Chemical disinfectants and antiseptics - Chemical-thermal textile disinfection - Test method and requirements (phase 2, step 2)

This document specifies a test method and the minimum requirements for the microbicidal activity of a defined disinfection process for the treatment of contaminated textile. This procedure is carried out by using a washing machine as defined in 5.3.2.18 and refers to the disinfection step without prewash. This procedure is not limited to certain types of textile. The suppliers instructions shall be sufficient to allow the method in the standard to be carried out fully (e.g. dosing disinfectant in whatever washing phase e.g. main wash, rinsing, disinfecting at 40 °C). 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. The method described is intended to determine the activity of a product or product combination under the conditions in which they are used. This is a phase 2, step 2 laboratory test that simulates the conditions of application of the product. NOTE This method corresponds to a phase 2, step 2 test (see EN 14885). EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”.

Chemisches Desinfektionsmittel und Antiseptika - Chemothermische Wäschedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die mikrobizide Wirkung eines festgelegten Desinfektionsprozesses zur Behandlung von kontaminierten Textilien fest. Dieses Verfahren wird unter Einsatz einer in 5.3.2.18 festgelegten Waschmaschine durchgeführt und bezieht sich auf den Desinfektionsschritt ohne Vorwaschen. Dieses Verfahren ist nicht auf bestimmte Arten von Textilien beschränkt. Die Anweisungen des Lieferanten werden als ausreichend angenommen, um die vollständige Durchführung (z. B. Dosierung des Desinfektionsmittels in einer beliebigen Waschphase, z. B. Haupt¬waschgang, Spülen, Desinfektion bei 40 °C) des in diesem Dokument beschriebenen Verfahrens zu ermöglichen.
Dieses Dokument gilt für Bereiche und unter Bedingungen, wo eine Desinfektion aus medizinischen Gründen angezeigt ist. Indikationen dieser Art liegen bei der Patientenbetreuung vor, z. B.:
—   in Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich;
—   in medizinischen Einrichtungen von Schulen, Kindergärten und Heimen;
und könnten auch am Arbeitsplatz und im häuslichen Bereich gegeben sein.
Eingeschlossen sein könnten auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung der Patienten dienen.
Das beschriebene Verfahren dient der Bestimmung der Wirksamkeit eines Produkts oder einer Kombination von Produkten unter den Bedingungen, unter denen es/sie angewendet wird/werden. Es ist eine Laborprüfung der Phase 2, Stufe 2, der die Bedingungen der Produktanwendung simuliert.
ANMERKUNG   Dieses Verfahren entspricht einer Prüfung der Phase 2, Stufe 2 (siehe EN 14885).
EN 14885 legt im Einzelnen die Beziehungen der verschiedenen Prüfungen untereinander und zu den „Anwendungsempfehlungen“ fest.

Désinfectants chimiques et antiseptiques - Désinfection thermochimique du textile - Méthode d'essai et prescriptions (phase 2, étape 2)

Le présent document spécifie une méthode d’essai et les exigences minimales relatives à l’activité microbicide d’un processus défini de désinfection destiné au traitement de textile contaminé. Cette procédure consiste à utiliser une machine à laver comme défini en 5.3.2.18 et se rapporte à l’étape de désinfection sans prélavage. Cette procédure ne se limite pas à certains types de textile. Il est escompté que les instructions du fournisseur seront suffisantes pour permettre de réaliser complètement la méthode indiquée dans le présent document (par exemple, le dosage du désinfectant quelle que soit l’étape de lavage, par exemple, le lavage, le rinçage, la désinfection à 40 °C).
Le présent document s’applique aux secteurs et situations pour lesquels la désinfection est médicalement préconisée. Ces préconisations touchent les soins délivrés aux patients, par exemple :
   dans les hôpitaux, les cabinets médicaux et dentaires ;
   dans les infirmeries des écoles, des écoles maternelles et des maisons de repos ;
et pourraient concerner les lieux de travail et le domicile des particuliers.
Le présent document pourrait également s’appliquer aux textiles entretenus par des blanchisseries et aux articles textiles de cuisine en contact direct avec les patients.
La méthode décrite a pour objet de déterminer l’activité d’un produit ou d’une combinaison de produits dans leurs conditions d’emploi. Il s’agit d’un essai en laboratoire de phase 2, étape 2 qui simule les conditions d’application du produit.
NOTE   Cette méthode correspond à un essai de phase 2, étape 2 (voir EN 14885).
L’EN 14885 spécifie en détail la relation entre les divers essais et la façon « d’appliquer les recommandations ».

