TABLE OF ABBREVIATIONS.................................................................................................... I-199
Document: REPLIES BY
Date: 28 January 2005
Document codes: WT/DS291/R/Add.7; WT/DS292/R/Add.7; WT/DS293/R/Add.7
Complainant: Argentina
Respondent: EU
Third parties: Australia, Brazil, Canada, Chile, China, Taiwan, Colombia, El Salvador, Honduras, Mexico, New Zealand, Norway, Paraguay, Peru, Thailand, Uruguay, U.S.
Dispute stage: Mutually Agreed Solution
Agreements cited: Art. 2.2, 2.3, 5.1, 5.2, 5.5, 5.6, 7, 8, 10.1, Annex B, Annex C  Sanitary and Phytosanitary Measures (SPS)Art. III:4, X:1, X:3(a), XI:1  GATT 1994Art. 2.1, 2.2, 2.8, 2.9, 2.11, 5.1, 5.2, 5.6, 5.8, 12  Technical Barriers to Trade (TBT)Art. 4.2  AgricultureArt. I:1  GATT 1994

TABLE OF ABBREVIATIONS

ACRE UK Government's Advisory Co mmittee on Releases to the Environment
ACT Australian Capital Territory
AIA procedure Advance Informed Agreement procedure
ASEAN Association of South East Asian Nations
BINAS Biosafety Information Network and Advisory Service
Bt Bacillus thuringiensis
CA Competent authority
CTFBT Codex Alimentarius Commission established an Ad Hoc
Intergovernmental Task Force on Foods derived from Biotechnology
Directive 2001/18 Directive 2001/18/EC of the European Parliament and of the Council of
12 March 2001 on the deliberate release into the environment of
genetically modified organisms and repealing Council Directive
90/220/EEC
Directive 90/220 Council Directive 90/220/EEC of 23 April 1990 on the deliberate release
of genetically modified organisms
DNA Deoxyribonucleic Acid
ECJ European Court of Justice
EFSA European Food Safety Authority
EPA United States Environmental Protection Agency
FAO Food and Agriculture Organization of the United Nations
FDA Food and Drug Administration
FSE Crops and Farm Scale Evaluations
GATT 1994 General Agreement on Tariffs and Trade 1994
GEF Global Environment Facility
GILSP Good industrial large-scale practice
GM foods Food products containing, consisting or produced from GMOs
GM products Genetically modified products
GMHT Genetically Modified Herbicide Tolerant
GMOs Genetically modified organisms
HT Herbicide Tolerant
IANB UN Inter-Agency Network for Safety in Biotechnology
ICPM Interim Commission on Phytosanitary Measures
IOE The International Office of Epizootics
IPPC International Plant Protection Convention

LMO-FFPs Living modified organism intended for direct use as food or feed, or for
processing
LMOs Living modified organisms
NAS United States National Academy of Science
OECD Organisation for Economic Cooperation and Development
OSR Oilseed rape
Regulation 258/97 Regulation (EC) N° 258/97 of the European Parliament and of the Council
of 27 January 1997 concerning novel foods and novel food ingredients
SCP Scientific Committee on Plants
SPS Agreement Agreement on the Application of Sanitary and Phytosanitary Measures
TBT Agreement Agreement on Technical Barriers to Trade
UNEP United Nations Environmental Programme
UNIDO The United Nations Industr ial Development Organisation
WHO World Health Organisation
ORF Open Reading Frame (Protein Coding Sequence of the DNA)

I. INTRODUCTION
1. In this submission the European Communities responds to the invitation by the Panel to
comment on the advice provided by the Panel's experts, in the form of comments on their replies, and,
where appropriate, in the form of further scientific and technical evidence in relation to the Panel's
questions.
2. The European Communities has taken very seriously its task of providing science based
comments, to contribute to the Panel's understanding of the expert replies. In this regard the European
Communities has sought to rely on the most up-to-d ate science available. For the purpose of ensuring
scientific and technical accuracy of its comment s, the European Communities has also relied upon
independent scientific advice from internationally renowned scientists, as appropriate.
3. By way of introduction, the European Commun ities is pleased to note that the replies
provided by the Panel's experts very largely conf irm the Communities' view that the scientific and
technical issues involved in considering whether to authorise the release into the environment and the
marketing of GM products are very complex and of ten controversial. The independent experts have
confirmed that the science has developed considerably over the last ten years and is still evolving at a
fast pace. And most significantly they have confirmed that each case is to be taken on its own merits,
and indeed that each case was taken on its own merits . This in turn confirms the approach taken by
the European Communities and the Member States and is inconsistent with any claim as to a
'moratorium'.
4. The European Communities does not however agr ee with all the advice and every answer (or
specific parts thereof) provided by the experts, and will comment in detail below. These comments
follow the order of the Panel's questions and are divided into sections corresponding to the main
issues as identified by the Panel (Sections IV to VIII below).
5. At this stage, the European Communities will provide comments only in relation to a number
of replies, as, on these, it considers appropriate to submit them in advance of the meeting with experts.
This approach is intended to contribute to the Pane l's understanding of the experts' replies, having
regard also to the time resources available to comment within the rather tight deadlines.
6. One of the consequences of the lack of time is that this submission is not as concise and well
prepared as the European Communities would have wished. Some repetition has been unavoidable
and although this submission is long, it is not complete. The European Communities reserves its right
to revisit its replies in due course, as well as other replies and questions, and provide further
comments and scientific or technical evidence, as prov ided for in paragraphs 13 and 14 of the Panel's
additional working procedures for consultations with scientific and/or technical experts . The absence
of any response or comment in respect of an answer to any question should not necessarily be taken as
acceptance on the part of the Communities of the adequacy or accuracy of the response to the
question.
7. Before commenting on the replies to the qu estions, however, the European Communities
comments on the varying approaches taken by the va rious experts in undertaking their task and on
some general and methodological issues (Sections II and III). As will be explained below, this has a
bearing on the interpretation of the advice, on the wei ght to be given to the various expert opinions,
and consequently on the Panel's ability to make findings of fact on that basis.

8. In this submission, the European Communities does not discuss the questions of definitions
on which international organisations have recently pr ovided advice. As requested by the Panel in its
letter of 22 December 2004, these issues will be the s ubject of comment in a separate submission that
the European Communities will present to the Panel on 4 February.
9. The European Communities welcomes the oppor tunity to discuss these scientific and
technical issues further with the Panel and the experts on 17 and 18 February. On this occasion it
reserves its right to provide the Panel with further scientific and technical evidence in response to the
many aspects of the questions that remain unans wered. The European Communities may bring some
additional scientific and technical experts to that meeting as part of its delegation.
10. The European Communities is providing only one exhibit to this submission, Exhibit EC-156,
containing the unpublished scientific li terature referred to herein. Ot her documents that are publicly
available are not exhibited.
II. THE ADVICE AVAILABLE AND THE VARYING APPROA CHES OF THE
EXPERTS TO THEIR TASK
11. The European Communities wishes, through the Panel, to thank the Panel's experts for their
valuable contributions, which was extremely difficult in the light of the amount of information to
process, the number of issues to address, as well as the number of scientific and technical disciplines
to cover. The Communities is especially conscious of the fact that responses were provided within the
very short timeframe provided for in DSU proceedings.
A. T
HE INCOMPLETENESS OF THE RESPONSES
12. It is therefore not surprising that the Panel's experts could not cover all necessary disciplines
and aspects of the Panel's questions, and provided in several instances answers only on certain aspects
(or occasionally no answer at all), in accordance with what they had already made clear in their
correspondences from last November. They provided re plies, sometimes with detailed and extensive
elements on some specific aspects, which, all togeth er, left a large number of elements of the Panel's
questions unanswered.
13. There are at least 18 questions which are either not answered at all (e.g. 2, 26, 114), or for
which necessary elements of the replies are explicitly omitted by the experts. There are many more, as
identified below in the detailed comments of the European Communities, where necessary elements
of the replies are implicitly omitted by the experts, in accordance with their own scientific or technical
expertise, and/or their initial commitment identified in their correspondence of last November.
14. The European Communities has endeavoured to collect the necessary scientific and technical
evidence to provide the Panel with complementary elements of replies to the many aspects that remain
unanswered. It has not, however, had the necessary time to submit all this information at this stage,
and will seek to provide further evidence at the meeting with the Panel and the experts, if appropriate.
15. The European Communities also re grets, in light of the complexity of the advice sought by
the Panel, and more generally of this case, that often only one opinion (and sometimes a partial one)
has been made available to the Panel on many qu estions, or parts thereof. For many questions a
number of scientific disciplines and issues are invo lved, and these do not allow for much overlap
between the limited number of selected experts. It is doubtful whether the Panel can make definitive
findings of fact on the basis of advice which is, on its own terms, so limited and incomplete.

B. T
HE DIFFERENT APPROACHES OF THE EXPERTS
16. The Communities notes that the Panel's experts have taken very different approaches, and
frequently disagree among themselves, as indicated below. This confirms that there is legitimate room
for disagreement on many of the issues. Such disag reement contributes to uncertainty and is bound to
be taken into account by the Panel as it carries out its tasks.
17. After consultation and peer review with several independent scientists which all concur with
the same opinion, the European Co mmunities has come to the firm view that, among the four experts
which were allocated the whole set of questions , only three (Dr. Andow, Snow and Squire) have
adopted a balanced and fully reasoned approach , including an indication of their processes of
thinking. These experts have provided for transpar ency on their conclusions and well-structured
replies. They have critically evaluated the ava ilable information, questioned claims, and presented
their own rigorous and logic scientific thinking, based on their own expertise, and each within the
limited framework of their respective scientific disciplines.
18. Dr. Andow has been very thorough and plainly has carried out extensive work in preparing
his detailed replies, which are often accurate a nd relevant. He may however on some aspects, as
explained below, have overstepped his scientific expert mandate. Dr . Snow and Squire have covered
fewer issues, and often in less de pth than Dr. Andow, but they have nevertheless also given quite a
number of accurate, sensible and fair replies.
19. In contrast, the responses of Dr. Nutti are much less scientific, and largely superficial in
character. For the most part, no detailed explanati on has been given into the basis for her thinking.
Often Dr. Nutti has taken the information provided at face value without personal critical scientific
assessment regarding its data basis and validity. As a consequence, her replies, which miss references
of the most appropriate work, including as re gards Codex references, seem unbalanced and
incomplete as they often reach an identical conclu sion (usually a systematic pattern of disagreement
with any request for more information from Memb er States). The European Communities is bound to
conclude that her replies are frequently inconsistent and often lack proper scientific argumentation.
20. One aspect of her replies is also the low leve l of information requirements for food safety
assessments. Dr. Nutti advocates these in general terms, without proper consideration of the context of
the complete information available for each product at that time, and in particular the possible
unintended effects that can not be excluded in th e face of difficulties, such as missing data, missing
assessment or biased analysis, related to the mol ecular characterization of the specific GM product
being considered. As a consequence, her a pproach addresses only minor parts of possible
toxicological, nutritional or other potential food or f eed safety problems, and is therefore of limited
relevance. Coupled with Dr. Nutti's stated abse nce of expertise on environmental impacts the
European Communities has concluded that the views of this expert should be subject to careful and
critical scrutiny.
21. The European Communities judges that the responses by Dr. Andow, Dr. Snow and
Dr. Squire provide a more scientific and valuable source of expertise, addressing the issues that justify
comment and attention, and that the Panel should most valuably use and take into account to make its
findings.
22. As regards the two other experts, who have addressed a limited number of questions, they
have provided a reasonable insight into their own scientific thinking. The European Communities is
struck by the number of disagreements identified in their respective replies, even in relation to the
small number of issues they have addressed. It is noteworthy that they have very different and

opposite requirement in terms of the informa tion necessary to properly assess the molecular
characterization of the products in dispute: Dr . Healy seemingly has a much lower threshold of
requirement than Dr. Snape. Furthermore, acco rding to the assessment of scientists which are
specialized in and working on detection methods, Dr. Healy is often inaccurate and has almost a
systematic pattern of advice against the European Communities in her few replies addressing this
issue of detection methods.
C. T
HE INDEPENDENCE OF THE EXPERTS
23. The European Communities would like here to note that both Dr. Nutti and Dr. Healy are
employed by Government agencies working for regula tory authorities, including one of a third Party
in these disputes on the side of the complainants . By contrast, the other scientific experts are
independent. Dr. Nutti and to a lesser extend Dr. Healy (for instance on Q25), are also the only two
experts who have touched upon issues in a more gene ral way, going beyond their mandate or their
own field of scientific expertise.
D. THE ASSESSMENT OF THE REASONABLENESS OF TIME TAKEN
24. Dr. Andow is providing advice outside his field of expertise when he opines on the length of
time that would be appropriately taken to formulate an opinion or a request for further information. It
is obvious from his own advice that he has demonstrated his ability to process in a very short time and
in detail, a very large amount of relevant inform ation. However, it cannot be assumed that such
efficiency can be a benchmark for the facts involved in this dispute, which involved complex
regulatory frameworks and interaction between multipl e authorities within different jurisdictions, or
where a single relevant regulatory authority or legislator had to understand complex scientific
information on all issues at stake, call for its ow n scientific independent advice, including through
multiple external advisory bodies, process its or their opinions, interact with an applicant, and finally
formulate a request for further scientific information.
III. GENERAL AND METHODOLOGICAL ISSUES
25. There are a number of general and methodologi cal issues that the European Communities
finds more convenient to deal with separately before embarking on a detailed commentary on the
responses submitted by the Panel's experts to each of the Panel's questions. These are dealt with in
this Section. Some can be dealt with briefly, but one, relating to the evolution of risk assessment
techniques (sub-section P below), needs to be discussed in some detail.
A. COMPLEXITY
26. It is clear from the experts' replies that the science behind any assessment of the products in
dispute is complex, and how many different scientific fields of expertise must be involved to address
the issues to give a complete picture. In partic ular, the potential impacts of the products in dispute
may apparently only be uncovered and assessed by th e scientific analysis of complex networks of
food, agro-environmental or ecological webs, involving direct and indirect interactions between many
living organisms and systems. This has obviously raised significant methodological challenges for the
scientific community, with all that implies for governmental policy and decision-making.
B. C
ASE BY CASE ASSESSMENT
27. There is overwhelming evidence from the experts' scientific advice that each of the products
in dispute raises scientific and technical issues that are specific to the particular construct and genetic
modification that has been engineered in each of them, and to their intentional or unintentional use.

28. The generic trait (or traits) such as herbicide resistance, or disease resistance, introduced by
genetic modification in the GM products in disput e may relate to a common "basic" set of generic
issues (which may even be sometimes similar to so me issues raised by non genetically engineered
products). But it is also very clear from the experts replies that each product's specific characteristics
(e.g.: glufosinate or glyphosate resistance, Bt toxin expression, or a combination of them) raises for
each individual situation and product a range of new and extremely specific scientific questions. This
applies for each species at stake ( e.g.: sugar beet, o ilseed rape, maize, potato, etc…), and has to take
into account the genetic construction, the combin ation and the expression of these characters in each
product's genetic makeup, as well as how and in wh ich specific environment the product is going to
be intentionally or unintentionally used.
29. These case-specific considerations, which cannot be automatically derived from a common
set of basic and generic questions, are due in part to the fact that most of the specific characters
introduced in the GM products at stake (e.g.: AR MG, glufosinate or glyphosate resistance, systemic
endogenous Bt toxin expression, etc…) do not exist, ca nnot be obtained or have not yet been found, in
non genetically modified plants. The advice before th e Panel illustrates the need to take into account
the international consensus that a case by case and thorough approach is justified for each and every
genetically modified product. Each individual product has to be considered rigorously and assessed on
the basis of its own individual merits, taking into account the relevant set of specific information
pursuant to that very product, and its intentional or unintentional specific recipient environment.
C. S
YSTEMIC ISSUES (INTERACTIONS AND CUMULATIVE EFFECTS)
30. The advice before the Panel also confirms th at a case by case assessment may need to be
supplemented by the assessment of "systemic" impacts. Such impacts are specific to the combined use
of GM products in dispute, i.e. issues which are originating from potential impacts on "systems" (e.g.
agricultural or agro environmental, for instance) , namely potential changes induced in different
"systems" by the use, in combination or in tempor al sequence, of several different GM products, and
which can not be identified by a case by case approach.
31. Having regard to the experts' advice, these sy stemic questions often open up large areas of
scientific uncertainty. This is becau se the scientific or technical know ledge is often not complete, due
to the novel character of the GM products in disp ute, or the fact that the empirical and limited
experience gained in one type of system (for in stance in one of the three complainants' systems)
cannot be transposed to a corresponding system elsewhere.
D. FAMILIARITY
32. The scientific advice provided to the Panel clearly shows, in particular in the light of the
amount of scientific issues that were or are still unr esolved or unclear, that some issues have not yet
been studied at all, that the products in dispute ha ve regularly given rise to many new questions in the
last ten years, and that they remain at the forefr ont of scientific knowledge, bot h in terms of risks and
benefits. The Communities considers that the lack of familiarity with the issues raised must be taken
into account by the Panel in understanding the way new problems and corresponding requests for
further information have arisen over time, in the face of developing scientific understanding.
E. EVOLVING SCIENCE
33. It is also striking that the experts confir m how little was known on so many of the relevant
issues only 10 to 15 years ago, and how much the scientific understanding of many of these issues has
developed since then, including in international fora such as the Codex. Developments identified by

the experts include the identification of previous ly unsuspected areas of risks and impacts, or
identification of flaws in the way risk assessments may have been conducted in the past, both issues
which are still moving very fast forward today.
34. One of the explanations for the evolution of th e scientific understanding of the relevant issues
before the Panel is that the sience, by being co nfronted with the necessity to address such new
complex impacts and networks of interactions, for wh ich no or little data was available, had to make
necessary shortcuts and to take a reductionist appr oach. It thereby initiated the process of assessing
the relevance and likelihood of some potential impact s that were first identified, but as a consequence
of the shortcuts and the necessary reductionism of the scientific process, it largely overlooked many
issues which are now on the table in most countries confronted with the GM products in dispute,
including the three complainants. This has a majo r consequence, which is that a scientific risk
assessment opinion is only to be considered relevant at a certain point in time, within the context of
the specific impacts it had looked at, and within th e context of the scientific considerations and
methodologies that were available to it.
F. S
CIENTIFIC EVIDENCE OF RISKS OR ABSENCE OF RISKS
35. As noted by Dr. Andow and Dr. Squire, an ab sence of scientific evidence does not constitute
evidence of an absence of impacts or risks. This is in particular important in the case of these novel
products, when a relevant new scientific issue, raised in the context of these GM products'
assessments, has even not yet been studied, or not studied with proper scientific methodologies. Even
though science cannot be asked to prove an absence of risk or a negative result, the novel character of
the products in dispute and the issues they raise justifies the requests for new scientific information, in
a context where scientific understanding is constantly evolving and new issues of potential impacts of
GM products are continuously arising.
G. CONTROVERSY
36. In the limited areas of expertise in which there is overlap between the Panel's experts, or
where independent scientific opinion has been s ought by the European Communities on these expert
replies, it is noteworthy that there is extensive di sagreement between the experts or with independent
scientists. This indicates a clear lack of consensus in the scientific circles on the issues at stake in
these proceedings. This lack of consensus is partly explained by the questions addressed below. The
European Communities considers that the Panel cannot make definitive findings of fact in the face of
such conflict of views or uncertainty, beyond indicating the existence of such differences or
uncertainty.
H. ABSENCE OF AGREED SCIENTIFIC CRITERIA
37. As stated by Dr. Squire in his general notes on ecological and environmental standards, one of
the problems in addressing issues of science in these proceedings, is the absence of agreed criteria on
many issues (in scientific and regulatory circles), in cluding in respect of the information necessary to
perform a risk assessment and, also, the manner in which to interpret the relevant data. The novel
character of many of the issues at stake, which of ten emerged only within the past decade, is largely
responsible for the absence of agreed criteria. This contributes to further scientific uncertainty, in
particular as regards the impacts on the environment and the agro-ecosystem at large.
I. JUDGING WHEN THE SCIENTIFIC INFORMATION IS SUFFICIENT
38. It is apparent from the scientific advice now before the Panel, that there is no unique,
absolute, scientific cut off threshold available to d ecide whether a GM product is safe or not (the risk

assessment end point). From the experts opinion it appears that there is a general trend, as the
scientific data accumulate, that indicates a move towards the formulation of a clearer scientific
opinion. However, to decide when the available data is sufficient to conclude that an element of the
risk assessment is adequate or not still involves th e judgement of experts (ideally this should be
expertise which is independent of the regulatory au thorities and the legislator, as recommended by the
Codex guidance on risk analysis). More importan tly, experts may disagree on whether there is a
sufficiency of information (as they do in th e present case very frequently) without necessarily
disagreeing on the science. There may be a legitimate difference of view not on what is the state of
scientific knowledge, but how it is to be applied to a particular set of facts. It is clear that, expert
judgement and subjectivity are important elements of a science based risk assessment. In this regard,
it appears rather clearly from Dr. Healy's considera tions that she often believes that the information
available for a specific dossier may have been su fficient to reach a positive conclusion; yet she may
still agree remaining uncertainties exist and have be en identified. In her approach these are of low
significance – she may be less cautious (or precautionary ) than another expert -, while other experts,
on the same data, may have an equally valid judge ment and reach a different conclusion as to the
consequences to be drawn from such uncertainties. They may, for example, conclude that further
information is necessary to try eliminate uncertainties to a greater extent.
J. I
NTERPRETATION OF SCIENTIFIC INFORMATION
39. Another source of the experts divergent c onclusions derives from differences in data
interpretation – disagreements on the science. Diffe rences of individual experts' arguments and
approaches leading to their scientific advice, which is sometimes conflicting despite being faced with
the same sets of scientific facts and data, illustrates the subjective character of the Panel's experts' and
other scientists' opinions in this field. This indicat es that the scientific data at stake requires a
substantial amount of scientific interpretation. It also confirms that the novel character of these GM
products and the new issues they raise may require a different approach, and that any interpretation
cannot be based on agreed criteria and methodologies or on a non-existent scientific consensus.
K. AVAILABILITY OF RISK MITIGATION AND RISK MANAGEMENT TOOLS
40. There are two general points stemming from the experts' advice that merit attention in relation
to risk assessment and risk management in the c ontext of availability of mitigation and monitoring
tools. First, as for the science, it appears clearly from the experts advice that the availability and
appropriateness of mitigation measures has evolved over time (particularly since 1998) in parallel
with the evolution of scientific understanding of th e issues. Even if mitigation measures may be
available today for many of the potential impacts at stake, they are not available for all of them, and
they were not available only a few years back. Furthermore, even when they have been available for
some time, it has often been necessary to revise them as evolving science identified them as being
inappropriate.
41. Second, it is apparent that the introduction of any GM product into the environment may
cause an irreversible effect under certain conditions , in particular if a particular product, or a
particular genetic modification, has the ability to maintain or spread itself into the environment.
Therefore, if uncertainties remain for a particular product that may have these characteristics, any
available mitigation measure may be considered on policy grounds to be inadequate. This is especially
the case if the effects are irreversible.

L. P
OST MARKET MONITORING AND GENERAL SURVEILLANCE
42. Even if there is a degree of consensus among th e Panel's experts as regards the necessity for
post market monitoring of potential adverse impacts, they sometimes refer only to the monitoring of
risks identified in the process of risk assessment.
43. A number of the experts indicate that there mi ght be no need for monitoring if a particular
risk has not been identified. The European Communi ties disagrees with this view. It considers that it
may be necessary, in the face of the lack of fa miliarity with the new GM products at stake, to
implement monitoring plans as a general post marketing surveillance tool to identify unexpected
effects. This is consistent with international instruments. As scientific understanding of the issues has
evolved, the discovery of new potential impacts that had not been thought of, or identified in earlier
risk assessments, is an ample demonstration of th e validity of such approach. Dr. Andow, Dr. Snow
and Dr. Squire agree that some ecological or agro -environmental hazards predicted in the late 1990's
are only beginning to emerge now. This is the case for the molecular basis of the transformation,
horizontal gene flow, Bt and herbicide resistance management, and non target species.
44. Furthermore, if a risk is found to be of limited or no impact in an expert opinion, it is still
appropriate to consider the availa bility of general surveillance and sp ecific monitoring plan to check
the validity of that expert opinion, and to ensure that practices do not lead to more harmful outcomes
than anticipated. This may be th e case, for example, when monitori ng the impact of the occurrence,
maintenance or spread of occasional escapes from non cultivated crops with high weediness potential,
such as oilseed rape. In this respect, the Europ ean Communities would like to draw the attention of
the Panel to the latest international expert advi ce provided to the FAO in this field, which has
recommended that any responsible deployment of genetically modified crops needs to comprise the
whole technology development process, from pre-release risk assessment to post release monitoring.1
M. SURVEILLANCE AND FOOD SAFETY
45. As regards food safety, even if some GM pr oducts have been found to be safe and approved
on a large scale, for instance by the three compla inants, the lack of general surveillance and
consequently of any exposure data and assessment, means that there is no data whatsoever available
on the consumption of these products – who has eat en what and when. Consequently, one can accept
with a high degree of confidence that there is no acute toxicological risk posed by the relevant
products, as this would probably not have gone undetected – even if one cannot rule out completely
acute anaphylactic exceptional episodes. However, in the absence of exposure data in respect of
chronic conditions that are common, such as allergy and cancer, there simply is no way of
ascertaining whether the introduction of GM products has had any other effect on human health.
N. R
EMOVING UNCERTAINTIES WITH LARGE SCALE CULTIVATION
46. Some of the opinions put forward by the e xperts address the need to collect further
information, through large scale cultivation, in order to remove some remaining scientific
uncertainties. They touch in particular on large scale impacts on biodiversity and the agro-ecosystems,
in the light of the recently available info rmation on these issues. The European Communities
considers that it may be appropriate to request furt her information on the relevant issues prior to the
grant of the relevant authorization to cultivate th e product. Another regulatory regime, dealing with
consents for deliberate releases of genetically modified organisms for purposes other than placing on

1 FAO expert consultation on monitoring the environmental effects of GM crops. Available at:
<http://www.fao.org/newsroom/en/news/2005/89259/index.html>.

the market,
2 has already enabled several such studies to be conducted (see in particular the Farm Scale
Evaluation studies). Other such studies are currently under way.
O. DETECTION METHODS
47. On the availability of detection methods, and th eir relationship with risk assessment and risk
management, some of the Panel's experts seem to indicate that they are not related to the risk
assessment process, and hence, with the risk manage ment process, and that they are only available for
the purpose of identifying individual products.
48. The European Communities would like to make clear its view that detection methods,
including event-specific and quantitative methods, ar e necessary for the purpose of risk management,
even in the absence of an identified risk in the process of risk assessment. First, and independently of
any other identification purposes, they are necessar y to ensure compliance and control of compliance
with the legislative framework that provides for th e risk assessment (only products satisfying specific
legislative criteria for assessment and consents, pos itive opinion of advisory bodies, and/or specific
thresholds of presence, may be found to be presen t). This requires the help of these quantitative,
event-specific, detection methods.
49. Second, they may be necessary to enable the withdrawal of a specific product from the
market. This could be in case of the emergence or o ccurrence of a previously unanticipated risk, or in
case of new uncertainties as regards some significant ri sks that still need to be assessed, or in case of
illegal placing on the market of a product. The two latter conditions occurred in the United States
when the Starlink product had to be withdrawn from the food and feed chains a few years ago, even
when found only in minute traces.
P. N
EW DEVELOPMENTS OF RISK ASSESSMENT CONCEPTS FOR GENETICALLY MODIFIED
PRODUCTS
50. The development of risk assessment concepts in the last 10 to 15 years reflected the progress
in the understanding of unintended effects of GM pr oducts, and of biotechnological methods in plant
breeding. Experiences drawn from a growing body of risk assessment processes, for different GM
products, often indicate similar underlying problems . Early regulations (e.g. Directive 90/220/EEC)
for genetically modified organisms did not differen tiate between systemic e nvironmental and product
specific risks assessments, whereas most modern regulations differentiate between a general
environmental assessment and specific assessments for specific products, such as GM plants for
pharmaceuticals, foods and/or feeds, seeds, chemical s or even fibre products. Consequently, specific
risk assessment procedures were or are currently being developed for these products. These new
specifications resulted in a diversification for th e risk assessments. However, experience drawn from
the accumulated evidence identified several underl ying problems, most notably the need for a
molecular (genotypic) and phenotypic character isation and assessment of potential unintended
molecular effects was identified as the basis for the assessment in all fields.3

2 "Part B" or Directives 90/220/EEC and 2001/18/EC.
3 Andow, D.A., Hilbeck, A., 2004: Science-based risk assessment for non-target effects of transgenic
crops. – BioScience 54(7): 637-649. FAO/WHO, Safety assessment of foods derived from genetically modified
animals, including fish, a joint FAO/WHO expert consultation on food derived from biotechnology, Rome,
Italy, 17-21 November 2003. Available at
<http://www.who.int/foodsafety/biotech/meetings/en/gmanimal_reportnov03_en.pdf>.

1. The "substantial equivalence concept"
51. The concept that a comparison of a final produc t with one having an acceptable standard of
safety would provide an important element of safety assessments of GMOs was a commonly used
basis for the development of both food safety and environmental risk assessment.
4 This concept was
elaborated by FAO, WHO and OECD in the early 1990s and referred to as 's ubstantial equivalence'
for the assessment of GM foods. But already in 2000 an FAO/WHO consultation acknowledged that
this concept of substantial equivalence had attracted cr iticism from the perception that it was the end-
point of a safety assessment rather than being the starting point. 5 The consultation concluded that
consideration of compositional changes is not the so le basis for a valid determination of safety and
that safety can only be determined when the results of all necessary aspects under comparison, and not
merely comparisons of key constituents, are integr ated. Recently, the concept has further evolved to
the Comparative Safety Assessment for GMO foods. 6 By 2003/2004, most international frameworks
and guidance covering living modified organisms beca me effective, notably on environmental safety
(Cartagena Protocol on Biosafety), on food safety (Codex) and on plant health safety of genetically
modified products; with all systems being based on the concept of a case by case approach. Most
recently, the need for a very comprehensive molecu lar characterisation of each transformation event,
including the analysis of integrated constructs a nd the flanking region, as well as the need to address
potential unintended effects, wa s appreciated for food safety and environmental assessments. 7 These
ideas were also reinforced by several general reco mmendations of expert advice, such as for instance
a United States EPA FIFRA recent expert panel.8
2. Codex principles for the risk assessment of GM foods
52. The Codex Alimentarius Commission adopted the Principles for the Risk Analysis of Foods
Derived from Modern Biotechnology, and the dr aft Guidelines for the Conduct of Food Safety
Assessment of Foods Derived from Recomb inant-DNA Plants and Microorganisms in 2003. 9 T h e
Principles for the Risk Analysis – sets out important principles, such as those related to whole food

4 WHO, 1991. Strategies for Assessing the Safety of Foods Produced by Biotechnology, Report of A
Joint FAO/WHO Consultation. World Health Organisation, Geneva. Available at
<http://www.who.int/foodsafety/publications/biotech/1990/en/>.
5 FAO/WHO, 2000. Safety Aspects of Genetically M odified Foods of Plant Origin. Report of a Joint
FAO/WHO Expert Consultation on Foods Derived from Biotechnology, Geneva, Switzerland, 29 May - 2 June
2000. Food and Agriculture Organisation of the United Nations, Rome.
ftp://ftp.fao.org/es/esn/food/gmreport.pdf; Millstone, E. , Brunner, E., Mayer, S., 1999. Beyond 'substantial
equivalence'. Nature 401, 525-526; Schenkelaars, P. , 2002. Rethinking substantial equivalence. Nature
Biotechnology 20, 2, 119.
6 Kok, E.J., Kuiper, H.A., 2003. Comparative sa fety assessment for biotech crops. Trends in
Biotechnology 21, 439-444.
7 FAO/WHO, Safety assessment of foods derived from genetically modified animals, including fish, a
joint FAO/WHO expert consultation on food derived from biotechnology, Rome, Italy, 17 - 21 November 2003.
Available at <http://www.who.int/foodsafety/biotech/meetings/en/gmanimal_reportnov03_en.pdf>.
8 SAP Report No. 2004-05. MINUTES of Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA)
Scientific Advisory Panel (SAP) Meeting, June 8-10, 2004, Arlington, Virginia. A Set of Scientific Issues Being
Considered by the Environmental Protection Agency Regarding: Product Characterization, Human Health Risk,
Ecological Risk, And Insect Resistance Management For Bacillus thuringiensis (Bt) Cotton Products. 84 pages.
9 Principles for the Risk Analysis of Foods Derived from Modern Biotechnology; CAC/GL 44-2003;
Guideline for the Conduct of Food Safety Assessment of Foods Derived from Recombinant-DNA Plants;
CAC/GL 45-2003; Guideline for the Conduct of Food Safety Assessment of Foods Produced using
Recombinant-)DNA Microorganisms; CAC/GL 46-2003. Codex Alimentarius Commission, 2003. These were
provided to the Panel by the European Communities as part of Exhibit EC-44 to the first written submission.
Available at <ftp://ftp.fao.org/codex/standard/en/CodexTextsBiotechFoods.pdf>.

assessment, uncertainties, sustainability of data provi ded to the risk assessment, and review of current
risk assessment processes.
53. Common agreed principles for the safety assess ment dictate a case-by-case pre-market
assessment on the basis of a Comparative Safety Assessment (CSA). The CSA is basically a two-
tiered approach. The initial step is comprised of a thorough comparison (not limited to a simple
comparison of major key nutrients or phenotypes) with the closely related conventional food organism
counterpart (there is however continuous debate as to what may constitute a suitable control
counterpart), to identify any differences that may have safety implications for the consumer. This
comparison includes both thorough phenotypic characteristics as well as a thorough compositional
analysis (primary and secondary metabolites known to be bio-active). The second step comprises the
toxicological and nutritional evaluation of the identified differences between the food derived from
the GMO and its comparator. Hazard identification and characterization are typically the first steps in
any risk assessment, and an extensive molecular ch aracterisation of the inserted genetic material
construct is required as a prerequisite to enable a sound hazard identification and characterization.
54. The safety of newly introduced gene produc ts must be assessed on a case-by-case basis.
Following the phase of hazard identification, charact erization and food intake -assessment (including,
where necessary, target animal and/or whole feed studies), an integrated toxicological evaluation
(potentially covering both acute and chronic exposures) will combine all the information with relation
to the food safety of the GMO-derived food.
55. As is discussed elsewhere (see discussion in our comments on Q111), genetic modification
may induce dramatic unintended changes in th e composition of foods, which may have gone
undetected if only key nutriments comparison had been performed.
10 For the identification of
potentially occurring unintended effects, metabolic profiling methods have been proposed, and
different possibilities for profiling me thods have been characterized. 11 In addition to investigating
health risks directly associated with food prod uction, the Codex risk assessment has and is still
broadening to include indirect effects, and now also encompasses effects of new products on the
environment that may have an indirect impact on human health.12

3. The environmental safety assessment of GMOs

56. A case by case assessment considering each GM organism derived from a single
transformation event, or from each combination of them, as well as considering the different receiving
environments (intentionally or unintentionally), is broadly recognised as the best basic framework for
assessing environmental risks of GMOs. Internationally , in particular within the OECD, a concept of
familiarity was developed also in the framework of environmental safety of transgenic plants. The
concept may facilitate risk assessments, because achieving familiarity means having enough and
thorough information, in order to then be able to make a sound and experienced judgement on safety
or risk.
13 Unfortunately, as has been shown eloquently by the experts' replies, there is still

10 See, for instance, Metabolic Profiling Allows Comprehensive Phenotyping of Genetically or
Environmentally Modified Plant System s, Ute Roessner, Alexander Luedema nn, Doreen Brust, Oliver Fiehn,
Thomas Linke, Lothar Willmitzer, and Alisdair R. Fernie; The Plant Cell, Vol. 13, 11–29, January 2001.
11 Kuiper, H.A., Kok, E.J. , Engel, K.H. (2003) Exploitation of molecular profiling techniques for GM
food safety assessment. Current Opinion in Biotechnology 14, 238-243.
12 Haslberger, A.G., 2003. Codex guidelines for GM foods include the analysis of unintended effects.
Nature Biotechnology 21, 7, 739-741.
13 NAS (National Academy of Sciences) (2002) Envi ronmental Effects of Transgenic Plants:the Scope
and Adequacy of Regulation. Washington, DC: National Academy Press
http://books.nap.edu/books/0309082633/html/index.html. Co nner, A.J., Glare, T.R. and Nap.J.P. 2003. The

considerable lack of familiarity with many ecolo gical systems and interactions occurring both in
cultivated and natural environments.
57. Familiarity could then also be used to indi cate appropriate management practices, including
the evaluation whether standard agricultural practices are adequate or other management practices are
needed to manage the risk.
14 Familiarity depends also on having existing knowledge on the range of
environments in Europe, their agro-ecosystems, and the relevant ecological webs, in order to
determine the interaction of introduced organisms with these environments and to conduct the risk
assessments. In addition, risk ass essments will need to consider th at agricultural practices in one
specific geographical regions, may differ from that in another region. Furthermore, as discussed above
and in the relevant experts' replies, there are al so broader systemic agro-environmental risk issues
which need to be assessed on top of each individu al case by case assessment for a specific organism,
in a given environment. For example, to assess impacts of GM crops and associated management
practices on soil ecosystems it is now considered more appropriate to measure changes in soil
ecological functions (e.g. soil nutrient cycling) rather than monitor an individual indicator species.
15
58. Currently, the Cartagena Protocol on Biosafety (CPB) to the Convention on Biological
Diversity is the only internationa l regulatory instrument which addresses specifically the potential
adverse effects of GMOs on "the conservation and th e sustainable use of biological diversity", taking
also into account effects on human health. 16 This instrument calls fo r scientifically sound risk
assessments, and the establishment of appropriate risk management measures to prevent adverse
effects of living modified organisms (LMOs) on the conservation and the sustainable use of biological
diversity, taking also into account risks to human h ealth. In doing so, it provides in its Annex III for a
general set of risk assessment principles, met hodology, steps and points to consider. However, it
should be noted that the development of detailed guidance on risk assessment and risk management of
LMOs will only happen in the future, as it is curren tly on the agenda of the next meetings of the
parties to the CPB.
59. Annex III of the CPB focuses especially on id entification of any novel genotypic and
phenotypic characteristics that may have adverse eff ects on biological diversity in the likely potential
receiving environment. The information on the recei ving environment includes data on the location,
geographical, climatic and ecological characteristic s, including relevant information on biological
diversity and centres of origin of species containe d within the likely potential receiving environment
(region or country). As the focus of the CPB is bi odiversity, its consideration of human health safety
is limited, concentrating on situations in which the LMO itself may end up in the food supply, such as
might happen via trade of crop seeds, or as regards indirect human health effects that may arise from
direct impact of the GM crop and/or its management system on local biodiversity.
60. Recent work for the implementation of the CP B recommends, as regards the assessment of
non target environmental risks, the analysis of eff ects of GMOs on important indicator species in the
environment, before an assessment of effects on the biodiversity. This is mainly because a better
access is available to species assessment and there are serious methodological limitations for an

release of genetically modified crops into the Environment. The Plant Journal 33, 19–46. Poppy & Sutherland
2004; Physiol Ent 29: 257-268.
14 OECD 1993 SAFETY EVALUATION OF FOODS DERIVED BY MODERN
BIOTECHNOLOGY, CONCEPTS A ND PRINCIPLES. Available at
<http://www.oecd.org/dataoecd/57/3/1946129.pdf>.
15 Birch et al., 2004 In: Environmental Risk Assessment of Genetically Modified Organisms vol 1. a
case study of Bt maize in Kenya. Eds Hilbeck and Andow. CABI Publishing. pp 117-1850.
16 See the discussion in the first written submission of the European Communities, paras 87 to 112.

analysis of effects to biodiversity and the diversity of species in general.
17 Specifically, a non target
individual species risk assessment, for assessing the non target effects of GM crops, was considered to
be more useful than the conventionally used eco -toxicology or non-indigenous-species model: this
new ecological risk assessment model requires info rmation about the intended receiving environment
and aims to focus on the analysis of possible effects of a GMO on local species which are assessed as
having a specific and regionally-important ecologi cal function in this system or have special
conservation or heritage value to the country.
61. However, toxicity tests are obviously consider ably more difficult to conduct with endangered
species. Where, for instance, nativ e insects may have particular cultural value, studies to understand
their distribution and ecology in agricultural habitats, and methods for conducting laboratory
bioassays with them, may help with GM plant risk assessment.
18 However, not all species are
amenable to such bioassays, because they are often protected.
4. The plant health assessment of GMOs
62. Recently, within the framework of the International Plant Protection Convention, (hereinafter
"IPPC") guidance on the plant health risk assessment of LMOs has been developed in harmony and
mutual supportiveness with the principles of the CP B. This guidance (ISPM11), provides a useful and
detailed guidance to address essentially plant pest risk s (risks to plant health) that may arise from the
GM product itself. It is noteworthy, however, that the framework of the IPPC, although addressing
plant health in general, has focused its technical e xpertise and work on crop plant health, rather that
on health of any wild plant health to be found in the ecosystem, nor on the ecological food webs
involving non cropped wild plants to be found in na tural ecosystems in the same region as the GM
crop.
5. Starting points for assessing effects of GM Os on food safety, mediated through the
environment
63. Both the Codex principles for food safety and the risk assessment provided for by the CPB,
provide opportunities to more explicitly consider in teractions between food safety, feed safety and
environmental safety. For instance, the broadening of the Codex risk assessment to include indirect
effects, provides for assessing effects on the environment that may have an indirect impact on human
health.
19
64. Indirect effects on human health or the envi ronment may be described as effects occurring
through a causal chain of events, through mechanisms such as interactions with other organisms,
transfer of genetic material, or changes in use or management associated with the GM crop in the
region or country of use. 20 Indirect effects are likely to be de layed and possibly involve sub-chronic,
long term effects. Given examples include im pacts which can arise from changed agricultural
practices associated with the management of a geneti cally modified (GM) crop rather than directly
from the genetically modified crop itself. The a bove mentioned explanatory guide suggests that

17 Andow & Hilbeck (2004). Science-ba sed risk assessment for non-target effects of transgenic crops.
BioScience 54: 637-649. Birch et al., 2004 In: Environmental Risk Assessment of Genetically Modified
Organisms vol 1. a case study of Bt maize in Kenya. Eds Hilbeck and Andow. CABI Publishing. pp 117-1850.
18 O'Callaghan, Glare, Burgess and Malone (2005). E ffects of plants genetically modified for insect
resistance on nontarget organisms. Annu. Rev. Entomol. 50: 271-292.
19 Haslberger, A.G., 2003. Codex guidelines for GM foods include the analysis of unintended effects.
Nature Biotechnology 21, 7, 739-741.
20 CPB, explanatory guide. Available at <http://www.biodiv.org/doc/books/2003/B-01669.pdf> (last
visited on 31 January 2005).

questions related to human health effects cover the human health aspects that are linked to the state of
use and conservation of biological diversity, or the envi ronment in general. Such a direct link exists if
the health effect is consequent to an exposure to the risk in situ , for instance, if a farmer were to
develop an allergenic reaction to pollen from genetically modified plants; but it also exists indirectly
if the health effect resulted fro m effects or changes on biological diversity (indirect or secondary
effect). Direct effects on human health (e.g. cau sed by consumption of GM food, or exposure to
pharmaceuticals for humans or animals) would, however, be mainly covered by human health focused
instruments (Codex, WHO, OIE).
6. The animal health assessment of GMOs
65. It is finally critical to note that, as regards animal health impacts of GM plant or other GM
products (impacts on animals used for food, br eeding animals, or even on all relevant non
domesticated organisms from the animal kingdom), or as regards target or non target animal feed
safety, there is not yet much specific risk assess ment guidance developed by international expert
consultation or organisations. It is important to consider that the lack of identification of human health
risks in the risk assessment of a GM plant used as food, does not necessarily correlate with an absence
of risks on target or non target animals in the fr amework of feed safety, in particular for non
mammals. This relates to, among others, differe nces in animal physiology and metabolism,
parts/products of crop plants that are consumed, pr ocessing, and exposure (intake) levels. As has been
indicated by the OIE in its recent submission, the OI E's international standards are not linked directly
with modern biotechnology. Consequently, indirect environmental or human health effect that may
arise from direct impacts on animal health or GM plant induced imbalance in the animal interactions
with the ecosystem is still a largely an unexplored area.
Q. O
THER GENERAL ISSUES – DEALT WITH IN THE COMMENTS ON THE QUESTIONS
66. There are a number of other general issues that arise from the advice provided to the Panel
that the European Communities discu sses in commenting on the questi ons to which they relate, as
identified below in brackets after each issue. These include:
– Unintended effects, and general molecula r aspects of genetic modification, and
transgenics (Q111);

– Molecular characterization requirements (Q9);

– Correlation between molecular characterization and toxicological assessment,
according to substantial equivalence and comparative assessments (Q9 and Q111);

– Allergenicity assessment (Q111);

– Horizontal gene transfer (Q1 and Q2);

– Cross breeding and assessment of stacking of genetic modification (Q111);

IV. THE PANEL'S GENERAL QUESTIONS (N°S 1 TO 9 AND 110 TO 114)
Question 1

On the basis of the information before the Pane l, is there any scientific evidence to support the
hypothesis that antibiotic resistance marker genes (A RMG) pass from a biotech food or feed product
to bacteria or other micro-organisms present in the human or animal gut?

(a) If scientific evidence indicates that such an event could occur, what risks, if any,
would arise from that event? What is the comparative relevance or magnitude of this
risk in relation to the likelihood of such a transfer from other sources of antibiotic
resistance not involving the use of recombinant DNA technology?
(b) If such risks have been identified, what is the likelihood of adverse effects to human
or animal health, in light of the processing of raw biotech products into human food
or animal feedstuffs?
(c) Are these consequences relevant to the specific types of ARMG currently used in the
products at issue in this dispute? Please explain.
(d) If such risks have been identified, what risk management options are available to
mitigate those risks and what is their efficacy?
General comments

67. The Panel's first general questi on sought advice on the issue of whether, how, and what are
the consequences if antibiotic resistance marker genes (ARMG) could pass from GM food or feed
product to bacteria or other micro-organisms present in the human or animal gut. However, it sought
also in its next question equally important advice as to whether other routes than food or feed, and
persistence into the environment and in soil of plant DNA, may also lead to the development of
antibiotic resistance.
68. Unfortunately only one of the Panel's expert s, Dr. Nutti, has addressed the issue of ARMG,
and then has limited her comments to question 1 with the exception of sub-questions c) and d). Also,
Dr. Nutti does not respond to question 2, stating that it is a question relating to environmental safety
on which she does not have expertise.
69. Dr. Nutti's response is however characterised by a lack of detailed arguments and failure to
cite and discuss the most relevant and updated sci entific evidence, and also presents an erroneous
conclusion. It therefore is seriously misleading to those unfamiliar with the true state of scientific
knowledge and understanding. Dr. Nutti is definitively not correct in her statement that
To date, there are no reports that marker genes in plant DNA transfer to these
(microbial and mammalian) cells.
70. She is equally seriously misleading by not citing the most recent and relevant Codex guidance
as regards ARMG.
71. The only other contribution that Dr. Nutti ma kes is to seek to minimise with sweeping
statements the adverse consequences that would be ca used by a transfer of antibiotic resistance. This
is again very superficial, and she does not answer all parts of the Panel's question 1.
72. Because of the inadequacy, incorrectness and in completeness of the advice provided to the
Panel on related Questions 1 and 2, the European Communities provides its own analysis below on
each question. The European Comm unities has prepared this info rmation with the assistance of

scientists, experts in the field of antibiotic resistance and ARMG, and presents it as objectively as
possible so as to complete the information available to the Panel.
21
73. On the basis of this analysis, it can be concluded that:
 – The demonstration has been made that antibiotic resistance genes from GM plants
can be captured and expressed by bacteria, including pathogenic bacteria.

 – Based only on the few studies that exist, th e occurrence in nature of such an event is
considered as low but not zero.

21 The following are the complete references relating to the discussion in this section:
Bertolla F, Kay E, Simonet P. Potential dissemination of antibiotic resistance genes from transgenic
plants to microorganisms. Infect Control Hosp Epidemiol. 2000, 21:390-3.
Coelho J, Woodford N, Turton J, Livermore DM. Multiresistant Acinetobacter in the UK: how big a
threat? J Hosp Infect. 2004, 58:167-9.
de Vries J, Heine M, Harms K, Wackernagel W. Spread of recombinant DNA by roots and pollen of
transgenic potato plants, identified by highly specific biomonitoring using natural transformation of an
Acinetobacter sp. Appl Environ Microbiol. 2003,69:4455-62.
de Vries J, Herzfeld T, Wackernagel W. Transfer of plastid DNA from tob acco to the so il bacterium
Acinetobacter sp. by natural transformation. Mol Microbiol. 2004;53:323-34.
de Vries J, Wackernagel W. Integration of foreign DNA during natural transformation of Acinetobacter
sp. by homology-facilitated illegitimate recombination. Proc Natl Acad Sci U S A. 2002, 99:2094-9.
Droge M, Puhler A, Selbitschka W. Horizontal gene transfer as a biosafety issue: a natural
phenomenon of public concern. J Biotechnol. 1998, 64:75-90.
Duggan PS, Chambers PA, Heritage J, Forbes JM. Survival of free DNA encoding antibiotic resistance
from transgenic maize and the transformation activity of DNA in ovine saliva, ovine rumen fluid and silage
effluent. FEMS Microbiol Lett. 2000, 191:71-7.
Duggan PS, Chambers PA, Heritage J, Michael Forbes J. Fate of genetically modified maize DNA in
the oral cavity and rumen of sheep. Br J Nutr. 2003, 89:159-66.
Gebhard F, Smalla K. Monitoring field releases of genetically modified sugar beets for persistence of
transgenic plant DNA and horizontal gene transfer. FEMS Microbiol Ecol 1999, 28;261-72.
Gebhard F, Smalla K. Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet
DNA. Appl Environ Microbiol. 1998, 64:1550-4.
Gordon KA, Jones RN; SENTRY Participant Group s (Europe, Latin America, North America).
Susceptibility patterns of orally admi nistered antimicrobials among urinar y tract infection pathogens from
hospitalized patients in North America: comparison repor t to Europe and Latin America. Results from the
SENTRY Antimicrobial Surveillance Program (2000). Diagn Microbiol Infect Dis. 2003, 45:295-301.
Jain R, Danziger LH. Multidrug-resistant Acinetobacter infections: an emerging challenge to clinicians.
Ann Pharmacother. 2004,38:1449-59.
Kay E, Vogel TM, Bertolla F, Nalin R, Simonet P. In situ transfer of antibiotic resistance genes from
transgenic (transplastomic) tobacco plants to bacteria. Appl Environ Microbiol. 2002, 68:3345-51.
Mercer DK, Scott KP, Bruce-Johnson WA, Glover LA, Flint HJ. Fate of free DNA and transformation
of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Appl Environ Microbiol.
1999, 65:6-10.
Mercer DK, Scott KP, Melville CM, Glover LA, Flin t HJ. Transformation of an oral bacterium via
chromosomal integration of free DNA in the presence of human saliva. FEMS Microbiol Lett. 2001,200:163-7.
Nielsen KM, Bones AM, Smalla K, van Elsas JD. Horizontal gene transfer from transgenic plants to
terrestrial bacteria--a rare event? FEMS Microbiol Rev. 1998, 22:79-103.
Nielsen KM, van Elsas JD, Smalla K. Transformation of Acinetobacter sp. strain
BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection
of transformants. Appl Environ Microbiol. 2000, 66:1237-42.
Opinion of the Scientific Panel on Genetically Modi fied Organisms on the use of antibiotic resistance
genes as marker genes in genetically modified plants. The EFSA Journal 2004:48;1-18
Paget E, Simonet P. On the track of natural tr ansformation in soil. FEMS Microbiol Ecol 1994,
15:109-18.

 – The risk for human health should be an alysed case by case. The kanamycin resistance
gene presents the lowest level of risk, th e ampicillin and streptomycin/spectinomycin
resistance genes present a higher degree of risk (although still limited). In particular,
the fact that the resistance genes are already naturally present limits the risk.

Detailed comments

74. Dr. Nutti refers the Panel to an old FAO/WH O expert consultation from 2000 to state that
DNA transfer from food derived from plants to microbial or mammal cells, under normal
circumstances of dietary exposure, would require all the following events to occur:
 – the relevant gene(s) would have to be released, probably as a linear fragment;
 – the gene(s) would have to survive nucleas es in the plant and in the gastrointestinal
tract;
 – the gene(s) would have to compete for uptake with dietary DNA;
 – the recipient bacteria or mammalian cells would have to be competent for
transformation
 – and the gene(s) would have to survive their restriction enzymes; and
 – the gene(s) would have to be inserted in the host DNA by rare repair or recombinant
events.

75. This citation may be correct, but Dr. Nutti omits to cite the most relevant and recent Codex
guidance as regards ARMG. The Codex guidelines for the conduct of food safety assessment of foods
derived from recombinant-DNA plants
22, provide detailed guidance on the use of antibiotic resistance
marker genes in its paragraphs 55 to 58:
55. Alternative transformation technologies that do not result in antibiotic
resistance marker genes in foods should be used in the future development of
recombinant-DNA plants, where such technol ogies are available and demonstrated to
be safe.
56. Gene transfer from plants and their food products to gut microorganisms or
human cells is considered a rare possi bility because of the many complex and
unlikely events that would n eed to occur consecutively. Nevertheless, the possibility
of such events cannot be completely discounted.
57. In assessing safety of foods containing antibiotic resistance marker genes, the
following factors should be considered:
A) the clinical and veterinary use and importance of the antibiotic in question;
(Certain antibiotics are the only drug availabl e to treat some clinical conditions (e.g.
vancomycin for use in treating certain st aphylococcal infections). Marker genes
encoding resistance to such antibiotics s hould not be used in recombinant-DNA
plants.)
B) whether the presence in food of the enzyme or protein encoded by the
antibiotic resistance marker gene would comp romise the therapeutic efficacy of the
orally administered antibiotic; and (This assessment should provide an estimate of the
amount of orally ingested antibiotic that could be degraded by the presence of the

22 CAC/GL 45-2003.

enzyme in food, taking into account fact ors such as dosage of the antibiotic, amount
of enzyme likely to remain in food following exposure to digestive conditions,
including neutral or alkaline stomach cond itions and the need fo r enzyme cofactors
(e.g. ATP) for enzymatic activity and estim ated concentration of such factors in
food.)
C) safety of the gene product, as woul d be the case for any other expressed gene
product.
58. If evaluation of the data and information suggests that the presence of the
antibiotic resistance marker gene or gene product presents risks to human health, the
marker gene or gene produc t should not be present in the food. Antibiotic resistance
genes used in food production that encode resistance to clini cally used antibiotics
should not be present in foods [emphasis added, footnote omitted].
76. The Codex Guidelines clearly addresses the ri sk management options that should be
considered, and, as will be discussed below, many of the ARMG present in the products at stake in
these disputes encode resistance to antibiotics that ar e clinically used, even if in a limited number of
pathologies, and therefore require a more careful scrutiny than advocated by Dr. Nutti.
77. Similarly, the Codex guidelines for the conduc t of food safety assessment of foods produced
using recombinant-DNA Micro-organisms (CAC /GL 46-2003), provide detailed guidance on
antibiotic and gene transfer in its paragraphs 51 to 54:
51. In general, traditional strains of microorganisms developed for food
processing uses have not been assessed for antibiotic resistance. Many
microorganisms used in food production possess intrinsic resistance to specific
antibiotics. Such properties need not excl ude such strains from consideration as
recipients in constructing recombinant- DNA microorganisms. However, strains in
which antibiotic resistance is encoded by transmissible genetic elements should not
be used where such strains or these genetic elements are present in the final food.
Any indication of the presence of plasmids , transposons, and integrons containing
such resistance genes should be specifically addressed.
52. Alternative technologies, demonstrated to be safe, that do not rely on
antibiotic resistance marker genes in viab le microorganisms present in foods should
be used for selection purposes in recombinant-DNA microorganisms. In general, use
of antibiotic resistance markers for constr ucting intermediate strains should pose no
significant hazards that would exclude th e use of the ultimate strains in food
production, provided that the antibiotic resi stance marker genes have been removed
from the final construct.
53. Transfer of plasmids and genes between the resident intestinal microflora and
ingested recombinant-DNA microorgani sms may occur. The possibility and
consequences of gene transfer from recombinant-DNA microorganisms and food
products produced by recombinant-DNA mi croorganisms to gut microorganisms or
human cells should also be considered. Transferred DNA would be unlikely to be
maintained in the absence of selective pressu re. Nevertheless, the possibility of such
events cannot be completely discounted.
54. In order to minimize the possibility of gene transfer, the following steps
should be considered:

– chromosomal integration of the inserted genetic material may be preferable to
localization on a plasmid;
– where the recombinant-DNA microorganism will remain viable in the
gastrointestinal tract, genes should be avoided in the genetic construct that
could provide a selective advantage to recipient organisms to which the
genetic material is unintentionally transferred; and
– sequences that mediate integration in to other genomes shoul d be avoided in
constructing the introduced genetic material.

78. As indicated earlier, Dr. Nutti's statement that:
To date, there are no reports that marker genes in plant DNA transfer to these
(microbial and mammalian) cells.
is patently not correct, as several scientis ts have demonstrated such gene transfer in natura23 or under
simulated environmental conditions.24 Extracts from work cited follows:

Uptake of DNA from GM plants and transformation of micro-organisms may occur
along the complete chain of events, extending from the field to food processing and
storage, to the digestive tract and finally again to the environment. The highest
probability of transformation events to occur can be expected when the
"concentrations" of the "reactants", i.e. transformable DNA and competent bacteria
are high. Highest microbial counts in a food of plant origin are found in food
fermentation processes such as they occur in the production of Sauerkraut, fermented
olives, tomatoes, cucumbers, egg-plants,
25 beer, sourdough, and many Asian foods
based on soy, beans, peanuts, cereals or coconut. Fermentation processes are finally
also important in post harvest treatment of coffee and cacao. The numbers of bacteria
involved in these processes may exceed 10 9 cells per gram, and the species involved
comprise bacteria from virtually all genera included in the group of lactic acid
bacteria, bacilli (in Asian food), acetic acid bacteria, enterobacteria. Representative
forms of many of these organisms of these groups have been shown to be
transformable with DNA (Lorenz and Wackernagel, 1994)
26.
Examples of the insertion of GM plant DNA into bacterial chromosomes under
simulated environmental c onditions have all involved highly homologous regions
between an antibiotic resistance marker ge ne and an identical gene present in the
recipient bacterial chromosome, known as marker rescue experiments. 27 The key

23 Simonet et al, 2004.
24 G. van den Eede, H. Aarts, H.-J. Buhk, G. Corthier, H.J. Flint, W. Hammes, B. Jacobsen, T.
Midtvedt, J. van der Vossen, A. von Wright, W. Wackernagel, A. Wilcks. Food and Chemical Toxicology 42
(2004), 1127-1156.
25 Buckenhüskes, H.J., Hammes, W.P., 1990. Starterkulturen bei der Verarbeitung von Obst und
Gemüse. Bioengineering 2, 34–42. Buckenhüskes, H.J., 1 993. Selection criteria for lactic acid bacteria to be
used as starter cultures for various food commodities. FEMS Microbiology Reviews 12, 253–272.
26 Lorenz, M.G., Wackernagel, W., 1994. Bacterial gene transfer by natural genetic transformation in
the environment. Microbiological Reviews 58, 563–602.
27 Gebhard, F., Smalla, K., 1998. Transformation of Acinetobacter sp. strain BD413 by transgenic
sugar beet DNA. Applied and Environmental Microbiology 64, 1550–1554. Nielsen, K.M., van Elsas, J.D.,
Smalla, K., 2000. Transformation of Acinetobacter sp. strain BD413 (pFG4 nptII) with transgenic plant DNA in

question must be whether such events can lead to the capture of adjacent genes
flanking the region of homology. Currently we can state that this possibility does
exist where the homologous region is present in a circular molecule, or where
multiple regions of homology are present. B ased on most recent findings in different
bacterial species
28 a "homology facilitated illegitimate recombination" can increase
the frequency of a basically illegitimate in corporation of genes when heterologous
DNA of up to 2.9 kb is flanked by a s hort sequence homologous to the integration
site. These findings show that the incorporation of a foreign gene and its expression is
basically possible, although such an event has not yet been shown to take place in
environments in which the DNA is released from the plant genome as it occurs e.g. in
the intestinal tract.
79. There is now wide international agreement th at horizontal transfer of genes, especially
ARMG, from GM plants or products thereof to micro-or ganisms, especially in the gut, is unlikely, but
not impossible. Therefore a case by case risk ass essment is necessary. Work under the EU 5
th
framework project GMOBILITY has shown in in vitro models that transformation and integration of
plasmids is obviously possible in any part of the g astrointestinal tract, and that stability of DNA from
GM plants is sufficient to enable small amounts of DNA fragments to survive the passage to distal
parts of the gastrointestinal tract. 29 However, no transfer could be detected in in vivo digestive tract
models, reflecting also small lik elihood. Two papers recently pub lished in Nature Biotechnology
indicate that current methods for monitoring horizonta l gene transfer from transgenic crops to micro-
organisms are very problematic and too insensitive to detect horizontal gene transfer. As such, even
though efforts so far have largely failed to observe horizontal gene transfer in the field or have
deemed frequencies too low or too rare to pose risks, this may not be true and the reality may clearly
be different. The analysis by scientists from th e New Zealand and Norwegian Institutes of Gene
Ecology criticizes contemporary horizontal gene transfer risk assessment of transgenic crops.30
80. Equally, the Codex guidelines cited above indicat es that gene transfer from plants and their
food products to gut micro-organi sms or human cells is considered a rare possibility, but that
nevertheless, the possibility of such events cannot be completely discounted.

soil microcosms and effects of kanamycin on selection of transformants. Applied and Environmental
Microbiology 66, 1237–1242. de Vries, J., Wackernagel, W., 1998. Detection of nptII (kanamycin resistance)
genes in genomes of transgenic plants by marker-rescue transformation. Molecular and General Genetics 257,
606–613.
28 de Vries, J., Wackernagel, W., 2002. Integration of foreign DNA during natural transformation of
Acinetobacter sp. by homology-facilita ted illegitimate recombination. Pro ceedings of the National Academy of
Sciences of the United States of America 99, 2094–2099. Prudhomme, M., Libante, V., Claverys, J.P., 2002.
Homologous recombination at the border: insertion-deletions and the trapping of foreign DNA in Streptococcus
pneumoniae. Proceedings of the National Academy of Sciences of the United States of America 99, 2100–2105.
Meier, P., Wackernagel, W., 2003. Monitoring the spread of recombinant DNA from field plots with transgenic
sugar beet plants by PCR and natural transformation of Pseudomonas stutzeri. Transgenic Research (in press)
29 GMOBILITY. G. van den Eede, H. Aarts, H.-J. Buhk, G. Corthier, H.J. Flint, W. Hammes, B.
Jacobsen, T. Midtvedt, J. van der Vossen, A. von Wright, W. Wackernagel, A. Wilcks. Food and Chemical
Toxicology 42 (2004), 1127-1156.
30 Heinemann JA and Traavik T. 2004 "Problems in mon itoring horizontal gene transfer in field trials
of transgenic plants" Nature Biotechnology 22, 1105-1109. Nielsen, K.M. and Townsend,J.P., 2004,
Monitoring and modeling horizontal gene transfer. Nat.Biotechnol., 2004, 22, 9, 1110-1114.

81. More recently, the latest FAO/WHO expert consultation on foods derived from GM animals
31
agreed that the DNA construct used should be car efully considered within an assessment, since
horizontal transfer or recombination may occur. Ad ditionally, bacterial vector-derived materials may
include additional sequence fragments unrelated to the target gene.32 There was also the recognition of
potential for horizontal transfer of the gene c onstruct as food-ingested foreign DNA may not be
completely degraded in the gastro intestinal tract of mice and pigs. 33 For the food safety assessment, it
is therefore prudent to assume that DNA fragments may survive the human gast rointestinal tract and
be absorbed by either the gut microflora or somatic cells lining the intestinal tract.
82. Several commercially available GMOs are fitted with antibiotic resistance genes which have
been used as marker genes in th e construction of genetically modifi ed plants. Concerns have arisen
which are of two types:
 – The antibiotics administered orally in a patient could be inactivated by antibiotic
resistance proteins present in food derived from a GM plants.

 – The resistance genes could escape to be integrated in pathogen bacteria and
expressed.

83. The first concern does not seem to be medically important on the basis that: resistance genes
are generally not expressed in plants and that inactiv ating enzymes are for the most part inactivated in
the gut. Anyway, these inactivating enzymes are al so produced by resistant bacteria present as
colonisers of gut, oral cavity and other mucosal su rfaces and there is no clinical evidence for any
impact in terms of therapy failure.
84. Only the second concern is the object of the general question 1. The European Communities
will discuss below, first the detaile d mechanisms and likelihood of such gene transfer, and thereafter
the risks that this would pose for human (or animal) health, as well as risk management issues.
85. The transfer of cloned ARMG from genetically modified plants to pathogenic bacteria
requires numerous steps (WHO/FAO, 2000, see Dr. Nutti's reply) for the process to occur under
natural conditions. These may be summarized as being:
• A. DNA from GMOs has to be released from plan ts and to persist in an intact and active
form in soils or in the gut of humans or animals fed with GM plants.
• B. DNA from GMOs present in soils or in th e oral cavity of humans or animals has to
penetrate into bacteria, the foreign gene has to be integrated and should be able to achieve
production of resistance proteins.
• C. The foreign DNA integrated into the genome of soil bacteria or non pathogenic
bacteria has to be transferred to pathogenic bacteria.

31 FAO/WHO, Safety assessment of foods derived from genetically modified animals, including fish, a
joint FAO/WHO expert consultation on food derived from biotechnology, Rome, Italy, 17 - 21 November 2003.
Available at <http://www.who.int/foodsafety/biotech/meetings/en/gmanimal_reportnov03_en.pdf>.
32 Environmental Effects of Transgenic Plants. The Scope and Adequacy of Regulation. National
Research Council, 2002. National Academy Press, Washington, 320 pp.
33 Chowdhury EH, Shimada N, Murata H, Mikami O, Sultana P, Miyazaki S, Yoshioka M, Yamanaka
N, Hirai N & Nakajima Y (2003): Detection of Cry1Ab protein in gastrointestinal contents but not visceral
organs of genetically modified Bt11-fed calves, VETERINARY AND HUMAN TOXICOLOGY 45, pp. 71-75.
Schubbert et al., 1997; Schubbert et al., 1998.

A. The DNA from GMOs has to be released from plants and to persist in an intact and
active form in soils or in the gut of humans or animals fed with GM plants

86. What do we know? We know that there is DNA in the soil which resu lts from the lysis of
cells. For the most part this DNA is degraded. However, a detectable quantity can still be found after a
long period of time. Intact DNA of GM plant was detected by a highly sensitive molecular technique
(PCR) more than two years after the harvesting of the crops
34. The longest persistence of DNA in field
soil was observed with soil from a potato field in 1997 sampled in the following year in April and
then stored moist at 4 degrees C in the dark for 4 years prior to extract preparation and
transformation35. The quality of the DNA has been testified by its capacity to penetrate into bacteria
and to be integrated into their genome experimentally.
87. In the oral cavity of humans, again the DN A was rapidly degraded but a few intact DNA
persisted which was demonstrated to be active 36. Similarly, Duggan et al. 37 found that in sheep saliva
and rumen fluid active DNA still persisted but was ra pidly degraded in the gastro-intestinal tract.
Also, intact transgenes from silage are unlikely to survive significan tly in the rumen whereas DNA in
maize grains persists for a significant time and may, therefore, provide a source of transforming DNA
in the rumen
38.
88. In conclusion therefore, there is convincing ev idence that DNA sufficiently intact to penetrate
into bacteria can persist at low levels in the soil , in the oral cavity of humans and animals but that
DNA is nearly completely destroyed in the lower intestinal tract.
B. The DNA from GMOs present in soils or in the oral cavity of humans or animals has to
penetrate into bacteria, the foreign gene has to be integrated and should be able to
achieve production of resistance proteins

89. The way of penetration of extra-cellular DNA into bacteria is called "natural transformation".
Only a limited number of bacteria have been iden tified (nearly 40 species) which have the cellular
machinery to be transformed. These species include a few pathogenic or non pathogenic (commensal)
bacterial species which are usual hosts of the oral cavity of humans and animals ( Streptococcus,

34 Paget E, Simonet P. On the track of natural tr ansformation in soil. FEMS Microbiol Ecol 1994,
15:109-18. Gebhard F, Smalla K. Monitoring field releases of genetically modified sugar beets for persistence
of transgenic plant DNA and horizontal gene transfer. FEMS Microbiol Ecol 1999, 28;261-72. Gebhard F,
Smalla K. Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet DNA. Appl Environ
Microbiol. 1998, 64:1550-4. Kay E, Vogel TM, Bertolla F, Nalin R, Simonet P. In situ transfer of antibiotic
resistance genes from transgenic (tra nsplastomic) tobacco plants to bacteria. Appl Environ Microbiol. 2002,
68:3345-51.
35 de Vries J, Heine M, Harms K, Wackernagel W. Spread of recombinant DNA by roots and pollen of
transgenic potato plants,identified by highly specific biomonitoring using natural transformation of an
Acinetobacter sp. Appl Environ Microbiol. 2003,69:4455-62.
36 Mercer DK, Scott KP, Bruce-Johnson WA, Gl over LA, Flint HJ. Fate of free DNA and
transformation of the oral bacterium Streptococcus gordonii DL1 by plasmid DNA in human saliva. Appl
Environ Microbiol. 1999, 65:6-10. Mercer DK, Scott KP, Melville CM, Glover LA, Flint HJ. Transformation of
an oral bacterium via chro mosomal integration of free DNA in the pr esence of human saliva. FEMS Microbiol
Lett. 2001,200:163-7.
37 Duggan PS, Chambers PA, Heritage J, Forbes JM. Survival of free DNA encoding antibiotic
resistance from transgenic maize and the transformation activity of DNA in ovine saliva, ovine rumen fluid and
silage effluent. FEMS Microbiol Lett. 2000, 191:71-7.
38 Duggan PS, Chambers PA, Heritage J, Michael Forbes J. Fate of genetically modified maize DNA in
the oral cavity and rumen of sheep. Br J Nutr. 2003, 89:159-66.

