Inactivation kinetics of benzalkonium chloride and ethanol-based hand sanitizers against a betacoronavirus and an alphacoronavirus

Summary

Background

Hand hygiene is critical to lower the potential for the spread of SARS-CoV-2 and other infectious agents by direct contact. When running water and soap are not available for hand hygiene, ethanol-based hand sanitizers are currently the recommended standard of care [1–3]. Though recently published data showed comparable in vitro effectiveness of benzalkonium chloride (BAK)-based and ethanol-based hand sanitizers against SARS-CoV-2 virus, a paucity of peer-reviewed data on the effectiveness of these formulations against other types of infective coronaviruses remains. This work assessed human coronavirus HCoV-229E (genus Alphacoronavirus) concurrently with SARS-CoV-2, Isolate USA-WA1/2020 (genus Betacoronavirus) to fill this gap.

Methods

The test was conducted according to EN14476:2013-A2:2019 [EN14476] Quantitative Suspension Test for the Evaluation of Virucidal Activity in the Medical Area [4]. Two BAK-based hand sanitizers, five ethanol-based hand sanitizers, and an 80% ethanol reference formulation were tested for antiviral activity against SARS-CoV-2 and HCoV-229E at 15- and 30- second contact times.

Results

Both SARS-CoV-2 and HCoV-229E were reduced by greater than 4.00-log₁₀ within 15 seconds of contact. Virus decay constants (k) following first-order kinetics were similar for BAK and ethanol-based formulations against both test viruses. The SARS-CoV-2 results reported herein mirrored previous data reported by Herdt et al. (2021).

Conclusion

BAK and ethanol hand sanitizer formulations inactivate SARS-CoV-2 and HCoV-229E at similar rates. This data supports previously published effectiveness data for both chemistries and indicates that additional coronavirus strains and variants would demonstrate similar inactivation trends.

Introduction

Despite the availability of effective vaccines, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants remain a substantial threat to public health and economies worldwide. More than 766 million infections and over 6.9 million deaths have been attributed to COVID-19 at the time of this writing [5]. The distribution of vaccines will continue to contribute to lower rates of illness and death, and it has been proposed that SARS-CoV-2 variants circulating endemically within human populations will contribute substantially to population immunity [6]. Nevertheless, the ability to respond effectively to currently circulating and emerging viruses that demonstrate pandemic-threat potential remains of paramount concern. The Centers for Disease Control and Prevention [CDC] and the World Health Organization [WHO] continue to recommend the use of ethanol-based hand sanitizers for personal hygiene and infection control during the COVID-19 pandemic [1,7].

The vast majority of hand sanitizer available for use by both healthcare professionals and the general public include either ethanol or BAK as their active ingredient. Mechanistically, both ingredients are categorized as denaturants by Block (2001) [22] though the specific targets for deactivation within a viral particle may be variable, for example the surfactant nature of BAK can target the lipid membrane surrounding an enveloped virus where Ethanol is believed to target the protein based viral capsid. A thorough review of hand hygiene ingredients by Golin et al. (2020) [23] provides a more detailed review of mechanisms of action for these active ingredients particularly around Coronavirus.

Evidence of the in vitro effectiveness of ethanol-based hand sanitizers against SARS-CoV-2 and previously-circulating strains of pathogenic coronaviruses including the 2003 SARS-CoV pandemic strain and the Middle Eastern Respiratory Syndrome coronavirus (MERS-CoV), was previously reported by Kratzel et al. (2020) [2] and Siddharta et al. (2017) [8]. Chin et al. (2020) [9], Ogilvie et al. (2021) [10], and Ijaz et al. (2020) [11] subsequently demonstrated the effectiveness of BAK to inactivate SARS-CoV-2, and a review published by Schrank et al. (2020) [3] urges further research to assess the efficacy of BAK against SARS-CoV-2. Newly published work by Herdt et al. (2021) [12] further supported the in vitro effectiveness of commercially-available BAK hand sanitizer formulations and an 80% ethanol-based WHO hand sanitizer against SARS-CoV-2.

