EN    EN   
 
 EUROPEAN COMMISSION 
Brussels, XXX   
SANCO/12462/2011 Rev. 2 (POOL/E1/2011/12462/12462R2-EN.doc) […](2012) XXX  draft 
  
COMMISSION IMPLEMENTING REGULATION (EU) No …/.. 
of XXX  
on applications for authorisat ion of genetically modified food and feed in accordance 
with Regulation (EC) No 1829/2003 of the European Parliament and of the Council and 
amending Regulations (EC) No  641/2004 and (EC) No 1981/2006  
(Text with EEA relevance) 
  
EN 1   EN COMMISSION IMPLEMENTING REGULATION (EU) No …/.. 
of XXX 
on applications for authorisation of geneti cally modified food and feed in accordance 
with Regulation (EC) No 1829/2003 of the Eu ropean Parliament and of the Council and 
amending Regulations (EC) No 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), 11(5), 17(7)  and 23(5) thereof, 
After consulting the European Food Safety Authority, Whereas: (1) Regulation (EC) No 1829/2003 la ys down Union procedures for the authorisation and 
supervision of genetically modified food a nd feed, including rules for the labelling of 
such food and feed. That Regulation provides fo r a scientific evaluation to be carried 
out on the risks that the genetically modifi ed food or feed may present for human and 
animal health and, as the case may be, for the environment. It also, provides that a 
genetically modified food or f eed must not mislead the consumer or the user and must 
not differ from the food or feed which it is intended to replace to such an extent that its 
normal consumption would be nutritionally di sadvantageous for humans or animals. 
(2) Regulation (EC) No 1829/2003 provides, in  particular, that applications for 
authorisation are to adequately and sufficiently demonstrate that the genetically modified food and feed satisfy the requireme nts laid down in that Regulation, in 
respect of their proposed uses. 
(3) In the interest of consistency of Union le gislation, certain definitions laid down in 
Regulation (EC) No 178/2002 of the European  Parliament and of the Council of 
28 January 2002 laying down the general prin ciples and requireme nts of food law, 
establishing the European Food Safety Authority and laying down procedures in 
matters of food safety
2 should also apply to this Regulation. 
                                                 
1 OJ L 268, 18.10.2003, p. 1. 
2 OJ L 31, 1.2.2002, p. 1.  
EN 2   EN (4) Commission Regulation (EC) No 641/2004 of 6 April 2004 on detailed rules for the 
implementation of Regulation (EC) No 1829/20033 provides for certain detailed rules 
concerning applications for authorisation submitted in accordance with Regulation 
(EC) No 1829/2003. To facilitate the preparatio n of applications an d ensure that they 
contain all the information needed for their assessment, it is necessary to provide for 
more comprehensive and systematic rules c oncerning applications  for authorisation, 
which should also be specific to each type of genetically modified organisms (GMO), 
namely plants, animals and micro-organisms. 
(5) The rules laid down in this Regulation should only cover applications concerning 
genetically modified plants for food or feed uses, food or feed containing or consisting 
of genetically modified plants and food or feed produced from such plants. Genetically 
modified plants constitute the vast major ity of current applications and for which 
sufficient experience is available to date. 
(6) The rules laid down in this Regulation shoul d specify the general requirements for the 
presentation and preparation of applicati ons, namely requirements to provide general 
and scientific information, including methods  for detection, and id entification, as well 
as reference material so as to ensure that applications comply with the conditions laid 
down in Article 5, 17 and 30 of Regulation (EC) No 1829/2003. 
(7) The applicant should also take into consid eration the scientific information to be 
provided in the application as regards the environmental risk assessment of GMOs or 
food and feed containing or consisting of GM Os, as set out in th e principles for the 
environmental risk assessment in Annex II to Directive 2001/18/EC of the European 
Parliament and of the Council of 12 Marc h 2001 on the deliberate release into the 
environment of genetically modified or ganisms and repealing Council Directive 
90/220/EEC4, as well as the applicable guidance published by the European Food 
Safety Authority (EFSA) in this regard. 
(8) In addition to the general requirements for the presentation and preparation of 
applications, it is appropriate to provide for specific rules to ensure that the scientific 
information required in the application ade quately and sufficiently demonstrates that 
the genetically modified food or feed satis fy the requirements laid down in Regulation 
(EC) No 1829/2003, in respect of their proposed uses. 
(9) These rules should therefore provide for a set of studies that should be included in all 
applications, as well as the test methods to be followed to perform such studies, whilst taking into account relevant in ternational standards, such as the guideline of the Codex 
Alimentarius for the conduct of safety assessment of foods derived from the recombinant-DNA plant 
5. 
(10) In accordance with the applic able guidance of the EFSA6, the safety assessment of the 
genetically modified food or feed should include studies relate d to new components 
resulting from the genetic modification, the molecular characterisation of the 
genetically modified plant, the compara tive analysis of the composition and the 
                                                 
3 OJ L 102, 7.4.2004, p. 14. 
4 OJ L 106, 17.4.2001, p. 1. 
5 Codex Alimentarius Commission, GL 45-2003.  
6 EFSA Journal 2011; 9(5):2150.  
EN 3   EN phenotype of the genetically modified plant compared to its conventional counterpart. 
Depending on the characteristics of the genetic ally modified plant and on the outcome 
of that first set of studies, the EFSA guida nce indicates that it may be necessary to 
perform additional studies. In that respect, th e EFSA considers that notwithstanding its 
limitations, a 90-day feeding study in rode nts with whole food or feed is, when 
justified, the primary additional study to addr ess uncertainties identified in the course 
of the safety assessment. 
(11) It has, however, not been proved possible to  define with the necessary precision the 
level of uncertainties which would require  the submission of 90- day feeding studies. 
In addition, some food and feed assessm ent bodies of Member States are of the 
opinion that such study shoul d be performed in all app lications for genetically 
modified plants containing single transformation events. Considering these diverging 
views, as well as to improve consumer confidence, such st udies should be, for the time 
being, requested in all applica tions related to ge netically modified plants with single 
transformation events and, where appropr iate, on genetically modified plants 
containing stacked events. 
(12) Studies to demonstrate that a genetically m odified food or feed fulfils the requirements 
of Regulation (EC) No 1829/2003 involving the use of laboratory animals should be 
carried out in accordance with Council Directive 86/609/EEC of 24 November 1986 
on the approximation of laws, regulations and administrative provisions of the 
Member States regarding the protection of the animals used for experimental and other 
scientific purposes7 which will be repealed and replaced by Directive 2010/63/EU of 
the European Parliament and Council of 22 September 2010 regarding the protection 
of the animals used for scientific purposes8, and should be kept to a minimum while 
ensuring an adequate demonstr ation of the safety of the genetically modified food or 
feed. The current uncertainties in relation to the need and design of 90-day feeding 
trials will be addressed by a large resear ch project under the 2012 work programme of 
Theme 2 " Food, Agriculture and Fisherie s, and Biotechnologies" of the seventh 
Framework Programme for Research (FP7). The requirements regarding animal feeding trials in the context of GMO risk assessments should be reviewed in the light 
of the outcome of this project expected to be available by the end of 2015 at the latest. 
Other credible scien tific knowledge which mi ght be available at that time should also 
be taken into account. 
(13) While the rules laid down in this Regulati on should be valid for all applications for 
genetically modified plants, the type and necessity of the studies to evaluate the 
characteristics and safety of genetically modi fied food or feed subject to an application 
may vary, depending on the nature of the ge netic modification or of the product. For 
example, genetic modifications which have negligible impact on the composition of a genetically modified food or feed or highly refined products  that may be proven to be 
identical to products produced from the c onventional counterpart  require different 
studies than a product resulting from comp lex genetic modification aiming to modify 
its nutritional characteristics. 
                                                
 
7 OJ L 358, 18.12.1986, p. 1.  
8 OJ L 276, 20.10.2010, p. 33.  
EN 4   EN (14) The requirements laid down in this Regulati on regarding the studies which have to be 
included in an applicati on for authorisation under Regulation (EC) No 1829/2003 
should not prevent the EFSA to request , where appropriate, the applicant to 
supplement the particulars accompanying the application in accordance with 
Articles 6(2) and 18(2) of  Regulation (EC) No 1829/2003. 
(15) In order to ensure that studies are of high quality and documented in a transparent 
way, it is essential that they  are performed under appropriate  quality assurance systems 
and raw data should be provided in all cases in a suitable  electronic format. 
Toxicological studies should be performed in accordance with the quality assurance 
principles laid down by Direct ive 2004/10/EC of the European Parliament and Council 
of 11 February 2004 on the harmonisation of  laws, regulations and administrative 
provisions relating to the appl ication of the principles of  good laboratory practice and 
the verification of their applications  for tests on chemical substances9. If such studies 
are carried out outside the Union, they should follow th e state of the art OECD 
Principles on Good Laboratory Practice (GLP ). With regard to studies other than 
toxicological studies, they should be conducted under ISO or GLP standards. 
(16) It is also necessary to define the require ments regarding the submission of additional 
information related to the safety of the GMO and scientific peer-reviewed literature 
related to the potential effects on health  and on the environment of the products 
covered by the application. 
(17) During the process of the genetic modificatio n of plants and other organisms, marker 
genes are often used to facilitate the se lection and identification of genetically 
modified cells, containing the gene of inte rest inserted into the genome of the host 
organism, among the vast majority of untrans formed cells. Such marker genes should 
be carefully selected. In addition, it is now  possible to develop GMOs without the use 
of antibiotic resistance marker  genes. Against this backgr ound and in accordance with 
Article 4(2) of Directive 2001/18/EC, the a pplicant should therefore aim to develop 
GMOs without the use of antibiotic resistan ce marker genes . 
(18) The harvest of segregating genetically modifi ed plants (segregating crops) containing 
stacked transformation events contains va rious sub-combinations of transformation 
events. In addition, current control procedur es do not allow identifying the origin of 
combinations of transformation events. Therefore, in order to ensure that authorisations are coherent with the produc ts of which the placing on the market is 
unavoidable and for the feasibility of cont rols, the applications for genetically 
modified food and feed from segregating crops should include all sub-combinations 
independently of their origin.
 
(19) Regulation (EC) No 1829/2003 provides that a proposal for post-market monitoring of 
the use of the genetically modified food or feed shall only be submitted by the applicant where it is appropr iate. It is therefore necessa ry to set out the conditions 
under which such a proposal should, according to the outcome of the risk assessment, accompany the application. Post-market m onitoring should only be considered in 
cases where, notwithstanding th e fact that the safety of genetically modified food and 
feed has been demonstrated, it is appropriate to confirm the expected consumption, the 
                                                
 
9 OJ L 50, 20.2.2004, p. 44.  
EN 5   EN application of conditions of uses or identi fied effects. This is for example the case 
when the genetically modified food or f eed has altered nutri tional composition or 
when that its nutritional valu e differs from the conventiona l food or feed that it would 
replace or when there is a likelihood of increased allergenicity due to the genetic 
modification. 
(20) This Regulation should take account of the international trade commitments of the 
Union and of the requirements of the Ca rtagena Protocol on Biosafety to the 
Convention on Biological Diversity (the Ca rtagena Protocol), approved by Council 
Decision 2002/628/EC of 25 June 2002 concer ning the conclusion, on behalf of the 
European Community, of the Ca rtagena Protocol on Biosafety10 as well as the 
provisions of Regulation (EC) No 1946/ 2003 of 15 July 2003 of the European 
Parliament and of the Council on transbounda ry movements of genetically modified 
organisms11. 
(21) In order to ensure that te st methods included in the ap plication are adequate to 
demonstrate that the food or feed complies with the requirements for authorisation set 
out in Regulation (EC) No 1829/2003, they shou ld be carried out in accordance with 
the present Regulation, or internationally ag reed guidelines such as  those described by 
the OECD, when available. To ensure that  applications for renewal meet the same 
standards as regards tests methods, it is appr opriate that these requirements also apply 
to application for renewal of aut horisation of GM food and feed.  
(22) In order to provide an accura te designation of the GM food or feed subject to an 
application under Regulation (EC) No 1829/2003, applications should include 
proposals for a unique identifier for each GMO concerned in accordance with 
Commission Regulation (EC) No 65/2004 of 14 January 2004 establishing a system 
for the development and assignment of uniqu e identifiers for genetically modified 
organisms12. 
(23) This Regulation replaces certain provi sions of Regulation (EC) No 641/2004 as 
regards genetically modified plants for food or feed uses, food or feed containing or 
consisting of genetically modified plants and food or feed produced from genetically 
modified plants. However, Regulation (EC)  No 641/2004 should continue to apply as 
regards other types of genetically modifi ed products, namely genetically modified 
animals and genetically modified micro-or ganisms. Moreover, certain provisions of 
that Regulation are obsolete. Regulati on (EC) No 641/2004 should therefore be 
amended accordingly. 
(24) Commission Regulation (EC)  No 1981/2006 of 22 December 2006 on detailed rules 
for the implementation of Article 32 of  Regulation (EC) No 1829/2003 of the 
European Parliament and of the Counc il as regards the Co mmunity reference 
laboratory for genetically modified organisms13 should be amended to include 
references to this Regulation. 
                                                 
10 OJ L 201, 31.7.2002, p. 48. 
11 OJ L 287, 5.11.2003, p. 1. 
12 OJ L 10, 14.1.2004, p. 5. 
13 OJ L 368, 22.12.2006, p. 99.  
EN 6   EN (25) Regulation (EC) No 1829/2003 pr ovides that the Commission is to consult the EFSA 
before establishing implementing rules with regard to the applications for authorisation under that Re gulation. The EFSA has been  consulted on those rules 
accordingly. 
(26) This Regulation has been drawn up on the ba sis of current scientific and technical 
knowledge. Therefore, the Commission should m onitor any developments in this field 
and the publication of new or a dditional guidance by the EFSA. 
(27) This Regulation applies to applications submitted after its entry into force. 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. 
(28) The measures provided for in this Regulati on 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 applicati ons submitted under Articles 5, 11, 17 and 23 of 
Regulation (EC) No 1829/2003 for the authorisation of: 
(a) genetically modified plan ts for food or feed uses; 
(b) food or feed containing or consis ting of genetically modified plants; 
(c) food produced from or containing ingred ients produced from ge netically modified 
plants or feed produced from such plants. 
Article 2 
Definitions 
For the purposes of this Regulation, the defi nitions in Regulation (EC) No 1829/2003 apply.  
The definitions of 'risk', 'risk assessment' and 'hazard' applicable for the purposes of this 
Regulation are those provided in Ar ticle 3 of Regulation (EC) No 178/2002.  
EN 7   EN CHAPTER II 
 
General requirements  
Article 3 
Preparation and presentation of applicati ons submitted under Artic les 5(1) and 17(1) 
1. The application submitted under Article 5(1) and 17(1) of Regulation (EC) No 
1829/2003 shall: 
(a) be submitted in accordance with the requirements for the preparation and 
presentation of applicati ons set out in Annex I;  
(b) contain all the information required by A nnex I, in accordance with the specific 
requirements of Articles 4, 5 and 6. 
2. The application shall include, for each of  the specific requirements laid down in 
Articles 4, 5 and 6: 
(a) the summaries and results of the studies referred to in the application; (b) annexes where detailed inform ation on those studies is provided. 
3. The application shall contain a checklist de monstrating that the information required 
under Articles 4, 5 and 6 is complete. 
4. Where an application is limited to either f ood 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. The application shall, at the time of submission, clearly state which parts of the 
application are claimed to be confidential  and provide verifiab le justification in 
accordance with Article 30 of Regulation (EC) No 1829/2003.  
Additional information submitted during the authorisation procedure shall, at the 
time of submission, clearly state which parts of this additional information is claimed 
to be confidential and provide verifiable ju stification in accordance with Article 30 
of Regulation (EC) No 1829/2003. 
6. When studies have been already submitte d to the European Food Safety Authority 
(EFSA) for the purposes of an application and, where relevant, to th e extent that they 
may be used by the applicant in accordance with Article 31 of Regulation (EC) No 
1829/2003, a reference to such studies and the results of the EFSA 's assessment may, 
with the agreement of EFSA , be made in the framework of another application.  
EN 8   EN CHAPTER III 
 
