ASTM E1052-20

Standard Practice to Assess the Activity of Microbicides against Viruses in Suspension

SIGNIFICANCE AND USE
5.1 This practice is to determine if a test substance can inactivate viruses in suspension.  
5.2 Regulatory agencies may require additional testing using in vitro (Practice E1053, Test Method E2197) or in vivo (Test Method E1838) carrier tests for product registration purposes.
SCOPE
1.1 This practice is intended to demonstrate the virucidal activity of test substances with viruses in suspension.  
1.2 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).  
1.3 Refer to the appropriate regulatory agency for performance standards of virucidal efficacy.  
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. The user should consult a reference for the laboratory safety recommendations.2  
1.6 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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 E1052-11 - Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension
• Effective Date: 01-Jan-2020
• Refers: ASTM E1053-20 - Standard Practice to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental Surfaces
• Effective Date: 01-Jan-2020
• 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 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 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 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 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 E1053-11 - Standard Test Method to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental Surfaces
• Effective Date: 01-Jan-2011
• 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

Overview

ASTM E1052-20 is the internationally recognized standard practice developed by ASTM International for assessing the activity of microbicides against viruses in suspension. This standard provides systematic procedures to determine whether a test substance possesses virucidal activity in a controlled laboratory environment. Its application is crucial for the evaluation of disinfectants, sanitizers, and other microbicidal products intended to combat viral pathogens. Regulatory agencies often refer to ASTM E1052-20 as a key method for establishing product efficacy as part of registration or approval processes.

Key Topics

• Virucidal Activity Assessment: The standard outlines the protocols for evaluating the effectiveness of microbicides on viruses suspended in solution, simulating real-life contamination events.
• Good Laboratory Practices (GLP): Investigators must determine if GLP compliance is required under regulations such as 40 CFR Part 160 (EPA) or 21 CFR Part 58 (FDA), and follow them as appropriate.
• Safety and Compliance: Users of ASTM E1052-20 are responsible for ensuring laboratory safety, environmental protection, and compliance with relevant regulations before conducting tests.
• Standardization of Test Parameters: The standard prescribes SI units for all measurements, ensuring global consistency in results. It calls for the use of appropriate negative and positive controls, and careful measurement of parameters such as temperature, contact time, and virus-host combinations.
• Neutralization Procedures: Guidance is provided on the neutralization of microbicidal activity post-exposure to prevent ongoing action after the intended contact period.
• Viruses and Host Systems: A list of representative enveloped and non-enveloped viruses and corresponding host cell lines is included for validating microbicidal spectrum claims.
• Control and Replicates: The practice emphasizes the importance of multiple replicates and controls (virus control, cytotoxicity, and neutralization control) to validate the outcomes.

Applications

ASTM E1052-20 is widely adopted in several practical scenarios, including:

• Product Registration and Regulatory Approval: Manufacturers of disinfectants and microbicidal formulations use this standard to generate efficacy data for submission to regulatory agencies globally.
• Quality Control in Manufacturing: The practice supports internal quality assurance of antiviral products by providing a robust framework for periodic batch testing.
• Healthcare & Public Health: Cleaners, sanitizing products, and handrubs intended for use in healthcare settings are assessed using this method to confirm their ability to inactivate a spectrum of viruses in suspension.
• Research and Development: R&D laboratories utilize the standard for the development and comparative assessment of new antiviral chemicals and formulations.

Related Standards

To provide a comprehensive approach to virucidal testing, ASTM E1052-20 references and complements several other standards, including:

• ASTM E1053: Practice to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental Surfaces.
• ASTM E2197: Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and Sporicidal Activities of Chemicals.
• ASTM E1838: Test Method for Determining the Virus-Eliminating Effectiveness of Hygienic Handwash and Handrub Agents Using the Fingerpads of Adults.
• ASTM E1482: Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization.
• ASTM E2756: Terminology Relating to Antimicrobial and Antiviral Agents.
• Regulatory References: U.S. EPA 40 CFR Part 160 and FDA 21 CFR Part 58 for Good Laboratory Practice.

ASTM E1052-20 plays a pivotal role in the global effort to standardize the evaluation of microbicidal and virucidal activities, promoting reliable comparison of products and ensuring effective control of viral contamination in various industries. Utilize this standard to meet regulatory demands and to ensure scientific credibility in antiviral product claims.

