kSIST FprEN ISO 16140-3:2020

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/FDIS 16140-3:2020)

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 von Lebensmitteln und Futtermitteln - Verfahrensvalidierung - Teil 3: Arbeitsvorschrift für die Prüfung von Referenz- und alternativer Verfahren in einem einzelnen Labor

Dieses Dokument legt die Arbeitsvorschriften fest, die bei der Verifizierung der Referenzverfahren und der validierten Alternativverfahren 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 sind auf andere (Mikro )Organismen oder deren Metaboliten anwendbar. 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 de méthodes de référence et alternatives validées appliquées dans un laboratoire (ISO/DIS 16140-3:2017)

Mikrobiologija v prehranski verigi - Validacija metode - 3. del: Protokol za preverjanje referenčnih in validiranih alternativnih metod, izvedenih v posameznem laboratoriju (ISO/FDIS 16140-3:2020)

Standards Content (sample)

SLOVENSKI STANDARD
oSIST prEN ISO 16140-3:2018
01-marec-2018
0LNURELRORJLMDYSUHKUDQVNLYHULJL9DOLGDFLMDPHWRGHGHO3URWRNRO]D
SUHYHUMDQMHUHIHUHQþQLKLQYDOLGLUDQLKDOWHUQDWLYQLKPHWRGL]YHGHQLKY
SRVDPH]QHPODERUDWRULMX,62',6

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

reference and validated alternative methods implemented in a single laboratory (ISO/DIS

16140-3:2017)

Mikrobiologie von Lebensmitteln und Futtermitteln - Verfahrensvalidierung - Teil 3:

Arbeitsvorschrift für die Prüfung von Referenz- und alternativer Verfahren in einem

einzelnen Labor

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

pour la vérification de méthodes de référence et alternatives validées appliquées dans

un laboratoire (ISO/DIS 16140-3:2017)
Ta slovenski standard je istoveten z: prEN ISO 16140-3
ICS:
07.100.30 Mikrobiologija živil Food microbiology
oSIST prEN ISO 16140-3:2018 en

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

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oSIST prEN ISO 16140-3:2018
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oSIST prEN ISO 16140-3:2018
DRAFT INTERNATIONAL STANDARD
ISO/DIS 16140-3
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2017-12-15 2018-03-09
Microbiology of the food chain — Method validation —
Part 3:
Protocol for the verification of reference and validated
alternative methods implemented in a single laboratory
Microbiologie de la chaîne alimentaire — Validation des méthodes —

Partie 3: Protocole pour la vérification de méthodes de référence et alternatives validées appliquées dans

un laboratoire
ICS: 07.100.30
THIS DOCUMENT IS A DRAFT CIRCULATED
This document is circulated as received from the committee secretariat.
FOR COMMENT AND APPROVAL. IT IS
THEREFORE SUBJECT TO CHANGE AND MAY
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IN ADDITION TO THEIR EVALUATION AS
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Reference number
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ISO/DIS 16140-3:2017(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
PROVIDE SUPPORTING DOCUMENTATION. ISO 2017
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oSIST prEN ISO 16140-3:2018
ISO/DIS 16140-3:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland

All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized otherwise in any form

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Contents

Foreword .......................................................................................................................................................................... 5

Introduction..................................................................................................................................................................... 6

1 Scope ............................................................................................................................................................................... 8

2 Normative references ............................................................................................................................................... 8

3 Terms and definitions .............................................................................................................................................. 8

4 Principle ..................................................................................................................................................................... 11

4.1 General ...................................................................................................................................................................... 11

4.2 Implementation verification ............................................................................................................................ 11

4.3 (Food) item verification ..................................................................................................................................... 12

4.4 Implementation verification and (food) item verification .................................................................. 12

4.5 Performance characteristics ............................................................................................................................ 15

5 Qualitative methods — Technical protocol for verification .................................................................... 15

5.1 Estimated LOD (eLOD ) determination .................................................................................................. 15

50 50

5.2 Experimental design ........................................................................................................................................... 15

5.3 Selection of (food) items .................................................................................................................................... 16

