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Detection of Para-Chloroaniline, Reactive Oxygen Species, and 1-Chloro-4-Nitrobenzene in High Concentrations of Chlorhexidine and in a Mixture of Chlorhexidine and Calcium Hydroxide
Basic Research—Technology
Detection of Para-Chloroaniline, Reactive Oxygen Species, and 1-Chloro-4-Nitrobenzene in High Concentrations of Chlorhexidine and in a Mixture of Chlorhexidine and Calcium Hydroxide Luiz Eduardo Barbin, DDS, MSc, PhD,* Carlos Estrela, DDS, MSc, PhD,† Debora Fernandes Costa Guedes, BCH, MSc, PhD,‡ J ulio Cesar Emboava Span o, DDS, MSc, PhD,* Manoel Dami~ ao Sousa-Neto, DDS, MSc, PhD,§ and Jesus Djalma Pecora, DDS, MSc, PhD§ Abstract Introduction: Chlorhexidine (CHX) is likely to decompose into reactive by-products. This study evaluated the generation of 4-chloroaniline (pCA), reactive oxygen species (ROS), and 1-chloro-4-nitrobenzene in high concentrations of CHX and in a mixture of CHX and calcium hydroxide at different time points. Methods: A gas chromatography method was developed to detect pCA and CHX by-products. Mass spectroscopy was used to elucidate the structure of compounds. The samples, which were kept at 36.5 C and 95% relative humidity during the study, were analyzed immediately and 7 days after preparation. Results: pCA was detected in the 2% CHX solution and in the mixture of CHX and calcium hydroxide at all time points. pCA concentrations increased after storing under those conditions. The 2% CHX solution alone and the mixture of CHX and calcium hydroxide released ROS at all time points, but 1-chloro4-nitrobenzene was not found. Conclusions: pCA and ROS were identified as by-products of the 2% CHX aqueous solution alone and as ointment base of calcium hydroxide paste. (J Endod 2013;39:664–668)
Key Words Calcium hydroxide, chlorhexidine, p-chloroaniline, 1-chloro-4-nitrobenzene, reactive oxygen species
From the *Department of Semiology and Clinics, Dental School, Federal University of Pelotas, Pelotas, Rio Grande do Sul; †Department of Stomatologic Sciences, Dental School, Federal University of Goias, Goi^ania, Goias; ‡Laboratory of Dental Waste Management, Department of Restorative Dentistry, Dental School, University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo; and §Department of Restorative Dentistry, Dental School, University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil. Supported by a grant from the Brazilian agency FAPERGS (0902351). Address requests for reprints to Prof Dr Carlos Estrela, Department of Stomatologic Sciences, Dental School, Federal University of Goias, Prac¸a Universitaria s/n, Setor Universitario CEP 74605220, Goi^ania, GO, Brazil. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.10.018
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arious antimicrobial strategies have been recommended to eliminate microorganisms from the root canal system, including root canal instrumentation and the use of different irrigants, three-dimensional intracanal dressings, and coronal seals (1–4). The success of endodontic infection treatment is directly associated with the control of microorganisms and the disruption of the bacterial biofilm (5–7). The best choice of irrigants and the precise definition of their concentrations remain challenges in endodontics. Sodium hypochlorite, chlorhexidine (CHX), and calcium hydroxide, evaluated in a large number of studies, are antimicrobial agents often used for root canal irrigation or intracanal dressing during endodontic therapy (1–7). These agents have different chemical characteristics and trigger different tissue responses. The effectiveness of their antimicrobial action varies, depending on the experimental models of endodontic infection: in vivo infected human teeth, in vivo infected canine teeth, in vitro infected human teeth, in vitro infected bovine teeth, agar diffusion tests, direct contact tests including tests with planktonic bacteria, and biofilm models (1, 3–5, 7–11). CHX has often been recommended as an endodontic irrigant in concentrations of 0.2%–2% (5, 12–16) or in combination with calcium hydroxide (14, 15). CHX, also called di-D-gluconate or D-gluconic acid, is a compound with n, n-bis(4chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediimidamide and molecular mass of 505.4 g/mol (17, 18). It has been widely used as an antiseptic agent for routine dental plaque control and reduction of endodontic microbiota during root canal irrigation (3, 10, 12, 13, 16, 19) but does not detoxify endotoxins (12). However, the structure of the CHX molecule, in addition to the high pH values caused by the presence of calcium hydroxide, poses a systemic risk because CHX is likely to decompose into reactive by-products such as 4-chloroaniline (pCA) (17, 20). CHX may release pCA and reactive oxygen species (ROS) as a function of time, alkaline environment (high pH), and heat (21–24). The International Agency for Research on Cancer classifies pCA in their 2B group ‘‘possibly carcinogen to humans,’’ which means that this agent is a possible human carcinogen (25). The interaction of CHX and calcium hydroxide may generate ROS (24), which significantly affects the cell wall and membrane structures of microorganisms. Waris and Ahsan (26) reported that several carcinogens might also exert part of their effect by generating ROS during metabolism. Oxidative damage to cell DNA may lead to mutations and play an important role in the initiation and progression of multistage carcinogenesis. Tissue damage should be considered when CHX is used, particularly at higher concentrations, because of its toxic effects, which lead to necrotic changes in the epidermis, dermis, and subcutaneous tissue, as well as to reactive inflammatory responses (22, 27). This study evaluated the generation of pCA and other derivates, such as ROS and 1-chloro-4-nitrobenzene (pCNO), in CHX at a high concentration and in a mixture of CHX and calcium hydroxide at different time points.
