Vattenundersökningar Bestämning av genotoxicitet genom bildning av mikrokärnor Del 2: Metod med V79-celler från kinesisk hamster (ISO :2006)

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1 SVENSK STANDARD SS-EN ISO :2009 Fastställd/Approved: Publicerad/Published: Utgåva/Edition: 1 Språk/Language: engelska/english ICS: Vattenundersökningar Bestämning av genotoxicitet genom bildning av mikrokärnor Del 2: Metod med V79-celler från kinesisk hamster (ISO :2006) Water quality Evaluation of genotoxicity by measurement of the induction of micronuclei Part 2 Mixed population method using the cell line V79 (ISO :2006) SWEDISH STANDARDS INSTITUTE

2 Hitta rätt produkt och ett leveranssätt som passar dig Standarder Genom att följa gällande standard både e ektiviserar och säkrar du ditt arbete. Många standarder ingår dessutom ofta i paket. Tjänster Abonnemang är tjänsten där vi uppdaterar dig med aktuella standarder när förändringar sker på dem du valt att abonnera på. På så sätt är du säker på att du alltid arbetar efter rätt utgåva. e-nav är vår online-tjänst som ger dig och dina kollegor tillgång till standarder ni valt att abonnera på dygnet runt. Med e-nav kan samma standard användas av era personer samtidigt. Leveranssätt Du väljer hur du vill ha dina standarder levererade. Vi kan erbjuda dig dem på papper och som pdf. Andra produkter Vi har böcker som underlättar arbetet att följa en standard. Med våra böcker får du ökad förståelse för hur standarder ska följas och vilka fördelar den ger dig i ditt arbete. Vi tar fram många egna publikationer och fungerar även som återförsäljare. Det gör att du hos oss kan hitta över 500 unika titlar. Vi har även tekniska rapporter, speci kationer och workshop agreement. Matriser är en översikt på standarder och handböcker som bör läsas tillsammans. De nns på sis.se och ger dig en bra bild över hur olika produkter hör ihop. Standardiseringsprojekt Du kan påverka innehållet i framtida standarder genom att delta i någon av SIS ca 400 Tekniska Kommittéer. Find the right product and the type of delivery that suits you Standards By complying with current standards, you can make your work more e cient and ensure reliability. Also, several of the standards are often supplied in packages. Services Subscription is the service that keeps you up to date with current standards when changes occur in the ones you have chosen to subscribe to. is ensures that you are always working with the right edition. e-nav is our online service that gives you and your colleagues access to the standards you subscribe to 24 hours a day. With e-nav, the same standards can be used by several people at once. Type of delivery You choose how you want your standards delivered. We can supply them both on paper and as PDF les. Other products We have books that facilitate standards compliance. ey make it easier to understand how compliance works and how this bene ts you in your operation. We produce many publications of our own, and also act as retailers. is means that we have more than 500 unique titles for you to choose from. We also have technical reports, speci cations and workshop agreements. Matrices, listed at sis.se, provide an overview of which publications belong together. Standardisation project You can in uence the content of future standards by taking part in one or other of SIS s 400 or so Technical Committees.

3 Europastandarden EN ISO :2009 gäller som svensk standard. Detta dokument innehåller den officiella engelska versionen av EN ISO :2009. The European Standard EN ISO :2009 has the status of a Swedish Standard. This document contains the official English version of EN ISO :2009. Copyright/Upphovsrätten till denna produkt tillhör SIS, Swedish Standards Institute, Stockholm, Sverige. Användningen av denna produkt regleras av slutanvändarlicensen som åter nns i denna produkt, se standardens sista sidor. Copyright SIS, Swedish Standards Institute, Stockholm, Sweden. All rights reserved. The use of this product is governed by!the end-user licence for this product. You will nd the licence in the end of this document. Upplysningar om sakinnehållet i standarden lämnas av SIS, Swedish Standards Institute, telefon Standarder kan beställas hos SIS Förlag AB som även lämnar allmänna upplysningar om svensk och utländsk standard. Information about the content of the standard is available from the Swedish Standards Institute (SIS), tel Standards may be ordered from SIS Förlag AB, who can also provide general information about Swedish and foreign standards. SIS Förlag AB, SE Stockholm, Sweden. Tel: Fax: sis.sales@sis.se Internet:

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5 EUROPEAN STANDARD NORME EUROPÉENNE EUROPÄISCHE NORM EN ISO March 2009 ICS English Version Water quality - Evaluation of genotoxicity by measurement of the induction of micronuclei - Part 2: Mixed population method using the cell line V79 (ISO :2006) Qualité de l'eau - Évaluation de la génotoxicité par le mesurage de l'induction de micronoyaux - Partie 2: Méthode de la population mélangée à l'aide de la lignée de cellules V79 (ISO :2006) Wasserbeschaffenheit - Bestimmung der Gentoxizität mit dem In-vitro-Mikrokerntest - Teil 2: Verwendung einer nichtsynchronisierten V79-Zellkulturlinie (ISO :2006) This European Standard was approved by CEN on 1 March 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 ISO :2009: E

6 SS-EN ISO :2009 (E) Provläsningsexemplar / Preview ISO :2006(E) Contents Page Foreword... iv 1 Scope Normative references Terms and definitions Principle Interferences Reagents and media Apparatus Test facility criteria Procedure Evaluation and assessment Precision Test report Annex A (informative) Bromodeoxyuridine (BrdU) method Annex B (informative) Evaluation schemes Annex C (normative) S9 fraction Annex D (informative) Precision data Bibliography iii

7 SS-EN ISO :2009 (E) EN ISO :2009 (E) Foreword The text of ISO :2006 has been prepared by Technical Committee ISO/TC 147 Water quality of the International Organization for Standardization (ISO) and has been taken over as EN ISO :2009 by Technical Committee CEN/TC 230 Water analysis the secretariat of which is held by DIN. 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 September 2009, and conflicting national standards shall be withdrawn at the latest by September Attention is drawn to the possibility that some of the elements of this document may be the subject of patent rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights. 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. Endorsement notice The text of ISO :2006 has been approved by CEN as a EN ISO :2009 without any modification. iv

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9 SS-EN ISO :2009 (E) Water quality Evaluation of genotoxicity by measurement of the induction of micronuclei Part 2: Mixed population method using the cell line V79 WARNING Persons using this part of ISO should be familiar with normal laboratory practice. This standard does not purport to address all of the safety problems, if any, associated with its use. It is the responsibility of the user to establish appropriate safety and health practices and to ensure compliance with any national regulatory conditions. IMPORTANT It is absolutely essential that tests conducted according to this part of ISO be carried out by suitably trained staff. 1 Scope This part of ISO specifies a method for the determination of genotoxicity of water and waste water using a mammalian in vitro test which detects damage, induced by water-soluble substances, to the chromosomes or the mitotic apparatus of V79 cells from the Chinese hamster. The micronucleus test allows the identification of substances that cause cytogenetic damage which results in the formation of micronuclei containing lagging chromosome fragments and/or whole chromosomes. The assay is based on the increase in the frequency of micronucleated cells after incubation with and without metabolic activation. 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. ISO , Water quality Sampling Part 16: Guidance on biotesting of samples 3 Terms and definitions For the purposes of this document, the following terms and definitions apply: 3.1 cell lines distinct families of cells grown in culture originated from a single clone 3.2 cofactor solution aqueous solution of chemicals (e.g. NADP, Glucose-6-phosphate and inorganic salts) needed for the activity of the enzymes in the S9 fraction ISO 2006 All rights reserved 1

