The Open Antimicrobial Agents Journal, 2010, 2, 71-78

71Open Access

Antiviral Effect of Chlorine Dioxide against Influenza Virus and Its
Application for Infection Control
Takanori Miura* and Takashi Shibata
Taiko Pharmaceutical Co., Ltd, 3-34-14 Uchihonmachi, Suita, Osaka, 564-0032 Japan
Abstract: Influenza is a respiratory tract infection, causing pandemic outbreaks. Spanish flu (A/H1N1), a pandemic occurred between 1918 and 1919, tolled patients and fatalities of 500 million and 50 million, respectively. Recently, humaninfection with highly pathogenic avian influenza A/H5N1 and swine influenza [Pandemic (H1N1) 2009] was reported.
Because of the population explosion and busy global aircraft traffics, Pandemic (H1N1) 2009 is rapidly spreading worldwide. In addition, it is seriously concerned that H5N1 influenza pandemic would emerge in the very near future. The
pandemic will cause the freeze of social activity and the crisis of business continuity, having a serious impact on
the global economy consequently. It is fervently desired that efficient methods of infection control against influenza
pandemic be developed.
Chlorine dioxide (ClO2) has a strong antiviral effect, and can disinfect the surface of object and the air in space. In recent
study on interaction between ClO2 and protein, ClO2 oxidatively modified tyrosine and tryptophan residues, and the protein was structurally denatured. Since hemagglutinin and neuraminidase of influenza virus A/H1N1 were inactivated by
the reaction with ClO2, it is likely that denaturation of the proteins caused inactivation of the virus. A low concentration(0.03 ppm) of ClO2 gas, where people can stay for a long period of time without any harmful effect, prevented the deathof mice caused by infection of influenza virus delivered as aerosol. We review current information based on the efficiencyof ClO2 solution and gas, and also discuss the application of ClO2 against influenza pandemics outbreak.

Keywords: Chlorine dioxide, influenza, pandemic.
1. INTRODUCTION
We, human being, experienced pandemics caused byinfluenza virus many times in the history. In the 20th century, three pandemics occurred in 1918, 1957, and 1968.
Each pandemic was named as Spanish flu, Asian flu, andHong Kong flu, among which the subtype of influenza A
virus differs as H1N1, H2N2, and H3N2, respectively [1].
The Spanish flu (A/H1N1) pandemic occurred between 1918
and 1919 with three waves, recorded more than the 500
million infected and 50 million deaths, which subsequently
reduced the lethality with antigen drift, then it was settled asa seasonal influenza [2]. Thirty eight years after the settlement of the Spanish flu, by antigen shift, the Asian flu(A/H2N2) erupted and became a pandemic once again. The
Asian flu also became a seasonal flu and was settled, however, 10 years later, by antigen shift, the Hong Kong flu(A/H3N2) occurred and became a pandemic, which, by antigen drift, became a seasonal influenza and was settled once
again. Through the pandemics in the 20th century, it is obvious that, by antigen shift that occurs every few decades, a
new subtype of virus was generated and caused a pandemic.
Also, it is thought that all these pandemics were originatedfrom the Spanish flu A/H1N1 subtype [3].

*Address correspondence to this author at the Research Institute, Taiko
Pharmaceutical Co., Ltd. 3-34-14 Uchihonmachi, Suita, Osaka, 564-0032,
Japan; Tel: +81 6 6382 3100; Fax: +81 6 6382 1152;
E-mail: miura@seirogan.co.jp
1876-5181/10

In this new century, a pandemic caused by the infectionof influenza virus occurred once again [4]. Since April 2009,
an influenza derived from the porcine type A influenza virus(H1N1) originated in Mexico has spread worldwide almost
instantly, and resulted in the declaration by the World HealthOrganization (WHO) of a pandemic on June 11, 2009, 41
years after the Hong Kong flu [5]. According to a surveillance by WHO as of November 22, 2009, the new type influenza [Pandemic (H1N1) 2009] tolled over 622,482 diagnosed infected patients and at least 7,826 deaths, and theinfection is still spreading worldwide [6]. The geographicalbreakdown of the number of diagnosed patients are: 15,503
in African region, 190,765 in American region, 38,359 in the
East Mediterranean region, over 154,000 in European region
where the spread of infection is still ongoing in Ukraina,
47,059 in Southeast Asian region, and 176,796 in Western
Pacific region, indicating a rapid transmission of infection
worldwide [6]. Since these numbers are those with a positive
result of examination of Pandemic (H1N1) 2009 influenza
virus, it is assumed that, among the huge number of influenza-like illnesses (ILIs), a considerable portion of Pandemic(H1N1) 2009 influenza patients are included. It may be said
that this is a real example of a long concerned worldwide
spread of infection in the contemporary world where theway of transportation by airplane and railroad, etc. is highly
developed.
In May 1997, prior to the Pandemic (H1N1) 2009 influenza, a human infection of a highly pathogenic avian influ2010 Bentham Open

