ASTM E2011-21

Standard Test Method for Evaluation of Hygienic Handwash and Handrub Formulations for Virus-Eliminating Activity Using the Entire Hand

SIGNIFICANCE AND USE
5.1 This test method is designed to evaluate the virus-eliminating activity of hygienic handwash and handrub agents from experimentally-contaminated hands. Such formulations may be further assessed in a clinical trial for their effectiveness in the field. This test method incorporates whole-hand exposure and reflects actual use conditions such as friction during hand decontamination, and enables alternative product forms such as alcohol- or non-alcohol-based liquids, gels, and foams to be tested according to label directions. It is meant to extend, if required, the results of testing with Test Method E1838, which gives precise reductions in viral infectivity on a limited area of the hands. It may also serve as an alternative test method when product form is not amenable to testing by Test Method E1838.  
5.2 This test method is not meant for use with surgical hand scrubs or preoperative skin preparations.
Note 2: Application of viruses on the entire surface of both hands entails a greater risk to the subjects than using fingerpads only. Therefore, greater care is needed to ensure that the hands of the participants are free from any apparent damage. Also, virus preparations must be thoroughly screened for, or documented to be free from, extraneous or adventitious pathogens before use in such tests.
SCOPE
1.1 This test method is designed to evaluate handwash or handrub agents for their ability to reduce or eliminate viable viruses from the skin of human hands.
Note 1: A knowledge of virological techniques is required for this test method.  
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.3 This standard may involve hazardous materials, operations and equipment. 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, health, and environmental practices and determine the applicability of regulatory limitations prior to use. The user should consult a reference for laboratory safety recommendations. (3-5)  
1.4 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.

General Information

• Status: Published
• Publication Date: 31-Mar-2021
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• ICS: 71.100.70 - Cosmetics. Toiletries
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Refers: ASTM E2756-19 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Nov-2019
• Refers: ASTM E2756-18 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-Apr-2018
• Refers: ASTM E1838-17 - Standard Test Method for Determining the Virus-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults
• Effective Date: 01-Apr-2017
• Refers: ASTM E1482-12 - Standard Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization
• Effective Date: 01-Oct-2012
• Refers: ASTM E2756-10 - Standard Terminology Relating to Antimicrobial and Antiviral Agents
• Effective Date: 01-May-2010
• Refers: ASTM E1838-10 - Standard Test Method for Determining the Virus-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults
• Effective Date: 01-Apr-2010
• Refers: ASTM E1482-04 - Standard Test Method for Neutralization of Virucidal Agents in Virucidal Efficacy Evaluations
• Effective Date: 01-Oct-2004
• Refers: ASTM E1482-92(2004) - Standard Test Method for Neutralization of Virucidal Agents in Virucidal Efficacy Evaluations
• Effective Date: 01-Apr-2004
• Refers: ASTM E1838-02 - Standard Test Method for Determining the Virus-Eliminating Effectiveness of Liquid Hygienic Handwash and Handrub Agents Using the Fingerpads of Adult Volunteers
• Effective Date: 10-Apr-2002
• Refers: ASTM E1482-92(1998) - Standard Test Method for Neutralization of Virucidal Agents in Virucidal Efficacy Evaluations
• Effective Date: 10-Oct-1998

Overview

ASTM E2011-21 is the internationally recognized standard test method for evaluating the virus-eliminating activity of hygienic handwash and handrub formulations using the entire surface of both hands. Developed by ASTM International, this method is used to assess the effectiveness of various hand hygiene products-such as soaps, handrubs, gels, foams, and non-alcohol-based formulations-in reducing or removing viable viruses from hands under conditions that closely replicate real-world use.

This standard provides a robust evaluation approach by simulating actual practices, including whole-hand exposure and friction, which are critical for determining the true efficacy of products in virus elimination. While designed for laboratory testing, products that perform well may be further evaluated in field clinical trials to confirm real-world effectiveness.

Key Topics

• Whole-hand Testing Approach: Unlike fingerpad-only tests, ASTM E2011-21 covers both the palmar and dorsal surfaces of the hands, reflecting how these products are used in practice.
• Evaluation of Virus Reduction: The method measures reduction or elimination of infectious virus from contaminated hands after product use, comparing test product performance against appropriate controls.
• Product Versatility: Supports testing of a wide range of products, including alcohol-based handrubs, non-alcohol-based handrubs, gels, and foams, following manufacturer instructions.
• Realistic Test Conditions: Incorporates variables such as friction and contact time, and uses a “whole-hand” contamination model to better simulate typical user scenarios in hospitals, food processing, daycare, and similar settings.
• Safety Protocols: Stresses the need for participant safety, thorough screening of viral preparations, and the absence of skin damage before testing.
• Not for Surgical Scrubs: Specifically excludes use with surgical hand scrubs or preoperative skin preparations, focusing instead on hygienic hand hygiene agents for general use.

