Efficient removal of 2,6-xylidine precipitate using different agitation protocols: An in vitro field emission scanning electron microscopic study.

Background: Any solution of chemical nature when combined will result in the formation of a precipitate, which in the root canal system will interfere with the hermetic seal. In clinical practice presence of any precipitate, especially carcinogenic in nature, should be eliminated. Aim: To evaluate whether final irrigation with different agitation protocols will remove the precipitate formed following lidocaine hydrochloride and sodium hypochlorite combination at the coronal, middle, and apical-root thirds. Materials and
Methods: Forty-four uniradicular teeth were standardized at 17 mm. All specimens were injected with 2% lidocaine hydrochloride and 2.5% sodium hypochlorite, followed by mechanical instrumentation using rotary files. Further, specimens were arbitrarily divided into four groups (n = 11) based on the final irrigation protocol: Group 1: No irrigation; Group 2: Manual irrigation; Group 3: Mechanical irrigation; Group 4: Ultrasonic irrigation. Samples were sectioned and Field Field Emission Scanning Electron Microscopic (FESEM) analysis of the canal wall was done at coronal, middle, and apical thirds.
Results: FESEM images revealed occluded dentinal tubules with the presence of precipitate seen in all-thirds of each specimen.
Conclusion: No irrigation protocol was successful in removing the precipitate formed; but when compared ultrasonic irrigation had the least precipitate seen at-coronal,-middle and apical thirds. Copyright:

INTRODUCTION

Pain is a complex multifactorial and biopsychosocial event which can be attributed to real or plausible harm to tissue, with varying perceptions, anxiety, and manifestations between different individuals.[12] Even in this era of advanced medical sciences, the terms “root canal” and “pain” are considered alike by a layman. Every practitioner must tackle this misconception since the dexterity of the dentist is often judged chiefly by their ability to control pain.[3] However, there are certain techniques to manage pain successfully such as psychological strategies, preprocedural medications, and anesthesia. This enforces the need for effectual anesthesia and hemostasis to be followed for every endodontic procedure.

Due to its low allergic response and high efficiency, 2% lidocaine hydrochloride with adrenaline (LA) in 1:100,000 concentration is most commonly administered via various routes among which local infiltration and nerve block are most frequently practiced.[4] However, supplemental anesthetic techniques like intraligamentary/intraosseous methods, and/or intrapulpal injections can also be used to ensure the profound anesthetic effect. In endodontic management of “hot tooth” or symptomatic irreversible pulpitis in mandibular molars adjuvant intrapulpal administration of local anesthesia is effective when the pain still persists.[124] This technique ensures complete analgesia for pulpal extirpation and chemomechanical preparation of the root canal system.

To attain an effective aseptic environment in the canal system, irrigation with sodium hypochlorite (NaOCl, 0.5%–0.25%) is still considered the gold standard. However, a certain degree of toxicity ranging from a mild allergic response to genotoxicity and mutagenicity is observed in a plethora of dental materials, including sodium hypochlorite.[5] Recently, an organic carcinogen named 2,6-xylidine was reported by Vidhya et al. formed on the interaction between lidocaine hydrochloride and sodium hypochlorite.[6] The presence of this precipitate leads to blocking of dentinal tubules which subsequently affects the hermetic seal and prognosis of the concerned tooth. The existence of any carcinogen in the root canal system over a longer duration under constant function will lead to its eventual seepage into the periapical environment.[5] Saravanakarthikeyan et al. conducted a study, which showed the persistence of this precipitate in the canal even after chemo-mechanical preparation.[7]

In endodontic literature, there are no published reports evaluating the repercussion of different agitation protocols on this precipitate formed on root canal dentin. The objective of this in vitro study was to evaluate whether final irrigation along with different agitation protocols will remove the precipitate formed following LA and NaOCl combination at the coronal- middle- and apical-root thirds.

MATERIALS AND METHODS

A total of 44 uniradicular teeth were collected, teeth that had more than one canal, incompletely formed apices, or any pathological alterations were rejected for the study. The teeth were carefully cleansed of debris and calculus and stored in 0.1% thymol solution at ambient temperature. After this stage of sample selection, the selected teeth were stored and surfaced-adhering to the infection control protocol. To eliminate root length as a variable, the specimens were decoronated using a water-cooled safe-ended diamond disc (Kerr Corporation, California, United States) to standardize the length of all specimens at 17 mm.

