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Free Active Chlorine in Sodium Hypochlorite Solutions Admixed with Octenidine, SmearOFF, Chlorhexidine, and EDTA
Basic Research—Technology
Free Active Chlorine in Sodium Hypochlorite Solutions Admixed with Octenidine, SmearOFF, Chlorhexidine, and EDTA Unni Krishnan, BDS, MDS, MSc, FRACDS (GDP), FRACDS (Endo),* Sreeja Saji, BSc, MSc,† Roger Clarkson, BDSc, MDSc,* Ratilal Lalloo, BChD, BSc Med(Hons), MChD, PhD,* and Alex J. Moule, BDSc, PhD* Abstract Introduction: The therapeutic effects of sodium hypochlorite (NaOCl) solutions are dependent on the levels of free available chlorine (FAC). Mixing these solutions with irrigants can result in significant reductions in FAC. Although the effect of some irrigants on FAC is known, the effect of other commonly used irrigants is not. Thus, the therapeutic ramifications of the concurrent use of these on the efficiency of NaOCl solutions is not known. Methods: Aliquots of 5.2% (w/v) NaOCl solutions were admixed in proportions of 90:10, 80:20, and 50:50 with the following irrigants: octenidine dihydrochloride (OCT); SmearOFF (Vista Dental Products, Racine, WI), 17% EDTA; and 0.2%, 2%, and 5% chlorhexidine (CHX) solutions. Changes in FAC were measured by iodometric titration. Statistical differences between means were determined using a post hoc Tukey analysis test after an analysis of variance. Results: OCT appeared not to affect FAC and was significantly different than all other irrigants, except for 90:10 and 80:20 mixtures of low concentration (0.2%) CHX. CHX solutions showed a marked concentration- and mixture proportion–dependent detrimental effect on FAC. The reduction of FAC between different concentrations of CHX was statistically significant in 80:20 and 50:50 proportions, with 50:50 mixtures of 5% CHX having the greatest influence. Mixtures containing even small proportions of SmearOFF or EDTA exhibited significant losses in FAC. Conclusions: OCT has little effect on FAC and can be used concurrently with NaOCl solutions. Higher concentrations of CHX significantly affect FAC. Their combined use with NaOCl solutions should be avoided. EDTA and SmearOFF should not be mixed with NaOCl solutions. (J Endod 2017;-:1–6)
Key Words Chlorhexidine, ethylenediaminetetraacetic acid, free active chlorine, octenidine, SmearOFF, sodium hypochlorite
S
odium hypochlorite Significance (NaOCl) is an oxidizing Octenidine can be used in combination with NaOCl agent that exists in solution because it has a minimal effect on FAC. Chlorhexas hypochlorous acid and idine has a concentration- and proportionhypochlorite ion in various dependent decrease on FAC. SmearOFF and proportions according to EDTA should not be mixed with NaOCl. the pH of the solution. Together they constitute the free active chlorine (FAC) content (1). It is this FAC that is responsible for protein breakdown and inhibition of bacterial enzymes (2). The efficacy of NaOCl solutions quickly decreases in contact with organic matter, especially in the presence of inflammatory exudate and dentin (3). NaOCl is widely accepted as an irrigant in endodontics because of its tissue dissolution potential and antibacterial effect (3). Recently, however, concerns have been raised that NaOCl cannot predictably eradicate biofilm (4) or diffuse completely into biofilm even at a 2% concentration (5) and that the potential exists for subantimicrobial concentrations of NaOCl to actually increase biofilm formation (6). This may be of clinical significance, considering variations in the use of NaOCl between countries and between practitioners within the same country (7, 8). Although in vitro tests using higher concentrations of NaOCl have mostly demonstrated its antibacterial effect, the presence of dentin collagen, exudate, and microbial biomass inside root canals rapidly reduce its efficacy, resulting in bacterial persistence (3). The collateral tissue damage seen with high concentrations of NaOCl (eg, a proportional increase in cytotoxicity and a decrease in the mechanical properties of dentin) is an additional concern (9, 10). The use of additional irrigation with secondary antibacterial irrigants is 1 of the proposed strategies to enhance bacterial eradication (3). Using some of the currently available irrigants in combination with NaOCl has its drawbacks, such as the formation of toxic reaction products and antagonistic interactions (11). Our group has previously shown that FAC is dramatically reduced when NaOCl and EDTA are mixed together, even in small proportions (1). However, there are no data that support the use of any particular secondary irrigant solution. Nevertheless, there is a need to identify secondary irrigants with antibacterial and antibiofilm properties that can be used safely and effectively in root canal systems in combination with NaOCl without reducing the FAC. This research investigates one such irrigant, octenidine dihydrochloride (OCT), measuring its effect on FAC and comparing it with 3 other commercially available irrigants. OCT is a potentially useful root canal irrigant and antimicrobial agent with antibiofilm properties (12, 13). It has been extensively used in wound disinfection and in the
From the *School of Dentistry, University of Queensland, Brisbane, Australia; and †School of Dentistry, Kuwait University, Jabriya, Kuwait. Address requests for reprints to Dr Unni Krishnan, University of Queensland School of Dentistry, 288 Herston Road, Brisbane 4006, Australia. E-mail address: drunni@ yahoo.com 0099-2399/$ - see front matter Copyright ª 2017 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2017.03.034
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Free Active Chlorine in NaOCl Solutions
