SIST EN ISO 16140-6:2020

Microbiology of the food chain - Method validation - Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation and typing procedures (ISO 16140-6:2019)

This document specifies the general principle and the technical protocol for the validation of alternative confirmation methods for microbiology in the food chain. This document compares the result of the alternative confirmation method against the confirmation procedure of a reference method or, if needed, a reference confirmation method (e.g. whole genome sequencing).
This document is applicable to the validation of alternative confirmation methods used for the analysis (detection or quantification) of isolated microorganisms in:
—          products intended for human consumption;
—          products intended for animal feeding;
—          environmental samples in the area of food and feed production, handling;
—          samples from the primary production stage.
Validated alternative confirmation methods can be used to replace (partly or completely) the confirmation procedure described in:
—          the reference method;
—          an alternative method validated in accordance with ISO 16140-2 only if one of the isolation agars specified in the validation study of the alternative confirmation method is used.
This document is also applicable to the validation of alternative typing methods, where the reference method can be, for example, a serological method (e.g. serotyping of Salmonella) or a molecular method (e.g. typing of Shiga toxin-producing E. coli).
This document is, in particular, applicable to bacteria and fungi. Some clauses can be applicable to other (micro)organisms, to be determined on a case-by-case basis.
Validation studies in accordance with this document are primarily intended to be performed by organizations or expert laboratories involved in method validation, but can also be used by a single laboratory, especially when performing in-house validation under certain conditions (see ISO 16140-4).

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 6: Arbeitsvorschrift für die Validierung (urheberrechtlich geschützter) Alternativverfahren für die Bestätigungs- und Typisierungsprüfung (ISO 16140-6:2019)

Dieses Dokument legt das allgemeine Prinzip und die technische Arbeitsvorschrift für die Validierung alternativer Bestätigungsverfahren in der Mikrobiologie der Lebensmittelkette fest. Dieses Dokument vergleicht die Ergebnisse des alternativen Bestätigungsverfahrens mit denen des Bestätigungsvorgangs eines Referenzverfahrens oder, falls erforderlich, eines Referenzbestätigungsverfahrens (z. B. Gesamtgenom¬sequenzierung).
Dieses Dokument ist anwendbar auf die Validierung von alternativen Bestätigungsverfahren zur Analyse (Nachweis oder quantitative Bestimmung) von isolierten 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.
Validierte alternative Bestätigungsverfahren können verwendet werden, um das Bestätigungsverfahren (ganz oder teilweise) zu ersetzen, das beschrieben ist
   im Referenzverfahren,
   in einem Alternativverfahren, das nach ISO 16140 2 validiert worden ist, sofern dabei eines der in der Validierungsuntersuchung des alternativen Bestätigungsverfahrens angegebenen Isolierungs Agars verwendet wird.
Darüber hinaus ist dieses Dokument auch anwendbar auf die Validierung von alternativen Typisierungsverfahren, bei denen das Referenzverfahren beispielsweise ein serologisches Verfahren (z. B. zur Serotypisierung von Salmonella) oder ein molekulares Verfahren ist (z. B. zur Typisierung von Shigatoxin bildenden E. coli).
Dieses Dokument gilt insbesondere für Bakterien und Pilze. Einige Abschnitte sind auch auf andere (Mikro )Organismen anwendbar. Die Bestimmung muss im Einzelfall erfolgen.
Validierungsuntersuchungen nach diesem Dokument sollen in erster Linie von Organisationen oder Expertenlaboren durchgeführt werden, die an der Verfahrensvalidierung beteiligt sind. Sie können aber auch von einem Einzel-Labor verwendet werden, insbesondere bei der laborinternen Validierung unter bestimmten Bedingungen (siehe ISO 16140 4).

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 6: Protocole pour la validation de méthodes alternatives (commerciales) pour la confirmation microbiologique et le typage (ISO 16140-6:2019)