Specific Requirements 
Article 4 
Requirements for the performance of studi es submitted under Artic les 5(3) and 17(3) 
1. Toxicological studies sha ll be conducted in facilitie s which comply with the: 
(a) requirements of Directive 2004/10/EC; or (b) "OECD Principles on Good Laboratory Practice" (GLP),  if carried out outside 
the Union. 
The applicant shall provide evidence to demonstrate such compliance. 
2. Studies, other than toxico logical studies, shall: 
(a) comply with the principles of Good Laboratory Practice (GLP) laid down in 
Directive 2004/10/EC ;or 
(b) be conducted by organisations accred ited under the relevant ISO standard. 
3. Information on the study protocols and the results obtained from the studies referred 
to in paragraphs 1 and 2 shall be compre hensive and include the raw data in an 
electronic format, suitable for carrying out statistical or  other analysis. 
Article 5 
Scientific requirements for the risk assessm ent of genetically modified food and feed 
submitted under Articles 5(3) and 17(3) 
1. Information, including studies, required to accompany the application as referred to 
in Article 5(3)(a) to (f) a nd (h) and in Article 17(3)(a) to (f) and (h) of Regulation 
(EC) No 1829/2003 shall be provided in acco rdance with the scientific requirements 
for the risk assessment of genetically modified food and feed set out in Annex II to this Regulation. 
2. By way of derogation from paragraph 1, an application may be submitted that does 
not satisfy all the requirements of  that paragraph provided that: 
(a) particular information is not necessary owing to the nature of the genetic 
modification or of the product; or 
(b) it is not scientifically necessary, or technically possible to supply such 
information. 
The applicant shall submit reasoned justification for the derogation.  
EN 9   EN 3. Paragraphs 1 and 2 shall not prevent th e EFSA to request, where appropriate, the 
applicant to supplement the particulars acco mpanying the application as provided for 
in Articles 6(2) and 18(2)  of Regulation (EC) 1829/2003. 
Article 6 
Additional information related to risk asse ssment submitted under Ar ticles 5(3) and 17(3) 
1. In addition to the information required in accordance with Article 5 and Annex II, the 
application shall include a systematic review  of studies published in the scientific 
literature and studies performed by the appl icant within the peri od of 10 years prior 
to the date of submission of the dossier on the potential effect s on human and animal 
health of the genetically modified fo od and feed covered by the application; 
 
2. During the authorisation pro cedure, the applicant shall su bmit, without delay, to the 
EFSA additional information which might influence the risk assessment of the genetically modified food or feed generated followi ng the submission of the 
application. In particular, the applicant shall submit to the EFSA information regarding any prohibition or restriction imposed by a co mpetent authority of any 
third country on the basis of a risk assessm ent of the genetically modified food and 
feed. 
Article 7 
Requirements applicable for post-market monito ring of genetically modified food or feed 
submitted under Articles 5(3) and 17(3) 
1. The applicant shall submit a proposal for post-market monitoring regarding the use 
of the food and feed as referred to in Ar ticles 5(3)(k) and 17(3)(k) of Regulation 
(EC) No 1829/2003 when the information provided in accordance with Articles 4, 5 
and 6 demonstrates that the genetically m odified food and feed comply with Articles 
4(1) and 16(1) of Regulation (EC) No 1829/2003 and when, in accordance with the 
outcome of the risk assessment, it is appropriate to confirm: 
(a) that specific recommendations of us es are followed by the consumer/animal 
owner; 
(b) the predicted consumption of the ge netically modified food or feed; or 
(c) the relevance and intensity of effect s and unintended effects detected during 
the pre-market risk assessment which can only be further characterised by post-market monitoring. 
2. The applicant shall ensure that  the post-market monitoring is: 
(a) developed to collect reliable informati on with respect to one or several of the 
aspects set out in paragraph 1. This information shall allow the detection of indications on whether any (adverse) effect on heal th may be related to 
genetically modified food or feed consumption;  
EN 10   EN (b) based on strategies aiming at collec ting relevant information from specific 
stakeholders including consumers and on a reliable and validated flow of 
information between the different stakehol ders. More specific strategies shall 
be included when data on individual intakes of a spec ific food item or intakes 
of particular age groups have to be collected; 
(c) accompanied by adequate justifica tion and a thorough description of the 
selected methodologies for the proposed post-market monitoring including aspects related to the analysis  of the collected information. 
 
Article 8 
Requirements concerning the methods of det ection, identification and quantification and for 
control samples and reference material submitte d under Articles 5(3), 11(2),17(3) and 23 (2) 
The application submitted under Articles 5(1) , and 17(1) of Regula tion (EC) No 1829/2003 
shall comply with the following requirements, as referred to in Articles 5(3)(i) and (j) and 17(3)(i) and (j) of that Regulation (EC) No 1829/2003, and set out in Annex III to this Regulation, for: 
(a) the methods for detection  and identi fication of the transformation event;  
(b) samples of food or feed and their contro l samples, and information as to the place 
where the reference material can be accessed. 
2. For applications submitted under Articles 11(1) and 23(1) of Regulation (EC) No 
1829/2003 , the requirements set out in Annex III to this Regulation for : 
(a) the methods for detection  and identi fication of the transformation event;  
(b) samples of food or feed and their contro l samples, and information as to the place 
where the reference material can be accessed.  
shall apply only for the purposes of app lication of Articles 11( 2)(d) and 23(2)(d). 
 
CHAPTER IV 
 
Transitional and final provisions 
Article 9 
Transitional provisions  
1. Until [fixed date corresponding to 6 months after the publication in the Official 
Journal], applicants may choose to submit a pplications falling under the scope of this  
EN 11   EN Regulation under Regulation (E C) No 641/2004 in the versi on of that Regulation in 
force on [fixed date corresponding to the publication in the Official Journal. 
2. By way of derogation from Article 4(2), in the case of studies launched prior to the 
date of entry into force of this Regulation and conducted under quality assurance systems other than GLP and ISO, the applicant shall provide: 
(a) a thorough description of the quality  assurance system under which such 
studies were performed, and; 
(b) comprehensive information on the protoc ols and the results obtained from the 
studies including the raw data. 
Article 10 
Amendments to Regulation (EC) No 641/2004 
Regulation (EC) No 641/2004 is amended as follows: 
(1) Article 1 is replaced by the following: 
"Article 1  
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 applications covered by Commission Regulation [XXX/2012 – 
reference to this Regulation] ."  
(2) Articles 5 to 19 are deleted. 
Article 11 
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 tr ial involving national reference 
laboratories of the method performan ce criteria set by the applicant as 
compliant with the document en titled ‘Definition of minimum 
performance requirements for analytical methods of GMO testing’ referred to:  
– in the case of genetically modified  plants for food or feed uses, 
food or feed containing or  consisting of genetically modified plants 
and food produced from or contai ning ingredients produced from 
genetically modified plants or feed produced from genetically 
modified plants, in point 3.1.C.4. of Annex III to Regulation (EC) No XXX/2012;   
EN 12   EN – in all other cases, in point 1(B)  of Annex I to Regulation (EC) 
No 641/2004; 
and (ii) the assessment of the precision an d trueness of the method provided by 
the applicant." 
(2) In Article 3(2), the first and second s ubparagraphs are replaced by the following: 
"2. The CRL shall request the applicant to pay an additional contribution of 
EUR 60 000 where a full validation pro cedure of a method of detection and 
identification for a single GMO event acco rding to the requirements laid down 
in the following provisions is required:  
(a) Annex III to Regulation (EU) No XXX/2012, when the application is 
related to:  
(i) genetically modified plan ts for food or feed uses;  
(ii) food or feed containing or consisting of genetically modified 
plants;  
(iii) food produced from or cont aining ingredients produced from 
genetically modified plants or f eed produced from such 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." 
Article 12 
Review 
1. The Commission shall monito r the application of this Regulation, the developments 
in scientific knowledge on re placement, reduction and refinement of animal use in 
scientific procedures and the publication of new guidance from EFSA. 
2. The Commission shall review the requi rements of point 1.4.4.1 of Annex II on the 
basis of new scientific information. The resu lts of this review shall be published by 
30.6.2016 at the latest. 
Article 13 
Entry into force 
This Regulation shall enter into force on the twentieth day following that of its publication in 
the Official Journal of  the European Union .   
EN 13   EN Done at Brussels,  
 For the Commission 
 The President  José Manuel BARROSO  
EN 14   EN ANNEX I  
 
PREPARATION AND PRESENTATION OF APPLICATIONS  
The application shall contai n the following information: 
PART I: GENERAL 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 gene tically modified plant and its products; 
4. Scope of the application 
(a) Genetically modified food 
 Food containing or consisting of  genetically modified plants 
 Food produced from genetically m odified plants or containing 
ingredients produced from gene tically modified plants 
(b) Genetically modified feed 
 Feed containing or consisting of  genetically modified plants 
 Feed produced from genetically modified plants 
(c) Genetically modified plan ts for food or feed uses 
 Products other than food and feed co ntaining or consisting of genetically 
modified plants with the exception of cultivation 
 Seeds and other plant propagating ma terial for cultiva tion in the Union. 
5. Unique identifier 
A proposal for a unique identifier for the genetically modified plant developed in 
accordance with Regulation (EC) No 65/2004.  
EN 15   EN 6. Where applicable, a detailed descriptio n of the method of production and 
manufacturing.  
This description would include, for exampl e, a detailed description of specific 
methods of production of food or feed which would be due to the nature of the 
genetic modification or which would l ead to food or feed  with specific 
characteristics. 
7. Where appropriate, the conditions for the placing on th e market of the 
genetically modified food(s) or feed(s),  including specific conditions for use and 
handling. 
8. Where applicable, the status of the food or feed or of related substances under 
other provisions of Union law 
Additional authorisatio n requirements provided for in  Union law, related to the 
placing on the market of the food or feed or applicable ‘maxim um residue level’ 
(MRL) where the food or feed is likely to  contain residues of plant protection 
products. 
PART II: SCIENTIFIC INFORMATION  
All the requirements of Part II shall be pr ovided in the application except where such 
requirements are not justified by the scope of  the application (for example, where the 
application is limited to food or feed produced from GMOs).  
1. H AZARD IDENTIFICATION AND CHARACTERISATION  
1.1. Information relating to the recipient or  (where appropriate) parental plants 
(a) Complete name: 
(i) family name; (ii) genus; (iii) species; (iv) subspecies; (v) cultivar, breeding line; (vi) common name; 
(b) Geographical distributio n and cultivation of the plant 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 plant, such as history of safe 
use for consumption as food or feed, in cluding information on how the plant is  
EN 16   EN typically cultivated, transported and stored, whether special processing is 
required to make the plant safe to eat, a nd the plant normal role in the diet (for 
example, which part of the plant is  used as a food source, whether its 
consumption is important in particul ar subgroups of the population, what 
important macro- or micro-nutrients  it contributes to the diet);  
(e) Additional information relating to the r ecipient or parental plants required for 
the environmental safety aspects: 
(i) Information concerning reproduction:  
– mode(s) of reproduction;  
– specific factors affecting reproduction (if any);  
– generation time;  
(ii) Sexual compatibility with other cultivated or wild plant species; 
(iii) Survivability:  
– ability to form structures for survival or dormancy;  
– specific factors, if any, affecting survivability; 
(iv) Dissemination:  
– ways and extent of disseminati on (to include, for example, an 
estimation of how viable pollen and/or seed declines with 
distance);  
– special factors affecti ng dissemination, if any; 
(v) Geographical distribution within the Union of the sexually compatible 
species; 
(vi) Where a plant species is not grow n in the Union, a description of the 
natural habitat of the plant, includ ing information on natural predators, 
parasites, competitors and symbionts; 
(vii) Other potential interactions of the genetically modified plant with 
organisms in the ecosystem where it is usually grown, or used elsewhere, including information on toxic eff ects 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  
EN 17   EN 1.2.1.3. Source of donor nucleic acid(s) used for transformation, size and intended function 
of each constituent fragment of the region intended for insertion 
1.2.2. Information relating to the genetically modified plant 
1.2.2.1. General description of the tr ait(s) and characteristics wh ich 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 phenot ypic stability of the genetically modified 
plant 
1.2.2.5. Potential risk associated w ith horizontal gene transfer 
1.2.3. Additional information relating to the ge netically modified plant required for the 
environmental safety aspects 
1.2.3.1. Information on how the genetically modified plant differs from the recipient plant in 
reproduction, dissemination, survivability or other properties. 
1.2.3.2. Any change to the ability of the geneti cally modified plant to transfer genetic 
material to other organisms, namely: 
(a) Plant to bacteria gene transfer;  
(b) Plant to plant gene transfer.  
1.2.4 Conclusions of the molecular characterisation 1.3. Comparative analysis 
1.3.1. Choice of the conventional 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 
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 
1.3.7. Effects of processing 1.3.8. Conclusion   
EN 18   EN 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 genetic ally modified food or feed 
1.4.4.1. 90-day feeding study in rodents 
1.4.4.2. Animal studies with respect  to reproductive, developmental or chronic toxicity 
1.4.4.3. Other animal studies to examine the safe ty and the characteristics of genetically 
modified food and feed 
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 th e whole genetically  modified plant 
1.5.3. Conclusion of the allergenicity assessment 
1.6. Nutritional assessment 
1.6.1. Nutritional assessment of th e genetically modified food 
1.6.2. Nutritional assessment of th e genetically modified feed 
1.6.3. Conclusion of the nutritional assessment 
2. E XPOSURE ASSESSMENT - ANTICIPATED INTAKE OR EXTENT OF USE  
3. R ISK CHARACTERISATION  
4. P OST-MARKET MONITORING ON THE GENETICALLY MODIFIED FOOD OR FEED  
5. E NVIRONMENTAL ASSESSMENT  
6. E NVIRONMENTAL MONITORING PLAN 
7. A DDITIONAL INFORMATION RELATED TO THE SAFETY OF THE GENETICALLY 
MODIFIED FOOD OR FEED   
EN 19   EN A systematic review of studies published in th e scientific literature and studies performed by 
the applicant within the period of  10 years prior to the date of submission of the dossier on the 
potential effects on human and animal health of the genetically modified food and feed 
covered by the applicati on shall be included in the application. This systematic review shall 
be carried out by taking into account the guida nce of EFSA on application of systematic 
review methodology to food and feed safety assessments to support decision making14. 
Where the information obtained from those studi es is not coherent with the information 
obtained from the studies performed in accordance with the requirements set out in Annex II, the applicant shall provide a thor ough analysis of the respective  studies and provide plausible 
explanations for the observed discrepancies. 
Additional information which might influence the evaluation of the safety of the genetically 
modified food or feed generated following the submission of the appli cation, as well as any 
information regarding any prohibi tion or restriction imposed by a competent authority of any 
third country on the basis of a safety asse ssment shall be provided by the applicant. 
P
ART III:  CARTAGENA PROTOCOL  
The application shall provide the inform ation required under Article 5(3)(c) and 
Article 17(3)(c) of Regulation (EC) No 1829/ 2003 for the purpose of complying with Annex 
II to the Cartagena Protocol on Biosafety to  the Convention on Biological Diversity.  
The provided information shall contain as a mini mum the information specified in Annex II to 
Regulation (EC) No 1946/200315: 
(a) The name and contact details of the applicant for a decision for domestic use; 
(b) The name and contact details of the authority responsible for the decision; 
(c) Name and identity of the GMO; (d) Description of the gene modificati on, the technique use d, and the resulting 
characteristics of the GMO; 
(e) Any unique identification of the GMO; (f) Taxonomic status, common name, point  of collection or acquisition, and 
characteristics of recipient organism or parental organi sms related to biosafety; 
(g) Centres of origin and centres of genetic diversity, if known, of th e recipient organism 
and/or the parental organism s and a description of the habitats where the organisms 
may persist or proliferate; 
(h) Taxonomic status, common name, point  of collection or acquisition, and 
characteristics of the donor organism or organisms related to biosafety; 
(i) Approved uses of the GMO; 
                                                
