ASTM E2011-1999 Standard Test Method for Evaluation of Handwashing Formulations for Virus-Eliminating Activity Using the Entire Hand 《评价全部用手工消除病毒活性的人工洗涤方法的标准试验方法》.pdf

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1、Designation: E 2011 99Standard Test Method forEvaluation of Handwashing Formulations for Virus-Eliminating Activity Using the Entire Hand1This standard is issued under the fixed designation E 2011; 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.INTRODUCTIONPhysical removal, inactivation in situ, elimination and a combination of these actions to

3、reduce thespread of viral infections with effective handwashing has become an achievable goal. Artificialcontamination of hands with selected test viruses provides a usable model. This guide is closely relatedto another ASTM test of handwash agents which restricts contamination and sampling to a sma

4、llerarea of the hand, Test Method E 1836, Determining the Virus Eliminating Effectiveness of LiquidHygienic Handwash Agents Using the Fingerpads of Adult Volunteers. This test method tests a largerarea of the hands than the fingerpad method; however, reported results are comparable. (1)21. Scope1.1

5、This test method is designed to evaluate antimicrobialagents in formulations for utility and effectiveness for virus-eliminating activity using human subjects.31.2 This standard may involve hazardous materials, opera-tions and equipment. This standard does not purport to addressall of the safety con

6、cerns, if any, associated with its use. It isthe responsibility of the user of this standard to establishappropriate safety and health practices and determine theapplicability of regulatory limitations prior to use. The usershould consult a reference for laboratory safety recommenda-tions. (2, 3)2.

7、Referenced Documents2.1 ASTM Standards:42.1.1 Use the most current editions of the standards refer-enced herein.E 1052 Test Method for Efficacy of Virucidal AgentsAgainst Viruses in SuspensionE 1053 Test Method for Efficacy of Virucidal Agents In-tended for Inanimate Environmental SurfacesE 1482 Tes

8、t Method for Neutralization of Virucidal Agentsin Virucidal Efficacy EvaluationsE 1836 Test Method for Determining the Virus-EliminatingEffectiveness of Liquid Hygienic Handwash Agents Usingthe Fingerpads of Adult Volunteers2.2 AOAC Standard:5AOAC 960.93. Summary of Test Method3.1 This test method i

9、s conducted on subjects selected froma group of adult volunteers who have provided a writteninformed consent and whose hands have been determined to befree from any apparent damage. All subjects should haverefrained from the use of any antimicrobials for at least oneweek prior to initiation of the t

10、est and be supplied with selectedproducts free from antimicrobials for use during this week. Atleast 12 to 15 subjects are selected from this group for the test.The number of required subjects may vary with the virustested.3.2 Aprepared suspension of the selected test virus is grownand diluted or co

11、ncentrated to produce a titer with a minimumof 108infective units/mL. The contaminating virus is applied tothe hands and the hands are washed with the test formulationaccording to the manufacturers directions or with a set testregimen.3.3 The virus titer recovered after washing with the testproduct

12、is compared to a control titer of virus. For the control,a titer of virus is applied to the hands and recovered fromsubjects washing with standard hard water (200 ppm ascalcium carbonate) or vehicle, or both, instead of the formu-lation.1This test method is under the jurisdiction of ASTM Committee E

13、35 onPesticides and is the direct responsibility of Subcommittee E35.15 on AntimicrobialAgents.Current edition approved March 10, 1999. Published September 1999.2The boldface numbers in parentheses refer to a list of references at the end ofthis test method.3A knowledge of virological techniques is

14、required for this test method.4For referenced 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.5Available from Association of

15、Organic and Analytical Chemists International,Gaithersburg, MD.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.4 The virus is exposed to the virucide for the length oftime that is representative of actual use conditions of theprodu

16、ct, for example, from 10 to 20 s for a handsoap. The virusto be recovered after exposure to the test germicide is assayedin a cell culture system appropriate to the test virus. The virustiter of the stock, test samples and controls is determined by thea suitable infectivity assay. Cytotoxicity of th

17、e host cell culturesystem caused by the test germicide at the tested concentrationis also determined. The virus-germicide mixture is assayed innumerous units of the host system at a dilution just beyond thecytotoxicity range of the germicide. At least three replicatedeterminations are performed on c

18、ontrols (untreated) and testsamples (treated) to confirm virus elimination by a sample ofthe test germicide. Results are recorded as the median value oflog10- reduction in virus infectivity.3.4.1 This test method is designed to be performed by atrained microbiologist or virologist who is responsible

19、 forchoosing the appropriate host system for the test virus, andapplying the techniques necessary for propagation and main-tenance for host and test virus. For a reference text, see Ref.(4).4. Significance and Use4.1 This test method should be used to evaluate the virus-eliminating effectiveness of

