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Efficacy of ethanol against viruses in hand disinfection

  • G. Kampf
    Correspondence
    Corresponding author. Address: University Medicine Greifswald, Institute for Hygiene and Environmental Medicine, Walter-Rathenau-Straße 49A, 17475 Greifswald, Germany.
    Affiliations
    University Medicine Greifswald, Institute for Hygiene and Environmental Medicine, Greifswald, Germany
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Published:September 04, 2017DOI:https://doi.org/10.1016/j.jhin.2017.08.025

      Summary

      Ethanol is used worldwide in healthcare facilities for hand rubbing. It has been reported to have a stronger and broader virucidal activity compared with propanols. The aim of this review was to describe the spectrum of virucidal activity of ethanol in solution or as commercially available products. A systematic search was conducted. Studies were selected when they contained original data on reduction of viral infectivity from suspension tests (49 studies) and contaminated hands (17 studies). Ethanol at 80% was highly effective against all 21 tested, enveloped viruses within 30 s. Murine norovirus and adenovirus type 5 are usually inactivated by ethanol between 70% and 90% in 30 s whereas poliovirus type 1 was often found to be too resistant except for ethanol at 95% (all test viruses of EN 14476). Ethanol at 80% is unlikely to be sufficiently effective against poliovirus, calicivirus (FCV), polyomavirus, hepatitis A virus (HAV) and foot-and-mouth disease virus (FMDV). The spectrum of virucidal activity of ethanol at 95%, however, covers the majority of clinically relevant viruses. Additional acids can substantially improve the virucidal activity of ethanol at lower concentrations against, e.g. poliovirus, FCV, polyomavirus and FMDV although selected viruses such as HAV may still be too resistant. The selection of a suitable virucidal hand rub should be based on the viruses most prevalent in a unit and on the user acceptability of the product under frequent-use conditions.

      Keywords

      Introduction

      Ethanol is widely used in hand rubs, gels, and foams for hand hygiene in healthcare settings [
      • Goroncy-Bermes P.
      Hand disinfection according to the European Standard EN 1500 (hygienic handrub): a study with gram-negative and gram-positive test organisms.
      ,
      • Goroncy-Bermes P.
      • Koburger T.
      • Meyer B.
      Impact of the amount of hand rub applied in hygienic hand disinfection on the reduction of microbial counts on hands.
      ,
      • Kampf G.
      • Ostermeyer C.
      • Werner H.-P.
      • Suchomel M.
      Efficacy of hand rubs with a low alcohol concentration listed as effective by a national hospital hygiene society in Europe.
      ,
      • Kramer A.
      • Rudolph P.
      • Kampf G.
      • Pittet D.
      Limited efficacy of alcohol-based hand gels.
      ,
      • Kampf G.
      • Rudolf M.
      • Labadie J.-C.
      • Barrett S.P.
      Spectrum of antimicrobial activity and user acceptability of the hand disinfectant agent Sterillium Gel.
      ,
      • Macinga D.R.
      • Shumaker D.J.
      • Werner H.P.
      • Edmonds S.L.
      • Leslie R.A.
      • Parker A.E.
      • et al.
      The relative influences of product volume, delivery format and alcohol concentration on dry-time and efficacy of alcohol-based hand rubs.
      ,
      • Kampf G.
      • Marschall S.
      • Eggerstedt S.
      • Ostermeyer C.
      Efficacy of ethanol-based hand foams using clinically relevant amounts: a cross-over controlled study among healthy volunteers.
      ]. The World Health Organization has even listed ethanol at 80% (v/v) as an essential medicine in the category ‘alcohol-based hand rub’ [
      • World Health Organization
      WHO model list of essential medicines.
      ]. Since 1994 the US Food and Drug Administration considers ethanol between 60% and 95% as generally safe and effective for hand rubbing, although the safety aspect has been under review again since 2015 [
      • Department of Health and Human Services; Food and Drug Administration
      Tentative final monograph for health care antiseptic products; proposed rule.
      ]. Recent studies indicated that there may be a risk from systemic absorption and long-term exposure. The aim of the new proposed rule is to demonstrate systemic safety under daily and long-term use [
      • Department of Health and Human Services; Food and Drug Administration
      Safety and effectiveness of healthcare antiseptics. Topical antimicrobial drug products for over-the-counter human use; proposed amendment of the tentative final monograph; reopening of administrative record; proposed rule.
      ]. The spectrum of antimicrobial activity of a hand rub used in healthcare must include bacteria and yeasts according to EN 14885; virucidal activity, however, is not mandatory [
      • EN 14885:2015
      Chemical disinfectants and antiseptics. Application of European standards for chemical disinfectants and antiseptics.
      ]. However, ∼5% of all healthcare-associated infections are caused by viruses, so virucidal activity of hand rubs has relevance in some units with a high prevalence of viral disease and during viral outbreaks [
      • Aitken C.
      • Jeffries D.J.
      Nosocomial spread of viral disease.
      ]. The European terminology allows three different claims on virucidal activity: ‘active against enveloped viruses’; ‘limited spectrum virucidal activity’ which includes enveloped viruses, adenoviruses, noroviruses and rotaviruses; and ‘virucidal activity’ which includes all viruses relevant in human medicine [
      • EN 14476:2013
      Chemical disinfectants and antiseptics. Virucidal quantitative suspension test for chemical disinfectants and antiseptics used in human medicine.
      ]. Numerous studies have been published on the virucidal activity of ethanol solutions and products but the spectrum of virucidal activity of ethanol has so far not been described in detail. The aim of this review is therefore to describe the spectrum of virucidal activity of ethanol in vitro and in vivo. In addition, the European claims are evaluated regarding specific viruses that may not be covered by the test viruses selected for a specific claim.

      Methods

      A systematic literature search was conducted via the National Library of Medicine (PubMed) on October 10th, 2016, and updated on June 22nd, 2017, using the following terms: virucidal activity alcohol (85 hits), virucidal activity ethanol (41 hits), virucidal efficacy alcohol (38 hits), virucidal efficacy ethanol (22 hits), alcohol-based hand rub virus (22 hits), alcohol-based hand rub viruses (19 hits), ethanol-based hand rub virus (three hits), ethanol-based hand rub viruses (one hit), ethanol virucidal disinfectant (50 hits), ethanol-based sanitizer virucidal (four hits), alcohol-based sanitizer virucidal (three hits), virus inactivation ethanol hand (15 hits), virus inactivation ethanol hands (16 hits) and virus inactivation alcohol hand (24 hits). In addition, studies deemed suitable for this review were also included. Data were extracted from the publications and were selected when they provided original data on reduction of viral infectivity in suspension tests (49 studies) and on contaminated hands (17 studies). A solution or product was considered sufficiently effective against a virus in a suspension test when it achieved ≥4 log10 reduction or the limit of detection was reached. There was no minimum criterion for the limit of detection so that solutions or products could have passed in an assay, although the initial viral titre may have been low or the cytotoxicity of the product may have been high, resulting in an overall low log10 reduction of viral infectivity. Studies were not included when they provided data on the reduction of viral infectivity on inanimate surfaces or used other methods to determine efficacy (e.g. reduction of viral RNA).

