Deliberate decision making during the structuring of microbial challenges, product loads, and biological indicators can provide a validation process for EtO sterilization that ensures accuracy, the absence of microbes, and a smooth testing procedure.
A companion to this article, EtO Sterilization: Principles of Process Design, discussed the components of each phase of two 100% EtO with nitrogen processes, focusing on the engineering aspects of designing EtO cycles.1 This article focuses on various approaches to medical device sterilization cycle validation from a microbiological standpoint. The discussion assumes that the first two stages of the validation—the commissioning and the physical performance qualification of the sterilization chamber—have been successfully completed. Therefore, this discussion deals solely with the third stage of EtO validation: the microbiological performance qualification (MPQ). The topics that are discussed include how to choose the appropriate microbial challenge for an EtO sterilization process, approach to developing the EtO cycle, product load, placement of biological indicators in/on the product load, method for determining cycle lethality, and calculations to determine the D-value.
Information is also given on the documentation for the report on validation and certification of the process, and revalidation is discussed briefly. Some suggestions exceed the current requirements presented in international standards, but they can enhance a validation process, resulting in a more thorough and accurate study.
International standards ISO 9001 and 9002 present the quality system requirements for the design, development, production, installation, and servicing of healthcare products. The ISO 9000 series treats medical device sterilization as a special manufacturing process because the results cannot be verified by inspecting and testing 100% of the product at the conclusion of the cycle. Sterilization processes must, therefore, be assessed for special considerations, validated prior to use (or during use in certain situations), and routinely monitored.
To design an effective validation and routine control program for a sterilization process, the bioburden on the product and packaging must be considered. Bioburden is defined as "the population of viable microorganisms on a product and/or a package" and is characterized in terms of number, identity, and resistance. A validated test method must demonstrate that it can consistently and adequately remove the bioburden from the product and packaging. There are various bioburden test methods and associated validation procedures from which to choose.2
Many factors can contribute to the bioburden on product and packaging, including the origin of the raw materials and components, transit and storage conditions, and the manufacturing environment in which the finished products are assembled and packaged. European standards place particular emphasis on controlling the processes used to manufacture sterile products.3
Sterility is defined as the "state of being free from viable microorganisms."4 Microbial death relating to the gaseous sterilization of healthcare products is an exponential function typically defined as the probability of a nonsterile item existing per a given number of units in a batch. This probability, called the sterility assurance level (SAL), defines the "probability of a viable microorganism being present on a product unit after sterilization."4 While sterilization can reduce the bioburden on a given product to a very low number, that probability can never be reduced to zero. Therefore, in order to achieve the desired bioburden levels, it is critical to design a validation program that provides a high degree of confidence for consistent sterilization.
DETERMINING THE APPROPRIATE MICROBIAL CHALLENGE
A biological indicator (BI) is an "inoculated carrier contained within its primary pack providing a known resistance to the relevant process."4 There are many different types of BIs but the most common include:
- Commercial units supplied with the manufacturer's certification, such as bacterial spore strips individually packaged in glassine envelopes or pieces of filter paper impregnated with a certified population of a challenge organism that has a known resistance to EtO, such as the spores of Bacillus subtilis var. niger.
- A liquid bacterial spore suspension commercially sold and certified by the manufacturer. The suspension is placed on or in the product, which is then referred to as inoculated product.
- A liquid bacterial spore suspension made by a device manufacturer using a commercial, certified strain of a bacterium.
- Strains of microorganisms that have been isolated from a device manufacturing facility. These strains represent the most resistant organisms found in or on the devices and are typically used for the combined BI/bioburden method of validation.
It is crucial to ensure that the type of BI used to validate or routinely monitor a given sterilization process is the most appropriate indicator for that process. In addition to identity, quantitation, resistance, storage, general directions for use, and disposal conditions, the manufacturers of BIs are required to provide information regarding the optimal culturing conditions, such as temperature and type of growth media.
Irrespective of which BI is chosen, the methods used to recover the challenge organism must be validated. This recovery is expressed in terms of the percent recovery of the original inoculum. These recovery studies can be especially challenging when using liquid spore suspensions because of the interaction between the suspension and the material onto which it is inoculated. The material substrate can alter the resistance characteristics of the inoculum because of such anomalies as spore clumping or the physical sheltering of spores in certain sites within the product.
The goal is to kill the microbes, which means disabling their ability to reproduce even in their most favorable growth conditions as described by their manufacturer. There have been records of seemingly killed microbes that regenerated when conditions become favorable.5 The user must validate that the incubation time under the prescribed conditions is sufficient to recover delayed growth of the organisms after exposure to a given sterilization process. For routine processing, this time period is typically 7 days unless validated for a shorter time period in accordance with current national requirements.6 In such cases, periodic checks should be run to confirm that the shorter time period yields equivalent recoveries to those obtained from the longer incubation period. It is also important to ensure that the incubation time is sufficient to recover growth from injured organisms exposed to sublethal cycles. In some cases, this may mean using a 14-day incubation period. This incubation period is also required by the U.S. Pharmacopeia for product sterility testing.7
Just as the BI must provide a defined resistance to a specified process, it is necessary to prove that the inherent bioburden on the product does not have a greater resistance than the BI. Characterizing bioburden involves quantitation, identity, and resistance of the bioburden. Several methods can be employed to determine which BI is appropriate for a specific situation.
