Guidelines for the validation of qualitative screening methods for the detection of residues of veterinary drugs in milk and milk products (ISO/TS 23758:2021)

This document describes general workflows and protocols for the validation and the verification
of qualitative screening tests for the detection of residues of veterinary drugs in liquid milk (raw,
pasteurized, UHT and reconstituted milk powders and whey protein extracts) including biological
methods. This guideline does not cover the validation of residue analysis by HPLC, UHPLC or LC-MS/MS.
This document is intended to be useful for manufacturers of screening test kits, laboratories validating
screening methods or tests, competent authorities and dairies or end users of reagents or tests for the
detection of veterinary drug residues in milk products. This document facilitates and improves the
validation and verification of screening methods. The goals of this document are a harmonization in
validation of methods or test kits in order for all stakeholders to have full trust in the result of residue
screening and to limit the overlap and multiplication of validation work in different laboratories by
sharing the validation results generated by an independent laboratory. Furthermore, a harmonized
validation and verification procedure allows for comparison of the performance of different screening
methods.
This document does not imply that all end users are bound to perform all verification work proposed.
The verification of the correct use of reagents/kits for the detection of antimicrobials is not part of the
scope of this document.

Leitlinie für die Validierung qualitativer Screening-Methoden zur Detektion von Tierarzneimittelrückständen in Milch und Milcherzeugnissen (ISO/TS 23758:2021)

Dieses Dokument beschreibt allgemeine Arbeitsabläufe und Arbeitsvorschriften für die Validierung und Verifizierung von qualitativen Screening-Tests zum Nachweis von Tierarzneimittelrückständen in Flüssigmilch (roh, pasteurisiert, UHT sowie rekonstituierte Milchpulver und Molkeneiweißextrakte), einschließlich biologischer Verfahren. Diese Leitlinie umfasst nicht die Validierung der Rückstandsanalyse durch HPLC, UHPLC oder LC-MS/MS.
Dieses Dokument soll für Hersteller von Screening-Testkits, Labore, die Screening-Verfahren oder  Tests validieren, zuständige Behörden und Molkereien oder Endanwender von Reagenzien oder Tests zum Nachweis von Tierarzneimittelrückständen in Milcherzeugnissen nützlich sein. Dieses Dokument erleichtert und verbessert die Validierung und Verifizierung von Screening-Verfahren. Die Ziele dieses Dokuments sind eine Harmonisierung der Validierung von Verfahren oder Testkits, damit alle Beteiligten volles Vertrauen in das Ergebnis des Rückstand-Screenings haben, und die Überschneidung und Vervielfachung der Validierungsarbeit in verschiedenen Laboren durch das zur Verfügung stellen der von einem unabhängigen Labor erzielten Validierungsergebnisse zu begrenzen. Darüber hinaus ermöglicht eine harmonisierte Validierungs  und Verifizierungsverfahrensweise einen Vergleich der Leistungsfähigkeit verschiedener Screening-Verfahren.
Dieses Dokument impliziert nicht, dass alle Endanwender verpflichtet sind, alle vorgeschlagenen Verifizierungsarbeiten durchzuführen.
Die Verifizierung der korrekten Anwendung von Reagenzien/Kits zum Nachweis antimikrobiell wirksamer Substanzen ist nicht Teil des Anwendungsbereichs dieses Dokuments.

Lignes directrices pour la validation des méthodes qualitatives de dépistage des résidus de médicaments vétérinaires dans le lait et les produits laitiers (ISO/TS 23758:2021)

Smernice za validacijo kvalitativnih presejalnih metod za detekcijo ostankov veterinarskih zdravil v mleku in mlečnih proizvodih (ISO/TS 23758:2021)

General Information

Status
Published
Public Enquiry End Date
04-Jul-2021
Publication Date
26-Sep-2021
Current Stage
6060 - National Implementation/Publication (Adopted Project)
Start Date
06-Sep-2021
Due Date
11-Nov-2021
Completion Date
27-Sep-2021

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SLOVENSKI STANDARD
SIST-TS CEN ISO/TS 23758:2021
01-december-2021
Smernice za validacijo kvalitativnih presejalnih metod za detekcijo ostankov
veterinarskih zdravil v mleku in mlečnih proizvodih (ISO/TS 23758:2021)

Guidelines for the validation of qualitative screening methods for the detection of

residues of veterinary drugs in milk and milk products (ISO/TS 23758:2021)
Leitlinie für die Validierung qualitativer Screening-Methoden zur Detektion von
Tierarzneimittelrückständen in Milch und Milcherzeugnissen (ISO/TS 23758:2021)

Lignes directrices pour la validation des méthodes qualitatives de dépistage des résidus

de médicaments vétérinaires dans le lait et les produits laitiers (ISO/TS 23758:2021)

