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This laboratory study aimed to evaluate the accumulated hard tissue debris (AHTD) and shaping performance following root canal instrumentation with a novel stainless steel rotary system (Gentlefile; MedicNRG, Kibbutz Afikim, Israel) compared with two single-file nickel-titanium instruments of different kinematics through micro–computed tomography (micro-CT) analysis. Thirty isthmus-containing mesial roots of human mandibular molars with a curvature of 15°–35° were scanned with micro-CT at an isotropic resolution of 12 μm and randomly assigned to three groups (n = 10) according to the instruments used for canal preparation: Gentlefile (GF; #23/.04), One Curve (OC; #25/.06; Micro-Mega, Besancon, France), and Reciproc Blue R25 (RB; #25/.08; VDW, Munich, Germany). The AHTD, unprepared canal surface area, volume changes, surface area changes, and transportation were evaluated by comparing preoperative and postoperative micro-CT images. In addition, the time required for canal preparation was recorded. Data were statistically analyzed using the one-way analysis of variance (ANOVA) test or the Kruskal-Wallis test at a significance level of 5%. No significant differences were observed in the percentage of AHTD or in the unprepared canal surface area between the three groups (P >.05). The RB group demonstrated a significantly greater percentage increase in volume and surface area than the GF group (P <.05). The GF and OC groups showed significantly less transportation at 3 mm from the apex than the RB group (P <.001). The OC group required significantly less time for instrumentation than the RB and GF groups (P <.05). Instrumentation with GF (#23/.04), OC, and RB yielded similar levels of AHTD and unprepared surface area in isthmus-containing curved canals. GF achieved this with less dentin removal and apical transportation, at the expense of a longer preparation time. [ABSTRACT FROM AUTHOR]