A Systematic Review of the Effect of Oral Rinsing with H2O2 on Clinical and Microbiological Parameters Related to Plaque, Gingivitis, and Microbes.

BACKGROUND: Hydrogen peroxide (H2O2) has been used for more than a century clinically to control plaque and gingival inflammation, with unclear supporting evidence. AIM: The aim of the present systematic review of the literature is to assess the effect of mouth rinses with H2O2 on dental plaque, gingival inflammation, and oral microorganisms.
METHODS: Five databases (PubMed, Scopus, Embase, Cochrane Library, and Web of Science) were searched with the following focused question: what is the effect of hydrogen peroxide, in comparison to chlorhexidine or to a placebo solution, in oral microbiota control, dental plaque, and gingival inflammatory outcomes? Two independent examiners retrieved the articles and evaluated the evidence.
RESULTS: The majority of included studies were performed with 1.5% H2O2. Results related to plaque accumulation generally demonstrate a slightly better effect of H2O2 as compared to placebo mouth rinses, however with a lower performance as compared to chlorhexidine. In terms of gingival inflammation, H2O2 performs better than placebo and more clearly demonstrates an anti-inflammation effect. No studies evaluated the effect of H2O2 against viruses or fungi. In terms of bacteria, H2O2 demonstrates an antibacterial effect.
CONCLUSION: Rinsing with H2O2 has the potential to affect plaque, gingivitis, and oral bacteria, as compared to placebo. However, the antibacterial results are not comparable to the performance of chlorhexidine.

1. Introduction

Hydrogen peroxide (H2O2) mouthwashes have been used for a long time [1]. They have been utilized in an attempt to complement mechanical plaque control methods as well as to prevent/control oral infections [2]. However, the evidence supporting its use is not unequivocal even though it is still used by a number of professionals.

In 2011, Hossainian et al. [3] published a systematic review to evaluate the effect of H2O2 mouthwashes on the prevention of plaque and gingival inflammation. The focused question of such review was as follows: “what are the effects of oxygenating mouthwashes on plaque accumulation and gingival inflammation parameters in adults, when compared with positive or negative controls mouthwashes or no oral hygiene, when used as a monotherapy or as an adjunct in daily oral hygiene?” Surprisingly, the number of included studies was relatively low (n = 12, in which only 5 were specifically formulated with H2O2). The other 7 studies were related to other oxygenating agents. The results of the review demonstrated that mouthwashes containing H2O2 do not consistently prevent plaque accumulation when used as a short-term monotherapy.

Recently, the effect of H2O2 on viruses has become a renewed interest due to the COVID-19 pandemic. H2O2 mouth rinses are being recommended as a preprocedural rinse, as well as a regular rinsing solution with the aim of diminishing contamination possibilities by the new coronavirus. A number of associations, including the American Dental Association, are recommending the use of H2O2 mouth rinses as prerinses prior to procedures [4-6]. However, the evidence for the use of H2O2 for oral antiviral purposes is virtually nonexistent. Most of the evidence only demonstrates the potential of H2O2 to disinfect surfaces [7].

New studies have been conducted and published after the systematic review of Hossainian et al. [3]. Hence, the existing review [3] could be broadened to consider these additional studies, especially in this particular moment. Therefore, the aim of this study is to systematically review the literature, assessing the effects of H2O2 mouth rinses in controlling dental plaque, gingival inflammation, and oral microbiota.

2. Materials and Methods

The focused question of the present study was as follows: “what is the effect of hydrogen peroxide, in comparison to chlorhexidine or to a placebo solution, in oral microbiota control, dental plaque, and gingival inflammatory outcomes?”

In order to be included, the study must fulfill all of the following inclusion criteria:

Clinical trials with humans of any age.

Test group: individuals that used, at least one time per day, hydrogen peroxide mouthwash. Any concentration of hydrogen peroxide was accepted.

Control group: individuals that used, at least one time per day, a placebo or chlorhexidine mouthwash. Any concentration of chlorhexidine was accepted.

Outcomes: any oral microbiological, plaque index, or gingival index analysis.

No restriction to language or date of publication was imposed. Studies that used both chlorhexidine and hydrogen peroxide in the same group were excluded. Studies that involved outcomes assessed in dental implants were also excluded.

A search strategy was performed, up to April 23, 2020, in five databases: PubMed, Scopus, Embase, Web of Science, and Cochrane Central Register of Controlled Trials (CENTRAL). The search strategy performed in PubMed database is expressed as follows:

Terms for hydrogen peroxide: Hydrogen Peroxide [MeSH terms] OR Hydrogen Peroxide [text word] OR Peroxides [text word] OR H2O2 [text word] OR Hydroperoxide [text word] OR Superoxol [text word] OR Oxydol [text word] OR Perhydrol [text word] OR oxidizing agent [text word] OR Bocasan [text word] OR Amosan [text word] OR Peroxyl [text word] OR Ascoxal [text word] OR peroxyborate [title/abstract]

Terms for mouthwashes: Mouthwashes [MeSH terms] OR mouthwashes [text word] OR mouthrinses [text word] OR mouthwash [text word] OR mouthrinse [text word] OR rinse [text word] OR Mouthwashes [pharmacological action] OR mouth rinse [text word] OR mouth rinses [text word] OR Mouth Bath [text word] OR Mouth Baths [text word] OR Mouth Wash [text word] OR Mouth Washes [text word] OR Oral Sprays [MeSH terms] OR Oral Sprays [text word] OR spray [text word]

Terms to assess all outcomes: microbiology [MeSH terms] OR microbiology [text word] OR Bacteria [MeSH terms] OR Bacteria [text word] OR Streptococcus mutans [text word] OR Porphyromonas gingivalis [text word] OR Aggregatibacter actinomycetemcomitans [text word] OR Tannerella forsythia [text word] OR Fusobacterium nucleatum [text word] OR Treponema denticola [text word] OR streptococcus sobrinus [text word] OR Streptococcus sanguis [text word] OR bacterial strain [text word] OR fungi [text word] OR fungi [MeSH terms] OR fungus [text word] OR Mold [text word] OR Candida [text word] OR Oral pathogens [text word] OR oral microorganisms [text word] OR antimicrobial [text word] OR antibacterial [text word] OR minimal inhibitory concentration [text word] OR MIC [text word] OR ATCC [text word] OR Type Culture Collection [text word] OR Saliva [MeSH terms] OR saliva [text word] OR dental caries [MeSH terms] OR Dental Caries [text word] OR dental plaque [MeSH terms] OR Dental plaque [text word] OR plaque [text word] OR biofilms [MeSH terms] OR biofilm [text word] OR dental deposit [text word] OR antiplaque [text word] OR Dental Plaque Index [MeSH terms] OR dental plaque index [text word] OR Oral Hygiene [MeSH terms] OR Oral hygiene [text word] OR Quigley-Hein Index [text word] OR Silness Loe index [text word] OR Oral Hygiene Index [MeSH terms] OR Oral hygiene index [text word] OR antigingivitis [text word] OR Gingivitis [MeSH terms] OR gingivitis [text word] OR gingival inflammation [text word] OR Bleed∗ [text word] OR gingival index [text word] OR gingival bleeding [text word] OR bleeding on probing [text word] OR papillary bleeding [text word] OR Periodontal Index [MeSH terms] OR Periodontal index [text word] OR periodontal diseases [MeSH terms] OR periodontal diseases [text word] OR periodontitis [text word]

Terms for hydrogen peroxide AND terms for mouthwashes AND terms to assess all outcomes

An adaptation of the abovementioned search strategy was performed in the other databases. Two researchers independently selected the studies and extracted the data in a spreadsheet specifically developed for this study (CKR and FWMGM). Regarding study selection, both screening (title and abstract analyses) and eligibility (full-text analysis) phases were performed independently. All discrepancies were solved between the researchers by discussion.

