Chemical disinfectants and antiseptics - Quantitative non-porous surface test for the evaluation of virucidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 2)

This European Standard 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 - with water.
This European Standard applies to products that are used in the veterinary area on non-porous surfaces without mechanical action i.e. in the breeding, husbandry, production, veterinary care facilities, transport and disposal of all animals except when in the food chain following death and entry to the processing industry.
EN 14885 specifies in detail the relationship of the various tests to one another and to "use recommendations".
NOTE 1   The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used.
NOTE 2   This method corresponds to a Phase 2 Step 2 test.
NOTE 3   Using this European Standard, it is possible to determine the virucidal activity of the undiluted product.
NOTE 4   This standard uses Porcine Parvovirus because Bovine Enterovirus Type 1 (ECBO) virus used in the suspension test EN 14675 cannot be used for surface testing because of its loss of titre during drying. Porcine Parvovirus has comparable resistance to ECBO virus.

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur Bestimmung der viruziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich auf nicht-porösen Oberflächen - Prüfverfahren und Anforderungen - Phase 2, Stufe 2

Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die viruzide Wirkung von chemischen Desinfektionsmitteln fest, die bei Verdünnung mit Wasser standardisierter Härte oder – im Falle gebrauchsfertiger Produkte – mit Wasser als homogene und physikalisch stabile Zubereitung vorliegen.
Dieses Dokument gilt für Produkte, die ohne mechanische Einwirkung zur Desinfektion von nicht porösen Oberflächen im Veterinärbereich verwendet werden, d. h. bei der Aufzucht, Haltung, Produktion, in veterinärmedizinischen Gesundheitseinrichtungen und beim Transport von Tieren sowie bei der Tierkörperbeseitigung, außer wenn die Tiere nach der Tötung durch Zuführung in die weiterverarbeitende Industrie in die Lebensmittelkette gelangen.
EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den „Anwendungsempfehlungen“ fest.
ANMERKUNG 1   Das beschriebene Verfahren ist zur Bestimmung der Wirkung von handelsüblichen Zubereitungen oder Wirkstoffen unter deren Anwendungsbedingungen vorgesehen.
ANMERKUNG 2   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 2.
ANMERKUNG 3   Bei Anwendung dieses Dokuments ist es möglich, die viruzide Wirkung des unverdünnten Produkts zu bestimmen.
ANMERKUNG 4   Für dieses Dokument wird das Porzine Parvovirus verwendet, da das für den Suspensionsversuch EN 14675 eingesetzte Bovine Enterovirus E (früher Bovine Enterovirus Typ 1 (ECBO)) aufgrund der Abnahme des Virentiters beim Trocknen nicht für Oberflächenprüfungen verwendet werden kann. Das Porzine Parvovirus und das ECBO-Virus weisen eine vergleichbare Resistenz auf.

Antiseptiques et désinfectants chimiques - Essai quantitatif de surfaces non poreuses pour l'évaluation de l'activité virucide des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions - Phase 2, étape 2

Le présent document spécifie une méthode d’essai et les prescriptions minimales relatives à l’activité virucide des désinfectants chimiques qui forment une préparation homogène et physiquement stable lorsqu’ils sont dilués dans de l’eau dure ou – dans le cas de produits prêts à l’emploi – dans l’eau.
Le présent document s’applique aux produits utilisés pour désinfecter sans action mécanique des surfaces non poreuses dans le domaine vétérinaire, à savoir pour la reproduction, l’élevage, la production, les établissements de soins vétérinaires, le transport et l’abattage de tous les animaux, sauf au cours de la chaîne alimentaire qui suit l’abattage et l’entrée dans l’industrie de transformation.
L’EN 14885 spécifie de façon détaillée les relations des différents essais entre eux et avec les « recommandations d’emploi ».
NOTE 1   La méthode décrite a pour objet de déterminer l’activité de formulations commerciales ou de substances actives dans leurs conditions d’emploi.
NOTE 2   Cette méthode correspond à un essai de phase 2, étape 2.
NOTE 3   L’utilisation du présent document permet de déterminer l’activité virucide d’un produit non dilué.
NOTE 4   Le présent document utilise le parvovirus porcin, car l’entérovirus bovin E [anciennement entérovirus bovin de type 1 (ECBO)] utilisé dans l’essai de suspension de l’EN 14675 ne peut pas être employé pour les essais de surface en raison de sa perte de titre pendant le séchage. Le parvovirus porcin présente une résistance comparable au virus ECBO.

