The overkill method (AAMI/ISO 11135 Method C) is most commonly used when performing an EtO sterilization validation. The overkill method is based on demonstrating that the sterilization of a microbial challenge (biological indicator) exceeds the challenge posed by the bioburden of the product.

Ethylene Oxide (ETO) Cycle Validation – Microbiological Requirements

1. Validation Protocol
2. Bioburden Method Validation
3. Bioburden Enumeration
4. Bacteriostasis/Fungistasis Test
5. Microbial Challenge – Use of Biological Indicators
6. Product vs. B.I. Resistance to EO
7. Inclusion of External B.I.s
8. Product Load Configuration

EO Cycle Parameters and Monitoring

All EO cycles must be run using sterilization equipment that has been calibrated and for which IQ and OQ processes have been completed.  Verify equipment status with the contract sterilizer before initiating the validation cycles. The parameters for the fractional, half and full cycles must be established in consultation with the contract sterilizer. During validation cycles, the temperature of the product at several widely distributed locations should be monitored and compared with the chamber control temperature. The monitored locations should include somewhere product with B.I. test samples are located. The sterilizer chamber EO concentration and the chamber and product humidity should also be monitored.

1. Microbial Validation – Sample Processing
2. Routine Cycle Monitoring
3. Routine Bioburden Monitoring
4. Revalidation

Validation Protocol

For an initial validation, a protocol should be prepared which outlines the overall validation requirements. The protocol should describe the medical device and specify the test procedures that will be used.

Please submit one sample to Pacific BioLabs for evaluation

Bioburden Method Validation

The method that will be used for routine determination of product bioburden levels must be validated to insure that it is effective in recovering microorganisms from the product and that it allows for adequate growth of the recovered microorganisms. Typically validating the bioburden test method involves two phases of testing: adverse substance screening and recovery testing.

Recovery Validation using the Repetitive (Exhaustive) Method (5 nonsterile samples are recommended: a minimum of 3 samples are required) -OR- Product Inoculation (Simulated) Method (5 sterile samples are recommended: a minimum of 3 samples are required). This study will determine if it is necessary to apply a recovery factor to routine bioburden test results.

Bioburden Enumeration

The bioburden level of the product must be determined. For most products, aerobic bacteria and fungi bioburden are appropriate. AAMI/ISO recommends the testing of 10 samples from each of three production lots. It is most appropriate to sample the production lots that will be used in the three validation cycles. The bioburden samples may be selected to represent various production or packaging times. The samples should be collected immediately prior to sterilization.

10 nonsterile samples from each of three production lots are recommended.

Bacteriotasis/Fungistasis Test

The procedure that will be used to perform product sterility testing must be validated by means of bacteriostasis/fungistasis testing. This testing will ensure that false negative results will not occur in the sterility test. A false negative result allows a nonsterile sample to appear sterile due to inhibition of the microbial growth. This type of reaction is caused by certain materials utilized in some medical devices, but can be overcome by modifications to the sterility test procedure. Sterile samples are required for B/F testing. If sterile samples are not initially available, the B/F samples can be run with the fractional or half-cycles.

6 sterile samples are required.

Microbial Challenge – Use of Biological Indicators

For the validation study, biological indicators (B.I.s) are used to challenge EO cycles. The indicator organism is Bacillus atrophaeus (formerly subtilis var. niger), a spore forming bacteria with well characterized resistance to the EO sterilization process. Most commonly, B. atrophaeus spore strips with a population of 106 colony forming units (cfu) per strip are used. The population label claim from each lot of biological indicators used for validation testing must be verified. Three biological indicator replicates per lot used for validation purposes should be tested.

Product vs. B.I. Resistance to EO

One purpose of the validation process is to support the use of B. atrophaeus biological indicators as a release mechanism for the product following routine EO processing. This can be done by means of one or more fractional cycles. After EO processing in the fractional cycle(s), both the product and the spore strips will be tested for sterility. Sterility test results must indicate that the B. atrophaeus organisms on the spore strips are more resistant to the EO process than the organisms that make up the bioburden of the product. If this is verified, and if product tests sterile in the fractional cycles, it is only necessary to test the spore strips after EO processing in the half cycles. For the half cycles, the spore strips must be placed in the product in same manner that was used in the fractional cycle. + B.I. Placement and Quantity Required

Before processing in EO cycles, the biological indicators must be placed within the product inside the product package. The product must be evaluated to determine appropriate locations to place the B.I.s. The area of the device that presents the greatest potential challenge to gas penetration should be selected.

