Antimicrobial and biocompatible potential of peracetic acid as a novel root canal irrigant

Antimicrobial and biocompatible potential of peracetic acid as a novel root canal irrigant | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Antimicrobial and biocompatible potential of peracetic acid as a novel root canal irrigant Rikako Takami, Ryota Yamasaki, Ayako Washio, Yoshie Yoshioka, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7911128/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Sodium hypochlorite (NaClO) is currently the most widely used irrigant in root canal treatment; however, it exerts cytotoxic effects on the periapical tissues. To reduce the biological risk, we aimed to investigate the potential usefulness of a peracetic acid (PAA)-based disinfectant as an alternative to root canal irrigants. Methods The antibacterial activity of the PAA-based disinfectant, Actril (MEDIVATORS, Minneapolis, MN, USA)—against oral pathogenic bacteria—was evaluated by determining its minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm inhibitory concentration (MBIC). Cytotoxicity toward periodontal tissue-related cells was assessed using a Cell Counting Kit-8 assay. The bactericidal effect against Enterococcus faecalis biofilms was further examined using live/dead staining. To simulate the clinical environment, E. faecalis biofilms were established on extracted human teeth, and the irrigation efficacy of Actril in root canals was evaluated using scanning electron microscopy (SEM) and quantitative polymerase chain reaction (qPCR). Data were analyzed using one-way analysis of variance followed by Tukey’s test or the Kruskal–Wallis test with Steel–Dwass post-hoc comparisons, as appropriate. Results Actril demonstrated strong antibacterial activity against oral pathogenic bacteria at a PAA concentration of 9.4×10⁻⁴%, which was significantly lower than the effective concentration of NaClO, while showing reduced cytotoxicity against periodontal tissue-related cells. Live/dead staining revealed that Actril exerted bactericidal effects against E. faecalis within the biofilms. Furthermore, SEM observations demonstrated the removal of biofilm structures from the root canal surface, and qPCR analysis confirmed a significant reduction in the number of viable E. faecalis cells. These findings indicate that Actril is not only effective against pathogenic microorganisms but also exhibits superior biocompatibility compared with NaClO solution. Conclusions The PAA-based disinfectant shows promise as a safe and effective alternative root canal irrigant to conventional NaClO solutions. Peracetic acid-based disinfectant Biofilm Enterococcus faecalis Root canal treatment Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 BACKGROUND The success of endodontic treatment relies not only on mechanical debridement but also on the elimination of microorganisms and biofilms within the root canal system using chemical irrigants. This necessity arises from the complex anatomy of the root canal system, which includes intricate structures—such as dentinal tubules, lateral canals, isthmuses, and fins—that mechanical instruments are often unable to reach [ 1 ]. In such complex areas, debris tends to remain after root canal treatment [ 2 ], and biofilms protected by extracellular polymeric substances can become infected [ 3 ], further complicating the cleaning process. Sodium hypochlorite (NaClO), the most commonly used irrigant, possesses strong antimicrobial activity and is considered the gold standard for root canal irrigation [ 4 ]. However, its high cytotoxicity, the risk of tissue damage from extrusion beyond the apical foramen, and reduced activity in the presence of organic matter have raised concerns [ 5 , 6 ]. Peracetic acid (PAA) is a high-level disinfectant widely used for the disinfection of dialysis equipment and medical devices [ 7 ]. Moreover, because its decomposition products—oxygen, water, and acetic acid—are harmless, they impose minimal environmental and biological burdens and are attracting attention as an alternative to NaClO not only in the medical field but also for wastewater disinfection and the control of foodborne pathogens [ 8 , 9 ]. Chemically, PAA is an equilibrium mixture of acetic acid and hydrogen peroxide; its peroxy bond (-O–O-) is highly unstable and releases reactive oxygen species. This structural property accounts for its strong oxidizing capacity, which enables the efficient disruption of microbial membranes and oxidation of essential enzymes and proteins [ 10 , 11 ]. Importantly, PAA exhibits a broad antimicrobial spectrum, demonstrating potent activity against a wide variety of microorganisms, including gram-positive and gram-negative bacteria, viruses, fungi, and even resistant forms such as bacterial spores and biofilms [ 12 – 16 ]. These characteristics make PAA a versatile disinfectant across diverse environment. This study aimed to evaluate the antimicrobial activity of Actril® (MEDIVATORS, Minneapolis, MN, USA), a low-concentration PAA-based disinfectant, against representative oral pathogenic bacteria, as well as its cytotoxicity toward periodontal tissue-related cells. Furthermore, focusing on Enterococcus faecalis , which is often detected in refractory-infected root canals [ 3 , 17 ], we used a human extracted tooth infection model to comprehensively assess the irrigation and disinfection performance of Actril in comparison with a NaClO solution and explored its potential for clinical application. MATERIAL AND METHODS Preparation of disinfectants The primary sterilizing reagent used in this study was the PAA-based disinfectant, Actril (MEDIVATORS). Additionally, NaClO solution—Antiformin (NISHIKA, Yamaguchi, Japan)—was used for comparison. Actril concentrate contains 0.06% CH₃COOOH, 0.8% H₂O₂, and 5% CH₃COOH. Antiformin concentrate contains 3–6% NaClO. Two-fold serial dilutions of these were prepared and used. All dilution procedures were performed under aseptic conditions, and the prepared solutions were used immediately after preparation. Culture conditions for oral pathogenic bacteria The oral pathogens used were E. faecalis NBRC 100482, Streptococcus mutans UA159, and Porphyromonas gingivalis ATCC 33277. E. faecalis was cultured at 37 ℃ under 5% CO₂ for 24 h in 1.6% nutrient broth (BD, Franklin Lakes, NJ, USA), 0.5% yeast extract (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 2% D-(+)-glucose (TCI, Tokyo, Japan). For agar and liquid media, S. mutans was cultured in 3.7% brain heart infusion (BHI; BD) and 1% yeast extract. For the determination of the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm inhibitory concentration (MBIC), 0.1% sucrose (Sigma-Aldrich, St. Louis, MO, USA) sterilized using a 0.2-µm filter (Minisart, Sartorius, Göttingen, Germany) was added to the medium, and the cultures were incubated in 96-well plates (Wuxi NEST Biotechnology, Jiangsu, China) at 37 ℃ under 5% CO₂ for 24 h. P. gingivalis were cultured at 37 ℃ for 72 h using Anaero Pack (MGC, Tokyo, Japan) on ABHK agar medium (SHIMADZU, Tokyo, Japan), and the liquid medium comprised BHI supplemented with hemin (FUJIFILM Wako Chemicals, Osaka, Japan) to a final concentration of 5 mg/L and menadione (FUJIFILM Wako Chemicals) to a final concentration of 0.5 mg/L. The cultures were incubated anaerobically. Evaluation of efficacy against oral pathogenic bacteria Two-fold serial dilutions of the disinfectants were prepared in 96-well plates. The control comprised 200 µL of liquid medium without added disinfectant. A bacterial suspension of each oral pathogenic bacterium was prepared to a final OD 600 of 0.05 and added, then cultured under the respective conditions as described above. After the designated incubation period, the absorbance was measured at 620 nm using a Multiskan FC (Thermo Fisher Scientific) and the lowest concentration of the agent that inhibited bacterial growth was defined as the MIC. Additionally, 10 µL of bacterial suspension from each well showing growth inhibition was plated onto agar medium and incubated for the designated period, and the MBC was determined based on the presence or absence of colony formation. After measuring the absorbance at 620 nm, the supernatant was discarded, and the cells were washed three times with water purified by reverse osmosis (RO water, Milli-RO system; Merck Millipore, Darmstadt, Germany). The wells were stained with 0.1% crystal violet solution (FUJIFILM Wako Chemicals) for 20 min. They were then washed three times with RO water and incubated in 95% ethanol for 5 min. After the 5-min incubation, the wells were pipetted, and the absorbance at 540 nm was measured to determine the MBIC. Evaluation of cytotoxicity toward periodontal tissue-related cells Mouse-derived gingival epithelial cells (GE-1; Riken Cell Bank, Ibaraki, Japan) were cultured at 37°C in serum-free medium (SHIMADZU) supplemented with 1% fetal bovine serum (FBS; SIGMA, St. Louis, MO, USA), 100 U/mL penicillin G, 100 µg/mL streptomycin (FUJIFILM Wako Chemicals), and 1 µg/mL epithelial growth factor (PeproTech, Cranbury, NJ, USA) under 5% CO₂ for 24 h. After incubation, the cells were harvested using a 0.25% of trypsin–ethylenediaminetetraacetic acid (EDTA) solution (0.5 g/L trypsin and 0.53 mmol/L EDTA; Nacalai Tesque, Kyoto, Japan). Twenty percent of the total recovered cells were seeded into new dishes for passage and cultured for 48 h. After passage, cells were treated with trypsin-EDTA and seeded into a 96-well plate at a density of 8.0 × 10⁴ cells/well. After 24 h, Actril or Antiformin diluted in a two-fold serial dilution was applied for 2 min. Two minutes later, the disinfectants were aspirated, and the samples were washed. Then, 100 µL of culture medium and 10 µL of reagent from Cell Counting Kit-8 (CCK-8; DOJINDO, Kumamoto, Japan) were added, and the cells were cultured at 37 ℃ under 5% CO₂ for 2 h. The absorbance of each well at 540 nm was measured after incubation. Human periodontal ligament fibroblasts (HPLF; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in MEM-α (FUJIFILM Wako Chemicals) supplemented with 10% FBS and 1% penicillin G/streptomycin at 37 ℃ under 5% CO₂. The cells after passage were seeded into a 96-well plate at a density of 1.0×10⁴ cells/well. The disinfectant was applied as described above, and the CCK-8 reagent was added. The cells were then incubated at 37 ℃ under 5% CO₂ for 3 h. After incubation, the absorbance of each well at 450 nm was measured. Live/dead staining for E. faecalis in biofilm Totally, 200 µL of liquid medium was added to each well of a µ-96 well plate (ibidi, Gräfelfing, Germany). Subsequently, E. faecalis was adjusted to a final OD 600 of 0.05 and added to each well. The plates were incubated under the conditions described above to allow biofilm formation. 48 h, the supernatant was discarded. Each sample was then rinsed with 2 mL of the solution for 2 min using a 27G side-port soft needle and aspirator according to the disinfection groups listed in Table 1 . After 2 min, the sample was washed once with 600 µL of 0.85% saline solution. The Antiformin group was neutralized using 600 µL of 5% sodium thiosulfate to prevent bleaching of the live/dead staining reagent. As a control, wells were also prepared in which only the washing process with 0.85% saline solution was performed, in the absence of any disinfectant. Additionally, to serve as a reference for determining the exposure time, wells were also prepared in which the formed biofilm was treated with 200 µL of undiluted isopropanol for 1 h to sterilize the biofilm. Equal volumes of the SYTO 9 (1.67 mM)/Propidium iodide (1.67 mM) mixture and the SYTO 9 (1.67 mM)/Propidium iodide (18.3 mM) mixture from the LIVE/DEAD® BacLight™ Bacterial Viability Kit L7007 (Invitrogen, MA, USA) were combined and added at a volume of 3 µL per 1 mL of 0.85% saline solution. Subsequently, 200 µL of the prepared reagent was added to each well, and the plates were incubated in the dark for 15 min and protected from light with aluminum foil. After incubation, the supernatant was discarded, and the wells were washed once with 250 µL of 0.85% saline solution. Observations were performed using an all-in-one fluorescence microscope BZ-X810 (KEYENCE, Osaka, Japan) with a 10× objective lens, capturing both green and red fluorescence at an exposure time of 1.5 s. Three-dimensional rendering was performed using the BZ-H4R/3D analysis application (KEYENCE). Table 1 Concentration and pH of irrigating solutions used in this study Irrigation Ingredient concentration during use (%) pH Actril PAA* 1 : 9.4×10⁻⁴ 2.96 Antiformin NaClO: 4.5* 2 12.22 saline NaCl: 0.85 5.66 *1 Peracetic acid *2 Antiformin concentration (3–6% per product information) is calculated using the median value of 4.5%. Evaluation of the efficacy against E. faecalis in infected root canals Preparation of extracted teeth Single-rooted extracted human permanent teeth were prepared, and to confirm that they had single root canals, radiographs were obtained in the mesial and distal directions (Hatela instant film ISO speed D, Instant DQE; HANSHIN TECHNICAL LABORATORY, Hyogo, Japan). After autoclave sterilization, crowns were removed at 9 mm from the apex using an Isomet low-speed saw (BUEHLER, Lake Bluff, IL, USA). The root canals were filled with saline solution, and the working length was set to 1 mm shorter than the canal length. The canals were then enlarged to size #40/10 using Nickel-Titanium (Ni-Ti) rotary files in M7 mode (Endo Wave, MORITA, Osaka/Tokyo, Japan). After preparing the extraction site, the tips of 27G soft needles (NIPRO, Osaka, Japan) were set to 1 mm short of the working length, and irrigation was performed. First, 1 mL of EDTA (Smearclen, NISHIKA, Yamaguchi, Japan) was used for rinsing for 30 s, and ultrasonic irrigation was performed for 1 min using a SUPRASSON P-max+ (ACTEON EQUIPMENT [SATELEC], Merignac, FRANCE) in E1 mode with Irisafe files (HAKUSUI, Osaka, Japan) to remove the smear layers. This procedure was performed twice and subsequently repeated twice using Antiformin. In addition, the canals were rinsed with 0.1 mL dental etching agent (Green Activator, SUNMEDICAL, Shiga, Japan) for 