Microbiology of the food chain - Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp. - Part 1: Detection method (ISO 11290-1:2017)

ISO 11290-1:2017 specifies a horizontal method for
-      the detection of L. monocytogenes, and
-      the detection of Listeria spp. (including L. monocytogenes).
ISO 11290-1:2017 is applicable to
-      products intended for human consumption and for the feeding of animals, and
-      environmental samples in the area of food production and food handling.

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die Zählung von Listeria monocytogenes und von Listeria spp. - Teil 1: Nachweisverfahren (ISO 11290-1:2017)

Dieses Dokument legt ein horizontales Verfahren fest für
-   den Nachweis von L. monocytogenes und
-   den Nachweis von Listeria spp. (einschließlich L. monocytogenes).
Dieses Dokument gilt für
-   Produkte, die für den menschlichen Verzehr oder als Futtermittel bestimmt sind und
-   Umgebungsproben aus dem Bereich der Herstellung von Lebensmitteln und beim Umgang mit Lebensmitteln.
Es ist möglich, dass bestimmte, zusätzlich beschriebene Listeria Arten nicht unbedingt mit diesem Verfahren nachweisbar oder bestätigbar sind [5], [10], [12], [14], [25], [26], [27].

Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le dénombrement de Listeria monocytogenes et de Listeria spp. - Partie 1: Méthode de recherche (ISO 11290-1:2017)

ISO 11290-1:2017 spécifie une méthode horizontale pour:
-      la recherche de L. monocytogenes; et
-      la recherche de Listeria spp. (y compris L. monocytogenes).
ISO 11290-1:2017 s'applique aux:
-      produits destinés à la consommation humaine ou à l'alimentation animale; et
-      échantillons de l'environnement de production et de distribution des aliments.

Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje prisotnosti in števila Listeria monocytogenes in Listeria spp. - 1. del: Metoda za ugotavljanje prisotnosti (ISO 11290-1:2017)

Ta standard opisuje način ugotavljanja prisotnosti Listeria monocytogenes (referenčni dokument: EN/ISO 11290 -1 vklj./dod. 1)

General Information

Status
Published
Publication Date
13-Jun-2017
Current Stage
6060 - Definitive text made available (DAV) - Publishing
Due Date
03-Mar-2014

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SLOVENSKI STANDARD
SIST EN ISO 11290-1:2017
01-september-2017
1DGRPHãþD
SIST EN ISO 11290-1:1997
SIST EN ISO 11290-1:1997/A1:2005
Mikrobiologija v prehranski verigi - Horizontalna metoda za ugotavljanje

prisotnosti in števila Listeria monocytogenes in Listeria spp. - 1. del: Metoda za

ugotavljanje prisotnosti (ISO 11290-1:2017)

Microbiology of the food chain - Horizontal method for the detection and enumeration of

Listeria monocytogenes and of Listeria spp. - Part 1: Detection method (ISO 11290-

1:2017)

Mikrobiologie der Lebensmittelkette - Horizontales Verfahren für den Nachweis und die

Zählung von Listeria monocytogenes und von Listeria spp. - Teil 1: Nachweisverfahren

(ISO 11290-1:2017)

Microbiologie de la chaîne alimentaire - Méthode horizontale pour la recherche et le

dénombrement de Listeria monocytogenes et de Listeria spp. - Partie 1: Méthode de

recherche (ISO 11290-1:2017)
Ta slovenski standard je istoveten z: EN ISO 11290-1:2017
ICS:
07.100.30 Mikrobiologija živil Food microbiology
SIST EN ISO 11290-1:2017 en

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

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SIST EN ISO 11290-1:2017
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SIST EN ISO 11290-1:2017
EN ISO 11290-1
EUROPEAN STANDARD
NORME EUROPÉENNE
June 2017
EUROPÄISCHE NORM
ICS 07.100.30 Supersedes EN ISO 11290-1:1996
English Version
Microbiology of the food chain - Horizontal method for the
detection and enumeration of Listeria monocytogenes and
of Listeria spp. - Part 1: Detection method (ISO 11290-
1:2017)

Microbiologie de la chaîne alimentaire - Méthode Mikrobiologie der Lebensmittelkette - Horizontales

horizontale pour la recherche et le dénombrement de Verfahren für den Nachweis und die Zählung von

Listeria monocytogenes et de Listeria spp. - Partie 1: Listeria monocytogenes und von Listeria spp. - Teil 1:

Méthode de recherche (ISO 11290-1:2017) Nachweisverfahren (ISO 11290-1:2017)
This European Standard was approved by CEN on 6 April 2017.

