EN 17846:2023
(Main)Chemical disinfectants and antiseptics - Quantitative test method for the evaluation of sporicidal activity against Clostridioides difficile on non-porous surfaces with mechanical action employing wipes in the medical area (4-field test) - Test method and requirements (phase 2, step 2)
Chemical disinfectants and antiseptics - Quantitative test method for the evaluation of sporicidal activity against Clostridioides difficile on non-porous surfaces with mechanical action employing wipes in the medical area (4-field test) - Test method and requirements (phase 2, step 2)
requirements for sporicidal activity against spores of Clostridioides difficile 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 is applicable to products that are used in the medical area for disinfecting non-porous surfaces including surfaces of medical devices by wiping - regardless if they are covered by the 93/42/EEC Directive on Medical Devices or not.
Due to the new methods of application of surface disinfectants like pre-impregnated wipes this document was established to cover the different application method.
The document is applicable for four method of application of products for wiping and/or mopping:
a) soaking any non-specified wipe or mop with product;
b) spraying the product on any non-specified wipe and / or mop or a specified wipe or mop.
c) impregnation of specified wipes or mops by the user with the product according to the manufacturer’s recommendation;
d) pre-impregnation of specified wipes or mops by the manufacturer as ready-to-use wipes or mops.
In all types of application the water control has to be done with the standard wipe [5.3.2.17 a)], because it is a process or method control.
This document does not apply to products that are sprayed on or flooding surfaces, then left until the contact application phase 2, step 2 standards without mechanical action should be used and their methods performed.
The test surface (5.3.2.16) was selected as standard surface and should cover all non-porous surfaces. It was not intended to cover the influence of each different surface.
This document is applicable 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 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 - Quantitatives Prüfverfahren zur Bestimmung der sporiziden Wirkung gegen Clostridioides difficile auf nicht-porösen Oberflächen mit mechanischer Einwirkung mit Hilfe von Tüchern im humanmedizinischen Bereich (4‑Felder-Test) - Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
Dieses Dokument legt ein Prüfverfahren für und die Mindestanforderungen an die sporizide Wirkung gegen Sporen von Clostridioides difficile von chemischen Desinfektionsmitteln fest, die bei Verdünnung mit Wasser standardisierter Härte – bzw. bei gebrauchsfertigen Produkten bei der Verdünnung mit Wasser – als homogene, physikalisch stabile Zubereitung vorliegen.
Dieses Dokument ist anwendbar für Produkte, die im medizinischen Bereich zur Desinfektion von nicht-porösen Oberflächen, einschließlich Oberflächen auf Medizinprodukten, durch Abwischen verwendet werden – unabhängig davon, ob sie in der Richtlinie 93/42/EWG über Medizinprodukte erfasst sind oder nicht.
Aufgrund der neuen Verfahren zur Anwendung von Oberflächendesinfektionsmitteln wie vorgetränkten Tüchern wurde dieses Dokument erstellt, um die verschiedenen Anwendungsmethoden zu behandeln.
Das Dokument ist anwendbar für vier Verfahren zur Anwendung von Produkten zum Wischen und/oder Abputzen:
a) Tränken eines nicht-spezifizierten Tuchs oder Putzlappens mit dem Produkt;
b) Sprühen des Produkts auf ein nicht-spezifiziertes Tuch und/oder einen Putzlappen oder ein spezifiziertes Tuch oder Putzlappen;
c) Tränken spezifizierter Tücher oder Putzlappen mit dem Produkt entsprechend den Herstellerempfehlungen durch den Anwender;
d) Vortränken spezifizierter Tücher oder Putzlappen als gebrauchsfertige Tücher oder Putzlappen durch den Hersteller.
Bei allen Anwendungsarten muss mit dem Standardtuch [5.3.2.17 a)] der Kontrollversuch mit Wasser durchgeführt werden, da es sich um eine Prozess- oder Verfahrenskontrolle handelt.
Dieses Dokument ist nicht anwendbar für Produkte, die auf Oberflächen gesprüht oder geschwemmt und bis zur Einwirkungsanwendung darauf belassen werden; dann sollten Normen der Phase 2, Stufe 2 ohne mechanische Einwirkung und die zugehörigen Verfahren durchgeführt werden.
Die Prüffläche (5.3.2.16) wurde als Standardoberfläche gewählt und sollte alle nicht-porösen Oberflächen abdecken. Es war nicht beabsichtigt, den Einfluss der einzelnen unterschiedlichen Oberflächen zu behandeln.
Dieses Dokument ist anwendbar für Bereiche und unter Bedingungen, wo eine Desinfektion aus medizinischen Gründen angezeigt ist. Indikationen dieser Art liegen z. B. bei der Patientenbetreuung in:
Krankenhäusern, kommunalen medizinischen Einrichtungen und im Dentalbereich,
medizinischen Einrichtungen in Schulen, Kindergärten und Heimen
vor und können auch am Arbeitsplatz und im häuslichen Bereich gegeben sein. Eingeschlossen sein können auch Einrichtungen wie Wäschereien und Küchen, die der direkten Versorgung der Patienten dienen.
