Bactericidal Efficacy of So-Called Sanitizers in Japan

Abstract

Commercially available so-called sanitizers in Japan are often touted as having remarkable “sanitizing (jokin)” effect, “virus-removal” capabilities, and “99.99%” removal rate of microbes and pathogens. In this study, we investigated the bactericidal efficacy of these so-called sanitizers for environmental surfaces against Enterococcus faecalis and Pseudomonas aeruginosa. Of note, out of 43 products, 24 (55.8%) did not exhibit bactericidal effects on either Enterococcus or Pseudomonas. Among these 43 products, there were no bactericidal effects in 9 (47.4%) of 19 products that stated as containing “alcohol” as the formula; as well as 4 (80%) of 5 products stating only “benzalkonium chloride”; similarly no effect in 5 (83.3%) of 6 stating only“chlorine chemicals.” Furthermore, 6 (46.2%) of 13 products that stated components other than alcohol, benzalkonium chloride, and chlorine chemicals on the product container or with no description of constituent components failed to show any bactericidal effects. Four disinfectants (alcohol for disinfection, 0.1% benzalkonium chloride, 0.05% (500 ppm) hypochlorite, 0.1% (1000 ppm) hypochlorite) as control showed bactericidal effect. The lack of bactericidal activity in nearly half of sanitizers may be explained by the low concentration of the effective ingredient such as alcohol, benzalkonium chloride, and hypochlorite. In sanitizers containing hypochlorite, degradation of hypochlorite with prolongation of time after manufacturing may be another reason.

INTRODUCTION

With the coronavirus disease 2019 (COVID-19) pandemic, the general population in addition to the medical staff has become increasingly aware of the importance of proper disinfection of the environmental surfaces. In Japan, so-called sanitizers are ubiquitous and widely available for purchase. Most products are labeled with phrases such as “eradication (Jokin, in Japanese),” “sanitization,” “antibacterial action,” “virus removal,” “99.99%,” and “alcohol” on the product package, suggesting strong effect towards eliminating many surface contaminants including bacteria and virus. In fact, the sanitizer industry is regulated by the Japanese Society of Detergent Association (JSDA), which sets the criteria to allow products to have certain labels. One example is to allow the product to be labeled as sanitizer (jokin) in case where Staphylococcus species or Escherichia coli can be decreased by 2 log₁₀. Of note, JSDA does not specify the contact time required between the chemical and its target pathogen. This omission of contact time criterion pose some difficulty in consistent evaluation of chemical efficacy in objective measurement. As a result, many sanitizer products are routinely used for disinfection by the lay population despite its lacking consistency in rigorous testing. However, as they differ from disinfectants (products approved by the government for sale as medical products after considering efficacy and safety) and are commercially sold as miscellaneous goods, their disinfection capacity are by default not subject to meticulous scrutiny. In fact, most consumers in Japan may not understand the distinction between disinfectants versus the so-called sanitizers because the distinction is not always clear. As a result, most consumers may purchase these so-called sanitizers under the impression of it having proven qualities for disinfection. On the other hand, the U.S. Environmental Protection Agency (EPA), which regulates antimicrobial surface products, make clear distination between the two categories of sanitizers and disinfectants. Broadly speaking, sanitizers under the EPA are restricted to making efficacy claims only against bacteria; disinfectants are able to make efficacy claims against viruses and bacteria.^1,2)

One survey in Japan studying the effects of so-called sanitizers found that 13 (46.4%) of 28 products did not show antibacterial activity.³⁾ Thus, we further examined the effectiveness of these so-called sanitizers by increasing the number of products to be surveyed, and examined in detail the chemical components contained in these products.

MATERIALS AND METHODS

Test Agents

A total of 43 sanitizers were tested. These general household products were purchased at convenience stores or drug stores in Yamaguchi Prefecture in Japan between May and August 2021. Impregnated cloth products were tested by extracting out the solutions from the products. As control agents, we used ethanol for disinfection (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 0.1% benzalkonium chloride (Osvan^® S, Takeda Pharmaceuticals Co., Ltd., Tokyo, Japan), and 0.05% (500 ppm) and 0.1% (1000 ppm) sodium hypochlorite (Milton^®, Kyorin Pharmaceutical Co., Ltd., Tokyo, Japan).

