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Abstract The purpose of the present in vitro study was to compare the shaping and cleaning abilities of the 2Shape, Neolix, and ProTaper Next rotary NiTi systems in terms of canal transportation, centering ability, amount of untouched canal walls, smear layer removal, and canal cleanliness in mesiobuccal canals of human extracted first mandibular permanent molar teeth. A total of one hundred and twenty-nine mesiobuccal canals of first mandibular permanent molars were selected with an angle of curvature ranging between 20{u00B0}- 40{u00B0} degrees according to Schneider{u2019}s method. All teeth were decoronated to obtain 16 mm uniform root lengths. Distal root of each sample was resected. For the evaluation of canal transportation and centering ability, fifty-seven mesiobuccal roots were randomly divided into 3 equal groups (n = 19) as follows: group I: 2Shape, group II: Neolix and group III: ProTaper Next. The samples of each group were mounted in transparent auto-polymerizing acrylic resin using plastic moulds. Then, the mounted samples were scanned, before and after mechanical preparation, using CBCT, to obtain 3 tomograms for each specimen representing the coronal, middle and apical root thirds. Pre and post-instrumentation scans were superimposed, using OnDemand 3D software program, to evaluate the degree of transportation as well as the centering ability of the tested instruments. For the evaluation of the amount of untouched canal walls, fifty-seven mesiobuccal roots were randomly divided into 3 equal groups as mentioned before. The samples were embedded in a clear epoxy resin blocks using custom-made Teflon mould. Each specimen was sectioned into three locations; 3,6 and 9 mm from the apex using ISOMETTM precision sectioning saw |