SIST EN 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 (ISO 16140-2:2016)

This part of ISO 16140 specifies the general principle and the technical protocol for the validation
of alternative, mostly proprietary, methods for microbiology in the food chain. Validation studies
according to this part of ISO 16140 are intended to be performed by organizations involved in method
validation.
This part of ISO 16140 is applicable to the validation of methods for the analysis (detection 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 of this part of
ISO 16140 could be applicable to other (micro) organisms or their metabolites on a case-by-case-basis.
In the future, guidance for other organisms (e.g. viruses and parasites) will be included in either this
part or a separate part of ISO 16140.

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 2: Arbeitsvorschrift für die Validierung von alternativen (urheberrechtlich geschützten) Verfahren anhand eines Referenzverfahrens (ISO 16140-2:2016)

Dieser Teil von ISO 16140 legt die allgemeinen Benennungen und Definitionen in Bezug auf die Verfahrensvalidierung auf dem Gebiet der Mikrobiologie der Lebensmittelkette fest.
Der vorliegende Teil von ISO 16140 ist anwendbar auf die Validierung von Verfahren zur Untersuchung (Nachweis oder quantitative Bestimmung) von Mikroorganismen in:
   Erzeugnissen, die für den menschlichen Verzehr und als Futtermittel vorgesehen sind;
   Umgebungsproben im Bereich der Herstellung und Handhabung von Lebensmitteln;
   Proben aus dem Bereich der Primärproduktion.

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 2: Protocole pour la validation de méthodes alternatives (commerciales) par rapport à une méthode de référence (ISO 16140-2:2016)

L'ISO 16140-2 :2016 établit le principe général ainsi que le protocole technique de validation des méthodes alternatives, qui sont pour la plupart commerciales, dans le domaine de la microbiologie de la chaîne alimentaire. Les études de validation conformément à l'ISO 16140-2 :2016 sont destinées à être effectuées par des organismes impliqués dans la validation de méthode.
Elle est applicable à la validation de méthodes pour l'analyse (détection ou quantification) 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, et
-      les échantillons au stade de la production primaire.
L'ISO 16140-2 :2016 est notamment applicable aux bactéries et aux champignons. Certains paragraphes de l'ISO 16140-2 :2016 peuvent être applicables à d'autres micro-organismes ou à leurs métabolites au cas par cas. À l'avenir, des préconisations concernant d'autres organismes (par exemple, virus et parasites) seront incluses dans l'ISO 16140 (toutes les parties).

Mikrobiologija v prehranski verigi - Validacija metode - 2. del: Protokol za validacijo alternativnih (lastniških) metod glede na referenčno metodo (ISO 16140-2:2016)

Ta del standarda ISO 16140 določa splošno načelo in tehnični protokol za validacijo
alternativnih, večinoma lastniških metod za mikrobiologijo v prehranski verigi. Študije validacije
v skladu s tem delom standarda ISO 16140 so namenjene organizacijam, ki se ukvarjajo z validacijo metod.
Ta del standarda ISO 16140 se uporablja za validacijo metod za analizo (odkrivanje ali kvantifikacijo) mikroorganizmov v:
– proizvodih, namenjenih za prehrano ljudi;
– proizvodih, namenjenih za živalsko krmo;
– okoljskih vzorcih na področju proizvodnje hrane in krme; ter
– vzorcih iz faze primarne proizvodnje.
Ta del standarda ISO 16140 se uporablja zlasti za bakterije in glive. Nekatere točke tega dela
standarda ISO 16140 se lahko uporabljajo za druge (mikro-) organizme ali njihove metabolite za vsak posamezen primer.
V prihodnosti bodo v ta del ali ločen del standarda ISO 16140 vključeni napotki za druge organizme (npr. viruse in parazite).

General Information

• Status: Published
• Public Enquiry End Date: 14-Apr-2013
• Publication Date: 13-Sep-2016
• ICS: 07.100.30 - Food microbiology
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 28-Jul-2016
• Due Date: 02-Oct-2016
• Completion Date: 14-Sep-2016
• Ref Project: EN 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 (ISO 16140-2:2016)

Relations

• Replaces: SIST EN ISO 16140:2003/A1:2012 - Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods - Amendment 1 (ISO 16140:2003/AMD 1:2011)
• Effective Date: 01-Oct-2016
• Replaces: SIST EN ISO 16140:2003 - Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods (ISO 16140:2003)
• Effective Date: 01-Oct-2016
• Replaces: SIST EN ISO 16140:2003 - Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods (ISO 16140:2003)
• Effective Date: 08-Jun-2022

Standards Content (Sample)

