Microbiology of the food chain - Method validation - Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods (ISO 16140-5:2020)

The proposed deliverable specifies an alternative technical protocol for the validation of mostly non-proprietary methods in the field of microbiological analysis of food, animal feed, and environmental and primary production stage samples.
It is closely related to ISO 16140-2. The latter specifies the technical protocol for the validation of proprietary methods, including a classical interlaboratory study and a method comparison study to be conducted in one laboratory. The realization of classical interlaboratory studies demands a sufficient number of participating laboratories (at least 8 laboratories are required). There are many occasions where a sufficient number of participating laboratories is not available (e.g. when a new method is required quickly after an outbreak of a new microorganism). In this case, the validation cannot be considered as reliable any longer.
The proposed deliverable uses a modified protocol based on orthogonal, factorial studies. 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 collaborating laboratories can be reduced up to a minimum of 4.
This validation protocol can be used in different ways. If the 4 collaborators can be considered a “random sample” of independent and competent laboratories and from different organizations, the test method can be considered as being validated in the sense that accurate and precise measurements are to be expected from any competent laboratory. If the 4 collaborators can be considered a “random sample” of independent and competent laboratories from one organization, the test method can be considered as being validated in the sense that accurate and precise measurements are to be expected from any competent laboratory in this organization.

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 5: Arbeitsvorschrift für eine faktorielle Ringversuch-Verfahrensvalidierung (ISO 16140-5:2020)

Dieses Dokument legt (auf der Grundlage von orthogonalen, faktoriellen Untersuchungen) die allgemeinen Grundsätze und technischen Arbeitsvorschriften für die Validierung nicht urheberrechtlich geschützter Verfahren in der Mikrobiologie der Lebensmittelkette fest.
Dieses Dokument ist anwendbar auf die 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.
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.
Dieses Dokument legt Arbeitsvorschriften für die Validierung mit Bezug auf ein Referenzverfahren sowohl für quantitative als auch qualitative Verfahren fest. Darüber hinaus stellt dieses Dokument eine Arbeitsvorschrift für die Validierung von quantitativen Verfahren ohne Bezug auf ein Referenzverfahren bereit. Qualitative Verfahren können nach diesem Dokument ohne Bezug auf ein Referenzverfahren nicht validiert werden.
ANMERKUNG ISO 16140 2 legt das allgemeine Prinzip und die technische Arbeitsvorschrift für die Validierung alternativer, zum Großteil urheberrechtlich geschützter Verfahren mit Bezug auf ein Referenzverfahren fest.
Dieses Dokument ist nur für die Validierung solcher Verfahren anwendbar, bei denen alle relevanten Parameter vollständig angegeben sind (einschließlich der Toleranzen zu Temperaturen und der Festlegungen zu Nährmedien) und die bereits optimiert wurden.
Verfahren, die nach diesem Dokument validiert wurden, können von den Laboren verwendet werden, die zu der festgelegten Grundgesamtheit der Labore gehören.

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 5: Protocole pour la validation interlaboratoires de méthodes non commerciales par plan factoriel (ISO 16140-5:2020)

Le présent document établit les principes généraux ainsi que les protocoles techniques (fondé sur des études factorielles orthogonales) de validation de méthodes non commerciales, dans le domaine de la microbiologie de la chaîne alimentaire.
Le présent document est applicable à la validation 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.
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.
Le présent document spécifie des protocoles pour la validation de méthodes quantitatives et qualitatives par rapport à une méthode de référence. Il établit également un protocole pour la validation de méthodes quantitatives sans méthode de référence. Les méthodes qualitatives ne peuvent pas être validées sans méthode de référence conformément au présent document.
NOTE       L'ISO 16140‑2 spécifie le principe général et le protocole technique pour la validation de méthodes alternatives, majoritairement commerciales, par rapport à une méthode de référence.
Le présent document est uniquement applicable à la validation de méthodes dont les paramètres pertinents sont intégralement spécifiés (notamment les tolérances sur les températures et les spécifications sur les milieux de culture) et qui ont déjà été optimisées.
Les méthodes qui ont été validées conformément au présent document peuvent être utilisées par les laboratoires de la population de laboratoires spécifiée.