Le présent document établit le principe général ainsi que le protocole technique pour la validation des méthodes alternatives de confirmation dans le domaine de la microbiologie de la chaîne alimentaire. Le présent document compare le résultat de la méthode alternative de confirmation avec celui du mode opératoire de confirmation d'une méthode de référence ou, si nécessaire, d'une méthode de confirmation de référence (par exemple, séquençage de génome complet).
Le présent document est applicable à la validation de méthodes alternatives de confirmation utilisées pour l'analyse (recherche ou quantification) de micro-organismes isolés présents dans:
—          les produits destinés à la consommation humaine;
—          les produits destinés à l'alimentation animale;
—          les échantillons environnementaux dans les domaines de la production et de la manutention de produits alimentaires;
—          les échantillons au stade de la production primaire.
Des méthodes alternatives de confirmation validées peuvent être utilisées pour remplacer (en partie ou en totalité) le mode opératoire de confirmation décrit dans:
—          la méthode de référence;
—          une méthode alternative validée conformément à l'ISO 16140‑2 uniquement si l'une des géloses d'isolement spécifiées dans l'étude de validation de la méthode de confirmation alternative est utilisée.
Le présent document est également applicable à la validation de méthodes alternatives de typages, dans lesquelles la méthode de référence peut, par exemple, être une méthode sérologique (par exemple, sérotypage de Salmonella) ou une méthode moléculaire (par exemple, typage d'E. coli produisant des shigatoxines).
Le présent document est notamment applicable aux bactéries et aux champignons. Certains articles peuvent être applicables à d'autres (micro)organismes, qui doivent être déterminés au cas par cas.
Les études de validation conformément au présent document sont principalement destinées à être effectuées par des organismes ou des laboratoires experts impliqués dans la validation des méthodes, mais peuvent également être utilisées par un seul laboratoire, notamment lors de la réalisation d'une validation interne sous certaines conditions (voir ISO 16140‑4).

Mikrobiologija v prehranski verigi - Validacija metode - 6. del: Protokol za validacijo alternativnih (lastniških) metod za postopke mikrobiološke potrditve in tipizacije (ISO 16140-6:2019)

Ta del standarda ISO 16140 določa splošno načelo in tehnični protokol za validacijo alternativnih, večinoma lastniških metod za potrditev mikrobiološke analize hrane, krme za živali ter vzorcev okoljske in primarne proizvodnje. Ta postopek je omejen na validacijo alternativnih (lastniških) metod za potrditev, ki naj bi delno ali v celoti nadomestile postopek potrditve, opisan v standardni metodi štetja ali zaznavanja specifičnih (skupin) mikroorganizmov. »Vzorec« za potrditev je sumljiva kolonija, ki je bila pridobljena s postopkom za pridobivanje referenčne ali alternativne kulture. Postopek ni namenjen potrditvi z uporabo (čiste) kolonije neznanega izvora. Validativne študije v skladu s tem standardom naj bi izvajale organizacije, ki sodelujejo pri validaciji metod.

General Information

• Status: Published
• Public Enquiry End Date: 04-Mar-2018
• Publication Date: 12-Jan-2020
• ICS: 07.100.30 - Food microbiology
• Technical Committee: KŽP - Agricultural food products
• Current Stage: 6060 - National Implementation/Publication (Adopted Project)
• Start Date: 12-Dec-2019
• Due Date: 16-Feb-2020
• Completion Date: 13-Jan-2020
• Directive: 882/2004 - Regulation (EC) No 882/2004 of the European Parliament and of the Council of 29 April 2004 onofficial controls performed to ensure the verification of compliance with feed and food law, animal health and animal welfare rules
• Ref Project: EN ISO 16140-6:2019 - Microbiology of the food chain - Method validation - Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation and typing procedures (ISO 16140-6:2019)

Overview

EN ISO 16140-6:2019 - Microbiology of the food chain - Method validation - Part 6 - specifies a protocol for validating alternative (proprietary) confirmation and typing methods used in food- and feed-chain microbiology. The standard describes how to compare an alternative confirmation or typing procedure with a reference confirmation method (or an appropriate reference such as whole genome sequencing) to demonstrate equivalent performance for isolated microorganisms. It is applicable primarily to bacteria and fungi and to samples from products for human or animal consumption, environmental monitoring in food/feed production, and primary production stages.

Key topics and requirements

• Scope and applicability: Validation of alternative confirmation and typing methods for detection or quantification of isolated microorganisms in food, feed, environmental and primary production samples.
• Comparison to reference methods: Results from the alternative method are compared against the reference confirmation procedure or a validated reference typing method (e.g., serotyping, molecular typing, WGS).
• Study design elements:
  • Strain selection and documented criteria for inclusivity (target strains) and exclusivity (non‑target strains).
  • Method comparison study (single-laboratory stage) covering expression and interpretation of results and evaluation criteria.
  • Interlaboratory study (when required) with defined data sets, protocol, result expression and statistical interpretation.
• Performance characteristics: Requirements for evaluation, acceptability limits and decision rules are provided (see normative annexes for options and limits).
• Examples and annexes: Informative examples (e.g., Listeria species confirmation, Salmonella serovar typing) and guidance for strain selection are included to support practical implementation.

