Effect of octenidine mouthwash on plaque, gingivitis, and oral microbial growth: A systematic review

Abstract Objective Octenidine dihydrochloride is an antimicrobial cationic surfactant compound. We conducted a systematic review to determine the efficacy of octenidine‐based mouthwash on plaque formation, gingivitis, and oral microbial growth in subjects with or without periodontal disease. Materials and Methods PubMed/MEDLINE, ScienceDirect, Google Scholar, and Cochrane Library were searched for relevant studies. The review was conducted per PRISMA guidelines. Only randomized controlled trials and observational studies comparing octenidine with placebo or other mouthwashes in healthy subjects with or without periodontal disease, were considered for this review. The endpoints included percentage reduction in plaque index (PI), gingival index (GI), absolute reduction in the mean number of colony‐forming units (CFU/ml [log10]) and adverse effects (AEs; tooth staining/mucosal tolerance). Results Ten randomized controlled and six observational studies fulfilled the selection criteria. Twice or thrice daily rinsing with 0.1% octenidine for 30–60 s produced significant reduction in plaque, gingivitis and oral microbial growth. Compared to control mouthwash or baseline, 0.1% octenidine inhibited plaque formation by ~38.7%–92.9%, which was either equal or greater than that of chlorhexidine gluconate. 0.1% octenidine reduced gingivitis by ~36.4%–68.37% versus control mouthwash or baseline and microbial growth by 0.37–5.3 colony‐forming units (vs. chlorhexidine: 0.4–4.23 colony‐forming units). Additional benefits of 0.1% octenidine were significant reduction in the number of bleeding sites, papilla bleeding index, sulcus bleeding index, and gingival fluid flow. Conclusion Within the limitations of this study, there exists moderate evidence that 0.1% OCT was found to be an effective antiplaque agent. Octenidine inhibited plaque formation upto 93% and gingivitis upto 68% versus placebo and was either superior or comparable to chlorhexidine. Octenidine was well‐tolerated and safe and can be an effective alternative to CHX and other contemporary mouthwashes.


| INTRODUCTION
Dental caries and periodontal disease are common oral conditions, though frequently neglected, affect 3.58 billion individuals worldwide (Vos et al., 2017). Although mechanical methods of plaque control (tooth brushing or flossing) are reasonably effective in maintaining oral hygiene, these alone are insufficient for complete plaque removal, especially in inaccessible areas of the oral cavity (Arora et al., 2014). Therefore, a chemical means to achieve optimum oral hygiene such as daily use of an antimicrobial mouthwash is recommended, particularly in individuals who are at risk of developing periodontitis (Barnett, 2006). Unlike a toothpaste, mouthwash being liquid can significantly reduce total oral microbial load as it rinses the entire oral cavity including inaccessible areas and soft and hard oral surfaces (Ciancio, 2015). To maintain the oral hygiene antimicrobial mouthwash is useful in older age people, patients who are unable to brush their teeth. It is particularly useful in the maintenance of oral hygiene in patients unable to brush their teeth due to illness or surgery, or in the elderly and in patients with special needs (Prasad et al., 2016). Commercial mouthwashes have antimicrobial and breath-freshening properties, and contain a combination of antiseptics, astringents, breath fresheners, essential oils (EOs), flavorings, and so on (Sykes et al., 2016).
Chlorhexidine gluconate (CHX) is a widely used, time-tested agent in mouthwashes effective against plaque formation, gingivitis, and oral microbial growth (Van Strydonck et al., 2012). However, the associated side-effects such as tooth discoloration, mucosal irritation, supragingival calculus formation, cytotoxicity, and taste alteration limit its benefits when it is required to be used for the long term. A novel antimicrobial cationic surfactant compound, octenidine dihydrochloride (OCT) was developed at the Sterling-Winthrop Research Institute, Rensselaer, NY in the 1980s (Al-Doori et al., 2007;Slee & O'Connor, 1983). OCT disrupts the cell membrane of fungi, bacteria, and yeast through strong adherence to lipid components and binding to negatively charge microbial surfaces (Brill et al., 2006;Kodedová & Sychrová, 2017). Data indicate that systemic absorption following cutaneous or oral administration of OCT is negligible. However, no data have been reported for secondary pharmacodynamics, drug interactions, metabolism and microbial resistance (Al-Doori et al., 2007;EPAR, 2009). It is mainly eliminated in the feces, no accumulation in the body has been reported (EPAR, 2009). 0.1% OCT mouthwash with excipients, additives and flavorings is approved for maintaining oral hygiene in pre-and post-periodontal or oral surgical interventions, gingivitis treatment, gingival bleeding upon probing and halitosis prevention (Sykes et al., 2016).
With a broad spectrum of activity, OCT is effective against several bacterial strains and fungi in vitro (Bailey et al., 1984). It acts at an extremely broad range of pH (1.6-12.2; Ellabib et al., 1990). While comparing the antimicrobial activity, OCT was found to be more effective than CHX, polyvinylpyrrolidone-iodine (PVP-I), polyhexamethylene biguanide, and triclosan in an in-vitro study (Koburger et al., 2010). Additionally in a randomized cross-over study, OCT was more efficacious than EOs, acriflavine hydrochloride, cetylpyridinium digluconate, and hydrogen peroxide in reducing the oral aerobic bacterial growth (Pitten & Kramer, 1999). Octenidine was superior to CHX and alexidine in inhibiting plaque-forming enzymes in the oral cavity in an in vitro study (Bailey et al., 1984). Importantly, OCT was less cytotoxic than CHX, EOs or PVP-I against gingival fibroblasts and epithelial cells in an in vitro study (Schmidt et al., 2016). In an in vitro study, octenidine effectively inhibits colony-forming microbe coaggregation thereby preventing plaque formation (Smith et al., 1991).
Although several clinical studies have reported antibacterial and antiplaque efficacy of OCT against established antimicrobial mouthwashes, there are no systematic evaluations on the effectiveness of OCT-based mouthwash. Therefore, this review systematically evaluated the evidence on the effectiveness of 0.1% OCT mouthwash, either as a monotherapy or as an adjunct, to regular oral hygiene against plaque formation, gingivitis and oral microbial load in subjects with or without periodontal disease.

