0GAS STERILIZATION WITH ETHYLEA-\NE OXIDE,

p)hen1YmannoSid(ase Of Streptlo iyces grisetus. Biochem..J., 57, 93-98.

PERLMAN, 1). AND LANGLYKKE, A. F. 1948 The occuirreineof mannosidostreptomvcinase. J. Am. Chemn. Soc., 70,3968.

TAKEDA, K. AND WATARt, S. 1949 OIn the effect of i'oIn aII(other metals acnd oils on the production of streptomvcinaind chloromN-cetin. J. Antibiotics (Japan), Ser. A, 2,88-91.

UMEZAWA, H., KAMETANI, R., OSATO, T., KANARI, H.,KAWAHARA, A., WADA, R., AND ()KAMI, Y. 1950 StuidiesoIn the p)ilot plant scale prodtictioni of streptomycin invariouis fermentors. J. Antibiotics (Japan), Ser. A, 3,16-19.

WOODRUFF, H. B. AND AIC1)ANIEL, L. E. 1954 StreptomyciIn.In Industrial fetrmentations, Vol. II, p. 270. L,dite(1 byL. A. Undeikofler and R. J. Hickey. Chemical Publish-ing Co., Inc., New York, New York.

Simple Improvised Chambers for Gas Sterilization withEthylene Oxide

DONALD G. SCHLEY, ROBERT K. HOFFMAN, AND CHARLES R. PHILLIPS

U. S. Aitioy Chenmical Corps, Fort Detrick, Maryland

Received for publication June 22, 1959

This paper describes readily available simiiple ex-posure chambers in which material or equipment maybe sterilized by ethylene oxide gas. Although specializedautomatic equipment is now available for such purposes(Perkins, 1956; Stryker, 1958), occasions may arisewhere it it desirable (a) to adapt laboratory equipmenit,such as a standard autoclave so that it can be used withethylene oxide, without interfering with its regular iuse,or (b) to have cheap, mobile equipment that can bereadily transported to outside locations where one hasmaterial to sterilize. The present availability of nioni-flammable ethylene oxide mixtures packaged in lightweight aerosol bomb type cans makes it quite feasibleto utilize such simple exposure chambers and, as thedata presented show, material can be readily sterilizedin such devices.

Ethylene oxide is a gas at room temperature and 760mm Hg pressure, but is readily liquefied because ofits relatively high boiling point (10.8 C). Keeping theliquefied compound in simple containers presents asafety hazard in both storage and use, since the vaporis highly flammable in air in any concentration between3 and 80 per cent by volume. For many years thecompound has been commercially available, for fumi-gation or sterilization purposes, in combination withcarbon dioxide,1 an inert diluent which renders themixture nonflammable when present in air in anly pro-portion (Jones and Kennedy, 1930). Because of thehigh internal pressure exerted by the carbon dioxide,this mixture can be conitained onily in heavy steelcyiinders which are both costly and awkward to handle.Numerous improvised experimental setups have been

described in the literature on the use of ethylene oxide,I Carboxide; product of Carbide and Carbon Chemical

Companiy, New York, New York.

alone or in combination with carbon dioxide, to sterilizematerials or equipmenit. For example, Yesair anidWilliams (1942) sterilized spices under vacuum in analuminum kettle containing ethylene oxide gas anid Iioinert diluent. Kaye and Surkiewicz (1952) sterilizedequipment in bags or under tarpaulins made of gastight coated fabrics. The pure ethylene oxide gas wasadmitted into the system by crushing glass ampules ofthe liquid chemical or by releasing it from metal cyl-inders. Fulton and MIitchell (1952) used an ethyleneoxide-carboni dioxide mixture when experimenting withsuch improvised chambers as (a) a wooden box, (b) ahypochlorite bleach drum, and (c) a segment of anautomobile rubber inner tube in which footwear wassterilized. Newman et al. (1955) sterilized articles in astandard autoclave by use of an ethylene oxide-carbondioxide mixture admitted to 20 lb pressure from heavycylinders. In all of these experiments either a flamma-bility hazard existed when using undiluted ethyleneoxide or a second problem was created when admittinigan ethylene oxide-carbon dioxide mixture unider highpressure from awkward heavy cylinders inito smallconfined spaces which would not hold all of the vaporshould the entire contents of the cylinder be accidentlyreleased.The sterilant mixture used in the experiments de-

scribed herein was dev,eloped for the U.S. ArmyChemical Corps by the IDepartment of Agriculture(Haenni et al., 1959). It contains 11 per cent (by weight)ethylene oxide, 44.5 per cent dichlorodifluoromethane,and 44.5 per cent trichloromonofluoromethane. It ispackaged2 in 16 oz low pressure aerosol cans containing66 g (75 ml) of ethylene oxide. Fach aerosol can is

