In safety data sheet No. 123, the Institut National de la Recherche Scientifique (INRS) reports Occupational Exposure Limit Values authorized over 8 hrs  (OELV-8h) in the air of working areas ranging from 0.5 ppm in Germany to 1 ppm (1.5 mg.m^-3) in France (INRS, 2022). Thus, it is recommended that over a one-day period workers should not be exposed to more than five times the value of 1 ppm for 15 minutes for a total duration of one and a half hours exposure in the air. However, this exposure value cannot be taken into consideration since the isolator is a closed leak-tight system.

Moreover, this chemical process is a function of the concentration of vaporized H₂O₂ present in the ambience of the chamber, then condensed at the workload surface. The relative humidity at the start of the cycle, the temperature, the efficacy of mixing and the homogeneity of distribution of the decontaminating fluid are parameters that influence a repeatable and validated biodecontamination cycle. Biodecontamination may be broken down into several phases namely a leak-tightness test,  conditioning, injection, stabilization and an aeration phase which reduces the H₂O₂ concentration to a level compatible with the activities taking place in the isolator. This residual  H₂O₂ concentration potentially affects the active molecules of pharmaceutical products. Consequently, the authorities, as for OELV, recommend weighted exposure limits over time of between 0.5 and 1.0 ppm.

As a reminder, the User Requirement Specifications (URS) for sterility tests for isolators requiring biodecontamination with H₂O₂ did not previously include any specific requirement for the rinsing phase, not even measurement of the residual level of sterilizing agent. “Appropriate rinsing” was generally stipulated in these URS without mention of the H₂O₂ concentration in ppm. The important thing was more to check the compatibility of the sterilizing agent with the materials and to show a 6-log spore reduction within the time allocated than to check the residual level. Residual levels of 5, 3 then 1 ppm were then demanded with a rapid rinsing phase without worrying about desorption from polymer or Tyvek packaging. Nevertheless to achieve these low residual concentrations values, the rinsing and waiting time may be long generating immobilization costs.

2. Aim of the study

Based on paragraph 2.6.1. of the European Pharmacopoeia (2020), the aim of this preliminary study was to assess whether a sterility test generated false negatives when it was performed in an isolator with a residual H₂O₂ level above 1 ppm, following its biodecontamination.

3. Materials and Methods

3.1 Equipment used

a. Isolator used

To do this three sterility tests at a residual H₂O₂ concentration of 5 ppm were performed in a 3 glove isolator incorporating a biodecontamination airlock. A reference test at a residual H₂O₂ concentration of 0 ppm was also performed, the residual concentration being measured by a Dräger sensor H₂O₂ LC.

b. Products to be tested

These sterility tests were performed on sterile wipes (Ecolab KlerwipeTM Product reference 3035500).

c. Preparation and sterilization of the culture media used

The sterility of each batch of culture medium made was ensured in compliance with the standard EN ISO 17665-1: 2006, “Sterilization of health care products”.

The fertility of each batch of culture medium was checked before use in accordance with the paragraph “effectiveness of culture media” on testing, chap. 2.6.12 of the European Pharmacopoeia 10^th Edition (2020), the culture media used being Trypticase-soy agar, Sabouraud agar with chloramphenicol, casein hydrolysate and soy broth, Thioglycollate broth with resazurin.

d. Microorganismes testés et conditions de culture

Table 1 presents the microorganisms tested and their culture conditions. These microorganisms were used in the form of calibrated inocula, titrated at between 10 and 100 CFU /0.1ml-1, developed by the company ICARE (see Table 1).

3.2 Methods

a. Preparation

For each sterility test the following were placed in the isolator before decontamination:

– 8 jars containing 500 ml of casein hydrolysate and soy broth

– 8 jars containing 500 ml of thioglycollate broth with resazurin

– 1 packet of 10 sterile wipes

– 8 pairs of sterile forceps

– 1 20-200 μl micropipette packaged in a sterile sachet

– 1 box of 200 μl micropipette tips packaged in a sterile sachet

Once this equipment was deposited, 3 Geobacillus stearothermophilus ATCC 7953 biological indicators with 106 spores/biological indicator were placed in the isolator. In fact, the European Ph. 10^th  edition and the USP 31 recommend the use of Geobacillus stearothermophilus for validation of the sterilization method, as it is the strain most resistant to H₂O₂. It is more resistant than Bacillus subtilis spores, Bacillus being gram+ aerobic bacteria that produce endospores known for their high level of resistance to heat, to chemical products and to dryness. Their viability in low nutrient media is also well known. An H₂O₂ biodecontamination cycle of the isolator chamber was then started.

