ASTM E1882-2005 Standard Test Method for Evaluation of Antimicrobial Formulations by the Agar Patch Technique《用琼脂斑贴技术评价抗菌剂的标准试验方法》.pdf

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1、Designation: E 1882 05Standard Test Method forEvaluation of Antimicrobial Formulations by the Agar PatchTechnique1This standard is issued under the fixed designation E 1882; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year

2、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.1. Scope1.1 This test method determines the antibacterial activityand persistence of test formulations, as measured by theinhi

3、bition of a test organism on an agar surface exposed to testsites on human skin treated with the formulations.1.2 A knowledge of microbiological techniques is requiredfor these procedures.1.3 It is the responsibility of the investigator to determine ifGood Laboratory Practice (GLP) and Good Clinical

4、 Practice(GCP) are required and to adhere to these practices, asappropriate.1.4 In this test method, metric units are used for allapplications except linear measure. In that case, inches are usedand metric units follow in parentheses.1.5 This standard does not purport to address all of thesafety con

5、cerns, 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. Performance of thisprocedure requires the knowledge of regulations pertaining to

6、the protection of human subjects (see 21 CFR, Ch. I, Parts 50and 56 ).2. Referenced Documents2.1 ASTM Standards:2E 1874 Test Method for Evaluation of Antibacterial Washesby Cup Scrub Technique2.2 Federal Standard321 CFR, Ch. I, Parts 50 and 56 Protection of HumanSubjects3. Terminology3.1 active test

7、 formulationa substance containing activeingredient(s).3.2 active ingredienta substance performing a functiondefined in the test method; in this test method, a substanceadded to a formulation specifically for the inhibition orinactivation of microorganisms.3.3 active plateinoculated agar plate that

8、has been at-tached to a skin site treated with an active formulation.3.4 antibacterial activitykilling of bacteria or supressionof their growth or reproduction.3.5 control formulationa formulation that does not con-tain an active ingredient.3.6 control plateinoculated agar plate that has been at-tac

9、hed to an untreated skin site, or one treated with a controlformulation.3.7 inhibitionprevention of bacterial population growth,either through lethality or through prevention of bacterialreproduction.3.8 inoculum determination platean inoculated plate thathas not been exposed to any skin test site.3

10、.9 persistenceeffectiveness of a test formulation in in-hibiting bacteria, defined in terms of time elapsed betweenapplication of test formulation and application of test plates.3.10 resident microorganismsmicroorganisms that liveand multiply on skin, forming a permanent population.3.11 transient mi

11、croorganismsmicroorganisms from theenvironment that contaminate, but do not normally perma-nently colonize skin.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and is the direct responsibility of Subcommittee E35.15 on AntimicrobialAgents.Current edition approved Nov.

12、1, 2005. Published November 2005. Originallyapproved in 1997. Last previous edition approved in 2000 as E 1882 00.2For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to th

13、e standards Document Summary page onthe ASTM website.3Available from Superintendent of Documents, U.S. Government PrintingOffice, Washington, D.C. 20402.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.12 volar aspect of the forearm

14、sthe inside of the forearmon the same plane as the palm of the hand.4. Summary of Test Method4.1 This test method is conducted on subjects selected froma group of volunteers who have refrained from using topicalantimicrobials for at least one week and have minimal hair onthe test site. The test site

15、 should normally have a low numberof resident microorganisms (approximately 104CFU/cm2orfewer) and be easily sampled.4.2 The surfaces of agar contact plates are inoculated withthe selected organism and placed in contact with skin sites thathave been treated with active or control formulations, or le

16、ftuntreated. After contact with the treated skin sites, these platesare incubated and the colonies enumerated. Inhibitory activityof the active test formulation is measured by comparingdifferences in microbial colony counts between plates that werein contact with sites treated with an active formula

17、tion andplates that were in contact with untreated sites, or sites treatedwith a control formulation. Results are expressed as percentinhibition.45. Significance and Use5.1 This procedure can be used to evaluate formulationscontaining ingredients intended to inhibit growth of bacteria onintact skin

18、and measures the difference, post-product-exposure,between numbers of bacterial colonies on active test formula-tion plates and numbers on control plates, expressed as percentinhibition.5.2 This procedure may also be used to test for persistenceof activity, as a function of time elapsed between appl

19、ication ofactive test formulation and application of active test plates.5.3 Because no procedure for neutralization of the antimi-crobial action of active ingredients can be included in the test,the agar patch method is limited to the extent that resultsexpressed as percent inhibition do not differe

