|Year : 2014 | Volume
| Issue : 1 | Page : 1-6
Comparative evaluation of the cytotoxicity of 5.25% sodium hypochlorite, 2% chlorhexidine and mixture of a tetracycline isomer, an acid and a detergent on human red blood corpuscles: An in-vitro study
Krishna Prasad Shetty1, Sarvepalli Venkata Satish1, Krishnarao Kilaru1, Kalyana Chakravarthi Ponangi1, Vijay Reddy Venumuddala2, P Ratnakar3
1 Department of Conservative Dentistry and Endodontics, Navodaya Dental College and Hospital, Raichur, Karnataka, India
2 Department of Conservative Dentistry and Endodontics, Hitech Dental College and Hospital, Bhubneswar, Odisha, India
3 Department of Conservative Dentistry and Endodontics, HKES Dental College, Gulbarga, Karnataka, India
|Date of Web Publication||28-Feb-2014|
Krishna Prasad Shetty
Department of Conservative Dentistry and Endodontics, Navodaya Dental College and Hospital, Navodaya Nagar, Mantralayam Road, Raichur 584 103, Karnataka
Source of Support: None, Conflict of Interest: None
Aim: The aim of this study was to analyze the cytotoxicity of various volumes of 5.25% of sodium hypochlorite, 2% of chlorhexidine gluconate and mixture of a tetracycline isomer, an acid and a detergent (MTAD) by checking for hemolysis of human red blood corpuscles. Materials and Methods: A total volume of 100 μl of diluted red blood corpuscles obtained through centrifugation was added to three irrigating solutions (5.25% of sodium hypochlorite, 2% of chlorhexidine and 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 3 min hemoglobin content was measured using an automated hemoanalyzer data was analyzed using one sample t-test. Results: Sodium hypochlorite is the most cytotoxic solution followed by MTAD and chlorhexidine. Conclusions: This study suggests that the three irrigating solutions do cause detrimental effects on the diluted red blood corpuscles. A great deal of care should therefore be exercised when using 5.25% sodium hypochlorite during endodontic irrigation.
Keywords: 2% chlorhexidine, 5.25% sodium hypochlorite, cytotoxicity, diluted red blood corpuscles, irrigating solution, mixture of a tetracycline isomer, an acid and a detergent
|How to cite this article:|
Shetty KP, Satish SV, Kilaru K, Ponangi KC, Venumuddala VR, Ratnakar P. Comparative evaluation of the cytotoxicity of 5.25% sodium hypochlorite, 2% chlorhexidine and mixture of a tetracycline isomer, an acid and a detergent on human red blood corpuscles: An in-vitro study. Saudi Endod J 2014;4:1-6
|How to cite this URL:|
Shetty KP, Satish SV, Kilaru K, Ponangi KC, Venumuddala VR, Ratnakar P. Comparative evaluation of the cytotoxicity of 5.25% sodium hypochlorite, 2% chlorhexidine and mixture of a tetracycline isomer, an acid and a detergent on human red blood corpuscles: An in-vitro study. Saudi Endod J [serial online] 2014 [cited 2019 May 19];4:1-6. Available from: http://www.saudiendodj.com/text.asp?2014/4/1/1/127979
| Introduction|| |
Successful endodontic treatment depends on effective mechanical and chemical cleaning of the root canal system and subsequently fill it in three dimension.  Endodontic therapy is primarily based on the removal of potentially noxious stimuli from the complex root canal system.  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 characteristic of the root canals system. , The persistence of residual pulp tissue, infected dentin or bacteria in the root canal system is responsible for treatment failure. 
Irrigation dynamics plays a significant role during root canal treatment , and the effectiveness depends on the working mechanism(s) of the irrigant and the ability to bring the irrigant in contact with the microorganisms and tissue debris in the root canal system. ,
Several irrigating solution have been used during root canal treatment. Sodium hypochlorite is the most commonly used irrigant in endodontics. It has an antimicrobial as well as solvent activity for both necrotic and vital tissues. , Severe irritations of the periradicular tissue usually resulted if concentrated solution is forced beyond the apex of the tooth. ,
Chlorhexidine is a cationic biguanide that possesses broad antibacterial activity in combination with relatively low toxicity. Nearly 2% has been suggested as an irrigant for the root canal system, which lacks the ability to dissolve the organic and inorganic material. ,
A mixture of 3% doxycycline, 4.25% citric acid and detergent-Tween 80 (mixture of a tetracycline isomer, an acid and a detergent [MTAD]) was introduced as an alternative to ehylenediamine tetra acetic acid (EDTA) to remove the smear layer. It is. It has a combined chelating and antibacterial property. 