The research reported herein addresses certain limitations outlined by Herdt et al. (2021) [12] including testing of the hand sanitizer products at two independent laboratories rather than conducting all tests at a single lab, and evaluating products multiple times on the same test day compared to testing the product independently over several days. In the current study, each product was tested in triplicate on a single day using the European Norm Standard 14476 methodology at a single laboratory. Virus decay rates (k) were then calculated to compare the inactivation kinetics of SARS-CoV-2 and HCoV-229E given exposure in suspension to each test formulation. This parallel testing in a single lab provides an additional methodological basis for evaluating effectiveness.

The purpose of the current investigation was to provide valuable new data assessing the antiviral efficacy of BAK-based and ethanol-based hand formulations at multiple contact times, concentrations and form factors against a strain from genus Betacoronavirus (SARS-CoV-2, Isolate USA-WA1/2020) and one from genus Alphacoronavirus (HCoV-229E) as examples of the subfamilies of Orthocoronavirinae with the seven known identified human infective coronaviruses, including the causative agents of the 2003 SARS-CoV pandemic and Middle Eastern Respiratory Syndrome (MERS) which was first reported in 2012 [13]. Formulations that demonstrate efficacy against both alphacoronaviruses and betacoronaviruses may provide insight as to their potential antiviral activity against other species and strains of coronaviruses, including future variants of SARS-CoV-2 as suggested by Ijaz et al., in 2020 and 2021 [19,20]. This work expands upon the research reported by Herdt et al. (2021) [12], and includes a new kinetic element and rate constant to potentially enable comparisons between this and other in vitro antiviral data sets.

Methods

Human coronavirus 229E and the two cell lines used in the study, MRC-5 and Vero E6, were procured from the American Type Culture Collection (ATCC; Manassas, VA). SARS-CoV-2 Isolate USA-WA1/2020 was deposited by the Centers for Disease Control and Prevention and obtained through BEI Resources, The National Institute of Allergies and Infectious Disease (NIAID), and The National Institutes of Health (NIH). All laboratory work involving SARS-CoV-2 was conducted within a Biosafety Level 3 laboratory at the University of Arizona campus. Stocks of HCoV-229E (ATCC VR-740) and SARS-CoV-2 (BEI NR-52281) were prepared in accordance with the coronavirus propagation protocol described by Gundy et al. [14] using the MRC-5 (ATCC CCL-171) and Vero E6 (ATCC CRL-1586) cell lines, respectively. Briefly, infected cell monolayers underwent three freeze-thaws followed by a polyethylene glycol (PEG) precipitation [12% (w/v) PEG (8,000 g/mol) with 0.5M NaCl] with stirring overnight at 4 °C. The suspension was centrifuged (10,000 x g for 60 minutes) and the virus pellet resuspended in 0.01M PBS to 5% of the original suspension volume. Aliquots were stored at -80 °C until used in the study. Stocks and test suspension titers (including controls) for HCoV-229E and SARS-CoV-2 were enumerated using their respective host cell lines seeded into multi-well trays, and the tissue culture infective dose at the 50% endpoint (TCID₅₀) values were calculated using the Spearman-Karber algorithm [15]. Ethics approval was not required as this study does not include human participants.

Eight hand sanitizer solutions were evaluated during the study: Hand Sanitizer Product A [BAK-based Ecolab Foaming Hand Sanitizer, Ecolab, St. Paul, USA]; Hand Sanitizer Product B [BAK-based Ecolab Concentrated Hand Sanitizer, Ecolab, St Paul, USA], and six Ecolab provided commercial ethanol-based hand sanitizers including Hand Sanitizer Product C [80% ethanol WHO hand sanitizer formulation] [16] and Hand Sanitizers D through H (Table I) representing a range of ethanol concentrations and product form factors.

Table I.

