Foodstuffs -- Nucleic acid based methods of analysis of genetically modified organisms and derived products -- Information to be supplied and procedure for the addition of methods to ISO 21569, ISO 21570 or ISO 21571

ISO/TS 21098:2005 defines the principles and specifies the nature of the information to be supplied for acceptance of a method as an annex to ISO 21569, ISO 21570 or ISO 21571. It also specifies the process for adding, amending and retaining methods annexed to these standards. ISO/TS 21098:2005 is necessary in order to attain consistency in methods that are to be employed as part of the standards. It does not cover the specifics of the development of a method or laboratory set-up.

Produits alimentaires -- Méthodes basées sur les acides nucléiques pour l'analyse des organismes génétiquement modifiés et des produits dérivés -- Informations à fournir et procédure pour l'addition de méthodes à l'ISO 21569, l'ISO 21570 ou l'ISO 21571

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Publication Date
05-Sep-2005
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05-Sep-2005
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6060 - International Standard published
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Completion Date
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ISO/TS 21098:2005 - Foodstuffs -- Nucleic acid based methods of analysis of genetically modified organisms and derived products -- Information to be supplied and procedure for the addition of methods to ISO 21569, ISO 21570 or ISO 21571
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TECHNICAL ISO/TS
SPECIFICATION 21098
First edition
2005-09-15
Foodstuffs — Nucleic acid based
methods of analysis of genetically
modified organisms and derived
products — Information to be supplied
and procedure for the addition of
methods to ISO 21569, ISO 21570 or
ISO 21571
Produits alimentaires — Méthodes basées sur les acides nucléiques
pour l'analyse des organismes génétiquement modifiés et des produits
dérivés — Informations à fournir et procédure pour l'addition de
méthodes à l'ISO 21569, l'ISO 21570 ou l'ISO 21571
Reference number
ISO/TS 21098:2005(E)
ISO 2005
---------------------- Page: 1 ----------------------
ISO/TS 21098:2005(E)
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ii © ISO 2005 – All rights reserved
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ISO/TS 21098:2005(E)
Contents Page

Foreword............................................................................................................................................................ iv

Introduction ........................................................................................................................................................ v

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

2 DNA-based analysis ............................................................................................................................. 1

3 Multi-laboratory studies ....................................................................................................................... 1

4 Description of information to be supplied about a method submitted to be annexed to

ISO 21569, ISO 21570 and ISO 21571.................................................................................................. 2

5 Validation of methods .......................................................................................................................... 3

5.1 General................................................................................................................................................... 3

5.2 Validation of quantitative methods..................................................................................................... 3

5.3 Validation of qualitative methods ....................................................................................................... 4

6 Process for adding, amending and retaining methods (as annexes) .............................................. 5

6.1 Expert group for consideration of the methods ................................................................................ 5

6.2 Addition of methods to the standards................................................................................................ 5

6.3 Amending of methods in the standards............................................................................................. 5

6.4 Retention of methods in the standards.............................................................................................. 6

Annex A (normative) Template for supplying the required information about a method to be

annexed to ISO 21569........................................................................................................................... 7

Annex B (normative) Template for supplying the required information about a method to be

annexed to ISO 21570......................................................................................................................... 11

Annex C (normative) Template for supplying the required information about a method to be

annexed to ISO 21571......................................................................................................................... 15

Bibliography ..................................................................................................................................................... 18

© ISO 2005 – All rights reserved iii
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ISO/TS 21098:2005(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.

In other circumstances, particularly when there is an urgent market requirement for such documents, a

technical committee may decide to publish other types of normative document:

⎯ an ISO Publicly Available Specification (ISO/PAS) represents an agreement between technical experts in

an ISO working group and is accepted for publication if it is approved by more than 50 % of the members

of the parent committee casting a vote;

⎯ an ISO Technical Specification (ISO/TS) represents an agreement between the members of a technical

committee and is accepted for publication if it is approved by 2/3 of the members of the committee casting

a vote.

An ISO/PAS or ISO/TS is reviewed after three years in order to decide whether it will be confirmed for a

further three years, revised to become an International Standard, or withdrawn. If the ISO/PAS or ISO/TS is

confirmed, it is reviewed again after a further three years, at which time it must either be transformed into an

International Standard or be withdrawn.