Ta slovenski standard je istoveten z: CEN ISO/TS 23758:2021
ICS:
11.220 Veterinarstvo Veterinary medicine
67.100.01 Mleko in mlečni proizvodi na Milk and milk products in
splošno general
SIST-TS CEN ISO/TS 23758:2021 en,fr,de

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

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SIST-TS CEN ISO/TS 23758:2021
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SIST-TS CEN ISO/TS 23758:2021
CEN ISO/TS 23758
TECHNICAL SPECIFICATION
SPÉCIFICATION TECHNIQUE
August 2021
TECHNISCHE SPEZIFIKATION
ICS 67.100.01
English Version
Guidelines for the validation of qualitative screening
methods for the detection of residues of veterinary drugs
in milk and milk products (ISO/TS 23758:2021)

Lignes directrices pour la validation des méthodes Leitlinie für die Validierung qualitativer Screening-

qualitatives de dépistage des résidus de médicaments Methoden zur Detektion von

vétérinaires dans le lait et les produits laitiers (ISO/TS Tierarzneimittelrückständen in Milch und

23758:2021) Milcherzeugnissen (ISO/TS 23758:2021)

This Technical Specification (CEN/TS) was approved by CEN on 29 July 2021 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to

submit their comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS

available promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in

parallel to the CEN/TS) until the final decision about the possible conversion of the CEN/TS into an EN is reached.

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. CEN ISO/TS 23758:2021 E

worldwide for CEN national Members.
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SIST-TS CEN ISO/TS 23758:2021
CEN ISO/TS 23758:2021 (E)
Contents Page

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

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SIST-TS CEN ISO/TS 23758:2021
CEN ISO/TS 23758:2021 (E)
European foreword

This document (CEN ISO/TS 23758:2021) has been prepared by Technical Committee ISO/TC 34 "Food

products" in collaboration with Technical Committee CEN/TC 302 “Milk and milk products - Methods of

sampling and analysis” the secretariat of which is held by NEN.

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.

Any feedback and questions on this document should be directed to the users’ national standards

body/national committee. A complete listing of these bodies can be found on the CEN websites.

According to the CEN-CENELEC Internal Regulations, the national standards organizations of the

following countries are bound to announce this Technical Specification: 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/TS 23758:2021 has been approved by CEN as CEN ISO/TS 23758:2021 without any

modification.
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SIST-TS CEN ISO/TS 23758:2021
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SIST-TS CEN ISO/TS 23758:2021
TECHNICAL ISO/TS
SPECIFICATION 23758
IDF/RM 251
First edition
2021-08
Guidelines for the validation of
qualitative screening methods for the
detection of residues of veterinary
drugs in milk and milk products
Lignes directrices pour la validation des méthodes qualitatives de
dépistage des résidus de médicaments vétérinaires dans le lait et les
produits laitiers
Reference numbers
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
ISO and IDF 2021
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SIST-TS CEN ISO/TS 23758:2021
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO and IDF 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 International Dairy Federation
CP 401 • Ch. de Blandonnet 8 Silver Building • Bd Auguste Reyers 70/B
CH-1214 Vernier, Geneva B-1030 Brussels
Phone: +41 22 749 01 11 Phone: +32 2 325 67 40
Fax: +32 2 325 67 41
Email: copyright@iso.org Email: info@fil-idf.org
Website: www.iso.org Website: www.fil-idf.org
Published in Switzerland
ii © ISO and IDF 2021 – All rights reserved
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SIST-TS CEN ISO/TS 23758:2021
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
Contents Page

Forewords .....................................................................................................................................................................................................................................iv

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

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

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

4 Principle ........................................................................................................................................................................................................................ 4

5 General requirements for the test/kit ........................................................................................................................................... 5

6 Reagents ........................................................................................................................................................................................................................ 5

6.1 Standard blank matrix ...................................................................................................................................................................... 5

6.2 Antibiotics ................................................................................................................................................................................................... 6

6.3 Standard stock solution ................................................................................................................................................................... 6

6.4 Working stock solutions ................................................................................................................................................................. 6

6.5 Spiked sample .......................................................................................................................................................................................... 6

7 Apparatus ..................................................................................................................................................................................................................... 7

8 Sample Preparation........................................................................................................................................................................................... 7

8.1 Stock solution preparation ........................................................................................................................................................... 7

8.2 Working stock solution preparation .................................................................................................................................... 8

8.3 Blank matrix sample selection .................................................................................................................................................. 8

8.4 Spiked sample creation .................................................................................................................................................................... 8

9 Procedure..................................................................................................................................................................................................................... 8