2 Pennsylvania Engineering Companv, Philadelphia, l?enni-sylvania.

19360] 15

D. G. SCHLEY, R. K. HOFFMAN, AND C. R. PHILLIPS

fitted with a 1-in. flat cap to which a needle valve witha can holder and hollow needle3 is attached to releasethe sterilant mixture (figure 1).The substitution of fluorinated hydrocarbons for

carbon dioxide as a diluent permits the ethylene oxideto be packaged as a nonflammable mixture in lightweight aerosol-bomb type cans. The use of the fluo-rinated compounds with their higher molecular weightsalso permits a higher concentration of ethylene oxidein the vapor state than can be achieved with the carbondioxide mixture. Although the weight percentage fornonflammable formulations are approximately the samein the two mixtures, the molar percentage of ethyleneoxide and hence the volume percentage of its vapor isabout 2.7 times as high with the fluorinated hydro-carbon mixture as it is with Carboxcide.The use of an ethylene oxide-fluorinated hydrocarbon

mixture was first reported by Skeehan et al. (1956), intheir paper on the sterilization of ophthalmic instru-ments in an autoclave simply converted for the purposeby the Fort Detrick investigator in this joint study.Since then, at least two ethylene oxide-fluorinatedhydrocarbon mixtures have become available on thecommercial market.4 Each of these mixtures containsapproximately the same amount of ethylene oxide asthat used for the work reported herein but containsdifferent proportions of the two fluorinated hydrocarbondiluents.

Three quite simple exposure chambers, which utilize

3 Can-O-Gas Valve; manufactured by the Virginia SmeltingCompany, West Norfolk, Virginia. Dual Charging Can Valve;manufactured by the Superior Valve and Fittings Company,Pittsburgh, Pennsylvania.

4Cryoxcide; product of American Sterilizer Company, Erie,Pennsylvania. Steroxcide; product of Wilmot Castle Company,Rochester, New York.

Figure 1. Can holder, aerosol can, and holder attached tocan in operating position.

the low-pressure nonflammable ethylene oxide mixture,are described below with data on their performance.

MATERIALS AND METHODSExposure Chambers

Autoclave. The autoclave was selected as aii ethyleneoxide sterilization chamber because of its universalavailability and ease of modification. The autoclave wasmodified to permit the use of either steam or ethyleneoxide gas. A valve was installed on each pipeline at-tached to the autoclave if the line was not equipped witha valve. By means of these valves all exhaust and drainlines could be closed to make the autoclave a sealedchamber. A pipe "tee" was installed on one of the ex-haust lines between the cutoff valve and the chamber.A needle valve with can holder and hollow needle wasattached to the pipe "tee" with 14 in. (o.d.) coppertubing. The can holder was attached in such a way thatit held the can of sterilant in an inverted position(figure 2). This position is mandatory as the cans arenot equipped with eductor tubes. The volume of theautoclave used in these experiments was 120 L so thatthe injection of 66 g of ethylene oxide gave a concentra-tion of 550 mg of the oxide per L of space.The procedure for sterilizing materials in the auto-

clave simply involved placing the contaminated itemsin the autoclave, closing the door and all valves and thenadmitting the sterilant. Air was not evacuated fromthe autoclave, nor was the temperature raised beforeadmitting the ethylene oxide. The sterilant mixture.s

L vg,>_L̂ ___. _ >_i. s_

s_- : . -|_ Ar_. N^ ¢_ 1 E | 1s1| i ....... l a _

19I11.. ..

Figure 2. Autoclave modified for gaseous #terilization

16 [VOL. 8

GAS STERILIZATION WITH ETHYLENE OXIDE

was admitted by attaching the can to the holder,screwing the can down until the hollow needle piercedthe cap, and then opening the needle valve.Drums. The two drums tested were unlined steel

drums of flash welded seam construction. The prinmarydifference between the two drums was the method ofclosure. The lid of one drum was sealed by a metal rinigwith a lever type lock known as Quik-Lox. The seconiddrum also was equipped with a steel ring but the rirngwas tightened with a nut and bolt. Both drums weresatisfactory as ethylene oxide sterilizing chambers.Each drum was modified by cutting two, u-in. holes

