ASTM E3058-2016 Standard Test Method for Determining the Residual Kill Activity of Hand Antiseptic Formulations《测定手消毒制剂的剩余杀菌活性的标准试验方法》.pdf

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1、Designation: E3058 16Standard Test Method forDetermining the Residual Kill Activity of Hand AntisepticFormulations1This standard is issued under the fixed designation E3058; 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 () indicates an editorial change since the last revision or reapproval.1. Scope1.1 This test method is designed to determine the residualkilling activity of skin antiseptics against transient microb

3、ialskin flora on the hands .2It may be used to evaluate productsthat are used with the aid of water and rinsed off and those thatare used without the aid of water and not rinsed off.1.2 Performance of this procedure requires the knowledgeof regulations pertaining to the protection of human subjects(

4、see 21 CFR Parts 50 and 56).1.3 This test method should be performed by persons withtraining in microbiology, in facilities designed and equippedfor work with potentially infectious agents at biosafety level 2.1.4 UnitsThe values stated in SI units are to be regardedas standard. No other units of me

5、asurement 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 user of this standard to establish appro-priate safety and health practices and determine the applica-bility of regulatory l

6、imitations prior to use. For more specificprecautionary statements see 8.1.2. Referenced Documents2.1 ASTM Standards:3E1054 Test Methods for Evaluation of Inactivators of Anti-microbial AgentsE2752 Guide for Evaluation of Residual Effectiveness ofAntibacterial Personal Cleansing ProductsE1882 Test M

7、ethod for Evaluation of Antimicrobial Formu-lations by the Agar Patch TechniqueE2197 Quantitative Disk Carrier Test Method for Determin-ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,and Sporicidal Activities of ChemicalsE2756 Terminology Relating to Antimicrobial and AntiviralAgents2.2 F

8、ederal Standards:421 CFR Part 50 Protection of Human Subjects21 CFR Part 56 Institutional Review Boards3. Terminology3.1 DefinitionsFor definitions of terms used in thisdocument, see Terminology E2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 healthcare personnel hand rub, nan antimic

9、robialgel, foam, liquid, spray, or wipe, applied by rubbing to reducethe transient microbial skin flora on hands that are not visiblysoiled, and which does not require a post-treatment water rinse.Such agents may also be referred to as hand rubs, hygienichand rubs, hand sanitizers, or hand antisepti

10、cs.3.2.2 healthcare personnel hand wash, na cleanser requir-ing rinsing or a non-rinse agent intended to reduce transientmicrobial skin flora on the hands.3.2.3 room temperature, ntemperature in the range of 20to 25C (68 to 77F).3.2.4 test bacteria, nan applied suspension of bacteriahaving character

11、istics that permit ready identification of colo-nies. Test bacteria are used to simulate a transient topicalmicrobial contaminant. These may also be referred to as testorganisms, marker organisms, simulants, or contaminants.3.2.5 test material, na product or formulation that incor-porates an antimic

12、robial ingredient(s).4. Summary of Test Method4.1 This test method uses adult subjects who have provideda written informed consent and whose hands have beendetermined to be free from any clinical evidence of skindisorders, dermatosis, cuts, lesions, or hangnails at the time of1This test method is un

13、der the jurisdiction of ASTM Committee E35 onPesticides, Antimicrobials, and Alternative Control Agents and is the directresponsibility of Subcommittee E35.15 on Antimicrobial Agents.Current edition approved Nov. 15, 2016. Published December 2016. DOI:10.1520/E3058162Rutter J.D., Angiulo K., Macinga

14、 D.R., Measuring residual activity of topicalantimicrobials: is the residual activity of chlorhexidine an artefact of laboratorymethods?J. Hosp. Infect. 88:113-115, 20143For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annua

15、l Book of ASTMStandards volume information, refer to the standards Document Summary page onthe ASTM website.4Available from U.S. Government Printing Office, Superintendent ofDocuments, 732 N. Capitol St., NW, Washington, DC 20401-0001, http:/www.access.gpo.gov.Copyright ASTM International, 100 Barr

16、Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United StatesThis international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for theDevelopment of International Standards, Guides and Recommen

17、dations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.1participation in the study. Subjects are to refrain from use ofany antimicrobials for at least 7 days prior to the initiation ofthe test procedure (see 12.3).4.2 Subjects hands are pre-treated with the test m

18、aterial toload the antimicrobial onto the skin. Test material remains onthe hands for a pre-determined time (the time selected todemonstrate the test products residual kill activity) prior tocontamination.4.3 Subjects press each fingerpad onto a stainless steel disccontaminated with approximately 7

19、log10CFU of test organism(using one disc per fingerpad), which transfers approximately 6log10CFU of test organism to each fingerpad (that is, approxi-mately 10% transfer). The test organism, Staphylococcusaureus (ATCC 6538), remains viable upon drying and is stableon both the stainless steel discs a

