WO2000053150A1

WO2000053150A1 - Compositions and method for the disinfection of dental root canals

Info

Publication number: WO2000053150A1
Application number: PCT/FI2000/000188
Authority: WO
Inventors: Markus Haapasalo; Eva Siren; Dag ØRSTAVIK; Tuomas Waltimo
Original assignee: Markus Haapasalo; Eva Siren; Oerstavik Dag; Tuomas Waltimo
Priority date: 1999-03-11
Filing date: 2000-03-10
Publication date: 2000-09-14
Also published as: FI990529A; AU3294100A; SE0102999L; SE523704C2; SE0102999D0; FI990529A0

Abstract

A method and compositions for the antimicrobial treatment of dental root canals are disclosed. Benefits are achieved by combining calcium hydroxide with iodine-potassium iodide, chlorhexidine or mixtures of these. The antibacterial effect of iodine-potassium iodide and/or chlorhexidine in compositions with calcium hydroxide has been shown to be an advantage in the treatment of dental root canal infections either in primary or retreatment cases. The compositions according to the invention may be applied at the site of treatment in suitable, pharmaceutically acceptable carriers, in the form of solutions, pastes or gels. They may also be included in a matrix of guttapercha or a corresponding material, as used e.g. for the medication of a root canal in the course of endodontic treatment.

Description

Compositions and method for the disinfection of dental root canals

Introduction Micro-organisms and their products are major etiologic factors in pulpal and periapical disease (1-3). Today, more than 300 bacterial species are recognised as normal inhabitants of the oral cavity (4). However, when infected root canals are sampled they contain only a limited assortment of the total available oral flora (3-7). The infection is usually polymicrobial and is dominated by anaerobic bacteria that use tissue remnants and serum proteins as a nutritional supply. The endodontic treatment of teeth with periapical lesions must therefore be directed at the elimination of bacteria and any sources of nutrient supply. Several studies have shown a higher success rate in cases where the canal is free from bacteria when it is obturated (8-10). Treatment strategies that are designed to eliminate this microflora must include agents that can effectively disinfect the root canal. In vitro and in vivo studies have shown that most bacteria isolated from infected root canals are susceptible to calcium hydroxide (11-14). When the ecological conditions in the root canal change during treatment, only those microbes survive, that can tolerate alkalinity, lack of nutrients and increased oxygen level. In these rare cases the root canal infection changes from a polymicrobial anaerobic towards a facultative flora and monoinfections occur more frequently. Enterococcus faecalis has been reported to survive an alkaline environment (11,15,17) and it is the species most often connected to persistent endodontic infections (18- 21).

