International Dental Journal 2012; 62: 208–212

doi: 10.1111/j.1875-595X.2011.00111.x

Effects of 0.05% sodium hypochlorite oral rinseon supragingival biofilm and gingival inflammation

Rodrigo De Nardo1, Veronica Chiappe1, Mariel Gomez1, Hugo Romanelli1 and Jørgen Slots2

1Department of Periodontology, School of Dentistry, Maimonides University, Buenos Aires, Argentina; 2Department of Periodontology, School ofDentistry, University of Southern California, Los Angeles, CA, USA.

Objective: This study aimed to evaluate the clinical effects of 0.05% sodium hypochlorite mouth rinse on supragingivalbiofilm and gingival inflammation. Methods: The study was performed as a controlled, randomised, investigator-blinded,parallel group trial in 40 prison inmates. Following a preparatory period to obtain a plaque- and gingivitis-free dentition,tooth-brushing was substituted for 21 days by supervised twice daily rinsing with either 15 ml of fresh solution 0.05%sodium hypochlorite or 15 ml of distilled water. Clinical outcomes were assessed using the Quigley–Hein Plaque Index(QHPI), the Loe and Silness Gingival Index (L&SGI) and bleeding on probing. Adverse events were evaluated byquestionnaire, visual examination and clinical photographs. Results: At day 21, the average QHPI score had increased to3.82 in the water rinse group and 1.98 in the sodium hypochlorite rinse group. The average L&SGI score had increased to2.1 in the water rinse group and 1.0 in the sodium hypochlorite rinse group, and the average percentage of sites that bled onprobing had increased to 93.1% in the water rinse group and 56.7% in the sodium hypochlorite rinse group. Differenceswere statistically significant (P = 0.001). A brown extrinsic tooth stain along the gingival margin appeared in 100% ofparticipants in the sodium hypochlorite rinse group and in 35.0% of participants in the water rinse group (P < 0.05).Conclusions: An oral rinse with 0.05% sodium hypochlorite resulted in significant reductions in supragingival biofilmaccumulation and gingival inflammation. Dilute sodium hypochlorite may represent an efficacious, safe and affordableantimicrobial agent in the prevention and treatment of periodontal disease.

Keywords: Sodium hypochlorite, household bleach, biofilm, plaque index, gingival index, mouth rinse, periodontal treatment

Periodontal disease and dental caries are associatedwith specific pathogenic bacteria harboured in oralbiofilms. The successful prevention and treatment of thetwo major dental diseases are contingent upon theeffective control of odontopathic biofilms. Basic treat-ment in periodontics and cariology includes themechanical removal of dental biofilms, but mechanicaltreatment alone may sometimes fail to resolve odonto-pathic infections.

Scaling and root planing have long represented thereference standard in periodontal treatment. However,it is difficult to remove periodontopathic bacteria fromdeep periodontal pockets or furcation defects, and,although commercial antibiotic products for subgingi-val placement have become available, they are not idealbecause their clinical efficacy is limited and acquisitioncosts are high1. The drawbacks of topical antibiotictherapy include an insufficient range of antimicrobialactivity for even broad-spectrum antibiotics and therisk that resistance to the antibiotic employed and tomultiple drugs will develop. In addition, no antibiotic

against bacteria will cover all periodontopathic speciesor affect periodontal viruses and yeasts.

Antiseptics attack multiple components of bacteria,viruses and yeasts, practically eliminating the risk forresistance development, and do not interact withprescription medications. Antiseptics are especiallyimportant in the treatment of biofilm infections, whichmay be unresponsive to even high concentrations ofsystemic antibiotics2. Furthermore, as relatively smallamounts of antimicrobial agents are applied subgingi-vally and the content of inflamed periodontal pockets isemptied into the oral cavity every 90 s3, the risk thatantiseptics might enter the gingival tissue and causesystemic damage is virtually non-existent.

Research has begun to examine the utility of low-costantiseptic agents as adjuncts to mechanical periodontaltherapy. Sodium hypochlorite has a century-long his-tory of use as a root canal irrigant at concentrationsranging from 1.0% to 5.25%4. Sodium hypochloriterinse also exerts high antimicrobial activity against oralbiofilms5,6. Lobene et al.7 used college students who

208 ª 2012 FDI World Dental Federation

O R I G I N A L A R T I C L E

were required to abstain from oral hygiene to study theclinical effects of an investigator-administered rinsingof tooth surfaces with 0.5% (5,000 ppm) sodiumhypochlorite (Carrel–Dakin solution) in a water-pressure cleansing device. The sodium hypochloriterinse produced a 47% greater reduction in the amountof dental plaque compared with the water rinse, andlow pretreatment gingivitis scores were maintainedaround teeth rinsed in sodium hypochlorite, whereasgingivitis scores for water-rinsed sites increased by50%7. Lobene et al.7 also found that sodium hypo-chlorite rinse interfered for ‡ 24 h with the ability ofdental plaque to produce acid following a sucrosechallenge, which suggests a potential anti-caries effect.

