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

Biological activities of different triclosan-detergent combinationsV. Kjaerheim*, SM. Waaler, G. Riilla

Dentalfaculty, 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 triclosanis important for its clinical plaque-inhibiting effect. The aim of the present study was to compare the clinical effect oftwo detergents, sodium lauryl sulphate (SLS) and sodium lauryl sarcosinate (LS), when used as solubilizers of triclosanin mouthrinses, and furthermore to examine if the critical micelle concentration (CMC) of the detergents used inmouthrinses with triclosan had an influence on the clinical effect. The antibacterial activity of the mouthrinses in vitrowas also tested.

The results of the clinical trials showed that the SLS-containing mouthrinse had a better antiplaque effect than theone containing LS. SLS has a higher CMC than LS. When the CMC of SLS was reduced by the addition of salt, theclinical effect of this mouthrinse was also reduced. A higher CMC thus appeared to be associated with a good clinicaleffect. It seems that the concentration of monomers in the solution (which are probably associated with triclosanmolecules) is significant, whereas the triclosan trapped within the micelles is probably less clinically active. Theopposite seemed to apply concerning the antibacterial in vitro effect of detergent-triclosan combinations: a decreasedCMC increased the antibacterial effect. It is suggested that the nature of receptor sites and the shear forces are differentin 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-hydroxydiphenylether), a lipid-soluble antibacterial agent, is addedto toothpastes and mouthrinses to reduce thedeposition of bacterial plaque on teeth, known tobe associated with dental diseases [l-3]. It isnecessary to add detergents and/or organicsolvents to dental products containing triclosan tosolubilize this agent. There are indications that thenature of the detergents or the organic solventsused influence the clinical effects of the triclosan-*Corresponding author. Department of Pedodontics and CariesProphylaxis, Faculty of Dentistry, University of Oslo,Geitmyrsveien 71, N-0455 Oslo, Norway.

containing products: Tween 80, which can solubi-lize triclosan, reduces its clinical effect, whereassodium lauryl sulphate (SLS), appears to be wellsuited as a solubilizing detergent [4]. Triclosancan be dissolved in alkali [S] but this reduced itsclinical effect, presumably because the triclosanexhibits a negative charge at high pH, the pK ofthe hydroxyl group being 7.9. Poly(ethylene glycol)or glycerol are less favourable solvents than propyl-ene glycol 161, whereas oils (olive, soya etc.) totallyinhibit the clinical effect of triclosan [ 71. Theseobservations are of clinical interest since tooth-pastes and mouthrinses contain both organicsolvents and oils.

The aim of the present study was to comparethe clinical effect of SLS and sodium lauryl sarcosi-

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20 V. Kjcerheim et al/Cl&ids Surfaces B: Biointerfaces 3 (1994) 19-24

nate (LS) as solubilizers for triclosan inmouthrinses. Both of these detergents are used incommercial toothpastes and rinses. The mecha-nisms involved in the plaque-inhibiting effect oftriclosandetergent combinations were studied byreducing the critical micelle concentration (CMC)of the detergents, and its effect on the antiplaqueefficacy was examined. The CMC effect was alsostudied on the antibacterial activity in vitro.

2. Materials and methods2.1. Cli ni cal stud ies

Twelve dental students of both sexes volunteeredfor this study. They were between 22 and 29 yearsof age and had excellent oral health status with aminimum of 24 teeth without caps or large fillings.Prior to each experimental period, their teeth wereprofessionally cleaned, and scaled when necessary.The participants were asked to rinse with 10 ml ofthe allocated mouthrinse for 1 min twice daily.

To enhance plaque formation the test panel wasgiven sucrose-containing gum to be chewed for5 min six times a day. No oral home care was tobe performed during the test periods which lastedfor 4 days. At the end of each period, plaque wasrecorded according to the Silness and Lije PlaqueIndex [ 81. This index, which is widely used toassess dental bacterial plaque, consists of fourscores from 0 to 3. Score 0 indicates that no plaqueis visible either on the tooth surface or on theprobe after it has been used to scrape the toothalong the gingival margin. Score 1 indicates thatplaque is not visible on the tooth but on the probe,score 2 indicates plaque visible directly on thetooth surface and score 3 is used where there is anabundance of plaque. All surfaces of all teeth exceptthird molars were scored (giving a maximum of112 scores). The distribution of the different scoreswas also examined.

