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8.6.2013 Official
Journal
of
the
European
Union L
157/1
II
(Non-legislative acts)
REGULATIONS
COMMISSION IMPLEMENTING REGULATION (EU) No 503/2013
of 3 April 2013
on applications for authorisation of genetically modified food and feed in accordance with
Regulation (EC) No 1829/2003 of the European Parliament and of the Council and amending
Commission Regulations (EC) No 641/2004 and (EC) No 1981/2006
(Text with EEA relevance)
THE EUROPEAN COMMISSION,
modified food and feed satisfy the requirements laid
down in that Regulation, in respect of their proposed
uses.
Having regard to the Treaty on the Functioning of the European
Union,
(3)
In the interest of consistency of Union legislation, certain
definitions laid down in Regulation (EC) No 178/2002 of
Having regard to Regulation (EC) No 1829/2003 of the
the European Parliament and of the Council of 28 January
European Parliament and of the Council of 22 September
2002 laying down the general principles and
2003 on genetically modified food and feed ( 1 ), and in
requirements of food law, establishing the European
particular Articles 5(7), 11(5), 17(7) and 23(5) thereof,
Food Safety Authority and laying down procedures in
matters of food safety ( 2 ) should also apply to this Regu
lation.
After consulting the European Food Safety Authority,
(4) Commission
Regulation
(EC)
No
641/2004
( 3 ) on
Whereas:
detailed rules for the implementation of Regulation (EC)
No 1829/2003 provides for certain detailed rules
concerning applications for authorisation submitted in
accordance with Regulation (EC) No 1829/2003. To
(1) Regulation
(EC)
No
1829/2003
lays
down
Union
facilitate the preparation of applications and ensure that
procedures for the authorisation and supervision of
they contain all the information needed for their
genetically modified food and feed, including rules for
assessment, it is necessary to provide for more compre
the labelling of such food and feed. That Regulation
hensive and systematic rules concerning applications for
provides for a scientific evaluation to be carried out on
authorisation, which should also be specific to each type
the risks that the genetically modified food or feed may
of genetically modified organisms (GMO), namely plants,
present for human and animal health and, as the case
animals and micro-organisms.
may be, for the environment. It also provides that a
genetically modified food or feed must not mislead the
consumer or the user and must not differ from the food
or feed which it is intended to replace to such an extent
that its normal consumption would be nutritionally
(5) The
rules
laid
down
in
this
Regulation
should
only
cover
disadvantageous for humans or animals.
applications concerning genetically modified plants for
food or feed uses, food or feed containing or consisting
of genetically modified plants and food or feed produced
from such plants. Genetically modified plants, for which
(2) Regulation
(EC)
No
1829/2003
provides,
in
particular,
sufficient experience is available to date, constitute the
that applications for authorisation are to adequately
vast majority of current applications.
and sufficiently demonstrate that the genetically
( 2 ) OJ L 31, 1.2.2002, p. 1.
( 1 ) OJ L 268, 18.10.2003, p. 1.
( 3 ) OJ L 102, 7.4.2004, p. 14.

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Official Journal of the European Union
8.6.2013
(6) The
rules
laid
down
in
this
Regulation
should
specify
the (11) It
has,
however,
not
been
proved
possible
to
define
with
general requirements for the presentation and prep
the necessary precision the level of uncertainties which
aration of applications, namely requirements to provide
would require the submission of 90-day feeding studies.
general and scientific information, including methods for
In addition, some food and feed assessment bodies of
detection, and identification, as well as reference material
Member States are of the opinion that such study
so as to ensure that applications comply with the
should be performed in all applications for genetically
conditions laid down in Articles 5, 17 and 30 of Regu
modified plants containing single transformation events.
lation (EC) No 1829/2003.
Considering these diverging views, as well as to improve
consumer confidence, such studies should be, for the
time being, requested in all applications related to
genetically modified plants with single transformation
(7) The
applicant
should
also
take
into
consideration
the
events and, where appropriate, on genetically modified
scientific information to be provided in the application
plants containing stacked transformation events.
as regards the environmental risk assessment of GMOs or
food and feed containing or consisting of GMOs, as set
out in the principles for the environmental risk
assessment in Annex II to Directive 2001/18/EC of the
European Parliament and of the Council of 12 March
(12) Studies to demonstrate that a genetically modified food
or feed fulfils the requirements of Regulation (EC)
2001 on the deliberate release into the environment of
No 1829/2003 involving the use of laboratory animals
genetically modified organisms and repealing Council
should be carried out in accordance with Directive
Directive 90/220/EEC ( 1 ), as well as the applicable
2010/63/EU of the European Parliament and of the
guidance published by the European Food Safety
Council of 22 September 2010 regarding the protection
Authority (EFSA) in this regard.
of the animals used for scientific purposes ( 4 ), and should
be kept to a minimum while ensuring an adequate
demonstration of the safety of the genetically modified
(8)
In addition to the general requirements for the presen
food or feed. The current uncertainties in relation to the
tation and preparation of applications, it is appropriate to
need and design of 90-day feeding trials will be addressed
provide for specific rules to ensure that the scientific
by a large research project under the 2012 work
information required in the application adequately and
programme of Theme 2 ‘Food, Agriculture and Fisheries,
sufficiently demonstrates that the genetically modified
and Biotechnologies’ of the seventh Framework
food or feed satisfy the requirements laid down in Regu
Programme for Research (FP7). The requirements
lation (EC) No 1829/2003, in respect of their proposed
regarding animal feeding trials in the context of GMO
uses.
risk assessments should be reviewed in the light of the
outcome of this project expected to be available by the
end of 2015 at the latest. Other credible scientific
knowledge which might be available at that time
(9) These
rules
should
therefore
provide
for
a
set
of
studies
should also be taken into account.
that should be included in all applications, as well as the
test methods to be followed to perform such studies,
whilst taking into account relevant international stan
dards, such as the guideline of the Codex Alimentarius
(13) While
the
rules
laid
down
in
this
Regulation
should
be
for the conduct of safety assessment of foods derived
valid for all applications for genetically modified plants,
from the recombinant-DNA plant ( 2 ).
the type and necessity of the studies to evaluate the char
acteristics and safety of genetically modified food or feed
subject to an application may vary, depending on the
(10) In accordance with the applicable guidance of the
nature of the genetic modification or of the product.
EFSA (
For example, genetic modifications which have negligible

3 ), the safety assessment of the genetically
modified food or feed should include studies related to
impact on the composition of a genetically modified
new components resulting from the genetic modification,
food or feed or highly refined products that may be
the molecular characterisation of the genetically modified
proven to be identical to products produced from the
plant, the comparative analysis of the composition and
conventional counterpart require different studies than
the phenotype of the genetically modified plant
a product resulting from complex genetic modification
compared to its conventional counterpart. Depending
aiming to modify its nutritional characteristics.
on the characteristics of the genetically modified plant
and on the outcome of that first set of studies, the
EFSA guidance indicates that it may be necessary to
perform additional studies. In that respect, the EFSA
(14) The
requirements
laid
down
in
this
Regulation
regarding
considers that notwithstanding its limitations, a 90-day
the studies which have to be included in an application
feeding study in rodents with whole food or feed is,
for authorisation under Regulation (EC) No 1829/2003
when justified, the primary additional study to address
should not prevent the EFSA to request, where appro
uncertainties identified in the course of the safety
priate, the applicant to supplement the particulars
assessment.
accompanying the application in accordance
with Articles 6(2) and 18(2) of Regulation (EC)
No 1829/2003.
( 1 ) OJ L 106, 17.4.2001, p. 1.
( 2 ) Codex Alimentarius Commission, GL 45-2003.
( 3 ) EFSA Journal 2011; 9(5):2150.
( 4 ) OJ L 276, 20.10.2010, p. 33.

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(15) In
order
to
ensure
that
studies
are
of
high
quality
and
monitoring should only be considered in cases where,
documented in a transparent way, it is essential that they
notwithstanding the fact that the safety of genetically
are performed under appropriate quality assurance
modified food and feed has been demonstrated, it is
systems and raw data should be provided in all cases
appropriate to confirm the expected consumption, the
and be in a suitable electronic format. Toxicological
application of conditions of uses or identified effects.
studies should be performed in accordance with the
This is for example the case when the genetically
quality assurance principles laid down by Directive
modified food or feed has altered nutritional composition
2004/10/EC of the European Parliament and of the
or when that its nutritional value differs from the
Council of 11 February 2004 on the harmonisation of
conventional food or feed that it would replace or
laws, regulations and administrative provisions relating to
when there is a likelihood of increased allergenicity due
the application of the principles of good laboratory
to the genetic modification.
practice and the verification of their applications for
tests on chemical substances ( 1 ). If such studies are
carried out outside the Union, they should follow the
(20) This
Regulation
should
take
account
of
the
international
state-of-the-art OECD Principles on Good Laboratory
trade commitments of the Union and of the requirements
Practice (GLP). With regard to studies other than toxico
of the Cartagena Protocol on Biosafety to the Convention
logical studies, they should be conducted under ISO or
on Biological Diversity (the Cartagena Protocol),
GLP standards.
approved by Council Decision 2002/628/EC of 25 June
2002 concerning the conclusion, on behalf of the
European Community, of the Cartagena Protocol on
(16) It
is
also
necessary
to
define
the
requirements
regarding
Biosafety ( 2 ) as well as the provisions of Regulation
the submission of additional information related to the
(EC) No 1946/2003 of 15 July 2003 of the European
safety of the GMO and scientific peer-reviewed literature
Parliament and of the Council on transboundary
related to the potential effects on health and on the
movements of genetically modified organisms ( 3 ).
environment of the products covered by the application.
(21) In
order
to
ensure
that
test
methods
included
in
the
(17) During the process of the genetic modification of plants
application are adequate to demonstrate that the food
and other organisms, marker genes are often used to
or feed complies with the requirements for authorisation
facilitate the selection and identification of genetically
set out in Regulation (EC) No 1829/2003, they should be
modified cells, containing the gene of interest inserted
carried out in accordance with the present Regulation, or
into the genome of the host organism, among the vast
internationally agreed guidelines such as those described
majority of untransformed cells. Such marker genes
by the OECD, when available. To ensure that applications
should be carefully selected. In addition, it is now
for renewal meet the same standards as regards tests
possible to develop GMOs without the use of antibiotic
methods, it is appropriate that these requirements also
resistance marker genes. Against this background and in
apply to application for renewal of authorisation of GM
accordance with Article 4(2) of Directive 2001/18/EC,
food and feed.
the applicant should therefore aim to develop GMOs
without the use of antibiotic resistance marker genes.
(22) In
order
to
provide
an
accurate
designation
of
the
GM
food or feed subject to an application under Regulation
(EC) No
1829/2003, applications should include
(18) The
harvest
of
segregating
genetically
modified
plants
(segregating crops) containing stacked transformation
proposals for a unique identifier for each GMO
events contains various subcombinations of trans
concerned in accordance with Commission Regulation
formation events. In addition, current control procedures
(EC) No 65/2004 of 14 January 2004 establishing a
do not allow identifying the origin of combinations of
system for the development and assignment of unique
transformation events. Therefore, in order to ensure that
identifiers for genetically modified organisms ( 4 ).
authorisations are coherent with the products of which
the placing on the market is unavoidable and for the
feasibility of controls, the applications for genetically
(23) This
Regulation
replaces
certain
provisions
of
Regulation
modified food and feed from segregating crops should
(EC) No 641/2004 as regards genetically modified plants
include all subcombinations independently of their origin
for food or feed uses, food or feed containing or
and not yet authorised.
consisting of genetically modified plants and food or
feed produced from genetically modified plants.
However, Regulation (EC) No
641/2004 should
continue to apply as regards other types of genetically
(19) Regulation
(EC)
No
1829/2003
provides
that
a
proposal
modified products, namely genetically modified animals
for post-market monitoring of the use of the genetically
and genetically modified micro-organisms. Moreover,
modified food or feed shall only be submitted by the
certain provisions of that Regulation are obsolete. Regu
applicant where it is appropriate. It is therefore
lation (EC) No 641/2004 should therefore be amended
necessary to set out the conditions under which such a
accordingly.
proposal should, according to the outcome of the risk
assessment, accompany the application. Post-market
( 2 ) OJ L 201, 31.7.2002, p. 48.
( 3 ) OJ L 287, 5.11.2003, p. 1.
( 1 ) OJ L 50, 20.2.2004, p. 44.
( 4 ) OJ L 10, 14.1.2004, p. 5.

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(24) Commission Regulation (EC) No
1981/2006 of
Article 2
22 December 2006 on detailed rules for the implemen
tation of Article 32 of Regulation (EC) No 1829/2003 of
Definitions
the European Parliament and of the Council as regards
For the purposes of this Regulation, the definitions in Regu
the Community reference laboratory for genetically
lation (EC) No 1829/2003 apply.
modified organisms ( 1 ) should be amended to include
references to this Regulation.
The definitions of ‘risk’, ‘risk assessment’ and ‘hazard’ applicable
for the purposes of this Regulation are those provided in
(25) Regulation (EC) No
1829/2003 provides that the
Article 3 of Regulation (EC) No 178/2002.
Commission is to consult the EFSA before establishing
implementing rules with regard to the applications for
authorisation under that Regulation. The EFSA has been
CHAPTER II
consulted on those rules accordingly.
GENERAL REQUIREMENTS
Article 3
(26) This
Regulation
has
been
drawn
up
on
the
basis
of
Preparation and presentation of applications submitted
current scientific and technical knowledge. Therefore,
under Articles 5(1) and 17(1)
the Commission should monitor any developments in
this field and the publication of new or additional
1. The
application
submitted
under
Articles
5(1)
and
17(1)
guidance by the EFSA.
of Regulation (EC) No 1829/2003 shall:

(a) be submitted in accordance with the requirements for the
(27) This
Regulation
applies
to
applications
submitted
after
its
preparation and presentation of applications set out in
entry into force. It is necessary to provide for transitional
Annex I;
measures in order to enable the applicants to comply
with those rules and for the current applications or the
applications close to being submitted to proceed without
(b) contain all the information required by Annex I, in
unnecessary delays.
accordance with the specific requirements of Articles 4, 5
and 6.
(28) The
measures
provided
for
in
this
Regulation
are
in
2. The application shall include, for each of the specific
accordance with the opinion of the Standing
requirements laid down in Articles 4, 5 and 6:
Committee on the Food Chain and Animal Health,
(a) the summaries and results of the studies referred to in the
HAS ADOPTED THIS REGULATION:
application;
(b) annexes where detailed information on those studies is
CHAPTER I
provided.
GENERAL PROVISIONS
Article 1
3. The
application
shall
contain
a
checklist
demonstrating
that the information required under Articles 4, 5 and 6 is
Scope
complete.
This Regulation shall apply to applications submitted under
Articles 5, 11, 17 and 23 of Regulation (EC) No 1829/2003
for the authorisation of:
4. Where
an
application
is
limited
to
either
food
or
feed
use,
it shall contain a verifiable justification explaining why the auth
orisation shall not cover both uses in accordance with Article 27
of Regulation (EC) No 1829/2003.
(a) genetically modified plants for food or feed uses;
5. The
application
shall,
at
the
time
of
submission,
clearly
(b) food or feed containing or consisting of genetically modified
state which parts of the application are claimed to be
plants;
confidential and provide verifiable justification in accordance
with Article 30 of Regulation (EC) No 1829/2003.
(c) food produced from or containing ingredients produced
Additional information submitted during the authorisation
from genetically modified plants or feed produced from
procedure shall, at the time of submission, clearly state which
such plants.
parts of this additional information is claimed to be confidential
and provide verifiable justification in accordance with Article 30
( 1 ) OJ L 368, 23.12.2006, p. 99.
of Regulation (EC) No 1829/2003.

