ASTM E2897-12(2017) Standard Guide for Evaluation of the Effectiveness of Hand Hygiene Topical Antimicrobial Products using ex vivo Porcine Skin.pdf

1、Designation: E2897 12 (Reapproved 2017)Standard Guide forEvaluation of the Effectiveness of Hand Hygiene TopicalAntimicrobial Products using ex vivo Porcine Skin1This standard is issued under the fixed designation E2897; 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.INTRODUCTIONUse of ex vivo animal skin models such as pigskin has widely been us

3、ed as surrogate for humanskin. Pigskin model is a safe, inexpensive, accurate, and reliable platform of testing antisepticefficacy.2,3,4The test guide described here utilizes sterilized pigskin to evaluate the effectiveness ofhand hygiene topical antimicrobial products. The pigskin substrate is used

4、 to overcome limitationsposed by exposure of human subjects to potentially pathogenic microorganisms, while offering thebenefit of applicability to a wide variety of hand-washing conditions that cannot be simulated in testtubes. The microbial reduction is the difference in log10value obtained from a

5、rtificially contaminatedpigskins after use of test formulation to the log10value obtained from contaminated pigskins notexposed to the test formulation.1. Scope1.1 This guide is designed to demonstrate the effectivenessof hand hygiene topical antimicrobial products using pigskin asa surrogate model.

6、1.2 Knowledge of microbiological techniques is requiredfor these procedures.1.3 This standard guide can be used to evaluate topicalantimicrobial handwash or handrub formulations.1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandar

7、d.1.5 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.1.6 This inte

8、rnational standard was developed in accor-dance with internationally recognized principles on standard-ization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recom-mendations issued by the World Trade Organization TechnicalBarriers to Trade (TBT)

9、Committee.2. Referenced Documents2.1 ASTM Standards:5E1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE1174 Test Method for Evaluation of the Effectiveness ofHealth Care Personnel Handwash FormulationsE1874 Test Method for Recovery of Microorganisms FromSkin using the Cup Sc

10、rub Technique3. Terminology3.1 Definitions:3.1.1 antimicrobial ingredient, na substance added to aformulation specifically for the inhibition or inactivation ofmicroorganisms.3.1.2 neutralization, nthe process for inactivating orquenching the activity of a microbicide, often achieved throughphysical

11、 (for example, filtration or dilution) or chemical means.3.1.3 resident microorganisms, nmicroorganisms that sur-vive and multiply on the skin, forming a stable population.1This guide is under the jurisdiction of ASTM Committee E35 on Pesticides,Antimicrobials, and Alternative Control Agents and is

12、the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2017. Published April 2017. Originallyapproved in 2012. Last previous edition approved in 2012 as E289712. DOI:10.1520/E2897-12R17.2Woolwine, J. D., and Gerberding, J. L., “Effect of Testing Met

13、hod on ApparentActivities of Antiviral Disinfectants and Antiseptics,” Antimicrobial Agents andChemotherapy, Vol 39, 1999, pp. 921923.3Bush, L. W., Benson, L. M., and White, J. H., “Pigskin as a Test Substrate forEvaluating Topical Antimicrobial Activity,” J. Clinical Microbiology, Vol 24, 1986,pp.

14、343348.4McDonnel, G., Haines, K., Klein, D., Rippon, M., Walmsley, R., and Pretzer,D., “Clinical Correlation of a Skin Antisepsis Model,” J. Microbiological Methods,Vol 35, 1999, pp. 3135.5For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at service

15、astm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with

16、 internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.13.1.4 transient microorganisms, nmicroorga

17、nisms thatcontaminate the skin but do not form a stable population.3.1.5 test organism, nan applied inoculum of an organismthat has characteristics which allow it to be readily identified.The test organism is used to simulate a transient topicalmicrobial contaminant. It may also be referred to as a

18、markerorganism, bacterial simulant, or bacterial contaminant.3.1.6 test substance, na leave-on or wash-off product orformulation which incorporates antimicrobial ingredient(s).4. Summary of Guide4.1 This guide describes procedures for testing using non-living pigskin substrates that have been shaved

19、 to remove thehair and sterilized using gamma radiation prior to use intesting. The specific procedures allow use of a test microor-ganism of the investigators choice.Activity of a test substanceis measured by comparing the number of test microorganismsrecovered from artificially contaminated pigski

20、ns after use ofan antimicrobial formulation to the number of test microorgan-isms recovered from contaminated pigskins not exposed to thetest formulation. The antimicrobial activity of the test sub-stance can be measured following a single wash or multiplewashes, or both, in a single day. Sampling i