CEN members are bound to comply with the CEN/CENELEC Internal Regulations which stipulate the conditions for giving this

European Standard the status of a 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 European Standard 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, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,

Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, 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: Avenue Marnix 17, B-1000 Brussels

© 2017 CEN All rights of exploitation in any form and by any means reserved Ref. No. EN ISO 11290-1:2017 E

worldwide for CEN national Members.
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SIST EN ISO 11290-1:2017
EN ISO 11290-1:2017 (E)
Contents Page

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

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SIST EN ISO 11290-1:2017
EN ISO 11290-1:2017 (E)
European foreword

This document (EN ISO 11290-1:2017) 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 European Standard shall be given the status of a national standard, either by publication of an

identical text or by endorsement, at the latest by December 2017, and conflicting national standards

shall be withdrawn at the latest by December 2017.

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.

This document supersedes EN ISO 11290-1:1996.

This document has been prepared under a mandate given to CEN by the European Commission and the

European Free Trade Association.

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, Former Yugoslav Republic of Macedonia,

France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta,

Netherlands, Norway, Poland, Portugal, Romania, Serbia, Slovakia, Slovenia, Spain, Sweden, Switzerland,

Turkey and the United Kingdom.
Endorsement notice

The text of ISO 11290-1:2017 has been approved by CEN as EN ISO 11290-1:2017 without any

modification.
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SIST EN ISO 11290-1:2017
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SIST EN ISO 11290-1:2017
INTERNATIONAL ISO
STANDARD 11290-1
Second edition
2017-05
Microbiology of the food chain —
Horizontal method for the detection
and enumeration of Listeria
monocytogenes and of Listeria spp. —
Part 1:
Detection method
Microbiologie de la chaîne alimentaire — Méthode horizontale pour
la recherche et le dénombrement de Listeria monocytogenes et de
Listeria spp. —
Partie 1: Méthode de recherche
Reference number
ISO 11290-1:2017(E)
ISO 2017
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SIST EN ISO 11290-1:2017
ISO 11290-1:2017(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2017, Published in Switzerland

All rights reserved. Unless otherwise specified, 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
Ch. de Blandonnet 8 • CP 401
CH-1214 Vernier, Geneva, Switzerland
Tel. +41 22 749 01 11
Fax +41 22 749 09 47
copyright@iso.org
www.iso.org
ii © ISO 2017 – All rights reserved
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SIST EN ISO 11290-1:2017
ISO 11290-1:2017(E)
Contents Page

Foreword ..........................................................................................................................................................................................................................................v

Introduction ................................................................................................................................................................................................................................vi

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

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

3 Terms and definitions ..................................................................................................................................................................................... 2

4 Principle ........................................................................................................................................................................................................................ 2

4.1 General ........................................................................................................................................................................................................... 2

4.2 Primary enrichment in a selective liquid enrichment medium with reduced

concentration of selective agents (half-Fraser broth) .................. ......................................................................... 2

4.3 Secondary enrichment with a selective liquid enrichment medium with full

concentration of selective agents (Fraser broth) ...................................................................................................... 3

4.4 Plating out and identification ..................................................................................................................................................... 3

4.5 Confirmation ............................................................................................................................................................................................. 3

5 Culture media and reagents ...................................................................................................................................................................... 3

6 Equipment and consumables .................................................................................................................................................................. 3

7 Sampling ........................................................................................................................................................................................................................ 4

8 Preparation of test sample ......................................................................................................................................................................... 4

9 Procedure..................................................................................................................................................................................................................... 4

9.1 Test portion and initial suspension ....................................................................................................................................... 4

9.2 Primary enrichment ........................................................................................................................................................................... 4

9.3 Secondary enrichment ..................................................................................................................................................................... 5