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6. When
studies
have
been
already
submitted
to
the (a) particular information is not necessary owing to the nature
European Food Safety Authority (EFSA) for the purposes of
of the genetic modification or of the product; or
an application and, where relevant, to the extent that they
may be used by the applicant in accordance with Article 31
of Regulation (EC) No 1829/2003, a reference to such studies
(b) it is not scientifically necessary, or technically possible to
and the results of the EFSA’s assessment may, with the
supply such information.
agreement of EFSA, be made in the framework of another
application.
The applicant shall submit reasoned justification for the dero
gation.
CHAPTER III
SPECIFIC REQUIREMENTS
3. Paragraphs 1 and 2 shall not prevent the EFSA to request,
Article 4
where appropriate, the applicant to supplement the particulars
Requirements for the performance of studies for
accompanying the application as provided for in Articles 6(2)
applications submitted under Articles 5(3) and 17(3)
and 18(2) of Regulation (EC) No 1829/2003.
1. Toxicological
studies
shall
be
conducted
in
facilities
which
comply with the:
Article 6
Additional information related to the risk assessment of
(a) requirements of Directive 2004/10/EC; or
genetically modified food or feed for applications
submitted under Articles 5(3) and 17(3)
1. In
addition
to
the
information
required
in
accordance
with
(b) ‘OECD Principles on Good Laboratory Practice’ (GLP), if
Article 5 and Annex II, the application shall include a
carried out outside the Union.
systematic review of studies published in the scientific literature
and studies performed by the applicant within the period of 10
years prior to the date of submission of the dossier on the
The applicant shall provide evidence to demonstrate such
potential effects on human and animal health of the genetically
compliance.
modified food and feed covered by the application.
2. Studies,
other
than
toxicological
studies,
shall:
2. During the authorisation procedure, the applicant shall
submit, without delay, to the EFSA additional information
(a) comply with the principles of Good Laboratory Practice
which might influence the risk assessment of the genetically
(GLP) laid down in Directive 2004/10/EC; or
modified food or feed generated following the submission of
the application. In particular, the applicant shall submit to the
EFSA information regarding any prohibition or restriction
(b) be conducted by organisations accredited under the relevant
imposed by a competent authority of any third country on
ISO standard.
the basis of a risk assessment of the genetically modified food
and feed.
3. Information on the study protocols and the results
obtained from the studies referred to in paragraphs 1 and 2
Article 7
shall be comprehensive and include the raw data in an elec
Requirements applicable for post-market monitoring of
tronic format, suitable for carrying out statistical or other
genetically modified food or feed for applications
analysis.
submitted under Articles 5(3) and 17(3)
1. The
applicant
shall
submit
a
proposal
for
post-market
Article 5
monitoring regarding the use of the food and feed as referred
to in Articles 5(3)(k) and 17(3)(k) of Regulation (EC)
Scientific requirements for the risk assessment of
No 1829/2003 when the information provided in accordance
genetically modified food and feed for applications
with Articles 4, 5 and 6 demonstrates that the genetically
submitted under Articles 5(3) and 17(3)
modified food and feed comply with Articles 4(1) and 16(1)
1. Information,
including
studies,
required
to
accompany
the of Regulation (EC) No 1829/2003 and when, in accordance
application as referred to in Article 5(3)(a) to (f) and (h) and in
with the outcome of the risk assessment, it is appropriate to
Article 17(3)(a) to (f) and (h) of Regulation (EC) No 1829/2003
confirm:
shall be provided in accordance with the scientific requirements
for the risk assessment of genetically modified food and feed set
out in Annex II to this Regulation.
(a) that specific recommendations of uses are followed by the
consumer/animal owner;
2. By
way
of
derogation
from
paragraph
1,
an
application
may be submitted that does not satisfy all the requirements of
(b) the predicted consumption of the genetically modified food
that paragraph provided that:
or feed; or

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(c) the relevance and intensity of effects and unintended effects
shall apply only for the purposes of application of Articles
detected during the pre-market risk assessment which can
11(2)(d) and 23(2)(d).
only be further characterised by post-market monitoring.
CHAPTER IV
2. The
applicant
shall
ensure
that
the
post-market
moni
toring is:
TRANSITIONAL AND FINAL PROVISIONS
Article 9
(a) developed to collect reliable information with respect to one
Transitional provisions
or several of the aspects set out in paragraph 1. This
1. Until
8
December
2013,
applicants
may
choose
to
submit
information shall allow the detection of indications on
applications falling under the scope of this Regulation under
whether any (adverse) effect on health may be related to
Regulation (EC) No 641/2004 in the version of that Regulation
genetically modified food or feed consumption;
in force on 8 June 2013.
(b) based on strategies aiming at collecting relevant information
2. By
way
of
derogation
from
Article
4(2),
in
the
case
of
from specific stakeholders including consumers and on a
studies launched prior to the date of entry into force of this
reliable and validated flow of information between the
Regulation and conducted under quality assurance systems
different stakeholders. More specific strategies shall be
other than GLP and ISO, the applicant shall provide:
included when data on individual intakes of a specific
food item or intakes of particular age groups have to be
collected;
(a) a thorough description of the quality assurance system
under which such studies were performed; and
(c) accompanied by adequate justification and a thorough
description of the selected methodologies for the proposed
(b) comprehensive information on the protocols and the results
post-market monitoring including aspects related to the
obtained from the studies including the raw data.
analysis of the collected information.
Article 10
Article 8
Amendments to Regulation (EC) No 641/2004
Requirements concerning the methods of detection,
identification and quantification and for control samples
Regulation (EC) No 641/2004 is amended as follows:
and reference material of genetically modified food or
feed for applications submitted under Articles 5(3), 11(2),
(1) Article 1 is replaced by the following:
17(3) and 23(2)
1. Applications
submitted
under
Articles
5(1)
and
17(1)
of
Regulation (EC) No 1829/2003 shall comply with the following
‘Article 1
requirements, as referred to in Article 5(3)(i) and (j) and
Article 17(3)(i) and (j) of that Regulation, and set out in
Annex III to this Regulation, for:
This chapter provides detailed rules concerning applications
for authorisation submitted in accordance with Articles 5
and 17 of Regulation (EC) No 1829/2003 except for those
(a) the methods for detection and identification of the trans
applications covered by Commission Implementing Regu
formation event;
lation (EU) No 503/2013 (*).
___________
(b) samples of food or feed and their control samples, and
(*) OJ L 157, 8.6.2013, p. 1.’;
information as to the place where the reference material
can be accessed.
(2) Articles 5 to 19 are deleted.
2. For
applications
submitted
under
Articles
11(1)
and
23(1)
of Regulation (EC) No 1829/2003, the requirements set out in
Annex III to this Regulation for:
Article 11
Amendments to Regulation (EC) No 1981/2006
(a) the methods for detection and identification of the trans
Regulation (EC) No 1981/2006 is amended as follows:
formation event;
(1) in Article 2, point (a) is replaced by the following:
(b) samples of food or feed and their control samples, and
information as to the place where the reference material
can be accessed,
‘(a) “full validation procedure” means:

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(i) the assessment through a ring trial involving
(ii) food or feed containing or consisting of genetically
national reference laboratories of the method
modified plants;
performance criteria set by the applicant as
compliant with the document entitled “Definition
of minimum performance requirements for
(iii) food produced from or containing ingredients
analytical methods of GMO testing” referred to:
produced from genetically modified plants or feed
produced from such plants; or
— in the case of genetically modified plants for
food or feed uses, food or feed containing or
(b) point 1(B) of Annex I to Regulation (EC) No 641/2004,
consisting of genetically modified plants and
in all other cases.
food produced from or containing ingredients
produced from genetically modified plants or
feed produced from genetically modified plants,
That amount shall be multiplied by the number of GMO
in point 3.1.C.4 of Annex III to Commission
events to be fully validated.’.
Implementing Regulation (EU) No 503/2013 (*),
— in all other cases, in point 1(B) of Annex I to
Article 12
Regulation (EC) No 641/2004;
Review
and
1. The
Commission
shall
monitor
the
application
of
this
Regulation, the developments in scientific knowledge on
replacement, reduction and refinement of animal use in
(ii) the assessment of the precision and trueness of the
scientific procedures and the publication of new guidance
method provided by the applicant.
from EFSA. The Commission shall in particular monitor the
___________
outcome of the research project called GRACE (GMO Risk
(*) OJ L 157, 8.6.2013, p. 1.’;
Assessment and Communication of Evidence) under the 2012
work programme of the seventh Framework Programme for
Research (FP7).
(2) in Article 3(2), the first and second subparagraphs are
replaced by the following:
2. The
Commission
shall
review
the
requirement
to
perform
‘2. The
CRL
shall
request
the
applicant
to
pay
an 90-day feeding studies in rodents with whole genetically
additional contribution of EUR 60 000 where a full vali
modified food/feed (point 1.4.4.1 of Annex II) on the basis of
dation procedure of a method of detection and identifi
new scientific information. The results of this review shall be
cation for a single GMO event according to the
published by 30 June 2016 at the latest.
requirements laid down in the following provisions is
required:
Article 13
(a) Annex III to Implementing Regulation (EU)
Entry into force
No 503/2013, when the application is related to:
This Regulation shall enter into force on the twentieth day
following that of its publication in the Official Journal of the
(i) genetically modified plants for food or feed uses;
European Union.
This Regulation shall be binding in its entirety and directly applicable in all Member States.
Done at Brussels, 3 April 2013.
For the Commission
The President
José Manuel BARROSO

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8.6.2013
ANNEX I
PREPARATION AND PRESENTATION OF APPLICATIONS
The application shall contain the following information:
PART I
GENERAL INFORMATION
1. Name and address of the applicant (company or institute).
2. Name, qualification and experience of the responsible scientist(s) and contact details of the responsible person for all
dealings with the European Food Safety Authority (EFSA).
3. Designation and specification of the genetically modified plant and its products.
4. Scope of the application:
(a) Genetically modified food
Food containing or consisting of genetically modified plants
Food produced from genetically modified plants or containing ingredients produced from genetically modified
plants
(b) Genetically modified feed
Feed containing or consisting of genetically modified plants
Feed produced from genetically modified plants
(c) Genetically modified plants for food or feed uses
Products other than food and feed containing or consisting of genetically modified plants with the exception
of cultivation
Seeds and other plant propagating material for cultivation in the Union.
5. Unique identifier.
A proposal for a unique identifier for the genetically modified plant developed in accordance with Regulation (EC)
No 65/2004.
6. Where applicable, a detailed description of the method of production and manufacturing.
This description would include, for example, a detailed description of specific methods of production of food or feed
which would be due to the nature of the genetic modification or which would lead to food or feed with specific
characteristics.
7. Where appropriate, the conditions for the placing on the market of the genetically modified food(s) or feed(s),
including specific conditions for use and handling.
8. Where applicable, the status of the food or feed or of related substances under other provisions of Union law.
Additional authorisation requirements provided for in Union law, related to the placing on the market of the food or
feed or applicable ‘maximum residue level’ (MRL) where the food or feed is likely to contain residues of plant
protection products.

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PART II
SCIENTIFIC INFORMATION
All the requirements of Part II shall be provided in the application except where such requirements are not justified by the
scope of the application (for example, where the application is limited to food or feed produced from GMOs).
1. HAZARD
IDENTIFICATION
AND
CHARACTERISATION
1.1. Information
relating
to
the
recipient
or
(where
appropriate)
parental
plants
(a) Complete name:
(i) family name;
(ii) genus;
(iii) species;
(iv) subspecies;
(v) cultivar, breeding line;
(vi) common name;
(b) Geographical distribution and cultivation of the plant within the Union;
(c) Information on the recipient or parental plants relevant to their safety, including any known toxicity or
allergenicity;
(d) Data on the past and present use of the recipient plant, such as history of safe use for consumption as food
or feed, including information on how the plant is typically cultivated, transported and stored, whether
special processing is required to make the plant safe to eat, and the plant normal role in the diet (for
example, which part of the plant is used as a food source, whether its consumption is important in particular
subgroups of the population, what important macro- or micro-nutrients it contributes to the diet);
(e) Additional information relating to the recipient or parental plants required for the environmental safety
aspects:
(i) information concerning reproduction:
— mode(s) of reproduction;
— specific factors affecting reproduction (if any);
— generation time;
(ii) sexual compatibility with other cultivated or wild plant species;
(iii) survivability:
— ability to form structures for survival or dormancy;
— specific factors, if any, affecting survivability;
(iv) dissemination:
— ways and extent of dissemination (to include, for example, an estimation of how viable pollen
and/or seed declines with distance);
— special factors affecting dissemination, if any;
(v) geographical distribution within the Union of the sexually compatible species;

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(vi) where a plant species is not grown in the Union, a description of the natural habitat of the plant,
including information on natural predators, parasites, competitors and symbionts;
(vii) other potential interactions of the genetically modified plant with organisms in the ecosystem where it is
usually grown, or used elsewhere, including information on toxic effects on humans, animals and other
organisms.
1.2. Molecular
Characterisation
1.2.1. Information
relating
to
the
genetic
modification
1.2.1.1. Description of the methods used for the genetic modification
1.2.1.2. Nature and source of vector used
1.2.1.3. Source of donor nucleic acid(s) used for transformation, size and intended function of each constituent fragment
of the region intended for insertion
1.2.2. Information
relating
to
the
genetically
modified
plant
1.2.2.1. General description of the trait(s) and characteristics which have been introduced or modified
1.2.2.2. Information on the sequences actually inserted/deleted
1.2.2.3. Information on the expression of the insert(s)
1.2.2.4. Genetic stability of the insert and phenotypic stability of the genetically modified plant
1.2.2.5. Potential risk associated with horizontal gene transfer
1.2.3. Additional information relating to the genetically modified plant required for the environmental safety aspects
1.2.3.1. Information on how the genetically modified plant differs from the recipient plant in reproduction, dissemi
nation, survivability or other properties.
1.2.3.2. Any change to the ability of the genetically modified plant to transfer genetic material to other organisms,
namely:
(a) Plant to bacteria gene transfer;
(b) Plant to plant gene transfer.
1.2.4. Conclusions
of
the
molecular
characterisation
1.3. Comparative
analysis
1.3.1. Choice
of
the
conventional
counterpart
and
additional
comparators
1.3.2. Experimental
design
and
statistical
analysis
of
data
from
field
trials
for
comparative
analysis
1.3.2.1. Description of the protocols for the experimental design
1.3.2.2. Statistical analysis
1.3.3. Selection
of
material
and
compounds
for
analysis
1.3.4. Comparative
analysis
of
composition
1.3.5. Comparative
analysis
of
agronomic
and
phenotypic
characteristics
1.3.6. Effects
of
processing
1.3.7. Conclusion

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1.4. Toxicology
1.4.1. Testing
of
newly
expressed
proteins
1.4.2. Testing
of
new
constituents
other
than
proteins
1.4.3. Information
on
natural
food
and
feed
constituents
1.4.4. Testing
of
the
whole
genetically
modified
food
or
feed
1.4.4.1. 90-day feeding study in rodents
1.4.4.2. Animal studies with respect to reproductive, developmental or chronic toxicity
1.4.4.3. Other animal studies to examine the safety and the characteristics of genetically modified food and feed
1.4.5. Conclusion
of
the
toxicological
assessment
1.5. Allergenicity
1.5.1. Assessment
of
allergenicity
of
the
newly
expressed
protein
1.5.2. Assessment
of
allergenicity
of
the
whole
genetically
modified
plant
1.5.3. Conclusion
of
the
allergenicity
assessment
1.6. Nutritional
assessment
1.6.1. Nutritional
assessment
of
the
genetically
modified
food
1.6.2. Nutritional
assessment
of
the
genetically
modified
feed
1.6.3. Conclusion
of
the
nutritional
assessment
2. EXPOSURE
ASSESSMENT
—
ANTICIPATED
INTAKE
OR
EXTENT
OF
USE
3. RISK
CHARACTERISATION
4.
POST-MARKET MONITORING ON THE GENETICALLY MODIFIED FOOD OR FEED
5. ENVIRONMENTAL
ASSESSMENT
6. ENVIRONMENTAL
MONITORING
PLAN
7. ADDITIONAL
INFORMATION
RELATED
TO
THE
SAFETY
OF
THE
GENETICALLY
MODIFIED
FOOD
OR
FEED
A systematic review of studies published in the scientific literature and studies performed by the applicant within the
period of 10 years prior to the date of submission of the dossier on the potential effects on human and animal health of
the genetically modified food and feed covered by the application shall be included in the application. This systematic
review shall be carried out by taking into account the guidance of EFSA on application of systematic review methodology
to food and feed safety assessments to support decision making ( 1 ).
Where the information obtained from those studies is not coherent with the information obtained from the studies
performed in accordance with the requirements set out in Annex II, the applicant shall provide a thorough analysis of the
respective studies and provide plausible explanations for the observed discrepancies.
Additional information which might influence the evaluation of the safety of the genetically modified food or feed
generated following the submission of the application, as well as any information regarding any prohibition or restriction
imposed by a competent authority of any third country on the basis of a safety assessment shall be provided by the
applicant.
PART III
CARTAGENA PROTOCOL
The application shall provide the information required under Articles 5(3)(c) and 17(3)(c) of Regulation (EC)
No 1829/2003 for the purpose of complying with Annex II to the Cartagena Protocol on Biosafety to the Convention
on Biological Diversity.
( 1 ) EFSA Journal 2010; 8(6):1637.

