BSL-3 and BSL-4 Laboratory Fumigation Strategies

Containment Talk 10

Containment talk articles discuss risk and safety issues in and around microbiological and biomedical laboratories (BSL-2, 3, and 4). Feel free to contact me via LinkedIn messaging if you have any questions or comments about laboratory safety, biosafety, biocontainment, design & equipment, engineering, etc.

1 Introduction

BSL-3 and BSL-4 facilities must be designed and constructed in such a way that the containment and the contaminated parts of the ventilation system, including the exhaust HEPA-filter stations and enclosed systems (biosafety cabinets Class III, ducts, etc.) can be safely decontaminated by fumigation.

In this Containment Talk article, possible fumigation strategies and methods are discussed with their advantages and disadvantages. Various cost-effective and safe fumigation strategies for BSL-3 and BSL-4 facilities are possible based on the risk-based design as proposed by WHO [1], [2].

2 Legal Requirements and Guideline Recommendations

Regardless of the regulatory framework and applicable biosafety standards or guidelines, a BSL-3 and BSL-4 facility must be sealed or sealable for fumigation, as explained in a previous Containment Talk article [3].

In general, fumigation is required for:

• Annual maintenance and (temporary) decommissioning
• Managing spills outside primary containment systems
• Replacement of HEPA filters
• Creation of a safe working environment for training, inspections, troubleshooting, certification, etc.

3 Fumigation Chemicals and Systems

Chemicals

As formaldehyde has been phased out for safety reasons, facilities often use hydrogen peroxide (H2O2, [4]-[6]) or chlorine dioxide (ClO2, [7], [8]). This article focuses on hydrogen peroxide systems, but the same approach can be applied to chlorine dioxide systems.

Systems

In general, the lightweight, portable devices generate H2O2 aerosols, mists, fogs, or sprays. They can be used to decontaminate rooms, but not ducts, HEPA filter stations and closed systems such as Class III biosafety cabinets (BSC Class III).

Larger, but still mobile units usually have hose connections for the supply and return of hydrogen peroxide from the outside into closed systems or ducts (H2O2 loop).

For this purpose, ducts must be equipped with shut-off dampers and supply/return ports at strategic points to facilitate decontamination of the duct section concerned. BSL-3/4 compliant HEPA filter stations, typically of the BIBO type, and BSC Class III are fitted with these ports and isolation valves as standard [9].

In addition to these manual systems, there are automatic systems. Automatic systems are primarily utilised by the pharmaceutical industry, which frequently requires decontamination of its facilities. They are sophisticated and costly and may not offer optimal value for money for typical BSL-3/4 laboratories. They are fully integrated into the HVAC system and require minimal effort to operate. Manual portable and mobile units, on the other hand, are typically employed in general BSL-3/4 facilities. This article will focus exclusively on manually operated portable and mobile units.

4 Fumigation Strategy

The term “fumigation strategy” refers to the method used to fumigate the entire containment area, including individual rooms, zones, ducts, HEPA-filter stations, and enclosed devices or systems (Biosafety Cabinet Class III, isolators).

5 Physical BSL-3/4 Containment Prerequisites

Solutions for

• the tightness of the containment and partitions, including the doors,
• the design of the HVAC system, and
• the fumigation strategy

must be determined at a very early stage of concept planning. The three systems are interdependent to a certain extent. In addition, the space available in the building for the laboratory, containment and technical centre (mechanical space) is a decisive factor. This means that the physical containment concept design should include a holistic planning approach and a cost comparison between the options under consideration.

As outlined in Section 2, biosafety regulations and guidelines require containment that is sealed or sealable for fumigation. The question is what “sealed” or “sealable” means, i.e. how leaktight the containment and duct sections intended for fumigation must be.

In a previous Containment Talk article [3], we described the classic definition of what leaktightness means in the context of the design and construction of BSL-3 and BSL-4 laboratories. Thus, the containment barrier with penetrations, the HEPA filter housing and the duct sections intended for fumigation should comply with the relevant standards [10]-[14]. Does this solve the problem? Let's take a closer look at the issue.

