1、Designation: E1053 11Standard Test Method toAssess Virucidal Activity of Chemicals Intended forDisinfection of Inanimate, Nonporous EnvironmentalSurfaces1This standard is issued under the fixed designation E1053; the number immediately following the designation indicates the year oforiginal adoption
2、 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.1. Scope1.1 This test method is used to evaluate the virucidalefficacy of liquid, aeroso
3、l, or trigger-spray microbicides in-tended for use on inanimate, nonporous environmental sur-faces. This test method may be employed with most viruses,which can be grown in cultured cells.2However, other hostsystems (for example, embryonic eggs) may be used withproper justification and documentation
4、.1.2 This test method should be performed only by thosetrained in microbiological and virological techniques in facili-ties designed and equipped for work with infectious agents atthe appropriate biosafety level.1.3 It is the responsibility of the investigator to determinewhether Good Laboratory Pra
5、ctice regulations (GLPs) arerequired and to follow them where appropriate (40 CFR, Part160 for EPA submissions and 21 CFR, Part 58 for FDAsubmissions). Refer to the appropriate regulatory agency forperformance standards of virucidal efficacy.1.4 This standard does not purport to address all of thesa
6、fety 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. The user shouldconsult a reference for laboratory safety recommendations
7、.22. Referenced Documents2.1 ASTM Standards:3D1129 Terminology Relating to WaterE1153 Test Method for Efficacy of Sanitizers Recom-mended for Inanimate Non-Food Contact SurfacesE1482 Test Method for Neutralization of Virucidal Agentsin Virucidal Efficacy EvaluationsE2197 Standard Quantitative Disk C
8、arrier Test Method forDetermining Bactericidal, Virucidal, Fungicidal, Mycobac-tericidal, and Sporicidal Activities of Chemicals2.2 Federal Standards:4Title 21, Code of Federal Regulations (CFR), Food andDrug Administration, Part 58, Laboratory Practice forNonclinical Laboratory StudiesTitle 40, Cod
9、e of Federal Regulations (CFR), Environmen-tal Protection Agency, Subchapter E, Pesticide Programs;Part 160, Good Laboratory Practice Standards3. Terminology3.1 DefinitionsFor definitions of general terms used inthis test method, refer to Terminology D1129.3.2 Definitions of Terms Specific to This S
10、tandard:3.2.1 hard water, nwater with a standard hardness ascalcium carbonate.3.2.2 neutralization, na process which results in quench-ing the microbicidal activity of a test substance. This may beachieved through dilution of the test substance to reduce themicrobicidal activity, or through the use
11、of chemical agents,called neutralizers, to eliminate microbicidal activity.3.2.3 soil load, na solution of one or more organic and/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 sub
12、stance or test formulation, na formulationwhich incorporates microbicidal ingredients.4. Summary of Test Method4.1 The virus suspension is dried on an inanimate, nonpo-rous surface. The test substance is added over the dried film atits use-dilution or sprayed from an aerosol can or trigger-sprayer f
13、ollowing the manufacturers directions. Control carri-ers receive an equivalent volume of a buffer harmless to the test1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Methods and is the directresponsibility of Subcommittee E35.15
14、 on Antimicrobial Agents.Current edition approved Jan. 1, 2011. Published March 2011. Originallyapproved in 1985. Last previous edition approved in 2002 as E1053 97 (2002).DOI: 10.1520/E1053-11.2Public Health Service, Centers for Disease Control and Prevention, andNational Institutes of Health, Bios
15、afety in Microbiological and BiomedicalLaboratories, U.S. Department of Health and Human Services, Washington, DC,December 2009, 422 pp.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume in
16、formation, refer to the standards Document Summary page onthe ASTM website.4Available from U.S. Government Printing Office Superintendent of Documents,732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http:/www.access.gpo.gov.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C
17、700, West Conshohocken, PA 19428-2959, United States.virus and its host cells. After exposure at the appropriatetemperature (usually 22 6 2C) for the recommended time, theeluates from control and test carriers are assayed for infectivity.4.2 This test method is designed to be performed by aperson tr
18、ained in culturing and assaying infectious viruses whois responsible for choosing the appropriate host system for thetest virus and applying the techniques necessary for propaga-tion and maintenance of the host system and test virus. For areference text, refer to Lennette et al.55. Significance and
19、Use5.1 This test method may be used to determine the effec-tiveness of liquid, aerosols/foams, and trigger-spray productsagainst designated prototype viruses.5.2 The number of lots of the test substance and the numberof replicates in each test will depend on the requirements of thetarget regulatory
20、agency.5.3 Certain regulatory agencies may require additional test-ing using other carrier tests for product registration purposes.6. Materials and Reagents6.1 Host System and Assay of Infectious VirusSee Note 2.6.1.1 Cell Cultures, appropriate for test virus.6.1.2 Growth and Maintenance Media, any
21、growth andmaintenance media suitable for work with the virus and its hostcells.NOTE 1Materials and reagents for cell culture may be purchased frombiological supply houses.6.1.3 Diluent, Earles Balanced Salt Solution (EBSS) orother appropriate media.6.1.4 Plastic Cell Culture Ware.NOTE 2Plastic cell
22、culture ware may be purchased from mostlaboratory supply houses.6.1.5 Incubator, capable of maintaining 36 6 1C or othertemperature appropriate for replication of the specific testvirus; an incubator with 5 to 7 % CO2will be needed if an opensystem is being used for cell culture and virus assay.6.1.
