1、Designation: E2946 13Standard Test Method forDetermining the Bacteria-Reducing Effectiveness of Food-Handler Handwash Formulations Using Hands of Adults1This standard is issued under the fixed designation E2946; 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.1. Scope1.1 This test method is designed to determine the activity offood-handler handwas
3、hes against transient bacterial flora onthe hands.1.2 Performance of this procedure requires the knowledgeof regulations pertaining to the protection of human sub-jects(1)2.1.3 This test method should be performed by persons withtraining in microbiology, in facilities designed and equippedfor work w
4、ith potentially infectious agents at biosafety level 2(2).1.4 UnitsThe values stated in SI units are to be regardedas standard. No other units of measurement are included in thisstandard.1.5 This standard does not purport to address all of thesafety concerns, if any, associated with its use. It is t
5、heresponsibility 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. For more specificprecautionary statements see 8.1.1.2. Referenced Documents2.1 ASTM Standards:3E1054 Test Methods for Evaluatio
6、n of Inactivators of Anti-microbial AgentsE1174 Test Method for Evaluation of the Effectiveness ofHealth Care Personnel Handwash FormulationsE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 AATCC Standards:4Test Method 147 Antibacterial Activity Assessment of Tex-tile Materials: Pa
7、rallel Streak Method3. Terminology3.1 DefinitionsSee Terminology E2756.3.2 food-handler handwash, na water or non-water-aidedformulation for use in food service settings intended to kill orremove transient bacteria, or both from the hands.3.3 test material, na product or formulation that incorpo-rat
8、es one or more antimicrobial ingredients.4. Summary of Test Method4.1 This test method is performed using adult subjects whohave provided written informed consent, and whose handshave been determined to be free of any apparent damage at thetime of participation in the study. Subjects are to refrain
9、fromuse of any antimicrobials for at least one week prior to theinitiation of test procedures (Section 11).4.2 Subjects contaminate hands with E. coli incorporatedinto a medium or heavy organic soil (beef broth or hamburger,respectively), which then is distributed over all surfaces of thehands and f
10、ingers to result in a minimum baseline recoverypopulation of 107colony forming units (CFU)/hand.4.3 Test material effectiveness is measured by comparingthe number of challenge bacteria recovered from contaminatedhands after a single application of the test material to thenumber recovered from contam
11、inated hands not exposed to thetest material.5. Significance and Use5.1 Hand hygiene is considered one of the most importantmeasures for preventing the spread of infectious microorgan-isms and is critical for reducing the incidence of food-bornedisease. Food-handling settings are unique in that mode
12、rate toheavy soil load present on hands often can influence the abilityof a product to remove or kill microorganisms (3, 4). Test1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommit
13、tee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2013. Published November 2013. DOI:10.1520/E2946132The boldface numbers in parentheses refer to a list of references at the end ofthis standard.3For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Cus
14、tomer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Available from American Association of Textile Chemists and Colorists(AATCC), P.O. Box 12215, Research Triangle Park, NC 27709, http:/www.aatcc.org.
15、Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1methods are needed for assessing the efficacy of hand hygieneproducts under conditions representative of those encounteredin a food-handling environment.5.2 This test method is specifica
16、lly designed to evaluate theeffectiveness of food-handler products to kill and removebacteria from experimentally-contaminated hands under con-ditions of moderate to heavy organic soil load. The inclusion ofsoils typical of food service setting makes this a methodologymore appropriate than Test Meth
17、od E1174, which was de-signed to evaluate healthcare personnel hand washes and doesnot include an option to include soil (4).6. Apparatus6.1 Colony CounterAny of several types may be used; forexample, Quebec colony counters and similar devices.Automated, computerized plater/counter systems may also
18、beused.6.2 Gloves, sterile, loose-fitting, unlined, powder-freegloves possessing no antimicrobial properties. Perform a zoneof inhibition test, such asAATCC Test Method 147, to evaluatefor antibacterial activity.6.3 Handwashing Sink, sufficient in size to permit hand-washing without the touching of
19、hands to sink surface or othersubjects.6.3.1 Water Faucet(s)Located above the sink at a heightto permit hands to be held higher than the elbow during thewashing procedure.6.3.2 Tap Water Temperature Regulator and TemperatureMonitorTo set and maintain the tap water temperature at 406 2C.6.4 Incubator
20、, capable of maintaining temperatures of 35 62C.6.5 Miscellaneous LabwareContinuously adjustable pi-petters (1-mL and 0.2-mL capacity) and sterile pipette tips,sterile serological pipettes (5.0-mL capacity), sterile culturetubes, sterile disposable Petri dishes, sterile syringes, Erlen-meyer flasks,
