ASTM E1589-2005(2015) Standard Test Method for Evaluation of First Aid Antiseptic Drug Products《评定急救抗菌药品的标准试验方法》.pdf

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1、Designation: E1589 05 (Reapproved 2015)Standard Test Method forEvaluation of First Aid Antiseptic Drug Products1This standard is issued under the fixed designation E1589; 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 The tests described in this test method are designed toevaluate antimicrobial agents in formulations intended for usea

3、s first aid antiseptic products for their ability to reduce orsuppress the growth, or both, of the skin microflora.1.2 A knowledge of microbiological techniques is requiredfor these procedures.1.3 Performance of this procedure requires the knowledgeof regulations pertaining to the protection of huma

4、n subjects.(See 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 does not purport to address all of thesafety concerns, if any, associated with its use. It is theresponsibility of the us

5、er of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:2D1193 Specification for Reagent WaterE1054 Test Methods for Evaluation of Inactivators of Anti-microbial Agents

6、2.2 Federal Standards:3CFR Parts 50 and 563. Terminology3.1 active ingredient, na substance performing a functiondefined by this method.3.2 neutralization, na process which results in quenchingor inactivating inactivation of the antimicrobial activity of aformulation. This may be achieved with dilut

7、ion of theformulation, or with the use of chemical agents, called neu-tralizers.3.3 neutralizer, na procedure or chemical agent used toinactivate, neutralize, or quench the microbiocidal properties ofan antimicrobial agent.3.4 resident microorganisms, nmicroorganisms that liveand multiply on skin, f

8、orming a permanent population.3.5 sampling fluid, na recovery fluid that may or may notcontain a neutralizer to inactivate the active ingredients in testand internal reference formulations.3.6 test formulation, na formulation containing an activeingredient(s).3.7 transient microorganisms, nmicroorga

9、nisms that con-taminate but do not normally permanently colonize the skin.4. Summary of Test Method4.1 These test methods describe standard in vivo techniquesto determine the following:4.1.1 Effect of the Test Formulation to Reduce an ArtificiallyEnhanced Skin Microbial FloraThe forearms of subjects

10、 areoccluded for 48 h prior to application of the test formulation toincrease the microbial population on the skin of the volarforearm surface. At treatment the occlusion material is re-moved and the skin is allowed to dry, the test formulation isthen applied to selected sites. At a pre-determined t

11、ime(s)following application, the sites are microbiologically sampledand the samples plated for total aerobic bacteria count. Thecounts obtained from the treated sites are compared to countsobtained from untreated occluded sites.4.1.2 Effect of the Test Formulation to Suppress the Growthof Normal Ski

12、n Flora When Applied As a DressingThedressings are applied to the forearm for 24 h. The density of theresident microorganisms that develop under the dressings arecompared to the population that develops on a similar untreatedoccluded site. Following 24 h of occlusion, the sites aremicrobiologically

13、sampled and the samples plated for totalaerobic bacteria count.4.2 The principal of the test is that the microflora of forearmskin is sparse. The impermeable dressing will increase surface1This test method is under the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative

14、Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Oct. 1, 2015. Published November 2015. Originallyapproved in 1994. Last previous edition approved in 2010 as E1589 05(2010).DOI: 10.1520/E1589-05R15.2For referenced ASTM standards,

15、visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.3Available from U.S. Government Publishing Office, 732 N. Capitol St., NW,Washington DC, 2040

16、1-0001, http:/www.gpo.gov.Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1moisture by preventing diffusional water loss and thus expandtransient resident skin microorganisms population. A signifi-cant antimicrobial effect by the test

17、agent will be reflected bysignificantly lower population recovered from the nontreatedsite.5. Significance and Use5.1 The procedures in this test method should be used for invivo evaluation the antimicrobial activity of drug productsapplied topically to the skin that are intended to help preventinfe

18、ction in minor cuts, scrapes and burns.5.1.1 This test method is applicable for testing liquids,ointments, powders, films, or dressing containing or impreg-nated with an antimicrobial for their effect to reduce anenhanced skin microflora or their effects to suppress the growthof the skin flora, or b

19、oth.6. Apparatus6.1 Colony CounterAny of several types may be used, forexample, Quebec colony counter.6.2 IncubatorAny incubator capable of maintaining atemperature of 35 6 2C.6.3 SterilizerAny suitable steam sterilizer capable of pro-ducing the conditions or sterilization.6.4 Timer (Stop-Clock)One

