CEN ISO/TS 23758:2021

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

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.

Contents Page
European foreword . 3

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

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/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

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/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/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

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/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 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.
4 © ISO and IDF 2021 – All rights reserved

ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
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 level), 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 level).
— 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 current 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
−1
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 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 or twelve different blank milk samples should be used, respectively. At least four 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
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
previously the equivalence of results between raw milk and thawed or reconstituted lyophilized
milk, after the analysis of negative and positive milk samples.
Table 1 — Examples of reference data for the composition and quality of normal milk of
different animal species
b
TBC pH Antibiotics Lactating period
a d e
SCC FC PC
Species c
cfu per
cells per ml g/l g/l
ml
6,7 to
Target value < 150 000 < 30 000 40 33
Between 60 and
6,8
Cow Absence 200 days after
Acceptable 35 to 6,6 to
calving
< 400 000 < 100 000 30 to 36
range 45 6,9
6,7 to
Target value < 2 000 000 < 60 000 38 34
Between 20 and
6,8
Goat Absence 150 days after
Acceptable 30 to 6,6 to
kidding
28 to 40
range 50 6,9
6,7 to
Target value < 2 000 000 < 60 000 70 55
Between 20 and
6,8
Ewe Absence 150 days after
Acceptable 50 to 6,6 to
lambing
40 to 70
range 90 6,9
a
Somatic cell count.
b
Total bacterial count.
c
Colony forming units.
d
Fat content.
e
Protein content.
6.2 Antibiotics
Only use analytical grade or certified reference material for validation or verification purposes.
6.3 Standard stock solution
— Standard stock solutions of the antibiotic at 100 mg/l are made in water or a suitable solvent and
[14]
kept below 4 °C (refer to 8.1) . The shelf life depends on the stability of the molecule.
— In the preparation of the stock solution, correction for impurity and water content is performed.
— For each substance a single stock solution is prepared, but by preference for certain problematic
compounds (for example solubility problem, stability), at least two stock solutions should be
prepared to determine the detection capability. A list of problematic compounds is given in Annex C.
— If only one stock solution is used it should be either prepared from certified material or verified
with an independent physicochemical method.
— Some compounds like tetracyclines are light sensitive and need to be kept protected from light.
Other compounds can require specific requirements for the glassware used.
6.4 Working stock solutions
Dilutions of 10 mg/l to 0,1 mg/l are freshly prepared on a daily basis.
6.5 Spiked sample
For the preparation of end concentration, the final spiking is performed in the standard blank matrix.
6 © ISO and IDF 2021 – All rights reserved

ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
The blank milk will be spiked with each analyte.
The added volume of working stock solution should be below 5 % of the final volume of the milk sample
to be tested.
7 Apparatus
Any apparatus specified in the test kits procedure that is not provided by the test kit manufacturer.
7.1 Test kit, in which reagents of at least two and by preference three different production lots are
used.
Production lots should be determined randomly and be representative of the production level of the
product.
7.2 Incubator or water-bath, capable of maintaining the appropriate incubation temperature for the
test.
If the manufacturer has an incubator that is designed for the test, this incubator should be supplied by
the manufacturer for use in the validation or verification.
7.3 Automated readers. If the manufacturer provides an apparatus for evaluating the results from the
test, this apparatus should be supplied by the manufacturer for use and evaluation in the validation or
verification.
Calibration procedures for the automated reader shall be made available and all readings shall be taken
from only calibrated equipment.
7.4 Micropipettes, capable of delivering the appropriate amount of sample required for use in the
test.
If the test kit supplies micropipettes or other liquid transfer equipment these should be by preference
evaluated as part of the validation or verification.
8 Sample Preparation
8.1 Stock solution preparation
A 100 mg/l solution of each antibiotic is prepared by first calculating the amount of reference material
needed to give 10 mg ± 0,1 mg of active compound. This calculation is done using Formula (1) and the
information (purity, water content) from the certificate of analysis of the antibiotic.
100 100
   
mm=× × (1)
   
