SURFACES

ELSEVIER
Colloids and Surfaces B: Biointerfaces 3 (1994) 19-24

Biological activities of different triclosan-detergent

V. Kjaerheim*,
Dentalfaculty,

combinations

SM. Waaler, G. Riilla

University of Oslo, Oslo, Norway

(Received 23 December 1992; accepted 21 December 1993)

Abstract Several previous studies have shown that the nature of detergents and solvents used in conjugation with triclosan is important for its clinical plaque-inhibiting effect. The aim of the present study was to compare the clinical effect of two detergents, sodium lauryl sulphate (SLS) and sodium lauryl sarcosinate (LS), when used as solubilizers of triclosan in mouthrinses, and furthermore to examine if the critical micelle concentration (CMC) of the detergents used in mouthrinses with triclosan had an influence on the clinical effect. The antibacterial activity of the mouthrinses in vitro was also tested. The results of the clinical trials showed that the SLS-containing mouthrinse had a better antiplaque effect than the one containing LS. SLS has a higher CMC than LS. When the CMC of SLS was reduced by the addition of salt, the clinical effect of this mouthrinse was also reduced. A higher CMC thus appeared to be associated with a good clinical effect. It seems that the concentration of monomers in the solution (which are probably associated with triclosan molecules) is significant, whereas the triclosan trapped within the micelles is probably less clinically active. The opposite seemed to apply concerning the antibacterial in vitro effect of detergent-triclosan combinations: a decreased CMC increased the antibacterial effect. It is suggested that the nature of receptor sites and the shear forces are different in the oral cavity and in the in vitro systems and that these factors contribute to the difference. Key words: Chemical plaque inhibition; Clinical study; Critical micelle concentration; Detergents; Triclosan

1. Introduction Triclosan (2,4,4-trichloro-2-hydroxydiphenyl ether), a lipid-soluble antibacterial agent, is added to toothpastes and mouthrinses to reduce the deposition of bacterial plaque on teeth, known to be associated with dental diseases [l-3]. It is necessary to add detergents and/or organic solvents to dental products containing triclosan to solubilize this agent. There are indications that the nature of the detergents or the organic solvents used influence the clinical effects of the triclosan*Corresponding author. Department of Pedodontics and Caries Prophylaxis, Faculty of Dentistry, University of Oslo, Geitmyrsveien 71, N-0455 Oslo, Norway.
0927-7765/94/$07.00 0 1994 Elsevier Science B.V. All rights reserved SSDZ 0927-7765(93)01121-7

containing products: Tween 80, which can solubilize triclosan, reduces its clinical effect, whereas sodium lauryl sulphate (SLS), appears to be well suited as a solubilizing detergent [4]. Triclosan can be dissolved in alkali [S] but this reduced its clinical effect, presumably because the triclosan exhibits a negative charge at high pH, the pK of the hydroxyl group being 7.9. Poly(ethylene glycol) or glycerol are less favourable solvents than propylene glycol 161, whereas oils (olive, soya etc.) totally inhibit the clinical effect of triclosan [ 71. These observations are of clinical interest since toothpastes and mouthrinses contain both organic solvents and oils. The aim of the present study was to compare the clinical effect of SLS and sodium lauryl sarcosi-

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3 (1994) 19-24

nate (LS) as solubilizers for triclosan in mouthrinses. Both of these detergents are used in commercial toothpastes and rinses. The mechanisms involved in the plaque-inhibiting effect of triclosandetergent combinations were studied by reducing the critical micelle concentration (CMC) of the detergents, and its effect on the antiplaque efficacy was examined. The CMC effect was also studied on the antibacterial activity in vitro.

