Comparison of the SARS-CoV-2-inactivating activities of the differently manufactured hypochlorous acid water products with various pH

Since the first case of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, was reported in China in December 2019, a staggering number of cases have been reported globally. As of 16 March 2021, the number of infections and deaths is still rising (World Health Organization 2020), and the immediate expansion of infection control measures is an urgent, global issue. Inactivating SARS-CoV-2 in the environment and on people's hands is a key approach to prevent the spread of infection.

Like the other enveloped viruses, SARS-CoV-2 is sensitive to alcohol and can, therefore, be rapidly inactivated by alcohol-based formulations (Kratzel et al. 2020). Various disinfectants such as 10% sodium hypochlorite, 20% potassium peroxymonosulfate, 4% formaldehyde, 50% guanidine thiocyanate, and amphoteric surfactants can also inactivate SARS-CoV-2 in 1 min (Chan et al. 2020), while 7.5% povidone–iodine, 0.05% chloroxylenol, 0.05% chlorhexidine, and 0.1% benzalkonium chloride can inactivate SARS-CoV-2 in 5 min (Chin et al. 2020). Although alcohol-based disinfectants are useful because they do not induce adverse reactions in relatively large numbers of people, and they do not cause environmental pollution, these products were in short supply in the medical field when the COVID-19 pandemic occurred. On the other hand, the non-alcohol-based disinfectants mentioned above have disadvantageous characteristics, such as being harmful to people and the environment and being residual on exposed surfaces, making them unfavorable as surface disinfectants. Hence, there is a global demand for anti-SARS-CoV-2 disinfectants that are suitable for both people and the environment.

Acidic water containing HClO has strong bactericidal and virucidal activities. Hypochlorous acid water (HAW) is acidic water that contains HClO, ClO⁻, and Cl₂ (Wang et al. 2007). Acidic electrolyzed water (AEW), a type of HAW, has a broad spectrum of activities against influenza A virus, norovirus, hepatitis B virus, hepatitis C virus, human immunodeficiency virus, herpesvirus, hepadnavirus, and foot-and-mouth disease virus (Tanaka et al. 1999; Morita et al. 2000; Tagawa et al. 2000; Sakurai et al. 2003; Geun et al. 2007; Huang et al. 2008; Tamaki et al. 2014; Hakim et al. 2015; Bui et al. 2017) and also exhibits bactericidal activities (Huang et al. 2008). Furthermore, we have recently reported that a sufficient volume of AEW at pH 2.5 and a residual chlorine concentration (RCC) of more than 65 ppm showed potent virucidal activity within 1 min against SARS-CoV-2 (Takeda et al. 2020).

When HAW reacts with organic substances, the RC is consumed, leaving just water; therefore, HAW does not pollute the environment and leaves no residue (Huang et al. 2008). HAW is also safe for human use. A clinical study has shown that intraperitoneal lavage and wound washing using AEW with an RCC of ∼40–60 ppm did not cause any adverse effects (Kubota et al. 2015). In addition, animal safety and toxicity tests showed that 0.01–0.1% (w/v) HClO, generated by mixing of hydrochloric acid and sodium hypochlorite, did not irritate the eye and skin or exhibit systemic toxicity (Wang et al. 2007). Thus, HAW can be a useful anti-SARS-CoV-2 disinfectant. In addition, the United States Environmental Protection Agency has recommended using several HAW products for SARS-CoV-2 inactivation (United States Environmental Protection Agency 2020).

There are several methods of manufacturing HAW. This includes electrolyzing hypochloric acid or sodium chloride solutions, mixing acid and sodium hypochlorite solutions, running a sodium hypochloride solution through ion exchange, and dissolving sodium dichloroisocyanurate (NaDCC) in water (Wang et al. 2007; Huang et al. 2008; Makino & Terada 2011; Sen 2016). These different manufacturing methods have produced a large number of HAW products with various pH values that are now marketed in Japan. However, there is little information on the comparative virucidal activities of these HAWs.

In the present study, we compared the SARS-CoV-2-inactivating activities and chemical properties of various HAW products.