Neissseria, Haemophilus, and Moraxella)
39; the others are present in the environment. In addition, the
ability to be transformed depends on favourable envi ronmental circumstances which are not the same
according to the bacteria. Also, th e incoming DNA should not be degrad ed by the bacteria and it is
considered that integration in the chromosome by a process called recombination requires that the
foreign DNA has similarity within a cert ain length with the host chromosomal DNA 40. The required
length of similarity depends on the bacterial species.
90. Despite these difficulties, aut hors succeeded in transforming Acinetobacter calcoaceticus (an
environmental bacteria) in vitro and in soil with plant DNA bearing an antibiotic resistance gene
(kanamycin or streptomycin/spectinomycin resistance) 41. The frequencies of transformation were
relatively low but variable depending on the conditions. The three important observations are:
• 1/ there was integration of the DNA (reco mbination) even in regions of the host
chromosome with few similarity with th e foreign DNA; only the presence of few and
short "hotspots" of similarity were sufficient
• 2/ the integrated DNA segments often enco mpassed plant genes which were adjacent to
the antibiotic resistance gene and
• 3/ the bacteria expressed the plant gene.

91. However, most experiments were carried out in vitro and a major factor limiting
transformation of genes from transgenic plants is th e acquisition of a state for the bacteria where it is
naturally transformable. The soil conditions are probably not optimal and the frequency of
transformation is very low under natural conditions
42. However, it should be mentioned that evidence
that plant genes (from natural, "non modified" plants) were transferred during evolution from plants to
bacteria was demonstrated by nucleotide and protein sequences43.
92. Therefore, it may be concluded that:
• 1/ integration of resistance genes from a plan t DNA into the genome of a soil bacteria by
natural transformation is possible at a lo w frequency but more easily than initially
thought, and

39 Reviewed in Bertolla F, Kay E, Simonet P. Potential dissemination of antibiotic resistance genes
from transgenic plants to microorganisms. Infect Control Hosp Epidemiol. 2000, 21:390-3.
40 Ibidem.
41 Gebhard F, Smalla K. Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet
DNA. Appl Environ Microbiol. 1998, 64:1550-4. Nielsen KM, Bones AM, Smalla K, van Elsas JD. Horizontal
gene transfer from transgenic plants to terrestrial bacteria--a rare event? FEMS Microbiol Rev. 1998, 22:79-103.
Nielsen KM, van Elsas JD, Smalla K. Transformation of Acinetobacter sp. strain BD413(pFG4DeltanptII) with
transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants. Appl Environ
Microbiol. 2000, 66:1237-42. de Vries J, Herzfeld T, Wackernagel W. Transfer of plastid DNA from tobacco to
the soil bacterium Acinetobacter sp. by natural transformation. Mol Microbiol. 2004;53:323-34. de Vries J,
Wackernagel W. Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-
facilitated illegitimate recombination. Proc Natl Acad Sci U S A. 2002, 99:2094-9
42 Nielsen KM, van Elsas JD, Smalla K. Transformation of Acinetobacter sp. strain
BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on selection
of transformants. Appl Environ Microbiol. 2000, 66:1237-42.
43 Nielsen KM, Bones AM, Smalla K, van Elsas JD. Horizontal gene transfer from transgenic plants to
terrestrial bacteria--a rare event? FEMS Microbiol Rev. 1998, 22:79-103. Droge M, Puhler A, Selbitschka W.
Horizontal gene transfer as a biosafety issue: a natural phenomenon of public concern. J Biotechnol. 1998,
64:75-90.

• 2/ the presence of resistance genes facilitate s as "anchors" the integration into the
bacteria of adjacent gene plants ; these genes are expressed and can be disseminated via
the bacteria in the environment. The effi ciency of the gene transfer under natural
conditions appears to be weak. However, th is statement is based on a small number of
experimental studies found in the literature.

C. The foreign DNA integrated into the genome of soil bacteria or non pathogenic bacteria
has to be transferred to pathogenic bacteria

93. There are numerous evidences which show th at DNA is easily exchanged through various
ways between bacteria, including pathogens and non pathogens.
94. There may also be further considerations necess ary to perform a thorough risk assessment of
three ARMG, such as the presence, integrated into the plant DNA and genetically linked with the
ARMG, of remaining important bacterial plasmi d sequences and functions (origin of autonomous
replication, horizontal mobility f unctions) of the vector initially u sed to transform the plant. These
sequences may further confer autonomous replication of the ARMG if transferred to bacteria, or
worst, wide host range transfer plasmid functions would enable the rapid spread of the ARMG to
bacteria from other species or genera.
95. The general conclusion on the transfer of clone d ARMG from genetically modified plants to
pathogenic bacteria is therefore that since each re quired step has been demonstrated as possible, a
plausible scenario in which the cloned ARMG is tr ansferred from genetically modified plants to
pathogen bacteria can be built. This conclusion is supported by circumstantial evidence that indicates
travelling of genes from plants to bacteria duri ng evolution and confirms the likelihood of the
scenario. However, in terms of risk analysis, the ri sk has not been properly quantified and is probably
very low.
96. The next sub question is then, if ARMG from plan ts happen to be transferred to the bacteria,
which risk for human health? Which risks for animal health? This relates to the antibiotics concerned
by the ARMG from GM plants.
97. Since the risk will depend on the clinical a nd veterinary importance of antibiotics toward
which resistance is conferred, a case-by-case analysis of antibiotics should be carried out. The major
antibiotic resistance markers commonly used in GM plants are:
Kanamycin resistance gene: nptII gene

98. This gene is widely used as a marker. It c onfers resistance to kanamycin and neomycin by
inactivation of the antibiotics. The use of kanamycin is marginal in therapy and resistance is of very
weak clinical importance. However, amikacin is an important antibiotic for therapy in humans which
is derived from kanamycin. Mutations in the nptII gene can extend the activity of the produced
inactivating enzyme to amikacin. However, two mutations are required a nd have only be obtained
under laboratory conditions and never detected so far in clinical is olates although there are clinical
isolates which harbour the nptII gene.
Streptomycin resistance gene: aadA gene

99. The confers resistance to streptomycin and sp ectinomycin by inactivation of the antibiotics.
The use of these antibiotics is margin al in therapy and resistance is of weak clinical importance. The
only indications are multidrug resistant tuberculosis for streptomycin and uret ritis due to gonococus

(gonorrhoea) for spectinomycin which are narrow but important indications. Streptomycin is used as a
pesticide in agriculture, mostly in the USA and in Japan.
Ampicillin resistance gene: bla
(TEM) gene

100. The gene confers resistance to ampicillin a nd related antibiotics by inactivation of the
antibiotics due to an enzyme called penicilinase. Ampi cillin is an important antibiotic for humans and
animals. However, resistance to ampicillin is already widespread in pathogenic Gram-negative
bacteria responsible for urinary tract and intra-abdo minal (peritonitis) infections and this antibiotic is
mostly used in respiratory tract infections mostly due to Gram-positive bacteria which do not produce
beta-lactamases. 30%-50% of E. coli , the most common bacteria responsible for urinary tract
infections, isolated from urines in Europe, the USA and Latin America produce penicillinases
44. Most
of the penicillinase-producing isolates contain a bla(TEM) gene.
101. These ARMG have also to be carefully considered as regards the relevant veterinary uses of
the antibiotic concerned to treat animal diseases, in particular any epizootics or zoonosis.
102. Are all the antibiotics concerned by the resistance genes from GM plants of equal
importance? As explained above, it appears that risk is different according to the usage of antibiotic. It
is also different according to the previous spread of the resistance genes in nature. It appears that the
nptII gene is already widely distributed among soil a nd enteric bacteria and that it confers resistance
to non important antibiotics. The aadA gene is similar in importance; however, it is used in a very
limited but important indications in therapy. The bla gene confers resistance to clinically important
antibiotics but is already spread in nature.
103. Furthermore, there are other ARMG present in the GM products in dispute that may have
different risk assessments, in the light of othe r important therapeutic uses of the corresponding
antibiotics. This is for instance the case for the nptIII gene, conferring resistance to amikacin, as
explained in our comments in relation to Q 98.
104. A scientific panel of the European Food Safety Authority 45 has evaluated the potential risks
associated with specific antibiotic resistance genes and has recommended to classify the antibiotics in
three risk groups, from the lowest to the highest: group 1 which contains antibiotic resistance genes
already widely distributed among soil and enteric bact eria and confer resistance to antibiotics which
have no or only minor therapeutic relevance, gr oup 2 which contains antibiotic resistance genes
already widely distributed in nature and confer r esistance to antibiotics which are used for therapy in
defined areas of medicine and group 3 which co ntains antibiotic resistance genes which confer
resistance to antibiotics highly relevant for human therapy, irrespective of considerations about the
realistic value of the threat. The group 1 contained the nphII gene, the group 2, the bla and the aadA
genes and the group 3 other genes not discussed he re. The panel recommended that the use of
antibiotic resistance gene of group 2 should be rest ricted to field trial purposes whereas the panel
found that there was no rationale for restricting the use of group 1 antibiotic resistance genes.

44 Gordon KA, Jones RN; SENTRY Participant Gr oups (Europe, Latin America, North America).
Susceptibility patterns of orally admi nistered antimicrobials among urinar y tract infection pathogens from
hospitalized patients in North America: comparison repor t to Europe and Latin America. Results from the
SENTRY Antimicrobial Surveillance Program (2000). Diagn Microbiol Infect Dis. 2003, 45:295-301.
45 Opinion of the Scientific Panel on Genetically Modified Organisms on the use of antibiotic
resistance genes as marker ge,es in genetically modified plants. The EFSA Journal 2004:48;1-18. Available at
<http://www.efsa.eu.int>

105. The notion that there were so far no reports in bacterial isolates from humans or animals that
marker genes have transferred from GM plants to b acteria is not based on sufficient evidence: since
the marker genes are the same as those already spread in nature, it is difficult to trace them.
106. This scientific evidence is the basis of the conc lusions that have been outlined above by the
European Communities.
Risk management considerations

107. Although the risk to human health from the tran sfer of antibiotic resi stance to pathogenic
bacteria is considered low, it is nonetheless si gnificant, as the Codex Alimentarius has recommended
that the presence of ARMG be avoided. The hazard from the gene transfer of ARMG clearly exists,
and it is hard, if not currently impossible, to qua ntify precisely the corresponding risks by the current
risk analysis methods. Accordingly, ARMG should be avoided.
108. This recommendation also takes into account the fact that these genes, which are required for
the construction of the genetic modification, are no longer useful in the commercially available GM
plants, and that techniques exist to remove the ARMG genes from the GM plants at the terminal steps
of the construction.
109. This risk management conclusion is also justifie d by the fact that there is general policy in
Europe, and to some extend more globally, to reduce the incidence of bacterial antibiotic resistance in
pathogen bacteria. This is reflected in particular in the efforts that are made to monitor and control
antibiotic consumption in humans and animals (c ampaigns to reduce consumption, controlled
prescription in hospitals, ban of antibiotics u sed as growth promoters in animals) and
recommendations for the approval of pro-biotics wh ich should be devoid of "dangerous" resistance
genes. These efforts should not be undermined by the use of ARMGs in GMOs.
Question2

On the basis of the information before the Pane l, is there any scientific evidence to support the
hypothesis that antibiotic resistance develops through ways other than the uptake of food or feed by
humans or animals, that is, due to the potential p ersistence of plant-derived DNA in the environment
during crop cultivation and harvesting, and in soil residues?

(i) If scientific evidence indicates that su ch an event could occur, what risks, if
any, would arise from that event? What is the comparative relevance or
magnitude of this risk in relation to the likelihood of such a transfer from
other sources of antibiotic resistance no t involving the use of recombinant
DNA technology?
(ii) Are these consequences relevant to the specific types of ARMG currently used
in the products at issue in this dispute?
(iii) If such risks have been identified, what risk management options are
available to mitigate those risks and what is their efficacy?
General comments

110. The Panel's second general question sought equa lly important advice as to whether other
routes than food or feed, and persistence into the environment and in soil of plant DNA, may also lead
to the development of antibiotic resistance.

111. Unfortunately no expert has responded to this important general question, Dr. Nutti stating
that it is a question relating to environmental safety on which she does not have expertise. As it is a
question intimately related to Question 1, the European Communities will endeavour to provide as
objectively as possible scientific evidence to address the issue raised by the Panel.
112. From the scientific evidence presented below, it can be concluded that:
• antibiotic resistance may develop through ways other than uptake of food/feed by humans
and animals and transfer to gut microorganisms;
• the demonstration has been obtained that resistance genes from GM plants can be
captured and expressed by bacteria from the environment, probably at a low frequency;
• the risk for human or animal health (infection in immuno-compromised patients directly
by the environment bacteria) may be low, but the hazard is significant.
• the same case-by-case analysis as in the co mments on question 1 should be applied, as
different ARMG, different genetic constructi ons, and different environments may bear
significant differences in the associated risks.

Detailed comments

113. There is clear evidence that antibiotic resistance develops not only through the uptake of food
but also through the circulation of bacteria in the environment and between humans and animals
directly (by contact) or indirectly (by inert vectors). An example is the case of nosocomial infections
(infections acquired in the hospital) which are most ly related to the acquisition by the patients of
multiply resistant bacteria from other hospitali sed patients or from insufficiently sterilized
instruments. Another example is the acquisition of resistant bacteria through the consumption of water
contaminated by bacteria from the environment, or from soil contamination of wounds for farmer or
other workers exposed to soil. As regards to the question 2, this last example is the most relevant.
114. It must be remembered that we know only a minority of the bacterial and microbial flora in
the soil. Probably up to more than 95 to 99% of the species are still unk nown. Consequently, the
consequences of a the presence of ARMG DNA in the soil, and its possible transfer to bacteria, cannot
be fully assessed at this stage.
115. In soil bacteria, there are already a significant num ber of antibiotic resistant strains, including
among pathogenic bacteria. On one ha nd, this would make that adding some more resistance genes,
and probably in low frequency, has no heavy impact . But on the other hand, all efforts aiming at
reducing the proportion of resistance genes in popula tions of bacteria, and in particular in unknown
bacterial ecosystems, should be highly encouraged. Moreover, if it occurred that a pathogenic strain
had become resistant as a result of the cultivation of GM plants, and that these bacteria caused fatal
diseases, there would be no way to assess the origin of this event. There is no tracing of the resistance
genes introduced in GMOs. From that point of view, adding ARMG genes in any part of the
environment is certainly not acceptable.
116. On top of that, the genetic linkage in the GM plant of ARMG with other genetic elements
might enhance the maintenance or amplification of ARMG DNA derived from GM plants in the
environment. Indeed, these genetic elements linked to the ARMG, whether vol untarily present or not,
such as bacterial plasmid origin of replication, bacterial plasmid origin of transfer and mobility
functions, including wide host range transfer, or plant genes that would confer a positive selective
advantage to that plant under specific conditions (e .g. an herbicide resistance gene in the presence of
the herbicide) might confer selective advantages to the maintenance of the ARMG in the different
cells and organisms where it might be integrated.

117. Furthermore, largely unexplored physico-chemical properties of the soil, such as its pH, may
be very diverse and would have a very important impact on stability and transformability of naked
DNA in the soil. In certain environmental and physico-chemical conditions, intact DNA has been
showed to be able to persist for very long period of time in nature.
118. As mentioned in Question 1 above, we know that Acinetobacter calcoaceticus , an
environmental bacteria, has been successfully tr ansformed with plant DNA bearing an antibiotic
resistance gene (kanamycin or streptomycin/spectinomycin resistance)
46. The frequencies of
transformation were relatively low. It should be noted that Acinetobacter is an important source of
infections (particularly in fragilized and imm uno-compromised patients) originating from the
environment in hospitals and is considered as an emerging pathogen 47. However, in hospitals, this
bacteria is already resistant to a large panel of antibiotics and an additional resistance coming from
plants would not significantly change the dramat ic problem posed by multiple resistance of this
bacteria.
119. This single example may however be the tip of the iceberg, that has gone uncovered because
it was looked for, while other examples may also prov e to occur and be more problematic in terms of
therapeutic impact, but have not been discovered ye t because they were not properly searched for. In
addition the risk for spread of resi stance in nature should be significant only if an antibiotic selective
pressure was exerted by the use of antibiotics: th e use of a selector antibiotic would amplify the
number of bacteria with the resistance gene by eliminating the susceptible bacteria. This, nevertheless,
does not exclude maintenance and movement of the AR GM in nature in the absence of the selection,
or its amplification due to the selection of another genetic element linked to the ARMG.
120. Also, there is no absolute barrier between b acteria which could prevent gene transfer.
Therefore, resistance could be transferred from plant to environmental bacteria and then to pathogenic
bacteria in multi-step events. Again the probability of occurrence of this event should be very low,
unless there is an advantage to maintain or transfer the ARMG.
121. Another important point is that, as already mentioned, the DNA segments integrated in
Acinetobacter often encompasses plant genes which were ad jacent to the ARMG gene, and that the
bacteria expressed the plant gene 48. The presence of ARMG genes facilitates as "anchors" the
integration into the bacteria of adjacent plan t genes; these genes can be expressed and can be
disseminated via the bacteria in the environment. Again, in case of use of the selector antibiotic in
agriculture, the presence of ARMG and any geneti cally linked gene might lead to a significant
amplification of the number of bacteria and significant spread of the corresponding genes.

46 Gebhard F, Smalla K. Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet
DNA. Appl Environ Microbiol. 1998, 64:1550-4. Nielsen KM, Bones AM, Smalla K, van Elsas JD. Horizontal
gene transfer from transgenic plants to terrestrial bacteria--a rare event? FEMS Microbiol Rev. 1998, 22:79-103.
Nielsen KM, van Elsas JD, Smalla K. Transformation of Acinetobacter sp. strain BD413(pFG4DeltanptII) with
transgenic plant DNA in soil microcosms and effects of kanamycin on selection of transformants. Appl Environ
Microbiol. 2000, 66:1237-42. de Vries J, Herzfeld T, Wackernagel W. Transfer of plastid DNA from tobacco to
the soil bacterium Acinetobacter sp. by natural transformation . Mol Microbiol. 2004;53:323-34. de Vries J,
Wackernagel W. Integration of foreign DNA during natural transformation of Acinetobacter sp. by homology-
facilitated illegitimate recombination. Proc Natl Acad Sci U S A. 2002, 99:2094-9.
47 Coelho J, Woodford N, Turton J, Livermore DM. Multiresistant Acinetobacter in the UK: how big a
threat? J Hosp Infect. 2004, 58:167-9. Jain R, Danzig er LH. Multidrug-resistant Acinetobacter infections: an
emerging challenge to clinicians. Ann Pharmacother. 2004,38:1449-59.
48 de Vries J, Herzfeld T, Wackernagel W. Transfer of plastid DNA from tob acco to the soil bacterium
Acinetobacter sp. by natural transformation . Mol Microbiol. 2004;53:323-34.

122. Here again, the risk management options propo sed by the Codex as it relates to food safety,
may be applied more generally, namely that the pr esence of ARMG should by all means be avoided,
and should not be present in GM plants when the ge nes may potentially confer resistance to clinically
and veterinary important antibiotics.
Question 3

On the basis of the information before the Pane l, is there any scientific evidence to support the
hypothesis that wide-spread cultivation of Bt crops such as biotech maize of the Bt variety adversely
affects non-target organisms which may be exposed to such crops under typical agricultural practice?
(See, inter alia, EC-149, EC-150, EC-151, EC-152) If so, how does this risk compare with risks to
non-target organisms arising from non-biotech applications for Bt toxins (i.e., the use of Bt toxin as
an insecticide in conventional and organic farming)? What risk management options are available to
mitigate any resulting risks and what is their efficacy?

General comments

123. The European Communities notes that the two of the Panel's experts who address this issue
(Drs. Squire and Andow) agree that the e ffects of GMOs expressing the Bt toxin may have effects on
non-target organisms, which they believe is not yet confirmed, as small scale releases show little
immediate toxic or catastrophic effects. Howeve r the absence of observed catastrophic effects does
not mean in the view of the experts no effects on non target organisms, and Dr. Andow advocates
therefore that any risk management options shoul d be based on worst case scenarios. Dr. Squire
explains that the long term field scale monitoring will be necessary to determine whether there is such
a significant effect. Such tests will be specific to the crop and the environment in which it is to be
grown.
124. The European Communities has little detailed comm ent to add to this advice at the present
stage, but would like to point to a recent body of sci entific evidence of non target effects of Bt crops,
in particular on soil organisms and nematodes, as well as a number of selected references relevant to
non target organisms sensitivities to Bt toxins.
Detailed comments
125. As regards unintended effects of Bt maize on non target organisms, a very recent paper
49
shows some effects of the maize expressing the Bt toxin on soil nematodes, which are site and season
specific effects, supporting the arguments for a) cas e by case studies b) long term monitoring (3 years
minimum), c) region specific studies, d) and the incl usion of relevant comparisons (other crops and
soil management treatments).
126. The effects detected on nematodes was also found by other authors 50, although no one yet
understands why the effect happens (less nematode s under Bt maize in some soils/regions/tillage

49 See Exhibit EC-164: B. S. Griffiths, S. Caul, J. Thompson, A. N. E. Birch, C. Scrimgeour, M. N.
Andersen, J. Cortet, Antoine Messéan, Christophe Sausse, Bernard Lacroix, P. H. Krogh. A comparison of soil
microbial community structure, protozoa and nematodes in field plots of conventional and genetically modified
maize expressing the Bacillus thuringiensis CryIAb toxin. Manuscript accepted in Plant and Soil Journal.
50 B. Manachini, S., Landi, M. C. Fiore, M. Fe sta and S. Arpaia, IOBC wprs Bulletin, 2004, 27, 103.
Manachini B., Lozzia G.C., 2002 - First investigations in to the effects of Bt corn crop on Nematofauna. - Boll.
Zool. Agr. Bachic. Ser. II, 34(1): 85-96. Manachini B., 2003 - Effects of transgenic corn on Lydella thompsoni
Herting (Diptera:Tachinidae) parasitoid of Ostrinia nubi lalis Hb. (Lepidoptera Crambidae).- Boll. Zool. agr.
Bachic. Ser II, 35(2): 111-125.

regime combinations). It may be related to root exudation, plant composition, altered moisture below
Bt maize plants, altered trophic in teractions of a food source for ne matodes, or any other effect
directly or indirectly related to the expression of the Bt toxin. It strikingly shows evidence of
continued scientific uncertainties and regional differences on soil ecology as regards non target effects
of Bt maize. Some would say that the results are small and transitory, and could therefore be
dismissed, but this is not an appropriate risk asse ssment end point if the effects accumulate over time
due to repeated maize monoculture in the same fiel d, due to the ability to grow some GM crops with
no tillage and repeatedly over several seasons. In such circumstances, the risk to soil organisms and
soil ecosystem could then increase over time.
127. The European Communities would further like to submit scientific evidence on differences in
insect species/sensitivity to Bt toxins for a) pests, and b) non-pests (conservation or heritage species,
like butterflies), which are both relevant for this question and Question 4.
128. The current state of Bt environmental risk ass essment in Europe shows that there were and
still are considerable grounds for concern about the toxin Bt, especially non-target effects, which have
only been addressed in recent years and which still continue to produce large amount of data.
129. This additional scientific evidence on differen tial sensitivities to Bt toxins, available from
peer-reviewed publications, show that between pest species and conservation or heritage species there
is a wide range of sensitivities to Bt toxins expressed in GM crops. Hence it is possible to argue that:
 – Scientific uncertainties about the non-target species effects of Bt toxins, on regional
non-target insect sensitivities, still do exist (e .g. butterflies of regional cultural value
that could be exposed to Bt toxins via the GM crop in any one region of Europe). (Bt
toxin sensitivities for lepidopteran, non-pest species
 which could have regional- or
country-specific conservation or heritage value. i.e. non-target herbivores (butterflies)
that could be affected by Bt crops and show species differences in sensitivity to Bt
toxins)51

 – Even for target pest lepidopteran species , considerable differences in species
sensitivities exist, which can impact on Insect Resistance Management (IRM)
strategies for any particular EC country or region (Bt toxin sensitivities for
lepidopteran (caterpillar) pest
 species which are potential 'target' species for Bt crops,
showing Bt toxin sensitivity differences between species) 52. i.e. A 'high dose + refuge
strategy' for species X in one region may not be effective as a 'high dose + refuge
strategy' for species Y in another region, because species Y is less sensitive to Bt
toxin than species X. This effect has already been demonstrated
53 for Bt cotton grown
in USA vs Australia, where the Bt cott on event produced in USA was reasonably
effective in USA under the 'a high dose + ref uge strategy' but failed to be sufficiently

51 Wraight, Zangerl, Carroll & Berembaum (2001). Proc. Natl. Acad. Sci. USA 97: 7770-7773.
Zangerl, McKenna, Wraight, Carroll, Ficarello, et al., (2001). Proc. Natl. Acad. Sci. USA 98: 113-118. Sears,
Hellmich, Stanley-Horn, Oberhauser, Pleasants, et al., (2001). Proc. Natl. Acad. Sci. USA 98: 119317-42. Jesse
& Obrycki (2000) Oecologia 125: 241-248. Jesse & Obrycki (2002). J. Kansas Entomol. Soc. 75: 55-58. Jesse
& Obrycki (2003) Agric. Environ. 97: 225-233. Hellmich, siegfried, Sears, stanley-Horn, Daniels, et al., (2001).
Proc. Natl. Acad. Sci. USA 98: 11925-30.
52 Fitt et al., 2004 Report for International Cott on Advisory Committee, pp 1-64. Felke, Lorenz &
Langenbruch (2002). J. Appl. Entomol. 126: 320-325. Wolt, Peterson, Bystrak & Meade (2003). Environ.
Entomol. 32(2) 237- 246. Dutton, Romeis & Bigler (2003). BioControl 27: 441-447. Bernal, Griset & Gillogly
(2002). J. Entomol. Sci. 37: 27-40.
53 Fitt et al., 2004 Report for International Cotton Advisory Committee, pp 1-64

effective against important Australian cott on pest species in the IRM because they
were less sensitive to the Bt toxin.

130. Australia has now moved to a new Bt cotton event expressing two different Bt toxins to
achieve a better 'high dose' strategy for Australian species which were less sensitive to first generation
Bt cotton (especially at the end of the growing season).
131. This scientific evidence further points to regional differences and lack of sufficient
information for both target (pests) and non target (biodiversity and conservation species).
Question 4

On the basis of the information before the Pane l, is there any scientific evidence to support the
hypothesis that the wide-spread cultivation of Bt maize or other, non-biotech applications of Bt toxins,
leads to the emergence of Bt-resistant target or ganisms under field conditions? If so, what risk
management options exist to mitigate any resulting risks and what is their efficacy?

General comments

132. The European Communities notes that the two of the Panel's experts who address this issue
(Drs. Squire and Andow) both agree that the development of Bt resistance in target insects is
inevitable. They both agree that th ere are different risk mitigation techniques, and that there is still a
large amount of uncertainties as regards the pace and impact of resistance development. According to
Dr. Andow, the "high dose refuge" strategy is the most effective.
Detailed comments

133. The European Communities has nothing to add to this advice at the present stage, but would
like to point to the further evidence it had submitted in relation to the previous question, namely as
regards differences of Bt toxin sensitivity in differe nt target organisms, that may warrant regional
differences in risk management and integrated resistance management strategies (confer the
differences that occur between the United States and Australia, that necessitates differences in
mitigation approaches).
134. From an evolutionary and ecological point of view, it is clear, as pointed out by the experts,
that adding the same genes in the genome of numerous crops, cultivated on vast neighbouring areas
will, sooner or later, lead to the appearance of resistant organisms. How long it will take, is not
known. A parallel may be drawn with dangerous uses th at have developed in the past with antibiotics.
The overstated confidence in our ability to find new molecules when the existing ones become
inefficient has prevented a sustainable use of many chemicals. For example, antibiotics were a great
discovery, among the greatest in medical sciences. If they had been properly used and managed, they
would probably still be perfectly efficient. As soon as 1945, Flemming warned that unconscious use
and abuse of these molecules would lead to the lo ss of their efficiency. This example holds for
pesticides, and it is to be remembered that the exogenous Bt toxin is, in certain instances, the only
technique currently available to some agricultural practices, such as organic farming.
Question 5

On the basis of the information before the Pane l, is there any scientific evidence to support the
hypothesis that Bt maize varieties are any more to xic to humans or animals than conventional maize
under field conditions? If so, what risk management options exist to mitigate any resulting risks and
what is their efficiency?

General comments

135. In answering this question, Dr. Squire appropr iately examines the evidence of harm (or of
absence of harm) of growing Bt crops in Europe, an d opines that the evidence of Bt maize to humans
or animals is inconclusive at present, while acknowledging that the only sound indication that the
current state of knowledge provides, is that there is no evidence of acute toxicity to mammals and
most animals other than pests.
136. On the contrary, Dr. Nutti offers a characteris tically short, negative, unsubstantiated answer,
stating only that:
Based on the information before the Panel, there is no evidence to support the
hypothesis that Bt maize varieties are more toxic to humans or animals than
conventional maize under field conditions.
137. Dr. Nutti's response is scientifically inadequate in terms of coverage and lack of any
supporting evidence. There are numerous possible ways that Bt maize could be toxic (i.e. have an
adverse effect on the receiving organism following acute or chronic exposure) when Bt maize is
grown under European field conditions.
138. Because of the inadequacy, and incompleteness of the advice provided by Dr. Nutti to the
Panel, the European Communities provides below its own analysis. The European Communities has
prepared this information with the assistance of scientists, experts in the field of non target direct and
indirect impacts, toxicity to organisms, and ecological assessment, and presents it as objectively as
possible so as to complete the information availa ble to the Panel. In so doing, the European
Communities will interpret the term "animals" as incl uding the wide range of non target organisms
belonging to the animal kingdom (i.e., in a ddition to humans and domesticated mammals) which
could be adversely affected by Bt maize, as would many international scientists researching non target
effects of GMOs consider this term.
Detailed comments

139. It is not scientifically reasonable to simply translate and extrapolate the limited risk
assessment results on the toxicity of Bt maize to human and non-target organisms from USA,
Australia or some other non-European countries, because the
 – regional growing environments,
 – scales of farm fields,
 – crop management practices,
 – local/regional target and non-target speci es considered most important in the agro-
ecosystem,
 – interactions between cultivated crops, and
 – surrounding biodiversity,

could each be different from published non-European studies and could differ substantially between
regions and countries within the EC. Case-by-case risk assessment (including interactions between the
GM crop and its local environment) is widely accepted as a valid approach for assessing possible
environmental and health impacts of GM crops and foodstuffs.

140. Studies illustrating non-target effects and scientific uncertainties over non-target effects from
the longer term exposure of Bt crops to appropriate ly selected non-target organisms, above and below
ground, include the following:
141. Effects (either directly to the non-target organism, or indirectly to non-target organisms via
simple or complex trophic interactions in the food web) of the Bt toxin itself, or of the Bt crop as a
whole, on non-target insects which provide important 'ecological services' in agro-ecosystems.
54
142. In several of the above non-target studies on Bt crops, the possibility is raised from a
scientific viewpoint that sub-lethal effects detected in small scale or short term studies (e.g. lab or
contained glasshouse) could possibly be exacerbated by longer term exposure (i.e. over a growing
season encompassing multiple generations of non-targ et insect species) and by toxic interactions
between expressed Bt toxins and other components of the non-target target organisms normal diet. For
an herbivore, this intake of other toxic compounds which the Bt toxin could interact with neutrally,
additively, synergistically or antagonistically, would include defensive plant chemicals produced
constitutively or induced by plant herbivore damage. 55 These complex toxicological and behavioural
interactions mean that simple risk assessment tests on non-target organisms without using the growing
GM crop in a realistic and appropriate environment are not conclusive to indicate a meaningful degree
of risk (or not).
143. Several examples (cited in the studies referenced previously) of unintended or unanticipated
effects arising from transformation of transgenic crops, show that plant metabolites involved in
ecological interactions with non-target organisms can be changed during production of Bt crops, and
have the potential to influence the toxicity of Bt toxins by unpredictable toxicological interactions as
part of the complex diet consumed by herbivores.
144. From many ecological and eco-toxicological studies 56 it is clear that plant toxins and other
plant defensive chemicals can in general have mark edly different effects on the receiving organism,
depending on the quantity of both toxin dose and exposure time (termed the 'hormesis' effect). Thus,
quantitative changes (increases or decreases) in expressed Bt toxins and/or in plant metabolites
affected by the transformation process have the potential to influence the overall 'toxicity' or
environmental impact of the Bt plant for the he rbivore and other non-target organisms at higher
trophic levels in a local agro-ecosystem (mean ing the Bt crop and the non-cultivated vegetation
associated with the local cropping system of the Bt crop).
145. Scientific uncertainty is increased due to the ongoing scientific debate over the most
appropriate testing methods and scales (spatially and temporally) useful to determine realistic
ecological effects of Bt crops. 57 According to Levidow's review 58, a precautionary approach identifies

54 References: Hilbeck et al., 1998, Environ. Entomol. 27: 480-487. Hilbeck et al., 1999, Ent. Expt.
Appl. Et Appl 91: 305-316. Saxena, Flores and Stotsky 1999; Nature 402: 480. Lovei 2001; Proc. 54th conf NZ
plant prot Soc 54: 93-100. Bernal et al 2002; Ent Expl et Appl 102: 21-28. Groot & Dicke 2002; Plant Journal
31(4): 387-406. Oberhauser & Rivers, 2003; Agbionet 5: 1-7. Zwahlen et al., 2003; Mol Ecol 12: 765-775.
Knols and Dicke 2003; Nature Biotech 21(9): 973-974. Fitt 2003; Proc. 3rd World Cotton Research Conf, Cape
Town, pp 371-381. Conner et al 2003; Plant Journal 33: 19-46. Poppy & Sutherland 2004; Physiol Ent 29: 257-
268. Wilson et al 2004; Technical report for EcoNexus. Andow and Hilbeck 2004; BioScience 54: 637-649.
Lovei and Arpaia 2005; Ent Expt et Appl. In press. Andow and Zwahlen 2005 Ecology Letters In press.
55 Birch et al 2001; Ann Appl Biol 140: 143-149. Haslberger 2003 Nature Biotechnology 21 (7)
739-741.
56 See for a review and summary, Calabrese and Baldwin 2003 Nature 421:691-692.
57 Levidow 2003 J Invert Path 83: 113-117. Dutton et al., 2003; BioControl 48: 611-636. Andow and
Hilbeck 2004; BioScience 54: 637-649. Poppy & Sutherland 2004; Physiol Ent 29: 257-268.

new unknowns, and scientific questions, generat es different criteria for evidence, and make
judgements on environmental risk assessment more transparent. In his review, this is illustrated by the
questions raised by the use of "surrogate" Bt toxin (i.e. a toxin artificially isolated from an
heterologous expression systems which is different from the Bt maize considered), in order to test the
toxicity of the isolated Bt toxins on non target or ganisms. In reality, the non target herbivore will
consume the Bt toxin produced by the homologous Bt-expressing plant, which may therefore be
different in molecular structure, size, posttranslational modifications, and biological activity to the
artificially produced "surrogate" Bt toxin
59.
146. Bt toxin exposure routes for non target orga nisms, like insect pollinators, predators and
parasitoids, are frequently multi-trophic 60, not simply bi-trophic that is, they arise from indirect
complex and network interactions. Simplified bi-t rophic testing systems, based on eco-toxicological
models (as featured in many non target methods us ing a purified Bt toxin in artificial diets for non
target insects) are now not considered by many e xperts to be ecologically realistic for assessing multi-
trophic interactions of Bt crops over several insect generations and spanni ng at least three trophic
levels (see eg following references 61).
147. The views summarised in these cited publications focus on the need for multi-trophic testing,
using real Bt crops in realistic testing conditions, tested with regionally appropriate non-target species,
likely to be exposed to Bt toxin or metabo lites. These views arise from accumulating scientific
knowledge over the period from the mid 1990s until today. Scientific uncertainties on effects of Bt
crops on non-target species or ecological functio ns were (and still are) identified and are being
addressed by many international scientists.
148. Other scientific uncertainties arise from incomplete knowledge of the transgene locus
structure and expression, of different sized Bt toxins, which can interact in uncertain ways in different
environments. GM crops should be tested under regionally representative environments and
appropriate crop management practices,62 and without precise molecular characterisation data, it is not
possible to be certain about correct doses and exposure routes for non-target organisms in the local
agro-ecosystem.
149. Often the measured 'effect' (positive, negative, or neutral) of the GM crop on selected non-
target species or ecological functions (e.g. predation, parasitism, pollination) is compared with one or
more other treatments in the experi ment (e.g. an isogenic line with/w ithout chosen pesticides, or local
varieties +/- application of chosen pesticides, or a local crop variety grown under Integrated Pest
Management or under organic production, etc). This wide range of comparators, as 'baselines', to
determine any change caused by growing the GM crop, from 'norm' equating to the selected baseline,
means it is very difficult or impossible to generali se or extrapolate between studies, even within one
country and for one GM crop event. This enforces the scientific arguments for regionally specific,
case-by-case studies of GM crop impacts performed over several (preferably 3 or more) growing
seasons in the same region.

58 Levidow 2003 J Invert Path 83: 113-117
59 Levidow 2003 J Invert Path 83: 113-117. Freese and Schubert 2004 Biotechnology and Genetic
Engineering 21: 299-324.
60 Groot & Dicke 2002; Plant Journal 31(4): 387-406.
61 Birch et al. 1998, 199. Hilbeck et al., 1998, Environ. Entomol. 27: 480-487. Hilbeck et al., 1999,
Ent. Expt. Appl. Et Appl 91: 305-316. Groot & Dicke 2002; Plant Journal 31(4): 387-406.Oberhauser & Rivers,
2003; Agbionet 5: 1-7.Capalbo et al., 2003; J Invert Path 83: 104-106. Andow and Hilbeck 2004; BioScience
54: 637-649. Birch et al., 2004 In: Environmental risk assessment of Genetically Modified Organisms Vol 1: a
case study of Bt maize in Kenya. Ch4,. Eds Hilbeck and Andow, CABI, UK, pp117-187
62 Andow and Hilbeck 2004; BioS cience 54: 637-649. Freese and Sc hubert 2004 Biotechnology and
Genetic Engineering 21: 299-324. Wilson et al 2004; Technical report for EcoNexus.

150. Question 5 presupposes suitable access to scien tific knowledge applicable under 'field
conditions'. Scale of testing has a major impact
63 on environmental impact assessment (e.g. toxicity of
Bt crops to non-target organisms). Scale-dependent effects of Bt crops can thus only be determined by
studying their impacts over a wide range of spatial (labs, glasshouses, fields, landscapes) and temporal
(hours, days, seasons) scales, under the localised e nvironmental conditions (regions within countries)
and using crop management systems regionally appropr iate to were the Bt crop event is proposed to
be grown. Generation of data to see the 'bigger scientific picture' generally takes several years of
detailed research in several countries (see for in stance the EU ECOGEN project as an example of
suitable testing scales over multiple regions, coun tries and seasons, with Bt maize management
practices compared, e.g. conventional versus mini mum tillage). This project will only produce final
reports in 2005/6 after 3-4 years of GM crop field trials in Denmark and France.
151. The European Communities would also like to address the comparison of Bt maize toxicity
with conventional maize under field conditions. As must now be self evident to the Panel, biology is a
complex and empirical science. As such, it allows to predict situations when they have been
repeatedly tested (in order to make the necessary statistics). "Natural", sexual, crosses give results
which we cannot predict, but we have evidence that , statistically, they produce harmless genotypes.
They are not harmless because they are natural, but because they are familiar.
152. On the contrary, a GM crop, where a new Bt gene is introduced into its genome, leads to lots
of unpredictable interactions. One of the impor tant discoveries of genomics is that complex
organisms, such as a human beings, can be produced by a number of genes which is not more than
that of a plant such as maize. One of the conclu sions from this understanding is that natural gene
interactions are numerous and important, that they form complex webs of ge ne regulations, and that
these interactions statistically reach genome-wide equilibrium in "conventional" organisms. No one
can scientifically claim to be able to predict all consequences of the presence and functioning of a new
gene (and even less for several) in a genome which has never been exposed or contained this gene.
The potential hazard here is not a consequence of the action of modification the plant genome, but of
the fact that it generates high levels of unpredicta bility. The risk here may not come from the genetic
modification itself, but from the extreme unpredictab ility of the direct and indirect effects of the
introduction of a new gene(s) and ge ne product(s) into the plant genome and its gene expression. This
cannot be compared to conventional maize, where such new combinations have ever occurred.
153. In the face of this unpredictability, one po ssible way forward is the implementation of
thorough risk management and monitoring programs. However, this leads also to very significant
methodological problems, as it is first necessary to collect extensive "baseline" data, before a
monitoring plan can be meaningfully implemented for such complex effects. Without appropriate
control data (namely the state of the ecosystem and the environment prior to the introduction of the Bt
maize), which is very difficult, expensive, tedi ous and time consuming to collect, monitoring plans
and general surveillance may not be able to detect and identify significant unintended effects, or long
term, sub-chronic toxicity on non target animals.
Question 6

On the basis of the information before the Panel, is there any scientific evidence to suggest that
herbicide tolerant crops (whether biotech or devel oped through mutagenesis) are more persistent in
the agricultural environment or more persistent in the non-agri cultural environment than their
conventional counterparts? If so, do herbicide tolera nt crops qualify as a potential "pest" as the term
is used in International Standard for Phytosanitary Measures (ISPM) 11?

63 Joao 2002 Environmental Impact Assessment Review 22: 289-310

(i) What is the potential for the establishment and spread of herbicide tolerant
plants arising from handling, spillage during transport of the plant/plant
parts, or any other means outside of cu ltivation in the absence of application
of the herbicide? How is any potentia l for establishment and spread affected
by environmental conditions, the presen ces of wild or conventional relatives
of the herbicide tolerant plants in an area, or other factors?
(ii) What is the potential for the establishment and spread of herbicide tolerant
plants in the presence of herbicide applic ation (in fields, urban, domestic or
other environments)? How is any poten tial affected by the existence of feral
related plant species; infertile wild relatives; seed survival in relevant
pedoclimatic conditions; the reproduction biology of the species; or other
factors?
(iii) Is this potential different for biotech crops tolerant to two wide-spectrum
herbicides? Please explain.
(iv) If significant risks of establishment an d spread have been identified, what
risk management options exist to mitigate any resulting risks and what is
their efficacy?
(v) What types of post-market monitoring and data collection activities could be
envisaged on the basis of the monitoring and review principles described in
ISPM 11?
General comments

154. Drs. Snow and Squire have provided long and useful answers to the question and all sub-
questions, with a significant amount of detailed evid ence to substantiate their respective opinions. Dr.
Andow has also provided a useful, substantiated answer, although he only addresses the theoretical
framework of the main question, and the last sub- question on post market monitoring. Together, even
if they do not always agree among themselves, all three experts have provided convincing evidence in
the field of the potential difficulties that are linked with herbicide tolerant (HT) crops. Their replies
provide in particular useful evidence as regards the issues arising from the combined use of the HT
crop and the corresponding herbicides, or from the comb ination of several HT traits, and in particular
in the context of glyphosate and/or glufosinate HT, for which HT crops can only be currently obtained
by way of genetic modification.
155. Some of the points made by the experts that the European Communities would like to note
are: all three experts indicate that the available ev idence demonstrates persistence of HT crops in the
presence of herbicides, and Dr. Snow points also to the available evidence of the emerging problems
with HT weeds. She also points to the difficulties to eliminate the current problems of spread of some
glyphosate or multiple resistant HT crops in Cana da. Dr. Squire identifies the existence of risk
management options, but stresses the main difficulties arising with oilseed rape in Europe. He also
indicates that monitoring schemes do not yet exist, a lthough research is in progress, and that he is not
certain whether it would be feasible to monitor ev ents with low frequency occurrences. Dr. Andow
indicates also that any ecological effects would be scale dependent.
156. By way of contrast, Dr. Nutti has again provid ed a short and unsubstantiated answer, which
contradicts the three other experts, and addresses only the "pest" qualification. She only states, after
recalling the pest definition in ISPM 11:

In my opinion, herbicide tolerant crops can not be qualified as "pest", according to
this definition.
157. Dr. Nutti has probably misunderstood the question as asking her to provide a judgement on
the overall risk of GM HT crops on plant or plan t products, while other experts have answered the
question as to whether they could potentially qualify as a pest. Dr. Squire states that:
Oilseed rape and beet are both crops and pests, as are many other crop plants (pests as
defined in ISPM-11, page 6).
158. Dr. Snow also states that, while each type of herbicide-tolerant crop should be considered
individually and that it is not possible to generalize across all herbicide-tolerant crops,:
A crop with herbicide tolerance can qualify as a potential "pest" under the ISPM 11
definition if 1) it transmits herbicide resistance to weedy volunteer plants or the crop's
sexually-compatible wild relatives, and 2) this leads to worse weed problems.
Volunteer oilseed rape and its sexually-compatible relative, Brassica rapa, are
considered to be weeds because they compete with food crops and require
management.
159. Although Dr. Snow does not address in this statement the potential weediness and pest
problems of volunteers in crop rotation, the European Communities agrees with these experts that GM
HT crops do qualify as pests as defined in ISPM11.
160. The European Communities notes Dr. Andow's remark that:
Thus I conclude that GM HT crops can qualify as a potential "pest" as the term is
used in the International Plant Conven tion's (IPPC) International Standard for
Phytosanitary Measures (ISPM) 11 (EC-130). However, not all of the risks associated
with GM HT crops can be considered phytosanitary risks.
161. Dr. Andow is correct in noting that not all of the risks associated with GM HT crops can be
considered phytosanitary risks. Some are environmental or other risks rather than phytosanitary risks.
162. Considering the complexity of the question and sub-questions of the Panel, the importance of
the issues raised for the Eur opean Communities, and the disagre ement between the experts, the
European Communities provides below its own anal ysis. The European Co mmunities has prepared
this information with the assistance of scientists, e xperts in the field of agricultural management and
agro-ecological impacts of HT crops. The Europ ean Communities may address some sub-elements of
the replies separately, where necessary.
Detailed comments

163. As a first general comment, the European Communities would like to clarify the following: as
indicated by the experts, a plant with an herbicide resistance will easily become a pest if the herbicide
is widely used. The consequence is that the same HT should not be introduced in different species that
may be used in the same agricultural system, if no potential adverse consequences are to be expected.
This is obvious, and is a systemic risk rather th an a case-by-case problem. This is the issue that
recently came to light when HT wheat was considered for approval in Canada and the United States,
while several other crops had already been cultivat ed with the same GM HT character, including

oilseed rape. Furthermore, herbicides are not only u sed to remove weeds or volunteers in the fields
but also on road verges, railways, gardens, industrial plants, etc.
164. Some species, like rapeseed, are already very a ggressive outside and inside cultivated fields.
As discussed by the experts, introducing resistan ce to important herbicides in certain species may
certainly lead to significant spread and impacts. A problematic (worst) strategy would then be to
introduce HT to several of the same relevant herbic ides in one plant species with weediness potential.
Cross hybridization would rapidly lead to the existence of multi-resistant plants which would then
become major pests. As discussed by Dr. Snow, the fast appearance of such plants has already been
demonstrated in Canada, and proves extremely difficult to manage.
165. Concerning sugar beets, their easy hybridization with wild relatives give birth to very
invasive weeds in Europe. Introducing resistances to herbicides in cultivated beets would also lead to
significant agro-environmental impacts, as it would also rapidly lead to HT weeds, probably within a
couple of years.
166. A weed is an unwanted plant in the field that competes successfully with the crop plant for
resources and thereby interferes with our goal of maximizing yields. A weed becomes more noxious
the better it can escape measures of control by the farm er. There are various strategies, by which such
noxious plants can do that. One is to mimic the crop plant and thereby being indistinguishable for the
farmer from the crop during the time period it could be controlled (typically early season). Thus,
many crop plants have close relatives that beca me tough weeds/competitors of them. Examples are:
rice/weedy rice, maize/teosinte, oilseed rape a nd several oilseed rape-relatives, sugar beet/beta
maritima etc.
167. The major means for control in in dustrialized, large-scale farming is by using chemicals, i.e.
herbicides. Therefore, another very effective stra tegy for competitive weeds is to develop resistance
against herbicides. The more intensive the control measure the larger the selection pressure to develop
resistance and the quicker it develops. Weeds ha ve and still do exhibit an astounding capacity of
developing resistances against almost all compounds ever used against them and this happens in a
way highly correlated to the amount and types of he rbicides used against them (One of the most
authoritative sources of information on the current status of resistant weeds worldwide is the
"International Survey of Herbicide Resistant Weeds" that can be found at
http://www.weedscience.org

168. However, until recently, the plants had to deve lop these resistance mechanisms at least by
themselves. Since the introduction of HT crop plan ts, we actually put HT-resistance genes actively
out into the agro-ecosystem and make them availabl e to any crop relative. All it takes ever since for
related weeds, notably often bad competitors already, is to take these offered HT genes up, they do
not even have to develop them anymore. This s hortcuts the resistance development process of related
and unrelated weeds significantly. In addition, we increase the selection pressure tremendously by
exclusively spraying the complementary herbicide.
169. As already indicated, HT crops are only more pe rsistent in environments where the specific
herbicide or a closely related chemical with sim ilar physiological activity is used. There is no
indication that they are more fit or competitive in other environments. 64 Indeed the continuous
production of enzymes to degrade or inactivate the herbicides could reduce resources available to the
crop/plant. Studies of isogenic lines with and wi thout GM HT suggest no difference in crop

64 Crawley et al. 2001.

performance and thus no differences in ecological fitness.
65 But then of course the maintenance in the
agro-ecosystem and the seed bank of the HT charac ter leaves it available for any situation in the
future where the selection pressure will again be applied.
170. The persistence of the crop is case specific and depends largely on the crop species and
variety. In Europe the oilseed rape grown is predom inantly winter type (which requires vernalisation)
and persists much more in milder European conditi ons. There is no winter kill and high persistance of
seed banks in soil. This is a different crop from North American spring oilseed rape which does not
survive winters except in seed banks and is not so invasive.
171. It also depends on the regions in Europe wher e crops are grown. For example maize survives
very poorly in regions with colder winters. But in warm maritime regions maize seeds and volunteer
seedlings will persist and survive during winter and so could occur in subsequent crops or their field
margins.
172. If so, do herbicide tolerant crops qualify as a potential "pest" as the term is used in
International Standard for Phytosanitary Measures (ISPM) 11 ?
173. With HT crops there is two different risks: a) the HT crop becomes itself a weed in situations
where the respective broad spectrum herbicide is used , b) the HT gene is conferred to related weed
species exacerbating their weediness by the acquired new resistance.
174. All crops can be weeds (pests) when they grow as volunteers in subsequent crops and
compete with them. Different crop species vary in their competitiveness and ease of control. Often
they are controlled by the herbicide programmes us ed to control weeds. However specific treatments
may be required to remove some of them. HT crops (especially oilseed rape) have become
problematic when they have occurred adventitiously in other crops without farmers realising it and
having tried to use the specific herbicide to control them without success. This means that they have
suffered losses of yield or quality when they norm ally would not. In some cases different GMHT
oilseed rape types have hybridised resulting in volunteers with multiple herbicide tolerance and can be
more difficult to manage
66.
175. Adventitious presence is particularly a potential problem in Europe where GM oilseed rape
seeds shed at harvest may persist in soil for sever al years and emerge in subsequent non-GM rape
fields. These GM plants will then cross pollinate with conventional rape to the extent that thresholds
for presence of GM seeds in conventional crops may be exceeded67.
176. Glyphosate is widely used in agriculture today in combination with sterile seed bed
techniques (i.e. cultivation of gr ound to encourage weed germin ation and then spraying with
glyphosate to remove the weeds) as a means of reducing weed pressure on farms. In addition
glyphosate and glufosinate are used as dessicants of crops with th e added advantage that they kill
weeds maturing in the crops just before harvest and reducing weed seed return to soil. Growing
glufosinate and glyphosate tolerant crops would rest rict farmer's ability to conduct these practices and

65 SIMPSON, E & SWEET J B (2002). Consequence analysis of herbicide tolerant oilseed rape. Report
for DEFRA, Project RG 0217., 55pp.
66 ORSON J (2002): Gene stacking in oilseed rape: lessons from the North American Experience.
Report for English Nature 2002: 17pp.
67 SWEET, J. B., SIMPSON E. LAW., J, LUTMAN, P.J., BERRY, K., PAYNE, R., CHAMPION, G.,
MAY, M. WALKER, K., WIGHTMAN, P., & LAINSBURY, M. (2004). Botanical and Rotational Implications
of Genetically Modified Herbicide Tolerance in winter oilseed rape and sugar beet (BRIGHT Project). HGCA
Project Report No 353, 265 pp. ( www.hgca.com)

they would have to find other methods for mana ging weeds on their farms, which may entail extra
costs or use of herbicides with a worse environmental profile.
177. However, Sweet et al (2004) also concluded that HT volunteers of rape and beet could be
potentially controlled in non-related crops in rotation with appropriate management, assuming farmers
were fully aware of their presence and the potential problems, and were prepared to accept the
additional management load and possible economic consequences. This may however prove to not
always be the case.
(a) First sub-question
(a) What is the potential for the establishm ent and spread of herbicide tolerant plants
arising from handling, spillage during transpor t of the plant/plant parts, or any other
means outside of cultivation in the absence of application of the herbicide? How is
any potential for establishment and spread affected by environmental conditions, the
presences of wild or conventional relatives of the herbicide tolerant plants in an area,
or other factors?

178. The European Communities believe that this question warrants an assessment highly specific
to the case at hand. Dr. Squire states that:
In the absence of the specific he rbicide to which the plants are tolerant plants having
HT and non-HT traits should act similarly.
179. But he further points out that:
Species would differ (…) and spread depends on local conditions in ways that are not
clear.
which justifies a thorough in-depth analysis of each considered crop.

180. As indicated above, the potential for establishmen t and spread is the same as for conventional
crops and will only increase in the presence of th e herbicide. The potential for establishment will
depend on the crop species and the environment. The crops with the greatest potential are crops like
oilseed rape which can readily colonised disturbed ground, e.g. building sites, road verges etc.
181. One should consider that some crops have a high potential for dispersal and long term
establishment, especially crops like oilseed rape, which can be influenced by a range of environmental
and agronomic factors. GM oilseed rape will behave in the same way. In addition crops like oilseed
rape and beet have wild relatives with which they can hybridise.
68 Thus the presence of these related
species will result in hybrid formation and introgres sion of genes into these wild species. The wild

68 BARTSCH, D, CUGUEN, J, BIANCARDI, E, and SWEET, J (2003) Environmental implications of
gene flow from sugar beet to wild beet – current status and future research needs. Environ. Biosafety Res. 2,
123-127 . Anne-Marie Chèvre, Henrie tte Ammitzbøll, Broder Breckling, Antj e Dietz Pfeilstetter, Frédérique
Eber, Agnès Fargue, César Gomez-Campo, Eric Jenczewski, Rikke Jørgensen, Claire Lavigne, Matthias S.
Meier, Hans C.M. den Nijs, Kathrin Pascher, Ginette Séguin-Swartz, Jeremy Sweet, C. Neal Stewart Jr. &
Suzanne Warwick (2004). A review on interspecific gene fl ow from oilseed rape to wild relatives. In: Hans
C.M. den Nijs, Detlef Bartsch & Jeremy Sweet (Editors 2004) Introgression from genetically modified plants
into wild relatives and its consequences. Proceedings of an ESF AIGM conference, Amsterdam, 21-24 January,
2003, 235-252.

population can then act as a potential reservoir over space and time for transgenes
69 or can become a
weed in HT crops.70
182. Crop specific cases are discussed below:
Case of rapeseed

183. A retrospective study has been carried sin ce 1996 in Selommes (Loir et Cher, France). The
aim was to determine the origin of feral popul ations in road and field borders around a silo 71 and the
impact of border management practices on their persistence. 72 In summary, this study demonstrates
that seeds do disperse a lot. A field of rapeseed produces about 75000 seeds per square meter. About
10% are lost during harvest and transportation. It has been shown that these seeds establish all around
the fields and roads where they are transported and that the plants could live and reproduce outside of
the fields. It was demonstrated that some plants found outside the fields corresponded to genotypes
which had not been cultivated for more than 10 years.
184. Several hypothesis concerning the origins of th ese plants were raised: spillage during sowing
or harvest of adjacent fields, losses during transport, 73 "auto recruitment". A great diversity of
situations and practices was observed, 74 depending on farming systems and landscape patterns. A
complementary study determining the fitness of these populations was also carried out (Deville et al,
2002; 2003). Although these populations seems to have a lower fitness than those issued from the
fields (Deville, 2003b), they produce seeds and persist in the environment.
185. Indeed, Pessel et al (2001) showed evidence that seeds could persist 8 years in road verges
near cultivated area. Single-low rape seed plants (low erucic/high glucosinolates) were still observed
eight years after corresponding varieties had been withdrawn from the market. Lutman et al .75
observed persistence of buried seeds of rape over time. They concluded in 2003 that " Appreciable

69 EASTHAM, K & SWEET, J B (2001) Genetically Modified Organisms: the significance of gene
flow through pollen transfer. European Environment Agency, Environmental Issue Report 28, 75 pp.
70 Norris, Carol, Jeremy Sweet, John Parker & J ohn Law (2004) Implications for hybridization and
introgression between oilseed rape (Brassica napus) and w ild turnip (B. rapa) from an agricultural perspective.
In: Hans C.M. den Nijs, Detlef Bartsch & Jeremy Sweet (E ditors 2004) Introgression from genetically modified
plants into wild relatives and its consequences. Pro ceedings of an ESF AIGM conference, Amsterdam, 21-24
January, 2003,107-124
71 Pessel, F. D., J. Lecomte, V. Emeriau, M. Krouti, A. Messean, and P. H. Gouyon. (2001). Persistence
of oilseed rape (Brassica napus L.) outside of cultivated fields. Theoretical and Applied Genetics 102:841-846.
72 Deville A. Garnier A., Lecomte J., Adamczyk K. Huet S., Merrien A., Messéan A., 2003a. Origin
and dynamics of feral oilseed rape populations. Proceedings of the First European Conference on the Co-
existence of Genetically Modified Crops with Conventional and Organic Crops, Borupsgaard, 13-14 November
2003, pp 100-102. The Fate of Feral Plants; Jane LECOMTE In Commission du Génie Biomoléculaire, 2003.
séminaire du 28 novembre 2003, 80p.; English version in press.
73 Pessel, F. D., J. Lecomte, V. Emeriau, M. Krouti, A. Messean, and P. H. Gouyon. (2001). Persistence
of oilseed rape (Brassica napus L.) outside of cultivated fields. Theoretical and Applied Genetics 102:841-846.
Crawley M.J., Brown S.L., 1995. Seed limitation and the dynamics of feral oilseed rape on the M25 motorway.
Proc. of the Royal Society of London, B 259:49-54.
74 Deville A. Monod H., Lecomte J., 2003b. Are rapeseed feral populatio ns adapted to their
environment? Proceedings of the First European Conference on the Co-existence of Genetically Modified Crops
with Conventional and Organic Crops, Borupsgaard, 13-14 November 2003, p 208.
75 Lutman P. J. W.,Freeman S. E. Pekrun C., 2003. The long-term persistence of seeds of oilseed rape
(Brassica napus) in arable fields The Journal of Agricultural Science, 141:231-240

numbers of rape seeds will persist up to 4 years, in normal cropping conditions and in the absence of
cultivation, one experiment has confirmed persistence for over 11 years".
186. These results are consistent with those obtained in the BRIGHT study
76. Gene flow between
feral populations and fields was demonstrated by Devaux et al (2003). 77 Therefore, rapeseed and
consequently HT oilseed rape plants has a potentia l to spread and persist over time in non-cultivated
areas. Crop management practices in cultivated fields as well as road verges management have a great
influence on survival and development of such feral populations (see below).
Case of weed beets

187. In Europe, four forms of the species Beta vulgaris exist:
 – Wild or ruderal beet ( B. vulgaris ssp. maritima)
 – Sea beet ( B. vulgaris ssp. maritime and ssp. adanensis in the eastern Mediterranean
area)
 – Cultivated beet ( B. vulgaris ssp vulgaris)
 – Weed beet ( B. vulgaris ssp. maritima).

188. These forms of beet are interfertile, as shown by the gene flow detected between wild and
cultivated beets78 and by the transfer of useful genes of ssp maritima to new varieties of sugar beet.79
189. Forms of Beta vulgaris L. were first recognised as weeds in the 1950's,80 but this problem was
not really taken seriously in many European countries and the USA until the 1970's, when a large
number of studies were initiated.
81 The presence of weed beet in suga r beet crops results in decreases
in sugar yield (approximately 10% sugar yield loss per weed beet plant per m²) 82 and difficulties with
harvest and sugar extraction. These problems resu lt from differences in the reproductive cycle, as
sugar beet is biennial and weed beets are annuals . The management of weed beet infestations has
varied greatly and resulted in a large range of weed beet densities. No traditional chemical treatment

76 SWEET, J. B., SIMPSON E. LAW., J, LUTMAN, P.J., BERRY, K., PAYNE, R., CHAMPION, G.,
MAY, M. WALKER, K., WIGHTMAN, P., & LAINSBURY, M. (2004). Botanical and Rotational Implications
of Genetically Modified Herbicide Tolerance in winter oilseed rape and sugar beet (BRIGHT Project). HGCA
Project Report No 353, 265 pp. Available at <www.hgca.com>.
77 Devaux C., Klein E. K., Lavigne C. 2003. A first step for modelling pollen dispersal at landscape
level: determining the shape of dispersal functions at long-distance. The case of oilseed rape. Proceedings of the
First European Conference on the Co-existence of Genetically Modified Crops with Conventional and Organic
Crops, Borupsgaard, 13-14 November 2003, pp 172-174.
78 Santoni S. and Bervillé A., 1992: Evidence for gene exchanges between sugar beet (Beta vulgaris L.)
and wild beets: consequences for transgenic sugar beets. Plant Molecular Biology 20: 578-580. Boudry P.,
Mörchen M., Saumitou-Laprade P., Vernet Ph., Van Dijk H., 1993. The origin and evolution of weed beets:
consequences for the breeding and release of herbicide-resistant transgenic sugar beets. Theoretical and Applied
Genetics, 87: 471-478. Bartsch D. and Schmidt M., 1997. Influence of sugar beet breeding on populations of
Beta vulgaris ssp. maritima in Italy. Journal of Vegetation Science, 8: 81-84. Arnaud J.F., Viard F., Delescluse
M., Cuguen J., 2003. Evidence for gene flow via seed dispersal from crop to wild relatives in Beta vulgaris
(Chenopodiaceae): consequences for the release of gene tically modified crops sp ecies with weedy lineages.
Proc. Roy. Soc., London, B270, 1565-1571.
79 Debock T.S.M., 1986. The genus Beta: domesticati on, taxonomy and interspecific hybridization for
plant breeding. Acta Horticulturae 182: 335-343
80 Longden P.C., 1980. Weed beet. Agricultural progress 55: 17-25.
81 Hornsey K.G. and Arnold M.H., 1979. The origins of weed beet. Annals of Applied Biology 92:
279-285
82 Longden P.C., 1989. Effects of increasing weed-b eet density on sugar-beet yield and quality. Annals
of Applied Biology 114: 527-532.

can eliminate weed beets as they belong to the same species as sugar beet and are resistant to the same
herbicides. In France, 4.5% of the sugar beet cropp ing area (20,000 ha) is infested with weed beet to
such an extent that sugar beet crop production has had to cease because yields are too low. Weed beet
has been reported in crops other than sugar beet since 1974.
83 Weed beet plants have never been
considered troublesome as a weed in cereal crops because they are not very competitive and are easily
controlled by herbicides. 84 They have also been identi fied as a common weed in pea ( Pisum sativum
L.) and potato (Solanum tuberosum L.) crops.85
190. The origin of weed beet was dete rmined in 1993 from genetic studies 86: accidental
hybridisation seems to have o ccurred in the seed production area between a plant with cytoplasmic
male sterility and a wild pollen donor from the maritima subspecies. Whereas most cultivated forms
of beet are biennial, wild beets are mostly annual (dominant B allele). Hybrids between the two forms
contaminating seed lots are sown at the same time as the seeds of the cultivated variety and grow in
the row. It becomes possible to detect them when they begin to bolt and to flower. Weed beets may
also originate in two other ways87: 1) Possible bolting of cultivated sugar beet due to low temperatures
in the spring, vernalising the most sensitive plants so that they bolt and flower instead of producing
reserves of sucrose; 2) Potential re-growth of th e roots and neck, which remain on the ground after
harvest. The plants resulting from such re-growth ar e in their second year of development and they
bolt and produce flowers and seeds in the crop afte r sugar beet if they are not managed. These
flowering beets then produce seeds, thereby mainta ining the seed bank in the soil. Weed beet seeds
have been demonstrated to survive in soil for considerable periods of time and it is therefore
imperative for growers to prevent seed set.
Case of maize

191. Grains dispersed during harvesting, which could theoretically germinate, flower and pollinate
the next maize crop, do not generally survive for more than a year. Maize is susceptible to cold and
frost, and climatic conditions in Europe minimise its chances of long-term survival. Indeed, in south-
west France, where maize is frequently grown in continuous cropping systems and where winter
temperatures are mild, the maximum number of volunteers observed is 100 plants per ha. This number
might be higher in southern countries like Spain where surveys are being carried out. This level of
infestation would be sufficient to cause 0.9% cont amination, but the volunteers are easy to pick out
and to kill because they are located outside of the sowing lines in the field.
(b) Second sub-question
(b) What is the potential for the establishment and spread of herbicide tolerant plants in
the presence of herbicide application (in fields, urban, domestic or other
environments)? How is any potential affected by the existence of feral related plant

83 Gunn J.S., 1979. Weed beet in other crops of the arable rotation. British Sugar Beet Review, 7-10.
84 Gestat de Garambé, 2000. Gestion des conséquences agronomiques et environnementales de la
Mauvaises Herbes. Dijon, Septembre 2000.
85 Gunn J.S., 1982. Population dynamics of w eed beet. In: Proceedings 1982 British Crop Protection
Conference-Weeds, Brighton, UK, 61-66.
86 Boudry P., Mörchen M., Saumitou-Laprade P., Vernet Ph., Van Dijk H., 1993. The origin and
evolution of weed beets: consequences for the breeding and release of herbicide-resistant transgenic sugar beets.
Theoretical and Applied Genetics, 87: 471-478.
87 Longden P.C., 1993: Weed beet resurgence in 1993: the bolters return. British Sugar Beet Review
vol 61, no 3.

species; infertile wild relatives; seed survival in relevant pedoclimatic conditions; the
reproduction biology of the species; or other factors?

192. From the above, it is clear that these plants which can establish survive and reproduce without
any advantage would become invasive if an advantage was given to them.
193. The European Communities therefore agrees with Dr. Squire that:
applying the herbicide would favour the HT pl ants, lead to greater seed set on HT
plants and their increase in the seed bank.
194. The European Communities equally agrees with Dr. Snow when she states that:
In the absence of exposure to the herbicide in question, herbicide-tolerant plants will
not have any selective advantage over their non-transgenic counterparts. But when
the herbicide is used repeatedly, it will select very quickly for plants that are resistant
to the herbicide. The scientific literature in weed science is full of examples of rapid
evolution and spread of herbicide resistant weeds.
195. It is important to note that spread of tran sgenes can occur not only via pollen, but by seeds
and vegetative parts, and human activity most importantly. A model based approach
88 was used in a
study dealing with the co-existence of GM and non-GM rapeseed crops in European Agriculture. 89
The aim of the Genesys-model90 is to evaluate the effect of cropping system and rapeseed varieties on
gene escape from rapeseed crops to rapeseed volunteers in neighbour plots and in subsequent crops.
196. Results of this study pointed out to the importance of border and set-aside management on the
harvest purity,91 as well as field spatial distribution (see be low). Indeed, when borders and set-aside

88 The input variables of the model were (1) the regional field pattern comprising waysides, field edges
and fields; (2) the crop succession of each field; (3) the ag ricultural practices used to manage each crop (stubble
breaking, tillage, sowing date and density, herbicides, harvest conditions) and (4) rapeseed variety
characteristics (genotype, self-pollination rates, diff erences between GM and non-GM varieties in pollen
emission and yield). The main output variables are, for each year, adult rapeseed plants, newly produced seeds
and seed bank. For each of these variables, the density of individuals and their genotypic proportions are given.
The model describes the annual life-cy cle of volunteer or cropped rapeseed plants, which is simulated for each
plot and year. It comprises seed banks, seedlings, adult plants, flowers, pollen dispersal, newly produced seeds
and seed dispersal. These stages depend on crop type and management. Pollen and seed exchanges between
plots depend on plot areas, forms and distances as well as on flowering dates. The model is presently being
evaluated. The first results show that it correctly ranks cropping systems according to their rapeseed volunteer
infestation. However, pollen and seed dispersal is fre quently underestimated and it was kept in mind when
analysing the results.
89 Bock, A.-K., Lheureux K,. Libeau-Dulos M. Nils agard H., Rodriguez-Cer ezo E., 2002. "Scenarios
for co-existence of genetically modified, conventional and organic crops in European agriculture", Technical
Report Series of the Joint Research Center of the European Commission, EUR 20394 EN., 133p.
90 Colbach N., Clermont-Dauphin C., Meynard J.M., 2001a. GeneSys: A model of the influence of
cropping system on gene escape from herbicide tolerant rapeseed crops to rape volunteers. I. Temporal
evolution of a population of rapeseed volunteers in a field. Agric. Ecosyst. Environ., 83, 235-253. Colbach N.,
Clermont-Dauphin C., Meynard J.M., 2001b. GeneSys-Rape, a model of the influence of cropping system on
gene escape from herbicide tolerant rapeseed crops to rape volunteers. II. Genetic exchanges among volunteer
and cropped populations in a small region. Agric. Ecosyst. Environ., 83, 255-270.
91 Angevin F., Colbach N., Meynard, J. M., 2003. Coexistence of Rapeseed Varieties in Time and
Space: Using GeneSys Model to Adapt Cropping Systems. 11th International Rapeseed Congress, Copenhagen,
6-10 July 2003, pp 732-734. Colbach N., Angevin F., Meynard J. M., Messéan A., 2004 Using the GeneSys

with feral populations (whether glyphosate tolera nt or not) were treated by spraying glyphosate
instead of mowing or cutting, rates of contamina tion in rapeseed harvests would be almost 15 times
higher than the basic situation (current practices) giving evidence of the potential influence of feral
populations on development of HT plants in non GM fields.
197. There is potential for the establishment and spread of GM HT crops, particularly for
glyphosate (Roundup) or sulphonyl urea (eg Imidazo linone IMI) tolerant crops as these herbicides are
widely used in agricultural syst ems for controlling weeds outside of the crop growing season, before
crops have emerged and when they are used as crop dessicants. (See the reply of Dr. Snow on this).
Also glyphosate is widely used on railway lines, ro ad verges, parks, nurseries and gardens so that
there is a potential for greater survival of feral HT plants and related HT wild species. However this is
not so much of a problem with glufosinate (Liberty) as it is currently not widely used in agriculture
except as a crop dessicant.
198. Resistance to the herbicides glufosinate and glypho sate is also an issue that has been raised in
the procedures in dispute. Glyphosate is already one of the most widely used herbicides in the world
and so how significantly the additional usage on HT crops would speed up the evolution of tolerant
weeds is difficult to predict. Some resistance ha s already been found and some have argue that
increased usage on HT crops will accelerate the evolu tion of resistance. This would initially be an
agricultural problem but could be become an environm ental issue if it results in an increased usage of
herbicides with much worse enviromental profiles. (See also the reply of Dr. Snow). Farmers may
have a range of alternative herbicides they can u se to control HT volunteers or ferals, provided they
know they are likely to be present.
199. It is noteworthy to consider the reply of Dr . Andow in his paragraph 06.15, as it touches upon
an issue which has been discussed at length in the procedures in dispute.
200. His response does indeed point to an ambiguity in so far as HT crop plants may only partly be
assessed for environmental impact, because the impact arises also through the application of the
herbicide, which is often assessed separately. Howeve r, both components are to a large extent an in-
separable package in the GM HT technology, and, as systemic issues may arise from the combination
of both, some have argued that both should be assess ed as a package. By separ ating them, some view
that a component of the environmental impact may be excluded, and this may be at the heart of the
confusion and, sometimes, unspecificity, of some requests for further risk assessment information.
These requests were expressed by some that believe d that assessing the environmental impact of HT
crops without the effects of the corresponding herbic ide may essentially be an incomplete, if not a
moot exercise that might miss important aspects of the risk assessment.
201. It is also important to consider that one of the main agronomic advantages of HT systems is
that you can use HT systems to c ontrol volunteers of the same species and closely related weeds in
HT crops (e.g. weed beet in beet and wild turnip and radish in oilseed rape ). If these species become
HT themselves through gene flow then they revert to being problematic weeds in HT crops with a
high potential to spread in the presence of the herb icide and to act as a reservoir of transgenes for
other crops.
202. In beet seed crops there is currently an issue of crops being pollinated by wild or weedy beet.
This results in annual beet seed that produces no ha rvestable root but which can grow as a weed in
crops competing with them and shedding seeds to cr eate further weed beet problems (see above). If

model quantifying the effect of cropping systems on gene escape from GM rape varieties to evaluate and design
cropping systems. OCL, 11: 11-20.

