The Hitchhiker’s Guide to Hydrogen Peroxide Fumigation,Part 2: Verifying and Validating Hydrogen Peroxide Fumigation Cycles

Abstract

Introduction:

Part 1 of this two-part series describes the use of hydrogen peroxide as a fumigant and compares it with other fumigants on the market. Technical requirements are outlined while considering physical and biological limitations of the system. This second part focuses primarily on the use of process controls to verify and validate hydrogen peroxide fumigations. Finally, a model encompassing the entire fumigation process is presented.

Methods:

Part 2 of the series focuses on the authors’ long-time personal experiences in room and filter fumigation using various fumigation systems and is supplemented with relevant literature searches.

Results:

The reader is introduced to the planning and implementation of fumigation process validations. Biological indicators help users develop safe and efficient processes. Chemical indicators can be used as process controls, while measuring physical parameters will help avoid condensation of hydrogen peroxide. How many biological and chemical indicators and what type should be applied for cycle development are additionally explained.

Discussion:

It is important to consider numerous technical requirements when planning to implement hydrogen peroxide fumigation at an institution. Also, considerable thought needs to go into the verification and validation of the fumigation process.

Conclusions:

Part 1 of this series presents an overview of different fumigation systems based on hydrogen peroxide on the market and their technical requirements. Part 2 focuses on validation and verification of hydrogen peroxide fumigation while considering the entire fumigation process. The two parts together will serve users as a guide to establishing hydrogen peroxide fumigations at their facilities.

Introduction

This is the second of a two-part series looking at hydrogen peroxide fumigation from various angles. In the first part, ¹ we look at hydrogen peroxide as a fumigant, compare it with other fumigants, and present various systems that deploy hydrogen peroxide for fumigation. Furthermore, we detail some of the technical requirements to be considered when fumigating with hydrogen peroxide. Physical characteristics of the fumigant as well as the fumigation zone are considered while looking at its biological activity.

To complete our overview and to provide the reader with a complete guide to hydrogen peroxide fumigation, the second part further defines the requirements for process controls and their use in verification and validation. Ultimately, we outline the fumigation process in its entirety to remind users of its complexity and to remind them of process steps going beyond the simple fumigation of an area.

Together, the two parts serve as a guide to hydrogen peroxide fumigation and help users choose the right system for their institutions and implement a process suited for their needs.

Verification and Validation of Hydrogen Peroxide Fumigations

Definitions

Fumigations serve to decontaminate laboratory suites, animal or production rooms, patient or surgery rooms, and other areas. ^{2 -5} Fumigations are therefore critical processes. They ideally guarantee worker and environmental safety. Workers or patients entering a fumigated area will no longer be exposed to infectious agents previously present. At the same time, infectious agents can no longer escape to the environment once they have been inactivated during a fumigation process or contaminate a clean process once eliminated from a production area. It would therefore seem evident that such a critical process is monitored and results checked prior to giving the all clear. The newly published international standard ISO 35001 ⁶ on biorisk management for laboratories and other related organizations defines two important terms in this regard: verification and validation. Verification is defined as “demonstration that a validated method functions in the user’s hands according to the method’s specifications determined in the validation study and is fit for purpose,” while validation is the “establishment of the performance characteristics of a method and provision of objective evidence that the performance requirements for a specified intended use are fulfilled.” These definitions appear rather complicated at first and may confuse end users. For the sake of this publication, we would like to offer the following alternative definitions for the two terms: (1) verification is the periodic confirmation of previous validation results, and (2) validation is the confirmation of a predefined process result for a specific intended use or application in 3 or more consecutive runs. Here we would like to propose, as others have done before, ^7,8 that fumigation processes should be validated and periodically verified.

Validation as Part of the Commissioning Process

The validation process is essential as part of the commissioning of a newly built or refurbished facility. Figure 1 depicts the V diagram. It illustrates the design, construction, and commissioning process for a facility. The process is well established and used extensively in the pharmaceutical and information technology industries but is still relatively new to the science or health care industry. What is evident in the diagram is the fact that users are one responsible for the specification of the user requirements but on the other hand also for validation. The user requirement specifications form the basis for the design and ultimately the validation tests. Often, facilities are handed over to users and/or operators prior to completing the validations. Sometimes, design teams perform a process qualification in addition to the installation and operational qualification. However, this is not to be confused with a (biological) validation, which generally goes much further. For example, process qualification for an autoclave may at best include testing a run once with a standard load. During the validation, autoclave runs will ideally be performed under worst-case conditions and repeated at least 3 times as defined earlier.