All of the HAW products tested in this study were provided by the National Institute of Technology and Evaluation (NITE) for blind testing. The name of the companies providing the HAW products remains confidential. Table 1 lists the information on the HAW products tested.

As per the definition by the Japanese Ministry of Health, Labor, and Welfare, AEW samples were divided into the weakly AEW (WAEW; pH 2.7–5.0) and slightly AEW (SAEW; pH 5.0–6.5). HAW samples with an RCC of ∼50 and 30 ppm were prepared by diluting the test samples with ultrapure water (UPW). The RCC, pH, and oxidation-reduction potential (ORP) were measured using AQUAB AQ-202 (Sibata Scientific Technology Ltd, Tokyo, Japan), Compact pH meter (Horiba Co., Ltd, Kyoto, Japan), and Water Proof ORP meter (Custom Co., Tokyo, Japan), respectively. For several experiments, HAW samples with an RCC of ∼50 ppm were stored at 22 °C in the dark with the tube lid closed or loosened for 21 days.

The JPN/TY/WK-521 strain of SARS-CoV-2 was obtained from the National Institute of Infectious Diseases (Tokyo, Japan). VeroE6/TMPRSS2 cells (Nao et al. 2019) were obtained from the Japanese Collection of Research Bioresources (Cell No.: JCRB1819, Osaka, Japan). SARS-CoV-2-infected VeroE6/TMPRSS2 cells were cultured in virus growth medium (VGM) containing Dulbecco's modified Eagle's medium (Nissui Pharmaceutical Co., Ltd, Tokyo, Japan) and 1% fetal bovine serum (FBS), 2 mM L-glutamine (Fujifilm Wako Pure Chemical Co., Ltd, Osaka, Japan), 100 μg/mL of kanamycin (Meiji Seika Pharma Co., Ltd, Tokyo, Japan), 2 μg/mL amphotericin B (Bristol-Myers Squibb Co., New York, NY), and 10 mM of sodium thiosulfate, a chlorine neutralizer.

VGM supernatant containing SARS-CoV-2, of which the viral titer of 7.25 log₁₀ 50% tissue culture infective dose (TCID₅₀)/mL, was mixed with HAW samples in virus-to-HAW solution ratio of 1:9 or 1:19. Thus, the virus was exposed to a smaller volume of HAW in the 1:9 mixture than that in the 1:19 mixture. In the control group, the virus solution was mixed with UPW. The RCC of test HAW products was immediately measured before mixing with virus supernatant.

For AEW samples and Non-EW type HAW samples, the mixture of virus and HAW was placed at 22 °C for 20 s or 5 min. Then, 20 μL of the mixture was added into 180 μL of VGM in which VeroE6/TMPRSS2 cells were cultured.

For NaDCC, the mixture of virus and NaDCC was incubated at 22 °C for 20 s and then diluted 1:7 with phosphate-buffered saline supplemented with 5% FBS, 2 mM L-glutamine, 100 μg/mL of kanamycin, 2 μg/mL amphotericin B, 10 mM of sodium thiosulfate, and 50% Diaion^® HP-20 resin (Sigma-Aldrich, Inc., Saint Louis, MO). After 5 min incubation at 22 °C to remove the NaDCC cytotoxicity with Diaion^® HP-20 resin, 100 μL of the supernatant of the SARS-CoV-2 and NaDCC mixture was added into 100 μL of VGM in which VeroE6/TMPRSS2 cells were cultured.

Then, a tenfold serial dilution of the added mixture was performed on VeroE6/TMPRSS2 cells. After a 3-day incubation at 37 °C, the viral titer (log₁₀ TCID₅₀/mL) was calculated with the Behrens–Kärber method (Kärber 1931) to determine the virucidal effect of each HAW sample.

The Student's t-test was performed to analyze the statistical significance of the differences between the control and HAW samples; p values of less than 0.05 were considered significantly different. The number of biological replicates was 3–4 per group. The degree of freedom based on biological replicates was 4–6. All the experiments were repeated two times to confirm the reproducibility.