72 The Open Antimicrobial Agents Journal, 2010, Volume 2

Miura and Shibata

enza A/H5N1 virus was reported [7, 8], which has been continuously spreading. Influenza A (H5N1) is infected by adirect contact with sick or dead poultry, and a large numberof human deaths has been reported. Between November
2003 and December 2007, 349 cases in 14 countries were
infected [9], among whom 62% died [10]. Recently, in
Egypt, incidence of the influenza A (H5N1) is increasing,and among the 88 accumulated patients, 27 died [11]. So far,the infectivity of the influenza A (H5N1) to human seems
relatively low, however, if a mutant virus which is more
adaptable to human should emerge, it is easy to expect thatthe infection spreads worldwide almost instantly, like thecase of the Pandemic (H1N1) 2009 influenza. Since the lethality of the influenza A (H5N1) is alarmingly high, it isconcerned that there will be a huge number of deaths cases,
unprecedented health hazards, and subsequent corruption ofsocial functions as a whole.
In this paper, we focus on chlorine dioxide (ClO2) which
has a potent virucidal activity against influenza virus, andintroduce the confirmative results of antiviral effects in thein vitro experiments. In addition, we report the details of
preventive effect of ClO2 gas in influenza infection experiments in mice, as well as a recently reported ameliorating
effect on the cumulative school absenteeism. Finally, we
propose new preventive measures against the flu pandemic
utilizing ClO2 in addition to normal preventive methods.
2. CHLORINE DIOXIDEClO2 (CAS. No. 10049-04-4) is a molecule with the molecular weight of 67.46, and it forms a stable radical [12].
Dauy, a British chemist, first discovered this moleculein 1811, by oxidizing potassium perchlorate [13]. ClO 2
is yellowish gas at room temperature, and relatively easily
dissolved in water [solubility in water is 20 g/L (0-5oC
and 70-100 mmHg)] [13]. However, since ClO2 gas
dissolved in water dissipates within a short period of time,the dissolved level of ClO2 gas decreases as time passes [13].
Consequently, ClO2 solution has been used by generation on
site.
ClO2 is an oxidizer, which is reduced to chlorite ion(ClO2-) by capturing an electron (ClO2 + e-  ClO2-). The
redox potential (Eº) is relatively high as 0.95 V. ClO2-, in thepresence of water, captures four electrons and is reduced tochloride ion (Cl-)(ClO2- + 2H2O + 4e-  Cl- + 4OH-), whose
Eº is 0.78 V which is lower than that of ClO2. Consequently,
the oxidative activity of ClO2 is stronger than ClO2-. The Eºof ClO2 is lower than that of hydroxyl radical (·OH, 2.8 V),
ozone (O3, 2.07 V), and hypochlorous acid (HClO, 1.49 V),
therefore, it undergoes more selective oxidative reaction thanthese other molecules [14, 15].
So called “stabilized” chlorine dioxide, which, in fact, iscolorless solution of ClO2- as the principal ingredient, hasbeen on the market in the Western countries, which is causing a confusion of exact nomenclature [16, 17]. Generally
speaking, ClO2 is synthesized by the reaction formula of (1) (3) as below [13, 18] ((g) means gas).
2NaClO2 + Cl2(g) = 2ClO2(g) + 2NaCl(1)

2NaClO2 + HOCl = 2ClO2(g) + NaCl + NaOH

(2)

5NaClO2 + 4HCl = 4ClO2(g) + 5NaCl + 2H2O

(3)

As a result, the “so called” stabilized chlorine dioxide inwhich ClO2- is the principal ingredient, cannot produce ClO2
if chlorine gas, hypochlorous acid or acid does not coexist,
and the “so called” stabilized chlorine dioxide itself does notpossess the selective oxidative activity of ClO2. Therefore,
the efficacy data of the “so called” stabilized chlorine dioxide on microorganisms differ from those of ClO2 itself, andthey need to be carefully interpreted [19-21].
ClO2 may be used as gas as well as solution, therefore,
the field of its application is considerably wide. Since longtime ago, principally in the Western countries, ClO2 has beenutilized for the bleaching of paper pulp [22]. ClO2, which isdifferent from Cl2, oxidizes lignin selectively among various
kinds of carbohydrates existing in the pulp [23, 24]. In addition, ClO2 has an advantage of reducing the production ofdioxins compared with other chlorine type bleaching agents.
Therefore, ClO2, by virtue of its ECF (Elemental Chlorine
Free) property without producing dioxins, serves as an alternative to the bleaching by Cl2 [22]. Also, ClO2 is allowed by
the U.S. Food and Drug Administration (FDA) to be used asa food additive for bleaching of cereal flours [25] and disinfection of vegetables and fruits [26]. Furthermore, the U.S.
Environmental Protection Agency (EPA) has allowed ClO 2
to be used as a disinfectant of tap water (less than 0.8 ppm
for targeted water quality) [27], and the molecule is exploited as a disinfection method which produce little amountof trihalomethane, a carcinogen, at water purification plants
in the U.S.A. [28, 29]. In addition, the EPA has allowed ahigh concentration ClO2 gas as a sterilizer for manufacturing
and experimental apparatus, clean rooms, environmental
surfaces, and tools [30], and recently, the gas was standardized by Annex G of NSF/ANSI 49 as an alternative to formalin fumigation for the sterilization of biosafety cabinets[31]. As will be described later, we confirmed a preventive
effect of the low concentration of ClO2 gas against the influenza virus infection [32]. In the future, we hope that the legal
legislation not only for high concentration ClO2 gas but also
for low concentration ClO2 gas as a preventive measure of
infection will take place.
In these current circumstances for ClO2, we at Taiko
Pharmaceutical Co., Ltd. manufacture and market a series of
uniquely developed antimicrobial and deodorant consumer
products under the brand name of “Cleverin” in Japan, utilizing ClO2 [33]. At present, as a products lineup, we have
“CleverinG” series which disseminate low concentration of
ClO2 gas continuously, “CleverinS” and “CleverinL”
which maintain the ClO2 concentration (approximately 100
ppm and 500 ppm, respectively) stably in solution for a long
period of time. These products look yellowish which is acharacteristic of ClO2 as describe formerly, and exploit ClO 2
gas itself. Also, “CleverinS” is a ready-to-use product,
without necessitating the ClO2 gas generation on site. Furthermore, in addition to these products, we manufacture andmarket LISPASS, a device which continuously releases low
concentration (below 0.1 ppm) of ClO2 gas. There are two
types of LISPASS: LISPASSS is a small size portable
type for the floor area up to 200 m2. The other is LISPASSNEO, which can cover the large floor area up to 16,000 m2.
Recently, we conducted a real size room experiment, andconfirmed that, although the level of released ClO2 gas is
affected by humidity and lighting, the apparatus can run