Kemična razkužila in antiseptiki - Kvantitativni preskus na neporoznih površinah za vrednotenje virucidnega delovanja kemičnih razkužil in antiseptikov v veterini - Preskusna metoda in zahteve (faza 2, stopnja 2)

Ta evropski standard določa preskusno metodo in minimalne zahteve za virucidno delovanje kemičnih razkužil in antiseptikov, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčeni s trdo vodo oziroma z vodo pri proizvodih, ki so pripravljeni za uporabo.
Ta evropski standard se uporablja za izdelke v veterini na neporoznih površinah brez mehanskega delovanja, tj. pri vzreji, živinoreji, proizvodnji, v veterinarskih ambulantah, pri prevozu in odstranjevanju vseh živali, razen če so v prehrambeni verigi po smrti in so del predelovalne industrije.
Standard EN 14885 podrobno določa razmerje med različnimi preskusi in »priporočili za uporabo«.
OPOMBA 1: Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi pod pogoji, v katerih se uporabljajo.
OPOMBA 2 Ta metoda ustreza preskusu faze 2, stopnje 2.
OPOMBA 3: S tem evropskim standardom je mogoče določiti virucidno delovanje kemičnih razkužil pri nerazredčenem izdelku.
OPOMBA 4: V tem standardu je uporabljen prašičji parvovirus, saj govejega enterovirusa tipa 1 (ECBO), ki je uporabljen v preskusu suspenzije EN 14675, ni mogoče uporabiti za površinsko preskušanje zaradi izgube titra med sušenjem. Prašičji parvovirus ima primerljivo odpornost na virus ECBO.

General Information

Status
Published
Public Enquiry End Date
04-Apr-2017
Publication Date
03-Feb-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
25-Oct-2019
Due Date
30-Dec-2019
Completion Date
04-Feb-2020

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SLOVENSKI STANDARD
SIST EN 17122:2020
01-marec-2020
Kemična razkužila in antiseptiki - Kvantitativni preskus na neporoznih površinah

za vrednotenje virucidnega delovanja kemičnih razkužil in antiseptikov v veterini -

Preskusna metoda in zahteve (faza 2, stopnja 2)

Chemical disinfectants and antiseptics - Quantitative non-porous surface test for the

evaluation of virucidal activity of chemical disinfectants and antiseptics used in the

veterinary area - Test method and requirements (phase 2, step 2)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Oberflächenversuch zur

Bestimmung der viruziden Wirkung chemischer Desinfektionsmittel und Antiseptika für

den Veterinärbereich auf nicht-porösen Oberflächen - Prüfverfahren und Anforderungen

- Phase 2, Stufe 2

Antiseptiques et désinfectants chimiques - Essai quantitatif de surfaces non poreuses

pour l'évaluation de l'activité virucide des désinfectants chimiques utilisés dans le

domaine vétérinaire - Méthode d'essai et prescriptions - Phase 2, étape 2
Ta slovenski standard je istoveten z: EN 17122:2019
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
11.220 Veterinarstvo Veterinary medicine
SIST EN 17122:2020 en,fr,de

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 17122:2020
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SIST EN 17122:2020
EN 17122
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2019
EUROPÄISCHE NORM
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Quantitative non-
porous surface test for the evaluation of virucidal activity
of chemical disinfectants and antiseptics used in the
veterinary area - Test method and requirements - Phase2,
step2

Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -

quantitatif de surfaces non poreuses pour l'évaluation Quantitativer Oberflächenversuch zur Bestimmung der

de l'activité virucide des désinfectants et antiseptiques viruziden Wirkung chemischer Desinfektionsmittel

chimiques utilisés dans le domaine vétérinaire - und Antiseptika für den Veterinärbereich auf nicht-

Méthode d'essai et prescriptions - Phase 2, étape 2 porösen Oberflächen - Prüfverfahren und

Anforderungen - Phase 2, Stufe 2
This European Standard was approved by CEN on 23 September 2019.

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. Up-to-date lists and bibliographical references

concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN

member.