9.1 Validation ..................................................................................................................................................................................................... 8

9.1.1 General...................................................................................................................................................................................... 8

9.1.2 Detection capability (CCβ)....................................................................................................................................... 9

9.1.3 Test selectivity/specificity ....................................................................................................................................13

9.1.4 Robustness testing ......................................................................................................................................................14

9.1.5 Reader and test repeatability .............................................................................................................................18

9.1.6 Participation in a(n) (inter)national ring trial ....................................................................................20

9.2 V erification testing of a transferred screening method ....................................................................................20

9.2.1 General...................................................................................................................................................................................20

9.2.2 Detection capability ...................................................................................................................................................21

9.2.3 Test selectivity/specificity ....................................................................................................................................21

9.2.4 Robustness testing ......................................................................................................................................................21

9.2.5 Reader and test repeatability .............................................................................................................................21

9.2.6 Participation in a(n) (inter)national ring trial ....................................................................................23

Annex A (informative) European legislation on veterinary drugs in cow milk .....................................................24

Annex B (informative) USA legislation on animal drug residues in milk ....................................................................28

Annex C (informative) List of problematic compounds in the preparation of stock solutions .............29

Annex D (informative) Summary of the stability of antibiotics in solution and in matrix ........................30

Bibliography .............................................................................................................................................................................................................................33

© ISO and IDF 2021 – All rights reserved iii
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SIST-TS CEN ISO/TS 23758:2021
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
Forewords

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 5,

Milk and milk products, and the International Dairy Federation (IDF), in collaboration with the European

Committee for Standardization (CEN) Technical Committee CEN/TC 302, Milk and milk products —

Methods of sampling and analysis, in accordance with the Agreement on technical cooperation between

ISO and CEN (Vienna Agreement). It is being published jointly by ISO and IDF.

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 and IDF 2021 – All rights reserved
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SIST-TS CEN ISO/TS 23758:2021
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)

IDF (the International Dairy Federation) is a non-profit private sector organization representing the

interests of various stakeholders in dairying at the global level. IDF members are organized in National

Committees, which are national associations composed of representatives of dairy-related national

interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and

governments/food control authorities.

ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis

and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International

Standards using the logos and reference numbers of both organizations.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. IDF 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.

This document was prepared by the IDF Standing Committee on Analytical Methods for Additives and

Contaminants and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and

milk products, in collaboration with the European Committee for Standardization (CEN) Technical

Committee CEN/TC 302, Milk and milk products — Methods of sampling and analysis, in accordance

with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). It is being

published jointly by ISO and IDF.

This IDF Reviewed method is equal to an ISO Publicly Available Specification (ISO/PAS) or an ISO

Technical Specification (ISO/TS) and is therefore published jointly under ISO conditions.

The work was carried out by the IDF-ISO Action Team on A10 of the Standing Committee on Analytical

Methods for Additives and Contaminants under the aegis of its project leader Dr W. Reybroeck (BE).

© ISO and IDF 2021 – All rights reserved v
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SIST-TS CEN ISO/TS 23758:2021
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SIST-TS CEN ISO/TS 23758:2021
ISO/TS 23758:2021(E)
TECHNICAL SPECIFICATION
IDF /RM 251:2021(E)
Guidelines for the validation of qualitative screening
methods for the detection of residues of veterinary drugs
in milk and milk products
1 Scope

This document describes general workflows and protocols for the validation and the verification

of qualitative screening tests for the detection of residues of veterinary drugs in liquid milk (raw,

pasteurized, UHT and reconstituted milk powders and whey protein extracts) including biological

methods. This guideline does not cover the validation of residue analysis by HPLC, UHPLC or LC-MS/MS.

This document is intended to be useful for manufacturers of screening test kits, laboratories validating

screening methods or tests, competent authorities and dairies or end users of reagents or tests for the

detection of veterinary drug residues in milk products. This document facilitates and improves the

validation and verification of screening methods. The goals of this document are a harmonization in

validation of methods or test kits in order for all stakeholders to have full trust in the result of residue

screening and to limit the overlap and multiplication of validation work in different laboratories by

sharing the validation results generated by an independent laboratory. Furthermore, a harmonized

validation and verification procedure allows for comparison of the performance of different screening

methods.

This document does not imply that all end users are bound to perform all verification work proposed.

The verification of the correct use of reagents/kits for the detection of antimicrobials is not part of the

scope of this document.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
biological method
method that is used to detect cellular responses to analytes
EXAMPLE Inhibition of bacterial growth, immunological test, and receptor test.
3.2
qualitative method

method that gives a yes/no response, with no indication of the concentration of the putative analyte

EXAMPLE 1 Bacterial growth inhibition tests which give a result of either “no zone” or “zone of inhibition”.

