| Abstract|| |
Introduction: Chlorhexidine (CHX) interacts with sodium hypochlorite (NaOCl) and herbal irrigants such as neem and tulsi to form precipitate which contains para-chloroaniline (PCA). No studies till date have reported about metal elements present in this combination as well as in irrigants.
Aim: The aim of this study was to evaluate the precipitate formed on combination of different irrigants, weigh the amount of precipitate formed, and to analyze 35 different metal elements in each irrigant, precipitate formed as well as in PCA.
Materials and Methods: Seven irrigants, namely 2% CHX gluconate, 3% NaOCl, 17% ethylenediaminetetraacetic acid (EDTA), 5% neem, 5% tulsi, 5% Aloe vera, and 5% garlic were taken in different test tubes. Group (1–6): 1 ml of CHX is mixed with 1 ml of 3% NaOCl/17% EDTA/5% neem/5% tulsi/5% A. vera/5% garlic. Group (7–11): 1 ml of 3% NaOCl is mixed with 1 ml of 17% EDTA/5% neem/5% tulsi/5% A. vera/5% garlic. Group (12–15): 1 ml of 17% EDTA is mixed with 1 ml of 5% neem/5% tulsi/5% A. vera/5% garlic. Group (16–18): 1 ml of 5% neem is mixed with 1 ml of 5% tulsi/5% A. vera/5% garlic. Group (19 and 20): 1 ml of 5% tulsi is mixed with 5% A. vera/5% garlic. Group 21 includes 1 ml of 5% A. vera and 5% garlic. Each group is observed for any precipitate formation, and precipitate formed was weighed. Samples such as 2% CHX gluconate, 3% NaOCl, 17% EDTA, 5% neem, 5% tulsi, 5% A. vera, PCA, and precipitate formed in each group were analyzed for 35 different metal elements using inductively coupled plasma mass spectrometry (ICP-MS).
Statistical Analysis: One-way ANOVA and Post hoc Tukey's test for the precipitate formed.
Results: Precipitate formation was seen in CHX + NaOCl (reddish-brown), CHX + EDTA (white), CHX + neem (light green), CHX + A. vera (green), CHX + tulsi (dark green), CHX + garlic (beige). ICP-MS analysis showed the presence of International Agency for Research on Cancer Group 1 carcinogens in NaOCl, CHX, EDTA, and PCA.
Conclusion: Carcinogenic metals are undetected in herbal irrigants which is found to be risk free alternatives in near future.
Keywords: Aloe vera extract; chlorhexidine; ethylenediaminetetraacetic acid; garlic extract; neem extract; para-chloroaniline; sodium hypochlorite and tulsi extract
|How to cite this article:|
Siddique R, Nivedhitha MS, Jacob B. Quantitative analysis for detection of toxic elements in various irrigants, their combination (precipitate), and para-chloroaniline: An inductively coupled plasma mass spectrometry study. J Conserv Dent 2019;22:344-50
|How to cite this URL:|
Siddique R, Nivedhitha MS, Jacob B. Quantitative analysis for detection of toxic elements in various irrigants, their combination (precipitate), and para-chloroaniline: An inductively coupled plasma mass spectrometry study. J Conserv Dent [serial online] 2019 [cited 2020 May 31];22:344-50. Available from: http://www.jcd.org.in/text.asp?2019/22/4/344/270507
| Introduction|| |
The success of endodontic therapy is chiefly accredited to biomechanical preparation, microbial control and complete obturation of the root canal space. Pulpal and periapical pathologies are a mainstay unless the microorganisms are completely eradicated from the root canal systems. Chemo-mechanical debridement plays a crucial role in reducing or possibly ousting the microbial load from within the root canal complexities. No irrigating solution possess all ideal properties pertaining to tissue dissolution and antibacterial efficacy, making it mandatory to combine different irrigants.
Sodium hypochlorite (NaOCl), an irrigant widely used in endodontics, conferred with the eponym of being the gold standard, exerts both antimicrobial and tissue dissolution properties at the same time. However, the potential drawbacks of NaOCl pertaining to toxicity, unpleasant taste, and above all, accidents arising from negligent or inexperienced usage, cannot be overlooked  Even though chlorhexidine (CHX), an irrigant also used in endodontic therapies, have found its mark against NaOCl-resistant bacterial strains owing to its substantivity; however, CHX has been shown to induce a cytotoxic effect on human blood lymphocytes.