Name, form factor, active ingredient and concentrations for products used in study

Name	Form factor	Active ingredient	Labelled active concentration
Product A	Ecolab Foaming BAK Hand Sanitizer	Benzalkonium chloride (BAK)	0.1% by weight
Product B	Ecolab Concentrated BAK Hand Sanitizer	Benzalkonium chloride (BAK)	0.089% by weight at ready to use dilution
Product C	WHO 80% Ethanol	Water Thin Alcohol	80% by volume
Product D	Ecolab Ethanol Gel #1	Ethanol Gel	70% by weight
Product E	Ecolab Ethanol Gel #2	Ethanol Gel	70% by weight
Product F	Ecolab Foaming Alcohol	Foaming Alcohol	70% by weight
Product G	Ecolab Foaming Alcohol	Foaming Alcohol	62% by weight
Product H	Ecolab Ethanol Gel	Ethanol Gel	60% by volume

The CAS identity for the BAK in both Product A and Product B is 68424-85-1. For Products C through H, the ethanol CAS identity is 64-17-5. Hand Sanitizer Product B is sold commercially as a concentrate that is diluted using an automated dilution system at a ratio of 1:10 using water available on site at the time of use, generally not exceeding 10 grain per gallon [gpg] hardness, producing a ready-to-use solution of hand sanitizer containing 0.089% BAK. For the current study, a 1:10 dilution in 10 grain per gallon AOAC synthetic hard water (171 ppm) was made. All other samples were provided as ready-to-use finished product. Products A through C are the same as those tested in Herdt et al. (2021) [12], providing an additional replicate of those test conditions.

All eight products were tested for virucidal activity against SARS-CoV-2 and HCoV-229E using the quantitative suspension test protocol as described in the European Standard EN14476:2013-A2:2019 [EN14476] Quantitative Suspension Test for the Evaluation of Virucidal Activity in the Medical Area [4], with modifications as appropriate. An aliquot of virus stock (0.1 mL) was combined with 0.1 mL of 0.3 g/L bovine serum albumin simulating “clean conditions,” and then exposed to 0.8 mL of Hand Sanitizer Products A through H (Table I) for 15 and 30 seconds at 20–21^oC. Following exposure, 0.1 ml of each test mixture was removed and neutralized using Letheen Broth Base (0.9 ml) followed immediately by passage of the neutralized suspension (1 ml) through two Sephadex G-10 gel filter columns in sequence. Control suspensions were concurrently assessed and processed as described at the longest contact time of 30 seconds, with 0.8 ml of 0% FBS MEM employed in lieu of the test formulation(s).

The final filtrate volumes underwent serial dilutions (1:10) to enumerate surviving viruses. Aliquots of each dilution (0.1 ml) were transferred in replicates of six to microtiter plates containing Vero E6 or MRC-5 cells. Neutralization of the test formulations was also validated, data available Herdt (2023) on Mendeley data [21], and the formaldehyde control [1.4% (v/v)] was evaluated as specified in reference method EN14476 to assess antiviral efficacy against HCoV-229E and SARS-CoV-2 for the study contact times of 15 and 30 seconds. The multi-well plates were incubated at 36 ± 1^oC in a 5 ± 1% CO₂ atmosphere and monitored regularly for the development of viral cytopathogenic effects (CPE) and product-specific cytotoxicity using inverted light microscopy. Virus titers were determined using the TCID₅₀ (tissue culture infective dose at the 50% endpoint) technique and calculated using the Spearman-Karber method followed by log₁₀ transformation. Inactivation kinetics were assessed and virus decay rates calculated according to first-order kinetics as described by Hiatt [17] and shown in Equation 1, where N₀ is the mean virus concentration (TCID₅₀ per mL) at Time Zero, N = the mean virus concentration (TCID₅₀ per mL) after the contact time of 15 seconds (t). The values for N₀ and N per virus can be found in the Supplemental Information.

$$\mathit{log}10\left(\frac{N}{No}\right)=-kt$$ (Eq. 1)

Results

Antiviral effectiveness testing was performed according to EN14476 against SARS-CoV-2 and HCoV-229E over the course of two days, and are identified herein as Series 1 and Series 2. Series 1 testing against SARS CoV-2 began with a mean log₁₀ viral count of 6.2 per ml as measured in the controls, and testing was conducted with BAK-based Products A and B and ethanol-based Product H. Series 1 testing against HCoV-229E was initiated with a mean log₁₀ viral count of 6.6 per ml as measured in the controls, and was conducted using BAK-based Products A and B and ethanol-based Products D, E, and H.

