Microbiology of the food chain - Method validation - Part 3: Protocol for the verification of reference methods and validated alternative methods in a single laboratory (ISO 16140-3:2021)

This document deals with verification of methods for the detection and/or the enumeration of microorganisms, with particular emphasis on the implementation of a reference/alternative method in the user laboratory and verification of a reference/alternative method using items included in the scope of the method and tested routinely but not tested in the original validation study

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 3: Arbeitsvorschrift für die Verifizierung von Referenz- und alternativen Verfahren in einem einzelnen Labor (ISO 16140-3:2021)

Dieses Dokument legt die Arbeitsvorschrift fest, die bei der Verifizierung von Referenzverfahren und von validierten alternativen Verfahren zur Umsetzung im Anwenderlabor eingesetzt werden.
Dieses Dokument ist anwendbar auf die Verifizierung von Verfahren zur Untersuchung (Nachweis bzw. quantitative Bestimmung), Bestätigung und Typisierung von Mikroorganismen in:
- Erzeugnissen, die für den menschlichen Verzehr vorgesehen sind;
- Erzeugnissen, die als Futtermittel vorgesehen sind;
- Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln und Futtermitteln;
- Proben aus dem Bereich der Primärproduktion.
Dieses Dokument gilt insbesondere für Bakterien und Pilze. Einige Abschnitte können auf andere (Mikro )Organismen oder deren Metaboliten angewendet werden. Die Bestimmung muss im Einzelfall erfolgen.
Die technischen Arbeitsvorschriften für die Verifizierung validierter qualitativer Verfahren und validierter quantitativer Verfahren sind in Abschnitt 5 und Abschnitt 6 beschrieben. Die technische Arbeitsvorschrift für die Verifizierung von validierten alternativen Bestätigungs- und Typisierungsverfahren ist in Abschnitt 7 beschrieben. Die Arbeitsvorschriften für die Verifizierung von nicht validierten Referenzverfahren werden in Anhang F beschrieben.

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 3: Protocole pour la vérification dans un seul laboratoire de méthodes de référence et de méthodes alternatives validées (ISO 16140-3:2021)

Le présent document spécifie le protocole pour la vérification de méthodes de référence et de méthodes alternatives validées destinées à être mises en œuvre dans le laboratoire utilisateur.
Le présent document est applicable à la vérification de méthodes utilisées pour l'analyse (recherche et/ou quantification), la confirmation et le typage de micro-organismes présents dans:
—          les produits destinés à la consommation humaine;
—          les produits destinés à l'alimentation animale;
—          les échantillons environnementaux dans le domaine de la production et de la manutention de produits alimentaires;
—          les échantillons au stade de la production primaire.
Le présent document est notamment applicable aux bactéries et aux moisissures. Certains articles peuvent être applicables à d'autres (micro-)organismes ou à leurs métabolites, qui doivent être déterminés au cas par cas.
Les protocoles techniques pour la vérification de méthodes qualitatives validées et de méthodes quantitatives validées sont décrits aux Articles 5 et 6. Le protocole technique pour la vérification de méthodes alternatives validées de confirmation et de typage est décrit à l'Article 7. Les protocoles pour la vérification de méthodes de référence non validées sont décrits à l'Annexe F.

Mikrobiologija v prehranski verigi - Validacija metode - 3. del: Protokol za preverjanje referenčnih in validiranih alternativnih metod, izvedenih v posameznem laboratoriju (ISO 16140-3:2021)

General Information

Status
Published
Public Enquiry End Date
04-Mar-2018
Publication Date
02-Mar-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
11-Feb-2021
Due Date
18-Apr-2021
Completion Date
03-Mar-2021

