SIST EN ISO 16140-2:2016/oprA1:2023
(Amendment)Microbiology of the food chain - Method validation - Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method
Microbiology of the food chain - Method validation - Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 2: Arbeitsvorschrift für die Validierung von alternativen (urheberrechtlich geschützten) Verfahren anhand eines Referenzverfahrens
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 2: Protocole pour la validation de méthodes alternatives (commerciales) par rapport à une méthode de référence
Mikrobiologija v prehranski verigi - Validacija metode - 2. del: Protokol za validacijo alternativnih (lastniških) metod glede na referenčno metodo - Dopolnilo A1
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 16140-2:2016/oprA1:2023
01-oktober-2023
Mikrobiologija v prehranski verigi - Validacija metode - 2. del: Protokol za
validacijo alternativnih (lastniških) metod glede na referenčno metodo - Dopolnilo
A1
Microbiology of the food chain - Method validation - Part 2: Protocol for the validation of
alternative (proprietary) methods against a reference method
Mikrobiologie der Lebensmittelkette - Verfahrensvalidierung - Teil 2: Arbeitsvorschrift für
die Validierung von alternativen (urheberrechtlich geschützten) Verfahren anhand eines
Referenzverfahrens
Microbiologie de la chaîne alimentaire - Validation des méthodes - Partie 2: Protocole
pour la validation de méthodes alternatives (commerciales) par rapport à une méthode
de référence
Ta slovenski standard je istoveten z: EN ISO 16140-2:2016/prA1
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16140-2:2016/oprA1:2023 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 16140-2:2016/oprA1:2023
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SIST EN ISO 16140-2:2016/oprA1:2023
DRAFT AMENDMENT
ISO 16140-2:2016/DAM 1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
2023-07-18 2023-10-10
Microbiology of the food chain — Method validation —
Part 2:
Protocol for the validation of alternative (proprietary)
methods against a reference method
AMENDMENT 1: Revision of qualitative MCS data evaluation,
RLOD calculations in the ILS, calculation and interpretation
of the RT study, and inclusion of a commercial sterility testing
protocol for specific products
ICS: 07.100.30
This document is circulated as received from the committee secretariat.
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
NOT BE REFERRED TO AS AN INTERNATIONAL
STANDARD UNTIL PUBLISHED AS SUCH.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL,
TECHNOLOGICAL, COMMERCIAL AND
USER PURPOSES, DRAFT INTERNATIONAL
STANDARDS MAY ON OCCASION HAVE TO
BE CONSIDERED IN THE LIGHT OF THEIR
POTENTIAL TO BECOME STANDARDS TO
WHICH REFERENCE MAY BE MADE IN
Reference number
NATIONAL REGULATIONS.
ISO 16140-2:2016/DAM 1:2023(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
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PROVIDE SUPPORTING DOCUMENTATION. © ISO 2023
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
DRAFT AMENDMENT
ISO 16140-2:2016/DAM 1
ISO/TC 34/SC 9 Secretariat: AFNOR
Voting begins on: Voting terminates on:
Microbiology of the food chain — Method validation —
Part 2:
Protocol for the validation of alternative (proprietary)
methods against a reference method
AMENDMENT 1: Revision of qualitative MCS data evaluation,
RLOD calculations in the ILS, calculation and interpretation
of the RT study, and inclusion of a commercial sterility testing
protocol for specific products
ICS: 07.100.30
This document is circulated as received from the committee secretariat.
COPYRIGHT PROTECTED DOCUMENT
THIS DOCUMENT IS A DRAFT CIRCULATED
FOR COMMENT AND APPROVAL. IT IS
© ISO 2023
ISO/CEN PARALLEL PROCESSING
THEREFORE SUBJECT TO CHANGE AND MAY
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
NOT BE REFERRED TO AS AN INTERNATIONAL
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on STANDARD UNTIL PUBLISHED AS SUCH.
