oSIST prEN 17914:2022

SLOVENSKI STANDARD
oSIST prEN 17914:2022
01-december-2022
Kemična razkužila in antiseptiki - Kvantitativna suspenzijska preskusna metoda za
vrednotenje virucidnega delovanja kemičnih razkužil in antiseptikov v živilski in
drugih industrijah, gospodinjstvu in javnih ustanovah - Preskusna metoda in
zahteve (faza 2, stopnja 1)
Chemical disinfectants and antiseptics - Quantitative suspension test method for the
evaluation of virucidal activity of chemical disinfectants and antiseptics in food, industrial,
domestic and institutional areas - Test method and requirements (phase 2, step 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur
Bestimmung der viruziden Wirkung chemischer Desinfektionsmittel und Antiseptika in
den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen -
Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
Antiseptiques et désinfectants chimiques - Méthode d’essai quantitatif de suspension
pour l’évaluation de l’activité virucide des antiseptiques et des désinfectants chimiques
dans l’agroalimentaire, l’industrie, la sphère domestique et les collectivités - Méthode
d’essai et exigences (phase 2, étape 1)
Ta slovenski standard je istoveten z: prEN 17914
ICS:
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
oSIST prEN 17914:2022 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 17914:2022


DRAFT
EUROPEAN STANDARD
prEN 17914
NORME EUROPÉENNE

EUROPÄISCHE NORM

October 2022
ICS 71.100.35
English Version

Chemical disinfectants and antiseptics - Quantitative
suspension test method for the evaluation of virucidal
activity of chemical disinfectants and antiseptics in food,
industrial, domestic and institutional areas - Test method
and requirements (phase 2, step 1).
Antiseptiques et désinfectants chimiques - Méthode Chemische Desinfektionsmittel und Antiseptika -
d'essai quantitatif de suspension pour l'évaluation de Quantitativer Suspensionsversuch zur Bestimmung der
l'activité virucide des antiseptiques et des viruziden Wirkung chemischer Desinfektionsmittel
désinfectants chimiques dans l'agroalimentaire, und Antiseptika in den Bereichen Lebensmittel,
l'industrie, la sphère domestique et les collectivités - Industrie, Haushalt und öffentliche Einrichtungen -
Méthode d'essai et exigences (phase 2, étape 1) Prüfverfahren und Anforderungen (Phase 2, Stufe 1)
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 216.

If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2022 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 17914:2022 E
worldwide for CEN national Members.

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Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 7
3 Terms and definitions . 8
4 Requirements . 9
5 Test methods . 10
5.1 Principle . 10
5.2 Materials and reagents, including cell cultures . 11
5.2.1 Test organisms . 11
5.2.2 Culture media, reagents and cell cultures . 11
5.3 Apparatus and glassware . 14
5.4 Preparation of test organism suspensions and product test solutions . 16
5.4.1 Test organism suspension (test virus suspension) . 16
5.4.2 Product test solutions . 16
5.5 Procedure for assessing the virucidal activity of the product . 17
5.5.1 General . 17
5.5.2 Test procedure . 18
5.5.3 Modified method for ready-to-use products . 19
5.5.4 Cytotoxicity caused by product test solutions . 19
5.5.5 Control of efficiency of suppression of product’s activity . 21
5.5.6 Reference test for virus inactivation . 21
5.5.7 Titration of the virus control . 21
5.5.8 Titration of test samples . 21
5.6 Experimental data and calculation . 21
5.6.1 Protocol of results . 21
5.6.2 Calculation of infectivity titre (TCID50 or PFU) . 22
5.7 Verification of the methodology . 22
5.8 Expression of results . 23
5.8.1 General . 23
5.8.2 Calculation of the limited spectrum virucidal activity of products . 23
5.8.3 Calculation of the virucidal activity of products . 23
5.8.4 Calculation of the virucidal activity against enveloped viruses . 24
5.9 Test report . 24
Annex A (informative) Detoxification of test mixtures by molecular sieving . 26
TM
A.1 Molecular sieving with Sephadex LH 20 . 26
A.1.1 Principle . 26
A.1.2 Sephadex suspension . 26
A.1.3 Procedure. 26
A.2 Molecular sieving using MicroSpin™ S 400 HR . 28
A.3 Determination of the residual virus titre by the large-volume-plating (LVP) method . 28
A.3.1 General . 28
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A.3.2 Example for the calculation of titres and the reduction according to the large-
volume-plating Method . 29
Annex B (informative) Calculation of the viral infectivity titre . 31
B.1 Quantal tests — Example of TCID determination by the Spearman-Kärber method . 31
50
B.2 Plaque test . 32
B.3 Biometrical evaluation of experimental approaches and assessment of the
disinfecting effect on the virus (reduction [R]) . 32
B.3.1 General . 32
B.3.2 Calculating the virus titre . 32
B.3.3 Calculating the reduction . 33
B.3.4 Practical example . 33
Annex C (informative) Presentation of test results of one active concentration . 35
Bibliography . 37

