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Understanding ISO 11138-2:2017 – Biological Indicators for Ethylene Oxide Sterilization
Key requirements, testing protocols, and compliance guidelines for healthcare and sterilization industries
Scope and Application of ISO 11138-2:2017
ISO 11138-2:2017, adopted in Canada as CAN/CSA-ISO 11138-2-17, defines the specific requirements for biological indicators (BIs) used to validate and monitor ethylene oxide (EO) sterilization processes. The standard is part of the ISO 11138 series, which covers general requirements (Part 1) and BIs for other sterilants such as steam (Part 3) and low-temperature steam formaldehyde (Part 4).
This standard applies to BIs that are inoculated with viable microorganisms of known resistance to EO. These indicators are essential for assessing the lethality of sterilization cycles, performing routine process control, and making release decisions. The intended users include sterilization service providers, healthcare facilities, medical device manufacturers, and regulatory bodies that require evidence of sterilization efficacy.
Tip: ISO 11138-2:2017 should be used in conjunction with ISO 11138-1:2017, which covers general requirements for biological indicators, including packaging, labeling, and quality assurance provisions.
The standard explicitly covers BIs for EO processes that operate within the typical parameter ranges: ethylene oxide gas concentrations from 300 to 1200 mg/L, temperatures from 30°C to 60°C, relative humidity from 40% to 80%, and exposure times of 30 minutes to 6 hours. It is not intended for BIs used in direct gas or plasma sterilization methods outside these ranges.
Technical Requirements and Performance Testing
Microbiological and Resistance Specifications
ISO 11138-2:2017 mandates the use of Bacillus atrophaeus (formerly Bacillus subtilis var. niger) as the standard test organism. The BI must contain a defined population of spores with known resistance characteristics. Key performance parameters include:
- D-value: The time (in minutes) required to achieve a 1-log (90%) reduction in the viable spore population under specified EO exposure conditions. The minimum D-value shall be ≥ 1.0 minute when tested at 50°C, 600 mg/L EO, and 60% relative humidity.
- Population: The labeled spore count per carrier shall be between 1.0 × 10⁵ and 1.0 × 10⁶ colony-forming units (CFU). The actual count must be within ±50% of the labeled count.
- Resistance profile: A survival curve (number of survivors vs. exposure time) must be established, showing a consistent log-linear decline with no shoulder effect.
- Purity: The inoculum shall be free from contamination by other microorganisms.
| Parameter | Specification | Test Method |
|---|---|---|
| Test organism | Bacillus atrophaeus | Identification by morphological and biochemical tests |
| Spore population | 1.0 × 10⁵ to 1.0 × 10⁶ CFU/carrier | Serial dilution and plating |
| D-value (minimum) | ≥ 1.0 minute at 50°C, 600 mg/L EO, 60% RH | Fraction-negative or direct count method |
| Survival time (ST) | ≥ D-value × 4 minutes | Calculated from D-value |
| Kill time (KT) | ≤ D-value × 16 minutes | Calculated from D-value |
| Resistance stability | No significant change after 2 years at 25°C/60% RH | Accelerated aging and D-value re-test |
Testing Conditions and Methodology
The standard specifies precise test conditions for determining D-values and resistance profiles. Testing must be performed in a defined EO atmosphere, with gas concentration controlled to ±10% by volume, temperature within ±1°C, and relative humidity within ±5%. The recovery medium (e.g., soybean casein digest agar or broth) and incubation conditions (typically 35°C for 7 days) are also standardized to ensure reproducibility.
Warning: Deviations from the specified test conditions can alter the measured D-value significantly. Always document test parameters and validate any variations in your laboratory protocol.
Implementation Highlights and Best Practices
Integration with Sterilization Validation
ISO 11138-2:2017 BIs are integral to the three-stage validation process for EO sterilization: installation qualification (IQ), operational qualification (OQ), and performance qualification (PQ). During PQ, BIs placed in worst-case locations within the load must be inactivated to demonstrate the cycle’s lethality. The standard requires that BIs used for validation meet the manufacturer’s labeled D-value and population to ensure the safety margin.