JOE — Volume 39, Number 5, May 2013
Basic Research—Technology Material and Methods Chemical Reagents The research-grade chemical reagents used in this study were 20% chlorhexidine digluconate aqueous solution (product number C939425ml, CAS number 18472-51-0 and batch 0001440607; SigmaAldrich Corp, Steinheim am Albuch, BW, Germany), $95.0% calcium hydroxide, A. C. S. reagent (product number 239232 - 100g, CAS number 1305-62-0 and batch 22602JO-433; Sigma-Aldrich Corp), and $99% sublimed 4-chloroaniline (product number 477222-1G, CAS number 106-47-8 and batch 69996LJ; Sigma-Aldrich Corp). Other researchgrade chemicals and reagents were also used or analyzed: acetonitrile (Solusorb; J. T. Baker, Phillipsburg, NJ), 98% 4-chloroaniline (Acros Organics, Fair Lawn, NJ), and 99% 1-chloro-4-nitrobenzene (Acros Organics). Sample Preparation The substances were diluted in deionized water purified by reverse osmosis by using germicide ultraviolet irradiation and 5.0-mm filters (Q842; Quimis Industria Diadema, S~ao Paulo, Brazil). The pCA solution was prepared by dilution of 2.5 mg/mL 1 (1.96 10 5 mol) pCA in 1.0 mL deionized water (1.96 10 2 mol L 1). The 2% CHX solution was prepared by dilution of 200 mL 20% CHX in 2.0 mL deionized water. The paste was prepared by mixing 116 mg Ca(OH)2 and 150 mL 2% CHX. A gas chromatography/mass spectrometry (GC/MS) method was performed by using a QP2010 mass spectrometer fitted with a GC17A gas chromatographer (Shimadzu Scientific Instruments, Tokyo, Japan). Ionization voltage was 70 eV. GC was conducted in the temperature-programming mode with a DB-5MS column (30 m 0.25 mm 0.25 mm). First, the column temperature was 120 C for 2 minutes; then it was increased linearly at a rate of 15 C min 1 to 200 C and kept at that temperature for 10 minutes. The temperature of the injection port was 225 C, and the GC/MS interface was stored at 300 C. The helium carrier gas flow rate was 1.0 mL/min 1. A GC method was developed to detect pCA and other possible CHX by-products in the high concentrations of CHX used in clinical endodontics (2% in aqueous solution) and in a paste produced by mixing 2% CHX solution and calcium hydroxide. MS was used to determine compound structures. The samples were analyzed immediately and 7 days after preparation. During the intervals of the experiment, the samples were kept at 36.5 C and 95% relative humidity. All assays were performed in triplicate under aseptic conditions. GC/MS analysis, where the effluent to the GC instrument is the feed to the MS instrument, is in wide use for confirmation testing of substances. Drug testing, manufacturing quality control, and environmental testing are some typical uses. When an analyst uses the GC instrument to separate compounds before analysis with an MS instrument, a complementary relationship exists, making an effective combination for chemical analysis.
Results GC/MS Analysis of Standard pCA Solution GC/MS was first used to determine a standard reference to detect pCA. The chromatographic analysis of compounds in the standard pCA solution immediately after preparation showed that pCA eluted at a retention time of 7.8 minutes (Fig. 1A). The MS analysis of the peak at 7.8-minute retention of pCA, immediately after its preparation, revealed a relation between mass and load (m/z) of ionized molecules in the pCA standard, with peaks at 45, 65, and 127 m/z, which confirmed the presence of pCA (Fig. 1B). JOE — Volume 39, Number 5, May 2013
Chromatography and MS Analysis of 2% CHX at Two Time Points pCA detection was confirmed by chromatography of the compounds of the standard 2% CHX solution immediately after preparation, which revealed a peak at a retention time of 7.8 minutes with a magnitude of about 40,000 (Fig. 1C), and by MS, which showed peaks at 45, 65, and 127 m/z (Fig. 1D). The chromatographic analysis of compounds in the 2% CHX standard solution 7 days after preparation revealed a peak at a retention time of 7.8 minutes, with a magnitude greater than 75,000 (Fig. 1E). For 2% CHX 7 days after preparation, the peak in the chromatogram (Fig. 1E) at the 7.8-minute retention time and the MS peaks at 45, 65, and 127 m/z confirmed the presence of pCA (Fig. 1F). The magnitude of the peak at the 7.8-minute retention time plotted on the y-axis of a chromatogram for 2% CHX immediately after preparation, which was about 40,000 (Fig. 1C), was lower than that of the peak at the 7.8-minute retention time of 2% CHX 7 days after preparation, which was larger than 75,000 (Fig. 1E). Such findings suggested that storing the 2% CHX solution at 36.5 C and 95% relative humidity for 7 days increased pCA generation. GC/MS Analysis of Paste at Two Time Points pCA detection was confirmed by chromatography of the compounds of a paste produced by mixing 2% CHX solution and calcium hydroxide immediately after preparation, which revealed a peak at a retention time of 7.8 minutes, with a magnitude of about 40,000 (Fig. 1G), and by MS, which showed peaks at 45, 65, and 127 m/z (Fig. 1H). Seven days after preparation, chromatography revealed a peak at the 7.8-minute retention time, with a magnitude of close to 80,000 (Fig. 1I), and MS showed peaks at 45, 65, and 127 m/z (Fig. 1J), which also confirmed the presence of pCA. The magnitude of the peak at the 7.8-minute retention time plotted on the y-axis of a chromatogram of the paste immediately after its preparation, which was about 40,000 (Fig. 1G), was lower than the magnitude of the peak at the 7.8-minute retention time of the paste 7 days after preparation, which was close to 80,000 (Fig. 1I). Such findings suggested that storing the mixture of 2% CHX solution and Ca(OH)2 at 36.5 C and 95% relative humidity for 7 days increased pCA generation. pCA and pCNO were observed in control by using standard solutions of these substances. Data of pCNO were not detected.
Discussion pCA and ROS were detected in 2% CHX and in the combination of 2% CHX and calcium hydroxide at all time points, but pCNO was not found. GC/MS was used to evaluate the production of potentially toxic factors such as pCA, which originate from high concentrations of CHX (2% aqueous solution), as well as from mixing CHX and calcium hydroxide. pCA may be analyzed by using chromatography (6, 21, 23, 24, 28). Barbin et al (24) detected pCA in solutions with low concentrations of CHX (0.2% aqueous solution) stored at 36.5 C and 95% relative humidity 14 days after preparation, but no traces of pCA were found in the paste produced by mixing 0.2% CHX and calcium hydroxide at different time points, although ROS were generated by 0.2% CHX alone and CHX mixed with calcium hydroxide at all time points. Those authors also found that there were no traces of CHX in the paste (0.2% CHX + calcium hydroxide) immediately and 7 and 14 days after preparation when stored at 36.5 C and 95% relative humidity. This finding suggests that the addition of calcium hydroxide to 0.2% CHX leads to an immediate and total degradation of lowconcentration CHX (0.2% aqueous solution). The correlation of those
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Figure 1. (A) Chromatogram of standard pCA solution immediately after preparation; elutes from column at retention time of 7.8 minutes; x-axis shows retention time in minutes, and y-axis, signal intensity in total ion current (TIC) (1,000,000). (B) Mass spectrum of stock solution of pCA immediately after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-to-charge ratio (m/z), and y-axis, relative abundance (percentage). (C) Chromatogram of compounds in standard 2% CHX solution immediately after preparation: peak at 7.8-minute retention time and magnitude of about 4.0 10,000; x-axis shows retention time in minutes, and y-axis, signal intensity in TIC (10,000). (D) Mass spectrum of stock solution of 2% CHX immediately after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-tocharge ratio (m/z), and y-axis, relative abundance (percentage). (E) Chromatogram of compounds in standard 2% CHX solution 7 days after preparation; peak at 7.8minute retention time and magnitude greater than 0.75 100,000; x-axis shows retention time in minutes, and y-axis, signal intensity in TIC (100,000).