- Challenge organisms that make up BIs typically resist a particular sterilization process far more than do common bioburden organisms. The resistance of the bioburden cannot be adequately evaluated by quantitation only, yet it must be determined if the products to be processed might contain organisms that are more resistant than the challenge organism. After the microbial identifications and quantitation of the bioburden have been completed and analyzed, comparative resistance determinations of the most resistant bioburden component or product must be calculated. A literature review is also required.
- When microbial identifications of the bioburden are not performed, at least one sublethal cycle should be run to compare the relative inactivation rates of the bioburden with that of the challenge organism. Product sterility testing, after exposure to at least one sublethal cycle under appropriate experimental conditions, can ensure that the product's bioburden is not more resistant than the challenge organism.
- If the quantity, identity, and resistance of the product's bioburden are known, it might be possible to validate and routinely monitor the sterilization process by combining BI and bioburden methods. It must be demonstrated that the BI's degree of challenge to the sterilization process is adequate to ensure that the process will attain the desired SAL for the bioburden. Combining the BI and bioburden methods to determine the appropriateness of the BI can be time-consuming and result in additional testing costs. However, the required sterilization parameters can be more accurately determined, which can result in reduced processing time and reduced exposure to the sterilant.
BIs can be configured in many different ways depending on the cycle development method chosen.
Inoculated Product. The actual product, configured and packaged as it is intended to be sold, can be inoculated with spores of a microorganism such as Bacillus subtilis var. niger. Direct inoculation usually uses spores suspended in liquid, then placed on the product and dried. The product's surface characteristics will affect the distribution of spores and may lead to a difference in resistance behavior compared with other challenge systems.4 It is, therefore, important to achieve an even distribution of spores on the product's surface. Indirect inoculation involves placement of a carrier, such as filter paper that has been impregnated with spores, in the product or its package.
Inoculated Unit. A carrier, such as a filter paper strip or disk, can be inoculated with a population of a resistant organism, such as Bacillus subtilis var. niger, that has been extensively characterized and certified by the manufacturer. The resistance of this inoculated carrier must be compared to that of the inherent bioburden of the product being validated or the equivalent simulated product. An inoculated unit is usually used when there is the potential that the bioburden on the product is more resistant than the indicator organism and is required for the combined bioburden and BI cycle development method.
Inoculated Simulated Product. A simulated product that comprises the most difficult to sterilize portions of a device or that configuratively represents a device family can also be directly or indirectly inoculated. This simulated product must present the greatest challenge to the process in order to be considered an adequate microbial challenge. Each unit must contain a certified inoculum either in liquid form or on a carrier.
Natural Product. The inherent bioburden on the product can also be used as the microbial challenge during validation and for routine monitoring when the absolute bioburden method is employed for cycle development (see "EtO Cycle Development Approaches", below).
All validation methods for EtO sterilization require that the BI used for validation and to monitor routine cycles must be more resistant than the bioburden of the product and be placed in a location that is more difficult to sterilize. Comparative resistance testing is an effective means of selecting the BI and its location in the product that presents the greatest challenge to the sterilization process. Such an assessment should be made prior to validation as part of determining the appropriateness of the BI. These studies are usually carried out in small chambers that are capable of delivering rapid ramp rates, e.g., the times required to achieve specific pressure set points.
Products should be exposed to cycles in which the only variable is the gas exposure time period. The data obtained from this testing can be used to justify the choice of a specific actual or simulated product to inoculate and use for the BI. If the design of the product is such that a BI unit cannot be placed in the part of the device that is the most difficult to sterilize, the product should be inoculated with a liquid spore suspension to provide a known number of viable spores. The spore suspension, materials, and techniques used should comply with ISO 11138, parts 1 and 2.8,9
Many device manufacturers include an additional objective in their validation plan that involves the use of external BI monitoring systems. Often referred to as process challenge devices (PCDs), they assess the lethality of the EtO process after the cycle has been designed. The PCDs are geometrically distributed around the load rather than in internal locations in the case cartons. Direct comparisons can then be made between the sterility test data obtained from these external PCDs and the BIs placed in internal locations. A PCD must be shown through comparative resistance studies to provide more of a challenge to the process when it is placed in external locations in the load than do the the BIs placed in internal locations. They usually, therefore, bear no resemblance to the product. Examples of external PCDs are spore strips double-packaged in plastic bags, in sealed plastic tubing, or in syringes. There are also commercially available PCDs that are sold as ready-to-use packaged systems. It is advisable during the validation studies to evaluate different PCD configurations during the comparative resistance studies to determine the best candidate. To monitor routine sterilization cycles, it must be shown at the time of validation that the PCDs in the external locations comply with the same requirements for resistance to sterilization.
ETO CYCLE DEVELOPMENT APPROACHES
There 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 CHALLENGE
Microbiological 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 LOAD
After 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:
- At least 20 BIs for usable chamber volumes up to 5 m3.
- Increase the number of BIs by two for every additional 1 m3 of usable sterilizer chamber volume between 5 and 10 m3.
- Increase the number of BIs by two more for every additional 2 m3 of usable sterilizer chamber volume above 10 m3.
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 LETHALITY
Results 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.