Virucidal Properties of Molecular Iodine Oral Rinse Against SARS-CoV-2

There are at least three separate routes by which SARS-CoV-2 can infect the oral cavity: the lower and upper respiratory tracts produce liquid droplets that connect with the oral cavity¹; infected blood can enter the mouth via gingival crevicular fluid; and SARS-CoV-2 infections of the salivary gland can be introduced through the salivary ducts.²

RNA sequencing of human salivary glands and gingiva indicates saliva, oral mucosa, and epithelium are susceptible to viral infection and carry the virus, even in asymptomatic individuals. (COVID-19 salivary diagnostics make this self-evident.) Research from Huang et al in 2021 indicates mucosal scrapings are infectious and retain their ability to replicate even after shedding³: "Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission… The oral cavity represents a robust and underappreciated site for SARS-CoV-2 infection."

In their 2019 meta‐analysis, Marui et al showed that preprocedural mouthrinses can significantly reduce the microbial load of dental aerosols.⁴ However, saliva, which includes upper respiratory tract secretions of mucous/glycoproteins, can reduce the effectiveness of mouthrinses not only by neutralizing the rinses' antimicrobial components⁵ but also by coating viruses so they are "protected" from exposure (Figure 1). Therefore, in order to understand whether a mouthrinse is effective against SARS-CoV-2, definitive in vitro tests must include human saliva.

A relatively new (2017) molecular iodine oral rinse (MIOR) (ioRinse^™ RTU, IoTech International, iotechinternational.com) has shown promise as a SARS-CoV-2 virucidal.⁶ Therefore, an in vitro test of MIOR against SARS-CoV-2 in saliva suspension was performed to more accurately understand MIOR's potential effect on SARS-CoV-2 infectivity.

Testing was performed at BioScience Laboratories (biosciencelabs.com) in Bozeman, Montana, and based on ASTM E1052-20 ("Standard Practice to Assess the Activity of Microbicides against Viruses in Suspension"). Assays were conducted in the presence of 5% human saliva, with results recorded at 30 and 60 seconds.

The virucidal suspension test (in vitro time-kill method) was performed in accordance with Good Laboratory Practices, as specified in 21 CFR Part 58. A 1.5 ml solution of human saliva was used as the "organic soil load" (OSL), which is approximately 50% greater by volume than that found in average adults (average female 0.96 ml, and average male approximately 1.19 ml of saliva), providing a more conservative saliva "buffer" and more critical test.⁷ The amount of the test antimicrobial MIOR used was 30 ml, as recommended by the manufacturer's Instructions for Use (IFU).

MIOR was provided to the testing facility by the study sponsor, which retained determination of its identity, strength, purity, composition, solubility, and stability. The challenge virus SARS-CoV-2 strain (USA-WA1/2020) was provided by the Biological and Emerging Infections Resource Program, National Institute of Allergy and Infectious Diseases (BEI Resources #NR-52281). Host cells were Vero E6 (ATCC #CRL-1586 - green money kidney, epithelial). (See Table 1 through Table 3.) Under informed consent and according to inclusion/exclusion criteria (age 21 to 70, good general health), human saliva was donated by 10 adults on the day of testing; male:female ratio was 50:50. (Internal Review Board approval was not required for saliva donation.) Two donors were tobacco smokers (20%) in order to mimic national smoking incidence. Collected saliva was "unstimulated" or "resting" saliva. The saliva was collected from the donors on the morning of the efficacy testing. Lyra Direct RT-PCR SARS-CoV-2 Assay (Quidel Corp., quidel.com) was used to ensure saliva samples were negative for SARS-CoV-2. Samples were pooled and refrigerated at 2ºC to 8ºC until used.

Testing was performed using RT-PCR Instrument 7500 Fast Dx Series (Applied Biosystems, thermofisher.com) with Lyra Direct RT-PCR SARS-CoV-2 Assay (FDA Emergency Use Authorization for COVID-19 diagnostics). Growth medium was Eagle's Minimal Essential Medium (EMEM) with 10% fetal bovine serum (FBS) along with antibiotics, 1% penicillin-streptomycin-amphotericin B (10,000; 10,000; and 25 µg/ml PSAB, respectively). Maintenance medium was EMEM with 2% FBS and 2% antibiotic (20,000; 20,000; and 50 µg/ml PSAB, respectively). Dey-Engley Neutralizing Broth was used as a neutralizer for MIOR activity (Table 4).

