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

The proposed deliverable specifies the procedure for single-laboratory validation of mainly non-proprietary methods in the fields of microbiological analysis of food, feed, and environmental and primary production stage samples. Single-laboratory validation is required if an interlaboratory validation according to ISO 16140-2 is not appropriate, e.g. for in-house methods or when the required number of participating laboratories is not available. Single-laboratory validation is not part of the optimization of methods. It can be applied only for methods that are fully specified with regard to all relevant parameters (including tolerances on temperatures and specifications on nutrient media).
The proposed deliverable describes two protocols for single-laboratory validation, a conventional protocol, and a factorial protocol. The conventional protocol is a stepwise procedure; both the study design and the performance measures are derived from ISO 16140-2. The performance measures of the factorial protocol are also derived from ISO 16140-2; however, it is using an orthogonal, factorial study design. By selection of suitable influencing factors (technician, nutrient media, sample preparation, temperature, duration) a high certainty of the determined method validation parameters is obtained, so that the number of required individual tests can be reduced by more than 50 %.

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
Public Enquiry End Date
04-Mar-2018
Publication Date
08-Oct-2020
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
08-Sep-2020
Due Date
13-Nov-2020
Completion Date
09-Oct-2020

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SLOVENSKI STANDARD
SIST EN ISO 16140-4:2020
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
SIST EN ISO 16140-4:2020 en

2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST EN ISO 16140-4:2020
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SIST EN ISO 16140-4:2020
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.
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SIST EN ISO 16140-4:2020
EN ISO 16140-4:2020 (E)
Contents Page

European foreword ....................................................................................................................................................... 3

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SIST EN ISO 16140-4:2020
EN ISO 16140-4:2020 (E)
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.
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SIST EN ISO 16140-4:2020
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SIST EN ISO 16140-4:2020
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
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SIST EN ISO 16140-4:2020
ISO 16140-4:2020(E)
COPYRIGHT PROTECTED DOCUMENT
© 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
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2020 – All rights reserved
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SIST EN ISO 16140-4:2020
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 2020 – All rights reserved iii
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SIST EN ISO 16140-4:2020
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

organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.

ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of

electrotechnical standardization.

The procedures used to develop this document and those intended for its further maintenance are

described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the

different types of ISO documents should be noted. This document was drafted in accordance with the

editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of

any patent rights identified during the development of the document will be in the Introduction and/or

on the ISO list of patent declarations received (see www .iso .org/ patents).

Any trade name used in this document is information given for the convenience of users and does not

constitute an endorsement.

For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and

expressions related to conformity assessment, as well as information about ISO’s adherence to the

World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT) see www .iso .org/

iso/ foreword .html.

This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC 9,

Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical

Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical

cooperation between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 16140 series can be found on the ISO website.

Any feedback or questions on this document should be directed to the user’s national standards body. A

complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2020 – All rights reserved
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SIST EN ISO 16140-4:2020
ISO 16140-4:2020(E)
Introduction
0.1 The ISO 16140 series

The ISO 16140 series has been expanded in response to the need for various ways to validate or verify

test methods. It is the successor to ISO 16140:2003. The ISO 16140 series consists of six parts with the

general title, Microbiology of the food chain — Method validation:
— Part 1: Vocabulary;

— Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method;

— Part 3: Protocol for the verification of reference methods and validated alternative methods in a single

laboratory;
— Part 4: Protocol for method validation in a single laboratory;

— Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods;

— Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation

and typing procedures.

ISO 17468 is a closely linked International Standard, which establishes technical rules for the

development and validation of standardized methods.
In general, two stages are needed before a method can be used in a laboratory.

— The first stage is the validation of the method. Validation is conducted using a study in a single

laboratory followed by an interlaboratory study (see ISO 16140-2, ISO 16140-5 and ISO 16140-6).

In the case when a method is validated within one laboratory (as described in this document), 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.”.

This document, 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. This document

and ISO 16140-5 can be used for validation against a reference method. This document (regarding

qualitative and quantitative methods) and ISO 16140-5 (regarding quantitative methods only) can also

be used for validation without a reference method.
© ISO 2020 – All rights reserved v
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SIST EN ISO 16140-4:2020
ISO 16140-4:2020(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.

ISO 16140-6 is somewhat different from the other parts in the ISO 16140 series in that it relates to

a very specific situation where only the confirmation procedure of a method is to be validated [e.g.

the biochemical confirmation of Enterobacteriaceae (see ISO 21528-2)]. The confirmation procedure

advances a suspected (presumptive) result to a confirmed positive result. The validation of alternative

typing techniques (e.g. serotyping of Salmonella) is also covered by ISO 16140-6. The validation study

in ISO 16140-6 clearly defines the selective agar(s) from which strains can be confirmed using the

alternative confirmation method. If successfully validated, the alternative confirmation method can

only be used if strains are recovered on an agar that was used and shown to be acceptable within the

validation study. Figure 2 shows the possibilities where an alternative confirmation method validated

in accordance with ISO 16140-6 can be applied (see text in the boxes).
vi © ISO 2020 – All rights reserved
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SIST EN ISO 16140-4:2020
ISO 16140-4:2020(E)
Figure 2 — Use of validated alternative confirmation methods (see ISO 16140-6)

EXAMPLE An example application of a validated alternative confirmation method is as follows.