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The provided information shall contain as a minimum the information specified in Annex II to Regulation (EC)
No 1946/2003 of the European Parliament and of the Council ( 1 ):
(a) The name and contact details of the applicant for a decision for domestic use;
(b) The name and contact details of the authority responsible for the decision;
(c) Name and identity of the GMO;
(d) Description of the gene modification, the technique used, and the resulting characteristics of the GMO;
(e) Any unique identification of the GMO;
(f) Taxonomic status, common name, point of collection or acquisition, and characteristics of recipient organism or
parental organisms related to biosafety;
(g) Centres of origin and centres of genetic diversity, if known, of the recipient organism and/or the parental organisms
and a description of the habitats where the organisms may persist or proliferate;
(h) Taxonomic status, common name, point of collection or acquisition, and characteristics of the donor organism or
organisms related to biosafety;
(i) Approved uses of the GMO;
(j) A risk assessment report consistent with Annex II to Directive 2001/18/EC;
(k) Suggested methods for the safe handling, storage, transport and use, including packaging, labelling, documentation,
disposal and contingency procedures, where appropriate.
PART IV
LABELLING
The application shall include:
(a) a proposal for labelling in all official languages of the Union, where a proposal for specific labelling is required in
accordance with Articles 5(3)(f) and 17(3)(f) of Regulation (EC) No 1829/2003;
(b) either a reasoned statement that the food or feed does not give rise to ethical or religious concerns or a proposal for
labelling in all official languages of the Union as required by Articles 5(3)(g) and 17(3)(g) of Regulation (EC)
No 1829/2003;
(c) when appropriate a proposal for labelling complying with the requirements of point A(8) of Annex IV to Directive
2001/18/EC.
PART V
METHODS OF DETECTION, SAMPLING AND IDENTIFICATION AND REFERENCE MATERIAL
The applicant shall provide methods for detection, sampling and identification, as well as samples of the food or feed and
their controls samples to the European Union Reference Laboratory (EURL) as referred to in Article 32 of Regulation (EC)
No 1829/2003.
The application shall include a copy of the completed form for the submission of those samples to the EURL and proof
of sending to the EURL.
The application shall include information as to the place where the reference material can be accessed.
The applicant shall follow the instructions for the preparation and the sending of the samples provided by the EU
Reference laboratory (EURL) as referred to in Article 32 of Regulation (EC) No 1829/2003. These instructions are
published on the following webpage: http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm
( 1 ) OJ L 287, 5.11.2003, p. 1.

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PART VI
ADDITIONAL INFORMATION TO BE PROVIDED FOR GENETICALLY MODIFIED PLANTS AND/OR FOOD
OR FEED CONTAINING OR CONSISTING OF GENETICALLY MODIFIED PLANTS
The information required in the notification as set out in Annex III to Directive 2001/18/EC shall be provided where it is
not covered by the requirements of other parts of the application.
PART VII
SUMMARY OF APPLICATIONS
This Part specifies the standardised form, which the summary of the application dossier must follow.
Depending on the scope of the application, some of the requested information may not be applicable.
The summary shall not contain parts considered to be confidential in accordance with Article 30 of Regulation (EC)
No 1829/2003.
1. GENERAL
INFORMATION
1.1. Details
of
application
(a) Member State of application
(b) Application number
(c) Name of the product (commercial and any other names)
(d) Date of acknowledgement of valid application
1.2. Applicant
(a) Name of applicant
(b) Address of applicant
(c) Name and address of the representative of the applicant established in the Union (if the applicant is not
established in the Union)
1.3. Scope
of
the
application
(a) Genetically modified food
Food containing or consisting of genetically modified plants
Food produced from genetically modified plants or containing ingredients produced from genetically
modified plants
(b) Genetically modified feed
Feed containing or consisting of genetically modified plants
Feed produced from genetically modified plants
(c) Genetically modified plants for food and feed use
Products other than food and feed containing or consisting of genetically modified plants with the
exception of cultivation
Seeds and plant propagating material for cultivation in the Union

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1.4. Is the product or the uses of the associated plant protection product(s) already authorised or subject to
another authorisation procedure within the Union?
No
Yes (in that case, specify)
1.5. Has
the
genetically
modified
plant
been
notified
under
Part
B
of
Directive
2001/18/EC?
Yes
No (in that case, provide risk analysis data on the basis of the elements of Part B of Directive
2001/18/EC)
1.6. Has
the
genetically
modified
plant
or
derived
products
been
previously
notified
for
marketing
in
the
Union under Part C of Directive 2001/18/EC?
No
Yes (in that case, specify)
1.7. Has
the
product
been
subject
to
an
application
and/or
authorised
in
a
third
country
either
previously
or
simultaneously to this application?
No
Yes In that case, specify the third country, the date of application and, where available, a copy of the risk
assessment conclusions, the date of the authorisation and the scope of the application
1.8. General
description
of
the
product
(a) Name of the recipient or parental plant and the intended function of the genetic modification
(b) Types of products planned to be placed on the market according to the authorisation applied for and any
specific form in which the product must not be placed on the market (such as seeds, cut-flowers, vegetative
parts) as a proposed condition of the authorisation applied for
(c) Intended use of the product and types of users
(d) Any specific instructions and recommendations for use, storage and handling, including mandatory restrictions
proposed as a condition of the authorisation applied for
(e) If applicable, geographical areas within the Union to which the product is intended to be confined under the
terms of the authorisation applied for
(f) Any type of environment to which the product is unsuited
(g) Any proposed packaging requirements
(h) Any proposed labelling requirements in addition to those required by other applicable EU legislation than
Regulation (EC) No 1829/2003 and when necessary a proposal for specific labelling in accordance with
Article 13(2) and (3), Article 25(2)(c) and (d) and Article 25(3) of Regulation (EC) No 1829/2003
In the case of products other than food and feed containing or consisting of genetically modified plants, a
proposal for labelling which complies with the requirements of point A(8) of Annex IV to Directive
2001/18/EC must be included.
(i) Estimated potential demand
(i) In the EU
(ii) In EU export markets
(j) Unique identifier in accordance with Regulation (EC) No 65/2004.

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1.9. Measures suggested by the applicant to take in the case of unintended release or misuse of the product as
well as measures for its disposal and treatment
2. INFORMATION
RELATING
TO
THE
RECIPIENT
OR
(WHERE
APPROPRIATE)
PARENTAL
PLANTS
2.1. Complete
name
(a) Family name
(b) Genus
(c) Species
(d) Subspecies
(e) Cultivar/breeding line
(f) Common name
2.2. Geographical distribution and cultivation of the plant, including the distribution within the Union
2.3. Information
concerning
reproduction
(for
environmental
safety
aspects)
(a) Mode(s) of reproduction
(b) Specific factors affecting reproduction
(c) Generation time
2.4. Sexual
compatibility
with
other
cultivated
or
wild
plant
species
(for
environmental
safety
aspects)
2.5. Survivability
(for
environmental
safety
aspects)
(a) Ability to form structures for survival or dormancy
(b) Specific factors affecting survivability
2.6. Dissemination
(for
environmental
safety
aspects)
(a) Ways and extent of dissemination
(b) Specific factors affecting dissemination
2.7. Geographical distribution within the Union of the sexually compatible species (for environmental safety
aspects)
2.8. In the case of plant species not normally grown in the Union description of the natural habitat of the
plant, including information on natural predators, parasites, competitors and symbionts (for environ
mental safety aspects)
2.9. Other
potential
interactions,
relevant
to
the
genetically
modified
plant,
of
the
plant
with
organisms
in
the
ecosystem where it is usually grown, or used elsewhere, including information on toxic effects on
humans, animals and other organisms (for environmental safety aspects)
3. MOLECULAR
CHARACTERISATION
3.1. Information
relating
to
the
genetic
modification
(a) Description of the methods used for the genetic modification
(b) Nature and source of the vector used
(c) Source of donor nucleic acid(s) used for transformation, size and intended function of each constituent
fragment of the region intended for insertion
3.2. Information
relating
to
the
genetically
modified
plant
3.2.1. Description of the trait(s) and characteristics which have been introduced or modified

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3.2.2. Information on the nucleic acid(s) sequences actually inserted or deleted
(a) The copy number of all detectable inserts, both complete and partial
(b) In the case of deletion(s), size and function of the deleted region(s)
(c) Subcellular location(s) of insert(s) (nucleus, chloroplasts, mitochondria, or maintained in a non-integrated form)
and methods for its/their determination
(d) The organisation of the inserted genetic material at the insertion site
(e) In the case of modifications other than insertion or deletion, describe function of the modified genetic material
before and after the modification, as well as direct changes in expression of genes as a result of the modi
fication
3.2.3. Information on the expression of the insert
(a) Information on developmental expression of the insert during the life cycle of the plant
(b) Parts of the plant where the insert is expressed
3.2.4. Genetic stability of the insert and phenotypic stability of the genetically modified plant
3.2.5. Information (for environmental safety aspects) on how the genetically modified plant differs from the recipient plant in:
(a) Mode(s) and/or rate of reproduction
(b) Dissemination
(c) Survivability
(d) Other differences
3.2.6. Any change to the ability of the genetically modified plant to transfer genetic material to other organisms (for environmental
safety aspects)
(a) Plant to bacteria gene transfer
(b) Plant to plant gene transfer
4. COMPARATIVE
ANALYSIS
4.1. Choice
of
the
conventional
counterpart
and
additional
comparators
4.2. Experimental
design
and
statistical
analysis
of
data
from
field
trials
for
comparative
analysis
Description of the experimental design (number of locations, growing seasons, geographical spread, replicates and
number of commercial varieties in each location) and of the statistical analysis.
4.3. Selection
of
material
and
compounds
for
analysis
4.4. Comparative
analysis
of
agronomic
and
phenotypic
characteristics
4.5. Effect
of
processing
5. TOXICOLOGY
(a) Toxicological testing of newly expressed proteins
(b) Testing of new constituents other than proteins
(c) Information on natural food or feed constituents
(d) Testing of the whole genetically modified food and feed

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6. ALLERGENICITY
(a) Assessment of allergenicity of the newly expressed protein
(b) Assessment of allergenicity of the whole genetically modified plant
7. NUTRITIONAL
ASSESSMENT
(a) Nutritional assessment of the genetically modified food
(b) Nutritional assessment of the genetically modified feed
8. EXPOSURE
ASSESSMENT
—
ANTICIPATED
INTAKE/EXTENT
OF
USE
9. RISK
CHARACTERISATION
10. POST-MARKET MONITORING ON THE GENETICALLY MODIFIED FOOD OR FEED
11. ENVIRONMENTAL
ASSESSMENT
11.1. Mechanism of interaction between the genetically modified plant and target organisms
11.2. Potential changes in the interactions of the genetically modified plant with the biotic environment
resulting from the genetic modification
(a) Persistence and invasiveness
(b) Selective advantage or disadvantage
(c) Potential for gene transfer
(d) Interactions between the genetically modified plant and target organisms
(e) Interactions of the genetically modified plant with non-target organisms
(f) Effects on human health
(g) Effects on animal health
(h) Effects on biogeochemical processes
(i) Impacts of the specific cultivation, management and harvesting techniques
11.3. Potential interactions with the abiotic environment
11.4. Risk characterisation
12. ENVIRONMENTAL
MONITORING
PLAN
(a) General (risk assessment, background information)
(b) Interplay between environmental risk assessment and monitoring
(c) Case-specific genetically modified plant monitoring (approach, strategy, method and analysis)
(d) General surveillance of the impact of the genetically modified plant (approach, strategy, method and analysis)
(e) Reporting the results of monitoring
13. DETECTION AND IDENTIFICATION TECHNIQUES FOR THE GENETICALLY MODIFIED PLANT
14. INFORMATION
RELATING
TO
PREVIOUS
RELEASES
OF
THE
GENETICALLY
MODIFIED
PLANT
(FOR
ENVI
RONMENTAL RISK ASSESSMENT ASPECTS)

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14.1. History of previous releases of the genetically modified plant notified under Part B of Directive
2001/18/EC or under Part B of Council Directive 90/220/EEC ( 1 ) by the same notifier
(a) Notification number
(b) Conclusions of post-release monitoring
(c) Results of the release with respect to any risk to human health and the environment, submitted to the
competent authority in accordance with Article 10 of Directive 2001/18/EC
14.2. History of previous releases of the genetically modified plant carried out outside the Union by the same
notifier
(a) Release country
(b) Authority overseeing the release
(c) Release site
(d) Aim of the release
(e) Duration of the release
(f) Aim of post-releases monitoring
(g) Duration of post-releases monitoring
(h) Conclusions of post-release monitoring
(i) Results of the release with respect to any risk to human health and the environment
( 1 ) OJ L 117, 8.5.1990, p. 15.

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ANNEX II
SCIENTIFIC REQUIREMENTS FOR THE RISK ASSESSMENT OF GENETICALLY MODIFIED FOOD AND FEED
I. INTRODUCTION
1. DEFINITIONS
For the purpose of this Annex, the following definitions shall apply:
1. ‘hazard identification’ means the identification of biological, chemical and physical agents capable of causing
adverse health effects and which may be present in a particular food and feed or group of foods and feeds;
2. ‘hazard characterisation’ means the qualitative and/or quantitative evaluation of the nature of the adverse
health effects associated with biological, chemical and physical agents which may be present in food and feed;
3. ‘risk characterisation’ means the qualitative and/or quantitative estimation, including attendant uncertainties, of
the probability of occurrence and severity of known or potential adverse health effects in a given population
based on hazard identification, hazard characterisation and exposure assessment.
2. SPECIFIC
CONSIDERATIONS
2.1.
Insertion of marker genes and other nucleic acid(s) sequences not essential to achieve the desired trait
In order to facilitate the risk assessment, the applicant shall endeavour to minimise the presence of inserted
nucleic acid(s) sequences not essential to achieve the desired trait.
During the process of genetic modification of plants and other organisms, marker genes are often used to
facilitate the selection and identification of genetically modified cells, containing the gene of interest inserted into
the genome of the host organism, among the vast majority of untransformed cells. The applicant shall carefully
select such marker genes and Article 4(2) of Directive 2001/18/EC shall be respected. Against that background,
the applicant shall therefore aim to develop GMOs without the use of antibiotic resistance marker genes.
2.2. Risk
assessment
of
genetically
modified
food
and
feed
containing
stacked
transformation
events
For the risk assessment of genetically modified food and feed containing stacked transformation events obtained
by conventional crossing of genetically modified plants containing one or several transformation event(s), the
applicant shall provide a risk assessment of each single transformation event or, in accordance with Article 3(6)
of this Regulation, refer to already submitted application(s). The risk assessment of genetically modified food and
feed containing stacked transformation events shall also include an assessment of the following aspects:
(a) stability of the transformation events;
(b) expression of the transformation events;
(c) potential synergistic or antagonistic effects resulting from the combination of the transformation events shall
be subject to an assessment in accordance with Sections 1.4 (Toxicology), 1.5 (Allergenicity) and 1.6
(Nutritional assessment).
For genetically modified food and feed containing, consisting of or produced from genetically modified plants,
whose cultivation is associated with the production of genetically modified material containing various subcom
binations of transformation events (segregating crops), the application shall include all subcombinations indepen
dently of their origin which have not yet been authorised. In such a case, the applicant shall provide a scientific
rationale justifying that there is no need to provide experimental data for the concerned subcombinations or, in
the absence of such scientific rationale, provide the experimental data.
For genetically modified food and feed containing, consisting of or produced from genetically modified plants,
the cultivation of which does not lead to the production of genetically modified material containing various
combinations of transformation events (non-segregating crops), the application shall only cover the combination
which is to be placed on the market.
The rules set out in this Section apply mutatis mutandis to transformation events which are combined by other
means such as co- and retransformation.

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II. SCIENTIFIC REQUIREMENTS
1. HAZARD
IDENTIFICATION
AND
CHARACTERISATION
1.1. Information
relating
to
the
recipient
or
(where
appropriate)
parental
plants
1.1.1. The applicant shall provide comprehensive information relating to the recipient or (where appropriate) the
parental plants in order to:
(a) evaluate all issues of potential concern, such as the presence of natural toxins or allergens;
(b) identify the need for specific analyses.
1.1.2. For the purposes referred to in point 1.1.1, the applicant shall provide the following information:
(a) Complete name:
(i) family name;
(ii) genus;
(iii) species;
(iv) subspecies;
(v) cultivar/breeding line or strain;
(vi) common name.
(b) Geographical distribution and cultivation of the plant, including its distribution within the Union.
(c) Information on the recipient or parental plants relevant to their safety, including any known toxicity or
allergenicity.
(d) Data on the past and present use of the recipient plant. This information shall include the history of safe use
for consumption as food or feed, information on how the plant is typically cultivated, transported and
stored, whether special processing is required to make the plant safe to eat, and describe the normal role of
the plant in the diet (such as which part of the plant is used as a food or feed source, whether its
consumption is important in particular subgroups of the population, what important macro- or micro-
nutrients it contributes to the diet).
1.2. Molecular
Characterisation
1.2.1. Information
relating
to
the
genetic
modification
The applicant shall provide sufficient information on the genetic modification:
(a) to identify the nucleic acid(s) intended for transformation and related vector sequences potentially delivered
to the recipient plant;
(b) to characterise the nucleic acid(s) actually inserted in the plant.
1.2.1.1. Description of the methods used for the genetic modification
The applicant shall provide information on the following elements:
(a) the method of genetic transformation including relevant references;
(b) the recipient plant material;
(c) the species and the strain of Agrobacterium and other microbes, if used during the genetic transformation
process;
(d) the helper plasmids, if used during the genetic transformation process;
(e) the source of carrier nucleic acid(s), if used during the genetic transformation process.

