Chemical analysis of the flocculate formed by the association of sodium hypochlorite and chlorhexidine Melissa Andr��ia Marchesan, DDS, MSc,a Braulio Pasternak J��nior, DDS, MSc,a M��rcia Maisa de Freitas Afonso, MSc, PhD,b Manoel Dami��o Sousa-Neto, DDS, MSc, PhD,a Cristina Paschoalato, MSc, PhD,b Sao Paulo, Brazil UNIVERSITY OF RIBEIR��O PRETO Objective. The association of chlorhexidine (CHX) and sodium hypochlorite (NaOCl) to enhance their chemical properties during root canal biomechanical preparation can lead to a brown flocculate being formed. Therefore, this study evaluated the metals present in their association by atomic absorption spectrophotometry, and evaluated a possible reagent that could dissolve the flocculate, allowing its clinical use. Study design. Five concentrations of CHX and NaOCl were mixed (2.5% NaOCl 0.2% CHX, 2% CHX 0.5% NaOCl, 2% CHX 5% NaOCl, 0.2% CHX 0.5% NaOCl, and 0.2% CHX 5% NaOCl). Methanol and hexane, acetic acid, and vinegar were evaluated to dissolve the flocculate. Results. A flocculate was formed with all proportions and concentrations. Spectrophotometry analysis showed the presence of Ca, Fe, and Mg. Conclusion. It can be concluded that the association proposed led to the formation of brown flocculate regardless of concentration and proportion. (Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007 103:e103-e105) Among the auxiliary products used during the biome- chanical preparation of root canals, irrigating solutions are vital for the removal of preexisting debris, dentine chips, bacteria, toxic products, and substrates necessary for bacterial growth.1-4 Sodium hypochlorite at different concentrations is the most widely accepted irrigant because of its effective antibacterial action, dissolution of organic materials, clearing, transformation of amines into chloramines, and deodorizing effects5,6 however, high concentrations are toxic and irritate periapical tissues.7,8 Therefore, other chemical solutions have also been recommended for irri- gation. Among these, chlorhexidine digluconate, at con- centrations ranging from 0.1% to 2%, shows a large- spectrum antibacterial effect, substantivity, and low toxicity, but does not dissolve organic material, which is an important property for adequate root canal therapy.9-12 The antibacterial effect of chlorhexidine is comparable to sodium hypochlorite and is effective against some resis- tant bacterial strains that lead to treatment failure.13 Some authors report more favorable results with chlorhexidine for disinfecting the root canal system when compared to sodium hypochlorite.14,15 Others, using similar methods, report better results with sodium hypochlorite.3,16,17 A third group of researchers shows similar results when using either product for root canal irrigation.18,19 In light of this lack of agreement, Kuruvilla and Ka- math20 proposed the microbiological evaluation of the root canal system after irrigation with sodium hypochlo- rite and chlorhexidine digluconate alone or associated. They found not only an increase in the antibacterial ac- tivity by the association, but also tissue dissolution due to sodium hypochlorite and lower toxicity due to chlorhexi- dine. According to the authors, these effects can be attrib- uted to the formation of chlorhexidine chlorite, which increased the ionization capacity of the chlorhexidine molecule. However, Cathro9 suggests that this association can form a dense brown flocculate, which is difficult to remove from the root canal and could cause darkening of the dental structures. Thus, this study associated these substances at dif- ferent concentrations and proportions, trying not to form the flocculate, and evaluated the metals present by atomic absorption spectrophotometry. A possible re- agent that could dissolve the flocculate, allowing its clinical use, was also evaluated. MATERIAL AND METHODS Chlorhexidine digluconate was prepared at 0.2% and 2.0% and sodium hypochlorite was titrated at 0.5%, 2.5%, and 5.0% at the Dental Research Laboratory, University of Ribeir��o Preto (UNAERP). Kuruvilla and Kamatt20 recommend the association of 0.2% chlorhexidine digluconate and 2.5% sodium aDepartment of Dentistry, Endodontic Department, University of Ribeir��o Preto (UNAERP). bDepartment of Chemistry, University of Ribeir��o Preto (UNAERP). Received for publication Jul 27, 2006 returned for revision Oct 23, 2006 accepted for publication Nov 4, 2006. 1079-2104/$ - see front matter �� 2007 Mosby, Inc. All rights reserved. doi:10.1016/j.tripleo.2006.11.008 e103

