Measurement of antiviral activity on plastics and other non-porous surfaces

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.

Mesure de l'activité antivirale sur les matières plastiques et autres surfaces non poreuses

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Status
Published
Publication Date
06-May-2019
Current Stage
6060 - International Standard published
Start Date
07-May-2019
Completion Date
07-May-2019
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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 21702:2019(E)
ISO 2019
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ISO 21702:2019(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2019

All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may

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Published in Switzerland
ii © ISO 2019 – All rights reserved
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ISO 21702:2019(E)
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

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ISO 21702:2019(E)

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
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ISO 21702:2019(E)
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.
© ISO 2019 – All rights reserved v
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ISO 21702:2019(E)
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
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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
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ISO 21702:2019(E)
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
Host cell MDCK cell CRFK cell
(dog kidney cell origin) (cat kidney cell origin)
ATCC CCL-34 ATCC CCL-94
Growth medium EMEM (4.3.8) RPMI 1640 (4.3.8)

The other host cells can be used after appropriate validations regarding the sensitivity against each virus.

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.
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ISO 21702:2019(E)
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.

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ISO 21702:2019(E)

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.
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.

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ISO 21702:2019(E)
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.

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ISO 21702:2019(E)
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
...

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