Use of chlorine dioxide gas as a chemosterilizing agent

A method for sterilizing a substantially gas impermeable surface which is contaminated with spores comprises the steps of exposing the surface to a humid gaseous environment to enhance the susceptibility of the spores to subsequent chemosterilization, and then exposing the spores to an amount of gaseous chlorine dioxide in an inert carrier gas effective to sterilize the surface and a method for sterilizing an article contaminated with spores which comprises exposing the surface to a gaseous atmosphere comprised of chlorine dioxide gas and water vapor, wherein the amount of water vapor in said atmosphere is adapted to enhance the susceptibility of said spores to the sporicidal action of chlorine dioxide.

BACKGROUND OF THE INVENTION 
The continuous introduction into technical use of new materials which 
cannot be radiation or heat sterilized or sterilized by exposure to liquid 
systems has necessitated the development of other means of sterilization. 
A major modern method for this purpose is based on the use of gaseous 
chemical agents. Such chemical compounds must be employed selectively, 
however, as only those which kill spores can be classified as chemical 
sterilizing agents. A wide variety of antimicrobial agents are available, 
but in most instances they do not kill resistant bacterial spores. 
Microbiocides are specifically limited to the destruction of the type of 
organism suffixed by "cide", e.g., bactericide refers to killing of 
bacteria, fungicide to fungi, viricide to viruses and sporicide to spores, 
both bacterial and fungal. Since bacterial spores are generally the most 
difficult to destroy, only sporicides may be considered synonymous with 
chemosterilizers. These may be defined as chemical agents which, when 
utilized properly, can destroy all forms of microbiological life, 
including bacterial and fungal spores and viruses. 
Gaseous ethylene oxide and formaldehyde are used at many hospitals and 
medical research facilities to sterilize equipment or work areas that 
cannot be readily heat- or liquid-sterilized. Formaldehyde, if applied in 
high concentrations, is likely to leave a residue of solid 
paraformaldehyde. For this reason, it is often avoided in the 
sterilization of delicate equipment or in situations in which allergic 
reactions to this substance may occur. Ethylene oxide, which, unlike 
formaldehyde, penetrates well into porous materials, is strongly absorbed 
by rubber and by many plastics so that the vapors are not readily 
eliminated by brief aeration. 
The publication of research relating to the mutagenicity and oncogenicity 
of both ethylene oxide and formaldehyde threatens to lead to severe 
limitations, if not outright bans, on the use of these compounds as 
sterilizing agents. The limitations would significantly increase the costs 
associated with ethylene oxide and formaldehyde sterilization. 
Apart from its potential health hazards, ethylene oxide is difficult to 
handle at the concentrations and temperatures required for effective 
sterilization. Ethylene oxide at a 3-80% concentration in air is violently 
explosive and so ethylene oxide is commonly employed in admixture with an 
inert gas such as a fluorocarbon, for example, 12% ethylene oxide and 88% 
Freon 12 (E.I. duPont Co.). In the sterilization of medical products, 
temperatures as high as 130.degree.-140.degree. F. are commonly employed 
to ensure sterility at chamber concentrations of 300-1200 mg/L (milligrams 
per liter) of ethylene oxide. Prehumidification followed by gas exposure 
times of at least 4.0 hours are commonly employed. Also, ethylene oxide is 
more effective in killing dry spores on porous materials, such as paper or 
fabrics, than on nonporous materials such as glass, ceramics, hard 
plastics and metals. See C. W. Bruch and M. K. Bruch, Gaseous 
Disinfection, in Disinfection, M. A. Benarde, Ed., Marcel Decker, Pub., 
New York (1970) at pages 149-207. 
Chlorine dioxide has long been recognized as being biologically active and 
early studies indicate that it possesses bactericidal, viricidal and 
sporicidal properties when applied in aqueous solution at minimum 
concentrations of about 0.20-0.25 mg/L. See W. J. Masschelein in Chlorine 
Dioxide: Chemistry and Environmental Impact of Oxychlorine Compounds, R. 
C. Rice, ed., Ann Arbor Science Pub. (1979); G. M. Ridenour, et al., Water 
& Sewage Works, 96, 279 (1949). However, more recent patents have stated 
that aqueous chlorine dioxide alone is not sporicidal unless used in the 
presence of stabilizers and/or activators. See Synder, U.S. Pat. No. 
4,073,888. Sterilization with aqueous chlorine dioxide suffers from all of 
the general disadvantages associated with the use of aqueous sterilizing 
agents, including formulation and handling difficulties, the inability to 
sterilize moisture-sensitive equipment or substances, and the deposition 
of residues upon drying. 
Little is known of the gas-phase chemistry of chlorine dioxide in air. At 
concentrations above about 10% (i.e., at about 300 mg per liter), the 
compound is unstable and sometimes detonates--probably in a shock or 
light-catalyzed decomposition. For this reason, chlorine dioxide gas 
cannot be stored. At the same concentration by weight in aqueous solution, 
it is quite stable. 