| METHODS
We did a systematic search based on the standard procedure adhering

| Objectives
The primary objective was to determine the effectiveness of OCT mouthwash alone or as an adjunct to regular oral hygiene in controlling plaque, gingivitis, and oral microbial growth against a control mouthwash or only regular oral hygiene in subjects with/without periodontal disease. The secondary objective was to compare OCT versus CHX mouthwash in plaque, gingivitis and oral microbial growth reduction. Adverse effects (AEs) associated with OCT mouthwash use were also identified. The endpoints included percentage reduction in plaque and gingival index (GI), absolute reduction in the mean number of colony-forming units (CFU/ml [log 10 ]) for cariogenic bacteria and AEs (tooth staining/mucosal tolerance).

| Inclusion and exclusion criteria
Only randomized controlled trials (RCTs) and observational studies that compared any concentration of OCT against a control mouthwash/mouthwashes containing CHX, EOs or PVP-I in healthy subjects with or without periodontal disease, were included. Preclinical studies, case series and patents were excluded.

| Search strategy
A comprehensive search using combinations of the terms "octenidine," "plaque," "gingivitis," and "antimicrobial efficacy" was performed

| Quality assessment
The quality of the methodologies of all included studies were assessed per modified Jadad scale criteria (Jadad et al., 1996)

| Ethics Statement
No ethical approval per se was needed for this work as data only from the previous published studies in which informed consent and/or ethical approval was duly obtained by primary investigators had been systematically reviewed and analyzed. No human subject participants are directly involved in the conduct of this work.

| Search results
Electronic searches yielded 2737 hits; 2721 studies were rejected (not meeting selection criteria; Figure 1). Inter-observer agreement of a Cohen's kappa (K) of 0.77 was achieved for selection of studies.

