Ethylene Oxide Sterilization
Craig A. Wallace
Ethylene oxide (EO), also known as epoxyethane or EO, is a colorless gas that is used as a fumigant to treat food products such as spices and as a chemical sterilant for medical devices and other health care products. For many years, EO sterilization was the primary low-temperature process used by hospitals for sterilization of medical devices that were not compatible with the elevated temperature and high humidity of steam sterilization (Figure 31.1). The EO is still used in hospitals around the world, although vaporized hydrogen peroxide-based processes have replaced EO in many facilities because of faster cycle times and control of personnel safety requirements. The EO is used extensively in industrial sterilization of medical devices that are not compatible with radiation or steam sterilization (Figure 31.2). EO is an excellent sterilant because EO molecules exhibit an ability to permeate through most polymeric materials while retaining molecular integrity and producing only negligible changes in the wide variety of materials used in medical devices (Figure 31.3).1 The physical properties of EO are provided in Table 31.1.
The EO was discovered in 1859, but its biocidal properties were not recognized until the 1920s and then primarily as an insecticide.2 The ability of EO to kill microorganisms was first noted in the 1940s and was first applied as a treatment for spices and gums. This antimicrobial property was expanded for use as a sterilant for heat and moisture sensitive medical devices in the 1950s.3 The development of medical devices made of heat-sensitive thermoplastic materials drove the need for the further development and optimization of EO sterilization processes.
BIOCIDAL MECHANISM AND EFFICACYThe primary biocidal mechanism for EO is alkylation or the replacement of a hydrogen atom with an alkyl group.4,5 The primary targets of the alkylation process are sulfhydryl, amino, carboxyl, phenolic, and hydroxyl groups contained in cellular macromolecules such as nucleic acids and proteins.3 The alkylation of nucleic acids was confirmed in studies on Salmonella6 and Clostridium.7 Alkylation of these critical macromolecules disrupts cellular replication.
The EO has been shown in many studies to be a highly efficacious biocide, having a broad spectrum of activity against vegetative bacteria, bacterial spores, and viruses.4,8,9,10,11,12,13,14,15,16,17,18,19 One study indicated that Pyronema domesticum, a type of fungus, had a higher than expected resistance to EO.20 EO is used as a chemical sterilant and, like all chemical sterilants, requires direct contact with the microorganism to inactivate it. Studies have demonstrated that the presence of organic soil (10% fetal bovine serum) and 0.65% salt reduces the effectiveness of EO on exposed surfaces and inside lumens, with similar effects noted for other chemical sterilants such as vaporized hydrogen peroxide and vaporized peracetic acid.21 In another lumen study, a liquid chemical sterilant system was more effective than gaseous EO in tests involving organic soil and salt. But EO was found to be effective in inoculated lumen testing where the soil challenge included only fetal bovine serum but no salt.22 EO was not found to be efficacious in the sterilization of dental handpieces where the internal surfaces of the devices were inadequately cleaned.23 Another study demonstrated greater EO efficacy in lumen challenge testing when compared to one vaporized hydrogen peroxide system, whereas EO was equivalent in performance to a second vaporized hydrogen peroxide system.17
The high diffusivity of the EO molecule enables penetration through many polymers and into narrow lumens. Studies have confirmed the ability of EO to penetrate lumens24 and to penetrate some tissue matrices used for human tissue allografts.25 A number ofcarbapenem-resistant Enterobacteriaceae infections and outbreaks related to use of contaminated duodenoscopes
in endoscopic retrograde cholangiopancreatography procedures have been reported in the literature. These endoscopes contain long narrow lumens and are typically reprocessed by high-level disinfection. Several health care facilities reported that implementation of EO sterilization of their endoscopes halted the outbreaks.26,27,28,29,30
in endoscopic retrograde cholangiopancreatography procedures have been reported in the literature. These endoscopes contain long narrow lumens and are typically reprocessed by high-level disinfection. Several health care facilities reported that implementation of EO sterilization of their endoscopes halted the outbreaks.26,27,28,29,30
 FIGURE 31.1 Small chamber ethylene oxide sterilizer for hospital and industrial use. For a color version of this art, please consult the eBook. Photo courtesy of 3M. |