SLOVENSKI STANDARD
SIST EN ISO 16140-2:2016
01-oktober-2016
1DGRPHãþD
SIST EN ISO 16140:2003
SIST EN ISO 16140:2003/A1:2012
0LNURELRORJLMDYSUHKUDQVNLYHULJL9DOLGDFLMDPHWRGHGHO3URWRNRO]D
YDOLGDFLMRDOWHUQDWLYQLKODVWQLãNLKPHWRGJOHGHQDUHIHUHQþQRPHWRGR,62

Microbiology of the food chain - Method validation - Part 2: Protocol for the validation of
alternative (proprietary) methods against a reference method (ISO 16140-2:2016)
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 2: Arbeitsvorschrift für
die Validierung von alternativen (urheberrechtlich geschützten) Verfahren anhand eines
Referenzverfahrens (ISO 16140-2:2016)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 2: Protocole
pour la validation de méthodes alternatives (commerciales) par rapport à une méthode
de référence (ISO 16140-2:2016)
Ta slovenski standard je istoveten z: EN ISO 16140-2:2016
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16140-2:2016 en
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16140-2:2016

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SIST EN ISO 16140-2:2016


EN ISO 16140-2
EUROPEAN STANDARD

NORME EUROPÉENNE

July 2016
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 16140:2003
English Version

Microbiology of the food chain - Method validation - Part 2:
Protocol for the validation of alternative (proprietary)
methods against a reference method (ISO 16140-2:2016)
Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -
méthodes - Partie 2: Protocole pour la validation de Verfahrensvalidierung - Teil 2: Arbeitsvorschrift für die
méthodes alternatives (commerciales) par rapport à Validierung von alternativen (urheberrechtlich
une méthode de référence (ISO 16140-2:2016) geschützten) Verfahren anhand eines
Referenzverfahrens (ISO 16140-2:2016)
This European Standard was approved by CEN on 12 May 2016.

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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, 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: Avenue Marnix 17, B-1000 Brussels
© 2016 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-2:2016 E
worldwide for CEN national Members.

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SIST EN ISO 16140-2:2016
EN ISO 16140-2:2016 (E)
Contents Page
European foreword . 3
2

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SIST EN ISO 16140-2:2016
EN ISO 16140-2:2016 (E)
European foreword
This document (EN ISO 16140-2:2016) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods”
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 January 2017, and conflicting national standards shall
be withdrawn at the latest by January 2017.
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.
This document supersedes EN ISO 16140:2003.
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, Former Yugoslav Republic of Macedonia,
France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,
Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland,
Turkey and the United Kingdom.
Endorsement notice
The text of ISO 16140-2:2016 has been approved by CEN as EN ISO 16140-2:2016 without any
modification.

3

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SIST EN ISO 16140-2:2016
INTERNATIONAL ISO
STANDARD 16140-2
First edition
2016-06-15
Microbiology of the food chain —
Method validation —
Part 2:
Protocol for the validation of
alternative (proprietary) methods
against a reference method
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 2: Protocole pour la validation de méthodes alternatives
(commerciales) par rapport à une méthode de référence
Reference number
ISO 16140-2:2016(E)
©
ISO 2016

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ISO 16140-2:2016(E)

COPYRIGHT PROTECTED DOCUMENT
© ISO 2016, 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. [phone]
Fax [phone]
[e-mail]
www.iso.org
ii © ISO 2016 – All rights reserved

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Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 General principles for the validation of alternative methods . 1
5 Qualitative methods — Technical protocol for validation . 2
5.1 Method comparison study . 2
5.1.1 General considerations . 2
5.1.2 Paired or unpaired study . 2
5.1.3 Sensitivity study . 2
5.1.4 Relative level of detection study . 7
5.1.5 Inclusivity and exclusivity study . 9
5.2 Interlaboratory study .10
5.2.1 General considerations .10
5.2.2 Measurement protocol .10
5.2.3 Calculations and summary of data .12
5.2.4 Interpretation of data .15
6 Quantitative methods — Technical protocol for validation .16
6.1 Method comparison study .16
6.1.1 General considerations .16
6.1.2 Relative trueness study .16
6.1.3 Accuracy profile study.20
6.1.4 Limit of quantification study .24
6.1.5 Inclusivity and exclusivity study .24
6.2 Interlaboratory study .26
6.2.1 General considerations .26
6.2.2 Measurement protocol .26
6.2.3 Calculations, summary, and interpretation of data .27
Annex A (informative) Classification of sample types and suggested target combinations for
validation studies .30
Annex B (normative) Order of preference for use of naturally and artificially contaminated
samples in validation studies .46
Annex C (informative) General protocols for contamination by mixture and artificial
contamination of foods .47
Annex D (informative) Models for RLOD calculations using data from the method
comparison study .50
Annex E (normative) Points to be considered when selecting strains for testing inclusivity
and exclusivity .52
Annex F (informative) Considerations for calculations of the relative level of detection
(RLOD) between laboratories as obtained in an interlaboratory study .54
Annex G (informative) Principle of the accuracy profile for validation of quantitative models .57
Annex H (informative) Application of the accuracy profile in the method comparison study .59
Annex I (informative) Example of the application of the accuracy profile for an
interlaboratory study .62
Bibliography .66
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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 meaning of ISO specific terms and expressions related to conformity
assessment, as well as information about ISO’s adherence to the WTO principles in the Technical
Barriers to Trade (TBT) see the following URL: Foreword - Supplementary Information
The committee responsible for this document is ISO/TC 34, Food products, Subcommittee SC 9,
Microbiology.
This first edition of ISO 16140-2, together with ISO 16140-1, cancels and replaces ISO 16140:2003, which
has been technically revised. It also incorporates the Amendment ISO 16140:2003:Amd.1:2011.
ISO 16140 consists of the following parts, under 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
The following parts are under preparation:
— 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 of non-proprietary methods
— Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation
and typing
iv © ISO 2016 – All rights reserved

