ASTM E2613-08
(Test Method)Standard Test Method for Determining Fungus-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using Fingerpads of Adults
Standard Test Method for Determining Fungus-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using Fingerpads of Adults
SIGNIFICANCE AND USE
This in vivo procedure is designed to test the ability of hygienic handwash or handrub agents to eliminate fungal contamination from experimentally-contaminated hands. Since the two thumbpads and all eight fingerpads can be used in any given test, it allows for the incorporation of an input control (two), control for viable cells of the test fungus remaining after the inoculum has been allowed to dry (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 more than 100 µL of the test fungal suspension is required to complete one test.
Whereas this test method is designed to work with fungi, similar ASTM standards exist for testing against viruses (Test Method E 1838) and vegetative bacteria (Test Method E 2276).
Infectious microorganisms left on hands 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 included to avoid fungal removal by the drying process itself.
This test method is not meant for testing surgical hand scrubs or preoperative skin preps.
The level of contamination with viable fungi on each fingerpad after the drying of the inoculum should be at least 104 CFU so that it would permit the detection of up to a 4-log10 reduction in the viability titer of the test organism by a test formulation under the conditions of this test. This in itself does not represent the product performance criterion, which may vary depending on the jurisdiction and the nature of the formulation being evaluated.
SCOPE
1.1 This test method is designed to assess the ability of hygienic handwash and handrub agents to reduce levels of fungal contamination on hands (3). This test method is not meant for use with surgical hand scrubs (Test Method E 1115) or preoperative skin preps (Test Method E 1173).
1.2 Performance of this procedure requires the knowledge of regulations pertaining to human experimentation.
1.3 The test method should be performed by persons with training in microbiology in facilities designed and equipped for work with infectious agents at biosafety level 2 (4).
1.4 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 responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: E2613 − 08
StandardTest Method for
Determining Fungus-Eliminating Effectiveness of Hygienic
Handwash and Handrub Agents Using Fingerpads of
Adults
This standard is issued under the fixed designation E2613; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision. A number in parentheses indicates the year of last reapproval. A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Human hands are frequently in contact with other surfaces and can readily acquire transient
microbial contamination. Fungi are common among these types of contaminants (1, 2), particularly
in 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 method
addresses the gap.
1. Scope 2. Referenced Documents
1.1 This test method is designed to assess the ability of 2.1 ASTM Standards:
hygienic handwash and handrub agents to reduce levels of D1129 Terminology Relating to Water
fungal contamination on hands (3). This test method is not E1054 Test Methods for Evaluation of Inactivators of Anti-
meant for use with surgical hand scrubs (Test Method E1115) microbial Agents
or preoperative skin preps (Test Method E1173). E1115 Test Method for Evaluation of Surgical Hand Scrub
Formulations
1.2 Performance of this procedure requires the knowledge
3 E1173 Test Method for Evaluation of Preoperative,
of regulations pertaining to human experimentation.
Precatheterization, or Preinjection Skin Preparations
1.3 The test method should be performed by persons with
E1838 Test Method for Determining the Virus-Eliminating
traininginmicrobiologyinfacilitiesdesignedandequippedfor
Effectiveness of Hygienic Handwash and HandrubAgents
work with infectious agents at biosafety level 2 (4).
Using the Fingerpads of Adults
1.4 The values stated in SI units are to be regarded as E2276 Test Method for Determining the Bacteria-
Eliminating Effectiveness of Hygienic Handwash and
standard. No other units of measurement are included in this
standard. Handrub Agents Using the Fingerpads of Adults
1.5 This standard does not purport to address all of the
3. Terminology
safety concerns, if any, associated with its use. It is the
3.1 Definitions—Fordefinitionsofgeneraltermsusedinthis
responsibility of the user of this standard to establish appro-
test method, refer to Terminology D1129.
priate safety and health practices and determine the applica-
bility of regulatory limitations prior to use.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 active ingredient, n—a substance added to a formula-
1 tion specifically for the inhibition or inactivation of microor-
This test method is under the jurisdiction of ASTM Committee E35 on
ganisms.
Pesticides and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2008. Published May 2008. DOI: 10.1520/
E2613-08. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
The boldface numbers in parentheses refer to a list of references at the end of contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
this standard. Standards volume information, refer to the standard’s Document Summary page on
Federal Register, Vol 46, No. 17, Jan. 27, 1991. the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2613 − 08
3.2.2 dry control—a control to determine the number of formulations are being compared in the same test, one of them
colonyformingunits(CFU)ofthetestfungusremainingviable may be designated as a reference and used in place of the hard
after the initial drying of the inoculum on the skin. water control.
3.2.3 handrub, n—aliquid,gel,orfoam,whichisappliedby
5. Significance and Use
rubbing to decontaminate lightly soiled hands between hand-
5.1 This in vivo procedure is designed to test the ability of
washings and generally does not require a post-treatment water
hygienic handwash or handrub agents to eliminate fungal
rinse; such agents usually contain alcohol alone or with other
contamination from experimentally-contaminated hands. Since
active ingredients.
the two thumbpads and all eight fingerpads can be used in any
3.2.4 hard water, n—water with a defined level of hardness
given test, it allows for the incorporation of an input control
as calcium carbonate.