N variety of antimicrobial agents have been tested for their ability to eradicate calcium hydroxide resistant micro-organisms, especially E. faecalis, from root canal and dentinal tubules. Medicaments such as camphorated paramonochlorophenol, camphorated phenol, combinations of steroids and antibacterial agents and irrigants such as iodine potassium iodide (IKI), chlorhexidine (CHX) and sodium hypochlorite have been tested (11,14-17,22- 25). In these in vitro studies the phenol compounds, IKI and CHX have been shown to kill E. faecalis in the dentinal tubules more effectively than calcium hydroxide (14,17,23,24). The shortcoming of many intracanal medicaments in comparison with calcium hydroxide, however, is a relatively short lasting antimicrobial action and the toxicity associated with phenol based medicaments (26-29). Chlorhexidine, which was syntethized in search of antimalarial drugs, is bactericidal in topical use. Chlorhexidine is effective in vitro against a wide range of both gram-negative and gram-positive bacteria (30,31). It is also fungicide (30-32). The mode of action of chlorhexidine is by adsorption onto bacterial cell surface and reaction with negatively charged groups on the cell surface, causing a reduction of the surface charge (33). Chlorhexidine seems to have a low level of both local and systemic toxicity (34). Clinical side effects noted after oral use have been bitter taste and discoloration of teeth and fillings. Iodine potassium iodide has long been used as an intracanal antimicrobial agent in endodontics. Iodine potassium iodide is effective against a variety of micro-organisms found in root canals and 2 % solution is least toxic to tissue culture cells compared with intracanal medicaments other than calcium hydroxide (35-38). Iodine is a strong oxidising agent, it reacts with free sulphydryl groups of bacterial enzymes, resulting in disulphide linkages (39). The major disadvantage to the use of iodine solutions is that occasionally patients may have allergic reactions. While in vivo studies have indicated calcium hydroxide to be the most effective all purpose intracanal medicament (11), IKI and CHX may be able to kill calcium hydroxide resistant bacteria, at least in vitro (14,17,23-26). Supplementing the antibacterial activity of calcium hydroxide with IKI or CHX preparations may therefore be a way to improve intracanal medicament efficacy. The aim of this in vitro study was to measure the antibacterial effect of combinations of calcium hydroxide with IKI or CHX against E. faecalis and to evaluate the cytotoxicity of the combinations as compared to their components alone. As alkalinity is considered important to the antimicrobial effect of calcium hydroxide in the root canal, the effect of IKI and CHX on the pH in combinations was also studied. Several studies have shown a higher success rate in cases where the canal is free from bacteria when it is obturated. Treatment strategies that are designed to eliminate this micro flora must include agents that can effectively disinfect the root canal. Enterococcus faecalis has been reported to survive an alkaline environment and it is the species most often connected to persistent endodontic infections. While in vivo studies have indicated calcium hydroxide to be the most effective all purpose intracanal medicament, iodine potassium iodide and chlorhexidine may be able to kill calcium hydroxide resistant bacteria. Supplementing the antibacterial activity of calcium hydroxide with iodine potassium iodide or chlorhexidine preparations was studied in bovine dentin blocks. The results suggest that calcium hydroxide does not inhibit the disinfecting effect of iodine potassium iodide and chlorhexidine. As the addition of chlorhexidine or iodine potassium iodide did not effect the alkalinity of the products it can be assumed that combinations have potential to be used as long term medication as well. Cytotoxicity tests with neutral red method indicate that the mixtures presented in this study are no more toxic than their pure components.

Material and methods Antibacterial effect Dentine test specimens: Freshly extracted bovine incisors were used for the experiment. The teeth were kept in 0.5 % NaOCl for surface disinfection overnight. The apical 1-2 mm and the crown were removed and the root dentin machined into cylindrical test pieces, approximately 4 mm high and 6 mm wide with a central pulpal lumen diameter of 2.1 mm. The root cementum was not removed. Organic and inorganic debris including smear layer were removed by ultrasonic treatment in 17 % EDTA for 10 min. The test pieces were then sterilised by autoclaving (121 °C, 15 min) in Tryptone Soya Broth (TSB, 30 g/L Oxoid Ltd. Basinstoke, England). Details of the procedure have been described previously by Haapasalo and 0rstavik (15). Infection of the dentin specimens: A clinical strain of Enterococcus faecalis ( A 197 A) was used as a test organism. The strain was isolated from a persistent root canal infection and identified in the Microbiological Service Laboratory in University of Helsinki (API 20 E test kit, BioMerieaux sa, Marcy/Etoile, France). For infection, four test blocks were kept in each test tube with 10 ml TSB in 37° C. The broth was changed at 2-3 days intervals and infection continued for two weeks. Strict asepsis was used to avoid contamination and the purity of the cultures was checked regularly by Gram staining and Bile-Esculin test agar (Bile Esculin Agar 64 g/L, Difco Laboratories, Detroit, MI, USA).

Medicaments: The following root canal medicaments were tested: 2. chlorhexidine acetate (CHX) 0.5 % in water (Ulleval apotek, Oslo, Norway) 3. iodine (2 %) in potassium iodide (4 %) water solution (IKI) (Yliopiston apteekki, Helsinki, Finland) 4. calcium hydroxide (Riedel-De Haen AG, Seelze, Germany) in distilled water (lg/ml). 5. calcium hydroxide in IKI (lg/ml)

6. calcium hydroxide in CHX (lg/ml).

