## 2 Materials and Methods
### Group III: Passive Ultrasonic Irrigation (PUI)  
The samples in this group were sequentially shaped to a 30 size and 0.6 taper (Protaper gold, Dentsply Mallefer, Ballagues, Switzerland). In due course of instrumentation, a 30-gauge side-vented needle fixed to a 5 mL barrel syringe was used for irrigation. An amount of 2 mL of 5.25% NaOCl (Parcan, Septodont, France) was used after each instrumentation. Alternative irrigation was carried out using 2 mL of 17% EDTA (MD Cleanser, Metabioped, South Korea) after every change of instrument. The needle was oscillated at a frequency of 1 Hz, at an amplitude of 3 mm continuously until the end of the irrigant delivery. At the end of instrumentation and manual irrigation, activation was carried out using passive ultrasonic irrigant activation (PUI). An amount of 1 mL of 5.25% NaOCl was activated for 20 s, which was performed for 3 consecutive cycles using an IRRI S ultrasonic tip (VDW), attached to an ultrasonic device (Ultra Device, VDW), at a power setting at 30, placed 1 mm short of the working length. Following the activation, the final rinse was carried out using 4 mL of 5.25% NaOCl, 5 mL of 17% EDTA, and 5 mL of distilled water. A total of 20 mL of NaOCl was used per specimen [18].  
Following the experimental instrumentation and irrigation, decoration was performed until the CEJ. The roots were then split buccolingually using a microtome (Leica SP1600; Leica, Wetzlar, Germany). The roots were then sputter coated with gold and examined under scanning electron microscopy (SEM) (FE-SEM; Sigma, Carl Zeiss, Jena, Germany; secondary electron emission mode, 5 KV accelerated voltage) at 2000\(\times\). The Hulsmann scoring system includes a score from 1 to 5 with regard to the residual smear layer and debris [19]. A score of 1-5 was given independently by the examiners using the reference images. Separate evaluations were undertaken for the debris and smear layer with a five score index for each using reference photographs. Debris was defined as dentin chips, pulp remnants, and particles loosely attached to the root canal wall [19]. Below is the reported Hulsmann criteria for debris scoring:  
* Score 1: Clean root canal wall, only few small debris particles.
* Score 2: Few small agglomerations of debris.
* Score 3: Many agglomerations of debris covering less than 50% of the root canal wall.
* Score 4: More than 50% of the root canal wall covered by debris.
* Score 5: Complete or nearly complete root canal wall covered by debris.  
Figure 2: Automated root canal irrigation device [13].  
Smear layer was defined as proposed by the American Association of Endodontists' glossary \"Contemporary Terminology for Endodontics\" [19]: a surface film of debris retained on dentin or other surfaces after instrumentation with either rotary instruments or endodontic files; consists of dentin particles, remnants of vital or necrotic pulp tissue, bacterial components, and retained irrigant. Below is the reported [PERSON] criteria for smear layer scoring:  
* Score 1: No smear layer, dentinal tubules open.
* Score 2: Small amount of smear layer, some dentinal tubules open.
* Score 3: Homogeneous smear layer covering the root canal wall, only few dentinal tubules open.
* Score 4: Complete root canal wall covered by a homogeneous smear layer, no open dental tubules.
* Score 5: Heavy, inhomogeneous smear layer covering the complete root canal wall.  
Two independent evaluators (KR, KVT) with calibration on debris and smear layer scores performed the scoring of all the samples. The debris and smear layers were scored in 2000\(\times\) magnification.  
IBM SPSS Statistics for Windows, Version 23.0 (IBM Corp, Armonk, NY, USA) was used to assess the data statistically. Independent groups were assessed using the Kruskal-Wallis test. [PERSON]'s pairwise test was used for intragroup comparison.