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1.2.1.2. Nature and source of vector used
The applicant shall provide the following information:
(a) a physical map of the functional elements and other plasmid/vector components together with the relevant
information needed for the interpretation of the molecular analyses (such as restriction sites, the position of
primers used in polymerase chain reaction (PCR), the location of probes used in Southern analysis). The
region intended for insertion shall be clearly indicated;
(b) a table identifying each component of the plasmid/vector (including the region intended for insertion), its
size, its origin and its intended function.
1.2.1.3. Source of nucleic acid(s) used for transformation, size and intended function of each constituent fragment of the
region intended for insertion
The applicant shall provide information on the donor organism(s) and on the nucleic acid(s) sequence(s) intended
to be inserted in order to determine whether the nature of the donor organism(s) or the nucleic acid sequence(s)
may trigger any safety issue.
Information regarding the function of the nucleic acid region(s) intended for insertion shall comprise the
following elements:
(a) the complete sequence of the nucleic acid(s) intended to be inserted, including information on any deliberate
alteration(s) to the corresponding sequence(s) in the donor organism(s);
(b) the history of safe use of the gene product(s) arising from the regions intended for insertion;
(c) data on the possible relationship of the gene products with known toxins, anti-nutrients and allergens.
Information regarding each donor organism shall comprise:
— taxonomic classification;
— history of use regarding food and feed safety.
1.2.2. Information
relating
to
the
genetically
modified
plant
1.2.2.1. General description of the trait(s) and characteristics which have been introduced or modified
Information provided under this point may be limited to a general description of the introduced trait(s) and the
resulting changes to the phenotype and metabolism of the plant.
For example, when the introduced trait is herbicide tolerance, the applicant shall provide information on the
mode of action of the active substance and its metabolism in the plant.
1.2.2.2. Information on the sequences actually inserted/deleted
The applicant shall provide the following information:
(a) the size and copy number of all detectable inserts, both complete and partial; this is typically determined by
Southern analysis.
Probe/restriction enzyme combinations used for this purpose shall provide complete coverage of sequences
that could be inserted into the genetically modified plant, such as any parts of the plasmid/vector or any
carrier or foreign nucleic acid(s) remaining in the genetically modified plant.
The Southern analysis shall span the entire transgenic locus(i) as well as flanking sequences and include all
appropriate controls.
For the determination of copy number of the insert, complementary methods may also be used (such as real-
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(b) the organisation and sequence of the inserted genetic material at each insertion site in a standardised
electronic format, with the aim of identifying changes in the inserted sequences compared to the
sequence intended for insertion;
(c) in the case of deletion(s), size and function of the deleted region(s), whenever possible;
(d) subcellular location(s) of insert(s) (nucleus, chloroplasts, mitochondria, or maintained in a non-integrated
form) and methods for its/their determination;
(e) sequence information in a standardised electronic format for both 5’ and 3’ flanking regions at each insertion
site, with the aim of identifying interruptions of known genes.
Bioinformatic analyses shall be conducted using up-to-date databases with the aim of performing both
intraspecies and interspecies similarity searches.
In the case of genetically modified plants containing stacked transformation events, the safety of potential
interactions between any unintended modifications at each insertion site shall be assessed;
(f) Open Reading Frames (hereafter referred to as ‘ORFs’ and defined as any nucleotide sequence that contains a
string of codons that is uninterrupted by the presence of a stop codon in the same reading frame) created as
a result of the genetic modification either at the junction sites with genomic DNA or due to internal
rearrangements of the insert(s).
The ORFs shall be analysed between stop codons, not limiting their lengths. Bioinformatic analyses shall be
conducted to investigate possible similarities with known toxins or allergens using up-to-date databases.
The characteristics and versions of the databases shall be provided.
Depending on the information gathered, further analyses (such as transcription analysis) may be needed to
complete the risk assessment.
1.2.2.3. Information on the expression of the insert(s)
The applicant shall provide information:
— to demonstrate whether the inserted/modified sequence results in intended changes at the protein, RNA
and/or metabolite levels;
— to characterise the potential unintended expression of new ORFs identified under point 1.2.2.2(f) as raising a
safety concern.
For those purposes, the applicant shall provide the following information:
(a) The method(s) used for expression analysis together with their performance characteristics;
(b) Information on developmental expression of the insert during the life cycle of the plant.
The requirement for information on developmental expression shall be considered on a case-by-case basis
taking into account the promoter used, the intended effect(s) of the modification and scope of the appli
cation;
(c) Parts of the plant where the insert/modified sequences are expressed;
(d) Potential unintended expression of new ORFs identified under point 1.2.2.2(f) which raise a safety concern;
(e) Protein expression data, including the raw data, obtained from field trials and related to the conditions in
which the crop is grown.
Data on expression levels from those parts of the plant used for food and feed purposes shall be provided in
all cases.

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In addition, information shall also be provided on the expression of target genes in other parts of the plant
when tissue-specific promoters have been used and when this is relevant for the safety assessment. The
minimum requirement for protein expression shall be data provided from three growing sites or from one
site over three seasons. Permutations of the sites and seasons shall be acceptable provided that the minimum
requirement is met. When justified by the nature of the insert (such as silencing approaches or where
biochemical pathways have been intentionally modified), specific RNA(s) or metabolite(s) shall be analysed.
For silencing approaches by RNAi expression, potential ‘off target’ genes should be searched by in silico
analysis to assess if the genetic modification could affect the expression of other genes which raise safety
concerns;
(f) With regard to the stacking of transformation events by conventional crossing, expression data shall be
provided to assess the potential interactions between the events which may raise any additional safety
concerns over protein and trait expression compared with the single transformation events. The comparison
shall be carried out with data obtained from plants grown in the same field trials. On a case-by-case basis,
and where concerns arise, additional information may be necessary.
1.2.2.4. Genetic stability of the insert and phenotypic stability of the genetically modified plant
The applicant shall provide information:
(a) to demonstrate the genetic stability of the transgenic locus(i) and the phenotypic stability and inheritance
pattern(s) of the introduced trait(s);
(b) in case of stacked transformation events, to establish that each of the transformation events stacked in the
plant has the same molecular properties and characteristics as in the plants with the single transformation
events.
For the purposes of that information, applicants shall provide data which demonstrate stability over multiple
(normally five) generations or vegetative cycles for plant containing single transformation events. Data from the
first and the last generations of vegetative cycles are sufficient. The source of the material used for the analysis
shall be specified. Data shall be analysed using appropriate statistical methods.
For stacked transformation events comparisons between the original transformation events and the stacked
transformation events shall be carried out using plant materials representative of those designed for commercial
production. The applicant shall provide adequate justification for the plant materials used. Comparisons shall
include comparisons of sequences of the inserts and the flanking regions obtained from GM plants containing
single events and plants containing stacked transformation events.
To assess genetic stability of the transformation event(s), applicants shall use appropriate molecular approaches
referred to in Section 1.2.2.2.
1.2.2.5. Potential risk associated with horizontal gene transfer
The applicant shall assess the probability of horizontal gene transfer from the product to humans, animals and
micro-organisms and any potential associated risk when intact and functional nucleic acid(s) remains in the
genetically modified food and feed.
1.2.3. Conclusions
of
the
molecular
characterisation
The molecular characterisation shall provide data on the structure and expression of the insert(s), and on the
stability of the intended trait(s). This shall also apply to situations where transformation events have been stacked
by conventional breeding.
It shall be specifically indicated whether the molecular characterisation of the genetic modification(s) raises safety
concerns with regard to the interruption of endogenous genes or regulatory sequences.
The molecular characterisation shall also aim to identify whether the genetic modification(s) raise(s) any issues
regarding the potential for producing proteins/substances other than those intended and in particular new toxins
or allergens.
The potential unintended changes identified in this Section shall be addressed in the relevant complementary
part(s) of the safety assessment.

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1.3. Comparative
analysis
The comparative analysis of composition and agronomic as well as phenotypic characteristics shall constitute,
together with the molecular characterisation, the starting point to structure and conduct the risk assessment of a
new genetically modified food and feed.
It shall aim at identifying similarities and differences:
(a) in composition, agronomic performance and phenotypic characteristics (intended and unintended alterations)
between the genetically modified plant and its conventional counterpart;
(b) in composition between the genetically modified food and feed and its conventional counterpart.
Where no appropriate conventional counterpart can be identified, a comparative safety assessment cannot be
made and consequently a safety and nutritional assessment of the genetically modified food or feed shall be
carried out as for novel foods falling within the scope of Regulation (EC) No 258/97 of the European Parliament
and of the Council ( 1 ) that do not have conventional counterparts (such as where the genetically modified food
or feed is not closely related to a food or feed with a history of safe use or where a specific trait or specific traits
are introduced with the intention of bringing complex changes in the composition of the genetically modified
food or feed).
1.3.1. Choice
of
the
conventional
counterpart
and
additional
comparators
In the case of vegetatively propagated crops, the conventional counterpart shall, in principle, be the near-isogenic
variety used to generate the transgenic line.
In the case of crops that reproduce sexually, the conventional counterpart shall have a genetic background
comparable to the genetically modified plant. When the GM plant has been developed using back-crossing, a
conventional counterpart with a genetic background that is as close as possible to the genetically modified plant
shall be selected.
In addition, the applicant may include a comparator having a closer genetic background to the genetically
modified plant than the conventional counterpart (such as a negative segregant).
In the case of herbicide tolerant genetically modified plants and in order to assess whether the expected
agricultural practices influence the expression of the studied endpoints, three test materials shall be compared:
the genetically modified plant exposed to the intended herbicide; the conventional counterpart treated with
conventional herbicide management regimes; and the genetically modified plant treated with the same conven
tional herbicide management regimes.
In the case of stacked transformation events, it is not always possible to use a conventional counterpart with a
genetic background as close to the genetically modified plant as with conventional counterpart normally used for
single transformation events. In such circumstances, the applicant shall provide reasoned justification on the
choice of the conventional counterpart and assess its limitations for the risk assessment. In addition, single
parental genetically modified lines or genetically modified lines containing a subcombination of the stacked
transformation events for which an application has been submitted or negative segregants derived from these
genetically modified lines may also be included as additional comparators. The applicant shall provide detailed
information justifying the choice of additional comparators.
In all cases, the applicant shall provide information on the breeding scheme (pedigree) in relation to the
genetically modified plant, the conventional counterpart and, where appropriate, additional comparators
together with an adequate justification of their selection. The history of safe use of the conventional counterpart
shall be adequately supported by both qualitative and quantitative data.
More detailed guidance for the application of the requirements of this Section is available in the EFSA scientific
opinion ‘Guidance on selection of comparators for the risk assessment of genetically modified plants and derived
food and feed’ ( 2 ).
( 1 ) OJ L 43, 14.2.1997, p. 1.
( 2 ) EFSA Journal 2011; 9(5):2149.

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1.3.2. Experimental
design
and
statistical
analysis
of
data
from
field
trials
for
comparative
analysis
1.3.2.1. Description of the protocols for the experimental design
(a) Principles of experimental design
Field trials used for the production of material for the comparative analysis shall be performed in order to
determine whether the genetically modified plant and/or the genetically modified food and feed is different
from its conventional counterpart and/or equivalent to non-genetically modified reference varieties with a
history of safe use.
For each endpoint, the comparative analysis shall involve the following two approaches:
(i) a test of difference, to verify whether the genetically modified plant is different from its conventional
counterpart and might therefore be considered a hazard depending on the type of the identified
difference, and on the magnitude and type of exposure;
(ii) a test of equivalence to verify whether the genetically modified plant is equivalent or not to non-
genetically modified reference varieties, apart from the introduced trait(s).
In testing for difference, the null hypothesis shall be that there is no difference between the GMO and its
conventional counterpart against the alternative hypothesis that a difference exists.
Where additional comparator(s) are used for the risk assessment, a test of difference shall be carried out
between the genetically modified plant and each of the additional comparator(s) following the requirements
set out in Section 1.3.2.2 for the test of difference between the genetically modified plant and its conven
tional counterpart.
In testing for equivalence, the null hypothesis shall be that the difference between the GMO and the set of
reference varieties is at least as great as a specified minimum size (see Section 1.3.2.2) against the alternative
hypothesis that there is no difference or a smaller difference than the specified minimum between the GMO
and the set of reference varieties.
Rejection of the null hypothesis shall be required in order to conclude that the GMO and the set of reference
varieties are unambiguously equivalent for the endpoint considered. The equivalence limits used for the test
of equivalence shall represent appropriately the range of natural variation expected for reference varieties with
a history of safe use.
(b) Specific protocols for experimental design
Natural variation may have several sources: variation within a variety arises due to environmental factors and
variation between varieties arises due to a combination of both genetic and environmental factors. In order
to identify and estimate differences attributable only to genotypes, it is essential to control environmental
variability. Therefore, non-genetically modified reference varieties shall be included in the experimental design
of the field trials and in sufficient numbers to ensure an adequate estimate of the variability required to set
the equivalence limits. All test materials consisting of genetically modified plants, conventional counterpart,
reference varieties and, where appropriate, additional comparator(s) shall be randomised to plots within a
single field at each site, usually in a completely randomised or randomised block experimental design. The
different sites selected for the field trials shall reflect the different meteorological and agronomic conditions
under which the crop is to be grown; the choice shall be explicitly justified. The choice of non-genetically
modified reference varieties shall be appropriate for the chosen sites and shall be justified explicitly. In the
case the sites cover a restricted range of growing conditions, the applicant shall replicate the field trials over
more than one year.
Within each site the test materials consisting of genetically modified plants, conventional counterpart and,
where appropriate, additional comparator(s) shall be identical for all replicates. In addition, unless there is
explicit justification for not doing so, at each site there shall be at least three appropriate non-genetically
modified reference varieties of the crop that have a known history of safe use, which shall also be identical
between replicates. The replication at each site is the number of results obtained for each test material; the
replication should never be less than four at any site. However, if only two appropriate reference varieties are
available at a particular site, then the replication shall be six at that site; if only one is available then the
replication shall be eight.

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Each field trial shall be replicated at a minimum of eight sites, chosen to be representative of the range of
likely receiving environments where the plant is to be grown. The field trials may be conducted in a single
year, or spread over multiple years. The non-genetically modified reference varieties may vary between sites
and at least six different reference varieties shall be used over the entire set of field trials.
When the genetically modified plant is tested together with other genetically modified plants of the same
crop species (such as Zea mays) the production of material for the comparative assessment of these different
genetically modified plant may be produced simultaneously at the same site and within the same field trial by
the placing of the different genetically modified plants and their appropriate comparator(s) in the same
randomised block. This shall be subject to the following two strict conditions:
(i) the conventional counterpart and, where appropriate, additional comparator(s) shall always occur
together with the genetically modified plant in the same block;
(ii) all the different genetically modified plants and their comparator(s) and all the non-genetically modified
reference varieties used to test equivalence with those genetically modified plants shall be fully
randomised within each block.
If the number of plots per block required for such a field trial were to exceed 16, then a partially balanced
incomplete block design may be used, to reduce the number of plots per block, by excluding some of the
genetically modified plants and their appropriate comparator(s) from each block. This shall be subject to the
following two strict conditions:
(i) the conventional counterpart shall always occur together with its particular genetically modified plant in
the same block;
(ii) all of the non-genetically modified reference varieties shall appear in each of the incomplete blocks and
be fully randomised with the plants and their comparator(s).
The field trials shall be adequately described, giving information on important parameters such as
management of the field before sowing, date of sowing, soil type, herbicide use, climatic and other culti
vation/environmental conditions during growth and time of harvest, as well as the conditions during storage
of the harvested material.
More detailed guidance for the application of the requirements of this Section is available in the EFSA
opinion on ‘Statistical considerations for the safety evaluation of GMOs’ ( 1 ).
1.3.2.2. Statistical analysis
Analysis of data shall be presented in a clear format, using standardised scientific units. The raw data and the
programming code used for the statistical analysis shall be given in an editable form.
Data transformation may be necessary to ensure normality and to provide an appropriate scale on which
statistical effects are additive. For many endpoint response variables, a logarithmic transformation is expected
to be appropriate. In such cases, any difference between the genetically modified material and any other test
material shall be interpreted as a ratio on the natural scale. However, when logarithmic transformation does not
provide appropriate results, the natural scale or another scale shall be considered.
The total variability of each endpoint observed in the field trials shall be estimated and partitioned using
appropriate statistical models in order to derive two sets of confidence limits and to set a lower and upper
equivalence limit based on the variability observed among the reference varieties. One set of confidence limits
shall be used in the test of difference; the other set and the equivalence limits shall be used in the test of
equivalence.
A linear mixed statistical model shall be used for calculation of the confidence limits for both tests (that is to say,
the difference and equivalence tests); a slightly different model shall be used to estimate the equivalence limits to
be used in the equivalence test.
( 1 ) EFSA Journal 2010; 8(1):1250.

