1、Designation: E 1053 97 (Reapproved 2002)Standard Test Method forEfficacy of Virucidal Agents Intended for InanimateEnvironmental Surfaces1This standard is issued under the fixed designation E 1053; the number immediately following the designation indicates the year oforiginal adoption or, in the cas
2、e of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. Asuperscript epsilon (e) indicates an editorial change since the last revision or reapproval.1. Scope1.1 This laboratory test method is used to evaluate thevirucidal efficacy of liquid, aerosol,
3、or trigger spray antimicro-bial solutions on inanimate nonporous environmental surfaces.This test method may be employed with most viruses and isdesigned for cell culture host systems.1.2 This test method should be performed only by thosetrained in microbiological or virological techniques.1.3 This
4、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 and health practices and determine the applica-bility of regulatory limitations prior to use. The user shouldconsult
5、a reference for the laboratory safety recommenda-tions.21.4 It is 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). Refe
6、r to the appropriate regulatory agency forperformance standards of virucidal efficacy.2. Referenced Documents2.1 ASTM Standards:3E 1052 Test Method for Efficacy of Virucidal AgentsAgainst Viruses in SuspensionsE 1153 Test Method for Efficacy of Sanitizers Recom-mended for Inanimate Non-Food Contact
7、SurfacesE 1482 Test Method for Neutralization of Virucidal Agentsin Virucidal Efficacy Evaluations2.2 Federal Standards:Title 40, Code of Federal Regulations (CFR), Environmen-tal Protection Agency, Subchapter E, Pesticide Programs;Part 160, Good Laboratory Practice Standards4Title 21, Code of Feder
8、al Regulations (CFR), Food andDrug Administration, Part 58, Laboratory Practice forNonclinical Laboratory Studies43. Summary of Test Method3.1 The virus suspension is dried on an inanimate, nonpo-rous surface. The antimicrobial is added over the dried film asa use dilution solution or sprayed from a
9、n aerosol can or triggerspray container following the recommended directions. Afterexposure at the appropriate temperature (usually 22 6 2C) forthe recommended time, the virus-antimicrobial mixture isassayed in a host system appropriate for the test virus. Thevirus titer of an untreated surface is d
10、etermined by the medianinfective dose (ID50) method of virus titration. Cytotoxicity tothe host system of the antimicrobial at the tested concentrationis determined by an LD50method. The virus-antimicrobialmixture is assayed in numerous units of the host system at adilution just beyond the cytotoxic
11、ity range of the antimicrobial.The extent of virus inactivation by the antimicrobial is deter-mined. Results are recorded as log10-virus inactivated.3.2 This test method is designed to be performed by atrained virologist who is responsible for choosing the appro-priate host system for the test virus
12、 and applying the techniquesnecessary for propagation and maintenance of host and testvirus. For a reference text, refer to Schmidt and Emmons.54. Significance and Use4.1 This test method may be used to determine the effec-tiveness of liquid and aerosol antimicrobial products againstdesignated proto
13、type viruses.4.2 The effective antimicrobial concentration should bedetermined utilizing cell cultures as the host system for specificviruses.4.3 This test method is applicable for testing of liquid andpressurized antimicrobial products against viruses on inani-mate nonporous environmental surfaces.