 
14 EFSA Journal 2010; 8(6):1637. 
15 OJ L 287, 5.11.2003, p. 1.  
EN 20   EN (j) A risk assessment report consistent  with Annex II to Directive 2001/18/EC; 
(k) Suggested methods for the safe handli ng, storage, transport and use, including 
packaging, labelling, documentation, dis posal and contingenc y procedures, where 
appropriate. 
PART IV: LABELLING  
The application shall include: 
(a) A proposal for labelling in all official  languages of the Union, where a proposal for 
specific labelling is required in accordance  with Articles 5(3 )(f) and 17(3)(f) of 
Regulation (EC) No 1829/2003;  
(b) Either a reasoned statement that the food or feed does not give rise to ethical or 
religious concerns or a proposal  for labelling in all offici al languages of the Union as 
required by Articles 5(3)(g) and 17(3 )(g) of Regulation (EC) No 1829/2003;  
(c) When appropriate a proposal  for labelling complying with the requirements of point 
A(8) of Annex IV to Directive 2001/18/EC.  
PART V: METHODS OF DETECTION , SAMPLING AND IDENTIFICATION AND REFERENCE 
MATERIAL  
The applicant shall provide methods for dete ction, sampling and iden tification, as well as 
samples of the food or feed and their contro ls samples to the European Union Reference 
Laboratory (EURL) as referred to in Article 32 of Regulation (EC) 1829/2003. 
The application shall include a copy of the completed form for the submission of those 
samples to the EURL and pr oof of sending to the EURL. 
The application shall include information as to the place where the reference material can be 
accessed.  
The applicant shall follow the instructions fo r the preparation and the sending of the samples 
provided by the EU Reference laboratory (EURL)  as referred to in Article 32 of Regulation 
(EC) 1829/2003. These instructions are publis hed on the following webpage: http://gmo-
crl.jrc.ec.europa.eu/guidancedocs.htm. 
PART VI: ADDITIONAL INFORMATION TO BE PROVIDED FOR GENETICALLY MODIFIED 
PLANTS AND /OR FOOD OR FEED CONTAINING OR CO NSISTING OF GENETICALLY MODIFIED 
PLANTS   
The information required in the notification as set out in Annex III to  Directive 2001/18/EC 
shall be provided where it is not covered by the requirements of other parts of the application. 
PART VII:  SUMMARY OF APPLICATIONS  
This Part specifies the standardised form, whic h the summary of the application dossier must 
follow.   
EN 21   EN Depending on the scope of the ap plication, some of the reque sted information may not be 
applicable.  
The summary shall not contain pa rts considered to be confiden tial 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 any other names) (d) Date of acknowledgement of valid application 
1.2. Applicant 
(a) Name of applicant 
(b) Address of applicant (c) Name and address of the 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) Genetically modified food 
 Food containing or consisting of  genetically modified plants 
 Food produced from genetically m odified plants or containing 
ingredients produced from gene tically modified plants 
(b) Genetically modified feed 
 Feed containing or consisting of  genetically modified plants 
 Feed produced from genetically modified plants 
(c) Genetically modified pl ants for food and feed use 
 Products other than food and feed co ntaining or consisting of genetically 
modified plants with the exception of cultivation 
 Seeds and plant propagating material for cultivation in the Union 
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?   
EN 22   EN No   
Yes  (in that case, specify) 
1.5. Has the genetically modified plant b een notified under Part B of Directive 
2001/18/EC? 
Yes  
No  (in that case, provide risk analysis data on the basis of the elements of Part B 
of Directive 2001/18/EC) 
1.6. Has the genetically modified plant or de rived products been previously notified 
for marketing in the Union under Part C of Directive 2001/18/EC?  
No   
Yes  (in that case, specify) 
1.7. Has the product been subject to an a pplication and/or authorised in a third 
country either previously or simultaneously to this application? 
No  
Yes  In that case, specify the third country, the date of application and, where 
available, a copy of the risk asse ssment conclusions, the date of the 
authorisation and the sc ope of the application 
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 (such as seeds,  cut-flowers, vegetative parts,) as a 
proposed condition of the authorisation applied for 
(c) Intended use of the product and types of users (d) Any specific instructions and recommendations for use, storage and handling, 
including mandatory restrictions propos ed as a condition of  the authorisation 
applied for 
(e) If applicable, geographical areas w ithin the Union 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 other 
applicable EU legislation than Regulation (EC) No 1829/2003 and when  
EN 23   EN necessary a proposal for spec ific labelling in accordan ce with Articles 13(2) 
and (3), Article 25(2)(c) and (d) and Article 25(3) of Regulation (EC) No 
1829/2003 
In the case of products other than food and feed containing or consisting of 
genetically modified plants, a proposal  for labelling which complies with the 
requirements of point A(8) of Annex IV to Directive 2001/18/EC must be 
included.  
(i) Estimated potential demand 
(i) In the EU (ii) In EU export markets 
(j) Unique identifier in accordan ce with Regulation (EC) No 65/2004 
1.9. Measures suggested by the a pplicant to take in the case  of unintended release or 
misuse of the product as well as meas ures for its 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  (f) Common name 
2.2. Geographical distribution and cultivation of the plant, including the 
distribution within the Union 
2.3. Information concerning reproduction (for environmental safety aspects) 
(a) Mode(s) of reproduction 
(b) Specific factors affecting reproduction (c) Generation time 
2.4. Sexual compatibility with other cu ltivated or wild plant species (for 
environmental safety aspects) 
2.5. Survivability (for environmental safety aspects)  
EN 24   EN (a) Ability to form structures for survival or dormancy 
(b) Specific factors affecting survivability 
2.6. Dissemination (for environmental safety aspects) 
(a) Ways and extent of dissemination 
(b) Specific factors affecting dissemination 
2.7. Geographical distribution within the Unio n of the sexually compatible species 
(for environmental safety aspects) 
2.8. In the case of plant species not normally grown in the Union  description of the 
natural habitat of the pl ant, including information on natural predators, 
parasites, competitors and symbionts (for environmental safety aspects) 
2.9. Other potential interactions , relevant to the genetically  modified plant, of the 
plant with organisms in the ecosystem where it is usually grown, or used 
elsewhere, including information on toxi c effects on humans,  animals and other 
organisms (for environmental safety aspects) 
3. M OLECULAR CHARACTERISATION  
3.1. Information relating to the genetic modification 
(a) Description of the methods us ed for the genetic modification 
(b) Nature and source of the vector used 
(c) Source of donor nucleic acid(s) used  for transformation, size and intended 
function of each constituent fragment of the region intended for insertion 
3.2. Information relating to the genetically modified plant  
3.2.1. Description of the trait(s) and charac teristics which have been introduced or 
modified 
3.2.2. Information on the nucleic acid(s) seque nces actually inserted or deleted 
(a) The copy number of all detectable inserts, both complete and partial 
(b) In the case of deletion(s), size and function of the deleted region(s) 
(c) Sub-cellular location(s) of insert(s) (nucleus, chloroplasts, mitochondria, or 
maintained in a non-integrated form), and methods for its/their determination 
(d) The organisation of the inserted ge netic material at the insertion site 
(e) In the case of modificat ions other than insertion or  deletion, describe function 
of the modified genetic material before  and after the modification, as well as 
direct changes in expression of genes as a result of the modification  
EN 25   EN 3.2.3. Information on the expression of the insert 
(a) Information on developmental expression of the insert during the life cycle of 
the plant 
(b) Parts of the plant where the insert is expressed.  
3.2.4. Genetic stability of the in sert and phenotypic stability of  the genetica lly modified 
plant 
3.2.5. Information (for environmental safety asp ects) on how the genetically modified plant 
differs from the recipient plant in: 
(a) Mode(s) and/or rate of reproduction 
(b) Dissemination (c) Survivability (d) Other differences 
3.2.6. Any change to the ability of the genetica lly modified plant to transfer genetic 
material to other organisms (for environmental safety aspects) 
(a) Plant to bacteria gene transfer 
(b) Plant to plant gene transfer 
4. C
OMPARATIVE 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 varieties in each location) 
and of the statis tical analysis.  
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  
EN 26   EN (c) Information on natural food or feed constituents 
(d) Testing of the whole genetic ally modified food and feed. 
6. A LLERGENICITY  
(a) Assessment of allergenicity of the newly expressed protein 
(b) Assessment of allergenicity of th e whole genetically modified plant 
7. N UTRITIONAL ASSESSMENT  
(a) Nutritional assessment of the genetically modified food 
(b) Nutritional assessment of the genetically modified feed 
8. E XPOSURE ASSESSMENT – ANTICIPATED INTAKE /EXTENT OF USE  
9. RISK CHARACTERISATION  
10. P OST-MARKET MONITORING ON THE GENETICALLY MODIFIED FOOD OR FEED  
11. E NVIRONMENTAL ASSESSMENT  
11.1. Mechanism of interaction between the ge netically modified plant and target 
organisms  
11.2. Potential changes in the interactions of  the genetically modi fied plant with the 
biotic environment resulting fr om the genetic modification 
(a) Persistence and invasiveness 
(b) Selective advantage or disadvantage  
(c) Potential for gene transfer  (d) Interactions between th e genetically modified pl ant and target organisms 
(e) Interactions of the genetically modi fied plant with non-target organisms  
(f) Effects on human health  (g) Effects on animal health  (h) Effects on biogeochemical processes  (i) Impacts of the specific cultivation, ma nagement and harvesting techniques   
EN 27   EN 11.3. Potential interactions with the abiotic environment 
11.4. Risk characterisation 
12. E NVIRONMENTAL MONITORING PLAN  
(a) General (risk assessment, background information) 
(b) Interplay between environmenta l risk assessment and monitoring 
(c) Case-specific genetically modified  plant monitoring (approach, strategy, 
method and analysis) 
(d) General surveillance of the impact of  the genetically modi fied plant (approach, 
strategy, method and analysis) 
(e) Reporting the results of monitoring 
13. D ETECTION AND IDENTIFICATION TECHNI QUES FOR THE GENETICALLY MODIFIED 
PLANT  
14. I NFORMATION RELATING TO PREVIOUS RELEASES OF THE GENETICALLY 
MODIFIED PLANT (FOR ENVIRONMENTAL RISK ASSESSMENT ASPECTS ) 
14.1. History of previous releases of the gene tically modified plan t notified under part 
B of Directive 2001/18/EC or under Part B of Directive 90/220/EEC16 by the 
same notifier  
(a) Notification number 
(b) Conclusions of post-release monitoring (c) Results of the release with respect to any risk to hum an health and the 
environment, submitted to the competen t authority in accordance with Article 
10 of Directive 2001/18/EC 
14.2. History of previous releases of the gene tically modified plan t 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 
                                                
 
16 OJ L 117, 8.5.1990, p. 15.  
EN 28   EN (e) Duration of the release 
(f) Aim of post-releases monitoring (g) Duration of post-releases monitoring (h) Conclusions of post-release monitoring (i) Results of the release with respect to any risk to hum an health and the 
environment  
EN 29   EN ANNEX II  
 