Figure 1.

V diagram depicting the design, installation, and commission process of a facility.

What happens, though, when a validation cannot be performed successfully or runs fail repeatedly? This may be remedied by adapting the process. Often, though, it may require changes to the facility, which are generally very costly. Who will pay for these changes? Will that be covered by the construction budget or will new funds have to be opened? Regardless, should changes be required, it will take a lot of time and be costly and frustrating. An example of such an occurrence was described by Kümin et al. ⁹ It is thus in users’ interest to include the validation process during commissioning of their facilities.

Also, users need to clearly specify their desired validation outcome for each process. Some of the following questions will need to be answered prior to outlining the validation process: (1) What are the required turnaround times following room fumigation? How long can a facility be out of service because of a fumigation taking place before it must be handed back over to its actual use? (2) How will validation results be obtained? Is biological inactivation the desired outcome? Will chemical indicators be sufficient, or will physical parameters suffice? (3) When looking for biological inactivation, at what level will the cutoff line be drawn? Is a 6-log reduction required, or will a lower log reduction suffice? (4) How many indicators will be needed per fumigation cycle? These are just some of the questions, and others may need to be addressed additionally. It is clearly desirable to develop a validation concept that includes the goals, time plan, process description, and a definition of the desired results and how they will be confirmed. Below, we will look at some of the process parameters we have been interested in and how we have used them in validations and verifications.

Biological Indicators

Biological indicators are the gold standard for confirmation of a successful fumigation, as they actually show whether inactivation was achieved. As with indicators for autoclave validation and verification, bacterial spores are the agents of choice. ¹⁰ There are several providers on the market. However, most use either spores of Geobacillus stearothermophilus (ATCC 12980 or 7953) or Bacillus atrophaeus (ATCC 9372). Spores are present in various forms but are sold most often plated and dried onto stainless steel discs, which in turn are packaged in Tyvek. Some manufacturers provide the spores on filter paper, which may not be ideal, as the filter paper possibly adsorbs the fumigant and releases it over time, thus giving false-negative results (ie, falsely negative for growth). ^11,12 Additionally, Macellaro et al ¹³ showed that regardless of whether formaldehyde or vaporized hydrogen peroxide is used for fumigation, the choice of the correct indicator to show inactivation of the desired agents is of outmost importance.

Use of bacterial spores as biological indicators is based on the assumption that they are more resistant to inactivation than most other agents and thus represent a worst-case scenario. ¹⁴ However, this may not always be the case. It has been shown that foot-and-mouth disease virus, a nonenveloped virus of the family Picornaviridae, genus Aphtovirus, is more resistant to vaporized hydrogen peroxide than bacterial spore indicators. ¹² We thus recommend validating all fumigations with the agents likely present within the fumigation zone to compare the results with those for bacterial spore indicators. This may offer certain challenges. First, one may not want to contaminate a fumigation zone with pathogenic agents to validate the process. If an agent has not been validated before and data are not available either in the literature or from colleagues, it may well not be possible to go ahead with this. Certainly, in new facilities that have never been fumigated, this poses an unsurmountable obstacle. Authorities are unlikely to allow spreading pathogenic agents within a fumigation zone, if an alternative, proven decontamination process is not available, should a fumigation fail. Second, certain agents are difficult to plate and dry onto stainless steel discs, as is done for bacterial spore indicators initial titers are too low because thawing and drying are too high, or the agents are difficult to culture following fumigation. There are certain protocols available that outline procedures for viruses. ^12,15,16 Others may well have to be developed specifically for a particular validation protocol.

Regardless of the aforementioned recommendation, we also support the use of biological indicator spores. As stated previously, a comparison between the agents to be fumigated and the spore indicators gives an idea of the efficiency of inactivation. In addition, should results be comparable and in the same range, one can use biological spore indicators for future runs as controls, facilitating the process.