30 s, followed by ultrasonic irrigation for 1 min. Subsequently, the canals were rinsed with 1 mL of 0.85% sodium chloride (FUJIFILM Wako Chemicals) for 30 s. Holes were made in the caps of 5 mL tubes using straight steel round burs ISO 027 (DENTSPLY, MAILLEFER, Ecublens, Canton de Vaud, Switzerland). The extracted teeth were coated at the apex and around the root using a self-curing resin (Provinice Fast; SHOFU, Kyoto, Japan). Subsequently, four horizontal lines were made on the side of each tube using straight fissure burs (DENTSPLY), which were mounted with self-curing resin, and autoclave sterilization was performed (Fig. 1 ). Preparation of E. faecalis-infected root canals E. faecalis bacterial suspension was adjusted to an OD 600 of 0.05, and 4.5 mL was added to 5 mL tubes in which extracted teeth had been mounted with self-curing resin. The tubes were then incubated at 37°C under 5% CO₂ for 7 days. To prevent air bubbles from entering the root canal, 20 µL of the bacterial suspension was initially introduced into the canal before adding the remainder. After culturing E. faecalis for 7 days to allow biofilm formation, the self-curing resin blocks with teeth were removed from the 5 mL tubes. They were then placed on utility wax that had been disinfected with 70% ethanol, with the coronal side of the root canal facing downward. Each side of the root was rinsed with 1 mL of Antiformin and left for 2 min to disinfect the bacteria on the external surface. Subsequently, both sides of the extracted teeth were rinsed with 0.85% saline solution to remove Antiformin, followed by further disinfection with UV light irradiation (wavelength: 254 nm) for 5 min, after which the teeth were mounted in 5 mL tubes with utility wax. The extracted teeth were randomly divided into groups, and root canal irrigation was performed with each disinfectant. Using a 27-gauge side-vented soft needle bent 1 mm short of the working length, 2 mL of the solution was flushed into the root canal over 2 min while the needle tip was moved gently up and down. After 2 min, 1 mL of 0.85% saline solution was flushed into the root canal for 1 min to rinse out the irrigant. The concentrations of the irrigants were determined based on the results of antibacterial efficacy and cytotoxicity evaluations. A non-irrigated group served as the control group. In both the control and experimental groups, the bacterial suspension remaining within the root canal was absorbed and removed using a paper point; however, irrigation was applied only to the experimental groups. Scanning electron microscopy (SEM) observation SEM images were obtained prior to E. faecalis inoculation, after E. faecalis biofilm formation, and after root canal irrigation. Specimens were fixed in 1% glutaraldehyde (Nacalai Tesque), followed by 2% osmium tetroxide (VIII) solution (FUJIFILM Wako Chemicals). After dehydration through a graded series of acetone (50%, 70%, 90%, 95%, and absolute; KANTO-CHEMICAL, Tokyo, Japan), notches were made using an isomet saw. A flat-head screwdriver was inserted into the notches, and the specimens were split along the tooth axis by striking them with a hammer. After exchanging with t-butanol (FUJIFILM Wako Chemicals), the specimens were freeze-dried using a freeze dryer (EIKO, Tokyo, Japan). A platinum coating was then applied using a magnetron sputter coater MSP-1S (VACUUM DEVICE, Ibaraki, Japan; 40–45 mA, 1 min pre-evacuation, 30 s coating), and the middle portion of the root canal walls was observed using SEM (S-4300/HITACHI, Tokyo, Japan). DNA Extraction from E. faecalis Biofilms on Root Canal Walls After root canal irrigation, the Ni-Ti file Endo Wave Lax (reverse taper #50, MORITA, Osaka, Japan) was advanced to approximately the working length in the M4 mode, followed by one complete circumferential filing. The root canal was not filled with solution during this step. A 50 mL tube was prefilled with 20 mL of 0.85% saline, and the Endo Wave Lax carrying dentin debris was placed into the tube. In addition, the extracted tooth root canal was filled with enough of the same saline to cover the canal, and this solution was then added to the 50 mL tube. The tubes were sealed with Parafilm (Amcor, Zurich, Switzerland). Ultrasonic treatment was performed for 10 min using an ultrasonic device (BRANSONIC 2510 J-DTH; 42 kHz, BRANSON, Danbury, CT, USA), followed by vortexing at 3,000 rpm for 1 min. To concentrate the sample 20-fold, 10 mL was collected and centrifuged at 10,000 × g for 20 min at 4°C (MX-305, TOMY, Tokyo, Japan). The supernatant was discarded, and 500 µL of phosphate buffered saline (PBS) was added. The PBS solution was transferred to a 1.5 mL tube and centrifuged at 10,000 × g for 1 min at 4°C. DNA was extracted from the resulting pellet using a DNeasy UltraClean Microbial Kit (QIAGEN, Hilden, Germany). Nucleic acid concentration was measured with a NanoDrop (Thermo Fisher Scientific), and the DNA was stored at − 30°C. Quantitative polymerase chain reaction (qPCR) assay Reactions were performed using the AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, CA, USA). PCR reagents were prepared so that each well contained 3.8 µL of RNase-free water, non-DEPC (BDL, Tokyo, Japan), 0.1 µL each of forward primer (10 µM, 5’-CGCTTCTTTCCTCCCGAGT-3’) and reverse primer (10 µM, 5’-GCCATGCGGCATAAACTG-3’), and 5 µL of SYBR Green qPCR Master Mix (Thermo Fisher Scientific). To each well, 1 µL of the extracted DNA was added. As a control, a well containing 1 µL of RNase-free water, non-DEPC instead of DNA was also prepared. A standard curve relating bacterial counts (colony forming units, CFU) to Cq values was constructed using a pure culture of E. faecalis (Table S1 ). The CFU of the bacterial suspension were determined by spotting the culture onto agar plates and counting the resulting colonies. The obtained standard curve was used to calculate the number of CFU remaining after drug treatment. The thermal cycling conditions were as follows: an initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. Subsequently, a melting curve analysis was performed with steps of 95°C for 30 s, 65°C for 30 s, and 95°C for 30 s to confirm the specificity of the E. faecalis genome [ 18 ]. Statistical analysis Statistical analyses were performed using EZR (R Commander version 2.9-1, R version 4.3.1, Jichi Medical University Saitama Medical Center, Saitama, Japan). All data were expressed as mean ± standard deviation (SD). The MIC, MBC, MBIC, and CCK-8 assay data were analyzed using one-way analysis of variance, followed by Tukey’s post-hoc test. qPCR data were evaluated using the Kruskal–Wallis test, followed by the Steel–Dwass post-hoc test. RESULTS Evaluation of antibacterial activity against oral pathogenic bacteria Figures 2 and 3 present the MIC and MBIC, respectively, with the pink frame indicating the respective values in each figure. Growth was inhibited when the concentration of PAA contained in Actril was at least 4.7×10⁻⁴% for E. faecalis , 9.4×10⁻⁴% for S. mutans , and 2.3×10⁻⁴% for P. gingivalis . At the same concentrations, biofilm formation was also inhibited in E. faecalis and S. mutans . Furthermore, in P. gingivalis , a tendency toward inhibition of biofilm formation was observed at the same concentration. In E. faecalis , growth was inhibited when the NaClO concentration in Antiformin was at least 0.14%, in S. mutans at 0.28%, and in P. gingivalis at 0.018%. Biofilm formation was also inhibited at the same concentrations. Table 2 shows the results of the MBC. In E. faecalis and S. mutans , the detection of viable cells was suppressed when the PAA concentration in Actril was at least 9.4 ×10⁻⁴%, and in P. gingivalis when it was at least 2.3×10⁻⁴%. In contrast, the detection of viable cells was suppressed at NaClO concentrations of 0.14% for E. faecalis , 0.28% for S. mutans , and 0.035% for P. gingivalis in Antiformin. Table 2 MBC of PAA and NaClO against oral pathogens Bacteria PAA (%) NaClO (%) Enterococcus faecalis 0.00094 0.14 Streptococcus mutans 0.00094 0.28 Porphyromonas gingivalis 0.00023 0.035 MBC (%) of PAA and NaClO against E. faecalis , S. mutans , and P. gingivalis . Concentrations are calculated based on the dilution ratios of PAA in Actril and NaClO in Antiformin. MBC, minimum bactericidal concentration; NaClO, sodium hypochlorite; PAA, peracetic acid. Evaluation of cytotoxicity toward periodontal tissue-related cells Figure 4 shows the results of the CCK-8 assay. The red lines represent the MBC of each irrigant against E. faecalis. Cytotoxic effects on cellular metabolic activity were observed in GE-1 cells when the PAA concentration in Actril reached 7.5×10⁻³%, and in HPLF cells when it reached 1.9×10⁻³%, both of which were higher than the MBC against E. faecalis . In contrast, cytotoxicity appeared in Antiformin at NaClO concentrations of 0.14% or higher for GE-1 cells, and 8.8×10⁻³% or higher for HPLF cells, which were lower than or comparable to the MBC against E. faecalis. Live/dead staining for E. faecalis in biofilm Figure 5 presents the results of live/dead staining. In the control and saline groups, the biofilms predominantly exhibited green fluorescence with minimal red fluorescence indicating dead cells, suggesting that most of the bacterial population remained viable. In the Actril group, the proportion of dead cells was markedly higher than that in the control and saline groups. Nevertheless, the effect of Actril on biofilm bacteria was variable, and in some instances, its efficacy was limited (Supplementary Fig. 1). No live or dead cells were detected in the Antiformin group. SEM analysis of root canal wall after irrigation Figure 6 shows SEM images of the central root canal wall at 1,000× magnification. Comparison of the root canal before biofilm formation under sterile conditions (A) with that after 7 days of biofilm cultivation (B) revealed that the root canal wall was covered with biofilm, and the dentinal tubule orifices were occluded following the 7-day culture. Panels (C), (D), and (E) show the root canal walls after 2 min of syringe irrigation with 9.4×10⁻⁴% peracetic acid Actril, undiluted Antiformin, and 0.85% saline, respectively. These results indicate that Actril removed the biofilm from the root canal wall more effectively than Antiformin or saline, allowing the dentinal tubule orifices to become visible again. qPCR assay Figure 7 shows the results of the qPCR assay used to quantify the number of bacteria remaining in the root canal walls. No significant difference was observed between the Actril and Antiformin groups; however, both treatments significantly reduced CFU compared with the non-irrigated group (control). DISCUSSION PAA-based disinfectants—such as Actril—are commonly employed for the disinfection of medical devices, including dialysis equipment, and for cleanroom sanitation; however, they have not yet been applied as disinfectants in the oral cavity. Actril contains PAA, hydrogen peroxide, and acetic acid at defined concentrations that may influence the persistence of its antibacterial effects. Nevertheless, most studies have utilized PAA solutions rather than commercial formulations, often at relatively high concentrations compared with those of Actril. Recent in vitro studies have reported that 1–2% PAA exhibits antibacterial activity against E. faecalis [ 19 ]. In addition, 2.25% PAA exhibited effects comparable to that of 17% EDTA in the removal of the smear layer from root canal walls, suggesting the potential to act on both biofilms and the smear layer with a single agent [ 20 ]. However, some reports have indicated that even high concentrations of PAA show only limited bactericidal efficacy against mature biofilms, with the effectiveness depending on factors such as concentration, exposure time, and bacterial species [ 21 , 22 ]. Collectively, these findings suggest that, although PAA is a promising root canal irrigant, its antibacterial activity, safety, and reproducibility in clinically relevant models remain unclear. Therefore, this study focused on evaluating the inhibitory effects of Actril on oral pathogenic bacteria and its potential for biofilm removal from infected root canals. In this study, the PAA present in Actril exhibited MIC, MBC, and MBIC values approximately 1/100 to 1/1000 of those of NaClO, the active ingredient in Antiformin, demonstrating a potent inhibitory effect, even at low concentrations (Fig. 2 , 3 and Table 2 ). These results suggest that Actril may be more effective than Antiformin, possibly due, at least in part, to differences in the duration of action between PAA and NaClO. To measure the MIC, MBC, and MBIC, the disinfectants were allowed to act for more than 24 h. During this period, the effectiveness of the active ingredients may gradually decrease; however, PAA is considered to have a longer duration of action than NaClO. This is because Actril contains hydrogen peroxide and acetic acid, which allows PAA to be regenerated even after decomposition. Next, the cytotoxicity of Actril on periodontal tissue-related cells was evaluated and compared with that of Antiformin. In this study, Actril exerted a smaller effect on cellular metabolic activity than that of Antiformin (Fig. 4 ). Previous studies have reported that when 1% PAA and 2.5% NaClO were diluted and applied to the mouse fibroblast cell line L929 for 10 min, PAA resulted in lower cell viability than NaClO at dilutions below 0.03%, whereas no significant difference was observed at dilutions of 0.03% or higher [ 23 ]. Another study examined the cytotoxicity of disinfectants on FG11 and FG15 fibroblasts after exposure for up to 4 h. It was reported that 2.5% NaClO showed no cytotoxicity only at a 0.01% dilution, whereas 1% PAA showed no cytotoxicity not only at 0.01% but also at a 0.05% dilution [ 24 ]. Based on these findings, PAA at a concentration of 5.0×10⁻⁴% showed no cytotoxicity in the previous study, whereas in this study, even at a higher PAA concentration of 9.4×10⁻⁴%, the impact on GE-1 and HPLF cells was minimal. The results presented in Figs. 2 , 3 , and 4 and Table 2 indicate that PAA at a concentration of 2.3–9.4× 10⁻⁴% exerted