Evaluation of H1N1 Antiviral Properties of an Essential Oil-Containing Mouthrinse

Objective:  The purpose of this in vitro study was to evaluate the virucidal properties of an antiseptic essential oil (EO)-containing mouthrinse (COOL MINT^® LISTERINE^® Antiseptic), when challenged with the pandemic A/California/04/2009 strain of H1N1 Influenza A (CDC ID #2009712047). Methods: ASTM E 1052-96, Standard Test Method for Efficacy of Antimicrobial Agents against Viruses in Suspension, was used to evaluate an EO-containing antiseptic mouthrinse.  The A/California/04/2009 strain of Influenza A H1N1 was exposed to the EO-containing antiseptic mouthrinse (N=3) for 30s at 90% concentration (v/v) to approximate in vivo usage conditions. Following exposures, samples were immediately neutralized with Dey/Engley Neutralizing Broth.  A dilution series of both control (untreated virus) and test samples were plated (4 replicates for each dilution) onto ≤48h-old confluent monolayers of Madin Darby Canis Kidney cells in 24-well tissue culture plates and incubated in a CO₂ incubator for 5-14 days at 37±2^◦C.  The cytopathic/cytotoxic effects of treated and control samples were visualized using an inverted compound microscope, to determine the surviving viral population in each. Viral titers were calculated using the Spearman-Kärber Method, and were expressed as log₁₀ of the 50% tissue culture infectious dose (log₁₀ TCID₅₀).  Standard deviation (N-1) was determined for 3 test replicates.  Results:  Following a 30 second exposure, an EO-containing antiseptic mouthrinse, at 90% (v/v) concentration, reduced infectivity of a pandemic strain of Influenza A H1N1 by 4.67log₁₀ ± 0.144, a >99.99% reduction, compared to non-treated virus. Conclusion:  An EO-containing antiseptic mouthrinse at concentrations similar to in vivo usage demonstrated significant virucidal activity against the A/California/04/2009 strain of a pandemic H1N1 Influenza in vitro.