Applications and users

EN ISO 16140-6:2019 is intended for:

• Test kit and alternative method developers validating proprietary confirmation or typing products.
• Expert and validation laboratories conducting validation studies and interlaboratory trials.
• Single laboratories performing in‑house validation under ISO 16140-4 conditions.
• Food and feed producers, contract testing labs and regulatory bodies evaluating or adopting validated alternative confirmation/typing methods for routine testing, outbreak investigation and surveillance.

Practical uses include demonstrating equivalence to standard confirmation procedures, replacing part or all of a reference confirmation workflow, and validating newer typing approaches (serological or molecular) such as WGS-based confirmation and typing.

Related standards

• ISO 16140 series: Part 1 (vocabulary), Part 2 (validation vs reference), Part 3 (verification), Part 4 (single-lab validation), Part 5 (factorial interlaboratory validation).
• ISO 17468 (technical rules for development/validation of standardized methods).

Keywords: EN ISO 16140-6:2019, microbiology of the food chain, method validation, alternative confirmation methods, typing procedures, interlaboratory study, inclusivity, exclusivity, proprietary methods.

Standards Content (Sample)

SLOVENSKI STANDARD
01-februar-2020
Mikrobiologija v prehranski verigi - Validacija metode - 6. del: Protokol za
validacijo alternativnih (lastniških) metod za postopke mikrobiološke potrditve in
tipizacije (ISO 16140-6:2019)
Microbiology of the food chain - Method validation - Part 6: Protocol for the validation of
alternative (proprietary) methods for microbiological confirmation and typing procedures
(ISO 16140-6:2019)
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 6: Arbeitsvorschrift für
die Validierung (urheberrechtlich geschützter) Alternativverfahren für die Bestätigungs-
und Typisierungsprüfung (ISO 16140-6:2019)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 6: Protocole
pour la validation de méthodes alternatives (commerciales) pour la confirmation
microbiologique et le typage (ISO 16140-6:2019)
Ta slovenski standard je istoveten z: EN ISO 16140-6:2019
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 16140-6
EUROPEAN STANDARD
NORME EUROPÉENNE
December 2019
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Method validation - Part 6:
Protocol for the validation of alternative (proprietary)
methods for microbiological confirmation and typing
procedures (ISO 16140-6:2019)
Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -
méthodes - Partie 6: Protocole pour la validation de Verfahrensvalidierung - Teil 6: Arbeitsvorschrift für die
méthodes alternatives (commerciales) pour la Validierung (urheberrechtlich geschützter)
confirmation microbiologique et le typage (ISO 16140- Alternativverfahren für die Bestätigungs- und
6:2019) Typisierungsprüfung (ISO 16140-6:2019)
This European Standard was approved by CEN on 10 September 2019.

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
© 2019 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-6:2019 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 16140-6:2019) 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 June 2020, and conflicting national standards shall be
withdrawn at the latest by June 2020.
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 16140-6:2019 has been approved by CEN as EN ISO 16140-6:2019 without any
modification.
INTERNATIONAL ISO
STANDARD 16140-6
First edition
2019-11
Microbiology of the food chain —
Method validation —
Part 6:
Protocol for the validation of
alternative (proprietary) methods
for microbiological confirmation and
typing procedures
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 6: Protocole pour la validation de méthodes alternatives
(commerciales) pour la confirmation microbiologique et le typage
Reference number
ISO 16140-6:2019(E)
©
ISO 2019
ISO 16140-6:2019(E)
© ISO 2019
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: [phone]
Fax: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2019 – All rights reserved