Mikrobiologija v prehranski verigi - Validacija metode - 5. del: Protokol za medlaboratorijsko validacijo nelastniških metod (ISO 16140-5:2020)

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 16140-5:2020
01-november-2020
Mikrobiologija v prehranski verigi - Validacija metode - 5. del: Protokol za
medlaboratorijsko validacijo nelastniških metod (ISO 16140-5:2020)

Microbiology of the food chain - Method validation - Part 5: Protocol for factorial

interlaboratory validation for non-proprietary methods (ISO 16140-5:2020)

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 5: Arbeitsvorschrift für

eine faktorielle Ringversuch-Verfahrensvalidierung (ISO 16140-5:2020)

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 5: Protocole

pour la validation interlaboratoires de méthodes non commerciales par plan factoriel

(ISO 16140-5:2020)
Ta slovenski standard je istoveten z: EN ISO 16140-5:2020
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16140-5:2020 en

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

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SIST EN ISO 16140-5:2020
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SIST EN ISO 16140-5:2020
EN ISO 16140-5
EUROPEAN STANDARD
NORME EUROPÉENNE
August 2020
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Method validation - Part 5:
Protocol for factorial interlaboratory validation for non-
proprietary methods (ISO 16140-5:2020)

Microbiologie de la chaîne alimentaire - Validation des Mikrobiologie der Lebensmittelkette -

méthodes - Partie 5: Protocole pour la validation Verfahrensvalidierung - Teil 5: Arbeitsvorschrift für

interlaboratoires de méthodes non commerciales par eine faktorielle Ringversuch-Verfahrensvalidierung

plan factoriel (ISO 16140-5:2020) (ISO 16140-5:2020)
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 16140-5:2020 E

worldwide for CEN national Members.
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SIST EN ISO 16140-5:2020
EN ISO 16140-5:2020 (E)
Contents Page

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

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SIST EN ISO 16140-5:2020
EN ISO 16140-5:2020 (E)
European foreword

This document (EN ISO 16140-5:2020) 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 16140-5:2020 has been approved by CEN as EN ISO 16140-5:2020 without any

modification.
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SIST EN ISO 16140-5:2020
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SIST EN ISO 16140-5:2020
INTERNATIONAL ISO
STANDARD 16140-5
First edition
2020-07
Microbiology of the food chain —
Method validation —
Part 5:
Protocol for factorial interlaboratory
validation for non-proprietary
methods
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 5: Protocole pour la validation interlaboratoires de méthodes
non commerciales par plan factoriel
Reference number
ISO 16140-5:2020(E)
ISO 2020
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SIST EN ISO 16140-5:2020
ISO 16140-5:2020(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 16140-5:2020
ISO 16140-5:2020(E)
Contents Page

Foreword ........................................................................................................................................................................................................................................iv

Introduction ..................................................................................................................................................................................................................................v

1 Scope ................................................................................................................................................................................................................................. 1

2 Normative references ...................................................................................................................................................................................... 1

3 Terms and definitions ..................................................................................................................................................................................... 2

4 General principles for the factorial interlaboratory validation of non-proprietary

methods ......................................................................................................................................................................................................................... 3

4.1 General ........................................................................................................................................................................................................... 3

4.2 Validation against a reference method ............................................................................................................................... 3

4.3 Validation without a reference method ............................................................................................................................. 3

5 Qualitative methods — Technical protocol for factorial interlaboratory validation ....................3

5.1 In-house validation study .............................................................................................................................................................. 3

5.2 Interlaboratory validation study against a reference method ....................................................................... 4

5.2.1 General considerations ............................................................................................................................................... 4

5.2.2 Measurement protocol ............................................................................................................................................... 4

5.2.3 Selection of the factors to be studied ............................................................................................................. 5

5.2.4 Experimental design ..................................................................................................................................................... 6

5.3 Calculations and summary of data ......................................................................................................................................... 6

5.4 Interpretation of data ........................................................................................................................................................................ 8

5.4.1 Paired study ......................................................................................................................................................................... 8

5.4.2 Unpaired study .................................................................................................................................................................. 9

5.4.3 Analysis of factorial effects with respect to RLOD .............................................................................. 9

6 Quantitative methods — Technical protocol for factorial interlaboratory validation .............10

6.1 In-house validation study ...........................................................................................................................................................10

6.2 Interlaboratory validation study against a reference method ....................................................................10

6.2.1 General considerations ............................................................................................................................................10

6.2.2 Measurement protocol ............................................................................................................................................10

6.2.3 Experimental design ..................................................................................................................................................11

6.3 Calculations, summary, and interpretation of data ..............................................................................................12

6.3.1 Summary of test results ..........................................................................................................................................12

6.3.2 Precision data ..................................................................................................................................................................13

6.3.3 Accuracy profile .............................................................................................................................................................14

6.4 Interlaboratory validation study without a reference method ..................................................................14

Annex A (informative) List of factors and factor levels for factorial validation....................................................15

Annex B (informative) Example of a factorial interlaboratory study for a quantitative method .......17

Annex C (informative) Example of a factorial interlaboratory study for a qualitative method ...........28

Bibliography .............................................................................................................................................................................................................................34

© ISO 2020 – All rights reserved iii
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SIST EN ISO 16140-5:2020
ISO 16140-5:2020(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 16140-5:2020
ISO 16140-5:2020(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 16140-6, 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. This document, 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 this

document can be used for validation against a reference method. ISO 16140-4 (regarding qualitative

and quantitative methods) and this document (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 16140-5:2020
ISO 16140-5:2020(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 16140-5:2020
ISO 16140-5:2020(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.