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Basic Research—Technology treatment of diabetic foot ulcers, burn injuries, and orthopedic implant infections. It has excellent tissue tolerability on open wounds (14). Although OCT has been shown to be specifically effective against endodontic pathogens and is capable of disinfection of dentin tubules, it has limited pulp dissolution ability (15–17). Its use as an irrigant in combination with NaOCl, which has excellent tissue dissolution properties, has potential. A recent study reported that no apparent deleterious products are formed when OCT and NaOCl solutions are mixed together (18). However, there is limited information on the effect of the combination of these 2 irrigants on the active chlorine content in NaOCl solutions. Chlorhexidine (CHX) is a positively charged divalent biguanide with the ability to bind to dentin and negatively charged bacterial surfaces, resulting in prolonged antibacterial and antifungal effects when used in a root canal (19). Even though it is a popular root canal irrigant, CHX lacks tissue dissolving property. It is also inactivated by serum albumin and has a relatively limited effect on gram-negative bacteria (19, 20). CHX forms a precipitate when mixed with NaOCl, the composition and toxicity of which are still controversial (21, 22). The effect of CHX on the FAC when it is mixed with NaOCl is unknown. SmearOFF (Vista Dental Products, Racine, WI) is a new irrigant containing, among other things, CHX gluconate <1% wt and tetrasodium ethylenediaminetetraacetate dihydrate 18% wt. It is marketed as an irrigant that does not produce a precipitate with NaOCl (23). The manufacturer suggests it can be used with NaOCl as a 2-step procedure instead of the 3-step procedure usually involving the removal of NaOCl from the canal by irrigation with saline or alcohol before other irrigants are used (20, 23, 24). Tartari et al (25) have recently shown that alkaline tetrasodium salts of EDTA do not have a substantial effect on the FAC, even in 1:1 proportions of mixture with NaOCl, but they do have an effect when the pH is lowered. For NaOCl solutions to be effective in dissolving proteins and inhibiting bacterial enzymes, a high level of FAC is necessary. Thus, it is important to know what effect mixing other solutions with NaOCl has on FAC. The aim of this investigation was to measure the effect of OCT, CHX, SmearOFF, and EDTA on the FAC content of NaOCl solutions when mixed at varying proportions. Although the effect of EDTA on the FAC when added to NaOCl solutions has been well reported previously (1, 20, 26), this solution was retested as a positive control to confirm that the results were consistent with previous studies.
80:20, 20 mL 5.2% NaOCl was mixed with 5 mL secondary solution 50:50, 12.5 mL 5.2% NaOCl was mixed with 12.5 mL secondary solution The baseline FAC of the primary solution was established by averaging 3 iodometric titrations immediately before mixing with the secondary solution. The FAC content of the mixtures was also established immediately after mixing. Three independent assessments were made for each 25-mL aliquot of each mixture in each of the 3 proportions for each test solution and averaged.
Data Analysis Statistical analysis was performed using SPSS 22.0 (SPSS Inc, Armonk, NY). A post hoc Tukey test analysis was performed after analysis of variance to compare the mean FAC between mixtures of NaOCl with different secondary solutions at different proportions. The level of significance was P < .05.
Results The mean FAC in 5.2% (w/v) NaOCl at the commencement of each experiment was 4.3% (w/v). The mean FAC of each mixture and the percentage of the remaining FAC in each mixture are graphically illustrated (Figs. 1–3). Mixing NaOCl with other test solutions resulted in a decrease of FAC in all mixtures. There was a statistically significant difference (P < .05) in the mean FAC between different proportions of the same secondary solution. With OCT, the loss of FAC was almost identical to what would be expected because of dilution, indicating that little if any reaction occurred between solutions. With all other mixtures, the reduction in FAC was much greater than would have been expected from dilution, indicating that a chemical reaction occurred between the solutions. The percentages provided later in parentheses represent the remaining percentage of the original FAC at proportions of 90:10, 80:20, and 50:50.
OCT When NaOCl was mixed with OCT, the FAC was 3.91% (w/v) (90.9%), 3.44% (w/v) (81%), and 2.15 % (w/v) (51.2%), respectively, almost the same as would be expected from dilution only.
Materials and Methods
CHX 0.2% The FAC after mixing was 3.8% (w/v) (88.4%), 3.3% (w/v) (76.8%), and 1.95% (w/v) (45.4%), respectively.
The NaOCl solution used (primary solution) was Clorox regular bleach (The Clorox Company, Oakland, CA), which had a nominal free chlorine content of 5.2% (w/v). The 6 secondary solutions tested were the following:
CHX 2% The FAC after mixing was 3.68% (w/v) (85.6%), 3.05% (w/v) (70.9%), and 1.23% (w/v) (28.2%).
1. 0.1% OCT (Octenisept, Sch€ulke & Mayr, Nordersdedt, Germany) 2. SmearOFF (Vista Dental Products, Racine, WI), a proprietary mixture of CHX and tetrasodium salt of EDTA 3. Three CHX gluconate solutions prepared from concentrate (J&J Medical Pty Ltd Australia, North Ryde, NSW, Australia) at 5%, 2%, and 0.2% 4. 17% EDTA (Vista Dental Products)
Preparation of Mixtures The test solutions were made by diluting the primary solution with the secondary solution to make up 25-mL aliquots of different proportions. The proportions and their composition were as follows: 90:10, 22.5 mL 5.2% NaOCl was mixed with 2.5 mL secondary solution 2
Krishnan et al.
