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Year : 2018  |  Volume : 21  |  Issue : 4  |  Page : 428-432
Efficacy of various solutions in preventing orange-brown precipitate formed during alternate use of sodium hypochlorite and chlorhexidine: An in vitro study

1 Department of Conservative Dentistry and Endodontics, K M Shah Dental College and Hospital, Sumandeep Vidyapeeth, Vadodara, Gujarat, India
2 Department of Conservative Dentistry, Faculty of Dentistry, MAHSA University, Kuala Lumpur, Malaysia

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Date of Submission15-Mar-2018
Date of Decision03-Apr-2018
Date of Acceptance22-May-2018
Date of Web Publication27-Jul-2018


Aim: The study evaluated the effectiveness of three intermediate endodontic irrigating solutions in eliminating the residual sodium hypochlorite (NaOCl), thus preventing the formation of the orange-brown precipitate when 2% chlorhexidine (CHX) is used as the final irrigant.
Materials and Methods: A total of 40 extracted human maxillary anterior teeth were selected, disinfected, and decoronated to obtain a standardized length of 10 mm. The teeth were prepared with Protaper universal rotary files until size F4 using 2.5% NaOCl as an irrigant during instrumentation. The teeth were then randomly divided into four groups of 10 samples each based on the irrigating solutions used during final wash sequence as follows: Group A - (2.5% NaOCl and 2% CHX), Group B - (2.5% NaOCl followed by 70% Isopropyl Alcohol and 2% CHX), Group C - (2.5% NaOCl followed by 6.25% sodium metabisulfite and 2% CHX), and Group D - (2.5% NaOCl followed by 3.86% sodium Thiosulfate and 2% CHX). The roots were sectioned longitudinally and the canal surface was evaluated under dental operating microscope (×16) for the presence of orange-brown precipitate. The results were tabulated as per scoring criteria and statistically analyzed.
Statistical Analysis Used: One-way ANOVA test and post hoc Tukey's test.
Results: The lowest mean score was observed in Group C, followed by Group D and Group B, respectively. In comparison, there was a statistically significant (P < 0.001) difference in results between Group C and the other experimental groups. However, there was no statistically significant difference between Group B and Group D.
Conclusion: Sodium metabisulfite was found to be very effective in preventing the formation of orange-brown precipitate.

Keywords: Chlorhexidine; isopropyl alcohol; sodium hypochlorite; sodium metabisulfite; sodium thiosulfate

How to cite this article:
Chhabra N, Gangaramani S, Singbal KP, Desai K, Gupta K. Efficacy of various solutions in preventing orange-brown precipitate formed during alternate use of sodium hypochlorite and chlorhexidine: An in vitro study. J Conserv Dent 2018;21:428-32

How to cite this URL:
Chhabra N, Gangaramani S, Singbal KP, Desai K, Gupta K. Efficacy of various solutions in preventing orange-brown precipitate formed during alternate use of sodium hypochlorite and chlorhexidine: An in vitro study. J Conserv Dent [serial online] 2018 [cited 2019 Dec 10];21:428-32. Available from:

   Introduction Top

The key objective of endodontic therapy is the absolute abolition of microbiota from the root canal system. Although the mechanical instrumentation is able to decrease the microbial loading, it is difficult to attain the comprehensive canal disinfection due to the complex internal root canal anatomy.[1]

The profound use of an antimicrobial solution like sodium hypochlorite (NaOCl) is strongly recommended during biomechanical preparation to obtain thorough elimination of the necrotic pulp as well as microorganisms. NaOCl is opted as the irrigant of first choice during root canal therapy due to its excellent antimicrobial efficacy and tissue dissolving potential.[2]

A broad-spectrum antimicrobial solution, chlorhexidine (CHX) gluconate in various concentrations has also been used as a substitute or in conjunction with NaOCl due to its lesser toxicity [3] and antimicrobial substantivity.[4] It has been found to be greatly effective against several strains of Enterococcus fecalis. Few researches have advocated the alternate use of NaOCl and CHX to take advantage of the beneficial qualities of both solutions.[5]

