1、Designation: E2752 10Standard Guide forEvaluation of Residual Effectiveness of AntibacterialPersonal Cleansing Products1This standard is issued under the fixed designation E2752; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the
2、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 guide is designed to demonstrate the effectivenessof an antibacterial personal cleansing product in reduc
3、ing thenumbers of a marker organism (representing transients) bothimmediately and after prolonged exposure to (cleansing) wash-ing when used as recommended under simulated use condi-tions. The method demonstrates the effect of residual antibac-terial activity by means of inhibition of proliferation
4、of bacteriaon the skin after the contact period. Antimicrobial activity iscompared with a vehicle or to a baseline organism count.1.2 A knowledge of microbiological techniques is requiredfor these procedures.1.3 Performance of this procedure requires the knowledgeof regulations pertaining to the pro
5、tection of human subjects(21 CFR Parts 50 and 56).1.4 The values stated in SI units are to be regarded asstandard. No other units of measurement are included in thisstandard.1.5 This standard may involve hazardous materials, opera-tions, and equipment. This standard does not purport toaddress all of
6、 the safety concerns, if any, associated with itsuse. It is the responsibility of the user of this standard toestablish appropriate safety and health practices and deter-mine the applicability of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2E1054 Test Methods for Ev
7、aluation of Inactivators of An-timicrobial AgentsE1874 Test Method for Recovery of Microorganisms FromSkin using the Cup Scrub Technique2.2 Federal Document:321 CFR Parts 50 and 56 Code of Federal Regulations:Protection of Human Subjects; Institutional ReviewBoards3. Terminology3.1 Definitions of Te
8、rms Specific to This Standard:3.1.1 marker organism, nan applied inoculum of anorganism that has characteristics that allow it to be readilyidentified or differentiated. Marker organisms are used tosimulate transient microorganisms.3.1.2 occlusion, ncovered and sealed from the outsideenvironment.3.1
9、.3 occlusive chamber, na covering that protects thesampling surface without contacting the sampling surface.3.1.4 personal cleansing products, nproducts used forpersonal hygiene such as soaps, hand sanitizers, towelettes,body washes, and so forth.3.1.5 transient organisms, norganisms from the enviro
10、n-ment that contaminate but do not normally permanentlycolonize skin.3.1.6 vehicle, na formulation for delivery of the antimi-crobial agent.4. Summary of Test Method4.1 This guide is conducted on a group of volunteer subjectswho refrained from using oral and topical antimicrobials for atleast one we
11、ek.4.2 The test sites are washed multiple times with thecleansing product or vehicle. After washing, the sites areinoculated with a marker organism and occluded for a specifiedperiod of time after which the sites are sampled and enumer-ated for the marker organism. Activity of the cleansing producti
12、s measured by comparing microbial counts from treated sitesto those derived from the sites treated with vehicle or to anuntreated baseline organism count.1This guide is under the jurisdiction of ASTM Committee E35 on Pesticides andAlternative Control Agents and is the direct responsibility of Subcom
13、mittee E35.15on Antimicrobial Agents.Current edition approved April 1, 2010. Published August 2010. DOI: 10.1520/E275210.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refe
14、r to the standards Document Summary page onthe ASTM website.3Available from Standardization Documents Order Desk, DODSSP, Bldg. 4,Section D, 700 Robbins Ave., Philadelphia, PA 19111-5098, http:/dodssp.daps.dla.mil.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken,
15、PA 19428-2959, United States.5. Significance and Use5.1 The procedure is used to evaluate personal cleansingproducts containing antibacterial ingredients that are intendedto reduce the number of organisms on intact skin. It also maybe used to demonstrate the effect of residual antibacterialactivity
16、by means of inhibition of the proliferation of bacteriaon the skin after contact.6. Apparatus6.1 Colony CounterUse any of several types.6.2 IncubatorAny incubator capable of maintaining asuitable temperature 62C.6.3 SterilizerAny suitable steam sterilizer capable ofproducing the conditions of steril
