EN ISO 11350:2025

SLOVENSKI STANDARD
01-april-2025
Kakovost vode - Določanje genotoksičnosti za vodo in odpadno vodo -
Salmonella/mikrosomski fluktuacijski preskus (Amesov fluktuacijski preskus)
Water quality - Determination of the genotoxicity of water and waste water -
Salmonella/microsome fluctuation test (Ames fluctuation test) (ISO 11350:2012)
Wasserbeschaffenheit - Bestimmung der Gentoxizität von Wasser und Abwasser -
Verfahren mittels Salmonella/Microsomen-Fluktuationstest (Ames-Fluktuationstest) (ISO
11350:2012)
Qualité de l'eau - Évaluation de la génotoxicité des eaux résiduaires - Essai de
Salmonella/microsome (essai d'Ames-fluctuation) (ISO 11350:2012)
Ta slovenski standard je istoveten z: prEN ISO 11350
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.

INTERNATIONAL ISO
STANDARD 11350
First edition
2012-05-15
Water quality — Determination of the
genotoxicity of water and waste water —
Salmonella/microsome fluctuation test
(Ames fluctuation test)
Qualité de l’eau — Évaluation de la génotoxicité des eaux résiduaires —
Essai de Salmonella/microsome (essai d’Ames-fluctuation)
Reference number
ISO 11350:2012(E)
©
ISO 2012
ISO 11350:2012(E)
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Published in Switzerland
ii © ISO 2012 – All rights reserved

ISO 11350:2012(E)
Contents Page
Foreword .iv
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 Interferences . 3
5 Principle . 4
6 Apparatus and materials . 4
7 Reagents, media and dilutions . 5
8 Sampling and samples . 9
9 Procedure . 9
9.1 Overnight culture . 9
9.2 Preparation of S9 mix .10
9.3 Testing of water samples .10
9.4 Measurement of revertant growth .13
9.5 Calculation of cytotoxicity .13
10 Validity criteria .14
11 Assessment criteria .14
12 Test report .14
Annex A (normative) Nutrient broth and agar .15
Annex B (normative) Preparation of ampicillin agar plates and stock cultures.16
Annex C (normative) Checking of genotype .17
Annex D (normative) S9 fraction .18
Annex E (informative) Example for application of samples on a 24 well plate .19
Annex F (informative) Example for reporting .21
Annex G (informative) Testing of chemicals .22
Annex H (informative) Precision data.25
Annex I (informative) Statistical assessment .27
Annex J (informative) Measurement of the lowest ineffective dilution (LID) of a waste water —
A simplified evaluation for testing of waste water .33
Annex K (informative) Use of additional tester strains .35
Bibliography .36
ISO 11350:2012(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 11350 was prepared by Technical Committee ISO/TC 147, Water quality, Subcommittee SC 5,
Biological methods.
iv © ISO 2012 – All rights reserved

INTERNATIONAL STANDARD ISO 11350:2012(E)
Water quality — Determination of the genotoxicity of water and
waste water — Salmonella/microsome fluctuation test (Ames
fluctuation test)
WARNING — Persons using this International Standard 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 International Standard
be carried out by suitably trained staff.
1 Scope
This International Standard specifies a method for the determination of the genotoxic potential of water and
waste water using the bacterial strains Salmonella enterica subsp. enterica serotype Typhimurium TA 98 and
TA 100 in a fluctuation assay. This combination of strains is able to measure the genotoxicity of chemicals that
induce point mutations (base pair substitutions and frameshift mutations) in genes coding for enzymes that are
involved in the biosynthesis of the amino acid, histidine.
[8]
NOTE 1 ISO 13829 applies for the measurement of genotoxicity of samples containing DNA-crosslinking agents.
This method is applicable to:
— fresh water;
— waste water;
— aqueous extracts and leachates;
— eluates of sediments (fresh water);
— pore water;
— aqueous solutions of single substances or of chemical mixtures;
— drinking water.
NOTE 2 When testing drinking water, extraction and pre-concentration of water samples can prove necessary.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are indispensable
for its application. For dated references, only the edition cited applies. For undated references, the latest edition
of the referenced document (including any amendments) applies.
ISO 3696, Water for analytical laboratory use — Specification and test methods
ISO 7027, Water quality — Determination of turbidity
ISO 11350:2012(E)
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
3.1
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
[10]
[Source: ISO 21427-2:2006, definition 3.2]
3.2
culture medium
nutrients presented in a form and phase (liquid or solidified) which support microbiological growth
[6]
[Source: ISO 6107-6:2004, definition 24]
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 1 to entry: For undiluted water or waste water, this coefficient per definition is 1→1. [In this International Standard,
the arrow indicates the transition from initial total volume to final total volume.] The corresponding and smallest possible
value of D is 1.
[6]
[Source: ISO 6107-6:2004, definition 28]
3.4
lowest ineffective dilution
LID
lowest dilution within a test batch which does not show any effect, i.e. no statistically significant increase in the
number of revertant wells compared with the negative control
NOTE 1 to entry: LID is determined for each incubation condition (strain, ±S9 mix). The highest LID value is decisive for
the overall assessment.
3.5
induction rate
difference between the mean value of wells with revertant growth counted on the plates treated with a dose
of the test sample or with a positive control, and the mean value of the corresponding wells treated with the
negative control using the same strain under identical conditions
[6]
[Source: ISO 6107-6:2004, definition 43, modified: “wells with revertant growth” replaces “mutant colonies”;
“corresponding wells” replaces “corresponding plates”]
3.6
inoculum
fraction of a culture of microorganisms used to start a new culture, or an exponentially growing preculture,
in fresh medium
[6]
[Source: ISO 6107-6:2004, definition 44]
3.7
negative control
dilution water without test sample
[6]
[Source: ISO 6107-6:2004, definition 51]
2 © ISO 2012 – All rights reserved

ISO 11350:2012(E)
3.8
revertant growth
visible mutant colonies on the microplate at the end of the respective test
3.9
overnight culture
culture started late in the afternoon and incubated overnight (usually about 16 h) to be ready during the following
morning for purposes such as the inoculation of a preculture
[6]
[Source: ISO 6107-6:2004, definition 54]
NOTE 1 to entry For specification, see 9.1.
3.10
positive control
any well characterized material and/or substance, which, when evaluated by a specific test method, demonstrates
the suitability of the test system to yield a reproducible, appropriate positive or negative response in the test system
[7]
[Source: ISO 10993-12:—, definition 3.12]
NOTE 1 to entry The positive controls mentioned in this International Standard are dissolved in dimethyl sulfoxide (DMSO)
prior to use. For the purposes of this International Standard, the positive controls are known mutagens which are suitable
for the verification of the sensitivity of the method and/or the activity of the S9 mix.
3.11
S9 fraction
supernatant at 9 000g of a tissue homogenate in 0,15 mol/l KCl, obtained from livers of male rats (200 g to
300 g) pretreated with a substance or substance combination appropriate for enzyme induction
[6]
[Source: ISO 6107-6:2004, definition 74]
3.12
S9 mix
mixture of S9 fraction and cofactor solution
[6]
[Source: ISO 6107-6:2004, definition 75]
3.13
stock culture
culture of a strain of organisms maintained under conditions to preserve original features such as
nucleotide sequences
[6]
[Source: ISO 6107-6:2004, definition 87]
3.14
test sample
undiluted, diluted or otherwise prepared portion of a sample to be tested, after completion of all pr
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