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SLOVENSKI STANDARD
SIST EN ISO 16140-3:2021
01-april-2021
Mikrobiologija v prehranski verigi - Validacija metode - 3. del: Protokol za
preverjanje referenčnih in validiranih alternativnih metod, izvedenih v
posameznem laboratoriju (ISO 16140-3:2021)
Microbiology of the food chain - Method validation - Part 3: Protocol for the verification of
reference methods and validated alternative methods in a single laboratory (ISO 16140-
3:2021)
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 3: Arbeitsvorschrift für
die Verifizierung von Referenz- und alternativen Verfahren in einem einzelnen Labor
(ISO 16140-3:2021)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 3: Protocole
pour la vérification dans un seul laboratoire de méthodes de référence et de méthodes
alternatives validées (ISO 16140-3:2021)
Ta slovenski standard je istoveten z: EN ISO 16140-3:2021
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16140-3:2021 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16140-3:2021

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SIST EN ISO 16140-3:2021


EN ISO 16140-3
EUROPEAN STANDARD

NORME EUROPÉENNE

February 2021
EUROPÄISCHE NORM
ICS 07.100.30
English Version

Microbiology of the food chain - Method validation - Part 3:
Protocol for the verification of reference methods and
validated alternative methods in a single laboratory (ISO
16140-3:2021)
Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -
méthodes - Partie 3: Protocole pour la vérification dans Verfahrensvalidierung - Teil 3: Arbeitsvorschrift für die
un seul laboratoire de méthodes de référence et de Verifizierung von Referenz- und alternativen Verfahren
méthodes alternatives validées (ISO 16140-3:2021) in einem einzelnen Labor (ISO 16140-3:2021)
This European Standard was approved by CEN on 28 December 2020.

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-CENELEC 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-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, Turkey 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
© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-3:2021 E
worldwide for CEN national Members.

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SIST EN ISO 16140-3:2021
EN ISO 16140-3:2021 (E)
Contents Page
European foreword . 3

2

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SIST EN ISO 16140-3:2021
EN ISO 16140-3:2021 (E)
European foreword
This document (EN ISO 16140-3:2021) 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 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 August 2021, and conflicting national standards shall
be withdrawn at the latest by August 2021.
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.
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, Turkey and the
United Kingdom.
Endorsement notice
The text of ISO 16140-3:2021 has been approved by CEN as EN ISO 16140-3:2021 without any
modification.

3

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SIST EN ISO 16140-3:2021

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SIST EN ISO 16140-3:2021
INTERNATIONAL ISO
STANDARD 16140-3
First edition
2021-01
Microbiology of the food chain —
Method validation —
Part 3:
Protocol for the verification of
reference methods and validated
alternative methods in a single
laboratory
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 3: Protocole pour la vérification dans un seul laboratoire de
méthodes de référence et de méthodes alternatives validées
Reference number
ISO 16140-3:2021(E)
©
ISO 2021

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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2021
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 2021 – All rights reserved

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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)

Contents Page
Foreword .v
Introduction .vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General principles of verification of qualitative (detection) methods and
quantification methods . 5
4.1 General . 5
4.2 Implementation verification . 5
4.3 (Food) item verification . 6
4.4 Requirements for implementation verification and (food) item verification . . 6
4.5 Performance characteristics. 9
5 Qualitative methods — Technical protocol for verification . 9
5.1 Estimated LOD (eLOD ) determination . 9
50 50
5.2 Experimental design . 9
5.3 Selection of (food) items .10
5.4 Artificial contamination .10
5.4.1 Selection of strains .10
5.4.2 Inoculation of the test portions .11
5.5 Evaluation of results .13
5.5.1 Determination of eLOD using protocol 1 .13
50
5.5.2 Determination of eLOD using protocol 2 .16
50
5.5.3 Use of protocol 3 .17
5.6 Acceptability limits .18
5.7 Root cause analysis .18
6 Quantitative methods — Technical protocol for verification .19
6.1 Intralaboratory reproducibility standard deviation determination .19
6.1.1 General.19
6.1.2 Experimental design .19
6.1.3 Selection of the (food) item .21
6.1.4 Natural contamination .21
6.1.5 Artificial contamination .21
6.1.6 Evaluation of results .22
6.1.7 Acceptability limit .23
6.1.8 Root cause analysis .24
6.2 Estimated bias (eBias) determination .25
6.2.1 General.25
6.2.2 Experimental design .25
6.2.3 Selection of (food) items .25
6.2.4 Artificial contamination .25
6.2.5 Evaluation of results .27
6.2.6 Acceptability limit .27
6.2.7 Root cause analysis .28
7 Validated alternative confirmation and typing methods — Technical protocol for
verification.28
7.1 General .28
7.2 Implementation verification .28
7.3 Experimental design .28
7.3.1 General.28
7.3.2 Strain selection .29
7.4 Evaluation of results .29
7.5 Acceptability limit .30
© ISO 2021 – All rights reserved iii