2. Materials and methods

2.1. Clinical studies

Twelve dental students of both sexes volunteered for this study. They were between 22 and 29 years of age and had excellent oral health status with a minimum of 24 teeth without caps or large fillings. Prior to each experimental period, their teeth were professionally cleaned, and scaled when necessary. The participants were asked to rinse with 10 ml of the allocated mouthrinse for 1 min twice daily. To enhance plaque formation the test panel was given sucrose-containing gum to be chewed for 5 min six times a day. No oral home care was to be performed during the test periods which lasted for 4 days. At the end of each period, plaque was recorded according to the Silness and Lije Plaque Index [ 81. This index, which is widely used to assess dental bacterial plaque, consists of four scores from 0 to 3. Score 0 indicates that no plaque is visible either on the tooth surface or on the probe after it has been used to scrape the tooth along the gingival margin. Score 1 indicates that plaque is not visible on the tooth but on the probe, score 2 indicates plaque visible directly on the tooth surface and score 3 is used where there is an abundance of plaque. All surfaces of all teeth except third molars were scored (giving a maximum of 112 scores). The distribution of the different scores was also examined. In experiment 1, two solutions of 0.3% triclosan which was solubilized in aqueous solutions of either 1.5% SLS or 1.5% LS were compared. A positive and a negative control, chlorhexidine and water, respectively, were included. In experiment 2, the plaque-inhibiting effect of a solution of 0.3%

triclosan in 1.5% SLS was compared to the efficacy of the same solution containing 0.6 M NaCl. The salt was added to reduce the CMC of the ionic detergents [9]. All the mouthrinses, except the controls, contained l/S propylene glycol (PG) to enhance the solubility of triclosan. The rinses contained no flavouring component. The two clinical experiments were performed with different test panels, and scored by two different clinicians because the second trial was carried out 1 year after the first. The scoring had, however, been calibrated. These experiments were evaluated individually and not compared for the reasons described above. The study design was cross-over and double blind. Students t-test was applied on the mean of the differences between the paired results from each individual. The significance level was set at a=o.o5. 2.2. Bacteriological tests The phenol coefficient test was chosen as the appropriate technique since triclosan is a bis phenol and is almost insoluble in the bacteriological medium [ 41. Twofold dilutions of the triclosancontaining solutions were prepared. A loopful of a freshly grown culture of Streptococcus sobrinus (omz 176) was exposed to the different dilutions for 40 s and then transferred to a 5% glucose broth. The cultures were incubated for 24 h at 37C and the highest dilution which caused complete inhibition of bacterial growth was recorded. Spot tests on blood agar with bacteria from whole saliva were also performed. Blood agar plates were covered with human whole saliva which had been diluted with three parts of sterile saline. Two millilitres of this mixture were distributed over the surface of a plate and allowed to dry for 1 h at 37 C. Twofold dilutions of the test solutions (drops of 30 ~1) were placed on the dried plates and incubated for 24 h at 37C. The highest dilution causing bacterial inhibition was recorded. 2.3. Determination of detergent CMC The CMC of the mouthrinses was determined according to a modification of a method described

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et al/Colloids

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3 (1994) 19-24

21

by Vulliez-Le Normand and EiselC [lo] which is based on the solubilization of dye by detergents. This appeared to be the method of choice since the lipid-soluble dye can be considered a visible model of the triclosan and its solubilization by the detergents. Sudan black B (certified for use in fat staining) was used as the dye, and the colour was read at 630 nm in a spectrophotometer (Shimadzu UV-240 spectrophotometer, Shimadzu, Kyoto, Japan). One milligram of Sudan black B was placed in each glass test-tube and 3 ml of a serial dilution of SLS or LS with and without propylene glycol, and with and without 0.6 M NaCl were tested. The tubes were shaken at room temperature and the results were recorded after 24 h. After the incubation period the test-tubes were centrifuged to eliminate excess dye. The experiments were repeated at least twice and the results were found to be highly reproducible. (Sodium lauryl sulphate was purchased from BDH Biochemical, Kebo Lab., UK, sodium lauryl sarcosinate from Sigma Chemical Comp., St. Louis, USA, propylene glycol from Fluka Chemia AG, Switzerland, chlorhexidine from ICI, Macclesfield, UK, and Sudan black B from Allied Chemical Corp., New York, USA.)