ma
PW100−
   
where
m is the mass of the material required, in mg;
m
m is the mass of the analyte required, in mg;
a
P is the purity, in %;
W is the water content, in %.
Weigh this amount directly in a weight boat and transfer into a 100 ml volumetric flask and make up to
100 ml using the appropriate solvent.
ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
The samples are vortexed or sonicated until dissolved. These can be kept at 4 °C for up to 4 weeks or
at −18 °C or lower for up to 12 months. Stability of different antimicrobials in solution and in matrix at
different storage temperatures are given in Table D.1 and Table D.2.
8.2 Working stock solution preparation
If necessary, intermediate stocks of 100 000 µg/l, 10 000 µg/l, 1 000 µg/l or 100 µg/l, respectively (or
other appropriate concentrations) are made up in distilled water and stored at 4 °C to 6 °C for maximum
one day.
8.3 Blank matrix sample selection
For each antibiotic to be tested, the number of different samples of standard blank matrix from
different sources to be tested depends on the closeness of the predicted detection capability (CCβ) to
the regulatory limit:
— If CCβ is below or equal to half MRL: at least two different batches of milk for 20 replicates.
— If CCβ is between 50 % and 90 % of MRL: at least four different batches of milk for 40 replicates.
— If CCβ is near MRL (≥90 % to 100 % of MRL): at least six different batches of milk for 60 replicates.
— If CCβ is above MRL: at least two different batches of milk for 20 replicates.
The sample should be of sufficient size to provide multiple test portions of the size specified by the test
protocol. When selecting the source of the milk, samples covering the range of “normally” produced
milk should be selected. This could cover factors like breed of animal, feed source, geographic region,
etc.
The samples should be checked to ensure the absence of beta-lactamase or para-aminobenzoic acid
by adding a small but detectable quantity of penicillin or sulfonamide to the milk, respectively and
incubate and test. If a negative result is obtained it indicates the presence of beta-lactamase or para-
aminobenzoic acid (PABA), respectively. This check is only needed if beta-lactams or sulfonamides
are included in the validation, respectively. The pH of the sample should be recorded and a subsample
should be kept and stored at −25 °C ± 5 °C. In case of questionable results during validation or
verification (false-positive results, CCβ lower than the CCβ announced by the manufacturer or obtained
with different milk), the laboratory thaws the sample for screening or confirmatory analyses.
8.4 Spiked sample creation
The appropriate working stock solution should be added to the blank matrix sample to produce a
sample at the required level of antibiotic. The added volume of working stock solution should be below
5 % of the final volume.
Furthermore, the last working solution can be done in milk, except for tetracyclines.
NOTE For tetracyclines, it is recommended that only the added volume of working stock solution be below
1 % or 2 % of the final volume. It is recommended to prepare all the dilutions of working solutions in water and
only the final dilution in milk to avoid binding to milk proteins and to calcium.
9 Procedure
9.1 Validation
9.1.1 General
The validation is a procedure applied to characterize the performances of a test, in the originator
laboratory (manufacturer’s laboratory) or in an independent laboratory. The validation demonstrates
8 © ISO and IDF 2021 – All rights reserved

ISO/TS 23758:2021(E)
IDF /RM 251:2021(E)
that the method is fit for purpose. The originator laboratory could be the laboratory (or a group of
laboratories) which developed the new analytical method or the first laboratory (or group of
laboratories) performing a full validation study. The originator laboratory performing the validation
should by preference be an independent laboratory with experience in the field and a quality control
system (e.g. ISO/IEC 17025) in place and accredited for analogue methods for the same matrix.
Laboratory staff should have access to all required equipment (incubator and reader system when
applicable) and should be fully trained to run the test.
The validation of a test covers:
— the detection capability;
— test selectivity/specificity;
— test robustness; and
— reader and test repeatability.
All factors should be present in the final validation report.
During the validation, the detection capabilities are determined as precisely as possible.
9.1.2 Detection capability (CCβ)
9.1.2.1 General
The detection capability should be determined in the specific matrix/species for which the test was
developed, which in most cases will be raw cow milk. If the test is claimed to be appropriate for use
in testing an alternative matrix/species then the validation is required to cover this matrix as well. In
such a case two approaches are possible:
Option 1: The CCβ is determined in the main matrix as such (for example in raw cow milk) then other
matrices/species are studied as part of the applicability and/or robustness testing. In the applicability
and/or robustness testing, the samples may be spiked up to levels of CCβ + 20 %. For as far as positive
results are obtained, the CCβ determined in the main matrix is also valid in the new matrix/species. If
negative results are obtained, the new matrix/species should be fully validated to determine the CCβ,
or the conclusion is that the method is not applicable to the new matrix/species with the same CCβ.
Option 2: The detection capabilities are in the same study determined directly for the different
matrices with an equal number of replicates for each different matrix (e.g. cows’ milk, goats’ milk and
ewes’ milk; UHT milk, sterilized milk and reconstituted milk powder).
Reconstituted milk powder should not be mixed with raw milk when testing microbial inhibitor tests
since they require a different incubation time. Only use similar matrices with equal incubation times in
the same run.
This last procedure can increase the CCβ if the CCβ is different for each matrix. The highest CCβ is the
final value. The same phenomenon can be observed when different lots of test reagents are used in
combination for the determination of CCβ.
9.1.2.2 Compounds involved in the study
All substances relevant for end users in their routine application (whether defined by regulation,
registration or actual use) should be validated.
The compounds to be tested for detection capability are determined by the type of test to be validated.
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