1、Designation: F 1608 00 (Reapproved 2004)Standard Test Method forMicrobial Ranking of Porous Packaging Materials (ExposureChamber Method)1This standard is issued under the fixed designation F 1608; the number immediately following the designation indicates the year oforiginal adoption or, in the case
2、 of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is used to determine the passage ofairborne bacteria through porous materi
3、als intended for use inpackaging sterile medical devices. This test method is designedto test materials under conditions that result in the detectablepassage of bacterial spores through the test material.1.1.1 A round-robin study was conducted with eleven labo-ratories participating. Each laboratory
4、 tested duplicate samplesof six commercially available porous materials to determinethe LRV. Materials tested under the standard conditions de-scribed in this test method returned average values that rangefrom LRV 1.7 to 4.3.1.1.2 Results of this round-robin study indicate that cautionshould be used
5、 when comparing test data and ranking materi-als, especially when a small number of sample replicates areused. In addition, further collaborative work (such as describedin Practice E 691) should be conducted before this test methodwould be condsidered adequate for purposes of setting perfor-mance st
6、andards.1.2 This test method requires manipulation of microorgan-isms and should be performed only by trained personnel. TheU.S. Department of Health and Human Services publicationBiosafety in Microbiological and Biomedical Laboratories(CDC/NIH-HHS Publication No. 84-8395) should be con-sulted for g
7、uidance.1.3 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Refe
8、renced Documents2.1 ASTM Standards:2E 177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE 691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3. Terminology3.1 Definition:3.1.1 porous packaging material, na material used inmedical pack
9、aging which is intended to provide an environ-mental and biological barrier, while allowing sufficient air flowto be used in gaseous sterilization methods (for example, EO,steam, gas plasma).4. Summary of Test Method4.1 Samples of porous materials are subjected to an aerosolof Bacillus subtilis var.
10、 niger spores within an exposurechamber. Spores which pass through the porous sample arecollected on membrane filters and enumerated. The logarithmreduction value (LRV) is calculated by comparing the loga-rithm of the number of spores passing through the porousmaterial with the logarithm of the micr
11、obial challenge.4.2 Standard Set of ConditionsThis test method specifiesa standard set of conditions for conducting the exposurechamber test method.Astandard set of conditions is required toenable evaluation of materials between laboratories. The con-ditions stated in this test method were chosen fo
12、r severalreasons. First, it is difficult to maintain an aerosol of sporesover long periods of time. (Also, if the spore challenge time islong, the cost of the test increases). Second, to determine thedifferences between materials, it is necessary to test thematerials under conditions which allow pas
13、sage of bacterialspores. If a material does not allow any passage of spores, allthat can be stated is that it has better resistance to penetrationthan the severity of the challenge conditions. Third, it isnecessary to have a large spore challenge level to be able todetect the passage of spores throu
14、gh the entire range ofcommercially available porous packaging materials. The stan-dard conditions stated in this test method are based upon thesefactors. (Additional information may be found in the Refer-ences section). However, since many factors influence thedetermination of an appropriate porous
15、material (outlined in5.1.1-5.1.4), each user may modify these conditions (that is,1This test method is under the jurisdiction ofASTM Committee F02 on FlexibleBarrier Materials and is the direct responsibility of Subcommittee F02.15 onChemical/Safety Properties.Current edition approved Oct. 1, 2004.