3. Results The results of the first clinical trial are shown in Table 1. It can be seen that the SLS-containing

Table 1 Mean plaque indices and 0 scores [8] Rinse

n 5 Plaque

0 score

index Water 0.3% triclosan, 1.5% SLS 0.3% triclosan, 1.5% LS 0.2% chlorhexidine 12 12 12 12 1.42 k 0.18 0.86 k 0.32 1.09 _+0.30 0.15 + 0.11 11.7 + 4.87 38.0 f 16.91 23.4 _+13.05 94.4 f 13.53

The values, which are given with were obtained after rinsing with a positive control (0.2% chlorhexidine) ing 0.3% triclosan and a detergent. a Signiticantly different from the (p<O.O5).

their standard deviations, negative control (water), a and two solutions containLS-containing mouthrinse

mouthrinse was significantly more effective than the LS rinse (p = 0.03 12). When the distribution of individual scores in the same groups was compared, a significant difference between the number of 0 scores of the two test solutions was found (p=O.O089), SLS again being better than LS. The control and the chlorhexidine rinses showed values comparable with previous results [ 111. The CMC of the mouthrinsing solutions is shown in Figs. 1(A) and l(B). It can be seen that the CMC of SLS in this system was 0.2% (Fig. 1(a)) whereas the CMC of LS was 0.1% (Fig. 1(b)). No micelle formation was found in chlorhexidine solutions, presumably due to the lack of head-andtail structure in this molecule. The presence of propylene glycol did not change the CMC in any of the detergents, but increased the solubility of the hydrophobic dye (and presumably also that of triclosan). The addition of salt (0.6 M NaCl), however, reduced the CMC of both detergents markedly, as expected [9]. The first clinical experiment (Table 1) demonstrated that SLS-triclosan was superior to LS-triclosan as a plaque inhibitor. Since SLS has a higher CMC than LS, a second experiment was designed to examine the significance of CMC as such on the plaque-inhibiting efficacy. This second clinical experiment compared a mouthrinse containing SLS with a mouthrinse containing SLS and 0.6 M NaCl. It was found that the NaClcontaining mouthrinse (i.e. with a lower CMC) was clinically less effective than the mouthrinse without NaCl, indicated by a higher mean plaque index, although only a borderline statistical significance was found (p = 0.0589) (Table 2). However, the number of 0 scores was significantly lower in the rinse containing 0.6 M NaCl (p = 0.0304). Both clinical experiments thus indicate an association between high CMC and good clinical effect. The results from the phenol coefficient test and the spot test on blood agar are shown in Table 3. It can be seen that the LS- containing triclosan solution was effective at a l/S dilution and the SLS-containing solution at a l/4 dilution in the first test. The spot test with salivary bacteria from whole saliva showed no difference between SLS and LS. The addition of 0.6 M NaCl increased the antibacterial activity of both solutions in both

V. Kjcerheim er al/Colloids Surfaces B: Biointerfaces 3 (1994) 19-24

OD SLS + 0.6 M NaCl

* , 0.8 0.8

,= SLS + PG ,I *

systems, indicating that a decreased CMC enhanced the antibacterial effect of the triclosandetergent combination.

4. Discussion The first clinical experiment (Table 1) showed that the solubilization of triclosan with SLS gave a significantly better clinical result than when triclosan was solubilized with LS. Both the mean plaque score and the number of 0 scores were significantly better in the SLS group. The 0 score is presumably a particularly important parameter, because the distinction between this score and score 1 is more reliable than between the other scores, owing to its clear definition (no plaque). This allows a high reproducibility of the 0 scores, and this score, indicating clean tooth surfaces, can also be directly related to dental health (no caries or gingivitis without plaque). These data and the data concerning the CMC of the detergents used in the mouthrinses (Figs. l(a) and 1(b)) were the basis for the speculation that a higher CMC may be associated with a better clinical effect. This hypothesis was examined in the second clinical experiment where the CMC of SLS was reduced by the addition of 0.6 M NaCl. This solution was compared to a SLS solution without the addition of salt. It was found that the lowering of the CMC reduced the clinical effect. The clinical effect of combinations of detergents and triclosan thus seems to be associated with the CMC of the detergents in both clinical experiments: sodium lauryl sulphate has a higher CMC than sodium lauroyl sarcosinate and SLS also has a higher CMC than SLS + 0.6 M NaCl (Fig. 1). The presence of small amounts of propylene glycol in the solutions did not appear to affect the CMC of the detergents (Fig. l), and since the amount was similar in all the tests, this factor is probably of no significance. The clinical effect of the triclosan-detergent combinations may thus be directly related to the concentration of detergent monomers in the combinations. It is conceivable that triclosan binds to the hydrocarbon tail of these monomers (or dimers or trimers) and provides clinically active forms of triclosan-detergent

