In almost all countries within the European Union, products for hygienic and surgical hand disinfection are considered biocidal products (product type 1, intended for human hygiene). Products for surface disinfection belong to product type 2 (disinfectants and algaecides not intended for direct application to humans or animals) or product type 4 (food and feed area). Isopropanol has been approved in 2015 as an active biocidal agent for product types 1, 2, and 4.²⁴ n-Propanol has also been approved (November 2017) as an active biocidal agent for product types 1, 2, and 4.²⁵ Ethanol remains under review (June 2018) as an active biocidal agent for product types 1, 2, 4, and 6 (preservatives for products during storage).²⁶

Skin antiseptics are commonly regulated as medicinal products. The efficacy requirements for a disinfectant depend on the intended use. In human medicine, a product for disinfection has to show at least bactericidal and yeasticidal activity. Additional claims such as fungicidal, tuberculocidal, mycobactericidal, virucidal (three levels of efficacy), and sporicidal activity are possible but not mandatory. The application of European efficacy standards is summarized in EN 14885.²⁷

In the United States the regulation of alcohols depends mostly on the product application. Skin antiseptics fall under the purview of the US Food and Drug Administration and are regulated as drugs, either as an over-the-counter under a drug monograph guideline or as a new drug approval, whereas alcohol-based antimicrobials for inanimate surface applications are regulated by the US Environmental Protection Agency.

Alcohol-based hand rubs are recommended as the first choice treatment for the decontamination of clean hands in health care since 2009.²⁸ In 2015, the World Health Organization (WHO) described alcohol-based hand rubs as essential medicines,²⁹ referencing the simple formulations using 80% ethanol or 75% isopropanol,²⁸ although they do not meet European efficacy requirements when applied
with 3 mL for 30 seconds.³⁰ In 2016, WHO recommended the use of alcohol-chlorhexidine-skin-antiseptics for surgical site preparation in patients undergoing surgical procedures.³¹ In the same guideline, “suitable” alcohol-based hand rubs are recommended next to surgical hand scrubs for the preoperative treatment of hands.³¹

Other regions of the world may regulate alcohol use for antimicrobial purposes differently. Other national specific guidelines are not considered further in this review.

The spectrum of antimicrobial activity is usually determined in suspension tests or time kill tests. The test principle is the same. The disinfectant or antiseptic must demonstrate a minimum log₁₀ reduction of culturable microorganisms or reduction of viral infectivity within a specific exposure time compared to a control (eg, pre-exposure or exposure to negative control).

In Europe, the spectrum of antimicrobial activity must include bactericidal activity according to EN 13727 and yeasticidal activity according to EN 13624. The same spectrum of antimicrobial activity is required in the United States, although the number of bacterial species to be investigated to determine bactericidal activity is much higher with at least 20 species compared to 4 species in EN 13727.²³ A mycobactericidal or virucidal activity is optional.²⁷ In veterinary medicine, the products must be at least bactericidal according to EN 13727.²⁷ Hygienic hand rub products used in food, industrial, domestic, and institutional areas have in Europe no specific minimum spectrum of antimicrobial activity. Tests are available to demonstrate bactericidal (EN 1276) and yeasticidal (EN 1650) activity.²⁷

In the following sections, the alcohol concentrations are given as wt/wt unless otherwise indicated.

Ethanol at 78% or more has comprehensive bactericidal activity within 30 seconds against Acinetobacter baumannii,³² Acinetobacter lwoffii,³² Bacteroides fragilis,³² Burkholderia cenocepacia,³³ Burkholderia cepacia,³² Campylobacter jejuni,³⁴ the vegetative cell form of Clostridium difficile,³² Corynebacterium jeikeium,³⁵ Enterobacter aerogenes,³² Enterobacter cloacae,³² Enterococcus faecalis,³²,³⁶ E faecium,³²,³⁶ Enterococcus hirae,³⁷,³⁸ E coli,³²,³⁷,³⁸ Haemophilus influenzae,³² Haemophilus parasuis,³⁹ Helicobacter pylori,⁴⁰ Klebsiella pneumoniae,³² Klebsiella oxytoca,³² Listeria monocytogenes,³² Micrococcus luteus,³² Proteus mirabilis,³² Pseudomonas aeruginosa,³²,³⁷,³⁸,⁴¹ Serratia marcescens,³² Staphylococcus aureus,³²,³⁷,³⁸,⁴¹ Staphylococcus epidermidis,³²,³⁵ Staphylococcus haemolyticus,³² Staphylococcus hominis,³² Staphylococcus saprophyticus,³² Streptococcus pneumoniae,³² Streptococcus pyogenes,³² Salmonella enteritidis,³² Salmonella Typhimurium,³²,⁴² and Shigella sonnei.³² Lower concentration of ethanol may require longer exposure times to reach an equivalent efficacy, depending on the formulation.

