A new method for measuring porous microbial barriers: Part II A closer look at ASTM international standard test method F2638 

By Paul F. Herman & Curtis L. Larsen, CPP, Fellow

Background

Balloting on a new test method for use in the ranking of porous packaging materials used in
sterile packaging applications by their ability to provide bacterial holdout was completed in July
2007 by ASTM International Committee F02 Flexible Barrier Packaging. As a result of this
balloting, there is a new test method available for evaluating the barrier capabilities of porous
materials intended for use in the packaging of terminally sterilized products. This new test
method, ASTM F2638-07 Standard Test Method for Using Aerosol Filtration for Measuring the
Performance of Porous Packaging Materials as a Surrogate Microbial Barrier, utilizes an
aerosol of 1.0µm diameter polystyrene spheres to measure the filtration efficiency of porous
materials. Unlike its predecessor, ASTM F1608-00(2004), Standard Test Method for Microbial
Ranking of Porous Packaging Materials (Exposure Chamber Method), which uses actual spores
of a nominal 1.0 µm size for testing and can take several days to produce results, the apparatus
defined in the new ASTM F2638-07 test method can provide almost instantaneous test results.

Method Summary

A porous packaging test specimen is placed into a sample holder to create a filter between the
challenge and filtrate aerosol streams. On the challenge side of the sample chamber an aerosol of
particles is presented to the surface of the test specimen. Air flow is generated through the test
specimen and laser particle counters are used to enumerate the particles in both the challenge and
filtrate aerosol streams. Enumeration of particles in the challenge and filtrate streams can be
performed sequentially using only one particle counter or concurrently using two particle
counters. A percent penetration value can be calculated from the challenge and filtrate particle
count data. Monitoring and plotting percent penetration vs. flow rate through the sample
generates a typical filtration efficiency curve. The maximum percent penetration value and the
flow rate at which it occurred is determined from the peak value of the curve.

Significance and Use

This new test method has been developed as a result of the research conducted by Air Dispersions
Ltd. (ADL) of Manchester, UK, and funded by the Barrier Test Consortium Ltd. (BTC).

Members of the Barrier Test Consortium Ltd. (BTC)
• Amcor Flexibles (formerly Rexam Medical Packaging)
• Billerud (formerly Henry Cooke)
• DuPont Medical Packaging
• Kimberly-Clark
• Oliver Products
• Perfecseal, a Division of Bemis Corp.
• Westfield Medical Packaging

The results of this research have demonstrated that testing the barrier performance of porous
packaging materials using microorganisms correlates with measuring the filtration efficiency of
the materials. The new method does not require the use of microbiological methods and can be
conducted in a rapid manner. The incumbent test method for measuring microbial barrier, ASTM
F1608, challenges test specimens at only one flow rate, a rate which is considered by many to be
unrealistically high. In contrast, the new method generates filtration efficiency data over a range
of flow rates that are considered to be more representative of the environment encountered by
sealed packages during normal handling and distribution cycles.

When measuring the filtration efficiency of a porous packaging material, a typical filtration
efficiency curve is generated. Because the arc of the curve is dependent upon the characteristics
of the individual test material, the appropriate way to compare materials is to use the parameter
that measures the maximum penetration through the material, the flow rate at which the most
particles pass through the sample.

Testing Apparatus

The main components which comprise the new system are:

• an aerosol generator, which uniformly introduces polystyrene particles into the challenge
air stream at a desired concentration;
• the sample holder;
• a manometer for measuring pressure drop across the test specimen;
• one laser particle counter for enumerating the particles in both the challenge and filtrate
aerosol streams;
• a means of recording the data from the particle counters and pressure manometer;

While this is the most economical apparatus for conducting the new test method, the use of only
one particle counter requires the challenge and filtrate aerosol streams to be monitored
alternately. This doubles the amount of time required to perform the testing. In addition, the use
of only one counter means the value of the challenge counts must be calculated vs. measured
during the interval when filtrate counts are being measured. This increases data reduction
difficulty and time. The addition of a second particle counter allows for simultaneous
measurement of the challenge and filtrate aerosol streams. This reduces both testing and data
reduction time.

Some additional instrumentation in the form of mass flow meters and pressure transducers can
make the test system more user friendly. While adding some cost, this additional instrumentation
can provide real-time information regarding aerosol generator flow, filtrate aerosol flow, vacuum
generator pressure and challenge vent pressure. Constant monitoring of these various system
parameters can alert the operator to possible system malfunctions, which could generate
erroneous data. In addition, the direct measurement of the filtrate aerosol flow rate practically
eliminates the need for converting pressure drop across the sample to flow through the sample,
further reducing data reduction difficulty and time.

Development of the Standard Test Method

The development of a standard test method was conducted in parallel with the work to “finetune” the test unit. ASTM International was selected as the standards organization to develop the
test because it is a consensus standards organization; it has a well-defined protocol for determining precision and bias; and it has a plethora of standards commonly used in the medical device packaging community.

