Microbiology of the food chain - Method validation - Part 3: Protocol for the verification of reference methods and validated alternative methods in a single laboratory (ISO 16140-3:2021)

This document deals with verification of methods for the detection and/or the enumeration of microorganisms, with particular emphasis on the implementation of a reference/alternative method in the user laboratory and verification of a reference/alternative method using items included in the scope of the method and tested routinely but not tested in the original validation study

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 3: Arbeitsvorschrift für die Verifizierung von Referenz- und alternativen Verfahren in einem einzelnen Labor (ISO 16140-3:2021)

Dieses Dokument legt die Arbeitsvorschrift fest, die bei der Verifizierung von Referenzverfahren und von validierten alternativen Verfahren zur Umsetzung im Anwenderlabor eingesetzt werden.
Dieses Dokument ist anwendbar auf die Verifizierung von Verfahren zur Untersuchung (Nachweis bzw. quantitative Bestimmung), Bestätigung und Typisierung 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 gilt insbesondere für Bakterien und Pilze. Einige Abschnitte können auf andere (Mikro )Organismen oder deren Metaboliten angewendet werden. Die Bestimmung muss im Einzelfall erfolgen.
Die technischen Arbeitsvorschriften für die Verifizierung validierter qualitativer Verfahren und validierter quantitativer Verfahren sind in Abschnitt 5 und Abschnitt 6 beschrieben. Die technische Arbeitsvorschrift für die Verifizierung von validierten alternativen Bestätigungs- und Typisierungsverfahren ist in Abschnitt 7 beschrieben. Die Arbeitsvorschriften für die Verifizierung von nicht validierten Referenzverfahren werden in Anhang F beschrieben.

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 3: Protocole pour la vérification dans un seul laboratoire de méthodes de référence et de méthodes alternatives validées (ISO 16140-3:2021)

Le présent document spécifie le protocole pour la vérification de méthodes de référence et de méthodes alternatives validées destinées à être mises en œuvre dans le laboratoire utilisateur.
Le présent document est applicable à la vérification de méthodes utilisées pour l'analyse (recherche et/ou quantification), la confirmation et le typage de micro-organismes présents dans:
—          les produits destinés à la consommation humaine;
—          les produits destinés à l'alimentation animale;
—          les échantillons environnementaux dans le domaine de la production et de la manutention de produits alimentaires;
—          les échantillons au stade de la production primaire.
Le présent document est notamment applicable aux bactéries et aux moisissures. Certains articles peuvent être applicables à d'autres (micro-)organismes ou à leurs métabolites, qui doivent être déterminés au cas par cas.
Les protocoles techniques pour la vérification de méthodes qualitatives validées et de méthodes quantitatives validées sont décrits aux Articles 5 et 6. Le protocole technique pour la vérification de méthodes alternatives validées de confirmation et de typage est décrit à l'Article 7. Les protocoles pour la vérification de méthodes de référence non validées sont décrits à l'Annexe F.

Mikrobiologija v prehranski verigi - Validacija metode - 3. del: Protokol za preverjanje referenčnih in validiranih alternativnih metod, izvedenih v posameznem laboratoriju (ISO 16140-3:2021)

General Information

Status
Published
Public Enquiry End Date
04-Mar-2018
Publication Date
02-Mar-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
11-Feb-2021
Due Date
18-Apr-2021
Completion Date
03-Mar-2021

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SLOVENSKI STANDARD
SIST EN ISO 16140-3:2021
01-april-2021
Mikrobiologija v prehranski verigi - Validacija metode - 3. del: Protokol za
preverjanje referenčnih in validiranih alternativnih metod, izvedenih v
posameznem laboratoriju (ISO 16140-3:2021)

Microbiology of the food chain - Method validation - Part 3: Protocol for the verification of

reference methods and validated alternative methods in a single laboratory (ISO 16140-

3:2021)

Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 3: Arbeitsvorschrift für

die Verifizierung von Referenz- und alternativen Verfahren in einem einzelnen Labor

(ISO 16140-3:2021)

