ASTM E1174-21

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it 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 version
of the standard as published by ASTM is to be considered the official document.
Designation: E1174 − 13 E1174 − 21
Standard Test Method for
Evaluation of the Effectiveness of Health Care Healthcare
Personnel Handwash Formulations
This standard is issued under the fixed designation E1174; 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.
1. Scope
1.1 This test method is designed to determine the effectiveness of antimicrobial handwashing agents for the reduction of transient
microbial skin flora when used in a handwashing procedure.
1.2 A knowledge of microbiological techniques is required for these procedures.
1.3 This test method may be used to evaluate topical antimicrobial handwash formulations.
1.4 Performance of this procedure requires the knowledge of regulations pertaining to the protection of human subjects.
1.5 The values stated in SI units are to be regarded as standard; except for distance, in which case inches are used and metric units
follow in parentheses.
1.6 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 safety, health, and healthenvironmental practices and determine the
applicability of regulatory limitations prior to use.For more specific precautionary statements see 8.2.
1.7 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.
2. Referenced Documents
2.1 ASTM Standards:
E1054 Practices for Evaluation of Inactivators of Antimicrobial Agents
E2755 Test Method for Determining the Bacteria-Eliminating Effectiveness of Healthcare Personnel Hand Rub Formulations
Using Hands of Adults
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
This test method is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15Antimicrobial Agents.
Current edition approved Feb. 15, 2013Oct. 1, 2021. Published February 2013October 2021. Originally approved in 1987. Last previous edition approved in 20062013
as E1174 – 06.E1174 – 13. DOI: 10.1520/E1174-13.10.1520/E1174-21.
Federal Register,This version Vol 46, No. 17,of the standard has Jan. 27, 1991.been revised to align with current United States Food and Drug Administration
requirements for healthcare personnel handwash formulations. The procedure for multiple washes present in earlier versions of this standard has been moved to Appendix
X1 of this version.
45CFR Part 46 Protection of Human Subjects (Effective July 19, 2018)
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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E1174 − 21
3. Terminology
3.1 Definitions—For definitions of terms used in this method, refer to Terminology E2756.
3.2 Definitions: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.
3.1.2 cleansing wash, n—a non-antimicrobial wash intended to remove gross soil or residues from the hands of the panelists prior
to the conduct of the study and as noted throughout the study. This may also be referred to as a cosmetic wash.
3.2.2 healthcare personnel handwash, n—a cleanser or waterless agent intended to reduce transient bacteriamicroorganisms on the
hands.
3.1.4 neutralization, n—a process which results in quenching the antimicrobial activity of a test material. This may be achieved
through dilution of the test material(s) to reduce the antimicrobial activity, or through the use of chemical agents, called
neutralizers, to eliminate antibacterial activity.
3.2.3 resident microorganisms, n—microorganisms that live and multiply on the skin, forming a permanent population.
3.2.4 test formulation, n—a formulation which incorporates antimicrobial ingredient(s).
3.2.5 test organism—organism, n—an applied inoculum of an organismbacterial culture that has characteristics which allow it to
be readily identified. The test organism is used to simulate a transient topical microbial contaminant. It may also be referred to as
a marker organism, bacterial simulant, or bacterial contaminant.
3.2.5.1 Discussion—
The test organism is used to simulate a transient topical microbial contaminant. It may also be referred to as a marker organism,
bacterial simulant, or bacterial contaminant.
3.2.6 transient microorganisms—microorganisms, n—organisms from the environment that contaminate but do not normally
colonize the skin.
4. Summary of Test Method
4.1 This test method is conducted on a group of volunteer panelists who have refrained from using topical antimicrobial
formulations for at least one week prior to the initiation of the test. Activity of the test material is measured by comparing the
number of test organisms recovered from artificially contaminated hands after use of a handwashing formulation to the number
recovered from contaminated hands not exposed to the test formulation. The method describes specific procedures to be followed
using Serratia marcescens as the test organism. The activity of the test material is measured following a single wash and may be
measured following multiple washes in a single day using a neutralization recovery method.wash.
4.2 An alternative test organism that can be used is Escherichia coli.coli, if deemed appropriate. Culture media and incubation
conditions appropriate for this organism should be employed. The investigator should also be aware that there may be health risks
associated with the use of this organismbacterium and precautions similar to those referenced in 8.2 should be undertaken.
5. Significance and Use
5.1 The procedure may be is used to test the antimicrobial effectiveness of antimicrobial handwashing agents.formulations. The
test formulations may begenerally are designed for frequent use to reduce the transient bacterial flora on hands. Alcohol-based hand
rubs and other leave-on formulations used without the aid of water mayshould be tested using Test Method E2755.
