Defining an Acceptable Equilibration Time After Cycle Development Runs Allowed? Which Rationale?

Dear all,

Let’s say, after weeks of pre-vacuum phase engineering cycles to develop a sterilization program, the equilibration time for a complicated tubeset was able to be reduced from 10 minutes to let’s say 2 minutes. Every time, a bioindicator test was included at the mapping point of the thermocouple, and only the first trial (the one with 10 minutes equilibration time) the bioindicator test was not passed and showed growth. All other tests thereafter showed a inactivated bioindicator.

Since the requirements is 30 seconds (for large 800L+ autoclaves), can we rationalize 2 minutes as an acceptable equilibration time for validation? If so, which other supporting data would be desired for inspections?

If 2 minutes if accepted, how could we defend in case of 2 min 5 seconds equilibration time during validation?


The 30 second equilibration requirement came from EN285 (from Europe, but being adopted world-wide).

The purpose of the requirement is to show that all the air has been removed from the equipment, and that steam has consistent concentration throughout the load.

You have a few options here to meet that requirements. (1) Break the load into smaller parts (so steam can effectively get to every nook/cranny), but then you’ll have to aseptically re-assemble the parts under hood or using sterile connects. (2) Replace some of the tubing to have larger diameter. (3) Shorten some of the lengths of tubing.

Another thing to look at to help see if there is adequate steam penetration is temperature uniformity within the autoclave. There are additional requirements in steam sterilization industry standards that all temperatures are uniform or within +/- of the mean. Have those requirements been met as well? If so, then you have a better argument that the 2 minute equilibration time is adequate. If not, then you still might have air pockets within the chamber.

Overall, I would strongly recommend modifying the load somehow, as your original 10 minutes equilibration time is a quite long, and shows a very tortuous path for steam to have to penetrate to get everywhere.

Also, another thing to consider with tubing assemlies, is that arrangement of the assembly within the sterlizer can have a huge impact on results. A coil of tubing with a low point could cause a puddle to form in the tubing during a run, blocking steam, and therefore causing the sterilization to be innefective. In this case, something might pass validation then when set-up by someone else, there might be less caution to avoid low points, then the load might not get sterilized, and potentially hurt/kill someone due to microbial contamination.

Not to preach. But typically steam sterilization is considered a high risk operation. As the results of incomplete sterilization for some products is serious hurt to the patient.

Regarding the 2 minute equilibration time. Some people have modified the last pre-vac cycle, so that it is less extreme and ramps to the final set-point a little more slowly - if everything else is robust and reliable.

This cycle with 2-10 minutes of equilibration time is not acceptable. The period is too long and you do not have steam sterilization, you have sterilization with hot air because steam can not penetrate enough, steam is heating air and air transfer heat . Maybe it can not be sterilize with steam simple…

Thanks for the replies! I was just wondering, since the bioindicator D-value = 1.8 was inactivated with this long equilibration time (2-8 minutes), can’t this be a “proof” that somehow saturated steam conditions were present inside the tubeset?

I am pretty positive that this bioindicator (exposed to 121 C dry heat) would not be inactivated after 30 minutes. What is your opinion?


Dear Monepok, yes, there are situation when even you have long equilibration time, there are no survival BIs. But, you and we do not know how your process ia strong? Do you have just killed BIs or you have safety margine to have repetability of process. Do we know how strong is your sterilization process…? This is the reason that we want to have good autoclave practice - all parameters that can affect sterilization (steam quality, holding time, eq time) are in acceptable limits…

Agreed. Monepok, another question: what is the concentration of your BI’s? I;ve always used 10^6 for autoclave and using the overkill method.

Also autoclave “development” should have also been done prior to qualification. Has development been done? If you need to shorten your equilibration time to 30 seconds, what steps have you already taken?