EN    EN EN EN    EN  COMMISSION OF THE E UROPEAN COMMUNITIES 
Brussels,  
C(2009)  
Draft 
COMMISSION REGULATION 
of 
on implementing rules concerning applications  for authorisation of genetically modified 
food and feed in accordance with Regu lation (EC) No 1829/2003 of the European 
Parliament and of the Council and amendi ng Regulations No (EC) 641/2004 and (EC) 
No 1981/2006  
(Text with EEA relevance) EN 2   EN Draft 
COMMISSION REGULATION 
of 
on implementing rules concerning applications  for authorisation of genetically modified 
food and feed in accordance with Regu lation (EC) No 1829/2003 of the European 
Parliament and of the Council and amendi ng Regulations No (EC) 641/2004 and (EC) 
No 1981/2006  
(Text with EEA relevance) 
THE EUROPEAN COMMISSION, 
Having regard to the Treaty on the Functioning of the European Union, Having regard to Regulation (EC) No 1829/2003 of the European Parliament and of the 
Council of 22 September 2003 on gene tically modified food and feed
1, and in particular 
Articles 5(7) and 17(7) thereof, 
After consulting the European Food Safety Authority in accordance with Articles 5(7) and 
17(7) of Regulation (EC) No 1829/2003, 
Whereas:  
(1) Regulation (EC) No 1829/2003 lays dow n Community procedures for the 
authorisation and supervision of genetic ally modified (GM) food and feed and 
provisions for the labelling of such food a nd feed, after a scientific evaluation is 
undertaken on the risks they may present fo r human and animal health and, as the 
case may be, for the environment, and afte r ensuring that they do not mislead the 
consumer or user and do not differ from the food or feed which is intended to replace to such an extent that its normal consumption would be nutritionally 
disadvantageous for animals or humans. 
(2) It provides in particular th at applications for authoris ation should adequately and 
sufficiently demonstrate that the products  covered by the Regulation satisfy the 
requirements laid down in that Regulati on, in respect of their proposed uses.  
(3) Commission Regulation (EC) No 641/2004 on detailed rules for the 
implementation of Regulation (EC) No  1829/2003 of the European Parliament 
and of the Council as re gards the applica tion for the authorisation of new 
genetically modified food and feed, the notification of existing products and 
                                                
 
1 OJ L 268, 18.10.2003, p. 1. EN 3   EN adventitious or technically unavoidable pres ence of genetically modified material 
which has benefited from a favourable risk evaluation2 provides for certain rules 
concerning applications for authorisation submitted in accordance with Regulation (EC) No 1829/2003. To facilitate the preparation of the applications 
by the applicant and to ensure that the applications will contain all the 
information needed for their assessment , it is necessary to provide for more 
comprehensive and systematic rules concer ning the applications for authorisation 
which should also be specific to each t ype of GMO (plants, animals and micro-
organisms).  
(4) These rules should only apply to applica tions concerning GM plants for food or 
feed uses, food or feed containing or cons isting of GM plants and food or feed 
produced from GM plants which constitute the vast majority of current applications and for which sufficient experience is available to date. More specific rules than those provided in Re gulation (EC) No 641 /2004 as regards the 
other types of GM products may be adopted  in the future taki ng into account the 
experience gained in those fields.  
(5) These rules should specify for the atte ntion of the app licant the general 
requirements for the presentation and preparation of applications, namely 
requirements to provide for general and sc ientific information, including methods 
of detection, sampling and identification as  well as reference material so as to 
ensure that the application meets the conditions referred to in Article 5 and 17 of Regulation (EC) No 1829/2003. The applicant should also take into consideration 
the scientific information to be provided in the application as concerns the environmental assessment of genetically modified organisms (GMOs) or food and feed containing or consisting of GM Os set out in Annex II to Directive 
2001/18/EC of the European Parliament  and of the Council of 12 March 2001 on 
the deliberate release into the environmen t of genetically modified organisms and 
repealing Council Directive 90/220/EEC
3 as well as the applicable guidance 
published by the European Food Safety Authority in this regard. 
(6) It is appropriate to provide for specific rules to ensure that the scientific 
information required in the application ad equately and sufficiently demonstrates 
that the product does not have adverse effects on human or animal health, does 
not mislead the consumer or user and does not differ from the food or feed which 
it is intended to replace to such an exte nt that its normal consumption would be 
nutritionally disadvantageous for animals or humans. These rules should take into 
account relevant international standards,  such as the guide line of the Codex 
Alimentarius for the conduct of food safety assessment derived from 
recombinant-DNA plant4.  
(7) Depending on the nature of the GM food and feed or their conditions of use, the 
type and necessity of the studies needed to evaluate their proprieties or their 
effects may vary. Some degree of flexib ility should therefore be granted to 
applicants regarding the type of studies to be presented in the applications to 
                                                 
2 OJ L 102, 7.4.2004, p. 14.  
3 OJ L 106, 17.4.2001, p. 1. 
4 Codex Alimentarius Commission, GL 45-2003.  EN 4   EN demonstrate the safety of the geneti cally modified products concerned. 
Appropriate verifiable justification should be given in such cases. 
(8) It is essential to apply appropriate qualit y assurance systems to facilities in which 
toxicological tests are performed in order to ensure that the results are of high 
quality. Such principles are laid down by Directive 2004/10/EC of the European 
Parliament and Council of 11 Februa ry 2004 on the harmonisation of laws, 
regulations and administrative provisions  relating to the application of the 
principles of good laboratory practice and th e verification of their applications for 
tests on chemical substances.5 If such tests are carried out outside the Union, they 
should follow “the OECD Principles of Good Laboratory Practice” (GLP). With 
regard to studies other than toxicologi cal studies, they should be conducted under 
ISO or GLP standards or other appropriate quality assurance. 
(9) In order to demonstrate that a GM food or feed fulfils the requirements of 
Regulation (EC) No 1829/2003, experimental testing may be necessary involving 
laboratory animals. This should be carried out in accordance with Directive 86/609/EEC of 24 November 1986 on the appr oximation of laws, regulations and 
administrative provisions of the Member States regarding the protection of the 
animals used for experimental  and other scientific purposes
6, and should be kept 
to a minimum while ensuring an adequate demonstration of the safety of the GM 
food and feed. 
(10) This Regulation should define the cond itions under which a proposal for post-
market monitoring of the use of the GM food and feed should be submitted.  
(11) The content of this Regulation take s account of the in ternational trade 
commitments of the Union and of the requ irements of the Cartagena Protocol on 
Biosafety to the Convention on Biological Diversity (the Cartagena Protocol), 
approved by Council Decision 2002/628/EC7 and the provisions of Regulation 
(EC) No 1946/2003 of 15 July 2003 of the European Parliament and of the 
Council on transboundary movements of genetically modified organisms8.  
(12) Studies presented in applications should be carried out in accordance with this 
Regulation, internationally agreed protocols and the test methods described by 
the OECD when available.  
(13) Applications should include proposals for a unique identifier for each GMO 
concerned in accordance with Commi ssion Regulation (EC) No 65/2004 of 14 
January 2004 establishing a system for the development and assignment of 
unique identifiers for genetically modified organisms9. 
(14) This Regulation replaces provisions laid  down in Regulation (EC) No 641/2004 
as regards GM plants for food or feed us es, food or feed containing or consisting 
of GM plants and food or feed produced  from GM plants. However, Regulation 
                                                 
5 OJ L 50, 20.2.2004, p. 44. 
6 OJ L 358, 18.12.1986, p. 1.  
7 OJ L 201, 31.7.2002, p. 48. 
8 OJ L 287, 5.11.2003, p. 1. 
9 OJ L 10, 14.1.2004, p. 5. EN 5   EN (EC) No 641/2004 should continue to ap ply as regards ot her types of GM 
products (GM animals, GM micro-organi sms). Moreover, certain provisions of 
Regulation (EC) No 641/2004 are obsolete. It follows that Regulation (EC) 
No 641/2004 should be amended accordingly. 
(15) Commission Regulation (EC) No 1981/ 2006 of 22 December 2006 on detailed 
rules for the implementation of Articl e 32 of Regulation (EC) No 1829/2003 of 
the European Parliament and of the Council as regards the Community reference laboratory for genetically modified organisms
10 should be amended to include 
references to this Regulation. 
(16) According to Articles 5 (7) and 17 (7) of Regulation (EC) No 1829/2003, the 
Commission shall consult the Europ ean Food Safety Authority before 
establishing implementing rules with regard to the applications for authorisations 
under that Regulation.  In accordance with the indicated Articles, the European 
Food Safety Authority has been consulted on those rules.  
(17) The implementing rules have been drawn up on the basis of current scientific and 
technical knowledge. Th erefore, the Commission should monitor any 
developments in this field and the publication of new or additional guidance by the European Food Safety Authority.  
(18) It is necessary to provide for transitional 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 unnecessary delays. 
(19) The measures provided for in this Regul ation are in accordance with the opinion 
of the Standing Committee on the Food Chain and Animal Health,  
HAS ADOPTED THIS REGULATION: 
CHAPTER I 
 
General provisions  
 
Article 1  
Scope 
This Regulation shall apply to applications submitted under Regulation (EC) No 1829/2003 
for authorisation of genetically modified (GM)  plants for food or feed uses, food and feed 
containing or consisting of GM  plants, and food and feed produced from GM plants.  
                                                 
10 OJ L 368, 22.12.2006, p. 99. EN 6   EN Article 2 
Definitions 
For the purposes of this Regulation the definiti ons set out in Articles 2 and 3 of Regulation 
(EC) No 178/2002 of the European Parliament and the Council11 shall apply.  
CHAPTER II 
 
General requirements  
Article 3 
Preparation and presentation of the application 
1. The application shall be submitted in accord ance with Annex I and shall contain all 
the information and particulars required th erein, in accordance with the requirements 
set out in Articles 4 to 6. 
2. The application, as well as any supplementary information submitted during the 
authorisation procedure, sha ll clearly state which parts of  the application are claimed 
to be confidential, providing verifiable justification in accordance with Article 30 of Regulation (EC) No 1829/2003. Parts claimed to  be confidential sh all be included at 
the time of submission of the application but  separately from the main application.  
CHAPTER III 
 
Specific Requirements 
Article 4 
Scientific Requirements for the safe ty assessment of GM food and feed 
1. Information, including studies, required fo r the risk assessment as referred to in 
Article 5(3)(a) to (f) and (h) and  in Article  17(3)(a) to (f) and (h ) of Regulation (EC) 
No 1829/2003 to be included in the applicati on shall be provided in accordance with 
the requirements set out in Annex II. 
2. By way of derogation from paragraph 1, an  application may be accepted even if it 
does not satisfy all the requirements set out  in that paragraph, provided that the 
applicant submits verifiable justification for each element not complying with those 
requirements. 
3. Toxicological studies shal l be conducted in facilities  which comply with the 
requirements of Directive 2004/ 10/EC or, if they are carried out outside the Union, 
                                                 
11 OJ L 31, 1.2.2002, p. 1. EN 7   EN they shall follow “the OECD Principles of Good Laboratory Practice” (GLP). The 
applicant shall provide evidence to demonstrate that those requirements are complied with. 
4. Studies other than toxicological studi es shall be conducte d under ISO or GLP 
standards or other appropriate quality a ssurance. In case where quality assurance 
standards other than ISO or GLP standard s are used, the applicant shall provide a 
thorough description of the quality assura nce system under which they have been 
conducted.  
5. Where trials involve animals as referre d to in Council Dir ective 86/609/EEC, the 
results of those trials shall not be c onsidered confidential in accordance with 
Article 30(3)(d) of Regul ation (EC) No 1829/2003.  
6. Without prejudice to Article 31 of Regul ation (EC) No 1829/2003 on data protection, 
when studies have been already submitted for the purposes of an application to the European Food Safety Authority, a reference to such studies and the results of the 
evaluation may, with the agr eement of the Authority, be made in the framework of 
another application.  
Article 5 
Requirements applicable for post-mar ket monitoring of GM food and feed 
When necessary, a proposal for post-market mo nitoring regarding the use of food for human 
consumption and/or the use of feed for animal  consumption shall be submitted in accordance 
with Annex III.  
Article 6 
Requirements concerning the methods of de tection, sampling, identification and reference 
material  
The application shall meet the specific requirements concerni ng the methods of detection, 
sampling and identification as well as the reference material referred to in Articles 5.3 (i) and 
(j) and 17.3 (i) and (j) of Regulation (E C) No 1829/2003, as set out in Annex IV.  
CHAPTER IV 
 
Final provisions 
Article 7 
Amendments to Regulation (EC) No 641/2004 
Regulation (EC) No 641/2004 is amended as follows: 
1) Article 1 shall be replaced by the following: 
"Article 1  EN 8   EN This chapter provides detailed rules c oncerning applications for authorisation 
submitted in accordance with Articles 5 and 17 of Regulation (EC) No 1829/2003 except for those covered by Commission Regulation [XXX/2009 – reference to this 
Regulation] ."  
2) Articles 5 to 19 shall be deleted. 
Article 8 
Amendments to Regulation (EC) No 1981/2006  
Regulation (EC) No 1981/2006 is amended as follows: 
1) In Article 2, point (a) is replaced by the following: 
"(a) ‘full validation procedure’ means  : 
(i)  the assessment through a ring trial involving national reference laboratories of 
the method performance criteria set by the applicant as compliant with the document entitled ‘Definition of mi nimum performance requirements for 
analytical methods of GM O testing’ referred to  
-  in the case of GM plants for food or  feed uses, food or feed containing or 
consisting of GM plants and food or feed produced from GM plants, in 
point 1.1 (B) of Annex IV to Regulation (EC) No XXX/2009; 
 - in all other cases, in point 1), B)  of Annex I to Regulation (EC) No 
641/2004" 
and (ii)  the assessment of the repeatability and tr ueness of the method  provided by the 
applicant;  
2) In Article 3, paragraph 2 is replaced by the following: 
"2.  The CRL shall request the applicant to pay an additional contribution of EUR 60 000 
where a full validation procedure of a met hod of detection and identification for a 
single GMO event according to the requirements laid down in the following 
provisions is required:  
(a) Annex IV of Regulation (EC) No XXX/2009 , when the application is related to 
GM plants for food or feed uses, food or feed containing or consisting of GM plants and food or feed pr oduced from GM plants, or 
(b) point 1(B) of Annex I to Regulati on (EC) No 641/2004, in all other cases."  
That amount shall be multiplied by the number of GMO events to be fully validated. The CRL shall reduce the amount of the addi tional contribution, in proportion of the 
costs saved: EN 9   EN (a) where the material needed to perform the full validation procedure is supplied 
by the applicant; and/or 
(b) where the applicant provides data that refer to modules, such as DNA 
extraction protocols and species specific  reference systems, already validated 
and published by the CRL."  
Article 9 
Monitoring 
The Commission shall monitor the application of  this Regulation and the publication of new 
or additional guidance from the European Food Safety  Authority as well as the availability of 
updated guidance on products falling under the scope of Regulation (EC) No 1829/2003 
which have not yet been covered by this Regulation.  
Article 10  
Transitional provisions  
1. Until [ fixed date corresponding to 6 months after the publication in the Official 
Journal ], applicants may choose to submit a pplications under Regulation (EC) No 
641/2004 in the version in force on [ fixed date corresponding to  the publication in 
the Official Journal ]. 
2. For applications submitted until [ fixed date corresponding to  18 months after the 
publication in the Official Journal ] compliance with point 1.3.2. of Annex II shall be 
voluntary. 
Article 11 
Entry into force 
This Regulation shall enter into force on the 20th day following that of its publication in the 
Official Journal of the European Union .  
 
