ASTM E1053-20

Standard Practice to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental Surfaces

SIGNIFICANCE AND USE
5.1 This practice may be used to determine the effectiveness of liquid, aerosols/foams, and trigger-spray products against designated prototype viruses.  
5.2 The number of lots of the test substance and the number of replicates in each test will depend on the requirements of the target regulatory agency.  
5.3 Certain regulatory agencies may require additional testing using other carrier tests for product registration purposes.
SCOPE
1.1 This practice is used to evaluate the virucidal efficacy of liquid, aerosol, or trigger-spray microbicides intended for use on inanimate, nonporous environmental surfaces. This practice may be employed with most viruses, which can be grown in cultured cells.2 However, other host systems (for example, embryonic eggs) may be used with proper justification and documentation.  
1.2 This practice should be performed only by those trained in microbiological and virological techniques in facilities designed and equipped for work with infectious agents at the appropriate biosafety level.  
1.3 It is the responsibility of the investigator to determine whether Good Laboratory Practice regulations (GLPs) are required and to follow them where appropriate (40 CFR, Part 160 for EPA submissions and 21 CFR, Part 58 for FDA submissions). Refer to the appropriate regulatory agency for performance standards of virucidal efficacy.  
1.4 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.2  
1.5 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-Dec-2019
• ICS: 65.100.01 - Pesticides and other agrochemicals in general
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Replaces: ASTM E1053-11 - Standard Test Method to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental Surfaces
• Effective Date: 01-Jan-2020
• Refers: ASTM D1129-13(2020)e2 - Standard Terminology Relating to Water
• Effective Date: 01-May-2020
• Refers: ASTM E2197-17e1 - Standard Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals
• Effective Date: 01-Dec-2017
• Refers: ASTM E2197-17 - Standard Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals
• Effective Date: 01-Dec-2017
• Refers: ASTM E1153-14 - Standard Test Method for Efficacy of Sanitizers Recommended for Inanimate, Hard, Nonporous Non-Food Contact Surfaces
• Effective Date: 01-Apr-2014
• Refers: ASTM E1482-12 - Standard Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization
• Effective Date: 01-Oct-2012
• Refers: ASTM E2197-11 - Standard Quantitative Disk Carrier Test Method for Determining the Bactericidal, Virucidal, Fungicidal, Mycobactericidal and Sporicidal Activities of Liquid Chemical Germicides
• Effective Date: 01-Jan-2011
• Refers: ASTM E1153-03(2010)e1 - Standard Test Method for Efficacy of Sanitizers Recommended for Inanimate Non-Food Contact Surfaces
• Effective Date: 01-Apr-2010
• Refers: ASTM D1129-10 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2010
• Refers: ASTM D1129-06ae1 - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1129-06a - Standard Terminology Relating to Water
• Effective Date: 01-Sep-2006
• Refers: ASTM D1129-06 - Standard Terminology Relating to Water
• Effective Date: 15-Feb-2006
• 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 D1129-04e1 - Standard Terminology Relating to Water
• Effective Date: 01-Mar-2004

Overview

ASTM E1053-20, "Standard Practice to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental Surfaces," defines standardized procedures for determining the effectiveness of chemical disinfectants against viruses on nonporous surfaces. Developed by ASTM International, this standard supports the evaluation of liquid, aerosol, and trigger-spray microbicides intended for use in diverse environments. The methodology assists manufacturers, regulatory agencies, and laboratories in ensuring disinfectant efficacy, safety, and regulatory compliance.

Key Topics

• Virucidal Efficacy Testing: The standard specifies procedures for evaluating whether a chemical disinfectant effectively inactivates viruses dried on hard, nonporous surfaces.
• Test Methods: The practice outlines how to apply virus suspensions, expose them to disinfectants (either as liquids or sprays), and measure infectivity post-treatment.
• Controls and Neutralization: Essential controls-including cytotoxicity controls, neutralization controls, and plate recovery controls-are integral to accurate results. Neutralization procedures ensure that microbial activity is accurately measured and not masked or exaggerated by residual disinfectant action.
• Selection of Test Viruses: The standard provides guidance on choosing relevant test viruses and cell lines, with emphasis on including at least one non-enveloped virus to demonstrate broad-spectrum efficacy.
• Safety and Laboratory Requirements: Only trained personnel using biosafety-compliant facilities should perform the procedures. It is also necessary to adhere to Good Laboratory Practice (GLP) regulations according to EPA or FDA requirements.
• Soil Load Simulation: Incorporates organic and inorganic soil loads to mimic real-world contamination, ensuring disinfectants are evaluated under practical conditions.

