May 11, 2026
Evaluation of the Antibacterial Efficacy of SmearOFF and Sodium Hypochlorite Against Enterococcus faecalis: An In Vitro Study
- Ahmed Mohamed Adly Ali1,
- Sarah Samir Abouelenien2,
- Laila Zakria3
- 1Master degree candidate, Department of Endodontics, Faculty of Dentistry, Cairo University.;
- 2Assoc. Professor of Endodontics, Faculty of Dentistry, Cairo University.;
- 3Lecturer in Endodontics, Faculty of Dentistry, Cairo University.
- cairo university
Protocol Citation: Ahmed Mohamed Adly Ali, Sarah Samir Abouelenien, Laila Zakria 2026. Evaluation of the Antibacterial Efficacy of SmearOFF and Sodium Hypochlorite Against Enterococcus faecalis: An In Vitro Study. protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl84yx7g2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: May 08, 2026
Last Modified: May 11, 2026
Protocol Integer ID: 316652
Keywords: antibacterial efficacy of smearoff, sodium hypochlorite against enterococcus faecali, in vitro study endodontic treatment, endodontic treatment, evaluation of the antibacterial efficacy, antibacterial activity, antibacterial efficacy, root canal system, pulp infection, current disinfection method, complicated shape of the root canal system, sodium hypochlorite as irrigant, ability of current disinfection method, effect of smearoff, organic components of the smear layer, enterococcus faecali, enhanced smear layer removal, detergent, greater dentin tubule clearance, smear layer removal, in vitro study, sodium hypochlorite, bacteria, future bacterial invasion, chlorhexidine, complete cleaning
Abstract
Endodontic treatment aims to remove pulp infection and stop future bacterial invasion. The ability of current disinfection methods to kill bacteria is limited. Another challenge is the complicated shape of the root canal system, which makes complete cleaning difficult Sodium hypochlorite (NaOCl) has been the gold standard irrigant due to its antibacterial and tissue-dissolving properties, However, it is cytotoxic, removes only organic components of the smear layer. SmearOFF was recently introduced as a single irrigant containing EDTA, chlorhexidine, and a detergent, that has superior chelating properties, resulting in enhanced smear layer removal and significantly greater dentin tubule clearance compared with traditional EDTA formulations, without precipitate formation when combined with NaOCl. Limited studies have focused on the effect of SmearOFF on smear layer removal. However, given the limited evidence regarding its antibacterial activity, further investigation is needed. Accordingly, the aim of current study is to assess the antibacterial efficacy of SmearOFF compared to sodium hypochlorite as irrigants against Enterococcus faecalis.
Guidelines
- Access Preparation: Access cavities will be prepared using a diamond round bur. The cavity walls will then be refined with a tapered cylindrical diamond bur.
- Canal Negotiation: K-files of sizes 10 and 15 will be used to negotiate each canal to the full working length until patency is achieved. The working length will be confirmed when the tip of the size 15 file is visible at the apical foramen.
- Canal Preparation: Each root canal will be prepared with rotary files and will be enlarged to size 40, taper 0.04 using the crown-down technique to the full working length, operated with Endo motor (speed 350 rpm, torque 2 N/cm). 3 ml sodium hypochlorite irrigation will be performed with a 30-gauge side-vented needle positioned 1 mm short of the working length between each file until total volume of irrigant will be 24 ml. Upon completion of instrumentation, a final rinse with 5 ml of distilled water will be performed, followed by drying of the canals with sterile paper points.
- All teeth will be stored in saline after preparation and will be autoclaved for 20 minutes at 121 °C.
- The prepared canals will be inoculated with Enterococcus faecalis biofilms under standardized laboratory conditions. A pure culture of E. faecalis will be cultivated in Brain Heart Infusion (BHI) broth and incubated at 37 °C for 24 hours. The teeth will then be immersed in the bacterial suspension and incubated for 21 days to allow mature biofilm formation, with the culture medium refreshed every 48 hours. This procedure will ensure uniform contamination of all specimens with mature biofilms prior to the application of the irrigant regimens.
- The specimens will be randomly allocated into three experimental groups, with 18 specimens in each group.