SCIENTIFIC REQUIREMENTS FOR THE RISK ASSESSMENT OF 
GENETICALLY MODIFIED FOOD AND FEED  
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 and f eed or group of foods and feeds;  
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 and feed; 
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 characte rization and exposure assessment. 
2. S PECIFIC CONSIDERATIONS  
2.1. Insertion of marker genes and other nuc leic acid(s) sequences  not essential to 
achieve the desired trait 
In order to facilitate  the risk assessment, the applicant shall e ndeavour to minimise 
the presence of inserted nucle ic acid(s) sequences not esse ntial to achieve the desired 
trait. 
During the process of genetic modification of plants and other organisms, marker 
genes are often used to facilitate the se lection and identificat ion of genetically 
modified cells, containing the gene of inte rest inserted into the genome of the host 
organism, among the vast majority of unt ransformed cells. The applicant shall 
carefully select such marker genes and Article 4(2) of Directive 2001/18 shall be 
respected. Against that background, the a pplicant shall therefore aim to develop 
GMOs without the use of antibiotic resist ance marker genes . 
2.2. Risk assessment of gene tically modified food and feed containing stacked 
transformation events  
For the risk assessment of genetically modified food and feed containing stacked 
transformation events obtained by conven tional crossing of genetically modified 
plants containing one or seve ral transformation event(s), the applicant shall provide a 
risk assessment of each single transformati on event or, in accordance with Article 
3(6) of this Regulation, refer to already su bmitted application(s). The risk assessment  
EN 30   EN of genetically modified food and feed cont aining stacked transformation events shall 
also include an assessment of the following aspects: 
(a) stability of the transformation events;  (b) expression of the transformation events;  (c) potential synergistic or antagonistic effects resulting from the combination of 
the transformation events shall be subject  to an assessment in accordance with 
Sections 1.4 (Toxicology), 1.5 (Allergen icity) and 1.6 (Nutriti onal assessment). 
For genetically modified food and feed c ontaining, consisting of or produced from 
genetically modified plants, whose cultivat ion is associated with the production of 
genetically modified material  containing various sub-combinations of transformation 
events (segregating crops), the applica tion shall include all sub-combinations 
independently of their origin. In such a cas e, the applicant shall provide a scientific 
rationale justifying that there is no need to  provide  experimental data  with the 
concerned sub-combinations or, in the abse nce of such scientific rationale, provide 
the experimental data. 
For genetically modified food and feed c ontaining, consisting of or produced from 
genetically modified plants, the cultivati on of which does not lead to the production 
of genetically modified mate rial containing various combinations of transformation 
events (non segregating crops), the app lication shall only cover the combination 
which is to be placed on the market.  
The rules set out in this Section apply mutatis mutandis  to transformation events 
which are combined by other means such as co- and retransformation.  
II. SCIENTIFIC REQUIREMENTS 
1. H
AZARD IDENTIFICATION AND CHARACTERISATION  
1.1. Information relating to the recipient or  (where appropriate) parental plants 
1.1.1 The applicant shall provide comprehensiv e information relating to the recipient 
or (where appropriate ) the parental plants in order to:  
(a) evaluate all issues of potential co ncern, such as the presence of natural 
toxins or allergens; 
(b) identify the need for specific analyses. 
1.1.2 For the purposes referred to in poi nt 1.1.1, the applicant shall provide the 
following information: 
(a) Complete name; 
(i) family name; (ii) genus;  
EN 31   EN (iii) species; 
(iv) subspecies; (v) cultivar/breeding line or strain; (vi) common name. 
(b) Geographical distribution and cult ivation 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 plant. This information 
shall include the history of safe use for consumption as food or feed, information on how the pl ant is typically cultivated, transported and 
stored, whether special processing is required to make the plant safe to 
eat, and describe the normal role of the plant in the diet (such as which part of the plant is used as a food or  feed source, whether its consumption 
is important in particular subgroup s of the population, what important 
macro- or micro-nutrients it contributes to the diet). 
1.2. Molecular Characterisation 
1.2.1. Information relating to the genetic modification 
The applicant shall provide sufficient information on the genetic modification: 
(a) to identify the nucleic acid(s) intended for transformation and related vector 
sequences potentially delivered  to the recipient plant; 
(b) to characterise the nucleic acid(s) actually inserted in the plant. 
1.2.1.1. Description of the methods used for the genetic modification  
The applicant shall provide information on the following elements: (a) the method of genetic transforma tion including relevant references; 
(b) the recipient plant material;  (c) the species and the strain of Agrobacterium and other microbes , if used during 
the genetic transformation process; 
(d) the helper plasmids, if used duri ng the genetic transformation process; 
(e) the source of carrier nucleic acid(s),  if used during the genetic transformation 
process.  
EN 32   EN 1.2.1.2. Nature and source of vector used  
The applicant shall provide the following information: (a) a physical map of th e functional elements and other plasmid/vector 
components together with the rele vant information needed for the 
interpretation of the molecular analyses (such as restriction sites, the position 
of primers used in polymerase chai n reaction (PCR), the location of probes 
used in Southern analysis). The region intended for insertion shall be clearly 
indicated;  
(b) a table identifying each component of the plasmid/vector (including the region 
intended for insertion), its size, its  origin and its intended function. 
1.2.1.3. Source of nucleic acid(s) us ed for transformation, size a nd intended function of each 
constituent fragment of the region intended for insertion  
The applicant shall provide information on the donor organism(s) and on the nucleic 
acid(s) sequence(s) intended to  be inserted in order to determine whether the nature 
of the donor organism(s) or the nucleic acid sequence(s) may trigger any safety issue. 
Information regarding the function of the nuc leic acid region(s) intended for insertion 
shall comprise the following elements: 
(a) the complete sequence of the nucleic ac id(s) intended to be inserted, including 
information on any deliberate alteration(s)  to the corresponding sequence(s) in 
the donor organism(s); 
(b) the history of safe use of the gene product(s) arising from the regions intended 
for insertion; 
(c) data on the possible 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 th e genetically modified 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. 
For example, when the introduced trait is herbicide tolerance, the applicant shall 
provide information on the mode of action of the active substance and its metabolism 
in the plant.  
EN 33   EN 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 purp ose shall provide 
complete coverage of sequences that c ould be inserted into the genetically 
modified plant, such as any parts of the plasmid/vector or any carrier or foreign 
nucleic acid(s) remaining in the genetically modified plant. 
The Southern analysis shall span the entire transgenic locus(i) as well as 
flanking sequences and include all appropriate controls.  
For the determination of copy number of the insert, complementary methods 
may also be used (such as real-time PCR); 
(b) the organisation and sequence of the inse rted genetic material at each insertion 
site in a standardised electronic format , with the aim of identifying changes in 
the inserted sequences compared to the sequence intended for insertion; 
(c) in the case of deletion(s), size and f unction of the deleted region(s), whenever 
possible; 
(d) Sub-cellular location(s) of insert(s) (nucleus, chloroplasts, mitochondria, or 
maintained in a non-integrated form), a nd methods for its/their determination; 
(e) sequence information in a standardised electronic format for both 5’ and 3’ 
flanking regions at each insertion site, w ith the aim of identifying interruptions 
of known genes.  
Bioinformatic analyses shall be conduc ted using up-to-date databases with the 
aim of performing both intraspecies a nd interspecies similarity searches. 
In the case of genetically modified plan ts containing stacked events, the safety 
of potential interactions between a ny unintended modifications at each 
insertion site shall be assessed; 
(f) Open Reading Frames (hereafter refe rred to as "ORFs" and defined as any 
nucleotide sequence that contains a string of codons that is uninterrupted by the 
presence of a stop codon in the same r eading frame) created as a result of the 
genetic modification either at the junc tion sites with genomic DNA or due to 
internal rearrangements of the 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 ve rsions of the databases shall be provided.   
EN 34   EN Depending on the information gathered, furt her analyses (such as transcription 
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 inserted/modified sequence results in intended changes 
at the protein, RNA and/or metabolite levels ; 
– to characterise the potential unintende d expression of new ORFs identified 
under point 1.2.2.2(f) as rais ing a safety concern. 
For those purposes, the applicant shal l provide the following information: 
(a) The method(s) used for expression an alysis together with  their performance 
characteristics; 
(b) Information on developmental expression of the insert during the life cycle of 
the plant.  
The requirement for information on de velopmental expression shall be 
considered on a case-by-cas e basis taking into account the promoter used, the 
intended effect(s) of th e modification and scope of the application;  
(c) Parts of the plant where the inse rt/modified sequences is expressed.  
(d) Potential unintended expression of ne w ORFs identified under point 1.2.2.2(f) 
which raise a safety concern; 
(e) Protein expression data, including the raw data, obtai ned from field trials and 
be related to the conditions in which the crop is grown. 
Data on expression levels from those part s of the plant used for food and feed 
purposes shall be provided in all cases.  
In addition, information sh all also be provided on th e expression of target 
genes in other parts of the plant when tissue-specific promoters have been used 
and when this is relevant for the safe ty assessment.  The minimum requirement 
for protein expression shall be data provi ded from three growing sites or from 
one site over three seasons. Permutations of the sites and seasons shall be acceptable provided that the minimum requirement is met. When justified by the nature of the insert, (such as sile ncing approaches or where biochemical 
pathways have been intentionally modifi ed) specific RNA(s) or metabolite(s) 
shall be analysed.  
For silencing approaches  by RNAi expression, pot ential "off target" gene 
should be searched by in silico  analysis to assess if the genetic modification 
could affect the expression of other genes which raise safety concerns. 
 (f) With regard to the stacking of tr ansformation events by conventional crossing, 
expression data shall be provided to assess the potential interactions between  
EN 35   EN the events which may raise any additional safety concerns over protein and trait 
expression compared with the single transformation events. The comparison shall be carried out with data obtained from plants grown in the same field trials. On a case-by-case basis, a nd where concerns arise, additional 
information may be necessary. 
1.2.2.4. Genetic stability of the insert and phenot ypic stability of the genetically modified 
plant  
The applicant shall provide information: (a) to demonstrate the genetic stability of the transgenic locus(i) and the 
phenotypic stability and inheritance pa ttern(s) of the introduced trait(s); 
(b) in case of stacked transformation ev ents, to establish that each of the 
transformation events stacked in the pl ant has the same molecular properties 
and characteristics as in the plants with the single transformation events. 
For the purposes of that information, applic ants shall provide data which demonstrate 
stability over multiple (normally five) generations or vegetative cycles for plant containing single transformation events. Data  from the first and the last generations 
of vegetative cycles are sufficient. The sour ce of the material used for the analysis 
shall be specified. Data shall be analys ed using appropriate statistical methods. 
For stacked transformation events comparis ons between the original transformation 
events and the genetically modified stacks shall be carried out using plant materials 
representative of those designed for co mmercial production. The applicant shall 
provide adequate justificatio n for the plant materials used . Comparisons shall include 
comparisons of sequences of the inserts and the flanking regions obtained from GM 
plants containing single events and plants containing stacked events. 
To assess genetic stability of the transformation event(s), applicants shall use 
appropriate molecular approaches  referred to in Section 1.2.2.2. 
1.2.2.5. Potential risk associated w ith horizontal gene transfer 
The applicant shall assess the probability of horizontal gene transfer from the product 
to humans, animals and micro-organisms and any potential associated risk when intact and functional nucleic acid(s) rema ins in the genetically modified food and 
feed.  
 
1.2.3. Conclusions of the molecular characterisation 
The molecular characterisation shall provide data on the structure and expression of 
the insert(s), and on the stability of the in tended trait(s). This shall also apply to 
situations where transformation events ha ve been stacked by conventional breeding. 
It shall be specifically indi cated whether the molecular ch aracterisation of the genetic 
modification(s) raises safety concerns w ith regard to the in terruption of endogenous 
genes or regulatory sequences.  
EN 36   EN The molecular characterisation shall also aim to identify whether the genetic 
modification(s) raise(s) any issues regarding the potential for producing 
proteins/substances other than those inte nded and in particular new toxins or 
allergens. 
The potential unintended changes identified in  this Section shall be addressed in the 
relevant complementary part(s) of the safety assessment. 
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 genetically 
modified food and feed.  
It shall aim at identifying similarities and differences: (a) in composition, agronomic perfor mance and phenotypic characteristics 
(intended and unintended alterations) be tween the genetically modified plant 
and its conventional counterpart;  
(b) in composition between the genetica lly modified 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 genetically modified food or feed shall be carried out as for novel 
foods falling within the scope of Regulat ion (EC) No 258/97 of the Council and the 
European Parliament
17 that do not have conventional counterparts (such as where the 
genetically modified food or feed is not closely related to a food or feed with a 
history of safe use or where a specific trai t or specific traits are introduced with the 
intention of bringing complex changes in th e composition of the genetically modified 
food or 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 gene tically modified plant. When the GM 
plant has been developed using back-cross ing, a conventional counterpart with a 
genetic background that is as close as possibl e to the genetically modified plant shall 
be selected. 
In addition, the applicant may include a comparator having a closer genetic 
background to the genetically modified plan t than the conventional counterpart (such 
as a negative segregant). 
                                                 
17 OJ L 043, 14.2.1997, p. 1.  
EN 37   EN In the case of herbicide tolerant genetically modified plants and in order to assess 
whether the expected agricultural practices influence the expressi on of the studied 
endpoints, three test materials shall be compared: the genetica lly modified plant 
exposed to the intended he rbicide; the conventional counterpart treated with 
conventional herbicide management regime s; and the genetically modified plant 
treated with the same conventiona l herbicide management regimes. 
In the case of stacked transformation ev ents, it is not always possible to use a 
conventional counterpart with a genetic background as close to the genetically 
modified plant as with conventional counterpart normally used for single 
transformation events. In such circumstan ces, the applicant shall provide reasoned 
justification on the choice of  the conventional counterpart  and assess its limitations 
for the risk assessment. In addition, single parental genetically modified lines or genetically modified lines containi ng a sub-combination of the stacked 
transformation events for which an application has been submitted or negative segregants derived from these genetically modified lines may also be included as 
additional comparators. The applicant shal l provide detailed information justifying 
the choice of additional comparators. 
In all cases, the applicant shall provi de information on the breeding scheme 
(pedigree) in relation to the genetically m odified plant, the c onventional counterpart 
and, where appropriate, additional compar ators together with an adequate 
justification of their selecti on. The history of safe use of  the conventional counterpart 
shall be adequately supported by both  qualitative and quantitative data.  
More detailed guidance for the application of the requirements of this section is 
available in the EFSA scientific opinion "Guidance on selection of comparators for 
the risk assessment of genetically modi fied plants and derived food and feed"
18. 
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 the production of material for the comparative analysis 
shall be performed in order to determine whether the genetically modified plant and/or the genetically modified food and feed is different from its conventional 
counterpart and/or equivale nt to non genetically modified reference varieties 
with a history of safe use. 
For each endpoint, the comparative analys is shall involve the following two 
approaches: 
(i) a test of difference, to verify wh ether the genetically modified plant is 
different from its conventional c ounterpart and might therefore be 
considered a hazard depending on the t ype of the identified difference, 
and on the magnitude and type of exposure; 
                                                
 
18 EFSA Journal 2011; 9(5):2149.  
EN 38   EN (ii) a test of equivalence to verify whether the genetically modified plant is 
equivalent or not to non  genetically reference varieties, apart from the 
introduced trait(s). 
In testing for difference, th e null hypothesis shall be th at there is no difference 
between the GMO and its conventional counterpart against the alternative 
hypothesis that a difference exists. 
Where additional comparator(s) are used for the risk assessment, a test of 
difference shall be carried out between the genetically modified plant and each 
of the additional comparator(s) followi ng the requirements set out in Section 
1.3.3.2 for the test of difference between th e genetically modified plant and its 
conventional counterpart.  
In testing for equivalen ce, the null hypothesis shall be that the difference 
between the GMO and the set of reference varieties is at least as great as a 
specified minimum size (see Section 1.3.2.2)  against the alternative hypothesis 
that there is no difference or a smaller difference than the specified minimum between the GMO and the set of reference varieties. 
Rejection of the null hypothesi s shall be required in or der to concl ude that the 
GMO and the set of reference varieties are unambiguously equivalent for the 
endpoint considered. The equivalence limits  used for the test of equivalence 
shall represent appropriately the range  of natural variation expected for 
reference varieties with a history of safe use. 
(b) Specific protocols for experimental design 
Natural variation may have several sour ces: variation within a variety arises 
due to environmental factors and varia tion between varieties arises due to a 
combination of both genetic and environmen tal factors. In order to identify and 
estimate differences attributable only to genotypes, it is es sential to control 
environmental variability. Therefore, non genetically modified reference varieties shall be included in the experime ntal design of the field trials and in 
sufficient numbers to ensure an adequate  estimate of the variability required to 
set the equivalence limits. All test materi als consisting of ge netically modified 
plants, conventional counter part, reference varietie s and, where appropriate, 
additional comparator(s) shall be randomi sed to plots within a single field at 
each site, usually in a completely randomised or randomised block experimental design. The different sites sele cted for the field tr ials shall reflect 
the different meteorological and agr onomic conditions under which the crop is 
to be grown; the choice shall be e xplicitly justified. The choice of non 
genetically modified reference varietie s shall be appropriate for the chosen 
sites and shall be justified explicitly. In  the case the sites cover a restricted 
range of growing conditions, the applicant shal l replicate the field trials over 
more than one year.  
Within each site the test materials cons isting of genetically modified plants, 
conventional counterpart and, where appr opriate, additional comparator(s) 
shall be identical for al l replicates. In addition, unless there is explicit 
justification for not doing so, at each site there shall be at least three  
EN 39   EN appropriate non genetically modified refere nce varieties of the crop that have a 
known history of safe use, which shall al so be identical between replicates. The 
replication at each site is the number of results obtained for each test material; 
the replication should never be less than four at any site. However, if only two 
appropriate reference varie ties are available at a pa rticular site, then the 
replication shall be six at that site; if only one is available then the replication 
shall be eight. 
Each field trial shall be re plicated at a minimum of eight sites, chosen to be 
representative of the range  of likely receiving envir onments where the plant is 
to be grown. The field trials may be c onducted in a single year, or spread over 
multiple years. The non genetically modified reference varieties may vary between sites and at least si x different reference varietie s shall be used over the 
entire set of field trials.  
When the genetically modified plant is tested together with other genetically 
modified plants of the same crop species (such as Zea mays ) the production of 
material for the comparative assessment of  these different genetically modified 
plant may be produced simultaneously at the same site and within the same 
field trial by the placing of the different  genetically modified plants and their 
appropriate comparator(s) in the same randomised block. This shall be subject 
to the following two strict conditions: 
(i) the conventional counterpart and, where appropriate, additional 
comparator(s) shall always occur together with the genetically modified plant in the same block; 
(ii) all the different genetically modified plants and their comparator(s) and 
all the non genetically modified re ference varieties used to test 
equivalence with those genetically modified plants shall be fully 
randomized within each block. 
If the number of plots per block required  for such a field trial were to exceed 
16, then a partially balanced incomplete  block design may be used, to reduce 
the number of plots per block, by excludi ng some of the genetically modified 
plants and their appropriate comparat or(s) from each block. This shall be 
subject to the following two strict conditions: 
(i) the conventional counterpart shal l always occur t ogether with its 
particular genetically modified plant in the same block; 
(ii) all of the non genetically modified  reference varieties shall appear in 
each of the incomplete blocks and be  fully randomised with the plants 
and their comparator(s). 
The field trials shall be adequately described, giving information on important 
parameters such as management of the field before sowing, date of sowing, soil type, herbicide use, climatic and othe r cultivation/environmental conditions 
during growth and time of harvest, as well as the conditions during storage of 
the harvested material.  
EN 40   EN More detailed guidance for the application of the requirements of this section is 
available in the EFSA opinion on "Statis tical considerations for the safety 
evaluation of GMOs"19. 
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 logarith mic transformation is expect ed to be appropriate. In 
such cases, any difference between the gene tically modified material and any other 
test material shall be interpreted as a ratio on the natural scale. However, when logarithmic transformation does not provide appropriate results, the natural scale or 
another scale shall be considered.  
The total variability of each endpoint 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 reference vari eties. One set of confidence limits shall 
be used in the test of difference; the other set and the equivalence limits shall be used in the test of equivalence. 
A linear mixed statistical model shall be us ed for calculation of the confidence limits 
for both tests (that is to say, the difference and equivalence tests); a slightly different 
model shall be used to estimate the equivale nce 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 non- GM reference varieties, and I=0 otherwise. Then the 
random factors for model 1 should be, but not  necessarily be restricted to, those 
representing the variation: (i ) between the test materials (a set that includes the GM 
plant, its conventional count erpart, each of the non-GM re ference 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 
model 1 except that the random factor repr esenting the interacti on between the test 
materials and I is omitted.   
The fixed factor for both models should ha ve as many levels as there are test 
materials and represent the contrasts between  the means of the test materials.  The 
test materials are as defined above: the GM plant; its conventi onal counterpart; the 
set of non-GM reference varieties; and any additional test materi als.  The set of non-
GM reference varieties is considered as a single level 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 betwee n, respectively, the GM plant and its 
                                                
