ASTM E2197-2017e1 Standard Quantitative Disk Carrier Test Method for Determining Bactericidal Virucidal Fungicidal Mycobactericidal and Sporicidal Activities of Chemicals《测定化学药品杀菌、.pdf

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1、Designation: E2197 171Standard Quantitative Disk Carrier Test Method forDetermining Bactericidal, Virucidal, Fungicidal,Mycobactericidal, and Sporicidal Activities of Chemicals1This standard is issued under the fixed designation E2197; the number immediately following the designation indicates the y

2、ear oforiginal adoption or, in the case of 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.1NOTESections 8.1 and 11.8 were editorially corrected in January

3、2018.INTRODUCTIONThe quantitative test method described here uses disks of stainless steel (1 cm in diameter) ascarriers. It employs the same basic set of materials and procedures to assess the ability of liquidchemicals to inactivate vegetative bacteria, viruses, fungi, mycobacteria, and bacterial

4、spores (1-7).2Performance standards for test substances, the level of water hardness, the type and level of a soil load,the test organism(s), and other test conditions may vary depending on the target regulatory agency.This basic test can also be adapted for use with other carrier materials of simil

5、ar dimensions.The development of this test method was made possible with financial support from theAntimicrobials Division of the U.S. Environmental Protection Agency.1. Scope1.1 This test method is designed to evaluate the ability oftest substances to inactivate vegetative bacteria, viruses, fungi,

6、mycobacteria, and bacterial spores (1-7) on disk carriers ofbrushed stainless steel that represent hard, nonporous environ-mental surfaces and medical devices. It is also designed to havesurvivors that can be compared to the mean of no less thanthree control carriers to determine if the performance

7、standardhas been met. For proper statistical evaluation of the results,the number of viable organisms in the test inoculum should besufficiently high to take into account both the performancestandard and the experimental variations in the results.1.2 The test protocol does not include any wiping or

8、rubbingaction. It is, therefore, not designed for testing wipes.1.3 This test method should be performed by persons withtraining in microbiology in facilities designed and equipped forwork with infectious agents at the appropriate biosafety level(8).1.4 It is the responsibility of the investigator t

9、o determinewhether Good Laboratory Practice Regulations (GLPs) arerequired and to follow them where appropriate (40 CFR, Part160 for EPA submissions and 21 CFR, Part 58 for FDAsubmissions).1.5 In this test method, SI units are used for all applications,except for distance in which case inches are us

10、ed and metricunits follow.1.6 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, health, and environmental practices and deter-mine the applicability of regulator

11、y limitations prior to use.1.7 This international standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organi

12、zation TechnicalBarriers to Trade (TBT) Committee.2. Referenced Documents2.1 ASTM Standards:3A967/A967M Specification for Chemical Passivation Treat-ments for Stainless Steel PartsD1129 Terminology Relating to WaterD1193 Specification for Reagent Water1This test method is under the jurisdiction of A

13、STM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Dec. 1, 2017. Published December 2017. Originallyapproved in 2002. Last previous edition approved in 2011 as E2197 11

14、. DOI:10.1520/E2197-17E01.2The boldface numbers in parenthesis refer to the list of references at the end ofthis standard.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, ref

15、er 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 StatesThis international standard was developed in accordance with internationally recognized principles on standardization establis

16、hed in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1E2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 CFR Standard:421 CFR, Part 58 Laboratory

17、Practice for Nonclinical Labo-ratory Studies40 CFR, Part 160 Good Laboratory Practice Standards2.3 CEN Standard:5EN 10088-2 1J/2J Stainless steels - Part 2: Technical deliv-ery conditions for sheet/plate and strip of corrosionresisting steels for general purposes3. Terminology3.1 DefinitionsFor defi

18、nitions of general terms used in thistest method, refer to Terminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 carrier, nan inanimate surface or object inoculatedwith the test organism.3.2.2 eluate, nan eluent, which contains the recoveredorganism(s).3.2.3 eluent, nany solutio

19、n that is harmless to the testorganism(s) and that is added to a carrier to recover theorganism(s) in or on it.3.2.4 neutralization, na process to quench the antimicro-bial activity of a test substance. This process may be achievedby dilution of the organism/test substance mixture and/or byadding to