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Introduction
Today, many alternative, mostly proprietary, methods exist that are used to assess the microbiological
quality of raw materials and finished products and the microbiological status of manufacturing
procedures. These methods are often faster and easier to perform than the corresponding standardized
method. The developers, end users, and authorities need a reliable common protocol for the validation
of such alternative methods. The data generated will also provide potential end users with performance
data for a given method, thus, enabling them to make an informed choice on the adoption of a particular
method. The data generated can also be the basis for the certification of a method by an independent
organization.
This part of ISO 16140
— is intended to provide a specific protocol and guidelines for the validation of proprietary
methods intended to be used as a rapid and/or easier method to perform than the corresponding
reference method,
— can also be used for the validation of other non-proprietary methods that are used instead of the
reference method,
— is intended as the successor of the validation protocol published in the first version of ISO 16140
(ISO 16140:2003), and
— is mainly written for the validation of methods that are capable of culturing the target microorganism,
but can also be applied to methods for microorganisms that cannot be cultured such as viruses (e.g.
Norovirus) and protozan parasites (e.g. Cryptosporidium or Giardia). In these cases, some wordings
are to be interpreted so as to fit the situation for non-culturable organisms.
The use of this part of ISO 16140 involves expertise on relevant areas such as microbiology, statistical
design, and analysis as indicated in the respective sections. The statistical expertise encompasses
overview of sampling theory and design of experiments, statistical analysis of (qualitative and
quantitative) microbiological data, and overview of statistical concepts on random sampling, sample
heterogeneity, sample stability, design of experiments, and variance components.
When this part of ISO 16140 is next reviewed, account will be taken of all information then available
regarding the extent to which the guidelines have been followed and the reasons for deviation from
them in the case of particular products.
The harmonization of validation methods cannot be immediate and for certain groups of products,
International Standards and/or national standards may already exist that do not comply with this part
of ISO 16140. It is hoped that when such standards are reviewed, they will be changed to comply with
ISO 16140 so that eventually, the only remaining departures from this part of ISO 16140 will be those
[3]
necessary for well-established technical reasons. For example, ISO 16297 deals with a very specific
validation for a specific subject (the hygienic status of raw milk samples) and will remain as a vertical
standard besides ISO 16140. If such a validation is needed, the vertical standard is more important.
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SIST EN ISO 16140-2:2016
INTERNATIONAL STANDARD ISO 16140-2:2016(E)
Microbiology of the food chain — Method validation —
Part 2:
Protocol for the validation of alternative (proprietary)
methods against a reference method
1 Scope
This part of ISO 16140 specifies the general principle and the technical protocol for the validation
of alternative, mostly proprietary, methods for microbiology in the food chain. Validation studies
according to this part of ISO 16140 are intended to be performed by organizations involved in method
validation.
This part of ISO 16140 is applicable to the validation of methods for the analysis (detection 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 of this part of
ISO 16140 could be applicable to other (micro) organisms or their metabolites on a case-by-case-basis.
In the future, guidance for other organisms (e.g. viruses and parasites) will be included in either this
part or a separate part of ISO 16140.
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 16140-1, Microbiology of the food chain— Method validation — Part 1: Vocabulary
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 16140-1 apply.
4 General principles for the validation of alternative methods
The validation protocol comprises two phases:
— a method comparison study of the alternative (proprietary) method against the reference method
carried out in the organizing laboratory;
— an interlaboratory study of the alternative (proprietary) method against the reference method
carried out in different laboratories.
The technical rules for performing the method comparison study and the interlaboratory study are
given in Clause 5 and Clause 6, depending upon whether the alternative (proprietary) method is
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qualitative or quantitative in nature. The data generated in some parts of the validation study are
evaluated using the so-called Acceptability Limits (AL) and no statistical evaluation of the data are
conducted. These AL are based on experts’ opinion and data generated in existing validation studies.
5 Qualitative methods — Technical protocol for validation
5.1 Method comparison study
5.1.1 General considerations
The method comparison study is the part of the validation process that is performed in the organizing
laboratory. It consists of three parts namely the following:
— a comparative study of the results of the reference method to the results of the alternative method
in (naturally and/or artificially) contaminated samples (so-called sensitivity study);
— a comparative study to determine the relative level of detection (RLOD) in artificially contaminated