21、s performed usingthe cup scrub technique and a fluid shown to neutralizeeffectively the antimicrobial activity of the test formulation,and to be non-toxic to the test microorganism.5. Significance and Use5.1 The guide may be used to demonstrate the effectivenessof topical antimicrobial products usin

22、g pigskin as a surrogatefor human skin and the cup scrub technique for sampling.5.2 The techniques described can be used to simulate TestMethod E1174 and will use the pigskin substrate to overcomelimitations posed by exposure of human subjects to potentiallypathogenic microorganisms, while offering

23、the benefit ofapplicability to a wide variety of hand-washing conditions thatcannot be simulated in test tubes.5.3 Use of the pigskin surrogate offers less expensive andhigher throughput screening.6. Apparatus6.1 Colony CounterAny of several types may be used, forexample, Quebec Colony Counter or si

24、milar devices.6.2 IncubatorAny incubator capable of maintaining de-sired temperature range.6.3 SterilizerAny suitable steam sterilizer capable of pro-ducing the conditions of sterilization.6.4 Timer (Stop-Clock)Type that can be read for minutesand seconds.6.5 SinkA sink of sufficient size to permit

25、the washing ofpigskins without touching the sink surface.6.6 Water Faucet(s)To be located above the sink at heightwhich permits the rubbing of the pigskins during the washingprocedure. Faucet should maintain a constant flow rate.6.7 Water Temperature Regulator and TemperatureMonitorTo set and mainta

26、in the water temperature at 40 62C.6.8 Vortex MixerAny suitable vortex mixer capable ofmixing sample and diluent.6.9 SpectrophotometerAn instrument that can measureoptical density at a wavelength of 620 nm.7. Reagents and Materials7.1 Sterile Bacteriological Pipettes10-mL capacity; 1-mLcapacity and

27、0.1-mL capacity.7.2 Inoculating Loops or Sterile Swabs.7.3 Gamma Sterilized PigskinsPig hides can be obtainedfrom local source, defatted, washed with water and sterilizedby gamma-irradiation (or other acceptable method) and storedin a freezer (20C) prior to use.7.4 Industrial Grade AdhesiveAny of se

28、veral types can beused; for example, epoxy or other suitable glue.7.5 Suitable CarrierFor holding pigskin in place allowingmechanical manipulation (washing, rubbing, and so forth); forexample, phenolic caps/closures.7.6 ScalpelOr any other appropriate cutting tool.7.7 Tissues or Paper TowelsAny ster

29、ile tissue or papertowel that can be used to dry the pigskins.7.8 Sterile ContainerAny sterile or sterilizable containerhaving the capacity to culture the volume of inoculum requiredfor testing.7.9 Scrub CupsSterile plastic/polypropylene or other suit-able cylinders, height approximately 2.5 cm, ins

30、ide diameterapproximately 4.2 cm. Useful sizes range from approximately1.5 to 4.0 cm.7.10 Sterile Polytetrafluoroethylene (PTFE) ScraperCanbe fashioned in the laboratory or purchased.7.11 Sterile Culture Tubes, or equivalent.7.12 Appropriate Bacterial Cultures.7.13 Test Formulation/SubstanceManufact

31、urer directionsfor use of the test substance should be utilized and, prior totesting, the appropriate volume to be applied to the surface areaof the pigskin substrate should be calculated as a ratio of theestimated volume that would exist on the hands of a humansubject. If directions are not availab

32、le, the investigator mustdetermine an appropriately realistic volume.7.14 Sampling and Dilution FluidSterile Butterfieldsphosphate buffered diluent,6containing an antimicrobial inac-tivator specific for the test formulation.NOTE 1Neutralizer validation should be conducted according to TestMethod E10

33、54 prior to the study. Test Method E1054 provides a list ofneutralizers appropriate for commonly used antimicrobial agents. In somecases neutralization may be achieved by dilution alone.6Horowitz, W., (Ed.), Offcial Methods of Analysis of the AOAC, 17th Ed., Sec.6.3.03 A.(f), Chapter 6, 2000, p. 10.