| Study characteristics
Detailed study characteristics are presented in Table 1. There was a considerable heterogeneity in the design of the included studies.
Overall, OCT was used twice or thrice daily with 30-60 s of rinsing.
The number, gender, age and type of participants, and dentifrice use varied considerably among the studies. Ten studies enrolled healthy adults, two included children with orthodontic appliances; and four studies included children with poor oral hygiene, HIV patients with periodontal disease, adults with periodontal pockets, and adults with gingivitis, respectively. We classified the studies as those assessing the short-term (immediately post-rinsing up to 120 min, n = 4) and long-term (2 days to 3 months, n = 12) effects of OCT on study outcomes. Table 2 represents the estimated risk of bias of the included RCTs. In five RCTs (Beiswanger et al., 1990;Lorenz et al., 2018;Patters et al., 1983Patters et al., , 1986Welk et al., 2016), the potential risk of bias was low. The risk was moderate for three RCTs (Hemanth et al., 2017;Jain et al., 2017;Mutters et al., 2015); and it was unclear for two studies that were available only as abstracts (Koertge et al., 1986;Lobene et al., 1985). Only five RCTs clearly the described adequate random sequence generation and allocation concealment, while seven reported adequate blinding of study participants. Seven RCTs adequately reported withdrawal rates by number and reason per arm (Table 3). Table 4 presents the quality assessment of the six observational studies. The overall quality of two studies was good (Guši c et al., 2016;Pitten & Kramer, 1999); and that of three, was fair (Dogan et al., 2008(Dogan et al., , 2009Kocak et al., 2009). One study could not be appraised as the full text was unavailable in English (Kramer et al., 1998).
Two studies compared the effectiveness of OCT and CHX on plaque growth (Hemanth et al., 2017;Welk et al., 2016

| Gingivitis
The effect of OCT on GI was evaluated in six studies (Table 6; Beiswanger et al., 1990;Koertge et al., 1986;Lobene et al., 1985;Lorenz et al., 2018;Patters et al., 1986;Guši c et al., 2016). All studies reported a significant reduction in GI with OCT versus control mouthwash (Table 6). When compared to control mouthwash or baseline,  (9) 0.12% CHX (9) Antimicrobial enzymatic rinse (9) -Rinsing with mouthwash for 2 min OCT had a significant effect on S. mutans and preserved antimicrobial efficiency even after 60 min. OCT was found to be more effective than other mouth rinses over S. mutans (p < 0.05)  Patters et al., 1986). A significant decrease in GI by 65.27% at 1 month and 67.07% at 3 months from baseline was observed in a study by Guši c et al. (2016). Octenidine also demonstrated a favorable effect on GI in studies by Lobene et al. (1985) and Koertge et al. (1986).

| Additional benefits of OCT-based mouthwashes
The number of bleeding sites significantly reduced (60%) after 3 months of 0.1% OCT use versus control mouthwash with routine toothbrushing with a dentifrice (Beiswanger et al., 1990 Pitten and Kramer (1999) Representativeness of the exposed cohort Selected group Selected group Truly representative Truly representative Truly representative Selection of the nonexposed cohort ND ND Drawn from the same community as the exposed cohort Drawn from the same community as the exposed cohort Drawn from the same community as the exposed cohort  (Patters et al., 1983). All formulations containing different concentrations of OCT significantly reduced crevicular fluid flow after 7 days of twice-daily rinsing (Lobene et al., 1985). Thus, 0.1% OCT effectively prevented mucositis in susceptible patients (Mutters et al., 2015; Table 1).
The other reported AEs were decreased taste, tongue dorsum discoloration, bitter aftertaste, mild tingling of the tongue, and poor mucosal tolerance with varied incidence across the included studies (Table 8). A higher proportion of subjects using OCT in aqueous solution experienced mucosal intolerance in the studies by Koertge et al., 1986;Patters et al., 1983;Patters et al., 1986; however, the OCT formulation in vehicle was well tolerated by the oral mucosa and no significant AEs were observed.

| DISCUSSION
Oral health is an integral part in general health. Thus, to enjoy optimum quality of life it is essential to maintain a better oral health status T A B L E 5 Effect on plaque index (PI) Beiswanger et al. (1990) After 3 months of treatment with 0.1% OCT, there was a 38.7% reduction in the PI compared to placebo mouth rinse, and difference between means was significant at α = 0.05 Only the abstract were available and required quantitative details were not available from the abstracts Koertge et al. (1986) Abbreviations: CHX, chlorhexidine; OCT, octenidine; PI, plaque index.