In experiment 1, two solutions of 0.3% triclosanwhich was solubilized in aqueous solutions ofeither 1.5% SLS or 1.5% LS were compared. Apositive and a negative control, chlorhexidine andwater, 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 efficacyof the same solution containing 0.6 M NaCl. Thesalt was added to reduce the CMC of the ionicdetergents [9]. All the mouthrinses, except thecontrols, contained l/S propylene glycol (PG) toenhance the solubility of triclosan. The rinses con-tained no flavouring component.The two clinical experiments were performedwith different test panels, and scored by twodifferent clinicians because the second trial wascarried out 1 year after the first. The scoring had,however, been calibrated. These experiments wereevaluated individually and not compared for thereasons described above.

The study design was cross-over and doubleblind. Students t-test was applied on the mean ofthe differences between the paired results fromeach individual. The significance level was set ata=o.o5.2.2. Bacteri ological tests

The phenol coefficient test was chosen as theappropriate technique since triclosan is a bisphenol and is almost insoluble in the bacteriologi-cal medium [ 41. Twofold dilutions of the triclosan-containing solutions were prepared. A loopful of afreshly grown culture of Streptococcus sobrinus(omz 176) was exposed to the different dilutionsfor 40 s and then transferred to a 5% glucose broth.The cultures were incubated for 24 h at 37C andthe highest dilution which caused complete inhibi-tion of bacterial growth was recorded.

Spot tests on blood agar with bacteria fromwhole saliva were also performed. Blood agarplates were covered with human whole saliva whichhad been diluted with three parts of sterile saline.Two millilitres of this mixture were distributedover the surface of a plate and allowed to dry for1 h at 37 C. Twofold dilutions of the test solutions(drops of 30 ~1) were placed on the dried platesand incubated for 24 h at 37C. The highest dilu-tion causing bacterial inhibition was recorded.2.3. Determi nat i on of detergent CMC

The CMC of the mouthrinses was determinedaccording to a modification of a method described


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V. Kjwheim et al/Colloids Surfaces B: Biointerfaces 3 (1994) 19-24 21by Vulliez-Le Normand and EiselC [lo] which isbased on the solubilization of dye by detergents.This appeared to be the method of choice sincethe lipid-soluble dye can be considered a visiblemodel of the triclosan and its solubilization by thedetergents. Sudan black B (certified for use in fatstaining) was used as the dye, and the colour wasread at 630 nm in a spectrophotometer (ShimadzuUV-240 spectrophotometer, Shimadzu, Kyoto,Japan). One milligram of Sudan black B wasplaced in each glass test-tube and 3 ml of a serialdilution of SLS or LS with and without propyleneglycol, and with and without 0.6 M NaCl weretested. The tubes were shaken at room temperatureand the results were recorded after 24 h. After theincubation period the test-tubes were centrifugedto eliminate excess dye. The experiments wererepeated at least twice and the results were foundto be highly reproducible. (Sodium lauryl sulphatewas purchased from BDH Biochemical, Kebo Lab.,UK, sodium lauryl sarcosinate from SigmaChemical Comp., St. Louis, USA, propylene glycolfrom Fluka Chemia AG, Switzerland, chlorhexi-dine from ICI, Macclesfield, UK, and Sudanblack B from Allied Chemical Corp., New York,USA.)

3. ResultsThe results of the first clinical trial are shown inTable 1. It can be seen that the SLS-containing

Table 1Mean plaque indices and 0 scores [8]Rinse n 5 Plaque 0 scoreindex

Water 12 1.42 k 0.18 11.7 + 4.870.3% triclosan, 1.5% SLS 12 0.86 k 0.32 38.0 f 16.910.3% triclosan, 1.5% LS 12 1.09 _+0.30 23.4 _+13.050.2% chlorhexidine 12 0.15 + 0.11 94.4 f 13.53The values, which are given with their standard deviations,were obtained after rinsing with a negative control (water), apositive control (0.2% chlorhexidine) and two solutions contain-ing 0.3% triclosan and a detergent.a Signiticantly different from the LS-containing mouthrinse(p


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V. Kjcerheim er al/ Coll oi ds Surf aces B: Bi oint erfaces 3 (1994) 19-24

ODSLS + 0.6 M NaCl ,= SLS + PG

,I**,0. 8

0. 8

(4

OD1. 2 -

1. 0 - I LS + 0.6 M NaCl p LS+PG,,*#0. 8 -

0. 8 -

,** LS8*

ii /

,0. 4

0

CONC.

