ABSTRACT

OBJECTIVE

This study aimed to investigate the effect of mouthwash use on the development of oral cancer.

METHODS

Observational studies with adult/older adult populations that have examined the association between mouthwash use and oral cancer were included. Electronic search was performed in July 2022, with no time or language restrictions. PubMed/Medline, Embase, and Web of Science databases were used, and the search was extended to theses and dissertations libraries, Google Scholar, reference lists, and other sources. Methodological quality was assessed using the Newcastle-Ottawa Scale and quantitative data synthesis was performed by random effects meta-analysis, with different subgroup analyses and meta-regression. This revision was registered in Prospero (CRD42020143307).

RESULTS

Of the 4,094 studies identified in the search, 15 case-control studies were included in the review, totaling 6,515 cases and 17,037 controls. The meta-analysis included 17 measures of effect from 15 case-control studies. The pooled OR was 1.00 (95%CI: 0.79–1.26, n = 17 studies), but it was 2.58 (95%CI: 1.38–4.82, n = 2 studies) among those who had used mouthwashes three times or more times a day, and 1.30 (95%CI: 1.10–1.54, n = 4 studies) among those who had used mouthwashes for more than 40 years.

CONCLUSIONS

We found evidence that a high frequency of mouthwash use may be associated with an increased risk of oral cancer. However, despite the biological plausibility for this association, we suggest caution upon interpretation of our findings due to the few number of studies that have investigated the mouthwash use frequency, which should be considered. Therefore, we recommend that future studies assess, in detail, the frequency, duration, and content of mouthwashes to increase the strength of evidence for a possible dose-response effect of mouthwashes on oral cancer risk.

Mouthwashes; Mouth Neoplasms; Risk Factors; Meta-Analysis

INTRODUCTION

Oral cancer (OC) comprises tumors of the lip, oral cavity, and oropharynx¹1. Choi SW, Thomson P. Increasing incidence of oral cancer in Hong Kong-Who, where…and why? J Oral Pathol Med. 2019 Jul;48(6):483-90. https://doi.org/10.1111/jop.12868
https://doi.org/10.1111/jop.12868... . It is considered a major public health problem worldwide²2. Fan J, Liu W, Zhang M, Xing C. A literature review and systematic meta-analysis on XRCC3 Thr241Met polymorphism associating with susceptibility of oral cancer. Oncol Lett. 2019 Sep;18(3):3265-73. https://doi.org/10.3892/ol.2019.10609
https://doi.org/10.3892/ol.2019.10609... , being responsible for 476,125 new cases in 2020³3. Ferlay J, Ervik M, Lam F, Colombet M, Mery L, Piñeros M, et al. Global cancer observatory: cancer today. Lyon, International Agency for Research on Cancer; 2018 [cited 2022 Aug 7]. Available from: https://gco.iarc.fr/today
https://gco.iarc.fr/today... . Squamous cell carcinoma represents more than 90% of this total⁴4. Cooper JS, Porter K, Mallin K, Hoffman HT, Weber RS, Ang KK, et al. National Cancer Database report on cancer of the head and neck: 10-year update. Head Neck. 2009 Jun;31(6):748-58. https://doi.org/10.1002/hed.21022
https://doi.org/10.1002/hed.21022... , commonly affecting men after the fifth decade of life⁵5. Montero PH, Patel SG. Cancer of the oral cavity. Surg Oncol Clin N Am. 2015 Jul;24(3):491-508. https://doi.org/10.1016/j.soc.2015.03.006
https://doi.org/10.1016/j.soc.2015.03.00... . OC is a complex and multifactorial etiology disease⁵5. Montero PH, Patel SG. Cancer of the oral cavity. Surg Oncol Clin N Am. 2015 Jul;24(3):491-508. https://doi.org/10.1016/j.soc.2015.03.006
https://doi.org/10.1016/j.soc.2015.03.00... , in which cells accumulate oncogenic stimuli and deviation from homeostatic mechanisms. Thus, a transition process from a normal to a dysplastic epithelium can be triggered by potentially malignant precursor disorders for the carcinoma⁶6. Leemans CR, Braakhuis BJM, Brakenhoff RH. The molecular biology of head and neck cancer. Nat Rev Cancer. 2011;11(1):9-22. https://doi.org/10.1038/nrc2982
https://doi.org/10.1038/nrc2982... . Some of the major risk factors are tobacco use⁷7. Asthana S, Labani S, Kailash U, Sinha DN, Mehrotra R. Association of smokeless tobacco use and oral cancer: a systematic global review and meta-analysis. Nicotine Tob Res. 2019 Aug;21(9):1162-71. https://doi.org/10.1093/ntr/nty074
https://doi.org/10.1093/ntr/nty074... ,⁸8. Gupta B, Johnson NW. Systematic review and meta-analysis of association of smokeless tobacco and of betel quid without tobacco with incidence of oral cancer in South Asia and the Pacific. PLoS One. 2014 Nov;9(11):e113385. https://doi.org/10.1371/journal.pone.0113385
https://doi.org/10.1371/journal.pone.011... , alcohol consumption¹1. Choi SW, Thomson P. Increasing incidence of oral cancer in Hong Kong-Who, where…and why? J Oral Pathol Med. 2019 Jul;48(6):483-90. https://doi.org/10.1111/jop.12868
https://doi.org/10.1111/jop.12868... ,⁹9. Mello FW, Melo G, Pasetto JJ, Silva CA, Warnakulasuriya S, Rivero ER. The synergistic effect of tobacco and alcohol consumption on oral squamous cell carcinoma: a systematic review and meta-analysis. Clin Oral Investig. 2019 Jul;23(7):2849-59. https://doi.org/10.1007/s00784-019-02958-1
https://doi.org/10.1007/s00784-019-02958... , age¹⁰10. Aghiorghiesei O, Zanoaga O, Nutu A, Braicu C, Campian RS, Lucaciu O, et al. The World of Oral Cancer and its risk factors viewed from the aspect of microRNA expression patterns. Genes (Basel). 2022 Mar;13(4):594. https://doi.org/10.3390/genes13040594
https://doi.org/10.3390/genes13040594... , and sex¹¹11. Ram H, Sarkar J, Kumar H, Konwar R, Bhatt ML, Mohammad S. Oral cancer: risk factors and molecular pathogenesis. J Maxillofac Oral Surg. 2011 Jun;10(2):132-7. https://doi.org/10.1007/s12663-011-0195-z
https://doi.org/10.1007/s12663-011-0195-... , as well as oral human papillomavirus infection, diet, genetics¹²12. Alqahtani WS, Almufareh NA, Al-Johani HA, Alotaibi RK, Juliana CI, Aljarba NH, et al. Oral and oropharyngeal cancers and possible risk factors across gulf cooperation council countries: a systematic review. World J Oncol. 2020 Aug;11(4):173-81. https://doi.org/10.14740/wjon1283
https://doi.org/10.14740/wjon1283... , and persistent exposure to pathological or environmental cytotoxics¹³13. Calderón-Montaño JM, Jiménez-Alonso JJ, Guillén-Mancina E, Burgos-Morón E, López-Lázaro M. A 30-s exposure to ethanol 20% is cytotoxic to human keratinocytes: possible mechanistic link between alcohol-containing mouthwashes and oral cancer. Clin Oral Investig. 2018 Nov;22(8):2943-6. https://doi.org/10.1007/s00784-018-2602-z
https://doi.org/10.1007/s00784-018-2602-... , without consensus about the mouthwashes use.

Mouthwashes have been used for centuries as breath fresheners, medicines, and antiseptics¹⁴14. Adams D, Addy M. Mouthrinses. Adv Dent Res. 1994 Jul;8(2):291-301. https://doi.org/10.1177/08959374940080022401
https://doi.org/10.1177/0895937494008002... but the safety of their use and a likely association with OC have been widely discussed¹⁵15. Eliot MN, Michaud DS, Langevin SM, McClean MD, Kelsey KT. Periodontal disease and mouthwash use are risk factors for head and neck squamous cell carcinoma. Cancer Causes Control. 2013 Jul;24(7):1315-22. https://doi.org/10.1007/s10552-013-0209-x
https://doi.org/10.1007/s10552-013-0209-... . Different hypotheses have been investigated for the mechanisms involved in the carcinogenicity of alcohol-based mouthwashes, such as (1) intraoral oxidation of ethanol to its toxic metabolite acetaldehyde¹⁷17. Lachenmeier DW, Gumbel-Mako S, Sohnius EM, Keck-Wilhelm A, Kratz E, Mildau G. Salivary acetaldehyde increase due to alcohol-containing mouthwash use: a risk factor for oral cancer. Int J Cancer. 2009 Aug;125(3):730-5. https://doi.org/10.1002/ijc.24381
https://doi.org/10.1002/ijc.24381... ,²²22. Seitz HK, Matsuzaki S, Yokoyama A, Homann N, Väkeväinen S, Wang XD. Alcohol and cancer In: Verster JC, Brady K, Galanter M, Conrod P, eds. Drug abuse and addiction in medical illness: causes. consequences and treatment. 2001;25(5):137S-43S. https://doi.org/10.1007/978-1-4614-3375-0_35
https://doi.org/10.1007/978-1-4614-3375-... , and (2) an accentuated local cytotoxic effect on human epithelial keratinocytes of the oral mucosa¹³13. Calderón-Montaño JM, Jiménez-Alonso JJ, Guillén-Mancina E, Burgos-Morón E, López-Lázaro M. A 30-s exposure to ethanol 20% is cytotoxic to human keratinocytes: possible mechanistic link between alcohol-containing mouthwashes and oral cancer. Clin Oral Investig. 2018 Nov;22(8):2943-6. https://doi.org/10.1007/s00784-018-2602-z
https://doi.org/10.1007/s00784-018-2602-... ,²³23. Guillén-Mancina E, Calderón-Montaño JM, López-Lázaro M. Avoiding the ingestion of cytotoxic concentrations of ethanol may reduce the risk of cancer associated with alcohol consumption. Drug Alcohol Depend. 2018 Feb;183(183):201-4. https://doi.org/10.1016/j.drugalcdep.2017.11.013
https://doi.org/10.1016/j.drugalcdep.201... . Cytotoxicity occurs when ethanol, in contact with the cells, induces deeper-layers stem cells to divide more often than normal to replace the damaged epithelium, leading to a variety of cancer-related errors, thereby increasing the risk of malignant transformation²³23. Guillén-Mancina E, Calderón-Montaño JM, López-Lázaro M. Avoiding the ingestion of cytotoxic concentrations of ethanol may reduce the risk of cancer associated with alcohol consumption. Drug Alcohol Depend. 2018 Feb;183(183):201-4. https://doi.org/10.1016/j.drugalcdep.2017.11.013
https://doi.org/10.1016/j.drugalcdep.201... .