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
IN ADDITION TO THEIR EVALUATION AS
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CH-1214 Vernier, Geneva
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WHICH REFERENCE MAY BE MADE IN
Reference number
Email: copyright@iso.org
NATIONAL REGULATIONS.
Website: www.iso.org ISO 16140-2:2016/DAM 1:2023(E)
RECIPIENTS OF THIS DRAFT ARE INVITED
Published in Switzerland
TO SUBMIT, WITH THEIR COMMENTS,
NOTIFICATION OF ANY RELEVANT PATENT
RIGHTS OF WHICH THEY ARE AWARE AND TO
ii
© ISO 2023 – All rights reserved
PROVIDE SUPPORTING DOCUMENTATION. © ISO 2023
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO's adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 34, Food products, Subcommittee SC
9, Microbiology, in collaboration with the European Committee for Standardization (CEN) Technical
Committee CEN/TC 463, Microbiology of the food chain, in accordance with the Agreement on technical
cooperation between ISO and CEN (Vienna Agreement).
A list of all parts in the ISO 16140 series can be found on the ISO website.
Any feedback or questions on this document should be directed to the user’s national standards body.
A complete listing of these bodies can be found at www.iso.org/members.html.
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SIST EN ISO 16140-2:2016/oprA1:2023
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
Microbiology of the food chain — Method validation —
Part 2:
Protocol for the validation of alternative (proprietary)
methods against a reference method
AMENDMENT 1: Revision of qualitative MCS data evaluation,
RLOD calculations in the ILS, calculation and interpretation
of the RT study, and inclusion of a commercial sterility testing
protocol for specific products
Introduction
Replace the text with the following:
Introduction
0.1 The ISO 16140 series
The ISO 16140 series has been expanded in response to the need for various ways to validate or verify
test methods. It is the successor to ISO 16140:2003. The ISO 16140 series consists of six parts with the
general title, Microbiology of the food chain — Method validation:
— Part 1: Vocabulary;
— Part 2: Protocol for the validation of alternative (proprietary) methods against a reference method;
— Part 3: Protocol for the verification of reference methods and validated alternative methods in a single
laboratory;
— Part 4: Protocol for method validation in a single laboratory;
— Part 5: Protocol for factorial interlaboratory validation for non-proprietary methods;
— Part 6: Protocol for the validation of alternative (proprietary) methods for microbiological confirmation
and typing procedures.
ISO 17468 is a closely linked International Standard, which establishes technical rules for the
development and validation of standardized methods.
In general, two stages are needed before a method can be used in a laboratory.
— The first stage is the validation of the method. Validation is conducted using a study in a single
laboratory followed by an interlaboratory study (see ISO 16140-2, ISO 16140-5 and ISO 16140-6).
In the case when a method is validated within one laboratory (see ISO 16140-4), no interlaboratory
study is conducted.
— The second stage is method verification, where a laboratory demonstrates that it can satisfactorily
perform a validated method. This is described in ISO 16140-3. Verification is only applicable to
methods that have been validated using an interlaboratory study.
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
In general, two types of methods are distinguished: reference methods and alternative methods.
A reference method is defined in ISO 16140-1:2016, 2.59, as an “internationally recognized and widely
accepted method”. The note to entry clarifies that “these are ISO standards and standards jointly
published by ISO and CEN or other regional/national standards of equivalent standing”.
In the ISO 16140 series, reference methods include standardized reference (ISO and CEN) methods as
defined in ISO 17468:2016, 3.5, as a “reference method described in a standard”.
An alternative method (method submitted for validation) is defined in ISO 16140-1:2016, 2.4, as a
“method of analysis that detects or quantifies, for a given category of products, the same analyte as
is detected or quantified using the corresponding reference method”. The note to entry clarifies that:
“The method can be proprietary. The term ‘alternative’ is used to refer to the entire ‘test procedure
and reaction system’. This term includes all ingredients, whether material or otherwise, required for
implementing the method.”.