CONC.
(4

OD
1.2 1.0 0.8 0.8 0.4

LS + 0.6 M NaCl ,
, **

p LS+PG

,* # LS

8 , *

ii
0.2

llrJ I
I I I I

0.1

0.2

0.3

0.4

0.5

(b)

CONC.

Fig. l.(a) CMC determinations for three SLS-containing solutions. The optical density was read at 630 nm; the concentrations are given in per cent (w/v). (b) CMC determinations for three LS-containing solutions. The optical density was read at 630 nm; the concentrations are given in per cent (w/v).

V. Kjarrheim et alfColloids

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3 (1994) 19-24

23

Table 2 Mean plaque with 0 scores after rinsing with two triclosar-SLS Rinse n

mouthrinses with different CMCs X Plaque index 1.07 k 0.32 1.23+ 0.23 0 score

0.3% triclosan, 1.5% SLS 0.3% triclosan, 1.5% SLS, 0.6 M NaCl Values are given with their standard deviations. p=O.O589. b Significantly different from the control (p<O.O5).

10 9

34.3 + 14.84 24.2b k 12.23

Table 3 Antibacterial effect of triclosan solutions containing SLS and LS Solution tested Reciprocal titre

Phenol Water, Water, Water, Water,

coefficient test PG, 0.3% triclosan, PG, 0.3% triclosan, PG, 0.3% triclosan, PG, 0.3% triclosan,

1.5% SLS 1.5% LS 1.5% SLS, 0.6 M NaCl 1.5% LS, 0.6 M NaCl

4 8 16 16

Spot test Water, PG, Water, PG, Water, PG, Water, PG,

0.3% 0.3% 0.3% 0.3%

triclosan, triclosan, triclosan, triclosan,

1.5% SLS 1.5% LS 1.5% SLS, 0.6 M NaCl 1.5% LS, 0.6 M NaCl

8 8 32 32

Propylene glycol.

molecules in the mouthrinses. Triclosan trapped inside the detergent micelles may thus be less available for biological effect in the oral cavity for the short period of rinsing (1 min) during which the oral surfaces are exposed to the rinsing solution before it is expectorated. Triclosan may be transferred from the detergent monomers to the oral receptor sites directly, or the detergents may bind to the oral surfaces and triclosan molecules could be retained through the adsorbed detergent monomers. It is well established that SLS molecules bind to the oral surfaces for a considerable time [ 121. The retention of the monomers in the oral cavity is probably due to a combination of hydrophilic and hydrophobic forces between the detergent and the oral surfaces. The retention of micelles is mainly ionic and thus less strong. The smaller detergent monomers are more likely to be exposed to lower shear forces in the oral cavity than the 80 times larger micelles [ 91. This effect may contribute to

a higher retention of detergent monomers. The concept concerning the significance of the monomers rather than the micelles for the clinical effect of triclosandetergent combinations is new, since previous studies have indicated the importance of the micelles for the clinical effect of such combinations [4, 131. The significance of CMC in triclosan-detergent-containing mouthrinses is supported by the observed negligible clinical effect of triclosan in Tween 80. Tween 80 is an uncharged detergent with a low CMC compared to that of the ionic detergents. Furthermore, it was shown in a previous study that a reduced concentration of triclosan did not reduce the clinical effect if the SLS concentration was unchanged [4]. In light of the results from the present study, this could then be explained by the fact that the amount of detergent monomers would be the same. Chlorhexidine solutions have a superior effect as plaque inhibitors. This supports the significance of a high CMC indirectly, since chlorhexidine molecules do not form conventional micelles. This is due to the nature of the chlorhexidine molecule which consists of two heads with a hydrocarbon bridge between them, rather than a head-and-tail configuration. Chlorhexidine molecules are thus always present in mouthrinses as monomers, whereas the rinses with other cationic detergents, to which chlorhexidine has been compared, contain micelles. Another conceivable mechanism by which a high CMC detergent may be superior to a detergent with lower CMC is as follows. The micellar solution with a high CMC is diluted in the oral cavity while rinsing. The concentration thus drops below the CMC, and triclosan is released and deposited on the oral mucosal surfaces. For the low CMC