Kemična razkužila in antiseptiki - Termokemično razkuževanje tekstila - Preskusna metoda in zahteve (faza 2, stopnja 2)

General Information

Status
Not Published
Public Enquiry End Date
01-Feb-2021
Current Stage
4020 - Public enquire (PE) (Adopted Project)
Start Date
22-Oct-2020
Due Date
11-Mar-2021
Completion Date
03-Feb-2021

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SLOVENSKI STANDARD
oSIST prEN 16616:2021
01-januar-2021

Kemična razkužila in antiseptiki - Termokemično razkuževanje tekstila - Preskusna

metoda in zahteve (faza 2, stopnja 2)

Chemical disinfectants and antiseptics - Chemical-thermal textile disinfection - Test

method and requirements (phase 2, step 2)
Chemisches Desinfektionsmittel und Antiseptika - Chemothermische
Wäschedesinfektion - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)

Désinfectants chimiques et antiseptiques - Désinfection thermochimique du textile -

Méthode d'essai et prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: prEN 16616
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
71.100.35 Kemikalije za dezinfekcijo v Chemicals for industrial and
industriji in doma domestic disinfection
purposes
oSIST prEN 16616:2021 en,fr,de

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

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oSIST prEN 16616:2021
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oSIST prEN 16616:2021
DRAFT
EUROPEAN STANDARD
prEN 16616
NORME EUROPÉENNE
EUROPÄISCHE NORM
December 2020
ICS 11.080.20 Will supersede EN 16616:2015
English Version
Chemical disinfectants and antiseptics - Chemical-thermal
textile disinfection - Test method and requirements (phase
2, step 2)

Désinfectants chimiques et antiseptiques - Désinfection Chemisches Desinfektionsmittel und Antiseptika -

thermochimique du textile - Méthode d'essai et Chemothermische Wäschedesinfektion - Prüfverfahren

prescriptions (phase 2, étape 2) und Anforderungen (Phase 2, Stufe 2)

This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee

CEN/TC 216.

If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations

which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other

language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC

Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,

Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,

Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and

United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are

aware and to provide supporting documentation.

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

notice and shall not be referred to as a European Standard.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels

© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16616:2020 E

worldwide for CEN national Members.
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prEN 16616:2020 (E)
Contents Page

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

Introduction .................................................................................................................................................................... 7

1 Scope .................................................................................................................................................................... 8

2 Normative references .................................................................................................................................... 8

3 Terms and definitions ................................................................................................................................... 9

4 Requirements ................................................................................................................................................... 9

5 Test methods ................................................................................................................................................. 10

5.1 Principle .......................................................................................................................................................... 10

5.2 Materials and reagents ............................................................................................................................... 10

5.2.1 Test organisms .............................................................................................................................................. 10

5.2.2 Culture media and reagents ..................................................................................................................... 11

5.3 Apparatus and glassware .......................................................................................................................... 13

5.3.1 General ............................................................................................................................................................. 13

5.3.2 Usual microbiological laboratory equipment .................................................................................... 13

5.4 Preparation of test organism suspensions (test suspension) ...................................................... 16

5.4.1 General ............................................................................................................................................................. 16

5.4.2 Preservation and stock cultures of test organisms.......................................................................... 16

5.4.3 Working culture and test organisms ..................................................................................................... 16

5.4.4 Test suspension (N) ..................................................................................................................................... 17

5.4.5 Inoculation of the carriers ........................................................................................................................ 21

5.5 Procedure for assessing the microbicidal activity of the product .............................................. 21

5.5.1 General ............................................................................................................................................................. 21

5.5.2 Method ............................................................................................................................................................. 23

5.6 Experimental data and calculation ........................................................................................................ 25

5.6.1 Explanation of terms and abbreviations ............................................................................................. 25

5.6.2 Calculation ...................................................................................................................................................... 25

5.7 Verification of methodology ..................................................................................................................... 28

5.7.1 General ............................................................................................................................................................. 28

5.7.2 Control of weighted mean counts ........................................................................................................... 28

5.7.3 Basic limits ..................................................................................................................................................... 28

5.8 Expression of results and precision ...................................................................................................... 29

5.8.1 Reduction ........................................................................................................................................................ 29

5.8.2 Repetitions ..................................................................................................................................................... 29

5.9 Interpretation of results – conclusion .................................................................................................. 29

5.9.1 General ............................................................................................................................................................. 29

5.9.2 Microbicidal activity ................................................................................................................................... 29

5.10 Test report ...................................................................................................................................................... 30

Annex A (informative) Referenced strains in national collections ........................................................... 32