ISO 16140-4 addresses validation within a single laboratory. The results are therefore only valid for
the laboratory that conducted the study. In this case, verification (as described in ISO 16140-3) is not
applicable. ISO 16140-5 describes protocols for non-proprietary methods where a more rapid validation
is required or when the method to be validated is highly specialized and the number of participating
laboratories required by ISO 16140-2 cannot be reached. ISO 16140-4 and ISO 16140-5 can be used for
validation against a reference method. ISO 16140-4 (regarding qualitative and quantitative methods)
and ISO 16140-5 (regarding quantitative methods only) can also be used for validation without a
reference method.
The flow chart in Figure 0.1 gives an overview of the links between the different parts mentioned
above. It also guides the user in selecting the right part of the ISO 16140 series, taking into account the
purpose of the study and the remarks given above.
Figure 0.1 — Flow chart for application of the ISO 16140 series
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
NOTE In this document, the words “category”, “type” and/or “item” are sometimes combined with “(food)”
to improve readability. However, the word “(food)” is interchangeable with “(feed)” and other areas of the food
chain as mentioned in Clause 1.
ISO 16140-6 is somewhat different from the other parts in the ISO 16140 series in that it relates to
a very specific situation where only the confirmation procedure of a method is to be validated [e.g.
the biochemical confirmation of Enterobacteriaceae (see ISO 21528-2)]. The confirmation procedure
advances a suspected (presumptive) result to a confirmed positive result. The validation of alternative
typing techniques (e.g. serotyping of Salmonella) is also covered by ISO 16140-6. The validation study
in ISO 16140-6 clearly defines the selective agar(s) from which strains can be confirmed using the
alternative confirmation method. If successfully validated, the alternative confirmation method can
only be used if strains are recovered on an agar that was used and shown to be acceptable within the
validation study. Figure 0.2 shows the possibilities where an alternative confirmation method validated
in accordance with ISO 16140-6 can be applied (see text in the boxes).
Figure 0.2 — Use of validated alternative confirmation methods (see ISO 16140-6)
EXAMPLE An example application of a validated alternative confirmation method is as follows.
An alternative confirmation method based on ELISA has been validated (in accordance with
ISO 16140-6) to replace the biochemical confirmation for Salmonella as described in ISO 6579-1. In
the validation study, XLD (mandatory agar in accordance with ISO 6579-1) plus BGA and a specified
chromogenic agar (two optional agars for second plating in accordance with ISO 6579-1) were used
as the agars to start the confirmation. The validated confirmation method can be used to replace the
biochemical confirmation under the following conditions:
— by laboratories using the ISO 6579-1; or
— by laboratories using an ISO 16140-2 validated alternative method that refers to ISO 6579-1 for
confirmation; or
— by laboratories using an ISO 16140-2 validated alternative method that starts the confirmation
from XLD and/or BGA agar and/or the specified chromogenic agar.
The validated confirmation method cannot be used under the following conditions:
— by laboratories using an ISO 16140-2 validated alternative method that refers only to agars other
than those included in the validation to start the confirmation (e.g. Hektoen agar and SS agar only);
or
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
— by laboratories using an ISO 16140-2 validated alternative method that refers only to a confirmation
procedure that does not require isolation on agar.
0.2 Validation protocols in the ISO 16140 series
ISO 16140-2 describes the general approach to method validation in the field of microbiology of the food
chain and serves as a fundamental basis to the other parts of the ISO 16140 series, which cross-refer to
it. An understanding of the performance characteristics, the (food) categories, the technical protocol
and data analysis as outlined in ISO 16140-2, will provide support in the application of the ISO 16140
series in general.
Clause 4
Add the following text at the end of the clause:
For the validation of an alternative qualitative method, a corresponding qualitative reference method
is selected for carrying out the validation study. This is commonly done using test portions of 10 gram,
25 gram or higher. In some cases it can be of interest to validate a qualitative alternative method against
a quantitative reference method, using smaller test portion sizes.