The preponderant role of ethanol in the carcinogenic potential of alcoholic mouthwashes does not exclude the possibility that other components may also be involved in OC¹³13. Calderón-Montaño JM, Jiménez-Alonso JJ, Guillén-Mancina E, Burgos-Morón E, López-Lázaro M. A 30-s exposure to ethanol 20% is cytotoxic to human keratinocytes: possible mechanistic link between alcohol-containing mouthwashes and oral cancer. Clin Oral Investig. 2018 Nov;22(8):2943-6. https://doi.org/10.1007/s00784-018-2602-z
https://doi.org/10.1007/s00784-018-2602-... . The impact of the complex mixture on oral cell’s cytotoxicity and antimicrobial activity is largely unknown²⁴24. Müller HD, Eick S, Moritz A, Lussi A, Gruber R. Cytotoxicity and Antimicrobial Activity of Oral Rinses In Vitro. BioMed Res Int. 2017;2017:4019723. https://doi.org/10.1155/2017/4019723
https://doi.org/10.1155/2017/4019723... . Various molecules included in commercial mouthwashes are preparations created and proposed for the market²⁵25. Tartaglia GM, Kumar S, Fornari CD, Corti E, Connelly ST. Mouthwashes in the 21st century: a narrative review about active molecules and effectiveness on the periodontal outcomes. Expert Opin Drug Deliv. 2017 Aug;14(8):973-82. https://doi.org/10.1080/17425247.2017.1260118
https://doi.org/10.1080/17425247.2017.12... . In this way, it is possible that active antibacterial ingredients, other than ethanol, such as phenolic compounds²⁶26. Vlachojannis C, Al-Ahmad A, Hellwig E, Chrubasik S. Listerine ® Products: an update on the efficacy and safety. Phytother Res. 2016 Mar;30(3):367-73. https://doi.org/10.1002/ptr.5555
https://doi.org/10.1002/ptr.5555... , triclosan²⁷27. Sangroula S, Baez Vasquez AY, Raut P, Obeng B, Shim JK, Bagley GD, et al. Triclosan disrupts immune cell function by depressing Ca2+ influx following acidification of the cytoplasm. Toxicol Appl Pharmacol. 2020 Oct;405(June):115205. https://doi.org/10.1016/j.taap.2020.115205
https://doi.org/10.1016/j.taap.2020.1152... ,²⁸28. Ruszkiewicz JA, Li S, Rodriguez MB, Aschner M. Is Triclosan a neurotoxic agent? J Toxicol Environ Health B Crit Rev. 2017;20(2):104-17. https://doi.org/10.1080/10937404.2017.1281181
https://doi.org/10.1080/10937404.2017.12... , cetylpyridinium chloride²⁹29. Fromm-Dornieden C, Rembe JD, Schäfer N, Böhm J, Stuermer EK. Cetylpyridinium chloride and miramistin as antiseptic substances in chronic wound management - prospects and limitations. J Med Microbiol. 2015 Apr;64(Pt 4):407-14. https://doi.org/10.1099/jmm.0.000034
https://doi.org/10.1099/jmm.0.000034... , and chlorhexidine³⁰30. Coelho AS, Laranjo M, Gonçalves AC, Paula A, Paulo S, Abrantes AM, et al. Cytotoxic effects of a chlorhexidine mouthwash and of an enzymatic mouthwash on human gingival fibroblasts. Odontology. 2020 Apr;108(2):260-70. https://doi.org/10.1007/s10266-019-00465-z
https://doi.org/10.1007/s10266-019-00465... may increase the risk of OC by changing the diversity of oral bacteria¹⁵15. Eliot MN, Michaud DS, Langevin SM, McClean MD, Kelsey KT. Periodontal disease and mouthwash use are risk factors for head and neck squamous cell carcinoma. Cancer Causes Control. 2013 Jul;24(7):1315-22. https://doi.org/10.1007/s10552-013-0209-x
https://doi.org/10.1007/s10552-013-0209-... and causing cell damage²⁴24. Müller HD, Eick S, Moritz A, Lussi A, Gruber R. Cytotoxicity and Antimicrobial Activity of Oral Rinses In Vitro. BioMed Res Int. 2017;2017:4019723. https://doi.org/10.1155/2017/4019723
https://doi.org/10.1155/2017/4019723... .

A previous systematic review³³33. Ustrell-Borràs M, Traboulsi-Garet B, Gay-Escoda C. Alcohol-based mouthwash as a risk factor of oral cancer: a systematic review. Med Oral Patol Oral Cir Bucal. 2020 Jan;25(1):e1-12. https://doi.org/10.4317/medoral.23085
https://doi.org/10.4317/medoral.23085... and meta-analyses³⁴34. Hostiuc S, Ionescu IV, Drima E. Mouthwash use and the risk of oral, pharyngeal, and laryngeal câncer: a meta-analysis. Int J Environ Res Public Health. 2021 Aug;18(15):8215. https://doi.org/10.3390/ijerph18158215
https://doi.org/10.3390/ijerph18158215... have investigated the association between mouthwash use and OC, but none of them found any evidence. The authors did not perform subgroup analyses considering adjusted and unadjusted estimates, type of controls, or frequency and duration of mouthwash use. Only Houstiuc et al.³⁴34. Hostiuc S, Ionescu IV, Drima E. Mouthwash use and the risk of oral, pharyngeal, and laryngeal câncer: a meta-analysis. Int J Environ Res Public Health. 2021 Aug;18(15):8215. https://doi.org/10.3390/ijerph18158215
https://doi.org/10.3390/ijerph18158215... performed analyses in terms of duration and frequency of mouthwash use and alcohol content, but they only considered upper aerodigestive tract cancers, not OC.

