Choosing a PCD Configuration for Your Cycle

By Crystal Hostler

Ethylene Oxide (EO) is an effective and widely used sterilant that has been successfully employed for largescale industrial sterilization of medical products for decades. One of the tools utilized for the development, validation and routine monitoring of the process is the process challenge device (PCD). A PCD is a device or test pack containing a microbiological challenge that represents the most difficult to sterilize location in the load. Mesa manufactures a family of PCDs that have nominal D-values ranging from 3 to 58 minutes. This wide window allows the user to select the PCD configuration that performs in a similar manner as the microbially challenged product.

This Spore News will assist the user in selecting an appropriate PCD configuration.

Choosing an appropriate PCD Configuration Overkill half-cycle approach
When selecting a process challenge device (PCD), you must first develop your cycle using biological indicators (BIs) embedded in the most difficult-to-sterilize location(s) in the load. This can be done using an internal process challenge device (iPCD).

1. The overkill half-cycle approach is conducted by first running fractional cycles, in which some of the BIs embedded in the load survive and some of the BIs are killed.
2. Data from the fractional cycle is used to calculate the Spore Log Reduction (SLR) achieved at that exposure time.
3. The cycle time is then extended incrementally to obtain total kill of all the embedded BIs. Total kill of all BIs is obtained at approximately an 8 SLR of a BI with a 10⁶ spore population.
4. This exposure time, where all of the BIs were killed, would then become the time of the half-cycle.
5. The half-cycle time is demonstrated upon completion of three consecutive exposures, each of which results in complete kill of all embedded BIs. The half- cycle exposure time can be doubled to obtain the overkill cycle.

6 does not equal 8
ISO 11135:2014, Sterilization of health care products - Ethylene oxide - Requirements for development, validation, and routine control of a sterilization process for medical devices, states that complete kill must be obtained in the half-cycle. Those who are not familiar with BIs may think that complete kill is obtained within 6 SLRs. The logic being that the “overkill method” is centered around achieving 12 SLRs, which is equivalent to a Sterility Assurance Level (SAL) of 10^-6; therefore, half of 12 is 6. So, it is automatically assumed that in 6 SLRs all spores are killed, but this is NOT true.

Complete kill occurs at 8 SLRs
Theoretically, for a BI with a 1 x 10⁶ spore population (at 6 SLRs of the population), there would be one surviving spore per BI. However, there is not going to be exactly one spore per BI—some are going to contain one while others may contain zero, two or three spores. Statistically, this works out to 63% of the BIs being positive.

At the log of the population + 1 (or 7 SLRs), statistically 10% of the exposed BIs would be positive.

At the log of the population + 2 (or 8 SLRs), statistically 1% of the exposed BIs would be positive. This would be the exposure time where complete kill would consistently appear because the likelihood of observing that one positive would be small unless there are 100 or more replicate BIs.

If the target is to have complete kill at the half-cycle, you should expose the units until the time point at which 8 SLR is achieved.

Exposing the BIs to this time period (8 SLR) is approximate because it is not known at which time point prior to the cycle time the BIs were actually killed.

For this reason, we suggest you run a cycle in which there is fractional kill and then slowly increase the cycle until complete kill is achieved. However, total kill at 8 SLR is sufficient if the legwork in developing a cycle has been completed for which the internal or imbedded BI (iPCD) is killed in the half-cycle.

Calculating a process D-value
Once the cycle development work has been completed, the D-value can be calculated. D-value is the time it takes to reduce the BI population by one log. Take the exposure time at the half-cycle and divide by 8, because 8 SLR is where total kill of a BI with a 10⁶ spore population has occurred.

One may purposely target 6 SLR when conducting initial studies and the performance of the iPCD or ePCD is unknown. In this case, the fraction negative results, where some units are positive and some negative, can be accurately calculated using the exact SLRs achieved with the MPN method where MPN =ln(n/r). For more information see Addendum at the end of this article.

Example: D-value calculation
Let’s assume that you have already performed the initial testing with imbedded BIs, and this is what you have used to structure the half-cycles. Let’s also assume you have a total cycle time of 4 hours (or 240 minutes).

If you simply want to prove kill at the “half-cycle,” then we would be looking for kill at 120 minutes.

If we then take 120 minutes and divide it by 8 SLR needed for kill, we obtain 15 minutes. This is the approximate D-value needed to achieve kill at the half-cycle.

120 min / 8 SLR = 15 min D-value

Nominal D-values on Mesa PCD
Mesa Labs PCDs are supplied with a nominal D-value which is determined in an ISO 18472 compliant resistometer. A
resistometer uses the parameters of 600 mg/L EO, 54°C and 60% relative humidity (RH) with the PCDs being the only objects in the chamber. A commercial sterilizer may use different parameters and will be loaded, so the PCD will likely perform differently than in a resistometer. PCD resistance can be calculated using the resistometer data and EO parameters; however, the data is only good for that exact EO cycle in a small resistometer chamber. In the field, medical device companies use many different EO cycles, and the EO cycles are much different than those in a resistometer. The large EO sterilizers do not perform the same as a resistometer. Sterilizer temperature and, especially, product temperature can vary. Vacuum removal times, %RH, EO charging times and post-vacuum rates can affect the results, leaving an exact comparison between the PCD in a resistometer and a PCD in a large industrial EO sterilizer somewhat variable.

How to choose a PCD – The PCD Selection Set
The Mesa selection set is designed to identify the most appropriate external PCD (ePCD) for routine monitoring of the validated cycle. The set consists of four PCD types each with an increasing resistance to the process. The set is attached to the outside of the load packaging and processed in the half cycle exposure. The PCD configuration in the set that exhibits slightly greater resistance as compared to the iPCD is the most appropriate for use as the ePCD. The PCD Selection Set – Tech Sheet contains information on the nominal D-values.

