1、Designation: E 2613 08Standard Test Method forDetermining Fungus-Eliminating Effectiveness of HygienicHandwash and Handrub Agents Using Fingerpads ofAdults1This standard is issued under the fixed designation E 2613; the number immediately following the designation indicates the year oforiginal adopt
2、ion or, in the case of revision, the year 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.INTRODUCTIONHuman hands are frequently in contact with other surfaces and can readil
3、y acquire transientmicrobial contamination. Fungi are common among these types of contaminants (1, 2),2particularlyin healthcare settings and where food is handled. Standardized methods to assess the fungus-eliminating potential of handwash and handrub agents have not been available and this test me
4、thodaddresses the gap.1. Scope1.1 This test method is designed to assess the ability ofhygienic handwash and handrub agents to reduce levels offungal contamination on hands (3). This test method is notmeant for use with surgical hand scrubs (Test Method E 1115)or preoperative skin preps (Test Method
5、 E 1173).1.2 Performance of this procedure requires the knowledgeof regulations pertaining to human experimentation.31.3 The test method should be performed by persons withtraining in microbiology in facilities designed and equipped forwork with infectious agents at biosafety level 2 (4).1.4 The val
6、ues 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 user of this standard to establish appro-priate sa
7、fety and health practices and determine the applica-bility of regulatory limitations prior to use.2. Referenced Documents2.1 ASTM Standards:4D 1129 Terminology Relating to WaterE 1054 Test Methods for Evaluation of Inactivators ofAntimicrobial AgentsE 1115 Test Method for Evaluation of Surgical Hand
8、 ScrubFormulationsE 1173 Test Method for Evaluation of Preoperative, Pre-catheterization, or Preinjection Skin PreparationsE 1838 Test Method for Determining the Virus-EliminatingEffectiveness of Liquid Hygienic Handwash and HandrubAgents Using the Fingerpads of Adult VolunteersE 2276 Test Method fo
9、r Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Han-drub Agents Using the Fingerpads of Adult Subjects3. Terminology3.1 DefinitionsFor definitions of general terms used inthis test method, refer to Terminology D 1129.3.2 Definitions of Terms Specific to This Standard:3.
10、2.1 active ingredient, na substance added to a formula-tion specifically for the inhibition or inactivation of microor-ganisms.3.2.2 dry controla control to determine the number ofcolony forming units (CFU) of the test fungus remaining viableafter the initial drying of the inoculum on the skin.3.2.3
11、 handrub, na liquid, gel, or foam, which is appliedby rubbing to decontaminate lightly soiled hands betweenhandwashings and generally does not require a post-treatmentwater rinse; such agents usually contain alcohol alone or withother active ingredients.3.2.4 hard water, nwater with a defined level
12、of hardnessas calcium carbonate.1This test method is under the jurisdiction of ASTM Committee E35 onPesticides and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2008. Published May 2008.2The boldface number
13、s in parentheses refer to a list of references at the end ofthis standard.3Federal Register, Vol 46, No. 17, Jan. 27, 1991.4For referenced ASTM standards, visit the ASTM website, www.astm.org, orcontact ASTM Customer Service at serviceastm.org. For Annual Book of ASTMStandards volume information, re
14、fer to the standards Document Summary page onthe ASTM website.1Copyright ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959, United States.3.2.5 hygienic handwash agent, nan agent generally usedfor handwashing by personnel in hospitals, other health-carefaciliti
15、es, day-care centers, nursing homes, and food-handlingestablishments to eliminate transient microorganisms fromintact skin.3.2.6 input control, na control to determine the number ofcolony forming units of the test fungus placed on each digit.3.2.7 neutralizationa process which results in quenchingth
16、e antifungal activity of a test material. This may be achievedthrough dilution of the test material(s) to reduce the antifungalactivity, or through the use of chemical agents, called neutral-izers, to eliminate antifungal activity.3.2.8 non-medicated soap, na soap or detergent for hand-washing that
17、is mild to the skin and does not contain anyantimicrobial chemicals.3.2.9 soil lead, na solution of one or more organic and/orinorganic substances added to the suspension of the testorganism to simulate the presence of body secretions, excre-tions or other extraneous substances.3.2.10 test formulati
18、on, na formulation which incorpo-rates antimicrobial ingredients.3.2.11 test organism, nan applied inoculum of an organ-ism that has characteristics which allow it to be readilyidentified. The test organism is used to simulate a transienttopical fungal contaminant. It may also be referred to as amar