The apical foramen was sealed with resin composite to avert the ejection of experimental solutions. With the aid of #10- and #15-size K-files (Mani Inc., Tochigi Ken, Japan) canal patency was achieved. All specimens were rinsed with 5 ml of 17% ethylenediaminetetraacetic acid (EDTA) (Prime Dental Products Pvt Ltd., Thane, India) for 1 min; following which 0.5 mL of 2% LA was administered using a 27G stainless steel beveled needle, under pressure and left for 1 min in the pulpal space. Following this, 2 ml of 2.5% sodium hypochlorite (Vishal Dentocare Pvt Ltd., Gujarat, India) was injected into the canal and left for 1 min. The samples were subjected to cleaning and shaping using ProTaper Universal rotary system (Dentsply, Pennsylvania, United States) sequentially up to F3 with continuous movement at a speed of 300 rpm, torque of 3.5 Ncm, and irrigation was done using 2 ml of 2.5% NaOCl and 2 ml of 17% EDTA for 1 min each using 29G – double side vented closed-end needle (Vishal Dentocare Pvt Ltd., Gujarat, India) kept 1 mm short of the working length, with the change of each instrument.

Experimental groups

The teeth were then arbitrarily divided into four groups (n = 11) based on their final irrigation protocol:

Group 1 (control group): No irrigation was done

Group 2 (manual irrigation): Irrigation was done with 5 ml of 5.25% NaOCl for 1 min followed by rinsing with 5 ml of distilled water using 29G – double side vented closed-end needle

Group 3 (mechanical irrigation): Irrigation was done with 5 ml of 5.25% NaOCl agitated using XP-endo Finisher (FKG Dentaire, La Chaux de Fonds, Switzerland) for 1 min followed by rinsing with 5 ml of distilled water using 29G – double side vented closed-end needle

Group 4 (ultrasonic irrigation): Irrigation was done with 5 ml of 5.25% NaOCl agitated using ultrasonic for 1 min followed by rinsing with 5 ml of distilled water using 29G – double side vented closed-end needle.

The samples were then dried using absorbent paper points (Dentsply, Pennsylvania, United States) and access cavities were closed with a cotton pellet.

Field emission scanning electron microscopic (FESEM) evaluation

Longitudinal grooves were given along the buccolingual direction in each specimen with water-cooled diamond-coated safe-ended disc without disturbing the canal space. A chisel and surgical hammer was used to split the samples into two halves to unveil the canal walls. The superior bisection was selected, dried in a desiccator, and sputter coated. Evaluation at the coronal-(12 mm from apex), middle-(7 mm from apex), and apical-(2 mm from apex) third under a Field Emission Scanning Electron Microscope (Carl Zeiss AG, Oberkochen, Germany) at ×3000 was performed.

The percentage of the precipitate for each image was calculated by using Adobe Photoshop CS5, Adobe Systems. The ×3000 micrographics in TIFF format, standardized at 47 μm × 63 μm and the number of pixels of the entire image was determined. A magnetic pointer was used to indicate the resulting precipitate and the number of pixels was assessed. By dividing these two values, the percentage was achieved.

Statistical analysis

Data processing was done using the statistic program SPSS v 26.0 (IBM Corp., Armonk, NY, USA). The normality of numerical data was checked using Shapiro–Wilk test and since the data followed a normal curve; parametric tests have been used for comparisons. Analysis between and within the groups was done using one-way ANOVA and post hoc Bonferroni tests. Significance was set at P < 0.05.

RESULTS

Figure 1 illustrates precipitate occlusion at coronal, middle, and apical root thirds of all the specimens studied. However, the percentage of precipitate covering the root surface differed significantly (P < 0.05) with each agitation protocol used. In any irrigation protocol, the coronal third had the least levels of precipitate (P < 0.05) than the middle and apical third, whereas the apical third had the highest levels of precipitate [Tables 1 and 2]. Intergroup comparison showed that the middle third of manual irrigation had a higher mean value of precipitate than the no irrigation group [Tables 1-3]. Ultrasonic agitation showed promising results in reducing the extent of precipitate remaining after final irrigation [Table 3]. However, the precipitate levels at the apical thirds of XP-endo Finisher and ultrasonic irrigation showed no significant difference (P > 0.05).