ISO 16140-6:2019(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 General principles for the validation of confirmation and typing methods.3
5 Strains . 4
6 Method comparison study . 4
6.1 General . 4
6.2 Selection of test strains . 4
6.3 Inclusivity study . 4
6.3.1 Testing of target strains . 4
6.3.2 Family level (non-Salmonella) . 5
6.3.3 Genus level (non-Salmonella) . 5
6.3.4 Species level (non-Salmonella) . 5
6.3.5 Microbial (sub)type level (non-Salmonella) . 5
6.3.6 Salmonella genus or species level . 5
6.3.7 Salmonella serovar level . 6
6.4 Exclusivity study . 6
6.4.1 Testing of non-target strains . 6
6.4.2 Family level (non-Salmonella) . 6
6.4.3 Genus level (non-Salmonella) . 6
6.4.4 Species level (non-Salmonella) . 6
6.4.5 Microbial (sub)type level (non-Salmonella) . 6
6.4.6 Salmonella genus or species level . 7
6.4.7 Salmonella serovar level . 7
6.5 Expression and interpretation of results . 7
6.6 Evaluation . 9
7 Interlaboratory study .10
7.1 General .10
7.2 Data sets to be obtained .10
7.3 Protocol .11
7.4 Expression of results .12
7.5 Interpretation and evaluation .12
Annex A (normative) Points to be considered when selecting strains for testing inclusivity
and exclusivity .14
Annex B (informative) Example of the validation of an alternative confirmation method to
the species level (Listeria monocytogenes) .16
Annex C (informative) Example of the validation of an alternative typing method to the
Salmonella serovar level (15 different serovars claimed) .19
Annex D (normative) Overview of the various options for validation under this document
and the acceptability limits .23
Bibliography .25
ISO 16140-6:2019(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.
A list of all parts of 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.
iv © ISO 2019 – All rights reserved

ISO 16140-6:2019(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 validated 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 as described in
this document). 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 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 ISO 16140-3) 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.
ISO 16140-6:2019(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 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.
This document, 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 this document. The validation study
in this document 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 this document can be applied (see text in the boxes).
vi © ISO 2019 – All rights reserved