      Results

      The concentrations of ethanol are described in w/w and were sometimes determined by calculation when they were originally in v/v. Some studies, however, did not specify whether the ethanol concentration was by volume or weight. In these cases, the ethanol concentration is described without w/w.

      Efficacy against enveloped viruses

      Ethanol has been shown to be effective against various enveloped viruses [
      • Klein M.
      • Deforest A.
      Antiviral action of germicides.
      ]. Beginning at a concentration of 42.6% (w/w) ethanol is effective within 30 s against SARS coronavirus, MERS coronavirus, ebolavirus, influenza A virus including the human type H3N2, the avian type H3N8 and human type H1N1, influenza B virus, HIV, HBV, vacciniavirus, duck hepatitis B virus, togavirus, pseudorabies virus, Newcastle disease virus, bovine viral diarrhoea virus, zikavirus, herpes simplex viruses type 1 and 2 and RSV [
      • Kampf G.
      • Rudolf M.
      • Labadie J.-C.
      • Barrett S.P.
      Spectrum of antimicrobial activity and user acceptability of the hand disinfectant agent Sterillium Gel.
      ,
      • Klein M.
      • Deforest A.
      Antiviral action of germicides.
      ,
      • Rabenau H.F.
      • Kampf G.
      • Cinatl J.
      • Doerr H.W.
      Efficacy of various disinfectants against SARS coronavirus.
      ,
      • Siddharta A.
      • Pfaender S.
      • Vielle N.J.
      • Dijkman R.
      • Friesland M.
      • Becker B.
      • et al.
      Virucidal activity of World Health Organization-recommended formulations against enveloped viruses, including Zika, Ebola, and emerging coronaviruses.
      ,
      • Eggers M.
      • Eickmann M.
      • Kowalski K.
      • Zorn J.
      • Reimer K.
      Povidone-iodine hand wash and hand rub products demonstrated excellent in vitro virucidal efficacy against Ebola virus and modified vaccinia virus Ankara, the new European test virus for enveloped viruses.
      ,
      • Groupe V.
      • Engle C.C.
      • Gaffney P.E.
      Virucidal activity of representative antiinfective agents against influenza A and vaccinia virus.
      ,
      • Jeong E.K.
      • Bae J.E.
      • Kim I.S.
      Inactivation of influenza A virus H1N1 by disinfection process.
      ,
      • Kampf G.
      • Steinmann J.
      • Rabenau H.
      Suitability of vaccinia virus and bovine viral diarrhea virus (BVDV) for determining activities of three commonly-used alcohol-based hand rubs against enveloped viruses.
      ,
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      ,
      • Martin L.S.
      • Meoougal J.S.
      • Loskoski S.L.
      Disinfection and inactivation of the human T lymphotropic virus type III/lymphadenopathy associated virus.
      ,
      • Spire B.
      • Barre-Sinoussi F.
      • Montagnier L.
      Inactivation of lymphadenopathy associated virus by chemical disinfectants.
      ,
      • van Engelenburg F.A.
      • Terpstra F.G.
      • Schuitemaker H.
      • Moorer W.R.
      The virucidal spectrum of a high concentration alcohol mixture.
      ,
      • van Bueren J.
      • Larkin D.P.
      • Simpson R.A.
      Inactivation of human immunodeficiency virus type 1 by alcohols.
      ,
      • Bond W.X.V.
      • Favero M.S.
      • Petersen N.J.
      • Ebert J.W.
      Inactivation of hepatitis B virus by intermediate to high-level disinfectant chemicals.
      ,
      • Kobayashi H.
      • Tsuzuki M.
      • Koshimizu K.
      Susceptibility of hepatitis B virus to disinfectants or heat.
      ,
      • Bingel K.F.
      • Hermann C.
      Die experimentelle Desinfektion des Vakzinevirus als Grundlage für die klinische Pockenimpfung.
      ,
      • Grossgebauer K.
      Zur Desinfektion der mit Pocken kontaminierten Hand.
      ,
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      ,
      • Rabenau H.F.
      • Rapp I.
      • Steinmann J.
      Can vaccinia virus be replaced by MVA virus for testing virucidal activity of chemical disinfectants?.
      ,
      • Sauerbrei A.
      • Schacke M.
      • Gluck B.
      • Bust U.
      • Rabenau H.F.
      • Wutzler P.
      Does limited virucidal activity of biocides include duck hepatitis B virucidal action?.
      ,
      • Bucca M.A.
      The effect of various chemical agents on eastern equine encephalomyelitis virus.
      ,
      • Cunningham C.H.
      The effect of certain chemical agents on the virus of Newcastle disease of chicken.
      ,
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Paulmann D.
      • Friesland M.
      • Pietschmann T.
      • et al.
      Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.
      ]. Ethanol is effective at 73.6% (w/w) against HCV in 15 s and 30 s but not at 40% [
      • Siddharta A.
      • Pfaender S.
      • Vielle N.J.
      • Dijkman R.
      • Friesland M.
      • Becker B.
      • et al.
      Virucidal activity of World Health Organization-recommended formulations against enveloped viruses, including Zika, Ebola, and emerging coronaviruses.
      ,
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Paulmann D.
      • Friesland M.
      • Pietschmann T.
      • et al.
      Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.
      ,
      • Ciesek S.
      • Friesland M.
      • Steinmann J.
      • Wedemeyer H.
      • Manns M.P.
      • Steinmann J.
      • et al.
      How stable is the hepatitis C virus (HCV)? Environmental stability of HCV and its susceptibility to chemical biocides.
      ].

      Efficacy against non-enveloped viruses in suspension tests

      EN 14476 allows determining a partial activity against non-enveloped viruses described as ‘limited spectrum virucidal activity’ tested with adenovirus type 5 and murine norovirus. A hand rub with limited spectrum virucidal activity is considered to be effective against enveloped viruses, adenovirus, norovirus, and rotavirus. In a first step, data from suspension tests according to the test principle of EN 14476 are reviewed for those viruses covered by the claim of ‘limited spectrum virucidal activity’. These viruses have particular clinical importance as outbreak viruses. In 2016 a total of 1444 nosocomial outbreaks have been notified in Germany with 76.0% being caused by noroviruses and 6.4% being caused by rotaviruses [
      • Robert Koch-Institut
      Infektionsepidemiologisches Jahrbuch meldepflichtiger Krankheiten für 2016.
      ].

