Quantitative analysis for detection of toxic elements in various irrigants, their combination (precipitate), and para-chloroaniline: An inductively coupled plasma mass spectrometry study.

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. Copyright:

INTRODUCTION

The success of endodontic therapy is chiefly accredited to biomechanical preparation, microbial control and complete obturation of the root canal space.[1] 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.[2] No irrigating solution possess all ideal properties pertaining to tissue dissolution and antibacterial efficacy, making it mandatory to combine different irrigants.[3]

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.[4] However, the potential drawbacks of NaOCl pertaining to toxicity, unpleasant taste, and above all, accidents arising from negligent or inexperienced usage, cannot be overlooked[5] 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.[6]

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.[7] 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.[8] Tulsi is composed of 71% eugenol and 20% methyl eugenol which exhibits antibacterial, antioxidant, analgesic, antipyretic, anti-ulcer, antidiabetic and anti-cancer activities.[9] 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.[10] 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.[11]

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.

Null hypothesis

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 [Tables 2 and 3].

Table 1

Groups

Groups	Irrigants	Precipitate formed	Color	Mean weight (mg)
Group 1	1 ml 2% CHX + 1 ml 3% NaOCl	Yes	Reddish-brown	5.50
Group 2	1 ml 2% CHX + 1 ml 17% EDTA	Yes	White	4.80
Group 3	1 ml 2% CHX + 1 ml 5% neem	Yes	Light green	3.70
Group 4	1 ml 2% CHX + 1 ml 5% tulsi	Yes	Dark green	3.20
Group 5	1 ml 2% CHX + 1 ml 5% Aloe vera	Yes	Green	4.20
Group 6	1 ml 2% CHX + 1 ml 5% garlic	Yes	Beige	4.00
Group 7	1 ml 3% NaOCl + 1 ml 17% EDTA	No	-	-
Group 8	1 ml 3% NaOCl + 1 ml 5% neem	No	-	-
Group 9	1 ml 3% NaOCl + 1 ml 5% tulsi	No	-	-
Group 10	1 ml 3% NaOCl + 1 ml 5% Aloe vera	No	-	-
Group 11	1 ml 3% NaOCl + 1 ml 5% garlic	No	-	-
Group 12	1 ml 17% EDTA + 1 ml 5% neem	No	-	-
Group 13	1 ml 17% EDTA + 1 ml 5% tulsi	No	-	-
Group 14	1 ml 17% EDTA + 1 ml 5% Aloe vera	No	-	-
Group 15	1 ml 17% EDTA + 1 ml 5% garlic	No	-	-
Group 16	1 ml 5% neem + 1 ml 5% tulsi	No	-	-
Group 17	1 ml 5% neem + 1 ml 5% Aloe vera	No	-	-
Group 18	1 ml 5% neem + 1 ml 5% garlic	No	-	-
Group 19	1 ml 5% tulsi + 1 ml 5% Aloe vera	No	-	-
Group 20	1 ml 5% tulsi + 1 ml 5% garlic	No	-	-
Group 21	1 ml 5% Aloe vera + garlic	No	-	-

CHX: Chlorhexidine, NaOCl: Sodium hypochlorite, EDTA: Ethylenediaminetetraacetic acid

Figure 1

Irrigants on combination

Table 2

One - way ANOVA

Weight of precipitate (mg)	Sum of Squares	df	Mean Square	F	Sig.
Between Groups	33.333	5	6.667	9.137	0.000
Within Groups	39.400	54	0.730
Total	72.733	59

Table 3

Post hoc Tukey’s test

Groups (I)	Groups (J)	Mean Difference (I-J)	Std Error	Sig.
CHX + NaOCl	CHX + EDTA	0.700	0.382	0.454
	CHX + NEEM	1.800	0.382	0.000
	CHX + ALOEVERA	1.300	0.382	0.015
	CHX + GARLIC	1.500	0.382	0.003
	CHX + TULSI	2.300	0.382	0.000
CHX + EDTA	CHX + NaOCl	-0.700	0.382	0.454
	CHX + NEEM	1.100	0.382	0.060
	CHX + ALOEVERA	0.600	0.382	0.621
	CHX + GARLIC	0.800	0.382	0.306
	CHX + TULSI	1.600	0.382	0.001
CHX + NEEM	CHX + NaOCl	-1.800	0.382	0.000
	CHX + EDTA	-1.100	0.382	0.060
	CHX + ALOEVERA	-0.500	0.382	0.779
	CHX + GARLIC	-0.300	0.382	0.969
	CHX + TULSI	0.500	0.382	0.779
CHX + ALOEVERA	CHX + NaOCl	-1.300	0.382	0.015
	CHX + EDTA	-0.600	0.382	0.621
	CHX + NEEM	0.500	0.382	0.779
	CHX + GARLIC	0.200	0.382	0.995
	CHX + TULSI	1.000	0.382	0.110
CHX + GARLIC	CHX + NaOCl	-1.500	0.382	0.003
	CHX + EDTA	-0.800	0.382	0.306
	CHX + NEEM	0.300	0.382	0.969
	CHX + ALOEVERA	-0.200	0.382	0.995
	CHX + TULSI	0.800	0.382	0.306
CHX + TULSI	CHX + NaOCl	-2.300	0.382	0.000
	CHX + EDTA	1.600	0.382	0.001
	CHX + NEEM	-0.500	0.382	0.779
	CHX + ALOEVERA	-1.000	0.382	0.110
	CHX + GARLIC	-0.800	0.382	0.306

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.

