SIST EN 16777:2019

Chemical disinfectants and antiseptics - Quantitative non-porous surface test without mechanical action for the evaluation of virucidal activity of chemical disinfectants used in the medical 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 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 European Standard applies to products that are used in the medical area for disinfecting non-porous surfaces including surfaces of medical devices without mechanical action.
This European Standard applies to areas and situations where disinfection is medically indicated. Such indications occur in patient care, for example:
-   in hospitals, in community medical facilities, and in dental institutions;
-   in clinics of schools, of kindergartens, and of nursing homes;
and may occur in the workplace and in the home.
It may also include services such as laundries and kitchens supplying products directly for the patients.
NOTE 1   The method described is intended to determine the activity of commercial formulations or active substances on viruses in the conditions in which they are used.
NOTE 2   This method corresponds to a phase 2, step 2 test.
EN 14885 specifies in detail the relationship of the various tests to one another and to "use recommendations".

Chemische Desinfektionsmittel und Antiseptika-Quantitativer Versuch auf nicht porösen Oberflächen ohne mechanische Einwirkung zur Bestimmung der viruziden Wirkung im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die viruzide Wirkung von chemischen Desinfektions¬mitteln fest, die bei Verdünnung mit Wasser standardisierter Härte als homogene, physikalisch stabile Zubereitung vorliegen, beziehungsweise bei gebrauchsfertigen Produkten bei Verdünnung mit Wasser.
Dieses Dokument gilt für Produkte, die im medizinischen Bereich zur Desinfektion von nicht porösen Oberflächen, einschließlich Oberflächen auf Medizinprodukten, ohne mechanische Einwirkung verwendet werden.
Dieses Dokument gilt für Bereiche und unter Bedingungen, wo eine Desinfektion aus medizinischen Gründen angezeigt ist. Indikationen dieser Art liegen z. B. vor bei der Patientenbetreuung:
-   in Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich,
-   in medizinischen Bereichen in Schulen, Kindergärten und Heimen
und können auch am Arbeitsplatz oder im privaten Bereich gegeben sein.
Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung von Patienten dienen.
ANMERKUNG 1   Das beschriebene Verfahren dient zur Bestimmung der Wirkung von handelsüblichen Zubereitungen oder Wirkstoffen auf Viren unter den Bedingungen, unter denen sie angewendet werden.
ANMERKUNG 2   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 2.
EN 14885 legt im Einzelnen die Beziehung der verschiedenen Prüfungen untereinander sowie zu den "Anwendungsempfehlungen" fest.

Antiseptiques et désinfectants chimiques - Essai quantitatif de surface non-poreuse sans action mécanique pour l'évaluation de l'activité virucide désinfectants chimiques utilisés dans le domaine humain médicine - Méthode d'essai et prescriptions (phase 2/étape 2)

Le présent document décrit une méthode d’essai et les exigences minimales relatives à l’activité virucide des désinfectants chimiques qui forment une préparation homogène, 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 en médecine humaine pour la désinfection des surfaces non poreuses, y compris les surfaces des dispositifs médicaux, sans action mécanique.
Le présent document s’applique aux domaines et situations où la désinfection est indiquée en médecine humaine. De telles indications se rencontrent dans le cadre des soins apportés aux patients, par exemple :
-   dans des hôpitaux, dans des centres de soins médicaux et des cabinets dentaires ;
-   dans des infirmeries d’écoles, de jardins d’enfants et de maisons de retraite ;
et peuvent aussi se rencontrer sur les lieux de travail ou à domicile.
Elles peuvent également concerner des services tels que des blanchisseries et des cuisines qui fournissent des produits directement aux patients.
NOTE 1   La méthode décrite vise à déterminer l’activité des formulations commerciales ou des substances actives sur les virus dans les conditions dans lesquelles elles sont utilisées.
NOTE 2   Cette méthode correspond à un essai de phase 2, étape 2.
L’EN 14885 spécifie de façon détaillée les relations des différents essais entre eux et avec les « recommandations d’emploi ».

