Evaluation of new Seawater-based Mouth Rinse Versus Chlorhexidine 0.2% Reducing Plaque and Gingivitis Indexes. A Randomized Controlled Pilot Study

Featured Application: The use of this seawater mouth rinse reduce the e ﬀ ect of aggressive oral bacteria, taste, and numbness problems. Abstract: For a long time, Chlorhexidine digluconate (CHX) has been considered the most used mouth-rinse for reducing plaque and gingivitis. Sea4 ® Encias is a new seawater-based mouth rinse with a similar action to CHX. Its prolonged use produces fewer side e ﬀ ects. This study compared the e ﬀ ects of two oral rinses: Chlorhexidine 0.20% and Sea 4 ® Encias (seawater) for reducing plaque and gingivitis indices. This double-blind crossover study recruited and monitored 93 volunteer dental students, hygienists and doctors from the Universidad Cat ó lica de Murcia (UCAM), for 1 month, and compared the e ﬃ cacy of Chlorhexidine 0.20% (Group A); Sea4 ® Enc í as (Group B); and a placebo saline solution (Group C) mouth-rinses for reducing plaque and gingivitis indices. Plaque and gingival inﬂammation (Löe and Silness test) were evaluated at baseline and after each study stage. inhibitory activity on plaque (1.32 ± 0.22) compared with CHX (1.97 ± 0.34) and saline (2.78 ± 0.11). Sea4 Encias and Chlorhexidine 0.20% mouth-rinses signiﬁcantly reduce plaque growth and G.I. compared with saline mouth-rinse; Sea4 Enc í as mouth-rinse is more e ﬀ ective against plaque regrowth than Chlorhexidine in this pilot study.


Introduction
Many bacteria present in the mouth cannot be eliminated properly with daily brushing, and can be present in the preservation of healthy dental and periodontal tissues [1]. Gingivitis begins with plaque build-up, which will end in periodontitis [2]. In this context, much research has investigated the efficacy of several types of mouthwash, which are designed to improve plaque and gingivitis levels. V-Sol containing alcohol, as well as essential oils (E.O.), have been shown to offer beneficial effects on plaque and gingivitis index, where it seems to significantly improve oral health six months after use [3,4]. Different mouthwashes containing natural compounds (NCCM) versus E.O. mouthwash (Listerine®) demonstrated their effectiveness for plaque control [4,5]. Another study evaluated the effects of E.O. that reduces plaque and gingivitis compared to cetylpyridinium chloride (CPC) and a placebo. Essential oils showed a better effect than mechanical plaque control (MPC) and CPC, reducing plaque and gingival inflammation in patients who may suffer gingivitis. Therefore, mouthwashes containing E.O. should be the first option for mechanical plaque control [6][7][8]. The therapeutic application of natural compounds showed that these compounds have some efficacy in the fight against gingivitis and periodontitis [9][10][11][12][13]. However, evidence that the efficiency and effectiveness of NCCM, as routine methods that help mechanical oral hygiene practices for plaque control and gingivitis has remained low, and it seems that some NCCMs may offer various oral health benefits by reducing the plaque and gingivitis [14]. The main side effects of alcohol-containing mouthwashes (ACM) were oral pain, burning sensation, and taste disturbances [14][15][16][17]. When the clinical efficacy of two 0.2% chlorhexidine mouthwash (CHX) formulations are compared, the alcohol-free chlorhexidine rinse demonstrated action levels similar to the use of frequently used alcohol-formed rinses [18]. Some of the most frequent side effects from the use of CHX are staining of the tongue and teeth, as well as taste alteration, as CHX is a rinse that is very effective in reducing plaque and gingivitis. Some authors describe moderate evidence when oxygenating agent (OA) and CHX work together to reduce teeth staining, without interfering with the inhibition of plaque growth [19,20]. Mouthwashes with seawater could offer a promising alternative for CHX to overcome its possible adverse effects. Balneotherapy with spa water or the Dead Sea water can be used to treat inflammatory skin diseases (psoriasis and atopic dermatitis). This effect of seawater has been attributed to the preservation of the skin barrier induced by NaCl and KCl [21]. Only one study has investigated the use of a mouthwash solution with sea salt in street children in Manila, demonstrating its effectiveness in reducing mild to severe forms of periodontal disease. The authors proposed sea salt mouthwash for its antimicrobial action, which is highly effective and well-tolerated by the user and could be implemented worldwide using low-cost resources [22]. This is considered to be the first pilot study t which compares the clinical efficacy of 0.2% chlorhexidine with a new mouthwash formulated with seawater (analyzed for the first time) and a saline solution, in reducing gingiva inflammation and plaque formation.