The VDI standard 2083 sheet 19 [10] requires tightness Class 4 for BSL 3 laboratories (Class 5 for BSL-4), but depending on the area and room configuration, walls, penetrations, and special doors that fulfil this tightness class can be very expensive. However, the VDI standard allows tightness Class 2 for rooms that are under controlled negative pressure during fumigation. Maintaining a negative pressure in the containment during fumigation in combination with a surrounding safety corridor with its own once-through ventilation system (no recirculation) is at least as safe, if not safer, than the classic approach. This option is discussed in more detail in section 5.4.

Please note: Whether this design is approved by the competent authorities or a certifier must be clarified during concept development. A project-specific cost estimate must be carried out to determine which option offers the best cost-benefit ratio.?

There are four basic design options, which are explained in more detail below:

1. Portable units for in-room fumigation
2. Mobile units used in containment with supply and return lines (H2O2 loop)
3. Mobile units operated from the mechanical space (H2O2 loop)
4. Fumigation of the whole containment in re-circulation mode (H2O2 loop)

The option that best suits the needs and complies with local regulations must be customised for the specific project in each case. The user requirements in relation to the purpose of the laboratory must be considered: Research and development (single or multiple users), diagnostics, 24/7 availability, animal testing, small-scale production, etc.

5.1 Option 1: Portable Units for In-Room Fumigation

A portable unit that generates hydrogen peroxide vapour (e.g. VHP?), aerosol, mist, fog, or spray ([4]-[6]) is brought into the room(s) to be fumigated. Of course, mobile units can also be used. It should be noted again that it is not possible to fumigate HEPA filter housings, ducts, etc. with portable and mobile units without the possibility of forming a loop, even if ducts and these systems have fumigation ports (Figure 1).

For room or zone fumigation as shown in Figure 1, the shut-off dampers of the room or zone are closed during the process. Depending on the laboratory’s room configuration and the HVAC specifications, the ventilation is on or off. The holding time is timer controlled. After the holding time has elapsed, the shut-off dampers are opened to wash out the fumigant (ventilation on).

The unit’s capacity must be sufficient for the space. For process validation several trial-and-error runs may be necessary. It may be necessary to enhance the distribution of the vapour, aerosol, mist, of fog by means of electric fans.

• Advantages: Portable units offer a simple, effective solution with low CAPEX and OPEX.
• Disadvantages: These units are not suitable for decontaminating BSC Class III, ducts, or HEPA filter housings. These units require a supply and return hose to maintain a loop. They can be rented or borrowed for this purpose. Incidentally, BSC Class II can be fumigated together with the room (BSC switched on during fumigation).

5.2 Option 2: Mobile Units Used in Containment With Supply and Return Lines (H2O2 Loop)

A unit with supply and return hoses is used for fumigation of rooms, enclosed devices and systems. Because of the hose option, in addition of using it inside a room, this device allows room fumigation from outside, e.g. from a contamination-free corridor or an area that has already been fumigated (Figure 3). Wall-mounted hose couplings or fumigation ports must be provided for this purpose. When not in use, the ports are tightly capped (see Figure 3 room to the right).

It is essential to place shut-off dampers and fumigation ports/couplings at strategic locations in order to successfully fumigate the ductwork. In cases where the duct segments are long and the ambient temperature can be low, it may not be possible to validate the process due to the potential for H2O2 to condense on the cold inner surface of the duct. In such instances, it is necessary to fit the ducts with heating tapes to ensure the process is completed effectively.

For the procedure and validation see option 1.

• Advantages: Mobile supply/return systems are simple and effective, can be operated from neighbouring, contamination-free rooms or zones and can be used for ducts, enclosed systems, and the HEPA filter station.
• Disadvantages: For HEPA-filter housing and duct fumigation the unit must be transferable to the mechanical space. Duct fumigation requires ports at strategic points. This option has higher CAPEX and OPEX than option 1 (unit costs, in-wall fumigation connections/coupling, duct ports). A cost-effective solution would be to outsource the service or borrow a unit. For urgent room fumigation a portable unit may be used.