9.4 Plating out and identification ..................................................................................................................................................... 5

9.4.1 General .................................................................................................................................................................................... 5

9.4.2 Agar Listeria according to Ottaviani and Agosti ................................................................................... 5

9.4.3 Second selective medium ......................................................................................................................................... 6

9.5 Confirmation of Listeria monocytogenes or Listeria spp. ................................................................................... 6

9.5.1 Selection of colonies for confirmation .......................................................................................................... 6

9.5.2 Confirmation of L. monocytogenes .... ............................................................................................................. 6

9.5.3 Confirmation of Listeria spp. ............................................................................................................................. 10

9.6 Interpretation of morphological and physiological properties and of the

biochemical reactions ....................................................................................................................................................................11

9.7 Additional characterization of isolated strains (optional) .............................................................................11

10 Expression of results .....................................................................................................................................................................................11

11 Performance characteristics of the method ..........................................................................................................................11

11.1 Method validation studies ..........................................................................................................................................................11

11.2 Sensitivity .................................................................................................................................................................................................11

11.3 Specificity .................................................................................................................................................................................................11

11.4 Level of detection (LOD ) .........................................................................................................................................................11

12 Test report ................................................................................................................................................................................................................12

13 Quality assurance .............................................................................................................................................................................................12

Annex A (normative) Diagram of procedure .............................................................................................................................................13

Annex B (normative) Composition and preparation of culture media and reagents .....................................14

Annex C (informative) Distinction of Listeria spp. from other genera ...........................................................................27

Annex D (informative) Reactions for the identification of Listeria species ..............................................................28

Annex E (informative) Listeria selective agars ........................................................................................................................................30

© ISO 2017 – All rights reserved iii
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SIST EN ISO 11290-1:2017
ISO 11290-1:2017(E)
Annex F (informative) Results of the interlaboratory studies for detection of

Listeria monocytogenes ...............................................................................................................................................................................31

Bibliography .............................................................................................................................................................................................................................35

iv © ISO 2017 – All rights reserved
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SIST EN ISO 11290-1:2017
ISO 11290-1:2017(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 on 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 the following

URL: w w w . i s o .org/ iso/ foreword .html.

This document was prepared by the European Committee for Standardization (CEN) Technical

Committee CEN/TC 275, Food analysis — Horizontal methods, in collaboration with ISO Technical

Committee ISO/TC 34, Food products, Subcommittee SC 9, Microbiology, in accordance with the

agreement on technical cooperation between ISO and CEN (Vienna Agreement).

This second edition cancels and replaces the first edition (ISO 11290-1:1996), which has been technically

revised. It also incorporates the amendment ISO 11290-1:1996/Amd.1:2004.
The main changes, compared to ISO 11290-1:1996, are the following.
— The detection of Listeria monocytogenes has been modified as listed below.
— Primary enrichment in half-Fraser broth: incubation for 25 h ± 1 h.
[29]
— Secondary enrichment in Fraser broth: incubation for 24 h ± 2 h.

— Half-Fraser and Fraser broths may be refrigerated before transfer or isolation on selective agar for

a maximum of 72 h.

— Storage of isolation plates: incubated plates can be refrigerated for a maximum of two days before

reading.

— Microscopic aspect for confirmation is optional if the isolation agar allows distinction between

pathogenic and non-pathogenic Listeria spp.
— CAMP test and catalase test are optional.
— Inclusion of new performance characteristics.

— Moreover, detection of Listeria spp. has been included in the scope and the title changed accordingly.

A list of parts in the ISO 11290 series can be found on the ISO website.
© ISO 2017 – All rights reserved v
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SIST EN ISO 11290-1:2017
ISO 11290-1:2017(E)
Introduction

The main changes, listed in the foreword, introduced in this document compared to ISO 11290-1:1996

[28]

are considered as major (see ISO 17468 ). The technical changes were assessed and were considered

to have no significant effect on the method performance characteristics or test results.

Because of the large variety of food and feed products, this horizontal method may not be appropriate

in every detail for certain products for which it may be necessary to use different or specific methods.

Nevertheless, in all cases, this horizontal method is intended to be applied as far as possible and

deviations from this only be made for justified technical reasons.