inhibitory effects on oral pathogenic bacteria, which is approximately one-tenth of the concentration that results in cytotoxicity in cells. The concentrations of NaClO that exhibited inhibitory effects on oral pathogenic bacteria ranged from 0.018% to 0.28%, which were comparable to the concentrations that induced cytotoxicity in cells. At concentrations effective against oral pathogenic bacteria, Antiformin was shown to be cytotoxic to periodontal tissue-related cells, whereas Actril, at a PAA concentration of 9.4×10⁻⁴%, was suggested to be effective against oral pathogenic bacteria while exhibiting low cytotoxicity. Actril at this concentration was tested for its effect on E. faecalis biofilms using live/dead staining. Notably, Actril effectively exerted a bactericidal effect on bacteria within the biofilm (Fig. 5 ). It is thought that the bactericidal components of Actril penetrate the biofilm and exert their effects. However, as shown in Supplementary Fig. 1, Actril did not exhibit consistent bactericidal effects. This may be due to the PAA concentration. The PAA concentration used in this study, 9.4 × 10⁻⁴%, may represent a threshold concentration: if it can penetrate the biofilm, it exhibits a bactericidal effect, whereas if penetration is not achieved, no bactericidal effect is observed. In the Antiformin-washed group, neither live nor dead bacteria were observed, suggesting that the biofilms were entirely dispersed. One possible reason is the surface modification effect of NaClO on polystyrene (PS) [ 25 ]. The biofilm on the PS substrate of the µ-96 well plate used in this study may have largely detached because NaClO treatment chlorinated and oxidized the substrate surface, roughening it (altering the surface energy) and thereby reducing adhesion. As live/dead staining suggested that Actril at a PAA concentration of 9.4×10⁻⁴% exerts a bactericidal effect on E. faecalis within biofilms, we next evaluated its cleaning efficacy by forming E. faecalis biofilms on extracted human teeth. To simulate the clinical conditions, the extracted teeth were not sectioned into blocks but were used in their original root canal morphology, and the experiment was conducted under root canal irrigation. This study suggested that Actril, at a PAA concentration of 9.4×10⁻⁴%, had a superior irrigation effect on biofilms formed on the root canal walls compared with that of Antiformin (Fig. 6 ). The NaClO solution is extensively consumed in the presence of organic matter [ 26 ] and exhibits limited penetration into the biofilm matrix [ 27 ]. In contrast, PAA was effective even in the presence of extracellular polysaccharides produced by E. faecalis , and its acidic properties are thought to decalcify calcium on the root canal wall surface, thereby detaching the biofilm. A previous study reported that 3% NaClO solution could remove patient-derived bacterial biofilms formed on root canal dentin [ 28 ], which is inconsistent with our results. The extended application time of 15 min and the larger contact area in that study were considered to have allowed for greater interaction between NaClO and the biofilm. Because the application time of irrigants in clinical practice is generally limited to 1–2 min, the marked irrigation efficacy of Actril within 2 min, as revealed in this study, is considered highly valuable. The qPCR analysis revealed no significant differences in bacterial counts between the Actril and Antiformin irrigation groups (Fig. 7 ). Nevertheless, a reduction in bacteria was also observed in the saline irrigation group compared with the control, indicating that the observed effect reflects not only the antimicrobial activity of the irrigants but also the physical effect of fluid movement within the root canal. During clinical root canal irrigation, irrigants also flow continuously through the canal; thus, these results are thought to more closely reflect clinical conditions and suggest that Actril exhibits an effect comparable to that of Antiformin. While SEM images suggested a superior irrigation effect of Actril compared with Antiformin, the lack of a significant difference in the qPCR results was thought to be due to the sample collection method. Previous literature has also demonstrated that treatment with 0.6% NaClO for 2 h still leaves viable bacteria, as shown by live/dead staining [ 29 ]. In this study, samples were centrifuged to remove lysed bacteria and their intracellular contents prior to qPCR analysis. Therefore, the genomes detected by qPCR were considered to originate solely from viable bacteria. That is, although biofilms were collected using Ni-Ti files after irrigation in this study, some bacteria that remained alive following Antiformin irrigation may have been killed during collection with the Ni-Ti files, resulting in the number of recovered viable bacteria being comparable to that observed in the Actril group. However, because the collection of bacteria using files after irrigation is a standard method [ 30 , 31 ], a significant difference between Actril and Antiformin might be detectable if the bacterial load in the biofilm is increased. Furthermore, the large error bars may have been another factor contributing to the absence of significant differences. This variability is largely attributable to individual differences among the extracted human teeth [ 3 ]. Differences in root canal morphology [ 1 ] and dentin hardness [ 32 ] may result in variations in biofilm formation. CONCLUSION In this study, the PAA-based disinfectant—Actril—exhibited antibacterial activity against oral pathogenic bacteria at lower concentrations than that of the NaClO solution—Antiformin—while showing lower cytotoxicity toward cells associated with periodontal tissues. Moreover, at concentrations that are both effective against oral pathogenic bacteria and non-cytotoxic to periodontal tissue-related cells, Actril demonstrated superior irrigation efficacy in an E. faecalis -infected root canal model using extracted human teeth compared with the NaClO solution. Therefore, Actril has the potential to serve as a safe and effective root canal irrigant compared with the NaClO solution. However, this study did not fully address certain aspects of clinical relevance such as the efficacy of the disinfectant in the apical region, its effects on bacteria within the dentinal tubules, or its tissue-dissolving ability in the pulp. Future studies are warranted to evaluate its safety in vivo and verify its effectiveness through clinical trials. Abbreviations GE-1, mouse-derived gingival epithelial cells HPLF, human periodontal ligament fibroblasts MIC, minimum inhibitory concentration MBC, minimum bactericidal concentration MBIC, minimum biofilm inhibitory concentration NaClO, sodium hypochlorite PAA, peracetic acid qPCR, quantitative polymerase chain reaction Declarations Ethics approval and consent to participate This study was approved by the Public University Corporation Kyushu Dental University Research Ethics Committee (approval number: 24-13). The committee waived the need for written informed consent because the study was retrospective in nature and used anonymized extracted human teeth that had been obtained following routine dental treatment and were not removed for research purposes. The procedures conducted in this study were carried out in accordance with the ethical guidelines of the institution and domestic regulations, as well as the principles of the 1964 Declaration of Helsinki and its later amendments. Consent for publication Not applicable. Availability of data and materials Radiographic images other than those shown in Figure 1 (A) are available from the corresponding author upon reasonable request. All other data supporting the findings of this study are included in the main manuscript and the supplementary files. Competing interests The authors declare that they have no competing interests. Funding This work was partially supported by the JSPS KAKENHI (grant number 24K12888). Authors’ contributions RT: Conceptualization, methodology, investigation, original draft writing, manuscript review and editing. RY: Conceptualization, methodology, data curation, supervision, manuscript review and editing. AW: Pilot experiments, supervision, manuscript review and editing. YY: Supervision. WA: Methodology and supervision. CK: Conceptualization and supervision. All authors contributed to data collection and interpretation and critically reviewed the manuscript. All authors have read and approved the final version of the manuscript. Acknowledgements We would like to thank Dr. Takashi Toyono for their guidance on using the SEM. We would like to thank Editage ( www.editage.jp ) for English language editing. References Versiani MA, Martins J, Ordinola-Zapata R. Anatomical complexities affecting root canal preparation: a narrative review. Aust Dent J. 2023;68 Suppl 1:S5-S23. Babeer A, Bukhari S, Alrehaili R, Karabucak B, Koo H. Microrobotics in endodontics: A perspective. Int Endod J. 2024;57(7):861-71. Swimberghe RCD, Coenye T, De Moor RJG, Meire MA. Biofilm model systems for root canal disinfection: a literature review. Int Endod J. 2019;52(5):604-28. Cai C, Chen X, Li Y, Jiang Q. Advances in the role of sodium hypochlorite irrigant in chemical preparation of root canal treatment. Biomed Research International. 2023;2023(1):8858283. Cho-Kee D, Basrani BR, Vera J, Ordinola-Zapata R, Aguilar RR. Sodium Hypochlorite Accidents: A Retrospective case-series analysis of CBCT Imaging and Clinician Surveys. J Endod. 2025. HÜLSMANN M, RÖDIG T, Nordmeyer S. Complications during root canal irrigation. Endodontic Topics. 2007;16(1):27-63. Zhang N, Guo J, Liu L, Wu H, Gu J. Study on the Efficacy of Peracetic Acid Disinfectant (Type III) on Gastrointestinal Endoscopy Disinfection. Surg Laparosc Endosc Percutan Tech. 2021;31(4):395-8. Kitis M. Disinfection of wastewater with peracetic acid: a review. Environ Int. 2004;30(1):47-55. Stearns R, Freshour A, Shen CJJoA, Research F. Literature review for applying peroxyacetic acid and/or hydrogen peroxide to control foodborne pathogens on food products. J Agric Food Res. 2022;10:100442. Butucel E, Balta I, Ahmadi M, Dumitrescu G, Morariu F, Pet I, et al. Biocides as Biomedicines against Foodborne Pathogenic Bacteria. Biomedicines. 2022;10(2). McDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev. 1999;12(1):147-79. Chino T, Nukui Y, Morishita Y, Moriya K. Morphological bactericidal fast-acting effects of peracetic acid, a high-level disinfectant, against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in tubing. Antimicrob Resist Infect Control. 2017;6:122. Singh A, Kaur H, Sawale M, Ozadali F, Benyathiar P, Mishra DKJL. Bacillus cereus spore inactivation by peracetic acid for aseptic packaging application. LWT. 2025;224:117811. Ledwoch K, Maillard JY. Candida auris Dry Surface Biofilm (DSB) for Disinfectant Efficacy Testing. Materials (Basel). 2018;12(1). Oettler MJ, Conraths FJ, Roesler U, Reiche S, Homeier-Bachmann T, Denzin N. Efficiency of Virucidal Disinfectants on Wood Surfaces in Animal Husbandry. Microorganisms. 2024;12(5). Kapes T, Quinn C, Cragun AE, House T, Nims RW, Zhou SS. Differing Susceptibilities to Certain Microbicidal Chemistries among Three Representative Enveloped Viruses. Microorganisms. 2024;12(3). Barbosa-Ribeiro M, De-Jesus-Soares A, Zaia AA, Ferraz CC, Almeida JF, Gomes BP. Antimicrobial Susceptibility and Characterization of Virulence Genes of Enterococcus faecalis Isolates from Teeth with Failure of the Endodontic Treatment. J Endod. 2016;42(7):1022-8. Pinheiro ET, Candeiro GT, Teixeira SR, Shin RC, Prado LC, Gavini G, et al. RNA-based Assay Demonstrated Enterococcus faecalis Metabolic Activity after Chemomechanical Procedures. J Endod. 2015;41(9):1441-4. Briseno-Marroquin B, Callaway A, Shalamzari NG, Wolf TG. Antibacterial efficacy of peracetic acid in comparison with sodium hypochlorite or chlorhexidine against Enterococcus faecalis and Parvimonas micra . BMC Oral Health. 2022;22(1):119. Elsamra AH, Darrag AM, Ghoneim WMJTDJ. Efficacy of different chelating agents in smear layer removal. Tanta dent J. 2023;20(1):27-33. Brandao-Neto DO, Mello JVZ, Marceliano-Alves MFV, Carvalho Coutinho TM, Marceliano EFV, Galhardi MPW, et al. Final Endodontic Irrigation with 2% Peracetic Acid: Antimicrobial Activity and Cytotoxicity. Eur J Dent. 2021;15(3):533-8. Lee SHI, Cappato LP, Corassin CH, Cruz AG, Oliveira CAF. Effect of peracetic acid on biofilms formed by Staphylococcus aureus and Listeria monocytogenes isolated from dairy plants. J Dairy Sci. 2016;99(3):2384-90. Viola KS, Rodrigues EM, Tanomaru-Filho M, Carlos IZ, Ramos SG, Guerreiro-Tanomaru JM, et al. Cytotoxicity of peracetic acid: evaluation of effects on metabolism, structure and cell death. Int Endod J. 2018;51 Suppl 4:e264-e77. Teixeira PA, Coelho MS, Kato AS, Fontana CE, Bueno CE, Pedro-Rocha DG. Cytotoxicity assessment of 1% peracetic acid, 2.5% sodium hypochlorite and 17% EDTA on FG11 and FG15 human fibroblasts. Acta Odontol Latinoam. 2018;31(1):11-5. Guo C, Zou Q, Wang J, Wang H, Chen S, Zhong Y. Application of surface modification using sodium hypochlorite for helping flotation separation of acrylonitrile-butadiene-styrene and polystyrene plastics of WEEE. Waste Manag. 2018;82:167-76. Zhang C, Brown PJB, Hu Z. Higher functionality of bacterial plasmid DNA in water after peracetic acid disinfection compared with chlorination. Sci Total Environ. 2019;685:419-27. De Beer D, Srinivasan R, Stewart PS. Direct measurement of chlorine penetration into biofilms during disinfection. Appl Environ Microbiol. 1994;60(12):4339-44. Clegg MS, Vertucci FJ, Walker C, Belanger M, Britto LR. The effect of exposure to irrigant solutions on apical dentin biofilms in vitro. J Endod. 2006;32(5):434-7. Rosen E, Tsesis I, Elbahary S, Storzi N, Kolodkin-Gal I. Eradication of Enterococcus faecalis Biofilms on Human Dentin. Front Microbiol. 2016;7:2055. Kranz S, Guellmar A, Braeutigam F, Tonndorf-Martini S, Heyder M, Reise M, et al. Antibacterial Effect of Endodontic Disinfections on Enterococcus Faecalis in Dental Root Canals-An In-Vitro Model Study. Materials (Basel). 