In addition, although the searches have included the grey literature and reference lists, they were restricted to the main online databases, especially PubMed/ Medline, Web of Science, and Scopus. The PICO, PECO, or PEO strategies were not mentioned and few descriptors were inserted, and only studies published in English³⁴34. Hostiuc S, Ionescu IV, Drima E. Mouthwash use and the risk of oral, pharyngeal, and laryngeal câncer: a meta-analysis. Int J Environ Res Public Health. 2021 Aug;18(15):8215. https://doi.org/10.3390/ijerph18158215
https://doi.org/10.3390/ijerph18158215... or English and Spanish³⁵35. Aceves Argemí R, González Navarro B, Ochoa García-Seisdedos P, Estrugo Devesa A, López-López J. Mouthwash with alcohol and oral carcinogenesis: systematic review and meta-analysis. J Evid Based Dent Pract. 2020 Jun;20(2):101407. https://doi.org/10.1016/j.jebdp.2020.101407
https://doi.org/10.1016/j.jebdp.2020.101... were included. Furthermore, some of these meta-analyses³⁴34. Hostiuc S, Ionescu IV, Drima E. Mouthwash use and the risk of oral, pharyngeal, and laryngeal câncer: a meta-analysis. Int J Environ Res Public Health. 2021 Aug;18(15):8215. https://doi.org/10.3390/ijerph18158215
https://doi.org/10.3390/ijerph18158215... ,³⁵35. Aceves Argemí R, González Navarro B, Ochoa García-Seisdedos P, Estrugo Devesa A, López-López J. Mouthwash with alcohol and oral carcinogenesis: systematic review and meta-analysis. J Evid Based Dent Pract. 2020 Jun;20(2):101407. https://doi.org/10.1016/j.jebdp.2020.101407
https://doi.org/10.1016/j.jebdp.2020.101... included studies that may have contained overlapping samples³⁷37. Wynder EL, Kabat G, Rosenberg S, Levenstein M. Oral cancer and mouthwash use. J Natl Cancer Inst. 1983 Feb;70(2):255-60.. This potential duplication occurred because these studies were part of multicenter research⁴⁴44. Ahrens W, Pohlabeln H, Foraita R, Nelis M, Lagiou P, Lagiou A, et al. Oral health, dental care and mouthwash associated with upper aerodigestive tract cancer risk in Europe: the ARCAGE study. Oral Oncol. 2014 Jun;50(6):616-25. https://doi.org/10.1016/j.oraloncology.2014.03.001
https://doi.org/10.1016/j.oraloncology.2... ,⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... or were smaller in scale³⁷37. Wynder EL, Kabat G, Rosenberg S, Levenstein M. Oral cancer and mouthwash use. J Natl Cancer Inst. 1983 Feb;70(2):255-60.. Moreover, the meta-analyses incorporated various types of studies, such as case series⁴⁶46. Weaver A, Fleming SM, Smith DB. Mouthwash and oral cancer: carcinogen or coincidence? J Oral Surg. 1979 Apr;37(4):250-3., meta-analysis³⁶36. Gandini S, Negri E, Boffetta P, La Vecchia C, Boyle P. Mouthwash and oral cancer risk quantitative meta-analysis of epidemiologic studies. Ann Agric Environ Med. 2012;19(2):173-80., and studies focused on outcomes or objectives unrelated to oral cancer¹⁷17. Lachenmeier DW, Gumbel-Mako S, Sohnius EM, Keck-Wilhelm A, Kratz E, Mildau G. Salivary acetaldehyde increase due to alcohol-containing mouthwash use: a risk factor for oral cancer. Int J Cancer. 2009 Aug;125(3):730-5. https://doi.org/10.1002/ijc.24381
https://doi.org/10.1002/ijc.24381... ,⁴⁷47. Morse DE, Katz RV, Pendrys DG, Holford TR, Krutchkoff DJ, Eisenberg E, et al. Mouthwash use and dentures in relation to oral epithelial dysplasia. Oral Oncol. 1997 Sep;33(5):338-43. https://doi.org/10.1016/S1368-8375 (97)00019-5
https://doi.org/10.1016/S1368-8375 (97)0... . Therefore, since some studies indicate an association between mouthwash use and OC¹⁵15. Eliot MN, Michaud DS, Langevin SM, McClean MD, Kelsey KT. Periodontal disease and mouthwash use are risk factors for head and neck squamous cell carcinoma. Cancer Causes Control. 2013 Jul;24(7):1315-22. https://doi.org/10.1007/s10552-013-0209-x
https://doi.org/10.1007/s10552-013-0209-... ,⁴⁴44. Ahrens W, Pohlabeln H, Foraita R, Nelis M, Lagiou P, Lagiou A, et al. Oral health, dental care and mouthwash associated with upper aerodigestive tract cancer risk in Europe: the ARCAGE study. Oral Oncol. 2014 Jun;50(6):616-25. https://doi.org/10.1016/j.oraloncology.2014.03.001
https://doi.org/10.1016/j.oraloncology.2... ,⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... ,⁵⁵55. Sharma AP, Naqvi A, Sharma N, Haider K. Assessment of the association between oral health and hygiene practices and oral câncer: a hospital-based case-control study. Eur J Mol Clin Med. 2020;7(8):1206-11., whereas other studies do not show such association, and considering the gaps left behind by previous meta-analyses, we propose to estimate the pooled effect of mouthwash use on OC depending on duration and frequency, type of control, and adjustment for confounding factors.

METHODS

Protocol and Registration

This systematic review with meta-analysis was reported following the recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (Prisma)⁵⁸58. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. J Clin Epidemiol. 2021 Jun;134:178-89. https://doi.org/10.1016/j.jclinepi.2021.03.001
https://doi.org/10.1016/j.jclinepi.2021.... and Meta-Analysis of Observational Studies in Epidemiology (MOOSE)⁵⁹59. Brooke BS, Schwartz TA, Pawlik TM. MOOSE Reporting Guidelines for Meta-analyses of Observational Studies. JAMA Surg. 2021 Aug;156(8):787-8. https://doi.org/10.1001/jamasurg.2021.0522
https://doi.org/10.1001/jamasurg.2021.05... guidelines. The detailed protocol (CRD42020143307) was registered in the International Prospective Register of Systematic Reviews (Prospero – Available at www.crd.york.ac.uk/PROSPERO).

Context

This research aimed to answer the following questions: 1) Can mouthwash use be associated with OC? 2) Do mouthwashes have a dose-response relationship with OC? 3) How does the association behave depending on the alcohol content?

Outcome

The primary outcome was the occurrence of OC (oral cavity and oropharynx) according to the International Classification of Diseases (ICD), 11^th Revision, 2B6E.0⁶⁰60. World Health Organization. International classification of diseases for mortality and morbidity statistics (11th Rev). Geneva: World Health Organization; 2018.. The anatomical subsites of the oral cavity consist of the labial mucosa, buccal mucosa, floor of the mouth, alveolar crest, gingiva, two anterior thirds of the tongue (anterior to the circumvented papillae), hard palate, and retromolar trigone, whereas the oropharynx consists of the soft palate, base (or posterior third) of the tongue, palatine tonsils, palatoglossal folds, epiglottic vallecula, and posterior pharyngeal wall⁶¹61. Chi AC, Day TA, Neville BW. Oral cavity and oropharyngeal squamous cell carcinoma: an update. CA Cancer J Clin. 2015;65(5):401-21. https://doi.org/10.3322/caac.21293
https://doi.org/10.3322/caac.21293... .

Databases and Search Strategy

Systematic searches were performed in the following indexed databases: PubMed/Medline, Embase, Web of Science, Science Direct, Scopus (Elsevier), Biblioteca Brasileira de Odontologia (BBO), Dentistry and Oral Sciences Source - DOSS (EBSCO), Scientific Electronic Library Online (SciELO), LILACS, WHO Global Health Library, Directory of Open Access Journals – DOAJ, and Cumulative Index to Nursing and Allied Health Literature (CINAHL). Searches were also conducted using Google Scholar and grey literature from the Brazilian Digital Library of Theses and Dissertations. The reference lists of the included papers were also evaluated. The industries were contacted to request studies and data included in this study.

Initially two examiners were responsible for the search (JSSA, EBAFT). The PEO search strategy [Population (adults or older adults), Exposure (mouthwash use), and Outcome (OR)] was used. Thus, objective-related keywords, and MeSH terms (Medical Subject Headings) combined with Boolean operators (OR/AND/NOT) were used to ensure that the search strategy was comprehensive. The titles were searched in July 2022. Year of publication and language were unrestricted. The search strategy by database is detailed in Supplementary Table 1^aaAvailable from: https://drive.google.com/file/d/1XhrSZK83w25gs21xPjPXbBngWiBQ8aXf/view?usp=sharing . The searched study titles and their respective information were included in a Microsoft Excel^® 365 software spreadsheet (Microsoft Corporation, Washington, USA) to check for duplicity and to apply the eligibility criteria. Duplicate studies were excluded. The searches were compared, and any disagreement was resolved by the third reviewer (MCFNC).

Eligibility Criteria

We included primary studies with adult or older adult populations that aimed to analyze the association between mouthwashes and OC. The excluded criteria included: 1) studies with specific populations with syndromes or congenital changes; 2) studies with more susceptible populations to the development cancer such as those previously exposed to chemotherapy or radiotherapy, and patients with specific genetic mutations; 3) publications involving the same population sample – in this case, the study with the major sample was selected; 4) studies with outcomes defined as dysplasia, cell damage, or nuclear alterations; and 5) letters to the editor, conference and congress abstracts, case series, case reports, in vitro studies, experimental studies in animals, review studies, and meta-analyses.

Selection of Studies

An independent selection of studies was performed by two examiners (JSSA, EBAFT) and disagreements were resolved by consensus with the third reviewer (MCFNC). The first selection was based on the title and abstract, hiding the journal and author’s names, avoiding possible bias and conflicts of interest. Studies not selected at this stage or in the subsequent stages were registered in the spreadsheet as excluded, with their respective reasons. In cases where the study seemed to be eligible, but presented insufficient data in the title and abstract, the text was fully read and evaluated following the inclusion criteria afterwards. The full texts of the remaining studies were recovered and those eligible for this review were identified.

Data Extraction

Relevant data from the selected articles were extracted, processed, and tabulated in a data collection form pre-developed in Microsoft Excel^® 365 (Microsoft Corporation, Washington, USA) by two reviewers (JSSA, EBAFT). All included articles were case-control studies, and the following data were recorded: authors of the studies, year of publication, country, recruitment period, sample size, age, gender (only one or two genders), type of exposure (mouthwash use – yes or no – and according to the frequency of use, alcohol content, and use duration over the years), type of outcome (OC site and ICD), type of controls (community or hospital), effect size (odds ratio), case-controls ratio, and variables considered in the adjustment for confounding (whether in pairing, sample restriction, or adjusted analysis).

For studies that reported measures of effect according to the cancer involvement site (oral cavity, pharynx, larynx, esophagus), those located in the oral cavity or oral cavity and pharynx were selected (when the measure of effect was simultaneously presented at both sites). For studies that reported effect sizes by categories regarding frequency of use or time of use, these measures were considered in subgroup analysis for dose response, sometimes being recategorized to allow comparability with other studies. For results stratified by gender, the measures of effect from each stratum were considered in the meta-analysis by inserting the letters a (men) and b (women). For studies that presented adjusted estimates for different confounding variable arrangements, the effect size adjusted for the largest number of variables was considered instead of potential mediators. Considering the possibility of residual confounding, subgroup analysis was performed considering three categories of adjustment: adjusted, when adjusted, at least, for age, gender, and tobacco and alcohol consumption; partially adjusted, when adjusted only for some of these variables; or unadjusted. Alcohol content could not be categorized, as information was missing in some studies, possibly because it was self-reported data. Data missing from the studies were disregarded. When the study did not present enough data to be included in the quantitative analysis, e-mails were sent to the authors to retrieve the data.

