1、Designation: E2613 08E2613 14Standard Test Method forDetermining Fungus-Eliminating Effectiveness of HygienicHandwash and Handrub Agents Using Fingerpads ofAdults1This standard is issued under the fixed designation E2613; the number immediately following the designation indicates the year oforiginal
2、 adoption or, in the case of revision, the year 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.INTRODUCTIONHuman hands are frequently in contact with other surfaces and can r
3、eadily acquire transientmicrobial contamination. Fungi are common among these types of contaminants (1, 2),2 particularlyin 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 t
4、est methodaddresses the gap.1. Scope1.1 This test method is designed to assess the ability of hygienic handwash and handrub agents to reduce levels of fungalcontamination on hands (3). This test method is not meant for use with surgical hand scrubs (Test Method E1115) or preoperativeskin preps (Test
5、 Method E1173).1.2 Performance of this procedure requires the knowledge of regulations pertaining to human experimentation.31.3 The test method should be performed by persons with training in microbiology in facilities designed and equipped for workwith infectious agents at biosafety level 2 (4).1.4
6、 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibilityof the user of this standard to establish appr
7、opriate safety and health practices and determine the applicability of regulatorylimitations prior to use.2. Referenced Documents2.1 ASTM Standards:4D1129 Terminology Relating to WaterE1054 Test Methods for Evaluation of Inactivators of Antimicrobial AgentsE1115 Test Method for Evaluation of Surgica
8、l Hand Scrub FormulationsE1173 Test Method for Evaluation of Preoperative, Precatheterization, or Preinjection Skin PreparationsE1838 Test Method for Determining the Virus-Eliminating Effectiveness of Hygienic Handwash and HandrubAgents Using theFingerpads of AdultsE2197 Quantitative Disk Carrier Te
9、st Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, andSporicidal Activities of ChemicalsE2276 Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Usingthe Fingerpads of Adults1 This test method is under the jurisdic
10、tion of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility ofSubcommittee E35.15 on Antimicrobial Agents.Current edition approved April 1, 2008April 1, 2014. Published May 2008May 2014. Originally approved in 2008. Last previous edition
11、approved in 2008 as E261308.DOI: 10.1520/E2613-08.10.1520/E2613-14.2 The boldface numbers in parentheses refer to a list of references at the end of this standard.3 Federal Register, Vol 46, No. 17, Jan. 27, 1991.4 For referencedASTM standards, visit theASTM website, www.astm.org, or contactASTM Cus
12、tomer Service at serviceastm.org. For Annual Book of ASTM Standardsvolume information, refer to the standards Document Summary page on the ASTM website.This document is not an ASTM standard and is intended only to provide the user of an ASTM standard an indication of what changes have been made to t
13、he previous version. Becauseit may not be technically possible to adequately depict all changes accurately, ASTM recommends that users consult prior editions as appropriate. In all cases only the current versionof the standard as published by ASTM is to be considered the official document.Copyright
14、ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States1E2756 Terminology Relating to Antimicrobial and Antiviral Agents3. Terminology3.1 DefinitionsFor definitions of general terms used in this test method, refer toTerminologyTerminologies D1129 and E
15、2756.3.2 Definitions of Terms Specific to This Standard:3.2.1 active ingredient, na substance added to a formulation specifically for the inhibition or inactivation of microorganisms.3.2.2 dry controla control to determine the number of colony forming units (CFU) of the test fungus remaining viable
16、afterthe initial drying of the inoculum on the skin.3.2.3 handrub, na liquid, gel, or foam, which is applied by rubbing to decontaminate lightly soiled hands betweenhandwashings and generally does not require a post-treatment water rinse; such agents usually contain alcohol alone or with otheractive
17、 ingredients.3.2.4 hard water, nwater with a defined level of hardness as calcium carbonate.3.2.5 hygienic handwash agent, nan agent generally used for handwashing by personnel in hospitals, other health-carefacilities, day-care centers, nursing homes, and food-handling establishments to eliminate t
18、ransient microorganisms from intactskin.3.2.6 input control, na control to determine the number of colony forming units of the test fungus placed on each digit.3.2.7 neutralizationa process which results in quenching the antifungal activity of a test material. This may be achievedthrough dilution of