As has been stated previously, not all spore preparations are equal. There are differences among manufacturers and even between spore preparations. ^12,13 Certain products were developed specifically for a certain process. This raises the question, though, of whether these “optimized” spore preparations reflect actual results seen with other agents and whether they provide a true reflection of the fumigant’s activity and efficacy. Once again, indicators, whether from commercial providers or prepared by oneself, require evaluation to determine their value for their intended use.

Generally, manufacturers of biological spore indicators recommend incubation for up to 5 to 7 days following exposure to the fumigant. We have found that in most cases, growth will occur immediately and can be observed following an overnight incubation. In very rare cases, growth could be observed after 3 days of incubation (data not shown). We attributed this to a single spore that is severely crippled but still surviving and slowly regenerating to replicate and thus giving a positive growth result.

Chemical Indicators

In addition to biological indicators, we also advocate the use of chemical indicators. The market for these is much smaller and does not provide the same choice as for biological indicators. Chemical indicators change color once they have been in contact with the fumigant for a certain amount of time. We have found that the results for chemical indicators do not necessarily correspond with those for the biological ones (data not shown). Often chemical indicators will show a color change and thus exposure to the fumigant, whereas biological indicators may not confirm the result and instead show growth. Despite the lack of a clear correlation, we have always included chemical indicators in our verifications and validations. The chemical indicators can be read out immediately, and the color change may even be followed during the fumigation cycle when placed conveniently at windows for example. Furthermore, they will give an indication of the fumigant distribution within the fumigation zone and will therefore provide a measure for the success of the fumigation. In case not all chemical indicators show a color change, there is a good chance that a problem occurred during the fumigation. Generally, this is confirmed later by the biological indicators.

Positioning Indicators

The placement of any indicators, be they biological or chemical, will influence the results of a fumigation. Generally speaking, the more convenient the placement of the indicators, the more likely the results will correspond with the desired outcome of the process. However, very likely, this will not depict the true efficacy of the fumigation process. Contrary to others (eg, Coppens et al ⁷ ), we have always observed the following rules for determining the ideal placement of indicators for the validation of a fumigation:

All corners of a fumigation zone at ceiling and floor level. Spreading the indicators over all corners of a fumigation zone will offer the best indication on the distribution efficacy for the fumigant.

On, under, behind, and inside large pieces of equipment, such as biosafety cabinets (BSCs), incubators, freezers, and so on, and furniture. Equipment and furniture may hinder the distribution of fumigant and thus negatively influence the outcome of the fumigation. To control whether the fumigant also reaches areas around these obstacles, it is important to check with indicators.

Any other hard-to-reach areas within the fumigation zone. These could be areas that may have to be covered because of other risk factors. For instance, the bioseals of a pass-through autoclave had to be covered with fire-resistant plates because of the estimated fire hazard within the laboratory. Thus, indicators were placed behind the cover to confirm that sufficient amounts of fumigant reached the area and led to the inactivation of the biological spore indicators. ⁹

If equipment, such as BSCs or individually ventilated or containment rodent cages, is fumigated together with the fumigation zone, ⁹ indicators are placed within the equipment at locations deemed to be relevant to the process and hard to reach.

Positions that can be observed from outside the fumigation zone, such as windows or in front of surveillance cameras, to follow the process in real time.

Depending on the room layout and the room load, a large number of indicators may have to be placed on the basis of these guidelines. It is difficult to say what the required number of indicators is to produce a valid validation result. Generally, it may be argued that the more indicators are placed within the fumigation zone, the better the result of the fumigation process can be interpreted. Also, a single failure among, for example, a total of 10 indicators may be much more severe than 1 among 100 indicators. Examples of what has been used for the validation of room and HEPA filter fumigations are presented by Kümin et al. ^9,12,17 Obviously, there is a cost factor involved. Indicators are not cheap, and it may well be possible that a large number will have to be used during cycle development and validation. Thus, cost must be factored into the design of the validation process. However, we would like to express our concerns and point out that financial burdens should never be the cause for improperly conducted validations.

Measuring Physical Parameters

Besides the aforementioned biological and chemical indicators, we also promote the observation of physical parameters during and following a fumigation process. These include the following: (1) data logger(s) to measure the hydrogen peroxide concentration during the fumigation cycle, (2) data loggers for relative humidity (rH) and temperature, and (3) measurement of the hydrogen peroxide end concentration following aeration of the fumigation zone.