The chemistry of chlorine dioxide in water is thought to be influenced by 
the formation of hydrates. At low temperatures (near 0.degree. C.), high 
concentrations of chlorine dioxide precipitate out as hydrates of somewhat 
variable composition; warming permits these to redissolve. It is likely 
that chlorine dioxide in these warmed solutions still has some water 
molecules clustered about it. Such hydrates would not, of course, occur in 
the vapor phase. 
In general, both the distance of molecules from one another in the gas 
phase and the absence of polar solvent effects must profoundly alter the 
chemistry of chlorine dioxide in air. Finally, not many large molecules 
have sufficient vapor pressure to co-exist with chlorine dioxide gas. 
Thus, compounds frequently available for reaction in natural water (e.g., 
proteins, certain amino acids, fumic acids and fulvic acids as well as 
most stabilizers) would not be found in the vapor state. 
Lovely (U.S. Pat. No. 3,591,515) discloses powdered compositions which may 
be formulated to release 10-10,000 ppm of chlorine dioxide gas. The 
liberated chlorine dioxide gas is disclosed to be useful to kill bacteria 
and prevent fungus growth on fruit during shipment. 
Due to the handling difficulties associated with chlorine dioxide, the 
differences in its gas phase and solution chemistry, and the 
inconsistencies in the above-cited work, chlorine dioxide gas has not been 
demonstrated to possess utility as a chemosterilizing agent at any 
concentration. 
Accordingly, it is an object of the present invention to utilize chlorine 
dioxide gas as a chemosterilizing agent, i.e., as a sporicide, for a 
variety of materials commonly used for medical and dental implements and 
products. 
It is another object of the present invention to utilize chlorine dioxide 
gas as a chemosterilizer at short exposure times and at near ambient 
temperature and near ambient pressures. 
It is a further object of this invention to provide a method for 
sterilizing surfaces contaminated with spores by subjecting the surface to 
an atmosphere of controlled humidity either concurrently with or prior to 
application to the surface of sporicidal amounts of chlorine dioxide gas. 
It is another object of the present invention to utilize chlorine dioxide 
as a chemosterilizing agent for materials such as medical implements which 
are sealed within gas permeable wrappings. 
It is a further object of the present invention to utilize chlorine dioxide 
gas as a chemosterilizer for impermeable surfaces, which may be dried 
prior to sterilization. 
Other objects, advantages and novel features of the present invention will 
be apparent to those skilled in the art from the following description and 
appended claims. 
DETAILED DESCRIPTION OF THE INVENTION 
This invention provides a method for sterilizing microbiologically 
contaminated articles, such as the dry and gas impermeable surfaces of 
medical or dental implements or other articles contaminated with live 
bacteria and bacterial spores. 
The sterilant employed in the method described herein is gaseous chlorine 
dioxide, which is preferably employed in the form of a gaseous mixture of 
the chlorine dioxide in an inert carrier gas. A preferred inert carrier 
gas is nitrogen. In general, the concentration of chlorine dioxide 
sterilant in the inert carrier gas (e.g. nitrogen) may range from about 
1.0 mg/L (milligrams per liter) to about 300 mq/L, preferably from about 8 
mq/L to about 100 mg/L, and most preferably from about 10 mg/L to about 40 
mg/L. As will be described in greater detail below, the particular 
concentration of chlorine dioxide in the carrier gas selected for use will 
be a function of several factors, including the inherent ability of the 
particular spores or live bacteria to resist the action of the sterilant, 
as well as the exposure time, and humidity conditions under which the 
object to be sterilized is contacted with the gaseous sterilant. 
The relative humidity (RH) of an indoor environment rarely rises above 
about 60%, and is often below about 25% RH. Bacterial spores present on an 
essentially moisture-free substrate and exposed to low indoor ambient 
humidities will be in a low moisture or desiccated state. It is well 
recognized in the art that desiccated spores possess a high degree of 
resistance to chemical sterilizing agents. Thus, surfaces contaminated 
with desiccated spores will require substantially more rigorous 
sterilization conditions (e.g. higher sterilant concentration, longer 
exposure times, etc.), than would be required to sterilize the same type 
of spore in a non-desiccated state. 
In accordance with an embodiment of this invention, the susceptibility of 
desiccated spores to chemosterilization with chlorine dioxide gas is 
enhanced by exposing the spores to a gaseous atmosphere of controlled 
humidity immediately prior to and/or during exposure of the spores to the 
chlorine dioxide gaseous sterilant. By enhancing the susceptibility of the 
spores to the sterilant, one may advantageously employ lower 
concentrations of chlorine dioxide gas and/or shorter exposure times than 
would be required if the foregoing humidification procedure were not 
employed. The relative humidity of the gaseous atmosphere employed in 
accordance with the humidification procedure of this invention will, of 
course, be substantially higher (e.g., 10%-15% higher) than the ambient 
humidity conditions to which the desiccated spores were exposed prior to 
sterilization. 
