Evaluation of the effect of irrigants on the accuracy and repeatability of three electronic apex locators: an ex vivo study

Objective: To evaluate the effect of 2% chlorhexidine (CHX) and 2.5% sodium hypochlorite (NaOCl) on the accuracy and repeatability of three Electronic Apex Locators (EALs). Methodology: Thirty one human teeth were connected to a platform, irrigated with 2.0% CHX, and obtained two readings, again irrigated with 2.5% NaOCl and obtained two more readings. The distance between the tip of the file and the apical constriction was measured. Repeatability was calculated using the intraclass correlation coefficient (ICC) and repeatability coefficient (CR). Results: For CHX 2%, with the Mini Apex Locator, 25 of 31 electronic measurements were accurate (80.6%), 28 of 31 measurements for iPex (90.3%) and for Root ZX II 26 (83, 9%). For 2.5% NaOCl, with the Mini Apex Locator, 25 of the 31 measurements were accurate (80.6%), 28 of the 31 measurements for the iPex (90.3%) and for the Root ZX II, 24 (77, 4%). There were no differences between the two irrigants (p>0.05). The values (ICC) were 0.975 and 0.994 for the Mini Apex Locator, 0.981 and 0.971 for the i Pex and 0.994 and 0.995 for the Root ZX II. When using CHX, the (CR) was 0.196 for the Mini Apex Locator, 0.152 for the iPex and 0.088 for the Root ZX II. When using 2.5% NaOCl, the (CR) was 0.088 for the Mini Apex Locator, 0.196 for the iPex and 0.088 for the Root ZX II. Conclusions: The use of 2% CHX and 2.5% NaOCl did not affect the accuracy and repeatability of the three devices.


Introduction
The success of endodontic therapy depends on the correct performance of all treatment stages, such as the determination of working length, a challenge to dentists because of the anatomy of the apical region (Mazzi-Chaves et al., 2020). Root canal preparation and filling should be performed, as much as possible, close to the apical constriction in the root canal at the level of the cementodentinal junction (CDJ), which is about 1 to 2 mm from the radiographic apex of the root canal (Ricucci & Langeland, 1998).
Electronic apex locators (EALs) are widely utilized to determine working length during root canal treatment.
Introduced in the classic study of Sunada in 1962, the EALs has become an invaluable tool in current endodontic practice.
These devices, which work with an attached file, are able to detect the point at which the file leaves the root canal and enters the periodontal ligament. Early electronic apex locators relied on the principle that the electrical resistance between the oral mucous membranes and the periodontal ligament remained constantly at 6.5 kΩ regardless of patient age and tooth type or shape (Sunada, 1962).
Since its development, numerous electronic devices have been introduced in the market, and their accuracy has been analyzed. Current models can perform readings under different conditions inside the root canal (Çinar & Üstüm 2020;Bernardes et al., 2021;Saritha et al., 2021;Suguro et al. 2021). However, studies have found conflicting results for EALs using different irrigants (Baruah et al. 2018;Marek et al., 2020).
The Mini Apex Locator, developed by SybronEndo (Anaheim, CA, USA), has a multifrequency measurement system and digital signal. According to the manufacturers this device locates the apical constriction without any influence of the root canal contents (D'Assunção et al., 2014).
The iPex (NSK, Tochigi, Japan) is an apex locator that measures capacitance and resistance simultaneously, in order to determine the location of the file tip inside the root canal system (Stöber et al., 2011).
The Root ZX II (J Morita Corp, Tokyo, Japan) is a commercial device that uses two different alternating currents with different frequencies. It calculates the ratio of impedances of two different frequencies. At the apical terminus, there is a sharp change in the impedance, and Root ZX II recognizes it as the apex. This device is composed of two parts: the root canal measurement module and the slow-speed handpiece (Nekoofar et al., 2006).
An electronic apex locator must be both accurate and reliable. In statistics, a reliable measurement means measuring something consistently with minimal variance. However, according to Jung et al., (2011), most authors only evaluated the accuracy of EALs to determine the length and did not assess the reliability of the measuring method.
Root ZX is considered the gold standard for locators, several studies have shown satisfactory results (Piasecki et al. 2016;Melo et al. 2020), even with its use with different irrigating solutions (Bolbolian et al. 2018). However, when comparing the influence of different irrigation solutions, commonly utilized in endodontics on the accuracy of Root ZX and Mini Apex Locator, different accuracy results were found (Mull et al. 2012).
Therefore, using a mounting model, the aim of this study was to evaluate and compare the accuracy and repeatability coefficient of measurements obtained by the Mini Apex Locator, iPex and Root ZX II, using two different irrigants: 2.5% sodium hypochlorite (NaOCl) and 2.0% chlorhexidine gluconate (CHX).