ANMERKUNG 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 - Méthode d’essai quantitative pour l’évaluation de l’activité sporicide contre Clostridioides difficile sur des surfaces non poreuses, avec action mécanique à l’aide de lingettes dans le domaine médical (essai à 4 zones) - 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 sporicide contre les spores de Clostridioides difficile des désinfectants chimiques qui forment une préparation homogène, physiquement stable, lorsqu’ils sont dilués dans l’eau dure ou — dans le cas de produits prêts à l’emploi — dans l’eau.
Ce document est applicable aux produits utilisés dans le secteur médical pour désinfecter les surfaces non poreuses, y compris les surfaces de dispositifs médicaux par essuyage — qu’elles soient couvertes ou non par la Directive 93/42/CEE relative aux dispositifs médicaux.
En raison des nouvelles méthodes d’application de désinfectants de surface, par exemple les lingettes pré-imprégnées, ce document a été rédigé pour couvrir les différentes méthodes d’application.
Le document est applicable à quatre méthodes d’application de produits pour l’essuyage et/ou le lavage :
a) imprégnation d’une lingette ou lavette non spécifiée avec le produit ;
b) pulvérisation du produit sur une lingette et/ou lavette non spécifiée ou sur une lingette ou lavette spécifiée ;
c) imprégnation des lingettes ou lavettes spécifiées par l’utilisateur avec le produit, conformément aux recommandations du fabricant ;
d) pré-imprégnation des lingettes ou lavettes spécifiées par le fabricant sous forme de lingettes ou lavettes prêtes à l’emploi.
Dans tous les types d’application, le témoin eau doit être réalisé avec la lingette standard [5.3.2.17 a)], car il s’agit d’un témoin du processus ou de la méthode.
Ce document ne s’applique pas aux produits pulvérisés sur des surfaces inondables puis laissés jusqu’à ce que les normes de phase 2, étape 2 sur l’application de contact sans action mécanique soient utilisées et leurs méthodes effectuées.
La surface d’essai (5.3.2.16) a été choisie comme surface standard et il convient qu’elle englobe toutes les surfaces non poreuses. Le présent document n’est pas destiné à traiter de l’influence de chaque surface différente.
Le présent document est applicable aux secteurs et situations où la désinfection est médicalement préconisée. Ces indications relèvent des soins aux patients, par exemple :
dans les hôpitaux, les établissements médicaux, les centres de soins dentaires ;
dans les infirmeries d’écoles, de jardins d’enfants et de crèches ;
et peuvent également concerner le lieu de travail et le domicile. Il peut également s’agir de services, comme des blanchisseries ou des cuisines, fournissant directement des produits pour le patient.
NOTE Cette méthode correspond à un essai de type phase 2, étape 2.
L’EN 14885 précise de manière détaillée la relation entre les différents essais et les « recommandations d’utilisation ».
Kemična razkužila in antiseptiki - Kvantitativna preskusna metoda za vrednotenje sporocidnega delovanja na Clostridioides difficile na neporoznih površinah z mehanskim delovanjem z odvzemom brisa v humani medicini (4-področni preskus) - Preskusna metoda in zahteve (faza 2, stopnja 2)
Ta dokument določa preskusno metodo in minimalne zahteve za sporocidno delovanje kemičnih razkužil na spore Clostridioides difficile, ki tvorijo homogen, fizikalno stabilen pripravek, če je razredčen s trdo vodo oziroma z vodo pri izdelkih, ki so pripravljeni za uporabo.
Dokument se uporablja za izdelke, ki se v humani medicini uporabljajo za razkuževanje neporoznih površin z brisanjem, vključno s površinami medicinskih pripomočkov – tudi če niso zajeti v Direktivi EGS/93/42 o medicinskih pripomočkih.
Zaradi novih načinov uporabe izdelkov za površinsko razkuževanje, kot so predhodno impregnirani robčki, je bil ta dokument pripravljen tako, da obravnava različne načine uporabe.
Dokument se uporablja za štiri načine uporabe izdelkov za brisanje in/ali čiščenje tal:
a) namakanje neopredeljenih robčkov ali krp v izdelku;
b) pršenje izdelka po neopredeljenih ali opredeljenih robčkih in/ali krpah;
c) impregnacija opredeljenih robčkov ali krp z izdelkom, ki jo izvede uporabnik v skladu s priporočilom proizvajalca;
d) predhodna impregnacija opredeljenih robčkov ali krp, ki jo izvede proizvajalec, pri čemer so takšni robčki oziroma krpe že pripravljene za uporabo.
Pri vseh vrstah uporabe je treba nadzor vode izvesti s standardnim robčkom [5.3.2.17 a)], saj gre pri tem za nadzor postopka oziroma načina uporabe.
