EN 1276:2009

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.Chemische Desinfektionsmittel und Antiseptika - 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 food, industrial, domestic and institutional areas - Test method and requirements (phase 2, step 1)71.100.35Kemikalije za dezinfekcijo v industriji in domaChemicals for industrial and domestic disinfection purposesICS:Ta slovenski standard je istoveten z:EN 1276:2009SIST EN 1276:2010en,fr,de01-januar-2010SIST EN 1276:2010SLOVENSKI
STANDARDSIST EN 1276:20011DGRPHãþD

EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM
EN 1276
October 2009 ICS 71.100.35 Supersedes EN 1276:1997English Version
Chemical disinfectants and antiseptics -Quantitative suspension test for the evaluation of bactericidal activity of chemical disinfectants and antiseptics used in food, industrial, domestic and institutional areas - 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 de l'agro-alimentaire, dans l'industrie, dans les domaines domestiques et en collectivité - Méthode d'essai et prescriptions (Phase 2, étape 1)
Chemische Desinfektionsmittel und Antiseptika - 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) 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 1276:2009: ESIST EN 1276:2010

Referenced strains in national collections . 30Annex B (informative)
Neutralizers and rinsing liquids . 31Annex C (informative)
Graphical representations of dilution neutralization method and membrane filtration method . 33C.1Dilution-neutralization method . 33C.2Membrane filtration method . 35Annex D (informative)
Example of a typical test report . 37Test results (bactericidal suspension test) . 39Annex E (informative)
Precision of the test result . 41Bibliography . 44 SIST EN 1276:2010

 Escherichia coli ATCC 10536;
 Staphylococcus aureus ATCC 6538;
 Enterococcus hirae ATCC 10541. If required for specific applications, additional strains may be chosen, for example from:  Salmonella Typhimurium ATCC 13311;
 Lactobacillus brevis DSM 6235;
 Enterobacter cloacae DSM 6234. NOTE See Annex A for strain references in some other culture collections. 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 European Standard refer to the anhydrous salts. Hydrated forms may be used as an alternative, but the weights required shall be adjusted to allow for consequent molecular weight differences. The reagents shall be of analytical grade and/or appropriate for microbiological purposes. They shall be free from substances that are toxic or inhibitory to the test organisms. 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 limitation for use should be fixed.
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 1276:2010

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 the 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. 5.2.2.4 Diluent Tryptone sodium chloride solution, consisting of: 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 (5.3.2.1 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. SIST EN 1276:2010

(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. 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 and detergents) shall be defined. NOTE The term “interfering substance” is used even if it contains more than one substance. 5.2.2.8.2 Clean conditions (bovine albumin solution – low concentration) Dissolve 0,3 g of bovine albumin fraction V (suitable for microbiological purposes) in 100 ml of water (5.2.2.2). Sterilize by membrane filtration (5.3.2.7), keep in the refrigerator (5.3.2.8) and use within one month. The final concentration of bovine albumin in the test procedure (5.5) is 0,3 g/l. SIST EN 1276:2010

2) Disposable sterile equipment is an acceptable alternative to reusable glassware. 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 1276:2010

(5.5.1.1 a) and use within 2 h.
4) cfu/ml = colony forming unit(s) per millilitre. SIST EN 1276:2010

(in °C):  the obligatory temperature to be tested is θ = 20 °C;  additional temperatures may be chosen from 4 °C, 10 °C, 30°C or 40 °C;  the allowed deviation for each chosen temperature is ± 1 °C; b) contact time t (in min):  the obligatory contact time to be tested is t = 5 min or t = 1 min (hands disinfection);  additional contact times may be chosen from 1 min, 15 min, 30 min or 60 min;  the allowed deviation for each chosen contact time is ± 10 s (except for 1 min, for which it is ± 5 s); c) interfering substance:  the obligatory interfering substance to be tested is 0,3 g/l bovine albumin (5.2.2.8.2) for clean conditions or 3 g/l bovine albumin (5.2.2.8.3) for dirty conditions according to practical applications;  additional interfering substances as given in 5.2.2.8 shall be chosen according to the field of application specified for the product; d) test organisms (5.2.1):  the obligatory test organisms are: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus and Enterococcus hirae. If required for specific applications, additional test organisms may be chosen
for example from: Salmonella Typhimurium, Lactobacillus brevis, Enterobacter cloacae. 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. If both neutralizers are found to be invalid, the membrane filtration method (5.5.3) may be used. NOTE In special circumstances, it may be necessary to add neutralizer to MEA (5.2.2.3). 5.5.1.3 General instructions for validation and control procedures SIST EN 1276:2010

(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 through 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.
5) For a graphical representation of this method, see Annex C.1. SIST EN 1276:2010

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 θ 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. SIST EN 1276:2010
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