Decontamination methods for Bio Safety cabinets

Bio Safety Cabinets (BSC) are the correct laboratory equipment to work safely with hazardous biological material. When internal decontamination is required for several reasons, users can choose between different available methods. This article will analyse them to guide users in the selection process.

Previous concepts

• What exactly does ‘decontamination’ mean?

Decontamination is a term used to describe a process or treatment that renders a medical device, instrument, or environmental surface safe to handle. A decontamination procedure can range from sterilisation to simple cleaning with soap and water. Sanitisation, disinfection and sterilisation are all forms of decontamination.
Sanitisation: It’s the process of making something (usually an inanimate object) clean. This is typically defined as a 2 log reduction which means the number of germs is reduced by 99%.
Disinfection: The process of eliminating pathogenic organisms or making them inert, i.e., to kill the germs and bacteria or render them harmless. This is typically defined as a 2-5 log reduction. (max. 99.999% reduction)
Sterilisation: The process of completely eliminating microbial viability, i.e., to kill all non-pathogenic and pathogenic spores, fungi and viruses.
As this is an absolute statement, and cannot be measured, a probabilistic approach is accepted by different industries, defining the concept of Sterility Assurance Level (SAL) of 10-6. This value is showing that one microorganism out of 106 could be viable, typically defined as 6 log reduction of 106 population of microorganisms.
     SANITISATION                          DISINFECTION                                STERILISATION
     Two log – 10-2                             Five log – 10-5                                  Six log – 10-6
    

Sterilisation describes a process that destroys or eliminates all forms of microbial life and is carried out in healthcare facilities by physical or chemical methods. Steam under pressure, dry heat, EtO gas, hydrogen peroxide gas plasma, and liquid chemicals are the principal sterilising agents used in healthcare facilities.
When talking about BSC decontamination, the objective is to reach the maximum decontamination level.

• When is it necessary to decontaminate a BSC?

There are 5 basic reasons why it may be fundamental to carry out a decontamination procedure in BSCs:
1. Before performing any service activity which requires access to potentially biologically contaminated areas of the BSC such as absolute filters or fan replacement.
2. Prior to relocation or movement of the biological safety cabinet in another room or building to avoid any biological contamination risk during the operation.
3. In the event of an accidental spill of a hazardous microorganism in the working area.
4. When working procedures are drastically changed and a different microorganism is used inside the BSC.
5. As part of a routine decontamination protocol when BSL-3 or BSL-4 organisms are
• How often should a decontamination procedure be carried out?
In most research facilities and hospitals, decontamination will be performed once or twice during the life of a cabinet, especially in those BSC providing additional pre-filters that can extend HEPA filter life up to 7 years.
In pharmaceutical laboratories it could be the need to conduct decontamination cycles between batches and numerous experiments. This could be a weekly activity.
• How do I decontaminate a BSC?
BSC manufactures must define a recommended decontamination procedure that has to be validated to proof the 6 log reduction. The validation is done by placing chemical and biological indicators in several places in the contaminated areas, and they are incubated to check if the BI has been sterilised. Validation is considered successful when at least 3 different tests results are the same. This procedure will be included in the user manual provided with the equipment.
Decontamination must be performed using a chemical disinfectant in a gas or vapour state. The main reason for this is because it must pass through HEPA filter media and decontaminate external and internal surfaces to avoid any contamination risk.
Most manufacturers offer accessories to facilitate the decontamination process such as covers and a connection for vapour hoses. However, any cabinet can be decontaminated using plastic film/bag and sealing tape.
Decontamination specialists will assure proper sealing to avoid any leak and will consider the permitted national exposure limits to the chemical according to local authorities.

Formaldehyde
The use of formaldehyde for BSC decontamination has been the most common procedure in the market because both NSF/ANSI 49 and EN-12469 (actually under review) are describing a process using this chemical, and it’s proved to be effective reaching a 6 log reduction when using Bacillus Atrophaeus biological Indicators (BI). However, this chemical has been proven to be carcinogen and was forbidden in Europe in 2016. It is still accepted in other regions. Its toxicity makes critical the sealing of the cabinet during the decontamination, and it may be necessary to cover the cabinet with a full plastic bag sealed towards the floor with sealing tape.
Decontamination with formaldehyde gas is performed either by vaporising formalin solution, (37% water-based solution called formalin) or by depolymerisation of solid paraformaldehyde. It is necessary to circulate the gas in the BSC for up to 12 hours and afterwards neutralisation is usually achieved using ammonia gas. This process will leave residue on working area surfaces that has to be manually cleaned (which is difficult to accomplish in the plenums, fans, etc.), extending the total decontamination process up to 24 hours before its ready to be used once again.