in the lid. To each hole was brazed a 14- to 5A-in. pipereducer. A pressure gauge was attached to one fitting(figure 3) and a needle valve, with can holder and aninductor tube, was attached to the second fitting. Aseries of tests was made to determine the maximumpressure to be encountered when the sterilant mixturewas added. The maximum drum pressure encounteredwas 6 psig; above this pressure, leakage occurredaround the drum head gasket. Preliminary experimentsindicated that more consistent bacteriological resultswere obtained if the drum was fitted with an inductortube. This tube permitted the halogenated hydrocar-bon-ethylene oxide formulation to be released in thebottom of the drum. As the formulation expandedwithin the drum, pressure increased and air was forcedout around the gasket. If no inductor tube were present,

more ethylene oxide would be forced out of the drumand a longer exposure time would be required.The adequacy of the drum to serve as a container

for ethylene oxide sterilization was found to dependupon the sealing qualities of the drum head gasket.Three types of gaskets were tested. One, made of5 6-in. thick sponge rubber was used only once or twicebefore it became compressed and thus nonserviceable.The second gasket was a hollow, hemispherical, seam-less, rubber or pliable plastic gasket made with fourlongitudinal ridges along its flattened side. It was usedeight times with no observable loss of sealing quality.The third gasket, a round hollow rubber type, was usedthree times with no apparent loss in serviceability.The 55-gallon drum has a volume of approximately210 L and therefore the addition of 66 g of ethyleneoxide gives a theoretical concentration of approxi-mately 320 mg of the oxide per L of space. Actually,the concentration of ethylene oxide within the drumwoWld be somewhat less than the theoretical 320 mg perL since some of the vapor will be forced out of thedrum with the air when the pressure surpasses 6 psigupon admitting the sterilant to the system.The procedure used to sterilize material in the drum

was somewhat similar to that used with the autoclave.Clothing and cloth patches contaminated with Bacillus-subtilis var. niger (B. globigii) spores were placed inthe drum, the drum closed, and the ethylene oxide canattached to the holder. The can was screwed down onthe hollow needle and the sterilant mixture admittedby opening the needle valve. The drum was then rolledon its side a few times to insure a homogenous distribu-tion of the gas throughout the container. The drumwas placed in an upright position during the steriliza-tion period.

Polyethylene bag. The simplest device used forethylene oxide sterilization was a large polyethylenebag. Preliminary tests with various plastics showedthat a 4 to 6 mil thick polyethylene film would retainethylene oxide gas for a period of time sufficient toachieve sterilization. Bags made from this polyethylene

Figure 3. Drum modified for gaseous sterilizationFigure 4. Polyethylene bags, empty and used as a sterilizing

container.

19601 17

8D. G. SCHLEY, R. K. HOFFM1AN, AND C. R. PHILLIPS

film were selected because they were commerciallyavailable as chemical drum liners, could be usedrepeatedly, stored for a long period of time, and re-usedwithout the bag losing its flexibility and gas holdingqualities. The bags used in these tests were 6 ft longand 2/1 ft wide when flat (figure 4). They were con-structed of tubular polyethylene and had a seam onlyat the bottom. The bag when closed had a volume ofapproximately 145 L and therefore the addition of66 g of ethylene oxide gave a concentration of approxi-mately 450 mg of the oxide per L of space.The procedure used to sterilize the material in the

bag was also quite simple. The clothing or equipmentwas placed in the bag. A needle valve with a can holder

TABLE 1Recover.y of Bacillus subtitiis tar. niger spores front instrumients

before and after exposuire to ethylene oxide

Polvethylene Bag Autoclave

Expo- No. of spores No. of sporessure Instrument reco, ered* Instrument recovered*Time

Beoe After Beoe Aftertreatment treat- treatment terment ment

hr

2 Hygrother- 110,000 2,140 Centri- 325,0001 53mograph fuge

2 it 830,000 20, 175 it 442,500 2254 it 159,000 0 " 460,000 04 19,500 0 " 24,350 06 i 46,500 0 " 58,500 0

2 pH meter 167,500 1,064 Colony 20,000 0coun-ter

2 i 985,000 18,000 it 1,085,000 04 tt 290,000 0 " 660,000 04 i 310,000 0 " 99,000 06 395,000 0 i 104,000 0

* Average of two samples.

