In-vitro evaluation of intermediate irrigants for removal of para-chloroaniline from the tooth during root canal treatment

Introduction

Root canal cleaning requires removing all potential irritants such as bacteria, organic/inorganic debris, vital and necrotic pulp tissues, as well as blood. Acceptable cleaning of the root canal can be achieved through irrigation and instrumentation. Irrigation of root canal is essential during chemomechanical preparation due to its role in the eradication of microorganism, tissue dissolution, removal of debris as well as smear layer.^1,2 The profound use of an antimicrobial solution like sodium hypochlorite (NaOCl) are found to very essential in elimination of the necrotic pulp as well as microorganisms.

Sodium hypochlorite is considered as first choiceand most commonly used irrigant during root canal therapy due to its excellent antimicrobial efficacy as well as tissue dissolving property³ . From, earlier studies it was found to be greatly effective against several strains of Enterococcus fecalisand other dental pathogens. SinceNaOCl was allergic to some patients CHXgluconate was also widely used antimicrobial solution at various concentrations and also as a substitute or in conjunction with Sodium hypochlorite due to its lesser toxicity⁴ . CHXimpartsantimicrobial substantivelyto dentin as well⁵ . NaOClwhen used with CHX results in formation of orange brown precipitate known as parachloroaniline which might be carcinogenic and has immune toxic effect^6,7. This precipitate occludes the dentinal tubule⁸ , may also hamper radicular seal⁷ . To minimize the formation of orange brown precipitate various intermediate intra-canal medicaments are used and stated⁹ .

70%alcohol as an intermediate intra-canal flush can prevent the formation of precipitate because of itss volatile,tensioactive agent,highly electronegative. It can also penetrate deeply to remove residual Sodium hypochlorite present in canal¹⁰. 17% EDTA is the most commonly used irrigating solution by chemo-mechanical preparation. Due to its chelating action it produces smooth and clean canal walls so the sealer can adhere to the canal walls resulting in accurate root canal filling and proper seal^11,12. Citrus aurantifoliaextract that contains citric acid is used as an intermediate intracanalirrigant. Sinice citric acid is a weak organic acid, and has chelating properties which is thought to be able to prevent the formation of orange brown precipitate.¹³

Our current study aim is to evaluate the spectrophotometric analysis of Para-chloroaniline formed when sodium hypochlorite and chlorhexidine are mixed, and prevention of orange brown precipitate by three different intermediate irrigating solutions in varying concentration gradients.

Materials and methods

This is a prospective experimental study. The study was conducted at Dextrose Technologies Pvt Ltd., Bangalore, Karnataka, India. All the chemicals, test samples were procured from Himedia (Mumbai). 3% Sodium hypochlorite, 2% Chlorhexidine, 14.25% EDTA, 70% Ethanol and Distilled water,Fresh lime juice extract was prepared just before the analysis.

λmax estimation of Parachloroaniline Inorder to estimate

the λmaxof Parachloroaniline, we mixed1 ml of 3% sodium hypochlorite and various concentrations of chlorhexidine solution (10-100µl) to find the concentration of chlorohexidine at which orange-brown precipitation, i.e. Para-chloroaniline is formed. Following which that particular concentration will be obtained and analysed spectro photometrically at a wavelength range between 350-550 nm and λmaxwill be calculated.

Experimental design for testing irrigating solutions

Three different irrigating solutions; Fresh lime juice extract, 14.25% EDTA and 70% Ethanol were grouped as A,B,C respectively.Two trialsfor each group of irrigating solution (test samples) was carried out, spectrophotometrically using centrifuged test samples. The test mixture were centrifuged at 5000 rpm for 20 minutes .

Group A: 100µl of 3% Sodium hypochlorite and 60µl of 2% Chlorhexidine was taken as control and was tested against centrifuged test solution containing 100µl of 3% Sodium hypochlorite, 60µl of 2% Chlorhexidine and different volumes of Fresh lime juice extract(10-100µl) by measuring the OD

Group B: 100µl of 3%Sodium hypochlorite and 60µl of2% Chlorhexidine was taken as control and was tested against centrifuged test solution containing 100µl of 3% Sodium hypochlorite, 60µl of 2% Chlorhexidine and different concentrations of 14.25% of EDTA (10-100µl) by measuring the OD

Group C: 100µl of 3% Sodium hypochlorite and 60µl of 2% Chlorhexidine was taken as control and was tested against centrifuged test solution containing 100µl of 3%Sodium hypochlorite, 60µl of 2% Chlorhexidine and different concentrations of 70% of Ethanol (10-100µl) by measuring the OD

In vitro analysis:

In vitro analysis was carried out using single rooted teeth. After washing with distilled water and ultrasonic scaling, the specimens were immersed in 0.5% Chloramine T solution until use. The teeth weredecoronated to obtain a standardized root length . Canal patency was evaluated using #10 K file and teeth with canal obstructions discarded. The actual length of each tooth was determined with #10 K file, which was introduced into the canal until its tip emerged through the major apical foramen. The working length was established by subtracting 1 mm from the actual length. The apices of the specimens was sealed with wax to prevent extrusion of irrigating solutions. All the canals were prepared by the using NiTi rotary files until F4 size as per the manufacturer’s instructions. And the canals were irrigated with 2.5% NaOCl between each instrumentation sequence using a 30G side vented needle introduced 2 mm short of the apex. After complete mechanical instrumentation all the samples were thoroughly flushed with 2.5 ml of 3% NaOCl using 30G side vented needle. The samples was randomly allocated to three groups of 15 samples each based on the use of 2.5 ml of intermediate irrigating solution for 60 s as specified below Group A –fresh lime juice extract ,Group B-14.25% edta,GroupC - 70% ethanol.After that, all the group samples were irrigated with 2 ml of 2% CHX gluconate solution as final wash. The root canals were dried using paper points of corresponding apical preparation size..

Two longitudinal grooves were made along the buccal and lingual surfaces of the roots with watercooled diamond disc. The roots were then sectioned and was observed under stereomicroscope .

Statistical analysis: Statistics was carried out using Statistical Package for Social Sciences [SPSS] for Windows, Version 22.0. Released 2013. Armonk, NY: IBM Corp. Descriptive Statistics was taken to correlate expression of the OD values in terms of Mean and SD. And Inferential Statistics One-way ANOVA followed by Tukey's Post hoc Analysis was used to compare the mean Optical Density values between 3 groups. The level of significance [P-Value] was set at P<0.05

Results

λmax estimation of Parachloroaniline

Parachloroanilineformation (orange-brown precipitation ) was found at a concentration of 60µl of chlorhexidine and 1ml of Sodium hypochlorite (Figure 1). The spectrophotometric analysis showed peak value at 450nm that is the intensity of orange brown precipitate formation.

Testing of irrigating solutions

Group A: Fresh lime juice extract:Parachloroaniline formation was decreasing upon increasing the volume of lime juice extract and it was found that at a volume of 40µl of lime juice extract it decreased the orange- brown precipitate formation. (Fig 2)

Group B: 14.25% EDTA: Parachloroaniline formation was decreasing upon increasing the volume of 14.25% EDTA and it was found that, at a volume of 100µl of 14.25% EDTA decreased the orange-brown precipitate formation.(Fig 3)

Group C: 70% Ethanol : Parachloroaniline formation was decreasing upon increasing the volume of 70% ethanol and it was found that, at a volume of 30µl of 70% ethanol decreased the orange-brown precipitate formation.(Fig 4)Stereomicroscopic image of a) control b) Lime extract b) 14.25%EDTA c) 70% ethanol

Lime extract group could remove the precipitate along the enter root canal followed by 14.25% EDTA in the ethanolalcohol group orange brown precipitate was present along the coronal ,middle and apical one third.

Statistical analysis

The test demonstrates the mean Optical Density values measured for each study group. The mean Optical Density values for Lime Juice group was 0.7106 ± 0.0906, for Ethanol group, it was 0.8684 ± 0.0970 and for EDTA group, it was 0.8343 ± 0.1209. This difference in the mean Optical Density values between 03 groups was statistically significant at P=0.005. [Refer Table no. 1 and Fig. no. 1]

Multiple comparison of mean differences between different groups revealed that Ethanol group showed significantly higher mean Optical Density values as compared to Lime Juice group at P=0.006. This was followed by EDTA showing significantly higher mean Optical Density values as compared to Lime Juice group at P=0.03. However, no significant differences were observed between the Ethanol and EDTA group [P=0.75]. This infers that the mean Optical Density values was least for lime juice group followed by EDTA group and highest in ethanol group. [ table no. 2 & Fig. no. 2]

λmax determination and quantification of PCA

One way Anova test showed that group A exihibited the least OD value among all the experimental groups followed by group B and group C (Table no: 1). Post hoc analysis showed significant difference on group A as compared to other groups (Table 2).Group A gave the best results.Although Group C performed better than Group B the difference was not statistically significant.

Discussion

The main aim of this study was to evaluate the intensity of parachloroaniline formation when 60µl of chlorhexidine is used as final irrigant and also to prevent the formation of parachloroanilineby different intermediary intracanalirrigants. The intensity of parachlroaniline formed was measured by spectrophotometer²¹. Spectrophotometry is a method to measure how much a chemical substance absorbs light by measuring the intensity of light as a beam of light passes through sample solutionandit works on the principle of Beer Lambert’s Law in which - max determination of PCA was done using a wavelength scan between 350nm to 550nm . Each compound absorbs or transmits light over a certain range of wavelength. This measurement can also be used to measure the amount of a known chemical substance.