GM HT seed crops are pollinated by wild/weed beet then the annual beet contaminants would be HT
and thus not controllable using the specific herbicide. This would then nullify a major benefit of HT
beet is that the herbicide can be used to control weed beet.
92
Infertile wild relatives

203. Infertile wild relatives are often difficult to c ontrol with existing herbicides which is why the
HT systems may be attractive. However it will be impor tant to manage the use of the herbicide so that
resistant populations do not develop due to over use and selection pressure.
Seed survival in relevant pedoclimatic conditions

204. Seed survival is already high in many species (e .g. Brassica spp) and so provides a reservoir
seed bank population to carry over contaminant GM into subsequent crops. Seed survival also
depends on the regions in Europe where crops are grown. For example maize survives very poorly in
regions with colder winters. But in warm and some maritime regions maize seeds and volunteer
seedlings will persist and survive during winter and so could occur in subsequent crops or their field
margins (see above).
The reproduction biology of the species

205. Outcrossing species (e.g. oilseed rape) are likely to disseminate genes more widely through
cross pollination. Crops and weeds producing large quantities of small robust seeds (e.g. brassicae)
also have better dispersal characteris tics. (See comments above on cross fertility) 93. However, the
reproduction biology of crops may not always be well enough known. For instance, pollen dispersal
of some major crop species is now known to be at a relatively constant density above a field over
several hundreds of meters. This part of the atmos phere moves a lot and this results in very long
distance dispersal of pollen. It took scientists a l ong time to realize this, because most of the pollen
fall near the plant which produced them. But those pollen grain that do not can travel very far from
their source. It was easy to find the pollen grains which did not migrate far, but it has been far more
difficult to show the existence of the pollen at much further distances. This is done now.
Or other factors

206. Multiple physical and biological factors influence gene flow – (see reviews).
94 These may be
biological factors: seed dormancy, seed longevity , seed vectors, pollen size, pollen viability, pollen
vectors etc…, as well as physical factors: scale of GM crop, size of recipient crops, distance between
donor and recipient crops, wind speed and direction, insect activity, air temperature, humidity, soil
moisture and temperature , …
207. One expert addressed these issues in detail, and her line of argument is stringent and
supported by science. But as of now, only the near est steps of out-crossing to the next and most

92 EASTHAM, K & SWEET, J B (2001) Genetically Modified Organisms: the significance of gene
flow through pollen transfer. European Environment Agency, Environmental Issue Report 28, 75 pp.
93 SWEET, J.B. (2003) Pollen dispersal and cross po llination. Proceedings of the First European
Conference on the Co-existence of Genetically Modified crops with Conventional and Organic crops, Denmark.
Danish Institute for Agricultural Sciences, 21-32.
94 EASTHAM, K & SWEET, J B (2001) Genetically Modified Organisms: the significance of gene
flow through pollen transfer. European Environment Agency, Environmental Issue Report 28, 75 pp. SWEET,
J.B. (2003) Pollen dispersal and cros s pollination. Proceedings of the First European Conference on the Co-
existence of Genetically Modified crops with Conventional and Organic crops, Denmark. Danish Institute for
Agricultural Sciences, 21-32.

compatible relatives (for example, Brassica rapa for oilseed rape) have been considered and
researched in the context of GM plants. However, multiple transgene out-crossing events via several
relatives of crop plants have not been studied in any great detail to our knowledge and none of the
experts addressed this gap of knowledge.
208. All of the relevant scientific issues were th eoretically known and discussed before and up to
1998
95, and such problems were all anticipated at th at time by many experts but it had not happened
yet at that time, since HT plants had only been recently released. However, similar processes were
known from other cases before and that is why expe rts were anticipating them. However, the first
peer-reviewed publication did not appear until early 2000s but it does in retrospect confirm those who
did warn of these developments and re-enforce t hose who called for precaution. Today, Canadian
farmers do have to carefully select the herbicides they can use and with no resistance management
plan in place, this situation will worsen from year to year. For Europe and oilseed rape, the gene flow
issue and its possible consequences for ecosystems (s pread of transgenes, reservoir function, multiple
pathways of spread) and agricultural practice (resist ance issues, stacking, weediness, coexistence) are
still being fully and rigorously thoug ht through in all the consequen ces. The situation in Europe for
oilseed rape is sufficiently complex, so that ther e remains uncertainties which are likely not to allow
experts to get a full grasp of all the consequences in the limited time available.
(c) Third sub-question
(c) Is this potential different for biotech crops tolerant to two wide-spectrum herbicides?
Please explain ?

209. The European Communities notes that Dr. Squire implicitly calls for a case-by-case analysis:
this will be highly specific to the agronomy of a farm or region
210. This question indeed depends mainly on the herbicides and the type of crop, e.g. in the case of
oilseed rape (high potential to disperse and surviv e) with tolerance to herbicides widely used in
cultivation, e.g. Imidazolinone (related to sulph onyl urea) and glyphosate (Roundup): then these have
greater potential to become weedy and survive treatme nts in a range of farmi ng situations. Herbicides
from different groups to these would be needed to control them. However if the tolerance is to
glufosinate and atrazine, which are only used in particular cropping circumstances, then weediness
potential is generally less but could be a problem in certain farming systems dependant on these
herbicides.
211. Only one of the experts, Dr. Snow, has pointed out the problem of stacking of different HT
resistance genes in invasive crops, making it an increasingly uncontrollable weed within shortest
period of time. At this time, triple resistant oilseed rape plants are confirmed in Canada.
96 These
plants become increasingly difficult to control, and most certainly farmers have again to resort to
'herbicides which may have a negative environmental impact.

95 Rissler J & Mellon M (1996): The ecological risks of engineered crops. Cambridge, MA: MIT Press.
96 Friesen LF, Nelson AG & Van Acker RC (2003): Evidence of contamination of pedigreed canola
(Brassica napus) seedlots in Western Canada with ge netically engineered herbicide resistance traits: -
AGRONOMY JOURNAL 95(5): 1342-1347. Beckie HJ, Seguin-Swartz G, Nair H, Warwick SI, Johnson E
(2004): Multiple herbicide-resistant canola can be co ntrolled by alternative herbicides. - WEED SCIENCE
52(1): 152-157.

212. Generally speaking, the same type of poten tial impacts should be addressed; however, the
magnitude of those impacts may sometimes differ si gnificantly, as pointed out by Dr. Andow in his
paragraphs 7.08 to 7.12 in his reply to the next question.
(d) Fourth sub-question
(d) If significant risks of establishment and spread have been identified, what risk
management options exist to mitigate any resulting risks and what is their efficacy?
213. The European Communities notes Dr. Squire's comment on risk management differences
between GM crops and non-GM crops, depending upon the agricultural system, that:
… need to be more stringently applied if cropping was to ensure the proportion of
GM in non-GM remained below a threshold
214. The European Communities notes also Dr. Snow's comment:
it is expected that transgenes that confer herbicide tolerance will not be associated
with negative effects on the weedy plants that inherit them. This means that the
transgenes could persist indefinitely in weed populations, even in the absence of
exposure to herbicides.
215. Risk management options are obviously critical in this area, and the European Communities
concur with the approach of the experts, and agre es that the listed measures could help reducing the
risk of maintaining and spreading the HT genes, while noting however that they would have several
shortcomings ecologically and agronomically. Know ing agricultural practice, compliance would be
low and hardly to enforce. Not least of all, it w ould put quite a burden on the farmers, and potentially
also increase the exposure of farmers to less innocuous chemicals, with all the related human health
consequences, and it would not eliminate the risk after all.
216. The risk at stake relates mainly to farmi ng and economics. Management may also include
seed purity, segregation (temporal and spatial), labe lling, separation of crops and products, careful
recording of farm operations and management, crop hygiene, crop rotational management, careful
planning of weed and volunteer control, monitoring of presence of HT volunteers and weeds etc.
97
217. Properly adopted measures can be effective at minimising the building up of HT volunteers
and weeds on farms. However they are liable to require changes to farming systems and have
economic consequences. The measures for maintaining low levels of HT oilseed rape on farms will be
the most difficult to implement and will require very stringent measures to maintain volunteers at low
levels and restrict gene flow. Temporal separa tion of non-GM rape from GM rape will be several
years (8-10) and spatial separation and sanitary measures will be needed.
98
218. The European Communities will elaborate below on risk management options:

97 TOLSTRUP, K., ANDERSEN, S.V., BOELT, B., BUUS,M., GYLLI NG,M., HOLM,P.B.,
KJELLSON,G., PEDERSEN,S., OSTERGARD,H. & MIKK ELSEN, S.A. (2003). Report of the Danish
Working Group on the Co-existence of Genetically Modified Crops with conventional and organic crops.
Danish Institute of Agricultural Sciences Report Plant Production No. 94 November 2003 275pp. Boelt B
(2003) Proceedings of the First Euro pean Conference on the Co -existence of GM crop s with conventional and
organic crops, Denmark, November 2003. Danish Institute for Agricultural Sciences, 228 pp.
98 SWEET, J. B., SIMPSON E. LAW., J, LUTMAN, P.J., BERRY, K., PAYNE, R., CHAMPION, G.,
MAY, M. WALKER, K., WIGHTMAN, P., & LAINSBURY, M. (2004). Botanical and Rotational Implications
of Genetically Modified Herbicide Tolerance in winter oilseed rape and sugar beet ( BRIGHT Project). HGCA
Project Report No 353, 265 pp. ( www.hgca.com)

Case of sugar beets
99

219. Weed beets are already a real problem in sugar beet fields as they belong to the same species
as the cultivated form and are not sensitive to herb icides used in sugar beet crops. The technical
institute in France estimated that one bolter in a suga r beet field leads to 18 bolters in the next sugar
beet crop. In most European countries, situations have been observed in which sugar beet cultivation
has been abandoned due to the yield losses inflicted by unmanaged weed beet infestations. Even in the
first sugar beet crop of a field, specific practices are required to control bolters. Various aspects of the
cropping system influence the evolution of weed beet populations:
 – Bolter-specific practices, such as hand pu lling in sugar beet crops, local herbicide
application, cutting etc.;

 – Weed management in the other crops of the rotation affect the weed beet seed bank
between two successive sugar beet crops . For example, the seed bank decreases if
groundskeepers and weed beet are eliminated by herbicides before crop production.

 – The number of years between two sugar beet crops in the crop rotation also affects
the weed beet population as weed beets pr oduce their seeds mostly in sugar beet
crops and the weed beet seed bank decreases in the other parts of the crop rotation.

 – The choice of the appropriate sugar beet variety limits the appearance of weed beets,
according to the number of hybrid seeds in the seed lot and the resistance of the
plants to vernalisation.

Case of oilseed rape

220. From the available scientific evidence, it can be concluded that species like rapeseed has a
potential for spreading and establishment as an HT crops. Risk management options would aim at
achieving two main objectives: (i) the development or extension of practices aiming at reducing, in
time and space, the persistence of undesirable plan ts (HT volunteers and hybrids with wild relatives)
and (ii) the avoidance of selection pressure on these undesirable plants.
221. The establishment of GM HT oilseed rape can be reduced by:
 – favouring the immediate emergence of seeds remaining on the soil after harvesting, in
order to withdraw them from the seed bank: no tillage until the first rain then repeated
minimum soil tillage avoiding seed dormancy;

 – increasing the control of rapeseed volunteers within the subsequent crops in order to
reduce the seed bank;

 – not growing other crops resistant to the same herbicide within the rotation making it
easier to control tolerant volunteers in the subsequent crops;

 – organizing the spatial location of crops through adequate isolation distances and/or
through regional specialization;

transgénique et adventice apparentée. Cas de la betterave sucrière (Beta vulgaris L.). Mémoire de thèse de
l'Université de Bourgogne. 153pp.

 – and dedicating to tolerant crops the use of broad spectrum herbicides with their active
matter used alone (glyphosate or glufosinat e) and associating another active matter to
these for their non-selective uses (pre-harvest applications, fallow land management).

222. As stated earlier, these measures as well as othe r measures such as feral plant control within
edges, set-aside management or cropping systems ch anges, have been assessed, through simulations
with the G
ENESYS®-rape modelling system. 100 Table 1 below illustrates the results for one specific
situation. From this example, it turns out that th ree major factors must be taken into account when
aiming at reducing the establishment of HT rapeseed plants: seed purity (effect of farm-saved seeds),
management of fallow lands and road verges, a nd spatial distribution of rapeseed fields over
landscape. Changing existing agricultural practices related to these factors will prove difficult to
achieve as they would suppose a more integrated management of cultivated and non-cultivated areas
(e.g. coordination between farmers and public road au thorities) and could lead to additional costs or
time requirements which are difficult to estimate as they will depend on each local farm structure and
cropping systems.

100 Angevin F., Colbach N., Meynard, J. M., 2003. Coexistence of Rapeseed Varieties in Time and
Space: Using GeneSys Model to Adapt Cropping Systems. 11th International Rapeseed Congress, Copenhagen,
6-10 July 2003, pp 732-734.Bock, A.-K., Lheureux K,. Libeau-Dulos M. Nilsagard H., Rodriguez-Cerezo E.,
2002. "Scenarios for co-existence of genetically modified, conventiona l and organic crops in European
agriculture", Technical Report Series of the Joint Research Center of the European Commission, EUR 20394
EN., 133p.

Table 1. Effect of changes in cropping system on the level of impurities in hybrid seed production
Farm 1: 0% GM on farm - 50% GM outside
Typical rotation: rape/wheat/wheat/fallow/wheat/barley

G
ENESYS®-rape simulation Relative impact on GM
HT impurities
Basic system (Table 1) 1
Harvest loss in rape crops: 10% vs 5% 1.15
Farm seeds vs. certified seeds 4.025
Tillage before rape: plough vs. chisel 0.725
 before other crops: plough vs. chisel 1.975
Rape sowing: early non-GM + late GM vs. simultaneous 3
 late non-GM + early GM vs. simultaneous 0.275
Border cutting: mid-April vs. uncut 0.625
Border herbicides: glyphosate vs. none 14.9
Set-aside: spring sown vs. unsown 0.025
Rotation: spring barley added 0.425
Crop location: fields with past rape >200m vs.0 m 0.015
Farm field location: clustered vs. dispersed 0.025

223. According to some agronomists, it is always possible to control rapeseed volunteers and weed
beets in cereal crops with "older" herbicides such as 2,4D (auxines), diquat, paraquat that are
registered in European lists and some aminotriazol ( not registered for this use yet). Profiles of these
active ingredients are quite different from those of the new registered ones and it would be necessary
to take into account differences in their environmental and human and animal health impact.
224. The European Communities note that Dr. Snow indicates:
Management options become more challengi ng and more complicated when the pest
population has genes for several types of herb icide resistance. In some cases, it may
be necessary to revert to the use of herbicides that have greater toxicity and longer
persistence in the environment (e.g., 2,4-D).
(e) Fifth sub-question
(e) What types of post-market monitoring and data collection activities could be
envisaged on the basis of the monitori ng and review principles described in
ISPM 11?

225. In ISPM 11, monitoring is primarily to identify novel or changed pest situations or to discover
failures of pest management measures. Thus, accord ing to ISPM11, post marketing monitoring could
include monitoring populations of HT feral crop pl ants and weeds on farms to look for development
of herbicide resistant populations. ISPM11 giv es little emphasis on monitoring for environmental
impacts, such as destruction of non-targets and be neficial organisms. However these are issues which
have now become much more important in Integrated Crop Management systems as widely advocated
throughout agriculture and one could argue that ISPM 11 is lagging behind in this respect (see in
particular the recent FAO expert consultation on m onitoring the environmental effects of GM crops
referred above in the European Communities' comments).

226. The European Communities notes Dr. Squire's co mments in that respect, on the difficulty of
developing and implementing appropriate monitoring strategies, and the lack of details of ISPM 11:
ISPM 11 does not give great detail on monitoring. The type of monitoring in this
instance would differ depending on purpose: for example, monitoring the effect of a
HT cropping on biodiversity or ecosystem functioning would require a different set of
measurements from monitoring the presence or abundance of a HT trait. What is clear
from existing data and work in progress is th at monitoring of this type is far from
simple and needs much time and effort, especially if the aim to measure low
frequencies (e.g. 1%, 0.1%) or small effects (e.g. 1.5-fold effects on populations or
ecological processes). Recently introduced populations are highly aggregated or
clumped and this increases the number of samples and area over which samples have
to be taken. At present, there are no reliable and accepted monitoring schemes for the
presence or impact of biotech-derived plants: research is in progress to develop such
schemes [ ], but it is not even certain that it would be feasible or practicable to
monitor low frequency occurrences or ecological effects routinely in the field.
227. The European Communities would equally note Dr. Snow's comments in that respect:
In cases where the persistence and prolifer ation of herbicide-resistant weeds could
pose problems, post-market monitoring could be used to develop an early warning
system for alleviating the problem. Weed scientists are already acutely aware of
emerging problems with herbicide-resistant weeds that develop in response to heavy
herbicide use. Many farmers are also familiar with these problems. Stewardship
programs can be developed for risk management, for example by encouraging
farmers to report cases of herbicide failure to the proper authorities. Studies of
reported cases could be carried out to test for resistance due to transgenes, and to
determine whether the stewardship programs are working as planned.
However, by the time the spread of herbicid e resistance is detected in free-living pest
populations, such as weeds or volunteer plan ts, it may be too late to prevent these
plants from proliferating further. By this time, continuous efforts may be needed to
keep their populations as low as possible. After a transgene for glyphosate has spread
widely in oilseed rape, as occurred in Cana da, there is no easy way to eliminate the
problem.
228. What is obviously needed most urgently, is a coherent resistance management strategy for
weeds and their proper monitoring, similar to the efforts that went into developing strategies to
mitigate insect resistance against Bt toxins. The European Communitie s agree with the experts, this
task is far from being simple and cheap and quick . However with Bt plants, we have not yet seen
significant resistance developing in insects; as w ith HT crops and glyphosate, we do see resistance
developing in weeds. Once glufosinate resistant plants are grown on a similar scale as glyphosate-
resistant crops, we can reasonably expect to observe developments also with them which might be of
the same type.
229. As pointed out by Dr. Snow, the issue is to de tect as early as possible th e spread of herbicide
tolerance in weeds and volunteers/feral populations . If one knows it exists, mitigation measures can
be applied in order to minimize the appearance of such populations (see Snow and Squire above).
That would certainly be the case for GM HT grow ers (although it has been observed that farmers not
always comply with good agricultural practices recommended by companies or technical institutes).

230. However, due to pollen dispersal, such populations can also be present in non-GM farmer
fields. Several studies have been carried out in various countries to assess the long distance pollen
flow.
101 Over one kilometer distance, outcrossing rate s are generally below 0.1 % but not equal to
zero. As the number of seed losses at harvest is high (1,000 up to 6,000 seeds/m²), the presence of
GM seeds in non-GM fields is not a rare event (around 1 GM seed per m² at long distance, thus
10,000 seeds/ha). In the absence of use of the specific herbicide, th is will however represent a very
small proportion of the volunteers (less than the outcrossing rate as seedbank already contains seeds).
231. Any application of the specific herbicide woul d led to inconvenience but not necessarily to a
selection pressure:
 – application of herbicide on volunteers (GM HT/non-GM HT) will leave GM HT
volunteers alive but not always lead to seed production (e.g. pre-sowing spray of
glyphosate before winter wheat sowing);

 – the most sensitive compartments are fa llow lands (if no use of mowing) and road
verges and other non cultivated areas that could act as a reservoir.

232. This would mean that all farmers (both GM and non-GM) should comply with mitigation
measures if we intend to mitigate the spread of herb icide resistance. This is technically feasible but
not easy to implement under the current legal framewo rk. This would also lead to extra costs for non-
GM farmers.
233. The functioning of these field volunteer plant populations is now relatively well-known. A
high volume of seeds (1000 to 6000 seeds per m
2 i.e. approximately 100 times the sowing dose)
remains in the soil of the crop plot after harvesting. Once buried in the soil, these seeds remain viable,
possibly for several years (between 5 and 10 years on average) and can re-grow in the rotation crops.
Volunteer plants then represent a potential new source of pollen and seed emission. The number of
volunteer plants per unit of surface area depends partic ularly on the moisture content of the soil and
farming practices during the period between crops. The size and fertility of these volunteer plants
decreases with the vigour of the farmed crops with which they are in competition.
102 Their frequency
in subsequent oilseed rape crops appears to decline with the length of the rotation.
Question 7

On the basis of the information before the Pane l, is there any scientific evidence to support the
hypothesis that repeated use of a given biotech h erbicide tolerant crop has adverse effects on flora
and fauna, including soil micro- and macro-fauna? If so, how does this compare with any similar
risks of adverse effects from the repeated use of a non-biotech herbicide tolerant crop (i.e, one
developed through mutagenesis)?

101 e.g. Rieger et al., 2002. SQUIRE, G.S., BROOKS, D.R., BOHAN, D.A., CHAMPION, G.T. et al.
(2003) On the rationale and interpretation of the Fa rm Scale Evaluation of genetically modified herbicide-
tolerant crops. Philosophical Transactions of the Royal Society B, 358, 1779-1800.
102 Fargue, A.; Meynard, J. M.; Colbach, N.; Va llee, P.; Grandeau, G.; Renard, M. , 2004.
Contamination of rapeseed harvest by volunteers of other varieties: a study of intergenotypic competition.
European Journal of Agronomy 21 (2): 193-207.

General comments

234. The European Communities notes that the Pane l's question does not touch upon the use of
different crops in rotation with the same herbicide tolerance, such as glyphosate or glufosinate, that
are only used with GM HT plants, and its potential adverse effects.
235. The European Communities notes that two of th e Panel's experts have addressed this issue
(Drs. Andow and Squire). Dr. Squire's reply indi cates that effects may be possible, and calls for a
case-by-case assessment, but that further research is needed and ongoing, and that effects on
biodiversity also relates to the effects of intensiv e agriculture. Dr. Andow agrees that there may be an
effect in certain cases, and refers to the Farm Scal e Evaluation study, but identifies as well that there
are gaps in knowledge, as no study has been pe rformed on soil micro-oganisms. Dr. Andow also
indicates that GM HT crops are more likely to spread than conventional crops.
236. Dr. Squire has indicated in his reply, with respect to the Farm Scale Evaluation:
The direction of effect differed depending on the severity of the conventional
management. In maize, where conventional management uses persistent, highly toxic
herbicides, GMHT increased the flora and fauna; in spring oilseed rape and beet,
where current practice was less effective against arable plants, it had the opposite
effect, reducing the flora and fauna. The cr ucial point about the FSE – and one that
has been missed or misrepresented by many international commentators – is that the
effects on the arable flora or weeds (though small by international standards) were
important in the context of the UK's arable scene in the early 21st century.
237. The European Communities agrees th at such effects have to be looked at for each GM crop,
in each receiving environment.
Detailed comments

238. The repeated use of a given GM HT crop may have indirect effects through changes of
farming practices. All agricultural practices constitute a significant and complex ecological
disturbance, even if limited to a single and simple action. Indeed, whatever the nature and objective of
the action, a large number of ecological processes are affected, and numerous discontinuities may
occur within the agro-ecosystem, in both time and space.
239. Any change in technical practice (e.g. the introduction of GM crops, new conventional
varieties, new chemical inputs, changes in the croppi ng system or in soil tillage) may therefore lead to
changes in ecosystems, of various degrees of significance, through ecological processes and
interactions with other agricultural practices. Such changes may have widespread environmental or
socio-economical consequences. Th ese changes affect not only the cultivated areas, but also the
ecosystem as a whole and the natural environment at large. Furthermore, as successive operations
within the field are interdependent, any change in one operation may necessitate modifications of
various extents to the other operations or to the cropping system. The introduction of a new variety
with a high level of resistance to a major disease may lead to decreases in chemical applications, or
may even render chemical application entirely u nnecessary. It may also make shorter rotations
feasible. When assessing ecological impact, it is not su fficient to focus only on the direct effect of
innovation; it is also necessary to take into account indirect and systemic effects.
240. As to HT crops, the risk of spread and est ablishment in agro-ecosystems is linked with
changes in the practices associated with herbicide-tolerant transgenic crops and the use of the related
herbicides. HT crops not only lead to a change of herbicide (with consequences on flora and fauna as

demonstrated in the FSE UK study) but also make easier other practices, such as minimum soil tillage
or no tillage at all or lead to changes in croppi ng systems as observed in Argentina (substitution of
grasses by soybean). Such indirect effects have environmental implications that could bear more
impacts (positive and/or negative) than the direct effect of HT crops.
241. As this is likely to occur equally in European farming systems, an ex-ante evaluation of such
indirect effects is a pre-requisite to set up appropriate risk mitigation measures as well as post-
marketing monitoring programmes.
242. The ecologically adverse effects of HT tolerant crops may also arise significantly through the
repeated large-scale use of the corresponding broad-sp ectrum herbicide, in particular those that can
only be currently used on a large scale with GM HT crops. Other adverse effects could arise through
unintended and unexpected changes in the GM plants metabolism through the process of genetic
engineering, due to the expression of the transgene. That such effects can occur are known and have
been documented in the peer-re viewed scientific literature.
103 Such effects can arise through the
insertion of the transgene and depend on the posit ion of the transgene in the plant genome, the
neighbouring gene sequences, ectopic effects, gene silencing etc.
243. This is recognized by Dr. Andow, who states that:
Most of the GM HT crops tolerate glufosinate and glyphosate, while most of the non-
GM HT crops tolerate imidazolinone and sulf onylurea herbicides. It is possible that
there are important differences in these herbic ides that result in different risks to flora
and fauna. I am unaware of studies that a ddress this possibility. In addition, the
options for managing resistant weeds ar e likely to differ among the different
herbicides.
244. However, the European Communities disagrees with the statement of Dr. Squire in the last
paragraph of its response, that "a general knowle dge of the macro- and micro-fauna in the soil
suggests they will be much less sensitive to GM HT cropping than will the above-ground flora and
fauna". The European Communities believes that the level of knowledge in the scientific community
does not exist to date to support that thinking. In f act, it might be the very lack of data and general
neglect of soil ecosystems that might create the notion that soil systems are less sensitive. In the cases,
where soil scientists look into it, they often find th e very opposite. Soil systems react quite sensitively
to chemical inputs, but because agricultural soils ar e already highly disturbe d and often quite poor in
biodiversity, and only productive with significant ex ternal inputs of agrochemicals, changes are not
easily detectable, certainly not for lay people.
245. This is where this particular conclusion of th is expert leads to a normative judgement rather
than scientific ones. He acknowledges the widespr ead use of other agrochemicals and that in
comparison to these, 'the herbicides glyfosate and glufosinate ammonium being much less directly
toxic to soil fauna than other agrochemicals and the GM HT plants themselves'. While, it might be
true that the two named herbicides are less toxic than say Atrazine, it is a normative judgement to
imply that this would justify the more intensive use of a less toxic one. This may sound like a choice
between two adverse situations, fo r which it is not clear whether one is better than the other.
However, no long-term data (> 3 years) and systema tically recorded field experience is available to

103 Saxena D & Stotzky G (2001): Bt corn has a higher lignin content than non-Bt corn. - AMERICAN
JOURNAL OF BOTANY 88(9): 1704-1706. Birch ANE, Geoghegan IE, Griffiths DW & McNicol JW (2002):
The effect of genetic transformations for pest resistance on foliar solanidine-based glycoalkaloids of potato
(Solanum tuberosum). - ANNALS OF APPLIED BIOLOGY 140(2): 143-149

date on development of soil microflora and –f auna under large-scale use of GM HT crops and
repeated applications of glufosinate or glyphosate.
246. Some data however, do emerge from the use of glyphosate resistant soybeans in the US and
some of these findings do rather point in the direction of a change in soil microbial activity towards
favouring fungi over bacteria. For example Kremer et al. (2000)
104 found that in soils repeatedly
treated with glyphosate and grown to glyphosate resist ant soybeans, soybeans significantly fell victim
to a Fusarium fungus causing 'damping off'. It would in fact be rather surprising if such intensive use
of one chemical would NOT cause a change in the microbial communities. The experience from
Canada and the US also clearly show that the u se of the respective herbicides complementary to GM
HT crops do increase significantly with the production of the respective HT crops105.
247. Finally, as regards the statement of Dr. Andow in his paragraph 7.05 that
It may be possible that there are significant sources of genetic diversity in oilseed
rape and beets in Europe.
248. The European Communities would like to provide some scientific evidence to clarify this
issue, at least for the latter species mentioned. 106 Although the origin of the different species is not
entirely clear, B. napus must be derived from cross between B. rapa and B. oleracea. Since the latter
was originally confined to the Med iterranean region, it is believed that B. napus must have originated
southern Europe from where it was intr oduced to into Asia in the early 18 th century 107. B. rapa is
believed to be the oldest species and to have had the widest distribution. It could be found at least 200
years ago over an area extending from the west of Europe to the east of China and Korea and from
Norway to the north of the Sahara and India. 108 Burkhill 109 proposed that B. rapa originated
somewhere in Europe as a biennial form and that the annual form evolve d later. Central Asia,
Afghanistan or India may have bee another centre of origin.110
Question 8

What are the different detection methods currently av ailable for testing for the presence of material
from genetically modified plants?

 (a) Have commercially available detecti on methods changed since the mid- 1990's?
Were methods available in the mid-90's event specific?

 (b) Please outline the steps necessary to validate a detection method, including the
determination of what types of refe rence materials needed and differences in
validation steps for qualitative and quantitative detection methods.

104 Kremer RL, Donald PA & Keaster AJ (2000): Herb icide impact on Fusarium spp. and soybean cyst
nematode in glyphosate-tolerant soybean. American Society of Agronomy publication [online] Available from:
http://www.biotech-info.net/fungi_buildup_abstract.html [Accessed 19 January 2005]
105 C. Benbrook, 2004.
106 Kimber D. S., McGregor D. I., 1995. The species and their origin, cultivation and world production.
In "Brassica oilseeds. Production and Utilization", Kimber D. S., McGregor D. I. Eds, CAB International
Publishing, pp 1-7.
107 Downey & Röbbelen, 1989.
108 Hedge, 1976.
109 Burkhill, 1930 cited in Prakash, 1980.
110 Sinskaia, 1928; Vavilov, 1949.

 (c) What are the differences in the intende d uses of qualitative and quantitative detection
methods? What are the differences betw een event specific and non-event specific
detection methods? How does the availability of different types of detection methods
relate to risk assessment and risk management processes?

General comments

249. The European Communities regrets that only one expert has provided a reply to that
important question. The European Communities no tes that Dr. Nutti has provided some general,
mainly descriptive, elements of reply, although in answering some parts of the question, she has stated
that she is "not an expert in this area", and has limited herself to provide "the information that she
could find" (she provides a list of references and of the information she found), "but that she did not
express her personal opinion".
250. The European Communities will comment in detail below on the information she has
provided, which is descriptive and factual, but woul d like to point already now that it does not agree
with all her statements, such as the following one, wh ich obviously contains an internal contradiction,
that:
The protein based methods, while not even t-specific, can identify uniquely most
commercial events.
251. The European Communities will therefore end eavour to provide further scientific and
technical evidence to the Panel, in order for it to understand the reply of the expert and the state of
play on detection methods as fully and objectiv ely as possible. The Eu ropean Communities has
prepared this information with the assistance of scien tists, experts in the fiel d of the development and
validation of detection methods.
252. The European Communities would also like to refe r back to its initial comment in the section
on general and methodological issues, on the relationship between detection methods of GM products,
and risk management.
Detailed comments

253. Detection methods can be classified on the b asis of the target, e.g. seeds, protein or DNA
molecules. They can also be classi fied on the basis of their specificity relative to the genetic material
they detect, e.g. screening, trait/construct and even t specific methods. Finally, they can be classified
on the basis of their ability to quantify or not the GM material. A comprehensive overview of
detection methodologies and their applicability range and limitations is given in Miraglia et al. (2004).
254. Detection methods can be classified on the basis of the kind of material they can detect: the
GMP itself may be detected by application of a wide range of methods, while processing will reduce
the range of methods suitable for detection of GM P derived material. The by far most commonly
applied currently available detection methods target e ither the modified genetic information itself, i.e.
a DNA sequence, or a product of the modified ge netic information, i.e. the transcript (an RNA
sequence) or more often a translation of the transcript (a protein or an amino acid sequence). It is also
possible to apply e.g. herbicide sprays to test for herbicide tolerant traits, but this is not widely
applied.
255. Both DNA and some proteins are persistent to certain types of processing, and therefore both
DNA and proteins may be detectable in processed material. Protein methods target particular

structures or epitopes of the proteins by application of immunological techniques; Western blots,
ELISA (Enzyme linked immunosorbent assays), or lateral flow-strips. DNA methods target particular
sequence motifs, usually by application of PCR (p olymerase chain reaction) amplification of the
specific DNA sequence followed by de tection and/or identification of the amplification product(s). If
DNA of high quality is available and from a low or u nprocessed pure plant material, it is also possible
to detect GMP derived material by digestion of the DNA with restriction enzymes followed by
Southern bloting and hybridisation with a labelle d probe corresponding to a particular sequence of
interest.
256. With PCR detection/identification and quantification can be done simultaneously in real-time.
Methods to detect GMP derived material may be classified into categories depending on their
specificity.
257. Screening methods can detect material that is likely to be derived from GMP, but that may
also be derived from a non-GM naturally occurring source such as soil bacter ia or the Cauliflower
mosaic virus (CaMV). These methods typically targ et commonly used regulatory sequences (e.g. the
CaMV 35S promoter [P -35S] or terminator [T-35S], or the Agrobacterium tumefaciens nopaline
synthase terminator [T-nos]), marker genes (e.g . neophosphinotrin transferase [nptII] or kanamycin
[cam]), or parts of commonly applied cloning vectors (e.g. pUC18/pUC19 derived sequences).
258. Gene or trait specific methods can detect material from GMP or naturally occurring non-GM
organisms containing a gene encoding the protein of interest. These methods typically target genes
inserted in multiple GMPs (e.g. CryIA(b), EPSPS, Pat or Bar genes). Notably, the inserted genes may
have been subject to modifications, so that th e specific DNA sequence may be synthetic, in which
case the detection method may be more specific (see also next category). Particular proteins or
epitopes may also fall into this category.
259. Construct specific methods can detect material that is derived only from synthetic genetic
constructs, e.g. a virus derived promoter in combination with a gene from one type of bacterium and a
terminator from another type of bacterium.
260. Event specific methods can detect material that is derived fro m a single transformation event,
e.g. the unique sequence motif created when an in serted genetic construct is fused to a recipient
chromosome in the plant that is subject to genetic modification.
261. Notably, a regulatory sequence, gene or a genetic construct, naturally occurring or synthetic,
can be inserted into a GMP in one or several comp lete or partial/truncated copies in the modified
haploid genome. These sequences may therefore be cons idered unreliable with respect to their fitness
for quantification purposes. Furthermore, these types of sequences can be introduced into more than
one recipient plant, i.e. they may be found in more than one transformation event now or in the future.
These sequences may therefore also be considered unreliable with respect to their fitness for
identification purposes. In contrast, event specific target sequences are unique to a single transformed
plant (the elite event) and its descending lines, and it is always present in only a single copy in the
modified haploid genome. These sequences may therefor e be considered reliable with respect to their
fitness for quantification and identification purposes.
First sub-question

 (a) Have commercially available detecti on methods changed since the mid- 1990's?
Were methods available in the mid-90's event specific?

262. The answer to the first Panel Sub-question is yes, available commercial and non commercial
detection methods have significantly changed since the mid 1990. DNA based methods available
before then were not event specific; quantita tive DNA based methods mainly applied competitive
PCR compared to extensive use of real-time PCR, since the turn of the millennium. Finally, lateral
flow-strip tests for detection of transgenic proteins were not available.
263. In the mid 1990, there were practically no me thods commercially available for detection of
GM plants, although restriction analysis combined with Southern bloting and DNA hybridisation was
an option for molecular characterisation and detectio n, provided that a DNA probe was available (e.g.
screening targets or gene/trait specific targets could be used since these were easily available).
264. The PCR technique was well established, but no suitable primers or probes were publicly
available for the purpose of detecting GMP derived material, apart from those typically developed for
amplification of sequences inserted into commerc ial cloning vectors. The first PCR methods
developed specifically for the purpose of detecting GMP derived material were published late in the
1990's. The first method for real-time quantificati on of GMP derived material was published in 1999
and the first event specific PCR methods were publis hed in 2000 and 2001 ( see e.g. Holst-Jensen et
al., 2003). In general, availability of detection me thods has been restricted by lack of available
sequence data and reference materi al required for method development and validation. There has also
been significant developments, since the end of the 1990's of DNA isolation methods from various
plant and food samples, in order to overcome technical barriers due to the presence of inhibitors of the
PCR reaction, or to overcome the poor quality of the isolated DNA.
Second sub-question

 (b) Please outline the steps necessary to validate a detection method, including the
determination of what types of reference mat erials needed and differences in validation steps
for qualitative and quantitative detection methods.?

265. The European Communities agree with the explan ation of Dr Nutti as regards the importance
given to appropriate certified reference materials. For further information, it refers to documents
available on the website of the Community Reference Laboratory
111.
266. Method validation is a very complex process, but simply outlined it consists of the following
steps:
 1. Method development and optimisation, in cluding the determination of the method
performance in relation to the scope of the method.

 2. Method transferability studies (pre-validation trial in small scale).

 3. Collaborative trial validation according to international harmonised standard and
protocol.

267. First, it is necessary to define the term "met hod", because the term is used inconsistently.
Relative to validation in the context of detection of GMP derived material, a method usually consists
of a DNA extraction step and two PCR steps, one targeting a reference sequence (usually a single
copy housekeeping species specific sequence) and the other targeting a GMP derived sequence.
Sometimes the instructions associated with only one of these steps is referred to as a method. In the

111 http://crl-gmo.jrc.it

following, a method will refer to the combination of instructions for all three steps. In the case of
protein based methods there are usually only two step s involved, i.e. the extraction of protein and the
detection/identification of the GMP derived protein. Protein based methods are usually applied semi-
quantitatively, i.e. a qualitative test is performed with a sensitivity corresponding to a desired
threshold. Because the vast majority of validated methods for detection of GMP derived material
target DNA sequences, the following description of method validation is gene rally referring to how
DNA based methods are validated (see also Miraglia et al., 2004).
268. Method development and optimisation . Method validation is a process starting with the
method development on the basis of a particular scope and a particular reference material. The scope
includes definition of the specificity (detection or identification), the sensitivity (lower limit of
detection/quantification) and the application (matrix limitations, qualitative and/or quantitative).
269. After method development and optimisation, th e performance characteristics of the method
are described and evaluated. If the performance ch aracteristics are deemed fit for purpose then it is
strongly recommended to perform a small scale pre- validation trial including at least one or two
laboratories familiar with the technology applied but not with the particular method. The purpose of
this is to assess the method transf erability prior to a full scale collabor ative trial, in order to identify
gaps in the protocol, sources of possible errors and to avoid wasting resources on sub-optimal
methods.
270. Method transferability studies. The objective of the second step is to assess whether it is
likely that the method will meet the desired perform ance values in a full collaborative trial. This
phase includes preparative steps like selection of par ticipants, production of reference materials and
test samples, reagent quality testing, as well as evaluation and reporting of the resulting data.
271. Planning and preparing for the validation trial also includes deciding what kind of test and
reference material is going to be used, and productio n of the test and reference material of appropriate
quality in sufficient quantity. Test materials are the samples to be treated as unknown in the
validation study. Reference materials are the sample s to be treated as known re ferences or calibrants
in the validation study. Quality here refers to i nherent characteristics of the material such as
homogeneity, presence of the analyte of interest (e.g . DNA or protein) in a condition that allows for
its detection and eventually for its quantificati on, and correctly assigned quantity of GMP derived
material.
272. Most validation studies have used seeds or kernels as the basis for production of test and/or
reference material, mixed on a weight: weight b asis, and ground and blende d to homogeneity.
However, the grinding and blending process may affect the analyte of interest, e.g. by considerable
degradation. Furthermore, a weight: weight based reference material may have an assigned GMP
concentration considerably different from that determined analytically (e.g. a DNA: DNA based
concentration). It is therefore important to ensu re correspondence between the reference material and
the test material, depending on the scope of the method.
273. If the method shall determine the weight: weight based concentration, then both the reference
material and the test material should be made on the basis of weight units. If the method shall
determine the analyte: analyte based concentration, then both the reference material and the test
material should be made on the basis of analyte units. To further complicate, it is important that test
material and reference material are produced inde pendently. A problem with weight: weight based
quantiation is that the same quantity can be obtained with the use of both homozygous and
heterozygous seeds/kernels. Thus, if homozygous ma terial is used to produce the reference material,
and heterozygous material is used to produce the test material, a 50% measurement error will be
introduced if the plant concerned is diploid and a DNA based detection method is applied. This

potential error is also relevant in the context of application of the method si nce it is not possible for
the analyst to assess for a sample received the homo- vs. hete rozygous status of its GMP derived
material.
274. Therefore the most recent validation studies have focused only on the PCR steps, and
prepared reference materials and test materials from purified DNA, in which the assigned
concentration of GMP derived material is determ ined on the basis of target sequence copies.
However, this also means that the DNA extraction st ep is somewhat detached from the method. This
approach is referred to as the modular approach, and is currently subject to considerable debate. It is
based on the assumption that DNA extraction is linked with the matrix, e.g. a flour produced from
ground seeds/kernels, and that if a particular in struction for DNA extraction from e.g. a soya bean
flour has been subject to successful validation then it may be applicable to other soya bean flour from
substantially equivalent soya beans.
275. Presently, there are consequently two favoured options: either to 1) produce matrix based
reference and test materials with assigned concen trations based on weight concentrations (which
means there is a considerable source of error in th e application of the method), or 2) produce analyte
based reference and test materials with assigned c oncentrations based on analyte ratios (which means
that if there is an analytical bias introduced by analyte extraction it is ignored erroneously).
276. Collaborative trial validation . This includes preparative step s like selection of participants,
production of reference materials and test samples, reagent quality testing, as well as evaluation and
reporting of the resulting data. The detailed listing of parameters etc. in Dr. Nutti's reply is indeed
correct, but one of the major problems with GMO detection methods is to reach consensus on the
values that must be achieved for each of the parame ters for the methods to be considered fit for
purpose. While the European Network of GMO La boratories reached an agreement which was first
published on the website of the Community Refe rence Laboratory in the summer of 2003, other
bodies like the European Committee for Normalisati on (CEN), TC 275/WG 11 and the International
Standardization Organization (ISO), TC 34/WG 7, are still discussing on method acceptance criteria.
Indeed the ISO WG recently proposed to est ablish a separate work item on defining method
acceptance criteria in relation to the achieved specifi c values of the parameters in collaborative trial
validation studies (ISO, 2004).
277. Prior to launching a validation trial all reag ents must be quality checked, to avoid that
impurities in e.g. bathces of PCR primers or probes co mpromise the result. Furthermore, it is critical
to identify qualified and willing pa rticipants. A full scale collaborativ e trail must according to the
internationally harmonised protocol for method validation (Horwitz, 1995) include at least 8
succesfull participants, and therefore it is common to include between 12 and 15 laboratories in a
collaborative trail (buffering for non-replying particip ating laboratories and outliers). Inexperienced
laboratories sometimes confuse method validation with proficiency testing and volunteer to
participate in validation studies. To ensure that the method and not the laboratories are trialed,
selection of participant laboratories is in itself an important task. The typical labour effort contributed
to a method validation study by a participating labo ratory, including reporting of data etc. is 2-5
person workdays per method. This may affect availability of volunteer laboratories.
278. On the basis of the defined scope of the met hod, the validation study is set up with test
materials and reference materials covering a range of quantities of analytes. It is most common to
consider the concentration of GMP derived material , and to prepare the materials to cover values in
the range of 0% to 5% GMP derived material. However, the limit of detection and quantitation in
PCR analyses is usually linked with the number of am plifiable copies of the target sequence. Recent
validation studies therefore also consider the numbe r of target copies in the test and reference

material. In this case the values may e.g. c over the range between 40 and 5000 copies for GMP
specific DNA sequences and between 1000 and 10 0000 copies for the reference DNA sequences in
each single PCR. Thus, the validation study may result in expression of method performance both in
relative values (concentration, expressed in percen t), and in absolute values (number of target
sequence copies).
279. The planning, preparation and conduct of a valid ation study also includes defining how e.g.
PCR plates are to be set up to determine the so called plate effect, the number of replicates and
independent runs (plates) to determine the so ca lled run effects and assess the repeatability standard
deviation RSD
r. Data processing also includes treatment of outliers.
280. If a validation study shall also include assessment of the method specificity, this will require
setting up negative target control samples. This is usually considered only in the context of method
development. Method robustness is another parameter that is usua lly not subject to testing in
collaborative trial validation. This would require t esting the effect of intended modifications, e.g.
altered reagent concentrations or altered temper ature profiles in PCRs. This too is normally
considered during method validation only.
Third sub-question

 (c) What are the differences in the intended uses of qualitative and quantitative detection
methods? What are the differences between event specific and non-event specific detection
methods? How does the availability of different types of detection methods relate to risk
assessment and risk management processes?

281. Qualitative methods are used to assess whether GM material is present or not, and to identify
the GMO from which the GM the material is derive d, including assessment of its status as authorised
or not. A qualitative detection method is typically inte nded for quick determination of the status of a
material, i.e. does it contain or not a particular item (above the lowest limit of detection of the item)?
Such a method would typically be used to assess whet her e.g. unauthorised items are present in the
material, or to assess whether further use of quant itative detection methods are required (e.g. in
relation to authorised material and particular thresholds).
282. A quantitative detection method is typically inte nded for determination of the actual quantity
of an item, e.g. in relation to a particular contractual or legal threshold. The unit of measurement plays
a key role in defining quantitative methods and the resulting values of applying the methods. For
example will a batch consisting of 100 GM maize kernels contain 100% GM if kernels or weight units
are used, while it will contain between 40% and 100% GM if haploid genome equivalents [DNA copy
numbers] are used, depending on the homo- vs. hetero zygous status of each individual kernel, and
whether the maternal and/or paternal gametes were GM or not.
283. Event specific methods can be u sed to establish with absolute certainty that a material is
derived from a particular transf ormation event, and to quantify very reliably the haploid genome
(DNA) based quantity of the derived material. Othe r methods like trait or construct specific methods
may sometimes be used to establish with acceptabl e certainty that a material is derived from a
particular transformation event, and to quantify with acceptable re liability the haploid genome based
quantity of the derived material.
284. While event specific methods target a DNA se quence motif created as a unique consequence
of a particular transformation event, all other me thods (screening, gene/trait and construct specific)
target DNA sequence motifs that may be present in more than one transformation event existing or

potentially created in the future. This is due to th e nature of the transformation process itself (see e.g.
Holst-Jensen et al., 2003).
285. Furthermore, in a haploid GM genome, each event specific sequence motif is present in only
one copy, while any other sequence involved in the genetic modification may be present in more than
one copy, and the number of copies may vary from one GMO to another. Consequently, event specific
methods are ideal for identification and quantification purposes.
286. Two objections may be raised: that event specifi c motifs will also appear in stacked events,
and that DNA based quantification may yield result s significantly different from e.g. weight or seed
number based calculations. Consequently, if stack ed events are considered unique and should be
distinguished from their parental counterparts, then there is presently no truly useful quantification
method. And if weight or seed number based calcula tions shall prevail, then it is necessary to either
test on single unprocessed seeds, or alternatively for processed material to know in advance the actual
degree of homo- and heterozygous material, and th e actual frequency of GM male and female germ
cells that gave rise to the original material.
287. However, the reliability of event specific met hods both with respect to identification and
quantification purposes is superior to any other altern ative. In relation to risk assessment and risk
management, highly specific and quantitative methods can be very useful tools if a material is to be
identified e.g. to be removed successively, in case of occurrence of unforeseen risks. To be able to
develop an event specific detection method it is n ecessary to have access to the DNA sequence of the
junction region between the insertion locus and the inserted DNA. This will include detailed
characterisation of the inserted DNA, not just the original construct that was intended to be inserted.
288. This is important since it is well known th at significant rearrangements of DNA in or
associated with genetic constructs take place during tr ansformation. And, equally important, this also
implies that the insertion locus itself will be charact erised. This in effect will allow for a better
evaluation of the potential effects of the genetic m odification, such as whether there is a risk of
unintended genetic effects, and whether the copy numbe r and location of insert is likely to be stable.
Such data may also provide additional and valuab le information about the different levels of
predictability associated with different transformatio n technologies. Such information may later be
used to give recommendations concerning the effectiveness and precision of alternative
transformation technologies
289. However, sometimes, the gene product may be of particular relevance. Development of
detection methods is often closely linked with mo lecular characterisation, and consequently may
provide important background information to risk assessors.
Question 9

In what ways does molecular characterization info rm the risk assessment for any particular biotech
product? Can a risk assessment be carried out in the absence of a comprehensive molecular
characterization of each transformation event?

General comments

290. The European Communities notes that all expe rts agree upon the fact that a molecular
characterisation is useful and necessary in a safety evaluation. As rightly pointed out by all experts,
this has been established in a number of internati onal consensus documents and notably in the Codex
Guideline CAC/GL 45-2003.

291. Dr. Andow emphasises that "a risk assessment cannot be carried out in the absence of a
comprehensive molecular characterization of each transformation event." He considers it necessary
for the prediction and verification of transgenic expression as well as genetic stability. Dr. Healy
concludes that currently the molecular characterisa tion is an important element of safety assessment,
providing verification that the intended genetic modification has occurred and information about
unintended effects." Dr. Nutti confirms the "fundame ntal importance" of molecular characterisation
for the food safety assessment of a GM product. Fr om his perspective as an environmental expert, on
the other hand, Dr. Squire considers that it is "not always" necessary when considering spread or
persistence of GMOs or their effect on the ecosystem. He concludes, nevertheless that it is
"reasonable" for an assessing body to request full characterisation.
292. Generally opinions tend to differ, however, as to the level of molecular characterisation that is
needed for a safety assessment. There is now some guidance in international consensus documents as
to what is reasonable and necessary to include on mo lecular characterisation, but the issue has been
controversial throughout and remains in part controversial. Among scientists, on the other hand, there
is considerable agreement on the kind of informati on required to sustain formulated conclusions. This
fact is reflected in the experts' replies.
Detailed comments

293. The experts' general conclusion that a molecula r characterisation is necessary is in line with
Codex guideline CAC/GL 45-2003 which states in its paragraph 30.
In order to provide clear understanding of the impact on composition and safety of
foods derived from recombinant-DNA pl ants, a comprehensive molecular and
biochemical characterization of the genetic modification should be carried out.
294. Molecular data provide insight into whether th e intended modification has been achieved and
is stably inherited, and also whether uninte nded effects may have occurred, for example by
rearrangements of the inserted DNA
112.
295. This is part of the internationally acknowl edged comparative approach of safety assessment
of GMOs (also known as "substantial equivalence"). In this appr oach, the differences between a
GMO and a conventional counterpart with a history of safe use are established. Based upon these
differences, it can be decided upon wh ich safety tests are further needed
113. Molecular
characterization will aid to clarify which changes have taken place in the GMO at a molecular level as
caused by the genetic modification. Molecular char acterization is therefore an essential part of the
safety assessment of a GMO for environmental release, food and feed applications.
296. Dr. Squire's statement may cast a doubt on this generally agreed fact as he considers that
molecular characterisation mzy "not always" be need for the purpose of assessing spread and
persistence. First of all it should be pointed ou t that this statement is made from the specific
perspective of an ecological/environmental expert. Indeed it may even have been made from the
specific perspective of what is needed for the purposes of research rather than for the purposes of a
safety assessment. Second, even if that were to be the case the European Communities is not quite in
agreement with Dr. Squire on this point. To mon itor spread and persistence of a new GM event,
detection methodologies based on phenotypic trait(s), protein(s) and/or DNA are necessary. A

112 See, for example, Kuiper, H.A ., Kleter, G.A. (2003) The scientif ic basis for risk assessment and
regulation of genetically modified foods. Trends in Food Science and Technology 14: 277-293.
113 See also paragraph 13, of the Codex guideline CAC/GL 45-2003.

complete molecular characterisation forms the basis for all further monitoring tests. Especially in
environments where different GM events may grow and occur next to each other.
297. Equally Dr. Squire's states that:
for some aspects of comparing GM and non-GM plants, detailed knowledge of the
molecular construct is not necessary. For instance, if comparing the ecological effects
of GMHT or Bt plants, the studies could be done equally well without detailed
molecular knowledge of the construct.
298. This statement is misleading. A GM plant coul d not be compared to a non-GM plant, if no
molecular data at all were available about the inser ted genes. Before the long-term ecological impact
of e.g. a Bt plant can be assessed, detailed data are required on the exact structural composition of this
Bt-transgene construct, e.g. the type of promotor (constitutive or not?), type of terminator, the type,
length and exact part(s) of the Bt-gene(s) inserted, the copy number of inserted transgene construct
and the number of insertion loci, and the stability of the inserted transgene construct copies. All these
characteristics will influence the expression of the Bt gene(s) and thus the effect on ecology. E.g. one
Bt-plant can have other ecological effects than another Bt-plant, depending on which Bt-gene(s)
is(are) inserted, the construction of the T-DNA insert, the locus of integration, number of integrations
and number of transgene copies per integration, an d other genetic factors determining the expression
of the gene(s).
299. There are differing views as to the level of de tail for molecular characterisation needed.
There is some guidance at the level of internati onal consensus documents. Thus, the Codex Guideline
CAC/GL 45-2003 recommends that
a comprehensive molecular and biochemical characterization of the genetic
modification should be carried out and in formation on the DNA insertions into the
plant genome should include: A) the characterization and description of the inserted
genetic materials; B) the number of insertion sites; C) the organisation of the inserted
genetic material at each insertion site including copy number and sequence data of the
inserted material and of the surrounding regi on, sufficient to identify any substances
expressed as a consequence of the inserted material, or, where more appropriate,
other information such as analysis of transcripts or expression products to identify
any new substances that may be present in the food; and D) identification of any
open reading frames within the inserted DNA or created by the insertions with
contiguous plant genomic DNA including those that could result in fusion proteins.
114
300. Furthermore, the latest F AO/WHO expert consultation on GM animals which updated the
entire field in this area concluded that
An extensive molecular characterization of the inserted genetic material construct will
generally be required, both before and after the insertional event. The molecular
characterization should furthermore comprise an analysis of the copy number and a
sequence analysis of the flanking regions of th e place of insertion in order to identify
any unintended effects [emphasis added]115

115 FAO/WHA, 2003 in Transgene Locus Structure and Expression of Bt Maize, Environ. Risk. Chap
04 13/5/04.

301. In the scientific literature, this need has been confirmed. Thus, Andow et al., recommend that
DNA sequencing is necessary to characterize th e fine structure of the transgene locus
and its flanking genomic region. With sequence information, it is possible to identify
rearrangements that may produce unexpected protein products, to design probes
necessary to establish the presence or ab sence of unexpected RNA or protein
products, to evaluate the risk of combin ations or other rearrangements, and to
evaluate the possibility that plan t gene expression is disturbed. None of the other
methods for characterizing transgene locus structure can enable assessment of all of
these potential risk. [emphasis added]
116
V. THE PANEL'S QUESTIONS ON ISSUE 1 ("DELAY") (NOS 10 TO 58)
Bayer oilseed rape (Falcon GS40/90)
C/DE/96/05 (EC chronology 62)
Question 10

Given the information before the Panel, including the notification by AgrEvo (EC-62/At.1-30) and the
EC SCP's opinion (EC-62/At.74), was the informati on to assess the long-term effect of the newly
expressed protein on the biogeochemical cycle and the food chain requested by the Italian CA
(EC-62/At.95) necessary to ensure that conclusions of the safety assessment were valid?

General comments

302. Two Panel's experts have answered this qu estion, Dr. Nutti and Dr. Andow. Although
Dr. Nutti focused on safety assessment in the food ch ain and Dr. Andow analysed the environmental
aspect, their assessment of the scien tific evidence submitted by the applicant differs substantially.
Dr. Nutti considers it "sound scientific evidence".
117 Dr. Andow points in several instances to the
limited scientific value, or to the absence of, scientific studies. 118 The European Communities finds
that Dr. Nutti's response, by not reviewing the eviden ce actually in front of the SCP and by not citing
and discussing any relevant scientific evidence, prese nts a conclusion which is not only erroneous but
also unmotivated. Dr. Andow, on the contrary, cr itically evaluates the available information and
presents his own conclusions. Nonetheless, the Eu ropean Communities does not fully share all of
Dr. Andow's reasoning and conclusions.
Detailed comments

303. In its only paragraph on this question, Dr. Nutti agrees with the conclusion of the SCP that
"the protein was safe for human consumption in the food chain and was based on sound scientific
evidence, which was presented by the applicant".
304. The 'sound scientific evidence' to which Dr. Nu tti refers consists only on checking protein
homologies with existing allergens and a study of the isolated purified transgene product in a
simulated gastric fluid.119 Based on this, Bt proteins are degrad ed within minutes. However, today we

116 Andow, D.A., Somers, D.A. Amugune,N., Aragao, F., Ghosh,K., Gudu,S., Magiri.E., Moar W.J.,
Osir (2004). Environ. Risk., Chapt 4 in CAB Intern ational 2004. Environmental Risk Assessment of
Genetically Modified Organisms: Vol. 1. A Case Study of Bt Maize in Kenya (eds A. Hilbeck and D. Andow).
117 Answers by Dr. Nutti of 7 January 2005, p. 12.
118 Responses to Questions Submitted by Dr. Andow of 6 January 2005, para. 10.03, 10.04, 10.09.
119 See, SCP Opinion, (EC-62/Att.74).

know that when embedded within transgenic plan t material Bt-proteins can pass even through the
intestinal tract of cows.
120 The request made by the Italian authority was, therefore, well founded. In
reality, some experts consider that the data requ ested by the Italians on food chain should become
routine information provided in approval documentation.
305. In paragraph 10.08 of his response, Dr. Andow concurs with the finding of the SCP at that
time, that the provided information was 'not scientifically convincing that the environmental effects of
Falcon GS 40/90 are in no way different from non-transg enic cultivars.' Plot sizes were apparently so
small that no statistical analyses could be conducte d by the notifier and, thus, could hardly detect
differences anyway. The European Communities consid ers correct to conclude that the scientific
value from such small studies is rather limited. Theref ore, it is appropriate to request more data from
the notifier which the notifier in tu rn did on relevant aspects of the risk assessment, 'dispersal of OSR
pollen', the 'potential invasiveness' and 'a number of aspects related to fitness, weediness and out-
crossing' – although the expert does note that neither the data nor the sources were provided which is
crucial for an independent assessment.
306. In paragraph 10.09 , Dr. Andow notes the contradiction of the SCP which then concluded
that there is no evidence to indicate that Falcon GS 40/90 is likely to cause adverse effects on human
health and the environment while admitting that only 'few studies have been conducted on the safety
of modified OSR to other organisms' – and no st udies on geochemical cycling or the food chain were
done.
307. In paragraph 10.10, it is quite appropriate of the expert to point out at this point that 'lack of
evidence does not imply that there is a lack of an effect.' Statements on 'no evidence to indicate …
adverse effects …' clearly need to be substantiated more than just basing it on lack of studies. This is
not satisfactory from a scientific committee. Such a conclusion is not logically supported by the
provided information.
308. Paragraphs 10.13 and 10.14 relate to the problem of unspecified requests by the CA and
insufficient information delivered in turn by the no tifier. The European Co mmunities agrees that the
request from Italian CA is rather general and thus difficult to address but Dr. Andow clearly states
that food chain and biogeochemical had not been addressed by the notifier.121 Therefore, the European
Communities disagree with its conclusion that "unl ess the Italian CA can point to prior regulator
precedents where these terms are clarified, the phrasing of the request is not scientifically justified".
309. With regard to the comment by Dr. Andow, contained in paragraph 10.14, on the length of
time it took the Italian authorities to review the in formation received and to pose new questions, the
European Communities refer the Panel to its General comments above.
Question 12

Given the information before the Panel, including the notification by AgrEvo and the conclusions of
the EC SCP, was the information regarding the mol ecular characterization of this product requested

120 Chowdhury EH, Shimada N, Murata H, Mikami O, Sultana P, Miyazaki S, Yoshioka M, Yamanaka
N, Hirai N & Nakajima Y (2003): Detection of Cry1Ab protein in gastrointestinal contents but not visceral
organs of genetically modified Bt11-fed calves, VETERINARY AND HUMAN TOXICOLOGY 45, pp. 71-75;
Einspanier R, Lutz B, Rief S, Berezina O, Zverlov V, Schwarz W & Mayer J (2004): Tracing residual
recombinant feed molecules during digestion and rumen bacterial diversity in cattle fed transgenic maize,
EUROPEAN FOOD RESEARCH AND TECHNOLOGY 218, pp. 269-273.
121 See paragraph 10.09.

by the lead CA (EC-62/At.106) necessary to ensure that conclusions of the safety assessment were
valid?

General comments

310. Two Panel's experts have answered this qu estion, Dr. Healey and Dr. Snape, reaching
opposite conclusions. The two experts seem to a ddress the question in a different way. While
Dr. Healy addresses this question more from a histori cal perspective, Dr. Snape takes into account the
current state-of-the-art. In fact, the dossier w as submitted in 1996 and at that moment, molecular
characterisation was focused on the inserted DNA fragment. However, the European Communities
cannot concur with Dr. Healy because already then, (1) a number of scientists evaluating the dossiers
insisted for more information on the molecular char acterisation and were convinced of the value of
this information for safety assessment;
122 (2) the RoundupReady soybean case was known and could
have been taken into account;123 and (3) it had already been proven that, technically, it was possible to
carry out the experiments needed to address the question of the leading CA in 2002.124
311. For what concerns safety assessment, the Europ ean Communities wants to stress that a safety
evaluation of a new event cannot be called 'com plete' as long as some aspects of molecular
characterization, such as the screening for inserted fragments and the sequencing of the plant genome
DNA flanking these insert(s), are missing. In this case, no definitive statement can be done on the
absence of rearrangements and unintended effects, a nd thus neither on the safety of the product. The
European Communities, therefore, notes that some expe rts, such as Dr. Snape, are of the opinion that
"more information is still required", which obviously necessitates further clarification and information
before the issue can be definitively resolved. Some specific comments on the responses are provided
below.
Detailed comments

312. In the first paragraph of page 5, Dr. Healy states that:
The molecular characterisation submitted included a sequence analysis of the pat
gene isolated from the soil bacterium, full DNA sequence analysis of the DNA
construct containing the pat gene and Southern blot and Polymerase Chain Reaction
(PCR) analysis of the transgene. The latter demonstrated that the pat gene was stably
inserted into two separate locations within the recipient strain and that vector
sequences outside the border regions had not integrated.
This factual assessment is wrong. In reality, the information (EC-62 At.3, 13, 14) provided before
2 April 2002 (date of request for additional information, see EC-62/At.106) failed to identify the exact

122 Windels P, Theuns I, Dendauw J, Depicker A, Van Bockstaele E, De Loose M (1999), Development
of a line specific GMO detection method: a case study, Med. Fac. Landbouwk. Wet., 64/5b, pp.459-462.
Windels P, Depicker A, Van Bockstaele E, De Loose M (2000), Characterisation of the 3'NOS junction of
Roundup Ready soy, Med. Fac. Landbouww. Univ. Gent, 65/3b, pp.463-465;
123 Windels P, Taverniers I, Depicker A, Van Bock staele E, De Loose M (2001), Characterisation of
the Roundup Ready soybean insert, Eur. Food Res. Technol., 213, pp.107-112.
124 Depicker A, De Buck S, Van Montagu M, Theuns I, Huyghebaert A, Van Bockstaele E, De Loose
M (1998), Detection and characterizatio n of transgene inserts into plants, Med. Fac. Landbouww. Univ. Gent,
63/4b, pp.1479-1484; Theuns I, De Buck S, Depicker A, Van Gaver D, Huyghebaert A, Van Bockstaele E, De
Loose M (1998), Anchored PCR method as alternative to detect and identify GMOs, Med. Fac. Landbouww.
Univ. Gent, 63/4b, pp.1539-1542; Theuns I, Windels P, De Buck S, Depicker A, Van Bockstaele E, De Loose M
(2002), Identification and characterization of T-DNA inserts by T-DNA fingerprinting, Euphytica, 123,
pp.75-84.

number of copies of transgenic sequences into GS 40/90. Initially, two intact copies of the pat gene
construct were believed to be inserte d. Later, as correctly noted by Dr. Snape,
125 it was determined,
following the requested information, that the total copy number of the pat gene was shown to be
three.126 According to the European Communities, th is underlies the need for complete molecular
characterization from the beginning and justifies the CA request for more information on this topic.

313. In the last paragraph of page 6, Dr. Healy states that:
Impact of the Additional Information on the Conclusions of the Safety Assessment
The additional information requested by th e Lead CA is unlikely to influence the
conclusions of the safety assessment carried out by the SCP.
The European Communities submits that this would only be the case if no deletions, insertions or any
other rearrangements in the flanking plant ge nome DNA had occurred. However, as no sequence
information was submitted originally on the genom ic DNA adjacent to the insert, but only on the
inserted genes, at that moment, no evidence could be given of the absence or rearrangements. As long
as no evidence is given on the absence of rearrangements, also outside the inserted construct, no
conclusions on the safety of the event can be drawn.

314. In the following bullet-points on pages 6 and 7 , Dr. Healey gives the rationale for her
conclusions. In the first bullet-point, she affirms that, as "the transgene was inserted into the Falcon
GS40/90 line via agrobacterium mediated transformati on techniques, this technique is less likely to
result in DNA rearrangements and other modifications of the inserted DNA". The European
Communities disagrees. There is substantial evid ence in literature that Agrobacterium-mediated
transformation induces severe changes in the plant chromosomes and therefore may be the cause of
many unintended effects. In fact, truncations , rearrangements, etc., cannot be avoided with
Agrobacterium-mediated transformation.
127

125 Report by Dr. Snape of 5 January 2005, p. 1, point (1).
126 The information (EC-62 At.3, 13, 14) provided before 2 April 2002 (date of request for additional
information, see EC-62/At.106) shows that oilseed rape event Falcon GS40/90 was transformed using plasmid
pHoe6/Ac by means of Agrobacterium- mediated transfer. The T-DNA of the plasmid pHoe6/Ac carries a pat
gene construct conferring resistance to the herbicide glufosinate ammonium. Southern Blot analysis indicated
that two intact copies of the pat gene were inserted at two individual functional loci (At.14). To prove that no
region outside the two border sequences of the inserts was present, Southern Blot analysis was conducted.
However, the Southern Blot analysis undertaken seems to have been conducted on an oilseed rape line
containing only one copy of the pat cassette (At. 113). The additional information (EC-62 At. 114) requested by
the lead CA on 2 April 2002 gives more information on the transgene loci, denoted EE1 and EE2. It is said that
(data proving this are lacking) one of the two functional lo ci contains two pat expression units in inverted tail-
to-tail orientation, bringing the total copy number of the pat gene to three instead of two. The sequence of locus
EE1 and its 5-prime and 3-prime flanking sequences, together with the DNA sequence of its pre-insertion site
were determined. For locus EE2, no sequence data were gi ven on the insert itself, but the 5-prime and 3-prime
flanking sequences, together with the DNA sequence of its pre-insertion site were determined. Sequence
analysis of the EE1 locus showed th at this sequence contains one T-DNA copy and lacks the 65 sub-terminal
bases at the RB and ends at the break point of the le ft border. A BLAST search on the 5-prime and 3-prime
flanking sequences of both transgene loci revealed no meaningful similarities. Alignment of the 5-prime and 3-
prime flanking sequences of the transg ene loci with the DNA isolated from wild type oilseed rape revealed
minor and major deletions and the pres ence of filler DNA. For EE1, a fra gment of 1715 bp was deleted upon
integration of the T-DNA. In addition, at the 3-prime junction of EE1, eleven nucleotides 'filler' DNA were
inserted at the transgene locus. Sequence alignment with EE2 revealed a loss of 16 nucleotides at the target site.
127 Windels P, De Buck S, Van Bockstaele E, De Loose M, Depicker A (2003), T-DNA integration in
Arabidopsis chromosomes. Presence and origin of filler DNA sequences, Plant Physiology 133, pp.2021-2068.