findings with the results of our study may indicate that higher CHX concentrations (2% aqueous solution) increase pCA generation in both substances (2% CHX alone and in combination with calcium hydroxide) immediately and 7 days after preparation. This study detected the presence of pCA in 2% CHX and in a paste produced by mixing CHX with calcium hydroxide both immediately after preparation and after storage for 7 days at a temperature of 36.5 C and 666
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95% relative humidity and suggests that pCA concentrations increased after storing under those conditions. Palarettia et al (29) developed a high-performance liquid chromatography method for CHX and its metabolites (pCA and pCNO) produced by oxidation reactions mediated by metalloporphyrins such as cytochrome P-450 models for metabolite prediction. They found that the presence of metalloporphyrins in the reaction substantially increased the formation of pCA (up to 58%) as JOE — Volume 39, Number 5, May 2013
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Figure 1. (continued) (F) Mass spectrum of stock solution of 2% CHX 7 days after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-to-charge ratio (m/z), and y-axis, relative abundance (percentage). (G) Chromatogram of compounds in paste produced by mixing 2% CHX solution with calcium hydroxide immediately after preparation; peak at 7.8-minute retention time and magnitude of about 0.4 100,000; x-axis represents retention time in minutes, and yaxis, signal intensity in TIC (100,000). (H) Mass spectrum of paste produced by mixing 2% CHX solution with calcium hydroxide immediately after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-to-charge ratio (m/z), and y-axis, relative abundance (percentage). (I) Chromatogram of compounds in paste produced by mixing 2% CHX solution with calcium hydroxide 7 days after preparation; peak at 7.8-minute retention time and magnitude of about 0.8 100,000; xaxis shows retention time in minutes, and y-axis, signal intensity in TIC (100,000). (J) Mass spectrum of paste produced by mixing 2% CHX solution with calcium hydroxide 7 days after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-to-charge ratio (m/z), and y-axis, relative abundance (percentage).
a CHX metabolite, especially at a neutral pH. Their findings provided evidence of a possible in vivo formation of pCA as a CHX metabolite mediated by cytochrome P-450 metabolism, which might even increase the risks to the health of patients treated with a 2% CHX solution or a paste produced by mixing 2% CHX and calcium hydroxide. The formation of chloroaniline from all chloronitrobenzene isomers was also mediated by cytochrome P-450 metabolism. In addition, pCA is a common chemical intermediate produced from pCNO (30), which may explain why pCNO was not found in this study, because there may have been a total conversion of pCNO into pCA. Cutaneous absorption of CHX is not significant (31), which minimizes its toxicity when used topically, but the possible presence of CHX in host tissue during endodontic treatment, when CHX may extrude through the apical foramen, may lead to the spreading of CHX or its toxic by-products, JOE — Volume 39, Number 5, May 2013
such as pCA, pCNO, and ROS, in the periapical tissue and the lymphatic and circulatory systems. Some chemical interactions may be observed between substances used in endodontics (21–24, 32–35). CHX degrades into pCA and several reactive by-products, and its substantial antimicrobial activity and residual effects have been demonstrated (19, 36, 37). Particularly when applied in clinical endodontics at high concentrations for 7 days or longer (38), it may prolong pCA generation, and both CHX and its by-products may remain in host tissues for long periods of time. The combination of these effects may increase the potential systemic risk of CHX. pCA is a persistent environmental degradation product found in some herbicides and fungicides, has potential long-term toxicity and carcinogenicity in male rats and male mice, and may cause
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Basic Research—Technology methemoglobinemia (21, 30). In addition, pCA induces DNA damage in bacteria. However, the results of gene mutation are still inconclusive. Gene mutation, but not mitotic recombination, has been induced in fungi, and gene mutation, sister chromatid exchange, and chromosomal aberrations have been induced in cultured mammalian cells, but conflicting data were obtained for cell transformation. Although pCA carcinogenicity in experimental animals has been demonstrated, there is not enough evidence of it in humans. However, results indicate that it may also be carcinogenic to humans, and the International Agency for Research on Cancer has listed it under group 2B ‘‘possibly carcinogen to humans’’ (21, 30). Kacmar et al (39) evaluated the toxic and immunotoxic effects of a pCA metabolite of a herbicide (monolinuron) in suspensions of peripheral blood leukocytes of 5 sheep by using a migration-inhibition test. The toxic effects of pCA were recorded at concentrations of 1.0–0.1 mg/mL 1 and immunotoxic effects at 0.01–0.001 mg/mL 1. Results confirmed total inhibition of leukocyte migration. Immunotoxic effects, defined as mitogenetic activation of leukocytes by phytohemagglutinin, concanavalin A, and lipopolysaccharide, were detected at pCA concentrations 10–100 times lower than those that resulted in toxic effects. The costs (systemic risks) and benefits of choosing CHX as a firstchoice agent to control endodontic infection must be weighed against those of other substances with antimicrobial properties, so that the goals of endodontic treatment may be achieved predictably and without unnecessary risks of systemic damage. Further studies should evaluate which CHX concentrations are safe for use in humans by assessing how much pCA may be generated when using 0.12%–2% CHX solutions, gel, and combinations with other substances.
Acknowledgments The authors deny any conflicts of interest related to this study.
