ASTM E1838-2002 Standard Test Method for Determining the Virus-Eliminating Effectiveness of Liquid Hygienic Handwash and Handrub Agents Using the Fingerpads of Adult Volunteers《利用成.pdf

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1、Designation: E 1838 02Standard Test Method forDetermining the Virus-Eliminating Effectiveness of LiquidHygienic Handwash and Handrub Agents Using theFingerpads of Adult Volunteers1This standard is issued under the fixed designation E 1838; the number immediately following the designation indicates t

2、he year oforiginal adoption or, in the case 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.INTRODUCTIONHands play an important role in the spread of ma

3、ny viruses, thus proper and regular handwashingis considered crucial in preventing such spread, particularly in health-care settings, day-care centers,and food-handling establishments. Many viruses that are known to spread through contaminated handscan remain infectious for several hours on human ha

4、nds, and also may be more resistant than thebacteria commonly used to evaluate the germicidal activity of handwash and handrub agents (1,2, 3).2Contaminated hands also can readily transfer infectious virus to other surfaces (1,2). Hand antisepsishas been shown to interrupt the spread of viral infect

5、ions (4). Standardized methods to assess thevirus-eliminating potential of handwash and handrub agents have not been available and this testmethod addresses the gap.1. Scope1.1 Human skin does not carry viruses as a part of itsresident flora. Hands transiently contaminated with viruses,however, can

6、act as vehicles for the spread of many types ofviral infections. Hygienic hand washing is meant to reduce theload of viruses and other transient microorganisms on hands,thereby reducing the risk of disease transmission. Such reduc-tion in the virus load may be due to a combination of virusinactivati

7、on and removal of infectious virus from the skin.1.2 Standard test methods to assess the capacity of hygienichandwash and handrub agents to reduce virus levels on handsare not presently available. This test method, therefore, hasbeen designed to determine the comparative virus-eliminatingeffectivene

8、ss of germicidal or non-germicidal formulations.This test method is not meant for use with surgical hand scrubsor preoperative skin preps.NOTE 1The test method should be performed by persons with trainingin virology in facilities designed and equipped for work with infectiousagents at biosafety leve

9、l 2 (5).1.3 This 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.2. Refe

10、renced Documents2.1 ASTM Standards:3D 1129 Terminology Relating to WaterE 1115 Test Method for Evaluation of Surgical Hand ScrubFormulationE 1173 Test Method for Evaluation of a Pre-Operative SkinPreparationE 1174 Test Method for Evaluation of Health Care Person-nel Handwash FormulationE 2011 Test M

11、ethod for Evaluation of Handwashing For-mulations for Virus-Eliminating Activity Using the EntireHand3. Terminology3.1 DefinitionsFor definitions of general terms used inthis test method, refer to Terminology D 1129.3.2 Definitions of Terms Specific to This Standard:3.2.1 hygienic (health-care perso

12、nnel) handwash agents,nagents generally used for handwashing by personnel inhospitals, other health-care facilities, day-care centers, nursing1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee

13、E35.15 on Antibacterial Agents.Current edition approved April 10, 2002. Published July 2002. Originallypublished as E 183896. Last previous edition E 183896.2The boldface numbers in parentheses refer to the list of references at the end ofthis standard.3For referenced ASTM standards, visit the ASTM

14、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.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United

15、States.homes, and food-handling establishments should be safe forrepeated use, nonirritating, fast-acting, and efficient in elimi-nating transient microorganisms from intact skin.3.2.2 nonmedicated soap, na soap or detergent that ismild to the skin and does not contain any germicidal chemicals.3.2.3

16、 soil(organic) load, 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.3.2.4 virus-eliminating (killing/removing) agent, nanyagent that rids hands of viruses

17、by either killing them on theskin or by dislodging them for subsequent wash-off.3.2.5 virus inactivating agent, nany agent that renders avirus noninfectious.4. Summary of Test Method4.1 This test method is conducted on a group of adultvolunteers who have provided informed consent and the skin ofwhos