14、1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and is the direct responsibility of Subcommittee E35.15 onAntimicrobialAgents.Current edition approved April 10, 1997. Published June 1997. Originallypublished as E 1053 85. Last previous edition E 1053 91.2CDC-NIH, Biosa
15、fety in Microbiological and Biomedical Laboratories, 3rd ed.,U.S. Department of Health and Human Services, Washington, DC, May 1993.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume inform
16、ation, refer to the standards Document Summary page onthe ASTM website.4Available from U.S. Government Printing Office, Superintendent of Docu-ments, Washington, DC 20402.5Diagnostic Procedures for Viral and Rickettsial Infections, N. J. Schmidt and R.W. Emmons, eds., 6th ed., American Public Health
17、 Association, Washington, DC,1989.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.5. Materials and Reagents5.1 Cell Culture Technique:55.1.1 Cell Culture System, appropriate for test virus.5.1.2 Growth Media/Maintenance Media, Medium
18、 199, Ea-gles minimal essential medium (EMEM) or equivalent,supplemented with appropriate concentration of serum (inac-tivated and mycoplasma-free), antibiotics, and other growthfactors as needed.65.1.3 Diluent, the media listed in 5.1.2, phosphate bufferedsaline, trypticase soy broth supplemented w
19、ith serum or othersimilar buffered solutions.5.1.4 Plastic Cell Culture Ware.75.1.5 Incubator, capable of maintaining 37 6 1C or othertemperature appropriate for replication of the specific testvirus.5.1.6 Refrigerator,46 2C.5.1.7 Test Tubes, screw-capped.5.1.8 Pipettes, serological, 10, 1, and 0.5
20、mL.5.1.9 Microtitration Kit.85.1.10 Petri Plates, glass, 60-mm diameter, 1 cm deep.5.2 Additional or equivalent materials and reagents specificto the host recovery system may be necessary. The trainedmicrobiologist or virologist is responsible to choose accord-ingly as needed.6. Test Viruses6.1 To d
21、etermine virucidal efficacy, a prototype strain froma particular virus family must be tested. Because new strains ofviruses are being discovered continuously and methods ofisolation and growth are being improved, the following proto-types and the cell cultures in which to grow and test them aresugge
22、sted. Other strains within a family may be substituted astesting prototypes for specific marketing claims.6.2 To demonstrate the range of antiviral activity of anantimicrobial, the formulation should be tested against virusesrepresenting a range of resistances to germicides. A possiblegroup of virus
23、es includes a poliovirus (representative of thoseviruses most resistant to chemical germicides), a herpes virus(representative of those most easily inactivated), and an aden-ovirus (representative of intermediate resistance to germi-cides). The following is a list of suggested virus strains thattypi
24、cally are assayed, as well as cell cultures that support theirgrowth.6.3 Typical test virus strains and cell cultures.6.3.1 Poliovirus, Type 1, Chat strain, ATCC VR-192.Cell line options: Monkey Kidney Cells (VERO): HumanEpidermoid.Carcinoma, Larynx (HEp-2):African Green Monkey Kid-ney (CV-1).6.3.2
25、Hepatitis A Virus, HM-175 strain, ATCC VR-2093.Cell line options: Fetal Kidney, Rhesus Monkey, Continu-ous (FRhK-4).6.3.3 Herpes Simplex, Type 1, Strain F (1), ATCC VR-733.Cell line options: VERO, HEp-2.6.3.4 Cytomegalovirus, strain AD-169, ATCC VR-538.Cell line options: Human Diploid Lung (MRC-5 or
26、WI-38).6.3.5 Adenovirus, Type 2, Adenoid 6 strain, ATCC VR-2.Cell line options: Human Lung Carcinoma (A549),HEp-2.6.3.6 Influenza A2, Hong Kong Strain, ATCC VR-544.Cell line options: Canine Kidney (MDCK); Rhesus Mon-key Cells, Continuous (LLC-MK2).6.3.7 Respiratory Syncytial Virus, Long strain, ATCC
27、 VR-26.Cell line options: HEp-2, MRC-5.6.3.8 Vaccinia, WR strain, ATCC VR-119.Cell line options: VERO, HEp-2.6.3.9 Rhinovirus, Type 37, Strain 151-1, ATCC VR-1147.Cell line options: MRC-5, WI-38.NOTE 1Rhinovirus-infected cultures require incubation at 33 6 1C.6.3.10 Rotavirus, Wa strain, ATCC VR-201
28、8.Cell line options: Rhesus Monkey Kidney, Continuous(MA-104) orAfrican Green Monkey Kidney, Continuous (CV-1).NOTE 2Some lots of fetal calf serum may be inhibitory to rotavirus.6.4 Other Viral GroupsVirucidal efficacy against certaintypes of viruses such as Human Immunodeficiency Virus mustbe subst
29、antiated in a laboratory having Biosafety Level 3Facilities.27. Virus Stock7.1 The titer of the test virus suspension must be sufficientlyhigh so that at least 104infective units may be recovered fromthe dried films as described as follows. Utilize an appropriatehost to prepare virus suspensions wit
30、h minimum infectivitytiters of about 107to 108infective units/mL. The host systememployed for the virus pool need not be the same system usedfor virus recovery following virus challenge of the antimicro-bial. The virus titer of the stock virus is determined by theCCID50, plaque assay or other quanti