The inactivating activities of various HAW samples at a pH of 2.8–6.2 and an RCC (before the mixing with virus supernatant) of 51–56 ppm were tested against SARS-CoV-2. These HAW samples were mixed with virus supernatants at a virus-to-HAW sample ratio of 1:19. All the HAW samples demonstrated potent virucidal activities by causing a decrease in viral titers of more than 4.00 log₁₀ TCID₅₀/mL in 20 s reaction time; the virucidal activity of the NaDCC solution seemed to be slightly lower than those of other HAW samples (Figure 1). Thus, the different manufactured HAW samples at various pH values all achieved a virus inactivation of more than 99.99%.

The viral titer of each mixture was measured after a reaction time of 20 s. Results are indicated as mean ± SD. A Student's t-test was performed to analyze the significant difference between the UPW group and each HAW group; **p < 0.01; ***p < 0.001.

Next, Non-EW type-I HAW at pH 5.6 or SAEW at pH 5.0–5.4 was mixed with virus supernatants at a virus-to-HAW sample ratio of 1:9, and thus, the virus was exposed to a smaller volume of the Non-EW type-I HAW or SAEW. At an HAW sample RCC of ∼50 ppm and reaction time of 20 s, the virucidal activities of Non-EW type-I HAW and SAEW at the virus-to-HAW 1:9 ratio were weaker than those at the 1:19 ratio (Figures 1 and 2(a)). In addition, even after 5 min reaction time, the viral titers were not below the detection limit at the 1:9 ratio (Figure 2(a)). Moreover, when the RCC was ∼30 ppm, the reduction in viral titers in 5 min reaction time was less than 2.00 log₁₀ TCID₅₀/mL in both Non-EW type-I HAW and SEWA groups (Figure 2(b)).

The viral titer of each mixture was measured after a reaction time of 20 s and 5 min (Figure 2(a) and 2(b)). Results are indicated as mean ± SD. A Student's t-test was performed to analyze the significant difference between the UPW group and each HAW group; *p < 0.05; **p < 0.01; ***p < 0.001.

The changes in the RCC, pH, and ORP of various HAW samples were evaluated over time. The level of RCC slowly reduced in the slightly acidic HAW samples (pH 5.0–6.4). At day 21 under the loosened lid condition, the RCC level remained higher than 30 ppm, whereas in the open-lid condition, the RCC of WAEW (pH ∼ 3.0) decreased to 0 ppm on day 7. However, in the closed-lid condition, there was less reduction of RCC in WAEW, and the RCC level remained at 13 ppm after 21 days in storage (Figure 3(a), left). After 21 days of storage, the pH did not change dramatically in any of the HAW samples with a pH of ∼3.0 or 5.0–6.4 (Figure 3(a), middle). Simultaneously, there was little change in the ORP in Non-EW type-I HAW or SAEW, even in the open-lid condition (Figure 3(a), right).

The SARS-CoV-2 inactivating activity of Non-EW type-I HAW and SAEW was evaluated after 21 days of storage. At the virus-to-HAW sample ratio of 1:19 and in the open-lid condition, Non-EW type-I HAW (RCC 43 ppm) and SAEW (RCC 50 ppm) showed a ∼4.00 log₁₀ TCID₅₀/mL reduction of viral titers in 20 s reaction time (Figure 3(b) and 3(c)).

HAW samples with RCC at ∼50 ppm were stored at room temperature in the dark with a closed or an open tube lid for 21 days (Figure 3(a)). During the storage, RCC (left), pH (middle), and ORP (right) were measured. After the reaction time of 20 s, each viral titer was measured (Figure 3(b) and 3(c)). The titers of virus treated with the Non-EW type-I HAW sample stored for 21 days under the open-lidded condition (Figure 3(b)) and the SAEW sample stored for 21 days under the open-lidded condition (Figure 3(c)) were measured. Results are indicated as mean ± SD. A Student's t-test was performed to analyze the significant difference between the UPW group and each HAW group; ***p < 0.001.