Risk of Bias and Grading Quality of Evidence

The individual risk of bias of each study included in the systematic review was assessed by the Newcastle Ottawa Scale (NOS) for case-control studies by two independent examiners (JSSA, EISM). Differences were resolved by consensus in the presence of the third reviewer (MCFNC). The quality of evidence of the studies included in the meta-analysis was assessed following the GRADEpro Guideline Development Tool (GDT)⁶²62. GRADEpro GDT: GRADEpro Guideline Development Tool [Software]. London: Cochrane; 2020.,⁶³63. Ryan R, Hill S. How to GRADE the quality of the evidence. Cochrane Consumers and Communication Group. 2016;Version 3 :1-24. https://cccrg.cochrane.org/author-resources
https://cccrg.cochrane.org/author-resour... .

Statistical Analysis

Stata 14.0 software (StataCorp, College Station, USA) was used for the meta-analysis. Since the heterogeneity evaluated by the I²2. Fan J, Liu W, Zhang M, Xing C. A literature review and systematic meta-analysis on XRCC3 Thr241Met polymorphism associating with susceptibility of oral cancer. Oncol Lett. 2019 Sep;18(3):3265-73. https://doi.org/10.3892/ol.2019.10609
https://doi.org/10.3892/ol.2019.10609... test was high (77.1%), the DerSimonian-Laird Random-Effect method was chosen. Subgroup analyses were done with the studies that reported duration and frequency of mouthwash use to assess a likely dose-response, as well as to evaluate the subgroup according to the type of control (hospital or community) and the variables considered in the confounding adjustment.

Crude and multivariable meta-regressions were used to assess the contribution (%) of the co-variables [gender (men only; women only; and men and women), setting (low/middle-income or high-income country), sample size (up to 500; 501 to 1,000; and over 1,000 subjects), cancer site (only oral cavity or oral/pharyngeal/larynx sites), control type (hospital or community), and case-controls ratio (at least one case to two controls “1:2” or one case to one control “1:1”), OR adjustment] on the heterogeneity among the studies. Co-variables with p-value < 0.20 in crude meta-regression were included in the multivariable meta-regression. A funnel plot associated with the Egger regression asymmetry test was used to investigate the possibility of publication bias. OR were estimated and weighted by the study sample size and by their respective 95% confidence intervals (95%CI).

RESULTS

Searching Results

We identified 4,094 records in the bibliographic search. After excluding duplicates, 3,517 titles and abstracts were read. Of these, 50 studies were selected for full-text reading, and 14 studies were included in our review, with one more paper identified after searching in the reference lists. Thus, 15 papers were included in the qualitative and quantitative analyses, totalizing 6,515 cases and 17,037 controls. The reasons for the 36 full-text articles excluded were: sample already included in other multicenter studies⁴⁴44. Ahrens W, Pohlabeln H, Foraita R, Nelis M, Lagiou P, Lagiou A, et al. Oral health, dental care and mouthwash associated with upper aerodigestive tract cancer risk in Europe: the ARCAGE study. Oral Oncol. 2014 Jun;50(6):616-25. https://doi.org/10.1016/j.oraloncology.2014.03.001
https://doi.org/10.1016/j.oraloncology.2... ,⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... (n = 7); letter to editor (n = 4); insufficient data (n = 2); other outcome/ objective (n = 12); in vitro studies (n = 3); review (n = 5); conference abstract (n = 3) (Figure 1).

Description of the Studies

Chart presents the main characteristics of the included studies. All studies featured the same design: case-control. Two studies were characterized as multicentric⁴⁴44. Ahrens W, Pohlabeln H, Foraita R, Nelis M, Lagiou P, Lagiou A, et al. Oral health, dental care and mouthwash associated with upper aerodigestive tract cancer risk in Europe: the ARCAGE study. Oral Oncol. 2014 Jun;50(6):616-25. https://doi.org/10.1016/j.oraloncology.2014.03.001
https://doi.org/10.1016/j.oraloncology.2... ,⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... – one in different European countries (The ARCAGE study)³⁹39. Garrote LF, Herrero R, Reyes RM, Vaccarella S, Anta JL, Ferbeye L, et al. Risk factors for cancer of the oral cavity and oro-pharynx in Cuba. Br J Cancer. 2001 Jul;85(1):46-54. https://doi.org/10.1054/bjoc.2000.1825
https://doi.org/10.1054/bjoc.2000.1825... and the other⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... was a compilation of published and unpublished case-control studies in countries from America, Europe, and Asia. Other studies using these multicentric data⁴¹41. Guha N, Boffetta P, Wünsch Filho V, Eluf Neto J, Shangina O, Zaridze D, et al. Oral health and risk of squamous cell carcinoma of the head and neck and esophagus: results of two multicentric case-control studies. Am J Epidemiol. 2007 Nov;166(10):1159-73. https://doi.org/10.1093/aje/kwm193
https://doi.org/10.1093/aje/kwm193... were not included to avoid duplication of sample from the same survey. Studies were conducted in United States¹⁵15. Eliot MN, Michaud DS, Langevin SM, McClean MD, Kelsey KT. Periodontal disease and mouthwash use are risk factors for head and neck squamous cell carcinoma. Cancer Causes Control. 2013 Jul;24(7):1315-22. https://doi.org/10.1007/s10552-013-0209-x
https://doi.org/10.1007/s10552-013-0209-... ,⁶⁴64. Blot WJ, Winn DM, Fraumeni JF Jr. Oral cancer and mouthwash. J Natl Cancer Inst. 1983 Feb;70(2):251-3. https://doi.org/10.1093/jnci/70.2.251
https://doi.org/10.1093/jnci/70.2.251... , Brazil⁵⁷57. Marques LA, Eluf-Neto J, Figueiredo RA, Góis-Filho JF, Kowalski LP, Carvalho MB, et al. Oral health, hygiene practices and oral cancer. Rev Saude Publica. 2008 Jun;42(3):471-9. https://doi.org/10.1590/S0034-89102008000300012
https://doi.org/10.1590/S0034-8910200800... ,⁶⁹69. Assunção Junior N, Teixeira VP, Lemos CA. Use of mouthwashes in patients with oral and oropharyngeal cancer. Clinical and Laboratorial Research in Dentistry. 2015;21(2):91-8. https://doi.org/10.11606/issn.2357-8041.clrd.2015.102956
https://doi.org/10.11606/issn.2357-8041.... , Italy⁷⁰70. Talamini R, Vaccarella S, Barbone F, Tavani A, La Vecchia C, Herrero R, et al. Oral hygiene, dentition, sexual habits and risk of oral cancer. Br J Cancer. 2000 Nov;83(9):1238-42. https://doi.org/10.1054/bjoc.2000.1398
https://doi.org/10.1054/bjoc.2000.1398... , China⁷¹71. Chang JS, Lo HI, Wong TY, Huang CC, Lee WT, Tsai ST, et al. Investigating the association between oral hygiene and head and neck cancer. Oral Oncol. 2013 Oct;49(10):1010-7. https://doi.org/10.1016/j.oraloncology.2013.07.004
https://doi.org/10.1016/j.oraloncology.2... , Australia⁷²72. Alnuaimi AD, Wiesenfeld D, O’Brien-Simpson NM, Reynolds EC, McCullough MJ. Oral Candida colonization in oral cancer patients and its relationship with traditional risk factors of oral cancer: a matched case-control study. Oral Oncol. 2015 Feb;51(2):139-45. https://doi.org/10.1016/j.oraloncology.2014.11.008
https://doi.org/10.1016/j.oraloncology.2... , Pakistan⁵⁶56. Saira, Ahmed R, Malik S, Khan MF, Khattak MR. Epidemiological and clinical correlates of oral squamous cell carcinoma in patients from north-west Pakistan. J Pak Med Assoc. 2019 Aug;69(8):1074-8., and India⁵⁵55. Sharma AP, Naqvi A, Sharma N, Haider K. Assessment of the association between oral health and hygiene practices and oral câncer: a hospital-based case-control study. Eur J Mol Clin Med. 2020;7(8):1206-11.. Most studies have used hospital controls and only three had community controls¹⁵15. Eliot MN, Michaud DS, Langevin SM, McClean MD, Kelsey KT. Periodontal disease and mouthwash use are risk factors for head and neck squamous cell carcinoma. Cancer Causes Control. 2013 Jul;24(7):1315-22. https://doi.org/10.1007/s10552-013-0209-x
https://doi.org/10.1007/s10552-013-0209-... ,⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... ,⁶⁷67. Winn DM, Blot WJ, McLaughlin JK, Austin DF, Greenberg RS, Preston-Martin S, et al. Mouthwash use and oral conditions in the risk of oral and pharyngeal cancer. Cancer Res. 1991 Jun;51(11):3044-7.. Seven studies had a proportion of at least two controls for each case¹⁵15. Eliot MN, Michaud DS, Langevin SM, McClean MD, Kelsey KT. Periodontal disease and mouthwash use are risk factors for head and neck squamous cell carcinoma. Cancer Causes Control. 2013 Jul;24(7):1315-22. https://doi.org/10.1007/s10552-013-0209-x
https://doi.org/10.1007/s10552-013-0209-... ,⁴⁴44. Ahrens W, Pohlabeln H, Foraita R, Nelis M, Lagiou P, Lagiou A, et al. Oral health, dental care and mouthwash associated with upper aerodigestive tract cancer risk in Europe: the ARCAGE study. Oral Oncol. 2014 Jun;50(6):616-25. https://doi.org/10.1016/j.oraloncology.2014.03.001
https://doi.org/10.1016/j.oraloncology.2... ,⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... ,⁵⁷57. Marques LA, Eluf-Neto J, Figueiredo RA, Góis-Filho JF, Kowalski LP, Carvalho MB, et al. Oral health, hygiene practices and oral cancer. Rev Saude Publica. 2008 Jun;42(3):471-9. https://doi.org/10.1590/S0034-89102008000300012
https://doi.org/10.1590/S0034-8910200800... ,⁶⁵65. Mashberg A, Barsa P, Grossman ML. A study of the relationship between mouthwash use and oral and pharyngeal cancer. J Am Dent Assoc. 1985 May;110(5):731-4. https://doi.org/10.14219/jada.archive.1985.0422
https://doi.org/10.14219/jada.archive.19... ,⁶⁸68. D’Souza G, Kreimer AR, Viscidi R, Pawlita M, Fakhry C, Koch WM, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med. 2007 May;356(19):1944-56. Available from: www.nejm.org https://doi.org/10.1056/NEJMoa065497
https://doi.org/10.1056/NEJMoa065497... ,⁷²72. Alnuaimi AD, Wiesenfeld D, O’Brien-Simpson NM, Reynolds EC, McCullough MJ. Oral Candida colonization in oral cancer patients and its relationship with traditional risk factors of oral cancer: a matched case-control study. Oral Oncol. 2015 Feb;51(2):139-45. https://doi.org/10.1016/j.oraloncology.2014.11.008
https://doi.org/10.1016/j.oraloncology.2... , and the others have used a 1:1 ratio. Six studies considered cancers in the oral cavity¹⁵15. Eliot MN, Michaud DS, Langevin SM, McClean MD, Kelsey KT. Periodontal disease and mouthwash use are risk factors for head and neck squamous cell carcinoma. Cancer Causes Control. 2013 Jul;24(7):1315-22. https://doi.org/10.1007/s10552-013-0209-x
https://doi.org/10.1007/s10552-013-0209-... ,⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... ,⁵⁵55. Sharma AP, Naqvi A, Sharma N, Haider K. Assessment of the association between oral health and hygiene practices and oral câncer: a hospital-based case-control study. Eur J Mol Clin Med. 2020;7(8):1206-11.,⁵⁷57. Marques LA, Eluf-Neto J, Figueiredo RA, Góis-Filho JF, Kowalski LP, Carvalho MB, et al. Oral health, hygiene practices and oral cancer. Rev Saude Publica. 2008 Jun;42(3):471-9. https://doi.org/10.1590/S0034-89102008000300012
https://doi.org/10.1590/S0034-8910200800... ,⁷¹71. Chang JS, Lo HI, Wong TY, Huang CC, Lee WT, Tsai ST, et al. Investigating the association between oral hygiene and head and neck cancer. Oral Oncol. 2013 Oct;49(10):1010-7. https://doi.org/10.1016/j.oraloncology.2013.07.004
https://doi.org/10.1016/j.oraloncology.2... ,⁷²72. Alnuaimi AD, Wiesenfeld D, O’Brien-Simpson NM, Reynolds EC, McCullough MJ. Oral Candida colonization in oral cancer patients and its relationship with traditional risk factors of oral cancer: a matched case-control study. Oral Oncol. 2015 Feb;51(2):139-45. https://doi.org/10.1016/j.oraloncology.2014.11.008
https://doi.org/10.1016/j.oraloncology.2... , eight studies included oropharynx⁴⁴44. Ahrens W, Pohlabeln H, Foraita R, Nelis M, Lagiou P, Lagiou A, et al. Oral health, dental care and mouthwash associated with upper aerodigestive tract cancer risk in Europe: the ARCAGE study. Oral Oncol. 2014 Jun;50(6):616-25. https://doi.org/10.1016/j.oraloncology.2014.03.001
https://doi.org/10.1016/j.oraloncology.2... ,⁶⁴64. Blot WJ, Winn DM, Fraumeni JF Jr. Oral cancer and mouthwash. J Natl Cancer Inst. 1983 Feb;70(2):251-3. https://doi.org/10.1093/jnci/70.2.251
https://doi.org/10.1093/jnci/70.2.251... , and one study also included the larynx as the outcome⁵⁶56. Saira, Ahmed R, Malik S, Khan MF, Khattak MR. Epidemiological and clinical correlates of oral squamous cell carcinoma in patients from north-west Pakistan. J Pak Med Assoc. 2019 Aug;69(8):1074-8..