hypochlorite for root canal irrigation. Thus, in the present laboratory study a mixture of 0.2% chlorhexi- dine digluconate and 2.5% sodium hypochlorite was made at a 1:1 proportion. The formation of a brownish flocculate occurred at the surface. To remove this floc- culate from the solution, quantitative filter paper (JP41 JProlab, Sao Jose do Pinhal, PR, Brazil), 12.5 cm in diameter with 28- m pore, was used. Subsequently, atomic absorption spectrophotometry (model Analisty 100, ICP-inductively coupled plasma, PerkinElmer, Wellesley, MA) was performed and col- orimetric methods were applied. Detection values for different metals were calculated for 2.5% sodium hy- pochlorite, 0.2% chlorhexidine digluconate, and also for the brownish flocculate that was retained on the filter paper. Other concentrations and proportions were also associ- ated with trying not to form the flocculate: 2% chlorhexi- dine 0.5% sodium hypochlorite, 2% chlorhexidine 5% sodium hypochlorite, 0.2% chlorhexidine 0.5% sodium hypochlorite, and 0.2% chlorhexidine 5% so- dium hypochlorite. However, the brownish flocculate was formed at the contact of the first drop of the solutions regardless of the concentration or proportion. Therefore, a third substance was added to this asso- ciation to dissolve the flocculate: methanol (Merck, Darmstadt, Germany) or hexane (Merck) because they are universal solvents (���similar dissolves similar���) for polar and nonpolar substances, respectively. Because only methanol dissolved the flocculate and cannot be used in vivo, pure acetic acid (PA) (Merck), which has low polarity, was used. As expected, it also dissolved the flocculate. Therefore, common vinegar (acetic acid) was used at 0.1 mol/L because of its low toxicity and biocompatibility. RESULTS Results of the concentrations of the metals analyzed are reported in Table I. Pure acetic acid dissolved the flocculate however, when the lower concentration of 0.1 mol/L was used, the flocculate was dissolved but the brown coloring of the solution continued. DISCUSSION Different solutions are associated and mixed during root canal biomechanical preparation to enhance the chemical properties that each solution presents.12 In endodontics, the possibility of preparing mixtures and associations has been consecrated by many research- ers.21-23 Although Kuruvilla and Kamath20 recommend the association of sodium hypochlorite and chlorhexi- dine for root canal irrigation, the physico-chemical properties involved are still not clear. In the present study, we examined only the flocculate with spectrophotometry and not the entire solution of the association of 0.2% chlorhexidine and 2.5% sodium hypochlorite because, at a proportion of 1:1, the result- ing filtrate flocculated again with the addition of chlo- rhexidine. This leads us to suggest that the filtrate was sodium hypochlorite. In other dental specialties, such as periodontology and preventive dentistry, there are many reports on staining of enamel or restorations in patients using chlorhexidine to control dental plaque, because of the interaction with dentifrices or chromogens contained in foods and beverages.24 These studies showed that be- cause of the cationic nature of chlorhexidine, it cannot be associated to anionic substances, anionic detergents, phosphates, or sulfates and has competitive inhibition with calcium.25-27 Therefore, the association of chlo- rhexidine and sodium hypochlorite is also contraindi- cated because sodium hypochlorite tends to be an an- ionic substance and showed a relatively high quantity of calcium by the atomic absorption spectrophotomet- ric analysis. Also based on the spectrophotometric analysis, 0.678 mg/L iron was found in sodium hypochlorite. These data are in accordance with the Cumulative Exposure Project of the U.S. Environmental Protection Agency28 of acceptable percentage of mass in solutions of sodium hypochlorite (3.0 mg/L). However, iron leads to the formation of iron salts, which are soluble in water, easily oxidized, and form iron hydroxides, which tend to flocculate, leading to a brown solution.29 These flocculations were seen in the mixture of sodium hy- pochlorite and chlorhexidine. Additionally, Barkvoll et al.30 reported incompatibil- ity of sodium monofluorophosphate, a constituent of toothpaste, and chlorhexidine through the formation and precipitation of insoluble salts in vitro. This was Table I. Metals found by atomic absorption spectro- photometry reported in mg/L* Metals Chlorhexidine Sodium hypochlorite Flocculate Cd 0.001 0.001 0.001 Cu 0.001 0.42000 0.09900 Zn 0.00120 0.10600 0.02300 Cr 0.001 0.001 0.001 K 0.001 8.56000 0.001 Fe 0.05800 0.67800 0.10030 Mn 0.00900 0.08300 0.01900 Mg 0.05600 10.25300 0.18300 Ca 0.12300 25.01230 0.42300 Ag 0.001 0.02700 0.001 Pb 0.001 0.001 0.001 *Values 0.001 indicate no significant expression. OOOOE e104 Marchesan et al. May 2007