- Group A: Sodium Hypochlorite and EDTA: Irrigation will be performed with 5 ml of 5 % NaOCl. The solution will be delivered with a 30-gauge side-vented needle positioned 1 mm short of the working length. Activation will be carried out using ultrasonic activation device for 60 seconds, followed by 5 ml of 17% EDTA.
Materials
- Diamond round bur (Komet Dental, Lemgo, Germany)
- Tapered cylindrical diamond bur (Komet Dental, Lemgo, Germany)
- K-Files sizes 10 and 15 (MANI, Inc., Utsunomiya, Japan)
- Rotary files (Rogin Dental, Shenzhen, China)
- Endo motor (Wismy, Bondent Group, Shenzhen, China)
- Sodium hypochlorite (5%, JK Dental Vision, Mansoura, Egypt)
- 30-gauge side-vented needle
- Ultrasonic activation device (Ultra X, Changzhou, China)
- EDTA (17%, JK dental)
Safety warnings
Ethical approval was obtained in accordance with institutional guidelines and the Declaration of Helsinki.
Scientific background
Endodontic treatment is a complex process, but its main goal is always to remove pulp infection and stop future bacterial invasion. The ability of current disinfection methods to kill bacteria is limited. Another challenge is the complicated shape of the root canal system, which makes complete cleaning difficult [1].
There are several approaches to managing endodontic infection, but irrigation plays a central role in removing microorganisms from the root canal. It is an essential step for thorough canal cleaning and for eliminating bacterial toxins and biofilm, while also compensating for the limitations of instruments and anatomical challenges. Irrigation serves multiple purposes, including flushing out debris, lubricating canal walls, dissolving organic tissue, and providing antibacterial action [2].
Studies have shown that when instrumentation is performed without irrigation, root canals retain nearly 70% more debris compared to those that are irrigated. The mechanical and chemical actions of irrigants play a crucial role in canal cleaning and disinfection. Microbial infection within the root canal system is recognized as the primary cause of pulpal and periapical disease, and mature biofilms are particularly resistant to antimicrobial agents. Since current cleaning and shaping methods alone cannot achieve a bacteria-free canal, the use of effective chemical irrigants is essential for eliminating microorganisms and their toxins [2].
Enterococcus faecalis is a microorganism frequently identified in asymptomatic, persistent endodontic infections, with reported prevalence ranging from 24% to 77%. This persistence can be attributed to several survival and virulence mechanisms, including its ability to compete with other microorganisms, penetrate dentinal tubules, withstand nutritional deprivation, and survive chemo-mechanical instrumentation as well as intracanal medication, allowing it to reinfect previously treated canals. Given these characteristics, E. faecalis is considered one of the most resistant species in endodontic infections, making its eradication a critical challenge in root canal therapy [3].
Maintaining strict aseptic technique, enlarging apical preparation sizes, and using 2% chlorhexidine in combination with sodium hypochlorite are regarded as among the most effective strategies for eliminating Enterococcus faecalis within root canal systems [3].
Sodium hypochlorite (0.5–5.25%) has long been the gold standard irrigant in endodontics due to its potent antibacterial and tissue-dissolving properties. However, it is cytotoxic, removes only organic components of the smear layer. Moreover, NaOCl reduces dentin microhardness, alters mechanical properties, and causes irreversible erosion of dentin. These drawbacks highlighted the need for alternative irrigants that maintain antimicrobial efficacy while minimizing adverse effects on dentin and surrounding tissues [2].
Studies have shown that sodium hypochlorite alone results in incomplete eradication of root canal micro biota. SmearOFF is a recently introduced irrigant formulated with ethylenediaminetetraacetic acid (EDTA), chlorhexidine, and a detergent. [4]
Rationale
SmearOFF was recently introduced as a single irrigant that has superior chelating properties, resulting in enhanced smear layer removal and significantly greater dentin tubule clearance 27% more than that achieved with the leading 17% EDTA formulation and present at lower concentrations, making it less aggressive toward dentin [5][6]. Importantly, when combined with sodium hypochlorite (NaOCl), the solution did not produce precipitation [8]. Its improved calcium suspension capacity, low surface tension, and incorporation of chlorhexidine (CHX) further contribute to its efficacy and clinical applicability. This formulation may be compared to Dentsply Sirona QMix 2in1 Irrigating Solution [8]. It increases depth of sealer penetration into the dentinal tubules [12].
