ASTM E1482-23

Standard Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization

SIGNIFICANCE AND USE
5.1 This practice is to be used for the removal of virucidal agents from test product-virus mixtures, or from test product-neutralizer-virus mixtures, at or after the contact period and before the inoculation of these mixtures into host systems for assay of viral infectivity.  
5.2 The purpose of the practice is to reduce the concentration of the cytotoxic properties of the test product and neutralizers in order to permit the evaluation of viral infectivity at dilutions that would otherwise be toxic to the host cells.  
5.3 The practice is applicable to the testing of liquid, pre-saturated towelettes, and pressurized disinfectant products, as well as handwash/rub products.  
Note 3: When testing products, the ability of the solution to pass through the column must be verified prior to testing. Certain products with high viscosities are unable to pass through columns. If the product is determined to be too viscous, alternative neutralization methods should be employed.  
5.4 This practice is compatible with organic soil loads, hard water, disinfectants containing organic solvents, and chemical neutralizers.
SCOPE
1.1 This practice is intended to be used to reduce the cytotoxic level of the virus-test product mixture prior to assaying for viral infectivity. It is used in conjunction with evaluations of the virucidal efficacy of disinfectant solutions, wipes, trigger sprays, or pressurized disinfectant spray products intended for use on inanimate, nonporous environmental surfaces. This practice may also be used in the evaluation of hygienic handwashes/handrubs, or for other special applications. The practice may be employed with all viruses and host systems.
Note 1: Gel filtration columns may impact virus titer and their use should be taken into consideration when selected for use.  
1.2 This practice should be performed only by persons trained in virology techniques.  
1.3 This practice utilizes gel filtration technology. The effectiveness of the practice is dependent on the ratio of gel bed volume to sample size and uniformity in the preparation of columns as well as the conditions of centrifugation. The effectiveness of this practice is maximized by investigator practice and experience with gel filtration techniques.  
1.4 This practice will aid in the reduction, but not necessarily elimination, of test product toxicity while preserving the titer of the input virus.  
1.5 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.  
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use.  
1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

General Information

• Status: Published
• Publication Date: 31-Mar-2023
• ICS: 11.080.20 - Disinfectants and antiseptics
• ICS: 71.100.35 - Chemicals for industrial and domestic disinfection purposes
• Technical Committee: E35 - Pesticides, Antimicrobials, and Alternative Control Agents
• Drafting Committee: E35.15 - Antimicrobial Agents

Relations

• Refers: ASTM E1052-20 - Standard Practice 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 E1052-11 - Standard Test Method to Assess the Activity of Microbicides against Viruses in Suspension
• Effective Date: 01-Oct-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 E1053-97(2002) - Standard Test Method for Efficacy of Virucidal Agents Intended for Inanimate Environmental Surfaces
• Effective Date: 10-Apr-1997
• Refers: ASTM E1053-97 - Standard Test Method for Efficacy of Virucidal Agents Intended for Inanimate Environmental Surfaces
• Effective Date: 10-Apr-1997
• Refers: ASTM E1052-96(2002) - Standard Test Method for Efficacy of Antimicrobial Agents Against Viruses in Suspension (Withdrawn 2011)
• Effective Date: 10-Oct-1996
• Refers: ASTM E1052-96 - Standard Test Method for Efficacy of Antimicrobial Agents Against Viruses in Suspension
• Effective Date: 10-Oct-1996

Overview

ASTM E1482-23 is the internationally recognized standard practice for the use of gel filtration columns for cytotoxicity reduction and neutralization in virology testing. Developed by ASTM International, this standard outlines procedures to decrease the cytotoxic properties of test product mixtures, enabling more accurate assay of viral infectivity. By removing or reducing residual disinfectants or neutralizing agents that could harm host cells, researchers can evaluate virucidal efficacy without interference from cytotoxicity. This standard is essential for laboratories evaluating disinfectant efficacy and is commonly referenced in the context of surface disinfectant, hand hygiene product, and antiviral testing on environmental surfaces.