20、nd on the fingerpads over thecourse of the experiment. The fingerpads are exposed to thechallenge organism for pre-determined times.4.4 The test bacterium is then recovered from the fingerpadsby rubbing each fingerpad for 30 s in a Petri dish containing 10ml neutralizer (one Petri dish per fingerpad

21、).4.5 Residual killing by the test material is measured bycomparing the number of test bacteria recovered from contami-nated fingerpads at specific time intervals after contaminationto the number recovered at time zero (treated fingerpad withzero time to allow for reduction in microorganism afterapp

22、lication).5. Significance and Use5.1 Many marketed hand antiseptics make claims of “long-lasting protection” or “extended kill” (for example 6 hours),which are typically based on results of testing as described inTest Method E1882 or Guide E2752, or both.At this time thereare no standard methods for

23、 evaluating a hand antisepticformulation for its ability to kill microorganisms on handswhen a “dry” contamination event occurs at some time afterproduct use. This test method provides a method to substantiateresidual kill claims for hand antiseptics.6. Apparatus6.1 Aluminum barsDiscs are attached t

24、o these to avoidmovement and / or sticking of the discs to the fingerpads duringcontamination (see Fig. 1). Any of several types may be used,for example, multipurpose 6061 aluminum rectangular,38 in. 12 in. 6 in.5.6.2 Colony CounterAny of several types may be used; forexample, Quebec darkfield colon

25、y counters and similar de-vices. Automated, computerized plater/counter systems mayalso be used.6.3 Discs1 cm diam. and 0.7 mm thick made from sheetsof brushed stainless steel, AISI Type 430 (E2197).6.4 Handwashing SinkSufficient in size to permit hand-washing without the touching of hands to sink s

26、urface or othersubjects.6.5 Humidity ChamberCapable of maintaining 50-60%relative humidity in the chamber for 24 h at room temperature.6.6 Humidity Monitor (Hygrometer)Calibrated and ca-pable of displaying relative humidity in 1% increments6.7 Water Faucet(s)Located above the sink at a height toperm

27、it hands to be held higher than the elbow during thewashing procedure.6.8 Tap Water Temperature Regulator and TemperatureMonitorTo set and maintain the tap water temperature at40 6 2C6.9 IncubatorCapable of maintaining a temperature of35 6 2C.6.10 Biological Safety Cabinet.6.11 Miscellaneous Labware

28、Continuously adjustable pi-petters (1-mL and 0.2-mL capacity) and sterile pipette tips,sterile serological pipettes (5.0-mL capacity), sterile culturetubes, sterile disposable Petri dishes, sterile syringes, Erlen-meyer flasks, sterile loops and beakers.6.12 Sampling Petri dishesSterile dishes measu

29、ring 100mm 15 mm, and able to hold 10 mL sampling solution (see7.7).6.13 Absorbance MeterCapable of reading at 625 nm witha 1 cm path length.6.14 SterilizerAny steam sterilizer capable of processingculture media and reagents.5The sole source of supply of the apparatus known to the committee at this

30、timeis available from McMaster Carr, part number 8975K614. http:/ If you are aware of alternative suppliers, please provide thisinformation to ASTM International Headquarters. Your comments will receivecareful consideration at a meeting of the responsible technical committee,1whichyou may attend.FIG

31、. 1 Aluminum BarE3058 1626.15 Timer (Stop-Clock)Type that can be read for minutesand seconds.6.16 Vortex MixerAny vortex that will ensure propermixing of culture.7. Reagents and Materials7.1 Antibiotic OintmentA topical, triple-antibiotic oint-ment for application to the hands after the final decont

32、amina-tion.7.2 Cleansing WashA mild, proven non-antimicrobial liq-uid soap. May be purchased commercially or prepared accord-ing to the instructions: Soft Soap, 200 g/L: Linseed oil 50 partsby weight Potassium hydroxide 9.5 parts Ethanol 7 partsDistilled or high purity water as needed Add linseed oi

33、l to asolution of potassium hydroxide in 15 parts water and heat upto approximately 70C while constantly stirring. Add theethanol and continue heating while stirring until the saponifi-cation process is completed and a sample dissolves clearly inwater and almost clearly in alcohol. The weight of the

34、 soft soapis then brought up to 100 parts by addition of hot water. Take200 g of the soft soap in 1 L of water. Dispense in toappropriate containers and sterilize in an autoclave.7.3 Chlorhexidine Skin CleanserAntiseptic skin cleansercontaining 4 % chlorhexidine gluconate (w/v) for hand decon-tamina

