key: cord-0691103-w4nop99i
authors: Komine, Akihiko; Yamaguchi, Erika; Okamoto, Nako; Yamamoto, Kazushi
title: Virucidal activity of oral care products against SARS-CoV-2 in vitro
date: 2021-02-22
journal: J Oral Maxillofac Surg Med Pathol
DOI: 10.1016/j.ajoms.2021.02.002
sha: 8df22da765b25f5ee41f46ec663377b205cfea7a
doc_id: 691103
cord_uid: w4nop99i

OBJECTIVE: Coronavirus disease 2019 (COVID-19) caused by infection by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide. Since reducing the amount of virus in saliva is considered to prevent broader infection, the Center for Disease Control (CDC) and American Dental Hygienists’ Association (ADHA) have recommended use of CPC- or CHX-containing oral care products before the dental procedure. However, there is no certified evidence. So, we examined inactivation of SARS-CoV-2 by oral care products in several countries in vitro. METHODS: 0.05% Cetylpyridinium chloride (CPC) mouthwash, 0.05% CPC toothpaste and 0.30% CPC spray in Japan; 0.06% chlorhexidine gluconate (CHX) + 0.05% CPC mouthwash and 0.12% CHX + 0.05% CPC mouthwash in Europe; 0.075% CPC mouthwash, 0.12% CHX mouthwash, and 0.20% delmopinol hydrochloride mouthwash in the USA; and 0.04% CPC mouthwash in China were assessed for their virucidal activity with ASTM E1052. RESULTS: The virus was inactivated in vitro by the contact time in directions for use of all oral care products containing CPC or delmopinol hydrochloride as anticeptics. CONCLUSIONS: These results suggest that these oral care products in each country may reduce the viral load in the mouth.

Coronavirus disease 2019 due to infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has spread worldwide from December 2019, and COVID-19 was declared a pandemic by the World Health Organization (WHO) in March 2020. On November 18 th , 2020 the WHO reported that 54,771,888 people had been infected, with the result of 1,324,249 deaths [1] .

SARS-CoV-2 is a beta coronavirus that is about 50 to 200 nm in size, has an envelope as a lipid bilayer, and a single-stranded positive-sense RNA genome of about 30,000 bases [2] . Urgent research and development on cell models of SARS-CoV-2 infection, therapeutic agents, and vaccines is underway. The infection route is contact infection or droplet infection by saliva, with infection by saliva droplets being most common [3, 4] . The oral cavity is considered to be a reservoir for viruses, and reducing the amount of virus in the oral cavity can lead to prevention of infection in other individuals [4] . Povidone-iodine (PVP-I),

Cetylpyridinium chloride (CPC), chlorhexidine gluconate (CHX), and delmopinol hydrochloride are often added to oral care products as antiseptics. PVP-I is an excellent fungicide that has an inactivating effect on SARS-CoV-2 in vitro and in vivo [5] [6] [7] [8] . However, PVP-I and CHX carry a risk of anaphylactic shock and also PVP-I cannot be used by people with impaired thyroid function [9, 10] .

The Center for Disease Control (CDC) and American Dental Hygienists' Association (ADHA) have recommended use of CPC-or CHX-containing mouthwash before the dental procedure against COVID-19 [11, 12] . However, only a few reports have examined an inactivating effect of CHX on SARS-CoV-2 [7, 13] and some have suggested effects of CPC on other viruses [3, 14, 15, 16, 17] , but with no certified data on direct inactivation effects of CPC on SARS-CoV-2 [18] . On the other hands, in previous studies, virucidal activities were tested against some Coronaviruses [19, 20] . There are some different anti-viral sensitivities J o u r n a l P r e -p r o o f depend on Coronavirus species, for example Beazalkonium chloride and hydrogen peroxide [7, 19] . Therefore, in this study, we evaluated SARS-CoV-2 inactivation in vitro by oral care products made under the regulations of quasi-drugs, drugs, medical device or cosmetics standards of several different countries.

All tests in the study were performed by QTEC (Japan Textile Products Quality and Technology Center, https://www.qtec.or.jp/en) as a test contract facility. 

The test solutions are shown in Table 1 were evaluated for cytotoxicity, interference and virucidal activity.