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.

ISO/TS 21098 was prepared by Technical Committee ISO/TC 34, Food products.

The reasons why this document is published as a Technical Specification are given in the Introduction.

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ISO/TS 21098:2005(E)
Introduction

ISO has an obligation to ensure that the international standards it develops, adopts and publishes are globally

relevant. Among the criteria detailed in Annex 4, paragraph 10, of the Second Triennial review of the operation

and implementation of the Barriers to Trade Agreement, dated 13 November 2000, it is stated that a globally

relevant standard should be performance based as opposed to design prescriptive. Thus any method

submitted for inclusion in an International Standard should contain sufficient information for its performance to

be judged.

Although ISO 24276 states “The criteria for the selection of methods are listed in the standards on the

detection of genetically modified organisms and derived products, ISO 21568, ISO 21569, ISO 21570 and

ISO 21571. Acceptable levels of performance for methods included in the annexes are those which have

preferably been collaboratively trialed/single laboratory validated. Methods selected for inclusion in the

annexes have either been validated according to ISO 5725, or the Harmonized Protocol (Horwitz 1995) or

according to Thompson et al. (2002)”, there is insufficient guidance in these documents to allow the analyst to

test whether a method is specifically suitable for inclusion in the annexes. It is important that an International

Standard or Technical Specification should be performance based. For a standard to be performance based,

a clear definition of performance characteristics must be available.

It was noted at the 5th meeting of ISO/TC 34/WG 7, held 18th to 20th February 2004 in Seoul, Korea, that

there is no formal process for submitting methods for inclusion in the standards. Although a number of specific

methods have been proposed as part of the proposed standards (ISO 21569, ISO 21570 and ISO 21571) and

associated general document (ISO 24276), there is not sufficient clarity for submitters to be able to judge

whether a method meets the standard, and no mechanism is in place to govern acceptability and/or adoption

of such method or for retaining methods in the standards.

Therefore, this Technical Specification was developed in order to provide guidance and to define the

performance characteristics that should be supplied for each method in order to ensure the global relevance

of these standards, and to delineate the process for adding, amending and retaining methods annexed to the

standards.
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TECHNICAL SPECIFICATION ISO/TS 21098:2005(E)
Foodstuffs — Nucleic acid based methods of analysis of
genetically modified organisms and derived products —
Information to be supplied and procedure for the addition of
methods to ISO 21569, ISO 21570 or ISO 21571
1 Scope

This Technical Specification defines the principles and specifies the nature of the information to be supplied

for acceptance of a method as an annex to ISO 21569, ISO 21570 or ISO 21571. It also specifies the process

for adding, amending and retaining methods annexed to these standards. This Technical Specification is

necessary in order to attain consistency in methods that are to be employed as part of the standards. It does

not cover the specifics of the development of a method or laboratory set-up. The operation of laboratories is

covered in ISO/IEC 17025.

Method validation is instrumental in assessing the reliability of a test method. Its central role is to establish

numerical values for the performance criteria that are to be established. ISO 24276 includes details on method

validation, taking into consideration specific technical issues related to the detection of genetically modified

organisms and derived products. Given the attention to, and widespread use of deoxyribonucleic acid-based

tests or protein-based tests, and the implications to trade of any discrepancies in test results, a

single-laboratory validation is most likely not warranted in this case and a multi-centre method validation could

be performed according to the international guidelines.
2 Deoxyribonucleic acid (DNA)-based analysis

DNA-based analysis is commonly performed using polymerase chain reaction (PCR), although ISO 21569,

ISO 21570 and ISO 21571 also allow for other methods. DNA is a high-molecular weight polymer that may be

degraded during food processing by, for example, heat, enzymes and mechanical shearing. In addition, the

DNA may be chemically altered by the formation of adducts, or by loss of the bases. Any degradation of the

DNA shall be considered when assessing method validation and applying performance criteria. Degradation of

DNA will affect the limit of detection and the limit of quantitation of the tests. It is important that the

performance criteria for a method consider this effect. Additionally, it is important to point out the restrictions

that method(s) may have in certain food matrices.

