ASTM F1608-00(2009)
(Test Method)Standard Test Method for Microbial Ranking of Porous Packaging Materials (Exposure Chamber Method)
Standard Test Method for Microbial Ranking of Porous Packaging Materials (Exposure Chamber Method)
SIGNIFICANCE AND USE
The exposure-chamber method is a quantitative procedure for determining the microbial-barrier properties of porous materials under the conditions specified by the test. Data obtained from this test are useful in assessing the relative potential of a particular porous material to contribute to the loss of sterility to the contents of the package versus another porous material. This test method is not intended to predict the performance of a given material in a specific sterile-packaging application. The maintenance of sterility in a particular packaging application will depend on a number of factors, including, but not limited to the following:
The bacterial challenge (number and kinds of microorganisms) that the package will encounter in its distribution and use. This may be influenced by factors such as shipping methods, expected shelf life, geographic location, and storage conditions.
The package design, including factors such as adhesion between materials, the presence or absence of secondary and tertiary packaging, and the nature of the device within the package.
The rate and volume exchange of air that the porous package encounters during its distribution and shelf life. This can be influenced by factors including the free-air volume within the package and pressure changes occurring as a result of transportation, manipulation, weather, or mechanical influences (such as room door closures and HVAC systems).
The microstructure of a porous material which influences the relative ability to adsorb or entrap microorganisms, or both, under different air-flow conditions.
SCOPE
1.1 This test method is used to determine the passage of airborne bacteria through porous materials intended for use in packaging sterile medical devices. This test method is designed to test materials under conditions that result in the detectable passage of bacterial spores through the test material.
1.1.1 A round-robin study was conducted with eleven laboratories participating. Each laboratory tested duplicate samples of six commercially available porous materials to determine the LRV. Materials tested under the standard conditions described in this test method returned average values that range from LRV 1.7 to 4.3.
1.1.2 Results of this round-robin study indicate that caution should be used when comparing test data and ranking materials, especially when a small number of sample replicates are used. In addition, further collaborative work (such as described in Practice E691) should be conducted before this test method would be condsidered adequate for purposes of setting performance standards.
1.2 This test method requires manipulation of microorganisms and should be performed only by trained personnel. The U.S. Department of Health and Human Services publication Biosafety in Microbiological and Biomedical Laboratories (CDC/NIH-HHS Publication No. 84-8395) should be consulted for guidance.
1.3 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.4 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: F1608 − 00(Reapproved 2009)
Standard Test Method for
Microbial Ranking of Porous Packaging Materials (Exposure
Chamber Method)
This standard is issued under the fixed designation F1608; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
1. Scope 2. Referenced Documents
2.1 ASTM Standards:
1.1 This test method is used to determine the passage of
E177Practice for Use of the Terms Precision and Bias in
airborne bacteria through porous materials intended for use in
ASTM Test Methods
packagingsterilemedicaldevices.Thistestmethodisdesigned
E691Practice for Conducting an Interlaboratory Study to
to test materials under conditions that result in the detectable
Determine the Precision of a Test Method
passage of bacterial spores through the test material.
1.1.1 Around-robin study was conducted with eleven labo-
3. Terminology
ratories participating. Each laboratory tested duplicate samples
3.1 Definitions:
of six commercially available porous materials to determine
3.1.1 porous packaging material, n—a material used in
the LRV. Materials tested under the standard conditions de-
medical packaging which is intended to provide an environ-
scribed in this test method returned average values that range
mentalandbiologicalbarrier,whileallowingsufficientairflow
from LRV 1.7 to 4.3.
to be used in gaseous sterilization methods (for example, EO,
1.1.2 Results of this round-robin study indicate that caution
steam, gas plasma).
should be used when comparing test data and ranking
materials,especiallywhenasmallnumberofsamplereplicates
4. Summary of Test Method
are used. In addition, further collaborative work (such as
4.1 Samples of porous materials are subjected to an aerosol
described in Practice E691) should be conducted before this
of Bacillus subtilis var. niger spores within an exposure
test method would be condsidered adequate for purposes of
chamber. Spores which pass through the porous sample are
setting performance standards.
collected on membrane filters and enumerated. The logarithm
1.2 This test method requires manipulation of microorgan- reduction value (LRV) is calculated by comparing the loga-
isms and should be performed only by trained personnel. The rithm of the number of spores passing through the porous
U.S. Department of Health and Human Services publication material with the logarithm of the microbial challenge.