35、tion.7.4 Culture Media:7.4.1 BrothSoybean-casein digest broth (tryptic soybroth) is recommended.7.4.2 Agar Plating Media:7.4.2.1 Plating MediumSoybean-casein digest agar (tryp-tic soy agar TSA) containing an effective inactivator for thetest material, if necessary is recommended.NOTE 1Ensure that st

36、ock culture of S. aureus and any subsequentsubculture used produces golden-colored colonies on soybean-caseindigest agar.7.4.2.2 S. aureus Plating MediumHardyCHROM6, con-taining an effective inactivator for the test material, ifnecessary, may be used as an alternative to the standard platingmedia. O

37、ther indicator media for S. aureus may be appropriatebut should be validated prior to use.NOTE 2S. aureus forms smooth, deep pink to fuchsia-colored colonieswhen grown on HardyCHROM.6The growth of most other organisms,including Staphylococcus epidermidis are partially to completely inhib-ited.7.5 Di

38、lution and Sampling FluidDissolve 0.4 g KH2PO4,10.1 g Na2HPO4, 1.0 g isooctylphenoxypolyethoxyethanol(for example, Triton X-100), and appropriately validatedneutralizers, if necessary (see Note 3), in distilled water.AdjustpH to 7.8 6 0.1 with 0.1 N HCl or 0.1 N NaOH and bringvolume to 1 L with dist

39、illed water.NOTE 3Aneutralizer validation should be conducted according toTestMethods prior to the study. Test Methods E1054 provides a list ofneutralizers appropriate for commonly used antimicrobial agents. In somecases (for example, some alcohol-based hand rubs) neutralization isachieved by diluti

40、on alone, therefore, inclusion of an inactivator is onlyrequired if neutralization of the test material cannot be achieved upondilution (see 7.5).7.6 Ethanol Solution70 % ethanol in water (v/v) for handdecontamination.7.7 Test MaterialUse directions provided with the testmaterial. If directions are

41、not provided, use the directions givenin this method.7.8 Negative Control70 % ethanol in water (v/v).8. Hazards8.1 Application of microorganisms to the skin may involvea health risk. Determine the antibiotic sensitivity profile of thetest bacteria prior to applying to the skin. After the test hasbee

42、n completed, decontaminate the subjects hands and followproper procedures to reduce infection risk (13.11). If aninfection occurs, provide the antibiotic susceptibility profile tothe attending clinician.9. Test Bacteria9.1 Staphylococcus aureus ATCC 6538 (methicillin-sensitive) is the recommended te

43、st bacterial species. S. aureusis differentiated from resident microbial skin flora (includingStaphylococcus epidermidis) colonies by colony morphologyand pigmentation on standard plating media (see 7.4.2.1)orwith chromogenic indicator medium (see 7.4.2.2).10. Preparation of Test Bacteria Suspension

44、10.1 Preparation of S. aureus:10.1.1 Prepare a stock culture of S. aureus ATCC 6538 (nomore than 5 transfers from original ATCC vial) by inoculatingapproximately 5 mL of soybean-casein digest broth (see 7.4.1)from a frozen stock or lyophilized vial and incubate for 18-24hat356 2C.10.1.2 Using a ster

45、ile bacteriological loop inoculate a suf-ficient number of soybean-casein digest agar plates (see7.4.2.1) from the overnight culture for colony isolation andincubate for 18-24 h at 35 6 2C.10.1.3 Using a sterile bacteriological loop, gently scrape orrub surface of agar to remove golden-colored colon

46、ies andsuspend in fresh soybean-casein digest broth to an absorbanceof 0.2-0.25 at 625 nm at a path length of 1 cm (or otherappropriate measurement based on your absorbance meterspecifications) to approximate a titer of 8.5-9.0 log10CFU/ml.Remove an aliquot from the suspension, dilute and plate forc

47、ounting. An isolation streak plate should be made to confirmpurity.11. Inoculation of Stainless Steel Discs11.1 Mix the test bacterial suspension using a vortex for 30s to homogenize it.11.2 Use a calibrated pipette to transfer 10 L of thebacterial inoculum to the center of each sterile disc (6.3).

48、Donot spread the inoculum to avoid operator variability and alsoto maintain uniform thickness of the inoculum on all discs. Forconsistency, the same pipette tip should be used when inocu-lating a given batch of discs. If possible, the inoculation should6Trademarked HardyCHROMStaph aureus, available

49、from Hardy DiagnosticsE3058 163be done in the laminar flow hood to avoid disturbance of theinoculum during transport to the hood.11.3 Transfer the inoculated discs to a biological safetycabinet for approximately 1 hour to dry the inocula.11.4 Transfer the dried inoculated discs to a 50-60% relativehumidity chamber and maintain at room temperature for 17 to24 h.NOTE 4Variation in the relative humidity within the test facility canaffect the rate of transfer of the test organism to the fingerpads. Storage oft