Antiviral Effect of Chlorine Dioxide Against Influenza Virus

within the safety level below 0.03 ppm [34]. Also, as to theefficacy against viruses, bacteria, and fungi as well as thesafety of our ClO2 products, we have published the research
results in the world leading journals [32, 35-39]. In the following section, among these various research results, we
focus on the influenza infection, introduce the efficacy dataof ClO2, and, together with the safety, we overview them.
Also, we introduce a totally new concept of “three phase
protection” to control the influenza infection.

The Open Antimicrobial Agents Journal, 2010, Volume 2

At various pH (5-6) ranges, the Cleverin solution at 1 ppm
and sodium hypochlorite solution at 10 ppm showed the antiviral activity (Table 4). When we increased the ClO2 concentration of the Cleverin solution up to 10 ppm, 99.99% ofantiviral activity was observed up to pH 10.
Table 1.

3. POSITIVE EFFICIENCY OF CHLORINE DIOXIDEAGAINST INFLUENZA VIRUS AND INFENCTIOUS
DISEASE

Relation between Disinfectant Concentration and
FBS Added to Inactivate 99.999% Influenza A
Virus without Added NaCl for 1 min at 25°C

Disinfectant Name

[Disinfectant] (ppm)[FBS] (%)

1

NDa

10

1

100

10

10

NIb

100

1

3.1. Chlorine Dioxide Solution
There are many reports that ClO2 solution has a virucidal
activity [40-44]. The inactivation concentration against various viruses are: 1-2 ppm in poliovirus [40, 41]，2.19 ppm incoronavirus which causes SARS [42]，7.5 ppm in hepatitis
A virus [43]，and 0.2 ppm in rotavirus [44]. Recently, weused ten types of viruses and reported the antiviral activity ofCleverinS and L (hereinafter referred to as Cleverin solution) [36]. The Cleverin solution, at 10 ppm ClO2 concentration, showed 99.99% or greater antiviral activity against feline calicivirus, human influenza virus A, measles virus, canine distemper virus, human herpesvirus-1, human herpesvirus-2, human adenovirus type 2, canine adenovirus type 2,
canine parvovirus and human immunodificiency virus type 1[36]. Furthermore, under various conditions of the Cleverin
solution, we measured the antiviral activity against influenza
virus A, and reported the results in comparison with sodium
hypochlorite solution [45]. Therefore, in this article, we describe the comparative antiviral activity of the Cleverin solution and sodium hypochlorite solution.
First, in the comparative antiviral activity experiment
loaded with fetal bovine serum (FBS), the Cleverin solution,even in the presence of 1% FBS as an interfering organic
compound, ClO2 at 10 ppm showed 99.999% or greater antiviral activity against influenza virus A (Table 1). On theother hand, under the same condition, 100 ppm of sodium
hypochlorite solution was necessary to achieve the sameantiviral activity (Table 1). Therefore, it was shown that,
under the serum loaded condition, the Cleverin solution was
ten times more effective than sodium hypochlorite solution.
Furthermore, since the Cleverin solution containing 100 ppm
ClO2 showed 99.999% or greater antiviral activity under thecondition of 10% FBS loading, this product may well show
the antiviral activity in the contaminated household environment.
Next, when we compared the antiviral activity in thepresence of the high concentration of salt (3 M or greater of
NaCl) against influenza virus A, the Cleverin solution at 10ppm, and sodium hypochlorite solution at 100 ppm showed
the antiviral activity (Table 2). Therefore, same as the FBS
loading, in the presence of high concentration of salt, theefficacy of the Cleverin solution was ten times higher thanthat of sodium hypochlorite solution.
Furthermore, when we compared the antiviral activity
at various temperatures (4-50°C) against influenza virus A,
the Cleverin solution at 10 ppm and sodium hypochlorite
solution at 100 ppm showed the antiviral activity (Table 3).

73

Cleverin

NaClO
a

b

Not done, Not inactivated.

Table 2.

Relation between Disinfectant Concentration and
NaCl Added to Inactivate 99.999% Influenza A
Virus without Added FBS for 1 min at 25°C

Disinfectant Name

[Disinfectant] (ppm)[NaCl] (M)

1

1

10

>3

100

>3

10

NIa

100

>3

Cleverin

NaClOa

Not inactivated.

Table 3.

Relation between Disinfectant Concentration andReaction Temperature to Inactivate 99.999%
Influenza A Virus without Added FBS and NaCl for
1 min

Disinfectant Name

Cleverin[Disinfectant] (ppm)

Temperature (°C)

1

10-50

10

4-50

100

4-50

10

NIa

100

4-50

NaClO
a

Not inactivated.

From these results, we confirmed that the Cleverin solution used in the case of contact infection of influenza virus
showed ten times higher antiviral activity than sodium hypochlorite solution which is a chlorine type bleaching agent,and its activity as a virucidal agent was not significantly af-

74 The Open Antimicrobial Agents Journal, 2010, Volume 2

Miura and Shibata

fected by the presence of organic compounds, salt concentration, temperature, and pH.
Table 4.

