ISO 21702:2019

INTERNATIONAL ISO
STANDARD 21702
First edition
2019-05
Measurement of antiviral activity
on plastics and other non-porous
surfaces
Mesure de l'activité antivirale sur les matières plastiques et autres
surfaces non poreuses
Reference number
©
ISO 2019
© ISO 2019
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Published in Switzerland
ii © ISO 2019 – All rights reserved

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Materials . 2
4.1 Virus and host cell to be used for the tests . 2
4.2 Reagents. 2
4.3 Culture medium and solutions . 2
4.3.1 General. 2
4.3.2 Eagle's minimum essential medium (EMEM) . 2
4.3.3 RPMI 1640 medium . 2
4.3.4 7,5 % sodium bicarbonate solution . 3
4.3.5 Formaldehyde solution. 3
4.3.6 Methylene blue solution . 3
4.3.7 Inactivated fetal bovine serum (FBS) . 3
4.3.8 Growth medium . 3
4.3.9 Maintenance medium . 3
4.3.10 Double concentration of the maintenance medium . 4
4.3.11 Phosphate buffered saline [PBS (-)] . 4
4.3.12 Trypsin derived from beef pancreas and PBS (-) solution . 4
4.3.13 Trypsin EDTA solution . 4
4.3.14 DEAE-dextran solution . 4
4.3.15 Agar medium for plaque assay . 5
4.3.16 Soybean casein digest broth with lecithin and polyoxyethylene sorbitan
monooleate (SCDLP broth) . 5
5 Apparatus . 5
6 Preparation . 6
6.1 Restoration of host cell from cryopreservation . 6
6.2 Subculture of host cell . 7
6.3 Cell culture for measuring virus infectivity titer . 7
6.4 Preparation of test inoculums. 7
6.4.1 Influenza virus . 7
6.4.2 Feline calicivirus. 8
6.5 Preparation of test specimens . 8
6.6 Control test . 9
6.6.1 General. 9
6.6.2 Verification of cytotoxic effect on host cell . 9
6.6.3 Verification of cell sensitivity to virus and the inactivation of antiviral activity . 9
7 Test procedure .11
7.1 Preparation of test specimen .11
7.2 Inoculation of test specimens .11
7.3 Contact of the inoculated test specimens.12
7.4 Recovery of virus from test specimens .12
7.4.1 Test specimens immediately after inoculation .12
7.4.2 Test specimens after contact .12
7.5 Determining the infectivity titer of virus by plaque assay .12
8 Expression of results .13
8.1 Determination of the infectivity titer of virus .13
8.2 Conditions for a valid test .13
8.3 Calculation of the antiviral activity .14
8.4 Effectiveness of the antiviral agent .14
9 Repeatability and reproducibility .14
10 Test report .14
Annex A (informative) Composition of media .16
Annex B (informative) Repeatability and reproducibility .18
Bibliography .20
iv © ISO 2019 – All rights reserved

Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see the following
URL: www .iso .org/iso/foreword .html.
This document was prepared by Technical Committee ISO/TC 61, Plastics, Subcommittee SC 6, Ageing,
chemical and environmental resistance.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/members .html.
Introduction
Antibacterial-treated porous and non-porous products have been widely accepted and used among
general consumers as their new choices to purchase for the additional function, which are different
from what traditional materials had in terms of material protection.
Recently, antiviral-treated porous and non-porous products have been also in the market.
The measuring test method of antibacterial activity on non-porous products is described in ISO 22196.
The measuring test method of antibacterial activity on porous products (textiles) is described in
ISO 20743.
The measuring test method of antiviral activity on porous products (textiles) is described in ISO 18184.
This document is the test method of antiviral activity on non-porous products. It is written based on
ISO 22196 and ISO 18184.
