Bactericidal and virucidal activity of ethanol and povidone‐iodine

Abstract Ethanol and povidone‐iodine (PVP‐I) are important microbicides that inactivate bacteria and viruses. The present study provides a review of literature data on the concentration‐dependent bactericidal and virucidal activity of ethanol and PVP‐I in vitro. A systematic search was performed using the meta‐database for biomedicine PubMed. Eventually, 74 studies with original data on the reduction of bacterial and viral infectivity using in vitro tests were analyzed. A safe bactericidal effect of ethanol can be expected at concentrations between 60% and 85%, and the exposure times vary between ≤0.5 and ≥5 min. Within an exposure of up to 5 min, 80%–90% ethanol also exerts virucidal/low‐level activity, which includes its action against enveloped viruses plus adeno‐, noro‐, and rotaviruses. For PVP‐I, the best bactericidal and virucidal/high‐level effect is present at a concentration range of approx. 0.08%–0.9% depending on the free iodine concentration. The maximum exposure times are 5 min for bacteria and 60 min for viruses. The available data may help optimize the significant inactivation of bacteria and viruses in various areas. However, as the conditions in application practice can vary, concrete recommendations for the application can only be derived to a limited extent.

Hollingsworth, 2008). In comparison, several studies have tested virucidal efficacy including limited virucidal activity (active against enveloped viruses), a low-level of virucidal activity (active against enveloped viruses plus adeno-, noro-, and rotaviruses), and a highlevel of virucidal activity (active against enveloped and non-enveloped viruses). In most studies, only a limited virucidal activity was detected for higher ethanol concentrations.
The iodophor PVP-I, consisting of elementary iodine bound to the carrier poly (1-vinyl-2-pyrrolidone), is regarded as a microbicide that exerts broad-spectrum activity against bacteria, fungi, protozoa, and viruses (Görtz, Reimer, & Neef, 1996). Due to its excellent antiseptic properties, it is used particularly for wound, skin, and throat disinfection. The number of experimental studies testing different concentrations of PVP-I from <0.001% to 10% for inactivating efficacy against gram-positive and gram-negative bacteria is extensive. Significantly, the inactivating effect is dependent on the concentration of free iodine, which decreases with the increasing concentration of PVP-I especially within the range of 5%-10% (Atemnkeng, Plaizier-Vercammen, & Schuermans, 2006). Similarly, an increasing number of studies in the literature are testing the spectrum of virucidal efficacy from limited virucidal activity to a highlevel of virucidal activity.
The objective of the present study was to describe the bactericidal and virucidal activity of ethanol and PVP-I without the addition of interfering substances (organic load) based on the data available in the literature. Particular attention should be given to an exposure temperature of 22 ± 3°C and an exposure time of up to 60 min.