Applications

• Hand Hygiene Product Validation: Used by manufacturers to validate the antiviral claims of their products in healthcare, food service, child care, hospitality, and other environments requiring strict hand hygiene.
• Regulatory Compliance: Helps meet regulatory requirements for demonstrating the effectiveness of handwash and handrub formulations in eliminating viruses on human skin.
• Product Comparative Assessment: Enables comparison across different brands and types of hand hygiene products to determine their relative effectiveness against viruses.
• Research & Development: Essential in R&D for optimizing formulations and supporting claims with data from standardized, reproducible tests.

Types of Products Tested

• Alcohol-based and non-alcohol-based handrubs
• Antiviral handwashes and soaps
• Sanitizing gels and foams suitable for frequent use

Typical Use Scenarios

• Healthcare facilities, hospitals, and nursing homes
• Food-handling and processing environments
• Daycare centers and educational institutions
• Public places requiring high standards of infection control

Related Standards

• ASTM E1838: Provides a fingerpad method for evaluating virus-eliminating activity of hand hygiene agents on a smaller skin area. ASTM E2011-21 extends and complements E1838’s findings for whole-hand applications.
• ASTM E1482: Practice for use of gel filtration columns for cytotoxicity reduction and neutralization, referenced for supporting test accuracy.
• ASTM E2756: Terminology relevant to antimicrobial and antiviral agents.
• AOAC 960.9: Official methods for preparing control solutions and reagent water hardness.

Practical Value

Choosing ASTM E2011-21 ensures that claims regarding hand hygiene product effectiveness are verified using a rigorous, transparent, and internationally respected method. This standard is crucial for public health initiatives aiming to reduce viral transmission and for organizations committed to supplying safe and proven hand hygiene solutions.

Keywords: hygienic handwash, handrub, virus elimination, hand hygiene, ASTM E2011-21, antiviral hand sanitizer, standard test method, healthcare infection control, hand decontamination, virus removal, regulatory compliance, whole-hand testing.

Standards Content (Sample)