As sodium hypochlorite occurs naturally in humanneutrophils, monocytes and macrophages8, it does notevoke allergic reactions, is not a mutagen, carcinogenor teratogen, and has a century-long safety record9. TheAmerican Dental Association Council on Dental Ther-apeutics has designated 0.1% sodium hypochlorite a‘mild antiseptic mouth rinse’ and suggested its use fordirect application to mucous membranes10. Dilutesodium hypochlorite has no contraindications. Its highdegree of safety permits frequent and broad usage byboth dentists and patients. Sodium hypochlorite isavailable globally at exceptionally low cost as house-hold bleach in concentrations of 5–6%.

Theusefulnessofdilutesodiumhypochloriteasamouthrinse in the treatment of periodontal disease warrantsfurther study. The specific aim of the present study was toevaluate the effects of 0.05% sodium hypochlorite onsupragingival biofilm and gingival inflammation. Thestudy was designed as a variant of the experimentalgingivitis protocol with administration a supervisedtwice-daily sodium hypochlorite oral rinse for 21 days.

METHODS

Study population

A total of 44 male inmates at the Men’s Prison of theProvince of Formosa, Argentina were entered into thestudy. Study subjects were required to have at least 20natural teeth, healthy gingivae or slight periodontitiswith clinical attachment loss of £ 2 mm. Exclusioncriteria were the use of systemic or topical antibiotictherapy within 6 months prior to the initiation of thestudy, the presence of systemic diseases such asdiabetes, clotting disorder or human immunodeficiencyvirus (HIV) infection, acute necrotising gingival disease,immunosuppressive drug therapy or use of medicationsproducing gingival enlargement, smoking of > 10cigarettes ⁄ day, reduced salivary flow, current ortho-dontic treatment, or failure to consent to participate inthe study. The study was conducted according to theprotocol outlined by the Research Committee at

Maimonides University and was approved by the EthicsCommittee of Maimonides University School of Den-tistry. The study subjects signed informed consent afterthe nature and the risks of the study had beenthoroughly explained. The research was conducted infull accordance with the World Medical AssociationDeclaration of Helsinki 1975 (revised in 2008).

Sodium hypochlorite

A 10% (101 g ⁄ l) sodium hypochlorite stock solutionwas purchased from a chemical drugstore. The 0.05%working solution was obtained by mixing 5 ml of thestock solution with 995 ml distilled water. A freshsodium hypochlorite working solution was made every24 h and stored in dark disposable bottles.

Study design

This study was performed as a randomised, controlled,single-blinded, clinical trial in parallel groups accordingto the CONSORT criteria11. Participants receivedperiodontal treatment over a pre-experimental periodof 30 days in order to establish gingival health. Studysubjects underwent initially supra- and then subgingivalscaling, as well as professional polishing with a rubbercup and dentifrice and interproximal cleaning usingdental floss every 2 days. In addition, all individuals wereenrolled in an oral hygiene motivation session, whichemphasised use of the Bass tooth-brushing techniquetwice per day and flossing once per day. Subjects wereeligible to participate in the study if ‡ 75% of all toothsites showed absence of bleeding on probing. Shortlybefore the baseline examination, all subjects receiveddental prophylaxis to remove any remaining supragingi-val plaque and stain. Teeth with prosthetic restorationsor non-restored dental caries were not included inthe study.

Clinical assessment was based on the Quigley–HeinPlaque Index (QHPI)12 as modified by Turesky et al.13,a modified Loe and Silness Gingival Index (L&SGI) todetermine visual signs of inflammation14, and thepresence or absence of bleeding on probing to thebottom of the pocket, which was examined as anindependent variable. Probing was carried out using aMarquis CP 12 probe at four sites at each tooth(mesiofacial, midfacial, distofacial, midlingual). Clini-cal photographs of the anterior teeth were used toevaluate the presence or absence of dental stain.Clinical measurements and photographs were obtainedat baseline, every 7 days, and at the termination of thestudy on day 21. Measurements were obtained by onecalibrated examiner (RDN), who was masked to themouth rinse used by the subjects. An evaluation ofintra-examiner calibration, performed in five patients,yielded an agreement of > 85%.