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Denote by I an indicator variable (uncentered in the mixed model) such that I = 1 for a field plot having any of
the non-GM reference varieties, and I = 0 otherwise. Then the random factors for model 1 should be, but not
necessarily be restricted to, those representing the variation: (i) between the test materials (a set that includes the
GM plant, its conventional counterpart, each of the non-GM reference varieties and any additional comparators);
(ii) in the interaction between the test materials and I; (iii) between sites; and (iv) between blocks within sites.
Model 2 should be identical to model 1 except that the random factor representing the interaction between the
test materials and I is omitted.
The fixed factor for both models should have as many levels as there are test materials and represent the
contrasts between the means of the test materials. The test materials are as defined above: the GM plant; its
conventional counterpart; the set of non-GM reference varieties; and any additional test materials. The set of non-
GM reference varieties is considered as a single level of the fixed factor. For the difference test, the component of
the fixed factor of interest is the single degree-of-freedom contrast between the GM plant and its conventional
counterpart. For the equivalence test, the component of the fixed factor of interest is the single degree-of-freedom
contrast between the GM plant and the set of non-GM reference varieties.
Both the difference test and the equivalence test shall be implemented using the correspondence between
hypothesis testing and the construction of confidence limits. In the case of equivalence testing, the approach
used shall follow the two one-sided tests (TOST) methodology by rejecting the null hypothesis of non-equiv
alence when the both confidence limits fall between the equivalence limits. The choice of 90 % confidence limits
corresponds to the customary 95 % level for statistical testing of equivalence.
The results of the difference and equivalence tests shall be represented visually for all the endpoints simulta
neously, on a single graph or a few graphs.
The graph(s) shall show the line of zero difference between the genetically modified material and its conventional
counterpart, and for each endpoint: the lower and upper adjusted equivalence limits; the mean difference
between the genetically modified material and its conventional counterpart; and the confidence limits for this
difference (see the set of possible outcomes for a single endpoint in the graph in Figure 1).
When, in addition to the conventional counterpart, another test material is used as comparator, the mean
difference between the genetically modified material and that comparator, its confidence limits and its
adjusted equivalence limits shall be displayed on the graph(s), for all such additional comparators, by
referring this to the same zero baseline as defined by the conventional counterpart. The line of zero difference
on the logarithmic scale corresponds to a multiplicative factor of unity on the natural scale. The horizontal axis
shall be labelled with values that specify the change on the natural scale. In the case of logarithmic trans
formation, changes of 2x and x will appear equally spaced on either side of the line of zero difference.
Despite the expected proportion of spurious significant differences, the applicant shall report and discuss all
significant differences observed between the genetically modified crop, its conventional counterpart and, where
applicable, any other test material, focusing on their biological relevance (see Section 3 on Risk Characterisation).
For reporting, full details shall be given for each endpoint analysed, listing:
(a) the assumptions underlying the analysis;
(b) full specification of the mixed models chosen, including fixed and random effects;
(c) results of any test of interaction between the test materials and sites;
(d) fixed effects, together with the appropriate estimated residual variation with which they are compared, and
variance components for the random factors;
(e) estimated degrees of freedom;
(f) any other relevant statistics.
A discussion on the likely impact of other growing conditions not tested in the field trial shall be provided.

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Figure 1: simplified version of a graph for comparative assessment showing the seven outcome types possible for
each single endpoint. After adjustment of the equivalence limits, a single confidence limit (for the difference)
serves visually for assessing the outcome of both tests (difference and equivalence). Here, only the upper adjusted
equivalence limit is considered. Shown are: the mean of the genetically modified crop on an appropriate scale
(square); the confidence limits (whiskers) for the difference between the genetically modified crop and its
conventional counterpart (bar shows confidence interval); a vertical line indicating no difference (for test of
difference); and vertical lines indicating adjusted equivalence limits (for test of equivalence). For outcome types 1,
3 and 5, the null hypothesis of no difference cannot be rejected: for outcomes 2, 4, 6 and 7, the genetically
modified crop is different from its conventional counterpart. Regarding interpretation of equivalence, four
categories (i) to (iv) are identified: in category (i), the null hypothesis of non-equivalence is rejected in favour
of equivalence; in categories (ii), (iii) and (iv), non-equivalence cannot be rejected.
A. Regarding test of difference, each outcome from the graph shall be categorised as follows and the respective
appropriate conclusion shall be drawn.
(i) Outcome types 1, 3 and 5: the confidence interval bar overlaps with the line of no-difference. The null
hypothesis of no difference cannot be rejected and the appropriate conclusion is that there is insufficient
evidence that the genetically modified crop and its conventional counterpart differ.
(ii) Outcome types 2, 4, 6 and 7: the confidence interval bar does not overlap with the line of no-difference.
The null hypothesis of no difference must be rejected and the appropriate conclusion is that the
genetically modified crop is significantly different from its conventional counterpart.
B. Regarding test of equivalence, each outcome from the graph shall be categorised as follows, and the
respective appropriate conclusion shall be drawn.
(i) Outcome types 1 and 2 (category (i), Figure 1): both confidence limits lie between the adjusted equiv
alence limits and the null hypothesis of non-equivalence is rejected. The appropriate conclusion is that
the genetically modified crop is equivalent to the set of non-genetically modified reference varieties.
(ii) Outcome types 3 and 4 (category (ii), Figure 1): the mean of the genetically modified crop lies between
the adjusted equivalence limits, but the confidence interval bar overlaps at least one of the adjusted
equivalence limits on the graph. Non-equivalence cannot be rejected and the appropriate conclusion is
that equivalence between the genetically modified crop and the set of non-genetically modified reference
varieties is more likely to be the case than lack of equivalence.
(iii) Outcome types 5 and 6 (category (iii), Figure 1): the mean of the genetically modified crop lies outside
the adjusted equivalence limits, but the confidence interval bar overlaps with at least one of the adjusted
equivalence limits. Non-equivalence cannot be rejected and the appropriate conclusion is that lack of
equivalence between the genetically modified crop and the set of non-genetically modified reference
varieties is more likely to be the case than equivalence.

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(iv) Outcome type 7 (category (iv), Figure 1): both confidence limits lie outside the adjusted equivalence
limits. The appropriate conclusion is that there is lack of equivalence between the genetically modified
crop and the set of non-genetically modified reference varieties.
In the case of significant difference and/or lack of equivalence for any particular endpoint, further statistical
analysis shall be carried out to assess whether there are interactions between any of the test materials and site,
possibly using a simple standard ANOVA approach. Whatever approach is adopted, details shall be given, for
each endpoint analysed, listing: (a) the assumptions underlying the analysis, and, when appropriate: (b)
degrees of freedom; (c) the estimated residual variation for each source of variation, and variance components;
(d) any other relevant statistics. These additional analyses are intended to aid the interpretation of any
significant differences found and to study potential interactions between test materials and other factors.
More detailed guidance for the application of the requirements of this Section is available in the EFSA
opinion on ‘Statistical considerations for the safety evaluation of GMOs’ ( 1 ).
1.3.3. Selection
of
material
and
compounds
for
analysis
Analysis of the composition of plant material is crucial when comparing the genetically modified food and feed
with its conventional counterpart. The material to be used for the comparative assessment shall be selected while
taking into account the uses of the genetically modified plant and the nature of the genetic modification. In the
case of herbicide tolerant genetically modified plants, three test materials shall be used: the genetically modified
plant exposed to the intended herbicide; the conventional counterpart treated with conventional herbicide
management regimes; and the genetically modified plant treated with the same conventional herbicide
management regimes. Unless duly justified, analysis shall be carried out on the raw agricultural commodity,
as this usually represents the main point of entry of the material into the food and feed production and
processing chain. Additional analysis of processed products (such as food and feed, food ingredients, feed
materials, food and feed additives or food flavourings) shall be conducted, where appropriate and on a case-
by-case basis (see also Section 1.3.6). The sampling, analysis and preparation of the tested material shall be
carried out according to appropriate quality standards.
1.3.4. Comparative
analysis
of
composition
In addition to the analysis on the level of the newly expressed proteins (see Section 1.2.2.3), the compositional
analysis shall be carried out on an appropriate range of compounds. In each case, the applicant shall provide at
least analysis on proximates (including moisture and total ash), key macro- and micro-nutrients, anti-nutritional
compounds, natural toxins, and already identified allergens, as well as other secondary plant metabolites char
acteristic for specific crop plant species, as referred to in the Organisation for Economic Cooperation and
Development (OECD) consensus documents on compositional considerations for new plant varieties (OECD
consensus documents) ( 2 ). The vitamins and minerals selected for analysis shall be those which are present at
levels which are nutritionally significant and/or which make nutritionally significant contributions to the diet at
the levels at which the plant is consumed. The specific analyses required shall depend on the plant species
examined, but shall include a detailed assessment appropriate to the intended effect of the genetic modification,
the considered nutritional value and use of the plant. The applicant shall pay particular attention to key nutrients
such as proteins, carbohydrates, lipids/fats, fibre, vitamins and minerals. For example, a fatty acid profile shall be
included for oil-rich plants (main individual saturated, mono-unsaturated and poly-unsaturated fatty acids) and an
amino acid profile (individual protein amino acids and main non-protein amino acids) for plants used as an
important protein source. Analysis of plant cell wall components are also required for the vegetative parts of
plants used for feed purposes.
The applicant shall also provide an analysis on key toxins inherently present in the recipient plant which may
adversely affect human/animal health depending on their toxic potency and levels. The concentrations of such
compounds shall be assessed according to plant species and the proposed use of the food and feed product.
Similarly, anti-nutritional compounds, such as digestive enzyme inhibitors, and already identified allergens shall
be studied.
The characteristics of the introduced trait may trigger further analysis of specific compounds including meta
bolites of potentially modified metabolic pathways. The applicant shall consider when appropriate, the inclusion
of compounds other than the key nutrients, key toxins, anti-nutrients and allergens identified by the OECD
consensus documents and justify the selection of these compounds.
( 1 ) EFSA Journal 2010; 8(1):1250.
( 2 ) http://www.oecd.org/document/15/0,3746,en_2649_34385_46726799_1_1_1_1,00.html

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1.3.5. Comparative
analysis
of
agronomic
and
phenotypic
characteristics
The applicant shall provide a comparison between the genetically modified plant and its conventional
counterpart. This comparison shall enable the applicant to identify unintended effects resulting from the
genetic modification and shall address also plant biology and agronomic traits, including common breeding
parameters (such as yield, plant morphology, flowering time, day degrees to maturity, duration of pollen viability,
response to plant pathogens and insect pests, sensitivity to abiotic stress). The protocols of these field trials shall
follow the specifications set out in Section 1.3.2.
Where transformation events are stacked by conventional crossing, there may also be changes to agronomic and
phenotypic characteristics. Possible differences in phenotypic characteristics and agronomic properties of stacked
transformation events shall be assessed in field trials. Where appropriate, the applicant shall provide additional
information on agronomic traits of the stacked transformation events from additional field trials.
1.3.6. Effects
of
processing
The applicant shall assess whether or not the processing and/or preserving technologies applied are likely to
modify the characteristics of genetically modified end products compared with their respective conventional
counterpart. The applicant shall provide a description of the different processing technologies in sufficient detail,
paying special attention to the steps which may lead to significant changes in the product content, quality or
purity.
Genetic modification can target metabolic pathways resulting in changes in the concentration of non-protein
substances or in new metabolites (such as in nutritionally enhanced foods). Processed products may be assessed
together with the assessment of the genetically modified plant for the safety of the genetic modification, or a
processed product may be assessed separately. The applicant shall provide the scientific rationale for the risk
assessment of these products. On a case-by-case basis, the submission of additional experimental data shall be
considered by the applicant.
When appropriate, depending on the product, information shall be necessary on the composition, level of
undesirable substances, nutritional value and metabolism, as well as on the intended use.
When appropriate, depending on the nature of the newly expressed protein(s), it shall be necessary to assess the
extent to which the processing steps lead to the concentration or to the elimination, denaturation and/or
degradation of these protein(s) in the final product.
1.3.7. Conclusion
The conclusion of the comparative analysis shall clearly state:
(a) whether agronomic and phenotypic characteristics of the genetically modified plant are, except for the
introduced trait(s), different to the characteristics of its conventional counterpart and/or equivalent to the
reference varieties, taking into account natural variation;
(b) whether compositional characteristics of the genetically modified food and feed are, taking into account
natural variation, different to the characteristics of its conventional counterpart and/or equivalent to the
reference varieties, except for the introduced trait(s);
(c) characteristics for which the genetically modified plant or the genetically modified food and feed are different
to the characteristics of its conventional counterpart and/or not equivalent to the reference varieties taking
into account natural variation, which need further investigation;
(d) whether, in the case of transformation events stacked by conventional crossing, there are indications of
interactions between the combined transformation events.
1.4. Toxicology
The toxicological impact of any changes on the whole genetically modified food/feed resulting from the genetic
modification such as the introduction of new genes, gene silencing or over-expression of an endogenous gene
shall be assessed.
Toxicological assessment shall be performed in order to:
(a) demonstrate that the intended effect(s) of the genetic modification has no adverse effects on human and
animal health;

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(b) demonstrate that unintended effect(s) of the genetic modification(s) identified or assumed to have occurred
based on the preceding comparative molecular, compositional or phenotypic analyses, have no adverse
effects on human and animal health;
(c) identify potential adverse effects of new constituents and determine their highest dose level that does not
result in adverse effects. From data obtained from an appropriate animal study, an acceptable daily intake
(ADI) of single compounds by humans may be derived by using uncertainty or safety factors that take into
account differences between test animal species and humans, and inter-individual variations among humans;
(d) identify potential adverse effects on the whole genetically modified food/feed or address remaining uncer
tainties through the performance of 90-day feeding studies.
The applicant shall consider the nature of toxicological testing to be conducted on new constituents and whole
genetically modified food/feed based on the outcome of the molecular and comparative analysis referred to in
Sections 1.2 and 1.3, namely the differences identified between the genetically modified product and its
conventional counterpart, including intended as well as unintended changes. The applicant shall also assess
the results of the performed toxicological tests to consider the need to conduct additional testing on new
constituents or on whole genetically modified food/feed as set out in Sections 1.4.4.2 and 1.4.4.3.
The applicant shall take into account the presence of: newly expressed proteins, the potential presence of other
new constituents and/or possible changes in the level of natural constituents beyond normal variation. The
specific information requirements and testing strategies are set out in Sections 1.4.1 to 1.4.4.
As regards applications of which the scope include or is restricted to genetically modified food and feed
produced from genetically modified plants, toxicological studies with the processed products shall be provided
except if the applicant provides a risk assessment of the genetically modified plant (or relevant parts of it)
demonstrating its safety and there are no indications that the processed genetically modified food and feed would
be any different from their respective conventional counterpart. The applicant shall provide adequate justification
in this regard.
Toxicology studies designed to evaluate risks to human and/or animal health shall complement each other. Most
studies required for the assessment of the safety of the genetically modified food are also valid for the assessment
of genetically modified feed.
Besides the exposure of consumers and animals through intake of food and feed, any adverse effect(s) on
individuals that could be due to their exposure to genetically modified food and feed material as part of their
professional activities, for example, farming, seed processing, shall be reported by the applicant. Appropriate
studies shall be performed to further characterise these indications of potential adverse effects.
The applicant shall use internationally agreed protocols and test methods for toxicity testing (see Tables 1 and 2
of Section 1.7). Adaptations of these protocols or use of any methods that differ from such protocols shall be
justified in the application.
1.4.1. Testing
of
newly
expressed
proteins
The applicant shall provide an evaluation of all newly expressed proteins. The studies required to investigate the
potential toxicity of a newly expressed protein shall be selected on a case-by-case basis, depending on the
knowledge available with respect to the protein’s source, function or activity and history of human or animal
consumption. As regards proteins expressed in the genetically modified plant, in the case where the history of
safe use for consumption as food and/or feed of both the plant and the newly expressed proteins is duly
documented, specific toxicity testing as provided for in this Section shall not be required. In such case, the
applicant shall provide the necessary information regarding the history of safe use of the proteins.
Where specific testing is required, the tested protein shall be equivalent to the newly expressed protein as it is
expressed in the genetically modified plant. If, due to the lack of sufficient amount of test materials from the
plant, a protein produced by micro-organisms is used, the structural, biochemical and functional equivalence of
this microbial substitute to the newly expressed plant protein shall be demonstrated. In particular, comparisons
of the molecular weight, amino acid sequence, post-translational modification, immunological reactivity and, in
the case of enzymes, the enzymatic activity are needed to provide evidence for the equivalence. In case of
differences between the plant expressed protein and its microbial substitute, the significance of these differences
for the safety studies shall be evaluated.