In addition, hand search was performed in the list of references of the included studies and in previously published literature reviews [3]. Studies from the last 30 years were hand searched in the following journals: Journal of Clinical Periodontology, Journal of Periodontology, Journal of Periodontal Research, and Journal of Dental Research. Searches for grey literature were also performed in the Clinical Trials (clinicaltrials.gov) and Google Scholar databases, using an adaptation of the abovementioned search strategy. All the corresponding authors of the included studies were contacted by e-mail in order to detect other potentially relevant clinical trials. In addition, manufactures were contacted to supply information about other published, unpublished, or ongoing research studies using H2O2.

The risk of bias of all randomized clinical trials was assessed by the RoB2 tool, as recommended by Cochrane [8]. Randomization process, deviations from the intended interventions, missing outcome data, measurement of the outcome, selection of the reported results, and overall risk of bias were assessed by two reviewers (FWMGM and CKR). In case of disagreements, a consensus was made between reviewers. A positive sign was given for an item when sufficient information was available, indicating low risk of bias, and a negative mark was used, for high risk of bias, when information was lacking. When risk of bias could not be assessed, the item was classified as unclear. For the nonrandomized trials, the ROBINS-I tool was used [9]. Several sources of bias were assessed, such as confounding, selection of participants, classification of interventions, deviations from intended interventions, missing data, measurement of outcomes, selection of the reported result, and overall bias.

3. Results and Discussion

3.1. Main Characteristics of the Included Studies

The search strategy and flowchart of articles retrieval is demonstrated in Figure 1. The reasons for exclusion of the identified studies are reported in Table S1. It should be noted that five databases were searched. The additional search strategies did not add any study to the present review, except for one additional study identified in the Google Scholar database [10]. The retrieved studies were very distinct in all aspects, preventing the possibility of a meta-analysis. Therefore, descriptive information will be given, according to design and outcome (experimental gingivitis or not; plaque, gingival inflammation, or microbiological parameters).

Figure 1

Flowchart of the studies during the review.

3.1.1. Risk of Bias

Figure 2 demonstrates the analysis of risk bias of the randomized clinical trials included in this review according to the RoB2 instrument. It may be detected that only one study presented low risk of bias in all criteria analyzed [11]. Four other studies presented an overall high risk of bias [10, 12–14]. The criteria randomization process showed unclear risk of bias in almost all included studies. The other sources of bias comprised mainly lack of information of reproducibility.

Figure 2

Risk of bias assessment for the randomized clinical trials.

The risk of bias for the nonrandomized trials included in the present review is demonstrated in Table 1. Bias due to confounding was critical in all studies. The other analyses mostly demonstrate moderate to low risk of bias.

Table 1

Risk of bias of the nonrandomized clinical trial, assessed by ROBINS-I tool, included in the present systematic review.

Author, year	Bias due to confounding	Bias in selection of participants into the study	Bias in classification of interventions	Bias due to deviations from intended interventions	Bias due to missing data	Bias in measurement of outcomes	Bias in selection of the reported result	Overall bias
Boyd, 1989 [15]	Critical	Low	Moderate	Low	Moderate	Moderate	Low	Moderate
Gusberti, 1988 [16]	Critical	Low	Moderate	Low	Low	Serious	Low	Critical
Pitten, 1999 [17]	Critical	Low	Moderate	Moderate	Low	Serious	Low	Critical
Wennström, 1979 [18]	Critical	Low	Moderate	Low	Low	Moderate	Low	Serious

3.1.2. Plaque Index

(1) Nonexperimental Gingivitis Studies. The plaque index measurements are demonstrated in Table 2. It should be highlighted that 10 studies evaluated plaque parameters, of which six were performed as clinical trials allowing mechanical plaque control [11, 12, 15, 19–21]. In one, it was not possible to determine if mechanical plaque control was possible [10]. All studies that used H2O2 as adjunct to mechanical oral hygiene were performed with the concentration of 1.5%. Those studies demonstrate a higher antiplaque efficacy of chlorhexidine in comparison to H2O2, except two, in which H2O2 presented similar efficacy to chlorhexidine [10, 21]. Generally, very little differences from negative controls were detected.

Table 2

Main characteristics and results for the studies that assess plaque index.