Material and Methods
Undergraduate students, hygienists and doctors of the Universidad Católica de Murcia (UCAM, Catholic University of Murcia), School of Medicine and Dentistry, volunteered to take part in the study. The sample included 39 males and 54 females aged between 19 and 42 years, as a pilot study. This double-blind crossover study recruited 93 volunteer dental students (eighty one), hygienists (four) and doctors (eight). The UCAM Research and Ethics Committee at the Faculty of Medicine and Dentistry approved the study protocol, and subjects gave their informed consent to participate. The study was conducted in accordance with the CONSORT statement.
The inclusion criteria were as follows: -Healthy patients with at least 26-32 permanent teeth; -Non-smokers, with good oral health, presenting probing depths ≤ 3 mm, no active dental caries, no fixed or removable prostheses, and orthodontic devices.
Exclusion criteria Exclusion criteria were as follows: -Subjects with systemic diseases, caries, or active periodontal disease; -Subjects with crowns, or an orthodontic treatment; -Subjects taking antibiotics or anti-inflammatory drugs, or any other medication; -Subjects with an allergy to any of the ingredients of the mouth rinse.
The presence of plaque was evaluated using the Plaque Index (PI Silness and Löe). These index measurements were performed at three points of both, the lingual and buccal surfaces of all teeth, classifying the data as a 0-3 score: 0 = absence of plaque; 1 = no plaque; 2 = moderate accumulation of plaque; 3 = abundant accumulation of plaque on the dental surfaces. Gingival Inflammation was evaluated using the Gingival Index (GI Löe and Silness) on the same surfaces as the P.I., classifying the results with the same 0-3 score: (0 = no inflammation; 1 = light swelling but lack of bleeding during probing; 2 = redness, swelling, and bleeding during probing; 3 = intense redness, swelling, and spontaneous bleeding.

Experimental Protocol
The twenty-one volunteers completed a pre-treatment phase that included early prophylaxis (thorough mechanical cleaning) and, after receiving instruction in oral hygiene practices, performed the recommended regime for one week (without mouth rinse). Plaque and gingival indexes were registered at baseline and the end of the pre-treatment phase ( Figure 1). The randomization process was carried out, and in the flowchart, explain the method of our study.
Appl. Sci. 2019, 9, x FOR PEER REVIEW 3 of 10 -Non-smokers, with good oral health, presenting probing depths ≤ 3 mm, no active dental caries, no fixed or removable prostheses, and orthodontic devices . Exclusion criteria Exclusion criteria were as follows: -Subjects with systemic diseases, caries, or active periodontal disease; -Subjects with crowns, or an orthodontic treatment; -Subjects taking antibiotics or anti-inflammatory drugs, or any other medication; -Subjects with an allergy to any of the ingredients of the mouth rinse.
The presence of plaque was evaluated using the Plaque Index (PI Silness and Löe). These index measurements were performed at three points of both, the lingual and buccal surfaces of all teeth, classifying the data as a 0-3 score: 0 = absence of plaque; 1 = no plaque; 2 = moderate accumulation of plaque; 3 = abundant accumulation of plaque on the dental surfaces. Gingival Inflammation was evaluated using the Gingival Index (GI Löe and Silness) on the same surfaces as the P.I., classifying the results with the same 0-3 score: (0 = no inflammation; 1 = light swelling but lack of bleeding during probing; 2 = redness, swelling, and bleeding during probing; 3 = intense redness, swelling, and spontaneous bleeding.

