ASTM E1838-2010 Standard Test Method for Determining the Virus-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults《使用成人用指垫测定卫生洗手和擦手剂的病毒.pdf

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1、Designation: E1838 10Standard Test Method forDetermining the Virus-Eliminating Effectiveness of HygienicHandwash and Handrub Agents Using the Fingerpads ofAdults1This standard is issued under the fixed designation E1838; the number immediately following the designation indicates the year oforiginal

2、adoption 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.INTRODUCTIONHands play an important role in the spread of many viruses. Thus, pr

3、oper and regular hand hygieneis crucial in preventing such spread, particularly in health-care settings, day-care centers, andfood-handling establishments. Many viruses that are known to spread through contaminated hands canremain infectious for several hours on human hands, and also may be more res

4、istant than the bacteriacommonly used to evaluate the microbicidal activity of handwash and handrub agents (1, 2, 3, 4).2Contaminated hands also can readily transfer infectious virus to other surfaces (1, 2, 3). Handantisepsis has been shown to interrupt the spread of viral infections (5). Standardi

5、zed methods toassess the virus-eliminating potential of handwash and handrub agents have not been available and thistest method addresses the gap.1. Scope1.1 Human skin is not known to carry viruses as a part of itsresident microbiota. Hands transiently contaminated with vi-ruses can, however, act a

6、s vehicles for the spread of many typesof viral infections. Hand hygiene is meant to reduce the load ofviruses and other transient microorganisms on hands, therebyreducing the risk of disease transmission. Such reductions inthe virus load may be due to a combination of virus inactiva-tion and mechan

7、ical removal of infectious virus from the skin.1.2 This test method is designed to determine the compara-tive virus-eliminating effectiveness of microbicidal or non-microbicidal formulations. This test method is not meant foruse with surgical hand scrubs or preoperative skin preps.NOTE 1The test met

8、hod should be performed by persons with trainingin virology in facilities designed and equipped for work with infectiousagents at biosafety level 2 (6).1.3 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.4 This standard does n

9、ot 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.2. Referenced Documents2.1 ASTM Standar

10、ds:3E2011 Test Method for Evaluation of Hygienic Handwashand Handrub Formulations for Virus-Eliminating ActivityUsing the Entire HandE2276 Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Han-drub Agents Using the Fingerpads of Adult SubjectsE2613 Test Meth

11、od for Determining Fungus-EliminatingEffectiveness of Hygienic Handwash and Handrub AgentsUsing Fingerpads of Adults3. Terminology3.1 Definitions of Terms Specific to This Standard:3.1.1 hygienic (health-care personnel) handwash agents,nagents generally used for handwashing by personnel in1This test

12、 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, 2010. Published May 2010. Originallyapproved in 1996. Last previous edition approved in

13、 2002 as E1838 02. DOI:10.1520/E1838-10.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.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

14、formation, refer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.hospitals, other health-care facilities, day-care centers, nursinghomes, and food-handling establishments shou

15、ld be safe forrepeated use, nonirritating, fast-acting, and efficient in elimi-nating transient microorganisms from intact skin.3.1.2 nonmedicated soap, na soap or detergent that ismild to the skin and does not contain any germicidal chemicals.3.1.3 soil(organic) load, na solution of one or moreorga

16、nic and/or inorganic substances added to the suspension ofthe test organism to simulate the presence of body secretions,excretions or other extraneous substances.3.1.4 virus-eliminating (killing/removing) agent, nanyagent that rids hands of viruses by either killing them on theskin or by dislodging

17、them for subsequent wash-off.3.1.5 virus inactivating agent, nany agent that renders avirus noninfectious.4. Summary of Test Method4.1 This test method is conducted on a group of adultsubjects who have provided informed consent and the skin ofwhose hands has been determined to be free from any appar

18、entdamage. The subjects are to refrain from using any productscontaining antimicrobial agents for at least one week prior tothe test.Aknown volume of the test virus suspension is placedon a demarcated area on each fingerpad and the inoculumallowed to dry. The contaminated area then is exposed to the

19、control (standard hard water) or test agent for the desiredcontact time and virus remaining on the fingerpad is eluted andthe eluates titrated for infectious virus along with the requiredcontrols. Percent and/or log10reductions in the levels ofinfectious virus after treatment with the control and te