19、 the test material(s) to reduce the antifungal activity, or through the use of chemical agents, called neutralizers,to eliminate antifungal activity.3.2.8 non-medicated soap, na soap or detergent for handwashing that is mild to the skin and does not contain anyantimicrobial chemicals.3.2.8 soil lead
20、,load, na solution of one or more organic and/or inorganic substances added to the suspension of the testorganism to simulate the presence of body secretions, excretions or other extraneous substances.3.2.9 test formulation, na formulation which incorporates antimicrobial ingredients.3.2.10 test org
21、anism, nan applied inoculum of an organism that has characteristics which allow it to be readily identified. Thetest organism is used to simulate a transient topical fungal contaminant. It may also be referred to as a marker organism, fungalsimulant/surrogate or fungal contaminant.3.2.11 test vehicl
22、e, nthe test agent without an active ingredient.3.2.12 unmedicated soap, na soap or detergent for handwashing that is mild to the skin and does not contain any antimicrobialchemicals.4. Summary of Test Method4.1 This test method is conducted using uses a group of adult subjects who have provided inf
23、ormed consent and the skin ofwhose hands has been determined to be free from any apparent damage. Subjects are to refrain from using any products containingantimicrobial agents for at least one week prior to the test.Aknown volume of the test fungal suspension is placed on a demarcatedarea on each f
24、ingerpad and the inoculum allowed to dry. dried. The contaminated area is then exposed to the control (standard hardwater) (water with a standard level of hardness) or test agent for the desired contact time and time, organisms remaining on thefingerpad are eluted and the eluates assayed for fungal
25、CFU. Reductions in the numbers of CFU after treatment with the controland test agentsagent(s) are then determined. If two different formulations are being compared in the same test, one of them maybe designated as a reference and used in place of the hard water control.5. Significance and Use5.1 Thi
26、s in vivo procedure is designed to test the ability of hygienic handwash or handrub agents to eliminate fungalcontamination from experimentally-contaminated hands. Since the two thumbpads and all eight fingerpads can be used in anygiven test, it allows for the incorporation of an input control (two)
27、, control for viable cells of the test fungus remaining after theinoculum has been allowed to dry dried (two), fungal cells eliminated after treatment with a control or reference solution (two),and up to four replicates to assess the fungus-eliminating efficiency of the formulation under test. No mo
28、re than 100 L of the testfungal suspension is required to complete one test.5.2 Whereas this test method is designed to work with fungi, similar ASTM standards exist for testing against viruses (TestMethod E1838) and vegetative bacteria (Test Method E2276).5.3 Infectious microorganisms left on hands
29、 after washing can be reduced further by drying the washed hands with paper, cloth,or warm air (5). A step for the drying of fingerpads after exposure to the control or test solution, therefore, has not been includedto avoid fungal removal by the drying process itself.5.4 This test method is not mea
30、nt for testingdesigned to test surgical hand scrubs or preoperative skin preps.E2613 1425.5 The level of contamination with viable fungi on each fingerpad after the drying of the inoculum should be at least 104 CFUso that it would permit the detection of up to a 4-log10 reduction in the viability ti
31、ter of the test organism by a test formulationunder the conditions of this test. This in itself does not represent the product performance criterion, which may vary dependingon the jurisdiction and the nature of the formulation being evaluated.6. Equipment and Apparatus6.1 Colony counter, any of sev
32、eral types may be used, for example, Quebec Colony Counter.6.2 Freezer, a freezer at 70C70 6 2C or lower is required to store fungal stocks.6.3 Handwashing sink, a sink of sufficient size to permit subjects to wash hands without touching hands to sink surface.6.4 Incubator, any incubator that can ma
33、intain a temperature suitable for the growth of Candida albicansthe fungal andspeciesAspergillus niger.under test.6.5 Laminar flow cabinet, a Class II biological safety cabinet.6.6 Magnetic stirrer and magnets, Llargelarge enough to hold a 5-L beaker or Erlenmeyer flask for preparing culture media o
34、rother solutions.6.7 Membrane filtration system, a membrane filtration system and membranes with a pore diameter of 0.45 m or 0.22 m arerequired to sterilize heat-sensitive media/solutions.6.8 Positive displacement pipette, a pipette and pipette tips that accurately can dispense 10-L volumes.6.9 Ref