In ISO 22196, the scope has been expanded to include surfaces made of plastics and other non-porous
materials, thus this document is intended to be applicable to products such as plastics, coating materials,
ceramics, natural and artificial leathers, stainless, rubbers, etc.
vi © ISO 2019 – All rights reserved

INTERNATIONAL STANDARD ISO 21702:2019(E)
Measurement of antiviral activity on plastics and other
non-porous surfaces
WARNING — Handling and manipulation of viruses and host cells which are potentially
hazardous requires a high degree of technical competence and may be subject to current
national legislation and regulations. Only personnel trained in biological techniques should
carry out such tests. Appropriate practices for disinfection, sterilization and personal hygiene
must be strictly observed.
1 Scope
This document specifies proper methods for measuring antiviral activity on plastics and other
non-porous surfaces of antiviral-treated products against specified viruses. Due to the individual
sensitivities, the results of one test virus might not be applicable for other viruses.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https: //www .iso .org/obp
— IEC Electropedia: available at http: //www .electropedia .org/
3.1
antiviral
state where the number of infectious virus particles on surfaces of products is reduced
3.2
antiviral agent
agent that reduces the number of infectious virus on surface of products
3.3
antiviral activity
difference in the logarithm of the infectivity titer of virus found on an antiviral-treated product and an
untreated product after inoculation with and contact to virus
3.4
cytopathic effect
morphological change or destruction of the host cells as a result of the virus multiplication
3.5
infectivity titer of virus
number of infectious viral particles present per unit volume in a suspension
3.6
plaque
lysis formed area in a cell monolayer under semisolid medium due to infection by and multiplication of
a single infectious virus
3.7
plaque forming units
PFU
unit expressed as the concentration of the infectious viral particle per unit volume (ml)
3.8
plaque assay
assay to determine the infectivity titer of virus (3.5) from PFU by using the series of dilution
4 Materials
4.1 Virus and host cell to be used for the tests
The example species of virus and host cell to be used are shown in Table 1.
Other species of virus and host cells can be used after appropriate validations, as the important virus
may differ depending on the target application. If the other species are used, the name of the species
and the specific reason for their use shall be included in the test report.
Table 1 — Examples of viruses, virus strains, host cells and media to be used
Virus name Influenza virus Feline calicivirus
Virus strain Influenza A virus (H3N2): A/Hong Kong/8/68: Feline calicivirus; Strain: F-9 ATCC VR-782
TC adapted ATCC VR-1679
a
Host cell MDCK cell CRFK cell
(dog kidney cell origin) (cat kidney cell origin)
ATCC CCL-34 ATCC CCL-94
b
Growth medium EMEM (4.3.8) RPMI 1640 (4.3.8)
a
The other host cells can be used after appropriate validations regarding the sensitivity against each virus.
b
The other media can be used after appropriate validations for the growth of cells.
4.2 Reagents
Any water used shall be deionized and/or distilled and/or ultra-filtered and/or filtered with RO [reverse
osmosis] and have a conductivity of <1 ìS/cm.
All reagents shall be of analytical grade and/or of a grade appropriate for microbiological purposes.
4.3 Culture medium and solutions
4.3.1 General
The culture medium specified below shall be used. The medium may be obtained from commercial
suppliers which shall be prepared for use in accordance with the manufacturer’s instructions.
4.3.2 Eagle's minimum essential medium (EMEM)
The composition is described in Annex A. If there are any components missing from the composition,
they can be added according to Table A.1.
4.3.3 RPMI 1640 medium
The composition is described in Annex A. If there are any components missing from the composition,
they can be added according to Table A.2.
2 © ISO 2019 – All rights reserved

4.3.4 7,5 % sodium bicarbonate solution
Select and prepare the solution using one of the two following options:
— Option 1: Prepare a 7,5 % sodium bicarbonate solution by dissolving 75 g of sodium bicarbonate in
1 000 ml of water. Sterilize the solution by using a 0,22 μm membrane filter.
— Option 2: Prepare a 7,5 % sodium bicarbonate solution by sterilizing 75 g of sodium bicarbonate
in a culture container with a cap closed tightly in an autoclave. Sterilize 1 000 ml of water in the
autoclave. Dissolve the sterilized sodium bicarbonate in the sterilized water well.