References 1. Nair PNR, Henry S, Cano V, Vera J. Microbial status of apical root canal system of human mandibular first molars with primary apical periodontitis after ‘‘one-visit’’ endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 99:231–52. 2. Paquette L, Legner M, Fillery ED, Friedman S. Antibacterial efficacy of chlorhexidine gluconate intracanal medication in vivo. J Endod 2007;33:788–95. 3. Estrela C, Holland R, Bernabe PFE, et al. Antimicrobial potential of medicaments used in healing process in dogs’ teeth with apical periodontitis. Braz Dent J 2004;15:181–5. 4. 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. 5. Siqueira JF Jr, Paiva SSM, R^oc¸as IN. Reduction in the cultivable bacterial populations in infected root canals by a chlorhexidine-based antimicrobial protocol. J Endod 2007;33:541–7. 6. Wang CS, Arnold RR, Trope M, Teixeira FB. Clinical efficiency of 2% chlorhexidine gel in reducing intracanal bacteria. J Endod 2007;33:1283–9. 7. Estrela C, Sydney GB, Figueiredo JAP, Estrela CRA. Antibacterial efficacy of intracanal medicaments on bacterial biofilm: a critical review. J Appl Oral Sci 2009;17:1–7. 8. Bystr€om A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Dent Traumatol 1985;1:170–5. 9. Haapasalo M, Ørstavik D. In vitro infection and of dentinal tubules. J Dent Res 1987; 66:1375–9. 10. Gomes BPFA, Souza SFC, Ferraz CCR, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267–75. 11. Ohara PK, Torabinejad M, Kettering JD. Antibacterial effects of various endodontic irrigants on selected anaerobic bacteria. Endod Dent Traumatol 1993;9:95–100. 12. Buck RA, Cai J, Eleazer PD, et al. Detoxification of endotoxin by endodontic irrigants and calcium hydroxide. J Endod 2001;27:325–7. 13. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98.
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14. Kontakiotis EG, Tsatsoulis LN, Papanakou SI, Tzanetakis GN. Effect of 2% chlorhexidine gel mixed with calcium hydroxide as an intracanal medication on sealing ability of permanent root canal filling: a 6-month follow-up. J Endod 2008;34: 866–70. 15. Estrela CRA, Estrela C, Reis C, et al. Control of microorganisms in vitro by endodontic irrigants. Braz Dent J 2003;14:187–92. 16. Estrela C, Estrela CRA, Decurcio DA, et al. Antimicrobial efficacy of ozonated water, gaseous ozone, sodium hypochlorite and chlorhexidine in infected human root canals. Int Endod J 2007;40:85–93. 17. Havlıkova L, Matysova L, Novakova L, et al. HPLC determination of chlorhexidine gluconate and p-chloroaniline in topical ointment. J Pharm Biomed Anal 2007; 43:1169–73. 18. Denton GW. Chlorhexidine. In: Block SS, ed. Disinfection, Sterilization and Preservation. 4th ed. Philadelphia: Lea & Febiger; 1991:274–89. 19. Basrani B, Santos JM, Tj€aderhane L, et al. Substantive antimicrobial activity in chlorhexidine-treated human root dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:240–5. 20. Usui K, Hishinuma T, Yamaguchi H, et al. Determination of chlorhexidine (CHD) and nonylphenolethoxylates (NPEOn) using LC-ESI-MS method and application to hemolyzed blood. J Chromatogr B 2006;831:105–9. 21. World Health Organization. WHO Library Cataloguing-in-Publication Data: Concise International Chemical Assessment Document—4-Chloroaniline. Geneva, Switzerland: World Health Organization; 2003. 1–58. 22. Yeung SY, Huang CS, Chan CP, et al. Antioxidant and pro-oxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions. Int Endod J 2007;40: 837–44. 23. Basrani BR, Manek S, Sodhi RNS, et al. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33:966–9. 24. Barbin LE, Saquy PC, Guedes DFC, et al. Determination of para-chloroaniline and reactive oxygen species in chlorhexidine and chlorhexidine associated with calcium hydroxide. J Endod 2008;34:1508–14. 25. World Health Organization. International Agency for Research on Cancer: Monographs on the Evaluation of Carcinogenic Risks to Humans—Occupational Exposures of Hairdressers and Barbers and Personal Use of Hair Colourants; Some Hair Dyes, Cosmetic Colourants, Industrial Dyestuffs and Aromatic Amines. Lyon, France: International Agency for Research on Cancer; 1993. 1–436. 26. Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinog 2006;5:1–8. 27. Faria G, Celes MRN, Rossi A, et al. Evaluation of chlorhexidine toxicity injected in the paw of mice and added to cultured L929 fibroblasts. J Endod 2007;33: 715–22. 28. Ciarlone AE, Gangarosa LP, Fong BC. Detection of p-chloroaniline in chlorhexidine solutions using thin-layer chromatography. J Dent Res 1976;55:918. 29. Palarettia V, Faria AL, Santos JS, et al. Metaloporfirinas como catalisadores na oxidac¸~ao do agente antimicrobiano clorexidina. XXII CICAT, Congresso Iberoamericano de Catalise, 2010. 30. World Health Organization. International Agency for Research on Cancer: ParaChloroaniline—Monographs on the Evaluation of Carcinogenic Risks to Humans: Occupational Exposures of Hairdressers and Barbers and Personal Use of Hair Colourants; Some Hair Dyes, Cosmetic Colourants, Industrial Dyestuffs and Aromatic Amines. Lyon, France: International Agency for Research on Cancer; 1993. 305–321. 31. Karpanen TJ, Worthington T, Conway BR, et al. Permeation of chlorhexidine from alcoholic and aqueous solutions within excised human skin. Antimicrob Agents Chemother 2009;53:1717–9. 32. Kim HS, Zhu Q, Baek SH, et al. Chemical interaction of alexidine and sodium hypochlorite. J Endod 2012;38:112–6. 33. Nowicki JB, Sem DS. An in vitro spectroscopic analysis to determine the chemical composition of the precipitate formed by mixing sodium hypochlorite and chlorhexidine. J Endod 2011;37:983–8. 34. Thomas JE, Sem DS. An in vitro spectroscopic analysis to determine whether parachloroaniline is produced from mixing sodium hypochlorite and chlorhexidine. J Endod 2010;36:315–7. 35. 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. 36. Rosenthal S, Sp angberg L, Safavi K. Chlorhexidine substantivity in root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:488–92. 37. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229–31. 38. Komorowski R, Grad H, Wu XY, Friedman S. Antimicrobial substantivity of chlorhexidine-treated bovine root dentin. J Endod 2000;26:315–7. 39. Kacmar P, Pistl J, Mikula I. The effect of p-chloroaniline on leucocytes of sheep peripheral blood under the migration-inhibition test conditions. Immunopharmacol Immunotoxicol 1995;17:577–84.