When this document is next reviewed, account will be taken of all information then available regarding

the extent to which this horizontal method has been followed and the reasons for deviations from it in

the case of particular products.

The harmonization of test methods cannot be immediate, and for certain groups of products

International Standards and/or national standards may already exist that do not comply with this

horizontal method. It is hoped that when such standards are reviewed, they will be changed to comply

with this document so that eventually the only remaining departures from this horizontal method will

be those necessary for well-established technical reasons.
vi © ISO 2017 – All rights reserved
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SIST EN ISO 11290-1:2017
INTERNATIONAL STANDARD ISO 11290-1:2017(E)
Microbiology of the food chain — Horizontal method for
the detection and enumeration of Listeria monocytogenes
and of Listeria spp. —
Part 1:
Detection method

WARNING — In order to safeguard the health of laboratory personnel, it is essential that tests

for detecting L. monocytogenes and Listeria spp. are only undertaken in properly equipped

laboratories, under the control of a skilled microbiologist, and that great care is taken in the

disposal of all incubated materials. Persons using this document should be familiar with

normal laboratory practice. This document does not purport to address all of the safety aspects,

if any, associated with its use. It is the responsibility of the user to establish appropriate

safety and health practices. In particular, it is strongly recommended that tests for detecting

L. monocytogenes are undertaken in laboratories providing biosafety level 2 conditions. It is

strongly recommended that female laboratory staff are made aware of the particular risk to the

developing foetus presented by infection of the mother through exposure to L. monocytogenes

and Listeria spp., and that pregnant personnel and persons with recognized underlying

conditions or diseases that impair cell-mediated immunity do not manipulate cultures of

L. monocytogenes and Listeria spp.
1 Scope
This document specifies a horizontal method for
— the detection of L. monocytogenes, and
— the detection of Listeria spp. (including L. monocytogenes).
This document is applicable to
— products intended for human consumption and for the feeding of animals, and
— environmental samples in the area of food production and food handling.

It is possible that certain additionally described Listeria species may not be detected or confirmed by

[5],[10],[12],[14],[25],[26],[27]
this method.
2 Normative references

The following documents are referred to in the text in such a way that some or all of their content

constitutes requirements of this document. For dated references, only the edition cited applies. For

undated references, the latest edition of the referenced document (including any amendments) applies.

ISO 6887 (all parts), Microbiology of the food chain — Preparation of test samples, initial suspension and

decimal dilutions for microbiological examination

ISO 7218, Microbiology of food and animal feeding stuffs — General requirements and guidance for

microbiological examinations

ISO 11133, Microbiology of food, animal feed and water — Preparation, production, storage and

performance testing of culture media
© ISO 2017 – All rights reserved 1
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SIST EN ISO 11290-1:2017
ISO 11290-1:2017(E)
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:

— IEC Electropedia: available at http:// www .electropedia .org/
— ISO Online browsing platform: available at http:// www .iso .org/ obp
3.1
Listeria monocytogenes

microorganisms which form typical colonies on solid selective media and which display the

morphological, physiological and biochemical characteristics described when tests are carried out in

accordance with this document
3.2
detection of Listeria monocytogenes

determination of the detection/non detection of Listeria monocytogenes (3.1), in a given mass or volume

of product or a specified surface, when tests are carried out in accordance with this document

3.3
Listeria spp.

microorganisms which form typical colonies on solid selective media and which display the

morphological, physiological and biochemical characteristics described when tests are carried out in

accordance with this document
3.4
detection of Listeria spp.

determination of the detection/non detection of Listeria spp. (3.3), in a given mass or volume of product

or a specified surface, when tests are carried out in accordance with this document

4 Principle
4.1 General

Listeria spp. may be present in small numbers and are often accompanied by considerably larger

numbers of other microorganisms, therefore selective enrichment is necessary. It is also necessary to

detect injured and stressed Listeria spp. and the primary selective enrichment medium, with reduced

inhibitor concentration, fulfils at least part of this function.

