EN 1656:2009

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Antiseptika und Desinfektionsmittel - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika in den Bereichen Lebensmittel, Industrie, Haushalt und öffentliche Einrichtungen - Prüfverfahren und Anforderungen (Phase 2, Stufe 1)Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (phase 2, étape 1)Chemical disinfectants and antiseptics - Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)11.220VeterinarstvoVeterinary medicine11.080.20Dezinfektanti in antiseptikiDisinfectants and antisepticsICS:Ta slovenski standard je istoveten z:EN 1656:2009SIST EN 1656:2010en,fr,de01-januar-2010SIST EN 1656:2010SLOVENSKI
STANDARDSIST EN 1656:20011DGRPHãþD

EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 1656
November 2009 ICS 71.100.35 Supersedes EN 1656:2000English Version
Chemical disinfectants and antiseptics -Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in the veterinary area - Test method and requirements (phase 2, step 1)
Antiseptiques et désinfectants chimiques - Essai quantitatif de suspension pour l'évaluation de l'activité bactéricide des antiseptiques et des désinfectants chimiques utilisés dans le domaine vétérinaire - Méthode d'essai et prescriptions (phase 2, étape 1)
Chemische Desinfektionsmittel und Antiseptika - Quantitativer Suspensionsversuch zur Bestimmung der bakteriziden Wirkung chemischer Desinfektionsmittel und Antiseptika für den Veterinärbereich - Prüfverfahren und Anforderungen (Phase 2, Stufe 1) This European Standard was approved by CEN on 20 September 2009.
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 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 Management Centre has the same status as the official versions.
CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre:
Avenue Marnix 17,
B-1000 Brussels © 2009 CEN All rights of exploitation in any form and by any means reserved worldwide for CEN national Members. Ref. No. EN 1656:2009: ESIST EN 1656:2010

Referenced strains in national collections . 25Annex B (informative)
Examples of neutralizers of the residual antimicrobial activity of chemical disinfectants and antiseptics and rinsing liquids . 27Annex C (informative)
Graphical representations of dilution-neutralization method and membrane filtration method . 28Annex D (informative)
Example of a typical test report . 32Bibliography . 37 SIST EN 1656:2010

According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following countries are bound to implement this European Standard: Austria, Belgium, Bulgaria, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom.
This European Standard applies to products that are used in the veterinary area – e.g. in the breeding, husbandry, transport and disposal of all animals except when in the food chain following death and entry to the processing industry. EN 14885 specifies in detail the relationship of the various tests to one another and to “use recommendations”. NOTE 1 The method described is intended to determine the activity of commercial formulations or active substances under the conditions in which they are used. NOTE 2 This method corresponds to a phase 2 step 1 test. 2 Normative references The following referenced documents are indispensable for the application 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, mycobactericidal, sporicidal and fungicidal activity EN 14885:2006, 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 14885:2006 apply. 4 Requirements The product shall demonstrate at least a five-decimal log (lg) reduction when diluted with hard water (5.2.2.7) or – in the case of ready-to-use products – with water (5.2.2.2) and tested in accordance with Clause 5 under simulated low-level soiling (3,0 g/l bovine albumin solution – 5.2.2.8.2) or simulated high-level soiling (10 g/l bovine albumin solution and 10 g/l yeast extract – 5.2.2.8.3) (or 10 g/l skimmed milk for teat disinfectants – 5.2.2.8.4) according to its practical applications and under the other obligatory test conditions four or three [for teat disinfectants] selected test organisms, 10 °C [30 ûC for teat disinfectants], 30 min [5 min for teat disinfectants]). The bactericidal activity shall be evaluated using the following organisms: a) Products for general disinfection: b) Teat disinfectants:  Enterococcus hirae;  Escherichia coli;  Proteus vulgaris;  Staphylococcus aureus;  Pseudomonas aeruginosa;  Streptococcus uberis. SIST EN 1656:2010