23、6 Refrigerator,46 2C.6.1.7 Test Tubes, screw-capped.6.1.8 Pipettes, serological, 10, 1, and 0.5 mL.6.1.9 Micropipettors and Tips, (96-well assay only).6.1.10 96-Well Dilution Plates, (96-well assay only).6.1.11 Microtitration Kit.NOTE 3Microtitration kits may be purchased from most laboratorysupply
24、houses.6.1.12 Petri Plates, glass, 100-mm diameter, 1-cm deep.7. Test Viruses7.1 Appendix X1 lists viruses and their respective host cellsas examples for use in this test method.7.2 To demonstrate that the test substance has broad viru-cidal activity, it should be shown to be effective against at le
25、astone non-enveloped virus.8. Virus Stock8.1 The titer of the test virus suspension must be sufficientlyhigh so that at least 104infective units/carrier can be recoveredfrom the inoculated carriers after the inoculum has dried. Thehost system employed for virus propagation need not be thesame as tha
26、t used for virus recovery and the infectivity assay.9. Operating Technique9.1 Test Substance DiluentFor test substances requiringdilution in water to obtain a use-dilution, water with astandardized and specified level of hardness, such as 400 ppmas CaCO3, shall be used as the diluent.69.2 Cytotoxici
27、ty ControlThe objective of this control is to(1) determine the dilution of the test substance post-neutralization at which it causes no apparent degeneration(cytotoxicity) of the cell line to be used for measuring virusinfectivity and (2) assess if the neutralizer in any way reducesor enhances such
28、cytotoxicity. Make an initial 1:2 dilution ofthe use-dilution of the test substance in the neutralizer andthree further ten-fold dilutions of the neutralized test substancein the diluent. Remove the culture medium from the monolay-ers of the host cell line(s) and put into each test monolayerseparate
29、ly the same volume of inoculum as used in virustitration; control monolayers receive an equivalent amount ofdiluent (without any neutralizer) only. Another set of mono-layers should be exposed to the neutralizer alone. Hold thecultures for the same period of time as used in virus titrationand examin
30、e them under an inverted microscope for anycytotoxicity. In case of cytotoxicity, a different neutralizer orgel filtration (see Test Method E1482) of the neutralizedvirus-test substance mixture may be needed.NOTE 4If gel filtration is used in the virucidal activity test runs, theneutralized and gel-
31、filtered test substance should be evaluated for thecytotoxicity control.9.3 Test Substance Neutralization ControlTo determinethe dilution at which neutralization of the test substance hasoccurred, prepare and inoculate an additional set of cytotoxic-ity controls with the neutralizer added to the tes
32、t substance. Tovalidate the neutralization, add equal volumes of the neutral-ized test substance, the neutralizer alone and a control fluid (forexample, PBS) a relatively low number (for example, 1000 to5000) infective units of the test virus and hold the mixtures for10 to 20 min at room temperature
33、. Titrate the mixtures forinfectious virus. Comparable levels of infective units must berecovered from the control, the neutralizer alone, and theneutralized test substance for the neutralization to be success-ful. In case of incomplete neutralization, either another neu-tralizer may be needed or th
34、e gel filtration method (TestMethod E1482) may be used.9.4 Plate Recovery ControlVortex the virus suspensionthoroughly and place 0.2-mL on the inside bottom surface ofeach glass petri dish. Allow the virus inoculum to dry underambient conditions in a laminar flow hood or other suitablechamber with t
35、he petri dish cover removed. The drying time of5Schmidt, N. J., , Lennette, D. A., Lennette, E. T., and Lennette, E. H. eds.,Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, SeventhEdition, American Public Health Association, Washington, DC, 1995.6AOAC International, Offcial M
36、ethods of Analysis of the AOAC, Arlington,VA, 1990.E1053 112this control should be consistent with that for the test runs. Arecovery of at least 104infective units/control dish should beachieved for the test to be considered valid. Pools of certaintypes of viruses may require concentration by ultra-