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To demonstrate the safety of newly expressed proteins, the applicant shall provide:
(a) A molecular and biochemical characterisation of the newly expressed protein, including determination of the
primary structure, molecular weight (for example using mass spectrometry), studies on post-translational
modifications and a description of its function. In the case of newly expressed enzymes, information on the
enzyme activities including the temperature and pH range for optimum activity, substrate specificity, and
possible reaction products shall also be provided. The potential interaction with other plant constituents shall
also be evaluated.
(b) An up-to-date search for homology to proteins known to cause adverse effects, such as toxic proteins. A
search for homology to proteins exerting a normal metabolic or structural function may also contribute
valuable information. The database(s) and the methodology used to carry out the search shall be specified.
(c) A description of the stability of the protein under relevant processing and storage conditions and the
expected treatment of the food and feed. The influences of temperature and pH changes shall be
examined and potential modification(s) of the proteins (such as denaturation) and/or production of stable
protein fragments generated through such treatments shall be characterised.
(d) Data concerning the resistance of the newly expressed protein to proteolytic enzymes (such as pepsin), such
as by in vitro investigations using appropriate and standardised tests. Stable breakdown products shall be
characterised and evaluated with regard to the potential to cause adverse health effects linked to their
biological activity.
(e) A repeated-dose 28-day oral toxicity study with the newly expressed protein in rodents. When appropriate
depending on the outcome of the 28-day toxicity study, further targeted investigations shall be provided,
including an analysis of immunotoxicity.
Acute toxicity testing of the newly expressed proteins of genetically modified plants is of little additional value
for the risk assessment of the repeated human and animal consumption of genetically modified food and feed
and shall not be provided as part of the studies performed under this point.
The applicant shall perform studies with combined administration of proteins when the genetic modification
results in the expression of two or more proteins in the genetically modified plant and when, based on scientific
knowledge, a possibility of synergistic or antagonistic interactions of safety concerns is identified.
1.4.2. Testing
of
new
constituents
other
than
proteins
The applicant shall provide a risk assessment of identified new constituents other than proteins. This shall
include, on a case-by-case basis, an evaluation of their toxic potency and of the need of toxicological testing
as well as a determination of their concentration in the genetically modified food and feed. To establish the safety
of new constituents having no history of safe use for consumption in food and feed, the applicant shall provide
information analogous to that described in the ‘Guidance for submissions for food additive evaluations by the
EFSA Panel on Food Additives and Nutrient Sources added to Food’ of 16 August 2012 ( 1 ) and Commission
Regulation (EC) No 429/2008 of 25 April 2008 on detailed rules for the implementation of Regulation (EC)
No 1831/2003 as regards the preparation and the presentation of applications and the assessment and the
authorisation of feed additives ( 2 ). This shall include the submission of information on a core set of studies such
as on metabolism/toxicokinetics, subchronic toxicity, genotoxicity, chronic toxicity, carcinogenicity and repro
duction and developmental toxicity, accompanied by any other appropriate type of study. For specific guidelines
for animal tests, see Table 1 of Section 1.7 of this Annex. Genotoxicity test protocols are provided for in Table 2
of Section 1.7 of this Annex.
1.4.3. Information
on
altered
levels
of
food
and
feed
constituents
This Section shall apply only in the case where the intended or unintended effect of the genetic modification
would result in an alteration of the levels of food and feed constituents beyond the natural variation.
( 1 ) http://www.efsa.europa.eu/en/efsajournal/pub/2760.htm
( 2 ) OJ L 133, 22.5.2008, p. 1.

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To demonstrate the safety of the altered levels of food and feed constituents such as macro- and micro-nutrients,
anti-nutrients, and natural toxins as well as other secondary plant metabolites, the applicant shall submit a
detailed risk assessment based on the knowledge of the physiological function and/or toxic properties of these
constituents.
The result of that risk assessment shall determine if, and to what extent, the applicant shall provide additional
toxicological tests to the 90-day feeding study in rodents with whole genetically modified food/feed on selected
food and feed constituents.
1.4.4. Testing
of
the
whole
genetically
modified
food
and
feed
The applicant shall primarily base its risk assessment of the genetically modified food and feed on molecular
characterisation, comparative agronomic, phenotypic and comprehensive compositional analysis, and the toxi
cological evaluation of the identified intended and unintended effects, including a 90-day feeding study in
rodents with whole genetically modified food/feed as specified in Section 1.4.4.1. Under the circumstances set
out in points 1.4.4.2 and 1.4.4.3 of this Section, additional specific toxicological studies with the whole
genetically modified food and feed shall be carried out.
1.4.4.1. 90-day feeding study in rodents with whole genetically modified food/feed
The applicant shall include a 90-day feeding study with whole food and feed in rodents for the assessment of
food and feed containing, consisting of or produced from genetically modified plants with a single trans
formation event or with stacked transformation events which are not obtained by conventional crossing of
genetically modified plants containing a single transformation event.
In the case of stacked transformation events obtained by conventional crossing of genetically modified plants
containing one or several transformation event(s), a 90-day feeding study with whole food and feed in rodents
shall be included for each of the genetically modified plant with a single transformation event which was used.
An additional 90-day feeding study with whole food and feed in rodents with the genetically modified plant with
the stacked transformation events shall be included where indications of potential adverse effects are identified
during the assessment of: (i) the stability of the inserts; (ii) the expression of the inserts; and (iii) the potential
synergistic or antagonistic effects resulting from the combination of the transformation events.
The design of the toxicity study with genetically modified food and feed should be performed according to the
‘subchronic oral toxicity test repeated-dose 90-day oral toxicity study in rodents’ (see Table 1) following an
adapted protocol. In principle a minimum of two test doses and a negative control shall be used. The highest
dose shall be the maximum achievable without causing nutritional imbalance; the lowest dose shall contain the
tested food and/or feed in an amount always above the anticipated human/target animal intake level. The
genetically modified food and feed analysed should be relevant to the product to be consumed. In the case
of herbicide tolerant genetically modified plants, the tested material should come from the genetically modified
plant exposed to the intended herbicide. Whenever possible, information on natural variation of test parameters
shall be derived from historical background data rather than from the inclusion of reference varieties, consisting
of commercially available food and feed derived from non-GM plants with a history of safe use, in the experi
ments. The statistical analysis shall focus on the detection of possible differences between the test material and its
control. A power analysis to estimate a sample size capable of detecting a pre-specified biologically relevant effect
size with a specified power and significance level should be used. More detailed guidance for performing this
study is provided in the EFSA Guidance on conducting repeated-dose 90-day oral toxicity study in rodents on
whole food/feed ( 1 ).
1.4.4.2. Animal studies with respect to reproductive and developmental toxicity testing
When information required in Sections 1.4.1, 1.4.2 and 1.4.3 on the genetically modified food and feed suggest
the potential for reproductive, developmental or chronic toxicity or in case of indications of adverse effects from
the 90-day feeding study in rodents (such as functional and/or histological modifications of nervous, endocrine,
reproductive or immunological tissues/organs), appropriate testing shall be performed. Protocols for reproductive,
developmental and chronic toxicity testing (see Table 1 of Section 1.7) may be adapted for the purposes of
testing the whole genetically modified food and feed.
Given that the 90-day feeding study in rodents is only designed to detect effects on adult reproductive organ
weights and histopathology and that it does not detect other effects on reproduction or development, testing of
the whole food and feed beyond a 90-day rodent feeding study shall be conducted where hazards in this respect
have been identified.
( 1 ) EFSA Journal 2011; 9(12):2438.

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1.4.4.3. Other animal studies to examine the safety and the characteristics of genetically modified food and feed (see also
Sections 1.6.1 and 1.6.2)
Feeding studies with target animal species shall be submitted when indications of adverse effects are drawn from
information required in Sections 1.4.1, 1.4.2 and 1.4.3 on the genetically modified food and feed or from the
outcome of the 90-day feeding study in rodents. Their focus shall be on the safety of new constituents (newly
expressed proteins and other new constituents), on the identification and characterisation of unintended effects,
and on the nutritional impact of any intentional, substantial, compositional modifications of the genetically
modified plant (see also Section 1.6).
Studies of that type shall be limited to plant materials suitable for inclusion in their diets and which can be
nutritionally matched to a suitable control diet.
1.4.4.4. Interpretation of relevance of animal studies
Relevant effects observed in the animal trials shall be evaluated in order to identify potential consequences for
the health of humans and animals and to assess their relevance for the safety of the genetically modified food
and feed. This evaluation may be supported by additional information and considerations. Attention should be
paid to the fact that certain effects may be specific for the test animal, but not for humans due to interspecies
differences.
The applicant shall, in particular, consider dose-response relationships in parameters that have been changed
(that is to say, commensurate increases in changes at increased doses) since they provide a strong indication for
an effect of the tested compound. When a difference is noted only at the highest dose applied, other factors shall
be considered to determine whether there is a relationship with treatment. Information on the background
variability in a given parameter may be obtained by the applicant from data from other animals of the same
species/strain tested in the same or other experiments, or from internationally harmonised databases.
In tests where animals of both genders are used, changes occurring in animals of one gender only may still be
relevant indicators of an effect, depending on the parameter being changed and the mechanism by which the
change may have been caused. For example, animals of one gender may be more or even specifically prone to
changes caused by a given constituent than animals of the other gender, such as in the case of endocrine effects.
The applicant shall also identify possible inter-relationships between observed changes in single parameters which
may strengthen the indication that an effect has occurred. For example, liver damage, which may be observed in
the liver itself as a change in histopathology, gross pathology, and organ weights, may also be evident from the
changed levels of certain liver-derived compounds, such as enzymes or bilirubin in serum.
With regard to the potential cause for an observed effect, the likelihood of causality shall be taken into account,
not only for the test compound, but also for other factors that may have also influenced the outcomes (such as
body weight decrease due to reduced intake of less palatable diet). Supportive data for a hypothesis of causality
between the test compound and effects in test animals may include, for example, predictive data for plausible
effects from in vitro and in silico experiments and dose-response relationships observed in the animal test.
1.4.5. Conclusion
of
the
toxicological
assessment
The conclusion of the toxicological assessment shall indicate whether:
(a) potential adverse effects identified in other parts of the safety assessment have been confirmed or discarded;
(b) the available information on the newly expressed protein(s) and other new constituents resulting from the
genetic modification gives indications of potential adverse effects in particular, whether and at which dose
levels adverse effects were identified in specific studies;
(c) the information on natural constituents of which the levels are different from those in its conventional
counterpart provides indications of potential adverse effects, in particular, whether and at which dose levels
adverse effects were identified in specific studies;
(d) adverse effects have been identified from the studies made on the whole genetically modified food and feed
and at which dose levels.
The applicant shall evaluate the result of the toxicological assessment in the light of anticipated intake of the
genetically modified food and feed (see Section 2).

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1.5. Allergenicity
Food allergy is an adverse reaction to food and represents an important public health problem. Food allergy is
different from toxic reactions and intolerance. Allergy is a pathological deviation of the immune response to a
particular substance, which affects only some individuals where a combined effect of variations in the
environment and genetic predisposition has resulted in allergic sensitisation.
In allergic individuals, sometimes minute amounts of a food that is well tolerated by the vast majority of the
population can cause serious symptoms and death. It is not the allergen per se, but the allergic person’s
abnormal reaction to the allergen that causes the adverse health effect.
Food allergy can be caused by various immune mechanisms. However, IgE-mediated food allergy represents the
main form of food allergy, that causes the most severe reactions and the only form causing life-threatening
reactions. This IgE-mediated food allergy has been the focus in the risk assessment of allergenicity of GMOs.
Importantly, food allergy consists of two separate phases: first sensitisation where no symptoms occur while the
capacity of the immune system to react increases dramatically, and later elicitation (provocation) with clinical
manifestations.
When ingested, the allergen(s), that is to say, the sensitising food or food constituent is to some extent degraded
by digestive enzymes, absorbed by the gut mucosa (small amounts even by the oral mucosa), processed in
specialised cells of the immune system and then presented to the reactive immune cells that produce an immune
response. Sensitisation can also occur if the food allergen comes into contact with the skin or is inhaled.
The majority of the constituents that are responsible for allergenicity of foods, as well as of pollens, are proteins.
Some protein breakdown products, that is to say, peptide fragments, may conserve part of the allergenicity of the
native protein and thus can also be considered as allergens.
The specific allergy risk of GMOs is associated with: (i) exposure to newly expressed protein(s) that can be
present in edible parts of the plants or in the pollen; this point is related to the biological source of the
transgene; and (ii) alterations to the allergenicity of the whole plant and its products, for example, due to
over-expression of natural endogenous allergens as an unintended effect of the genetic modification; this
point is related to the biology of the recipient plant itself.
More detailed guidance for the application of the requirements of this Section is available in the EFSA scientific
opinion on the assessment of allergenicity of GM plants and micro-organisms and derived food and feed adopted
on 30 June 2010 ( 1 ).
1.5.1. Assessment
of
allergenicity
of
the
newly
expressed
protein
Allergenicity is not an intrinsic, fully predictable property of a given protein but is a biological activity requiring
an interaction with individuals with a pre-disposed genetic background. Allergenicity therefore depends upon the
genetic diversity and variability in atopic humans. Frequency, severity and specificity of allergic reactions also
depend upon geographic and environmental factors. Given this lack of complete predictability, it is necessary to
consider several aspects in the assessment of allergenicity to obtain a cumulative body of evidence which
minimises any uncertainty with regard to the protein(s) in question.
When studying the structural characteristics and the biological and physicochemical properties of a newly
expressed protein, it is essential that the tested protein is equivalent with respect to structure and activity to
the newly expressed protein in the genetically modified plant. Studies carried out using purified target proteins
prepared by expression in organisms such as Escherichia coli shall be acceptable provided that the properties of the
microbial substitute protein are identical to those of the protein expressed in the plant, thus taking into account
all post-translational modifications that specifically occur in the plant.
The applicant shall verify whether the source of the transgene is allergenic. When the introduced genetic material
is obtained from wheat, rye, barley, oats or related cereal grains, the applicant shall also assess the newly
expressed proteins for a possible role in the elicitation of gluten-sensitive enteropathy or other enteropathies
which are not IgE-mediated. Where transformation events have been stacked, the applicant shall provide an
assessment of any potential for increased allergenicity to humans and animals on a case-by-case approach. These
potential effects may arise from additive, synergistic or antagonistic effects of the gene products.
( 1 ) EFSA Journal 2010; 8(7):1700.

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The applicant shall follow an integrated, case-by-case approach, that is to say, a weight-of-evidence approach, in
the assessment of possible allergenicity of newly expressed proteins. This approach shall include:
(a) Amino acid sequence homology comparison between the newly expressed protein and known allergens
In every case, a search for sequence homologies and/or structural similarities between the expressed protein
and known allergens shall be performed to identify potential IgE cross-reactivity between the newly expressed
protein and known allergens. The applicant shall ensure that the quality and the comprehensiveness of the
databases are state of the art. The alignment-based criterion involving 35 % sequence identity to a known
allergen over a window of at least 80 amino acids is considered a minimal requirement. All sequence
alignment parameters used in the analysis shall be provided including calculation of percent identity
(PID). The calculation of PID shall be performed on a window of 80 amino acids with gaps so that
inserted gaps are treated as mismatches. In some cases, for assessing short peptidic fragments such as
ORFs, a search for sequences of contiguous identical or chemically similar amino acid residue can be
conducted. However, this search shall not be carried out routinely for the identification of potential linear
IgE binding epitopes because of its poor sensitivity or specificity.
(b) Specific serum screening
When there is an indication of sequence homology or structure similarities, an important procedure for
assessing the potential that exposure to the newly expressed proteins might elicit an allergic reaction in
individuals already sensitised to cross-reactive proteins, is based on in vitro tests that measure the capacity of
specific IgE from serum of allergic patients to bind the test protein(s). There is inter-individual variability in
the specificity and affinity of the human IgE response. In particular, the specificity of the IgE antibodies to the
different allergens present in a given food/source and/or to the different epitopes present on a given protein
may vary amongst allergic individuals. In order to optimise the sensitivity of the test, individual sera from
well-characterised allergic individuals shall be used. The applicant shall perform specific serum screening in
the following cases:
(i) the source of the introduced gene is considered allergenic, even if no sequence homology of the newly
expressed protein to a known allergen is demonstrated; or
(ii) the source is not known to be allergenic, but there are indications of a relationship between the newly
expressed protein and a known allergen, based on sequence homology or structure similarity.
Specific serum screening shall be undertaken with individual sera from individuals with a proven and well-
characterised allergy to the source or to the potentially cross-reacting allergen using relevant immuno
chemical tests. IgE-binding assays (such as Radio or Enzyme Allergosorbent Assay (RAST or EAST),
Enzyme Linked Immunosorbent Assay (ELISA) and electrophoresis followed by immunoblotting with
specific IgE-containing sera) are adequate methods.
(c) Pepsin resistance and in vitro digestibility tests
Stability to digestion by proteolytic enzymes has long been considered a characteristic of allergenic proteins.
Although it has been established that no absolute correlation exists, resistance of proteins to pepsin digestion
is an additional criterion to be considered in the weight-of-evidence approach for the assessment of aller
genicity. The pepsin resistance test is generally performed under quite standardised conditions, at low pH
values and high pepsin:protein ratios. It is recognised that the pepsin resistance test does not reflect the
physiological conditions of the digestion. The digestibility of the newly expressed proteins in specific
segments of the population, such as infants and individuals with impaired digestive functions, may be
assessed using in vitro digestibility tests using different conditions. Also, since the protein encoded by the
newly introduced genes will be present in the product as a complex matrix, the impact of the possible
interaction between the protein and other components of the matrix, as well as the effects of the processing,
shall be taken into account in additional in vitro digestibility tests. Depending on the outcome of the in vitro
digestibility test, a comparison of the intact, the heat-denatured and the pepsin-digested proteins for IgE
binding shall be assessed since an altered digestibility may impact on the allergenicity of the newly expressed
protein.
(d) Additional tests
Although additional tests including in vitro cell based assays or in vivo tests on animal models have not been
validated to date for regulatory purposes, they may provide useful additional information, for example, on
the potential of the newly expressed protein for de novo sensitisation.

