Millipore Technical Publications |
 | Tech Note - TB1012EN00 | |
The Effect of Peracetic Acid on Steritest Sterility Testing Devices | ||
| Lit Number: | TB1012EN00 | |
| Year: | 2001 | |
Introduction
Closed, disposable Steritest sterility test devices reduce the risk of false positives during the sterility testing of pharmaceuticals. To further reduce the risk of false positives, some manufacturers use the devices inside an isolator. Typically, the filtration devices are placed in the isolator while the isolator is decontaminated using sterilization gases.The purpose of this study was to compare the fertility of standard Steritest devices with HA membrane (TLHALV210) after exposure to a peracetic acid atmosphere in an isolator to nonexposed TLHALV210 Steritest units. The study was conducted in two phases:
- In Phase 1, we compared the fertility of non-exposed Steritest TLHALV210 units to Steritest TLHALV210 units exposed to a peracetic acid atmosphere for 6 hours
followed by 12 hours flushing.
- In Phase 2, we again compared exposed and non-exposed units but pre-rinsed each canister with 50 mL of sterile peptone solution to determine the effect of mem brane pre-rinsing on the elimination of peracetic acid residues.
Figure 1. Pseudomonas aeruginosa shows little resistance to peracetic acid.
Pseudomonas aeruginosa was selected for the fertility tests because of the microorganism's high sensitivity to peracetic acid. (Figure 1). The fertility tests were conducted by:
- Inoculating 10 and 100 germs into 100 mL of broth and transferring the broth into Steritest canisters.
- Inoculating 10 and 100 germs into 10 mL of agar medium and transferring the medium into Steritest units with colony counts.
- Inoculating 10 or 100 germs into a solution, filtering the solution through the Steritest units, and then cutting the membranes from the canisters and placing them in Petri dishes with colony counts.*
* Because Pseudomonas aeruginosa is an aerobic germ, we assumed that its growth can be inhibited in agar medium. Therefore, we conducted this experiment in Phase 1 to determine whether we obtained the same counts with a membrane aerated in a Petri dish. The Phase 1 experiment showed no significant difference in colony counts between the two methods after 7 days incubation and it was decided to keep just the agar medium method for Phase 2.
Experimental Design
Phase 1
Growth promotion of Steritest units TLHALV210 versus Steritest TLHALV210 exposed to a peracetic atmosphere.
| Sterility Control | Fertility at ~ 10 CFU | Fertility at ~ 100 CFU |
ETO-Sterilized Steritest | <p><b>1 Unit 1 canister with 100 mL broth 1 canister with ~ 10 mL agar medium (colony count) | <p><b>10 Units 10 canisters with 100 mL broth 5 canisters with ~10 mL agar medium (colony count) 5 canisters with membrane cut out and placed in a Petri dish (colony count) | <p><b>10 Units 10 canisters with 100 mL broth 5 canisters with ~10 mL agar medium(colony count) 5 canisters with membrane cut out and placed in a Petri dish (colony count) |
| ETO-Sterilized Steritest + 6 Hours Isolator + 12 Hours Flushing | <p><b>1 Unit 1 canister with 100 mL broth 1 canister with ~ 10 mL agar medium (colony count) | <p><b>10 Units 10 canisters with 100 mL broth 5 canisters with ~10 mL agar medium (colony count) 5 canisters with membrane cut out and placed in a Petri dish (colony count) | <p><b>10 Units 10 canisters with 100 mL broth 5 canisters with ~10 mL agar medium (colony count) 5 canisters with membrane cut out and placed in a Petri dish (colony count) |
Phase 2
Growth promotion of Steritest devices versus Steritest units exposed to a peracetic atmosphere followed by a pre-rinsing step with 50 mL sterile peptone solution per canister.
Pre-Rinsing with 50 mL Sterile Peptone Solution Per Canister
| Sterility Control | Fertility at ~ 10 Germs | Fertility at ~ 100 Germs |
ETO-Sterilized Steritest | 1 Unit (with pre-rinsing step) 1 canister with 100 mL broth 1 canister with ~ 10 mL agar medium (colony count) | 5 Units 5 canisters with 100 mL broth 5 canisters with ~10 mL agar medium (colony count) | 5 Units 5 canisters with 100 mL broth 5 canisters with ~10 mL agar medium (colony count) |
| ETO-Sterilized Steritest + 6 Hours Isolator + 12 Hours Flushing | 1 Unit (with pre-rinsing step) 1 canister with 100 mL broth 1 canister with ~ 10 mL agar medium (colony count) | 5 Units 5 canisters with 100 mL broth 5 canisters with ~10 mL agar medium (colony count) | 5 Units 5 canisters with 100 mL broth 5 canisters with ~10 mL agar medium (colony count) |
Materials and Methods
Materials
The following materials were used:
- La Calh‘ne Isolator
- ETO-sterilized Steritest filtration devices in blister packages (Millipore, cat. no. TLHALV210 from one lot number)
- Biological indicators (spore strips)
- Indicator strips for determination of peracetic acid levels (Merckoquant, 5-50 mg, 1,10084,0001)
Methods
The isolator was loaded with Steritest units and 10 spore strips were placed inside the isolator taking into consideration the critical points of the isolator (e.g., in the corners of the isolator chamber and between the blister packages).