2021;14(9). Shimaoka T, Maezono H, Ono S, Asahi Y, Kawanishi Y, Klanliang K, et al. Application of on-demand aqueous chlorine dioxide solution for non-surgical root canal treatment. Sci Rep. 2025;15(1):36215. Ozdemir HO, Buzoglu HD, Calt S, Stabholz A, Steinberg D. Effect of ethylenediaminetetraacetic acid and sodium hypochlorite irrigation on Enterococcus faecalis biofilm colonization in young and old human root canal dentin: in vitro study. J Endod. 2010;36(5):842-6. Additional Declarations No competing interests reported. Supplementary Files Supplementary.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7911128","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":542353281,"identity":"b701fbd4-42ab-4078-99a9-096c53661cc0","order_by":0,"name":"Rikako Takami","email":"","orcid":"","institution":"Kyushu Dental University","correspondingAuthor":false,"prefix":"","firstName":"Rikako","middleName":"","lastName":"Takami","suffix":""},{"id":542353282,"identity":"310fa581-c88d-458a-9209-7ad8535be397","order_by":1,"name":"Ryota Yamasaki","email":"data:image/png;base64,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","orcid":"","institution":"Kyushu Dental University","correspondingAuthor":true,"prefix":"","firstName":"Ryota","middleName":"","lastName":"Yamasaki","suffix":""},{"id":542353283,"identity":"c0179c2c-db86-4f9d-88c8-e2c9fa7d7cb2","order_by":2,"name":"Ayako Washio","email":"","orcid":"","institution":"Kyushu Dental University","correspondingAuthor":false,"prefix":"","firstName":"Ayako","middleName":"","lastName":"Washio","suffix":""},{"id":542353284,"identity":"911a7321-db3d-47c5-8c00-2ca4e6f1aadc","order_by":3,"name":"Yoshie Yoshioka","email":"","orcid":"","institution":"Kyushu Dental University","correspondingAuthor":false,"prefix":"","firstName":"Yoshie","middleName":"","lastName":"Yoshioka","suffix":""},{"id":542353285,"identity":"bd788215-4d8f-4a70-957a-b94723b9af62","order_by":4,"name":"Wataru Ariyoshi","email":"","orcid":"","institution":"Kyushu Dental University","correspondingAuthor":false,"prefix":"","firstName":"Wataru","middleName":"","lastName":"Ariyoshi","suffix":""},{"id":542353286,"identity":"dfc24b63-1a83-47a9-81af-14ab103f9ef8","order_by":5,"name":"Chiaki Kitamura","email":"","orcid":"","institution":"Kyushu Dental University","correspondingAuthor":false,"prefix":"","firstName":"Chiaki","middleName":"","lastName":"Kitamura","suffix":""}],"badges":[],"createdAt":"2025-10-21 06:38:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7911128/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7911128/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":95657454,"identity":"112b1720-1d1d-45fc-a28e-5b7a60e2b847","added_by":"auto","created_at":"2025-11-11 16:20:54","extension":"tif","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1804602,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/d069ae4a4b4556a0a8d06181.tif"},{"id":95629755,"identity":"c21994f7-d49d-4ddc-be72-6a273ef9657e","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1044008,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/bfe4b0619af727b7d9465766.tif"},{"id":95656806,"identity":"05f2d610-f5ab-423b-9b92-6afaf1ad16ad","added_by":"auto","created_at":"2025-11-11 16:19:38","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":105810,"visible":true,"origin":"","legend":"","description":"","filename":"Revisedmaintext.docx","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/105e376809c407d662ce2d3e.docx"},{"id":95657871,"identity":"52ccad56-8aa7-4ab4-b18f-831c51227541","added_by":"auto","created_at":"2025-11-11 16:22:18","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":995302,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/be348bb21a316ef45be1b118.tif"},{"id":95629759,"identity":"9f28d6d9-8186-4548-9e50-728504b3779e","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1095400,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/59f9c24027dd3c47ef90ace4.tif"},{"id":95629764,"identity":"73fb013e-1fa9-4753-809a-d8d4ccd71140","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2167314,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/78ae040f85a564347894bd3b.tif"},{"id":95657154,"identity":"d58d9019-bb10-4865-a206-8c9e9ac3255c","added_by":"auto","created_at":"2025-11-11 16:20:11","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4286736,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/328a8107ac76c165bcbf4471.tif"},{"id":95629763,"identity":"8d6a5e04-4391-42b3-8a22-3c502debc5af","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61596,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.7.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/7d4826c176eb79c39fc436a4.tif"},{"id":95658185,"identity":"c939911b-aa50-40a8-8c1f-8b167dfcf8a5","added_by":"auto","created_at":"2025-11-11 16:23:33","extension":"json","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":8407,"visible":true,"origin":"","legend":"","description":"","filename":"281034c4411d400cbb5a30b0e897a623.json","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/360021d9635d4eedeff129cf.json"},{"id":95657517,"identity":"7598e0ff-8cdd-4ec6-b153-391ecd9bdf67","added_by":"auto","created_at":"2025-11-11 16:21:03","extension":"docx","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":163105,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/64c96f97fcd980ed9d3af8af.docx"},{"id":95657469,"identity":"209fe0b2-8b0b-43a5-9c8d-ae71eec1846e","added_by":"auto","created_at":"2025-11-11 16:20:55","extension":"xml","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107388,"visible":true,"origin":"","legend":"","description":"","filename":"281034c4411d400cbb5a30b0e897a6231enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/de671fd6c7f2b842953c0283.xml"},{"id":95629766,"identity":"db2f4048-f583-4d0c-8165-c544cd394ec0","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"tif","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1804602,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/2975965e01f39a051abb7104.tif"},{"id":95629772,"identity":"97803fb3-668d-47db-86d2-26d1bb965793","added_by":"auto","created_at":"2025-11-11 11:20:24","extension":"tif","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1044008,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/c0fe2ebd824bd0cf61255753.tif"},{"id":95657179,"identity":"01755a7e-7019-41e1-80d0-6caa5dd59d1c","added_by":"auto","created_at":"2025-11-11 16:20:15","extension":"tif","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":995302,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/01ed68dbdf70b35f1040d4f9.tif"},{"id":95629768,"identity":"59c32503-7938-4fc7-bf14-3b101300003c","added_by":"auto","created_at":"2025-11-11 11:20:24","extension":"tif","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1095400,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.4.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/65dc8de6655a1188865ce8ee.tif"},{"id":95658070,"identity":"30c1ad18-ba27-4fdd-ad62-beb23dec50fb","added_by":"auto","created_at":"2025-11-11 16:22:52","extension":"tif","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2167314,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.5.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/88a0334443f4286fb1aff80d.tif"},{"id":95629781,"identity":"56d041af-3e9e-4714-a367-adea3257d675","added_by":"auto","created_at":"2025-11-11 11:20:24","extension":"tif","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4286736,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.6.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/d7508b86f69dc58669e2550c.tif"},{"id":95629774,"identity":"05ca6481-5057-4ce2-8446-b70bcdfea8ce","added_by":"auto","created_at":"2025-11-11 11:20:24","extension":"tif","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":61596,"visible":true,"origin":"","legend":"","description":"","filename":"Fig.7.tif","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/a208873c58fc6bc00ccf4ba1.tif"},{"id":95629784,"identity":"925d58be-0fe2-4c1a-9ce1-53e5c1de6063","added_by":"auto","created_at":"2025-11-11 11:20:24","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":166041,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/f87f79258298607a4e68cf28.png"},{"id":95657661,"identity":"872146d9-1edb-4167-9ea4-21ea382c88ee","added_by":"auto","created_at":"2025-11-11 16:21:34","extension":"png","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":74386,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/ebae63632865cc995bfc4239.png"},{"id":95629777,"identity":"2fda9f1a-4266-4ba8-84a1-00516c13a06c","added_by":"auto","created_at":"2025-11-11 11:20:24","extension":"png","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":67224,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/e8e2ce2ec2d24ef3d1652a4a.png"},{"id":95629775,"identity":"543f5327-ef14-431d-a3a0-3365cec0cf49","added_by":"auto","created_at":"2025-11-11 11:20:24","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97517,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/55c33f2e3e1bce7e7585a316.png"},{"id":95656601,"identity":"6d2ec493-4ac5-4060-b792-3ae415c61bc2","added_by":"auto","created_at":"2025-11-11 16:19:10","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":246256,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/974999a45bd27c5fd7855f2c.png"},{"id":95657901,"identity":"aff4807f-1d11-4c88-b8a2-8ab3c54abd30","added_by":"auto","created_at":"2025-11-11 16:22:23","extension":"png","order_by":23,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2409200,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/5d112c5274cb373fc09c91e6.png"},{"id":95657872,"identity":"b3c65215-b615-4ff4-8d83-a372be331edf","added_by":"auto","created_at":"2025-11-11 16:22:18","extension":"png","order_by":24,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7980,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/109eda97ea0ccd34cf952858.png"},{"id":95657886,"identity":"76f1debc-b5e5-4800-b50b-bdac2c3ec4a7","added_by":"auto","created_at":"2025-11-11 16:22:20","extension":"xml","order_by":25,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":106472,"visible":true,"origin":"","legend":"","description":"","filename":"281034c4411d400cbb5a30b0e897a6231structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/02a7b2d120592cf54d0a42ef.xml"},{"id":95629783,"identity":"98451092-e9c8-4004-9a3f-2b4e9689757d","added_by":"auto","created_at":"2025-11-11 11:20:24","extension":"html","order_by":26,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":114738,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/409cba7fdc14718c56dd2d27.html"},{"id":95629753,"identity":"cc41dbde-41ae-4f46-8fc9-5e0386ca15d4","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2767794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental procedures for root canal preparation and irrigation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Periapical X-rays were taken from mesial and distal directions to confirm that the extracted teeth were single-rooted. Scale bar = 1.0 cm. (B) Crowns were sectioned 9.0 mm from the apex, and the canal lengths were measured. Working lengths were determined 1 mm shorter than these measurements, and the canals were prepared using Nickel-Titanium (Ni-Ti) files up to size #40 with a 10° taper. (C) Root canals were irrigated 1 mm short of the working length with gentle vertical motions according to the following protocol: 30 s flow irrigation with 1 mL of EDTA followed by 1 min ultrasonic irrigation (repeated twice); 30 s flow irrigation with 1 mL of NaClO followed by 1 min ultrasonic irrigation (repeated twice); 30 s flow irrigation with 0.1 mL etchant followed by 1 min ultrasonic irrigation; and finally, 30 s flow irrigation with 1 mL saline. (D) After cleaning, the apices and periapical areas were sealed with self-curing resin, and each tooth was fixed in a 5-mL tube. For aerobic culture, a hole was perforated in the tube cap using a round bur. EDTA, ethylenediaminetetraacetic acid; NaClO, sodium hypochlorite.\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/059fb709d35aa91a3f1b7156.png"},{"id":95629751,"identity":"54180583-d5aa-4d33-9376-ea78173ba8a6","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":689050,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAntimicrobial activity of PAA and NaClO against oral pathogens\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) MIC of PAA and (B) NaClO against \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (left), \u003cem\u003eStreptococcus mutans\u003c/em\u003e (middle), and \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e (right), with the MIC values highlighted by the pink frame. The x-axis indicates the actual concentrations calculated from the dilution ratios of PAA in Actril and NaClO in Antiformin. Data represent n = 3–6. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s post-hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01 compared with the control. MIC, minimum inhibitory concentration; NaClO, sodium hypochlorite; PAA, peracetic acid.\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/1f4df33280709e1f122bfc1d.png"},{"id":95656534,"identity":"85c16cac-59f1-4d0f-a45e-c9e27f50d6e9","added_by":"auto","created_at":"2025-11-11 16:18:58","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":604101,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibitory effects of PAA and NaClO on biofilm formation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) MBIC of PAA and (B) NaClO against \u003cem\u003eEnterococcus faecalis\u003c/em\u003e (left), \u003cem\u003eStreptococcus mutans\u003c/em\u003e (middle), and \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e (right), with the MBIC values highlighted by the pink frame. The x-axis indicates the actual concentrations calculated from the dilution ratios of PAA in Actril and NaClO in Antiformin. Data represent n = 3–6. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s post-hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01 compared with the control. MBIC, minimum biofilm inhibitory concentration; NaClO, sodium hypochlorite; PAA, peracetic acid.