ASTM E2784-2010(2015) Standard Test Method for Evaluation of the Effectiveness of Handwash Formulations Using the Paper Towel (Palmar) Method of Hand Contamination《使用手污染纸巾 (掌) 法评定洗.pdf

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1、Designation: E2784 10 (Reapproved 2015)Standard Test Method forEvaluation of the Effectiveness of Handwash FormulationsUsing the Paper Towel (Palmar) Method of HandContamination1This standard is issued under the fixed designation E2784; the number immediately following the designation indicates the

2、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 () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method covers the determination of the eff

3、ec-tiveness of antimicrobial handwashing agents for the reductionof transient microbial flora when used in a handwashingprocedure.1.2 A knowledge of microbiological techniques is requiredfor these procedures.1.3 This test method may be used to evaluate topicalantimicrobial handwash formulations.1.4

4、Performance of this procedure requires the knowledgeof regulations pertaining to the protection of human subjects.21.5 In this test method, SI units are used for all applications,except for distance in which case inches are used and SI unitsfollow in parentheses.1.6 This standard does not purport to

5、 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. For more specificprecautionary statements see 8.5.2

6、. Referenced Documents2.1 ASTM Standards:3E1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE1174 Test Method for Evaluation of the Effectiveness ofHealth Care Personnel Handwash Formulations2.2 Other Standards:AATCC Test Method 147 Antibacterial Assessment of Tex-tile Materi

7、als: Parallel Streak Method43. Terminology3.1 Definitions:3.1.1 active ingredient, na substance added to a formula-tion specifically for the inhibition or inactivation of microor-ganisms.3.1.2 cleansing wash, na procedure intended to removesoil or residue.This may also be referred to as a cosmetic w

8、ash.3.1.3 healthcare personnel handwash, na cleanser orwaterless agent intended to reduce transient bacteria on thehands.3.1.4 neutralization, nthe process for inactivating orquenching the activity of a microbiocide. Often achievedthrough chemical or physical means (for example, filtration ordilutio

9、n).3.1.5 resident microbial skin flora, nmicroorganisms thatsurvive and multiply on the skin, forming a stable population.3.1.6 test material, na formulation which incorporatesantimicrobial ingredient(s).3.1.7 test organism, nan applied inoculum of an organismthat has characteristics which allow it

10、to be readily identified.The test organism is used to simulate a transient topicalmicrobial contaminant. It may also be referred to as a markerorganism, bacterial simulant, or bacterial contaminant.3.1.8 transient microbial skin flora, nmicroorganisms thatcontaminate the skin but do not normally for

11、m a stablepopulation.4. Summary of Test Method4.1 This test method is conducted on a group of volunteersubjects who have refrained from using topical antimicrobialformulations for at least one week prior to the initiation of thetest.Activity of the test material is measured by comparing the1This tes

12、t method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2015. Published November 2015. Originallyapproved in 2010. Last previo

13、us edition approved in 2010 as E278410. DOI:10.1520/E278410R15.2Federal Register, Vol 46, No. 17, Jan 27, 1991.3For 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 s

14、tandards Document Summary page onthe ASTM website.4Technical Manual of the American Association of Textile Chemists andColorists, P.O. Box 12215, Research Triangle Park, North Carolina 27709.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United St

15、ates1number of test organisms recovered from artificially contami-nated hands after use of a handwashing formulation to thenumber recovered from contaminated hands not exposed to thetest formulation. The method describes specific procedures tobe followed using Serratia marcescens as the test organis

16、m.The activity of the test material is measured following a singlewash in a single day using a neutralization recovery method.4.2 Alternative test organisms which may be used areEscherichia coli, Shigella flexneri, and Staphylococcus aureus.Culture media and incubation conditions appropriate for the

17、alternative organisms should be employed.4.3 The investigator should be aware that there may behealth risks associated with the use of the test organisms andprecautions similar to those referenced in 8.5 should beundertaken.5. Significance and Use5.1 This procedure has been designed to evaluate hand

18、washproducts using a palmar surface only contamination method.This method is an alternative contamination procedure to thatlisted in Test Method E1174. The current contaminationprocedure in Test Method E1174 describes a standardizedprocedure for contaminating the entire hand, palmar surfaceand back,

19、 directly using a marker organism. The contaminationprocedure in Test Method E1174 does not necessarily representreal world hand contamination. During routine activities it isonly the palmar surface, comprising palms, fingers, and fingerpads of the hands that becomes contaminated by contact withtran

20、sient microorganisms. These microorganisms can then betransferred to food or objects. Methods to measure the amountof microorganisms transferred to food or objects can be foundin Fischler et al5and Fuls et al6and will be developed into afuture ASTM standard.6. Apparatus6.1 Colony CounterAny of sever

