ASTM E1052-2011 7500 Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension《抗悬浮病毒杀菌剂性能评估的标准试验方法》.pdf

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1、Designation: E1052 11Standard Test Method toAssess the Activity of Microbicides against Viruses inSuspension1This standard is issued under the fixed designation E1052; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of las

2、t 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 intended to demonstrate the virucidalactivity of test substances with viruses in suspension.1.2 It is

3、 the responsibility of the investigator to 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).1.3 Refer to the appropriate regulatory agency for perfor-mance standa

4、rds of virucidal efficacy.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 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

5、standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use. The user shouldconsult a reference for the laboratory safety recommenda-tions.22. Referenced Documents2.1 ASTM Standards:3E1053 Test Method to Assess Virucidal Acti

6、vity of Chemi-cals Intended for Disinfection of Inanimate, NonporousEnvironmental SurfacesE1482 Test Method for Neutralization of Virucidal Agentsin Virucidal Efficacy EvaluationsE1838 Test Method for Determining the Virus-EliminatingEffectiveness of Hygienic Handwash and Handrub AgentsUsing the Fin

7、gerpads of AdultsE2197 Quantitative Disk Carrier Test Method for Determin-ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,and Sporicidal Activities of ChemicalsE2756 Terminology Relating toAntimicrobial andAntiviralAgents2.2 Federal Standards:421 CFR Code of Federal Regulations (CFR), Food

8、 and DrugAdministration, Part 58, Laboratory Practice for Nonclini-cal Laboratory Studies40 CFR Code of Federal Regulations (CFR), EnvironmentalProtection Agency, Part 160, Good Laboratory PracticeStandard3. Terminology3.1 DefinitionsFor definitions of general terms used inthis test method, refer to

9、 Terminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 hard water, nwater with a standard hardness ascalcium carbonate.3.2.2 neutralization, nthe process for inactivating orquenching the activity of a microbicide, often achieved throughphysical (for example, filtration or diluti

10、on) or chemical means.3.2.2.1 DiscussionThis neutralization may be achievedthrough dilution of the test substance to reduce the microbi-cidal activity, or through the use of chemical agents, calledneutralizers, to eliminate microbicidal activity.3.2.3 soil load, na solution of one or more organic an

11、d/orinorganic substances added to the suspension of the testorganism to simulate the presence of body secretions, excre-tions, or other extraneous substances.3.2.4 test substances or test formulation, na formulationwhich incorporates microbicidal ingredients.4. Summary of Test Method4.1 One part of

12、the virus suspension is added to nine partsof the test substance, the mixture held at the desired tempera-ture for the required contact time and then assayed for viablevirus in an appropriate host system. For control, one part of thevirus is added to nine parts of a buffer harmless to the virus and1

13、This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2011. Published October 2011. Originallyapproved in 1985. Last

14、 previous edition published in 2002 as E1052 96 (2002),which was withdrawn in July 2011 and reinstated in October 2011. DOI:10.1520/E1052-11.2CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories, FifthEdition, U.S. Department of Health and Human Services, Washington, DC, May2009.3For re

15、ferenced 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.4Available from U.S. Government Printing Office, Superintendent of D

16、ocu-ments, Washington, DC 20402.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.its host cells. Cell culture, cytotoxicity, and virus susceptibilitycontrols must also be included in each test.4.2 This test method must be performed by

17、 a trainedmicrobiologist or virologist who is responsible for choosing theappropriate host system for the test virus, and applying thetechniques necessary for propagation and maintenance of hostcell lines and test virus. For a reference text, refer to Schmidtet al.55. Significance and Use5.1 This te

18、st method is to determine if a test substance caninactivate viruses in suspension.5.2 Regulatory agencies may require additional testing us-ing in vitro (Test Methods E1053, E2197)orin vivo (TestMethod E1838) carrier tests for product registration purposes.6. Materials and Reagents6.1 Cell Culture T

19、echnique.56.1.1 Cell Culture System appropriate for test virus.6.1.2 Growth Media/Maintenance Media, Eagles minimalessential medium (EMEM) or equivalent, supplemented withappropriate concentration of serum (inactivated andmycoplasma-free), antibiotics, and other growth factors asneeded. See Note 1.N