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1.5.2. Assessment
of
allergenicity
of
the
genetically
modified
food
or
feed
When the recipient plant is known to be allergenic, the applicant shall assess any potential change in the
allergenicity of the genetically modified food or feed by comparison of the allergen repertoire with that of its
conventional counterpart. The potential over-expression of natural endogenous allergen(s) in the genetically
modified plant shall, in particular, be investigated.
The applicant shall follow a case-by-case approach depending on the available information on the allergenic
potential of the recipient plant. It is generally performed by analytical methodologies such as proteomics in
association with the use of allergic human sera as probes. Sera from clinically well-characterised allergic indi
viduals that are the reference material for IgE binding studies may be available in limited number and quantity. In
order to minimise the use of human sera, preliminary important information on the likelihood of an unintended
alteration of the overall allergenicity of the genetically modified plant can be obtained by using sera of animals
experimentally sensitised in well-defined conditions and by including relevant identified endogenous allergens in
the comparative compositional analysis.
In addition, the applicant shall provide, where available, information on the prevalence of allergy in persons
working with, coming into contact with or in the vicinity of genetically modified plant cultivation.
1.5.3. Adjuvanticity
Adjuvants are substances that, when co-administered with an antigen, increase the immune response to the
antigen and therefore might increase as well the allergic response. In cases when known functional aspects of the
newly expressed protein or structural similarity to known strong adjuvants may indicate possible adjuvant
activity, the applicant shall assess the possible role of these proteins as adjuvants. As for allergens, interactions
with other constituents of the food matrix and/or processing may alter the structure and bioavailability of an
adjuvant and thus modify its biological activity.
1.5.4. Conclusion
of
the
allergenicity
assessment
The conclusion of the allergenicity assessment shall indicate:
(a) whether the novel protein(s) is likely to be allergenic;
(b) whether the genetically modified food or feed is likely to be more allergenic than its conventional
counterpart.
When there is a likelihood of increased allergenicity due to the genetic modification, the genetically modified
food or feed shall be further characterised in the light of its anticipated intake (see Section 2). The applicant shall
propose appropriate conditions for placing on the market (such as post-market monitoring and labelling).
1.6. Nutritional
assessment
1.6.1. Objectives
of
the
nutritional
assessment
The applicant shall provide a nutritional evaluation to demonstrate that:
(a) the introduction of the genetically modified food and feed into the market is not nutritionally disadvan
tageous to humans and animals, respectively. This evaluation shall include the relevance for the nutrition of
newly expressed proteins, other new constituents, and changes in the levels of food and feed constituents, as
well as potential alterations in the total diet of the consumer or the animal;
(b) unintended effects of the genetic modification that were identified or that may be assumed to have occurred
based on the preceding molecular, compositional or phenotypic analyses, in accordance with Sections 1.2
and 1.3, have not adversely affected the nutritional value of the genetically modified food and feed.
For stacked transformation events combined by conventional crossing, an assessment of the potential changes in
nutritional value that might arise from synergistic or antagonistic effects of the gene products including
compositional changes shall be provided by the applicant. This may be particularly relevant where the
combined expression of the newly introduced genes has unexpected effects on biochemical pathways.

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1.6.2. Points to consider for the nutritional assessment of genetically modified food and feed
The nutritional assessment of genetically modified food and feed shall consider:
(a) the composition of the genetically modified food and feed with regard to the levels of nutrients and anti-
nutrients (see compositional studies as described in Section 1.3);
(b) the bioavailability and biological efficacy of nutrients in the food and feed taking into account the potential
influences of transport, storage and expected treatment of the food and feed;
(c) the anticipated dietary intake of the food and feed (see Section 2) and resulting nutritional impact.
When the comparative analysis has identified compositional characteristics of the genetically modified food and
feed that are different from its conventional counterpart and/or not equivalent to the characteristics of the
reference varieties, their nutritional relevance shall be assessed on the basis of current scientific knowledge. If
that assessment does conclude to the nutritional equivalence between the genetically modified food and feed and
its conventional counterpart, no further studies shall be carried out. By contrast if, on the basis of the assessment
of the information obtained from the comparative analysis, it is not possible to conclude on the nutritional
equivalence, further nutritional studies shall be carried out. Comparative growth studies shall be conducted with
young rapidly growing animal species (such as broiler chicks as animal model for non-ruminants; lambs for
ruminants; or other rapidly growing species).
1.6.3. Nutritional
studies
of
genetically
modified
food
The applicant shall determine the necessity and design of nutritional studies on the basis of the introduced
trait(s), the outcome of the comparative analysis, and of the 90-day feeding study, where available. Supple
mentary information regarding the nutritional value may be obtained from comparative growth performance
studies conducted with other animal species, such as broiler chickens, addressing the nutritional assessment of
genetically modified feed. When nutritional studies are conducted, the control diet(s) shall include the conven
tional counterpart and where appropriate additional comparator(s). In the case of herbicide tolerant genetically
modified plants, the tested material should come from the genetically modified plant exposed to the intended
herbicide.
Genetically modified foods modified to provide additional health benefits to the consumer as compared to
conventional foods, may benefit specific populations or subpopulations while others may be at risk from the
same food. In cases where an altered bioavailability needs to be established and may raise concern for subpopu
lation(s), the level of the nutrient in the food shall be determined, taking into account all the different forms of
the compound. The methods to test for bioavailability shall be selected on a case-by-case basis depending on the
nutrient or other constituent, the food containing these constituents, as well as the health, nutritional status and
dietary practices of the specific population(s) anticipated to consume the food.
1.6.4. Nutritional
studies
of
genetically
modified
feed
The applicant shall determine the necessity and design of further nutritional studies on the basis of the
introduced trait(s), the outcome of the comparative analysis, and the 90-day feeding study, where available.
Supplementary information regarding the nutritional value may be obtained from comparative growth
performance studies conducted with other animal species, such as broiler chickens, addressing the nutritional
assessment of genetically modified feed. When nutritional studies are conducted, the control diet(s) shall include
the conventional counterpart and where appropriate additional comparator(s).
In the case of genetically modified feed with improved nutritional characteristics, feeding studies with target
animal of food producing species shall be conducted to assess the impact on the feed. In the case of genetically
modified plants modified for improved content and bioavailability of nutrients, studies with target food
producing animal species shall be conducted to determine the bioavailability of individual nutrients in the
genetically modified plant compared to its conventional counterpart. In the case of genetically modified
plants specifically modified with traits to enhance animal performance through increased nutrient density
(such as increased oil content) or an enhanced level of a specific nutrient (such as an essential amino acid or
a vitamin), an appropriate control diet using its conventional counterpart shall be formulated by supplementing
it with the specific nutrient to the extent of the change effected in the genetically modified plant. Regarding co-
products (such as oilseeds meals), from which the ingredient targeted by the genetic modification has been
extracted, these may be compared with co-products produced from the conventional counterpart.

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Target animal feeding studies shall span either the growing and/or finishing period to slaughter for chickens,
pigs, and cattle for fattening or a major part of a lactation cycle for dairy cows, or laying cycle for laying hens or
quails. For feedstuffs intended only for aquaculture, growth studies with aquatic species such as carp, catfish,
salmonidae or typical herbivores shall be chosen.
When appropriate, tests with various experimental designs shall be provided to demonstrate that the nutritionally
improved genetically modified plant fulfils the expected nutritional value. The exact experimental design and
statistical approaches of feeding experiments in food producing animals to test the nutritional value of genetically
modified feed, modified for enhanced nutritional characteristics, shall depend on the targeted animal species, type
of plant trait(s) studied and the size of the expected effect. The experimental diets shall be formulated in such a
way that the key measured endpoints are responsive to a difference in the quantity and/or availability of the
nutrient in question. Endpoint measurements shall vary with the target species used in the study, but shall
include feed intake, body weight, animal performance and bioavailability of nutrients.
More detailed guidance for the application of the requirements of this Section is available in the Report of the
EFSA GMO Panel Working Group on Animal Feeding Trials ( 1 ).
1.6.5. Conclusion
of
the
nutritional
assessment
The conclusion of the nutritional assessment of genetically modified food and feed shall indicate whether the
genetically modified food and feed is nutritionally equivalent to its conventional counterpart, taking natural
variations into account.
The applicant shall evaluate the result of the nutritional assessment in the light of anticipated intake of the
genetically modified food and feed (see Section 2).
1.7. Standardised
guidelines
for
toxicity
tests
The applicant shall use for toxicity testing internationally agreed guidelines and test methods described by
Commission Regulation (EC) No 440/2008 of 30 May 2008 laying down test methods pursuant to Regulation
(EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Auth
orisation and Restriction of Chemicals (REACH) ( 2 ) (see Tables 1 and 2). A non-exhaustive list of validated
test methods which, where necessary, shall be used in a possibly adapted form for GMO toxicological testing
is provided in Tables 1 and 2.
The performance of test methods depends on the type of genetically modified food and feed, type of the genetic
modification and resulting intended and unintended alterations, intended use and exposure/intake, and the
available knowledge. Some of the tests were developed for the assessment of risks at the workplace (see
Sections 1.4 and 1.5).
Table 1
Non-exhaustive list of validated test methods for chemicals as provided by Regulation (EC) No 440/2008
and which may be used in a possibly adapted form for GMO toxicological testing
Reference of the method in Part B of
Title
the Annex to Regulation (EC)
No 440/2008
ACUTE TOXICITY (DERMAL) B.3.
SKIN SENSITISATION B.6.
REPEATED-DOSE (28 DAYS) TOXICITY (ORAL) B.7.
REPEATED-DOSE (28 DAYS) TOXICITY (DERMAL) B.9.
SUBCHRONIC ORAL TOXICITY TEST REPEATED-DOSE 90-DAY ORAL
B.26.
TOXICITY STUDY IN RODENTS
CHRONIC TOXICITY TEST B.30.
( 1 ) EFSA, 2008 Report of the EFSA GMO Panel Working Group on Animal Feeding Trials, 2008. Safety and nutritional assessment of GM
plants and derived food and feed. The role of animal feeding trials. Food and Chemical Toxicology 46 (2008) S2-S70.
( 2 ) OJ L 142, 31.5.2008, p. 1.

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Reference of the method in Part B of
Title
the Annex to Regulation (EC)
No 440/2008
CARCINOGENICITY TEST B32.
COMBINED CHRONIC TOXICITY/CARCINOGENICITY TEST B.33.
ONE-GENERATION REPRODUCTION TOXICITY TEST B.34.
TWO-GENERATION REPRODUCTION TOXICITY STUDY B.35.
TOXICOKINETICS B.36.
NEUROTOXICITY STUDY IN RODENTS B.43.
Table 2
Genotoxicity tests as provided by Regulation (EC) No 440/2008
Reference of the method in Part B of
Title
the Annex to Regulation (EC)
No 440/2008
MUTAGENICITY — IN VIVO MAMMALIAN BONE MARROW CHRO
B.11.
MOSOME ABERRATION TEST
MUTAGENICITY — IN VIVO MAMMALIAN ERYTHROCYTE MICRO
B.12.
NUCLEUS TEST
MUTAGENICITY: REVERSE MUTATION TEST USING BACTERIA B.13/14.
MUTAGENICITY TESTING AND SCREENING FOR CARCINOGENICITY
B.15.
GENE MUTATION — SACCHAROMYCES CEREVISIAE
MITOTIC RECOMBINATION — SACCHAROMYCES CEREVISIAE B.16.
DNA DAMAGE AND REPAIR — UNSCHEDULED DNA SYNTHESIS —
B.18.
MAMMALIAN CELLS IN VITRO
MUTAGENICITY — IN VITRO MAMMALIAN CELL GENE MUTATION
B.17.
TEST
SISTER CHROMATID EXCHANGE ASSAY IN VITRO B.19.
IN VITRO MAMMALIAN CELL TRANSFORMATION TESTS B.21.
MAMMALIAN SPERMATOGONIAL CHROMOSOME ABERRATION TEST B.23.
2. EXPOSURE
ASSESSMENT
—
ANTICIPATED
INTAKE/EXTENT
OF
USE
An estimate of the expected intake shall be an essential element in the risk assessment of genetically modified
food and feed and shall also be required for the nutritional evaluation. Information shall be provided by the
applicant on the intended function, the dietary role, and the expected level of use of the genetically modified
food and feed in the EU. In addition, the expected range of concentrations of newly produced proteins or
existing plant proteins deliberately modified in the genetically modified food(s) and feed(s) to be placed on the
market shall be provided.
On the basis of representative consumption data for products obtained from the respective conventional plants,
the applicant shall estimate the anticipated average and maximum intake of the genetically modified food and
feed. Probabilistic methods may be used to determine ranges of plausible values rather than single values or point
estimates. The applicant shall identify and consider particular groups of the EU population with an expected
higher exposure and shall consider this higher exposure within the risk assessment. Any assumptions made in
the exposure assessment shall be described. Recent developments in methodologies and appropriate consumption
data shall be used. Data on import and production quantities may provide additional information for the intake
assessment.

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The applicant shall determine by appropriate methods the concentrations of the newly expressed proteins, other
new constituents and endogenous food and feed constituents, of which the levels have been altered as a result of
the genetic modification (for example, due to changes in metabolic pathways) in those parts of the genetically
modified plant intended for food or feed use. Expected intake of these constituents shall be estimated taking into
account the influences of processing, storage and expected treatment of the food and feed in question, for
example, potential accumulation or reduction. In cases where the genetic modification has resulted in an altered
level of a natural constituent, or if a new constituent occurs naturally in other food and feed products, the
anticipated change in total intake of this constituent shall be assessed considering realistic as well as worst case
intake scenarios.
The applicant shall provide information on known or anticipated human/animal intake of analogous genetically
modified food and feed and on other routes of exposure to the respective new and natural constituents,
including amount, frequency and other factors influencing exposure.
3. RISK
CHARACTERISATION
3.1. Introduction
The applicant shall base its risk characterisation of genetically modified plants and foods and feed on data from
hazard identification, hazard characterisation, and on exposure/intake data. The applicant shall ensure that the
risk characterisation is comprehensive by considering all the available evidence from several analysis including
molecular analysis, phenotypic, agronomical and compositional analysis, toxicity and allergenicity testing. The
applicant shall consider indications resulting from the risk characterisation that may require specific activities for
post-market monitoring of genetically modified food and feed.
In performing its risk characterisation, the applicant shall demonstrate that the hazard identification and hazard
characterisation are complete. The applicant shall discuss the quality of existing data and information. The
discussion shall clearly indicate how this body of information has been taken into account in the determination
of the final risk characterisation.
The applicant shall provide estimations of the uncertainties associated to each test as well as to the different
stages of the risk assessment. The applicant shall quantify them to the possible extent. A distinction shall be
made between uncertainties that reflect natural variations in biological parameters (including variations in
susceptibility in populations), and variation amongst different species’ responses.
Depending on the issue to be addressed and the available data, the applicant shall perform a qualitative and,
where possible, quantitative risk characterisation. The conditions for the estimated risk, and associated uncer
tainties, shall be as precise as possible.
3.2. Issues
to
be
considered
for
risk
characterisation
When appropriate and depending on the type of genetic modification, the applicant shall carry out a risk
assessment of genetically modified plants in an integrative manner in accordance with Section 3.1. This risk
assessment shall be performed on a case-by-case basis depending on the modified plant and the type of genetic
modification, the cultivation practices of the genetically modified plant and uses of the genetically modified food
and feed. The applicant shall take into account the different issues considered in hazard identification and hazard
characterisation and exposure steps. The outcomes of these issues shall be considered by the applicant together
in the risk characterisation step. The list of issues provided in this Section shall not be exhaustive.
3.2.1. Molecular
characterisation
Evaluation of the characteristics and previous use of the donor and the recipient plant shall be a key element to
identify the need for specific analyses, such as the occurrence of specific toxins, or allergens in the unmodified
recipient plant which may be unintentionally increased as result of the genetic modification.
Transformation protocols, molecular characterisation strategies and the specificity and sensitivity of the methods
used shall be discussed by the applicant in relation to the intentional and possibly unintentional insertion and
expression of gene sequences.
Where sequence analysis has identified a potential hazard, the applicant shall demonstrate how approaches like
bioinformatic analysis, compositional/agronomical analysis and possibly animal feeding trials with the whole
genetically modified food and feed contribute to the safety assessment. The value of the results obtained shall be
evaluated in the light of the available knowledge on the structure and function of genomic databases of the crop
species in question or related species.