EXAMPLE 2 Inhibition tests which give a colour change of growth medium.
© ISO and IDF 2021 – All rights reserved 1
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SIST-TS CEN ISO/TS 23758:2021
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)

EXAMPLE 3 Immunochemical/ligand binding tests, where a response is considered as “above” or “below” a

cut-off level; or where analytes with different cross-reactivities are included within the method scope.

EXAMPLE 4 Biosensors.
3.3
matrix
non-analyte portion of the sample
Note 1 to entry: Matrices are included in the scope.
3.4
detection capability
CCβ

smallest content of the analyte that can be detected, identified and/or quantified in a sample with an

error probability of β

Note 1 to entry: The β error is the probability that the tested sample is truly non-conformant even though a

conformant measurement has been obtained.
3.5
cut-off level

response or signal from a screening test which indicates that a sample contains an analyte at or above

the screening target concentration
3.6
blank matrix sample
negative control sample

sample from animals with known history of treatment which have not been exposed to the substance

in question

Note 1 to entry: If samples from such animals are not available, samples which have been previously confirmed

as conformant and not containing residues of the substance of interest by suitably sensitive physicochemical

tests can also be acceptable.
Note 2 to entry: See Table 1.
3.7
positive control sample

control sample that is spiked with the test analyte at the screening target concentration

Note 1 to entry: This can, however also be an incurred-positive sample (i.e. sample taken from animals which

have been treated with the substance in question) or Certified Reference Material.

3.8
screening target concentration

concentration at which a screening test categorizes the sample as “screen positive” (potentially non-

conformant)
Note 1 to entry: This should always be lower than the regulatory limit.
3.9
validation

confirmation, through the provision of objective evidence, that the requirements for a specific intended

use or application, such as a test or measurement method, have been fulfilled

EXAMPLE Procedure applied in the originator laboratory (manufacturer’s laboratory) or in an independent

laboratory.

Note 1 to entry: Validation often determines the fitness for purpose of a method.

2 © ISO and IDF 2021 – All rights reserved
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SIST-TS CEN ISO/TS 23758:2021
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
3.10
verification

procedure applied to a method which has been previously validated in the case of a transfer validation

Note 1 to entry: The verification procedure is applied by a receptor laboratory for the same matrix as initially

validated, to demonstrate that the method will work reliably in that laboratory with locally sourced milk and is

fit for purpose.
3.11
originator laboratory
laboratory that has performed the complete validation of the method

Note 1 to entry: This is by preference an ISO/IEC 17025 accredited independent laboratory and preferably not the

laboratory that developed the method. The laboratory should have experience in residue testing and in validation

of screening tests for the detection of residues of veterinary drugs in milk.
3.12
receptor laboratory
laboratory that will perform the verification of the method
Note 1 to entry: This could be any laboratory interested in using the method.
3.13
spectrum
range of substances that a test can detect

Note 1 to entry: Some tests detect several classes of antibiotics and a large number of substances, whereas others

are more specific.
3.14
regulatory limit
level defined by food legislation for residues in food

Note 1 to entry: Regulatory limits can be MRL (see 3.15), MRPL (see 3.16), RPA (see 3.17).

3.15
maximum residue limit for veterinary drugs
MRL

maximum concentration of residue resulting from the use of veterinary drugs that is recommended by

the Codex Alimentarius Commission to be legally permitted or recognized as acceptable in food

Note 1 to entry: Antibiotics are used to treat and prevent diseases in animal husbandry and as a result, low

residues of antibiotics can be present in food. MRLs are set for pharmacologically active substances used or

intended to be used in veterinary medicinal products placed on the market. In the EU the MRLs are set by EMA

(European Medicines Agency).
3.16
minimum required performance limit
MRPL

minimum content of an analyte in a sample, which at least has to be detected and confirmed

Note 1 to entry: MRPL is intended to harmonize the analytical performance of methods for substances for which

no permitted limit has been established.
3.17
reference point for action
RPA

level of a residue of a pharmacologically active substance established for control reasons in the case

of certain substances for which a maximum residue limit has not been laid down following certain EU

regulations

Note 1 to entry: EU Regulation 470/2009 is applicable for maximum residue limits.

© ISO and IDF 2021 – All rights reserved 3
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SIST-TS CEN ISO/TS 23758:2021
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)

Note 2 to entry: RPAs are currently based on analytical considerations (i.e. the lowest concentration that can

be quantified using a validated analytical method). The aim is “to define an analytical concentration for a non-

allowed pharmacologically active substance that can be determined by official control laboratories and that is

[19]

low enough to adequately protect the consumers of food commodities which contain that substance” .