In a preferred embodiment of the humidification procedure of this 
invention, desiccated spores are briefly humidified by exposure to highly 
humid air having a relative humidity of above about 60%, e.g., 70% to 
about 95% for at least about 15 minutes, and preferably for about 20 
minutes to about one or more hours, immediately prior to the step of 
exposing the spores to the gaseous sterilant. Humidification may be 
conducted at about room temperature, although lower or higher temperatures 
(e.g. 25.degree.-30.degree. C.) may be employed if desired. It should be 
noted that although humidification is preferably conducted with humid air, 
other humid gases, such as humidified nitrogen, etc., may be employed. 
The concentration of chlorine dioxide gas employed in conjunction with the 
foregoing humidification procedure preferably ranges from about 10 mg/L to 
about 40 mg/L. The article undergoing sterilization is preferably exposed 
to the gaseous sterilant for at least about 1 to about 4 hours. Moreover, 
as described in greater detail below, when humidification is conducted in 
a closed exposure chamber, the chlorine dioxide gas may be introduced into 
the chamber while it still contains the humid air employed during the 
humidification procedure. 
The humidification procedure may be carried out using conventional 
apparatus. For example, the article contaminated with spores and/or live 
bacteria may be placed in a closed chamber and a vacuum drawn on the 
chamber. Water may then be injected into the evacuated chamber and 
vaporized to provide the desired humidity level in the chamber. After the 
spore contaminated article has been exposed to the humid atmosphere 
provided in the chamber for at least about 15 minutes, the chlorine 
dioxide sterilant gas is introduced into the humidified atmosphere of the 
chamber. The bacterially contaminated material is exposed to a gaseous 
environment of the gaseous sterilant for a period sufficient to sterilize 
the materials being treated, e.g., for about 30 minutes to about 24 hours, 
and preferably about 1 hour to about 3 hours. 
In further embodiments of the humidification procedure of this invention, a 
stream of moist air and a separate stream of chlorine dioxide in an inert 
carrier gas may be introduced at the same time into an exposure chamber 
containing the spore-contaminated article. Alternatively, chlorine dioxide 
in an inert gas carrier may be mixed with water vapor or moist air, and 
the humid gaseous chlorine dioxide mixture introduced into the exposure 
chamber to sterilize the spore-contaminated article. Sterilization with 
the humid gaseous chlorine dioxide mixture is preferably conducted at 
about room temperature to about 30.degree. C., and the relative humidity 
of the gaseous chlorine dioxide is preferably above about 60%, and most 
preferably about 70-95% RH. The contaminated article is preferably 
contacted with the humid sterilant for about 1 hour to about 4 hours, 
although longer exposure times may be employed if desired. 
For commerical applications, it is desirable to employ a relatively narrow 
range of exposure times and gaseous chlorine dioxide concentrations to 
obtain about a 100% confidence level that the article has been 
sterilized--without the need to adjust exposure times and/or chlorine 
dioxide concentrations on the basis of the ambient humidity conditions to 
which the article has been exposed prior to sterilization. The 
humidification procedure disclosed herein aids in standardizing the 
susceptibility of spores to sterilization. This facilitates commercial 
usage of a narrow range of chlorine dioxide concentrations and a narrow 
range of exposure times to reproducibly sterilize articles, without regard 
to the ambient humidity conditions to which the spores contaminating the 
article have been exposed prior to sterilization. 
Light catalyzes the decomposition of chlorine dioxide to chlorine and 
oxygen, and possibly other species. The oxygen and chlorine decomposition 
species of chlorine dioxide are far less effective sterilants than 
chlorine dioxide itself. In addition, chlorine is a corrosive substance 
which is incompatible with rubbers, plastics, and other materials which 
may be sterilized in accordance with this invention. By conducting the 
sterilization process of this invention in the dark or in very subdued 
light, the potential reduction in efficacy of the gaseous sterilant due to 
decomposition of the chlorine dioxide into less effective species as well 
as the disadvantages associated with the corrosive properties of chlorine 
are minimized. 
The process disclosed in this application may be employed to sterilize a 
wide variety of microbiologically contaminated articles. In particular, 
the process of this invention may be employed to sterilize articles formed 
from glass, cellulosics, plastics, or the like which provide an 
essentially moisture-free substrate (e.g., a substrate with a less than 
about 10% moisture content or a substrate having some desiccated spores) 
for bacterial growth under ambient conditions. For example, medical or 
dental or other articles formed from any one or more of the following 
commonly employed materials may be sterilized in accordance with the 
process of this application: aluminum, aluminum oxide, chromed brass, 
cotton, gauzes (or cellulosics), copper, polyesters, ethylene vinyl 
acetate, latex, "Mylar", "Neoprene", nickel plated cold formed steel, 
"Nylon", platinum, polycarbonates, polyethylene, polymethylmethacrylate, 
polypropylene, styrene, Teflon, polyurethane, polyvinylalcohol, 
polyvinylacetate, polyvinyl chloride, pyrolytic and vitreous carbons, 
silicones, stainless steels, sterling silver, titanium, tungsten carbide, 
"Tygon", glass, ceramics, etc. 