18、e hands has been determined to be free from any apparentdamage. Panelists are to refrain from any products containingantimicrobial agents for one week prior to the test. A knownvolume of the test virus suspension is placed on a demarcatedarea on each fingerpad and the inoculum allowed to dry. Thecon

19、taminated area then is exposed to the control (standard hardwater) or test agent for the desired contact time and virusremaining on the fingerpad is eluted and the eluates are titratedfor infectious virus along with the required controls. Percentreductions in the amounts of infectious virus after tr

20、eatmentwith the control and test agents are then determined. Thefingerpad method gives results that are comparable to thoseobtained using a whole-hand procedure (1,6). If two differentformulations are being compared in the same test, one of themmay be designated as a reference and used in place of t

21、he hardwater control. If desired, one also may use tap water in parallelwith the hard water control to determine the influence of waterhardness on the test products virus eliminating activity.5. Significance and Use5.1 This in vivo procedure is designed to test the ability ofhygienic handwash agents

22、 to reduce levels of selected infec-tious viruses from experimentally contaminated fingerpads ofadult volunteers. Since the two thumbpads and all eightfingerpads can be used in any given test, it allows for theincorporation of input virus control (two), amount of virusremaining after the inoculum ha

23、s been allowed to dry (two),virus eliminated after treatment with a control or referencesolution (two), and up to four replicates to assess the virus-eliminating efficiency of the product under test. No more than100 L of the virus suspension are required to complete onetest. The results of testing w

24、ith this test method may form thebasis for confirmatory tests using a suitable whole-hand testprotocol.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 applying the techniquesnecessary for pro

25、pagation and maintenance of host and testvirus. For a reference text, refer to Schmidt and Emmons (7).5.3 Whereas, this test method relates to testing with virusesof human origin, it can be readily adapted to work withbacteria, fungi, protozoa and bacteriophages.5.4 Infectious microorganisms left on

26、 hands after washingcan be reduced further by drying the washed hands with paper,cloth, or warm air (8). A step for the drying of fingerpads afterexposure to the control or test solution, therefore, has not beenincluded to avoid virus removal by the drying process itself.5.5 This test method is not

27、meant for use with surgical handscrubs or preoperative skin preps.5.6 The amount of virus on each fingerpad after the dryingof the inoculum should not be less than 104infectious units thatwould permit the detection of up toa4log10reduction in theinfectivity titer of the virus by a given product unde

28、r theconditions of this test method.6. Equipment and Apparatus6.1 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).6.2 IncubatorAn incubator at 36 6 1C is needed forgrowing host c

29、ells 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.6.3 Positive Displacement PipetteA pipette and pipettetips that accurately can dispense 10-L volumes.6.4 SterilizerAn

30、y 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 Sterilization SystemA membrane or cartridgefiltration system (0.22-m pore diameter) is required forsterilizing hea

31、t-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 lower is requiredto store viruses.6.7 RefrigeratorA refrigerator at 46 2C for storage ofprepared cell culture media a

32、nd 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 mediaor other solutions.6.10 Handwashing SinkA sink of sufficient size to permitpanelists to wash hands without tou

33、ching 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 with electronic sensorsor those that are wrist-, elbow-, knee-, or foot-operated arepreferred to avoid recontaminat

34、ion of the washed hands.6.10.2 Tap Water Temperature Regulator and TemperatureMonitor, to monitor and regulate water temperature at 40 62C.6.11 Liquid Nitrogen Storage for CellsA proper liquidnitrogen container and liquid nitrogen for cryopreservation ofthe stocks of cell lines.E18380226.12 Inverted

35、 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, and 1.0-mL capacity.7.2 Cell Culture Flasks4Plastic cell culture flasks of 25 or75-cm2capacity for culturing cel

36、ls and for preparing viruspools.NOTE 2Each flask for growing cell monolayers can be reused ten ormore times before being discarded.7.3 Cell Culture Media and Supplements5Culture mediaand the types and ratios of supplements will vary depending onthe cell line. Eagles minimal essential medium (EMEM) w

