Development and virucidal activity of a novel alcohol-based hand disinfectant supplemented with urea and citric acid

Virus transfer via human hands is a major route of infection inside and outside medical settings [1]. The efficiency of virus transfer from hands to hands (direct transmission) or via contaminated surfaces (indirect transmission) is closely connected with virus persistence [2]. Detailed studies regarding virus persistence are still lacking however non-enveloped viruses generally are persisting much longer than enveloped-viruses [3‐5]. Viruses of the respiratory tract like the non-enveloped rhinovirus and the enveloped viruses like influenza virus and coronavirus can persist on surfaces for several days [6]. When dried, enveloped blood-borne viruses as hepatitis C virus can be infectious for more than a week [7]. Non-enveloped viruses like hepatitis A virus (HAV), adenovirus (AdV) and human norovirus may even persist for several weeks [6, 8]. The actual level of the viral contamination in the environment is likely underestimated due to limited detection of known viruses and presence of unknown viruses [9, 10]. Often the detection of viral genomes by nucleic acid assays is the only way to get insights: Carducci et al. detected viral nucleic acid in 16.7 % of all surface samples in healthcare settings [11]. A disinfectant for hand hygiene therefore would ideally possess broad antiviral spectrum covering non-enveloped in addition to enveloped viruses offering increased protection against persistent, unexpected or unknown viruses [12, 13]. The recommendation of broad spectrum alcohol-based hand rubs (ABHRs) for preventing virus transmission by hands is the most important feature of current guidelines [14].

Worldwide there are differences regarding the number of test viruses needed to be inactivated in standardized tests before a broad spectrum claim including non-enveloped viruses can be given by manufacturers for a hand disinfectant [15]. Regulatory authorities in USA admit activity against certain stable reference viruses using methods of American Society for Testing and Materials (ASTM) (Table 1). The choice of reference test virus is left to the manufacturer [16]. Hand antiseptics can be tested on artificially contaminated hands or fingerpads with test viruses such as AdV, rhinovirus type 14 or 37, human rotavirus, surrogates of human norovirus like MNV or feline calicivirus (FCV) and HAV [17‐19]. Yet cases are well documented in which a disinfectant active against a reference virus was not active against an non-enveloped virus like parvovirus [20]. Regulatory authorities in Germany have established a minimum set of test viruses [21] which are not only difficult to inactivate, but also vary in their susceptibility to disinfectants and thus are thought to be representative of the whole known virus families. Under the Guideline of Deutsche Vereinigung zur Bekämpfung der Viruskrankeiten e.V. and the Robert Koch-Institute (DVV/RKI Guideline) [22], disinfectants achieving at least 4 log₁₀ titer reduction factor (RF of 4) against vaccinia virus and bovine viral diarrhea virus (BVDV) are active against all enveloped viruses (limited spectrum virucidal) [23, 24]. Disinfectants also inactivating poliovirus (PV), AdV and polyomavirus SV40 (SV40) and since 2015 also MNV can claim activity against all viruses (virucidal) according DVV/RKI Guideline [22]. The discrimination between enveloped only / all viruses was proved successful and has been taken up in the European EN 14476 for hand rubs being valid in whole Europe (Table 1) [25]. In 2012, an additional DVV Guideline for testing the antiviral activity of disinfectants on stainless steel disks carriers simulating practical situations was established [26] and the discrimination limited spectrum virucidal / virucidal claim exists there as well (Table 1). At present, work is in progress to develop a EN Norm for the virucidal carrier test testing MNV and AdV as test viruses [27].

Most biocidal active substances used in hand hygiene have no difficulty inactivating enveloped viruses [1], which are sensitive to alcohol-based hand rubs even in the presence of interfering substances [24]. Achieving the virucidal claim of the DVV/RKI Guideline is considerably more difficult and products that can claim inactivation of all non-enveloped viruses according to the German regulatory model are rare in hand hygiene [28]. The fulfilment of DVV/RKI Guideline and EN 14476 in hand hygiene area is even complicated by the practical requirement of a short exposure time. A user will normally not wait more than about 30–60 s for a ABHRs to act and will not reapply the product if it has dried up before the target exposure time [29].