Except for Sharma et al.⁵⁵55. Sharma AP, Naqvi A, Sharma N, Haider K. Assessment of the association between oral health and hygiene practices and oral câncer: a hospital-based case-control study. Eur J Mol Clin Med. 2020;7(8):1206-11., Mashberg et al.⁵⁸58. Page MJ, McKenzie JE, Bossuyt PM, Boutron I, Hoffmann TC, Mulrow CD, et al. The PRISMA 2020 statement: an updated guideline for reporting systematic reviews. J Clin Epidemiol. 2021 Jun;134:178-89. https://doi.org/10.1016/j.jclinepi.2021.03.001
https://doi.org/10.1016/j.jclinepi.2021.... , and Young et al.⁵⁹59. Brooke BS, Schwartz TA, Pawlik TM. MOOSE Reporting Guidelines for Meta-analyses of Observational Studies. JAMA Surg. 2021 Aug;156(8):787-8. https://doi.org/10.1001/jamasurg.2021.0522
https://doi.org/10.1001/jamasurg.2021.05... , the other studies were matched minimally by gender and age. Other prevalent confounding variables included in multivariable analyses comprised tobacco and alcohol consumption, and, less often, fruit and vegetable consumption, ethnicity, socioeconomic conditions, among others. Human papillomavirus (HPV) was not included in the regression analyses of the identified studies. One study restricted the sample to people aged 40 years or older with no history of tobacco use⁶⁵65. Mashberg A, Barsa P, Grossman ML. A study of the relationship between mouthwash use and oral and pharyngeal cancer. J Am Dent Assoc. 1985 May;110(5):731-4. https://doi.org/10.14219/jada.archive.1985.0422
https://doi.org/10.14219/jada.archive.19... . One study provided only crude effects, i.e., no matching, no restriction, and no multivariable analyses⁵⁵55. Sharma AP, Naqvi A, Sharma N, Haider K. Assessment of the association between oral health and hygiene practices and oral câncer: a hospital-based case-control study. Eur J Mol Clin Med. 2020;7(8):1206-11..

Risk of Bias and Grading Quality of Evidence

According to NOS, eight studies presented a low risk¹⁵15. Eliot MN, Michaud DS, Langevin SM, McClean MD, Kelsey KT. Periodontal disease and mouthwash use are risk factors for head and neck squamous cell carcinoma. Cancer Causes Control. 2013 Jul;24(7):1315-22. https://doi.org/10.1007/s10552-013-0209-x
https://doi.org/10.1007/s10552-013-0209-... ,⁴⁴44. Ahrens W, Pohlabeln H, Foraita R, Nelis M, Lagiou P, Lagiou A, et al. Oral health, dental care and mouthwash associated with upper aerodigestive tract cancer risk in Europe: the ARCAGE study. Oral Oncol. 2014 Jun;50(6):616-25. https://doi.org/10.1016/j.oraloncology.2014.03.001
https://doi.org/10.1016/j.oraloncology.2... ,⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... ,⁵⁷57. Marques LA, Eluf-Neto J, Figueiredo RA, Góis-Filho JF, Kowalski LP, Carvalho MB, et al. Oral health, hygiene practices and oral cancer. Rev Saude Publica. 2008 Jun;42(3):471-9. https://doi.org/10.1590/S0034-89102008000300012
https://doi.org/10.1590/S0034-8910200800... ,⁶⁴64. Blot WJ, Winn DM, Fraumeni JF Jr. Oral cancer and mouthwash. J Natl Cancer Inst. 1983 Feb;70(2):251-3. https://doi.org/10.1093/jnci/70.2.251
https://doi.org/10.1093/jnci/70.2.251... ,⁶⁷67. Winn DM, Blot WJ, McLaughlin JK, Austin DF, Greenberg RS, Preston-Martin S, et al. Mouthwash use and oral conditions in the risk of oral and pharyngeal cancer. Cancer Res. 1991 Jun;51(11):3044-7.,⁷⁰70. Talamini R, Vaccarella S, Barbone F, Tavani A, La Vecchia C, Herrero R, et al. Oral hygiene, dentition, sexual habits and risk of oral cancer. Br J Cancer. 2000 Nov;83(9):1238-42. https://doi.org/10.1054/bjoc.2000.1398
https://doi.org/10.1054/bjoc.2000.1398... ,⁷¹71. Chang JS, Lo HI, Wong TY, Huang CC, Lee WT, Tsai ST, et al. Investigating the association between oral hygiene and head and neck cancer. Oral Oncol. 2013 Oct;49(10):1010-7. https://doi.org/10.1016/j.oraloncology.2013.07.004
https://doi.org/10.1016/j.oraloncology.2... , and seven presented a moderate risk of bias⁵⁵55. Sharma AP, Naqvi A, Sharma N, Haider K. Assessment of the association between oral health and hygiene practices and oral câncer: a hospital-based case-control study. Eur J Mol Clin Med. 2020;7(8):1206-11.,⁵⁶56. Saira, Ahmed R, Malik S, Khan MF, Khattak MR. Epidemiological and clinical correlates of oral squamous cell carcinoma in patients from north-west Pakistan. J Pak Med Assoc. 2019 Aug;69(8):1074-8.,⁶⁵65. Mashberg A, Barsa P, Grossman ML. A study of the relationship between mouthwash use and oral and pharyngeal cancer. J Am Dent Assoc. 1985 May;110(5):731-4. https://doi.org/10.14219/jada.archive.1985.0422
https://doi.org/10.14219/jada.archive.19... ,⁶⁶66. Young TB, Ford CN, Brandenburg JH. An epidemiologic study of oral cancer in a statewide network. Am J Otolaryngol. 1986;7(3):200-8. https://doi.org/10.1016/S0196-0709 (86)80007-2
https://doi.org/10.1016/S0196-0709 (86)8... ,⁶⁸68. D’Souza G, Kreimer AR, Viscidi R, Pawlita M, Fakhry C, Koch WM, et al. Case-control study of human papillomavirus and oropharyngeal cancer. N Engl J Med. 2007 May;356(19):1944-56. Available from: www.nejm.org https://doi.org/10.1056/NEJMoa065497
https://doi.org/10.1056/NEJMoa065497... ,⁶⁹69. Assunção Junior N, Teixeira VP, Lemos CA. Use of mouthwashes in patients with oral and oropharyngeal cancer. Clinical and Laboratorial Research in Dentistry. 2015;21(2):91-8. https://doi.org/10.11606/issn.2357-8041.clrd.2015.102956
https://doi.org/10.11606/issn.2357-8041.... ,⁷²72. Alnuaimi AD, Wiesenfeld D, O’Brien-Simpson NM, Reynolds EC, McCullough MJ. Oral Candida colonization in oral cancer patients and its relationship with traditional risk factors of oral cancer: a matched case-control study. Oral Oncol. 2015 Feb;51(2):139-45. https://doi.org/10.1016/j.oraloncology.2014.11.008
https://doi.org/10.1016/j.oraloncology.2... (Chart; Supplementary Table 2^bbAvailable from: https://drive.google.com/file/d/10bjMWcTHo McovuRhrxkEzN9_eD2oGQLK/view?usp=sharing ). In part, the methodological weakness of the investigated studies is their control selection since they present hospital controls. In addition, none of the studies reported the blinding of cases and controls in regarding exposure, which could have generated measurement bias.