The annexes in ISO 21569, ISO 21570 and ISO 21571 should contain information on performance criteria

from which methods fit for ISO purposes may be selected. It is possible that two different methods for the

same event/sequence, both fulfilling the performance criteria, once established, will be included in the

annexes.

It should be noted that, due to the limitations of the instruments and other factors, quantitative PCR methods

in general do not follow a Gaussian distribution for blank values around the zero. Thus, the determination of

the limit of detection and limit of quantitation cannot be carried out assuming such a distribution, and will not

follow the procedures outlined, for example, in ISO 11843-1 Thus detection limits for quantitative PCR

methods cannot be determined using the mean and standard deviation of the blank samples, and shall be

determined experimentally, or methods shall be performed at levels which are significantly above the

detection limit.
3 Multi-laboratory studies

Under certain circumstances (i.e. when the conduct of a formal collaborative trial is not practicable), methods

may be validated via single laboratory validation (see Reference [11]). The methods used for determination of

the presence of material originating from biotechnology-derived crops and food are able to be, and are

intended to be, performed at multiple laboratories and shall therefore be validated by multi-laboratory

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ISO/TS 21098:2005(E)

collaborative studies. The results of such studies may be incorporated by reference to the relevant scientific

publication(s), in which case copies of the publications shall be submitted to the expert group when submitting

the method.

At the time that ISO 21569, ISO 21570 and ISO 21571 are being prepared, few methods have completed a

full multi-laboratory validation on a multi-regional basis. Therefore, as an interim measure, methods may be

appended as informative annexes to the standards, after a properly conducted single-laboratory validation, or

after validation in a small number of laboratories, providing that all the other criteria have been met. Methods

which have been validated on a limited basis but are lacking full interlaboratory validation data, may be

temporarily endorsed for a period not exceeding 3 years. Such methods will be reviewed within 3 years, at

which time they should have been properly validated in a full interlaboratory collaborative study.

4 Description of information to be supplied about a method submitted to be
annexed to ISO 21569, ISO 21570 and ISO 21571

The proposed informative annex should contain information about the performance characteristics of a

method. This includes specific information on the multi- or single-laboratory trial, including relevant information

obtained during prevalidation of the method (e.g. variation of parameters, reagents).

The method should be in a format that conforms to the template and be validated according to internationally

accepted norms (see Reference [12]). Templates are given in Annexes A, B and C. A complete and detailed

description of all the components of the method shall be included. In particular, the description shall address

the following.

a) Scientific basis: An overview of the principles of the method, such as DNA molecular biology based (e.g.

for real-time PCR) information should be provided. References to relevant scientific publications are

useful.

b) Scope/Applicability: The objective of the method and the relevance of the method should be indicated.

The matrix is of particular concern for these methods. Thus the method shall in particular give an

indication of the matrix (e.g. processed food, raw materials, DNA solution), the type of samples (e.g.

seeds, flour, pizza, cookies) and the range to which the method can be applied (range of use). Relevant

limitations of the method should also be addressed (e.g. interference by other analytes or inapplicability

to certain situations). Limitations may include possible restrictions due to the costs, equipment or specific

and non-specific risks implied for either the operator and/or the environment (see Reference [13]). The

allelic and copy number stability of the target sequence should be considered for cultivars of different

geographic and phylogenic origins as per ISO 21570.

c) Selectivity: Empirical results from testing the method with non-target transgenic events and

non-transgenic plants should be provided where possible. This testing should include closely related

events and cases were the limits of the selectivity are truly tested.

d) Reference materials: Reference materials are not readily available for many events. However, as with any

method, their use is encouraged. When a reference material becomes available, method submitters are

encouraged to test the methods against these materials and include the information by amendment to the

standard.

e) Analytical controls: Controls shall be clearly specified and their interpretation recorded. These may

include positive and negative controls, their detailed contents, the extent to which they should be used,

and the interpretation of the results obtained.

f) Trueness and precision: Information on the trueness and precision of the method shall be supplied as far

as is feasible. “Trueness” refers to the closeness of agreement between the arithmetic mean of a large

number of test results and the true or accepted reference value. “Precision” refers to the closeness of

agreement between test results (see ISO 5725-1 for details).