20、ntiate betweenbacteriostatic and bacteriocidal effects and, hence, must not beportrayed as “reductions.”6. Apparatus6.1 Colony CounterAny of several types may be used. Amagnifying device, such as a dissecting microscope, may beused for manual enumeration of colonies.6.2 IncubatorAny incubator capabl

21、e of maintaining asuitable temperature 6 2C may be used.6.3 SterilizerAny steam sterilizer capable of producingthe conditions of sterility.6.4 Timer (Stop Watch)One that can be read for hours,minutes, and seconds.7. Reagents and Materials7.1 Bacteriological Pipettes, 10.0 and 2.2 or 1.1 mL capac-ity

22、.NOTE 1Presterilized/disposable bacteriological pipettes are availablefrom most laboratory supply houses.7.2 Pipetter, with disposable tips capable of delivering 10L.7.3 Plating Medium, soybean-casein digest agar, or equiva-lent.57.4 Dilution Fluid, Butterfields phosphate buffer6,orequivalent.7.5 Is

23、opropanol or Ethanol, 60 to 75% (v/v)7.6 Sterile Disposable Culture Dishes, 1.4 3 0.4 in (35 mmby 10 mm) and 4.0 3 0.8 in (100 mm by 20 mm).7.7 Sterile Test Tubes.7.8 Surgical Adhesive Tape, or equivalent.7.9 Disposable Examining Gloves.7.10 Inoculating Loop or Glass Spreader.7.11 Appropriate Bacter

24、ial Cultures.7.12 Test FormulationsDirections for application of ac-tive and control formulations should be followed, if available.If directions are not available, the directions provided in thistest method may be applied.8. Test and Control Skin Sites8.1 The volar aspect of the forearm is commonly

25、used as thelocation of the skin sites, but other areas such as the back orforehead may be used for test sites. Application of active testand control formulations (or no treatment) will be assigned bya predetermined randomization so that either forearm (or eitherside, right or left, of other anatomic

26、al areas) may receive activeor control formulations (or none).WarningApplication ofagar patch plates and alcohol to the forehead risks contaminat-ing the eyes, and extra precautions must be exercised.9. Subjects9.1 Number of SubjectsThe number of subjects used inthe test depends on the statistical s

27、ignificance required for theexpected results, the sampling variability encountered in thestudy, and the relative efficacy of the active formulation beingevaluated.9.1.1 Recruit a sufficient number of healthy adult subjectswho have no clinical evidence of dermatoses, open wounds, orother skin disorde

28、rs that may affect the integrity of the test.9.2 Instruct the subjects to avoid contact with antimicrobialsfor at least the week prior to testing and, other than the activeformulation, for the duration of the test. This restrictionincludes spray antiperspirants and deodorants, shampoos, lo-tions, di

29、shwashing detergents, and soaps containing antimicro-bial compounds, and materials such as acids, bases, andsolvents. Bathing in biocide-treated pools, hot tubs, or spasshould be avoided. Subjects may be provided with a kit ofnon-antimicrobial personal hygiene products for exclusive useduring the te

30、st period and rubber gloves to be worn whencontact with antimicrobial agents cannot be avoided.4Yackovich, F., C.A. Wagner, and J.E. Heinze. 1989. Validity of the agar patchtest with an antibacterial liquid soap and comparison with the finger imprint method.J. Soc. Cos. Chem. 40:263-271.5U.S. Pharma

31、copeia XXIV, NF 19. 2000. United States Pharmacopeial Con-vention Inc., Rockville, MD. Chapter 61, entitled “ Microbial Limits Test.”6Horowitz, W. (Ed.). 1980. Offcial Methods of Analysis of the AOAC,13thEd.Sec. 46.013(m), p. 825. Assoc. of Official Analytical Chemists, Washington, D.C.1018 pp.E1882

32、05210. Procedure10.1 Preparation of Agar Patch Contact Plates:10.1.1 Aseptically place three 35 mm by 10 mm Petri disheswith lids removed into a sterile 100 mm by 20 mm Petri dish.NOTE 2Three 35 mm by 10 mm plates are needed per active orcontrol formulation per organism per subject. Three additional

33、 plates areneeded per organism for an inoculum determination count.10.1.2 Using a sterile pipette, aseptically dispense approxi-mately 11.5 mL of soybean-casein digest agar (or otherappropriate solid medium) into each of the small Petri dishes.The dishes are filled to form a convex meniscus elevated

34、 abovethe rim of the plate.10.1.3 Allow the agar to solidify and the surface to drybefore inoculation.10.2 Preparation of Test Organisms:10.2.1 The test organisms selected should be representativeof the microbial flora of the skin or of transient microorganismsthat may contaminate human skin under c