      Adenovirus

      Ethanol as a solution has largely sufficient activity in 30 s against adenovirus type 5 (test virus of EN 14476) at concentrations between 45% (w/w; contains additional active ingredients) and 95% (w/w) although one study indicates sufficient activity only in 1 min with 77% (w/w) ethanol [
      • Kampf G.
      • Ostermeyer C.
      • Werner H.-P.
      • Suchomel M.
      Efficacy of hand rubs with a low alcohol concentration listed as effective by a national hospital hygiene society in Europe.
      ,
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      ,
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Paulmann D.
      • Friesland M.
      • Pietschmann T.
      • et al.
      Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.
      ,
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      ,
      • Okunishi J.
      • Okamoto K.
      • Nishihara Y.
      • Tsujitani K.
      • Miura T.
      • Matsuse H.
      • et al.
      Investigation of in vitro and in vivo efficacy of a novel alcohol based hand rub, MR06B7.
      ]. Ethanol at 70% (w/w) as a gel was sufficiently active in 2 min [
      • Kampf G.
      • Ostermeyer C.
      • Werner H.-P.
      • Suchomel M.
      Efficacy of hand rubs with a low alcohol concentration listed as effective by a national hospital hygiene society in Europe.
      ].
      Adenovirus type 2 is less susceptible. Ethanol needs 2 min at 55% (w/w) and 85% (w/w) [
      • Kampf G.
      • Rudolf M.
      • Labadie J.-C.
      • Barrett S.P.
      Spectrum of antimicrobial activity and user acceptability of the hand disinfectant agent Sterillium Gel.
      ,
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      ]. In 30 s, ethanol at 62.4% (w/w) was not sufficiently effective [
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      ]. Ethanol at 50% required a 10 min exposure time to be effective [
      • Klein M.
      • Deforest A.
      Antiviral action of germicides.
      ]. The combination of ethanol 73.6% (w/w) and peracetic acid (0.2%), however, was effective in 30 s [
      • Wutzler P.
      • Sauerbrei A.
      Virucidal efficacy of a combination of 0.2% peracetic acid and 80% (v/v) ethanol (PAA-ethanol) as a potential hand disinfectant.
      ].
      Data with other adenoviruses do not provide a clear picture. Ethanol at 72.5–77% (w/w) was effective in 60 s against adenovirus type 7 but not against adenovirus type 8 [
      • Iwasawa A.
      • Niwano Y.
      • Kohno M.
      • Ayaki M.
      Virucidal activity of alcohol-based hand rub disinfectants.
      ].

      Rotavirus

      Gels based on 85% ethanol (w/w) or the combination of 62.4% (w/w) ethanol with additional citric acid were effective against rotavirus in 30 s as well as a hand rub based on 55% ethanol (w/w), 0.7% phosphoric acid and three other alcohols [
      • Kampf G.
      • Rudolf M.
      • Labadie J.-C.
      • Barrett S.P.
      Spectrum of antimicrobial activity and user acceptability of the hand disinfectant agent Sterillium Gel.
      ,
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      ,
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      ].

      Norovirus

      Today, the murine norovirus (MNV), a gastrointestinal tract virus, is widely used to determine the activity against noroviruses. Ethanol at concentrations between 62.4% (w/w) and 85.8% (w/w) is usually effective against MNV in 30 s or 1 min [
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      ,
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Paulmann D.
      • Friesland M.
      • Pietschmann T.
      • et al.
      Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.
      ,
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      ,
      • Paulmann D.
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Virucidal activity of different alcohols against murine norovirus, a surrogate of human norovirus.
      ,
      • Park G.W.
      • Barclay L.
      • Macinga D.
      • Charbonneau D.
      • Pettigrew C.A.
      • Vinje J.
      Comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and GII.4 norovirus.
      ,
      • Tung G.
      • Macinga D.
      • Arbogast J.
      • Jaykus L.A.
      Efficacy of commonly used disinfectants for inactivation of human noroviruses and their surrogates.
      ]. Lower concentrations were less effective. A gel based on 53.2% (w/w) ethanol was only effective in 1 h [
      • Belliot G.
      • Lavaux A.
      • Souihel D.
      • Agnello D.
      • Pothier P.
      Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants.
      ]. According to one study, ethanol at 42.6% (w/w) reduced MNV in 1 min by 0.3 log10 and in 5 min by 0.4 log10 [
      • Park G.W.
      • Barclay L.
      • Macinga D.
      • Charbonneau D.
      • Pettigrew C.A.
      • Vinje J.
      Comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and GII.4 norovirus.
      ]. In another study, ethanol at 42.6% (w/w) reached ∼3 log10 reduction in 30 s whereas ethanol at 24.7% (w/w) or 8% (w/w) is ineffective within 3 min [
      • Paulmann D.
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Virucidal activity of different alcohols against murine norovirus, a surrogate of human norovirus.
      ].
      In the past, the feline calicivirus (FCV) was often used as a surrogate for human noroviruses although it is a respiratory tract virus. FCV is difficult to inactive by ethanol. Ethanol solutions at 42.5% and 62.4% (both w/w) were effective at 3 min; ethanol at 73.6% (w/w) required 5 min [
      • Gehrke C.
      • Steinmann J.
      • Goroncy-Bermes P.
      Inactivation of feline calicivirus, a surrogate of norovirus (formerly Norwalk-like viruses), by different types of alcohol in vitro and in vivo.
      ]. Formulations with 72.5% ethanol (w/w) did not reach a 4 log10 reduction in 60 s [
      • Iwasawa A.
      • Niwano Y.
      • Kohno M.
      • Ayaki M.
      Virucidal activity of alcohol-based hand rub disinfectants.
      ]. A solution based on 77% ethanol (w/w) showed only little activity against FCV in 60 s (<1 log10) [
      • Iwasawa A.
      • Niwano Y.
      • Kohno M.
      • Ayaki M.
      Virucidal activity of alcohol-based hand rub disinfectants.
      ]. Even after 5 min the efficacy was still insufficient (<2 log10) [
      • Okunishi J.
      • Okamoto K.
      • Nishihara Y.
      • Tsujitani K.
      • Miura T.
      • Matsuse H.
      • et al.
      Investigation of in vitro and in vivo efficacy of a novel alcohol based hand rub, MR06B7.
      ]. A hand rub based on >85.8% (w/w) ethanol was almost ineffective in 30 s (<1 log10) [
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      ]. Finally, ethanol at 42.6%, 62.4%, and 85.8% (all w/w) revealed insufficient efficacy in 5 min with the highest log10 reduction of 2.6 [
      • Park G.W.
      • Barclay L.
      • Macinga D.
      • Charbonneau D.
      • Pettigrew C.A.
      • Vinje J.
      Comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and GII.4 norovirus.
      ].
      Whenever different types of acid are added, ethanol in hand rubs has been described to be effective in 30 s at 45%, 50.2%, 55%, 62.4%, and 67.9% (all w/w) [
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      ,
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      ,
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      ,
      • Shimizu-Onda Y.
      • Akasaka T.
      • Yagyu F.
      • Komine-Aizawa S.
      • Tohya Y.
      • Hayakawa S.
      • et al.
      The virucidal effect against murine norovirus and feline calicivirus as surrogates for human norovirus by ethanol-based sanitizers.
      ]. Malic acid at 0.35% has also been reported to improve the efficacy of ethanol against FCV to some extent [
      • Akasaka T.
      • Shimizu-Onda Y.
      • Hayakawa S.
      • Ushijima H.
      The virucidal effects against murine norovirus and feline calicivirus F4 as surrogates for human norovirus by the different additive concentrations of ethanol-based sanitizers.
      ].