Biosafety in Microbiological and Biomedical Laboratories
4.2 Standard Set of Conditions —This test method specifies
(CDC/NIH-HHS Publication No. 84-8395) should be con-
a standard set of conditions for conducting the exposure
sulted for guidance.
chambertestmethod.Astandardsetofconditionsisrequiredto
enable evaluation of materials between laboratories. The con-
1.3 The values stated in SI units are to be regarded as
ditions stated in this test method were chosen for several
standard. No other units of measurement are included in this
reasons. First, it is difficult to maintain an aerosol of spores
standard.
over long periods of time. (Also, if the spore challenge time is
1.4 This standard does not purport to address all of the
long, the cost of the test increases). Second, to determine the
safety concerns, if any, associated with its use. It is the
differences between materials, it is necessary to test the
responsibility of the user of this standard to establish appro-
materials under conditions which allow passage of bacterial
priate safety and health practices and determine the applica-
spores. If a material does not allow any passage of spores, all
bility of regulatory limitations prior to use.
that can be stated is that it has better resistance to penetration
than the severity of the challenge conditions. Third, it is
necessary to have a large spore challenge level to be able to
ThistestmethodisunderthejurisdictionofASTMCommitteeF02onFlexible
Barrier Packaging and is the direct responsibility of Subcommittee F02.15 on
Chemical/Safety Properties. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved Oct. 1, 2009. Published November 2009. Originally contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
approved in 1995. Last previous edition approved in 2004 as F1608–00(2004). Standards volume information, refer to the standard’s Document Summary page on
DOI: 10.1520/F1608-00R09. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
F1608 − 00 (2009)
detect the passage of spores through the entire range of 5.1.2 The package design, including factors such as adhe-
commercially available porous packaging materials. The stan- sion between materials, the presence or absence of secondary
dard conditions stated in this test method are based upon these
and tertiary packaging, and the nature of the device within the
factors. (Additional information may be found in the Refer-
package.
ences section). However, since many factors influence the
5.1.3 The rate and volume exchange of air that the porous
determination of an appropriate porous material (outlined in
package encounters during its distribution and shelf life. This
5.1.1 – 5.1.4), each user may modify these conditions (that is,
can be influenced by factors including the free-air volume
bacterial challenge, time, flow rate) after first conducting
within the package and pressure changes occurring as a result
studiesatthespecifiedstandardconditions.Thestandardsetof
of transportation, manipulation, weather, or mechanical influ-
targetparametersforconductingthetestmethodareasfollows:
ences (such as room door closures and HVAC systems).
4.2.1 Flow Rate Through Sample—2.8 L/min.
5.1.4 The microstructure of a porous material which influ-
4.2.2 Exposure Time— 15 min.
6 ences the relative ability to adsorb or entrap microorganisms,
4.2.3 Target Microbial Challenge —1×10 colony forming
or both, under different air-flow conditions.
units (CFU)/sample port.
5. Significance and Use 6. Apparatus
5.1 The exposure-chamber method is a quantitative proce-
6.1 This procedure should be conducted in a microbiologi-
dure for determining the microbial-barrier properties of porous
cal laboratory by trained personnel. As a result, it is assumed
materials under the conditions specified by the test. Data
thatbasicmicrobiologicalequipmentandsuppliesforconduct-
obtained from this test are useful in assessing the relative
ing routine microbiological manipulations (that is, standard
potentialofaparticularporousmaterialtocontributetotheloss
plate counts, sterilization with an autoclave, and so forth) will
ofsterilitytothecontentsofthepackageversusanotherporous
be available.
