1、Designation: F1608 00 (Reapproved 2009)Standard Test Method forMicrobial Ranking of Porous Packaging Materials (ExposureChamber Method)1This standard is issued under the fixed designation F1608; the number immediately following the designation indicates the year oforiginal adoption or, in the case o
2、f revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon () 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 materials
3、 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 te
4、sted 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 wh
5、en 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 E691) should be conducted before this test methodwould be condsidered adequate for purposes of setting perfor-mance standa
6、rds.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 guida
7、nce.1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 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
8、appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E177 Practice for Use of the Terms Precision and Bias inASTM Test MethodsE691 Practice for Conducting an Interlaboratory Study toDetermine the P
9、recision of a Test Method3. Terminology3.1 Definition:3.1.1 porous packaging material, na material used inmedical packaging 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
10、 plasma).4. Summary of Test Method4.1 Samples of porous materials are subjected to an aerosolof Bacillus subtilis var. niger spores within an exposurechamber. Spores which pass through the porous sample arecollected on membrane filters and enumerated. The logarithmreduction value (LRV) is calculated
11、 by comparing the loga-rithm of the number of spores passing through the porousmaterial with the logarithm of the microbial 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 req
12、uired toenable evaluation of materials between laboratories. The con-ditions stated in this test method were chosen for 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
13、, to determine thedifferences between materials, it is necessary to test thematerials under conditions which allow passage of bacterialspores. If a material does not allow any passage of spores, allthat can be stated is that it has better resistance to penetration1This test method is under the juris
14、diction ofASTM Committee F02 on FlexibleBarrier Packaging and is the direct responsibility of Subcommittee F02.15 onChemical/Safety Properties.Current edition approved Oct. 1, 2009. Published November 2009. Originallyapproved in 1995. Last previous edition approved in 2004 as F1608 00(2004).DOI: 10.
15、1520/F1608-00R09.2For 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 standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harb
16、or Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.than the severity of the challenge conditions. Third, it isnecessary to have a large spore challenge level to be able todetect the passage of spores through the entire range ofcommercially available porous packaging materials. Th
17、e 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 material (outlined in5.1.1-5.1.4), each user may modify these conditions (t
18、hat is,bacterial challenge, time, flow rate) after first conductingstudies at the specified standard conditions. The standard set oftarget parameters 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 1
19、06colony formingunits (CFU)/sample port.5. Significance and Use5.1 The exposure-chamber method is a quantitative proce-dure for determining the microbial-barrier properties of porousmaterials under the conditions specified by the test. Dataobtained from this test are useful in assessing the relative
20、potential of a particular porous material to contribute to the lossof sterility to the contents of the package versus another porousmaterial. This test method is not intended to predict theperformance of a given material in a specific sterile-packagingapplication. The maintenance of sterility in a p
21、articular pack-aging application will depend on a number of factors, includ-ing, but not limited to the following:5.1.1 The bacterial challenge (number and kinds of micro-organisms) that the package will encounter in its distributionand use. This may be influenced by factors such as shippingmethods,
22、 expected shelf life, geographic location, and storageconditions.5.1.2 The package design, including factors such as adhe-sion between materials, the 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
23、the porouspackage encounters during its distribution and shelf life. Thiscan be influenced by factors including the free-air volumewithin the package and pressure changes occurring as a resultof transportation, manipulation, weather, or mechanical influ-ences (such as room door closures and HVAC sys
24、tems).5.1.4 The microstructure of a porous material which influ-ences the relative ability to adsorb or entrap microorganisms,or both, under different air-flow conditions.6. Apparatus6.1 This procedure should be conducted in a microbiologi-cal laboratory by trained personnel. As a result, it is assu
25、medthat basic microbiological equipment and supplies for conduct-ing routine microbiological manipulations (that is, standardplate counts, sterilization with an autoclave, and so forth) willbe available.6.2 Exposure Chamber, constructed primarily from acrylicsheeting and consists of two major sectio
26、ns, as illustrated inFig. 1. The bottom section contains a six-place manifoldconnected to six flowmeters, one per port, containing hosesattached to six filtering units. The port to the manifold isattached to a vacuum source. A vacuum gage is mountedbetween the manifold and the vacuum source. The upp
27、erFIG. 1 Exposure ChamberF1608 00 (2009)2chamber contains a fan for dispersion of the bacterial aerosol,a port for attachment of the nebulizer, a port for exhausting thechamber, and a plate for attachment of disposable or steriliz-able filter units. The chamber may use disposable filter units orreus
28、able filter units, or both.7. Materials7.1 Bacillus subtilis var. niger (ATCC9372), aqueous sporesuspension in water.7.2 Soybean Casein Digest AgarBottles 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
29、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, (Falcon-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
30、or 50-mm diameter, depending uponfilter unit specification.7.9 Sterile Gloves.7.10 Sterile Syringe, 3-cm3with needle or micropipette.7.11 Sterile Pipettes, 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 Time
31、r.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 Sterile 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 andd
32、iameters.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 filter.7.24 Biocontainment Hood.7.25 Chlorine Bleach, or suitable sporocide.8. Sample Preparation8.1 Cut random samples of material into disks in accordancew
33、ith 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 errors during the procedure. Typically, thesample disks are sterilized prior to testing using a test methodappropriate for the specific material. Materi
34、als may also betested before or after they are subjected to other conditionssuch as heat or cold, relative humidity, different sterilizationprocesses, real time, or accelerated aging. The samples may bestored in sterile petri plates or other suitable sterile containersbefore testing.8.2 The minimum
35、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 samples be used toimprove precision and bias (Section 14).9. Apparatus Preparation9.1 Since aerosols containing bacterial spores are formedduring the use of
36、 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 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 filt
37、ered 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 Using 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
38、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 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
39、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 nebulizer. 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 tubi
40、ng 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 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 vac
41、uum 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 units as specified by the manufac-turer. Presterilized filter units do not need to be resterilized.10. Apparatus Validation10.1 The test apparatus (see
42、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 runsshould be conducted. The following description outlines vali-dation of the test procedure for a challenge of 1 3 106colonyforming units (CFU) per port
43、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.10.1.1 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 o
44、f 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 desired aqueous sporesuspension into the nebulizer. When using the DeVilbiss #40nebulizer, a volume of 3.0 mL at a concentration of 5 3 107F1608 00 (2009)3spo
45、res/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 is importantthat all ports be set to the same flow and monitored during theexposure period. Before adjusting each flowmeter, open eachva
46、lve completely, then slowly open the vacuum and fine adjustuntil the desired flow is achieved.10.1.6 Adjust the nebulizer flow rate as recommended bythe nebulizer manufacturer to produce droplets that are withinthe appropriate particle size range. When using the DeVilbiss#40 nebulizer, a flow rate o
47、f 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 the 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 E
48、vacuate the chamber for 15 min by connecting thevacuum source to the chamber through a microbial filterassembly.10.1.10 Disinfect the outside of each filtering unit. Usediluted chlorine (5 mL bleach to 245 mL water, prepared freshdaily) or other suitable sporicide.10.1.11 Disconnect the hoses from e
49、ach 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, and 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