The sterilization process of this invention may also be employed to 
sterilize articles contained in packaging which is permeable to gaseous 
chlorine dioxide, and preferably packaging which is also permeable to 
moisture. For example, this process may be employed to sterilize medical 
or dental implements which have been packaged in gas permeable packaging 
under non-sterile conditions. A wide variety of conventional packaging 
materials are readily permeated by chlorine dioxide gas, including coated 
and uncoated paper, plastic sheeting, etc. 
The chlorine dioxide gas may be prepared by any of the methods known in the 
art. A preferred method involves passing a stream of air diluted chlorine 
gas or nitrogen diluted chlorine gas at a metered rate through a column of 
finely divided sodium chlorite, and into a partially evacuated chamber. 
That procedure is disclosed in H. Grubitsch, E. Suppan, Monatsh., Vol. 93, 
p. 246 (1962), which is incorporated herein by reference. 
A second suitable method for generating chlorine dioxide gas is the 
reaction of sodium chlorite solutions in the presence of acids. In one 
embodiment of this method a dilute solution of aqueous potassium 
persulfate is treated with a dilute solution of aqueous sodium chlorite at 
ambient temperatures, i.e., at 20.degree.-30.degree. C., in a closed 
reaction vessel. See Rosenblatt et al., J. Org. Chem., 28, 2790 (1963). 
The temperature of the chloride dioxide atmosphere which forms in the 
space above the stirred reaction may be adjusted by external heating or 
cooling. 
The desired amount of chlorine dioxide gas is admitted into a suitable 
exposure chamber which preferably has been partially evacuated, and which 
contains the objects to be sterilized. The chlorine dioxide gas is 
admitted into the exposure chamber in admixture with a carrier gas which 
is inert to (i.e. nonreactive with) chlorine dioxide at the concentrations 
which are used for sterilization. The final internal pressure may be 
adjusted, i.e., to near one atmosphere, with nitrogen, argon or another 
inert gas. At the end of the exposure period, the exposure chamber is 
evacuated to remove the chlorine dioxide and flushed with filtered inert 
gas or air. The evacuated chlorine dioxide may be easily destroyed by 
passing it through a reducing agent, for example, by passing it through a 
column of sodium thiosulfate chips. 
The composition of the chlorine dioxide atmosphere employed for various 
sterilization runs may be determined colorimetrically by any of the 
standard methods, for example by the method of Wheeler, et al., Microchem. 
J., 23, p. 160, (1978). A sample of the atmosphere inside the exposure 
chamber is obtained via a septum port using a gas-tight syringe. The 
volume of the sample is varied depending on the anticipated concentration 
of chlorine dioxide in the atmosphere. The atmosphere is preferably 
monitored at the beginning, and at the end of the exposure period. The 
syringe contents are injected into a suitable container, i.e., a cuvette, 
holding an appropriate volume of chemicals which react to result in a 
chlorine dioxide concentration-dependent color. After completion of the 
reaction, the absorbance of the solution at an appropriate wavelength is 
measured and the concentration of chlorine dioxide determined via a 
reference curve. The method may generally be adapted to employ any of the 
well known colorimetric methods of analyzing for chlorine dioxide. 
The spores of the standard test organism employed to determine the 
effective sterilizing concentration of chlorine dioxide gas in certain of 
the specific examples set forth below were those of Bacillus subtilis var. 
niger (ATCC 9372). The dry spores of this organism are known to be 
extremely resistant to sterilization and have been often used to measure 
the effectiveness of gaseous sterilizing agents. See, e.g. P. M. Borick 
and R. E. Pepper, The Spore Problem, in Disinfection, M. A. Benarde, Ed., 
Marcel Decker, Publ., N.Y. (1970) at pages 85-102 and A. M. Cook and M. R. 
W. Brown, J. Appl. Bact., 28, 361 (1965), the disclosures of which are 
incorporated herein by reference. Therefore, a given concentration of 
chlorine dioxide may be rated effective as a sterilizing agent if an 
initial population of 10.sup.5 -10.sup.7 spores showed no growth on the 
nutrient medium after nine days observation following exposure to said 
concentration. 
In the detailed Examples which follow, standard suspensions of spores of B. 
subtilis var. niger were prepared as described by Dadd and Daley in J. 
Appl. Bacteriol., 49, 89 (1980), which is incorporated herein by 
reference. Test paper strips for incubation were prepared by adding 0.2 ml 
of a methanolic suspension of the spores to 7.times.35 mm strips of 
presterilized Whatman 3 mm paper in glass Petri dishes. The papers were 
vacuum-dried (30 min. at 30.degree. C. and 28 in. Hg) and kept at ambient 
temperature and humidity (20.degree.-30.degree. C., 40-60% relative 
humidity) prior to use. The spore load on each strip prepared in this way 
was approximately 1.4.times.10.sup.6 spores. 
Metal foil test pieces were prepared by fashioning 18.times.28 mm square 
aluminum foil into small cups. These were sterilized in glass Petri 
dishes. To each cup was added 0.2 ml of a methanolic suspension of the 
spores. The cups were dried at ambient temperature and held at ambient 
temperature and humidity prior to use. The spore load on each cup was 
approximately 1.4.times.10.sup.6 spores. 