EXAMPLE 1 Enterobacteriaceae criterion for pasteurized milk and other liquid pasteurized products in
REGULATION (EC) No 2073/2005 is < 10 cfu/ml and refers to the quantitative method ISO 21528-2.
In such situations, it is of interest to validate the performance of qualitative alternative methods against
the specified (quantitative) reference method. To that end, the technical protocol for the validation of
qualitative methods (Clause 5) is to be used. For such validation study, the quantitative results of the
reference method have to be converted into qualitative results prior to interpretation according to
Clause 5.
-2
EXAMPLE 2 When one or more colonies are observed on a plate using 1 ml of a 10 dilution this result
corresponds to a positive detection in 0,01 gram.
NOTE Annex J provides the special case of validation of a method for commercial sterility testing of sterilized
or Ultra High Temperature (UHT) dairy and plant-based liquid products.
If a technical change in a validated alternative (proprietary) method is evaluated as being major, a re-
validation of this alternative method in accordance with ISO 16140-2 is needed.
When the re-validation of the alternative method is conducted, the impact on the performance
characteristics shall be evaluated to determine if the changes are to be regarded as major (performance
characteristics have substantially changed) or minor (no impact on performance characteristics
observed). In certain cases, a major technical change in the method can be considered to be minor, if
the re-validation study shows that it has no significant impact on the performance characteristics or
test results. A major (technical) change that, after re-validation, has a major impact on the performance
characteristics of the alternative method, requires re-verification of the method by the user laboratory
in accordance with ISO 16140-3.
5.1.1
Add the following text at the end of the subclause:
The organizing laboratory shall be competent to perform both the reference method as well as the
alternative method.
NOTE Competence can be demonstrated in different ways, e.g. for the reference method an ISO 17025
accreditation and for the alternative method a documented training.
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
5.1.2
Add the following text at the end of the subclause:
NOTE When the reference and alternative methods are based on two different principles and are performed
with the same test portion, but do not share a common enrichment procedure, an unpaired data study is
considered. For example, when a qualitative alternative method is validated against a quantitative reference
method at a limit of 100 cfu/g. In this case a suspension of the (food) item can be used to inoculate both culture
media for the reference method and the alternative method before any enrichment/multiplication of the
microorganism.
5.1.3.3
Add the following text at the end of the subclause:
The alternative method shall be evaluated for a defined test portion size (e.g. 25 g, 200 g, 375 g)
during the validation study. The method is considered to be validated for any test portion size up to
the validated test portion size if the testing protocol (dilution ratio, incubation time and incubation
temperature) is the same as that used during the validation study.
EXAMPLE 1 A reference method used in an ISO 16140-2 validation study was validated for a ‘broad range of
foods’ using 25 g test portion and a 1:10 dilution ratio. The alternative method was validated for the category of
“Raw meat and ready to cook meat products (except poultry)” using 375 g test portion and 1:10 dilution ratio at a
determined incubation time. In practice, a user laboratory can use the alternative method for the “Raw meat and
ready to cook meat products (except poultry)” with test portion sizes up to 375 g with a 1:10 dilution ratio at a
validated incubation time (unless stated differently by the organization involved in the method validation).
EXAMPLE 2 A reference method used in an ISO 16140-2 validation study was validated for a ‘broad range of
foods’ using 25 g test portion and a 1:10 dilution ratio. The alternative method was validated for a ‘broad range
of foods’ using 25 g test portion and a 1:5 dilution ratio. In practice, a user laboratory can use the alternative
method for all food items (broad range of foods) using a test portion size up to 25 g test portion and a 1:5 dilution
ratio (unless stated differently by the organization involved in the method validation).