3.18
positive / negative result

result of the test after interpretation of the reading of the test taking into account the (pre-set) cut-off

level

Note 1 to entry: Positive result: presence of antimicrobial residues (microbial inhibitor test) or presence of

residues of veterinary drugs.

Note 2 to entry: Negative result: absence of antimicrobial residues (microbial inhibitor test) or absence of

residues of veterinary drugs. Since only screening tests are involved, no judgement about ‘conformant’ or ‘non-

conformant’ can be made.
3.19
repeatability limit

value less than or equal to which the absolute difference between two measurement results obtained

under repeatability conditions is expected with a probability of 95 %
3.20
probability of detection
POD

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

level or concentration
[7]
Note 1 to entry: POD is concentration dependent (AOAC, 2014 ).
4 Principle

Samples of matrix spiked with known levels of analyte are run on the test under validation or

verification to determine the detection capability, sensitivity and robustness of the test. Evaluation of

the test results determines the tests' suitability for routine use in screening milk for the presence of

veterinary residues.

NOTE Annex B provides information on FDA tolerances and/or safe levels of animal drug residues in milk.

The key requirement for a screening method is its ability to reliably detect the analyte in question at the

chosen screening target concentration. The screening target concentration should be chosen to avoid

false-negative results, i.e. low enough to ensure that if the analyte in question is present in the sample

at the Regulatory Limit, the sample will be classified as 'Screened Positive'.

Both validation and verification should provide the objective evidence that this key requirement is met.

Validation should cover the entire matrix/species/analyte combinations claimed within the scope of

the method standard operating procedure (SOP). Validation should be as broad as possible to cover the

scope of all end users.

Verification should cover the matrix/species/analyte combinations included in the scope of the

implementing (receptor) laboratory. The extent of validation required is variable, depending on whether

it is a validation or a verification of a transferred method.

The verification does not need to cover the entire spectrum if the implementing laboratory is to be

applicable to only a limited scope (e.g. some species an
...

SLOVENSKI STANDARD
kSIST-TS FprCEN ISO/TS 23758:2021
01-julij-2021
Smernice za validacijo kvalitativnih informativnih metod za detekcijo ostankov
veterinarskih zdravil v mleku in mlečnih proizvodih (ISO/PRF TS 23758:2021)

Guidelines for the validation of qualitative screening methods for the detection of

residues of veterinary drugs in milk and milk products (ISO/PRF TS 23758:2021)
Leitlinie für die Validierung qualitativer Screening-Methoden zur Detektion von

Tierarzneimittelrückständen in Milch und Milcherzeugnissen (ISO/PRF TS 23758:2021)

Lignes directrices pour la validation des méthodes qualitatives de dépistage des résidus

de médicaments vétérinaires dans le lait et les produits laitiers (ISO/PRF TS
23758:2021)
Ta slovenski standard je istoveten z: FprCEN ISO/TS 23758
ICS:
11.220 Veterinarstvo Veterinary medicine
67.100.01 Mleko in mlečni proizvodi na Milk and milk products in
splošno general
kSIST-TS FprCEN ISO/TS 23758:2021 en,fr,de

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

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kSIST-TS FprCEN ISO/TS 23758:2021
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kSIST-TS FprCEN ISO/TS 23758:2021
TECHNICAL ISO/TS
SPECIFICATION 23758
IDF/RM 251
First edition
Guidelines for the validation of
qualitative screening methods for the
detection of residues of veterinary
drugs in milk and milk products
Lignes directrices pour la validation des méthodes qualitatives de
dépistage des résidus de médicaments vétérinaires dans le lait et les
produits laitiers
PROOF/ÉPREUVE
Reference numbers
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
ISO and IDF 2021
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kSIST-TS FprCEN ISO/TS 23758:2021
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
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ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
Contents Page

Forewords .....................................................................................................................................................................................................................................iv

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

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

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

4 Principle ........................................................................................................................................................................................................................ 4

5 General requirements for the test/kit ........................................................................................................................................... 5

6 Reagents ........................................................................................................................................................................................................................ 5

6.1 Standard blank matrix ...................................................................................................................................................................... 5

6.2 Antibiotics ................................................................................................................................................................................................... 6

6.3 Standard stock solution ................................................................................................................................................................... 6

6.4 Working stock solutions ................................................................................................................................................................. 6

6.5 Spiked sample .......................................................................................................................................................................................... 6

7 Apparatus ..................................................................................................................................................................................................................... 7

8 Sample Preparation........................................................................................................................................................................................... 7

8.1 Stock solution preparation ........................................................................................................................................................... 7

8.2 Working stock solution preparation .................................................................................................................................... 8

8.3 Blank matrix sample selection .................................................................................................................................................. 8