If the solution is not intended to be used immediately after preparation, then preserve it at 5 °C to
10 °C. Never use 7,5 % sodium bicarbonate solution that has been kept for longer than one month after
preparation.
4.3.5 Formaldehyde solution
Prepare the formaldehyde solution by adding 100 ml of 37 % formaldehyde solution to 900 ml of water.
If it is not intended to be used immediately after preparation, preserve it at 20 °C to 25 °C. Never use the
formalin solution that has been kept for longer than one month after preparation.
NOTE The other solution for cell fixation can be used after appropriate validation for cell fixation.
4.3.6 Methylene blue solution
Prepare the methylene blue solution by dissolving 0,375 g of the methylene blue and 62,5 μl of 1 N
sodium hydroxide solutions in 1 000 ml of water. If it is not intended to be used immediately after
preparation, then preserve it at 20 °C to 25 °C. Never use the methylene blue solution that has been kept
for longer than one month after preparation.
4.3.7 Inactivated fetal bovine serum (FBS)
Put a freezed cryopreserved fetal bovine serum in a package into a water bath at 37 °C and keep it until
it defrosts. Then, raise the temperature of the water bath to 56 °C and keep it for 30 min to inactivate.
Divide it into several tubes. Put them in a freezer at a temperature less than −20 °C. Just before using,
put it in the water bath at 37 °C and keep it until it defrosts.
4.3.8 Growth medium
Dissolve 9,53 g of the Eagle's minimum essential medium or 10,4 g of RPMI 1640 medium and 60 mg
of kanamycin sulfate in 800 ml of water. Add water to make 1 000 ml of solution in total. Sterilize the
solution by using a 0,22 μm membrane filter.
When L-glutamine is not included in the EMEM or RPMI 1640 medium purchased in the market,
sterilizing in the autoclave may be applied. Then, before use, add L-glutamine as listed in the example of
composition in Annex A.
Add 15 ml of 7,5 % sodium bicarbonate solution and 100 ml of the inactivated fetal bovine serum in
the solution and mix well. If it is not intended to be used immediately after preparation, then preserve
it at 5 °C to 10 °C. Never use a growth medium that has been kept for longer than one month after
preparation.
4.3.9 Maintenance medium
Dissolve 9,53 g of the Eagle's minimum essential medium and 60 mg of kanamycin sulfate in 800 ml
of water. Add water to make 1 000 ml of solution in total. Sterilize the solution by using a 0,22 μm
membrane filter.
When L-glutamine is not included in the EMEM purchased in the market, sterilizing by autoclaving may
be applied. Then, before use, add L-glutamine as listed in the example of composition in Annex A.
Add 15 ml of 7,5 % sodium bicarbonate solution in the solution and mix well. If it is not intended to be
used immediately after preparation, then preserve it at 5 °C to 10 °C. Never use a maintenance medium
that has been kept for longer than one month after preparation.
4.3.10 Double concentration of the maintenance medium
Dissolve 19,06 g of the Eagle's minimum essential medium and 120 mg of kanamycin sulfate in 800 ml
of water. Add water till there are 1 000 ml of solution in total. Sterilize the solution by using a 0,22 μm
membrane filter. If it is not intended to be used immediately after preparation, then preserve it at 5 °C
to 10 °C. Never use a growth medium that has been kept for longer than one month after preparation.
When L-glutamine is not included in the EMEM purchased on market, sterilizing by autoclaving could
be applied. Then, before use, add L-glutamine as listed in the example of composition in Annex A.
4.3.11 Phosphate buffered saline [PBS (-)]
Prepare PBS (-) by dissolving 8,0 g of sodium chloride, 0,2 g of potassium chloride, 2,9 g of phosphoric
acid hydrogen 2 sodium 12 hydrate and 0,2 g of phosphoric acid 2 hydrogen potassium in 1 000 ml of
water. Sterilize by autoclaving (see 6.2). If it is not intended to be used immediately after preparation,
preserve it at 5 °C to 10 °C. Never use a PBS (-) that has been kept for longer than one month after
preparation.