Annex B (informative) Suitable neutralizers and rinsing liquids .............................................................. 34

B.1 B.1 General ..................................................................................................................................................... 34

B.2 B.2 Neutralizers ............................................................................................................................................ 34

B.3 B.3 Neutralizer added to the agar for counting ................................................................................. 35

Annex C (informative) Graphical representations of the test method ..................................................... 36

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Annex D (informative) Example of washing machine specification .......................................................... 37

Annex E (informative) Preparation of hard water for using in the test and reference

procedures ...................................................................................................................................................... 38

Annex F (informative) Test report (example) ................................................................................................... 39

Annex G (informative) Example for loading the washing machine ........................................................... 46

Annex ZA (informative) Relationship between this European Standard and the and the

General Safety and Performance Requirements of Regulation (EU) 2017/745 aimed

to be covered .................................................................................................................................................. 48

Bibliography ................................................................................................................................................................. 50

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oSIST prEN 16616:2021
prEN 16616:2020 (E)
European foreword

This document (prEN 16616:2020) has been prepared by Technical Committee CEN/TC 216 “Chemical

disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This document is currently submitted to the CEN Enquiry.
This document will supersede EN 16616:2015.

This document has been prepared under a standardization request given to CEN by the European

Commission and the European Free Trade Association, and supports essential requirements of

EU Directive.

For relationship with EU Directive, see informative Annex ZA, which is an integral part of this

document.

The document was revised to adapt it to the latest state of science, to correct errors and ambiguities, to

harmonize the structure and wording with other tests of CEN/TC 216 existing or in preparation and to

improve the readability of the standard and thereby make it more understandable. The following is a

list of significant technical changes since the last edition:
— the scope is adapted to the scope of the WG 1;
— in Clause 4 the requirements for phase 2, step 1 tests were deleted;
— in 5.1. some editorial changes were done;
— in 5.2.2.4 water for the test and reference control were added;
— old 5.2.2.6 was deleted, because it was not used in the standard;

— the example for washing machines in 5.3.2.18 of the previous version was switched to Annex D;

— Potter S1 apparatus was added (5.3.2.21);
— 5.4.3.1 to 5.4.3.3 were adapted to other current standards;
— 5.4.4 was reviewed (editorial changes and better description);

— the description of the neutralization (5.5.1.2) was re-worded, a reference to phase 2, step 1 tests

was added;

— in 5.4.3.1 and 5.4.3.2 an information on using a spectrophotometer for counting cell numbers of

mycobacteria was added (NOTE);
— in 5.4.5 some editorial changes were done;
— 5.4.2 (old version) was deleted;

— the choice of the neutralizer shall be documented and justified in the test report (5.5.1.2);

— 5.5.1.4 was deleted, no longer necessary;
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prEN 16616:2020 (E)
— in 5.5.2.1 a new NOTE was added;

— in 5.5.2.2 a NOTE and the reference to Annex G was added, c) is deleted, no longer necessary;

— the reference control was added (5.5.2.3);
— RII was deleted (5.5.2.5);

— 5.5.2.6 to 5.5.2.8 were deleted, a reference to phase 2, step 1 tests was added;

— in 5.5.2.9 some editorial changes were done;
— in 5.6.1.1 the table was corrected and adapted to the current tests;
— in 5.6.2.1 two paragraphs are added (former 5.6.2.3);
— in 5.6.2.2 the V -values will be expressed per carrier (former per ml);
— In 5.6.2.3 the calculation of N and N was added, the formula was corrected;

— in 5.6.2.4 the calculation was changed to values per carrier, the formula was corrected and the

weighted mean was added in all calculations;
— in 5.6.2.4 the requirements for N were corrected;
— 5.6.2.5 was deleted, a reference to phase 2, step 1 test was added;

— 5.6.2.6 calculation was changed: only N will be calculated, RI is not counted and RII, B and C are no

longer used in the standard;
— the example in 5.7.2 was corrected;
— 5.7.3 was adapted to the tests in the current version;
— in 5.8.1 some editorial changes were done;
— in 5.10 a new test report was added;
— Annex A was adapted to EN 12353;
— Annex C was corrected;
— addition of a new Annex D “Example of a washing machine”;

— addition of a new Annex E “Preparation of hard water for using in the test and reference

procedure”;
— addition of a new Annex F “Example of a test report”;
— addition of a new Annex G “Example for loading the washing machine”.
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prEN 16616:2020 (E)

The changes of this revision have no impact on the test results obtained with reference to the version

EN 16616:2015. Those results are still valid except the reduction of the reference control N was

higher than 3 lg and/or the calculation of the results followed the wrong way of version 2015.