| MATERIAL AND ME THODS
First, a search and analysis of the existing literature were carried out from January to March 2019 using PubMed (the Englishlanguage text-based meta-database for biomedicine) with the keywords "bactericidal activity/efficacy of ethanol", "virucidal activity/efficacy of ethanol", "bactericidal activity/efficacy of povidone-iodine", "virucidal activity/efficacy of povidone-iodine".
About 600 entries were found under these keywords. All studies with original data on the reduction of bacterial and viral infectivity using in vitro tests were selected. After the analysis of the respective abstracts, 148 publications were shortlisted, and their full text had to be evaluated. In the cited literature of these articles, another 50 relevant papers were found; the full text of these papers was also analyzed. From these 198 papers, 74 publications resulted, which were of essential importance in defining the bactericidal and virucidal activity of ethanol and PVP-I. To be able to make a statement about the concentration-dependent antimicrobial effect of ethanol and PVP-I, their respective concentrations analyzed in the literature were evaluated with respect to their bactericidal and virucidal effect. Only studies that tested the bactericidal or virucidal efficacy of ethanol or PVP-I in liquids were included. Studies that analyzed disinfectants based on ethanol or PVP-I, but with additives that may influence the microbicidal effect, were excluded from the present review.
Methodologically, only those studies were considered that had examined the listed results in in vitro tests. A compilation of these methods and the corresponding references are given in Table 1 for the determination of bactericidal efficacy and in Table 2 for the determination of virucidal efficacy. The most frequently used method for determining the bactericidal and virucidal effect of ethanol and PVP-I was the quantitative suspension test, which was often carried out in the standardized form following European standards or national guidelines. In a few cases, carrier tests of practical relevance using glass or metal carriers or ex vivo skin tests with pigskin were also used.
In the evaluation of the data obtained, primarily results were considered that were obtained without interfering additives to aggravate the disinfection effect. If such findings were not available, the obtained results were analyzed with the addition of interfering substances (organic load, e.g., bovine serum albumin or erythrocytes), as shown in Tables 1 and 2. The exposure temperature in the studies considered was 22 ± 3°C in most of the cases. In some studies, only the term "room temperature" was used; alternatively, no precise information on the exposure temperature was given, in which case room temperature was assumed. Deviations from the specified temperature range are also noted in Tables 1 and 2.
For the analysis of the microbicidal activity of ethanol and PVP-I in the listed studies, various initial compounds in the form of commercial disinfectants or antiseptics were used, as noted in the tabular lists of the results on antimicrobial activity using footnotes.
Where no note is given, either ethanol or PVP-I were used as chemical reagents. The concentration of ethanol was given by most investigators in volume percent (vol%, v/v-volume per volume), and, in very rare cases, in weight percent (weight%, w/w-weight per weight). In numerous studies, however, the tested concentrations of ethanol were not specified in greater detail (see Tables 1 and 2 , n.d.-no data). The stated concentrations of PVP-I generally refer to w/v (weight per volume).
The range of activity of disinfectants against enveloped/lipophilic viruses is called "limited virucidal," and the range of activity against enveloped/lipophilic, as well as non-enveloped/hydrophilic, viruses is called "virucidal" (Rabenau et al., 2014). As per current German guidelines or recommendations (Rabenau, Schwebke, Steinmann, Eggers, & Rapp, 2012), the "virucidal" range is further subdivided into "virucidal/low-level" or "limited virucidal plus" (enveloped viruses in addition to adeno-, noro-, and rotaviruses, but excluding enteroand parvoviruses) and "virucidal/high-level" (all viruses mentioned as virucidal/low-level plus entero-and parvoviruses). As per the European terminology, there are also three different claims on virucidal activity: "active against enveloped viruses"; "limited spectrum of virucidal activity" including against enveloped viruses plus adeno-, noro-, and rotaviruses"; and "virucidal activity," which includes action against all relevant human viruses (EN, 14476, 2019).  & Wedmore, 2010;McLure & Gordon, 1992;Reimer et al., 2000;Salvatico, Feuillolay, Mas, Verrière, & Roques, 2015) generally assume a safe bactericidal effect if the tested substance causes a reduction in the bacterial count by 4-5 powers of ten (4-5 log 10 ) corresponding to 99.99%-99.999% (see Tables A1 and A3). In several cases, a reduction in the bacterial count by 3 powers of ten (3 log 10 ) corresponding to 99.9% (Anagnostopoulos et al., 2018;Rikimaru et al., 2002) or complete germ inactivation (100%) (Berkelman, Holland, & Anderson, 1982;Kampf & Hollingsworth, 2008;Koshiro & Oie, 1984;Tavichakorntrakool et al., 2014) is also given in the literature. According to the current guidelines, a virucidal effect is defined as a reduction of the virus titer by at least 4 decimal powers (≥4 log 10 ) resulting in virus titer reduction of ≥99.99% (Eggers et al., 2018;Kawana et al., 1997;Noda, Watanabe, Yamada, & Fujimoto, 1981 Tables A2 and   A4). Only one study describes a complete (100%) virus inactivation by the electron microscopic analysis of human norovirus-like particles (Sato et al., 2016). When analyzing the results in relation to the concentrations of the active substance, it must be taken into account that when using the quantitative suspension test to determine the virucidal effect, the final concentration of the formulation tested is usually 80% (EN, 14476, 2019;Rabenau et al., 2014). Table 3 that a safe bactericidal effect of ethanol, including inactivation of vegetative forms of spores, is given in concentrations of 60%-85%, with the optimal effective concentration being 80%-85%. In the latter concentration range, exposure times are a maximum of 30 s, and for 60%-70% ethanol, a longer exposure of ≥5 min is necessary. Concentrations of 30%-50% ethanol have a significantly lower bactericidal activity, whereas the tested exposure times of 5-30 min are partly insufficient for a significant bactericidal effect. A concentration of 80%-90% ethanol also exerts virucidal/low-level activity, which includes action against enveloped viruses plus adeno-, noro-, and rotaviruses. For a titer reduction of 4 log 10 , a time interval of up to 5 min is required, depending on the virus structure, whereby a safe virucidal effect against enteroviruses could not be demonstrated. In comparison, lower concentrations of TA B L E 1 Methods for evaluation of bactericidal efficacy of ethanol and PVP-I

| D ISCUSS I ON
The present article aimed to describe the bactericidal and virucidal activity of ethanol and PVP-I as a function of substance concentration without the addition of organic load at an exposure  (Kampf & Hollingsworth, 2008); alternatively, it is effective as low-concentration ethanol only in combination products, for instance in combination with propanol (Marchetti, Kampf, Finzi, & Salvtorelli, 2003). However, 100% of ethanol has no safe microbicidal effect, as the denaturation of proteins is difficult to achieve in the absence of water (Gold & Avva, 2020). Nevertheless, a study published by Koshiro and Oie (1984)

ACK N OWLED G M ENTS
The article was funded by corelife oHG, Hannover, Germany.

CO N FLI C T O F I NTE R E S T
None declared.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All literature data associated with this article are provided in full in this paper.

TA B L E A 1 (Continued)
TA B L E A 2 Virucidal efficacy of ethanol at concentrations of 30%-100%