Limited studies have focused on the effect of SmearOFF on smear layer removal [5][6][8]. However, given the limited evidence regarding its antibacterial activity, further investigation is warranted. Accordingly, the aim of current study is to assess the antibacterial efficacy of SmearOFF compared to sodium hypochlorite as irrigants against Enterococcus faecalis.
Review of literature
Swati Srivastava (2019) compared the antibiofilm efficacy of 0.1% octenidine, SmearOFF, 1% alexidine, and 5.25% sodium hypochlorite against E. faecalis biofilm formed on 80 single-rooted human mandibular premolars. The samples were allocated into four experimental groups. Each group was treated for 10 minutes with 3 mL of one of the irrigants. All samples were evaluated for E. faecalis growth and the number of colony-forming units. It was concluded that 0.1% octenidine (OCT) exhibited the greatest antibiofilm activity against E. faecalis significantly stronger than all others. Both 1% alexidine (ALX) and 5.25% sodium hypochlorite (NaOCl) demonstrated no significant difference in efficacy against E. faecalis. In contrast, SmearOFF showed only significantly lower antibiofilm effects against this organism. [7]
Jaynit et al. (2022) evaluated the presence of aerobic and anaerobic bacterial species, including E. faecalis, F. nucleatum, Propionibacteria sp., and Actinomyces sp., at different stages of endodontic retreatment. Twenty-eight patients who fulfilled the eligibility criteria were selected for root canal retreatment and were randomly allocated into two groups. Group 1 followed the conventional protocol, employed 5.25% sodium hypochlorite (NaOCl) as the irrigant in conjunction with calcium hydroxide (Ca(OH)₂) as the intracanal medicament. Group 2 followed the advocated protocol, utilized SmearOFF as the irrigant combined with 2% chlorhexidine (CHX) gel as the intracanal medicament. All samples were subjected to qualitative analysis using PCR, and quantification was performed by colony-forming unit (CFU) analysis. It was demonstrated that SmearOFF was more effective than 5.25% sodium hypochlorite in reducing the bacterial counts of E. faecalis and F. nucleatum. Furthermore, 2% chlorhexidine (CHX) gel exhibited superior antimicrobial efficacy against E. faecalis and was advocated as an intracanal medicament in secondary endodontic treatment. [4]
Nidambur et al. (2023) assessed the efficacy of three 2-in-1 root canal irrigants in smear layer removal from canal walls, their interaction-induced precipitation, antibacterial activity, and cytotoxicity. Forty single rooted teeth were mechanically instrumented followed by irrigation with QMix, SmearOFF, Irritrol, or 0.9% saline as a control, with subsequent evaluation of smear layer removal efficacy in each specimen with scanning electron microscope. Precipitate formation following the interaction of the irrigating solutions with NaOCl was assessed. In addition, confocal laser scanning microscopy was employed to evaluate the antimicrobial activity of the irrigants against E. faecalis biofilms. It was found that there was no significant difference between QMix and SmearOFF in eliminating smear layers from the coronal-third and middle-third of the canal spaces. In the apical-third, SmearOFF removed smear layers effectively. QMix demonstrated a higher E. faecalis cell death percentage and a smaller biovolume. SmearOFF exhibited a larger decrease in biovolume compared with Irritrol. In contrast, QMix and Irritrol exhibited higher levels of cytotoxicity when compared with SmearOFF. Furthermore, Irritrol was found to be associated with precipitate formation following its interaction with NaOCl. [8]
Castagnola et al. (2024) evaluated the smear layer, debris removal, and antimicrobial activity of different irrigating solutions. Thirty single-rooted, single-canal teeth were divided into three groups and irrigated with Triton, Dual Rinse HEDP mixed with 6% NaOCl, and a conventional dual-step irrigation protocol using 6% NaOCl followed by 17% EDTA. The teeth were examined under a scanning electron microscope (SEM) to assess canal wall cleanliness. Samples were also evaluated for E. faecalis and Candida albicans growth, as well as the number of colony-forming units. Triton demonstrated statistically greater efficacy than Dual Rinse HEDP and NaOCl/EDTA in debris removal, except in the coronal third where NaOCl/EDTA performed similarly. In terms of smear layer removal, Triton was significantly more effective than Dual Rinse HEDP in the apical and middle thirds. All irrigation protocols produced a significant reduction in E. faecalis counts. All tested irrigating solutions killed 100% of E. faecalis, and SEM evaluation confirmed the absence of bacteria or the presence of small amounts of bacteria not possible to detect by CFU analysis. Notably, the Triton group exhibited the lowest number of residual Candida albicans. [9]
