1、August 2009DEUTSCHE NORM English price group 13No part of this standard may be reproduced without prior permission ofDIN Deutsches Institut fr Normung e. V., Berlin. Beuth Verlag GmbH, 10772 Berlin, Germany,has the exclusive right of sale for German Standards (DIN-Normen).ICS 13.060.50!$Y6“1541998ww
2、w.din.deDDIN EN ISO 21427-2Water quality Evaluation of genotoxicity by measurement of the induction ofmicronuclei Part 2: Mixed population method using the cell line V79(ISO 21427-2:2006)English version of DIN EN ISO 21427-2:2009-08Wasserbeschaffenheit Bestimmung der Gentoxizitt mit dem In-vitro-Mik
3、rokerntest Teil 2: Verwendung einer nicht-synchronisierten V79-Zellkulturlinie (ISO 21427-2:2006)Englische Fassung DIN EN ISO 21427-2:2009-08www.beuth.deDocument comprises 27 pagesDIN EN ISO 21427-2:2009-08 2 National foreword This standard has been prepared by Technical Committee ISO/TC 147 “Water
4、quality” in collaboration with Technical Committee CEN/TC 230 “Water analysis” (Secretariat: DIN, Germany). The responsible German body involved in its preparation was the Normenausschuss Wasserwesen (Water Practice Standards Committee), Technical Committees NA 119-01-03-07-03 AK Gentoxizitt and NA
5、119-01-03 AA Wasseruntersuchung. This standard is part of the series Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammunter-suchung Suborganismische Testverfahren (Gruppe T) (German standard methods for the examination of Designation of method T 5 “Evaluation of genotoxicity by measureme
6、nt of the induction of micronuclei Part 2: Mixed population method using the cell line V79”: Method DIN EN ISO 21427-2 T 5 This standard has been published to implement the Water Framework Directive (WFD), Directive 2000/60/EC of the European Parliament and of the Council of 23 October 2000 establis
7、hing a framework for Community action in the field of water policy*). In the English version of ISO 21427-2, subclauses 6.2.2 and 6.2.15, the wrong formulae are given. These have been corrected in the German version of DIN EN ISO 21427-2 and correspondingly in the English version of the standard. Th
8、e DIN Standards corresponding to the International Standards referred to in this document are as follows: ISO 5667-1 DIN EN ISO 5667-1 ISO 5667-3 DIN EN ISO 5667-3 ISO 5667-10 DIN 38402-11 ISO 5667-16 DIN EN ISO 5667-16 ISO 13829 DIN 38415-3 ISO 16240 DIN 38415-4 Expert assistance and specialized la
9、boratories will be required to perform the analysis described in this standard. Existing safety regulations are to be taken into account. Depending on the objective of the analysis, a check shall be made on a case-by-case basis as to whether and to what extent additional conditions will have to be s
10、pecified. *) Registered in the DITR database of DIN Software GmbH, obtainable from: Beuth Verlag GmbH, 10772 Berlin. As a result of national implementation in Germany, the following action has been taken: water, waste water and sludge Bio-assays with microorganisms (group T) and describes method T 5
11、. DIN EN ISO 21427-2:2009-08 3 German standard methods for the examination of water, waste water and sludge Standard methods published as DIN Standards are obtainable from Beuth Verlag GmbH, either individually or grouped in volumes. The standard methods included in the loose-leaf publication entitl
12、ed Deutsche Einheits-verfahren zur Wasser-, Abwasser- und Schlammuntersuchung will continue to be published jointly by Wiley-VCH Verlag and Beuth Verlag GmbH. All standard methods relevant to the Abwasserverordnung (Waste Water Regulation) (AbwV1) included in the new Regulation on Section 7a of the
13、Gesetz zur Ordnung des Wasserhaushaltes (German Water Management Act1)the Abwasserverordnung and the Gesetz zur Ordnung des Wasserhaushalts have been published by Beuth-Verlag GmbH as a loose-leaf collection Analysenverfahren in der Abwasserverordnung Rechtsvorschriften Standards or draft standards
14、bearing the group title “German standard methods for the examination of water, waste water and sludge” are classified under the following categories (main titles): General information (group A) (DIN 38402) Sensory analysis (group B) (DIN 38403) Physical and physicochemical parameters (group C) (DIN
15、38404) Anions (group D) (DIN 38405) Cations (group E) (DIN 38406) Substance group analysis (group F) (DIN 38407) Gaseous constituents (group G) (DIN 38408) Parameters characterizing effects and substances (group H) (DIN 38409) Biological-ecological methods of analysis (group M) (DIN 38410) Microbiol
16、ogical methods (group K) (DIN 38411) Test methods using water organisms (group L) (DIN 38412) Individual constituents (group P) (DIN 38413) Sludge and sediments (group S) (DIN 38414) Bio-assays with microorganisms (group T) (DIN 38415) In addition to the methods described in the DIN 38402 to DIN 384
17、15 series of standards, there are a number of European and International Standards available as DIN EN, DIN EN ISO and DIN ISO Standards, which also form part of the collection of German standard methods. Information on Parts of these series of standards that have already been published can be obtai
18、ned from the offices of the Normenausschuss Wasserwesen, telephone +49 30 2601-2448, or from Beuth Verlag GmbH, Burggrafenstr. 6, 10787 Berlin. 1) Registered in the DITR database of DIN Software GmbH, obtainable from: Beuth Verlag GmbH, 10772 Berlin. und Normen (Supplements). ) concerning Anforderun