Dokument se ne uporablja za izdelke, s katerimi se poškropi ali prekrije površine, nato pa se pusti do faze kontaktne uporabe 2 (2. korak); v tem primeru je priporočljivo uporabiti standarde brez mehanskega delovanja in njihove metode.
Preskusna površina (5.3.2.16) je bila izbrana kot standardna površina, ki naj zajema vse neporozne površine. Dokument ni bil namenjen za obravnavo vpliva posameznih različnih površin.
Ta dokument se uporablja za področja in primere, ko obstajajo zdravniške indikacije za razkuževanje. Te indikacije se pojavljajo pri negi bolnikov, na primer:
– v bolnišnicah, javnih zdravstvenih in zobozdravstvenih ustanovah;
– v ambulantah šol, vrtcev in domov za starejše;
ter lahko se pojavljajo na delovnem mestu ali doma. Vključene so lahko tudi storitve v pralnicah in kuhinjah, ki izdelke dostavljajo neposredno bolnikom.
OPOMBA: Ta metoda ustreza 2. stopnji preskusa faze 2.
Standard EN 14885 podrobno določa razmerje med različnimi preskusi in »priporočili za uporabo«.
General Information
Standards Content (Sample)
SLOVENSKI STANDARD
01-december-2023
Kemična razkužila in antiseptiki - Kvantitativna preskusna metoda za vrednotenje
sporocidnega delovanja na Clostridioides difficile na neporoznih površinah z
mehanskim delovanjem z odvzemom brisa v humani medicini (4-področni
preskus) - Preskusna metoda in zahteve (faza 2, stopnja 2)
Chemical disinfectants and antiseptics - Quantitative test method for the evaluation of
sporicidal activity against Clostridioides difficile on non-porous surfaces with mechanical
action employing wipes in the medical area (4-field test) - Test method and requirements
(phase 2, step 2)
Chemische Desinfektionsmittel und Antiseptika - Quantitatives Prüfverfahren zur
Bestimmung der sporiziden Wirkung gegen Clostridioides difficile auf nicht-porösen
Oberflächen mit mechanischer Einwirkung mit Hilfe von Tüchern im
humanmedizinischen Bereich (4-Felder-Test) - Prüfverfahren und Anforderungen (Phase
2, Stufe 2)
Antiseptiques et désinfectants chimiques - Méthode d’essai quantitative pour l’évaluation
de l’activité sporicide contre Clostridioides difficile sur des surfaces non poreuses, avec
action mécanique à l’aide de lingettes dans le domaine médical (essai à 4 zones) -
Méthode d'essai et prescriptions (phase 2, étape 2)
Ta slovenski standard je istoveten z: EN 17846:2023
ICS:
11.080.20 Dezinfektanti in antiseptiki Disinfectants and antiseptics
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
EN 17846
EUROPEAN STANDARD
NORME EUROPÉENNE
November 2023
EUROPÄISCHE NORM
ICS 11.080.20
English Version
Chemical disinfectants and antiseptics - Quantitative test
method for the evaluation of sporicidal activity against
Clostridioides difficile on non-porous surfaces with
mechanical action employing wipes in the medical area (4-
field test) - Test method and requirements (phase 2, step
2)
Antiseptiques et désinfectants chimiques - Méthode Chemische Desinfektionsmittel und Antiseptika -
d'essai quantitative pour l'évaluation de l'activité Quantitatives Prüfverfahren zur Bestimmung der
sporicide contre Clostridioides difficile sur des surfaces sporiziden Wirkung gegen Clostridioides difficile auf
non poreuses, avec action mécanique à l'aide de nicht-porösen Oberflächen mit mechanischer
lingettes dans le domaine médical (essai à 4 zones) - Einwirkung mit Hilfe von Tüchern im
Méthode d'essai et prescriptions (phase 2, étape 2) humanmedizinischen Bereich (4-Felder-Test) -
Prüfverfahren und Anforderungen (Phase 2, Stufe 2)
This European Standard was approved by CEN on 4 September 2023.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this
European Standard the status of a national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This European Standard exists in three official versions (English, French, German). A version in any other language made by
translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management
Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATIO N
EUROPÄISCHES KOMITEE FÜR NORMUN G
CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2023 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN 17846:2023 E
worldwide for CEN national Members.
Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Terms and definitions . 6
4 Requirements . 6
5 Test methods . 7
5.1 Principle . 7
5.2 Materials and reagents . 7
5.2.1 Test organism . 7
5.2.2 Culture media and reagents . 8
5.3 Apparatus and glassware . 10
5.3.1 General . 10
5.3.2 Usual microbiological laboratory equipment . 10
5.4 Preparation of test organism suspensions and product test solutions . 14
5.4.1 Test organism suspensions . 14
5.4.2 Product test solution . 15
5.5 Procedure for assessing the sporicidal activity against C. difficile of the product . 16
5.5.1 General . 16
5.5.2 Method . 18
5.6 Experimental data and calculation . 21
5.6.1 Explanation of terms and abbreviations . 21
5.6.2 Calculation . 21
5.7 Verification of methodology . 27
5.7.1 General . 27
5.7.2 Control of weighted mean counts . 27
5.7.3 Basic limits test microorganisms for sporicidal activity . 27
5.8 Expression of results and precision . 27
5.8.1 Overview of the different suspensions / test mixtures . 27
5.8.2 V -values . 28
C
5.8.3 Limiting test organism and sporicidal concentration . 28
5.8.4 Precision, repetitions . 29
5.9 Interpretation of results – conclusion . 29
5.10 Test report . 29
Annex A (informative) Referenced strain in national collections . 32
Annex B (informative) Neutralizers . 33
Annex C (informative) Graphical representations of the test method . 35
Annex D (informative) Example of a typical test report. 37
Bibliography . 42
European foreword
This document (EN 17846:2023) has been prepared by Technical Committee CEN/TC 216 “Chemical
disinfectants and antiseptics”, the secretariat of which is held by AFNOR.
This European Standard shall be given the status of a national standard, either by publication of an
identical text or by endorsement, at the latest by May 2024, and conflicting national standards shall be
withdrawn at the latest by May 2024.
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.
Any feedback and questions on this document should be directed to the users’ national standards body.
A complete listing of these bodies can be found on the CEN website.
According to the CEN-CENELEC Internal Regulations, the national standards organisations of the
following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Croatia,
Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland,
Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of North
Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the United
Kingdom.
Introduction
This document specifies a carrier test for establishing whether a chemical disinfectant for use on surfaces
administered with wipes has a sporicidal activity against Clostridioides difficile in the fields described in
the scope.
The laboratory test closely simulates practical conditions of application such as contact time,
temperature and interfering substances, including pre-drying specified test organisms on a test-surface
as carrier and wiping the product on the test-surface with a wipe. The conditions are intended to cover
general purposes. However, if for some applications the recommendations of use of a product differ
additional test conditions may be or need to be used.
Each utilization concentration of the product found by this test corresponds to specified experimental
conditions.
1 Scope
This document specifies a test method and the minimum requirements for sporicidal activity against
spores of Clostridioides difficile 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 is applicable to products that are used in the medical area for disinfecting non-porous
surfaces including surfaces of medical devices by wiping – regardless if they are covered by the
93/42/EEC Directive on Medical Devices or not.
Due to the new methods of application of surface disinfectants like pre-impregnated wipes this document
was established to cover the different application method.
The document is applicable for four method of application of products for wiping and/or mopping:
a) soaking any non-specified wipe or mop with product;
b) spraying the product on any non-specified wipe and / or mop or a specified wipe or mop;
c) impregnation of specified wipes or mops by the user with the product according to the
manufacturer’s recommendation;
d) pre-impregnation of specified wipes or mops by the manufacturer as ready-to-use wipes or mops.
In all types of application the water control has to be done with the standard wipe [5.3.2.17 a)], because
it is a process or method control.
This document does not apply to products that are sprayed on or flooding surfaces, then left until the
contact application phase 2, step 2 standards without mechanical action should be used and their
methods performed.
The test surface (5.3.2.16) was selected as standard surface and should cover all non-porous surfaces. It
was not intended to cover the influence of each different surface.
This document is applicable 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 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 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 17126:2018, Chemical disinfectants and antiseptics — Quantitative suspension test for the evaluation
of sporicidal activity of chemical disinfectants 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 and the following apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp/
— IEC Electropedia: available at https://www.electropedia.org/
3.1
pre-impregnated wipe
ready-to-use wipe
wipe containing disinfectant added by the wipe manufacturer at the manufacturing site
3.2
impregnated wipe
wipe containing disinfectant added by the user
Note 1 to entry: Examples include a wipe soaked in disinfectant, a wipe sprayed with disinfectant.
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) or
simulated dirty conditions (3,0 g/l bovine albumin + 3,0 ml/l sheep erythrocytes) according to its
practical applications and under the following test conditions: temperature between 4 °C and 30 °C,
1)
contact time min. 1 min and max. either 30 min or 60 min shall demonstrate at least a decimal log (lg)
reduction in counts of 4 on test field 1. The mean of the number of cfu per 25 cm on the test fields 2 to 4
shall be equal or less than 50, the mean of the number of cfu on test fields 1 to 4 of the water control shall
be equal or more than 10. Details on the precision and repetition are given in 5.8.4 and EN 14885.
The sporicidal activity against Clostridioides difficile spores shall be evaluated using the following test
organism: Clostridioides difficile R027.
Where indicated, additional specific sporicidal activity shall be determined applying other contact times
and test organisms in order to take into account intended specific use conditions.
NOTE For these additional conditions, the concentration specified as a result can be lower than the one
obtained under the minimum test conditions.
1)
See 5.5.1.1 b).