The interaction between NaOCl when followed by CHX results in orange-brown precipitate formation, known as para-chloroaniline which is possibly carcinogenic to humans and has toxic and immune-toxic effects.[6],[7] This precipitate may hamper the radicular seal during obturation.[7] It may considerably augment the likelihood of leaching of unidentified chemicals into the periradicular tissue and also result in tooth discoloration.[6]

An intermediate intracanal flush with distilled water, isopropyl alcohol, and 50% citric acid has been suggested to eliminate the residual NaOCl before using CHX to prevent the formation of a precipitate.[6],[7],[8],[9]

Sodium metabisulfite is a chemical reagent which is used as a disinfectant, antioxidant, and preservative agent.[10],[11] However, its use in dentistry is restricted as a preservative in local anesthetic solution to prevent oxidation of vasoconstrictor adrenaline.[12]

Sodium thiosulfate has been used as an antidote to cyanide poisoning,[13] and treatment of calciphylaxis in hemodialysis patients with end-stage kidney disease.[14] It has also been effectively used for neutralizing NaOCl in the laboratory studies.[15]

Therefore, the potential of both sodium metabisulfite and sodium thiosulfate in minimizing the formation of orange-brown precipitate when used as an intermediate endodontic irrigant while using CHX after NaOCl appears to be self-evident.

The null hypothesis of the present research stated that there would not be any difference between the effectiveness of sodium metabisulfite, sodium thiosulfate, and isopropyl alcohol in preventing the precipitate formation when used as an intermediary intracanal flush between NaOCl and CHX.

   Materials and Methods Top

Forty human permanent, intact and mature maxillary anterior teeth with single canals, recently extracted for periodontal reasons were selected by direct clinical examination for this study. After washing with distilled water and ultrasonic scaling, the specimens were immersed in 0.5% Chloramine T solution until use.

The teeth were decoronated to obtain a standardized root length of 10 mm. Canal patency was evaluated using #10 K file and teeth with canal obstructions were discarded. The actual length of each tooth was determined with #10 K file, which was introduced into the canal until its tip emerged through the major apical foramen. The working length was established by subtracting 1 mm from the actual length. The apices of the specimens were sealed with wax to prevent extrusion of irrigating solutions. All the canals were prepared by the same operator using NiTi rotary files (Protaper Universal, Dentsply, India) until F4 size as per the manufacturer's instructions. Glyde Prep Canal™ (Dentsply India Pvt. Ltd., New Delhi, India) was used as a lubricant during instrumentation and the canals were irrigated with 2.5% NaOCl between each instrumentation sequence using a 30G side vented needle (RC Twents Irrigation Needle, Prime dental Products Pvt. Ltd., Bhiwandi, India) introduced 2 mm short of the apex. After complete mechanical instrumentation all the samples were thoroughly flushed with 2.5 ml of 2.5% NaOCl using 30G side vented needle.

The samples were randomly allocated to four groups of 10 samples each based on the use of 2.5 ml of intermediate irrigating solution for 60 s as specified below: Group A - No intermediary intracanal irrigant used (control group); Group B - 70% Isopropyl alcohol; Group C - 1% sodium metabisulfite; Group D - 3.86% sodium thiosulfate. After that, all the group samples were irrigated with 2.5 ml of 2% CHX gluconate solution as final wash.