 
19 EFSA Journal 2010; 8(1):1250.  
EN 41   EN conventional counterpart, and the GM plant and the set of non-GM reference 
varieties. 
Both the difference test and the equivale nce test shall be implemented using the 
correspondence between hypothesis testing a nd the construction of confidence limits. 
In the case of equivalence testing, the a pproach used shall fo llow the two one-sided 
tests (TOST) methodology by rejecting the null hypothesis of non- equivalence when 
the both confidence limits fall between th e equivalence limits. The choice of 90% 
confidence limits corresponds to the customary 95% level for statistical testing of 
equivalence. 
The results of the difference and equivalen ce tests shall be represented visually for 
all the endpoints simultaneously, on  a single graph or a few graphs. 
The graph(s) shall show the line of zero di fference between the genetically modified 
material and its conventional counterpart  and, for each endpoint: the lower and upper 
adjusted equivalence limits; the mean di fference between the genetically modified 
material and its conventional counterpart; and the confidence limits for this difference (see the set of possible outcome s for a single endpoint in the graph in 
Figure 1). 
When, in addition to the conventional counterp art, another test material is used as 
comparator, the mean difference between the genetically modified material and that comparator, its confidence limits and its adjusted equivalence limits shall be 
displayed on the graph(s) , for all such a dditional comparators, by referring this to 
the same zero baseline as defined by the conventional counterpart . The line of zero 
difference on the logarithmic scale corres ponds 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 transformation, changes of 2x 
and ½x will appear equally spaced on eith er side of the line of zero difference.  
Despite the expected proportion of spurious  significant differences, the applicant 
shall report and discuss all significant di fferences observed between the genetically 
modified crop, its conventiona l counterpart and, where applicable, any other test 
material, focusing on their biologica l relevance (see Section 3 on Risk 
Characterisation). 
For reporting, full details shall be give n for each endpoint analysed, listing:  
(a) the assumptions underlying the analysis; (b) full specification of the mixed mode ls 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 appr opriate estimated residual variation with 
which they are compared, and variance components for the random factors; 
(e) estimated degrees of freedom; (f) any other relevant statistics.  
EN 42   EN A discussion on the likely impact of other growing conditions not tested in the field 
trial shall be provided. 
Figure 1. Simplified version of a graph for comparative assessment showing the 
seven outcome types possible for each si ngle endpoint. After adjustment of the 
equivalence limits, a single confidence limit (f or the difference) serves visually for 
assessing the outcome of both tests (di fference and equivalence). Here, only the 
upper adjusted equivalence limit is cons idered. Shown are: the mean of the 
genetically modified crop on an appropriate scale (square); the confidence limits 
(whiskers) for the difference between the genetically modified crop and its 
conventional counterpart (bar shows confid ence interval); a vertical line indicating 
no difference (for test of difference); and vertical lines indicating adjusted 
equivalence limits (for test of equivale nce). For outcome types 1, 3 and 5, the null 
hypothesis of no difference cannot be re jected: for outcomes 2, 4, 6 and 7, the 
genetically modified crop is different from its conventio nal counterpart. Regarding 
interpretation of equivalence, four categories (i) to (iv) are identified: in category (i), 
the null hypothesis of non-equivalence is re jected in favour of  equivalence; in 
categories (ii), (iii) and (iv), non- equivalence cannot be rejected.  
 
 
     
Line of no 
difference  
lower    
 Outcome 
type  1   
2   
3   
4   
5   
6   
7   
Adjusted 
lower 
equivalence 
limit Adjusted 
upper 
equivalence 
limit Equivalence 
category (i) 
(ii) 
(iii) 
(iv) 
 
A. Regarding test of differe nce, each outcome from the graph shall be categorised 
as follows and the respective appr opriate conclusion shall be drawn.  
EN 43   EN (i) Outcome types 1, 3 and 5: the conf idence interval bar overlaps with the 
line of no-difference. The null hypot hesis of no difference cannot be 
rejected and the appropriate conclu sion is that there is insufficient 
evidence that the genetically modi fied crop and its conventional 
counterpart differ. 
(ii) Outcome types 2, 4, 6 and 7: the co nfidence interval ba r does not overlap 
with the line of no-difference. The null hypothesis of no difference must 
be rejected and the a ppropriate conclusion is  that the genetically 
modified crop is significantly differe nt from its conventional counterpart. 
B. Regarding test of equivalence, e ach outcome from the graph shall be 
categorised as follows, and the respect ive appropriate co nclusion shall be 
drawn. 
(i) Outcome types 1 and 2 (category (i ), Figure 1): both confidence limits lie 
between the adjusted equivalence limits and the null hypothesis of non-
equivalence is rejected. The appropriate  conclusion is that the genetically 
modified crop is equivalent to th e set of non genetically modified 
reference varieties. 
(ii) Outcome types 3 and 4 (categor y (ii), Figure 1): the mean of the 
genetically modified cr op lies between the adjusted equivalence limits, 
but the confidence interval bar overla ps at least one of the adjusted 
equivalence limits on the graph. Non-e quivalence cannot be rejected and 
the appropriate conclusion is that equivalence between the genetically modified crop and the set of non genetic ally modified reference varieties 
is more likely to be the case than lack of equivalence. 
(iii) Outcome types 5 and 6 (category (iii), Figure 1): the mean of the 
genetically modified crop lies outside the adjusted equivalence limits, but the confidence interval bar overlaps with at least one of the adjusted equivalence limits. Non-equivalence cannot be rejected and the appropriate conclusion is that lack of  equivalence between the genetically 
modified crop and the set of non genetic ally modified reference varieties 
is more likely to be the case than equivalence.  
(iv) Outcome type 7 (category (iv), Figure 1): both confidence limits lie 
outside the adjusted equivalence lim its. The appropriate conclusion is 
that there is lack of equivalence between the genetically modified crop and the set of non genetically modified reference varieties.  
In the case of significant difference and/or lack of equivalence for any 
particular endpoint, further statistical an alysis shall be carr ied out to assess 
whether there are interactions between any of the test materials and site, 
possibly using a simple standard ANOVA approach. Whatever approach is adopted, details shall be given, for each  endpoint analysed, listing: (a) the 
assumptions underlying the analysis, a nd, when appropriate: (b) degrees of 
freedom, (c) the estimated residual vari ation for each source of variation, and 
variance components, (d) any other re levant statistics. These additional 
analyses are intended to aid the interp retation of any significant differences  
EN 44   EN found and to study potential interactions between test materials and other 
factors. 
More detailed guidance for the application of the requirements of this section is 
available in the EFSA opinion on "Statis tical considerations for the safety 
evaluation of GMOs"20. 
1.3.3. Selection of material and compounds for analysis 
Analysis of the composition of plant ma terial is crucial when comparing the 
genetically modified food and feed with its conventional co unterpart. The material to 
be used for the comparative assessment shall be selected while taking into account the uses of the genetically m odified plant and the nature of the genetic modification. 
In the case of herbicide tolerant genetically  modified plants, three test materials shall 
be used: the genetically modified plant exposed to the intended herbicide; the 
conventional counterpart treated with conve ntional herbicide ma nagement regimes; 
and the genetically modified plant treate d with the same conventional herbicide 
management regimes. Unless duly justified, analysis shall be carried out on the raw 
agricultural commodity, as this usually represents the main point of entry of the material into the food and feed production and processing chain. Additional analysis 
of processed products (such as food and fee d, food ingredients, fe ed materials, food 
and feed additives or food flavourings), shall be conducted, where appropriate, and 
on a case-by-case basis (see also Sec tion 1.3.6). The sampling, analysis and 
preparation of the tested material shall be  carried out according to appropriate quality 
standards. 
1.3.4. Comparative analysis of composition 
In addition to the analysis on the level of the newly expressed pr oteins (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 for specific crop plant species, as referred 
to in the Organisation for Economic  Co-operation and Development (OECD) 
consensus documents on compositional cons iderations for new plant varieties 
(OECD consensus documents)
21. The vitamins and minerals selected for analysis 
shall be those which are present at levels  which are nutritionally significant and/or 
which make nutritionally signifi cant contributions to the diet at the levels at which 
the plant is consumed. The specific analyses require d shall depend on the plant 
species examined, but shall include a deta iled assessment appropriate to the intended 
effect of the genetic modification, the cons idered nutritional va lue and use of the 
plant. The applicant shall pay particular a ttention to key nutrients  such as proteins, 
carbohydrates, lipids/fats, fibre, vitamins and minerals. For example, a fatty acid profile shall be included for oil-rich  plants (main individual saturated, mono-
unsaturated and poly-unsaturated fatty aci ds) and an amino acid profile (individual 
protein amino acids and main non-protei n amino acids) for plants used as an 
                                                
 
20 EFSA Journal 2010; 8(1):1250. 
21 http://www.oecd.org/document/15/0,3746,en_2649_34385_46726799_1_1_1_1,00.html  
EN 45   EN important protein source. Analysis of plan t cell wall components are also required for 
the vegetative parts of plants  used for feed purposes. 
The applicant shall also provide an analysis  on key toxins inherently present in the 
recipient plant which may 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, anti-nutrients and allergens identified by the OECD 
consensus documents and justify the selection of these compounds. 
1.3.5. Comparative analysis of agronomic  and phenotypic characteristics 
The applicant shall provide a comparison be tween the genetically modified plant and 
its conventional counterpart. This comparis on shall enable the applicant to identify 
unintended effects resulting from the gene tic modification and shall address also 
plant biology and agronomic traits, including common breeding parameters (such as 
yield, plant morphology, flowering time, day degrees to maturity, duration of pollen 
viability, response to plant pathogens and inse ct pests, sensitivity to abiotic stress). 
The protocols of these field trials shall follow the specifications set out in Section 1.3.2. 
Where transformation events are stacked by conventional crossing, there may also be 
changes to agronomic and phenotypic char acteristics. Possible differences in 
phenotypic characteristics and agronomic properties of stac ks shall be assessed in 
field trials. Where appropriate, the applican t shall provide additi onal information on 
agronomic traits of the stacked transformation events from additional field trials.  
 1.3.6. Effects of processing 
The applicant shall assess whether or not the processing and/or preserving 
technologies applied are likely to modify the characteristic s of genetically modified 
end products compared with their respectiv e conventional counterpart. The applicant 
shall provide a description of the differe nt processing technologies in sufficient 
detail, paying special atten tion to the steps which may lead to significant changes in 
the product content, quality or purity. 
Genetic modification can target metabolic pathways resulting in changes in the 
concentration of non-protein substances  or in new metabolites (such as in 
nutritionally enhanced foods). Processed pr oducts may be assessed together with the 
assessment of the genetically modified plant for the safety of the genetic modification, or a processed product may be assessed sepa rately. The applicant shall 
provide the scientific rationa le for the risk assessment of these products. On a case-
by-case basis, the submission of additional experimental data shall be considered by the applicant.  
EN 46   EN 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. 
1.3.7. Conclusion 
The conclusion of the comparative analysis shall clearly state: 
(a) whether agronomic and phenotypic charac teristics of the genetically modified 
plant are, except for the introduced trait(s) , different to the characteristics of its 
conventional counterpart and/or equivalent  to the reference varieties, taking 
into account natural variation;  
(b) whether compositional characteristics of the genetically modified food and 
feed are, taking into account natural variation, different to the characteristics of 
its conventional counterpart and/or equiva lent to the reference varieties, except 
for the introduced trait(s); 
(c) characteristics for which the genetically modified plant or the genetically 
modified food and feed are different to  the characteristics of its conventional 
counterpart and/or not equivalent to th e reference varieties taking into account 
natural variation, which need  further investigation; 
(d) whether, in the case of transformati on events stacked by conventional crossing, 
there are indications of interactions  between the combined transformation 
events. 
1.4. Toxicology  
The toxicological impact of any changes re sulting from the expression of introduced 
genes or any other type of genetic modifi cation, (such as gene silencing or over-
expression of an endogenous gene) shall be assessed. 
Toxicological assessment shall be performed in order to: 
(a) identify potential adverse effects of single compounds and determine their 
highest dose level that does not result in  adverse effects. From data obtained 
from an appropriate animal study, an acceptable daily intake (ADI) of single 
compounds by humans may be derived by us ing uncertainty or safety factors 
that take into account differences between test animal species and humans, and inter-individual variations among humans. 
(b) demonstrate that the intended effect (s) of the genetic modification has no 
adverse effects on human and animal health.  
(c) demonstrate that unintended effect(s) of the genetic modification(s) identified 
or assumed to have occurred based on the preceding comparative molecular,  
EN 47   EN 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 comparative analysis referred to in Sec tion 1.2 and 1.3, namely 
the differences identified between the genetically modified product and its conventional counterpart, including inte nded as well as unintended changes.  
The applicant shall take into account the presence of: newly expressed proteins, the 
potential presence of other new constituents and/or possible changes in the level of natural constituents beyond normal variati on. The specific information requirements 
and testing strategies are se t out in Sections 1.4.1 to 1.4.4. 
As regards applications of which the scope include or is restricted to genetically 
modified food and feed produced from gene tically modified plants, toxicological 
studies with the processed pr oducts shall not be provided under the condition that the 
applicant provides a risk assessment of the genetically modified plant (or relevant parts of it) demonstrating its safety and pr ovided that there are no indications that the 
processed genetically modifi ed food and feed would be any different from their 
respective conventional counterpart. Th e applicant shall provide adequate 
justification in this regard.  
Toxicology studies designed to evaluate risks to human a nd/or animal health shall 
complement each other. Most studies required for the assessment of the safety of the genetically modified food are also valid for the assessment  of genetically modified 
feed.  
Besides the exposure of consumers and anim als through intake of food and feed, any 
adverse effect(s) on individuals that could be due to their exposure to genetically 
modified food and feed material as part of  their professional activities, for example, 
farming, seed processing, shall be reported by the applicant. Appropriate studies 
shall be performed to further characteris e these indications of potential adverse 
effects. 
The applicant shall use internationally agreed  protocols and test methods for toxicity 
testing (see Tables 1 and 2 of Section 1.7). Adaptations of  these protocols or use of 
any methods that differ from such protocol s shall be justified in the application.  
EN 48   EN 1.4.1. Testing of newly expressed proteins 
The applicant shall provide an evaluati on of all newly expressed proteins. The 
studies required to investig ate the potential toxicity of  a newly expressed protein 
shall be selected on a case-by-case basis,  depending on the know ledge available with 
respect to the protein's source, function or  activity and history of human or animal 
consumption. As regards proteins expressed in  the genetically modified plant, in the 
case where the history of safe use for cons umption as food and/ or feed of both the 
plant and the newly expressed proteins is duly documente d, specific toxicity testing 
as provided for in this Section shall not be  required. In such case, the applicant shall 
provide the necessary informa tion regarding the history of safe use of the proteins.  
Where specific testing is required, the tested  protein shall be equi valent to the newly 
expressed protein as it is e xpressed in the genetically modi fied plant. If, due to the 
lack of sufficient amount of test mate rials from the plant , a protein produced by 
micro-organisms is used, the structural, biochemical and functi onal equivalence of 
this microbial substitute to the newly expressed plant protein shall be demonstrated. In particular, comparisons of the mol ecular weight, amino acid sequence, post-
translational modification, i mmunological reactivity and, in  the case of enzymes, the 
enzymatic activity, are needed to provide ev idence for the equivalence. In case of 
differences between the plant expressed protein and its microbial substitute, the significance of these differences for th e 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 structure, molecular weight (for example using mass spectrometry), studies  on post-translational modifications 
and a description of its function . In the case of newly expressed enzymes, 
information on the enzyme activities 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, 
such as toxic proteins. A search fo r homology to proteins 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 relevant processing and 
storage conditions and the expected treatment of the food and feed. The 
influences of temperature and pH changes shall be examined and potential modification(s) of the proteins (such as denaturation) and/or production of 
stable protein fragments generated through such treatments shall be 
characterised. 
(d) Data concerning the resistance of the newly expressed protein to proteolytic 
enzymes (such as pepsin), such as by in vitro  investigations using appropriate 
and standardised tests. Stable breakdow n products shall be characterised and 
evaluated with regard to the potential to  cause adverse health effects linked to 
their biological activity.   
EN 49   EN (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 expresse d proteins of genetically modified plants 
is of little additional value for the risk assessment of the repeated human and animal 
consumption of genetically modified food and feed and shall not be provided as part of the studies performed under this Point.  
The applicant shall perform st udies with combined admini stration of proteins when 
the genetic modification results in the expr ession of two or more  proteins in the 
genetically modified plant and when, based on scientific knowledge , a possibility of 
synergistic or antagonistic interactions of safety concerns is identified.  
1.4.2. Testing of new constituen ts other than proteins  
The applicant shall provide a risk assessment of identified new constituents other 
than proteins. This shall include, on a case- by-case basis an evaluation of their toxic 
potency and of the need of toxicological te sting as well as a determination of their 
concentration in the genetically modified food and feed. To esta blish the safety of 
new constituents having no history of safe use for consumption in food and feed, the 
applicant shall provide information analogous  to that described in the "Guidance on 
submissions for food additive evaluations by the Scientific Committee on Food” of 
11 July 2001
22 and Commission Regulation (EC) No 429/2008 on detailed rules for 
te implementation of Regulation (EC) No  1831/2003 as regards the preparation and 
the presentation of applica tions and the assessment and the authorisation of feed 
additives23. This shall include the submission of information on a core set of studies 
such as on metabolism/toxicokinetics, s ub-chronic toxicity, genotoxicity, chronic 
toxicity, carcinogenicity a nd reproduction and developmental toxicity, accompanied 
by any other appropriate type of study. For specific guidelin es for animal tests, see 
Table 1 of Section 1.7 of this Annex. Genotoxi city test protocols are provided for in 
Table 2 of Section 1.7 of this Annex.  
1.4.3. Information on altered levels of food and feed constituents 
This Section shall apply only in the case wh ere the intended or unintended effect of 
the genetic modification would result in an alteration of the levels of food and feed 
constituents beyond the natural variation. 
To demonstrate the safety of the altered leve ls of food and feed constituents such as 
macro- and micronutrients, anti-nutrients , and natural toxins as well as other 
secondary plant metabolites, the applicant shall submit a detailed risk assessment 
based on the knowledge of the physiological fu nction and/or toxic properties of these 
constituents. 
The result of that risk assessment shall determine if, and to what extent, the applicant 
shall provide toxicological tests on selected food and feed constituents. 
                                                 