Meta-Analysis

Figure 2A shows the summarization of the 17 OR from the 15 studies included in the meta-analysis. Mouthwash use, regardless of alcohol content or frequency/duration of use, was not associated with OC (OR = 1.00; 95%CI: 0.79–1.26) and the heterogeneity among studies was substantial (I²2. Fan J, Liu W, Zhang M, Xing C. A literature review and systematic meta-analysis on XRCC3 Thr241Met polymorphism associating with susceptibility of oral cancer. Oncol Lett. 2019 Sep;18(3):3265-73. https://doi.org/10.3892/ol.2019.10609
https://doi.org/10.3892/ol.2019.10609... : 77.1%). The funnel plot suggests a possible effect of the smaller studies, as they are more concentrated on the bottom right, but the Egger tests were not statistically significant (p = 0.651), indicating symmetry in the distribution of studies, and therefore a low possibility of publication bias (Figure 2B). When considering only the five effect estimates (OR) of the studies that analyzed alcohol-containing mouthwash versus no mouthwash use (Figure 2C), the overall weighed random effect increased but remained non-significant (OR = 1.20; 95%CI: 0.93–1.55).

Subgroup analyses according to the subset of variables considered in the adjustment for confounding (OR = 1.00; 95%CI: 0.79–1.26) (Figure 3A) and control type (OR = 1.13; 95%CI: 0.95–1.35) (Figure 3B) did not show any association between mouthwash use and OC. However, when considering the frequency of use among mouthwash users (Figure 3C), the overall weighed random effect was significant (OR = 1.30; 95%CI: 1.10–1.54), showing that the use longer than 40 years was associated with 44% increased odds of OC compared with people who did not use mouthwash (OR = 1.44; 95%CI: 1.10–1.90).

Mouthwash use less than once a day (OR = 0.85; 95%CI: 0.65–1.12) (Figure 4A) and 1-2 times a day (OR = 1.13; 95%CI: 0.93–1.37) (Figure 4B), compared to no use, was not associated with OC. However, mouthwash use 3 or more times daily (Figure 4C) was associated with an increased chance of OC (OR = 2.58; 95%CI: 1.38–4.82).

Meta-Regression

In the non-adjusted analysis, the co-variable ‘setting’ and ‘case-control ratio’ presented a p < 0.20 in association with OC, and explained 23.8% and 26.3% of the heterogeneity among the studies, respectively. Multivariable meta-regression showed that these variables, together, explained 39.4% of heterogeneity among the studies (Supplementary Table 3^ddAvailable from: https://drive.google.com/file/d/1jFB_FOL8yj NnFgdEtuFgYFN3Ug8xeXsp/view?usp=sharing ).

DISCUSSION

In this systematic review and meta-analysis of 15 case-controls and 17 OR estimates including 6,515 cases and 17,037 controls, we observed no association between mouthwash use (any versus no use) and OC (OR = 1.00; 95%CI: 0.79–1.26). Three previous meta-analyses also did not find association³⁴34. Hostiuc S, Ionescu IV, Drima E. Mouthwash use and the risk of oral, pharyngeal, and laryngeal câncer: a meta-analysis. Int J Environ Res Public Health. 2021 Aug;18(15):8215. https://doi.org/10.3390/ijerph18158215
https://doi.org/10.3390/ijerph18158215... . When Hostiuc et al.³⁴34. Hostiuc S, Ionescu IV, Drima E. Mouthwash use and the risk of oral, pharyngeal, and laryngeal câncer: a meta-analysis. Int J Environ Res Public Health. 2021 Aug;18(15):8215. https://doi.org/10.3390/ijerph18158215
https://doi.org/10.3390/ijerph18158215... evaluated the overall risk of upper aerodigestive tract cancers associated with mouthwash use in 17 studies, the authors reported that the difference in risk between cases and controls was not significant. Argemi et al.³⁷37. Wynder EL, Kabat G, Rosenberg S, Levenstein M. Oral cancer and mouthwash use. J Natl Cancer Inst. 1983 Feb;70(2):255-60. also did not find association between mouthwash use and OC, neither when considered mouthwashes with alcohol in five case-control studies, nor without alcohol in four studies. Similarly, Gandini et al.³⁶36. Gandini S, Negri E, Boffetta P, La Vecchia C, Boyle P. Mouthwash and oral cancer risk quantitative meta-analysis of epidemiologic studies. Ann Agric Environ Med. 2012;19(2):173-80. estimated a non-significant relative risk summarized from nine studies. These authors also considered any frequency/duration of mouthwash use.

However, when we investigated the frequency of use, the odds of developing OC in individuals who frequently used mouthwashes (three or more times a day) was 1.30 times higher than in those who never used (OR = 1.30; 95%CI: 1.10–1.54); additionally, it was 158% higher among those who used mouthwashes for more than 40 years when compared to non-users (OR = 2.58; 95%CI: 1.38–4.82). This could suggest a dose-response effect. Gandini et al.³⁶36. Gandini S, Negri E, Boffetta P, La Vecchia C, Boyle P. Mouthwash and oral cancer risk quantitative meta-analysis of epidemiologic studies. Ann Agric Environ Med. 2012;19(2):173-80., however, estimated the relative risk with the frequency of use once, twice, or thrice a day and found no significant trend in risk with increasing daily use. Comparably, Hostiuc et al.³⁴34. Hostiuc S, Ionescu IV, Drima E. Mouthwash use and the risk of oral, pharyngeal, and laryngeal câncer: a meta-analysis. Int J Environ Res Public Health. 2021 Aug;18(15):8215. https://doi.org/10.3390/ijerph18158215
https://doi.org/10.3390/ijerph18158215... found a non-statistically significant risk difference on the incidence of cancers in upper aerodigestive tract according to the frequency of use. We were not able to identify other meta-analyses that had assessed the dose-response effect related to OC.

Tobacco, alcohol, and betel consumption, diet, nutrition, as well as immunosuppression, environmental, and genetic factors are considered risk factors for OC⁶¹61. Chi AC, Day TA, Neville BW. Oral cavity and oropharyngeal squamous cell carcinoma: an update. CA Cancer J Clin. 2015;65(5):401-21. https://doi.org/10.3322/caac.21293
https://doi.org/10.3322/caac.21293... ,⁷³73. Kumar M, Nanavati R, Modi TG, Dobariya C. Oral cancer: etiology and risk factors: A review. J Cancer Res Ther. 2016;12(2):458-63. https://doi.org/10.4103/0973-1482.186696
https://doi.org/10.4103/0973-1482.186696... . When we performed subgroup analyses considering studies that reported both crude and adjusted associations, we reduced the probability that confounding biased the pooled estimates. However, the possibility of unmeasured confounding cannot be completely disregarded since important confounding factors could have been disregarded, such as HPV infection (not considered in any of the studies), tobacco and alcohol consumption, diet/nutrition, and socioeconomic conditions (considered only in some of the association estimates). Additionally, if a confounding factor is poorly measured or inadequately defined, residual confounding may also occur. However, we can suppose that the effect of the time of mouthwash use could be confounded by the age of the participants since increasing age is associated with increasing OC risk¹⁰10. Aghiorghiesei O, Zanoaga O, Nutu A, Braicu C, Campian RS, Lucaciu O, et al. The World of Oral Cancer and its risk factors viewed from the aspect of microRNA expression patterns. Genes (Basel). 2022 Mar;13(4):594. https://doi.org/10.3390/genes13040594
https://doi.org/10.3390/genes13040594... . However, all studies included in this subgroup analysis have been adjusted for age and other potential confounders⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... ,⁶⁷67. Winn DM, Blot WJ, McLaughlin JK, Austin DF, Greenberg RS, Preston-Martin S, et al. Mouthwash use and oral conditions in the risk of oral and pharyngeal cancer. Cancer Res. 1991 Jun;51(11):3044-7.,⁶⁹69. Assunção Junior N, Teixeira VP, Lemos CA. Use of mouthwashes in patients with oral and oropharyngeal cancer. Clinical and Laboratorial Research in Dentistry. 2015;21(2):91-8. https://doi.org/10.11606/issn.2357-8041.clrd.2015.102956
https://doi.org/10.11606/issn.2357-8041.... .