Chlorhexidine 5% The FAC after mixing was 3.38% (w/v) (78.6%), 2.5% (w/v) (58.4%), and 0.39% (w/v) (9.1%) for the 90:10, 80:20, and 50:50 proportions, respectively. Although the addition of OCT resulted in the least amount of FAC loss, this loss was not statistically significantly different compared with NaOCl/0.2% CHX at 90:10 and 80:20 proportions. However, the loss of FAC with NaOCl/OCT was statistically significant compared with the 50:50 proportion of NaOCl/0.2% CHX mixture and with all other concentrations of CHX in all proportions. The greatest loss (90%) of FAC resulted when NaOCl was mixed with 5% CHX at a 50:50 proportion and was statistically significantly lower than all other secondary solutions, except the NaOCl/SmearOFF mixture. Comparing the results among the CHX groups, FAC loss between the different concentrations of CHX was statistically significant, except JOE — Volume -, Number -, - 2017
Basic Research—Technology
Figure 1. The mean (standard deviation) values of the FAC content of mixtures of NaOCl with SmearOFF (orange), OCT (gray), EDTA (yellow), and 2% CHX (blue) in different proportions because of the combined effect of dilution and the chemical reaction. The calculated reduction caused by dilution alone is also shown (green).
between 90:10 and 80:20 proportions of NaOCl/0.2% CHX and NaOCl/ 2% CHX mixtures.
SmearOFF The FAC after mixing was 1.29% (w/v) (30%), 1.04% (w/v) (24.2%), and 0.53% (w/v) (12%) for the 90:10, 80:20, and 50:50 proportions, respectively. EDTA The FAC after mixing was 1.47% (w/v) (35.2%), 1.07% (w/v) (24.9%), and 0.69% (w/v) (14.7%), respectively. The loss with SmearOFF in all proportions was much greater than was expected from dilution and more than that measured from EDTA, but the difference between the 2 was not statistically significant. However, the loss of FAC with SmearOFF and EDTA was statistically significant compared with all the other groups, except between SmearOFF and the 50:50 proportion of NaOCl/5% CHX mixture, which were not statistically significant.
Discussion In considering FAC in mixtures of irrigants, it must be remembered that any reduction is a combined effect of dilution and a chemical reaction (1). The effect of dilution alone on the FAC of NaOCl has been preJOE — Volume -, Number -, - 2017
viously confirmed by Clarkson et al (1). The expected reduction in values caused by dilution alone is indicated in Figures 1 and 2. The effect of dilution would be more clinically significant if solutions are added to NaOCl solutions of lower strengths (1). Any reduction in FAC beyond that expected from dilution can be assumed to be a result of a chemical reaction. The decline of FAC in all proportions of the NaOCl/OCT mixture was almost identical to this effect of dilution, indicating that a minimal chemical reaction occurred between the 2 compounds. This confirms a proposal in a recent article that identified the cloudy precipitate formed when NaOCl and OCT were mixed to be phenoxyethanol (PE) and not a reaction by-product. PE is already present in the commercially available OCT (18). Interestingly, PE has also been shown to be an antibacterial agent known to have a synergistic effect with OCT (27). The fact that FAC is not affected in the NaOCl/OCT combination and the finding of the previous article (18) suggest that OCT can be safely used in combination with NaOCl with potentially synergistic activity. Our results showed that the reduction of FAC with all concentrations and combinations of CHX was more than the effect of dilution, confirming that NaOCl and CHX reacted with each other. The effect of 0.2% CHX in smaller proportions on the FAC was not much greater than that expected with dilution and not significantly greater than with OCT. This is likely because at this concentration the CHX content is so low that it
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Basic Research—Technology
Figure 2. The effect of dilution and chemical reaction of different concentrations of CHX in various proportions on the mean (standard deviation) FAC of 5.2% NaOCl.
Figure 3. The percentage of the remaining FAC with different combinations and proportions of irrigants.
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Basic Research—Technology would be almost immediately exhausted when mixed and thus would have little effect on the overall FAC. At this low concentration, CHX is also ineffective as a root canal irrigant (20, 28). Because larger proportions and higher concentrations of CHX produced a significant reduction in FAC, the poor clinical outcome reported when CHX was used concurrently with NaOCl (29) is likely because of the combined effect of the reduced FAC nullifying any antimicrobial effect of the mixture and in part because of the precipitate (22), which could affect the obturation seal (30). Dentin tubule disinfection is an important step, especially in retreatment cases associated with persistent apical periodontitis, because bacteria have been found to penetrate into deep dentin closer to the cementum (31). At room temperature, 3% NaOCl is ineffective in achieving dentin tubule disinfection, with 60% of live bacteria remaining within the tubules at 300 mm (32). Even if 6% NaOCl is used at 45 C for 20 minutes, the intratubular penetration of NaOCl is limited to 300 mm in dentin slab models in vitro (33). On the other hand, OCT has been shown to be able to significantly disinfect dentinal tubules (17, 34) and reduce the colony-forming units at a depth of 400 mm compared with controls and CHX (35). Considering the retention of still more than 50% of FAC when NaOCl and OCT are mixed in equal proportions, the potential for combined irrigation of NaOCl with OCT exists, with a likelihood of better dentin tubule disinfection. Further studies are underway to evaluate the effect of activation and temperature on the disinfection potential of combined irrigation with OCT and NaOCl. Previous studies have shown that EDTA can reduce up to 80%– 88% of available chlorine from NaOCl solutions, which was mirrored in the results of this study (1, 26). What was surprising was the substantial reduction in FAC observed with SmearOFF. Recently, Tartari et al (25) showed