Where indicated, additional specific bactericidal activity shall be determined applying other contact times, temperatures, interfering substances and test organisms (in accordance with 5.2.1, 5.2.2.8 and 5.5.1.1) in order to take into account intended specific use conditions. NOTE For these additional conditions, the concentration defined as a result can be lower than the one obtained under the obligatory test conditions. 5 Test method 5.1 Principle 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 bacteria in a solution of an interfering substance. The mixture is maintained at (10 ± 1)
°C (or (30 ± 1) °C for teat disinfectants) for 30 min ± 10 s (5 min ± 10 s for teat disinfectants) (obligatory test conditions). At the end of this contact time, an aliquot is taken, and the bactericidal and/or the bacteriostatic activity 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 bacteria in each sample are determined and the reduction is calculated. 5.1.2 For general disinfectant products, the test is performed using Enterococcus hirae, Proteus vulgaris, Pseudomonas aeruginosa and Staphylococcus aureus as test organisms. For teat disinfectants the test is performed using Escherichia coli, Staphylococcus aureus and Streptococcus uberis as test organisms. 5.1.3 Additional and optional contact times and temperatures are specified. Additional test organisms can be used. 5.2 Materials and reagents 5.2.1 Test organisms The bactericidal activity shall be evaluated using the following strains as test organisms1):
a) General disinfection products:
 Enterococcus hirae ATCC 10541  Proteus vulgaris ATCC 13315
 Pseudomonas aeruginosa ATCC 15442
 Staphylococcus aureus ATCC 6538
b) Teat disinfectants:
 Escherichia coli ATCC 10536
 Staphylococcus aureus ATCC 6538
 Streptococcus uberis ATCC 19436
1) 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 standard and does not constitute an endorsement by CEN of the product named. SIST EN 1656:2010

The required incubation temperature for these test organisms is (36 ± 1) °C or (37 ± 1) °C (5.3.2.3). The same temperature (either 36 °C or 37 °C) shall be used for all incubations performed during a test and its control and validation. If additional test organisms are used, they shall be incubated under optimum growth conditions (temperature, time, atmosphere, media) noted in the test report. If the additional test organisms selected do not correspond to the specified strains, their suitability for supplying the required inocula shall be verified. If these additional test organisms are not classified at a reference centre, their identification characteristics shall be stated. In addition, they shall be held by the testing laboratory or national culture collection under a reference for five years. 5.2.2 Culture media and reagents 5.2.2.1 General All weights of chemical substances given in this standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. NOTE 1 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. NOTE 2 For each culture medium and reagent, a shelf life should be fixed (see ISO/IEC 17025). 5.2.2.2 Water The water shall be freshly glass-distilled water and not demineralized water. Sterilize in the autoclave (5.3.2.1 a). 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 [1] in the bibliography) can be used. NOTE 3 See 5.2.2.7 for the procedure to prepare hard water. 5.2.2.3 Tryptone Soya Agar (TSA) Tryptone soya agar, consisting of: Tryptone, pancreatic digest of casein
15,0 g Soya 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 (5.3.2.1 a). After sterilization the pH of the medium shall be equivalent to 7,2 ± 0,2 when measured at (20 ± 1) °C. NOTE In case of encountering problems with neutralization (5.5.1.2 and 5.5.1.3) it may be necessary to add neutralizer to the TSA. Annex B gives guidance on the neutralizers that may be used. SIST EN 1656:2010

1,0 g Sodium chloride (NaCl)
8,5 g Water (5.2.2.2)
to 1 000,0 ml Sterilize in the autoclave (5.3.2.1 a). After sterilization, the pH of the diluent shall be equivalent to 7,0 ± 0,2 when measured at (20 ± 1) °C. 5.2.2.5 Neutralizer The neutralizer shall be validated for the product being tested in accordance with 5.5.1.2, 5.5.1.3 and 5.5.2. It shall be sterile. NOTE Information on neutralizers that have been found to be suitable for some categories of products is given in Annex B. 5.2.2.6 Rinsing liquid (for membrane filtration) 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 membrane under the test conditions described in 5.5.3. NOTE Information on rinsing liquids that have been found to be suitable for some categories of products is given in Annex B. 5.2.2.7 Hard water for dilution of products For the preparation of 1 000 ml of hard water, the procedure is as follows:  Prepare solution A: dissolve 19,84 g magnesium chloride (MgCl2) and 46,24 g calcium chloride (CaCl2) 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 (5.3.2.8) for no longer than one month;  Prepare solution B: dissolve 35,02 g sodium bicarbonate (NaHCO3) 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) 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 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 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. NOTE 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 is lower than 300 mg/l of calcium carbonate (CaCO3) in the test tube. 5.2.2.8 Interfering substance 5.2.2.8.1 General The interfering substance shall be chosen according to the conditions of use laid down for the product. SIST EN 1656:2010

2) Disposable sterile equipment is an acceptable alternative to reusable glassware. SIST EN 1656:2010

