ASTM E1482-12
(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 and health practices and determine the applicability of regulatory limitations prior to use.
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Designation: E1482 − 12
Standard Practice for
Use of Gel Filtration Columns for Cytotoxicity Reduction
1
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.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
2
NOTE 1—The title was formerly Standard Test Method for Neutraliza-
2.1 ASTM Standards:
tion of Virucidal Agents in Virucidal Efficacy Evaluations.
E1052Test Method to Assess the Activity of Microbicides
against Viruses in Suspension
1.1 This practice is intended to be used to reduce the
E1053Test Method to Assess Virucidal Activity of Chemi-
cytotoxic level of the virus-test product mixture prior to
cals Intended for Disinfection of Inanimate, Nonporous
assaying for viral infectivity. It is used in conjunction with
Environmental Surfaces
evaluations of the virucidal efficacy of disinfectant solutions,
wipes, trigger sprays, or pressurized disinfectant spray prod-
3. Summary of Test Methods
ucts intended for use on inanimate, nonporous environmental
3.1 After the exposure of a virus to a test product (or
surfaces. This practice may also be used in the evaluation of
handwash/rub product), the virus-product suspension is added
hygienic handwashes/handrubs, or for other special applica-
3 3
to a column of Sephadex LH-60, Sephadex LH-20, or
tions. The practice may be employed with all viruses and host
3
Sephacryl S-1000 Superfine. The column (encased within a
systems.
sterile centrifuge tube in order to capture the filtrate) is placed
1.2 This practice should be performed only by persons
in a centrifuge and centrifuged to separate the virus from the
trained in virology techniques.
test product by gel filtration. Alternatively, samples may be
hand-plunged using a syringe plunger.The filtrate (the column
1.3 This practice utilizes gel filtration technology. The
flow-through which contains the virus) is assayed in the
effectivenessofthepracticeisdependentontheratioofgelbed
appropriate host system. The untreated virus control suspen-
volume to sample size and uniformity in the preparation of
sionisgel-columnfiltered,usingthesamemethods/techniques,
columns as well as the conditions of entrifugation. The
and the virus titer of the filtrate is determined by assay of
effectiveness of this practice is maximized by investigator
infectivity. The residual cytotoxicity of the disinfectant is
practice and experience with gel filtration techniques.
determinedbygelfiltrationofthetestproductcontrolunderthe
1.4 This practice will aid in the reduction, but not necessar- same conditions as those which were used in the test. Results
ily elimination, of test product toxicity while preserving the for the virus inactivation and test product cytotoxicity of
gel-column filtrates are recorded in the same manner as
titer of the input virus.
described in Test Methods E1052 and E1053. The gel-column
1.5 The values stated in SI units are to be regarded as
filtration procedures described in this practice are a modifica-
standard. No other units of measurement are included in this
4
tion of the method of Blackwell and Chen.
standard.
NOTE 2—Alimitation of utilizing columns in virological assays is that
1.6 This standard does not purport to address all of the
safety concerns, if any, associated with its use. It is the
2
For referenced ASTM standards, visit the ASTM website, www.astm.org, or
responsibility of the user of this standard to establish appro-
contact ASTM Customer Service at service@astm.org. For Annual Book of ASTM
priate safety and health practices and determine the applica-
Standards volume information, refer to the standard’s Document Summary page on
bility of regulatory limitations prior to use.
the ASTM website.
3
Sephadex is a registered trademark ofAmersham 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 informa-
1
This practice is under the jurisdiction ofASTM Committee E35 on Pesticides, tion to ASTM International Headquarters. Your comments will receive careful
1
Antimicrobials, and Alternative Control Agentsand is the direct responsibility of consideration at a meeting of the responsible technical committee, which you may
Subcommittee E35.15 on Antimicrobial Agents. attend.
4
Current edition approved Oct. 1, 2012. Published November 2012. Originally Blackwell,H.H.,andChen,J.H.S.,“EffectsofVariousGermicidalChemicals
approved in 1992. Last previous edition approved in 2004 as E1482–04). DOI: on H.EP.2 Cell Culture and Herpes simplex Virus,” J
...
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 − 04 E1482 − 12
Standard Test Method Practice for
Neutralization of Virucidal Agents in Virucidal Efficacy
EvaluationsUse of Gel Filtration Columns for Cytotoxicity
1
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
1.1 This test method covers the use, in conjunction with evaluations of the virucidal efficacy, of disinfectant solutions or
pressurized disinfectant spray products intended for use on inanimate nonporous environmental surfaces or for other special
applications. The test method may be employed with all viruses and host systems.
1.2 This test method should be performed only by persons trained in microbiology and virology.
1.3 This test method utilizes gel filtration technology. The effectiveness of the test method is dependent on the ratio of gel bed
volume to sample size and uniformity in the preparation of columns and centrifugation conditions. The effectiveness of this test
method is maximized by investigator practice and experience with gel filtration techniques.
1.4 This test method will reduce, but not necessarily eliminate, disinfectant toxicity while preserving the titer of input virus.
1.5 The values stated in SI units are to be regarded as the 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 and health practices and determine the applicability of regulatory
limitations prior to use.
2. Referenced Documents
2
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 Method
3
3.1 After the exposure of a virus to a disinfectant, the virus-disinfectant suspension is applied to a column of Sephadex
LH60-120, or Sephacryl S-1000 Superfine. The column is placed in a centrifuge and centrifuged to separate the virus from the
disinfectant by gel filtration. The filtrate (the column flow-through that contains the virus) is assayed in the appropriate host system.
The untreated virus control suspension is similarly gel filtered, 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 disinfectant control under the same conditions.
Results for the virus inactivation and disinfectant cytotoxicity of gel filtrates are recorded in the same manner as described in Test
Methods E1052 and E1053. The gel filtration procedures described in this test method are a modification of the method of
4
Blackwell and Chen.
1
This test method 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, 2004Oct. 1, 2012. Published October 2004November 2012. Originally approved in 1992. Last previous edition approved in 2004 as
E1482 - 92 (2004).E1482 – 04). DOI: 10.1520/E1482-04.10.1520/E1482-12.
2
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.
3
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
1
of the responsible technical committee, which you may attend.
4
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.
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E1482 − 12
4. Significance and Use
4.1
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