Relation between Disinfectant Concentration andReaction pH to Inactivate 99.99% Influenza A
Virus without Added FBS and NaCl for 1 min at
25°C

Disinfectant Name

[Disinfectant] (ppm)

pH

1

5-6

10

5-10

100

NDa

1

NIb

10

5-6

100

NDa

Cleverin

NaClOa

In addition to this animal model, a retrospective humanstudy revealed that the low concentration ClO2 gas decreased
the cumulative absenteeism of primary school children [35].
When three CleverinGs were placed in a primary schoolclassroom (space volume 230 m3) for the purpose deodorization (0.01-0.03 ppm), in classes where CleverinG was notplaced, the cumulative absenteeism rate during 38 consecutive days was 4.0%, while it was reduced to 1.5% in a classroom where CleverinGs were placed (P < 0.00001, Table
6). The primary reason of absenteeism was cold or influenza.
From these experiments, it was suggested that the low concentration of ClO2 gas prevented the infection of influenza ina semi-closed space.

Not done, bNot inactivated.

3.2. Chlorine Dioxide Gas
In addition to the Cleverin solution, we focused on thepotent virucidal activity of ClO2 gas, and are extending
unique research activities to confirm its efficacy and also to
elucidate its mechanism of action. As to the infection of influenza virus, we used an animal (mice) model of infection
experiment, and found that ClO2 gas far below the eight hour
average weighted mean exposure level (0.1 ppm) defined by
the Occupational Safety and Health Administration (OSHA,
U.S.A.) suppressed the infection of influenza virus A[32]. We housed 15 mice (CD-1) in a semi-closed cage,and introduced ClO2 gas (final average concentration: 0.032ppm）for 15 minutes simultaneously introducing the aerosolof influenza virus strain A/PR/8/34 (H1N1) (approximately 1
LD50) generated by a nebulizer (treated group). As the control group, fresh air instead of ClO2 gas was introduced.
Three days later, we picked up five mice and measured thepulmonary virus titer (TCID50). In the control group, TCID50
was 6.7 ± 0.2 log (mean ± SD), while in the treated group, it
Table 5.

was significantly reduced to 2.6 ± 1.5 log (P = 0.003, Table
5). Also, when we compared the mortality of the remaining
ten mice until day 16, in the control group, it was 7/10
(number of dead animals/total number of animals), whereas,in the treated group, it was significantly reduced to 0/10 (P =0.002, Table 5). From these results, it was suggested that as
low as 0.03 ppm of ClO2 gas prevented the influenza infection in the mouse model.

3.3. Virucidal Mechanism of Chlorine Dioxide
As a mechanism of action of antiviral effect of ClO2,
denaturation of surface proteins, such as hemagglutinin (HA)
and neuraminidase (NA), was postulated. Therefore, we investigated the effect of various concentrations of ClO2 on theactivity of HA and NA. As a result, it was revealed that, in
accordance with the increase in the ClO2 level, the activity of
HA and NA was decreased [32]. Furthermore, a detailed
physicochemical analysis was performed to reveal the interaction of protein and ClO2, utilizing bovine serum albumin(BSA) and glucose-6-phosphodehydronase (G6PD) as model
proteins [46]. As a result, an analysis by nuclear magnetic
resonance (NMR) spectroscopy and high performance liquid
chromatography-mass spectrometry (HPLC-MS) revealed
that ClO2 performed an oxidative modification of tyrosine
residue and tryptophan residue in the protein, and changed
the tyrosine residue to 3,4-dihydroxyphenylalanine (DOPA)
and 2,4,5-trihydroxyphenylalanine (TOPA), and the trypto-

Effect of ClO2 Gas on Pulmonary Titer, Mortality and Body Mass of Mouse Challenged with Influenza A Virus[ClO2 gas] (ppm)

Pulmonary Virus Titera (log10)

Mortalityb

Relative Body Massc0

6.7±0.2d

7/10e

0.9±0.04e

0.03

2.6±1.5d

0/10e

1.09±0.08eaVirus titer (TCID50) was measured 72 hours after a challenge of virus aerosols (n = 5 in each group). bMortality was measured until 16 days after the challenge (n = 10 in eachgroup). cRatio of body mass on day 7 to that on day 0 in each group (n = 5 in each group).dP = 0.003, eP = 0.002.

Table 6.

a

P < 0.00001.

Effect of ClO2 Gas on Cumulative Absenteeism Rate
Cleverin® G

Cumulative Attendance Rate (%)

Cumulative Absenteeism Rate (%)