20、that can be read for hours andminutes.7. Reagents and Materials7.1 Bacteriological Pipette5.0 and 2.2 mL or 1.1 mLcapacity.NOTE 1Presterilized/disposable bacteriological pipettes are availablefrom most laboratory supply houses.7.2 Water Dilution BottlesAny sterilizable container hav-ing a 150 to 200

21、 mL capacity and tight closure.7.3 Scrubbing CupsSterile cylinders, (Recommendedheight approximately 2.5 cm, inside diameter 23 cm.7.4 Rubber PolicemanCan be fashioned in the laboratoryor purchased from most laboratory supply houses.7.5 Test FormulationWith directions for use.7.6 Occlusive Plastic W

22、rapUsed to occlude skin sites.7.7 Sampling SolutionDissolve 0.4 g KH2PO4, 10.1 gNa2HPO4and 1.0 g octylphenoxypolyethoxyethanol (see Note2) in 1 L of distilled water or higher purity water that meets orexceeds, Specification D1193, Type III or better. Include in thisformulation a neutralizer specific

23、 for the antimicrobial in thetest formulation (see Test Methods E1054) if appropriate.Adjust to pH 7.8 6 0.1. Dispense appropriate volumes andsterilize .NOTE 2Also known as Triton X-100.7.8 Dilution FluidButterfields phosphate buffered water4adjusted to pH 7.2, and containing an antimicrobial activa

24、torspecific for the test formulation. (See Test Methods E1054.)7.9 Plating MediumSoybean-casein digest agar medium5or commercial equivalent.7.10 Personal Hygiene Kitcontents may include variousnon-antimicrobial formulations such as shampoo, hand soap,non-aerosol deodorant, and gloves at the discreti

25、on of theinvestigator.7.11 Adhesive Tapesurgical or other appropriate adhesivetape.8. Procedure8.1 Reduction of Microbial Flora by Products That Are NotApplied Under Dressings.8.1.1 Number of SubjectsSample size calculations shouldbe done to determine the number of subjects necessary to findstatisti

26、cally significant differences (reductions) from baseline.The number of subjects required depends on the statisticalconfidence required for the expected results, the variabilityencountered in the data collection (for example, variability inreductions from baseline), and the expected efficacy of the t

27、estproduct (for example, its expected reduction from baseline).The minimum number of subjects (n) required for each testformulation can be estimated from the following equation:n.S2SZ/21Z!2D2 D(1)S2= estimate of variance (of reductions from baselinebased on in-house data pool),(Z/2= cumulative proba

28、bility of the standard normaldistribution, = 1.96 for =0.05,Z= power of the test = 0.842 for = 0.80, andD = expected efficacy (expected reduction frombaseline).NOTE 3Experience has shown that a range from 1218 subjectsprovides acceptable data.8.1.2 Subject Inclusion Criteria:8.1.2.1 Individuals betw

29、een the ages of 18 and 65 yearsbeing preferably both male and female,8.1.2.2 Hands and forearms free of dermatoses, lesions,open wounds hangnails, or other skin disorders, and8.1.2.3 Are in general good health as evidenced by historyand limited medical examination.8.1.3 Subject Exclusion Criteria:8.

30、1.3.1 Exposure to antimicrobial agents, medicated soaps,medicated shampoos or medicated lotions during the two weekwashout period or test period,8.1.3.2 Exposure of hands or forearms to strong detergents,solvents or other irritants during the two week pre-wash periodor test period,8.1.3.3 Currently

31、receiving a typical or systemic antibiotic,and8.1.3.4 Not willing to fulfill the requirements of the proto-col.8.1.4 Subject InstructionsSubjects are to refrain fromusing any product containing an antimicrobial agent for at leasttwo weeks prior to the test. Kits containing non-medicated bar4Downes,

32、F.P. and K. Ito, Compendium of Methods for the MicrobiologicalExamination of Foods,American Public HealthAssociation, Washington, DC, 2001,p. 637 and p. 643.5U.S. Pharmacopeia 2567, United States Pharmacopeial Convention, Inc.,Rockville, MD,200234, see Chapter entitled “Microbial Limits Test.”E1589

33、05 (2015)2soap, shampoo, and roll-on or stick antiperspirant for useduring this time are provided. Subjects are instructed not to usemedicated creams, ointments, or take antibiotics. Bathing inbiocide treated pools, hot tubs, and spas is not permitted.Subjects are instructed not to shower in the 48