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.
Designation: E2011 − 21
Standard Test Method for
Evaluation of Hygienic Handwash and Handrub
Formulations for Virus-Eliminating Activity Using the Entire
Hand
This standard is issued under the fixed designation E2011; the number immediately following the designation indicates the year of
original adoption or, in the case of revision, the year of last revision.Anumber in parentheses indicates the year of last reapproval.A
superscript epsilon (´) indicates an editorial change since the last revision or reapproval.
INTRODUCTION
Mechanicalremovaland/orinsituinactivationofvirusesbyhygienichandwashandhandrubagents
can be assessed using artificially-contaminated hands of adults. This test method uses the entire
surface of both hands (including both the palmar and dorsum sides of the hands) in contrast to only
the fingerpads in the procedure described in Test Method E1838. However, the reported results from
these two methods are comparable. (1, 2)
1. Scope 2. Referenced Documents
2.1 ASTM Standards:
1.1 This test method is designed to evaluate handwash or
E1482Practice for Use of Gel Filtration Columns for Cyto-
handrub agents for their ability to reduce or eliminate viable
toxicity Reduction and Neutralization
viruses from the skin of human hands.
E1838Test Method for Determining the Virus-Eliminating
NOTE1—Aknowledgeofvirologicaltechniquesisrequiredforthistest
EffectivenessofHygienicHandwashandHandrubAgents
method.
Using the Fingerpads of Adults
1.2 The values stated in SI units are to be regarded as
E2756Terminology Relating toAntimicrobial andAntiviral
standard. No other units of measurement are included in this
Agents
standard.
2.2 AOAC Standard:
1.3 This standard may involve hazardous materials, opera-
AOAC 960.9 Official Methods of Analysis (2007)
tions and equipment. This standard does not purport to address
all of the safety concerns, if any, associated with its use. It is
3. Terminology
the responsibility of the user of this standard to establish
3.1 For definitions of terms used in this method refer to
appropriate safety, health, and environmental practices and
Terminology E2756.
determine the applicability of regulatory limitations prior to
3.2 Definitions of Terms Specific to This Standard:
use. The user should consult a reference for laboratory safety
3.2.1 hygienic handwash agents, n—agents generally used
recommendations. (3-5)
for handwashing by personnel in hospitals, other health-care
1.4 This international standard was developed in accor-
facilities, day-care centers, nursing homes, and food-handling
dance with internationally recognized principles on standard-
establishments; should be safe for repeated use, non-irritating,
ization established in the Decision on Principles for the
fast-acting, and efficient in eliminating transient microorgan-
Development of International Standards, Guides and Recom-
isms from intact skin.
mendations issued by the World Trade Organization Technical
3.2.2 hygienic handrub agents (that is, hand sanitizers),
Barriers to Trade (TBT) Committee.
n—agents not requiring rinsing and generally used for hand
hygiene by personnel in hospitals, other health-care facilities,
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 E35.15 on Antimicrobial Agents. For referenced ASTM standards, visit the ASTM website, www.astm.org, or
Current edition approved April 1, 2021. Published June 2021. Originally contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
approved in 1999. Last previous edition approved in 2013 as E2011–13. DOI: Standards volume information, refer to the standard’s Document Summary page on
10.1520/E2011-21. the ASTM website.
2 4
The boldface numbers in parentheses refer to a list of references at the end of Available from AOAC International, 481 North Frederick Ave., Suite 500,
this test method. Gaithersburg, Maryland 20877-2417, http://www.aoac.org.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2011 − 21
day-care centers, nursing homes, and food-handling establish- the host system at a dilution just beyond the cytotoxicity range
ments; should be safe for repeated use, non-irritating, fast- of the formulation tested. At least three replicate determina-
acting, and efficient in eliminating transient microorganisms tions are performed on controls (untreated) and test samples
from intact skin. (treated) to confirm the extent of virus elimination by the
number of lots of the test substance required by the target
3.2.3 non-medicated soap, n—a soap or detergent that is
regulatory agency. Results are recorded and log and/or
mildtotheskinanddoesnotcontainanygermicidalchemicals.
percent reduction in virus infectivity are calculated.
3.2.4 soil (organic) load, n—a solution of one or more
4.5.1 This test method is designed to be performed by a
organicand/orinorganicsubstancesaddedtothesuspensionof
person trained and experienced in working with human patho-
the test organism to simulate the presence of body secretions,
genic viruses and their host cells. Such an individual will also
excretions or other extraneous substances.
beresponsibleforchoosingtheappropriatehostsystemforthe
3.2.5 virus-eliminating (inactivating/removing) agent,
test virus, and applying the techniques necessary for propaga-
n—any agent that rids hands of viruses by either inactivating
tion and maintenance for host system and test virus. For a