The root canals were dried using paper points of corresponding apical preparation size. Two longitudinal grooves were made along the buccal and lingual surfaces of the roots with water-cooled diamond disc. The roots were then sectioned using mallet and chisel. The exposed surfaces of the root canal were examined for the orange-brown precipitate using a dental operating microscope at ×16 magnification [Figure 1]. The images were then transferred to a computer and evaluated using image editing software (Adobe Photoshop CS5; Adobe Systems, San Jose, CA, USA) by a blinded independent examiner. The quantum of orange-brown precipitate was scored as per criteria suggested by Arslan et al.[9] as follows: 0 - The root canal surface was completely free of orange-brown precipitate; 1 - Orange-brown precipitate present in < half of the root canal surface; 2 - Orange-brown precipitate covered more than half of the root canal surface; and 3 - The root canal surface completely covered with orange-brown precipitate. The obtained data were subjected to one-way ANOVA test and post hoc Tukey test for statistical analysis.
Figure 1: Images of representative specimen from each group: (a) Control Group, (b) isopropyl alcohol Group, (c) sodium metabisulfhate group, (d) sodium thiosulfate group

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   Results Top

The one-way ANOVA test showed that Group C exhibited the least mean scores amongst all the experimental groups (P< 0.001), followed by Group D, B, and A, respectively [Table 1]. The post hoc Tukey's test showed a statistically significant difference between Group C on one hand as compared to all the other experimental groups, whereas Group A revealed the maximum amount of orange-brown precipitate [Table 2]. Although the Group B and Group D performed better than Group A, the difference was not statistically significant. The difference between Group B and Group D was also statistically insignificant. Thus Group C exhibited the least amount of orange-brown precipitate when compared with all the experimental groups (P< 0.001).
Table 1: ANOVA values for the presence of orange-brown precipitate

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Table 2: Post hoc scores for the presence of orange-brown precipitate

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   Discussion Top

The essentiality of the irrigation is paramount during chemo-mechanical preparation due to its noteworthy role in the eradication of microorganism, tissue dissolution, removal of debris as well as smear layer, especially in areas untouched during chemomechanical preparation which amount to almost 40%–60% of the total surface area.[16]

The precipitate formed, when 2% CHX is used as a final irrigating solution after NaOCl, covers the surface of the root canal dentin and adversely affects the dentin permeability. This is detrimental to medicament diffusion and sealer adaptation.[17]

To mimic the scenario of precipitate formation in vitro, the maxillary anterior teeth were selected for this study since they provided sufficient radicular bulk and wider canal diameter. It also facilitated splitting of the roots to observe the radicular surface of the canal.[18]

The extracted teeth were stored in 0.5% Chloramine T solution to provide adequate disinfection and prevent dehydration while having no adverse effect on the organic phase (collagen) of dentin.[19]

The instrumentation was done up to size 40 and 0.06 taper to permit the deeper penetration of the irrigant in the apical portion of the root canal.[20],[21]

During chemomechanical preparation as per established protocol suggested by the Zehender,[5] Glyde was used as a lubricant and 2.5% NaOCl was used as the irrigating solution. This protocol is widely used in contemporary endodontic practice; although, there have been concerns related to the reduced efficacy of tissue dissolving capability of NaOCl.[22]

To maximize the beneficial effect from the mutually exclusive properties of NaOCl and CHX, namely tissue dissolution and substantivity, respectively, alternative use of these solutions has been proposed. The antimicrobial effect of a combination of NaOCl and CHX has been shown to be superior to either of the components individually.[4]

The flocculate resulting from the interaction of NaOCl and CHX is possibly carcinogenic to humans (Group2B-International Agency for Research on Cancer 1993) and has toxic and immunotoxic effects.[8] Moreover, para-chloroaniline occludes dentinal tubules and induces color change in teeth.[23]

The present study was designed to assess the inhibition of precipitate formation specifically on the radicular dentinal surface and thus, the prepared specimens were evaluated using dental operating microscope at magnification setting of ×16. This effectively provided a well magnified two-dimensional view for quantifying the surface precipitate formed.

To minimize the formation of this precipitate, the use of ethyl alcohol as an intermediate irrigating solution has been advocated. Since ethyl alcohol is highly electronegative and a tensioactive agent it can penetrate deep into the dentinal tubules and remove the residual NaOCl present in the canals.[9] About 70% isopropyl alcohol has been shown to have similar properties as of ethyl alcohol validating its use in the present study.[24] However, the volatility of alcohol-based irrigants restricts their shelf life.