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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)

7.6 Root cause analysis .30
8 Summary of acceptability limits for the verification of validated methods .30
Annex A (informative) Classification of (food) categories and suggested target
combinations for verification studies .31
Annex B (informative) Guidance on how to choose challenging (food) item(s) for (food)
item verification .45
Annex C (informative) Qualitative method verification — Example .47
Annex D (informative) Quantitative method verification — Example .55
Annex E (informative) Validated alternative confirmation or typing method verification —
Examples .60
Annex F (normative) Protocol for the verification of non-validated reference methods
in a single laboratory .63
Bibliography .70
iv © ISO 2021 – All rights reserved

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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(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.
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 documents 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).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
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.
© ISO 2021 – All rights reserved v

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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)

Introduction
0.1  The ISO 16140 series
The ISO 16140 series has been expanded in response to the need for various ways to validate or verify
test methods. It is the successor to ISO 16140:2003. The ISO 16140 series consists of six parts with the
general title, Microbiology of the food chain — Method validation:
— Part 1: Vocabulary;
— Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method;
— Part 3: Protocol for the verification of reference methods and validated alternative methods in a single
laboratory;
— Part 4: Protocol for method validation in a single laboratory;
— Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods;
— Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation
and typing procedures.
ISO 17468 is a closely linked International Standard, which establishes technical rules for the
development and validation of standardized methods.
In general, two stages are needed before a method can be used in a laboratory.
— The first stage is the validation of the method. Validation is conducted using a study in a single
laboratory followed by an interlaboratory study (see ISO 16140-2, ISO 16140-5 and ISO 16140-6).
In the case when a method is validated within one laboratory (see ISO 16140-4), no interlaboratory
study is conducted.
— The second stage is method verification, where a laboratory demonstrates that it can satisfactorily
perform a validated method. This is described in this document (i.e. ISO 16140-3). Verification is
only applicable to methods that have been validated using an interlaboratory study.
In general, two types of methods are distinguished: reference methods and alternative methods.
A reference method is defined in ISO 16140-1:2016, 2.59, as an “internationally recognized and widely
accepted method”. The note to entry clarifies that “these are ISO standards and standards jointly
published by ISO and CEN or other regional/national standards of equivalent standing”.
In the ISO 16140 series, reference methods include standardized reference (ISO and CEN) methods as
defined in ISO 17468:2016, 3.5, as a “reference method described in a standard”.
An alternative method (method submitted for validation) is defined in ISO 16140-1:2016, 2.4, as a
“method of analysis that detects or quantifies, for a given category of products, the same analyte as
is detected or quantified using the corresponding reference method”. The note to entry clarifies that:
“The method can be proprietary. The term ‘alternative’ is used to refer to the entire ‘test procedure
and reaction system’. This term includes all ingredients, whether material or otherwise, required for
implementing the method”.
ISO 16140-4 addresses validation within a single laboratory. The results are therefore only valid for
the laboratory that conducted the study. In this case, verification (as described in this document) is not
applicable. ISO 16140-5 describes protocols for non-proprietary methods where a more rapid validation
is required or when the method to be validated is highly specialized and the number of participating
laboratories required by ISO 16140-2 cannot be reached. ISO 16140-4 and ISO 16140-5 can be used
for validation against a reference method. ISO 16140-4 (qualitative and quantitative) and ISO 16140-5
(quantitative only) can also be used for validation without a reference method.
vi © ISO 2021 – All rights reserved