that alkaline tetrasodium salts of EDTA did not have a substantial effect even in 1:1 proportions at a pH of 12. Nevertheless, tetrasodium salts of EDTA in Tartari et al’s pilot study showed a significant reduction in tissue dissolution at a neutral pH, which is likely because of the reduced presence of hypochlorite ion or free chlorine (25). The marked reduction of FAC observed with admixtures of SmearOFF may be caused by the reaction of NaOCl with CHX and because the product contains 4% wt sodium bisulfite (23). This is a reducing agent known to rapidly react with NaOCl to form sodium bisulfate and salt (36). Thus, the fact that admixtures of SmearOff and NaOCl do not form a precipitate is likely caused by the rapid exhaustion of available chlorine before a precipitate can occur. Although a case could be made for the concomitant use of tetrasodium salt of EDTA with NaOCl at a pH of 12 to perform organic matter dissolution and smear layer removal simultaneously (25), the commercial product SmearOFF, which has a pH of only 8 to 9, appears not to retain any significant active chlorine when mixed with NaOCl. Hence, the intended use of SmearOFF should be clear; it should only be used alone as a final irrigant. Even though it does not produce a precipitate when mixed with NaOCl, SmearOFF should not be used concurrently with NaOCl because of the rapid displacement of available chlorine. On the other hand, the potential for OCT as a final irrigant requires serious consideration because of its excellent tissue tolerability; its lack of reaction with NaOCl; and its ability to retain antimicrobial effectiveness even in the presence of albumin, blood, and mucin (27, 37). Further studies are underway combining OCT and various salts of EDTA at different pH levels to explore the antimicrobial and smear layer removing ability of such combinations. This article is not without limitations. The chlorine levels determined using the iodometric titration methods are generally higher than those that are clinically achievable because of the inherent instability of NaOCl and the neutralizing effect of dentin and organic matter (3). In addition, the inclusion of chlorate, chlorite, perchlorate, and JOE — Volume -, Number -, - 2017
bromate ions in FAC measurements in the iodometric titration makes it less accurate compared with FAC measurement with liquid chromatography and tandem mass spectrometry (38). Because these ions do not have any therapeutic effect, there may be an overestimation of clinically active chlorine with the iodometric method (39). Thus, the measured levels of FAC may have been higher than if other more complex titration methods had been used (1). Because solutions vary in their density, a wt/wt mixture of the irrigants would have been ideal. However, a volume-based measurement of the mixture as previously reported by Clarkson et al (1) was used because it is a clinically relevant way of testing irrigant interaction. Water was not used as a positive control. This additional step was considered unnecessary because dilution of NaOCl with water is proven to reduce the FAC proportionally (1). In conclusion, when mixed with NaOCl solutions, SmearOFF appears to cause a marked reduction in FAC in a similar manner to that of EDTA, and, thus, its combined use with NaOCl cannot be recommended. CHX showed a marked concentration- and mixture proportion– dependent effect on FAC. On the other hand, OCT has no demonstrable effect on the available chlorine levels in all mixed proportions other than the effect of dilution. Its use as an irrigant in combination with NaOCl is a promising development. Further studies are required to evaluate the extent of synergism achievable using OCT as a root canal irrigant in combination with NaOCl and also with EDTA at various pH levels. The ideal proportion and minimal concentration of NaOCl with OCT to achieve optimal antimicrobial and tissue dissolution actions require further investigation.
Acknowledgments The authors deny any conflicts of interest related to this study.
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Basic Research—Technology 15. Arslan D, Guneser MB, Kustarci A, et al. Pulp tissue dissolution capacity of QMix 2in1 irrigation solution. Eur J Dent 2015;9:423–7. 16. Tirali RE, Turan Y, Akal N, et al. In vitro antimicrobial activity of several concentrations of NaOCl and Octenisept in elimination of endodontic pathogens. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2009;108:e117–20. 17. Tandjung L, Waltimo T, Hauser I, et al. Octenidine in root canal and dentine disinfection ex vivo. Int Endod J 2007;40:845–51. 18. Thaha KA, Varma RL, Nair MG, et al. Interaction between octenidine-based solution and sodium hypochlorite: a mass spectroscopy, proton nuclear magnetic resonance, and scanning electron microscopy–based observational study. J Endod 2017;43:135–40. 19. Mohammadi Z, Abbott PV. The properties and applications of chlorhexidine in endodontics. Int Endod J 2009;42:288–302. 20. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98. 21. Orhan EO, Irmak O, Hur D, et al. Does para-chloroaniline really form after mixing sodium hypochlorite and chlorhexidine? J Endod 2016;42:455–9. 22. Basrani BR, Manek S, Mathers D, et al. Determination of 4-chloroaniline and its derivatives formed in the interaction of sodium hypochlorite and chlorhexidine by using gas chromatography. J Endod 2010;36:312–4. 23. Vista Dental Products. SmearOFF 2-in-1. Available at: http://vista-dental.com/ smearoff-2-in-1/. Accessed September 6, 2016. 24. Basrani BR, Manek S, Sodhi RN, et al. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33:966–9. 25. Tartari T, Oda DF, Zancan RF, et al. Mixture of alkaline tetrasodium EDTA with sodium hypochlorite promotes in vitro smear layer removal and organic matter dissolution during biomechanical preparation. Int Endod J 2017;50: 106–14. 26. Grawehr M, Sener B, Waltimo T, et al. Interactions of ethylenediamine tetraacetic acid with sodium hypochlorite in aqueous solutions. Int Endod J 2003;36:411–7. 27. Hubner NO, Siebert J, Kramer A. Octenidine dihydrochloride, a modern antiseptic for skin, mucous membranes and wounds. Skin Pharmacol Physiol 2010;23: 244–58.