Measuring the concentration of hydrogen peroxide is not a very sensitive or accurate science. Some electrochemical sensors have an accuracy of ±20%. Thus, results will seldom reflect the true concentration in the fumigation zone. In addition, sensors generally cover two different concentration ranges, a low range of approximately 0 to 20 ppm and a high range of up to 2000 pm, with each either not working at all or not being very accurate in the other range. Despite these restrictions, we have often used hydrogen peroxide sensors to log the concentration during fumigation. We were interested in comparing one run for a certain fumigation zone with another in the same zone. Absolute values may have slightly differed between runs, but the curves should look similar and exclude any phenomena, such as, for example, additional peaks due to condensed hydrogen peroxide evaporating again, ⁹ never before observed. Thus, concentration curves may, despite their lower accuracy, still give a rapid indication of whether a run may have been successful or whether a problem occurred.

Similar to the hydrogen peroxide concentration curves, rH values during a fumigation run reflect the fumigation cycle. ¹⁸ For example, when using vaporized hydrogen peroxide, rH will initially decrease before slowly increasing again as hydrogen peroxide is injected into the fumigation zone, reaching a plateau and decreasing again as the fumigation zone is purged. ^9,17 Similar observations can be seen with aerosolized hydrogen peroxide. As for the concentration data, we are interested in comparing individual runs for a specific fumigation zone among one another and want to see matching curves. It is also interesting to use more than one sensor to follow the rH at different spots in the fumigation zone. ¹⁷ Interestingly, even with a so-called dry system such as vaporized hydrogen peroxide, we often observed values of greater than 90% rH.

Temperature is another factor to be considered. Temperature is directly related to room pressure, which is especially meaningful in airtight environments such as will be found in fumigation zones. Gay-Lussac’s law states that for a given mass and constant volume of an ideal gas, the pressure exerted on the sides of its container is directly proportional to its absolute temperature. Assuming ideal gas conditions, a temperature rise of 1 K will result in a pressure increase of approximately 354 Pa. Why is this relevant? If one is to use vaporized hydrogen peroxide, there will be a temperature input into the fumigation zone due to the vapor created by the generator. As was stated previously, hydrogen peroxide vapor is ejected from the generator at a temperature of up to 80°C. Especially in smaller fumigation zones, this will have an impact. In addition, if one is to leave equipment, such as freezers, running during the fumigation, room temperature will increase and with it room pressure. Ideally, fumigations are started at negative pressure as first supply dampers are shut followed by the exhaust dampers of the fumigation zone. At best, one will reach the same negative pressure as is created by and thus present in the exhaust ventilation system. However, long fumigation cycles may lead to an increase in room temperature and may reach positive values in extreme cases. To get an idea of the temperature curve and thus the pressure during a fumigation cycle, we propose the use of temperature loggers. Should a cycle lead to positive pressure in the course of a fumigation, additional measures to contain the fumigant or making it leak inward ¹⁹ may have to be taken as a fumigant leak to adjacent rooms shall always be avoided.

Finally, because hydrogen peroxide poses a threat to workers’ health, it is advised to measure the end-concentration of hydrogen peroxide following aeration and prior to giving the all-clear for entering the fumigation zone. Maximum workplace concentrations for hydrogen peroxide are generally in the range of 1 ppm. Ideally, aeration should occur until said level is reached.

Validating and Verifying Fumigation of HEPA Filters

Fumigation of HEPA filters poses an additional challenge in confirming successful completion of the fumigation cycle. Once a laboratory is operational, HEPA filter housings may not be opened before they have been properly decontaminated without risk for contaminating the area where the boxes are situated, as unfiltered air may exit containment even were the fan still running (eg, turbulences). A bag-in/bag-out system can be used. However, if this is not feasible, fumigation is the only option. How then can one confirm whether a fumigation run was successful or not? Generally, HEPA filter housings are not equipped with sample ports where biological or chemical indicators may be placed during fumigation. Even if sample ports were installed on HEPA filter housings, where would they be located? One could easily assume that locating the sample ports after the filter should be safe. However, what if the filter or its seal is damaged? Kümin et al ¹⁷ proposed what was called the IndicatorSafe as an option for the safe confirmation of a successful fumigation cycle. The IndicatorSafe is made from polycarbonate. Its size offers enough space to place biological and chemical indicators as well as a data logger for rH and temperature. It is placed after the HEPA filter housing in the return line for the fumigant. It is assumed that a positive result observed in the IndicatorSafe (ie, no growth of the biological indicator and color change for the chemical indicator) confirms that the fumigant has passed the HEPA filter and thus inactivated any contamination present in the filters. Although this is not direct proof of a successful fumigation, it still gives an indication of whether sufficient amounts of fumigant have been used. Additionally, the fumigation cycle can be followed in real time as the IndicatorSafe is transparent, thus allowing the observation of any color changes with the chemical indicator and the read-out of the physical parameters.