Vero E6 cells were maintained as monolayers in disposable cell culture labware. Host cells were seeded onto 24 well cell culture plates. Vero E6 monolayers (passage 4) were 80% confluent and less than 48 hours old before inoculation with SARS-CoV-2. Growth medium was replaced with maintenance medium to support virus propagation. SARS-CoV-2 was propagated in Vero E6 cells and stored in a -70ºC freezer. Upon use, the aliquots of stock virus were thawed, and 0.5 ml aliquot of pooled saliva were added to 0.5 ml of virus stock and mixed thoroughly.

Aliquots of MIOR, saliva, and maintenance medium were pre-equilibrated to 37ºC. The MIOR/virus/saliva suspension was examined to make sure there were no conglomerates following testing. The viral titers were determined using a 50% tissue culture infectious dose (TCID50) calculation-the Quantal test (Spearman-Kärber Method). Log₁₀ infectivity reductions were calculated as (log₁₀ TCID50 of the virus control) - (log₁₀ TCID50 of the virucidal test suspension). Percent reductions were calculated as [1- (TCID50 virucidal test/TCID50 virus control)] x 100.

The study was initiated January 19, 2021, and completed March 24, 2021. No deviations from the study protocol or applicable BioScience Laboratories Standard Operating Procedures occurred during the course of the evaluation. Test acceptance criteria, as follows, were all met:

· ≥6.5 log₁₀ TCID50 was recovered from the virus control.

· Cells in the cell culture control wells were viable and attached to the bottom of the wells.

· Medium was free of contamination.

· MIOR test product was fully neutralized after the timed exposures such that the difference between virus titer for the neutralization and virus controls did not exceed 1.0 log₁₀. (Actual difference was zero, as both neutralization and virus controls were equivalent in the presence of saliva.) Neutralized MIOR displayed no cell cytotoxicity.

· Saliva (OSL) and neutralizer alone (controls) did not have any significant effect on virus viability.

In the presence of MIOR with saliva, virus load reductions compared with baseline were 4.75 log₁₀ (>99.99% reduction) after 30 seconds exposure and ≥5.25 log₁₀ (>99.99% reduction) after 60 seconds. Testing in the absence of human saliva, infectivity was reduced by 5.00 log₁₀ (>99.99% reduction) and ≥5.75 log₁₀ (> 99.99% reduction) following 30- and 60-second exposures, respectively (Table 5). For additional results, including virus infectivity, with and without saliva, and infectivity of controls and cytotoxicity, see Table 1 through Table 3.

This study evaluated the percent log₁₀ reductions of initial populations of SARS-CoV-2 in the presence of 5% human saliva after exposure to a molecular iodine oral rinse for 30 and 60 seconds. Virucidal effectiveness was ≥99.99% SARS-CoV-2 reduction in fresh human, whole saliva.

Many infection control products have not been tested for their efficacy against SARS-CoV-2, because it is extremely contagious. In the United States, in vitro testing of SARS-CoV-2 is limited by the Centers for Disease Control and Prevention (CDC) and performed only at BioContainment Level 3 or higher laboratories.⁸ To date, in vivo testing of mouthrinse effectiveness against SARS-CoV-2 has been limited, unrealistic for in-office use and ineffective.^9,10 When actually tested against SARS-CoV-2, several rinses recommended early in the pandemic have subsequently been shown to be ineffective (or poorly effective), and, to date, none have been tested in vitro in the presence of saliva. Initially, povidone iodine and hydrogen peroxide both were suggested as pretreatment oral rinses for SARS-CoV-2 management. While the American Dental Association at one time recommended 1.5% hydrogen peroxide as an antiviral preprocedural rinse, the CDC has published test data showing 18 to 20 minutes are required for hydrogen peroxide to inactivate rhinovirus.^11,12 However, neither povidone iodine, hydrogen peroxide, nor chlorhexidine have confirmed SARS-CoV-2 virus inactivation in the presence of fresh human saliva.⁵

Consequently, clinicians are left with insufficient evidence and well-intentioned but confusing guidance about the antiviral efficacy of oral rinses against SARS-CoV-2. To help clarify the confusion and sometimes conflicting advice, Table 2 compiles laboratory test results for popular antimicrobials.¹³ Unfortunately, these mouthrinses were not tested in combination with saliva, and even when tested without saliva, at 60 seconds MIOR provided a greater log₁₀ SARS-CoV-2 reduction than 3.8% "foaming" and 1.5% non-foaming hydrogen peroxide, povidone iodine or 0.12% chlorhexidine. (Note that the "foaming" hydrogen peroxide results were reported by a different laboratory than the results for the other three antimicrobials.)

