1、Designation: E2011 13Standard Test Method forEvaluation of Hygienic Handwash and HandrubFormulations for Virus-Eliminating Activity Using the EntireHand1This standard is issued under the fixed designation E2011; 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.INTRODUCTIONMechanical removal and/or in situ inactivation of viruses by hygienic handwas
3、h and handrub agentscan be assessed using artificially-contaminated hands of adults. This test method uses the entiresurface of both hands (including both the palmar and dorsum sides of the hands) in contrast to onlythe fingerpads in the procedure described in Test Method E1838. However, the reporte
4、d results fromthese two methods are comparable. (1, 2)21. Scope1.1 This test method is designed to evaluate handwash orhandrub agents for their ability to reduce or eliminate viableviruses from the skin of human hands.NOTE 1Aknowledge of virological techniques is required for this testmethod.1.2 The
5、 values stated in SI units are to be regarded asstandard. No other units of measurement 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
6、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. (3-5)2. Referenced Documents2.1 ASTM Standards:3E148
7、2 Practice for Use of Gel Filtration Columns for Cyto-toxicity Reduction and NeutralizationE1838 Test Method for Determining the Virus-EliminatingEffectiveness of Hygienic Handwash and HandrubAgentsUsing the Fingerpads of Adults2.2 AOAC Standard:AOAC 960.9 Official Methods of Analysis (2007)43. Term
8、inology3.1 Definitions of Terms Specific to This Standard:3.1.1 hygienic handwash agents, nagents generally usedfor handwashing by personnel in hospitals, other health-carefacilities, day-care centers, nursing homes, and food-handlingestablishments; should be safe for repeated use, non-irritating,fa
9、st-acting, and efficient in eliminating transient microorgan-isms from intact skin.3.1.2 hygienic handrub agents (that is, hand sanitizers),nagents not requiring rinsing and generally used for handhygiene by personnel in hospitals, other health-care facilities,day-care centers, nursing homes, and fo
10、od-handling establish-ments; should be safe 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) l
11、oad, na solution of one or moreorganic and/or inorganic substances added to the suspension ofthe test organism to simulate the presence of body secretions,excretions or other extraneous substances.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alt
12、ernative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2013. Published May 2013. Originallyapproved in 1999. Last previous edition approved in 2009 as E2011 09. DOI:10.1520/E2011-13.2The boldface numbers in parentheses
13、 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 Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.
14、4Available from AOAC International, 481 North Frederick Ave., Suite 500,Gaithersburg, Maryland 20877-2417, http:/www.aoac.org.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States13.1.5 virus-eliminating (inactivating/removing) agent,nany a
15、gent that rids hands of viruses by either inactivatingthem on the skin or by dislodging them for subsequentwash-off.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 conse
16、nt and whose hands have beendetermined to be free from any apparent damage at the time oftheir participation in the study.4.1.1 Since both hands, including nail beds, of the testsubject are exposed to high-titer suspensions of virus, eachsubject shall be carefully examined for any skin irritations,m
17、icro-breaches, or breaks in the hand skin and around the nailsusing a magnifying glass under well-lighted conditions. Thosewith any breaches, breaks, or other apparent skin damages shallnot participate in the test.4.1.2 While no fewer than six subjects are recommended foreach virus-test substance co
18、mbination to be evaluated, thenumber required may vary depending on the intended use ofthe data and the target regulatory 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
19、 the selected test virus is grownand diluted or concentrated to produce a titer with a minimumof 107infective units/mL.The contaminating virus is applied tothe hands and the hands are treated with the test substanceaccording to the manufacturers directions or with a set testregimen.4.4 The virus tit
20、er recovered after treatment with the testsubstance is compared to a control. For the control, the testvirus is applied 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 v
21、irus on experimentally contaminated hands isexposed to the test substance for the length of time that isrepresentative 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 ass
22、ayed in a cell culture system appropriate to thetest virus.The virus titer of the stock, test samples, and controlsis 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. Theviru
23、s-test substance mixture is assayed using multiple replicatewells or flasks of the host system at a dilution just beyond thecytotoxicity range of the formulation tested. At least threereplicate determinations are performed on controls (untreated)and test samples (treated) to confirm the extent of vi
24、ruselimination by the number of lots of the test substance requiredby the target regulatory agency. Results are recorded and log10and/or percent reduction in virus infectivity are calculated.4.5.1 This test method is designed to be performed by aperson trained and experienced in working with human p
25、atho-genic viruses and their host cells. Such an individual will alsobe responsible for choosing the appropriate host system for thetest virus, and applying the techniques necessary for propaga-tion and maintenance for host system and test virus. For areference text, see Ref (6).5. Significance and
26、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 effectivenessin the field. This test method incorporates whole-hand exp
27、o-sure and reflects actual use conditions such as friction duringhand decontamination, and also enables alternative productforms such as alcohol- or non-alcohol-based liquids, gels, andfoams to be tested according to label directions. It is meant toextend, if required, the results of testing with Te
28、st MethodE1838, which gives precise reductions in viral infectivity on alimited area of the hands. It may also serve as an alternativetest method when product form is not amenable to testing byTest Method E1838.5.2 This test method is not meant for use with surgical handscrubs or preoperative skin p
29、reparations.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 apparent damage. Also, virus preparations must be thoroughlysc
30、reened 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 ofsuch cabinets are given in Ref (3, 4).6.2 Incubatoran inc
31、ubator at 35 6 2C or other appropri-ate temperature for growing host cells and for incubatingvirus-infected cultures. If an open system is used for cellculture, a CO2incubator will be required.6.3 Positive Displacement Pipettea pipette and pipettetips that can accurately dispense 10 to 20-L volumes.