For routine monitoring, BIs are placed in each load and subjected to a partial cycle or the full cycle. The standard recommends that the BI’s resistance is verified at least annually by the user or manufacturer to confirm stability. Additionally, the recovery of any surviving spores after exposure must be interpreted using defined acceptance criteria (e.g., no growth after full cycle indicates sterility).
Handling and Storage
BIs must be stored in their original packaging at controlled room temperature (15°C–30°C) and relative humidity below 60%. Avoid exposure to ethylene oxide outside of testing, which could degrade the resistance characteristics. The standard advises against using BIs past their expiration date, and each lot must be verified upon receipt via visual inspection and a resistance performance test if the user requires high assurance.
Best Practice: Use BIs with a known D-value that matches your process lethality requirements. For high-risk products (e.g., implants), consider including BIs with higher D-values as part of overkill cycles to demonstrate a safety margin.
Compliance Notes and Regulatory Acceptance
Compliance with ISO 11138-2:2017 is widely recognized by international regulatory authorities, including Health Canada (recognizing CAN/CSA-ISO 11138-2-17), the US Food and Drug Administration (FDA), and the European Union’s Notified Bodies for CE marking of medical devices. The standard provides a basis for showing that sterilization processes are effective and that the BIs used for monitoring are reliable.
Organizations seeking compliance must establish a quality management system that covers BI purchasing, incoming inspection, use, and performance documentation. ISO 11138-2:2017 also requires that the BI manufacturer provide a certificate of compliance and detailed instructions for use, including safety precautions related to the test organism and the handling of EO residuals.
Important: Using BIs that do not meet the ISO 11138-2 specifications (e.g., non-standard organisms, incorrect population, or unverified D-value) can lead to false-positive or false-negative sterilization results, jeopardizing patient safety and regulatory approval.
Common compliance gaps include failure to verify the D-value upon receipt, using expired BIs, and inadequate recovery conditions (e.g., insufficient incubation time or inappropriate media). Auditors will review the traceability of BIs to their certificates and the documented evidence of resistance stability. The standard also recommends participation in inter-laboratory comparison programs to validate testing competence.
Q: Why is Bacillus atrophaeus specified for ethylene oxide BIs?
A: This organism produces spores with high and consistent resistance to EO, making it suitable for demonstrating the lethality of the sterilization process. Its resistance characteristics are well-documented and allow for reliable comparison across laboratories and protocols.
A: This organism produces spores with high and consistent resistance to EO, making it suitable for demonstrating the lethality of the sterilization process. Its resistance characteristics are well-documented and allow for reliable comparison across laboratories and protocols.
Q: Can I use ISO 11138-2:2017 BIs for other sterilization methods like steam or hydrogen peroxide?
A: No. Each part of the ISO 11138 series specifies BIs for a specific sterilant. Steam BIs are covered in ISO 11138-3, and low-temperature vaporized hydrogen peroxide BIs are in ISO 11138-5. Using the wrong BI can lead to incorrect lethality results and potential sterility failures.
A: No. Each part of the ISO 11138 series specifies BIs for a specific sterilant. Steam BIs are covered in ISO 11138-3, and low-temperature vaporized hydrogen peroxide BIs are in ISO 11138-5. Using the wrong BI can lead to incorrect lethality results and potential sterility failures.
Q: How often should I verify the D-value of the BIs I use?
A: The standard recommends at least annual verification of resistance performance by the BI manufacturer, and after any significant change in the manufacturing process. Users are encouraged to perform periodic verification according to their quality assurance policies, especially when the BIs are used for critical load release.
A: The standard recommends at least annual verification of resistance performance by the BI manufacturer, and after any significant change in the manufacturing process. Users are encouraged to perform periodic verification according to their quality assurance policies, especially when the BIs are used for critical load release.
This article is based on ISO 11138-2:2017 (Sterilization of health care products — Biological indicators — Part 2: Biological indicators for ethylene oxide sterilization processes) and its Canadian adoption CAN/CSA-ISO 11138-2-17. Always refer to the latest version of the standard for official requirements.
Published 2026