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

Ta evropski standard določa preskusno metodo in minimalne zahteve za virucidno delovanje kemičnih razkužil, ki tvorijo homogen, fizikalno stabilen pripravek, če so razredčena s trdo vodo oziroma z vodo pri proizvodih, ki so pripravljeni za uporabo.
Ta evropski standard se uporablja za izdelke, ki se na zdravstvenem področju uporabljajo za razkuževanje neporoznih površin, vključno s površinami medicinskih pripomočkov, brez mehanskega delovanja.
Ta evropski standard se uporablja za področja in primere, ko obstajajo zdravniške indikacije za dezinfekcijo. Te indikacije se pojavljajo pri negi bolnikov, na primer:
– v bolnišnicah, javnih zdravstvenih in zobozdravstvenih ustanovah;
– v ambulantah šol, vrtcev in domov za starejše;
in lahko se pojavljajo na delovnem mestu ali doma.
Vključujejo lahko tudi storitve, kot so pralnice in kuhinje, ki proizvode dostavljajo neposredno bolnikom.
OPOMBA 1: Opisana metoda je namenjena določevanju dejavnosti komercialnih oblik ali aktivnih snovi na virusih pod pogoji, v katerih se uporabljajo.
OPOMBA 2: Ta metoda ustreza preskusu stopnje 2 faze 2.
Standard EN 14885 podrobno določa razmerje med različnimi preskusi in »priporočili za uporabo«.

General Information

• Status: Published
• Public Enquiry End Date: 09-Feb-2017
• Publication Date: 13-Jan-2019
• ICS: 11.080.20 - Disinfectants and antiseptics
• Technical Committee: KDS - Cosmetics, chemical disinfectants and surface active agents
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 09-Jan-2019
• Due Date: 16-Mar-2019
• Completion Date: 14-Jan-2019
• Ref Project: EN 16777:2018 - Chemical disinfectants and antiseptics - Quantitative non-porous surface test without mechanical action for the evaluation of virucidal activity of chemical disinfectants used in the medical area - Test method and requirements (phase 2/step 2)

Relations

• Revised: oSIST prEN 16777:2014 - Chemical disinfectants and antiseptics - Quantitative non-porous surface test without mechanical action for the evaluation of virucidal activity of chemical disinfectants used in the medical area - Test method and requirements (phase 2/step 2)
• Effective Date: 01-Feb-2019

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN 16777:2019
01-februar-2019
.HPLþQDUD]NXåLODLQDQWLVHSWLNL.YDQWLWDWLYQLSUHVNXVQDQHSRUR]QLKSRYUãLQDK
EUH]PHKDQVNHJDGHORYDQMD]DYUHGQRWHQMHYLUXFLGQHJDGHORYDQMDNHPLþQLK
UD]NXåLOYKXPDQLPHGLFLQL3UHVNXVQDPHWRGDLQ]DKWHYHID]DVWRSQMD
Chemical disinfectants and antiseptics - Quantitative non-porous surface test without
mechanical action for the evaluation of virucidal activity of chemical disinfectants used in
the medical area - Test method and requirements (phase 2/step 2)
Chemische Desinfektionsmittel und Antiseptika-Quantitativer Versuch auf nicht porösen
Oberflächen ohne mechanische Einwirkung zur Bestimmung der viruziden Wirkung im
humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Antiseptiques et désinfectants chimiques - Essai quantitatif de surface non-poreuse sans
action mécanique pour l'évaluation de l'activité virucide désinfectants chimiques utilisés
dans le domaine humain médicine - Méthode d'essai et prescriptions (phase 2/étape 2)
Ta slovenski standard je istoveten z: EN 16777:2018
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
SIST EN 16777:2019 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN 16777:2019

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SIST EN 16777:2019


EN 16777
EUROPEAN STANDARD

NORME EUROPÉENNE

December 2018
EUROPÄISCHE NORM
ICS 11.080.20
English Version

Chemical disinfectants and antiseptics - Quantitative non-
porous surface test without mechanical action for the
evaluation of virucidal activity of chemical disinfectants
used in the medical area - Test method and requirements
(phase 2/step 2)
Antiseptiques et désinfectants chimiques - Essai Chemische Desinfektionsmittel und Antiseptika -
quantitatif de surface non poreuse sans action Quantitativer Versuch auf nicht porösen Oberflächen
mécanique pour l'évaluation de l'activité virucide des ohne mechanische Einwirkung zur Bestimmung der
désinfectants chimiques utilisés dans le domaine viruziden Wirkung im humanmedizinischen Bereich -
médical - Méthode d'essai et exigences (phase 2/étape Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
2)
This European Standard was approved by CEN on 24 September 2018.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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
© 2018 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 16777:2018 E
worldwide for CEN national Members.