In addition, calcium carbonate (CaCO₃, Merck AG, Darmstadt, Germany) mixed with distilled water (lg/ml) was used as negative control.

The solubility of chlorhexidine gluconate in water is substantially greater than that of the acetate. Chlorhexidine gluconate may be used instead of or in combination with chlorhexidine acetate. Preferably, solutions with concentrations in the range of about 0.01 to about 5 % may be used; more preferably, concentrations in the range of about 0,5 to about 2

%.

Iodine-potassium iodide solutions of other concentrations than those indicated may be used as well; solutions of 2, 4, 5 and 10 % are readily available.

The solubility of calcium hydroxide in water is low; the amount used thus serves to provide a saturated environment.

Procedures for testing the medicaments for antibacterial activity:

Dentin test specimen infected with E. faecalis were blotted dry from the culture media and mounted with epoxy glue to the bottom of tissue culture dishes. The glue was tested and found not to impair the growth of E. faecalis. The medicaments were applied to the pulpal lumen, filling the lumen and covering the top surface of the specimen. The specimens were incubated in air at 37° C for periods of 1 and 7 days. Drying of the specimens during the incubation was avoided by keeping the culture dishes in 100 % humidity. The bacteriological samples were taken by shaving the dentine inside the lumen with round burs ranging in size from ISO 023 to 040 (Table 1). The method used in bacteriological sampling allowed for a sequential removal of 100 μm thick zones of dentin from the central canal towards the periphery. CaCO₃ control specimens were uniformly infected and yielded growth in bur samples at every depth. After sampling, the residual block was also cultivated to check for growth outside the sampling area. The dentine powder and residual blocks were collected in separate test-tubes containing TSB medium and incubated up to 5 days to detect bacterial growth. The results were confirmed by culturing 100 μl from each test-tube in Tryptone Soya agar plates (TSB 30 g/L and Bacteriological Agar 15 g/L, Oxoid Ltd. Basinstoke, England). When growth occurred, the purity of the cultures was tested as described above.

In vitro cytotoxicity test The neutral red assay was used to test the cytotoxicity of the medicaments. Neutral red test can be used with a variety of cell types in culture, to provide quantitative data that can be used to rank test agents according to their cytotoxic potential (40,41). Mouse fibroblasts (l,5xl0⁵ cells/well), suspended in 1 ml Minimum Essential medium (MEM) containing 5% fetal bovine serum and 2 mM L-glutamine (MEM, L-glutamine and foetal bovine serum from Life Technologies Ltd., Scotland, UK) were seeded into individual wells of 24-well microtitre tissue culture plates, to achieve 60-70% confluence at the time of the addition of the test agents. The plates were incubated at 37° C for 24 hours. Dilutions of test agents were added to the wells and incubated for 24 hours. The test agents were: 1. calcium hydroxide saturated distilled water (10, 50, 100, 200 and 300 μl)

