Chemical disinfectants and antiseptics - Quantitative carrier test for the evaluation of virucidal activity for instruments used in the medical area - Test method and requirements (phase 2, step 2)

This document specifies a test method and the minimum requirements for virucidal activity of chemical disinfectant 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 document applies to products that are used in the medical area for disinfecting instruments by immersion.
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 under 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 Keimträgerversuch zur Prüfung der viruziden Wirkung für Instrumente im humanmedizinischen Bereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

Dieses Dokument legt ein Prüfverfahren und die Mindestanforderungen an die viruzide Wirkung von chemischen Desinfektionsmitteln fest, die bei Verdünnung mit Wasser standardisierter Härte als homogene, physikalisch stabile Zubereitung vorliegen bzw. bei gebrauchsfertigen Produkten mit Wasser verdünnt werden.
Dieses Dokument gilt für Produkte, die zur Instrumentendesinfektion im humanmedizinischen Bereich durch Eintauchen 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 Einrichtungen in Schulen, Kindergärten und Heimen
-   und können auch am Arbeitsplatz oder im häuslichen 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 kommerziell erhältlichen Zubereitungen oder Wirkstoffen 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.

Désinfectants chimiques et antiseptiques - Essai quantitatif de porte-germe pour l'évaluation de l'activité virucide pour instruments utilisés en médecine - Méthode d'essai et exigences (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 pour la désinfection des instruments par immersion.
Le présent document s’applique aux domaines et situations où la désinfection est indiquée en médecine. De telles indications se rencontrent dans le cadre des soins apportés aux patients, par exemple :
-   dans les hôpitaux, les centres de soins médicaux et les cabinets dentaires ;
-   dans les infirmeries d’écoles, de crèches/garderies 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 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 la relation entre les différents essais et les « recommandations d’utilisation ».

Kemična razkužila in antiseptiki - Kvantitativni preskus s steklenim nosilcem za vrednotenje virucidnega delovanja kemičnih razkužil in antiseptikov za instrumente, ki se uporabljajo 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 ali, pri proizvodih, ki so pripravljeni za uporabo, z vodo. Ta evropski standard se uporablja za izdelke, ki se uporabljajo v humani medicini za dezinfekcijo instrumentov s potopitvijo – tudi če niso zajeti v Direktivi EGS/93/42 o medicinskih pripomočkih. Ta evropski standard se uporablja za področja in primere, ko obstajajo zdravniške indikacije za dezinfekcijo. Te indikacije se pojavljajo pri negi bolnikov, na primer: a) v bolnišnicah, skupnih zdravstvenih in zobozdravstvenih ustanovah; b) v klinikah šol, vrtcev in domov za starejše; c) in lahko nastanejo 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 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

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Published
Publication Date
30-Oct-2018
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
31-Oct-2018
Completion Date
31-Oct-2018

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SLOVENSKI STANDARD
SIST EN 17111:2019
01-januar-2019
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YUHGQRWHQMHYLUXFLGQHJDGHORYDQMDNHPLþQLKUD]NXåLOLQDQWLVHSWLNRY]D
LQVWUXPHQWHNLVHXSRUDEOMDMRYKXPDQLPHGLFLQL3UHVNXVQDPHWRGDLQ]DKWHYH
ID]DVWRSQMD

Chemical disinfectants and antiseptics - Quantitative carrier test for the evaluation of

virucidal activity for instruments used in the medical area - Test method and
requirements (phase 2, step 2)

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Keimträgerversuch zur

Prüfung der viruziden Wirkung für Instrumente im humanmedizinischen Bereich -
Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Désinfectants chimiques et antiseptiques - Essai quantitatif de porte germe pour

l'évaluation de l'activité virucide pour instruments utilisés en médecine - Méthode d'essai

et prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: EN 17111:2018
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
SIST EN 17111:2019 en,fr,de

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

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SIST EN 17111:2019
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SIST EN 17111:2019
EN 17111
EUROPEAN STANDARD
NORME EUROPÉENNE
October 2018
EUROPÄISCHE NORM
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Quantitative
carrier test for the evaluation of virucidal activity for
instruments used in the medical area - Test method and
requirements (phase 2, step 2)

