DATA AND METHODS Data source The first author searched Medline (1999-01/ 2011-12) to retrieve the relevant articles with “chelating agent, smear layer, root canal irrigation, root canal preparation” as key words. Later, manual search was carried out by all the three authors independently and the articles in English language were reviewed. Inclusion criteria (1) Literature content having significant correlation with the objective of the review. (2) Arguments having significant basis on the original literature. (3) Definitive conclusions and opinions and comprehensive analysis of the references. Exclusion criteria (1) The research content and the objective of the review have little or no correlation. (2) Repetition of the research content. Quality assessment Literature selection and its evaluation were cross checked independently by the three authors and the differences in opinions were resolved through discussion, reasoning and communication with the corresponding author. Data extraction The initial search revealed 80 articles. After prescreening, i.e., reading the title, abstract and content by the corresponding author, 30 literatures were found to have no correlation with the review and thus excluded. Repetition of the content was found in five articles, hence not included. Later, by manual searching, six articles were found relevant. Finally a total of 50 articles were included in the review."

RESULTS Chelating agents Chelating agents are combined with hydroxyapatite crystals to form stable complexes causing dentin demineralization[7]. Commonly used chelating agents include 15% to 17% ethylenediaminetetraacetic acid, ethylene glycoltetraacetic acid (EGTA), RC Prep (root canal preparation cream), citric acid, a mixture of doxycycline, citric acid and a detergent [Tween 80](Dentsply, Tulsa, OK, USA), smear clear (17% ethylenediaminetetraacetic acid, cetrimide, surfactant), chlorhexidine digluconate, etc. Mixture of tetre cycline isomer, an acid and a detergent is an aqueous solution of 3% doxycycline, a broad-spectrum antibiotic; 4.25% citric acid, a dematerializing agent; and 0.5% polysorbate 80 detergents (Tween 80)[8]. It represents an effective solution for removal of smear layer from instrumented canal walls without adversely affecting the physical properties of the tooth, favors adhesive restorative procedures and is biocompatible. It possesses reasonable, antimicrobial property against enterococcus facials; a facultative gram-positive anaerobe, predominantly associated with persistent per apical infections in endodontic treated teeth, which is extremely resistant to current treatment modalities[1]. It is widely accepted that the most effective method to remove smear layer is alternate irrigation of the root canal with ethylenediaminetetraacetic acid and NaOCl which flushes out the debris. NaOCl with its photolytic capabilities acts as a tissue solvent dissolving the organic portion of the smear layer, i.e. pupil remnants and dentin debris and at the same time acts as a bactericidal agent. Ethylenediaminetetraacetic acid forms calcium chelae with hydroxyapatite crystals which in turn changes the permeability, micro hardness and solubility of root canal dentin[9-11] by causing dentin demineralization[12]. A study by Clarkson et al [13] demonstrated that active chlorine content of NaOCl was greatly reduced by mixing with ethylenediaminetetraacetic acid. Even small admixtures of ethylenediaminetetraacetic acid caused substantial reduction in the active chlorine content irrespective of the concentration of NaOCl. Thus to retain their respective therapeutic benefits, NaOCl and ethylenediaminetetraacetic acid should not be mixed in the canal and it should be evacuated and/or dried between solutions. The order in which the two solutions should be used remains a matter of ongoing debate. Factors affecting the role of chelating agent The volume of the fluid There is always a study going on in search for the best method to remove the smear layer. Volume of the irrigate is also one of the most debated elements. A recent study by Uroz-Torres et al [14] reported no significant improvement in smear layer removal with end activator which may be attributed to the lower volume of the irrigate used (1 mL 17% ethylenediaminetetraacetic acid, 3 mL 4% NaOCl). Kuah et al [15] demonstrated that a greater volume of 17% ethylenediaminetetraacetic acid (5 mL) followed by 1% NaOCl (5 mL) provided more effective smear layer and debris removal in the apical region of straight root canals, which was further enhanced by ultrasonic agitation. Crumpton et al [16] demonstrated that the ideal volume of 17% ethylenediaminetetraacetic acid for debris and smear layer removal was 1 mL with Max-I- probe irrigating tip. The study concluded that 1 mL ethylenediaminetetraacetic acid solution was as effective as 10 mL ethylenediaminetetraacetic acid solution. On increasing the volume of ethylenediaminetetraacetic acid over 1 mL, no further debris removal was