Table 4

Inductively coupled plasma mass spectrometry analysis data

	Individual irrigants and PCA								Groups (1-6) precipitate formed
	Chemical irrigants			Herbal irrigants				PCA
	CHX	NaOCl	EDTA	Neem	Tulsi	Aloe vera	Garlic		CHX + NaOCl	CHX + EDTA	CHX + neem	CHX + tulsi	CHX + Aloe vera	CHX + garlic
As	BLQ	0.25	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Cd	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Cr	1.65	BLQ	0.4	BLQ	BLQ	BLQ	BLQ	0.64	BLQ	0.5	BLQ	BLQ	BLQ	BLQ
Ni	0.4	BLQ	1.3	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Pb	0.2	BLQ	0.7	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Co	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Se	BLQ	7.0	BLQ	BLQ	BLQ	BLQ	BLQ	0.21	1.75	BLQ	BLQ	BLQ	BLQ	BLQ
V	BLQ	1.0	BLQ	BLQ	BLQ	BLQ	BLQ	1.5	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Na	0.1	1702	0.9	15.75	51.25	69.75	15.75	56.0	632.8	2.62	9.37	27.12	38.0	14.75
K	0.4	16.75	4.6	123	166	30.25	67.7	BLQ	7.12	3.0	59.6	78.8	13.37	43.5
Fe	BLQ	1.25	BLQ	4.25	16.25	BLQ	8.75	3.21	11.62	BLQ	6.5	2.37	4.5	BLQ
Ca	4.0	BLQ	8.85	83	36.25	51.5	30.0	9.54	14.25	3.5	41.12	18.12	37.62	16.75
Mg	BLQ	BLQ	0.7	120.5	107.25	232.5	51.5	51.2	23.0	0.25	60.37	48.5	128.12	35.75
Mn	BLQ	BLQ	0.15	3.5	2.75	2.0	2.5	0.429	2.62	BLQ	1.87	3.5	2.75	1.5
Cu	1.45	BLQ	4.7	BLQ	BLQ	BLQ	BLQ	BLQ	0.75	BLQ	BLQ	BLQ	BLQ	0.5
Zn	1.5	BLQ	8.2	BLQ	0.5	1.0	BLQ	BLQ	0.75	BLQ	BLQ	5.25	BLQ	BLQ
Hg	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Sb	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Al	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Bi	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Ce	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Ga	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Au	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Pt	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Ag	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Te	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Tl	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Sn	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Mo	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	0.42	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Ba	BLQ	BLQ	BLQ	4.0	2.25	BLQ	1.0	BLQ	0.37	BLQ	2.12	1.12	1.0	0.75
Be	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
In	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Li	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Ti	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ
Ge	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ	BLQ

BLQ: Below the Limit of quantification, ppm: Parts per million, CHX: Chlorhexidine, NaOCl: Sodium hypochlorite, EDTA: Ethylenediaminetetraacetic acid, PCA: Para-chloroaniline, As: Arsenic, Cd: Cadmium, Cr: Chromium, Ni: Nickel, Be: Beryllium, Pb: Lead, Co: Cobalt, Se: Selenium, V: Vanadium, Na: Sodium, K: Potassium, Fe: Iron, Ca: Calcium, Mg: Magnesium, Mn: Manganese, Cu: Copper, Zn: Zinc, Hg: Mercury, Sb: Antimony, Al: Aluminum, Bi: Bismuth, Ce: Cerium, Ga: Gallium, Au: Gold, Pt: Platinum, Ag: Silver, Te: Tellurium, Ti: Titanium, Sn: Tin, Mo: Molybdenum, Ba: Barium, In: Indium, Li: Lithium, Ge: Germanium, Tl: Thallium

Statistical analysis

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) [Figures 2 and 3]

Figure 2

Samples collected for inductively coupled plasma mass spectrometry analysis

Figure 3

Precipitate

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.

DISCUSSION

Inductively coupled plasma mass spectrometry is most commonly used in biological and environmental sciences to quantify target elemental analytes.[11] 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

IARC groups	Description	Elements
Group 1	Carcinogenic to humans	As, Cd, Cr, Ni, Be
Group 2A	Probably carcinogenic to humans	Pb
Group 2B	Possibly carcinogenic to humans	Co, PCA
Group 3	Not classifiable as to its carcinogenicity to humans	Hg, Se
Group 4	Probably not carcinogenic to humans	-

As: Arsenic, Cd: Cadmium, Cr: Chromium, Ni: Nickel, Be: Beryllium, Pb: Lead, Co: Cobalt, PCA: Para-chloroaniline, Hg: Mercury, Se: Selenium, IARC: International Agency for Research on Cancer

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).[12] 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.[13] 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.[14]

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.[1516] 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.[17] 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.[18] 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.[17] 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.[19] 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.[1720] 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.[21] 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.[22] 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.[23] 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.[24] 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.

Future scope

Studies should focus on herbal irrigants which are not toxic and moreover, does not impart any precipitate formation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.