22 http://ec.europa.eu/food/fs/sc/scf/out98_en.pdf 
23 OJ L 133, 22.5.2008, p. 1.  
EN 50   EN 1.4.4. Testing of the whole genetica lly modified food and feed  
The applicant shall primarily base its risk assessment of the genetically modified 
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 effects. Under the circumstances set out in Points 
1.4.4.1, 1.4.4.2 and 1.4.4.3 of this Section, specific toxicological studies with the whole genetically modified food a nd feed shall be carried out.  
1.4.4.1. 90-day feeding study in rodents with w hole genetically modified food/feed:  
The applicant shall include a 90-day f eeding study with whole food and feed in 
rodents for the assessment of food and f eed containing, consisting of or produced 
from genetically modified plants with a si ngle transformation event or with stacked 
transformation events which are not obtaine d by conventional crossing of genetically 
modified plants containing a single transformation event. 
In the case of stacked transformation even ts obtained by conve ntional crossing of 
genetically modified plants containing one  or several transformation event(s), a 90-
day feeding study with whole food and feed in rodents shall be included for each of 
the genetically modified plant with a singl e transformation event which was used. An 
additional 90-day feeding study with whole food and feed in rodents with the 
genetically modified plant w ith the stacked transformatio n events shall be included 
where indications of potential adverse eff ects are identified duri ng the assessment of 
(i) the stability of the inserts, (ii) the expression of the inserts and (iii) the potential synergistic or antagonistic effects resulting from the combination of the transformation events. 
 The design of the toxicity study with gene tically modified food and feed  should be 
performed according to the "sub-chronic oral toxicity test repeated dose 90-day - oral 
toxicity in rodents" (see Table 1) follo wing an adapted protoc ol. In principle a 
minimum of two test doses and a negative control shall be used. The highest dose 
shall be the maximum achievable without cau sing nutritional imbalance; the lowest 
dose shall contain the tested food and/or  feed in an amount always above the 
anticipated human/target anim al intake level. The geneti cally modified food and feed 
analysed should be rele vant to the product to be consum ed. In the case of herbicide 
tolerant genetically modified plants, th e tested material should come from the 
genetically modified plant exposed to the intended herb icide. Whenever possible, 
information on natural variation of test para meters shall be deri ved from historical 
background data rather than from the inclus ion of reference vari eties, consisting of 
commercially available food and feed derive d from non-GM plants with a history of 
safe use, in the experiments. The statistic al analysis shall focus on the detection of 
possible differences between the test materi al and its control. The use of a power 
analysis to estimate a sample size capable of detecting a pre-sp ecified biologically 
relevant effect size with a specified power and significance  level should be done to 
determine an appropriate sample size. Mo re detailed guidance for performing this 
study is provided in the EFSA Guidan ce on conducting repeated -dose 90-day oral 
toxicity study in r odents on whole food/feed
24. 
                                                 
24 EFSA Journal 2011; 9(12):2438.  
EN 51   EN 1.4.4.2. Animal studies with respect to reprod uctive and developmental toxicity testing 
When information required in Sections 1.4.1, 1.4.2 and 1.4.3 on the genetically 
modified food and feed sugge st the potential for reprodu ctive, developmental or 
chronic toxicity or in case of indications of adverse effects from the 90-day feeding 
study in rodents (such as functional and/or  histological modifications of nervous, 
endocrine, reproductive or im munological tissues/organs), appropriate testing shall 
be performed. Protocols for reproductive, de velopmental and chronic toxicity testing 
(see Table 1 of Section 1.7) may be adapte d for the purposes of testing the whole 
genetically modified food and feed. 
Given that the 90-day feeding study in rode nts is only designed to detect effects on 
adult reproductive organ weights and histopa thology and that it does not detect other 
effects on reproduction or development, te sting of the whole food and feed beyond a 
90-day rodent feeding study shall be conducte d where hazards in this respect have 
been identified.  
1.4.4.3. Other animal studies to examine the safe ty and the characteristics of genetically 
modified food and feed (see also Sections 1.6.1 and 1.6.2) 
Feeding studies with target animal species  shall be submitted when indications of 
adverse effects are drawn from inform ation required in Sections 1.4.1, 1.4.2 and 
1.4.3 on the genetically modified food and feed or from the outcome of the 90-day feeding study in rodents. Their focus shall be on the safety of new constituents (newly expressed proteins and other new constituents), on the identification and 
characterisation of unintended effects,  and on the nutritional impact of any 
intentional, substantial, compositional m odifications of the ge netically modified 
plant (see also Section 1.6). 
Studies of that type shall be limited to plant materials suitable for inclusion in their 
diets and which can be nutritionally matched to a suitable control diet.  
1.4.4.4. Interpretation of rele vance of animal studies 
Relevant effects observed in the animal trials  shall be evaluated in order to identify 
potential consequences for the health of  humans and animals and to assess their 
relevance for the safety of the genetically modified f ood and feed. This evaluation 
may be supported by additional information and considerations. Attention should be paid to the fact that certain  effects may be specific for the test animal, but not for 
humans due to interspecies differences. 
The applicant shall, in particular, consider dose-response relationships in parameters 
that have been changed (that is to say, commensurate increases in changes at increased doses) since they provide a str ong indication for an effect of the tested 
compound. When a difference is noted only at  the highest dose applied, other factors 
shall be considered to determine whether there is a relationship with treatment. Information on the background variability in a given pa rameter may be obtained by 
the applicant from data from other animals of the same species/strain tested in the same or other experiments, or from  internationally harmonised databases.   
EN 52   EN 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 given constituent 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 liver -derived compounds, such as enzymes or 
bilirubin 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 outco mes (such as body weight decrease due to 
reduced intake of less palatable diet). Supportive data for a hypothesis of causality 
between the test compound and effects in test animals may include, for example, 
predictive data for plausible effects from in vitro  and in silico  experiments and dose-
response relationships observed in the animal test. 
1.4.5. Conclusion of the 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 indicati ons of potential adverse 
effects, in particular, whether and at which dose levels adverse effects were 
identified in specific studies; 
(d) adverse effects have been identified from the studies made on the whole 
genetically modified food and f eed and at which dose levels.  
The applicant shall evaluate the result of the toxicological assessment in the light of 
anticipated intake of the genetically m odified food and feed (see Section 2).  
1.5. Allergenicity 
Food allergy is an adverse reaction to food a nd represents an important public health 
problem. Food allergy is differe nt from toxic reactions and intolerance. Allergy is a 
pathological deviation of th e immune response to a pa rticular substance, which 
affects only some individuals where a co mbined effect of variations in the 
environment and genetic predisposition has resulted in allerg ic sensitisation.   
EN 53   EN In allergic individuals, sometimes minute amoun ts of a food that is  well tolerated by 
the vast majority of the population can cause  serious symptoms and death. It is not 
the allergen per se , but the allergic person’s abnormal reaction to the allergen that 
causes the adverse health effect.  
Food allergy can be caused by various imm une mechanisms. However, IgE-mediated 
food allergy represents the ma in form of food allergy, th at causes the most severe 
reactions and the only form causing life-th reatening reactions. This IgE-mediated 
food allergy has been the focus in the ri sk assessment of allergenicity of GMOs. 
Importantly, food allergy consists of two separate phases: first sensitisation  where no 
symptoms occur while the capacity of the immune system to react increases 
dramatically, and later elicitation (provocation) with clinical manifestations.  
When ingested, the allergen(s), that is to  say, the sensitising f ood or food constituent 
is to some extent degraded by digestive enzymes, absorbed by the gut mucosa (small amounts even by the oral mucosa), processe d in specialised cells of the immune 
system and then presented to the reactiv e immune cells that  produce an immune 
response. Sensitisation can also  occur if the food allergen comes into cont act with the 
skin or is inhaled. 
The majority of the constituents that are responsible for allerg enicity of foods, as 
well as of pollens, are proteins. Some pr otein breakdown products, that is to say, 
peptide fragments, may conserve part of the allergenicity of the native protein and 
thus can also be considered as allergens.  
The specific allergy risk of GMOs is associated with: (i) exposure to newly 
expressed protein(s) that can be present in ed ible parts of the plants or in the pollen; 
this point is related to the bi ological source of the transgene;  and (ii) alterations to the 
allergenicity of the whole plant and its pr oducts, for example, due to over-expression 
of natural endogenous allergens as an unintended effect of  the genetic modification; 
this point is related to the biol ogy of the recipient plant itself. 
More detailed guidance for the application of the requirements of this Section is 
available in the EFSA scientific opinion on the assessment of allergenicity of GM plants and microorganisms and derive d food and feed adopted on 30 June 2010
25.  
1.5.1. Assessment of allergenicity of  the newly expressed protein 
Allergenicity is not an intrinsic, fully pred ictable property of a gi ven protein but is a 
biological activity requiring an interacti on with individuals with a pre-disposed 
genetic background. Allergenicity therefor e depends upon the gene tic diversity and 
variability in atopic humans. Frequency, severity and specificity of allergic reactions 
also depend upon geographic and environmental factors. Given this lack of complete 
predictability, it is necessary to consid er several aspects in the assessment of 
allergenicity to obtain a cumulative body of evidence which minimises any uncertainty with regard to the protein(s) in question.  
When studying the structural characteristic s and the biological and physicochemical 
properties of a newly expresse d protein, it is essential th at the tested protein is 
                                                