315. In the second bullet-point, Dr. Healey asserts that
The molecular characterisation originally submitted did include a PCR analysis,
which indicated that only the DNA sequences associated with the pat gene had
integrated into the recipient strain. In pa rticular, the analysis verifies that that no
sequences from the vector, others than intended, integrated into the recipient strain.
Now, PCR analysis of inserted genes only canno t provide any information on rearrangements,
truncations, presence of vector backbone DNA, etc. which occurred outside of the amplified genes.
This highlights the importance of complete molecu lar characterization, including sequencing of the
complete T-DNA insert and adjacent plant genome seque nces, and thus justifies the additional request
of the lead CA. Also, as seen above, the inform ation on molecular characterization submitted after
additional request by the CA was different than the molecular data origin ally submitted. Therefore, as
some aspects were not known originally , some aspects of the risk assessment were missing at the
moment of the risk assessment.

316. As with regard to Dr. Healy's conclusion that:
The additional molecular characterisation re quested by the Lead CA is unlikely to
affect the conclusions of the safety assessme nt and was not necessary to ensure that
the conclusions of the safety assessment were valid.
128
The European Communities would like to note that, if no sequence data of the T-DNA/plant junction
regions are available, no evidence can be given of the absence of any rearrangement at the locus of
integration in the plant, and thus neither of the absence of any unintended effect and thus ecological
impact. As long as this evidence is lacking, th e risk/safety assessment of the event cannot be
considered as being complete.

Question 13

Given the information before the Panel, including the notification by AgrEvo and the conclusions of
the EC SCP in relation to the potential persist ence or invasiveness of Ba yer oilseed rape (Falcon
GS40/90), would this product qualify as a potential "pest" as the term is used in ISPM 11?

General comments

317. Four Panel's experts provided a response to th is question: Dr. Andow, Dr. Nutti, Dr. Snow
and Dr. Squire. Dr. Nutti states, without much of an explanation on why, that Bayer oilseed rape
(Falcon GS40/90) is not dangerous and thus cannot qualify as a "pest". Both Dr. Snow and Dr. Squire,
on the contrary, recognise that Bayer oilseed rape ha s the potential to be a "pest", but Dr. Snow adds
that other methods of weed control could manage this problem. Dr. Andow considers that the
information in front of the Panel, both as submitted by the notifier and as contained in the SCP
opinion, are insufficient to determine the "pest" status of this product.

See also, for instance, Genome Scrambling - Myth or Reality?; Transformation-Induced Mutations in
Transgenic Crop Plants by Allison Wilson and Jonathan Latham and Ricarda Steinbrecher available at
<http://www.econexus.info/pdf/ENx-Genome-Scrambling-Summary.pdf> (last visited on 25 january 2005):

"This report identifies the insertio n-site and genome-wide mutations created by plant transformation
procedures as potentially major, but poorly understood, sources of hazard associated with the production and use
of commercial transgenic cultivars".
128 Responses by Dr. Healy of 6 January 2005, p. 7.

318. For its general view on the persistence of HT products and their potential as "pests", the
European Communities refers the Pa nel to its comments under Question 6 above. With regard to the
specifics of this question, the European Communities considers oilseed rape (OSR) a crop with weedy
potential, especially in other broad leaved crops in arable rotations. Introduction of herbicide tolerance
into OSR further reduces options for managing it and thus it could be argued that, under certain
circumstances (e.g. existing use of the specific herbicid e), its pest or weed status is increased by the
introduction of HT traits and requires additional measures to manage it. Likewise some related species
are also weeds and their weediness could be enhanced by the introduction of HT traits via gene flow.
The European Communities thus concurs with Dr. Snow's acknowledgment of the fact that specific
measures should be taken to control a "potential p est" and that "management options become more
challenging and more complicated when the pest po pulation has genes for several types of herbicide
resistance".
129 Furthermore, as this "potential pest" could spread out of the GM farms, control should
also be applicable by non-GM farmers in order to be efficient. However, some of these management
options may be difficult to implement (especially in non-GM farms), have higher costs and/or less
desirable environmental impacts.
Detailed comments

319. As said in the comments under Question 6 above, all crops are weeds to a greater or lesser
extent when they grow in other crops. Oilseed rape has many characteristics of a weed: high seed set,
high seed dormancy, variable germination, compet itive under fertile growing conditions, etc. In
addition GMHT rape has the ability to grow in sub sequent non-GM rape crops even when grown after
several years interval, resulting in potential impurity problems 130. In addition if the HT gene transfers
to existing related weeds, they in turn can b ecome problematic weeds of the HT crop since the
specific herbicide will not control them.131
320. Glufosinate is currently used as a herbicide a nd a dessicant in certain crops (e.g. potato).
Introduction of glufosinate tolerant oilseed rape will mean that glufosinate cannot be used to control
HT rape volunteers in these crops. Glufosinate is also widely used as a dessicant in conventional
oilseed rape crops. This practice would have to change in order to manage conventional crops
containing admixtures of glufosinate tolerant crops . In addition another dessicant would be required
on glufosinate tolerant oilseed rape crops.
321. Glufosinate tolerance has been developed in maize and sugar beet. Glufosinate tolerant
oilseed rape would be a problematic weed in these crops requiring additional weed control
measures.132

129 See also Responses to Scientific Questions from the Panel of Dr. Snow of 5 January 2005, page 14,
question 6d.
130 SWEET, J. B., SIMPSON E. LAW., J, LUTMAN, P.J., BERRY, K., PAYNE, R., CHAMPION, G.,
MAY, M. WALKER, K., WIGHTMAN, P., & LAINSBURY, M. (2004). Botanical and Rotational Implications
of Genetically Modified Herbicide Tolerance in winter oilseed rape and sugar beet (BRIGHT Project). HGCA
Project Report No 353, 265 pp. Available at <www.hgca.com>.
131 Norris, Carol, Jeremy Sweet, John Parker & John Law (2004) Implications for hybridization and
introgression between oilseed rape (Brassica napus) and w ild turnip (B. rapa) from an agricultural perspective.
In: Hans C.M. den Nijs, Detlef Bartsch & Jeremy Sweet (E ditors 2004) Introgression from genetically modified
plants into wild relatives and its consequences. Pro ceedings of an ESF AIGM conference, Amsterdam, 21-24
January, 2003, pp. 107-124.
132 See Sweet et al (2004), cited above. MESSEAN, A. (2002). Monitoring case report: impact of
transgenic plants within cropping systems. In LMO' s and the Environment: proceedings of an international
conference. November 27-30, 2001. Raleigh, USA. Edited by Craig Roseland. pp 207-215.

322. All of these above issues were not raised by the SCP in its Opinion (EC62/Att.74) who
focused very much on whether the HT rape would be a problem in crops other than oilseed rape or
other HT crops. However the SCP did comment that gl ufosinate tolerant rape should not be grown in
proximity to rape with other HT traits, since combination of HT genes in a single rape plant might
make it harder to manage. This is based on the Ca nadian experiences of gene stacking referred to in
Dr. Snow's submission.
323. SCP also commented that: Glufosinate drift in to field margins may enhance establishment of
feral HT rape populations in these areas. This conclusion may have been based on the report by Sweet
et al
133.
324. Although most of these above issues are manage able by changing agronomic practices and
using different herbicide programmes, some of these changes (e.g. extending crop rotations between
HT and non-HT crops) may not be economically justif ied, environmentally desirable or even difficult
to implement (changes in non-GM farms).
325. Furthermore, the SCP qualified its response by saying that oilseed rape is a relatively new
crop in Europe, information on weediness, gene flow and invasiveness (especially of related species)
is limited and that caution and close monitoring is required.
Bayer hybrid oilseed rape (MS8/RF3)

C/BE/96/01 (EC chronology 63)

Question 15

Given the information before the Panel, including the conclusions of the EC SCP, was the information
regarding the assessment of the long-term effect of the newly expressed protein on the biogeochemical
cycle and the food chain requested by the Italia n CA (EC-63/At.87) necessary to ensure that
conclusions of the safety assessment were valid?

General comments

326. Two Panel's experts answered this question: Dr. Andow and Dr. Nutti. As under question 10,
where the issues at stake were similar, their conclusions diverge substantially, on the basis of the same
information before them. While the lack of evid ence – no additional evidence was put forward in
addition to the limited amount contained in th e notifier's documentation – was interpreted as
indication for safety by Dr. Nutti, Dr. Andow stated correctly that it is not sound science to interpret
lack of evidence as evidence for lack of an effect and even less so as evidence for safety. While this
may seem subtle, it is yet of great bearing for the safety discussion and often the reason for dissent.
Detailed comments

327. Dr. Nutti focuses on the bar-gene and PAT gene expression and transgene product. She
concludes that since the bar-gene is controlled by plant promoter, it would not be functional in a
bacteria. "Consequently, its expression in the un likely event of transformation would not occur"
134.
While Dr. Nutti may be correct to state that in the unlikely event of horizontal gene transfer of a bar-
gene, PAT product expression might no t have negative effects as long as the substrate, the herbicide

133 Sweet J.B., Shepperson R., Thomas J.E., Simpson E. (1997) The impact of releases of genetically
modified herbicide tolerant oilseed rape in the UK. Proceedings of the Brighton Crop Protection Conference -
Weeds. pp 291-302.
134 Answers by Dr. Nutti of 7 January 2005, p. 14, first paragraph of Answer 15.

glufosinate, is missing. However, the argument th at the bar gene would not be expressed because the
plant promotor controlling this transgene in the plan t would not work in bacteria is insufficient. The
bacterial promoters can be expected to be able to do the job even better than the plant promoters.
Expression of a transferred transgene of former bacterial origin cannot be excluded in bacteria.
328. Further, the expert speaks of "weight of ev idence available elsewhere concerning the safety
…"
135, however the evidence is missing and no refere nces are provided. Arguments like these can
often be heard in the discussions about safety of GM food and products. However, when researching
for the 'weight of evidence' one finds more often than not that this evidence is actually meagre,
unpublished, informal, contained in grey literature or notifier documentation. If such points become
crucial for the case, the weight of evidence should actually be requested to be put forward in
verifiable form.
Question 16

Given the information before the Panel, including th e conclusions of the SC P, was the information
regarding molecular characterization of this pr oduct requested by the lead CA (EC-63/At.107)
necessary to ensure that conclusions of the safety assessment were valid?

General comments

329. Two Panel's experts have answered this qu estion, Dr. Healey and Dr. Snape, reaching
different conclusions. While for Dr. Healy the l ead CA had enough information to conclude on the
safety of Bayer hybrid oilseed rape (MS8/RF3) already in 2002, accordin g to Dr. Snape "more
information is still required".
330. The European Communities general comments on the experts' replies on question 16 are the
same as those made on question 12, with the differe nce that here the missing molecular data – that
were requested by the lead CA in 2002 – con cern the comparison between the GM event and the
isogenic line. Thus, complete characterization of GM events at the molecular level, including a
comparison at DNA level between the insertion locus in the GM event and the corresponding locus in
the isogenic line, gives detailed and highly valuab le information on (1) the exact composition of
transgene inserts, (2) the exact number of inserts a nd number of transgene copies per insert (including
inverted repeats of a transgene copy at one site of integration), (3) the presence of vector backbone
DNA sequences within the insert or outside the insert, (4) the occurrence of deletions, insertions or
any other rearrangement within the transgene insert( s) and/or the adjacent plant genome DNA, (5) the
stability of transgene inserts. Un expected and unintended effects of transgenes cannot be excluded
without detailed molecular data on all above me ntioned topics. Some specific comments on the
responses are provided below.
Detailed comments

331. With regard to molecular characterisation data, in the first and second paragraph of page 9 ,
Dr. Healy concludes that no homology with known seque nces was found using the 3' and the 5'
flanking sequences. The fact that no sequence homologies with known sequences were found for the
3' flanking sequence of MS8 and the 5' flanking seque nce of RF3, and the fact that the sequence data
of T-DNA/plant junction regions have not been comp ared to the isogenic line, cannot exclude that
unexpected and unwanted truncations or rearrangeme nts occurred at these sites of integration.
Providing this type of information gives the CA a more comfortable position to come to a positive
evaluation of the safety. Absence of this information does not necessarily lead to a negative report.

135 Ibidem, second paragraph of Answer 15.

332. With regard to the impact of additional information on the conclusions of the safety
assessment, Dr. Healy concludes that
The additional molecular characterisation da ta sought by the lead CA in 2002 were
not required to demonstrate the safety of food products derived from MS8, RF3 or
MS8xRF3 hybrid lines.
The European Communities submits that, if th e sequenced plant DNA regions, adjacent to the
transgene inserts, are provided and compared to th e corresponding sequences at the site of integration
in the isogenic lines, this additional information a llows to conclude on the fact that besides the
insertion of the intended fragment, no other changes in the DNA sequence have taken place during the
transformation process. If this is the case, this is a positive element to conclude on the safety of the
GMO.

333. In the following bullet-points on pages 9 and 10 , Dr. Healey gives the rationale for her
conclusions. In the last bullet-point, she affirms that,
The most significant elements of the assessment of MS8 and RF3 oilseed rape lines
more appropriately focus on the molecular characterisation of the insertion events and
the resultant novel gene products, particularly in terms of their potential toxicity and
allergenicity. The food products derived from transgenic oilseed rape are likely to
contain at most only trace amounts of plant proteins due to processing. Comparative
compositional data from the seeds of bot h transgenic lines and the corresponding
isogenic lines therefore provides necessary information to determine unintended
effects.
According to the European Commun ities, risk assessment has to include more than only taking into
account the impact caused by the main product from a crop. In other words, also non-intended effects
in side products and on the environment as a whole have to be considered. As with regard to the last
sentence, comparative sequence data between th e transgenic lines and the corresponding isogenic
lines is exactly what the lead CA requested. Thus, th is paragraph justified th is additional request by
the lead CA. The European Commun ities respectfully submits that this conclusion is in contradiction
with the reasoning of Dr. Healy.

334. As with regard to the analysis of Dr. Snape of Exhibit EC063/Att.98 , the European
Communities notes that the molecular data were incomp lete, as sequences at integration loci were not
compared to sequences at corresponding sites in wild-type isogenic lines.
335. Finally, in the conclusion by Dr. Snape , the European Communities notes that the repeated
reference to "MsR8" is not clear and would appreciate clarifications from this expert.
Question 17

Were detection methods commercia lly available in 2001 sufficient to enable the detection of the
transgenic proteins expressed by the plant line hybrid oilseed rape MS8/RF3? Given the information
before the Panel, including the SNIF (EC-63/At .109) and the updated environmental risk assessment
(EC-63/At.110-140), was additional informatio n regarding a quantitative detection method
(EC-63/At.141) necessary to ensure that conclusions of the safety assessment were valid?

General comments

336. Question 17 addresses two issues: whether the de tection methods commercially available at
the time of the request of the lead CA for add itional information regarding a quantitative detection

method sufficient to enable the detection of the tran sgenic proteins expressed by the plant line hybrid
oilseed rape MS8/RF3; and whether this request for information was necessary to ensure that
conclusions of the safety assessment were valid. Two Panel's experts answered this question:
Dr. Healey and Dr. Nutti. With regard to the first issue, they both conclude that such a method was
available, but Dr. Nutti specifies that it had not been validated. On the second issue, they agree that a
quantative detection method as required by the lead CA was not necessary for safety conclusions,
however Dr. Nutti points out that such a method would have provided relevant information for
labelling and the consumer.
337. The European Communities respectfully consider s that the question posed by the Panel is
vitiated by a misunderstanding. As it is clear from EC-63/At.141, the lead CA points to the need of a
quantitative detection method "to facilitate post-mark eting control and inspection" and does not refer
to the need for such in a safet y assessment as suggested by the questi on. As remarked by Dr. Nutti,
the necessity of having a detection method is li nked to monitoring and traceability of GMOs after
release. For post-marketing control and inspection, as explained by many of the experts, (1) an event-
specific method is required and (2) this can onl y be the case when the method is based upon the
presence of foreign DNA and not upon the presence of fore ign protein. Therefore, the conclusions of
the experts with regard to the necessity of such a method for safety assessment are of no help in
understanding the scientific value of the request of the lead CA with regard to post-marketing
monitoring and control.
338. The European Communities provides below Detailed comments with regard to the experts'
opinions on the preference for protein- versus DNA-based methods. However, in the context of the
lead CA's request, this is not of high relevance.
Detailed comments

339. With regard to the conclusion of Dr. Healy on the first part of question 17 , the European
Communities underlines that it is not possible to conc lude on the availability of a commercial kit for
PAT detection in 2001. As Dr. Nutti rightly points out in its first paragraph of her answer to
question 17, the method was not validated and, presumably, not commercially available.
340. The European Communities respectfully disagrees with Dr. Healy statement in the
penultimate paragraph of page 12 that
Quantitative PCR methods have been deve loped for a range of food commodities
including soybean and maize products but validation of these methods is not
straightforward.
341. In fact, the recently accepted modular validation approach for GMO methodologies perfectly
allows to validate quantitative PCR methods, acc ording to internationally accepted, harmonized
guidelines
136. Collaborative trial validation of quantita tive PCR methods presently included in the
draft international standards has been done in a number of slightly different ways (prEN ISO 21571
Foodstuffs - Methods of analysis for the detecti on of genetically modified organisms and derived
products - Quantitative nucleic acid based methods). However, essentially the principles are the
same: Samples of unknown content (test samples) are compared to samples with assigned
concentrations (reference materials). In some cases the DNA has to be extracted from the samples (in
which case validation includes validation also of a DNA extraction component), while in other cases

136 See ISO 5725, 1994; Horwitz W (1995) Protocol for the design, conduct and interpretation of
method performance studies. Pure Appl Chem 67, pp.331-343.

the samples distributed consist of pre-extracted DNA (in which case validation includes only the PCR
components). A recent paper
137 provides justification for separate validation of DNA extraction and
PCR components, - primarily linked with cost efficiency and improved flexibility in the application of
methods. The number of methods included in pr EN ISO 21571 is currently 13 (all collaboratively
validated), while the number of methods include d in EN ISO 21572 (Foodstuffs – Methods of
analysis for the detection of genetically modified organisms and derived pr oducts - Protein based
methods) for comparison is one!
342. Not only does this fact demonstrate the feasibility of validating quantitative PCR methods, it
also demonstrates that quantitative protein-based methods of detection are less directly accessible, in
contrast to the statements of Dr. Healy. that
… it is generally recognised that quantitativ e protein-based methods of detection are
more directly accessible.
343. Notably, the protein based methods also have a much more narrow application range than
PCR based methods. To the European Communitie s's knowledge world-wide there is no single
quantitative protein-based method that has been va lidated and considered fit for the purpose.
Dr. Nutti seems to agree with this viewpoint when she affirms that: "I can infer that there was no
general validation of the method used by the notif ier". The European Communities also wishes to
underline that accessibility can be interpreted in di fferent ways. First, it can be discussed whether
ELISA versus real-time PCR is favourable in di fferent conditions. This largely depends on the
tradition and historical perspective concerning th ese technologies. Second, it can be discussed on the
accessibility of the target to be detected: (1) DNA can be readily isolated from most samples; (2)
DNA, in contrast to proteins, is much more stable and generally present in different types of matrices
and thus enable a more horizontal applicability; (3) PCR is recognized and accepted as the reference
method for GMO identification and quantification for regulatory purposes in the EU, because of those
advantages; (4) DNA-based methods for GMO quantif ication, i.e. DNA extraction protocols and
quantitative PCR assays can be easily found in the lite rature and in annexes of the ISO norms for
DNA-based GMO detection.138
Question 18

Given the information before the Panel, including the SNIF and th e updated environmental risk
assessment (referenced above), was the information regarding molecular data requested by the CA
(EC-63/At.144) necessary to ensure that conclusions of the safety assessment were valid?

General comments

344. This question was answered by two Panel's expe rts: Dr. Healy and Dr. Snape. Dr. Snape has
answered this question together with question 16. Hi s conclusion is, therefore, the same, which is to
say that "more information is still required". On the contrary, Dr. Healy concludes her assessment
considering that the additional information sought by the CA was not necessary to ensure that that
conclusions of the safety assessment were valid. Dr. Healey adds that "given the pace of advancing
knowledge in the field of molecular biology an d plant biotechnology and the rapidly evolving
technical capabilities, for a foreseeab le time, there will often be a di screpancy between the scientific
data submitted in a dossier for assessment, and the information possible from the most recently
developed experimental techniques".

137 Holst-Jensen A, Berdal, KG (2004) The modular analytical procedure and validation approach and
the units of measurement for genetically modified materials in foods and feeds. J AOAC Int 87, pp.927-936.
138 ISO/FDIS 21571, 2004; ISO/DIS 21569, 2004; ISO/DIS 21570, 2003.

345. The European Communities respectfully disagr ees with the conclusion of Dr. Healy and
points to the fact that it is the duty of a regulatory authority to continually update its requirements
taking new methodologies into account. In particul ar, the Belgian guidelines on the requirements on
molecular information were devel oped with the help of a panel of scientists specialised in gene
technology and experts from the European Associat ion of Bioindustries ("EuropaBio", the association
of biotech industries in Europe). The need for the demands for mol ecular data in these guidelines has
been thoroughly discussed in the light of safety assessment and is purely science based. Bio-
informatic analysis enables one to check for the presence of novel chimeric open reading frames at the
transition between plant and insert DNA or in the region of rearranged DNA fragments. This way, the
risk of production of allergens, toxins or other pharmacologically active proteins in the transgenic
crop can be assessed. This approach can therefore be a valuable tool for biosafety evaluation as long
as other (genomic, proteomic or metabolomic) techniques aimed at the assessment of potential
unintended effects of the transformation are not operati onal. In addition, bio-informatic analysis can
also reveal if a transgene has inserted inside a plant ORF. It must be noted that bio-informatic analysis
is not only required according to the Belgian Guid elines for molecular characterisation of GM higher
plants to be placed on the market, but have also been accepted as a tool in safety assessment by the
EuropaBio
139 and the European Food safety Authority. 140 In addition, the guidelines for conducting
safety assessment of GM foods that were a dopted by the Codex Alimentarius Commission 141 require
information sufficient to identify any new substanc es that may be included in food. DNA sequence
information on the junction regions of the transgene is one mechanism to obtain this information.
346. In the specific of this case, the new data provided by the notifier in September 2003 in
response to the request from the lead CA was nec essary to ensure that conclusions of the safety
assessment were valid. The original sequence data describing the integrated inserts were incorrect,
and several errors were detected when the transf orming plasmids and the integrated inserts were
sequenced for verification (cf. Exhibit EC63/At.1 31 and EC63/At.132). Fortunately, these errors
were concluded insignificant with respect to the conclu sions of the risk evaluation. On the contrary, a
significant unintended rearrangement of an inserted truncated gene cassette in Rf3 was observed and
characterised (EC63/At.115). Without a detailed sequence based characterisation of this
rearrangement also with respect to its flanking sequences, it would not be possible to predict the
potential risk associated with the rearrangement. The nature of the integration sites in the recipient
genome was determined through sequence character isation of the 5-prime and 3-prime flanking
sequences of the inserts in Ms8 (EC63/At.114) a nd Rf3 (EC63/At.115), and these were confirmed to
be of host plant origin with a high degree of probability (EC63/At.116-119, EC63/At.133 and
EC63/At.134). However, no f unctional assignments of these sequences were provided in the reply
from the notifier. To this date, two of the fl anking sequences are believed to represent genes coding
for proteins for which it is claimed that " overexpression causes pleiotropic phenotypic changes ".
Pleiotropic phenotypic changes means unpredicte d or unexpected changes in the appearance of
presumably unrelated or unlinked characters induced by the character under study. Such a statement
would therefore undoubtedly attract the attention of risk assessors, if it had been provided by the
notifier. It is, however, likely that this partic ular information was unavailable to the notifier by
September 2003. Information that was available wi thout being forwarded until specifically requested
concerned the sequence of the flanking sequen ces (cf. EC63/At.114 and EC63/At.115). The

139 EuropaBio, 2003. Molecular Characterisation in Guidance document for Safety Assessment of GM
Crops.
140 EFSA, 2004. Guidance document of the scientific panel on genetically modified organisms for the
risk assessment of genetically modified plants and derived food and feed. The EFSA Journal 99, 1-94.
141 Codex Alimentarius Commission (2003), Principles for the risk analysis of foods derived from
modern biotechnology. Codex Guideline for the Conduct of Food Safety Assessment of Foods derived from
Recombinant-DNA plants, CAC/GL 45-2003.

publication of genetic rearrangements and the flanking sequences of GTS 40-3-2 (RoundupReady®)
soybeans
142 led to rapid establishment of consensus co ncerning the relevance of detailed (sequence
based) genetic maps of the inserted and flanki ng sequences. The detection methods provided
(EC63/At.136 and EC63/At.137) are not event specific and not quantitative. Furthermore, the
specificity of the methods was only assessed against Brassica napus samples (EC63/At.138). It is
common practice in peer review journals to require considerably wider taxon samples in this kind of
specificity test.
347. More Detailed comments on the information relating to molecu lar data in this notification to
complete the assessment of Dr. Healy are provided below. For comments to Dr. Snape's response,
the European Communities refers to its comments to answer 16.
Detailed comments

348. The EC chronology includes several exhibits which are relevant for this issue.
349. In particular, EC-63/At.114, which forms an integrated part of the updated risk assessment of
Sept 2003, was completed on November 26 th 2001, and shows that the flanking sequences of Ms8
were available and known to Aventis CropScience almo st two years before making it available to the
CA. See page 4 of the exhibit:
To demonstrate the nature of the flanking sequences of Brassica napus elite event
Ms8, PCR analysis was performed on a numbe r of templates using different primer-
pairs. In a first PCR reaction the identity of the Ms8 template was confirmed using
the primer-pairs described in the Ms8 Di scriminating PCR protocol. A primer-pair
targeting the flanking sequence was used to demonstrate the nature of the flanking
sequence. The PCR analysis demonstrat ed unequivocally that the characterized
flanking sequences are present in all tested Brassica napus entries and are thus of
Brassica napus plant origin.
350. Exhibit EC63/At.115 on confidential information on the characterisation of Rf3, which forms
an integrated part of the updated risk assessm ent of September 2003, was completed on November
26th 2001, and shows that the flanking sequences of Rf3 were available and known to Aventis
CropScience almost two years before making it available to the CA.See from page 4 of the exhibit:
To demonstrate the nature of the flanking sequences of Brassica napus elite event
Rf3, PCR analysis was performed on a numbe r of templates using different primer-
pairs. In a first PCR reaction the identity of the Rf3 template was confirmed using
the primer-pairs described in the Rf3 Di scriminating PCR protocol. A primer-pair
targeting the flanking sequence was used to demonstrate the nature of the flanking
sequence. The PCR analysis demonstrat ed unequivocally that the characterized
flanking sequences are present in all tested Brassica napus entries and are thus of
Brassica napus plant origin.
351. Genetic rearrangements as a result of the integration event are common.143 The nature of such
rearrangements is not predictable, and each case may therefore deserve examination as part of the risk

142 Windels P., Tavernier I., Depicker A., Van Bock staele E. & De Loose M. (2001). Characterisation
of the Roundup Ready soybean insert. Eur. Food Res. Technol. 213:107-112.
143 Qpcrgmofood (2004). Final report of workpackage 3 "Sequence characterisation of transformation
events (junction between insert and insertion site in the genome)" QLK1-1999-01301. Available at
<http://www.vetinst.no/Qpcrgmofood/Deliverable3.pdf> (last visited on 27 January 2005).

assessment procedure. In Rf3 rearrangement was observed at the 3-prime end, as described in
paragraph 4.2 on page 8 of the document:
The integration of the T-DNA resulted in a large plant DNA duplication: the flanking
sequences at the 3-prime end (1275 bp in tota l) consist of 816 bp duplication of 5-
prime flanking sequences ('duplicated 5-prime flanking sequence') and 459 bp ('3-
prime flanking sequence'). The identity of the 'duplicated 5-prime flanking sequence'
is described in 4.1.).
352. Also Exhibit EC63/At.116
, containing confidential alignment with transformation plasmid of
Ms8 and which forms an integrated part of the updated risk assessment of September 2003, was
completed on October 10th 2001. It shows that the flanking sequences were not derived from the Ms8
transforming plasmid.
353. Exhibit EC63/At.117 on confidential BLAST with flanking sequences of Ms8, which forms
an integrated part of the updated risk assessm ent of September 2003, was completed on September
26th 2001, and shows that no similarity to known sequences was observed, with exception for some
similarity to an 87 bp sequence derived from the closely related species Arabidopsis thaliana .
Apparently, no attempts were made to determin e if any function was assigned to the sequence,
although this would be permitted in connection with the BLAST sear ch. The similar sequence motif
in Arabidopsis thaliana is not assigned any function in the NCBI/GenBank database as per this date.
354. Exhibit EC63/At.118 on confidential alignment with transformation plasmid of Rf3, which
forms an integrated part of the updated risk assessment of September 2003, was completed on
October 10th 2001, and shows that the flanking sequences we re not derived from the Rf3 transforming
plasmid.
355. Exhibit EC63/At.119 on confidential BLAST with flanking sequences of Rf3, which forms an
integrated part of the updated risk assessment of September 2003, was completed on October 8th 2001,
and shows that both the 5-prime and 3-prime flanki ng sequences show similarity to known sequences
from the closely related species Arabidopsis thaliana . The 5-prime flanking sequence shows
similarity to two 97 bp and 33 bp sequences derived from Arabidopsis thaliana . Apparently, no
attempts were made to determine if any functions were assigned to the sequences, although this would
be permitted in connection with the BLAST search . Both the 97 bp and 33 bp sequence motifs in
Arabidopsis thaliana are assigned function in the NCBI/GenBank database as per January 18 th, 2005,
cf. Box 1 and Box 2, and both are coding for proteins. Notably, it is stated with the accession in Box
1 in a description of the function of the protein that " overexpression causes pleiotropic phenotypic
changes". The European Communities has not been ab le to determine whether these accessions were
released by September 2003, although the accession described in Box 1 was submitted to GenBank on
March 11th 2003. The 3-prime flanking sequence shows similarity to a 52 bp sequence derived from
Arabidopsis thaliana. Apparently, no attempt was made to de termine if any function was assigned to
the sequence, although this would be permitted in connection with the BLAST search. The 52 bp
motif in Arabidopsis thaliana is assigned function in the NCBI /GenBank database as per January 18 th
2005, cf. Box 3, and it is coding for a protein. Notably, the retrieved accession is very similar to the
accession shown in Box 1 , and for both it is believed that " overexpression causes pleiotropic
phenotypic changes ". The European Communities has not be en able to determine if the accession
described in Box 3 was released by September 2003, although it was submitted on March 11th, 2003.

Box 1. Sequence retrieved from Blast search on the NCBI server January 18
th 2005, applying
the 97 bp sequence motif from Arabidopsis thaliana as a probe.

LOCUS AY254319 123 bp mRNA linear PLN 04-MAR-2004
DEFINITION Arabidopsis thaliana DVL3 mRNA, complete cds.
ACCESSION AY254319
VERSION AY254319.1 GI:37955425
KEYWORDS .
SOURCE Arabidopsis thaliana (thale cress)
 ORGANISM Arabidopsis thaliana
 Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
 Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots;
 rosids; eurosids II; Brassicales; Brassicaceae; Arabidopsis.
REFERENCE 1 (bases 1 to 123)
 AUTHORS Wen,J., Lease,K.A. and Walker,J.C.
 TITLE DVL, a novel class of small polypeptides: overexpression alters
 Arabidopsis development
 JOURNAL Plant J. 37 (5), 668-677 (2004)
 PUBMED 14871303
REFERENCE 2 (bases 1 to 123)
 AUTHORS Wen,J., Lease,K.A. and Walker,J.C.
 TITLE Direct Submission
 JOURNAL Submitted (11-MAR-2003) Biological Sciences, University of
 Missouri, 308 Tucker Hall, Columbia, MO 65211-7400, USA
FEATURES Location/Qualifiers
 source 1..123
 /organism="Arabidopsis thaliana"
 /mol_type="mRNA"
 /db_xref="taxon:3702"
 /chromosome="2"
 /map="BAC F1N21"
 CDS 1..123
 /note="similar to DVL1; DVL gene family member;
 overexpression causes pleiotropic phenotypic changes"
 /codon_start=1
 /product="DVL3"
 /protein_id="AAP13818.1"
 /db_xref="GI:37955426"
 /translation="MKGTKKKTPCNKKLGGYLKEQKGRLYIIRRCVVMLICWHD"
ORIGIN
 1 atgaaaggta ccaagaagaa gacgccatgc aacaaaaagc ttggaggata cttgaaagag
 61 caaaagggaa ggctttacat catcagaaga tgtgtggtca tgctcatttg ttggcatgac
 121 taa

Box 2. Sequence retrieved from Blast search on the NCBI server January 18 th 2005, applying
the 33 bp sequence motif from Arabidopsis thaliana as a probe.

LOCUS CNS0AEJL 607 bp mRNA linear HTC 06-FEB-2004
DEFINITION Arabidopsis thaliana Full-length cDNA Complete sequence from clone
 GSLTSIL6ZB03 of Silique of strain col-0 of Arabidopsis thaliana
 (thale cress).
ACCESSION BX818257
VERSION BX818257.1 GI:42475241
KEYWORDS HTC; GSLT_cDNA.
SOURCE Arabidopsis thaliana (thale cress)
 ORGANISM Arabidopsis thaliana
 Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
 Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots;
 rosids; eurosids II; Brassicales; Brassicaceae; Arabidopsis.
REFERENCE 1 (bases 1 to 607)

 AUTHORS Castelli,V., Aury,J.M., Jaillon,O., Wincker,P., Clepet,C.,
 Menard,M., Cruaud,C., Quetier,F., Scarpelli,C., Schachter,V.,
 Temple,G., Caboche,M., Weissenbach,J. and Salanoubat,M.
 TITLE Whole Genome Sequence Comparisons and 'Full-Length' cDNA Sequences:
 A Combined Approach to Evaluate and Improve Arabidopsis Genome
 Annotation
 JOURNAL Unpublished
REFERENCE 2 (bases 1 to 607)
 AUTHORS Genoscope.
 TITLE Direct Submission
 JOURNAL Submitted (18-NOV-2003) Genoscope - Centre National de Sequencage:
 BP 191 91006 EVRY cedex - FRANCE (E-mail: seqref@genoscope.cns.fr
 - Web: www.genoscope.cns.fr)
COMMENT The sequences are based on single pass reads.
 Life Technologies (a division of Invitrogen) members carried out
 full-length librairies construction: Temple G.
 Genoscope members carried out sequencing and annotation: Castelli
 V., Aury J.M., Jaillon O., Wincker P., Menard M., Cruaud C.,
 Schachter V., Weissenbach J., Salanoubat M.
 URGV INRA: Clepet C., Caboche M.
 Annotation is based on the June 2003 version of the Arabidopsis
 genome released by MIPS (Munich Information center for Protein
 Sequences). 5 prime and 3 prime are assembled with Phrap.
 http://www.genoscope.cns.fr/externe/sequences/Banque_Projet_EF/Full
 _length
 http://www.genoscope.cns.fr/cgi-bin/ggb/ggb?source=Arabidopsis.
FEATURES Location/Qualifiers
 source 1..607
 /organism="Arabidopsis thaliana"
 /mol_type="mRNA"
 /strain="Col-0"
 /db_xref="taxon:3702"
 /clone="GSLTSIL6ZB03"
 /tissue_type="Silique"
 /plasmid="pCMVSPORT_6"
ORIGIN
 1 caacatctct atgtgctaag gcatctcttt ctctcccttt ctcaccaaat actctctcaa
 61 tttctctctg ccactctctt tctctagaga ttttcataat gaaaggtacc aagaagaaga
 121 cgccatgcaa caaaaagtct tggaggatac ttcgaaagag caaaagggaa ggctttacat
 181 catcagaaga tgtgtggtca tgctcatttg ttggcatgac taatttacaa cttatatgtg
 241 ttatatatac atgcatatgc acatgcatat tatagacaat atctacgttg tactacaacg
 301 gtttgattga agaaagggat cggatcttcg tggttgtggc gatttgtgat gtgagcggag
 361 ttcgcaagat agagatataa gggcttccaa ccattcatat catgtatatg agtttttact
 421 cgtatatgag ttaagataat aatttttgag ttagtatgaa aatttatatg tagaatcttc
 481 tctaatgtta gtgtatgata tagagtaagg agagaaaaat tataagctta gtttgtatat
 541 gcacacagag agtagttgtt agttgaattt ccttgtctct aattaatcca agtcttcctt
 601 cttgtta

Box 3. Sequence retrieved from Blast search on the NCBI server January 18 th 2005, applying
the 52 bp sequence motif from Arabidopsis thaliana as a probe.

LOCUS AY254203 156 bp mRNA linear PLN 04-MAR-2004
DEFINITION Arabidopsis thaliana DVL1 mRNA, complete cds.
ACCESSION AY254203
VERSION AY254203.1 GI:37955419
KEYWORDS .
SOURCE Arabidopsis thaliana (thale cress)
 ORGANISM Arabidopsis thaliana
 Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
 Spermatophyta; Magnoliophyta; eudicotyledons; core eudicots;
 rosids; eurosids II; Brassicales; Brassicaceae; Arabidopsis.
REFERENCE 1 (bases 1 to 156)

 AUTHORS Wen,J., Lease,K.A. and Walker,J.C.
 TITLE DVL, a novel class of small polypeptides: overexpression alters
 Arabidopsis development
 JOURNAL Plant J. 37 (5), 668-677 (2004)
 PUBMED 14871303
REFERENCE 2 (bases 1 to 156)
 AUTHORS Wen,J., Lease,K.A. and Walker,J.C.
 TITLE Direct Submission
 JOURNAL Submitted (11-MAR-2003) Biological Sciences, University of
 Missouri, 308 Tucker Hall, Columbia, MO 65211-7400, USA
FEATURES Location/Qualifiers
 source 1..156
 /organism="Arabidopsis thaliana"
 /mol_type="mRNA"
 /db_xref="taxon:3702"
 /chromosome="5"
 /map="between At5g16020 and At5g16030"
 CDS 1..156
 /note="DVL gene family member; overexpression causes
 pleiotropic phenotypic changes"
 /codon_start=1
 /product="DVL1"
 /protein_id="AAP13816.1"
 /db_xref="GI:37955420"
 /translation="MEMKRVMMSSAERSKEKKRSISRRLGKYMKEQKGRIYIIRRCMV
 MLLCSHD"
ORIGIN
 1 atggaaatga agagggtcat gatgagctct gcagagagat caaaggagaa gaagagatca
 61 ataagtagaa gattggggaa gtatatgaag gaacaaaagg gaaggattta catcatcaga
 121 agatgtatgg tcatgctcct ttgttcgcat gattga

356. Exhibit EC63/At.120
 on confidential description of Rf3 transgene locus, which forms an
integrated part of the updated risk assessment of September 2003, was completed on April 8 th 2002,
and shows that 5 bp 'filler' has been introduced between the 5-prime flanking sequence and the 5-
prime end of the inserted sequence.
357. Exhibit EC-63/At.131 on confidential determination of th e inserted sequences in Ms8, which
forms an integrated part of the updated risk asse ssment of September 2003, was completed on August
15th 2001, and includes an alignment of the seque nce of the putative transforming plasmid pTHW107
with the sequence of the inserted sequence present in Ms8. This was apparently a re-analysis and
verification of earlier data, and pr oved to demonstrate several minor errors in the earlier data. The
errors observed were the following. For the firs t fragment, covering the 5-prime flanking sequence
and the bar gene cassette, two insertions of one nucleo tide each, one deletion of two nucleotides, and
three (one plus two) nucleotide substitutions, were observed. N one of these observed differences
occurred in coding regions. For the second fragmen t, covering the 3-prime flanking sequence and the
barnase gene cassette, three single nucleotide inserti ons and one deletion of two nucleotides were
observed. The deletion was located in the 5- prime end of the PTA29 promoter sequence which
regulates the transcription of the barnase gene cassette. The reanalysis also revealed that the inserted
sequence was identical with the true sequence of the transforming plasmid, i.e. different from the
putative plasmid sequence included in the original data submitted by the notifier.
358. Exhibit EC-63/At.132
 on confidential determination of th e inserted sequences in Rf3, which
forms an integrated part of the updated risk assessment of September 2003, was completed on
September 11th 2001, and includes an alignment of the se quence of the putative transforming plasmid
pTHW118 with the sequence of the inserted sequence present in Rf3. This was apparently a re-
analysis and verification of earlier data, and proved to demonstrate several minor errors in the earlier
data. The errors were the following. For the fi rst fragment, covering the 5-prime flanking sequence
and the bar gene cassette, three single nucleotide and one double nucleotides insertions, one double

nucleotides deletion and one single and two double nucleotides substitutions were observed. The
double nucleotides insertion was located in the pSsuAr a promoter which regulates the transcription of
the bar gene cassette. For the second fragment, covering the TA29 promoter, no deviations from the
putative transforming plasmid sequence were observed. For the third fragment, covering the promoter
region of the anter specific TA29 gene a double nuc leotide deletion and a single nucleotide insertion
was observed. The deletion was located at the 5- prime end of the PTA29 promoter sequence. For the
fourth fragment, no deviations from the putative tr ansforming plasmid sequence were observed. For
the fifth fragment, covering the 3-prime flanki ng sequences and the second (inverted) of the two
integrated barstar gene cassettes, two single nucleotide in sertions, one double nucleotide deletion and
a single nucleotide substitution was observed. The deletion was located at the 5-prime end of the
3'nos which defines the terminus of the transcription of the second (inverted) barstar gene cassette.
359. Exhibit EC-63/At.133
 on confidential information analys is of integration sequences Ms8,
which forms an integrated part of the updated ri sk assessment of September 2003, was completed on
September 26th 2003, and
"revealed meaningful sequence similarity of the flanking plant DNA fragments of
oilseed rape event Ms8 with published Brassica oleracea sequences. None of these
Brassica oleracea sequence entries in the database are annotated. The intrinsic
bioinformatics methods used (Open Reading Frame search; analysis of the first ATG
codon context sequences; core promoter a nd 3'UTR regulatory motifs search) did not
produce evidence that a novel transcript might arise at either junction of the oilseed
rape Ms8 insert."
360. Exhibit EC-63/At.134
 on confidential information analys is of integration sequences Rf3,
which forms an integrated part of the updated ri sk assessment of September 2003, was completed on
September 26th 2003, and
"revealed meaningful sequence similarity of the flanking plant DNA fragments of
oilseed rape event Rf3 with published Brassica oleracea and Arabidopsis thaliana
sequences. None of these sequence entri es are annotated in the database. The
intrinsic bioinformatics methods used (Open Reading Frame search; analysis of the
first ATG codon context sequences; core promoter and 3'UTR regulatory motifs
search) did not produce evidence that a novel transcript might arise at either junction
of the oilseed rape Rf3 insert."
361. Exhibit EC-63/At.136
 on confidential Ms8 discriminating method shows that this method is
not event specific, it is qualitative and it has onl y been assessed with DNA extracted from leaf punch
or single seed material, i.e. it is not certain whet her it would work with lo w concentrations of GM
material.
362. Exhibit EC-63/At.137 on confidential Rf3 discriminating method shows that this method is
not event specific, it is qualitative and it has onl y been assessed with DNA extracted from leaf punch
or single seed material, i.e. it is not certain whet her it would work with lo w concentrations of GM
material.
363. Exhibit EC-63/At.138 on confidential demonstration of sp ecificity of methods for Ms8 and
Rf3 shows that the methods are here only tested on Brassica napus samples (event Ms1, Ms8, Rf1,
Rf2, Rf3 and wild type). It is not certain whether it would be specific with other samples, e.g. other
GM events.

Question 19

Given the information before the Panel, includi ng the SNIF, the updated environmental risk
assessment and the clarification provided by the notifier (EC-63/At.147), was the information
regarding ecological effects of this product on agricultural systems requested by the lead CA
(EC-63/At.149) necessary to ensure that conclusions of the safety assessment were valid?

General comments

364. Three Panel's experts answered this question: Dr. Andow, Dr. Snow and Dr. Squire. They all
concur that the information on ecological effects re quested by the lead CA was not available in the
notification. Dr. Snow considers that "the information on the indirect effects of introducing herbicide-
tolerant crops could be considered as necessary
 because they are still being investigated in the UK".
However, she questions "the feasibility of obtaining scientific data on all of the processes listed by the
CA prior to deregulation without extensive multi- year studies and underlines the vagueness of the
request of the lead CA asking for some unnecessary data". Dr. Squire writes that "it is legitimate to
ask what such effects might be since cropping wi th transgenic oilseed rape (MS8/RF3) and its
herbicide has potentially new effects on arable flora (i.e. different from those of other oilseed rape
varieties with other herbicides), which may be se verely depleted in any case. The need for an
appropriate comparator against which the new technology should be judged is noted". And
Dr. Andow states that "...although the UK-FSE trials will not be published until later in 2003,
everyone involved was aware of the experiments an d the possibility that th e associated herbicide use
would have adverse environmental effects. These issues are not addressed in the response of the
notifier. Hence, I conclude that the request by the lead CA for more information on point 9 was
necessary to ensure that conclusions of the safety assessment were valid."
365. The European Communities concurs with the overall conclusion of the experts that it was
legitimate to ask what such effects could be. On e of the principles of an environmental risk
assessment is that it should be carried out in a scientifically sound and transparent manner based on
available scientific and technical data. For this reason, an environmental risk assessment has to be up-
to-date and should cover all the relevant issues even where uncertainty remains. Moreover, if new
information on the GMO and its effects on human hea lth or the environment becomes available, the
environmental risk assessment may need to be re-addr essed in order to 1) determine whether the risk
has changed, and/or 2) determine whether it is necessary to amend the risk management accordingly.
366. For its general comments on the persistence of HT products and their potential as "pests", the
European Communities refers the Pa nel to its comments under Question 6 above. In the specifics of
this case, it can easily be taken for granted that th e large scale application of broad spectrum herbicide
in farmland area will cause wide-spread and serious disruption of trophic structures and food webs as
the food basis of all species feeding on anything but the crop is eliminated at least temporarily and
locally. The severity of this effect will be a function of the area sprayed and the frequency of
applications. Such effects were all anticipated at that time and before 1998, however, solid data on
GMHT crop production systems were lacking at time th at would have allowed to better quantify such
effects. The publication of the first conclusions of the FSE trials on 8 October 2003 was a sufficient
reason to support the request of the lead CA. One could criticise the way the question has been
formulated, the type of information asked, and the le vel of detail of the questions but fundamentally it
was legitimate to ask the notifier to complement its environmental risk assessment with the available
data on the ecological impact of the novel weed ma nagement regime. In fact, data on safety issues
related to combined use of the GM HT crop with th e herbicide, including indirect effects, could have
been missing from the original environmental risk assessment because of a di fferent interpretation of
the regulatory framework by the notifier and the lead CA. According to the lead CA, the indirect
effects of herbicide treatments on the farmland biodi versity through the weed population and, more in

general, novel weed management associated with the commercial release of transgenic herbicide-
tolerant crops should be considered as a novel ag ronomic and management technique and should be
assessed under any legal framework, and could therefore be addressed under Directive 2001/18/EC.
Detailed comments

367. With regard to the remark of Dr. Andow in paragraphs 19.05 and 19.06, it is true that the
data on epigaeic predators and honeybees was weak at that time and remains so until today. It is
questionable whether with some more studies pr ovided the knowledge would have improved greatly.
This field was and remains to be quite understudied and highly complex.
368. With regard to the remark of Dr. Andow in paragraph 19.08 , the European Communities
notes that
 the "feeding" study on rabbit was, in fact a single digestibility study. This study only
compared the nutritional values of Ms8*Rf3 as a feed with respect to its conventional counterpart
through estimation of the digestible energy and the digestibility coefficients and is therefore neither a
toxicological study nor a feeding study. A chicken feeding study is mentioned in the environmental
risk assessment but the specific report was not included in the application. In fact, the lead CA made a
mistake in this statement as no chicken feeding st udy has been carried out with the event Ms8*Rf3.
The chicken feeding studies carried out by BCS included other events.
369. As per comments included in paragraphs 19.09 and 19.10, OSR indeed participates in
geochemical cycling. Of course, like anything else in nature, also OSR participates in geochemical
cycling and there are simple methodologies and experi ments that can be conducte d to provide at least
some initial basic insights.
370. The European Communities also concurs with the assessment of Dr. Andow in paragraph
19.12 of his response, that one 'benefit' of GMHT cr ops used for promotion of the products is that no-
tillage cultivation becomes possible. This is a very significant change in agricultural practice indeed.
More information on this is necessary if that is to be evaluated in the approval process.
371. With regard to the comment by Dr. Andow, contained in paragraph 19.13, on the length of
time it took the lead CA to review the informat ion received and to articulate the questions, the
European Communities refer the Panel to its General comments above.
Trifolium/Monsanto/Danisco Roundup Ready fodder beet (A5/15)
C/DK/97/01 (EC chronology 64)
Question 21

Given the information before the Panel, including the lead CA's positive opinion (EC 64/At. 29-30)
and the conclusions of the EC SCP (EC-64/At.83), di d the additional risk assess ment provided by the
notifier (EC-64/At.104-105) in response to requests by the Dutch CA for a theoretical safety
assessment address outstanding scientific concerns relate d to the potential risks associated with this
product?

General comments

372. Two Panel's experts have addressed this questi on: Dr. Andow and Dr. Nutti, and they reach
opposite conclusions. According to Dr. Andow, "the additional risk assessment would have addressed

outstanding scientific concern related to the potential risks associated with this product".
144 On the
contrary, Dr. Nutti, without much of an explanation on the reasons, concludes that the additional risk
assessment provided by the notifier "does not address outstanding scientific concern since they are not
related to the potential risks associated with th is product", and "was not necessary in the first
place".145 The European Communities agrees with Dr. Andow.
373. The European Communities provides below mo re detailed comments on the information
relating to molecular data in this notifica tion. As with regard to the comment by Dr. Andow,
contained in paragraph 21.04 , on the length of time it took th e Dutch authorities to review the
information received and request additional risk assessment, the European Communities refer the
Panel to its general comments above.
Detailed comments

374. The EC chronology includes several exhibits which are relevant for this question, in particular
the following ones.
375. Exhibit EC-64/At.029, containing a short summary report from the Danish Environmental
Protection Agency [EPA] on the dossier, dated 7 October 1997, states
Southern blot and PCR analysis made on the genetic modified line A5/15 and
following generations show that only one gene , cp4-epsps, from the vector is inserted
and that the inserted DNA is stable. Sub sequently the evaluation was restricted to
insertion of the one gene, cp4-epsps, giving tolerance to glyphosate.
376. Exhibit EC-64/At.083 containing the report from EC Scientific Committee on Plants, dated
23 June 1998, states that
2. Terms of reference. The Scientific Committee on Pl ants is asked to consider
whether there is any scientific reason to believe that the placing on the market of
genetically modified fodder beet tolerant to glyphosate with the purpose to be used as
any other fodder beet is likely to cause an y adverse effects on human health and the
environment.
…
6.1.3. Transgenic construct in the genetically modified plant: Vector pMON17204
was designed to transfer DNA located between the right and left borders. In the
A5/15 construct it was determined that only part of the DNA between the borders
were transferred. Molecular analysis b ased on the Southern blot technique showed
that the insert contains only the cp4 epsps gene. The uidA, gox and nptII genes
located between the borders in the vector we re not incorporated into line A5/15. By
PCR experiments it was stated that the pl asmid origins of replication (oriV and
oriColE1) were not incorporated into line A5/15. The T-DNA was truncated in the
E9 3' element before the 35S promoter and the uidA gene resulting in a fully
functional cp4 epsps gene and no other elements of pMON17204 are inserted into
line A5/15. Southern blot analysis showed that there is one copy of T-DNA inserted
into line A5/15.

144 Responses to Questions Submitted by Dr. Andow of 6 January 2005, para. 21.03.
145 Answers by Dr. Nutti of 7 January 2005, p. 16.

Stability of the insert was determined in two ways:
(a) Testing of multiple generations of RR hybrids for tolerance suggested that the
levels of tolerance are consistent between generations.
(b) Physical stability testing by Southern blot and PCR walking experiments
were performed on the original transformation event (T0) and on 5-6 plants from each
of the subsequent 3 generations (T1 to T3). It is indicated that no differences in the
banding pattern were observed among the generations.
The fact that no meaningful differences be tween the ranges and mean levels of CP4
EPSPS in A5/15 were observed over 2 years in samples from field trials is consistent
with stable insertion and expression of the RR gene over generations.
377. Exhibits EC-64/At.104 and 105, containing a c onfidential letter from Monsanto to Dutch CA,
dated 19 October 1999, where it is stated that:
During our last discussion in your office on August 4, you have confirmed that the
Dutch experts wanted to see new information: 1) either molecular characterization
data demonstrating the absence of backbone sequences in the beet genome; 2) or a
theoretical safety assessment based on th e hypothesis that the entire backbone
sequences are present in the beet genome.
We are currently in the process of generati ng further data to confirm the absence of
the backbone sequences, and this informa tion should be available in the following
months. In the interim we are providing a risk assessment as requested under point 2
(See attachment).
378. It is not uncommon to conduct a theoretical sa fety assessment based on a worst case scenario.
The lack of available detailed documentation de monstrating experimentally the absence of the
backbone (plasmid/vector) sequences precluded a more accurate and reliable safety assessment. In
this situation a theoretical assessment could logi cally be performed on a worst case scenario, i.e.
under the assumption that the backbone sequences were present in the GMO. Since such sequences at
least theoretically could lead to establishment of chimeric open reading frames (ORFs) encoding
proteins or other gene products of unknown function, the theoretical safety assessment addressed
outstanding scientific concerns related to potential risks associated with the product.
379. Up until late in the 1990's little attention was pa id to detailed molecula r characterization of
GMOs, primarily because of technological limita tions. As technological solutions were made
available that would allow for more detailed char acterization, i.e. improved PCR, cloning kits and
automated sequencing tools, the genetic maps became more detailed. Parallel to this development, the
literature grew rapidly on genetic rearrangements and unintended ge netic effects associated with
transformation. When the CEN/TC 275/WG 11 had its first meeting in Berlin in February 1999,
researchers from several European countries were independently already planning projects that would
focus on detailed characterization of the inserted and flanking sequences (often referred to as the
border sequences of the transformation event), and development of event specific quantitative
detection methods. Previous communication with biotechnology companies and competent
authorities by some of these researchers had made it clear that the companies were generally
unwilling to provide any kind of sequence information. Furthermore, the competent authorities were
sometimes either unable to communicate requests fo r verified insert and border sequence information
to the biotechnology companies, or they were some times hesitant to do so. In effect, requests were

not always forwarded to the biotech companies, or they were sometimes lacking sufficient precision.
However, given the high level of awareness in the biotechnology industry in relation to gene
technology, it is very unlikely that the notif ier was unaware of the growing demand for such
information. The technology required to determin e the border sequences was available since the mid-
1990's,
146 and that to determine the insert sequences was available several years earlier.
Question 22

Given the information before the Panel, including th e conclusions of the SCP's risk assessment, was
the information requested by the Italian CA in March 2000 (EC-64/At.116) necessary to ensure that
conclusions of the safety assessment were valid?

General comments

380. Two Panel's experts addressed this question: Dr. Andow and Dr. Nutti. Dr. Nutti, without
explaining the reasons for her answer, comes to the c onclusion that the request of the Italian authority
was not necessary, as far as food safety was concerne d. Dr. Andow briefly elaborates on each of the
aspects and finally considers that only the question on transfer and recombinant in natural conditions
was necessary to ensure that conclusions of the safety assessment were valid.
381. The European Communities considers that the It alian CA was justified in requesting this
further information in order to have the latest information on these issues and to determine likely
frequency of such gene flow in order that risk management strategies could be developed.
Detailed comments

382. The Italian CA had asked for additional info rmation on the efficacy of glyphosate, gene
transfer and gene flow to wild relatives. Italy is a major beet seed producing country and thus is
concerned to protect the production of high qualit y seeds by maintaining high levels of seed crop
purity and quality.
383. Efficacy of glyphosate is an issue because it would be n eeded to control contamination of
beet seed crops with non GM and off-type beets, as well as weeds. However, information on this
could also have been gleaned from data on effi cacy of glyphosate from other sources (eg pesticide
registration).
384. There would also have been concerns about crop safety and that seed yields or quality would
not be reduced by applying glyphosate to HT seed crops. Such information was not provided in the
application though there were comments on crop (root) yields and performance showing that it is
often enhanced compared with conventional herb icides. This was also shown by some experts
work
147.
385. On the issue of gene flow to wild relatives, the SCP said that cross pollination and gene flow
are known to occur between cultivated beets and w ild beet types. Different forms of beet are

146 Liu et al. 1995, TAIL-PCR, The Plant Journal 8(3): pp. 457-463.
147 MAY, M.J. (2003) Economic consequences for UK farmers of growing GM herbicide tolerant sugar
beet. Annals of Applied Biology, 142: 41-48.

interfertile, as shown by the gene flow de tected between wild and cultivated beets
148 and by the
transfer of useful genes of ssp maritima to new varieties of sugar beet149.
386. Studies showed that gene introgression occurred 150 potentially creating additional problems
for conventional beet seed producers. This is because the introduction of GM beet may result in flow
of transgenes to wild beets, where they are incorporated into these wild populations 151. Subsequent
flow of transgenes from wild beet to non-GM beet seed crops could introduce these genes into beet
seed and weed beet contaminants of beet seed. Th is could have important implications for the seed
industry.
387. Also, the SCP did not address in its Opinion the management of re-growth of HT beet roots
left in the soil after harvest which can flower and cross pollinate with other beets and weed beet. This
is especially important in seed production areas wh ich are often close to sugar beet production areas
in Italy. Glyphosate herbicide is often used in co nventional crop rotations to destroy such volunteer
plants regrowing from root residues and conventional beet growers should change their practice as
well.
388. It was also important to know the tendancy fo r bolting (adventitious flowering in the first
season) of the beet varieties likely to be transformed particularly as, in the past, fodder beets used to
show more tendancy than sugar beet varieties.
Amylogene starch potato
C/SE/96/3501 (EC chronology 67)

Question 25

Given the information before the Panel, including the application (EC-67/At.13-44), and additional
information provided by the notifier (EC-67/At.51, 57, 61-63, 75-83, 87, 92-93, 94-95, 101, and 103),
was the information regarding molecular characterisation, toxicity, protein analysis, animal feed
trials, effects on non-target organisms, bleomycin resistance, and substantial equivalence requested
by the Scientific Committee for Food (SCF) (EC-67/At .84-86, 96, 98, 100, 10 2, 104, 105 and 106),
necessary to support a valid safety assessment?

General comments

389. Four Panel's experts have answered this qu estion: Dr. Andow, Dr. Healy, Dr. Nutti, and
Dr. Snape. Dr. Nutti considers that "the inform ation required was necessary to support a valid safety
assessment. The notifier failed to provide inform ation on several fundamental issues concerning

148 Santoni S. and Bervillé A., 1992: Evidence for gene exchanges between sugar beet (Beta vulgaris
L.) and wild beets: consequences fo r transgenic sugar beets. Plant Molecular Biology 20: 578-580; Boudry P.,
Mörchen M., Saumitou-Laprade P., Vernet Ph., Van Dijk H., 1993. The origin and evolution of weed beets:
consequences for the breeding and release of herbicide-resistant transgenic sugar beets. Theoretical and Applied
Genetics, 87: 471-478; Bartsch D. and Schmidt M., 1997. Influence of sugar beet breeding on populations of
Beta vulgaris ssp. maritima in Italy. Journal of Vegetation Science, 8: 81-84.
149 Debock T.S.M., 1986. The genus Beta: domestication, taxonomy and interspecific hybridization for
plant breeding. Acta Horticulturae 182: 335-343.
150 Bartsch D., Cuguen J., Biancardi E., SweetJ. 2003. Environmental implications of gene flow from
sugar beet to wild beet – current status and future research needs. Environ. Biosafety Res. 2: 123-127.
151 Arnaud J.F., Viard F., Delescluse M., Cuguen J., 2003. Evidence for gene flow via seed dispersal
from crop to wild relatives in Beta vulgaris (Chen opodiaceae): consequences for the release of genetically
modified crops species with weedy lineages. Proc. Roy. Soc., London, B270, 1565-1571.

safety such as substantial equivalence, toxicity, prot ein analysis and animal feeding trials." Dr. Snape
reinforces this point with regard to molecular characterisation by concluding that "more information is
still required". Similarly Dr. Andow, who only comment ed on the "non-target organism" part of the
question, states that "a request for additional in formation was scientifically justified in 1999 and
remains justifiable even today". On the contra ry, Dr. Healy concludes that: "The information
requested after [20 July 2000] (and much of the inform ation requested prior to this date) is unlikely to
influence the conclusions of the safety assessment."
390. The European Communities shares the conclusi ons reached by Dr. Andow, Dr. Nutti, and
Dr. Snape. Detailed comments on the reply by Dr. Healy are provided below.
Detailed comments

391. With regard to molecular characterisation , the European Communities concurs with the
analysis of Dr. Snape. The questions of the SCP address problems of elements in the flanking region
("confusing and inconclusive PCR analyses to de monstrate absence of vector sequences flanking the
border regions") as well as the antibio tic specificity of the nptII gene
152. The question of the SCP to
the molecular characterisation of the backbone of th e plasmid as well as the question of the quality of
the southern blot (overlapping probes, analysis of nos gene region, contro ls) are according to the
present state of the art of a characterisation. The question to the detailed level of molecular
characterisation including an analysis of potential ORFs and expression of genes is in accordance with
the present state of the art for a molecular characte risation of a transformation event as defined by
Codex, FAO/WHO consultations and scientific literature, 153 which requires a precise and detailed
characterisation including sequencing in flanking re gions outside the boarders (where it is not agreed
how many bps outside the boarder). Although this w as probably not so clearly defined at the time of
the assessment by the SCP, the SCP reacted to upcoming evidences about possible unintended effects
of insertions asking for a detailed molecular charact erisation of the product. The requests of the SCP
are confirmed in their validity by the standards presently accepted.
392. As far as toxicity and feeding trial are concerned, the identification of an ORF with
transcription and unclear translation was a consequence of the inefficient molecular characterisation at
this time and could have been of safety releva nce. The argument of Dr. Healy on compositional
equivalence does not add to safety as a risk assessmen t. Looking for substantial equivalence at this
time would not likely have detected hazards from such an ORF event. In fact , the requested feeding
trials might have not added subs tantially to the safety assessment bu t have been the best possible
reaction to an unsolved problem and the available tools at this time.
393. On the issue of protein analysis , Dr. Healy agrees with the requests for additional
information formulated before July 2000. On the contrary, on the SCP requests of 20 July 2000
(EC-67/At.100), 1 February 2001 (EC-67/At.102), 14 March 2001 (EC-67/At.104), 3 April 2001
(EC-67/At.105) and 8 November 2001 (EC-67/At.106), she concludes that "sufficient information is
available" and that " further analysis including Western blotting requiring ORF4 specific
antibodies is not justified".

152 See Exhibit EC-67/At.84.
153 Codex Guideline for the Conduct of Food Safety Assessment of Foods derived from Recombinant-
DNA plants, CAC/GL 45-2003. FAO/WHO expert cons ultation, 2003. D.A. ANDOW, D.A. SOMERS, N.
AMUGUNE, F. ARAGAO,K. GHOSH, S. GUDU, E. MAGIRI, W.J. MOAR AND E. OSIR, Transgene Locus
Structure and Expression of Bt Maize, Environ. Risk. Chap 04 in: CAB International 2004. Environmental Risk
Assessment of Genetically Modified Organisms: Vol. 1. A Case Study of Bt Maize in Kenya (eds A. Hilbeck
and D. Andow).

394. The European Communities considers the requ est for detailed sequence comparison as very
important because it shows, inter alia, the still exis ting different point of view on the assessment of
allergenicity. The Codex Guideline for the Conduct of Food Safety Assessment of Foods Derived
from Recombinant DNA plants explains that
… sequence homology searches have certain li mitations. In particular, comparisons
are limited to the sequences of known allerg ens in publicly available databases and
the scientific literature. There are also limita tions in the ability of such comparisons
to detect non-contiguous epitopes capable of binding themselves specifically with IgE
antibodies. A negative sequence homology result indicates that a newly expressed
protein is not a known allergen and is unlikely to be cross-reactive to known
allergens. A result indicating absence of significant sequence homology should be
considered along with the other data outlined under this strategy in assessing the
allergenic potential of newly expressed prot eins. Further studies should be conducted
as appropriate.
395. A report of a Joint FAO/WHO Expert Consulta tion on Allergenicity of Foods Derived from
Biotechnology, 2001, concluded that:
5.6.1. Level of expression
Highly allergenic proteins are often expr essed at relatively high levels. However,
allergens can sensitize susceptible individuals at less than milligram levels, possibly
at less than microgram levels (Sorva et al., 1994; Jarvinen et al., 1999). ….Thus, level
of expression cannot yet be incorporated into the assessment of the allergenicity of
genetically modified foods.
In achieving the objective of conferring a spec ific target trait (intended effect) to the
host organism by the insertion of DNA sequences, additional traits could,
theoretically, be acquired or existing traits lost or enhanced (unintended effects).
Unintended effects may be due to factors such as random insertion events, which
might result in disruption of existing genes and modification of protein expression.
6.1. Sequence Homology as Derived from Allergen Databases
The commonly used protein databases (PIR, SwissProt and TrEMBL) contain the
amino acid sequences of most allergens for which this information is known.
However, these databasesare currently not fully up-to-date…… Structural similarity
with known allergens may still be important if significant amino acid identity is
found, but it is below 35 %. In this case significant cross-reactivity is
unlikely…..Since identity of 6 contiguous amino acids has an appreciable risk of
occurring by chance, verification of poten tial crossreactivity is warranted when
criterion (1) is negative, but criterion (2) is positive. In this situation suitable
antibodies (from human or animal source) have to be tested to substantiate the
potential for crossreactivity.
Conclusion of the Consultation:
The original decision tree from the FAO/WHO 2000 Consultation served as a basis
for this consultation. The C onsultation concurred that this decision tree be modified
as a consequence of more recent research and which is reflected in the FAO/WHO

2001 decision tree. When the expressed prot ein is derived from a source with no
known allergenicity, the FAO/WHO 2001 decision tree proposes that the initial
investigation would also be analysis of sequence homology to known allergens from
food and environmental sources. If positive matches are found with known allergens,
then the protein is considered likely al lergenic. If no significant sequence homology
is identified, then targeted serum screeni ng is conducted with serum samples that
contain high levels of IgE antibodies with a specificity that is broadly related to the
gene source. If the targeted serum screening is positive, then the protein is considered
likely allergenic. If the targeted serum screen ing is negative, then pepsin resistance of
the expressed protein and the immunogenicity of the expressed protein in suitable
animal models.
396. The latest FAO/WHO consultation on GM foods of 2003 agrees that
It has been recognized that there is no singl e parameter that can predict the allergenic
potential of a substance. A strategy to asses s allergenicity of biotechnology products
has been formulated (FAO/WHO, 2001; Codex Alimentarius Commission, 2003),
which relies on the parameters: source of the gene, sequence homology, serum testing
of patients known to be allergenic to the source organism or to sources distantly
related, pepsin resistance, the prevalen ce of the trait and assessment using animal
models"
397. The Consultation recommended that additional e fforts should be directed to the further
development and validation of models and that there is a need to improve the accessibility and
interconnectivity of existi ng databases or to establish a centralized database on allergenic linear and
conformational epitopes and tools for screening transg enes for allergenic potential. Even the present
strategy based on a sequence prediction model established by FAO/WHO was contradicted by
important groups.
154
398. These facts demonstrate that there is no agreed detailed, standardized common system for
safety assessment of allergencity and even nowad ays many experts feel that assessment schedules
need to be improved. Therefore the conclusion of Dr. Healy that "sufficient information is available to
support the conclusions of the safety assessment" wit hout additional information as required by the
SCP, cannot be accepted. Already at the time of the debate, the SCP adhered to scientific
uncertainties in the assessment of a potential allerg enicity of GM foods which are even valid and
more concrete nowadays. The requirement of tests including antibodies/sera is widely accepted in
unclear situations nowadays. The assessment of the consequences of a new ORF with a potential
protein with no history in risk assessment or food saf ety is certainly a case where additional testing is
required according to the Codex Guideline.
399. With regard to Dr. Healy's discussion of the SCP request for "in vitro data on the survival of
the APH(3')II protein from leaf tissue in the prese nce of rumen micro-organisms (and of ORF4, if
expressed)" (EC-67/At.85), the European Communities r espectfully submits that Dr. Healy is
overstepping her mandate to analyse molecular ch aracterisation by discussing the risks associated
with antibiotic resistance genes. The inte rnational understanding these issues has evolved
substantially over the last years (see the Europ ean Communities' comments under questions 1 and 2
above) and there is now wide international agreemen t that horizontal transfer of genes, especially
antibiotic resistance genes, from GM plants or produc ts derived from such plants to micro-organisms,

154 Stadler and Stadler, Allergenicity prediction by protein sequence, FASEB, J 2003, 17, 9, 1141-3.
Difficulties in the concept are summarised in Jank and Haslberger, Improved evaluation of potential allergens in
GM food, Trends Biotechnol., 2003, 21, 6, 249-250.

especially in the gut, is unlikely, but not impossible. In consequence of this state of the art, an
appropriate risk assessment, based on a case by case a pproach, in respect to a possible transfer of
genes from a GMO to micro-organisms of the GI tract or in other relevant ecosystems is necessary
and has to include aspects of the gene such as the resistance and the consequence of a possible
transfer of the resistance. Dr. Healy agreed with th e relevance of this evaluation to determine whether
the potential ORF4 polypeptide would confer bleomycin resistance (on bleomycin, see more below).
400. Similarly, the European Communities respectfully submits that Dr. Healy has overstepped her
mandate when assessing the SCP request for data on the safety of modified crops to non-target
organisms (EC-67/At.84).
401. With regard to bleomycin resistance, the dossier submitted contained strong indication that
(a) an unknown protein may have been synthesised in the potato tubers and (b) that this protein may
have encoded resistance against the antibiotic bleo mycin which is a chemotherapeutic used in cancer
treatment. The potential risk was due to the use of a transformation vector, pBin19, which is broadly
used in plant transformation. Therefore, this dossier could have had consequences beyond this specific
file. In this light, the SCP has scrutinised well all data, has had several meetings with the applicant
and has concluded only when all no further doubt s existed. Consequently, the process might have
been lengthy but the delays where all based on safety considerations.
402. On the issue of substantial equivalence, the European Communities notes that, in principle
the expert agrees with the need of this kind of assess ment. This discussion needs also to be seen in the
light of developments and changes of concepts at th is time. The concept – that a comparison of a final
product with one having an acceptable standard of safety provides an important element of safety
assessments of GMOs – was a commonly used basis for the development of both, food safety and the
environmental risk assessment.
155 This concept was elaborated by FAO, WHO and OECD in the early
1990s and referred to as substantial equivalence for the assessment of GM foods. But in 2000, a
FAO/WHO consultation acknowledged that this concept had attracted criticism from the perception
that it was the end-point of a safety asse ssment rather than the starting point. 156 Furthermore,
evidences gathered about considerable variati on in constituents in GMOs in response to
environmental conditions. 157 The Consultation concluded that a consideration of compositional
changes is not the sole basis for determination of safety and that safety can only be determined when
the results of all aspects under comparison, ad not merely comparisons of key constituents are
integrated. More recently the concept has evolve d to a Comparative Safety Assessment for GMO
foods.158
403. Finally, with regard to unintended changes in GM crops detected by metabolic profiling, the
European Communities would like to refer the Pa nel to a study using four independent potato

155 World Health Organization. 1991. Strategies for assessing the safety of foods produced by
biotechnology. Report of a Joint FAO/WHO Consultation. World Health Organization, Geneva. Switzerland.
156 FAO/WHO 2000; Safety aspects of genetically m odified foods of plant origin, a joint FAO/WHO
consultation on foods derived from biotechnology, Geneva, Switzerland 29 May - 2 June 2000. Millstone et
al.,1999; Millstone E, Brunner E, Mayer S. Beyond 'substantial equivalence' Nature. 1999 Oct 7;401(6753):525-
6. Schenkelaars, 2002: Schenkelaars P. Rethinking substantial equivalence. Nat Biotechnol. 2002
Feb;20(2):119.
157 Novak,W.K.; Haslberger,A.G., Substantial equivalence of antinutrients and inherent plant toxins in
genetically modified novel foods., Food Chem.Toxicol., 2000, 38, 6, 473-483.
158 Kok EJ, Kuiper HA., Comparative safety assessmen t for biotech crops., Trends Biotechnol. 2003
Oct;21(10):439-44.

genotypes characterized by modifications in sucrose metabolism
159. This research has general
implications for food/feed safety and for environm ent (target and non-target organisms could be
affected in unpredictable ways) but also supports case-by-case assessments, as each GM event could
be uniquely different to the parental line or is ogenic (non-GM line). Nine lines for altered sucrose
metabolism were transformed, a nd 88 metabolites analyzed. Of the 88, the majority exhibited
significant changes in levels in 1 or more of the 9 transgenic events v. conventional potatoes. In
addition, 9 novel metabolites (not present in conventi onal potatoes) were discovered in some or all of
the transgenic lines. This seems to be a striking indication of how genetic engineering can cause
widespread alterations in cellular metabolism. The study also has an estimate of 90,000 to 200,000
for the number of different molecules in the co mbined plant kingdoms; while Arabadopsis has
roughly 5,000. If 5,000 is typical for a sp ecies, and if the proportion of unmeasured
potato compounds whose level is altered is anything like the "over half" of the 88 this study measured,
one could be talking significant alterations in the e xpression levels of hundreds to thousands of genes,
plus dozens or hundreds of novel compounds. Grante d that most of these changes would likely not
have any adverse impacts, it is still a large pool of al terations that, it seems to me, would be likely to
harbour a few nasty ones.
Monsanto Roundup Ready oilseed rape (GT73)
C/NL/98/11 (EC chronology 70)

Question 27

Given the information before the Panel, including the notification, was the information regarding feed
safety aspects of this product requested by the Netherlands (EC-70/At.8 and 13) necessary to ensure
that conclusions of the safety assessment were valid?