\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/e921b67a5d79c8313287a4ba.png"},{"id":95629758,"identity":"99dbd64a-f2bb-41db-b361-22757eeeaa6a","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1157312,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCytotoxic effects of PAA and NaClO on periodontal tissue-related cells\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Effects of PAA and (B) NaClO on gingival epithelial cells (left) and human periodontal ligament fibroblasts (right). The red lines indicate the MBC of each irrigant against \u003cem\u003eE. faecalis\u003c/em\u003e. The x-axis indicates the actual concentrations calculated from the dilution ratios of PAA in Actril and NaClO in Antiformin. Data represent n = 3. Statistical analyses were performed using one-way analysis of variance followed by Tukey’s post-hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01 compared with the control. NaClO, sodium hypochlorite; PAA, peracetic acid.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/8a98e56e23941b3b2d60878c.png"},{"id":95629762,"identity":"dfe101bc-44aa-477a-b9dc-d28c28bb8010","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3462545,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLive/dead staining of bacteria after treatment with irrigation agents\u003c/strong\u003e\u003cbr\u003e\n Representative images obtained after treatment with each irrigation agent at 100× magnification. Green fluorescence indicates live bacteria, and red fluorescence indicates dead bacteria. Two-dimensional (2D) image (above); three-dimensional (3D) reconstruction (below). Scale bar = 100 µm.\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/03de52cf602fd9794058edcc.png"},{"id":95656801,"identity":"6208bdec-d08b-4b95-a4f1-3a8252dbdd4b","added_by":"auto","created_at":"2025-11-11 16:19:36","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":5886320,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSEM observation of root canal walls after biofilm formation and irrigation treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSEM images of the inner wall of the central portion of the root canal (1000× magnification). (A) Before biofilm formation. (B) After biofilm formation without irrigation. (C) After Actril treatment. (D) After Antiformin treatment. (E) After saline treatment. At least three samples were examined for each condition. Scale bar = 10 µm. SEM, scanning electron microscope.\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/f28521bff0ecc94bc76c0ecf.png"},{"id":95657596,"identity":"1f03e933-c51e-4fd8-b4be-90e790c304af","added_by":"auto","created_at":"2025-11-11 16:21:18","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":76310,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eQuantification of bacterial viability after irrigation treatments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCFU counts after treatment with each irrigant were determined using qPCR. Data represent at least 5 teeth. Statistical analyses were performed using the Kruskal–Wallis test, followed by the Steel–Dwass post-hoc test. *p \u0026lt; 0.05, **p \u0026lt; 0.01 compared with the non-irrigated group (control).CFU, colony forming unit; qPCR, quantitative polymerase chain reaction.\u003c/p\u003e","description":"","filename":"Fig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/87dff1a6e166e869b9b6e32b.png"},{"id":100883516,"identity":"97bfdc25-a36e-456a-b8f6-f3ad890f7cc2","added_by":"auto","created_at":"2026-01-22 11:39:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15245238,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/fde4b0bf-4440-4e83-9113-53f7ef9f2818.pdf"},{"id":95629756,"identity":"daa22582-7f67-4f36-8a19-dadb64394c00","added_by":"auto","created_at":"2025-11-11 11:20:23","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":163105,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementary.docx","url":"https://assets-eu.researchsquare.com/files/rs-7911128/v1/17c6baacd4722b3efbbd6172.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Antimicrobial and biocompatible potential of peracetic acid as a novel root canal irrigant","fulltext":[{"header":"BACKGROUND","content":"\u003cp\u003eThe success of endodontic treatment relies not only on mechanical debridement but also on the elimination of microorganisms and biofilms within the root canal system using chemical irrigants. This necessity arises from the complex anatomy of the root canal system, which includes intricate structures\u0026mdash;such as dentinal tubules, lateral canals, isthmuses, and fins\u0026mdash;that mechanical instruments are often unable to reach [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In such complex areas, debris tends to remain after root canal treatment [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and biofilms protected by extracellular polymeric substances can become infected [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], further complicating the cleaning process. Sodium hypochlorite (NaClO), the most commonly used irrigant, possesses strong antimicrobial activity and is considered the gold standard for root canal irrigation [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, its high cytotoxicity, the risk of tissue damage from extrusion beyond the apical foramen, and reduced activity in the presence of organic matter have raised concerns [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003ePeracetic acid (PAA) is a high-level disinfectant widely used for the disinfection of dialysis equipment and medical devices [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Moreover, because its decomposition products\u0026mdash;oxygen, water, and acetic acid\u0026mdash;are harmless, they impose minimal environmental and biological burdens and are attracting attention as an alternative to NaClO not only in the medical field but also for wastewater disinfection and the control of foodborne pathogens [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Chemically, PAA is an equilibrium mixture of acetic acid and hydrogen peroxide; its peroxy bond (-O\u0026ndash;O-) is highly unstable and releases reactive oxygen species. This structural property accounts for its strong oxidizing capacity, which enables the efficient disruption of microbial membranes and oxidation of essential enzymes and proteins [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Importantly, PAA exhibits a broad antimicrobial spectrum, demonstrating potent activity against a wide variety of microorganisms, including gram-positive and gram-negative bacteria, viruses, fungi, and even resistant forms such as bacterial spores and biofilms [\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These characteristics make PAA a versatile disinfectant across diverse environment.\u003c/p\u003e\u003cp\u003eThis study aimed to evaluate the antimicrobial activity of Actril\u0026reg; (MEDIVATORS, Minneapolis, MN, USA), a low-concentration PAA-based disinfectant, against representative oral pathogenic bacteria, as well as its cytotoxicity toward periodontal tissue-related cells. Furthermore, focusing on \u003cem\u003eEnterococcus faecalis\u003c/em\u003e,\u003c/p\u003e\u003cp\u003ewhich is often detected in refractory-infected root canals [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], we used a human extracted tooth infection model to comprehensively assess the irrigation and disinfection performance of Actril in comparison with a NaClO solution and explored its potential for clinical application.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of disinfectants\u003c/h2\u003e\u003cp\u003eThe primary sterilizing reagent used in this study was the PAA-based disinfectant, Actril (MEDIVATORS). Additionally, NaClO solution\u0026mdash;Antiformin (NISHIKA, Yamaguchi, Japan)\u0026mdash;was used for comparison. Actril concentrate contains 0.06% CH₃COOOH, 0.8% H₂O₂, and 5% CH₃COOH. Antiformin concentrate contains 3\u0026ndash;6% NaClO. Two-fold serial dilutions of these were prepared and used. All dilution procedures were performed under aseptic conditions, and the prepared solutions were used immediately after preparation.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eCulture conditions for oral pathogenic bacteria\u003c/h3\u003e\n\u003cp\u003eThe oral pathogens used were \u003cem\u003eE. faecalis\u003c/em\u003e NBRC 100482, \u003cem\u003eStreptococcus mutans\u003c/em\u003e UA159, and \u003cem\u003ePorphyromonas gingivalis\u003c/em\u003e ATCC 33277. \u003cem\u003eE. faecalis\u003c/em\u003e was cultured at 37 ℃ under 5% CO₂ for 24 h in 1.6% nutrient broth (BD, Franklin Lakes, NJ, USA), 0.5% yeast extract (Gibco, Thermo Fisher Scientific, Waltham, MA, USA), and 2% D-(+)-glucose (TCI, Tokyo, Japan). For agar and liquid media, \u003cem\u003eS. mutans\u003c/em\u003e was cultured in 3.7% brain heart infusion (BHI; BD) and 1% yeast extract. For the determination of the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm inhibitory concentration (MBIC), 0.1% sucrose (Sigma-Aldrich, St. Louis, MO, USA) sterilized using a 0.2-\u0026micro;m filter (Minisart, Sartorius, G\u0026ouml;ttingen, Germany) was added to the medium, and the cultures were incubated in 96-well plates (Wuxi NEST Biotechnology, Jiangsu, China) at 37 ℃ under 5% CO₂ for 24 h. \u003cem\u003eP. gingivalis\u003c/em\u003e were cultured at 37 ℃ for 72 h using Anaero Pack (MGC, Tokyo, Japan) on ABHK agar medium (SHIMADZU, Tokyo, Japan), and the liquid medium comprised BHI supplemented with hemin (FUJIFILM Wako Chemicals, Osaka, Japan) to a final concentration of 5 mg/L and menadione (FUJIFILM Wako Chemicals) to a final concentration of 0.5 mg/L. The cultures were incubated anaerobically.\u003c/p\u003e\n\u003ch3\u003eEvaluation of efficacy against oral pathogenic bacteria\u003c/h3\u003e\n\u003cp\u003eTwo-fold serial dilutions of the disinfectants were prepared in 96-well plates. The control comprised 200 \u0026micro;L of liquid medium without added disinfectant. A bacterial suspension of each oral pathogenic bacterium was prepared to a final OD\u003csub\u003e600\u003c/sub\u003e of 0.05 and added, then cultured under the respective conditions as described above. After the designated incubation period, the absorbance was measured at 620 nm using a Multiskan FC (Thermo Fisher Scientific) and the lowest concentration of the agent that inhibited bacterial growth was defined as the MIC. Additionally, 10 \u0026micro;L of bacterial suspension from each well showing growth inhibition was plated onto agar medium and incubated for the designated period, and the MBC was determined based on the presence or absence of colony formation. After measuring the absorbance at 620 nm, the supernatant was discarded, and the cells were washed three times with water purified by reverse osmosis (RO water, Milli-RO system; Merck Millipore, Darmstadt, Germany). The wells were stained with 0.1% crystal violet solution (FUJIFILM Wako Chemicals) for 20 min. They were then washed three times with RO water and incubated in 95% ethanol for 5 min. After the 5-min incubation, the wells were pipetted, and the absorbance at 540 nm was measured to determine the MBIC.\u003c/p\u003e\n\u003ch3\u003eEvaluation of cytotoxicity toward periodontal tissue-related cells\u003c/h3\u003e\n\u003cp\u003eMouse-derived gingival epithelial cells (GE-1; Riken Cell Bank, Ibaraki, Japan) were cultured at 37\u0026deg;C in serum-free medium (SHIMADZU) supplemented with 1% fetal bovine serum (FBS; SIGMA, St. Louis, MO, USA), 100 U/mL penicillin G, 100 \u0026micro;g/mL streptomycin (FUJIFILM Wako Chemicals), and 1 \u0026micro;g/mL epithelial growth factor (PeproTech, Cranbury, NJ, USA) under 5% CO₂ for 24 h. After incubation, the cells were harvested using a 0.25% of trypsin\u0026ndash;ethylenediaminetetraacetic acid (EDTA) solution (0.5 g/L trypsin and 0.53 mmol/L EDTA; Nacalai Tesque, Kyoto, Japan). Twenty percent of the total recovered cells were seeded into new dishes for passage and cultured for 48 h. After passage, cells were treated with trypsin-EDTA and seeded into a 96-well plate at a density of 8.0 \u0026times; 10⁴ cells/well. After 24 h, Actril or Antiformin diluted in a two-fold serial dilution was applied for 2 min. Two minutes later, the disinfectants were aspirated, and the samples were washed. Then, 100 \u0026micro;L of culture medium and 10 \u0026micro;L of reagent from Cell Counting Kit-8 (CCK-8; DOJINDO, Kumamoto, Japan) were added, and the cells were cultured at 37 ℃ under 5% CO₂ for 2 h. The absorbance of each well at 540 nm was measured after incubation.\u003c/p\u003e\u003cp\u003eHuman periodontal ligament fibroblasts (HPLF; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in MEM-α (FUJIFILM Wako Chemicals) supplemented with 10% FBS and 1% penicillin G/streptomycin at 37 ℃ under 5% CO₂. The cells after passage were seeded into a 96-well plate at a density of 1.0\u0026times;10⁴ cells/well. The disinfectant was applied as described above, and the CCK-8 reagent was added. The cells were then incubated at 37 ℃ under 5% CO₂ for 3 h. After incubation, the absorbance of each well at 450 nm was measured.\u003c/p\u003e\u003cp\u003e\u003cb\u003eLive/dead staining for\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003ein biofilm\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTotally, 200 \u0026micro;L of liquid medium was added to each well of a \u0026micro;-96 well plate (ibidi, Gr\u0026auml;felfing, Germany). Subsequently, \u003cem\u003eE. faecalis\u003c/em\u003e was adjusted to a final OD\u003csub\u003e600\u003c/sub\u003e of 0.05 and added to each well. The plates were incubated under the conditions described above to allow biofilm formation. 