According to the manufacturer, MIOR contains 100 ppm molecular iodine (the only biocidal form of iodine), water, potassium iodate, citric acid, zinc gluconate, flavoring, and sodium saccharin. Ten percent povidone iodine contains approximately 31,600 ppm total iodine but only 3 ppm molecular iodine. Compared with povidone iodine, MIOR's 100 ppm molecular iodine (free iodine) increases biocidal activity while decreasing cytotoxicity.^13,14 Pioneering work by Wada et al has demonstrated that the virucidal efficacy of povidone iodine solution is directly dependent on the level of molecular iodine (free iodine) present.¹⁵ They further validated that increasing the level of molecular iodine within povidone iodine increases its virucidal efficacy in a dose-dependent manner. The other species of iodine present in povidone iodine account for almost all of the total 31,600 ppm iodine. They contribute to toxicity and staining but are not biocidal. It is the 3 ppm of molecular iodine alone that provides povidone iodine with its antiseptic activity. Molecular iodine (I₂) is an essential nutrient, while iodide (I^-), which is not present in MIOR, is taken up by the body more quickly and completely and can adversely affect the thyroid gland while also being the component of concern in radiocontrast media.^16-18 Also, shellfish allergies, mistakenly thought to be iodine allergies, are actually reactions to the protein tropomyosin.¹⁹

According to its IFU, MIOR should be used as packaged; it should not be diluted or stored in other containers, which can result in potency problems and iodine vapor leakage. Mouthrinse dosing and irrigation instructions are included. Though MIOR appears to be a safe and effective rinse and irrigant, it is only one facet of a full personal protective equipment protocol. The manufacturer cautions MIOR should not be used on patients with known sensitivity to its components, on children <6 years, or patients with underlying conditions contraindicated by their physicians. Clinicians should also remember the importance of using an oral rinse even if patients are fully vaccinated, as they can still be SARS-CoV-2 carriers.

Other antimicrobials should be tested and compared with MIOR in the presence of human saliva, using methods outlined by this study. Although the study was conducted in vitro, not in vivo, the urgency and need for COVID-19 therapeutics require practical and rapid testing in order to provide timely healthcare solutions. Although the study reported here was not a controlled and randomized, in vivo clinical trial, the time required for and the ethical considerations (treating COVID-19 patients) of running such a trial leave the authors with little alternative and the opinion that an in vitro, saliva suspension test is the most expedient and practical evaluation for SARS-CoV-2 antimicrobials.

Under the conditions of this evaluation-SARS-CoV-2 infectivity in the presence of human saliva-the test product MIOR reduced the infectivity of SARS-CoV-2 by 4.75 log₁₀ (>99.99%) following 30-second exposure and ≥5.25 log₁₀ (> 99.99%) following 60-second exposure. In the absence of saliva, infectivity was reduced by 5.00 log₁₀ (>99.99%) and 5.75 log₁₀ (> 99.99%) after 30 and 60 seconds, respectively. MIOR appears effective in reducing SARS-CoV-2 infectivity and to date is the only oral rinse virucidal tested in vitro and in the presence of human saliva. To reduce SARS-CoV-2 infectivity, molecular iodine oral rinse may be an effective mouthrinse and irrigant.

The authors thank Rella Christensen, PhD, dental microbiologist team leader of TRACResearch and co-founder of Clinical Research Associates, for her advice and direction on the testing preformed in this study.

This study was funded in part by a grant from ioTech International^™. The authors report no conflicts of interest.

Volha Teagle, PhD
Principal Scientist, BioScience Laboratories, LLC, Bozeman, Montana

Donald S. Clem, DDS
Adjunct Professor, University of Texas Health Science Center at San Antonio, San Antonio, Texas; Private Practice in Periodontics and Dental Implant Surgery, Fullerton, California; Member, McGuire Institute (iMc) Practice-Based Clinical Research Network

Thomas Yoon, DDS
Associate Dean, Chairman, Department of Periodontology, Director of Research, Director of Specialists, Lake Erie College of Osteopathic Medicine, Bradenton, Florida; Chief Resident, Department of Periodontology, College of Dentistry, University of Florida, Gainesville, Florida