Measurement of Concentration of Ingredients

We measured the concentration of ethanol, benzalkonium chloride, and free residual chlorine contained in the sanitizing products. The ethanol content of undiluted or 10-fold diluted solution was determined with SHIMADZU prominence HPLC system (Shimadzu Corporation, Kyoto, Japan). Chromatographic separation was achieved using a Shodex SH 1011 column (Resonac Corporation, Tokyo, Japan) maintained at 50 °C and the eluate was monitored using a RID-10A. We used 5 × 10⁻³ mol/L sulfuric acid aq. as the mobile phase and the total flow rate was set at 1.0 mL/min. The benzalkonium chloride of undiluted or 10-fold diluted solution was determined with LC2030C 3D Plus HPLC system (Shimadzu Corporation). Chromatographic separation was achieved using a Unison UK-C18 column (50 × 2.0 mm i.d., 3 µm, Imtakt Corporation, Kyoto, Japan) maintained at 35 °C and mobile phase consisted of 20 mM perchloric acid aq. and acetonitrile set at 0.5 mL/min of total flow rate. The eluate was monitored at 210 nm using a photodiode array (PDA) detector. The concentration of the free residual chlorine was measured using a N,N-diethyl-p-phenylenediamine (DPD) reagent, and the concentration of the free residual chlorine in the samples contained 0 to 300 ppm was measured using a SIBATA handy water quality analysis meter AQUAB AQ-202 (Shibata Co., Ltd., Tokyo, Japan). The concentration of the free residual chlorine in the samples contained 0 to 2.00 ppm was measured using a Pocket Colorimeter (HACH Company, Loveland, CO, U.S.A.).

Evaluation of Microbicidal Efficacy

Enterococcus faecalis ATCC29202 and Pseudomonas aeruginosa ATCC27853 were grown on trypticase soy agar (TSA) (Eiken Chemical, Tokyo, Japan) for 24 h at 35 °C and scraped into sterile phosphate-buffered saline (PBS), and centrifuged three times at 3000 rpm for 10 min to remove the growth medium. Resuspension was carried out in PBS, yielding a concentration of approximately 10⁸ colony forming units (CFU)/mL. Efficacy of sanitizers against E. faecalis and P. aeruginosa was examined with 2 assay conditions: albumin-poor ‘clean’ medium (8 : 1 : 1 of sanitizers: 0.3% bovine albumin: bacterial suspension) and albumin-rich ‘dirty’ medium (8 : 1 : 1 of sanitizers: 3% bovine albumin: bacterial suspension) at 20 ± 1 °C. After standing for five minutes, 0.1 mL aliquot of the suspension were collected and added to 9.9 mL of soybean-casein digest broth with lecithin and polysorbate (SCDLP) (Eiken Chemical) as inactivators for 10 min. Sanitizers containing chlorine chemicals were inactivated by nutrient broth (Eiken Chemical) containing 0.1% sodium thiosulfate. Samples were serially diluted ten-fold with sterile physiologic saline. The undiluted or diluted samples (0.2 mL) were plated on nutrient agar (Eiken Chemical), streaked with a plastic “hockey stick” and incubated at 35 °C for 48 h to count viable cells. Experiments were repeated twice. Evaluation guideline on environmental disinfectant issued by Japanese Society for Infection Prevention and Control was consulted to determine the effects of microbicidal properties of the sanitizing products.⁴⁾ According to the guideline, we indicated the positive bactericidal effect with 〇 for ≧4 log₁₀ reduction in CFU and indicated negative bactericidal effect with × for <4 log₁₀ reduction in CFU_. The final concentration of the test solutions assayed was estimated to be 80% of the original product, keeping in line with approximate dilution the sanitizer solutions undergo while using on ‘dirty’ surfaces.

RESULTS

With respect to the total of 43 sanitizers, the stated purpose of use, component name/actual concentrations, and bactericidal effects are presented in Table 1. Of these 43 products, the specification of use for these products were stated as for: “fingers and environment” (twenty six), and “environment” (seventeen). Of the 43 products tested, 20 were “liquid” and 23 were in “impregnated cloth” form. The 4 control disinfectants were liquid. The packaging of most products detailed the names of components with likely microbicidal effects in the ingredient column. Briefly, both alcohol and benzalkonium chloride were mentioned on 8 products; ethanol alone on 7; benzalkonium chloride alone on 5; chlorine chemicals, including hypochlorous acid solution, on 6, and other components on 7 products. There were no component names on 6 of the products. Additionally, the actual concentrations of ethanol, benzalkonium chloride, and free residual chlorine concentrations in these 43 products were 1.30–62.7 vol%, 0.0008–0.09% (w/v), and 0.02–318 ppm, respectively.