ISO 16140-6:2019(E)
Figure 2 — Use of validated alternative confirmation methods (described in this document)
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  Validation and verification of methods for the microbiological confirmation and typing
procedures
The procedure described in this document is intended for the “full” validation of alternative
(proprietary) methods for microbiological confirmation and/or typing, hereafter referred to as
“alternative confirmation methods”.
During the validation study, the performance of the alternative confirmation method is compared to
the performance of the reference confirmation procedure.
The procedure for verification of alternative confirmation methods in a single laboratory is described
in ISO 16140-3.
INTERNATIONAL STANDARD ISO 16140-6:2019(E)
Microbiology of the food chain — Method validation —
Part 6:
Protocol for the validation of alternative (proprietary)
methods for microbiological confirmation and typing
procedures
1 Scope
This document specifies the general principle and the technical protocol for the validation of alternative
confirmation methods for microbiology in the food chain. This document compares the result of the
alternative confirmation method against the confirmation procedure of a reference method or, if
needed, a reference confirmation method (e.g. whole genome sequencing).
This document is applicable to the validation of alternative confirmation methods used for the analysis
(detection or quantification) of isolated microorganisms in:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production, handling;
— samples from the primary production stage.
Validated alternative confirmation methods can be used to replace (partly or completely) the
confirmation procedure described in:
— the reference method;
— an alternative method validated in accordance with ISO 16140-2 only if one of the isolation agars
specified in the validation study of the alternative confirmation method is used.
This document is also applicable to the validation of alternative typing methods, where the reference
method can be, for example, a serological method (e.g. serotyping of Salmonella) or a molecular method
(e.g. typing of Shiga toxin-producing E. coli).
This document is, in particular, applicable to bacteria and fungi. Some clauses can be applicable to other
(micro)organisms, to be determined on a case-by-case basis.
Validation studies in accordance with this document are primarily intended to be performed by
organizations or expert laboratories involved in method validation, but can also be used by a single
laboratory, especially when performing in-house validation under certain conditions (see ISO 16140-4).
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 16140-1:2016, Microbiology of the food chain — Method validation — Part 1: Vocabulary
ISO 16140-6:2019(E)
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:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
acceptability limit
AL
maximum positive or negative acceptable difference between the reference value (or if not known, the
accepted reference value) of a sample and an individual result obtained when applying the operating
procedure of an analytical method
Note 1 to entry: Annex D provides further information on the use of AL for this document.
[SOURCE: ISO 16140-1:2016, 2.1, modified — Note 1 to entry has been replaced.]
3.2
alternative confirmation or typing method
confirmation or typing method submitted for validation
method of analysis that confirms or types the same analyte as is confirmed or typed using the
corresponding reference method
Note 1 to entry: 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.
Note 2 to entry: For clarity of reading, the text in this document generally describes validation of a confirmation
method (detailed examples are given in Annex B). If applicable, this can be read as validation of a typing method
(detailed examples are given in Annex C).
3.3
confirmation procedure
number of defined confirmation tests (3.4) that are performed on a strain, the combined results of which
are used to definitively confirm the identity of that strain
3.4
confirmation test
single test which is carried out to verify a presumptive result
Note 1 to entry: The result of a single test may not on its own be able to definitively confirm the identity of the strain.
[SOURCE: ISO 16140-1:2016, 2.17, modified — In the term and definition, “procedure or” has been
removed, “single” has been added and the Note 1 to entry has been replaced.]
3.5
microbial (sub)type
group of closely related microorganisms (within a species) distinguished by their shared specific
characteristics as determined by, for example, serological testing (serotype) or molecular testing
(genotype)
3.6
non-target strain
strain, defined according to the scope of the reference method, that would not reasonably be expected
to be confirmed by the alternative method
[SOURCE: ISO 16140-1:2016, 2.44, modified — In the definition, “confirmed” has replaced “detected or
enumerated”.]
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ISO 16140-6:2019(E)
3.7
reference confirmation or typing procedure
combination of the confirmation or typing tests that are claimed to be replaced by the alternative
confirmation or typing method (3.2)
Note 1 to entry: The number of confirmation tests (3.4) depends on the reference method for the specific
microorganisms. The number of confirmation tests can also be one.
3.8
target strain
strain, defined according to the scope of the reference method, that is expected to be confirmed by the
alternative method
[SOURCE: ISO 16140-1:2016, 2.74, modified — In the definition, “confirmed” has replaced “detected or
enumerated”.]
3.9
typing procedure
process of determining a particular microbial (sub)type (3.5)
4 General principles for the validation of confirmation and typing methods
In the validation study, the alternative confirmation method is compared to the confirmation
procedure described in the reference method for the enumeration or detection of specific (groups of)
microorganisms.
The validation protocol comprises two phases:
— a method comparison study, of the alternative confirmation method against the reference
confirmation procedure, carried out in the organizing laboratory;
— an interlaboratory study.
NOTE It is possible, if relevant, to include inclusivity or exclusivity data obtained in an ISO 16140-2 validation
study into a study related to this document.
