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TITLE PAGE

Title of the article: Comparative evaluation of the cytotoxicity of MTAD, 2% chlorhexidine and 5.25% sodium hypochlorite on human red blood corpuscles.

Contributors 1. Dr. Krishna Prasad Shetty MDS Professor and Head Department of Conservative dentistry and Endodontics Navodaya Dental College & Hospital. Raichur Karnataka. India Email: kprasad11@yahoo.com 2. Dr. Sarvepalli Venkata Satish MDS Professor Department of Conservative dentistry and Endodontics Navodaya Dental College & Hospital. Raichur Karnataka. India Email: drsatishdent@gmail.com 3. Dr. Krishnarao Kilaru MDS, DNB Reader Department of Conservative dentistry and Endodontics Navodaya Dental College & Hospital. Raichur Karnataka. India. Email: drkrishna2@yahoo.com 4. Dr. Ratnakar P. MDS Reader Department of Conservative dentistry and Endodontics H K E S Dental College. Gulbarga Karnataka. India

2 Email: dr.ratnakarchoudary@gmail.com 5. Dr. Vijay Reddy Venumuddala MDS Professor and Head Hitech Dental College and Hospital. Bhubneswar Orissa. India Email: vijdent@yahoo.com 6. Dr. Kalyana Chakravarthi Ponangi BDS Post Graduate Student. Department of Conservative dentistry and Endodontics Navodaya Dental College & Hospital. Raichur Karnataka. India Email: drkalyandent@gmail.com

Department and institution Department of Conservative dentistry and Endodontics. Navodaya Dental College & Hospital. Raichur Karnataka. India

Running title: cytotoxicity of three endodontic irrigants on human red blood corpuscles: MTAD, 2% chlorhexidine and 5.25% sodium hypochlorite. Key-words: Cytotoxicity-Endodontic irrigants; Red Blood Corpuscles; MTADcytotoxicity; Chlorhexidine-cytotoxicity; Sodium Hypochlorite-cytotoxicity.

Corresponding Author: Name:Dr. Krishna Prasad Shetty Address: Department of Conservative dentistry and Endodontics Navodaya dental college & hospital. Navodaya nagar. Mantralayam road. Raichur. PIN 584103 Karnataka. India. Phone numbers: 919611282811 Facsimile numbers: 08532-223043 E-mail address: kprasad11@yahoo.com

4 Title of the article: Comparative evaluation of the cytotoxicity of 5.25% Sodium hypochlorite, 2% Chlorhexidine & MTAD on human red blood corpuscles: An in-vitro study

Abstract:

Aim: The aim of this study was to analyse the cytotoxicity of various volumes of 5.25% Sodium hypochlorite, 2% Chlorhexidine gluconate and MTAD by checking for haemolysis of human red blood corpuscles. Methodology: Hundred micro litres of diluted Red Blood Corpuscles obtained through centrifugation was added to three groups (Group I: 5.25% Sodium hypochlorite, Group II: 2% Chlorhexidine, Group III: MTAD) of six test tubes each. Individual irrigants per group were added in increasing volume starting from the second test tube keeping the first one as the control. After incubation for three minutes haemoglobin content was measured using an automated haemoanalyser. Data was analyzed using one sample t test.

Results: Sodium hypochlorite is the most cytotoxic followed by MTAD and chlorhexidine the least.

Conclusions: This study suggests that these irrigating fluids do cause detrimental effects on vital tissues. Sodium hypochlorite though an irrigant of choice should be used cautiously. Chlorhexidine should be used when excessive extrusion is expected. MTAD

5 is the irrigant of choice because of its antimicrobial efficacy and smear layer removing ability.

Key Messages: 2% Chlorhexidine is less cytotoxic when compared to 5.25% Sodium Hypochlorite and MTAD. Therefore 2% Chlorhexidine is the irriganting solution of choice in teeth with incomplete root development and perforations.

6 Text Introduction:

Successful endodontic treatment depends on the ability of the operator to mechanically and chemically clean the root canal system and subsequently obturate it three dimensionally (1). Endodontic therapy is primarily based on the removal of potentially noxious stimuli from the complex root canal system (2).