Published data with isopropanol indicates a strong bactericidal activity of a 70% solution beginning after 15 seconds against C jejuni,³⁴ Enterococcus species,⁴³ H parasuis,³⁹ S aureus,⁴⁴ and S epidermidis.⁴⁵ Additional data with mixed propanols (eg, 45% isopropanol plus 30% n-propanol) indicate comprehensive bactericidal activity within 30 seconds.³⁷,⁴⁶

n-Propanol at 60% has a strong bactericidal activity beginning after 15 seconds against A baumannii,⁴⁷ Enterococcus species,⁴³ and S aureus.⁴⁸ Additional data with mixed propanols (eg, 30% n-propanol plus 45% isopropanol) indicate comprehensive bactericidal activity within 30 seconds.³⁷,⁴⁶ The bactericidal activity of 60% n-propanol is considered to be equal to isopropanol at 70%, whereas lower concentrations such as 50% or 40% have a lower bactericidal activity.⁴⁹

Ethanol is usually effective at 70% to 90% within 30 seconds to 5 minutes against fungi typically found in health care setting such as C albicans,³⁸,⁵⁰ Aspergillus niger,³⁸ Cryptococcus neoformans,⁵⁰ or Cryptococcus uniguttulatus.⁵⁰ Food-related fungi such as ascospores,⁵¹ conidia,⁵¹ or yeasts⁵¹,⁵² were often more resistant to ethanol with a variable efficacy of 70% ethanol in 10 minutes. In mixed suspensions of environmental isolates (Rhodotorula rubra, C albicans, C uniguttulatus) and clinical isolates (R rubra, C albicans, C neoformans), 70% ethanol is still fungicidal (log₁₀ reduction ≥6) in 5 minutes, although the effect was somewhat less against the environmental mix.⁵⁰

Isopropanol at 60% or 70% was mostly effective with 5 to 10 minutes against various types of yeasts including
food-associated yeasts.⁵¹,⁵² The efficacy of 70% isopropanol against food-associated fungal spores such as ascospores or conidia is low even at 10-minute exposure.⁵¹

Fourteen percent n-propanol has been described to inhibit multiplication of C albicans suggesting a levurostatic activity at that concentration.⁵³ At 89.5% (vol/vol), n-propanol is effective against C albicans.⁵⁴ A commercial product based on n-propanol (>30%) and isopropanol (15%-30%) has been investigated for yeasticidal activity against 25 strains isolated from food or food processing. Within 5 minutes, the product reduced the number of yeast cells by at least 4 log₁₀ steps of 19 species; some species were less susceptible.⁵²

Ethanol of 70% or more has sufficient activity against selected mycobacterial species such as Mycobacterium chelonae,³⁵ Mycobacterium nonchromogenicum,³⁵ Mycobacterium smegmatis,³⁵,⁵⁵ Mycobacterium terrae,³⁸,⁵⁶ and Mycobacterium tuberculosis⁵⁶,⁵⁷ within 1 to 5 minutes. Only few data were found against aquatic nontuberculous mycobacteria. For example, Mycobacterium marinum was effectively reduced by ethanol at 50% and 70% in 1 minute.⁵⁸

Early data from 1953 indicate a tuberculocidal activity of isopropanol at 50% to 70%.⁵⁹ In suspension tests, little has been published on the mycobactericidal activity of isopropanol. At 60%, it was mostly effective against both M tuberculosis and M terrae within 5 minutes.⁵⁶ Whether the tuberculocidal efficacy covers other mycobacterial species is not clear.

n-Propanol at 20% has been described to reduce Mycobacterium avium on frosted glass strips by >6 log in 20 minutes.⁶⁰ No other data were found to describe the efficacy of n-propanol against mycobacteria.

