FprEN 13624

SLOVENSKI STANDARD
oSIST prEN 13624:2019
01-oktober-2019
Kemična razkužila in antiseptiki - Kvantitativni suspenzijski preskus za
vrednotenje fungicidnega delovanja ali delovanja na kvasovke v humani medicini -
Preskusna metoda in zahteve (faza 2, stopnja 1)

Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation

of fungicidal or yeasticidal activity in the medical area - Test method and requirements

Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur

Bestimmung der fungiziden oder levuroziden Wirkung im humanmedizinischen Bereich -

l'évaluation de l'activité fongicide ou levuricide en médecine - Méthode d'essai et

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

Désinfectants chimiques et antiseptiques - Essai Chemische Desinfektionsmittel und Antiseptika -

quantitatif de suspension pour l'évaluation de l'activité Quantitativer Suspensionsversuch zur Bestimmung der

fongicide ou levuricide en médecine - Méthode d'essai fungiziden oder levuroziden Wirkung im

et prescriptions (phase 2, étape 1) humanmedizinischen Bereich - Prüfverfahren und

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

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

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

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

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

© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 13624:2019 E

European foreword ....................................................................................................................................................... 3

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

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

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

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

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

5 Test method ...................................................................................................................................................... 7

5.1 Principle ............................................................................................................................................................. 7

5.2 Materials and reagents .................................................................................................................................. 8

5.3 Apparatus and glassware .......................................................................................................................... 11

5.4 Preparation of test organism suspensions and product test solutions .................................... 13

5.5 Procedure for assessing the fungicidal and yeasticidal activity of the product .................... 18

5.6 Experimental data and calculation ........................................................................................................ 26

5.7 Verification of methodology ..................................................................................................................... 32

5.8 Expression of results and precision ...................................................................................................... 33

5.9 Interpretation of results – conclusion .................................................................................................. 34

5.10 Test report ...................................................................................................................................................... 35

Annex A (informative) Referenced strains in national collections ........................................................... 37

Annex B (informative) Neutralizers and rinsing liquids ............................................................................... 38

Annex C (informative) Graphical representation of test procedures ....................................................... 40

C.1 Dilution-neutralization method ............................................................................................................. 40

C.2 Membrane filtration method ................................................................................................................... 42

C.3 Dilution-neutralization method (modified method for ready-to-use products) .................. 44

C.4 Membrane filtration method (modified method for ready-to-use products) ........................ 46

Annex D (informative) Example of a typical test report................................................................................ 48

Annex E (informative) Precision of the test result .......................................................................................... 53

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

covered............................................................................................................................................................. 56

Bibliography ................................................................................................................................................................. 58

This document (prEN 13624:2019) has been prepared by Technical Committee CEN/TC 216 “Chemical

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

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

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

— new Annex ZA was added and the reference was changed to the Medical Device Regulation (instead

— The amounts for the dirty conditions for the modified method for ready-to-use products were

— Clarification that a neutralization time of 10 s shall be used for all products with contact times of 10

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

This document specifies a suspension test for establishing whether a chemical disinfectant or an

antiseptic has a fungicidal or yeasticidal activity in the area and fields described in the scope.

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 can

influence its action in practical situations. Each utilization concentration of the chemical disinfectant or

This document specifies a test method and the minimum requirements for fungicidal or yeasticidal

activity of chemical disinfectant and antiseptic products that form a homogeneous, physically stable

preparation when diluted with hard water, or – in the case of ready-to-use products – with water.

Products can only be tested at a concentration of 80 % or less (97 % with a modified method for special

cases) as some dilution is always produced by adding the test organisms and interfering substance.

This document applies to products that are used in the medical area in the fields of hygienic handrub,

hygienic handwash, surgical handrub, surgical handwash, instrument disinfection by immersion, and

This document applies to areas and situations where disinfection or antisepsis is medically indicated.

and can occur in the workplace and in the home. It can also include services such as laundries and

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

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

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

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

ISO 4793:1980, Laboratory sintered (fritted) filters — Porosity grading, classification and designation

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:

The product shall demonstrate at least a 4 decimal logarithms (lg) reduction (for hygienic handwash at

Minimum Candida albicans Candida albicans a) fungicidal a) fungicidal a) fungicidal

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

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

of the manufacturer. Products intended to disinfect surfaces that are likely to come into contact with the patient

and/or the medical staff and surfaces, which are frequently touched by different people, leading to the

transmission of microorganisms to the patient, shall be tested with a contact time of maximum 5 min. The same

applies where the contact time of the product shall be limited for practical reasons. Products for other surfaces

Hygienic and surgical handrub shall be tested as a minimum under clean conditions.