34、h periodproceeding the sampling period; however, a sponge bathexcluding the test areas is permitted.8.1.5 Test Site OcclusionFollowing a two-week wash-outperiod, the forearms of each of the human subjects areoccluded with occlusive plastic wrap patches. The occlusiveplastic wrap is cut to fashion a

35、patch that will cover the volaraspect of the forearm, approximately 8 to 10 cm by 18 to 20cm. A patch is placed in the mid-volar surface of each arm andanchored with adhesive tape.8.1.6 Treatment ProcedureA test formulation treatmentand two control sites, no treatment and vehicle control (for-mula w

36、ithout the active), are located on the volar aspect ofeach forearm, each site measures 3.5 by 3.5 cm. The assign-ment of the treatment and two controls to the three sites oneach forearm is made according to a predetermined random-ization scheme. Approximately 48 h following occlusion,individual site

37、s are exposed to air prior to treatment, untilvisibly dry. The test formulation and vehicle control sites aretreated with the appropriate material according to the productlabel instructions. The treated test formulation sites and vehiclecontrol sites are to be sampled after a 30 min exposure period.

38、(Other appropriate time intervals may be used.) The untreatedcontrol sites are sampled without treatment at the samepost-treatment time interval.8.1.7 Bacterial Sampling (Cup Scrubbing Method):8.1.7.1 Delineate the area to be sampled by a sterile sam-pling cylinder. Firmly hold it against the skin d

39、uring samplingto ensure that the washing fluid does not leak from thesampling site.8.1.7.2 Three mL of sterile sampling fluid is pipeted into thecup, and the entire area is scrubbed with moderate pressure forone minute using a sterile rubber “policeman.”8.1.7.3 The sampling fluid is removed with a s

40、terile pipetteand retained.8.1.7.4 The scrubbing procedure is repeated and the fluidsfrom the two washes are pooled.8.1.7.5 Dilution fluid is Butterfields phosphate buffer thatcontains a neutralizer for the antimicrobial agent.8.1.7.6 Plating medium: Soybean casein digest agar usingstandard pour or

41、spread plate procedure may be used.8.1.7.7 Promptly after the collection of the washing fluid,aliquots are plated 100through 10-4for all samples.8.1.7.8 All plating is in duplicate, and plates are incubatedaerobically at 35 6 2C for 48 6 4 h before counting.8.1.8 Analysis of DataTransform counts fro

42、m each testsite on each arm to log10and use the log10counts for statisticalevaluation. Evaluate differences between treatments and per-form an overall analysis to test the null hypothesis of nodifference among treatments. Test treatment differences forstatistical significance at the 95 % confidence

43、level, usingappropriate parametric or non-parametric procedures, or both.8.2 Suppression of Growth Under a Dressing:8.2.1 Number of SubjectsSame as 8.1.1.NOTE 4Experience has shown that at least 10 subjects providesacceptable data.8.2.2 Eligibility of Test SubjectsSame as 8.1.2 and 8.1.3.8.2.3 Subje

44、ct InstructionsSame as 8.1.4.8.2.4 Treatment Procedure:8.2.4.1 Application SitesA test product treatment and twocontrol sites, no treatment and vehicle control (formula withoutthe active), are located on the volar aspect of each forearm.Following a two-week washout period, application of the testpro

45、duct and controls to the arms are made according to apredetermined randomization.8.2.4.2 Application of Liquid, Ointment and PowdersOnemLor1gofthetest material is spread over each 25 cm2site.The product treated site, vehicle control site, and the untreatedcontrol site are immediately covered with a

46、25 cm2ofocclusive wrap. Each site is occlusively sealed with occlusivewrap and tape. A strip of adhesive tape is placed between eachsite to prevent translocation of the test agents and the micro-organisms from one site to another.8.2.4.3 Application of Impregnated DressingsApplydressings according t

47、o label directions.8.2.4.4 Each site is quantitatively sampled after 24 h ofocclusion using the procedure described in 8.1.6.8.2.5 Analysis of DataSame as 8.1.8.9. Neutralizer Validation9.1 When neutralizers are added to culture media or dilutingfluids, or both, validate the effectiveness of the sys

48、temsaccording Test Methods E1054.10. Precision and Bias10.1 A precision and bias statement cannot be made for thistest method at this time.11. Keywords11.1 antiseptic; bacteria; bandages; cup scrub; dressing; firstaid; efficacy; first aid; impregnated dressing; occlusion; skinmicrofloraE1589 05 (201

49、5)3ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedin this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the riskof infringement of such rights, are entirely their own responsibility.This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years andif not revised, either reapproved or withdrawn. Your comments are