8.4 Spiked sample creation .................................................................................................................................................................... 8

9 Procedure..................................................................................................................................................................................................................... 8

9.1 Validation ..................................................................................................................................................................................................... 8

9.1.1 General...................................................................................................................................................................................... 8

9.1.2 Detection capability (CCβ)....................................................................................................................................... 9

9.1.3 Test selectivity/specificity ....................................................................................................................................13

9.1.4 Robustness testing ......................................................................................................................................................14

9.1.5 Reader and test repeatability .............................................................................................................................18

9.1.6 Participation in a(n) (inter)national ring trial ....................................................................................20

9.2 V erification testing of a transferred screening method ....................................................................................20

9.2.1 General...................................................................................................................................................................................20

9.2.2 Detection capability ...................................................................................................................................................21

9.2.3 Test selectivity/specificity ....................................................................................................................................21

9.2.4 Robustness testing ......................................................................................................................................................21

9.2.5 Reader and test repeatability .............................................................................................................................21

9.2.6 Participation in a(n) (inter)national ring trial ....................................................................................23

Annex A (informative) European legislation on veterinary drugs in bovine milk .............................................24

Annex B (informative) USA legislation on animal drug residues in milk ....................................................................28

Annex C (informative) List of problematic compounds in the preparation of stock solutions .............29

Annex D (informative) Summary of the stability of antibiotics in solution and in matrix ........................30

Bibliography .............................................................................................................................................................................................................................32

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ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
Forewords

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 5,

Milk and milk products, and the International Dairy Federation (IDF), in collaboration with the European

Committee for Standardization (CEN) Technical Committee CEN/TC 302, Milk and milk products —

Methods of sampling and analysis, in accordance with the Agreement on technical cooperation between

ISO and CEN (Vienna Agreement). It is being published jointly by ISO and IDF.

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.
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ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)

IDF (the International Dairy Federation) is a non-profit private sector organization representing the

interests of various stakeholders in dairying at the global level. IDF members are organized in National

Committees, which are national associations composed of representatives of dairy-related national

interest groups including dairy farmers, dairy processing industry, dairy suppliers, academics and

governments/food control authorities.

ISO and IDF collaborate closely on all matters of standardization relating to methods of analysis

and sampling for milk and milk products. Since 2001, ISO and IDF jointly publish their International

Standards using the logos and reference numbers of both organizations.

Attention is drawn to the possibility that some of the elements of this document may be the subject of

patent rights. IDF 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.

This document was prepared by the IDF Standing Committee on Analytical Methods for Additives and

Contaminants and ISO Technical Committee ISO/TC 34, Food products, Subcommittee SC 5, Milk and

milk products, in collaboration with the European Committee for Standardization (CEN) Technical

Committee CEN/TC 302, Milk and milk products — Methods of sampling and analysis, in accordance

with the Agreement on technical cooperation between ISO and CEN (Vienna Agreement). It is being

published jointly by ISO and IDF.

This IDF Reviewed method is equal to an ISO Publicly Available Specification (ISO/PAS) or an ISO

Technical Specification (ISO/TS) and is therefore published jointly under ISO conditions.

The work was carried out by the IDF-ISO Action Team on A10 of the Standing Committee on Analytical

Methods for Additives and Contaminants under the aegis of its project leader Dr W. Reybroeck (BE).

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ISO/TS 23758:2021(E)
TECHNICAL SPECIFICATION
IDF /RM 251:2021(E)
Guidelines for the validation of qualitative screening
methods for the detection of residues of veterinary drugs
in milk and milk products
1 Scope

This document describes general workflows and protocols for the validation and the verification

of qualitative screening tests for the detection of residues of veterinary drugs in liquid milk (raw,

pasteurized, UHT and reconstituted milk powders and whey protein extracts) including biological

methods. This guideline does not cover the validation of residue analysis by HPLC, UHPLC or LC-MS/MS.

This document is intended to be useful for manufacturers of screening test kits, laboratories validating

screening methods or tests, competent authorities and dairies or end users of reagents or tests for the

detection of veterinary drug residues in milk products. This document facilitates and improves the

validation and verification of screening methods. The goals of this document are a harmonization in

validation of methods or tests kits in order for all stakeholders to have full trust in the result of residue

screening and to limit the overlap and multiplication of validation work in different laboratories by

sharing the validation results generated by an independent laboratory. Furthermore, a harmonized

validation and verification procedure allows for comparison of the performance of different screening

methods.

This document does not imply that all end users are bound to perform all verification work proposed.

The verification of the correct use of reagents/kits for the detection of antimicrobials is not part of the

scope of this document.
2 Normative references
There are no normative references in this document.
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.