10 SS-EN ISO :2009 (E) ISO :2006(E) Provläsningsexemplar / Preview 3.3 dilution level D denominator of the dilution coefficient (using the numerator 1) of a mixture of water or waste water with dilution water as integral number NOTE value is 1. For undiluted water or waste water, this coefficient is per definition 1:1. The corresponding smallest possible D 3.4 D value smallest value of D at which, under the conditions of this part of ISO 21427, no increase in the number of micronuclei per culture is detected NOTE In the case of more than one D value (at maximum two are possible, see 9.2), the highest D value is decisive. 3.5 karyotype characteristic of the nucleus of a cell, including its size, form and chromosome number 3.6 micronuclei small particles consisting of acentric fragments of chromosomes and/or entire chromosomes which lag behind at anaphase stage of cell division and form, after telophase, single or multiple micronuclei in the cytoplasm 3.7 mitotic index percentage of cells of a cell population under division at a particular time of observation 3.8 plating efficiency measure of the number of colonies originated from single cells 3.9 proliferation index rate at which cells are dividing within the culture 3.10 proliferation rate rate with which cells replicate, calculated by a formula which takes into account 1, 2, 4 and 8 cell stages of clones 3.11 S9 fraction g supernatant of a tissue homogenate in 0,15 mol/l KCl, obtained from livers of male rats (200 g to 300 g) pretreated with an appropriate substance or substance combination for enzyme induction 3.12 S9 mix mixture of the S9 fraction and the cofactor solution 3.13 stock culture frozen culture for the preservation of the characteristics of V79 cells 3.14 survival index percentage of surviving cells compared to all cells, used as index of toxicity 3.15 test culture culture of cells used for the study 2 ISO 2006 All rights reserved

11 SS-EN ISO :2009 (E) ISO :2006(E) 4 Principle The possible clastogenic and/or aneugenic activity of the test sample is detected by comparing, for the respective activation condition, the number of micronucleated cells in cultures treated with the negative control and the number in cultures treated with undiluted and diluted test samples, respectively. During cell division, chromatid fragments without centromers will not move to the nuclei of the daughter cells and will stay within the cytoplasm. Part of the chromosome aberrations induced by the test item will be chromatid fragments without centromers and will, therefore, not be incorporated in the nuclei of the daughter cells. In addition, spindle disorders may lead to chromosomes which are not incorporated into the nucleus. These particles will form micronuclei in the plasma. V79 cells are exposed for 24 h (4 h with the S9 mix) to a range of concentrations of a test sample. Thereafter, slides are prepared, and cells are stained and evaluated for the presence of micronucleated cells. An increased incidence of these micronucleated cells in comparison to the negative control indicates that the test item may cause chromosome breaks or spindle disorders in V79 cells in vitro. 5 Interferences Biologically relevant alterations of the culture conditions may induce chromosome aberration due to secondary mechanisms resulting in artificial positive and, therefore, irrelevant results [16]. Those factors are, e.g. stronger changes in osmolality or ph, precipitation of test sample and phagocytosis thereof, and strong cytotoxic effects of the test sample. Therefore, test samples should be monitored at least for changes in ph or osmolality of the cultures using the same proportion of test item per culture as will be used later under test conditions. If there is a shift in ph in the culture, the test item should be adjusted to ph 7,0 ± 0,2. If there is a change in osmolality, the highest concentration used in the test has to be reduced so that no relevant alteration of osmolality occurs in the cultures. To avoid artifacts based on phagocytosis or severe cytotoxicity, limitations are given for the highest concentration, which should be used for testing (see 9.1 and 9.2). 6 Reagents and media As far as possible use reagent grade chemicals. If chemicals with different amounts of water of crystallization are used, calculate the needed amounts accordingly. Always perform autoclaving for 20 min at 121 C ± 2 C. Cover vessels loosely (e.g. with aluminium foil). Never seal air-tight. 6.1 Water. Prepare all aqueous solutions with water of a conductivity of u 5 μs/cm. 6.2 Reagents Glucose-6-phosphate dihydrate, C 6 H 11 O 9 PNa 2 2 H 2 O Nicotinamide adenine dinucleotide phosphate disodium salt, NADP, C 21 H 26 N 7 Na 2 O 17 P Magnesium chloride hexahydrate, MgCl 2 6 H 2 O Potassium dihydrogenphosphate, KH 2 PO di-sodium hydrogenphosphate dihydrate, Na 2 HPO 4 2 H 2 O Ethanol (absolute), C 2 H 5 OH. ISO 2006 All rights reserved 3