Need for Validation and Verification

We recommend that validations be performed for all containment-relevant processes, including fumigations. They should be required during commissioning of a facility and following relevant changes to a facility (eg, refurbishment), to equipment and/or the process used. Validation results need to be documented. This ideally includes a plan for future verifications, such as periodical verification of validation results or controls for individual cycles.

On the basis of a risk assessment, considering the work performed at a facility, the agents used, the results of the validation, and so on, validations need to be verified periodically. Generally, this is done annually, but we have also defined processes in which validation results are verified only every 5 years. Whether such a verification is done exactly as outlined for the validation of the process should be risk-based as well. One could argue either way: (1) confirm the results of the validation exactly as previously done in a single run or (2) concentrate on areas observed to pose a higher risk for failure on the basis of the validation results. Regardless of which approach is used, the reasons for it need to be documented and outlined.

Individual cycles should be verified as well. This is not the same as a verification of a validation result, but it should give the user the certainty that a process has worked as defined and that a fumigation zone can be safely returned to its intended use. It is unlikely that one would use the exact same setup and numbers of indicators as for the validation itself. Rather, one defines process-relevant areas that require controls for individual cycles. Once again, we recommend a risk-based approach, considering the results and experiences of the validation.

The Hydrogen Peroxide Fumigation Process

Consideration and careful implementation of all the aforementioned statements are crucial to the successful establishment of a fumigation protocol. The complexity of all the required steps and sequences integral to setting up a fumigation and the subsequent release and clearance of a fumigation zone (on the basis of successful process controls) clearly illustrates that a fumigation must be carefully planned and prepared. It is therefore important to realize that the fumigation itself, meaning the production and distribution of the fumigant in the fumigation zone, is just one of several other important steps in a fumigation. We propose the following sequence of tasks involved in performing a successful fumigation (Figure 2).

Figure 2.

Fumigation cycle summarizing the different steps of the entire process.

The fumigation cycle depicted in Figure 2 is based on the fumigation of a laboratory suite but may be adapted to fumigation of animal rooms, production facilities, hospital wards, or equipment, such as BSCs and HEPA filter housings. It may be noted that this sequence can be adapted for use with other fumigation systems.

Maintenance of Fumigation Equipment

Fumigation equipment needs to be ready to use at all times. Although some fumigations can be planned ahead (eg, annual maintenance shut-down), others cannot (eg, an accident involving the release of infectious agents). It is for these situations that we need to be ready. Fumigation generators should be maintained and calibrated, if required by the manufacturer. Additional equipment, such as hoses, valves, fans, and other air handling devices (eg, gas-tight dampers), needs to be intact and working properly. Data loggers used to document physical parameters need to be calibrated. Sterilant solutions and biological indicators with the corresponding growth medium as well as chemical indicators need to be available in sufficient numbers.

It is recommended to include the equipment used for fumigation in a facility’s (annual) maintenance plan.

Event Possibly Requiring Fumigation

Fumigations are not performed for the sake of it; an event occurs that will initiate the fumigation process. Some examples are (1) a biological incident with possible release of aerosols containing (highly) infectious agents, (2) the end of an animal experiment in which animals could not be housed in individually ventilated cages, (3) (periodic) shut-down for maintenance, (4) repurposing a facility, and (5) decommissioning a facility and/or its equipment.