Applications

• Disinfectant Product Registration: Results obtained using ASTM E1053-20 are often required by regulatory bodies, such as the US EPA and FDA, for registering new disinfectant products making antiviral claims.
• Healthcare and Public Facility Maintenance: Hospitals, laboratories, and other institutions use products validated under this standard to ensure the inactivation of potentially harmful viruses on surfaces.
• Product Development and Quality Control: Manufacturers employ this standard in research and development to substantiate antiviral claims, support labeling, and maintain rigorous quality assurance.
• Environmental Hygiene Programs: The practice helps design and monitor disinfection protocols for surfaces in settings where infection control is paramount.

Related Standards

• ASTM E1153: Test Method for Efficacy of Sanitizers Recommended for Inanimate, Hard, Nonporous Non-Food Contact Surfaces.
• ASTM E2197: Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals.
• ASTM E1482: Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization.
• ASTM D1129: Terminology Relating to Water.
• Good Laboratory Practice Standards: Title 40 CFR, Part 160 (EPA) and Title 21 CFR, Part 58 (FDA).

Practical Value

• Credible and Comparable Data: The harmonized, internationally recognized methodology allows data comparability across products, suppliers, and regulatory authorities.
• Supports Public Health Goals: Adoption of proven virucidal surface disinfectants mitigates the spread of infectious diseases in public and private environments.
• Compliance and Safety Assurance: The standard underpins regulatory submission requirements and validation procedures for disinfectant efficacy, enhancing consumer safety and product reliability.

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Keywords: ASTM E1053-20, virucidal activity, disinfectant efficacy testing, surface viruses, microbicide evaluation, nonporous surfaces, infection control, regulatory compliance, EPA, FDA, biosafety.

