Comparative efficacy evaluation of disinfectants against severe acute respiratory syndrome coronavirus-2

Background Disinfection is one of the most effective ways to block the rapid transmission of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Due to the prolonged coronavirus disease 2019 (COVID-19) pandemic, disinfectants have become crucial to prevent person-to-person transmission and decontaminate hands, clothes, facilities and equipment. However, there is a lack of accurate information on the virucidal activity of commercial disinfectants. Aim To evaluate the virucidal efficacy of 72 commercially available disinfectants constituting 16 types of ingredients against SARS-CoV-2. Methods SARS-CoV-2 was tested with various concentrations of disinfectants at indicated exposure time points as recommended by the manufacturers. The 50% tissue culture infectious dose assay was used to calculate virus titre, and trypan blue staining and CCK-8 were used to assess cell viability after 3–5 days of SARS-CoV-2 infection. Findings This study found that disinfectants based on 83% ethanol, 60% propanol/ethanol, 0.00108–0.0011% sodium dichloroisocyanurate and 0.497% potassium peroxymonosulfate inactivated SARS-CoV-2 effectively and safely. Although disinfectants based on 0.05–0.4% benzalkonium chloride (BAC), 0.02–0.07% quaternary ammonium compound (QAC; 1:1), 0.4% BAC/didecyldimethylammonium chloride (DDAC), 0.28% benzethonium chloride concentrate/2-propanol, 0.0205–0.14% DDAC/polyhexamethylene biguanide hydrochloride (PHMB) and 0.5% hydrogen peroxide inactivated SARS-CoV-2 effectively, they exhibited cytotoxicity. Conversely, disinfectants based on 0.04–4% QAC (2:3), 0.00625% BAC/DDAC/PHMB, and 0.0205–0.14% and 0.0173% peracetic acid showed approximately 50% virucidal efficacy with no cytotoxicity. Citric acid (0.4%) did not inactivate SARS-CoV-2. Conclusion These results indicate that most commercially available disinfectants exert a disinfectant effect against SARS-CoV-2. However, re-evaluation of the effective concentration and exposure time of certain disinfectants is needed, especially citric acid and peracetic acid.

Comparative efficacy evaluation of disinfectants against severe acute respiratory syndrome coronavirus-2 Introduction Coronavirus disease 2019  is caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) [1]. The major symptoms of COVID-19 are fever, shortness of breath, coughing and atypical pneumonia within 2 weeks of transmission [2e7]. The World Health Organization (WHO) declared the SARS-CoV-2 outbreak a global pandemic on 11 March 2020, and it is still affected the public health community worldwide [8]. SARS-CoV-2 is transmitted directly through droplets and indirectly via contaminated surfaces [9e12]. Previous studies have shown that SARS-CoV-2 remains viable in aerosols for up to 3 h, and can survive for >3 days on wood, metal, glass, plastic, paper and clothes [13e15]. The persistence of SARS-CoV-2 on contaminated surfaces is one possible means of transmission, meaning that the use of disinfectants to inactivate SARS-CoV-2 on surfaces is a key control measure [15]. Chemical disinfectants including alcohol, peroxide, aldehyde and quaternary ammonium compounds (QACs) have been developed to inactivate or eliminate viruses and bacteria. Previous reports showed that disinfectants based on 70% ethanol or isopropanol are highly effective against coronaviruses. Furthermore, 80% ethanol combined with a 5% isopropanol mixture rapidly inactivates human immunodeficiency virus, hepatitis B virus and hepatitis C virus [16,17]. Several studies demonstrated that 62e71% ethanol, 0.5% hydrogen peroxide and 0.1% sodium hypochlorite could inactivate human coronaviruses effectively on contaminated inanimate surfaces [18,19]. Moreover, disinfectants comprising acids or alkalis, such as citric acid and sodium carbonate, could inactivate several viruses [20,21]. However, chemical disinfectants have certain drawbacks, such as being required at a high concentration for complete inactivation of viruses, and causing harm to public health and the environment [22]. This study evaluated the virucidal activity of commercially available disinfectants composed of different active ingredients against SARS-CoV-2, and presented guidelines for the use of appropriate disinfectants.