Ethanol has been shown to be effective against various enveloped viruses.⁶¹ Beginning at a concentration of 42.6%, ethanol was effective within 30 seconds against severe acute respiratory syndrome coronavirus⁶²; Middle East respiratory syndrome coronavirus⁶³; Ebola virus⁶³; influenza A virus,⁶¹,⁶⁴,⁶⁵ including the human type H3N2,⁶⁶ the avian type H3N8,⁶⁶ and human type H1N1⁶³; influenza B virus⁶⁷; human immunodeficiency virus (HIV)³⁸,⁶⁸,⁶⁹,⁷⁰,⁷¹; hepatitis B virus (HBV)⁷²,⁷³; vaccinia virus⁶¹,⁶⁴,⁷⁴,⁷⁵,⁷⁶,⁷⁷,⁷⁸; duck HBV⁷⁷; togavirus⁷⁹; pseudorabies virus⁷¹; Newcastle disease virus⁸⁰; bovine viral diarrhea virus⁷¹,⁷⁸,⁸¹; Zika virus⁶³; herpes simplex viruses (HSVs)⁶⁶ types 1 and 2; and respiratory syncytial virus (RSV).⁶⁷ Ethanol is effective at 73.6% against hepatitis C virus (HCV) in 15 seconds⁸¹ and 30 seconds⁶³ but not in 40%.⁸²

Ethanol as a solution has mostly sufficient activity in 30 seconds against adenovirus type 5 (test virus of EN 14476) at concentrations between 45% (contains additional active ingredients) and 95%,⁷⁸,⁸¹,⁸³,⁸⁴ although one study indicates sufficient activity only in 1 minute with 77% ethanol.⁸⁵ Ethanol at 70% as a gel was sufficiently active in 2 minutes.⁸⁴ Adenovirus type 2 was less susceptible, requiring 2 minutes at 55%⁶⁷ and 85%.³⁸ In 30 seconds, 62.4% ethanol was not sufficiently effective⁸⁶ and at 50% required a 10-minute exposure time to be effective.⁶¹ The combination of ethanol 73.6% and peracetic acid (0.2%) was effective in 30 seconds,⁸⁷ but this would be expected based on the potency of peracetic acid alone (see chapter 18). Data with other adenoviruses suggests that ethanol at 72.5% to 77% was effective in 60 seconds against adenovirus type 7 but not against adenovirus type 8.⁸⁸