48 h, the supernatant was discarded. Each sample was then rinsed with 2 mL of the solution for 2 min using a 27G side-port soft needle and aspirator according to the disinfection groups listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. After 2 min, the sample was washed once with 600 \u0026micro;L of 0.85% saline solution. The Antiformin group was neutralized using 600 \u0026micro;L of 5% sodium thiosulfate to prevent bleaching of the live/dead staining reagent. As a control, wells were also prepared in which only the washing process with 0.85% saline solution was performed, in the absence of any disinfectant. Additionally, to serve as a reference for determining the exposure time, wells were also prepared in which the formed biofilm was treated with 200 \u0026micro;L of undiluted isopropanol for 1 h to sterilize the biofilm. Equal volumes of the SYTO 9 (1.67 mM)/Propidium iodide (1.67 mM) mixture and the SYTO 9 (1.67 mM)/Propidium iodide (18.3 mM) mixture from the LIVE/DEAD\u0026reg; BacLight\u0026trade; Bacterial Viability Kit L7007 (Invitrogen, MA, USA) were combined and added at a volume of 3 \u0026micro;L per 1 mL of 0.85% saline solution. Subsequently, 200 \u0026micro;L of the prepared reagent was added to each well, and the plates were incubated in the dark for 15 min and protected from light with aluminum foil. After incubation, the supernatant was discarded, and the wells were washed once with 250 \u0026micro;L of 0.85% saline solution. Observations were performed using an all-in-one fluorescence microscope BZ-X810 (KEYENCE, Osaka, Japan) with a 10\u0026times; objective lens, capturing both green and red fluorescence at an exposure time of 1.5 s. Three-dimensional rendering was performed using the BZ-H4R/3D analysis application (KEYENCE).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eConcentration and pH of irrigating solutions used in this study\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIrrigation\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eIngredient concentration during use (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003epH\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eActril\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePAA*\u003csup\u003e1\u003c/sup\u003e: 9.4\u0026times;10⁻⁴\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2.96\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAntiformin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNaClO: 4.5*\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e12.22\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esaline\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNaCl: 0.85\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5.66\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"3\"\u003e\u003csup\u003e*1\u003c/sup\u003e Peracetic acid\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003csup\u003e*2\u003c/sup\u003e Antiformin concentration (3\u0026ndash;6% per product information) is calculated using the median value of 4.5%.\u003c/p\u003e\u003cp\u003e\u003cb\u003eEvaluation of the efficacy against\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003ein infected root canals\u003c/b\u003e\u003c/p\u003e\n\u003ch3\u003ePreparation of extracted teeth\u003c/h3\u003e\n\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eSingle-rooted extracted human permanent teeth were prepared, and to confirm that they had single root canals, radiographs were obtained in the mesial and distal directions (Hatela instant film ISO speed D, Instant DQE; HANSHIN TECHNICAL LABORATORY, Hyogo, Japan). After autoclave sterilization, crowns were removed at 9 mm from the apex using an Isomet low-speed saw (BUEHLER, Lake Bluff, IL, USA). The root canals were filled with saline solution, and the working length was set to 1 mm shorter than the canal length. The canals were then enlarged to size #40/10 using Nickel-Titanium (Ni-Ti) rotary files in M7 mode (Endo Wave, MORITA, Osaka/Tokyo, Japan). After preparing the extraction site, the tips of 27G soft needles (NIPRO, Osaka, Japan) were set to 1 mm short of the working length, and irrigation was performed. First, 1 mL of EDTA (Smearclen, NISHIKA, Yamaguchi, Japan) was used for rinsing for 30 s, and ultrasonic irrigation was performed for 1 min using a SUPRASSON P-max+ (ACTEON EQUIPMENT [SATELEC], Merignac, FRANCE) in E1 mode with Irisafe files (HAKUSUI, Osaka, Japan) to remove the smear layers. This procedure was performed twice and subsequently repeated twice using Antiformin. In addition, the canals were rinsed with 0.1 mL dental etching agent (Green Activator, SUNMEDICAL, Shiga, Japan) for 30 s, followed by ultrasonic irrigation for 1 min. Subsequently, the canals were rinsed with 1 mL of 0.85% sodium chloride (FUJIFILM Wako Chemicals) for 30 s. Holes were made in the caps of 5 mL tubes using straight steel round burs ISO 027 (DENTSPLY, MAILLEFER, Ecublens, Canton de Vaud, Switzerland). The extracted teeth were coated at the apex and around the root using a self-curing resin (Provinice Fast; SHOFU, Kyoto, Japan). Subsequently, four horizontal lines were made on the side of each tube using straight fissure burs (DENTSPLY), which were mounted with self-curing resin, and autoclave sterilization was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003ePreparation of E. faecalis-infected root canals\u003c/h2\u003e\u003cp\u003e\u003cem\u003eE. faecalis\u003c/em\u003e bacterial suspension was adjusted to an OD\u003csub\u003e600\u003c/sub\u003e of 0.05, and 4.5 mL was added to 5 mL tubes in which extracted teeth had been mounted with self-curing resin. The tubes were then incubated at 37\u0026deg;C under 5% CO₂ for 7 days. To prevent air bubbles from entering the root canal, 20 \u0026micro;L of the bacterial suspension was initially introduced into the canal before adding the remainder. After culturing \u003cem\u003eE. faecalis\u003c/em\u003e for 7 days to allow biofilm formation, the self-curing resin blocks with teeth were removed from the 5 mL tubes. They were then placed on utility wax that had been disinfected with 70% ethanol, with the coronal side of the root canal facing downward. Each side of the root was rinsed with 1 mL of Antiformin and left for 2 min to disinfect the bacteria on the external surface. Subsequently, both sides of the extracted teeth were rinsed with 0.85% saline solution to remove Antiformin, followed by further disinfection with UV light irradiation (wavelength: 254 nm) for 5 min, after which the teeth were mounted in 5 mL tubes with utility wax. The extracted teeth were randomly divided into groups, and root canal irrigation was performed with each disinfectant. Using a 27-gauge side-vented soft needle bent 1 mm short of the working length, 2 mL of the solution was flushed into the root canal over 2 min while the needle tip was moved gently up and down. After 2 min, 1 mL of 0.85% saline solution was flushed into the root canal for 1 min to rinse out the irrigant. The concentrations of the irrigants were determined based on the results of antibacterial efficacy and cytotoxicity evaluations. A non-irrigated group served as the control group. In both the control and experimental groups, the bacterial suspension remaining within the root canal was absorbed and removed using a paper point; however, irrigation was applied only to the experimental groups.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eScanning electron microscopy (SEM) observation\u003c/h3\u003e\n\u003cp\u003eSEM images were obtained prior to \u003cem\u003eE. faecalis\u003c/em\u003e inoculation, after \u003cem\u003eE. faecalis\u003c/em\u003e biofilm formation, and after root canal irrigation. Specimens were fixed in 1% glutaraldehyde (Nacalai Tesque), followed by 2% osmium tetroxide (VIII) solution (FUJIFILM Wako Chemicals). After dehydration through a graded series of acetone (50%, 70%, 90%, 95%, and absolute; KANTO-CHEMICAL, Tokyo, Japan), notches were made using an isomet saw. A flat-head screwdriver was inserted into the notches, and the specimens were split along the tooth axis by striking them with a hammer. After exchanging with t-butanol (FUJIFILM Wako Chemicals), the specimens were freeze-dried using a freeze dryer (EIKO, Tokyo, Japan). A platinum coating was then applied using a magnetron sputter coater MSP-1S (VACUUM DEVICE, Ibaraki, Japan; 40\u0026ndash;45 mA, 1 min pre-evacuation, 30 s coating), and the middle portion of the root canal walls was observed using SEM (S-4300/HITACHI, Tokyo, Japan).\u003c/p\u003e\n\u003ch3\u003eDNA Extraction from E. faecalis Biofilms on Root Canal Walls\u003c/h3\u003e\n\u003cp\u003eAfter root canal irrigation, the Ni-Ti file Endo Wave Lax (reverse taper #50, MORITA, Osaka, Japan) was advanced to approximately the working length in the M4 mode, followed by one complete circumferential filing. The root canal was not filled with solution during this step. A 50 mL tube was prefilled with 20 mL of 0.85% saline, and the Endo Wave Lax carrying dentin debris was placed into the tube. In addition, the extracted tooth root canal was filled with enough of the same saline to cover the canal, and this solution was then added to the 50 mL tube. The tubes were sealed with Parafilm (Amcor, Zurich, Switzerland). Ultrasonic treatment was performed for 10 min using an ultrasonic device (BRANSONIC 2510 J-DTH; 42 kHz, BRANSON, Danbury, CT, USA), followed by vortexing at 3,000 rpm for 1 min. To concentrate the sample 20-fold, 10 mL was collected and centrifuged at 10,000 \u0026times; g for 20 min at 4\u0026deg;C (MX-305, TOMY, Tokyo, Japan). The supernatant was discarded, and 500 \u0026micro;L of phosphate buffered saline (PBS) was added. The PBS solution was transferred to a 1.5 mL tube and centrifuged at 10,000 \u0026times; g for 1 min at 4\u0026deg;C. DNA was extracted from the resulting pellet using a DNeasy UltraClean Microbial Kit (QIAGEN, Hilden, Germany). Nucleic acid concentration was measured with a NanoDrop (Thermo Fisher Scientific), and the DNA was stored at \u0026minus;\u0026thinsp;30\u0026deg;C.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eQuantitative polymerase chain reaction (qPCR) assay\u003c/h2\u003e\u003cp\u003eReactions were performed using the AriaMx Real-Time PCR System (Agilent Technologies, Santa Clara, CA, USA). PCR reagents were prepared so that each well contained 3.8 \u0026micro;L of RNase-free water, non-DEPC (BDL, Tokyo, Japan), 0.1 \u0026micro;L each of forward primer (10 \u0026micro;M, 5\u0026rsquo;-CGCTTCTTTCCTCCCGAGT-3\u0026rsquo;) and reverse primer (10 \u0026micro;M, 5\u0026rsquo;-GCCATGCGGCATAAACTG-3\u0026rsquo;), and 5 \u0026micro;L of SYBR Green qPCR Master Mix (Thermo Fisher Scientific). To each well, 1 \u0026micro;L of the extracted DNA was added. As a control, a well containing 1 \u0026micro;L of RNase-free water, non-DEPC instead of DNA was also prepared. A standard curve relating bacterial counts (colony forming units, CFU) to Cq values was constructed using a pure culture of \u003cem\u003eE. faecalis\u003c/em\u003e (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The CFU of the bacterial suspension were determined by spotting the culture onto agar plates and counting the resulting colonies. The obtained standard curve was used to calculate the number of CFU remaining after drug treatment. The thermal cycling conditions were as follows: an initial denaturation at 95\u0026deg;C for 10 min, followed by 40 cycles of 95\u0026deg;C for 15 s and 60\u0026deg;C for 1 min. Subsequently, a melting curve analysis was performed with steps of 95\u0026deg;C for 30 s, 65\u0026deg;C for 30 s, and 95\u0026deg;C for 30 s to confirm the specificity of the \u003cem\u003eE. faecalis\u003c/em\u003e genome [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eStatistical analyses were performed using EZR (R Commander version 2.9-1, R version 4.3.1, Jichi Medical University Saitama Medical Center, Saitama, Japan). All data were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). The MIC, MBC, MBIC, and CCK-8 assay data were analyzed using one-way analysis of variance, followed by Tukey\u0026rsquo;s post-hoc test. qPCR data were evaluated using the Kruskal\u0026ndash;Wallis test, followed by the Steel\u0026ndash;Dwass post-hoc test.\u003c/p\u003e\u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of antibacterial activity against oral pathogenic bacteria\u003c/h2\u003e\u003cp\u003eFigures \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e present the MIC and MBIC, respectively, with the pink frame indicating the respective values in each figure. Growth was inhibited when the concentration of PAA contained in Actril was at least 4.7\u0026times;10⁻⁴% for \u003cem\u003eE. faecalis\u003c/em\u003e, 9.4\u0026times;10⁻⁴% for \u003cem\u003eS. mutans\u003c/em\u003e, and 2.3\u0026times;10⁻⁴% for \u003cem\u003eP. gingivalis\u003c/em\u003e. At the same concentrations, biofilm formation was also inhibited in \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eS. mutans\u003c/em\u003e. Furthermore, in \u003cem\u003eP. gingivalis\u003c/em\u003e, a tendency toward inhibition of biofilm formation was observed at the same concentration. In \u003cem\u003eE. faecalis\u003c/em\u003e, growth was inhibited when the NaClO concentration in Antiformin was at least 0.14%, in \u003cem\u003eS. mutans\u003c/em\u003e at 0.28%, and in \u003cem\u003eP. gingivalis\u003c/em\u003e at 0.018%. Biofilm formation was also inhibited at the same concentrations.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the results of the MBC. In \u003cem\u003eE. faecalis\u003c/em\u003e and \u003cem\u003eS. mutans\u003c/em\u003e, the detection of viable cells was suppressed when the PAA concentration in Actril was at least 9.4 \u0026times;10⁻⁴%, and in \u003cem\u003eP. gingivalis\u003c/em\u003e when it was at least 2.3\u0026times;10⁻⁴%. In contrast, the detection of viable cells was suppressed at NaClO concentrations of 0.14% for \u003cem\u003eE. faecalis\u003c/em\u003e, 0.28% for \u003cem\u003eS. mutans\u003c/em\u003e, and 0.035% for \u003cem\u003eP. gingivalis\u003c/em\u003e in Antiformin.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eMBC of PAA and NaClO against oral pathogens\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBacteria\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePAA (%)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eNaClO (%)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eEnterococcus faecalis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.14\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003eStreptococcus mutans\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00094\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.28\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e\u003cb\u003ePorphyromonas gingivalis\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00023\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.035\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eMBC (%) of PAA and NaClO against \u003cem\u003eE. faecalis\u003c/em\u003e, \u003cem\u003eS. mutans\u003c/em\u003e, and \u003cem\u003eP. gingivalis\u003c/em\u003e. Concentrations are calculated based on the dilution ratios of PAA in Actril and NaClO in Antiformin. MBC, minimum bactericidal concentration; NaClO, sodium hypochlorite; PAA, peracetic acid.