them on the skin or by dislodging them for subsequent
reference text, see Ref (6).
wash-off.
5. Significance and Use
3.2.6 virus-inactivating agent, n—any agent that renders a
5.1 This test method is designed to evaluate the virus-
virus noninfectious.
eliminating activity of hygienic handwash and handrub agents
from experimentally-contaminated hands. Such formulations
4. Summary of Test Method
maybefurtherassessedinaclinicaltrialfortheireffectiveness
4.1 This test method uses adult subjects who have provided
in the field. This test method incorporates whole-hand expo-
a written informed consent and whose hands have been
sure and reflects actual use conditions such as friction during
determined to be free from any apparent damage at the time of
hand decontamination, and enables alternative product forms
their participation in the study.
such as alcohol- or non-alcohol-based liquids, gels, and foams
4.1.1 Since both hands, including nail beds, of the test
to be tested according to label directions. It is meant to extend,
subject are exposed to high-titer suspensions of virus, each
if required, the results of testing with Test Method E1838,
subject shall be carefully examined for any skin irritations,
which gives precise reductions in viral infectivity on a limited
micro-breaches,orbreaksinthehandskinandaroundthenails
area of the hands. It may also serve as an alternative test
using a magnifying glass under well-lighted conditions. Those
method when product form is not amenable to testing by Test
withanybreaches,breaks,orotherapparentskindamagesshall
Method E1838.
not participate in the test.
5.2 Thistestmethodisnotmeantforusewithsurgicalhand
4.1.2 While no fewer than six subjects (one evaluated for
scrubs or preoperative skin preparations.
baselineandfiveevaluatedpost-applicationoftestproduct)are
NOTE 2—Application of viruses on the entire surface of both hands
recommended for each virus-test substance combination to be
entails a greater risk to the subjects than using fingerpads only.Therefore,
evaluated, the number required may vary depending on the
greater care is needed to ensure that the hands of the participants are free
intended use of the data and the target regulatory agency.
from any apparent damage. Also, virus preparations must be thoroughly
screened for, or documented to be free from, extraneous or adventitious
4.2 All subjects should refrain from using any antimicrobi-
pathogens before use in such tests.
als starting at least one week prior to the experimental
contamination of their hands. 6. Equipment and Apparatus
4.3 Apreparedsuspensionoftheselectedtestvirusisgrown 6.1 Laminar Flow Cabinet—a Class II biological safety
and diluted or concentrated to produce a titer with a minimum cabinet.The procedures for the proper maintenance and use of
of10 infectiveunits/mL.Thecontaminatingvirusisappliedto such cabinets are given in Ref (3, 4).
the hands and the hands are treated with the test substance
6.2 Incubator—an incubator at 35°C 6 2°C or other
according to the manufacturer’s directions or with a set test
appropriate temperature for growing host cells and for incu-
regimen.
batingvirus-infectedcultures.Ifanopensystemisusedforcell
4.4 The virus titer recovered after treatment with the test culture, a CO incubator will be required.
substance is compared to a baseline sample.
6.3 Positive Displacement Pipette—a pipette and pipette
4.5 The virus on experimentally contaminated hands is tips that can accurately dispense 10-µL to 20-µL volumes.
exposed to the test substance for the length of time that is
6.4 Sterilizer—any steam sterilizer suitable for processing
recommended by the product manufacturer and is representa-
cell culture media and reagents. The steam supplied to the
tive of actual use conditions of the product. The virus to be
sterilizer must be free from additives toxic to cell cultures.
recovered after exposure to the test substance is assayed in a
6.5 Filter Sterilization System—a membrane or cartridge
cell culture system appropriate to the test virus. The virus titer
filtration system (0.22-µm pore diameter) is required for
of the stock, test samples, and controls is determined by a
sterilization of heat-sensitive media and solutions.
suitable infectivity assay. Cytotoxicity of the host cell culture
system caused by the test substance or vehicle at the tested 6.6 Freezers—a freezer at –20°C 6 2°C for the storage of
concentration is also determined. The virus-test substance serum and other additives for cell culture media. A second
mixture is assayed using multiple replicate wells or flasks of freezer at –70°C or lower is required to store viruses.
E2011 − 21
NOTE 4—Serum is considered unsuitable for use as a soil load with
6.7 Refrigerator—a refrigerator at 4°C 6 2°C is necessary
rotaviruses because of its rotavirus-inhibitory and trypsin-neutralizing
for storage of prepared cell culture media and reagents.
activity.
6.8 Timer—any calibrated stopwatch that can be read in
7.2.2 A tripartite soil load, as an alternative to serum, is
minutes and seconds.
prepared from the following stock solutions in phosphate
6.9 Magnetic Stirrer and Magnets—magnetic stirrer and
buffer (pH 7.2 to 7.4).
magnets must be large enough to hold a 5-L beaker or
7.2.2.1 Add 0.5 g of tryptone or yeast extract to 10 mL of
Erlenmeyer flask for preparing cell culture media or other
