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 hu- midity—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 valida- tion 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 indi- cator of the worst-case location within the loaded EtO sterilizer. Once the worst- case location is identified, the validation studies are conducted with the goal of inactivating a known concentration of indicator micro-organisms in the worst- case 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 perme- ation? How dense are the materials through which EtO gas must per- meate? 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 valida- tion process.

2. Use a laboratory-sized EtO sterilizer during early phases of the valida- tion 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 consis- tent 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 indi- cators 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 se- quence of events should all be based upon prior knowledge and expe- rience. 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 em-

ployed?

g. Does the selected cycle sterilize the product, and what is the esti- mated probability of nonsterility?

7. Tests should be conducted on the final packaged product. The proto- col 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 inte- grated into a single protocol. An example of one protocol is as fol- lows:

a. Use approximately 10 biological indicators per 100 cubic feet of chamber space.

b. Place these indicators throughout the load along with thermocou- ples at the same locations.

c. Use at least three sublethal exposure cycle times, each in tripli- cate; then define the required EtO exposure times using D value calculations. The exposure time should be increased by an addi- tional 50% to add a safety factor.

d. Perform three or more fully loaded sterilization cycles at the se- lected exposure time, monitoring these cycles with thermocouples and biological indicators.

e. Concomitantly, perform EtO residual tests on the materials ex- posed to the desired exposure cycle times from full-load runs. 8. Institute a documented monitoring system primarily relying on bio-

logical indicators, with lesser reliance on end-product sterility testing.

VII. VALIDATION OF VAPOR PHASE HYDROGEN