ISO and IEC maintain terminological databases for use in standardization at the following addresses:

— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
biological method
method that detects cellular responses to analytes
EXAMPLE Inhibition of bacterial growth, immunological test, and receptor test.
3.2
qualitative method

method that gives a yes/no response, with no indication of the concentration of the putative analyte

Note 1 to entry: Bacterial growth inhibition tests which give a result of either “no zone” or “zone of inhibition”.

EXAMPLE 2 Inhibition tests which give a colour change of growth medium.
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EXAMPLE 3 Immunochemical/ligand binding tests, where a response is considered as “above” or “below” a

cut-off level; or where analytes with different cross-reactivities are included within the method scope.

EXAMPLE 4 Biosensors.
3.3
matrix

non-analyte portion of the sample Note 1 to entry: Matrices are included in the Scope.

3.4
detection capability
CCβ

smallest content of the analyte that can be detected, identified and/or quantified in a sample with an

error probability of β

Note 1 to entry: The β error is the probability that the tested sample is truly non-conformant even though a

conformant measurement has been obtained.
3.5
cut-off level

response or signal from a screening test which indicates that a sample contains an analyte at or above

the screening target concentration
3.6
blank matrix sample
negative control sample

sample from animals with known history of treatment which have not been exposed to the substance

in question

Note 1 to entry: If samples from such animals are not available, samples which have been previously confirmed

as conformant and not containing residues of the substance of interest by suitably sensitive physicochemical

tests can also be acceptable.
Note 2 to entry: See Table 1.
3.7
positive control sample

control sample that is spiked with the test analyte at the screening target concentration

Note 1 to entry: This may, however also be an incurred-positive sample (i.e. sample taken from animals which

have been treated with the substance in question) or Certified Reference Material.

3.8
screening target concentration

concentration at which a screening test categorizes the sample as “screen positive” (potentially non-

conformant)
Note 1 to entry: This should always be lower than the regulatory limit.
3.9
validation

confirmation, through the provision of objective evidence, that the requirements for a specific intended

use or application, such as a test or measurement method, have been fulfilled

Note 1 to entry: Procedure applied in the originator laboratory (manufacturer’s laboratory) or in an independent

laboratory.

Note 2 to entry: Validation often determines the fitness for purpose of a method.

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3.10
verification

procedure applied to a method which has been previously validated in the case of a transfer validation

Note 1 to entry: The verification procedure is applied by a receptor laboratory for the same matrix as initially

validated, to demonstrate that the method will work reliably in that laboratory with locally sourced milk and is

fit for purpose.
3.11
originator laboratory
laboratory that has performed the complete validation of the method

Note 1 to entry: This is by preference an IEC/ISO 17025 accredited independent laboratory and preferably not

the laboratory that developed the method. The laboratory should have experience in residue testing and in

validation of screening tests for the detection of residues of veterinary drugs in milk.

3.12
receptor laboratory
laboratory that will perform the verification of the method
Note 1 to entry: This could be any laboratory interested in using the method.
3.13
spectrum
range of substances that a test can detect

Note 1 to entry: Some tests detect several classes of antibiotics and a large number of substances, whereas others

are more specific.
3.14
regulatory limit
level defined by food legislation for residues in food
Note 1 to entry: Regulatory limits can be MRL, MRPL, RPA.
3.15
maximum residue limit for veterinary drugs
MRL

maximum concentration of residue resulting from the use of veterinary drugs that is recommended by

the Codex Alimentarius Commission to be legally permitted or recognized as acceptable in food

Note 1 to entry: Antibiotics are used to treat and prevent diseases in animal husbandry and as a result, low

residues of antibiotics can be present in food. MRLs are set for pharmacologically active substances used or

intended to be used in veterinary medicinal products placed on the market. In the EU the MRLs are set by EMA

(European Medicines Agency).
3.16
minimum required performance limit
MRPL

minimum content of an analyte in a sample, which at least has to be detected and confirmed

Note 1 to entry: MRPL is intended to harmonize the analytical performance of methods for substances for which

no permitted limit has been established.
3.17
reference point for action
RPA

level of a residue of a pharmacologically active substance established for control reasons in the case

of certain substances for which a maximum residue limit has not been laid down following certain EU

regulations

Note 1 to entry: EU Regulation 470/2009 is applicable for maximum residue limits.

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Note 2 to entry: RPAs are currently based on analytical considerations (i.e. the lowest concentration that can

be quantified using a validated analytical method). The aim is “to define an analytical concentration for a non-

allowed pharmacologically active substance that can be determined by official control laboratories and that is

[19]

low enough to adequately protect the consumers of food commodities which contain that substance” .