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In cases of GM plants containing stacked transformation events, the additional risks possibly arising from the
combined effects of the stacked genes shall be evaluated.
3.2.2. Comparative
analysis
The first goal of the comparative analysis is to identify possible differences between the GM plant and its
conventional counterpart and, where appropriate, additional comparators. The second goal of the comparative
analysis is to identify possible lack of equivalences between the GM plant and its reference varieties. These
differences and/or lack of equivalences should be assessed with respect to their possible impact on food and feed
safety and nutritional properties, taking into account natural variation. The estimated risk and associated uncer
tainties should be as precise as possible and taken into account.
The applicant shall demonstrate that the comparative analysis between the genetically modified plant and its
conventional counterpart with respect to agronomic, morphological and compositional characteristics has been
carried out in accordance with the requirements of this Regulation. The selection of the conventional counterpart
and, where appropriate, additional comparators shall be justified.
3.2.3. Food
and
feed
safety
in
relation
to
intake
The applicant shall evaluate the data generated to estimate possible short-term and long-term risks to human or
animal health associated with the consumption of genetically modified food or feed with respect to the
expression of new proteins/metabolites, as well as significantly altered levels of original plant proteins/metabolites
in genetically modified foods/feed. This evaluation shall include a thorough analysis of the relevance and
limitations of each test as well as of the whole information.
The applicant shall consider the range of levels observed for the compounds known to occur in the conventional
counterpart and in reference varieties. This variability may be caused by differences that are genotype-dependent,
environmentally dependent, or caused by genotype x environment interactions. In addition, the range of levels
observed in a broad spectrum of food and feed representative for the human and animal diet may be taken into
account given that it reflects the levels of the specific compound to which consumers may be exposed.
If single constituents and/or whole genetically modified food and feed are found to induce adverse effects in
specific studies, information on dose response relationships, threshold levels, delayed onset of adverse effects,
risks for certain groups in the population, and use of uncertainty factors in extrapolation of animal data to
humans shall be submitted.
The applicant shall consider data on the characteristics of the new compounds present in the genetically
modified plant including potential biological effects in humans and animals. If the compounds have known
adverse health effects and maximum levels for the presence of these compounds in the plant or its products were
laid down in specific legislation, these maximum levels shall be taken into account. Otherwise, reference values
for acceptable or tolerable levels of intake, such as the acceptable daily intake (ADI) or tolerable upper intake
level (UL), shall be considered in relation to the anticipated intake. In cases where the compound has been safely
consumed in food, the intake levels of consumers from a conventional diet shall be considered as safe.
The applicant shall evaluate the information on the effects of processing on the new compounds. Potential
accumulation/depletion in food and feed products entering the human or animal diet shall be considered. The
applicant shall also evaluate the relevance of differences resulting from chemical reactions known to occur under
processing conditions.
In cases where more complex genetic modifications are produced, for example, via transfer of multiple genes in a
single construct, retransformation of pre-existing genetically modified lines, and stacking of transformation
events through conventional breeding of genetically modified parents, the applicant shall discuss strategies for
the assessment of any risk(s) associated with possible interactions between the newly expressed proteins, new
metabolites and original plant constituents. The assessment shall take into account all available information
including the mode of action of the newly expressed proteins, the molecular and compositional/agronomical
characteristics of the genetically modified plant, and the outcome of animal toxicity studies and feeding trials.
The applicant shall evaluate data obtained to assess the allergenic potential of newly expressed proteins in
genetically modified plants with respect to introduction of new allergenic proteins into the food and feed
plants, a possible provocation of allergic reactions of susceptible individuals, as well as information to demon
strate that the genetic modification does not cause unintended changes in the characteristics and/or levels of
expression of endogenous allergenic proteins in the genetically modified food. In particular, the choice of the test
models shall be justified with respect to specificity, predictability and validation status.

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With respect to intake estimations of genetically modified foods, the applicant shall evaluate the applied
methodologies with respect to uncertainties associated with the prediction of long-term intake. Specific
attention shall be paid to those genetically modified plants which are aimed at modifying nutritional char
acteristics of the food and feed. For those genetically modified products, the requirement for post-market
monitoring shall be discussed as a mechanism for determining actual changes to overall dietary intake
patterns of the genetically modified food, to what extent this has occurred and whether or not the product
induces known (side) effects or unexpected side effects. If the performance of post-market monitoring is deemed
necessary, the reliability, sensitivity and specificity of the proposed methods shall be provided.
3.3. The
result
of
risk
characterisation
In accordance with the requirements of Articles 4 and 16 of Regulation (EC) No 1829/2003, the applicant shall
ensure that the final risk characterisation clearly demonstrates that:
(a) the genetically modified food and feed has no adverse effects on human and animal health;
(b) the genetically modified food does not differ from the food which it is intended to replace to such an extent
that its normal consumption would be nutritionally disadvantageous for the consumer;
(c) the genetically modified food does not mislead the consumer;
(d) the genetically modified feed does not harm or mislead the consumer by impairing the distinctive features of
the animal products;
(e) the genetically modified feed does not differ from the feed which it is intended to replace to such an extent
that its normal consumption would be nutritionally disadvantageous for animals or humans.
The applicant shall clearly indicate what assumptions have been made during the risk assessment in order to
predict the probability of occurrence and severity of adverse effect(s) in a given population, and the nature and
magnitude of uncertainties associated with establishing these risks.
The applicant shall also include detailed information justifying the inclusion or not of a proposal for labelling in
the application, in accordance with Article 13(2)(a), Article 13(3), Article 25(2)(c) and Article 25(3) of Regulation
(EC) No 1829/2003.

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ANNEX III
VALIDATION OF METHODS FOR THE DETECTION, IDENTIFICATION AND QUANTIFICATION OF THE
TRANSFORMATION EVENT, AND REQUIREMENTS FOR CONTROL SAMPLES AND THE CERTIFIED
REFERENCE MATERIAL
1. INTRODUCTION
1. For the purposes of implementing Article 5(3)(i) and (j) and Article 17(3)(i) and (j) of Regulation (EC)
No 1829/2003, this Annex sets out requirements on:
(a) the performance characteristics of the submitted method(s);
(b) technical requirements regarding the type of information that the applicant must submit so as to verify that
those requirements are met;
(c) samples of the food and feed and their control samples;
(d) certified reference material.
2. The applicant must include information on the method as such and on the method testing carried out by the
applicant.
3. The applicant shall also consider further guidance and information about the operational procedures of the
validation process that is made available by the EU Reference Laboratory (EURL) as referred to in Article 32
of Regulation (EC) No 1829/2003, assisted by the European Network of GMO Laboratories ( 1 ).
2. DEFINITIONS
For the purpose of this Annex, the following definitions shall apply:
(a) ‘certified reference material’ means reference material as referred to in Articles 5(3)(j) and 17(3)(j) of Regulation
(EC) No 1829/2003 and corresponds to any material or substance, one or more of whose property values are
certified for calibration or quality control of methods. It is accompanied by a certificate that provides value of the
specified property, its associated uncertainty and a statement of metrological traceability;
(b) ‘method performance requirements’ means the minimum performance criteria that the method shall demonstrate
upon completion of the validation study carried out by the EURL, according to internationally accepted technical
provisions.
3. METHOD
VALIDATION
3.1. Information about the method
A. The method(s) shall refer to all the methodological steps needed to analyse the relevant food and feed material in
accordance with Articles 5(3)(i) and 17(3)(i) of Regulation (EC) No 1829/2003.
For a particular food or feed material, the methodological steps shall include the methods for DNA extraction
and the subsequent quantification in a real-time Polymerase Chain Reaction (PCR) System. In such a case, the
whole process from extraction up to the PCR-technique shall constitute a method. The applicant shall provide
information about the whole method.
B. The applicant shall be allowed to refer to validated protocols, if available and appropriate, for method modules
used in the analytical procedure such as a DNA extraction protocol from a certain matrix.
In that case, the applicant shall provide experimental data from an in-house validation in which the method
module has been successfully applied in the context of the application for authorisation.
( 1 ) http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm

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C. The applicant shall demonstrate that the method(s) fulfils the following requirements:
1. The method(s) shall be specific to the transformation event (hereafter referred to as ‘event-specific’) and thus
shall only be functional with the genetically modified organism or genetically modified based product
considered and shall not be functional if applied to other transformation events already authorised;
otherwise the method cannot be applied for unequivocal detection/identification/quantification. This shall
be demonstrated with a selection of non-target transgenic authorised transformation events and conventional
counterparts. This testing shall include closely related transformation events.
2. The method(s) shall be applicable to samples of the food or feed, to the control samples and to the certified
reference material.
3. The applicant shall take into consideration the following documents for the development of the detection
method:
(a) Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — General requirements and definitions: ISO 24276;
(b) Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Nucleic acid extraction: ISO 21571;
(c) Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Quantitative nucleic acid based methods: ISO 21570;
(d) Foodstuffs — Methods of analysis for the detection of genetically modified organisms and derived
products — Qualitative nucleic acid based methods: draft European standard ISO 21569.
4. The method shall also take into consideration the more detailed requirements set out in the common criteria
set by the EURL, and the ENGL for minimum performance requirements for analytical methods for GMO
testing. These criteria are part of the guidance provided by the EURL.
D. For the purpose of implementing Articles 5(3)(i) and 17(3)(i) of Regulation (EC) No 1829/2003, the applicant
shall provide the event-specific quantitative detection method(s) of the genetically modified material. The
applicant shall discuss the validity and limitations of the detection methods in the various types of foods and
feeds (the various matrixes) that are expected to be placed on the market.
E. The applicant shall provide a complete and detailed description of the method.
The following points shall be clearly addressed by the applicant:
1. Scientific basis: the applicant shall provide an overview of the principles of how the method works. This
overview shall include references to relevant scientific publications.
2. Scope of the method: the applicant shall indicate the matrix(es) (for example, processed food, raw materials),
the type of samples and the percentage range to which the method may be applied.
3. Operational characteristics of the method: the required equipment for the application of the method shall be
specified, with regard to the analysis as such and the sample preparation. Further information of any specific
aspects crucial for the application of the method shall also be included.
4. Protocol: the applicant shall provide a complete optimised protocol of the method. The protocol shall present
all the details as required to transfer and apply the method independently in other laboratories.
5. A prediction model (or a similar tool) needed to interpret results and to make inferences shall be described in
full details. Instructions for the correct application of the model shall be provided by the applicant.
6. Breeding schemes that are to be applied for the production of genetically modified food and feed and their
impact on the interpretation of results shall be provided by the applicant.

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3.2. Information about the method testing carried out by the applicant
A. The applicant shall provide all the available and relevant data of the method optimisation and testing carried out.
These data and results shall be presented, where possible and appropriate, by using the performance parameters
as referred to under point 3.1.C.4. The applicant shall also provide a summary of the testing carried out and the
main results as well as all the data including the outliers.
B. The applicant shall ensure that the provided information demonstrates the robustness of the method for inter-
laboratory transferability. For this purpose, the applicant shall provide the results of the testing of the method by
at least one laboratory that is different from the laboratory which has developed the method.
C. The applicant shall provide the following information about the method development and the method optimis
ation:
1. primer pairs tested and probe, if appropriate, including a justification as to how and why the proposed primer
pair has been selected;
2. stability testing, which shall be established through the submission of experimental results from testing the
method with different plant varieties;
3. specificity, which shall be established through the submission of the full sequence of the insert(s) in a
standardised electronic format, together with the base pairs of the host flanking sequences so as to enable
the EURL to assess the specificity of the proposed method by running homology searches in a molecular
database;
4. precision, the relative repeatability standard deviation shall be less than or equal to 25 % related to mass
fraction over the whole dynamic range of the method.
D. The applicant shall, in addition to the information required under Sections A, B and C provide the following
information regarding the testing:
1. participating laboratories, time of the analysis and outline of the experimental design, including the details
about the number of runs, samples, replicates, etc.;
2. description of the laboratory samples (such as size, quality, date of sampling), positive and negative controls as
well as certified reference material, plasmids and alike used;
3. description of the approaches that have been used to analyse the test results and outliers;
4. any particular points observed during the testing;
5. references to relevant literature or technical provisions used in the testing.
3.3. Samples of the food and feed and their control samples
For the purpose of implementing Articles 5(3)(j) and 17(3)(j) of Regulation (EC) No 1829/2003, the applicant shall,
together with the information required under Sections 1, 2 and 3 of this Annex, also provide samples of the food
and feed and their control samples of a type and amount to be specified by the EURL for the specific application for
authorisation.
The information accompanying the control samples shall include information on the breeding of the plant which
has been used for the production of the control samples and on the zygosity of the insert(s).
The applicant may use the same raw material for the production of certified reference material and for the
production of control samples.
4. CERTIFIED
REFERENCE
MATERIAL
The certified reference material shall be produced under ISO Guide 34 (General requirements for the competence of
reference material producers) by a producer accredited to ISO Guide 34.

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The applicant shall provide information as regards the place where the certified reference material can be accessed.
This shall be accompanied by adequate information demonstrating that the availability of the certified reference
material will be maintained throughout the period of validity of the authorisation. For verification and value
assignment, a method that has been properly validated (see ISO/IEC 17025: General requirements for the
competence of testing and calibration laboratories) shall be used.
Uncertainties shall be estimated according to the ISO Guide to the Expression of Uncertainty in Measurement (GUM).
The main characteristics of those internationally accepted technical provisions are the following:
1. Genetically modified reference material containers:
(a) genetically modified reference material container (such as bottles, vials, ampoules) shall be tight and contain
not less than the stated amount of material;
(b) the commutability of the genetically modified reference material must be assured;
(c) packaging shall be appropriate to the purpose;
(d) labelling shall be of good aspect and quality.
2. Homogeneity testing:
(a) samples shall have appropriate homogeneity;
(b) between-bottle homogeneity shall be examined;
(c) any possible between-bottle heterogeneity shall be accounted for in the overall estimated reference material
uncertainty. This requirement shall apply even when no statistically significant between-bottle variation is
present. In this case, the method variation or the actual calculated between-bottle variation, whichever is
larger, shall be included in the overall uncertainty.
3. Stability testing:
(a) samples shall have appropriate stability;
(b) stability shall be positively demonstrated by appropriate statistical extrapolation for the genetically modified
reference material shelf-life to be within the stated uncertainty; the uncertainty related to this demonstration is
part of the estimated reference material uncertainty. Assigned values are valid only for a limited time and shall
be subject to a stability monitoring.
4. Batch characterisation:
1. The methods used for verification and for certification shall:
(a) be applied under metrologically valid conditions;
(b) have been properly technically validated before use;
(c) have precision and trueness compatible with the target uncertainty.
2. Each set of measurements shall:
(a) be traceable to the stated references;
(b) be accompanied by an uncertainty statement whenever possible.
3. Participating laboratories shall:
(a) have the required competence for the execution of the task;

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L 157/48
EN
Official Journal of the European Union
8.6.2013
(b) be able to achieve traceability to the required stated references;
(c) be able to estimate their measurement uncertainty;
(d) have in place a sufficient and appropriate quality assurance system.
5. Final storage:
1. To avoid degradation after sample production, all samples shall be stored under conditions designated for the
final storage of the genetically modified certified reference material before measurements are started.
2. Otherwise, they shall be transported from door to door keeping them at all times under such storage
conditions for which it has been demonstrated that there is no influence on the assigned values.
6. Establishment of a certificate for certified reference material:
A certificate complemented by a certification report shall be established, containing all information relevant to
and needed by the user.
The certificate and report shall be made available when the genetically modified certified reference material is
distributed.
The information accompanying the certified reference material shall include information on the breeding of the
plant which has been used for the production of the certified reference material and on the zygosity of the
insert(s).
The certified value of the GMO content shall be given in mass fraction and, where available, in copy number per
haploid genome equivalent.
Certified values (such as quantity of genetically modified material expressed in mass fraction) shall be traceable to
stated references and be accompanied by an expanded uncertainty statement valid for the entire shelf-life of the
genetically modified certified reference material.

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