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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)

The flow chart in Figure 1 gives an overview of the links between the different parts mentioned above.
It also guides the user in selecting the right part of the ISO 16140 series, taking into account the purpose
of the study and the remarks given above.
Figure 1 — Flow chart for application of the ISO 16140 series
NOTE 1 In this document, the words “category”, “type” and/or “item” are sometimes combined with “(food)”
to improve readability. However, the word “(food)” is interchangeable with “(feed)” and other areas of the food
chain as mentioned in Clause 1.
NOTE 2 The general principle for method verification is that the method to be verified (either alternative or
reference) has been validated. However, some reference methods (including ISO or CEN standards) are not yet
(fully) validated. For verification of these methods, the protocols are described in Annex F.
ISO 16140-6 is somewhat different from the other parts in the ISO 16140 series in that it relates to
a very specific situation where only the confirmation procedure of a method is to be validated [e.g.
the biochemical confirmation of Enterobacteriaceae (see ISO 21528-2)]. The confirmation procedure
advances a suspected (presumptive) result to a confirmed positive result. The validation of alternative
typing techniques (e.g. serotyping of Salmonella) is also covered by ISO 16140-6. The validation study
in ISO 16140-6 clearly defines the selective agar(s) from which strains can be confirmed using the
alternative confirmation method. If successfully validated, the alternative confirmation method can
only be used if strains are recovered on an agar that was used and shown to be acceptable within the
validation study. Figure 2 shows the possibilities where an alternative confirmation method validated
in accordance with ISO 16140-6 can be applied (see text in the boxes).
© ISO 2021 – All rights reserved vii

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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)

Figure 2 — Use of validated alternative confirmation methods (see ISO 16140-6)
EXAMPLE An example application of a validated alternative confirmation method is as follows.
An alternative confirmation method based on ELISA has been validated to replace the biochemical confirmation
for Salmonella as described in ISO 6579-1. In the validation study, XLD (mandatory agar in accordance with
ISO 6579-1) plus BGA and a specified chromogenic agar (two optional agars for second plating in accordance with
ISO 6579-1) were used as the agars to start the confirmation. The validated confirmation method can be used to
replace the biochemical confirmation under the following conditions:
— by laboratories using the ISO 6579-1; or
— by laboratories using an ISO 16140-2 validated alternative method that refers to ISO 6579-1 for confirmation; or
— by laboratories using an ISO 16140-2 validated alternative method that starts the confirmation from XLD
and/or BGA agar and/or the specified chromogenic agar.
The validated confirmation method cannot be used under the following conditions:
— by laboratories using an ISO 16140-2 validated alternative method that refers only to agars other than those
included in the validation to start the confirmation (e.g. Hektoen agar and SS agar only); or
— by laboratories using an ISO 16140-2 validated alternative method that refers only to a confirmation
procedure that does not require isolation on agar.
0.2  Verification versus validation
ISO 16140-1:2016 defines the terms for validation and verification, as follows:
— validation: establishment of the performance characteristics of a method and provision of objective
evidence that the performance requirements for a specified intended use are fulfilled;
— verification: demonstration that a validated method performs, in the user’s hands, according to the
method’s specifications determined in the validation study and is fit for its intended purpose.
NOTE 1 The user’s hand means the user laboratory.
Method verification applies to methods that are:
— reference methods, including ISO or CEN standards, that are validated using at least an
interlaboratory study;
viii © ISO 2021 – All rights reserved

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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)

