SIST EN ISO 16140:2003/A1:2012
(Amendment)Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods - Amendment 1 (ISO 16140:2003/AMD 1:2011)
Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative methods - Amendment 1 (ISO 16140:2003/AMD 1:2011)
This EN ISO 16140 defines the general principle and the technical protocol for the validation of alternative methods in the field of microbiological analysis of food, animal feeding stuff and environmental and veterinary samples (see 5.1.1.2.1) for:
- the validation of alternative methods which can be used in particular in the framework of the official control;
- the international acceptance of the results obtained by the alternative method.
It also gives the general principles of certification of these alternative methods, based on the validation protocol defined in this EN ISO 16140 (see 4.2).
Where an alternative method is used on a routine basis for internal laboratory use without the requirement to meet (higher) external criteria of quality assurance, a less stringent comparative validation of the alternative method than that set in this standard may be appropriate.
Mikrobiologie von Lebensmitteln und Futtermitteln - Arbeitsvorschrift für die Validierung alternativer Verfahren (ISO 16140:2003/AMD 1:2011)
Microbiologie des aliments - Protocole pour la validation des méthodes alternatives - Amendement 1: Étude interlaboratoire sur les méthodes quantitatives (ISO 16140:2003/AMD 1:2011)
Mikrobiologija živil in krme - Protokol za validacijo alternativnih metod - Dopolnilo 1: Medlaboratorijske raziskave o kvantitativnih metodah (ISO 16140:2003/AMD 1:2011)
Ta EN ISO 16140 določa splošni princip in tehnični protokol za validacijo alternativnih metod na področju mikrobiološke analize živil, krme ter okoljskih in veterinarskih vzorcev (glej 5.1.1.2.1) za:
- validacijo alternativnih metod, ki se lahko uporablja predvsem v okviru uradnih kontrol;
- mednarodno sprejemljivost rezultatov, pridobljenih z alternativno metodo.
Prav tako podaja splošna načela certifikacije teh alternativnih metod, zasnovanih na validacijskem protokolu, določenem v tem EN ISO 16140 (glej 4.2).
Kjer se na ustaljen način za interno uporabo v laboratoriju uporablja alternativna metoda brez zahtev za izpolnitev (višjih) zunanjih meril zagotavljanja kakovosti, je lahko primerna manj strožja primerljiva validacija alternativne metode, kot je tista, podana v tem standardu.
General Information
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Standards Content (Sample)
SLOVENSKI STANDARD
SIST EN ISO 16140:2003/A1:2012
01-januar-2012
Mikrobiologija živil in krme - Protokol za validacijo alternativnih metod - Dopolnilo
1: Medlaboratorijske raziskave o kvantitativnih metodah (ISO 16140:2003/AMD
1:2011)
Microbiology of food and animal feeding stuffs - Protocol for the validation of alternative
methods - Amendment 1 (ISO 16140:2003/AMD 1:2011)
Mikrobiologie von Lebensmitteln und Futtermitteln - Arbeitsvorschrift für die Validierung
alternativer Verfahren (ISO 16140:2003/AMD 1:2011)
Microbiologie des aliments - Protocole pour la validation des méthodes alternatives -
Amendement 1: Étude interlaboratoire sur les méthodes quantitatives (ISO
16140:2003/AMD 1:2011)
Ta slovenski standard je istoveten z: EN ISO 16140:2003/A1:2011
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 16140:2003/A1:2012 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.
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SIST EN ISO 16140:2003/A1:2012
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SIST EN ISO 16140:2003/A1:2012
EUROPEAN STANDARD
EN ISO 16140:2003/A1
NORME EUROPÉENNE
EUROPÄISCHE NORM
August 2011
ICS 07.100.30
English Version
Microbiology of food and animal feeding stuffs - Protocol for the
validation of alternative methods - Amendment 1 (ISO
16140:2003/AMD 1:2011)
Microbiologie des aliments - Protocole pour la validation Mikrobiologie von Lebensmitteln und Futtermitteln -
des méthodes alternatives - Amendement 1 (ISO Arbeitsvorschrift für die Validierung alternativer Verfahren -
16140:2003/AMD 1:2011) Änderung 1 (ISO 16140:2003/AMD 1:2011)
This amendment A1 modifies the European Standard EN ISO 16140:2003; it was approved by CEN on 29 July 2011.
CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for inclusion of this
amendment into the relevant national standard without any alteration. Up-to-date lists and bibliographical references concerning such
national standards may be obtained on application to the CEN-CENELEC Management Centre or to any CEN member.
This amendment exists in three official versions (English, French, German). A version in any other language made by translation under the
responsibility of a CEN member into its own language and notified to the CEN-CENELEC Management Centre has the same status as the
official versions.
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, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland and United Kingdom.
EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION
EUROPÄISCHES KOMITEE FÜR NORMUNG
Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2011 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 16140:2003/A1:2011: E
worldwide for CEN national Members.
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SIST EN ISO 16140:2003/A1:2012
EN ISO 16140:2003/A1:2011 (E)
Contents Page
Foreword .3
2
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SIST EN ISO 16140:2003/A1:2012
EN ISO 16140:2003/A1:2011 (E)
Foreword
This document (EN ISO 16140:2003/A1:2011) has been prepared by Technical Committee ISO/TC 34 "Food
products" in collaboration with Technical Committee CEN/TC 275 “Food analysis - Horizontal methods” the
secretariat of which is held by DIN.
This Amendment to the European Standard EN ISO 16140:2003 shall be given the status of a national
standard, either by publication of an identical text or by endorsement, at the latest by February 2012, and
conflicting national standards shall be withdrawn at the latest by February 2012.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN/CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to implement this European Standard: 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, Romania, Slovakia, Slovenia, Spain,
Sweden, Switzerland and the United Kingdom.
Endorsement notice
The text of ISO 16140:2003/AMD 1:2011 has been approved by CEN as a EN ISO 16140:2003/A1:2011
without any modification.
3
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SIST EN ISO 16140:2003/A1:2012
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SIST EN ISO 16140:2003/A1:2012
INTERNATIONAL ISO
STANDARD 16140
First edition
2003-05-01
AMENDMENT 1
2011-08-15
Microbiology of food and animal feeding
stuffs — Protocol for the validation of
alternative methods
AMENDMENT 1
Microbiologie des aliments — Protocole pour la validation des
méthodes alternatives
AMENDEMENT 1
Reference number
ISO 16140:2003/Amd.1:2011(E)
©
ISO 2011
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SIST EN ISO 16140:2003/A1:2012
ISO 16140:2003/Amd.1:2011(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2011
All rights reserved. Unless otherwise specified, no part of this publication may be reproduced or utilized in any form or by any means,
electronic or mechanical, including photocopying and microfilm, without permission in writing from either ISO at the address below or
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Published in Switzerland
ii © ISO 2011 – All rights reserved
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SIST EN ISO 16140:2003/A1:2012
ISO 16140:2003/Amd.1:2011(E)
Contents Page
Foreword . iv
6.3 Interlaboratory study . 2
6.3.1 General . 2
6.3.2 Terms and definitions . 2
6.3.3 Measurement protocol and samples . 4
6.3.4 Determination of the trueness and precision characteristics . 5
6.3.5 Scrutiny of the measurement results for consistency . 7
6.3.6 Comparison of the trueness and precision characteristics of the reference method and
the alternative method . 8
Annex Q (normative) Calculation of Rousseeuw's scale estimator Q . 9
n
Annex V (informative) Indicators for Mandel's robust h- and k-statistics . 11
Annex W (informative) Example: Determination of E. coli . 12
Bibliography additions . 18
© ISO 2011 – All rights reserved iii
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SIST EN ISO 16140:2003/A1:2012
ISO 16140:2003/Amd.1:2011(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.
International Standards are drafted in accordance with the rules given in the ISO/IEC Directives, Part 2.
The main task of technical committees is to prepare International Standards. Draft International Standards
adopted by the technical committees are circulated to the member bodies for voting. Publication as an
International Standard requires approval by at least 75 % of the member bodies casting a vote.
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.
Amendment 1 to ISO 16140:2003 was prepared by Technical Committee ISO/TC 34, Food products,
Subcommittee SC 9, Microbiology.
iv © ISO 2011 – All rights reserved
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SIST EN ISO 16140:2003/A1:2012
ISO 16140:2003/Amd.1:2011(E)
Microbiology of food and animal feeding stuffs — Protocol for
the validation of alternative methods
AMENDMENT 1
Page 22, 6.3
Replace the existing text with that commencing on p. 2.