This European Standard exists 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, Turkey and

United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17122:2019 E

worldwide for CEN national Members.
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SIST EN 17122:2020
EN 17122:2019 (E)
Contents Page

European Foreword ...................................................................................................................................................... 3

Introduction .................................................................................................................................................................... 4

1 Scope .................................................................................................................................................................... 5

2 Normative references .................................................................................................................................... 5

3 Terms and definitions ................................................................................................................................... 5

4 Requirements for virucidal activity on surfaces.................................................................................. 6

5 Test method ...................................................................................................................................................... 6

5.1 Principle ............................................................................................................................................................. 6

5.2 Materials and reagents, including cell cultures ................................................................................... 7

5.3 Apparatus and glassware .......................................................................................................................... 10

5.4 Preparation of test organism suspensions and product test solutions .................................... 12

5.5 Procedure for assessing the virucidal activity of the product ..................................................... 13

5.6 Experimental data and calculation ........................................................................................................ 17

5.7 Verification of the methodology ............................................................................................................. 17

5.8 Expression of results ................................................................................................................................... 18

5.9 Test report ...................................................................................................................................................... 18

Annex A (informative) Referenced strains of national collections ......................................................... 20

Annex B (informative) .............................................................................................................................................. 21

Annex C (informative) Calculation of virus infectivity titre ....................................................................... 24

Annex D (informative) Example of a typical test report ............................................................................. 31

Annex E (informative) Full virucidal activity and activity against enveloped viruses only ........... 33

Bibliography ................................................................................................................................................................. 34

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SIST EN 17122:2020
EN 17122:2019 (E)
European Foreword

This document (EN 17122:2019) has been prepared by Technical Committee CEN/TC 216 “Chemical

disinfectants and antiseptics”, the secretariat of which is held by AFNOR.

This European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by May 2020, and conflicting national standards shall be

withdrawn at the latest by May 2020.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. CEN shall not be held responsible for identifying any or all such patent rights.

According to the CEN-CENELEC Internal Regulations, the national standards organisations of the

following countries are bound to implement this European Standard: 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, Turkey and the United

Kingdom.
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SIST EN 17122:2020
EN 17122:2019 (E)
Introduction

This document describes a surface test method for establishing whether a product proposed as a

disinfectant in the fields described in Clause 1 has or does not have virucidal activity on non-porous

surfaces.

The laboratory test closely simulates practical conditions of application. Chosen conditions (contact time,

temperature, organisms on surfaces etc.) reflect parameters which are found in practical situations

including conditions which may influence the action of disinfectants. Each use concentration found from

this test corresponds to defined experimental conditions.

The conditions are intended to cover general purposes and to allow reference between laboratories and

product types. Each utilization concentration of the chemical disinfectant found by this test corresponds

to defined experimental conditions.

However, for special applications the recommendations for use of a product can differ and therefore

additional test conditions might be needed, which cannot be covered by this document.

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SIST EN 17122:2020
EN 17122:2019 (E)
1 Scope

This document specifies a test method and the minimum requirements for virucidal activity of chemical

disinfectants that form a homogeneous physically stable preparation when diluted with hard water, or –

in the case of ready-to-use-products – with water.

This document applies to products that are used for disinfecting without mechanical action non-porous

surfaces in the veterinary area - i.e. in the breeding, husbandry, production, veterinary care facilities,

transport and disposal of all animals except when in the food chain following death and entry to the

processing industry.

EN 14885 specifies in detail the relationship of the various tests to one another and to “use

recommendations”.

NOTE 1 The method described is intended to determine the activity of commercial formulations or active

substances under the conditions in which they are used.
NOTE 2 This method corresponds to a Phase 2 Step 2 test.

NOTE 3 Using this document, it is possible to determine the virucidal activity of the undiluted product.

NOTE 4 This document uses Porcine Parvovirus because Bovine Enterovirus E (former Bovine Enterovirus Type

1 (ECBO)) virus used in the suspension test EN 14675 cannot be used for surface testing because of its loss of titre

during drying. Porcine Parvovirus has comparable resistance to ECBO virus.
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 14675, Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of

virucidal activity of chemical disinfectants and antiseptics used in the veterinary area – Test method and

requirements (Phase 2, step 1)

EN 14885, Chemical disinfectants and antiseptics — Application of European Standards for chemical

disinfectants and antiseptics

EN 10088-2, Stainless steels — Part 2: Technical delivery conditions for sheet/plate and strip of corrosion

resisting steels for general purposes
3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 14675 and EN 14885 apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

