1、Designation: E 2011 09Standard Test Method forEvaluation of Hygienic Handwash and HandrubFormulations for Virus-Eliminating Activity Using the EntireHand1This standard is issued under the fixed designation E 2011; the number immediately following the designation indicates the year oforiginal adoptio
2、n 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.INTRODUCTIONMechanical removal and/or in situ inactivation of viruses by hygienic handw
3、ash and handrub agentscan be assessed using artificially-contaminated hands of adults. This test method uses the entiresurface of both hands in contrast to only the fingerpads in the procedure described in Test MethodE 1838. However, the reported results from these two methods are comparable. (1, 2)
4、21. Scope1.1 This test method is designed to evaluate handwash orhandrub agents for their ability to reduce viral contaminationfrom hands.NOTE 1Aknowledge of virological techniques is required for this testmethod.1.2 The values stated in SI units are to be regarded asstandard. No other units of meas
5、urement are included in thisstandard.1.3 This standard may involve hazardous materials, opera-tions and equipment. This standard does not purport to addressall of the safety concerns, if any, associated with its use. It isthe responsibility of the user of this standard to establishappropriate safety
6、 and health practices and determine theapplicability of regulatory limitations prior to use. The usershould consult a reference for laboratory safety recommenda-tions. (3-5)2. Referenced Documents2.1 ASTM Standards:3E 1482 Test Method for Neutralization of Virucidal Agentsin Virucidal Efficacy Evalu
7、ationsE 1838 Test Method for Determining the Virus-EliminatingEffectiveness of Liquid Hygienic Handwash and HandrubAgents Using the Fingerpads of Adult Volunteers2.2 AOAC Standard:AOAC 960.9 Official Methods of Analysis (1990)43. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 hy
8、gienic (health-care personnel) handwash agents,nagents generally used for handwashing by personnel inhospitals, other health-care facilities, day-care centers, nursinghomes, and food-handling establishments; should be safe forrepeated use, non-irritating, fast-acting, and efficient in elimi-nating t
9、ransient microorganisms from intact skin.3.1.2 hygienic handrub agents (that is, hand sanitizers),nagents generally used for hand hygiene by personnel inhospitals, other health-care facilities, day-care centers, nursinghomes, and food-handling establishments that do not requirerinsing; should be saf
10、e for repeated use, non-irritating, fast-acting, and efficient in eliminating transient microorganismsfrom intact skin.3.1.3 non-medicated soap, na soap or detergent that ismild to the skin and does not contain any germicidal chemicals.3.1.4 soil (organic) load, na solution of one or moreorganic and
11、/or inorganic substances added to the suspension ofthe test organism to simulate the presence of body secretions,excretions or other extraneous substances.3.1.5 virus-eliminating (killing/removing) agent, nanyagent that rids hands of viruses by either killing them on theskin or by dislodging them fo
12、r subsequent wash-off.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2009. Published April 2009. Originallyapproved in 1999.
13、 Last previous edition approved in 1999 as E 2011 99, whichwas withdrawn in August 2008 and reinstated in April 2009.2The boldface numbers in parentheses refer to a list of references at the end ofthis test method.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM C
14、ustomer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Available from AOAC International, 481 North Frederick Ave., Suite 500,Gaithersburg, Maryland 20877-2417, http:/www.aoac.org.1Copyright ASTM Inter
15、national, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.1.6 virus inactivating agent, nany agent that renders avirus noninfectious.4. Summary of Test Method4.1 This test method uses adult subjects who have provideda written informed consent and whose hands hav
16、e beendetermined to be free from any apparent damage at the time oftheir participation in the study. While no fewer than sixsubjects are recommended for each virus-test substance com-bination to be evaluated, the number required may varydepending on the intended use of the data and the targetregulat
17、ory agency.4.2 All subjects should refrain from using any antimicrobi-als starting at least one week prior to the experimentalcontamination of their hands.4.3 Aprepared suspension of the selected test virus is grownand diluted or concentrated to produce a titer with a minimumof 108infective units/mL
18、. The contaminating virus is applied tothe hands and the hands are treated with the test formulationaccording to the manufacturers directions or with a set testregimen.4.4 The virus titer recovered after treatment with the testsubstance is compared to a control. For the control, the testvirus is app
