Advancing the Sustainable Use of Ethylene Oxide through Process Validation

Ethylene oxide (EO) was first prepared by Charles Adophe Wurtz, a French chemist, in 1859. Its origins as a sterilant began in the late 1930s, when Paul M. Gross and Lawrence F. Dixon obtained a U.S. Patent (no. 2,075,845). This patent describes a process involving an EO sterilant, temperature, vacuum, and spore-forming indicator organism, as is used today.

Similar to many other sterilization technologies, EO is an effective bactericidal, virucidal, fungicidal, and sporicidal agent. Microbial inactivation is achieved through the alkylation of cellular constituents such as nucleic acids, proteins, and enzymes. The addition of alkyl groups, via binding to sulfhydryl, hydroxyl, amino, and carboxyl groups, prevents normal cellular reproduction and growth.¹ 

EO is differentiated as an effective, flexible sterilization method because its compatibility “with a wide range of materials and its chemical molecule penetration properties in not so aggressive environments, compared with dry heat or steam, made EO sterilization the most suitable process for the majority of heat- and/or moisture-sensitive medical products.”¹  In addition, EO often is suitable for radiation-sensitive products. This level of material compatibility is the key factor contributing to its widespread use. Currently, EO is used to sterilize about 50% of single-use medical devices manufactured globally,²  accounting for more than 20 billion devices sold each year in the United States.³ 

A typical EO sterilization process consists of three phases: preconditioning, sterilization, and aeration. The preconditioning phase of the process consists of subjecting product to controlled temperature and relative humidity conditions for a defined duration. This phase is followed by sterilization, where the sterilant is exposed to the product at a specific temperature and time. The last phase of the process is aeration, where EO residuals are removed from the product at a defined time, temperature, and exhaust rate.

Although EO is the microbicidal agent used to deliver lethality, several interdependent variables (e.g., temperature, relative humidity, duration of exposure, EO gas concentration) aid in delivering an efficient sterilization process. Typically, if one factor is decreased, another factor must be increased to achieve the same sterility assurance level (SAL). Certain factors have a greater importance than others. For example, Q₁₀ (i.e., factor by which the inactivation rate changes for every 10°C change in temperature) values of approximately two have been reported, whereas if relative humidity is maintained between 30% and 90%, the effect on microbial inactivation is generally “constant.”¹  Kereluk et al.⁴  also demonstrated that EO gas concentration was of greater importance than relative humidity. In historical EO cycle design, the typical EO concentrations used were 400 to 1,200 mg/L, often depending on material compatibility or load densities.

When validating an EO process for sterilization of a medical device, a device manufacturer may adopt one of two methods, as outlined in AAMI/ANSI/ISO 11135:2014⁵ : (1) biological indicator (BI)/bioburden approaches or (2) overkill approaches. The overkill half-cycle approach has been favored greatly by industry because of its simplicity of application and added lethality (beyond the required SAL).

Although many benefits to optimization exist, two main factors influence the current industry focus on optimization: epidemiological data and environmental sustainability. The International Agency for Research on Cancer has categorized EO as a Group 1 agent (i.e., carcinogenic to humans).⁶  The toxicity of EO, particularly its carcinogenic properties and effects, has been studied from a number of perspectives.^7–9  Regulations from various countries and continents that directly affect the continued use of EO are of particular relevance to the healthcare sterilization industry. Examples include the Integrated Risk Information System from the Environmental Protection Agency (EPA)¹⁰  and guidance from the Agence Nationale de Sécurité du Médicament et des Produits de Santé regarding EO residuals (tested in accordance with ISO 10993-7) on neonatal healthcare products.¹¹  These measures prompted the 2019 amendment of 10993-7¹²  and its forthcoming comprehensive revision, which is likely to include additional guidance for consideration of special populations.

Recognizing the need to address public health concerns, the Food and Drug Administration (FDA) launched an Innovation Challenge program and Ethylene Oxide Sterilization Master File Pilot Program in late 2019 to encourage industry to explore alternatives to EO and improve and optimize current EO processes.

EO sterilization is validated in accordance with the 11135 internationally recognized consensus standard, in which validations are classified into two categories (BI/bioburden method and overkill method), with each method having several approaches that may be used.