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: E1052 − 20
Standard Practice to
Assess the Activity of Microbicides against Viruses in
Suspension
This standard is issued under the fixed designation E1052; 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 E1053 Practice to Assess Virucidal Activity of Chemicals
Intended for Disinfection of Inanimate, Nonporous Envi-
1.1 This practice is intended to demonstrate the virucidal
ronmental Surfaces
activity of test substances with viruses in suspension.
E1482 Practice for Use of Gel Filtration Columns for Cyto-
1.2 It is the responsibility of the investigator to determine
toxicity Reduction and Neutralization
whether Good Laboratory Practice regulations (GLPs) are
E1838 Test Method for Determining the Virus-Eliminating
required and to follow them where appropriate (40 CFR, Part
Effectiveness of Hygienic Handwash and HandrubAgents
160 for EPA submissions and 21 CFR, Part 58 for FDA
Using the Fingerpads of Adults
submissions).
E2197 Quantitative Disk Carrier Test Method for Determin-
ing Bactericidal, Virucidal, Fungicidal, Mycobactericidal,
1.3 Refer to the appropriate regulatory agency for perfor-
mance standards of virucidal efficacy. and Sporicidal Activities of Chemicals
E2756 Terminology Relating to Antimicrobial and Antiviral
1.4 The values stated in SI units are to be regarded as
Agents
standard. No other units of measurement are included in this
2.2 Federal Standards:
standard.
21 CFR Code of Federal Regulations (CFR), Food and Drug
1.5 This standard does not purport to address all of the
Administration, Part 58, Laboratory Practice for Nonclini-
safety concerns, if any, associated with its use. It is the
cal Laboratory Studies
responsibility of the user of this standard to establish appro-
40 CFR Code of Federal Regulations (CFR), Environmental
priate safety, health, and environmental practices and deter-
Protection Agency, Part 160, Good Laboratory Practice
mine the applicability of regulatory limitations prior to use.
Standard
The user should consult a reference for the laboratory safety
recommendations.
3. Terminology
1.6 This international standard was developed in accor-
3.1 Definitions—Fordefinitionsofgeneraltermsusedinthis
dance with internationally recognized principles on standard-
test method, refer to Terminology E2756.
ization established in the Decision on Principles for the
Development of International Standards, Guides and Recom-
3.2 Definitions of Terms Specific to This Standard:
mendations issued by the World Trade Organization Technical
3.2.1 hard water, n—water with a standard hardness as
Barriers to Trade (TBT) Committee.
calcium carbonate.
3.2.2 neutralization, n—the process for inactivating or
2. Referenced Documents
quenchingtheactivityofamicrobicide,oftenachievedthrough
2.1 ASTM Standards:
physical(forexample,filtrationordilution)orchemicalmeans.
3.2.2.1 Discussion—This neutralization may be achieved
through dilution of the test substance to reduce the microbi-
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides,
cidal activity, or through the use of chemical agents, called
Antimicrobials, and Alternative Control Agents and is the direct responsibility of
neutralizers, to eliminate microbicidal activity.
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Jan. 1, 2020. Published March 2020. Originally
3.2.3 soil load, n—a solution of one or more organic and/or
approved in 1985. Last previous edition published in 2011 as E1052 – 11. DOI:
inorganic substances added to the suspension of the test
10.1520/E1052-20.
CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories, Fifth
organism to simulate the presence of body secretions,
Edition, U.S. Department of Health and Human Services, Washington, DC, May
excretions, or other extraneous substances.
2009.
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 Available from U.S. Government Printing Office, Superintendent of
the ASTM website. Documents, Washington, DC 20402.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1052 − 20
NOTE 3—Microtitration kit may be purchased from most laboratory
3.2.4 test substances or test formulation, n—a formulation
supply houses.
which incorporates microbicidal ingredients.
6.2 Additional or equivalent materials and reagents specific
4. Summary of Practice
to the host recovery system may be necessary. The trained
microbiologist or virologist is responsible to choose accord-
4.1 One part of the virus suspension is added to nine parts
ingly as needed.
of the test substance, the mixture held at the desired tempera-
ture for the required contact time and then assayed for viable
7. Test Viruses
virus in an appropriate host system. For control, one part of the
virusisaddedtoninepartsofabufferharmlesstothevirusand
7.1 To demonstrate the spectrum of virucidal activity of the
its host cells. Cell culture, cytotoxicity, and virus susceptibility
test substance, it should be tested against viruses with varying
controls must also be included in each test.
levels of resistance to microbicides. Appendix X1 lists sug-
gested viruses and their host cells.
4.2 This practice must be performed by a trained microbi-
ologist or virologist who is responsible for choosing the
8. Virus Stock
appropriate host system for the test virus, and applying the
techniques necessary for propagation and maintenance of host
8.1 Use an appropriate host to prepare virus suspensions.
cell lines and test virus. For a reference text, refer to Schmidt
The host system for titrating virus infectivity may be different
et al.
from that used for preparing the virus pool. A virus endpoint
titer of at least 10 infective units should be recovered for the
5. Significance and Use
test to be considered valid. Pools of certain types of viruses
may require concentration by ultra-centrifugation to obtain
5.1 This practice is to determine if a test substance can
titers high enough to give a minimum of 10 infective units.
inactivate viruses in suspension.
However,anysuchconcentratedvirusmustbevortexedwellto
5.2 Regulatory agencies may require additional testing us-
reduce the presence of viral aggregates.
ing in vitro (Practice E1053, Test Method E2197)or in vivo
(Test Method E1838) carrier tests for product registration NOTE 4—If the virus control has a virus endpoint titer of >10 infective
units the virus culture may be standardized by dilution to target a virus
purposes.
4 6
endpoint titer of 10 to 10 infective units.
6. Materials and Reagents
9. Operating Technique
6.1 Cell Culture Technique.
9.1 The test must include the parameters given in Table 1.
6.1.1 Cell Culture System appropriate for test virus.
6.1.2 Growth Media/Maintenance Media, Eagle’s minimal 9.2 Please refer to Test Method E2197 for details on
essential medium (EMEM) or equivalent, supplemented with
cytotoxicity and other controls.
appropriate concentration of serum (inactivated and
9.3 Thoroughly mix virus suspension and then add one part
mycoplasma-free), antibiotics, and other growth factors as
to nine parts of the test substance in a sterile medication tube
needed. See Note 1.
held at the appropriate exposure temperature (usually
–1
NOTE1—Materialsandreagentsforcellculturemaybepurchasedfrom 22 6 2
...