20、 it one or more chemical neutralizers.3.2.5 soil load, na solution of one or more organic, orinorganic substances, or both, added to the suspension of thetest organism to simulate the presence of body secretions,excretions, or other extraneous substances.3.2.6 test organism, nan organism that has ch

21、aracteristicsthat allow it to be readily identified. It also may be referred toas a surrogate, a simulant, or a marker organism.3.2.7 test substance, na formulation that incorporatesantimicrobial ingredients.4. Summary of Test Method4.1 Each disk (1 cm in diameter) receives 10 L of the testorganism

22、with a soil load. The inoculum is dried, and then thedisk is placed on the inside bottom surface of a sterile plasticvial prior to contact with 50 L of the use-dilution of testsubstance. The contact time and temperature may vary asrequired. Control carriers receive 50 L of a fluid harmless tothe tes

23、t organism(s) and its host cells, if any, but are otherwisetreated in the same way as test carriers.4.2 For tests against vegetative bacteria, fungi,mycobacteria, and bacterial spores, the test substance is thenneutralized and the inoculum eluted.The eluate and subsequentrinses of the carrier and it

24、s vial are membrane filtered. Cultureplates with the filters are incubated, colonies counted, and log10reductions calculated.4.3 For tests with viruses, appropriate dilutions of the eluateare inoculated into suitable cell cultures, the cultures areexamined for cytopathology/infectious foci, which ar

25、e esti-mated as the most probable number (MPN) or counted as focior plaques, and log10are calculated.5. Significance and Use5.1 The design of this test eliminates any loss of viableorganisms through wash off, thus making it possible to producestatistically valid data using many fewer test carriers t

26、hanneeded for methods based on simple MPN estimates.5.2 The stringency in the test is provided by the use of a soilload, the microtopography of the brushed stainless steel carriersurface, and the smaller ratio of test substance to surface areatypical for many disinfectant applications. Thus, the tes

27、tsubstance being assessed is presented with a reasonable chal-lenge while allowing for efficient recovery of the test organ-isms from the inoculated carriers. The metal disks in the basictest are also compatible with a wide variety of actives.5.3 The design of the carriers makes it possible to place

28、 ontoeach a precisely measured volume of the test organism (10 L)as well as the control fluid or test substance (50 L).5.4 The inoculum is placed at the center of each diskwhereas the volumes of the test substance covers nearly theentire disk surface, thus virtually eliminating the risk of anyorgani

29、sms remaining unexposed.5.5 In all tests, other than those against viruses, the additionof 10 mL of an eluent/diluent gives a 1:200 dilution of the testsubstance immediately at the end of the contact time. Whilethis step in itself may be sufficient to arrest the microbicidalactivity of most actives,

30、 the test protocol permits the additionof a specific neutralizer to the eluent/diluent, if required.Except for viruses, the membrane filtration step also allowsprocessing of the entire eluate from the test carriers and,therefore, the capture and subsequent detection of even lownumbers of viable orga

31、nisms that may be present. Subsequentrinsing of the membrane filters with saline also reduces the riskof carrying any inhibitory residues over to the recoverymedium. Validation of the process of neutralization of the testsubstance is required by challenge with low numbers of the testorganism.5.6 In

32、tests against viruses, addition of 1 mL of buffer at theend of the contact time achieves a 1:20 dilution of the testsubstance while keeping the volume of the eluate reasonablysmall to allow for the titration of most or all of the eluate in cellcultures. Confirmation of neutralization of the test sub

33、stance isrequired by challenge of a residual disinfection load with lownumbers of infective units of the test virus. Since the virusassay system is indirect, an additional step is required todemonstrate that prior exposure of the appropriate cell line toany residual disinfectant or disinfectant/neut

34、ralizer mixturedoes not interfere with the detection of a low level of viruschallenge (See Appendix).NOTE 1In 5.5 and 5.6, volumes of 10 mL and 1 mL are recommendedinstead of 9.95 mL and 950 L, respectively, for ease of dispensing theeluent.4Available from U.S. Government Printing Office Superintend

35、ent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.5Available from European Committee for Standardization (CEN), AvenueMarnix 17, B-1000, Brussels, Belgium, http:/www.cen.eu.E2197 17125.7 The soil load in this test is a mixture of three types ofpr