The effectiveness of ethanol on nonenveloped viruses has also been found to vary depending on the virus type. Gels based on 85% ethanol or the combination of 62.4% ethanol with additional citric acid were effective against rotavirus in 30 seconds³⁸,⁸⁶ as well as a hand rub based on 55% ethanol, 0.7% phosphoric acid, and three other alcohols.⁶⁷ Today, murine norovirus (MNV), an enteric (gastrointestinal virus), is commonly used as a surrogate to assess activity of disinfectants and antiseptics against human noroviruses. Ethanol at concentrations between 62.4% and 85.8% is usually effective in 30 seconds⁷⁸,⁸¹,⁸³,⁸⁹,⁹⁰,⁹¹ or 1 minute⁹¹; lower concentrations were less effective. A gel based on 53.2% ethanol was only effective in 1 hour.⁹² Ethanol at 42.6% reduces MNV according to one study in 1 minute by 0.3 log₁₀ and in 5 minutes by 0.4 log₁₀.⁹¹ In another study, 42.6% ethanol demonstrated approximately 3 log₁₀ reduction in 30 seconds, whereas ethanol at 24.7% or 8% was ineffective within 3 minutes.⁸⁹ Feline calicivirus (FCV) has often been used in the past as a surrogate for human noroviruses, although it is not preferred because it is a respiratory tract virus and is highly sensitive to acidity.⁹³ The FCV is difficult to inactive by ethanol. Ethanol solutions at 42.5% and 62.4% were effective at 3 minutes, whereas 73.6% required 5 minutes.⁹⁴ Formulations with 72.5% ethanol did not reach a 4 log₁₀ reduction in 60 seconds.⁸⁸ A solution based on 77% ethanol showed little activity against FCV in 60 seconds (<1 log₁₀).⁸⁸ Even after 5 minutes, the efficacy was still insufficient (<2 log₁₀).⁸⁵ A hand rub based on >85.8% ethanol was practically ineffective in 30 seconds (<1 log₁₀).⁷⁸ Finally, ethanol at 42.6%, 62.4%, and 85.8% had insufficient efficacy in 5 minutes with the highest log₁₀ reduction of 2.6.⁹¹ Whenever different types of acid are added, ethanol in hand rubs has been described to be effective in 30 seconds at 45%,⁷⁸ 50.2%,⁶⁷,⁷⁸ 55%,⁹⁵ 62.4%,⁸⁶ and 67.9%.⁹⁵ Malic acid at 0.35% has also been described to improve the efficacy of ethanol against FCV to some extent.⁹⁶ Data on the efficacy of ethanol against coxsackievirus are conflicting. Ethanol at 72.5% to 92.4% has been described to be effective against coxsackievirus B5 within 60 seconds⁸⁸,⁹⁷ and also against coxsackievirus B1 within 10 minutes but not against coxsackievirus A7.⁸⁸ Higher
ethanol concentrations (85%-90%) are effective against coxsackievirus B3 in 15 to 60 seconds.⁹⁸ The efficacy of ethanol against echoviruses is rather good. One study shows that ethanol at 92.4% is effective against echovirus 11 in 20 seconds.⁹⁹ Another study found a log₁₀ reduction ≥3 within 1 minute for ethanol at 92.4% against the same virus, whereas ethanol at 67.9% was almost ineffective (<1 log₁₀) at the same exposure time.¹⁰⁰ Against echovirus 6, ethanol was effective at 50% within 10 minutes.⁶¹ With human enterovirus 71, ethanol had only little activity at 62.4% and 67.9% within 10 minutes (<1 log₁₀). At 79.6%, a 3.2 log₁₀ reduction was achieved in the same exposure time, and at 92.4%, this increased to 5.8 log₁₀.¹⁰¹

Formulations with ethanol up to 80% and without acid were not sufficiently effective in up to 5 minutes against poliovirus type 1 when tested under standard conditions with 80% product proportion in the suspension. When the product proportion is increased in the suspension test to 97%, some formulations with 73.5% or 80% ethanol were effective within 1 minute. One formulation with 95% ethanol was effective in 30 seconds. Gels were mostly less effective compared to solutions. 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 seconds.¹⁰² Poliovirus type 2 is somewhat less susceptible to ethanol compared to poliovirus type 1.¹⁰³ A hand rub based on 55% ethanol, 0.7% phosphoric acid, and three other alcohols was effective in 30 seconds against rhinoviruses.⁶⁷

A gel based on 54.2% ethanol showed no effect at all (0 log₁₀) against hepatitis A virus (HAV) after 30 seconds.⁸⁶ Ethanol at 80% or 95% was not sufficiently effective within 2 minutes.¹⁰⁴ A hand rub based on 80% ethanol showed only little reduction of viral infectivity within 30 seconds (0.47) but increased efficacy on exposure at 2 minutes (≥2.2 log₁₀).⁷¹ When 0.7% phosphoric acid and three other alcohols are added to ethanol at 55%, the formulation was effective against HAV in 30 seconds.⁶⁷ Ethanol at 62.4% with additional 0.25% citric acid, however, was not sufficiently effective in 30 seconds with 1.75 log₁₀.⁸⁶ The infectivity of the foot-and-mouth disease virus was insufficiently reduced by ethanol between 55.2% and 72.5% within 5 minutes.¹⁰⁵ Hand rubs with 70% to 75.2% ethanol and additional phosphoric acid (0.6%) or 50% ethanol and additional citric acid (0.5%) were effective within 30 seconds.¹⁰⁵