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: E1052 − 11 E1052 − 20
Standard Test Method Practice to
Assess the Activity of Microbicides against Viruses in
Suspension
This standard is issued under the fixed designation E1052; 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 intended to demonstrate the virucidal activity of test substances with viruses in suspension.
1.2 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).
1.3 Refer to the appropriate regulatory agency for performance standards of virucidal efficacy.
1.4 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.
1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory
limitations prior to use. The user should consult a reference for the laboratory safety recommendations.
2. Referenced Documents
2.1 ASTM Standards:
E1053 Practice to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental
Surfaces
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
E2197 Quantitative Disk Carrier Test Method for Determining Bactericidal, Virucidal, Fungicidal, Mycobactericidal, and
Sporicidal Activities of Chemicals
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
2.2 Federal Standards:
21 CFR Code of Federal Regulations (CFR), Food and Drug Administration, Part 58, Laboratory Practice for Nonclinical
Laboratory Studies
40 CFR Code of Federal Regulations (CFR), Environmental Protection Agency, Part 160, Good Laboratory Practice Standard
3. Terminology
3.1 Definitions—For definitions of general terms used in this test method, refer to Terminology E2756.
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—the process for inactivating or quenching the activity of a microbicide, often achieved through physical
(for example, filtration or dilution) or chemical means.
3.2.2.1 Discussion—
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 Oct. 1, 2011Jan. 1, 2020. Published October 2011March 2020. Originally approved in 1985. Last previous edition published in 20022011 as
E1052 – 96 (2002)E1052 – 11., which was withdrawn in July 2011 and reinstated in October 2011. DOI: 10.1520/E1052-11. DOI: 10.1520/E1052-20.
CDC-NIH, Biosafety in Microbiological and Biomedical Laboratories, Fifth Edition, U.S. Department of Health and Human Services, Washington, DC, May 2009.
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, Washington, DC 20402.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1052 − 20
This neutralization 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 substances or test formulation, n—a formulation which incorporates microbicidal ingredients.
4. Summary of Test Method
4.1 One part of the virus suspension is added to nine parts of the test substance, the mixture held at the desired temperature
for the required contact time and then assayed for viable virus in an appropriate host system. For control, one part of the virus is
added to nine parts of a buffer harmless to the virus and its host cells. Cell culture, cytotoxicity, and virus susceptibility controls
must also be included in each test.
4.2 This test method must be performed by a trained microbiologist or virologist who is responsible for choosing the appropriate
host system for the test virus, and applying the techniques necessary for propagation and maintenance of host cell lines and test
virus. For a reference text, refer to Schmidt et al.
5. Significance and Use
5.1 This test method is to determine if a test substance can inactivate viruses in suspension.
5.2 Regulatory agencies may require additional testing using in vitro (Test Methods E1053, E2197) or in vivo (Test Method
E1838) carrier tests for product registration purposes.
6. Materials and Reagents
6.1 Cell Culture Technique.
6.1.1 Cell Culture System appropriate for test virus.
6.1.2 Growth Media/Maintenance Media, Eagle’s minimal essential medium (EMEM) or equivalent, supplemented with