Fortea et al. (2024) conducted a systematic review on chelating agents in endodontics, assessed their antimicrobial activity, efficacy, cytotoxicity, and influence on treatment outcomes. Evidence supported that 17% EDTA is the most effective for smear layer removal and growth factor release, though its use with sodium hypochlorite is contraindicated. More recent studies highlight etidronic acid (9–18%) as a promising alternative due to its compatibility with sodium hypochlorite and ability to prevent smear layer formation through continuous chelation. While encouraging, further research is needed to confirm its clinical applicability and establish optimal protocols. [10]
Specific objectives
Aim of the study: This aim is to assess the antibacterial efficacy of SmearOFF compared to sodium hypochlorite as irrigants against Enterococcus faecalis in single-rooted anterior extracted human teeth.
Research Questions: Will the use of SmearOFF result in a greater antibacterial effect compared to sodium hypochlorite irrigation against Enterococcus faecalis in single-rooted anterior extracted human teeth?
Hypotheses: Null Hypothesis (H0): is that there is no significant difference in the antibacterial efficacy of SmearOFF and sodium hypochlorite as irrigants in single-rooted anterior extracted human teeth.
PICO:
P (Population): Single-rooted anterior extracted human teeth infected with Enterococcus faecalis.
I (Intervention): Irrigation using SmearOFF solution.
C (Comparison): Irrigation using Sodium Hypochlorite (NaOCl).
O (Outcome): Antibacterial efficacy
Methods
Calculated sample size: Our study will measure the antibacterial efficacy of SmearOFF irrigant by quantifying colony-forming units (CFU) in independent experimental and control groups, with a 1:1 allocation ratio. Based on results derived from a previous study [11], the response variable within each group was assumed to follow a normal distribution with a standard deviation of 1.37. Assuming a true mean difference of 7.97 between the experimental and control groups, a sample size of 18 subjects per group was determined, yielding a total of 54 extracted teeth. This sample size was calculated to achieve a statistical power of 0.8 for rejecting the null hypothesis of equal population means. The calculation was performed using G*Power version 3.1.9.7, with a significance level (α) of 0.05. A p-value ≤ 0.05 will be considered statistically significant.
Description of study sample: A total of 54 single-rooted completely erupted human anterior teeth Will be collected from the surgical department of the Faculty of Dentistry Cairo University. Extracted for orthodontic, periodontal or prosthodontic reasons and ethical approval was obtained in accordance with institutional guidelines and the Declaration of Helsinki.
Inclusion criteria:
Fully formed single-rooted teeth.
Teeth with no caries.
Teeth with no fissures.
Teeth with no internal or external root resorption.
Exclusion criteria:
Teeth with severe curvature.
Teeth with resorption or perforations.
Teeth with calcification.
Teeth with open apices.
Any prior root canal treatment, posts, or extensive restorations compromising canal integrity.
Intervention for each group: A total of 54 single-rooted, completely erupted human anterior teeth will be utilized and stored in saline. The external root surfaces will be cleaned of calculus and periodontal ligament, then they will be stored in sodium hypochlorite for 30 minutes. Afterwards, they will be coated with nail polish, serving as a protective barrier to prevent bacterial penetration and to restrict material diffusion into the dentin.
Access Preparation: Access cavities will be prepared using a diamond round bur (Komet Dental, Lemgo, Germany). The cavity walls will then refined with a tapered cylindrical diamond bur (Komet Dental, Lemgo, Germany).
Canal Negotiation: K-files of sizes 10 and 15 (K-Files, MANI, Inc., Utsunomiya, Japan) will be used to negotiate each canal to the full working length until patency is achieved. The working length will be confirmed when the tip of the size 15 file is visible at the apical foramen.
