Glycative Stress Research
Online edition : ISSN 2188-3610
Print edition : ISSN 2188-3602
Received : March 26, 2022
Accepted : May 17, 2022
Published online : June 30, 2022
doi:10.24659/gsr.9.2_93

Original article

Antibacterial efficacy of stable sodium hypochlorite
Kyle Haasbroek, Masayuki Yagi, Yoshikazu Yonei
Anti-Aging Medical Research Center and Glycative Stress Research Center,
Faculty of Life and Medical Sciences, Doshisha University, Kyoto, JapanAbstract

The bactericidal efficacy of stable sodium hypochlorite (s-SH, AirRish) was evaluated through in vitro testing against
planktonic solutions of Staphylococcus aureus and Staphylococcus epidermidis. After an exposure time of 5 minutes viable
colony forming units of both species fell below the limit of detection with both 100 ppm and 200 ppm s-SH, a greaterthan 6 log10 reduction (p < 0.001). Additionally, the antimicrobial effect of s-SH was also examined in hand sanitization
applications. In a 60s hand rub, 200 ppm s-SH achieved a 96 % reduction in hand CFUs, of similar effect as a typical alcohol
sanitizer (p = 0.28). s-SH maintained considerable bactericidal efficacy at the tested concentrations.

KEY WORDS: stable sodium hypochlorite, antibacterial, sanitization, Staphylococcus aureus

Introduction

Frequent handwashing is of vital importance for theprevention of illness and reducing the spread of communicabledisease. However, frequent handwashing with detergents
damages the protective proteins of the stratum corneum,removes beneficial lipids from the skin, and may trigger
contact dermatitis 1). Irritation and desiccation from repeatedhandwashing causes cracked and broken skin that is ultimately
more vulnerable to infection in spite of the employed hygienic
practice. For repeated everyday use, sanitizing hand rubs arean effective method for hand sanitization that produce lessirritation 2). Alcohol based sanitizing solutions are highly
effective at eliminating pathogens, but cause significant
desiccation 3) without the addition of emollients. Alcohols
such as isopropanol and n -propanol may also perturb
keratinocyte functioning and cause irritation 4). Ethanol does
not typically cause skin irritation, but can cause erythemain individuals with dysfunctional aldehyde dehydrogenase 5).
Additionally, during periods of high demand, manufacturing
and supply chains may struggle to meet high demand forsanitizers, as occurred during the early stages of the ongoing
SARS- CoV-2 pandemic. There is a need for a varied supplyof safe, non-irritating, and effective hand sanitizing agents.
Sodium hypochlorite (NaClO) is a chemical compoundwith broad antimicrobial activity that is used for cleaning anddeodorization in hospital, food processing, and household
environments. NaClO also sees use in a broad variety ofcontexts, including water sanitization and dental procedures,such as endodontic treatment.
NaClO rapidly degrades at high temperatures andunder exposure to ultraviolet light, oxygen, and contact withorganic contaminants. These drawbacks complicate thelong-term storage and practical use of NaClO. However,a new formulation of a stable sodium hypochlorite (s-SH)
solution has been developed which is better able to withstanddegradation and extends the shelf life of s-SH when stored
in a cool, dark environment. S-SH is used as an industrial
surface sanitizer at concentrations of 1,000 and 2,000 ppm
where its antimicrobial efficacy is well established.
One hundred ppm and 200 ppm formulations have beendeveloped for use in personal hand and skin sanitization, andnon-industrial surface sanitization. Recently we evaluatedthe skin safety of 200 ppm s-SH in a 24-hour closed pathtest 6), and observed no adverse effects of skin contact withs-SH. The bactericidal efficacy of these lower concentration
formulations requires confirmation. In this study weevaluated the bactericidal efficacy of 100 and 200 ppm s-SH.

Contact information: Professor Yoshikazu Yonei, MD, PhDAnti-Aging Medical Research Center / Glycative Stress Research Center,Graduate School of Life and Medical Sciences, Doshisha University
1- 3, Tatara Miyakodani, Kyotanabe, Kyoto, 610- 0394, Japan
TEL & FAX: + 81-774 -65- 6394 e-mail: yyonei@mail.doshisha.ac.jp
Co-authors: Haasbroek K, cygd2001@mail.doshisha.ac.jp;
Yagi M, myagi@mail.doshisha.ac.jp

Glycative Stress Research 2022; 9 (2) : 93-96(c) Society for Glycative Stress Research_ 93 _

Antibacterial Efficacy of Stable Sodium HypochloriteMethods

Test Materials
One hundred ppm and 200 ppm S-SH was provided byAirRish Co., Ltd. (Osaka, Japan). An alcohol solution wasused as a positive control, consisting of 63.5 % ethanol and10.5 % isopropanol (Yamazen; Osaka).

In Vitro Bactericidal Test
Overnight stock of Staphylococcus aureus (ATCC12600) and Staphylococcus epidermidis (ATCC 14990) was
prepared in Tryptic Soy Broth (Becton, Dickinson, andCompany; MD, USA) and incubated for 24 hours at 37 °C
with 250 rpm shaking. 100 µL aliquots of bacterial stock
were inoculated into 9.9 mL of 100 ppm s-SH, 200 ppm s-SH,alcohol, or 0.1 M phosphate buffer (pH 7.4) as a control.
Solutions were vortexed and incubated at room temperature(~ 25 °C ) for 5 minutes before neutralization and serial
dilution in 0.1 M phosphate buffer. 100 µL aliquots of dilutions
were plated on Tryptic Soy Agar ( Becton, Dickinson, andCompany; MD, USA) in triplicate and colony counting wasperformed after 72 hours of incubation at 37 °C.