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: E1053 − 20
Standard Practice to
Assess Virucidal Activity of Chemicals Intended for
Disinfection of Inanimate, Nonporous Environmental
Surfaces
This standard is issued under the fixed designation E1053; 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.
1. Scope 2. Referenced Documents
1.1 Thispracticeisusedtoevaluatethevirucidalefficacyof 2.1 ASTM Standards:
liquid, aerosol, or trigger-spray microbicides intended for use D1129Terminology Relating to Water
oninanimate,nonporousenvironmentalsurfaces.Thispractice E1153TestMethodforEfficacyofSanitizersRecommended
may be employed with most viruses, which can be grown in for Inanimate, Hard, Nonporous Non-Food Contact Sur-
cultured cells. However, other host systems (for example, faces
embryonic eggs) may be used with proper justification and E1482Practice for Use of Gel Filtration Columns for Cyto-
documentation. toxicity Reduction and Neutralization
E2197Quantitative Disk Carrier Test Method for Determin-
1.2 This practice should be performed only by those trained
ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
in microbiological and virological techniques in facilities
and Sporicidal Activities of Chemicals
designed and equipped for work with infectious agents at the
2.2 Federal Standards:
appropriate biosafety level.
Title21,CodeofFederalRegulations(CFR),FoodandDrug
1.3 It is the responsibility of the investigator to determine
Administration,Part58,LaboratoryPracticeforNonclini-
whether Good Laboratory Practice regulations (GLPs) are
cal Laboratory Studies
required and to follow them where appropriate (40 CFR, Part
Title40,CodeofFederalRegulations(CFR),Environmental
160 for EPA submissions and 21 CFR, Part 58 for FDA
Protection Agency, Subchapter E, Pesticide Programs;
submissions). Refer to the appropriate regulatory agency for
Part 160, Good Laboratory Practice Standards
performance standards of virucidal efficacy.
1.4 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, health, and environmental practices and deter-
3.2 Definitions of Terms Specific to This Standard:
mine the applicability of regulatory limitations prior to use.
3.2.1 hard water, n—water with a standard hardness as
The user should consult a reference for laboratory safety
calcium carbonate.
recommendations.
1.5 This international standard was developed in accor-
3.2.2 neutralization, n—a process which results in quench-
dance with internationally recognized principles on standard-
ing the microbicidal activity of a test substance. This may be
ization established in the Decision on Principles for the
achieved through dilution of the test substance to reduce the
Development of International Standards, Guides and Recom-
microbicidal activity, or through the use of chemical agents,
mendations issued by the World Trade Organization Technical
called neutralizers, to eliminate microbicidal activity.
Barriers to Trade (TBT) Committee.
3.2.3 soil load, n—a solution of one or more organic and/or
inorganic substances added to the suspension of the test
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Jan. 1, 2020. Published March 2020. Originally For referenced ASTM standards, visit the ASTM website, www.astm.org, or
approved in 1985. Last previous edition approved in 2011 as E1053–11. DOI: contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
10.1520/E1053-20. Standards volume information, refer to the standard’s Document Summary page on
Public Health Service, Centers for Disease Control and Prevention, and the ASTM website.
National Institutes of Health, Biosafety in Microbiological and Biomedical AvailablefromU.S.GovernmentPrintingOfficeSuperintendentofDocuments,
Laboratories, U.S. Department of Health and Human Services, Washington, DC, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
December 2009, 422 pp. www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1053 − 20
organism to simulate the presence of body secretions, excre- 6.1.8 Pipettes, serological, various volumes.
tions or other extraneous substances. 6.1.9 Micropipettors and Sterile Tips
6.1.10 96-Well Dilution Plates, (only required for use in a
3.2.4 test substance or test formulation, n—a formulation
96-well assay).
which incorporates microbicidal ingredients.
6.1.11 Microtitration Kit, if applicable.
4. Summary of Practice
NOTE 3—Microtitration kits may be purchased from most laboratory
supply houses.
4.1 The virus suspension is dried on an inanimate, nonpo-
rous surface. The test substance is added over the dried film at
6.1.12 Petri Plates, glass, 100-mm diameter, 10-15 mm
its use-dilution or sprayed from an aerosol can or trigger-
deep.
sprayer following the manufacturer’s directions. Control carri-
6.1.13 Microscope.
ersreceiveanequivalentvolumeofabufferharmlesstothetest
6.1.14 Biosafety cabinet.
virus and its host cells. After exposure at the appropriate
temperature (usually 20 6 2°C) for the recommended time, 7. Test Viruses
the eluates from control and test carriers are assayed for
7.1 AppendixX1listsvirusesandtheirrespectivehostcells
infectivity.
asexamplesforuseinthispractice.Othervirusesandcelllines
4.2 This practice is designed to be performed by a person may be used.
trained in culturing and assaying infectious viruses who is
7.2 To demonstrate that the test substance has broad viru-
responsible for choosing the appropriate host system for the
cidalactivity,itshouldbeshowntobeeffectiveagainstatleast
test virus and applying the techniques necessary for propaga-
one non-enveloped virus.
tion and maintenance of the host system and test virus. For a
reference text, refer to Lennette et al.
8. Virus Stock
8.1 Thetiterofthetestvirussuspensionmustbesufficiently
5. Significance and Use
4.8
high so that at least 10 infective units/carrier can be
5.1 Thispracticemaybeusedtodeterminetheeffectiveness
recovered from the inoculated carriers after the inoculum has
of liquid, aerosols/foams, and trigger-spray products against
dried. The host system employed for virus propagation need
designated prototype viruses.
not be the same as that used for virus recovery and the
5.2 Thenumberoflotsofthetestsubstanceandthenumber
infectivity assay.
ofreplicatesineachtestwilldependontherequirementsofthe
9. Operating Technique
target regulatory agency.
9.1 Test Substance Diluent—For test substances requiring
5.3 Certain regulatory agencies may require additional test-
dilution in water to obtain a use-dilution, water with a
ing using other carrier tests for product registration purposes.
standardized and specified level of hardness, or otherwise
6. Materials and Reagents
recommended by manufacturer, shall be used as the diluent.
6.1 Host System and Assay of Infectious Virus—See Note 2.
9.2 Cytotoxicity Control—The objective of this control is to
6.1.1 Cell Cultures in multi-well plates, appropriate for test
(1) determine the dilution of the test substance post-
virus.
neutralization at which it causes no apparent degeneration
6.1.2 Growth and Maintenance Media, any growth and
(cytotoxicity) of the cell line to be used for measuring virus
