EN ISO 21427-2:2009

SLOVENSKI STANDARD
01-maj-2009
.DNRYRVWYRGH9UHGQRWHQMHJHQRWRNVLþQRVWL]PHUMHQMHPLQGXNFLMH
PLNURQXNOHXVRYGHO0HWRGD]QHVLQKURQL]LUDQRSRSXODFLMRFHOLF9FHOLþQH
OLQLMH,62
Water quality - Evaluation of genotoxicity by measurement of the induction of micronuclei
- Part 2: Mixed population method using the cell line V79 (ISO 21427-2:2006)
Wasserbeschaffenheit - Bestimmung der Gentoxizität mit dem In-vitro-Mikrokerntest -
Teil 2: Verwendung einer nicht-synchronisierten V79-Zellkulturlinie (ISO 21427-2:2006)
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 21427-2:2006)
Ta slovenski standard je istoveten z: EN ISO 21427-2:2009
ICS:
13.060.70 Preiskava bioloških lastnosti Examination of biological
vode properties of water
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EUROPEAN STANDARD
EN ISO 21427-2
NORME EUROPÉENNE
EUROPÄISCHE NORM
March 2009
ICS 13.060.70
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 21427-2:2006)
Qualité de l'eau - Évaluation de la génotoxicité par le Wasserbeschaffenheit - Bestimmung der Gentoxizität mit
mesurage de l'induction de micronoyaux - Partie 2: dem In-vitro-Mikrokerntest - Teil 2: Verwendung einer nicht-
Méthode de la population mélangée à l'aide de la lignée de synchronisierten V79-Zellkulturlinie (ISO 21427-2:2006)
cellules V79 (ISO 21427-2:2006)
This European Standard was approved 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 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 Ref. No. EN ISO 21427-2:2009: E
worldwide for CEN national Members.

Contents Page
Foreword .3

Foreword
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 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 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 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 21427-2:2006 has been approved by CEN as a EN ISO 21427-2:2009 without any
modification.
INTERNATIONAL ISO
STANDARD 21427-2
First edition
2006-11-15
Water quality — Evaluation of
genotoxicity by measurement of the
induction of micronuclei —
Part 2:
Mixed population method using the cell
line V79
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
Reference number
ISO 21427-2:2006(E)
©
ISO 2006
ISO 21427-2:2006(E)
PDF disclaimer
This PDF file may contain embedded typefaces. In accordance with Adobe's licensing policy, this file may be printed or viewed but
shall not be edited unless the typefaces which are embedded are licensed to and installed on the computer performing the editing. In
downloading this file, parties accept therein the responsibility of not infringing Adobe's licensing policy. The ISO Central Secretariat
accepts no liability in this area.
Adobe is a trademark of Adobe Systems Incorporated.
Details of the software products used to create this PDF file can be found in the General Info relative to the file; the PDF-creation
parameters were optimized for printing. Every care has been taken to ensure that the file is suitable for use by ISO member bodies. In
the unlikely event that a problem relating to it is found, please inform the Central Secretariat at the address given below.

©  ISO 2006
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
ISO's member body in the country of the requester.
ISO copyright office
Case postale 56 • CH-1211 Geneva 20
Tel. + 41 22 749 01 11
Fax + 41 22 749 09 47
E-mail copyright@iso.org
Web www.iso.org
Published in Switzerland
ii © ISO 2006 – All rights reserved

ISO 21427-2:2006(E)
Contents Page
Foreword. iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions. 1
4 Principle. 3
5 Interferences . 3
6 Reagents and media. 3
7 Apparatus . 7
8 Test facility criteria . 7
9 Procedure . 8
10 Evaluation and assessment. 12
11 Precision. 14
12 Test report . 14
Annex A (informative) Bromodeoxyuridine (BrdU) method. 15
Annex B (informative) Evaluation schemes. 17
Annex C (normative) S9 fraction . 18
Annex D (informative) Precision data. 19
Bibliography . 20

ISO 21427-2:2006(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards bodies
(ISO member bodies). The work of preparing International Standards is normally carried out through ISO
technical committees. Each member body interested in a subject for which a technical committee has been
established has the right to be represented on that committee. International organizations, governmental and
non-governmental, in liaison with ISO, also take part in the work. ISO collaborates closely with the
International Electrotechnical Commission (IEC) on all matters of electrotechnical standardization.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. ISO shall not be held responsible for identifying any or all such patent rights.
ISO 21427-2 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
ISO 21427 consists of the following parts, under the general title Water quality — Evaluation of genotoxicity by
measurement of the induction of micronuclei:
⎯ Part 1: Evaluation of genotoxicity using amphibian larvae
⎯ Part 2: Mixed population method using the cell line V79

iv © ISO 2006 – All rights reserved

INTERNATIONAL STANDARD ISO 21427-2:2006(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 21427 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 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 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 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
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 21427-2:2006(E)
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 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
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
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 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