19、lied to the hands and recovered after the subject hastreated the hands with standard hard water (200 ppm ascalcium carbonate) or vehicle, or both, instead of the testsubstance.4.5 The virus on experimentally-contaminated hands isexposed to the test substance for the length of time that isrepresentat
20、ive of actual use conditions of the product, forexample, from 10 to 20 s for a handsoap and 20 to 30 s for ahandrub. The virus to be recovered after exposure to the testsubstance is assayed in a cell culture system appropriate to thetest virus. The virus titer of the stock, test samples, and control
21、sis determined by a suitable infectivity assay. Cytotoxicity ofthe host cell culture system caused by the test substance orvehicle at the tested concentration is also determined. Thevirus-test substance mixture is assayed in numerous units of thehost system at a dilution just beyond the cytotoxicity
22、 range ofthe formulation tested. At least three replicate determinationsare performed on controls (untreated) and test samples (treated)to confirm the extent of virus elimination by the number of lotsrequired by the target regulatory agency. Results are recordedand log10and/or percent reduction in v
23、irus infectivity arecalculated.4.5.1 This test method is designed to be performed by atrained microbiologist or virologist who is responsible forchoosing the appropriate host system for the test virus, andapplying the techniques necessary for propagation and main-tenance for host system and test vir
24、us. For a reference text, seeRef (6).5. Significance and Use5.1 This test method is designed to evaluate the virus-eliminating activity of hygienic handwash and handrub agentsfrom experimentally-contaminated hands. Such formulationsmay be further assessed in a clinical trial for their effectivenessi
25、n the field. This test method incorporates whole-hand expo-sure and reflects actual use conditions such as friction duringhand decontamination, and also enables alternate productforms such as alcohol- or non-alcohol-based liquids, gels, andfoams to be tested according to label directions. It is mean
26、t toextend, if required, the results of testing with Test MethodE 1838, which gives precise reductions in virus infectivity ona limited area of the hands. It may also serve as an alternativetest method when product form is not amenable to testing byTest Method E 1838.5.2 This test method is not mean
27、t for use with surgical handscrubs or preoperative skin preparations.NOTE 2Application of viruses on the entire surface of both handsentails a greater risk to the subjects than using fingerpads only. Therefore,greater care is needed to ensure that the hands of the participants are freefrom any appar
28、ent damage. Also, virus preparations must be thoroughlyscreened for, or documented to be free from, extraneous or adventitiouspathogens before use in such tests.6. Equipment and Apparatus6.1 Laminar Flow Cabineta Class II biological safetycabinet. The procedures for the proper maintenance and use of
29、such cabinets are given in Ref (3, 4).6.2 Incubatoran incubator at 36 6 1C for growing hostcells and for incubating virus-infected cultures. If an opensystem is used for cell culture, a CO2incubator will berequired.6.3 Positive Displacement Pipettea pipette and pipettetips that can accurately dispen
30、se 10 to 20 L volumes.6.4 Sterilizerany steam sterilizer suitable for processingcell culture media and reagents. The steam supplied to thesterilizer must be free from additives toxic to cell cultures.6.5 Filter Sterilization Systema membrane or cartridgefiltration system (0.22 m pore diameter) is re
31、quired forsterilization of heat-sensitive media and solutions.6.6 Freezersa freezer at 20 6 2C for the storage of fetalbovine serum and other additives for cell culture media. Asecond freezer at 70C or lower is required to store viruses.6.7 Refrigeratora refrigerator at 4 6 2C is necessary forstorag
32、e of prepared cell culture media and reagents.6.8 Timerany stopwatch that can be read in minutes andseconds.6.9 Magnetic Stirrer and Magnetsmagnetic stirrer andmagnets must be large enough to hold a 5-L beaker orErlenmeyer flask for preparing cell culture media or othersolutions.6.10 Handwashing Sin
33、ka sink of sufficient size to permitsubjects to wash hands without touching hands to sink surface.6.10.1 Water faucet(s) are to be located above the sink at aheight that permits the hands to be held higher than the elbowduring the washing procedures. Faucets with electronic sensorsor those that are