5 Test methods
5.1 Principle
5.1.1 A test-surface is marked with 4 squares of 5 cm × 5 cm, the “test fields”, in a row. Test field 1 on
the test-surface is inoculated with a test suspension of Clostridioides difficile (C. difficile) spores in a
solution of interfering substances. The inoculum is dried. A wipe is soaked with a sample of the product
as delivered and/or diluted with hard water (for ready to use products: water). The test-surface is wiped
with the soaked wipe across the four marked test fields, starting in front of test field 1, turning
immediately after test field 4 and wiped back to the starting point. In parallel a water control is
performed: a wipe is soaked with hard water [5.5.2.2 e)] instead of the product.
NOTE For the purposes of this document references to wiping, wipe and wiped can be equated to mopping,
mop and mopped when the standard method is used to test a mopping application.
Temperature, soiling and contact time are employed as recommended by the manufacturer. At the end of
the contact time, the test organisms are recovered from each test field with moistened swabs. The swabs
are brought into a tube containing diluent and neutralizer and the test organisms are to be severed from
the swab by shaking. The numbers of surviving test organisms in each sample are determined, and the
reduction is calculated by comparing the results of the drying control D and the results obtained with
Ct
the product. In parallel to the test with the product water is applied in the same way to ensure that the
test organisms are spread on the 4 fields and their number reaches a certain level. The test is performed
using C. difficile as test organism (minimum test conditions).
5.1.2 Additional test organisms (only sporicidal strains), contact times and interfering substances can
be used.
5.2 Materials and reagents
5.2.1 Test organism
2)
The sporicidal activity against C. difficile shall be evaluated using the following strain as test organism :
— Clostridioides difficile R027 NCTC 13366
See Annex A for strain references in some other culture collections.
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, their suitability for supplying the required inoculum
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 required incubation temperature for these test bacteria is 36 °C ± 1 °C or 37 °C ± 1 °C (5.3.2.3) under
anaerobic conditions. The same temperature (36 °C or 37 °C) and anaerobic conditions shall be used for
all incubations performed during its control and validation.
2)
The NCTC numbers are the collection numbers of strains supplied by the National Collection of Type Cultures
(NCTC). This information is given for the convenience of users of this document and does not constitute an
endorsement by CEN of the product named.
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 test organism.
To improve reproducibility, it is recommended that commercially available dehydrated material is used
for the preparation of culture media if it complies with the formulas given below. 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.
All specified pH values are measured at 20 °C ± 1 °C (5.3.2.4).
5.2.2.2 Water
The water shall be freshly glass-distilled or deionized and demineralized water. If distilled water or
deionized and demineralized water of adequate quality is not available, water for injections (see [1]) may
be used.
Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used, e.g. for preparation
of culture media and subsequently sterilized.
See 5.2.2.7 for the procedure to prepare hard water.
5.2.2.3 Medium
a) BHIYT-L Agar
— Brain heart infusion 37,0 g
— Yeast extract 5,0 g
— L-Cysteine 1,0 g
— Sodium taurocholate 1,0 g
— Agar 15,0 g
— Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave [5.3.2.1 a)]. After sterilization the pH (5.3.2.4) of the medium shall be equivalent
to 7,0 ± 0,2. Let the medium cool down to 48 °C ± 2 °C. Dissolve 200 000 units of lysozyme in 10 ml water
(5.2.2.2). Sterilize the enzymatic solution by membrane filtration (5.3.2.7).
In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add
neutralizer to BHIYT-L. Annex B gives guidance on the neutralizers that may be used. It is recommended
not to use a neutralizer that causes opalescence in the agar.
5.2.2.4 Diluent
a) General Diluent
Tryptone Sodium Chloride Solution:
— Tryptone, pancreatic digest of casein 1,0 g
— Sodium chloride (NaCl) 8,5 g
— Water (5.2.2.2) to 1 000,0 ml
Sterilize in the autoclave (5.3.1). After sterilization the pH (5.3.2.4) of the general diluent shall be
equivalent to 7,0 ± 0,2.
5.2.2.5 Neutralizer
The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2 and 5.5.2. It
shall be sterile.
Information on neutralizer that has been found to be suitable for some categories of products is given in
Annex B.
5.2.2.6 Sterile defibrinated sheep blood
The sterile defibrinated sheep blood can be acquired from a commercial supplier.
5.2.2.7 Hard water for dilution of products - General
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 [5.3.2.1 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 a refrigerator (5.3.2.8)
for no longer than one month.
— Prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO ) in water and dilute to 1 000 ml.
Sterilize by membrane filtration (5.3.2.7). Store the solution in a refrigerator (5.3.2.8) for no longer
than one week.
— Place 600 ml to 700 ml water (5.2.2.2) in a 1 000 ml volumetric flask (5.3.2.12) and add 6,0 ml
(5.3.2.9) of solution A, then 8,0 ml of solution B. Mix and dilute to 1 000 ml with water (5.2.2.2). The
pH (5.3.2.4) of the hard water shall be 7,0 ± 0,2. If necessary, adjust the pH by using a solution of
approximately 40 g/l (about 1 mol/l) of sodium hydroxide (NaOH) or approximately 36,5 g/l (about
1 mol/l) of hydrochloric acid (HCl).