Risk Assessment

The decision whether a fumigation is needed in the first place, and what needs to be fumigated, should always be risk based. Reasons for a fumigation are outlined above. Any fumigation that can be planned ahead should be documented in the biorisk management plan of a facility, including the relevant risk assessment. Unplanned fumigations (eg, biological spills) require a risk assessment of the specific situation at hand. Ideally, this is documented as part of the incident report.

Definition of Fumigation Zone and Fumigation Order

The fumigation zone should be defined as part of the risk assessment. However, additional thought may need to be given to the fumigation order. As outlined above, rooms need to be prepared for fumigation. This involves bringing in material such as air-handling equipment (eg, fans) and process controls. This may become complicated when we consider that rooms are still operational. As the room and therefore its atmosphere must be seen as potentially contaminated, this conceptually influences the fumigation process, as additional fumigation zones may have to be defined. When personnel open a door between 2 connecting rooms (of which at least 1 is operational), both rooms then are to be seen as potentially contaminated. This may be clearer when considering one of the operational rooms to be an animal room ²⁰ or a laboratory following a spill of biologically active material outside a BSC generating aerosols. Because an air exchange between the rooms cannot be excluded anymore, consequently both rooms, after being connected by an open door, become potentially contaminated and require decontamination. The risk for potential contamination of rooms by air exchange needs to be assessed individually for each institution on the basis of the facility design, experimental requirements, and the governing legislation.

To further explain the process, we will discuss the example of a maximum-containment laboratory with its adjacent rooms (Figure 3). So-called suit laboratories ²¹ have a chemical shower through which the laboratory is accessed and exited by personnel under normal operation. When exiting, a chemical shower process is automatically initiated and decontaminates the protective suits of the personnel together with the room surfaces independently of the activities that were performed in the laboratory. Thus, the chemical shower process serves as a containment barrier and prevents the potential contamination, as discussed earlier, of the suit room. Therefore, the laboratory space behind the chemical shower can be easily prepared and set up as a single-room fumigation, because the laboratory was connected only to the chemical shower room with its own decontamination system. However, this situation is different when fumigating a room other than the chemical shower but adjacent to the laboratory main room, such as a laboratory storage room. Such a room can be fumigated only in sequence or together with the laboratory main room. To simplify this thought, Figure 3 shows an overview of an example maximum-containment suite containing a suit room (A), a chemical shower (B), a laboratory (C), a storage room (D), and an airlock (E). If, for example, the storage room (D) needs to be fumigated, a subsequent fumigation of the laboratory (C) needs to be performed in addition, as indicated with sequential numbers in brackets. Consequently, both rooms need to be prepared for fumigation at the same time. Fumigations are then performed sequentially, and process controls for both rooms are retrieved following the fumigations. Alternatively, both rooms are temporarily connected by leaving the door between them open during the fumigation process, and the rooms in this case are prepared and set up as one large fumigation zone. Depending on the validated requirements for air handling during the fumigation process, sometimes large, heavy distribution equipment needs to be brought into the rooms when preparing them for fumigation. In maximum-containment laboratories, this would mean that all the equipment needed for the complete sequential room fumigations would need to be brought into the laboratory over the chemical shower. Depending on the design and available space of the chemical shower room, this may not be possible. In such a case, the equipment can be brought into the laboratory via the airlock as described above. The airlock (E) can be loaded from the outside and later be unloaded from the inside into the laboratory (C) when preparing the rooms for fumigation. Obviously, in this case 3 sequential fumigations would be required, similarly to what was described above.

Figure 3.

Example maximum-containment suite layout. A potential room fumigation sequence is denoted with numbers (1, 2 and 3) and for the fumigation of the storage room (D) with fumigation equipment and material to be brought in via the airlock (E).

Preparation of Fumigation Zone (Part 1)

Once the decision to fumigate has been taken, the fumigation zone needs to be prepared as outlined in the validation protocols (eg, maximum load). All laboratory work needs to be stopped and biological material safely stored and locked away as required in defined storage equipment (ie, freezers or refrigerators). The overall room load is to be reduced and distributed according to the validated maximum room load. Laboratory waste is autoclaved and brought out. Hydrogen peroxide–adsorbing materials (eg, paper and cardboard) is reduced or better yet eliminated. Equipment and devices such as centrifuge rotors, pipettes, racks, metal buckets, and so on, that can be autoclaved should be treated accordingly and exited from the fumigation zone to further reduce the room load. The necessity of whether removal of said equipment is required should be addressed during the validation of the fumigation process.