g) Instrument specificity: The required equipment for application of the method should be clearly described,

with regards to the analysis per se and also to the sample preparation. An indication of costs, timing,

practical difficulties, and of any other factor that could be of importance for the operators should also be

indicated. Instrument specificity can also be critical to a method. It is however, the policy of ISO not to

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ISO/TS 21098:2005(E)

specify to a particular instrument wherever possible. Therefore, method developers and submitters are

encouraged to validate methods on a variety of instruments. They should indicate in the method the

instrument(s) on which the method was validated.

h) Practicability of the method: information should also be given on the practicability of the method, where

known, such as an estimate of the time and resources required to perform the method on a specific

number of samples.

i) Specification of the prediction model/mathematical model needed for the method: If the derivation of the

results relies upon a mathematical relationship, this shall be outlined and recorded (e.g. a regression line

or calibration curve obtained by other means). Instructions for the correct application of the model should

be provided. These should include, depending on the method, a recommended number and range of

levels to be analysed, minimum number of replicates to be included or the means to evaluate the fitness

for purpose, and criteria for setting instrument thresholds.

j) Criteria for acceptance of data: The criteria adopted to interpret results and to make inferences shall be

described in full detail. This includes the handling of, for example, conflicting results from replicate

samples.

k) Sensitivity and range: The sensitivity of a method is defined as its ability to determine small differences in

analyte concentrations. Empirical results from testing the method at different concentrations shall be

provided in order to demonstrate the range of use of the method [see b)].

l) Robustness testing: The method provider should describe empirical results from testing the method

against small but deliberate variations in method parameters.

m) Performance requirement: If the performance of a particular component of the method (e.g. the amplitaq

enzyme) is particularly critical to performance of a method, then information as to how an operator can

independently test the performance of the component should be included. This information will be

important for the in-house validation that is necessary upon adoption of a method in any particular

laboratory.

n) Intellectual property and related issues: Any intellectual property and related issues should be indicated.

5 Validation of methods
5.1 General

The method performance study or method validation establishes the performance characteristic for a specific

method application, i.e. a specific analytical procedure for a well-defined scope. For a detailed discussion and

explanation of the descriptions, refer to the Procedural Manual of Codex Alimentarius (see Reference [14])

and pertinent International Standards (e.g. the ISO 5725 series). Some of the most pertinent terms are

outlined in Clause 4.

A method should be validated using the conditions under which it will be performed. Thus, it should not be

tested using an unreasonable number of amplification cycles. Most PCRs can be expected to result in

analytical artefacts if operated with too many cycles and/or under non-optimal conditions. Validation can

include limit of detection (LOD) and range of use and, if applicable, the limit of quantitation (LOQ) for DNA

detection methods.
5.2 Validation of quantitative methods

The validation of methods is described in ISO 5725-2 and the IUPAC/ISO/AOAC harmonized protocol (see

Reference [12]). It is worth noting that a determination of an LOD or LOQ is not necessarily needed to

establish the validity of a method for a given application. For example, if the method is to be used for

concentrations in grams per kilogram, it does not add much value if an LOD is determined to be 1 ng/kg. In

this and similar cases, the reliability of the method will be proven by the other parameters. Similar

considerations apply for the LOQ. However, it is always necessary to determine the range of use of the

method in the validation study. The method will only be applicable in that range.

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ISO/TS 21098:2005(E)

If it is desired to determine the LOD, it is common practice to estimate it as the value of the blank increased by

three times the standard deviation of the blank. However, this approach relies on normal Gaussian distribution

of the blank measurements around zero. Its use is not valid for many instrument-based methods such as

quantitative PCR, in which the distribution of measurement values for blanks is typically truncated at zero and

therefore not normally distributed. The LOD in these cases shall be experimentally determined unless the

range of use is well above the LOD.

For a quantitative method, it is important to know if the LOQ is close to the values to be measured in a

particular matrix. The traditional approach, in which the LOQ is estimated to be the blank increased by 6 to

10 times the standard deviation of the blank, is not valid for many instrument-based methods such as

quantitative PCR.

It is important to note that a method should be validated using the conditions under which it will be performed.