5.4 Artificial contamination ..................................................................................................................................... 16

5.4.1 Selection of strains ............................................................................................................................................... 16

5.4.2 Inoculation of the test portions ...................................................................................................................... 16

5.5 Results ....................................................................................................................................................................... 17

5.6 Acceptance criteria .............................................................................................................................................. 19

5.7 Root cause analysis .............................................................................................................................................. 19

6 Quantitative methods — Technical protocol for verification ............................................................ 20

6.1 Precision (intralaboratory reproducibility) determination ............................................................... 20

6.1.1 General ...................................................................................................................................................................... 20

6.1.2 Experimental design ........................................................................................................................................... 20

6.1.3 Selection of (food) item ...................................................................................................................................... 22

6.1.4 Natural contamination ....................................................................................................................................... 22

6.1.5 Artificial contamination ..................................................................................................................................... 22

6.1.6 Results ....................................................................................................................................................................... 23

6.1.7 Acceptance criteria .............................................................................................................................................. 24

6.1.8 Root cause analysis .............................................................................................................................................. 25

6.2 Estimated bias determination ......................................................................................................................... 26

6.2.1 General ...................................................................................................................................................................... 26

6.2.2 Experimental design ........................................................................................................................................... 26

6.2.3 Selection of (food) items .................................................................................................................................... 26

6.2.4 Artificial contamination ..................................................................................................................................... 26

6.2.5 Results ....................................................................................................................................................................... 28

6.2.6 Acceptance criteria .............................................................................................................................................. 28

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

7 Summary of acceptance criteria ................................................................................................................... 29

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

for verification studies.............................................................................................................................................. 30

Annex B (normative) —Requirements and guidance on how to choose challenging (food) item(s)

for (food) item verification ..................................................................................................................................... 47

B.1 General ...................................................................................................................................................................... 47

B.2 Matrix effects to consider .................................................................................................................................. 47

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B.2.1 Microbial characteristics ................................................................................................................................... 47

B.2.2 Physical and chemical characteristics ......................................................................................................... 47

B.2.3 Food process characteristics ........................................................................................................................... 48

B.3 Selection of (food) items for verification .................................................................................................... 48

Annex C (informative) — Protocol for QUALITATIVE method verification ........................................... 49

Annex D (informative) — Protocol for QUANTITATIVE method verification ....................................... 52

D.1 Protocol for precision (intralaboratory reproducibility) determination ...................................... 52

D.2 Protocol for estimated bias (eBias) determination ................................................................................ 53

Bibliography ................................................................................................................................................................. 56

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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 on 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 the following

URL: www.iso.org/iso/foreword.html.

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

Microbiology (Working Group WG 3, Method validation).
A list of all parts of the ISO 16140 series can be found on the ISO website.
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Introduction

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

test methods. It is the successor of ISO 16140:2003, Microbiology of food and animal feeding stuffs —

Protocol for the validation of alternative methods. ISO 16140 series consists of several 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 and validated alternative methods implemented in a

single laboratory
 Part 4: Protocol for single-laboratory (in-house) method validation

 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, Microbiology of the food chain — Technical requirements and guidance on establishment or

[2]

revision of a standardized reference method , is a closely linked International Standard. This

International Standard, which establishes technical rules for the development and validation of

standardized methods, is intended for the development of standardized methods by ISO/TC 34, Food

products, Subcommittee SC 9, Microbiology and CEN/TC 275/WG 6, Microbiology of the food chain.

In general, two stages are needed before a method can be used in a laboratory:

 The first stage is the validation of the method. This is either conducted in several laboratories

(parts 2 and 5 of ISO 16140) or in one laboratory (part 4 of ISO 16140).

 The second stage is method verification, where a laboratory demonstrates that it can satisfactorily

perform a validated method. This is described in part 3 of ISO 16140 (method verification). In

part 3, a separation is made between verification of (food) items that are included in the validation

study and (food) items that are not tested in the validation study but belong within the scope of

validation.

NOTE 1 Standardized reference methods (with and without published validation data) only require verification

before implementation in the laboratory.