17、ization.6.4 Timer (Stop Clock)One that displays hours and min-utes.6.5 TableAny elevated surface, such asa1by2-mtablewith mattress or similar padding to allow the subject to recline,when applicable.6.6 Handwashing SinkA sink of sufficient size to permitpanelist to wash without touching hands to the
18、sink surface orother panelist.6.7 Water Faucet(s)To be located above the sink at aheight that permits the hands to be held higher than the elbowsduring the washing procedure.6.8 Tap Water Temperature Regulator and TemperatureMonitorTo monitor and regulate water temperature.7. Reagents and Materials7
19、.1 Bacteriological PipettesSterile, of appropriate capac-ity.NOTE 1Presterilized/disposable bacteriological pipettes are availablefrom most laboratory supply houses.7.2 Water Dilution BottlesAny sterilizable container hav-ing a 100 to 200-mL capacity and tight closure.NOTE 2Milk dilution bottles of
20、160-mLcapacity have a screw-cappedclosure and are available from most local laboratory supply houses.7.3 Scrub CupsSterile cylinders, height approximately2.5 cm, inside diameter of convenient size. Useful sizes rangefrom approximately 1.5 to 4.0 cm.7.4 Teflon Policeman or Rubber PolicemanCan be fash
21、-ioned in the laboratory or purchased.7.5 PipettorWith disposable tips capable of delivering 10L.7.6 Graduated CylindersSterile, of appropriate capacity.7.7 BeakersSterile, of appropriate capacity.7.8 Occlusive ChamberFor covering inoculated test sites.NOTE 3Occlusive chambers or plastic weigh boats
22、 of appropriate sizeavailable from laboratory supply houses7.9 Adhesive DressingFor securing the occlusive cham-ber.NOTE 4Adhesive dressings such as adhesive tape, surgical tape, orothers secural devices.7.10 Bacterial CulturesSuch as Staphylococcus aureusATCC 27217 or Escherichia coli ATCC 11229, o
23、r others asappropriate.7.11 Test FormulationsWith directions for use.7.12 Sampling and Dilution FluidSterile Butterfieldsphosphate buffered water, containing an antimicrobial inacti-vator specific for the test formulation as determined by TestMethod E1054.7.13 Plating MediumSoybean-casein digest aga
24、r orequivalent as appropriate with neutralizers, as determined byTest Method E1054.7.14 Sterile Culture Tubes, or equivalent with closures ofappropriate capacity.8. Test Control and Baseline Skin Sites8.1 Select skin sites appropriate for target flora and the testmaterials intended use. Where possib
25、le, a contralateral site isrecommended for application of the vehicle or for the micro-organism count control.NOTE 5Forearms are a convenient site for most applications.9. Subjects9.1 Number of SubjectsThe number of subjects requireddepends on the statistical confidence for the expected testresults,
26、 the variability encountered in the study, and the relativeefficacy of the antibacterial agent evaluated.9.1.1 Recruit a sufficient number of healthy adult volunteerswho have no clinical evidence of dermatoses, open wounds, orother skin disorders that affect the integrity of the test.9.2 Instruct th
27、e subjects to avoid contact with antimicrobialsfor at least one week prior to the test. This restriction includesantimicrobial-containing antiperspirants, deodorants, sham-poos, lotions, and soaps, also such material as acids bases andsolvents. Bathing in biocide-treated pools, hot tubs, spas, andso
28、 forth, should be avoided. Provide volunteers with a kit ofnon-antimicrobial personal care products for exclusive useduring the test. Volunteers must wear rubber gloves whencontact with antimicrobial agents cannot be avoided.10. Procedure10.1 Treatment(s) Application Procedure:10.1.1 Application of
29、test material is assigned by a prede-termined randomized application schedule. Each subject willhave an active and a vehicle or no vehicle treatment site.10.1.2 Application of test and vehicle material consists ofan equal number of washes. For demonstration of residualactivity, more than one test ma
30、terial application may berequired. Schedule applications at least one hour apart.10.1.3 Perform all washes under supervision of a techniciantrained in the methodology. The following are suggestedtreatment procedures for evaluating two rinse-off products, abar soap product, and a liquid soap product;
31、 and two no-rinseproducts, a liquid gel product and a pre-wetted towelette.10.1.4 Bar Soap Products and VehicleCheck, record, andmaintain the temperature of the water at 35 to 38C before eachwash. Water flow should be 4L/min. Remove all jewelry fromhands and wrists prior to start of wash procedure.