b. Biodecontamination of the isolator

The biodecontamination system used for this study was a STELEC* H₂O₂  generator that vaporizes H₂O₂ via a thermal action. The decontamination agent 35% H₂O₂ (RAIDOX) was vaporized via a vector gas in the volume to be biodecontaminated according to predefined parameters incorporating time, pressure, humidity level, temperature, volume and the rate and speed of injection, which allowed a 6-log spore destruction to be achieved. The duration of the biodecontamination phase for this test was 60 min.

A continuous recording of the following parameters was performed via HMI (Siemens, France): humidity level, quantity of sterilizing agent, evaporation temperature, diffusion temperature, diffusion time, contact and aeration time, diffusion pressure, flow of diffused air, high and low H₂O₂ concentration.

The isolator was ventilated for a duration of 10 minutes via a catalytic system until the target concentration of residual H₂O₂ to be tested, that is 5%, was reached. Following these operations, a sterility test was performed.

c. Performance of the sterility test

As soon as the target concentration of residual H₂O₂ was reached in the isolator (5ppm), sterility tests by direct inoculation were performed following the methodology in paragraph 2.6.1. of the European Pharmacopoeia (2020): for each microorganism tested, a jar of culture medium appropriate for the microorganism tested was opened for 6 seconds in order to place a sterile wipe inside (= Tests), another jar was opened then closed again without placing a sterile wipe inside (=Validation controls). It should be reported  that this contact time with the air is identical to what may be recorded during sterility tests by direct inoculation performed in laboratories.

For each type of culture medium used, negative controls were run in the same way as Tests and Validation controls (= Test negative controls and Validation negative controls respectively). All jars of culture media were then closed again pending complete ventilation of the isolator.

During the return to a residual H₂O₂ concentration of 0 ppm in the isolator (around 15 minutes), calibrated inocula of the microbial strains to be tested were placed in the isolator transfer airlock, packaged in a sterile plastic bag. A decontamination cycle of the H₂O₂ transfer airlock was then performed before passing the calibrated inocula into the isolator chamber. This decontamination cycle lasted 75 minutes.

Ten to 100 microorganisms of each microbial strain to be tested (see Table 1) were then inoculated, each into one of the jars of appropriate culture medium containing a wipe (“Test”) and into an appropriate culture medium receptacle without a wipe (“Validation control”). The “Test negative controls” and “Validation negative controls” were not contaminated by calibrated inocula.

d. Tests performed

Simultaneously, for each microbial strain tested, a test of the number of microorganisms inoculated was performed by counting on an agar plate appropriate for each microorganism (see Table 1).

This was performed by depositing the  volume used in μl of calibrated inocula of each microorganism on the appropriate agar culture medium (2 dishes per strain).

For each decontamination cycle performed, Geobacillus stearothermophilus ATCC 7953 biological indicators were placed in the isolator to check the efficacy of the disinfection process, as well as an unexposed biological indicator (positive control). These biological indicators were cultured in tubes of liquid casein hydrolysate and soy medium (Spordex culture media, STERIS).

e. Incubation

After performance of the sterility tests by direct inoculation, the jars of liquid media and agar culture media were incubated in ovens at controlled temperatures, in accordance with the culture conditions set out in  Table 1. For the bacterial strain Clostridium sporogenes ATCC 11437, the agar culture media were placed in hermetically sealed plastic sachets, containing anaerobic generators (GENbag anaer kit, Biomérieux) and anaerobic indicators (ThermoScientific).

For each decontamination cycle performed, the Geobacillus stearothermophilus ATCC 7953 biological indicators placed in the isolator to monitor the efficacy of the disinfection process, and an unexposed biological indicator (positive control), were cultured in tubes of liquid casein hydrolysate and soy medium (Spordex culture media, STERIS) and incubated at 58°C ± 2°C for 7 days.

4. Results 

4.1. Reference test. Validation of a sterility test method in an isolator decontaminated with H₂O₂.

Residual value after decontamination: 0 ppm.

After 5 days of incubation, (Table 2), each bacterial strain studied gave rise to microbial growth. Thus a sterilization cycle in a decontaminated isolator with a residual H₂O₂ concentration of 0 ppm does not generate false negatives.