ISO 16140-4:2020/Amd 1
(Amendment)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
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
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
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FINAL DRAFT
Amendment
ISO 16140-4:2020/
FDAM 1
ISO/TC 34/SC 9
Microbiology of the food chain —
Secretariat: AFNOR
Method validation —
Voting begins on:
2024-04-29
Part 4:
Protocol for method validation in a
Voting terminates on:
2024-06-24
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
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/CEN PARALLEL PROCESSING LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
ISO 16140-4:2020/FDAM 1:2024(en) © ISO 2024
FINAL DRAFT
ISO 16140-4:2020/FDAM 1:2024(en)
Amendment
ISO 16140-4:2020/
FDAM 1
ISO/TC 34/SC 9
Microbiology of the food chain —
Secretariat: AFNOR
Method validation —
Voting begins on:
Part 4:
Protocol for method validation in a
Voting terminates on:
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
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2024
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/CEN PARALLEL PROCESSING
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
or ISO’s member body in the country of the requester.
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland Reference number
ISO 16140-4:2020/FDAM 1:2024(en) © ISO 2024
ii
ISO 16140-4:2020/FDAM 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/FDAM 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.
Annex H
Add the following annex after Annex G, before the Bibliography.
ISO 16140-4:2020/FDAM 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 portion size has been validated, all test portions smaller than the largest validated test
portion can be
...
rd
3 draft FDAmd.1 for ISO 16140-4:20202016/FDAM 1:2024(en)
Date: 2024-04-11
ISO/TC 34/SC 9/WG 3/N 912
Secretariat: NENAFNOR
Project leaders: Adrianne Klijn, Danièle Sohier, Paul in ‘t Veld
Date: 2024-02-16
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
rd
3 draft ISO 16140-4:2020/FDAmd.1
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 the internet or an intranet, without prior written
permission. Permission can be requested from either ISO at the address below or ISO’s member body
in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2024 – All rights reserved
ISO 16140-4:2020/FDAM 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/FDAM 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 definitionterminological 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.
Annex H
Add the following annex after Annex G, before the Bibliography.
rd
3 draft ISO 16140-4:2020/FDAmd.1
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 and, 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 portion size has been validated, all test portions smaller than the largest validated
test portion can be used for routine testing for this particular (food) category at the same sample/diluent
ratio. For example, a method that has been validated for 375 g test portions can be used for 25 g, 100 g,
etc., up to 375 g test portions.
The protocol is intended to demonstrate that a larger test portion size provides a similar or lower level
of detection (LOD ) compared to the LOD of the (validated) test portion size as specified in the method.
50 50
The relative level of detection (RLOD) approach specified in 6.1.1.3 shall be used. For calculation of the
data, the larger test portion size corresponds to the alternative method and the original test portion size
corresponds to the reference method.
2 © ISO 2024 – All rights reserved
ISO 16140-4:2020/FDAM 1:2024(en)
a
The “one laboratory” is the laboratory that performed the validation in accordance with this document.
Figure H.1 — Steps to validate a larger test portion size and the imp
...
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