In addition, 12 sealed test tubes containing trypticase soy medium were placed in the isolator before the sterilization cycle (10 for the 10 spore strips, one for sterility control, one for fertility control).
After the sterilization cycle and flushing, spore strips were placed into 10 tubes (one spore strip per tube) and the tubes were incubated. The sterility control tube remained closed and the fertility control tube was incubated with an unsterilized spore strip from the same lot.
These steps were performed to ensure the efficacy of the isolator sterilization cycle.
Two additional sealed tubes were also included: one with a dry peracetic indicator strip and one with a sterile peptone solution used for wetting the peracetic indicator strip before reading.
Sterilization of the isolator
The isolator was connected to an automated sterilizer. The volume of peracetic acid required for sterilization was 50mL/hour plus 150 mL residual volume. The sterilization time was set at 6 hours with a timer. The peracetic acid flow rate (as vapor) was adjusted to 45 to 50 mL per minute. The pressure within the isolator was set at 6 to 7 mm water.
Flushing of the isolator
The 12 hour flushing phase was automatically activated after the sterilization phase. Flushing was ensured by the circulation of filtered air through the bubble. All manipulations were then performed by the operator using the glove box under constant filtered airflow (flow rate adjusted to 6 to7 mm of water).
Confirming isolator sterility after flushing
The spore strips confirmed the capability of peracetic acid to kill Bacillus subtilis spores (106). The spore strips previously inserted in various locations of the isolator were put into 10 test tubes containing trypticase soy broth. The tubes were incubated for 14 days at 37oC (10 test tubes with spore strips, one fertility control, and one sterility control).
Confirming acidity levels after flushing
After the isolator was flushed with air, the tubes containing the Merckoquant 5-50 mg peracetic acid indicator strips were opened, the pads were wetted, and readings were performed after exposure to the isolator atmosphere for 5 seconds. The acidity within the isolator was to remain at or below 5 ppm.
Results and Discussion
Peracetic Acid Levels Inside theIsolator
The Merckoquant 5-50 mg peracetic acid indicator strips indicated that the peracetic acid level remained <5 ppm after each sterilization cycle.
Isolator Load Sterility
Isolator sterilization was effective because the 10 spore strips that were placed in different areas within the isolator showed no growth after 14 days incubation in the two sterilization cycles.
Experiment 1:
Growth Promotion of Steritest Units TLHALV210 Versus Steritest TLHALV210 Exposed to a Peracetic Atmosphere
At about 10 CFU, the Steritest units exhibited total growth inhibition in 30% of the cases in liquid medium after 7 days and total growth inhibition in 80% of the cases on solid medium (10 mL agar medium in the canister or membrane cut out and placed in a Petri dish).
At about 150 CFU, the Steritest units exhibited slightly delayed growth in liquid medium and a partial growth inhibition on solid medium (10 mL agar medium in the canister or membrane cut out and placed in a Petri dish). See Tables 1 and 2.
Table 1
Table 2
Experiment 2:
Growth Promotion of Steritest Units Versus Steritest Units Exposed to a Peracetic Atmosphere Followed by a Pre-rinsing Step with 50 mL Sterile Peptone Solution Per Canister
The Steritest units exhibited good growth after 1 day and 7 days in liquid medium and on solid medium (10 ml agar medium in the canister) at both about 5 and 50 CFU (Tables 3 and 4).
Conclusion
The fertility tests done with Steritest units using Pseudomonas aeruginosa, which is known to be highly sensitive to peracetic acid, showed no evidence of inhibitory residuals after rinsing the membrane with 50 mL of sterile peptone solution immediately after peracetic acid sterilization within the isolator.
These conclusions are only valid for the conditions evaluated in this study.Each isolator must be validated independently because each isolator's configuration, contents, and sterilization cycle protocols vary.
If growth inhibition is detected during validation of a sterility testing procedure within an isolator sterilized with peracetic acid, Millipore recommends that customers incorporate and optimize a pre-rinsing step of the Steritest canisters before contact with pharmaceutical product.
Table 3
Table 4