The complexity of the root canal system is, however, one of the determining factors in the failure of root canal treatment even in properly treated teeth, due to irregular and sometimes unpredictable spaces that characterize root canals (3, 4). The persistence of residual pulp tissue, infected dentin or bacteria in the root canal system is responsible for treatment failure (5). Irrigation, therefore, plays a crucial role in determining the outcome of root canal treatment (6). Irrigants flush debris from canals and assist in reducing microbial flora of infected canals and help to dissolve the necrotic tissues.

An endodontic irrigant should be non toxic when it comes in contact with vital tissues and non caustic to the periodontal tissues (7). A potential complication of irrigation is the forced extrusion of the irrigant and debris through the apex. Tissue cytotoxicity is therefore a major concern when choosing an endodontic irrigant for root canal treatment.

Sodium hypochlorite has solvent activity for both necrotic and vital tissues (2). It is known in medical fields since the beginning of 20 th century and was introduced as a part

7 of endodontic treatment in 1936 by Walker. Literature is full of studies showing severe irritations when concentrated solution were inadvertently forced into the periapical tissues beyond the apex of the tooth under endodontic treatment (8, 9).

Chlorhexidine was developed in the late 1940s in the research laboratories of Imperial Chemical Industries Ltd (7). Chlorhexidine is a cationic biguanide that possesses broad antibacterial activity in combination with relatively low toxicity and the ability to bind to skin and mucous membranes.

MTAD was introduced as an alternative to EDTA to remove the smear layer by Torabinejad et al. It is a mixture of 3% doxycycline, 4.25% citric acid and detergentTween 80. It has a combined chelating and antibacterial property (10).

So the main objective of this study is to compare the cytotoxicity of 5.25%sodium hypochlorite, 2%chlorhexidine and MTAD.

8 Materials and Methods: The informed consent of all human subjects who participated in the experimental investigation reported or described in this manuscript was obtained after the nature of the procedure and possible discomforts and risks had been fully explained. And the institutional IRB approved the protocol.

Red Blood Corpuscles are chosen as the convenient cell type as they are readily available and whose intracellular haemoglobin content can be easily measured. Fresh blood was drawn from a human volunteer and collected in EDTA bottles. Centrifugation of the blood is done, plasma removed and packed cell volume of Red Blood Corpuscles is obtained. Red Blood Corpuscles are washed with saline and centrifuged several times to remove white cells and any traces of plasma. One millilitre of packed cell volume is added to four millilitres of saline to increase the volume of blood to five millilitres. Hundred micro litres of this diluted Red Blood Corpuscles is added to 18 test tubes with six test tubes in each group. The groups are as mentioned below.

Group I: 5.25% Sodium hypochlorite (VENSONS INDIA) Group II: 2% Chlorhexidine gluconate (RC-CHLOR, DEOR, INDIA) Group III: MTAD (DENTSPLY Tulsa Dental, United States) For all the three groups, the first test tube is kept as a control in which no irrigant is added. In the second test tube ten micro litres of the irrigant is added. Twenty micro litres is added to third test tube, thirty micro litres to the fourth test tube, forty micro litres to the fifth test tube and fifty micro litres to the sixth test tube.

9 After an incubation time of three minutes, haemoglobin percentage after haemolysis of red blood corpuscles is noted using an automated haemoanalyser (ABX MICROS 60). The haemoanalyser measures the haemoglobin content of the remaining red blood corpuscles after haemolysis. For all the three groups the experiment is repeated three times and the mean value is taken. Data obtained in the present study is subjected to statistical analysis using one sample t test. A p value of less than .05 is considered as significant. Statistical analysis is done using Minitab V.14 software.

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Results:

Group I (5.25% Sodium Hypochlorite) showed significant cytotoxicity in comparison to the control group. The cytotoxicity of the irrigant is directly proportional to the increase in the volume and the exposure time. At a volume of fifty micro litres, Sodium Hypochlorite showed complete haemolysis of the Red Blood Corpuscles as shown in Table 1.

GROUP I 5.25% Sodium Hypochlorite Volume of Percentage of Percentage of Percentage of the haemoglobin haemoglobin haemoglobin irrigant in Trial I Trial II Trial III micro litres 0(control) 4.4 4.4 4.4 10 1.9 2.0 2.0 20 1.0 0.9 1.1 30 0.3 0.4 0.3 40 0.1 0.2 0.1 50 0 0 0 Table 1: Sodium Hypochlorite shows complete haemolysis at a volume of 50 micro litres. Group II (2% Chlorhexidine gluconate), regarded as the safest among the three irrigants showed cytotoxicity which is statistically significant. The cytotoxicity is least when compared to MTAD and Sodium Hypochlorite. The test results are shown in Table 2.