seen. Similarly, Mello et al [17] demonstrated that a final rinse with 5 mL of ethylenediaminetetraacetic acid was as efficient for removing smear layer as 10 mL or 15 mL. The study suggested high-volume irrigates to be unnecessary for root canal debris removal. Most of the studies have suggested better smear layer removal with higher volume of root canal irrigate used. As the volume is more, the chelating solution gets better contact with the root canal walls thus enhancing the chelating action, mostly in the apical thirds of the root canals. The above mentioned literatures also suggest that the volume of the solution has no significant correlation with the dentinal erosion. The ability of an irrigating solution to remove smear layers from the coronal third, middle third, and apical third of a canal wall depends on the aggressiveness of the irrigate and the manner in which the irrigate is delivered[18]. For example, the presence of a vapor lock in a closed-canal system precludes optimal delivery of irrigate to the apical third of the canal wall[12]. Although the presence of a vapor lock in a closed-canal system[19] may affect the efficacy of smear layer removal from the apical third of the canal well, the presence of a film of irrigate between the air bubble and the canal wall still permits some form of smear layer removal albeit in a less efficient manner. On the contrary, the ability to clear debris from the canal walls is more dependent on the flow of the manner in which irrigate is agitated[12] instead of the aggressiveness of the irritants. Because there is only limited flow of irrigates by manual delivery of irrigate through a side-vented needle without additional agitation[20]. In the future, the efficiency of debris clearance from the canal space should be evaluated in a closed-canal system in conjunction with agitation devices such as sonic and ultrasonic agitation systems as well as devices that incorporate an apical negative pressure approach. The washing time Studies have shown that the optimal working time for ethylenediaminetetraacetic acid is 15 minutes after which no chelating action can be expected, because of the self-limiting decalcifying action of ethylenediaminetetraacetic acid [21]. Calt et al [21] from their study concluded that 10 mL of 17% ethylenediaminetetraacetic acid followed by NaOCl completely removed the smear layer in 1 minute. The applications of ethylenediaminetetraacetic acid for 10 minutes caused erosion of peritubular and intertubular dentin. Thus they recommended not prolonging the washing time to longer than 1 minute. In a comparative study of 17% ethylenediaminetetraacetic acid, 10% citric acid, and 37% phosphoric acid, with time period of 30 seconds, 1 minute and 3 minutes, none of the experimental solutions could remove the smear layer effectively within 30 seconds. In the 1-minute time period, the phosphoric acid solution showed better results than other substances evaluated. In the 3-minute time period, all the substances worked well in the middle and cervical thirds although phosphoric acid solution showed excellent smear layer removal even in the apical thirds[22]. Studies on NaviTip FX demonstrated them to be effective in smear layer removal with both the 17% ethylenediaminetetraacetic acid solution[24] and 19% ethylenediaminetetraacetic acid viscous gel (FileEze)[23] in 1 minute. It may be attributed to the effective penetration of the irrigation needle combined with longer irrigation time which exposed the root canal to a higher volume and better flow of irrigates. Study by Kuah et al [15] also showed that a 1-minute application of ultrasonically agitated 17% ethylenediaminetetraacetic acid efficiently removed the smear layer and debris in the apical region of the root canal. A study by Salman et al [25] in human molar root canals showed 30-second passive agitation of 17% ethylenediaminetetraacetic acid with the Sonic is Canal Brush improved root canal cleanliness via debris removal and smear layer reduction. In contrast to this using the Max-Ⅰ-Probe irrigating tip, Saito et al [26] demonstrated that reducing the irrigation time with 1 mL of 17% ethylenediaminetetraacetic acid to 30 or 15 seconds significantly decreased smear layer removal compared with a 1-minute rinse. Depending on the canal master apical size, curvature, and taper, the optimal regimen suggested for effectively removing the smear layer in root canals was a final rinse with 1 mL of 17% ethylenediaminetetraacetic acid for 1 minute followed by a 3 mL rinse with 6% NaOCl. When the exposure time of 17% ethylenediaminetetraacetic acid and 2.5% NaOCl was increased from 1 minute to 5 minutes, there was more calcium ion removal from the root. This may decrease the micro hardness of the root canal and the dentinal erosion may interfere with proper adhesion of endodontic sealers and adhesive cements[27]. Thus the majority of the studies suggest 1 minute being the most appropriate time for application of