EN ISO 16140-4:2020

Microbiology of the food chain - Method validation - Part 4: Protocol for method validation in a single laboratory (ISO 16140-4:2020)

This document specifies the general principles and the technical protocols for single-laboratory validation of methods for microbiology in the food chain. The protocols in this document only validate the method for the laboratory conducting the study.
This document is applicable to single-laboratory validation of:
—     methods used in 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;
—     samples from the primary production stage;
—     methods for the confirmation or typing of microorganisms. This validation will replace only the confirmation or typing procedure of a specified method (see Annex G).
This document 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.
Single-laboratory validation is required if an interlaboratory validation in accordance with ISO 16140-2 is not appropriate. Possible applications are:
—     validation of an in-house method;
—     method evaluation study in the validation process of a reference method in accordance with ISO 17468;
—     extension of the scope of an ISO 16140-2 validated method, e.g. category extension or test portion size;
—     modifications of existing methods.
Single-laboratory validation is the second step in the standardization of a reference method (see ISO 17468). It is only applicable to methods that are fully specified with regard to all relevant parameters (including tolerances on temperatures and specifications on culture media) and that have already been optimized.

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 4: Arbeitsvorschrift für Einzel-Labor-Verfahrensvalidierung (ISO 16140-4:2020)

Dieses Dokument legt die allgemeinen Grundsätze und technischen Arbeitsvorschriften für die Einzel-Labor-Validierung von Verfahren in der Mikrobiologie der Lebensmittelkette fest. Die Arbeitsvorschriften in diesem Dokument validieren das Verfahren nur für das Labor, in dem die Untersuchung durchgeführt wird.
Dieses Dokument ist anwendbar auf die Einzel-Labor-Validierung von:
- Verfahren zur Analyse (Nachweis oder Quantifizierung) 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;
- Verfahren zur Bestätigung oder Typisierung von Mikroorganismen. Diese Validierung wird nur das Bestätigungs  oder Typisierungsprüfung für ein festgelegtes Verfahren ersetzen (siehe Anhang G).
Dieses Dokument ist insbesondere auf Bakterien und Pilze anwendbar. Einige Abschnitte können auf andere (Mikro )Organismen oder deren Metabolite angewendet werden. Die Bestimmung muss im Einzelfall erfolgen.
Eine Einzel-Labor-Validierung ist dann erforderlich, wenn ein Ringversuch nach ISO 16140 2 nicht angemessen ist. Mögliche Anwendungsbereiche sind:
- Validierung eines laborinternen Verfahrens;
- Untersuchung zur Verfahrensbewertung im Rahmen des Validierungsprozesses eines Referenzverfahrens nach ISO 17468;
- Erweiterung des Anwendungsbereichs eines nach ISO 16140 2 validierten Verfahrens, z. B. Erweiterung einer Kategorie oder Änderung der Größe einer Prüfmenge;
- Änderungen bereits bestehender Verfahren.
Die Einzel-Labor-Validierung ist der zweite Schritt bei der Standardisierung eines Referenzverfahrens (siehe ISO 17468). Sie ist nur für solche Verfahren anwendbar, bei denen alle relevanten Parameter angegeben sind (einschließlich der Toleranzen zu Temperaturen und der Festlegungen zu Nährmedien) und die bereits optimiert wurden.

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 4: Protocole pour la validation de méthodes dans un seul laboratoire (ISO 16140-4:2020)

Le présent document établit les principes généraux ainsi que les protocoles techniques pour la validation dans un seul laboratoire des méthodes applicables à la microbiologie de la chaîne alimentaire. Les protocoles du présent document valident la méthode uniquement pour le laboratoire effectuant l'étude.
Le présent document est applicable à la validation dans un seul laboratoire de:
—          méthodes utilisées 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 les domaines de la production et de la manutention de produits alimentaires;
—   les échantillons au stade de la production primaire;
—          méthodes de confirmation ou de typage de micro-organismes. Cette validation remplacera uniquement le mode opératoire de confirmation ou de typage d'une méthode spécifiée (voir l'Annexe G).
Le présent document est notamment applicable aux bactéries et aux champignons. Certains articles peuvent être applicables à d'autres (micro-)organismes ou à leurs métabolites, qui doivent être déterminés au cas par cas.
La validation dans un seul laboratoire est requise si une validation interlaboratoires conformément à l'ISO 16140‑2 n'est pas appropriée. Les applications possibles sont les suivantes:
—          validation d'une méthode interne;
—          étude d'évaluation de méthode lors du processus de validation d'une méthode de référence conformément à l'ISO 17468;
—          extension du domaine d'application d'une méthode validée de l'ISO 16140‑2, par exemple extension de catégorie ou taille de la prise d'essai;
—          modifications de méthodes existantes.
La validation dans un seul laboratoire est la deuxième étape de la normalisation d'une méthode de référence (voir l'ISO 17468). Elle est uniquement applicable aux méthodes qui sont intégralement spécifiées par rapport à tous les paramètres pertinents (notamment les tolérances sur les températures et les spécifications sur les milieux de culture) et qui ont déjà été optimisées.