36、oteins (high molecular weight proteins, low molecularweight peptides, and mucous material) designed to representbody secretions, excretions, or other extraneous substances thatmicrobicidal chemicals may encounter under field conditions.It is suitable for working with all types of test organismsinclu

37、ded here. The components of the soil load are readilyavailable and subject to much less variability than animal sera.5.8 If distilled water or other diluent is not to be specified onthe product label, the diluent for the test substance is assumedto be tap water. Since the quality of tap water varies

38、 consid-erably both geographically and temporally, this test methodincorporates the use of water with a specified and documentedlevel of hardness to prepare use-dilutions of test substance thatrequire dilution in water before use. While water with ahardness of at least 300 ppm as CaCO3is recommended

39、consult local regulations regarding use of hard water prior totesting.5.9 The Annex contains a list of those organisms that areoften used in assessing the microbicidal activities of disinfec-tants for use on environmental surfaces or medical devices.Culture conditions for each organism are also incl

40、uded in theAnnex. Depending on the label claim(s) desired and therequirements of the target regulatory agency, one or more ofthe organisms listed may be selected for the testing. Iforganisms other than those listed are to be used (for example,in the dairy or brewing industries), a clear justificatio

41、n must beprovided and details of the culture media and growth condi-tions must be validated and clearly specified in test reports.6. General Equipment and Labware6.1 Air Displacement Pipettes, Eppendorf or equivalent,100 to 1000 L with disposable tips.6.2 Analytical Balance, to weigh chemicals and t

42、o standard-ize inoculum delivery volumes by pipettes.6.3 Cell Culture Flasks and Other Plastic-ware for Viruses,(see Note 2) plastic cell culture flasks of 25- and 75-cm2capacity for culturing cells and for preparing virus pools;12-well or 96-well plastic plates for titrating virus infectivity.NOTE

43、2Plastic culture ware may be purchased from most laboratorysupply houses.6.4 Centrifuge, to allow for the sedimentation of the cells/spores of the test organism(s) for concentration, or washing, orboth.6.5 Colony Counter, for example, Quebec Colony Counter.6.6 Desiccator, recommended size is 25 cm w

44、ide by 20 cmdeep, with an active desiccant for drying the inocula on thecarriers.6.7 Dissecting Microscope, for the screening of the metaldisks for damage to surface topography.6.8 Environmental Chamber or Incubator, to hold the car-riers at the desired test temperature.6.9 Filter Sterilization Syst

45、em for Media and Reagents, amembrane or cartridge filtration system (0.22-m pore diam-eter) is required for sterilizing heat-sensitive solutions.6.10 Forceps, straight or curved, (1) with smooth tips tohandle membrane filters, and (2) to pick up the metal diskcarriers for placement in plastic vials.

46、6.11 Freezers, a freezer at 20 6 2C is required for thestorage of media and additives. A second freezer at 70C orlower is required to store the stocks of test organisms.6.12 Glassware, 1-L flasks with a side-arm and appropriatetubing to capture the filtrates from 47-mm diameter membranefilters; 250-

47、mL Erlenmeyer flasks for culture media.6.13 Hemocytometer, for counting fungal conidia, and/or foruse in the preparation of suitable cell numbers for seedingmonolayers.6.14 Hot Air Oven, an oven at 60C to dry clean and sterileglassware.6.15 Incubators, an ordinary incubator, an anaerobicincubator, a

48、nd a CO2incubator to incubate cell cultures in a5% CO2atmosphere. If only one ordinary incubator isavailable, its temperature will require adjustment depending onthe type of organism under test.6.16 Inverted Microscope, an inverted microscope with 10eyepiece and 5, 10, and 40 objectives to examine c

49、ellcultures.6.17 Laminar Flow Cabinet, a Class II (Type A) biologicalsafety cabinet. The procedures for the proper maintenance anduse of such cabinets are given in Ref (8).6.18 Liquid Nitrogen Storage for Cells, a proper liquidnitrogen container and liquid nitrogen supply for cryopreser-vation of the stocks of cell lines.6.19 Magnetic Stir Plate and Stir Bars, large enough for a5-L beaker or Erlenmeyer flask for preparing culture media orother solutions.6.20 Markers, for permanent marking of labware.6.21 Membrane Filtration System for Capture of the