21、al types may be used, forexample, Quebec Colony Counter.6.2 IncubatorAny incubator capable of maintaining thefollowing temperatures: S. marcescens (25 6 2Cthis tem-perature is required to ensure pigment production for S.marcescens); S. aureus, E. coli, S. flexneri (35 6 2C).6.3 SterilizerAny suitabl

22、e steam sterilizer capable of pro-ducing the conditions of sterilization is acceptable.6.4 Timer (Stop-Clock)One that can be read for minutesand seconds.6.5 Handwashing SinkA sink of sufficient size to permitsubjects to wash without touching hands to sink surface orother subjects.6.5.1 Water Faucet(

23、s)To be located above the sink at aheight which permits the hands to be held higher than theelbow during the washing procedure. Faucet should maintain aflow rate of 4 L per minute, as determined in (10.3).6.5.2 Tap Water Temperature Regulator and TemperatureMonitorTo monitor and regulate water tempe

24、rature of40 6 2C.6.6 Vortex MixerAny suitable vortex mixer capable ofmixing sample and diluent.6.7 Sterile Bacteriological Pipets1.1, 2.2, 5.0, and 30.0mL capacity.6.8 Adjustable or Fixed Volume Pipets and Sterile Tips0.1mL and 1.0 mL capacity.6.9 Sampling ContainersAny sterile or sterilizable con-t

25、ainer having tight closures and sufficient capacity to hold 75mL sampling solution (7.3).6.10 Sterile ContainerAny sterile or sterilizable containerhaving the capacity to culture the amount of inoculum requiredfor testing.6.11 GlovesLoose-fitting, unlined, powder-free gloveswhich possess no antimicr

26、obial properties, or equivalent.Perform a zone of inhibition test, such asAATCC Test Method147, to evaluate the antibacterial activity. (Plastic bags (6.12)with low bioburden may be used in place of gloves.)6.12 Plastic BagsMay be used in place of gloves (6.11).Bags should be approximately 29 by 31

27、cm, possess noantimicrobial properties, and have a low bioburden. Perform azone of inhibition test, such as AATCC Test Method 147, toevaluate the antibacterial activity.6.13 Wrist Ties or TourniquetsAny item which will allowthe plastic bags (6.12) or gloves (6.11) to be secured to thesubjects wrist.

28、6.14 Sterile Paper Towel PouchesEach pouch consists oftwo single-ply paper towels, each of which measures approxi-mately 20 by 32 6 5 cm, encased in aluminum foil.6.14.1 Fold two single-ply paper towels in half, lengthwiseto form a rectangle approximately 20 by 16 cm. Place onepaper towel inside of

29、the other.6.14.2 Place the paper towels in a piece of aluminum foilwhich has been folded in half, widthwise. The aluminum foilshould measure approximately 38 by 23 cm, after folding.Aluminum foil which is rated as “Heavy Duty” and has aminimum thickness of 0.2 mm is recommended to minimizethe risk o

30、f tearing during handling. Fold the edges of thealuminum foil together to form a pouch ensuring that the papertowels remain flat. Sterilize the pouch by autoclaving.6.15 Sterile Centrifuge TubesMinimum of 50 mL capac-ity.7. Reagents and Materials7.1 Cleansing WashA mild, proven, non-antimicrobialsof

31、t soap. The formula in Table 1 can be used if a mild,non-antimicrobial soft soap is not commercially available.7.1.1 Add linseed oil to a solution of potassium hydroxidein 15 parts water and heat up to approximately 70C while5Fischler, et al, “Effect of Hand Wash Agents on Controlling the Transmissi

32、onof Pathogenic Bacteria from Hands to Food,” Journal of Food Protection, Vol 70,No. 12, 2007, pp. 28732877.6Fuls, et al, “Alternative Hand Contamination Techniques to Compare theActivities of Antimicrobial and Nonantimicrobial Soaps Under Different TestConditions,” Applied and Environmental Microbi

33、ology , Vol 74, No. 12, June 2008,pp. 37393744.E2784 10 (2015)2constantly 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 1

34、00 parts byaddition of hot water. Take 200 g of the soft soap in 1 L ofwater. Dispense in to appropriate containers and sterilize in anautoclave.7.2 Test MaterialManufacturer directions for use of thetest material should be utilized. If directions are not available,use the directions provided in thi

35、s test method.7.3 Sampling SolutionDissolve 0.4 g monopotassiumphosphate (KH2PO4), 10.1 g disodium hydrogen phosphate(Na2HPO4), 1.0 g isooctylphenoxypolyethoxyethanol (forexample, Triton X-100), and appropriately validated neutraliz-ers in 1 L distilled water. Adjust pH with 0.1 N hydrochloricacid (