Series 2 testing against SARS-CoV-2 began with a mean log₁₀ challenge of 5.8 per ml to assess the effectiveness of ethanol-based Products C, D, E, F, and G. Series 2 testing against HCoV-229E began with a mean log₁₀ count of 6.7 per ml and was run using ethanol-based Products C, F, and G.

Products A through H inactivated infectious SARS-CoV-2 by ≥ 4.0-log₁₀ within 15 seconds of exposure (Figure 1). Reductions of SARS-CoV-2 measured ≥ 4.3-log₁₀ by the 30-second contact time and complete inactivation was observed (Figure 1). Antiviral effectiveness against SARS-CoV-2 was similar between BAK-based products and ethanol-based formulations, with reductions ranging from ≥ 4.4-log₁₀ to ≥ 4.7-log₁₀ and ≥ 4.0-log₁₀ to ≥ 4.7-log₁₀, respectively. Figure 1 shows the in vitro effectiveness of BAK-based and ethanol-based hand sanitizers or rubs against SARS-CoV-2 in comparison with a no treatment baseline where 0% FBS MEM was employed in lieu of the test formulations.

Figure 1.

In vitro effectiveness of BAK- and Ethanol-based hand sanitizers (Table I) against SARS CoV-2 at contact times of 15 seconds and 30 seconds. (Series 1 mean control titer = 6.2 log 10; Series 2 mean control titer = 5.8 log10; Limit of detection = ≤ 1.50 log10; and ≤ = bold outline around bar indicates complete inactivation during the tested timeframe).

Products A through H reduced levels of viable human coronavirus 229E by greater than 4-log₁₀ following a 15 second contact time, and the virus was inactivated to levels below the limit of detection for all formulations within 30 seconds (Figure 1). Cytotoxic effects observed on MRC-5 cells for Product B increased the limit of detection (≤2.5 log₁₀) compared to that observed for Products A and C through H (≤1.5 log₁₀); however, no viral cytopathogenic effects (CPE) were observed beyond the levels of toxicity after the 15- and 30-second contact times, indicating complete inactivation. Inactivation of HCoV-229E (Figure 2) was consistent across products and contact times similar to SARS-CoV-2. Antiviral effectiveness was similar between the tested products, with log₁₀ reductions ranging from 4.1 to 5.1 for the BAK-based products, and 5.1 to 5.2 for the ethanol-based products over the course of the 15- and 30-second contact times.

Figure 2.

In vitro effectiveness of BAK- and Ethanol-based hand sanitizers (Table I) against HCoV-229E at contact times of 15 seconds and 30 seconds. (Series 1 mean control titer = 6.7 log 10; Series 2 mean control titer = 6.6 log10; Limit of detection for Products A and C – H = ≤ 1.50 log10; a Limit of detection for Product B = ≤ 2.50 log10; and ≤ = bold outline around bar indicates complete inactivation during the tested timeframe).

Figure 3 shows the comparative effectiveness of the BAK and ethanol actives against SARS-CoV-2, a betacoronavirus, and the alphacoronavirus HCoV-229E, with both genera exhibiting similar inactivation trends. The BAK and ethanol hand sanitizers each achieved the 4-log₁₀ reduction success criterion specified in the EN 14476 standard method for both HCoV-229E and SARS-CoV-2 within the contact time of 15 seconds. The log₁₀ reductions measured coupled with the similarity of the calculated virus decay rates (Table II) indicate the high degree of kinetic consistency of BAK and ethanol-based hand treatments against agents from the genera Alphacoronavirus (HCoV-229E) and Betacoronavirus (SARS-CoV-2), with complete inactivation observed.

Figure 3.

Comparison of in vitro effectiveness of BAK-based hand sanitizers (Products A and B) and 80% ethanol-based sanitizer (Product H) against SARS-CoV-2 and HCoV-229E (Series 1 starting titres); Limit of detection for SARS-CoV2 = ≤ 1.50 log 10; Limit of detection for HCoV2-229E = ≤ 1.50 log10 Products A and H and = ≤ 2.50 log10 for Product B; and ≤ = bold outline around bar indicates complete inactivation during the tested timeframe).

