SIST EN ISO 16140-4:2020

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.

---------------------- Page: 1 ----------------------
SIST EN ISO 16140-4:2020

---------------------- Page: 2 ----------------------
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.

---------------------- Page: 3 ----------------------
SIST EN ISO 16140-4:2020
EN ISO 16140-4:2020 (E)
Contents Page
European foreword . 3

2

---------------------- Page: 4 ----------------------
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.

3

---------------------- Page: 5 ----------------------
SIST EN ISO 16140-4:2020

---------------------- Page: 6 ----------------------
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

---------------------- Page: 7 ----------------------
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

---------------------- Page: 8 ----------------------
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
50
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
50
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

---------------------- Page: 9 ----------------------
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

---------------------- Page: 10 ----------------------
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

---------------------- Page: 11 ----------------------
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

---------------------- Page: 12 ----------------------
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

---------------------- Page: 13 ----------------------
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
50
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

---------------------- Page: 14 ----------------------
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

---------------------- Page: 15 ----------------------
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
...