ethylenediaminetetraacetic acid. Increasing the washing time has been found to be deleterious to the root canal dentin. However, the protocols followed by different studies might be different and the similarity in results is just not sufficient to lead to conclusions. Further studies are still necessary to be carried out in search for the ideal washing time of the root canal irrigate. The concentration of chelating agent Different studies have compared the smear layer removal capacity of different concentrations of ethylenediaminetetraacetic acid. Concentration as high as 24% to as low as 1% of ethylenediaminetetraacetic acid has been studied for efficient smear layer removal and to minimize the dentinal erosion. Studies have shown that the ability of ethylenediaminetetraacetic acid to remove the smear layer has no significant correlation with the concentration. 3% ethylenediaminetetraacetic acid and 24% ethylenediaminetetraacetic acid have the same efficiency in smear layer removal. Concentration as low as 1% has the capacity to remove the smear layer[28]. Irrespective of their concentration, ethylenediaminetetraacetic acid can cause erosion of the dentin when used as a root canal irrigate except 1% ethylenediaminetetraacetic acid which demonstrates limited erosion[29]. However 10%-17% ethylenediaminetetraacetic acid has been shown to be particularly effective in the removal of organic and inorganic debris without weakening the tooth when used in combination with NaOCl (2.5%-5%)[18]. In another study by Silva et al [30], Smear Clear (17% ethylenediaminetetraacetic acid + cetrimide + surfactant) was able to remove the smear layer from the root canals of permanent teeth as effectively as 14.3% ethylenediaminetetraacetic acid. From the results of above studies, it is seen that irrespective of concentrations ethylenediaminetetraacetic acid could remove the smear layer if other elements such as time, pH were appropriate and limited erosion was seen with low concentration of ethylenediaminetetraacetic acid. However, these data are not sufficient to reach to a conclusion and still further studies are necessary to find the appropriate concentration of ethylenediaminetetraacetic acid. Acid solutions have been recommended for removing the smear layer, including sodium salt of ethylenediaminetetraacetic acid, most active at a concentration of 15%–17% and pH of 7–8; citric acid solutions used at concentrations of 10%, 25%, and 50%; and orthophosphoric acid at concentrations of 10%, 32%, and 37%[31]. Currently, the chelating agents like ethylenediaminetetraacetic acid are extensively being used to remove the smear layer formed during the chemo-mechanical preparation of the root canals. However, there are few studies comparing the action of these chelating agents in their liquid and gel forms. Wadhwani’s study[32] evaluated the ability of 17% ethylenediaminetetraacetic acid solution and 19% ethylenediaminetetraacetic acid gel to remove debris and smear layer produced during root canal preparation with two NiTi files systems, Mtwo and Protaper. The experimental results showed that there was no statistically significant difference in the varied instruments used (Mtwo and Protaper files), and 17% ethylenediaminetetraacetic acid solution and 19% ethylenediaminetetraacetic acid gel. They believed that both the NiTi instruments produced a similar dentin surface on the root canal wall when used with ethylenediaminetetraacetic acid gel and ethylenediaminetetraacetic acid solution. The use of lubricant gel is recommended with all rotary endodontic instruments, as it acts as a cheater as well as a lubricant. As most of the gels available have ethylenediaminetetraacetic acid as one of the main constituents, it is recommended that instead of 17% liquid ethylenediaminetetraacetic acid, 19% gel should be preferred as the lubricant and smear layer removal. Although the action of gel and solution forms of ethylenediaminetetraacetic acid are the same, the advantage of lubricant in gel form is that it does not extrude from the root canal. Hence, gel should be preferred over liquid ethylenediaminetetraacetic acid because of its thyrotrophic nature. The pH of chelating agent The chelating efficiency of ethylenediaminetetraacetic acid at high pH is reported to be greater due to a higher ratio of ionized to no ionized molecules in the solution. At high pH values, the excess number of hydroxyl groups will slow down the dissociation of hydroxyapatite, thus limiting the number of Ca2+ available. At low or neutral pH, the binding of Ca2+ will tend to increase the dissociation of hydroxyapatite and its availability for chelating[33]. In a comparative study of 17% ethylenediaminetetraacetic acid (pH-7) and 7% malefic acid (pH-1.04) for the evaluation of decalcifying effect on root canal dentin, malefic acid reduced the Ca2+/PO43- ratio significantly more than ethylenediaminetetraacetic acid up to 5 minutes. At 10 and 15 minutes, ethylenediaminetetraacetic acid also significantly