 
25 EFSA Journal 2010; 8(7):1700.  
EN 54   EN equivalent with respect to st ructure and activity to the ne wly expressed protein in the 
genetically modified plant. Studies carried out using purified target  proteins prepared 
by expression in organisms such as Escherichia coli  shall be acceptable provided that 
the properties of the microbial substitute protein are identical to those of the protein expressed in the plant, thus taking into account all post-translational modifications 
that specifically occur in the plant.  
The applicant shall verify whether the source of the transgene is allergenic. When the 
introduced genetic material is obtained from wh eat, rye, barley, oats or related cereal 
grains, the applicant shall also assess the newly expressed proteins for a possible role 
in the elicitation of gluten-sensitive enteropathy or other enteropathies which are not IgE-mediated. Where transformation events have been stacked, the applicant shall provide an assessment of any potential for increased allergenicity to humans and 
animals on a case-by-case approach. These potential effects may arise from additive, synergistic or antagonistic e ffects of the gene products. 
The applicant shall follow an integrated, case-by-case approach, that is to say, a 
weight-of-evidence approach, in the assessm ent of possible allergenicity of newly 
expressed proteins. This approach shall include: 
(a) Amino acid sequence homology comp arison between the newly expressed 
protein and known allergens 
In every case, a search for sequence homo logies and/or structural similarities 
between the expressed protein and know n allergens shall be performed to 
identify potential IgE cross-reactivity be tween the newly expressed protein and 
known allergens. The applicant shall ensure that the quality and the 
comprehensiveness of the databases are  state of the art. The alignment-based 
criterion involving 35 % sequence identi ty to a known alle rgen over a window 
of at least 80 amino acids is consid ered a minimal requirement. All sequence 
alignment parameters used in the an alysis shall be provided including 
calculation of percent identity (PID). Th e calculation of PID shall be performed 
on a window of 80 amino acids with gaps so  that inserted gaps are treated as 
mismatches. In some cases, for assessing short peptidic fragments such as ORFs, a search for sequences of conti guous identical or chemically similar 
amino acid residue can be conducted. Howeve r, this search shall not be carried 
out routinely for the identification of potential linear IgE binding epitopes 
because of its poor sens itivity or specificity.  
(b) Specific serum screening 
When there is an indication of sequence homology or structure similarities, an 
important procedure for assessing the potential that expos ure to the newly 
expressed proteins might elicit an al lergic reaction in individuals already 
sensitised to cross-reactive proteins, is based on in vi tro tests that measure the 
capacity of specific IgE from serum of allergic patients to bind the test protein(s). There is inter-i ndividual variability in the specificity and affinity of 
the human IgE response. In particular, th e specificity of the IgE antibodies to 
the different allergens present in a gi ven food/source and/or to the different 
epitopes present on a given protein may vary amongst allergic  individuals. In 
order to optimise the sensitivity of the test, individual sera from well- 
EN 55   EN characterised allergic indi viduals shall be used. Th e applicant shall perform 
specific serum screening in the following cases: 
(i) the source of the introduced gene is  considered allergenic, even if no 
sequence homology of the newly expre ssed protein to a known allergen 
is demonstrated; or  
(ii) the source is not known to be allergenic, but there are indications of a 
relationship between the newly expre ssed protein and a known allergen, 
based on sequence homology or structure similarity.  
Specific serum screening shall be unde rtaken with individual sera from 
individuals with a proven and well-characterised allergy to the source or to the 
potentially cross-reacting allergen usi ng relevant immunochemical tests. IgE-
binding assays (such as Ra dio or Enzyme Allergosorbent Assay (RAST or 
EAST), Enzyme Linked Immunosorbent Assay (ELISA) and electrophoresis 
followed by immunoblotting with specific IgE-containing sera) are adequate methods. 
(c) Pepsin resistance and in vitro digestibility tests. 
Stability to digestion by proteolytic enzymes has long been considered a 
characteristic of allergen ic proteins. Although it has been established that no 
absolute correlation exists, resistance of  proteins to pepsin digestion is an 
additional criterion to be considered in the weight-of-evidence approach for the 
assessment of allergenicity. The pepsin resistance test is generally performed 
under quite standardised conditions, at low pH values and high pepsin:protein ratios. It is recognized that the pepsin resistance test does not reflect the 
physiological conditions of the diges tion. The digestibility of the newly 
expressed proteins in speci fic segments of the populat ion, such as infants and 
individuals with impaired digestive f unctions, may be assessed using in vitro 
digestibility tests using different cond itions. Also, since the protein encoded by 
the newly introduced genes will be presen t in the product as a complex matrix, 
the impact of the possible interact ion between the protein and other 
components of the matrix, as well as th e effects of the processing, shall be 
taken into account in additional in v itro digestibility tests. Depending on the 
outcome of the in vitro digestibility te st, A comparison of the intact, the heat-
denatured and the pepsin-digested prot eins for IgE binding shall be assessed 
since an altered digestibility may impact on the allergenicity of the newly expressed protein.   
EN 56   EN (d) Additional tests 
Although additional tests including in vitro cell based assays or in vivo tests on 
animal models have not been validated  to date for regulatory purposes, they 
may provide useful additional informati on, for example, on the potential of the 
newly expressed protein for de novo  sensitisation.  
1.5.2. Assessment of allergenicity of the genetically modified food or feed 
When the recipient plant is known to be allergenic, the applic ant shall assess any 
potential change in the alle rgenicity of the genetically modified food or feed by 
comparison of the allergen repertoire with that of its conventional counterpart. The 
potential over-expression of  natural endogeneous allerg en(s) in the genetically 
modified plant shall, in pa rticular, be investigated.  
The applicant shall follow a case-by- case approach depending on the available 
information on the allergenic potential of  the recipient plant. It is generally 
performed by analytical methodologies such as proteomics in a ssociation with the 
use of allergic human sera as probes. Sera from clinically well-characterised allergic 
individuals that are the reference material for IgE binding studies may be available in 
limited number and quantity. In order to minimize the use of human sera, preliminary 
important information on the likelihood of  an unintended alteration of the overall 
allergenicity of the genetically modified  plant can be obtained by using sera of 
animals experimentally sensitised in well-defined conditions and by including 
relevant identified endogenous allergens in the comparative compositional analysis. 
In addition, the applican t shall provide, where available, information on the 
prevalence of allergy in persons working w ith, coming into contact with or in the 
vicinity of genetically m odified plant cultivation. 
1.5.3. Adjuvanticity 
Adjuvants are substances that, when co-a dministered with an antigen increase the 
immune response to the antigen and theref ore might increase as well the allergic 
response. In cases when know n functional aspects of the newly expressed protein or 
structural similarity to  known strong adjuvants may indicate possible adjuvant 
activity, the applicant shall assess the possible role of these proteins as adjuvants. As 
for allergens, inter actions with other constituents  of the food matrix and/or 
processing may alter the structure and bioavail ability of an adjuvant and thus modify 
its biological activity. 
1.5.4. Conclusion of the allergenicity assessment 
The conclusion of the allergenic ity assessment shall indicate:  
(a) whether the novel protein(s) is likely to be allergenic; 
(b) whether the genetically modified food or  feed is likely to be more allergenic 
than its conventional counterpart. 
When there is a likelihood of  increased allergenicity due to the genetic modification, 
the genetically modified food or feed shall be  further characterised in the light of its  
EN 57   EN anticipated intake (see Section 2). The appl icant shall propose appropriate conditions 
for placing on the market (such as post-market monitoring and labelling). 
1.6. Nutritional assessment 
1.6.1. Objectives of the nutritional assessment 
The applicant shall provide a nutritio nal evaluation to demonstrate that: 
(a) the introduction of the genetically modi fied food and feed 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  food and feed constituents, as well as potential alterations in the total diet of the consumer or the animal;  
(b) unintended effects of the genetic modifi cation that were identified or that may 
be assumed to have occurred based on the preceding molecular, compositional 
or phenotypic analyses, in accordance with Sections 1.2 and 1.3, have not adversely affected the nutritional valu e of the genetically modified food and 
feed. 
For stacked transformation ev ents combined by conventional crossing, an assessment 
of the potential changes in nutritional value that might arise from synergistic or 
antagonistic effects of the gene products  including compositional changes shall be 
provided by the applicant. This may be pa rticularly relevant where the combined 
expression of the newly introduced gene s has unexpected effects on biochemical 
pathways. 
1.6.2. Points to consider for the nutritional assessment of genetica lly modified food and 
feed 
The nutritional assessment of genetically modified food and feed shall consider: 
(a) the composition of the genetically modi fied food and feed w ith regard to the 
levels of nutrients and anti-nutrients (see compositional studies as described in Section 1.3); 
(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 th e food and feed; 
(c) the anticipated dietary intake of the food and feed (see Section 2) and resulting 
nutritional impact. 
When the comparative analysis has identif ied compositional characteristics of the 
genetically modified food and feed that  are different from its conventional 
counterpart and/or not equivale nt to the characteristics of the reference varieties, their 
nutritional relevance shall be assessed on the basis of curre nt scientific knowledge. If 
that assessment does conclude to the nutri tional equivalence betw een the genetically 
modified food and feed and its conventional counterpart, no further studies shall be 
carried out. By contrast if , on the basis of the assessment of the information obtained  
EN 58   EN from the comparative analysis, it is not  possible to conclude on the nutritional 
equivalence, further nutritional studies sh all be carried out. Comparative growth 
studies shall be conducted with young rapi dly growing animal species (such as 
broiler chicks as animal model for non-ru minants; lambs for ruminants; or other 
rapidly growing species).  
1.6.3. Nutritional studies of genetically modified food 
The applicant shall determine the necessity  and design of nutritional studies on the 
basis of the introduced trait(s), the outcome  of the comparative analysis, and of the 
90-day feeding study, where available. Supplementary information regarding the nutritional value may be obtained from co mparative growth performance studies 
conducted with other animal species, such as broiler chickens, addressing the 
nutritional assessment of ge netically modified feed.  When nutritional studies are 
conducted, the control diet(s) shall include  the conventional counterpart and where 
appropriate additional comparat or(s). In the case of herb icide tolerant genetically 
modified plants, the tested material should come from the genetically modified plant 
exposed to the intended herbicide. 
Genetically modified foods modified to provide additional health benefits to the 
consumer as compared to conventional f oods, may benefit specif ic populations or 
sub-populations while others may be at risk  from the same food. In cases where an 
altered bioavailability needs to be es tablished and may raise concern for sub-
population(s), the level of the nutrient in the food shall be dete rmined, taking into 
account all the different forms of th e compound. The methods to test for 
bioavailability shall be selected on a cas e-by-case basis depending on the nutrient or 
other constituent, the food containing these constituents, as well as the health, nutritional status and dietary practices of the specific pop ulation(s) anticipated to 
consume the food. 
1.6.4. Nutritional studies of genetically modified 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. Supplementary information regarding the nutritional value may be obtained from co mparative growth performance studies 
conducted with other animal species, such as broiler chickens, addressing the 
nutritional assessment of ge netically modified feed.  When nutritional studies are 
conducted, the control diet(s) shall include  the conventional counterpart and where 
appropriate additiona l comparator(s). 
In the case of genetically modified feed  with improved nutritional characteristics, 
feeding studies with target animal of food producing sp ecies shall be conducted to 
assess the impact on the feed. In the case of genetically modified plants modified for 
improved content and bioavailab ility of nutrients, studies with target food producing 
animal species shall be conducted to dete rmine the bioavailability of individual 
nutrients in the genetically modified plant compared to its conventional counterpart. 
In the case of genetically modified plants sp ecifically modified with traits to enhance 
animal performance through increased nut rient density (such as increased oil 
content) or an enhanced level of a specific nutrient (such as an essential amino acid or a vitamin), an appropriate control diet using its conventional counterpart shall be  
EN 59   EN formulated by supplementing it with the speci fic nutrient to the extent of the change 
effected in the genetically modified plan t. Regarding co-produc ts (such as oilseeds 
meals), from which the ingredient target ed by the genetic modification has been 
extracted, these may be compared with co -products produced from the conventional 
counterpart. 
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 studi es with aquatic spec ies such as carp, 
catfish, salmonidae  or typical herbivores shall be chosen.  
When appropriate, tests with various expe rimental designs shall be provided to 
demonstrate that the nutritionally improve d genetically modified  plant fulfils the 
expected nutritional value. The exact expe rimental design and statistical approaches 
of feeding experiments in food producing an imals to test the nutritional value of 
genetically modified feed, modified for e nhanced nutritional characteristics, shall 
depend on the targeted animal species, type of plant trait(s) studied and the size of 
the expected effect. The experimental diets sh all be formulated in such a way that the 
key measured endpoints are responsive to a difference in the quantity and/or 
availability of the nutrient in question. E ndpoint measurements shall vary with the 
target species used in the study, but sha ll include feed intake, body weight, animal 
performance and bioavailability of nutrients. 
More detailed guidance for the application of the requirements of this section is 
available in the Report of the EFSA GM O Panel Working Group on Animal Feeding 
Trials26. 
1.6.5. Conclusion of the nutritional assessment 
The conclusion of the nutritional assessmen t of genetically modified food and feed 
shall indicate whether the genetically modified food and feed is nutritionally 
equivalent to its conventional counterpart,  taking natural vari ations into account. 
The applicant shall evaluate the result of the nutritional assessment in the light of 
anticipated intake of the genetically m odified food and feed (see Section 2).  
1.7. Standardised guidelin es for toxicity tests 
The applicant shall use for toxicity testing internationally agreed guidelines and test 
methods described by Council Regulati on (EC) No 440/2008 laying down test 
methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and 
of the Council on the Registration, Evalua tion, Authorisation and Restriction of 
Chemicals (REACH)27 (see Tables 1 and 2). A non-exha ustive list of validated test 
methods which, where necessary, shall be us ed in a possibly adapted form for GMO 
toxicological testing is pr ovided in Tables 1 and 2. 
                                                 
26 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. 
27 OJ L 142, 31.5.2008, p. 1.  
EN 60   EN The performance of test methods depends on the type of genetically modified food 
and feed, type of the genetic modifica tion and resulting intended and unintended 
alterations, intended use and exposure/inta ke, and the available knowledge. Some of 
the tests were developed for the assessment of risks at the workplace (See Sections 1.4 and 1.5). 
Table 1: Non-exhaustive list of validated test methods for chemicals as provided by 
Regulation (EC) No 440/2008 and which may be  used in a possibly adapted form for 
GMO toxicological testing 
Title Reference of the method in 
Part B of the Annex of 
Regulation (EC) 
No 440/2008 
ACUTE TOXICITY (DERMAL) B.3. 
SKIN SENSITISATION B.6. 
REPEATED DOSE (28 DAYS) TOXICITY 
(ORAL) B.7. 
REPEATED DOSE (28 DAYS) TOXICITY 
(DERMAL) B.9. 
SUB-CHRONIC ORAL  TOXICITY TEST 
REPEATED DOSE 90 — DAY ORAL 
TOXICITY STUDY IN RODENTS B.26. 
CHRONIC TOXICITY TEST B.30. 
CARCINOGENICITY TEST B32. 
COMBINED CHRONIC 
TOXICITY/CARCINOGENICITY TEST B.33. 
ONE-GENERATION REPRODUCTION 
TOXICITY TEST B.34. 
TWO-GENERATION REPRODUCTION 
TOXICITY STUDY B.35. 
TOXICOKINETICS B.36. 
NEUROTOXICITY STUDY IN RODENTS B.43.  
EN 61   EN Table 2: Genotoxicity tests as pr ovided by Regulation (EC) No 440/2008. 
Title Reference of the method 
in Part B of the Annex of 
Regulation (EC) 
No 440/2008 
MUTAGENICITY - IN VIVO MAMMALIAN 
BONE MARROW CHROMOSOME 
ABERRATION TEST B.11. 
MUTAGENICITY - IN VIVO MAMMALIAN 
ERYTHROCYTE MICRONUCLEUS TEST B.12. 
MUTAGENICITY: REVERSE MUTATION 
TEST USING BACTERIA B.13/14. 
MUTAGENICITY TESTING AND 
SCREENING FOR CA RCINOGENICITY 
GENE MUTATION - SACCHAROMYCES 
CEREVISIAE B.15. 
MITOTIC RECOMBINATION - 
SACCHAROMYCES CEREVISIAE B.16. 
DNA DAMAGE AND REPAIR - 
UNSCHEDULED DNA SYNTHESIS - 
MAMMALIAN CELLS IN VITRO B.18. 
MUTAGENICITY - IN VITRO MAMMALIAN 
CELL GENE MUTATION TEST B.17. 
SISTER CHROMATID EXCHANGE ASSAY 
IN VITRO B.19. 
IN VITRO MAMMALIAN CELL 
TRANSFORMATION TESTS B.21. 
MAMMALIAN SPERMATOGONIAL 
CHROMOSOME ABERRATION TEST B.23. 
 