NOTE Presence of L. monocytogenes can be masked by the presence of other Listeria species, in particular

L. innocua or L. ivanovii.

Within the limits of this document, the detection of L. monocytogenes and of Listeria spp. necessitates

four successive stages, as described in the flowchart in Annex A.
4.2 Primary enrichment in a selective liquid enrichment medium with reduced
concentration of selective agents (half-Fraser broth)

Inoculation of a selective primary enrichment medium containing half the concentrations of

acriflavine and nalidixic acid (half-Fraser broth, see B.1), which is also used as a dilution fluid for the

test portion (9.1).
Incubation of the initial suspension at 30 °C for 24 h to 26 h.
2 © ISO 2017 – All rights reserved
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SIST EN ISO 11290-1:2017
ISO 11290-1:2017(E)
4.3 Secondary enrichment with a selective liquid enrichment medium with full
concentration of selective agents (Fraser broth)

Inoculation of full-strength secondary liquid enrichment medium (Fraser broth) with a culture

obtained from 4.2.
[29]
Incubation of the Fraser broth at 37 °C for 24 h.
4.4 Plating out and identification

From the cultures obtained in 4.2 and 4.3, plating out on the two selective solid media:

— Agar Listeria according to Ottaviani and Agosti (see References [16] and [17] and B.3);

— any other solid selective medium at the choice of the laboratory complementary to Agar Listeria

according to Ottaviani and Agosti, using a different substrate and/or principle than the one used in

Listeria agar according to Ottaviani and Agosti (see B.4). See Annex E for information about media.

Incubation of the Agar Listeria according to Ottaviani and Agosti at 37 °C for a total of 48 h. If colonies of

presumptive L. monocytogenes or Listeria spp. are evident at 24 h the incubation may be stopped at this

stage. Incubation of the second selective medium at the appropriate temperature and examination after

the appropriate time.
4.5 Confirmation

Subculturing of the colonies of presumptive L. monocytogenes or Listeria spp., plated out as described

in 4.4, and confirmation by means of appropriate morphological and/or biochemical tests.

5 Culture media and reagents
For current laboratory practices, refer to ISO 11133.

Composition and performance testing of culture media and reagents and their preparation are

described in Annex B.
6 Equipment and consumables

Usual microbiological equipment (as specified in ISO 7218) and, in particular, the following.

6.1 Apparatus for dry sterilization (oven) or wet sterilization (autoclave).
As specified in ISO 7218.

6.2 Drying cabinet or incubator, capable of being maintained between 25 °C and 50 °C.

6.3 Incubators, capable of operating at 30 °C ± 1 °C, 37 °C ± 1 °C and at 25 °C ± 1 °C (optional).

6.4 Water bath, capable of operating at 47 °C to 50 °C.

6.5 Sterile loops approximately 3 mm in diameter or 10 µl, and inoculating needle or wire.

6.6 pH meter, capable of being read to the nearest 0,01 pH unit at 25 °C, enabling measurements to be

made which are accurate to ± 0,1 pH unit.

6.7 Graduated pipettes or automatic pipettes, of nominal capacities 1 ml and 10 ml.

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SIST EN ISO 11290-1:2017
ISO 11290-1:2017(E)
6.8 Petri dishes, for example of diameter 90 mm.
6.9 Microscope, preferably with phase-contrast, and with slides and cover slips.
6.10 Refrigerator, capable of operating at 5 °C ± 3 °C.
7 Sampling

Sampling is not part of the method specified in this document. If there is no specific International

Standard dealing with sampling of the product concerned, it is recommended that the parties

[3]

concerned come to an agreement on this subject. For food and feed samples, refer to ISO/TS 17728 .

[2]
For environmental samples, use ISO 18593 and see Reference [24].

It is important that the laboratory receives a sample which is truly representative and has not been

damaged or changed during transport or storage (see ISO 7218).
8 Preparation of test sample

Prepare the test sample in accordance with the specific International Standard dealing with the product

[2]

concerned [see ISO 6887 (all parts) and ISO 18593 ]. If there is no specific International Standard, it is

recommended that the parties concerned come to an agreement on this subject.
9 Procedure
9.1 Test portion and initial suspension

Refer to ISO 6887 (all parts) and any specific International Standard appropriate to the product

concerned
...

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