NOTE 2 In this part of ISO 16140, the words ‘category’, ‘type’ and ‘item’ are sometimes combined with ‘food’ to

improve the readability of this document. However, the word ‘food’ is interchangeable with ‘feed’ and the other

areas of the food chain as mentioned in the Scope of ISO 16140-3.

Part 4 of ISO 16140 addresses validation within a single laboratory. The results are therefore only valid

in the laboratory which conducted the study. In this case, verification (part 3 of ISO 16140) is not

required.

Part 5 of ISO 16140 describes protocols for situations where a more rapid validation is required or

when the method to be validated is highly specialised, and, the number of participating laboratories

required by ISO 16140-2 cannot be reached.
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The flow chart in Figure 1 gives an overview of the links between the different parts mentioned above.

It also guides the users in selecting the right part of the ISO 16140 series, taking into account the

purpose of the study and the remarks given above. For this, it is important to distinguish between

'reference method' and 'standardized reference method'. A reference method is an internationally

recognized and widely accepted method (term 2.59 of ISO 16140-1:2016) and a standardized reference

method is a reference method described in a standard (term 3.5 of ISO 17468:2016). In the ISO 16140

series, reference method includes standardized reference method. The flow diagram acknowledges that

published validation data may not be available for some standardized reference methods.

Figure 1 — Flow chart for application of the different parts of the ISO 16140 series

Part 6 of ISO 16140, 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 validated. The

confirmation procedure advances a suspected (presumptive) result to a confirmed positive result. The

typing of pure strains (e.g. serotyping of Salmonella) is included in part 6 of ISO 16140.

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Microbiology of the food chain — Method validation — Part 3:
Protocol for the verification of reference and validated alternative
methods implemented in a single laboratory
1 Scope

This part of ISO 16140 describes the protocol for the verification of reference methods, standardized

reference methods and validated alternative methods for implementation in the user laboratory.

Method verification does not apply to non-validated alternative methods.

This part of ISO 16140 is applicable to the verification of methods used for the analysis (detection

and/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, and
 samples from the primary production stage.

This part of ISO 16140 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.

The verification focuses on those (food) items that are within the scope of validation and are tested in

the user laboratory.
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 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
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:
— IEC Electropedia: available at http://www.electropedia.org/
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— ISO Online browsing platform: available at http://www.iso.org/obp
3.1
bias
measurement bias

estimate of a systematic measurement error, or the systematic difference between the quantitative

assigned value and the average of measurement replicate results
[SOURCE: ISO 16140-1:2016, 2.9]
3.2
category
group of (food) types of the same origin
EXAMPLE Heat-processed milk and dairy products.
Note 1 to entry: The (food) categories are listed in Annex A of this document.
[SOURCE: ISO 16140-1:2016, 2.11, modified]
3.3
estimated bias (eBias)

determination of the bias based on the experimental design described in this document

Note 1 to entry: An accurate determination of the bias is not possible as the number of samples tested is small.

Therefore, the term estimated bias (eBias) is used in this document.
3.4
estimated LOD (eLOD )
50 50

determination of the LOD (level of detection at 50 % probability of detection) based on the

experimental design described in this document

Note 1 to entry: An accurate determination of the LOD is not possible as the number of samples tested is small.

Therefore, the term estimated LOD (eLOD ) is used.
50 50
Note 2 to entry: LOD50 is defined in ISO 16140-1:2016, 2.35.
3.5
item
single specified food, feed, environmental, or primary production matrix

EXAMPLE Food category: heat-processed milk and dairy products; food type: pasteurised dairy product;

food item: crème brûlée.
[SOURCE: ISO 16140-1:2016, 2.34]
3.6
laboratory sample

sample prepared for sending to the laboratory and intended for inspection or testing

[SOURCE: ISO 6887-1:2017, 3.1]
3.7
matrix (product)
all the components of the sample
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[SOURCE: ISO 16140-1:2016, 2.38]
3.8
reference material

material, sufficiently homogeneous and stable with respect to one or more specified properties, which

has been established to be fit for its intended use in a measurement process

Note 1 to entry: Properties can be quantitative or qualitative, e.g. identity of substances or species.