35、ertain conditions. Apartial list of organisms that have been used or recommendedfor use in this test includes Staphylococcus epidermidis ATCC14990, S. epidermidis ATCC 12228, S. aureus ATCC 6538,Escherichia coli ATCC 11229, and Klebsiella pneumoniaeATCC 10031. This list is not intended to be exhaust

36、ive. Otherorganisms can be used at the discretion of the investigator. Theorganisms used should be differentiable from the test subjectsown normal resident microorganisms by using the inoculumdetermination plate as a resource for colony morphology or byusing differential or selective media. However,

37、 if no differencebetween resident and test microorganisms can be detected(e.g., S. epidermidis), then it may be assumed that the colonycounts on the control plates due to contamination with residentmicroorganisms are in relative proportion to those on the activeplates, and effectively cancel out.NOT

38、E 3A recent antibiotic sensitivity profile for each test organismused in testing is required for purposes of subject safety.10.2.2 Sequentially transfer culture(s) twice (once every 18to 24 h) into appropriate liquid growth media. The secondtransfer must be into a volume of medium sufficient to perf

39、ormthe test.10.2.3 Alternatively, the second transfer may be onto anagar plate.10.2.4 If preparing inoculum from an agar plate, suspendorganisms in dilution fluid before use.10.2.5 The final concentration of inoculum should be ad-justed to 1.03.0 3 108CFU/mL. Inoculum should be wellmixed to break up

40、 clumps.NOTE 4If the inoculum is from a plate, additional care must be takento mix the sample well.10.2.6 Prepare ten-fold serial dilutions from the 1.0 3.0 3108CFU/mLsuspension using 9 mLof dilution fluid to achievea final inoculum of 1.0 3.0 3 104CFU/mL.10.2.7 Pipette 10 L of the final inoculum pr

41、eparation ontothe surface of each of the prepared agar plates.10.2.8 Spread the drop evenly across the surface of the plateusing a bent sterile glass spreader or other suitable device. Theinoculum should be allowed to soak into the agar.NOTE 5Steps 10.2.7 and 10.2.8 should be performed no more than

42、30min before applying plates to the test sites.11. Decontamination of Test Sites11.1 Prior to testing, apply ethanol or isopropanol (60 to75 % v/v) to the test sites to reduce populations of resident ortransient microorganisms.11.2 Allow the alcohol to air-dry completely before pro-ceeding with test

43、ing.12. Application12.1 Application sites will be located on the volar aspects ofthe forearms or on other suitable areas such as the forehead orback.Application will be assigned by a predetermined random-ization such that the forearms (or either side, right or left, ofother anatomical areas) of each

44、 subject are equally likely to beused for treatment with the active or control formulation (orleft untreated).12.2 Treatment will consist of one or more washes orapplications with either active test formulation or controlformulation.NOTE 6More than one treatment may be required for some testformulat

45、ions to display antibacterial activity.13. Application of Bar-Form Wash Products (to forearmsites, for example)13.1 Place a clean disposal glove on the right hand. Brieflywet the volar aspect of the left forearm with warm (38 6 2C)tap water.13.2 Wet the bar (active or control formulation) with tapwa

46、ter, using only the gloved hand.13.3 Rub the bar on the left volar forearm for 15 6 2s.Place bar aside. Lather arm with gloved hand for 45 6 5s.Iflather becomes too dry, a small amount of water may be addedto maintain lather.13.4 Rinse the arm under warm (38 6 2C) tap water forapproximately 15 s to

47、remove all lather and allow arm toair-dry.13.5 If a control formulation is used, repeat procedure forright forearm using a new, clean disposable glove on the lefthand to apply the formulation (active or control) not used inSection 13.2.14. Application of Liquid or Gel Wash Products (toforearm sites,

48、 for example)14.1 Place a clean disposable glove on the right hand.Briefly wet the volar aspect of the left forearm with warm (386 2C) tap water.14.2 Deliver the amount of product specified by labelinstructions into the gloved hand. If instructions are notavailable, 2.0 mL is a commonly used amount.

49、14.3 Lather the left volar aspect of the forearm for 60 6 5s.14.4 Rinse the arm under warm tap water (38 6 2C) forapproximately 15 s to remove all lather and allow arm toair-dry.E188205314.5 If a control formulation is used, repeat procedure forright forearm using a new, clean disposable glove on the lefthand to apply the formulation (active or control) not used inSection 14.2.15. Application of Leave-On (No-Rinse) Products (toforearm sites, for example)15.1 It is suggested that preliminary testing be performed todetermine the amount of produc