SIST EN ISO 16140-4:2020/A1:2024

SLOVENSKI STANDARD
01-november-2024
Mikrobiologija v prehranski verigi - Validacija metode - 4. del: Protokol za
validacijo metode v posameznem laboratoriju (hišne metode)- Dopolnilo A1:
Validacija testnega vzorca večje velikosti za kvalitativne metode (ISO 16140-
4:2020/Amd 1:2024)
Microbiology of the food chain - Method validation - Part 4: Protocol for method validation
in a single laboratory - Amendment 1: Validation of a larger test portion size for
qualitative methods (ISO 16140-4:2020/Amd 1:2024)
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 4: Arbeitsvorschrift für
Einzel-Labor-Verfahrensvalidierung - ÄNDERUNG 1: Validierung größerer Prüfmengen
für qualitative Verfahren (ISO 16140-4:2020/Amd 1:2024)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 4: Protocole
pour la validation de méthodes dans un seul laboratoire - Amendement 1: Validation
d’une taille de prise d’essai plus grande pour des méthodes qualitatives (ISO 16140-
4:2020/Amd 1:2024)
Ta slovenski standard je istoveten z: EN ISO 16140-4:2020/A1:2024
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 16140-4:2020/A1
EUROPEAN STANDARD
NORME EUROPÉENNE
September 2024
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Method validation - Part 4:
Protocol for method validation in a single laboratory -
Amendment 1: Validation of a larger test portion size for
qualitative methods (ISO 16140-4:2020/Amd 1:2024)
Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -
méthodes - Partie 4: Protocole pour la validation de Verfahrensvalidierung - Teil 4: Arbeitsvorschrift für
méthodes dans un seul laboratoire - Amendement 1: Einzel-Labor-Verfahrensvalidierung - Änderung 1:
Validation d'une taille de prise d'essai plus grande Validierung größerer Prüfmengen für qualitative
pour des méthodes qualitatives (ISO 16140- Verfahren (ISO 16140-4:2020/Amd 1:2024)
4:2020/Amd 1:2024)
This amendment A1 modifies the European Standard EN ISO 16140-4:2020; it was approved by CEN on 25 July 2024.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for inclusion of
this amendment into the relevant national standard without any alteration. Up-to-date lists and bibliographical references
concerning such national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN
member.
This amendment 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-CENELEC Management Centre has the
same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway,
Poland, Portugal, Republic of North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and
United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Rue de la Science 23, B-1040 Brussels
© 2024 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-4:2020/A1:2024 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 16140-4:2020/A1:2024) has been prepared by Technical Committee ISO/TC 34
"Food products" in collaboration with Technical Committee CEN/TC 463 “Microbiology of the food
chain” the secretariat of which is held by AFNOR.
This Amendment to the European Standard EN ISO 16140-4:2020 shall be given the status of a national
standard, either by publication of an identical text or by endorsement, at the latest by March 2025, and
conflicting national standards shall be withdrawn at the latest by March 2025.
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. CEN shall not be held responsible for identifying any or all such patent rights.
Any feedback and questions on this document should be directed to the users’ national standards
body/national committee. A complete listing of these bodies can be found on the CEN website.
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,
Croatia, Cyprus, Czech Republic, Denmark, Estonia, Finland, France, Germany, Greece, Hungary, Iceland,
Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Republic of
North Macedonia, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland, Türkiye and the
United Kingdom.
Endorsement notice
The text of ISO 16140-4:2020/Amd 1:2024 has been approved by CEN as EN ISO 16140-
4:2020/A1:2024 without any modification.

International
Standard
ISO 16140-4
First edition
Microbiology of the food chain —
2020-07
Method validation —
AMENDMENT 1
Part 4:
2024-09
Protocol for method validation in a
single laboratory
AMENDMENT 1: Validation of a larger
test portion size for qualitative methods
Microbiologie de la chaîne alimentaire — Validation des
méthodes —
Partie 4: Protocole pour la validation de méthodes dans un seul
laboratoire
AMENDEMENT 1: Validation d’une taille de prise d’essai plus
grande pour des méthodes qualitatives
Reference number
ISO 16140-4:2020/Amd.1:2024(en) © ISO 2024