Recently, in our previous study, it was demonstrated that a combination of CHX with NaOCl/neem/tulsi not only formed a precipitate each but also contained para-chloroaniline (PCA) as well. The HPLC analysis showed the percentage dry weight of PCA as 14.00 ppm (CHX + NaOCl), 10.57 ppm (CHX + neem), and 9.00 ppm (CHX + tulsi). The International Agency for Research on Cancer (IARC, 2006) classified PCA as Group 2B carcinogens which imply that there is sufficient evidence of carcinogenicity in animals but limited evidence in humans.
Natural irrigants such as neem, tulsi, Aloe vera, and garlic have been tested of their antimicrobial effectiveness and biocompatibility, in vitro. The biological effect of neem is mainly due to its active component nimbidin and other isolated components such as nimbin, nimbinin, nimbidinin, nimbolide, and nimbidic acid. Tulsi is composed of 71% eugenol and 20% methyl eugenol which exhibits antibacterial, antioxidant, analgesic, antipyretic, anti-ulcer, antidiabetic and anti-cancer activities. A. vera possess both anti-bacterial and anti-fungal activity owing to the presence of alloins and barbadoins. The main active component of garlic is allicin which have been implicated in destroying the cell wall and cell membrane of root canal bacteria, can be used as an alternative to NaOCl. Concentrated garlic (95%) extract contains 34% allicin, 44% total thiosulfinates and 20% vinyldithins.
Arsenic (As), cadmium (Cd), chromium (Cr), and nickel (Ni) are classified as Group 1 heavy metals, carcinogenic to humans according to the IARC. The elemental analysis for endodontic irrigants and their combinations are limited although Marchesan et al. 2007, reported the presence of Calcium (Ca), Iron (Fe), and Magnesium (Mg) in reddish brown flocculate.
Therefore, the aim of this study was to evaluate the precipitate formed following interaction of CHX with NaOCl, ethylenediaminetetraacetic acid (EDTA), neem, tulsi, A. vera and garlic, weigh the amount of precipitate formed and finally analyze each irrigant, the precipitate formed and PCA for 35 different metal elements employing inductively coupled plasma mass spectrometry.
No toxic heavy metals were detected in any irrigant and its combination which forms precipitate.
| Materials and Methods|| |
Seven irrigants, namely 2% CHX gluconate (Asep-RC), 3% NaOCl (VENSONS INDIA), 17% EDTA (META BIOMED CO. LTD), 5% neem (Naturmed's), 5% tulsi (Naturmed's), 5% A. vera (Naturmed's) and 5% garlic (Health VIT) were used in this study.
Neem, tulsi, A. vera and garlic extract were supplied in the form of powder, readily soluble in distilled water. These extracts were prepared in 5% concentration by mixing 5 g of powder in 100 ml of distilled water and kept in place for 24 h. The prepared solutions were filtered using WhatMann's filter paper No 1. The seven irrigants were then grouped into 21 groups of various combinations [Table 1]. Group (1–6) 1 ml of CHX was mixed with 1 ml of 3% NaOCl/17% EDTA/5% neem/5% tulsi/5% A. vera/5% garlic. Group (7–11) 1 ml of 3% NaOCl was mixed with 1 ml of 17% EDTA/5% neem/5% tulsi/5% A. vera/5% garlic. Group (12–15) 1 ml of 17% EDTA was mixed with 1 ml of 5% neem/5% tulsi/5% A. vera/5% garlic. Group (16–18) 1 ml of 5% neem was mixed with 1 ml of 5% tulsi/5% A. vera/5% garlic. Group (19 and 20) 1 ml of 5% tulsi was mixed with 5% A. vera/5% garlic. Group 21 includes 1 ml of 5% A. vera, and 5% garlic. Each group was observed for any precipitate formation [Figure 1], precipitate formed was filtered, dried, and weighed using weighing electronic balance. The data obtained from weight of precipitate (mg) were statistically analyzed using one-way ANOVA and Post hoc Tukey's test [Table 2] and [Table 3].
Samples from each irrigant, PCA and precipitate were analyzed for 35 different metals using inductively coupled plasma mass spectrometry [Table 4]. Commercially available 98% PCA (4-Chloroaniline; Sigma-Aldrich) was used in this study.