The practice of the invention will be further illustrated by reference to 
the following detailed examples.

EXAMPLE 1 
A 1000 ml 2-necked round-bottomed flask was equipped with a dropping funnel 
and magnetic stirring. A inlet tube for nitrogen gas equipped with a glass 
wool filter and a needle valve was positioned so that nitrogen could be 
admitted below the surface of the reaction mixture. An outlet tube was 
equipped with a needle valve and positioned so that gas could be allowed 
to pass from the top of the reaction vessel into the exposure vessel. 
A 2000 ml glass reaction kettle equipped with a septum-capped port, a 
manometer, and inlet and outlet ports was employed as the exposure vessel. 
The outlet tube of the 1000 ml flask was connected to the inlet port of 
the exposure vessel. 
In a typical run the 1000 ml flask was charged with 100 ml of an 8% aqueous 
sodium chlorite solution under nitrogen. All of the valves were closed and 
a solution of 2.0 g potassium persulfate in 100 ml of water was added 
dropwise with stirring. The reaction mixture was stirred for 30-45 minutes 
at 27.degree. C. to complete the generation of the chlorine dioxide gas. 
The relative humidity of the chlorine dioxide gas generated by the 
foregoing procedure was about 60%. 
The exposure chamber was loaded with 3-6 spore-coated paper strips or 
aluminum foil cups, each contained in an individual glass Petri dish. The 
spore-coated paper strips and aluminum foil cups were prepared from 
methanolic suspensions of spores of B. subtilis, in the manner described 
on page 11. It has been reported in the literature that exposure to 
methanol may enhance the sensitivity of the spores to chemosterilization. 
The chamber was swept with nitrogen, closed and then evacuated (approx. 28 
in. Hg). The outlet valve on the tube leading from the reaction vessel was 
opened, and the amount of chlorine dioxide gas admitted from the reaction 
vessel was controlled by following the increased pressure readings on the 
manometer. The outlet valve was closed and the pressure in the exposure 
vessel was then brought to one atmosphere by admission of nitrogen. 
The atmosphere in the exposure vessel was immediately sampled by removal of 
0.5-2.0 ml of the atmosphere by means of a gas-tight syringe via the 
septum. The chlorine dioxide concentration was determined by the method of 
Wheeler, et al., Microchem. J., 23, 160 (1978). After 60 minutes had 
elapsed the atmosphere was sampled again. The exposure chamber was then 
evacuated and refilled with filtered air. The evacuation and refilling 
steps were repeated, the chamber was opened and the contents removed under 
sterile conditions. 
The paper strips were aseptically transferred to individual tubes of 
trypticase soy broth and incubated at 37.degree. C. Observations to 
determine the presence or absence of spore growth were made after 24 and 
48 hours. Those tubes which did not show growth after 48 hours were 
incubated for one week and observed every 24 hours. If no growth was 
observed after one week, the strip was recorded as negative, or 
sterilized. 
After exposure, the foils were transferred into individual tubes containing 
20 ml of sterile water and a few glass beads. After vigorous shaking to 
dislodge and suspend the spores, 0.1 ml of the suspension was placed in 
duplicate on a plate of trypticase soy agar. The plates were incubated at 
37.degree. C. and observed as described above for the paper strips. 
Appropriate control strips and foils were run for these determinations. 
The outcome of 18 specific runs is summarized in Table I as Examples 2-19. 
TABLE I 
______________________________________ 
CHLORINE DIOXIDE STERILIZATION 
Chlorine Results* 
Example Dioxide (mg/L) Strips Foil Cups 
______________________________________ 
2 11 0/6 0/6 
3 12 0/6 0/6 
4 25 0/6 0/6 
5 31 1/6 0/6 
6 34 0/6 5/6 
7 35 1/6 0/6 
8 40 0/6 0/6 
9 41 0/6 0/6 
10 44 0/5 0/6 
11 45 0/6 0/6 
12 46 0/6 0/6 
13 65 0/6 0/6 
14 69 1/6 0/6 
15 78 0/6 0/6 
16 84 0/6 0/6 
17 94 0/6 0/6 
18 98 0/6 0/6 
19 113 0/6 0/6 
______________________________________ 
*Exposure time 1 hr. Results in number of strips or cups on which growth 
is observed/number of strips or cups exposed. 
The results of Examples 2-19 demonstrate that a chlorine dioxide 
concentration of at least 40 mg/L was effective to sterilize paper strips 
contaminated with dry B. subtilis spores, and thus, presumably, to kill 
any other microorganisms present. The scattered incidences of growth 
observed in Examples 5, 7 and 14 may be largely discounted as due to 
random experimental error. It is expected that more rigorous control of 
the laboratory procedures of the biological standards would demonstrate 
effective sterilization over the complete range of gas concentrations 
employed. Similar concentrations would be expected to sterilize other 
types of porous organic surfaces, such as rubber, gas permeable plastic, 
sponge, plant material, wood and the like, without causing appreciable 
decomposition or residue deposition. 