6. Apparatus
6.1 Colony Counter—Any of several types may be used, for example, Quebec Colony Counter.
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6.2 Incubator—Any incubator capable of maintaining the following temperatures: S. marcescens (25(25 °C 6 2°C)2 °C) or E. coli
(35 6 2°C). This temperature (35 °C 6 2 °C). The former temperature range is required to ensure pigment production for
pigment-production by S. marcescens.
6.3 Sterilizer—Any suitable steam sterilizer capable of producing the conditions of sterilization is acceptable.
6.4 Timer (Stop-clock)—One that can be read for minutes and seconds.
6.5 Handwashing Sink—A sink of sufficient size to permit panelists to wash without touching hands to sink surface or other
panelists.
6.5.1 Water Faucet(s)—To be located above the sink at a height whichthat permits the hands to be held higher than the elbow
during the washing procedure.
6.6 Tap Water Temperature Regulator and Temperature Monitor—To monitorregulate and regulatemoniter water at a temperature
of 4040 °C 6 2°C.2 °C.
7. Reagents and Materials
7.1 Bacteriological Pipettes—10.010.0 mL and 2.2-mL2.2 mL or 1.1-mL1.1 mL capacity.
NOTE 1—Presterilized/disposable bacteriological pipettes are available from most local laboratory supply houses.
7.2 Water Dilution Bottles—Any sterilizable glass container having a 150150 mL to 200 mL capacity and tight closures maycan
be used.
NOTE 2—Milk dilution bottles of 160-mL160 mL capacity having a screw-cap closure are available from most local laboratory supply houses.
7.3 Erlenmeyer Flask—2-L2 L capacity for culturing test organism.
7.4 Cleansing Wash—A mild, non-antimicrobial soft soap.
Soft Soap, 200 g/L
Linseed oil 50 parts by weight
Potassium hydroxide 9.5 parts
Ethanol 7 parts
Distilled or high purity water as needed
7.4.1 Add linseed oil to a solution of potassium hydroxide in 15 parts water and heat up to approximately 70°C70 °C while
constantly stirring. Add the ethanol and continue heating while stirring until the saponification process is completed and a sample
dissolves clearly in water and almost clearly in alcohol. The weight of the soft soap is then brought up to 100 parts by addition
of hot water. TakeAdd 200 g of the soft soap into 1 L of water. Dispensewater, dispense in to appropriate containers, and sterilize
in an autoclave.
7.5 Test Material—Directions for use of the test material mayshould be utilized. If directions are not available, use directions
provided in this test method.
7.6 Gloves—Loose-fitting, unlined, powder-free gloves whichthat possess no antimicrobial properties, or equivalent. (Plastic bags
with low bioburden maycan be used in place of gloves.)
A zone of inhibition test such as AATCC Test Method 90-1965 may be used to evaluate antimicrobial properties of gloves, AATCC Test Methods, American Association
of Textile Chemists and Colorist, 1968 Technical Manual, Section B-75.
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7.7 Sampling Solution—Dissolve 0.4 g KH PO , 10.1 g Na HPO , and 1.0 g isooctylphenoxypolyethoxyethanol, and with
2 4 2 4
appropriately validated neutralizers neutralizing additives in 1 L of distilled water. Adjust pH to 7.8 with 0.1 N HCl or 0.1 N NaOH.
Dispense so that final volume after sterilization is 75 mL, sterilized at 121°C.mL after sterilization at 121 °C.
7.8 Dilution Fluid—Sterile Butterfield’s Buffer , or other suitable diluent, adjusted to pH 7.2 with effective neutralizer that
includes neutralizing additives proved effective for the test material. Adjust to pH approximately 7.2 with 0.1N HCl or 0.1N NaOH.
See Test Methods E1054.
7.9 Agar—Soybean-casein digest agar, or other solid mediamedium appropriately validated to support growth of the test organism
with appropriate neutralizers organism, including appropriate neutralizing additives, if needed.
7.10 Broth—Soybean-casein digests brothdigest broth, or other liquid media appropriate medium appropriately validated to
support growth of the test organism.
7.11 Ethanol Solution—70 % ethanol in water (v/v) for hand decontamination
7.12 Chlorhexidine Skin Cleanser—Antiseptic skin cleanser containing 4 % chlorhexidine gluconate (w/v) for hand decontami-
nation.
7.13 Antibiotic Ointment—A topical, triple-antibiotic ointment for application to the hands and forearms after the final
decontamination. If necessary, consult with a physician prior to use.
8. Test Organism
8.1 Serratia marcescens (ATCC 14756) is to be used as the test organism. This is a strain having that produces stable pigmentation
at 25°C.25 °C. (Warning—The application of this bacterium to the skin may involve a health risk to healthy humans. Prior to
application, the antibiotic susceptibility profile of the strain should be determined. The strain must be susceptible to gentamicin.
If an infection occurs, the antibiotic sensitivity profile must be made available to the attending physician. Following the subject’s
last contamination and wash with the formulation, the subject’s hands are to be decontaminated as described in Section 14. The
purpose of this decontamination is to destroy residual test bacteria on the skin.)