31、fiable measure of infec-tivity.8. Operating Technique8.1 The test must include the following parameters.Parameter Summary ReplicatesCell Culture Control Medium alone 4 per groupVirus Control Virus surface + 2 mLmedium4 per dilutionVirucidal Test Virus surface + 2 mLantimicrobial10 per dilutionCytoto
32、xicity Control 2 mL antimicrobial +medium4 per dilutionNeutralization Control Neutralizedantimicrobial + virus4 per dilution8.2 Cytotoxicity ControlPrior to studying the effects ofthe antimicrobial on viruses, determine its cytotoxic effect onthe host system by an LD50determination. Use serial 2-fol
33、ddilutions and inoculate 4 units/dilution. At least two replicatedeterminations are performed as cytotoxicity controls.6Materials and reagents for tissue culture may be purchased from biologicalsupply houses.7Plastic tissue culture ware may be purchased from most laboratory supplyhouses.8Microtitrat
34、ion kit may be purchased from most laboratory supply houses.E 1053 97 (2002)28.3 Germicide Neutralization ControlIf excessive cyto-toxicity cannot be eliminated by dilution of the virus/germicidemixture, follow Test Method E 1482. Use serum or otherchemicals to neutralize the cytotoxic effect of the
35、 germicide.Todetermine the dilution at which neutralization of the germicidehas occurred, prepare and inoculate an additional set ofcytotoxicity controls with the neutralizer added to the diluent.Following inoculation of cell cultures, add 0.1 mL (or volumeinoculated previously) of the diluted stock
36、 virus at approxi-mately 100 to 1000 infectious units to each dilution. Thosedilutions that are toxic to the cells or do not exhibit virusreplication, or both, are not included in the log10reductioncalculations of the germicidal activity.8.4 Virus ControlAfter cytotoxicity is known, performtest agai
37、nst viruses. Shake virus suspension thoroughly andplace 0.2-mL amounts on bottoms of four 60-mm petri plates.Allow the virus films to dry for 30 6 5 min at 37 6 2C or 606 5 min at 22 6 2C. Do not use a humidified CO2incubatorfor drying. A recovery of at least 104infectious units/surfaceshould be ach
38、ieved. Use two films as the virus controls; add 2.0mL PBS or other suitable neutralizing diluent over film, letstand for the same contact time as for the experimentals andthen scrape surface with sterile rubber policeman to resuspendfilm.This solution is the 101dilution of the virus. Immediatelyprep
39、are serial 10-fold dilutions and inoculate 0.1-mL amountsinto 4 host system units/each dilution.8.5 Virucidal TestEach of the remaining films is treatedwith the antimicrobial, 2.0 mL of the use dilution of a liquidproduct or the amount of product released during recom-mended use of the aerosol or tr
40、igger spray container. After theappropriate time contact at 22 6 2C (usual room temperaturerange), add sufficient PBS or neutralizing diluent to each plateto dilute beyond the cytotoxicity of the antimicrobial; then,scrape surface with sterile rubber policeman to resuspend film.Inoculate 0.1 mL of e
41、ach virus-antimicrobial dilution onto tentissue culture test tubes (macro), or 0.05 mL onto 10 mono-layers in microculture (if used). Allow the virus to adsorb onehour at 37C and then add 0.9-mL (macro) or 0.10-mL (micro)maintenance media/monolayer. Incubate at 37C or otherappropriate temperature an
42、d observe cell sheet for evidence ofvirus-specific cytopathic effect.9. Organic Soil or Hard Water9.1 To simulate an organic soil load, if required in testing,add calf serum or other serum to virus suspensions. The serumshould be tested for absence of antiviral inhibitors, see 6.3.10.Pancreatic dige
43、st of casein may be used as an alternative toserum; 7.6 g of tryptone/L of physiological saline (0.85 %)contains 2.0 g of total protein, which is approximately equiva-lent to the protein content of a 5 % solution of serum.9.2 If tests are to be performed with hard water as a diluentof liquid disinfe
44、ctants, follow the methods listed in TestMethod E 1153.10. Calculation10.1 Use the method of Reed and Muench or Spearman-Karber2to calculate the control virus titer and cytotoxicity.10.2 Report the titer of the stock virus (virus control),degree of cytotoxicity (cytotoxicity control), the degree ofv
45、irus inactivation (results of virucide test), and the dilution atwhich neutralization occurred (neutralization control).11. Precision and Bias11.1 A precision and bias statement cannot be made for thistest method at this time.12. Keywords12.1 carrier test; cell cultures; disinfectant; fomites; germi
46、-cide; infection control; surface test; virucidal test; virus;viruses on dried environmental surfacesASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that det
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48、r withdrawn. Your comments are invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of theresponsible technical committee, which you may attend. If you feel th
49、at 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 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).E 1053 97 (2002)3
