North American
Sterilization & Packaging
Company
17 Park Drive, Franklin, New Jersey 07416 USA
Phone:1.973.209.4388
1.800.392.6310
Fax:1.973.209.6374
Email: info@naspco.com
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ETO CYCLE DEVELOPMENT APPROACHESThere are three basic approaches to developing EtO sterilization cycles—the overkill method, the combined bioburden and BI method, and the absolute bioburden method. The overkill method is probably the most widely used because it is relatively easy to use and it results in a robust SAL. The method ensures that the sterilization process will inactivate a specific number of microorganism spores known to be resistant to the EtO sterilization process. The organism most commonly used to monitor the overkill process is Bacillus subtilis var. niger. A certified preparation consisting of a stated population of Bacillus subtilis var. niger spores is inactivated through exposure to specific cycle parameters that have been assessed to be significantly higher than those required to kill the inherent bioburden on the product. The parameters are increased on a routine basis to provide the desired SAL (see "Methods for Determining Cycle Lethality," below). The combined bioburden and BI method is used when the two are equally resistant. This method requires routine bioburden and BI testing in addition to a considerable amount of routine sterility testing to develop a cycle that will inactivate the BI challenge population. The BI must be sufficiently resistant to ensure that the EtO process will deliver the desired SAL relative to the bioburden on the product. The absolute bioburden method is used less frequently in cycle development because it requires extensive testing in both the development phase and routine processing. However, it must be used when the product's bioburden is more resistant than the BI. Such bioburden resistance to the EtO process can be caused by any number of factors, such as the configuration of the product, the quantity or location of the microorganisms, or the bioburden's intrinsic resistance. Since the bioburden on the product constitutes the essential microbial challenge for the process, the bioburden test method must be validated and strictly controlled. The resistant microorganisms are screened through bioburden testing and may be isolated and propagated for use in cycle development studies. One negative of this method is that the microorganisms' resistance can change as a result of how they are cultured, which can adversely affect the results of the cycle development studies. The absolute bioburden method also requires extensive controls of the manufacturing environment in addition to routine product bioburden monitoring and resistance studies. DETERMINING THE APPROPRIATE PRODUCT LOAD CHALLENGEMicrobiological performance qualification (MPQ) should be performed using specified products and packaging configured in the same manner in which they will be routinely sterilized. For the cycle to be accurate, the product load must represent the greatest challenge intended for future routine sterilization. If a device manufacturer intends to use multiple load configurations on an ongoing basis, the densest configuration should be used for the MPQ. Each type of configuration must be documented in terms of the number of product units per case, the number of cases per pallet, the stacking patterns on the pallet, and the density. This documentation should be included with the validation data. Some testing should also be conducted on the least dense configuration, which, theoretically, presents less of a challenge to the process. This testing can be as simple as placing thermocouples throughout the least dense load on a routine cycle and comparing the temperature distribution with that of the densest load. In other cases, additional microbial challenge studies might be required. Changes in the product load must be evaluated carefully because seemingly innocuous changes, such as changing the shrink wrap or corrugate on the load, can have a significant effect on the cycle's efficacy from the perspective of product sterilization. BI PLACEMENT IN THE PRODUCT LOADAfter the product load challenge has been identified, the BI positioning and placement can be determined. BIs should be distributed throughout the product load and, as much as possible, in the same orientation (e.g., vertical). The placement must include those locations that are considered to present the greatest challenge to the process and can be the same as those used for temperature monitoring. The ANSI/AAMI/ISO 11135-1994 standard suggests placing two BIs at each location with a temperature-monitoring device in order to obtain additional information on process efficacy. It also provides the following recommendation for the number of BIs to be included in each validation cycle:
The AAMI technical information report "Contract Sterilization for Ethylene Oxide" can provide additional information on the number of BIs and monitoring devices recommended based on product load volume.10 METHODS FOR DETERMINING CYCLE LETHALITYResults obtained from commissioning and physical performance qualification and monitoring devices should be used to identify critical features of the equipment or process that can be investigated during the MPQ. For example, it is critical that the sterilant injection time is consistent among the MPQ cycles to ensure a uniform delivery from one cycle to the next. Even minor changes in the sterilant injection time can result in significant differences in lethality. The MPQ should be performed in the industrial chamber that will also be used for routine processing unless equivalency can be demonstrated between the industrial chamber and whatever chamber is used for the qualification. Maintaining the precise and consistent delivery of the sterilization cycle parameters is more difficult to accomplish in large industrial chambers than in small test chambers. It is also important to conduct these studies using the actual product load intended for routine sterilization. Hence, these studies are usually conducted in large industrial chambers rather than in small test chambers. The MPQ can be performed by determining the lethality of the cycle on the basis of the number of D-values applied. The D-value is defined as "the time required to reduce a specific microbial population by 90% or one logarithm."4 The survivor curve construction or fraction-negative methods (described below) may be used as outlined in current standards.4 Another means of evaluating the MPQ is the half-cycle method, based on the number of times required to completely inactivate the BI microorganisms with an added margin of safety. The ultimate objective of each method is to determine the full cycle to