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GROUP II 2% Chlorhexidine gluconate Volume of Percentage of Percentage of Percentage of irrigant in haemoglobin haemoglobin haemoglobin micro litres Trial I Trial II Trial III 0(control) 4.4 4.4 4.4 10 3.7 3.5 3.7 20 3.5 3.5 3.4 30 3.3 3.3 3.2 40 3.0 2.8 3.0 50 2.8 2.6 2.8 Table 2: Reduction in the haemoglobin percentage as the irrigant volume increases

Group III (MTAD) showed cytotoxicity which is statistically significant but lesser when compared to sodium hypochlorite and more when compared to chlorhexidine. The test results are shown in Table 3.

GROUP III MTAD Volume of Percentage of Percentage of Percentage of irrigant in haemoglobin haemoglobin haemoglobin micro litres Trial I Trial II Trial III 0(control) 4.4 4.4 4.4 10 3.5 3.6 3.6 20 3.5 3.4 3.4 30 3.1 3.2 3.0 40 2.9 3.0 2.9 50 2.7 2.6 2.7 Table 3: Reduction in the haemoglobin percentage as the irrigant volume increases. Chlorhexidine is the least cytotoxic of the compared irrigants.

The mean and standard deviation of the percentage of haemoglobin for all the three irrigants using one sample t test, depicting significance is shown in Tables 4, 5 and 6.

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Group I - 5.25% Sodium Hypochlorite Volume of the irrigant in micro litres 10 20 30 40 Table Mean differe nce 95% CI differenc e 2.29 2.57 3.15 3.65 3.92 4.21 4.12 4.41

Min

Max

Mean SD

tvalue

p-value

Remarks

1.97 0.06 1 0.9 1.1 0.1 0.33 0.3 0.4 0.06 0.13 0.1 0.2 0.06 4: Statistically significant 1.9 2.0

2.43 3.40 4.07 4.27

73 58.8 9 122 128

P<0.000 2 P<0.000 3 P<0.000 1 P<0.000 1

Significance Significance Significance Significance

cytotoxicity shown by sodium hypochlorite at all the

volumes tested.

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Group II - 2% Chlorhexidine gluconate Volume of the irrigant in micro litres 10 20 30 40 50 Table Mean differenc e 95% CI differenc e

Min

Max

Mean SD

tvalue

p-value

Remarks

3.63 0.47 0.76 11.5 P<0.007 Significance 1.05 0.11 3.47 0.79 3.4 3.5 0.93 28 P<0.001 Significance 1.07 0.06 3.27 0.99 3.2 3.3 1.13 34 P<0.0009 Significance 1.27 0.06 2.93 1.18 2.8 3.0 1.47 22 P<0.002 Significance 1.75 0.11 2.73 1.38 2.6 2.8 1.67 25 P<0.002 Significance 1.95 0.11 5: Statistically significant cytotoxicity shown by chlorhexidine gluconate at all the 3.5 3.7

volumes tested.

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Group III MTAD Volume of the irrigant in micro litres 10 Mean differenc e 95% CI differenc e

Min

Max

Mean SD

tvalue

p-value

Remarks

3.57 0.68 0.83 25 P<0.002 Significance 0.97 0.06 3.43 0.82 20 3.4 3.5 0.97 29 P<0.001 Significance 1.11 0.06 3.1 1.05 30 3.0 3.2 1.30 22.52 P<0.002 Significance 0.06 1.55 2.93 1.32 40 2.9 3.0 1.47 44 P<0.0005 Significance 1.61 0.06 2.63 1.62 50 2.6 2.7 1.77 53 P<0.0004 Significance 1.91 0.06 Table 6: Statistically significant cytotoxicity shown by MTAD at all the volumes tested. 3.5 3.6 Graphical representation of the fall in the percentage of haemoglobin with the increase in the volume of the irrigant for all the three groups is shown in Graph 1.

15 Discussion:

Sodium hypochlorite is considered as an ideal irrigant in endodontics (Grossman et al., 1941) (11). Hypochlorite preparations are sporicidal, virucidal and show far greater tissue dissolving effects on necrotic than on vital tissues. These features prompted the use of aqueous sodium hypochlorite in endodontics as the main irrigant as early as 1920 (12).