Studies have reported the occurrence of a colour change and precipitation when 3% Sodium hypochlorite and 2% Chlorhexidine are combined. This precipitate known as Parachloroaniline, which is possibly carcinogenic to humans and has toxic and immunotoxic effects. The Para-chloroaniline precipitate formed, when 2% Chlorhexidine is used as a final irrigating solution after 3% Sodium hypochlorite , covers the surface of the root canal dentin and adversely affects the dentin permeability. This is detrimental to medicament diffusion and sealer adaptation¹⁴. A study evaluated the chemical nature of this precipitate and found that there was an intermediate reaction when 2% chlorhexidine was combined with 3% Sodium hypochlorite.⁶

In this study we evaluated minimum amount of 2% chlorhexidine that can lead to Parachloroaniline formation. in graph 1, it shows the peak value of Para-chloroaniline formed was at 450nm. The optical density at 450nm is 1.91 .Above this wavelength it is observed that OD value decreases and shows minimum value at 550nm. In figure 1 it was seen that at OD 450, minimum of 60µl of chlorhexidine when combined with 100 µl of 3% Sodium hypochlorite was required for the total precipitation to occur. At 60µl concentration of chlorhexidine precipitation starts appearing.

In this study 70% of ethanol as an intermediate flush between sodium hypochlorite and chlorhexidine could prevent the formation of precipitate . Because alcohol is a volatile,tensioactive agent it is highly electronegative and can penetrate deeply to remove the residual sodium hypochlorite present in the canal. Since alcohol is a volatile agent it also helps in drying the canal .Alcohol helps in reducing the thickness of precipitate due to dilution of Sodium hypochlorite and flushing away Sodium hypochlorite¹⁰. However the biocompatibility of alcohol with periapical tissues isof concern and further investigations has to be done for its use as an endodontic irrigant. The study also shows the faster settling of the orange-brown precipitate upon centrifugation.

EDTA is a chelating agent used in endodontics for preparation of narrow and calcified canals. EDTA in liquid solution chemically soften the root canal dentine and dissolve smear layer and increase the dentine permeability.Studies shows that , irrigation with 14.25% EDTA followed by 3% Sodium hypochlorite could demineralize the dentine and produce erosions in coronal as well as the middle part of the root canal .and it is due to the chelating action of EDTA.¹⁵ Thus this property of EDTA was able to remove the residual 3% Sodium hypochlorite in the rootcanaland prevent Parachloroaniline formation when 2% chlorhexidine is used.Whereas some studies also shows that the simultaneous use of 14.25% EDTA and 3% Sodium hypochlorite reduces the amount of 3% Sodium hypochlorite, which may decrease the ability to dissolve tissue and antimicrobial activity, so such solutions should not mixed together^17,18. When EDTA and Chlorhexidine is mixed together a pink precipitate is formed which might reduce the property of EDTA to remove smear layer.^16,17

Citric acid belongs to the chelating agents category, is a weak organic acid¹⁹ Citrus aurantifolia (lime juice) is used as an intermediate irrigant, because it consists of citric acid along with an added antimicrobial feature. Lime juice consists of 88% water, 6-8% citric acid, 2% potassium citrate and calcium, 0.4-0.6% and other substances²⁰. Because citrus aurantifolia extract has citric acid, it is able to remove the smear layer and open the dentinal tubules.In this study fresh lime extract is used to prevent the formation of Para-chloroaniline. When lime juice is used in full concentration the citric acid present which is a weak organic acid remove the residual 3% Sodium hypochlorite in the canal walls and prevent the formation of Para-chloroaniline. Braumgartner and Mader demonstrated that sodium hypochlorite and EDTA combination have more antimicrobial action than when sodium hypochlorite is used alone. Citric acid present in lime juice has antimicrobial action thus when combined with 3% Sodium hypochlorite have more antimicrobial actionthan when used alone^19,20.

Conclusion

Within the limitation of this study through spectrophotometric analysis freshly prepared limeextract was found to be very effective in prevention of Para-chloroaniline, followed by 14.25% EDTA and 70% ethanol. At a volume of 40µl of lime juice extract, 100µl of 14.25% EDTA and 30µl of 70% Ethanol Para-chloroaniline formation was decreased. Group A (lime group) exhibited the least OD value among all the experimental groups followed by group B (14.25% EDTA) and group C(70% Ethanol) and also statistically proved.Since lime juice is a natural agent no undesirable effects were observed and can be safely used as an intermediate irrigating solution. Hence it can be used as an alternative to other intermediate irrigating solutions currently used and gives insight about the analysis and novel ways of prevention of PCA during root canal treatment.

Acknowledgement

We thank everyone who supported this study, especially Dr. SumangalaBhat and Miss Aiswarya from Dextrose Technologies pvt.Ltd. for allowing to carrying out the study at their company and helping in the article as well.

Conflict of Interest:

There is no conflict of interest.