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EN 16777:2018 (E)
Contents Page
European foreword . 4
Introduction . 5
1 Scope . 6
2 Normative references . 6
3 Terms and definitions . 7
4 Requirements for virucidal activity on surfaces. 7
5 Test methods . 8
5.1 Principle . 8
5.2 Materials and reagents, including cell cultures . 8
5.2.1 Test organisms . 8
5.2.2 Culture media, reagents and cell cultures . 9
5.3 Apparatus and glassware . 12
5.3.1 General . 12
5.3.2 Usual microbiological laboratory equipment . 13
5.3.3 Test surfaces . 14
5.4 Preparation of test organism suspensions and product test solutions . 14
5.4.1 Test organisms suspensions (test virus suspension) . 14
5.4.2 Product test solution . 14
5.5 Procedure for assessing the virucidal activity of the product . 15
5.5.1 Experimental conditions . 15
5.5.2 Test procedure . 16
5.5.3 Cytotoxicity caused by product solutions . 17
5.5.4 Control of efficiency for suppression of disinfectant virucidal activity . 18
5.5.5 Reference test for virus inactivation . 19
5.5.6 Titration of the virus control . 19
5.6 Experimental data and calculation . 19
5.6.1 Protocol of the results . 19
5.6.2 Calculation of infectivity titre (TCID PFU) . 19
50 –
5.7 Verification of the methodology . 20
5.8 Expression of results . 20
5.8.1 General . 20
5.8.2 Calculation of the virucidal activity of products . 20
5.9 Test report . 21
Annex A (informative) Examples of viruses sorted according to their presence in the
human body in case of virus infection . 23
Annex B (normative) Detoxification of test mixtures by molecular sieving . 25
B.1 Molecular sieving with Sephadex™ LH 20 . 25
B.1.1 Principle . 25
B.1.2 Sephadex suspension . 25
B.1.3 Procedure. 25
B.2 Molecular sieving using MicroSpin™ S 400 HR . 27
2

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B.3 Determination of the residual virus titre by the large-volume-plating (LVP) method . 27
B.3.1 General . 27
B.3.2 Example for the calculation of titres and the reduction according to the large-
volume-plating Method . 28
Annex C (informative) Calculation of the viral infectivity titre . 30
C.1 Quantal tests - Example of TCID determination by the Spaerman-Kärber method . 30
50
C.2 Plaque test . 30
C.3 Biometrical evaluation of experimental approaches and assessment of the
disinfecting effect on the virus (reduction [R]): . 31
C.3.1 General . 31
C.3.2 Calculating the virus titre with 95 % confidence interval - Example . 32
C.3.3 Calculating the reduction and its 95 % confidence interval . 32
C.3.4 Calculating the average reduction (R ) and its 95 % confidence interval . 33
(mi)
C.3.5 Practical example . 34
Bibliography . 37

3

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SIST EN 16777:2019
EN 16777:2018 (E)
European foreword
This document (EN 16777:2018) 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 June 2019, and conflicting national standards shall be
withdrawn at the latest by June 2019.
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
4

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EN 16777:2018 (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 or antiseptic found by this test
corresponds to defined experimental conditions.
However for special applications the recommendations of use of a product can differ and therefore
additional test conditions might be needed, which cannot be covered by this document.
5

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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 in the medical area for disinfecting non-porous
surfaces including surfaces of medical devices without mechanical action.
This document applies to areas and situations where disinfection is medically indicated. Such
indications occur in patient care, for example:
— in hospitals, in community medical facilities, and in dental institutions;
— in clinics of schools, of kindergartens, and of nursing homes;
and may occur in the workplace and in the home.
It may also include services such as laundries and kitchens supplying products directly for the patients.
NOTE 1 The method described is intended to determine the activity of commercial formulations or active
substances on viruses in the conditions in which they are used.
NOTE 2 This method corresponds to a phase 2, step 2 test.
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 14476, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of
virucidal activity in the medical area — Test method and requirements (Phase 2/Step 1)
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
EN 10088-1, Stainless steels — Part 1: List of stainless steels
EN 10088-2, Stainless steels — Part 2: Technical delivery conditions for sheet/plate and strip of corrosion
resisting steels for general purposes
6