21、 and beakers.6.6 Sampling Containers, sterile or sterilizable containershaving tight closures and sufficient capacity to hold 75 mL ofsampling solution (7.7)6.7 Sterilizer, any steam sterilizer capable of processingculture media and reagents.6.8 Timer (Stop-Clock), type that can be read for minutesa
22、nd seconds.6.9 Tourniquets, child size or any style capable of securinggloves to the wrist.6.10 Vortex MixerAny vortexing device that will ensureproper mixing of culture tubes.7. Reagents and Materials57.1 Cleansing WashAny mild, proven non-antimicrobialliquid soap. It may be purchased commercially
23、or as anexample it may be prepared according to instructions formaking soft soap.Soft Soap, 200 g/LLinseed oil 50 parts by weightPotassium hydroxide 9.5 partsEthanol 7 partsDistilled or high purity water as neededAdd linseed oil to a solution of potassium hydroxide in 15parts water and heat up to ap
24、proximately 70C while con-stantly stirring. Add the ethanol and continue heating whilestirring until the saponification process is completed and asample dissolves clearly in water and almost clearly in alcohol.The weight of the soft soap is then brought up to 100 parts byaddition of hot water. Take
25、200 g of the soft soap in 1 L ofwater. Dispense in to appropriate containers and sterilize in anautoclave.7.2 Chlorhexidine Skin CleanserAntiseptic skin cleansercontaining 4% chlorhexidine gluconate (w/v) for hand decon-tamination.7.3 Culture Media:7.3.1 BrothSoybean-casein digest broth (tryptic soy
26、broth) is recommended.7.3.2 Agar Plating Media:7.3.2.1 MacConkey Agar is recommended. E. coli (ATCC#11229) will produce purple colonies on this agar.7.4 Beef BrothAny standard, sterile liquid beef broth, suchas Swanson brand beef broth or other similar product, would besuitable, low sodium or other
27、modified versions are notrecommended.7.5 HamburgerGamma-irradiated 90% lean ground beefto ensure that the ground beef contains no contaminatingmicroorganisms prior to use in testing; ground beef can bepurchased pre-irradiated from a standard meat supplier.7.6 Dilution FluidSterile Butterfields buffe
28、red phosphatediluent (5) (or other suitable diluent) adjusted to pH 7.2 6 0.1and containing an effective inactivator of the antimicrobialchemistry of the test material, if necessary.NOTE 1Inactivator is required only if neutralization of the testmaterial cannot be achieved upon dilution into the Sam
29、pling Solution(7.8).7.7 Ethanol Solution70% ethanol in water (v/v) for handdecontamination.5Reagent Chemicals, American Chemical Society Specifications, AmericanChemical Society, Washington, DC. For Suggestions on the testing of reagents notlisted by the American Chemical Society, see Annual Standar
30、ds for LaboratoryChemicals, BDH Ltd., Poole, Dorset, U.K., and the United States Pharmacopeiaand National Formulary, U.S. Pharmacopeial Convention, Inc. (USPC), Rockville,MD.E2946 1327.8 Sampling SolutionDissolve 0.4 g KH2PO4, 10.1 gNa2HPO4, 1.0 g isooctylphenoxypolyethoxyethanol,6and ap-propriately
31、 validated neutralizers, if necessary, in distilledwater. Adjust pH to 7.8 6 0.1 with 0.1 N HCl or 0.1 N NaOHand bring volume to 1 L with distilled water. Sterilize in anautoclave and aseptically dispense 75-mL aliquants into sterilesampling containers (6.6)(6).NOTE 2Aneutralizer validation should b
32、e conducted according toTestMethod E1054 prior to the study. Test Method E1054 provides a list ofneutralizers appropriate for commonly used antimicrobial agents. In somecases neutralization can be achieved by dilution alone.7.9 Test MaterialUse directions provided with the testmaterial. If direction
33、s are not provided, use the directionsprovided in this method.8. Test Bacteria Species8.1 Escherichia coli ATCC 11229.8.1.1 WarningApplication of microorganisms to the skinmay involve a health risk. Determine the antibiotic sensitivityprofile of the test bacteria prior to applying to the skin. After
34、testing has been completed, decontaminate the subjects handsand follow proper procedures to reduce infection risk (12.1 12.2). If an infection occurs, provide the antibiotic susceptibil-ity profile to the attending clinician.9. Preparation of Test Bacteria Suspension9.1 Method 1( for moderate soil m
35、atrix with beef broth): :9.1.1 Prepare a stock culture by transferring E. coli (ATCC11229) from a cryogenic stock or lyophilized vial or pellet intoapproximately 5 mL of soybean-casein digest broth (7.3.1) andincubate for 24 6 2hat356 2C. Inoculate a volume ofsoybean-casein digest broth with 1 mL of
36、 the stock culture per1000 mL of broth to yield a volume of suspension sufficient tocomplete the study. The volume of the suspension should notexceed one half of the capacity of the Erlenmeyer flask.Incubate for 24 6 2hat356 2C to yield a titer ofapproximately 1.0 108CFU/mL.NOTE 3The frozen or lyoph
37、ilized stock should be at least two, but nomore than four, 24-h soybean-casein digest broth transfers from theoriginal ATCC culture.9.1.2 Transfer the culture to appropriately sized sterilecentrifuge tubes or bottles and centrifuge to sediment theculture completely. Recommended conditions are 4750 6
38、 50rpm for 30 min. Decant the supernatant and resuspend thepellet in a volume of beef broth equal to the original culturevolume (7.4) to yield a homogeneous challenge suspensioncontaining approximately 1.0 108CFU/mL.9.1.3 Swirl or shake suspension prior to withdrawal of eachaliquot used for testing.