NOTE 2 However, some reference methods (including ISO or CEN standards) are not yet (fully) validated.
For verification of these methods, the protocols are described in Annex F.
— alternative methods, proprietary or otherwise, when the validation included an interlaboratory
study. The method has been validated in accordance with
— ISO 16140-2 for alternative (proprietary) methods,
— ISO 16140-5 for non-proprietary methods, or
— ISO 16140-6 for alternative (proprietary) confirmation and typing methods.
In a validation study, it is not possible to test all existing foods; the diversity and number of samples
used in any validation study is limited. In most cases, the validation is based on five different food
categories (categories as defined in ISO 16140-1:2016, 2.11, and specified in ISO 16140-2:2016, Annex A).
Sometimes the validation is supplemented with additional (other) categories such as pet food and
animal fee
...

SLOVENSKI STANDARD
oSIST prEN ISO 16140-3:2018
01-marec-2018
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Microbiology of the food chain - Method validation - Part 3: Protocol for the verification of
reference and validated alternative methods implemented in a single laboratory (ISO/DIS
16140-3:2017)
Mikrobiologie von Lebensmitteln und Futtermitteln - Verfahrensvalidierung - Teil 3:
Arbeitsvorschrift für die Prüfung von Referenz- und alternativer Verfahren in einem
einzelnen Labor
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 3: Protocole
pour la vérification de méthodes de référence et alternatives validées appliquées dans
un laboratoire (ISO/DIS 16140-3:2017)
Ta slovenski standard je istoveten z: prEN ISO 16140-3
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 16140-3:2018 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN ISO 16140-3:2018

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oSIST prEN ISO 16140-3:2018
DRAFT INTERNATIONAL STANDARD
ISO/DIS 16140-3
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2017-12-15 2018-03-09
Microbiology of the food chain — Method validation —
Part 3:
Protocol for the verification of reference and validated
alternative methods implemented in a single laboratory
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 3: Protocole pour la vérification de méthodes de référence et alternatives validées appliquées dans
un laboratoire
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
ISO/CEN PARALLEL PROCESSING
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO/DIS 16140-3:2017(E)
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 SUPPORTING DOCUMENTATION. ISO 2017

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oSIST prEN ISO 16140-3:2018
ISO/DIS 16140-3:2017(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland
All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved

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oSIST prEN ISO 16140-3:2018
ISO/DIS 16140-3:2017(E)
Contents
Foreword . 5
Introduction. 6
1 Scope . 8
2 Normative references . 8
3 Terms and definitions . 8
4 Principle . 11
4.1 General . 11
4.2 Implementation verification . 11
4.3 (Food) item verification . 12
4.4 Implementation verification and (food) item verification . 12
4.5 Performance characteristics . 15
5 Qualitative methods — Technical protocol for verification . 15
5.1 Estimated LOD (eLOD ) determination . 15
50 50
5.2 Experimental design . 15
5.3 Selection of (food) items . 16
5.4 Artificial contamination . 16
5.4.1 Selection of strains . 16
5.4.2 Inoculation of the test portions . 16
5.5 Results . 17
5.6 Acceptance criteria . 19
5.7 Root cause analysis . 19
6 Quantitative methods — Technical protocol for verification . 20
6.1 Precision (intralaboratory reproducibility) determination . 20
6.1.1 General . 20
6.1.2 Experimental design . 20
6.1.3 Selection of (food) item . 22
6.1.4 Natural contamination . 22
6.1.5 Artificial contamination . 22
6.1.6 Results . 23
6.1.7 Acceptance criteria . 24
6.1.8 Root cause analysis . 25
6.2 Estimated bias determination . 26
6.2.1 General . 26
6.2.2 Experimental design . 26
6.2.3 Selection of (food) items . 26
6.2.4 Artificial contamination . 26
6.2.5 Results . 28
6.2.6 Acceptance criteria . 28
6.2.7 Root cause analysis . 28
7 Summary of acceptance criteria . 29
Annex A (informative) — Classification of (food) categories and suggested target combinations
for verification studies. 30
Annex B (normative) —Requirements and guidance on how to choose challenging (food) item(s)
for (food) item verification . 47
B.1 General . 47
B.2 Matrix effects to consider . 47
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B.2.1 Microbial characteristics . 47
B.2.2 Physical and chemical characteristics . 47
B.2.3 Food process characteristics . 48
B.3 Selection of (food) items for verification . 48
Annex C (informative) — Protocol for QUALITATIVE method verification . 49
Annex D (informative) — Protocol for QUANTITATIVE method verification . 52
D.1 Protocol for precision (intralaboratory reproducibility) determination . 52
D.2 Protocol for estimated bias (eBias) determination . 53
Bibliography . 56