Study, year, country (reference)Study design	Systemic conditionsDiagnosis of the oral conditionPlaque index assessed	H2O2 group (concentration)Rinsing protocolN (smokers)AgeBaselineN(M)/N(F)EndN(M)/N(F)	CHX group (concentration) Rinsing protocolN (smokers)AgeBaselineN(M)/N(F)EndN(M)/N(F)	Control groupRinsing protocolN (smokers)AgeBaselineN(M)/N(F)EndN(M)/N(F)	Main results (follow-up)
					Baseline scoresMean ± SD	End scores (follow-up)Mean ± SD	∆ (%)	Author conclusions
Studies without experimental gingivitis
Boyd, 1989, USA [15];Non-RCT	Systemically healthy;Adolescents with initial oral health scheduled to have fixed orthodontic treatment;PI (S & L)	H2O2 1.5% + 0.05% NaF + toothbrush;Once-a-day (1/2 ounce per one minute) during 18 months;Smokers NR;Mean age:13.2 yrs;Baseline: 9/17;End: 24 individuals in total (proportion of M/F NR).	NA	0.05% NaF + toothbrush;Once-a-day (1/2 ounce per one minute) during 18 months;Smokers NR;Mean: 13.5 yrs;Baseline: 11/23;End: 32 individuals in total; (proportion of M/F NR).	Percent of sites with PI (S & L) >1:A:13.15 ± 17.40B: NAC: 19.05 ± 26.00	(18 months)Percent of sites with PI (S & L) >1:A: 18.50 ± 13.18B: NAC: 36.79 ± 35.60	PI (S & L)A: 5.35 (40.68%)B: NAC: 17.74 (93.12%#)∗∗	When the percentage of sites with plaque index greater than 1 was considered, the group that used H2O2 showed significantly lower percentage when compared to the control group
Clark, 1989, USA [19]RCT (parallel)	Systemically healthy;Gingivitis (at least 8 sites with marginal gingival bleeding)PIS (Q & H)	H2O2 1.5% (use of toothbrush and dentifrice NR);Once-a-day (20 ml for 30 s) during 21 weeks;2 smokersMean age: 28.9 yrsBaseline: 9/16End: 22 individuals in total (proportion of M/F NR)	NA	Water (use of toothbrush and dentifrice NR);Once-a-day (20 ml for 30 s) during 21 weeks;No smokersMean age 27.8 yrsBaseline: 8/17End: 22 individuals in total (proportion of M/F NR)	A: 2.87 ± 0.38B: NAC: 2.90 ± 0.25	(6 months)A: 1.72 ± 0.58B: NAC: 1.74 ± 0.46	A: −1.15 (−40.07%)B: NAC: −1.16 (−40.00%)	No statistically significant difference between groups
Gomes, 1984, USA [20]RCT (parallel)	Nonhandicapped staff and handicapped patients—both of them were free of systemic diseasesIndividuals without periodontitis (probing depth <5 mm)PI (L)	H2O2 1.5% (use of toothbrush and dentifrice NR)Three times per day (10 ml per 60 s) during 7 daysStaff: smokers NR Mean age: 30.34 yrsBaseline: 5/34End: NR/NRPatients: smokers NRMean age: 28.65 yrsBaseline: 30/24End: NR/NR	NA	Placebo mouth rinse (use of toothbrush and dentifrice NR)Three times per day (10 ml per 60 s) during 7 daysStaff: smokers NR Mean age: 29.94 yrsBaseline: 5/30End: NR/NRPatients: smokers NRMean age: 28.62 yrsBaseline: 25/25End: NR/NR	Staff—maleA: 0.68 ± 0.54B: NAC: 0.66 ± 0.38Staff—femaleA: 0.61 ± 0.50B: NAC: 0.43 ± 0.24Patients—maleA: 1.29 ± 0.55B: NAC: 1.53 ± 0.69Patients—femaleA: 1.30 ± 0.55B: NAC: 1.23 ± 0.59	(7 days)Staff—maleA: 0.54 ± 0.49B: NAC: 0.72 ± 0.52Staff—femaleA: 0.45 ± 0.50B: NAC: 0.41 ± 0.26Patients—maleA: 1.05 ± 0.55B: NAC: 1.51 ± 0.55Patients—femaleA: 1.07 ± 0.41B: NAC: 1.19 ± 0.57	Staff—maleA: −0.14 (−20.59%#)B: NAC: 0.06 (9.09%)Staff—femaleA: −0.16 (−26.22%#)B: NAC: −0.02 (−4.65%)Patients—maleA: −0.24 (−18.60%#)B: NAC: −0.02 (−1.31%)Patients—femaleA: −0.23 (−17.69%#)B: NAC: −0.04 (−3.25%)	In the within group comparison, a significantly lower plaque score was detected, for both staff and patient groups, only for H2O2
Hasturk, 2004; USA [11]RCT (parallel)	Systemically healthyGingivitis (gingival index >2.0 and >10% sites with bleeding). In the first 28 days, only the mouth rinses were used.PIS (Q & H)	H2O2 1.5% + 0.05 sodium fluoride toothpaste + toothbrushTwice daily (quantity not reported for 30 s) during 6 months;3.9% smokers (N not provided)Mean age: 31.4 yrs;Baseline: 19%/51% (N not provided)End: NR/NR	NA	Placebo mouth rinse 0.05 sodium fluoride toothpaste + toothbrushTwice daily (quantity not reported for 30 s) during 6 months18.8% smokers (N not provided)Mean age: 31.0 yrsBaseline: 45.8%/54.2% (N not provided)End: NR/NR	A: 1.03 ± 0.81B: NAC: 0.87 ± 0.58	(6 months)A: 0.99 ± NRB: NAC: 0.99 ± NR	A: −0.04 (−3.88%)B: NAC: 0.12 (13.79%)	No statistically significant difference between groups
Porwal, 2018, India [10]RCT (parallel)	Systemically healthyGingivitis (modified gingival index of 3 and plaque index of 4)PIS (Q & H)	H2O2 3% with distilled water (ratio 1 : 1)Twice daily (10 ml and time not reported) during 15 daysSmokers NRMean age: NRBaseline: 10 individuals in total (proportion of M/F NR)End: 10 individuals in total (proportion of M/F NR)	CHX 0.2% with distilled water (ratio 1 : 1)Twice daily (10 ml and time not reported) during 15 daysSmokers NRMean age: NRBaseline: 10 individuals in total (proportion of M/F NR)End: 10 individuals in total (proportion of M/F NR)	NA	A: 3.12 ± 0.44B: 3.26 ± 0.53C: NA	(28 days)A: 1.28 ± 0.86B: 0.82 ± 0.54C: NA	A: −1.84 (−58.97%#)B: −2.44 (−74.85%#)C: NA	No statistically significant difference between groups
Rashed, 2016, Saudi Arabia [12]RCT (parallel)	Systemically healthyPeriodontitis (clinical attachment loss >4 mm in >30% of the sites)PI (O'L)	H2O2 1.5% + scaling and root planning + mechanical oral hygieneTwice daily (quantity and time not reported) during 10 daysNo smokersAge range: 30–50 yrsBaseline: 15/0End: 15/0	CHX 0.2% + scaling and root planing + mechanical oral hygieneTwice daily (quantity and time not reported) during 10 daysNo smokersAge range: 30−50 yrsBaseline: 15/0End: 15/0	Scaling and root planing only + mechanical oral hygieneNot applicableNo smokersAge range: 30−50Baseline: 15/0End: 15/0	A: NR ± NRB: NR ± NRC: NR ± NR	(3 months)A: NR ± NRB: NR ± NRC: NR ± NR	A: NR (NR#)B: NR (NR#)C: NR (NR#)	No statistically significant difference among groups
Sharma, 2019; India [21]RCT (parallel)	Systemically healthy;Halitosis (patients with organoleptic score >1, in a scale of 0 to 5);PI (S & L)	H2O2 1.5% (use of toothbrush and dentifrice NR)Twice daily (10 ml and time not reported) during 15 daysNo smokersMean age: 24.78 yrs (whole-sample)Baseline: NR/NREnd: NR/NR	CHX 2% (use of toothbrush and dentifrice NR)Twice daily (10 ml and time not reported) during 15 daysNo smokersMean age: 24.78 yrs (whole-sample)Baseline: NR/NREnd: NR/NR	NA	A: 1.82 ± 0.13B: 1.78 ± 0.22C: NA	(15 days)A: 1.68 ± 0.12B: 1.50 ± 0.26C: NA	A: −0.14 (−7.69%#)B: −0.28 (−15.73%#)∗C: NA	CHX group showed significantly lower plaque in comparison to H2O2
Studies with experimental gingivitis
Gusberti, 1988, Switzerland [16]Non-RCT (parallel)	Not reportedNot reportedPI (S & L)	H2O2 1%Twice daily (15 ml for 30 s) during 21 daysSmokers NRMean age NRBaseline: NR/NREnd: NR/NR	CHX 0.12%Twice daily (15 ml for 30 s) during 21 daysSmokers NRMean age NRBaseline: NR/NREnd: NR/NR	Flavored alcoholic placebo solutionTwice daily (15 ml for 30 s)Smokers NRMean age NRBaseline: NR/NREnd: NR/NR	A: 0.00 ± 0.00B: 0.00 ± 0.00C: 0.00 ± 0.00	(21 days)A: 1.40 ± NRB: 0.35 ± NRC: 1.56 ± NR	A: 1.40B: 0.35∗C: 1.56	Significantly lower plaque index detected in the CHX group when compared to both groupsNo statistically significant difference was detected between H2O2 and control groups
Hoenderdos, 2009 [22] NetherlandsRCT (parallel)	Systemically healthyPeriodontally healthy (no sites with probing depth >5mm)PIS (Q & H)	H2O2 0.013% + 0.004% glycerol dissolved in demineralized waterTwice daily (15–20 ml for 20 s) during 3 daysSmokers NRMean: 24.5 yrs (whole-sample)Baseline: NR/NREnd: NR/NR	NA	0.004% glycerol dissolved in demineralized waterTwice daily (15–20 ml for 20 s) during 3 daysSmokers NRMean: 24.5 yrs (whole-sample)Baseline: NR/NREnd: NR/NR	A: 0.00 ± 0.00B: NAC: 0.00 ± 0.00	(3 days)A: 2.66 ± 0.29B: NAC: 2.70 ± 0.32	A: 2.66B: NAC: 2.70	No statistically significant difference was detected between groups
Wennström, 1979; Sweden [18]Non-RCT (cross-over)	Systemically healthyOral healthPI (S & L)	H2O2 (1.7 g dissolved in 30 ml of tap water)Three times a day (30 ml per 2 minutes) during 14 daysSmokers NRMean age NRBaseline: NR/NR (total n = 14)End: NR/NR (total n = 14).	NA	Placebo mouthwashThree times a day (30 ml per 2 minutes) during 14 daysSmokers NRMean age NRBaseline: NR/NR (total n = 14)End: NR/NR (total n = 14).	Percent of sites with score 0A: 98.90 ± 0.50B: NAC: 98.70 ± 0.50Percent of sites with score 1A: 1.00 ± 0.40B: NAC: 1.30 ± 0.50Percent of sites with score 2A: 0.10 ± 0.10B: NAC: 0.00 ± 0.00Percent of sites with score 3A: 0.00 ± 0.00B: NAC: 0.00 ± 0.00	(14 days)Percent of sites with score 0A: 46.40 ± 4.30B: NAC: 17.20 ± 2.90Percent of sites with score 1A: 45.20 ± 3.60B: NAC: 37.00 ± 4.80Percent of sites with score 2A: 8.70 ± 3.60B: NAC: 44.40 ± 6.20Percent of sites with score 3A: 0.00 ± 0.00B: NAC: 1.40 ± 0.90	Sites with score 0A: −52.50 (−53.08%)B: NAC: −81.50 (−82.57%)∗∗Sites with score 1A: 44.20 (4420%)B: NAC: 35.70 (2,746%)Sites with score 2A: 8.6 (8,600%)B: NAC: 44.4∗∗Sites with score 3A: 0 (0%)B: NAC: 1.40∗∗	Significantly higher percentage of score 0 was detected in the H2O2 group in comparison to the control groupNo statistically significant difference was detected between groups of score 1Significantly lower percentage of score 2 was detected in the H2O2 group in comparison to the control groupSignificantly lower percentage of score 2 was detected in the H2O2 group in comparison to the control group