Placed98.5

1.5a

Not placed

96.0

4.0a

Antiviral Effect of Chlorine Dioxide Against Influenza Virus

The Open Antimicrobial Agents Journal, 2010, Volume 2phan residue to N-formylkynurenine. Furthermore, in the
peptide derived from HA and NA, we observed that the tyrosine residue and tryptophan residue were oxidized [32].
Therefore, it was suggested that the mechanism of action of
ClO2 to inactivate the influenza virus was specific oxidative
modification of these two amino acid residues. Both tyrosine
residue and tryptophan residue are contained in HA and NA
in Pandemic (H1N1) 2009 virus and highly pathogenic influenza A/H5N1 virus [47-50], suggesting the antiviral effect ofa low concentration of ClO2 gas and ClO2 solution.
4. SAFETY OF CHLORINE DIOXIDE
4.1. Chlorine Dioxide Solution
ClO2 has been used to disinfect the tap water in the
U.S.A., and a review article was published by EPA [51].
Hereinafter, we extracted the content of this report.
As a result of oral administration of ClO2 solution for 90
days in rats, the lowest-observed-adverse-effect level(LOAEL) was 25 ppm (2 mg/kg/day). Also, from anotherexperiment in which ClO2 solution was orally administered
for two years in rats, the no-observed-adverse-effect level(NOAEL) was 10 ppm (1.3 mg/kg/day) [51-53]. In an experiment in which African green monkeys were orally administered with ClO2 solution for six weeks, NOAEL was 30
ppm (3.5 mg/kg/day) and LOAEL was 100 ppm (9.5 mg/kg/day) [51, 54]. Also, a human study was conducted and there
was no toxic signs observed after oral intake of tap watercontaining 5 ppm of ClO2 for 12 weeks [51, 55]. As to themetabolism of ClO2, an investigation was made by a single
oral administration of radiolabeled 36ClO2 in rats, which revealed that the plasma 36Cl level reached the peak two hours
after the administration, and 30% of the radioactivity was
excreted in urine by 72 hours after the administration [51,56]. Approximately 80% of 36Cl were in the form of Cl-, andmost of the remaining radioactivity was ClO2-, with a small
amount of ClO3- [57].
We also confirmed the toxicity of Cleverin solution in
animals, resulting in the acute oral toxicity more than 5,000
Table 7.

a

mg/kg, and the inhalation toxicity was more than 12,000
mg/m3. No dermal irritation was observed and the mucosal
irritation was minimal (Table 7). These experimental results
suggest that the safety of both ClO2 solution and the Cleverin
solution containing ClO2 is relatively high, and may be usedin households.
4.2. Chlorine Dioxide Gas
Dalhamn performed an inhalation toxicity study in rats[58]. Some rats died after the exposure of as high as 260
ppm of ClO2 gas for two hours. On the other hand, after the
exposure of ClO2 gas at 0 or 0.1 ppm (average level during
ten weeks, ranging from 0.05 to 0.3 ppm) for five hours a
day and seven days a week for ten weeks, neither death nor
any toxic signs were observed. Our ClO2 products have an
advantage of controlling the concentration of ClO2 below 0.1
ppm in the space. This low concentration is lower than theweighted average of eight hours per day allowed by OSHA
as the long term exposure, the weighted average of ten hourlabor allowed by the National Institute for Occupation Safety
and Health (NIOSH, U.S.A.) [59], and the standard level foreight hours a day for 40 hours a week allowed by the American Conference of Governmental Industrial Hygienist(ACGIH, U.S.A.) [60]. From these findings, it may be assumed that there is little toxicity concern about the use of the
low concentration of ClO2 gas below 0.1 ppm.
5. THREE PHASE PROTECTION FOR INFECTIONCONTROL OF INFLUENZA
In order to prevent the infection in offices, hospitals, andhousehold rooms, disinfection of the hard surface may not be
sufficient to control all the infection routes, and an application which can be applied to every infection route becomes
necessary. It is said that the main infection routes of influenza virus to human being are physical contact with the infected individual or via the intermediate materials (directcontact infection) and inhalation of droplets containing thevirus and exhausted from the infected individual (droplet
infection). As to the infection caused by the dried droplets or
the aerosol with the size of 5 m or smaller containing the

Toxicology Study of Cleverin Solutiona

Test

AnimalResultAcute oral toxicity

Mouse

LD50 > 5000 mg/kg

Acute inhalation toxicity

Mouse

LC50 > 12000 mg/m3

Single dose skin irritation

Rabbit

No irritation

Multiple dose skin irritation

Rabbit

No irritation

Ophthalmological eye irritation

Rabbit

No irritation

Single dose mucosal irritation

RabbitMinimal irritation

Subchronic oral toxicity

Rat

LD50 > 1000 mg/kg

Skin allergic reaction

Guinea pig

No allergic reaction

Micronucleous test

Mouse (bone marrow)

No induction

ClO 2 concentration: 100 ppm.

75

76 The Open Antimicrobial Agents Journal, 2010, Volume 2virus (airborne infection), although a lot of discussion havebeen made, the conclusion is yet to be reached [61-65].
However, the infection experiments in Guinea Pigs [66] andFerrets [67] confirmed the airborne infection via aerosol.
Also, influenza virus was detected in the aerosol sample
taken from the ambulatory hospital [68]. Therefore, the existence of influenza virus in the air and the airborne infection
by the virus cannot be denied. Hence, we proposed an ideaof the three phase protection as a measure to prevent the infection of influenza virus. This concept of infection control
system is consisted with the physical surface disinfection
against the contact infection (the first phase protection), disinfection of the space against the droplet and airborne infection (the second phase protection), and the control of infection and growth of virus taken into the body (the third phase
protection). Examples of the physical protection are the frequent hand washing, the physical shielding of the infection
route by wearing a mask or a protective cloth, and the hardsurface disinfection of the contact infection routes [69]. For
the hard surface disinfection, we can utilize CleverinS andCleverinL which show highly efficient antiviral effects. As
to the space protection, examples are the physical removal ofthe virus by HEPA filter, and the chemical removal of thevirus by gas with a virucidal effect. For the chemical removal of the virus, the choice of CleverinG, LISPASSS,
and LISPASSNEO, all of which generate ClO2 gas, may
be recommended according to the size of the space tobe disinfected. For the human body protection, examples are
the therapy by administration of anti-influenza drugs [70],the prevention by vaccination [71, 72] and the high dose
vitamin D administration which is reported to be effective[73].
Pandemic (H1N1) 2009 influenza is expected to further
spread worldwide. Also, highly pathogenic influenza A(H5N1) infection keeps increasing in Egypt [11]. Furthermore, it is not unlikely that a new type of influenza virus
may emerge by antigen shift and antigen drift of these twoviruses and acquire high morbidity and mortality. We believe firmly that, under the concept of the three phase protection, both the ClO2 solution and the low concentration of
ClO2 gas which can be used effectively and safely will become an effective measure to prevent not only the current
influenza infection but also the new emerging influenza
virus.
REFERENCES
[1]
[2]