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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 documents 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).
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. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation on 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 the following
URL: www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology (Working Group WG 3, Method validation).
A list of all parts of the ISO 16140 series can be found on the ISO website.


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Introduction
The ISO 16140 series has been elaborated in response to the need for various ways to validate or verify
test methods. It is the successor of ISO 16140:2003, Microbiology of food and animal feeding stuffs —
Protocol for the validation of alternative methods. ISO 16140 series consists of several parts with the
general title, Microbiology of the food chain — Method validation:
 Part 1: Vocabulary
 Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method
 Part 3: Protocol for the verification of reference and validated alternative methods implemented in a
single laboratory
 Part 4: Protocol for single-laboratory (in-house) method validation
 Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods
 Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological
confirmation and typing procedures
ISO 17468, Microbiology of the food chain — Technical requirements and guidance on establishment or
[2]
revision of a standardized reference method , is a closely linked International Standard. This
International Standard, which establishes technical rules for the development and validation of
standardized methods, is intended for the development of standardized methods by ISO/TC 34, Food
products, Subcommittee SC 9, Microbiology and CEN/TC 275/WG 6, Microbiology of the food chain.
In general, two stages are needed before a method can be used in a laboratory:
 The first stage is the validation of the method. This is either conducted in several laboratories
(parts 2 and 5 of ISO 16140) or in one laboratory (part 4 of ISO 16140).
 The second stage is method verification, where a laboratory demonstrates that it can satisfactorily
perform a validated method. This is described in part 3 of ISO 16140 (method verification). In
part 3, a separation is made between verification of (food) items that are included in the validation
study and (food) items that are not tested in the validation study but belong within the scope of
validation.
NOTE 1 Standardized reference methods (with and without published validation data) only require verification
before implementation in the laboratory.
NOTE 2 In this part of ISO 16140, the words ‘category’, ‘type’ and ‘item’ are sometimes combined with ‘food’ to
improve the readability of this document. However, the word ‘food’ is interchangeable with ‘feed’ and the other
areas of the food chain as mentioned in the Scope of ISO 16140-3.
Part 4 of ISO 16140 addresses validation within a single laboratory. The results are therefore only valid
in the laboratory which conducted the study. In this case, verification (part 3 of ISO 16140) is not
required.
Part 5 of ISO 16140 describes protocols for situations where a more rapid validation is required or
when the method to be validated is highly specialised, and, the number of participating laboratories
required by ISO 16140-2 cannot be reached.

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The flow chart in Figure 1 gives an overview of the links between the different parts mentioned above.
It also guides the users in selecting the right part of the ISO 16140 series, taking into account the
purpose of the study and the remarks given above. For this, it is important to distinguish between
'reference method' and 'standardized reference method'. A reference method is an internationally
recognized and widely accepted method (term 2.59 of ISO 16140-1:2016) and a standardized reference
method is a reference method described in a standard (term 3.5 of ISO 17468:2016). In the ISO 16140
series, reference method includes standardized reference method. The flow diagram acknowledges that
published validation data may not be available for some standardized reference methods.