Cleaning

Because the fumigant will reach only accessible surfaces, all surfaces in the laboratory need to be clean and free of dust. This step is fundamental for a successful surface decontamination by fumigation. Special attention is needed to get all the small laboratory equipment clean and accessible for fumigation (ie, pipettes, vortexes, centrifuges, microscopes, computers, etc). Additionally, it is crucial that all surfaces that cannot be reached by the fumigant (eg, spots where equipment stands on a table, contact points with the floor of chairs, or other movable furniture or equipment) be cleaned to remove organic material and then properly decontaminated by manual surface decontamination like spraying and/or wiping. Cleaning is even more relevant in large animal rooms where bedding and manure need to be removed, rooms washed down and manually disinfected prior to fumigation.

Preparation of Fumigation Zone (Part 2)

Once the fumigation zone has been cleaned, equipment required for fumigation can be brought into the fumigation zone and set up as outlined. Also, all process controls, such as biological indicators, chemical indicators, and data loggers are placed as defined.

Fumigation Setup

The fumigation generator is installed (either in- or outside the fumigation zone), and parameters for fumigation are set. The fumigation zone is isolated, and all equipment controlled by remotely activated electrical power sockets is turned on.

Fumigation

The fumigation cycle is initiated. It is recommended to monitor the progress of the fumigation as outlined earlier.

Aeration

The fumigation zone is aerated using the building’s heating, ventilation, and air conditioning system, allowing a much faster purge of the fumigant and thus shorter turnaround time for the fumigation zone.

Process Controls

The fumigation zone is reentered to collect all process indicators for documentation and incubation. As the success of the fumigation is, at this point in time, not yet officially confirmed, one must decide how to enter the fumigation zone. If one trusts the validation of the fumigation and no obvious failures were observed during the fumigation process, one can enter the fumigation zone without any further precautions. If, however, the risk assessment determines that staff is still at risk for a possible exposure, one would have to enter the fumigation zone wearing full protective gear. In the example shown in Figure 3, the laboratory will be accessed in full protective equipment via the chemical shower (B), and process control indicators will directly be read out (chemical indicators) or put in culture media (biological indicators) inside the laboratory (C) before being taken out through the chemical shower (B) in a transport box for later incubation and read-out.

Clearance

Following confirmation of the successful fumigation, the fumigation zone may be cleaned out, all fumigation equipment removed, and the zone returned to the users for its intended purpose.

Documentation

The final step includes a written report of the fumigation process. The report summarizes the results of the process, including possible deviations from the expected result, and is stored for future reference. The document may be shown to enforcement authorities, external contractors, or maintenance personnel.

Conclusions

This two-part series was written as a guide for those involved in planning and performing fumigations. Its focus has been on hydrogen peroxide as a fumigant. Hydrogen peroxide, although it has its limitations, has obvious advantages over other fumigants. It is environmentally friendly and offers increased personnel safety, as no toxic end products are formed. Short cycle times lead to short turnaround times and, thus, to increased availability of fumigation zones to their users and intended purpose. Hydrogen peroxide shows good material compatibility. Hydrogen peroxide fumigations can be validated and have been shown to work successfully in many situations and with various agents.

It is important to consider hydrogen peroxide’s limitations as a fumigant, as these will affect the necessary technical installations of a facility. We have attempted to outline a decision process for future users of fumigation systems to determine what system best suits their purpose.

All containment-relevant processes require validation and verification. Both may be achieved by different means with various process controls. The second part gives an overview of process controls available to users and outlines strategies to confirm successful fumigations.

Ultimately, a fumigation process, regardless of what system is used, is very complex and involves further steps besides the actual fumigation. The process, as we see it, has been outlined and will help users define their processes on the basis of their facility layout and requirements.

Although hydrogen peroxide has been used as a fumigant for many years, and more and more data are available supporting the notion that it is a fumigant with a broad spectrum of activity and a wide variety of applications, it is only recently that it could be shown to be a low cost, versatile, and robust fumigant. These recent developments may well pave the way for further applications and wider use of hydrogen peroxide fumigations. With this series of articles, we hope to have supported these developments and look forward to future successes.