the buffer.
solutions.
7.2.2.2 Add0.5gofbovineserumalbumin(BSA)to10mL
of the buffer.
6.10 Handwashing Sink—a sink of sufficient size to permit
7.2.2.3 Add 0.04 g of bovine mucin to 10 mLof the buffer.
subjectstowashhandswithouttouchinghandstosinksurface.
7.2.2.4 Prepare the stock solutions separately and sterilize
6.10.1 Water faucet(s) are to be located above the sink at a
by passage through a 0.22-µm pore diameter membrane filter,
height that permits the hands to be held higher than the elbow
aliquot and store at either 4°C 6 2°C or –20°C 6 2°C. Use
duringthewashingprocedures.Faucetswithelectronicsensors
within a validated shelf-life.
or those that are wrist-, elbow-, knee-, or foot-operated are
7.2.2.5 To obtain a 500-µL inoculum of the test inoculum,
preferred to avoid recontamination of the washed hands.
add to 340 µLof the microbial suspension 25 µLBSA, 100 µL
6.10.2 Mild, proven non-antimicrobial soap, preferably liq-
mucin,and35µLoftryptone/yeastextractstocksolutions.This
uid.
mixturecontainsapproximately2goftotalprotein/L,whichis
6.10.3 Tap water temperature regulator and temperature
approximately equivalent to the protein content of a 5%
monitor to monitor and regulate water temperature at 40°C 6
solution of fetal bovine serum.
2°C.
7.3 Standard Hard Water—Water prepared according to
6.11 Liquid Nitrogen Storage for Cells—an appropriate
AOAC 960.9 to a standard hardness of 200 ppm as calcium
liquid nitrogen container and liquid nitrogen for cryopreserva-
carbonate is used for dilution of test substance. This is the
tion of cell line stocks.
control solution to determine the baseline level of virus
6.12 Inverted Microscope—an inverted microscope with
elimination, and to rinse the hands after exposure to the test
10× eye pieces and 5×, 10×, and 40× objectives.
substance.
6.13 Serological Pipettes—sterile reusable or single-use
7.4 Number of Test Substance Lots to be Used—Thenumber
pipettes of 10.0-, 5.0-, and 1.0-mL capacity or other suitable
of separate manufactured lots (batches) of each test formula-
capacity.
tiontobetestedwilldependonthespecificrequirementsofthe
6.14 Cell Culture Flasks—plastic cell culture flasks of
target regulatory agency.
2 2
25cm or 75cm or other suitable capacity for culturing cells
7.5 Diluent for Virus Titration—Earle’s balanced salt solu-
and for preparing virus pools.
tion(EBSS)orotherappropriatedilutionmediumwithapHof
NOTE 3—Each plastic flask for growing cell monolayers can be reused 7.2 to 7.4.
byreseedingwithnewcellculturesupto10timesbeforebeingdiscarded.
7.6 Eluent for Virus Recovery from Hands—EBSS or other
6.15 Plastic and Glass Vials, Medication (Medicant)—
appropriate dilution medium containing 1% peptone and
sterile screw-capped vials will be required for storage of
0.1% Polysorbate 80 at final concentrations.
samples.
7.7 Sterile Disposable Gloves—Loose-fitting, unlined,
6.16 Miscellaneous Labware—automatic pipettes, pipette
powder-free gloves which possess no antiviral or cytotoxic
tips, plastic vials for storing cell and virus stocks, dilution
properties,orequivalent.(Plasticbagswithlowbioburdenmay
tubes, cluster plates or flasks for virus titration.
be used in place of gloves.)
6.17 Sterile Glass Beads—3.5 mm in diameter.
8. Test Viruses and Cell Cultures
6.18 Glass or Plastic Funnel—27 cm in diameter.
8.1 See Appendix X1 for suggested viruses and host cells.
6.19 Glass or Plastic Beaker—200 mL in capacity.
8.2 Virus stocks as well as host cells used for virus
7. Materials and Reagents
propagation may contain adventitious viruses or other patho-
gens potentially harmful to human subjects. Therefore, great
7.1 Cell Culture Media and Supplements—Culture media
care should be used in the selection and use of such materials
andthetypesandratiosofsupplementswillvarydependingon
to be applied on human hands.
the cell line. For example, Eagle’s minimal essential medium
(EMEM) with 5% to 10% fetal bovine serum (virus- and
9. Preparation of Virus Stocks and Determination of
mycoplasma-tested)isusedforgrowingawidevarietyofcells
Infectivity Titer
(see Note 4). Antibiotics may be required in the medium to
suppress bacterial contamination. 9.1 Useappropriatehostcellstopreparetheviruspool.The
virus pool should contain ≥10 infective unit/mL.
7.2 Soil Load:
7.2.1 Bovine serum, at a final concentration of 5% in the 9.2 Remove growth or maintenance medium and inoculate
virus inoculum (see Note 4), if required for the test. 0.1mLofvirus(controlflasksreceive0.1mLofEBSSinstead)
E2011 − 21
into each flask (for example, 75 cm ) with a confluent cell monolayer in an inverted microscope for any signs of cytotox-
monolayer and allow 60 to 120 min for virus adsorption. Place icity. Absence of any apparent degradation of the cells indi-
15 mL of maintenance medium into each inoculated flask and cates freedom from cytotoxicity that could interfere with the
reincubate until about 75% to 95% of each infected cell scoring for viral infectivity. If cytotoxicity is detected, a
monolayer shows virus-induced cytopathology. Control mono- different neutralizer or alternative approaches (7) to removal/
layers must remain free from any apparent degeneration or reduction of cytotoxicity may be needed.
contamination. Freeze (–20 to –90°C) and thaw (room tem-
10.4 Control for Interference with Viral Infectivity—This
perature)theinfectedflasksthreetimestodis
...