5.1.3.4
Add the following text before the last sentence of the second paragraph:
The interpretation of the results (positive agreement, negative agreement, etc.) is based on a comparison
of the reference method result (column 1 in Tables 1 and 2) and the alternative method result, including
any confirmations as described in the alternative method protocol, (column 2 in Tables 1 and 2). When
positive or negative deviations are obtained, a footnote should be included at the end of each table
to provide additional explanations for the interpretation of the deviations. The footnotes indicate if
the result is due to a false positive or false negative result of the alternative method. The footnote is a
comparison of the results of the alternative method (including any confirmations as described in the
alternative-method protocol) (column 2 in Tables 1 and 2) and the confirmed alternative method (by
any means) (column 3 in Tables 1 and 2).
5.1.3.4, after the second paragraph
Replace the text with the following:
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
Table 1 — Comparison and interpretation of sample results between the reference and
alternative methods for a paired study
Result of the (reference or alternative) method per sample
Reference Alternative method Confirmed Interpretation
method (including any confirmations alternative (based on the confirmed
as described in the method alternative-method result)
a
alternative-method protocol) (by any means)
b
+ + Not needed Positive Agreement (PA)
b
- - Not needed Negative Agreement (NA)
Negative Deviation due to false
b
+ - Not needed negative alternative-method result
(ND )
FN(alt)
- + + Positive Deviation (PD)
Positive Deviation due to false
- + - positive alternative-method result
c
(PD )
FP(alt)
a
Confirmation of the alternative-method result is done according to 5.1.3.3.
b
No need for additional confirmation test(s). Confirmed alternative-method result is the same as the alternative-method
result.
c
This false-positive result (FP) shall also be used to calculate the false positive ratio.
Table 2 — Comparison and interpretation of sample results between the reference and
alternative methods for an unpaired study
Result of the (reference or alternative) method per sample
Reference Alternative method Confirmed Interpretation
method (including any confirmations alternative method (based on the confirmed
a,b
as described in the (by any means) alternative-method result)
alternative-method protocol)
+ + + Positive Agreement (PA)
Positive Agreement due to false
+ + - positive alternative-method result
c
(PA )
FP(alt)
- - - Negative Agreement (NA)
Negative Agreement due to false
- - + negative alternative-method result
(NA )
FN(alt)
+ - - Negative Deviation (ND)
Negative Deviation due to false
+ - + negative alternative-method result
(ND )
FN(alt)
- + + Positive Deviation (PD)
Positive Deviation due to false
- + - positive alternative-method result
c
(PD )
FP(alt)
a
Confirmation of the alternative-method result is done according to 5.1.3.3
b
Confirmation by any means is only required when the result of the alternative method does not produce viable
organisms. This will be used as the confirmed alternative method result in comparison to the reference method result.
c
These false-positive results (FP) shall also be used to calculate the false positive ratio.
Determine the Total Negative Deviation (TND) and Total Negative Agreement (TNA) for the validation
study.
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
Paired evaluation: Total Negative Deviation: TND = ND
FN(alt)
Total Negative Agreement: TNA = NA + PD
FP(alt)
Unpaired evaluation: Total Negative Deviation: TND = ND + ND + PA
FN(alt) FP(alt)
Total Negative Agreement: TNA = NA + NA + PD
FN(alt) FP(alt)
Table 3 — Summary of results obtained with the reference and alternative methods (after
confirmation) of all samples for each category
Reference-method positive Reference-method negative
(R+) (R-)
+/+ -/+
Alternative-method positive (A+)
Positive Agreement (PA) Positive Deviation (PD)
+/- −/−
Alternative-method negative (A-)
Total Negative Deviation (TND) Total Negative Agreement (TNA)
Based on data summarized in Table 3 for the combined categories per category and per type, calculate
the values for sensitivity of the alternative method (formula 1) and of the reference method (formula 2),
as well as the relative trueness (formula 3) and false positive ratio for the alternative method after the
additional confirmation of the results (two formulae under 4) as follows:
Sensitivity for the alternative method:
()PA+PD
SE = ×100% (1)
alt
PA++TNDPD
()
Sensitivity for the reference method:
()PA+TND
SE = ×100% (2)
ref
()PA+TND+PD
Relative trueness:
()PA+TNA
RT= ×100% (3)
N
False positive ratio for the alternative method:
PD
FP()alt
Paired evaluation: FPR=×100 %
TNA
(4)
PA + PD
FP()altFPa()lt
Unpaired evaluationn: FPR= ×100 %
TNA
where N is the total number of samples (PA + PD + TND + TNA) and FP is the false-positive results. For
explanation of the abbreviations used, see Table 1 to Table 3.