20、these formulations after handwash-ing. Effective formulations can be further evaluated in aclinical trial on human subjects. Published data have shown (1)that results of in vitro tests do not accurately reflect whatoccurs when this class of products is used in the health carefacility. This test meth

21、od involves the incorporation of wholehand exposure and friction from washing, reflecting actual useconditions in human subjects. It is meant to confirm the resultsof testing with Test Method E 1836. This method gives precisereductions on a limited area of the finger, the fingerpads.4.2 This test me

22、thod is not meant for use with surgical handscrubs or preoperative skin preparations.5. Equipment and Apparatus5.1 Laminar Flow CabinetA Class II biological safetycabinet is required for virus work. The procedures for theproper maintenance and use of such cabinets are given in Ref.(5).5.2 IncubatorA

23、n incubator at 36 6 1C is needed forgrowing host cells and for incubating virus-infected cultures. Ifan open system is used for cell culture, a CO2incubator will berequired. Work with rhinoviruses will require an incubator at33 6 1C.5.3 Positive Displacement PipetteA pipette and pipettetips that can

24、 accurately dispense 10 to 20 L volumes isrequired.5.4 SterilizerAny steam sterilizer suitable for processingcell culture media and reagents is acceptable. The steamsupplied to the sterilizer must be free from additives toxic tocell cultures.5.5 Filter Sterilization SystemA membrane or cartridgefilt

25、ration system (0.22 m pore diameter) is required forsterilization of heat-sensitive media and solutions.5.6 FreezersA freezer at 20 6 2C is required for thestorage of fetal bovine serum and other additives for cellculture media. A second freezer at 70C or lower is requiredto store viruses.5.7 Refrig

26、eratorArefrigerator at 4 6 2C is necessary forstorage of prepared cell culture media and reagents.5.8 TimerAny stop watch that can be read in minutes andseconds is acceptable.5.9 Magnetic Stirrer and MagnetsMagnetic stirrer andmagnets must be large enough to hold a 5-L beaker orErlenmeyer flask for

27、preparing cell culture media or othersolutions.5.10 Handwashing SinkA sink of sufficient size to permitsubjects to wash hands without touching hands to sink surfaceis required.5.10.1 Water faucet(s) are to be located above the sink at aheight that permits the hands to be held higher than the elbowdu

28、ring the washing procedures. Faucets with electronic sensorsor those that are wrist-, elbow-, knee-, or foot-operated arepreferred to avoid recontamination of the washed hands.5.10.2 Bland, proven non-antimicrobial soap, preferablyliquid, is needed.5.10.3 Tap water temperature regulator and temperat

29、uremonitor to monitor and regulate water temperature at 40 6 2Cis needed.5.11 Liquid Nitrogen Storage for CellsAn appropriateliquid nitrogen container and liquid nitrogen for cryopreserva-tion of the stocks of cell lines are needed.5.12 Inverted MicroscopeAn inverted microscope with10X eye pieces an

30、d 5X, 10X and 40X objectives.5.13 Serological PipettesSterile reusable or single-usepipettes of 10.0, 5.0 and 1.0 mL capacity are needed.5.14 Cell Culture Flasks6Plastic cell culture flasks of 25cm2or 75 cm2capacity for culturing cells and for preparingvirus pools are needed.NOTE 1Each flask for gro

31、wing cell monolayers can be reused byreseeding with new cell cultures 10 or more times before being discarded.5.15 Plastic and Glass Vials, Medication (Medicant)Sterile screw-capped vials will be required or post test sam-pling of hands.5.16 Miscellaneous LabwareRequired are: automatic pi-pettes, pi

32、pette tips, plastic vials for storing cell and virusstocks, dilution tubes, cluster plates or flasks for virus titration.5.17 Sterile Glass beadsBeads that are 3.5 mm in diam-eter are needed.6. Materials and Reagents6.1 Cell Culture Media and SupplementsCulture mediaand the types and ratios of suppl

33、ements will vary depending onthe cell line. Eagles minimal essential medium (EMEN) with5 to 10 % fetal bovine serum (virus- and mycoplasma-tested) isused for growing a wide variety of cells (see Note 2).Antibiotics may be required in the medium to suppressbacterial growth.6Plastic cell culture ware

34、and other related supplies may be purchased from mostlaboratory supply houses.E20119926.2 Organic Load:6.2.1 Fetal bovine serum, at a final concentration of 5 % inhe virus inoculum (see Note 2), if required for the test.6.2.2 Peptone, a pancreatic digest of casein as an alternativeto serum, is made

