key: cord-0714769-5ricjw70
authors: Statkute, Evelina; Rubina, Anzelika; O’Donnell, Valerie B; Thomas, David W.; Stanton, Richard J.
title: Brief Report: The Virucidal Efficacy of Oral Rinse Components Against SARS-CoV-2 In Vitro
date: 2020-11-18
journal: bioRxiv
DOI: 10.1101/2020.11.13.381079
sha: 1f97ad9b879febe44b2497e9ecc2766fb3848e3f
doc_id: 714769
cord_uid: 5ricjw70

The ability of widely-available mouthwashes to inactivate SARS-CoV-2 in vitro was tested using a protocol capable of detecting a 5-log10 reduction in infectivity, under conditions mimicking the naso/oropharynx. During a 30 second exposure, two rinses containing cetylpyridinium-chloride and a third with ethanol/ethyl lauroyl arginate eliminated live virus to EN14476 standards (>4-log10 reduction), while others with ethanol/essential oils and povidone-iodine (PVP-I) eliminated virus by 2-3-log10. Chlorhexidine or ethanol alone displayed little or no ability to inactivate virus. Studies are warranted to determine whether these formulations can inactivate virus in the human oropharynx in vivo, and whether this might impact transmission risk.

Background 35

The lipid membranes of enveloped viruses are sensitive to disruption by lipidomimetic agents and 36 surfactants. Thus, we hypothesised that the SARS-CoV-2 virus would be susceptible to inactivation by 37 components in widely available mouthwashes, such as ethanol/essential oils, cetylpyridinium chloride 38 (CPC) and povidone-iodine (PVP-I) [1] . Indeed Since only one of the in vitro studies has used the SARS-CoV-2 pathogen to date, here, we extended 50 this work by testing the virucidal activity against SARS-CoV-2 of a range of mouthwashes including CPC 51 (0.05-0.1%w/v Dentyl Dual Action, 0.05-0.1% w/v Dentyl Fresh Protect, 0.10% w/v SCD Max) 52 ethanol/essential oils (Listerine Cool Mint, 21.7% v/v ethanol), ethanol/ethyl lauroyl arginate 53 (Listerine Advanced Gum Treatment, 23% v/v ethanol), chlorhexidine (0.2% w/v; Corsodyl) and 54 povidone iodine (0.5% w/v; Videne). We found that three products had sufficient activity to pass 55 EN14476 against SARS-CoV2. We also investigated the contribution of ethanol to the observed 56 virucidal activity, to inform future studies as to which products are most likely to provide the greatest 57 benefit against SARS-CoV2, and to provide information as to how future virucidal formulations might 58 be optimised. In initial experiments, we examined the effect of mouthwashes on the VeroE6/ACE2/TMPRSS2 83 monolayers used to detect live virus, following serial dilution in DMEM. Four of the seven products 84 demonstrated toxicity to the monolayer, which was not eliminated until they were diluted at least 85 100-1000-fold, limiting the sensitivity of the assay to measure residual infectivity. We therefore used 86 size-exclusion chromatography (SEC) to rapidly purify virus away from the products. When virus was 87 purified on S-400 HR microspin columns, only minimal loss of infectivity was observed (Fig 1A) , 88 however toxicity from the mouthwashes against the cell monolayer was virtually eliminated (Fig 1B) . 89 SEC was therefore used for all assays from this point forwards. This approach also ensures that the 90 activity of the mouthwashes against the virus was rapidly stopped after the desired co-incubation 91 time. In comparison, our results suggest that 'stopping' the reaction by serial 10-fold dilution would 92 leave sufficient mouthwash activity to have continued biological activity against the virus. Combined 93 with the use of VeroE6/ACE2/TMPRSS2 cells for titration, which SARS-COV2 enters >1log more 94 efficiently than parental VeroE6, the assay was capable of detecting a 5-log10 decrease in virus titre. 95 This is more than sufficient to detect the 4-log10 reduction in activity specified by EN14476. 96

Having optimised a sensitive protocol for the detection of virucidal activity, we tested the ability of a 97 wide range of commercially available mouthwash formulations (Table 1) (Fig 1C) . Ethanol alone at <23 % had no effect on virus infectivity, 104 thus the inclusion of essential oils (Listerine Cool Mint) or LAE (Listerine Advanced) appears to be 105 required for optimal efficacy. Lastly, chlorhexidine was relatively inactive (<2 log fold reduction). 

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