Over the years, the main hypothesis for the link between mouthwashes and OC was the alcohol composition of these products. The carcinogenesis process would occur inducing a marked cytotoxic effect in human epithelial keratinocytes¹³13. Calderón-Montaño JM, Jiménez-Alonso JJ, Guillén-Mancina E, Burgos-Morón E, López-Lázaro M. A 30-s exposure to ethanol 20% is cytotoxic to human keratinocytes: possible mechanistic link between alcohol-containing mouthwashes and oral cancer. Clin Oral Investig. 2018 Nov;22(8):2943-6. https://doi.org/10.1007/s00784-018-2602-z
https://doi.org/10.1007/s00784-018-2602-... ,²³23. Guillén-Mancina E, Calderón-Montaño JM, López-Lázaro M. Avoiding the ingestion of cytotoxic concentrations of ethanol may reduce the risk of cancer associated with alcohol consumption. Drug Alcohol Depend. 2018 Feb;183(183):201-4. https://doi.org/10.1016/j.drugalcdep.2017.11.013
https://doi.org/10.1016/j.drugalcdep.201... , previously investigated in vitro with two commercially available mouthwash brands¹⁴14. Adams D, Addy M. Mouthrinses. Adv Dent Res. 1994 Jul;8(2):291-301. https://doi.org/10.1177/08959374940080022401
https://doi.org/10.1177/0895937494008002... . For each brand, an alcohol-free and an alcohol-containing version (96 mg/mL and 213.03 mg/mL, respectively) were tested on human oral keratinocytes with and without a mild dysplasia. The authors concluded that alcohol-based mouthwashes were genotoxic to both normal and dysplastic oral keratinocytes, inducing generalized changes in gene expression in vitro.

Similar results were also found in clinical trials evaluating the effect of alcohol-containing and alcohol-free mouthwashes on exfoliated oral cells⁷⁴74. Carlin V, Matsumoto MA, Saraiva PP, Artioli A, Oshima CT, Ribeiro DA. Cytogenetic damage induced by mouthrinses formulations in vivo and in vitro. Clin Oral Investig. 2012 Jun;16(3):813-20. https://doi.org/10.1007/s00784-011-0559-2
https://doi.org/10.1007/s00784-011-0559-... ,⁷⁵75. Zamora-Perez AL, Mariaud-Schmidt RP, Fuentes-Lerma MG, Guerrero-Velázquez C, Gómez-Meda BC, López-Verdín S, et al. Increased number of micronuclei and nuclear anomalies in buccal mucosa cells from people exposed to alcohol-containing mouthwash. Drug Chem Toxicol. 2013 Apr;36(2):255-60. https://doi.org/10.3109/01480545.2012.737804
https://doi.org/10.3109/01480545.2012.73... . In this context, the authors found an increased frequency of micronuclei and cellular abnormalities in the group exposed to the alcohol-containing mouthwash. Due to the superficial and intracellular characteristics of the oral mucosa epithelium, the detection of DNA damage and cell death in desquamated epithelial cells requires the genotoxic agent to overcome the permeability barrier of the basal layer and induce DNA damage, later converting them into micronuclei during cell division⁷⁶76. Hernandes KC, da Silva CA, Schmidt VK, Boeira JM. Assessment of effects of mouthwash by buccal micronucleus cytome assay: a preliminary study. Rev Eletr Cient UERGS. 2016;2(3):267-75. https://doi.org/10.21674/2448-0479.23.267-275
https://doi.org/10.21674/2448-0479.23.26... . The correlation between the number of stem cell divisions that occurred in a tissue during a person’s life and the risk of cancer diagnosis in that tissue is highly positive and statistically significant⁷⁷77. Tomasetti C, Vogelstein B. Cancer etiology. Variation in cancer risk among tissues can be explained by the number of stem cell divisions. Science. 2015 Jan;347(6217):78-81. https://doi.org/10.1126/science.1260825
https://doi.org/10.1126/science.1260825... .

When considering only the use of alcohol containing mouthwashes versus no use, the association in our meta-analysis did not remain significant (OR = 1.20; 95%CI: 0.93–1.55). Argemí et al.³⁵35. Aceves Argemí R, González Navarro B, Ochoa García-Seisdedos P, Estrugo Devesa A, López-López J. Mouthwash with alcohol and oral carcinogenesis: systematic review and meta-analysis. J Evid Based Dent Pract. 2020 Jun;20(2):101407. https://doi.org/10.1016/j.jebdp.2020.101407
https://doi.org/10.1016/j.jebdp.2020.101... also summarized data referred to the alcohol content of nine studies and showed a non-significant association (OR = 1.48; 95%CI: 0.85–2.56). Although the composition of mouthwashes and the alcohol content were not well described in all studies, the supposition that these non-alcoholic products with antimicrobial activity may also be cytotoxic should be mentioned²⁵25. Tartaglia GM, Kumar S, Fornari CD, Corti E, Connelly ST. Mouthwashes in the 21st century: a narrative review about active molecules and effectiveness on the periodontal outcomes. Expert Opin Drug Deliv. 2017 Aug;14(8):973-82. https://doi.org/10.1080/17425247.2017.1260118
https://doi.org/10.1080/17425247.2017.12... ,⁷⁸78. Schmidt J, Zyba V, Jung K, Rinke S, Haak R, Mausberg RF, et al. Effects of octenidine mouth rinse on apoptosis and necrosis of human fibroblasts and epithelial cells - an in vitro study. Drug Chem Toxicol. 2018 Apr;41(2):182-7. https://doi.org/10.1080/01480545.2017.1337124
https://doi.org/10.1080/01480545.2017.13... . A wide variety of antiseptics containing different active ingredients are available and widely used in dentistry³⁰30. Coelho AS, Laranjo M, Gonçalves AC, Paula A, Paulo S, Abrantes AM, et al. Cytotoxic effects of a chlorhexidine mouthwash and of an enzymatic mouthwash on human gingival fibroblasts. Odontology. 2020 Apr;108(2):260-70. https://doi.org/10.1007/s10266-019-00465-z
https://doi.org/10.1007/s10266-019-00465... . These products are regulated as cosmetic products, thereby not requiring ingredients declaration²⁶26. Vlachojannis C, Al-Ahmad A, Hellwig E, Chrubasik S. Listerine ® Products: an update on the efficacy and safety. Phytother Res. 2016 Mar;30(3):367-73. https://doi.org/10.1002/ptr.5555
https://doi.org/10.1002/ptr.5555... . Thus, we can assume that other components are also involved in cell damage²⁴24. Müller HD, Eick S, Moritz A, Lussi A, Gruber R. Cytotoxicity and Antimicrobial Activity of Oral Rinses In Vitro. BioMed Res Int. 2017;2017:4019723. https://doi.org/10.1155/2017/4019723
https://doi.org/10.1155/2017/4019723... or oral microflora alterations, harboring the potential to alter the balance of immune tolerance, further contributing to the genesis and promotion of OC¹⁵15. Eliot MN, Michaud DS, Langevin SM, McClean MD, Kelsey KT. Periodontal disease and mouthwash use are risk factors for head and neck squamous cell carcinoma. Cancer Causes Control. 2013 Jul;24(7):1315-22. https://doi.org/10.1007/s10552-013-0209-x
https://doi.org/10.1007/s10552-013-0209-... .