35、rigerator, a refrigerator at 4 6 2C for storage of prepared culture media and reagents.6.10 Sterilizer, any suitable steam sterilizer that produces the conditions of sterilization is acceptable.6.11 Timer (stop clock), one that can be read for minutes and seconds.6.12 Tap water temperature regulator
36、 and temperature monitor, to monitor and regulate water temperature at 40 6 2C.6.13 Water faucet(s), to be located above the sink at a height that permits the hands to be held higher than the elbow duringthe washing procedure. Faucets with electronic sensors or those that are wrist-, elbow-, knee-,
37、or foot-operated are preferred toavoid recontamination of the washed hands.7. Reagents and Materials7.1 Serological pipettes, sterile reusable or single-use pipettes of 10.0, 5.0, and 1.0-mL capacity.7.2 Culture plates, Petri plates of 100 15 mm diameter for culturing the fungus.7.3 Soil Load:7.3.1
38、A tripartite soil load, load Test Method E2197, prepared from the following three stock solutions, as an alternative toserum.7.3.2 Add 0.5 g of tryptone or yeast extract to 10 mL of phosphate buffer.7.3.3 Add 0.5 g of bovine serum albumin (BSA) to 10 mL of phosphate buffer.7.3.4 Add 0.04 g of bovine
39、 mucin to 10 mL of phosphate buffer.7.3.5 Prepare the stock solutions separately and sterilize by passage through a 0.22 m pore diameter membrane filter, aliquotand store at either 4 6 2C or 20 6 2C.7.3.6 To obtain a 500-L inoculum of the test inoculum, add to 340 L of the fungal suspension 35 L of
40、tryptone or yeastextract (7.3.2), 25 L BSA (7.3.3), and 100 L mucin (7.3.4) stock solutions. This mixture contains approximately 2 g of totalprotein/L, which is roughly equivalent to the protein content of a 5 % solution of fetal bovine serum.7.4 Standard Hard WaterThe quality and disinfectant (for
41、example, chlorine) residual in tap water can vary from site to siteand also at different times at the same site. The use of standard hard water, therefore, is recommended here to avoid variations inresults due to differences in tap water quality. Water prepared in accordance withAOAC 960.09 E and F
42、(6) to a standard hardnessof 200 ppm as calcium carbonate is used for dilution of test products, as the control solution to determine the baseline level offungal elimination, and to rinse the fingerpads after exposure to the test product. The standard hard water and tap water (if used)must first be
43、tested to ensure that they do not have any activity against the test fungus. If water with a higher level of hardnessis used for making the use-dilution of the test formulation, this change must be clearly justified and documented in the report.7.5 Test agents, at least two samples of the test formu
44、lation shall be evaluated.NOTE 1Water with a standard level of hardness is also recommended as a control in this test procedure to determine any mechanical removal of thetest 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 bu
45、ffer.7.7 Eluent for fungal recovery from fingerpads, normal saline.E2613 1437.8 Plastic vials, sterile screw-capped 2.0-mL vials with an inside diameter of about 8 mm will be required for demarcation ofthe fingerpads and to hold various test solutions.7.9 Miscellaneous laboratory ware, automatic pip
46、ettes, pipette tips, and plastic vials for storing stock cultures.7.10 Broth, trypticase soy broth (TSB) or equivalent to prepare the inoculum of Candida albicans.7.11 Agar, sabouraud dextrose agar or equivalent to prepare the inoculum of Aspergillus niger and to recover and count thecolonies of the
47、 two test organisms in control and test samples. The addition of any neutralizer(s) in such recovery media must firstbe properly validated (Test Methods E1054).8. Test Fungi8.1 TheAppendix X1 selection of the following test fungi is based on their (contains a) relative safety to the subjects as well
48、as experimenters, ( list b) ability to grow to titers sufficiently high for testing, (c) ease of recovery and identification inof suggestedtest fungi. WarningThe application of microorganisms to the skin may involve a health risk. Prior to applying the test organismto the skin, the antibiotic sensit
49、ivity profile of the strain should be determined. If the applied organism causes an infection, theantibiotic sensitivity profile should be made available to the attending clinician. the laboratory, and (d) potential to spread throughcontaminated hands. Other strains or types of fungi may be substituted provided they meet the preceding criteria and are approvedby the relevant institutional review board.8.1.1 Candida albicans (ATCC 10231). This represents non-filamentous fungi (yeast-like) and is among the most commonspecies of Candida recover