0.01% Tween20) and firmly brushed 10 times against a4 cm2 section of the palm and immediately plated. Sanitizing
hand rub was performed based on a modified protocol of theEuropean Standard 7). Roughly 3 mL of sanitizing solution
was applied to the hands and rubbed into the skin with ahand washing motion for 60 seconds. Post- sanitization,an additional sample swab was collected as previouslydescribed. Plates were incubated for 24 hours at 37 °C before
colony counting was performed. Plates were incubated again
for an additional 48 hours to observe any slow growingcolonies. Control swabs were dipped in extraction buffer andexposed to the air of the testing environment before plating.

Results

In Vitro Bactericidal EffectSkin microflora samples were collected from the skinof the hand via swabbing. Sterile cotton sample collection
swabs were dipped in extraction buffer (0.15 M NaCl andAll test solutions demonstrated strong bactericidal activity
against planktonic S. aureus and S. epidermidis (Fig. 1). After
exposure to sanitizing solutions, bacterial stock of an initialconcentration of ~ 4 x10 8 CFU/mL (S. aureus) and ~ 6x10 8
CFU/mL (S. epidermidis) experienced a total loss of cell
viability. No colonies were observed at any dilution, with aminimum detection level of 100 CFU/mL, corresponding
to a reduction of at least 6 log 10 compared to control. Withan exposure time of 5 minutes, s-SH showed equal in vitro
bactericidal efficacy to the alcohol rub mix at both 100 ppm
and 200 ppm concentrations.

a)b)S. aureus

S. epidermidis

1.0E + 08

1.0E + 08

1.0E + 06

1.0E + 06

CFUs/mL

CFUs/mL

Skin Sanitization Test

1.0E + 04

1.0E + 02

1.0E + 00

1.0E + 04

1.0E + 02

0.1 M PB

100 ppms-SH

200 ppm
s-SH

1.0E + 00

Alcohol

0.1 M PB

100 ppms-SH200 ppm
s-SH

Alcohol

Fig. 1. In vitro bactericidal efficacy.
Cell viability of S. aureus (a ) and S. epidermidis ( b) after 5-minute exposure to sanitizing solutions. 1.0E + 02 CFUs/mL (100 CFU/mL)

is the theoretical minimum detection limit, although no viable colonies were observed. n = 3, mean ± SD. Statistical difference calculatedby Student’s t-test, *** p < 0.001. CFUs, colony forming unit; SD, standard deviation.

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Skin Sanitization
Immediately prior to sanitizing hand rub, sampled skinshowed a mean microbial count of 178.4 ± 127.3 CFUs.
After application and washing, all solutions demonstrated
a substantial decrease of skin colony forming units ( Fig. 2 ).
The 100 ppm s-SH hand rub resulted in an 81% reductionof CFUs, significantly less than 200 ppm s-SH at 96 % ( p <0.01) and the alcohol solution at 98 % (p < 0.01). The efficacy
of 200 ppm s-SH and the alcohol solution did not significantly
differ (p = 0.28)

1% CFU Reduction

0.96
0.8

0.98

0.810.6Conclusions
Stable sodium hypochlorite at concentrations of 100
ppm and 200 ppm is safe for use on the skin and demonstrates
strong antibacterial activity. Exposure to s-SH in planktonic
liquid solution caused a total loss of cell viability in both S.
aureus and S. epidermis within 5 minutes of contact time. Inskin sanitization applications, s-SH significantly reduced theamount of colony forming units on the skin of the hands, andefficacy did not significantly differ from the alcohol-based
solution at a concentration of 200 ppm. It was suggested
that this test product may be a disinfectant with a sterilizing
action equivalent to that of alcohol products.

0.40.20decrease in CFUs was capable of being observed. Skin withlower baseline microbial content at the time of testing resulted
in a smaller reduction of CFUs, despite low final CFU counts(e.g., 3 CFU decreasing to 1 after washing). Samples withextremely low baseline CFUs (below 10) were excluded fromanalysis.
Due to the ongoing SARS-CoV-2 pandemic, stricter
than usual hygiene and hand sanitization standards arebeing adhered to, which may explain some of the lower thantypical CFU counts on pre-sanitization skin. For the same
reason, repeated use of desiccating alcohol-based hand rubs
and frequent hand washing have increased the incidence ofdry and broken skin 10). Therefore, direct contamination ofhands with bacterial broth prior to sanitization (which may
have better demonstrated efficacy against higher bacterialconcentrations) was avoided due to of the risk of infection via
open wounds on the skin of the hands. Thus, the sanitization
effects were tested against resident hand microbes only.

100 ppm s-SH

200 ppm s-SH

AlcoholFig. 2. Reduction of CFUs after sanitizing hand rub.

Percent reduction in colony forming units after 60-second
hand rub with 3 mL of sanitizing solution. n = 3, mean ± SD.
Statistical difference calculated by Student’s t-test, * p < 0.01.
CFUs, colony forming units; SD, standard deviation.

Conflict of interest disclosure statement
Research support was provided by AirRish Corporation.

Discussion

Mechanism of Microbicidal Action
NaClO has a long history of use as a cleaning anddisinfection agent. It’s microbicidal activity is well established,although the mechanism of these effects has been less wellunderstood until recent years. NaClO damages lipids, DNA,and inhibits essential enzyme activity 8). In solution NaClO
forms hypochlorous acid (HClO) at varying concentrations
depending on pH, which is the primary microbicidal agent.
HClO is permeable to bacterial membranes and readily
denatures essential bacterial proteins, leading to the formationof large intracellular aggregates 9).

Skin Sanitization Experiments
Resident microbial content of the skin before testing wasconsiderably variable between tests, and the antimicrobial
effects of the sanitizing solutions were more readily apparent
against skin with higher microbial content, where a larger_ 95 _Antibacterial Efficacy of Stable Sodium Hypochlorite

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