ª 2012 FDI World Dental Federation 209

Sodium hypochlorite and supragingival biofilm

Treatment protocol

The study subjects were instructed to halt all oralhygiene measures for 21 days. Twenty subjects wererandomly assigned to rinsing with 0.05% sodiumhypochlorite and 20 to rinsing with distilled water.Subjects rinsed twice daily for 60 s with 15 ml of theassigned study solution. Rinsing was supervised by aprofessional dental practitioner other than the dentalexaminer.

Scores on the QHPI and L&SGI and the percentage ofsites that bled on probing were recorded at baseline and atthe termination of the study. Subjects were also moni-tored weekly using a questionnaire and a visual test inorder to determine possible adverse drug effects on hardand soft tissues. After completing the study, the subjectsreceived professional prophylaxis and oral hygiene rein-forcement followed by topical fluoride application.

Statistical analysis

The difference between baseline and post-rinsing obser-vationswasdeterminedforeachtoothsite,andthemediandifference was calculated for each patient. Differencesbetween the test and control groups were analysed usingthe Mann–Whitney test. P-values of £ 0.05% wereconsidered to indicate statistical significance.

RESULTS

Forty of the 44 enrolled subjects completed the study.Excluded from the study were three individuals who didnot achieve healthy gingival status and one individualwho required antibiotic medication for medical reasonsduring the study. The average age of the studyparticipants was 27.8 ± 5.6 years.

Table 1 lists the key clinical findings of the study.Baseline data showed no difference between the twostudy groups for any of the variables evaluated. Plaqueand staining were absent after the professional prophy-laxis. At day 21, the average QHPI score had increasedto 3.82 in the water rinse group and to 1.98 in thesodium hypochlorite rinse group; the average L&SGI

score had increased to 2.1 in the water rinse group andto 1.0 in the sodium hypochlorite rinse group, and theaverage percentage of sites that bled on probing hadincreased to 93.1% in the water rinse group and to56.7% in the sodium hypochlorite rinse group. Alldifferences were statistically significant (P = 0.001).

Extrinsic brown tooth stains (Figure 1) appeared in100% of the subjects in the sodium hypochlorite groupand in 35.0% of the subjects in the water rinse group(P < 0.05). Stain removal was relatively easy and wasoften accomplished by intensive conventional oralhygiene measures. Examination of the oral mucosarevealed redness of the tongue in 35.0% (95% confi-dence interval [CI] 15.3–77) of subjects using sodiumhypochlorite and no detectable changes in the rest ofthe mouth. Tooth decalcifications were not observed.

All (95% CI 83.1–100) participants in the sodiumhypochlorite group reported ‘bleach taste’. A total of85.0% (95% CI 62.1–96.8) of subjects in this groupdescribed the mouth rinse as ‘tolerable’, 15.0% (95%CI 3.2–37.9) described it as ‘moderately tolerable’ and45.0% (95% CI 23.0–68.5) reported a burning sensa-tion. Subjects in the sodium hypochlorite groupreported a cleaner mouth and less bad breath despitenot brushing their teeth for 21 days.

DISCUSSION

The present study was based on the experimental gingi-vitis study model15 and closely followed the design ofLobene et al.7, but involved more subjects and a longerstudy period. Captive populations, such as inmates orstudents, are ideal for investigations of investigator-administered medication but their use demands height-ened ethical scrutiny by institutional review boards. Alleligible inmates consented to participate in the presentstudy.

The major finding of the study was that 0.05% sodiumhypochlorite constitutes an efficacious mouth rinse inperiodontal health care. Compared with the water rinsegroup, the sodium hypochlorite group demonstratedreductions of 48.2% in scores on the QHPI, 52.4% inscores on the L&SGI and 39.1% in the percentage of sites

Table 1 Clinical index values at days 0 and 21 in subjects using a sodium hypochlorite or distilled water oral rinse

Day 0 Day 21

QHPI score,mean(SD)

L&SGI score(visual assessment),

mean (SD)

Percentage ofsites that bled

on probingQHPI score,mean (SD)

L&SGI score(visual assessment),

mean (SD)

Percentage ofsites that bled

on probing

Sodium hypochloriterinse (n = 20)

0 (0) 0.12 (0.05) 7.7% 1.98* (0.51) 1.00* (0.2) 56.7%*

Water rinse (n = 20) 0 (0) 0.08 (0.04) 7.3% 3.82 (0.61) 2.10 (0.2) 93.1%

QHPI scores: 2 = a thin and continuous band of plaque (£ 1 mm) at the cervical margin; 4 = plaque covering at ‡ 33% but £ 67% of the surface.L&SGI scores: 1 = mild inflammation, slight change in colour, slight oedema; 2 = moderate inflammation, redness, oedema.*Statistically significant at P = 0.001.SD, standard deviation; QHPI, Quigley–Hein Plaque Index; L&SGI, Loe and Silness Gingival Index.