samples (so-called RLOD study);
— an inclusivity/exclusivity study of the alternative method.
The results (tables and calculations) of the different parts and the interpretation of the results, including
discrepant results, shall be given in a study report.
Test portions size shall be used as written in the reference method.
5.1.2 Paired or unpaired study
The reference and alternative methods shall be performed with, as far as possible, exactly the same
sample (same test portion). However, a distinction is made between studies where the same test portion
can be used for both the reference and the alternative method due to both methods having exactly the
same first step in the (enrichment) procedure and those where different test portions need to be used
for the reference and the alternative method (e.g. due to different enrichment broths). In the case where
the same test portion is used for both methods, the results from both methods are highly related to
each other. For example, when the sample is not contaminated, both methods should find the result of
that sample negative. Due to this relationship, the data produced by the reference and the alternative
method are named paired or matched. In this part of ISO 16140, the wording “paired study” will be
used for this type of study.
The opposite situation where there is no shared initial (enrichment) step for both the reference and
the alternative method is also possible. In this case, different test portions coming from the same batch
or lot of product have to be used for the two methods and the resulting data are named unpaired or
unmatched. In this part of ISO 16140, the word “unpaired study” will be used for this type of study. The
choice of having a paired study or an unpaired study depends on the protocols of the reference and
alternative method. If there is a common initial step in the (enrichment) procedures, a paired study
design is mandatory.
This clause describes the method comparison study if the reference and alternative method have a
joint initial step in the (enrichment) procedures (paired study) and if the reference and alternative
method do not have a joint initial (enrichment) step (unpaired study). Differences between both types
of studies are indicated in the text where appropriate.
5.1.3 Sensitivity study
The sensitivity study aims to determine the difference in sensitivity between the reference and the
alternative method. This study is conducted using naturally and/or artificially contaminated samples.
Different categories and types shall be tested for this. Acceptability Limits have been defined for the
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maximum acceptable difference depending on the type of study (paired/unpaired) and the number of
categories tested.
5.1.3.1 Selection of categories to be used
The selection of categories and types used within the validation will depend on the type or group of
microorganism and the scope of the validation.
If the method is to be applied for a broad range of foods, then at least five categories of food shall be
studied. The validation study report shall state the food categories used in the study. If the method
is to be validated for a restricted number of food categories, e.g. “ready-to-eat, ready-to-reheat meat
products”, and “heat-processed milk and dairy products”, then only these categories need to be studied.
In addition to food, feed samples, environmental samples, and primary production stage samples can be
included as additional categories. This will broaden the application of the use of the alternative method
for these additional categories.
For all selected categories (food and others), at least three different types per category shall be
included in the study. Annex A presents an overview of the relevant types and categories for specific
microorganisms that might be relevant for the validation. Annex A should be used to facilitate the
selection of categories, types, and items for the specific microorganism involved. It should not be
regarded as a mandatory choice.
When selecting samples for the study, it is of the highest priority to find those that are naturally
contaminated. If it is not possible to acquire a sufficient number of naturally contaminated samples,
artificial contamination of samples is permissible (see Annex B and Annex C). Details on the preparation
of the artificially inoculated samples should be given in the validation study report. It is desirable that
food samples come from as wide a distribution as possible in order to reduce any bias from local food
specialities and to broaden the range of validation.
It shall be ensured that with the selection of the different types, both high and low (natural) background
microflora, different types of stresses due to processing, and raw (unprocessed) items are included in
the study.
EXAMPLE For the validation of a method for detection of Listeria monocytogenes and the category “ready-
to-eat, ready-to-reheat meat products”, the types can be (1) cooked meat products (lower background flora, heat
stress), (2) fermented or dried meat products (high background flora, pH stress), and (3) raw cured (smoked)
(a <0,92) (intermediate background flora, a stress).
w w
In some cases, for example, for an alternative method that is applicable for a broad range of foods, it is
possible to combine the “ready-to-eat” and “raw” categories from the same product group. For example,
the categories raw and ready-to-eat meat (products) can be combined into one category having
three types divided over relevant raw and ready-to-eat food types. The selection of (combined) food
categories should be based on risk analy
...