Figure 1

Representative FESEM images of the root canal surfaces of Group 1 (No irrigation), Group 2 (Manual irrigation), Group 3 (XP-endo irrigation) and Group 4 (Ultrasonic irrigation) observed at coronal (C), middle (M) and apical (A) thirds at ×3000 (a-l). All the samples revealed occlusion of dentinal tubules with precipitate formation

Table 1

Inter group comparison of percentage values of precipitate formed (n=11 per group) between 4 groups at 3 levels using one - way ANOVA test

Groups	Levels	Mean (%)	SD	Minimum	Maximum	P
No irrigation	Coronal	13.841818	1.9479056	10.5000	17.4500	0.000*
	Middle	33.877273	2.1604633	31.2900	37.6500
	Apical	59.650909	6.1850084	49.1200	72.0100
Manual irrigation	Coronal	7.690000	1.2112886	5.9800	9.4200	0.000*
	Middle	39.539091	3.0738232	34.8200	45.3200
	Apical	48.862727	4.1762378	41.0900	55.0200
XP-endo irrigation	Coronal	3.730000	1.0253975	2.5600	5.4900	0.000*
	Middle	5.390000	1.1230939	3.7600	7.2900
	Apical	8.154545	2.0788668	5.0500	11.0900
Ultrasonic irrigation	Coronal	1.023636	0.3997067	0.0000	1.5500	0.000*
	Middle	2.170909	0.8174650	1.0400	3.4500
	Apical	4.034545	1.2899796	1.9500	6.2900

*Significant. SD: Standard deviation

Table 2

Inter group pair wise comparison of percentage values of precipitate formed (n=11 per group) within 4 groups at each level using post-hoc test

Groups	Levels		Mean difference	P
No irrigation	Coronal	Middle	−20.0354545*	0.000*
	Coronal	Apical	−45.8090909*	0.000*
	Middle	Apical	−25.7736364*	0.000*
Manual irrigation	Coronal	Middle	−31.8490909*	0.000*
	Coronal	Apical	−41.1727273*	0.000*
	Middle	Apical	−9.3236364*	0.000*
XP-endo irrigation	Coronal	Middle	−1.6600000*	0.036*
	Coronal	Apical	−4.4245455*	0.000*
	Middle	Apical	−2.7645455*	0.000*
Ultrasonic irrigation	Coronal	Middle	−1.1472727*	0.016*
	Coronal	Apical	−3.0109091*	0.000*
	Middle	Apical	−1.8636364*	0.000*

*Significant

Table 3

Inter group pair wise comparison of percentage values of precipitate formed (n=11 per group) between different irrigation methods at each level

Levels	Groups		Mean difference	P
Coronal	No irrigation	Manual irrigation	6.1518182*	0.000*
	No irrigation	XP-endo irrigation	10.1118182*	0.000*
	No irrigation	Ultrasonic irrigation	12.8181818*	0.000*
	Manual irrigation	XP-endo irrigation	3.9600000*	0.000*
	Manual irrigation	Ultrasonic irrigation	6.6663636*	0.000*
	XP-endo irrigation	Ultrasonic irrigation	2.7063636*	0.000*
Middle	No irrigation	Manual irrigation	−5.6618182*	0.000*
	No irrigation	XP-endo irrigation	28.4872727*	0.000*
	No irrigation	Ultrasonic irrigation	31.7063636*	0.000*
	Manual irrigation	XP-endo irrigation	34.1490909*	0.000*
	Manual irrigation	Ultrasonic irrigation	37.3681818*	0.000*
	XP-endo irrigation	Ultrasonic irrigation	3.2190909*	0.003*
Apical	No irrigation	Manual irrigation	10.7881818*	0.000*
	No irrigation	XP-endo irrigation	51.4963636*	0.000*
	No irrigation	Ultrasonic irrigation	55.6163636*	0.000*
	Manual irrigation	XP-endo irrigation	40.7081818*	0.000*
	Manual irrigation	Ultrasonic irrigation	44.8281818*	0.000*
	XP-endo irrigation	Ultrasonic irrigation	4.1200000	0.082+

*Significant, +Not significant

DISCUSSION

A wide range of complex variations in root canal anatomy exists, including root canal configuration type, developmental anomalies, and more minor canal morphology such as accessory canals and apical deltas. During cleaning and shaping, debridement of complex anatomical intricacies cannot be achieved solely by mechanical instrumentation. Irrigation of the canal systems plays an important role to debride such complexities which may be left uninstrumented, aiding in achieving an aseptic environment. No single irrigant (NaOCl, CHX, EDTA) has ideal characteristics to provide the desired effect on the root canal, hence a combination of irrigants is advocated. However, when in contact with each other, they are known to chemically react and form a precipitate. Endodontic literature states that para-chloroaniline precipitate, which is a known carcinogen formed due to interaction between NaOCl and CHX, as reported by Basrani et al.; and a nontoxic white powdery precipitate on the combination between 17% EDTA and CHX as reported by Rasimick et al.[89]