removed more Ca2+ and PO43-. The faster action of malefic acid has been attributed to its acidic pH compared with that of ethylenediaminetetraacetic acid [34]. Serper et al [29, 35] had similar conclusions that ethylenediaminetetraacetic acid is more effective at neutral pH than when it is applied at pH 9.0. Then, Serper[29] confirmed that the dematerializing effect measured as the amount of released phosphorus is stronger for solutions with a pH of 7.5 as compared to solutions with pH of 9.0. In another experiment by Calt et al [21] using 10-minute application of 17% ethylenediaminetetraacetic acid, pH 7.4 excessive per tubular and inter tubular dentinal erosion was observed. pH of the ethylenediaminetetraacetic acid solution showed little or no alteration throughout the observation period. Thus they concluded that to reduce erosive effects of ethylenediaminetetraacetic acid solutions during prolonged cleaning and shaping of root canals, lower concentrations of ethylenediaminetetraacetic acid should be used at neutral pH. The root canal preparation size and taper of the apical Literature has shown that apical enlargement and deeper positioning of the irrigation needle are required to clean the apical third of the root canal[36-37]. Khademi et al [38] found that apical instrumentation to a #30 size file with 0.06 coronal taper was effective for the removal of debris and smear layer from the apical portion of root canals. In a study on straight root canal tooth by Goel et al, the NaviTipFX brushes showed almost complete removal of the smear layer and debris at the apical third, with no significant difference between the apical, middle and coronal thirds[24]. Continuous delivery of irrigate (17% ethylenediaminetetraacetic acid) at the apical third and better mechanical debridement with active scrubbing may be the reason for the better cleaning. The design of the NaviTipFX allows it to reach up to the apex and at the same time actively scrubs the canal wall while concomitantly delivering the irrigates. This mechanical activation might have enhanced the chemical action of the irrigate in removing the smear layer and debris simulating the cleansing effect of a bottle brush. This result is supported by Batista et al [39] who proposed that neither time of application nor concentration of the irrigates was responsible for removal of the smear layer; it was the method of application that was more important. Similarly, the Self Adjusting File operated with the continuous flow of irrigates alternating between NaOCl and ethylenediaminetetraacetic acid, resulted in root canals that were free of debris and almost free of smear layer even in the apical third of the canal[40]. Huang et al [41] devised a test model to test the efficacy of irrigation (dynamic/static) in removing a bio-molecular film from root canal walls and found that both the dynamic and static irrigation could not completely remove the collagen bio-molecular film. Factors influencing its removal, in descending order of priority, were: corona-apical level, apical size and taper of canal preparation, and dynamic/static irrigation. The first finding of Arvaniti’s study[42] was that root canal taper did not affect its debridement. The experimental result can be compared with those of some previous studies. Lee and Van der Sluis et al [43-44] prepared root canals with GT files Series 20 and studied the influence of root canal taper on debris removal. According to their results, the increase in taper led to better debridement. Albrecht et al [45] reported that when canals were prepared with GT files size 20 (DENTSPLY/Maillefer), the increase in taper led to better debridement, whereas when the apical preparation size was 40, taper had no influence on debris removal. It was concluded that root canal debridement is mainly affected by final instrument size and to a lesser extent by canal taper. The influence of final instrument size on root canal cleanliness was also studied by Usman et al [46], who reported that root canal instrumentation with GT file 40/0.06 led to significantly better debris removal than with GT file 20/0.06. In Arvaniti’s study[42], root canals were prepared with GT files size 30, and no statistically significant differences were found between groups of different tapers. They could conclude that the root canal taper can affect its debridement only when final instrument size is smaller than 30. However, we believe that this conclusion needs further investigation. Action of chelating agents on root canal dentin and per apical tissues In a comparative study on micro hardness of root canal lumen dentin using chelating solutions: 15% ethylenediaminetetraacetic acid, 10% citric acid, 5% malice acid, 5% acetic acid, apple vinegar and 10% sodium citrate, 15% ethylenediaminetetraacetic acid and 10% citric acid were found to be more effective. The solutions reduced the micro hardness of the most superficial dentin layer of the root canal lumen facilitating the