NOTE 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 Shaker a) Electromechanical agitator, e.g. Vortex® mixer3); b) Mechanical shaker. 5.3.2.7 Membrane filtration apparatus, constructed of a material compatible with the substances to be filtered. The apparatus shall have a filter holder of at least 50 ml volume. It shall be suitable for use with filters of diameter 47 mm to 50 mm and 0,45 µm pore size for sterilization of hard water (5.2.2.7), bovine albumin (5.2.2.8.2 and 5.2.2.8.3) and if the membrane filtration method (5.5.3) is used. The vacuum source used shall give an even filtration flow rate. In order to obtain a uniform distribution of the micro-organisms over the membrane and to prevent overlong filtration, the device shall be set so as to obtain the filtration of 100 ml of rinsing liquid in 20 s to 40 s. 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, or calibrated automatic pipettes. 5.3.2.10 Petri dishes (plates) of size 90 mm to 100 mm. 5.3.2.11 Glass beads, 3 mm to 4 mm in diameter. 5.3.2.12 Volumetric flasks 5.4 Preparation of test organism suspensions and product test solutions 5.4.1 Test organism suspensions (test and validation suspension) 5.4.1.1 General For each test organism, two different suspensions have to be prepared: the “test suspension” to perform the test and the “validation suspension” to perform the controls and method validation.
3) Vortex® is an example of a suitable product available commercially. This information is given for the convenience of users of this standard and does not constitute an endorsement by CEN of this product. SIST EN 1656:2010

4) cfu/ml = colony forming unit(s) per millilitre. SIST EN 1656:2010

(in °C): SIST EN 1656:2010
5.5.1.2 Choice of test method (dilution-neutralization or membrane filtration) The method of choice is the dilution-neutralization method (5.5.2). To determine a suitable neutralizer, carry out the validation of the dilution neutralization method (5.5.2.3, 5.5.2.4 and 5.5.2.5 in connection with 5.5.2.6) using a neutralizer, chosen according to laboratory experience and published data. If this neutralizer is not valid, repeat the validation test using an alternative neutralizer taking into account the information given in Annex B. NOTE In special circumstances, it may be necessary to add neutralizer to TSA (5.2.2.3). If neutralizer is added to TSA the same amount shall be added to TSA used in the test procedure.
5.5.1.3 General instructions for validation and control procedures The neutralization and/or removal of the bactericidal and/or bacteriostatic activity of the product shall be controlled and validated – only for the highest product test concentration – for each of the used test organisms and for each experimental condition (interfering substance, temperature, contact time). These procedures (experimental condition control, neutralizer or filtration control and method validation) shall be performed at the same time with the test and with the same neutralizer – or rinsing liquid – used in the test. In the case of ready-to-use-products, use water (5.2.2.2) instead of hard water. If because of problems with neutralization, a neutralizer has been added to TSA (5.5.1.2) used for the validation and control procedures, the TSA used for the test shall contain the same amount of this neutralizer as well. SIST EN 1656:2010

(5.5.1.1 a) using the water bath (5.3.2.2) controlled at θ. Check that the temperature of the reagents is stabilized at θ. The neutralizer (5.2.2.5) or the rinsing liquid (5.2.2.6) and water (5.2.2.2) shall be equilibrated at a temperature of (20 ± 1) °C. In the case of ready-to-use-products, water (5.2.2.2) shall be additionally equilibrated to θ. 5.5.1.5 Precautions for manipulation of test organisms Do not touch the upper part of the test tube sides when adding the test or the validation suspensions (5.4.1). 5.5.2 Dilution-neutralization method5) 5.5.2.1 General The test and the control and validation procedures (5.5.2.2 to 5.5.2.5) shall be carried out in parallel and separately for each experimental condition (5.5.1.1). 5.5.2.2 Test "Na" – determination of bactericidal concentrations The procedure for determining bactericidal concentrations is as follows: a) Pipette 1,0 ml of the interfering substance (5.2.2.8) into a tube. Add 1,0 ml of the test suspension (5.4.1.4). Start the stopwatch (5.3.2.5) immediately, mix (5.3.2.6 a) and place the tube in a water bath controlled at the chosen test temperature θ (5.5.1.1 a) for 2 min ± 10 s. At the end of this time, add 8,0 ml of one of the product test solutions (5.4.2). Restart the stopwatch at the beginning of the addition. Mix (5.3.2.6 a) and place the tube in a water bath controlled at θ for the chosen contact time t
(5.5.1.1 b). Just before the end of t, mix (5.3.2.6 a) again. b) At the end of t, take a 1,0 ml sample of the test mixture "Na" and transfer into a tube containing 8,0 ml neutralizer (5.2.2.5) and 1,0 ml water (5.2.2.2). Mix (5.3.2.6 a) and place in a water bath controlled at (20
± 1) °C. After a neutralization time of 5 min ± 10 s, immediately take a sample of 1,0 ml of the neutralized test mixture "Na" (containing neutralizer, product test solution, interfering substance and test suspension) in duplicate and inoculate using the pour plate or spread plate technique. 1) When using the pour plate technique, pipette each 1,0 ml sample into separate Petri dishes and add 15 ml to 20 ml of melted TSA (5.2.2.3), cooled to (45 ± 1) °C.
2) When using the spread plate technique, spread each 1,0 ml sample – divided into portions of approximately equal size – on an appropriate number (at least two) of surface dried plates containing TSA (5.2.2.3). For incubation and counting, see 5.5.2.6. c) Perform the procedures a) and b) using the other product test solutions at the same time. d) Perform the procedures a) to c) applying the other obligatory and – if appropriate – other additional experimental conditions (5.5.1.1).
5) For a graphical representation of this method, see C.1. SIST EN 1656:2010