Experimental Protocol
The twenty-one volunteers completed a pre-treatment phase that included early prophylaxis (thorough mechanical cleaning) and, after receiving instruction in oral hygiene practices, performed the recommended regime for one week (without mouth rinse). Plaque and gingival indexes were registered at baseline and the end of the pre-treatment phase ( Figure 1). The randomization process was carried out, and in the flowchart, explain the method of our study.

Calibration Trial
One single doctor (J.L.C-G) recorded probing pocket depth (PPD) and soft tissue recession (G.R.) in all 93 subjects. These measurements were repeated 1 week later. The researcher checked all the measurements; ± 2 mm tolerance value is accepted as appropriate. All repeated measurements of the Gingival index (G.I.) and plaque index (P.I.) were performed at the same time, every week for four weeks of the study. During the treatment phase, the subjects used the three mouth rinses as part of their oral hygiene regime as follows: First mouth rinse used for 10 days followed by a resting period of 10 days; the second mouth rinse for another 10 days followed by a resting period of 10 days; the

Calibration Trial
One single doctor (J.L.C-G) recorded probing pocket depth (PPD) and soft tissue recession (G.R.) in all 93 subjects. These measurements were repeated 1 week later. The researcher checked all the measurements; ± 2 mm tolerance value is accepted as appropriate. All repeated measurements of the Gingival index (G.I.) and plaque index (P.I.) were performed at the same time, every week for four weeks of the study. During the treatment phase, the subjects used the three mouth rinses as part of their oral hygiene regime as follows: First mouth rinse used for 10 days followed by a resting period of 10 days; the second mouth rinse for another 10 days followed by a resting period of 10 days; the third mouth rinse during the last 10 days, followed by one week of plaque removal through prophylaxis according to oral hygiene instruction. The mouth rinse groups under investigation were as follows: Group A (n = 7) solution with seawater Sea4 ® Encias (Blue Sea Laboratories, Alicante, Spain); Group B (n = 7) Chlorhexidine Digluconate 0.20 % (Laboratorios KIN, S.A., Barcelona, Spain) and Group C (n = 7) placebo/control saline solution (Betafar, Madrid, Spain). The mouth rinses were given to blinded the study subjects at each respective period of using, in the same packaging with only the following indications: Bottle A, Bottle B and Bottle C (Figure 2). The 93 volunteers rinsed three times a day for 1 min after meals, with 10 ml of solution provided randomly in Bottle A, Bottle B or Bottle C. After the end of each mouth rinse period of ten days, initial and final clinical measurements were obtained (Day 0 -Day 10: P.I., G.I.). Afterwards, and before the beginning of the next ten day period, a resting phase of 10 days was scheduled when subjects exercised prophylaxis (according to instruction in oral hygiene) for plaque removal. This allowed some time to eliminate the effects of the previous mouth rinse. Rinsing was unsupervised throughout the study period.
Appl. Sci. 2019, 9, x FOR PEER REVIEW 4 of 10 third mouth rinse during the last 10 days, followed by one week of plaque removal through prophylaxis according to oral hygiene instruction. The mouth rinse groups under investigation were as follows: Group A (n = 7) solution with seawater Sea4 ® Encias (Blue Sea Laboratories, Alicante, Spain); Group B (n = 7) Chlorhexidine Digluconate 0.20 % (Laboratorios KIN, S.A., Barcelona, Spain) and Group C (n = 7) placebo/control saline solution (Betafar, Madrid, Spain). The mouth rinses were given to blinded the study subjects at each respective period of using, in the same packaging with only the following indications: Bottle A, Bottle B and Bottle C (Figure 2). The 93 volunteers rinsed three times a day for 1 min after meals, with 10 ml of solution provided randomly in Bottle A, Bottle B or Bottle C. After the end of each mouth rinse period of ten days, initial and final clinical measurements were obtained (Day 0 -Day 10: P.I., G.I.). Afterwards, and before the beginning of the next ten day period, a resting phase of 10 days was scheduled when subjects exercised prophylaxis (according to instruction in oral hygiene) for plaque removal. This allowed some time to eliminate the effects of the previous mouth rinse. Rinsing was unsupervised throughout the study period.