Table 1. Components and Bactericidal Effects of So-Called Sanitizers in Japan Whose Microbicidal Effects Were Described with Phrases Including “Eradication,” “Sanitization (Jokin),” “Antibacterial Action,” “Virus Removal,” “99.99%,” and “Alcohol”

Sample No.	Purpose of use described on products	Type	Component name described in the ingredient display columna)	Actual value of component			Bactericidal effects on Enterococcus faecalis		Bactericidal effects on Pseudomonas aeruginosa
				Ethanol (vol%)	Benzalkonium chloride (% (w/v))	Free residual chlorine (ppm)	Cleanliness conditionsb)	Pollution conditionsc)	Cleanliness conditions	Pollution conditions
1	Fingers/environment	Liquid	Ethanol	61.2			〇e)	〇	〇	〇
2	Fingers/environment	Impregnated cloth	Ethanol, benzalkonium chloride	49.2	0.01		〇	〇	〇	〇
3	Fingers/environment	Impregnated cloth	Ethanol, sanitizer	19.3			〇	〇	〇	〇
4	Fingers/environment	Impregnated cloth	Ethanol, benzalkonium chloride	18.9	0.006		〇	〇	×f)	×
5	Fingers/environment	Impregnated cloth	Ethanol, benzalkonium chloride	18.8	0.03		〇	〇	×	×
6	Fingers/environment	Impregnated cloth	Ethanol, benzalkonium chloride	18.5	0.04		〇	〇	〇	〇
7	Fingers/environment	Impregnated cloth	Ethanol, benzalkonium chloride	17.6	0.01		〇	〇	×	×
8	Fingers/environment	Impregnated cloth	Ethanol	17.1			×	×	×	×
9	Fingers/environment	Impregnated cloth	Ethanol	15.4			〇	〇	〇	〇
10	Fingers/environment	Impregnated cloth	Ethanol, benzalkonium chloride				〇	〇	×	×
			Sanitizer	12.6	0.09
11	Fingers/environment	Impregnated cloth	Ethanol, benzalkonium chloride	11.3	0.01		〇	〇	×	×
12	Fingers/environment	Liquid	Alcohol	2.6			×	×	×	×
13	Fingers/environment	Impregnated cloth	Ethanol, benzalkonium chloride	2.4	0.03		〇	〇	〇	×
14	Fingers/environment	Impregnated cloth	Benzalkonium chloride		0.02		〇	〇	×	×
15	Fingers/environment	Impregnated cloth	Benzalkonium chloride		0.017		〇	〇	〇	〇
16	Fingers/environment	Impregnated cloth	Benzalkonium chloride		0.01		〇	〇	×	×
17	Fingers/environment	Impregnated cloth	Benzalkonium chloride		0.002		×	×	×	×
18	Fingers/environment	Impregnated cloth	Benzalkonium chloride		0.0008		〇	〇	×	×
19	Fingers/environment	Liquid	Hypochlorous acid water			0.76	×	×	×	×
20	Fingers/environment	Liquid	Hypochlorous acid water			0.02	×	×	×	×
21	Fingers/environment	Impregnated cloth	—d)				〇	〇	×	×
22	Fingers/environment	Impregnated cloth	Sanitizer				〇	〇	〇	〇
23	Fingers/environment	Impregnated cloth	—				〇	〇	〇	〇
24	Fingers/environment	Impregnated cloth	Sanitizer				〇	〇	×	×
25	Fingers/environment	Impregnated cloth	Sanitizer				〇	〇	〇	〇
26	Fingers/environment	Impregnated cloth	—				×	×	〇	〇
27	Environment	Liquid	Ethanol	62.7			〇	〇	〇	〇
28	Environment	Liquid	Ethanol	58			〇	〇	〇	〇
29	Environment	Liquid	Ethanol, surface active agent, sanitizer	38.8			〇	〇	〇	〇
30	Environment	Liquid	Ethanol, sanitizer	12.4			〇	〇	〇	〇
31	Environment	Liquid	Ethanol, sanitizer				〇	×	〇	〇
			Amphoteric surface active agent	8.04
32	Environment	Impregnated cloth	Ethanol	1.3			〇	〇	〇	〇
33	Environment	Liquid	Chlorous acid water			318	〇	〇	〇	〇
34	Environment	Liquid	Sodium hypochlorite			99	〇	×	〇	×
35	Environment	Liquid	Chlorous acid water			76	〇	×	〇	×
36	Environment	Liquid	HClO2			62	〇	×	〇	×
37	Environment	Liquid	Sanitizer				〇	〇	〇	〇
38	Environment	Liquid	Surface active agent				〇	〇	〇	〇
39	Environment	Liquid	Antibacterial agent				〇	〇	〇	〇
40	Environment	Liquid	—				〇	〇	×	×
41	Environment	Liquid	Sanitizer, surface active agent				〇	〇	〇	〇
42	Environment	Liquid	—				×	×	×	×
43	Environment	Liquid	—				×	×	×	×

a) Components with likely microbicidal effects were detailed on product. b) Containing 0.03% bovine serum albumin. c) Containing 0.3% bovine serum albumin. d) —: Components with likely microbicidal effects were not detailed on product. e): 〇: ＞4log₁₀ reduction in CFU. f): ×: ＜4log₁₀ reduction in CFU.