 
 
Done at Brussels, 
 For the Commission     
 José Barroso President of the Commission   EN 10   EN ANNEX I  
 
PREPARATION AND PRESENTATION OF APPLICATIONS  
The application shall contai n the following information: 
PART I: G ENERAL INFORMATION  
1. Name and address of the applic ant (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 GM plant and derived product; 
4. Scope of the application 
(a) GM food 
 Food containing or consisting of GM plants 
 Food produced from GM plants or containing ingredients produced from 
GM plants 
(b) GM feed 
 Feed containing or consisting of GM plants 
 Feed produced from GM plants 
(c) GM plants for food or feed uses 
 Products other than food and feed co ntaining or consisting of GM plants 
with the exception of cultivation 
 Seeds and plant propagating material for cultivation in the EU 
Where an application is limited to either food or feed use, it sha ll contain a verifiable 
justification explaining why the authorisati on shall not cover both uses in accordance 
with Article 27 of Regulation (EC) No 1829/2003.  
5. Unique identifier 
A proposal for a unique identifier for the GM plant and derived products in question, 
developed in accordance with Co mmission Regulation (EC) No 65/2004. 
6. Where applicable and where relevant to  the risk assessment, a detailed 
description of the method of production and manufacturing.  EN 11   EN This would include, for example, a descrip tion of methods used to process the GM 
plant materials during the preparation of f ood/feed, food/feed ingr edients, food/feed 
additives or food flavourings. 
7. Where appropriate, the conditions for placing on the market of the food(s) or 
feed(s) produced from it, including specific conditions for use and handling. 
PART II: S CIENTIFIC INFORMATION  
All the following requirements shall be provided in the application except where it is not 
justified by the scope (e.g. limited to derived products),  
1. H AZARD IDENTIFICATION AND CHARACTERISATION  
1.1. Information relating to the recipient or (where appropriate) parental plants 
(a) Complete name; (a) family name, (b ) genus, (c) species, (d) subspecies, (e) 
cultivar/breeding line or strain, (f) common name; 
(b) Geographical distribution and cultiva tion of the plant, including its 
distribution within the Union; 
(c) Information on the recipient or paren tal plants relevant to their safety, 
including any known toxicity or allergenicity; 
(d) Data on the past and present use of the recipient organism, such as history of 
safe use for consumption as food or feed, including information on how the 
plant is typically cultivated, tran sported and stored, whether special 
processing is required to make the plant safe to eat, and the plant normal role 
in the diet (e.g. which part of the plant  is used as a food source, whether its 
consumption is important in particu lar subgroups of the population, what 
important macro- or micro-nutrien ts it contributes to the diet) 
(e) Additional information relating to the recipient or parental plants required for 
the environmental safety aspects:  
(a) Information concerning reproduction:  (i) mode(s) of reproduction, (ii) 
specific factors affecting reproduction (if any), (iii) generation time;  
(b) Sexual compatibility with other cultivated or wild plant species; 
(c) Survivability: (a) ability to form structures for survival or dormancy, 
and (b) specific factors, if any, affecting survivability; 
(d) Dissemination: (a) ways and exte nt of dissemination (to include, for 
example, an estimation of how viable  pollen and/or seed declines with 
distance), and (b) spec ial factors affecting dissemination, if any. 
(e) Geographical distribution within th e Union of the sexually compatible 
species. EN 12   EN (f) In the case of a plant species not grown in the Member State(s), 
description of the natural habitat of th e plant, including information on natural 
predators, parasites, competitors and symbionts; 
(g) Other potential interactions of the GM 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 DNA used for transformation, size and intended  function of each 
constituent fragment of the region intended for insertion 
1.2.2. Information relating to the GM 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 expr ession of the insert(s) 
1.2.2.4. Genetic stability of the insert and phenotypic stability of the GM plant 
1.2.3. Additional information relating to the GM  plant required for the environmental 
safety aspects 
1.2.3.1. Information on how the GM plant di ffers from the recipient plant in 
reproduction, dissemination, survivability 
1.2.3.2. Any change to the ability of the GM plan t to transfer genetic material to other 
organisms 
● Plant to bacteria gene transfer  
● Plant to plant gene transfer  
1.2.4 Conclusions of the Molecular characterisation 
1.3. Comparative analysis 
1.3.1. Choice of the conventional c ounterpart 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 protocol s for the experimental design EN 13   EN 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. Potential risk associated w ith horizontal gene transfer 
1.3.7. Effects of processing 1.3.8. Conclusion 
1.4. Toxicology 
1.4.1. Testing of newly expressed proteins 
1.4.2. Testing of new constituen ts other than proteins 
1.4.3. Information on natural f ood and feed constituents 
1.4.4. Testing of the whole GM food/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 GM food 
and feed EN 14   EN 1.4.5. Conclusion of the to xicological assessment 
1.5. Allergenicity 
1.5.1. Assessment of allergenicity of  the newly expressed protein 
1.5.2. Assessment of allergenicity of  the whole GM plant or crop 
1.5.3. Conclusion of the allergenicity assessment 
1.6. Nutritional assessment 
1.6.1. Nutritional assessment of GM food 
1.6.2. Nutritional assessment of GM feed 1.6.3. Conclusion of the nutritional assessment 
2. E
XPOSURE ASSESSMENT - ANTICIPATED INTAKE /EXTENT OF USE  
3. R ISK CHARACTERISATION  
4. P OST-MARKET MONITORING ON GM  FOOD /FEED  
5. E NVIRONMENTAL ASSESSMENT  
6. E NVIRONMENTAL MONITORING PLAN 
PART III: C ARTAGENA PROTOCOL  
The application shall provide information requi red under Article 5(3)(c) and Article 17(3)(c) 
of Regulation (EC) No 1829/2003 for the purpose of complying with th e Cartagena Protocol. 
Depending on the scope of the application, the provided info rmation shall contain as a 
minimum the information specified in Annexe s II or III to Regulation (EC) No 1946/200312. 
PART IV: L ABELLING  
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;  
                                                 
12 OJ L 287, 5.11.2003, p. 1. EN 15   EN (b) Either a reasoned statemen t that the food/feed does not give rise to ethical or 
religious concerns or a proposal  for labelling in all offici al languages of the Union as 
required by Articles 5(3)(g) and 17(3)(g ) of Regulation (EC) No 1829/2003; and,  
(c) When appropriate a proposal for labellin g complying with the requirements of 
Annex IV, A(8) to Directive 2001/18/EC. 
PART V: M ETHODS OF DETECTION , SAMPLING AND IDENTIFICATION AND REFERENCE 
MATERIAL  
The applicant shall provide methods for dete ction, sampling and iden tification as well as 
samples of the food/feed, their controls samples to the CRL. A copy of the completed form 
for submission of the samples to the CRL and a proof of sending to the CRL shall be provided 
in the application.  
– Information as to the place where the reference material can be accessed shall be 
provided in the application.  
– The applicant shall follow the following instructions in the preparation and the 
sending of the samples:  
(a) The preparation of the samples and control samples shall follow the 
specifications laid down in: http://gmo-crl.jrc.ec.europa.eu  
(b) The parcel shall be specified to cont ain “Free samples”, and it shall include the 
list of all items and their storage instructions.  
PART VI: A DDITIONAL INFORMATION TO BE PROVIDED FOR GM  PLANTS AND /OR 
FOOD /FEED CONTAINING OR CONSISTING OF GM  PLANTS   
The information required by Annex III to Dir ective 2001/18/EC shall be provided where it is 
not covered by the requirements of  other parts of the application.  
PART VII: S UMMARY OF APPLICATIONS  
This part specifies the standa rdised form, which the summary of the dossier applications 
should follow. Depending on the scope of the app lication, some of the requested information 
may not be applicable. The summary shall not c ontain parts considered to be confidential in 
accordance with Article 30 of Regulation (EC) No 1829/2003.  
1. G ENERAL INFORMATION  
1.1. Details of application 
(a) Member State of application 
(b) Application number (c) Name of the product (commercial and other names) EN 16   EN (d) Date of acknowledgement of valid application 
1.2. Applicant 
(a) Name of applicant 
(b) Address of applicant (c) Name and address of the representativ e of the applicant established in the 
Union (if the applicant is not  established in the Union) 
1.3. Scope of the application 
(a) GM food 
 Food containing or consisting of GM plants 
 Food produced from GM plants or containing ingredients produced from 
GM plants 
(b) GM feed 
 Feed containing or consisting of GM plants 
 Feed produced from GM plants 
(c) GM plants for food or feed use 
 Products other than food and feed co ntaining of consisting of GM plants 
with the exception of cultivation 
 Seeds and plant propagating material for cultivation in the EU 
1.4. Is the product or the uses of the associ ated plant protection  product(s) already 
authorised or subject to another author isation procedure within the Union?  
No   
Yes  (in that case, specify) 
1.5. Has the GM 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 GM plant or derived products b een previously notifi ed for marketing in 
the Union under Part C of Directive 2001/18/EC? 
No   
Yes  (in that case, specify) EN 17   EN 1.7. Has the product been notified/authorised in a third country either previously or 
simultaneously? 
No  
Yes  (in that case, specify the third country and provide a copy of the risk 
assessment conclusions, the date of  the authorisation and the scope) 
1.8. General description of the product 
(a) Name of the recipient or parental pl ant 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 specifi c form in which the product must not 
be placed on the market (seeds, cut-fl owers, vegetative parts, etc.) as a 
proposed condition of the authorisation applied for 
(c) Intended use of the product and types of users (d) Any specific instructions and/or recommendations for use, storage and 
handling, including mandatory restrictions proposed as a condition of the authorisation applied for 
(e) If applicable, geographical areas w ithin the EU to which the product is 
intended to be confined under the term s 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 law and 
when necessary a proposal for specifi c labelling in accordance with Articles 
13(2), (3) and 25(2)(c), (d) and 25(3) of Regulation (EC) No 1829/2003. In the 
case of GMO plants, food and/or feed c ontaining or consis ting of GMO plants, 
a proposal for labelling has to be included complying with the requirements of 
Annex IV, A(8) of Directive 2001/18/EC.  
(i) Estimated potential demand 
(i) In the Union (ii) In export markets for EU supplies 
(j) Unique identifier in accordan ce with Regulation (EC) No 65/2004 EN 18   EN 1.9. Measures suggested by the applicant to take in case of unintended release or 
misuse as well as measures for disposal and treatment 
2. I NFORMATION 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 or strain (f) Common name 
2.2. Geographical distribution and cultiv ation 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 cu ltivated 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 EN 19   EN 2.7. Geographical distribution within the Unio n of the sexually compatible species 
(for environmental safety aspects) 
2.8. In the case of plant species not normally grown in the Member State(s), 
description of the natural ha bitat of the plant, includi ng information on natural 
predators, parasites, competitors and symbionts (for environmental safety 
aspects) 
2.9. Other potential interactio ns, relevant to the GM 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. M
OLECULAR CHARACTERISATION  
3.1. Information relating to the genetic modification 
3.1.1. Description of the methods us ed for the genetic modification 
(a) Nature and source of the vector used 
(b) Source of donor DNA, si ze and intended function of each constituent fragment 
of the region intended for insertion 
3.2. Information relating to the GM plant  
3.2.1 Description of the trait(s) and charac teristics which have been introduced or 
modified 
3.2.2. Information on the sequences actually inserted or deleted 
(a) The copy number of all detectable inserts, both complete and partial 
(b) In case of deletion(s), size a nd function of the deleted region(s) 
(c) Chromosomal location(s) of insert(s) (nucleus, chloroplasts, mitochondria, or 
maintained in a non-integrated form),  and methods for its determination 
(d) The organisation of the inserted ge netic material at the insertion site 
(e) In case of modifications other than inserti on 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 modification 
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  EN 20   EN 3.2.4. Genetic stability of the insert and phenotypic stability of the GM plant 
3.2.5. Information (for environmental safety asp ects) on how the GM 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 GM 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 counter part and additional comparators 
4.2. Experimental design and statistical an alysis 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 varietie s in each location).  
4.3. Selection of material and compounds for analysis 
4.4. Comparative analysis of agronom ic and phenotypic characteristics 
4.5. Effect of processing 
5. T OXICOLOGY  
(a) Toxicological testing of  newly expressed proteins 
(b) Testing of new constituents other than proteins (c) Information on natural food and feed constituents (d) Testing of the whole GM food/feed 
6. A
LLERGENICITY  
(a) Assessment of allergenicity of the newly expressed protein 
(b) Assessment of allergenicity of the whole GM plant or crop EN 21   EN 7. N UTRITIONAL ASSESSMENT  
(a) Nutritional assessment of GM food 
(b) Nutritional assessment of GM feed 
8. E XPOSURE ASSESSMENT – ANTICIPATED INTAKE /EXTENT OF USE  
9. RISK CHARACTERISATION  
10. P OST-MARKET MONITORING ON GM  FOOD /FEED  
11. E NVIRONMENTAL ASSESSMENT  
11.1. Mechanism of interaction between th e GM plant and target organisms 
11.2. Potential changes in the interactions  of the GM 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 GM  plant and target organisms 
(e) Interactions of the GM plan t 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, ma nagement and harvesting techniques  
11.3. Potential interactions with the abiotic environment 
12. E
NVIRONMENTAL MONITORING PLAN  
(a) General (risk assessment, background information) 
(b) Interplay between environmenta l risk assessment and monitoring 
(c) Case-specific GM plant monitoring (approach, strategy, method and analysis) 
(d) General surveillance of the impact of  the GM plant (app roach, strategy, method 
and analysis) EN 22   EN (e) Reporting the results of monitoring 
13. D ETECTION AND EVENT -SPECIFIC IDENTIFICATION TECHNIQUES FOR THE GM  
PLANT  
14. I NFORMATION RELATING TO PREVIOUS RELEASES OF THE GM  PLANT (FOR 
ENVIRONMENTAL SAFETY ASPECTS ) 
14.1. History of previous rele ases of the GM plant notified under Part B of the 
Directive 2001/18/EC and under Part B of  Directive 90/220/EEC by the same 
notifier 
(a) Notification number 
(b) Conclusions of post-release monitoring (c) Results of the release in respect to any risk to human health and the 
environment (submitted to the Competent Authority according to Article 10 of Directive 2001/18/EC) 
14.2. History of previous releases of the GM  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 in respect to any risk to human health and the 
environment EN 23   EN ANNEX II  
 