5.3 Option 3: Mobile Units Operated From the Mechanical (H2O2 loop)

Option 3 is comparable to option 2 in that it employs a mobile supply/return unit. However, the unit is always situated in the mechanical space, which is optimally located above the containment (see Figure 4).

For the procedure and validation see option 1. Depending on the fumigation unit’s capacity a single room or a row of rooms connected by open doors can be fumigated in one go. To fumigate the HEPA-filter housing, both shut-off dampers are closed and the upstream and downstream ports are connected to the unit. This requires shutting down HVAC system, except a parallel backup HEPA-filter station is available.

• Advantages: Mobile supply/return systems are simple and effective, can be operated from the mechanical space without the need to enter the containment. Ducts, closed equipment (e.g., BSC Class III) and systems including the HEPA filter housing can also be fumigated.
• Disadvantages: This option has higher CAPEX and OPEX than option 1 (unit costs, fumigation connections/coupling) but has about the same cost as option 2. A cost-effective solution would be to outsource the service or borrow a device for larger fumigation tasks. Option 1 for urgent room fumigation can be used.

5.4 Option 4: Fumigation of the Whole Containment in Re-Circulation Mode (H2O2 Loop)

This option can only be used in facilities that allow a temporary complete shutdown of all activities for fumigation. It is generally used as a manual process in the pharmaceutical industry between different production campaigns, both in the production areas and in their quality control laboratories.

The main feature is a recirculation loop that allows the fumigant to be recirculated for the required time and concentration. For this purpose, the HVAC mode (100 % fresh air) used in normal operation is switched to recirculation mode for fumigation, as shown in Figure 5.

Left: Normal HVAC operation. The fan of the fumigation loop is switched off and its shut-off dampers are closed.

Right: Fumigation mode. The HVAC system's air handling units are switched off except for the exhaust air handler (or a small exhaust fan). A bypass with a valve is used to regulate the negative pressure in the containment during fumigation. The fumigation loop is open and the circulation of the decontamination agent is maintained by the recirculation fan. See text for further explanations.

For fumigation, mobile or even portable units are placed in each room. The effectiveness of fumigation in the loop can be easily validated (and monitored) by placing chemical and biological indicators at strategic locations in all rooms and along the loop.

This fumigation strategy makes it possible to design and build the BSL-3 containment significantly less leaktight, if the competent authorities, the certifier, or both agree. It can comply with leaktightness Class 2 [10], which is intended for rooms that are under controlled negative pressure during fumigation. Class 2 BSL-3 containment is significantly cheaper than Class 4 containment. As an additional safety measure, these facilities almost always have a safety corridor around the containment boundary with viewing panels that allow monitoring and inspection from the outside. This corridor should be supplied by a dedicated once-through (100 percent fresh air) HVAC system.

• Advantages: Mobile or portable units are simple and effective. They are brought into the containment via material locks. Ducts, enclosed systems (e.g. BSC Class III) and systems including ducts and the HEPA filter station are fumigated in a single operation. This option is cost-effective and very safe. The fumigation service is often outsourced or the equipment is borrowed or rented.
• Disadvantages: This option can only be used in facilities designed for complete shutdown and simultaneous fumigation of all containment rooms. It is generally only used by pharmaceutical manufacturing companies, not the typical BSL-3 and BSL-4 laboratories. Procuring a large number of fumigation units is expensive, so this service is often outsourced. It requires agreement with the competent authorities, the certifier, or both, as the "sealed" or "sealable" requirement for fumigation must be interpreted and confirmed.

6 Conclusions

For new BSL-3 facilities the fumigation strategy requires a holistic approach considering:

1. The intended fumigation frequency and fumigation method,
2. the configuration of the HVAC system,
3. the leaktightness of the containment boundary and separation walls including doors, and
4. the agreement with the competent authorities, the certifier, or both.