36、HCl) or 0.1 N sodium hydroxide (NaOH). Sterilize in anautoclave. Final pH after sterilization is 7.8 6 0.1 Dispense sothat final volume after sterilization is 75 mL.7Perform TestMethod E1054 to determine what neutralizers are required.7.4 Dilution FluidSterile Butterfields buffered phosphatediluent8

37、or other suitable diluent with appropriately validatedneutralizers. Perform Test Method E1054 to determine whatneutralizers are required. The addition of neutralizer is onlyrequired if inactivation of the test material cannot be achievedupon dilution into the sampling solution (7.3). Adjust pH with0

38、.1 N HCl or 0.1 N NaOH. Final pH after sterilization 7.2 60.1.7.5 Soybean-Casein Digest AgarSterile tryptic soy agar orother solid media appropriately validated to support growth ofSerratia species. With appropriate neutralizers if required perTest Method E1054.7.6 Hektoen Enteric AgarUsed for the r

39、ecovery andgrowth of Shigella species. With appropriate neutralizers ifrequired per Test Method E1054.7.7 Mannitol Salt AgarSterile. Used for the recovery andgrowth of Staphylococcus species. With appropriate neutraliz-ers if required per Test Method E1054.7.8 Soybean-Casein Digest Agar with MUG9Ste

40、rile tryp-tic soy agar with MUG, used for the indication, recovery andgrowth of Escherichia species or other solid media appropri-ately validated to support the growth of the test organism. Withappropriate neutralizers if required per Test Method E1054.7.9 BrothSterile soybean-casein digest broth (t

41、ryptic soybroth) or other liquid media appropriate to support growth ofthe test organism.7.10 Ethanol or Isopropyl Solution70 % ethanol or iso-propyl alcohol in water (v/v) for hand decontamination.7.11 Antibiotic OintmentA topical triple-antibiotic oint-ment for application to the hands after the f

42、inal decontamina-tion.7.12 Chlorhexidine Skin CleanserAntiseptic skin cleansercontaining 4 % chlorhexidine gluconate (w/v) for hand decon-tamination.7.13 Physiological SalineSterile. Used to dilute inoculumif lower levels are desired.8. Test Organisms8.1 Serratia marcescens (ATCC 14756) is to be use

43、d as thetest organism. This is a strain having stable pigmentation at25C. The plating agar should be soybean-casein digest agar(7.5).8.2 Escherichia coli (ATCC 11229) is an alternative testorganism. When E. coli is used, the plating agar should besoybean-casein digest agar with MUG (7.8) or another

44、suitableindicator.8.3 Shigella flexneri (ATCC 700930) is an alternative testorganism. When S. flexneri is used, the plating agar should beHektoen enteric agar (7.6).8.4 Staphylococcus aureus (ATCC 6538) is an alternativetest organism. When S. aureus is used, the plating agar shouldbe Mannitol salt a

45、gar (7.7).8.5 (WarningThe application of microorganisms to theskin may involve a health risk. Prior to applying the testorganism to the skin, the antibiotic sensitivity profile of thestrain should be determined. If an infection occurs, the antibi-otic sensitivity profile should be made available to

46、the attend-ing clinician. Following the subjects last contamination andwash with the formulation, a decontamination procedureshould be performed (Section 13.)8.6 Preparation of Test Organism Suspension:8.6.1 Serratia marcescens (ATCC 14756)A homogeneousculture is used to inoculate the hands. The sto

47、ck culture, frozenor lypholized, should be at least two 24 h soybean-casein digestbroth (7.9) transfers from the original ATCC culture, but thereshould be no more than five transfers removed from the ATCCculture. From the stock, inoculate the appropriate volume of7Peterson, A. F., “The Microbiology

48、of the Hands: Evaluating the Effects of theSurgical Scrubs,” Developments in Industrial Microbiology, Vol 14, 1973, pp.125130.8Horowitz, W., (Ed.), Offcial Methods of Analysis of the AOAC, 17th Ed., Sec.6.3.03 A.(f), Chapter 6, 2000, p.10. Official Methods of Analysis of AOACInternational, Gaithersb

49、urg, MD.9United States Pharmacopeia 32: United States Pharmacopeial Convention,Inc., Rockville, MD, Chapter entitled “Microbial Limits Test.” The MUG (4-methylumbelliferyl-D-gluconride) substrate is hydrolyzed by -D-gluconridase toyield a fluorescent end product, 4-methylumbelliferone. -D-gluconridase is pos-sessed by E. coli (ATCC 11229). MUG is incorporated into the appropriate growthmedium at 0.05 g/L.TABLE 1 Formula for Mild, Non-Antimicrobial Liquid Soft SoapSoft Soap, 200 g/LLinseed oil 50 parts by weightPotassium hydro