37、centrifugation to obtain titers high enough to give a minimumof 104infective units/dish after the inoculum has been dried.However, any such concentrated virus must be vortexed well toreduce the presence of viral aggregates.NOTE 5The volume of virus inoculum per carrier may be increaseddepending on t
38、he titer of the virus. This volume must be consistentbetween the plate recovery control and test substance runs. It should benoted, however, that an increased volume will prolong the drying of theinoculum and may lead to increased losses in virus infectivity.9.4.1 After drying, overlay each dried co
39、ntrol film with 2.0mL of PBS or another buffered solution harmless to the virusand its host cells. Let stand for the same contact time as usedfor the test carriers and then add an equal amount of neutralizer(2.0 mL). Scrape the inside bottom surface with a sterile cellscraper to resuspend the viral
40、film. This suspension may beconsidered the 101dilution of the virus. Prepare serial 10-folddilutions using diluent and inoculate an amount appropriate tothe test format to no less than four replicate cell monolayers/dilution.9.5 Test for Virucidal ActivityFor each lot of the testsubstance, treat a d
41、ried film carrier with 2.0 mL of the use-dilution of a liquid product or the amount of product releasedduring recommended use of the aerosol or trigger spray. Holdfor the required contact time. Upon completion of the contacttime, immediately add an equal volume of neutralizer (2.0 mL)to the carrier
42、and mix well. Scrape the film to resuspend thevirus/test substance/neutralizer mixture. This suspension maybe considered the 101dilution of the virus. Prepare serial10-fold dilutions using diluent and inoculate an amount appro-priate to the test format to no less than four replicate cellmonolayers/d
43、ilution starting from 102.10. Soil Load (refer to Test Method E2197)10.1 The soil load to be incorporated in the suspension ofthe test organism may consist of a mixture of the followingstock solutions in phosphate buffer (pH 7.2):10.2 Add 0.5 g of tryptone or yeast extract to 10 mL ofphosphate buffe
44、r.10.3 Add 0.5 g of BSA to 10 mL of phosphate buffer.10.4 Add 0.04 g of bovine mucin to 10 mL of phosphatebuffer.10.5 Prepare the solutions separately and sterilize by pas-sage through a 0.22-m pore diameter membrane filter, aliquot,and store at either 4 6 2C or 20 6 2C; such solutions canbe stored
45、in the refrigerated for about three months and frozenfor at least one year.10.6 To obtain 500 L of the inoculum, add 25 L of BSA,100 L of mucin, and 35 L of tryptone or yeast extract stockto 340 L of the virus suspension.NOTE 6Other types of soil load such as animal sera may be useddepending on the
46、target regulatory agency and recommended use of thetest substance. The soil load mixture given above contains a level ofprotein roughly equal to that in 5 % serum. Preliminary screening ofalbumin and mucin is recommended to ensure compatibility with testorganism(s).10.7 If the use-dilution of the te
47、st substance is to beprepared in water, follow the methods listed in Test MethodE1153 for making the hard water to be used. The test reportmust clearly specify the level of hardness used in testing forvirucidal activity.11. Additional Controls11.1 Cell Culture ControlTo ensure that the host cells ar
48、enot contaminated with bacteria, fungi, or any cytopathogenicviruses other than those used in the test and to confirm theviability of the cells during the incubation period of the assay,at least four host cell monolayers are left untreated in each testand examined first at the end of the incubation
49、period. Anyobvious contamination or degeneration in such monolayerswould invalidate the virus titration.11.2 Virus Stock Titer ControlAn aliquot of the stockvirus used in the test is serially diluted in diluent and inoculatedonto the host culture. This is to confirm that the host cells aresusceptible to the virus and that the viral infection assay isperformed appropriately. This control will also confirm that thetiter of the stock virus is appropriate for use in the test. A lackof obvious and typical virus-induced cytopathic effects in thelower