The validation protocol shall clearly define the selective agar(s) from which strains can be confirmed
using the alternative confirmation method. All inclusivity and exclusivity strains shall be tested. In
cases where some strains are unable to grow on the specified selective agar(s), a non-selective agar
plate shall also be included in the method comparison study and the interlaboratory study.
The technical rules for performing the method comparison study and the interlaboratory study
are given in Clauses 6 and 7. The following six cases are covered; a distinction is made between the
confirmation/typing of Salmonella and that of other microorganisms:
— validation of methods used for confirmation to the family level (non-Salmonella);
— validation of methods used for confirmation to the genus level (non-Salmonella);
— validation of methods used for confirmation to the species level (non-Salmonella);
— validation of methods used for confirmation/typing to the microbial (sub)type level (non-Salmonella);
— validation of methods used for confirmation/typing to the Salmonella genus or species level;
— validation of methods used for confirmation/typing to the Salmonella serovar level.
ISO 16140-6:2019(E)
5 Strains
The pure strains used for determining the inclusivity and the exclusivity shall be well-characterized
in line with the purpose of the validation study. The identification information of each strain will be
used to (additionally) confirm the result in cases of discrepancies between the results of the reference
confirmation procedure and the alternative confirmation method.
NOTE National, regional or international reference laboratories could be contacted during such
investigations.
6 Method comparison study
6.1 General
The method comparison study is the part of the validation that is performed in one laboratory.
It consists of an inclusivity and exclusivity study of the alternative confirmation method. The results
are then compared to those of the reference confirmation procedure.
6.2 Selection of test strains
A range of strains shall be used. Criteria for selecting test strains are given in Annex A. The strains
selected should take into account the measurement principle (e.g. culture-based, immunological,
molecular-based) of the alternative method. Different measurement principles may require the use
of panels of different test strains, representing the diversity of the studied microorganism(s). It is
important to include non-target microorganisms that may grow on the media used for the reference and
for the alternative method, including those that produce suspect colonies (i.e. look like those produced
by the target strains).
The rationale for the choice of the strains and their characteristics shall be included in the validation
study report.
Each strain shall be characterized biochemically and/or serologically and/or genetically in sufficient
detail for its identity to be known. Strains should preferably have been isolated from foods, feed, the
food-processing environment or from primary production; depending on the scope of the validation.
However, clinical, environmental and culture collection strains can also be used. The original source
of all strains should be known, and they should be held in a local (e.g. expert laboratory), national or
international culture collection to enable them to be used in future testing if required. See ISO 11133
for guidance on the local maintenance of stock cultures.
Results generated by a specialized reference laboratory, using the reference method, can be used if
the laboratory performing the validation study is not able to perform the confirmation/typing of rare
strains according to the reference method. For example, the use of serotyping results of a Salmonella
reference laboratory is allowed in cases of rare Salmonella serovars.
6.3 Inclusivity study
6.3.1 Testing of target strains
Pure cultures of all target strains shall be tested with both the reference confirmation procedure and
the alternative confirmation method. It is not necessary to repeat the reference confirmation procedure
along with the alternative confirmation method if the required data for the reference procedure are
available. As all inclusivity strains shall be tested, subculture the strains on a non-selective agar
plate, along with the clearly defined selective agar(s) from which strains can be confirmed using the
alternative confirmation method. This will ensure that a viable strain is available for confirmation.
The number of strains to be tested under the various options (see 6.3.2 to 6.3.7) are summarized in
Tables D.1 and D.2.
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ISO 16140-6:2019(E)
In cases where the required number of specific target strains are not available, the number of strains
to be tested could be reconsidered, taking into account factors such as the frequency of occurrence
and the public health or spoilage significance of these specific strains. There should be a minimum of
5 strains per microbial (sub)type.
6.3.2 Family level (non-Salmonella)
For inclusivity, a minimum of 200 different target strains shall be tested.
EXAMPLE The alternative method claims to confirm Enterobacteriaceae. A minimum of 200 different strains
of Enterobacteriaceae are included in the inclusivity study.
6.3.3 Genus level (non-Salmonella)
For inclusivity, a minimum of 150 different target strains shall be tested.
EXAMPLE The alternative method claims to confirm Listeria spp. A minimum of 150 different strains of
Listeria spp. are included in the inclusivity study.
6.3.4 Species level (non-Salmonella)
For inclusivity, a minimum of 100 different target strains per claimed species shall be tested.
EXAMPLE The alternative method claims to confirm L. monocytogenes. A minimum of 100 different strains
of L. monocytogenes are included in the inclusivity study.
6.3.5 Microbial (sub)type level (non-Salmonella)
For inclusivity, a minimum of 25 different target strains per claimed microbial (sub)type shall be tested.
If 5 or more microbial (sub)types are claimed, then a minimum of 100 strains shall be tested. These
should be proportionally divided according to the distribution and availability of the various microbial
(sub)types under study, with a minimum of 5 strains per microbial (sub)type.
EXAMPLE The alternative method claims to confirm E. coli O157, E. coli O111, E. coli O26, E. coli O103 and
E. coli O145. Therefore, at least 20 different strains of each of these 5 serogroups are included in the inclusivity