maintenancemediasuitableforworkwiththevirusanditshost
infectivity and (2) assess if the neutralizer in any way reduces
cells.
or enhances such cytotoxicity. Make an initial 1:2 dilution of
the use-dilution of the test substance in the neutralizer and
NOTE1—Materialsandreagentsforcellculturemaybepurchasedfrom
threefurtherten-folddilutionsoftheneutralizedtestsubstance
biological supply houses.
in the diluent. Remove the culture medium from the monolay-
6.1.3 Diluent, Earle’s Balanced Salt Solution (EBSS) or
ers of the host cell line(s) and put into each test monolayer
other appropriate media.
separately the same volume of inoculum as used in virus
6.1.4 Plastic Cell Culture Ware.
titration; control monolayers receive an equivalent amount of
NOTE 2—Plastic cell culture ware may be purchased from most
diluent (without any neutralizer) only. Another set of mono-
laboratory supply houses.
layers should be exposed to the neutralizer alone. Hold the
6.1.5 Incubator, capable of maintaining 36 6 1°C or other
cultures for the same period of time as used in virus titration
temperature appropriate for replication of the specific test
and examine them under an inverted microscope for any
virus;anincubatorwith5to7%CO willbeneededifanopen
cytotoxicity. In case of cytotoxicity, a different neutralizer or
system is being used for cell culture and virus assay.
gel filtration (see Test Method E1482) of the neutralized
6.1.6 Refrigerator, 4 6 2°C.
virus-test substance mixture may be needed.
6.1.7 Test Tubes, screw-capped or snap capped or similar.
NOTE 4—If gel filtration is used in the virucidal activity test runs, the
Schmidt, N. J., , Lennette, D. A., Lennette, E. T., and Lennette, E. H. eds.,
Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, Seventh AOACInternational, Offıcial Methods of Analysis of the AOAC,Arlington,VA,
Edition, American Public Health Association, Washington, DC, 1995. 1990.
E1053 − 20
neutralized and gel-filtered test substance should be evaluated for the
(2.0 mL). Scrape the inside bottom surface with a sterile cell
cytotoxicity control.
scrapertoresuspendtheviralfilm.Ifgelfiltrationcolumnswill
9.3 Test Substance Neutralization Control—To determine beusedinadditiontotheliquidneutralizer,followingscraping
the dilution at which neutralization of the test substance has the suspension will be added to a previously prepared gel
occurred, prepare and inoculate an additional set of filtration column. The solution is pushed through the gel
neutralization/cytotoxicity controls following the same proce- filtration column utilizing the syringe plunger or by centrifu-
dureasthetestsubstanceefficacyevaluationrunsusingamock gation.Thissuspensionmaybeconsideredthe10–1dilutionof
inoculum (for example,dilution medium) in lieu of virus. To the virus. Prepare serial 10-fold dilutions using diluent and
validate the neutralization, add equal volumes of the neutral- inoculate an amount appropriate to the test format to no less
ized test substance, and a control fluid (for example, PBS) a than four replicate cell monolayers/dilution. Alternatively, in
relatively low number (for example, 1000 to 5000) infective lieuofaddingliquidneutralizerfollowingthecontacttime,just
units of the test virus and hold the mixtures for at least the prior to the end of the exposure time the dish is scraped and
contacttimeatthecontacttemperature.Titratethemixturesfor immediatelyfollowingthecontacttimethesolutionisaddedto
infectious virus. Comparable levels of infective units must be a gel filtration column which was prepared previously. The
recovered from the control and the neutralized test substance solution is pushed through the gel filtration column utilizing
for the neutralization to be successful. In case of incomplete the syringe plunger or by centrifugation. This suspension may
–1
neutralization, either another neutralizer may be needed or the be considered the 10 dilution of the virus. See Practice
gel filtration method (Test Method E1482) may be used. If gel E1482.
filtration columns are used as the only neutralization method,
9.5 Test for Virucidal Activity—For each lot of the test
the test substance will be added to a previously prepared gel
substance, treat a dried film carrier with 2.0 mL of the use-
filtration column. The solution is pushed through the column
dilution of a liquid product or the amount of product released
utilizing the syringe plunger or by centrifugation. Then a
during recommended use of the aerosol or trigger spray. Hold
relatively low number (for example, 1000 to 5000) infective
for the required contact time. Upon completion of the contact
units of the test virus will be added to the eluate; and the
time,immediatelyaddanequalvolumeofneutralizer(2.0mL)
mixturesheldforatleastthecontacttimeatroomtemperature.
to the carrier and mix well. Scrape the film to resuspend the
Titrate the mixtures for infectious virus. Comparable levels of
virus/test substance/neutralizer mixture. If gel filtration col-
infective units must be recovered from the control and the
umns will be used in addition to the liquid neutralizer,
post-column eluate for the neutralization to be successful.
followingscrapingthesuspensionwillbeaddedtoapreviously
9.4 Plate Recovery Control—Vortex the virus suspension prepared gel filtration column. The solution is pushed through
thoroughly and place 0.2-mL on the inside bottom surface of
the gel filtration column utilizing the syringe plunger or by
each glass petri dish. Allow the virus inoculum to dry under centrifugation If gel filtration columns are used as the only
ambient conditions in a laminar flow hood or other suitable
neutralization method, the test substance will be added to a
chamberwiththepetridishcoverremoved.Thedryingtimeof previously prepared gel filtration column. The solution is
this control should be consistent with that for the test runs. A
pushed through the column utilizing the syringe plunger or by
4.8-6.3
recovery of at least 10 infective units/control dish should
centrifugation. Prepare serial 10-fold dilutions using diluent
beachievedforthetesttobeconsideredvalid.Poolsofcertain
and inoculate an amount appropriate to the test format to no
types of viruses may require concentration by ultra-
less than four replicate cell monolayers/dilution starting from
centrifugation to obtain titers high enough to give a minimum the first ten-fold dilution of the post-neutralized sample.
4.8
of 10 infective units/dish after the inoculum has been dried.
However,anysuchconcentratedvirusmustbevortexedwellto
10. Soil Load (refer to Test Method E2197)
reduce the presence of viral aggregates. If the plate recovery
10.1 Fetal Bovine Serum, Horse Serum, or other animal
6.3
control has a virus endpoint titer of >10 infective units/
serum as desired, may be used depending on the target
control dish the virus culture may be standardized by dilution
regulatory agency. The soil load mixture given in 10.2 – 10.7
4.8 6.3
to target a virus endpoint titer of 10 to 10 infective
contains a level of protein roughly equal to that in 5% ser
...

