ASTM E1882-2010 Standard Test Method for Evaluation of Antimicrobial Formulations by the Agar Patch Technique《使用琼脂贴片法评估抗菌配方的标准试验方法》.pdf

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1、Designation: E1882 10Standard Test Method forEvaluation of Antimicrobial Formulations by the Agar PatchTechnique1This standard is issued under the fixed designation E1882; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of

2、 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 determines the antibacterial activityand persistence of test formulations, as measured by theinhibit

3、ion 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 Pr

4、actice(GCP) are required and to adhere to these practices, asappropriate.1.4 In this test method, SI units are used for all applicationsexcept linear measure. In that case, inches are used and SI unitsfollow in parentheses.1.5 This standard does not purport to address all of thesafety concerns, if a

5、ny, 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 tothe protect

6、ion of human subjects (see 21 CFR, Ch. I, Parts 50and 56 ).2. Referenced Documents2.1 Federal Standard221 CFR, Ch. I, Parts 50 and 56 Protection of HumanSubjects3. Terminology3.1 active test formulationa substance containing activeingredient(s).3.2 active ingredienta substance added to a formulation

7、specifically for the inhibition or inactivation of microorgan-isms.3.3 active plateinoculated agar plate that 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 formu

8、lation that does not con-tain an active ingredient.3.6 control plateinoculated agar plate that has been at-tached 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 bacterialrep

9、roduction.3.8 inoculum determination platean inoculated plate thathas not been exposed to any skin test site.3.9 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 residen

10、t microorganismsmicroorganisms that sur-vive and multiply on the skin, forming a stable population.3.11 transient microorganismsmicroorganisms that con-taminate the skin, but do not form a stable population.3.12 volar aspect of the forearmsthe surface of theforearm on the same side as the palm of th

11、e 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 should normally have a low numberof resident microorganisms

12、(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 leftuntreated. After contact with the treated skin sites, these

13、 platesare incubated and the colonies enumerated. Inhibitory activityof the active test formulation is measured by comparing1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicr

14、obial Agents.Current edition approved April 1, 2010. Published April 2010. Originallyapproved in 1997. Last previous edition approved in 2005 as E1882 05. DOI:10.1520/E1882-10.2Available from Superintendent of Documents, U.S. Government PrintingOffice, Washington, D.C. 20402.1Copyright ASTM Internat

15、ional, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.differences in microbial colony counts between plates that werein contact with sites treated with an active formulation andplates that were in contact with untreated sites, or sites treatedwith a control formu

16、lation. Results are expressed as percentinhibition.35. Significance and Use5.1 This procedure can be used to evaluate formulationscontaining ingredients intended to inhibit growth of bacteria onintact skin and measures the difference, post-product-exposure,between numbers of bacterial colonies on ac

17、tive 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 application ofactive test formulation and application of active test plates.5.3 Because no procedu

18、re 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 differentiate betweenbacteristatic and bactericidal effects and, hence, must not beportrayed as “redu

19、ctions.”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 capable of maintaining asuitable temperature 62C may be used.6.3 SterilizerAny steam sterilizer capabl

20、e 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.NOTE 1Presterilized/disposable bacteriological pipettes are availablefrom most laboratory supply

21、 houses.7.2 Pipetter, with disposable tips capable of delivering 10L.7.3 Plating Medium, soybean-casein digest agar, or equiva-lent.47.4 Dilution Fluid, Butterfields phosphate buffer5,orequivalent.7.5 Isopropanol or Ethanol, 60 to 75% (v/v)7.6 Sterile Disposable Culture Dishes, 1.4 3 0.4 in. (35 by1

22、0 mm) and 4.0 3 0.8 in. (100 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 Bacterial Cultures.7.12 Test FormulationsDirections for application of ac-tive and control formulations sho

23、uld 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 used as thelocation of the skin sites, but other areas such as the back orforehead may be used for te

24、st 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 anatomical areas) may receive activeor control formulations (or none). (WarningApplication ofagar patch plate

25、s 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 significance required for theexpected results, the sampling variability encountered in thestudy, an

26、d 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 disorders that may affect the integrity of the test.9.2 Instruct the subjects to avoid contact with antim

27、icrobialsfor 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, dishwashing detergents, and soaps containing antimicro-bial compounds, and materials such as acids,

28、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 test period and rubber gloves to be worn whencontact with antimicrobial agents cannot be avoided.10.

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

30、dedper 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 abovethe rim o

31、f 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 certain conditio

32、ns. A3Yackovich, F., Wagner, C. A., and Heinze, J. E., “Validity of the agar patch testwith an antibacterial liquid soap and comparison with the finger imprint method,”J. Soc. Cos. Chem. Vol 40, 1989, pp. 263271.4U.S. Pharmacopeia XXIV, NF 19. 2000. United States Pharmacopeial Con-vention Inc., Rock

33、ville, MD. Chapter 61, entitled “Microbial Limits Test.”5Horowitz, W. (Ed.), Offcial Methods of Analysis of the AOAC,13thEd. Sec.46.013(m), p. 825. Assoc. of Official Analytical Chemists, Washington, D.C. 1980,1018 pp.E1882 102partial list of organisms that have been used or recommendedfor use in th

34、is 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 exhaustive. Otherorganisms can be used at the discretion of the investigator. Theorganisms used shou

35、ld 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, if no differencebetween resident and test microorganisms can be detected(e.g., S. epidermidi

36、s), 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.NOTE 3A recent antibiotic sensitivity profile for each test organismused in testing is required

37、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 performthe test.10.2.3 Alternatively, the second transfer may be onto anagar plate.10.2.4 If prep

38、aring 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 clumps.NOTE 4If the inoculum is from a plate, additional care must be takento mix the sample

39、 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 preparation ontothe surface of each of the prepared agar plates.10.2.8 Spread the drop evenly a

40、cross 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 30min before applying plates to the test sites.11. Decontamination of Test Sites11.1 Prior to

41、 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 testing.12. Application12.1 Application sites will be located on the volar aspects ofthe forearms

42、 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 subject are equally likely to beused for treatment with the active or control formulation (o

43、rleft 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 testformulations to display antibacterial activity.13. Application of Bar-Form Wash Products (to forearms

44、ites, 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 tapwater, using only the gloved hand.13.3 Rub the bar on the left volar forearm for 15 6 2s.Place

45、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 remove all lather and allow arm toair-dry.13.5 If a control formulation is used, repeat proce

46、dure 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, for example)14.1 Place a clean disposable glove on the right hand.Briefly wet the volar aspe

47、ct 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.14.3 Lather the volar aspect of the left forearm for 60 6 5s.14.4 Rinse the arm under warm ta

48、p water (38 6 2C) forapproximately 15 s to remove all lather and allow arm toair-dry.14.5 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 Le

49、ave-On (No-Rinse) Products (toforearm sites, for example)15.1 It is suggested that preliminary testing be performed todetermine the amount of product (active or control formula-tion) that covers the test site adequately, without excess. Thatamount may then be expressed as volume (or weight) per cm2of test site surface area.15.2 Place a clean disposable glove on the right hand.NOTE 7Because of higher rates of evaporation, an alcohol-basedproduct usually can be applied in larger volume, without excess, to a skinsite than can a non-alcohol-base