To date all of the products that claim virucidal activity for hand hygiene under German DVV/RKI Guideline are alcohol-based formulations containing either high amounts of ethanol or an ethanol/1-propanol mixture supplemented with phosphoric acid [30]. Yet high alcohol content hand disinfectants are problematic for reasons of fire safety and toxicity [30] and it is also desirable to produce ABHRs with reduced acidity. Finally, alcohols were reported to inactivate AdV on carriers [31], but little is known regarding the ability of ABHRs to fulfil the DVV carrier test. Therefore, it was the aim to develop a hand disinfectant with a virucidal claim in suspension and carrier tests. We now present a novel ABHR based on ethanol (ca 70 %), supplemented with variable amounts of citric acid and urea additives that fulfils DVV/RKI Guideline and EN 14476 for virucidal activity in quantitative suspension tests. The formulation with the optimum ratio of additives was further characterized in detail showing to possess virucidal activity (without enteroviruses and parvoviruses) on carriers according to DVV Guideline [26].

The development of broad antiviral spectrum hand disinfectants with activity against all enveloped and non-enveloped viruses is limited by the small number of permissible active ingredients – broad spectrum hand rubs are generally based on alcohol. With ethanol alone in concentrations above 80 % v/v stable non-enveloped viruses like PV and AdV can be inactivated but not polyomavirus SV40 [36, 37]. In contrast, > 60 % v/v 1- and 2-propanol being often used in ABHRs can easily inactivate AdV and SV40 but not PV [36, 38]. Human enterovirus 71 was only inactivated by 95 % ethanol and not by 70 % and 75 % ethanol or any concentration of isopropanol [39]. The reasons for this differential sensitivity of viruses to alcohols are presumed to result from the hydrophobic / hydrophilic nature of the viral particles. The hydrophilic PV is more susceptible to ethanol and the more hydrophobic AdV and polyomavirus SV40 are more susceptible to both propanols [36, 40]. An improvement in activity can be achieved by re-formulation of alcohol solutions with additional ingredients that may enhance the activity. It was therefore a subject of recent developments to look for such additives that would produce a virucidal alcohol-based hand disinfectant. Yet products on the market, which are able to inactivate PV, AdV and MNV as required in the EN 14476 and also polyomavirus SV40 as required by the DVV/RKI Guideline are few. They are based either on 90–94 % ethanol, achieving an activity time of 2 min or about 70 % ethanol with the addition of 0.7 % phosphoric acid, achieving a sufficient activity time of 1 min [30].

We now report that urea in combination with citric acid can enhance the virucidal activity of ethanol solution (ca 70 %) and inactivate all reference viruses of the DVV/RKI Guideline within 1 min exposure time. Tests with bacteria and fungi are still under investigation for this antiseptic formulation which requires a broad spectrum as shown earlier for a product based on chlorine and alcohol [41]. The virucidal activity of concentrated aqueous urea solutions against PV is well known from early experiments with monkeys [42]. Organic acids used as a diluted aqueous solution are active against enveloped but not against non-enveloped viruses on their own [43]. The activity of 70 % ethanol solution against non-enveloped FCV increases from 2.6 log₁₀ to >4.4 log₁₀ reduction when the pH of the solution is lowered from 7.4 to 3.0 [44]. Citric acid has shown a virucidal efficacy against rhinovirus at artificially contaminated hands [45] and has already been incorporated in an alcohol-based hand rub [46]. Inorganic acids achieve the highest increase in virucidal activity of alcohol formulations [30].

A synergistic virucidal effect of urea and citric acid additives with ethanol was evaluated by measuring the activity against PV, AdV and polyomavirus SV40 according to Guideline of DVV/RKI in the presence of FCS. Keeping urea concentration stable and increasing acid and vice versa, as well as increasing the concentration of both compounds generally increased the antiviral activity of the mixture against all three non-enveloped test viruses. In the presence of FCS, the optimal concentrations of citric acid and urea for the new formulation were identified which resulted in a 4 log₁₀ reduction against all three test viruses (Table 2) within 30 to 60 s. Interestingly, we found a better activity in the quantitative suspension test against PV and polyomavirus SV40 compared to AdV with lower concentrations of urea and citric acid. A urea amount of 1.0 % and citric acid amount of 1.5 % was sufficient to inactivate PV and an urea amount of 1.5 % in combination with 1.5 % citric acid was sufficient to inactivate polyomavirus SV, but neither of these formulations was sufficiently active on AdV. These results are consistent with the data from the study of Kramer et al., who tested a virucidal alcoholic hand rub containing a low amount of ethanol and phosphoric acid and found AdV type 2 to be more stable than PV and SV40 [30]. These results strengthen the idea to test viruses from different virus families with various susceptibilities as found in the EN 14476 and DVV/RKI Guideline although it is known that important virus like Hepatitis A Virus and parvoviruses might be more stable than the test viruses used [34].