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V. KjErheim et al/Colloids Surfaces B: Biointerfaces 3 (1994) 19-24

systems the dilution does not bring the concentration below the CMC; all the micelles are still present, retaining their complete triclosan cargo. Thus very little triclosan is deposited on the oral mucosa and the main part is expectorated with the SLS micelles. The significance of a high CMC for a good clinical effect has some experimental support, but which of the two mechanisms outlined above is the most important is undecided. The bacteriological tests showed that the antibacterial activity of SLS- or LS-triclosan combinations was inversely related to the CMC of the detergents used to solubilize triclosan. A low CMC gave a high antibacterial effect. This was in contrast to the clinical results where a low CMC gave a reduced effect, as discussed above. The mechanisms involved in these two processes thus appear to be different. The bacteria in vitro are probably less protected against chemical traumata than the plaque bacteria. In vitro, bacteria are present as single bacteria or as small chains, whereas in plaque they constitute a multilayered biofilm which may be further protected by a coverage of polysaccharides and salivary protein present in plaque. The nature of the receptor sites in the oral cavity and on bacterial surfaces may well also be different. It has been suggested previously that detergent micelles adsorbed to bacterial surfaces are able to transfer antibacterial agents which reside inside the micelles to the bacteria [14]. However, studies indicate that a lowered CMC would lower the antibacterial activity of antibacterial agents [ 15-J. It appears likely that the enhancement or reduction of the effect of water-soluble or water-insoluble antibacterial agents by detergents depends on the nature of the ingredients, and that it may differ from case to case.

The concept presented above suggests an association between high CMC of the solubilizing detergent and good clinical effect of triclosan. If this concept is correct, an antiplaque product for clinical use should include a detergent with a high CMC. The detergent should presumably be present in concentrations close to the CMC and the addition of inorganic salts should be kept to a minimum.

5. References Cl1 C.A. Saxton, J. Periodontol., 57 (9) (1986) 555. PI B. Svatun, C.A. Saxton, G. Riilla and F. van der Ouderaa,
Stand. J. Dent. Res., 97 (1989) 242. c31 P. Gjermo and C.A. Saxton, J. Clin. Periodontol., 18 (1991) 468. c41 S.M. Waaler, G. Ralla, K.K. Skji)rland and B. Ogaard, Stand. J. Dent. Res., 101 (1993) 192. c51 S. Jenkins, M. Addy and R. Newcombe, J. Clin. Periodontal., 39 (1991) 145. C61V. Kjaerheim and S.M. Waaler, Adv. Dent. Res., 8( 1) 1994. c71 V. Kjrerheim, S.M. Waaler and G. Rolla, Stand. J. Dent. Res., 102 (1994). C81 J. Silness and H. Lae, Acta Odont. Stand., 22 (1964) 121. c91 P.C. Hieminz, Principles of Colloid and Surface Chemistry, Marcel Dekker, New York, 1986, p. 432. Cl01 B. Vulliez-Le Normand and J.-L. Eiselt, Anal. Biochem., 208 (1993) 241. Cl11 E. Giertsen, A.Aa. Scheie and G. RUa, Stand J. Dent. Res., 96 (1988) 541. Cl21 P. Barkvoll, G. Riilla and A.K. Svendsen, J. Clin. Periodontol., 16 (1989) 593. Cl31 S.M. Waaler, G. Rblla and V. Kjrerheim, Stand. J. Dent. Res., 102 (1994) 46. Cl41 H.S. Bean and H. Berry, J. Pharm. Pharmacol., 3 (1951) 639. Cl51 A. Agar and A.E. Alexander, Trans. Faraday Sot., 45 (1949) 528.