• IEC Electropedia: available at http://www.electropedia.org/
• ISO Online browsing platform: available at https://www.iso.org/obp
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SIST EN 17122:2020
EN 17122:2019 (E)
4 Requirements for virucidal activity on surfaces

The product shall demonstrate at least a decimal log (lg) reduction of 3 in virus titre of the parvovirus

and coronavirus test strains when tested in accordance with Table 1 and Clause 5. To claim virucidal

activity against enveloped viruses the product shall pass this standard with the coronavirus test strain

and to claim full virucidal activity the product shall pass both EN 14675 with the bovine enterovirus test

strain and this standard with the porcine parvovirus test strain.

NOTE See Annex E for further information on the appropriateness of claims of virucidal activity against

enveloped viruses and full virucidal activity
Table 1 — Minimum and additional test conditions
Minimum spectrum of test Full virucidal activity
organisms
Porcine Parvovirus, Strain NADL2
Virucidal activity against enveloped viruses
Feline Coronavirus, Strain Munich
Test temperature 10 °C ± 1 °C
Additional temperatures 4 °C ± 1 °C; 20 °C ± 1 °C; 40 °C ± 1 °C
Contact time The contact time(s) shall be selected from the values given below
Minimum contact time 1min ± 5 s
Other contact times 5min ± 10 s, 15 min ± 10 s, 30 min ± 10 s, 60 min ± 10 s
Maximum contact time 120 min ± 10 s
Interfering substances - low 3,0 g/l bovine albumin
level soiling
Interfering substances - high 10 g/l yeast extract and 10 g/l bovine albumin
level soiling
b Further contact time(s), interfering substance(s) or virus(es)
Additional conditions

The contact times for surface disinfectants stated in this table are chosen on the basis of the practical

conditions of the product. The recommended contact time for the use of the product is within the responsibility

of the manufacturer.

Where appropriate (specific purposes), additional specific virucidal activity shall be determined under other

conditions of time, temperature, and interfering substances (see 5.2.2.8) in accordance with 5.5, in order to take

into account intended specific use conditions. Additional virus(es) can be tested, if relevant. For the additional

conditions, the concentration defined as a result can be lower than the one obtained under the minimum test

conditions.

The determined virucidal concentration of the test product is suggested as being suitable for practical

situations of use.
5 Test method
5.1 Principle
5.1.1 Outline

A test suspension of viruses in a solution of interfering substances is inoculated onto a test surface and

dried. A prepared sample of the product under test is applied in a manner which covers the dried film.

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SIST EN 17122:2020
EN 17122:2019 (E)

The test surface is maintained at a specified temperature for a defined period of time. The test surface is

transferred to cell maintenance medium so that the action of the disinfectant is immediately neutralized.

The titre of the virus recovered from the test surface is determined.

The titre of the inoculum on a test surface treated with hard water in place of the disinfectant is also

determined and the reduction in virus titre attributed to the product is calculated by difference.

5.1.2 Test Organisms

The test is performed using the test organisms as specified in Clause 4, Table 1.

5.1.3 Variations

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.
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

to Clause 4, Table 1
a) Non-enveloped DNA virus
Porcine Parvovirus strain NADL2 (PPV)
b) Enveloped RNA virus
Feline Coronavirus, strain Munich (FeCoV)

NOTE Virus strains can be obtained from a national or international culture collection. PPV and FeCoV can be

obtained from the Friedrich-Loeffler-Insitut, Bundesforschungsinstitut für Tiergesundheit, Hauptsitz Insel Riems

Südufer 10, 17493, Greifswald-Insel Riems .

The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.12). 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 document 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.

This information is given for the convenience of users of this standard and does not constitute an endorsement by

CEN of this institute.
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EN 17122:2019 (E)

To improve reproducibility, it is recommended that commercially available – dehydrated if appropriate

- material 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 ± 1) °C.
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 [5.3.2.1 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 (Na HPO x 12H O) 2,89 g
2 4 2
Potassium phosphate, monobasic (KH PO ) 0,20 g
2 4
Water (5.2.2.2) to 1000,0 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

0,44 µm pore size filter and store at 4 °C in the dark.
5.2.2.5 Foetal calf serum (FCS)

FCS shall be certified free of viruses and mycoplasma. Extraneous viruses and mycoplasma may interfere

with cell and virus growth resulting in false results.
5.2.2.6 Trichloroacetic acid (10 % solution) (TCA)

Dissolve 10 g of TCA crystals in 80 ml of water (5.2.2.2), and 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

(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.1 c) or in the

autoclave [5.3.2.1 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 (5.3.2.6) for no

longer than one month;

Sigma N 7005 is an example of a suitable product available commercially. This information is given for the

convenience of users of this standard and does not constitute an endorsement by CEN of this product.