4.3.12 Trypsin derived from beef pancreas and PBS (-) solution
4.3.12.1 Dissolve 1,0 g of trypsin derived from pancreas in 100 ml of PBS (-) and mix well for 2 h by
using a mixer. Sterilize the solution by using 0,22 μm membrane filter. If it is not intended to be used
immediately after preparation, divide the solution in test tubes and preserve them in the freezer at a
temperature less than −80 °C. Just before using, put it in the water bath at 37 °C and keep it until it
defrosts.
4.3.12.2 Add 1,0 ml of the solution of 4.3.12.1 to 9,0 ml of PBS (-) and mix well. Divide the solution in
test tubes and preserve them in the freezer at a temperature less than −20 °C. Just before using, put it in
the water bath at 37 °C and keep it until it defrosts.
1)
4.3.13 Trypsin EDTA solution
Prepare Trypsin EDTA solution by dissolving 2,5 g of trypsin, 0,1 g of kanamycin sulfate, 0,1 g of
streptomycin sulfate, 2 mg of amphotericin B and 0,014 mol of EDTA in 1 000 ml of PBS (-). Sterilize the
solution by using a 0,22 μm membrane filter. Divide the solution in test tubes and preserve them in the
freezer at a temperature less than −20 °C. Just before using, put it in the water bath at 37 °C and keep it
until it defrosts.
NOTE Trypsin EDTA solution is available in the market. The products with different components could be
used after proper validations.
4.3.14 DEAE-dextran solution
Prepare DEAE-dextran solution by dissolving 20 g of DEAE-dextran in 1 000 ml of water. Sterilize the
solution by using 0,22 μm membrane filter. If it is not intended to be used immediately after preparation,
then preserve it at 5 °C to 10 °C. Never use a DEAE-dextran solution that has been kept for longer than
one month after preparation.
1) Trypsin EDTA solution is an example of a product available in the market. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of the product named.
Equivalent products may be used if they can be shown to lead to the same results.
4 © ISO 2019 – All rights reserved

4.3.15 Agar medium for plaque assay
4.3.15.1 A liquid
Add 10 ml of DEAE-dextran solution and 40 ml of 7,5 % sodium bicorbonate solution to 1 000 ml of
Double concentration of maintenance medium and mix well. Only for the influenza virus test, add 3,0 ml
of the Trypsin from pancreas and PBS (-) solution. Keep the solution in the water bath at 37 °C.
4.3.15.2 B liquid
Add 15 g of cell culture agar to 1 000 ml of water and mix well. Sterilize by autoclaving. Keep the
solution in the water bath at 60 °C.
4.3.15.3 Preparation of agar medium
Prepare agar medium for plaque assay with A liquid and B liquid with one to one amount and mix well
just before using.
4.3.16 Soybean casein digest broth with lecithin and polyoxyethylene sorbitan monooleate
(SCDLP broth)
Prepare the SCDLP broth by dissolving 17,0 g of casein peptone, 3,0 g of soybean peptone, 5,0 g of
sodium chloride, 2,5 g of disodium hydrogen phosphate, 2,5 g of glucose and 1,0 g of lecithin in 1 000 ml
of water. Mix well and add 7,0 g of nonionic surfactant. Adjust the pH to a value between 6,8 and 7,2 (at
25 °C) with sodium hydroxide or hydrochloric acid. Sterilize by autoclaving. If the broth is not intended
to be used immediately after preparation, preserve it at 5 °C to 10 °C. Never use the SCDLP broth if it
has been kept for longer than one month after preparation.
[7] [8]
NOTE SCDLP is a typical neutralizer. Refer to ASTM E 1054 and EN 1040 for further information about
the other neutralizer.