which the product load must be exposed. Survivor Curve Construction Method. The survivor curve construction method involves the direct enumeration of survivors in terms of colony forming units (CFUs) recovered after exposure to graded amounts of the sterilization cycle. A CFU is defined as "a visible outgrowth of a population of organisms arising from a single or multiple cells."2 A minimum of five cycles should be run, each using different graded time exposures to EtO.4 The parameters used, with the exception of the gas exposure time, must be kept consistent. The first cycle is a time zero study in which the initial CFU survival count of the BI is determined by exposing the BIs to all stages of the process, including preconditioning if used, prior to the EtO injection phase of the cycle. All BIs should survive because they will not be exposed to the sterilant. After each of the four or more additional cycles, all employing different gas exposure time periods, the number of BIs that survive the processes are counted. The BIs should be removed from the chamber and the load as soon as possible within the confines of worker exposure policies. The BIs should also be tested as soon as possible after being removed to reduce their exposure to EtO residuals, which can affect BI survival rates. In all cases, the time intervals between when the load is removed from the chamber, when the BIs are removed from the load, and when they are subjected to the enumeration process must be consistent among the cycles. Ideally, a final enumeration of the BIs from one cycle should be obtained before the next cycle is initiated to more accurately assess the exposure time to use. This is not always feasible because the enumeration process can take days or weeks to complete. The number of BIs used in each of the cycles in the study should be statistically significant to ensure obtaining dependable data. The number can also be based on the size of the chamber or the size of the product load. The data acquired in the study are used to calculate an EtO exposure time and the minimum process parameters expected to elicit a specific probability of survival of the challenge organism expressed in CFUs (see "The Full Cycle," below). Theoretically, because all other process parameters are the same, the statistical evaluation of the data should result in a plotted survivor curve or regression analysis that demonstrates a consistent relationship between the EtO gas exposure time and the number of survivors (positive BIs). Unfortunately, this is not always the case. In addition to the issue of consistency between the BIs, the integrity of the data is dependent on the consistent delivery of the process parameters for each cycle, including precise control of gas injection times that can be considered part of the gas exposure phase for the purposes of this study. Controlling the gas injection time in large industrial EtO chambers necessitates ensuring that the head space pressure in the gas tanks is consistent from one cycle to another and that the gas delivery lines are uniformly either full or purged of gas. A few minutes difference in gas inject time can significantly change the results in this study. Other variables to consider include the temperature of the gas volatilizer, evacuation rates and times, gas makeups, and air exchanges. If the survivor curve study is conducted in an industrial chamber, the data should elicit an accurate probability of survival of a specific challenge organism from which routine cycle parameters can be determined. The BI should be an inoculated carrier. For example, if the BI is a spore strip that contains a population of at least one million (106) spores of Bacillus subtilis var. niger and the construction of the survivor curves demonstrates that total kill was obtained at 2 hours of gas exposure time, the full cycle used to routinely sterilize the product to an SAL of 10–6 could employ 4 hours of gas exposure. To achieve a smaller SAL, for example 10–3, would require adding the appropriate additional gas exposure time to the original 2 hours. The BI testing process should be validated prior to initiation of the survivor curve study to obtain an acceptable and documented recovery method. The process begins with macerating the BIs in sterile water. The suspension is evaluated by plating specific dilution aliquots of serial dilutions of the suspension onto a selected agar medium and counting the number of CFUs after incubation. The number and extent of dilutions needed will be based on the duration of gas exposure and concentration in relation to the number of BI organisms. In other words, more dilutions are required when more survivors are expected. A statistical analysis made from each cycle of survival data should show the log10 of the surviving population plotted against EtO exposure time intervals. The best-fit rectilinear curve through the data can be drawn or determined by regression analysis using the method of least squares.8 The survivor curve method is complicated by the number of serial dilutions that must be prepared and the quality of the dilutions being dependent on the skill of the person performing the test as well as the precision of the equipment used. Only trained personnel who can adequately practice aseptic technique should conduct this test. Calibrated pipettes and dilution controls help ensure the test's accuracy. The dilutions should also be chosen to yield counts between 30 and 300 CFUs. It is generally assumed that numbers ranging from 30 to 100 CFUs should be used because it is thought that higher numbers of CFUs per plate could result in inaccurately low counts and that numbers lower than 10 CFUs per plate could give unreliable counts. For practical purposes, counts between 30 and 300 are generally acceptable. Fraction-Negative Method. The fraction-negative method also involves exposing BIs to multiple cycles of graded exposures to EtO. The differences between the two methods are the number of cycles recommended and the number used to enumerate the survivors (in terms of the positive BIs).4 A minimum of seven cycles should be employed in this study, each utilizing different gas exposure time periods. These seven cycles should elicit the following survivor data:
The method used to enumerate the number of positive BIs is more straightforward than in the survivor curve method. In the fraction-negative method, the BIs are immersed directly into the appropriate media and incubated. Results are recorded in terms of the total number of BIs demonstrating growth and the total number eliciting no growth for each set of test samples. Click here to proceed to page 4, Methods for Determining Cycle Lethality, Calculating D Values |
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