ASTM E1482-12(2017)
(Practice)Standard Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization
Standard Practice for Use of Gel Filtration Columns for Cytotoxicity Reduction and Neutralization
SIGNIFICANCE AND USE
4.1 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.
4.2 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.
4.3 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.
4.4 This practice is compatible with organic soil loads, hard water, disinfectants containing organic solvents, and chemical neutralizers.
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.
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. 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 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.
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Designation: E1482 − 12 (Reapproved 2017)
Standard Practice for
Use of Gel Filtration Columns for Cytotoxicity Reduction
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 Development of International Standards, Guides and Recom-
NOTE 1—The title was formerly Standard Test Method for Neutraliza-
mendations issued by the World Trade Organization Technical
tion of Virucidal Agents in Virucidal Efficacy Evaluations.
Barriers to Trade (TBT) Committee.
1.1 This practice is intended to be used to reduce the
cytotoxic level of the virus-test product mixture prior to
2. Referenced Documents
assaying for viral infectivity. It is used in conjunction with
2.1 ASTM Standards:
evaluations of the virucidal efficacy of disinfectant solutions,
E1052 Test Method to Assess the Activity of Microbicides
wipes, trigger sprays, or pressurized disinfectant spray prod-
against Viruses in Suspension
ucts intended for use on inanimate, nonporous environmental
E1053 Test Method to Assess Virucidal Activity of Chemi-
surfaces. This practice may also be used in the evaluation of
cals Intended for Disinfection of Inanimate, Nonporous
hygienic handwashes/handrubs, or for other special applica-
Environmental Surfaces
tions. The practice may be employed with all viruses and host
systems.
3. Summary of Test Methods
1.2 This practice should be performed only by persons
trained in virology techniques. 3.1 After the exposure of a virus to a test product (or
handwash/rub product), the virus-product suspension is added
1.3 This practice utilizes gel filtration technology. The
3 3
to a column of Sephadex LH-60, Sephadex LH-20, or
effectiveness of the practice is dependent on the ratio of gel bed
Sephacryl S-1000 Superfine. The column (encased within a
volume to sample size and uniformity in the preparation of
sterile centrifuge tube in order to capture the filtrate) is placed
columns as well as the conditions of entrifugation. The
in a centrifuge and centrifuged to separate the virus from the
effectiveness of this practice is maximized by investigator
test product by gel filtration. Alternatively, samples may be
practice and experience with gel filtration techniques.
hand-plunged using a syringe plunger. The filtrate (the column
1.4 This practice will aid in the reduction, but not necessar-
flow-through which contains the virus) is assayed in the
ily elimination, of test product toxicity while preserving the
appropriate host system. The untreated virus control suspen-
titer of the input virus.
sion is gel-column filtered, using the same methods/techniques,
1.5 The values stated in SI units are to be regarded as
and the virus titer of the filtrate is determined by assay of
standard. No other units of measurement are included in this
infectivity. The residual cytotoxicity of the disinfectant is
standard.
determined by gel filtration of the test product control under the
same conditions as those which were used in the test. Results
1.6 This standard does not purport to address all of the
for the virus inactivation and test product cytotoxicity of
safety concerns, if any, associated with its use. It is the
gel-column filtrates are recorded in the same manner as
responsibility of the user of this standard to establish appro-
described in Test Methods E1052 and E1053. The gel-column
priate safety, health, and environmental practices and deter-
mine the applicability of regulatory limitations prior to use.
1.7 This international standard was developed in accor-
dance with internationally recognized principles on standard- 2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
ization established in the Decision on Principles for the contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
Standards volume information, refer to the standard’s Document Summary page on
the ASTM website.
1 3
This practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Sephadex is a registered trademark of Amersham Biosciences. The sole source
Antimicrobials, and Alternative Control Agentsand is the direct responsibility of of supply of the apparatus known to the committee at this time is Amersham
Subcommittee E35.15 on Antimicrobial Agents. Biosciences. If you are aware of alternative suppliers, please provide this informa-
Current edition approved Nov. 1, 2017. Published December 2017. Originally tion to ASTM International Headquarters. Your comments will receive careful
approved in 1992. Last previous edition approved in 2012 as E1482 – 12. DOI: consideration at a meeting of the responsible technical committee, which you may
10.1520/E1482-12R17. attend.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1482 − 12 (2017)
filtration procedures described in this practice are a modifica- 5.2.3 Glass wool, sterilized.
tion of the method of Blackwell and Chen.
5.2.4 Centrifuge tube, 15- and/or 50-mL, conical, sterile,
and disposable.
NOTE 2—A limitation of utilizing columns in virological assays is that
they are unable to effectively neutralize all actives. Prior to testing, ensure
5.3 Labware:
the effectiveness of gel-filtration columns with the intended product
5.3.1 Pipettes, serological, 10-, 5-, and 2-mL.
chemistry. In addition, chemical neutralization is recommended to ensure/
5.3.2 Erlenmeyer Flask, sterile, 250-mL or other suitable
aid neutralization of certain difficult to neutralize product active(s) in
sterilizable container.
addition to the use of Sephadex columns.
