VALIDATION OF ETHYLENE OXIDE STERILIZATION CYCLES

Ethylene oxide (EtO) has been a sterilant for over 50 years. Yet, while much
attention in the literature has been focused on validation of heat sterilization
cycles, EtO cycle validation has received relatively little attention. Undoubtedly,a major reason is the inability to define accurately the kinetics of microbial
death upon exposure to EtO. This is a result of the complexity of the process,
in which not one but three variables—heat, EtO concentration, and relative humidity—
must be controlled in order to determine D values of microorganisms
when considering EtO sterilization.
The discussion of EtO validation in this section reflects largely what has
been written on this subject since 1977. Several good references [31–35] have
significantly contributed to the rationale, design, and implementation of validation
programs for EtO sterilization cycles.
Five variables are critical to the EtO process. They are EtO concentration,
relative humidity, temperature, time, and pressure/vacuum. Temperature is the
easiest variable to measure and monitor, therefore temperature is used as the indicator
of the worst-case location within the loaded EtO sterilizer. Once the worstcase
location is identified, the validation studies are conducted with the goal of
inactivating a known concentration of indicator micro-organisms in the worstcase
location using a specific loading pattern with a specific EtO cycle with all
variables defined and controlled.
The procedure for EtO cycle validation can be described in eight steps.
1. Address the products specifications and package design. What is the
chemical nature of the components of the product? Do there exist
long and/or narrow lumens that will represent barriers to EtO permeation?
How dense are the materials through which EtO gas must permeate?
What is the nature of the primary and secondary packaging?
Where are dead air spaces within the package and within the load?
By addressing questions such as these, the problems in validating the
EtO cycle can be anticipated and solved at an early stage in the validation
process.
2. Use a laboratory-sized EtO sterilizer during early phases of the validation
process as long as the sterilizer is equipped with devices allowing
variability in vacuum, relative humidity, temperature, gas pressure,
timing, and rate of gassing the chamber. Involve production sterilizer
experts in these early phases of the EtO validation process.
3. Verify the calibration of all instrumentation involved in monitoring
the EtO cycle. Examples include thermocouple and pressure gauge
calibration, gas leak testing equipment, relative humidity sensors, and
gas chromatographic instrumentation.
4. Perform an extensive temperature distribution study using an empty
sterilizer. Identify the zones of temperature extremes, then use these
locations for monitoring during loaded vessel runs. Monitoring will
be accomplished using both thermocouples and biological indicator
spore strips. The most common biological indicator for EtO cycle validation is B. subtilis var. niger. Concentration of these spores per
strip usually is 106. Significant spore survival results will indicate the
need to increase the cycle lethality parameters. It is also prudent to
analyze gas concentration at periodic intervals during the distribution
studies.
5. Do a series of repetitive runs for each sterilization cycle in an empty
vessel in order to verify the accuracy and reliability of the sterilizer
controls and monitoring equipment. Thermocouple locations should
be basically the same for all the heat-distribution studies.
6. Do a series of repetitive heat-distribution and heat-penetration runs
using a loaded EtO sterilizer. The sterilizer should be an industrial
unit in order to ascertain the cycle requirements that will yield consistent
and reliable assurance that all components of the load will be
sterile. The validation procedure should include data collected on both
partial- and full-load sizes. The loading design should be defined at
this point. Dummy loads closely resembling the actual packaging can
be used to test cyclic parameters. Thermocouples and biological indicators
should be placed in a statistically designed format throughout
the load, including areas within the dummy packaged products. The
number of loading patterns, repetitive runs, and the daily timing sequence
of events should all be based upon prior knowledge and experience.
At this point and before proceeding further, the data should
verify the following questions:
a. What is the concentration of EtO released into the vessel?
b. What is the concentration of water vapor in the vessel?
c. What is the range of temperature distribution throughout the
loaded vessel?
d. How much EtO is consumed during the cycle?
e. What are the rates of creating a vacuum and applying pressure?
f. What D value should be used for the biological indicator employed?
g. Does the selected cycle sterilize the product, and what is the estimated
probability of nonsterility?
7. Tests should be conducted on the final packaged product. The protocol
applied should be one that leads to minimal interruption of the
standard manufacturing operations of the facility. Intermediate pilot
plant studies should be carried out to simulate large-scale industrial
sterilization cycles. The EtO cycle documentation should be integrated
into a single protocol. An example of one protocol is as follows:
a. Use approximately 10 biological indicators per 100 cubic feet of
chamber space.b. Place these indicators throughout the load along with thermocouples
at the same locations.
c. Use at least three sublethal exposure cycle times, each in triplicate;
then define the required EtO exposure times using D value
calculations. The exposure time should be increased by an additional
50% to add a safety factor.
d. Perform three or more fully loaded sterilization cycles at the selected
exposure time, monitoring these cycles with thermocouples
and biological indicators.
e. Concomitantly, perform EtO residual tests on the materials exposed
to the desired exposure cycle times from full-load runs.
8. Institute a documented monitoring system primarily relying on biological
indicators, with lesser reliance on end-product sterility testing.