(two), control for viable cells of the test fungus remaining after
the inoculum has been allowed to dry (two), fungal cells
3.2.5 hygienic handwash agent, n—an agent generally used
eliminated after treatment with a control or reference solution
for handwashing by personnel in hospitals, other health-care
(two),anduptofourreplicatestoassessthefungus-eliminating
facilities, day-care centers, nursing homes, and food-handling
efficiency of the formulation under test. No more than 100 µL
establishments to eliminate transient microorganisms from
of the test fungal suspension is required to complete one test.
intact skin.
5.2 Whereasthistestmethodisdesignedtoworkwithfungi,
3.2.6 input control, n—a control to determine the number of
similar ASTM standards exist for testing against viruses (Test
colony forming units of the test fungus placed on each digit.
Method E1838) and vegetative bacteria (Test Method E2276).
3.2.7 neutralization—a process which results in quenching
5.3 Infectious microorganisms left on hands after washing
the antifungal activity of a test material. This may be achieved
can be reduced further by drying the washed hands with paper,
through dilution of the test material(s) to reduce the antifungal
cloth, or warm air (5).Astep for the drying of fingerpads after
activity, or through the use of chemical agents, called neutral-
exposure to the control or test solution, therefore, has not been
izers, to eliminate antifungal activity.
included to avoid fungal removal by the drying process itself.
3.2.8 non-medicated soap, n—a soap or detergent for hand-
5.4 This test method is not meant for testing surgical hand
washing that is mild to the skin and does not contain any
scrubs or preoperative skin preps.
antimicrobial chemicals.
5.5 The level of contamination with viable fungi on each
3.2.9 soil lead, n—a solution of one or more organic and/or
fingerpad after the drying of the inoculum should be at least
inorganic substances added to the suspension of the test
10 CFUsothatitwouldpermitthedetectionofuptoa4-log
organism to simulate the presence of body secretions, excre-
reduction in the viability titer of the test organism by a test
tions or other extraneous substances.
formulation under the conditions of this test.This in itself does
3.2.10 test formulation, n—a formulation which incorpo- not represent the product performance criterion, which may
rates antimicrobial ingredients. vary depending on the jurisdiction and the nature of the
formulation being evaluated.
3.2.11 test organism, n—an applied inoculum of an organ-
ism that has characteristics which allow it to be readily
6. Equipment and Apparatus
identified. The test organism is used to simulate a transient
6.1 Colony counter, any of several types may be used, for
topical fungal contaminant. It may also be referred to as a
example, Quebec Colony Counter.
marker organism, fungal simulant/surrogate or fungal contami-
6.2 Freezer, a freezer at –70°C or lower is required to store
nant.
fungal stocks.
3.2.12 test vehicle, n—the test agent without an active
6.3 Handwashing sink, a sink of sufficient size to permit
ingredient.
subjects to wash hands without touching hands to sink surface.
4. Summary of Test Method
6.4 Incubator,anyincubatorthatcanmaintainatemperature
suitable for the growth of Candida albicans and Aspergillus
4.1 This test method is conducted using a group of adult
niger.
subjects who have provided informed consent and the skin of
whose hands has been determined to be free from any apparent 6.5 Laminar flow cabinet, a Class II biological safety
cabinet.
damage. Subjects are to refrain from using any products
containing antimicrobial agents for at least one week prior to
6.6 Magnetic stirrer and magnets, Llarge enough to hold a
the test. A known volume of the test fungal suspension is
5-L beaker or Erlenmeyer flask for preparing culture media or
placed on a demarcated area on each fingerpad and the
other solutions.
inoculum allowed to dry. The contaminated area is then
6.7 Membrane filtration system, a membrane filtration sys-
exposed to the control (standard hard water) or test agent for
tem and membranes with a pore diameter of 0.45µmor 0.22
the desired contact time and organisms remaining on the
µm are required to sterilize heat-sensitive media/solutions.
fingerpad are eluted and the eluates assayed for fungal CFU.
Reductions in the numbers of CFU after treatment with the 6.8 Positive displacement pipette, a pipette and pipette tips
control and test agents are then determined. If two different that accurately can dispense 10-µL volumes.
E2613 − 08
NOTE 1—Water with a standard level of hardness is also recommended
6.9 Refrigerator, a refrigerator at 4 6 2°C for storage of
as a control in this test procedure to determine any mechanical removal of
prepared culture media and reagents.
the test organism(s) from the skin.
6.10 Sterilizer, any suitable steam sterilizer that produces
7.6 Diluent for fungal titration, normal saline (0.85 %
the conditions of sterilization is acceptable.
NaCl) at pH 7.2 to 7.4 or an appropriate buffer.