General comments

404. Only one Panel's expert, Dr. Nutti, answered this question. Her conclusion is that the studies
requested by the Dutch authorities were not necessar y to ensure the product's safety assessment. The
European Communities respectfully disagrees because, as it will be shown in details below, very
serious reserves on the interpretation of the results and on the conclusions of the rat experiments can
be formulated. This especially as Dr. Nutti addr esses only some aspects of the safety assessment. She
does not at all address the aspects of unintended e ffects based on sincere problems in the molecular
characterisation, which are at the centre of con cern of the CA. The analysis of potential unintended
effects is a central element in the guidelines for a safety assessment in CODEX.
Detailed comments

405. With regard to the feed safety aspects, the Dutch authority requested a whole report to
evaluate composition (Exhibit EC-70/At.08) as well as the experimental results of semi-chronic rat
and trout studies and an assessment of the relevanc e of the performed studies in experimental animals
to predict the safety of the product in target animals such as ruminants, pigs and poultry
(EC-70/At.13). This request for additional experiment s is particularly justified because these target
species are known to be particularly sensitive for the detection of anti-nutritional factors (ANFs) such
as goitrogenic effects of GT73, already suspected on rats.

159 Roessner U., Luedemann A., Brust D., Fiehn O. , Linke T., Willmitzer L., Fernie A.R., 2001.
Metabolic Profiling Allows Comprehensive Phenotyping of Genetically or Envir onmentally Modified Plant
Systems, in The Plant Cell, Vol. 13, 11–29.

406. It is clear from Exhibit EC-70/At.02 that very unsatisfactory experimental designs have been
used in rats experiments. The results on feeding studies were not accurately presented. The first 6-
week feeding study revealed an increase in the absolute liver weight (+ 5 / 20 %) in male rats fed with
GT 73. The second 4-week experiment confirmed th e observations of the previous experiment. An
increase in the relative liver weight (+ 12-16 % in males for a level of 15 % of toasted meal) has been
observed. In addition, a mixture of seeds of different GT 73 lines has been acknowledged in the
second experiment: "Inadvertent commingling of seeds …",
160 sic. Furthermore, the experiment was
conducted apparently with only one group of animals fed GT 73, without a control group.
407. What is also to be noted when considering th e answer provided to this question is that the
assessment of toxicology , such as requiring feeding studies, needs to be supported by important
aspects of molecular characterisation . Dr. Nutti does not at all consider the aspects of unintended
effects based on problems in the molecular characterisation, nor could she relied on an answer to
question 26, but – nonetheless – she comments on toxi cological relevant questions. This discrepancy
(especially in the possible understanding of the basi s of her toxicological comments) deserves further
examination.
408. As seen above, the question of the Dutch CA addresses sincere lacks in the safety assessment
of the product, especially significant flaws in th e molecular characterisation. If the molecular
characterisation of a product is not adequately pr ovided or if the characterisation indicates potential
hazards (such as rearrangements in flanking region s, unclear ORFs or not-conclusive results of
sequence analysis for toxicological assessment) at least toxicological testing using other methods,
such as feeding studies, are required to exclude hazards from unintended effects which can not be
clarified by the molecular characterisation. Howe ver, one might/should argue that results from non
observed effects in toxicological relevant feeding studies cannot serve as a substitute of necessary
conclusive results from a molecular characterisation.
409. Whereas the need for analysing potential uni ntended effects and an adequate molecular
characterisation was already discussed at the time of the CA request, recently important scientific
organisation confirmed and detaile d this procedure. In a FIFRA SAP meeting of the US-EPA, the
expert panel agreed on the relation between molecular characterisation and toxicology testing:
Screening for unintended effects is encouraged. Such a screening could use profiling
methods for unintended effects, e.g. expression microarrays or metabolomic studies
(e.g., as discussed in SAFOTEST). However, as these tests are still under
development, toxicology studies in rodents, looking for effects of unintended effects,
although unlikely, could be appropriate. Wh ile the targeted safety assessment system
in place today has apparently been effec tive in risk characterization, it cannot
eliminate the chance that unintended or une xpected consequences of plant breeding
will escape detection. It would be extrem ely useful to have comparative data
regarding the frequency and magnitude of unintended effects associated with
conventional plant breeding with or without the use of genetic engineering techniques
upon which a quantitative risk assessment could be based
161.

160 See page 73 of the ACNFP Review of Glyphosate-tolerant Oilseed Rape, annexed to the
Application contained in Exhibit EC-70/At.02.
161 Minutes of the FIFRA Scientific Advisory Panel Meeting Held June 8-10, 2004: Product
Characterization, Human Health Risk, Ecological Risk, And Insect Resistance Management.

410. As with regard to Codex, Dr. Nutti argues that
… according to the Codex Guideline for th e Conduct of Food Safety Assessment of
Foods Derived from Recombinant-DNA Plants (Page 4, paragraph 53), some foods
may require additional testing; regarding an imal feeding studies, extra studies may be
warranted if changes in the bioavailability of nutrients are expected or if the
composition is not comparable to conven tional foods. Although the product was
concluded to be substantially equivalent …
411. This summary of the Codex Guidelines is not comprehensive and is misleading. The cited
paragraph 53 needs to be analysed together with paragraphs 11-13 and 17, which states that:
… Detecting any potential adverse effects and relating these conclusively to an
individual characteristic of the food can therefore be extremely difficult. If the
characterization of the food indicates that the available data are insufficient for a
thorough safety assessment, properly designe d animal studies could be requested on
the whole foods. Another consideration in deciding the need for animal studies is
whether it is appropriate to subject experi mental animals to such a study if it is
unlikely to give rise to meaningful information.
Due to the difficulties of applying traditional toxicological testing and risk assessment
procedures to whole foods, a more focu sed approach is required for the safety
assessment of foods derived from food pl ants, including recombinant-DNA plants.
This has been addressed by the developm ent of a multidisciplinary approach for
assessing safety which takes into account bot h intended and unintended changes that
may occur in the plant or in the foods derived from it, using the concept of substantial
equivalence.
The concept of substantial equivalence is a key step in the safety assessment process.
However, it is not a safety assessment in itself; rather it represents the starting point
which is used to structure the safety assessment of a new food relative to its
conventional counterpart. This concept is u sed to identify similarities and differences
between the new food and its conventional counterpart
2. It aids in the identification of
potential safety and nutritional issues and is considered the most appropriate strategy
to date for safety assessment of foods derived from recombinant-DNA plants. The
safety assessment carried out in this way does not imply absolute safety of the new
product; rather, it focuses on assessing the safety of any identified differences so that
the safety of the new product can be c onsidered relative to its conventional
counterpart.
…
The safety assessment of foods derived from recombinant-DNA plants involves
methods to identify and detect such unint ended effects and procedures to evaluate
their biological relevance and potential impact on food safety. A variety of data and
information are necessary to assess unintended effects because no individual test can
detect all possible unintended effects or id entify, with certainty, those relevant to
human health.
412. The misleading conclusion that a risk assessme nt according to the substantial equivalent per
se can establish safety was corrected explic itly in 2000 when a FAO/WHO consultation
acknowledged that this concept had attracted criticism because it was the end-point of a safety

assessment rather than the starting point
162. The Consultation concluded that a consideration of
compositional changes is not the sole basis for de termination of safety and that safety can only be
determined when the results of all aspects under comparison, ad not merely comparisons of key
constituents are integrated. More recently the concept has evolved to a Comparative Safety
Assessment for GMO foods.163
413. The latest FAO/WHO expert consultation on GM animals, which updated the entire field
(where FAO/WHO expert consultations are usually the basis for Codex recommendations) concluded
that
An extensive molecular characterization of the inserted genetic material construct will
generally be required, both before and after the insertional event. The molecular
characterization should furthermore comprise an analysis of the copy number and a
sequence analysis of the flanking regions of the place of insertion in order to identify
any unintended effects. It is recommende d that the approach for the molecular
characterization should be further standardized to include the flanking regions.164
414. As the Dutch CA realised that fundament al problems were present in the molecular
characterisation, it requested supplementary studi es to investigate potential unintended effects as
requested in the Codex guidelines for GM plants. The argument that the substantial equivalent did not
see any significant changes cannot at all mean that in this case no further testing is necessary, even in
the absence of conclusive information from mol ecular characterisation. The safety assessment carried
out according to the substantial equivalent does not imply absolute safety of the new product; rather, it
focuses on assessing the safety of any identified differe nces so that the safety of the new product can
be considered relative to its conve ntional counterpart (Codex). Therefore, the requests of the Dutch
CA fully adhered to Codex Guidelines.
Question 28

Given the information before the Panel, including the notification and the SNIF (EC-70/At.49-53),
was the additional information provided in Monsanto's submission to the Commission
(EC-70/At.84-97) necessary to ensure that conclusions of the safety assessment were valid?

General comments

415. Two Panel's experts have provided answers to this questions: Dr. Andow and Dr. Nutti.
Dr. Andow considers that questions related to mon itoring that refer to specific parts of the risk
assessment are justifiable, including Italy's question related to the AMPA metabolite that is formed
from the glyphosate (Roundup) herbicide, whilst he pe rceives the requirements for the monitoring
procedures (e.g. Sweden, with regard to human and an imal health) as too strict, given that preferably

162 FAO/WHO 2000; Safety aspects of genetically m odified foods of plant origin, a joint FAO/WHO
consultation on foods derived from biotechnology, Geneva, Switzerland 29 May - 2 June 2000; Canadian Royal
Society: Canadian Royal Society, Elements of Precaution: Recommenda tions for the Regulation of Food
Biotechnology in Canada, an Expert Panel Report on the Future of Food Biotechnology prepared by The Royal
Society of Canada at the request of Health Canada, Canadian Food Inspection Agency and Environment
Canada, 2001; Millstone et al.,1999; Millstone E, Brunner E, Mayer S. Beyond 'substantial equivalence' Nature.
1999 Oct 7;401(6753):525-6; Schenkelaars, 2002: Schenkelaars P. Rethinking substantial equivalence. Nat
Biotechnol. 2002 Feb;20(2):119.
163 Kok EJ, Kuiper HA., Comparative safety assessmen t for biotech crops., Trends Biotechnol. 2003
Oct;21(10):439-44.
164 FAO/WHO Consultation, 2003.

"worst cases" should be monitored for. Dr. Nutti, on the contrary, regards the additionally requested
animal experiments as not necessary given the prev iously provided information and its (positive)
evaluation by the EFSA GMO Panel. More Detailed comments on the experts' replies are provided
below.
Detailed comments

416. The European Communities notes that question 28 pertains to additional information provided
in Monsanto's submission, whereas replies of the Pane l's experts pertain to th e validity of some (but
not all) of the questions that were addressed by Monsanto.
417. Thus, for example, with regard to Dr. Andow's comments on the strictness of the monitoring
pertaining to, among others, procedures requested by Sweden on 28 February 2003, for monitoring for
adverse effects to animals (consuming feed) and human health ( cf. EC-70/At.67-transl, pp. 3-4), the
European Communities notes that Sweden had only reite rated, albeit in different but similar wording,
the items mentioned in the monitoring plan s ubmitted by Monsanto itself on 13 December 2002
165.
Therefore, the Swedish request posed no new requirements on the applicant. It is also to be noted that
the pertinent monitoring plan was updated with more details of the animal feed and human health
surveillance by the applicant in July 2003 (EC-70/At. 92, pp. 19-26).
418. In addition, Dr. Nutti's notes that additional animal experiments have been requested.
However, this request is not exp licitly mentioned or addressed by the applicant in its reply to the
Member States comments (EC-70/At.85). One exhibit reviews toxicity studies with rats, which had,
however, previously been provided to the rapporteur Member State and therefore did not represent
truly new additional information (EC-70/At. 90).
419. With regard to additional information not cons idered by the Panel experts, Monsanto has
provided a certain quantity of additional information consisting of both replies and appendices.
420. Additional information pertaining to bioinformatic studies of open reading frames (ORFs,
i.e. part of potential new genes) in the DNA fla nking the insert created by the genetic modification
were requested by the United Kingdom (EC-70/At.85, p. 10, additional information in EC-70/At.88
and 94-96 (MSL18493). These bioinformatics studies of ORFs are in line with Codex guideline,
166 as
well as its Annex' paragraphs 8-11, regarding the presence of potential fusion proteins and the toxicity
and allergenicity of newly expressed proteins. Th e additional information was therefore necessary to
ensure that conclusions of the safety assessment were valid.
421. With regard to transgenic protein used for toxicity studies, a study showing the equivalence
of the recombinant GOX protein expressed in GM canola GT73 to that expressed in GM bacteria
(Escherichia coli) and purified for the purpose of safety testing was requested by Spain (EC-70/At.85,
p. 11, additional information in EC-70/At.89 (M SL12968). In addition, the applicability of studies
with bacterially produced GOX and EPSPS was doubted by Austria. The provided information is
relevant for the safety assessment of a "new substance" in a GM plant, as explained by Codex
guidelines, paragraph 40:

165 EC-70/At.64, pp. 96-97: "Additional information to complement oilseed rape notification
C/NL/98/11, as requested by the entering into force of the Directive 2001/18/EC", Annex VII, "Monitoring
plan", point 2.
166 Codex Guideline for the Conduct of Food Safety Assessment of Foods derived from Recombinant-
DNA plants, CAC/GL 45-2003, paragraphs 31D and 38.

This may require the isolation of the new substance from the recombinant DNA-plant
or the production of the substance from an alternative source, in which case, the
material should be shown to be bioche mically, structurally, and functionally
equivalent to that produced in the recombinant-DNA plant.167
The additional information was th erefore necessary to ensure th at conclusions of the safety
assessment were valid.

422. With regard to compositional analysis of glucosinolates (toxic), additional information
pertaining to the differences observed between GT73 canola and control canola was requested by
Austria (EC-70/At.85, p. 13; additional informa tion provided in EC-70/At.90). The comparative
safety assessment, also termed the prin ciple of "substantial equivalence",
168 focuses on differences
between the GM crop and its conventional counterpart with a history of safe use. Therefore, the
statistically significant differences in glucosinolat e levels should be considered as to whether they
would trigger a further safety assessment. The add itional information, providing compositional data
from later periods than previously, has an important role in support of the applicant's arguments that
the glucosinolate levels are well within safety limits and that their elevation in GM canola GT73 was
of a transient nature. The additional information was therefore necessary to ensure that conclusions of
the safety assessment were valid.
423. In addition, Monsanto has also provided rep lies without separate appendices that would have
contained original studies or full reports. These replies were provided in response to questions
supplementary to those mentioned above. These questions pertained to compositional analysis,
herbicide residues, and potential allergenicity.
424. For example, with regard to compositional analysis, Member States had sought clarifications
about statistical analysis, herbicides used in the experiments, size of samp les taken for analysis,
potential cross-contamination, and comparability of Ca nadian with European conditions. With regard
to phenotypic and agronomic characteristics, there was a question about the differences observed in
one location. These are details that are important in the identification and interpretation of differences
observed during the comparative safety assessme nt approach of "substantial equivalence" 169. The
Member States' questions were therefore relevant to ensure that conclusions of the safety assessment
were valid.
425. Some of the Member States' questions with regard to allergenicity pertained specifically to
identicalness of transgenic products (GOX, and from other ORFs) to known allergens and celiac-
disease-related proteins. The identicalness at stake c onsisted of 6- or 7- amino acid identities that had
been documented previously by the applicant, and that, in one case, had been identified in a peer-
reviewed article as part of an anti serum-binding segment of an allergen. 170 The Annex to the Codex
guideline,171 paragraph 8, recommends that:

167 Ibidem, paragraph 40.
168 Ibidem, paragraph 13.
169 Ibidem, paragraph 13.
170 Kleter, G.A., Peijnenburg, A.A.C.M. (2002) Screening of transgenic proteins expressed in
transgenic food crops for the presence of short amino acid sequences identical to potential, IgE - binding linear
epitopes of allergens. BMC Struct ural Biology 2: 8. Available at <http://www.biomed central.com/1472-
6807/2/8> (last visited on 28 January 2005).
171 Codex Guideline for the Conduct of Food Safety Assessment of Foods derived from Recombinant-
DNA plants, CAC/GL 45-2003, page 40.

Strategies such as stepwise contiguous id entical amino acid segment searches may
also be performed for identifying sequen ces that may represent linear epitopes. The
size of the contiguous amino acid segment should be based on a scientifically
justified rationale in order to minimi ze the potential for false positive or false
negative results
N.
And in the footnote to this, it reads as follows:

It is recognized that the 2001 FAO/WHO consultation suggested moving from 8 to 6
identical amino acid segments in searches. The smaller the peptide sequence used in
the stepwise comparison, the greater the likelihood of identifying false positives, the
larger the peptide sequence used, the greate r the likelihood of false negatives, thereby
reducing the utility of the comparison.
In the light of these recommendations by Codex, it was legitimate for the Member States to ask
questions about the 6- and 7- am ino acid segment identities. In its reply to these questions, the
applicant provided arguments for his c hoice for an 8-amino acid threshold, i.e. its "scientifically
justified rationale" as recommended by Codex. The Me mber States' questions were therefore relevant
to ensure that conclusions of the safety assessment were valid.

Pioneer/Dow AgroSciences Bt corn Cry1F (1507)

C/NL/00/10 (EC chronology 74)

Question 32

Given the information before the Panel, including the original notification (EC-74/At.1-15), was the
information regarding molecular characterizati on of this product requested by the lead CA
(EC-74/At.17 and 33) necessary to ensure that conclusions of the safety assessment were valid?

General comments

426. Only Dr. Nutti wrote three lines on this question. There she affirmed that "a lot of information
was provided", however she felt "not capable of j udging if such material wa s enough to ensure that
conclusions of the safety assessment were valid".
427. The molecular characterization provided by the notifier was based on Southern blot analysis
and assumptions about the specific sequence of the tr ansforming sequence and the insert. This is not
sufficient given the technological development in the 1990's and the accumulated literature on
unintended genetic rearrangements etc. that may accompany presumably successful transformations
of plants by the time the notification was su bmitted. The information regarding molecular
characterization of this product requested by the l ead CA was, therefore, necessary to ensure that
conclusions of the safety assessment were valid . The European Communities will provide below
some detailed comments on the information requested by the lead CA on molecular characterization.
Detailed comments

428. Table 4 of exhibit EC-74/At.005 summarizes the observed fragments during Southern
analyses of the DNA insert of 1507 maize and may be compared with the predicted fragments listed in
Table 3 of exhibit EC-74/At.005. There are sever al fragments listed in Table 4 that were not
predicted in Table 3, and that could not be referre d to similar fragments observed in negative control
genomic DNA (see footnotes to Table 4). These extr a bands may represent genetic rearrangements or
duplicate copies of insert elements. The Southern blots presented in Figures 4-7 of exhibit

EC-74/At.005 were not reproduced in sufficient quality to be evaluated. The data provided cannot be
said to demonstrate the absence of additional inserte d sequences. If such sequences were present, they
would deserve to be characterised in more detail to determine whether they are likely to pose a risk to
human or animal health or the environment.
429. Question 3 of exhibit EC-74/At.017 requires the nucleotide sequences of both the vector and
the integrated insert, i.e. the sequence that is actually present in the GMO (that may differ from that in
the vector). And questions 5-7 of exhibit EC-74/At. 017 appear to say that the notifier was required to
provide a sequence based characterisation of the DNA sequence(s) that is actually inserted in the
GMO. Furthermore, the detection method must be event specific to meet the requirement to confirm
the unique molecular identity of the maize line. All other types of detection method will fail to meet
the requirement.
172
430. Question 3 of exhibit EC-74/At.017, as presented in exhibit EC-74/At.019, and the
information provided in Figures 1, 2, and 3 of ex hibit EC-74/At.019 appear to give details on the
intended sequence of the vector but not of the in tegrated insert as it is present in the GMO, and
possibly also not verified through sequencing of the vector. This is similar to the situation discussed
in relation to question 18 (EC chronology 63, exhibits 63/131, 63/132 and possibly also 63/115
above).
431. The answers to questions 5 and 6 of exhibit EC-74/At.017, provided in exhibit
EC-74/At.019, clearly demonstrate that the characterization was based on Southern blot analysis and
not supported by DNA sequencing studies. This al so means that a range of rearrangements and
substitutions and/or insertions and deletions of short sequen ce motifs (e.g. single or double
nucleotides) were unlikely to be detected (see comment under this exhibit question 3, above).
432. Based on the answer to question 3 of exhibit EC-74/At.033 , provided in exhibit
EC-74/At.054, the European Communities believes that the inserted sequence in the 1507 maize
should be verified by sequencing of the maize, not ju st by assuming that the sequence of the vector is
unaltered. There are several reports of such r earrangements, including a well known and frequently
cited paper published a y ear before this reply,
173 suggesting that a full characterisation may be
justified.
433. Although Southern blot analysis with probes of the size applied here (cf. exhibit
EC-74/At.054, reply to question 4 of exhibit EC-74/At.033) is unlikely to detect short fragments that
may have been integrated in other parts of the genome, the lead CA according to exhibit
EC-74/At.002 seem to conclude that this is not a problem . In this context, Table 4 of exhibit
EC-74/At.005 should be noted, where several unpredicted bands were observed that have not been
explained. As commented above, it cannot be conc luded that the absence of unintended insertions is
demonstrated (exhibit EC-74/At.005).
434. Finally, the detection method seems to target a sequence motif that is not covering the
junction between the flanking sequence of the recipient genome and the inserted sequence, but instead
target a sequence resulting from a rearrangement of inserted sequences (cf. exhibit EC-74/At.057).
This sequence motif is likely to maintain its status as unique to 1507 maize also if the same vector is

172 See e.g. Miraglia, M., Berdal, K.G., Brera, C., Corbisier, P., Holst-Jensen, A., Kok, E., Marvin,
H.J.P., Schimmel, H., Rentsch, J., van Rie, J.P. & Zagon, J. (2004; alphabetical order). Detection and
traceability of genetically modified organisms in the fo od production chain. Food Chem. Toxicol. 42: 1157-
1180.
173 Windels P., Tavernier I., Depicker A., Van Bock staele E. & De Loose M. (2001). Characterisation
of the Roundup Ready soybean insert. Eur. Food Res. Technol. 213:107-112.

used to transform new maize or ot her plants in the future. The sequence is furthermore a single copy
sequence in the haploid genome that carries the geneti c modification, and as such could be fit for the
purpose of DNA based quantitation as well. The detec tion method is qualitative, not quantitative, and
the limit of detection is reported to be 0.1 % on kernel count basis (cf. exhibit EC-74/At.058). The
length of the amplification product is slightly longer than other typical event specific targets used in
real-time PCR assays, and this may potentially limit its applicability for analyses of processed
materials in comparison to methods that target s horter sequences. However, no specific requirements
concerning the length of amplicons have been rais ed in connection with the requests from the lead
CA.
Question 34

Given the information before the Panel, including the original notification and additional information
provided by Pioneer (EC-74/At. 18-13, 35-50, and 53 -59), was the information regarding feed safety,
detection methods and reference materials requested by the lead CA (EC-74/At.52 and 60) necessary
to ensure that conclusions of the safety assessment were valid?

General comments

435. Only Dr. Nutti addresses this question asserting that the requests from the Dutch CA were
correct and necessary. Only with regard to th e request on detection method (EC-74/At.60), she
considers herself not apt for judging.
436. On this regard, the European Communities woul d like to note that the data relating to the
detection has not yet been validated acc ording to international standards (see http://crl-gmo.jrc.it
) but
has only been tested in the laboratory where it has b een developed. This is a completion of the first
step of the process, i.e. the development and initial characterisation of the method. The second step is
the evaluation of the data by an independent laborat ory (under the current legislation EC legislation,
this is the Community Reference Laboratory) and the evaluation of the data in the light of the
"minimum performance criteria for carrying out a validation study". In the case of Event TC1507, the
full validation is currently still on going and therefor e, as of today there is no detection method that
can be considered as "fit for the purpose".
437. Furthermore, as for question 17, the European Co mmunities wishes to point out that the need
for a detection method has been requested in the c ontext of post-marketing surveillance and not in the
context of safety assessment.
438. This said, the request contained in exhibit EC-74/At.060 is very clear and detailed with
respect to the type of information sought and the scientific basis for requesting the information. Thus,
although the need for a detection method is usually considered to be more relevant to consumer policy
than to safety, the availability of a reliable detec tion method constitutes also a precautionary measure.
In fact, in the very unlikely event that an authori sed GMO is found to be harmful to human health, the
availability of a reliable detection method may fac ilitate efficient tracing and withdrawal of the GMO
and its derived products. Without reference materials, no detection method can be considered truly
reliable. The request for a detection method and re ference material is therefore necessary also from a
safety perspective.
Question 35

In your view can EC field trials, in France, Italy, and Chile, provide compositional data on maize
kernels that is relevant to evaluating cultivation areas exporting maize to the EC?

General comments

439. Two Panel's experts, Dr. Andow and Dr. Nutti, replied to this question. Their conclusions
diverge substantially. Dr. Nutti states that the com positional data on France, Italy, and Chile were
supplementary to other previously provided data (w hich, in fact, they were not) and that therefore,
they would not have been necessary. In so doi ng, she does not really address the question of the
Panel. Dr. Andow, on the contrary, clearly concludes that "maize from France, Italy, and Chile would
not provide compositional data on maize kernels th at is relevant to evaluating cultivation areas
exporting maize to the EC".
440. The European Communities conc urs with Dr. Andow's above conclusion and will provide
below some detailed comments on replies from both experts.
Detailed comments

441. In paragraph 35.07, Dr. Andow discusses the point that no explicit scientific rationale has
been provided for the lead CA's request mentione d above. Whereas the lead CA's request is not
mentioned in question 35, it is in line with th e Codex guideline, paragraph 45, pertaining to
compositional analysis of key components, which reads as follows:
The location of field trials should be representative of the range of environmental
conditions under which the plant varieties w ould be expected to be grown. The
number of trial sites should be sufficient to allow accurate assessment of
compositional characteristics over this range. Similarly, trials should be conducted
over a sufficient number of generations to a llow adequate exposure to the variety of
conditions met in nature.
174
Therefore, while no explicit rationale is provided in the lead CA's request, this is in line with Codex
recommendations.

442. Indeed, Dr. Nutti correctly notes that the com positional data should be in line with Codex
recommendations, citing paragraph 45 of these guidelin es with regard to the choice of field trial
location being representative for the various environm ental conditions for crop cultivation. However,
Dr. Nutti assumes that the compositional data from Fr ance, Italy, and Chile has been provided as a
supplement to previous data that would comply wi th Codex guidelines. Perhaps the answer by the
applicant (EC-74/At.36, p.5), recapitulating what has previously been provided, might have created
this confusion. However, the data from France, Italy, and Chile were the only compositional data
provided by the applicant at that time (summa rized on pp. 11-26 of EC74/At.7). Based upon the
Codex paragraph cited by Dr. Nutti, and given that the applicatio n concerned import of maize 1507,
the question whether France, Italy, and Chile are re levant for areas exporting maize to the EU would
therefore still be relevant. And, as discussed by Dr. Andow, as these countries are not among the
major exporters of maize in the world, the answer to the question should be negative.

174 Codex Guideline for the Conduct of Food Safety Assessment of Foods derived from Recombinant-
DNA plants, CAC/GL 45-2003.

Pioneer/Dow AgroSciences Bt corn Cry1F (1507)

C/ES/01/01 (EC chronology 75)

Question 36

Given the information before the Panel, includi ng (EC-75/At.1), was the information regarding
molecular characterization, toxicity, and environm ental impact of this product requested by the
National Biosafety Committee of Spain (NBC) (EC-75/At.5) necessary to ensure that conclusions of
the safety assessment were valid?

 (a) Given that Pioneer had notified this product under the regulation concerning the
deliberate release of this plant (cultivation) , were concerns raised by the lead CA on
allergenicity and toxicity of the proteins expressed by the inserted gene sequences
relevant to the risk assessment of this product?

General comments

443. Both Dr. Nutti and Dr. Andow have provided an swers to this question in their respective
fields of expertise. Their replies overlap on toxicity issues, on which they take widely differing
positions. Dr. Nutti's comments point to a factual misunderstanding dismissed. Dr. Andow's analysis
to the greatest extent confirms the lead CA's approa ch in this instance. Furthermore, the European
Communities notes that no expert has commented on the request for additional molecular
characterisation and will, therefore, offer a few brief comments itself.
Detailed comments

444. Dr. Nutti's reply on question 36 points to a mi sunderstanding on the lead CA's side. When
stating that "the protein used in the toxicology a nd allergenicity tests was obtained in a heterologous
system and not in other maize" Dr. Nutti bases herself on the data contained in EC 75 At. 1. These
data are actually from a later date (February 14
th, 2002) than the requests in EC 75 At.5 (November
28th, 2001). In fact, these are the data that the app licant provided in response to the lead CA's request
in At. 5.
445. In any event, specifically in the context of f ood safety the requested information with regard
to the source of the transgenic protein is relevant for the safety assessment of a "new substance" in a
GM plant, as explained by the Codex guideline CAC/GL 45-2003:
"This may require the isolation of the new substance from the recombinant DNA-
plant or the production of the substance from an alternative source, in which case, the
material should be shown to be bioche mically, structurally, and functionally
equivalent to that produced in the recombinant-DNA plant."175
446. Dr. Nutti's reply on question 36a which she links to the lead CA's question on degradation of
proteins (question 6 of At. 5) seems to be b ased on a misunderstanding regarding the chronological
order of data submitted as above, a nd the data discussed in the answ er may not have been known to
the lead CA at the time of the request. In a ddition, the lead CA's questions on toxicity and

175 Codex Alimentarius, 2003. Codex Principles and Guidelines on Foods Derived from Biotechnology.
Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture
Organization, Rome. ftp://ftp.fao.org/codex/standard/e n/CodexTextsBiotechFoods.pdf, {Codex alimentarius,
2003; par. 40}

allergenicity were relevant, since the applicati on for cultivation of maize 1507 also included animal
feed purposes.
447. In any event, Dr. Andow provides a thorough explanation on why data on degradation of
protein is necessary (Dr. Andow's reply on question 36, para. 36.06). Specifically in the context of
food safety, this is confirmed by the recommendations by Codex in its guideline for the safety
assessment of foods from plants derived through modern biotechnology:
In the case of proteins, the assessment of potential toxicity should focus on ……
stability to heat or processing and to degrad ation in appropriate representative gastric
and intestinal model systems."
176
The absolute exposure to the newly expressed protein and the effects of relevant
processing will contribute toward an overall conclusion about the potential for human
risk. In this regard, the nature of th e food product intended for consumption should
be taken into considerati on in determining the types of processing which would be
applied and its effects on the presence of the protein in the final product.177
448. Dr. Andow addresses the environmental issues in great detail. He concludes that most of the
requests of the Spanish CA were valid to ensu re the conclusions of the safety assessment were
relevant to Spain and rigorously conducted. Indeed , toxicity and environmental impact data on other
species (e.g. regionally appropriate non target in sects, including other non-do mesticated herbivores)
and regional environments (local gr owing regions in Spain) would be needed to accurately determine
toxicity and environmental impacts to local Span ish fauna of Bt corn Cry1F and its degradation
products (i.e. resulting from ingestion by herbivores and decomposition in the soil of plant material
and root exudates). Even for target pest species from different countries or regions, sensitivities to
expressed Bt toxins vary widely. Hence it can be r easonably expected that the same (species-specific
and even population-specific variability in sensitivit y to Bt toxins) will apply to local non target
species that could be affected by this Bt toxin e.g. local butterflies of conservation concern or of
heritage value. There is thus s upport for the argument that there still was scientific uncertainty about
impacts of Bt crops on regionally-appropriate target pest populations and non-target species.
Therefore there were scientifically justified envir onmental concerns (i.e. case-by-case and potentially
member state specific) and also unanswered scientific questions over the reliability of the 'high dose
and refugia' strategy to work if the particular Bt crop event has not been tested regionally. The point
of these specific tests are to ensure that a) th e regionally selected species of target pests are
susceptible to the expressed Bt t oxin AND that b) regionally selected non-target species are not
susceptible to the expressed Bt toxin or metabolites, if exposure is considered likely. 178

178 Publications on the issue of target pest and non- target lepidopterans differing in their sensitivity to
Bt toxins include Felke, Lorenz & Langenbruch (2002). J. Appl. Entomol. 126: 320-325; Wolt, Peterson,
Bystrak & Meade (2003). Environ. Entomol. 32(2) 237- 246; Dutton, Romeis & Bigler (2003). BioControl 27:
441-447; Fitt et al., 2004 Report for International Cotton Advisory Committee, pp 1-64; Fitt 2003; Proc. 3rd
World Cotton Research Conf, Cape Town, pp 371-381; Bern al, Griset & Gillogly (2002 ). J. Entomol. Sci. 37:
27-40. Publications showing species differences in Bt toxin sensitivities for lepidopteran, non-pest species
which could have regional- or country-specific conservation or heritage value. i.e. non-target herbivores
(butterflies) that could be affected by Bt crops include: Wraight, Zangerl, Carroll & Berembaum (2001). Proc.
Natl. Acad. Sci. USA 97: 7770-7773; Zangerl, McKenna, Wraight, Carroll, Ficarello, et al., (2001). Proc. Natl.
Acad. Sci. USA 98: 113-118; Sears, Hellmich, Stanley-Horn, Oberhauser, Pleasants, et al., (2001). Proc. Natl.
Acad. Sci. USA 98: 119317-42; Jesse & Obrycki (2000) Oecologia 125: 241-248; Jesse & Obrycki (2002). J.

449. Finally, as the issue of molecular characterisa tion has not been addressed by any expert, the
European Communities would like to offer the following brief comments:
450. The European Communities would refer to the in ternationally agreed principles regarding the
necessity of detailed molecular characterisation in a thorough safety assessment (see section III on
general and methodological issues, and the expert rep lies on Q 9, 111 et suiv). As regards the present
case, the National Biosafety Committee of Spain (NBC, EC-75/At.5) concluded that the issue of a
possible open reading frame (ORF) was not suffici ently addressed, and requested more information
on this, along with a verified sequence characterisation of the insert.
451. It should be noted that the dossier is dated July 6
th 2001, i.e. several months after the
publication of Windels et al. (2001). 179 The applicant, therefore, must have been familiar with the
issue of unintended rearrangements and the unin tended presence extra fragments. The original
dossier did not include essential data for the molecu lar characterization, such as a verified sequence
characterisation of the insert nor flanking sequences or a PCR method for detection of the GMO. The
CA's request for additional information was, theref ore, necessary to ensure that conclusions of the
safety assessment were valid.
Monsanto Roundup Ready corn (NK603)
C/ES/00/01 (EC chronology 76)

Question 37

Given the information before the Panel, including the notification (EC-76/At.1-2 and 27), was further
information regarding molecular characterisation, nutritional analysis, and environmental impact
requested by the lead CA (EC-76/At.6) necessary to ensure that conclusions of the safety assessment
were valid?

General comments

452. Dr. Nutti, Dr. Andow and Dr. Squire have pr ovided answers to this question in their
respective fields of expertise. Dr. Nutti's repl y seems to be based on an erroneous assumption due
perhaps to a misreading of the Spanish text. While disagreeing with some of the requests made by the
lead CA, Dr. Andow confirms the necessity of requesting information on gene escape/spillage. In that,
he is supported by Dr. Squire who adds further arguments on the relevance of such data for the case at
hand. Furthermore, the European Communities notes that no expert has provided comment on the
request for additional information on molecular charact erisation and will, therefore, offer some brief
comments itself.
Detailed comments

453. Dr. Nutti's
 comment relates to toxicity and nutritio nal studies in the context of determining
substantial equivalence. Her st atements acknowledge the importance of the animal studies that have
been carried out by the applicant on chicken broilers and rats. The point she makes is that no
additional studies beyond a broiler feeding study and a subchronic rat toxicity study are needed:
"Whatever studies were further requested by the lead CA in EC-76/At.6, such studies were not
necessary." Thus, she seems to assume that the lead CA had asked for additional studies, which would

Kansas Entomol. Soc. 75: 55-58; Jesse & Obrycki (2003) Agric. Environ. 97: 225-233; Hellmich, siegfried,
Sears, stanley-Horn, Daniels, et al., (2001). Proc. Natl. Acad. Sci. USA 98: 11925-30.
179 Windels P., Tavernier I., Depicker A., Van Bock staele E. & De Loose M. (2001). Characterisation
of the Roundup Ready soybean insert. Eur. Food Res. Technol. 213:107-112.

expand on the previous experiments with broilers a nd rats. However, the lead CA had not asked for
additional studies, but had merely repeat ed a request for submission of the reports of the chicken
broiler and rat studies (in Spanish):
pollos y los estudios de toxicidad de la pr oteína a dosis repetidas, tan pronto estén
disponibles," (EC76, At. 6, p. 2, par. 8, emphasis added).
454. These reports, which are the ones referred to as necessary by Dr. Nutti herself, were not
included in the original application. Following the above renewed request, they were submitted by
the applicant in September 2001 (Annexes 4 and 5 of EC76, At. 8_SCI, summary on pp. 20-31, title
pages on pp. 39-40).
455. Dr. Nutti, therefore, apart from the erroneous assumption she makes, is in complete
agreement with the lead CA in this instance.
456. Dr. Andow
 is rather critical of the need for certain data, on the basis of a request found in a
previous attachment (At5) which, as regards envi ronmental assessment and management plan, he
believes would only be needed in the context of cu ltivation, but not, as is the case here, where the
product in question is destined for import only. Two points should be made on this: First, it seems that
all three experts discuss different background docum ents and requests. Second, it may be safe to
assume, as does Dr. Andow, that the lead CA was an ticipating future cultiva tion of the product in
light of an impending application for cultivation. In light of this, even if one were to argue, that
requests relating to herbicide use, non target orga nisms etc. were outside the scope of the present
application, the request for information considered by Dr. Andow would have been relevant if the CA
had reasons to believe that the im ported grains could have escaped unintentionally and be maintained
into the environment, or more likely, that th ey wanted to better prepare the assessment of a
forthcoming application on deliberate release. That CA request does not state at any point that it is
"suspensive", and it may therefore simply be a nor mal cooperative exchange of information between
applicants and CA to prepare the ground ffor future or imminent applications.
457. In any event, Dr. Andow suggests that accidental spillage should be the focus of the
information provided by the applicant: "Some inform ation is necessary to consider how gene escape
can occur either during processing, storage or trans port, but detailed information is not necessary."
Dr. Squire's reply supports and reinforces this view for the particular case at hand (Spain): "On the
more specific question of whether GMHT maize persis ts (accidental germination), it is justified to
query the original statement since emergence has been recorded in some southern European areas."
458. Finally, as regards the request for additional information on molecular characterisation, on
which no expert has commented, the European Co mmunities would like to offer the following brief
comments:
459. The European Communities would refer to the in ternationally agreed principles regarding the
necessity of detailed molecular characterisation in a thorough safety assessment (see section III on
general and methodological issues, and the expert rep lies on Q 9, 111 et suiv). In the present case, it
should be noted that the notification is dated January 2 nd 2001. The applicant, therefore, was already
familiar with the contents of Windels et al. (2001) 180 and the issues of molecular characterisation of
inserts, flanking sequences and possible rearrangement s and extra fragments. According to the SNIF

180 Windels P., Tavernier I., Depicker A., Van Bock staele E. & De Loose M. (2001). Characterisation
of the Roundup Ready soybean insert. Eur. Food Res. Technol. 213:107-112.

(EC-76/At.2) the notification did not include verified sequence data for the full insert, only for the
flanking sequences and the 5' and 3' ends of the insert. The Spanish CA's request pertains to these
data ("la secuencia genética completa introducida, incluidas las secuencias flanqueantes"), and was,
therefore, necessary to complete the risk analysis, and in particular to enable the development of
appropriate detection methods. The applicant partially provided the data on September 5
th 2001
(EC-76/At.7-8), (but not the verified sequence data for the complete insert).
Question 38

Given the information before the Panel, including the notification and letter from Monsanto providing
additional information (EC-76/At.7-9), was additio nal information necessary regarding molecular
composition and environmental impacts associated with accidental germination requested by the lead
CA (EC-76/7-9 and 10) necessary to ensure that conclusions of the safety assessment were valid?

General comments

460. Dr. Andow and Dr. Squire both comment on this question in addressing the issue of
environmental impact of accidental dissemination or germination. This issue was already addressed
by both experts in question 37, where both conf irmed the necessity of a request for additional
information on this. The present comments add to the debate on the specificity of such a request.
Although perhaps generally more critical than Dr. Squire on the question of specificity, Dr. Andow
supports the lead CA's request as "necessary to ensure that conclusions of the safety assessment are
valid."
461. Furthermore, the European Communities notes that no expert has provided comment on the
request for additional information on molecular ch aracterisation. The Eu ropean Communities would
refer to the internationally agreed principles regarding the necessity of detailed molecular
characterisation in a thorough safety assessment (see section III on general and methodological issues,
and the expert replies on Q 9, 111 and following).
462. On October 10
th 2001 (EC-76/At.10) the request of the CA was for the application of PCR to
detect small fragments, and for the application of RT-PCR to detect possible transcripts of a possible
unintended open reading frame (ORF). The notifie r provides more data concerning the possible ORF
in EC-76/At.12. These data clearly indicate that the use of RT-PCR improved the sensitivity of the
molecular characterisation in comparison to the Nort hern blot data provided in the notification. A
previously undetected transcript was detected and characterised with RT-PCR, although this
apparently did not derive from the possible ORF. Therefore, although the notifier concludes that the
conclusion in relation to safety provided in the notifi cation is not altered, one can conclude that the
lead CAs request for additional information at this point was necessary to ensure that conclusions of
the safety assessment were valid.
Question 39

Given the information before the Panel, includi ng the notification and additional letter from
Monsanto providing additional in formation (referenced above and EC-76/At.11-12), was additional
information regarding molecular characterizati on and toxicology requested by the lead CA
(EC-76/At.14) necessary to ensure that conclusions of the safety assessment were valid?

General comments

463. Dr. Nutti and Dr. Squire have commented on parts of this question in their respective fields of
expertise. Dr. Nutti addresses the issue of safety marg ins in feeding studies. As she bases herself on

a document which she cannot have seen (as it has not been submitted to the Panel) her reply must be
dismissed. Dr. Squire restates his general reply to questions 37 through 39 with which the European
Communities is generally in agreement.
Detailed comments

464. Dr. Nutti refers to the lead CA's request for clarification on the titles of tables 2 and 3 on p. 24
concluding that that request was not necessary. Ho wever, the appendix that the lead CA is referring
to is not part of the documents submitted to the Pane l. EC 76 Att. 12 SCI only contains the title page
of the appendix. It is therefore not known to which details the request of the lead CA pertains.
Dr. Nutti's conclusion that the request was not necessary, thus, has no foundation.
465. In general, safety margins for animal feeding, i.e. the ratio between the minimum level where
adverse effects occur and the actual intake, are impo rtant in assessing the risks associated with the
intake of a potentially noxious compound. Prefer ably the safety margin is way above 1, in other
words: the intake of a noxious compound should be way below the minimum dose at which it would
exert adverse effects. This information was therefor e relevant to risk assessors in the interpretation of
toxicity data and is addressed, for example, by Codex guideline
181, paragraph 35:
The safety assessment should take into account the chemical nature and function of
the newly expressed substance and identify th e concentration of the substance in the
edible parts of the recombinant-DNA plant, including variations and mean values.
Current dietary exposure and possible effects on population sub-groups should also
be considered
466. In addition, paragraph 10 of the Codex guideline also addresses safety margins, as follows:
It is therefore relatively straightforward to feed such compounds to animals at a range
of doses some several orders of magnitude greater than the expected human exposure
levels, in order to identify any potential adverse health effects of importance to
humans. In this way, it is possible, in most cases, to estimate levels of exposure at
which adverse effects are not observed and to set safe intake levels by the application
of appropriate safety factors.
182
467. The same reasoning as for human food derived from GM crops (Codex) applies to animal
feed.
468. Furthermore, as identified in EC 76 Att. 27, th ere were detected chang es (two nucleotides) in
the CP4EPSPS, and CP4EPSPS L214 P genes, which con sequently coded for two new proteins which
had not been assessed before. As a consequence, ev en if it is judged a cautious approach, the CA
request proved fully justified a posteriori , in light of the subsequent information provided by the
applicant.

181 Codex Alimentarius, 2003. Codex Principles and Guidelines on Foods Derived from Biotechnology.
Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture
Organization, Rome. ftp://ftp.fao.org/codex/standard/en/CodexTextsBiotechFoods.pdf

Question 39bis

Given the information before the Panel, includi ng the notification and additional letter from
Monsanto providing additional information (previ ously referenced and EC-76/At.11-12), was
additional information regarding allergenicity studies and PCR tests requested by Austria
(EC-76/At.44) necessary or useful to ensure that conclusions of the safety assessment were valid?

General comments

469. Dr. Nutti simply concludes that the requ est was not necessary without providing any
explanation. Her reply, thus, is unfounded and must be dismissed. In making their request, in fact,
the Austrian authorities had followed the reco mmendations of the latest FAO/WHO Expert
Consultation on that issue at the time. The request was therefore relevant and necessary.
Detailed comments

470. The request for additional studies on allergenicity came in addition to the simulated stomach
degradation studies and computer-aided comparisons with allergens that had already been provided by
the applicant. This request had been made by Austria and addressed two issues (EC76, At. 44_transl):
A request for additional screening of the transgenic proteins for binding to sera from
allergy patients, and
Another request for testing potential changes in the intrinsic allergenicity of maize
caused by the genetic modification, using whole plant extracts for these studies
471. The request for this information was dated March 21st, 2003 (EC76, At. 44). At that moment,
an FAO/WHO Expert Consultation on the Allergen icity of Genetically Modified Foods had been
concluded.
183 This consultation provided input for the establishment of the pertinent Annex on
allergenicity testing in the Codex guideline for safety assessment of foods derived from GM crops, 184
which was published on July 9th, 2003.
472. The FAO/WHO Expert Consultation devised a decision tree approach for the testing of
potential allergenicity of GM foods, in which serum screening was one of the steps that had to be
followed. Serum screening could be either specific (if the foreign gene was from an allergenic source,
and therefore sera directed against this allergen s hould be used) or targeted (if the gene was from a
source unknown to be allergenic, and therefore sera ag ainst broadly related allergens should be used).
Within this approach, a targeted serum screening s hould have been carried out with the transgenic
EPSPS proteins from maize NK603. Austria's questi on for the serum binding tests was therefore
relevant at that time.
473. In addition, potential alterations of intrinsic al lergens (which were already present in the GM
organism before it became modified) should be considered during the comparative safety assessment
(also called "substantial equivalen ce"). This is because the geneti c modification might unintendedly

183 FAO/WHO, 2001. Joint FAO/WHO expert consultation on foods derived from biotechnology -
allergenicity of genetically modified foods - Rome, 22 - 25 January 2001. Food and Agriculture Organisation of
the United Nations, Rome.
http://www.who.int/foodsafety/publications/biotech/en/ec_jan2001.pdf
184 Codex Alimentarius, 2003. Codex Principles and Guidelines on Foods Derived from Biotechnology.
Codex Alimentarius Commission, Joint FAO/WHO Food Standards Programme, Food and Agriculture
Organization, Rome. Available at ftp://ftp.fao.org/codex/standard/en/CodexTextsBiotechFoods.pdf

have changed this intrinsic allergenicity of the plan t. Intrinsic allergens ar e mentioned, for example,
in the Codex guideline section dealing with compositional analysis of key components, as footnote 5
to paragraph 44:
"Key toxicants are those toxicologically significant compounds known to be
inherently present in the plant, such as those compounds whose toxic potency and
level may be significant to health (e.g. sola nine in potatoes if the level is increased,
selenium in wheat) and allergens
."
474. The experiments proposed by Austria for testin g the allergenicity of whole plant extracts
included binding tests with sera against a range of plants, as well as sensibilization studies in animals.
Both types of tests are in accordance with the decision tree approach recommended by the FAO/WHO
Expert Consultation {FAO/WHO, 2001} and are also mentioned in the Codex guidelines as methods
and tools that may be considered as sci entific knowledge and technology evolves. 185 Austria's
question for these tests for altered intrinsic allergenicity was therefore relevant at that time.
475. In EC76, At. 44, Austria also requested availab ility of the necessary data and material for an
analytical method, including the necessary PCR prim er sequences, and the reference material. This
request appears fully justified and necessary, as no detection method, specific for that transformation
event, had been made available at that time by the applicant.
Monsanto Roundup Ready corn (GA 21)
C/ES/98/01 (EC chronology 78), C/GB/97/M3/2 (EC chronology 85)

Question 40

Given the information before the Panel, including the application (EC-85/At.25-26), questions by
Denmark (EC-85/At.32) and responses to these qu estions (EC-85/At.41), was the additional
information requested by Denmark (EC-85/At.42) n ecessary to ensure that conclusions of the safety
assessment were valid?

General comments

476. Dr. Nutti only replies to one aspect of the Danish request for additional information, namely
the safety of the GM crop for animals, not, however, to the safety of the herbicide applied to maize
GA21. Her reply is based generally on a n on-comprehensive approach to safety based on
compositional analysis and animal toxicity tests with purified proteins (see also the comments on
broader risk assessment in the section on general and methodological issues, as well as comments on
Q111 to 113) and more specifically on a limited and misleading citation of the Codex Guidelines.
Detailed comments

477. The Danish request comprises two aspects, namely the safety of the GM crop for animals, and
the safety of the herbicide used on the GM crop ( i.e. feeding of herbicide-treated GM maize to
animals and measurement of transfer of herbicides to edible animal products, in this case milk of dairy
cattle). Dr. Nutti only addresses the first, but not the second aspect.
478. As regards the first aspect, Dr. Nutti states th at "[a]ccording to the Codex Alimentarius
Guideline for the Conduct of Food Safety Asse ssment of Foods Derived from Recombinant-DNA

185 Codex Alimentarius, 2003; Annex on "Assessment of Potential Allergenicity, para. 17

Plants [...], additional animal testing will be re quired if the composition is not comparable to
conventional foods, which is not the case of Monsanto's event corn GA 21."
479. Several points need to be made. First, Dr . Nutti's general approach to safety based on
compositional analysis and animal toxicity test s with purified proteins is non-comprehensive
as
explained in the comments on broader risk assessment in the section on general and methodological
issues, as well as comments on Q111 to 113. In f act, the very paragraph Dr. Nutti is quoting (i.e.
paragraph 53 of the Codex Guidelines) also links the issue of insufficient data on other aspects ( e.g.
molecular characterisation) to the requirements of animal studies, a link which Dr. Nutti herself does
not make. Thus paragraph 53 recommends that
If the characterization of the food indicates th at the available data are insufficient for
a thorough safety assessment, properly designed animal studies could be requested on
the whole foods.
480. Based on this recommendation and the consid erations provided belo w, it would still be
relevant to ask for additional animal feeding studies.
481. Second, it should be pointed out that the Codex Guidelines concern food safety, but not feed
safety. Thus, they do not apply directly to the issue at hand.
482. Third, contrary to what Dr. Nutti states, the C odex Guidelines, if applies by analogy, would
support a request for additional animal testing.
483. Cattle and pigs belong to the target animals of feed products derived from maize, such as
maize gluten. These animals therefore constitute "consumer subgroups" with a different physiology
than that of the broiler chicken previously used by the applicant for nutritional testing. The Codex
guidelines, when considering human food applications of GM crops (and not animal feed), address the
issue of different physiologies of subgroups. The fo llowing sentence, for example, has been extracted
from paragraph 49 of the Codex guideline section dealing with nutritional modifications:
Attention should be paid to the particular physiological characteristics and metabolic
requirements of specific population groups such as infants, children, pregnant and
lactating women, the elderly and those with chronic diseases or compromised
immune systems.
186
484. Whereas this paragraph deals with human subpopul ations, it can be envisioned that the same
applies also to animals with different physiologi es in the case of GM crops used for animal feed
purposes.
485. In addition, the genetically modified protein introduced by the applicant, the mutant maize
EPSPS protein, was different from the bacterial CP4 EPSPS protein previously introduced into other
GM crops. This GM crop would therefore be the fi rst crop with this particular modification to be
marketed within the EU. The concept of "familiari ty" would therefore not be applicable to maize
GA21.
486. Furthermore, an OECD document reviewing animal feeding studies, which was published in
2003, recommends nutritional testing with certain an imals in case it is indicated that unintended

effects might have occurred.
187 This includes, for example, the fast-growing broiler chicken, which
are considered to be most sensitive to alterations in nutritional value. Broile rs had already been used
by the applicant when the Danish CA made its re quest. The OECD document also mentions the use
of other species, such as lactating dairy cattle, of which the milk production can serve as a substitute
for growth rate.
487. Taken all these considerations (different ph ysiology, non applicability of "familiarity", and
usefulness of animal models) together, contrary to Dr. Nutti's assertions the data requested by the
Danish authorities concerning additional animal f eeding studies for no treated event maize would
have provided relevant information.
488. As regards the second aspect, na mely the safety of the herbic ide applied to maize GA21,
Dr. Nutti does not address it at all. The European Communities offers the following brief comments:
489. With this second issue, the Danish author ities address potential risks for human health:
Herbicide residues may remain in products of animals fed with herbicide treated GM crop (e.g. milk).
490. The Codex Guidelines consider this issue in paragraph 54 (Potential accumulation of
substances significant to human health):
Some recombinant-DNA plants may exhibit tr aits (e.g. herbicide tolerance) which
may indirectly result in the potential for accumulation of pesticide residues, altered
metabolites of such residues, toxic metabo lites, contaminants, or other substances
which may be relevant to human health . The safety assessment should take this
potential for accumulation into account. Conventional procedures for establishing the
safety of such compounds (e.g., procedures for assessing the human safety of
chemicals) should be applied.
188
491. The questions are therefore relevant from the point of view of safety of the GM crop and any
pesticides that might accumulate in it.
Question 40bis

Given the information before the Panel, including the first whole food study and agronomic
performance tests (attached, co ver letter provided in EC 78/85/At. 19), was a second animal whole
food study requested by Denmark, Austria and Italy (EC-78/85 /At. 67, 40 and 72) necessary or useful
to identify potential adverse effects that had not been previously identified?

General comments

492. Similar to her reply on question 40, Dr. Nu tti's reply is based on a non-comprehensive
approach to safety based on compositional analysis and animal toxicity tests with purified proteins
(see also comments on broader risk assessment in the section on general and methodological issues, as
well as comments on Q111 to 113) as well as limited reference to the Codex Guidelines. The reply,
therefore, should be dismissed.

187 OECD (2003) Considerations for the Safety Assessment of Animal Feedstuffs derived from
Genetically Modified Plants. Series on the Safety of Novel Foods and Feeds, No. 9. Organization for Economic
Cooperation and Development, Paris, 46 pp. Available at
http://www.olis.oecd.org/olis/2003doc.nsf/LinkTo/env-jm-mono(2003)10

Detailed comments

493. Dr. Nutti relies on paragraph 53 of the Codex Guidelines when stating that "my understanding
is that additional animal feeding studies may be warranted for GM foods if changes in the
biovailability of the nutrients are expected or if th e composition of the GM food is not comparable to
conventional food, and this was not the case of maize GA21."
494. This conclusion is overlooking important issues in at least two respects. First, as set out in the
comments on broader risk assessment in the secti on on general and methodological issues, as well as
in the comments on Q111 to 113, Dr. Nutti's approach to compositional analysis is generally non-
comprehensive. Indeed, the very paragraph, Dr . Nutti is quoting (i.e. paragraph 53 of the Codex
Guidelines) also links the issue of of insufficient data on other aspects ( e.g. molecular
characterisation) to the requirements of animal feeding studies, a link which Dr. Nutti herself does not
make. Thus paragraph 53 recommends that
If the characterization of the food indicates th at the available data are insufficient for
a thorough safety assessment, properly designed animal studies could be requested on
the whole foods.
495. Based on this recommendation and the consid erations provided belo w, it would still be
relevant to ask for additional animal feeding studies.
496. Second, as pointed already, Dr. Nutti cites the Codex guideline for foods derived from GM
crops, whereas the application at stake pertains to animal feed purposes (and incidental human
consumption). That said, if one is to draw elem ents from these guidelines by analogy, the following
consideration apply:
497. The target animals of feed products derived from maize, such as maize gluten, include, for
example, cattle, pigs, and poultry. These animal s therefore constitute "c onsumer subgroups", which
may have a different physiology than that of the broiler chicken previously used by the applicant for
nutritional testing. The Codex gui delines (in consider human food a pplications of GM crops and not
animal feed), address the issue of different physio logies of subgroups. The following sentence, for
example, has been extracted from paragraph 49 of the Codex guideline section dealing with
nutritional modifications:
Attention should be paid to the particular physiological characteristics and metabolic
requirements of specific population groups such as infants, children, pregnant and
lactating women, the elderly and those with chronic diseases or compromised
immune systems."
189
498. Whereas this paragraph deals with human subpopul ations, it can be envisioned that the same
applies also to animals with different physiologi es in the case of GM crops used for animal feed
purposes.
499. In addition, an OECD document reviewing anim al feeding studies, which appeared after the
request had been done, recommends nutritional testi ng with animals showing high production, which
are considered to be most sensitive to alte rations in nutritional value of the GM crops. 190 T h i s

190 OECD (2003) Considerations for the Safety Assessment of Animal Feedstuffs derived from
Genetically Modified Plants. Series on the Safety of Novel Foods and Feeds, No. 9. Organization for Economic
Cooperation and Development, Paris, 46 pp..

includes, for example, the rapidly-growing broile r chicken, which has alre ady been used by the
applicant. Also lactating dairy cattle, of which milk production and characteristics can be measured,
may serve as a model, in which case milk production would substitute for growth rate.
500. Furthermore, the genetically modified protein in troduced by the applicant, the mutant maize
EPSPS protein, was different from the bacterial CP4 EPSPS protein previously introduced into other
GM crops. This GM crop would therefore be the fi rst crop with this particular modification to be
marketed within the EU. The concept of "familiari ty" would therefore not be applicable to maize
GA21.
501. In light of these considerations, contrary to Dr. Nutti's conclusi on, the data requested by the
three Member States' CAs would have provided rele vant information to assure the safety of maize
GA21.
Question 41

Given the information before the Panel, including the notification (EC-87/At.1), additional letters
from Monsanto providing additional information (EC-87/At.3, 7, CBI part in 8, and 14) and the
conclusion of the Commission on Genetic Mod ification that this pr oduct did not present
environmental risks (EC-87/At.8-9), was additional in formation regarding data on the composition of
high-oleic soybeans and the alteration in the protein profile of this product requested by the lead CA
(EC-87/At.11, 13, and 15) necessary to ensure that conclusions of the safety assessment were valid?

General comments

502. Dr. Nutti and Dr. Squire commented on this qu estion. Given that Dr. Nutti agrees that the
request for additional information was necessary, th e European Communities will at this stage refrain
from commenting on this issue. As regards Dr. Squire, he states that "the requests for further
information (EC-87, At 11, 13, 15) appear to be mainly about safety as a feed." This concurs with the
lead CA's correspondence specifically mentioning th at the request pertained to the safety assessment
for animal feed purposes.
Question 42

Given the information before the Panel, incl uding the notification (EC-88/At.1) and additional
information provided by the notifier (EC-88/At.10- 11), was the information regarding allergenicity,
molecular characterisation, and gene transfer in digestive tracts requested by the lead CA
(EC-88/At.12) necessary to ensure that conclusions of the safety assessment were valid?

General comments

503. Dr. Nutti does not reply to the question. The qu estion is about the lead CA's very specific
request for additional information contai ned in question contained in EC 88 Att. 12. Dr. Nutti instead
offers (a less relevant) analysis on the lead CA's request for additional information contained in EC 88
Att. 9, to which the applicant provided answers in EC 88 Att. 11. Her comments, therefore, must be
dismissed. The European Communities will offer a brief reply to the actual question.

http://www.olis.oecd.org/olis/2003doc.nsf/LinkTo/env-jm-mono(2003)10

Detailed comments

504. Question 42 addressed at the Panel Experts pertained to the questions posed by the lead CA in
EC88, Att. 12. The request for add itional information contained in that attachment (letter from lead
CA of 01. 10.1999) very specifically addresses the risk to humans and animals of potential survival of
GM material during digestion in ruminants and sub sequent transfer to intestinal cells. Dr. Nutti,
however, addresses the questions to which the applicant has already provided answers in EC88, At. 11
and not the questions in EC88, At. 12. Her analys is on the answers in Att. 11, moreover, is non-
comprehensive, as she only considers refined suga r as a product for food and animal feed, whereas
by-products of sugar production, such as sugar beet pulp, are also used for animal feeding.
505. As regards the actual request for additional info rmation, horizontal gene transfer from GM
food and feed in digestive tracts, among others with regard to the use of antibiotic resistance genes
(see comments on Q1 and 2), has been debated in fo rums organized by international organizations
since 1990.
191 The same pertains to the digestibility of proteins derived from the expression of
transgenes.192 Recent publications show that a fraction of transgenic proteins and DNA in GM crops,
such as the Cry1Ab protein and its gene in GM maize, may survive the intestinal passage after
consumption of these GM crops by target animals in vivo .193 Therefore, consideration of the issue

191 IFBC (1990) Biotechnologies and food: Assuring the safety of foods produced by genetic
modification. Regulatory Toxicology and Pharmacology 12: S1-S196;FAO/WHO (1991) Strategies for
Assessing the Safety of Foods Produced by Biotechnology, Report of A Joint FAO/WHO Consultation. World
Health Organisation, Geneva. http ://www.who.int/foodsafety/publications/biotech/1990/en/; FAO/WHO (1996)
Biotechnology and Food Safety. Report of a Joint FAO/WHO Consultation, Rome, Italy, 30 September – 4
October 1996. FAO Food and Nutrition Paper 61. Food and Agriculture Organisation of the United Nations,
Rome. http://www.fao.org/es/ESN/food/pdf/biotechnology.pdf ; FAO/WHO (2000) Safety Aspects of
Genetically Modified Foods of Plant Origin. Report of a Joint FAO/WHO Expert Consultation on Foods
Derived from Biotechnology, Geneva, Switzerland, 29 Ma y - 2 June 2000. Food and Agriculture Organisation
of the United Nations, Rome. ftp://ftp.fao.org/es/esn/f ood/gmreport.pdf ; OECD (1993) Safety Evaluation of
Foods Derived by Modern Biotechnology. Organisation fo r Economic Co-operation and Development, Paris.
http://www.oecd.org/dataoecd/57/3/1946129.pdf ; OECD (1996) Food Safety Evaluation. Organisation for
Economic Co-operation and Development, Paris.; OECD (1998) Report of the OECD Workshop on the
Toxicological and Nutritional Testing of Novel Foods, Aussois, France, 5-8 March, 1997. Organisation for
Economic Co-operation and Development, Paris. http://www.olis.oecd.org/olis/1998doc.nsf/LinkTo/sg-
icgb(98)1-final
192 IFBC (1990) Biotechnologies and food: Assuring the safety of foods produced by genetic
modification. Regulatory Toxicology and Pharmacology 12: S1-S196; FAO/WHO (1995) Application of the
Principles of Substantial Equivalence to the Safety Evaluation of Foods or Food Components from Plants
Derived by Modern Biotechnology, Report of A WHO Workshop. World Health Organisation, Geneva;
FAO/WHO (1996) Biotechnology and Food Safety. Report of a Joint FAO/WHO Consultation, Rome, Italy, 30
September – 4 October 1996. FAO Food and Nutrition Paper 61. Food and Agriculture Organisation of the
United Nations, Rome. http://www.fao.org/es/ESN/ food/pdf/biotechnology.pdf.; FAO/WHO (2001)
Allergenicity of Genetically Modified Foods. Report of a Joint FAO/WHO Expert Consultation on Foods
Derived from Biotechnology. Rome, 22 - 25 January 2001. Food and Agriculture Organisation of the United
Nations, Rome. ftp://ftp.fao.org/es/esn/food/allergygm.pdf
193 Chowdhury, E.H., Shimada, N., Murata, H., Mikami, O., Sultana, P., Miyazaki, S., Yoshioka, M.,
Yamanaka, N., Hirai, N., Nakajima, Y. (2003) Detection of Cry1Ab protein in gastrointestinal contents but not
visceral organs of genetically modified Bt11-fed calves. Veterinary and Human Toxicology 45: 72-75;
Chowdhury, E.H., Mikami, O., Murata, H., Sultana, P., Shimada, N., Yoshioka, M., Guruge, K.S., Yamamoto,
S., Miyazaki, S., Yamanaka, N., Nakajima, Y. (2004) Fate of maize intrinsic and r ecombinant genes in calves
fed genetically modified maize Bt11. Journal of Food Protection 67: 365-370; Einspanier, R., Lutz, B., Rief, S.,
Berezina, O., Zverlov, V., Schwarz, W., Mayer, J. (2 004) Tracing residual recombinant feed molecules during
digestion and rumen bacterial diversity in cattle fed tr ansgene maize. European Food Research and Technology
218: 269-273.

raised by the Belgian lead CA in EC88, At. 12, w as highly relevant for the assessment of animal feed
safety of GM crops including Roundup Ready sugar beet.
Question 43

Given the information before the Panel, includi ng the notification and additional information
provided by the notifier (EC-88/At.10, 11, 13, 14, 15, 16, and 18-26), was the information regarding
molecular characterisation and allergenicity of event '77' requested by the lead CA (EC-88/At.27-28)
necessary to ensure that conclusions of the safety assessment were valid?