Page 61, Annex Q
Replace the existing text with that commencing on p. 9.
Page 74, Bibliography
Insert Annexes V and W commencing on p. 11 before the bibliography.
Additions to the bibliography are given on p. 18.
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SIST EN ISO 16140:2003/A1:2012
ISO 16140:2003/Amd.1:2011(E)
6.3 Interlaboratory study
6.3.1 General
An interlaboratory study aims to determine the comparative performance characteristics (trueness and
precision characteristics) of the alternative method against the reference method.
Guidelines and requirements for organizing, dispatching and conducting the interlaboratory study are given in
Annex H and in ISO 5725-2.
At least eight laboratories shall participate in an interlaboratory study.
The organizing laboratory is responsible for the preparation of the test protocol and a data sheet (see below)
to be used by each laboratory for recording all measurement results and critical experimental conditions
(see H.3).
The analyst in each collaborating laboratory shall demonstrate competence in the use of the alternative
method and of the reference method prior to participating in the study.
In microbiology, the data {y} of repeated measurements do not always show a normal (Gaussian) distribution.
Therefore, the distribution of these data should be checked for normality if more than 30 values are available
at the same level. In order to get a more symmetric distribution, take logarithms of the counts.
The data obtained from interlaboratory studies often contain outliers, i.e. measurement values that deviate so
much from comparable measurements that they are considered inconsistent. If they are retained in the data
set, the trueness and precision characteristics (averages, standard deviations, etc.) obtained with classical
methods of statistical analysis are unreliable. Therefore, ISO 5725-2 includes outlier tests (Cochran, Grubbs)
in order to detect and eventually discard outliers and to obtain reliable trueness and precision characteristics.
Often this causes disputes as to which of the outliers should be discarded from or retained in the statistical
analysis. In order to avoid such disputes, robust estimates of the trueness and precision characteristics are
used in this International Standard. Since they are insensitive to any extreme values, they always use the
complete data set obtained from the interlaboratory study.
However, each extreme value should be checked for a clerical error in transcribing the measurement result,
an error in computation, a slip in performing the measurement or an analysis of the wrong sample. If possible,
such values should be replaced by the correct values. Other extreme measurement results or laboratories
reporting extreme values are not excluded from the statistical analysis unless exclusion is based on
sound microbiological reasons.
6.3.2 Terms and definitions
For the purpose of this document, the following terms and definitions apply.
6.3.2.1
accuracy
closeness of agreement between a test result or measurement result and the true value
NOTE 1 In practice, the accepted reference value is substituted for the true value.
[13]
[ISO 3534-2:2006 , 3.3.1]
NOTE 2 Accuracy refers to a combination of trueness and precision.
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SIST EN ISO 16140:2003/A1:2012
ISO 16140:2003/Amd.1:2011(E)
6.3.2.2
trueness
closeness of agreement between the expectation of a test result or a measurement result and the true value
NOTE 1 The measure of trueness is usually expressed in terms of bias.
NOTE 2 In practice, the accepted reference value is substituted for the true value.
[13]
[ISO 3534-2:2006 , 3.3.3]
6.3.2.3
precision
closeness of agreement between independent test/measurement results obtained under stipulated conditions
NOTE Quantitative measures of precision depend critically on the stipulated conditions. Repeatability conditions and
reproducibility conditions are particular sets of extreme stipulated conditions.
[13]
[ISO 3534-2:2006 , 3.3.4]
6.3.2.4
repeatability
precision under repeatability conditions
[13]
[ISO 3534-2:2006 , 3.3.5]
6.3.2.5
repeatability conditions
observation conditions where independent test/measurement results are obtained with the same method on
identical test/measurement items in the same test or measuring facility by the same operator using the same
equipment within short intervals of time
[13]
[ISO 3534-2:2006 , 3.3.6]
6.3.2.6
repeatability standard deviation
standard deviation of test result or measurement results obtained under repeatability conditions
[13]
[ISO 3534-2:2006 , 3.3.7]
6.3.2.7
repeatability limit
r
value less than or equal to which the absolute difference between two measurement results obtained under
repeatability conditions is expected to be with a probability of 95 %
[13]
NOTE Adapted from ISO 3534-2:2006 , 3.3.9.