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28. Ringel AM, Patterson SS, Newton CW, et al. In vivo evaluation of chlorhexidine gluconate solution and sodium hypochlorite solution as root canal irrigants. J Endod 1982;8:200–4. 29. Ng YL, Mann V, Gulabivala K. A prospective study of the factors affecting outcomes of nonsurgical root canal treatment: part 1: periapical health. Int Endod J 2011;44: 583–609. 30. Homayouni H, Majd NM, Zohrehei H, et al. The effect of root canal irrigation with combination of sodium hypo-chlorite and chlorhexidine gluconate on the sealing ability of obturation materials. Open Dent J 2014;8:184–7. 31. Peters LB, Wesselink PR, Buijs JF, et al. Viable bacteria in root dentinal tubules of teeth with apical periodontitis. J Endod 2001;27:76–81. 32. Wong DT, Cheung GS. Extension of bactericidal effect of sodium hypochlorite into dentinal tubules. J Endod 2014;40:825–9. 33. Zou L, Shen Y, Li W, et al. Penetration of sodium hypochlorite into dentin. J Endod 2010;36:793–6. 34. de Lucena JM, Decker EM, Walter C, et al. Antimicrobial effectiveness of intracanal medicaments on Enterococcus faecalis: chlorhexidine versus octenidine. Int Endod J 2013;46:53–61. 35. Cherian B, Gehlot PM, Manjunath MK. Comparison of the antimicrobial efficacy of octenidine dihydrochloride and chlorhexidine with and without passive ultrasonic irrigation - an in vitro study. J Clin Diagn Res 2016;10:ZC71–7. 36. Tikkanen M, Schroeter HJ, Leong L, et al. Guidance Manual for Disposal of Chlorinated Water. Denver, CO: AWWA Research Foundation and American Water Works Association; 2001. 37. Pitten FA, Werner HP, Kramer A. A standardized test to assess the impact of different organic challenges on the antimicrobial activity of antiseptics. J Hosp Infect 2003; 55:108–15. 38. Pisarenko AN, Stanford BD, Qui~nones O, et al. Rapid analysis of perchlorate, chlorate and bromate ions in concentrated sodium hypochlorite solutions. Anal Chim Acta 2010;659:216–23. 39. Leonardo NG, Carlotto IB, Luisi SB, et al. Calcium hypochlorite solutions: evaluation of surface tension and effect of different storage conditions and time periods over pH and available chlorine content. J Endod 2016;42:641–5.
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Free Active Chlorine in Sodium Hypochlorite Solutions Admixed with Octenidine, SmearOFF, Chlorhexidine, and EDTA Unni Krishnan, BDS, MDS, MSc, FRACDS (GDP), FRACDS (Endo),* Sreeja Saji, BSc, MSc,† Roger Clarkson, BDSc, MDSc,* Ratilal Lalloo, BChD, BSc Med(Hons), MChD, PhD,* and Alex J. Moule, BDSc, PhD* Abstract Introduction: The therapeutic effects of sodium hypochlorite (NaOCl) solutions are dependent on the levels of free available chlorine (FAC). Mixing these solutions with irrigants can result in significant reductions in FAC. Although the effect of some irrigants on FAC is known, the effect of other commonly used irrigants is not. Thus, the therapeutic ramifications of the concurrent use of these on the efficiency of NaOCl solutions is not known. Methods: Aliquots of 5.2% (w/v) NaOCl solutions were admixed in proportions of 90:10, 80:20, and 50:50 with the following irrigants: octenidine dihydrochloride (OCT); SmearOFF (Vista Dental Products, Racine, WI), 17% EDTA; and 0.2%, 2%, and 5% chlorhexidine (CHX) solutions. Changes in FAC were measured by iodometric titration. Statistical differences between means were determined using a post hoc Tukey analysis test after an analysis of variance. Results: OCT appeared not to affect FAC and was significantly different than all other irrigants, except for 90:10 and 80:20 mixtures of low concentration (0.2%) CHX. CHX solutions showed a marked concentration- and mixture proportion–dependent detrimental effect on FAC. The reduction of FAC between different concentrations of CHX was statistically significant in 80:20 and 50:50 proportions, with 50:50 mixtures of 5% CHX having the greatest influence. Mixtures containing even small proportions of SmearOFF or EDTA exhibited significant losses in FAC. Conclusions: OCT has little effect on FAC and can be used concurrently with NaOCl solutions. Higher concentrations of CHX significantly affect FAC. Their combined use with NaOCl solutions should be avoided. EDTA and SmearOFF should not be mixed with NaOCl solutions. (J Endod 2017;-:1–6)
Key Words Chlorhexidine, ethylenediaminetetraacetic acid, free active chlorine, octenidine, SmearOFF, sodium hypochlorite
S
odium hypochlorite Significance (NaOCl) is an oxidizing Octenidine can be used in combination with NaOCl agent that exists in solution because it has a minimal effect on FAC. Chlorhexas hypochlorous acid and idine has a concentration- and proportionhypochlorite ion in various dependent decrease on FAC. SmearOFF and proportions according to EDTA should not be mixed with NaOCl. the pH of the solution. Together they constitute the free active chlorine (FAC) content (1). It is this FAC that is responsible for protein breakdown and inhibition of bacterial enzymes (2). The efficacy of NaOCl solutions quickly decreases in contact with organic matter, especially in the presence of inflammatory exudate and dentin (3). NaOCl is widely accepted as an irrigant in endodontics because of its tissue dissolution potential and antibacterial effect (3). Recently, however, concerns have been raised that NaOCl cannot predictably eradicate biofilm (4) or diffuse completely into biofilm even at a 2% concentration (5) and that the potential exists for subantimicrobial concentrations of NaOCl to actually increase biofilm formation (6). This may be of clinical significance, considering variations in the use of