      Enteroviruses

      EN 14476 allows determining a full virucidal activity (test viruses: adenovirus type 5, murine norovirus, and poliovirus type 1). In a second step, data from suspension tests according to the test principle of EN 14476 are reviewed for enteroviruses that should be covered by the claim ‘virucidal activity’.

      Coxsackievirus

      Data on the efficacy of ethanol against coxsackievirus are conflicting. Ethanol at 72.5–92.4% (w/w) has been reported to be effective against coxsackievirus B5 within 60 s and also against coxsackievirus B1 within 10 min but not against coxsackievirus A7 [
      • Iwasawa A.
      • Niwano Y.
      • Kohno M.
      • Ayaki M.
      Virucidal activity of alcohol-based hand rub disinfectants.
      ,
      • Drulak M.
      • Wallbank A.M.
      • Lebtag I.
      • Werboski L.
      • Poffenroth L.
      The relative effectiveness of commonly used disinfectants in inactivation of coxsackievirus B5.
      ]. Higher ethanol concentrations (85–90%) are effective against coxsackievirus B3 in 15–60 s [
      • Moldenhauer D.
      Quantitative evaluation of the effects of disinfectants against viruses in suspension experiments.
      ].

      Echovirus

      The efficacy of ethanol against echoviruses is rather good. One study shows that ethanol at 92.4% (w/w) is effective against echovirus 11 in 20 s [
      • Drulak M.
      • Wallbank A.M.
      • Lebtag I.
      The relative effectiveness of commonly used disinfectants in inactivation of echovirus 11.
      ]. Another study found a log10 reduction ≥3 within 1 min for ethanol at 92.4% (w/w) against the same virus, whereas ethanol at 67.9% (w/w) was almost ineffective (<1 log10) at the same exposure time [
      • Kurtz J.B.
      Virucidal effect of alcohols against echovirus 11.
      ]. Against echovirus 6 ethanol was effective at 50% within 10 min [
      • Klein M.
      • Deforest A.
      Antiviral action of germicides.
      ].

      Human enterovirus 71

      Against the human enterovirus 71 ethanol had only little activity at 62.4% (w/w) and 67.9% (w/w) within 10 min (<1 log10). At 79.6% (w/w) a 3.21 log10 reduction was achieved in the same exposure time, and at 92.4% (w/w) the log10 reduction was 5.78 [
      • Chang S.C.
      • Li W.C.
      • Huang K.Y.
      • Huang Y.C.
      • Chiu C.H.
      • Chen C.J.
      • et al.
      Efficacy of alcohols and alcohol-based hand disinfectants against human enterovirus 71.
      ].

      Poliovirus

      All formulations with ethanol up to 80% (w/w) and without acid were not sufficiently effective in up to 5 min against poliovirus type 1 when tested under standard conditions with 80% product proportion in the suspension (Table I). When the product proportion is increased in the suspension test to 97%, some formulations with 73.5% or 80% ethanol (w/w) were effective within 1 min. One formulation with 95% (w/w) ethanol was effective in 30 s. Gels were mostly less effective compared to solutions.
      Table IEfficacy of different formulations based on ethanol without additional acids in suspension tests against poliovirus type 1
      Ethanol concentration (w/w)Viscosity (type of product)Exposure timeMean log10 reductionReference
      62.4%
      Determined by calculation (originally in v/v).
      Solution (S)1 min2.3
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      5 min1.0
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      10 min5.0
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      70%Gel (C)3 min0–0.87
      Four experiments.
      • Kampf G.
      • Ostermeyer C.
      • Werner H.-P.
      • Suchomel M.
      Efficacy of hand rubs with a low alcohol concentration listed as effective by a national hospital hygiene society in Europe.
      Solution (C)3 min0.75–3.25
      Three experiments.
      • Kampf G.
      • Ostermeyer C.
      • Werner H.-P.
      • Suchomel M.
      Efficacy of hand rubs with a low alcohol concentration listed as effective by a national hospital hygiene society in Europe.
      73.5%
      Determined by calculation (originally in v/v).
      Solution (F)0.5 min0.9
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Paulmann D.
      • Friesland M.
      • Pietschmann T.
      • et al.
      Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.
      Solution (S)1 min2.9
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      Solution (F)1 min2.2
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Paulmann D.
      • Friesland M.
      • Pietschmann T.
      • et al.
      Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.
      Solution (F)2 min2.0
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      Solution (F)2 min2.8
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Paulmann D.
      • Friesland M.
      • Pietschmann T.
      • et al.
      Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.
      Solution (F)3 min3.1
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Paulmann D.
      • Friesland M.
      • Pietschmann T.
      • et al.
      Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.
      Solution (S)5 min2.9
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      Solution (F)5 min2.2
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      Solution (F)5 min3.5
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Paulmann D.
      • Friesland M.
      • Pietschmann T.
      • et al.
      Virucidal activity of 2 alcohol-based formulations proposed as hand rubs by the World Health Organization.
      Solution (S)10 min5.4
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      Solution (S)30 min<4
      • Wutzler P.
      • Sauerbrei A.
      Virucidal efficacy of a combination of 0.2% peracetic acid and 80% (v/v) ethanol (PAA-ethanol) as a potential hand disinfectant.
      73.5%
      Determined by calculation (originally in v/v).
      ,
      Tested in suspension with 97% product proportion.
      Solution (F)30 s3.4
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      1 min4.5
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      78.8%
      Determined by calculation (originally in v/v).
      Solution (S)30 s0.4
      • Okunishi J.
      • Okamoto K.
      • Nishihara Y.
      • Tsujitani K.
      • Miura T.
      • Matsuse H.
      • et al.
      Investigation of in vitro and in vivo efficacy of a novel alcohol based hand rub, MR06B7.
      1 min1.3
      • Okunishi J.
      • Okamoto K.
      • Nishihara Y.
      • Tsujitani K.
      • Miura T.
      • Matsuse H.
      • et al.
      Investigation of in vitro and in vivo efficacy of a novel alcohol based hand rub, MR06B7.
      5 min2.9
      • Okunishi J.
      • Okamoto K.
      • Nishihara Y.
      • Tsujitani K.
      • Miura T.
      • Matsuse H.
      • et al.
      Investigation of in vitro and in vivo efficacy of a novel alcohol based hand rub, MR06B7.
      80%Solution (F)2 min2.0
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      5 min2.8
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      80%
      Tested in suspension with 97% product proportion.
      Solution (F)30 s3.1
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      1 min4.2
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Magulski T.
      • Steinmann J.
      • Steinmann E.
      Virucidal activity of Formulation I of the World Health Organization’s alcohol-based handrubs: impact of changes in key ingredient levels and test parameters.
      85%Gel (C)3 min4.25
      • Kampf G.
      • Rudolf M.
      • Labadie J.-C.
      • Barrett S.P.
      Spectrum of antimicrobial activity and user acceptability of the hand disinfectant agent Sterillium Gel.
      85.7%
      Determined by calculation (originally in v/v).
      Solution (S)1 min1.6
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      Solution (S)5 min1.2
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      Solution (S)10 min1.7
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      95%Solution (C)30 s≥4
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      100%Solution (S)1 min1.5
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      5 min2.3
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      10 min2.3
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      (C), commercially available product; (F), formulation with auxiliary agents or additional active agents; (S), solution of ethanol.
      a Determined by calculation (originally in v/v).
      b Four experiments.
      c Three experiments.
      d Tested in suspension with 97% product proportion.
      The addition of various types of acids can substantially improve the activity of ethanol against poliovirus type 1 so that the formulations are often sufficiently effective in 30 s (Table II).
      Table IIEfficacy of different formulations based on ethanol with additional acids in suspension tests against poliovirus type 1
      Ethanol concentration (w/w)Type of acidViscosity (type of product)Exposure timeMean log10 reductionReference
      45%Phosphoric acidSolution (C)30 s≥4
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      55%
      With three additional alcohols.
      0.7% phosphoric acidSolution (C)30 s≥4
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      1 min≥4
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      62.4%
      Determined by calculation (originally in v/v).
      0.25% citric acidGel (C)30 s3.5
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      69.39%
      With one additional alcohol.
      0.2% citric acid, 0.2% uric acidSolution (F)1 min4.0
      • Ionidis G.
      • Hubscher J.
      • Jack T.
      • Becker B.
      • Bischoff B.
      • Todt D.
      • et al.
      Development and virucidal activity of a novel alcohol-based hand disinfectant supplemented with urea and citric acid.
      73.5%
      Determined by calculation (originally in v/v).
      0.2% peracetic acidSolution (F)30 s≥4
      • Wutzler P.
      • Sauerbrei A.
      Virucidal efficacy of a combination of 0.2% peracetic acid and 80% (v/v) ethanol (PAA-ethanol) as a potential hand disinfectant.
      78.8%
      Determined by calculation (originally in v/v).
      0.1% citric acid, 1% lactic acidSolution (F)30 s≥4
      • Okunishi J.
      • Okamoto K.
      • Nishihara Y.
      • Tsujitani K.
      • Miura T.
      • Matsuse H.
      • et al.
      Investigation of in vitro and in vivo efficacy of a novel alcohol based hand rub, MR06B7.
      (C), commercially available product; (F), formulation with auxiliary agents or additional active agents.
      a With three additional alcohols.
      b Determined by calculation (originally in v/v).
      c With one additional alcohol.
      Poliovirus type 2 is somewhat less susceptible to ethanol compared to poliovirus type 1 [
      • Schürmann W.
      • Eggers H.J.
      Antiviral activity of an alcoholic hand disinfectant: comparison of the in vitro suspension test with the in vivo experiments on hands, and on individual fingertips.
      ].