32、Subjects canwash their own arms. The start time of each wash should berecorded. The following wash procedure is recommended.10.1.4.1 Subjects wash their left arm first with the assignedtest product or vehicle formulation:E2752 102(1) Wet the volar surface of the forearm under the runningwater.(2) We
33、t the appropriate bar of soap under the runningwater.(3) The subjects rub the bar on their forearm, using anup-and-down motion from the wrist to the elbow, for 15 s.(4) Then set the bar down and continue to rub the lather ontheir forearm using the same up-and-down motion from thewrist to the elbow,
34、for an additional 45 s.(5) Rinse the forearm for 15 s. Do not rub!(6) Pat the forearm dry using a paper towel. Do not rub!(7) Repeat steps 1 to 6 on their right forearm with the otherformulation.10.1.4.2 The above wash procedure is conducted a total ofnine (9) times. There will be three (3) washes e
35、ach day on thefirst, second, and third days of the study. The washes are to beat least one (1) hour apart.10.1.5 Liquid Soap Products and VehicleCheck, record,and maintain the temperature of the water at 35 to 38C beforeeach wash. Water flow should be 4L/min. Remove all jewelryfrom hands and wrists
36、prior to start of wash procedure.Subjects can wash their own arms. The start time of each washshould be recorded. The following wash procedure is recom-mended.10.1.5.1 Subjects wash their left arm first with the assignedtest product or vehicle formulation:(1) Wet the volar surface of the forearm und
37、er the runningwater.(2) 1.8 mL of liquid soap product is dispensed from asyringe onto the volar surface of the forearm.(3) The subjects rub the liquid soap on their forearm, usingan up-and-down motion from the wrist to the elbow, for 45 s.(4) They rinse their forearm for 15 s. Do not rub!(5) Pat the
38、 forearm dry using a paper towel. Do not rub!(6) Repeat steps 1 to 5 on the right forearm with the otherformulation.10.1.5.2 The above wash procedure is conducted a total ofnine (9) times. There are three (3) washes each day on the first,second, and third days of the test period. The washes are to b
39、eat least one (1) hour apart.10.1.6 No-Rinse, Liquid Gel, and PlaceboRemove alljewelry from hands and wrists prior to start of procedure. Thetime of treatment should be recorded at the start of application.Subjects can treat their own arms. The start time of eachtreatment should be recorded. The fol
40、lowing procedure isrecommended.10.1.6.1 Subjects left forearm is treated first with theassigned test product or vehicle formulation:(1) 1.8 mL of product is dispensed from a syringe onto thevolar surface of the forearm.(2) The subjects rub the formulation on their forearm, usingan up-and-down motion
41、 from the wrist to the elbow, for 15 s.(3) Allow subjects forearm to air dry.(4) Repeat steps 1 to 3 on the right forearm with the otherformulation.10.1.6.2 The above procedure is conducted a total of three(3) times at least one (1) hour apart.10.1.7 Pre-Wetted Towelette or Wipe Product and VehicleR
42、emove all jewelry from hands and wrists prior to start ofprocedure. The time of treatment should be recorded at the startof wiping. A technician will wipe each subjects arm. Thetechnician will wear gloves for this procedure. The start time ofeach treatment should be recorded. The following procedure
43、 isrecommended.10.1.7.1 Subjects left forearm is treated first with theassigned test product or vehicle formulation:(1) The technician will unfold the test towelette and wipethe forearm in an up-and-down motion for 15s.(2) The technician will flip the towelette and wipe theforearm with the opposite
44、side of the towelette in an up-and-down motion for another 15 s.(3) Allow subjects forearm to air dry.(4) Repeat steps 1 to 3 on the right forearm with the otherformulation.10.1.7.2 The above wipe procedure is conducted a total ofthree (3) times at least one (1) hour apart.10.2 Evaluation ProcedureR
45、esidual activity is assessed atany selected time after the last treatment application, forexample, 1 hour, 6 hours, 24 hours, and so forth, by measuringthe survival of marker bacteria applied under occlusion to thetreated skin sites after pre-selected exposure times, for ex-ample, 30 minutes, two ho
46、urs, or five hours depending on studyobjectives. The following is an example of a procedure forassessing residual activity 24 h after the last treatment usingexposure times of 30 min,2hand5h.10.2.1 Approximately 24 h after the final treatment appli-cation, three (3) evenly spaced circular test sites
47、 are markedalong the midsection of the volar aspect of each forearm,avoiding the area on the wrist and elbow crease. The lower siteis near the wrist and the upper site is near the elbow. Mark thesites by pressing a 3.0 cm inside diameter glass cylinder, inkedwith a stamp pad, against the skin.10.2.2
48、 Using a micro pipettor (section 7.5), inoculate thecenter of each circular site with 10 L of the bacterial cultureto obtain 106to 107colony forming units (CFUs) of S. aureus.A sterile, disposable, inoculating loop is used to evenly spreadthe inoculum within the center of the test site while remaini
49、ng4 to 5 mm from the marked edge. The culture is prepared asdescribed in section 10.5.10.2.3 Each inoculated site is immediately occluded bycovering it with an occlusive chamber (section 7.8) andsecured to the skin with an adhesive dressing (section 7.9).10.3 Harvesting of the Surviving OrganismsAll inocu-lated sites are sampled for surviving organisms. One (1) testsite on each arm is harvested at each of the three (3) timeperiods 30 6 5min,2h6 5 min, and 5 h 6 5 min afterinoculation. The randomization will indicate the time periodf