material. This test method is not intended to predict the
6.2 Exposure Chamber, constructed primarily from acrylic
performance of a given material in a specific sterile-packaging
sheeting and consists of two major sections, as illustrated in
application. The maintenance of sterility in a particular pack-
Fig. 1. The bottom section contains a six-place manifold
aging application will depend on a number of factors,
connected to six flowmeters, one per port, containing hoses
including, but not limited to the following:
attached to six filtering units. The port to the manifold is
5.1.1 The bacterial challenge (number and kinds of micro-
attached to a vacuum source. A vacuum gage is mounted
organisms) that the package will encounter in its distribution
and use. This may be influenced by factors such as shipping between the manifold and the vacuum source. The upper
methods, expected shelf life, geographic location, and storage chamber contains a fan for dispersion of the bacterial aerosol,
conditions. a port for attachment of the nebulizer, a port for exhausting the
FIG. 1 Exposure Chamber
F1608 − 00 (2009)
chamber, and a plate for attachment of disposable or steriliz- processes, real time, or accelerated aging.The samples may be
ablefilterunits.Thechambermayusedisposablefilterunitsor stored in sterile petri plates or other suitable sterile containers
reusable filter units, or both. before testing.
8.2 The minimum sample size for a given material is two,
7. Materials
which was used in the round-robin study of this test method.
7.1 Bacillus subtilis var. niger (ATCC9372), aqueous spore
However,itisstronglysuggestedthatmoresamplesbeusedto
suspension in water.
improve precision and bias (Section 14).
7.2 Soybean Casein Digest Agar—Bottles for pour plates
9. Apparatus Preparation
and pre-poured plates (;25 mL in 100 by 15-mm plates)
prepared commercially or in accordance with standard tech-
9.1 Since aerosols containing bacterial spores are formed
niques.
duringtheuseofthisapparatus,theexposurechamber(seeFig.
7.3 Sterile Cellulose Nitrate Filters, 47 or 50-mm diameter, 1) should be assembled and used within a biological safety
cabinet.
depending upon filter unit specification, 0.45-µm pore size.
9.1.1 Place the top of the chamber on the bottom base.
7.4 Sterile Bottle-Top Filter Units, (Falcon-type 7104 or
9.1.2 Connect the top of each of the six flowmeters to the
filter holders with funnel 310-4000 or equivalent).
manifold using 0.65-cm inside diameter hoses. Connect the
7.5 Glass Nebulizer.
manifold to a filtered vacuum source.
7.6 Sterile Forceps.
9.1.3 Connect the bottom of each sample flowmeter to a
filter unit with 0.65-cm inside diameter hose using an end
7.7 Incubator, 30 to 35°C.
connector.
7.8 Disk Cutter, 47 or 50-mm diameter, depending upon
9.1.4 Using a rubber hose, attach the nebulizer to a tee
filter unit specification.
connector made of a 0.65-cm PVC and three pieces of 0.6-cm
7.9 Sterile Gloves.
inside diameter PVC piping approximately 7.5 cm long.
9.1.5 Attach the vertical leg of the tee to a trap jar using a
7.10 Sterile Syringe, 3-cm with needle or micropipette.
rubber stopper with a 0.65-cm diameter hole. The trap jar is
7.11 Sterile Pipettes, to deliver 0.1, 1, 10, and 25 mL.
intended to retain any unsuspended droplets produced by the
7.12 Blender, with sterile ⁄2-pt jar(s).
nebulizer.
7.13 Vortex Mixer. 9.1.6 Attach the second end of the tee to a 1.3-cm inside
diameterrubbertubingapproximately3.8cmlongandconnect
7.14 Vacuum Pump, with air filter.
to the front port of the chamber.
7.15 Calibrated Timer.
9.1.7 Attach a 1.3-cm inside diameter rubber tubing ap-
7.16 Calibrated Flowmeters—Oneeachwitharangefrom5 proximately16cmlongtothemouthofthenebulizer.Connect
to 30 L/min; six each with a range from 1.0 to 5.0 L/min.
the loose end of the tubing to the third end of the tee.