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SIST EN 17122:2020
EN 17122:2019 (E)

— prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water (5.2.2.2) and dilute to

1000 ml. Sterilize by membrane filtration (5.3.2.1 c). Store the solution in the refrigerator (5.3.2.6)

for no longer than one week;

— place 600 ml to 700 ml of water (5.2.2.2) in a 1000 ml volumetric flask (5.3.2.9) and add 6,0 ml of

solution A, then 8,0 ml of solution B. Mix and dilute to 1000 ml with water (5.2.2.2). The pH (5.3.2.4)

of the hard water shall be 7,0 ± 0,2. 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.

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 (CaCO ) 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.

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.

“Diluent” is generally used in the other European Standards in the veterinary area to prepare the

interfering substance. Since there is no experience in virucidal testing with diluent, water (5.2.2.2) is used

instead.

NOTE The term “interfering substance” is used even if it contains more than one substance.

5.2.2.8.2 Low level soiling (Bovine albumin solution)

Bovine serum albumin shall be used as commercially available or shall be prepared as follows:

Dissolve 3 g of bovine albumin fraction V (suitable for microbiological purposes) in 90 ml of water

(5.2.2.2) in a 100 ml volumetric flask (5.3.2.9). Make up to the mark with water (5.2.2.2).

Sterilize by membrane filtration (5.3.2.1 c). Keep in a refrigerator (5.3.2.6) and use within one month.

The final concentration of bovine albumin in the test procedure (5.5) is 3 g/l.

5.2.2.8.3 High level soiling (mixture of bovine albumin solution with yeast extract)

Dissolve 50 g yeast extract powder in 150 ml of water (5.2.2.2) in a 250 ml volumetric flask (5.3.2.9) and

allow foam to collapse. Make up to the mark with water (5.2.2.2). Transfer to a clean dry bottle and

sterilize in the autoclave (5.3.2.1 a). Allow to cool to 20 °C ± 1 °C.

Pipette 25 ml of this solution into a 50 ml volumetric flask (5.3.2.9) and add 10 ml of water (5.2.2.2).

Dissolve 5 g of bovine albumin fraction V (suitable for microbiological purposes) in the solution with

shaking and allow foam to collapse. Make up to the mark with water (5.2.2.2), sterilize by membrane

filtration (5.3.2.1 c), keep in a refrigerator (at 2 °C to 8 °C) (5.3.2.6) and use within one month.

The final concentration in the test procedure (5.5) is 10 g/l yeast extract and 10 g/l bovine albumin.

5.2.2.9 Medium for cell cultures

Eagle’s minimal essential medium (MEM) or equivalent, supplemented with FCS (5.2.2.5), antibiotics, and

other growth factors as needed shall be used.
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SIST EN 17122:2020
EN 17122:2019 (E)

a) A growth medium for cell multiplication is supplemented with 10 % FCS. Add 10 parts of FCS (5.2.2.5)

to 90 parts of MEM.

b) A maintenance medium to maintain the cell culture metabolism without stimulation of cell

proliferation is supplemented with 2 % FCS. Add 2 parts of FCS (5.2.2.5) to 98 parts of MEM.

Other media may be used if appropriate for certain cell lines.

See also bibliographic reference [2], EN 14675 and EN 12353 for more detailed descriptions.