An endodontic irrigant should be non-toxic when it comes in contact with vital tissues and non-caustic to the periodontal tissues. 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. Various methods have been employed to evaluate the cytotoxicity of endodontic irrigants. Pashley et al. evaluated the cytotoxicity on red blood corpuscles,  Faria et al.  and Zhang et al.  evaluated the cytotoxicity on L929 fibroblasts and Barnhart et al. on gingival fibroblasts. 
Previous studies have shown MTAD to be less cytotoxic than 5.25% sodium hypochlorite and Peridex (0.12% chlorhexidine).  However, the optimal concentration of chlorhexidine (2%), with which MTAD has not been compared regarding the toxic potential.
Therefore, the aim of this study was to analyze the cytotoxicity of various volumes of 5.25% sodium hypochlorite, 2% chlorhexidine gluconate and MTAD by checking for hemolysis of human red blood corpuscles.
| Materials and Methods|| |
The informed consent of the human subject who participated in the experimental investigation was obtained after the nature of the procedure was explained and the Institutional Review Board approved the protocol.
Red blood corpuscles are chosen as the convenient cell type as they are readily available and whose intracellular hemoglobin 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. A volume of 1 ml of packed cell is added to 4 ml of saline to increase the volume of blood to 5 ml. A total volume of 100 μl 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 (VIP Vensons India, Bangalore, India)
Group II: 2% chlorhexidine gluconate (Deor, Kochi, India)
Group III: MTAD (Dentsply Tulsa Dental, Johnson City, TN, United States of America).
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 10 μl of the irrigant is added. 20 μl is added to the third test tube, 30 μl to the fourth test tube, 40 μl to the fifth test tube and 50 μl to the sixth test tube.
After an incubation time of 3 min, hemoglobin percentage after hemolysis of red blood corpuscles is noted using an automated hemoanalyzer (ABX Micros 60, HORIBA ABX, Japan). The hemoanalyzer measures the intracellular hemoglobin content of the remaining red blood corpuscles after hemolysis. For all the three groups, the experiment is repeated 3 times and the mean value is taken.
Data obtained in the present study is subjected to statistical analysis using one sample t-test. P <0.05 is considered as significant. Statistical analysis is performed using Minitab V.14 software (Minitab, Ltd. Coventry, UK).
| 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 50 μl, sodium hypochlorite showed complete hemolysis of the red blood corpuscles. Group II
(2% chlorhexidine gluconate) showed cytotoxicity which is statistically significant, but the level of cytotoxicity was minimal when compared to sodium hypochlorite and MTAD. Group III (MTAD) showed cytotoxicity, which is statistically significant but lesser when compared to sodium hypochlorite and more when compared to chlorhexidine.
Graphical representation of the fall in the percentage of hemoglobin with the increase in the volume of the irrigant for all the three groups is shown in Graph 1. Representation of the mean hemoglobin percentage with respect to the three groups is shown Graph 2.
Comparison of three groups were done by ANOVA test followed by post-hoc Tukey-Kramer pairwise comparisons test [Table 1].
The mean and standard deviation of the percentage of hemoglobin for all the three irrigants using one sample t-test, done to compare the cytotoxicity with the control group depicting significance is shown in [Table 2], [Table 3], [Table 4].
|Table 2: Statistically significant cytotoxicity shown by sodium hypochlorite at all the volumes tested |
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|Table 3: Statistically significant cytotoxicity shown by chlorhexidine gluconate at all the volumes tested |
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|Table 4: Statistically significant cytotoxicity shown by MTAD at all the volumes tested |
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| Discussion|| |
The use of human red blood corpuscle as a cell type for cytotoxicity evaluation can be justified. Drug-induced hemolysis is a relatively rare, but serious toxicity liability. It occurs by two mechanisms, toxic hemolysis and allergic hemolysis. The toxic hemolysis referred to the direct toxicity of the drug, its metabolite, or an excipient in the formulation. It evaluates hemoglobin release in the plasma (as an indicator of red blood cell lysis) following test agent exposure. The US FDA recommended it for hemolytic potential.  This method was used in this in vitro study.
The irrigating solutions used in this study were isotonic and thus excluded an osmotic pressure gradient, so the observed hemolysis and loss of cellular protein was due to the oxidizing effect of the solutions on the cell membrane of red blood corpuscles. Our findings were similar to Pashley et al. who reported that sodium hypochlorite (5.25%) in 1:1000 dilution caused complete hemolysis of red blood cells in vitro. 