A concentration of chlorine dioxide of at least 35 mg/L was adequate to 
sterilize aluminum foil contaminated with dry spores. The growth observed 
on foil in Example 6 was probably due to a ramdom experimental error, 
since a range of lower gas concentrations consistently resulted in 
sterilization. These results led to the expectation that other nonporous 
surfaces normally impermeable to gas sterilizing agents would be readily 
sterilized under similar conditions, such as those of medical or dental 
instruments or implements formed from metals such as stainless steels, 
plated steel, aluminum and nickel or from nonporous plastics, porcelain, 
ceramics, or glass. 
Chlorine dioxide gas has also been successfully employed to sterilize 
commercially-available spore strips which are sealed in gas permeable 
paper envelopes. A procedure which may be used to sterilize such materials 
is described below. 
EXAMPLE 20 
Six Spordi.RTM. paper spore strips (American Sterilizer Corp., Erie, Pa.), 
each containing a mixture of spores of B. subtilis and B. 
stearothermophilus (NCTC 10003) and each enclosed in a sealed, sterile 
envelope of glassine paper are exposed to atmospheres containing 50 and 
100 mg/L of chlorine dioxide gas as described in Example 1. The sealed 
spore strips are removed from the exposure chamber, opened under sterile 
conditions and incubated as described in Example 1. Growth levels are 
observed after nine days of incubation which indicate that the strips are 
effectively sterilized under these conditions. 
It is, therefore, expected that chlorine dioxide will effectively sterilize 
contaminated surfaces which are sealed in gas permeable container 
materials such as coated and uncoated paper, plastic sheeting, and the 
like without significantly reacting with the container materials. The 
ability of effective concentrations of chlorine dioxide to readily 
permeate such enclosures would find application in the sterilization of 
medical products which are preferably sterilized after packaging so as to 
be maintained in a sterile condition during shipping and storage. 
EXAMPLE 21 
In this experiment 72 Spordi.RTM. paper spore strips (American Sterilizer 
Corp., Erie, Pa.) were employed. Each strip was enclosed in a glassine 
envelope, and the strips were contaminated with a mixture of spores of B. 
subtillis and B. stearothermphilus (NCTC 10003). The strips in their 
glassine envelopes were equally divided into three sets--Set I was stored 
in the normal laboratory atmosphere, Set II was placed in a chamber at a 
controlled relative humidity of 33%, while Set III was placed in a 
desiccator over Drierite. The strips were permitted to equilibrate with 
their environment for three days. Upon conclusion of the storage period, 
the strips in Sets I, II and III were equally divided into two Groups A 
and B, and then exposed to chlorine dioxide in the darkened interior of an 
exposure chamber in the manner described below. 
The strips in Group A were exposed to high humidity conditions in the 
exposure chamber at about 27.degree. C. Humidification was performed by 
drawing a vacuum of about 27 in. Hg on the two-liter exposure chamber, and 
then injecting about 0.1 ml of distilled water into the evacuated chamber. 
The relative humidity in the exposure chamber was above about 70%. The 
strips in Group A were exposed to the humid conditions in the chamber for 
about 20 minutes. Chlorine dioxide, at the desired concentration level, 
was introduced into the chamber without prior evacuation of the moist air 
from the chamber. The strips were exposed to the chlorine dioxide at about 
27.degree. C. for about 2 hours. 
The strips in Group B were exposed to chlorine dioxide in the same manner 
as those in Group A, with the exception that the strips in Group B were 
not subjected to the preliminary humidification procedure described above 
with respect to the strips in Group A. The relative humidity of the 
chlorine dioxide/carrier gas employed to treat Group B ws less than about 
10%. The chlorine dioxide employed to treat both the strips in Group A and 
Group B was generated by passage of chlorine gas through finely divided 
dry sodium chlorite. 
After sterilization the spore strips were removed from the exposure chamber 
under sterile conditions, and incubated in the manner described in Example 
1. The results are summarized in Table II, in terms of the number of 
strips in which bacterial growth was observed/number of strips treated. 
TABLE II 
______________________________________ 
Set I Set II Set III 
mg/L, ClO.sub.2 
(normal) (33% RH) (desiccator) 
______________________________________ 
Group A - (humidification) 
33.6 0/4 0/4 0/4 
29.0 0/4 0/4 0/4 
9.1 3/4 2/4 3/4 
Group B - (non-humidification) 
61.7 4/4 0/4 4/4 
59.9 4/4 0/4 4/4 
38.1 1/4 0/4 4/4 
______________________________________ 
Table II illustrates that the humidified spores in Group A were sterilized 
by the action of the chlorine dioxide gas at concentrations of about 29 
mg/L and above. In contrast, the spores in Group B (Sets I and III) which 
were treated with a dry chlorine dioxide gas and not humidified prior to 
application of the sterilant, were far more resistant to the chlorine 
dioxide gas. The bacterial growth noted with respect to the strips in 
Group B (Set I) is attributed to the low humidity conditions in the 
laboratory (estimated to be about 15% RH) to which the strips were exposed 
prior to sterilization, coupled with the fact that the strips were treated 
with a dry chlorine dioxide sterilant (less than about 10% RH). 