[3]
[4]

[5]

[6]

Kilbourne ED. Influenza pandemic of the 20th century. Emerg
Infect Dis 2006; 12: 9-14.
Johnson NPAS, Mueller J. Updating the account: global mortalityof the 1918-1920 “Spanish” influenza pandemic. Bull Hist Med
2002; 76: 105-15.
Taubenberger JK, Morens DM. 1918 influenza: the mother of all
pandemic. Emerg Infect Dis 2006; 12: 15-22.
Novel Swine-Origin Influenza A (H1N1) Virus Investigation
Team, Dawood FS, Jain S, Finelli L, et al. Emergence of a novelswine-origin influenza A (H1N1) virus in humans. N Engl J Med.
2009; 360: 2605-15.
World now at the start of 2009 influenza pandemic [homepage on
the internet]. Genève, Switzerland. World Health Organization;[updated 2009 June 11; cited 2009 Nov 21]. Available from:http://www.who.int/mediacentre/news/statements/2009/h1n1_pandemic_phase6_20090611/en/index.html/
Pandemic (H1N1) 2009 - update 76 [homepage on the internet].
Genève, Switzerland. World Health Organization; [updated 2009

Miura and Shibata

[7]

[8]

[9]

[10]

[11]

[12]

[13]
[14]
[15]

[16]

[17]

[18]

[19]

[20]

[21]

[22]
[23]

[24]

[25]

[26]

Nov 27; cited 2009 Nov 30]. Available from: http://www.who.int/
csr/don/2009_11_27a/en/index.html/
Claas EC, Osterhaus AD, van Beek R, De Jong JC, Rimmelzwaan
GF, Senne DA, Krauss S, Shortridge KF, Webster RG. Human
influenza A H5N1 virus related to a highly pathogenic avian
influenza virus. Lancet 1998; 351: 472-7.
Subbarao K, Klimov A, Katz J, et al. Characterization of an avian
influenza A (H5N1) virus isolated from a child with a fatal respiratory illness. Science. 1998; 279: 393-6.
Dudley JP. Age-specific infection and death rates for human A(H5N1) avian influenza in Egypt. Euro Surveill 2009 May 7; [cited
2009 Nov 27]; 14: [article 4]. Available from: http://www.eurosurveillance.org/ViewArticle.aspx?ArticleId=19198-13.
Uyeki TM. Global epidemiology of human infection with highly
pathogenic avian influenza A (H5N1) viruses. Respirology 2008;
13: S2-9.
Avian influenza - situation in Egypt - update 24 [homepage on theinternet]. Genève, Switzerland. World Health Organization; [updated 2009 Nov 20; cited 2009 Nov 21]. Available from: http://
www.who.int/csr/don/2009_11_20/en/index.html/
Ozawa T, Kwan T. Electron spin resonance studies of chlorine
dioxide (ClO2) in aqueous solutions. Chem Pharm Bull 1983; 31:
2864-7.
Gates, D. The Chlorine Dioxide Hand Book. Denver: Amer Water
Works Assn 1998.
McCarthy JA. Bromide and chlorine as water disinfectants. J. New
Engl Water Works Assoc 1944; 58: 55-68.
Fukuyama MY, Tan H, Wheeler WB, Wei C. Reaction of aqueous
chlorine and chlorine dioxide with model food compounds. Environ Health Perspect 1986; 69: 267-74.
Stabilized chlorine dioxide [homepage on the internet]. DuPont
Inc.; [cited 2009 Nov 13]. Available from: http://www2.dupont.
com/Chlorine_Dioxide/en_US/products/chemicals/stabilized.html/
CloxideTM Stabilised Chlorine Dioxide [homepage on the internet].
Clearwater Technology Ltd.; [cited 2009 Nov 13]. Available from:http://www.clearwater.eu.com/chlorine_dioxide/cloxide__stabilised_chlorine_dioxide.html/
U.S. Environmental Protection Agency (EPA): Alternative
disinfectants and oxidants guidance manual. Washington DC: Apr
2000.
Keskinen LA, Burke A, Annous BA. Efficacy of chlorine, acidic
electrolyzed water and aqueous chlorine dioxide solutions to decontaminate Escherichia coli O157:H7 from lettuce leaves. Int J
Food Microbiol 2009; 132: 134-40.
Lukasik J, Bradley ML, Scott TM, et al. Reduction of poliovirus 1,
bacteriophages, Salmonella montevideo, and Escherichia coli
O157:H7 on strawberries by physical and disinfectant washes. J
Food Prot 2003; 66: 188-93.
Podolak R, Elliott PH, Taylor BJ, Khurana A, Black DG. Destruction of Alicyclobacillus acidoterrestris spores in apple juice on
stainless steel surfaces by chemical disinfectants. J Food Prot 2009;72: 510-4.
Alliance for Environmental Technology (AET). ECF: The Sustainable Technology, Washington DC: 2005.
Svenson DR, Jameel H, Chang H, Kadla JF. Inorganic reactions inchlorine dioxide bleaching of softwood kraft pulp. J Wood Chem
Technol 2006; 26: 201-13.
Hamzeh Y, Izadyar S, Mortha, G. Elemental chlorine freedelignification of chemical pulp in flow through reactor. J Appl Sci
2007; 7: 3786-90.
U.S. Food and Drug Administration (FDA) [website on the internet]. Title 21—Food and Drugs Chapter I- Food and Drug Administration Department of Health and Human Services Subchapter B—
Food for Human Consumption Part 137 Cereal flours and related
products [updated 2009 Apr 1; cited 2009 Nov 15]. Available from:http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearc
h.cfm?CFRPart=137&showFR=1/
U.S. Food and Drug Administration (FDA) [homepage on theinternet]. Title 21—Food and Drugs Chapter I- Food and Drug
Administration Department of Health and Human Services Part 173- Secondary direct food additives permitted in food for human consumption - Table of Contents Subpart D--Specific Usage Additives
Sec. 173.300 Chlorine dioxide. [updated 2002 Apr 1; cited 2009