Ethanol has little activity against polyomavirus SV40, an additional test virus that was chosen due to its resistance to alcohols by experts for the German test method to determine virucidal activity.¹⁰⁶ A gel based on 85% ethanol revealed sufficient activity in 15 minutes,³⁸ but a hand rub based on 78.2% ethanol had insufficient activity within 10 minutes (approximately 2 log₁₀).¹⁰³ When 0.7% phosphoric acid and three other alcohols are added to ethanol at 55%, the formulation was able to reduce infectivity sufficiently in 60 seconds.⁶⁷ Ethanol at 73.6% in combination with 0.2% peracetic acid was effective in 30 seconds.⁸⁷ Ethanol has almost no virucidal activity against parvoviruses. At 80% ethanol, the infectivity of the canine parvovirus, as an example was reduced in 5 minutes only by 0.1 log₁₀.⁷¹

Isopropanol at 40% is effective against the enveloped viruses duck HBV, vaccinia virus,⁷⁶ and the modified vaccinia virus Ankara.⁷⁷ At 75%, isopropanol is effective within 15 seconds against bovine viral diarrhea virus (a surrogate for HCV).⁸¹ Seventy percent isopropanol was also shown to be effective against influenza A virus,⁶⁵ whereas 30% isopropanol was effective at a 10-minute exposure time.⁶¹ The HIV can easily be inactivated by 70% isopropanol.¹⁰⁷ For inactivation of RSV, 35% isopropanol was effective within 1 minute.¹⁰⁸ The HSVs seem somewhat more resistant. The HSV types 1 and 2 were inactivated by 70% isopropanol in 5 minutes,¹⁰⁹ whereas another study indicated sufficient efficacy against HSV type 1 within 1 minute for isopropanol at 60% and 70%.¹¹⁰ A concentration of 20% was still effective against HSV type 1 but required a 10-minute exposure.⁶¹

As for other alcohols, the results with other viruses vary depending on their structure. Isopropanol is considered to be ineffective against enteroviruses.¹¹¹ Against poliovirus type 1, human enterovirus 71, and coxsackieviruses B2 and B3, no efficacy was found at concentrations between 70% and 100% within 10 minutes.⁶¹,⁹⁸,¹⁰¹,¹¹² The efficacy against echovirus⁶¹,¹⁰⁰,¹¹³ and astrovirus¹¹³ is poor. Fifty percent isopropanol was, however, described as effective against adenovirus within 10 minutes.⁶¹ The data with FCV, used as a surrogate for human noroviruses before the advent of MNV culture in vitro, are overall conflicting. The FCV was inactivated by isopropanol between 50% and 70% in 3 minutes, but at 80%, no sufficient efficacy was found after 5 minutes.⁹⁴ Another study indicated that isopropanol at 50%, 70%, and 90% for 5 minutes gave a maximum reduction of 0.8 log₁₀.⁹¹ The data with MNV are also not consistent. One study shows that isopropanol at 80% is effective against MNV in 30 seconds, whereas 50%, 60%, 70%, and 90% were not,⁸⁹ indicating that 80% may be the optimum concentration. Another study describes isopropanol at 70% in 5 minutes as effective (log₁₀ reduction ≥2.6) and greater at 90% after 1 minute.⁹¹ A third study shows that 60% isopropanol is effective against MNV in 60 seconds, but concentrations of 30% and 10% are not.⁹² In contrast, rotaviruses have been found to be inactivated quite easily as shown with a mixture of 45% isopropanol and 30% n-propanol.⁹²

Thirty percent and 60% n-propanol are effective within 1 minute against enveloped viruses such as HCV.¹¹⁴ n-Propanol at 70% was described as effective against influenza A virus H1N1 within 1 minute.⁶⁵ A combination of 30% n-propanol and 45% isopropanol was effective within 15 seconds against various enveloped virus species.⁶⁶

The FCV is inactivated within 30 seconds by n-propanol at 50% to 70%, but at 80%, an exposure time of 1 minute was necessary.⁹⁴ The MNV is also inactivated within 30 seconds by n-propanol at 50% to 90%.⁸⁹ The type 1 poliovirus is inactivated by n-propanol at 33% in 4 minutes by 3 log₁₀.¹¹⁵ The addition of 0.2% peracetic acid improved the efficacy resulting in a 4 log₁₀ reduction within 1.5 minute.¹¹⁵

Bacterial spores are regarded to be resistant to ethanol,¹¹⁶ isopropanol,¹¹⁷ and n-propanol.