34、wrist-, elbow-, knee-, or foot-operated arepreferred to avoid recontamination of the washed hands.6.10.2 Mild, proven non-antimicrobial soap, preferably liq-uid.6.10.3 Tap water temperature regulator and temperaturemonitor to monitor and regulate water temperature at 40 62C.6.11 Liquid Nitrogen Stor
35、age for Cellsan appropriateliquid nitrogen container and liquid nitrogen for cryopreserva-tion of the stocks of cell lines.E20110926.12 Inverted Microscopean inverted microscope with103 eye pieces and 53,103, and 403 objectives.6.13 Serological Pipettessterile reusable or single-usepipettes of 10.0,
36、 5.0, and 1.0 mL capacity.6.14 Cell Culture Flasksplastic cell culture flasks of 25cm2or 75 cm2capacity for culturing cells and for preparingvirus pools.NOTE 3Each plastic flask for growing cell monolayers can be reusedby reseeding with new cell cultures up to 10 times before being discarded.NOTE 4P
37、lastic cell culture ware and other related supplies may bepurchased from most laboratory supply houses.6.15 Plastic and Glass Vials, Medication (Medicant)sterile screw-capped vials will be required for storage ofsamples. See Note 4.6.16 Miscellaneous Labwareautomatic pipettes, pipettetips, plastic v
38、ials for storing cell and virus stocks, dilutiontubes, cluster plates or flasks for virus titration.6.17 Sterile Glass Beads3.5 mm in diameter.6.18 Glass or Plastic Funnel27 cm in diameter.6.19 Glass or Plastic Beaker200 mL capacity.7. Materials and Reagents7.1 Cell Culture Media and SupplementsCult
39、ure mediaand the types and ratios of supplements 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 5).Antibiotics may be required in the medium to suppressba
40、cterial contamination.7.2 Soil Load:7.2.1 Bovine serum, at a final concentration of 5 % in thevirus inoculum (see Note 5), if required for the test.7.2.2 A tripartite soil load, as an alternative to serum, isprepared from the following stock solutions in phosphatebuffer (pH 7.2 to 7.4).7.2.2.1 Add 0
41、.5 g of tryptone or yeast extract to 10 mL ofthe buffer.7.2.2.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mLof the buffer.7.2.2.3 Add 0.04 g of bovine mucin to 10 mL of the buffer.7.2.2.4 Prepare the stock solutions separately and sterilizeby passage through a 0.22 m pore diameter membrane filte
42、r,aliquot and store at either 4 6 2C or 20 6 2C.7.2.2.5 To obtain a 500-L inoculum of the test inoculum,add to 340 L of the microbial suspension 25 L BSA, 100 Lmucin, and 35 Lof tryptone/yeast extract stock solutions. Thismixture contains approximately2goftotal protein/L, which isapproximately equiv
43、alent to the protein content of a 5 %solution of fetal bovine serum.NOTE 5Fetal bovine serum is considered unsuitable for use as a soilload with rotaviruses because of its rotavirus-inhibitory and trypsin-neutralizing activity.7.3 Standard Hard WaterWater prepared according toAOAC 960.09 E and F (4)
44、 to a standard hardness of 200 ppmas calcium carbonate is used for dilution of test substance. Thisis the control solution to determine the baseline level of viruselimination, and to rinse the hands after exposure to the testsubstance.7.4 Number of Lots of Test Substance to be UsedThenumber of separ
45、ate manufactured lots of each test formulationto be tested will depend on the specific requirements of thetarget regulatory agency.7.5 Diluent for Virus TitrationEarles balanced salt solu-tion (EBSS) with a pH of 7.2 to 7.4.7.6 Eluent for Virus Recovery from HandsEBSS contain-ing 1 % peptone and 0.1
46、 % Tween-80 at final concentrations.8. Test Viruses and Cell Cultures8.1 See the Appendix for suggested viruses and host cells.9. Preparation of Virus Stocks and Determination ofInfectivity Titer9.1 Use appropriate host cells to prepare the virus pool. Thevirus pool should contain $108infective unit
47、/mL.9.2 Remove growth or maintenance medium and inoculate0.1 mLof virus (control flasks receive 0.1 mLof EBSS instead)into each flask (75 cm2) with a confluent cell monolayer andallow 60 min for virus adsorption. Place 15 mL of maintenancemedium into each inoculated flask and reincubate till about75
48、 % of each infected cell monolayer shows virus-inducedcytopathology and while control monolayers remain free fromany apparent degeneration. Freeze (20C) and thaw (roomtemperature) the infected flasks three times to disrupt host cellsfor virus release. Centrifuge the cell suspension for 20 min atappr
49、oximately 1000 xg to sediment the cell debris, collectsupernatant and store it at 70C in suitable aliquots.9.3 A titer of $108infective units/mL is required for thetesting and ultra-centrifugation of virus pools may be needed toachieve such levels of infectivity for the contamination ofhands.9.4 Determine the baseline level of viable virus remainingon the hands of the subject after the rubbing and drying of theexperimentally-contaminated skin by first washing or rubbingand then eluting the hands with EBSS-peptone. This repr