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European foreword
This document (prEN 17914:2022) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
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Introduction
This document specifies a suspension test for establishing whether a chemical disinfectant or an
antiseptic has a virucidal activity in the area and fields described in the scope.
This laboratory test takes into account practical conditions of application of the product including
contact time, temperature, test organisms and interfering substances, i.e. conditions which may
influence its action in practical situations. Each utilization concentration of the chemical disinfectant or
antiseptic found by this test corresponds to the chosen experimental conditions.
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1 Scope
This document specifies a test method and the minimum requirements for virucidal activity of chemical
disinfectant and antiseptic products that form a homogeneous physically stable preparation when
diluted with hard water – or in the case of ready-to-use products, i. e, products that are not diluted
when applied – with water. Products can only be tested at a concentration of 80 % (97 %, with a
modified method for special cases) as some dilution is always produced by adding the test organisms
and interfering substance.
This document applies to products that are used in the food, industrial, domestic and institutional area
for hand disinfection, hygienic hand washing, surface disinfection by wiping, spraying, flooding or
otherwise, and for disinfection of textile and equipment, excluding areas and situations where
disinfection is medically indicated and excluding products used on living tissues.
This document applies at least to the following:
a) processing, distribution and retailing of:
— food of animal origin:
— milk and milk products;
— meat and meat products;
— fish, seafood, and related products;
— eggs and egg products;
— animal feeds;
— etc.;
— food of vegetable origin:
— beverages;
— fruits, vegetables and derivatives (including sugar, distillery, etc.);
— flour, milling and baking;
— animal feeds;
— etc.;
b) institutional and domestic areas:
— catering establishments;
— public areas;
— public transports;
— schools;
— nurseries;
— shops;
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— sports rooms;
— waste containers (bins, etc.);
— hotels;
— dwellings;
— clinically non sensitive areas of hospitals;
— offices;
— etc.;
c) industries other than food:
— packaging material;
— biotechnology (yeast, proteins, enzymes, etc.);
— pharmaceutical;
— cosmetics and toiletries;
— textiles;
— space industry, computer industry;
— etc.
NOTE EN 14885 specifies in detail the relationship of the various tests to one another and to “use
recommendations”.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
EN 12353, Chemical disinfectants and antiseptics - Preservation of test organisms used for the
determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal
(including bacteriophages) activity
EN 14885, Chemical disinfectants and antiseptics - Application of European Standards for chemical
disinfectants and antiseptics
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3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 and the following apply.
3.1
cytotoxicity
morphological alteration of cells and/or their destruction or their reduced sensitivity to virus
multiplication caused by the product
3.2
plaque forming units
PFU
number of infectious virus particles per unit volume (ml)
3.3
reference test for virus inactivation
test with a defined product (e.g. glutaraldehyde) in parallel with a product under test for the internal
control of the test
3.4
TCID
50
50 % infecting dose of a virus suspension or that dilution of the virus suspension that induce a CPE (3.5)
in 50 % of cell culture units
3.5
viral cytopathic effect
CPE
morphological alteration of cells and/or their destruction as a consequence of virus multiplication
3.6
viral plaque
area of lysis formed in a cell monolayer under semisolid medium due to infection by and multiplication
of a single infectious virus particle
3.7
virus titre
amount of infectious virus per unit volume present in a cell culture lysate or in a solution
3.8
virucidal activity
capability of a product to reduce the number of infectious virus particles of adenovirus and murine
norovirus for for hygienic handrub and handwash, for surfaces disinfection in applications other than
food processing, distribution and retailing, murine norovirus for food processing, distribution and
retailing areas, and murine parvovirus for uses at a temperature >40°C
3.9
limited spectrum virucidal activity
capability of a product to reduce the number of infectious virus particles of all enveloped viruses
(Annex A) and norovirus, rotavirus and adenovirus for hygienic handrub and handwash
3.10
virucidal activity against enveloped viruses
capability of a product to reduce the number of infectious virus particles of Vaccinia virus, covering
activity against all enveloped viruses
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4 Requirements
The product shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre when tested in
accordance with Table 1 and Clause 5.
Table 1 — Minimum and additional test conditions
Test Conditions Hygienic handrub Surfaces disinfection Surfaces disinfection Disinfection of
and handwash in industries, food in institutional and textile
processing, domestic areas