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: E1053 − 11 E1053 − 20
Standard Test Method Practice to
Assess Virucidal Activity of Chemicals Intended for
Disinfection of Inanimate, Nonporous Environmental
Surfaces
This standard is issued under the fixed designation E1053; 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 used to evaluate the virucidal efficacy of liquid, aerosol, or trigger-spray microbicides intended for use
on inanimate, nonporous environmental surfaces. This test method may be employed with most viruses, which can be grown in
cultured cells. However, other host systems (for example, embryonic eggs) may be used with proper justification and
documentation.
1.2 This test method should be performed only by those trained in microbiological and virological techniques in facilities
designed and equipped for work with infectious agents at the appropriate biosafety level.
1.3 It is the responsibility of the investigator to determine whether Good Laboratory Practice regulations (GLPs) are required
and to follow them where appropriate (40 CFR, Part 160 for EPA submissions and 21 CFR, Part 58 for FDA submissions). Refer
to the appropriate regulatory agency for performance standards of virucidal efficacy.
1.4 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. The user should consult a reference for laboratory safety recommendations.
2. Referenced Documents
2.1 ASTM Standards:
D1129 Terminology Relating to Water
E1153 Test Method for Efficacy of Sanitizers Recommended for Inanimate, Hard, Nonporous Non-Food Contact Surfaces
E1482 Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization
E2197 Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and
Sporicidal Activities of Chemicals
2.2 Federal Standards:
Title 21, Code of Federal Regulations (CFR), Food and Drug Administration, Part 58, Laboratory Practice for Nonclinical
Laboratory Studies
Title 40, Code of Federal Regulations (CFR), Environmental Protection Agency, Subchapter E, Pesticide Programs; Part 160,
Good Laboratory Practice Standards
3. Terminology
3.1 Definitions—For definitions of general terms used in this test method, refer to Terminology D1129.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 hard water, n—water with a standard hardness as calcium carbonate.
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 Jan. 1, 2011Jan. 1, 2020. Published March 2011March 2020. Originally approved in 1985. Last previous edition approved in 20022011 as
E1053 – 97 (2002).E1053 – 11. DOI: 10.1520/E1053-11.10.1520/E1053-20.
Public Health Service, Centers for Disease Control and Prevention, and National Institutes of Health, Biosafety in Microbiological and Biomedical Laboratories, U.S.
Department of Health and Human Services, Washington, DC, December 2009, 422 pp.
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.
Available from U.S. Government Printing Office Superintendent of Documents, 732 N. Capitol St., NW, Mail Stop: SDE, Washington, DC 20401, http://
www.access.gpo.gov.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1053 − 20
3.2.2 neutralization, n—a process which results in quenching the microbicidal activity of a test substance. This may be achieved
through dilution of the test substance to reduce the microbicidal activity, or through the use of chemical agents, called neutralizers,
to eliminate microbicidal activity.
3.2.3 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.
3.2.4 test substance or test formulation, n—a formulation which incorporates microbicidal ingredients.
4. Summary of Test Method
4.1 The virus suspension is dried on an inanimate, nonporous surface. The test substance is added over the dried film at its
use-dilution or sprayed from an aerosol can or trigger-sprayer following the manufacturer’s directions. Control carriers receive an
equivalent volume of a buffer harmless to the test virus and its host cells. After exposure at the appropriate temperature (usually
22 6 2°C) for the recommended time, the eluates from control and test carriers are assayed for infectivity.
4.2 This test method is designed to be performed by a person trained in culturing and assaying infectious viruses who is
responsible for choosing the appropriate host system for the test virus and applying the techniques necessary for propagation and
maintenance of the host system and test virus. For a reference text, refer to Lennette et al.
5. Significance and Use
5.1 This test method may be used to determine the effectiveness of liquid, aerosols/foams, and trigger-spray products against
designated prototype viruses.
5.2 The number of lots of the test substance and the number of replicates in each test will depend on the requirements of the
target regulatory agency.
5.3 Certain regulatory agencies may require additional testing using other carrier tests for product registration purposes.
6. Materials and Reagents
6.1 Host System and Assay of Infectious Virus—See Note 2.
6.1.1 Cell Cultures, appropriate for test virus.
6.1.2 Growth and Maintenance Media, any growth and maintenance media suitable for work with the virus and its host cells.
NOTE 1—Materials and reagents for cell culture may be purchased from biological supply houses.
6.1.3 Diluent, Earle’s Balanced Salt Solution (EBSS) or other appropriate media.