study, taking into account the minimum of 100 strains in total.
6.3.6 Salmonella genus or species level
For inclusivity, a minimum of 150 different target strains shall be tested.
— If the alternative confirmation method claims confirmation of Salmonella spp., target strains
include at least 2 strains each of S. bongori, S. enterica subsp. salamae, S. enterica subsp. arizonae,
S. enterica subsp. diarizonae, S. enterica subsp. houtenae, S. enterica subsp. indica. Supplement these
with strains of S. enterica subsp. enterica, covering common serovars. Preferably, include at least
one representative of each (somatic) O-antigen described (see ISO/TR 6579-3).
— If the alternative confirmation method claims confirmation of S. enterica, target strains include
at least 2 strains each of S. enterica subsp. salamae, S. enterica subsp. arizonae, S. enterica subsp.
diarizonae, S. enterica subsp. houtenae, S. enterica subsp. indica. Supplement these with strains of
S. enterica subsp. enterica covering common serovars. Preferably, include at least one representative
of each (somatic) O-antigen described (see ISO/TR 6579-3).
— If the alternative confirmation method claims confirmation of S. enterica subsp. enterica, target
strains include different strains (and serovars) of S. enterica subsp. enterica only, covering common
serovars. Preferably, include at least one representative of each (somatic) O-antigen described
(see ISO/TR 6579-3).
NOTE More information on common serovars can be found on the following websites: www .cdc .gov/ ,
www .ecdc .europa .eu/ and www .efsa .europa .eu/ .
ISO 16140-6:2019(E)
6.3.7 Salmonella serovar level
For inclusivity, a minimum of 25 different target strains per claimed Salmonella serovar shall be tested.
If 11 or more serovars are claimed, then a minimum of 250 strains shall be tested. These should be
proportionally divided, according to the general serovar distribution.
In cases where the required number of specific target strains are not available, the number of strains
to be tested could be reconsidered, taking into account factors such as the frequency of occurrence
and the public health significance of these specific strains. There should be a minimum of 5 strains per
serovar.
EXAMPLE The alternative method claims to confirm Salmonella Enteritidis and Salmonella Typhimurium. A
minimum of 25 different strains of Salmonella Enteritidis and 25 different strains of Salmonella Typhimurium are
included in the inclusivity study.
6.4 Exclusivity study
6.4.1 Testing of non-target strains
Pure cultures of all non-target strains shall be tested with both the reference confirmation procedure
and the alternative confirmation method. It is not necessary to repeat the reference confirmation
procedure along with the alternative confirmation method if the required data for the reference
procedure are available. As all exclusivity strains shall be tested, subculture the strains on a non-
selective agar plate, along with the clearly defined selective agar(s) from which strains can be
confirmed using the alternative confirmation method. This will ensure that a viable strain is available
for confirmation.
The number of strains to be tested under the various options (see 6.4.2 to 6.4.7) are summarized in
Tables D.1 and D.2.
Preferably, select non-target strains that are able to grow on the selective agar(s) as used in the
reference method.
6.4.2 Family level (non-Salmonella)
For exclusivity, a minimum of 100 different non-target strains shall be tested.
6.4.3 Genus level (non-Salmonella)
For exclusivity, a minimum of 100 different non-target strains shall be tested.
6.4.4 Species level (non-Salmonella)
For exclusivity, a minimum of 100 different non-target strains shall be tested, of which there are:
— 50 strains from non-target genus;
— 50 strains from non-target species within the target genus.
EXAMPLE The alternative method claims to confirm L. monocytogenes. A minimum of 50 different strains
of non-Listeria spp. and 50 different strains of Listeria spp., but not L. monocytogenes, are included in the
exclusivity study.
6.4.5 Microbial (sub)type level (non-Salmonella)
For exclusivity, a minimum of 100 different non-target strains shall be tested, of which there are:
— 25 strains from non-target family;
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ISO 16140-6:2019(E)
— 25 strains from target family or target genus (but not the target species);
— 50 strains from non-target microbial (sub)type within the target species.
EXAMPLE The alternative method claims separately to confirm E. coli O157, E. coli O111, E. coli O26, E. coli
O103 and E. coli O145. A minimum of 25 different strains of non-Enterobacteriaceae, 25 different strains of
Enterobacteriaceae (but non-E. coli) and 50 different strains of E. coli serogroups other than the 5 serogroups
mentioned above are included in the exclusivity study.
6.4.6 Salmonella genus or species level
For exclusivity, a minimum of 100 different non-target strains shall be tested.
— If the alternative confirmation method claims confirmation of S. enterica, include:
— at least 2 strains of S. bongori;
— at least 75 (to make up to the total of 100) strains from the target family (Enterobacteriaceae).
— If the alternative confirmation method claims confirmation of S. enterica subsp. enterica, include:
— at least 2 strains each of S. bongori, S. enterica subsp. salamae, S. enterica subsp. arizonae,
S. enterica subsp. diarizonae, S. enterica subsp. houtenae and S. enterica subsp. indica;
— at least 75 (to make up to the total of 100) strains from the target family (Enterobacteriaceae).
6.4.7 Salmonella serovar level
For exclusivity, a minimum of 100 different non-target strains shall be tested.
If the alternative method claims typing of all Salmonella serovars, a total of 100 different strains from
non-target genus, but within target family (Enterobacteriaceae), shall be tested.
If the alternative method claims typing of specific Salmonella serovars, include:
— at least 25 strains from non-target genus, but within target family (Enterobacteriaceae);
— at least 75 strains from non-target serovars within the target subspecies. These should include non-
target serovars with partly the same O-antigens or H-antigens as the target Salmonella serovars.
EXAMPLE The alternative method claims to confirm Salmonella Enteritidis and Salmonella Typhimurium.
A minimum of 25 different strains of
...