Table II.

First-order decay rate constants for SARS-CoV-2 and HCoV-229E given exposure to Test Formulations A through H for 15 seconds

Test formulation	Active ingredient Concentration	Virus decay rate (k·sec−1)		Decay rate difference
		SARS-CoV-2	HCoV-229E
A	BAK (0.1% wt)	0.30	0.33	0.03
B	BAK (0.089% wt)	0.30	0.28	0.02
C	Water Thin Alcohol (80% vol)	0.29	0.35	0.06
D	Ethanol Gel (70% wt)	0.29	0.34	0.05
E	Ethanol Gel (70% wt)	0.29	0.34	0.05
F	Foaming Alcohol (70% wt)	0.28	0.35	0.07
G	Foaming Alcohol (62% wt)	0.27	0.35	0.08
H	Ethanol Gel (60% vol)	0.32	0.34	0.06

Discussion and conclusions

As the COVID-19 pandemic continues to present a global challenge to public health, effective hand hygiene measures remain critical. The work reported here demonstrates in vitro effectiveness measuring ≥ 4-log₁₀ of two BAK-based and six ethanol-based hand sanitizer formulations within 15 seconds against SARS CoV-2, a betacoronavirus, and HCoV-229E, an alphacoronavirus. Data is also provided for eight unique hand rub and sanitizer formulations, including the WHO-recommended 80% ethanol-based chemistry at two short-range contact times, demonstrating effectiveness across a breadth of commercial product finished form factors.

This paper presents some of the first side-by-side test data documenting the in vitro effectiveness of BAK and ethanol-based hand sanitizers against SARS CoV-2 and HCoV-229E, and thereby provides valuable new data as SARS-CoV-2 continues to mutate and evolve. The effectiveness of these two categories of active ingredients against the genera Alphacoronavirus and Betacoronavirus have been demonstrated against other public health threats including the 2003 pandemic strain of SARS-CoV and MERS-CoV [13], and may also provide for protection against future emerging strains of coronavirus with pandemic potential. Inactivation kinetics were similar and consistent for SARS CoV-2 and HCoV-229E across the actives and formulations evaluated, with each reducing infectious virus levels by several log₁₀ orders of magnitude and at similar rates of decay within 15 seconds.

Products A and B were BAK-based hand sanitizers and Product C, the WHO-recommended 80% ethanol-based product, were the same as those evaluated in Herdt et al. (2021) [12]. The log₁₀ inactivation values were consistent between the Herdt et al. (2021) [12] work and the test results reported here. The limitations identified in Herdt et al. [12] are addressed in this study with triplicate testing of each product on a single day using the same EN 14476 standard methodology at a single laboratory, thus providing an additional methodological basis for evaluating effectiveness.

In conclusion, this study confirms the findings of Herdt et al. (2021) [12], Chin et al. (2020) [7], Ogilvie et al. (2020) [10], and Ijaz et al. (2020) [11], and Saknimit et al. (1988) [18] that commercial BAK hand hygiene formulations and ethanol formulations inactivate coronaviruses and demonstrates similar inactivation kinetics for SARS-CoV-2 and HCoV-229E.

Authorship statement

Brandon L. Herdt: Conceptualization, Methodology, Formal Analysis, Resources, Data Curation, Writing, – original draft preparation, Writing - review and editing, Visualization, Supervision, Funding acquisition; Luisa A. Ikner: Methodology, Validation, Investigation, Resources, Data Curation, Writing - review and editing, Project Administration.

Conflict of Interest Statement

Ecolab Inc. is a manufacturer and seller of the commercial hand sanitizers based on benzalkonium chloride [BAK] and Ethanol included in this manuscript.

Funding Statement

Ecolab Inc. provided financial compensation to the University of Arizona Water and Energy Sustainable Technology Center to conduct this research and to Ramboll for providing technical and editorial assistance.