39、Assay the suspension for the number ofbacteria at the beginning and at the end of the use period. Donot use a suspension for more than 8 h. The population shouldnot vary by more than 6 0.5 log10CFU/mL over the 8-hperiod.9.2 Method 2 (for heavy soil matrix with hamburger):9.2.1 Prepare a stock cultur
40、e by transferring E. coli ATCC11229 from a cryogenic stock or lyophilized vial or pellet intoapproximately 5 mL of soybean-casein digest broth (7.3.1) andincubate for 24 6 4hat356 2C. Inoculate a volume ofsoybean-casein digest broth with 1 mL of the stock culture per1000 mL of broth to yield a volum
41、e of suspension sufficient tocomplete the study. The volume of the suspension should notexceed one half of the capacity of the Erlenmeyer flask.Incubate for 24 6 4hat356 2C to yield a titer ofapproximately 1.0 108CFU/mL.9.2.2 Swirl or shake suspension prior to withdrawal of eachaliquot used for test
42、ing, and assay it for the number of bacteriapresent at the beginning and at the end of the use period. Donot use a suspension for more than 8 h. The population shouldnot vary by more than 6 0.5 log10CFU/mL over the 8-hperiod.9.2.3 Prior to testing, weigh approximately 4-ounce (113 g)portions of gamm
43、a-irradiated ground beef (7.5). Assay arepresentative sample of ground beef for viable E. coli at thebeginning of the use period, if the E. coli was found to becontaminated it is recommended that subjects that used thatinoculum are replaced. Immediately prior to each handcontamination, dispense 5.0
44、ml of the challenge suspension(9.2.1) into a ground beef portion and, using gloved hands,knead the beef portion in a manner to ensure even distributionof the suspension fluid evenly throughout the portion.10. Subjects10.1 Recruit a sufficient number of healthy adult humansubjects who have no clinica
45、l evidence of dermatoses, cuts,lesions, hangnails, or other skin disorders on the hands orforearms. A minimum of eight (8) subjects should be used foreach test material. The total number of subjects used willdepend on the number of test materials, the purpose of thestudy, and the regulatory requirem
46、ents governing the study.10.2 It is the responsibility of the user of this test method toobtain the necessary approval from an Institutional ReviewBoard (IRB) or Independent Ethics Commission (IEC) for theuse of adult human subjects for testing and to obtain informedand written consent from those se
47、lected for the study beforestarting the tests.10.3 Instruct subjects to avoid contact with antimicrobialproducts for the duration of the test and for at least one weekprior to the test. This restriction includes antimicrobial-containing antiperspirants, deodorants, shampoos, lotions, andsoaps. Bathi
48、ng in biocide-treated pools, hot tubs, or spasshould be avoided. Harsh chemicals such as acids, bases, andsolvents should also be avoided. Subjects may not use topicalor systemic antimicrobials, antibiotics, or steroids other thanfor contraception or post-menopausal indications, and mustagree to abs
49、tain from these materials until the completion ofthe study. Provide subjects with a kit of non-antimicrobialpersonal care products for exclusive use during the test andinclude rubber gloves to be worn when contact with antimi-crobial or harsh chemicals cannot be avoided.6Sold under severalTrade Names includingTriton X-100 (The Dow ChemicalCompany, Midland, MI), Igepal CA-630 (Rhodia, Inc., Cranbury, NJ) andProtachem OP-9 (Protameen Chemicals, Inc., Totowa, NJ).E2946 13311. Procedure11.1 Admission to TestingInstruct each subject to return t
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