ISO 21427-2:2006(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
[16]
mechanisms resulting in artificial positive and, therefore, irrelevant results . 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.
6.2.1 Glucose-6-phosphate dihydrate, C H O PNa · 2 H O.
6 11 9 2 2
6.2.2 Nicotinamide adenine dinucleotide phosphate disodium salt, NADP, C H N Na O P .
21 26 7 2 17 3
6.2.3 Magnesium chloride hexahydrate, MgCl · 6 H O.
2 2
6.2.4 Potassium dihydrogenphosphate, KH PO .
2 4
6.2.5 di-Sodium hydrogenphosphate dihydrate, Na HPO · 2 H O.
2 4 2
6.2.6 Ethanol (absolute), C H OH.
2 5
ISO 21427-2:2006(E)
6.2.7 Glacial acetic acid, CH COOH.
6.2.8 Formaldehyde, HCHO, 37 % volume fraction.
6.2.9 tri-Sodium citrate dihydrate, HOC(COONa)(CH COONa) ·2 H O.
2 2 2
6.2.10 di-Sodium hydrogenphosphate, Na HPO .
2 4
6.2.11 Sodium dihydrogenphosphate, NaH PO .
2 4
1)
6.2.12 May-Grünwald-solution, modified .
6.2.13 Hydrochloric acid, c(HCl) = 1 mol/l.
6.2.14 Sodium hydroxide solution, c(NaOH) = 1 mol/l.
6.2.15 Dimethyl sulfoxide (DMSO), C H SO .
2 6 4
6.2.16 Positive controls.
6.2.16.1 Cyclophosphamide, monohydrate, C H Cl N O P·H O.
7 15 2 2 2 2
CAS Registration No: 6055-19-2.
6.2.16.2 Ethyl-methane sulfonate (EMS), CH SO CH CH .
3 3 2 3
CAS Registration No: 62-50-0.
6.2.17 Sodium citrate solution for hypotonic treatment.
Prepare a 1,5 % solution of tri-sodium citrate in water.
6.2.18 Fixation solution.
Mix 50 ml of glacial acetic acid with 150 ml of ethanol, add 2,5 ml of a 37 % formaldehyde solution.
1)
6.2.19 Buffer solution according to WEISE (pH 7,2) .
This solution is commercially available in ampoules. Dilute the contents of one ampoule in water and, using a
1 000 ml measuring flask, bring to volume with water.
1)
6.2.20 Giemsa solution .
Prepare a 2,6 % Giemsa solution in buffer according to WEISE (pH 7,2) (6.2.19). Filter prior to use.
6.2.21 Phosphate buffer.
Dissolve 2,13 g of Na HPO in 1 l water. Dissolve 1,8 g of NaH PO in 1 l water. Mix both solutions at the
2 4 2 4
ratio of 4:1 and adjust to a final pH of 7,4.
1)
6.2.22 MEM-medium (= Minimal Essential Medium) with stabilized glutamine .
1)
6.2.23 Fetal bovine serum (= FCS) .

1) This reagent is commercially available. This information is given for the convenience of users of this part of ISO 21427
and does not constitute an endorsement by ISO of these products.
4 © ISO 2006 – All rights reserved