Table III presents data on B. subtilis contaminated paper strips and 
aluminum foil cups. The foil cups and paper strips were contaminated in 
the manner described above on page 11, except that an aqueous spore 
suspension was employed as the contaminant. The spore-contaminated paper 
strips and foil cups were exposed to the various concentrations of 
chlorine dioxide specified in Table III in the darkened interior of the 
exposure chamber. The exposure time was two hours. No control over 
humidity conditions was exercised. In this trial the chlorine dioxide gas 
was generated in a dry form by passage of chlorine through a column of dry 
finely divided sodium chlorite. In additon, the foil cups and paper strips 
were dry and maintained for several days at room temperature and 
relatively low ambient humidity conditions (less than about 15% RH). 
Accordingly, the apparent resistance of the spores to the sterilant as 
shown by Table III is believed to be due to the combination of the use of 
a dry sterilant gas to treat spores which were in a relatively desiccated 
form, and, hence resistant to sterilization. 
TABLE III 
______________________________________ 
TEST RESULTS - GRADED SPORE LOADS* 
Foil Cups 
ClO.sub.2 
mg/L 10.sup.6 * 10.sup.5 
10.sup.4 
10.sup.3 
10.sup.2 
______________________________________ 
Spore Load 
50.8 4/4** 4/4 4/4 0/4 0/4 
36.3 4/4 2/4 4/4 0/4 0/4 
23.4 4/4 0/4 0/4 0/4 0/4 
19.1 4/4 4/4 2/4 3/4 1/4 
14.5 4/4 3/4 4/4 1/4 0/4 
12.7 4/4 4/4 4/4 0/4 1/4 
7.3 4/4 3/4 4/4 4/4 0/4 
5.8 4/4 4/4 4/4 4/4 3/4 
4.4 4/4 4/4 4/4 4/4 4/4 
Paper Strips 
50.8 4/4 4/4 0/4 0/4 0/4 
36.3 4/4 4/4 0/4 0/4 0/4 
23.4 4/4 2/4 1/4 0/4 0/4 
19.1 4/4 4/4 2/4 0/4 0/4 
14.5 4/4 4/4 1/4 0/4 0/4 
12.7 4/4 3/4 0/4 0/4 0/4 
7.3 4/4 4/4 3/4 0/4 0/4 
5.8 4/4 4/4 0/4 1/4 0/4 
4.4 4/4 4/4 2/4 0/4 0/4 
______________________________________ 
*Pre-sterilization number of spores on the contaminated article. 
**number grew/number exposed. 
EXAMPLE 22 
390 glass cups coated with spores of B. subtilis were equally divided into 
Groups A and B. Group A was exposed to chlorine dioxide generated by the 
dry method described in Example 21, without any control over humidity 
conditions. The contaminated glass cups in Group B were humidified and 
then exposed to the chlorine dioxide gas in accordance with the procedure 
of Example 21. Both Groups A and B were exposed to the gaseous sterilant 
in a closed and darkened chamber for about 2 hours. The data is given in 
Table IV, wherein the numbers with superscripts (10.sup.6, etc.) indicate 
the pre-sterilization number of spores per cup, while the numbers given in 
the body of Table IV represent the number of glass cups out of the 6 cups 
sterilized that produced live bacteria upon culturing subsequent to 
sterilization. 
TABLE IV 
______________________________________ 
ClO.sub.2 
mg/L 10.sup.6 10.sup.5 
10.sup.4 
10.sup.3 
10.sup.2 
______________________________________ 
Group A 
NO PRE-HUMIDIFICATION 
114/3 6/6* 6/6 3/6 0/6 0/6 
74.4 6/6 6/6 6/6 5/6 0/6 
61.7 6/6 6/6 4/6 3/6 1/6 
45/4 6/6 6/6 6/6 5/6 0/6 
29.0 6/6 6/6 6/6 5/6 1/6 
24.6 6/6 6/6 6/6 6/6 4/6 
8.2 6/6 6/6 6/6 6/6 6/6 
3.6 6/6 6/6 6/6 6/6 6/6 
Group B 
PRE-HUMIDIFIED 
119.7 0/6 0/6 0/6 0/6 0/6 
47.2 0/6 0/6 0/6 0/6 0/6 
30.8 0/6 0/6 0/6 0/6 0/6 
6.3 0/6 0/6 0/6 0/6 0/6 
4.0 0/6 0/6 0/6 0/6 0/6 
______________________________________ 
*Number grew/number exposed; sterilization conducted at 27.degree. C. 
EXAMPLE 23 
In this experiment, the test items were prepared by placing 0.1 ml of an 
aqueous suspension of B. subtilis var. niger spores in small glass cups (9 
mm.times.14 mm). The cups were vacuum dried for 2 hours in sterile Petri 
plates each containing 6 test cups. Each test cup carried a spore load of 
approximately 1.times.10.sup.6 spores. The chlorine dioxide used for 
sterilizing was prepared by passing chlorine through dry sodium chlorite. 