Note 2 to entry: Uses may include the calibration of a measurement system, assessment of a measurement

procedure, assigning values to other materials, and quality control.
[SOURCE: ISO Guide 30:2015, 2.1.1, modified]
3.9
scope of laboratory application

analytes, matrices, and concentrations for an analytical method that a user laboratory claims to be

capable of satisfactorily testing in its laboratory

Note 1 to entry: A method may have been validated to a broader range (scope) of analytes, matrices and

concentrations than the scope that will be claimed by a user laboratory. The scope of laboratory application

is ≤ the scope of validation.
3.10
scope of validation

analytes, matrices and concentrations for which a validated method of analysis can be used

satisfactorily
[SOURCE: ISO 16140-1:2016, 2.70]
3.11
test portion

measured (volume or mass) representative sample taken from the laboratory sample for use in the

preparation of the initial suspension

Note 1 to entry: Sometimes preparation of a test sample from the laboratory sample is required before the test

portion is taken, but this is infrequently used in microbiological examinations.
[SOURCE: ISO 6887-1:2017, 3.5, modified]
3.12
test sample

sample prepared from the laboratory sample according to the procedure specified in the test method

and from which test portions are taken

Note 1 to entry: Preparation of the laboratory sample before the test portion is taken is infrequently used in

microbiological examinations.
[SOURCE: ISO 6887-1:2017, 3.4, modified]
3.13
type

for a given category, a group of (food) items processed in a similar way, with similar intrinsic

characteristics and a similar microbial ecology

EXAMPLE Food category: heat-processed milk and dairy products; food type: pasteurised dairy product.

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[SOURCE: ISO 16140-1:2016, 2.78, modified]
3.14
user laboratory

laboratory which implements a validated alternative method and/or a reference method

3.15
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

4 Principle
4.1 General

Before performing method verification, it is crucial to refer to the published validation data for the

method in order to confirm the scope of validation and to select the appropriate (food) items.

The verification is undertaken in two parts:
 implementation verification;
 (food) item verification.

User laboratories intending to verify methods that have published validation data for comparison (this

includes all validated alternative methods) shall perform both the implementation and the (food) item

verification. Some reference methods may not have published validation data. For those methods, the

user laboratory will only perform (food) item verification.

Alternative methods that have not been validated shall first be validated, before a user laboratory can

verify its use in their laboratory (see Figure 1).
4.2 Implementation verification

Implementation verification aims to demonstrate the competence of the user laboratory to perform the

validated method. It compares user laboratory performances to those obtained during the validation.

Implementation verification applies only to the following methods with published validation data:

 reference methods; and
 validated alternative methods.
The user laboratory shall:
 review the published validation data for the method;

 select one (food) item tested during the validation study that belongs within the scope of laboratory

application of the user laboratory, if possible, and;

 use this (food) item and the sample size used in the validation study to perform implementation

verification.
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4.3 (Food) item verification

(Food) item verification applies to reference methods, with and without published validation data, and

validated alternative methods with validation data. The (food) item verification sets out to demonstrate

the competence of the user laboratory to perform the validated method with (food) items that are

tested in the user laboratory.
4.4 Implementation verification and (food) item verification

Table 1 provides guidance on when to use implementation verification and when to use (food) item

verification.
Table 1 — Implementation verification and (food) item verification
Method with published validation data Method without published validation data
Reference method Alternative method Reference method Alternative method
Implementation   Not applicable Not applicable
verification
(Food) item    Not applicable
verification

Not applicable: the method shall first be validated to ISO 16140-2 or ISO 16140-5 (see Figure 1) after which validation data

are available.

Figure 2a to Figure 2d show the number of (food) items required for implementation verification and

(food) item verification under different circumstances.

Figure 2a — (Food) items required when verifying a reference method with published validation

data or a validated alternative method for a “broad range of foods” scope
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Figure 2b — (Food) items required when verifying a reference method with published validation

data or a validated alternative method for a “limited range of f
...