SCIENTIFIC REQUIREMENTS FOR RISK  ASSESSMENT CONCERNING FOOD 
AND FEED SAFETY ASPECTS  
I. INTRODUCTION 
1. D EFINITIONS  
For the purpose of this annex, the following definitions shall apply: 
1. 'hazard identification' means the identif ication of biological, chemical, and 
physical agents capable of causing adve rse health effects and which may be 
present in a particular food or group of foods ;  
2. 'hazard characterisation' means the qualita tive 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; 
3. 'risk characterisation' means the quali tative and/or quan titative estimation, 
including attendant uncertainties, of the probability of occurrence and severity 
of known or potential advers e health effects in a given population based on 
hazard identification, hazard charac terization and exposure assessment; 
2. S PECIFIC CONSIDERATIONS  
2.1. Insertion of marker genes and other DNA not essential to achieve the desired 
trait 
During the process of genetic modification of plants and other organisms, marker 
genes are used to facilitate the selection and identification of genetically modified 
cells, containing the gene of interest inse rted into the genome of the host organism, 
among the vast majority of untransformed ce lls. Such marker genes shall be carefully 
selected.  In case of use of Antibiotic Re sistance Marker genes, Article 4 (2) of 
Directive 2001/18/EC shall be respected.  The applicant, when considering the 
submission of an application related to a GM plant with such type of gene, shall also 
consider the opinion of EFSA on the matter13. 
The risk assessment may be facilitated if the presence of inserted DNA not essential 
to achieve the desired trait is minimised.  
 
                                                 
13  ARM Statement of EFSA on the consolidated presentation of opinions on the use of antibiotic 
resistance genes as marker genes in genetically modified plants, the EFSA Journal (2009) 1108, 1-8 EN 24   EN 2.2. Risk assessment of GM food and feed containing stacked tr ansformation events 
combined by conventional crossing 
A risk assessment of the single events is a pre-requisite for the assessment of stacked 
events. 
The assessment of GM food and feed c ontaining more than two transformation 
events combined by conventional crossing sh all cover all sub-comb inations of these 
events. In such a case, the applicant shal l either provide a scientific rationale 
justifying that there is no need for experi mental data obtained for the concerned sub-
combinations or provide the experimental data. 
The risk assessment of the stacked events sh all then mainly focus on issues related to 
(a) stability of the inserts,  (b) expression of the events and  (c) potential synergistic or antagonistic effects resulting from the combination of 
the events. 
 
II. SCIENTIFIC REQUIREMENTS 
1. H
AZARD IDENTIFICATION AND CHARACTERISATION  
1.1. Information relating to the recipient or (where appropriate) parental plants 
The applicant shall provide comprehensive information relating to the recipient or 
(where appropriate) th e parental plants:  
– to evaluate all issues of potential con cern, such as the presence of natural 
toxins or allergens; 
– to identify the need for specific analyses. 
For these purposes, the applicant shal l provide the following information: 
(a) Complete name; (a) family name, (b) genus, (c) species, (d) subspecies, (e) 
cultivar/breeding line or strain, (f) common name. 
(b) Geographical distribution a nd cultivation of the plant,  including its distribution 
within the Union. 
(c) Information on the recipient or parent al plants relevant to their safety, 
including any known toxici ty or allergenicity. 
(d) Data on the past and present use of the recipient organism. This information 
shall include the history of safe us e for consumption as food or feed, 
information on how the plan t is typically cultivate d, transported and stored, 
whether special processing is required to make the plant safe to eat, and EN 25   EN describe the normal role of the plant in the diet (e.g. which part of the plant is 
used as a food and feed source, whether its consumption is important in particular subgroups of the population, what important macro- or micro-
nutrients it contribu tes 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: 
– to identify the DNA intended for transformation and related vector sequences 
potentially delivered to the recipient plant; 
– to characterise the DNA 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 transforma tion including relevant references; 
(b) the recipient plant material;  (c) the strain of Agrobacterium  if used during the genetic  transformation process; 
(d) the helper plasmids, if used duri ng the genetic transformation process; 
(e) the source of carrier DNA if used dur ing the genetic transformation process. 
1.2.1.2. Nature and source of vector used  
The applicant shall provide the following information: – a physical map of the f unctional elements and other plasmid/vector 
components together with the rele vant information needed for the 
interpretation of the molecular analyses (e.g. restriction site s, the position of 
primers used in PCR, location of probes us ed in Southern anal ysis). The region 
intended for insertion sha ll be clearly indicated; 
– a table identifying each component of th e plasmid/vector (including the region 
intended for insertion), its size, its  origin and its intended function. 
1.2.1.3. Source of DNA used for transformati on, 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 DNA 
sequence(s) intended to be inserted in orde r to determine whether the nature of the 
donor organism(s) or the DNA sequence(s) may trigger any safety issue. Information 
regarding the function of the DNA region(s) intended for insertion shall comprise the 
following elements: EN 26   EN (a) the complete sequence of the DNA intended to be inserted, including 
information on any deliberate alteration(s)  to the corresponding sequence(s) in 
the donor organism(s); 
(b) history of safe use of the gene product( s) arising from the regions intended for 
insertion; 
(c) data on the possible relati onship 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 GM plant  
1.2.2.1. General description of the tr ait(s) and characteristics wh ich 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 cha nges to the phenotype and metabolism of the 
plant. 
When the introduced trait is herbicide tolerance, the applicant shall provide 
information on the mode of action and 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 detectab le 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 host plant, such as any parts of the plasmid/vector or any carrier or fore ign DNA remaining in the GM plant. The 
Southern analysis shall span the entire transgenic locus(i) as well as flanking 
sequences and include all appropriate c ontrols. For the dete rmination of copy 
number of the insert, complementary me thods can also be used (e.g. real-time 
PCR) 
(b) the organisation and sequence of the inse rted genetic material at each insertion 
site; 
(c) in the case of deletion(s), size and f unction of the deleted region(s), whenever 
possible; 
(d) sub-cellular location(s) of insert(s) (integrated in nuclear-, plastid-, or 
mitochondrial chromosomes, or mainta ined in a non-inte grated form) and 
methods for its determination; EN 27   EN (e) sequence information for both 5’ and 3’ flanking regions at each insertion site, 
with the aim of identifying interrup tions of known Open Reading Frames 
corresponding to any nucleotide sequence th at contains a string of codons that 
is uninterrupted by the presence of a stop codon in the same reading frame (hereafter, referred to as ORFs) or re gulatory regions. Bioinformatic analyses 
shall be conducted using up-to-date data bases with the aim of performing both 
intraspecies and interspecies homology searches; 
(f) ORFs created as a result of the genetic modification either at the junction sites 
with genomic DNA or due to internal r earrangements of th e insert(s). The 
ORFs shall be analysed between st op codons, not limiting their lengths. 
Bioinformatic analyses shall be conducte d 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 (e.g. transc ription analysis) may be needed to 
complete the risk assessment. 
1.2.2.3. Information on the expr ession of the insert(s)  
The applicant shall provide information:  – to demonstrate whether the intended  changes in expression have been 
achieved; 
– to characterise the potential unintende d expression of new ORFs identified 
under section 1.2.2.2 as raising a safety concern. 
For these purposes, the applicant shal l provide the following information: 
(a) Methods used for expression analys is together with the raw datasets; 
(b) Information on developmental expression of the insert during the life cycle of 
the plant. The requirement for informa tion on developmental expression shall 
be considered on a case-by-case basis ta king into account th e promoter used, 
the intended effect(s) of the modifica tion and scope of the application;  
(c) Parts of the plant where the insert is  expressed. Data on expression levels from 
those parts of the plant used for food a nd feed purposes sha ll be provided in all 
cases. Where tissue-specific promoters have been used, information may be 
requested on expression of target genes in other plant parts relevant for risk 
assessment; 
(d) Potential unintended expression of new ORFs identified under section as 
raising a safety concern; 
(e) The range of concentrations of newl y produced proteins or existing plant 
proteins deliberately modified in the GM  food(s) and feed(s) to be placed on 
the market; 
(f) Protein expression data shall be obtai ned from field trials as specified in 
section 3.3.2 and be related to the co nditions in which the crop is grown; EN 28   EN (g) When justified by the nature of the insert, (e.g. gene silencing through RNA 
interference) information on the expr ession of thetargeted gene(s) and on 
possible effects on other endogenous  genes (to be selected by in silico  analysis) 
shall be provided; 
(h) With regard to the stacking of even ts by conventional crossing, data shall be 
provided to establish that the combin ation of events does not raise any 
additional safety concerns over protein and trait expre ssion compared with the 
single events. 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 GM plant  
The applicant shall provide information: – to demonstrate the genetic stability of the transgenic locus(i) and the 
phenotypic stability and inheritance pa ttern(s) of the introduced trait(s); 
– in case of stacked events, to establish that each of the events stacked in the 
plant has the same molecular properties a nd characteristics as in the individual 
events separately. 
For this purpose, applicants shall provide data from multiple (normally five) 
generations or vegetative cycl es for single events. The source  of the material used for 
the analysis shall be specified. Data shall be analysed using appropriate statistical 
methods. 
For stacked events comparisons between the original events and the GM stacks shall 
be carried out using plant materials repres entative of those de signed for commercial 
production. The applicant shall provide ad equate justification for its choice. 
To assess genetic stability of the event(s), applicants shall use appropriate molecular 
approaches detailed in section 1.2.2.2. 
1.2.3. Conclusions of the Molecular characterisation 
The molecular characterisation shall provide data on the st ructure of the insert(s), 
expression and stability of th e intended trait(s). This shall also apply to situations 
where events have been st acked by conventional breeding. 
It shall be specifically indi cated whether the molecular ch aracterisation of the genetic 
modification(s) raises safety  concerns with regard to  the potential production of 
proteins/products other than those intended. 
The molecular characterisation shall specifi cally aim to identify whether the genetic 
modification(s) raise(s) any issues regarding the potenti al for producing 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. EN 29   EN 1.3. Comparative analysis 
The comparative analysis of compositi on and agronomic as well as phenotypic 
characteristics shall constitute, together with the molecular characterisation, the 
starting point to structure and conduct th e risk assessment of a new GM food and 
feed and its derived prod ucts. It shall aim at: 
– identifying similarities and differences  in composition, agronomic performance 
and phenotypic characteristi cs (intended and uninte nded alterations) between 
the GM plant and its co nventional counterpart;  
– identifying similarities and differences in composition between the GM food 
and feed and its conventional counterpart. 
Where no appropriate conventional counter part can be identified, a comparative 
safety assessment cannot be made and consequently a safety and nutritional 
assessment of the GM food and feed shall be  carried out as for other novel foods that 
do not have conventional counterparts (e.g. where the GM food and feed is not closely related to a food and feed with a hi story 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 GM food and feed. 
1.3.1. Choice of the conventional c ounterpart and additional comparators 
In the case of vegetatively propagated cr ops, the conventional c ounterpart sh all, in 
principle, be the near-iso genic variety used to gene rate the transgenic line. 
In the case of crops  that reproduce sexually, the conve ntional counterpart shall have 
a genetic background comparable to the GM plant. Since many crops used to 
produce food and feed are developed using back-crossing, a conve ntional counterpart 
with a genetic background that  is as close as possible to the GM plant shall be 
selected. 
In all cases, the applicant shall provi de information on the breeding scheme 
(pedigree) in relation to bot h the GM plant and the conventional counterpart and an 
adequate justification for the use of the selected c onventional counterpart. The 
history of safe use of the conventional c ounterpart shall be adequately supported by 
both qualitative and quantitative data
14. In addition, the app licant may include a 
comparator having a closer genetic background to the GM plant than the conventional counterpart (such as a negative segregant).  
In the case of herbicide tolerant GM 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 GM plant exposed  to the intended 
herbicide, the conventional counterpart  treated with conve ntional herbicide 
management regimes and the GM plant trea ted with the same conventional herbicide 
management regimes. 
                                                