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Detection of Para-Chloroaniline, Reactive Oxygen Species, and 1-Chloro-4-Nitrobenzene in High Concentrations of Chlorhexidine and in a Mixture of Chlorhexidine and Calcium Hydroxide Luiz Eduardo Barbin, DDS, MSc, PhD,* Carlos Estrela, DDS, MSc, PhD,† Debora Fernandes Costa Guedes, BCH, MSc, PhD,‡ J ulio Cesar Emboava Span o, DDS, MSc, PhD,* Manoel Dami~ ao Sousa-Neto, DDS, MSc, PhD,§ and Jesus Djalma Pecora, DDS, MSc, PhD§ Abstract Introduction: Chlorhexidine (CHX) is likely to decompose into reactive by-products. This study evaluated the generation of 4-chloroaniline (pCA), reactive oxygen species (ROS), and 1-chloro-4-nitrobenzene in high concentrations of CHX and in a mixture of CHX and calcium hydroxide at different time points. Methods: A gas chromatography method was developed to detect pCA and CHX by-products. Mass spectroscopy was used to elucidate the structure of compounds. The samples, which were kept at 36.5 C and 95% relative humidity during the study, were analyzed immediately and 7 days after preparation. Results: pCA was detected in the 2% CHX solution and in the mixture of CHX and calcium hydroxide at all time points. pCA concentrations increased after storing under those conditions. The 2% CHX solution alone and the mixture of CHX and calcium hydroxide released ROS at all time points, but 1-chloro4-nitrobenzene was not found. Conclusions: pCA and ROS were identified as by-products of the 2% CHX aqueous solution alone and as ointment base of calcium hydroxide paste. (J Endod 2013;39:664–668)
Key Words Calcium hydroxide, chlorhexidine, p-chloroaniline, 1-chloro-4-nitrobenzene, reactive oxygen species
From the *Department of Semiology and Clinics, Dental School, Federal University of Pelotas, Pelotas, Rio Grande do Sul; †Department of Stomatologic Sciences, Dental School, Federal University of Goias, Goi^ania, Goias; ‡Laboratory of Dental Waste Management, Department of Restorative Dentistry, Dental School, University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo; and §Department of Restorative Dentistry, Dental School, University of S~ao Paulo, Ribeir~ao Preto, S~ao Paulo, Brazil. Supported by a grant from the Brazilian agency FAPERGS (0902351). Address requests for reprints to Prof Dr Carlos Estrela, Department of Stomatologic Sciences, Dental School, Federal University of Goias, Prac¸a Universitaria s/n, Setor Universitario CEP 74605220, Goi^ania, GO, Brazil. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2013 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2012.10.018
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arious antimicrobial strategies have been recommended to eliminate microorganisms from the root canal system, including root canal instrumentation and the use of different irrigants, three-dimensional intracanal dressings, and coronal seals (1–4). The success of endodontic infection treatment is directly associated with the control of microorganisms and the disruption of the bacterial biofilm (5–7). The best choice of irrigants and the precise definition of their concentrations remain challenges in endodontics. Sodium hypochlorite, chlorhexidine (CHX), and calcium hydroxide, evaluated in a large number of studies, are antimicrobial agents often used for root canal irrigation or intracanal dressing during endodontic therapy (1–7). These agents have different chemical characteristics and trigger different tissue responses. The effectiveness of their antimicrobial action varies, depending on the experimental models of endodontic infection: in vivo infected human teeth, in vivo infected canine teeth, in vitro infected human teeth, in vitro infected bovine teeth, agar diffusion tests, direct contact tests including tests with planktonic bacteria, and biofilm models (1, 3–5, 7–11). CHX has often been recommended as an endodontic irrigant in concentrations of 0.2%–2% (5, 12–16) or in combination with calcium hydroxide (14, 15). CHX, also called di-D-gluconate or D-gluconic acid, is a compound with n, n-bis(4chlorophenyl)-3,12-diimino-2,4,11,13-tetraazatetradecanediimidamide and molecular mass of 505.4 g/mol (17, 18). It has been widely used as an antiseptic agent for routine dental plaque control and reduction of endodontic microbiota during root canal irrigation (3, 10, 12, 13, 16, 19) but does not detoxify endotoxins (12). However, the structure of the CHX molecule, in addition to the high pH values caused by the presence of calcium hydroxide, poses a systemic risk because CHX is likely to decompose into reactive by-products such as 4-chloroaniline (pCA) (17, 20). CHX may release pCA and reactive oxygen species (ROS) as a function of time, alkaline environment (high pH), and heat (21–24). The International Agency for Research on Cancer classifies pCA in their 2B group ‘‘possibly carcinogen to humans,’’ which means that this agent is a possible human carcinogen (25). The interaction of CHX and calcium hydroxide may generate ROS (24), which significantly affects the cell wall and membrane structures of microorganisms. Waris and Ahsan (26) reported that several carcinogens might also exert part of their effect by generating ROS during metabolism. Oxidative damage to cell DNA may lead to mutations and play an important role in the initiation and progression of multistage carcinogenesis. Tissue damage should be considered when CHX is used, particularly at higher concentrations, because of its toxic effects, which lead to necrotic changes in the epidermis, dermis, and subcutaneous tissue, as well as to reactive inflammatory responses (22, 27). This study evaluated the generation of pCA and other derivates, such as ROS and 1-chloro-4-nitrobenzene (pCNO), in CHX at a high concentration and in a mixture of CHX and calcium hydroxide at different time points.