Mikrobiologija v prehranski verigi - Validacija metode - 4. del: Protokol za validacijo posamezne metode v laboratoriju (hišne metode) (ISO 16140-4:2020)

General Information

• Status: Published
• Publication Date: 25-Aug-2020
• Withdrawal Date: 27-Feb-2021
• ICS: 07.100.30 - Food microbiology
• Technical Committee: CEN/TC 463 - Microbiology of the food chain
• Drafting Committee: CEN/TC 275/WG 6 - Microbiological contamination
• Current Stage: 6060 - Definitive text made available (DAV) - Publishing
• Start Date: 26-Aug-2020
• Completion Date: 26-Aug-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: SIST EN ISO 16140-4:2020 - Microbiology of the food chain - Method validation - Part 4: Protocol for method validation in a single laboratory (ISO 16140-4:2020)

Relations

• Amended By: EN ISO 16140-4:2020/FprA1 - Microbiology of the food chain - Method validation - Part 4: Protocol for method validation in a single laboratory - Amendment 1: Validation of a larger test portion size for qualitative methods (ISO 16140 4:2020/FDAM 1:2024)
• Effective Date: 23-Jun-2021

Standards Content (Sample)

SLOVENSKI STANDARD
01-november-2020
Mikrobiologija v prehranski verigi - Validacija metode - 4. del: Protokol za
validacijo posamezne metode v laboratoriju (hišne metode) (ISO 16140-4:2020)
Microbiology of the food chain - Method validation - Part 4: Protocol for method validation
in a single laboratory (ISO 16140-4:2020)
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 4: Arbeitsvorschrift für
Einzel-Labor-Verfahrensvalidierung (ISO 16140-4:2020)
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 4: Protocole
pour la validation de méthodes dans un seul laboratoire (ISO 16140-4:2020)
Ta slovenski standard je istoveten z: EN ISO 16140-4:2020
ICS:
07.100.30 Mikrobiologija živil Food microbiology
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

EN ISO 16140-4
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2020
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Method validation - Part 4:
Protocol for method validation in a single laboratory (ISO
16140-4:2020)
Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -
méthodes - Partie 4: Protocole pour la validation de Verfahrensvalidierung - Teil 4: Arbeitsvorschrift für
méthodes dans un seul laboratoire (ISO 16140-4:2020) Einzel-Labor-Verfahrensvalidierung (ISO 16140-
4:2020)
This European Standard was approved by CEN on 25 May 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
© 2020 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-4:2020 E
worldwide for CEN national Members.

Contents Page
European foreword . 3

European foreword
This document (EN ISO 16140-4:2020) 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 February 2021, and conflicting national standards
shall be withdrawn at the latest by February 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 16140-4:2020 has been approved by CEN as EN ISO 16140-4:2020 without any
modification.
INTERNATIONAL ISO
STANDARD 16140-4
First edition
2020-07
Microbiology of the food chain —
Method validation —
Part 4:
Protocol for method validation in a
single laboratory
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 4: Protocole pour la validation de méthodes dans un seul
laboratoire
Reference number
ISO 16140-4:2020(E)
©
ISO 2020
ISO 16140-4:2020(E)
© ISO 2020
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
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Phone: [phone]
Email: [e-mail]
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved

ISO 16140-4:2020(E)
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 2
4 General principles of the single-laboratory detection or quantification method
validation . 4
4.1 General . 4
4.2 Principles of the factorial approach . 5
4.3 Principles of the conventional approach . 5
5 Technical protocol for validation — Factorial approach . 7
5.1 Qualitative methods. 7
5.1.1 Single-laboratory method validation study against a reference method . 7
5.1.2 Single-laboratory method validation study without a reference method .13
5.2 Quantitative methods .15
5.2.1 Single-laboratory method validation study against a reference method .15
5.2.2 Single-laboratory method validation study without a reference method .18
6 Technical protocol for validation — Conventional approach .19
6.1 Qualitative methods.19
6.1.1 Single-laboratory method validation study against a reference method .19
6.1.2 Single-laboratory method validation study without a reference method .20
6.2 Quantitative methods .21
6.2.1 Single-laboratory method validation study against a reference method .21
6.2.2 Single-laboratory method validation study without a reference method .23
7 Summary of acceptability limits .26
Annex A (informative) List of factors and factor levels for factorial method validation .27
Annex B (informative) Calculation of in-house reproducibility for qualitative methods
based on the LOD study described in 6.1.2.3 .29
Annex C (informative) Example of a factorial single-laboratory method validation study for
a quantitative method against a reference method .30
Annex D (informative) Example of a factorial single-laboratory method validation study for
a qualitative method against a reference method .36
Annex E (informative) Example of a factorial single-laboratory method validation study for
the variability of the LOD for a qualitative method without a reference method .40
Annex F (informative) Determination of precision if the artificially contaminated samples
are unstable .43
Annex G (informative) Protocol for single-laboratory validation of alternative methods for
microbiological confirmation and typing procedures .45
Bibliography .46
ISO 16140-4:2020(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
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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.
iv © ISO 2020 – All rights reserved

SIST EN ISO 16140-4:202
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