Désinfectants chimiques et antiseptiques - Essai Chemische Desinfektionsmittel und Antiseptika -

quantitatif de porte-germe pour l'évaluation de Quantitativer Keimträgerversuch zur Prüfung der

l'activité virucide pour instruments utilisés en viruziden Wirkung für Instrumente im

médecine - Méthode d'essai et exigences (phase 2, humanmedizinischen Bereich - Prüfverfahren und

étape 2) Anforderungen (Phase 2, Stufe 2)
This European Standard was approved by CEN on 18 June 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 17111:2018 E

worldwide for CEN national Members.
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SIST EN 17111:2019
EN 17111:2018 (E)
Contents Page

European foreword ....................................................................................................................................................... 4

Introduction .................................................................................................................................................................... 5

1 Scope .................................................................................................................................................................... 6

2 Normative references .................................................................................................................................... 6

3 Terms and definitions ................................................................................................................................... 6

4 Requirements ................................................................................................................................................... 6

5 Test method ...................................................................................................................................................... 8

5.1 Principle ............................................................................................................................................................. 8

5.2 Materials and reagents .................................................................................................................................. 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 .................................................................................... 12

5.4 Preparation of test organism suspensions and product test solutions .................................... 14

5.4.1 Test organism suspensions (test suspension) ................................................................................... 14

5.4.2 Product test solution ................................................................................................................................... 14

5.5 Procedure for assessing the virucidal activity of the product ..................................................... 15

5.5.1 General ............................................................................................................................................................. 15

5.5.2 Method ............................................................................................................................................................. 16

5.5.3 Cytotoxicity caused by product solutions ........................................................................................... 18

5.5.4 Control of efficiency for suppression of disinfectant activity ...................................................... 19

5.5.5 Reference test for virus inactivation ..................................................................................................... 19

5.5.6 Titration of the virus control ................................................................................................................... 19

5.5.7 Titration of test samples ............................................................................................................................ 19

5.6 Experimental data and calculation ........................................................................................................ 20

5.6.1 Protocol of the results ................................................................................................................................ 20

5.6.2 Calculation of infectivity titre (TCID PFU) .................................................................................. 20

50 –

5.7 Verification of the methodology ............................................................................................................. 20

5.8 Explanation of terms and abbreviations ............................................................................................. 21

5.9 Expression of results ................................................................................................................................... 21

5.9.1 General ............................................................................................................................................................. 21

5.9.2 Calculation of the virucidal activity of products ............................................................................... 21

5.10 Calculation ...................................................................................................................................................... 21

5.10.1 Virucidal activity .......................................................................................................................................... 21

5.10.2 Claims ............................................................................................................................................................... 22

5.11 Test report ...................................................................................................................................................... 22

Annex A (informative) Example of a typical test report .............................................................................. 24

Annex B (informative) Examples of viruses sorted according to their presence in the

human body in case of virus infection .................................................................................................. 26

Annex C (normative) Detoxification of test mixtures by molecular sieving ........................................ 28

C.1 Molecular sieving with Sephadex™ LH 20 ........................................................................................... 28

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EN 17111:2018 (E)

C.1.1 Principle ........................................................................................................................................................... 28

C.1.2 Sephadex suspension .................................................................................................................................. 28

C.1.3 Procedure ........................................................................................................................................................ 28

C.2 Molecular sieving using MicroSpin™ S 400 HR................................................................................... 30

C.3 Determination of the residual virus titre by the large-volume-plating (LVP) method ....... 30

C.3.1 General ............................................................................................................................................................. 30

C.3.2 Example for the calculation of titres and the reduction according to the large-

volume-plating Method .............................................................................................................................. 31

Annex D (informative) Calculation of the viral infectivity titre ................................................................ 33

D.1 Quantal tests - Example of TCID determination by the Spaerman-Kärber method ........ 33

D.2 Plaque test ....................................................................................................................................................... 34

D.3 Biometrical evaluation of experimental approaches and assessment of the

disinfecting effect on the virus (reduction [R]): ................................................................................ 34

D.3.1 General ............................................................................................................................................................. 34