2. CHX undiluted 10 μl, 1:10 dilution 10,20,50 μl, 1 :100 dilution lOμl

3. IKI undiluted 10 μl, 1:10 dilution 10, 20, 50 μl, 1:100 dilution lOμl

4. CHX saturated with calcium hydroxide(10, 50, 100, 200 and 300 μl)

5. IKI saturated with calcium hydroxide (10, 50, 100, 200 and 300 μl) 6. MEM only, as control

A 0.4% aqueous stock solution of Neutral red dye was prepared. MEM containing penicillin (100 U/ml, Life Technologies Ltd.) and streptomycin (100 μg/ml, Life Technologies Ltd.) was added to the dye to give a final concentration of 50 μg/ml. The neutral red medium was incubated at 37 °C for 24 hours, then centrifuged for 10 min at 1650 G to remove fine precipitate and dye crystals, formed when neutral red is mixed with medium. Medium and test agents were aspirated from all wells and each well was rinsed with Earles Balanced Salt Solution (Life Technologies Ltd.). One ml of the neutral red medium was added to each well and incubated for 3 hours at 37 °C. This resulted in the uptake of dye into viable cells. The dye-medium was aspirated and the cells were washed rapidly with 4% formaldehyde in 1 % CaCl₂ to remove non-absorbed dye and to simultaneously enhance adhesion of the cells to the substratum. The dye was then extracted from the cells using 1% acetic acid in 50% ethanol, for 20 min. The absorbencies were then measured at 540 nm using a spectrophotometer (Spektralphotometer DM 4, Zeiss, Jena, Germany). Readings are expressed as % of control absorbency i.e. % of viability where no test agents were added. All measurements were done in quadruplicate.

Effect of CHX and IKI on the pH of calcium hydroxide

One half ml of each test material was diluted in 100 ml distilled water and titrated with 1 M HC1. The titration was done at room temperature and under constant stirring with a magnetic stirrer. Because of poor solubility of calcium hydroxide, titrations were done very slowly and an electrode especially designed for measuring slurries was used (Ross "Sure-Flow" Combination, model 8172 BN, 210, Orion Research Inc. Cambridge, MA, USA). The pH was recorded with a pH-meter (Model 210, Orion Research Inc. Cambridge, MA, USA) and the results are given as milliliters of 1 M HC1 required to lower the pH to 7.0.

Results The results of 24 h medication are shown in Table 1. Calcium carbonate and calcium hydroxide were totally ineffective against E. faecalis. The most effective medicament was IKI, which disinfected the testblocks to the depth of 700 μm in one day. CHX, CHX-calcium hydroxide and IKI-calcium hydroxide mixtures showed antibacterial activity to the depth of 200 μm or more in one day.

Table 1. 24 h medication in bovine dentin blocks infected with E. faecalis

μm 0-100 100-200 200-300 300-400 400-500 500-600

CaCO, -H- ++++ + I I H- ++++

Ca(OH)₂ -H- ++++ ++++ MM

IKI

CHX + + -

H)₂ +++ + -H-

>

CHX+

Ca(OH)₂ ++- +++ - -H- - - ++

The results of 7 day medication are shown in Table 2. Calcium carbonate and calcium hydroxide showed little or no antibacterial effect against E. faecalis. IKI, CHX-calcium hydroxide and IKI-calcium hydroxide mixtures were effective in the whole 950 μm sampled. Pure CHX killed all bacteria in two of four blocks.

Table 2. 7day medication in bovine dentin blocks infected with E. faecalis μm 0-100 100-200 200-300 300-400 400-500 500-600

CaCO, •+++ +-H-+ -H-

Ca(OH)₂ ++ - ^•I I I I +++ .

IKI+ Ca(OH)₂

CHX+ Ca(OH)₂

The cytotoxic effect of the root canal antiseptics alone and in combinations is shown in Table 3. CHX and IKI were considerably more toxic than calcium hydroxide. However, the cytotoxicity of CHX and IKI with calcium hydroxide was closer to that of pure calcium 5 hydroxide than to that of CHX and IKI alone. Cytotoxicity of CHX-calcium hydroxide was the same as of pure calcium hydroxide. IKI-calcium hydroxide mixture was somewhat more toxic to fibroblasts than calcium hydroxide alone, but clearly less than CHX or IKI.

Table 3. Neutral red adsorption in mouse fibroblasts. The figures show the percentage of l o viable cells compared to controls.