Various concentrations of sodium hypochlorite from 0.5% to 5.25% have been tried out (13, 14). Higher the concentration better will be the antimicrobial effect and the tissue dissolving capacity. At the same time higher concentration also carries the risk of toxicity and tissue reaction. It is shown that 1% sodium hypochlorite is sufficient to dissolve the pulp tissue (15). Higher concentrations of sodium hypochlorite are proven to have greater bactericidal effect (16). Sodium hypochlorite (5.25%) in 1:1000 dilution caused complete hemolysis of Red blood cells in vitro (Pashley et al., 1985) (17). On investigating various concentrations of Sodium Hypochlorite (0.25%, 0.025 % and 0.0125%) for antimicrobial activity and tissue toxicity at varying time intervals it is found that 0.025% of Sodium Hypochlorite is bactericidal and not tissue toxic and 0.25% concentration of Sodium Hypochlorite showed tissue toxicity (Heggers et al., 1991) (18).Sodium Hypochlorite in a concentration of 0.55% induced an inflammatory response (M.Tanomaru Filho et al., 2002) (19). Sodium hypochlorite is toxic enough to cause complete haemolysis according to this study.

16 Chlorhexidine is recommended as an endodontic irrigant because of its antimicrobial activity, presumed non toxicity and intracanal substantivity (20). At higher

concentrations, Chlorhexidine results in extensive cell damage, coagulation of cytoplasm, and precipitation of proteins and nucleic acids (21).The concentration often used in endodontic therapy is 2% as it is more effective in less time when compared to other concentrations of Chlorhexidine ranging from 0.002% to 2% (22).

Chlorhexidine is toxic to human gingival cells and the toxic potency is dependent on the length of exposure and the composition of the exposure medium (23). On comparing the inflammatory response of 0.5% Sodium Hypochlorite, 2% Chlorhexidine digluconate, and phosphate buffered saline, it is found that 2% Chlorhexidine injection is similar to the phosphate-buffered saline control at all times tested, while the 0.5% Sodium Hypochlorite injection resulted in significant inflammation (Tanamaru Filho et al., 2002) (20).On treating fifty pieces of bovine pulp tissue with normal saline, MTAD, 2%

Chlorhexidine digluconate and 2.5% Sodium Hypochlorite it is found that Chlorhexidine has the weakest tissue dissolution capacity(Khademi et al., 2007) (24).

Chlorhexidine is recommended as an alternative in patients allergic to sodium hypochlorite or in teeth with incomplete root formation (22) and crestal perforations to prevent inflammatory response in proximity to the epithelial attachment (25).

Bio PureMTAD (Dentsply, Tulsa, OK) is designed to be used as a final root canal rinse before obturation (11). Tetracycline has many unique properties of low pH and acts as a

17 calcium chelator and cause enamel and root surface demineralization (26). MTAD is effective in removing the smear layer along the whole length of the root canal and does not produce any signs of erosion or physical changes in dentin (27, 28 and 29). MTAD is effective against Enterococcus faecalis, and it is less cytotoxic than a range of endodontic medicaments, including eugenol, hydrogen peroxide (3%), EDTA, and calcium hydroxide paste (30). In this study MTAD is found to be less cytotoxic than sodium hypochlorite and more cytotoxic than chlorhexidine.

Acknowledgement We affirm that we have no financial affiliation or involvement with any commercial organization with direct financial interest in the subject or materials discussed in this manuscript, nor have any such arrangements existed in the past three years. We deny any conflict of interest related to this study.

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Conclusion:

The ideal functions of an irrigant are to dissolve both the organic as well as inorganic components, render the root canal free of microbes and be non toxic to the tissues. No single irrigant accomplishes all the functions. This study suggests that these irrigating fluids do cause detrimental effects on vital tissues. The clinical situation, concentration used, exposure time to the agent and the exposure surface area are important factors which affect the cytotoxicity of the irrigating solution. Therefore taking into account the limitations & merits of the irrigating solutions, if used judiciously, it adds to the success of root canal therapy.