9.1.8 Connect the nebulizer inlet port with a 0.5-cm inside
7.17 Sterile Petri Plates.
diameterrubbertubingtothetopportofacalibratedflowmeter
7.18 Sterile Water, 100 and 9.9-mLaliquots, or other appro-
(from 5 to 30-L/min range).
priate volumes for membrane grinding and dilutions.
9.1.9 Connect the bottom port of the nebulizer to a filtered
7.19 Hoses and Piping— See Section 9 for lengths and
air source.
diameters.
9.1.10 Attach the exhaust port of the chamber that is used
for evacuation to a 1.3-cm inside diameter tubing which, in
7.20 Rubber Stoppers with Holes—See Section 9 for sizes.
turn, leads to an air filter and to a vacuum source.
7.21 Trap Jar.
9.2 Filter Unit-Holder Preparation:
7.22 Calibrated Vacuum Gage.
9.2.1 Wrap each non-sterile sterilizable filter unit in a
7.23 Compressed Air Source, with air filter.
sterilizable wrap.
7.24 Biocontainment Hood.
9.2.2 Sterilize the filter units as specified by the manufac-
turer. Presterilized filter units do not need to be resterilized.
7.25 Chlorine Bleach, or suitable sporocide.
10. Apparatus Validation
8. Sample Preparation
8.1 Cutrandomsamplesofmaterialintodisksinaccordance 10.1 The test apparatus (see Fig. 1) must be validated for
with the size required for the filter holder being used (47 or 50 bacterialchallengetoeachport.Thisstepshould be performed
mm) using a disk cutter. It is suggested that additional samples upon first use of the chamber and a minimum of three runs
be cut to allow for errors during the procedure. Typically, the should be conducted. The following description outlines vali-
sample disks are sterilized prior to testing using a test method dation of the test procedure for a challenge of 1×10 colony
appropriate for the specific material. Materials may also be forming units (CFU) per port in 15 min at a flow rate of 2.8
tested before or after they are subjected to other conditions L/min. If testing is to be conducted using other parameters, a
such as heat or cold, relative humidity, different sterilization validation should be conducted using those parameters.
F1608 − 00 (2009)
10.1.1 Aseptically place a sterile 0.45-µm membrane filter 10.1.8 After exposure, turn off the flow to the nebulizer, the
on the base of each filter unit using sterile forceps and gloves vacuum, and the fan.
(Fig. 2). 10.1.9 Evacuate the chamber for 15 min by connecting the
10.1.2 Attach the top of the filter unit to the bottom of the vacuum source to the chamber through a microbial filter
exposure chamber. Then attach each filter unit to its respective assembly.
flowmeter. 10.1.10 Disinfect the outside of each filtering unit. Use
10.1.3 Dispense 3.0 mL of the desired aqueous spore diluted chlorine (5 mLbleach to 245 mLwater, prepared fresh
suspension into the nebulizer. When using the DeVilbiss #40 daily) or other suitable sporicide.
nebulizer, a volume of 3.0 mL at a concentration of 5×10 10.1.11 Disconnect the hoses from each of the filter units,
spores/mLis necessary to achieve a challenge of 1×10 CFU and remove the units from the bottom plate of the chamber.
(60.5 log) per port in 15 min. 10.1.12 Remove the filter membranes aseptically, one at a
10.1.4 Turn on the chamber fan. time,andenumeratetheorganismsoneachmembrane(Fig.3).
10.1.5 Adjust port flowmeters to 2.8 L/min. It is important Since more than 100 CFU are anticipated, the spores must be
that all ports be set to the same flow and monitored during the elutedfromthemembranebygrindingthemembranefor1min
exposure period. Before adjusting each flowmeter, open each in a ⁄2-pt sterile blender jar containing 100.0 mL of sterile
valve completely, then slowly open the vacuum and fine adjust water. Samples are then serially diluted prior to performing
until the desired flow is achieved. standard plate counts to accurately determine the number of
10.1.6 Adjust the nebulizer flow rate as recommended by spores. A dilution and plating scheme, which
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