19、ker organism, fungal simulant/surrogate or fungal contami-nant.3.2.12 test vehicle, nthe test agent without an activeingredient.4. Summary of Test Method4.1 This test method is conducted using a group of adultsubjects who have provided informed consent and the skin ofwhose hands has been determined
20、to be free from any apparentdamage. Subjects are to refrain from using any productscontaining antimicrobial agents for at least one week prior tothe test. A known volume of the test fungal suspension isplaced on a demarcated area on each fingerpad and theinoculum allowed to dry. The contaminated are
21、a is thenexposed to the control (standard hard water) or test agent forthe desired contact time and organisms remaining on thefingerpad are eluted and the eluates assayed for fungal CFU.Reductions in the numbers of CFU after treatment with thecontrol and test agents are then determined. If two diffe
22、rentformulations are being compared in the same test, one of themmay be designated as a reference and used in place of the hardwater control.5. Significance and Use5.1 This in vivo procedure is designed to test the ability ofhygienic handwash or handrub agents to eliminate fungalcontamination from e
23、xperimentally-contaminated hands. Sincethe two thumbpads and all eight fingerpads can be used in anygiven test, it allows for the incorporation of an input control(two), control for viable cells of the test fungus remaining afterthe inoculum has been allowed to dry (two), fungal cellseliminated afte
24、r treatment with a control or reference solution(two), and up to four replicates to assess the fungus-eliminatingefficiency of the formulation under test. No more than 100 Lof the test fungal suspension is required to complete one test.5.2 Whereas this test method is designed to work with fungi,simi
25、lar ASTM standards exist for testing against viruses (TestMethod E 1838) and vegetative bacteria (Test Method E 2276).5.3 Infectious microorganisms left on hands after washingcan be reduced further by drying the washed hands with paper,cloth, or warm air (5). A step for the drying of fingerpads afte
26、rexposure to the control or test solution, therefore, has not beenincluded to avoid fungal removal by the drying process itself.5.4 This test method is not meant for testing surgical handscrubs or preoperative skin preps.5.5 The level of contamination with viable fungi on eachfingerpad after the dry
27、ing of the inoculum should be at least104CFU so that it would permit the detection of up to a 4-log10reduction in the viability titer of the test organism by a testformulation under the conditions of this test. This in itself doesnot represent the product performance criterion, which mayvary dependi
28、ng on the jurisdiction and the nature of theformulation being evaluated.6. Equipment and Apparatus6.1 Colony counter, any of several types may be used, forexample, Quebec Colony Counter.6.2 Freezer, a freezer at 70C or lower is required to storefungal stocks.6.3 Handwashing sink, a sink of sufficien
29、t size to permitsubjects to wash hands without touching hands to sink surface.6.4 Incubator, any incubator that can maintain a tempera-ture suitable for the growth of Candida albicans and Aspergil-lus niger.6.5 Laminar flow cabinet, a Class II biological safetycabinet.6.6 Magnetic stirrer and magnet
30、s, Llarge enough to hold a5-L beaker or Erlenmeyer flask for preparing culture media orother solutions.6.7 Membrane filtration system, a membrane filtration sys-tem and membranes with a pore diameter of 0.45mor0.22m are required to sterilize heat-sensitive media/solutions.6.8 Positive displacement p
31、ipette, a pipette and pipette tipsthat accurately can dispense 10-L volumes.6.9 Refrigerator, a refrigerator at 4 6 2C for storage ofprepared culture media and reagents.6.10 Sterilizer, any suitable steam sterilizer that producesthe conditions of sterilization is acceptable.6.11 Timer (stop clock),
32、one that can be read for minutes andseconds.6.12 Tap water temperature regulator and temperaturemonitor, to monitor and regulate water temperature at 40 62C.6.13 Water faucet(s), to be located above the sink at a heightthat permits the hands to be held higher than the elbow duringthe washing procedu
33、re. Faucets with electronic sensors orthose that are wrist-, elbow-, knee-, or foot-operated arepreferred to avoid recontamination of the washed hands.7. Reagents and Materials7.1 Serological pipettes, sterile reusable or single-use pi-pettes of 10.0, 5.0, and 1.0-mL capacity.E26130827.2 Culture pla
34、tes, Petri plates of 100 mm diameter forculturing the fungus.7.3 Soil Load:7.3.1 A tripartite soil load, prepared from the followingthree stock solutions, as an alternative to serum.7.3.2 Add 0.5 g of tryptone to 10 mL of phosphate buffer.7.3.3 Add 0.5 g of bovine serum albumin (BSA) to 10 mLof phos