Additional considerations
ISO 11135 states that, at the half-cycle, all iPCDs must be killed, but some positive ePCDs are allowed. The reason for this is that if all iPCDs and all ePCDs are killed in the half-cycle, the user doesn’t know which was killed first nor if the ePCD is more resistant. If all the iPCDs are killed in the half-cycle and there are some positive ePCDs, the ePCD has demonstrated greater resistance. The full cycle must demonstrate total kill for both the iPCDs and ePCDs.

The iPCD and ePCD do not have to use the same BI. For example, the iPCD may consist of a microstrip placed into the device, and the ePCD may contain a self-contained BI, such as EZTest®. It’s important that the ePCD configuration provides a greater challenge than the iPCD.

Some EO sterilizers will include a pre-humidification phase, which is sometimes performed in a separate chamber. The maximum allowable time should be established and adhered to—from the end of the pre-humidification to the start of the cycle. In this case, you should establish that relative humidity is a critical parameter of an EO cycle, and, if the pre-humidification is reduced due to a long time elapsing between end of pre-humidification and beginning of the cycle, the materials may absorb some of the RH from the cycle, thus reducing the lethality. Keep the following three points in mind:

Load configuration must be consistent
Any change in load configuration can cause different conditions in different locations within the chamber, which may result in unexpected positive results. One suggestion is to build a load configuration that is a mirror image of itself (where the front and back are the same). This way, if the load is inadvertently placed into the chamber backwards, it will not affect the conditions in the chamber.

If a load is wrapped in plastic, you should place the ePCD on the outside of the plastic. The plastic will create a barrier. Even if the validation was performed with the ePCDs under one layer of plastic, there is a chance that a technician may use one layer of plastic, but another technician will use two or three layers of plastic, creating more of a barrier than before.

IPCDs and EPCDs must be handled consistently at all stages
For validation fractional cycles, validation half-cycles, validation full-cycles and routine cycles, the most critical time of consistent handling is after the cycle. The BIs should be removed from the iPCD and ePCD pouch consistently, such as immediately post exposure or just before the BIs are cultured/incubated. The design of the iPCD and ePCD pouch makes it difficult for the EO to enter and reach the BI, also making it difficult for the EO to leave the iPCD and ePCD pouch.

Consequently, there is potential for residual EO to remain in the iPCD and ePCD pouch, which would provide additional lethality to the BI. For example, if the BI isn’t usually removed from the PCD pouch until right before it is cultured/incubated, but one time the BI is removed from the PCD pouch immediately after it was removed from the sterilizer, there is the potential for a variance from previous results.

PCDs/BIs should be inspected before use, especially if it is a self-contained BI
Do not use PCD if the media ampoule of a self-contained BI is broken. The media will wet the cap and paper spore carrier and the EO will not penetrate properly. Additionally, the culture media will not perform as designed. Low-fill volume in the media ampoule, condensate inside the plastic body, filter paper under the cap appearing wet or discolored and the spore strip appearing wet or discolored are indications of a damaged media ampoule or PCD/BI. The PCD pouch should also be inspected for intact seals.

Ready to get started?
We are available to help you select an appropriate PCD and can guide you through the process to ensure your new PCDs are properly integrated into your sterilization protocols.

To expedite the process, please let us know:

+ The parameters of your cycle (EO concentration, RH, temperature and exposure time)
+ If you have a validated cycle for your internal BIs
+ The exposure time of your half-cycle or full-cycle

For more information visit mesalabs.com
Download our PCD selection Guide

Addendum
Spore Log Reduction (SLR)
Total kill of all BIs will appear approximately within an 8-log reduction, which is approximately at the theoretical kill time.

Most Probable Number (MPN)
The percent of positive BIs above comes from the MPN equation. MPN = ln(n/r), where “n” is the number of exposed BIs and “r” is the number of negative BIs.

For example, the BI above with a population of 1.0 x 10⁶ and a 6 SLR has a 63% survival rate. To determine a percentage, we set “n” equal to 100 and solve for “r”. The 63% number is for when the MPN = 1.0 (i.e., if our BI population was exactly 1.0 x 10⁶ and we applied exactly 6.0 SLRs to that BI).

Thus:
1.0 = ln (100/r)
r = 100/inv ln
1.0 r = 100/2.718
r = 36.79

Therefore, out of 100 exposed replicate BIs, we’d predict that approximately 37 BIs would be negative and 63 BIs would be positive or a 63% chance for survival.

If replicate BIs are used at each location, you can calculate the SLR achieved at each location when fractional results are observed. For example, at one location containing 10 BIs with a population =

2.3 x 10⁶ and 5 BIs were negative and 5 BIs were positive.

MPN = ln(10/5)
MPN = ln(2)
MPN = 0.693

Next, we use this information to calculate the SLR at this test location with the following equation:

SLR = Log₁₀ N₀ – Log₁₀ MPN₀

Where SLR equals spore log reduction, and N₀ equals the initial spore population of the non-exposed BI as listed on the COA.

SLR = Log₁₀ 2.3 x 10⁶ – Log₁₀ 0.693
SLR = 6.362 – (-0.159)
SLR = 6.521

This would mean that, at this test location, you have achieved a 6.521 SLR.

To calculate the SLR using the MPN equation, you need to use statistical equivalents or multiple BIs (at least 3 BIs) in one location. You cannot calculate an SLR at a specific location by using the results of BIs at different locations. Ideally, to develop a cycle, you would use multiple BIs at multiple locations, since different conditions, such as differing concentration, temperature or RH