CaOH, CaOH₂

CaOH₂ CHX IKI sat. CHX sat. IKI

10 μl (1 : 100 102.28 97.62

10 μl (1 :10) 101.36 95.42

20 μl (1 :10) 76.17 80.96

50 μl (1 :10) 42.35 71.32

10 μl (1 :1) 102.12 38.60 41.49 102.49 100.80

50 μl (1 :1) 97.79 104.13 81.41

100 μl (1 :1) 100.84 107.40 32.73

200 μl (1 :1) 101.68 89.87 4.25

300 μl (1:1) 63.17 78.61 3.78

The effect of CHX and IKI on the alkalinity of calcium hydroxide is shown in Table 4. Chemical reactions in the mixtures did not reduce the alkalinity of the medicament. This was 15 verified both with fresh and over 96 hours old mixtures. Table 4. Volume (ml) of 1M HC1 needed to reduce pH of the testmedicaments to 7.0

IKI+ IKI+ CHX+ CHX+

Ca(OH)₂ Ca(OH)₂ Ca(OH)₂ Ca(OH)₂ Ca(OH)₂ Ca(OH)₂ fresh mix 96 h mix fresh mix 96 h mix fresh mix 96 h mix

weight (g) 0.68 0.68 0.72 0.69 0.67 0.67 initial pH 13.57 13.55 13.54 13.53 13.53 13.53

HC1 (ml) 8.48 8.49 8.79 8.69 8.44 8.54

5 Discussion

The antibacterial effect of combination of calcium hydroxide with IKI or CHX against E. faecalis was studied here by using infected bovine root blocks (15,16). The method is often used in in vitro testing of different root canal medicaments (22-25) and makes it therefore easy to compare our results with previous studies. Bovine incisors were used as the basic l o morphology, lumen size and density of the dentine tubules are similar to human teeth. All samples came from the same age group of cows to obtain homogenous dentine tubular structure. Removal of the cementum facilitates bacterial growth into dentine tubules (15). It may also effect the penetration of the medicaments. For this study the cementum was not removed, but bacteria were tested to grow through the test blocks. The smear layer was

15 washed away with EDTA before incubation of the bacteria.

E. faecalis was chosen as the test organism because it is most often associated with persistent root canal infections (18-21), and it is resistant to the commonly used calcium hydroxide medication (11,15,17). It is also most frequently found in endodontic cases needing retreatment (42). It is easy to grow and identify, it rapidly invades dentine tubules and is

20 therefore widely used in previous in vitro studies (22-25). The incubation period of two weeks, in which the bacteria penetrate the dentine tubules, was chosen based on methodological studies by Haapasalo and ørstavik (15,16) and verified by a pilot test in the present study. The results of this study confide the previous observations that CHX and IKI are more effective against E. faecalis than pure calcium hydroxide in vitro (15,16,24,25). One day experiments (table 1) show somewhat slower disinfection in combinations. However, after one week the effect of IKI-calcium hydroxide mixture was the same as with pure CHX and IKI. CHX-calcium hydroxide mixture did disinfect the tubules to the depth of 600 μm in one week and was able to kill all bacteria on one of the four test blocks. Slower disinfection in combinations may be due to some inhibitory effect of calcium hydroxide on IKI and CHX, but may also demonstrate the lower concentration of IKI and CHX in the mixtures. The final depths of the disinfecting effect of the various medicaments alone and in combinations suggests that calcium hydroxide does not inhibit the disinfecting effect of IKI and CHX.

