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Introduction: Dentinal microcracks have been supposedly associated with unrestorable vertical root fractures and consequently long-term treatment failure. This study aimed to investigate whether in vivo root canal instrumentation in mandibular incisors with vital pulps causes dentinal microcracks using two different irrigating solutions., Methods: Five patients with four vital mandibular incisors indicated for extraction were included. In vivo root canal preparation was performed using Reciproc R40 (tip #40 and taper 0.06). From these, two teeth were randomly assigned for root canal instrumentation irrigated with 5.25 % sodium hypochlorite irrigation (n = 10) or 2 % chlorhexidine gel with saline solution irrigation (n = 10). In sequence, all teeth were carefully extracted, stored in saline solution until microtomography (µCT) scan. Images were reconstructed and assessed for the presence or absence of dentinal microcracks where microcracks originating from the root canal lumen would be considered. All reconstructed samples were analysed dynamically and rendered in videos through the entire extension of the teeth, evaluating the axial cuts considering each third separately from the apex to the enamel-dentinal junction. Teeth were analysed using the DataViewer software at 100 % magnification without filters by three examiners blinded to the condition allocation., Results: No complete dentinal microcracks were observed after root canal instrumentation of mandibular incisors with vital pulps using Reciproc R40 regardless the irrigating solutions, 5.25 % sodium hypochlorite or 2 % chlorhexidine gel., Conclusions: In vivo root canal instrumentation of mandibular incisors with vital pulps and bone/periodontal insertion does not cause dentinal microcracks and the irrigating solutions tested did not influence this occurrence. Microcrack evaluation must be performed in vivo conditions of dental tissue moist and periodontal support to avoid dryness dentinal alterations after extraction provoking false positive results., Clinical Relevance: Reciprocating instrumentation performed in vivo is safe and do not induce dentinal microcracks in mandibular incisors., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)