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eEvaluation of cytotoxicity toward periodontal tissue-related cells\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e shows the results of the CCK-8 assay. The red lines represent the MBC of each irrigant against \u003cem\u003eE. faecalis.\u003c/em\u003e Cytotoxic effects on cellular metabolic activity were observed in GE-1 cells when the PAA concentration in Actril reached 7.5\u0026times;10⁻\u0026sup3;%, and in HPLF cells when it reached 1.9\u0026times;10⁻\u0026sup3;%, both of which were higher than the MBC against \u003cem\u003eE. faecalis\u003c/em\u003e. In contrast, cytotoxicity appeared in Antiformin at NaClO concentrations of 0.14% or higher for GE-1 cells, and 8.8\u0026times;10⁻\u0026sup3;% or higher for HPLF cells, which were lower than or comparable to the MBC against \u003cem\u003eE. faecalis.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLive/dead staining for\u003c/b\u003e \u003cb\u003eE. faecalis\u003c/b\u003e \u003cb\u003ein biofilm\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e presents the results of live/dead staining. In the control and saline groups, the biofilms predominantly exhibited green fluorescence with minimal red fluorescence indicating dead cells, suggesting that most of the bacterial population remained viable. In the Actril group, the proportion of dead cells was markedly higher than that in the control and saline groups. Nevertheless, the effect of Actril on biofilm bacteria was variable, and in some instances, its efficacy was limited (Supplementary Fig.\u0026nbsp;1). No live or dead cells were detected in the Antiformin group.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eSEM analysis of root canal wall after irrigation\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e shows SEM images of the central root canal wall at 1,000\u0026times; magnification. Comparison of the root canal before biofilm formation under sterile conditions (A) with that after 7 days of biofilm cultivation (B) revealed that the root canal wall was covered with biofilm, and the dentinal tubule orifices were occluded following the 7-day culture. Panels (C), (D), and (E) show the root canal walls after 2 min of syringe irrigation with 9.4\u0026times;10⁻⁴% peracetic acid Actril, undiluted Antiformin, and 0.85% saline, respectively. These results indicate that Actril removed the biofilm from the root canal wall more effectively than Antiformin or saline, allowing the dentinal tubule orifices to become visible again.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eqPCR assay\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e shows the results of the qPCR assay used to quantify the number of bacteria remaining in the root canal walls. No significant difference was observed between the Actril and Antiformin groups; however, both treatments significantly reduced CFU compared with the non-irrigated group (control).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003ePAA-based disinfectants\u0026mdash;such as Actril\u0026mdash;are commonly employed for the disinfection of medical devices, including dialysis equipment, and for cleanroom sanitation; however, they have not yet been applied as disinfectants in the oral cavity. Actril contains PAA, hydrogen peroxide, and acetic acid at defined concentrations that may influence the persistence of its antibacterial effects. Nevertheless, most studies have utilized PAA solutions rather than commercial formulations, often at relatively high concentrations compared with those of Actril. Recent in vitro studies have reported that 1\u0026ndash;2% PAA exhibits antibacterial activity against \u003cem\u003eE. faecalis\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, 2.25% PAA exhibited effects comparable to that of 17% EDTA in the removal of the smear layer from root canal walls, suggesting the potential to act on both biofilms and the smear layer with a single agent [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, some reports have indicated that even high concentrations of PAA show only limited bactericidal efficacy against mature biofilms, with the effectiveness depending on factors such as concentration, exposure time, and bacterial species [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Collectively, these findings suggest that, although PAA is a promising root canal irrigant, its antibacterial activity, safety, and reproducibility in clinically relevant models remain unclear. Therefore, this study focused on evaluating the inhibitory effects of Actril on oral pathogenic bacteria and its potential for biofilm removal from infected root canals.\u003c/p\u003e\u003cp\u003eIn this study, the PAA present in Actril exhibited MIC, MBC, and MBIC values approximately 1/100 to 1/1000 of those of NaClO, the active ingredient in Antiformin, demonstrating a potent inhibitory effect, even at low concentrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These results suggest that Actril may be more effective than Antiformin, possibly due, at least in part, to differences in the duration of action between PAA and NaClO. To measure the MIC, MBC, and MBIC, the disinfectants were allowed to act for more than 24 h. During this period, the effectiveness of the active ingredients may gradually decrease; however, PAA is considered to have a longer duration of action than NaClO. This is because Actril contains hydrogen peroxide and acetic acid, which allows PAA to be regenerated even after decomposition.\u003c/p\u003e\u003cp\u003eNext, the cytotoxicity of Actril on periodontal tissue-related cells was evaluated and compared with that of Antiformin. In this study, Actril exerted a smaller effect on cellular metabolic activity than that of Antiformin (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Previous studies have reported that when 1% PAA and 2.5% NaClO were diluted and applied to the mouse fibroblast cell line L929 for 10 min, PAA resulted in lower cell viability than NaClO at dilutions below 0.03%, whereas no significant difference was observed at dilutions of 0.03% or higher [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Another study examined the cytotoxicity of disinfectants on FG11 and FG15 fibroblasts after exposure for up to 4 h. It was reported that 2.5% NaClO showed no cytotoxicity only at a 0.01% dilution, whereas 1% PAA showed no cytotoxicity not only at 0.01% but also at a 0.05% dilution [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Based on these findings, PAA at a concentration of 5.0\u0026times;10⁻⁴% showed no cytotoxicity in the previous study, whereas in this study, even at a higher PAA concentration of 9.4\u0026times;10⁻⁴%, the impact on GE-1 and HPLF cells was minimal.\u003c/p\u003e\u003cp\u003eThe results presented in Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e indicate that PAA at a concentration of 2.3\u0026ndash;9.4\u0026times; 10⁻⁴% exerted inhibitory effects on oral pathogenic bacteria, which is approximately one-tenth of the concentration that results in cytotoxicity in cells. The concentrations of NaClO that exhibited inhibitory effects on oral pathogenic bacteria ranged from 0.018% to 0.28%, which were comparable to the concentrations that induced cytotoxicity in cells. At concentrations effective against oral pathogenic bacteria, Antiformin was shown to be cytotoxic to periodontal tissue-related cells, whereas Actril, at a PAA concentration of 9.4\u0026times;10⁻⁴%, was suggested to be effective against oral pathogenic bacteria while exhibiting low cytotoxicity.\u003c/p\u003e\u003cp\u003eActril at this concentration was tested for its effect on \u003cem\u003eE. faecalis\u003c/em\u003e biofilms using live/dead staining. Notably, Actril effectively exerted a bactericidal effect on bacteria within the biofilm (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). It is thought that the bactericidal components of Actril penetrate the biofilm and exert their effects. However, as shown in Supplementary Fig.\u0026nbsp;1, Actril did not exhibit consistent bactericidal effects. This may be due to the PAA concentration. The PAA concentration used in this study, 9.4 \u0026times; 10⁻⁴%, may represent a threshold concentration: if it can penetrate the biofilm, it exhibits a bactericidal effect, whereas if penetration is not achieved, no bactericidal effect is observed. In the Antiformin-washed group, neither live nor dead bacteria were observed, suggesting that the biofilms were entirely dispersed. One possible reason is the surface modification effect of NaClO on polystyrene (PS) [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. The biofilm on the PS substrate of the \u0026micro;-96 well plate used in this study may have largely detached because NaClO treatment chlorinated and oxidized the substrate surface, roughening it (altering the surface energy) and thereby reducing adhesion.\u003c/p\u003e\u003cp\u003eAs live/dead staining suggested that Actril at a PAA concentration of 9.4\u0026times;10⁻⁴% exerts a bactericidal effect on \u003cem\u003eE. faecalis\u003c/em\u003e within biofilms, we next evaluated its cleaning efficacy by forming \u003cem\u003eE. faecalis\u003c/em\u003e biofilms on extracted human teeth. To simulate the clinical conditions, the extracted teeth were not sectioned into blocks but were used in their original root canal morphology, and the experiment was conducted under root canal irrigation. This study suggested that Actril, at a PAA concentration of 9.4\u0026times;10⁻⁴%, had a superior irrigation effect on biofilms formed on the root canal walls compared with that of Antiformin (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). The NaClO solution is extensively consumed in the presence of organic matter [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and exhibits limited penetration into the biofilm matrix [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In contrast, PAA was effective even in the presence of extracellular polysaccharides produced by \u003cem\u003eE. faecalis\u003c/em\u003e, and its acidic properties are thought to decalcify calcium on the root canal wall surface, thereby detaching the biofilm. A previous study reported that 3% NaClO solution could remove patient-derived bacterial biofilms formed on root canal dentin [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], which is inconsistent with our results. The extended application time of 15 min and the larger contact area in that study were considered to have allowed for greater interaction between NaClO and the biofilm. Because the application time of irrigants in clinical practice is generally limited to 1\u0026ndash;2 min, the marked irrigation efficacy of Actril within 2 min, as revealed in this study, is considered highly valuable.\u003c/p\u003e\u003cp\u003eThe qPCR analysis revealed no significant differences in bacterial counts between the Actril and Antiformin irrigation groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Nevertheless, a reduction in bacteria was also observed in the saline irrigation group compared with the control, indicating that the observed effect reflects not only the antimicrobial activity of the irrigants but also the physical effect of fluid movement within the root canal. During clinical root canal irrigation, irrigants also flow continuously through the canal; thus, these results are thought to more closely reflect clinical conditions and suggest that Actril exhibits an effect comparable to that of Antiformin. While SEM images suggested a superior irrigation effect of Actril compared with Antiformin, the lack of a significant difference in the qPCR results was thought to be due to the sample collection method. Previous literature has also demonstrated that treatment with 0.6% NaClO for 2 h still leaves viable bacteria, as shown by live/dead staining [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, samples were centrifuged to remove lysed bacteria and their intracellular contents prior to qPCR analysis. Therefore, the genomes detected by qPCR were considered to originate solely from viable bacteria. That is, although biofilms were collected using Ni-Ti files after irrigation in this study, some bacteria that remained alive following Antiformin irrigation may have been killed during collection with the Ni-Ti files, resulting in the number of recovered viable bacteria being comparable to that observed in the Actril group. However, because the collection of bacteria using files after irrigation is a standard method [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], a significant difference between Actril and Antiformin might be detectable if the bacterial load in the biofilm is increased. Furthermore, the large error bars may have been another factor contributing to the absence of significant differences. This variability is largely attributable to individual differences among the extracted human teeth [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Differences in root canal morphology [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e] and dentin hardness [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] may result in variations in biofilm formation.