6.11 Timer (stop clock),onethatcanbereadforminutesand
7.7 Eluent for fungal recovery from fingerpads, normal
seconds.
saline.
6.12 Tap water temperature regulator and temperature
7.8 Plastic vials, sterile screw-capped 2.0-mL vials with an
monitor, to monitor and regulate water temperature at 40 6
insidediameterofabout8mmwillberequiredfordemarcation
2°C.
of the fingerpads and to hold various test solutions.
6.13 Water faucet(s),tobelocatedabovethesinkataheight
7.9 Miscellaneous laboratory ware, automatic pipettes, pi-
that permits the hands to be held higher than the elbow during
pette tips, and plastic vials for storing stock cultures.
the washing procedure. Faucets with electronic sensors or
7.10 Broth, trypticase soy broth (TSB) or equivalent to
those that are wrist-, elbow-, knee-, or foot-operated are
prepare the inoculum of Candida albicans.
preferred to avoid recontamination of the washed hands.
7.11 Agar, sabouraud dextrose agar or equivalent to prepare
7. Reagents and Materials
the inoculum of Aspergillus niger and to recover and count the
colonies of the two test organisms in control and test samples.
7.1 Serological pipettes, sterile reusable or single-use pi-
The addition of any neutralizer(s) in such recovery media must
pettes of 10.0, 5.0, and 1.0-mL capacity.
first be properly validated (Test Methods E1054).
7.2 Culture plates, Petri plates of 100 mm diameter for
culturing the fungus.
8. Test Fungi
7.3 Soil Load:
8.1 The selection of the following test fungi is based on
7.3.1 A tripartite soil load, prepared from the following
their(a)relativesafetytothesubjectsaswellasexperimenters,
three stock solutions, as an alternative to serum.
(b) ability to grow to titers sufficiently high for testing, (c) ease
7.3.2 Add 0.5 g of tryptone to 10 mL of phosphate buffer.
of recovery and identification in the laboratory, and (d)
7.3.3 Add 0.5 g of bovine serum albumin (BSA) to 10 mL
potential to spread through contaminated hands. Other strains
of phosphate buffer.
or types of fungi may be substituted provided they meet the
7.3.4 Add 0.04 g of bovine mucin to 10 mL of phosphate precedingcriteriaandareapprovedbytherelevantinstitutional
review board.
buffer.
7.3.5 Prepare the stock solutions separately and sterilize by 8.1.1 Candida albicans (ATCC 10231). This represents
passage through a 0.22 µm pore diameter membrane filter, non-filamentous fungi (yeast-like) and is among the most
aliquot and store at either 4 6 2°C or –20 6 2°C. common species of Candida recovered as clinical isolates(7).
7.3.6 To obtain a 500-µLinoculum of the test inoculum, add 8.1.2 Aspergillus niger (ATCC 64958). This is an example
of a filamentous fungus that is considered safe for the experi-
to340µLofthefungalsuspension35µLoftryptone(7.3.2),25
µLBSA(7.3.3), and 100 µLmucin (7.3.4) stock solutions.This mental contamination of the skin of adult subjects.
mixture contains approximately2gof total protein/L, which is
8.1.3 Other fungal species may be used provided they are
roughly equivalent to the protein content of a 5 % solution of
safe for contamination of the hands of adult subjects and only
fetal bovine serum.
after permission has been received for their use from the
relevant institutional review board.
7.4 Standard Hard Water—The quality and disinfectant (for
example, chlorine) residual in tap water can vary from site to
9. Preparation of Test Inoculum
site and also at different times at the same site. The use of
standard hard water, therefore, is recommended here to avoid
9.1 To prepare C. albicans test inoculum, add 0.1 mL of a
variations in results due to differences in tap water quality.
frozen stock culture to 10 mL of TSB (7.10) and incubate for
Water prepared in accordance with AOAC 960.09 E and F (6)
48 6 4 h at the appropriate temperature.
toastandardhardnessof200ppmascalciumcarbonateisused
9.2 To prepare a suspension of the conidia of A. niger,
for dilution of test products, as the control solution to deter-
inoculate the center of a sabouraud dextrose agar plate with 0.1
mine the baseline level of fungal elimination, and to rinse the
mL of a frozen stock suspension and incubate for 10 days at
fingerpads after exposure to the test product.The standard hard
28°C. Harvest mycelial mats from the agar surface, homog-
water and tap water (if used) must first be tested to ensure that
enize with sterile glass beads in normal saline, and filter
they do not have any activity against the test fungus. If water
through sterile gauze to remove the hyphae.
with a higher level of hardness is used for making the
use-dilutionofthetestformulation,thischangemustbeclearly 9.3 Add soil load (7.3) to the test fungal cell suspension, if
justified and documented in the report. required.
7.5 Test agents, at least two samples of the test formulation 9.4 Swirl, vortex, or shake the test fungal cell suspension
shall be evaluated. before withdrawal of each aliquot.
E2613 − 08
10. Subjects 11.1.7 Exposethedriedinoculumontherequirednu
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