Sodium hypochlorite is considered as an ideal irrigant in endodontics. Hypochlorite preparations are sporicidal, virucidal and show far greater tissue dissolving effects on necrotic than on vital tissues. Various concentrations of sodium hypochlorite from 0.5% to 5.25% have been tried out. , The higher the concentration the 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. Low concentration (1%) of sodium hypochlorite was reported to be sufficient to dissolve the pulp tissue.  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 was found that 0.025% of sodium hypochlorite was bactericidal and not tissue toxic and 0.25% concentration of sodium hypochlorite showed tissue toxicity. 
Chlorhexidine is recommended as an endodontic irrigant because of its antimicrobial activity, presumed non-oxicity and intracanal substantively.  At higher concentrations, chlorhexidine results in extensive cell damage, coagulation of cytoplasm and precipitation of proteins and nucleic acids.  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%.
Chlorhexidine was reported to be toxic to human gingival cells and red blood cells in culture even in low concentration and the toxic potency is dependent on the length of exposure and the composition of the exposure medium. , Our results were in accordance with Giannelli et al.  They attributed its toxic effect to be consisted in the induction of apoptotic and autophagic/necrotic cell deaths and involved disturbance of mitochondrial function, intracellular Ca 2 + increase and oxidative stress. In addition, its lytic properties could be the reason for hemolysis. On comparing the inflammatory response of 0.5% sodium hypochlorite, 2% chlorhexidine digluconate and phosphate buffered saline, it was found that 2% chlorhexidine injection was similar to the phosphate-buffered saline control at all times tested, while the 0.5% sodium hypochlorite injection resulted in significant inflammation. 
Bovine pulp tissue was treated with normal saline, MTAD, 2% chlorhexidine digluconate and 2.5% sodium hypochlorite it was found that chlorhexidine has the weakest tissue dissolution capacity.  Chlorhexidine is recommended as an alternative in patients allergic to sodium hypochlorite or in teeth with incomplete root formation and crestal perforations to prevent inflammatory response in proximity to the epithelial attachment.  Bio Pure MTAD (Dentsply, Tulsa, OK) is designed to be used as a final root canal rinse before obturation. Tetracycline has many unique properties of low pH and acts as a calcium chelator and cause enamel and root surface demineralization.  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. ,, In this study MTAD is found to be less cytotoxic than sodium hypochlorite and more cytotoxic than chlorhexidine. Sharaf et al. evaluated the degree of inflammation of exposed pulp of dog's teeth that was irrigated with MTAD.  Extravasated red blood cells of highly inflamed pulp were observed after 1 week. The inflammation was dramatically reduced within 8 weeks.
| Conclusion|| |
This study suggests that these irrigating solutions do cause detrimental effects on the diluted red blood corpuscles. Sodium hypochlorite has been proven to be highly cytotoxic and a great deal of care should therefore be exercised when using sodium hypochlorite during endodontic irrigation. MTAD has also demonstrated significant cytotoxicity when compared to chlorhexidine.
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 findings of this study chlorhexidine has proven to have least cytotoxic potential when compared to MTAD and sodium hypochlorite.
| References|| |
|1.||Schilder H. Cleaning and shaping the root canal. Dent Clin North Am 1974;18:269-96. |
|2.||Yesilsoy C, Whitaker E, Cleveland D, Phillips E, Trope M. Antimicrobial and toxic effects of established and potential root canal irrigants. J Endod 1995;21:513-5. |
|3.||Nair PN. On the causes of persistent apical periodontitis: A review. Int Endod J 2006;39:249-81. |
|4.||Gulabivala K, Patel B, Evans G. Effects of mechanical and chemical procedures on root canal surfaces. Endod Topics 2005;10:103-22. |
|5.||Moorer WR, Wesselink PR. Factors promoting the tissue dissolving capability of sodium hypochlorite. Int Endod J 1982;15:187-96. |
|6.||Gu LS, Kim JR, Ling J, Choi KK, Pashley DH, Tay FR. Review of contemporary irrigant agitation techniques and devices. J Endod 2009;35:791-804. |
|7.||Walmsley AD. Ultrasound and root canal treatment: The need for scientific evaluation. Int Endod J 1987;20:105-11. |