Preservation of high alkalinity for longer periods of time is regarded as one of the major advantages of calcium hydroxide. The possible inhibitory effect of IKI or CHX on the alkalinity of the combination with calcium hydroxide was evaluated by measuring the amount of acid needed to neutralise the composition. This experiment (table 4), with both freshly made and 4 days old compositions, clearly indicated that the alkalinity of the compositions remained unchanged. As the addition of CHX or IKI did not effect the alkalinity of the products it can be assumed that combinations have potential to be used as long term medication as well. In our experiments we have been comparing the antimicrobial effect of chlorhexidine gluconate (CHX-G) and chlorhexidine acetate (CHX- A) against Enterococcus faecalis, as well as the inhibition of their antimicrobial activities by dentine, dentine demineralised by ethylenediamine terra-acetic acid (EDTA) or citric acid, dentine matrix, hydroxyapatite, serum albumin, and type I collagen. These experiments show that CHX-G is more effective against E. faecalis and is less affected by the inhibitory effect of the various chemical compounds and substances listed above. This is an indication that CHX-G may give an even better antibacterial effect than CHX-A when combined with calcium hydroxide. The choice of an intracanal medicament should balance the antibacterial efficacy with tissue toxicity. This is often difficult to obtain since medicaments that are bactericidal are also usually toxic to mammalian cells (34-36). The goal in combining different medicaments is to obtain additive or synergistic antimicrobial effects without increasing toxicity. This must be accompanied by toxicity studies to ensure that the host tissues are not compromised. The concern for the potential toxicity of the tested antimicrobial agents rise especially in this study, because of the uncertainty of the reaction patterns and end-products in calcium hydroxide and IKI or CHX mixtures. Very little is known of the chemical reactions in mixtures of calcium hydroxide with either IKI or CHX. Basic chemical knowledge indicates that IKI does not react with calcium hydroxide (43) and observations on similar alkalinity and antimicrobial activity in this study suggest the same for CHX. It is known that calcium can displace chlorhexidine from carboxyl groups in vitro but this seems to affect its binding to surfaces and not antibacterial efficacy (44,45). Cytotoxicity tests with neutral red method (table 3) indicate that the mixtures presented in this study are no more toxic than their pure components. Moreover, the test even indicate that calcium hydroxide may protect host cells against the cytotoxic effect of CHX and IKI in the mixtures.

According to the present invention, benefits in the antimicrobial treatment of dental root canals are achieved by combining calcium hydroxide with IKI, CHX or mixtures of these. The widespread use of calcium hydroxide for disinfection of dental root canals is mainly based on its long lasting alkalinity and blocking of nutrients. These abilities are not negatively affected by the addition of CHX and/or IKI, while the additions distinctively increase the antibacterial effect of the medicament. The antibacterial effect of IKI and/or CHX in compositions with calcium hydroxide has been shown to be an advantage in the treatment of dental root canal infections either in primary or retreatment cases. The compositions according to the invention may be applied at the site of treatment in suitable, pharmaceutically acceptable carriers, in the form of solutions, pastes or gels. They may also be included in a matrix of guttapercha or a corresponding material, as used e.g. for the medication of a root canal in the course of endodontic treatment.

References

1. KAKEHASHI GH, STANLEY HR, FITZGERALD RL. The effects of surgical exposure of dental pulps in germ-free and conventional laboratory rats. Oral Surg, Oral Med, Oral Path 1965; 20: 340-349.

2. FABRICIUS L, DAHLEN G, HOLM SE, MOLLER AJR. Influence of combinations of oral bacteria on periapical tissues of monkeys.Scan Dent Res 1982; 90: 200-206.

3. SUNDQVIST G. Bacteriological studies of necrotic pulps. PhD Thesis 1976 Umea University, Umea, Sweden.

4. MOORE WEC, MOORE LVH. The bacteria of periodontal diseases. Periodontol 2000 1994; 5: 66-77.

5. Kantz WE, Henry CA. Isolation and classification of anaerobic bacteria from intact pulp chambers of non-vital teeth in man. Arch Oral Biol 1974; 19: 91-96.

6. Wittgow WC, Sabiston CB. Microorganisms from pulpal chambers of intact teeth with necrotic pulps. J Endod 1975; 1: 168-171.

7. Sundqvist G. Taxonomy, ecology, and pathogenicity of the root canal flora. Oral Surg Oral Med Oral Pathology 1994; 78, 522-530.

8. Engstrόm B, Hard af Segerstad L, Ramstrόm G, Frostell G. Correlation of positive cultures with the prognosis for root canal treatment. Odont Revy 1964; 15: 257-270.

