FprEN ISO 16140-4

SLOVENSKI STANDARD
oSIST prEN ISO 16140-4:2018
01-marec-2018
Mikrobiologija v prehranski verigi - Validacija metode - 4. del: Protokol za
validacijo posamezne metode v laboratoriju (hišne metode) (ISO/DIS 16140-4:2017)

Microbiology of the food chain - Method validation - Part 4: Protocol for single-laboratory

Mikrobiologie von Lebensmitteln und Futtermitteln - Verfahrensvalidierung - Teil 4:

Arbeitsvorschrift für innerbetriebliche Einzellabor-Verfahrensvalidierung (ISO/DIS 16140-

Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 4: Protocole

pour la validation de méthodes internes dans un laboratoire (ISO/DIS 16140-4:2017)

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

All rights reserved. Unless otherwise specified, 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

Foreword .......................................................................................................................................................................... 4

Introduction..................................................................................................................................................................... 5

1 Scope ............................................................................................................................................................................... 9

2 Normative references ............................................................................................................................................... 9

3 Terms and definitions ........................................................................................................................................... 10

4 General principles of the single-laboratory method validation ............................................................ 12

4.1 General ................................................................................................................................................................................... 12

4.2 Principles for factorial approach ................................................................................................................................ 13

4.3 Principles for conventional approach ....................................................................................................................... 13

5 Factorial approach .................................................................................................................................................. 14

5.1 Qualitative methods ......................................................................................................................................................... 14

5.1.1 Single-laboratory method validation study against reference method ............................................. 14

5.1.2 Single-laboratory method validation study without a reference method ......................................... 19

5.2 Quantitative methods ...................................................................................................................................................... 21

5.2.1 Single-laboratory method validation study against a reference method .......................................... 21

5.2.2 Single-laboratory method validation study without a reference method ......................................... 24

6 Conventional approach ......................................................................................................................................... 25

6.1 Qualitative methods ......................................................................................................................................................... 25

6.1.1 Single-laboratory method validation study against reference method ............................................. 25

6.1.2 Single-laboratory method validation study without a reference method ......................................... 26

6.2 Quantitative methods ...................................................................................................................................................... 27

6.2.1 Single-laboratory method validation study against reference method ............................................. 27

6.2.2 Single-laboratory method validation study without a reference method ......................................... 29

Annex A (normative) — List of factors for factorial study design ............................................................. 32

Annex B (informative) — Single-laboratory precision study or qualitative methods ...................... 34

Annex C (informative) — Example of a single-laboratory method validation study for a

quantitative method against a reference method .......................................................................................... 36

C.1 Study design ........................................................................................................................................................................ 36

C.2 Calculations and summary of data ............................................................................................................................. 38

C.2.1 Summary of the results .......................................................................................................................................... 38

C.2.2 Relative trueness ...................................................................................................................................................... 39

C.2.2 Accuracy profile ......................................................................................................................................................... 40

C.2.3 Precision data ............................................................................................................................................................. 41

Annex D (informative) — Example of a single-laboratory method validation study for a qualitative

method against a reference method .................................................................................................................... 42

Annex E (informative) — Determination of precision in the case that the inoculum is unstable . 45

E.1 General ............................................................................................................................................................. 45

E.2 Adjustment of measurement values in the case of a linear trend ........................................... 45

E.3 Adjustment of measurement values by using a reference method ........................................ 46

Bibliography ................................................................................................................................................................. 47

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

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

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

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

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:

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

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

 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

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

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

ISO 17468, Microbiology of the food chain — Technical requirements and guidance on establishment or

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

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

 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

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

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

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.

An interlaboratory study, according to ISO 16140-2 (proprietary methods), requires at least

8 laboratories for quantitative methods and 10 laboratories for qualitative methods. ISO 16140-5 is

intended to be used for interlaboratory studies comprising 4-7 laboratories for quantitative methods and

4-9 laboratories for qualitative methods. ISO 16140-5 can only be used for non-proprietary methods.

1 Part 4 of ISO 16140: factorial or conventional Part 4 of ISO 16140: factorial or conventional

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

≥ 8 (quantitative method)/ Part 2 of ISO 16140 (for the interlaboratory Not available

The aim of the single-laboratory validation studies described in this part of ISO 16140 is to assess the

performance of a method within a single laboratory, typically across a number of (food) categories and

(food) types. The protocols in this part of ISO 16140 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 ISO 16140-4 is used as the first phase of validation, followed

The general principles and concepts for single-laboratory validations are the same as those described in

ISO 16140-2 for the validation of alternative (proprietary) methods against a reference method. Part 4

cannot be used without ISO 16140-2, as many definitions and procedures are given in part 2 of ISO 16140.