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oSIST prEN 16616:2021
prEN 16616:2020 (E)
Introduction

This document specifies a carrier test for establishing whether a single-wash disinfecting product or

combination of products for the treatment of contaminated textile has or does not have necessary

microbicidal activity. This document only intends to validate the disinfection part of the laundry

process.

This laboratory test takes into account practical conditions of application of the product including

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

influence its action in practice.

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

laboratories and types of detergents and disinfectants. Each effective dosage of the chemical

disinfectant found by this test corresponds only to the chosen experimental conditions. Where actual

conditions vary additional testing in microbiological laboratories is needed to determine the effective

dosage. Instructions for use are the responsibility of manufactures of detergents or disinfectants.

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oSIST prEN 16616:2021
prEN 16616:2020 (E)
1 Scope

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

defined disinfection process for the treatment of contaminated textile. This procedure is carried out by

using a washing machine as defined in 5.3.2.18 and refers to the disinfection step without prewash. This

procedure is not limited to certain types of textile. The suppliers' instructions are expected to be

sufficient to allow the method in this document to be carried out fully (e.g. dosing disinfectant in

whatever washing phase e.g. main wash, rinsing, disinfecting at 40 °C).

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 could occur in the workplace and in the home.

It could also include services such as laundries and kitchens supplying products directly for the

patients.

The method described is intended to determine the activity of a product or product combination under

the conditions in which they are used. This is a phase 2, step 2 laboratory test that simulates the

conditions of application of the product.
NOTE This method corresponds to a phase 2, step 2 test (see EN 14885).

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

recommendations”.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

EN 12353, Chemical disinfectants and antiseptics — Preservation of test organisms used for the

determination of bactericidal (including Legionella), mycobactericidal, sporicidal, fungicidal and virucidal

(including bacteriophages) activity

EN 13624, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of

fungicidal or yeasticidal activity in the medical area — Test method and requirements (phase 2, step 1)

EN 13727, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of

bactericidal activity in the medical area — Test method and requirements (phase 2, step 1)

EN 14348, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation of

mycobactericidal activity of chemical disinfectants in the medical area including instrument disinfectants -

Test methods and requirements (phase 2, step 1)

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

disinfectants and antiseptics
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oSIST prEN 16616:2021
prEN 16616:2020 (E)
3 Terms and definitions

For the purposes of this document, the terms and definitions given in EN 14885 and the following 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
3.1
liquor ratio

ratio of the weight of dry textile in kilograms and volume of wash liquor in litres (w/v)

3.2
disinfection process

process taking into account practical conditions of application of the product including contact time,

temperature, test organisms and interfering substances to disinfect the textile
3.3
treatment of contaminated textile

handling the textile according the disinfection process to obtain disinfected textile

4 Requirements
The test results shall fulfil the basic limits (see 5.7.3).
Table 1 — Minimum and additional test conditions
Test conditions Textile disinfection Textile disinfection
temperature < 60°C temperature ≥ 60°C
Test organism E. coli K12 b
E. faecium
E. hirae
P. aeruginosa
S. aureus
C. albicans
Additional test organism A. brasiliensis
Fungicidal activity M. terrae
Tuberculocidal activity M. terrae and M. avium
Mycobactericidal activity
Temperature As recommended by the As recommended by the
a a
manufacturer and < 60°C manufacturer and ≥ 60°C
Contact time As recommended by the As recommended by the
manufacturer manufacturer
Interfering substance Sheep blood Sheep blood

NOTE The implementation of bacterial spores and viruses was discussed. Further development is necessary to

make it technically feasible.

The temperature and the contact time shall be chosen on the basis of the practical conditions of the product application

and within the responsibility of the manufacturer.

This includes bactericidal, yeasticidal, fungicidal, tuberculocidal and mycobactericidal activity.

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5 Test methods
5.1 Principle

Carriers made of cotton fabric (5.3.2.16) are contaminated with a test suspension of microorganisms in

defibrinated sheep blood (5.2.2.12). After drying the carriers are transferred into cotton towels and

then the disinfection process in the washing machine is performed at test temperatures either t < 60 °C

or t ≥ 60 °C. The process refers to the disinfection step without prewash. At the end of the disinfection

step of the procedure, the towels with the carriers have to be taken out. Each carrier is transferred into

a separate tube containing neutralizer (5.2.2.11) and glass-beads (5.3.2.11). The microorganisms should

be recovered from the carriers by shaking. The number of surviving microorganisms in each sample is

determined and the reduction rate is calculated.
5.2 Materials and reagents
5.2.1 Test organisms