NaOCl between countries and between practitioners within the same country (7, 8). Although in vitro tests using higher concentrations of NaOCl have mostly demonstrated its antibacterial effect, the presence of dentin collagen, exudate, and microbial biomass inside root canals rapidly reduce its efficacy, resulting in bacterial persistence (3). The collateral tissue damage seen with high concentrations of NaOCl (eg, a proportional increase in cytotoxicity and a decrease in the mechanical properties of dentin) is an additional concern (9, 10). The use of additional irrigation with secondary antibacterial irrigants is 1 of the proposed strategies to enhance bacterial eradication (3). Using some of the currently available irrigants in combination with NaOCl has its drawbacks, such as the formation of toxic reaction products and antagonistic interactions (11). Our group has previously shown that FAC is dramatically reduced when NaOCl and EDTA are mixed together, even in small proportions (1). However, there are no data that support the use of any particular secondary irrigant solution. Nevertheless, there is a need to identify secondary irrigants with antibacterial and antibiofilm properties that can be used safely and effectively in root canal systems in combination with NaOCl without reducing the FAC. This research investigates one such irrigant, octenidine dihydrochloride (OCT), measuring its effect on FAC and comparing it with 3 other commercially available irrigants. OCT is a potentially useful root canal irrigant and antimicrobial agent with antibiofilm properties (12, 13). It has been extensively used in wound disinfection and in the
From the *School of Dentistry, University of Queensland, Brisbane, Australia; and †School of Dentistry, Kuwait University, Jabriya, Kuwait. Address requests for reprints to Dr Unni Krishnan, University of Queensland School of Dentistry, 288 Herston Road, Brisbane 4006, Australia. E-mail address: drunni@ yahoo.com 0099-2399/$ - see front matter Copyright ª 2017 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2017.03.034
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Basic Research—Technology treatment of diabetic foot ulcers, burn injuries, and orthopedic implant infections. It has excellent tissue tolerability on open wounds (14). Although OCT has been shown to be specifically effective against endodontic pathogens and is capable of disinfection of dentin tubules, it has limited pulp dissolution ability (15–17). Its use as an irrigant in combination with NaOCl, which has excellent tissue dissolution properties, has potential. A recent study reported that no apparent deleterious products are formed when OCT and NaOCl solutions are mixed together (18). However, there is limited information on the effect of the combination of these 2 irrigants on the active chlorine content in NaOCl solutions. Chlorhexidine (CHX) is a positively charged divalent biguanide with the ability to bind to dentin and negatively charged bacterial surfaces, resulting in prolonged antibacterial and antifungal effects when used in a root canal (19). Even though it is a popular root canal irrigant, CHX lacks tissue dissolving property. It is also inactivated by serum albumin and has a relatively limited effect on gram-negative bacteria (19, 20). CHX forms a precipitate when mixed with NaOCl, the composition and toxicity of which are still controversial (21, 22). The effect of CHX on the FAC when it is mixed with NaOCl is unknown. SmearOFF (Vista Dental Products, Racine, WI) is a new irrigant containing, among other things, CHX gluconate <1% wt and tetrasodium ethylenediaminetetraacetate dihydrate 18% wt. It is marketed as an irrigant that does not produce a precipitate with NaOCl (23). The manufacturer suggests it can be used with NaOCl as a 2-step procedure instead of the 3-step procedure usually involving the removal of NaOCl from the canal by irrigation with saline or alcohol before other irrigants are used (20, 23, 24). Tartari et al (25) have recently shown that alkaline tetrasodium salts of EDTA do not have a substantial effect on the FAC, even in 1:1 proportions of mixture with NaOCl, but they do have an effect when the pH is lowered. For NaOCl solutions to be effective in dissolving proteins and inhibiting bacterial enzymes, a high level of FAC is necessary. Thus, it is important to know what effect mixing other solutions with NaOCl has on FAC. The aim of this investigation was to measure the effect of OCT, CHX, SmearOFF, and EDTA on the FAC content of NaOCl solutions when mixed at varying proportions. Although the effect of EDTA on the FAC when added to NaOCl solutions has been well reported previously (1, 20, 26), this solution was retested as a positive control to confirm that the results were consistent with previous studies.
80:20, 20 mL 5.2% NaOCl was mixed with 5 mL secondary solution 50:50, 12.5 mL 5.2% NaOCl was mixed with 12.5 mL secondary solution The baseline FAC of the primary solution was established by averaging 3 iodometric titrations immediately before mixing with the secondary solution. The FAC content of the mixtures was also established immediately after mixing. Three independent assessments were made for each 25-mL aliquot of each mixture in each of the 3 proportions for each test solution and averaged.
Data Analysis Statistical analysis was performed using SPSS 22.0 (SPSS Inc, Armonk, NY). A post hoc Tukey test analysis was performed after analysis of variance to compare the mean FAC between mixtures of NaOCl with different secondary solutions at different proportions. The level of significance was P < .05.