To evaluate damage to cells, the cytotoxicity of the test solutions was investigated. A test mixture was made with 0.1 mL 5% FBS containing EMEM and 0.9 mL of test solution. A sample of 0.1 mL of the test mixture was added to a test tube containing 0.9 mL of neutralizer 

Virucidal activity was evaluated with an ASTM E1052-20 [22] . The virus suspension (viral titer of 8.49 Log PFU/mL) of 0.1 mL was added to 0.9 mL of a test solution and mixed well with a vortex mixer. This suspension was incubated for 20 s, 30 s or 3 min at 25℃ (Table 1) .

Then, 0.1 mL of the suspension was added to a tube containing 0.9 mL of neutralizer and mixed well to form an inactivated mixture. A series of 10-fold dilutions of this mixture was made using 2% FBS containing DMEM and the viral infectivity titer was measured per 0.1 mL of inactivated mixture and calculated per 1.0 mL. Three independent experiments were performed (n=3).

We tested the anti-viral activity of eleven test solutions including CPC, CHX or delmopinol hydrochloride in several countries.

Before the virucidal activity test, we examined the cytotoxicity and interference tests. There were no cytotoxic or interference effects at dilutions ranging from 1 to 1/100 ( Table 1 ).

The virucidal activity of the oral care products against SARS-CoV-2 was evaluated using 

A recent study showed that mouthwash containing dequalinium chloride and benzalkonium chloride had virucidal activity against SARS-CoV-2 [7] , which suggested that a quaternary ammonium cation has activity against SARS-CoV-2. CPC is known to be a quaternary ammonium cation and delmopinol hydrochloride is known to be a cationic surfactant. So, our findings also suggest that cationic surfactants may be effective against SARS-CoV-2, although octenidine dihydrochloride did not have activity in the previous study [7] .

In this study and the previous study, mouthwash including CHX had no sufficient virucidal effect against SARS-CoV-2 in vitro [7] . However, a previous clinical case study showed that CHX mouthwash transiently decreased the SARS-CoV-2 viral load in the saliva [13] . Since CHX can retain on a surface of the oral cavity for several hours due to its cationic feature, the long-lasting effect in vivo might lead to reduce the viral load compared to neutralize the antiseptic in a short contact time in vitro [23] . But, there are few researches about CHX mouthwash against SARS-CoV-2, so more in vivo investigations of the antiviral effect of CHX mouthwash are necessary.

This study showed that oral care products containing CPC or delmopinol hydrochloride have antiviral activity against SARS-CoV-2. This supports the recommendation for a preprocedual use of CPC-containing mouthwash for SARS-CoV-2 reduction in aerosol by the CDC and ADHA. We note that the data in this report were collected in vitro, and a further evaluation of the anti-SARS-CoV-2 activity of the products is required in vivo.

J o u r n a l P r e -p r o o f 

World Health Organization, WHO Coronavirus Disease (COVID-19) Dashboard

A novel coronavirus from patients with pneumonia in china

Is the oral cavity relevant in SARS-CoV-2 pandemic?

Ocular tropism of respiratory viruses

Povidone-iodine demonstrates rapid in vitro virucidal activity against SARS-CoV-2, the virus causing COVID-19 disease

Comparison of in vitro inactivation of SARS CoV-2 with hydrogen peroxide and povidone-iodine oral antiseptic rinses

Virucidal efficacy of different oral rinses against sever acute respiratory syndrome coronavirus 2

Is povidone iodine mouthwash effective against SARS-CoV-2? First in vivo tests

Anaphylactic shock following povidone

antibody-mediated shock reaction caused by topical application of chlorhexidine

Clinical significance of a high SARS-CoV-2 viral load in the saliva

Repurposing quaternary ammonium compounds as potential treatments for COVID-19

Use of mouthwashes against COVID-19 in dentistry

Lowering the transmission and spread of human coronavirus

In vitro assessment of the virucidal activity of four mouthwashes containing cetylpyridinium chloride, ethanol, zinc and a mix of enzyme and proteins against a human coronavirus

Efficacy of commercial mouth-rinses on SARS-CoV-2 viral load in saliva: Randomized Control Trial in Singapore

Persistence of coronaviruses on inanimate surfaces and their inactivation with biocidal agents

Viricidal treatments for prevention of coronavirus infection

Consensus and variations in cell lines specificity among human metapneumovirus strains

American Society for Testing and Materials, ASTM E1052-20 Standard Practice to Assess the Activity of Microbicides against Viruses in Suspension

A comparison between chlorhexidine and some quaternary ammonium compounds with regard to retention, salivary concentration and plaqueinhibiting effect in the human mouth after mouth rinses

We would like to thank QTEC for performance of virucidal tests.J o u r n a l P r e -p r o o f