Key Topics

• Purpose and Scope

  • Reduces cytotoxicity in virus-test product mixtures prior to viral infectivity assays
  • Applicable for disinfectant solutions, wipes, sprays, pre-saturated towelettes, handwashes, and handrubs
  • Enables testing across a wide range of viruses and host systems

• Gel Filtration Technology

  • Utilizes gel filtration columns, such as those packed with Sephadex or similar media
  • Effectively separates viruses from cytotoxic agents via size exclusion chromatography
  • Efficiency depends on the volume of gel bed, sample size, and consistent column preparation
  • Compatible with organic soils, hard water, products containing organic solvents, and various chemical neutralizers

• Operational Considerations

  • Method should be carried out by individuals trained in virology techniques
  • High-viscosity products may require alternative neutralization methods, as some cannot pass through gel columns
  • The gel filtration method reduces but does not necessarily eliminate cytotoxicity, so further processing or controls may be needed

• Quality and Safety

  • Ensures reduction of cytotoxic activity while striving to maintain viral titer for accurate efficacy measurement
  • Requires adherence to health, safety, and regulatory guidelines as part of laboratory best practices

Applications

ASTM E1482-23 is a critical standard for practitioners in microbiology, virology, and clinical laboratory settings, particularly those involved in:

• Efficacy Testing of Disinfectants

  • Evaluating the virucidal activity of liquid and aerosolized disinfectants, wipes, and sprays on nonporous environmental surfaces

• Personal Hygiene Product Evaluation

  • Testing of handwash or handrub products for their effect on virus inactivation efficacy

• Neutralization in Virucidal Testing

  • Essential for studies where chemical neutralizers are insufficient or require confirmation of cytotoxicity removal

• Regulatory Compliance

  • Supports compliance with international requirements for disinfectant and sanitizer product registration, especially regarding virucidal claims

• Research and Development

  • Facilitates accurate virological assays by ensuring cell health, allowing comparison of product performance across laboratories

Related Standards

For a comprehensive approach to virucidal efficacy and cytotoxicity assessment, consider these associated ASTM standards:

• ASTM E1052: Practice to Assess the Activity of Microbicides Against Viruses in Suspension
• ASTM E1053: Practice to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental Surfaces
• ASTM E2756: Terminology Relating to Antimicrobial and Antiviral Agents

Using ASTM E1482-23 in conjunction with these standards helps ensure valid, reproducible, and regulator-approved data for antiviral and disinfectant product testing.

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Keywords: cytotoxicity reduction, gel filtration columns, viral infectivity assay, disinfectant efficacy testing, neutralization, virology, ASTM E1482-23, virucidal, tissue culture, handwash evaluation, nonporous surface disinfection

