was only found between the negative control and 0.1% CHX. Except the 0.06% CHX, all test solutions showed significantly higher DI scores when compared to the control (Table 2). However, from a clinical point of view, regarding the DI, no important differences between the active groups were seen. When the different active groups were compared, both the 0.1% CHX as well as the 0.06% CHX showed significantly less plaque accumulation than the ASF, but the 0.1% resulted in stronger staining than the ASF and the 0.06% CHX (Table 3).

Results
Seventy-three participants finished this long-term study, which represents a dropout of eight volunteers (Table 1). The highest number of dropouts was recorded in the control group followed by the 0.1% CHX. The global non-parametric test revealed no differences between the groups at M0 for the three clinical parameters. In general, PlI scores decreased slightly between day 14 and M0 in all groups, as well as between M0, M1 and M2, respectively, in the active groups (i.e., ASF and CHX groups; Fig. 2). Thereafter, with 0.1% CHX, a relatively stable situation was maintained until M6, while a slight increase was documented in all other groups. A similar trend could be observed for the GI, although the changes were much smaller (Fig. 3). The most distinctive reduction in GI was observed at M3 in all groups. Between M3 and M6 an increase occurred

93 Table 4 Comparison of the test products with the water control at month 6 (M6). n. s. not significant, CHX chlorhexidine, CHX/F chlorhexidine with sodium fluoride, ASF amine fluoride/stannous fluoride. * Significant, i.e. P0.05 in case without Bonferroni correction, P0.0051620 in case with Bonferroni correction With Bonferroni correction

Results after 6 months (M6) Compared to the control, again, all test products differed significantly regarding the PlI and the DI, while concerning the GI, only the 0.1% CHX was different from the control (Table 4). These statistical differences in the oral indices between the control group, on the one hand, and the active groups, on the other hand, were stated as clinically relevant. Comparing the active groups at M6, the 0.1% CHX showed significant differences in the PlI and the GI, when compared to the ASF, and in the GI, when compared to 0.06% CHX/F (Table 5).
n.s. * * * 0.0081 0.0011 0.0017 0.0011 * * * *

Significance levels Medians P-values

Without Bonferroni correction

Discussion
0.38 0.89 1.06 1.02 1.13

DI

General aspects The study design resembled the former investigation of Brecx et al. [9]. However, the study populations differed in various aspects. While the PlI scores on day 14 were very similar (0.72 in [9] versus 0.75 in the present study), the volunteers attending the Brecx et al. [9] study presented with higher GI scores (0.93 versus 0.63) and DI values (0.90 versus 0.38). However, due to the professional tooth cleaning at day 14 and the mechanical oral hygiene, all parameters improved to reach similar values in both populations at the start of the rinsing period, i.e. at M0. In the Canadian participants [9], all these parameters increased again from M1 to M3. In contrast, the PlI and GI further decreased in the present Dresden student population by M3 but increased from M3 to M6. The DI scores increased rather uniformly from M0 to M6. It appears that the German students kept a higher standard of oral hygiene than the Canadian volunteers, who were made up of students and staff. Moreover, a stronger Hawthorne effect [10, 22, 32] occurred during the initial 3 months of the present study. The dropout rate of 10% was mainly caused by the dropout of four subjects from the control group (Table 1) After investigation, it was revealed that some of the participants of the control group did not understand the scientific sense of rinsing with tap water. This points out that water might not be a suitable placebo solution and that the taste and appearance of the placebo should have been similar to those of the experimental solutions. Furthermore, another consequence is that the present study was double-blind due to the active solutions, but could, in fact, only be considered as a single-blind investigation regarding the control. One subject in the control group, however, had to be taken out because of leukoplakia; two others did not show up at the last examination. Another two dropouts were in the 0.1% CHX group, but these cases were not due to any adverse effects. Furthermore, two dropouts occurred because of antibiotic medication during this long experimental time. In their 3-month study, Brecx et al. [9] used a 0.2% CHX solution as a positive control. However, due