Hygienic and surgical handwash shall be tested as a minimum under dirty conditions.

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

5.1.1 A sample of the product as delivered and/or diluted with hard water (or water for ready to use

products) is added to a test suspension of fungi (yeast cells or mould spores) in a solution of an

interfering substance. The mixture is maintained at the temperature and the contact time specified in

Clause 4 and 5.5.1.1. At the end of this contact time, an aliquot is taken; the fungicidal and/or the

fungistatic action in this portion is immediately neutralized or suppressed by a validated method. The

method of choice is dilution-neutralization. If a suitable neutralizer cannot be found, membrane

filtration is used. The numbers of surviving fungi in each sample are determined and the reduction is

NOTE Handwash products are always prediluted with hard water (5.2.2.7). The resulting solution is regarded

5.1.2 The test is performed using the vegetative cells of Candida albicans and the conidiospores of

Aspergillus brasiliensis (fungicidal activity) or only the vegetative cells of Candida albicans (yeasticidal

5.1.3 Additional contact times and temperatures are specified (Clause 4, Table 1). Additional

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

The required incubation temperature for these test organisms is (30 ± 1) °C (5.3.2.3).

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

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

The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be

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

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 [2]) can be used.

Sterilize in the autoclave [5.3.2.1 a)]. Sterilization is not necessary if the water is used, e.g. for

The ATCC numbers are the collection numbers of strains supplied by these culture collections. This

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

Sterilize in the autoclave (5.3.1). After sterilization, the pH (5.3.2.4) of the medium shall be equivalent to

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

neutralizer to MEA. Annex B gives guidance on the neutralizers that may be used. It is recommended

If there are problems with producing at least 75 % spiny conidiospores, see 5.4.1.4.2.

Sterilize in the autoclave [5.3.2.1 a)]. After sterilization, the pH (5.3.2.4) of the diluent shall be

The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and

Information on neutralizers that have been found to be suitable for some categories of products is given

The rinsing liquid shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and

5.5.3. It shall be sterile, compatible with the filter membrane and capable of filtration through the filter

Information on rinsing liquids that have been found to be suitable for some categories of products is

This Malt extract from OXOID 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

— 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 the refrigerator

— 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)

— 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 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

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 expressed as calcium

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 (50

The ionic composition (e.g. pH, calcium and/or magnesium hardness) and chemical composition (e.g.

mineral substances, protein, carbohydrates, lipids and detergents) shall be defined.

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

Dissolve 0,30 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of diluent

Sterilize by membrane filtration (5.3.2.7), keep in a refrigerator (5.3.2.8) and use within one month.

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

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

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

Prepare at least 8,0 ml fresh defibrinated sheep blood (5.2.2.9). Centrifuge the erythrocytes at 800 g

for 10 min (5.3.2.13). After discarding the supernatant, resuspend erythrocytes in diluent (5.2.2.4).

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

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

and kept in separate containers for a maximum of 7 d in a refrigerator (5.3.2.8).

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

5.2.2.8.4 Clean and dirty conditions for the modified method for ready-to-use products (5.5.4)

Follow the procedures for preparation according to 5.2.2.8.2 and 5.2.2.8.3, but prepare the interfering

substance in fivefold higher concentrations, for the dirty conditions maximum 50 ml to avoid problems

a) Clean conditions (5.2.2.8.2) – dissolve 1,50 g bovine albumin (instead of 0,3 g) in 100 ml of diluent;

b) Dirty conditions (5.2.2.8.3) – dissolve 7,5 g bovine albumin (instead of 1,5 g) in 42,5 ml of diluent

(instead of 48,5 ml). Prepare at least 20 ml (instead of 4,0 ml) sheep blood. Resuspend 7,5 ml

(instead of 1,5 ml) of the packed sheep erythrocytes in 42,5 ml of sterilized bovine albumin solution

The defibrinated sheep blood should be sterile (aseptic blood-letting and preparation), pooled from

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:

5.3.2 Usual microbiological laboratory equipment , and, in particular, the following:

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

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

5.3.2.2 Water baths, capable of being controlled at (20 ± 1) °C, at (45 ± 1) °C (to maintain melted

MEA in case of pour plate technique) and at additional test temperatures ± 1 °C (5.5.1).

5.3.2.4 pH-meter, having an inaccuracy of calibration of no more than ± 0,1 pH units at (20 ± 1) °C.

A puncture electrode or a flat membrane electrode should be used for measuring the pH of the agar