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: E1482 − 23
Standard Practice for
Use of Gel Filtration Columns for Cytotoxicity Reduction
1,2
and Neutralization
This standard is issued under the fixed designation E1482; 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 1.7 This international standard was developed in accor-
dance with internationally recognized principles on standard-
1.1 This practice is intended to be used to reduce the
ization established in the Decision on Principles for the
cytotoxic level of the virus-test product mixture prior to
Development of International Standards, Guides and Recom-
assaying for viral infectivity. It is used in conjunction with
mendations issued by the World Trade Organization Technical
evaluations of the virucidal efficacy of disinfectant solutions,
Barriers to Trade (TBT) Committee.
wipes, trigger sprays, or pressurized disinfectant spray prod-
ucts intended for use on inanimate, nonporous environmental
2. Referenced Documents
surfaces. This practice may also be used in the evaluation of
2.1 ASTM Standards:
hygienic handwashes/handrubs, or for other special applica-
E1052 Practice to Assess the Activity of Microbicides
tions. The practice may be employed with all viruses and host
against Viruses in Suspension
systems.
E1053 Practice to Assess Virucidal Activity of Chemicals
NOTE 1—Gel filtration columns may impact virus titer and their use
Intended for Disinfection of Inanimate, Nonporous Envi-
should be taken into consideration when selected for use.
ronmental Surfaces
1.2 This practice should be performed only by persons
E2756 Terminology Relating to Antimicrobial and Antiviral
trained in virology techniques.
Agents
1.3 This practice utilizes gel filtration technology. The
3. Terminology
effectiveness of the practice is dependent on the ratio of gel bed
volume to sample size and uniformity in the preparation of 3.1 Definitions:
columns as well as the conditions of centrifugation. The 3.1.1 For definitions of terms used in this guide, refer to
effectiveness of this practice is maximized by investigator Terminology E2756.
practice and experience with gel filtration techniques.
4. Summary of Test Practice
1.4 This practice will aid in the reduction, but not necessar-
4.1 After the exposure of a virus to a test product, the
ily elimination, of test product toxicity while preserving the
virus-product suspension is added to a column of Sephadex
titer of the input virus.
LH-60, Sephadex LH-20, or other appropriate gel filtration
1.5 Units—The values stated in SI units are to be regarded
medium. The column (encased within a sterile centrifuge tube
as standard. No other units of measurement are included in this
in order to capture the filtrate) is placed in a centrifuge and
standard.
centrifuged to separate the virus from the test product by gel
1.6 This standard does not purport to address all of the
filtration. Alternatively, samples may be hand-plunged using a
safety concerns, if any, associated with its use. It is the
syringe plunger. The filtrate (the column flow-through which
responsibility of the user of this standard to establish appro-
contains the virus) is assayed in the appropriate host system.
priate safety, health, and environmental practices and deter-
The untreated virus control suspension is gel-column filtered,
mine the applicability of regulatory limitations prior to use.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
contact ASTM Customer Service at [e-mail]. For Annual Book of ASTM
This practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Standards volume information, refer to the standard’s Document Summary page on
Antimicrobials, and Alternative Control Agents and is the direct responsibility of the ASTM website.
Subcommittee E35.15 on Antimicrobial Agents. Sephadex is a registered trademark of Amersham Biosciences. The sole source
Current edition approved April 1, 2023. Published April 2023. Originally of supply of the apparatus known to the committee at this time is Amersham