appropriate concentration of serum (inactivated and mycoplasma-free), antibiotics, and other growth factors as needed. See Note
1.
NOTE 1—Materials and reagents for cell culture may be purchased from biological supply houses.
6.1.3 Diluent, The media listed in 6.1.2, phosphate buffered saline, trypticase soy broth supplemented with serum, Earle’s
Balanced Salt Solution (EBSS), or other similar buffered solutions.
6.1.4 Plastic Cell Culture Ware. See Note 2.
NOTE 2—Plastic cell culture ware may be purchased from most laboratory supply houses.
6.1.5 Incubator, with a 5 to 7 % CO atmosphere, capable of maintaining 36 6 1°C or other temperature appropriate for the
specific test virus.
6.1.6 Refrigerator, 4 6 2°C or other appropriate temperature.
6.1.7 Test Tubes, screw-capped.
6.1.8 Pipettes, serological, 10, 1, 0.5 mL or calibrated pipettors, or both.
6.1.9 Microtitration Kit. See Note 3.
NOTE 3—Microtitration kit may be purchased from most laboratory supply houses.
6.2 Additional or equivalent materials and reagents specific to the host recovery system may be necessary. The trained
microbiologist or virologist is responsible to choose accordingly as needed.
7. Test Viruses
7.1 To demonstrate the spectrum of virucidal activity of the test substance, it should be tested against viruses with varying levels
of resistance to microbicides. Appendix X1 lists suggested viruses and their host cells.
8. Virus Stock
8.1 Use an appropriate host to prepare virus suspensions. The host system for titrating virus infectivity may be different from
that used for preparing the virus pool.
9. Operating Technique
9.1 The test must include the parameters given in Table 1.
9.2 Please refer to Test Method E2197 for details on cytotoxicity and other controls.
Schmidt, N. J., Lennette, D. A., and Lennette, E. T., and Lennette, E. H.,eds., Diagnostic Procedures for Viral, Rickettsial and Chlamydial Infections, 7th Edition, Am.
Pub. Hlth. Assoc., Washington, DC, 1995 .
E1052 − 20
TABLE 1 Parameters
Parameter Summary Replicates
Cell culture medium alone 4/group
Virus control 1 part virus + 9 parts medium 4/dilution
Virucidal test 1 part virus + 9 parts test substance 4/dilution
Cytotoxicity control 1 part medium + 9 parts test 4/dilution
substance
Neutralization control neutralized test substance + virus 4/dilution
9.3 Thoroughly mix virus suspension and then add one part to nine parts of the test substance in a sterile medication tube held
–1
at the appropriate exposure temperature (usually 22 6 2°C). Consider this the 10 dilution of the virus. Following the exposure
for the time chosen, immediately neutralize the microbicidal activity by serial ten-fold dilutions into a neutralization solution
appropriate for the test substance.
NOTE 4—Perform the virus control (one part of virus + nine parts EBSS) and cytotoxicity control (one part EBSS + nine parts test substance)
concurrently with the virucidal test described above. If dilution alone is insufficient to reduce cytotoxicity, gel filtration as described in Test Method E1482
may be used.
9.4 Virus Recovery—Inoculate at least four cell culture monolayers per dilution of the virus-test substance mixture with 0.1 mL
volumes of each test and control dilution separately onto monolayers of appropriate host cell cultures. Other volumes may be used
depending on the type of cell culture vessel employed; however, no less than four separate monolayers of the host cells must be
inoculated for each dilution tested. Incubate the cultures at the appropriate temperature and observe for evidence of virus
replication (e.g., cytopathic effects, hemagglutination, plaque assay) and/or cytotoxicity
9.5 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.
9.6 To validate the neutralization, add equal volumes of the neutralized test substance, the neutralizer alone and a control fluid
(for example, EBSS) 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 as well as the neutralized test substance for the neutralization to be considered