19 References:

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20 8. Witton R, Henthorn K, Ethunandan M, et al. Neurological complications following extrusion of sodium hypochlorite solution during root canal treatment. International Endodontic Journal 2005; 38:843-8. 9. Gursoy UK, Bostanci V, Kosger HH. Palatal mucosa necrosis because of accidental sodium hypochlorite injection instead of anesthetic solution. International Endodontic Journal 2006; 39:157-61. 10. Torabinejad M, Khademi A A, Babagoli j, Cho Y, Johnson W B, Bozhilov K (2003) A new solution for removal of smear layer. Journal of Endodontics 2003; 29:170-5. 11. Grossman L I, Meiman B W (1941) Solution of pulp tissue by chemical agents. Journal of American Dental Association 1941; 28:223-5. 12. Kandaswamy D, Venkateshbabu N. Root canal irrigants. Journal of Conservative Dentistry 2010; 13:256-64. 13. Gomes BPFA, Ferraz CCR, Vianna MEVB, Berber FB. In vitro antimicrobial activity of several concentrations of sodium hypochlorite and Chlorhexidine Gluconate in the elimination of Enterococcus Faecalis. International

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21 15. Sirtes G, Waltimo T, Schaetzle M, Zehnder M. The effect of temperature on sodium hypochlorite short-term stability, pulp dissolution capacity, and antimicrobial efficacy. Journal of Endodontics 2005; 31: 669-71. 16. Arieh Y Kaufman, Senia Keila. Hypersensitivity to sodium hypochlorite. Journal of Endodontics 1989; 15:224-6. 17. EL Pashley, N.L Birdsong, k Bowman and D.H pashley. Cytotoxic effects of Sodium hypochlorite on vital tissue. Journal of Endodontics 1985; 11:525-8. 18. Heggers JP, Sazy A J, Stenberg BD, Strock LL, Mc Cauley RL, Hernon DN, Robson MC. Bacterial and wound healing properties of sodium hypochlorite solutions. Journal of Burn Care Rehabilitation 1991; 12:420-4. 19. M Tanomaru Filho MR Leonardo L .A .B Silva, F .F Anibal and L.H Faccioli. Inflammatory response to different endotoxic irrigating solutions. International Endodontic Journal 2002; 35:735-9. 20. Leonard MR, Tanomaru Filho, M.Silva CAB, Nelson Filho N, Bonifacio KC. In vivo antimicrobial activity of 2% chlorhexidine used as a root canal irrigating solution. Journal of Endodontics 1999; 25:167-71. 21. M E Vianna, BPFA Gomes. In vitro evaluation of the antimicrobial activity of Chlorhexidine and sodium hypochlorite. Oral surgery Oral Medicine Oral Pathology 2004; 97:79-84. 22. Lessa, F C Rosetti, Nogueira,F Silveira. Direct and transdentinal antibacterial activity of chlorhexidine. American Journal of Dentistry 2010; 25:255-9.

22 23. Babich H, Wurzburger BJ, Rubin Y L, Sinensky MC, Blau L (1995) An in Vitro study on the cytotoxicity of Chlorhexidine digluconate to human gingival cells. Cell Biol Toxicol 1995; 11:79-88. 24. Abbasali Khademi, Ehsan usefian, Mahboobe F. Tissue dissolving ability of several endodontic irrigants on bovine pulp tissue. Iranian Endodontic Journal 2007; 2:65-67. 25. Fuss Z, Trope M. Root perforations classification and treatment choices based on prognostic factors. Endodontics and Dental Traumatology 1996; 12: 225-64. 26. K. Bjorvatn, N. Skaug, and K. A. Selvig. Tetracycline-impregnated enamel and dentin: duration of antimicrobial capacity. Scandinavian Journal of Dental Research 1985; 93:1927. 27. M. Torabinejad, Y. Cho, A. A. Khademi, L. K. Bakland, S. Shabahang. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. Journal of Endoddontics 2003; 29:2339. 28. R. E. Beltz, M. Torabinejad, M. Pouresmail. Quantitative analysis of the solubilizing action of MTAD sodium hypochlorite, and EDTA on bovine pulp and dentin. Journal of Endodontics 2003; 29:3347. 29. T. K. Machnick, M. Torabinejad, C. A. Munoz, S. Shabahang. Effect of MTAD on flexural strength and modulus of elasticity of dentin. Journal of Endoddontics 2003; 29:74750.

23 30. S. Shabahang, M. Pouresmail, M. Torabinejad. In vitro antimicrobial efficacy of MTAD and sodium hypochlorite. Journal of Endodontics 2003; 29:4502.

FIGURE LEGENDS: Figure 1 (graph 1): Graphical representation of the fall in the percentage of haemoglobin with the increase in the volume of the irrigant for all the three groups is shown in Graph 1.

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