To analyze the data SPSS (IBM SPSS Statistics for Windows, Version 23.0, Armonk, NY, USA: IBM Corp. Released 2015) is used. One-Way ANOVA was used to compare the mean differences between and within groups which formed precipitate. Post hoc-Tukey test was used to analyze the highest amount of precipitate formed among the groups (P < 0.05).
| Results|| |
- Precipitate formation was seen in CHX + NaOCl (reddish-brown), CHX + EDTA (white), CHX + neem (light green), CHX + A. vera (green), CHX + tulsi (dark green), CHX + garlic (Beige) [Figure 2] and [Figure 3]
- The amount of precipitate was higher in Group 1 (5.50 mg) followed by Group 2 (4.80 mg), Group 5 (4.20 mg), Group 6 (4 mg), Group 3 (3.70 mg), and Group 4 (3.20 mg) [Table 1]
- 0.25 ppm of As (carcinogen) was detected in NaOCl sample [Table 4]
- Lead (Pb) was observed in CHX (0.2 ppm) and EDTA (0.7 ppm)
- Cr was seen in CHX (1.65 ppm), EDTA (0.4 ppm), PCA (0.64 ppm), and CHX + EDTA (0.5 ppm)
- Presence of Copper (Cu) was reported in CHX (1.45 ppm), EDTA (4.7 ppm), CHX + NaOCl (0.75 ppm), CHX + garlic (0.5 ppm)
- Ni was demonstrated in CHX (0.4 ppm) and EDTA (1.3 ppm)
- Zinc (Zn) was found in CHX (1.5 ppm), EDTA (8.2 ppm), TULSI (0.5 ppm), A. vera (1.0 ppm), CHX + NaOCl (0.75 ppm) and CHX + TULSI (5.25 ppm)
- The presence of vanadium and selenium (Se) were noticed only in PCA, NaOCl, and CHX + NaOCl.
|Figure 2: Samples collected for inductively coupled plasma mass spectrometry analysis|
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| Discussion|| |
Inductively coupled plasma mass spectrometry is most commonly used in biological and environmental sciences to quantify target elemental analytes. In this study, we have used the following elements: Sodium (Na), Potassium (K), Fe, Ca, Mg, Cr, Manganese (Mn), Ni, Cobalt, Cd, As, Cu, Zn, Mercury, Pb, Antimony, Aluminum, Bismuth, Cerium, Gallium, Gold, Platinum, Silver, Tellurium, Titanium, Tin, Vanadium (V), Molybdenum (Mo), Se, Barium, Beryllium, Indium, Lithium, and Germanium. Elements used were grouped into IARC classification is given in [Table 5].
|Table 5: Elements used were grouped into International Agency for Research on Cancer classification|
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In the present study, ICP-MS analysis revealed that CHX comprised of Group 1 carcinogens (Cr and Ni), Group 2A carcinogen (Pb) followed by Cu, Na, K, Ca and Zn. NaOCl contained group 1 carcinogen (As), toxic elements (V and Se) followed by Na, K and Fe. EDTA similar to CHX, consisted of Group 1 carcinogens (Cr and Ni) as well as the toxic element Pb followed by Cu, Na, K, Ca, Mg, Mn, and Zn. PCA comprised of Cr, Se, V, Na, Fe, Ca, Mg, Mn, and Mo. Carcinogenic elements are not detected in herbal irrigants like neem, tulsi, A. vera and garlic.
Most heavy metals induce cancer and other diseases. The mechanism of heavy metal-induced damages depend upon oxidative stress which is caused by reactive oxygen species (ROS). As, Cd, Cr, and Ni are classified as Group 1 heavy metals, carcinogenic to humans according to the IARC. These compounds lead to disruptions in tumor suppressor gene expression, damage reparative processes and enzymatic activities concerned with metabolism via oxidative damage. Antioxidants help in the prevention and detoxification of heavy metal-induced damage. Consumption of phytochemicals from antioxidant substances of plants help in anti-oxidant related detoxification process.
As exists in two forms: organic and inorganic, capable of solubilizing in water to form weak acids and produce salts called arsenate. The main mechanism involved in As-related damage is oxidative stress. Numerous studies have reported the genotoxicity in mice leucocytes and humans., Arsenite gives rise to DNA damage, apoptosis, cell damage and cell death. As compounds are associated with cancer of liver, leukemia, promyelocytic, carcinoma, prostate cancer, cancer of skin, Kupffer cell cancer, etc. In the present study, 0.25 ppm of As (carcinogen) was detected in NaOCl samples.