      Rhinovirus

      A hand rub based on 55% ethanol (w/w), 0.7% phosphoric acid, and three other alcohols was effective in 30 s [
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      ].

      Other non-enveloped viruses

      In a third step, data from suspension tests according to the test principle of EN 14476 are reviewed for other non-enveloped viruses that should be covered by the claim ‘virucidal activity’.

      Hepatitis A virus

      A gel based on 54.2% ethanol (w/w) showed no effect at all (0 log10) against hepatitis A virus (HAV) after 30 s [
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      ]. Ethanol at 80% or 95% (both w/w) was not sufficiently effective within 2 min [
      • Wolff M.H.
      • Schmitt J.
      • Rahaus M.
      • König A.
      Hepatitis A virus: a test method for virucidal activity.
      ]. A hand rub based on 80% ethanol showed only little reduction of viral infectivity within 30 s (0.47), but it was higher after 2 min (≥2.2 log10) [
      • van Engelenburg F.A.
      • Terpstra F.G.
      • Schuitemaker H.
      • Moorer W.R.
      The virucidal spectrum of a high concentration alcohol mixture.
      ]. When 0.7% phosphoric acid and three other alcohols are added to ethanol at 55% (w/w) the formulation was effective against HAV in 30 s [
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      ]. Ethanol at 62.4% (w/w) with additional 0.25% citric acid, however, was not sufficiently effective in 30 s with 1.75 log10 [
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      ].

      Foot-and-mouth disease virus

      The infectivity of the foot-and-mouth disease virus (FMDV) was insufficiently reduced by ethanol between 55.2% and 72.5% (both w/w) within 5 min [
      • Harada Y.U.
      • Lekcharoensuk P.
      • Furuta T.
      • Taniguchi T.
      Inactivation of foot-and-mouth disease virus by commercially available disinfectants and cleaners.
      ]. Hand rubs with 70–75.2% ethanol (w/w) and additional phosphoric acid (0.6%) or 50% (w/w) ethanol and additional citric acid (0.5%), however, were effective within 30 s [
      • Harada Y.U.
      • Lekcharoensuk P.
      • Furuta T.
      • Taniguchi T.
      Inactivation of foot-and-mouth disease virus by commercially available disinfectants and cleaners.
      ].

      Polyomavirus

      Ethanol has only little activity against polyomavirus SV 40, an additional test virus that was chosen due to its resistance to alcohols by experts for the German test method to determine virucidal activity [
      • Schwebke I.
      • Eggers M.
      • Gebel J.
      • Geisel B.
      • Glebe D.
      • Rapp I.
      • et al.
      Prüfung und Deklaration der Wirksamkeit von Desinfektionsmitteln gegen Viren zur Anwendung im humanmedizinischen Bereich: Stellungnahme des Arbeitskreises Viruzidie beim Robert Koch-Institut.
      ]. A gel based on 85% ethanol (w/w) revealed sufficient activity in 15 min [
      • Kampf G.
      • Rudolf M.
      • Labadie J.-C.
      • Barrett S.P.
      Spectrum of antimicrobial activity and user acceptability of the hand disinfectant agent Sterillium Gel.
      ]. A hand rub based on 78.2% (w/w) ethanol had insufficient activity within 10 min (∼2 log10) [
      • Schürmann W.
      • Eggers H.J.
      Antiviral activity of an alcoholic hand disinfectant: comparison of the in vitro suspension test with the in vivo experiments on hands, and on individual fingertips.
      ]. When 0.7% phosphoric acid and three other alcohols are added to ethanol at 55% (w/w) the formulation was able to reduce infectivity sufficiently in 60 s [
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      ]. Ethanol at 73.6% (w/w) in combination with 0.2% peracetic acid was effective in 30 s [
      • Wutzler P.
      • Sauerbrei A.
      Virucidal efficacy of a combination of 0.2% peracetic acid and 80% (v/v) ethanol (PAA-ethanol) as a potential hand disinfectant.
      ].

      Parvovirus

      Ethanol has almost no virucidal activity against parvovirus. At 80% ethanol the infectivity of the canine parvovirus was reduced in 5 min only by 0.1 log10 [
      • van Engelenburg F.A.
      • Terpstra F.G.
      • Schuitemaker H.
      • Moorer W.R.
      The virucidal spectrum of a high concentration alcohol mixture.
      ].