NA: not applicable; NR: not reported; RCT: randomized clinical trial; non-RCT: nonrandomized clinical trial; CHX: chlorhexidine. M: male; F: female. A: H2O2 rinse; B: chlorhexidine rinse; C: control rinse. ∆: delta of mean (final−initial mean); %: percentage of reduction. PI (S & L): plaque index (Silness & Löe, 1964); PIS (Q & H): Quigley–Hein plaque modified by Turesky plaque index; PI (L): plaque index (Löe, 1967); PI (O'L): O'Leary plaque index. Statistically significant differences between groups (H2O2 vs chlorhexidine); statistically significant differences between groups (H2O2 vs control); #statistically significant differences within group (end vs baseline score).

(2) Experimental Gingivitis Studies. Among the studies that evaluated plaque parameters, 3 used the experimental gingivitis model [16, 18, 22]. The information coming from these studies gives an idea of efficacy of the mouth rinses in undisturbed dental biofilms. This enhances the proofs of principle of the antiplaque effect, which would give useful information, e.g., for areas where mechanical plaque control is not effective.

3.1.3. Gingival Inflammation

(1) Nonexperimental Gingivitis Studies. The results related to gingival inflammatory parameters are presented in Table 3. Nine studies were included in this outcome [10–12, 15, 16, 18–21], six allowed mechanical plaque control [11, 12, 15, 19–21]. In one study, it was not clear if mechanical plaque control was allowed [10]. In these parameters, H2O2 mouth rinse performs better than negative controls, however less than chlorhexidine. A possibility of a decrease in inflammation could be raised since it seems that H2O2 performs better in terms of gingivitis than it does in relation to plaque.

Table 3

Main characteristics and results for the studies that assess gingival inflammation.

