ASTM E2613-23

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Designation: E2613 − 14 E2613 − 23
Standard Test Method Practice for
Determining the Fungus-Eliminating Effectiveness of
Hygienic Handwash and Handrub Agents Using the
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
practice addresses the gap.
1. Scope Scope*
1.1 This test method practice is designed to assess the ability of hygienic handwash and handrub agents to reduce levels of fungal
contamination on hands (3). This test method practice is not meant for use with surgical hand scrubs (Test Method E1115) or
preoperative skin preps (Test Method E1173).
1.2 Performance of this procedure requires the knowledge of regulations pertaining to human experimentation.
1.3 The test method practice 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 Units—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.
1.6 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
This test method practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility
of Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2014Jan. 1, 2023. Published May 2014February 2023. Originally approved in 2008. Last previous edition approved in 20082014 as
E2613E2613 – 14.–08. DOI: 10.1520/E2613-14.10.1520/E2613-23.
The boldface numbers in parentheses refer to athe list of references at the end of this standard.
Federal Register, Vol 46, No. 17, Jan. 27, 1991.
*A Summary of Changes section appears at the end of this standard
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2613 − 23
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
E1115 Test Method for Evaluation of Surgical Hand Scrub Formulations
E1173 Test Method for Evaluation of Preoperative, Precatheterization, or Preinjection Skin Preparations
E1838 Test Method for Determining the Virus-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the
Fingerpads of Adults
E2197 Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and
Sporicidal Activities of Chemicals
E2276 Test Method for Determining the Bacteria-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using
the Fingerpads of Adults
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
3. Terminology
3.1 Definitions—For definitions of general terms used in this test method, practice, refer to Terminologies D1129 and E2756.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 active ingredient, n—a substance added to a formulation specifically for the inhibition or inactivation of microorganisms.
3.2.2 CFU, n—colony-forming units.
3.2.3 dry control—control, n—a control to determine the number of colony forming units (CFU) CFU of the test fungus remaining
viableculturable after the initial drying of the inoculum on the skin.
3.2.4 handrub, n—a liquid, gel, or foam, which is applied by rubbing to decontaminate lightly soiled hands between handwashings
and generally does not require a post-treatment water rinse; such agents usually contain alcohol alone or with other active
ingredients.
3.2.5 hard water, n—water with a defined level of hardness as calcium carbonate.
3.2.6 hygienic handwash agent, n—an agent generally used for handwashing by personnel in hospitals, other health-care facilities,
day-care centers, nursing homes, and food-handling establishments to eliminate transient microorganisms from intact skin.
3.2.7 input control, n—a control to determine the number of colony forming units of the test fungus placed on each digit.
3.2.8 neutralization—neutralization, n—a process which results in quenching the antifungal activity of a test material. This may
be achieved through dilution of the test material(s) to reduce the antifungal activity, or through the use of chemical agents, called
neutralizers, to eliminate antifungal activity.
3.2.8.1 Discussion—
This may be achieved through dilution of the test material(s) to reduce the antifungal activity, or through the use of chemical
agents, called neutralizers, to eliminate antifungal activity.
3.2.9 soil load, n—a solution of one or more organic and/or inorganic substances added to the suspension of the test organism to
simulate the presence of body secretions, excretions or other extraneous substances.
3.2.10 test formulation, n—a formulation which incorporates antimicrobial ingredients.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
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3.2.11 test organism, n—an applied inoculum of an organism that has characteristics which allow it to be readily identified. The
test organism is used to simulate a transient topical fungal contaminant. It may also be referred to as a marker organism, fungal
simulant/surrogate or fungal contaminant.
3.2.12 test vehicle, n—the test agent without an active ingredient.
3.2.13 unmedicated soap, n—a soap or detergent for handwashing that is mild to the skin and does not contain any antimicrobial
chemicals.
4. Summary of Test MethodPractice
4.1 This test method practice uses a group of adult subjects who have provided informed consent and the skin of whose hands
has been determined to be free from any apparent damage. Subjects are to refrain from using any products containing antimicrobial
agents for at least one week prior to the test. A known volume of the test fungal suspension is placed on a demarcated area on each
fingerpad and the inoculum dried. The contaminated area is then exposed to the control (water with a standard level of hardness)
or test agent for the desired contact time, organisms remaining on the fingerpad are eluted and the eluates assayed for fungal CFU.
colony-forming units (CFU). Reductions in the numbers of CFU after treatment with the control and test agent(s) are then
determined. If two different formulations are being compared in the same test, one of them may be designated as a reference and
used in place of the hard water control.
5. Significance and Use
5.1 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 viableculturable cells of the test fungus remaining after the inoculum
has 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 more than 100 µL of the test fungal suspension is required to
complete one test.
5.2 Whereas this test method practice is designed to work with fungi, similar ASTM standards exist for testing against viruses
(Test Method E1838) and vegetative bacteria (Test Method E2276).
5.3 Infectious The levels of culturable 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.
5.4 This test method practice is not designed to test surgical hand scrubs or preoperative skin preps.
5.5 The level of contamination with viableculturable fungi on each fingerpad after the drying of the inoculum should be at least
10 CFU so that it would permit the detection of up to a 4-log 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.
6. Equipment and Apparatus
6.1 Colony counter,Counter, any of several types may be used, for example, Quebec Colony Counter.
6.2 Freezer, a freezer at –70 6 2°C–70 6 2 °C or lower is required to store fungal stocks.
6.3 Handwashing sink,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 maintain a temperature suitable for the growth of the fungal species under test.