      Virucidal activity on hands

      Some data can be found for the enveloped influenza A virus. A gel based on 61.5% ethanol completely reduced viral infectivity of the H1N1 influenza A virus after one application [
      • Grayson M.L.
      • Melvani S.
      • Druce J.
      • Barr I.G.
      • Ballard S.A.
      • Johnson P.D.
      • et al.
      Efficacy of soap and water and alcohol-based hand-rub preparations against live H1N1 influenza virus on the hands of human volunteers.
      ]. The formulations with ethanol between 54.2% and 58.1% (both w/w) ethanol effectively reduced viral infectivity of the same virus within 30 s by 3.25–3.35 log10 [
      • Larson E.L.
      • Cohen B.
      • Baxter K.A.
      Analysis of alcohol-based hand sanitizer delivery systems: efficacy of foam, gel, and wipes against influenza A (H1N1) virus on hands.
      ].
      First results with non-enveloped viruses were reported in 1978. One millilitre of a solution based on 70% ethanol reduced rhinovirus on hands by 1.5 log10 [
      • Hendley J.O.
      • Mika L.A.
      • Gwaltney Jr., J.M.
      Evaluation of virucidal compounds for inactivation of rhinovirus on hands.
      ]. Table III shows data obtained for ethanol-based formulations with current methods for various non-enveloped viruses. Overall, the majority of non-enveloped viruses can only be reduced partially by ethanol-based formulations. Exceptions are rotavirus and MNV for which most data indicate sufficient activity. When ethanol is combined with different types of acids the efficacy on hands can be improved substantially with HAV as an exception (Table IV).
      Table IIIMean log10 reduction of viral infectivity on artificially contaminated hands or finger tips by treatment of hands with ethanol-based formulations without additional acids
      Type of virusEthanol concentration (w/w)Viscosity (type of product)Application timeMean log10 reductionReference
      Poliovirus type 174.5%Solution (S)10 min1.0
      Hand test.


      2.5
      Finger test.
      • Schürmann W.
      • Eggers H.J.
      Antiviral activity of an alcoholic hand disinfectant: comparison of the in vitro suspension test with the in vivo experiments on hands, and on individual fingertips.
      80%Solution (S)30 s0.42
      • Davies J.G.
      • Babb J.R.
      • Bradley C.R.
      • Ayliffe G.A.J.
      Preliminary study of test methods to assess the virucidal activity of skin disinfectants using poliovirus and bacteriophages.
      80%Solution (S)10 min2.2
      • Steinmann J.
      • Nehrkorn R.
      • Meyer A.
      Two in vivo protocols for testing virucidal efficacy of handwashing and hand disinfection.
      96.8%Solution (S)10 min3.2
      • Steinmann J.
      • Nehrkorn R.
      • Meyer A.
      Two in vivo protocols for testing virucidal efficacy of handwashing and hand disinfection.
      Poliovirus type 274.5%Solution (S)10 min0.2
      Hand test.


      0.7
      Finger test.
      • Schürmann W.
      • Eggers H.J.
      Antiviral activity of an alcoholic hand disinfectant: comparison of the in vitro suspension test with the in vivo experiments on hands, and on individual fingertips.
      Coxsackievirus B374.5%Solution (S)10 min1.1
      Hand test.


      2.9
      Finger test.
      • Schürmann W.
      • Eggers H.J.
      Antiviral activity of an alcoholic hand disinfectant: comparison of the in vitro suspension test with the in vivo experiments on hands, and on individual fingertips.
      Coxsackievirus B474.5%Solution (S)10 min1.3
      Hand test.


      3.0
      Finger test.
      • Schürmann W.
      • Eggers H.J.
      Antiviral activity of an alcoholic hand disinfectant: comparison of the in vitro suspension test with the in vivo experiments on hands, and on individual fingertips.
      Echovirus type 9 strain Hill74.5%Solution (S)10 min0.7
      Hand test.


      2.6
      Finger test.
      • Schürmann W.
      • Eggers H.J.
      Antiviral activity of an alcoholic hand disinfectant: comparison of the in vitro suspension test with the in vivo experiments on hands, and on individual fingertips.
      Echovirus type 9 strain Barty74.5%Solution (S)10 min1.3
      Hand test.


      2.2
      Finger test.
      • Schürmann W.
      • Eggers H.J.
      Antiviral activity of an alcoholic hand disinfectant: comparison of the in vitro suspension test with the in vivo experiments on hands, and on individual fingertips.
      Polyomavirus SV 4074.5%Solution (S)10 min0.9
      Hand test.