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Basic Research—Technology Material and Methods Chemical Reagents The research-grade chemical reagents used in this study were 20% chlorhexidine digluconate aqueous solution (product number C939425ml, CAS number 18472-51-0 and batch 0001440607; SigmaAldrich Corp, Steinheim am Albuch, BW, Germany), $95.0% calcium hydroxide, A. C. S. reagent (product number 239232 - 100g, CAS number 1305-62-0 and batch 22602JO-433; Sigma-Aldrich Corp), and $99% sublimed 4-chloroaniline (product number 477222-1G, CAS number 106-47-8 and batch 69996LJ; Sigma-Aldrich Corp). Other researchgrade chemicals and reagents were also used or analyzed: acetonitrile (Solusorb; J. T. Baker, Phillipsburg, NJ), 98% 4-chloroaniline (Acros Organics, Fair Lawn, NJ), and 99% 1-chloro-4-nitrobenzene (Acros Organics). Sample Preparation The substances were diluted in deionized water purified by reverse osmosis by using germicide ultraviolet irradiation and 5.0-mm filters (Q842; Quimis Industria Diadema, S~ao Paulo, Brazil). The pCA solution was prepared by dilution of 2.5 mg/mL 1 (1.96 10 5 mol) pCA in 1.0 mL deionized water (1.96 10 2 mol L 1). The 2% CHX solution was prepared by dilution of 200 mL 20% CHX in 2.0 mL deionized water. The paste was prepared by mixing 116 mg Ca(OH)2 and 150 mL 2% CHX. A gas chromatography/mass spectrometry (GC/MS) method was performed by using a QP2010 mass spectrometer fitted with a GC17A gas chromatographer (Shimadzu Scientific Instruments, Tokyo, Japan). Ionization voltage was 70 eV. GC was conducted in the temperature-programming mode with a DB-5MS column (30 m 0.25 mm 0.25 mm). First, the column temperature was 120 C for 2 minutes; then it was increased linearly at a rate of 15 C min 1 to 200 C and kept at that temperature for 10 minutes. The temperature of the injection port was 225 C, and the GC/MS interface was stored at 300 C. The helium carrier gas flow rate was 1.0 mL/min 1. A GC method was developed to detect pCA and other possible CHX by-products in the high concentrations of CHX used in clinical endodontics (2% in aqueous solution) and in a paste produced by mixing 2% CHX solution and calcium hydroxide. MS was used to determine compound structures. The samples were analyzed immediately and 7 days after preparation. During the intervals of the experiment, the samples were kept at 36.5 C and 95% relative humidity. All assays were performed in triplicate under aseptic conditions. GC/MS analysis, where the effluent to the GC instrument is the feed to the MS instrument, is in wide use for confirmation testing of substances. Drug testing, manufacturing quality control, and environmental testing are some typical uses. When an analyst uses the GC instrument to separate compounds before analysis with an MS instrument, a complementary relationship exists, making an effective combination for chemical analysis.
Results GC/MS Analysis of Standard pCA Solution GC/MS was first used to determine a standard reference to detect pCA. The chromatographic analysis of compounds in the standard pCA solution immediately after preparation showed that pCA eluted at a retention time of 7.8 minutes (Fig. 1A). The MS analysis of the peak at 7.8-minute retention of pCA, immediately after its preparation, revealed a relation between mass and load (m/z) of ionized molecules in the pCA standard, with peaks at 45, 65, and 127 m/z, which confirmed the presence of pCA (Fig. 1B). JOE — Volume 39, Number 5, May 2013
Chromatography and MS Analysis of 2% CHX at Two Time Points pCA detection was confirmed by chromatography of the compounds of the standard 2% CHX solution immediately after preparation, which revealed a peak at a retention time of 7.8 minutes with a magnitude of about 40,000 (Fig. 1C), and by MS, which showed peaks at 45, 65, and 127 m/z (Fig. 1D). The chromatographic analysis of compounds in the 2% CHX standard solution 7 days after preparation revealed a peak at a retention time of 7.8 minutes, with a magnitude greater than 75,000 (Fig. 1E). For 2% CHX 7 days after preparation, the peak in the chromatogram (Fig. 1E) at the 7.8-minute retention time and the MS peaks at 45, 65, and 127 m/z confirmed the presence of pCA (Fig. 1F). The magnitude of the peak at the 7.8-minute retention time plotted on the y-axis of a chromatogram for 2% CHX immediately after preparation, which was about 40,000 (Fig. 1C), was lower than that of the peak at the 7.8-minute retention time of 2% CHX 7 days after preparation, which was larger than 75,000 (Fig. 1E). Such findings suggested that storing the 2% CHX solution at 36.5 C and 95% relative humidity for 7 days increased pCA generation. GC/MS Analysis of Paste at Two Time Points pCA detection was confirmed by chromatography of the compounds of a paste produced by mixing 2% CHX solution and calcium hydroxide immediately after preparation, which revealed a peak at a retention time of 7.8 minutes, with a magnitude of about 40,000 (Fig. 1G), and by MS, which showed peaks at 45, 65, and 127 m/z (Fig. 1H). Seven days after preparation, chromatography revealed a peak at the 7.8-minute retention time, with a magnitude of close to 80,000 (Fig. 1I), and MS showed peaks at 45, 65, and 127 m/z (Fig. 1J), which also confirmed the presence of pCA. The magnitude of the peak at the 7.8-minute retention time plotted on the y-axis of a chromatogram of the paste immediately after its preparation, which was about 40,000 (Fig. 1G), was lower than the magnitude of the peak at the 7.8-minute retention time of the paste 7 days after preparation, which was close to 80,000 (Fig. 1I). Such findings suggested that storing the mixture of 2% CHX solution and Ca(OH)2 at 36.5 C and 95% relative humidity for 7 days increased pCA generation. pCA and pCNO were observed in control by using standard solutions of these substances. Data of pCNO were not detected.
Discussion pCA and ROS were detected in 2% CHX and in the combination of 2% CHX and calcium hydroxide at all time points, but pCNO was not found. GC/MS was used to evaluate the production of potentially toxic factors such as pCA, which originate from high concentrations of CHX (2% aqueous solution), as well as from mixing CHX and calcium hydroxide. pCA may be analyzed by using chromatography (6, 21, 23, 24, 28). Barbin et al (24) detected pCA in solutions with low concentrations of CHX (0.2% aqueous solution) stored at 36.5 C and 95% relative humidity 14 days after preparation, but no traces of pCA were found in the paste produced by mixing 0.2% CHX and calcium hydroxide at different time points, although ROS were generated by 0.2% CHX alone and CHX mixed with calcium hydroxide at all time points. Those authors also found that there were no traces of CHX in the paste (0.2% CHX + calcium hydroxide) immediately and 7 and 14 days after preparation when stored at 36.5 C and 95% relative humidity. This finding suggests that the addition of calcium hydroxide to 0.2% CHX leads to an immediate and total degradation of lowconcentration CHX (0.2% aqueous solution). The correlation of those
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Figure 1. (A) Chromatogram of standard pCA solution immediately after preparation; elutes from column at retention time of 7.8 minutes; x-axis shows retention time in minutes, and y-axis, signal intensity in total ion current (TIC) (1,000,000). (B) Mass spectrum of stock solution of pCA immediately after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-to-charge ratio (m/z), and y-axis, relative abundance (percentage). (C) Chromatogram of compounds in standard 2% CHX solution immediately after preparation: peak at 7.8-minute retention time and magnitude of about 4.0 10,000; x-axis shows retention time in minutes, and y-axis, signal intensity in TIC (10,000). (D) Mass spectrum of stock solution of 2% CHX immediately after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-tocharge ratio (m/z), and y-axis, relative abundance (percentage). (E) Chromatogram of compounds in standard 2% CHX solution 7 days after preparation; peak at 7.8minute retention time and magnitude greater than 0.75 100,000; x-axis shows retention time in minutes, and y-axis, signal intensity in TIC (100,000).