In this study, we showed that Non-EW type HAW and AEW samples with an RCC of 50 ppm potently inactivated SARS-CoV-2 at a virus-to-HAW sample ratio of 1:19 in 20 s (Figure 1). However, the virucidal activity of the NaDCC solution with a 50 ppm RCC was weaker than those of other HAW samples. This decreased virucidal activity could be because only ∼50% of RC exists as free available chlorine when NaDCC dissolves in water (Sen 2016) in comparison with that of the other HAW solutions where most of the RC exists as free available chlorine. However, when the free available chlorine in the NaDCC solution is consumed, additional free available chlorine is supplied to maintain the equilibrium. Thus, this continuous supply of free available chlorine could be an advantage of using the NaDCC solution.

Free available chlorine, such as HClO, is believed to achieve bactericidal and virucidal effects via mechanisms such as the oxidative unfolding and aggregation of proteins, breaking of DNA or RNA strands, and destruction of membranes via reactions with membrane lipids or cholesterols (Carr et al. 1996, 1997; Hawkins & Davies 2002; Winter et al. 2008). We previously showed that the SARS-CoV-2-inactivating activity of AEW at pH 2.5 depends on the amount of free available chlorine (Takeda et al. 2020). In this study, we also demonstrated that the virucidal activities of Non-EW type HAW and SAEW decreased significantly when their level of RCC was lower or when the volume of the reaction was smaller (Figure 2). Combined with our previous study, these results indicate that, regardless of manufacturing methods of HAW products, the absolute amount of free available chlorine may be the most critical factor determining the effectiveness of a HAW product as an anti-SARS-CoV-2 disinfectant. However, our study also has several limitations. We tested the impact of HAW products on unpurified virus supernatants that contain not only virus but also other components, such as FBS, and we were unable to test purified SARS-CoV-2 in the absence of these components. In addition, we could not measure the RCC after the viral exposure. Since chlorine is consumed during reactions with organic substance, the actual RCC and the virus-to-HAW ratio required for the inactivation of the purified virus remain unclear. However, as viruses coexist with organic substances in the environment, our studies using unpurified virus supernatants may partially reflect such conditions. This implies that the more free available chlorine present, as determined by both the RCC and the volume of HAW, the more potent virucidal activity will be exhibited in the presence of organic substances on environmental surfaces. Consistent with this assumption, our previous study showed that the virucidal activity of AEW against unpurified SARS-CoV-2 supernatants with FBS was inversely proportional to the FBS concentration (Takeda et al. 2020).

We also showed that the loss of RCC was slower in the slightly acidic HAW products than that in the more acidic WAEW (Figure 3(a)). Since Cl₂ could be easily formed and released into the air at a lower pH, the loss of free available chlorine may have been faster in the more acidic WAEW products (Wang et al. 2007). In Non-EW type-I HAW and SAEW, the RCC and ORP remained stable under the open-lid condition for 21 days (Figure 3(a)). High ORP contributes to the inactivation of pathogens (Kim et al. 2000), and therefore, the stability of both RCC and ORP in the slightly acidic HAW products will enable these products to remain effective in storage long after production.

Providing liquid volume and RCC remains sufficiently high, the various HAW products tested here can efficiently inactivate unpurified SARS-CoV-2 supernatants in 20 s regardless of pH or manufacturing method. The slightly acidic HAW products can maintain high RCC and ORP and thereby sustain their SARS-CoV-2-inactivating activities for an extended period. However, the level of RCC is reduced rapidly in WAEW products. Thus, WAEW products should be used quickly after production, whereas the slightly acidic HAW products can remain in long-term storage. WAEW can only be used at locations with an electrolyzer. On the other hand, the slightly acidic HAW products can be transported anywhere, such as ordinary homes without an electrolyzer.

All of the HAW products tested in this study were provided by NITE for blind testing to evaluate the SARS-CoV-2 inactivating activities of commercial HAW products. We thank Mr H. Takagi and Dr K. Hanaki of the National Institute of Infectious Diseases for providing the method for removing NaDCC cytotoxicity with Diaion^® HP-20 resin (contact them directly for more information; [email protected]). We thank Enago (https://www.enago.jp) for English language editing.

The authors declared no conflict of interest.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

All relevant data are included in the paper or its Supplementary Information.