35、phate buffer.7.3.4 Add 0.04 g of bovine mucin to 10 mL of phosphatebuffer.7.3.5 Prepare the stock solutions separately and sterilize bypassage through a 0.22 m pore diameter membrane filter,aliquot and store at either 4 6 2C or 20 6 2C.7.3.6 To obtain a 500-L inoculum of the test inoculum, addto 340
36、 Lof the fungal suspension 35 Lof tryptone (7.3.2), 25L BSA(7.3.3), and 100 L mucin (7.3.4) stock solutions. Thismixture contains approximately2goftotal protein/L, which isroughly equivalent to the protein content of a 5 % solution offetal bovine serum.7.4 Standard Hard WaterThe quality and disinfec
37、tant (forexample, chlorine) residual in tap water can vary from site tosite and also at different times at the same site. The use ofstandard hard water, therefore, is recommended here to avoidvariations in results due to differences in tap water quality.Water prepared in accordance with AOAC 960.09
38、E and F (6)to a standard hardness of 200 ppm as calcium carbonate is usedfor dilution of test products, as the control solution to deter-mine the baseline level of fungal elimination, and to rinse thefingerpads after exposure to the test product. The standard hardwater and tap water (if used) must f
39、irst be tested to ensure thatthey do not have any activity against the test fungus. If waterwith a higher level of hardness is used for making theuse-dilution of the test formulation, this change must be clearlyjustified and documented in the report.7.5 Test agents, at least two samples of the test
40、formulationshall be evaluated.NOTE 1Water with a standard level of hardness is also recommendedas a control in this test procedure to determine any mechanical removal ofthe test organism(s) from the skin.7.6 Diluent for fungal titration, normal saline (0.85 %NaCl) at pH 7.2 to 7.4 or an appropriate
41、buffer.7.7 Eluent for fungal recovery from fingerpads, normalsaline.7.8 Plastic vials, sterile screw-capped 2.0-mL vials with aninside diameter of about 8 mm will be required for demarcationof the fingerpads and to hold various test solutions.7.9 Miscellaneous laboratory ware, automatic pipettes, pi
42、-pette tips, and plastic vials for storing stock cultures.7.10 Broth, trypticase soy broth (TSB) or equivalent toprepare the inoculum of Candida albicans.7.11 Agar, sabouraud dextrose agar or equivalent to preparethe inoculum of Aspergillus niger and to recover and count thecolonies of the two test
43、organisms in control and test samples.The addition of any neutralizer(s) in such recovery media mustfirst be properly validated (Test Methods E 1054).8. Test Fungi8.1 The selection of the following test fungi is based ontheir (a) relative safety to the subjects as well as experimenters,(b) ability t
44、o grow to titers sufficiently high for testing, (c) easeof recovery and identification in the laboratory, and (d)potential to spread through contaminated hands. Other strainsor types of fungi may be substituted provided they meet thepreceding criteria and are approved by the relevant institutionalre
45、view board.8.1.1 Candida albicans (ATCC 10231). This representsnon-filamentous fungi (yeast-like) and is among the mostcommon species of Candida recovered as clinical isolates (7).8.1.2 Aspergillus niger (ATCC 64958). This is an exampleof a filamentous fungus that is considered safe for the experi-m
46、ental contamination of the skin of adult subjects.8.1.3 Other fungal species may be used provided they aresafe for contamination of the hands of adult subjects and onlyafter permission has been received for their use from therelevant institutional review board.9. Preparation of Test Inoculum9.1 To p
47、repare C. albicans test inoculum, add 0.1 mL of afrozen stock culture to 10 mL of TSB (7.10) and incubate for48 6 4 h at the appropriate temperature.9.2 To prepare a suspension of the conidia of A. niger,inoculate the center of a sabouraud dextrose agar plate with 0.1mL of a frozen stock suspension
48、and incubate for 10 days at28C. Harvest mycelial mats from the agar surface, homog-enize with sterile glass beads in normal saline, and filterthrough sterile gauze to remove the hyphae.9.3 Add soil load (7.3) to the test fungal cell suspension, ifrequired.9.4 Swirl, vortex, or shake the test fungal
49、cell suspensionbefore withdrawal of each aliquot.10. Subjects10.1 Recruit a sufficient number of healthy adults who haveno clinical evidence of dermatoses, open wounds, hangnails, orother skin disorders. The number of subjects required for a trialis dependent on the number of treatments within a study.10.2 It is the responsibility of the user of this test method toarrange the necessary clearance for the use of subjects fortesting and to obtain informed and written consent from thoseselected for the study before starting the tests.10.3 Instruct subjects t
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