The draft test method was submitted to ASTM Committee F02.0 Flexible Barrier Packaging
which is responsible for medical packaging materials and systems. The test method F2638-07,
entitled Standard Test Method for Determining the Microbial Barrier of Porous Packaging
Materials (Aerosol Filtration Method using Dual Particle Counters), was balloted three times at
the subcommittee and main committee levels before being accepted.

With the successful balloting and publication completed, additional test units are planned to be
built by other members of the healthcare industry and a full round robin study will follow to
establish inter- and intra-laboratory reproducibility.

The standard test method will be submitted to ISO technical committee TC198 (Sterilization)
/working group WG7 (Packaging), which is responsible for ISO standard ISO 11607-1:2006
Packaging for terminally sterilized medical devices - Part 1: Requirements for materials, sterile
barrier systems and packaging systems. This committee was recently responsible for activities
that harmonized the ISO 11607 standard with the CEN medical packaging standard EN 868 Part

Microbial Barrier Tests…Old vs. New

ASTM F1608-00 (2004) Standard Test Method for Microbial Ranking of Porous
Packaging Materials (Exposure Chamber Method), or the log reduction value (LRV) test
as it is commonly called, also produces an aerosol and challenges the test specimen by
generating flow through the specimens. However, this test is conducted at only one flow
rate -- 2.8 ℓ/min. -- and instead of polystyrene spheres, the LRV test is conducted with
live organisms of nominal 1 µm size. Spores that penetrate the test specimens are
collected for plating and enumeration on membrane filters. Subsequent to plating,
enumeration samples are incubated for a minimum of 24 hours. Colony forming units
(CFUs) and dilution factors, if any, are recorded for each sample. The LRV is then
calculated by comparing the logarithm of the number of spores passing through the
sample (the number of colonies on the plate) with the logarithm of the microbial
challenge (the number of spores in suspension above or upstream of the sample).
The use of live organisms for ASTM F1608 means that care must be taken when
handling specimens. The colonies are often difficult to maintain; they will sometimes die
off or become “unhealthy,” resulting in questionable test results. To reduce the possibility
of generating erroneous results due to contamination from normal bioburden, test
specimens are typically sterilized prior to performing the test. There is also a need for
constant decontamination of instruments and equipment. Thus, it is almost a requisite
that this testing be conducted at a biological testing facility or a wet lab.

In addition to sterilization and set-up time, the time required to conduct this test includes
15 minutes of actual sample exposure to microbial challenge; time to perform dilutions, if
necessary; time to prepare plates; a minimum of 24 hours to incubate; and time to
enumerate the CFUs. Then, data reduction can begin. Therefore, typical turnaround
time quoted by contract test labs (to conduct a test, generate the data and write a report) is three to four days. In addition, it is important to note that this test is generally perceived as a rather “noisy” test, which could be attributed to the dilution process and how the CFUs are counted.

The most critical shortcoming of the ASTM F1608 test is the flow rate used during the
challenge process. The flow rate for this test is fixed at 2.8 ℓ/min, which generates a face
velocity greater that 140 cm/min. when accounting for the sample area. This rate is Inertial impaction – occurs when a particle, as a result of its mass, deviates from the air stream flowing around a fiber and collides with it. The effectiveness of this method of capture is directly related to the mass of the particle and the speed of the air stream. The higher the velocity and the mass of the particle, the greater the chance of it colliding with a fiber.

unrealistically high when compared to theoretical and real flow rates that would be
encountered by packaging materials during actual package distribution and handling, as
shown in Table I. (At a flow rate of 2.8 ℓ/min., inertial impaction is the dominant
filtration mechanism.) At the face velocity caused by this flow rate, spores cannot
rapidly change direction with the air flowing through the sample. Their inertia tends to
cause collisions with filter fibers, entrapping the spores. Because of this phenomenon,
some porous materials tested via this method appear to provide a much better microbial
barrier than they actually do when tested at flow rates approaching real-world conditions
where the percent penetration is much higher.

The area of the ASTM F2638 test specimen is four times larger than the area of the
sample required for the ASTM F1608 test. Testing the same amount of surface area
using ASTM F2638 requires one fourth the number of replicates as compared to ASTM
F1608. Testing a larger area per sample also helps reduce sample to sample variability
therefore minimizing data scatter

Using two particle counters, the ASTM F2638 test method requires only three minutes to
obtain sufficient data for percent penetration calculation at a given flow rate or pressure
differential. If one counter is used to alternately monitor challenge and filtrate particles,
the testing time is doubled. Data can be generated at five different flow rates in fifteen
minutes using a two counter system. This is the same exposure time required to generate
data for only one flow rate when using the ASTM F1608 test method. When testing via
ASTM F2638, data reduction can begin immediately upon completion of a test. The
population numbers are known immediately for both sides of the sample, filter reduction
can be calculated and expressed in LRV; there is no wait time to count the downstream
side. Since there are no actual organisms or spores, there is no need for decontamination,
culture plating or incubation. In addition, particle counters do the actual enumeration.