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 the previous version. Because
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: E2011 − 13 E2011 − 21
Standard Test Method for
Evaluation of Hygienic Handwash and Handrub
Formulations for Virus-Eliminating Activity Using the Entire
Hand
This standard is issued under the fixed designation E2011; 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
Mechanical removal and/or in situ inactivation of viruses by hygienic handwash and handrub agents
can be assessed using artificially-contaminated hands of adults. This test method uses the entire
surface of both hands (including both the palmar and dorsum sides of the hands) in contrast to only
the fingerpads in the procedure described in Test Method E1838. However, the reported results from
these two methods are comparable. (1, 2)
1. Scope
1.1 This test method is designed to evaluate handwash or handrub agents for their ability to reduce or eliminate viable viruses from
the skin of human hands.
NOTE 1—A knowledge of virological techniques is required for this test method.
1.2 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.3 This standard may involve hazardous materials, operations and equipment. 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. The
user should consult a reference for laboratory safety recommendations. (3-5)
1.4 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:
E1482 Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization
E1838 Test Method for Determining the Virus-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the
Fingerpads of Adults
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 E35.15 on Antimicrobial Agents.
Current edition approved April 1, 2013April 1, 2021. Published May 2013June 2021. Originally approved in 1999. Last previous edition approved in 20092013 as
E2011 – 09.E2011 – 13. DOI: 10.1520/E2011-13.10.1520/E2011-21.
The boldface numbers in parentheses refer to a list of references at the end of this test method.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E2011 − 21
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 AOAC Standard:
AOAC 960.9 Official Methods of Analysis (2007)
3. Terminology
3.1 For definitions of terms used in this method refer to Terminology E2756.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 hygienic handwash agents, n—agents generally used for handwashing by personnel in hospitals, other health-care facilities,
day-care centers, nursing homes, and food-handling establishments; should be safe for repeated use, non-irritating, fast-acting, and
efficient in eliminating transient microorganisms from intact skin.
3.2.2 hygienic handrub agents (that is, hand sanitizers), n—agents not requiring rinsing and generally used for hand hygiene by
personnel in hospitals, other health-care facilities, day-care centers, nursing homes, and food-handling establishments; should be
safe for repeated use, non-irritating, fast-acting, and efficient in eliminating transient microorganisms from intact skin.
3.2.3 non-medicated soap, n—a soap or detergent that is mild to the skin and does not contain any germicidal chemicals.
3.2.4 soil (organic) 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.
3.2.5 virus-eliminating (inactivating/removing) agent, n—any agent that rids hands of viruses by either inactivating them on the
skin or by dislodging them for subsequent wash-off.
3.2.6 virus-inactivating agent, n—any agent that renders a virus noninfectious.
4. Summary of Test Method
4.1 This test method uses adult subjects who have provided a written informed consent and whose hands have been determined
to be free from any apparent damage at the time of their participation in the study.
4.1.1 Since both hands, including nail beds, of the test subject are exposed to high-titer suspensions of virus, each subject shall
be carefully examined for any skin irritations, micro-breaches, or breaks in the hand skin and around the nails using a magnifying
glass under well-lighted conditions. Those with any breaches, breaks, or other apparent skin damages shall not participate in the
test.
4.1.2 While no fewer than six subjects (one evaluated for baseline and five evaluated post-application of test product) are
recommended for each virus-test substance combination to be evaluated, the number required may vary depending on the intended
use of the data and the target regulatory agency.
4.2 All subjects should refrain from using any antimicrobials starting at least one week prior to the experimental contamination
of their hands.
4.3 A prepared suspension of the selected test virus is grown and diluted or concentrated to produce a titer with a minimum of
10 infective units/mL. The contaminating virus is applied to the hands and the hands are treated with the test substance according
to the manufacturer’s directions or with a set test regimen.
4.4 The virus titer recovered after treatment with the test substance is compared to a control. For the control, the test virus is
applied to the hands and recovered after the subject has treated the hands with standard hard water (200 ppm as calcium carbonate)
or vehicle, or both, instead of the test substance.baseline sample.
4.5 The virus on experimentally contaminated hands is exposed to the test substance for the length of time that is recommended
Available from AOAC International, 481 North Frederick Ave., Suite 500, Gaithersburg, Maryland 20877-2417, http://www.aoac.org.
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by the product manufacturer and is representative of actual use conditions of the product, for example, from 10 to 20 s for a