210 ª 2012 FDI World Dental Federation

De Nardo et al.

that bled on probing. Lobene et al.7 found 47% greaterplaque reduction with sodium hypochlorite irrigationthanwithwater rinsing.Thepresent studywasperformedwithout concomitant tooth-brushing. As sodium hypo-chlorite exerts a unique anti-biofilm effect by looseningthe attachment of microorganisms to solid surfaces16,tooth-brushing following a sodium hypochlorite rinsemay further enhance plaque removal and increase thedifference between outcomes in the two subject groups inthis study.

Post-treatment, the mean L&SGI score was 1.0 andthe percentage of sites that bled on probing averaged56.7% in the sodium hypochlorite group. According tothe L&SGI14, scores of 0 or 1 are not associated withgingival bleeding, but a score of 2 is. However, whenwe examined visual GI scores and sites of bleeding onprobing independently, we found that 17.1% of siteswith a visual GI score of 0 bled on probing, 42.3% ofsites with a visual GI score of 1 bled on probing, and5.7% of sites with a visual GI score of 2 did not bleedon probing17. Apparently, visual signs of gingivalinflammation and bleeding on probing may not be astightly correlated or constitute as straightforward acontinuum as implied in the L&SGI14.

The brown extrinsic tooth stain observed after sodiumhypochloriterinsinghasalsobeenreportedafter theuseofother oral antiseptics, including chlorhexidine18 andhexetidine19. All subjects receiving sodium hypochloriterinse demonstrated a brownish stain at the end of thestudy, but 35.0% of subjects in the water rinse group alsoexhibited brown tooth staining, although of a lowermagnitude.Thediscolourationofthe teethmaystemfromchromogenic products in food or beverages20 or fromovergrowth of non-periodontopathic Actinomyces spe-cies21. The sodium hypochlorite-associated stain wasrelatively easy to remove, which contrasts with the toothstainrelatedtochlorhexidine.Researchhasstill toidentifythe composition of antiseptic-associated tooth stains andmethods to minimise or prevent their development.

Sodium hypochlorite was tested at a concentration of0.05% and applied as a mouth rinse for 60 s twice perday. Preliminary taste tests in our clinics showed that asodium hypochlorite concentration of 0.05% was gen-erally well tolerated. A 0.05% sodium hypochloriteconcentration is five times above the minimal antibacte-rial concentration of 0.01%22, but 10 times lower thanthe 0.5% concentration used for supragingival irrigationby Lobene et al.7 and four times lower than a newly

Figure 1. Dental plaque and staining in subjects rinsing with 0.05% sodium hypochlorite. Subject A exhibited the greatest amount of plaqueaccumulation and subject B showed a pattern of plaque accumulation typical of the sodium hypochlorite group. The plaque-disclosing dye used on

day 21 was a solution of 0.8% brilliant blue and 0.4% erythrosine.

ª 2012 FDI World Dental Federation 211

Sodium hypochlorite and supragingival biofilm

recommended mouth rinse containing 0.2% sodiumhypochlorite to be used for 30 s two or three times perweek23. The protocol of the present study resulted inapproximately three times as much exposure to sodiumhypochlorite as the application of 0.2% sodium hypo-chlorite for 30 s two or three times per week. Furtherstudies are needed to determine if low concentrations butfrequent use of sodium hypochlorite will yield levels ofbiofilm removal and tooth staining similar to thoseafforded by a higher concentration of sodium hypochlo-rite and less frequent rinsing. Factors of importance interms of patient compliance include the provision of aless objectionable taste and a reduction in the requiredfrequency of usage, but it may be difficult to achieve bothof these objectives simultaneously.

Sodium hypochlorite, which is widely available ashousehold bleach, can benefit all periodontal patients,but its low price makes it particularly suitable forindividuals with low incomes. In 2008, the WorldBank24 estimated that 1.4 billion people in 115 low-income countries lived in extreme poverty as measuredby the poverty line defined by a purchasing power ofUS$1.25 ⁄ day. Low-income individuals are at elevatedrisk for developing periodontal disease25 and are unableto afford many dental self-care products26, and thus theneed to implement efficacious and low-cost periodontalhealth care in many parts of the world is urgent. Wesuggest that dilute sodium hypochlorite can serve as auseful antimicrobial agent in the prevention andtreatment of most types of periodontal disease.

