These recommendations were developed by comparing the various performance tests identified in the standards described in the past two blogs, in an attempt to provide guidance on the development of a comprehensive performance field test. Consideration has also been given to those factors that may affect the ability to conduct a performance test using values in any given specification. Selection of ‘required’ tests as opposed to ‘optional’ tests should be based on a risk assessment that considers safety as well as the work being conducted. Influences to the system include temperature, humidity, altitude (as compared to factory readings), and barometric pressure. These factors can have a large effect on system readings. Routine work with animals as opposed to cell culture may justify measuring noise levels and considering more stringent control of decibels produced. A nuisance noise level to humans may be loud enough to have adverse effects on some species of animal.
Annual tests to strongly consider* include: Visual inspection*; Cabinet Integrity Test (Leak rate)* or Negative Pressure Test (rate of rise test) or Positive Pressure Test (pressure decay test); HEPA Filter Leak Test*; In-Rush Protection Test (loss of glove); Air Flow Test (smoke pattern tests); Alarm Tests (airflow and interlocks)*; and measuring* Illumination, Vibration, Electrical Continuity, and Noise level.
Visual Inspection
Turn on the fluorescent light. Visually inspect gloves, bag-out assembly, pass through chambers and all gaskets and o-rings for signs of holes, cracks, wear or damage.
Inspect the view screen for cracks or damage, and ensure the dunk tank is filled to the appropriate level with the correct decontaminating agent. Replace any worn or damaged parts and ensure all components of the Class III BSC are in proper order before conducting work.
Leak Rates – Factory Testing vs. Field Testing
The BMBL and other standards describe acceptable leak rates. In the absence of one internationally or nationally accepted standard, it is important to base field testing on technically proven or sound leak rates to promote safety and to consider the practicality of conducting the test on-site.
The original factory test specified in the Lab Safety Monograph allows for no loss greater than 1 x 10-5 cc/sec with 100% tracer gas in the factory setting. In the field it is impractical to test using 100% tracer gas, and the test is performed using 1% tracer gas with the leak rate adjusted to 1 x 10-7 cc/sec to compensate for the two log reduction in gas. The use of a helium leak detector (mass spectrometer) is typically used to measure and locate leaks. Pressure tests can be used in lieu of the use of helium gas.
Differential Pressure
Differential pressure of 0.5” water gauge may not be adequate to accommodate glove use in small cabinets. The physical act of pulling glove into and out from the cabinet can cause swings in the internal differential pressure and could trigger pressure alarms and could lead to an unsafe condition. However, high differential pressure can result in hard to maneuver gloves and may cause safety concerns due to increased resistance to arm movements. Choose an operational differential pressure that works for the design of the Class III BSC. Treat the 0.5” wg recommendation of the BMBL as a minimum.
HEPA Filter Testing
Filters can be tested for integrity using a scan or probe method. Scan testing of HEPA filters is a better method of discovering filter leaks and identifying their location than using the probe method.
The BMBL and other standards specify testing challenge by Di-Octyl Phthalate (DOP), though due to risks associated with DOP it has largely been replaced by mineral oil and Poly Alpha Olefin (PAO). PAO has similar characteristics of DOP and creates a consistent particle size. These materials generate an appropriate concentration and size dispersion aerosol challenge using a Laskin Nozzle at a defined flow rate. It is important to note that these challenge materials are not interchangeable and the photometer must be calibrated to the particular challenge material used.
Illumination, Vibration, Electrical Continuity, and Noise Level Tests
These tests can be conducted as described in NSF 49. A decision should be made as to whether these tests are optional (for data collection) or mandatory. If they are mandatory the failure of any of these tests would warrant stopping work with the unit until the appropriate maintenance is performed. It is clearly beneficial to conduct the tests at a minimum to demonstrate any performance trends that indicate maintenance is needed.
Equipment Calibration and Certifier Credentials
All instruments used to certify Class III BSCs must be calibrated and NIST (or national equivalent) traceable. The calibration should be done on an annual basis. Certificates of calibration should be available from the certifier. NSF qualifies Class II BSC certifiers by both a written and practical test and a requirement for attending Continuing Education every 5 years with retesting after 10 years. However, there is no regulating authority or set of training or experience requirements for personnel certifying Class III BSCs. NSF does specify that if an individual in an organization performs Class III BSC certifications they must be NSF accredited, but this is for Class II BSC.
There is no one requirement or standard for annual field testing of a Class III BSC, and a risk assessment and analysis of the work being performed should help to drive the certification process. The institute biosafety professional or a qualified biosafety consultant should be involved in developing the certification requirements. The institute must be provided a copy of all data and readings gathered by the certifier for a matter of record as well as for future trending data and definitive proof that a certification was actually conducted.
1. NSF 49 Class II (Laminar Flow) Biosafety Cabinetry, NSF/ANSI 49-2008 Edition: 11th
2. Guideline For Gloveboxes, Third Edition AGS-G001, February 2007
3. Biosafety in Microbiological and Biomedical Laboratories (BMBL), 5th Edition, 2007 NIH/CDC
4. Laboratory Biosafety Guidelines, 3rd Edition, 2004, Public Health Agency, Canada,
5. Laboratory Safety Monograph: A Supplement to the NIH Guidelines for Recombinant DNA Research, 1979
6. Biotechnology- Performance criteria for microbiological safety cabinets; BS EN 12469:2000/EN 12469:2000, 2000, Dandy Booksellers Ltd.

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