Sodium metabisulfite and sodium thiosulfate may present an effective alternative to isopropyl alcohol due to their reducing effect. A literature search was done to identify and understand any possible adverse tissue reaction or interactions of the tested irrigating solution. The results showed that sodium thiosulfate or sodium metabisulfite have been widely used in different fields of biomedical science [12],[13],[14] suggesting minimal or no adverse reaction to soft tissues. Before designing the present study, the reaction of the tested irrigating solutions with NaOCl was assessed for any adverse reaction or precipitate formation in the laboratory.

The superior performance of sodium metabisulfite as an intermediate irrigating solution (Group C) in minimizing the precipitate formation could be related to its effectiveness in neutralizing NaOCl by removing free chlorine ions. The reaction between NaOCl and sodium metabisulfite (weak acid salt) results in the formation of hypochlorous acid which undergoes an oxidation reaction converting sulfite group to sulfate group.[25]

The reduction in the formation of orange-brown precipitate in Group D (NaOCl + sodium thiosulfate + CHX) may be attributed to its neutralizing action against NaOCl which is in accordance with the study conducted by Radcliffe et al. where 1.93% sodium thiosulfate was found to be effective in neutralizing 0.5% NaOCl and 3.86% successfully neutralized the concentrations >1.0% NaOCl.[15]

   Conclusion Top

Within the limitations of this in vitro study, sodium metabisulfite was found to be very effective in preventing the formation of orange-brown precipitate. Hence, it can be suggested as a viable alternative to other intermediate irrigating solutions which are currently used. To ascertain the elimination of precipitate from within the dentinal tubules, it would be necessary to follow through using a scanning electron microscope which was outside the scope of the study. Further studies are needed to evaluate the effect of sodium metabisulfite and sodium thiosulfate on the structural properties of radicular dentin.