5 Apparatus
Unless otherwise specified, use the following apparatus and materials.
5.1 Dry-heat sterilizer, capable of maintaining the temperature at a value between 160 °C and 180 °C
within ±2 °C of the set point at equilibrium conditions.
5.2 Autoclave, capable of maintaining a temperature of (121 ± 2) °C and a pressure of (103 ± 5) kPa.
5.3 Hotplate with stirrer, or hot-water bath.
5.4 pH-meter, capable of measuring to ±0,2 units.
5.5 Balance, capable of the available range of 100 g ± 0,1 g to 0,01 g ± 0,000 1 g.
5.6 Micro pipetter, sterile, with 1 000 µl and 200 µl tips.
5.7 Pipetter, capable of mounting the plastic pipettes.
5.8 Plastic pipette, sterile, with capacities of 50 ml ± 0,5 ml, 25 ml ± 0,25 ml, 10 ml ± 0,1 ml and
5 ml ± 0,05 ml.
5.9 Freezer, capable of operating at a temperature of –(80 ± 2) °C or –(20 ± 2) °C.
5.10 Liquid nitrogen bath, for the preservation approximately at −196 °C.
5.11 Membrane filter, sterile, with a pore size of 0,22 μm.
5.12 Inverted microscope, capable of being used for cultured cells observation.
5.13 Centrifuge, capable of being operated at a temperature of (4 ± 2) °C, and relative centrifugal force
of approximately 1 000 g.
5.14 Six wells plastic plate, sterile, with an adherent type, for plaque assay.
5.15 Flasks for cell culture use, sterile, with an adherent type, a cell culture area of 75 cm and with
the 0,2 μm filter vent capwhich can prevent bacterial contamination.
5.16 CO incubator, 5 % CO , at a temperature of (34 ± 2) °C and (37 ± 2) °C.
2 2
5.17 Incubator, capable of maintaining a temperature of (25 ± 1) °C.
5.18 Vortex mixer, if required.
5.19 Sonicator, if required.
5.20 Cover film that does not affect viral stability or absorb water (made of polyethylene, polypropylene
or polyester [poly(ethylene terephthalate)]. A film thickness of 0,05 mm to 0,10 mm is recommended.
NOTE Films cut from stomacher bags are also suitable.
5.21 Test tubes.
5.22 Centrifuge tube.
5.23 Gauze or absorbent cotton.
5.24 1 000 ml volumetric flask.
5.25 Stoppered Erlenmeyer flasks or media bottles, as required for preparation of media.
6 Preparation
6.1 Restoration of host cell from cryopreservation
Put a cryopreserved stock host cell in the water bath at 37 °C and defrost rapidly. Transfer the whole
ampule of the defrosted host cell into a new flask for cell culture use with 20 ml of growth medium.
Incubate the flask at 37 °C in the CO incubator for (24 ± 2) h. Then, observe the cell under an inverted
microscope if the cells are attached on the bottom of the flask. If the growth is confirmed, go to the next
step. If not, continue to keep the flask in the incubator.
Remove the growth medium from the flask of the host cell and add 20 ml of the new growth medium
to the flask. Incubate the flask at 37 °C in the CO incubator for (48 ± 2) h. Then, observe the monolayer
under the inverted microscope and confirm if the cells are cultured as a confluent cell monolayer on the
bottom of the flask. If the sufficient growth is not confirmed, continue to incubate until the sufficient
growth is confirmed. Then, proceed the subculture by taking the following steps of 6.2.
6 © ISO 2019 – All rights reserved

6.2 Subculture of host cell
Remove the growth medium from the flask and wash the cell monolayer with 5 ml of PBS (-) two times.
