RUBBO, 8 . D. & GARDNER, JOAN F. (1968). J . appl. Bact. 31,16P169.

Sterilization of Syringes by Ethylene Oxide

S. D. RUBBO AND JOAN F. GARDNER School of Microbiology, ZJniversity of Melbourne, Australia

(Received 5 September 1967)

SUMMARY. Three types of disposable syringe, all known to be sterilized commercially by ethylene oxide, were contaminated with Bacillus subtilis spores a t the most inaccessible site, between plunger and barrel, and processed under controlled conditions. Tho rate of failure in sterilization was related to the syringe dcsign as well as to the number of spores initially present. Rubber tipped plungers forming a double edge contact with the barrel failed to a degree that varied with the thickness of the contacting edges and the tightness of fit in the barrel. Although ethylene oxide may be used if initial contamination is low, gamma radiation would be more reliable for this type of syringe.

THE ESSENTIAL PREREQUISITES for sterilization by ethylene oxide (EtO) gas have been clearly defined in numerous publications (Opfell, Hohmann & Latham, 1959; Royce & Bowler, 1961; Ernst & Shull, 1962; Gilbert, Gambill, Spiner, Hoffman & Phillips, 1964). Briefly, they are access of gas and moisture in the correct concentrations to all the surfaces to be sterilized and absence of dried crystalline or proteinaceous matsrial that could protect the organisms from effective contact with the sterilizing agent.

Successful application depends 011 careful adjustment of EtO concentration, time of exposure, temperature and humidity, and many papers have dealt with its use in sterilizing medical equipment on a commercial scale or in hospitals (Bruch, 1961: Stierli, Reed & Billick, 1962; Cunliffe, 1966, 1967; Weymes, 1966; Belsey, 1967). There is no doubt about its potential usefulness for certain articles that cannot be sterilized by heat or ionizing radiation, but it is also clear that the process must be operated by people with full knowledge of its limitations.

Because of occasional failure to meet one or other of the essential prerequisites for EtO sterilization the authors expressed the opinion (Rubbo & Gardner, 1965) that the method was unsuitable for disposable equipment which provided the oppor- tunity to introduce organisms parenterally into the tissues. Thus, disposable syringes and needles, blood taking and giving sets and sutures come into this category.

This report deals with the sterilization of syringes by EtO under controlled con- ditions in the laboratory. The results denionstrate clearly that the dcsign of a disposable syringe can determine its suitability for gas sterilization.

Materials and Methods The ethylene oxide sterilizer

The sterilizer was a horizontal water jacketed cylinder of 75 1 capacity, coated internally with an epoxy resin. It was fitted with a vacuum pump, vapourizers for the EtO mixture and water, and a gas sampling port. Temperature was controlled by the

Sterilization of syringes by ethylene oxide '65

water jacket and measured in the chamber and load by thermocouples. The gas used wasanon-inflammable mixture of 12% of EtO and SS70 of Freon 12. The chamber concentration of EtO was monitored in 3 ways: by weight of charge, by chamber pressure and by gas chromatographic analysis. Humidity in the chamber wa6 measured by the Karl-Fischer method, passing a sample of the gas through the reagent.

Several EtO concentrations were used in this investigation, those studied in most detail being 1000 mg/l for 12 h at 30" and 750 mg/l for 6 h at 50": these are the procedures most frequently used in the commercial processing of disposable syringes. In each case the relative humidity (RH) was adjusted to 3 0 4 0 % by admitting 1 ml of water as vapour to the evacuated chamber c. 45 min before the EtO.

The process was controlled by bacteriological and chemical indicators sited in the chamber, in the heat sealed plastic packages and within the syringes.

Bacteriological indicator This consisted of spores of Bacillus subtilis var. niger (Camp Detrick strain, NCTC 10073), otherwise known as B. globigii, harvested from a 2 day culture on nutrient agar and washed and suspended in 90% methanol according to the method of Beeby & Whitehouse (1965). The suspension was adjusted by plate counts to a spore con- centration of lo8 viablc: unitslml. Aluminium foil test strips contaminated with lo6 spores (0.01 ml from a dropping pipette) were dried at 55" for 30 min. The same suspension, appropriately diluted, was used to contaminate the syringes as described below.

Chemical indicators Royce's sachets (Royce & Bowler, 1959), with a concentration-time product of 7600 mg/h 1 at 20" and 2250 mg/h 1 a t 56", were used to indicate adequate exposure in the chamber and within the packages. Paper indicators (Aseptic Thermo Indicator Co., U.S.A.) were sited similarly and also within the syringes, usually in the groove between barrel and rubber plunger.

Syringes The three types of syringes tested (Fig. 1) were all known to be sterilized com- mercially by EtO. Type A, of 5 ml capacity, had an all-plastic plunger of high density polythene; types B and C, of 2 ml capacity, had rubber tipped plungers, differing slightly in shape and in tightness of fit in the barrel.

The sites selected for contamination represented different degrees of accessibility to the gas. In type A syringes the edge of the plunger was contaminated, while in types B and C, with double contact plungers, the groove between the two ridges was chosen. In similar syringes which served as controls for the entry of lethal amounts of gas into the barrel, the distal end of the plunger was contaminated.

Contamination with loa, lo4 and lo6 spores was carried out by adding 0.01 ml of the appropriately diluted methanol suspension to the 'sides of the plungers after they had been racked in a horizontal position. After drying a t 55" for 30 min, the syringes were assembled with their needles and guards and sealed either in their original plastics bags or in new ones made of polythene 0.004 in thick.

I 66 S. D. Rubbo and Joan F. Gardner

Site of contamination

11

U

Fig 1 . Types of plunger used in disposable syringes.