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Conflicts of interest

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   References Top

Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321-8.  Back to cited text no. 1
Estrela C, Estrela CR, Barbin EL, Spanó JC, Marchesan MA, Pécora JD, et al. Mechanism of action of sodium hypochlorite. Braz Dent J 2002;13:113-7.  Back to cited text no. 2
Leonardo MR, Tanomaru Filho M, Silva LA, Nelson Filho P, Bonifácio KC, Ito IY. In vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating solution.J Endod 1999;25:167-71.  Back to cited text no. 3
Okino LA, Siqueira EL, Santos M, Bombana AC, Figueiredo JA. Dissolution of pulp tissue by aqueous solution of chlorhexidine digluconate and chlorhexidine digluconate gel. Int Endod J 2004;37:38-41.  Back to cited text no. 4
Zehender M. Root canal irrigants. J Endod 2006;32:389-98.  Back to cited text no. 5
Basrani BR, Manek S, Sodhi RN, Fillery E, Manzur A. Interaction between sodium hypochlorite and chlorhexidine gluconate.J Endod. 2007;33:966-9.  Back to cited text no. 6
Bui TB, Baumgartner JC, Mitchell JC. Evaluation of the interaction between sodium hypochlorite and chlorhexidine gluconate and its effect on root dentin. J Endod 2008;34:181-5.  Back to cited text no. 7
Krishnamurthy S, Sudhakaran S. Evaluation and prevention of the precipitate formed on interaction between sodium hypochlorite and chlorhexidine. J Endod 2010;36:1154-7.  Back to cited text no. 8
Arslan H, Uygun AD, Keskin A, Karatas E, Seçkin F, Yıldırım A, et al. Evaluation of orange-brown precipitate formed in root canals after irrigation with chlorhexidine and QMix and spectroscopic analysis of precipitates produced by a mixture of chlorhexidine/NaOCl and QMix/NaOCl. Int Endod J 2015;48:1199-203.  Back to cited text no. 9
Noorafshan A, Asadi-Golshan R, Monjezi S, Karbalay-Doust S. Sodium metabisulphite, a preservative agent, decreases the heart capillary volume and length, and curcumin, the main component of curcuma longa, cannot protect it. Folia Biol (Praha) 2014;60:275-80.  Back to cited text no. 10
Maia AM, Baby AR, Yasaka WJ, Suenaga E, Kaneko TM, Velasco MV, et al. Validation of HPLC stability-indicating method for Vitamin C in semisolid pharmaceutical/cosmetic preparations with glutathione and sodium metabisulfite, as antioxidants. Talanta 2007;71:639-43.  Back to cited text no. 11
McLure HA, Rubin AP. Review of local anaesthetic agents. Minerva Anestesiol 2005;71:59-74.  Back to cited text no. 12
Baskin SI, Horowitz AM, Nealley EW. The antidotal action of sodium nitrite and sodium thiosulfate against cyanide poisoning. J Clin Pharmacol 1992;32:368-75.  Back to cited text no. 13
Auriemma M, Carbone A, Di Liberato L, Cupaiolo A, Caponio C, De Simone C, et al. Treatment of cutaneous calciphylaxis with sodium thiosulfate: Two case reports and a review of the literature. Am J Clin Dermatol 2011;12:339-46.  Back to cited text no. 14
Radcliffe CE, Potouridou L, Qureshi R, Habahbeh N, Qualtrough A, Worthington H, et al. Antimicrobial activity of varying concentrations of sodium hypochlorite on the endodontic microorganisms Actinomyces israelii, A. naeslundii, Candida albicans and Enterococcus faecalis. Int Endod J 2004;37:438-46.  Back to cited text no. 15
Metzger Z, Zary R, Cohen R, Teperovich E, Paqué F. The quality of root canal preparation and root canal obturation in canals treated with rotary versus self-adjusting files: A three-dimensional micro-computed tomographic study. J Endod 2010;36:1569-73.  Back to cited text no. 16
Akisue E, Tomita VS, Gavini G, Poli de Figueiredo JA. Effect of the combination of sodium hypochlorite and chlorhexidine on dentinal permeability and scanning electron microscopy precipitate observation. J Endod 2010;36:847-50.  Back to cited text no. 17
Lee SJ, Wu MK, Wesselink PR. The effectiveness of syringe irrigation and ultrasonics to remove debris from simulated irregularities within prepared root canal walls. Int Endod J 2004;37:672-8.  Back to cited text no. 18
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Rödig T, Sedghi M, Konietschke F, Lange K, Ziebolz D, Hülsmann M, et al. Efficacy of syringe irrigation, RinsEndo and passive ultrasonic irrigation in removing debris from irregularities in root canals with different apical sizes. Int Endod J 2010;43:581-9.  Back to cited text no. 20
van der Sluis LW, Wu MK, Wesselink PR. The efficacy of ultrasonic irrigation to remove artificially placed dentine debris from human root canals prepared using instruments of varying taper. Int Endod J 2005;38:764-8.  Back to cited text no. 21
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Souza Souza M, Cecchin D, Barbizam JV, Almeida JF, Zaia AA, Gomes BP, et al. Evaluation O the colour change in enamel and dentine promoted by the interaction between 2% chlorhexidine and auxiliary chemical solutions. Aust Endod J 2013;39:107-11.  Back to cited text no. 23
Engel GT, Goodell GG, McClanahan SB. Sealer penetration and apical microleakage in smear-free dentin after a final rinse with either 70% isopropyl alcohol or peridex. J Endod 2005;31:620-3.  Back to cited text no. 24
Available from: [Last updated on 2008 May 18; Last accessed on 2017 Aug 10].  Back to cited text no. 25

Correspondence Address:
Dr. Naveen Chhabra
Department of Conservative Dentistry and Endodontics, K M Shah Dental College and Hospital, Sumandeep Vidyapeeth, Piparia, Waghodia, Vadodara - 391 760, Gujarat
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/JCD.JCD_72_18

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