The confirmed alternative-method results shall be used to determine whether the alternative method
produces comparable results to the reference method.
Calculate the difference between (TND – PD) for both paired and unpaired studies and the sum of
(TND + PD) for paired studies. Check whether the difference and/or sum of PD and TND conform to the
Acceptability Limit (AL) stated in Table 4 with respect to the type of study (paired or unpaired) and
the number of categories used in the evaluation.
NOTE 1 Acceptability Limits (AL) are based on data and consensus expert opinion. The AL are not based on
statistical analysis of the data.
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SIST EN ISO 16140-2:2016/oprA1:2023
ISO 16140-2:2016/DAM 1:2023(E)
The interpretation of results shall be done per category and for all categories used in the validation
study. An interpretation of results shall also be done per enrichment protocol in case different
protocols are used for different types of samples. A sensitivity study can also exist of a partly paired
and unpaired study. In that case the results for (TND + PD) shall be evaluated based on the number of
positive samples obtained for the categories tested using the paired study design. The results for (TND
– PD) shall be evaluated based on the number of positive samples obtained for the full study (so all
categories belonging to both the paired and unpaired study design).
The AL is not met when the observed value is higher than the AL. When the AL is not met, if the number
of positive samples is higher than expected according to the number of categories (e.g. having 60 or more
positive samples for one single category), it is possible to use the second column of Table 4 and switch
to higher AL. When the AL is not met, investigations should be made (e.g. root cause analysis) in order
to provide an explanation of the observed results. Based on the AL and the additional information, it is
decided whether the alternative method is regarded as not fit for purpose for the category or categories
involved. The reasons for acceptance of the alternative method in case the AL is not met shall be stated
in the study report.
Table 4 — Acceptability limit parameters and values for a paired and unpaired study design in
relation to the number of positive samples obtained
c
Paired study Unpaired study Mixed study
Number of Number of positive
a b
categories samples (N+)
(TND - PD ) (TND + PD) (TND - PD) (TND + PD) (TND - PD)
1 30 to 59 3 6 3 6 3
2 60 to 89 4 8 4 8 4
3 90 to 119 5 10 5 10 5
4 120 to 149 5 12 5 12 5
5 150 to 179 5 14 5 14 5
6 180 to 209 6 16 6 16 6
7 210 to 239 6 18 7 18 7
8 240 to 269 6 20 7 20 7
9 270 to 299 7 22 8 22 8
10 300 to 329 7 24 8 24 8
11 330 to 359 7 26 9 26 9
12 360 to 389 8 28 9 28 9
13 390 to 419 8 30 10 30 10
14 420 to 449 8 32 10 32 10
15 450 to 479 9 34 11 34 11
a
TND = total number of samples with Negative Deviation results.
b
PD = number of samples with Positive Deviation results.
c
Mixed study includes both paired and unpaired study design.
NOTE 2 A negative value for (TND – PD) is acceptable as this indicates a better performance of the alternative
method compared to the reference method.
NOTE 3 Information on differences observed between results of the alternative method before and after
confirmation of the results (step 1 and step 2) according to the alternative-method protocol should be presented
in the validation report as additional information but is not used in the overall assessment of the alternative-
method performance.
5.1.4, last sentence
Replace the text with the following:
The level of contamination shall be determined. This allows calculation of the LOD of the alternative
50
method, which is required in order to verify the performance of the alternative method upon
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SIST
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