After removing an extra PBS (-), add 0,5 ml of trypsin EDTA solution to the flask and spread the solution
over the whole surface. Incubate the flask at 37 °C in the CO incubator for 10 min to 20 min. Then,
observe the flask if the cells are starting to come off, tap the side of the flask and disperse the cells. Add
5 ml of the growth medium to the flask and pipette the cell suspension gently to avoid the damage to the
cells. Transfer 1 ml of the cell suspension into a new flask for cell culture use with 20 ml of the growth
medium. The ratio of the suspension and the growth medium may be changed as needed. Incubate the
flask at 37 °C in the CO incubator for 3 days to 5 days until confluent cell monolayer is confirmed. The
culture period may be changed as needed.
In case of continuously subculturing the cell, repeat the same procedure from the beginning of 6.2.
6.3 Cell culture for measuring virus infectivity titer
The cell culture in a 6 wells plate is required for measuring the infectivity titer of virus by the plaque
assay. Transfer 1 ml of the subcultured cell suspension prepared in 6.2 into a sterile media bottle with
20 ml of the growth medium.
The ration of the suspension and the growth medium could be changed as needed.
Transfer 3 ml of the cell suspension into each hole of the 6 wells plastic plate for the plaque assay.
Incubate the plate at 37 °C in the CO incubator for 3 days to 5 days. The culture period could be changed
as needed. Observe the condition of the cells under the inverted microscope and confirm if the cells are
cultured as a confluent cell monolayer on the bottom of each hole.
6.4 Preparation of test inoculums
6.4.1 Influenza virus
Put the cryopreserved stock virus suspension in the water bath at 37 °C and defrost rapidly. Dilute the
defrosted stock influenza virus suspension with the maintenance medium to obtain a virus suspension
3 4
of 10 PFU/ml to 10 PFU/ml.
Remove the growth medium from the flask of the host cell monolayer prepared in 6.2, wash the
monolayer with 5 ml of the maintenance medium two times after removing an extra maintenance
medium. Inoculate with 1 ml of the adjusted influenza virus suspension on the surface of the cell
monolayer and spread to the whole surface. Incubate the flask at 34 °C in the CO incubator for 1 h
to allow the virus to adsorb to the cells. Add 20 ml of the maintenance medium and 30 μl of Trypsin
derived from pancreas and PBS (-) solution to the flask. Incubate the flask at 34 °C in the CO incubator
for 1 day to 3 days until about 80 % of the monolayer shows virus-induced cytopathic effect under the
inverted microscope. Transfer the contents of the flask into a centrifugal tube. Centrifuge the virus
suspension at 4 °C and 1 000 g for 15 min to separate the cell debris. Take the supernatant from the
centrifugal tube after the centrifugation. This is to be the stock influenza virus suspension. Divide
the suspension into new test tubes appropriately and cryopreseave at −80 °C in the freezer. Check the
concentration of the infectivity titer of virus if it is more than 10 PFU/ml. If the concentration is less
than 10 PFU/ml, prepare it from the beginning.
Just before using, put the cryopreserved stock influenza virus suspension in the water bath at 37 °C and
8 8
defrost it rapidly. Adjust the concentration of the virus suspension to 1 × 10 PFU/ml to 5 × 10 PFU/
ml with the maintenance medium. Then, dilute the adjusted virus suspension with water to obtain 10-
fold dilution suspension. This is to be the test inoculum. If it is not intended to be used immediately,
preserve it in the refrigerator at 4 °C.
6.4.2 Feline calicivirus
Put the cryopreserved stock virus suspension in the water bath at 37 °C and defrost rapidly. Dilute
the defrosted stock feline calicivirus suspension with the maintenance medium to obtain a virus
suspension of about 10 PFU/ml.
The concentration of the virus suspension could be changed as needed.
Remove the growth medium from the flask of the host cell monolayer prepared in 6.2, wash the
monolayer with 5 ml of the maintenance medium two times after removing an extra maintenance
medium. Inoculate with 1 ml of the adjusted feline calicivirus suspension on the surface of the cell
monolayer and spread to the whole surface. Incubate the flask at 37 °C in the CO incubator for 1 h
to allow the virus to adsorb to the cells. Add 5 ml to 20 ml of the maintenance medium to the flask.