Unlike with ASTM F1608, there is no need for a biological test facility when using the
ASTM F2638 test method. Therefore, this test can be set up and run almost anywhere.
In addition to rapid sample turnaround, the new test method has the ability to rapidly test
at multiple flow rates. These flow rates can be adjusted to imitate conditions encountered
by packages during actual distribution and handling. Data have been generated
demonstrating that the maximum penetration point of porous materials typically used for
sterile packaging applications occurs at flow rates below those used in the ASTM F1608
test.

It has become apparent to many in the medical device industry that ASTM F2638 is a
more rigorous and realistic test for measuring porous microbial barriers than ASTM
F1608. More can be learned regarding the barrier performance of porous materials by
testing via the new method. Figures I and II show data using both test methods. Figure I
displays data obtained using the ASTM F2638 test method on several common porous
sterile barrier system materials. Figure II displays the LRVs obtained by using the
ASTM F1608 test method for the same materials.

The Importance of Flow Rates

Figure III displays data collected while performing ASTM F2638. Percent of penetration
is plotted versus test sample face velocity. These are basically the same data displayed in
Figure I. However, displayed with these data is the face velocity experienced by test
samples during testing conducted in accordance with ASTM F1608. Note the extremely
high face velocity used in the ASTM F1608 test. Refer to Table I and compare this face
velocity with the typical velocity generated by air transport or routine handling. In
contrast, the range for face velocity used in the ASTM F2638 test is much closer to realworld stresses listed in Table I. Notice in Figure I or III that the maximum penetration
points or curve peaks all occur at face velocities less than 5 cm/min. The maximum
penetration points occur at a face velocity that is practically the same as those generated
by real life stresses. Knowing this fact raises the question, why test at face velocities
more than 100 times greater (ASTM F1608) than what might be encountered under
typical transportation and handling environments?”
The point of maximum penetration is where the microbial barrier properties of a porous
material are challenged to their limit. Therefore, these are the flow rates or velocities
most important to know when selecting a barrier material.

When reviewing flow rates or face velocities, it is important to consider the various
conditions a package may be exposed to during its life cycle. Every package will
encounter many stresses and levels of stress during its life cycle. However, these levels or
face velocities are only part of the equation. Factors such as package volume also affect
rate of pressure differential equilibration in a porous sterile barrier system. For example:
a flat, two-dimensional package has very little air in the original configuration to
evacuate and, as a result, very little air enters during the equilibration phase.

The surface area of the porous package material also affects face velocity. Less surface
area for air exchange means greater face velocity during any equilibration process. Small
patches or vents limit the space through which a sterile barrier system can vent or
equilibrate. Therefore, the forces are stronger, causing an increase in the face velocity.
Another variable to consider is package dimensions -- LxWxD. A long, thin flexible
sterile barrier system, such as a chevron peel pouch, exhibits different equilibration rates
than a rectangular formed film design or a square rigid blister.

The clinical usage of a sterile barrier system also subjects it to different stresses. For
example: the pressure changes that occur to a flexible pouch when a nurse reaches into a
box of small disposables, removes a handful and carries them in her pocket are very
different from the pressure changes experienced by a rigid blister package opened in an
operating room setting.

Conclusion

What does all of this mean to a medical device packaging engineer? The new ASTM
F2638 test method is an initial step toward establishing criteria and determining
appropriate microbial barrier requirements for sterile packages. Additional work related
to the relationship between package design, package volume, secondary packaging,
clinical usage, environmental stresses and maximum penetration rates of materials will
move the industry one step closer to adopting a universal standard for microbial barrier.
Although it will take time to generate the data required to shift the industry paradigm
related to acceptable microbial barrier, the new ASTM F2638 test method gives
packaging engineers a valuable new tool to rank various materials and begin questioning
the appropriate level of microbial barrier for medical devices.

References
1. ISO 11607-1:2006 Packaging for terminally sterilized medical devices Part 1: Materials,
sterile barrier systems and packaging systems.
2. ASTM International F1608-00(2004) Standard Test Method for Microbial Ranking of Porous
Packaging Materials (Exposure Chamber Method).
3. ASTM F2638-07 Standard Test Method for Using Aerosol Filtration for Measuring the
Performance of Porous Packaging Materials as a Surrogate Microbial Barrier,
4. Microbiological Barrier Testing of Porous Medical Packaging Materials, Alan Tallentire and
Colin Sinclair at the Meeting of the Society of Plastics Engineers (Scandinavian Section),
May 1994.

Paul F. Herman is a Nonwovens Application Consultant for DuPont Medical and Industrial
Packaging in Richmond, VA. Curtis L. Larsen is a Packaging Consultant for DuPont Medical
Packaging and Spartan Design Group, LLC located in Tonka Bay, MN.