approved in 1992. Last previous edition approved in 2017 as E1482 – 12 (2017). Biosciences. If you are aware of alternative suppliers, please provide this informa-
DOI: 10.1520/E1482-23. tion to ASTM International Headquarters. Your comments will receive careful
2 1
The title was formerly Standard Test Method for Neutralization of Virucidal consideration at a meeting of the responsible technical committee, which you may
Agents in Virucidal Efficacy Evaluations. attend.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1482 − 23
using the same methods/techniques, and the virus titer of the 6.1.2 Phosphate Buffered Saline (PBS).
filtrate is determined by assay of infectivity. The residual 6.1.3 Sterile Distilled or Deionized Water.
cytotoxicity of the disinfectant is determined by gel filtration of 6.1.4 1% Albumin Solution (in PBS).
the test product control under the same conditions as those
6.2 Sephadex Gel Filtration Column Assembly:
which were used in the test. Results for the virus inactivation
6.2.1 Sephadex LH-60 or LH-20, compatible with organic
and test product cytotoxicity of gel-column filtrates are re-
solvents.
corded in the same manner as described in Test Methods E1052
6.2.2 Syringe, 5 cc or 10 cc, disposable.
and E1053. The gel-column filtration procedures described in
6.2.3 Glass wool, sterilized.
this practice are a modification of the method of Blackwell and
6.2.4 Centrifuge tube, 15 and/or 50 mL, conical, sterile, and
Chen.
disposable.
NOTE 2—A limitation of utilizing columns in virological assays is that
6.3 Labware:
they are unable to effectively neutralize all actives. Prior to testing, ensure
6.3.1 Pipettes, serological, 10, 5, and 2 mL.
the effectiveness of gel-filtration columns with the intended product
6.3.2 Erlenmeyer Flask, sterile, 250 mL or other suitable
chemistry. In addition, chemical neutralization is recommended to ensure/
sterilizable container.
aid neutralization of certain difficult to neutralize product active(s) in
addition to the use of Sephadex columns.
6.3.3 Test Tube Rack or Holder, for 15 and 50 mL tubes.
6.3.4 Test Tubes, 18 by 150 mm.
5. Significance and Use
6.3.5 Laboratory Film, or other sealing film. (Aluminum
foil may also be used to cover the syringe/glass-wool/tube
5.1 This practice is to be used for the removal of virucidal
agents from test product-virus mixtures, or from test product- assembly and then autoclaved).
neutralizer-virus mixtures, at or after the contact period and
6.4 Equipment:
before the inoculation of these mixtures into host systems for
6.4.1 Centrifuge, clinical, with rotor and shields capable of
assay of viral infectivity.
holding 15 and/or 50 mL centrifuge tubes, and running at a
5.2 The purpose of the practice is to reduce the concentra- r/min that generates 550 to 650 × g.
tion of the cytotoxic properties of the test product and 6.4.2 Refrigerator, 2 to 8 °C
neutralizers in order to permit the evaluation of viral infectivity 6.4.3 Autoclave.
at dilutions that would otherwise be toxic to the host cells.
7. Procedure
5.3 The practice is applicable to the testing of liquid,
7.1 Suspend the Sephadex in a large excess of sterile
pre-saturated towelettes, and pressurized disinfectant products,
distilled or deionized water in an Erlenmeyer flask or other
as well as handwash/rub products.
suitable sterilizable container. Use an amount of Sephadex
NOTE 3—When testing products, the ability of the solution to pass
sufficient for the number of columns to be prepared (approxi-
through the column must be verified prior to testing. Certain products with
mately 0.5 g of Sephadex per column) or prepare a larger
high viscosities are unable to pass through columns. If the product is
volume slurry to give a final suggested concentration of 5 to
determined to be too viscous, alternative neutralization methods should be
22 % Sephadex g/v. The user should determine the concentra-
employed.
tion of Sephadex to be u
...