5.3.3 Test Tube Rack or Holder, for 15- and 50-mL tubes.
4. Significance and Use
5.3.4 Test Tubes, 18 by 150 mm.
4.1 This practice is to be used for the removal of virucidal
5.3.5 Laboratory Film, or other sealing film. (Aluminum
agents from test product-virus mixtures, or from test product-
foil may also be used to cover the syringe/glass-wool/tube
neutralizer-virus mixtures, at or after the contact period and
assembly and then autoclaved).
before the inoculation of these mixtures into host systems for
5.4 Equipment:
assay of viral infectivity.
5.4.1 Centrifuge, clinical, with rotor and shields capable of
4.2 The purpose of the practice is to reduce the concentra-
holding 15- and/or 50-mL centrifuge tubes, and running at a
tion of the cytotoxic properties of the test product and
r/min that generates 550 to 650 × g.
neutralizers in order to permit the evaluation of viral infectivity
5.4.2 Refrigerator, 2 to 8°C
at dilutions that would otherwise be toxic to the host cells.
5.4.3 Autoclave.
4.3 The practice is applicable to the testing of liquid,
pre-saturated towelettes, and pressurized disinfectant products, 6. Procedure
as well as handwash/rub products.
6.1 Suspend the Sephadex in a large excess of sterile
distilled or deionized water in an Erlenmeyer flask or other
NOTE 3—When testing handwash/rub products, the ability of the
solution to pass through the column must be verified prior to testing.
suitable sterilizable container. Use an amount of Sephadex
Certain products with high viscosities are unable to pass through columns.
sufficient for the number of columns to be prepared (approxi-
If the product is determined to be too viscous, alternative neutralization
mately 0.5 g of Sephadex per column) or prepare a larger
methods should be employed.
volume slurry to give a final suggested concentration of 5 to
4.4 This practice is compatible with organic soil loads, hard
22 % Sephadex g/v. Sterilize slurry by autoclaving. (Example
water, disinfectants containing organic solvents, and chemical
parameters for autoclaving are 121°C at 15 psi (pounds of
neutralizers.
pressure per square inch) for at least 15 min. Autoc
...
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 E1482 − 12 (Reapproved 2017)
Standard Practice for
Use of Gel Filtration Columns for Cytotoxicity Reduction
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.
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. 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 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 safety, health, and healthenvironmental 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.
2. Referenced Documents
2.1 ASTM Standards:
E1052 Test Method to Assess the Activity of Microbicides against Viruses in Suspension
E1053 Test Method to Assess Virucidal Activity of Chemicals Intended for Disinfection of Inanimate, Nonporous Environmental
Surfaces
3. Summary of Test Methods
3.1 After the exposure of a virus to a test product (or handwash/rub product), the virus-product suspension is added to a column
3 3 3
of Sephadex LH-60, Sephadex LH-20, or Sephacryl S-1000 Superfine. 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
This practice is under the jurisdiction of ASTM Committee E35 on Pesticides, Antimicrobials, and Alternative Control Agentsand is the direct responsibility of
Subcommittee E35.15 on Antimicrobial Agents.
Current edition approved Oct. 1, 2012Nov. 1, 2017. Published November 2012December 2017. Originally approved in 1992. Last previous edition approved in 20042012
as E1482 – 04E1482 – 12.). DOI: 10.1520/E1482-12.10.1520/E1482-12R17.
For referenced ASTM standards, visit the ASTM website, www.astm.org, or contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM Standards
volume information, refer to the standard’s Document Summary page on the ASTM website.
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.
Copyright © ASTM International, 100 Barr Harbor Drive, PO Box C700, West Conshohocken, PA 19428-2959. United States
E1482 − 12 (2017)
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.
4. Significance and Use
4.1 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.
4.2 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.
4.3 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.
4.4 This practice is compatible with organic soil loads, hard water, disinfectants containing organic solvents, and chemical
neutralizers.
5. Reagents and Materials
5.1 Reagents:
5.1.1 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.
5.1.2 Phosphate Buffered Saline (PBS).
5.1.3 Sterile Distilled or Deionized Water.
5.1.4 1% Albumin Solution (in PBS).
5.2 Sephadex Gel Filtration Column Assembly:
5.2.1 Sephadex LH-60 or LH-20, compatible with organic solvents. (Sephacryl S-1000 Superfine may be substituted.)
5.2.2 Syringe, 5-cc or 10-cc, disposable.
5.2.3 Glass wool, sterilized.
5.2.4 Centrifuge tube, 15- and/or 50-mL, conical, sterile, and disposable.
5.3 Labware:
5.3.1 Pipettes, serological, 10-, 5-, and 2-mL.
5.3.2 Erlenmeyer Flask, sterile, 250-mL or other suitable sterilizable container.
5.3.3 Test Tube Rack or Hold
...
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