Figure 1 — Flow chart for application of the different parts of the ISO 16140 series
Part 6 of ISO 16140, is somewhat different from the other parts in the ISO 16140 series in that it relates
to a very specific situation where only the confirmation procedure of a method is validated. The
confirmation procedure advances a suspected (presumptive) result to a confirmed positive result. The
typing of pure strains (e.g. serotyping of Salmonella) is included in part 6 of ISO 16140.


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Microbiology of the food chain — Method validation — Part 3:
Protocol for the verification of reference and validated alternative
methods implemented in a single laboratory
1 Scope
This part of ISO 16140 describes the protocol for the verification of reference methods, standardized
reference methods and validated alternative methods for implementation in the user laboratory.
Method verification does not apply to non-validated alternative methods.
This part of ISO 16140 is applicable to the verification of methods used for the analysis (detection
and/or quantification) of microorganisms in
 products intended for human consumption,
 products intended for animal feeding,
 environmental samples in the area of food and feed production, handling, and
 samples from the primary production stage.
This part of ISO 16140 is, in particular, applicable to bacteria and fungi. Some clauses can be applicable
to other (micro)organisms or their metabolites, to be determined on a case-by-case basis.
The verification focuses on those (food) items that are within the scope of validation and are tested in
the user laboratory.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements 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 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and
decimal dilutions for microbiological examination
ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for
microbiological examinations
ISO 16140-1:2016, Microbiology of the food chain — Method validation — Part 1: Vocabulary
ISO 16140-2:2016, Microbiology of the food chain — Method validation — Part 2: Protocol for the
validation of alternative (proprietary) methods against a reference method
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16140-1 and the following
apply. ISO and IEC maintain terminological databases for use in standardization at the following
addresses:
— IEC Electropedia: available at http://www.electropedia.org/
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— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
bias
measurement bias
estimate of a systematic measurement error, or the systematic difference between the quantitative
assigned value and the average of measurement replicate results
[SOURCE: ISO 16140-1:2016, 2.9]
3.2
category
group of (food) types of the same origin
EXAMPLE Heat-processed milk and dairy products.
Note 1 to entry: The (food) categories are listed in Annex A of this document.
[SOURCE: ISO 16140-1:2016, 2.11, modified]
3.3
estimated bias (eBias)
determination of the bias based on the experimental design described in this document
Note 1 to entry: An accurate determination of the bias is not possible as the number of samples tested is small.
Therefore, the term estimated bias (eBias) is used in this document.
3.4
estimated LOD (eLOD )
50 50
determination of the LOD (level of detection at 50 % probability of detection) based on the
50
experimental design described in this document
Note 1 to entry: An accurate determination of the LOD is not possible as the number of samples tested is small.
50
Therefore, the term estimated LOD (eLOD ) is used.
50 50
Note 2 to entry: LOD50 is defined in ISO 16140-1:2016, 2.35.
3.5
item
single specified food, feed, environmental, or primary production matrix
EXAMPLE Food category: heat-processed milk and dairy products; food type: pasteurised dairy product;
food item: crème brûlée.
[SOURCE: ISO 16140-1:2016, 2.34]
3.6
laboratory sample
sample prepared for sending to the laboratory and intended for inspection or testing
[SOURCE: ISO 6887-1:2017, 3.1]
3.7
matrix (product)
all the components of the sample
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[SOURCE: ISO 16140-1:2016, 2.38]
3.8
reference material
material, sufficiently homogeneous and stable with respect to one or more specified properties, which
has been established to be fit for its intended use in a measurement process
Note 1 to entry: Properties can be quantitative or qualitative, e.g. identity of substances or species.
Note 2 to entry: Uses may include the calibration of a measurement system, assessment of a measurement
procedure, assigning values to other materials, and quality control.
[SOURCE: ISO Guide 30:2015, 2.1.1, modified]
3.9
scope of laboratory application
analytes, matrices, and concentrations for an analytical method that a user laboratory claims to be
capable of satisfactorily testing in its laboratory