7 References

[1]????? WHO (2020). Laboratory biosafety manual, fourth edition. Geneva: World Health Organization; (Laboratory biosafety manual, fourth edition and associated monographs). Internet: https://www.who.int/publications/i/item/9789240011311. Accessed August 2024.

[2]????? GAPIV (2022). WHO Global Action Plan for Poliovirus Containment (GAPIV). Internet: https://polioeradication.org/polio-today/preparing-for-a-polio-free-world/containment/containment-guidance-documents/. Accessed August 2024.

[3]????? Basler & Hofmann (2024). Understanding ‘Airtight’ and ‘Gastight’ in Relation to the Design and Construction of BSL-3 and BSL-4 Facilities. Internet: https://dx.doi.org/10.13140/RG.2.2.31256.57601. Accessed August 2024.

[4]????? Kümin, D. et al. 2019). Case Study: Room Fumigation Using Aerosolized Hydrogen Peroxide—A Versatile and Economic Fumigation Method. Internet: https://doi.org/10.1177/1535676019887049. Accessed August 2024.

[5]????? Kümin D. et al. (2020). The hitchhiker’s guide to hydrogen peroxide fumigation, part 1: introduction to hydrogen peroxide fumigation. Internet: https://doi.org/10.1177/1535676020921007. Accessed August 2024.

[6]????? Kümin D. et al. (2021). The Hitchhiker’s Guide to Hydrogen Peroxide Fumigation, Part 2: Verifying and Validating Hydrogen Peroxide Fumigation Cycles. Internet: https://doi.org/10.1089/apb.21.921099. Accessed August 2024.

[7]????? Lowe, J.J. et al. (2012). A Case Study on Decontamination of a Biosafety Level-3 Laboratory and Associated Ductwork Within an Operational Building Using Gaseous Chlorine Dioxide. Internet: https://doi.org/10.1080/15459624.2012.733592. Accessed August 2024.

[8]????? Girouard, D.J. and Czarneski, M.A. (2016). Room, Suite Scale, Class III Biological Safety Cabinet, and Sensitive Equipment Decontamination and Validation Using Gaseous Chlorine Dioxide. Internet: https://www.liebertpub.com/doi/10.1177/1535676016638750. Accessed August 2024.

[9]????? SECB (2022). Recommendation on structural and technical safety measures in laboratories. A tool for Stakeholders. Internet: https://www.efbs.admin.ch/en/the-committee/news/empfehlung-der-efbs-zu-baulich-technischen-sicherheitsmassnahmen-in-bsl-3-laboratorien. Accessed August 2024.

[10]?? VDI (2024). VDI 2083, Sheet 19. Cleanroom technology - Tightness of containments - Classification, planning and testing. VDI Society for Construction and Building Technology (GBG). Internet: https://connect.snv.ch/de/vdi-2083-blatt-19-2024. Accessed August 2024.

[11]?? ASME (2022). American Society of Mechanical Engineers (ASME): N511 - In-Service Testing of Nuclear Air-Treatment, Heating, Ventilating, and Air-Conditioning Systems. Available through ANSI Webstore, Internet: https://webstore.ansi.org/search/find?in=1&st=ASME+N511-2022. Accessed August 2024.

[12]?? SN EN ISO 14644-3:2020. Cleanrooms and associated controlled environments - Part 3: Test methods. (ISO 14644-3:2019, corrected version 2020-06). Available from Swiss Association for Standardization, SNV, 8404 Winterthur. Internet: https://connect.snv.ch/de/sn-en-iso-14644-3-2020. Accessed August 2024.

[13]?? DIN 25496:2013. Ventilation components in nuclear installations. Available from Swiss Association for Standardisation, SNV, 8404 Winterthur. Internet (D): https://connect.snv.ch/de/din-25496-2013. Accessed August 2024.

[14] KTA (2022). Ventilation systems in nuclear power plants. Internet: https://www.kta-gs.de/e/standards/3600/3601_engl_2022_11.pdf. Accessed August 2024.