The most common molecules contained in mouthwashes are chlorhexidine, essential oils, cetylpyridinium chloride, triclosan, octenidine, delmopinol, polyvinylpyrrolidone, hyaluronic acid, and natural compunds²⁵25. Tartaglia GM, Kumar S, Fornari CD, Corti E, Connelly ST. Mouthwashes in the 21st century: a narrative review about active molecules and effectiveness on the periodontal outcomes. Expert Opin Drug Deliv. 2017 Aug;14(8):973-82. https://doi.org/10.1080/17425247.2017.1260118
https://doi.org/10.1080/17425247.2017.12... . When exposed to human gingival fibroblasts at the concentration required to inhibit 50% of cellular metabolic activity (IC50), 0.2% chlorhexidine decreased the viable cells number and increased the number of cells undergoing apopstosis³⁰30. Coelho AS, Laranjo M, Gonçalves AC, Paula A, Paulo S, Abrantes AM, et al. Cytotoxic effects of a chlorhexidine mouthwash and of an enzymatic mouthwash on human gingival fibroblasts. Odontology. 2020 Apr;108(2):260-70. https://doi.org/10.1007/s10266-019-00465-z
https://doi.org/10.1007/s10266-019-00465... . Other in vitro studies⁷⁸78. Schmidt J, Zyba V, Jung K, Rinke S, Haak R, Mausberg RF, et al. Effects of octenidine mouth rinse on apoptosis and necrosis of human fibroblasts and epithelial cells - an in vitro study. Drug Chem Toxicol. 2018 Apr;41(2):182-7. https://doi.org/10.1080/01480545.2017.1337124
https://doi.org/10.1080/01480545.2017.13... corroborated these findings. Cetylpyridinium chloride was also found to exhibit severe cytotoxic effects against human keratinocytes and murine fibroblasts even at low concentrations²⁹29. Fromm-Dornieden C, Rembe JD, Schäfer N, Böhm J, Stuermer EK. Cetylpyridinium chloride and miramistin as antiseptic substances in chronic wound management - prospects and limitations. J Med Microbiol. 2015 Apr;64(Pt 4):407-14. https://doi.org/10.1099/jmm.0.000034
https://doi.org/10.1099/jmm.0.000034... . Listerine^®, a product that contains thymol, eucalyptus, methyl salicylate, and menthol, had its cytotoxicity evaluated²⁶26. Vlachojannis C, Al-Ahmad A, Hellwig E, Chrubasik S. Listerine ® Products: an update on the efficacy and safety. Phytother Res. 2016 Mar;30(3):367-73. https://doi.org/10.1002/ptr.5555
https://doi.org/10.1002/ptr.5555... and the authors have suggested all phenolic compounds may contribute, to some extent, to cell damage in vitro.

Triclosan is toxic to mitochondria, immune cells²⁷27. Sangroula S, Baez Vasquez AY, Raut P, Obeng B, Shim JK, Bagley GD, et al. Triclosan disrupts immune cell function by depressing Ca2+ influx following acidification of the cytoplasm. Toxicol Appl Pharmacol. 2020 Oct;405(June):115205. https://doi.org/10.1016/j.taap.2020.115205
https://doi.org/10.1016/j.taap.2020.1152... , and possibly to the neural system²⁸28. Ruszkiewicz JA, Li S, Rodriguez MB, Aschner M. Is Triclosan a neurotoxic agent? J Toxicol Environ Health B Crit Rev. 2017;20(2):104-17. https://doi.org/10.1080/10937404.2017.1281181
https://doi.org/10.1080/10937404.2017.12... . In 2017, the Colgate-Palmolive company removed triclosan from dentifrices, following a determination by the United States Food and Drug Administration⁸²82. U.S. Food and Drug Administration. 5 things to know about Triclosan. Published May 16, 2019 [cited 2022 Aug 16]. Available from: https://www.fda.gov/consumers/consumer-updates/5-things-know-about-triclosan
https://www.fda.gov/consumers/consumer-u... . In addition to triclosan, twenty-three other active ingredients have also been removed from over-the-counter antiseptic products, due to insufficient data on their safety and effectiveness.

Hereupon, a limitation of our meta-analysis was the failure to perform subgroup analyses according to the different proportions of mouthwashes alcohol content. Otherwise, we could assess whether the substances present in their formulations are important for OC regardless of the alcohol content since the available evidence is supported only by in vitro studies. Thus, new studies that present data regarding the alcohol content of mouthwashes and their main components are essential to investigate and clarify the impact these molecules have.

This meta-analysis was also the first to analyze the quality of evidence using the GRADEpro GDT⁶²62. GRADEpro GDT: GRADEpro Guideline Development Tool [Software]. London: Cochrane; 2020.,⁶³63. Ryan R, Hill S. How to GRADE the quality of the evidence. Cochrane Consumers and Communication Group. 2016;Version 3 :1-24. https://cccrg.cochrane.org/author-resources
https://cccrg.cochrane.org/author-resour... . The tool estimated the quality of evidence as low. This result is mainly due to the design of the included studies. Case-control is the most feasible type of study design to investigate this subject, but it presents more biases than clinical trials and cohort studies. In this context, the possibility of some confounding, measurement, and selection biases leads us to classify the risk of bias as ‘serious’ by GRADEpro, despite most studies being classified as moderate or low risk of bias according to the NOS criteria. However, due to the unfeasibility of randomization, we can admit certain risk of bias in the case-control studies, so we can suggest that the NOS instrument, adopted in this meta-analysis, could have underestimated the risk of bias in the included studies. However, NOS is one of the most used instruments⁸³83. Zhang Y, Huang L, Wang D, Ren P, Hong Q, Kang D. The ROBINS-I and the NOS had similar reliability but differed in applicability: a random sampling observational studies of systematic reviews/meta-analysis. J Evid Based Med. 2021 May;14(2):112-22. https://doi.org/10.1111/jebm.12427
https://doi.org/10.1111/jebm.12427... , and its content validity and interobserver reliability are well established⁸³83. Zhang Y, Huang L, Wang D, Ren P, Hong Q, Kang D. The ROBINS-I and the NOS had similar reliability but differed in applicability: a random sampling observational studies of systematic reviews/meta-analysis. J Evid Based Med. 2021 May;14(2):112-22. https://doi.org/10.1111/jebm.12427
https://doi.org/10.1111/jebm.12427... ,⁸⁴84. Hootman JM, Driban JB, Sitler MR, Harris KP, Cattano NM. Reliability and validity of three quality rating instruments for systematic reviews of observational studies. Res Synth Methods. 2011 Jun;2(2):110-8. https://doi.org/10.1002/jrsm.41
https://doi.org/10.1002/jrsm.41... . A recent meta-analysis on the topic³⁴34. Hostiuc S, Ionescu IV, Drima E. Mouthwash use and the risk of oral, pharyngeal, and laryngeal câncer: a meta-analysis. Int J Environ Res Public Health. 2021 Aug;18(15):8215. https://doi.org/10.3390/ijerph18158215
https://doi.org/10.3390/ijerph18158215... did also use the same instrument; moreover, NOS seems to provide the same reliability, varying in applicability, compared to the ROBINS-I tool recommended by Cochrane. Furthermore, the complexity of using the ROBINS-I tool can be a limiting factor for its adoption⁸³83. Zhang Y, Huang L, Wang D, Ren P, Hong Q, Kang D. The ROBINS-I and the NOS had similar reliability but differed in applicability: a random sampling observational studies of systematic reviews/meta-analysis. J Evid Based Med. 2021 May;14(2):112-22. https://doi.org/10.1111/jebm.12427
https://doi.org/10.1111/jebm.12427... . Another factor that decreases the quality of the evidence is the inconsistency of results since some studies have showed positive (risk)⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... ,⁶⁷67. Winn DM, Blot WJ, McLaughlin JK, Austin DF, Greenberg RS, Preston-Martin S, et al. Mouthwash use and oral conditions in the risk of oral and pharyngeal cancer. Cancer Res. 1991 Jun;51(11):3044-7.and others negative (protective)⁵⁵55. Sharma AP, Naqvi A, Sharma N, Haider K. Assessment of the association between oral health and hygiene practices and oral câncer: a hospital-based case-control study. Eur J Mol Clin Med. 2020;7(8):1206-11.,⁵⁶56. Saira, Ahmed R, Malik S, Khan MF, Khattak MR. Epidemiological and clinical correlates of oral squamous cell carcinoma in patients from north-west Pakistan. J Pak Med Assoc. 2019 Aug;69(8):1074-8. associations between mouthwash use and OC.

As strengths of our study, this meta-analysis was the first to consider the effect of the frequency and duration of mouthwash use over the years in OC. Despite pioneer, our findings should be carefully interpreted, given the small number of studies that considered the frequency (n = 2) and duration (n = 3) of mouthwash use. Another strength was the vast bibliographic search in a higher number of databases, including the grey literature, using the PEO strategy, without language and publication date restrictions. Therefore, we were able to reach studies that were not included in the previously published meta-analyses. In addition, we did not include, in this meta-analysis³⁷37. Wynder EL, Kabat G, Rosenberg S, Levenstein M. Oral cancer and mouthwash use. J Natl Cancer Inst. 1983 Feb;70(2):255-60., samples previously used in other larger studies⁴⁴44. Ahrens W, Pohlabeln H, Foraita R, Nelis M, Lagiou P, Lagiou A, et al. Oral health, dental care and mouthwash associated with upper aerodigestive tract cancer risk in Europe: the ARCAGE study. Oral Oncol. 2014 Jun;50(6):616-25. https://doi.org/10.1016/j.oraloncology.2014.03.001
https://doi.org/10.1016/j.oraloncology.2... ,⁴⁵45. Boffetta P, Hayes RB, Sartori S, Lee YC, Muscat J, Olshan A, et al. Mouthwash use and cancer of the head and neck: a pooled analysis from the International Head and Neck Cancer Epidemiology Consortium. Eur J Cancer Prev. 2016 Jul;25(4):344-8. https://doi.org/10.1097/CEJ.0000000000000179
https://doi.org/10.1097/CEJ.000000000000... . We considered the alcohol content of mouthwashes versus the non-use when conducting the analyses, and different subgroup analyses were also performed. Lastly, a meta-regression was performed to explain the heterogeneity.

CONCLUSIONS

This systematic review and meta-analysis showed no relationship between mouthwash use and OC, except for the mouthwash use for three or more times a day and for people who have used it for over 40 years, suggesting a possible dose-dependent effect. These findings, however, should be analyzed with caution given the small number of studies that consider the frequency of mouthwash use. Therefore, we recommend that future studies evaluate, in detail, the frequency, duration, and content of mouthwashes to increase the strength of evidence for a possible dose-response effect of this exposure on OC risk.

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