Study Reduction in GI
without gingivitis, periodontitis, tooth decay and tooth loss. The fundamental objective of periodontal therapy is subgingival plaque control. Since the mechanical removal of subgingival plaque is timeconsuming and technically demanding, use of adjunctive antimicrobial agents such as mouthwash is a simple and effective option for optimum oral hygiene (Tartaglia et al., 2017). OCT, being positively charged, exerts its bactericidal action by binding to the negatively charged bacterial cell membranes and to soft and hard oral surfaces. It disrupts the phospholipid bilayer and attacks the enzyme systems causing the cell wall to lose integrity and leak its cytoplasmic contents.
OCT exhibits high affinity for cardiolipin, a lipid exclusively present in the bacterial cell membranes and therefore damages bacterial cells  (Table 1).
All studies assessing effects of a 0.1% OCT mouthwash reported significant decrease in plaque formation versus control mouthwash.
Moreover, the effect of twice-daily rinsing was observed even after short-term use for 4 days in some studies, and a long-term use for up to 3 months in others. A significant reduction in GI following the use T A B L E 7 Effect of 0.1% Octenidine on oral microbial growth Study Reduction in total bacterial count Dogan et al. (2008) Rinsing with 0.1% OCT and 0.2% CHX mouthwashes, respectively, reduced the total bacterial count (log 10 CFU/ml) by 4.4 and 1 at 15 min; 3.9 and 1.6 at 30 min; 3.7 and 1.3 at 60 min; and 3.1 and 1.9 at 120 min (all, p < 0.001). Control mouthwash (physiological saline) did not reduce the total bacterial count at any time points Dogan et al. (2009) Rinsing with 0.1% OCT, 0.2% CHX and control (physiological saline) mouthwashes, respectively, reduced the total bacterial count (log 10 CFU/ml) by 4.43, 0.96 and 0.049 at 15 min; 3.23 and 2.04 at day 2; 4.13, 2.14, and −0.04 at day 3; 3.73, 1.9 and −0.05 at day 5. These reductions were statistically significant for OCT and CHX (p < 0.001) and for control the p was 0.041 Guši c et al. (2016) Reduction in total bacterial count (log 10 CFU/ml) from baseline was 5.29 and 5.3 for 0.1% OCT (periodontal therapy + OCT mouthwash), and 5.35 and 5.44 for control (periodontal therapy only) at 1 and 3 months, respectively (p < 0.01) Jain et al. (2017) Reduction in S. mutant count (log 10 CFU/ml) from baseline was 3.95 and 4.11 for 0.2% CHX and 0.1% OCT groups (p = 0.430), respectively at day 1. Corresponding values at day 3 was 4.18 and 4.32 (p = 0.916); and at day 5 were 4.23 and 4.36 (p = 0.121). Reduction in Lactobacillus count (log 10 CFU/ml) from baseline was 3.30, 3.36 for 0.2% CHX and 0.1% OCT groups (p = 1.000), respectively at day 1. Corresponding values at day 3 were 3.41 and 3.46 (p = 0.743); and at day 5 were 3.48 and 3.51 (0.725) Lorenz et al. (2018) At day 4, reduction in total bacterial count (log 10 CFU/ml) from baseline was 0.37, 0.86, and 1.13 with 0.1%, 0.15% and 0.2% OCT mouthwashes, respectively. The corresponding value for placebo (0.9% saline solution) was −0.75. Difference from placebo was statistically significant for all OCT concentrations Mutters, Neubert, Nieth, & Mutters et al. (2015) Rinsing with 0.1% OCT and 0.2% CHX mouthwashes, respectively, reduced the total bacterial count (log 10 CFU/ml) by 1.3 and 4.1 (p = 0.04) at day 3; 1 and 5 (p = 0.003) at day 7 Pitten and Kramer (1999)  ). The corresponding values at day 5 were 0.4 and −2.9. Reduction in total mucosal bacterial count from baseline (log 10 CFU/ml) was 1.6, 1.9, 0.3 with 0.1% OCT, 0.12% CHX and placebo, respectively, on day 1. Corresponding values on day 5 were 0.6, 0.6 and −2.8. Difference between placebo and OCT (p = 0.003 for tooth surface and p < 0.0001 for mucosal surface) and/or CHX (p < 0.0001 for tooth surface and p = 0.001 for mucosal surface) were statistically significant but the difference between OCT and CHX was not significant (p = 0.781 for tooth surface and p = 1 for mucosal surface) Kramer et al. (1998) The required quantitative details were not available from the published articles by Kocak et al., and the abstract by Kramer et al. Kocak et al. (2009) Abbreviations: CFU, colony forming unit; CHX, chlorhexidine; OCT, octenidine.
In the presence of toothbrushing, but without toothpaste, 0.1% OCT significantly reduced cariogenic bacterial growth including S. mutans and Lactobacillus species versus control mouthwash (Jain et al., 2017). Moreover, 0.1% OCT prevented oral microbial growth for 12-16 h after the last rinsing (Welk et al., 2016). Complete elimination of P. intermedia, a periodontal pathogen, was reported following a twice-daily week-long rinsing with 0.1% OCT after periodontal therapy (mechanical debridement) and toothbrushing (Guši c et al., 2016). Additionally, antibacterial effect of 0.1% OCT in the saliva was observed even during the suspension of toothbrushing.
Since the placement of orthodontic appliances, especially brackets and wires, obstructs maintenance of effective oral hygiene by mechanical means, rinsing with a 0.1% OCT mouthwash can maintain adequate hygiene in plaque-infected sites, especially around the bracket bases that protects tooth enamel integrity, prevents white spot lesion, and periodontal damage (Dogan et al., 2009). Additional benefits of 0.1% OCT use included a significant reduction in the number of bleeding sites (Beiswanger et al., 1990). Further, significant reduction in PBI with 0.1% adjunct OCT versus periodontal therapy alone suggests a pronounced reduction in inflammation with the former (Guši c et al., 2016).
Compared to most mouthwashes with effects lasting 15-30 min postrinsing, the 0.1% OCT-based mouthwash exerted its effects even after 120 min (Dogan et al., 2008). A significant reduction in oropharyngeal flora with 0.1% OCT than 0.2% CHX was seen in ventilated cardiothoracic surgical patients and in patients with hematooncological malignancies (Mutters et al., 2015). Also, 0.1% OCT was more effective than 0.2% CHX on the fifth day of use even in the absence of toothbrushing or brushing without toothpaste (p < 0.001; Jain et al., 2017;Dogan et al., 2009). Hence, a 0.1% OCT-based mouthwash is an effective alternative to CHX and other contemporary mouthwashes in maintaining optimal oral health.
Tooth staining was a commonly reported AE following use of 0.1% OCT, as most studies refrained subjects from oral hygiene measures during the study; however, stain removal with single toothbrushing was reported. A majority of subjects claimed to continue use of the 0.1% OCT-based formulation (Lorenz et al., 2018). Despite the fact that bitter taste and mucosal irritation caused by OCT in aqueous solutions was a significant concern in earlier studies, OCT formulations in a mouthwash vehicle (at all concentrations) were welltolerated by the oral mucosa and no significant AEs were observed.
Overall, OCT is a chemically stable formulation with a low toxicity profile, is easy and safe to handle, nonflammable, and well-tolerated in clinical use (Assadian, 2016).