The toxicity of Cr is attributed to its chemical state and exists in divalent to hexavalent compounds. Cellular damage is caused due to high concentrations of trivalent Cr. Reactive hydroxyl radicals are produced by hexavalent Cr which is a strong toxicant. Cr toxicity is associated with cancer of intestines, lung carcinoma, lung cancer, skin diseases, kidney diseases, lung injury, dermatitis, allergic contact, malignant transformation, allergy, cancer, ulcer, dermatitis, dermatitis contact, etc. The presence of Cr was noted in CHX (1.65 ppm), EDTA (0.4 ppm), PCA (0.64 ppm), and CHX + EDTA (0.5 ppm). These findings are suggestive of carcinogenicity.
Ni is a widely used alloy in various metals namely Cr, Pb and Cu, which are a major source of Ni exposure. Contaminated water is a rich source of Ni compounds which results in dermatitis and allergy. Skin and oral epithelium damage is mainly caused due to oral exposure. Ni toxicity induces cell damage, DNA damage, cell death, apoptosis, DNA methylation, ROS generation, oxidative stress, and DNA strand breakage. Ni toxicity is associated with lung cancer, lung toxicity, cancer of nose, cancer, dermatitis, kidney toxicity, skin diseases, liver toxicity, respiratory tract cancer, lung diseases, and allergic reaction., In the present study, Ni was observed in CHX (0.4 ppm) and EDTA (1.3 ppm).
Pb is the most important toxic heavy metal which affects all the organs of the human body. It is easily absorbed in the blood stream, exert ill effects on the central nervous system, the cardiovascular system, immune system and kidneys. The maximum daily intake of Pb is set to a limit of 1.0 μg/g; however, prolonged intake may contribute to toxicity in humans. Blood Pb level of 10 μg/Dl or above is a major cause of concern according to US Centers for Disease Control and Prevention and the World Health Organization. Chronic Pb poisoning may cause anemia, fatigue, problems with sleep, headaches, stupor and slurred speech. However, in our study, the presence of Pb was seen only in CHX (0.2 ppm) and EDTA (0.7 ppm).
High Cu content is the primary cause for hemolysis in many clinical scenarios. The methemoglobin, lipid peroxidation and hemolysis were reported when human erythrocyte (red blood cell) suspensions incubated with Cu. Hemolysis is greater when concentration of Cu is higher; hemoglobin alterations, namely precipitations are more pronounced. In our study, Cu was found in CHX (1.45 ppm), EDTA (4.7 ppm), CHX + NaOCl (0.75 ppm), CHX + garlic (0.5 ppm).
Se is a well-known micronutrient which has both beneficial as well as toxic attributes whereas vanadium has no beneficial use and is thereby regarded as an occupational or environmental pollutant. In a study, it was found that the presence of both vanadium and Se, induced cytoplasmic vacuolization, suggestive of deterioration of cell functioning in CHO-K1 cells. In the present study, both vanadium and Se were detected in NaOCl and PCA, whereas Se alone was present in CHX and NaOCl combination.
No literature has reported the presence of toxic heavy metals in irrigants till date.
The null hypothesis stands rejected simply for the fact that precipitate formation was seen in all groups in which CHX was incorporated, also, toxic heavy metals were detected in all chemical irrigants and in PCA as well.
| Conclusion|| |
- CHX in combination with NaOCl, EDTA, neem, tulsi, A. vera and garlic produced reddish brown, white, light green, dark green, green, and beige precipitate
- The highest amount of precipitate was seen in group 1 followed by group 2, group 5, group 6, group 3, and group 4
- The presence of toxic metals was detected in NaOCl, CHX, and EDTA
- Carcinogenic metals were undetected in herbal irrigants, hence proving to be risk free alternatives in the near future.
Based on the results of the present study, utmost care should be taken while using chemical irrigants like NaOCl, CHX and EDTA.
Studies should focus on herbal irrigants which are not toxic and moreover, does not impart any precipitate formation.
The authors would like to acknowledge the technical assistance provided to us by Hubert Enviro Care Systems (P) LTD, Chennai, Tamil Nadu, India.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Hargreaves KM, Berman LH. Cohen's Pathways of the Pulp. 11th
ed. Missouri: Elsevier Health Sciences; 2016.
Byström A, Sundqvist G. Bacteriologic evaluation of the efficacy of mechanical root canal instrumentation in endodontic therapy. Scand J Dent Res 1981;89:321-8.
Kuruvilla JR, Kamath MP. Antimicrobial activity of 2.5% sodium hypochlorite and 0.2% chlorhexidine gluconate separately and combined, as endodontic irrigants. J Endod 1998;24:472-6.