An alternative confirmation method based on ELISA has been validated (in accordance with ISO 16140-6) 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 protocols in the ISO 16140 series

An interlaboratory validation study, in accordance with ISO 16140-2, requires at least eight laboratories

for quantitative methods and at least ten laboratories for qualitative methods. ISO 16140-5 is intended

to be used for interlaboratory studies comprising four to seven laboratories for quantitative methods

and four to nine laboratories for qualitative methods. ISO 16140-5 can only be used for non-proprietary

methods. Table 1 provides an overview of the different protocols.
© ISO 2020 – All rights reserved vii
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SIST EN ISO 16140-4:2020
ISO 16140-4:2020(E)

Table 1 — Overview of different validation protocols described in the ISO 16140 series

Number of participating
With reference method Without reference method
laboratories
This document: This document:

1 — factorial (see 5.1.1 and 5.2.1), or — factorial (see 5.1.2 and 5.2.2), or

— conventional (see 6.1.1 and 6.2.1) — conventional (see 6.1.2 and 6.2.2)

4 to 7 (quantitative method)/ ISO 16140-5: for non-proprietary ISO 16140-5: for non-proprietary

4 to 9 (qualitative method) methods only quantitative methods only
≥ 8 (quantitative method)/ ISO 16140-2: for the interlaboratory
Not applicable
≥ 10 (qualitative method) study part

The aim of this document is to assess the performance of detection or quantification methods within

a single laboratory, typically across a number of (food) categories and (food) types. Single-laboratory

validation of alternative methods for microbiological confirmation and typing procedures can also

be performed under certain conditions: the general principles are the same as those described in

ISO 16140-6 for the validation of alternative (proprietary) methods for microbiological confirmation

and typing procedures (except there is no interlaboratory study). Further information is given in

Annex G.

The protocols in this document only validate the method for the particular laboratory. A generalization

to other laboratories is not within the scope of these protocols. However, extension to other laboratories

is possible if this document is used as the first phase of validation of a reference method, to be followed

by an interlaboratory study as described in ISO 17468.

If a reference method is available, the validation of a method is conducted by comparing the alternative

method to the reference method. This allows inclusion of naturally contaminated samples in the

validation process and thus provides a more realistic picture of the performance of the method. If no

reference method is available, the validation process is based on samples with known contamination

levels only. This document provides protocols for both situations.

The general principles for single-laboratory validations of detection and quantification methods are the

same as those described in ISO 16140-2 for the validation of alternative (proprietary) methods against

a reference method. This document cannot be used without ISO 16140-1 or ISO 16140-2, as many

definitions and procedures are given in these International Standards. In addition to the validation

parameters described in ISO 16140-2, this document describes the calculation of in-house repeatability

and in-house reproducibility. Calculation of these parameters is not required if an interlaboratory study

is to be conducted after the single-laboratory validation (i.e. if the single-laboratory validation is only

the first phase of validation). Reliability of performance parameters obtained with this document is

comparable to ISO 16140-2. This also means that the workload associated with the technical protocols

for the single laboratory is comparable with the method comparison study of ISO 16140-2.

This document provides two strategies for the single-laboratory method validation of detection and

quantification methods. The first strategy is based on a factorial approach while the second strategy

uses the conventional approach derived from the protocols of ISO 16140-2. In addition, protocols for the

determination of the in-house reproducibility for quantitative methods are described.

The advantages of using a factorial approach, over the conventional approach, are that it takes into

account specific conditions that the laboratory encounters during routine testing and provides more

information on the factors (technicians, culture media, etc.) that vary within the laboratory across

relevant (food) items, while using fewer samples to assess the performance of the method. The factorial

approach offers assessment of the precision of quantitative methods. It allows computation of reliable

and representative single-laboratory method validation parameters such as in-house reproducibility

standard deviation, LOD or RLOD values because it provides information on the variability of these

values under different measurement conditions. The factorial approach requires fewer test results in

order to obtain similar or higher levels of reliability compared to the conventional approach.

viii © ISO 2020 – All rights reserved
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SIST EN ISO 16140-4:2020
INTERNATIONAL STANDARD ISO 16140-4:2020(E)
Microbiology of the food chain — Method validation —
Part 4:
Protocol for method validation in a single laboratory
1 Scope

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.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and

decimal dilutions for microbiological examination
© ISO 2020 – All rights reserved 1
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SIST EN ISO 16140-4:2020
ISO 16140-4:2020(E)

ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for

microbiological examinations

ISO 16140-1:2016, Microbiology of the food chain — Method validation — Part 1: Vocabulary

ISO 16140-2:2016, Microbiology of the food chain — Method validation — Part 2: Protocol for the validation

of alternative (proprietary) methods aga
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