The hard water shall be freshly prepared under aseptic conditions and used within 12 h.
When preparing the product test solutions (5.4.2), the addition of the product to the hard water produces
a different final water hardness in each test tube. In any case the final hardness expressed as calcium
carbonate (CaCO ) is in the test tube lower than 375 mg/l.
5.2.2.8 Interfering substances
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 specified.
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 general
diluent [5.2.2.4 a)].
Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within 1 month.
The final concentration of the bovine albumin in the test procedure (5.5) is 0,3 g/l.
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 general
diluent [5.2.2.4 a)].
Sterilize by membrane filtration (5.3.2.7).
Prepare at least 8,0 ml fresh sterile defibrinated sheep blood (5.2.2.6). Centrifuge the sheep blood at 800
g for 10 min. After discarding the supernatant, resuspend erythrocytes in general diluent [5.2.2.4 a)].
N
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 for a
maximum of 7 days in a refrigerator at 2 °C to 8 °C.
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.
5.3 Apparatus and glassware
5.3.1 General
Sterilize all glassware and parts of the apparatus that will come into contact with the culture media and
reagents or the sample, except those which are supplied sterile, by one of the following methods:
a) by moist heat, in the autoclave [5.3.2.1 a)];
b) by dry heat, in the hot air oven [5.3.2.1 b)].
3)
5.3.2 Usual microbiological laboratory equipment
and in particular, the following:
5.3.2.1 Apparatus for sterilization:
+ 3
a) For moist heat sterilization, an autoclave capable of being maintained at for a minimum
121 °C
holding time of 15 min;
+ 5
b) for dry heat sterilization, a hot air oven capable of being maintained at ( 180 ) °C for a minimum
+ 5 + 5
holding time of 30 min, at ( 170 ) °C for a minimum holding time of 1 h or at ( 160 ) °C for a
0 0
minimum holding time of 2 h.
3)
Disposable sterile equipment is an acceptable alternative to reusable glassware.
5.3.2.2 Water baths, capable of being controlled at 20° C ± 1 °C and at 45 °C ± 1 °C [to maintain
melted agar in case of pour plate technique and at additional test temperatures ± 1 °C (5.5.1)].
5.3.2.3 Incubator, capable of being controlled at either 36 °C ± 1 °C or at 37 °C ± 1 °C (5.2.1). The
same temperature shall be used for all incubations of the C. difficile spores performed during a test and
its controls and validation.
5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at 20 °C ± 1 °C.
A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar-media
(5.2.2.3).
5.3.2.5 Stopwatch.
5.3.2.6 Shakers.
® 4)
a) Electromechanical agitator, e.g. Vortex mixer .
b) Mechanical shaker.
5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances
to be filtered, with a filter holder of at least 50 ml volume, and suitable for use of filters of diameter 47 mm
to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7) and bovine albumin (5.2.2.8.2
and 5.2.2.8.3).
The vacuum source used shall give an even filtration flow rate.
5.3.2.8 Refrigerator, capable of being controlled at 2 °C to 8 °C.
5.3.2.9 Graduated pipettes of nominal capacities 10 ml and 1 ml and 0,1 ml. Calibrated automatic
pipettes may be used.
5.3.2.10 Petri dishes (plates) of size 90 mm to 100 mm. (The Petri dishes are needed for pour plate
technique and for pre-moistening the standard wipes cloth with 16 ml product test solution or hard
water)
5.3.2.11 Glass beads (diameter: 3 mm to 4 mm).
5.3.2.12 Volumetric flasks.
5.3.2.13 Centrifuge capable to being controlled at 2 °C to 8 °C (4 000 g ).
N
5.3.2.14 Rectangular glass spatula (4 cm edge length).
5.3.2.15 Loop (metal or plastic).
5.3.2.16 Test-surface, PVC plate free foam, thickness 2 mm; measuring 20 cm × 50 cm. Reprocessing
of the test surface is not allowed. Clean the test-surfaces before using with 2-propanol 70 %. To ensure
uniform precleaning a standard wiping cloth is impregnated with 16 ml 2-propanol (v/v) and, using the
unitary weight, is wiped once back and forth over the test surface. After drying mark with a pencil or a
permanent marker four squares as test fields 1 to 4, each measuring 5 cm × 5 cm, figuring a row at a
4) ®
Vortex in 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.
distance of 5 cm from one another. The row should be approximately in the middle of the test-surface
(see Figures 1 and 2). The drying controls D and D are performed on a smaller test-surface measuring
C0 Ct
minimum 7 cm × 13 cm - marked with two squares of 5 cm × 5 cm.
Example for the test-surface:
a) PVC plate free foam (20 cm × 50 cm, thickness 2 mm) FOREX classic, white matt finished, one side
with foil, art. nr. SFSFOXC020RW1F, thyssenkrupp Plastics, Widdersdorfer Str. 158, 50825 Cologne,
5)
Germany .