6.5 Laminar flow cabinet,Flow Cabinet, a Class II biological safety cabinet.
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6.6 Magnetic stirrer and magnets,Stirrer and Magnets, large enough to hold a 5-L5 L beaker or Erlenmeyer flask for preparing
culture media or other solutions.
6.7 Membrane filtration system,Filtration System, a membrane filtration system and membranes with a pore diameter of 0.45 µ
m or 0.22 µm are required to sterilize heat-sensitive media/solutions.
6.8 Positive displacement pipette,Displacement Pipette, a pipette and pipette tips that accurately can dispense 10-µL10 µL
volumes.
6.9 Refrigerator, a refrigerator at 4 6 2°C2 °C 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),(Stop Clock), one that can be read for minutes and seconds.
6.12 Tap water temperature regulator and temperature monitor,Water Temperature Regulator and Temperature Monitor, to
monitor and regulate water temperature at 40 6 2°C.2 °C.
6.13 Water faucet(s),Faucet(s), to be located above the sink at a height that permits the hands to be held higher than the elbow
during the washing procedure. Faucets with electronic sensors or those that are wrist-, elbow-, knee-, or foot-operated are preferred
to avoid recontamination of the washed hands.
7. Reagents and Materials
7.1 Serological pipettes,Pipettes, sterile reusable or single-use pipettes of 10.0, 5.0, and 1.0-mL1.0 mL capacity.
7.2 Culture plates,Plates, Petri plates of 100 × 15 mm diameter for culturing the fungus.
7.3 Soil Load:
7.3.1 A tripartite soil load Test Method E2197, prepared from the following three stock solutions, as an alternative to serum.
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 mucin to 10 mL of phosphate buffer.
7.3.5 Prepare the stock solutions separately and sterilize by passage through a 0.22 µm pore diameter membrane filter, aliquot and
store at either 4 6 2°C2 °C or –20 6 2°C.2 °C.
7.3.6 To obtain a 500-µL500 µL inoculum of the test inoculum, add to 340 µL of the fungal suspension 35 µL of tryptone or yeast
extract (7.3.2), 25 µL BSA (7.3.3), and 100 µL mucin (7.3.4) stock solutions. This mixture contains approximately 2 g of total
protein/L, which is roughly equivalent to the protein content of a 5 % solution of fetal bovine serum.
7.4 Standard Hard Water—The quality and disinfectant (for example, chlorine) residual in tap water can vary from site to site and
also at different times at the same site. The use of standard hard water, therefore, is recommended here to avoid variations in results
due to differences in tap water quality. Water prepared in accordance with AOAC 960.09 E and F (6) to a standard hardness of
200 ppm as calcium carbonate is used for dilution of test products, as the control solution to determine the baseline level of fungal
elimination, and to rinse the fingerpads after exposure to the test product. The standard hard water and tap water (if used) must
first be tested to ensure that they doit does not have any activity against the test fungus. If water with a higher level of hardness
is used for making the use-dilution of the test formulation, this change must be clearly justified and documented in the report.
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7.5 Test agents,Agents, at least two samples of the test formulation shall be evaluated.
NOTE 1—Water with a standard level of hardness is also recommended as a control in this test procedure to determine any mechanical removal of the
test organism(s) from the skin.
7.6 Diluent for fungal titration,Fungal Titration, normal saline (0.85 % NaCl) at pH 7.2 to 7.4 or an appropriate buffer.
7.7 Eluent for fungal recovery from fingerpads,Fungal Recovery from Fingerpads, normal saline.
7.8 Plastic vials,Vials, sterile screw-capped 2.0-mL2.0 mL vials with an inside diameter of about 8 mm will be required for
demarcation of the fingerpads and to hold various test solutions.
7.9 Miscellaneous laboratory ware,Laboratory Ware, automatic pipettes, 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, sabouraudSabouraud dextrose agar or equivalent to prepare the inoculum of Aspergillus nigerbrasiliensis and to
recover and count the colonies of the two test organisms in control and test samples. The addition of any neutralizer(s) in such
recovery media must first be properly validated (Test Methods E1054).
8. Test Fungi
8.1 Appendix X1 contains a list of suggested test fungi. Warning—The application of microorganisms to the skin may involve
a health risk. Prior to applying the test organism to the skin, the antibiotic sensitivity profile of the strain should be determined.
If the applied organism causes an infection, the antibiotic sensitivity profile should be made available to the attending clinician.
9. Preparation of Test Inoculum
9.1 See Appendix X1.
10. Subjects
10.1 Recruit a sufficient number of healthy adults who have no clinical evidence of dermatoses, open wounds, hangnails, or other
skin disorders. The number of subjects required for a trial is dependent on the number of treatments within a study.
10.2 It is the responsibility of the user of this test method to arrange the necessary clearance for the use of subjects for testing and
to obtain informed and written consent from those selected for the study before starting the tests.
10.3 Instruct subjects to avoid contact with antimicrobial products, other than the test material(s), for the duration of their
involvement in the study and for at least one week prior to the first test. This restric
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