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn this study, the PAA-based disinfectant\u0026mdash;Actril\u0026mdash;exhibited antibacterial activity against oral pathogenic bacteria at lower concentrations than that of the NaClO solution\u0026mdash;Antiformin\u0026mdash;while showing lower cytotoxicity toward cells associated with periodontal tissues. Moreover, at concentrations that are both effective against oral pathogenic bacteria and non-cytotoxic to periodontal tissue-related cells, Actril demonstrated superior irrigation efficacy in an \u003cem\u003eE. faecalis\u003c/em\u003e-infected root canal model using extracted human teeth compared with the NaClO solution. Therefore, Actril has the potential to serve as a safe and effective root canal irrigant compared with the NaClO solution. However, this study did not fully address certain aspects of clinical relevance such as the efficacy of the disinfectant in the apical region, its effects on bacteria within the dentinal tubules, or its tissue-dissolving ability in the pulp. Future studies are warranted to evaluate its safety in vivo and verify its effectiveness through clinical trials.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eGE-1, mouse-derived gingival epithelial cells\u003c/p\u003e\n\u003cp\u003eHPLF, human periodontal ligament fibroblasts\u003c/p\u003e\n\u003cp\u003eMIC, minimum inhibitory concentration\u003c/p\u003e\n\u003cp\u003eMBC, minimum bactericidal concentration\u003c/p\u003e\n\u003cp\u003eMBIC, minimum biofilm inhibitory concentration\u003c/p\u003e\n\u003cp\u003eNaClO, sodium hypochlorite\u003c/p\u003e\n\u003cp\u003ePAA, peracetic acid\u003c/p\u003e\n\u003cp\u003eqPCR, quantitative polymerase chain reaction\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Public University Corporation Kyushu Dental University Research Ethics Committee (approval number: 24-13).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe committee waived the need for written informed consent because the study was retrospective in nature and used anonymized extracted human teeth that had been obtained following routine dental treatment and were not removed for research purposes.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe procedures conducted in this study were carried out in accordance with the ethical guidelines of the institution and domestic regulations, as well as the principles of the 1964 Declaration of Helsinki and its later amendments.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRadiographic images other than those shown in Figure 1 (A) are available from the corresponding author upon reasonable request. All other data supporting the findings of this study are included in the main manuscript and the supplementary files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was partially supported by the JSPS KAKENHI (grant number 24K12888).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRT: Conceptualization, methodology, investigation, original draft writing, manuscript review and editing. RY: Conceptualization, methodology, data curation, supervision, manuscript review and editing. AW: Pilot experiments, supervision, manuscript review and editing. YY: Supervision. WA: Methodology and supervision. CK: Conceptualization and supervision. All authors contributed to data collection and interpretation and critically reviewed the manuscript. All\u0026nbsp;authors have read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank Dr. Takashi Toyono for their guidance on using the SEM.\u003cem\u003eWe would like to thank Editage (\u003c/em\u003e\u003ca href=\"https://www.editage.jp/\"\u003e\u003cem\u003ewww.editage.jp\u003c/em\u003e\u003c/a\u003e\u003cem\u003e) for English language editing.\u003c/em\u003e\u003cbr\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVersiani MA, Martins J, Ordinola-Zapata R. Anatomical complexities affecting root canal preparation: a narrative review. Aust Dent J. 2023;68 Suppl 1:S5-S23.\u003c/li\u003e\n\u003cli\u003eBabeer A, Bukhari S, Alrehaili R, Karabucak B, Koo H. Microrobotics in endodontics: A perspective. Int Endod J. 2024;57(7):861-71.\u003c/li\u003e\n\u003cli\u003eSwimberghe RCD, Coenye T, De Moor RJG, Meire MA. Biofilm model systems for root canal disinfection: a literature review. Int Endod J. 2019;52(5):604-28.\u003c/li\u003e\n\u003cli\u003eCai C, Chen X, Li Y, Jiang Q. Advances in the role of sodium hypochlorite irrigant in chemical preparation of root canal treatment. Biomed Research International. 2023;2023(1):8858283.\u003c/li\u003e\n\u003cli\u003eCho-Kee D, Basrani BR, Vera J, Ordinola-Zapata R, Aguilar RR. Sodium Hypochlorite Accidents: A Retrospective case-series analysis of CBCT Imaging and Clinician Surveys. J Endod. 2025.\u003c/li\u003e\n\u003cli\u003eH\u0026Uuml;LSMANN M, R\u0026Ouml;DIG T, Nordmeyer S. Complications during root canal irrigation. Endodontic Topics. 2007;16(1):27-63.\u003c/li\u003e\n\u003cli\u003eZhang N, Guo J, Liu L, Wu H, Gu J. Study on the Efficacy of Peracetic Acid Disinfectant (Type III) on Gastrointestinal Endoscopy Disinfection. Surg Laparosc Endosc Percutan Tech. 2021;31(4):395-8.\u003c/li\u003e\n\u003cli\u003eKitis M. Disinfection of wastewater with peracetic acid: a review. Environ Int. 2004;30(1):47-55.\u003c/li\u003e\n\u003cli\u003eStearns R, Freshour A, Shen CJJoA, Research F. Literature review for applying peroxyacetic acid and/or hydrogen peroxide to control foodborne pathogens on food products. J Agric Food Res. 2022;10:100442.\u003c/li\u003e\n\u003cli\u003eButucel E, Balta I, Ahmadi M, Dumitrescu G, Morariu F, Pet I, et al. Biocides as Biomedicines against Foodborne Pathogenic Bacteria. Biomedicines. 2022;10(2).\u003c/li\u003e\n\u003cli\u003eMcDonnell G, Russell AD. Antiseptics and disinfectants: activity, action, and resistance. Clin Microbiol Rev. 1999;12(1):147-79.\u003c/li\u003e\n\u003cli\u003eChino T, Nukui Y, Morishita Y, Moriya K. Morphological bactericidal fast-acting effects of peracetic acid, a high-level disinfectant, against Staphylococcus aureus and Pseudomonas aeruginosa biofilms in tubing. Antimicrob Resist Infect Control. 2017;6:122.\u003c/li\u003e\n\u003cli\u003eSingh A, Kaur H, Sawale M, Ozadali F, Benyathiar P, Mishra DKJL. Bacillus cereus spore inactivation by peracetic acid for aseptic packaging application. LWT. 2025;224:117811.\u003c/li\u003e\n\u003cli\u003eLedwoch K, Maillard JY. Candida auris Dry Surface Biofilm (DSB) for Disinfectant Efficacy Testing. Materials (Basel). 2018;12(1).\u003c/li\u003e\n\u003cli\u003eOettler MJ, Conraths FJ, Roesler U, Reiche S, Homeier-Bachmann T, Denzin N. Efficiency of Virucidal Disinfectants on Wood Surfaces in Animal Husbandry. Microorganisms. 2024;12(5).\u003c/li\u003e\n\u003cli\u003eKapes T, Quinn C, Cragun AE, House T, Nims RW, Zhou SS. Differing Susceptibilities to Certain Microbicidal Chemistries among Three Representative Enveloped Viruses. Microorganisms. 2024;12(3).\u003c/li\u003e\n\u003cli\u003eBarbosa-Ribeiro M, De-Jesus-Soares A, Zaia AA, Ferraz CC, Almeida JF, Gomes BP. Antimicrobial Susceptibility and Characterization of Virulence Genes of \u003cem\u003eEnterococcus faecalis\u003c/em\u003e Isolates from Teeth with Failure of the Endodontic Treatment. J Endod. 2016;42(7):1022-8.\u003c/li\u003e\n\u003cli\u003ePinheiro ET, Candeiro GT, Teixeira SR, Shin RC, Prado LC, Gavini G, et al. RNA-based Assay Demonstrated \u003cem\u003eEnterococcus faecalis\u003c/em\u003e Metabolic Activity after Chemomechanical Procedures. J Endod. 2015;41(9):1441-4.\u003c/li\u003e\n\u003cli\u003eBriseno-Marroquin B, Callaway A, Shalamzari NG, Wolf TG. Antibacterial efficacy of peracetic acid in comparison with sodium hypochlorite or chlorhexidine against \u003cem\u003eEnterococcus faecalis\u003c/em\u003e and \u003cem\u003eParvimonas micra\u003c/em\u003e. BMC Oral Health. 2022;22(1):119.\u003c/li\u003e\n\u003cli\u003eElsamra AH, Darrag AM, Ghoneim WMJTDJ. Efficacy of different chelating agents in smear layer removal. Tanta dent J. 2023;20(1):27-33.\u003c/li\u003e\n\u003cli\u003eBrandao-Neto DO, Mello JVZ, Marceliano-Alves MFV, Carvalho Coutinho TM, Marceliano EFV, Galhardi MPW, et al. Final Endodontic Irrigation with 2% Peracetic Acid: Antimicrobial Activity and Cytotoxicity. Eur J Dent. 2021;15(3):533-8.\u003c/li\u003e\n\u003cli\u003eLee SHI, Cappato LP, Corassin CH, Cruz AG, Oliveira CAF. Effect of peracetic acid on biofilms formed by \u003cem\u003eStaphylococcus aureus \u003c/em\u003eand \u003cem\u003eListeria monocytogenes \u003c/em\u003eisolated from dairy plants. J Dairy Sci. 2016;99(3):2384-90.\u003c/li\u003e\n\u003cli\u003eViola KS, Rodrigues EM, Tanomaru-Filho M, Carlos IZ, Ramos SG, Guerreiro-Tanomaru JM, et al. Cytotoxicity of peracetic acid: evaluation of effects on metabolism, structure and cell death. Int Endod J. 2018;51 Suppl 4:e264-e77.\u003c/li\u003e\n\u003cli\u003eTeixeira PA, Coelho MS, Kato AS, Fontana CE, Bueno CE, Pedro-Rocha DG. Cytotoxicity assessment of 1% peracetic acid, 2.5% sodium hypochlorite and 17% EDTA on FG11 and FG15 human fibroblasts. Acta Odontol Latinoam. 2018;31(1):11-5.\u003c/li\u003e\n\u003cli\u003eGuo C, Zou Q, Wang J, Wang H, Chen S, Zhong Y. Application of surface modification using sodium hypochlorite for helping flotation separation of acrylonitrile-butadiene-styrene and polystyrene plastics of WEEE. Waste Manag. 2018;82:167-76.\u003c/li\u003e\n\u003cli\u003eZhang C, Brown PJB, Hu Z. Higher functionality of bacterial plasmid DNA in water after peracetic acid disinfection compared with chlorination. Sci Total Environ. 2019;685:419-27.\u003c/li\u003e\n\u003cli\u003eDe Beer D, Srinivasan R, Stewart PS. Direct measurement of chlorine penetration into biofilms during disinfection. Appl Environ Microbiol. 1994;60(12):4339-44.\u003c/li\u003e\n\u003cli\u003eClegg MS, Vertucci FJ, Walker C, Belanger M, Britto LR. The effect of exposure to irrigant solutions on apical dentin biofilms in vitro. J Endod. 2006;32(5):434-7.\u003c/li\u003e\n\u003cli\u003eRosen E, Tsesis I, Elbahary S, Storzi N, Kolodkin-Gal I. Eradication of \u003cem\u003eEnterococcus faecalis\u003c/em\u003e Biofilms on Human Dentin. Front Microbiol. 2016;7:2055.\u003c/li\u003e\n\u003cli\u003eKranz S, Guellmar A, Braeutigam F, Tonndorf-Martini S, Heyder M, Reise M, et al. Antibacterial Effect of Endodontic Disinfections on \u003cem\u003eEnterococcus Faecalis\u003c/em\u003e in Dental Root Canals-An In-Vitro Model Study. Materials (Basel). 2021;14(9).\u003c/li\u003e\n\u003cli\u003eShimaoka T, Maezono H, Ono S, Asahi Y, Kawanishi Y, Klanliang K, et al. Application of on-demand aqueous chlorine dioxide solution for non-surgical root canal treatment. Sci Rep. 2025;15(1):36215.\u003c/li\u003e\n\u003cli\u003eOzdemir HO, Buzoglu HD, Calt S, Stabholz A, Steinberg D. Effect of ethylenediaminetetraacetic acid and sodium hypochlorite irrigation on\u003cem\u003e Enterococcus faecalis\u003c/em\u003e biofilm colonization in young and old human root canal dentin: in vitro study. J Endod. 2010;36(5):842-6.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Peracetic acid-based disinfectant, Biofilm, Enterococcus faecalis, Root canal treatment","lastPublishedDoi":"10.21203/rs.3.rs-7911128/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7911128/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cb\u003eBackground\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSodium hypochlorite (NaClO) is currently the most widely used irrigant in root canal treatment; however, it exerts cytotoxic effects on the periapical tissues. To reduce the biological risk, we aimed to investigate the potential usefulness of a peracetic acid (PAA)-based disinfectant as an alternative to root canal irrigants.\u003c/p\u003e\u003cp\u003e\u003cb\u003eMethods\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe antibacterial activity of the PAA-based disinfectant, Actril (MEDIVATORS, Minneapolis, MN, USA)\u0026mdash;against oral pathogenic bacteria\u0026mdash;was evaluated by determining its minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and minimum biofilm inhibitory concentration (MBIC). Cytotoxicity toward periodontal tissue-related cells was assessed using a Cell Counting Kit-8 assay. The bactericidal effect against \u003cem\u003eEnterococcus faecalis\u003c/em\u003e biofilms was further examined using live/dead staining. To simulate the clinical environment, \u003cem\u003eE. faecalis\u003c/em\u003e biofilms were established on extracted human teeth, and the irrigation efficacy of Actril in root canals was evaluated using scanning electron microscopy (SEM) and quantitative polymerase chain reaction (qPCR). Data were analyzed using one-way analysis of variance followed by Tukey\u0026rsquo;s test or the Kruskal\u0026ndash;Wallis test with Steel\u0026ndash;Dwass post-hoc comparisons, as appropriate.\u003c/p\u003e\u003cp\u003e\u003cb\u003eResults\u003c/b\u003e\u003c/p\u003e\u003cp\u003eActril demonstrated strong antibacterial activity against oral pathogenic bacteria at a PAA concentration of 9.4\u0026times;10⁻⁴%, which was significantly lower than the effective concentration of NaClO, while showing reduced cytotoxicity against periodontal tissue-related cells. Live/dead staining revealed that Actril exerted bactericidal effects against \u003cem\u003eE. faecalis\u003c/em\u003e within the biofilms. Furthermore, SEM observations demonstrated the removal of biofilm structures from the root canal surface, and qPCR analysis confirmed a significant reduction in the number of viable \u003cem\u003eE. faecalis\u003c/em\u003e cells. These findings indicate that Actril is not only effective against pathogenic microorganisms but also exhibits superior biocompatibility compared with NaClO solution.\u003c/p\u003e\u003cp\u003e\u003cb\u003eConclusions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe PAA-based disinfectant shows promise as a safe and effective alternative root canal irrigant to conventional NaClO solutions.\u003c/p\u003e","manuscriptTitle":"Antimicrobial and biocompatible potential of peracetic acid as a novel root canal irrigant","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-11 11:20:18","doi":"10.21203/rs.3.rs-7911128/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f19a2f9c-de70-47a9-a303-3bef2d8758a1","owner":[],"postedDate":"November 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-22T11:38:13+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-11 11:20:18","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7911128","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7911128","identity":"rs-7911128","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}