Antiviral Effect of Chlorine Dioxide Against Influenza Virus

[27]

[28]
[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]
[40]

[41]

[42]

[43]

[44]

[45]

[46]

[47]

[48]

Nov 15]. Available from: http://edocket.access.gpo.gov/cfr_2002/
aprqtr/21cfr173.300.htm/
U.S. Environmental Protection Agency (EPA): 40 CFR Parts 9,
141, and 142 National Primary Drinking Water Regulations: Disinfectants and Disinfection Byproducts; Final Rule. Washington DC:
Dec 1998.
Lykins BW, Griese MH. Using chlorine dioxide for trihalomethane
control. Am Water Works Assoc J 1986; 78: 88-93.
Werdehoff KS, Singer PC. Chlorine dioxide effects on THMFP,TOXFP and the formation of inorganic by-products. Am Water
Works Assoc J 1987; 79: 107-13.
U.S. Environmental Protection Agency (EPA): Reregistration
Eligibility Decision (RED) for Chlorine Dioxide and Sodium Chlorite (Case 4023). Washington DC: Aug 2006.
Lorcheim K. Validating BSC Contamination. ALN magazine 2009
Apr; [cited 2009 Nov 27]. Available from: http://www.alnmag.
com/articles.asp?pid=426/
Ogata N, Shibata T. Protective effect of low-concentration chlorine
dioxide gas against influenza A virus infection. J Gen Virol 2008;
89: 60-7.
Infection Control Product Series [homepage on the internet]. Suita,
Osaka, Japan: Taiko Pharmaceutical Co., Ltd.; [cited 2009 Nov
15]. Available from: http://www.seirogan.co.jp/english/index2.
html/
Tanabe S, Tsutsumi H, Takeuchi H, Shibata T, Setsujima M, Nakahara K. Infection control against pandemic flu – Part.1 Disinfection method indoors. J Jpn Air Clean Assoc 2009; 47: 31-9.
Ogata N, Shibata T. Effect of chlorine dioxide gas of extremely
low concentration on absenteeism of schoolchildren. Int J Med
Med Sci 2009; 1: 288-9.
Sanekata T, Fukuda T, Miura T, et al. Evaluation of antiviral
activity for feline calicivirus, human influenza virus, measles virus,
canine distemper virus, human herpesvirus, human adenovirus,
canine adenovirus and canine parvovirus by chlorine dioxide andsodium hypochlorite. Biocontrol Sci 2010; in press.
Morino H, Matsubara A, Fukuda T, Shibata T. Inhibition of hyphal
growth of the fungus Alternaria alternata by chlorine dioxide
gas at very low concentrations. Yakugaku Zasshi 2007; 127: 7737.
Morino H, Fukuda T, Miura T, Lee C, Shibata T, Sanekata T.
Inactivation of feline calicivirus, a norovirus surrogate, by chlorine
dioxide gas. Biocontrol Sci 2009; 14: 147-53.
Miura T, Shibata T. Microbial disinfecting technologies by chlorine
dioxide. Lab Animal Tech Sci 2009; 21: 11-6.
Alvarez ME, O’Brien RT. Mechanism of inactivation of poliovirus
by chlorine dioxide and iodine. Appl Environ Microbiol 1982; 44:1064-71.
Tachikawa M, Saita K, Tezuka M, Sawamura R. Inactivation ofpoliovirus with chlorine dioxide. Jpn J Toxicol Environ Health
1993; 39: 572-6.
Wang XW, Li JS, Jin M, et al. Study on the resistance ofsevere acute respiratory syndrome-associated coronavirus. J Virol
Methods 2005; 126: 171-7.
Li JW, Xin ZT, Wang XW, Zheng JL, Chao FH. Mechanisms ofinactivation of hepatitis A virus in water by chlorine dioxide. Water
Res 2004; 38, 1514-9.
Chen YS, Vaughn JM. Inactivation of human and simianrotaviruses by chlorine dioxide. Appl Environ Microbiol 1990; 56:1363-6.
Ogata N. Chlorine dioxide against pandemic influenza. In: 5thInternational Bird Flu Summit; 2007 Sep 27-28: Las Vegas, Nevada, USA.
Ogata N. Denaturation of protein by chlorine dioxide: oxidative
modification of tryptophan and tyrosine residues. Biochemistry2007; 46, 4898-911.
Lim AP, Chan CE, Wong SK, Chan AH, Ooi EE, Hanson BJ.
Neutralizing human monoclonal antibody against H5N1 influenza
HA selected from a Fab-phage display library. Virol J 2008; 5:130.
Maurer-Stroh S, Ma J, Lee RT, Sirota FL, Eisenhaber F. Mapping
the sequence mutations of the 2009 H1N1 influenza A virus
neuraminidase relative to drug and antibody binding sites. Biol
Direct 2009; 4: 18.