In Table 2 it can be seen that the formulation containing 2 % citric acid and 2 % urea possesses the needed activity for all required test viruses at the lowest citric acid and urea contents, which was then adopted for further analysis. The virus testing of this formulation confirmed the activity against a broad spectrum of human pathogenic viruses in the quantitative suspension assay. Enveloped viruses like BVDV and vaccinia virus were inactivated within 15 s exposure time. PV, AdV and polyomavirus SV40 were inactivated with A. bidest. and FCS as interfering substances within one minute exposure time. Under clean conditions according to the EN 14476 an exposure time of 30 s was achieved with AdV, PV and MNV. The results also shows that with EN 14476 higher RFs were achieved compared to the DVV/RKI Guideline (Table 3).

Tests on carriers confirmed the activity of the formulation found in the suspension assays also against viruses dried on the surface. The disinfectant inactivated AdV, MNV and vaccinia virus within one minute, making it active against non-enveloped viruses at low level (without enteroviruses and parvoviruses) on carriers, as defined according to DVV Guideline. The activity of the formulation is sufficient for virucidal activity on carriers according to the current version of prEN 16777. Stainless steel carrier methods have shown a good overall reproducibility between different labs [31], but the results on carriers may be not directly transferable to in-vivo situation. Fingerpad methods may be a better alternative for ABHRs testing under practical conditions, however fingerpad methods seem to lack reproducibility, which may be in part due to the inability of the method to properly distinguish the washing out of virus by mechanical means from virus inactivation by disinfectant. When comparing a mixture of propan-1-ol and propan-2-ol (RF = 2.8) and a hand wash product (RF = 3.0) in the fingerpad test, Tuladhar et al. concluded that washing hands with soap and water is better than using hand rubs based on alcohol for removal of norovirus from hands [47]. Own data with the ASTM E1838-10 [17] including modifications derived from the EN 1500 [48] and MNV as test virus demonstrated RFs of 4.25 and 3.94 after 30 s for 2 ethanol-based disinfectants with addition of an acid, whereas even water was able to achieve a RF of 2.86 [49]. Other works report that ABHR in fingerpad tests achieve RFs of 3–4 against non-enveloped viruses, with hard-water rinse achieving RF of 1 [50]. Ethanol-based hand rub fortified with phosphoric acid achieved in the fingerpad test with PV a RF of 3.04 after 30 s [30], whereas 80 % ethanol alone was not active against PV on contaminated hands in earlier tests [51]. Further research should provide clear discrimination between mechanical removal (addition of water) and the additional inactivation by chemical biocides for fingerpad tests. A direct comparison of results from European stainless steel carrier method and artificially contaminated fingerpad or whole hand methods should be carried out.

Hygienic hand disinfection can only be done with intact skin. For dermal tolerance no data for the product developed are available. In another study with a formulation containing 62 % ethanol and 4 % citric acid 9 % of the panelists were not included due to skin irritation [52]. It can be expected that 2 % citric acid will lower the described rate of adverse effect. The amount of urea on human hands after application of the new formulation is unknown. But urea has a positive effect on transepidermal water loss and on skin barrier function [53, 54].

In conclusion, this new formulation with a low alcohol content, citric acid and urea is capable of inactivating all enveloped and non-enveloped viruses as indicated in EN 14476 and DVV/RKI Guideline in quantitative suspension tests and inactivates MNV, AdV and vaccinia virus on stainless disk carriers. The formulation contributes a valuable addition to the hand disinfection portfolio. It is of course not possible to test the activity of a hand disinfectant against each virus. The test viruses as mentioned in European Norms or German Guidelines analogous to bactericide testing are representatives for the whole spectrum of relevant viruses. Therefore, the new formulation will not only inactivate the test viruses from the European Norm or German Guideline but is also covering the whole spectrum of all enveloped and all non-enveloped viruses being directly or indirectly transferred by human hands.