Study, year, country (reference)Study design	Systemic conditions;Diagnosis of the oral conditionGingival index assessed	H2O2 group (concentration);Rinsing protocolN (smokers)AgeBaseline;N(M)/N(F)EndN(M)/N(F)	CHX group (concentration); Rinsing protocolN (smokers);AgeBaseline;N(M)/N(F)EndN(M)/N(F)	Control groupRinsing protocolN (smokers)AgeBaseline;N(M)/N(F)EndN(M)/N(F)	Main results (follow-up)
					Baseline scoresMean ± SD	End scores (follow-up)Mean ± SD	∆ (%)	Author conclusions
Studies without experimental gingivitis
Boyd, 1989, USA [15]Non-RCT	Systemically healthyAdolescents with initial good oral health scheduledto have fixed orthodontic treatmentGI (L & S) and BT (A)	H2O2 1.5% + 0.05% NaF + toothbrushOnce a day (1/2 ounce per one minute) during 18 monthsSmokers NRMean age:13.2 yrsBaseline: 9/17End: 24 individuals in total (proportion of M/F NR)	NA	0.05% NaF + toothbrushOnce a day (1/2 ounce per one minute) during 18 monthsSmokers NRMean: 13.5 yrsBaseline: 11/23End: 32 individuals in total (proportion of M/F NR)	Percent of sites with GI (L & S) >1A: 17.41 ± 20.74B: NAC: 21.61 ± 24.93Percent of sites with BT(A) >1:A: 17.69 ± 21.77B: NAC: 22.70 ± 25.24	(18 months)Percent of sites with GI (L & S) >1A: 27.04 ± 24.69B: NAC: 53.46 ± 29.38Percent of sites with BT (A) >1:A: 24.52 ± 25.77B: NAC: 53.19 ± 30.49	GI (L & S)A: 9.63 (55.31%)B: NAC: 31.85 (147.39%#)∗∗BT (A)A: 6.83 (38.61%)B: NAC: 30.49 (134.32%#)∗∗	When the percentage of sites with gingival index >1 was considered, the group that used H2O2 showed significantly lower percentage when compared to the placebo groupWhen the percentage of sites with bleeding tendency >1 was considered, the group that used H2O2 showed significantly lower percentage when compared to the placebo group
Clark, 1989, USA [19]RCT (parallel)	Systemically healthyGingivitis (at least 8 sites with marginal gingival bleeding)GI (L & S)	H2O2 1.5% (use of toothbrush and dentifrice NR)Once a day (20 ml for 30 s) during 21 weeks2 smokersMean age: 28.9 yrsBaseline: 9/16End: 22 individuals in total (proportion of M/F NR)	NA	Water (use of toothbrush and dentifrice NR)Once a day (20 ml for 30 s) during 21 weeksNo smokersMean age 27.8 yrsBaseline: 8/17End: 22 individuals in total (proportion of M/F NR)	A: 2.20 ± 0.35B: NAC: 2.31 ± 0.24	(6 months)A: 1.62 ± 0.61B: NAC: 1.88 ± 0.50	A: −0.58 (−26.367%)B: NAC: −0.43 (−18.61%)	No statistically significant difference between groups
Gomes, 1984, USA [20]RCT (parallel)	Nonhandicapped staff and handicapped patients—both of them were free of systemic diseasesIndividuals without periodontitis (probing depth <5 mm)GI (L & S)	H2O2 1.5% (use of toothbrush and dentifrice NR)Three times per day (10 ml per 60 s) during 7 daysStaff: Smokers NR Mean age: 30.34 yrsBaseline: 5/34End: NR/NRPatients: smokers NRMean age: 28.65 yrsBaseline: 30/24End: NR/NR	NA	Placebo mouth rinse (use of toothbrush and dentifrice NR)Three times per day (10 ml per 60 s) during 7 daysStaff: Smokers NR Mean age: 29.94 yrsBaseline: 5/30End: NR/NRPatients: Smokers NRMean age: 28.62 yrsBaseline: 25/25End: NR/NR	Staff—maleA: 1.28 ± 0.58B: NAC: 1.00 ± 0.12Staff—femaleA: 1.30 ± 0.46B: NAC: 0.89 ± 0.31Patients—maleA: 1.37 ± 0.48B: NAC: 1.64 ± 0.63Patients—femaleA: 1.36 ± 0.48B: NAC: 1.31 ± 0.59	(7 days)Staff—maleA: 0.73 ± 0.29B: NAC: 1.01 ± 0.32Staff—femaleA: 0.85 ± 0.41B: NAC: 0.86 ± 0.32Patients—maleA: 1.19 ± 0.49B: NAC: 1.58 ± 0.58Patients—femaleA: 1.18 ± 0.42B: NAC: 1.25 ± 0.55	Staff—maleA: −0.55 (−42.97%#)B: NAC: 0.01 (1.00%)Staff—femaleA: −0.45 (−34.62%#)B: NAC: −0.03 (−3.37%)Patients—maleA: −0.18 (−13.14%#)B: NAC: −0.06 (−3.66%)Patients—femaleA: −0.18 (−13.24%#)B: NAC: −0.06 (−4.58%)	In the within group comparison, a significantly lower plaque score was detected, for both staff and patient groups, only for H2O2
Hasturk, 2004; USA [11]RCT (parallel)	Systemically healthyGingivitis (gingival index >2.0 and >10% sites with bleeding)In the first 28 days, only the mouth rinses were usedEIBI; mGI (G) and BoP	H2O2 1.5% + 0.05 sodium fluoride toothpaste + toothbrushTwice daily (quantity not reported for 30 s) during 6 months3.9% smokers (N not provided)Mean age: 31.4 yrsBaseline: 19%/51% (N not provided)End: NR/NR	NA	Placebo mouth rinse 0.05 sodium fluoride toothpaste + toothbrushTwice daily (quantity not reported for 30 s) during 6 months18.8% smokers (N not provided)Mean age: 31.0 yrsBaseline: 45.8%/54.2% (N not provided)End: NR/NR	EIBIA: 0.05 ± 0.14B: NAC: 0.04 ± 0.09mGI (G)A: 1.81 ± 0.41B: NAC: 1.79 ± 0.46BoPA: 0.12 ± 0.17B: NAC: 0.10 ± 0.12	(6 months)EIBIA: 0.02 ± NRB: NAC: 0.02 ± NRmGI (G)A: 1.63 ± NRB: NAC: 1.83 ± NRBoPA: 0.08 ± NRB: NAC: 0.10 ± NR	EIBIA: −0.03 (−64.34%#)B: NAC: −0.02 (−38.42%#)mGI (G)A: −0.18 (−9.95%#)B: NAC: 0.04 (2.23%)∗∗BoPA: −0.02 (−20%)B: NAC: −0.02 (−16.81%)	The decrease in the H2O2 group was significant in comparison to change in the placebo group (P = 0.004) when considering the mGI (G)No other statistically significant difference between groups was observed
Porwal, 2018, India [10]RCT (parallel)	Systemically healthyGingivitis (modified gingival index of 3 and plaque index of 4)mGI (L)	H2O2 3% with distilled water (ratio 1:1)Twice daily (10 ml and time not reported) during 15 daysSmokers NRMean age: NRBaseline: 10 individuals in total (proportion of M/F NR)End: 10 individuals in total (proportion of M/F NR)	CHX 0.2% with distilled water (ratio 1:1)Twice daily (10 ml and time not reported) during 15 daysSmokers NRMean age: NRBaseline: 10 individuals in total (proportion of M/F NR)End: 10 individuals in total (proportion of M/F NR)	NA	A: 2.92 ± 0.31B: 3.04 ± 0.23C: NA	(28 days)A: 0.86 ± 0.11B: 0.54 ± 0.35C: NA	A: −2.06 (−70.55%#)B: −2.50 (−82.24%#)C: NA	No statistically significant difference between groups
Rashed, 2016, Saudi Arabia [12]RCT (parallel)	Systemically healthyPeriodontitis (clinical attachment loss >4 mm in >30% of the sites)GI (L & S)	H2O2 1.5% + scaling and root planing + mechanical oral hygieneTwice daily (quantity and time not reported) during 10 daysNo smokersAge range: 30–50 yrsBaseline: 15/0End: 15/0	CHX 0.2% + scaling and root planing + mechanical oral hygieneTwice daily (quantity and time not reported) during 10 daysNo smokersAge range: 30–50 yrsBaseline: 15/0End: 15/0	Scaling and root planing only + mechanical oral hygieneNot applicableNo smokersAge range: 30–50Baseline: 15/0End: 15/0	A: NR ± NRB: NR ± NRC: NR ± NR	(3 months)A: NR ± NRB: NR ± NRC: NR ± NR	A: NR (NR#)B: NR (NR#)C: NR (NR#)∗∗	No statistically significant difference between CHX and H2O2 groupsStatistically significant lower gingival index in H2O2 in comparison to the control group
Sharma, 2019; India [21]RCT (parallel)	Systemically healthyHalitosis (patients with organoleptic score >1, in a scale of 0 to 5)GI (L & S)	H2O2 1.5% (use of toothbrush and dentifrice NR)Twice daily (10 ml and time not reported) during 15 daysNo smokersMean age: 24.78 yrs (whole sample)Baseline: NR/NREnd: NR/NR	CHX 2% (use of toothbrush and dentifrice NR)Twice daily (10 ml and time not reported) during 15 daysNo smokersMean age: 24.78 yrs (whole sample)Baseline: NR/NREnd: NR/NR	NA	A: 1.83 ± 0.35B: 1.74 ± 0.17C: NA	(15 days)A: 1.80 ± 0.34B: 1.66 ± 0.17C: NA	A: −0.03 (−1.64%#)B: −0.08 (−4.60%#)∗C: NA	CHX group showed significantly lower gingival index scores in comparison to H2O2
Studies with experimental gingivitis
Gusberti, 1988, Switzerland [16]Non-RCT (parallel)	Not reportedNot reportedGI (L & S)	H2O2 1%Twice daily (15 ml for 30 s) during 21 daysSmokers NRMean age NRBaseline: NR/NREnd: NR/NR	CHX 0.12%Twice daily (15 ml for 30 s) during 21 daysSmokers NRMean age NRBaseline: NR/NREnd: NR/NR	Flavored alcoholic placebo solutionTwice daily (15 ml for 30 s)Smokers NRMean age NRBaseline: NR/NREnd: NR/NR	A: 0.00 ± 0.00B: 0.00 ± 0.00C: 0.00 ± 0.00	(21 days)A: 1.27 ± NRB: 0.06 ± NRC: 1.49 ± NR	A: 1.27B: 0.06∗C: 1.49∗∗	Statistically significant lower gingival index was detected in the CHX group when compared to both groupsStatistically significant lower gingival index was detected in the H2O2 group when compared to the placebo group
Wennström, 1979, Sweden [18]Non-RCT (cross-over)	Systemically healthyOral healthGI (L & S)	H2O2 (1.7g dissolved in 30 ml of tap water)Three times a day (30 ml per 2 minutes) during 14 daysSmokers NRMean age NRBaseline: NR/NR (total n = 14)End: NR/NR (total n = 14)	NA	Placebo mouthwashThree times a day (30 ml per 2 minutes) during 14 daysSmokers NRMean age NRBaseline: NR/NR (total n = 14);End: NR/NR (total n = 14)	Percent of sites with score 0A: 72.10 ± 1.80B: NAC: 74.00 ± 2.30Percent of sites with score 1A: 26.4 ± 1.70B: NAC: 25.4 ± 2.20Percent of sites with score 2A: 1.50 ± 0.60B: NAC: 0.60 ± 0.30Percent of sites with score 3A: 0.00 ± 0.00B: NAC: 0.00 ± 0.00	(14 days)Percent of sites with score 0A: 27.40 ± 7.30B: NAC: 13.00 ± 2.30Percent of sites with score 1A: 53.30 ± 1.40B: NAC: 54.90 ± 3.40Percent of sites with score 2A: 19.40 ± 2.40B: NAC: 31.60 ± 4.60Percent of sites with score 3A: 0.00 ± 0.00B: NAC: 0.00 ± 0.00	Sites with score 0A: −44.70 (−62.00%)B: NAC: −61.00 (−82.43%)∗∗Sites with score 1A: 26.90 (101.89%)B: NAC: 29.50 (116.14%)Sites with score 2A: 17.9 (1.193%)B: NAC: 31 (5,166%)∗∗Sites with score 3A: 0 (0%)B: NAC: 0 (0%)	Significantly higher percentage of score 0 was detected in the H2O2 group in comparison to the control groupSignificantly a lower percentage of score 1 was detected in the H2O2 group in comparison to the control groupSignificantly a lower percentage of score 2 was detected in the H2O2 group in comparison to the control groupNo statistically significant difference was detected between groups for percentage of score 3