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 3: Protocole

pour la vérification dans un seul laboratoire de méthodes de référence et de méthodes

alternatives validées (ISO 16140-3:2021)
Ta slovenski standard je istoveten z: EN ISO 16140-3:2021
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16140-3:2021 en

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

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SIST EN ISO 16140-3:2021
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SIST EN ISO 16140-3:2021
EN ISO 16140-3
EUROPEAN STANDARD
NORME EUROPÉENNE
February 2021
EUROPÄISCHE NORM
ICS 07.100.30
English Version
Microbiology of the food chain - Method validation - Part 3:
Protocol for the verification of reference methods and
validated alternative methods in a single laboratory (ISO
16140-3:2021)

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

méthodes - Partie 3: Protocole pour la vérification dans Verfahrensvalidierung - Teil 3: Arbeitsvorschrift für die

un seul laboratoire de méthodes de référence et de Verifizierung von Referenz- und alternativen Verfahren

méthodes alternatives validées (ISO 16140-3:2021) in einem einzelnen Labor (ISO 16140-3:2021)

This European Standard was approved by CEN on 28 December 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

© 2021 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140-3:2021 E

worldwide for CEN national Members.
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SIST EN ISO 16140-3:2021
EN ISO 16140-3:2021 (E)
Contents Page

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

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SIST EN ISO 16140-3:2021
EN ISO 16140-3:2021 (E)
European foreword

This document (EN ISO 16140-3:2021) 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 August 2021, and conflicting national standards shall

be withdrawn at the latest by August 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 16140-3:2021 has been approved by CEN as EN ISO 16140-3:2021 without any

modification.
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SIST EN ISO 16140-3:2021
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SIST EN ISO 16140-3:2021
INTERNATIONAL ISO
STANDARD 16140-3
First edition
2021-01
Microbiology of the food chain —
Method validation —
Part 3:
Protocol for the verification of
reference methods and validated
alternative methods in a single
laboratory
Microbiologie de la chaîne alimentaire — Validation des méthodes —
Partie 3: Protocole pour la vérification dans un seul laboratoire de
méthodes de référence et de méthodes alternatives validées
Reference number
ISO 16140-3:2021(E)
ISO 2021
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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2021

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 2021 – All rights reserved
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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

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

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

3 Terms and definitions ..................................................................................................................................................................................... 1

4 General principles of verification of qualitative (detection) methods and

quantification methods ................................................................................................................................................................................. 5

4.1 General ........................................................................................................................................................................................................... 5

4.2 Implementation verification ....................................................................................................................................................... 5

4.3 (Food) item verification .................................................................................................................................................................. 6

4.4 Requirements for implementation verification and (food) item verification ......... .......................... 6

4.5 Performance characteristics........................................................................................................................................................ 9

5 Qualitative methods — Technical protocol for verification .................................................................................... 9

5.1 Estimated LOD (eLOD ) determination ..................................................................................................................... 9

50 50

5.2 Experimental design .......................................................................................................................................................................... 9

5.3 Selection of (food) items ..............................................................................................................................................................10

5.4 Artificial contamination ...............................................................................................................................................................10

5.4.1 Selection of strains ......................................................................................................................................................10

5.4.2 Inoculation of the test portions ........................................................................................................................11

5.5 Evaluation of results ........................................................................................................................................................................13

5.5.1 Determination of eLOD using protocol 1 ............................................................................................13

5.5.2 Determination of eLOD using protocol 2 ............................................................................................16

5.5.3 Use of protocol 3 ...........................................................................................................................................................17

5.6 Acceptability limits ...........................................................................................................................................................................18

5.7 Root cause analysis ..........................................................................................................................................................................18

6 Quantitative methods — Technical protocol for verification ..............................................................................19

6.1 Intralaboratory reproducibility standard deviation determination ......................................................19

6.1.1 General...................................................................................................................................................................................19

6.1.2 Experimental design ..................................................................................................................................................19

6.1.3 Selection of the (food) item .................................................................................................................................21

6.1.4 Natural contamination .............................................................................................................................................21

