Gaseous Sterilization - Sterilization Methods

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Chapter: Pharmaceutical Microbiology : Sterilization Procedures And Sterility Assurance

The chemically reactive gases ethylene oxide [(CH 2) 2 O] and formaldehyde [(methanal, H.CHO)] possess broad-spectrum biocidal activity, and have found application in the sterilization of reusable surgical instruments, certain medical, diagnostic and electrical equipment, and the surface sterilization of powders.



The chemically reactive gases ethylene oxide [(CH 22 O] and formaldehyde [(methanal, H.CHO)] possess broad-spectrum biocidal activity, and have found application in the sterilization of reusable surgical instruments, certain medical, diagnostic and electrical equipment, and the surface sterilization of powders. Sterilization processes using ethylene oxide sterilization are far more commonly used on an international basis than those employing formaldehyde.


Ethylene oxide treatment can also be considered as an alternative to radiation sterilization in the commercial production of disposable medical devices . These techniques do not, however, offer the same degree of sterility assurance as heat methods and are generally reserved for temperature-sensitive items.


The mechanism of antimicrobial action of the two gases is assumed to be through alkylation of sulphydryl, amino, hydroxyl and carboxyl groups on proteins and imino groups of nucleic acids. At the concentrations employed in sterilization protocols, type A survivor curves (Figure 21.1) are produced, the lethality of these gases increasing in a non-uniform manner with increasing concentration, exposure temperature and humidity. For this reason, sterilization protocols have generally been established by an empirical approach using a standard product load containing suitable biological indicator test strips (section 12.3). Concentration ranges (given as weight of gas per unit chamber volume) are usually of the order of 800–1200 mg/L for ethylene oxide and 15–100 mg/L for formaldehyde, with operating temperatures in the region of 45–63 °C and 70–75 °C, respectively. Even at the higher concentrations and temperatures, the sterilization processes are lengthy and therefore unsuitable for the re sterilization of high-turnover articles. Further delays occur because of the need to remove toxic residues of the gases before release of the items for use. In addition, because recovery of survivors in sterility tests is more protracted with gaseous sterilization methods than with other processes, an extended quarantine period may also be required.


As alkylating agents, both gases are potentially mutagenic and carcinogenic (as is the ethylene chlorohydrin that results from ethylene oxide reaction with chlorine); they also produce symptoms of acute toxicity including irritation of the skin, conjunctiva and nasal mucosa. Consequently, strict control of their atmospheric concentrations is necessary and safe working protocols are required to protect personnel. Table 21.3 summarizes the comparative advantages afforded by ethylene oxide and low-temperature steam and formaldehyde (LTSF) processes.



a)     Ethylene Oxide


Ethylene oxide gas is highly explosive in mixtures of more than 3.6% v/v in air; in order to reduce this explosion hazard it is usually supplied for sterilization purposes as a 10% mix with carbon dioxide, or as an 8.6% mixture with HFC 124 (2-chloro-1,1,1,2 tetrafluoroethane), which has replaced fluorinated hydrocarbons (freons). Alternatively, pure ethylene oxide gas can be used below atmospheric pressure in sterilizer chambers from which all air has been removed.


The efficacy of ethylene oxide treatment depends on achieving a suitable concentration in each article and this is assisted greatly by the good penetrating powers of the gas, which diffuses readily into many packaging materials including rubber, plastics, fabric and paper. This is not without its drawbacks, however, as the level of ethylene oxide in a sterilizer will decrease due to absorption during the process and the treated articles must undergo a desorption stage to remove toxic residues. Desorption can be allowed to occur naturally on open shelves, in which case complete desorption may take many days, e.g. for materials like PVC, or it may be encouraged by special forced-aeration cabinets where flowing, heated air assists gas removal, reducing desorption times to between 2 and 24 hours.


Organisms are more resistant to ethylene oxide treatment in a dried state, as are those protected from the gas by inclusion in crystalline or dried organic deposits. Thus, a further condition to be satisfied in ethylene oxide sterilization is attainment of a minimum level of moisture in the immediate product environment. This requires a sterilizer humidity of 30–70% and frequently a preconditioning of the load at relative humidities of more than 50%.


i)  Sterilizer design and operation


An ethylene oxide sterilizer consists of a leak-proof and explosion-proof steel chamber, normally of 100–300 L capacity, which can be surrounded by a hot-water jacket to provide a uniform chamber temperature. Successful operation of the sterilizer requires removal of air from the chamber by evacuation, humidification and conditioning of the load by passage of sub atmospheric-pressure steam followed by a further evacuation period and the admission of preheated vaporized ethylene oxide from external pressurized canisters or single-charge cartridges. Forced gas circulation is often employed to minimize variations in conditions throughout the sterilizer chamber. Packaging materials must be air, steam-and gas-permeable to permit suitable conditions for sterilization to be achieved within individual articles in the load. Absorption of ethylene oxide by the load is compensated for by the introduction of excess gas at the beginning or by the addition of more gas as the pressure drops during the sterilization process. The same may also be true for moisture absorption, which is compensated for by supplementary addition of water to maintain appropriate relative humidity.


After treatment, the gases are evacuated either directly to the outside atmosphere or through a special exhaust system. Filtered, sterile air is then admitted either for a repeat of the vacuum/air cycle or for air purging until the chamber is opened. In this way, safe removal of the ethylene oxide is achieved, reducing the toxic hazard to the operator. Sterilized articles are removed directly from the chamber and arranged for desorption. The operation of an ethylene oxide sterilizer should be monitored and controlled automatically. A typical operating cycle for pure ethylene oxide gas is shown in Figure 21.7.


b) Formaldehyde


Formaldehyde gas for use in sterilization is produced by heating formalin (37% w/v aqueous solution of formaldehyde) to a temperature of 70–75 °C with steam, leading to the process known as LTSF. Formaldehyde has a similar toxicity to ethylene oxide and although absorption to materials appears to be lower, similar desorption routines are recommended. A major disadvantage of formaldehyde is low penetrating power, and this limits the packaging materials that can be employed to principally paper and cotton fabric.


i)  Sterilizer design and operation


An LTSF sterilizer is designed to operate with sub-atmospheric-pressure steam. Air is removed by evacuation and steam is admitted to the chamber to allow heating of the load and to assist in air removal. The sterilization period starts with the release of formaldehyde by vaporization from formalin (in a vaporizer with a steam jacket) and continues through either a simple holding stage or through a series of pulsed evacuations and steam and formaldehyde admission cycles. The chamber temperature is maintained by a thermostatically controlled water jacket, and steam and condensate are removed via a drain channel and an evacuated condenser. At the end of the treatment period formaldehyde vapour is expelled by steam flushing and the load is dried by alternating stages of evacuation and admission of sterile, filtered air. A typical pulsed cycle of operation is shown in Figure 21.8.


c) Peroxygen  Compounds


Hydrogen peroxide (H2O2), 30–35%w/v, and peracetic acid (CH3CO3H), 3.5%w/v, are used as highly effective oxidizing agents to kill microorganisms. The liquids are heated to vaporize them and are held in a sealed chamber where all surfaces which come into contact with the vapour will be sterilized. These methods are widely used for the sterilization of equipment used for aseptic preparation and manufacture such as isolators, with the vaporized hydrogen peroxide (VHP) often used for the routine decontamination of cleanrooms.


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