37、ith5 to 10 % fetal bovine serum (virus- and mycoplasma-tested) isused for growing a wide variety of cells (see Note 3).7.4 Soil Load:7.4.1 Fetal Bovine Serum, at a final concentration of 5 % inthe virus inoculum (see Note 3).7.4.2 A tripartite soil load, as an alternative to serum. Add0.5 g of trypt

38、one to 10 mL of phosphate buffer. Add 0.5 g ofbovine serum albumin (BSA) to 10 mL of phosphate buffer.Add 0.04 g of bovine mucin to 10 mL of phosphate buffer.Prepare the stock solutions separately and sterilize by passagethrough a 0.22 m pore diameter membrane filter, aliquot andstore at either 462C

39、 or 2062C. To obtain a 500-Linoculum of the test inoculum, add to 340 L of the microbialsuspension 25 L BSA, 100 L mucin and 35 L of tryptonestock solutions. This mixture contains approximately2goftotal protein/L, which is approximately equivalent to theprotein content of a 5 % solution of fetal bov

40、ine serum.NOTE 3Fetal bovine serum is considered unsuitable for use as anorganic load when working with rotaviruses because of its rotavirusinhibitory and trypsin-neutralizing activity.7.5 Standard Hard WaterThe quality and disinfectant (forexample, chlorine) residual in tap water can vary from site

41、 tosite and also at different 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 accordce with AOAC 960.09 E and F (9) toa standard hardness of 200 ppm as calcium carbonate is u

42、sedfor dilution of test products, as the control solution to deter-mine the baseline level of virus elimination, and to rinse thefingerpads after exposure to the test product. The standard hardwater and tap water (if used) must first be tested to ensure thatthey do not have any virucidal activity ag

43、ainst the testvirus(es).7.6 Test AgentsAt least two samples of the product shallbe tested.7.7 Diluent for Virus TitrationEarles balanced salt solu-tion (EBSS) with a pH of 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 i

44、nside diameter of about 8 mm will be required fordemarcation of the fingerpads and to hold various test solu-tions.7.10 Miscellaneous Laboratory WareAutomatic pipettes,pipette tips, plastic vials for storing cell and virus stocks,dilution tubes, cluster plates, or flasks for virus titration.8. Test

45、Viruses and Cell Cultures8.1 The selection of the following test viruses is based ontheir (a) relative safety to the volunteers as well as experiment-ers, (b) ability to grow to titers sufficiently high for testing, (c)property to produce cytopathic effects or plaques, or both, incell cultures, (d)

46、potential to spread through contaminatedhands, and (e) relative resistance to agents used in hygienichandwashing. Other strains or types of viruses may be substi-tuted provided they meet the preceding criteria.NOTE 4There is insufficient information on whether the passagehistory, culture conditions,

47、 and strain differences of viruses can influencethe efficiency of their elimination by hygienic handwash agents. Cautionmust be exercised, however, when substituting viruses as this may lead tovariations in results from one laboratory to another.8.2 Human Adenovirus Type 4 (ATCC VR-4): Recom-mended

48、lines for making virus pools and infectivity titrationsare 293 (CRL-1573) and Vero (ATCC CCL-81) cells, respec-tively.8.3 Hepatitis A Virus Strain HM-175 (ATCC VR-1402):Recommended cell line FRhK-4 (ATCC CRL-1688).8.4 Human Rotavirus Wa (ATCC VR-2018)Recommended cell line: CV-1 (ATCC CCL-70) or MA-1

49、04(CRL-2378).8.4.1 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 also must be free from serum. Most rotavirusesalso require the presence of trypsin in the medium for growthand plaques formation.8.5 Human Rhinovirus Type 37 (ATCC VR-1147) or Rhi-novirus 14 (ATCC VR-284)Recommended cell line: MRC-5(ATCC CCL-171), WI-38 (ATCC CCL-75) or HeLa T4+cells.(Incubation of infected cells at 33C is re