| Limitations
Although the 0.1% OCT-based mouthwash was efficacious in maintaining optimal oral hygiene for a short duration in all studies, evidence supporting long-term use is yet to be established. The sample size of all included studies was small and a study by Kramer et al. reporting on the microbial effects could not be accessed as a full text manuscript, thus supposes a careful interpretation and extrapolation to a larger population.

| CONCLUSIONS
Within the limitation of this systematic review, there is moderate quality of evidence that 0.1% OCT was found to be an effective antiplaque agent, as weighed on evidence based Grade recommendations (Guyatt et al., 2008). OCT was efficacious, and substantially reduced plaque formation, gingivitis and oral microbial growth. It was more effective than placebo and other common chemical agents used for plaque control. OCT was either superior or comparable to CHX-based mouthwashes in controlling dental plaque. Furthermore, it was also found to be effective in controlling gingivitis in patients with fixed orthodontic appliances. The use of 0.1% OCT mouthwash resulted in complete elimination of atypical oral microbe species, even at 1 month after therapy. Additional benefits included prevention of white spot lesions. OCT was well-perceived, tolerable, safe, and an effective alternative to CHX and other contemporary antibacterial mouthwashes. However, further studies assessing the long-term effects of a 0.1% OCT-based mouthwash, involving larger sample size, are required to confirm the results.

ACKNOWLEDGMENTS
The authors thank Drs. Shalini Nair and Yogesh Sharma (Abbott) and

DATA AVAILABILITY STATEMENT
The data that supports the findings of this study are available in the supplementary material of this article.