distribution and
retailing areas
Minimum Limited spectrum Virucidal activity for Virucidal activity for Virucidal activity for
spectrum of virucidal activity biopharmaceutical institutional and institutional and
test organisms industry domestic use domestic use
Adenovirus
Murine Parvovirus Adenovirus Adenovirus
Murine Norovirus
Virucidal activity for Murine Norovirus Murine Norovirus
Virucidal activity
industries other than
against enveloped Virucidal activity Virucidal activity
biopharmaceutical
viruses against enveloped
against enveloped
and food industry
viruses viruses
Vaccinia virus
Adenovirus
Vaccinia virus Vaccinia virus

Murine Norovirus
Virucidal activity
Virucidal activity for
Murine Parvovirus for
food industry
temperatures > 40°C
Murine Norovirus
Virucidal activity
against enveloped
viruses
Vaccinia virus
Virucidal activity
Murine Parvovirus for
temperatures > 40°C
additional Any relevant test organism
Test
according to the manufacturer’s recommendation, but at / between
temperature
 20 °C 4 °C and ≤ 40 °C 4 °C and ≤ 40 °C 10°C and 70°C for
main wash cycle
10°C and 20°C for
rinse cycle
Contact time according to the manufacturer’s recommendation
 but between  but no longer than but no longer than
 30 s and 60 s 60 min 60 min 60 min for wash cycle
e
30 s and 120s for high 20 min for rinse cycle
level virucidal activity
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Test Conditions Hygienic handrub Surfaces disinfection Surfaces disinfection Disinfection of
and handwash in industries, food in institutional and textile
processing, domestic areas

distribution and
retailing areas
Interfering substance
clean conditions 0,3 g/l bovine albumin 0,3 g/l bovine albumin 0,3 g/l bovine albumin 0,3 g/l bovine
solution (hygienic solution solution albumin solution

handrub) (rinse cycle)
and/or and/or
3,0 g/l bovine
albumin solution
(wash cycle)
dirty conditions 3,0 g/l bovine albumin 3,0 g/l bovine albumin 3,0 g/l bovine albumin
solution (hygienic solution solution
handwash)
Additional clean or dirty; any relevant substance any relevant substance any relevant
conditions any relevant substance substance
5 Test methods
5.1 Principle
5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use
products) is added to a test suspension of viruses in a solution of an interfering substance. The mixture
is maintained at one of the temperatures and the contact times specified in Clause 4, Table 1 and 5.5.1.1.
At the end of this contact time, an aliquot is taken; the virucidal action in this portion is immediately
suppressed by a validated method (dilution of the sample in ice-cold cell maintenance medium). The
dilutions are transferred into cell culture units (petri dishes, tubes or wells of microtitre plates) either
using monolayer or cell suspension. Infectivity tests are done either by plaque test or quantal tests.
After incubation, the titres of infectivity are calculated according to Spearman and Kärber (quantal
tests, C.1) or by plaque counting (plaque test, C.2) and evaluated. Reduction of virus infectivity is
calculated from differences of lg virus titres before (virus control) and after treatment with the product.
NOTE Handwash products are always prediluted with hard water (5.2.2.7) and the resulting solution is
regarded as a ready-to-use product (5.4.2).
5.1.2 The test is performed using the test organisms as specified in Clause 4, Table 1.
5.1.3 Other contact times and temperatures within the limits specified in Clause 4, Table 1 may be
used. Additional interfering substances and test organisms may be used.
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5.2 Materials and reagents, including cell cultures
5.2.1 Test organisms
The virucidal activity shall be evaluated using the following strains as test organisms selected according
1)
to Clause 4, Table 1 :
2)
a) Non-enveloped RNA virus
1) Murine norovirus, strain S99 Berlin
b) Non-enveloped DNA virus
1) Adenovirus type 5, strain Adenoid 75, ATCC VR-5
2) Murine Parvovirus, minute virus of mice, strain Crawford, ATCC VR-1346
c) Enveloped DNA virus
1) Modified Vaccinia virus, strain Ankara (MVA), ATCC VR-1508.
The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.1.3).
The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test
and its control and validation.
If additional test organisms are used, they shall be kept and used under optimum growth conditions
(temperature, time, atmosphere, media) noted in the test report. If these additional test organisms are
not classified at a reference centre, their identification characteristics shall be stated. In addition, they
shall be held by the testing laboratory or national culture collection under a reference for five years.
5.2.2 Culture media, reagents and cell cultures
5.2.2.1 General
All weights of chemical substances given in this European Standard refer to the anhydrous salts.
Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for
consequent molecular weight differences.
The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be
free from substances that are toxic or inhibitory to the test organisms.
To improve reproducibility, it is recommended that commercially available material – if appropriate- is
used for the preparation of culture media. The manufacturer's instructions relating to the preparation
of these products should be rigorously followed.
For each culture medium and reagent, a time limitation for use should be fixed.
All specified pH values are measured at 20 °C ± 1 °C.