Incubate the flask at 37 °C in the CO incubator for 1 day to 3 days until about 80 % of the monolayer
shows virus-induced cytopathic effect under the inverted microscope. Transfer the contents of the
flask into a centrifugal tube. Centrifuge the virus suspension at 4 °C and 1 000 g for 15 min to separate
the cell debris. Take the supernatant from the centrifugal tube after the centrifugation. This is to be
the stock feline calicivirus suspension. Divide the suspension into new test tubes appropriately and
cryopreseave at −80 °C in the freezer. Check the concentration of the infectivity titer of virus if it is
8 8
more than 10 PFU/ml. If the concentration is less than 10 PFU/ml, prepare it from the beginning.
Just before using, put the cryopreserved stock feline calicivirus suspension in the water bath at 37 °C
8 8
and defrost it rapidly. Adjust the concentration of the virus suspension to 1 × 10 PFU/ml to 5 × 10
PFU/ml with the maintenance medium. Then, dilute the adjusted virus suspension with water to obtain
10-fold dilution suspension. This is to be the test inoculum. If it is not intended to be used immediately,
preserve it in the refrigerator at 4 °C.
6.5 Preparation of test specimens
Main testing shall be performed using at least three specimens from each treated test material. At
least twelve specimens of the untreated material and nine specimens of treated material are required.
Three untreated test specimens are used to measure the infectivity titer of virus immediately after
inoculation. Three untreated test specimens and three treated test specimens are used to measure the
infectivity titer of virus after contacting for 24 h. Six untreated test specimens and six treated test
specimens are used for the control test in 6.6.
NOTE Use of more than three replicate specimens of the treated test material can help reduce variability,
especially for materials that show lower antiviral effects.
When testing a series of antiviral treatments for a single polymer, each antiviral treatment may be
compared to a single set of untreated specimens if all tests are conducted at the same time using the
same test inoculum.
Prepare flat (50 ± 2) mm × (50 ± 2) mm specimens of the treated and the untreated test materials.
Specimens should be no thicker than 10 mm. If it is difficult or impossible to cut the product into a
square of this size, then test specimens in other sizes and shapes may be used, as long as they can be
2 2
covered with a film which surface area is between 400 mm and 1 600 mm . It is preferable to prepare
test specimens from the final product itself. If not, then the test specimens may be prepared in a format
suitable for the testing using the same raw materials and processing methods as are normally used for
the product. If the test specimen is not a 50 mm × 50 mm square dimension, the used dimensions shall
be included in the test report.
When preparing specimens, be sure to avoid contamination with microorganisms or extraneous organic
debris. At the same time, be sure to avoid the specimens to contact each other. If any metal apparatus is
used to avoid cross-contamination, it is necessary to ensure that the metal does not have any antiviral
effect. If necessary, test specimens can be cleaned/disinfected/sterilized prior to the testing (e.g. by
wiping with 70 % ethanol resolved in water). Cleaning of the test specimen shall not be allowed to avoid
softening, dissolution of the surface coating or elution of components. If cleaning is required due to
gross contamination, the cleaning method shall be included in the test report.
8 © ISO 2019 – All rights reserved

6.6 Control test
6.6.1 General
The purpose of the control test is to confirm the suppressive efficiency of agent’s activity on the test
specimen. The suppressive efficiency means no cytotoxic effect on the host cell, no reduction of the cell
sensitivity to virus, and inactivation of the antiviral activity in the SCDLP broth (4.3.16). There is a case
that the agent which does not obviously show the cytotoxicity still reduces the infectious or replicating
ability of the virus, thus the agent interferes the evaluation of the antiviral activity. Therefore, the
control test shall be performed to exclude such a possibility of failing to evaluate properly.
6.6.2 Verification of cytotoxic effect on host cell
Add 10 ml of either the SCDLP broth or a suitable, validated neutralizer to each Petri dish with the
test specimens prepared in 6.5. It is important to ensure that the neutralizer completely washes the
specimens with pipetting the SCDLP broth at least four times. Three untreated test specimens and
three treated test specimens are used for this test.