In addition to the validation parameters described in ISO 16140-2, part 4 of ISO 16140 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

This part of ISO 16140 provides two strategies for the single-laboratory method validation, using one or

more strains of the target organism. The first strategy is based on a factorial plan while the second

strategy provides method comparison designs derived from the protocols of ISO 16140-2 together with

protocols for the determination of the in-house reproducibility. Protocols are provided for qualitative

Factorial experiments require more experimental control and planning, but involve a smaller number of

experiments compared to the conventional approach, while at the same time providing more information

about the sources of variation. The factorial design offers several advantages. Factorial approach takes

into account the conditions a laboratory encounters during routine testing and provides more

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

(food) matrices, while using fewer samples to assess the performance of the method. In short, it allows

greater efficiency: fewer test results are required in order to obtain comparable levels of reliability.

Different (food) types are included and all identified influence factors are explicitly taken into

consideration and systematically varied across their respective ranges. The design offers assessment of

the precision of quantitative methods. It allows computation of reliable and representative single-

laboratory validation parameters such as in-house reproducibility standard deviation, LOD or RLOD

values because it provides information on the variability of these values under different measurement

In short, it allows greater efficiency: fewer test results are required in order to obtain comparable levels

If a reference method is available, the validation of a method is conducted by comparing the 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 artificially contaminated samples only. Part 4 of ISO 16140

This part of ISO 16140 describes the protocols for single-laboratory validation of methods for

microbiology in the food chain. The protocols in this part of ISO 16140 only validate the method for the

This part of ISO 16140 is applicable to single-laboratory validation of methods used in the analysis

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.

Single-laboratory validation is required if an interlaboratory validation according to ISO 16140-2 is not

 extension of the scope of an ISO 16140-2 validated method: e.g. category extension or test portion

Within ISO 17468 , single-laboratory validation is the first step in the standardization of a method. It

can be applied only for methods that are fully specified with regard to all relevant parameters (including

tolerances on temperatures and specifications on culture media) and which have already been optimised.

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 (series), Microbiology of the food chain — Preparation of test samples, initial suspension and

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

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

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

group of settings which have to be conducted in parallel or in a short time interval, and which are used

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

Note 1 to entry: In this part of ISO 16140, only those factors that are in line with the prescription of the method are

Note 1 to entry: In this part of ISO 16140, each factor is varied at two factor levels, ‘a’ and ‘b’.

measurement precision under a set of in-house repeatability conditions of measurement in a particular

Note 1 to entry: In-house repeatability conditions include the same measurement procedure, same technicians,

same measuring system, same operating conditions and same location, and replicate measurements on the same or

measurement precision under a set of in-house reproducibility conditions of measurement in a particular

Note 1 to entry: In-house reproducibility conditions include different technicians, operating conditions, and

replicate measurements on the same or similar objects over a longer period of time in a particular laboratory.

‹qualitative methods› measured analyte concentration, obtained by a given measurement procedure, for

EXAMPLE LOD is the level of detection for which 50 % of tests give a positive result.

Note 1 to entry: The term 'level of detection' is used for qualitative methods in microbiology based on replicate

analyses with three different inoculation levels of the target analyte in a tested matrix. The replicates are analysed,

and the number of positive results is recorded (e.g. 20 %, 70 % and 100 %) respectively at each inoculation level.

These data are then used to determine the number of cells that would give 50 % positive using a generalised linear

model (see ISO 16140-2). This differs from the procedure used for chemical and physical methods for which a 'limit

of detection' is defined as the lowest quantity of an analyte that can be distinguished from the absence of that analyte

‹quantitative methods› the lowest analyte concentration that can be quantified with an acceptable level

proportion of positive analytical outcomes for a qualitative method for a given matrix at a given analyte

Note 1 to entry: For qualitative methods, POD represents the probability of detection.

level of detection at P = 0,50 (LOD ) of the alternative (proprietary) method divided by the level of

Note 1 to entry: For purposes of alternative-method acceptance, the derived RLOD is checked with the acceptability

establishment of the performance characteristics of a method for the one particular laboratory in which

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

A single-laboratory method validation study is the first step in the framework of general method

validation and is needed to assess the performance of the method across a large number of (food) types

and (food) items. The second step is an interlaboratory study to assess the performance of the method

across the required number of laboratories. A single-laboratory method validation study is used to

demonstrate the performance of the method in the laboratory that conducted the study. The results are

This part of ISO 16140 describes two approaches for single-laboratory validation.

Validation procedures are dependent on whether the method is qualitative or quantitative, and on