0. 2l l r J

(b)

II I I I I0. 1 0. 2 0. 3 0. 4 0. 5

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

systems, indicating that a decreased CMCenhanced the antibacterial effect of the triclosan-detergent combination.

4. DiscussionThe first clinical experiment (Table 1) showed

that the solubilization of triclosan with SLS gavea significantly better clinical result than whentriclosan was solubilized with LS. Both the meanplaque score and the number of 0 scores weresignificantly better in the SLS group. The 0 scoreis presumably a particularly important parameter,because the distinction between this score andscore 1 is more reliable than between the otherscores, owing to its clear definition (no plaque).This allows a high reproducibility of the 0 scores,and this score, indicating clean tooth surfaces, canalso be directly related to dental health (no cariesor gingivitis without plaque).These data and the data concerning the CMCof the detergents used in the mouthrinses (Figs.l(a) and 1 (b)) were the basis for the speculationthat a higher CMC may be associated with a betterclinical effect. This hypothesis was examined in thesecond clinical experiment where the CMC of SLSwas reduced by the addition of 0.6 M NaCl. Thissolution was compared to a SLS solution withoutthe addition of salt. It was found that the loweringof the CMC reduced the clinical effect. The clinicaleffect of combinations of detergents and triclosanthus seems to be associated with the CMC of thedetergents in both clinical experiments: sodiumlauryl sulphate has a higher CMC than sodiumlauroyl sarcosinate and SLS also has a higherCMC than SLS + 0.6 M NaCl (Fig. 1).

The presence of small amounts of propyleneglycol in the solutions did not appear to affect theCMC of the detergents (Fig. l), and since theamount was similar in all the tests, this factor isprobably of no significance. The clinical effect ofthe triclosan-detergent combinations may thus bedirectly related to the concentration of detergentmonomers in the combinations. It is conceivablethat triclosan binds to the hydrocarbon tail ofthese monomers (or dimers or trimers) and pro-vides clinically active forms of triclosan-detergent


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Table 2Mean plaque with 0 scores after rinsing with two triclosar-SLS mouthrinses with different CMCsRinse n X Plaque 0 score

index

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0.3% triclosan, 1.5% SLS 10 1.07 k 0.32 34.3 + 14.840.3% triclosan, 1.5% SLS, 0.6 M NaCl 9 1.23+ 0.23 24.2b k 12.23Values are given with their standard deviations.p=O.O589.b Significantly different from the control (p


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24 V. KjErheim et al/Colloids Surfaces B: Biointerfaces 3 (1994) 19-24systems the dilution does not bring the concen-tration below the CMC; all the micelles are stillpresent, retaining their complete triclosan cargo.Thus very little triclosan is deposited on the oralmucosa and the main part is expectorated with theSLS micelles.The significance of a high CMC for a goodclinical effect has some experimental support, butwhich of the two mechanisms outlined above isthe most important is undecided.

The bacteriological tests showed that the anti-bacterial activity of SLS- or LS-triclosan combi-nations was inversely related to the CMC of thedetergents used to solubilize triclosan. A low CMCgave a high antibacterial effect. This was in contrastto the clinical results where a low CMC gave areduced effect, as discussed above. The mechanismsinvolved in these two processes thus appear to bedifferent. The bacteria in vitro are probably lessprotected against chemical traumata than theplaque bacteria. In vitro, bacteria are present assingle bacteria or as small chains, whereas inplaque they constitute a multilayered biofilm whichmay be further protected by a coverage of poly-saccharides and salivary protein present in plaque.The nature of the receptor sites in the oral cavityand on bacterial surfaces may well also be different.

It has been suggested previously that detergentmicelles adsorbed to bacterial surfaces are able totransfer antibacterial agents which reside inside themicelles to the bacteria [14]. However, studiesindicate that a lowered CMC would lower theantibacterial activity of antibacterial agents [ 15-J.It appears likely that the enhancement or reductionof the effect of water-soluble or water-insolubleantibacterial agents by detergents depends on thenature of the ingredients, and that it may differfrom case to case.

The concept presented above suggests an associ-ation between high CMC of the solubilizingdetergent and good clinical effect of triclosan. Ifthis concept is correct, an antiplaque product forclinical use should include a detergent with a highCMC. The detergent should presumably be presentin concentrations close to the CMC and the addi-tion of inorganic salts should be kept to aminimum.

5. References

Cl1PIc31c41c51C61c71C81c91

Cl01Cl11Cl21Cl31Cl41Cl51

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

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