Figure 1 — PVC plate free foam (20 cm × 50 cm, thickness 2 mm) FOREX classic, white matt
finished, one side with foil marked with 4 fields
Key
Schematic representation of the test-surface
a = 50 cm b = 20 cm
with four test areas T to T (5 cm × 5 cm) and a given range of wiper wipe.
1 4
c = 5 cm d = 10 cm e = 5 cm
Size of the unitary weight:
f = 8,6 cm g = 12,1 cm
5)
This test-surface 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.
Test field 1 is inoculated with 0,05 ml of the test organisms / interfering substance mixture (1,5 to
7 5 6
5,0 × 10 cfu/ml, corresponding to a final number on test field 1 of 6,75 × 10 to 2,25 × 10 cfu). The
arrow shows the cleaning sweep with the wipe. The starting point is in front of test field 1 and the turn is
immediately after test field 4. The end point of the wiping process is the starting point after passing test
field 1 for the second time.
Figure 2 — Scheme of the markings and the wipe sweep over four test fields on the test-surface
5.3.2.17 Wiping cloth (wipe)
a) Standard wiping cloth (wipe): For testing according to 5.5.2.2 a) and for the water control [5.5.2.2
h)] a standard wiping cloth with the dimension of 16,5 cm × 30 cm and composition of 55 % pulp,
45 % polyethylene terephthalate (PET) is used; (example: “Tork Low-Lint Cleaning”, art. nr. 190491,
6)
supplier: “SCA Tork” . Wipes are used only once.
b) Specified wiping cloth (wipe): For ready-to-use systems or impregnated wipe systems the
specified wiping cloth is used in combination with the disinfectant. The manufacturer describes
precisely their composition and how they are to be used (e.g. the number of layers). The wipes are
used only once.
5.3.2.18 Unitary weight, granite block, 12,1 cm long, 8,6 cm wide and 8,6 cm high (the height may
differ with other materials), weighing (2,3 to 2,5) kg. The use of the unitary weight standardizes the
wiping procedure and simulates the average pressure when wiping is performed in practice.
5.3.2.19 Swabs (sterile for single use only), the soakable part shall be made from nylon or pure cotton,
free from substances which might inhibit or promote the action of the product test solution and from
7)
substances inhibiting the test organisms (Suggestion for nylon swabs: FLOQSwabs Copan Diagnostics
8)
Inc., art.no. 502CS01; for pure cotton swabs : Greiner Bio-One; art.no. 420161).
5.3.2.20 Parafilm (for single use only), to protect the lower horizontal surface and the vertical
surfaces of the unitary weight from any contamination during the wiping procedure a parafilm covering
these parts is used. This parafilm shall be replaced by a new one after each wiping procedure (example:
9)
Parafilm® M (100 mm) Art.Nr. 7016 05, BRAND GMBH + CO KG, Postfach 11 55, 97861 Wertheim,
Germany).
5.3.2.21 Rubber, to fix the wipe at the unitary weight (diameter approximately 8,5 cm).
5.3.2.22 Anaerobic jar with a gas generating kit or other anaerobic systems.
5.3.2.23 Microscope, obligatory a phase-contrast type with magnification of at least 400×.
6)
This tool 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.
7)
FLOQSwabs are examples 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.
8)
Cotton swabs areexamples 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.
9)
Parafilm 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.
5.4 Preparation of test organism suspensions and product test solutions
5.4.1 Test organism suspensions
5.4.1.1 General
For each test organism, two different suspensions shall be prepared: the “test suspension” to perform the
test and the “validation suspension” to perform the controls and method validation.
5.4.1.2 Preservation and stock cultures of test organisms
The test organism and its stock cultures shall be prepared and kept according to EN 12353 respectively
EN 17126:2018, 5.4.1.2.
5.4.1.3 Working culture of test organisms
The preparation of C. difficile spore stock suspension shall be prepared, checked and determined in
accordance to EN 17126:2018, 5.4.1.3). Before use check and document the purity and the chemical
susceptibility of the spore suspension. The chemical susceptibility should have been done with the
associated quantitative suspension test (EN 17126) 1 month ago maximum.
5.4.1.4 Test suspension (N)
a) Suspend mechanically for 3 min the spore suspension stored with glass beads.
7 10) 7
b) Adjust the number of cells in the suspension to 1,5 × 10 cfu/ml to 5,0 × 10 cfu/ml using the
water [5.2.2.2] estimating the numbers of units by any suitable means. Maintain this test suspension
in the water bath at 20 °C and use within 2 h. Adjust the temperature according to 5.5.1.1 a) and
5.5.1.4 only immediately before the start of the test.