D.3.2 Calculating the virus titre with 95 % confidence interval ............................................................. 35

D.3.3 Calculating the reduction and its 95 % confidence interval ......................................................... 35

D.3.4 Calculating the average reduction (R ) and its 95 % confidence interval ........................ 36

(mi)

D.3.5 Practical example ......................................................................................................................................... 37

Bibliography ................................................................................................................................................................. 40

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SIST EN 17111:2019
EN 17111:2018 (E)
European foreword

This document (EN 17111:2018) has been prepared by Technical Committee CEN/TC 216 “Chemical

desinfectants 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 April 2019, and conflicting national standards shall be

withdrawn at the latest by April 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.
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SIST EN 17111:2019
EN 17111:2018 (E)
Introduction

This European Standard specifies a carrier test for establishing whether a chemical disinfectant for use

on instruments (surgical instruments, anaesthesia material, endoscopes etc.) has a virucidal activity in

the fields described in the scope.

The laboratory test closely simulates practical conditions of application including pre-drying viruses on

a carrier, contact time, temperature, test organisms and interfering substances, i.e. conditions which

may influence the action of chemical disinfectants in practical situations. Each utilization concentration

of the chemical disinfectant found by this test corresponds to defined experimental conditions.

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SIST EN 17111:2019
EN 17111:2018 (E)
1 Scope

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

disinfectant 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 document applies to products that are used in the medical area for disinfecting instruments by

immersion.

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

disinfectants and antiseptics
3 Terms and definitions

For the purposes of this document, the terms and definitions given in 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 http://www.iso.org/obp
4 Requirements

The product, when diluted with hard water or – in the case of ready-to-use products – with water, and

tested in accordance with Clause 5 under simulated clean conditions (0,3 g/l bovine albumin solution)

or simulated dirty conditions (3 g/l bovine albumin solution, plus 3 ml/l washed sheep erythrocytes)

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EN 17111:2018 (E)

according to its practical applications and under the minimum test conditions shall demonstrate at least

a 4 decimal log (lg) reduction.
Table 1 — Minimum and additional test conditions
Virucidal activity
against enveloped a
Virucidal activity
Virucidal activity
viruses
Test
(Instrument
(Instrument
Conditions
disinfection when
(Pre-cleaning products disinfection when
temperature is ≥ 40 °C)
with a combined temperature is < 40 °C)
cleaner/disinfectant)
modified vaccinia virus
Minimum Ankara Adenovirus
spectrum of or and murine parvovirus
test organisms vaccinia virus strain murine norovirus
Elstree
additional Any relevant test organism
according to the manufacturer’s recommendation, but at / between
Test
temperature
20 °C 20 °C and < 40 °C ≥ 40 °C and 70 °C
Contact time according to the manufacturer’s recommendation, but no longer than
60 min 60 min 60 min
Interfering
substance
clean
0,3 g/l bovine albumin 0,3 g/l bovine albumin 0,3 g/l bovine albumin
conditions
solution solution solution
and/or and/or and/or
3,0 g/l bovine albumin 3,0 g/l bovine albumin 3,0 g/l bovine albumin
dirty solution plus 3,0 ml/l solution plus 3,0 ml/l solution plus 3,0 ml/l
conditions washed sheep washed sheep washed sheep
erythrocytes erythrocytes erythrocytes
Additional
any relevant substance any relevant substance any relevant substance
conditions

Poliovirus (as used in the corresponding suspension test) cannot be used for surfaces, because of drying

problems. To claim the virucidal activity the product shall pass standards EN 14476 with polio-, adeno- and

murine norovirus.

The test for “virucidal activity against enveloped viruses” will cover all enveloped viruses only

(Annex A).

For the additional conditions, the concentration defined as a result can be lower than the one obtained

under the minimum test conditions.
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SIST EN 17111:2019
EN 17111:2018 (E)
5 Test method
5.1 Principle

5.1.1 A test suspension of test viruses in a solution of interfering substances is spread on a glass

carrier (5.3.2.23). After drying the carrier is immersed into a sample of the product as delivered and/or

diluted with hard water (for ready to use products: water). In parallel, a second carrier is treated with

(hard) water instead of the product applying the same test conditions (water control). The carriers are

maintained at one of the temperatures and contact times specified in Clause 4 and 5.5.1.1. At the end of

this contact time, the carriers are transferred into a maintenance medium containing glass beads. The

viruses are to be severed from the surface by shaking (5.3.2.17). The numbers of surviving viruses in

each sample are determined and the reduction is calculated by comparing the results of the product and

the water control.