Significance levels

With Bonferroni correction Without Bonferroni correction With Bonferroni correction Without Bonferroni correction

P-values

Medians

Significance levels

P-values

Medians

Control ASF 0.06% CHX/F 0.06% CHX 0.10% CHX

Products

0.72 0.29 0.27 0.25 0.13

PlI

0.0150 0.0130 0.0077 0.0007

* * * *

n.s. n.s. n.s. *

0.45 0.36 0.34 0.29 0.15

GI

0.186 0.138 0.080 0.003

n.s. n.s. n.s. *

n.s. n.s. n.s. *

94 Table 5 Comparison of the products at month 6 (M6) a. n. s. not significant, CHX chlorhexidine, CHX/F chlorhexidine with sodium fluoride, ASF amine fluoride/stannous fluoride. * Significant, Parameters Compared products Median values i.e. P0.05 in case without Bonferroni correction, P0.0051620 in case with Bonferroni correction P-values Significance levels Without Bonferroni correction PlI GI
a

With Bonferroni correction n.s. n.s. n.s.

ASF/0.10% CHX ASF/0.10% CHX 0.06% CHX/F / 0.10% CHX

0.29/0.13 0.36/0.15 0.34/0.15

0.040 0.007 0.043

* * *

Only significant differences without Bonferroni correction are listed

to its potential side effects, calculus formation and rate of withdrawal especially at this concentration [32], a commercial 0.1% CHX digluconate product was chosen. As mentioned, no dropout occurred due to side effects of any of the mouthwash preparations. Clinical relevance As mentioned, two kinds of calculations (without and with Bonferroni adjustments) were listed in Tables 2, 3, 4, and 5. Because the statistical outcome without Bonferroni adjustments reflected in general the clinical situation, only these data were referred to in the results section. At M3 and M6 all preparations used as supplements to the mechanical hygiene measures significantly reduced the PlI when compared to the control. All the active products can be defined as plaque-reducing agents [30]. This efficacy of CHX and ASF products has been repeatedly proven [6, 7, 8, 29, 30, 37, 38, 43, 47]. When compared to ASF at M3, both the 0.1% as well as the 0.06% CHX had a superior plaque-reducing effect. Concerning the GI, a significant difference to the negative control was only seen with the 0.1% CHX. Thus, if a further decrease of the GI is wanted, a minimum concentration of 0.1% CHX for long-term use in association with habitual cleaning seems to be necessary. However, concerning a period of 6 months, this should be restricted to specific patients. If maintaining clinical health is the goal, the other solutions are a good alternative to 0.1% CHX because they exhibit less staining. As expected, the amount of staining increased in all groups during the course of the study. Staining is rarely documented during long-term studies [9, 28, 32, 43]. Of the test substances, the 0.1% CHX solution always showed the highest DI scores. It is assumed that a strong anti-plaque/anti-gingivitis solution is associated with strong staining. Dose-dependent anti-plaque effects were proven for the CHX [11, 12], but no data concerning a dose-dependence of staining are available from long-term trials. However, when different mouthrinse preparations were compared, the most potent antiplaque solutions were also the ones that stained the most [9].

Conclusions
Regarding a population with good oral hygiene, the following conclusions can be drawn: All test products led to a clinically relevant reduction of the PlI scores when used as supplements to the usual mechanical hygiene measures. However, concerning the GI, only the 0.1% CHX showed a significant difference compared to the control. If a further decrease of the GI is desired, a minimum concentration of 0.1% CHX for long-term use in association with habitual cleaning is needed. However, this should be limited to specific patients. If maintaining clinically healthy conditions is the goal, the other solutions are a good alternative to 0.1% CHX because they exhibit less staining.
Acknowledgements The authors acknowledge the substantial help of Dr. C. Heumann, Dept. of Statistics, LMU Munich, Germany.

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