findings with the results of our study may indicate that higher CHX concentrations (2% aqueous solution) increase pCA generation in both substances (2% CHX alone and in combination with calcium hydroxide) immediately and 7 days after preparation. This study detected the presence of pCA in 2% CHX and in a paste produced by mixing CHX with calcium hydroxide both immediately after preparation and after storage for 7 days at a temperature of 36.5 C and 666
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95% relative humidity and suggests that pCA concentrations increased after storing under those conditions. Palarettia et al (29) developed a high-performance liquid chromatography method for CHX and its metabolites (pCA and pCNO) produced by oxidation reactions mediated by metalloporphyrins such as cytochrome P-450 models for metabolite prediction. They found that the presence of metalloporphyrins in the reaction substantially increased the formation of pCA (up to 58%) as JOE — Volume 39, Number 5, May 2013
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Figure 1. (continued) (F) Mass spectrum of stock solution of 2% CHX 7 days after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-to-charge ratio (m/z), and y-axis, relative abundance (percentage). (G) Chromatogram of compounds in paste produced by mixing 2% CHX solution with calcium hydroxide immediately after preparation; peak at 7.8-minute retention time and magnitude of about 0.4 100,000; x-axis represents retention time in minutes, and yaxis, signal intensity in TIC (100,000). (H) Mass spectrum of paste produced by mixing 2% CHX solution with calcium hydroxide immediately after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-to-charge ratio (m/z), and y-axis, relative abundance (percentage). (I) Chromatogram of compounds in paste produced by mixing 2% CHX solution with calcium hydroxide 7 days after preparation; peak at 7.8-minute retention time and magnitude of about 0.8 100,000; xaxis shows retention time in minutes, and y-axis, signal intensity in TIC (100,000). (J) Mass spectrum of paste produced by mixing 2% CHX solution with calcium hydroxide 7 days after preparation; peaks at 45, 65, and 127 m/z; x-axis shows mass-to-charge ratio (m/z), and y-axis, relative abundance (percentage).
a CHX metabolite, especially at a neutral pH. Their findings provided evidence of a possible in vivo formation of pCA as a CHX metabolite mediated by cytochrome P-450 metabolism, which might even increase the risks to the health of patients treated with a 2% CHX solution or a paste produced by mixing 2% CHX and calcium hydroxide. The formation of chloroaniline from all chloronitrobenzene isomers was also mediated by cytochrome P-450 metabolism. In addition, pCA is a common chemical intermediate produced from pCNO (30), which may explain why pCNO was not found in this study, because there may have been a total conversion of pCNO into pCA. Cutaneous absorption of CHX is not significant (31), which minimizes its toxicity when used topically, but the possible presence of CHX in host tissue during endodontic treatment, when CHX may extrude through the apical foramen, may lead to the spreading of CHX or its toxic by-products, JOE — Volume 39, Number 5, May 2013
such as pCA, pCNO, and ROS, in the periapical tissue and the lymphatic and circulatory systems. Some chemical interactions may be observed between substances used in endodontics (21–24, 32–35). CHX degrades into pCA and several reactive by-products, and its substantial antimicrobial activity and residual effects have been demonstrated (19, 36, 37). Particularly when applied in clinical endodontics at high concentrations for 7 days or longer (38), it may prolong pCA generation, and both CHX and its by-products may remain in host tissues for long periods of time. The combination of these effects may increase the potential systemic risk of CHX. pCA is a persistent environmental degradation product found in some herbicides and fungicides, has potential long-term toxicity and carcinogenicity in male rats and male mice, and may cause
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Basic Research—Technology methemoglobinemia (21, 30). In addition, pCA induces DNA damage in bacteria. However, the results of gene mutation are still inconclusive. Gene mutation, but not mitotic recombination, has been induced in fungi, and gene mutation, sister chromatid exchange, and chromosomal aberrations have been induced in cultured mammalian cells, but conflicting data were obtained for cell transformation. Although pCA carcinogenicity in experimental animals has been demonstrated, there is not enough evidence of it in humans. However, results indicate that it may also be carcinogenic to humans, and the International Agency for Research on Cancer has listed it under group 2B ‘‘possibly carcinogen to humans’’ (21, 30). Kacmar et al (39) evaluated the toxic and immunotoxic effects of a pCA metabolite of a herbicide (monolinuron) in suspensions of peripheral blood leukocytes of 5 sheep by using a migration-inhibition test. The toxic effects of pCA were recorded at concentrations of 1.0–0.1 mg/mL 1 and immunotoxic effects at 0.01–0.001 mg/mL 1. Results confirmed total inhibition of leukocyte migration. Immunotoxic effects, defined as mitogenetic activation of leukocytes by phytohemagglutinin, concanavalin A, and lipopolysaccharide, were detected at pCA concentrations 10–100 times lower than those that resulted in toxic effects. The costs (systemic risks) and benefits of choosing CHX as a firstchoice agent to control endodontic infection must be weighed against those of other substances with antimicrobial properties, so that the goals of endodontic treatment may be achieved predictably and without unnecessary risks of systemic damage. Further studies should evaluate which CHX concentrations are safe for use in humans by assessing how much pCA may be generated when using 0.12%–2% CHX solutions, gel, and combinations with other substances.
Acknowledgments The authors deny any conflicts of interest related to this study.
References 1. Nair PNR, Henry S, Cano V, Vera J. Microbial status of apical root canal system of human mandibular first molars with primary apical periodontitis after ‘‘one-visit’’ endodontic treatment. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005; 99:231–52. 2. Paquette L, Legner M, Fillery ED, Friedman S. Antibacterial efficacy of chlorhexidine gluconate intracanal medication in vivo. J Endod 2007;33:788–95. 3. Estrela C, Holland R, Bernabe PFE, et al. Antimicrobial potential of medicaments used in healing process in dogs’ teeth with apical periodontitis. Braz Dent J 2004;15:181–5. 4. 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. 5. Siqueira JF Jr, Paiva SSM, R^oc¸as IN. Reduction in the cultivable bacterial populations in infected root canals by a chlorhexidine-based antimicrobial protocol. J Endod 2007;33:541–7. 6. Wang CS, Arnold RR, Trope M, Teixeira FB. Clinical efficiency of 2% chlorhexidine gel in reducing intracanal bacteria. J Endod 2007;33:1283–9. 7. Estrela C, Sydney GB, Figueiredo JAP, Estrela CRA. Antibacterial efficacy of intracanal medicaments on bacterial biofilm: a critical review. J Appl Oral Sci 2009;17:1–7. 8. Bystr€om A, Claesson R, Sundqvist G. The antibacterial effect of camphorated paramonochlorophenol, camphorated phenol and calcium hydroxide in the treatment of infected root canals. Dent Traumatol 1985;1:170–5. 9. Haapasalo M, Ørstavik D. In vitro infection and of dentinal tubules. J Dent Res 1987; 66:1375–9. 10. Gomes BPFA, Souza SFC, Ferraz CCR, et al. Effectiveness of 2% chlorhexidine gel and calcium hydroxide against Enterococcus faecalis in bovine root dentine in vitro. Int Endod J 2003;36:267–75. 11. Ohara PK, Torabinejad M, Kettering JD. Antibacterial effects of various endodontic irrigants on selected anaerobic bacteria. Endod Dent Traumatol 1993;9:95–100. 12. Buck RA, Cai J, Eleazer PD, et al. Detoxification of endotoxin by endodontic irrigants and calcium hydroxide. J Endod 2001;27:325–7. 13. Zehnder M. Root canal irrigants. J Endod 2006;32:389–98.