handsoap and 20 to 30 s for a handrub. product. The virus to be recovered after exposure to the test substance is assayed in a cell
culture system appropriate to the test virus. The virus titer of the stock, test samples, and controls is determined by a suitable
infectivity assay. Cytotoxicity of the host cell culture system caused by the test substance or vehicle at the tested concentration is
also determined. The virus-test substance mixture is assayed using multiple replicate wells or flasks of the host system at a dilution
just beyond the cytotoxicity range of the formulation tested. At least three replicate determinations are performed on controls
(untreated) and test samples (treated) to confirm the extent of virus elimination by the number of lots of the test substance required
by the target regulatory agency. Results are recorded and log and/or percent reduction in virus infectivity are calculated.
4.5.1 This test method is designed to be performed by a person trained and experienced in working with human pathogenic viruses
and their host cells. Such an individual will also be responsible for choosing the appropriate host system for the test virus, and
applying the techniques necessary for propagation and maintenance for host system and test virus. For a reference text, see Ref
(6).
5. Significance and Use
5.1 This test method is designed to evaluate the virus-eliminating activity of hygienic handwash and handrub agents from
experimentally-contaminated hands. Such formulations may be further assessed in a clinical trial for their effectiveness in the field.
This test method incorporates whole-hand exposure and reflects actual use conditions such as friction during hand
decontamination, and also enables alternative product forms such as alcohol- or non-alcohol-based liquids, gels, and foams to be
tested according to label directions. It is meant to extend, if required, the results of testing with Test Method E1838, which gives
precise reductions in viral infectivity on a limited area of the hands. It may also serve as an alternative test method when product
form is not amenable to testing by Test Method E1838.
5.2 This test method is not meant for use with surgical hand scrubs or preoperative skin preparations.
NOTE 2—Application of viruses on the entire surface of both hands entails a greater risk to the subjects than using fingerpads only. Therefore, greater
care is needed to ensure that the hands of the participants are free from any apparent damage. Also, virus preparations must be thoroughly screened for,
or documented to be free from, extraneous or adventitious pathogens before use in such tests.
6. Equipment and Apparatus
6.1 Laminar Flow Cabinet—a Class II biological safety cabinet. The procedures for the proper maintenance and use of such
cabinets are given in Ref (3, 4).
6.2 Incubator—an incubator at 3535 °C 6 2°C2 °C or other appropriate temperature for growing host cells and for incubating
virus-infected cultures. If an open system is used for cell culture, a CO incubator will be required.
6.3 Positive Displacement Pipette—a pipette and pipette tips that can accurately dispense 1010-μL to 20-μL volumes.
6.4 Sterilizer—any steam sterilizer suitable for processing cell culture media and reagents. The steam supplied to the sterilizer
must be free from additives toxic to cell cultures.
6.5 Filter Sterilization System—a membrane or cartridge filtration system (0.22-μm pore diameter) is required for sterilization of
heat-sensitive media and solutions.
6.6 Freezers—a freezer at –20–20 °C 6 2°C2 °C for the storage of serum and other additives for cell culture media. A second
freezer at –70°C–70 °C or lower is required to store viruses.
6.7 Refrigerator—a refrigerator at 44 °C 6 2°C2 °C is necessary for storage of prepared cell culture media and reagents.
6.8 Timer—any calibrated stopwatch that can be read in minutes and seconds.
6.9 Magnetic Stirrer and Magnets—magnetic stirrer and magnets must be large enough to hold a 5-L beaker or Erlenmeyer flask
for preparing cell culture media or other solutions.
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6.10 Handwashing Sink—a sink of sufficient size to permit subjects to wash hands without touching hands to sink surface.
6.10.1 Water faucet(s) are to be located above the sink at a height that permits the hands to be held higher than the elbow during
the washing procedures. Faucets with electronic sensors or those that are wrist-, elbow-, knee-, or foot-operated are preferred to
avoid recontamination of the washed hands.
6.10.2 Mild, proven non-antimicrobial soap, preferably liquid.
6.10.3 Tap water temperature regulator and temperature monitor to monitor and regulate water temperature at 4040 °C 6
2°C.2 °C.
6.11 Liquid Nitrogen Storage for Cells—an appropriate liquid nitrogen container and liquid nitrogen for cryopreservation of cell
line stocks.
6.12 Inverted Microscope—an inverted microscope with 10× eye pieces and 5×, 10×, and 40× objectives.
6.13 Serological Pipettes—sterile reusable or single-use pipettes of 10.0-, 5.0-, and 1.0-mL capacity or other suitable capacity.
2 2
6.14 Cell Culture Flasks—plastic cell culture flasks of 25 cm25 cm or 75 cm75 cm or other suitable capacity for culturing cells
and for preparing virus pools.
NOTE 3—Each plastic flask for growing cell monolayers can be reused by reseeding with new cell cultures up to 10 times before being discarded.
6.15 Plastic and Glass Vials, Medication (Medicant)—sterile screw-capped vials will be required for storage of samples.
6.16 Miscellaneous Labware—automatic pipettes, pipette tips, plastic vials for storing cell and virus stocks, dilution tubes, cluster