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: E1482 − 12 (Reapproved 2017) E1482 − 23
Standard Practice for
Use of Gel Filtration Columns for Cytotoxicity Reduction
1,2
and Neutralization
This standard is issued under the fixed designation E1482; 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
NOTE 1—The title was formerly Standard Test Method for Neutralization of Virucidal Agents in Virucidal Efficacy Evaluations.
1.1 This practice is intended to be used to reduce the cytotoxic level of the virus-test product mixture prior to assaying for viral
infectivity. It is used in conjunction with evaluations of the virucidal efficacy of disinfectant solutions, wipes, trigger sprays, or
pressurized disinfectant spray products intended for use on inanimate, nonporous environmental surfaces. This practice may also
be used in the evaluation of hygienic handwashes/handrubs, or for other special applications. The practice may be employed with
all viruses and host systems.
NOTE 1—Gel filtration columns may impact virus titer and their use should be taken into consideration when selected for use.
1.2 This practice should be performed only by persons trained in virology techniques.
1.3 This practice utilizes gel filtration technology. The effectiveness of the practice is dependent on the ratio of gel bed volume
to sample size and uniformity in the preparation of columns as well as the conditions of entrifugation.centrifugation. The
effectiveness of this practice is maximized by investigator practice and experience with gel filtration techniques.
1.4 This practice will aid in the reduction, but not necessarily elimination, of test product toxicity while preserving the titer of the
input virus.
1.5 Units—The values stated in SI units are to be regarded as standard. No other units of measurement are included in this
standard.
1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility
of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of
regulatory limitations prior to use.
1.7 This international standard was developed in accordance with internationally recognized principles on standardization
established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued
by the World Trade Organization Technical Barriers to Trade (TBT) Committee.
This 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 Nov. 1, 2017April 1, 2023. Published December 2017April 2023. Originally approved in 1992. Last previous edition approved in 20122017 as
E1482 – 12.E1482 – 12 (2017). DOI: 10.1520/E1482-12R17.10.1520/E1482-23.
The title was formerly Standard Test Method for Neutralization of Virucidal Agents in Virucidal Efficacy Evaluations.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1482 − 23
2. Referenced Documents
2.1 ASTM Standards:
E1052 Practice to Assess the Activity of Microbicides against Viruses in Suspension
E1053 Practice to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental
Surfaces
E2756 Terminology Relating to Antimicrobial and Antiviral Agents
3. Terminology
3.1 Definitions:
3.1.1 For definitions of terms used in this guide, refer to Terminology E2756.
4. Summary of Test MethodsPractice
4.1 After the exposure of a virus to a test product (or handwash/rub product), product, the virus-product suspension is added to
4 4
a column of Sephadex LH-60, Sephadex LH-20, or Sephacrylother S-1000 Superfine. appropriate gel filtration medium. The
column (encased within a sterile centrifuge tube in order to capture the filtrate) is placed in a centrifuge and centrifuged to separate
the virus from the test product by gel filtration. Alternatively, samples may be hand-plunged using a syringe plunger. The filtrate
(the column flow-through which contains the virus) is assayed in the appropriate host system. The untreated virus control
suspension is gel-column filtered, using the same methods/techniques, and the virus titer of the filtrate is determined by assay of
infectivity. The residual cytotoxicity of the disinfectant is determined by gel filtration of the test product control under the same
conditions as those which were used in the test. Results for the virus inactivation and test product cytotoxicity of gel-column
filtrates are recorded in the same manner as described in Test Methods E1052 and E1053. The gel-column filtration procedures
described in this practice are a modification of the method of Blackwell and Chen.
NOTE 2—A limitation of utilizing columns in virological assays is that they are unable to effectively neutralize all actives. Prior to testing, ensure the
effectiveness of gel-filtration columns with the intended product chemistry. In addition, chemical neutralization is recommended to ensure/aid
neutralization of certain difficult to neutralize product active(s) in addition to the use of Sephadex columns.
5. Significance and Use
5.1 This practice is to be used for the removal of virucidal agents from test product-virus mixtures, or from test product-
neutralizer-virus mixtures, at or after the contact period and before the inoculation of these mixtures into host systems for assay
of viral infectivity.
5.2 The purpose of the practice is to reduce the concentration of the cytotoxic properties of the test product and neutralizers in
order to permit the evaluation of viral infectivity at dilutions that would otherwise be toxic to the host cells.
5.3 The practice is applicable to the testing of liquid, pre-saturated towelettes, and pressurized disinfectant products, as well as
handwash/rub products.
NOTE 3—When testing handwash/rub products, the ability of the solution to pass through the column must be verified prior to testing. Certain products
with high viscosities are unable to pass through columns. If the product is determined to be too viscous, alternative neutralization methods should be
employed.
5.4 This practice is compatible with organic soil loads, hard water, disinfectants containing organic solvents, and chemical
neutralizers.
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.
Sephadex is a registered trademark of Amersham Biosciences. The sole source of supply of the apparatus known to the committee at this time is Amersham Biosciences.
If you are aware of alternative suppliers, please provide this information to ASTM International Headquarters. Your comments will receive careful consideration at a meeting
of the responsible technical committee, which you may attend.
Blackwell, H. H., and Chen, J. H. S., “Effects of Various Germicidal Chemicals on H.EP.2 Cell Culture and Herpes simplex Virus,” Journal of the AOAC, Vol 53, 1970,
pp. 1229–1236.
E1482 − 23
6. Reagents and Materials
6.1 Reagents:
6.1.1 Purity of Reagents—Reagent grade chemicals shall be used in all tests. Unless otherwise indicated, it is intended that all
reagents shall conform to the specifications of the Committee on Analytical Reagents of the American Chemical Society, where
such specifications are available. Other grades may be used, provided it is first ascertained that the reagent is of sufficiently high
purity to permit its use without lessening the accuracy of the determination.
6.1.2 Phosphate Buffered Saline (PBS).
6.1.3 Sterile Distilled or Deionized Water.
6.1.4 1% Albumin Solution (in PBS).
6.2 Sephadex Gel Filtration Column Assembly:
6.2.1 Sephadex LH-60 or LH-20, compatible with organic solvents. (Sephacryl S-1000 Superfine may be substituted.)
6.2.2 Syringe, 5-cc or 10-cc,5 cc or 10 cc, disposable.
6.2.3 Glass wool, sterilized.
6.2.4 Centrifuge tube, 15- and/or 50-mL,15 and/or 50 mL, conical, sterile, and disposable.
6.3 Labware:
6.3.1 Pipettes, serological, 10-, 5-, and 2-mL.10, 5, and 2 mL.
6.3.2
...