Methodology
This study was performed in accordance with the guidelines issued by the Department of Health of the state of Paraiba, Brazil and after approval by its Research Ethics Committee of the Federal University of Paraiba.
Forty single-rooted human teeth with straight roots, a single root canal and fully-formed apices were evaluated using a magnifying lens at 4x magnification and radiographed mesiodistally and buccolingually. Root canals with open apices, root fractures, obstructed foramen, root perforations and accessory canals were excluded from the sample. A k-type file #10 was introduced in the root canal to rule out possible root obstructions, and to ensure foramen opening at the root apex and foramen patency. The final sample after the exclusion criteria was 31 single-rooted human teeth, with single and straight root canals.
The crown of each tooth was removed using a diamond disk to standardize the specimen medium length at 13 mm.
The contents of each root canal were removed using a conventional k-type file with a diameter compatible with the root canal, and a k-type file # 10 (Dentsply-Maillefer, Baillaigues, Switzerland) was used to confirm apical patency by introducing it into the root canal and pulling it back to 1 mm from the apical foramen. The middle and cervical thirds were pre-flared with Gates-Glidden burs # 2, and 3 (Dentsply-Maillefer, Baillaigues, Switzerland) according to the diameter of the root canals up to 5 mm short of total root length.
A model similar to the Mounting Model designed by Elayouti and Löst (2006)  The EAL was turned on, and each root canal was irrigated with 2.0% chlorhexidine gluconate, inserted in a 5 ml disposable plastic syringe with Luer Lock (Ultradent, South Jordan, Utah, USA) and 27G Endo-Eze needles (Ultradent, South Jordan, Utah, USA). The apical constriction position was determined: the file was slowly introduced into the root canal and the micrometer connected to the file was moved apically up until the electronic apex locator displayed "apex" on the screen. After that, the file was pulled back to the point when the EAL reading was 0.5, which corresponded to the apical constriction. The Research, Society and Development, v. 10, n. 13, e599101321636, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i13.21636 measurement at 0.5 was recorded and the instrument was pulled back to the cervical end {R = 0.0}. The measurements obtained using the EAL connected to the micrometer were acquired by moving the file cervically to avoid inaccurate measurements caused by curvatures along the root canal. The electronic measurement at 0.5 was repeated to confirm its repeatability. After recording the electronic measurements with 2.0% chlorhexidine gluconate, the root canals were rinsed with saline solution and irrigated with 2.5% sodium hypochlorite; after that, two other electronic measurements were made in the same method as for chlorhexidine gluconate. All procedures performed with the Mini Apex Locator were repeated for each specimen, using the iPex and the Root ZX II.
After the last electronic measurement was recorded, the file was cemented in place by using cyanoacrylate adhesive (Super Bonder; Henkel Technologies, Diadema, São Paulo, Brazil). Subsequently, the electronic length was checked again to confirm that the file was in the same position, and the file was kept in position for 30 minutes until the adhesive had set completely. The teeth were removed from the mounting model and a 3195F bur (K.G. Sorensen, São Paulo, Brazil) was used to expose the apical 4 mm of each root along the long axis of the tooth in a plane that showed the best representation of the minor diameter in relation to the file. This procedure was similar to the process described by Fadel et al. (2012). The last layer of dentin was then carefully removed using a # 15 scalpel blade. Each root was photographed with a digital camera (Sony Cyber-Shot-W70) and the images were projected onto a LCD screen and analyzed using the Image Tool 1.28 software (San Antonio, TX, USA). Two investigators, blinded to groups and procedures, marked and recorded the distance from the file tip to the minor diameter for each specimen. Seven teeth were lost during specimen preparation and after apical sectioning, 31 of the 40 specimens were prepared, and their apical constriction was visible.
The most incisal and most apical limits of apical constriction did represent the beginning and the end of the apical constriction and the smallest distance between the tip of the file and these limits was the reference for the actual length. An exact correlation between the file tip and the apical constriction indicated that the electronic length was the same as the actual length. However, a negative value indicated that the distance from the file tip to the apical constriction was shorter than actual length, and a positive value, that it was longer.
When a disagreement occurred between the evaluators, the distances were checked again, and the two evaluators reached a consensus.