BI/bioburden approaches typically use a BI containing Bacillus atrophaeus, which is of known high resistance to the sterilant, to represent the native bioburden. The extent of sterilant exposure time is established by determining the resistance of this BI and calculating the exposure time needed to achieve the desired SAL from the native product starting population count. Thus, this method uses the quantitative challenge of the native bioburden and the more resistant qualitative challenge of the BI to qualify process lethality. This method can provide for a very short sterilant exposure time (e.g., <2 h). However, because of the extent of work involved in characterizing the bioburden (in terms of both population and resistance) represented on the product(s) and relating that to a BI challenge, a substantial amount of laboratory testing and time are needed for implementation. Therefore, the BI/bioburden method typically is not the first choice for qualifying a sterilization process. This method primarily is used when there is a sufficient product volume that supports the additional work required to validate this process.

Overkill validation approaches do not rely directly on native product bioburden to establish the extent of the sterilization process required to achieve the desired SAL. There are two overkill validation approaches: half-cycle and cycle calculation. As with the BI/bioburden method, both rely on the use of a BI with a known high resistance to the sterilant. However, the overkill approaches use a default challenge population of 10⁶ , with lethality exceeding that required to address the challenge of the native microflora on the product (Figure 1). Hence, the reliance on a BI to establish the SAL simplifies the native microbiological characterization and provides a conservative level of lethality. The simplicity, conciseness, and conservativeness of the half-cycle overkill approach has made it the most-used method of validation.

Half-cycle validation approach. The half-cycle approach requires demonstration of all-kill (i.e., no growth) from BIs processed in a cycle using half of the intended sterilant exposure time. This demonstrates a minimum 6 spore log reduction (SLR) when using a BI with a spore population of 10⁶. When doubling the exposure time for routine processing, the SLR increases to a minimum of 12, thereby demonstrating a 10⁻⁶ SAL. However, the conservativeness of the half-cycle approach can lead to an excessively long, nonoptimal exposure time.

For several reasons, half-cycle qualifications typically deliver far beyond 12 SLR (10⁻⁶ SAL). First, the number of BIs tested must be considered in the SLR math because of the logarithmic nature of microbial inactivation over time (under fixed lethal conditions). The minimum number of BIs to qualify a process (per 11135) is five (or 10¹, when rounded up on a logarithmic scale).⁵  Thus, achieving no growth from 10 BIs in a half exposure cycle, one will exceed an SAL of 10⁻¹ (beyond a one in 10 probability). It is more likely that a 10⁻² SAL (one in 100 probability) is achieved, thereby demonstrating an 8 SLR from a 10⁶ starting BI population (Figure 1). When doubled for the routine exposure time, a 16 SLR is delivered. This defines a sterilization process that delivers at minimum a 10⁻¹⁰ SAL.

Additional factors contributing to excessive exposure time in half-cycle validations include (1) half exposure times often are an estimation, (2) use of a BI exceeding the typical product bioburden number and resistance, and (3) use of a process challenge device (PCD) to increase the resistance of the BI to represent the most difficult-to-sterilize location of the product. With these additional “layers” of conservativeness, an excessive lethality beyond that required to deliver the desired SAL (10⁻⁶) can be demonstrated (Figure 2).

Cycle calculation approach. Cycle calculation is identical to the BI/bioburden method, with the exception that SLR is calculated from a BI with a spore population of 10⁶ rather than a reduced spore population that represents the native bioburden count. Thus, one can qualify a more precise SAL while maintaining a conservative overkill approach using BIs contained in PCDs. This method typically offers the best balance of qualification effort versus cycle time efficiency, whereby considerable process improvements may be realized.

The following case studies demonstrate efforts by the medical device industry to optimize EO processes through validation activity in accordance with 11135.⁵ 

In 2016, a contract sterilization service provider examined more than 150 EO cycles to assess consistency and variance in key parameters. In particular, EO concentration was examined and found to average 612 mg/L. Examination of the observed variance in EO concentrations used for similar products revealed that required SAL could be achieved with lower optimized concentrations. Closer scrutiny of such optimized processes, validated in accordance with 11135, revealed the importance of ensuring that PCDs used in validation are appropriately representative of the product being validated as “sterile.”⁵ 

This review of legacy processes and the described validation methods per 11135 revealed opportunities to reduce the target EO concentration to levels closer to 300 mg/L. It was observed that many of the legacy PCDs represented an excessive challenge compared with the product(s) they were qualified to represent. Because process lethality is explicitly defined by the PCD, additional lethality (through additional exposure to EO) was required to establish the target SAL. Moreover, most processes were qualified with half-cycle methods that typically overestimated exposure times needed for required lethality.

In 2017, the service provider launched a program targeted at reductions in EO concentration while continuing to use the conservative half-cycle approach. Through comparative resistance studies, the appropriateness of the PCD relative to the actual microbiological challenge of the device was demonstrated as a critical first step in validations.