 
14  See, for example, Constable A, Jonas D, Cockburn A, Davi A, Edwards G, Hepburn P, Herouet-
Guicheney C, Knowles M, Moseley B, Oberdörfer R, Samuels F. History of safe use as applied to the 
safety assessment of novel foods and foods derive d from genetically modified organisms. Food Chem 
Toxicol. 2007 45, 2513-2525. EN 30   EN The appropriate conventional counterpart fo r stacked events shal l be selected in 
accordance with the principles defined previo usly in the present section. In addition, 
single parental GM lines or GM lines contai ning previously stacked events that have 
been fully risk assessed may also be included as additional comparators. The 
applicant shall provide detailed information justifying the choice of 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 protocol s for the experimental design 
(a) Principles of experimental design 
Field trials used for production of material  for the comparative analysis shall be 
performed in order to assess similarities and differences between three test materials: the GM plant, its conventional counterpar t (selected in accordance to section 1.3.1) 
and reference varieties: the objective shall be to determine whether the GM plant and/or derived food and feed is different from its conventional counterpart and/or 
equivalent to reference varietie s with a history of safe use. 
For each endpoint, the comparative analysis shall involve two approaches:  (i) a proof of difference, to  verify whether the GM pl ant is different from its 
conventional counterpart and might therefor e be considered a hazard (potential 
risk) depending on the type of the identif ied difference, extent and pattern on 
exposure; and  
(ii) a proof of equivalence to  verify whether the GM plan t is equivalent or not to 
reference varieties with a history of safe  use, apart from the introduced trait(s). 
In testing for difference the null hypothe sis shall that ther e is no difference 
between the GMO and its conventional counterpart against the alternative 
hypothesis that a difference exists. In testing for eq uivalence the null 
hypothesis shall that the difference between the GMO and the set of reference 
varieties is at least as great as a specified minimu m size (see section 1.3.2.2) 
against the alternative hypothesis that there is no difference or a smaller 
difference than the specified minimu m between the GMO and the set of 
reference varieties. Rejection of the null hypothesis is re quired 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 repr esent appropriately the range of natural variation 
expected for reference varieties with a history of safe use.  
Natural variation may have several sources: variation within a variety arises due to 
environmental factors and vari ation between varieties arises  due to a combination of 
both genetic and environmental factors. In order to identify and estimate differences 
attributable only to genotype s it is essential to control environmental variability. 
Therefore reference varieties sh all be included in the experi mental design of the field 
trials and in sufficient numbers to ensure  an adequate estimate of the variability 
required to set the equiva lence limits. All test mate rial (GMO, conventional 
counterpart, reference varieties and additiona l comparator(s) where appropriate) shall 
be randomised to plots within a single fi eld at each site, usually in a completely EN 31   EN randomised or randomised block experimental  design. The different sites selected for 
the trials shall be representative of the range of receiving environments where the 
crop will be grown, thereby reflecting relevant meteorological, soil and agronomic conditions; the choice shall be explicitly ju stified. The choice of reference varieties 
shall be appropriate for the chosen sites and shall be justified explicitly. In the case that sites cover a very restricted geogra phic range, the applican t shall replicate the 
trials over more than one year.  
This experimental design aims at maxi mizing the efficiency within available 
resources and providing suffi cient statistical power for a wide variety of endpoints 
with differing variability. 
(b) Specific protocols for experimental design 
Within each site the test materials (G M plants, conventional counterpart and 
additional comparator(s), where appropriate) 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 reference vari eties of the crop that have a known history 
of safe use. The number of distinct test  materials plus the number of reference 
varieties shall be denoted by t. For example, if there are the GM plant, the conventional counterpart plus four refere nce varieties, then t=6. The number of 
results to be obtained for each test material  and reference variety at each site (the 
replication) shall be denoted  as r. The minimum level of  replication shall be an 
integer greater or equal to [ 15/(t-1)]+1. For example, if t= 5 (the minimum value) then 
r, the replication, shall be at least 5; if t=6 then r shall be at least 4, etc. 
Notwithstanding these rules, the replication for a field trial shall never be less than 
r=4 at any site. 
Each field trial shall be replicated at a minimum of eight sites, chosen to be 
representative of the range of likely receiving environm ents where the plant will be 
grown. The trials may be conducted in a single year, or spread over multiple years. 
The reference varieties may vary between s ites and at least six different reference 
varieties shall be used over the entire set of trials.  
When the GM plant is tested together with other GM plants of the same crop species 
(e.g. Zea mays ) the production of material for the comparative assessment of these 
different GM plant may be produced simultane ously at the same site and within the 
same field trial by the placing of the di fferent GM plants and their appropriate 
comparator(s) in the same randomised block.  This shall be subject to two strict 
conditions:  
(i) each of the appropriate comparator(s) shall always occur together with its 
particular GM plant in the same block; and  
(ii) all the different GM plants and their comparator (s) and all the reference 
varieties used to test equivalence w ith those GM plants shall be fully 
randomized within each block. 
If the number of plots per block required for such a trial were  to exceed 16, then a 
partially balanced incomplete block desi gn may be used, to reduce the number of 
plots per block, by excluding some of th e GM plants and their appropriate 
comparator(s) from each block. This shall be subject to two strict conditions:  EN 32   EN (i) each of the appropriate comparator(s) shall always occur together with its 
particular GM plant in the same block and  
(ii) all of the reference vari eties shall appear in each of  the incomplete blocks and 
be fully randomised with the GM 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 cultiva tion/environmental conditions during growth 
and time of harvest, as well as the conditions during storage of the harvested material. 
Further information for the application of the requirements of this section is available 
in the EFSA report on "Statistical considerations for the safety evaluation of GMOs"
15. 
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 logari thmic transformation may be appropriate. In such cases, 
any difference between the GM and any other te st material is interpreted as a ratio on 
the natural scale. However, for other e ndpoints the logarithmic transformation may 
not be optimal and the natural scale or another scale may be more suitable.  
The total variability of  each endpoint observe d in the field trials shall be estimated 
and partitioned using appropria te 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 commercial va rieties. One set of confidence limits is 
used in the test of difference; the other se t and the equivalence limits are used in the 
test of equivalence.   
A linear mixed statistical model (denoted model 1) shall be used for calculation of 
the confidence limits for both tests (differe nce and equivalence); a slightly different 
model (model 2) shall be used to estimate the equivalence limits to be used in the equivalence test.   
Denote by I an indicator variable (uncentere d in the mixed model) such that I=1 for a 
field plot having any of the commercial varieties, and I=0 otherwise.  Then the 
random factors for model 1 should include, but not necessarily be restricted to, those 
representing the variation: (i ) between the test materials (a set that includes the GM 
crop, its conventional counterpart, each of the commercial varieties and any 
additional comparators); (ii)  in the interaction between  the test materials and I; (iii) 
between sites; and (iv) between blocks with in sites.  Model 2 s hould be identical to 
                                                
 
15 EFSA report on "Statistical considerations for th e safety evaluation of GMOs" [NB: reference to be 
modified when report published] EN 33   EN model 1 except that the random factor repr esenting the interaction between the test 
materials and I is omitted.   
The fixed factor for both models shall 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 crop;  its conventional coun terpart; the set of 
commercial varieties; and any additional test materials.  The set of commercial varieties shall be considered as a single le vel of the fixed factor.  For the difference 
and equivalence tests, the component of the fixed factor of interest is the single 
degree-of-freedom contrast between, resp ectively, the GM crop and its conventional 
counterpart, and the GM crop and th e set of commercial varieties. 
Both the difference test and the equivalence test are implemented using the well-
known 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) met hodology (e.g. Schuirmann, 1987) by rejecting 
the null hypothesis of non-equivalence when  the both confidence limits fall between 
the equivalence limits. The choice of 90%  confidence limits corresponds to the 
customary 95% level for statis tical testing of equivalence
. 
For each endpoint, calculation of the conf idence limits, estimation of equivalence 
limits and associated statistical tests shall be performed as described below, using the 
following notation.  Sample means are denoted by m, with subscripts G, C and R for 
the GM crop, its conventional counterpart and the set of commercial (reference) 
varieties, respectively.  The variability encompassed in the standard error of the 
difference between the means of any two te st materials, X and Y, calculated using 
model i (i = 1,2), is denoted sed(XY; i).  The 100 a% point of Student's t distribution 
is denoted as t(df;i;a), where i denotes the model used and df is the appropriate 
number of degrees of freedom which is  recommended to be calculated by the 
Kenward-Roger method. The least significan t difference between the means of any 
two test materials, X and Y, using model i, shall be calculated as the product of 
t(df;i;a) and sed(XY; i), and is denoted lsd(XY; i;a).   
For the difference test, the two-sided 90% c onfidence limits shall be calculated about 
mG, as mG ± lsd(GC;1;95); the null hypothe sis of equality between mG and mC shall be 
rejected and the test deemed statistically significant if mC falls outside these limits.  
For the equivalence test, the two-sided 95% equivalence limits shall be estimated as m
R ± lsd(GR;2;97.5) and two-sided 90% confiden ce limits shall be calculated about 
mG, as mG ± lsd(GR;1;95); the null hypothesis of non- equivalence shall be rejected 
and the test deemed statistically significan t if and only if the confidence limits lie 
entirely inside the equivalence limits. 
It is convenient to assess visually the quant ities involved in the a bove tests for all the 
endpoints simultaneously, on a single graph or a few graphs. This may be done by 
shifting all relevant values for each particular endpoint to a scale that has mC, the 
mean of the conventional coun terpart for that endpoint, as its baseline zero value. 
Therefore, on this new scale, the valu es of the means of the GM crop, its 
conventional counterpart and the set of comm ercial varieties, become, respectively:  
mG - mC, 0, mR - mC.   EN 34   EN The confidence limits for the difference test become: mG - mC ± lsd(GC;1;95), the 
equivalence limits   mR - mC ± lsd(GR;2;97.5), and the confidence limits for the 
equivalence test mG - mC ± lsd(GR;1;95).  To facilitate vi sual interpretation, instead 
of using the two sets of confidence limits  in the graphs, only one, that for the 
difference test, shall be displayed. Without  some adjustment, the confidence limits 
for the difference test would not give a valid  visual representation for the equivalence 
test on the graph. This problem is ove rcome by making an adjustment to the 
displayed equivalence limits. After this adjustment the displayed confidence limits for the difference test may be used as a basi s also for the visual representation of the 
equivalence test. In this way, one confid ence limit may serve visually for assessing 
the outcome of both tests simultaneously. Th e adjustment of the equivalence limits 
consists of two steps: (1) scaling the basi c equivalence limits, so that the confidence 
limits required for the difference and equiva lence tests have the same width; and (2) 
an appropriate shift to facilitate displa y of the adjusted limits, together with m
G, on 
the scale that has m C as its baseline zero value. Th e adjusted equivalence limits for 
visual display shall be ca lculated by the formula:  
(mG - m C)   +   {[(m R - m G) ±  lsd(GR;2;97.5)] lsd(GC;1;95) / lsd(GR;1;95)} 
 
The graph shall show the line of zer o difference between the GM and its 
conventional counterpart and, for each endpoint: the lower and upper adjusted equivalence limits, the mean difference between the GM and its conventional 
counterpart, and the confidence limits for this difference (see the set of possible 
outcomes for a single endpoint in Figure 1). When, in addition to the conventional 
counterpart, another test mate rial is used as comparator , the mean difference between 
the GM and that comparator, its confidence limits and its adjusted equivalence limits 
shall be displayed on the same graph referred to above, for all such additional 
comparators, by referring this to the same zero baseline as defined by the 
conventional counterpart. Note that 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 tran sformation, changes of 2x and ½x will appear 
equally spaced on either side of the line of zero difference. 
It is a consequence of the simplified graphical display that confidence limits for the 
difference test were chosen as 90%, yiel ding a 10% size for th e difference test, in 
which 1 in 10 of such tests is expected to  yield a significant result by chance alone. 
Despite the expected proportion of spurious  significant differences, the applicant 
shall report and discuss all significant di fferences observed between the GM crop, its 
conventional counterpart and, where applicab le, any other test ma terial, focusing on 
their biological relevance (see s ection 3. on Risk Characterization). 
For reporting, full details shall be given fo r 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 it is compared, and variance components for 
the random factors, (e) estimated degrees of freedom, (f) any other relevant statistics. 
The likely impact of other growing conditi ons not tested in the trial shall be 
discussed. EN 35   EN  
Figure 1. Simplified version of a graph for co mparative assessment showing the 7 outcome 
types possible for a single endpoint. After adju stment of the equivalence limits, a single 
confidence limit (for the difference) serves vi sually for assessing the outcome of both tests 
(difference and equivalence).  Here, only the u pper adjusted equivalence limit is considered. 
Shown are: the mean of the GM crop on an ap propriate scale (square),  the confidence limits 
(whiskers) for the difference between the GM  crop and its conventional counterpart (bar 
shows confidence interval), a vertical line indi cating zero difference (for  proof of difference), 
and vertical lines indicating adjusted equivale nce limits (for proof 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 GM crop is different from  its conventional counterpart. Regarding 
interpretation of equivalence, four categories (i ) - (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 re jected.  See text for what a ppropriate conclusions may be 
drawn.  
Regarding proof of difference, each outcome from the graph shal l be categorised as follows 
and the respective appropriate conclusion shall be drawn. 
• Outcome types 1, 3 and 5: the confidence in terval bar overlaps with the line of no-
difference. The null hypothesis of no differen ce cannot be rejected and the appropriate 
conclusion is that there is insufficient evid ence that the GM cr op and its conventional 
counterpart differ.    
line of  
no  
differenceupper 
equivalence
limit  lower 
equivalence 
limit  outcome 
type 
 1  
2 
3  
4 
5 
6 
7 (i) 
(ii) 
(iii) 
(iv) 
equivalence 
category adjusted adjustedEN 36   EN • Outcome types 2, 4, 6 and 7: the confidence in terval bar does not overlap with the line 
of no-difference. The null hypo thesis of no difference mu st be rejected and the 
appropriate conclusion is that the GM crop is significantly different from its conventional counterpart.   
 
Regarding proof of equivalence, each outcome fro m the graph shall be categorised as follows, 
and the respective appropriate conclusion shall be drawn. 
• Outcome types 1 and 2 (categor y (i), Figure 1): both confid ence limits lie between the 
adjusted equivalence limits a nd the null hypothesis of non-eq uivalence is rejected. The 
appropriate conclusion is that the GM is equivalent to the set of commercial varieties. 
• Outcome types 3 and 4 (categor y (ii), Figure 1): the mean of  the GM crop lies between 
the adjusted equivalence limits, but the confid ence interval bar overlaps at least one of 
the adjusted equivalence limits on the gra ph. Non-equivalence canno t be rejected but 
the appropriate conclusion is that equi valence between the GM and the set of 
commercial varieties is more likely than not.  Further evaluation may be required. 
• Outcome types 5 and 6 (category (iii), Figure 1): the mean of the GM crop lies outside 
the adjusted equivalence limits, but the conf idence interval bar overlaps with at least 
one of the adjusted equivalence limits.  Non-equivalence cannot be rejected and the 
appropriate conclusion is that equivalence between the GM and the set of commercial 
varieties is less likely than not.  Further evaluation is required. 
• Outcome type 7 (category (iv), Figure 1) : both confidence limits lie outside the 
adjusted equivalence limits. The appropriate conclusion is that the evidence analysed 
here demonstrates non-equivalence between  the GM and the set of commercial 
varieties.  Further ev aluation is required.  
 