5.2.2.10 Cell cultures

Cell monolayers shall be > 90 % confluent before inoculation. Cell lines are selected in accordance with

their sensitivity to the test organisms (5.2.1). Cells for virus titration, if used as suspensions in quantal

tests, shall be added to the dilutions of the test mixture (5.5.2) in such a density as to enable the formation

of a monolayer no longer than 2 days in the cell control. Cell cultures can be used as cell monolayers or

in suspensions for quantal tests. For details of cell lines see 5.5.1 e).
5.3 Apparatus and glassware
5.3.1 General

Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and

reagents or the sample, except those which are supplied sterile, by one of the following methods:

a) by moist heat, in the autoclave [5.3.2.1 a];
b) by dry heat, in the hot air oven [5.3.2.1 b].
5.3.2 Usual microbiological laboratory equipment
And, in particular, the following:
5.3.2.1 Apparatus for sterilization (moist and dry heat)

a) For moist heat sterilization, an autoclave capable of being maintained at ( 121 ) °C for a minimum

holding time of 15 min;

b) for dry heat sterilization, a hot air oven capable of being maintained at ( 180 ) °C for a minimum

+5 +5

holding time of 30 min, at ( 170 ) °C for a minimum holding time of 1 h or at ( 160 ) °C for a

0 0
minimum holding time of 2 h.

c) for media sterilization, use suitable membrane filtration apparatus with filters of diameter 47 mm to

50 mm and membranes with 0,22 μm pore size.

5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, and at additional test

temperatures ± 1 °C (5.5.1).
5.3.2.3 Inverted microscope for reading cell cultures microscopically
5.3.2.4 pH meter, having an accuracy of calibration of 0,1 pH units at 20 °C.
5.3.2.5 Stopwatch

Disposable sterile equipment is an acceptable alternative to reusable glassware.

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SIST EN 17122:2020
EN 17122:2019 (E)
5.3.2.6 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.7 Electromechanical agitator e.g. Vortex mixer

5.3.2.8 Containers: Petri plates, sterile test tubes, culture bottles or flasks of suitable capacity.

5.3.2.9 Volumetric flasks, calibrated at 20 °C.

5.3.2.10 Microtitre plates or tubes, petri dishes and flasks for cell culture use.

5.3.2.11 Membrane filtration apparatus for filtration

5.3.2.12 CO incubator (95 % air, 5 % CO ), capable of being controlled at (36 ± 1) °C, for incubation

2 2

of cell cultures. An incubator at (37 ± 1) °C may be used if an incubator at (36 ± 1) °C is not available.

5.3.2.13 Graduated sterile pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml.

NOTE Calibrated automatic pipettes can be used.
5.3.2.14 Magnetic stirrer for keeping cells in suspension before seeding

5.3.2.15 Ice producing machine or commercially available ice to cool the cell maintenance medium

and the reaction mixtures during the test (see 5.5.2 and 5.5.4)
5.3.2.16 Basin as ice bath with ice and water
5.3.2.17 Mechanical shaker
5.3.2.18 Centrifuge
5.3.2.19 Biological safety cabinet, class II
5.3.2.20 Freezer, −70 °C or less

5.3.2.21 Temperature controlled cabinet, capable of being controlled at 10 °C ± 1 °C

5.3.2.22 Vacuum Diaphragm Pump (e.g. throughput max. 3,8 m /h final vacuum < 75 mbar)

5.3.2.23 Cryotubes
5.3.3 Test surface

These shall be 1.4301 (EN 10088-1) stainless steel discs (2 cm diameter discs) with Grade 2 B finish on

both sides, in accordance with the requirements of EN 10088-2. The discs shall be as flat as possible and

this is best achieved by using stainless steel of a gauge of 1,2 mm or 1,5 mm. The discs shall be handled

only with forceps and used only once.
4 ®

Vortex is an example of a suitable product available commercially. This information is given for the convenience

of users of this standard and does not constitute an endorsement by CEN of this product.

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SIST EN 17122:2020
EN 17122:2019 (E)

Prior to use the discs shall be placed in a container with an appropriate quantity of 5 % per volume Decon

90 , or of 1 % Blanisol-Pur for 60 min, in a manner so that they do not stick together and the surface is

not damaged. Rinse the discs with running freshly distilled water (5.2.2.2) or demineralized water for

10 s, preventing them from drying to any extent.

Rinse the discs with water (5.2.2.2) for a further 10 s to ensure complete removal of the surfactant. To

supply a satisfactory flow of water, a sterilized fluid dispensing pressure vessel with suitable hose and

connectors or other suitable method can be used and regulated to supply approximately 2000 ml per

min. Dip the disc in a bath containing isopropanol (IPA) for 15 min. Remove the discs and rinse them with

water (5.2.2.2) for at least 10 s. Sterilize by autoclaving.

NOTE Suitable stainless steel discs can usually be purchased from local engineering companies.

5.4 Pre
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