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 genetically modified food and feed and shall also be required for the 
nutritional evaluation. Informa tion shall be provided by the applicant on th e intended 
function, the dietary role, and the expected level of use of the genetically modified 
food and feed in the EU. In addition, the e xpected range of conc entrations of newly 
produced proteins or existing plant proteins deliberately modified in the genetically 
modified food(s) and feed(s) to be pl aced on the market shall be provided. 
On the basis of representative consump tion data for products obtained from the 
respective conventional plants , the applicant shall estima te the anticipated average 
and maximum intake of the genetically modified food and feed. Probabilistic 
methods may be used to determine ranges of plausible values rather than single  
EN 62   EN values or point estimates. The applicant shall identify and consider particular groups 
of the EU population with an expected highe r exposure and shall consider this higher 
exposure within the risk assessment.  Any assumptions made in the exposure 
assessment shall be described. Recent developments in methodologies and appropriate consumption data shall be  used. Data on import and production 
quantities may provide additional information for the intake assessment.  
The applicant shall determine by appropria te methods the concentrations of the 
newly expressed proteins, other new cons tituents and endogenous food and feed 
constituents, of which the levels have been altered as a re sult of the genetic 
modification (for example, due to changes in metabolic pathways) in those parts of 
the genetically modified plant intended for f ood or feed use. Expect ed intake of these 
constituents shall be estimated taking into  account the influences of processing, 
storage and expected treatment of the food and feed in question, for example, 
potential accumulation or reduction. In cases where the genetic modification has 
resulted in an altered level of a natural c onstituent, or if a new constituent occurs 
naturally in other food and feed products, th e anticipated change in total intake of 
this constituent shall be assessed consider ing realistic as well as worst case intake 
scenarios.  
The applicant shall provide information on known or anticipated human/animal 
intake of analogous genetica lly modified food and feed  and on other routes of 
exposure to the respective new and natura l constituents, including amount, frequency 
and other factors influencing exposure. 
3. R
ISK CHARACTERISATION  
3.1. Introduction 
The applicant shall base its risk characteri sation of genetically modified plants and 
foods and feed on data from hazard identi fication, hazard char acterisation, and on 
exposure/intake data. The applicant shall en sure that the risk characterisation is 
comprehensive by considering all the avai lable 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 re quire specific activities for post-market 
monitoring of genetically modified food and feed. 
In performing its risk characterisation, the applicant shall demonstrate that the hazard 
identification and hazard characterisation are complete. The applicant shall discuss 
the quality of existing data and informa tion. The discussion shall clearly indicate 
how this body of information has been taken into account in the determination of the final risk characterisation . 
The applicant shall provide estimations of the uncertainties associated to each test as 
well as to the different stages of the ri sk assessment. The applicant shall quantify 
them to the possible extent.  A distinction shall be made between uncertainties that 
reflect natural variations in biologica l parameters (incl uding variations in 
susceptibility in populations), and varia tion amongst different species responses.   
EN 63   EN 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 
possible.  
3.2. Issues to be considered for risk characterisation 
When appropriate and depending on the type  of genetic modification, the applicant 
shall carry out a risk assessm ent of genetically modified plants in an integrative  
manner in accordance with Section 3.1. This  risk assessment sha ll be performed on a 
case-by-case basis depending on the modifi ed plant and the type of genetic 
modification, the cultivation practices of th e genetically modified plant and uses of 
the genetically modified food and feed. Th e applicant shall take  into account the 
different issues considered in hazard id entification and hazard characterisation and 
exposure steps. The outcomes of these issu es shall be considered by the applicant 
together in the risk characterisation step. Th e list of issues provi ded in this Section 
shall not be exhaustive. 
3.2.1. Molecular characterisation 
Evaluation of the characteris tics and previous use of the donor and the recipient plant 
shall be a key element to identify the need for specific analyses, such as the 
occurrence of specific toxins, or allergens in the unmodified recipient plant which 
may be unintentionally increased as 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 sequence analysis has identified a potential hazard, the applicant shall 
demonstrate how approaches like bioinfor matic analysis, com positional/agronomical 
analysis and possibly animal feeding trials with the whole genetically modified food 
and feed contribute to the safety assessment. The value of the results obtained shall be evaluated in the light of the availabl e knowledge on the structure and function of 
genomic databases of the crop specie s in question or related species. 
In cases of GM plants containing stacked events, the additional risks possibly arising 
from the combined effects of the stacked genes shall be evaluated. 
3.2.2. Comparative analysis  
The first goal of the comparative analysis is to identify possible differences between 
the GM plant and its conventional counter part and, where appropriate, additional 
comparators. The second goal of the compara tive analysis is to identify possible lack 
of equivalences between the GM plant and its reference varieties. These differences 
and/or lack of equivalences should be assessed with resp ect to their possible impact 
on food and feed safety and nutritional pr operties, taking into account natural 
variation. The estimated risk and associated  uncertainties should be as precise as 
possible and taken into account.  
EN 64   EN The applicant shall demonstrate that the comparative analysis between the 
genetically modified plant and its conve ntional counterpart with respect to 
agronomic, morphological and compositional ch aracteristics has been carried out in 
accordance with the requirements of this Regulation. The selection of the conventional counterpart and, where approp riate, additional comparators shall be 
justified. 
3.2.3. Food and feed safety in relation to intake  
The applicant shall evaluate the data gene rated to estimate possible short-term and 
long-term risks to human or animal hea lth associated with the consumption of 
genetically modified food or feed with respect to  the expression of new 
proteins/metabolites, as well as significan tly altered levels of original plant 
proteins/metabolites in genetically modifi ed foods/feed. This evaluation shall include 
a thorough analysis of the relevance and limitations of each test as well as of the whole information. 
The applicant shall consider the range of levels observed for the compounds known 
to occur in the conventional counterpart and in reference 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.  
If single constituents and/or whole genetic ally modified food a nd feed are found to 
induce adverse effects in specific studies, information on dose response relationships, 
threshold levels, delayed onset of adverse effects, risks for certain groups in the 
population, use of uncertainty f actors in extrapolation of animal data to humans shall 
be submitted.  
The applicant shall consider data on th e characteristics of the new compounds 
present in the genetically modified plant including potential biological effects in 
humans and animals.  If the compounds have known adverse health effects and 
maximum levels for the presence of thes e compounds in the plant or its products 
were laid down in specific legislation, th ese maximum levels shall be taken into 
account. Otherwise, reference values for acceptable or tolerable levels of intake, such as the acceptable daily intake (ADI) or tolerable upper intake level (UL), shall be considered in relation to the anticipated intake.  In cases where the compound has 
been safely consumed in food, the intake levels of consumers from a conventional 
diet shall be considered as safe. 
The applicant shall evaluate the information on the effects of processing on the new 
compounds. Potential accumulation/depletion in food and feed products entering the 
human or animal diet shall be considered . The applicant shall also evaluate the 
relevance of differences resulting from chemical reactions known to occur under 
processing conditions. 
In cases where more complex genetic modifications are produced, for example, via 
transfer of multiple genes in a single construct, re-transformation of pre-existing genetically modified lines, and stackin g of transformation events through  
EN 65   EN conventional breeding of genetically modifi ed parents, the app licant shall discuss 
strategies for the assessment of any risk(s ) associated with possible interactions 
between the newly expressed proteins, new metabolites and original plant 
constituents . The assessment shall take into account all available information 
including the mode of acti on of the newly expressed proteins, the molecular and 
compositional/agronomical charac teristics of the genetically  modified plant, and the 
outcome of animal toxicity studies and feeding trials. 
The applicant shall evaluate data obtained to  assess the allergenic potential of newly 
expressed proteins in genetically modified plants with respect to introduction of new 
allergenic proteins into th e food and feed plants, a possi ble provocation of allergic 
reactions of susceptible individuals, as we ll as information to demonstrate that the 
genetic modification does not cause unintende d changes in the characteristics and/or 
levels of expression of endogenous allergenic  proteins in the genetically modified 
food. In particular, the choice of the test models shall be  justified with respect to 
specificity, predictability  and validation status. 
With respect to intake estimations of genetically modified foods, the applicant shall 
evaluate the applied methodologies with resp ect to uncertainties associated with the 
prediction of long-term intake. Specific atte ntion shall be paid to those genetically 
modified plants which are aimed at modify ing nutritional characte ristics of the food 
and feed. For those genetically modified products, the requirement for post-market 
monitoring shall be discussed as a mechan ism for determining actual changes to 
overall dietary intake pattern s of the genetically modified  food, to what extent this 
has occurred and whether or not the pr oduct 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 the requirements of Articles 4 and 16 of Regulation (EC) 
No 1829/2003, the applicant shall ensure that the final risk characterisation clearly 
demonstrates that: 
(a) The genetically modified food and f eed has no adverse effects on human and 
animal health;  
(b) The genetically modified food does not differ from the food which it is 
intended to replace to such an extent that its normal consumption would be 
nutritionally disadvantage ous for the consumer; 
(c) The genetically modified food does not mislead the consumer; (d) The genetically modified feed does not harm or mislead the consumer by 
impairing the distinct ive features of the animal products;  
(e) The genetically modified feed does not differ from the feed which it is intended 
to replace to such an extent that its normal consumption would be nutritionally 
disadvantageous for animals or humans.  
EN 66   EN 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 th e application, in accordance w ith Article 13(2)(a) and (3) 
and Article 25(2)(c) and (3) of Regulation (EC) No 1829/2003.  
EN 67   EN ANNEX III  
 
VALIDATION OF METHODS FOR THE DETECTION, IDENTIFICATION AND 
QUANTIFICATION OF THE TR ANSFORMATION EVENT, AND 
REQUIREMENTS FOR CONTROL SAMPLES AND THE CERTIFIED 
REFERENCE MATERIAL  
1. I NTRODUCTION  
1. For the purposes of implementing Articles 5(3)(i) and (j) and 17(3)(i) and (j) of 
Regulation (EC) No 182 9/2003, this Annex sets out requirements on: 
(a) the performance characteristics of the submitted method(s);  (b) technical requirements regarding the type of information that the 
applicant must submit so as to verify  that those requirements are met.  
(c) samples of the food and feed  and their control samples; 
(d) certified reference material.  
2. The applicant must include informa tion on the method as such and on the 
method testing carried out by the applicant. 
3. The applicant shall also consider fu rther guidance and information about the 
operational procedures of the validation process that is made available by the 
EU Reference Laboratory (EURL) as refe rred to in Article 32 of Regulation 
(EC) 1829/2003, assisted by the Europ ean Network of GMO Laboratories
28. 
2. D EFINITIONS  
For the purpose of this Annex, the following definitions shall apply: 
(a) ‘certified reference material’ means reference material as referred to in Articles 
5(3)(j) and 17(3)(j) of Regulation (EC) No 1829/ 2003 and corresponds to any 
material or substance, one or more of  whose property values are certified for 
calibration or quality control of methods. It  is accompanied by a certificate that 
provides value of the specified propert y, its associated uncertainty and a 
statement of metr ological traceability; 
(b) ‘method performance requirements’ m eans the minimum performance criteria 
that the method shall demonstrate upon completion of the validation study 
carried out by the EURL, according to internationally accepted technical 
provisions.  
                                                 
28 http://gmo-crl.jrc.ec.europa.eu/guidancedocs.htm  
EN 68   EN 3. M ETHOD VALIDATION  
3.1. Information about the method 
A. The method(s) shall refer to all the methodological steps needed to analyse the 
relevant food and feed material in accord ance with Articles 5(3)(i) and 17(3)(i) 
of Regulation (EC) No 1829/2003.  
For a particular food or feed material, the methodol ogical steps sh all include 
the methods for DNA extraction and the subsequent quantification in a real-
time Polymerase Chain Reaction (PCR) System. In such a case, the whole process from extraction up to the PCR-t echnique shall constitute a method. The 
applicant shall provide information about the whole method. 
B. The applicant shall be allowed to refer to validated protocols, if available and 
appropriate, for method modules used in  the analytical procedure such as a 
DNA extraction protocol from a certain matrix.  
In that case, the applicant shall provide  experimental data from an in-house 
validation in which the method module ha s been successfully applied in the 
context of the applicat ion for authorisation. 
C. The applicant shall demonstrate that  the method(s) fulfils the following 
requirements. 
1. The method(s) shall be specific to the transformation event (hereafter 
referred to as "event-specific") and t hus shall only be functional with the 
genetically modified organism or genetically modified based product 
considered and shall not be functional if applied to other transformation events already authorised; otherwis e the method cannot be applied for 
unequivocal detection/identificatio n/quantification. This shall be 
demonstrated with a selection of non-target transgenic authorised 
transformation events and conventiona l counterparts. Th is testing shall 
include closely related transformation events. 
2. The method(s) shall be applicable to  samples of the food or feed, to the 
control samples and to the certified reference material    
3. The applicant shall take into consideration the following documents for 
the development of the detection method: 
(a) Foodstuffs -- Methods of analysis  for the detection of genetically 
modified organisms and derived products - General requirements 
and definitions: ISO 24276, 
(b) Foodstuffs -- Methods of analysis  for the detection of genetically 
modified organisms and derived pro ducts - Nucleic acid extraction: 
ISO 21571, 
(c) Foodstuffs -- Methods of analysis  for the detection of genetically 
modified organisms and derived pr oducts Quantitative nucleic acid 
based methods: ISO 21570,  
EN 69   EN  (d) Foodstuffs -- Methods of analysis  for the detection of genetically 
modified organisms and derived products Qualitative nucleic acid based methods: draft European standard ISO 21569. 
4. The method shall also take into consideration the more detailed 
requirements set out in the common criteria set by the EURL, and the 
ENGL for minimum performance requirements for analytical methods for GMO testing. These criteria are part of the guidance provided by the EURL.  
D. For the purpose of implementing Artic les 5(3)(i) and 17(3)(i) of Regulation 
(EC) No 1829/2003, the applicant shall pr ovide the event-sp ecific quantitative 
detection method(s) of the genetically modified material. The applicant shall 
discuss the validity and lim itations of the detection methods in the various 
types of foods and feeds (the various ma trixes) 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 clea rly addressed by the applicant: 
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 applicant shall indicate the matrix(es) (for 
example, processed food, raw material s), the type of samples and the 
percentage range to which the method may be applied. 
3. Operational characteristics of the method: The required equipment for the 
application of the method shall be speci fied, with regard to the analysis 
as such and the sample preparation. Further information of any specific 
aspects crucial for the ap plication of the method shall also be included.  
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 indepe ndently in other laboratories.  
5. A prediction model (or a similar tool) needed to interpret results and to 
make inferences shall be described in full details. Instructions for the correct application of the model sh all be provided by the applicant. 
6. Breeding schemes that are to be applied for the production of genetically 
modified food and feed and their impact on the in terpretation of results 
shall be provided by the applicant.  
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 point 3.1.C.4. The applic ant shall also provide a summary of  
EN 70   EN the testing carried out and the main result s 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, 
the applicant shall provide the results of  the testing of the method by at least 
one laboratory that is different from the laboratory which has developed the 
method.  
C. The applicant shall provide the following information about the method 
development and the method optimisation:  
1. primer pairs tested and probe, if ap propriate, including a justification as 
to how and why the proposed pr imer 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) in a standa rdised electronic format, together 
with the base pairs of the host fla nking sequences so as to enable the 
EURL to assess the specificity of the proposed method by running 
homology searches in a molecular database; 
4. precision, the relative rep eatability standard devia tion shall be less than 
or equal to 25 % related to mass fr action over the whole dynamic range 
of the method. 
D. The applicant shall, in addition to th e information required under Sections A, B 
and C provide the following inform ation regarding the testing:  
1. participating laboratories, time of the analysis and outline of the 
experimental design, including the de tails about the number of runs, 
samples, replicates etc., 
2. description of the labo ratory samples (such as size, quality, date of 
sampling), positive and negative controls as well as certified reference material, plasmids and alike used, 
3. description of the approaches that have been used to analyse the test 
results and outliers, 
4. any particular points observed during the testing, 5. references to relevant literature or technical provisi ons used in the 
testing. 
3.3. Samples of the food and feed and their control samples 
For the purpose of implementing Articles 5(3)(j) and 17(3)(j) of Regulation (EC) 
No 1829/2003, the applicant shall, together  with the information required under  
EN 71   EN Sections 1, 2 and 3 of this Annex, also provi de samples of the food and feed and their 
control samples of a type and amount to be specified by the EURL for the specific 
application for authorisation.  
The information accompanying the control samples shall include information on the 
breeding of the plant which has been used  for the production of the control samples 
and on the zygosity of the insert(s).  
The applicant may use the same raw material for the production of certified reference 
material and for the production of control samples. 
4. C ERTIFIED REFERENCE MATERIAL  
The certified reference material shall be produced under ISO Guide 34 (General 
requirements for the competence of refere nce material producers) by a producer 
accredited to ISO Guide 34. 
The applicant shall provide information as regards the place where the certified 
reference material can be accessed. This  shall be accompanied by adequate 
information demonstrating that the availabil ity of the certified reference material will 
be maintained throughout the period of va lidity of the authoris ation. For verification 
and value assignment, a method that has been properly validated (see ISO/IEC 
17025: General requirements for the competence of testing and calibration 
laboratories) shall be used.  
Uncertainties shall be estimated accordi ng to the ISO Guide to the Expression of 
Uncertainty in Measurement (GUM).  
The main characteristics of  those internationally accepte d technical provisions are 
the following:  
1. Genetically modified refe rence material containers: 
(a) genetically modified reference materi al container (such as bottles, vials, 
ampoules) shall be tight and contain not less than the stated amount of material; 
(b) the commutability of the genetically modified reference material must be 
assured; 
(c) packaging shall be a ppropriate to the purpose; 
(d) labelling shall be of  good aspect and quality. 
2. Homogeneity testing: 
(a) samples shall have appropriate homogeneity; (b) between-bottle homogeneity shall be examined;  
EN 72   EN (c) any possible between-bottle heteroge neity shall be accounted for in the 
overall estimated reference material uncertainty. This requirement shall apply even when no statistically sign ificant between-bottle variation is 
present. In this case, the method variation or the actual calculated 
between-bottle variation, whichever is larger, shall be included in the overall uncertainty. 
3. Stability testing: 
(a) samples shall have appropriate stability; (b) stability shall be positively dem onstrated by appropriate statistical 
extrapolation for the genetically modifi ed reference material shelf-life to 
be within the stated uncertainty; the uncertainty related to this 
demonstration is part of the estima ted reference material uncertainty. 
Assigned values are valid only for a lim ited time and shall be subject to a 
stability monitoring.  
4. Batch characterisation: 
1. The methods used for verifica tion and for certification shall: 
(a) be applied under metrologically valid conditions; 
(b) have been properly techni cally validated before use; 
(c) have precision and trueness compa tible with the target uncertainty. 
2. Each set of measurements shall: 
(a) be traceable to the stated references.  (b) be accompanied by an uncertainty statement whenever possible. 
3. Participating laboratories shall: 
(a) have the required competence for the execution of the task; (b) be able to achieve traceability to the required stated references; 
(c) be able to estimate their measurement uncertainty; (d) have in place a sufficient and a ppropriate quality assurance system. 
5. Final storage: 
1 To avoid degradation after sample production, all samples shall be stored 
under conditions designated for the final storage of the genetically modified certified reference material  before measurements are started. 
2. Otherwise, they shall be transported from door to door keeping them at 
all times under such storage conditions for which it has been demonstrated that there is no influence on the assigned values.  
EN 73   EN 6. Establishment of a certificate for certified reference material: 
A certificate complemented by a certification report shall be established, 
containing all information relevant  to and needed by the user.  
The certificate and report shall be ma de available when the genetically 
modified certified reference material is distributed.  
The information accompanying the certified reference material shall include 
information on the breeding of the plant which has been used for the production of the certified reference ma terial and on th e zygosity of the 
insert(s).  
The certified value of the GMO conten t shall be given in mass fraction and, 
where available, in copy number per haploid genome equivalent. 
Certified values (such as qua ntity of genetically modifi ed material expressed in 
mass fraction) shall be traceable to stated  references and be accompanied by an 
expanded uncertainty statement valid for the entire shelf-life of the genetically modified certified reference material.