19、gen an das Einleiten von Abwasser in Gewsser together with DIN EN ISO 21427-2:2009-08 4 National Annex NA (informative) Bibliography DIN 38402-11, German standard methods for the examination of water, waste water and sludge General information (group A) Part 11: Sampling of waste water (A 11) DIN 38
20、415-3, German standard methods for the examination of water, waste water and sludge Sub-animal testing (group T) Part 3: Determination of the genotype potential of water and waste water components with the umu-test (T 3) DIN 38415-4, German standard methods for the examination of water, waste water
21、and sludge Sub-animal testing (group T) Part 4: Determination of the genotoxic potential using the Salmonella microsome test (Ames Test) (T 4) DIN EN ISO 5667-1, Water quality Sampling Part 1: Guidance on the design of sampling programmes and sampling techniques DIN EN ISO 5667-3, Water quality Samp
22、ling Part 3: Guidance on the preservation and handling of water samples DIN EN ISO 5667-16, Water quality Sampling Part 16: Guidance on biotesting of samples 1 REIFFERSCHEID, G., DILL, F., FIEBLINGER, D., GMINSKI, R., GRUMMT, H. J., HAFNER, C., HOLLERT, H., KUNZ, S., RODRIGO, G., STOPPER, H., ZIEMAN
23、N, C. und SELKE, D.: Measurement of genotoxicity in wastewater samples Results of a collaborative study on the in vitro micronucleus test in the context of standardisation according to ISO. Mutation Res. http:/dx.doi.org/10.1016/j.mrgentox.2007.07.015 EUROPEAN STANDARDNORME EUROPENNEEUROPISCHE NORME
24、N ISO 21427-2March 2009ICS 13.060.70English VersionWater quality - Evaluation of genotoxicity by measurement of theinduction of micronuclei - Part 2: Mixed population method usingthe cell line V79 (ISO 21427-2:2006)Qualit de leau - valuation de la gnotoxicit par lemesurage de linduction de micronoya
25、ux - Partie 2:Mthode de la population mlange laide de la ligne decellules V79 (ISO 21427-2:2006)Wasserbeschaffenheit - Bestimmung der Gentoxizitt mitdem In-vitro-Mikrokerntest - Teil 2: Verwendung einer nicht-synchronisierten V79-Zellkulturlinie (ISO 21427-2:2006)This European Standard was approved
26、by CEN on 1 March 2009.CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this EuropeanStandard the status of a national standard without any alteration. Up-to-date lists and bibliographical references concerning such nationalstandards
27、 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 translationunder the responsibility of a CEN member into its own language and notified to the
28、CEN Management Centre has the same status as theofficial 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, Netherland
29、s, Norway, Poland, Portugal,Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.EUROPEAN COMMITTEE FOR STANDARDIZATIONCOMIT EUROPEN DE NORMALISATIONEUROPISCHES KOMITEE FR NORMUNGManagement Centre: Avenue Marnix 17, B-1000 Brussels 2009 CEN All rights of exploitation in any for
30、m and by any means reservedworldwide for CEN national Members.Ref. No. EN ISO 21427-2:2009: E2 DIN EN ISO 21427-2:2009-08 EN ISO 21427-2:2009 (E) Contents Page Foreword .3 1 Scope4 2 Normative references4 3 Terms and definitions .4 4 Principle .6 5 Interferences 6 6 Reagents and media6 7 Apparatus.1
31、0 8 Test facility criteria10 9 Procedure.11 10 Evaluation and assessment .15 11 Precision 17 12 Test report17 Annex A (informative) Bromodeoxyuridine (BrdU) method .18 Annex B (informative) Evaluation schemes .20 Annex C (normative) S9 fraction .21 Annex D (informative) Precision data .22 Bibliograp
32、hy23 Foreword 3 DIN EN ISO 21427-2:2009-08 EN ISO 21427-2:2009 (E) The text of ISO 21427-2:2006 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 21427-2:2009 by Technical Committee CEN/TC
33、 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 b
34、y September 2009. 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 stan
35、dards 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, P
36、ortugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and the United Kingdom. Endorsement notice The text of ISO 21427-2:2006 has been approved by CEN as a EN ISO 21427-2:2009 without any modification. WARNING Persons using this part of ISO 21427 should be familiar with normal laboratory
37、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 esse
38、ntial that tests conducted according to this part of ISO 21427 be carried out by suitably trained staff. 1 Scope This part of ISO 21427 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 sub
39、stances, 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
40、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
41、undated references, the latest edition of the referenced document (including any amendments) applies. ISO 5667-16, 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 di
42、stinct 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 4 DIN EN ISO 21427-2:2009-08 EN ISO 21427-2:2009 (E) 3.3 dilutio
43、n 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 For undiluted water or waste water, this coefficient is per definition 1:1. The corresponding smallest possible D value is 1. 3.4 D value smalles
44、t 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
45、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 in
46、dex 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 ce
47、lls replicate, calculated by a formula which takes into account 1, 2, 4 and 8 cell stages of clones 3.11 S9 fraction 9 000 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
48、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
49、 cells used for the study 5 DIN EN ISO 21427-2:2009-08 EN ISO 21427-2:2009 (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 cel