Results The mean FAC in 5.2% (w/v) NaOCl at the commencement of each experiment was 4.3% (w/v). The mean FAC of each mixture and the percentage of the remaining FAC in each mixture are graphically illustrated (Figs. 1–3). Mixing NaOCl with other test solutions resulted in a decrease of FAC in all mixtures. There was a statistically significant difference (P < .05) in the mean FAC between different proportions of the same secondary solution. With OCT, the loss of FAC was almost identical to what would be expected because of dilution, indicating that little if any reaction occurred between solutions. With all other mixtures, the reduction in FAC was much greater than would have been expected from dilution, indicating that a chemical reaction occurred between the solutions. The percentages provided later in parentheses represent the remaining percentage of the original FAC at proportions of 90:10, 80:20, and 50:50.
OCT When NaOCl was mixed with OCT, the FAC was 3.91% (w/v) (90.9%), 3.44% (w/v) (81%), and 2.15 % (w/v) (51.2%), respectively, almost the same as would be expected from dilution only.
Materials and Methods
CHX 0.2% The FAC after mixing was 3.8% (w/v) (88.4%), 3.3% (w/v) (76.8%), and 1.95% (w/v) (45.4%), respectively.
The NaOCl solution used (primary solution) was Clorox regular bleach (The Clorox Company, Oakland, CA), which had a nominal free chlorine content of 5.2% (w/v). The 6 secondary solutions tested were the following:
CHX 2% The FAC after mixing was 3.68% (w/v) (85.6%), 3.05% (w/v) (70.9%), and 1.23% (w/v) (28.2%).
1. 0.1% OCT (Octenisept, Sch€ulke & Mayr, Nordersdedt, Germany) 2. SmearOFF (Vista Dental Products, Racine, WI), a proprietary mixture of CHX and tetrasodium salt of EDTA 3. Three CHX gluconate solutions prepared from concentrate (J&J Medical Pty Ltd Australia, North Ryde, NSW, Australia) at 5%, 2%, and 0.2% 4. 17% EDTA (Vista Dental Products)
Preparation of Mixtures The test solutions were made by diluting the primary solution with the secondary solution to make up 25-mL aliquots of different proportions. The proportions and their composition were as follows: 90:10, 22.5 mL 5.2% NaOCl was mixed with 2.5 mL secondary solution 2
Krishnan et al.
Chlorhexidine 5% The FAC after mixing was 3.38% (w/v) (78.6%), 2.5% (w/v) (58.4%), and 0.39% (w/v) (9.1%) for the 90:10, 80:20, and 50:50 proportions, respectively. Although the addition of OCT resulted in the least amount of FAC loss, this loss was not statistically significantly different compared with NaOCl/0.2% CHX at 90:10 and 80:20 proportions. However, the loss of FAC with NaOCl/OCT was statistically significant compared with the 50:50 proportion of NaOCl/0.2% CHX mixture and with all other concentrations of CHX in all proportions. The greatest loss (90%) of FAC resulted when NaOCl was mixed with 5% CHX at a 50:50 proportion and was statistically significantly lower than all other secondary solutions, except the NaOCl/SmearOFF mixture. Comparing the results among the CHX groups, FAC loss between the different concentrations of CHX was statistically significant, except JOE — Volume -, Number -, - 2017
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Figure 1. The mean (standard deviation) values of the FAC content of mixtures of NaOCl with SmearOFF (orange), OCT (gray), EDTA (yellow), and 2% CHX (blue) in different proportions because of the combined effect of dilution and the chemical reaction. The calculated reduction caused by dilution alone is also shown (green).
between 90:10 and 80:20 proportions of NaOCl/0.2% CHX and NaOCl/ 2% CHX mixtures.
SmearOFF The FAC after mixing was 1.29% (w/v) (30%), 1.04% (w/v) (24.2%), and 0.53% (w/v) (12%) for the 90:10, 80:20, and 50:50 proportions, respectively. EDTA The FAC after mixing was 1.47% (w/v) (35.2%), 1.07% (w/v) (24.9%), and 0.69% (w/v) (14.7%), respectively. The loss with SmearOFF in all proportions was much greater than was expected from dilution and more than that measured from EDTA, but the difference between the 2 was not statistically significant. However, the loss of FAC with SmearOFF and EDTA was statistically significant compared with all the other groups, except between SmearOFF and the 50:50 proportion of NaOCl/5% CHX mixture, which were not statistically significant.
Discussion In considering FAC in mixtures of irrigants, it must be remembered that any reduction is a combined effect of dilution and a chemical reaction (1). The effect of dilution alone on the FAC of NaOCl has been preJOE — Volume -, Number -, - 2017
viously confirmed by Clarkson et al (1). The expected reduction in values caused by dilution alone is indicated in Figures 1 and 2. The effect of dilution would be more clinically significant if solutions are added to NaOCl solutions of lower strengths (1). Any reduction in FAC beyond that expected from dilution can be assumed to be a result of a chemical reaction. The decline of FAC in all proportions of the NaOCl/OCT mixture was almost identical to this effect of dilution, indicating that a minimal chemical reaction occurred between the 2 compounds. This confirms a proposal in a recent article that identified the cloudy precipitate formed when NaOCl and OCT were mixed to be phenoxyethanol (PE) and not a reaction by-product. PE is already present in the commercially available OCT (18). Interestingly, PE has also been shown to be an antibacterial agent known to have a synergistic effect with OCT (27). The fact that FAC is not affected in the NaOCl/OCT combination and the finding of the previous article (18) suggest that OCT can be safely used in combination with NaOCl with potentially synergistic activity. Our results showed that the reduction of FAC with all concentrations and combinations of CHX was more than the effect of dilution, confirming that NaOCl and CHX reacted with each other. The effect of 0.2% CHX in smaller proportions on the FAC was not much greater than that expected with dilution and not significantly greater than with OCT. This is likely because at this concentration the CHX content is so low that it
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Figure 2. The effect of dilution and chemical reaction of different concentrations of CHX in various proportions on the mean (standard deviation) FAC of 5.2% NaOCl.