TABLE 2

Recovery of Bacillus subtilis var. niger spores from cloth patchesbefore and after exposure to ethylene oxide

No. of Organisms Recovered fromExposure Control No. of Cloth Patches after Exposure*

Container Time Organisms perPatch-

Top Middle Bottom

hr

Bag 4 5,000,000 71 4 11Bag 4 5,000,000 478 183 270Bag 4 5,000,000 1,119 356.5 83.5Bag 4 5,000,000 397.5 176 85Bag 6 9,400,000 0 0 0Bag 6 9,400,000 0 0 0Drum 4 5,000. 000 339 183 270Drum 4 9,000,000 20 10 18J)rum 6 3,700,000 0 0 0Drum 6 9,400,000 0 0 0

* Average of two samples.

was attached to an ethylene oxide container, the valveclosed and the canl screNwed down until the hollowneedle punctured the cap. The can with the needlevalve attached was placed in the center of the bag butto onie side. The bag was closed by twisting approxi-mately 2 ft of the neck, folding it over on itself andtying it tightly with a piece of string. The closed bagwas positioned on its side and the can of sterilantgrasped from the exterior of the bag and the valveopenied while holding the can in an inverted position.When the bag was used as a container in which tosterilize clothing, it was rolled through an arc of 3600a few times to insure a thorough distribution of thedisinfectanit. When sterilizing delicate equipment, ofcourse, the bag could not be rolled. Following therolling procedure the bag was stored undisturbed atroom temperature for the desired exposure period.

Biological MlethodsThe adequacy of the container to serve as an ethylene

oxide sterilization chamber was determined by itsability to retain the oxide long enough to sterilizecloth patches or instruments contaminated with aviable spore suspension of B. subtilis var. niger. Thedegree of decontamination obtained on the test objectsserved as an index of the decontamination expectedfor similar items.

Cotton twill coveralls were used for bulk in thesetests. The contaminated cloth patches were placedstrategically through the clothing in the respectivechambers so that it could be assured that the ethyleneoxide penetrated the bulky clothing. Samples weretaken before and after the sterilization procedure andassessed using the standard bacteriological pour platetechnique.

RESULTS

Table 1 shows that the laboratory instruments whichwere heavily contaminated with bacterial spores weresterilized in the autoclave or polyethylene bag after4 hr exposure at room temperature to a theoreticalconcentration of 550 and 450 mg of ethylene oxide perL of space, respectively. Although 2 hr exposuresterilized some contaminated sites on the instruments,viable spores were still recovered from other sites.

Table 2 shows that heavily contaminated clothingwould require about 6 hr exposure to ethylene oxidebefore sterility would be obtained in a polyethylenebag or drunm.

DISCUSSION

It is evident from the results presented that theautoclave, steel drum, and polyethylene bag can serveas chambers in which to sterilize laboratory equipmentor clothing with ethylene oxide. The fact that suchsimple equipment can be used does not signify thatthere may not be many occasions where more elab-

18 [VOL. 8

GAS STERILIZATION WITH ETHYLENE OXIDE

orate permanienit types of chamber inistallatioins maybe preferable, particularly when the v-olumes of mate-rial being routiniely sterilized are large. Neither is theethylenie oxide sterilizinig method recommenided as areplacement for the cheaper routine steam or heatsterilizatioin methods where they cain he used. Theethylenie oxide system does, however, furnish a meanisof sterilizing nonautoclavable heat and moisture senisi-tive materials in the laboratory, or of steriliziig almostainy type of nmaterial in the field wNhere steam is unl-avaailable.

Materials may be mainitaiined in a sterile coniditionafteer decontamination if they are wrapped in paperbefore exposure to the ethylenie oxide. The oxide rcadilypenietrates the paper and sterilizes the items whilethe paper in turn prevents recontamination.

Materials which are heavily cointaminated withresistant bacterial spores will require an exposure to300 to 500 mg ethylene oxide per L of air for about6 hr to insure sterilization at room temperature (25 C).Since each 10 C change in temperature chaniges the effec-tiveness of the chemical by a factor of 2 to 3 (Phillips,1949), attempts to sterilize outdoors in cold weathershould be avoided.No effort was made to evacuate the drum or auto-

clave before admitting the ethylene oxide mixture. Avacuum is not necessary to achieve penetration ofethylene oxide through porous materials since thishighly volatile gas diffuses rapidly through manylayers of fabric or other permeable materials (Phillips,1957). Also it had been noted that, when air was evac-uated from a chamber before admitting ethylene oxidevapor, sterilization often became more difficult, andsometimes was not achieved in the expected timeinterval. This has been attributed to the removal ofmoisture as well as air when the chamber was evac-uated. Apparently some moisture is necessary for thekilling process to proceed rapidly (Yesair and Williams,1942; Kaye and Phillips, 1949).