20、OTE 1Materials and reagents for cell culture may be purchased frombiological supply houses.6.1.3 Diluent, The media listed in 6.1.2, phosphate bufferedsaline, trypticase soy broth supplemented with serum, EarlesBalanced Salt Solution (EBSS), or other similar bufferedsolutions.6.1.4 Plastic Cell Cult

21、ure Ware. See Note 2.NOTE 2Plastic cell culture ware may be purchased from mostlaboratory supply houses.6.1.5 Incubator,witha5to7%CO2atmosphere, capableof maintaining 36 6 1C or other temperature appropriate forthe specific test virus.6.1.6 Refrigerator,46 2C or other appropriate tempera-ture.6.1.7

22、Test Tubes, screw-capped.6.1.8 Pipettes, serological, 10, 1, 0.5 mL or calibratedpipettors, or both.6.1.9 Microtitration Kit. See Note 3.NOTE 3Microtitration kit may be purchased from most laboratorysupply houses.6.2 Additional or equivalent materials and reagents specificto the host recovery system

23、 may be necessary. The trainedmicrobiologist or virologist is responsible to choose accord-ingly as needed.7. Test Viruses7.1 To demonstrate the spectrum of virucidal activity of thetest substance, it should be tested against viruses with varyinglevels of resistance to microbicides. Appendix X1 list

24、s sug-gested viruses and their host cells.8. Virus Stock8.1 Use an appropriate host to prepare virus suspensions.The host system for titrating virus infectivity may be differentfrom that used for preparing the virus pool.9. Operating Technique9.1 The test must include the parameters given in Table 1

25、.9.2 Please refer to Test Method E2197 for details oncytotoxicity and other controls.9.3 Thoroughly mix virus suspension and then add one partto nine parts of the test substance in a sterile medication tubeheld at the appropriate exposure temperature (usually22 6 2C). Consider this the 101dilution o

26、f the virus.Following the exposure for the time chosen, immediatelyneutralize the microbicidal activity by serial ten-fold dilutionsinto a neutralization solution appropriate for the test substance.NOTE 4Perform the virus control (one part of virus + nine partsEBSS) and cytotoxicity control (one par

27、t EBSS + nine parts testsubstance) concurrently with the virucidal test described above. If dilutionalone is insufficient to reduce cytotoxicity, gel filtration as described inTest Method E1482 may be used.9.4 Virus RecoveryInoculate at least four cell culturemonolayers per dilution of the virus-tes

28、t substance mixturewith 0.1 mL volumes of each test and control dilutionseparately onto monolayers of appropriate host cell cultures.Other volumes may be used depending on the type of cellculture vessel employed; however, no less than four separatemonolayers of the host cells must be inoculated for

29、eachdilution tested. Incubate the cultures at the appropriate tem-perature and observe for evidence of virus replication (e.g.,cytopathic effects, hemagglutination, plaque assay) and/orcytotoxicity9.5 Test Substance Neutralization ControlTo determinethe dilution at which neutralization of the test s

30、ubstance hasoccurred, prepare and inoculate an additional set of cytotoxic-ity controls with the neutralizer added to the test substance.9.6 To validate the neutralization, add equal volumes of theneutralized test substance, the neutralizer alone and a controlfluid (for example, EBSS) a relatively l

31、ow number (forexample, 1000 to 5000) infective units of the test virus andhold the mixtures for 10 to 20 min at room temperature. Titratethe mixtures for infectious virus. Comparable levels of infec-tive units must be recovered from the control, the neutralizeralone as well as the neutralized test s

32、ubstance for the neutral-ization to be considered valid. In case of incomplete neutral-ization, try another neutralizer or use the gel filtration method(Test Method E1482) to reduce cytotoxicity.5Schmidt, N. J., Lennette, D. A., and Lennette, E. T., and Lennette, E. H.,eds.,Diagnostic Procedures for

33、 Viral, Rickettsial and Chlamydial Infections, 7thEdition, Am. Pub. Hlth. Assoc., Washington, DC, 1995 .TABLE 1 ParametersParameter Summary ReplicatesCell culture medium alone 4/groupVirus control 1 part virus + 9 parts medium 4/dilutionVirucidal test 1 part virus + 9 parts test substance 4/dilution