−5 −6
c) For counting prepare 10 and 10 dilutions of the test suspension using water [5.2.2.2 a)]. Mix
[5.3.2.6 a)].
d) Take a sample of 1,0 ml of each dilution in duplicate and inoculate using the pour plate or the spread
plate technique:
1) when using the pour plate technique, transfer each 1 ml sample into separate Petri dishes (i.e. in
duplicate = two plates) and add 15 ml to 20 ml melted BHIYT-L agar [5.2.2.3] for C. difficile;
2) when using the spread plate technique spread each 1,0 ml sample – divided in portions of
approximately equal size – on an appropriate number (at least two – i.e. in duplicate at least four
plates) of surface dried plates containing pre-reduced BHIYT-L agar (5.2.2.3). The plates shall be
pre-reduced overnight.
For incubation and counting see 5.4.1.6.
The validation of C. difficile tests has been performed with the pour plate technique and this method
should be therefore preferred.
5.4.1.5 Validation suspension (N )
V
a) To prepare the validation suspension, dilute the test suspension (5.4.1.4) with the water [5.2.2.2 a)]
2 3
−4
to obtain 3,0 × 10 cfu/ml to 1,6 × 10 cfu/ml (about one fourth (1+3) of the 10 dilution).
cfu/ml = colony forming unit(s) per millilitre.
NOTE The neutralizer control in this test is different from the neutralizer control N in phase 2, step 1
VB
suspension tests (EN 17126) since the test organisms are not confronted with a higher concentration of
neutralizer in the test N . Therefore, no 10-fold higher validation suspension for the neutralizer control is
a
prepared.
b) Maintain and use the validation suspension the same way as the test suspension [5.4.1.4 b)].
-1
c) For counting prepare a 10 dilution with the water [5.2.2.2 a)]. Mix [5.3.2.6 a)]. Take a sample of
1,0 ml in duplicate and inoculate using the pour plate or the spread plate technique [5.4.1.4 d) 1) or
2)].
For incubation and counting see 5.4.1.6.
5.4.1.6 Incubation and counting of the test suspensions
a) Incubate (5.3.2.3) the plates with C. difficile in an anaerobic jar (5.3.2.22) for 5 d. Discard any plates
that are not countable for any reason. Count the plates and determine the number of cfu.
b) Note for each plate the exact number of colonies but record > 330 for any counts higher than 330 and
determine the V -values according to 5.6.2.2.
C
c) Calculate the numbers of cfu/ml in the test suspension N and the validation suspension N using the
V
method given in 5.6.2.3 and 5.6.2.5. Verify according to 5.7.
5.4.2 Product test solution
Product test solutions shall be prepared in hard water (5.2.2.7) at minimum two different concentrations
to include one concentration in the active range and one concentration in the non-active range.
Dilutions of ready-to-use products, i.e. products which are not diluted when applied, shall be prepared in
water (5.2.2.2) instead of hard water. The concentration of use (specified by the manufacturer) shall be
tested, as one of the obligatory test concentration.
For pre-impregnated wipes or ready-to-use wipes which cannot be diluted, the non-active range has to
be shown with a shorter contact time. If 1 min is the claimed contact time the 5 min contact time has to
be tested in addition. In this case it isn’t needed to show a non-active range. Details on the precision and
repetition are given in 5.8.4.
The shortest contact time to be tested is 1 min.
For solid products, dissolve the product as received by weighing at least 1 g ± 10 mg of the product in a
volumetric flask and filling up with hard water. Subsequent dilutions (i.e. lower concentrations) shall be
prepared in volumetric flasks (5.3.2.12) on a volume/volume basis in hard water (5.2.2.7).
For liquid products, dilutions of the product shall be prepared with hard water (or water for ready-to-
use products) on a volume/volume basis using volumetric flasks.
The product test solutions shall be prepared freshly and used in the test within 2 h. They shall give a
physically homogenous preparation, stable during the whole procedure. If during the procedure a visible
inhomogeneity appears due to the formation of a precipitate or flocculate (for example through the
addition of the interfering substance), it shall be recorded in the test report.
For impregnated wipes or ready-to-use wipes follow the instructions of use given by the manufacturer.
NOTE Counting microorganisms embedded in a precipitate or flocculate is difficult and unreliable.
Record the test concentration in terms of mass per volume or volume per volume and details of the
product sample as received.
5.5 Procedure for assessing the sporicidal activity against C. difficile of the product
5.5.1 General
5.5.1.1 Experimental conditions
Besides the temperature, contact time, interfering substance and test organism, additional experimental
conditions may be selected according to the practical use considered for the product (Clause 4):
a) temperature θ (in °C):
the temperature to be tested is either room temperature (21,5 °C ± 3,5 °C) or between 4 °C and
15,5 °C or between 27,5 °C and 30 °C; the deviation for any temperature within the two temperature
corridors is ± 2,5 °C;
b) contact time t (in min):
1) the contact times to be tested are those recommended by the manufacturer but not longer than
60 min; the allowed deviation for each chosen contact time is ± 10 s, except for 1 min where it
is ± 5 s;
2) the contact times for surface disinfectants are chosen on the basis of the practical conditions of
the product. The recommended contact time for the use of the product is within the
responsibility of the manufacturer. 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 30 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;
3) the minimum contact time is 1 min (from the beginning of the
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.
Loading comments...