Patient Variables
Patient data was assessed by J.L.C-G, to evaluate the residual liquid (ml) in the patient´s returned bottles from the subjects. Patients' perception of the product and possible adverse effects were registered in a questionnaire the subjects were asked to complete on the last visit by placing a mark on a 10 cm visual analog scale (VAS) ( Table 1). All study subjects were evaluated in a dental office by a principal investigator (J.L.C-G). G.I. and P.I. Indexes were registered at days 0 and 7 of the pre-treatment phase), day 17 (end of the first rinse period), day 27 (end of the first resting phase), day 37 (end of the second rinse period), day 47 (end second resting phase), and day 57 (end of the third rinse period) and day 67 (end of the fourth rinse period). After the experiment was concluded, the participants received the last week of plaque removal through prophylaxis (according to instructions in oral hygiene.

Patient Variables
Patient data was assessed by J.L.C-G, to evaluate the residual liquid (ml) in the patient´s returned bottles from the subjects. Patients' perception of the product and possible adverse effects were registered in a questionnaire the subjects were asked to complete on the last visit by placing a mark on a 10 cm visual analog scale (VAS) ( Table 1). All study subjects were evaluated in a dental office by a principal investigator (J.L.C-G). G.I. and P.I. Indexes were registered at days 0 and 7 of the pre-treatment phase), day 17 (end of the first rinse period), day 27 (end of the first resting phase), day 37 (end of the second rinse period), day 47 (end second resting phase), and day 57 (end of the third rinse period) and day 67 (end of the fourth rinse period). After the experiment was concluded, the participants received the last week of plaque removal through prophylaxis (according to instructions in oral hygiene.

Statistical Analysis
As this was a pilot study, the sample size of 31 patients per group was selected for convenience. Quantitative clinical variables were calculated per patient and per visit, and then by group. Means, standard deviations and 95% confidence intervals (CI) were calculated in clinical variables, such as per patient, per visit, and then by group. The Kolmogorov-Smirnov test was used to evaluate the normality and significant distribution of the samples, and ANOVA Test was used to identify significant differences between groups at the initial visit, and significant changes before, and after, treatment within each group. Wilcoxon signed-rank test was used to the non-normal distribution of the frequency of the indexes, with a significance level of p < 0.05. Statistical analyses were performed separately for the gingivitis and plaque evaluations. Comparisons, between the baseline scores for G.I. and P.I. of subjects in the three different groups, were performed separately using an independent t-test. Intra-group comparisons regarding baseline G.I. and P.I. Scores versus follow-up G.I. and P.I. Scores were performed using a paired t-test. Inter-group comparisons regarding baseline-adjusted G.I. and P.I. Scores at the follow-up examinations were performed using ANOVA. All statistical tests of hypotheses were two-sided and employed a level of significance of α = 0.05. All analyses were performed using the IBM software package SPSS, v.20 (IBM Corp., Armonk, NY, USA).

Patients' Perception of Mouth Rinses
The differences in patients' perception of the products tested between small groups and the reduction of plaque scores in SEA 4 and Chlorhexidine 0.20% when compared to saline after first week onwards; however, reduction of plaque is higher in group SEA 4 than Chlorhexidine in second, third and fourth week.
No clinical signs of mucosal changes were found after the use of the three mouth rinses at any point during the study. Most of the volunteers reported a mouthwash taste sensation in the mouth, which remained for 45 minutes after rinsing.
The periodontal status of all subjects was evaluated at the time of recruitment. Probing depth was controlled ranged between 1.3 and 3.1 mm, while plaque indexes ranged between 0.1 and 1.2 mm. All subjects (N = 21; SEA 4 Group N = 7; Chlorhexidine Group N = 7; Saline water Group N = 7) complied with the experimental protocol, and the data obtained was complete. The amounts of the products returned in the patient´s bottles when the study was finished suggested the right level of subject uses with the study protocol. No side effects were reported during the study.
There were significant differences in the Plaque index between the three groups. The Sea4 Group showed more significant inhibitory effect on plaque regrowth compared with the CHX group in the first week; in fact, the mean overall plaque index was 0.86 in the SEA 4 Group, compared with 1.26 in the Chlorhexidine Group, and 1.56 in the saline water Group. The follow-up analysis found no stained teeth or taste alteration in the SEA 4 Group or the Saline Water Group, but some patients showed some taste alteration reporting that food lacked flavor, and some dental staining was observed in the Chlorhexidine Group.
After using SEA 4 Encías, some patients reported a taste alteration from salty to sweet menthol. Significant differences were found between the mean P.I. measured at the beginning of the study period (habitual oral hygiene) and mean P.I. obtained with SEA 4 Encías and Chlorhexidine (p < 0.01). After the first and second-week, plaque and gingival scores decreased in Group A and Group B, and Gingival scores showed a greater reduction in Group A and Group B, than in Group C in the second, third and fourth weeks, but the reduction was more significant in Group A than Groups B and C (Figure 3). SEA 4 Encías and Chlorhexidine presented similar levels of efficacy in delaying plaque growth in the fourth week, which was higher with Chlorhexidine than with saline water. SEA 4 Encías produced a more significant reduction in plaque at 4 weeks than the other two mouth rinses (Figure 4). Gingival scores decreased significantly more in Groups A and B than Group C during the entire study period.