20、st agentsare then determined. The fingerpad method gives results thatare comparable to those obtained using a whole-hand proce-dure (2, 7), another ASTM standard (Test Method E2011). Iftwo different formulations are being compared in the same test,one of them may be designated as a reference and use

21、d in placeof the hard water control. If desired, one also may use tap waterin parallel with the hard water control to determine theinfluence of water hardness on the test products virus-eliminating activity.5. Significance and Use5.1 This in vivo procedure is designed to test the ability ofhygienic

22、handwash and handrub agents to reduce levels ofselected infectious viruses from experimentally contaminatedfingerpads of adults. Since the two thumbpads and all eightfingerpads can be used in any given test, it allows for theincorporation of input virus control (two), virus remainingviable after the

23、 inoculum has been allowed to dry (two), viruseliminated after treatment with a control or reference solution(two), and up to four replicates to assess the virus-eliminatingefficiency of the substance under test. No more than 100 L ofthe virus suspension are required to complete one test. Theresults

24、 of testing with this test method may form the basis forfurther tests using a suitable whole-hand test protocol (forexample, Test Method E2011).5.2 This test method is designed to be performed by atrained individual, who is responsible for choosing the appro-priate host system for the test virus and

25、 applying the techniquesnecessary for propagation and maintenance of host and testvirus. For a reference text, refer to Lennette et al (8).5.3 Whereas the method described here relates to testingwith viruses of human origin, it can be readily adapted to workwith animal pathogenic viruses as well as

26、bacteriophages.Standard methods for working with bacteria (Test MethodE2276) and fungi (Test Method E2613) are also available.5.4 Infectious microorganisms left on hands after washingcan be reduced further by drying the washed hands with paper,cloth, or warm air (9). A step for the drying of fingerp

27、ads afterexposure to the control or test substance, therefore, has notbeen included to avoid virus removal by the drying processitself.5.5 This test method is not meant for use with surgical handscrubs or preoperative skin preps.5.6 The level of viable virus on each fingerpad after thedrying of the

28、inoculum should not be less than 104infectiousunits which would permit the detection of up toa4log10reduction in the infectivity titer of the virus by the testsubstance under the conditions of this test method.6. Equipment and Apparatus6.1 Laminar Flow CabinetA Class II biological safetycabinet is r

29、equired for virus work. The procedures for theproper maintenance and use of such cabinets are given in Ref(6).6.2 IncubatorAn 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.6.3

30、 Positive Displacement PipetteA pipette and pipettetips that accurately can dispense 10-L volumes.6.4 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.6.5 Filter

31、Sterilization SystemA membrane or cartridgefiltration system (0.22-m pore diameter) is required forsterilizing heat-sensitive media and solutions.6.6 FreezersA freezer at 206 2C is required for thestorage of fetal bovine serum and other additives for cellculture media. A second freezer at 70C or low

32、er is requiredto store viruses.6.7 RefrigeratorA refrigerator at 46 2C for storage ofprepared cell culture media and reagents.6.8 TimerAny stopwatch that can be read in minutes andseconds.6.9 Magnetic Stirrer and MagnetsLarge enough to hold a5-Lbeaker or Erlenmeyer flask for preparing cell culture m

33、ediaor other solutions.6.10 Handwashing SinkA sink of sufficient size to permitsubjects to wash hands without touching hands to sink surface.6.10.1 Water Faucet(s), to be located above the sink at aheight that permits the hands to be held higher than the elbowduring the washing procedure. Faucets wi

34、th electronic sensorsor those that are wrist-, elbow-, knee-, or foot-operated arepreferred to avoid recontamination of the washed hands.E1838 1026.10.2 Tap Water Temperature Regulator and TemperatureMonitor, to monitor and regulate water temperature at 40 62C.6.11 Liquid Nitrogen Storage for CellsA

35、 proper liquidnitrogen container and liquid nitrogen for cryopreservation ofthe stocks of cell lines.6.12 Inverted MicroscopeAn inverted microscope with103 eye pieces and 53,103, and 403 objectives.7. Materials and Reagents7.1 Serological PipettesSterile reusable or single-use pi-pettes of 10.0, 5.0