Test cups were sterilized at the temperatures, exposure times and 
humidities shown in the following tables. 
TABLE V 
______________________________________ 
30 MINUTE EXPOSURE TO 
CHLORINE DIOXIDE GAS* 
ClO.sub.2 Temperature 
mg/L 27.degree. C. 
37.degree. C. 
______________________________________ 
20 0/6** 
17 6/6 
12 3/6 
10 6/6 
7 6/6 
6 4/5, 6/6 
5 3/6 6/6 
4 2/6 
3 6/6 
______________________________________ 
*Chamber and contents humidified with 0.1 ml water at 27 in. Hg vacuum fo 
20 minutes prior to introduction of chlorine dioxide at stated 
concentration into the chamber. The relative humidity in the chamber was 
about 80%. 
**Values given as number of cups showing growth/number of cups exposed. 
TABLE VI 
______________________________________ 
60 MINUTE EXPOSURE TO 
CHLORINE DIOXIDE GAS* 
ClO.sub.2 Temperature 
mg/L 15.degree. C. 
27.degree. C. 37.degree. C. 
______________________________________ 
32 5/6** 
27 2/6 
23 3/6 
22 1/6 
21 1/6 
19 3/6 1/6 
18 2/6 5/6 
17 1/6 
16 0/6 
15 6/6 
14 6/6 
11 6/8 
6 0/6 5/6 
5 4/6 
2 1/6 4/5 
1 3/6 3/6 5/6,6/6 
______________________________________ 
*Chamber and contents humidified with 0.1 ml water at 27 in. Hg vacuum fo 
20 minutes prior to introduction of chlorine dioxide gas into the chamber 
The relative humidity in the chamber was about 80%. 
**Values given as number of cups showing growth/number of cups exposed. 
TABLE VIII 
______________________________________ 
120 MINUTE EXPOSURE TO 
CHLORINE DIOXIDE GAS* 
ClO.sub.2 
Temperature 
mg/L 15.degree. C. 
27.degree. C. 
37.degree. C. 
37.degree. C.** 
______________________________________ 
67 2/12*** 
51 0/6,0/6 
47 0/12 
44 6/12 
39 0/6 
31 6/12 
30 1/12 
21 9/12 
20 0/6 
19 0/12 6/6**** 
18 7/12 
17 10/12 
16 6/6,0/6 
15 1/12 0/12,0/12, 
2/12 
0/12 
14 7/12 
13 6/6,2/6 
12 10/12 
10 0/6 8/12 
9 9/12 
8 2/12,0/12, 
12/12 0/6,0/6 
0/12 
7 5/12 
6 0/6,0/6 
5 0/7 
3 10/12,12/12, 
2 12/12 
1 12/12 
0.5 12/12 
0.2 11/12 
______________________________________ 
*Chamber and contents humidified with 0.1 ml water at 27 in. Hg vacuum fo 
20 minutes prior to introduction of the chlorine dioxide gas. The relativ 
humidity of the chamber was about 80%. 
**Humidification with 0.2 ml of wtaer at 27 in. Hg vacuum for 60 minutes, 
RH was about 90%. 
***Values given as number of cups showing growth/number of cups exposed. 
****Data believed to be due to experimental error. 
The data presented above indicates that under the humidification procedures 
employed, optimum sterilization was obtained at sterilization temperatures 
of 27.degree. C. with exposure times of two hours. Although bacterial 
growth was noted during the trials conducted at 15.degree. C. and 
37.degree. C., it is believed that sterilization could be achieved at 
those temperatures by extending the chlorine dioxide exposure time. The 
data presented also indicates that chemosterilization by chlorine dioxide 
is not enhanced either by conducting the process at temperatures above 
(37.degree. C.) or below (15.degree. C.) about room temperature (e.g. 
20.degree.-30.degree. C.). 
EXAMPLE 24 
A dry mixture of nitrogen and chlorine dioxide gas suitable for use as a 
gaseous chemosterilant is prepared as follows: 
Chlorine gas from a standard cylinder fitted with a needle valve regulator 
is introduced slowly by means of a tee into a stream of nitrogen. The 
stream of nitrogen diluted chlorine is then passed over finely divided 
sodium chlorite contained in a series of three columns. The first two 
glass columns consist of gas drying bottles packed with sodium chlorite to 
provide columns measuring 3.5.times.14 cm. The third column in the series 
consists of a glass tube packed to provide a column measuring 1.times.40 
cm. The gas exiting the last column may be introduced directly into an 
evacuated exposure chamber (e.g. approx. 27 in. Hg), or into an evacuated 
flask for later use. 
While certain representative embodiments of the present invention have been 
discussed herein for the purpose of illustrating the present invention, it 
will be appreciated by those of ordinary skill in this art that 
modifications thereof may be made without departing from the scope and 
spirit of the present invention.