6.1.4 Plastic Cell Culture Ware.
NOTE 2—Plastic cell culture ware may be purchased from most laboratory supply houses.
6.1.5 Incubator, capable of maintaining 36 6 1°C or other temperature appropriate for replication of the specific test virus; an
incubator with 5 to 7 % CO will be needed if an open system is being used for cell culture and virus assay.
6.1.6 Refrigerator, 4 6 2°C.
6.1.7 Test Tubes, screw-capped.
6.1.8 Pipettes, serological, 10, 1, and 0.5 mL.
6.1.9 Micropipettors and Tips, (96-well assay only).
6.1.10 96-Well Dilution Plates, (96-well assay only).
6.1.11 Microtitration Kit.
NOTE 3—Microtitration kits may be purchased from most laboratory supply houses.
6.1.12 Petri Plates, glass, 100-mm diameter, 1-cm deep.
7. Test Viruses
7.1 Appendix X1 lists viruses and their respective host cells as examples for use in this test method.
7.2 To demonstrate that the test substance has broad virucidal activity, it should be shown to be effective against at least one
non-enveloped virus.
8. Virus Stock
8.1 The titer of the test virus suspension must be sufficiently high so that at least 10 infective units/carrier can be recovered
from the inoculated carriers after the inoculum has dried. The host system employed for virus propagation need not be the same
as that used for virus recovery and the infectivity assay.
Schmidt, N. J., , Lennette, D. A., Lennette, E. T., and Lennette, E. H. eds., Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, Seventh Edition,
American Public Health Association, Washington, DC, 1995.
E1053 − 20
9. Operating Technique
9.1 Test Substance Diluent—For test substances requiring dilution in water to obtain a use-dilution, water with a standardized
and specified level of hardness, such as 400 ppm as CaCO , shall be used as the diluent.
9.2 Cytotoxicity Control—The objective of this control is to (1) determine the dilution of the test substance post-neutralization
at which it causes no apparent degeneration (cytotoxicity) of the cell line to be used for measuring virus infectivity and (2) assess
if the neutralizer in any way reduces or enhances such cytotoxicity. Make an initial 1:2 dilution of the use-dilution of the test
substance in the neutralizer and three further ten-fold dilutions of the neutralized test substance in the diluent. Remove the culture
medium from the monolayers of the host cell line(s) and put into each test monolayer separately the same volume of inoculum
as used in virus titration; control monolayers receive an equivalent amount of diluent (without any neutralizer) only. Another set
of monolayers should be exposed to the neutralizer alone. Hold the cultures for the same period of time as used in virus titration
and examine them under an inverted microscope for any cytotoxicity. In case of cytotoxicity, a different neutralizer or gel filtration
(see Test Method E1482) of the neutralized virus-test substance mixture may be needed.
NOTE 4—If gel filtration is used in the virucidal activity test runs, the neutralized and gel-filtered test substance should be evaluated for the cytotoxicity
control.
9.3 Test Substance Neutralization Control—To determine the dilution at which neutralization of the test substance has occurred,
prepare and inoculate an additional set of cytotoxicity controls with the neutralizer added to the test substance. To validate the
neutralization, add equal volumes of the neutralized test substance, the neutralizer alone and a control fluid (for example, PBS)
a relatively low number (for example, 1000 to 5000) infective units of the test virus and hold the mixtures for 10 to 20 min at room
temperature. Titrate the mixtures for infectious virus. Comparable levels of infective units must be recovered from the control, the
neutralizer alone, and the neutralized test substance for the neutralization to be successful. In case of incomplete neutralization,
either another neutralizer may be needed or the gel filtration method (Test Method E1482) may be used.
9.4 Plate Recovery Control—Vortex the virus suspension thoroughly and place 0.2-mL on the inside bottom surface of each
glass petri dish. Allow the virus inoculum to dry under ambient conditions in a laminar flow hood or other suitable chamber with
the petri dish cover removed. The drying time of this control should be consistent with that for the test runs. A recovery of at least
10 infective units/control dish should be achieved for the test to be considered valid. Pools of certain types of viruses may require
concentration by ultra-centrifugation to obtain titers high enough to give a minimum of 10 infective units/dish after the inoculum
has been dried. However, any such concentrated virus must be vortexed well to reduce the presence of viral aggregates.
NOTE 5—The volume of virus inoculum per carrier may be increased depending on the titer of the virus. This volume must be consistent between the
plate recovery control and test substance runs. It should be noted, however, that an increased volume will prolong the drying of the inoculum and may
lead to increased losses in virus infectivity.
9.4.1 After drying, overlay each dried control film with 2.0 mL of PBS or another buffered solution harmless to the virus and