The Open Antimicrobial Agents Journal, 2010, Volume 2
[49]
[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]
[58]
[59]

[60]

[61]

[62]
[63]

[64]

[65]
[66]

[67]

[68]

[69]

[70]

77

Gallaher WR. Towards a sane and rational approach to management of Influenza H1N1 2009. Virol J 2009; 6: 51.
Yan Y, Li S, Yang C, Luo W, Wang M, Chen Y, Luo H, Wu T,
Zhang J, Xia N. Prediction of a common neutralizing epitope of
H5N1 avian influenza virus by in silico molecular docking. Chin
Sci Bull 2008; 53: 868-77.
U.S. Environmental Protection Agency (EPA): Toxicology
review of chlorine dioxide and chlorite. Washington, DC, Sep.,
2000.
Daniel FB, Condie LW, Robinson M, Stober JA, York RG, Olson
GR, Wang SR. Comparative subchronic toxicity studies of three
disinfectants. J Am Water Works Assoc 1990; 82: 61-9.
Haag HB. The effect on rats of chronic administration of sodium
chlorite and chlorine dioxide in the drinking water. Report to the
Mathieson Alkali Works from the Medical College of Virginia. Feb
7, 1949.
Bercz JP, Jones LL, Garner L, Murray D, Ludwig A, Boston J.
Subchronic toxicity of chlorine dioxide and related compounds indrinking water in the nonhuman primate. Environ Health Perspect
1982; 46: 47-55.
Lubbers JR, Chauhan S, Miller JK, Bianchine JR. The effects ofchronic administration of chlorine dioxide, chlorite and chlorate tonormal healthy adult male volunteers. J Environ Pathol Toxicol
Oncol 1984; 5: 229-38.
Abdel-Rahman MS, Couri D, Bull RJ. Kinetics of ClO 2 and effectsof ClO2, ClO 2- and ClO3 in drinking water on blood glutathione andhemolysis in rat and chicken. J Environ Pathol Toxicol 1979; 3:
431-49.
Abdel-Rahman MS, Couri D, Jones JD. Chlorine dioxide metabolism in rat. J Environ Pathol Toxicol 1979; 3: 421-30.
Dalhamn T. Chlorine dioxide toxicity in animal experiments andindustrial risks. AMA Arch Ind Health 1957; 15: 101-7.
The National Institute for Occupation Safety and Health (NOISH):
Recommendation for occupational safety and health: Compendiumof policy documents and statements. U.S. Department of Healthand Human Services, Public Health Service, Centers for Disease
Control, National Institute for Occupational Safety and Health,
DHHS (NOISH) Publication No. 92-100, Cincinnati, OH, Jan.,
1992.
The American Conference of Governmental Industrial Hygienist(ACGIH): 1994-1995 Threshold limit values for chemical substances and physical agents and biological exposure indices.
American Conference of Governmental Industrial Hygienists, Cincinnati, OH, 1994.
Bridges CB, Kuehnert MJ, Hall CB. Transmission of influenza:
implications for control in health care settings. Clin Infect Dis
2003; 37: 1094-101.
Tellier R. Review of aerosol transmission of influenza A virus.
Emerg Infect Dis 2006; 12: 1657-62.
Brankson G, Gitterman L, Hirji Z, Lemieux C, Gardam M. Transmision of influenza A in human being. Lancet Infect Dis 2007; 7:
257-65.
Weber TP, Stilianakis NI. Inactivation of influenza A virus in theenvironment and modes of transmission: A critical review. J Infect2008; 57: 361-73.
Tellier R. Aerosol transmission of influenza A virus: a review of
new studies. J R Soc Interface 2009; 6(Suppl 6): S783-90.
Mubareka S, Lowen AC, Steel J, Coates AL, García-Sastre A,
Palese P. Transmission of influenza virus via aerosols and fomites
in the guinea pig model. J Infect Dis 2009; 199: 858-65.
Munster VJ, de Wit E, van den Brand JM, et al. Pathogenesis
and transmission of swine-origin 2009 A (H1N1) influenza virus inferrets. Science 2009; 325: 481-3.
Blachere FM, Lindsley WG, Pearce TA, et al. Measurement of
airborne influenza virus in a hospital emergency department. Clin
Infect Dis 2009; 48: 438-40.
Jefferson T, Del Mar C, Dooley L, Ferroni E, Al-Ansary LA,
Bawazeer GA, van Driel ML, Foxlee R, Rivetti A. Physical interventions to interrupt or reduce the spread of respiratory viruses:
systematic review. BMJ 2009; 339: b3675.
Antiviral drugs and pandemic (H1N1) 2009 [homepage on theinternet]. Genève, Switzerland. World Health Organization; [updated 2009 Oct 6; cited 2009 Nov 27]. Available from: http://www.

78 The Open Antimicrobial Agents Journal, 2010, Volume 2[71]

Miura and Shibata

who.int/csr/disease/swineflu/frequently_asked_questions/swineflu_
faq_antivirals/en/index.html/
Vaccines for pandemic influenza A (H1N1) [homepage on theinternet]. Genève, Switzerland. World Health Organization; [updated 2009 Oct 30; cited 2009 Nov 27]. Available from:http://www.who.int/csr/disease/swineflu/frequently_asked_questions/vaccine_preparedness/en/index.html/

Received: January 25, 2010

[72]

[73]

Revised: April 20, 2010

Treanor JJ, Campbell JD, Zangwill KM, Rowe T, Wolff M. Safety
and immunogenicity of an inactivated subvirion influenza A(H5N1) vaccine. N Engl J Med. 2006; 354: 1343-51.
Cannell JJ, Vieth R, Umhau JC, et al. Epidemic influenza andvitamin D. Epidemiol Infect. 2006; 134:1129-40.

Accepted: April 23, 2010

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