Remove the growth medium from the 6 wells plastic plates prepared in 6.3, wash the monolayer once
with 3 ml of maintenance medium. After removing the extra maintenance medium, inoculate 0,1 ml of
the SCDLP broth recovered from the test specimen in the 2 wells. Put the plate at 34 °C for influenza
virus or at 37 °C for feline calicivirus in the CO incubator for 1 h, in the meantime, tilte the plates
every 15 min so the cell monolayer will be kept moist. Then, wash the monolayer once with 3 ml of the
maintenance medium. After removing the extra maintenance medium, pour 3 ml of the agar medium
for plaque assay (4.3.15) into the wells. Close a lid and keep it at room temperature for about 10 min.
After confirming the agar coagulates, incubate the plate at 34 °C for influenza virus or at 37 °C for feline
calicivirus in the CO incubator for 2 days to 3 days.
After the incubation, add 3 ml of the formalin solution (4.3.5) to the wells, then keep it at room
temperature for more than 1 h to fix the cells. Remove the formalin solution and the agar medium from
the wells, add 3 ml of the methylene blue solution (4.3.6), then keep it at room temperature for 15 min
to dye the cells. Wash the extra methylene blue solution with tapped water.
If no cytotoxicity is observed, proceed to the next step (see 6.6.3).
When cytotoxicity is observed, neutralizer should be carefully modified, changed or the amount of
neutralizer be carefully increased. When it is difficult to proceed with 10 ml of the neutralizer due to
the size and characteristics of the test specimen, then the volume of the solution may be increased.
If the neutralizer is modified, changed or the amount of neutralizer is increased, the same condition on
neutralizer shall be applied at the next step (6.6.3).
6.6.3 Verification of cell sensitivity to virus and the inactivation of antiviral activity
6.6.3.1 Procedure for verification
Add 10 ml of either the SCDLP broth (4.3.16) or a suitable, validated neutralizer to each Petri dish with
the test specimens prepared in 6.5. It is important to ensure that the neutralizer completely washes the
specimens by pipetting the SCDLP broth at least four times. Three untreated test specimens and three
treated test specimens are used for this test.
Take 5 ml of the SCDLP broth from each Petri dish recovered from the test specimen into new test
tubes, and as for negative control, take 5 ml of the new SCDLP broth into new three test tubes. Then,
add 50 μl of a virus suspension prepared to be a concentration of (4 to 6) × 10 PFU/ml into the 9 tubes
and keep them at 25 °C for 30 min.
Remove the growth medium from the 6 wells plastic plates prepared in 6.3, wash the monolayer once
with 3 ml of maintenance medium. After removing the extra maintenance medium, inoculate 0,1 ml
of each test suspension in the 2 wells. Put the plate at 34 °C for influenza virus or at 37 °C for feline
calicivirus in the CO incubator for 1 h to allow the virus to adsorb to the cells, in the meantime, tilt
the plates every 15 min so the monolayer keeps moisture. Then, wash the monolayer once with 3 ml of
maintenance medium. After removing the extra maintenance medium, pour 3 ml of agar medium for
plaque assay (4.3.15) into the wells. Close a lid and keep at room temperature for about 10 min. After
confirming the agar coagulates, incubate the plate at 34 °C for influenza virus or at 37 °C for feline
calicivirus in the CO incubator for 2 days to 3 days.
After incubation, add 3 ml of the formalin solution (4.3.5) to the wells, then keep it at room temperature
for more than 1 h to fix the cells. Remove the formalin solution and the agar medium from the wells, add
3 ml of the methylene blue solution (4.3.6), then keep it at room temperature for 15 min to dye the cells.
Wash the extra methylene blue solution with tapped water.
Select the wells containing 6 to 60 plaques. If the number of plaques in the wells containing the 0,1 ml
aliquots of the test susp
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