To date, more than 200 validations have been performed, achieving an average EO concentration of approximately 350 mg/L. Of the validations performed and now in operation, the outcomes realized from a subset (n = 10) may be summarized as a 47% reduction in EO concentration, with an average time reduction of 1.7 hours in sterilant exposure phase and, more importantly, an average reduction in total processing time of 23 hours (which includes all EO chamber processing time and external preconditioning and aeration, if used).

As part of the development and investigative work, a collaboration between the EO sterilization provider and a medical device manufacturer assessed the reductions in EO residuals for common medical device components when EO concentrations were lowered by half (i.e., 600 vs. 300 mg/L EO) and all other conditions were maintained similarly. The results of the analysis are shown in Figure 3.

This comparison study demonstrated that the percentage reduction in EO concentration leads to an equivalent or greater percentage reduction in residuals. Although this test was limited to two materials (polypropylene and polyvinyl chloride) and five common components presented for EO processing, the overall trend is commensurate with that observed broadly in the service provider's program.

To quickly achieve cycle optimization for multiple product lines while maintaining a robust sterilization process that consistently achieves the minimum desired SAL of 10⁻⁶, two main prerequisites should be considered: (1) EO product families should be established, and (2) a comprehensive understanding of process limitations through process characterization should be achieved. Guidance on developing EO product families demonstrating process equivalency can be found in the AAMI technical information report, TIR28:2016.¹⁴ 

Master products should be identified for the following sterilization attributes: resistance, bioburden, residuals, and load profiling. Depending on product type, the master product may be the same for all sterilization attributes or require different representatives for each attribute type. For example, the product representing the resistance challenge for a product family may be different from the product representing the sterilant residuals (i.e., materials absorption/desorption) challenge from the same product family. During optimization of the EO gas concentration, additional cycle parameters may require optimization, such as lengthening the EO dwell time or preconditioning time or the aeration temperature. Having a master product identified can allow for easier comparison of the impact of cycle attributes on all products within a family. This is especially true for firms with large product portfolios in a shared cycle across multiple chambers and locations.

Comprehensive characterization of the sterilization process also aids in the optimization of EO cycles, which is particularly true when “limit challenge” or “edge-of-failure” testing has taken place. Because process temperature, relative humidity, and EO gas concentration affect the lethality of the process, understanding the limitations of the cycle has implications for sterility and for functionality of the product. Testing the upper and lower limits of a sterilization process defines the routine processing range, and data can be used for addressing excursions that may occur during commercialization. In addition, a well-characterized cycle can aid in the validation of the cycle in multiple chambers and allow for demonstrating process equivalency, which can reduce validation efforts. This is especially useful when applying a change to a single cycle processed in multiple chambers and locations. Process characterization also is important for establishing EO gas concentration and supports evaluation of the PCD resistance versus product resistance.

The principles outlined above were applied by a medical device manufacturer to achieve a lower gas concentration. First, the SAL of the candidate cycle was first calculated by processing several sublethal cycles where fractional kill was obtained. The EO gas concentration then was adjusted and confirmed to achieve the desired SAL through fractional testing and cycle calculation. An SAL that exceeded the minimum requirement of 10⁻⁶ was targeted. This was done to address potential for future changes in product bioburden. In addition, the higher SAL provides flexibility for future product family adoption of complex devices without having to alter the PCD.

Comparative resistance studies were performed using master products to confirm lethality with appropriate PCDs. Because the overkill half-cycle method was previously used with the candidate cycle, the calculated SAL under routine processing conditions was found to be in the triple digits. A reduction of approximately 250 mg/L gas concentration resulted in a 30% to 50% (dependent on chamber volume and load size) decrease in EO gas weight while still allowing for SALs calculated from the internal process challenge devices that were four to six times greater than the minimum SAL of 10⁻⁶. Product residuals were also measured and were found to be reduced, on average, by about 30%. Further, by quantifying the SAL through the cycle calculation approach, the PCDs used pre-optimization were found to remain valid despite the reduction in concentration.

Because comprehensive process characterization had occurred on the candidate cycle and process equivalence had been demonstrated in the multiple chambers where the cycle had been validated, a reduced EO cycle was broadly implemented in a relatively short amount of time. The result was that the medical device manufacturer was able to convert 74% of EO sterilized product volume (~35,000 pallets) to a lower concentration in less than one year.

Another medical device manufacturer pursued a lower EO gas concentration sterilization cycle for approximately 2.3 million medical devices produced annually (~15,000 pallets), which were sterilized at a contract sterilizer. The project included approximately 50 product families, which currently are sterilized in eight separate validated and approved EO cycles of varying EO concentrations (between 615 and 760 mg/L). The complexity of the device design and materials of construction restricted the ability to sterilize these devices by methods other than EO.