Sixty-three percent and 80% isopropanol and ethanol at 5-minute contact have been shown to have some effects against cysts of Giardia duodenalis and Entamoeba invadens (a nonpathogenic model for Entamoeba histolytica), resulting in an almost complete collapse of the cyst wall of Giardia. Treatment with 80% isopropanol also prevented an oral infection of gerbils by 1000 G duodenalis cysts.¹¹⁸

In typical disinfectant applications, alcohols have an advantage of acting and evaporating rapidly and not leaving residue that allows time for bacteria to develop resistance. Apart from bacterial spores, no other bacteria with a notable resistance to n-propanol or propanol have been reported so far.²⁸,¹¹⁹,¹²⁰ Conidia of Penicillium chrysogenum, Penicillium digitatum, and Penicillium italicum were shown to resist ethanol vapors under nonoptimal natural conditions probably caused by a reduced intracellular water activity of dry-harvested conidia,¹²¹ but differences in the intrinsic resistance profiles of bacteria/fungi to alcohols may be demonstrated at low-level exposures.

Ethanol at 1.25% to 2.5% has been shown to significantly enhance S aureus biofilm formation by upregulation of some proteins with adhesive functions and others with cell maintenance functions and virulence factor EsxA.¹²² Similar findings were reported with L monocytogenes. When cells were exposed to 5% (vol/vol) ethanol for 60 minutes, they were significantly more difficult to kill by ethanol at 17.5%.¹²³ The adapted cells were also more difficult to kill by 0.1% hydrogen peroxide.¹²³ Attachment of cells can be significantly increased in some L monocytogenes strains when exposed to 2.5% ethanol, mostly at 10°C.¹²⁴ When the Pseudomonas species strain DJ-12 was exposed to 5% ethanol for 10 minutes, the cells were significantly more difficult to kill by 20% ethanol.¹²⁵ Cells treated with ethanol displayed irregular rod shapes with wrinkled surfaces.¹²⁵ Exposure of a Pseudomonas putida strain to toluene increased the tolerance to ethanol, which was explained by an inhibitory effect of ethanol on the biosynthesis of saturated fatty acids.¹²⁶ In Bacillus subtilis cells, the transfer of the mobile genetic element Tn916, a conjugative transposon, and the prototype of a large family of related elements was increased 5-fold by exposure to 4% ethanol for up to 2 hours. This may also result in a transfer of Tn916-like elements and any resistance genes they contain.¹²⁷

The maximum ethanol tolerance of S cerevisiae has been described to be at 25% (vol/vol).¹²⁸ The presence of 2.5% ethanol was found to increase S cerevisiae colony growth by 20%, whereas 5% ethanol or more inhibited yeast colony growth.¹²⁹ When S cerevisiae cells were exposed for 30 minutes to sublethal concentrations of ethanol (8%, vol/vol), they became less susceptible to ethanol at a previously lethal concentration (14%, vol/vol).¹³⁰ Specific activities of glycolytic and alcohologenic enzymes within intact living cells remained high by the presence of sublethal ethanol.¹²⁸ Trehalose plays a role in ethanol tolerance at lethal ethanol concentrations but not at sublethal ethanol concentrations.¹³¹ Using atomic force microscopy, it was shown that challenge of S cerevisiae with 9% ethanol (vol/vol) for 5 hours reduced the observed stiffness of glucose-grown yeast cells, suggesting that changes in the cell membrane due to the presence of ethanol could modify the biophysical properties of yeast cells.¹³²

Less has been published on the effects of low-level exposure of isopropanol on microorganisms. One study indicated that attachment of cells can be significantly increased in some L monocytogenes strains when exposed to 2.5% isopropanol, mostly at 10°C.¹²⁴ Biofilm formation with 37 clinical icaADBC-positive S epidermidis isolates was investigated after exposure to isopropanol at 1%, 2%, 4%, or 6%. In 14 of the 37 strains, biofilm formation was inducible by isopropanol exposure.¹³³ With C albicans, it was described that 2% isopropanol inhibited to some extent biofilm development.¹³⁴ In E coli, it was shown that low-level exposure to isopropanol concentrations up to 2.7% for up to 24 days reduced the susceptibility of the six tested strains.¹³⁵