This document provides a protocol that addresses the special case where the number of laboratories

required in an interlaboratory validation of a method by ISO 16140-2 cannot be achieved. The protocol

allows a method validation based on a minimum of four laboratories. It applies, for example, in situations

where there is an urgent need for a validated method but the in-house and interlaboratory studies in

accordance with ISO 16140-2 take too long to complete. This document also addresses the problem of

method validation of highly specialized methods, for which only a few laboratories might be available

for a validation study. This document can only be used for non-proprietary methods. Table 1 provides

an overview of the different protocols.
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SIST EN ISO 16140-5:2020
ISO 16140-5:2020(E)

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

Number of participating
With reference method Without reference method
laboratories
1 ISO 16140-4 ISO 16140-4

4 to 7 (quantitative method)/ This document: for non-proprietary This document: 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

In order to reduce the number of required laboratories to a minimum of four, while maintaining the

applicability of the validation to all laboratories, the protocol is based on a factorial experimental design.

In the factorial design, factors such as the technician or culture medium are altered simultaneously,

and the method is used in a range of different factor settings. This approach allows a more detailed

analysis of the precision parameters of the method while, at the same time, requiring a smaller number

of laboratories and tests.
viii © ISO 2020 – All rights reserved
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SIST EN ISO 16140-5:2020
INTERNATIONAL STANDARD ISO 16140-5:2020(E)
Microbiology of the food chain — Method validation —
Part 5:
Protocol for factorial interlaboratory validation for non-
proprietary methods
1 Scope

This document specifies the general principles and the technical protocols (based on orthogonal,

factorial studies) for the validation of non-proprietary methods for microbiology of the food chain.

This document is applicable to the validation of methods used for the analysis (detection or

quantification) of microorganisms in:
— products intended for human consumption;
— products intended for animal feeding;
— environmental samples in the area of food and feed production, handling;
— samples from the primary production stage.

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.

This document specifies protocols for the validation against a reference method for both quantitative

and qualitative methods. This document also provides a protocol for the validation of quantitative

methods without a reference method. Qualitative methods cannot be validated without a reference

method in accordance with this document.

NOTE ISO 16140-2 specifies the general principle and the technical protocol for the validation of alternative,

mostly proprietary, methods against a reference method.

This document is only applicable to the validation of 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.

Methods that have been validated in accordance with this document can be used by the laboratories of

the specified population of laboratories.
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 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for

microbiological examinations

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

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

of alternative (proprietary) methods against a reference method
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SIST EN ISO 16140-5:2020
ISO 16140-5:2020(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
factor

qualitative or quantitative parameter within the method that can be varied at two or more levels within

the limits of the specified method
EXAMPLE Technician.

Note 1 to entry: In this document, only those factors that are in line with the protocol of the method are

considered.
3.2
factor level
value of the factors (3.1) within the experimental design
EXAMPLE Technician “a”, technician “b”, etc.

Note 1 to entry: In this document, each factor is varied at two factor levels: “a” and “b”.

Note 2 to entry: This definition is based on how ISO 3534-3:2013, 3.1.12, defines “factor level”. In ISO 3534-3:2013,

3.1.12, the definition is more general, but the statistical meaning is the same.
3.3
orthogonal design

factorial design, in which for every pair of factors (3.1), each combination of factor levels (3.2) occurs the

same number of times across the possible factor levels

Note 1 to entry: This definition is based on how ISO 3534-3:2013, 3.1.31, defines “orthogonal array”, but for

“orthogonal design”, a more general and more theoretical definition is used.
3.4
reference value

estimated concentration level obtained with the reference method

3.5
reference value

estimated concentration

level obtained from the concentration level of the inoculum
3.6
setting
combination of factor levels (3.2)
EXAMPLE Technician “a” + culture media “b” + temperature “a” + etc.

Note 1 to entry: These conditions can be described by the combination of levels of factors varied within the study.

2 © ISO 2020 – All rights reserved
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SIST EN ISO 16140-5:2020
ISO 16140-5:2020(E)
4 General principles for the factorial interlaboratory validation of non-
proprietary methods
4.1 General

This document uses a protocol based on orthogonal, factorial studies. A high certainty of the determined

method validation parameters is achieved by focusing on suitable factors (e.g. technician, culture media,

sample preparation, temperature, test duration) that can influence the test
...

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