It must be emphasized that the can of ethyleneoxide-halogenated hydrocarbon mixture must be heldin an inverted position to insure a rapid release (withinseveral minutes) of the sterilant,when the can isopened. The can is not equipped with an eductor tubeand, therefore, if the can is opened while in an uprightposition, it will require a much longer time (about24 hr) to dispense the entire contents.

Care must be exercised when using ethylene oxideand liquid halogenated hydrocarbon mixture sinceboth liquid ethylene oxide anid liquid trichloromono-fluoromethane are excellent solvents for certaini plastics.Therefore, it is imperative that droplets of the liquiddisinifectant mixture, before they ha-ve an opportunityto vaporize as they emerge from the canl, not be per-mitted to contact certain materials. Ethylenie oxideand trichloromonofluoromethane evaporate quite rap-idly after dissemination anid once in the gaseous state

they are noncorrosive. It is advisable to provide ashield or baffle over the poIrt through which the sterilantmixture eniters the autoclave or the drum. Wheni steril-izing e(uipmnent in a bag, it is desirable to direct theeffluenit from the dispenser inlto a towel or rag to pro-tect eqjuipment which might be damaged by the li(quidsterilant or propellanit.

SU,MMARY

A simple ethylenie oxide dispenser system and threeeasily devised exposure chambers are described. Thedispenser is a 16 oz, low-pressure aerosol can containiing11 per cent (by weight) ethylene oxide, 44.5 per centdichlorodifluoromethane, and 44.5 per cent trichloro-monofluoromethane. The aerosol cans are fitted with a1-in. flat cap to which a needle valve with a can holderand hollowineedle can be attached to release thesterilant mixture. This system was adapted for usewith an ordinary steam autoclave, a 55 gallon open-head steel drum, or a simple large polyethylene bag.Any one of these devices can be used as an ethyleneoxide sterilizing chamber in the laboratory and the drumand polyethylene bag are suitable for use in the field.

REFERENCES

FUTLTO N, J. D. AND MITCHELL , R. B. 1952 Sterilization offootwear. U.S. Armed Forces MIed. J., 3, 425-439.

HAENNI, E. O., AFFENS, W. A., LENTO, H. G., YEOMANS,A. H., AND FULTON, R. A. 1959 New nonflammableformulations for sterilizing sensitive materials. Ind. Eng.Chem., 51, 685-688.

JONES, C. W. AND KENNEDY, R. E. 1930 Extinction of ethyl-ene oxide fumes with carbon dioxide. Ind. Eng. Chem.,22, 146-147.

KAYE, S. AND PHILLIPS, C. R. 1949 The sterilizing action ofgaseous ethylene oxide. IV. The effect of moisture. Am.J. Hyg., 50, 296-306.

KAYE, S. AND SURKIEWICZ, B. F. 1952 Miethods and eqluip-mentforsterilization with ethyleie oxide vapor. Abstractof papers presented at the 122nd MIeeting, Am. Chem. Soc.,Atlantic City, New Jersey, Sept. 14-19.

NEWMAN, L. B., COLWELLJ C. A., AND JAMESON, E. L. 1955Decontamination of articles made by tuberculosis pa-tients in physical medicine and rehabilitation. Am. Rev.Tuberc. Pulmonary Diseases, 71, 272-279.

PERKINS, J. J. 1956 Gaseous sterilization with ethyleneoxide. In Principles and mtiethods of sterilization. CharlesC Thomas, Springfield, Illinois.

PHILLIPS, C. R. 1949 The sterilizing action of gaseotus ethyl-ene oxide. II. Sterilization of contaminated objects withethylene oxide and related compounds: time, concentrationand temperature relationships. Am. J. Hyg., 50, 280-288.

PHILLIPS, C. R. 1957 Gaseouis sterilization. In Antiseptics,disinfectants, fuingicides and sterilization. Edited by G. F.Reddish. Lea and Febiger, Phila(lelphia, Pennsylvania.

SKEEHAN, R. A., JR., KING, J. H., JR., AND KAYE, S. 1956Ethylene oxide sterilization in ophthalmology. Am. J.Ophthalmol., 42, 424-430.

STRYKER, W. H. 1958 Gas sterilizationi. Hospital Manage-ment. March, 74-77.

YESAIR, J. AND WILLIAMS, 0. B. 1942 Spice contamina-tion and its control. Food Research, 7, 118-126.

19601 19