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3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN 14885 and EN 14476 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 http://www.iso.org/obp
4 Requirements for virucidal activity on surfaces
The product shall demonstrate at least a decimal log (lg) reduction of 4 in virus titre of the adenovirus
and murine norovirus test strains when tested in accordance with Table 1 and Clause 5. As described in
EN 14885, to claim virucidal activity against enveloped virus the product shall pass both EN 14476 and
this standard with vaccinia virus and to claim limited spectrum virucidal activity the product shall pass
both EN 14476 and this standard with adenovirus and murine norovirus. However, to claim the
virucidal activity the product shall pass standards EN 14476 with poliovirus, adenovirus and murine
norovirus and this standard with adenovirus and murine norovirus, because poliovirus is not resistant
to drying.
Table 1 — Minimum and additional test conditions
Minimum spectrum of test a
Virucidal activity
organisms
adenovirus

murine norovirus
b
Limited spectrum virucidal activity
adenovirus
murine norovirus
c
Virucidal activity against enveloped viruses
vaccinia virus

Test temperature between 18 °C and 25 °C

Additional temperature between 4 °C and 30 °C
Contact time according to the manufacturer’s recommendation, but not longer than
d
5 min or 60 min
Interfering substances 0,3 g/l bovine serum albumin
a) clean and/or
b) dirty 3,0 g/l bovine serum albumin plus 3,0 ml erythrocytes
e Further contact time(s), interfering substance(s) or virus(es)
Additional conditions
a
Poliovirus (as used in the corresponding suspension test) cannot be used for surfaces, because of drying
problems.
b
The test for “limited spectrum virucidal activity” will cover all enveloped viruses (Annex A) and norovirus,
rotavirus and adenovirus.
c
The test for “virucidal activity against enveloped viruses” will cover all enveloped viruses only (Annex A).
d
The contact times for surface disinfectants stated in this table are chosen on the basis of the practical
7

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EN 16777:2018 (E)
conditions of the product. The recommended contact time for the use of the product is within the responsibility
of the manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient
and/or the medical staff and surfaces, which are frequently touched by different people, leading to the
transmission of microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same
applies where the contact time of the product shall be limited for practical reasons. Products for other surfaces
than stated above may be tested with a contact time of maximum 60 min.
e
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 methods
5.1 Principle
5.1.1 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.
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 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.
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
a) Non-enveloped RNA virus
Murine norovirus, strain S99 Berlin
NOTE Virus strains can be obtained from a national or international culture collection. Murine norovirus can
be obtained from Friedrich-Loeffler-Insitut Bundesforschungsinstitut für Tiergesundheit, Hauptsitz Insel Riems
Südufer 10, 17493, Greifswald-Insel Riems; phone: [phone], fax: [phone].
http://www.fli.bund.de.
b) Non-enveloped DNA virus

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.
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Adenovirus type 5, strain Adenoid 75, ATCC VR-5
c) Enveloped DNA virus
Vaccinia virus, strain modified vaccinia virus Ankara (MVA), ATCC VR-1508 or vaccinia virus strain
Elstree, ATCC VR-1549
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 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 – if appropriate the 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 [5.3.2.1a)]. 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)
2
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.

2
Sigma N 7005 is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by CEN of this product.
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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.
For RAW 264.7 cells, special FCS shall 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), 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
2
(CaCl ) in water (5.2.2.2) and dilute to 1 000 ml. Sterilize by membrane filtration (5.3.2.11) or in
2
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;
— 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 (5.3.2.11). 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. (5.3.2.4). 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.
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.
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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 medical 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 Clean conditions (bovine serum albumin)
Bovine serum albumin shall be used as commercially available product or shall be prepared as follows:
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SIST EN 16777:2019
EN 16777:2018 (E)
— dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of water
(5.2.2.2);
— sterilize by membrane filtration;
— keep in a refrigerator and use within one month.
The final concentration of bovine serum albumin (BSA) in the test is 0,3 g BSA per litre.
5.2.2.8.3 Dirty conditions
a) bovine serum albumin:
Bovine serum albumin shall be used as commercially available product or shall be prepared as follows:
— dissolve 3 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of
water (see 5.2.2.2);
— sterilize by membrane filtration;
— keep in a refrigerator and use within one month.
The final concentration of bovine serum albumin (BSA) in the control is 3 g BSA per litre.
b) sheep erthrocytes:
Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at
800 g for 10 min (5.3.2.18). After discarding the supernatant, resuspend erythrocytes i
...