|8.||van der Sluis LW, Versluis M, Wu MK, Wesselink PR. Passive ultrasonic irrigation of the root canal: A review of the literature. Int Endod J 2007;40:415-26. |
|9.||Siqueira JF Jr, Rôças IN, Favieri A, Lima KC. Chemomechanical reduction of the bacterial population in the root canal after instrumentation and irrigation with 1%, 2.5%, and 5.25% sodium hypochlorite. J Endod 2000;26:331-4. |
|10.||Witton R, Henthorn K, Ethunandan M, Harmer S, Brennan PA. Neurological complications following extrusion of sodium hypochlorite solution during root canal treatment. Int Endod J 2005;38:843-8. |
|11.||Gursoy UK, Bostanci V, Kosger HH. Palatal mucosa necrosis because of accidental sodium hypochlorite injection instead of anaesthetic solution. Int Endod J 2006;39:157-61. |
|12.||Zamany A, Safavi K, Spångberg LS. The effect of chlorhexidine as an endodontic disinfectant. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2003;96:578-81. |
|13.||Ribeiro DA, Scolastici C, De Lima PL, Marques ME, Salvadori DM. Genotoxicity of antimicrobial endodontic compounds by single cell gel (comet) assay in Chinese hamster ovary (CHO) cells. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2005;99:637-40. |
|14.||Torabinejad M, Khademi AA, Babagoli J, Cho Y, Johnson WB, Bozhilov K, et al. A new solution for the removal of the smear layer. J Endod 2003;29:170-5. |
|15.||Pashley EL, Birdsong NL, Bowman K, Pashley DH. Cytotoxic effects of NaOCl on vital tissue. J Endod 1985;11:525-8. |
|16.||Faria G, Celes MR, De Rossi A, Silva LA, Silva JS, Rossi MA. Evaluation of chlorhexidine toxicity injected in the paw of mice and added to cultured l929 fibroblasts. J Endod 2007;33:715-22. |
|17.||Zhang W, Torabinejad M, Li Y. Evaluation of cytotoxicity of MTAD using the MTT-tetrazolium method. J Endod 2003;29:654-7. |
|18.||Barnhart BD, Chuang A, Lucca JJ, Roberts S, Liewehr F, Joyce AP. An in vitro evaluation of the cytotoxicity of various endodontic irrigants on human gingival fibroblasts. J Endod 2005;31:613-5. |
|19.||FDA. Guidance for Industry - Nonclinical Studies for the Safety Evaluation of Pharmaceutical Excipients. Pharmacology/Toxicology. May 2005. p. 1-12 |
|20.||Vianna ME, Gomes BP, Berber VB, Zaia AA, Ferraz CC, de Souza-Filho FJ. In vitro evaluation of the antimicrobial activity of chlorhexidine and sodium hypochlorite. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2004;97:79-84. |
|21.||Sirtes G, Waltimo T, Schaetzle M, Zehnder M. The effects of temperature on sodium hypochlorite short-term stability, pulp dissolution capacity, and antimicrobial efficacy. J Endod 2005;31:669-71. |
|22.||Heggers JP, Sazy JA, Stenberg BD, Strock LL, McCauley RL, Herndon DN, et al. Bactericidal and wound-healing properties of sodium hypochlorite solutions: The 1991 Lindberg Award. J Burn Care Rehabil 1991;12:420-4. |
|23.||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. |
|24.||Babich H, Wurzburger BJ, Rubin YL, Sinensky MC, Blau L. An in vitro study on the cytotoxicity of chlorhexidine digluconate to human gingival cells. Cell Biol Toxicol 1995;11:79-88. |
|25.||Giannelli M, Chellini F, Margheri M, Tonelli P, Tani A. Effect of chlorhexidine digluconate on different cell types: A molecular and ultrastructural investigation. Toxicol In Vitro 2008;22:308-17. |
|26.||Khademi A, Usefian E, Mahboobe F. Tissue dissolving ability of several endodontic irrigants on bovine pulp tissue. Iran Endod J 2007;2:65-7. |
|27.||Fuss Z, Trope M. Root perforations: Classification and treatment choices based on prognostic factors. Endod Dent Traumatol 1996;12:255-64. |
|28.||Bjorvatn K, Skaug N, Selvig KA. Tetracycline-impregnated enamel and dentin: Duration of antimicrobial capacity. Scand J Dent Res 1985;93:192-7. |
|29.||Torabinejad M, Cho Y, Khademi AA, Bakland LK, Shabahang S. The effect of various concentrations of sodium hypochlorite on the ability of MTAD to remove the smear layer. J Endod 2003;29:233-9. |
|30.||Beltz RE, Torabinejad M, Pouresmail M. Quantitative analysis of the solubilizing action of MTAD, sodium hypochlorite, and EDTA on bovine pulp and dentin. J Endod 2003;29:334-7. |
|31.||Machnick TK, Torabinejad M, Munoz CA, Shabahang S. Effect of MTAD on flexural strength and modulus of elasticity of dentin. J Endod 2003;29:747-50. |
|32.||Sharaf N, Aboul-Enein N, Hassan M, Ayad M, Zaazou M, Farrag A. Effect of MTAD and tea tree oil irrigation before pulp capping on healing of exposed dental pulp. Aust J Basic Appl Sci 2012;6:553-7. |
[Table 1], [Table 2], [Table 3], [Table 4]