1、Designation: F1608 16Standard 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 of revision, the ye
2、ar 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 intended for use
3、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 tested duplicate sam
4、plesof six commercially available porous materials to determinethe Log Reduction Value (LRV) (see calculation in Section 12).Materials tested under the standard conditions described in thistest method returned average values that range from LRV1.7 to4.3.1.1.2 Results of this round-robin study indica
5、te that cautionshould be used when comparing test data and rankingmaterials, especially when a small number of sample replicatesare used. In addition, further collaborative work (such asdescribed in Practice E691) should be conducted before thistest method would be considered adequate for purposes o
6、fsetting performance standards.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) sho
7、uld be con-sulted for guidance.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
8、this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E691 Practice for Conducting an Interlaboratory Study toDetermine the Precision of a Test Method3. Terminology3.1 Def
9、initions: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,ethylene oxide, steam, gas plasma).4. Summary of Test Metho
10、d4.1 Samples of porous materials are subjected to an aerosolof Bacillus atrophaeus spores within an exposure chamber.Spores which pass through the porous sample are collected onmembrane filters and enumerated. The logarithm reductionvalue (LRV) is calculated by comparing the logarithm of thenumber o
11、f spores passing through the porous material with thelogarithm 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 required toenable evaluation of materials betwe
12、en 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, to determine thedifferences between materi
13、als, 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 penetrationthan the severity of the challenge conditions. Third, it isnecessary to have a l
14、arge spore challenge level to be able todetect the passage of spores through the entire range of1This test method is under the jurisdiction ofASTM Committee F02 on FlexibleBarrier Packaging and is the direct responsibility of Subcommittee F02.15 onChemical/Safety Properties.Current edition approved
15、May 1, 2016. Published June 2016. Originallyapproved in 1995. Last previous edition approved in 2009 as F1608 00 (2009).DOI: 10.1520/F1608-16.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards vol
16、ume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1commercially available porous packaging materials. The stan-dard conditions stated in this test method ar
17、e 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 (that is,bacterial challenge, time, flow rate) after f
18、irst 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 Challenge 1106colony formingunits (CFU)/sample port.5. Significanc
19、e 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 is useful in assessing the relativepotential of a particular porous material in contributi
20、ng to theloss of sterility to the contents of the package versus anotherporous material. This test 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 num
21、ber of factors,including, 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, expected shelf life, geographic location, and storagec
22、onditions.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 the porouspackage encounters during its distribution an
23、d 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 systems).5.1.4 The microstructure of a porous material whi
24、ch 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 assumedthat basic microbiological equipment and supplies fo
25、r 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 sections, as illustrated inFig. 1. The bottom section contain
26、s a six-place manifoldFIG. 1 Example of an Exposure ChamberF1608 162connected to six flowmeters, one per port, containing hosesattached to six filtering units. The port to the manifold isattached to a vacuum source. A vacuum gauge is mountedbetween the manifold and the vacuum source. The upperchambe
27、r 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 orreusable filter units, or both.7. Materials7.1 Baci
28、llus atrophaeus (ATCC9372), aqueous spore sus-pension in water.7.2 Soybean Casein Digest Agar/ Tryptic Soy AgarBottlesfor pour plates and pre-poured plates (;25 mL in 100 by15-mm plates) prepared commercially or in accordance withstandard techniques.7.3 Sterile Cellulose Nitrate Filters, 47 or 50-mm
29、 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 or 50-mm diameter, depending uponfilte
30、r 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 NIST Traceable Calibrated Timer.7.16 NIST Traceable C
31、alibrated FlowmetersOne pressureflowmeter with a range from 5 to 30 L/min; six vacuumflowmeters 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 other appro-priate volumes for membrane grinding and dilutions.7.19 Hoses and Piping See S
32、ection 9 for lengths anddiameters.7.20 Rubber Stoppers with HolesSee Section 9 for sizes.7.21 Trap Jar.7.22 NIST Traceable Calibrated Vacuum Gauge.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 samp
33、les 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 errors during the procedure. Typically, thesample disks are sterilized prior to testing using a test methodappr
34、opriate for the specific material. Materials 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
35、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 samples be used toimprove precision and bias (Section 14).9. Apparatus Preparation9.1 Since aerosols containing bac
36、terial 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 on the bottom base.9.1.2 Connect the top of each of the six flowmeters to themanifold using 0.65-cm inside diam
37、eter 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 Using a rubber hose, attach the nebulizer to a teeconnector made of 0.65-cm PVC and three pieces of 0.6-cminside
38、 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 any unsuspended droplets produced by thenebulizer.9.1.6 Attach the second end of the tee to a 1.3-cm insidediame
39、ter 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 nebulizer. Connectthe loose end of the tubing to the third end of the tee.9.1.8 Connect the nebulizer inlet port w
40、ith 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 flowmeter 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, intu
41、rn, leads to an air filter and to a vacuum source.9.2 Filter Unit-Holder Preparation:9.2.1 Wrap the non-sterile sterilizable filter unit in a steril-izable wrap.9.2.2 Sterilize the filter units as specified by the manufac-turer. Presterilized filter units do not need to be resterilized.F1608 16310.
42、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 runsshould be conducted. The following description outlines thevalidation of the test procedure for a chal
43、lenge of 1 106colony forming units (CFU) per port in 15 min at a flow rate of2.8 L/min. If testing is to be conducted using other parameters,a validation should be conducted using those parameters.10.1.1 Place the sterile filtering apparatus in a biologicalsafety cabinet.10.1.2 Aseptically prepare s
44、ix filter units by placing a sterile0.45-m membrane filter on the base of each filter unit usingsterile forceps and gloves (Fig. 2).10.1.3 Attach the top of each filter unit to the bottom of theexposure chamber. Then attach each filter unit to its respectiveflowmeter.10.1.4 Dispense 3.0 mL of the sp
45、ore suspension into thenebulizer. When using the DeVilbiss #40 nebulizer, a volumeof 3.0 mL at a concentration of 5 107spores/mL is necessaryto achieve a challenge of 1 106CFU (60.5 log) per port in 15min.10.1.5 Turn on the chamber fan.10.1.6 Adjust port flowmeters to 2.8 L/min. It is importantthat
46、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.10.1.7 Adjust the nebulizer flow rate as recommended bythe nebulizer manufacturer to pr
47、oduce droplets that are withinthe appropriate particle size range. When using the DeVilbiss#40 nebulizer, a flow rate of 8.5 L/min is used.10.1.8 Immediately start the 15-min timer. At regularintervals, observe and adjust (if necessary) all flowmeters tomaintain the appropriate flow rate settings du
48、ring the 15-mintest period.10.1.9 After exposure, turn off the vacuum, the fan, and theair flow to the nebulizer. Open the filtered exhaust port at theback of the chamber.10.1.10 Evacuate the chamber for 15 min by connecting thevacuum source to the front of the chamber through a microbialfilter asse
49、mbly.10.1.11 Disconnect the hoses from each of the filter unitsand remove the units from the bottom plate of the exposurechamber.10.1.12 Disinfect the outside of each filtering unit with asuitable sporicide being careful not to compromise the testmaterial.10.1.13 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 a suitable blender that has been validated containing 100.0mL of steril