Thus, it should not be tested using an unreasonable number of amplification cycles. Most PCR methods can

be expected to result in analytical artefacts if operated with too many cycles and/or under non-optimal

conditions.
5.3 Validation of qualitative methods

The validation of qualitative methods has not been discussed extensively nor harmonized.

A qualitative PCR shall be validated in the same way as it is intended to be used. The sensitivity of the

method shall be shown to be such that it can reliably detect one positive particle (e.g. a single grain) in a pool,

and does not give rise to a significant number of false positives. A concept of using false-positive and

false-negative rates to describe the accuracy and precision of a qualitative assay has been developed for

microbial assays (see ISO 16140 and References [15] and [16]). This concept may be applied to qualitative

PCR assays. A critical issue in the validation of this type of method is the availability of test materials that are

known to be positive and negative.

By their very nature, qualitative tests result only in yes/no answers. The measures of precision and accuracy

are the frequencies of false negative and/or false positive results. False negative results indicate the absence

of a given analyte when in fact the analyte is present in the sample; false positive results indicate the

presence of an analyte that is not present in the sample. An increase in false negative results will be observed

when the amount of analyte approaches the LOD of the method. Like the LOD for quantitative methods, the

LOD for a qualitative method may be defined as the concentration at which a positive sample yields a positive

result at least 95 % of the time. This results in a rate of false negative results of 5 % or less. During validation

of a qualitative PCR assay, it is also important to determine the number of false positive results (a positive

result obtained using a sample that is known to be negative).
Both false positive and false negative results are expressed as rates.
5.3.1 False-positive rate

This is the probability that a known negative test sample has been classified as positive by the method. The

false-positive rate, R , is the number of misclassified known negatives, M , divided by the total number of

fp n

negative test samples, N (misclassified positives plus the number of correctly classified known negatives),

obtained with the method.
For convenience this rate can be expressed as percentage:
R=× 100 %
5.3.2 False-negative rate

This is the probability that a known positive test sample has been classified as negative by the method. The

false-negative rate, R , is the number of misclassified known positives, M , divided by the total number of

fn p

positive test samples, N (misclassified positives plus the number of correctly classified known positives),

obtained with the method.
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ISO/TS 21098:2005(E)
For convenience this rate can be expressed as percentage:
R=× 100 %

In order to demonstrate the false-negative rate for a qualitative assay, a series of samples (e.g. grain pools)

with a constant, known concentration of positive material in a pool of negative material (e.g. 1 positive kernel

in 199 conventional maize kernels) shall be analysed and the results evaluated. It is important to note that the

concept of confidence intervals and statistical uncertainty shall be applied to the risk of false positive and/or

false negative results as well. The desired level of confidence determines the size and number of pools that

need to be tested. For example, 100 positive test results obtained from 100 independent measurements on

truly positive samples lead to the conclusion that the level of false-negative results is below 4,5 % at a

confidence level of 99 % for the tested concentration of positive kernels (expressed as the number of positive

kernels in a pool of negative kernels).

A method should be validated using the conditions under which it will be performed. Thus, it should not be

tested using an unreasonable number of amplification cycles. Most PCRs can be expected to result in

analytical artefacts if operated with too many cycles and/or under non-optimal conditions.

6 Process for adding, amending and retaining methods (as annexes)
6.1 Expert group for consideration of the methods

Within ISO/TC 34/WG 7 (or its successor) an ad-hoc expert group shall be formed, and shall convene (by

electronic or other means) to consider any submitted methods, and shall report to the secretariat at least

6 weeks prior to the next WG 7 meeting. The report shall be circulated to the WG 7 members at that time.

The expert group shall be composed of at least 5 experts nominated by member bodies. The group shall have

an international membership, with representation that is regionally balanced.
6.2 Addition of methods to the standards

Each method shall be included as a separate annex, so as to simplify the formatting and amendment of the

documents.

Methods intended for inclusion in the annexes should be submitted by the member bodies to the secretariat

for distribution to the expert group at least 12 weeks before the meeting of ISO/TC 34/WG 7. This would allow

the expert group to receive and consider the methods, and for their report to be circulated to the participants

before the meeting. The WG 7 shall consider the report of the working group at the following

...

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