The bactericidal activity shall be evaluated using the following strains as test organisms

— Pseudomonas aeruginosa ATCC 15442
— Escherichia coli (K12) NCTC 10538
— Staphylococcus aureus ATCC 6538
— Enterococcus hirae ATCC 10541
— Enterococcus faecium ATCC 6057

The yeasticidal/fungicidal activity shall be evaluated using the following test organisms:

— Candida albicans ATCC 10231
— Aspergillus brasiliensis
ATCC 16404
(formerly Aspergillus niger ATCC 16404)

The tuberculocidal/mycobactericidal activity shall be evaluated using the following test organisms:

— Mycobacterium terrae ATCC 15755
— Mycobacterium avium ATCC 15769
NOTE See Annex A for strain reference in some other culture collections.

The required incubation temperatures for these test organisms are (36 ± 1) °C (5.3.2.3) [C. albicans and

A. brasiliensis: (30 ± 1) °C]. The same temperature shall be used for all incubations performed during a

test and its controls and validation.

If additional test organisms are used, they shall be incubated under optimum growth conditions

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

selected do not correspond to the specified strains/species, 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 stored under a reference for five years.

The ATCC numbers are the collection numbers of strains supplied by the American Type Culture Collection (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.
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5.2.2 Culture media and reagents
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 growth of test organisms.

To improve reproducibility, it is recommended that commercially available dehydrated 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 limitation for use should be fixed.
5.2.2.2 Water used for preparation of media

The water shall be fresh distilled water and not just demineralized water. Sterilize in the autoclave

[5.3.2.1a)].

NOTE 1 Sterilization is not necessary if the water is used e.g. for preparation of culture media and subsequently

sterilized.

NOTE 2 If distilled water of adequate quality is not available, water for injections (see bibliographic reference

[1]) can be used.
5.2.2.3 Hard water for dilution of products for validation tests
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 (5.3.2.7) or in the

autoclave [5.3.2.1a)]. Autoclaving, if used, could 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

(5.3.2.8) at (2 to 8) °C 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

1 000 ml. Sterilize by membrane filtration (5.3.2.7). Store the solution in the refrigerator (5.3.2.8) at

(2 to 8) °C for no longer than one week.

— Place 600 ml to 700 ml of water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add with the

use of a pipette (5.3.2.9) 6,0 ml of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml

with water (5.2.2.2). The pH of the hard water shall be 7,0 ± 0,2, when measured at (20 ± 1) °C

(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.

5.2.2.4 Water for the test and reference control

Water with potable water quality is necessary (the water should contain less than 100 cfu/ml of

bacteria at 36 °C and 22 °C). Water hardness shall be logged and mentioned in the laboratory protocol

and shall be documented in the test report. The final hardness shall be equal or higher than 4 mmol/l

2+ 2+

alkaline earth ions (Mg and Ca ). The temperature of the water influx should be between (12 and

20) °C.
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NOTE 1 Annex E indicates how to adjust the water hardness for the test and reference procedure using

solutions A and B from 5.2.2.3. The water hardness could be measured using commercial available test kits.

NOTE 2 Water supplier data can be used to document water hardness.
5.2.2.5 Tryptone Soy Agar (TSA)
Tryptone, pancreatic digest of casein 15,0 g
Soy peptone, papaic digest of soybean meal 5,0 g
Sodium chloride (NaCl) 5,0 g
Agar 15,0 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to

7,2 ± 0,2 when measured at (20 ± 1) °C.

In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it could be necessary to add

neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used.

5.2.2.6 Tryptone Soy Broth (TSB)
Tryptone, pancreatic digest of casein 15,0 g
Soy peptone, papaic digest of soybean meal 5,0 g
Sodium chloride (NaCl) 5,0 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to

7,2 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.7 Brain Heart Infusion Agar (BHI)
EXAMPLE
Brain heart infusion 12,5 g
Beef heart infusion 5,0 g
Proteose-Peptone 10,0 g
Glucose 2,0 g
Sodium chloride (NaCl) 5,0 g
Disodiumhydrogen phosphate 2,5 g
Agar 10,0 g
Water (5.2.2.2) to 1 000,0 ml

Sterilize in the autoclave [5.3.2.1a)]. After sterilization the pH of the medium shall be equivalent to

7,2 ± 0,2 when measured at (20 ± 1) °C.
5.2.2.8 Malt Extract Agar (MEA)
Malt extract 30,0 g
Soy peptone, papaic digest of soybean meal 3,0 g
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oSIST prEN 16616:2021
prEN
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