The ISO/AAMI guideline recommends calculating the appropriate quantity of B.I. challenge locations based on usable chamber volume as follows: 20 B.I.s for the first 5 m3 of chamber volume, 2 B.I.s for each additional m3 between 5 m3 and 10 m3 and then 1 B.I. for each m3 beyond 10 m3. An unexposed positive control B.I. should be sent to the lab with each group of exposed B.I.s.

A minimum of 20 biological indicators and 1 positive control B.I. is required.

Inclusion of External B.I.s

The use of external B.I.s as a release mechanism for routine EO processed products may be validated as part of the initial sterilization validation. This could eliminate the loss of product associated with allocating samples for internal B.I. placement and sterility testing. In addition to the B.I.s placed within the product, a second set of B.I.s is placed outside of product packages and processed in the fractional cycles (some customized packaging of the B.I.s is usually required to increase their survivability.) Sterility test results must indicate that the external B.I. has a similar or greater resistance to the EO process than the B.I. located within the product.

Product Load Configuration

For validation cycles, the maximum quantity of product representing the densest load configuration that will be processed in full routine cycles must be loaded in the EO sterilizer chamber. The product with B.I. test samples must be widely distributed throughout the sterilizer chamber. A placement diagram must be prepared, typically by the contract sterilization facility in consultation with the device manufacturer. The diagram must indicate the location of the product with B.I. test samples plus temperature and humidity sensors in the loaded chamber.

Microbial Validation – Sample Processing

Each cycle load is preconditioned for the designated minimum time and then processed in the sterilizer chamber using the previously defined parameters. The product (if applicable) and B.I. test samples must be removed from the load either before aeration or after the minimum routine aeration time. The test samples should be sent to Pacific BioLabs for sterility testing via same day or next day delivery service.

1 or more fractional cycles – products and B.I.s (internal and, if desired, external) to be tested for sterility

3 half cycles – B.I.s (internal and, if desired, external) to be tested for sterility

3 full cycles – B.I.s (internal or, if applicable, external) to be tested for sterility

For the full cycles, data from the first 3 routine processing loads will be included to support the initial EO sterilization validation. The microbial validation is acceptable if the sterility test results for the product in the fractional cycle are negative and all of the B.I.s from the half cycle are negative (B.I. positive results in the fractional cycle are expected). The sterility testing of B.I.s from full cycles must verify half cycle results.+ Ethylene Oxide Residuals Testing

To complete the validation of the EO sterilization process, ethylene oxide residuals testing of the sterile medical device is required. AAMI/ISO has published standards for EO residuals limits. The testing can be conducted at the final desired aeration timepoint, or if desired an EO decay curve can be established. Periodic sampling and analysis of the product can establish an EO dissipation curve. This data can be used to establish quarantine times prior to product release, or to provide more information about the changes in product EO levels resulting from manufacturing, packaging or sterilization process changes. EO analyses of samples at three to five time points are normally recommended to determine an EO dissipation curve.

At least 2 sterile samples per testing timepoint and 1 unsterilized control sample are required.

Routine Cycle Monitoring

As a result of the validation process, the use of B. atrophaeus spore strips in release testing for routine sterilization loads will likely be validated. Routine sterilization loads should be monitored with 10 to 20 biological indicators. It is a common practice to calculate the appropriate quantity of B.I.s based on usable chamber volume as follows: 10 B.I.s for the first 5 m3 of chamber volume, 1 B.I. for each additional m3 between 5 m3 and 10 m3 and then 1 B.I. for each m3 beyond 10 m3. An unexposed positive control B.I. should be sent to the lab with each group of exposed B.I.s. The load can be released following acceptable sterility testing of the B.I.s.

A minimum of 10 biological indicators and 1 positive control B.I. is required.

Routine Bioburden Monitoring

Product bioburden is an important indication of the microbiological cleanliness and control of the manufacturing process. Control of product bioburden is required to maintain a validated sterility assurance level. Many factors can affect product bioburden. Among these are changes in materials, vendors, manufacturing personnel, procedures or equipment, water systems used in manufacturing and seasonal changes. Therefore, we strongly recommend a minimum of quarterly bioburden testing for all routinely produced sterile medical devices, regardless of the means of terminal sterilization.

Revalidation

An annual documented review of all manufacturing and sterilization processes should be performed to demonstrate that nothing has changed that will affect the performance of the validated sterilization process. In addition to the annual review, AAMI recommends revalidating the EO sterilization process at least every two years to verify the effectiveness of the sterilization process. This revalidation should consist of (at a minimum) bioburden testing, one sub-lethal cycle, one half-cycle, and ethylene oxide residual testing. If any significant changes are made in the product, packaging or manufacturing, a complete revalidation is required.