In case of significant difference and/or la ck of equivalence for any particular 
endpoint, further analysis shall be done to  assess whether there are interactions 
between any of the test ma terials and site, possibly us ing a simple standard ANOVA 
approach. Whatever approach is adopted, details shall be given, for each endpoint 
analysed, listing: (a) the assumptions underlying the anal ysis, and, when appropriate: 
(b) degrees of freedom, (c) the estimated  residual variation for each source of 
variation, and variance com ponents, (d) any other relevant  statistics. These additional 
analyses are intended to aid the interpretation of a ny significant differences found 
and to study potential interactions betw een test materials and other factors. 
 Further information for the appli cation of the requirements of this  section is available in the 
EFSA report on "Statistical considerati ons for the safety evaluation of GMOs"
16.  
                                                 
16 EFSA report on "Statistical considerations for the safety evaluation of GMOs" 
[http://www.efsa.europa.eu/cs/Satellite] EN 37   EN 1.3.3. Selection of material and compounds for analysis 
Analysis of the composition is crucial wh en comparing the GM food and feed with 
its conventional counterpart. Th e material to be used for the comparative assessment 
shall be selected while taking into account the uses of the GM plant and the nature of 
the genetic modification. Unless duly justif ied, 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 (food and feed, food ingr edients, 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 sa mpling, analysis and preparation of the 
tested material shall be carried out acc ording to appropriate quality standards. 
1.3.4. Comparative analysis of composition 
Besides the analysis on the level of th e 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 id entified allergens, as well as other 
secondary plant metabolites characteristic fo r specific crop plant sp ecies as referred 
to in the OECD consensus documents on compositional considerations for new plant varieties (OECD consensus documents)
17. 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 specifi c analyses required shall depend on the 
plant species examined, but shall include a detailed assessment appropriate to the 
intended effect of the genetic modificati on, 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, vita mins and minerals. For example, a fatty 
acid profile shall be included for oil-rich  plants (main individual saturated, mono-
unsaturated and poly-unsaturated fatty aci ds) and an amino acid profile (individual 
protein amino acids and main non-protei n amino acids) for plants used as an 
important protein source. Measures of pl ant cell wall components are also required 
for the vegetative parts of plants used for feed purposes. 
The applicant shall also provide analysis on key toxins inherently present in the 
recipient plant which may a dversely affect human/anima l health depending on their 
toxic potency and levels. The concentrati ons 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 metabolite s of potentially modified metabolic pathways. The 
applicant shall consider when  appropriate) the inclusion of compounds other than the 
key nutrients, key toxins, and anti-nutrients and allergen s identified by the OECD 
consensus documents and justify th e selection of these compounds.  
                                                
 
17 OECD consensus documents on compositional considerations for new plant varieties being published in 
the Series on the Safety of Novel Foods and Feeds. EN 38   EN The same conditions apply for events stacked by conventional crossing. Where 
appropriate, additiona l compounds may be selected fo r analysis depending upon the 
introduced traits. 
1.3.5. Comparative analysis of agronomic  and phenotypic characteristics 
The applicant shall provide a comparison be tween the GM plant and its conventional 
counterpart. This comparison shall enable th e applicant to identif y unintended effects 
during the risk assessment process a nd shall address also plant biology and 
agronomic traits, including common br eeding parameters (e.g. yield, plant 
morphology, flowering time, day degrees to maturity, duration of pollen viability, 
response to plant pathogens and insect pest s, sensitivity to abiotic stress). The 
protocols of these field trials shall follo w the specifications made under section 1.3.2 
Where events are stacked by conventional crossing there may also be changes to 
agronomic and phenotypic characteristic s. Possible differences in phenotypic 
characteristics and agronomic properties of stacks shall be assessed in field trials 
over at least one season. Where appropria te, additional information on agronomic 
traits of the stacked even ts shall be provided from  additional field trials. 
1.3.6. Potential risk associated w ith horizontal gene transfer 
The applicant shall assess any potential risk associated with horizontal gene transfer 
from the product to humans, animals a nd micro-organisms when intact and 
functional DNA remains in the GM food and feed.  
1.3.7. Effects of processing 
The applicant shall assess whether or not the processing and/or preserving 
technologies applied are likel y to modify the characteri stics of GM end products 
compared with their respectiv e conventional counterpart. The applicant shall provide 
a description of the different processing te chnologies in suffici ent 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  (e.g. nutritionally 
enhanced foods, functional foods). Processed products may be assessed together with 
the assessment of the GM plant for the sa fety of the genetic modification, or a 
processed product may be assessed separa tely. The applicant shall provide the 
scientific rationale fo r the risk assessment of these products. On a case-by-case basis, 
experimental data may be required. 
When appropriate, depending on the product,  information shall be necessary on the 
composition, level of undesira ble 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 el imination, denaturation and/ or degradation of these 
protein(s) in the final product. EN 39   EN 1.3.8. Conclusion 
The conclusion of the comparative analysis shall clearly state: 
(a) whether agronomic and phenotypic charac teristics of the GM plant are, except 
for the introduced trait(s), different to the characteristics of its conventional 
counterpart and/or equivalent to the re ference varieties, taking into account 
natural variation;  
(b) whether compositional characteristics of  the GM food and feed are, taking into 
account natural variation, different to th e characteristics of its conventional 
counterpart and/or equivalent to th e reference varieties, except for the 
introduced trait(s); 
(c) characteristics for which the GM plant or the GM food and f eed are different to 
the characteristics of its conventional counterpart and/or equivalent to the 
reference varieties taking into account  natural variation, which need further 
investigation; 
(d) whether, in the case of events st acked by traditional crossing, there are 
indications of interactions between the combined events. 
1.4. Toxicology  
The toxicological impact of any changes re sulting from the expression of introduced 
genes or any other type of genetic m odification, (e.g. gene silencing or over-
expression of an endogenous gene) shall be assessed. 
Toxicological assessment shall be performed: (a) to identify, adverse effects of single compounds and determine the highest dose 
level(s) that do not result in adverse effects. From data obtained from an 
appropriate animal study an acceptable daily intake (ADI) for humans may be derived by using uncertainty or safety fact ors that take into account differences 
between test animal species and humans,  and inter-individua l variations among 
humans.  
(b) to demonstrate that the intended eff ect(s) of the genetic modification has no 
adverse effects on human and animal health.  
(c) to demonstrate that unintended eff ect(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.  
The applicant shall consider the need of toxicological testing based on the outcome 
of the molecular and comp arative analysis referred  to in section 2.2 and 2.3, i.e. the 
differences identified between the GM product and its conventional counterpart, 
including intended as well as unintended changes. For the purposes of this 
Regulation the applicant shall take into acc ount the presence of (a) newly expressed 
proteins, (b) the potential presence of other new constituents and/or (c) possible EN 40   EN changes in the level of natural constituen ts beyond normal variation. The specific 
information requirements and testing strategi es are outlined in the following sections. 
As regards GM food and feed produced from  GM plants, further toxicological tests 
with the processed products  shall not be pr ovided under the c ondition that the 
applicant provides an assessment of the GM plant (or relevant parts of it) 
demonstrating its safety and provided that there are no indications that the processed 
GM food and feed would be any differe nt from their respective conventional 
counterpart. The applicant shall provide adequa te justification in th is regard. This is 
also the case when the product is assesse d separately and there is no reason to 
suspect that it would be any different from its conventiona l counterpart (e.g. oil from 
insect protected cottonseed). 
In case the applicant consid ers that a decision on safety can be taken without 
conducting some of the tests recommended in this chapter and/or that other tests are 
more appropriate it shall state the reasons for not submitting the required or recommended studies or for carrying out st udies other than those mentioned below. 
Toxicology studies designed to evaluate risks to human a nd/or animal health shall 
complement each other. Most studies reco mmended for the assessment of the safety 
of the GM food are relevant for the asse ssment of GM feed. Testing methodologies 
are basically the same and the same leve l of data quality shall be provided. 
Besides the exposure of consumers and anim als through intake of food and feed, any 
adverse effect(s) on individuals  that could be due to th eir exposure to GM food and 
feed material as part of their professi onal activities e.g. farming, seed processing 
shall be reported by the applicant. Appropria te 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 as described by the OECD (see Tables  1 and 2 of section 1.7). Adaptations of 
these protocols or use of any methods th at differ from such protocols shall be 
justified. 
1.4.1. Testing of newly expressed proteins 
The applicant shall provide an evaluati on of all newly expressed proteins. The 
studies required to investigate the potenti al toxicity of a ne wly expressed protein 
shall be selected on a case-by-case basis,  depending on the know ledge available with 
respect to the protein's source, functio n/activity and history of human/animal 
consumption. As regards proteins expressed in the GM plant, in the case where the history of safe use for consumption as f ood and feed of both the plant and the newly 
expressed proteins is duly documented
18, specific toxicity tes ting as provided for in 
the following paragraphs of this  section shall not be required. 
                                                 
18  See, in particular, Constable A, Jonas D, Cockburn A, Davi A, Edwards G, Hepburn P, Herouet-
Guicheney C, Knowles M, Moseley B, Oberdörfer R, Samuels F. History of safe use as applied to the 
safety assessment of novel foods and foods derive d from genetically modified organisms. Food Chem 
Toxicol. 2007 45, 2513-2525. EN 41   EN Where specific testing is required, the tested  protein shall be equi valent to the newly 
expressed protein as it is expressed in the GM  plant. If, due to the lack of sufficient 
amount of test materials (e.g. plant protei ns), a protein produced by micro-organisms 
is used, the structural, bi ochemical and functional equi valence of this microbial 
substitute to the newly expressed plant prot ein shall be demonstr ated. 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. 
To demonstrate the safety of newly expre ssed proteins, the app licant shall provide: 
(a) A molecular and biochemical characte risation of the newl y expressed protein, 
including determination of the primary st ructure, molecular weight (e.g. using 
mass spectrometry), studies on post-tr anslational modifications and a 
description of the functi on. In the case of ne wly expressed enzymes, 
information on the enzyme activities in cluding the temperature and pH range 
for optimum activity, substrate specific ity, and possible reaction products shall 
also be provided. The potential interac tion with other plant constituents shall 
also be evaluated. 
(b) An up-to-date search for homology to pr oteins known to cause adverse effects, 
e.g. toxic proteins. A s earch for homology to pr oteins exerting a normal 
metabolic or structural function may also  contribute valuable information. The 
database(s) and the methodology used to ca rry out the search shall be specified. 
(c) A description of the stability of the protein under processing and storage 
conditions and the expected treatment of  the food and feed. The influences of 
temperature and pH changes shall be ex amined and potential modification(s) of 
the proteins (e.g. denaturation) and/or production of stable protein fragments 
generated through such treatme nts shall be characterised. 
(d) Data concerning the resistance of the newly expressed protein to proteolytic 
enzymes (e.g. pepsin), e.g. by in vitro  investigations using appropriate and 
standardised tests. Stab le breakdown products shall be characterised and 
evaluated with regard to the potential risks linked to their biological activity. 
(e) A repeated dose 28-day or al toxicity study with the newly expresse d 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 expre ssed proteins of GM plants is of little 
additional value for the risk assessment of the repeated human and animal consumption of GM food and feed and is therefore discouraged.  
The applicant shall perform st udies with combined admini stration of proteins when 
the genetic modification results in the expre ssion of two or more proteins in the GM 
plant and when, based on scientific knowle dge, a possibility of synergistic or 
antagonistic interactions of sa fety concerns is identified..  EN 42   EN 1.4.2. Testing of new constituen ts other than proteins  
The applicant shall provide a safety assessment of identified new constituents other 
than proteins. This shall include, on a cas e-by-case basis an eval uation of their toxic 
potency and of the need of toxicological te sting as well as a determination of their 
concentration in the GM food and feed. To establish the safety of new constituents 
having no history of safe use for consump tion in food and feed, the applicant shall 
provide information analogous to that described in the Guidance on submissions for food additive evaluations by the Scientif ic Committee on Foods” and Commission 
Regulation (EC) No 429/ 2008 of 25 April 2008 on detailed rules for the 
implementation of Regulation (EC) No 1831/ 2003 of the European Parliament and of 
the Council as regards the pr eparation and the presentation  of applications and the 
assessment and the authorisation of feed additives
19. This shall include the 
submission of information on a co re set of studies such as on 
metabolism/toxicokinetics, sub-chronic t oxicity, genotoxicity, chronic toxicity, 
carcinogenicity and reproduction and devel opmental toxicity , accompanied by any 
other appropriate type of st udy. (for specific OECD guidelines for animal tests, see 
Table 1 of section 1.7). Genotoxicity test pr otocols are provided for in Table 2 of 
section 1.7. 
1.4.3. Information on natural f ood and feed constituents 
The present section shall apply only in the case where the intended or unintended 
effect of the modification would result in an  alteration of the content of such natural 
food and feed constituents beyond the natural variation. 
To demonstrate the safety of the altered co ntent of natural food a nd feed constituents 
such as macro- and micronutrients, anti-nutrie nts, 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 fu nction and/or toxic properties of these 
constituents. The result of this assessment shall determine if, and to what extent, the 
applicant shall provide toxicological tests. 
1.4.4. Testing of the whole GM food and feed  
The applicant shall primarily base its risk assessment of the GM plant and derived 
food and feed on molecular characterisati on, comparative agrono mic, phenotypic and 
comprehensive compositional analysis, a nd the toxicological evaluation of the 
identified intended and unintended effect s. Under the circumstances presented 
hereunder, specific toxicological studies w ith the whole GM food and feed shall be 
carried out.  
1.4.4.1. 90-day feeding study in rodents 
The applicant shall adapt the OECD 90-day rodent toxicity study for the purposes of 
conducting the 90-day rodent feeding study for assessment of the safety and 
nutritional properties of the GM food and feed (see Table 1 of section 2.7). The aim 
of the study shall be to establish whethe r the GM food and feed is as safe and 
                                                 
19 OJ L 133, 22.5.2008, p. 1. EN 43   EN nutritious as its traditional comparator, a nd to demonstrate the absence of unintended 
effects of toxicological concern. 
Further information on the interests and li mitations as regards the 90-day rodent 
feeding study is available in the Report  of the EFSA GMO Panel Working Group on 
Animal Feeding Trials20.  
When such studies are conducted, the contro l diet(s) shall incl ude the conventional 
counterpart and where appropria te additional comparator(s). 
The applicant shall include a 90-day feeding study in rodents in the following cases: 
(a) GM plants with extensive in tended genetic modifications  
 In case the composition of the GM plant is modified substantially, the testing 
program shall include at least a 90-day feeding study in rodents.  
 Examples of GM plants that have b een modified substantially include GM 
plants which have been extensively modified in order to cope with 
environmental stress conditions like dr ought or high salt conditions, and GM 
plants with quality or output traits with the purpose to improve human or 
animal nutrition and/or health. In the case of insertion of multiple genes or 
gene cassettes, the applicant shall pay at tention to the fact that the internal 
metabolism in these GM plants may ha ve changed signifi cantly, leading to 
profound compositional alterati ons which may have an impact on the health or 
nutritional status of the consumer. More over the applicant sh all also consider 
that besides intended alterations in the composition, unintended and 
unpredicted changes may take place, whic h may not always be detected by the 
usual compositional analyses of major macro and micro nutrients, or naturally occurring toxins, and which may impact on human/animal health or nutritional status.  
(b) Indications for unintended effects or remaining uncertain ties in risk assessment 
 If there are indications or remaining uncertainties regarding the potential 
occurrence of unintended effects, based on the preceding molecular, 
agronomical, phenotypical and/or compos itional analysis, the applicant shall 
include in the testing program at leas t a 90-day toxicity study in rodents. 
 Indications for unintended effect s from molecular characterization 
 The molecular characterisation of the GM event shall specifically identify 
whether the event(s) raise(s) any issues regarding the potential for alterations in 
metabolic pathways which may have a negative impact on the safety and nutritional value of the GM  plant and derived food a nd feed like for instance 
the production of new toxins. 
                                                