9. Heling B, Shapira J. Roentgenologic and clinical evaluation of endodontically treated teeth, with or without negative culture. Quintessence International 1978; 11 : 79-84. lO.Sjogren U, Figdor D, Persson S, Sundqvist G. Influence of infection at the time of root filling on the outcome of endodontic treatment of teeth with apical periodontitis. Int J Endod 1997; XXXXXXXX

H.BYSTROM A, CLAESSON R, SUNDQVIST G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Endod Dent Traumatoll985; 1 : 170-5.

12.Cvek M, Hollender L, Nord C-E. Treatment of non-vital permanent incisors with calcium hydroxide. VI. A clinical, microbiological and radiological evaluation of treatment in one sitting of teeth with mature or immature root. Odont Revy 1976; 27: 93-108. 13.Orstavik D, Kerekes K, Molven O. Effects of extensive apical reaming and calcium hydroxide dressing on bacterial infection during treatment of apical periodontitis: a pilot study. Int J Endod 1991; 24: 1-7. 14.Safavi KE, Dowden WE, Introcaso JH, Langeland K. A comparison of antimicrobial effects of calcium hydroxide and iodine-potassium iodide. J Endod 1985; 11 : 454-456. 15.Haapasalo M; Orstavik D; In vitro infection and disinfection of dentinal tubules. J Dent

Res 1987; 66: 1375-1379. lό.Orstavik D. Haapasalo M. Disinfection by endodontic irrigants and dressings of experimentally infected dentinal tubules. Endod Dent Traumatol 1990; 6(4):142-149. 17.Safavi KE, Spangberg LS, Langeland K. Root canal dentinal tubule disinfection. J

Endod 1990; 16: 207-210. 18. BENDER NA, SELZER S. Combination of antibiotics and fungicides used in treatment of the infected pulpless tooth. J Am Dent Assoc 1952; 45: 293-300. 19.ENGSTROM B. The significance of enterococci in root canal treatment. Odont Revy 1964;

15, 87-106. 20.GOLDMAN M, PEARSON AH. Postdebridement bacterial flora and antibiotic sensitivity.

Oral Surg Oral Med Oral Pathology 1969; 28: 897-905. 21.GRAHNEN H, KRASSE B.The effect of instrumentation and flushing of non-vital teeth in endodontic therapy. Odont Revy 1963; 14: 167-77. 22.Heling I. Pecht M. Efficacy of Ledermix paste in eliminating Staphylococcus aureus from infected dentinal tubules in vitro. Endod Dent Traumatol 1991 ;7(6) :251-254. 23.VAHDATY A, FORD TRP, WILSON RF. Efficacy of chlorhexidine in disinfecting dentinal tubules in-vitro. Endod Dent Traumatol 1993; 9: 243-248. 24.HELING I, SOMMER M, STEINBERG D, FRIEDMAN M, SELA MN. Microbiological evaluation of the efficacy of chlorhexidine in a sustained-release device for dentin sterilization. Int J Endod 1992; 25: 15-19. 25.HELING I, STEINBERG D, KENIG S, GAVRILOVICH I, SELA MN, FRIEDMAN M.

Efficacy of a sustained-release device containing chlorhexidine and ca(oh)2 in preventing secondary infection of dentinal tubules.Int J Endod 1992; 25 :20-24. 26.LEWIS BB, CHESTER SB. Formaldehyde in dentistry: Rewiev of mutagenic and carsinogenic potential. J Am Dent Assoc 1981; 103, 429-34. 27.MESSER HH, CHEN R-S. The duration of effectiveness of root canal medicaments. J Endod