Of the 43 products, 12 (27.9%) did not have any bactericidal effects on Enterococcus faecalis, and 22 (51.2%) did not show any such effects on Pseudomonas aeruginosa. Furthermore, 24 (55.8%) of the 43 products did not have any bactericidal effects on Enterococcus faecalis, Pseudomonas aeruginosa, or both species of bacteria.

When examining bactericidal effects with respect to usage, of 26 products for “fingers/environment,” 17 (65.4%) did not show any bactericidal effects. Of 17 products for the “environment,” 7 (41.2%) did not show any bactericidal effects.

When investigating bactericidal effects with respect to components, of 8 products containing both ethanol and benzalkonium chloride, 6 (75.0%) did not show any bactericidal effects. Of 7 products containing ethanol alone, 2 (28.6%) failed to exhibit any bactericidal effects. Of 5 products containing benzalkonium chloride alone, 4 (80%) did not show any bactericidal effects. Of 6 products containing chlorine chemicals alone, 5 (83.3%) failed to exhibit any bactericidal effects. Of 13 products describing components other than ethanol, benzalkonium chloride, and chlorine chemicals or with no description of components, 6 (46.2%) did not show any bactericidal effects. Experiments were conducted twice, and the same results were obtained. In this experiment, Enterococcus faecalis was selected to represent Gram-positive bacteria, and Pseudomonas aeruginosa for the Gram-negative. These species of bacteria are relatively resistant to low-level disinfectants among the vegetative bacteria.^5–9)

DISCUSSION

The sanitizers in Japan that claim microbicidal effects uses phrases on packages, such as “sanitizing (jokin),” “virus removal,” “alcohol,” and “99.99%.” These sanitizers are miscellaneous goods not intended for medical use, and are not medical-grade disinfectants. As a result, these products may not be held at a medical-grade standard and may not be stringently tested to have demonstrable disinfection effects. There are no current regulatory guidelines that ensure antimicrobial effects in sanitizers sold in Japan. However in the U.S., for example, sanitizers intended for environmental surfaces are required to undergo testing for antibacterial capacity. Many consumers do not recognize the differences between sanitizers and disinfectants. In addition, the names of disinfectant components, such as alcohol and benzalkonium chloride, are listed in the ingredient display column of most sanitizers. Therefore, consumers may purchase these sanitizing products, assuming that they have disinfection effects. In particular, many consumers may have purchased such sanitizers for the expressed puropose of removing the novel coronavirus (severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)) from environmental surfaces during the recent COVID-19 pandemic. Our previous screening of sanitizers in Japan showed that 13 (46.4%) of 28 products did not show any bactericidal effects.³⁾ In this study, we further demonstrated that 24 (55.8%) of 43 products that are commercially available did not have any significant bactericidal effects. This may be due to the product not having sufficient concentrations of alcohol, benzalkonium chloride, and chlorine chemicals. In this survey of products, we found that there were three of products with an ethanol concentration of ≥50 vol%, sixteen products had ethanol concentrations ranging from 1.30 to 49.2 vol%. Furthermore, the concentrations of benzalkonium chloride in 13 products were in the lower range, ranging from 0.0008 to 0.09% (w/v). In addition, the concentration of free residual chlorine in 1 product was 318 ppm, but its concentrations in the other 5 products ranged from 0.02 to 99 ppm, also being low.^10–14) In fact, some sanitizers contain the same components as medical-grade disinfectants, but the concentrations of these components are often comparably lower, which would explain the lower potency of bactericidal effect using the sanitizers. In the future, some regulation on microbicidal effects must be established for sanitizing products as miscellaneous goods so that so-called sanitizers do not become commercially available.

In conclusion, out of 43 sanitizers in Japan, 24 (55.8%) did not exhibit bactericidal effects on either Enterococcus or Pseudomonas.

Acknowledgments

We thank Yamaguchi Prefectural Industrial Technology Institute for useful cooperation.

Conflict of Interest

The authors declare no conflict of interest.

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