## 2 Experimental Section
### Experimental Procedure  
The crowns of the teeth were removed between 1 mm and 3 mm under the cement-enamel junction to standardize dental samples to 10 mm in length. The teeth were submitted to digital radiography in both buccolingual and mesiodistal directions to assess the RCS anatomy. The working length (WL) of the RCS was determined using an operative microscope at 12 \(\times\) magnification (Zeiss Dental Microscope, Oberkochen, Germany) until the emergence of a size 8 K-file (Dentsply Maillefer, Ballaigues, Switzerland) was visible at the apical foramen of the RCS. Then, the samples were randomly placed (Epidat 4.1, Galicia, Spain) into the following study groups: Group A, EndoVac endodontic irrigation system (Dentsply Maillefer, Ballaigues, Switzerland) (EV) (\(n=10\)); Group B, Endoactivator endodontic irrigation system (Dentsply Maillefer, Ballaigues, Switzerland) (EA) (\(n=10\)); Group C, RCS performed with F2 ProTaper Gold endodontic rotary system (Dentsply Maillefer, Baillagues, Switzerland) but not irrigated (Positive control) (\(n=10\)); Group D, RCS not performed or irrigated (Negative control) (\(n=10\)).  
The RCSs were randomly assigned to the above-mentioned study and control groups. Surgery for the EV and EA study groups and Positive control group was performed by using the ProTaper Gold endodontic rotary system up to an F2 endodontic rotary file (Dentsply Maillefer, Ballaigues, Switzerland), using a 6:1 reduction handpiece (X-Smart plus, Dentsply Maillefer, Ballaigues, Switzerland) at 300 rpm and 2 N/cm torque, according to the manufacturer's recommendations. The RCSs randomly assigned to the EV study group were irrigated with 5 mL of 5.25% NaOCl (Clorox, Oakland, CA, USA) with a 0.3-mm-diameter endodontic needle (Miraject Endo Luer, Hager and Werken, Duisburg, Germany) inserted 1 mm into the WL. Furthermore, the disinfection of the RCS was improved by using the Endoactivator endodontic irrigation system (Dentsply Maillefer, Ballaigues, Switzerland) between each endodontic file of the sequence. The final irrigation procedure was performed with 5 mL of 17% ethylenediaminetetraacetic acid (EDTA) (SmearClear, SybronEndo, CA, USA), 5 mL of 5.25% NaOCl, and 5 mL of sterile saline solution (Braun(r), Melsungen, Germany). However, the RCSs randomly assigned to the EA study group were irrigated with 5 mL of 5.25% NaOCl (Clorox, Oakland, CA, USA) with a 0.3-mm-diameter endodontic needle (Miraject Endo Luer, Hager and Werken, Duisburg, Germany) inserted 1 mm into the WL. Furthermore, the disinfection of the RCS was improved by using the EndoVac endodontic irrigation system (Dentsply Maillefer, Ballaigues, Switzerland) between each endodontic file of the sequence. The final irrigation procedure was performed with 5 mL of 17% EDTA (SmearClear, SybronEndo, CA, USA), left in the canal for 1 min with 5 mL of 5.25% activated NaOCl, and 5 mL of sterile saline solution (Braun(r), Melsungen, Germany). Then, the RCSs randomly assigned to the EV and EA study groups were dried with sterile paper points (Dentsply Maillefer, Ballaigues, Switzerland) and the teeth were then stored in an incubator (mco-18 aic, Sanyo, Moriguchi, Osaka, Japan) (37 \({}^{\circ}\)C, 100% relative humidity). The RCSs randomly assigned to the Negative control group were neither shaped nor cleaned with irrigation solutions. All of the root canal treatments were performed by a single clinician.