6.1.5 Artificial contamination ..........................................................................................................................................21

6.1.6 Evaluation of results ..................................................................................................................................................22

6.1.7 Acceptability limit ........................................................................................................................................................23

6.1.8 Root cause analysis .....................................................................................................................................................24

6.2 Estimated bias (eBias) determination .............................................................................................................................25

6.2.1 General...................................................................................................................................................................................25

6.2.2 Experimental design ..................................................................................................................................................25

6.2.3 Selection of (food) items ........................................................................................................................................25

6.2.4 Artificial contamination ..........................................................................................................................................25

6.2.5 Evaluation of results ..................................................................................................................................................27

6.2.6 Acceptability limit ........................................................................................................................................................27

6.2.7 Root cause analysis .....................................................................................................................................................28

7 Validated alternative confirmation and typing methods — Technical protocol for

verification...............................................................................................................................................................................................................28

7.1 General ........................................................................................................................................................................................................28

7.2 Implementation verification ....................................................................................................................................................28

7.3 Experimental design .......................................................................................................................................................................28

7.3.1 General...................................................................................................................................................................................28

7.3.2 Strain selection ...............................................................................................................................................................29

7.4 Evaluation of results ........................................................................................................................................................................29

7.5 Acceptability limit .............................................................................................................................................................................30

© ISO 2021 – All rights reserved iii
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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)

7.6 Root cause analysis ..........................................................................................................................................................................30

8 Summary of acceptability limits for the verification of validated methods ........................................30

Annex A (informative) Classification of (food) categories and suggested target

combinations for verification studies .........................................................................................................................................31

Annex B (informative) Guidance on how to choose challenging (food) item(s) for (food)

item verification .................................................................................................................................................................................................45

Annex C (informative) Qualitative method verification — Example ................................................................................47

Annex D (informative) Quantitative method verification — Example ............................................................................55

Annex E (informative) Validated alternative confirmation or typing method verification —

Examples ....................................................................................................................................................................................................................60

Annex F (normative) Protocol for the verification of non-validated reference methods

in a single laboratory ....................................................................................................................................................................................63

Bibliography ............................................................................................................................................................................................................................70

iv © ISO 2021 – All rights reserved
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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(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.
© ISO 2021 – All rights reserved v
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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(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 (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 this document (i.e. 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 this document) 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.
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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(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 1 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.

NOTE 2 The general principle for method verification is that the method to be verified (either alternative or

reference) has been validated. However, some reference methods (including ISO or CEN standards) are not yet

(fully) validated. For verification of these methods, the protocols are described in Annex F.

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).
© ISO 2021 – All rights reserved vii
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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(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 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 Verification versus validation
ISO 16140-1:2016 defines the terms for validation and verification, as follows:

— validation: establishment of the performance characteristics of a method and provision of objective

evidence that the performance requirements for a specified intended use are fulfilled;

— verification: demonstration that a validated method performs, in the user’s hands, according to the

method’s specifications determined in the validation study and is fit for its intended purpose.

NOTE 1 The user’s hand means the user laboratory.
Method verification applies to methods that are:

— reference methods, including ISO or CEN standards, that are validated using at least an

interlaboratory study;
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SIST EN ISO 16140-3:2021
ISO 16140-3:2021(E)

NOTE 2 However, some reference methods (including ISO or CEN standards) are not yet (fully) validated.

For verification of these methods, the protocols are described in Annex F.

— alternative methods, proprietary or otherwise, when the validation included an interlaboratory

study. The method has been validated in accordance with
— ISO 16140-2 for alternative (proprietary) methods,
— ISO 16140-5 for non-proprietary methods, or
— ISO 16140-6 for alternative (proprietary) confirmation and typing methods.

In a validation study, it is not possible to test all existing foods; the diversity and number of samples

used in any validation study is limited. In most cases, the validation is based on five different food

categories (categories as defined in ISO 16140-1:2016, 2.11, and specified in ISO 16140-2:2016, Annex A).

Sometimes the validation is supplemented with additional (other) categories such as pet food and

animal fee
...

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