16、Published October 2004. Originallyapproved in 1995. Last previous edition approved in 2000 as F 1608 00.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standard
17、s Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.bacterial challenge, time, flow rate) after first conductingstudies at the specified standard conditions. The standard set oftarget parameters
18、 for conducting the test method are as follows:4.2.1 Flow Rate Through Sample2.8 L/min.4.2.2 Exposure Time 15 min.4.2.3 Target Microbial Challenge1 3 106colony formingunits (CFU)/sample port.5. Significance and Use5.1 The exposure-chamber method is a quantitative proce-dure for determining the micro
19、bial-barrier properties of porousmaterials under the conditions specified by the test. Dataobtained from this test are useful in assessing the relativepotential of a particular porous material to contribute to the lossof sterility to the contents of the package versus another porousmaterial. This te
20、st method is not intended to predict theperformance of a given material in a specific sterile-packagingapplication. The maintenance of sterility in a particular pack-aging application will depend on a number of factors, includ-ing, but not limited to the following:5.1.1 The bacterial challenge (numb
21、er and kinds of micro-organisms) that the package will encounter in its distributionand use. This may be influenced by factors such as shippingmethods, expected shelf life, geographic location, and storageconditions.5.1.2 The package design, including factors such as adhe-sion between materials, the
22、 presence or absence of secondaryand tertiary packaging, and the nature of the device within thepackage.5.1.3 The rate and volume exchange of air that the porouspackage encounters during its distribution and shelf life. Thiscan be influenced by factors including the free-air volumewithin the package
23、 and pressure changes occurring as a resultof transportation, manipulation, weather, or mechanical influ-ences (such as room door closures and HVAC systems).5.1.4 The microstructure of a porous material which influ-ences the relative ability to adsorb or entrap microorganisms,or both, under differen
24、t air-flow conditions.6. Apparatus6.1 This procedure should be conducted in a microbiologi-cal laboratory by trained personnel. As a result, it is assumedthat basic microbiological equipment and supplies for conduct-ing routine microbiological manipulations (that is, standardplate counts, sterilizat
25、ion with an autoclave, and so forth) willbe available.6.2 Exposure Chamber, constructed primarily from acrylicsheeting and consists of two major sections, as illustrated inFig. 1. The bottom section contains a six-place manifoldconnected to six flowmeters, one per port, containing hosesattached to s
26、ix filtering units. The port to the manifold isattached to a vacuum source. A vacuum gage is mountedbetween the manifold and the vacuum source. The upperchamber contains a fan for dispersion of the bacterial aerosol,a port for attachment of the nebulizer, a port for exhausting thechamber, and a plat
27、e for attachment of disposable or steriliz-able filter units. The chamber may use disposable filter units orreusable filter units, or both.7. Materials7.1 Bacillus subtilis var. niger (ATCC9372), aqueous sporesuspension in water.FIG. 1 Exposure ChamberF 1608 00 (2004)27.2 Soybean Casein Digest AgarB
28、ottles for pour platesand pre-poured plates (;25 mL in 100 by 15-mm plates)prepared commercially or in accordance with standard tech-niques.7.3 Sterile Cellulose Nitrate Filters, 47 or 50-mm diameter,depending upon filter unit specification, 0.45-m pore size.7.4 Sterile Bottle-Top Filter Units, (Fal
29、con-type 7104 orfilter holders with funnel 310-4000 or equivalent).7.5 Glass Nebulizer.7.6 Sterile Forceps.7.7 Incubator, 30 to 35C.7.8 Disk Cutter, 47 or 50-mm diameter, depending uponfilter unit specification.7.9 Sterile Gloves.7.10 Sterile Syringe, 3-cm3with needle or micropipette.7.11 Sterile Pi
30、pettes, to deliver 0.1, 1, 10, and 25 mL.7.12 Blender, with sterile12-pt jar(s).7.13 Vortex Mixer.7.14 Vacuum Pump, with air filter.7.15 Calibrated Timer.7.16 Calibrated FlowmetersOne each with a range from 5to 30 L/min; six each with a range from 1.0 to 5.0 L/min.7.17 Sterile Petri Plates.7.18 Ster
31、ile Water, 100 and 9.9-mL aliquots, or otherappropriate volumes for membrane grinding and dilutions.7.19 Hoses and Piping See Section 9 for lengths anddiameters.7.20 Rubber Stoppers with HolesSee Section 9 for sizes.7.21 Trap Jar.7.22 Calibrated Vacuum Gage.7.23 Compressed Air Source, with air filte
32、r.7.24 Biocontainment Hood.7.25 Chlorine Bleach, or suitable sporocide.8. Sample Preparation8.1 Cut random samples of material into disks in accordancewith the size required for the filter holder being used (47 or 50mm) using a disk cutter. It is suggested that additional samplesbe cut to allow for
33、errors during the procedure. Typically, thesample disks are sterilized prior to testing using a test methodappropriate for the specific material. Materials may also betested before or after they are subjected to other conditionssuch as heat or cold, relative humidity, different sterilizationprocesse