Viruses and cells
African green monkey kidney epithelial (Vero E6) cells were cultured in Dulbecco's modified Eagle medium (DMEM) (GIBCO, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco). SARS-CoV-2 (BetaCoV/Korea/KCDC03/ 2020, NCCP43326) was received from Korea Disease Control and Prevention Agency. The titration of SARS-CoV-2 in Vero E6 cells was calculated by 50% tissue culture infectious dose (TCID 50 ) assay using the ReedeMuench method, as described previously [23]. Briefly, Vero E6 cells were infected with a dose of SARS-CoV-2 (100 TCID 50 ), and after 3e5 days of infection, the cytopathic effect was monitored by observing the morphological changes under a microscope (Leica Microsystems, Wetzlar, Germany). All SARS-CoV-2 infection-related experiments were conducted in a biosafety level 3 laboratory using personal protective equipment, according to the biosafety protocol of Korea Research Institute of Jeonbuk National University.

Chemical disinfectants
Seventy-two disinfectants were tested for virucidal activity against SARS-CoV-2 under the conditions recommended by the manufacturers, including working concentrations and exposure times (Table I and Table S1, see online supplementary material). Five types of wipes and 20 types of sprays, including ethanol, propanol and benzalkonium chloride (BAC) compounds, were tested without dilution. In total, two types of tablets, including sodium dichloroisocyanurate, two powders, including potassium peroxymonosulfate, and 43 liquids, including peracetic acid, hydrogen peroxide, citric acid, ethanol, propanol, BAC compounds and QACs, were diluted in ultrapure deionized water (Biosolution, Seoul, South Korea) and tested. The disinfectant effectiveness of QAC (1:1), sodium hypochlorite, ethanol and citric acid against SARS-CoV-2 was evaluated in organic and inorganic solutions. The inorganic solution contained 0.305 g of CaCl 2 and 0.139 g of MgCl 2 6H 2 O in 1 l of distilled water, and the organic solution contained 5% FBS in the inorganic solution.

SARS-CoV-2 susceptibility against the disinfectants
The disinfectants were diluted in ultrapure water and mixed with SARS-CoV-2 according to the product recommendations (Table S1, see online supplementary material). The mixtures were incubated for 3e15 min depending on the product, and neutralized with DMEM containing 10% FBS [24e31]. Next, the mixtures were serially diluted to 10 À1 e10 À7 with serum-free DMEM and added to Vero E6 cells (2 Â 10 4 cells/well). After 1 h of treatment, the mixture was removed from the cells and washed twice with serum-free (DMEM) (GIBCO, Grand Island, NY, USA) and DMEM containing 2% FBS filled in each well. The SARS-CoV-2 titre was calculated by the Reed-Muench method based on the cell death at 3-5 days post-infection. The percentage of SARS-CoV-2 reduction rate was quantified by the SARS-CoV-2 infectivity with disinfectants/without disinfectants, and the percentage variance was calculated by the standard deviation (SD).

Evaluation of disinfectant cytotoxicity
The disinfectants were diluted with ultrapure deionized water, mixed in a 1:1 ratio with DMEM without serum, and incubated at recommended time points (Table S2, see online supplementary material). Following incubation, they were neutralized using DMEM containing 10% FBS, and the mixture was serially diluted with DMEM without serum (10 À1 and 10 À2 ). Vero E6 cells (2 x 10 4 cells/well) were treated with diluents and incubated for 3 days, then harvested by trypsinization, stained with trypan blue, and the numbers of total and living cells were counted using a Luna-II auto cell counter (Logos Biosystems, Gyeonggi-do, South Korea). These experiments were performed in triplicate. In addition, the effect of disinfectants on cell viability was measured using the economical cell counting kit 8 (CCK-8) (Abbkine, Wuhan, China). Cell viability was indicated as mean AE SD of the triplicate samples.

Virucidal efficacy of disinfectants against SARS-CoV-2 between organic and inorganic conditions
In order to evaluate the virucidal efficacy of the disinfectants against SARS-CoV-2 in environmental conditions with low or high levels of organic matter, several concentrations of a disinfectant were tested in inorganic and organic conditions because the survival rate of the virus was higher in organic conditions than in inorganic conditions [32,33]. SARS-CoV-2 activity was decreased up to 4 log 10 by 0.000071% QACs in inorganic conditions without cytotoxicity. However, 0.001% QAC was required for SARS-CoV-2 inactivation of up to 4 log 10 in organic conditions, but this concentration was highly toxic (Table III and Table S3, see online supplementary material). In inorganic conditions, 0.016% sodium hypochlorite was required for the complete inactivation of SARS-CoV-2, but in organic conditions, 0.2% sodium hypochlorite was required (Table III  and Table S4, see online supplementary material). Conversely, 50% ethanol and 2% citric acid exhibited similar virucidal efficacy in organic and inorganic conditions (Table III and  Table S5, see online supplementary material). These results suggest that the virucidal efficacy of disinfectants can be