Note 1 to entry: A method may have been validated to a broader range (scope) of analytes, matrices and
concentrations than the scope that will be claimed by a user laboratory. The scope of laboratory application
is ≤ the scope of validation.
3.10
scope of validation
analytes, matrices and concentrations for which a validated method of analysis can be used
satisfactorily
[SOURCE: ISO 16140-1:2016, 2.70]
3.11
test portion
measured (volume or mass) representative sample taken from the laboratory sample for use in the
preparation of the initial suspension
Note 1 to entry: Sometimes preparation of a test sample from the laboratory sample is required before the test
portion is taken, but this is infrequently used in microbiological examinations.
[SOURCE: ISO 6887-1:2017, 3.5, modified]
3.12
test sample
sample prepared from the laboratory sample according to the procedure specified in the test method
and from which test portions are taken
Note 1 to entry: Preparation of the laboratory sample before the test portion is taken is infrequently used in
microbiological examinations.
[SOURCE: ISO 6887-1:2017, 3.4, modified]
3.13
type
for a given category, a group of (food) items processed in a similar way, with similar intrinsic
characteristics and a similar microbial ecology
EXAMPLE Food category: heat-processed milk and dairy products; food type: pasteurised dairy product.
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[SOURCE: ISO 16140-1:2016, 2.78, modified]
3.14
user laboratory
laboratory which implements a validated alternative method and/or a reference method
3.15
verification
demonstration that a validated method performs in the user’s hands according to the method’s
specifications determined in the validation study and is fit for its intended purpose
4 Principle
4.1 General
Before performing method verification, it is crucial to refer to the published validation data for the
method in order to confirm the scope of validation and to select the appropriate (food) items.
The verification is undertaken in two parts:
 implementation verification;
 (food) item verification.
User laboratories intending to verify methods that have published validation data for comparison (this
includes all validated alternative methods) shall perform both the implementation and the (food) item
verification. Some reference methods may not have published validation data. For those methods, the
user laboratory will only perform (food) item verification.
Alternative methods that have not been validated shall first be validated, before a user laboratory can
verify its use in their laboratory (see Figure 1).
4.2 Implementation verification
Implementation verification aims to demonstrate the competence of the user laboratory to perform the
validated method. It compares user laboratory performances to those obtained during the validation.
Implementation verification applies only to the following methods with published validation data:
 reference methods; and
 validated alternative methods.
The user laboratory shall:
 review the published validation data for the method;
 select one (food) item tested during the validation study that belongs within the scope of laboratory
application of the user laboratory, if possible, and;
 use this (food) item and the sample size used in the validation study to perform implementation
verification.
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4.3 (Food) item verification
(Food) item verification applies to reference methods, with and without published validation data, and
validated alternative methods with validation data. The (food) item verification sets out to demonstrate
the competence of the user laboratory to perform the validated method with (food) items that are
tested in the user laboratory.
4.4 Implementation verification and (food) item verification
Table 1 provides guidance on when to use implementation verification and when to use (food) item
verification.
Table 1 — Implementation verification and (food) item verification
Method with published validation data Method without published validation data
Reference method Alternative method Reference method Alternative method
a
Implementation   Not applicable Not applicable
verification
a
(Food) item    Not applicable
verification
a
Not applicable: the method shall first be validated to ISO 16140-2 or ISO 16140-5 (see Figure 1) after which validation data
are available.

Figure 2a to Figure 2d show the number of (food) items required for implementation verification and
(food) item verification under different circumstances.

Figure 2a — (Food) items required when verifying a reference method with published validation
data or a validated alternative method for a “broad range of foods” scope
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Figure 2b — (Food) items required when verifying a reference method with published validation
data or a validated alternative method for a “limited range of foods” scope

Figure 2c — (
...

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