      1.8
      Finger test.
      • Schürmann W.
      • Eggers H.J.
      Antiviral activity of an alcoholic hand disinfectant: comparison of the in vitro suspension test with the in vivo experiments on hands, and on individual fingertips.
      Murine norovirus54.1%
      Determined by calculation (originally in v/v).
      Gel (C)20 s2.8
      • Sattar S.
      • Ali M.
      • Tetro J.A.
      In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!.
      54.1%
      Determined by calculation (originally in v/v).
      Gel (C)30 s3.5
      • Sattar S.
      • Ali M.
      • Tetro J.A.
      In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!.
      62.4%
      Determined by calculation (originally in v/v).
      Solution (S)30 s4.69
      • Paulmann D.
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Virucidal activity of different alcohols against murine norovirus, a surrogate of human norovirus.
      68%
      Determined by calculation (originally in v/v).
      Solution (S)30 s0.91
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      67.8%
      Determined by calculation (originally in v/v).
      Solution (S)20 s3.0
      • Sattar S.
      • Ali M.
      • Tetro J.A.
      In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!.
      67.8%
      Determined by calculation (originally in v/v).
      Solution (S)30 s2.7
      • Sattar S.
      • Ali M.
      • Tetro J.A.
      In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!.
      73.5%
      Determined by calculation (originally in v/v).
      Solution (S)20 s1.7
      • Sattar S.
      • Ali M.
      • Tetro J.A.
      In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!.
      >90%Solution (C)30 s3.91
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      Human enterovirus 7168%
      Determined by calculation (originally in v/v).
      Solution (S)30 s0.86
      • Chang S.C.
      • Li W.C.
      • Huang K.Y.
      • Huang Y.C.
      • Chiu C.H.
      • Chen C.J.
      • et al.
      Efficacy of alcohols and alcohol-based hand disinfectants against human enterovirus 71.
      92.9%
      Determined by calculation (originally in v/v).
      Solution (S)30 s4.06
      • Chang S.C.
      • Li W.C.
      • Huang K.Y.
      • Huang Y.C.
      • Chiu C.H.
      • Chen C.J.
      • et al.
      Efficacy of alcohols and alcohol-based hand disinfectants against human enterovirus 71.
      Rotavirus70%
      No information on v/v or w/w.
      Solution (S)30 s2.85
      • Bellamy K.
      • Alcock R.
      • Babb J.R.
      • Davies J.G.
      • Ayliffe G.A.
      A test for the assessment of “hygienic” hand disinfection using rotavirus.
      Feline calicivirus54.1%
      Determined by calculation (originally in v/v).
      Gel (C)20 s1.4
      • Sattar S.
      • Ali M.
      • Tetro J.A.
      In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!.
      54.1%
      Determined by calculation (originally in v/v).
      Gel (C)30 s2.1
      • Sattar S.
      • Ali M.
      • Tetro J.A.
      In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!.
      62%
      No information on v/v or w/w.
      Solution (S)30 s0.50
      • Lages S.L.S.
      • Ramakrishnan M.A.
      • Goyal S.M.
      In-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus.
      62%
      No information on v/v or w/w.
      Solution (S)2 min0.55
      • Lages S.L.S.
      • Ramakrishnan M.A.
      • Goyal S.M.
      In-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus.
      62.4%
      Determined by calculation (originally in v/v).
      Solution (S)30 s3.78
      • Gehrke C.
      • Steinmann J.
      • Goroncy-Bermes P.
      Inactivation of feline calicivirus, a surrogate of norovirus (formerly Norwalk-like viruses), by different types of alcohol in vitro and in vivo.
      62.4%
      Determined by calculation (originally in v/v).
      Solution (S)30 s0.68
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      67.8%
      Determined by calculation (originally in v/v).
      Solution (S)20 s1.5
      • Sattar S.
      • Ali M.
      • Tetro J.A.
      In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!.
      67.8%
      Determined by calculation (originally in v/v).
      Solution (S)30 s2.2
      • Sattar S.
      • Ali M.
      • Tetro J.A.
      In vivo comparison of two human norovirus surrogates for testing ethanol-based handrubs: the mouse chasing the cat!.
      70%Solution (S)30 s2.66
      • Kampf G.
      • Grotheer D.
      • Steinmann J.
      Efficacy of three ethanol-based hand rubs against feline calicivirus, a surrogate for norovirus.
      70%Solution (S)30 s2.62
      • Kampf G.
      • Grotheer D.
      • Steinmann J.
      Efficacy of three ethanol-based hand rubs against feline calicivirus, a surrogate for norovirus.
      70%Solution (S)30 s1.18
      • Kampf G.
      • Grotheer D.
      • Steinmann J.
      Efficacy of three ethanol-based hand rubs against feline calicivirus, a surrogate for norovirus.
      70%Solution (S)30 s1.45
      • Kampf G.
      • Grotheer D.
      • Steinmann J.
      Efficacy of three ethanol-based hand rubs against feline calicivirus, a surrogate for norovirus.
      70%Solution (S)30 s1.33
      • Kampf G.
      • Grotheer D.
      • Steinmann J.
      Efficacy of three ethanol-based hand rubs against feline calicivirus, a surrogate for norovirus.
      75.1%Solution (C)30 s0.93
      • Kampf G.
      • Grotheer D.
      • Steinmann J.
      Efficacy of three ethanol-based hand rubs against feline calicivirus, a surrogate for norovirus.
      80%Solution (C)30 s1.34
      • Kampf G.
      • Grotheer D.
      • Steinmann J.
      Efficacy of three ethanol-based hand rubs against feline calicivirus, a surrogate for norovirus.
      85.7%
      Determined by calculation (originally in v/v).
      Solution (S)30 s2.84
      • Gehrke C.
      • Steinmann J.
      • Goroncy-Bermes P.
      Inactivation of feline calicivirus, a surrogate of norovirus (formerly Norwalk-like viruses), by different types of alcohol in vitro and in vivo.
      95%Solution (C)30 s1.90
      • Kampf G.
      • Grotheer D.
      • Steinmann J.
      Efficacy of three ethanol-based hand rubs against feline calicivirus, a surrogate for norovirus.
      99.5%
      No information on v/v or w/w.
      Solution (S)30 s1.00
      • Lages S.L.S.
      • Ramakrishnan M.A.
      • Goyal S.M.
      In-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus.
      99.5%
      No information on v/v or w/w.
      Solution (S)2 min1.30
      • Lages S.L.S.
      • Ramakrishnan M.A.
      • Goyal S.M.
      In-vivo efficacy of hand sanitisers against feline calicivirus: a surrogate for norovirus.
      (C), commercially available product; (S), solution of ethanol.
      a Hand test.
      b Finger test.
      c Determined by calculation (originally in v/v).
      d No information on v/v or w/w.
      Table IVMean log10 reduction of viral infectivity on artificially contaminated hands or finger tips by treatment of hands with ethanol-based formulations containing additional acids
      Type of virusEthanol concentration (w/w)Viscosity (type of product)Application timeMean log10 reductionReference
      Adenovirus type 562.4%
      Determined by calculation (originally in v/v).
      Gel (C)
      Additional phosphoric acid.
      15 s≥3.16
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      30 s≥3.12
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      Poliovirus type 155%Solution (C)
      Additional phosphoric acid.
      30 s3.04
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      1 min3.13
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      62.4%
      Determined by calculation (originally in v/v).
      Gel (C)
      Additional citric acid.
      30 s2.98
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      Murine norovirus45%Solution (C)
      Additional phosphoric acid.
      30 s3.94
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      55%Solution (C)
      Additional phosphoric acid.
      30 s3.91
      • Steinmann J.
      • Paulmann D.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo.
      62.4%
      Determined by calculation (originally in v/v).
      Gel (C)
      Additional citric acid.
      30 s2.48
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      Hepatitis A virus62.4%
      Determined by calculation (originally in v/v).
      Gel (C)
      Additional citric acid.
      30 s1.32
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      Rotavirus62.4%
      Determined by calculation (originally in v/v).
      Gel (C)
      Additional citric acid.
      15 s≥4.32
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      30 s≥3.84
      • Macinga D.R.
      • Sattar S.A.
      • Jaykus L.A.
      • Arbogast J.W.
      Improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer.
      Feline calicivirus55%Solution (C)
      Additional phosphoric acid.
      30 s2.38
      • Kramer A.
      • Galabov A.S.
      • Sattar S.A.
      • Döhner L.
      • Pivert A.
      • Payan C.
      • et al.
      Virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations.
      (C), commercially available product.
      a Determined by calculation (originally in v/v).
      b Additional phosphoric acid.
      c Additional citric acid.
      Non-quantitative data were found in addition for rhinovirus. When hands artificially contaminated with rhinovirus were treated with 1.5 mL ethanol (65%) once or twice, rhinovirus was no longer be detected on 13 of 15 hands or 13 of 16 hands, respectively [
      • Turner R.B.
      • Fuls J.L.
      • Rodgers N.D.
      Effectiveness of hand sanitizers with and without organic acids for removal of rhinovirus from hands.
      ]. A similar result was described after the application of 1.5 mL ethanol (83%) [
      • Turner R.B.
      • Fuls J.L.
      • Rodgers N.D.
      Effectiveness of hand sanitizers with and without organic acids for removal of rhinovirus from hands.
      ].