Pros    Cons
Cheap    Carcinogen chemical (forbidden in EU)
Efficiency proven since 1939    Very long process
    Residues to be manually cleaned

Hydrogen peroxide
The use of Vaporised hydrogen peroxide (Vhp) has been extended in Europe and other regions, and several companies devoted to sterilisation are using this technology, capable to decontaminate BSC and entire lab rooms in an easier and healthier way. It requires special equipment which is much more expensive than formaldehyde vaporisers.
Hydrogen peroxide works by producing destructive hydroxyl free radicals that can attack membrane lipids, DNA, and other essential cell components. Catalase, produced by aerobic organisms and facultative anaerobes that possess cytochrome systems, can protect cells from metabolically produced hydrogen peroxide by degrading hydrogen peroxide to water and oxygen. This defence is overwhelmed by the concentrations used for decontamination.
This method is mentioned as an alternative to the formaldehyde decontamination process, and it must be validated for the model and size, according to Annex G of NSF/NSI 49. Hydrogen peroxide is active against a wide range of microorganisms, including bacteria, yeasts, fungi, viruses, and spores. It has proved to be effective reaching a 6 log reduction when using Geobacillus Stearothermophilus biological Indicators (BI). The high oxidation potential of the Vhp can superficially affect some materials (copper or POM) in high concentrations with long period of exposure, but it has proven not to affect most of the materials used in a BSC when using  a low concentrated solution.
By having a higher exposure limit makes the sealing of the BSC enclosure a less critical aspect and the whole process is made easier, but an external sensor is recommended to detect the concentration (ppm) in the ambient.

Pros    Cons
Fast    Requires validation by BSC manufacturer
Efficiency proven    Material compatibility under continued use
Higher exposure limit (1ppm)    
No residues (H2O+O2)    

Chlorine dioxide
This substance is the most used in the USA and other NSF influenced countries due to the inclusion of a validated procedure for decontamination provided in NSF/ANSI 49, Annex G.
Chlorine dioxide is an effective sterilising gas, so can easily pass through filters. This gas is non-carcinogenic, non-flammable, and effective at ambient temperatures, but is highly toxic and corrosive with some materials and it is not recommended when required decontamination frequency is high. The accepted exposition limits (TWA PEL) are just 0.1 ppm, much lower than when comparing with Vhp limits. It is necessary to evacuate personnel from the room and the BSC frame sealing becomes a critical process.
A typical chlorine dioxide sterilisation process uses a sequence of preconditioning, conditioning dwell period, charge, and exposure, followed by aeration. Aeration can be done directly when the cabinet is ducted to an external exhaust system, otherwise the chlorine dioxide must be removed by an activated carbon packed scrubber or a sodium thiosulfate counter-current scrubber. Like Vhp, it does not leave any residue after the process.

Pros:    Cons:
Fast    Low exposure limit (0.1 ppm)
Efficiency proven    Material compatibility under continued use
Real gas    Room should be evacuated
No residues   

Conclusions
All the methods listed above can be effective reaching 6-log reduction, but BSC manufacturer recommendations must always be the main option because it has been properly validated as required by certification bodies like TÜV Nord. Of course, considering the strong and weak points of each process, some users may prefer to use another method, in which case it is their responsibility to perform a validation test to check the effectiveness If any doubts arise they must contact the BSC manufacturer for further clarification and assurance.

Sources
NSF/ANSI 49: 2002 - Class II (Laminar Flow) Biosafety Cabinetry.
EN-12469: 2000 - Performance criteria for microbiological safety cabinets.
DIN-12980:2017 - Laboratory installations - Safety cabinets and glove boxes for cytotoxic substances and other CMR drugs.
Hydrogen peroxide: Is material compatibility a real challenge for this decontamination technology? – I.Cantera/M.Capella – October 2019
Material compatibility with vaporized hydrogen peroxide (vhp®) sterilization -
Steris www.sterislifesciences.com
Handbook for cleaning/decontamination of surfaces - Ingegard Johansson, P. Somasundaran, Elsevier, 20 jun. 2007

About the author
Víctor Lázaro, Research and Medical R&D Manager at Telstar, holds a MEng in Industrial Engineering from the Universitat Politècnica de Catalunya (UPC) and an Executive MBA from EADA.
In 2011, Víctor joined Azbil Telstar Technologies SLU to lead the development of R&D projects for Clean Air, Cold storage and Lyolabs business. Together with an international multidisciplinary team, has renewed and increased the product portfolio and driven a wide variety of projects related to the Life Science industry.