34、Cytotoxicity control 1 part medium + 9 parts testsubstance4/dilutionNeutralization control neutralized test substance + virus 4/dilutionE1052 1129.6.1 Those dilutions that are toxic to the cells or do notexhibit virus replication, or both, are not included in the log10reduction calculations of micro

35、bicidal activity.10. Soil Load and Hard Water10.1 If a soil load is required, add to the virus suspensionbovine serum at a final concentration of 5 % or the tripartitemixture as described in Test Method E2197.10.2 If tests are to be performed in water of a specifichardness, follow the methods listed

36、 in Test Method E2197.11. Calculation of Results11.1 Use an appropriate method to calculate the control andtest samples to determine the level of virus inactivationachieved in relation to the dilution at which cytotoxicity wasobserved.11.2 Report the titer of the stock virus, degree of cytotox-icity

37、, the degree of virus inactivation, and the dilution at whichneutralization occurred.12. Precision and Bias12.1 A precision and bias statement cannot be made for thistest method at this time.13. Keywords13.1 cell cultures; microbicide; suspension test; virucidaltest; virucide; viruses in suspensionA

38、PPENDIX(Nonmandatory Information)X1. VIRUSESX1.1 Representative enveloped and non-enveloped to as-sess the virucidal activity of microbicides in suspension. TheATCC numbers of the viruses and their host cells are given inparenthesis, where available.X1.1.1 Adenovirus, Type 2 (VR-846) or Type 5 (VR-5

39、). Cellline options: Human Lung Carcinoma (A549) CCL-185,HEp-2. CCL-23, Vero CCL-81.X1.1.2 Canine Parvovirus, Cornell-78091680 strain VR-2017. Cell line option: A72 CRL-1542.X1.1.3 Cytomegalovirus, strain AD-169, VR-538. Cellline options: Human diploid lung (MRC-5 CCL-171 orWI-38 CCL-75).X1.1.4 Feli

40、ne calicivirus, strain F-9 VR-782. Cell lineoption: CRFK CCL-94.X1.1.5 Hepatitis A Virus, HM-175 strain VR-2093. Cellline options: FRhK-4 CRL-1688.X1.1.6 Herpes simplex virus, Type 1, strain F (1) VR-733.Cell line options: VERO CCL-81, HEp-2 CCL-23.X1.1.7 Influenza A, A/Hong Kong/8/68 VR-544, A/PR/8

41、/34 VR-95. Cell line options: Madin-Darby Canine kidney(MDCK) CCL-34; Rhesus monkey kidney (LLC-MK2)CCL-7.X1.1.8 Murine Norovirus, Cell line: RAW 264.7 TIB-71.X1.1.9 Respiratory syncytial virus, Long strain VR-26.Cell line options: HEp-2 CCL-23, MRC-5 CCL-171.X1.1.10 Rhinovirus, Type 14 VR-284 or 37

42、 VR-1147.Cell line options: MRC-5 CCL-171, WI-38 CCL-75, HeLaT4+.X1.1.11 Rotavirus, Wa strain VR-2018. Cell line options:MA-104 CRL-2378.1 or African green monkey kidney(CV-1) CCL-70.X1.1.12 Vaccinia, WR strain, VR-119. Cell line options:VERO CCL-81, HEp-2 CCL-23.NOTE X1.1Rhinoviruses grow optimally

43、 at 33 6 1C.NOTE X1.2Fetal bovine serum may be inhibitory to rotavirus and itmay also neutralize the trypsin often needed for rotavirus and influenza-virus growth.ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this s

44、tandard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be re

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46、ible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you shouldmake your views known to the ASTM Committee on Standards, at the address shown below.This standard is copyrighted by ASTM International, 100 Barr Harbor Drive, PO Box C700, West

47、Conshohocken, PA 19428-2959,United States. Individual reprints (single or multiple copies) of this standard may be obtained by contacting ASTM at the aboveaddress or at 610-832-9585 (phone), 610-832-9555 (fax), or serviceastm.org (e-mail); or through the ASTM website(www.astm.org). Permission rights to photocopy the standard may also be secured from the ASTM website (www.astm.org/COPYRIGHT/).E1052 113