General comments

506. Dr. Nutti comments only on the issue of allergenic ity. Her statement that "[the information
previously provided by the applicant] on this item covered all the points recommended by WHO/FAO
Expert Consultation 1996, and the Codex Alimentarius Guidelines for the Conduct of Food Safety
Assessment of Foods Derived from Recombinan t-DNA Plants (CAC/GL 45-2003) and its Annex on
Possible Allergenicity Assessment " is totally unsubstantiated and misleading as it does not explain
whether the request of additional data by the l ead CA was scientifically founded. Conveniently,
Dr. Nutti omits to mention that also the lead CA's request is entirely in accordance with Codex and
FAO/WHO recommendations.
507. Furthermore, the European Communities notes that no expert has provided comment on the
request for additional information on molecular char acterisation. The European Communities refers
to the internationally agreed principles regarding the necessity of detailed molecular characterisation
in a thorough safety assessment (see section III on ge neral and methodological issues, and the expert
replies on Q 9, 111 and following).
Detailed comments

508. As she has not provided any explanation, one can only assume that Dr. Nutti's general
approach to allergenicity assessments is based on the same assumptions the applicant had made,
namely that there are two common ch aracteristics shared by most allergens: first, food allergens are
generally abundant in food, usually greater than 1% of the total protein and, second, food allergens are
stable to gastric digestion, and a specific test fo r gastric stability has been established and validated
using a broad array of known food allergens.
509. In this view the assessment of allergen icity is possible solely on the basis of in silico and in
vitro parameters, such as sequence homology and stability.
510. However, this view is not adhered to by many ot her scientists who take the position that more
tests, especially using sera from patients and animal models, are needed to assess allergenicity.
511. The answers and requests of the lead CA refl ect this debate among sc ientists in a very
scientific way: According to the lead CA the method of homology comparison and sequence
comparison as well as stability testing alone is not relevant in all circumstances as, for example, three
dimensional epitope characteristics and instability of several known allergens need to be considered.
The CA refers to the need to identify possible cross-reactivities using methods such as "RAST".
512. The scientific debate about the assessment of a llergenicity has been discussed controversially
in international organisations such as OECD for a long time and concluded in increasingly
comprehensive models in FAO/WHA expert consultations and CODEX guidelines. The CA reflected

the consequences of these discussions accordingly. The Codex guideline CAC/GL 45-2003, Annex
on the Assessment of Possible Allergenicity explains that
[…]sequence homology searches have certain limitations. In particular, comparisons
are limited to the sequences of known allerg ens in publicly available databases and
the scientific literature. There are also limita tions in the ability of such comparisons
to detect non-contiguous epitopes capable of binding themselves specifically with IgE
antibodies. A negative sequence homology result indicates that a newly expressed
protein is not a known allergen and is unlikely to be cross-reactive to known
allergens. A result indicating absence of significant sequence homology should be
considered along with the other data outlined under this strategy in assessing the
allergenic potential of newly expressed prot eins. Further studies should be conducted
as appropriate.
194
513. Similarly, the 2001 FAO/WHO Expert Consultatio n on Allergenicity of Foods derived from
Biotechnology (Dr. Nutti refers to an older one of 1996) concluded that:
When the expressed protein comes from a sour ce that is not known to be allergenic,
the FAO/WHO 2001 decision tree focuses on (1) sequence homology to known
allergens (food and environmental), (2) targ eted serum screening for cross-reactivity
with sera from patients allergic to materials that are broadly related to the source
material for the gene, (3) pepsin resistan ce and (4) immunogenicity testing in animal
models (Annex 4). In this situation the search for homologous allergens is based on
two procedures.The first step is a database search for an allergen with a homologous
amino acidsequence, according to the principles described in Section 6.1. If this
search reveals a level of homology with a known allergen that suggests a potential for
cross-reactivity, the expressed protein is cons idered to be an allergenic risk. No
further evaluation for allergenicity would typically be necessary.The second step is
conducted if no such homologous protein is found. In such cases,cross-reactivity is
tested with a panel of serum samples that contain high levels of IgE antibodieswith a
specificity that is broadly related to the gene source (Section 6.3).
195
514. Finally, the latest FAO/WHO consultation on GM foods (2003) agrees that
It has been recognized that there is no singl e parameter that can predict the allergenic
potential of a substance. A strategy to asses s allergenicity of biotechnology products
has been formulated (FAO/WHO, 2001; Codex Alimentarius Commission, 2003),
which relies on the parameters: source of the gene, sequence homology, serum testing
of patients known to be allergenic to the source organism or to sources distantly
related, pepsin resistance, the prevalen ce of the trait and assessment using animal
models. The Consultation recommended that ad ditional efforts should be directed to
the further development and validation of m odels and that there is a need to improve
the accessibility and interconnectivity of existing databases or to establish a
centralized database on allergenic linear and conformational epitopes and tools for
screening transgenes for allergenic potential.
196

194 Section 3.2, Paras. 10 and 11.
195 2001 FAO/WHO Expert Consultation on Allergenicity of Foods derived from Biotechnology,
http://www.who.int/foodsafety/publications/biotech/ec_jan2001/en/, at para. 5.4.
196 FAO/WHO Consultation on Food from GM Animals, 2003,
http://www.fao.org/documents/show_cdr.asp?url_file=/DOCREP/006/Y5316E/y5316e01.htm, section 5.2.2.3.

515. In the present case, thus, whereas some data on sequence analysis and stability were provided
by the applicant, the request of the CA for further allergenicity tests reflected the scientific dispute for
a need of improved assessment methods as reflected in turn in the above documents. It is to be noted
that the Codex does not provide for a final harmoni sed and agreed guidance as regards the assessment
of allergenicity that would restrict the analysis to the two tests provided for by the applicant.
516. As regards additional informa tion on molecular characterisa tion, to which no expert has
replied, the European Communities would like to offer the following brief comments:
517. In the present case, the lead CA in its request (EC-88/At.27, November 16
th 2000) explained
very clearly the basis for the request for additional information: lack of quality of Southern blot
images and also an expressed concern regarding a fragment observed without sufficient explanation
about its nature. On the basis of the available info rmation by the date of the request, the requested
information regarding molecular characterisation was, therefore, necessary to ensure that conclusions
of the safety assessment were valid.
Question 44

Given the information before the Panel, incl uding the notification (EC-94/At.1-3), was the
information requested by the Netherlands(EC-94/At. 12) concerning molecular characterization, DNA
sequence analysis of the insertion event, analysis of pr otein levels, effect of glyphosphate treatment,
composition, toxicology and the request for a study on dairy cows necessary to ensure that
conclusions of the safety assessment were valid?

General comments

518. Dr. Nutti and Dr. Andow both comment on this question in their respective fields of expertise,
overlapping on questions 4 and 6, of the lead CA's request. The European Communities disagrees
with all of Dr. Nutti's conclusions, as they are e ither based on erroneous r eading of the documents
submitted (question 3) or actually inconclusive to the question (questions 4, 5) or unfounded
(question 6). The European Communities further di sagrees with one aspect of Dr. Andow's analysis
of question 6 of the lead CA's request.
519. Furthermore, as no one has offered comments on que stions 1 and 2 of the lead CA's request,
the European Communities will offer a brief explanation.
Detailed comments

520. Dr. Nutti's comments on question 3
 of the CA's request are based on an error as she takes the
applicant's reply to that request to be the data originally submitted.
521. Dr. Nutti concludes that the information was not necessary to ensure the validity of the safety
assessment, as "the explanation given by the notifier was acceptable, since in EC-94/ At 13 , the

Notte that even the present strategy based on a sequence prediction model established by FAO/WHO was shown
to be amenable to refinement by important groups (for example, Stadler and Stadler, Allergenicity prediction by
protein sequence, FASEB, J 2003, 17, 9, 1141-3; Kleter and Peijnenburg, Screening of transgenic proteins
expressed in transgenic food crops fo r the presence of short amino acid sequ ences identical to potential, IgE -
binding linear epitopes of allergens. BMC Struct Biol, 2002, 2, 1, 8; and Zorzet et al., Prediction of food protein
allergenicity: a bioinformatic learning systems approach, In Silico Biol, 2002, 2, 4, 525-534). Difficulties in the
concept are summarised in Jank and Haslberger, Improved evaluation of potential allergens in GM food, Trends
Biotechnol., 2003, 21, 6, 249-250).

notifier presents the results of second year study in order to test this assumption, and the results
confirmed that the expression levels were similar."
522. Indeed, the data presented by the applicant in At. 13 are precisely the missing data that the
CA had asked for. These data were submitted in 2002. The CA's request dates from 2000.
523. Implicitly, therefore, Dr. Nutti confirms that the request of the lead CA was justified and that
the additional information, thus, was necessary to reach a valid conclusion on this issue.
524. As regards question 4
 of the lead CA, Dr. Nutti and Dr. Andow address different aspects of
that question in their respective comments.
525. Dr. Nutti comments relate to the necessity of comparing herbicide sprayed and non herbicide-
sprayed GM maize. The lead CA had asked for such data in order to verify whether the herbicide-
sprays could have influenced the results of com positional data. The applicant, in its original
application had provided some data that collectiv ely compared both sprayed and non-sprayed samples
with other groups of samples (compositional data EC94; At.2, pp. 76-186; request EC94, At.12).
526. Dr. Nutti in her comment mentions that the co mparison of GM with non GM crops usually
does not entail herbicide treatment of th e GM crop. She also refers to the post-hoc information
provided by the applicant (EC94, At.13) indicating that in other GM crops (GA 21 maize, Roundup
Ready soybean), no effect of herbicide treatment on compositional data had been noted. In addition,
she concludes that the data on both herbicide-sp rayed and non-sprayed had been provided and that
therefore the requested additional information was not necessary for the safety assessment.
527. However, the arguments provided by Dr. Nutti are actually in support of the lead CA's request
for a verification of the absence of an effect of glyphosate. Since data on both sprayed and non-
sprayed samples are already available, spraying constitutes a variable and the data can be compared to
check whether this variable cause s differences or not, as has been previously been done for other GM
crops.
528. This issue is also addressed by the Code x Guideline 2003 under the heading Compositional
Analysis of Key Components, in which the comparison of a GM crop with both its sprayed and non-
sprayed counterparts is recommended:
Analyses of concentrations of key components of the recombinant-DNA plant and,
especially those typical of food, should be compared with an equivalent analysis of a
conventional counterpart grown and harvest ed under the same conditions. In some
cases, a further comparison with the recombinant-DNA plant grown under its
expected agronomic conditions may need to be considered ( e.g. application of an
herbicide).
197
529. Treatment of a GM crop (MaisGard Roudup Ready using HT and Bt genes stacked) with
applied herbicide (glyphosate) could trigger metabol ic changes in the GM maize event. The effects
on plant metabolism under regional co nditions of the herbicide applie d to this GM crop event were
unknown at the time. Thus toxic effects on non target organisms likely to be exposed to the herbicide
treated GM maize event ( e.g. humans, dairy cows and a range of other herbivores consuming
glyphosate-treated Maisgard Roundup Ready) were impo ssible to predict without testing. Therefore,
comparative data on treated and untreated GM maize was required to assess such effects.

530. Dr. Andow's comment on question 4 relates to the method of the statistical analysis requested
by the lead CA. As Dr. Andow rightly points out, wi th the statistical design and analysis used by the
notifier, it would not be possible to estimate an RR effect independent of a glyphosate effect (see
Dr. Andow's comments at para. 44.07). The data provi ded thus, is inadequate to reveal interactions
between the GM crop, local environment, herbicid e treatment(s) and any non-target organisms likely
to be exposed to GM gene products, the applied herbicides and their metabo lites induced in the HT
crop after glyphosate treatment.
531. In conclusion the request by th e lead CA for an additional statistical analysis to verify
whether herbicide sprays may have caused additional changes in composition was therefore justifiable
and necessary to reach a conclusion on the safety of maize MON810xGA21.
532. In her comments on question 5
 in the lead CA's request, Dr. Nutti addresses two different
issues, namely the request for additional compar isons of hybrid MON810xGA21 with non GM lines
and the request for data on additional secondary metabolites and antinutrients.
533. As regards the request for additional comp arisons of hybrid MON810xGA21 with non GM
lines, Dr. Nutti refers to "different approaches" that can be used to perform comparisons based on the
compositional analysis on a hybrid GM. She takes th e view that the one opted for by the applicant,
i.e. to compare the hybrid only to its parental lin es (but not to conventional counterparts, providing
such a comparison has already b een carried out only between the parental lines and conventional
counterparts) is sufficient.
534. The lead CA, on the other hand, has taken the vi ew that a (direct) comparison of the hybrid
with conventional counterparts would also be required.
535. In fact, the issue of exact data requirements for conventional crosses of two parent GM lines
is still being discussed in and outside the EU. Co mparison of the different national legislations
pertaining to GM crops shows that there are differ ences with regard to the scientific understanding of
the needs for further risk assessments, and with rega rd to the obligation to notify conventional crosses
of GM lines or not.198 The issue is not specifically addressed by the Codex guideline.
536. The safety of the parent lines is important, because the same genetic modifications can be
present in the hybrid as in these GM parent lines. However, information on the parent lines alone
cannot provide full reassurance that the cross will be safe as well. For example, the introduced
genetic material and the products that ar e derived from this genetic material ( e.g. enzymes), may
interact with each other within the conventional cross of GM lines.
537. For example, new genes may participate in diffe rent steps in the same pathway (cascade) of
enzymatic reactions (such as, for example, the forma tion of a vitamin or plant fibre). In case these
genes are introduced into separate GM crops, which are subsequently crossed with each other, the
cross may exhibit features that are distinctive from the parental lines (for ex ample, this has been
observed by Pincon et al. (2001) in GM tree plants with modified production of precursors for cell
fibre components).199 Another example is that of "gene silenc ing" (inactivation of gene function) by

198 Kuiper, H.A., Kleter, G.A., Noteborn, H.P.J.M., Ko k, E.J. (2001) Assessmen t of the food safety
issues related to genetically modified foods. Plant Journal 27: 503-528.
http://www.blackwellpublishing.com/plantgm/tpj1119.pdf
199 Pincon, G., et al. (2001) Simultaneous down-regulation of caffeic/5-hydroxy ferulic acid-O-
methyltransferase I and cinnamoyl-co enzyme a reductase in the progeny from a cross between tobacco lines
homozygous for each transgene. Consequences for plant development and lignin synt hesis. Plant Physiology
126: 145-155.

bringing together genes with similar sequences fr om different parents within the same plant (as
observed, for example, by Charrier et al. (2000) and De Wilde et al. (2001)).
200 Interestingly, the
British CA asked for data on the homology of the tran sgenic constructs inserted into the parental GM
maize lines MON810 and GA21 (EC94, At. 10). This in formation would also have been particularly
relevant for the latter example of "gene silenc ing", for which there would be a likelihood of
occurrence in the crossed line MON810xGA21 in case of homology between the constructs.
538. In exhibit 94/013, answer to question 1, clause B, second last bullet point, the notifier states:
Finally, there are no known mechanisms by which two inserts at different locations
on different chromosomes could stimulate recombination in each other (if they do not
express proteins involved in recombination pathways).
539. While this statement is basically correct, it is also misleading because it gives the impression
that only recombination in the functional inserts is relevant for the risk assessment. However, in
plants the genes interact with other genes in sometimes very complex patterns involving genes
distantly located on the same or other chromosomes. It is therefore possible that recombination may
occur among or within genes at other loci, and that these genes may interact with an inserted gene.
Furthermore, the size of sequence motifs involved in recombination events may be short. It can not
be excluded that small random unint ended and undetected insertion events can be present in the
parental GM maize events MON 810 and GA21. To en sure that the hybrid GM maize is safe, it is
therefore necessary to test the hybrid separately, and to compare it not only with the GM parental
events but also with conventional counterparts.201
540. While this issue is not specifically addressed in the Codex Guideline 2003 some support for
the lead CA's approach can be found there as well. Thus, whereas in para.44 of the Guidelines it is
recommended that:
"The comparator(s) used in this assessment should ideally be the near isogenic
parental line"[emphasis added]202
541. It is also recommended that:
Analyses of concentrations of key components of the recombinant-DNA plant and,
especially those typical of food, should be compared with an equivalent analysis of a
conventional counterpart grown and harvest ed under the same conditions."
203
[emphasis added]
542. The Codex guideline defines a conventional counterpart as:

200 Charrier, B., Scollan, C., Ross,S., Zubko, E., Meyer, P. (2000) Co-silencing of homologous
transgenes in tobacco. Molecular Breeding 6: 407-419; De Wilde, C., Podevin, N., Windels, P., Depicker, A.
(2001) Silencing of antibody genes in plants with single-copy transgene inserts as a result of gene dosage
effects. Molecular Genetics and Genomics 265: 647-653.
201 See Wilson et al 2004; Technical report for EcoNexus; Freese and Schubert 2004 Biotechnology
and Genetic Engineering 21: 299-324; The Committee on Identifying and Assessing Unintended Effects of
Genetically Engineered foods on human health (2004). "Safety of Genetically Engineered Foods: Approaches to
assessing Unintended Health Effect s". Publishers: The National academic press, Washington, D.C. National
academy of Sciences (purchasable pdf download from www.national-academies.org).
203 Ibid.

a related organism/variety, its component s and/or products for which there is
experience of establishing safety based on common use as food.
204
543. In the present case, the GM parental lines cannot be considered "conve ntional counterparts"
with a history of safe use according to these standa rds, since at the moment of the request by the
Dutch lead CA, MON810 had only been approved for several years in the EU, and GA21 was still in
the approval procedure. Therefore, the comparison with the GM parental lines was not sufficient and
the request for a comparison of the hybrid MON810xGA21 with non-GM lines was justifiable.
544. More generally, data regarding the safety of th e biotech parent plants are obviously relevant
to the safety assessment of the hybrid plant produ cts. However, while relevant, they are not
sufficient.
545. Note also that this approach has recently b een confirmed from a molecular characterization
point of view by an US EPA-FIFRA SAP panel report 205 which addressed this general problem in the
context of a specific new product (Bt Cry1F/Cry1Ac). The US expert panel stated that:
A molecular characterization of the new product should show that the recombinant
traits/ sequences in the new product are iden tical to the insertion /traits in their
parental lines. In principle, the method of breeding the two parental lines could affect
the genetic characteristics of the inserted tra its. Therefore, evidence is needed that no
such effect has occurred. For these pr oducts, a risk assessment combining evidence
from parental lines and the product should be requested.
546. As already explained in the previous comments, it can not be excluded that unintended effects
may result from hybridisation between the two pare ntal GM events. The data requirements for
assessment of individual transformation events do not normally include provisions for experimental
data on the effect of hybridisation between the event under study and other GM events previously
authorised or under separate evaluation. This ma y be justifiable given the assumption that such
unintended hybrids will only occur in low frequenc y under field conditions, and consequently the
exposure from these hybrids to humans, animals and the environment will be low. On the contrary,
authorised hybrids will by default occur at high frequency and consequently the exposure will be
significantly higher. Consequently, the intended hybrid must be subject to separate evaluation
including additional data from appropriate feeding trials.
Dr. Nutti also mentions that the comparison with non-GM parental lines may sometimes be
problematic because they might not have been deve loped. In fact, the original study on the
compositional data (EC94, At.2, pp. 76-186) mentions that besides the hybrid MON810xGA21, the
parental GM lines MON810 and GA21, also non-GM control lines and commercial reference lines
were grown in the same locations (EC94, At. 2, p. 82). However, compositional data of these
additional non-GM lines have not been included in the statistical comparison by the applicant.
Therefore, Dr. Nutti's concern about the possible una vailability of non-GM material for a comparison
of MON810xGA21 with non-GM maize lines does not apply to this case.

205 SAP Report No. 2004-05. MINUTES of Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) Scientific Advisory Panel (S AP) Meeting, June 8-10, 2004, Arlington, Virginia. A Set of Scientific
Issues Being Considered by the Environmental Protection Agency Regarding: Product Characterization, Human
Health Risk, Ecological Risk, And Insect Resistance Management For Bacillus thuringiensis (Bt) Cotton
Products. 84 pages.

547. As regards the lead CA's request for da ta on additional secondary metabolites and
antinutrients Dr. Nutti's assessment is being contradi cted by the standard set out in Codex Guideline
CAC/GL 45-2003.
548. The lead CA requested data on five additiona l, secondary substances that could provide an
indication of potential unintended alterations of metabolic pathways. This would supplement the data
that had already been provided for the secondary substance phytic acid, which also acts as an
"antinutrient" since it interferes, for example with mineral uptake from the intestines.
549. Dr. Nutti concludes that this additional analys is would not have been necessary, since the
previous compositional data provide d by the applicant indicated that the pathways leading to these
substances had not been affected. For example, leve ls of phytic acid, which is formed from inositol
(one of the recommended substances), had not been altered in MON810xGA21 maize.
550. In the Codex Guideline CAC/GL 45-2003, this i ssue is dealt with under "Unintended effects"
and under "Potential accumulation of substances significant to human health".
551. Thus paragraph 15 of the Codex Guideline CAC/GL 45-2003 states that
Unintended effects may also result in the formation of new or changed patterns of
metabolites.
552. Paragraph 54 of the Codex Guideline states:
Some recombinant-DNA plants may exhibit tr aits (e.g. herbicide tolerance) which
may indirectly result in the potential for accumulation of pesticide residues, altered
metabolites of such residues, toxic metabo lites, contaminants, or other substances
which may be relevant to human health . The safety assessment should take this
potential for accumulation into account. Conventional procedures for establishing the
safety of such compounds (e.g., procedures for assessing the human safety of
chemicals) should be applied.
553. Based upon these standards, the request of the lead CA was relevant from the point of view of
safety of the GM crop and any plant secondary meta bolites that have been changed as an unintended
side-effect of the genetic modification.
206
554. It may be noted in passing that the applicant has also provided data on such plant secondary
metabolites in a study on another GM maize line.207

206 Specifically from an environmental point of view unusual crop phenotypes arising from unintended
genetic changes could have a significant impact on one or more ecological interactions of the particular GM
cultivar (event). This is because crop plants produce a multiplicity of bio-active compounds that are individually
or as a mixture associated with positive and negative nutritional qualities when consumed by non target
organisms associated with the crop in any particular region and local agro-ecosystem. Because of unknown or
unpredictable dose responses to bio-active compounds in plants and in general, "hormesis demands a reappraisal
of the way risks are assessed" (Calabrese and Baldwin, 2003; Toxicology rethinks its central belief. Nature 421,
691-692). The Codex Guidelines for GM foods includes analysis of unintended an d indirect effects on the
environment due to increasing evidence of unanticipated changes in biologically-active plant compounds arising
unexpectedly in transgenic crops. (e.g. Shewmaker et al., 1999; Saxena & Stotsky 2001; Birch et al., 2001;
Haslberger, 2003
207 Monsanto (2002) Safety Assessment of Roundup Ready Corn Event NK603. Monsanto Co, St.
Louis. www.monsanto.com/monsanto/content/sci_tech/prod_safety/roundup_corn/pss_NK603.pdf; Ridley,

555. Finally, as regards question 6
 of the lead CA's request, the European Communities disagrees
both with Dr. Nutti's and Dr. Andow's comment.
556. In question 6, the lead CA considered that the method to genetically modify the parental
maize may have caused the unintended insertion of additional fragments into the maize DNA. Shortly
before the lead CA's request, it had become know n that Roundup Ready soybeans (developed by the
same applicant, Monsanto), which had previously been approved in the EU for food and feed
purposes, but contained additional DNA fragments. Th ese previously undetected fragments had been
revealed by molecular techniques to sequence DNA inserts.
557. Dr. Nutti concludes that the information that ha d already been submitted by the applicant was
sufficient for the safety assessment. With regard to the toxicology studies, she refers to studies ( e.g.
acute toxicity, in vitro stomach fluid digestion) that have been performed with purified proteins and
not with the whole maize product. However, whol e feed/food studies should be performed in order to
assess the potential health consequences of unintended effects, such as those may have been caused
by the insertion of additional DNA fragments, beyo nd the intended modifications (e.g. transgenic
proteins) that have been tested for toxicity. (See also EC Comments on Question 44bis).
558. Dr. Andow acknowledges the lead CA's concer n about additional DNA fragments are "valid
concern," (Dr. Andow's reply, para. 44.10), but commen ts in this regard that "it would seem that the
notifier would have other alternatives to the toxicity test to address the concern of the Netherlands
CA." ( ibid.). However, Dr. Andow does not give the de tails of the alternative techniques that he
claims may address the concerns of The Nether lands. These methods should therefore provide
reassurance about additional fragments having been inserted or not.
559. In literature, other ways to detect unintende d effects are described, such as the use of
advanced analytical technologies called "profiling" or "metabolomics". These techniques are still
under development and have not been validated yet for routine appli cation in the safety assessment of
GM foods.208
560. As previously mentioned, there has already b een a post-market discovery of an unintended
effect, i.e. additional DNA fragments having been inserte d into GM herbicide-resistant (Roundup
Ready) soybean. In this case, the applicant (Mon santo) itself argued that these additional fragments
could not pose a risk, since no adverse effects had been observed in previously published animal
feeding studies with whole feed products derived from Roundup Ready soybeans.209
561. The lead CA made its request shortly after the new information surrounding the issue of
fragments in GM soybean had become public. For this reason, as well as those given above, it is
justifiable that the lead CA asked an additional an imal feeding experiment with the whole product as
additional reassurance for the absence of adverse e ffects that might have been caused by unintended
insertion of additional DNA fragments.

W.P., Sidhu, R.S., Pyla, P.D., Nemeth, M.A., Breeze, M.L., Astwood, J.D. (2002) Comparison of the nutritional
profile of glyphosate-tolerant corn event NK603 with th at of conventional corn (Zea mays L.). Journal of
Agricultural and Food Chemistry 50: 7235-7243.
208 Chassy, B., Hlywka, J.J.,. Kleter, G.A., Kok, E.J., Kuiper, H.A., McGloughlin, M., Munro, I.C.,
Phipps, R.H., Re, E.B., Reid, J.E. (2004) Nutritional an d Safety Assessments of Foods and Feeds Nutritionally
Improved through Biotechnology, Prepared by a Task Force of the ILSI International Food Biotechnology
Committee. Comprehensive Reviews in Food Science and Food Safety 3: 35-104.
http://www.ift.org/pdfs/crfsfs/crfsfsv3n2p0035-0104ms20040106.pdf
209 Monsanto (2000) Updated Molecular Characteri zation and Safety Assessment of Roundup Ready
Soybean Event 40-3-2. Monsanto Co. St. Louis, 20 http://archive.food.gov.uk/pdf_files/acnfp/summary.pdf

562. Finally, the European Communities would like to briefly comment on the lead CA's request
for additional information in questions 1 and 2
 of At. 12, on which none of the experts has
commented.
563. The European Communities would refer to the in ternationally agreed principles regarding the
necessity of detailed molecular characterisation in a thorough safety assessment (see section III on
general and methodological issues, a nd the expert replies on Q 9, 111 and following). In the present
case, it should be noted that the notifier applied only Southern blots to establish the number and
composition of insert(s). However, by the date of the notification (EC-94/At.1-3, February 29 th 2000)
DNA sequencing was a standard method that could be applied to verify the composition of the insert.
To request such information was therefore necessary and justified.
Question 44bis

Given the information before the Panel, including the first whole food study and agronomic
performance tests (EC 94/At.3, pp. 40), was a second animal whole food study (EC 94/At.12)
necessary or useful to identify potential adverse effects, including those associated with small random
DNA insertions, that had not been previously identified?

General comments

564. The question refers to question 6 of the lead CA in its request of July 2000 (At. 12), which
was already covered by and addressed in question 44. Dr. Nutti's comments are identical to her
comments in question 44. Therefore, the Europ ean Communities refers to its above comments under
question 44.
Question 44ter

In the context of this corn product which was pr oduced through conventional breeding from biotech
parents, is data regarding the safety of the biotech parent plants relevant to the safety assessment of
the hybrid plant products? Given the results of the risk assessment studies of the biotech parents, were
additional studies necessary or useful to ensure that th e conclusions of the safety assessment related
to the hybrid plants were valid?

General comments

565. The question can be linked to question 5 of the l ead CA in its request of July 2000 (At. 12),
which was already covered by and addressed in ques tion 44. Dr. Nutti refers back to her comments
on that issue in question 44 and so does the European Communities.
Question 45

Given the information before the P anel, were there scientific or tec hnical reasons which explain why
the process of validating the detection method took at least fourteen m onths (June 2002-August
2003)? (See EC-91/At.49-56)?

General comments

566. Dr. Nutti acknowledges that detection methodolog y is not her main area of expertise.
Question 45 turns in fact on an overall delay of a pproximately 14 months. No specific expertise on
detection methods is actually required to see that th e applicant took more than 9 months out of these
14 months to submit any information (methods or mate rials) whatsoever (EC-91, At. 50). That that

information could not even be used to start work on the EU side is something Dr. Nutti does not seem
to have identified. Her " impression, that there were no scientific or technical reasons which explain
why the process of validating the detection method took at least fourteen months" is simply a wrong
impression as will be explained in more detail below.
Detailed comments

567. The process of method validation for GMO detection methods is both complex and time
consuming. Simply put, two sets of information are needed to develop a detection method. First, the
actual method (i.e. the protocol) is needed. The me thod consists of two components: (a) a method to
extract good quality of DNA and (b) a method to assess the amount of GM analytically. Second,
control samples are ne eded. Control samples serve as an intern al control for any analytical method.
Provision of precise control materials/samples for precision instrument calibration must be
simultaneously supplied by the notifier along with the detailed method description. Thus, the
development of the detection method cannot be exec uted without appropriate control samples. For
GMOs they can only be supplied by the applicant who constructed the transformed organism.
568. With respect to the present product in qu estion (Monsanto Roundup Ready corn GA21)
following initial contact with the notifier on 5 June 2002 (EC-91, At. 45) to discuss and agree the
method validation for GA21, the applicant and the Jo int Research Centre signed a Material Transfer
Agreement on February 20
th 2003 (EC-91, At. 49) and the applicant did not provide any of the above
information before 27 March 2003, i.e. approximately 9 and a half months after the initial contact
date.
569. Furthermore, and contrary to what Dr. Nutti claims, the information submitted on that date,
was incomplete with regard to both of the above set s of information needed. Therefore, the validation
process could not to be started.
570. On the one hand, the information submitted wa s incomplete with regard to the method
description and this in several respects. First, the applicant failed to provide component (a) as
described above, i.e. a method on how to extract the DNA from the control samples, which is in the
first step in DNA based GMO detection and quantif ication. Without that information no method
validation can start. The missing information with respect to a suitable method for the extraction of
DNA of good quality was actually provided by JRC: between April and July 2003 (EC-91, At. 56),
the JRC contributed to the further scientific and t echnical definition and completion of this method
through the supply of a draft technical prot ocol for DNA extraction for subsequent DNA
quantification and method application, thus completi ng the system in readiness for validation to begin
proper.210
571. Second, certain critical reagents (i.e. chemic al substances needed as ingredients in the
analysis) provided by the notifier for the purpo ses of the validation procedure were found to be
supplied not only in insufficient quantity, but faulty (EC-91, At. 59), further delaying progress.
572. Finally, the company failed to provide convinc ing empirical data regarding overall method
performance, a prerequisite of method acceptance (EC-91, At. 52).

210 It should be noted that the European Commission – Joint Research Centre bore all the costs
associated with this validation procedure.

573. On the other hand, the information submitted on 27 March 2003 was incomplete with regard
to the control samples. The applicant provided th e appropriate amount and quality of control samples
only 9 April 2003 (EC-91, At. 51)
574. It was not until July 2003, the information necessary to launch the validation process was
complete.
575. As soon as this was the case, the JRC launched an in-house validation in its own facilities,
followed by a pre-validation trial with three laboratories which took place between December 2003
and January 2004, and the collaborative tr ial took place between April and June 2004.
211 Dr. Nutti
does not comment on these delays, which are extraord inarily short delays given the complexity and
dimension of this process.
Question 46

Were detection methods commercia lly available in 2002 sufficient to enable the detection of the
transgenic proteins expressed by the plant line Bt11 sweet corn?

General comments

576. Both Dr. Nutti and Dr. Healy comment on this question. The European Communities has
already explained the need for an event specific as opposed to a trait specific detection method for
GMOs. Nobody would contest that there was no commercially available event specific detection
method for Bt11 sweet corn in 2002 . Such a method was only recently published in a peer reviewed
journal in April 2003,
212 and modified for event specific quantitative application (published on
22 May 2004),213 while the validated study of this method was made available on the internet pages of
the Community Reference Laboratory on 28 July 2004.214
577. It should be stressed that what both experts discuss here, are generic trait specific methods ,
i.e. methods that allow only for the detection of a specific expressed trait (such as the CryIA(b)
protein). Trait specific methods cannot distinguish one GMO product (i.e. transformation event) from
another if both are genetically modified with the sam e trait (such as for example Bt 11 sweet corn and
Bt176 maize. Therefore, whether or not trait specific methods existed for CryIA(b) and the PAT gene
in 2002 is irrelevant , as they would not have ser ved to specifically detect and identify Bt11 sweet
corn as such.
215
Question 47

Given the information before the P anel, were there scientific or tec hnical reasons which explain why
the process of validating the detection method took twelve months (EC-92/At.54-56, 57-65, and 66)?

211 Information has been made publicly available on 17 January 2005 on the Internet pages of the
Community Reference Laboratory (http://gmo-crl.jrc.it )
212 Rønning, S.B., Vaïtilingom, M, Berdal, K.G. & Ho lst-Jensen, A. 2003. Event specific real time
quantitative PCR for genetically modified Bt11 maize (Zea mays). Eur Food Res Technol 216: 347 – 354
213 Hernández M., Duplan M-N, Be rthier G., Vaitilingom M., Hauser W., Freyer R., Pla M. and
Bertheau, Y. Development and comparison of four RTi-PCR systems for specific detection and
quantification of Zea mays L. J. Agric. Food Chem. 2004. 52: 4632-4637
214 Information has been made publicly available on 28 July 2004 on the Internet pages of the
Community Reference Laboratory (http://gmo-crl.jrc.it ).
215 In any event it may be pointed out that while it is correct to say that there was a (trait specific)
detection method for CryIA(b) in 2002 this method had not yet been validated. As for a detection method for
the PAT gene, as discussed in Question 17, no such method exists to-date.

What would constitute an adequate amount of material to be used in the detection method validation?
What are adequate performance indicators for pre-validation results for this product?

General comments

578. As for question 45, Dr. Nutti here acknowledges th at detection methods are not her main field
of expertise. Indeed, this time she openly admits th at she has "no expertise or references to answer
about adequate amount of material to be used in the detection method validation and adequate
performance indicators for pre-validation results for th is product." In light of this, her conclusion that
there were no scientific or technical reasons which explain why the process of validating the detection
method took twelve months is clearly unsubstantiated. For, in order to be able to come to such a
conclusion, one precisely needs to have some know ledge about "adequate am ounts of material" and
"adequate performance indicators." Dr. Nutti her self establishes that all exchanges between the
applicant and the JRC (At. 56 through 66) were a bout the adequacy of the data submitted. As she
does not have the relevant expertise, her reply is, therefore, unfounded and must be dismissed.
Detailed comments

579. The European Communities refers to its comments in question 45: For a detection method to
be developed, two sets of information are need ed. (1) The protocol (method) including (a) a DNA
extraction method and (b) an event specific quantification method, and (2) control samples.
580. At its first meeting with the European Comm ission on 24 June 2002 the applicant confirmed
that they had a method ready and agreed to supply the necessary materials (exhibit 92/54). However,
materials (in this instance DNA) necessary to star t the validation procedure was only received on 22
January 2003, some 7 months after first contact with the European Commission, and 3 months
following a planning meeting (held on 23 October 2002), in which details of the validation and
materials (DNA from seed) which should be provided by were agreed.
581. However, by 28 January 2003 quality testing of this DNA material employing a range of
testing instrumentation by the JRC revealed either very low amounts or, in extreme cases, virtually no
DNA material (i.e. a virtual absence of DNA) in the tubes provided. This is very clear from exhibit
92/56. Such measurements were considered at ypical, especially from maize seed material, and
indicated a technical failure (and hence low confid ence with the material). Following some additional
guidance from the JRC on 30 January 2003, the app licant provided a replacement batch of DNA
material on 27 February 2003 (exhibit 92/58), e xhibiting adequate quantities in the expected
concentration range.
582. All materials and information was then complete to commence the pre-validation study,
which was subsequently launched on 10 March 2003, some 8 and a half months following the initial
meeting between the European Commission and the applicant. The pre-validation study was
completed 51 days later on the 30 April 2003, the re sults of which indicated the proposed method to
be unsuitable to proceed (exhibit 92/59). In fact, th e results indicated an inherent problem with the
detection system, experienced by all participating laboratories in the ring-trail and further indicated
that the applicant had not taken the preparative steps as agreed at the planning meeting in October
2002 to optimise the method.
583. On the 23 May 2003, following discussions with the QPCRFOOD
216 consortium, the JRC
proposed the use of a successfully pre-valid ated Bt11 event-specific quantitative method as

216 QPCRGMOFOOD EU share cost action project (contract no. QLK1–1999–01301).

developed, and offered, by the consortium. Based on the pre-validation results, it was clear that this
method did not exhibit the same inherent technica l limitations as the original applicants system
(exhibit 92/60). After reviewing the details, th e applicant agrees on 7 July 2003 to adopt the
QPCRFOOD method (exhibit 92/63) and, following r eceipt of requested reagents on 15 July 2003
(exhibit 92/65) the JRC launches a full validation study on 30 July 2003 (almost 13 months following
initial contact with the applicant, but just 3 w eeks after the decision to proceed with the better
QPCRFOOD method was agreed by the applicant). The JRC reported the positive result of the
validation study on 2 October 2003 (exhibit 92 /66), in a little over two months from proper
commencement.
Question 49

Given the information before the Panel, includi ng the application (EC-93/At.1-2), was additional
information regarding nutritional and biochemical c haracterization and toxicity of the transgenic
plant requested by the Greek and Italian author ities (EC-93/At.16-17) necessary to ensure that
conclusions of the safety assessment were valid?

General comments

584. Dr. Nutti concludes on almost all specific request s that they were necessary. It is only with
regard to questions related to the herbicide tr eatment that she finds the CA's requests unnecessary
based on the "understanding that th e risk assessment conducted here is related to GMO and not to the
herbicide." The potential effects of herbicide tr eatment on crop characteristics, however, are highly
relevant to the safety assessment of herbicide-to lerant GMOs. Dr. Nutti, therefore, is wrong in
assuming that such information would not be necessary , just because the risk assessment is performed
on the GMO and not on the herbicide itself. Fina lly, in commenting on the necessity of carrying out
studies of sub-acute toxicity, Dr. Nutti
confirms the CA's initiative to ask for more information.
Detailed comments

585. Dr. Nutti correctly asserts in her review of It alian CA's questions 1 and 2 that herbicide
residues have to comply with standards like those of the Codex alimentarius. However, information
about herbicide treatment of the GM crop was also requested in question 1. This is equally relevant
for the safety assessment of GMOs, since it provides information about the conditions in which the
tested crops have been cultivated. In fact, agronomic practices, including herbicide treatment, of GM
crops used for compositional analysis are also addressed by Codex guideline CAC/GL 45-2003 which
states:
Analyses of concentrations of key components

of the recombinant-DNA plant and,
especially those typical of the food, should be compared with an equivalent analysis
of a conventional counterpart grown and harvested under the same conditions. In
some cases, a further comparison with the recombinant-DNA plant grown under its
expected agronomic conditions may need to be considered ( e.g. application of an
herbicide).
217
586. Information on herbicide treatment therefore constitutes a relevant part of the data on the field
trials from which samples for the compositional analysis have been obtained.
587. Question 2 of the Italian CA pertained to th e "nature of possible residues" derived from
herbicide treatment. Whereas Dr. Nutti correctly notes that the levels of these residues should be

within the limits specified with standards, the qu estion is relevant from a safety point of view,
regardless whether the issue falls under a different legislation or not.
588. In fact, the Codex guideline CAC/GL 45-2003 also considers this issue under the heading
"Potential accumulation of substances significant to human health":
Some recombinant-DNA plants may exhibit tr aits (e.g. herbicide tolerance) which
may indirectly result in the potential for accumulation of pesticide residues, altered
metabolites of such residues, toxic metabo lites, contaminants, or other substances
which may be relevant to human health . The safety assessment should take this
potential for accumulation into account. Conventional procedures for establishing the
safety of such compounds (e.g., procedures for assessing the human safety of
chemicals) should be applied.
218
589. The questions are therefore relevant from the point of view of safety of the GM crop and any
pesticides that might accumulate in it.
590. On the issue of sub-acute toxicity studies, the Italian CA requested such information in order
to obtain a more detailed assessment of the toxicological aspects.
591. The animal experiments that had been summarized by the applicant in its application (EC-93,
At. 2) include a chicken broiler feeding study (nut ritional) and a repeated-dose acute toxicity study
(14 days) with the purified transgenic protein administ ered to in rats. Dr. Nutti concludes that if the
results of the composition analysis of Liberty Link soybean, as well as the acute toxicity animal study
that have been carried out with it, were satisfactor y, no additional sub-acute animal test would be
needed. However, in the absence of full data on these tests, she says to be unable to judge the request.
592. In fact, this inability to judge these data due to their incompleteness at the time of the Italian
lead CA's request justifies the requ est for an animal study. This i ssue is also addressed by the Codex
guideline CAC/GL 45-2003:
If the characterization of the food indicates th at the available data are insufficient for
a thorough safety assessment, properly designed animal studies could be requested on
the whole foods.219
593. At the time of the request, it was therefore releva nt to ask for an additional animal toxicity
study.
Question 51

Given the information before the Panel, including the application (EC-95/At.1-2) and the responses
from Pioneer/Dow AgroSciences (EC-95/At.10- 12), was information regarding molecular
characterization, compositional analyses and toxicol ogical analyses of the product requested by the
Health Council of the Netherlands (EC-95/At.8 and 13) necessary to ensure that conclusions of the
safety assessment were valid?

General comments

594. Dr. Nutti addresses issues of compositional analys is and toxicological analysis. While she
confirms the necessity of requesting data on the leve ls of certain nutritional elements and secondary
metabolites, she does not see the need to ask for three (instead of two) seasons of field data, a view
which she does not substantiate and which is not supported by Codex standards. Her comment on the
broiler chicken study seems misplaced as there w as actually no request for such data in the
attachments under consideration. Furthermore, th e European Communities disagrees with Dr. Nutti's
conclusion on the necessity of a 90-day toxicity study.
595. Finally, as Dr. Nutti has not addressed the issues relating to molecular characterization, the
European Communities will offer some brief comments.
Detailed comments

596. First, on the number of seasons for compositional data (question 2 of the lead CA), Dr. Nutti
notes that, whereas two seasons are generally acceptabl e, the Dutch CA asked for compositional data
from three seasons. Actually, the Codex guide line CAC/GL 45-2003 addresses this issue by stating
that field trials for compositional analysis should be carried out during a sufficient number of
generations, but does not mention a specific number of generations (or seasons):
Similarly, trials should be conducted over a sufficient number of generations to allow
adequate exposure to the variety of conditions met in nature.
220
597. At the time of the request, the applicant had provided data from the seasons 1998-1999 in
Chile (Southern hemisphere) and 1999 in France a nd Italy (Northern hemisphere). The lead CA
considered this to be one season (EC95, At.8). Given this opinion of the CA and the Codex guideline
CAC/GL 45-2003 recommendation on this issue, it therefore appears justifiable that, at the time of the
request, the Dutch CA asked for data from multiple (3) seasons.
598. In addition, the Dutch CA later (in reply to additional compositional data provided by the
applicant) asked for an additi onal study from the US, as this would be representative for the
commercial production of the new maize line.221
599. Furthermore, the lead CA's request is in line with Codex guideline CAC/GL 45-2003,
paragraph 45, pertaining to compositional analysis of key components, which reads as follows:
The location of field trials should be representative of the range of environmental
conditions under which the plant varieties would be expected to be grown.
600. Therefore, the data requested by the lead CA on compositional analyses from multiple (3)
seasons, as well as from regions representative of co mmercial maize cultivation (and export to the
EU) are in line with Codex recommendations and relevant to the safety assessment.
601. Second, Dr. Nutti comments on the necessity of additional data on a broiler chicken study.
There was no question on the chicken broiler study po sed in EC95, At. 8 and At. 13, neither was the
broiler study mentioned in the attachment, namely EC95, At. 11. Dr. Nutti's comment on the chicken
broiler study, therefore, cannot be verified, since no reference to this study could be found in the

221 Note that this issue was also dealt with under question 35, where Dr. Andow cited trade statistics
showing that the major nations exporting maize were the US, Argentina, Brazil, and the People's Republic of
China (see Dr. Andow's reply, para. 35.03).

documents mentioned [requests by Member Stat e CA (EC95, At. 8 and 13) and reply from the
applicant (EC95, At. 11)].
602. Third, Dr. Nutti seems to argue that the 90 day rat study requested by the lead CA was not
necessary as "additional testing will be required if th e composition is not comparable to conventional
foods, which is not the case of Pioneer/Dow AgroSciences Bt corn Cry1F (1507)."
603. At the moment of the request of the Dutch CA for an additional 90-day rat study, the
applicant had provided (summaries of) a nutritional st udy in chicken broilers fed whole products and
acute toxicity in rats with the purified transgenic proteins Cry1F and PAT. It should be stressed here
that the chicken broiler study is not a model for toxicology, but for nutrition (for example, see the
discussion on this issue in Chassy et al., 2004). During studies on broilers, the growth, bodyweight,
feed consumption, and weight and composition of ed ible parts after slaughter are usually measured.
Therefore, the toxicity animal studies had been limit ed to those on acute toxicity of purified Cry1F
and PAT, and no toxicity studies with the whole food.
604. According to Dr. Nutti, the additionally request ed 90-day toxicity study was not necessary,
since compositional equivalence had been demonstrated. The rationale provided by the Dutch CA for
its request for the 90-day study was that it woul d provide additional reassurance of no unintended
changes with adverse effects (EC-95, At.8).
605. Whole feed/food studies can be used in order to provide reassurance about the potential health
consequences of unintended effects, such as those that may have been caused by the insertion of
additional DNA fragments, beyond the intended modifica tions (e.g. transgenic proteins) that have
been tested for toxicity. In fact, other ways to de tect unintended effects, such as the use of advanced
analytical technologies called "profiling" or "meta bolomics", are in development, but have not been
validated yet for routine application in the safety assessment of GM foods.
222
606. An example of animal feeding studies serving as additional reassurance of no adverse effects
is Roundup Ready soybeans. It was discovered in 2000 that unintended insertions of DNA fragments
had occurred in these soybeans, which had alread y been approved as a GM food in a number of
countries, including the EU. The applicant (Monsan to) itself argued that these additional fragments
could not pose a risk, since, among others, no adverse effects had been observed in a previously
published peer-reviewed animal feeding study with whole feed produc ts derived from Roundup
Ready soybeans.
223
607. It is therefore justifiable of the lead CA that , in the absence of toxicity studies with whole
foods, it sought additional reassurance for the innoxi ousness of any unintended effects by asking for
an additional animal toxicity experiment.
608. Finally, as regards the CA's requests for additional molecular characterization (on which
Dr. Nutti has not commented), the European Communities would offer the following brief comments:

222 Chassy, B., Hlywka, J.J.,. Kleter, G.A., Kok, E.J., Kuiper, H.A., McGloughlin, M., Munro, I.C.,
Phipps, R.H., Re, E.B., Reid, J.E. (2004) Nutritional an d Safety Assessments of Foods and Feeds Nutritionally
Improved through Biotechnology, Prepared by a Task Force of the ILSI International Food Biotechnology
Committee. Comprehensive Reviews in Food Science and Food Safety 3: 35-104.
http://www.ift.org/pdfs/crfsfs/crfsfsv3n2p0035-0104ms20040106.pdf
223 Monsanto (2000) Updated Molecular Characteri zation and Safety Assessment of Roundup Ready
Soybean Event 40-3-2. Monsanto Co. St. Louis, 20 pp.
http://archive.food.gov.uk/pdf_files/acnfp/summary.pdf

609. The European Communities would refer to the in ternationally agreed principles regarding the
necessity of detailed molecular characterisation in a thorough safety assessment (see section III on
general and methodological issues, a nd the expert replies on Q 9, 111 and following). In the present
case, the notification contained no evidence of actual verification of the insert by sequencing. Note
that it had been submitted on February 15
th 2001, by which date sequencing had been routine for
almost a decade.
610. The Health Council of the Netherlands in EC-95/At.8 (June 28 th 2001) requested specifically
sequencing of approx. 1000 bp of genomic DNA flanking the insert, as well as sequencing of the
complete insert itself. It was not clear from the responses of the notifier (E C-95/At.10-12, February
2002-February 2003) if the full length insert had been characterised by sequencing of the transformed
maize, or whether only the flanking sequences were ve rified from the GM maize, while the rest of the
insert sequence was simply determined by sequencing the transforming plasmid. Furthermore, in the
responses from the notifier only 271 bp of the 3'-flanking sequence was provided. The need for as
much as 1000 bp was reasonable given the state-of-the-a rt concerning gene regulation in plants at the
time the request was made
Question 52

Given the information before the Panel, incl uding the application and the responses from
Pioneer/Dow AgroSciences (referenced above), was information regarding the potential unintended
expression of allergenic proteins requested by Gezondheistsraad (EC-95/At.1 5) necessary to ensure
that conclusions of the safety assessment were valid?

General comments

611. As explained in the European Communities comments on Dr. Nutti's reply to question 43,
Dr. Nutti's approach to issues of allergenicity does not take into account the state of the analysis of
molecular characterisation of the product. Th at analysis, however, is necessary to comment on
toxicology problems regarding allergenicity (and uni ntended effects). Dr. Nutti's conclusion ("as far
as my knowledge goes") therefore, may be valid as regards general toxicity aspects of the CRY1F
protein, but it does not apply to the actual considera tions (related to molecular characterisation) that
the lead CA had raised.
Detailed comments

612. As the CA clearly explains, the request fo r the further allergenicity assessment addresses
problems of unintended molecular effects of the flanking regions, especially the potential fusion
proteins formed by such effects.
613. This request is fully in line with modern st andards of assessment of unintended effects in
flanking regions. Thus Codex guideline CAC/GL 45-2003 states in its paragraph 31D:
31. Information should be provided on th e DNA insertions into the plant genome;
this should include:
D) identification of any open reading fra mes within the inserted DNA or created
by the insertions with contiguous plant genomic DNA including those that could
result in fusion proteins.
while it also states in paragraph 5 of its Annex on the Assessment of Potential Allergenicity that:

As there is no single test that can pred ict the likely human IgE response to oral
exposure, the first step to characterize newly expressed proteins should be the
comparison of the amino acid sequence and certain physicochemical characteristics of
the newly expressed protein with those of established allergens in a weight of
evidence approach.
614. The CA's request for additional information on put ative fusion proteins was in line with these
standards and therefore justified.
Question 53

Given the information before the Panel, includin g the application (EC-96/At.1-2), was information
regarding molecular characterization, toxicity e ffects of unintended chang es and compositional data
requested by Gezondheitsraad (EC-96/At.7) necessa ry to ensure that conclusions of the safety
assessment were valid?

General comments

615. Dr. Nutti comments on the aspects related to compositional analysis and toxicological
analysis. As has already been pointed out before , Dr. Nutti's approach does not take into account the
need for a thorough analysis of the molecular characterisation, which makes her conclusions
incomplete or flawed. More specifically, her conc lusion that a semi chronic toxicity study is not
necessary must be dismissed on the basis of the avai lable scientific evidence on the relevance of such
studies.
616. Finally, as she has not commented on the speci fic requests for additional information on
molecular charcterisation, the European Communities will offer brief comments.
Detailed comments

617. Given the publication year (2001) of the requ ested 90-day study on rats (Dudek, 2001, cited
in EG96, At.2, p. 117), the applicant must have co mpleted this report by the time the CA made its
request for additional inform ation (December 13th, 2001), i.e. this information could have been
almost available by that time. These data should th erefore have been taken into account, in line with
paragraph 15 of the Codex Principles for the Risk Analysis of Foods Derived from Modern
Biotechnology, which states that:
Risk assessment should take into account all available scientific data and information
derived from different testing procedur es, provided that the procedures are
scientifically sound and the parameters being measured are comparable. [Emphasis
added]
In addition to this requirement, arguments for requesting a 90-day toxicity study on whole products of
maize NK603 are provided below.

618. The rationale provided by CA for its request for a semi chronic toxicity study was that
unintended, additional modifica tions might have taken place in maize NK603, including
modifications that had already been describe by the applicant.
619. With regard to animal studies, the applicant had provided (a summary of) a nutritional study
in chicken broilers fed whole products and acute toxicity in rodents with the purified transgenic

proteins EPSPS. It should be stressed here th at the chicken broiler study is not a model for
toxicology, but for nutrition (for example, see the discussion on this issue in Chassy et al., 2004).
224
During studies on broilers, the growth, bodyweight, feed consumption, and weight and composition of
edible parts after slaughter are usually measured. Therefore, the toxicity animal studies had been
limited to those on acute toxicity of purified EPSPS, and no toxicity studies with the whole food.
620. According to Dr. Nutti, the additionally request ed 90-day toxicity study was not necessary,
since compositional equivalence had been demons trated and the intended modification (the EPSPS
protein) had also been tested for potential toxicity and allergenicity. The Dutch CA, on the other
hand, provided as rationale for its request for the 90-day study that it would provide additional
reassurance of no unintended effects undesired effects (EC96, At.7).
621. Whole feed/food studies can be used in order to provide reassurance about the potential health
consequences of unintended effects, such as those may have been caused by the insertion of additional
DNA fragments, beyond the intended modifications (e.g. transgenic proteins) that have been tested for
toxicity. In fact, other ways to detect unintende d effects, such as the use of advanced analytical
technologies called "profiling" or "metabolomics", ar e in development, but have not been validated
yet for routine application in the safety assessment of GM foods.225
622. An example of animal feeding studies serving as additional reassurance of no adverse effects
would be the case of Roundup Ready soybeans. It was discovered in 2000 that unintended insertions
of DNA fragments had occurred in these soybeans, which had already been approved as a GM food in
a number of countries, including the EU. The appli cant (Monsanto) itself argued that these additional
fragments could not pose a risk, since, among othe rs, no adverse effects had been observed in a
previously published peer-reviewed animal feed ing study with whole feed products derived from
Roundup Ready soybeans.226
623. The lead CA, therefore, was justified, in the absence of toxicity studies with whole foods, to
seek additional reassurance for the innoxiousness of any unintended effects by asking for an animal
toxicity experiment with the whole food. In addition, such a study (Dudek, 2001, At96, At.2,
p.117)
227 had already been published by the applicant at the time of the request (or shortly thereafter,
in 2001), and the report of this study could therefore have been requested for the sake of completeness
of the safety evaluation.

224 Chassy, B., Hlywka, J.J.,. Kleter, G.A., Kok, E.J., Kuiper, H.A., McGloughlin, M., Munro, I.C.,
Phipps, R.H., Re, E.B., Reid, J.E. (2004) Nutritional an d Safety Assessments of Foods and Feeds Nutritionally
Improved through Biotechnology, Prepared by a Task Force of the ILSI International Food Biotechnology
Committee. Comprehensive Reviews in Food Science and Food Safety 3: 35-104.
http://www.ift.org/pdfs/crfsfs/crfsfsv3n2p0035-0104ms20040106.pdf
225 Chassy, B., Hlywka, J.J.,. Kleter, G.A., Kok, E.J., Kuiper, H.A., McGloughlin, M., Munro, I.C.,
Phipps, R.H., Re, E.B., Reid, J.E. (2004) Nutritional an d Safety Assessments of Foods and Feeds Nutritionally
Improved through Biotechnology, Prepared by a Task Force of the ILSI International Food Biotechnology
Committee. Comprehensive Reviews in Food Science and Food Safety 3: 35-104.
http://www.ift.org/pdfs/crfsfs/crfsfsv3n2p0035-0104ms20040106.pdf
226 Monsanto (2000) Updated Molecular Characteri zation and Safety Assessment of Roundup Ready
Soybean Event 40-3-2. Monsanto Co. St. Louis, 20 pp.
227 Dudek, B.R., 2001. Amendment 1 to final report. 13 Week Feeding Study in Rats with Grain from
Roundup Ready® Corn (NK603) Preceded by a 1-Week Baseline Food Consumption Determination with PMI
Certified Rodent Diet #5002. Monsanto Technical Report MSL 17555 (Amendment to MSL 17423), St Louis,
Missouri

624. Finally, as regards the CA's request for add itional information on molecular characterisation
(on which Dr. Nutti has not commented), the Europ ean Communities would offer the following brief
comments:
625. The European Communities refers to the inte rnationally agreed principles regarding the
necessity of detailed molecular characterisation in a thorough safety assessment (see section III on
general and methodological issues, a nd the expert replies on Q 9, 111 and following). In the present
case, it should be noted that the request is dated December 13
th 2001, i.e. almost one year after the
publication of the paper on GTS 40-3-2 (Windels et al., 2001). 228 Thus, the scientific basis for
requesting full sequence data of appr ox. 1000 bp flanking each side of th e insert is justified, also on
the basis of state-of-the-art knowledge and tec hnology. Clearly, Southern blot analysis was
insufficient as evidence by this date. Therefore, the requested data was necessary to ensure that
conclusions of the safety assessment were valid.
Question 53bis

Given the information before the Panel, including the first whole food study and agronomic
performance tests (EC 96/At.2, pp. 102-103), was a second animal whole food study (EC 96/At. 7)
necessary or useful to identify potential adverse effects, including those associated with small random
DNA insertions, that had not been previously identified?

General comments

626. The question refers to the lead CA's request fo r a semi chronic toxicity study (question 2 in
EC-96/At.2), which was already covered by and addressed in question 53. Dr. Nutti's comments in
essence are identical to her comments in question 53 on that issue. Therefore, the European
Communities refers to its above under question 53.
Question 54

Given the information before the Panel, including th e application, was information requested by Italy
(EC-96/At.9) necessary to ensure that conclusions of the safety assessment were valid?

General comments

627. Dr. Nutti comments on one specific request the Ital ian CA made, namely a request for further
animal studies. Her conclusion that such data would not be necessary has to be dismissed on the same
grounds as have been put forward in question 40 on the issue of feeding studies with farm animals
with physiologies different from that of the bro iler chicken Therefore, the European Communities
refers to its above comment under question 40.
Question 57

Given the information before the Panel, includi ng the application (EC-101/At.1-3) and the additional
information provided by Pioneer (EC-101/At 13), was additional information molecular
characterization, field trials, secondary plant metabolites, and toxicological tests requested by the
Netherlands (EC-101At.14) necessary to ensure that conclusions of the safety assessment were valid?

228 Windels P., Tavernier I., Depicker A., Van Bock staele E. & De Loose M. (2001). Characterisation
of the Roundup Ready soybean insert. Eur. Food Res. Technol. 213:107-112.

General comments

628. Dr. Nutti, Dr. Andow and Dr. Squire comment on this question. Dr. Squire generally
confirms the CA's request as "consistent with the type of information indicated in the Codex
Guidelines for the conduct of food assessment of foods deri ved from recombinant DNA plants. "
Dr. Nutti and Dr. Andow provide specific comments on qu estions 2, 3 and 4 raised by the Dutch CA.
On questions 2 and 3 the two experts come to opposite conclusions. The European Communities
agrees with Dr. Andow's conclusions. On question 4, the debate on the necessity of certain animal
toxicity studies on hybrids is identical to the one in question 44. The European Communities,
therefore, refers to its comments in question 44.
Detailed comments

629. On the issue of data on herbicide treatment Dr. Nutti concludes that the required information
is not relevant to ensure that conclusions of the safety assessment were valid. In that she is
contradicted by Dr. Andow who concludes that "t here is a scientific justification for requiring
comparison of T25 with glufosinate, T25 without glufosinate and maize without T25 and without
glufosinate." The European Communities agrees with Dr. Andow. Dr. Andow adds that he believes
"that there is a legitimate scientific debate as to whether all three are necessary to ensure that
conclusions of the safety assessment were valid." Th e fact that there is a scientific debate does not
call into question the CA's request (which Dr. Andow himself recognises as scientifically justified).
For the reasons already set out in its earlier comme nts, eg on question 49, the European Communities
sides with the view that the CA has taken, namely that the comparison of all three sets of data, which
the applicant indicated to be readily available (EC-101, At.13, A2, p.2), is necessary.
630. On the request for data on secondary compounds Dr. Nutti is of the opinion "that information
required is not relevant to ensure that conc lusions of the safety assessment were valid, as the
compositional information supplied before leads to the conclusion that the product was substantial
equivalent." She adds that "in this case, we have a product that is a conventional cross of two GM
corns, but this product has not been submitted to a new genetic modification, so the possibility of
fortuitous changes in the plant seems to be vanishing small, if exists." In this assessment, Dr. Nutti is
contradicted by Dr. Andow who finds that "There is a legitimate concern that there are additional
transgenes incorporated into the T25 and Mon81 0 lines that could be expressing additional but
unknown gene products, and that elude detection by present molecular methods. In addition, it is also
possible that the main gene products from T25 or Mon810 could interact with plant metabolism,
changing the composition of the plant." While Dr. A ndow also speaks of a "small likelihood" of this
happening, he finds that "it is difficult to argue how small."
631. The European Communities ag rees with Dr. Andow's assessment. While the issue of
conventional crosses of GM lines is not specifically addressed by the Codex guideline, it does so for
the issue of unintended effects, for example in paragraph 15 (Unintended effects):
Unintended effects may also result in the formation of new or changed patterns of
metabolites"
632. Dr. Nutti states that compositional analysis would already have led to the conclusion that the
conventional cross is substantially equivalent. This compositional an alysis was, however, "targeted",
i.e. specific compounds have been measured. Any changes in compounds that have not been
measured would therefore remain unnoticed.

633. Dr. Nutti also asserts that additional unintended effects are hardly possible in conventional
crosses of GM lines. For example, the introduced GM traits from the particular parent plant may
interact with each other within the conventional cross of GM lines.
634. One example is that of "gene silencing" (inac tivation of gene function) by bringing together
genes with similar sequences from different parents within the same plant (as observed, for example,
by Charrier et al. (2000) and De Wilde et al. (2001)).
229
635. Based upon the considerations above, including the fact that unintended effects in
conventional crosses of GM lines cannot be excluded on beforehand, the request of the lead CA was
relevant from the point of view of safety of the cross of two GM crops and any secondary metabolites
that have been changed as an unintended side-effect of the combination of genetic modifications.
636. On the request for additional toxicity testing, both Dr. Nutti and Dr. Andow take the view that
there is no need for such tests albeit for differing reasons. Both have discussed their position in more
detail in questions 44 and 44bis. There are valid argu ments to refute either's position. The European
Communities has discussed those in question 44 and 44 bis, therefore, refers back to its comments
therein.
637. Finally, as regards the CA's request for add itional information on molecular characterisation
(on which no expert has commented), the Euro pean Communities would offer the following brief
comments:
638. The European Communities refers to the inte rnationally agreed principles regarding the
necessity of detailed molecular characterisation in a thorough safety assessment (see section III on
general and methodological issues, and the expert re plies on Q 9, 111 and following) as well as to its
comments in questions 44 to 44ter.
639. Generally, stacked hybrids will require a separat e molecular characterisation and complete
risk assessment, and can not only rely on evaluation of the parental GM events. Furthermore, the
request from the Netherlands (EC-101/At.14, dated April 23 rd 2001) was necessary to ensure that
conclusions of the safety assessment were valid. By this date DNA sequencing had been applied
routinely for characterisation for almost a de cade. Furthermore, the Windels et al. (2001) 230 paper
was published a few months prior to the request. Th is paper clearly demonstrated the relevance of
detailed sequence information including the flanking sequences.
Question 58

Given the information before the Panel, including the application (EC-102At.1-20) and the
information provided by Monsanto/Novartis (EC-102/At.22, 26 and 27-30), was additional
information regarding food safety assessment of d erived proteins requested by the Netherlands
(EC-102/At.32) necessary to ensure that conclusions of the safety assessment were valid? Did the
food safety assessments provided by the applicant follow the Codex Guide line for the Conduct of
Food Safety Assessment of Foods Derived from Recombinant-DNA Plants.

229 Charrier, B., Scollan, C., Ross,S., Zubko, E., Meyer, P. (2000) Co-silencing of homologous
transgenes in tobacco. Molecular Breeding 6: 407-419.
De Wilde, C., Podevin, N., Windels, P., Depicker, A. (2001) Silencing of antibody genes in plants with
single-copy transgene inserts as a result of gene dosage effects. Molecular Genetics and Genomics 265:
647-653.
230 Windels P., Tavernier I., Depicker A., Van Bock staele E. & De Loose M. (2001). Characterisation
of the Roundup Ready soybean insert. Eur. Food Res. Technol. 213:107-112.

General comments

640. Dr. Nutti concludes that "there was no need for requesting a semi chronic toxicity study in
mice or rats using the edible parts of the sugar beet in order to rule out possible undesired effects of
additional unidentified changes." The request for a semi chronic toxicity study had been one of the
CA's requests. The European Communities di sagrees with Dr. Nutti's conclusion on the grounds
already set out in questions 44, 51, 53, and 57. The CA had also raised specific concerns on the
potential presence of a certain fusion protein (pro tein 34550), a concern which Dr. Nutti does not
specifically address in her comments, presumably because that question pertains to issues of
molecular characterization, which would be outside the scope of Dr. Nutti's comments.
VI. THE PANEL'S QUESTIONS ON ISSUE 2 ("SAFEGUARD MEASURES")
(NOS 59 TO 95)
A. I
NTRODUCTORY REMARKS
641. The Panel's questions 59 to 95 concern the Me mber State safeguard measures. Generally, for
each measure, there are three questions, concerning: the sufficiency of scientif ic evidence; the risk
assessment, and its relation to inte rnational guidelines; and the jus tification for provisional measures,
in the light of other available risk management options.
642. The European Communities would like to make the following general comments.
643. First, the European Communities understands that what the Panel's question aims to elucidate
is the issue of the point at which the information or scientific evidence is sufficient for the authority to
decide definitively whether or not to approve the product; and the issue of whether or not that point
had been reached in the specific circumstances of the Member State safeguard measures.
644. Second, in science as in law, it may be pr oblematic to prove a negative, especially
prospectively. That is, it may be problematic for a scientist to certify definitively that mass
commercialisation of a new product will have no ad verse effects on human health. In making a risk
assessment, it is likely that the scientist may have to place the product and the issues associated with it
in context. The scientist may need to get some kind of "helicopter vision" on the problem. The new
product may be just one feature of the scientific landscape – and the scientist may need to consider the
landscape as a whole before taking a view on the relative safety of the product. In these
circumstances, the absence of info rmation or evidence about the context may be just as significant as
the absence of information or evidence about the product itself.
645. Third, the Appellate Body has made it clear that the concept of "sufficiency" is a relative one
– the scientific information must be sufficient fo r a specific purpose, that is, for the purposes of
deciding whether or not to approve the product defini tively, taking into account the legislator's chosen
level of protection. The higher the chosen level of protection, the more scientific evidence may be
necessary before the evidence is su fficient to make a definitive decision, and the longer the period of
time and the greater the resources that may reasonably be required.
646. Fourth, the evaluation will also be a function of the information or scientific evidence
available: the less information or scientific informa tion available, the less detailed the evaluation may
be.
647. Fifth, it may well be that the available information or scientific evidence and the evaluation or
assessment are insufficient to support a definitive refusal to authorise: but that it is still sufficient to

support a provisional measure, at least if the conditions of Article 5.7 of the SPS Agreement are
complied with. This is the position with regard to the Member State safeguard measures.
648. Sixth, as the European Communities has alread y explained in its submissions, to fairly
understand why the Member States acted as they did it is not sufficient to look only at the text of the
provisional measures themselves (which is often re latively succinct), and the notification to the
Commission. It is also necessary to consider th e history of the discussions, including the discussions
at Community level, in which all the Member States participated. From this perspective, it is
unnecessary to distinguish between safeguard measur es adopted by different Member States, except
insofar as there are additional considerations spec ific to that Member States (such as climate,
agricultural structures and practices, flora, fauna, et c.). In order to avoid unnecessary repetition, the
European Communities will, in these comments, refer back to the more general observations that have
already been made.
649. Seventh, the European Communities would note that the responses to the three types of
question often overlap to a significant extent. In su ch circumstances, where appropriate, the European
Communities will comment in general terms in relation to each product for which a safeguard
measure has been adopted.
650. With these general remarks in mind, the Eu ropean Communities will now comment on the
expert advice with respect to each of the products.
B. Q
UESTIONS 59 THROUGH 65
Question 59

Given the information before th e Panel, including the evaluat ions undertaken by the UK
(EC-161/At.1), the Scientific Committee on Pl ants in May 1998 and May 1999 (CDA-35-A and CDA-
69, respectively), and the European Commission in its Decision of February 1996 (CDA-62), as well
as the information submitted by France
 with respect to its safeguard measure (EC -161/At. 3 to 11;
CDA-68, CDA-70, CDA-71), is there any reason to believe that the scientific evidence available to
France in November 1998 and July 2001 was NOT sufficie nt to permit it to undertake an appropriate
assessment of potential risks to human, plant and animal health, and the environment from the
growing for seed production of oilseed rape MS1 x RF1? If so, what scientific evidence do you
believe was insufficient?

 (a) If the evidence was not sufficient in November 1998 and/or in July 2001, was there
sufficient evidence available to France in August 2003 to permit it to undertake a
more objective assessment of potential risks to human, plant and animal health, and
the environment from the growing for seed production of oilseed rape MS1 x RF1? If
not, what scientific evidence do you believe was insufficient?