 
20 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 EN 44   EN  These indications are in particular  obtained from the information on the 
sequences actually inserted/deleted in the GM plant, on the organisation of the 
inserted genetic material at the inse rtion site, and sequence information on 
flanking regions that may indicate po ssible interruptions of known ORF or 
regulatory regions and/or on the pot ential for producing novel chimeric 
proteins.  
 Indications for unintended effect s from the comparative analysis 
 Each of the outcomes of the comparat ive analysis shall be evaluated with 
respect to the possible impact on the safety  and/or nutritional  properties of the 
GM plant, in particular those situations where differences of composition between the GM plant and its conventional counterpart  have been observed and 
where equivalence cannot unambiguously be  established. The applicant shall 
assess the information on the type a nd function of the test compound, its 
relevance for human/animal health (essen tial nutrient), and its toxicological 
profile. The outcome of this assessment shall determine whether animal feeding trials with the whole f ood and feed shall be performed. 
(c) Indications of potential adverse interactions in stacked events 
 In the case of stacked events, possi ble interactions between the expressed 
proteins, new metabolites and original plant constituents shall be assessed. Indications for possible adverse interac tions shall be obtained from (i) the 
outcome of the molecular characterizati on, (ii) the knowledge of the mode of 
action of the newly expressed proteins, (iii) information on the response to 
combined administration of the proteins, and (iv) information on the effects on the activity of target enzymes.  
1.4.4.2. Animal studies with respect  to reproductive, and deve lopmental toxicity testing 
Given that the subchronic 90-day rodent feeding study is not designed to detect 
effects on reproduction or development, other than effects on adult reproductive 
organ weights and histopathology, the app licant shall, where appropriate, conduct 
testing of the whole food and feed beyond a 90-day rodent feeding study. 
In cases of indications of adverse effects from the subc hronic study (e.g. functional 
and/or histological modifications of  nervous, endocrine, reproductive or 
immunological tissues/organs) or other information on the GM food and feed suggest 
the potential for reproductive,  developmental or chronic toxicity, the performance of 
such testing shall be assessed by the a pplicant. OECD protoc ols for reproductive, 
developmental and chronic toxicity test ing (see Table 1 of section 1.7) can be 
adapted for the purposes of tes ting the whole GM food and feed. 
1.4.4.3. Other animal studies to examine the safe ty and the characteri stics of GM food and 
feed (see also sections 1.6.1 and 1.6.2) 
Supplemental information to 90-day feed ing studies in rodents on the possible 
occurrence of unintended effects may be obt ained from comparative growth studies 
conducted with young rapidly growing animal  species (broiler chicks as animal 
model for non-ruminants; lambs for rumina nts; or other rapidl y growing species). EN 45   EN Studies of this type are limited to those ma terials suitable for inclusion in their diets 
and which can be nutritionally matc hed to a suitable control diet. 
Livestock feeding studies with target an imal species shall be considered by the 
applicant, on a case-by-case basis and be hypothesis driven . The focus shall be on the 
safety of newly expressed constituents, on the identification and characterisation of 
unintended effects, and on the nutritional im pact of any intenti onal, substantial, 
compositional modifications of the GM plant (see also section 1.6).  
1.4.4.4. Interpretation of rele vance of animal studies 
Any effects observed in the animal trials shall be evaluated by experts in order to 
identify relevant effects with respect to potential consequences for the health of humans and animals and to assess their re levance for the safety of food and feed 
derived from the GM product. This ev aluation may be supported by additional 
information and considerations. Informati on on the background variability in a given 
parameter may be obtained by the applican t from data from other animals of the 
same species/strain tested in the same or other experiments, or from internationally 
harmonised databases. If the change observed in a certain parameter falls within this 
background range of variabilit y, further considerations sh all be provided from the 
applicant with respect to a dose-response relationship, gender specificity, linkage 
with other changes, or any plausible cause.  
The applicant shall in particular consider  dose-response relationships in parameters 
that have been changed (i.e. commensurate  increases in changes at increased doses) 
since they provide a strong indication for an effect of the tested  compound. The 
absence of such a dose-response relations hip may indicate that the effect is 
accidental or spurious.  
In tests where animals of both genders are used, changes occurring in animals of one 
gender only may still be relevant indicator s 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 certain compound than animals of the other gender, such as in the case of endocrine effects. 
The applicant shall also identify possibl e inter-relationships between observed 
changes in single parameters which may stre ngthen 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, an d organ weights, may also be evident 
from the changed levels of certain live r-derived compounds, such as enzymes, 
bilirubin, etc., in serum. 
With regard to the potential cause for an observed effect, the lik elihood of causality 
shall be taken into account, not only for th e test compound, but also for other factors 
that may have also influenced the out comes (e.g. 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. EN 46   EN 1.4.5. Conclusion of the to xicological 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 newl y expressed protein(s) and other new 
constituents resulting from the gene tic 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 c ounterpart 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 GM 
food and feed and at which dose levels; 
(e) the information provided and the testi ng strategy used to assess the intended 
and/or unintended changes of the GM f ood and feed are considered adequate. 
The applicant shall evaluate the result of the toxicological assessment in the light of 
anticipated intake of the GM food and feed (see section 2).  
1.5. Allergenicity 
For the purposes of this Regulation, the a pplicant shall focus its assessment on the 
individuals who have a gene tic predisposition to devel op allergic reactions when 
exposed to food and feed (and pollen) deri ved from GMOs. The applicant shall focus 
its assessment on those individuals as rega rds sensitisation or  elicitation of an 
allergic reaction. 
The majority of the constituents responsible for allergenicity of food and feed as well 
as of pollen are proteins. Some protein br eakdown products, i.e. peptide fragments, 
may conserve part of the allergenicity of  the native protein a nd thus can also be 
considered as allergens. Th e specific allergy risk of GM Os is associated i) with 
exposure to newly expressed protein(s) that  can be present in edible parts of the 
plants including pollen. This point is relate d to the biological s ource of the transgene 
and ii) with alterati ons to the allergenic ity of the whole plant and derived products 
e.g. due to over-expression of  natural endogenous allergen s as an unintended effect 
of the genetic modification. This point is related to the biology of the host itself.  
1.5.1. Assessment of allergenicity of  the newly expressed protein 
Given that allergenicity is not an intrin sic, fully predictable property of a given 
protein but is a biological act ivity requiring an interaction with individuals with a 
pre-disposed genetic background and that it depends upon the genetic diversity and 
variability in atopic humans, the applic ant shall provide a cumulative body of 
evidence which shall minimise any uncertain ty with regard to the protein(s) in 
question. In order to provide  this body of evidence, th e applicant shall follow an EN 47   EN integrated, stepwise, case-by-case approach  in line with the recommendations of the 
Codex ad hoc Intergovernmental Ta sk Force on Foods Derived from 
Biotechnology21 and that allows the assessment of  possible allergenicity of newly 
expressed proteins 
The applicant shall provide a thorough charac terisation of the source of the transgene 
enabling to identify whether it has the po tential to encode for an allergen. The 
provided information shall specify at which stage of the development of the plant and 
in which organs of the plant the allerg enic protein may be expressed. When the 
introduced genetic material is obtained from wh eat, rye, barley, oats or related cereal 
grains, the applicant shall assess the newly expressed proteins for a possible role in 
the elicitation of gluten-sensitive enteropa thy or other enteropa thies which are not 
IgE mediated.  
Where events have been stacked by c onventional crossing, th e 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 eff ects of the gene products.  
The first step in the assessment shall be a search for sequence homologies and/or 
structural similarities between the expr essed protein and known allergens using 
various algorithms to identify overall structural similarities. Strategies for 
identification of sequences that may correspond to pote ntial linear IgE binding 
epitopes shall be conducted by a search for id entical peptidic fragments in the amino 
acid sequence of the te st protein to peptidic frag ments of known allergens. The 
number of contiguous identical amino acid re sidues used in the search setting shall 
be based on a scientifically justified rationale in order to minimise the potential for false negative or false positive results. The use of different homology searching strategies based on the sequences available in relevant databases may identify several 
scenarios, including a high degree of homol ogy, with or without conservation of the 
allergenicity, or a low degree of homol ogy with conservation of allergenicity.  
The second step for assessing the potential  that exposure to the newly expressed 
proteins might elicit an al lergic reaction in individuals  already sensitised to cross 
reactive proteins, shall be based on in vitro  tests that measure the capacity of specific 
IgE from serum of allergic patien ts to bind the test protein(s). 
If the source of the introduced gene is  considered allerg enic, but no sequence 
homology of the newly expr essed protein to a known allergen is demonstrated, 
specific serum screening of the expresse d protein shall be undertaken with 
appropriate sera from patients allergic to the source materi al using relevant validated 
immunochemical tests. If a positive IgE res ponse occur, the newly expressed protein 
may be considered very likely to be alle rgenic. If no IgE binding is observed, the 
newly expressed protein shall undergo pepsin  resistance tests and additional testing. 
If the source is not known to be allergen ic but there are consis tent indications of 
sequence homology to a known allergen, th e specific serum screening shall be 
                                                
 
21 Codex Alimentarius, 2003. Codex principles and guidelines on foods derived from biotechnology. 
Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture 
Organisation, Rome. EN 48   EN conducted with sera from patient s sensitised to th is allergen in order to confirm or 
exclude an IgE cross-reactivity between the newly expressed protein and this 
allergen. The results of the screening shall be interpreted as in the previous paragraph. 
As a third step, the applicant shall consider the following additional tests: (a) Pepsin resistance test. Stability to digestion by proteolytic enzymes has long 
been considered a characteristic of allergenic proteins. Although it has now 
been established that no absolute corre lation exists, resistance of proteins to 
pepsin digestion is still proposed as an additional criterion to be considered in an overall risk assessment. In case that  a rapid and extensiv e degradation of a 
protein in the presence of pepsin is not confirmed under appropriate conditions, further analysis shall be conducted to  determine the likelihood of the newly 
expressed protein being allergenic. It c ould also be useful to compare intact, 
pepsin digested and heat dena tured proteins for IgE binding.  
(b) Targeted serum screening. Targeted serum screening aims to assess the 
capacity of the newly expressed protein to  bind to IgE in sera of individuals 
with clinically-va lidated allergic re sponses to categories of foods broadly 
related to the gene source. 
 Specific (as well as targeted) serum sc reening requires a sufficient number and 
sufficient volumes of relevant sera fr om allergic humans. These might not 
always be available either  because the allergy is no t frequent or for other 
reasons. The use of existing models and the development and validation of new alternative models that may substitute fo r and/or complement the use of human 
biological material for evidence of cross reactivity and elicit ation potency shall 
be considered. These appro aches would include the search for T-cell epitopes, 
structural motifs, in vitro cell based a ssays using animal or humanised-animal 
immune cells, etc. They also include appropriate in vivo  animal models. 
(c) Animal models. Animal models may also be useful tools for the assessment of 
the sensitising potential of newly expr essed proteins, i.e. their capacity to 
induce an allergic immune response w ith the synthesis of specific IgE in 
individuals that have never been exposed to those proteins nor to proteins that 
cross react with them.  
1.5.2. Assessment of allergenicity of  the whole GM plant or crop  
When the host of the introduced gene is know n to be allergenic, the applicant shall 
test any potential change in  the allergenicity of the whole GM food and feed by 
comparison of the allergen re pertoire with that of th e conventional counterpart.  
These approaches shall be applied on a case-by-case basis depending on the available 
information on the allergenic potential of the source and/or the host. The applicant 
may use modern analytical tools includi ng profiling techniques. These tools may 
provide, in association with human and anim al serum or cell-based assays, valuable 
additional information.  EN 49   EN The integrated process applies to the assessment of the allergenicity of the edible 
components including pollen of GM plants (i.e. covers both food and respiratory 
allergy risk). 
In addition, the applican t shall provide, where available, information on the 
prevalence of occupational a llergy in workers or in farmers who have significant 
exposure to GM plant and crops, or to th e airborne allergens they may contain. 
1.5.3. Conclusion of the allergenicity assessment 
The conclusion of the allergenic ity assessment shall indicate:  
– whether the novel protein(s) is  likely to be allergenic; 
– whether the GM food and feed is likely  to be more allergenic than its 
conventional counterpart. 
When there is a likelihood of allergenicity in one of the above mentioned cases, the 
GM food and feed shall be further characteri sed in the light of its  anticipated intake 
(see section 2) and the applicant shall pr opose appropriate cond itions for placing on 
the market, including labelling. 
1.6. Nutritional assessment 
The applicant shall provide nutritional evaluation: 
(a) to demonstrate that intr oduction of the GM food and f eed into the market is not 
nutritionally disadvantageous to humans  and animals, respectively. This 
evaluation shall include th e relevance for the nutrition of newly expressed 
proteins, other new constituents, and changes in the levels of natural constituents in the GM food and feed, as  well as potential alterations in the 
total diet of the consumer; and, 
(b) to demonstrate that unintended effect s of the genetic modi fication that were 
identified or that may be assumed to  have occurred based on the preceding 
molecular, compositional or phenotypic an alyses, in accordance with sections 
1.2 and 1.3, have not adversely affected the nutritional value of the GM food 
and feed. 
For stacked events combined by conventio nal breeding, an assessment of the 
potential changes in nutritional value that mi ght arise from synergistic or antagonistic 
effects of the gene produc ts including compositional ch anges shall be provided by 
the applicant. This may be particularly relevant where the combined expression of 
the newly introduced genes has unexpect ed effects on biochemical pathways. 
The nutritional assessment of GM food and feed shall consider: 
(a) the composition of the GM food and feed with regard to the levels of nutrients 
and anti-nutrients (see compositional studies as describe d in section 1.3); EN 50   EN (b) the bioavailability and biological effi cacy of nutrients in the food and feed 
taking into account the potential influences  of transport, storage and expected 
treatment of the foods; 
(c) the anticipated dietary intake of the food and feed (see section 2) and resulting 
nutritional impact. 
When the comparative analysis has identif ied compositional characteristics of the 
GM food and feed that are diffe rent and/or not equivalent to  the characteristics of its 
conventional counterpart, their nutritional re levance shall be assessed on the basis of 
current scientific knowledge. If this a ssessment does conclude to the nutritional 
equivalence between the GM food and feed and its conventional counterpart, no further studies shall be recommended. By c ontrast if, on the basi s of the assessment 
of the information obtained from the comparative analysis, it is not possible to conclude to nutritional equivalence, further studies shall be carried out. 
When the applicant is required to provide a 90-day feeding study in rodents using the 
whole GM food and feed in accordance with  section 1.4.4.1, the a pplicant shall also 
consider the information on nutritional aspects that is available since this study starts with juvenile animals in rapid growth phase  that are sensitive to effects on weight 
gain. Further information in this respect is available in the Report of the EFSA GMO Panel Working Group on Animal Feeding Trials
22.  
1.6.1. Nutritional assessment of GM 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. Supplemental information regarding the nutritional value may be obtained from co mparative growth performance studies 
conducted with other animal species, e.g. br oiler chickens, addre ssing the nutritional 
assessment of GM feed.  When nutritional studies are conducted, the control diet(s) 
shall include the conventional counterpa rt and where appropriate additional 
comparator(s). 
GM foods modified to provide additional health benefits to the consumer as 
compared to conventional foods, may bene fit specific populations  or sub-populations 
while others may be at risk from th e same food. In cases where an altered 
bioavailability needs to be established and may raise concern for sub-population(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 bioava ilability 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 specif ic population(s) anticipa ted to consume the food. 
                                                