Canal Preparation: Each root canal will be prepared with rotary files (Rogin Dental, Shenzhen, China) and will be enlarged to size 40, taper 0.04 using the crown-down technique to the full working length, operated with Endo motor (Wismy, Bondent Group, Shenzhen, China) (speed 350 rpm, torque 2 N/cm). 3 ml sodium hypochlorite irrigation (5%, JK Dental Vision, Mansoura, Egypt) will be performed with a 30-gauge side-vented needle positioned 1 mm short of the working length between each file until total volume of irrigant will be 24 ml. Upon completion of instrumentation, a final rinse with 5 ml of distilled water will be performed, followed by drying of the canals with sterile paper points.
All teeth will be stored in saline after preparation and will be autoclaved for 20 minutes at 121 °C.
The prepared canals will be inoculated with Enterococcus faecalis biofilms under standardized laboratory conditions. A pure culture of E. faecalis will be cultivated in Brain Heart Infusion (BHI) broth and incubated at 37 °C for 24 hours. The teeth will then be immersed in the bacterial suspension and incubated for 21 days to allow mature biofilm formation, with the culture medium refreshed every 48 hours. This procedure will ensure uniform contamination of all specimens with mature biofilms prior to the application of the irrigant regimens.
The specimens will be randomly allocated into three experimental groups, with 18 specimens in each group.
Group A: Sodium Hypochlorite and EDTA:
Irrigation will be performed with 5 ml of 5 % NaOCl. The solution will be delivered with a 30-gauge side-vented needle positioned 1 mm short of the working length. Activation will be carried out using ultrasonic activation device (Ultra X, Changzhou, China) for 60 seconds, followed by 5 ml of 17% EDTA (JK dental). with a 30-gauge side-vented needle positioned 1 mm short of the working length. Activation will be carried out using ultrasonic activation devices for 60 seconds.
Group B: Sodium Hypochlorite (NaOCl) (Control)
Irrigation will be performed with 5 ml of 5 % NaOCl. The solution will be delivered with a 30-gauge side-vented needle positioned 1 mm short of the working length. Activation will be carried out using ultrasonic activation device (Ultra X, Changzhou, China) for 60 seconds.
Group C: SmearOFF (Intervention)
Irrigation will be performed using 5 ml of SmearOFF solution. The solution will be delivered with a 30-gauge side-vented needle positioned 1 mm short of the working length. Activation will be carried out using ultrasonic activation device (Ultra X, Changzhou, China) for 60 seconds.
Microbiological Sampling
After completion of the irrigation protocols, all specimens will be subjected to microbial sampling to assess the residual bacterial load. The canals will be dried with sterile paper points, and microbiological samples will be collected using sterile paper points inserted to the full working length. These samples will then be transferred to Brain Heart Infusion (BHI) broth for culturing and colony-forming unit (CFU) quantification.
Outcome
• The primary outcome will be the antibacterial efficacy of the tested irrigants (Sodium hypochlorite and EDTA, sodium hypochlorite, and SmearOFF ) against Enterococcus faecalis in single-rooted extracted anterior teeth.
• Residual bacterial load will be assessed by collecting microbiological samples from the root canals after irrigation, culturing them in Brain Heart Infusion (BHI) broth, and quantifying colony-forming units (CFUs).
• Antibacterial effect will be compared among the three groups to determine the relative efficacy of SmearOFF and sodium hypochlorite against E. faecalis.
| Outcome Measure | Description | Unit of Measurement | Timepoint | |
| Antibacterial Efficacy | reduction in bacterial load from baseline to post‑irrigation | CFU per ml | Pre + immediately post-irrigation |
Assignment to intervention
Sequence generation
Randomallocation and sequence generation will be performed using computer randomsequence generator program. (https//:www.random.org).
Allocation concealment
To preserve allocation concealment, a sealed opaque envelope system will be utilized. Each envelope will specify the allocation group assigned to a particular tooth number. The opacity of the envelopes will prevent personnel from predicting the group assignment. Envelopes will be opened only after tooth preparation and readiness for intervention, thereby ensuring that the allocation sequence remains concealed until the intervention
stage.
Implementation
Random allocation, sequence generation and the allocation concealment will be performed by the Co-supervisor.