valid. In case of incomplete neutralization, try another neutralizer or use the gel filtration method (Test Method E1482) to reduce
cytotoxicity.
9.6.1 Those dilutions that are toxic to the cells or do not exhibit virus replication, or both, are not included in the log reduction
calculations of microbicidal activity.
10. Soil Load and Hard Water
10.1 If a soil load is required, add to the virus suspension bovine serum at a final concentration of 5 % or the tripartite mixture
as described in Test Method E2197.
10.2 If tests are to be performed in water of a specific hardness, follow the methods listed in Test Method E2197.
11. Calculation of Results
11.1 Use an appropriate method to calculate the control and test samples to determine the level of virus inactivation achieved
in relation to the dilution at which cytotoxicity was observed.
11.2 Report the titer of the stock virus, degree of cytotoxicity, the degree of virus inactivation, and the dilution at which
neutralization occurred.
12. Precision and Bias
12.1 A precision and bias statement cannot be made for this test method at this time.
13. Keywords
13.1 cell cultures; microbicide; suspension test; virucidal test; virucide; viruses in suspension
E1052 − 20
APPENDIX
(Nonmandatory Information)
X1. VIRUSES
X1.1 Representative enveloped and non-enveloped to assess the virucidal activity of microbicides in suspension. The ATCC
numbers of the viruses and their host cells are given in parenthesis, where available.
X1.1.1 Adenovirus, Type 2 (VR-846) or Type 5 (VR-5). Cell line options: Human Lung Carcinoma (A549) [CCL-185], HEp-2.
[CCL-23], Vero [CCL-81].
X1.1.2 Canine Parvovirus, Cornell-780916–80 strain [VR-2017]. Cell line option: A–72 [CRL-1542].
X1.1.3 Cytomegalovirus, strain AD-169, [VR-538]. Cell line options: Human diploid lung (MRC-5 [CCL-171] or WI-38
[CCL-75]).
X1.1.4 Feline calicivirus, strain F-9 [VR-782]. Cell line option: CRFK [CCL-94].
X1.1.5 Hepatitis A Virus, HM-175 strain [VR-2093]. Cell line options: FRhK-4 [CRL-1688].
X1.1.6 Herpes simplex virus, Type 1, strain F (1) [VR-733]. Cell line options: VERO [CCL-81], HEp-2 [CCL-23].
X1.1.7 Influenza A, A/Hong Kong/8/68 [VR-544], A/PR/8/34 [VR-95]. Cell line options: Madin-Darby Canine kidney (MDCK)
[CCL-34]; Rhesus monkey kidney (LLC-MK2) [CCL-7].
X1.1.8 Murine Norovirus, Cell line: RAW 264.7 [TIB-71].
X1.1.9 Respiratory syncytial virus, Long strain [VR-26]. Cell line options: HEp-2 [CCL-23], MRC-5 [CCL-171].
4+
X1.1.10 Rhinovirus, Type 14 [VR-284] or 37 [VR-1147]. Cell line options: MRC-5 [CCL-171], WI-38 [CCL-75], HeLa T .
X1.1.11 Rotavirus, Wa strain [VR-2018]. Cell line options: MA-104 [CRL-2378.1] or African green monkey kidney (CV-1)
[CCL-70].
X1.1.12 Vaccinia, WR strain, [VR-119]. Cell line options: VERO [CCL-81], HEp-2 [CCL-23].
NOTE X1.1—Rhinoviruses grow optimally at 33 6 1°C.
NOTE X1.2—Fetal bovine serum may be inhibitory to rotavirus and it may also neutralize the trypsin often needed for rotavirus and influenzavirus growth.
ASTM International takes no position respecting the validity of any patent rights asserted in connection with any item mentioned
in this standard. Users of this standard are expressly advised that determination of the validity of any such patent rights, and the risk
of infringement of such rights, are entirely their own responsibility.
This standard is subject to revision at any time by the responsible technical committee and must be reviewed every five years and
if not revised, either reapproved or withdrawn. Your comments are invited either for revision of this standard or for additional standards
and should be addressed to ASTM International Headquarters. Your comments will receive careful consideration at a meeting of the
responsible technical committee, which you may attend. If you feel that your comments have not received a fair hearing you
...