32、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 required forsterilizatio
33、n of heat-sensitive media and solutions.6.6 Freezersa freezer at 20 6 2C for the storage ofserum and other additives for cell culture media. A secondfreezer at 70C or lower is required to store viruses.6.7 Refrigeratora refrigerator at 4 6 2C is necessary forstorage of prepared cell culture media an
34、d reagents.6.8 Timerany calibrated stopwatch that can be read inminutes and seconds.E2011 1326.9 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 Sinka sink of suff
35、icient 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 wrist-, elbow-,
36、 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 Storage for Cellsan
37、 appropriateliquid nitrogen container and liquid nitrogen for cryopreserva-tion of cell line stocks.6.12 Inverted Microscopean inverted microscope with10 eye pieces and 5, 10, and 40 objectives.6.13 Serological Pipettessterile reusable or single-usepipettes of 10.0-, 5.0-, and 1.0-mL capacity or oth
38、er suitablecapacity.6.14 Cell Culture Flasksplastic cell culture flasks of 25cm2or 75 cm2or other suitable capacity for culturing cells andfor preparing virus pools.NOTE 3Each plastic flask for growing cell monolayers can be reusedby reseeding with new cell cultures up to 10 times before being disca
39、rded.6.15 Plastic and Glass Vials, Medication (Medicant)sterile screw-capped vials will be required for storage ofsamples.6.16 Miscellaneous Labwareautomatic pipettes, pipettetips, plastic vials for storing cell and virus stocks, dilutiontubes, cluster plates or flasks for virus titration.6.17 Steri
40、le Glass Beads3.5 mm in diameter.6.18 Glass or Plastic Funnel27 cm in diameter.6.19 Glass or Plastic Beaker200 mL in capacity.7. Materials and Reagents7.1 Cell Culture Media and SupplementsCulture mediaand the types and ratios of supplements will vary depending onthe cell line. For example, Eagles m
41、inimal essential medium(EMEM) with 5 to 10 % fetal bovine serum (virus- andmycoplasma-tested) is used for growing a wide variety of cells(see Note 4). Antibiotics may be required in the medium tosuppress bacterial contamination.7.2 Soil Load:7.2.1 Bovine serum, at a final concentration of 5 % in the
42、virus inoculum (see Note 4), if required for the test.NOTE 4Serum is considered unsuitable for use as a soil load withrotaviruses because of its rotavirus-inhibitory and trypsin-neutralizingactivity.7.2.2 A tripartite soil load, as an alternative to serum, isprepared from the following stock solutio
43、ns in phosphatebuffer (pH 7.2 to 7.4).7.2.2.1 Add 0.5 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 pa
44、ssage through a 0.22-m pore diameter membrane filter,aliquot and store at either 4 6 2C or 20 6 2C. Use withina validated shelf-life.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 solution
45、s.Thismixture contains approximately2goftotal protein/L, which isapproximately equivalent to the protein content of a 5 %solution of fetal bovine serum.7.3 Standard Hard WaterWater prepared according toAOAC 960.9 to a standard hardness of 200 ppm as calciumcarbonate is used for dilution of test subs
46、tance. This is thecontrol solution to determine the baseline level of viruselimination, and to rinse the hands after exposure to the testsubstance.7.4 Number of Test Substance Lots to be UsedThe numberof separate manufactured lots (batches) of each test formula-tion to be tested will depend on the s
47、pecific requirements of thetarget regulatory agency.7.5 Diluent for Virus TitrationEarles balanced salt solu-tion (EBSS) or other appropriate dilution medium with a pH of7.2 to 7.4.7.6 Eluent for Virus Recovery from HandsEBSS or otherappropriate dilution medium containing 1 % peptone and0.1 % Polyso
48、rbate 80 at final concentrations.7.7 Sterile Disposable GlovesLoose-fitting, unlined,powder-free gloves which possess no antiviral or cytotoxicproperties, or equivalent. (Plastic bags with low bioburden maybe used in place of gloves.)8. Test Viruses and Cell Cultures8.1 See Appendix X1 for suggested
49、 viruses and host cells.8.2 Virus stocks as well as host cells used for viruspropagation may contain adventitious viruses or other patho-gens potentially harmful to human subjects. Therefore, greatcare should be used in the selection and use of such materialsto be applied on human hands.9. Preparation of Virus Stocks and Determination ofInfectivity Titer9.1 Use appropriate host cells to prepare the virus pool. Thevirus pool should contain 107infective unit/mL.9.2 Remove growth or maintenance medium and inoculate0.1 mLof v