Figure 3. The percentage of the remaining FAC with different combinations and proportions of irrigants.
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Basic Research—Technology would be almost immediately exhausted when mixed and thus would have little effect on the overall FAC. At this low concentration, CHX is also ineffective as a root canal irrigant (20, 28). Because larger proportions and higher concentrations of CHX produced a significant reduction in FAC, the poor clinical outcome reported when CHX was used concurrently with NaOCl (29) is likely because of the combined effect of the reduced FAC nullifying any antimicrobial effect of the mixture and in part because of the precipitate (22), which could affect the obturation seal (30). Dentin tubule disinfection is an important step, especially in retreatment cases associated with persistent apical periodontitis, because bacteria have been found to penetrate into deep dentin closer to the cementum (31). At room temperature, 3% NaOCl is ineffective in achieving dentin tubule disinfection, with 60% of live bacteria remaining within the tubules at 300 mm (32). Even if 6% NaOCl is used at 45 C for 20 minutes, the intratubular penetration of NaOCl is limited to 300 mm in dentin slab models in vitro (33). On the other hand, OCT has been shown to be able to significantly disinfect dentinal tubules (17, 34) and reduce the colony-forming units at a depth of 400 mm compared with controls and CHX (35). Considering the retention of still more than 50% of FAC when NaOCl and OCT are mixed in equal proportions, the potential for combined irrigation of NaOCl with OCT exists, with a likelihood of better dentin tubule disinfection. Further studies are underway to evaluate the effect of activation and temperature on the disinfection potential of combined irrigation with OCT and NaOCl. Previous studies have shown that EDTA can reduce up to 80%– 88% of available chlorine from NaOCl solutions, which was mirrored in the results of this study (1, 26). What was surprising was the substantial reduction in FAC observed with SmearOFF. Recently, Tartari et al (25) showed that alkaline tetrasodium salts of EDTA did not have a substantial effect even in 1:1 proportions at a pH of 12. Nevertheless, tetrasodium salts of EDTA in Tartari et al’s pilot study showed a significant reduction in tissue dissolution at a neutral pH, which is likely because of the reduced presence of hypochlorite ion or free chlorine (25). The marked reduction of FAC observed with admixtures of SmearOFF may be caused by the reaction of NaOCl with CHX and because the product contains 4% wt sodium bisulfite (23). This is a reducing agent known to rapidly react with NaOCl to form sodium bisulfate and salt (36). Thus, the fact that admixtures of SmearOff and NaOCl do not form a precipitate is likely caused by the rapid exhaustion of available chlorine before a precipitate can occur. Although a case could be made for the concomitant use of tetrasodium salt of EDTA with NaOCl at a pH of 12 to perform organic matter dissolution and smear layer removal simultaneously (25), the commercial product SmearOFF, which has a pH of only 8 to 9, appears not to retain any significant active chlorine when mixed with NaOCl. Hence, the intended use of SmearOFF should be clear; it should only be used alone as a final irrigant. Even though it does not produce a precipitate when mixed with NaOCl, SmearOFF should not be used concurrently with NaOCl because of the rapid displacement of available chlorine. On the other hand, the potential for OCT as a final irrigant requires serious consideration because of its excellent tissue tolerability; its lack of reaction with NaOCl; and its ability to retain antimicrobial effectiveness even in the presence of albumin, blood, and mucin (27, 37). Further studies are underway combining OCT and various salts of EDTA at different pH levels to explore the antimicrobial and smear layer removing ability of such combinations. This article is not without limitations. The chlorine levels determined using the iodometric titration methods are generally higher than those that are clinically achievable because of the inherent instability of NaOCl and the neutralizing effect of dentin and organic matter (3). In addition, the inclusion of chlorate, chlorite, perchlorate, and JOE — Volume -, Number -, - 2017
bromate ions in FAC measurements in the iodometric titration makes it less accurate compared with FAC measurement with liquid chromatography and tandem mass spectrometry (38). Because these ions do not have any therapeutic effect, there may be an overestimation of clinically active chlorine with the iodometric method (39). Thus, the measured levels of FAC may have been higher than if other more complex titration methods had been used (1). Because solutions vary in their density, a wt/wt mixture of the irrigants would have been ideal. However, a volume-based measurement of the mixture as previously reported by Clarkson et al (1) was used because it is a clinically relevant way of testing irrigant interaction. Water was not used as a positive control. This additional step was considered unnecessary because dilution of NaOCl with water is proven to reduce the FAC proportionally (1). In conclusion, when mixed with NaOCl solutions, SmearOFF appears to cause a marked reduction in FAC in a similar manner to that of EDTA, and, thus, its combined use with NaOCl cannot be recommended. CHX showed a marked concentration- and mixture proportion– dependent effect on FAC. On the other hand, OCT has no demonstrable effect on the available chlorine levels in all mixed proportions other than the effect of dilution. Its use as an irrigant in combination with NaOCl is a promising development. Further studies are required to evaluate the extent of synergism achievable using OCT as a root canal irrigant in combination with NaOCl and also with EDTA at various pH levels. The ideal proportion and minimal concentration of NaOCl with OCT to achieve optimal antimicrobial and tissue dissolution actions require further investigation.
Acknowledgments The authors deny any conflicts of interest related to this study.
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