Guide to validation of terminal sterilization process of drugs (GUI-0074): Process validation, sterilization by ethylene oxide

On this page

• Introduction
• Product/material definition
• Sterilizing agent specifications
• EO sterilization parameters
• Design of EO sterilization cycles
• Equipment qualification

Introduction

Ethylene oxide (EO) is mainly used to sterilize items that are sensitive to moist heat or radiation. EO is a toxic, flammable and explosive substance, listed in a schedule 1 of the Canadian Environmental Protection Act.

For guidelines on EO emission, consult:

• Guidelines for the reduction of ethylene oxide releases from sterilization applications

Factors that influence EO sterilization include:

• bioburden
• temperature
• package density
• relative humidity
• EO gas concentration
• pre-cycle conditioning
• packaging/material type
  • includes insulation characteristics of materials
• product/package loading patterns
• exposure, gassing and evacuation times

Product/material definition

You should design the:

• product/material, packaging and loading pattern to allow air to be removed and heat, humidity and EO to penetrate to the most difficult-to-sterilize locations
• product to allow EO to be removed at the end of the process
• product/material and package to allow EO, heat and humidity to penetrate

For example, you should avoid:

• using non-permeable materials/configurations
• attaching labels with large surface areas to breathable materials
• using plastic or foam inserts/supports
• applying moisture-resistant coatings
• using pressure-relief valves, stopcocks, manifolds or occluded spaces
  • includes closed containers (such as vials, ampules) that restrict or prevent EO penetration
• applying bleaching agents containing free chlorine, which react with EO, ethylene chlorohydrin (ECH) or ethylene glycol (EG))

You should also consider the tolerance of the product/material for the required temperatures.

Note: EO must not affect product/material integrity (for example, by causing cracking, phase separation and bio-compatibility).

Sterilizing agent specifications

You should:

• use a defined sterilizing agent (pure EO or a gas mixture) to validate
• specify the storage conditions and shelf life for the sterilizing agent
• define ethylene oxide (EO) residues:
  • determine rates of dissipation of the major EO residues after being subjected to the EO sterilization cycle
  • specify the maximum allowable levels of EO residues on drugs (limits must be based on safety studies and published international safety standards)
  • validate analytical methods for determining EO and ECH

Note: You may use EO as a pure gas (100%) or in a mixture of gases (such as carbon dioxide or nitrogen).

EO sterilization parameters

Monitor the following parameters:

• dwell time
• temperatures
• vacuum/pressure levels
• air/nitrogen washes humidity
• steam and gas concentration (if applicable)
• transfer time from preconditioning room to sterilizer

Consider the following when designing your EO sterilization cycles:

• product/material preparation
• delivery of the sterilization parameters
• removal of the residual sterilizing agents

Include at least the following in your EO sterilization specification:

• a definition of the preconditioning, exposure and aeration phases of the sterilization cycle
• a description of process parameters and their tolerances
  • include cycle variables: humidity, temperature, EO concentration, pressure and time
• a description of means used to monitor, control and record the process variables and how entire sterilization process is conducted
• a description of controlled conditions to achieve specified temperature and humidity for pre-treating the product/material within the load

Note:

• Residues of ethylene oxide (EO) and its reaction products may be hazardous. Aeration process helps to desorb them.
• Temperature, dwell time, forced air circulation, load characteristics and product/material and packaging materials affect aeration efficiency.
• Aeration may be performed within the sterilizer or in a separate area or both.

Also note:

• Relative humidity:
  • Maintaining an appropriate humidity in the sterilization chamber increases the effectiveness of EO sterilization by increasing EO penetration through bacterial cell walls.
  • A relative humidity of about 35% is beneficial for EO sterilization. Increased humidity can cause condensation on the product, chamber walls and optical EO sensors. Relative humidity of less than 30% results in less effective EO sterilization.
• Temperature:
  • EO cycle effectiveness improves as the temperature increases. The temperature in the chamber must be high enough to prevent the EO from liquefying.
• Gas concentration:
  • At higher EO levels, the sterilization process is more effective and requires a shorter dwell time.
• Diffusion:
  • The higher the diffusion rate of EO from the chamber to the product in the load, the shorter the required dwell time. Diffusion is improved by creating a vacuum in the chamber before it's charged with EO.
• Time:
  • An increase in gas concentration and temperature may shorten the time needed to achieve sterilization.

Humidity used to precondition and condition the product/material should be generated by steam.

Design of EO sterilization cycles

You should define the process to support the validity of process parameters and their tolerances (as defined in your sterilization process specification).

There are 3 basic approaches for validating an EO sterilization cycle:

1. Overkill ½ cycle:

The cycle is developed by performing 3 consecutive studies resulting in total inactivation of the biological indicators to confirm the minimum exposure time. The specified exposure time for the sterilization process should be at least double this minimum time. A fractional cycle of short duration from which BI survivors can be recovered should also be run to demonstrate the recovery technique for BIs exposed to EO gas is adequate.

Overkill cycle calculation:

The cycle is developed by performing time-graded exposures to EO or population-graded BIs exposed to EO, with all other parameters staying the same. The sublethal exposures are then used to calculate the cycle to ensure a 12 log reduction. Following that, routine bioburden monitoring should be performed.

2. Biological indicator (BI)/bioburden cycle:

The cycle is used when the bioburden of the product/material before EO treatment is fairly consistent over time and less resistant than the biological indicator. This sterilization process involves using a microbial challenge population lower than 106 (but not less than 103). Bacillus atrophaeus is commonly used for EO sterilization because of its high resistance. The BI should be distributed throughout the product load and in the same orientation. Placement should include spots that present the greatest challenge to the sterilization cycle.

Note: When developing the EO sterilization cycle and validation studies, you should test biological indicators as soon as possible after exposure to the sterilization cycle. Microbial inactivation continues after the sterilization cycle has been completed due to the presence of EO residues.

3. Absolute bioburden cycle:

This cycle is used when the product bioburden resistance to the EO process is very high (product's bioburden is more resistant than the BI). This can be caused by a number of factors, such as the configuration of the product/material, the quantity or location of the microorganisms, or the bioburden's intrinsic resistance.

The cycle can also be used when the bioburden is very low in resistance and relatively consistent, allowing an optimized sterilization cycle. The absolute bioburden method requires extensive controls of the manufacturing environment in addition to routine product bioburden monitoring and resistance studies.

Cycle development includes:

• exposing representative samples to incremental exposures
• testing the exposed samples for recovery of survivors
• performing counts

Representative product is used in EO cycle development studies. An inactivation curve is established for the product bioburden to project the exposure time required to achieve the desired SAL.

For additional guidance on EO sterilization, consult:

• ISO 11135: Sterilization of health care products – Ethylene oxide – Requirements for the development, validation and routine control of a sterilization process for medical devices

Equipment qualification

Your specification for EO equipment should include equipment from 3 phases of the sterilization process:

• preconditioning
• sterilization
• aeration

For your EO equipment specification, include, at a minimum:

• a description of the equipment
• the composition of the sterilizing agent and how it will be delivered to the chamber
• a description of other gases used in the process and how they will be delivered to the chamber
• the purity and quality of steam and/or compressed gases
• a description of instruments used to monitor, control and record the sterilization process
  • include the characteristics and locations of sensors
• the safety features for personnel and environmental protection
• a description and validation status of software used to control/monitor the process
• a description of the materials used to make the equipment
• the location, space and environment where the equipment is being installed

Examples of instruments that need calibration:

• timers
• balances
• recorders
• gas analyzers
• thermocouples
• pressure and humidity sensors