34、s, real time, or accelerated aging. The samples may bestored in sterile petri plates or other suitable sterile containersbefore testing.8.2 The minimum sample size for a given material is two,which was used in the round-robin study of this test method.However, it is strongly suggested that more samp
35、les be used toimprove precision and bias (Section 14).9. Apparatus Preparation9.1 Since aerosols containing bacterial spores are formedduring the use of this apparatus, the exposure chamber (see Fig.1) should be assembled and used within a biological safetycabinet.9.1.1 Place the top of the chamber
36、on the bottom base.9.1.2 Connect the top of each of the six flowmeters to themanifold using 0.65-cm inside diameter hoses. Connect themanifold to a filtered vacuum source.9.1.3 Connect the bottom of each sample flowmeter to afilter unit with 0.65-cm inside diameter hose using an endconnector.9.1.4 U
37、sing a rubber hose, attach the nebulizer to a teeconnector made of a 0.65-cm PVC and three pieces of 0.6-cminside diameter PVC piping approximately 7.5 cm long.9.1.5 Attach the vertical leg of the tee to a trap jar using arubber stopper with a 0.65-cm diameter hole. The trap jar isintended to retain
38、 any unsuspended droplets produced by thenebulizer.9.1.6 Attach the second end of the tee to a 1.3-cm insidediameter rubber tubing approximately 3.8 cm long and connectto the front port of the chamber.9.1.7 Attach a 1.3-cm inside diameter rubber tubing ap-proximately 16 cm long to the mouth of the n
39、ebulizer. Connectthe 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 insidediameter rubber tubing to the top port of a calibrated flowmeter(from 5 to 30-L/min range).9.1.9 Connect the bottom port of the nebulizer to a filteredair source.9.1.10
40、 Attach the exhaust port of the chamber that is usedfor evacuation to a 1.3-cm inside diameter tubing which, inturn, leads to an air filter and to a vacuum source.9.2 Filter Unit-Holder Preparation:9.2.1 Wrap each non-sterile sterilizable filter unit in asterilizable wrap.9.2.2 Sterilize the filter
41、units as specified by the manufac-turer. Presterilized filter units do not need to be resterilized.10. Apparatus Validation10.1 The test apparatus (see Fig. 1) must be validated forbacterial challenge to each port. This step should be performedupon first use of the chamber and a minimum of three run
42、sshould be conducted. The following description outlines vali-dation of the test procedure for a challenge of 1 3 106colonyforming units (CFU) per port in 15 min at a flow rate of 2.8L/min. If testing is to be conducted using other parameters, avalidation should be conducted using those parameters.1
43、0.1.1 Aseptically place a sterile 0.45-m membrane filteron the base of each filter unit using sterile forceps and gloves(Fig. 2).10.1.2 Attach the top of the filter unit to the bottom of theexposure chamber. Then attach each filter unit to its respectiveflowmeter.10.1.3 Dispense 3.0 mL of the desire
44、d aqueous sporesuspension into the nebulizer. When using the DeVilbiss #40nebulizer, a volume of 3.0 mL at a concentration of 5 3 107spores/mL is necessary to achieve a challenge of 1 3 106CFU(60.5 log) per port in 15 min.10.1.4 Turn on the chamber fan.10.1.5 Adjust port flowmeters to 2.8 L/min. It
45、is importantthat all ports be set to the same flow and monitored during theexposure period. Before adjusting each flowmeter, open eachvalve completely, then slowly open the vacuum and fine adjustuntil the desired flow is achieved.F 1608 00 (2004)310.1.6 Adjust the nebulizer flow rate as recommended
46、bythe nebulizer manufacturer to produce droplets that are withinthe appropriate particle size range. When using the DeVilbiss#40 nebulizer, a flow rate of 8.5 L/min is used.10.1.7 Immediately start the 15-min timer. At regular inter-vals, observe and adjust (if necessary) all flowmeters tomaintain t
47、he appropriate flow rate settings during the 15-mintest period.10.1.8 After exposure, turn off the flow to the nebulizer, thevacuum, and the fan.10.1.9 Evacuate the chamber for 15 min by connecting thevacuum source to the chamber through a microbial filterassembly.10.1.10 Disinfect the outside of ea
48、ch filtering unit. Usediluted chlorine (5 mL bleach to 245 mL water, prepared freshdaily) or other suitable sporicide.10.1.11 Disconnect the hoses from each of the filter units,and remove the units from the bottom plate of the chamber.10.1.12 Remove the filter membranes aseptically, one at atime, an
49、d enumerate the organisms on each membrane (Fig. 3).Since more than 100 CFU are anticipated, the spores must beeluted from the membrane by grinding the membrane for 1 minin a12-pt sterile blender jar containing 100.0 mL of sterilewater. Samples are then serially diluted prior to performingstandard plate counts to accurately determine the number ofspores. A dilution and plating scheme, which was used in theround-robin study, includes plating 10.0, 1.0, and 0.1-mLaliquots of the blended membrane in duplicate.An additional 1to 100 dilution is prepared by placing 0.1 mL i