Discussion
For the first time, this pilot study investigated a new SEA 4 Encías® seawater-based mouthwash, compared to the most commonly used mouthwash, such as Chlorhexidine. As this is the first clinical trial of SEA 4, it is impossible to compare the results with any other published research. However, the results provided clear evidence that mouthwash with seawater reduced gingivitis and plaque formation more effectively than 0.2% chlorhexidine digluconate and a placebo saline solution throughout the study period.

Discussion
For the first time, this pilot study investigated a new SEA 4 Encías® seawater-based mouthwash, compared to the most commonly used mouthwash, such as Chlorhexidine. As this is the first clinical trial of SEA 4, it is impossible to compare the results with any other published research. However, the results provided clear evidence that mouthwash with seawater reduced gingivitis and plaque formation more effectively than 0.2% chlorhexidine digluconate and a placebo saline solution throughout the study period.

Discussion
For the first time, this pilot study investigated a new SEA 4 Encías®seawater-based mouthwash, compared to the most commonly used mouthwash, such as Chlorhexidine. As this is the first clinical trial of SEA 4, it is impossible to compare the results with any other published research. However, the results provided clear evidence that mouthwash with seawater reduced gingivitis and plaque formation more effectively than 0.2% chlorhexidine digluconate and a placebo saline solution throughout the study period.
Arunachalam et al. compared the intraoral distribution of 0.2% chlorhexidine without alcohol (CHX-Alc) and 0.1% chlorhexidine with alcohol (CHX + Alc) at different rinse times (10 s, 20 s, 30 s) and described that the shortest rinse-time will assist in monitoring and continuity of the patient´s compliance [26]. Another short-term study by Shiloah and Patters found no statistically significant differences between two commercially available CHX types of mouthwash (0.2% and 0.12%) in plaque inhibition with a 30-second rinse time, reporting side effects of spots and taste disturbances, limiting their potential for long-term use [25].
It has been clinically proven that three common therapeutic agents found in mouthwashes produce essential benefits for the control of gingivitis and plaque, such as Chlorhexidine, CPC, and essential oils. The SEA 4 Encías®mouthwash includes CPC in its formulation, demonstrating that in vitro and clinical studies are useful both, in biofilms and in the reduction of the common gingival inflammation and plaque index.
The use of seawater mouthwashes will be the best alternative to the use of Chlorhexidine, with the same essential anti-bacterial activity, reducing the side effects of dental staining and taste disturbances.

Conclusions
This clinical study tested a newly formulated seawater mouthwash SEA 4 Encías, containing CPC and hyaluronic acid, and compared it with 0.20% chlorhexidine and a placebo mouth-rinse. Our research with larger sample sizes confirmed the results, and provided clear evidence that seawater mouthwash was more effective against plaque and gingivitis than Chlorhexidine 0.20% rinse in 4 weeks study. It was also more effective against plaque regrowth than the chlorhexidine 0.20% rinse and placebo. These findings provide clinical evidence that this mouthwash may be recommended to patients as a useful adjunct to their oral hygiene practices, avoiding staining and taste disturbances.