Statistical Analysis
The Statistical Package for Social Sciences software (SPSS 13.0; SPSS Inc., Chicago, IL, USA) was used for statistical analyses.
The correlation coefficient was used to compare the reliability. The intraclass correlation coefficient (ICC) was calculated to demonstrate the strength of agreement between the apical constriction and the file tip, when using the two irrigating substances and the upper and lower limits of agreement for both measuring points. The repeatability of apex locators was determined by calculating the coefficient of repeatability for each electronic apex locator device. This corresponds to 2 standard deviations of the differences between the first and the second measurement (Elayouti & Löst, 2006). The paired t test at a 0.05 significance level was used to determine the accuracy of the three electronic locators when using the two irrigating solutions within a clinical range of ± 0.5 mm.

Distance to the apical constriction
For 2% CHX, with the Mini Apex Locator, 25 of the 31 electronic measurements were located at the apical constriction (80.6%), for the iPex, 28 measurements were located at the apical constriction (90.3%) and for the Root ZX II 26 Research, Society andDevelopment, v. 10, n. 13, e599101321636, 2021 (CC BY 4.0) | ISSN 2525-3409 | DOI: http://dx.doi.org/10.33448/rsd-v10i13.21636 5 electronic measurements were located at the apical constriction (83.9%). For 2.5% NaOCl, with the Mini Apex Locator, 25 of the 31 electronic measurements were located at the apical constriction (80.6%), and with the iPex, 28 measurements were located at the apical constriction (90.3%). For the Root ZX II, 24 electronic measurements were located at the apical constriction (77.4%). The results of the paired t test for the three devices revealed no statistically significant differences between the electronic measurements (p>0.05), when the two irrigants were used (Table 1).  Table 2 shows the descriptive values (mean and standard deviation) of the differences of the electronic measurements made by the Mini Apex Locator, iPex locator and the Root ZX II in relation to apical constriction. There were no statistically significant differences between accuracy of the two measurements for each of the irrigants, 2.0% CHX and 2.5% NaOCl in relation to the apical constriction determination (p>0.05). There was no influence of the irrigating solutions in the accuracy of the electronic apex locators.

Repeatability
The intraclass correlation values were 0.975 and 0.994 for the Mini Apex Locator, 0.981 and 0.971 for the iPex, and 0.994 and 0.995 for the Root ZX II, indicating an excellent agreement between measurements 1 and 2. When using 2.0% CHX, the coefficients of repeatability were 0.196 for the Mini Apex Locator, 0.152 for the iPex and 0.088 for the Root ZX II. When using 2.5% NaOCl, the coefficients of repeatability were 0.088 for the Mini Apex Locator, 0.196 for the iPex, and 0.088 for the Root ZX II. There were no significant differences between the coefficient of repeated measurements, and more than 95% of the differences between repeated measurements were below the coefficient of repeatability for the three devices, for both irrigating solutions. These results demonstrated excellent repeatability (Table 3).