 
22 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 EN 51   EN 1.6.2. Nutritional assessment of GM feed 
The applicant shall determine the necessity and design of furthe r nutritional studies 
on the basis of the introduced trait(s), th e outcome of the comparative analysis, and 
the 90-day feeding study, where available. 
In the case of GM feed with improved nut ritional characteristics , livestock feeding 
studies with target animal  species shall be conducted on a case-by-case basis to 
assess the impact on the feed. In the case of GM plants modified for improved 
content and bioavailability of nutrients, livestock studies wi th target species shall be 
conducted to determine the bioavailability of individual nutrients in the GM plant 
compared to its conventional counterpart and a range of conventiona l varieties. In the 
case of GM plants specifically modified w ith traits to enhance animal performance 
through increased nutrient dens ity (e.g. increased o il content) or an enhanced level of 
a specific nutrient (e.g. an e ssential amino acid or a vitami n), an appropriate control 
diet using its conventional counterpart shal l be formulated by supplementing it with 
the specific nutrient to the extent of the ch ange effected in the GM plant. Regarding 
co-products (e.g. oilseeds meals), from which the ingredient targeted by the genetic 
modification has been extracted, these may be compared with co-products derived 
from the conventional counterpart and ot her conventional vari eties as additional 
comparators (on the basis that all these pr oducts are low in the component targeted 
by the genetic modification). 
Target animal feeding studies shall span eith er 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 fo r laying hens or quails. For feedstuffs 
intended only for aquaculture, growth studies  with aquatic species such as carp or 
other typical herbivor es shall be chosen.  
When appropriate tests with various expe rimental designs shall be provided to 
demonstrate that the nutritionally improved GM plant fulfils the expected nutritional 
value. The exact experimental design and statistical feed approaches of feeding 
experiments in food producing animals to test the nutritional value of GM feed 
modified for enhanced nutritional characteri stics 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 quantit y and/or availability  of the nutrient in 
question. Endpoint measurements shall vary wi th the target species  used in the study, 
but shall include feed intake, body weight, an imal performance and bioavailability of 
nutrients. 
Further information for the application of the requirements of this section is available 
in the Report of the EFSA GMO Panel Working Group on Animal Feeding Trials23.  
1.6.3. Conclusion of the nutritional assessment 
The conclusion of the nutritional assessmen t of GM food and f eed shall indicate: 
                                                 
23 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 EN 52   EN – whether the GM food and feed is nutriti onally equivalent to its conventional 
counterpart, taking natural va riations into account. 
The applicant shall evaluate the result of the nutritional assessment in the light of 
anticipated intake of the GM food and feed (see section 2).  
1.7. Standardised guidelin es for toxicity tests 
The applicant shall use for toxicity testing internationally agreed protocols and test 
methods described by the OECD (see Tables 1 and 2). Use of any methods that differ 
from such protocols shall be justified. A non-exhaustive list of validated test 
protocols which, where necessary, shall be used in a possibly adapted form for GMO 
toxicological testing is provide d in Tables 1 and 2 below. 
The performance of test protocols depends on the type of GM f ood and feed, type of 
the genetic modification and resulting inte nded and unintended alterations, intended 
use and exposure/intake, and the availabl e knowledge. Some of  the tests were 
developed for the assessment of risks at the workplace. (See previous sections of this Annex). 
Table 1: Non-exhaustive list of  validated test pr otocols for chemicals as described by 
OECD guidelines and which may be used  in a possibly adapted form for GMO 
toxicological testing 
No. OECD Title  
407  Repeated Dose 28-day Oral Toxicity Study in 
Rodents  
408  Repeated Dose 90-Day Oral Toxicity Study in 
Rodents  
410  Repeated Dose Dermal Toxicity:21/28-Day  
415  One-Generation Reproduction Toxicity  
416  Two-Generation Reproduction Toxicity Study  
417  Toxicokinetics  
421  Reproduction/Developmental Toxicity Screening 
Test  
424  Neurotoxicity Study in Rodents  
451  Carcinogenicity Studies  
452  Chronic Toxicity Studies  
453  Combined Chronic Toxicity /Carcinogenicity Studies  
402  Acute Dermal Toxicity EN 53   EN 406  Skin Sensitisation  
 
Table 2: Genotoxicity tests as  described by OECD guidelines24 
No. Title 
OECD 471 Bacterial reve rse mutation test  
OECD 473 In vitro mammalian ch romosome aberration test  
OECD 474 Mammalian erythrocyte micronucleus test 
OECD 475 Mammalian bone marrow chromosome aberration test 
OECD 476 In vitro mammalian cell gene mutation test 
OECD 479 In vitro sister chromatid exchange (SCE) assay in 
mammalian cells 
OECD 480 Saccharomyces cerevisiae, gene mutation assay 
OECD 481 Saccharomyces cerevisiae, mitotic recombination assay 
OECD 482 DNA damage and repair, unscheduled DNA synthesis in 
mammalian cells in vitro  
OECD 487 Draft guideline on: 
In vitro mammalian cell micronucleus test 
 
2. E XPOSURE ASSESSMENT - ANTICIPATED INTAKE /EXTENT OF USE  
An estimate of the expected intake shal l be an essential element in the risk 
assessment of GM food and feed and shal l 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 GM plant-derived food and feed 
product(s). 
On the basis of representative consump tion data for products derived from the 
respective conventional plants , the applicant shall estima te the anticipated average 
and maximum intake of the GM food and f eed. Probabilistic methods may be used to 
determine ranges of plausible values rather than single values or point estimates. If 
possible, the applicant shall identify and consider particular sections of the 
population with an expected high exposure and shall within the risk assessment.  Any 
                                                 
24 OECD, 1995. OECD Guidelines fo r the Testing of Chemicals.  EN 54   EN assumptions made in the exposure a ssessment shall be described. Recent 
developments in methodologies and approp riate consumption data shall be used. 
Data on import and production quantities ma y provide additional information for the 
intake assessment.  
The applicant shall determine by appropria te methods the concentrations of the 
newly expressed proteins, other new constitu ents and natural cons tituents, of which 
the levels have been altered as a result of the genetic modification (e.g. due to changes in metabolic pathways) in those pa rts of the GM plant intended for food or 
feed use. Expected intake of these constitu ents shall be estimated taking into account 
the influences of processing, storage and exp ected treatment of the food and feed in 
question, e.g. potential accumulation or re duction. 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 GM 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. R
ISK CHARACTERISATION  
3.1. Introduction 
The applicant shall base its risk characterisation of GM plants and derived foods/feed 
on data from hazard identification, hazard characterisation, and on exposure/intake 
data. He 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 
GM food and feed. 
In performing his risk char acterisation, the applicant sh all demonstrate that the 
hazard identification and subsequent char acterisation are complete. The applicant 
shall discuss the quality of existing data and information. The discussion shall clearly 
indicate how this body of informati on has been taken in to account in the 
determination of the final risk characterisation.  
The applicant shall provide estimations of th e uncertainties associated to each test as 
well as to the different stages of the risk  assessment. He shall quantify them through 
proper statistical methods as much as possible.  Distinction shall be made between 
uncertainties that reflect natural variati ons in biological pa rameters (including 
variations in susceptibility in populations ), and possible differences in responses 
between species.  
Depending on the issue to be addressed and the available data, the applicant shall 
perform a qualitative and, wh ere possible, quantitative risk characterisation. The 
conditions for the estimated ri sk, and associated uncertainties, shall be as precise as EN 55   EN possible. For instance, expressions like ‘n o/negligible/acceptable/significant risk’ 
shall be accompanied by further numerical quantification in terms of probability of exposure and/or occurren ce of adverse effects.  
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  GM plants in an integrative  manner in accordance 
with section 3.1 above. This risk assessm ent shall be performed on a case-by-case 
basis depending on the modified plant a nd the type of genetic modification, the 
cultivation practices of the GM plant and uses of the GM food and feed. The 
applicant shall take into account the different issues considered in hazard identification and characterisation and expos ure 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 s ection shall not be exhaustive. 
3.2.1. Molecular characterisation 
Evaluation of the characteri stics and previous use of the donor and the recipient 
organism shall be a key element to iden tify the need for specific analyses e.g. 
occurrence of specific toxins, or allergens in the unmodified recipient plant which 
may be unintentionally increased as re sult of the genetic modification.  
Transformation protocols, molecular characterisation strategies and the specificity 
and sensitivity of the methods used shall be  discussed by the app licant in relation to 
the intentional and possibly unintentiona l insertion and expression of gene 
sequences. 
Where flanking sequence analysis has identified chimeric ORFs, the applicant shall 
demonstrate how approaches like bioinfor matic analysis, com positional/agronomical 
analysis and possibly animal feeding tria ls with the whole GM 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 specie s in question or related species. 
3.2.2. Comparative analysis  
The applicant shall demonstrate that the comparative analysis between the GM plant 
and its conventional counter part with respect to ag ronomic, morphological and 
compositional characteristics has been carried out according to this Regulation. The selection of the conventional counterpar t and additional comparators shall be 
justified in particular with respec t to their history of safe use. 
In performing his comparative safety asse ssment, the applicant shall identify possible 
differences between the GM plant and its conventional counterpart. The risk 
characterisation shall concen trate on statistically significant differences in the 
composition of the GM plant compared to its conventional count erpart and whether 
these differences are likely to have an im pact on food and feed safety or nutrition. 
Moreover, the applicant shall perform an anal ysis of the uncertainties associated with 
the comparative analysis. EN 56   EN Unintended effects may result in differences  or lack of equivalence that may be 
observed in field trials re presentative of the range of receiving environmental 
conditions. A difference or lack of equivale nce that is consistently observed under 
most conditions maybe an indicator of such an effect. Whilst sporadic differences or 
lack of equivalence  may reflect the inherent variability known to occur in the GM 
plant and the conventional counterpart or, for specific endpoints be due to chance 
alone, they may also highlight a strong infl uence of special environmental conditions 
on the expression of a difference. 
If statistically significant differences and/ or non-equivalences are observed, using the 
methodology as described under section 1.3.5, the applicant shall consider whether to 
provide the following background data and sh all put these observations into context 
with respect to their potential rele vance for the human/animal health. 
3.2.2.1. Data on variability inherent to the plan t, the plant variety and the environment. 
The applicant shall consider the range of levels observed for the compounds known 
to occur in the conventional counterpart and in reference varietie s. This variability 
may be caused by differences that ar e genotype-dependent, environmentally 
dependent, or caused by genot ype x environment interactio ns. In addition, the range 
of levels observed in a broa d spectrum of food a nd feed representative for the human 
and animal diet may be taken into account given that it reflect s the levels of the 
specific compound to which consumers may be exposed.  
3.2.2.2. Information of variation of constituents from databases. 
The applicant shall specify the databases used for comparison and adequately assess 
them for their quality (e.g. type of material analyze d, analytical method used, 
sampling methods and strategies). No formal statistical analysis shall be carried out, 
but ranges as well as mean values shall be reported and considered. These data would indicate whether the GM lines fall w ithin the natural range in component 
concentrations found in non-GM comparat ors. The influence of environmental 
factors on phenotypical and compositional charac teristics of plants shall be taken into 
account when comparing analytical data fr om field studies with literature data. 
Based upon the considerations above, the applicant shall esta blish whether the 
differences and/or lack of equivalence obs erved are to be considered relevant for 
further consideration in the risk assessment process or if the difference and/or lack of 
equivalence does not raise safety concerns. 
3.2.3. Food and feed safety in relation to intake  
The applicant shall evaluate the data ge nerated to estimate  possible risks to 
human/animal health associated with the consumption of GM plant derived 
foods/feed with respect to the expression of new proteins/metabolites as well as 
significantly altered levels of original plan t proteins/metabolites in GM foods/feed. If 
single constituents and/or whole GM f ood and feed are found to induce adverse 
effects in specific studies, dose response relationships, thres hold levels, delayed 
onset of adverse effects, risks for certain  groups in the population, use of uncertainly 
factors in extrapolation of animal data to humans shall be presented.  EN 57   EN The applicant shall provide adequate justification as regards the relevance of short-
term toxicity data in order to predict possible long-term adverse effects of newly 
expressed proteins/new metabolites in the GM food and feed as well as the absence 
of specific studies (e.g. on reproductive and developmental toxicity) if applicable. 
Moreover when feeding trials with whole GM food and feed have been carried out, 
the relevance of their outcome shall be evaluated with respec t to experimental 
limitations (e.g. dose range, dietar y composition, confounding factors). 
The applicant shall consider data on th e characteristics of the new compounds 
present in the GM plant including potential bi ological effects in humans and animals.  
If the compounds have known adverse health effects a nd maximum levels for the 
presence of these compounds in the plant or derived 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 c onsumers 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/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 modifi cations are produced, e.g. via transfer of 
multiple genes in a single construct, re-transformation of pre-existing GM lines, and trait stacking through conventional breeding of GM pa rents, the applicant shall 
discuss strategies for the assessment of  any risk(s) associated with possible 
interactions between the newly expresse d proteins, new metabolites and original 
plant constituents . A holistic approach for the assessment shall be demonstrated 
considering all available information on e.g. the mode of action of the newly expressed proteins, th e molecular and compositional/ag ronomical characteristics of 
the GM plant, and where applicable on the outcome of animal toxicity studies and feeding trials. Where animal feeding tria ls are not performed, the applicant shall 
provide an explanation as to why th ese were not considered necessary. 
The applicant shall evaluate data provided to assess the allergenic potential of newly 
expressed proteins in GM pl ants with respect to introduction of new allergenic 
proteins into the food and feed plants a po ssible provocation of a llergic reactions of 
susceptible individuals, as well as information to demonstrate that the genetic 
modification process does not cause unwante d changes in the characteristics and/or 
levels of expression of endogenous allergen ic proteins in the GM plant derived food. 
In particular the choice of the test models sh all be justified with respect to specificity, 
predictability and validation status. 
With respect to intake estimations of GM  foods, the applicant shall evaluate the 
applied methodologies with respect to uncerta inties associated with  the prediction of 
long-term intake. Specific attention shall be  paid to those GM foods which are aimed 
at modifying nutritional quality. For the GM  products in questions the requirement 
for post-market monitoring shall be di scussed as a necessary mechanism for EN 58   EN determining changes to overall dietary intake  patterns of the GM food, to what extent 
this has occurred and whether or not the product induces known (side) effects or 
unexpected side effects. If the performa nce of post-market monitoring is deemed 
necessary, the reliability, sensitivity and speci ficity of the proposed methods shall be 
provided. 
3.3. The result of risk characterisation  
In accordance with Articles 4 and 16 of Regulation (EC) No 1829/2003, the 
applicant shall ensure that the final risk characterisation clearly  demonstrates that: 
– The GM food and feed has no adverse effects on human and animal health ;  
– The GM food does not differ from the f ood which it is intended to replace to 
such an extent that its normal consumption would be nutritionally 
disadvantageous for the consumer; 
– The GM food does not mislead the consumer; 
– The GM feed does not harm or misl ead the consumer by impairing the 
distinctive features of the animal products; and, 
– The GM feed does not differ from the f eed which it is intended to replace to 
such an extent that its normal consumption would be nutritionally 
disadvantageous for animals and humans. 
The applicant shall clearly i ndicate what assumptions have  been made during the risk 
assessment in order to predict the probabil ity of occurrence and severity of adverse 
effect(s) in a given populat ion, and the nature and magnitude of uncertainties 
associated with establishing these risks. 
The applicant shall also include detailed info rmation justifying the inclusion or not of 
a proposal for labelling in the application, in accordance with Articles 5(3)(f) and 
17(3)(f) of Regulation (EC) No 1829/2003. EN 59   EN ANNEX III  
 