This project sought to validate a single sterilization process with an EO concentration less than 400 mg/L deployed across multiple chambers to improve flexibility and use of EO sterilization capacity. By using less EO, this cycle contributed to lowering EO residuals from the sterilization of the manufacturer's product. The executed validation approach (overkill half-cycle approach in accordance with 11135⁵ ) is consistent with how the existing sterilization processes has been previously validated and approved and was divided into three stages.

Stage 1 of the project was to define a reduced EO concentration cycle that still provided the required SAL. Multiple process definition trial cycles were executed to assess lethality at reduced EO concentration while maintaining product within defined conditions, in order not to affect product functionality. The initial trials focused on identifying an appropriate reduced EO concentration at which lethality could be achieved safely by adjusting EO concentration without changing other cycle parameters. Subsequent trial cycles assessed the impact of reduced EO on temperature conditions within varying loads and across multiple chambers. These trials were able to identify improvements to the steam conditioning phase of the cycle to achieve more uniform temperature and relative humidity penetration into the various load configurations. Further trial cycles also resulted in modification of chamber pressures needed for the nitrogen blanket in EO dwell.

Stage 2 of the project was to consolidate eight current commercially used EO sterilization cycles across multiple product families into a single reduced EO process. This activity included a critical review of products in scope and was completed using product adoption guidance from 11135⁵  and TIR28:2016.¹⁴  From this review, a series of fractional cycle studies were performed that successfully established a worst-case internal PCD and appropriate external PCD as representative of all products in scope of the validation.

In stage 3, validation testing conducted in accordance with 11135⁵  confirmed that products can be sterilized using less than 400 mg/L of 100% EO sterilization to a minimum SAL of 10⁻⁶. Additional benefits were realized, including:

• The duration of the longest legacy cycles was reduced from eight to three days.

• Product with longest aeration times were reduced from 13 to five days.

• Chamber capacity utilization could be improved by at least 10%.

This case study provided evidence supporting the benefits of optimizing an EO sterilization process using the overkill cycle calculation approach described in 11135 as an alternative to the more typically used half-cycle approach. The goal of cycle optimization was to reduce the EO gas concentration and decrease overall cycle times. Although the reduction in EO gas concentration would de facto lead to shorter aeration times because of the lower product residuals following sterilization, the use of the overkill cycle calculation approach provided the additional opportunity of reducing EO exposure time.

Four different EO cycles were optimized using the overkill cycle calculation approach and Stumbo-Murphy-Cochran procedure to calculate D-values. Using this approach, a minimum of three fractional cycles were processed to meet the microbiological performance qualification requirements per 11135.⁵  The calculated D-values were used to determine the 12 SLR time needed to meet an SAL of 10⁻⁶ and establish the full-cycle EO exposure time. The key reduction outcomes from this optimization are shown in Figure 4, where EO gas concentration, EO exposure time, and aeration times are shown to be reduced considerably.

Product materials, design, pallet configuration, and density largely affect how much a cycle can be optimized. A decrease in concentration can lead to longer exposure times for complex and dense products; however, by using the cycle calculation approach, most exposure times may be maintained or even shortened.

The greatest benefit observed from lowering EO gas concentration was a reduction in aeration time. All four cycles resulted in greater than 50% reduction in aeration time. A reduction in product EO residual levels is critical given that the 2019 amendment to 10993-7 includes requirements for special patient populations.¹² 

The case studies described in this article demonstrate opportunities for EO process optimization via use of the validation methods detailed in 11135.⁵  From appropriate definition of the PCD to use of cycle calculation–based approaches, each case study demonstrated benefits from reducing the amount of sterilant, ultimately leading to more efficient and sustainable EO sterilization processes.

Currently, manufacturers and sterilization providers are engaging with the FDA (through both the agency's Innovation Challenge and the Ethylene Oxide Sterilization Master File Pilot Program) to quantify benefits such as those described here. These initiatives strive to deliver the necessary improvements in the most widely used sterilization modality.

Although the cycle calculation approach is not commonly used, the case study in this article demonstrates the added benefits of such an approach. By combining any of the optimization approaches described here with the appropriate definition of PCDs and bioburden-based methods, overall EO gas use may be reduced even further.

The authors thank and acknowledge the Kilmer Collaboration Modalities group for collaborative efforts to consider and explore how additional sterilization modalities may be deployed and adopted by the healthcare product manufacturing industry, for the benefit of patient care.