ASTM E1589-2005 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

1、Designation: E 1589 05Standard Test Method forEvaluation of First Aid Antiseptic Drug Products1This standard is issued under the fixed designation E 1589; the number immediately following the designation indicates the year oforiginal adoption or, in the case of revision, the year of last revision. A

2、 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 The tests described in this test method are designed toevaluate antimicrobial agents in formulations intended for useas first aid ant

3、iseptic 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 The values stated in SI units are to be regarded as thestandard.1.4 This standard does not purport to address all of t

4、hesafety 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.1.5 Performance of this procedure requires the knowledgeof regulation

5、s pertaining to the protection of human subjects.(See CFR Parts 50 and 56.)2. Referenced Documents2.1 ASTM Standards:2D 1193 Specification for Reagent WaterE 1054 Test Methods for Evaluation of Inactivators ofAntimicrobial Agents2.2 Federal Standards:3CFR Parts 50 and 563. Terminology3.1 active ingr

6、edient, 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 dilution of theformulation, or with the use of chemical agents, called neu-t

7、ralizers.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, forming a permanent population.3.5 sampling fluid, na recovery fluid th

8、at 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, nmicroorganisms that con-taminate but do not normally permanently colonize the

9、skin.4. Summary of Test Methods4.1 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 areoccluded for 48 h prior to application of the test formulation t

10、oincrease 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 time(s)following application, the sites are microbiologically sampled

11、and 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 Skin Flora When Applied As a DressingThedressings are applied to the fo

12、rearm 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 sampled and the samples plated for totalaerobic bacteria count.1This

13、 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. 1, 2005. Published December 2005. Originallyapproved in 1994. Last previous edition approved in 1994 as E1589 - 94 wh

14、ich waswithdrawn in June 2003 and reinstated in November 2005.2For 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 the standards Document Summary page onthe ASTM website

15、.3Available from U.S. Government Printing Office, Superintendent of Docu-ments, Washington DC, 20402.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.4.2 The principal of the test is that the microflora of forearmskin is sparse. The i

16、mpermeable dressing will increase surfacemoisture by preventing diffusional water loss and thus expandtransient resident skin microorganisms population. A signifi-cant antimicrobial effect by the test agent will be reflected bysignificantly lower population recovered from the nontreatedsite.5. Signi

17、ficance 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 preventinfection in minor cuts, scrapes and burns.5.1.1 This test method is applicable for testing liquids,oin

18、tments, 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 both.6. Apparatus6.1 Colony CounterAny of several types may be used, forexample, Quebec colony count

19、er.6.2 IncubatorAny incubator capable of maintaining atemperature of 35 6 2C.6.3 SterilizerAny suitable steam sterilizer capable ofproducing the conditions or sterilization.6.4 Timer (Stop-Clock)One that can be read for hours andminutes.7. Reagents and Materials7.1 Bacteriological Pipette5.0 and 2.2

20、 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 mL capacity and tight closure.7.3 Scrubbing CupsSterile cylinders, (Recommendedheight approximately

21、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 WrapUsed to occlude skin sites.7.7 Sampling SolutionDissolve 0.4 g KH2PO4, 10.1 gNa2HPO4and 1.0 g octy

22、lphenoxypolyethoxyethanol4in1Lofdistilled water or higher purity water that meets or exceeds,Specification D 1193, Type III or better. Include in thisformulation a neutralizer specific for the antimicrobial in thetest formulation (see Test Methods E 1054) if appropriate.Adjust to pH 7.8 6 0.1. Dispe

23、nse appropriate volumes andsterilize .7.8 Dilution FluidButterfields phosphate buffered water5adjusted to pH 7.2, and containing an antimicrobial activatorspecific for the test formulation. (See Practices E 1054.)7.9 Plating MediumSoybean-casein digest agar medium6or commercial equivalent.7.10 Perso

24、nal Hygiene Kitcontents may include variousnon-antimicrobial formulations such as shampoo, hand soap,non-aerosol deodorant, and gloves at the discretion of theinvestigator.7.11 Adhesive Tapesurgical or other appropriate adhesivetape.8. Procedure8.1 Reduction of Microbial Flora by Products That Are N

25、otApplied Under Dressings.8.1.1 Number of SubjectsSample size calculations shouldbe done to determine the number of subjects necessary to findstatistically significant differences (reductions) from baseline.The number of subjects required depends on the statisticalconfidence required for the expecte

26、d results, the variabilityencountered in the data collection (for example, variability inreductions from baseline), and the expected efficacy of the testproduct (for example, its expected reduction from baseline).The minimum number of subjects (n) required for each testformulation can be estimated f