3.18
positive / negative result

result of the test after interpretation of the reading of the test taking into account the (pre-set) cut-off

Note 1 to entry: Positive result: presence of antimicrobial residues (microbial inhibitor test) or presence of

residues of veterinary drugs.

Note 2 to entry: Negative result: absence of antimicrobial residues (microbial inhibitor test) or absence of

residues of veterinary drugs. Since only screening tests are involved, no judgement about ‘conformant’ or ‘non-

conformant’ can be made.
3.19
repeatability limit

value less than or equal to which the absolute difference between two measurement results obtained

under repeatability conditions is expected with a probability of 95 %
3.20
probability of detection
POD

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

level or concentration
[7]
Note 1 to entry: POD is concentration dependent (AOAC, 2014 ).
4 Principle

Samples of matrix spiked with known levels of analyte are run on the test under validation or

verification to determine the detection capability, sensitivity and robustness of the test. Evaluation of

the test results determines the tests' suitability for routine use in screening milk for the presence of

veterinary residues.

The key requirement for a screening method is its ability to reliably detect the analyte in question at the

chosen screening target concentration. The screening target concentration should be chosen to avoid

false-negative results, i.e. low enough to ensure that if the analyte in question is present in the sample

at the Regulatory Limit, the sample will be classified as 'Screened Positive'.

Both validation and verification should provide the objective evidence that this key requirement is met.

Validation should cover the entire matrix/species/analyte combinations claimed within the scope of

the method standard operating procedure (SOP). Validation should be as broad as possible to cover the

scope of all end users.

Verification should cover the matrix/species/analyte combinations included in the scope of the

implementing (receptor) laboratory. The extent of validation required is variable, depending on whether

it is a validation or a verification of a transferred method.

The verification does not need to cover the entire spectrum if the implementing laboratory is to be

applicable to only a limited scope (e.g. some species and not others, some residues more relevant than

others, raw but not UHT [Ultra-High temperature] milk, etc.).

If a receptor laboratory wants to use the method for screening in a different matrix (IDF 2014) not tested

by the originator laboratory, the receptor laboratory should test all necessary validation parameters to

prove that the method functions for that specific matrix.
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5 General requirements for the test/kit

The developer or the manufacturer should provide information regarding methodology, test reagents,

additional chemicals not necessarily included in the kit, operating requirements (information about

the reading system, cut-off value), test specifications and documentation (extracted from ISO 18330

and ISO 13969). Additionally, the target country(ies) and its/their specific regulatory limits should be

known, in order for the test to be evaluated against the appropriate regulatory limits.

Elements of information to be provided by the manufacturer/distributor/lab manager (in case of an in-

house developed method) before starting the validation are as follows:

— Test principle, principle of reading and interpretation of the test (including cut-off level or calculation

of cut-off).
— Test formats, if relevant (e.g. ampoules/plates).
— Scope of the test:

— Matrices suitable to be tested: matrices in the scope of the document (see Clause 1).

— Animal species producing the milk.
— Matrices with potential impact (interference) on the result.
— Potential impact of the use of sample preservatives.

— Spectrum of the test: list of veterinary drugs and expected detection capabilities (so far known).

— List with the actual regulatory limits (RL) for the detectable veterinary drugs in the matrix(ces) of

concern in the country(ies) of concern.

— Detailed protocol in a language understood by laboratory staff: if minor modifications need to be

made to the method according to the matrix/species, they should be announced in the test protocol

(kit manual).
6 Reagents
6.1 Standard blank matrix

— The raw milk used is commingled milk coming from at least 4 animals not treated with veterinary

drugs within the last 2 months, in mid lactation, and delivering milk with a low to moderate number

of somatic cells (e.g. < 150 000 ml for bovine milk). The raw milk is collected in sterile containers

and kept below 4 °C. The maximum period for the cold storage of the fresh raw milk should be in line

with the definition of fresh raw milk as fixed locally.

— The milk used should be in line with the normal milk produced in the country or area of concern.

This means that the composition and quality of the milk should approach the average composition

of the milk of the country/region.

— Table 1 gives examples of parameters to consider for ‘normal’ milk. Actual figures are likely to vary

depending on country and region.

— Milk of at least 4 animals is commingled and is considered as a sample of standard blank matrix. At

least four (4) such samples should be used for the determination of the detection capability when

testing 20 replicates. If 40 or 60 replicates need to be tested to determine the detection capability,

eight (8) or twelve (12) different blank milk samples should be used, respectively. At least four (4)

different commingled milks should be sourced and used in the verification work (20 replicates).

— The use of thawed or reconstituted lyophilized milk could also be authorized, but strictly on

condition. The pre-requisite condition to work with these alternative solutions, is to demonstrate

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previously the equivalence of results between raw milk and thawed or reconstituted

...

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