biomechanical preparation[5]. Most of the studies have confirmed that ethylenediaminetetraacetic acid in combination with NaOCl causes peritubular and intertubular dentinal erosion when applied for the removal of smear layer. In a comparative study of ethylenediaminetetraacetic acid and a mixture of doxycycline, citric acid and a detergent found that both the solutions were effective in removing the smear layer. However, the middle and cervical thirds of the root dentin showed erosion of tubules when exposed to ethylenediaminetetraacetic acid for about 5 minutes [8]. An in vitro experiment by Verdelis et al [47] showed that dentin demineralization was higher at the middle and cervical thirds than at the apical one third. This may reduce the diffusion of irrigating solutions, root canal dressings and medicaments into the apical dentin which reduces the proper adhesion of sealers decreasing the resistance to bacterial penetration. Segura et al [48] demonstrated that ethylenediaminetetraacetic acid concentrations lower than those used in root canal preparation inhibited vocative intestinal peptide binding to macrophage membranes. This result suggests that ethylenediaminetetraacetic acid leakage during root canal preparations could modify inflammatory reactions in peripheral apical tissues. The study by Calt and Serper[49] suggested that the inhibitory effect of ethylenediaminetetraacetic acid on vocative intestinal peptide binding wa s caused not only by chelating of calcium ions, but also by chelating of other divalent cautions, such as Mg2+. Thus, during delivery of ethylenediaminetetraacetic acid caution should be taken to avoid apical extrusion. It is generally believed that the teeth after undergoing endodontic treatment becomes more weak and brittle. In an in vitro study by Machnick et al [50], dentin bars were exposed to 17% ethylenediaminetetraacetic acid, a mixture of doxycycline, citric acid and a detergent and 0.6% NaOCl for 2 hours, showing a significant reduction in the modulus of elasticity and flexural strength. However, the dentin bars exposed to the higher concentration of NaOCl did not show significant alteration in their modulus of elasticity and flexural strength. According to the above studies, it can be concluded that ethylenediaminetetraacetic acid has a wide range of influence both on the dentin and peripheral apical tissues. In the dentin, it decreases the micro hardness facilitating biomechanical preparation; causes tubular erosion which interferes with root canal fillings and restoration and decreases the modulus of elasticity rendering it more brittle. On the piratical tissues, ethylenediaminetetraacetic acid interferes with inflammatory process. Thus from the above mentioned studies, it can be concluded that the most deleterious effect that ethylenediaminetetraacetic acid has erosion of the dentin which can be minimized by reducing the exposure to 1 minute. This reduces the surface irregularities presenting on the dentin and enamel surfaces and when in contact with root canal filling materials and restorations the tissues closely adapt to them. The tight periodical seal thus decreases the bacterial penetration and finally enhances the success of the root canal treatment."

ADVANCES Chelating agents used to remove the smear layer cause disinfection of the deeper layers, increase thickness of root canal filling materials and increase the success of root canal treatment. (1) The effectiveness of the chelating agent to remove the smear layer is affected by different factors. Such as the chelating agent concentration, reaction time, the flushing fluid transport, and other fluid of synergy, the root canal morphology, including the operating time and concentration is a major factor. (2) The effect on dentin: The alternate irrigation of ethylenediaminetetraacetic acid and sodium hypochlorite can effectively remove the smear layer. Ethylenediaminetetraacetic acid simultaneously dissolves the inorganic components of the smear layer and causes peritubular and intertubular dentin erosion. Further studies are to be carried out to find out whether it has other deleterious effects on tooth, such as discoloration. (3) The joint problem: Till date no single root canal irrigating agent has been developed that can completely remove the smear layer, and therefore, a combination of two or three kinds of irrigating agents are always used. The combination of sodium hypochlorite or organic acid with chelating agents is the most widely used for the efficient removal of smear layer. In recent years, some new chelating agents like tetracycline have been the focus of attention as an ideal irrigate. Due to its short shelf life and high cost, the irrigate material is still on its experimental research stage. The chelating agent most widely used today is efficient in removing the smear layer and its ability is further enhanced when used in combination with other root canal irrigates though the best combination is still to be explored."