for 2 min ± 10 s. At the end of this time, add 8,0 ml of hard water (5.2.2.7) (in the case of ready-to-use products: water (5.2.2.2) instead of hard water). Restart the stopwatch at the beginning of the addition. Mix (5.3.2.6 a) and place the tube in a water bath controlled at
for t. Just before the end of t, mix (5.3.2.6 a) again. b) At the end of t, take a sample of 1,0 ml of this mixture "A" in duplicate and inoculate using the pour plate or the spread plate technique (5.5.2.2 b).
For incubation and counting, see 5.5.2.6. 5.5.2.4 Neutralizer control "B" – verification of the absence of toxicity of the neutralizer To verify the absence of toxicity of the neutralizer, the procedure is as follows: a) Pipette 8,0 ml of the neutralizer – used in the test (5.5.2.2) — and 1,0 ml of water (5.2.2.2) into a tube. Add 1,0 ml of the validation suspension (5.4.1.5). Start the stopwatch at the beginning of the addition, mix (5.3.2.6 a), and place the tube in a water bath controlled at (20 ± 1) °C for 5 min ± 10 s. Just before the end of this time, mix (5.3.2.6 a). b) At the end of this time, take a sample of 1,0 ml of this mixture "B" in duplicate and inoculate using the pour plate or the spread plate technique (5.5.2.2 b). For incubation and counting, see 5.5.2.6. 5.5.2.5 Method validation "C" – dilution-neutralization validation To validate the dilution neutralization method, the procedure is as follows: a) Pipette 1,0 ml of the interfering substance used in the test (5.5.2.2) into a tube. Add 1,0 ml of the diluent (5.2.2.4) and then, starting a stopwatch, add 8,0 ml of the product test solution only of the highest concentration used in the test (5.5.2.2). Mix (5.3.2.6 a) and place the tube in a water bath controlled at θ
for t. Just before the end of t, mix (5.3.2.6 a) again. b) At the end of t transfer 1,0 ml of the mixture into a tube containing 8,0 ml of neutralizer (used in 5.5.2.2). Restart the stopwatch immediately at the beginning of the addition. Mix (5.3.2.6 a) and place the tube in a water bath controlled at (20 ± 1) °C for 5 min ± 10 s. Add 1,0 ml of the validation suspension (5.4.1.5). Start a stopwatch at the beginning of the addition and mix (5.3.2.6 a). Place the tube in a water bath controlled at (20 ± 1) °C for the contact time t. Just before the end of this time, mix (5.3.2.6 a) again. At the end of this time, take a sample of 1,0 ml of the mixture C in duplicate and inoculate using the pour plate or the spread plate technique (5.5.2.2 b). For incubation and counting, see 5.5.2.6. 5.5.2.6 Incubation and counting of the test mixture and the control and validation mixtures For incubation and counting of the test mixture and the control and validation mixtures, the procedure is as follows: a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates which are not countable (for any reason). Count the cfu on the plates to determine the total number of colony forming units. Incubate the plates for a SIST EN 1656:2010

6) For a graphical representation of this method, see C.2. SIST EN 1656:2010

5.5.3.6 Incubation and counting of test mixture and the control and the validation mixtures For incubation and counting of the test mixture and the control and validation mixtures, the procedure is as follows: a) Incubate (5.3.2.3) the plates for 20 h to 24 h. Discard any plates which are not countable (for any reason). Count the colonies on the membranes. Incubate the plates for a further 20 h to 24 h. Do not recount plates that no longer show well separa
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