Xterility tests The processed syringes were withdrawn from the bags with aseptic precautions and the plungers transferred to tubes containing Difco Heart Infusion Broth, or Oxoid Dextrose Tryptone Broth with 0.3% of yeast extract added, and incubated for 5-7 days. If growth occurred the culture was examined to confirm its identity with the test strain of B. subtilis. Test strips were cultured in the same way.

Results The proportion of syringes sterilized by exposure to 1000 mg of EtOjl a t 30" for 12 h is shown in Table 1. In all experiments the control untreated plungers, yielded positive cultures, while assembled syringes contaminated on the distal end of the plunger only were negative, provided the needle guard allowed penetration of EtO.

TABLE 1 Eficacy of EtO in sterilization of disposable syringes*

Syringes sterilized with contamination load of A 7 ,

Syringe 102 spores lo4 spores 10' spores - I--- --Gi&-- No. % No. % Type 7-

A Single contact plunger (high density polythene) ND - ND - 40/40t 100

R Double contact plunger 40/40 100 54/55 98 145/150 96.6 (thin edge rubber)

C Double contact plunger 60/66 92.5 63/102 61.7 110/231 47.5 (thick edge rubber)

* The treatment was with 1000 mg of EtO/l of air space for 12 h at 30" and 38% RH. ND, not done. i. No of syringes sterile/no. tested I

Sterilization of syringes by ethylene oxide 167

Of the syringes contaminated on the side or edge of the plunger, those of type A were invariably sterilized as was also the case when they were processed with the lower concentration of 400 mg of EtO/l for 18 h a t 20". Type B syringes were sterilized at the lowest level of contamination (102 spores), but occasional failures were evident when the level of contamination was higher. Unsterilized type C syringes were found at all levels of contamination.

Paper indicator strips positioned a t the same site indicated clearly that in type B syringes access of EtO was usually achieved but that with type C this was by no means true, the indicators being unchanged or partly changed in c. 75% of the syringes tested. It was noticed, however, that within 12 h of removal from the sterilizer they gradually changed colour, due presumably to the action of EtO retained in the package or in the syringe itself.

Further experiments were conducted therefore, keeping type B and type C syringes in the laboratory for 12 h before culture in addition to their being cultured immediately after removal. These results (Table 2) make it evident that a much higher proportion of syringes was sterilized after the 12 h holding period. However, a t the highest level of contamination, failures still occurred in both types but more frequently with type C.

TABLE 2 Xterility of EtO processed syringes stored after treatment*

Syringes sterilized with contamination load of

Syringe lo2 spores lo4 spores 106 spores 7- && Type Design No % No % No

B Double contact plunger 30/30t 100 30/30 100 33/35 94 (thin edge rubber)

(thick edge rubber) C Double contact plunger 25/25 100 46/46 100 62/78 80

* The treatment was with 1000 mg of EtO/l of air space for 12 h at 30' and 38% RH, and the

t No. of syringes sterile/no. tested. storage period after treatment was 12 h.

In one experiment type C syringes contaminated with lo6 spores were processed in 1000 mg of EtO/l a t 30" for 24 h and 3 of 20 were nonsterile.

Only syringes of type B were subjected to the alternative process of 750 mg of EtO/l a t 50" for 6 h, this being the treatment they were known to receive com- mercially. Of the relatively small numbers tested, no failures were detected a t con- tamination levels of lo4 or 1 0 6 spores/ml.

The sterilization of contaminated needles was not extensively studied in this investigation. Uniform sterilization of spores on the plunger end in syringes fitted with needles and guards indicates that EtO gains entry via the needle bore. Needles might be nonsterile, however, if humidity were below the optimum level.

I 68 S. D. Rubbo and Joan F. Gardner

Discussion Of the types of syringes tested, all of which are commercially sterilized by EtO, type A is the only one that was invariably sterilized by the processes used in these experi- ments. This is possibly due to two factors: the unlikelihood of heavy contamination on the narrow contacting edge, and access of gas to this edge from both sides.

Type B and C syringes, with plungers that could be termed of the 'cotton reel' type, were not sterilized under all conditions of testing. Type B, however, failed less frequently, either because the two contacting edges were finer or because the plunger did not fit so tightly in the barrel.

Tests for leakage of air past the plungcr when a pressure difference was applied showed that this might occur in type B more readily than in type C syringes. The method was not sufficiently sensitive, however, to allow a correlation to be made between tightness of fit and sterilization of individual syringes of the same type. A preliminary analysis of the rubber materials did not indicate any feature that might account for differences in the permeability of the plungers to EtO.

An important point to be considered in assessing the safety of a sterilization method is the level of contamination likely to exist on the unprocessed articles. Christensen, Holm & Jnul (1966) report that the highest levels encountered in 629 disposable syringes examined nere lo2 organisms/syringe, but counts of 105-106 have been recorded on some units in an apparently 'clean' batch (Kelsey, 1961).

We conclude, therefore, that the use of EtO in a properly controlled process, monitored by bacteriological tests, may be acceptable for sterilization of disposable syringes known to have a low level of contamination. However, of those investigated in this study, type A with a single contact plunger is the only syringe for which EtO can be recommended unconditionally. Those types with double contact rubber plungers woiiltl be preferably sterilized bv gamma radiation.

The authors wish to thank Mr. W. Anderson of the Royal Melbourne Institute of Technology for estimations of EtO by gas chromatography; Mr. L. Bailey of Dunlop Rubber Australia Limited for analysis of rubber samples; and Mr. G. Erdmanis of Ensign Laboratories Pty. Ltd. for determining relative humidities. Acknowledge- ment is made to the Australian Research Grants Committee for financial assistance in this project.

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