ISO 16140-4:2020/Amd.1:2024(en)
© ISO 2024
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
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ISO copyright office
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Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
ISO 16140-4:2020/Amd.1:2024(en)
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.
The procedures used to develop this document and those intended for its further maintenance are described
in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the different types
of ISO document should be noted. This document was drafted in accordance with the editorial rules of the
ISO/IEC Directives, Part 2 (see www.iso.org/directives).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and expressions
related to conformity assessment, as well as information about ISO’s adherence to the World Trade
Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical Committee
CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical cooperation
between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 16140 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.

iii
ISO 16140-4:2020/Amd.1:2024(en)
Microbiology of the food chain — Method validation —
Part 4:
Protocol for method validation in a single laboratory
AMENDMENT 1: Validation of a larger test portion size for
qualitative methods
Clause 3
Add the following terminological entry:
3.13
larger test portion size
measured (volume or mass) representative sample taken from the laboratory sample or test sample for use
in the preparation of the initial suspension that is larger than the test portion that has been described in the
original method and/or validation document

4.1
Add the following text after the second paragraph:
The protocol used to validate a larger test portion size for qualitative methods in a single laboratory shall be
as given in Annex H.
5.1.1.4, note
Replace the text with the following:
NOTE An Excel®-based program is available for performing the RLOD calculations from https:// standards .iso
.org/ iso/ 16140/ -4/ ed -1/ en/ amd/ 1/ .

5.1.2.4, Note 2
Replace the text with the following:
NOTE 2 An Excel®-based program is available for performing the LOD calculations from https:// standards .iso
.org/ iso/ 16140/ -4/ ed -1/ en/ amd/ 1/ .

6.1.2.3, Note 2
Replace the text with the following:
NOTE 2 An Excel®-based program is available for performing the LOD calculations from https:// standards .iso
.org/ iso/ 16140/ -4/ ed -1/ en/ amd/ 1/ .

ISO 16140-4:2020/Amd.1:2024(en)

Annex H
Add the following annex after Annex G, before the Bibliography.

ISO 16140-4:2020/Amd.1:2024(en)
Annex H
(normative)
Validation of a larger test portion size for qualitative methods
H.1 General
This annex specifies a protocol for the validation of qualitative methods when using a larger test portion
size. This protocol is intended to demonstrate the effect of analysing a test portion larger than the test
portion initially used to validate the method. Validation of the larger test portion size applies only to the
laboratory conducting the study and only to the specific (food) category used.
Qualitative reference methods which were validated using a larger test portion size in accordance with
ISO 17468 and qualitative alternative (proprietary) methods which were validated using a larger test
portion size in accordance with ISO 16140-2 only need to be verified by a laboratory following ISO 16140-3.
The larger test portion size can be used in other laboratories once this has been validated in a study in
accordance with ISO 16140-2 or ISO 16140-5. See the flow diagram in Figure 1. Once validated in such a
study, any laboratory can implement the larger test portion size after verification following ISO 16140-3.
For the verification of a larger test portion size, the same (food) category shall be used. If not validated in
accordance with ISO 16140-2 or ISO 16140-5, validation in accordance with the protocol in this annex is
necessary for each laboratory wishing to use a larger test portion size.
Requirements with regards to pooling are specified in ISO 6887-1.
When testing a larger single test portion, pooled test portion or pooled (pre-)enrichment test portion, the
dilution ratio (sample/diluent) used in the validated method shall remain the same as well as the other
incubation conditions (e.g. time and temperature). This ratio may be increased to overcome the inhibitory
effects coming from certain food materials as those mentioned in ISO 6887-4:2017, 9.1.4.4 (e.g. onion powder,
garlic, oregano, peppers, certain teas and coffees, vitamin premixes, highly salted products).
A reduction in, for example, the dilution ratio (sample/diluent) requires a validation study (details are given
in Figure H.1).
Items can either be composited, pooled out of the laboratory or in the laboratory as a test portion or as a
(pre-)enriched test portion but not as two or more combinations [i.e. pooling of (pre-)enriched as well as
test portion is not allowed].
When a laboratory, for example, wants to pool test portions as well as (pre-)enriched test portions, two
separate validation studies shall be conducted in accordance with this protocol.
Once the larger test p
...