its host cells. Let stand for the same contact time as used for the test carriers and then add an equal amount of neutralizer (2.0 mL).
–1
Scrape the inside bottom surface with a sterile cell scraper to resuspend the viral film. This suspension may be considered the 10
dilution of the virus. Prepare serial 10-fold dilutions using diluent and inoculate an amount appropriate to the test format to no less
than four replicate cell monolayers/dilution.
9.5 Test for Virucidal Activity—For each lot of the test substance, treat a dried film carrier with 2.0 mL of the use- dilution of
a liquid product or the amount of product released during recommended use of the aerosol or trigger spray. Hold for the required
contact time. Upon completion of the contact time, immediately add an equal volume of neutralizer (2.0 mL) to the carrier and
–1
mix well. Scrape the film to resuspend the virus/test substance/neutralizer mixture. This suspension may be considered the 10
dilution of the virus. Prepare serial 10-fold dilutions using diluent and inoculate an amount appropriate to the test format to no less
–2
than four replicate cell monolayers/dilution starting from 10 .
10. Soil Load (refer to Test Method E2197)
10.1 The soil load to be incorporated in the suspension of the test organism may consist of a mixture of the following stock
solutions in phosphate buffer (pH 7.2):
10.2 Add 0.5 g of tryptone or yeast extract to 10 mL of phosphate buffer.
10.3 Add 0.5 g of BSA to 10 mL of phosphate buffer.
10.4 Add 0.04 g of bovine mucin to 10 mL of phosphate buffer.
10.5 Prepare the solutions separately and sterilize by passage through a 0.22-μm pore diameter membrane filter, aliquot, and
store at either 4 6 2°C or –20 6 2°C; such solutions can be stored in the refrigerated for about three months and frozen for at
least one year.
AOAC International, Offıcial Methods of Analysis of the AOAC, Arlington, VA, 1990.
E1053 − 20
10.6 To obtain 500 μL of the inoculum, add 25 μL of BSA, 100 μL of mucin, and 35 μL of tryptone or yeast extract stock to
340 μL of the virus suspension.
NOTE 6—Other types of soil load such as animal sera may be used depending on the target regulatory agency and recommended use of the test
substance. The soil load mixture given above contains a level of protein roughly equal to that in 5 % serum. Preliminary screening of albumin and mucin
is recommended to ensure compatibility with test organism(s).
10.7 If the use-dilution of the test substance is to be prepared in water, follow the methods listed in Test Method E1153 for
making the hard water to be used. The test report must clearly specify the level of hardness used in testing for virucidal activity.
11. Additional Controls
11.1 Cell Culture Control—To ensure that the host cells are not contaminated with bacteria, fungi, or any cytopathogenic viruses
other than those used in the test and to confirm the viability of the cells during the incubation period of the assay, at least four 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 virus titration.
11.2 Virus Stock Titer Control—An aliquot of the stock virus used in the test is serially diluted in diluent and inoculated onto
the host culture. This is to confirm that the host cells are susceptible to the virus and that the viral infection assay is performed
appropriately. This control will also confirm that the titer of the stock virus is appropriate for use in the test. A lack of obvious and
typical virus-induced cytopathic effects in the lower dilutions would invalidate the test.
11.3 Control for Interference with Virus Infectivity (optional)—Levels of the test substance which show no obvious cytotoxicity
could still reduce or enhance the ability of the challenge virus to infect or replicate in host cells, thus interfering with the estimation
of its virucidal activity. An interference control should, therefore, be included to rule out such a possibility. Remove the culture
medium from the host cells and inoculate each one of the test monolayers with the same volume of inoculum as used in virus
titration and a 1:10 dilution of the test substance in the neutralizer. Controls receive PBS alone (without the neutralizer). Another
set of monolayers should be exposed to the neutralizer alone. Hold the monolayers at room temperature for 30 to 60 min and
inoculate each with a low number (for example, 20 to 50) of infective units of the challenge virus. Incubate the monolayers for
virus adsorption, place maintenance medium in the cultures, incubate them for the time required for virus replication and examine
them for cytopathology or foci of virus infection. Any significant difference in virus infectivity compared to the control is indicative
of the test substance’s or the neutralizer’s ability to affect the virus susceptibility of the host cells. In such a case, a different
neutralizer or alternative approaches to the removal of the residual test product in the samples to be titrated for virus infectivity
may be needed.
12. Calculation
12.1 When a most probable number (MPN) assay for virus titration is employed, use the method of Reed and Muench or
Spearman-Kärber to calculate reductions in virus infectivity.
12.2 Report the titer of the stock virus, titer of recovered virus (plate recovery control), degree of cytotoxicity (cytotoxicity