651. The European Communities would draw the Panel's attention to the fact that, in response to
this question, Dr. Squire notes that: "The EC SPC and the information submitted by France therefore
agreed on the nature of this potential problem, but differed on its importance or extent"; and that
"There was insufficient knowledge at that time to be able to predict accurately, for a country such as
France, what the rates of spread and cross pollinati on would be (GM to non-GM) if a large part of the

rapeseed areas were GM". Dr. Squire further notes th at even at the end of 2003 no conclusions can be
drawn and that work is still in progress, citing to specific references
231.
652. Furthermore, Dr. Snow advises as follows:
Given that, 1) the SCP identified concerns a bout the spread of herbicide resistance to
volunteer weeds, even though they considered this to be more of an agronomic
problem than an "environmental" one; 2) th e SCP underestimated the extent of gene
flow that is to be expected to other oi lseed rape crops, volunteer weeds, and weedy
Brassica rapa; and 3) the SCP recommended that the introduction of herbicide-
tolerant crops should be accompanied by "ii) a monitoring programme with an agreed
design and implementation plan to detect the occurrence and the establishment of
herbicide-tolerant volunteers and weeds under field conditions in the EU;" I conclude
that France had valid reasons to follow the advice of its Biomolecular Engineering
Committee to carry out more research to "supplement existing scientific knowledge
and validate methods for managing the cultivation of genetically modified oilseed
rape." Scientists in France were well awar e of the fact that transgenes would be
dispersed by means of pollen and seeds, making it difficult to design management
plans that would prevent problems in the future. These problems could be
compounded if transgenes for glyphosate to lerance were also approved in oilseed
rape and other crops, as I explain elsewhere. One could argue that future events are
not relevant to this isolated product, but glyphosate-tolerant oilseed rape has also
been proposed for commercialization in Europe."
232
653. With regard to environmental issues, Dr. Nutti gives no advice 233. With regard to human,
plant and animal health, Dr. Nutti expresses th e opinion that the information submitted was
"adequate"; that there was "no evidence to indicate" that the product was "likely" to cause adverse
effects; and that there was "no scientific evidence" on the basis of which France could "ask for the
moratorium"234.
654. In the opinion of the European Communities, Dr. Nutti's advice on this point is of little if any
assistance in resolving the relevant issue. First, Dr. Nutti appears to have considered whether or not
the science could have justified a definitive decision not to approve the product – concluding that this
is not so. However, that is not the pertinent issu e. The pertinent issue is whether or not the science
could justify a provisional measure – a matter that Dr. Nutti does not consider at all. Second, the
question for an authority in such a situation is not whether or not the information is merely "adequate"
– the standard applied by Dr. Nutti. Rather, the ques tion is whether or not the science is sufficient for
a definitive decision to be taken. Depending on the a cceptable level of risk chosen by the legislator
"adequate" may well fall far short of "sufficient". Third, Dr. Nutti appears to have considered whether
or not the science established that the product "is likely to cause adverse effects". But that is not the
applicable standard. In order to provisionally exclude a product, an authority is not required to show
that the product "is likely" to cause adverse effect s. Rather, the authority must comply with the
conditions set out in Article 5.7 of the SPS Agreement , which set out a different standard. Fourth,
Dr. Nutti's conclusions are based on the alleged absence of evidence to indicate likely adverse effects.
That is an erroneous expression of the burden of proof issue.

231 Squire, pages 14 and 15.
232 Snow, page 21.
233 Nutti, page 43.
234 Nutti, page 43.

655. Having regard to these fundamental errone ous assumptions, the European Communities
considers Dr. Nutti's advice to be of no value in resolving the issues before the Panel.
656. The European Communities concludes that, on th e environmental issue, the expert advice
given unanimously supports the position of the EC; and that on the issue of human, plant and animal
health, no expert advice has been given capable of calling into question the lawfulness of the view
that, for France's purposes, the scientific evidence was insufficient to adopt a definitive decision.
Question 60

With reference to the definition of a risk assessment in the SPS Agreement (see Background above), to
what extent does the scientific evidence and other documentation submitted by France evaluate the
relevant risks to human, plant or animal health, and the environment from the growing of oilseed rape
MS1 x RF1 for seed production?

 (a) How does the scientific evidence and other documentation submitted by France
compare with the relevant international guidelines for risk assessment and analysis
identified above?

657. The European Communities would draw the atte ntion of the Panel to the observation of
Dr. Squire: "It could be argued that France's pos ition was compatible with the tone of the SPS
Agreement, Annex A, paragraph 4 (including economic as well as biological consequences) and
compatible also with ISPM-11 Annex 3 on 'Determining the potential for a LMO to be a pest'."
235
658. The European Communities would also refer to Dr. Snow: "To the extent that these concerns
are justified in the context of invoking "safeguard" measures (which I consider to be a legal question),
I conclude that France had valid reasons for de ciding that additional scientific research was
needed."236
659. With regard to Dr. Nutti, the comments outlined above also apply, mutatis mutandis.
660. Essentially, the conclusion with respect to ques tion 60 follows inevitably from the conclusion
with respect to question 59. Once the insufficiency of the scientific evidence is established, it follows
that, even on the Complainants' (erroneous) r easoning, there can no longer be any possible
controversy about the provisional nature of the meas ure. The relevant provision being Article 5.7 of
the SPS Agreement , what is required is that the authority act "on the basis of available pertinent
information", making an assessment appropriate to the circumstances. Thus, the extent of the
evaluation will always be a function of the availabl e information and science – a view that is also
reflected in the relevant international guidelines.
Question 61

Does the scientific evidence and othe r information submitted by Fran ce support the adoption of a
temporary prohibition on the growing of oilseed rape MS1 x RF1 for seed production? In light of any
potential risks identified by France, what other ri sk management options were available in November
1998 and/or July 2001? What other risk management options are now available?

235 Squire, page 15.
236 Snow, page 22.

661. The European Communities notes that, according to Dr. Squire, "The risk management
options were and are similar to those indicated gene rally for oilseed rape … The risk of spread and
cross-pollination can be reduced by such measures but not eliminated"
237.
662. Dr. Snow observes:
I do not have enough information to answer this question. If the question refers to
seed production on a very small scale, a temporary prohibition presumably would be
less urgent and possibly unnecessary. If the plants are grown on a fairly large scale,
for example on 1,000 hectares per year, the i ssues at hand are similar to those for the
previous questions.
The basic concern is that herbicide resist ance genes would spread in conjunction with
wider use of this herbicide over time, and that other types of herbicide resistance
genes might also be approved in the futu re (e.g., glyphosate resistance), thereby
compounding the problem. One could argue that future events are not relevant to this
isolated product, but glyphosate-tolerant oilseed rape has also been proposed for
commercialization in Europe.
238
663. According to Dr. Andow:
The adoption of a temporary prohibition can be justified on the basis of the scientific
evidence and other information submitted by France.
Several other risk management options coul d also have been justified in November
1998 and July 2001. Risk management strategies include risk avoidance, risk
mitigation, and risk tolerance. In 1998 and 2001 mitigation and tolerance strategies
were probably inappropriate. Here I consider only the risk that France expressed in
its original decision to impose a temporary prohibition. One risk avoidance strategy
would have been to allow limited planting in a restricted region. In the first year, this
could have been at a scale of a large field trial, and build up from there. This would
allow determination of scale effects. Another strategy is outlined in paragraph 3.19.
Today several alternative risk management op tions are available. In addition to risk
avoidance as in the previous paragraph, risk mitigation strategies may also be
possible to control HT volunteers and wild species. It would appear that France is
now convinced that if HT volunteers or wild species occur that they can be detected
rapidly enough and eliminated.
239
664. The position with regard to Dr. Nutti has already been outlined above.
665. The European Communities observes that c oncerns raised by France about rapeseed
applications in 1998 focused on the impact of cr opping systems and agricultural practices on the
spread and establishment of HT plants, via volunteers or hybrids with wild relatives. Although
scientific knowledge was already available on pollen flow and out-crossing rate to wild relatives,
several issues remained open:

237 Squire, page 15.
238 Snow, pages 23 and 24.
239 Andow, page 84.

• Information available on gene flow (e.g. long distance gene flow) was obtained from
small-scale experiments and was difficult to extrapolate to large-scale dissemination
of GM crops;
• The effect of the various cropping systems and agricultural practices one may find in
European agriculture and which would ex ert a selection pressure on HT plants was
not assessed;
• Inter-specific hybrids with very common wild relatives Raphanus (wild mustard) in
French cropping systems had been recently established
240);
• The precise contribution to gene flow of feral plants which are present in non-
cultivated areas was clearly not available.

666. This meant that, depending on the legislator's c hosen level of protection, it was possible that
the overall ecological and economic balance of grow ing HT rapeseed varieties could not be assessed.
This statement was clearly applicable to crop prod uction. As to the seed production, even if the scale
is lower than for crop production, the acreage of rapeseed production was 4,314 ha in 2004 in France.
Even if it was unlikely that seed production of MS1 x RF1 would cover all this acreage, the figure of
1,000 ha mentioned by Dr. Snow is realistic. As seed production is pe rformed in various regions, this
could be considered as a significant dissemination (at least much higher than field experiments).
667. Furthermore, if seeds are produced, it would be likely that crop production would occur later
and it seems reasonable to anticipate larger scale di ssemination (in fact, an application for cultivation
was already submitted before 1998). The same kind of argument is considered relevant for taking into
account the impact of herbicides on HT crops ev en if the herbicide is not yet approved for
commercialisation.
668. With respect to imports, the exposure may be lower as no intended dissemination through
cultivation would normally occur. Nevertheless, spillage from transport or during handling of
imported seeds can occur 241. Even if the potential for spread a nd establishment could be considered
quite low (especially in the case of glufosinate to lerance), there were uncertainties about out-crossing
to wild relatives, spread and establishment of fera l plants (see above) while the detection of such
events in the environment was not yet available.
669. Wild relatives that outcross with B. napus are present in various regions: wild radish
(raphanus) and cabbage (B. oleracea) whose European coastal zone is a centre of diversity.
670. The European Communities would also note that if imported seeds entered into cultivated
fields (directly through seed dispersal or by pollen flow from feral plants to cultivars), their
development could then be maintained in the case of farm-saved seeds (more than 30 % of the French
cultivars).
671. With regard to the assertion that imports w ould concern spring rapeseed and, consequently,
lead to a reduced risk of establishment, the Eu ropean Communities observes that, in fact, spring
cultivars are mainly characterized by a shorter cycl e, but their frost tolerance, although lower than
winter types, does allow them to survive winters in many parts of Europe.

240 Chèvre AM, Eber F, Baranger A, Renard M. 1997. Gene flow from transgenic crops. Nature 389:
924.
241 Crawley et al, 1993.

672. Since 1998, various studies have been carried out to address the specific concerns outlined
above and the initial scientific stat ement was updated in 2001 and 2003.
242 Long distance gene flow
is now better established even if the precise role of insects in cross-pollination is still under
investigation. The potential for long persistence of HT plants has been demonstrated through the post-
release monitoring of HT field trials: in one locati on site, and six years after a HT field trial, GMHT
volunteers still accounted for almost 2% of the plants in a non-GM rapeseed field; and this occurred
without any selection pressure as the glufosinate had not been used in that area 243. Such a
phenomenon was only observed in one of several locations. However, this, once again, means that it
was legitimate, as a matter of WTO law, including under the SPS Agreement, for France to take into
account the great diversity of agro-ecosystems.
673. Gene flow models have been designed to assess the effect of cropping systems and of
management practices leading to a better predictive capacity (see question 6). In that context, it has
been confirmed that, under some agricultural practi ces, feral plants could highly contribute to the
spread and establishment of HT crops
244.
674. In the light of these observations, the European Communities again concludes that the weight
of expert advice to the Panel overwhelmingly sup ports the position of the European Communities in
these panel proceedings with regard to the French safeguard measure for MS1 x RF1.
C. TOPAS 19/2 (NOTIFICATION C/UK/95/M5/1) (FRANCE AND GREECE) (QUESTIONS 66, 67
AND 68)
675. Essentially, many of the issues and expert comments with regard to Topas 19/2 are very
similar to those regarding MS1 x RF1, except that Topas 19/2 was approved for seed processing rather
than cultivation. The European Communities will not th erefore re-iterate the questions or the experts'
comments, it being sufficient to observe that, whilst the risks may not have been quite as acute, they
were nevertheless there. And in this context it shou ld not be forgotten that the relevant question is
whether or not the Member State authorities, applyi ng their chosen level of protection, were entitled
to adopt a provisional measure on the basis of available pertinent information.
676. The European Communities would in particular not e that Dr. Andow confirms that, at least in
1998, an authority would have been entitled to take the position that the scien ce was insufficient to
take a definitive view. Areas of concern include d molecular characterisation (a particularly
fundamental issue) and co-existence. Furthermore, the Member States could reasonably have taken
the view that the risk management measures proposed by the SCP were not supported sufficiently by
science. Dr. Andow observes that: "The scientific evidence in 1998 on the agricultural practices
necessary to eliminate HT volunteers and wild species resulting from spillage may not have been
sufficient to complete an accurate assessment of risk management practices."245

242 Opinion of the French Biomolecular Engineering Committee (BEC), 02-2001; BEC Opinion 02-
2004.
243 CETIOM, internal report.
244 Bock, A.-K., Lheureux K,. Libeau-Dulos M. Nilsagard H., Rodriguez-Cerezo E., 2002. "Scenarios
for co-existence of genetically modified, conventional and organic crops in European agriculture", Technical
Report Series of the Joint Research Center of the European Commission, EUR 20394 EN., 133p.. Angevin F.,
Colbach N., Meynard, J. M., 2003. Coexistence of Rapeseed Varieties in Time and Space: Using GeneSys
Model to Adapt Cropping Systems. 11th International Rapeseed Congress, Copenhagen, 6-10 July 2003, pp
732-734. Colbach N., Angevin F., Meynard J. M., Messéan A., 2004 Using the GeneSys model quantifying the
effect of cropping systems on gene escape from GM rape varieties to evaluate and design cropping systems.
OCL, 11: 11-20.
245 Andow, page 86.

677. The European Communities would further note that , at least at the time these measures were
adopted, gene/pollen flow uncertain ties and coexistence were still a si gnificant and relevant area of
scientific uncertainty, in particular for multiple undesired HT gene stacking in oilseed.
678. There was also a significant outstanding issue at the time regarding possible seed escapes
from seed imports and seed dormancy (in particular in the context of the particular seed properties of
oilseed (size, longevity, etc...), and the well known risk of feral escapes during transport from the field
to the crop or the processing plant. These risks had to be considered also in the context of the then
limited availability of proper management tools (including pollen flow understanding and surveillance
and monitoring).
679. Even today, the risk arising from possible seed es capes must still be placed in the context of
the climate in the region of import, including where spring rape is not normally agronomically grown,
and where winter frost may not be prevalent. Th ere may be significant differences, for example,
between Mediterranean ports and Scotland. The risk must also be placed in the context of possible
potential "systemic" HT risks, if several different HT characteristics become present in oilseed rapes
from undesired pollen sources, and giving due consider ation to HT oilseed in the context of agro
management issues, where there are several different HT crops in rotation, which in turn could be
considered to gradually increase prevalence of glufosinate use.
680. In this context, if there is long seed dormancy, and if there had been a general increase over
the years of the use of HT crops, potential oilseed escapes, even limited, may reasonably have been
perceived then to potentially become a problem later, given also that tools were not yet identified at
the time which would have allowed appropriate management or rever sal of the situation, if it had
arisen.
681. A related aspect of the problem is that once cultivation of some HT crops has been authorised,
a decision has also been implicitly taken in principl e regarding future HT crops – even before a full
risk assessment may have been completed. This wo uld, for example, be th e case when it comes to
authorising glufosinate wheat in the context of auth orised glufosinate oilseed, sugar beet and maize,
even if the issues regarding oilseed at that time would have been limited to escapes from imports.
682. With regard to Greece, the European Comm unities would also note that Dr. Andow does
consider that, with reference to the SPS Agreement, the Greek case did raise relevant risks to plant
health. Dr. Andow also considers that the "poten tial pest" concept derived from ISPM should be
considered, and that such argument holds for th is particular case because Greece did identify a
potential risk, and because other potential risks could be identified. He further states that, for those
risks within the scope of ISPM 11, the risk assess ment process was consistent with ISPM 11. In
addition, he notes that the science, documentati on and reasoning of Greece are consistent with
Annex III of the Cartagena Protocol on Biosafety to the Convention on Biological Diversity.
Specifically, Annex III, Section 8(f), which states: "W here there is uncertainty regarding the level of
risk, it may be addressed by requesting further info rmation on the specific issues of concern or by
implementing appropriate risk management strategies and/or monitoring the living modified organism
in the receiving environment."
246
683. The Mediterranean region (including Greece) is a ce ntre of diversity for wild crucifer species,
and gene flow from GM brassica crops to these wild sp ecies is therefore an issue for consideration in
a risk assessment in Greece (Chèvre et al 2004). If escap ed Topas 19/2 plants establish, then there is a
possibility of gene flow to these re lated wild crucifer species which occur in Greece particularly in

246 Andow, pages 89 to 91.

some coastal areas. There was (and still is) uncertainty about gene flow to wild relatives of the GM
crop and possible consequences for rare or uncommon species.
D. M
AIZE BT-176 (NOTIFICATION C/F/11-03) (AUSTRIA, GERMANY, LUXEMBOURG)
(QUESTIONS 69 TO 77)
1. Introductory remarks
684. The European Communities consid ers that Dr. Nutti's response to question 69 is seriously
flawed.
685. First, as the European Communities has explained, it considers that question 69 is aimed at
ascertaining whether or not the science was sufficient for an authority to adopt a definitive decision on
whether or not to approve the product. Dr. Nutti's c onclusion does not address this issue at all. Rather,
Dr. Nutti concludes only that the science was sufficient for an appr opriate assessment. That is self-
evident, the information or science being sufficient for Austria to adopt a provisional measure, based
on an appropriate assessment. Dr. Nutti's conclusi on therefore makes no contribution at all towards
resolving the dispute between the parties.
686. Second, Dr. Nutti offers no detailed statement of fact nor developed line of scientific
reasoning to support her conclusion, confining hersel f to references to certain opinions from certain
scientific committees. She fails completely to addr ess the issue from the pers pective of the specific
legislator's chosen level of protection.
687. Third, she extends her conclusion to environmen tal issues, even though she repeatedly states
that environmental issues are not her field, and th at she cannot therefore offer any advice in that
respect.
688. The European Communities considers that ques tion 70 is designed to assist the Panel in
determining whether or not, underpinning Austria' s safeguard measure, there was an appropriate
assessment or evaluation. The European Communities considers that there was, and has pointed in
this respect not only to the additional documents pr oduced by Austria, but al so to the existing risk
assessments. Dr. Nutti confirms this – re-itera ting what the European Communities has already
referred to, namely the adequacy of the relevant risk assessments. Dr. Nutti thus confirms the position
of the EC.
689. With respect to question 71, Dr. Nutti states that she considers that there is no support for a
temporary prohibition, but offers no explanation for this assertion. The assertion is, in any event,
profoundly perplexing, given that it is uncontroversia l that the initial temporary restrictions imposed
by the general legislation, which are not challenged in these proceedings, are not inconsistent with the
WTO Agreements. Placed in this context, Dr. Nutti's unqualified and unexplained comments offer no
assistance in the resolution of the present dispute. Similar comments apply with regard to Dr. Nutti's
assertions concerning allergenicity and toxicology, insofar as no consideration is given to the question
of whether or not Austria, acting provisionally, based itself on available pertinent information.
690. Similar comments apply with regard to Dr. Nutti's comments concerning Germany and
Luxembourg.
2. Anti-biotic resistance
691. On the question of anti-biotic resistance, the European Communities refers to its comments to
questions 1 and 2 above.

3. Non-target organisms
692. On the question of non-target organisms, apar t from the submissions that it has already made,
and taking into account the general comments that it has already submitted in these observations, the
European Communities would like to make th e following additional points. The European
Communities considers that it is now clear that Bt toxin could accumulate in Bt-resistant herbivores
(e.g. caterpillars which are able to ingest the Bt toxin and thus accumulate it and/or its metabolites
without dying), and so pass the Bt toxin and/or its metabolites to organisms higher up the food web
(e.g. to predators and parasitoids which feed on Bt-resistant herbivores). This point involving multi-
trophic interactions
247 was not dealt with by the SCP in its analysis of risks to non-target organisms in
the environment, at least not in a manner reflecting the specific legislator's concerns.
693. Another issue similarly not considered by the SCP in a manner reflecting the specific
legislator's chosen level of protection is the point that any increase in Bt-resistant herbivores (target or
non-target species) will in crease the need for additional pesticide applications to control them. This
'knock on' effect, arising from the need to contro l Bt-resistant pests using additional pesticides could
add additional environmental risk to local biodive rsity (i.e. there is the potential for adversely
affecting natural enemies, depe nding which pesticides were applie d), spray operators (pesticide
handling and exposure risks) and consumers (pesticide residue risks).
694. On economic grounds (cost: bene fit) the requirement of additional pesticides on Bt corn 176
to control any Bt-resistant insects could negate any initial economic advantage of using GM crop
event 176 (i.e. the savings on pesticide application could be lost and enviro nmental harm could be
increased).
695. The European Communities disagrees that the use of event 176 'and any progeny derived
from crosses with traditional varieties' should automa tically be considered unproblematic, taking into
account the specific legislator's chosen level of pr otection. The genetic background of each parent as
well as any unintended genetic changes during tr ansformation and back-crossing are likely to
introduce changes in plant metabolism and gene expr ession that can affect efficacy of the Bt toxin
(important in the high dose / refuge strategy for insect resistance management) and nutritional value,
via the potential for altered primary and/or secondary plant metabolites 248. Such risks are mitigated
(reduced) in plant breeding by several generations of back-crossing and careful selection of 'normal'
crop phenotypes under a range of appropriate environments - taking several years, normally.
696. The European Communities agrees with Dr. Andow's opinions on the likelihood of Bt toxin
entering the soil ecosystem (via plant debris and/or ro ot exudates) and being more (not less) likely to
cause harm to soil organisms (e.g. collembolan that consume plant remains in the soil) than residues
from Bt sprays applied to foliage, where UV br eaks down Bt toxin in foliar sprays quickly. The
publication by Zwahlen et al. (2003) on earthworm s was apparently criticised by EFSA in July 2004
as not being conclusive and definitive. The EFSA al so made comparisons between growing Bt crops
with risks from using Bt sprays (the latter are know n to be UV unstable, contain different toxins, only
present on leaf surfaces etc) that have been s ubsequently criticised. The cited criticisms by EFSA
should at least have required that further follo w-on scientific investigations were performed
(precautionary approach after some evidence of adverse effects to an important soil NT organism), not
that the scientific evidence shoul d be dismissed and the potential risk to earthworms ignored. The
EFSA also appeared to discount linked eviden ce that in GM maize lines lignin content was

247 Groot & Dicke 2002; Plant Journal 31(4): 387-406.
248 Haslberger 2001; Birch et al 2001; Ann Appl Biol 140: 143-149. See also the comments on the
responses to question 3 for further details and citations.

unintentionally altered, leaving open possible l ong term effects on GM crop decomposition and
nutrient cycling
249.
697. The European Communities agrees with Dr. Andow that by 2003 some, but not all, scientific
uncertainties had been resolved. In particular, not all the most regionally important non-target species
that could be exposed to Bt toxin via consumption of pollen from event 176 had been identified in
Austria or other EC member states. Also, the risk of gene flow to non-GM varieties was still
considered a valid risk by Austria in 2004 – an observation with which the European Communities
would agree. The risks of pests developing resistan ce to Bt toxin, of adverse effects on regionally
important non-target organisms a nd of gene flow to conventional corn, could all potentially be
managed by Member States. Once sufficient region- specific research had been conducted in relevant
Member States. The methods now being developed (e.g. GMO Guidelines project) may now (in 2005)
assist Austria in developing suitable risk assessment and risk management strategies, but this would
take some time to conclude and verify in each relevant Member State.
698. The EU project Bt-BioNoTa reported uninte nded effects of Cry1Ab proteins and Bt
corn/cotton leafworm/green lacewing tri-trophic interactions
250. These results have still to be
published but add to already known uncertainties a bout effects of Bt crops and Bt toxins on NT
organisms.
699. Specifically regarding question 69, Dr. Andow's comments that prior to 1997 impacts on non-
target organisms and the risk of resistance had not been considered effectively are correct. There are
at least two important issues here. First, the di fference between the situation as it was generally
understood in 1997 compared with 2003. Dr. Andow correctly points out that attitudes changed
between these two dates. However, a more fundame ntal change was the increase in both the quantity
and quality of scientific data on NTO effects. By 2003, better designed, more field-based studies had
been conducted. Examples include the extensive st udies on Monarchs by Hellmich, Sears and others
published in 2001. These data models have been further refined
251 and clearly show that effects exist,
even if they are small.
700. The second issue is the agricultural context. Th ere are undoubtedly effects on at least some
non-target organisms252. It may reasonably be anticipated that as more species are tested, more effects
will be found. This supports the view it is very di fficult to be sure that all appropriate NTO effects
have been tested for.
701. A second difficulty which hampers interpretation of the evidence is that findings relating to
NTO effects are often conflicting and may be highly specific to particular GM lines. For example
there was early concern that lacewings would be affected by Bt
253 but more recent work has
contradicted this254. Similarly with Collembola, there are at least 4 studies – 3 of which show no effect
and 1 that has suggested the small effects observed ar e very specific to the particular type of cry1
product.

249 Birch et al., 2004, in Biodiversity and non-target impacts: a case study of Bt maize in Kenya, pp
117-188, CABI Publishing , UK.
250 Scientific presentation by Ruud de Maagd at international scientific conference in Ascona,
Switzerland, Sept 2003 – 'Biodiversity Implications of GM Plants'.
251 E.g. Dively et al 2004.
252 E.g. the examples mentioned by Dr. Andow, indi rect effects on parasitoid abundance as in the
Steffey et al. 2004 study and many others.
253 Hillbeck et al.
254 Romeis 2004.

702. The discussion about soil organisms nicely illustrat es the difficulties in terms of sufficiency
and NTO effects. It is a reasonable and lawful pos ition to say that no Bt crops can be planted until
there is information on all potential non-target organism s in the soil, particularly given that scientists
do not know much about most of the organisms in the soil (they cannot be reared and it is not known
what they feed on).
703. Another issue raised by Dr. Squire is that there is almost no information on the impact of non-
GM Bt preparations, which is a real concern if this is suggested as a comparator in monitoring
schemes. The comments of Dr. Andow relating to the te mporary nature of Bt sprays must be seen in
the light of the fact that there has been almost no monitoring of the impact of these sprays on either
resistance or NTO effects.
704. In terms of how monitoring can help with mitig ation of the risks as suggested by Andow and
Squire, there is still a huge amount of disagreement about how best to proceed. Schemes for specific
monitoring, e.g. to detect resistance, are easy to design, as are those testing specific hypotheses (such
as, are there fewer parasitoids in Bt crops than conventional ones). However, significant problems
arise on the question of general surveillance to detect unforeseen effects: because the effects are
unanticipated it may remain unclear how they are to be detected and dealt with. EFSA has published
draft guidance on general surveillance and both EFS A and the EC working group on PMM are in the
process of designing improved schemes. For example EFSA have already drawn up an environmental
risk assessment and monitoring plan for Bt11 maize. Other suggestions are in the ACRE guidance on
PMM.
4. Resistance management
705. On the question of resistance management, apart from the submissions that it has already
made, and taking into account the general comme nts that it has already submitted in these
observations, the European Communities would like to make the following additional points. The
European Communities would recall, first, that there is a significant risk, given that this is a new issue
specifically relating to Bt crops (regarding continu ous exposure and so on). Second, experience in the
context of the use of conventional pesticides confirms how quickly problems can arise and how
difficult it can be to control the situation once it has developed.
706. Data on resistance management is improving and th ere is less uncertainty in both the results
and how to measure them. The European Commun ities agrees with Dr. Andow that understanding in
2003 is better than in 1997. The Spanish monitoring scheme had been in place for more than 5 years
by 2003 and the EC protocol was published in 2003. There is also improved information from the US,
China and Spain to show how the development of resistance may be controlled if fields are well
managed and there are standard protocols for mana ging the risk. For example, there is the 1998 EPA
resistance management protocol and the report form the EU working group on insect-tolerant crops
protocol
255. There is thus more information on baselin es, monitoring procedures and length of time
needed – although many of these studies are not fro m Europe. Baselines remain problematic in terms
of general surveillance (few are available and those that are are poorly characterised and movable).
The criteria for monitoring plans may be better understood, but there remain arguments about
indicator species, etc. and these will vary between locations. The European Communities agrees that
Dr. Andow's suggestion of a progressive determination of the risks is a sensible one, and is an idea
that has been suggested in the literature many times before256. This tiered approach moves from lab to
small scale field to larger scale field studies and there are several examples of it working very

255 Doc Nr ENV/03/24.
256 E.g. Poppy; Romeis.

effectively. It is also probably the only sensibl e way forward in terms of general surveillance and
indeed it has already begun with the crops in Spain, China and the US.
E. M
AIZE MON 810 (NOTIFICATION C/F/95/12-02) (AUSTRIA, ITALY) (QUESTIONS 78 TO 80)
707. The European Communities has al ready indicated above the funda mental criticisms it has of
the approach adopted by Dr. Nutti.
708. In particular, the European Communities consid ers that Field data on development of Bt-
resistant pests or adverse effects on NT species from cotton in USA and Australia is clearly not
appropriate to determine the risks of growing Mon 8 10 maize in European countries, such as Austria
and Italy. The crop is different (cotton v maize), the target pests are often different in different
continents and countries, the NT species in EC countries are different in different countries and
regions, the cultivation methods for Bt maize are diff erent (due to climate, pest and disease pressures,
scales of fields, etc), the regional environments are different (impacting on plant genotype x
environment interactions), the scales of cultivation ar e different (size of maize fields, connectivity to
non-cultivated flora and fauna), etc. Thus, such gross simplification (i.e. considering Bt cotton in USA
or Australia equivalent to Bt maize in Europe or Austria) is, without doubt, seriously scientifically
flawed.
709. The research by Cornell on Monarch butterflies and Bt maize pollen was considered
'inconclusive'. In these circumstances, it would be appropriate to identify knowledge gaps and
uncertainties, as appropriate to the specific envi ronment under consideration, as well as NT species
and cultivation methods which warrant further scientific testing - not to simply reject the results out of
hand because they are inconclusive.
710. The European Communities thus shares Dr. A ndow's conclusion that scientific evidence in
1999 and 2003 was insufficient to reduce scientific uncertainties surrounding environmental impacts
of Bt Mon810 if grown in the relevant Member State, to an acceptable level.
711. The European Communities would also observe that this product, like Bt 176, also contains
the Cry1ab gene and many of the issues raised are the same (non-target organisms; resistance
management; monitoring). The European Commun ities therefore here also comments on certain
issues that are specific to this pr oduct. One issue which is rather different between Bt-176 and Mon
810 is the molecular characterisation. It is clear that there was uncerta inty over the molecular
characterisation of Mon 810 in 1998, with mo re information being provided by late 2003
257.
Dr. Andow raises this issue in his consideration of Bt 176, to which consid erations he continuously
refers in his comments on Mon 810.
712. The issues relating to resistance management a nd monitoring and NTOs are very similar to
Bt 176. However, the post-market monitoring (P MM) plan was very poor. Even by 2003 ACRE
considered that the PMM plan was not proactive enough.
713. Dr. Andow comes to the conclusion that, as in the case of BT 176, the scientific evidence
available in June 1999 was NOT sufficient to perm it it to undertake an appropriate assessment of
potential risks to plants and the environment. Furthe rmore, in 2003, there remained some uncertainty
about non-target risks of Mon810. Dr. Nutti answer s these questions focussing mainly on resistance
induction and effects on target organisms, which ar e environmental questions, although she indicates
that this is not her field of expertise. Furthermor e she only cites the statements of various Scientific
Committees and agrees, but without discussing the issues.

257 See ACRE documents and UK opinions in the relevant attachments.

714. The European Communities considers that the SC P obviously endorsed the critical arguments
by asking for and encouraging fundamental new and massive instruments for safety management,
such as monitoring in this area to detect any delete rious impacts. Obviously the scientific uncertainty
was very high at this time. As already publicly stat ed, the SCP considers that it would be sensible to
conduct monitoring in post-release situations. Furt hermore, it strongly endorsed the practice of
monitoring with appropriate and adequately targeted methodology, the large-scale introduction of
such crops in order to detect any deleterious im pact on non-targeted Lepi doptera and other insect
populations. The SCP wished to be informed of the results of any such field monitoring studies and as
relevant information continues to become available would further advise the Commission and draw its
attention to any significant concerns that may arise.
715. The European Communities also notes from a US -EPA FIFRA/SAP panel that uncertainties
in Bt resistance induction, models and effects on non target mechanisms are still remarkable. The
European Communities also refers to the WHO/ ANPA report on environmental effects of GMO
where this report indicated significant concerns in the addressed areas at the relevant time.
716. In Exhibit EC 158/30, 1/2004 Austria discusses the principal problems of GM plant
assessment in the fields of non target effects or allergenicity where especially the arguments of
Valenta for difficulties in the assessment of allergen icity are of relevance. Austria argues (Exhibit EC
158/31) that, despite the contrary opinion of the sci entific committee involved at the time, Austria's
doubts for the most part continued to persist. In th e case of the Bt maize varieties admitted, further
doubts had in the meantime also been expressed by sc ientists regarding the possible allergenicity of a
number of Bt proteins, in particular Bt 176 and MON 810 (and also Bt 11). In this connection, a study
commissioned by Austria established that all previous applications for admission under Directive
2001/18/EC and also EC Regulation 258/1997 were inadequate in their safety assessment with respect
to possible toxicological, allergenic and chronic effe cts of proteins formed by genetic modification.
Austria therefore considered that they do not co mply with the new requirements in Annex II of
Directive 2001/18/EC or with the criteria of the new Food and Feed Regulation.
717. Even if a given protein per se does not represent an allergen, its expression in another host
organism may indirectly upregulate the expression of potential allergens. It is therefore recommended
to compare the engineered plant/plant product with that of the parent/wildtype plant/plant product
regarding IgE reactivity to establish whether the transgenic organism represents a more potent
allergen source than the parent/wildtype organism for already sensitized patients. The potentially
increased ability of the transgenic organism versus the parent/wildtype organism to induce de novo
IgE responses (i.e. allergic sensitization) needs to be compared by immunization experiments.
718. Concerning the results of the toxicological assessment of the companies, it must be stated that
the comprehensive toxicological risk assessment as described in Spök et.al. should be carried out. The
recommendations given for a standardized and harm onized approach to the generation, presentation
and interpretation of data concerning toxicology an d allergenicity of GM pr oducts are based on in
depth scientific studies, performed by experienced sci entists in the field. The proposed tests should be
performed by the notifier and the resulting data provi ded in order to guarantee a high level of safety
and public confidence in the approach taken.
719.
 Preliminary results of scientific research on Cry1Ab proteins and the Bt-corn/cotton
leafworm/green lacewing tritrophic interaction – unde rtaken within the scope of the EU-project "Bt-
BioNoTa" show the following unintended effects. Pote ntial effects on non-target organisms (level of
expression) were influenced by the diet chosen, i. e. artificial versus tritrophic diet. This "dietary
effect" is regarded as stronger that the effect of the toxin itself. Cry1Ab increased the mortality in

green lacewing larvae both when fed directly as well as through a preherbivor (when expressed in
cotton).
720. The European Communities would also note that this maize line contains an ampicillin
resistance (bla gene) controlled by a bacterial pr omoter. Considerations detailed in the answer to
general question 1 apply to this GMO. This GMO contains an antibiotic resistance gene already
widely distributed in nature but confers resistan ce to antibiotics which are important and used for
therapy in defined areas of medicine. Since it is rather difficult to have a quantitative estimation of the
risk for human health and since there are still contr oversy on the risk, it seems preferable to use this
GMO only for limited trials in field but not for commercialization.
721. There is a similar environmental case as for Mai ze Bt 176. This consent is for cultivation, so
comments on the environmental impact of Bt on target, and non-target organisms and the
development of resistance in corn borers have mu ch phytosanitary relevance, since they will be
exposed by the growing of Bt maize in Austria or Italy.
722. Austria expressed concern that these issues of the environmental impact of Bt on target, and
non-target organisms and the development of r esistance in corn borers had not been properly
addressed by various committees across Europe. Scien tific papers were appearing which appeared to
show some adverse impacts on non-targets. Resist ance development was considered an agricultural
problem by these committees where as Austria saw resi stance development as an environmental issue
(and under current EU regulations it is now considered as an environmental issue). Strategies for
managing resistance in corn borer were only beginning to be developed in Europe.
723. Dr. Andow observes that, with reference to the SPS Agreement, Austria has evaluated
relevant risks to plant health. Specifically, Au stria has taken into account available scientific
evidence. The "potential pest" concept of ISPM 11 mu st first be considered. This is a special case of
the general argument, and that argument holds for this case because Austria argues that there is a plant
pest risk. For those risks within the scope of I SPM 11 the risk assessment process is consistent with
ISPM 11. Furthermore, the reasoning of Austria is consistent with Annex III of the Cartagena
Protocol on Biosafety to the Convention on Biological Diversity.
724. In conclusion, Austria and Itlay had several phytosanitary grounds for requesting a ban on
cultivation of maize Mon810, which are mostly supported by Andow:
the environmental impact of Bt non-target organisms;
the development of resistance to Bt in corn borers; and
the lack of clear strategies for managing resistance in corn borer.
725. Austria and Italy felt that these issues had not been properly addressed by various committees
across Europe while scientific papers were appear ing which appeared to s how some adverse impacts
on non-targets and Resistance development was cons idered an agricultural problem by these
committees and guidelines were only beginning to be developed.
726. Dr. Nutti states that the issues at stake con cerned undesired effects on NT organisms and
development of resistance in insects based on studies of other Bt plants in other continents. Clearly
she admits that she is not an expert in this area and her answer is inadequate to address the scientific
concerns raised.

727. More recent studies on possible environmental impacts of Bt crops
258 may provide the basis
(risk assessment tools) for appropriate risk manageme nt studies in future, at least in a generic sense.
However, these would need to be modified and valid ated for each Member State so that they were
appropriate (i.e. used to identify most regionally important NT species or ecological functions, most
regionally appropriate cultivation and crop mana gement systems, most appropriate crop and
management systems as 'baselines' to compare levels of risk and benefit).
728. The Codex Guidelines for GM foods to include analysis of unintended effects. "The
broadening of the Codex risk assessment to includ e indirect effects (e.g. unintended changes in GM
crops) now encompass effects of novel foods on the environment that have an indirect impact on
human health". Human health is viewed as an 'integrating index of ecological and social
sustainability', as outlined by the WHO and the Nati onal Agency for Protection of the Environment in
Rome (2000) when reviewing potential environm ental hazards of GM crops. These findings were
based on peer reviewed studies of GM barley, canola, maize, oilseed rape, potato and rice, published
between 1994-2001259.
729. Hence the European Communities disagrees with Dr. Nutti's analysis. There were several
scientific uncertainties in 1998 regarding environm ental harm and animal nutrition (interpreting
animals in its broadest sense to include a range of NT organisms). Levels of protein in GM crop
events are only one of several indicators of unintended changes during transformation which could
affect Italian NT organisms (others would in clude sugars, amino acids and plant secondary
metabolites known to be ecologically important to most insect species).
730. These uncertainties were in part confirmed later in 2003
260, when several unintended effects
in GM crops were reported which would require testing under Italian cond itions (i.e. regionally
relevant NT species, crop management systems, environments etc – see my earlier responses for
references).
731. Dr. Nutti often concludes on environmental issues and refers to CODEX/ FAO/WHO
guidelines, materials. However, Codex Guidelines do not relate to the environmental issues. The
European Communities refers to a published paper
261 which addresses the point that Codex Guidelines
might include the assessment of unintended effects in the environment as a prerequisite for health
assessment. This could include e.g. the need of an assessment of effects in the environment (such as
gene flow) which is relevant for assessing GM health and food safety in case contamination from
products without foods safety assessment enter the food chain.
732. According to Haslberger, the development of ri sk assessment concepts reflected the progress
in the understanding of possible unintended effects of biotechnological methods in breeding. Early
regulations (e.g. EC, directive 90/220) for GMO di d not differentiate betw een environmental and
product specific risks assessments wh ereas most modern regulations differentiate between a general
environmental assessment and asse ssments for specific products, such as pharmaceuticals, foods and
feeds, seeds, chemicals or even fibre products262. Specific risk assessment procedures were developed

258 Eg. from the GMO Guidelines Project; Birch et al., 2004 In: Environmental Risk Assessment of
Genetically Modified Organisms vol 1. a case study of Bt maize in Kenya. Eds Hilbeck and Andow. CABI
Publishing. pp 117-1850, and further case studies in press.
259 See Haslberger, A.G., 2003. Codex guidelines fo r GM foods include the analysis of unintended
effects. Nature Biotechnology 21, 7, 739-741 for summary.
260 See Haslberger, A.G., 2003. Codex guidelines fo r GM foods include the analysis of unintended
effects. Nature Biotechnology 21, 7, 739-741.
261 Ibidem.
262 EC, Biotechnology, 2004.

for these products. This specification resulted in to a diversification for the assessment; however,
experiences drawn from a growing body of risk assessment processes often indicate similar
underlying problems. Notably the need for a mole cular characterisation and assessment of potential
unintended molecular effects was identified as the basis for the assessment in all fields.
733. The concept that a comparison of a final produc t with one having an acceptable standard of
safety provides an important element of safety assess ments of GMOs was a commonly used basis for
the development of both, food safety and the environmental risk assessment
263. This concept was
elaborated by FAO, WHO and OECD in the early 19 90s and referred to as substantial equivalence for
the assessment of GM foods. Bu t in 2000 an FAO/WHO consultati on acknowledged that this concept
had attracted criticism from the perception that it w as the end-point of a safet y assessment rather than
the starting point264. The consultation concluded that a consid eration of compositional changes is not
the sole basis for determination of safety and that safety can only be determined when the results of
all aspects under comparison, and not merely comp arisons of key constituents are integrated. More
recently the concept has evolved to a Comparative Safety Assessment for GMO foods 265. By 2003,
both international systems covering GMO, GM f ood safety (Codex) and environmental safety (CPB)
became effective, where both systems are base d on the concept of a case by case approach. More
recently, the need for a comprehensive molecular characterisation of each transformation event,
including the analysis of integrated constructs and the flanking region as well as the need to address
potential unintended effects was appreciated fo r food safety- and environmental assessments
266 and
this idea was also enforced by general recommendations of a US-FIFRA expert panel267.
734. The Codex Alimentarius Commission adopted the Principles for the Risk Analysis of Foods
Derived from Modern Biotechnology, and the Draf t Guidelines for the Conduct of Food Safety
Assessment of Foods Derived from Recombinan t-DNA Plants and Microorganisms (Codex). The
principles for the safety assessment dictate a cas e by case pre-market assessment on the basis of a
comparative safety assessment (CSA). The CSA is basi cally a two-tiered approach. The initial step is
comprised of a thorough comparison with the closely related conventional food organism counterpart
to identify any differences that may have safety implications for the consumer. This comparison
includes both phenotypic characteristics as well as a compositional analysis. The second step
comprises the toxicological and nutritional evaluation of the identified differences between the food
derived from the GMO and its comparator. Hazard identification and characterization are typically
the first steps in any risk assessment and an ex tensive molecular characterisation of the inserted

263 WHO, 1991. Strategies for Assessing the Safety of Foods Produced by Biotechnology, Report of A
Joint FAO/WHO Consultation. World Health Organisation, Geneva. Available at
<http://www.who.int/foodsafety/publications/biotech/1990/en/>.
264 FAO/WHO, 2000. Safety Aspects of Genetically M odified Foods of Plant Origin. Report of a Joint
FAO/WHO Expert Consultation on Foods Derived from Biotechnology, Geneva, Switzerland, 29 May - 2 June
2000. Food and Agriculture Organisation of the United Nations, Rome.
ftp://ftp.fao.org/es/esn/food/gmreport.pdf; Millstone, E. , Brunner, E., Mayer, S., 1999. Beyond 'substantial
equivalence'. Nature 401, 525-526; Schenkelaars, P. , 2002. Rethinking substantial equivalence. Nature
Biotechnology 20, 2, 119. Canadian Royal Society, 2002
265 Kok, E.J., Kuiper, H.A., 2003. Comparative sa fety assessment for biotech crops. Trends in
Biotechnology 21, 439-444.
266 FAO/WHO, Safety assessment of foods derived from genetically modified animals, including fish, a
joint FAO/WHO expert consultation on food derived from biotechnology, Rome, Italy, 17 - 21 November 2003.
Available at <http://www.who.int/foodsafety/biotech/meetings/en/gmanimal_reportnov03_en.pdf>.
267 SAP Report No. 2004-05. MINUTES of Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) Scientific Advisory Panel (S AP) Meeting, June 8-10, 2004, Arlington, Virginia. A Set of Scientific
Issues Being Considered by the Environmental Protection Agency Regarding: Product Characterization, Human
Health Risk, Ecological Risk, And Insect Resistance Management For Bacillus thuringiensis (Bt) Cotton
Products. 84 pages.

genetic material construct is required. The safet y of the gene product must be assessed on a case-by-
case basis. Following the phase of h azard identification, characterization and food intake-assessment
an integrated toxicological evaluation will combine all the information with relation to the food safety
of the GMO-derived food. For the identification of potentially occurring unintended effects, profiling
methods have been proposed and different possibilities for profiling methods have been
characterized
268. In addition to investigating health risks directly associated with food production, the
broadening of the Codex risk assessment to includ e indirect effects now encompass effects of novel
food on the environment that may have an indirect impact on human health269.
735. A case by case assessment considering any organisms derived from a transformation event as
well as different receiving environments is broa dly recognised as the best framework for assessing
environmental risks of GMOs. Internationally the c oncept of familiarity was developed also in the
concept of environmental safety of transgenic pl ants. The concept facilitates risk/safety assessments,
because to be familiar, means having enough information to be able to make a judgement of safety or
risk270. Familiarity can also be used to indicate appropriate management practices including the
evaluation whether standard agricultural practices are adequate or other management practices are
needed to manage the risk
271. As familiarity depends also on th e knowledge about the environment
and its interaction with introduced organisms, the risk/safety assessment in one region may not be
applicable in another.
736. Currently The Cartagena Protocol on Biosafety (CPB) to the Convention on Biological
Diversity is the only international regulatory instru ment which deals specifically with the potential
adverse effects of GMOs on "the conservation and the sustainable use of biological diversity", an
important amenity of the environment, taking al so into account effects on human health. The CPB
covers transboundary movements of an y genetically modified foods that meet its official definition of
living modified organisms (LMOs). Article 11 of the CPB asks for a risk assessment where annex III
of the Protocol specifies general principles and step s for environmental risk assessment of LMOs and
focuses especially on identification of any novel genotypic and phenotypic characteristics that may
have adverse effects on biological diversity in th e likely potential receiving environment. Annex III
recommends that the risk assessment should also take into account risks to human health and evaluate
the likelihood of these adverse effects. The information on the receiving environment includes data on
the location, geographical, climatic and ecological ch aracteristics, including relevant information on
biological diversity and centres of origin of the likely potential receiving environment. As the focus of
the CPB is biodiversity, in line with the scope of the Convention itself, its consideration of human
health safety is limited, concentrating on situati ons in which an LMO itself may end up in the food
supply, such as might happen via trade of crop seeds
272. Pharmaceuticals are explicitly excluded.
737. Recent work for the implementation of the CPB recommends the analysis of effects of GMOs
on species in the environment before an assessment of effects on the biodiversity for the assessment
of non target environmental risks. This mainly because of a better access to species assessment and

268 Kuiper et al., 2003.
269 Haslberger, A.G., 2003. Codex guidelines for GM foods include the analysis of unintended effects.
Nature Biotechnology 21, 7, 739-741..
270 Environmental Effects of Transgenic Plants. The Scope and Adequacy of Regulation. National
Research Council, 2002. National Academy Press, Washington, 320 pp.Conner et al., 2003
271 OECD, 1993.
272 Miraglia, M., Berdal, K.G., Brera, C., Corbisier, P., Holst-Jensen, A., Kok, E., Marvin, H.J.P.,
Schimmel, H., Rentsch, J., van Rie, J. P. & Zagon, J. (2004; alphabetical order). Detection and traceability of
genetically modified organisms in the food production chain. Food Chem. Toxicol. 42: 1157-1180.

methodological limitations of an an alysis of effects to diversity
273. Specifically a non target risk
assessment for non target effects of GM crops was discussed to be more useful than the
conventionally used eco-toxicology or non-indige nous-species model. This new model requires
information about the intended receiving environment and aims to focus on the analysis of possible
effects of local species with a specific ecological function in this system.
738. Both, the Codex principles for food safety a nd the risk assessment provisions of the CPB
provide opportunities to more explicitly consider interactions between food safety and environmental
safety. The broadening of the Codex Risk assessment to include indirect effects provides for assessing
effects on the environment that may have an indirect impact on human health274.
739. In the explanatory guide to the CPB (CPD, explanatory guide) indirect effects on the
environment are described as effects on human health or the environment occurring through a causal
chain of events, through mechanisms such as interactions with other organisms, transfer of genetic
material, or changes in use or management. Observati ons of indirect effects are likely to be delayed.
Given examples include impacts which can arise from changed agricultural practices associated with
the management of a genetically modified (GM) cr op rather than from the genetically modified crop
itself. The explanatory guide suggests that questions related to human health effects were intended to
be covered by the Protocol only if the human health aspect is linked to biological diversity. Such a
link exists if the health effect is consequent to exposure in situ , for instance, if a farmer were to
develop an allergenic reaction to pollen from genetically modified plants; it also exists if the health
effect resulted from effects on biological diversity (secondary effect). Direct effects on human health
(e.g. caused by consumption of GM food) would, however, not be covered by the Protocol.
F. MAIZE T25 (NOTIFICATION C/F/95/12-07) (AUSTRIA, ITALY) (QUESTIONS 84 TO 89)
740. The European Communiti es has already outlined above its fundamental criticisms of the
approach adopted by Dr. Nutti.
741. The European Communities considers each GM cr op event should be examined on a case by
case basis, as stated in the Cartegena Protocol. This means that Member States are justified in
requesting region specific testing, since each GM event can respond differently to regional differences
in environment (weather, soil types etc), regiona l pest and disease pressures, regional agronomic
practices (e.g. pesticide and herbicide use, tillage systems), regional abundance of flora (including
weeds which could become resistant to herbicides) and fauna (which could be affected by any
unintended changes in the GM crop).
742. The European Communities agrees with Dr. A ndow that secondary ecological effects (longer
term over several seasons and indirect effect s resulting from the GM crop and its management
system) need to be assessed for each Member State under regionally appropriate conditions.
743. For GMHT crops, the UK Farm Scale Evaluation (FSE) trials indicated particular risks of
using HT crops in combination with particular he rbicides and particular crop management systems.
For maize no adverse effects were detected for GMHT maize, but the comparison was with
conventional maize used with the herbicide atrazi ne. This herbicide has known adverse effects on the
environment and NT organisms and will be banned in the UK shortly, so was widely criticised as a

273 Andow & Hilbeck (2004). Science-based risk assessm ent for non-target effects of transgenic crops.
BioScience 54: 637-649. Birch et al., 2004 In: Environmental Risk Assessment of Genetically Modified
Organisms vol 1. a case study of Bt maize in Kenya. Eds Hilbeck and Andow. CABI Publishing. pp 117-1850..
274 Haslberger, A.G., 2003. Codex guidelines for GM foods include the analysis of unintended effects.
Nature Biotechnology 21, 7, 739-741.

poor/inappropriate choice as a 'control treatment' fo r comparison with GM HT maize in the UK FSE
trials.
744. Hence the European Communities considers that regionally appropriate combinations of the
GMHT crop, herbicides, pesticides and agronomic prac tices need to be tested in comparison with an
appropriate baseline combination of these factors (e.g . to detect beneficial or adverse changes in
regional biodiversity on herbivorous species fe eding on weeds affected by the GMHT crop
management system).
745. Co-existence (and related contamination levels in seeds) is a relatively new issue, although
even here scientific uncertainties about distances of GM gene flow and establishment for several GM
crops including maize were widely discussed and str ongly debated Europe for several years prior to
2003.
746. The European Communities would agree with Dr. Andow that in 2003 maize T25 was
inadequately characterised regarding Austria's n eeds on risk management in relation to possible
effects on regionally import NT species, on regional ge ne flow, on regionally important weed control
issues and agronomic practices and on possible contamination issues if GM genes are introgressed
into conventional or organic maize in Austria, or other Member States. Johnson
275 provides a set of
generic questions which EC Member States could now adapt to carry out a more scientifically
rigorous risk assessment of GMHT crops and a review of regionally-appropriate management
strategies.
747. The European Communities observes that the an alysis of Dr. Andow is differentiated and
science based. He argues that the amended SCP Opin ion (CDA-77, 20 July 2001) does not provide an
assessment of environmental risks beyond gene flow risks. These would include non-target and other
biodiversity risks. The amended SCP Opinion (CDA-77, 20 July 2001) does not provide an
assessment of this environmental risk. Even tho ugh the Member States did not provide any scientific
evidence that this risk exists for T25 maize, resistance risks are widely recognized, and the consistent
use of glufosinate with T25 maize would result in a resistance risk. Thus, there was insufficient
scientific evidence available to the SCP and Austri a and Italy to assess weed resistance risk and
appropriate risk management measures.
748. The Member States expressed the view that risks from gene flow (dispersal) is small but still
must be monitored. This is especially true for specific regions. Here a concept for specific regions,
sensitive areas, is specified, scientifically cited an d explains why a monitoring is especially necessary
in the light of risks to specific areas/regions. As no monitoring concept was available Austria's
concern was justified.
749. Furthermore, special measures monitoring the possi ble – mostly regarded as safe – spread of
pollen to fields in the surroundings cultivated with conventional maize are missing.
750. The lack of a monitoring programme regarding th e long term effects of genetically modified
plants and herbicides can be criticised especially because of the fact that the approval conditions do
not foresee a protection of sensitive areas. Fu rthermore, regional ecological aspects are not
differentiated: the use of herbicide resistant plants in areas of unavoidable applications of herbicide
seems to be useful, if good agricultural practice minimizes the danger of a resistance development.

275 Johnson, Blancas and Borem (2004) (CABI book: 'Environmental Risk Assessment of GMOs: A
case study of Bt maize in Kenya'.

751. This concerns of Austria and Italy were ev en more substantiated when science in the
following years elaborated hazards from gene flow and dispersal, also in maize, addressing especially
problems in the area of co-existence. Co-existence arguments were addressed by Austria in detail in
subsequent discussion. Some points of the opinion of the SCP presented in 2000 may need to be
reflected by subsequent scientific findings on gene flow.
752. At the time of this application, concerns about the ecological impacts of the herbicides used
on HT crops was high and UK had implemen ted the Farm Scale Evaluation programme
276 to examine
these effects in maize and other crops.
753. There are at least 6 reasons for the safeguard measures. The reasons are: (1) The
environmental risks of T25 have not been sufficientl y evaluated under realistic conditions in Europe.
(2) There is no post-commercialization monitoring program. (3) Although harm from pollen transfer
is likely absent, the potential for gene flow to conventional maize production fields needs to be
quantified and included in monitoring because of its agricultural consequences. (4) There is no
provision for the protection of ecologically sensitive regions. (5) There is a need for "good farming
practice" guidelines to minimize the danger of herbicide resistance. (6) There is a need to assess long-
term and secondary ecological effects, especially those due to changes in herbicide management.
754. With reference to the SPS Agreement, the Member States have evaluated a risk relevant to
plant health. The "potential pest" concept of ISPM 11 must first be considered. This is a special case
of the general argument, and that argument holds for this case because Austria argues that there is a
plant pest risk. For those risks within the scope of ISPM 11 the risk assessment process is consistent
with ISPM 11. The reasoning of Austria and Ital y is consistent with Annex III of the Cartagena
Protocol on Biosafety to the Convention on Biological Diversity.
G. M
AIZE MON 809 (NOTIFICATION C/F/95/12-01/B) (ITALY) (QUESTIONS 90 TO 92)
755. The reasons why Italy declines to authorise th e placing on the market of this product refer
especially to the risk assessment based on substantia l equivalence (see the previous discussion of that
question in these comments), to international discussi ons and criticisms of this principle also under
CODEX, and to changes of the use of the principle and the legislation:
"… Having regard to the subsequent opinion of the Superior Institute of Health, dated
28 July 2000, addressed to the Minister of Health, which addresses the concept of
"substantial equivalence" from a technical- scientific viewpoint and states that, if
interpreted from a legal viewpoint, the concept could be defined differently, given the
lack of clarity in the Community legislation;
Considering that the Superior Institute of Health, in its opinion, pointed out that, of
the seven notified products cited above, the four types of maize contain levels of
protein deriving from the genetic modifica tions ranging from 0.04 to 30 parts per
million and that, therefore, the foodstuff has been permanently affected by the
modified elements;
Considering also that, in the same opinion, it is reported that the technical
documentation available does not examine the GMOs in comparison to their
conventional counterparts with regard to th e presence of certain micro-constituents,

276 Firbank et al 2003 e.g. Rieger et al., 2002. SQUIRE, G.S., BROOKS, D.R., BOHAN, D.A.,
CHAMPION, G.T. et al. (2003) On th e rationale and interpretation of the Farm Scale Evaluation of genetically
modified herbicide-tolerant crops. Philosophical Transactions of the Royal Society B, 358, 1779-1800..

and it finds a substantial identification with the conventional counterparts from a
purely nutritional standpoint (micro and macronutrients) but not from the standpoint
of the composition of the product, as foreseen in the Community regulations, because
of the presence of modified molecules;
Whereas, in the aforementioned opinion, the S uperior Institute of Health also comes
to the conclusion that, in the light of current scientific knowledge, there are no
apparent risks to the health of humans or livestock from the consumption of
derivatives of the aforementioned GMOs, but that this conclusion is reached in a
context in which, from the correspondence with the President of the European
Commission and with the pertinent European Commissioner, it is evident that there
are inadequacies in the risk assessment procedures;
Whereas, furthermore, in the aforementioned opinion, the Superior Institute of Health
declines to express an opinion regarding the risk of possible "environmental release"
of the GMOs in question, even when it has been ascertained that residues of modified
components remain in the foodstuffs, so that the inadequacy of information deriving
from the previous phase of preliminary assessment of this environmental release for
the simplified authorization procedure appear s to be even more deleterious to the
precaution principle, generally adhered to in these matters;"
756. The European Communities notes that Dr. Nutti agrees with the opinion of the Italian CA
regarding the ambiguity of the term "substantial e quivalence", and agrees that this concept could be
defined differently. In this respect, the European Communities notes that the assessment of the Italian
arguments by the SCF (in CDA 86) did not directly address in detail the substantial equivalence issue.
757. The European Communities also not es that there are some important toxicological issues that
have not been addressed by Dr. Nutti. For instance, the recently published observation on
occupational allergy to Bt bacterium spores in farm ers using Bt pesticides mentioned by the SCF and
by Italy. Furthermore, the Italian argument concer ning the risk of possible environmental release of
the GMOs in question, even when it has been ascertain ed that residues of modified components
remain in the foodstuffs is not discussed by Dr . Nutti. Thus, in essence substantial disagreement
remains on the question of substantial equivalence. It would appear that Italy effectively anticipated a
change in the relevant scientific and legal elements.
758. The concept of substantial equivalence is a key step in the safety assessment process.
However, it is not a safety assessment in itself; rath er it represents the starting point which is used to
structure the safety assessment of a new food relative to its conventional counterpart. This concept is
used to identify similarities and differences between the new food and its conventional counterpart. It
aids in the identification of potential safety an d nutritional issues and is considered the most
appropriate strategy to date for safety assessme nt of foods derived from recombinant-DNA plants.
The safety assessment carried out in this way do es not imply absolute safety of the new product;
rather, it focuses on assessing the safety of any iden tified differences so that the safety of the new
product can be considered relative to its conventional counterpart.
759. The misleading conclusion that a risk assess ment according to substantial equivalence per se
can establish safety was corrected explicitly in 2000 when an FAO/WHO consultation acknowledged
that this concept had attracted criticism from th e perception that it was the end-point of a safety

assessment rather than the starting point
277. The Consultation concluded that a consideration of
compositional changes is not the sole basis for de termination of safety and that safety can only be
determined when the results of all aspects under comparison, and not merely comparisons of key
constituents, are integrated. More recently the concept has evolved to a Comparative Safety
Assessment for GMO foods278.
760. Thus, in the light of its chosen level of prot ection, the European Communities considers that,
as a matter of WTO law, Italy was justified, before carrying out analysis on substantial equivalence, to
require access to data describing the molecular make -up of the transgenic events. This is necessary in
order to have a good description of the plant material used in the comparative analysis (e.g. chemical
composition, nutritional value, etc.).
H. MAIZE BT-11 (REFERENCE C/GB/96/M4/1) (ITALY) (QUESTIONS 93 TO 95)
761. The European Communities refers to its observatio ns with regard to the preceding product, as
well as its general observations and its observations in the context of the alleged product specific
delays, which apply mutatis mutandis . The European Communities h as the following additional
remarks and conclusions.
762. The information requested and supplied from the company was mixed, scarce, delivered
consecutively all over years, and not convincing. The quality of the dossier can therefore be
considered as not sufficiently informative, taking in to consideration the specific legislator's chosen
level of protection. The major weaknesses of the dossier relate to:
No sufficient experimental evidence to assess the safety;
Compositional data insufficient for a product directly consumed by human;
No in vivo experiments conducted on laboratory or farm target animals with grain of
the event "sweet maize";
Field maize used as a control - grain materi al was spiked with Bt proteins (resulting
in poor and unsatisfactory experimental conditions);
Further experiments performed on ruminants using the whole plant silage or stalk
have no meaning for the safety assessment.
763. Taking into account the legislator's chosen level of protection, these issues could be
considered to have justified requests for further evidence on the safety of the product.

277 FAO/WHO, 2000. Safety Aspects of Genetically M odified Foods of Plant Origin. Report of a Joint
FAO/WHO Expert Consultation on Foods Derived from Biotechnology, Geneva, Switzerland, 29 May - 2 June
2000. Food and Agriculture Organisation of the United Nations, Rome.
ftp://ftp.fao.org/es/esn/food/gmreport.pdf; Millstone, E. , Brunner, E., Mayer, S., 1999. Beyond 'substantial
equivalence'. Nature 401, 525-526; Schenkelaars, P. , 2002. Rethinking substantial equivalence. Nature
Biotechnology 20, 2, 119. Canadian Royal Society, 2002
278 Kok, E.J., Kuiper, H.A., 2003. Comparative sa fety assessment for biotech crops. Trends in
Biotechnology 21, 439-444.

VII. THE PANEL'S QUESTIONS ON ISSUE 3 ("LIKENESS") (NOS 96 TO 109)
Question 96

Has there been a significant change in the und erstanding of physical, chemical and biological
characteristics of biotech products, gene interac tions, gene expression, gene silencing, molecular
characterization, and product specific detection since 1998?

General comments

764. The Panel has received responses from Dr. Nutti and Dr. Squire, indicating a difference of
approach. Dr. Nutti makes clear that she excludes from her response any view in relation to "any
environmental issue", which she states is "not my area of expertise". On the matters within her
expertise she states that "the approach remains essentially the same". The European Communities
considers that this does not provide a clear or unambiguous response to the question posed.
765. Dr. Squire considers that "most of the underl ying molecular and biochemical knowledge of
the subject has not changed substantially during th is period." However, he makes it clear that the
absence of substantial change should not be taken as an inherently positive development in the state of
scientific understanding. As he puts it: "There is still uncertainty over unintended side-effects
occurring in biotech products, and the continued stab ility of phenotype in biotech plants or continued
expression of their genes outwith the crop itself (i.e. in volunteers and feral plants)." The
Communities understands this to mean that the ab sence of substantial change indicates continued
levels of uncertainty.
766. The European Communities considers that th ere has been a significant change in the
understanding of the issues addressed by the question.
Detailed comments

767. Although, the basic principles of risk assessm ent remain unaltered, there has been a
significant change in the understanding of most of th e characteristics listed in the question. This is
primarily because of the accumulation of considerab ly more data and the development of more
effective analytical tools for characterization, data mining and data comparison.
768. The Communities would refer by way of example in the food safety domain to a research
project - "ENTRANSFOOD, European network - safety assessment of genetically modified food
crops" – which was launched in 2000 to identify ke y issues of the safety evaluation of genetically
modified food crops, and to examine whether current research methods were adequate to characterize
specific safety hazards. The project was conclude d in 2003, and a series of position papers were
published in a special volume of Food and Chemical Toxicology.
279
769. The first position paper 280 provides an updated approach for adapting the test strategy to the
characteristics of the modified crop and the introduced trait, and assessing potential unintended effects
from the genetic modification. The second position paper 281 focuses more specifically on detection
and characterization of unintended effects in GM crops . It pays particular attention to new profiling

279 G. van den Eede, H. Aarts, H.-J. Buhk, G. Corthier, H.J. Flint, W. Hammes, B. Jacobsen, T.
Midtvedt, J. van der Vossen, A. von Wright, W. Wackemagel, A. Wilcks. Food and Chemical Toxicology 42
(2004), 1127-1156. (Volume 42, issue 7, July 2004).
280 König et al., 2004.
281 Cellini et al., 2004

technologies, including metabolomics, proteomics and transcriptomics. The background for the
project was that significant developments had been made with respect to the scientific understanding
of underlying biological, physical and chemical m echanisms in general, and particularly biological,
physical and chemical characteristics of biological products, including biotech products. Considerably
more is known since 1998 about gene interactions and gene expression. The approaches applied to
molecular characterization are different, in particular they are much more efficient and sensitive, and
allow us more efficient data mining. Information made available in e.g. genome and protein sequence
databases has made possible assignment of function to numerous sequences characterized in e.g. plant
species by comparison with characterized sequen ces of related plant species. One example is
provided in this report under EC chronology 63, exhibit 63/119.
770. As is clear elsewhere, there has also been adva nces of several orders of magnitude in the
development and availability of product specifi c detection (see comments on question 8),
methodologies for genetic modification characterization (see comments on question 9), and
identification of unintended effects thereof (see comments on question 111).
Question 97

On the basis of the information before the Pane l, is there new scientific evidence since 1998 that
would suggest that the potential risks to human, plan t or animal health, or to the environment, from
any of the specific biotech products subject to this dispute (including products subject to the member
State safeguard measures), are different in nature or magnitude as compared to the scientific
understanding of the risks associated with such biotech products prior to 1998, taking into account:

• the intended use of each product (direct human or animal consumption, further processing for
consumption, planting or any other specified use);

• any potential risks that may arise from the combination or successive use of biotech products.

Does the information before the Panel support the vi ew that the potential risks from the products in
this dispute should be assessed differently than the risks from biotech products approved prior to
1998?

General comments

771. The European Communities notes that there is support amongst the independent experts for
the view that the potential risks to human, plant or animal health, or to the environment, from some of
the specific biotech products subject to this dispute (including products subject to the member State
safeguard measures), are different in nature or ma gnitude as compared to the scientific understanding
of the risks associated with such biotech products prior to 1998. The information before the Panel
supports the view that potential risks from some of the products in this dispute should be assessed
differently than the risks from biotech products approved prior to 1998.
Detailed comments

772. The Panel has received responses from Dr. S now, Dr. Andow and Dr. Squire which are
broadly consistent, and a response from Dr. Nutti wh ich adopts a different approach. Dr. Nutti once
again excludes any consideration of environmenta l risks from her response. In relation to the
remaining matters she concludes that:

"My opinion is that, based on the existing literature and knowledge on the subject,
and from a scientific perspective, there is no new evidence that would suggest that the
potential risks to human, animal or plant health, from any of the specific biotech
products subject to this dispute, are different in nature or magnitude as compared to
those products of biotechnology approved by the European Communities prior to
October 1998."
773. In relation to environmental risks Dr. Snow reaches a different conclusion:
Yes, new scientific evidence has become available since 1998 with regard to gene
flow from herbicide-tolerant crops and taking into account the environmental
concerns and management goals of EC Member States. New scientific studies
published during 1998-2003 showed that the dispersal of transgenes that confer
resistance to glufosinate and glyphosate will occur much more widely and more
quickly than was previously expected, as I discuss in Part I. Also, new studies
showed that the widespread use of gluf osinate-tolerant and glyphosate-tolerant
oilseed rape could lead to populations of volunteers and weeds that are more difficult
to manage than their nontransgenic pred ecessors. Adopting these herbicide-tolerant
crops could lead to greater dependence on these and other herbicides (see Part I and
Friesen et al. 2003). These problems would not arise without the use of transgenic
crops because the genes that confer resistance to glufosinate and glyphosate have not
been found to occur naturally in the crop's gene pool." She concludes her response as
follows: "In summary, the types of new scientif ic information that I discuss in Part I
of my answers can be used to support the view that potential risks from the products
in this dispute could be assessed differen tly than the risks from biotech products
approved prior to 1998.
Dr. Squire concludes that

knowledge of the ecological impacts of biotech products and their spread and
concentration in the agricultural environm ent has changed, particularly in that
emergent properties at the scales of the field and landscape are now much better
appreciated even if they are still far from fully understood.
774. He provides several examples to support that conclusion.
775. Dr. Andow provides a detailed response which concludes that "risk assessment methodologies
and evidentiary standards for what constitutes an objective environmental risk assessment had
changed substantially from 1998 to 2005." These cha nges have come about "from increased scientific
knowledge about transgenic crops. This has affected risk assessment for transgenic crops intended for
planting in the environment. Scientific investiga tion of risks of combination biotech products has
lagged behind." According to Dr. Andow the basi s for risk assessment of transgenic crops has
changed
because several significant scientific points had come to light. (1) It became widely
appreciated that the molecular basis of transformation was more complex than
originally thought, and the implications of these findings for risk assessment were
articulated. (2) Non-target risk assessment shifted from assessing indicators of
environmental risk to assessing actual identified potential environmental risks.
Presently this is done on an ad hoc basi s, as no systematic methodology has gained
widespread acceptance. (3) Gene flow ri sk assessment has shifted from being based

primarily on an assessment of the probabilit y of gene flow to being based on an
assessment of both the probability of gene flow and the conditional hazard
probability. (4) Resistance risk is cons idered an environmental risk, and science-
based resistance management measures are required.
776. The European Communities notes that the respon ses of Dr. Snow, Dr. Squire and Dr. Andow
are consistent with the concerns of the European Communities in relation to environmental risks, and
that they justify the case-by- case approach to which the Commun ities is committed. The consensus
amongst them is that the whole field is very complex, that we do not fully understand it, that some of
the hazards predicted pre-1998 are beginning to em erge. These effects include the molecular basis of
the transformation, horizontal gene flow, resistance and problems with non-target species.
777. With regards to non-environmental risks, the Communities does not share the views of
Dr. Nutti. In particular, on the basis of existing research (and the absence of any exposure data on
which Dr. Nutti or other might rely) it is impossible to know whether the introduction of GM food had
had any human health effects other than acute toxi c reactions. Therefore it is impossible to comment
on any changes that might have occurred since 1998.
778. As regards research which has been carried out , this indicates a growing understanding of the
inadequacies of risk assessments which have been carried out on GM foods, having regard to the
uncertainties already referred to: see Freese and Schubert.
282 The scientific concerns noted by Freese
and Schubert (and which have been shared by many other scientists for several years) relate to:
• the use of surrogate Bt toxins to test for allergic, toxic or environmental NT effects;

• unintended effects being ignored or deemed unimportant by US assessors 283 when
there are several peer reviewed publications on such effects in GM crops;

• test protocols for risk assessment of poten tial human health aspects of GM crops are
not considered rigorous by these authors;

• comparisons with Bt sprays are considered invalid because of major differences with
Bt crops in terms of Bt toxin types, expo sure routes, exposure times, thermostability,
digestive stability, etc;

• metabolic profiling methods deployed to da te are largely only semi-quantitative and
often fail to accurately measure smaller, biologically active secondary metabolites in
GM plants (which can change as a result of transformation);

• metabolic stress responses of GM crops grow n in particular envi ronments have been
largely ignored by US assessors;

• chronic (long term) tests on GM crops and GM products are often lacking or poorly
executed;

282 Review published in Nov 2004, covering literature from 1991-2003 – Biotechnology and Genetic
Engineering Reviews 21, 299-324
283 See also Haslberger, A.G., 2003. Codex guidelines for GM foods include the analysis of unintended
effects. Nature Biotechnology 21, 7, 739-741.