Abou-Rass M, Piccinino MV. The effectiveness of four clinical irrigation methods on the removal of root canal debris. Oral Surg Oral Med Oral Pathol 1982;54:323-8.
Faras F, Abo-Alhassan F, Sadeq A, Burezq H. Complication of improper management of sodium hypochlorite accident during root canal treatment. J Int Soc Prev Community Dent 2016;6:493-6.
Salimi A, Alami B, Pourahmad J. Analysis of cytotoxic effects of chlorhexidine gluconate as antiseptic agent on human blood lymphocytes. J Biochem Mol Toxicol 2017;31:e21918.
Siddique R, Sureshbabu NM, Somasundaram J, Jacob B, Selvam D. Qualitative and quantitative analysis of precipitate formation following interaction of chlorhexidine with sodium hypochlorite, Neem, and Tulsi. J Conserv Dent 2019;22:40-7.
] [Full text]
Pankaj S, Lokeshwar T, Mukesh B, Vishnu B. Review on neem (Azadirachta indica
): Thousand problems one solution. Int Res J Pharm 2011;2:97-102.
Pandey G, Madhuri S. Pharmacological activities of Ocimum sanctum
(tulsi): A review. Int J Pharm Sci Rev Res 2010;5:61-6.
Humans IWG on the E of CR to. IARC Monographs: Arsenic, Metals, Fibres, and Dusts. Volume 100C. A review of Human Carcinogens; 2012.
Marchesan MA, Pasternak Júnior B, Afonso MM, Sousa-Neto MD, Paschoalato C. Chemical analysis of the flocculate formed by the association of sodium hypochlorite and chlorhexidine. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2007;103:e103-5.
Tchounwou PB, Yedjou CG, Patlolla AK, Sutton DJ. Heavy metal toxicity and the environment. Exp Suppl 2012;101:133-64.
Bánfalvi G. Heavy metals, trace elements and their cellular effects. InCellular effects of heavy metals. Springer, Dordrecht; 2011. p. 3-28.
Lobo V, Patil A, Phatak A, Chandra N. Free radicals, antioxidants and functional foods: Impact on human health. Pharmacogn Rev 2010;4:118-26.
Gonsebatt ME, Vega L, Herrera LA, Montero R, Rojas E, Cebrián ME, et al.
Inorganic arsenic effects on human lymphocyte stimulation and proliferation. Mutat Res 1992;283:91-5.
Saleha Banu B, Danadevi K, Jamil K, Ahuja YR, Visweswara Rao K, Ishaq M.In vivo
genotoxic effect of arsenic trioxide in mice using comet assay. Toxicology 2001;162:171-7.
Kim HS, Kim YJ, Seo YR. An overview of carcinogenic heavy metal: Molecular toxicity mechanism and prevention. J Cancer Prev 2015;20:232-40.
Eastmond DA, Macgregor JT, Slesinski RS. Trivalent chromium: Assessing the genotoxic risk of an essential trace element and widely used human and animal nutritional supplement. Crit Rev Toxicol 2008;38:173-90.
Jacob SE, Goldenberg A, Pelletier JL, Fonacier LS, Usatine R, Silverberg N. Nickel allergy and our children's health: A review of indexed cases and a view of future prevention. Pediatr Dermatol 2015;32:779-85.
Küpper M, Weinbruch S, Skaug V, Skogstad A, Thornér EE, Benker N, et al.
Electron microscopy of particles deposited in the lungs of nickel refinery workers. Anal Bioanal Chem 2015;407:6435-45.
Bergeson LL. The proposed lead NAAQS: Is consideration of cost in the clean air act's future? Environ Qual Manag 2008;18:79-84.
Pearce JM. Burton's line in lead poisoning. Eur Neurol 2007;57:118-9.
Fernandes A, Mira ML, Azevedo MS, Manso C. Mechanisms of hemolysis induced by copper. Free Radic Res Commun 1988;4:291-8.
Zwolak I. Increased cytotoxicity of vanadium to CHO-K1 cells in the presence of inorganic selenium. Bull Environ Contam Toxicol 2015;95:593-8.
Dr. Malli Sureshbabu Nivedhitha
Department of Conservative Dentistry and Endodontics, Saveetha Dental College, Saveetha Institute of Medical and Technical Sciences, 162, Poonamallee High Road, Chennai - 600 077, Tamil Nadu
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]