plates or flasks for virus titration.
6.17 Sterile Glass Beads—3.5 mm in diameter.
6.18 Glass or Plastic Funnel—27 cm in diameter.
6.19 Glass or Plastic Beaker—200 mL in capacity.
7. Materials and Reagents
7.1 Cell Culture Media and Supplements—Culture media and the types and ratios of supplements will vary depending on the cell
line. For example, Eagle’s minimal essential medium (EMEM) with 55 % to 10 % fetal bovine serum (virus- and mycoplasma-
tested) is used for growing a wide variety of cells (see Note 4). Antibiotics may be required in the medium to suppress bacterial
contamination.
7.2 Soil Load:
7.2.1 Bovine serum, at a final concentration of 5 % in the virus inoculum (see Note 4), if required for the test.
NOTE 4—Serum is considered unsuitable for use as a soil load with rotaviruses because of its rotavirus-inhibitory and trypsin-neutralizing activity.
7.2.2 A tripartite soil load, as an alternative to serum, is prepared from the following stock solutions in phosphate buffer (pH 7.2
to 7.4).
7.2.2.1 Add 0.5 g of tryptone or yeast extract to 10 mL of the buffer.
7.2.2.2 Add 0.5 g of bovine serum albumin (BSA) to 10 mL of the buffer.
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7.2.2.3 Add 0.04 g of bovine mucin to 10 mL of the buffer.
7.2.2.4 Prepare the stock solutions separately and sterilize by passage through a 0.22-μm pore diameter membrane filter, aliquot
and store at either 44 °C 6 2°C2 °C or –20–20 °C 6 2°C.2 °C. Use within a validated shelf-life.
7.2.2.5 To obtain a 500-μL inoculum of the test inoculum, add to 340 μL of the microbial suspension 25 μL BSA, 100 μL mucin,
and 35 μL of tryptone/yeast extract stock solutions. This mixture contains approximately 2 g of total protein/L, which is
approximately equivalent to the protein content of a 5 % solution of fetal bovine serum.
7.3 Standard Hard Water—Water prepared according to AOAC 960.9 to a standard hardness of 200 ppm as calcium carbonate is
used for dilution of test substance. This is the control solution to determine the baseline level of virus elimination, and to rinse the
hands after exposure to the test substance.
7.4 Number of Test Substance Lots to be Used—The number of separate manufactured lots (batches) of each test formulation to
be tested will depend on the specific requirements of the target regulatory agency.
7.5 Diluent for Virus Titration—Earle’s balanced salt solution (EBSS) or other appropriate dilution medium with a pH of 7.2 to
7.4.
7.6 Eluent for Virus Recovery from Hands—EBSS or other appropriate dilution medium containing 1 % peptone and 0.1 %
Polysorbate 80 at final concentrations.
7.7 Sterile Disposable Gloves—Loose-fitting, unlined, powder-free gloves which possess no antiviral or cytotoxic properties, or
equivalent. (Plastic bags with low bioburden may be used in place of gloves.)
8. Test Viruses and Cell Cultures
8.1 See Appendix X1 for suggested viruses and host cells.
8.2 Virus stocks as well as host cells used for virus propagation may contain adventitious viruses or other pathogens potentially
harmful to human subjects. Therefore, great care should be used in the selection and use of such materials to be applied on human
hands.
9. Preparation of Virus Stocks and Determination of Infectivity Titer
9.1 Use appropriate host cells to prepare the virus pool. The virus pool should contain ≥10 infective unit/mL.
9.2 Remove growth or maintenance medium and inoculate 0.1 mL of virus (control flasks receive 0.1 mL of EBSS instead) into
each flask (for example, 75 cm ) with a confluent cell monolayer and allow 60 to 120 min for virus adsorption. Place 15 mL of
maintenance medium into each inoculated flask and reincubate until about 7575 % to 95 % of each infected cell monolayer shows
virus-induced cytopathology. Control monolayers must remain free from any apparent degeneration or contamination. Freeze (–20
to –90°C)–90 °C) and thaw (room temperature) the infected flasks three times to disrupt host cells for virus release. Centrifuge the
cell suspension at 4°C4 °C for 10 min to 20 min at approximately 1000 x×g to sediment the cell debris, collect supernatant, aliquot
if necessary, and store it at –6–6 °C to –90°C–90 °C in suitable aliquots.
NOTE 5—Alternative flask size, inoculum volume, and/or medium volume may be used as appropriate.
9.3 A titer of ≥10 infective units/mL is required for the testing and ultra-centrifugation of virus pools may be needed to achieve
such levels of infectivity for the contamination of hands.
10. Controls
10.1 Cell Control—To ensure that the host cells are not contaminated with bacteria, fungi, or any cytopathogenic viruses other than
those used in the test, at least two host cell monolayers are left untreated in each test and examined first at the end of the incubation
period. Any obvious contamination or degeneration in such monolayers would invalidate the assay.
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10.2 Virus Susceptibility Control—To ensure that the host cells remain susceptible to the test virus, at least three host cell
monolayers will receive a level of the test virus sufficient to produce cytopathology. A lack of obvious and typical virus-induced
cytopathic effects in such a monolayer would also invalidate the test.
10.3 Cytotoxicity Control—This control applies only in to neutralizers used in the test and for tests which utilize handrub agents.
Its objective is to determine if residues of the test substance in the neutralized eluates can produce any apparent degeneration
(cytotoxicity) of the cell line for measuring viral infectivity.
10.3.1 Place 0.5 mL
...