5.1.2 The test is performed using modified vaccinia virus Ankara or vaccinia virus strain Elstree (pre-

cleaning products with a combined cleaner/disinfectant), adenovirus type 5 and murine norovirus as

test-organisms (minimum spectrum of test organisms); in case of test temperatures of 40 °C or higher

only murine parvovirus shall be used.

5.1.3 Other contact times and temperatures within the limits specified in Clause 4 may be used.

Additional interfering substances may be used.
5.2 Materials and reagents
5.2.1 Test organisms

The virucidal activity shall be evaluated using the following strains as test organisms selected according

to Clause 4.
a) Enveloped DNA virus

Vaccinia virus, strain modified vaccinia virus Ankara (MVA), ATCC VR-1508 or vaccinia virus strain

Elstree, ATCC VR-1549
b) Non-enveloped RNA virus
Murine norovirus, strain S99 Berlin
c) Non-enveloped DNA virus
Adenovirus type 5, strain Adenoid 75, ATCC VR-5
Murine parvovirus, minute virus of mice, strain Crawford, ATCC VR-1346

The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collections

(ATCC). 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.

Virus strains may be obtained from a national or international culture collection. Murine Norovirus may be

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

Südufer 10, 17493, Greifswald-Insel Riems; phone: +49 (0) 38351 7-0, fax: +49 (0) 38351 7-1219.

https://www.fli.de/en/home/.
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The required incubation temperature for these test organisms is 36 °C ± 1 °C or 37 °C ± 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 incubated under optimum growth conditions

(temperature, time, atmosphere, media) noted in the test report. If the additional test organisms

selected do not correspond to the specified strains, their suitability for supplying the required inocula

shall be verified. 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. The source of the strains shall be indicated.

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 (5.3.2.4) are measured at 20 °C ± 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)

Prepare neutral red (Sigma N7005) stock solution at 0,1 mg/ml in water (5.2.2.2). Filter through a

0,45 µm pore size filter and store 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.
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EN 17111:2018 (E)

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 % w/V 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 (5.3.2.11) 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;

— 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 (5.3.2.11). 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 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.
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, 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 100 ml of water

(5.2.2.2).

Sterilize by membrane filtration (5.3.2.11), keep in a refrigerator (5.3.2.6) and use within one month.

The final concentration of the bovine albumin in the test procedure (5.5) shall be 0,3 g/l.

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SIST EN 17111:2019
EN 17111:2018 (E)

5.2.2.8.3 Dirty conditions (Mixture of bovine albumin solutions – high concentration with sheep

erythrocytes)

Dissolve 3,00 g of bovine albumin fraction V (suitable for microbiological purposes) in 97 ml of water

(5.2.2.2).
Sterilize by membrane filtration (5.3.2.11).

Prepare at least 8,0 ml fresh sterile defibrinated sheep blood (5.2.2.9). Centrifuge the sheep blood at

800 g for 10 min (5.3.2.18). After discarding the supernatant, resuspend erythrocytes in PBS (5.2.2.3).

Repeat this procedure at least 3 times, until the supernatant is colourless.

Resuspend 3 ml of the packed sheep erythrocytes in the 97 ml of sterilized bovine albumin solution (see

above). To avoid contamination this mixture should be split in portions probably needed per day and

kept in separate containers (5.3.2.8) for a maximum of 7 days in a refrigerator (5.3.2.6).

The final concentration of bovine albumin and sheep erythrocytes in the test procedure (5.5) shall be

3 g/l and 3 ml/l respectively.
5.2.2.9 Defibrinated sheep blood

The defibrinated sheep blood shall be sterile (aseptic blood-letting and preparation). The defibrinated

sheep blood can be pooled from more than one sheep and can be acquired from a commercial supplier.

5.2.2.10 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.

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
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