control), the degree of virus inactivation (results of virucide test), and the dilution at which neutralization and absence of viral
interference occurred (neutralization/viral interference control).
13. Precision and Bias
13.1 A precision and bias statement cannot be made for this test method at this time.
14. Keywords
14.1 carrier test; cell or other tissue cultures; disinfectant; infection control; microbicide; surface test; virucidal test; virus;
viruses on dried environmental surfaces
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APPENDIX
(Nonmandatory Information)
X1. RECOMMENDED TEST VIRUS STRAINS AND THEIR HOST CELLS
X1.1 ATCC numbers are given, when available, in parentheses.
X1.1.1 Adenovirus, Type 4 (VR-4) or Type 5 (VR-5). Cell line options for making virus pools and infectivity titrations are 293
(CRL-1573) and Vero (CCL-81) cells, respectively.
X1.1.2 Canine parvovirus, Cornell-780916-80 strain, (VR-2017). Cell line: A-72 (CRL-1542).
X1.1.3 Cytomegalovirus, strain AD-169 (VR-538). Cell line: MRC-5 (ATCC CCL-171) orWI-38 (CCL-75).
X1.1.4 Feline calicivirus, strain F9 (VR-782). Cell line: CRFK (CCL-94).
X1.1.5 Hepatitis A Virus, HM-175 strain (VR-2093). Cell line: Rhesus monkey kidney - FRhK-4 (CRL-1688).
X1.1.6 Herpes Simplex Virus, Type 1, strain F (1) (VR-733). Cell line: Vero (CCL-81), HEp-2 (CLL-23), primary rabbit kidney.
X1.1.7 Influenza A, Hong Kong Strain (VR-544); PR-8 (VR-95). Cell line: Canine kidney (MDCK); established (e.g., LLC-MK2)
or primary monkey kidney cells.
NOTE X1.1—Animal serum may be inhibitory to influenza viruses and also to trypsin used for influenza virus culture.
X1.1.8 Murine Norovirus, Cell line: RAW 264.7 (TIB-71).
X1.1.9 Respiratory Syncytial Virus, Long strain (VR-26). Cell line: HEp-2, MRC-5 (CCL-171), HeLa (CCL-2).
X1.1.10 Rhinovirus, type 37, strain 151-1 (VR-1147); Rhinovirus type 14 (VR-284). Cell line: MRC-5 (CCL-171), WI-38
(CCL-75), H1-HeLa (CRL-1958).
X1.1.11 Rotavirus, Wa strain, (VR-2018). Cell line: Rhesus monkey kidney (MA-104) (CRL-2378) or African green monkey
kidney - CV-1 (CCL-70).
NOTE X1.2—Animal serum may be inhibitory to rotaviruses and also to trypsin used for rotavirus culture.
X1.1.12 Vaccinia Virus, WR strain (VR-119). Cell line: Vero (CCL-81), HEp-2 (CLL23).
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1. Scope
1.1 This practice is used to evaluate the virucidal efficacy of liquid, aerosol, or trigger-spray microbicides intended for use on
inanimate, nonporous environmental surfaces. This practice may be employed with most viruses, which can be grown in cultured
cells. However, other host systems (for example, embryonic eggs) may be used with proper justification and documentation.
1.2 This practice should be performed only by those trained in microbiological and virological techniques in facilities designed
and equipped for work with infectious agents at the appropriate biosafety level.
1.3 It is the responsibility of the investigator to determine whether Good Laboratory Practice regulations (GLPs) are required
and to follow them where appropriate (40 CFR, Part 160 for EPA submissions and 21 CFR, Part 58 for FDA submissions). Refer
to the appropriate regulatory agency for performance standards of virucidal efficacy.
1.4 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.
1.5 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:
D1129 Terminology Relating to Water
E1153 Test Method for Efficacy of Sanitizers Recommended for Inanimate, Hard, Nonporous Non-Food Contact Surfaces
E1482 Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization
E2197 Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and
Sporicidal Activities of Chemicals
2.2 Federal Standards:
Title 21, Code of Federal Regulations (CFR), Food and Drug Administration, Part 58, Laboratory Practice for Nonclinical
Laboratory Studies
Title 40, Code of Federal Regulations (CFR), Environmental Protection Agency, Subchapter E, Pesticide Programs; Part 160,
Good Laboratory Practice Standards
3. Terminology
3.1 Definitions—For definitions of general terms used in this test method, refer to Terminology D1129.
3.2 Definitions of Terms Specific to This Standard:
3.2.1 hard water, n—water with a standard hardness as calcium carbonate.
3.2.2 neutralization, n—a process which results in quenching the microbicidal activity of a test substance. This may be achieved
through dilution of the test substance to reduce the microbicidal activity, or through the use of chemical agents, called neutralizers,
to eliminate microbicidal activity.
3.2.3 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.
3.2.4 test substance or test formulation, n—a formulation which incorporates microbicidal ingredients.
4. Summary of Practice
4.1 The virus suspension is dried on an inanimate, nonporous surface. The test substance is added over the dried film at its
use-dilution or sprayed from an aerosol can or trigger-sprayer following the manufacturer’s directions. Control carriers receive an
equivalent volume of a buffer harmless to the test virus and its host cells. After exposure at the appropriate temperature (usually
20 6 2 °C) for the recommended time, the eluates from control and test carriers are assayed for infectivity.
4.2 This practice is designed to be performed by a person trained
...