Discussion
Electronic apical locators are very important devices in Endodontics. When compared with radiography, EAL decreases exposure to radiation and determines the position of the apical constriction or apical foramen, rather than of the radiographic apex (Bernardes et al. 2021;Abidi et al. 2020). Some authors, following manufacturers' instructions, and using methods similar to those adopted in this study, analyzed the accuracy of EALs in locating apical constriction, or a distance 0.5mm short of the apical foramen (Piasecki et al. 2016;Connert et al. 2018;Serna-Peña et al. 2020).
The mounting model is simple, easy to use and provides close control over experimental conditions. Measurements were stable and coherent, and a large number of root canals could be evaluated in a short time period, which would not be possible in the clinical setting (Elayouti et al. 2006;Connert et al. 2018).
Studies evaluating electronic apex locators, which do not use a mounting model, may be conducted according to the adjustment of a silicone stop of the measurement file to a reference point in the dental crown, after which the distance from the silicone stop to the file tip is measured. This type of method has been utilized in many other studies (Piasecki et al. 2016;Melo et al. 2020;Saritha et al. 2020;Iparraguirre Nuñovero et al. 2021). However, may lead to inaccurate measurements due to the poor adaptation of the silicone stop to the reference point because of movements during measurement, the absence of a parallel relation between the long axis of the file and the reference.
The present study demonstrated the accuracy of the Mini Apex Locator, iPex and Root ZX II to identify the apical constriction in the presence of Chlorhexidine and sodium hypochlorite in a range of 77.4% to 90.3%,. Some authors concluded that EALs may accurately determine working length in 90% to 100% of root canals with completely formed apices (Bolbolian et al., 2018;Melo et al., 2020;Serna-Peña et al. 2020), however, these cited studies did not utilize a mounting model.
In the present research, irrigants, 2.0% chlorhexidine gluconate and 2.5% sodium hypochlorite, did not affect the accuracy of electronic measurements made by the Mini Apex Locator, iPex or Root ZX II, the mean distances from the file tip to the apical constriction were in a range of 0.21mm to 0.09mm. These results are considered very accurate. Some studies revealed that EALs are not affected by the presence of irrigants inside the root canals (Duran-Sindreu et al. 2013;Çinar & Üstüm 2020). However, Marek et al. (2020) concluded that EALs had higher accuracy in root canals containing chlorhexidine in gel or in solution form than in the root canal containing sodium hypochlorite.
Results using EAL's mark 0.5 demonstrate that the EALs tested tend to obtain measurements near to the apical constriction. Nevertheless, majority of measurements obtained by the three devices, with both irrigating solutions did not surpass the apical foramen. These results are similar to that found in other studies which showed that the using EALs to determine the major foramen, in 0.0 EAL's mark led to overestimation of the working length (Connert et al. 2018;Chukka et al. 2020).
In the present study, periapical radiographs were also obtained for analyzing anatomy of root canals. Although Electronic apex locators has a great accuracy, when obtaining working length, these devices does not presents images.
Therefore, periapical radiography remains essential for confirming working length during endodontic treatment (Mahmoud et al. 2021).
Working length was determined close to the apical constriction, these results are relevant because studies have already shown that working length must be determined inside the root canal to avoid treatments underestimated, which allow persistence of infections, and also, to avoid over-instrumentation and accidents during endodontic treatment, that leads to a negative prognosis for endodontic treatment's outcome (Ng et al. 2011;Ricucci et al. 2016;Aminoshariae et al. 2020) The use of the mounting model increases the accuracy of the study (Elayouti & Löst 2006, D'Assunção et al. 2014Connert et al. 2018), however, this research is an ex vivo study and, therefore, it has the limitations of a laboratory study.

Conclusion
The results of this study revealed that 2% chlorhexidine gluconate and 2.5% sodium hypochlorite did not affect the performance of the Mini Apex Locator, iPex or Root ZX II and these devices locate a very close point of the apical constriction.