ISO 21427-2:2006(E)
1)
6.2.24 Penicillin/Streptomycin solution, 10 000 E/10 000 µg/ml .
1)
6.2.25 Amphotericin-B solution, 250 µg/ml .
1)
6.2.26 Trypsin/EDTA solution, 0,25 % .
1)
6.2.27 Hanks' Balanced Salt Solution (= HBSS) .
2+ 2+ 1)
6.2.28 Hanks' Balanced Salt Solution (= HBSS) without Ca and Mg .
6.2.29 Potassium chloride solution.
Dissolve 4 g of potassium chloride, in 1 l water.
6.3 Preparation of culture media
6.3.1 Culture medium with FCS.
This medium is used as general culture medium and for treatment of cells without the S9 mix.
Mix 500 ml of MEM-medium, 50 ml of FCS, 5 ml of Penicillin/Streptomycin solution and 5 ml of
Amphotericin-B solution.
The medium is stable for up to 4 weeks if stored in a refrigerator at 4 °C ± 2 °C.
6.3.2 Culture medium without FCS.
This medium is used only for the treatment period of cells under activation condition (S9 mix).
Mix 500 ml of MEM-medium, 5 ml of Penicillin/Streptomycin solution and 5 ml of Amphotericin-B solution.
The medium is stable for up to 4 weeks if stored in a refrigerator at 4 °C ± 2 °C.
6.4 Cell system.
6.4.1 Cell line, storage
The V79 cell line is a permanent cell line of Chinese hamster lung cells with
⎯ a high proliferation rate (cell cycle length about 12 h to 16 h);
⎯ a high plating efficiency (W 90 %);
⎯ a stable karyotype (modal number of chromosomes = 22).
Store permanent cultures (1 ml samples including 7 % DMSO) in liquid nitrogen at about −196 °C. Prior to
freezing, check each batch for mycoplasma contamination. Karyotype and plating efficiency (colony-forming
ability) should be determined at least prior to the first use of a thawed culture.
6.4.2 Cultivation
To start a culture, thaw a permanent culture in a water bath at 37 °C and add 0,5 ml of this sample to a
25 cm culture flask filled already with approximately 5 ml of MEM (minimal essential medium; composed of
medium, glutamine and antibiotics) including 10 % FCS (fetal calf serum). Cultivate the cells at 37 °C, using
5 % carbon dioxide and a humidity of at least 90 %. Subcultivate the cells twice a week.
ISO 21427-2:2006(E)
Withdraw the flasks (25 cm ) from the incubator and place them on a clean bench. Open the flasks singly and
remove the medium by suction. Wash the cells once with 5 ml Hanks Balanced Salt Solution (HBBS, without
2+ 2+
Ca and Mg ) for about 5 min. Thereafter, remove the medium again.
Trypsinize the cells for about 5 min using about 1,0 ml of trypsine (0,25 %) and approximately 1,0 ml HBBS
2+ 2+
(without Ca and Mg ) to separate the cells from the bottom of the culture flask.
Stop this reaction by adding approximately 3 ml of MEM including 10 % FCS.
Pipette this mixture several times to separate the cells from the flask and to obtain homogenous single cell
suspensions.
2)
Count the number of cells in a 10 µl sample in a hemocytometer .
Dilute the suspension to the required cell density (30 000 to 80 000 per culture) using MEM including 10 % of
FCS.
6.4.3 Duration of cell cycle
The cell cycle length of the V79 cells is normally about 12 h to 16 h. Determine its laboratory specific length
3)
using the BrdU method (see Annex A).
6.5 Metabolic activation
6.5.1 S9 fraction
For the treatment of enzyme induction and preparation of the S9 fraction, see Annex C. If the S9 fraction is
commercially purchased, it shall have been prepared (including enzyme induction) according to Annex C.
6.5.2 S9 mix
Prepare the needed amount of the S9 fraction freshly on the day of test or, if stored frozen, thaw at room
temperature. Immediately thereafter, prepare the S9 mix by mixing the following compounds under sterile
conditions:
a) 1 aliquot of S9 fraction;
b) 9 aliquots of S9 supplement (cofactor solution).
Keep the S9 mix permanently on ice (e.g. in a double-walled separator funnel containing iced water in
between these walls) and use it only on the same day. At the end of this day, discard the remaining S9 mix.
The concentrations of cofactors in the S9 mix are as follows:
MgCl 8 mmol/l
KCl 33 mmol/l
Glucose-6-phosphate 5 mmol/l
NADP 4 mmol/l
Phosphate buffer (pH 7,4) 15 mmol/l

2) Alternatively, an automatic cell or particle counter device may be used.
3) BrdU stands for bromodeoxyuridine.
6 © ISO 2006 – All rights reserved

ISO 21427-2:2006(E)
7 Apparatus
7.1 Cryo-vials, 1 ml, 2 ml, 5 ml.
2 2
7.2 Cell culture flasks, 25 cm , 75 cm .
7.3 Culture chambers, appropriate for 4 microscope slides, approx. 9 cm × 13 cm.
7.4 Microscope slides with frosted part.
7.5 CO incubator.
7.6 Laminar-airflow work bench.
7.7 Water bath.
7.8 Vacuum pump.
7.9 Inverse microscope.
7.10 Light microscope.
7.11 Bunsen burner.
7.12 Centrifuge.
7.13 Freezer (−80 °C).
7.14 Analytical balance.
7.15 Glass pipettes, 1 ml, 5 ml, 10 ml.
7.16 Adjustable volume pipettes.
7.17 Neubauer counting chamber.
7.18 Photographic clips.
7.19 Dewar tank for storage of the cells in liquid nitrogen.
7.20 Tweezers.
7.21 Cuvettes including holders for staining.
7.22 Sterile filters, 0,22 µm.
8 Test facility criteria
The test facility is qualified for the execution of this part of ISO 21427 if the in vitro micronucleus test is
established in this facility according to the following criteria:
⎯ a couple of independent experiments shall already have been performed;
⎯ a couple of known mutagenic and non-mutagenic chemicals shall already have been tested.
It should be decided case by case whether and to what extent additional instructions may be necessary for the
application of this part of ISO 21427.
ISO 21427-2:2006(E)
...