27、rom the following equation:n.S2SZa/21Zb!2D2 D(1)S2= estimate of variance (of reductions from baselinebased on in-house data pool)(Za/2= cumulative probability of the standard normaldistribution, = 1.96 for a=0.05,Zb= power of the test = 0.842 for b = 0.80,D = expected efficacy (expected reduction fr

28、om base-line).NOTE 2Experience has shown that a range from 1218 subjectsprovides acceptable data.8.1.2 Subject Inclusion Criteria:8.1.2.1 Individuals between 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,

29、 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:8.1.3.1 Exposure to antimicrobial agents, medicated soaps,medicated shampoos or medicated lotions during the two weekwashout period or test period,8

30、.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 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

31、are to refrain fromusing any product containing an antimicrobial agent for at least4Also known as Triton X-100.5Downes, F.P. and K. Ito, Compendium of Methods for the MicrobiologicalExamination of Foods,American Public HealthAssociation, Washington, DC, 2001,p. 637 and p. 643.6U.S. Pharmacopeia 2567

32、, United States Pharmacopeial Convention, Inc.,Rockville, MD,200234, see Chapter entitled 9Microbial Limits Test.9E1589052two weeks prior to the test. Kits containing non-medicated barsoap, shampoo, and roll-on or stick antiperspirant for useduring this time are provided. Subjects are instructed not

33、 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 h periodproceeding the sampling period; however, a sponge bathexcluding the test areas is permitted.8.1.5 Test Site OcclusionF

34、ollowing 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 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-vol

35、ar 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 without the active), are located on the volar aspect ofeach forearm, each site measures 3.5 cm by 3.5 cm. Theassignment of the

36、treatment and two controls to the three siteson each forearm is made according to a predetermined ran-domization scheme. Approximately 48 h following occlusion,individual sites are exposed to air prior to treatment, untilvisibly dry. The test formulation and vehicle control sites aretreated with the

37、 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.(Other appropriate time intervals may be used.) The untreatedcontrol sites are sampled without treatment at the samepost-trea

38、tment 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 during samplingto ensure that the washing fluid does not leak from thesampling site.8.1.7.2 Three mL of sterile sampling fluid

39、 is pipeted into thecup, and the entire area is scrubbed with moderate pressure forone minute using a sterile rubber 9policeman.98.1.7.3 The sampling fluid is removed with a sterile pipetteand retained.8.1.7.4 The scrubbing procedure is repeated and the fluidsfrom the two washes are pooled.8.1.7.5 D

40、ilution 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 spread plate procedure may be used.8.1.7.7 Promptly after the collection of the washing fluid,aliquots are plated 100through

41、10-4for all samples.8.1.7.8 All plating is in duplicate, and plates are incubatedaerobically at 35C 6 2C for 48 6 4 h before counting.8.1.8 Analysis of DataTransform counts from each testsite on each arm to log10and use the log10counts for statisticalevaluation. Evaluate differences between treatmen

42、ts and per-form an overall analysis to test the null hypothesis of nodifference among treatments. Test treatment differences forstatistical significance at the 95 % confidence level, usingappropriate parametric or non-parametric procedures, or both.8.2 Suppression of Growth Under a Dressing:8.2.1 Nu

43、mber of SubjectsSame as 8.1.1.NOTE 3Experience 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 Subject InstructionsSame as 8.1.4.8.2.4 Treatment Procedure:8.2.4.1 Application SitesA test product treatment and twocontrol site

44、s, 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 testproduct and controls to the arms are made according to apredetermined randomization.8.2.4.2 Application of Liquid, Ointment and

45、 PowdersOnemLor1gofthetest material is spread over each 25 cm2site.The product treated site, vehicle control site, and the untreatedcontrol site are immediately covered with a 25 cm2ofocclusive wrap. Each site is occlusively sealed with occlusivewrap and tape. A strip of adhesive tape is placed betw

46、een 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 to label directions.8.2.4.4 Each site is quantitatively sampled after 24 h ofocclusion using the procedure described in 8.1.6

47、.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 systemsaccording Test Method E 1054.10. Precision and Bias10.1 A precision and bias statement cannot be made for thistest metho

48、d at this time.11. Keywords11.1 antiseptic; bacteria; bandages; cup scrub; dressing; firstaid; efficacy; first aid; impregnated dressing; occlusion; skinmicrofloraE1589053ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentionedi

49、n 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 invited either for revision of this standard or for additional standardsand should be addressed to ASTM International Headquarters. Your comment