Recently, Vidhya et al.,[6] conducted a nuclear magnetic resonance (NMR) spectroscopy evaluating the interaction between LA and NaOCl and concluded that a carcinogenic precipitate 2,6-xylidine was formed. Due to an acid hydrolytic reaction, the carbon atoms present in LA reacted with hypochlorous acid formed due to dissociation of NaOCl, leading to its severance and subsequent cleavage of the double bond. On further hydrolysis, a carcinogenic precipitate 2,6-xylidine is formed.

Even after thorough cleaning and shaping procedures, this precipitate can still be observed on the canal walls, as reported by Saravanakarthikeyan et al.[7] In the above-mentioned study, the methodology included a single final rinse protocol. However, in this current study, we have incorporated different agitation protocols along with the final rinse, to evaluate the ease of removal of the precipitate.

In this study, to remove the smear layer effectively and efficiently and visualize open dentinal tubules, 17% EDTA was used initially and as a copious irrigant between each instrumentation in all the samples.[10] In our results, the coronal-and middle-thirds of the samples displayed an essentially nonexistent smear layer, while a moderate amount of smear layer persisted in the apical-thirds of the specimens. This can be associated with root canal intricacies, complex anatomies, and the inability of EDTA to adequately penetrate the apical-thirds of the root canal.[1112]

In the current study, the precipitate was observed occluding dentinal tubules in all the samples irrespective of the irrigant agitation protocol followed. Ultrasonic irrigation proved to be the most effective, in accordance with previous literature.[131415] Although XP-endo Finisher and ultrasonic groups had no significant difference at the apical thirds, the mean values of precipitate observed were more for the XP-endo Finisher. It was observed that the coronal third of all samples had the least amount of precipitate as compared to the apical third which can be attributed to exposure of the coronal third to increased quantities of irrigant than the apical third and diverse canal intricacies.[71617] In contrast to existing literature, our study showed that the middle third of manual irrigation had a higher mean value of percentage of precipitate when compared to no irrigation group, this may be due to varying cross-sectional shapes of the canal system.[181920]

Chauhan et al. and Homayouni et al. in their respective research have stated that the presence of any precipitate will hinder the hermetic seal which is an essential requisite to promote the healing of the concerned tooth.[2122] The existence of any precipitate in the pulp chamber may impede the coronal seal of postendodontic restoration.[23] Chauhan et al.[21] recently reported that the carcinogenic precipitate 2,6-xylidine demonstrated high levels of microleakage, thus having a negative effect on the hermetic seal of obturation. This may lead to possible leaching of this precipitate into the periapical environment as well. Current evidence-based literature also reveals this precipitate to show certain degree of cytotoxicity, genotoxicity, metabolic overload and acute systemic toxicity of the liver.[2425] However, quantity of the precipitate formed in the canal system is negligible when compared to actual levels required to show any significant systemic effects in humans.

This is a clinically relevant study since intrapulpal injection is routinely administered during root canal therapies. However, the actual volume of intrapulpal LA (0.2–0.5 ml) injected into the pulp space is modest, hence the precipitate formed is not significant enough to cause any lasting systemic or local side effects. Nonetheless, the presence of any carcinogenic substance in the canal system can prove to be detrimental to the tooth and its environment over a period of time. Hence, we should avert the immediate use of NaOCl following intrapulpal anesthetic injection or employ the use of saline flush subsequent to intrapulpal injection. Birchfield and Rosenberg stated that the backpressure of the solution independent of the solution injected is the primary contributing factor for the profound anesthetic effect; thus 0.9% saline can be substituted for 2% LA in intrapulpal route for profound supplemental anesthesia.[26]

Further research is warranted to determine the possible side effects of 2,6-xylidine on root canal dentin and postendodontic coronal restorations.

CONCLUSION

Within the limitations of this in vitro study, we can state that:

A carcinogenic precipitate, 2,6-xylidine is formed on the interaction between intrapulpal LA and subsequent irrigation with NaOCl occluding the dentinal tubules at the coronal-, middle- and apical-thirds of the root canal which is not completely removed by any irrigation agitation protocol

Ultrasonic irrigation was the most successful in removing the highest levels of precipitate occluding the dentinal tubules.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.