36、, and 1.0-mL capacity.7.2 Cell Culture FlasksPlastic flasks of 25 or 75-cm2capacity for culturing cells and for preparing virus pools.NOTE 2Each flask for growing cell monolayers can be reused ten ormore times before being discarded.NOTE 3Plastic cell culture ware may be purchased from mostlaborator

37、y supply houses.7.3 Cell Culture Media and SupplementsCulture mediaand the types and ratios of supplements will vary depending onthe cell line. Eagles minimal essential medium (EMEM) with5 to 10 % fetal bovine serum (virus- and mycoplasma-tested) isused for growing a wide variety of cells (see Note

38、5).NOTE 4Materials and reagents for cell culture may be purchased frombiological supply houses.7.4 Soil Load:7.4.1 Bovine Serum, at a final concentration of 5 % in thevirus inoculum (see Note 5).7.4.2 A tripartite soil load, as an alternative to serum. Add0.5 g of tryptone or yeast extract to 10 mL

39、of phosphate buffer.Add 0.5 g of bovine serum albumin (BSA) to 10 mL ofphosphate buffer. Add 0.04 g of bovine mucin to 10 mL ofphosphate buffer. Prepare the stock solutions separately andsterilize by passage through a 0.22 m pore diameter mem-brane filter, aliquot and store at either 462C or 2062C.

40、Toobtain a 500-L inoculum of the test inoculum, add to 340 Lof the microbial suspension 25 L BSA, 100 L mucin and 35L of tryptone stock solutions. This mixture contains approxi-mately2goftotal protein/L, which is approximately equiva-lent to the protein content of a 5 % solution of fetal bovineserum

41、.NOTE 5Bovine serum is unsuitable for use as an organic load whenworking with rotaviruses because of its rotavirus inhibitory and trypsin-neutralizing activity.7.5 Standard Hard WaterThe quality and disinfectant (forexample, chlorine) residual in tap water can vary from site tosite and also at diffe

42、rent times at the same site. The use ofstandard hard water, therefore, is recommended here to avoidvariations in results due to differences in tap water quality.Water prepared in accordance withAOAC 960.09 E and F (10)to a standard hardness of 200 ppm as calcium carbonate is usedfor dilution of test

43、 substance, as the control solution todetermine the baseline level of virus elimination, and to rinsethe fingerpads after exposure to the test product. The standardhard water and tap water (if used) must first be tested to ensurethat they do not have any virucidal activity against the testvirus(es).

44、7.6 Test SubstanceAt least two separate manufacturerslots of the substance shall be tested. For handwash productsthat are used with water, prepare a 25 % solution by adding 1part product to 3 parts standard hard water.7.7 Diluent for Virus TitrationEarles balanced salt solu-tion (EBSS) with a pH of

45、7.2-7.4.7.8 Eluent for Virus Recovery from FingerpadsEBSS (pH7.27.4).7.9 Plastic VialsSterile screw-capped 2.0-mL vials withan inside diameter of about 8 mm are required for demarcationof the fingerpads and to hold various test solutions.7.10 Miscellaneous Laboratory WareAutomatic pipettes,pipette t

46、ips, plastic vials for storing cell and virus stocks,dilution tubes, cluster plates, or flasks for virus titration.8. Test Viruses and Cell Cultures8.1 See Appendix X1 for recommended viruses and theirhost cells.9. Subjects9.1 Recruit a sufficient number of healthy adult humanvolunteers who have no

47、clinical evidence of dermatoses, openwounds, or other skin disorders (see 4.1). The number ofvolunteers required for a trial is dependent on the number oftreatments within a study.9.2 It is the responsibility of the user of this test method toarrange the necessary clearance for the use of adult subj

48、ects fortesting and to obtain informed and written consent from thoseselected for the study before starting the tests.10. Procedure10.1 Fig. 1 shows the main steps for this test method. It isrecommended that virus from the two thumbpads be elutedsimultaneously. The simultaneous exposure and elution

49、of thetwo fingerpads to be used for the control or test substance isalso recommended.10.2 The subject will wash his/her hands with a nonmedi-cated soap for at least 10 s, rinse, and then dry them thoroughlywith a clean paper or cloth towel. This procedure reducesvariability in the test results by removing accumulated oil anddirt from the hands. Place about 5 mL of 70 % (v/v) ethanol inthe palm of one of the washed hands and instruct the subject torub it well over the entire surface of both hands until thealcohol and water have