Acknowledgements

The authors would like to thank Dr Ricardo Macchi,Argentine Dental Association, Buenos Aires City,Argentina, for his assistance in the statistical analysis,and the authorities of the Men’s Prison of the Provinceof Formosa for their cooperation.

Conflicts of interest

None declared.

REFERENCES

1. Jorgensen MG, Aalam A, Slots J. Periodontal antimicrobials –finding the right solutions. Int Dent J 2005 55: 3–12.

2. Roberts AP, Mullany P. Oral biofilms: a reservoir of transferable,bacterial, antimicrobial resistance. Expert Rev Anti Infect Ther2010 8: 1441–1450.

3. Goodson JM. Gingival crevice fluid flow. Periodontol 2000 200331: 43–54.

4. Zehnder M. Root canal irrigants. J Endod 2006 32: 389–398.

5. Chavez de Paz LE, Bergenholtz G, Svensater G. The effects of anti-microbialsonendodonticbiofilmbacteria.JEndod201036:70–77.

6. Gosau M, Hahnel S, Schwarz F et al. Effect of six different peri-implantitis disinfection methods on in vivo human oral biofilm.Clin Oral Implants Res 2010 21: 866–872.

7. Lobene RR, Soparkar PM, Hein JW et al. A study of the effects ofantiseptic agents and a pulsating irrigating device on plaque andgingivitis. J Periodontol 1972 43: 564–568.

8. Harrison JE, Schultz J. Studies on the chlorinating activity ofmyeloperoxidase. J Biol Chem 1976 251: 1371–1374.

9. Bruch MK. Toxicity and safety of topical sodium hypochlorite.Contrib Nephrol 2007 154: 24–38.

10. American Dental Association. Accepted Dental Therapeutics.Chicago, IL: ADA; 1984. p. 326.

11. Altman DG, Schulz KF, Moher D et al. The revised CONSORTstatement for reporting randomised trials: explanation and elab-oration. Ann Intern Med 2001 134: 663–694.

12. Quigley GA, Hein JW. Comparative cleansing efficiency ofmanual and power brushing. J Am Dent Assoc 1962 64: 26–29.

13. Turesky S, Gilmore ND, Glickman I. Reduced plaque formationby the chloromethyl analogue of victamine C. J Periodontol 197041: 41–43.

14. Loe H, Silness J. Periodontal disease in pregnancy. I. Prevalenceand severity. Acta Odontol Scand 1963 21: 533–551.

15. Theilade E, Wright WH, Jensen SB et al. Experimental gingivitisin man. II. A longitudinal clinical and bacteriological investiga-tion. J Periodontal Res 1966 1: 1–13.

16. Rutala WA, Weber DJ. Uses of inorganic hypochlorite (bleach) inhealth care facilities. Clin Microbiol Rev 1997 10: 597–610.

17. Gomez M, Chiappe V, Galeano A et al. Correlation betweenbleeding on probing and visual signs of gingival inflammation. JDent Res 1992 71: 974.

18. Jones CG. Chlorhexidine: is it still the gold standard? Periodontol2000 1997 15: 55–62.

19. Afennich F, Slot D, Hossainian N et al. The effect of hexetidinemouthwash on the prevention of plaque and gingivalinflammation: a systematic review. Int J Dent Hyg 2011 9:182–190.

20. Watts A, Addy M. Tooth discolouration and staining: a review ofthe literature. Br Dent J 2001 190: 309–316.

21. Slots J. The microflora of black stain on human primary teeth.Scand J Dent Res 1974 82: 484–490.

22. Rutala WA, Cole EC, Thomann CA et al. Stability and bacteri-cidal activity of chlorine solutions. Infect Control HospEpidemiol 1998 19: 323–327.

23. Slots J. Anti-infective agents in periodontal treatment. Expertcommentary in Medscape Dentistry and Oral Health. http://www.medscape.com/viewarticle/749509. [Accessed 15 Septem-ber 2011.]

24. World Bank. Poverty data: A supplement to World DevelopmentIndicators 2008. http://siteresources.worldbank.org/DATASTATISTICS/Resources/WDI08supplement1216.pdf. [Accessed 12September 2011.]

25. Slots J. Low-cost periodontal therapy. Periodontol 2000 2012 (inpress).

26. Moran JM. Chemical plaque control – prevention for the masses.Periodontol 2000 1997 15: 109–117.

Correspondence to:Dr Rodrigo De Nardo,

Department of Periodontology, School of Dentistry,Maimonides University,

Rivadavia No 1463,Rivadavia No. 1463 Formosa Capital CP 3600,

Formosa, Argentina.Email: rodrigodenardo@hotmail.com

212 ª 2012 FDI World Dental Federation

De Nardo et al.