34、 Official Methods of Analysis of AOACInternational, Gaithersburg, MD.E2897 12 (2017)27.15 Plating MediumSoybean-casein digest agar or othersolid media appropriately validated to support growth of thetest organism, with effective neutralizers, if needed.8. Test Substrate8.1 The pigskin substrate is p

35、repared by removing hair withelectronic clippers and/or razor blades prior to irridiation. Careshould be taken not to damage the skin or introduce anyointments or gels during preparation.8.2 Using a sterile scalpel or other suitable tool, cut thesterile pigskin hides into applicable shapes and equal

36、 sizes.NOTE 2Shape and size should accommodate the coverage area of thescrub cup (7.9).8.3 Mount the pigskin substrates to the surfaces of pre-cleaned carriers, by applying the epoxy or other suitableadhesive to the carrier. Follow the adhesive manufacturersinstructions for opening and usage of the

37、product.8.4 The area to be sampled is delineated by the scrubcup/sampling cylinder.8.5 Allow enough time for the pigskin to adhere to thesurface prior to step 9.2.9. Procedure9.1 Test Microorganism(s):9.1.1 Preparation of Test Microbial Suspension(s):9.1.1.1 Test species representative of the bacter

38、ial floraencountered under the conditions of use should be selected fortesting.9.1.1.2 Transfer culture(s) 2 times (once every 18 to 24 h)into appropriate liquid growth medium. The second transfermust be into a volume of medium that will be sufficient totesting.9.1.1.3 Alternatively, the second tran

39、sfer can be to an agarplate or slant.9.1.1.4 If preparing the challenge suspension from broth,wash culture two times by means of centrifugation andresuspend in Butterfields phosphate buffered water.9.1.1.5 If preparing the challenge suspension from agarplate or slant, resuspend organisms in Butterfi

40、elds phosphate-buffered diluent or equivalent.9.1.1.6 Using a spectrophotometer or other comparator,adjust the final titer of the challenge species to 1.0 107to 1.0109cfu/mL. Inoculum should be well mixed to disperseclumps.9.1.1.7 Determine the titer of the challenge suspension by astandard plate co

41、unt method, or equivalent.9.2 Treatment of Test Samples:9.2.1 Contaminate each test pair of pigskin substrates withan appropriate volume of the challenge species. The investiga-tor shall determine the appropriate number of replicates to beused to achieve the desired confidence level.9.2.2 Immediatel

42、y, rub the pigskins together for 15 s toevenly distribute the inoculum while avoiding excessive lossdue to dripping. Allow appropriate dry-time.9.2.3 Apply the appropriate volume of test formulation onthe pair of pigskins. See Appendix X1.9.2.4 Immediately rub the pigskins together for 30 s toevenly

43、 distribute the product while avoiding excessive loss dueto dripping. Expose for the specified contact time.9.2.5 For leave-on products, proceed to step 9.2.9.9.2.6 For wash-off products, rinse for 30 s under tap waterregulated at 40 6 2C.9.2.7 Use a paper towel or tissue to blot the pigskins dry.9.

44、2.8 Sample using the cup scrub technique (Section 10).9.2.9 If multiple sampling intervals are desired, procedurescan be repeated using separate pairs of skin to examine eachsampling interval.9.3 Treatment of Control Samples:9.3.1 Contaminate each control pair of pigskin substrateswith an appropriat

45、e volume of the challenge species.9.3.2 Rub the pigskins together for 15 s and allow appro-priate dry-time.9.3.3 Sample using the cup scrub technique (Section 10).NOTE 3When necessary, conduct a sterility control on a randomlyselected, prepared pigskin substrate by following the procedure outlined i

46、nSection 10.10. Sampling10.1 Quantitative microbial counts are obtained using thecup scrub technique. This procedure is used for test and controldetermination.10.2 The treated pigskins are positioned on a flat surface.10.3 The cylinder is pressed firmly against the pigskinsurface during sampling to

47、ensure that the sampling fluid doesnot leak from the delineated sampling site.10.4 A specified volume of sterile sampling fluid (7.14)istransferred into the cylinder on elapse of the contact time.10.5 The pigskin is then scrubbed with moderate pressurefor 60 6 6 s using a sterile polytetrafluoroethy

48、lene (PTFE)scraper. After scrubbing, an aliquot of the sampling fluid istransferred into a sample tube and serially diluted, as necessary.This procedure is repeated for all samples.10.6 Care must be taken during the sampling process toprevent the fluid from spilling.11. Microbial Counts11.1 Each sam

49、ple is mixed thoroughly. Ten-fold serialdilutions of each sample are prepared in dilution fluid, asnecessary. Appropriate dilutions are pour- or spread-plated induplicate using the appropriate medium with suitable neutral-izer and incubated at the appropriate temperature, 62C, for 24to 72 h, or until colonies are countable.12. Determination of Microbial Reduction12.1 Convert plate counts to Log10values, and average thevalues from each pair for each sampling interval.12.2 Determine Log10reductions at each sampling intervalusing the follow