NA: not applicable; NR: not reported; RCT: randomized clinical trial; non-RCT: nonrandomized clinical trial; CHX: chlorhexidine; M: male; F: female. A: H2O2 rinse; B: chlorhexidine rinse; C: control rinse. ∆: delta of mean (final−initial mean); %: percentage of reduction. GI (L & S): gingival index (Löe & Silness, 1963); BT (A): bleeding tendency (Armitage et al., 1982); EIBI: Eastman interdental bleeding index; mGI (G); modified gingival index (Gordon et al., 1985); BoP: bleeding on probing; mGI (L): modified gingival index (Lobene et al., 1989). Statistically significant differences between groups (H2O2 vs chlorhexidine); statistically significant differences between groups (H2O2 vs control); #statistically significant differences within group (end vs baseline score).

(2) Experimental Gingivitis Studies. Among the studies that evaluated gingival inflammation, two were based on the experimental gingivitis model [16, 18]. The experimental gingivitis model provides information on the effect of the mouth rinse in areas in which plaque control is not adequate. Also, it rules out the eventual confounding effect of the adjunct plaque control in the study of chemical substances.

3.1.4. Microbiological Outcomes

(1) Nonexperimental Gingivitis Studies. The results related to microbiological parameters are demonstrated in Table 4. Six studies were included with these outcomes [11, 13, 14, 16–18]. Four studies allowed mechanical control of biofilm [11, 13, 14, 17]. Better results with mouth rinses containing H2O2 when compared to a placebo were detected.

Table 4

Main characteristics and results for the studies that performed microbiological analysis.