 FIGURE 31.2 Industrial large chamber ethylene oxide sterilizers. |
 FIGURE 31.3 Ethylene oxide molecule. From Block.1(p580) |
STERILIZATION FORMULATIONSMedical device sterilization processes have used EO in pure form (100% EO) as well as in gas mixtures with EO combined with inert gases such as fluorocarbons, hydrofluorocarbons, and carbon dioxide (CO2).31,32 The mixtures are used to improve safety by reducing explosion and fire risk. Mixture processes operate at higher pressures than typically used for 100% EO systems, which allows processing of some packaged devices that cannot withstand the low pressures (vacuum) used in the 100% EO systems.3 The commonly used mixture of 12% EO and 88% chlorofluorocarbon (CFC) was phased out in the mid-1990s when the CFC gases themselves were phased out because of environmental concerns and regulations. Hydrochlorofluorocarbon (HCFC) replacements were used until similar environmental regulations stopped
their production in 2014 in the United States.33,34 Common EO and CO2 (EO/CO2) mixtures consist of 8.5% EO and 91.5% CO2 or 20% EO and 80% CO2.35
their production in 2014 in the United States.33,34 Common EO and CO2 (EO/CO2) mixtures consist of 8.5% EO and 91.5% CO2 or 20% EO and 80% CO2.35
TABLE 31.1 Physical properties of ethylene oxidea | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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STERILIZATION PROCESS VARIABLESProcess variables are defined as conditions within a sterilization process, changes in which alter microbicidal effectiveness.36 The primary process variables for EO sterilization are temperature, EO gas concentration, relative humidity (RH), and exposure time.3,5,32,33,37,38,39,40,41 Exposure time is determined based on the temperature, EO concentration and RH selected. One international standard also lists pressure as a sterilization process variable.42
Temperature
Typical EO sterilization process temperatures range from 37°C to 63°C. Inactivation of Bacillus atrophaeus spores at various temperatures are shown in Figure 31.4.43 The Q10 value for this data is approximately 1.9. This corresponds to the reported values of 1.8 for B atrophaeus44 and 2.18 for Bacillus subtilis (ATCC 9524).45 A Q10 value of 1.55 to 1.64
has been reported for spores of Bacillus coagulans46 and a Q10 of 1.7 to 2.2 for spores of Clostridium botulinum.47 These data indicate that, in general, for every 10°C change in temperature, the inactivation rate for spores will double when the EO concentration is not limiting.3 This value has been corroborated in other summary publications.5
has been reported for spores of Bacillus coagulans46 and a Q10 of 1.7 to 2.2 for spores of Clostridium botulinum.47 These data indicate that, in general, for every 10°C change in temperature, the inactivation rate for spores will double when the EO concentration is not limiting.3 This value has been corroborated in other summary publications.5
 FIGURE 31.4 Inactivation of Bacillus atrophaeus spores at different temperatures at 500 mg/L ethylene oxide and 40% relative humidity. From Block,1(p586) Figure 33.2. |
Concentration
Typical EO concentrations in sterilization processes are reported to range from 450 to 1200 mg/L.
Survivor curves for the inactivation of B atrophaeus spores at different EO concentrations are shown in Figure 31.5.43 As the gas concentration increases from 50 to 500 mg/L, the inactivation rate increases significantly. At gas concentrations greater than 500 mg/L, there is no significant increase in the rate of spore inactivation. For B atrophaeus spores, at gas concentrations of 200 to 1200 mg/L, the most dramatic decrease in resistance occurs when the given concentration is increased from 200 to 400 mg/L at 54.4°C.3,8 More recent investigations using more precisely controlled test equipment have shown that inactivation of B atrophaeus followed second-order kinetics over EO concentrations of 100 to 1200 mg/L and temperatures of 22°C to 60°C.93
It is expected that the lethality of 100% EO and EO blends would be equivalent when the concentration of EO in the processes are equivalent.1 A more recent publication that compared the effect of 100% EO to an EO/HCFC gas blend on B atrophaeus-based EO biological indicators found that 100% EO had greater lethality at equivalent EO concentrations. The authors speculate that when using an HCFC blend gas, the HCFC competes with the EO molecules for access to the critical binding sites on the spores. With HCFC present in the exposure chamber and blocking EO access to the critical binding sites, the result is fewer alkylation reactions and thus decreased lethal impact on the spores.34
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