35、by dissolving 7.6 g of tryptone powder in 1Lphysiological saline (0.85 % NaCl). Sterilize by autoclaving ormembrane filtration. This peptone solution should containapproximately 2.0 g of total protein/L, which is approximatelyequivalent to the protein content of a 5 % solution of fetalbovine serum.N

36、OTE 2Fetal bovine serum is considered unsuitable for use as anorganic load when working with rotaviruses because of its rotavirus-inhibitory and trypsin-neutralizing activity.6.3 Standard Hard WaterWater prepared according toAOAC 960.09 E and F (4) to a standard hardness of 200 ppmas calcium carbona

37、te is used for dilution of test products. Thisis the control solution to determine the baseline level of viruselimination, and to rinse the fingerpads after exposure to thetest product.6.4 Test ProductsDuplicate samples of the product shallbe tested.6.5 Diluent for Virus TitrationEarles balanced sal

38、inesolution (EBSS) with a pH of 7.2 to 7.4.6.6 Eluent for Virus Recovery from Hands and FingersUse EBSS containing peptone.7. Test Viruses and Cell Cultures7.1 The selection of the following test viruses is based ontheir (a) relative safety to the volunteers as well as experiment-ers, (b) ability to

39、 grow to titers sufficiently high for testing, (c)property to produce cytopathic effects or plaques, or both, incell cultures, (d) potential to spread through contaminatedhands, and (e) relative resistance to agents used in hygienichandwashing. Other strains or types of viruses may be substi-tuted p

40、rovided they meet the above criteria.NOTE 3There is insufficient information on whether the passagehistory, culture conditions and strain differences of viruses can influencethe efficiency of their elimination by hygienic handwash agents. There-fore, caution must be exercised when substituting virus

41、es as this may leadto variations in results from one laboratory to another.NOTE 4Poliovirus is scheduled for eradication. The goal is to achievea world free from polio. At that time, laboratory work with the polioviruswill not be possible.7.2 Human Rotavirus Wa (ATCC VR-201 8) of the cell lineCV-1 (

42、ATCC CCL-70) is recommended.7.2.1 Prior to rotavirus inoculation, cell monolayers must bewashed at least three times with EBSS to remove the serumfrom the growth medium. All diluents, maintenance media andagar overlays must also be free from serum. Most rotavirusesalso require the presence of trypsi

43、n in the medium for growthand plaques formation.7.3 Human Rhinovirus Type 37 (ATCC VR-I 147) orRhinovirus 14 (ATCC VR-284) of the cell line MRC-5 (ATCCCCL-171) or WI-38 (ATCC CCL-75) is recommended.8. Virus Stock Titer Determinations8.1 Utilize the appropriate host to prepare the virus pool.The host

44、 system employed for the virus pool should be thesame system to be used for virus recovery following viruschallenge of the test germicide. The virus pool should containnot less than 108infective unit/mL.8.2 Cell Culture Technique1.0 mL of virus is allowed toadsorb on the cell sheet of a 0.75 cm2flas

45、k for about1hatanappropriate incubation temperature after which 25 mL ofmaintenance media is added. The flask is then re-incubateduntil about 75 % of the cell monolayer shows virus cytopathol-ogy. The flask is frozen and thawed three times in a dryice-alcohol bath and the disrupted cells centrifuged

46、 20 min atabout 1000 xg. The supernatant is collected and divided intoappropriate volumes for test and may be used fresh or stored at70C.8.3 Virus titer of the stock is the titer of the test virus aftergrowth and dilution or concentration. A titer of 108infectiveunits/1 mL is required for the test.

47、The control virus titer isdetermined on subjects using hard water (200 ppm as calciumcarbonate) or EBSS (with or without peptone) or the vehicle ofthe test product for handwashing of virus-contaminated hands.The initial titer of the virus is the virus recovered by samplingthe inoculated hands after

48、washing and recovery with EBSSsolution.8.4 The initial or control titers, or both, can be determinedusing the virus application techniques and recovery samplesafter washing with buffer, hard water or the vehicle of the testproduct.9. Cytotoxicity Testing Antimicrobial9.1 Prior to studying the effect

49、s of the test product onviruses, determine its cytotoxic effect on the host system. Useserial 2-fold dilutions and inoculate a minimum of 4 units/dilution.9.2 A technique recommended for reduction of cytotoxiceffects is based on gel filtration (see Test Method E 1482 and(5). The eluates from virus-contaminated hands are passedthrough separate columns of Sephadex LH-20 gel7to removeresidues of the antimicrobial which may be cytotoxic. Thefiltrates are centrifuged at 10 000 xg to remove bacteria beforevirus infectivity assay.10. Conditioning10.1