8.2 Escherichia coli (ATCC 11229) areis an alternative test organism. When E. coli is used, the plating agar should include a
suitable indicator (for example, MUG ). (Warning—The application of this bacterium to the skin may involve a health risk to
healthy humans. Prior to application, the antibiotic susceptibility profile of the strain should be determined. If the strain is not
susceptible to gentamicin, it must not be used. If an infection occurs, the antibiotic sensitivity profile must be made available to
the attending physician. Following the subject’s last contamination and wash with the formulation, the subject’s hands are to be
decontaminated as described in Section 14. The purpose of this decontamination is to destroy residual test bacteria on the
skin.Warning—)The application of microorganisms to the skin may involve a health risk. Prior to applying the test organism to
the skin, the antibiotic susceptibility profile of the strain should be determined. If the strain is not susceptible to gentamicin, do
not use it. If an infection occurs, the antibiotic sensitivity profile should be made available to the attending clinician. Following
the subject’s last contamination and wash with the formulation, the subject’s hands are to be sanitized by scrubbing with 70%
isopropanol solution or equivalent. The purpose of this alcohol scrub is to destroy residual test organisms on the skin.)
8.3 Preparation of Test OrganismBacterium Suspension
8.3.1 S. marcescens—A homogeneous culture is to be used to inoculate the hands. The stock culture, frozen or lyopholized,
lyophilized, should be at least two 24-h24 h soybean-casein digest broth (7.10) transfers from the original ATCC culture, but there
Peterson, A. F., “The Microbiology of the Hands: Evaluating the Effects of the Surgical Scrubs,” Developments in Industrial Microbiology, Vol 14, 1973, pp.
125–130.Peterson, A. F. 1973The Microbiology of the Hands: Evaluating the Effects of the Surgical Scrubs, Developments in Industrial Microbiology, Vol 14 pp. 125–130.
Horowitz, W., (Ed.), Offıcial Methods of Analysis of the AOAC, 17th Ed., Sec. 6.3.03 A.(f), Chapter 6, 2000, p. 10. Official Methods of Analysis of AOAC International,
Gaitherburg, MD.Horowitz, W., (Ed.), Offıcial Methods of Analysis of the AOAC International, 17th Ed. 2000. Chap. 6, p. 10. Gaitherburg, MD.
United States Pharmacopeia 28: United States Pharmacopeial Convention, Inc., Rockville, MD, Chapter entitled “Microbial Limits Test.” The MUG (4-
methylumbelliferyl-β-D-gluconride)(4-methylumbelliferyl-β-D-glucuronidase) substrate is hydrolyzed by β-D-gluconridase to yield a fluorescent end product,
4-methylumbelliferone. β-D-gluconridase is possessed by E. coli (ATCC 11229). MUG is incorporated into the appropriate a growth medium at 0.05 g/L.
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should be no more than four transfers removed from the ATCC culture. From the stock culture of culture, Serratia marcescens
(ATCC 14756), inoculate the appropriate volume of soybean-casein digest broth (7.10) with 0.1 mL of stock culture of S.
marcescens/100 mL of broth to yield the volume necessary to complete the study. Incubate for 2424 h 6 4 h at 2525 °C 6 2°C.
Broth should develop a red pigment.
8.3.2 E. coli—A homogeneous culture is to be used to inoculate the hands. The stock culture should be at least two 24-hour broth
transfers from the original ATCC culture, but no more than fivefour transfers removed from the ATCC culture. From the stock
culture of Escherichia coli (ATCC 11229), inoculate the appropriate volume of soybean-casein digest broth (7.10) with 0.1 mL of
stock culture/100 mL of broth to yield the volume necessary to complete the study. Incubate for 2424 h 6 4 h at 3535 °C 6 2°C.
8.4 Swirl or shake suspension before the withdrawal of each aliquot. aliquot for testing. Assay the suspension for number of
organismsbacteria at the beginning and end of the use period. Do not use a suspension for more than 8 h. The suspension may not
vary more than 60.5 log cfu/mL over an 8 h period.
9. Subjects
9.1 Recruit a sufficient number of healthy adult human volunteers who have no clinical evidence of dermatoses, open wounds,
hangnails, or other skin disorders.disorders on the hands and forearms.
9.2 Instruct subjects to avoid contact with antimicrobial products (other than the test material as dispensed for eachthe test wash)
for the duration of the test and for at least one week prior to the test. This restriction includes antimicrobial-containing
antiperspirants, deodorants, shampoos, lotions, and soaps, alsoand such materials as acids, bases, and solvents. Bathing in biocide
treated pools, hot tubs, or spas should must also be avoided. Subjects are to be provided with a kit of nonantimicrobial personal
care products for exclusive use durin
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