1)
The ATCC numbers are the collection numbers of strains supplied by these culture collections. This information is given
for the convenience of users of this European Standard and does not constitute an endorsement by CEN of the product named.
2)
Murine Norovirus may be obtained from Friedrich-Loeffler-Insitut “Bundesforschungsinstitut" für Tiergesundheit,
Hauptsitz Insel Riems Südufer 10, 17493, Greifswald-Insel Riems; phone: +49 38351 7-0, fax: +49 038351 7-121.
http://www.fli.bund.de.
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5.2.2.2 Water
The water shall be freshly glass-distilled water and not demineralized water. If distilled water of
adequate quality is not available, water for injections (see bibliographic reference [1]) may be used.
Sterilize in the autoclave [5.3.1.1 a)]. Sterilization is not necessary if the water is used e.g. for
preparation of culture media and subsequently sterilized.
See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Phosphate buffered saline (PBS)
Sodium chloride (NaCl) 8,00 g
Potassium chloride (KCl) 0,20 g
Disodium hydrogen phosphate, 12-hydrate 2,89 g
(Na2HPO4 × 12H2O)
Potassium phosphate, monobasic (KH2PO4) 0,20 g
Water (5.2.2.2) to 1 000,0 ml
5.2.2.4 Neutral Red (1:1000 solution)
Prepare neutral red (Sigma N7005) stock solution at 0,1 mg/ml in water (5.2.2.2). Filter through a
0,40 µm pore size filter and store at 4 °C in the dark.
5.2.2.5 Foetal calf serum (FCS)
FCS has to be certified free of viruses and mycoplasma. Extraneous viruses and mycoplasma may
interfere with cell and virus growth resulting in false results.
For RAW 264.7 cells, special FCS has to be used due to the cells’ high sensitivity to endotoxins.
5.2.2.6 Trichloroacetic acid (10 % solution) (TCA)
Dissolve 10 g of TCA crystals in 80 ml of water (5.2.2.2), then adjust the volume to 100 ml with water.
Stir to complete solution.
5.2.2.7 Hard water for dilution of products
For the preparation of 1 l of hard water, the procedure is as follows:
— prepare solution A: dissolve 19,84 g magnesium chloride (MgCl ) and 46,24 g calcium chloride
2
(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.1.7) or in the
2
autoclave [5.3.1.1 a)]. Autoclaving – if used - may cause a loss of liquid. In this case make up to
1 000 ml with water (5.2.2.2) under aseptic conditions. Store the solution in the refrigerator
(5.3.1.8) for no longer than one month;
— prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to
3
1000 ml. Sterilize by membrane filtration (5.3.1.7). Store the solution in the refrigerator (5.3.1.8)
for no longer than one week;
— place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.1.12) and add 6,0 ml
(5.3.1.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The
pH (5.3.1.4) of the hard water shall be 7,0 ± 0,2. (5.3.1.4). If necessary, adjust the pH by using a
solution of approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately
36,5 g/l (about 1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
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NOTE When preparing the product test solutions (5.4.2), the addition of the product to the hard water
produces different final water hardness in each test tube. In any case, the final hardness in the test tube expressed
as calcium carbonate (CaCO3) is lower than 375 mg/l.
5.2.2.8 Interfering substance
5.2.2.8.1 General
The interfering substance shall be chosen according to the conditions of use laid down for the product.
The interfering substance shall be sterile and prepared at 10 times its final concentration in the test (50
times in the case of the modified method, 5.2.2.8.4).
The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.
mineral substances, protein, carbohydrates, lipids and detergents) shall be defined.
NOTE The term “interfering substance” is used even if it contains more than one substance.
5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration)
Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in a final volume of
100 ml of water (5.2.2.2).
Sterilize by membrane filtration (5.3.1.7), keep in a refrigerator (5.3.1.8) and use within one mon
...