      Discussion

      The evidence for sufficient activity of ethanol against enveloped viruses is very broad. Hand rubs must fulfil the EN 1500 efficacy requirement, hence they often contain ≥80% ethanol when applied for 30 s [
      • Kramer A.
      • Rudolph P.
      • Kampf G.
      • Pittet D.
      Limited efficacy of alcohol-based hand gels.
      ,
      • Suchomel M.
      • Kundi M.
      • Pittet D.
      • Weinlich M.
      • Rotter M.L.
      Testing of the World Health Organization recommended formulations in their application as hygienic hand rubs and proposals for increased efficacy.
      ]. At this concentration hand rubs have comprehensive activity against different types of enveloped viruses.
      Hand rubs with ‘limited spectrum virucidal activity’ are deemed suitable for inactivation of noroviruses and adenoviruses. Based on data obtained for MNV, hand rubs with 70–90% ethanol are usually effective against norovirus within 30 s. For adenovirus, however, the situation is somewhat different. The EN 14476 test virus (adenovirus type 5) is mostly inactivated by ethanol between 45% and 95% in 30 s. Other types of adenovirus, however, are less susceptible to ethanol such as adenovirus types 2 and 8. Both types of adenovirus have been reported before to have only little susceptibility to other biocidal agents such as povidone iodine or peracetic acid, indicating a general reduced susceptibility [
      • Sauerbrei A.
      • Sehr K.
      • Brandstadt A.
      • Heim A.
      • Reimer K.
      • Wutzler P.
      Sensitivity of human adenoviruses to different groups of chemical biocides.
      ]. The in-vitro data are supported by the description of an outbreak with adenovirus type 8 which was successfully controlled by various measures including use of a virucidal hand rub [
      • Chaberny I.E.
      • Schnitzler P.
      • Geiss H.K.
      • Wendt C.
      An outbreak of epidemic keratoconjunctivtis in a pediatric unit due to adenovirus type 8.
      ].
      Virucidal activity according to EN 14476 aims to include all enveloped and non-enveloped viruses relevant in human medicine [
      • EN 14476:2013
      Chemical disinfectants and antiseptics. Virucidal quantitative suspension test for chemical disinfectants and antiseptics used in human medicine.
      ]. After reviewing published data, some viral species are unlikely to be sufficiently inactivated by ethanol alone, even at high concentrations: polyomavirus and coxsackieviruses B5 and B1. Other viruses are only sufficiently inactivated by ethanol at 95% such as poliovirus, echovirus 11, human enterovirus, and coxsackievirus B3 (≥85% ethanol). The majority of ethanol-based hand rubs with concentrations up to 90% are therefore likely to yield <4 log10 reduction against these viruses, suggesting insufficient efficacy. The addition of acids to the formulation such as phosphoric acid, citric acid, or peracetic acid can substantially improve the virucidal efficacy of ethanol, as shown with adenovirus type 2, poliovirus type 1, polyomavirus, HAV, FMDV, and FCV. Although the efficacy is better, the dermal tolerance is likely to be worse. Post-marketing surveillance of an ethanol-based hand rub with phosphoric acid revealed frequent and significant skin lesions among the users [
      • Kampf G.
      • Reichel M.
      Gehäufte Hautirritationen durch ein viruzides Händedesinfektionsmittel mit hohem Phosphorsäuregehalt.
      ]. Improved formulations, however, may have better user acceptability [
      • Stauffer F.
      • Griess M.
      • Pleininger G.
      • Zhumadilova A.
      • Assadian O.
      Acceptability and tolerability of liquid versus gel and standard versus virucidal alcohol-based hand rub formulations among dental students.
      ,
      • Conrad A.
      • Grotejohann B.
      • Schmoor C.
      • Cosic D.
      • Dettenkofer M.
      Safety and tolerability of virucidal hand rubs: a randomized, double-blind, cross-over trial with healthy volunteers.
      ]. It would be beneficial in any case to look at the user acceptability of a virucidal hand rub locally before healthcare workers are asked to use it [
      • Larson E.
      • Girard R.
      • Pessoa-Silva C.L.
      • Boyce J.
      • Donaldson L.
      • Pittet D.
      Skin reactions related to hand hygiene and selection of hand hygiene products.
      ].
      A European work item (WI 00216088) exists with the aim of measuring the virucidal activity on hands artificially contaminated with murine norovirus. Ethanol, probably at 70%, has been selected as the reference alcohol. Ethanol as an alcohol has a superior efficacy against non-enveloped viruses compared with the propanols. Against MNV, for example, most studies indicate that iso-propanol is considerably less effective compared with ethanol [
      • Paulmann D.
      • Steinmann J.
      • Becker B.
      • Bischoff B.
      • Steinmann E.
      • Steinmann J.
      Virucidal activity of different alcohols against murine norovirus, a surrogate of human norovirus.
      ,
      • Park G.W.
      • Barclay L.
      • Macinga D.
      • Charbonneau D.
      • Pettigrew C.A.
      • Vinje J.
      Comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and GII.4 norovirus.
      ,
      • Belliot G.
      • Lavaux A.
      • Souihel D.
      • Agnello D.
      • Pothier P.
      Use of murine norovirus as a surrogate to evaluate resistance of human norovirus to disinfectants.
      ]. Against FCV iso-propanol has overall only poor virucidal activity [
      • Park G.W.
      • Barclay L.
      • Macinga D.
      • Charbonneau D.
      • Pettigrew C.A.
      • Vinje J.
      Comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and GII.4 norovirus.
      ,
      • Gehrke C.
      • Steinmann J.
      • Goroncy-Bermes P.
      Inactivation of feline calicivirus, a surrogate of norovirus (formerly Norwalk-like viruses), by different types of alcohol in vitro and in vivo.
      ]. And enteroviruses have been reported to be more or less resistant to iso-propanol at various concentrations within 10 min [
      • Klein M.
      • Deforest A.
      Antiviral action of germicides.
      ,
      • Moldenhauer D.
      Quantitative evaluation of the effects of disinfectants against viruses in suspension experiments.
      ,
      • Chang S.C.
      • Li W.C.
      • Huang K.Y.
      • Huang Y.C.
      • Chiu C.H.
      • Chen C.J.
      • et al.
      Efficacy of alcohols and alcohol-based hand disinfectants against human enterovirus 71.
      ,
      • Tyler R.
      • Ayliffe G.A.J.
      • Bradley C.
      Virucidal activity of disinfectants: studies with the poliovirus.
      ,
      • Eggers H.-J.
      Experiments on antiviral activity of hand disinfectants. Some theoretical and practical considerations.
      ].
      A look at the efficacy data of ethanol against non-enveloped viruses, however, shows that MNV is one of the most susceptible non-enveloped viruses. In addition, ethanol at 70% would not be considered to be virucidal according to EN 14476 due to the insufficient activity against poliovirus. A non-virucidal reference alcohol may therefore be found in the future as a positive control for the phase 2, step 2 test for virucidal efficacy on hands, which may reduce the credibility and acceptance of the upcoming standard. Perhaps it will be an option to select MNV for a phase 2, step 2 test to support ‘limited spectrum virucidal activity’ and another more resistant non-enveloped virus to support virucidal activity under practical conditions.
      In conclusion, the spectrum of virucidal activity of ethanol at high concentrations covers the majority of clinically relevant viruses. Additional acids can substantially improve the virucidal activity against, for example, poliovirus, FCV, polyomavirus, and FMDV, although selected viruses such as HAV may still be too resistant. The selection of a suitable virucidal hand rub should be based on the most prevalent viruses in a unit and on the user acceptability of the product under frequent-use conditions.

      Conflict of interest statement

      None declared.

      Funding sources

      None.

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