Study, year, country (reference)Study design	Systemic conditionsDiagnosis of the oral conditionMicrobiological analysis performed	H2O2 group (concentration)Rinsing protocolN (smokers)AgeBaselineN(M)/N(F)EndN(M)/N(F)	Chlorhexidine group (concentration), rinsing protocolN (smokers)AgeBaselineN(M)/N(F)EndN(M)/N(F)	Control groupRinsing protocolN (smokers)Mean ageBaselineN(M)/N(F)EndN(M)/N(F)	Main results
					Baseline scoresMean ± SD	End scores (follow-up)Mean ± SD	∆ (%)	Author conclusions
Studies without experimental gingivitis
Hasturk, 2004; USA [11]RCT (parallel)	Systemically healthyGingivitis (gingival index >2.0 and >10% sites with bleeding). In the first 28 days, only the mouth rinses were used.F. nucleatum ss. vincenti, C. concisus, C. rectus, T. forsythensis, P. gingivalis, P. intermedia, P. nigrescens, C. sputigena, S. oralis, A. naeslundii, T. denticola, C. curva, and E. corrodens	H2O2 1.5% + 0.05 sodium fluoride toothpaste + toothbrushTwice daily (quantity not reported for 30 s) during 6 months3.9% smokers (N not provided)Mean age: 31.4 yrsBaseline: 19%/51% (N not provided)End: NR/NR	Not applicable	Placebo mouth rinse 0.05 sodium fluoride toothpaste + toothbrushTwice daily (quantity not reported for 30 s) during 6 months18.8% smokers (N not provided)Mean age: 31.0 yrsBaseline: 45.8%/54.2% (N not provided)End: NR/NR	A: NRB: NAC: NR	(6 months)A: NRB: NAC: NR	A: NRB: NAC: NR	Various degrees of reductions in F. nucleatum ss. vincentii, C. concisus, C. rectus, T. forsythensis, P. gingivalis, P nigrescens, C. sputigina, and E. corrodens, with the use of the test rinse but not the placeboNone of these changes, however, were statistically significant
Matula, 1988,Austria [13]RCT (cross-over)	Systemically healthyNot reportedTotal anaerobic/aerobic microbial counts	H2O2 3% aqueous solutionSingle usage (12 ml for 60 s)Smokers NRMean age: 23–49 yrsBaseline: NR/NR (total n = 12)End: NR/NR (total n = 12)	Not applicable	WaterSingle usage (12 ml for 60 s)Smokers NRMean age: 23–49 yrsBaseline: NR/NR (total n = 12)End: NR/NR (total n = 12)	Mean aerobic bacteriaA: 100%B: NAC: 100%Mean anaerobic bactériaA: 100%B: NAC: 100%	(60 minutes)Mean aerobic bacteriaA: 49.60% ± NRB: NAC: 103.10% ± NRMean anaerobic bactériaA: 41.40% ± NRB: NAC: 112.00% ± NR	Mean aerobic bacteriaA: 50.40%B: NAC: 3.10%∗∗Mean anaerobic bactériaA: 58.60%B: NAC: 12∗∗	H2O2 presented higher reductions in both aerobic and anaerobic bacteria as compared to control, except for aerobic bacteria at 30 minutes
Menendez, 2005, USA [14]RCT (cross-over)	Systemically healthyNot reportedTotal Streptococci levels and S. mutans levels in saliva	H2O2 1.5%Twice daily (15 ml for 60s) during 21 daysSmokers NRMean age: 26–55 yrsBaseline: NR/NR (total n = 16)End: NR/NR (total n = 16)	CHX 0.12%Twice daily (15 ml for 60 s) during 21 daysSmokers NRMean age: 26–55 yrsBaseline: NR/NR (total n = 16)End: NR/NR (total n = 16)	PlaceboTwice daily (15 ml for 60 s) during 21 daysSmokers NRMean age: 26–55 yrsBaseline: NR/NR (total n = 16)End: NR/NR (total n = 16)	Total StreptococciA: NRB: NRC: NRS. mutans (in CFU/ml)A: NRB: NRC: NR	(21 days)Total StreptococciA: 885857 ± 218478B: 121465 ± 273913C: 908645 ± 211957S. mutans (in CFU/ml)A: 10442 ± 7845B: 11614 ± 7685C: 1696 ± 1933	Total StreptococciA: NRB: NRC: NRS. mutans (in CFU/ml)A: NRB: NRC: NR	Chlorhexidine performed better in total level of Streptococcus, as compared to H2O2, that did not differ from placeboNo statistically significant differences were observed in S. mutans among groups
Pitten, 1999, Germany [17]Non-RCT	Systemically healthyNot reportedReduction factors of log CFU	H2O2 3%Single usage (20 ml, 30 seconds, followed by 20 ml of sterile water for additional 30 seconds)Smokers NRMean age: NRBaseline: NR/NR (total n = at least 10)End: NR/NR (total n = at least 10)	CHX 0.2%Single usage (20 ml, 30 seconds, followed by 20 ml of sterile water for additional 30 seconds)Smokers NRMean age: NRBaseline: NR/NR (total n = at least 10)End: NR/NR (total n = at least 10)	Distilled sterile waterSingle usage (20 ml, 30 seconds, followed by another 20 ml of sterile water for additional 30 seconds)Smokers NRMean age: NRBaseline: NR/NR (total n = at least 10)End: NR/NR (total n = at least 10	A: NRB: NRC: NR	(60 minutes)A: 0.35 ± NRB: 1.38 ± NRC: −0.06 ± NR	A: NRB: NR∗C: NR∗∗	Mean values of the reduction factor of log10 CFU demonstrated a higher reduction with chlorhexidine, followed by H2O2, which also performed better than distilled sterile water
Studies with experimental gingivitis
Gusberti, 1988, Switzerland [16]Non-RCT (parallel)	Not reportedNot reportedTotal facultative anaerobes, total cultivable microbiota, Streptococci, actinomyces, A. naeslundii, A. viscosus, Fusobacteria, Veillonella, and Capnocytophaga	H2O2 1%Twice daily (15 ml for 30 s) during 21 daysSmokers NRMean age NRBaseline: NR/NR (total n = 10)End: NR/NR (total n = 10)	CHX 0.12%Twice daily (15 ml for 30s) during 21 daysSmokers NRMean age NRBaseline: NR/NR (total n = 11)End: NR/NR (total n = 11)	Flavored alcoholic placebo solutionTwice daily (15 ml for 30 s)Smokers NRMean age NRBaseline: NR/NR (total n = 11)End: NR/NR (total n = 11)	To all microbiological analysesA: NRB: NRC: NR	(60 minutes)All analyses are reported in log10 CFU/teethTotal facultative anaerobesA: 8.35 ± 0.08B: 7.28 ± 0.11C: 8.05 ± 0.17Total cultivable microbiotaA: 8.57 ± 0.09B: 7.55 ± 0.09C: 8.33 ± 0.15StreptococciA: 8.14 ± 0.08B: 7.34 ± 0.12C: 7.73 ± 0.15ActinomycesA: 8.14 ± 0.09B: 5.61 ± 0.29C: 7.72 ± 0.22A. naeslundiiA: 7.49 ± 0.21B: 5.31 ± 0.29C: 6.70 ± 0.39A. viscosusA: 7.75 ± 0.22B: 4.74 ± 0.29C: 7.57 ± 0.24FusobacteriaA: 5.17 ± 0.32B: 4.87 ± 0.20C: 6.36 ± 0.23VeillonellaA: 6.66 ± 0.18B: 5.98 ± 0.20C: 7.23 ± 0.23CapnocytophagaA: 5.55 ± 0.34B: 5.10 ± 0.18C: 6.60 ± 0.26	A: NRB: NR∗C: NRA: NRB: NR∗C: NRA: NRB: NR∗C: NRA: NRB: NR∗C: NRA: NRB: NR∗C: NRA: NRB: NR∗C: NRA: NRB: NR∗C: NR∗∗A: NRB: NR∗C: NR∗∗A: NRB: NR∗C: NR∗∗	Chlorhexidine 0.12% demonstrated a broad-spectrum activity with significant reduction in the number of both facultative and strict anaerobe However, H2O2 1% did not affect total cultivable microbiota or facultative bacterial species such as Streptococci and ActinomycesIn relation to strict anaerobes (e.g., Fusobacterium and Veillonella), H2O2 1% was significantly less effective as compared to 0.12% chlorhexidine
Wennström, 1979 [18], SwedenNon-RCT (cross-over)	Systemically healthyOral healthCocoid cells/straight rods, filaments, fusiforms, motile/curved rods, and spirochetes	H2O2 (1.7 g dissolved in 30 ml of tap water)Three times a day (30 ml per 2 minutes) during 14 daysSmokers NRMean age NRBaseline: NR/NR (total n = 14)End: NR/NR (total n = 14)	Not applicable	Placebo mouthwashThree times a day (30 ml per 2 minutes) during 14 daysSmokers NRMean age NRBaseline: NR/NR (total n = 14)End: NR/NR (total n = 14)	Coccoid cells +straight rodsA: 88.20 ± 4.60B: NAC: 84.20 ± 3.90FilamentsA: 4.70 ± 1.80B: NAC: 7.00 ± 1.80FusiformsA: 4.00 ± 1.30B: NAC: 4.30 ± 2.00Motile + curved rodsA: 3.00 ± 1.20B: NAC: 4.50 ± 1.30SpirochetesA: 0.10 ± 0.10B: NAC: 0.10 ± 0.10	(14 days)Coccoid cells + straight rodsA: 84.90 ± 4.70B: NAC: 40.40 ± 6.00FilamentsA: 6.90 ± 2.00B: NAC: 17.70 ± 1.80FusiformsA: 5.40 ± 1.80B: NAC: 14.90 ± 1.80Motile + curved rodsA: 2.60 ± 1.80B: NAC: 22.10 ± 4.00SpirochetesA: 0.10 ± 0.10B: NAC: 4.90 ± 1.80	Coccoid cells + straight rodsA: 3.30 (−3.74%)B: NAC: −43.80 (−52.02%#)∗∗FilamentsA: 2.20 (46.81%)B: NAC: 10.70 (152.86%#)∗∗FusiformsA: 1.40 (35.00%)B: NAC: 10.60 (246.51%#)∗∗Motile + curved rodsA: −0.40 (−13.33%)B: NAC: 17.60 (391.11%#)∗∗SpirochetesA: 0.00 (0.00%)B: NAC: 17.6 (17600%#)∗∗	H2O2 mouthwash prevented the colonization of filaments, fusiforms, motile, and curved rods as well as spirochetesStatistically significant differences were observed in relation to placebo

NA: not applicable; NR: not reported; RCT: randomized clinical trial; non-RCT: nonrandomized clinical trial; CHX: chlorhexidine; M: male; F: female. A: H2O2 rinse; B: chlorhexidine rinse; C: control rinse. ∆: delta of mean (final−initial mean); %: percentage of reduction. Statistically significant differences between groups (H2O2 vs chlorhexidine); statistically significant differences between groups (H2O2 vs control); #statistically significant differences within group (end vs baseline score).