6.3.2.8
reproducibility
precision under reproducibility conditions
[13]
[ISO 3534-2:2006 , 3.3.10]
6.3.2.9
reproducibility conditions
observation conditions where independent test/measurement results are obtained with the same method on
identical test/measurement items in different test or measuring facilities with different operators using different
equipment
[13]
[ISO 3534-2:2006 , 3.3.11]
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SIST EN ISO 16140:2003/A1:2012
ISO 16140:2003/Amd.1:2011(E)
6.3.2.10
reproducibility standard deviation
standard deviation of test results or measurement results obtained under reproducibility conditions
[13]
[ISO 3534-2:2006 , 3.3.12]
6.3.2.11
reproducibility limit
R
value less than or equal to which the absolute difference between two measurement results obtained under
reproducibility conditions is expected to be with a probability of 95 %
[13]
NOTE Adapted from ISO 3534-2:2006 , 3.3.14.
6.3.3 Measurement protocol and samples
The protocol is the following.
One relevant food matrix is used (see Annex B).
The analyte concentrations should be chosen to cover at least the lower, middle and upper levels of the
entire range of the alternative method. The organizing laboratory shall assure that the samples are
homogeneous (Reference [14]). A negative control should also be included.
Artificial contamination of a food sample with the target analyte may be used.
To compare the alternative method with the reference method the same samples shall be used for each
method. Four sub-samples from each level (or two aliquots, each measured by both methods) are
prepared for each laboratory. These are blind coded but labelled so that two are measured by the
reference method and two are measured by the alternative method.
Liquid samples (compared to solid samples) give greater assurance of homogeneity if prepared and
dispatched without change in microbiological content and used correctly. In specific cases, it could be
necessary to subdivide the samples immediately before measurement with both methods.
The measurements shall be performed in each collaborative laboratory and the organizing laboratory at a
stipulated date using common batches of media and kits.
The rounding of results shall be set by the organizing laboratory.
For each level j, the results of the interlaboratory study shall be presented as shown in Table 9.
Table 9 — Presentation of the results of the interlaboratory study at level j
Laboratory Reference method (coded) Alternative method (coded)
i
Duplicate 1 Duplicate 2 Duplicate 1 Duplicate 2
1
2
…
p
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SIST EN ISO 16140:2003/A1:2012
ISO 16140:2003/Amd.1:2011(E)
6.3.4 Determination of the trueness and precision characteristics
6.3.4.1 General
For each level j, the following trueness and precision characteristics are determined:
the median of the laboratory means of the measurement results of the reference method and of the
alternative method (for the estimation of bias);
the repeatability standard deviation of the reference method and of the alternative method based on the
calculation of Rousseeuw's Q (see Annex Q);
n
the reproducibility standard deviation of the reference method and of the alternative method based on
Rousseeuw's Q (see Annex Q).
n
6.3.4.2 Calculations
For each level j, perform the calculations according to the following steps. In the following text, the subscript j
is omitted.
Step 1. With the measurement results for the reference method (Table 9, columns 2 and 3), prepare Table 10
for each level j.
Table 10 — Measurement results, means and deviations at level j
(for the reference method or for the alternative method)
1 2 3 4 5 6
Laboratory Measurement Mean Deviation from the mean
1 y y y d d
11 12 11 12
1
2 y y y d d
21 22 2 21 22
… … … … … …
i y y y d d
i1 i2 i i1 i2
… … … … … …
p y y y d d
p1 p2 p1 p2
p
In Table 10,
yy
ii12
y
i
2
is the mean of the two measurements in laboratory i and
yy
ii12
dyy
ii11 i
2
yy
ii21
dyy
ii22 i
2
are the deviations of these two measurements from their mean.
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SIST EN ISO 16140:2003/A1:2012
ISO 16140:2003/Amd.1:2011(E)
Step 2. Use the deviations of Table 10, columns 5 and 6 as a series of n 2p values and calculate
Rousseeuw's bias corrected scale estimator Q :
intra
Q c Q with n 2p
intra n n
as described in Annex Q.
Step 3. Calculate Rousseeuw's bias corrected scale estimator Q :
inter
Q c Q with n p
inter n n
of the laboratory means of Table 10, column 4 as described in Annex Q.
Step 4. Calculate the median m of the laboratory means of the results of the reference method (Table 10,
column 4).
Step 5. Calculate the repeatability s
...
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