Discussion
The main route of infection for SARS-CoV-2 is transmission through respiratory droplets, but contact with contaminated surfaces, such as plastic, glass, paper and metal, may also be important. Several recent studies have reported that SARS-CoV-2 can survive for up to 3 days on various inanimate surfaces [13,15]. WHO and the Centers for Disease Control and Prevention recommend the use of safe and effective disinfectants when cleaning and disinfecting surfaces. Various ingredients, known as biocidal agents, have been applied widely in commercial disinfectants. Among them, ethanol is a traditional ingredient for cleaning surfaces or eliminating microbes. Disinfectants comprising 62e71% ethanol can reportedly inactive coronaviruses efficiently on surfaces after 1 min of exposure [19,34]. The present study showed that all alcohol-based disinfectants inactivated SARS-CoV-2 completely without causing cytotoxicity (Tables I and II). As alcohol-based disinfectants decompose to oxygen, water and acetic acid, these oxidizing agents are recognized as safe disinfectants. Unlike alcohol-based disinfectants, hydrogen peroxide triggers cytotoxicity in various cell types [35,36]. Previous studies reported that cell damage occurs following exposure to >100 mM hydrogen peroxide [37]. The present study showed complete SARS-CoV-2 inactivation by a high concentration of hydrogen peroxide (w150 mM) after 10 min of exposure; however, this exhibited cytotoxicity. (Tables I  and II). In the cytotoxicity assessment, 1% of peracetic acid induced cell death, predominantly by necrosis [38]. In contrast, cell damage could not be observed in cells treated with 0.0173% peracetic acid during SARS-CoV-2 inactivation (6.8e56.28%) after 10 min of exposure (Table II). An oxidizing agent, potassium peroxymonosulfate, and the chemical compound sodium dichloroisocyanurate have been used to disinfect water in the food industry [39,40]. Both products comprising 0.497% potassium peroxymonosulfate or 0.00108e0.0011% sodium dichloroisocyanurate inactivated SARS-CoV-2 sufficiently without triggering any cell damage (Tables I and II). Citric acid is an approved disinfectant against foot-and-mouth disease virus in the Republic of Korea [28]. Citric acid reportedly exhibits low acute toxicity and no genotoxicity due to rapid degradation because of high environmental mobility [41]. Despite the lack of cytotoxicity, a commercial product comprising 0.4% citric acid did not show any significant effect, while 2.6e10% citric acid inactivated SARS-CoV-2 completely without cytotoxicity (Table S5, see online supplementary  material). QACs, comprised as single compounds or mixtures, are widely used as biocidal and virucidal agents, applied in commercial products including personal hygiene, domestic and cosmetic products [42,43]. However, the toxicity of QACs to humans and the environment has been hotly debated [44,45]. In addition, various mixed products, including or excluding BAC, showed differences in cell viability and SARS-CoV-2 inactivation according to the mixture of ingredients (Table II). The differing effects of mixed products may be caused by the generation of toxic substances during the manufacturing process, or by high concentrations and exposure times.
All experiments determined the virucidal effect of various commercial disinfectants against SARS-CoV-2 under inorganic conditions. The efficacy of a disinfectant against viruses could change under organic experimental conditions [28,46]. In the present study, the effective concentrations of four representative chemical compounds were evaluated as disinfectant components in organic and inorganic conditions. QACs and sodium hypochlorite required a 10-fold higher concentration to reduce SARS-CoV-2 activity by up to 4 log 10 in organic conditions compared with inorganic conditions. However, ethanol and citric acid exhibited similar virucidal activity at the same concentration in organic and inorganic conditions. Organic matter could interfere with virucidal activity, depending on the ingredients in the disinfectant. The virucidal activities of all disinfectants were assessed by an ASTM E1052-20, known as the suspension test method [47].
The surface test method supported incorrect results due to the low recovery rate of the reacting solution containing SARS-CoV-2 in preliminary tests (data not shown). In conclusion, the efficacy of various commercially formulated disinfectants against SARS-CoV-2, and their cytotoxic effects, were evaluated. The results indicate that currently available ingredients can be used to prevent SARS-CoV-2 infection. In addition, these results will be available as essential data in the disinfection guidelines for use by disinfectant ingredients.