(2) Experimental Gingivitis Studies. Two studies performed microbiological analysis using an experimental gingivitis design [16, 18]. The information coming from such studies supports the quality/quantity of different germs when plaque is accumulating overtime. It also rules out the effect of the uncontrolled mechanical plaque removal.

3.1.5. Qualitative Results—Safety

Among the 13 included studies, only five of them assessed for side effects. All of these five studies reported no side effects in individuals that used H2O2 mouthwashes [11, 14–16, 19]. Additionally, no side effects were reported in those that used chlorhexidine [14, 16]. Conversely, an increased tendency for desquamation of the mucosal lining was reported in individuals that used a placebo solution [16]. The other studies that used a negative control group reported no side effect in this group [11, 14, 15, 19].

3.2. Strengths and Limitations of SR

The present systematic review aimed to assess the eventual effects of mouth rinses with H2O2 on plaque, gingivitis, and different germs in the oral cavity. For that, five databases were searched, and 13 articles were methodologically appraised. In general, H2O2 mouth rinses demonstrated an effect on the three parameters under the study in different degrees.

Regarding the risk of bias of both randomized and nonrandomized clinical trials, it is important to highlight that most of the included studies presented an unclear or high risk of bias. Only one study demonstrated an overall low risk of bias [11]. This randomized clinical trial demonstrated a superior antigingivitis efficacy of H2O2 mouthwash in comparison to a placebo solution. However, no significant difference was observed for the antiplaque efficacy. The overall high risk of bias must be put into perspective when interpreting the results of the present study. This means that the use in clinical practice should be indicated with caution and not performed routinely since the support is not robust.

The strengths of the present systematic review were based on the importance of the topic, especially because the mouth is a very contaminated cavity and mouth rinses are used to reduce different degrees of contamination. In addition, with the COVID-19 pandemic, the use of mouth rinses has been considered an additional way for reducing all sorts of contamination. The limitations are related to the quality of the evidence. Therefore, the information contained herein should be cautiously interpreted. Also, in an attempt to decrease the time for publication of this information, no registration was performed and it was not possible to make a post hoc registration.

3.3. Quality of Evidence and Strength of Recommendation

Initially, the focused question included both a negative and a positive control group. The negative control could be either placebo, water, or no solution, whereas the control group should include the gold standard in terms of oral rinse—chlorhexidine. The results of this systematic review should be put into the perspective that H2O2 is widely used in oral care despite the lack of a large number of studies, especially in some of the aforementioned indications. We looked at the systematic review published by Hossainian et al. [3] that critically appraised the evidence until the beginning of this decade. Such work led to the conclusion that H2O2 does not consistently prevent plaque accumulation in short-term periods. Therefore, we expanded the search criteria, not restricting age, including microbiological parameters, updating the publication year to 2020, and including five databases instead of the two previously searched databases. Due to the higher usage of H2O2, we restricted the search to only include this substance and not any other oxygenating agent.

H2O2 has been used clinically for more than a century, and recently, H2O2 containing mouth rinse are being recommended, especially due to a possible antiviral effect and the pandemic of COVID-19. To the best of the authors' knowledge and making a systematic search in the same databases, no studies have observed any antiviral effect of H2O2 in the mouth. However, associations are supporting its use [5, 6]. The present systematic review used the most strict quality criteria for retrieving the studies. However, the interpretation will be contextualized in the moment that the world is facing a pandemic in which any kind of effort should be at least collated to make the sense of any preventive guideline.

In terms of plaque, one study [15] was performed in adolescents and the others in adults. One of them also included handicapped individuals [20]. Four of the six studies that allowed oral hygiene compared 1.5% H2O2 with a negative control [11, 15, 19, 20] and 2 of them with chlorhexidine [12, 21], and one of them was also compared to a negative control [12]. Among the studies that used the experimental gingivitis model [16, 18, 22], two were compared with a negative control [18, 22] and the other included a positive control [16]. In one study, the effect of H2O2 was compared to chlorhexidine, but it was not possible to determine if mechanical plaque control was allowed [10]. In these studies, different concentrations of H2O2 were used.

It is clear from the encountered results that 1.5% H2O2 is the most studied concentration in the formula of a mouth rinse. This result is in accordance with the previously published review [3]. For the publications evaluating the effect of H2O2 on plaque, only one study (which evaluated the antiplaque effect over an 18-month time period) demonstrated improved results when compared to a placebo [15]. The other studies, which evaluated the effect over shorter periods, did not find statistically significant differences. Also, in the studies that used the experimental gingivitis model, only one study demonstrated the superiority of H2O2 in comparison to placebo [18].

The same cited publications that evaluated plaque also evaluated the effect of H2O2 on gingival inflammation. Although only a single study demonstrated the antiplaque benefit of H2O2, more studies clearly point to a better antigingivitis effect of H2O2 mouth rinses as compared to placebo [11, 15, 20]. In fact, for one of the studies, no difference was observed between the H2O2 mouth rinse and the positive control [12]. Because the participants of these studies were allowed for routine mechanical oral hygiene, an effect on clinical inflammation alone (without having the associated plaque reduction benefit) should be highlighted. These results suggest that H2O2 might perform differently in terms of plaque and gingivitis, which is of great clinical interest.

Also, it is of high importance to evaluate the effect of mouth rinses on the oral microbiome. This includes not only bacteria but also other germs, such as viruses and fungi. However, despite completing a broad search of the literature, no studies were identified that evaluated the effect of H2O2 oral microorganisms other than bacteria. The comparisons of the effect of rinses on oral bacteria with H2O2 and with the positive control generally demonstrate a better effect of the latter. However, the differences in terms of the composition of the oral microbiome when H2O2 is compared to placebo are clear in a variety of bacterial species. The present study evaluated risk of bias both for the nonrandomized and randomized trials. As expected, the randomized clinical trials presented a higher quality, with decreased risk of bias. The nonrandomized studies in general present a higher risk of bias. This is inherent to the chosen design. Randomized studies tend to present a lower risk of bias.

A systematic review was recently published by Marui et al. [23] describing the effect of preprocedural rinses with different substances on dental office-generated aerosols. They demonstrated that rinses with chlorhexidine, essential oils, and cetylpyridinium chloride are effective. No studies with H2O2 were included.

3.4. Implications for Further Research

Meanwhile, taking into consideration the precautionary principle [24], even without the qualified evidence, due to the high levels of morbimortality, it is of interest to see other potentials of the use of H2O2. In such conditions, the use of “collateral evidence” is recommended, always with a surveillance look. Therefore, in the present moment, further studies including oral rinses with H2O2 and other substances are warranted. Studies with the antiviral effect of H2O2 are also needed.

4. Conclusions

In conclusion, rinsing with 1.5% H2O2 has demonstrated an antigingivitis effect as compared to placebo, with also greater reductions in oral bacteria. Chlorhexidine has demonstrated, up to now, the best antiplaque and antigingivitis effect on the oral microbiome.