POST-MARKET MONITORING OF GM FOOD/FEED –  
The applicant shall submit a proposal for Post Market Monitoring (PMM): 
– when the safety assessment carried out in accordance with Article 4 of this 
Regulation concludes th at it is not possible to addr ess remaining uncertainties by 
collecting additional information within the framework of the safety assessment and; 
– when these uncertainties are too high with respect to the following aspects: 
(i) the food/feed consumption is difficult to predict or it is necessary to ensure that 
specific recommendations of uses  are followed by the consumer; 
(ii) the relevance and intensity of effect s and side-effects detected during the pre-
market risk assessment are difficult to be predicted,  and; 
(iii) potential side effects ar e identified but can not be studied in the framework of 
the safety assessment. 
The applicant shall in particular consider th e submission of a proposal for a PMM when the 
GM (functional) food/feed has altered nutritiona l composition and its nutritional value differs 
from the conventional food/f eed that it would replace. 
The applicant shall design the PMM to collect reliable information with respect to one or 
several of the thr ee aspects outlined above. The PMM shall be based on  strategies aiming at 
collecting relevant information from specifi c stakeholders includi ng consumers and on a 
reliable and validated flow of information be tween the different stakeholders. The applicant 
shall also submit more specific strategies when estimations of individual intakes of a specific 
food item or intakes of particular age groups have  to be collected. The applicant shall ensure 
that collected information shall allow to detect  indications on whether any (adverse) effect on 
health may be related to GM food and feed  consumption. The applicant shall provide 
adequate justification and a thorough descripti on of the selected design of the proposed PMM 
including aspects related to the analysis of the collected information.  EN 60   EN ANNEX IV  
 
VALIDATION OF METHODS OF DETECTION, SAMPLING, IDENTIFICATION 
AND REFERENCE MATERIAL   
1. I NTRODUCTION  
1. For the purposes of implementing Artic les 5(3)(i) and 17(3)(i) of Regulation 
(EC) No 1829/2003, this Annex shall provide requirements on the performance characteristics of the submitted method as well as technical provisions on the 
type of information for the applicant to present so as to verify that these requirements are met. This includes information about the method as such and about the method testing carried out by the applicant. 
2. The applicant shall also consider fu rther information about the operational 
procedures of the validation process that is made available by the CRL, 
assisted by the European ne twork of GMO laboratories. 
2. D
EFINITIONS  
For the purpose of this annex, the following definitions shall apply: 
1. 'reference material' (RM) means referen ce 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 sufficiently 
homogenous and well-established to be used for calibration of an apparatus, the assessment of a measurement method, or for assigning values to materials;  
2. 'certified reference material' (CRM) means reference material, accompanied by 
a certificate, one or more of whose property values ar e certified by a procedure 
which establishes its traceability to an accurate realisation of the unit in which 
the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence; 
3. ‘method performance requirements’ mean s the minimum performance criteria 
that the method shall demonstrate upon completion of a validation study 
carried out by the CRL according to internationally accepted technical provisions and this in orde r to certify that the met hod validated is fit for the 
purposes of enforcing Regulation (EC) No 1829/2003. 
3.  M
ETHOD VALIDATION  
3.1. Information about the method 
A. The method shall refer to all the met hodological steps needed to analyse the 
relevant material in accordance with Articles 5(3)(i) and 17(3)(i) of Regulation (EC) No 1829/2003. EN 61   EN  For a particular material  this shall include the me thods for DNA extraction and 
the subsequent quantification in a poly merase chain reaction (PCR) system. In 
such a case, the whole process from extraction up to the PCR-technique (or 
equivalent) shall constitute a method. The applicant shall provide information 
about the whole method. 
B. The applicant shall be allowed to refer to existing methods for a certain 
module(s), if available and appropriate such as a DNA extraction method from 
a certain matrix. In such a case, the a pplicant 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. 
C. The applicant shall demonstrate th at the method fulfils the following 
requirements. 
1. The method shall be event-specific a nd thus shall only be functional with 
the GMO or GM based product consider ed and shall not be functional if 
applied to other events already au thorised; otherwise the method cannot 
be applied for unequivocal detection /identification/quantification. This 
shall be demonstrated with a selectio n of non-target tran sgenic authorised 
events and conventional counterparts, in the case of GM plants. This 
testing shall include closely relate d events, where relevant, and cases 
where the limits of the detection are truly tested. 
2. The method shall be applicable to samples of the food or feed, to the 
control samples and to the reference material, which is referred to in Articles 5(3)(j) and 17(3)(j) of Regulation (EC) No 1829/2003. 
3. The applicant shall take into consideration the following documents for 
the development of the detection method: 
– Foodstuffs -- Methods of analysis for the detection of genetically 
modified organisms and derived products - General requirements 
and definitions: ISO 24276:2006, 
– Foodstuffs -- Methods of analysis for the detection of genetically 
modified organisms and derived pro ducts - Nucleic acid extraction: 
ISO 21571:2005, 
– Foodstuffs -- Methods of analysis for the detection of genetically 
modified organisms and derived pro ducts  Quantitative nucleic acid 
based methods: ISO 21570:2005, 
– Foodstuffs -- Methods of analysis for the detection of genetically 
modified organisms and derived products Protein based methods: 
ISO 21572:2004, 
– Foodstuffs -- Methods of analysis for the detection of genetically 
modified organisms and derived pr oducts Qualitative nucleic acid 
based methods: draft European standard ISO 21569:2005. EN 62   EN 4. The method shall also take into consideration the more detailed 
requirements set out in the common criteria set by the CRL and ENGL 
for minimum performance requirements for analytical methods for GMO testing
25. 
D. For the purpose of implementing Artic les 5(3)(i) and 17(3 )(i) of Regulation 
(EC) No 1829/2003, the applicant shall provide: 
(a) in the case of an application for authorisation coveri ng GM food or feed 
containing or consisting of a GMO, the event-specific quantitative detection method of the GM material; and, 
(b) in the case of an application for authorisation coveri ng GM food or feed 
produced from a GMO where the gene tically modified material is 
detectable, the event-specific quant itative detection method in the GM 
foods or feeds produced from the GM O. The applicant shall discuss the 
validity and limitations of the detecti on 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: 
1. Scientific basis: The applicant shall provide an overview of the principles 
of how the method works. This overv iew shall include references to 
relevant scientific publications. 
2. Scope of the method: The applican t shall indicate the matrix(es) (e.g. 
processed food, raw materials), the t ype 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 cl early mentioned, with  regard to the 
analysis per se and the sample preparation. Further information of any 
specific aspects crucial for the appli cation of the method shall also be 
mentioned here. 
4. Protocol: The applicant shall provide  a complete optimised protocol of 
the method. The protocol shall pres ent all the details as required to 
transfer and apply the method inde pendently in other laboratories. 
Further guidance regarding the subm ission of this information is 
provided by the CRL. 
5. The prediction model (or alike) 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. 
                                                
 
25  http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requir_Analyt_methods_131008.pdf EN 63   EN 3.2. Information about the method test ing carried out by the applicant 
A. The applicant shall provide all the av ailable and relevant data of the method 
optimisation and testing carried out. These data and results shall be presented, where possible and appropriate, by us ing the performance parameters as 
referred to under section 1.2. 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-laborat ory transferability. For this purpose, 
he shall provide the resu lts of the testing of th e method by at least one 
laboratory that is indepe ndent from the laboratory which has developed the 
method.  
C. The applicant shall provide the following information required about the 
method development and the method optimisation:  
1. primer pairs tested (in the case of a PCR-based test), 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 testin g the method with different plant 
varieties; 
3. specificity, which shall be established through the submission of the full 
sequence of the insert(s), together with  the base pairs of the host flanking 
sequences so as to enable the CRL to assess the specificity of the proposed method by running homology s earches in a molecular database. 
D. The applicant shall, besides the inform ation requested in th e previous sections 
of this annex, provide the following in formation regarding the testing:  
– participating laboratories, time of the analysis and outline of the 
experimental design, including the de tails about the number of runs, 
samples, replicates etc., 
– description of the laboratory samp les (e.g. size, quality, date of 
sampling), positive and negative controls as well as reference material, plasmids and alike used, 
– description of the approaches that ha ve been used to analyse the test 
results and outliers, 
– any particular points observed during the testing, 
– references to relevant literature or technical provisions used in the 
testing. EN 64   EN 3.3. Samples of the food and feed and their control samples 
In view of implementing Articles 5( 3)(j) and 17(3)(j) of Regulation (EC) 
No 1829/2003, the applicant sha ll, together with the in formation specified under 
sections 1, 2 and 3 of this Annex, also provi de samples of the f ood and feed and their 
control samples of a type and amount to be specified by the CRL for the specific 
application for authorisation. 
4. REFERENCE MATERIAL  
The reference material shall be produ ced under ISO Guide 34 (laying down the 
general requirements for the competence of reference material producers) by a 
producer accredited to ISO Guide 34. 
The applicant shall provide information as regards the place where the reference 
material can be accessed. This shall be  accompanied by adequate information 
demonstrating that the availability of th e reference material will be maintained 
throughout the period of valid ity of the authorisation. 
The reference material shall be pr oduced from a single genotype. Breeding 
information describing the parental orig in of the genetic modification shall be 
provided.  
For verification and value assignment, a met hod that has been properly validated (see 
ISO/IEC 17025:5.4.5) shall be used. Uncertainties have to be estimated according to the ISO Guide to the Expression of Uncer tainty in Measurement (GUM). Main 
characteristics of these internationally accep ted technical provisions are provided in 
the following sections.  
1. GM RM containers: 
– GM RM container (bottles, vials, ampoules, etc.) shall be tight and 
contain not less than the st ated amount of material, 
– samples shall have appropriat e homogeneity and stability, 
– the commutability of the GM RM has to be assured, 
– packaging shall be appr opriate to the purpose, 
– labelling shall be of good aspect and quality. 
2. Homogeneity testing: 
– between-bottle homogeneity shall be examined; 
– any possible between-bottle heterogene ity shall be accounted for in the 
overall estimated RM uncertainty. Th is 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; EN 65   EN 3. Stability testing: 
– Stability shall be positively demons trated by appropriate statistical 
extrapolation for the GM RM shelf-life to be within the stated uncertainty; the uncertainty related to this demonstration is part of the estimated RM uncertainty. Assigned va lues are valid only for a limited 
time and shall be subject to a stability monitoring.  
4. Batch characterisation:  The methods used for verifica tion and for certification shall: 
– be applied under metrologically valid conditions, 
– have been properly technically validated before use, 
– have precision and accuracy compatib le with the target uncertainty. 
 Each set of measurements shall: 
– be traceable to the stated references, and 
– be accompanied by an uncertainty statement whenever possible. 
 Participating laboratories shall: 
– have the required competence for the execution of the task, 
– be able to achieve traceability to  the required stated references, 
– be able to estimate its measurement uncertainty, 
– have in place a sufficient and appr opriate quality a ssurance system. 
5. Final storage: 
– To avoid a posteriori degradation, all samples shall be stored under 
conditions designated for the final storage of the GM RM before 
measurements are started, 
– 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 CRMs: 
– 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 GM CRM is 
distributed,The certified value of the quantity of GM material shall be 
given in mass fraction. It may be co mplemented by a value in GM-DNA 
copy number in relation to target ta xon specific copy num bers calculated 
in terms of haploid genomes. EN 66   EN – The certificate shall mention the parental origin of the genetic 
modification (either paternal and ma ternal or only paternal or only 
maternal) as verified experiment ally during the certification.  
– Certified values (e.g. quantity of GM 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 GM 
CRM.