Blinding
Assessor Blinding: Laboratory staff responsible for performing microbial assays test will remain unaware of the group allocations. They will be informed only that the samples originate from an experimental study, without disclosure of the specific irrigant used for each specimen.
Blinding Method: Each specimen will be assigned a unique identification code that conceals its group designation. This coding system ensures that outcome assessments are conducted without knowledge of the treatment received, thereby minimizing measurement bias and safeguarding the validity of the results.
Statistical methods
All data generated during the study will be recorded, tabulated, and statistically analysed using appropriate statistical software. Numerical variables will be tested for normality through distribution assessment. For normally distributed data, results will be reported as mean ± standard deviation. For non-normally distributed data, results will be presented as median and range. A significance threshold of p < 0.05 will be adopted for all statistical tests. The choice of statistical tests will be determined based on the distribution of the data and the homogeneity of variances.
Ethics
The research will be submitted to the ethics committee for review. Upon completion of the experiment and receipt of the results, all instruments and tooth samples will be sterilized and disposed of in a specialized incinerator under the supervision of the Microbiology Department at Cairo University.
References
1- Elhady A, Obeid M, Yehia T. Efficiency of different irrigation
protocols on cleanliness and disinfection of root canal: An in-vitro study. AinShams Dent J. 2024;33(1):114-20.
2- El Ashry SH, Nagy MM, Elsewify TMA, Ali EBEA. Efficiency of newly introduced root canal irrigants based on nanoparticles: An in-vitro study. Ain Shams Dent J. 2022;27(3):46-52.
3- Stuart CH, Schwartz SA, Beeson TJ, Owatz CB. Enterococcus faecalis: Its role in root canal treatment failure and current concepts in retreatment. J Endod. 2006;32(2):93-8.
4- Tandon J, Taneja S, Bhalla VK, Jain A. Evaluation of bacterial reduction at various stages of endodontic retreatment after use of different disinfection regimens: An in vivo study. Eur Endod J. 2022;7(3):210-6.
5- Mobaraki B, Yeşildal Yeter K. Quantitative analysis of SmearOFF and different irrigation activation techniques on removal of smear layer: a scanning electron microscope study. Microsc Res Tech. 2020;83(12):1480-6.
6- Khudhur HA, Bakr DK, Hamasaeed NH, Saleem SS, Mahdi SF, Tawfiq HF. Unveiling SmearOFF efficacy in smear layer removal through ultrasonic activation examined by scanning electron microscopy. Int J Biomater. 2024;2024:8188413.
7- Srivastava S. Antibiofilm efficacy of 0.1% Octenidine, SmearOFF, 1% Alexidine and 5.25% Sodium Hypochlorite against Enterococcus faecalis biofilm formed on tooth substrate. IAIM. 2019;6(11):1-8.
8- Ballal NV, et al. Biological and chemical properties of 2-in-1
calcium-chelating and antibacterial root canal irrigants. J Dent.
2023;135:104575.
9- Castagnola R, Martini C, Colangeli M, Pellicciotta I, Marigo L, Grande NM, Bugli F, Plotino G. In vitro evaluation of smear layer and debris removal and antimicrobial activity of different irrigating solutions. Eur Endod J. 2024;9:81-8.
10- Fortea L, Sanz-Serrano D, Luz LB, Bardini G, Mercade M. Update on chelating agents in endodontic treatment: A systematic review. J Clin Exp Dent. 2024;16(4):e516-e538.
11- Gadekar T, Shetty R, Yadadi S, Adtani P, Desai V, Afrashtehfar K. Comparative evaluation of the antimicrobial efficacy of 20% chlorhexidine, 3% sodium hypochlorite, and dexamethasone acetate with thymol as a root canal disinfectant against Enterococcus faecalis: An in-vitro feasibility study. Open Dent J. 2025;19:e18742106333707.
12- Abusteit OE. Evaluation of resin sealer penetration of dentin following different final rinses for endodontic irrigation using confocal laser scanning microscopy. Aust Endod J. 2021;47(2):195-201.
Protocol references
[1] [2] [3] [4] [5] [6] [8] [12] [7] [4] [8] [9] [10] [11]
Acknowledgements
Self-funded study.