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Barbin et al.
14. Kontakiotis EG, Tsatsoulis LN, Papanakou SI, Tzanetakis GN. Effect of 2% chlorhexidine gel mixed with calcium hydroxide as an intracanal medication on sealing ability of permanent root canal filling: a 6-month follow-up. J Endod 2008;34: 866–70. 15. Estrela CRA, Estrela C, Reis C, et al. Control of microorganisms in vitro by endodontic irrigants. Braz Dent J 2003;14:187–92. 16. Estrela C, Estrela CRA, Decurcio DA, et al. Antimicrobial efficacy of ozonated water, gaseous ozone, sodium hypochlorite and chlorhexidine in infected human root canals. Int Endod J 2007;40:85–93. 17. Havlıkova L, Matysova L, Novakova L, et al. HPLC determination of chlorhexidine gluconate and p-chloroaniline in topical ointment. J Pharm Biomed Anal 2007; 43:1169–73. 18. Denton GW. Chlorhexidine. In: Block SS, ed. Disinfection, Sterilization and Preservation. 4th ed. Philadelphia: Lea & Febiger; 1991:274–89. 19. Basrani B, Santos JM, Tj€aderhane L, et al. Substantive antimicrobial activity in chlorhexidine-treated human root dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2002;94:240–5. 20. Usui K, Hishinuma T, Yamaguchi H, et al. Determination of chlorhexidine (CHD) and nonylphenolethoxylates (NPEOn) using LC-ESI-MS method and application to hemolyzed blood. J Chromatogr B 2006;831:105–9. 21. World Health Organization. WHO Library Cataloguing-in-Publication Data: Concise International Chemical Assessment Document—4-Chloroaniline. Geneva, Switzerland: World Health Organization; 2003. 1–58. 22. Yeung SY, Huang CS, Chan CP, et al. Antioxidant and pro-oxidant properties of chlorhexidine and its interaction with calcium hydroxide solutions. Int Endod J 2007;40: 837–44. 23. Basrani BR, Manek S, Sodhi RNS, et al. Interaction between sodium hypochlorite and chlorhexidine gluconate. J Endod 2007;33:966–9. 24. Barbin LE, Saquy PC, Guedes DFC, et al. Determination of para-chloroaniline and reactive oxygen species in chlorhexidine and chlorhexidine associated with calcium hydroxide. J Endod 2008;34:1508–14. 25. World Health Organization. International Agency for Research on Cancer: Monographs on the Evaluation of Carcinogenic Risks to Humans—Occupational Exposures of Hairdressers and Barbers and Personal Use of Hair Colourants; Some Hair Dyes, Cosmetic Colourants, Industrial Dyestuffs and Aromatic Amines. Lyon, France: International Agency for Research on Cancer; 1993. 1–436. 26. Waris G, Ahsan H. Reactive oxygen species: role in the development of cancer and various chronic conditions. J Carcinog 2006;5:1–8. 27. Faria G, Celes MRN, Rossi A, et al. Evaluation of chlorhexidine toxicity injected in the paw of mice and added to cultured L929 fibroblasts. J Endod 2007;33: 715–22. 28. Ciarlone AE, Gangarosa LP, Fong BC. Detection of p-chloroaniline in chlorhexidine solutions using thin-layer chromatography. J Dent Res 1976;55:918. 29. Palarettia V, Faria AL, Santos JS, et al. Metaloporfirinas como catalisadores na oxidac¸~ao do agente antimicrobiano clorexidina. XXII CICAT, Congresso Iberoamericano de Catalise, 2010. 30. World Health Organization. International Agency for Research on Cancer: ParaChloroaniline—Monographs on the Evaluation of Carcinogenic Risks to Humans: Occupational Exposures of Hairdressers and Barbers and Personal Use of Hair Colourants; Some Hair Dyes, Cosmetic Colourants, Industrial Dyestuffs and Aromatic Amines. Lyon, France: International Agency for Research on Cancer; 1993. 305–321. 31. Karpanen TJ, Worthington T, Conway BR, et al. Permeation of chlorhexidine from alcoholic and aqueous solutions within excised human skin. Antimicrob Agents Chemother 2009;53:1717–9. 32. Kim HS, Zhu Q, Baek SH, et al. Chemical interaction of alexidine and sodium hypochlorite. J Endod 2012;38:112–6. 33. Nowicki JB, Sem DS. An in vitro spectroscopic analysis to determine the chemical composition of the precipitate formed by mixing sodium hypochlorite and chlorhexidine. J Endod 2011;37:983–8. 34. Thomas JE, Sem DS. An in vitro spectroscopic analysis to determine whether parachloroaniline is produced from mixing sodium hypochlorite and chlorhexidine. J Endod 2010;36:315–7. 35. 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. 36. Rosenthal S, Sp angberg L, Safavi K. Chlorhexidine substantivity in root canal dentin. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;98:488–92. 37. White RR, Hays GL, Janer LR. Residual antimicrobial activity after canal irrigation with chlorhexidine. J Endod 1997;23:229–31. 38. Komorowski R, Grad H, Wu XY, Friedman S. Antimicrobial substantivity of chlorhexidine-treated bovine root dentin. J Endod 2000;26:315–7. 39. Kacmar P, Pistl J, Mikula I. The effect of p-chloroaniline on leucocytes of sheep peripheral blood under the migration-inhibition test conditions. Immunopharmacol Immunotoxicol 1995;17:577–84.
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