1984; 10: 240-245. 28.TRONSTAD L, YANG Z-P, TROPE M, BARNETT F, HAMMOND BF. Controlled release of medicaments in endodontic therapy. Endod Dent Traumatol 1985; 1 : 130-134. 29.ENGSTROM B. On duration of the antibacterial efficiency of four anticeptics used in root canal treatment in situ. Svenska Tandlakaretidskrift 1958; 51 : 1-6. 30.DAVIES GE, FRANCIS J, MARTIN AR, ROSE FL, SWAIN G. l :6-di-4- chlorophenyldiguanidohexane ("Hibitane"). Laboratory investigation of a new antibacterial agent of high potency. Brittish Journal of pharmacology 1954; 9: 192-196. 31.LAWRENCE C A. Antimicrobial activity, in vitro, of chlorhexidine. Journal of American

Pharmacological Association 1960; 49: 731-734. 32.VAN DER MEIREN L, MORIAME G, ACHTEN G, LEDOUX-CORBUSIER M. La chlorhexidine en dermatologie. (English summary). Archs Belg Derm Syph 1958; 14: 14-18. 33.Hugo WB, Longworth AR X. Some aspects of the mode of action of chlorhexidine. J

Pharm Pharmacol 1958; 16: 655-662. 34.F0ULKES DM. Some toxicological observations on chlorhexidine. J Periodontal Res 1973;

12: 55-60. 35.ENGSTROM B, SPANGBERG L X. Studies on root canal medicaments V. Toxic and antimicrobial effects of antiseptics in vitro. Svenska Tandlakaretidskrift 1958; 62: 543-

XXXXXXX. 36.SPANGBERG L, ENGSTROM B. Studies on root canal medicaments I. Cytotoxic effect of root canal antiseptics. Acta Odontol Scan 1967; 25: 77-XXXXX. 37.SPANGBERG L, ENGSTROM B. Studies on root canal medicaments IV. Antimicrobial effect of root canal medicaments. Odontol Revy 1968; 19: 187-XXXXXX 38.SPANGBERG L, ENGSTROM B, LANGELAND K. Biologic effect of dental materials III.

Toxicity and antimicrobial effects of endodontic antiseptics in vitro. Oral Surg Oral Med

Oral Pathology 1973; 36: 856-XXXXXX. 39JAWETZ E, MELNICKJL, ADELBERG EA. Review of medical microbiology, 16* edition.

Los Altos: Medical Publications 1984. 40.BORENFREUND E, PUERNER JA. Toxicity determined in vitro by morphological alterations and neutral red adsorption. Toxicology Letters 1985; 24: 119-124. 41.BABICH H, BORENFREUND E. Applications of the neutral red cytotoxicity assay to in vitro toxicology. ATLA 1990; 18: 129-144. 42.Molander A, Reit C, Dahlen G, Kvist T. Microbiological status of root-filled teeth with apical periodontitis. Int J Endod 1998; 31: 1-8. 43.KILPI S, TOMULA Es. Kvantitatiivisen analyysin oppikirja.WSOY, Helsinki, 1954; 332-

335. 44.DAVIES A. The mode of action of chlorhexidine. J Periodont Res 1973; 12: 68-75. Gjermo P. Bonesvoll P. Hjeljord LG. Rolla G. Influence of variation of pH of chlorhexidine mouth rinses on oral retention and plague-inhibiting effect. Caries Research 1975; 9(1): 74-82.

Claims

1. A composition for the disinfection of a dental root canal, said composition comprising calcium hydroxide as an active agent, characterised in that the composition further comprises iodine potassium iodide and/or chlorhexidine as active agents.

2. A composition according to claim 1, characterised in the chlorhexidine being in the form of chlorhexidine gluconate or chlorhexidine acetate.

3. A composition according to claim 1 or 2, characterised in the calcium hydroxide concentration thereof being saturated.

4. A composition according to any claim 1-3, characterised in the composition being in the form of a solution, a paste or a gel.

5. A composition according to any claim 1-4, characterised in the composition being included in a matrix of a material used for medicament during endodontic treatment.

6. A method of disinfecting a dental root canal comprising the application in said root canal of a composition containing calcium hydroxide and an agent selected from the group consisting of chlorhexidine, iodine potassium iodide and a combination thereof.