• the effects of different genetic backgr ounds and growing conditions (determining
plant genotype x environment interactions) for GM crops has been largely ignored,
although many scientists have expressed concerns about lack of data in this area; and

• the effects on human health post-release requires properly conducted epidemiological
studies, comparing GM food intake as a treatment with intake of conventional food
not contaminated with GM products, or with organic food not contaminated with GM
products. There do not appear to be any such epidemiological studies on human
populations having been published in peer reviewed journals.

Question 98

From a scientific perspective, is there a significant difference in risks to human, animal or plant
health or the environment arising from the use of a bacterial antibiotic resistance marker gene, or
part thereof, in any biotech product at issue in this dispute (e.g., Monsanto Bt cotton (531), Monsanto
Roundup Ready cotton (RRC1445), Amylogene starch potato) compared to those products of
biotechnology approved by the European Communities prior to October 1998?

General comments

779. The European Communities notes that there is support amongst the independent experts for
the view that there may be significant difference in risks to human, animal or plant health or the
environment arising from the use of a bacterial antibio tic resistance marker gene, or part thereof, in
some of the biotech products at issue in this dispute as compared to those products of biotechnology
approved by the European Communities prior to Oct ober 1998, having regard to changes in the state
of scientific understanding.
Detailed comments

780. The Panel has received a response from Dr. Nutti, which does not address environmental
risks. She states that "There have been numerous experiments aimed at evaluating the possibility of
transfer of plant DNA to microbes and mammalian ce lls. To date, there are no reports that marker
genes in plant DNA transfer to th ese cells." As noted and commented under question 1, this statement
which is taken from her reply to question 1, is ab solutely not correct. She notes, however, that "Even
so, the use of alternative tr ansformation methods, which do not use ARMG, is encouraged. If
alternative marker genes are used, they also must be evaluated regarding their safety." And she then
concludes:
My opinion is that, based on the existing literature and knowledge on the subject, and
from a scientific perspective, there is no new evidence that would suggest that the
potential risks to human, animal or plant h ealth arising from the use of a bacterial
antibiotic resistance marker gene, or part th ereof, from any biotech product at issue
in this dispute (e.g., Monsanto Bt cotton - 531, Monsanto Roundup Ready cotton -
RRC1445, Amylogene starch potato) are different in nature or magnitude as
compared to those products of biotechnology approved by the European
Communities prior to October 1998.
781. The European Communities notes that Dr. Nutti acc epts that the use of marker genes other
than ARMG "is encouraged". The Communities consid ers that the evidence indicates that the use of a
bacterial antibiotic resistance marker gene, or part thereof, may contribute to a significant difference

in risk including by means of transfer,
284 and that precautionary measures are therefore justified. The
Communities notes that evidence of the risk of tr ansfer only began to emerge in the scientific
literature in 1998 and in following years, and that no studies were available prior to 1998. A lot of the
relevant research is referred to in the ENTRANSFOOD publication. 285 The European Communities
refers to its comments on questions 1 and 2 above.
782. The presence of antibiotic resist ance genes in some of the biot ech products at issue in the
dispute (Monsanto Bt Cotton, Monsanto Round Read y Cotton, Amylogene starch potato, Maize T25,
Maize MON 810, Maize Bt-176, Topas 19/2, clearly makes a difference as compared to older
products, and this difference has been examined for its medical impact. The evidence is still
controversial.
783. For risk analysis, the risk should not be take n in general but analysed on a case-by-case basis.
Two parameters are to be considered: the existing spread of the antibiotic resistance marker in
bacteria isolated in man, in animals and in th e environment, and the relevance of the concerned
antibiotic(s) for human therapy. Based on these para meters, the lowest degree of risk (although not
zero) is represented by the nptII gene (kanamycin resistance), and a higher degree of risk by the bla
and the aadA genes. The qualification of "higher" risk for the later category is justified by the fact that
these genes confer resistance to important antibiotics. These genes have already spread, but so far, no
experiment has provided data to quantify the cont ribution of these resistance genes to the increase of
resistance genes in pathogenic b acteria. The assertion that the transfer is null or very low is not
documented and this question remains open. The bla Topas 19/2 and Amylogen Starch potato contain
the nptII gene and Maize MON 810, Maize T25, Maize Bt-176 contain the bla gene (ampicillin
resistance).
784. The Communities considers, in accordance with the relevant Codex guidelines, – and
Dr. Nutti appear to agree, albeit less strongly – th at there is no justification to maintain these
unnecessary resistance genes where alternatives exis t, and that ARMG coding for resistance to
antibiotics of clinical use, should not be present in GM products.
785. Finally, the European Communities wold like to indicate, that new ARMG were to be present
in GM product assessed between 98 and now, some of them being though to present significant
human health threads, such as nptIII, encoding fo r amykacin resistance, an antibiotic of critical
importance in certain pathological conditions.
Question 99

For those biotech products at issue in this disput e for which no significantly different nature or level
of risk has been identified, does the information be fore the Panel provide a scientific or technical
rationale for monitoring the occurrence of potential adverse effects, or of unintentional effects,

284 See: de Vries J, Wackernagel W. Integration of foreign DNA during natural transformation of
Acinetobacter sp. by homology-facilitated illegitimate recombination. Proc Natl Acad Sci U S A. 2002,
99:2094-9; Gebhard F, Smalla K. Transformation of Acinetobacter sp. strain BD413 by transgenic sugar beet
DNA. Appl Environ Microbiol. 1998, 64:1550-4 ; Jain R, Danziger LH. Multidrug-resistant Acinetobacter
infections: an emerging challenge to clinicians. Ann Pharmacother. 2004,38:1449-59; Nielsen KM, Bones AM,
Smalla K, van Elsas JD. Horizontal gene transfer from tr ansgenic plants to terrestri al bacteria--a rare event?
FEMS Microbiol Rev. 1998, 22:79-103; Nielsen KM, van Elsas JD, Smalla K. Transformation of Acinetobacter
sp. strain BD413(pFG4DeltanptII) with transgenic plant DNA in soil microcosms and effects of kanamycin on
selection of transformants. Appl Environ Microbiol. 2000, 66:1237-42.
285 G. van den Eede, H. Aarts, H.-J. Buhk, G. Corthier, H.J. Flint, W. Hammes, B. Jacobsen, T.
Midtvedt, J. van der Vossen, A. von Wright, W. Wackernagel, A. Wilcks. Food and Chemical Toxicology 42
(2004), 1127-1156.

arising from the consumption or use of these produc ts compared to those products of biotechnology
approved by the European Communities prior to October 1998?

General comments

786. The European Communities notes that there is general s upport amongst the independent
experts for the rationale for monitoring, and suppor t amongst some for the view that monitoring may
be appropriate for unanticipated risks having regard to the state of scientific understanding (noting in
particular that it has developed significantly since 1998).
Detailed comments

787. The Panel has received responses to this question from Dr. Nutti, Dr. Snow and Dr. Andow.
788. Dr. Nutti considers that
"the biotech products at issue in this dis pute should be treated in the same way as the
biotech products approved by the European Communities prior to 1998. The products
approved prior to 1998 are the so called "first generation" products. Most of them had
the new trait introduced in order to become insect resistant or herbicide tolerant, and
they were assessed as equivalent to th eir conventional counterparts. The products
approved prior to October 1998 have been c onsumed in different regions of the world
(USA, Canada, Argentina, Australia, New Zeal and) and, no adverse effects related to
their consumption have been reported in any of these countries." Dr. Nutti does not
appear to object to monitoring or consider that there is no scientific or technical
rationale under certain circumstances: "C ontinuous use of risk assessments based on
the Codex principles and, where appropriate, including post market monitoring,
should form the basis for evaluating the safety of GM foods."
789. Dr. Snow notes that this question refers to pro ducts for which no significant risks have been
identified, whereas many of the questions above re fer to products for which one or more Member
States have identified potential risks. She states that
"the scientific basis for which environmental consequences to monitor and how to do
this in a scientifically rigorous manne r are not well developed (e.g., NRC 2002).
There are certain types of environmental effects for which monitoring can be justified
on scientific or technical grounds. Specific and plausible environmental harms should
be identified before allocating time and money to these efforts."
790. In his response Dr. Andow begins by noting that "For all of the products at issue in this
dispute, there are some member countries that di sagree with the assessment of the relevant SCP (for
example) that there are no significant identified risks. This disagreement is sometimes related to
different standards of acceptable risk being applied by the disagreeing parties." He considers that this
disagreement provides "the only rationale for monitori ng potential adverse effect s" and that "[i]f all
parties agreed that there were no significant iden tified risks, there would no need to consider
monitoring for potential adverse effects." He identifies the various mechanisms by which an
unanticipated risk could arise, and states that "All of these possible sources unanticipated effects are
equally likely to occur for plant biotech products before 1998 and plant biotech products after 1998."
In his view all of these factors "provides a rationale for monitoring".

791. The European Communities notes that none of the responses indicate any a priori objection to
the principle of monitoring. There is a differen ce of emphasis, however, as to the circumstances in
which it might be justified. Dr. Andow's comments focus on unanticipated ri sks in a manner which
confirms the pertinence of the Community's appro ach. In light of her other responses, Dr. Snow
appears to accept that post-release monitoring will be especially well-justified where pre-release risk
assessments have not been carried out as rigorously or as completely as desirable.
792. The Communities considers that these responses are consistent with the approach it has taken,
namely that environmental monitoring is requi red when risk assessments identify potential risks
associated with factors such as time and scale of the release (e.g. large scale and delayed impacts)
and changes in management associated with cultivati on of GMOs. Monitoring will also be justified if
the environmental risk assessment identifies gaps in knowledge which are likely to be filled by studies
of commercial crops. And monitoring is also used to observe unanticipated effects caused by the
cultivation of GM crops such as significant chang es in crop production or management practices
which may have an environmental impact.
793. Prior to 1998 the extent of knowledge and unders tanding of potential impacts was less than it
is now, so that the approach to environmental ri sk assessments (ERA) was rather narrowly focused.
These earlier approaches have been criticised by some Member States and by Dr. Snow and
Dr. Andow. Experience of the methods and appro aches to ERA has developed and there is now a
greater understanding of complex ecological inte ractions, allowing consideration of broader
environmental issues, even if considerable uncerta inties remain. This is the logic which justifies
monitoring, in the face of changing scientific know ledge, particularly in relation to environmental
risks. This scientific and technical rationale is reflected in the numerous international instruments
which endorse the use of monitoring.
Question 100

For those biotech products at issue in this di spute for which an approval has been sought for
environmental release (notifications submitted under Directives 90/220 or 2001/18), and for which no
significantly different nature or level of risk has been identified, does the information before the Panel
provide a scientific or technical rationale for requiring specific agr icultural management practices
that differ from those for products of biotechnology approved by the European Communities prior to
October 1998?

General comments

794. The European Communities notes that there is general s upport amongst the independent
experts for the rationale for requiring specifi c agricultural management practices, and support
amongst some for the view that such practices ma y be appropriate to address unanticipated risks
having regard to the state of scientific understand ing (noting in particular that it has developed
significantly since 1998).
Detailed comments

795. The panel has received comments from Dr. S now, Dr. Andow and Dr. Squire. Dr. Nutti
considers that this question relates to envir onmental risks and accordingly does not provide a
response.
796. Dr. Snow and Dr. Andow refer to their earlier responses to Question 99. Dr. Squire provides a
response that is consistent with the approach taken by the European Communities. He states that

the innate biological qualities and behaviour of a product may not have changed, but
it is quite possible that the context in whic h that product would operate has changed,
in two ways. (A) The physical or agronomic environment is different. The agronomy
of these crops changes rapidly: examples of factors that have changed markedly in
Europe in recent times are the proportion of autumn-sown crops, the area covered by
oilseed rape, and the type and effectiven ess of pesticides used. An example, given
already, of the change in pesticides is the rise in use of glyphosate as an arable
herbicide in some countries in the past few years. (B) The understanding of the GM
product's role in the ecosystem has changed. It was also reported previously that some
major effects of intensification on arable ecosystems have become well established
scientifically only in the last few years. The way arable plants and their food webs are
considered is now different from the way they were generally considered in the early
1990s. The question is sometimes referenced back to before the early 1990s: why was
the large scale change to autumn-sown cropping in some parts of Europe not
scrutinised with the same rigour as the poten tial change to GM cropping? The answer
is that it was thought not to be important, while it is now known to have been very
important for farmland food webs.
797. The European Communities agrees with this approach. Since 1998 there is a greater
awareness of the impacts that the management of GM crops – especially HT crops - can have on the
environment. There has also been a greater desire to ensure that innovations in agriculture are
sustainable. Thus there has been a change in attitudes to what is environmentally acceptable in
agriculture in the late 1990's, and this has im pacted on the evaluation of GMOs in Europe and
elsewhere.
798. Beyond the examples given by Dr. Squire, the Communities notes that there are other
examples of changes in crop production which may be of importance in this case, including the
development of surfaces with no or simplified tillage (due to the increase of useful agricultural
acreage per farmer), as well as the significant devel opment of organic farming (which benefits from
aids at the national and European Communities leve ls on the grounds that these farming practices
bring environmental benefits). More significantly is the growth in acreage under GM cultivation,
giving rise to conditions under which the results of ea rlier risk assessments may no longer hold true.
The scale of use has a significant effect on risk.
286 Some GMHT crops have gained significant spatial
scales of use that dwarf those of non-GMHT crops. For example, RoundUp Ready ® soybean now
occupies >60 of the US soybean area (>20 million hectares), while no non-GMHT trait has come
close. With greater scale, small effects can rein force each other and become more apparent. This
would be especially true for non-target effects, such as those found in the farm-scale evaluations
carried out in the UK. Relatedly, between 1998 a nd 2001 new research results were published on
spatially explicit models which aim is to simula te the impact of GM crops in cropping systems.
287
This kind of models allows: th e ranking of cropping systems according to risk of gene flow
(diagnosis); testing the effect of different practices; and determining best crop management to control
it.288 This allows to adapt practices according to risk intensity, technical constraints, spatial
distribution of fields in the agricultural landscape e.g. to adapt practices to the diversity of situations.

286 NRC (National Research Coun cil). (1987). Introduction of recombinant DNA-engineered
organisms into the environment: key issues. National Academy Press, Washington, DC
287 Colbach et al, 2001 a et b.
288 See Bock et al, 2002, Angevin et al, 2003, Colbach et al, 2004.

Question 101

Does the information before the Panel support the ar gument that any potential risks from any of the
biotech products at issue in this dispute should be mitigated in a manner different than the products of
biotechnology approved by the European Communities pr ior to October 1998? If so, what means of
risk mitigation might be envisaged?

General comments

799. The European Communities notes that there is support amongst the independent experts for
the view that potential risks from some of the biotech products at issue in this dispute can and should
be mitigated in a manner different than the produc ts of biotechnology approved by the European
Communities prior to October 1998.
Detailed comments

800. The Panel received responses from Dr. Squi re, Dr. Snow and Dr. Andow. These are
consistent with the view of the European Communiti es that developments in scientific knowledge as
well as techniques of risk assessment and mitigation mean that new and different approaches to
mitigation are required, as compared with the situation which existed in 1998.
801. Dr. Squire's response indicated that there had b een a change of approach since 1998: "While
products might have very similar qualities, mitiga tion might differ as a result of new information
informing risk or a new perception of the importance of a particular risk. Among ecological topics,
the perceived mitigation to reduce the 'severity' of a risk to biodiversity and ecosystem functioning
has changed because of a greater appreciation that arable systems have been affected by intense
agriculture. Mitigation is unlikely to remain consta nt. For example, some current mitigation measures
which leave margins round the cropped area of land are insufficient when it is appreciated that
biodiversity in the cropped areas is necessary to main ecological function there." He added that new
research has developed new knowle dge: "That risk has not remained the same over time is an
inevitable consequence of scientific information on arable systems being collected at increasingly
larger scales since the mid-1990s."
802. Dr. Snow's response recognised the possibility th at although several of the products that were
approved before 1998 are the same as (or similar to ) those in the present dispute the possibility could
not be excluded that post-1998 scientific knowledge could result in a different approach:
"The answer to this question hinges on which potential environmental risks might
require mitigation, and whether any new scientific knowledge has become available
to justify changes to any mitigation plans that were required prior to October 1998. I
am not sure which mitigation plans may ha ve been required at that time, but new
scientific knowledge that was gained after 1998 certainly could be relevant to risks
related to gene flow (see above) and the evol ution of resistance in target pests of Bt
crops (e.g., NRC 2000)."
803. In a similar vein, Dr. Andow confirms that "there are new risk methodologies and assessment
standards being applied to biotech products today than prior to 1998. Thus, it is possible that risks
will be identified for new products that were not ev en considered in the older products. Under such
conditions, differences could be justified."

Question 102

Does the information before the Panel support the vi ew that the biotech products at issue in this
dispute (including products subject to the member State safeguard measures) give rise to the same
types of potential risks to human, plant or animal hea lth or to the environment as novel non-biotech
products, such as plant products produced by selective breeding, cross-breeding and induced
mutagenesis? If so, for any biotech product at i ssue in this dispute are there significant differences,
from a scientific perspective, in the nature or m agnitude of any potential risks from these products
compared to comparable novel non-biotech products taking into account:

• the specific genetic modification introduced and the resulting product;

• the intended use of each product (direct human or animal consumption, further processing for
consumption, planting or other use);

• any potential risks that may arise from the comb ination or successive use of biotech products
or comparable novel non-biotech products.

Please explain with reference to specific products at issue in this dispute.

General comments

804. The European Communities notes that there is support amongst the independent experts for
the view that the biotech products at issue in this dispute (includi ng products subject to the member
State safeguard measures) give rise to different a nd new types of potential risks to human, plant or
animal health or to the environment as compared with novel non-biotech products.
Detailed comments

805. The Panel received responses from Dr. Andow, Dr. Snow and Dr. Nutti. The responses from
the first two indicate that biotech products give rise to new risks as compared with non-biotech
products. This is consistent w ith the approach taken by the European Communities in these
proceedings.
806. Dr. Andow provided a detailed response on the differences between conventional breeding
and "molecular breeding," referring to the differences between clonal species (e.g. potato and banana)
and sexual species (e.g. oilseed rape, maize and soybean) and, as regard sexual species, between
outcrossing species (e.g. maize and oilseed rape) and inbreeding species (e.g. soybean and wheat). As
regards the type of risk, Dr. Andow stated that "the re are no differences in the types or kinds of risks
posed by biotech crops compared with their non-bi otech counterparts. The kinds of risks include
toxicity to humans and animals, allergenicity, nu trition, potential for prod ucing disease, gene flow
risks, non-target and biodiversity risks, and resist ance risks." However, he concludes that "Within
these kinds of risk, there are new risks of biotech plants." Dr. Andow states clearly that "Nearly all
risks associated with novel toxins (e.g., all Bt cr ops) introduced into crop plants are new risks", and
refers to "other risks of a new nature" identified in responses to other questions. He confirms that the
European Communities is correct in consider ing that new open reading frames (ORFs) "could
theoretically produce a new protein, which could cause or influence a new risk" and that insertional
mutagenesis is "another outcome of transgenesis that is new to breeding, and could cause or influence
a new risk". He clearly rejects Canada's claim th at transgenesis allows more precise control than
selective breeding. And he concludes that although the US, Canada and Argentina may be correct in
saying that translocations and other genomic disr uptions can occur in conventional breeding, such

genomic disruptions "are normally rare in conventional breeding" . Dr. Andow refers to ongoing
research which may lead to technical improvements which could "alter" these concerns.
807. In relation to environmental risks, Dr. Snow adopted a similar approach:
In my opinion … the biotech products in this dispute could be considered as different
from non-biotech products in the some cases". Her response referred in particular to
pesticide-producing crop plants and to he rbicide-tolerant crop plants with feral
(volunteer) populations or wild relatives that can hybridise with the crop.
808. In relation to non-environmental risks, Dr. Nutti stated that: "I am of the opinion that both
novel non-biotech products and biotech products shou ld be assessed in a comprehensive, scientific,
step by step , case by case bases, so the same safety assessment principles should be applied in all
cases." To the extent that this response suggests equiva lence of risk it is not consistent with the views
of Dr. Andow and Dr. Snow.
809. The European Communities would like to point out to the Panel, however, that there is wide
international agreement that each GM product should be assessed on its individual perits, taking into
account the individual transformation event and the par ticular genes that have been introduced. In that
respect, potential risks, in particular unintended or n on target effects, are certa inly specific to each of
the biotech products. The European Communities w ould like to refer to the comments it provided on
the evolution and the broader issues of risk anal ysis of GM products in Section III on general and
methodological issues.
810. But more strikingly, this being taken into account , it is noteworthy that most of the products
in dispute contained traits and genes that are specific to GM products, as they have not net been found
or obtained without the use of genetic engineering techniques (glyphosate, gluphosinate resistance, Bt
resistance). Consequently, they present unique protential risks, which may not compare with non GM
products.
Question 103

Does the information before the Panel support the view that any of the biotech products at issue in
this dispute poses a substantially greater risk as regards the direct or indirect consequences of
unintentional "contamination" of other plant vari eties than a comparable novel non-biotech products,
such as one of the 2300 different crop varie ties that have been developed using induced
mutagenesis?
289 If so, what means of risk mitigation might be envisaged?

General comments

811. The European Communities notes that there is support amongst the independent experts for
the view that the biotech products at issue in this dispute may pose a substantially greater risk, in
particular as regards indirect consequences of unintentional "contamination".

289 FAO/IAEA (Food and Agriculture Organization of the United Nations/International Atomic Energy
Agency). 2001. FAO/IAEA Mutant Varieties Database. Available online at http://www-
infocris.iaea.org/MVD/.

Detailed comments

812. The Panel received responses from Dr. Snow, Dr . Squire, Dr. Andow and Dr. Nutti. These
responses indicate scientific support for the view that some of the biotech products at issue in the
dispute may pose a substantially greater risk by mean s of indirect contamination. The extent of the
risk will depend on definitions of harm (including economic harm) and the nature and extent of the
activity. This confirms the approach adopted by the European Communities.
813. Dr. Snow indicated that "The present dispute involves only a few crop species with a limited
number of transgenic traits, and the question is whethe r the dispersal of these traits to other plants, by
means of pollen and seed movements, could pose subs tantially greater risks as compared to non-GM
traits." She stated that some of the biotech products at issue in the dispute posed "risks that are greater
in certain GM crops as compared to their non-GM predecessors" (referring to her responses to Part I
and answers to Question 6 and others).
814. Dr. Andow considered that risk and "contami nation" "depend on the scale of release and the
nature of the adverse effect". Ri sk depends on exposure and adverse e ffect. He states that "There is
little evidence to suggest that ceteris parabis, gene flow will be greater from a transgenic variety than
a conventional one", but that "There has been little discussion and less agreement over the nature of
the adverse effects of contaminati on". Depending on the definition of harm, "the risk associated with
biotech crops is substantially greater than the risk associated with any of the conventionally produced
varieties". He suggests that "the probability of cr oss-contamination rises slowly with spatial scale for
very small scale production, but once it reaches a large-size threshold, the probability rises much
faster".
815. Dr. Squire concludes that as between certain crops
"Given present knowledge of the life cycle and reproductive behaviour of the crops,
there is no reason to suppose that biotech crops confer different degrees of impurity
compared with crops produced from, say, induced mutagenesis."
816. Dr. Nutti's response implies that there is no di fference of risk between plants that were
developed using induced mutagenesis and tho se produced from the Recombinant-DNA technology.
Unlike other responses, however, and in particular that of Dr. Andow, she provides no indication of
the reasoning which might assist the reader form a view as to the basis upon which she reaches such
an implied conclusion.
Question 104

From a scientific perspective, is there a significant difference in risks to human, animal or plant
health or the environment arising from the use of a bacterial antibiotic resistance marker gene, or
part thereof, in any biotech product at issue in this dispute (e.g., Monsanto Bt cotton (531), Monsanto
Roundup Ready cotton (RRC1445), Amylogene starch potato) compared to novel non-biotech
products, such as comparable plant products pr oduced by selective breeding, cross-breeding and
induced mutagenesis?

General comments

817. Consistently with the approach set out above , the European Communities notes that there is
support amongst the independent experts for the view th at there is a significant difference in risks to
human, animal or plant health or the environment arising from the use of a bacterial antibiotic

resistance marker gene in any biotech product at issue, including as regards other non-biotech
products.
Detailed comments

818. The Panel has received a response only from Dr. Nutti.
819. Dr. Nutti states that
"the use of antibiotic resistant marker genes has been recognized as a safe tool and its
employment should be evaluated on a case by case basis. The same rule should be
applied to novel non-biotech products, such as comparable plant products produced
by selective breeding, cross-breeding and induced mutagenesis. I mean, these
products should be evaluated on a case by case basis, so the same safety assessment
principles should be applied in all cases."
820. In relation to other questions Dr. Nutti has indicated that she has no expertise in
environmental risk, so this response must be take n to be limited only to non-environmental risks.
Even if, as with other responses she provides no indication of the reasoning which might assist the
reader form a view as to the basis upon which she reaches such an implied conclusion, the European
Communities strongly disagrees with Dr. Nutti's vi ews that ARMG has been "recognized as a safe
tool" for the reasons outlined in its comments to Questions 1, 2 and 98, and in accordance to the
relevant latest Codex guidelines which recomme nd not to use such ARMG. Furthermore, the
European Communities notes the apparent inconsiste ncy between Dr. Nutti's response to this question
and that of Question 98, where she recognises that "the use of alternative transformation methods,
which do not use ARMG, is encouraged".
821. The European Communities refers to its co mments under Questions 1 and 2 for further
detailed considerations regarding the use of ARMG, or part thereof may pose a significant risk of
harm.
822. Furthermore, the European Communities would lik e to note the potetntial inconsistency of
Dr. Nutti's response, when she states that the same rule (namely that the use of antibiotic resistant
marker genes has been recognized as a safe tool and its emplyment should be evaluated on a case by
case basis) should be applied to novel non-biot ech products, such as comparable plant products
produced by selective breeding, cross-breeding and induced mutagenesis. Indeed, ARMG are
bacterial genes, and although it may not be excluded that such genes may be seldomly found in plant
genomes, as nobody has systematically searched for it, they can only be present in the plant biotech
products in dispute through genetic modification technique. There does not exist yet any novel non
biotech plant product produced by selective breedi ng, cross-breeding and induced mutagenesis which
has been found to contain ARMG, and therefore Dr. Nutti's comparison does not make sense.
Question 105

For those biotech products at issue in this disput e for which no significantly different nature or level
of risk has been identified, is there a scientific or technical rationale for monitoring the occurrence of
potential adverse effects, or of unintentional effects, arising from the consumption or use of these
products compared to novel non-biotech products, such as plant products produced by selective
breeding, cross-breeding and induced mutagenesis? If so, would such monitoring relate to the
specific genes or traits introduced into a biotech product, and how would this compare with the
monitoring of induced changes in novel non-biotech products?

General comments

823. The European Communities notes that there is support amongst the independent experts for
the view that there may be a scientific or technical rationale for monitoring the occurrence of potential
adverse effects, or of unintentional effects, aris ing from the consumption or use of biotech products
(as compared to novel non-biotech products) even where no significantly different nature or level of
risk has yet been identified, having regard to the possibility of unanticipated consequences.
Detailed comments

824. The Panel received responses from Dr. Snow, Dr. Nutti and Dr. Andow. Taken together these
are consistent with the growing support now evident in international instruments and around the world
for monitoring of unanticipated effects of biot ech products, including thr ough general surveillance
schemes, without prejudice to whatever monitori ng may be required for non-biotech products. These
responses support developments in the European Co mmunities towards greater monitoring in the face
of uncertainties.
290
825. Dr. Snow's response takes the question as stated that "It does not seem logical to require
monitoring if no risk has been identified (see Question 99)." (emphasis added). Conversely, it would
appear that Dr. Snow accepts that if a risk h as been identified it would be logical to require
monitoring. Dr. Snow's response cannot be taken as an indication of her view that the products which
are the subject of this dispute fall within the cate gory of products for which no significantly different
nature or level of risk has been identified. Fu rthermore, her answer does not address all potential
unanticipated and non target effects which have b een discussed elsewhere by the experts and in the
EC comments, which may indeed require also mon itoring and general surveillance programs, even if,
by definition, these portential effects have gone unde tected at the stage of risk assessment because of
limited scientific knowledge.
826. Dr. Nutti states that
"the monitoring of occurrence of potential adverse effects or of unintended effects
should be carried out based on scientific pa rameters in both cases, that is, for those
biotech products at issue in this dispute, for which no significantly different nature or
level of risk has been identified, and fo r novel non-biotech products, such as plant
products produced by selective breeding, cross-breeding and induced mutagenesis. In
both cases, I am considering that the products were assessed as safe."
She recognises the difficulty of monitoring for "any e ffect that we are not aware of" and proposes that
the subject should be "introduced in the agenda of the Codex Alimentarius Task Force on Foods
Derived from Biotechnology, which will start working in September 2005."

827. Dr. Andow considers that "Monitoring can be used to look for unanticipated effects" and that
"monitoring may be partially substitutable for mo lecular and biochemical characterization". He
considers that "Monitoring may also substitute for identifying all possible effects, by covering various
categories of unanticipated effects". As regards the objectives of monitoring, he states that monitoring
related to a specific potential adverse effect "would have to relate to the specific genes/ traits in the
transgenic crop", whereas for unanticipated effects "a more general approach is needed".

290 See Wilson, Latham and Steinbrecher, 'Genom e Scrambling – Myth or Reality? Transformation-
Induced Mutations in Transgenic Crop Plants', EcoNexus technical Summary Report, October 2004.

Question 106

For those biotech products at issue in this di spute for which an approval has been sought for
environmental release (notifications submitted under Directives 90/220 or 2001/18), and for which no
significantly different nature or level of risk has been identified, does the information before the Panel
provide any scientific or technical rationale for requiring specific agricultural management practices
that differ from those for novel non-biotech products , such as plant products produced by selective
breeding, cross-breeding and induced mutagenesis?

General comments

828. The European Communities notes that there is support amongst the independent experts for
the view that there may be a scientific or tec hnical rationale for requiring specific agricultural
management practices in respect of biotech produc ts (as compared to novel non-biotech products)
even where no significantly different nature or level of risk has yet been identified, having regard to
the possibility of unanticipated consequences.
Detailed comments

829. The Panel has received responses from Dr. Squire and Dr. Andow.
830. Dr. Squire states that if no significant level or type of risk has been detected, then no
particular change in practice should be needed. Howe ver, he recognises that if thresholds are imposed
(e.g. 0.9%, specifically for GM varieties in non-GM varieties) "then there will need to be different
agricultural practices for those GM varieties th at leave volunteers or spread genes by pollen to
neighbouring, sexually compatible crops." He also concludes that
"Under a system of coexistence in whic h a threshold (GM in non-GM) was imposed,
the agricultural practice might well have to ch ange in a crop such as oilseed rape to
ensure that threshold would be met. L onger intervals than normal between oilseed
rape crops, some regional segregation of GM and non-GM crop types and the
dropping of varietal associations (80% ma le sterile, 20% own pollen) from general
use would probably be necessary. Given present knowledge, these changes would be
in consequence of an imposed threshold of GM in non-GM product, not of any
inherent food-risk in the GM product itself."
831. Dr. Andow concludes that disagreement as to different standards of acceptable risk being
applied by the disagreeing parties "provides the onl y rationale for risk management. If all parties
agreed that there were no significant identified risk s, there would no need to consider monitoring for
potential adverse effects. If only some of the parties recognized the risk, then some kind of
conditional risk management could be justified. For example, one condition for the management
measures could be the country of use."
832. The European Communities generally agrees w ith these responses (although it considers that
Dr. Squire's response is premised on an assumption that the initial risk assessments were correct in
their methodology and outcome (a point which is re futed by Dr. Andow, Dr. Snow and Dr. Squire in
some of their earlier responses). The Communities recognises that both conventional and GM crops
have shown unintended and undesirabl e effects after release (e.g. reduced efficacy of pest resistance,
boll drop, decreased yields, increased requirements for fertilisers etc), so the screening systems are
obviously not always able to detect unintended effect s prior to large scale rele ase. However, because
of the vastly increased scale of GM crop productio n relative to most conventional crop counterparts,
the Communities considers that post-release monito ring is even more important for large scale

production GM crops than for convent ional crops grown on smaller scales (because the extent of any
harm would be much greater due to scaling effects) . This approach is supported by the responses to
the Panel.
Question 107

Does the information before the Panel support the vi ew that the biotech products at issue in this
dispute (including products subject to the member State safeguard measures) give rise to the same
types of potential risks to human, plant or animal he alth or to the environment as foods produced
using biotech processing aids, including yeasts, bac teria or enzymes that have been modified using
recombinant DNA technology? If so, for any biotech product at issue in this dispute are there
significant differences, from a scientific perspective, in the nature or magnitude of any potential risks
from the products at issue in this dispute compa red to foods produced with biotech processing aids,
taking into account the intended use of each pr oduct (direct human or animal consumption, further
processing for consumption, release into the environment, any other use).

General comments

833. The European Communities notes that there is support amongst the independent experts for
the view that the biotech products at issue in this dispute (includi ng products subject to the member
State safeguard measures) may give rise to potential ri sks to human, plant or animal health or to the
environment, if the food is made available to the en vironment, although the nature and extent of that
risk may be lower.
Detailed comments

834. The Panel has received responses from Dr. Andow and Dr. Nutti.
835. Dr. Andow expresses the opinion that since in general, foods produced with biotech
processing aids "are not expected to be able to self-reproduce in the environment", so that the kinds
of risk associated with these foods "do not include any of the environmental risks associated with
transgenic plants." However, he also believes th at foods produced with biotech processing aids "can
have some non-target effects if the food is availa ble in the environment" and that these effects "are
more similar to the effects of chemicals rather th an the effects of living biological organisms." In
respect of these products unanticipated effects could arise from a transgene by a number of
mechanisms, although in each case "the likelihood of it occurring in a transgenic bacterium or yeast
(that is typically used as a food processing aids) is much lower than in a transgenic plant."
836. Dr. Nutti addresses only non-environmental risks. In her view "the safety of the new
microorganisms and of the product thereo f should be assessed according to the FAO/WHO Codex
Guideline for the Conduct of Food Safety Assess ment of Foods Derived from Recombinant-DNA
Microorganisms."
837. The European Communities generally agrees w ith the approach taken by Dr. Andow. As
regards environmental issues, the modified food produc t is non-viable and the environmental impacts
of the transgenes would be restricted to wildlife and environments exposed to these food products or
the GM processing aids. Risks associated with these products would be case specific depending on
the traits produced by the processing aid, the inter action with the crop raw material and levels of
environmental, human and animal exposure. Sin ce the processing is largely done in contained
facilities (fermenters etc) the risk of environmenta l exposure will generally be low. However, gene
products may pass directly into the environment from human activity or factory discharges or may

pass indirectly into the environment after consum ption by humans and animals. Release of the GM
bacteria or yeasts which are the source of processing aid could have some environmental impacts
depending on the species of organism and the trait. If they can survive as free living organisms in soil
or water and/or mate with free living microorgani sms then there may be some ecological impacts
which would need to be evaluated.
Question 108

Is there a significant difference in risks to human, anima l or plant health or the environment arising
from the use of a bacterial antibiotic resistance ma rker gene, or part thereof, in any biotech product
at issue in this dispute (e.g., Monsanto Bt co tton (531), Monsanto Roundup Ready cotton (RRC1445),
Amylogene starch potato) compared to foods produced with biotech processing aids, including yeasts,
bacteria or enzymes that have been modified using recombinant DNA technology?

General comments

838. The Panel has received a response from Dr. Nutt i. It raises the same points as arise in
questions 1, 2, 98 and 104, and the European Communities refers to its comments thereunder.
Question 109

For those biotech products at issue in this disput e for which no significantly different nature or level
of risk has been identified, does the information be fore the Panel provide a scientific or technical
rationale for monitoring the occurrence of potential adverse effects, or of unintentional effects,
arising from the consumption or use of these pr oducts compared to foods produced with biotech
processing aids? Does the information before the Panel provide a scientific or technical rationale for
mitigating any potential risks arising from the biotech products at issue in this dispute in a manner
differently than products produced with biotech processing aids?

General comments

839. The European Communities notes that there is support amongst the independent experts for
the view there is a scientific or technical rationa le for monitoring the occurrence of potential adverse
effects, or of unintentional effects, arising from th e consumption or use of biotech products compared
to foods produced with biotech pr ocessing aids, even where no significantly different nature or level
of risk has yet been identified, having regard inte r alia to unanticipated effect, and that monitoring
may be justified.
Detailed comments

840. The Panel has received responses from Dr. Andow and Dr. Nutti.
841. Dr. Andow states that sources of sources unanticipated effects
are more likely to occur for plant biotech products than for foods produced with
biotech processing aids. This provides a rati onale for monitoring, but no rationale for
mitigation, as there are no concrete effects that can be mitigated.
842. Dr. Nutti states that
For those biotech products at issue in this dispute for which no significantly different
nature or level of risk has been identi fied and for foods produced with biotech

processing aids, where the processing aid and the Recombinant-DNA microorganism
have been assessed as safe, I am of the opini on that the monitoring of occurrence of
potential adverse effects or of unintended effects, in both cases, should be conducted
based on scientific parameters.
843. The European Communities notes that neither e xpert expresses an objection in principle to
monitoring, even where no significantly different nature or level of risk has been identified, and that
Dr. Andow has identified a positive rationale on the basis of unanticipated effects. As regards
Dr. Nutti's approach, based on a
symmetry between both types of products, the European
Communities considers that this may apply to GM yeasts and bacteria based on Codex guidelines for
their safety assessment but that it does not apply to processing aids consisting of GM enzymes, based
on the considerations of the diffe rent nature, function, dissemination, viability, activity, complexity,
and consumer exposure of the GM products at issue.
VIII. THE PANEL'S ADDITIONAL QUESTIONS
Question 111

Please provide an assessment of the US statements regarding the evaluation of the safety of biotech
products in paragraphs 128-133 of the US Supplem entary Rebuttal Submission, and in particular of
the statement in the last sentence of paragraph 133 th at: "... where all of the data consistently provide
no indication of adverse effects, and there is no sp ecific indication that the data submitted are
inadequate, there is generally no reason to expect that any remaining risk as gone undetected, and
that further studies are warranted". What relevanc e does the foreseen end product use(s) have in the
context of the evaluation of the safety of that product?

General comments

844. With question 111, the Panel's sought advice on th e statements in paragraphs 128-133 of the
US supplementary rebuttal submission, where the US describes its approach to assess the risk of
biotech products, and in particular, on the sweepi ng statement of the US, in paragraph 133, which
seeks to minimize the amount of information to be supplied by an applicant, omitting the difficulties
and the safety problems that may arise when following the US federal approach.
845. Dr. Nutti and Dr. Snow both answered that ques tion succinctly, but with different approaches
and emphasis; Dr. Snow addressed certain environmental concerns and flaws of the proposed US
approach, while Dr. Nutti presented her own approa ch to food safety assessment. Both, again with
different emphasis, not only agree with the relevance of end products uses in risk assessment, but also
consider it necessary in the assessment.
846. The European Communities agrees with Dr. Snow when she concludes that
"The last sentence of paragraph 133 does not acknowledge that important scientific
information about environmental consequen ces could be lacking in specific cases, in
which case further scientific studies would be warranted"
847. The European Communities believes that that stat ement is equally applicable to other aspects
of the risk assessment, such as food or feed safety, and, as Dr. Snow herself notes, the inherent
problems and shortcuts of the US proposed approach generally apply to the products in dispute, where
further information was sought by one or the other au thority, in order to complete its risk assessment
in accordance with the lack of important safety information.

848. The issues raised by the Panel's question, albeit of a general nature, are very important as they
are issues of principle for conducting a risk assessment, applicable throughout all products in dispute
and their respective applications, when considering wh ether there was a delay, and, if so, whether it
was undue.
849. Therefore, because of the succinct character of both replies, which didn't cover all necessary
aspects of the inappropriateness of the US approach , and of the general inadequacy of Dr. Nutti's
reply as regards food safety, the European Communiti es will comment in detail below and provide its
own analysis. It has prepared this information with the assistance of independent scientists, experts in
this field, and presents it as objectively as possible so as to complete the information available to the
Panel.
850. It will explain why the precise requirements and the principles for the ad hoc risk assessment
of a GMO have got to be decided on a case-by-case, in light of the specific information supplied (both
its content, and its quality), but also taking into account the likelihood of unintended effects and the
particular geographical and environmental conditions of the foreseen intended or unintended uses.
851. As widely explained by the experts replies and the comments submitted by the European
Communities, there was and still is a vast amount of open and unresolved issues in the assessment of
the biotech products in dispute. These uncertainties warrant the call for certainly more studies than
advocated by the US, in order to conduct a pr oper science-based risk assessment, and to reach a
rigorous conclusion, with a reasonable level of certa inty, as regards the safety of the biotech products
in dispute.
852. The European would like, in this context, to re fer the Panel to its general comments in section
III of this submission, on genera l and methodological issues, which ar e extremely relevant here, in
particular on case by case assessments, systemic issu es, evolving science, scientific evidence of risks
or absence of risks, absence of agr eed scientific criteria, judging wh en the scientific information is
sufficient, interpretation of scientific informa tion, surveillance and food safety, and on new
developments of broader risk assessment concepts for genetically modified products.
Detailed comments

853. The US statements essentially describes its vi ews as regards, not the conduct of a risk
assessment, but the endpoints of information and da ta requirements necessary for each isolated and
independent component of the risk assessment it describes (the safety of the new substance,
comparisons made with conventional products, molecular characterization, stability of the
modification, field trials analysis, and environmental impacts). The difficulty is not as much the risk
assessment paradigm itself, as described, but rather the studies claimed to be warranted by the US to
implement each step in this paradigm.
854. The approach presented by the US seems to be essentially inspired by food safety
considerations, in light of the little emphasis given to environmental and non target effects. The US
risk assessment paradigm is more or less roughly similar to the one used anywhere as regards food
safety, even if there is intense debate worldwide as to how to test each of the individual step, and what
further components and methods may be necessary to improve it, for instance to better integrate the
different steps of the risk assessmen t, or to assess the GM plant or th e GM food as a whole, in its real
environment or in its real uses.
855.
The US approach is based on the assumption that it is necessary to benchmark the assessment
of biotech products against the approaches to assess "conventional" products and techniques,
including the most unpredictable plant breeding t echnology, with old risk assessment techniques,

mainly developed to test the safety and eco-toxi cology of individual compounds in the chemical
industry.
856. This assumption was the one initially advocated by the industry and became the basis for the
regulation of transgenic crops in th e US (see for instance, for the FDA,
291). It hasn't changed since,
despite the large number of new issues and the many weaknesses of the US approach, as largely
explained by the experts. Each will be addressed below.
857. It is noteworthy that the inherent shortcuts and limitations of the US federal approach to the
risk assessment of genetically engineered products have forced the US federal authorities themselves
to review frequently their approach in recent years, and sometimes to improve coordination or revise
it, albeit scarcely and unwillingly, in the light of many identified flaws and recommendations from its
own advisory structures,292 even from the least progressive ones.
858. Even so, the last paragraph of the US statement is qualified (emphasis added):
… where all of the data consistently provide no indication of adverse effects, and
there is no specific indication that the data submitted are inadequate , there is
generally no reason to expect that any remain ing risk has gone undetected, and that
further studies are warranted.
859. As has been indicated in the comments under issu e 1, there was almost systematically, in the
biotech product applications at stake, missing data, inconsistency of the data provided, inadequacy or
bad quality of the data submitted, or specificities of the product or of its in tended or unintended uses,
which all warranted, according to this very US statement, the requests for further studies.
860. Dr. Snow, in her comments, indicates the lack of relevance of the tests proposed by the US to
assess some environmental risks, such as out-cro ssing impacts. The European Communities would
add that this applies in particular to unintended effects and non target organi sms. Furthermore, on top
of the lack of identification by the US of the met hods that could be used as regards "the effects on
other organisms in the ecosystem", that could include "indirect impacts", it is doubtful, in light of the
succinct methods proposed for food safety, that the US would address multi trophic (ie involving a
chain of organisms in complex food webs), sub chr onic, or delayed effects of the organisms in its
natural environment. Dr. Andow, although not repl ying to this question, has provided a ample
evidence to support such a view.

291 Kessler DA, Taylor MR, Maryanski JH, Flamm EL, Kahl LS (1992) The safety of foods developed
by biotechnology. Science 256: 1747-1832.
292 See for instance the many findings and recommendations regarding the analysis of the US APHIS
policy and assessment, of GMO monitoring, and of future approaches, stated by the US Committee on
Environmental Impacts Associated w ith Commercialization of Transgenic Plants (Environmental Effects of
Transgenic Plants – the scope and adequacy of regulation; 2002; National Research Council; National Academy
Press, Washington, D.C.; 320pp.), or the advise given to the US EPA by a FIFRA expert panel, in order to
address in its assessment approach for Bt cotton, in particular, unintended effects of GMO (SAP Report No.
2004-05. MINUTES of Federal Insecticide, Fungicide, an d Rodenticide Act (FIFRA) Scientific Advisory Panel
(SAP) Meeting, June 8-10, 2004, Arlington, Virginia. A Set of Scientific Issues Being Considered by the
Environmental Protection Agency Regarding : Product Characterization, Human Health Risk, Ecological Risk,
And Insect Resistance Management For Bacillus thuringiensis (Bt) Cotton Products. 84 pages.), or the advise
given recently to the US FDA to revise in depth its approach to the food safety assessment of transgenic
products.

861. Dr. Nutti addresses only the toxicological and allergenicity assessment of the food safety, not
even of the whole plant, but of the newly expressed character (mainly the protein). In doing so, while
quoting the relevant paragraph of the recent C odex guidelines (CAC/GL 45-2003), she presents what
tests, in her view, would satisfy these guidelines. She concludes in general terms that if these few tests
are satisfactory, no risk is to be expected, whic h by and large concurs with the US statements
scrutinized. She does not address feed safety, animal health, plant health, environmental safety or
agro-environmental impacts, which are obviously all part of a necessary risk assessment.
862. While it is clear that her analysis on risks s hould be dismissed on the grounds of critically
missing elements on other aspects than food safety, it is also substantially misleading on the latter.
863. First, she does not address the other important safety aspects referred to in the above-
mentioned Codex guidelines, namely the evalua tion of metabolites, food processing, nutritional
safety, accumulation of other potentially hazardous substances (e.g. pesticides), or antibiotic
resistance marker genes. These all have bearing on the safety of foods and may require further testing,
as shown below.
864. Second, she does not at all take into account that the assessment of the newly expressed
substance requires, by definition, its identification. For that, as an imperative pre-condition, a perfect
molecular characterisation is required to fulfil this initial test of the toxici ty assessment. As has been
shown before, she has often, in specific product appl ications, dismissed request for further food safety
information, when in most cases the molecular characterisation of the GMO has left substantial doubts
as regards the integrated construct and/or the expressed genes, hence on the newly expressed
substance(s), and not to say anything about unin tended genetic changes at, or near, the site of
integration of the modification. According to her own words, these doubts about the safety of the new
substances remaining would have warranted the use of toxicology or other studies.
865. The points of view of the US and Dr. Nutti are not fully comprehensive, as they imply that the
safety assessment is static by not taking into account that our knowledge about biotechnology and
safety issues is still evolving. For example, a DNA sequencing study revealed in 2000 that the insert
in GM herbicide-resistant soybean, which had al ready been approved for marketing, contained
additional fragments of "foreign DNA". For the safet y assessment of the additional insertions, among
others, the applicant (Monsanto
293) argued that these additional fragments could not pose a risk, since
no adverse effects had been observed in previously published animal feeding studies with whole feed
products derived from Roundup ready soybeans. Had the applicant only carried out the compositional
and toxicity studies mentioned by the US and Dr . Nutti (and thus not the aforementioned animal
studies), no data would have been available to judge the safety of these additional, previously
unsuspected modifications.
866. The US statements call for assessing the "stability of the transferred genetic material and the
demonstration of the Mendelian inheritance of the introduced genetic material", as important safety
considerations. Once done, it would be a static ass essment, as presented by the US. However, more
and more often, it is apparent that many GM crops that have been previously assessed for the stability
of the genetic modification appear to have undergone significant changes in their genetic modification
structure. This does not come as a surprise, as the plant genome is not a fixed object, and the plant has
to adapt to the presence of the newly inserted constr uct, but it raises important safety questions if the
initial assessment is not properly reviewed.

293 Monsanto (2000) Updated Molecular Characteri zation and Safety Assessment of Roundup Ready
Soybean Event 40-3-2. Monsanto Co. St. Louis, 20 pp.
http://archive.food.gov.uk/pdf_files/acnfp/summary.pdf

867. Third, as regards the toxicity and allergenicity studies she advocates, she only supports the
use of acute toxicity on mice (to assess the acute toxicity of the newly expressed protein only), and
very limited investigation of allergenicity potential. Not only, as regards the allergenic potential which
can not be assessed with certainty, does this not align with the most recent views and methods to
assess allergenicity, (see also the annex to the C odex guidelines she cites), but she also does not
mention the use of chronic tests and other t ests, which may often be necessary to assess acute and
chronic and sub-chronic toxicity of new substances with no known history of safe food use. Examples
of relevant tests are mentioned in paragraph 39 of the mentioned Codex guidelines.
868. Another example of evolution of knowledge th at is relevant for safety assessment is the
assessment of potential allergenicity based on computer-aided comparison of the structures of
transgenic "foreign" proteins with the struct ures of allergens. The knowledge on structures of
allergens is still growing and new allergens are identif ied. Therefore, it is not unlikely that a protein
at present may not show a relevant similarity to known allergens, but will do so in the future.
869. There is an obvious discrepancy in the allerg enicity assessment proposed by the US and by
Dr. Nutti with other relevant views. The US a nd Dr. Nutti assess the potential allergenicity of a
product with the focus of newly introduced protei ns, in line with their endorsed principle of
substantial equivalenve, without caring for possible unintended effects and the need for a molecular
characterisation. From 1996 to now it became gradually more evident that insertion of constructs into
plant genomes often causes unintended effects, esp ecially at the recombination boarders/ flanking
region of insertion. Because of these changes in open reading frame (the segment of DNA coding fro
protein), expression of new or alte red proteins is possible. These consequences of molecular events
need to be assessed by the molecular characterisati on in combination with allergenicity testing of the
potentially new expressed /changed proteins, or wher e this is not sufficiently clearly defined, by
assessment of the whole product. Dr. Nutti doses not seem to adhere to this approach.
870. There has also been di screpancy between experts in gene ral about allergenicity testing
schemes. Even the present strategy based on a sequence prediction model established by FAO/WHO
was contradicted by important groups
294. Difficulties raised by the current approach are summarised
in Jank and Haslberger, 2003 295. Whereas a general decision tree is generally accepted many experts
consider it often sufficient to do sequence homolo gy search and stability testing, whereas some
experts also in some of these cases ask for more testing. Also the ways to perform the sequence
homology search is under dispute. This idea is not fully explained in CODEX but there are elements
in it and the latest FAO/WHO expert consultations address it more clearly (CAC/ GL 45-2003 and its
annex; Report of a Joint FAO/WHO Expert Cons ultation on Allergenicity of Foods Derived from
Biotechnology, 2001).
871. The latest FAO/WHO consultation on GM foods (2003) agrees that "It has been recognized
that there is no single parameter that can predict th e allergenic potential of a substance. A strategy to
assess allergenicity of biotechnology products has been formulated (FAO/WHO, 2001; Codex
Alimentarius Commission, 2003), which relies on the parameters: source of the gene, sequence
homology, serum testing of patients known to be a llergenic to the source organism or to sources
distantly related, pepsin resistance, the prevalen ce of the trait and assessment using animal models.
The Consultation recommended that additional effort s should be directed to the further development
and validation of models and that there is a need to improve the accessibility and interconnectivity of

294 Stadler and Stadler, Allergenicity prediction by protein sequence, FASEB, J 2003, 17, 9, 1141-3
295 Jank and Haslberger, 2003. Improved evaluation of potential allergens in GM food, Trends
Biotechnol., 2003, 21, 6, 249-250 )

existing databases or to establish a centralized database on allergenic lin ear and conformational
epitopes and tools for screening transgenes for allergenic potential.
872. Fourth, Dr. Nutti does not refer to the fact that the toxicology of the newly expressed proteins
in the GM products at stake, was of ten tested with "surrogate" protei ns (ie isolated from heterologous
systems, different from the GM plant, see review by Freese and Schubert (2004))
296, without proper
demonstration of biochemical, structural, or functi onal equivalence of the surrogate protein to its
counterpart (for instance as regards mutational changes, post translational modifications, or others), as
recommended in Paragraph 40 of the Codex guidelines.
873. Fifth, she states that exposure to human diet that has no indication of adverse effects would be
a criteria to take into account. Notwithstanding th e ethical question of performing pre marketing
toxicity tests on humans, that statement is not scientifically sound for anything else than acute
toxicological risk, as explained by the Europ ean Communities in the section of general and
methodological issues, on surveillance and food safety. Standard epidemiology says that, in the
absence of exposure data with respect to chronic conditions, there is simply no way of ascertaining
any effect – or lack thereof – on human health.
874. Sixth, she refers to properly conducted substantia l equivalence tests, while it is clear from the
US proposed methods, that these would be limited to key components; for example, carbohydrates,
proteins, and fatty acids, amino acid and vitami n content, as well as naturally-occurring toxicants,
antinutrients, and allergens. Substantial equivalence as applied in such a way has received significant
criticism297, including within the relevant Codex work, as it would obviously miss important changes
for the safety of the product.
875. The safety assessment of selected compounds and the "targeted" compositional analysis, also
on selected compounds {e.g. Codex alimentarius; pa ragraph 16}, leaves room for "unpredictable"
unintended effects, whose likelihood will be gr eater in GM crops with complex or major
modifications {e.g. Codex alimentarius; paragraph 15}.
876. The difficulties with substantial equivalence is at least three fold: (i) it may largely overlook
significant and numerous differences in composition th at may remain undetected, (ii) it is often used
as an endpoint, rather than a starting point of the risk assessment, and (iii) it is a essentially only a
series of chemical tests, which establish gross differences or similarities (but within the broad range of
values for that species, including the less safe ones as regards toxicants or antinutrients), but by no
means able to test or demonstrate biological effects298, which would be the most relevant criteria.
877. Very large number of changes of endogenous metabolites may occur as a consequence of the
genetic modification. Such unintended changes in GM crops could be detected by metabolic
profiling299, but would remain silent according to a sta ndard substantial equivalence test, as proposed
by the US. For instance, Roessner et al. (2001) 300 have detected by metabolic profiling a very large
amount of significant unintended changes in GM potato, many more than previously assumed. 9 novel

296 Freeze W & Schubert D (2004). Safety testing an d regulation of genetica lly engineered foods.
Biotechnology and Genetic Reviews. 21: 299-324.
297 See relevant references in the comments on subs tantial equivalence in section III on general and
methodological issues.
298 The brain from a mad cow showing advanced spongiform encephalopathy would pass a test of
substantial equivalence undetected!
299 A relatively new technique which looks at a wider range of plant chemicals than before
300 Metabolic Profiling Allows Comprehensive Phenotyping of Genetically or Environmentally
Modified Plant Systems, Ute Roessner, Alexander Luedem ann, Doreen Brust, Oliver Fiehn, Thomas Linke,
Lothar Willmitzer, and Alisdair R. Fernie; The Plant Cell, Vol. 13, 11–29, January 2001.

compounds were detected in GM potato tubers not f ound in the control plants, and more than half of
the 88 plant chemicals measured had been alte red (levels) as a consequence of the genetic
modification.
878. It is not clear how significant these results are, but it seems to be a striking indication of how
some genetic modifications can cause widespread alte rations in cellular metabolism. If Roessner et al
has measured a representative sample of all pot ato compounds, the proportion of all unmeasured
potato compounds whose level could be altered could be anything lik e the "over half" found in this
study. If 5,000 is typical estimate of the number of different molecules for a species, one could be
talking significant alterations in the expression leve ls of hundreds of genes, plus dozens or hundreds
of novel compounds. It might be assumed that most of these changes would likely not have any
adverse impacts, but it would still be a very large pool of alterations, that, it seems, with regards to the
novelty in the plant genome of the newly introduced character, could bring safety concerns, hence the
need for further testing methods.
879. In literature, other ways to detect unintended effects than whole feed studies on animals are
described, such as the use of adva nced analytical technologies called "profiling" or "metabolomics".
These techniques are in development, but have not b een validated yet for routine application in the
safety assessment of GM foods {reviewed by Chassy et al., 2004
301}. These new techniques
(metabolic profiling) are helping to confirm many scientists' historical views that genetic modification
does not just result in addition of one gene with no reason to expect any further changes that could
result in potential risks (the 'clean technology' paradigm). This type of research has general
implications for food and feed safety, but also fo r the environment (target and non-target organisms
could be affected in unpredictable ways); it fu rther supports case-by-case assessments, as each GM
event could be uniquely different to the parental or isogenic line (the closest non-GM line that the
Codex recommends to use as a control). This type of new knowledge also seriously questions and
undermines the safety approach advocated the US as regards food safety.
880. Seventh, Dr. Nutti does not consider feed saf ety concerns. As explained in section III on
general and methodological issues, there is little guida nce as regards feed safety testing. The lack of
identification of human safety concern does not necessar ily correlate with a lack of safety concern for
target animal of the feed deri ved from the GM plant (or even l ess for non target animal), as
physiology, metabolism, plant parts consumed, pro cessing and exposure can each and all be different,
in particular for non mammal target species. In the li ght of the requirement to take into account the
end use of the GM product, feeding studies on targ et animals would be highly recommended for feed
uses, and have rarely been provided.
881. For instance, there has been reports 302 of significant changes in the lignin content of some Bt
maize; while this may have limited or no beari ng for the safety of the processed product in food (and
may also have gone undetected following the safety approach proposed by the US and Dr. Nutti), it
could have significant nutritional, if not toxicologi cal, impact on target animals and on wild animals
exposed to the maize in the field, including soil organisms, which would warrant further testing.

301 Chassy, B., Hlywka, J.J.,. Kleter, G.A., Kok, E.J., Kuiper, H.A., McGloughlin, M., Munro, I.C.,
Phipps, R.H., Re, E.B., Reid, J.E. (2004) Nutritional an d Safety Assessments of Foods and Feeds Nutritionally
Improved through Biotechnology, Prepared by a Task Force of the ILSI International Food Biotechnology
Committee. Comprehensive Reviews in Food Science and Food Safety 3: 35-104.
http://www.ift.org/pdfs/crfsfs/crfsfsv3n2p0035-0104ms20040106.pdf
302 See for instance Saxena D. and Stotzky G. (2001). Bt corn has a higher lignin content than non-Bt
corn. American Journal of Botany. 88(9): 1704-1706.

882. Finally, Dr. Nutti, as the US, dismisses by her approach the likelihood of significant impacts
from genome-wide or physiological unintended chang es. There are however several reports of such
wide-genome induced changes. A recent EcoNexus technical report
303, although it has not been
published as a peer review publication, very seriously and even-handedly presents the identification of
insertion-site and genome-wide mutations created by plant transformation procedures as "potentially
major, but poorly understood" sources of hazard associated with the production and use of GM crops.
883. The significant potential for genome-wide and transformation specific unintended effects
were recognised 304 in a recent FAO/WHO (2003) expert consultation on the food safety of GM
animals, or in a recent FIFRA expert panel report (see attached305).
884. That latest FAO/WHO expert consultation on GM animals updated the entire field of
assessing the food safety of GM products (FAO/WHO e xpert consultations are usually the basis for
CODEX recommendations) and concl uded that "An extensive molecular characterization of the
inserted genetic material construct will generally be required, both before a nd after the insertional
event. The molecular ch aracterization should furthermore comprise an analysis of the copy number
and a sequence analysis of the flanking regions of the place of insertion in order to identify any
unintended effects. It is recommended that the appr oach for the molecular characterization should be
further standardized to include the flanking regions"
885. This EcoNexus report and the other expert advice available clearly calls for radical changes in
the safety approach proposed by the US. There are three basic reasons why one can arrive at the
conclusion that there is "no evidence" of any remaining risk: (i) one did not look; (ii) one did look, but
the investigations were designed in such a way that they can not find the answer; (iii) on e did look,
with appropriate and well designed experiments, wi th negative results. Unfortunately, with all the
difficulties inherent to the need to test the safety of whole organisms "in real situations", because of
the novelty of the introduced charact ers, the majority of the potential questions relating to human
health come under (i): we did not look. And most of the science available to the regulators to assess
these products, because of lack of proper criteri a, harmonised and scientifically sound methodologies
to perform these new safety tests, fall under (ii), i. e. they are often poorly designed and inconclusive,
and very little comes under (iii).

303 See for example: A. Wilson et al. 2004. Genome scrambling – myth or reality; transgenic-induced
mutations in transgenic crop plants. Econexus technical report (October 2004). Available at www.econexus.info.
304 Introduction of a transgene into an animal is not a precisely controlled process, and can result in a
variety of outcomes regarding integration, expression and stability of the transgene in the host. The desired
outcome generally is stable integration of a single copy of the transgene into a single location in the genome,
and not in a functional gene or a regulatory elemen t. However, other outcomes are frequently observed,
including integration of multiple copies of the transgene at one locus or in sertion of the transgene at multiple
locations in the genome. Insertion of the transgene into a host gene may turn the host gene off, sometimes
affecting the viability or health of the host. Insertion of a transgene sometimes can affect expression of another
gene(s). A transgene may become rearranged before integration, thereby becoming non-functional. During the
process of transgenesis, undesired DNA sequences may become inserted into the genome, such as marker genes
or selectable markers from the expr ession vector or contaminating bact erial DNA left over from vector
production. Hazards stemming from insertional events or genetic instability can be identified by screening and
managed by culling individuals that have undesired events during the course of development of the transgenic
line. Expression of the transgene ideally should have no undesired effects on the expression of other host genes
or health of the host. Other outcomes, however, have been observed. The transgene can be silenced by
methylation or through other mechanisms.
305 SAP Report No. 2004-05. MINUTES of Federal Insecticide, Fungicide, and Rodenticide Act
(FIFRA) Scientific Advisory Panel (SAP) Mee ting, June 8-10, 2004, Arlington, Virginia. A Set of Scientific
Issues Being Considered by the Environmental Protection Agency Regarding : Product Characterization, Human
Health Risk, Ecological Risk, And Insect Resistance Management For Bacillus thuringiensis (Bt) Cotton
Products. 84 pages

886. The approach advocated by th e US is essentially designed to look the one new substance,
enzyme, or protein. But it should be clear now to the Panel that plants, ti ssues, and products derived
from GM crops are in many instances more complex th an purified enzymes, or proteins. Therefore,
additional issues, like for instan ce the occurrence of unintended ch anges in the GM crop likely
deserve greater attention than the single new protein.
887. The methodological difficulties arising from missing, emerging or new tests to assess such a
wide area of complex and uncertain organism-w ide potential risks, leaves the assessor and the
scientist alike to decide, subjectively, what is th e end point of data requirement and risk assessment,
and what constitutes a reasonable, science-based, evidence of lack of risks.
888. A last issue, although not explicitly apparent in the US statements on methods to assess
safety, merits consideration: the issue of whethe r and how to assess the genetic modification stacking
(an hybrid between two individual GM parents ca rrying the double modification). Following the US
and Dr. Nutti's approach, there would be no n eed to assess any new hybrid, as all individual
components would have been assessed separately ( each new gene products and each parent). And
indeed, the US deregulates GM crops, which can then be bred without any further assessment.
889. The EPA FIFRA expert panel report, previ ously mentioned, addresses this issue and
concludes as follow:
In principle, traditionally bred transforme d lines should have the same characteristics
as the parent organisms. However, analyses of events have pointed to potential
different outcomes in some cases where the basis of these events is not presently fully
understood. Two specific issues for consideration are:
(1) A molecular characterization of the new product should show that the
recombinant traits/ sequences in the new pr oduct are identical to the insertion /traits
in their parental lines. In principle, the method of breeding the two parental lines
could affect the genetic characteristics of the inserted traits. Therefore, evidence is
needed that no such effect has occurr ed. For these products, a risk assessment
combining evidence from parental lines and the product should be requested.
(2) Stability is another concern. For ot her similar products, it is uncertain how
many generations need to be observed to establish stability of inserted traits.
890. Consistent findings were made in the report on adequacy of the [US APHIS] regulation of
transgenic plants, by the US Committee on Environmental Impacts Associated with
Commercialisation of Transgenic Plants, and p ublished by the National Research Council and
mentioned earlier:
Finding 7.5: The current APHIS appro ach for deregulation does not assess the
environmental effects of stack ed genes for non additive or synergistic effects on the
expression of individual genes, nor does it assess stacked genes for cumulative
environmental effects at the field level.
Finding 7.6: There are at least two levels at which scientists and regulators must
look for interactions between inserted genes with regard to environmental effects:
(1) the individual plant phenotype and (2) the whole-field or farming system level.

891. Finally, as regards the use and validity of field tests data, there is much to debate, as the
validity and conditions of the tests, and their relevance for other regions and different agro-
environmental conditions, may lead to significant re quest for further risk assessment information.
These may as well relate to important safety questions , such as, for instance, the different value of
data collected on GM HT crop sprayed with the herbicide or not.
892. The debated question of the relative hazard posed between conven tional products and
genetically engineered ones will certainly remain an open question after this case; however, it is clear
that old, reductionist risk assessment approaches, are found inadequate to assess GM crops, by more
and more scientists, and more and more globally, in particular to assess their agro-environmental
direct and indirect effects, their non target imp acts, and their unintended characteristics, including
genome-wide potential changes and their associated possible health or environmental effects.
893. In the light of the expert advice and the above, it is clear that, under today's best science-based
understanding, the proposed US approach is flawed, incomplete and, by all means, an unacceptable
standard to properly assess the risks of GMOs.
Question 112

Please provide an assessment of the statements regar ding chronic toxicity t esting in paragraph 43 of
Canada's Third Submission and paragraphs 134-138 of the US Supplementary Rebuttal Submission.
What relevance does the foreseen end product use(s) have in the context of the evaluation of the
toxicity of that product?

General comments

894. The Panel sought advice on the relevance of the Ca nadian and US assertions referred to in the
question. Canada's text does not refer to a particular scientific argumentation and is essentially a legal
argument to justify that EC's approval proce dures are SPS measures, and hence is not to be
commented in this submission. The US text is ess entially an illustration, of the disconnected risk
assessment approaches, applied respectively to the novel protein and to the substantial equivalence of
the GM crop, and as outlined in the US methodology presented in the previous question.
895. Only Dr. Nutti has replied, simply repeating mo re briefly her line of argument in her previous
reply, and dismissing that such tests might be necessary. The European Communities disagree with
her statement.
Detailed comments

896. The European Communities will refer the Pane l to its detailed comments on the previous
question and in section III on general and methodol ogical issues, where ther e is ample scientific
explanation why these studies may be justified. May it simply say here that there are many reasons
why chronic or sub chronic studies may be requi red, including for instance when the inserted
construct has been not appropriately character ised and there might be a doubt on the exact
identity/modification of the expressed protein, or if previous acute toxicity tests had been performed
with surrogate proteins, and the equivalence of the proteins is not sufficiently proven, as requested by
the Codex guidelines (CAC/GL 45-2003).
897. The Codex guidelines, in paragraph 37, clearly states that the use of appropriate conventional
toxicology or other studies on the new substance ma y be necessary if, taking into account its function
and exposure, doubts about the safety of the new substance remain. This is certainly the case when the

construct is poorly characterised, potential unintende d effects of the genetic modification, or when
there are doubts on the acute gavage studies (protein used, methodology, interpretation, …).
898. There are generally no accepted clear distinctions available between classic (long-, short term,
chronic toxicology studies as developed for testi ng of pure proteins, pharmaceuticals or chemicals)
and whole food toxicology studies (new, and often confused) and feeding studies (developed often for
testing of feeds ). Furthermore, acute toxicity on mammals would only not assess the toxicity on other
animals, which may react very differently and be very important for the assessment of the
environmental impact (eg birds, fishes, insects, ..).
899. Acute toxicity testing may be highly inappropriate to find unintended effects because of its
lack of sensitivity. Chronic toxicity testing has been proposed by many scientific reports as a tool
(together with other profiling methods such as gene- expression micro arrays, biochemical, or
proteomic screening ). However limitations of w hole food studies have been identified and discussed
extensively.
900. In fact, many CA authorities started to ask fo r these studies because of uncertainties in the
molecular characterisation. The EU research proj ect on Entransfood has looked at it, and there is
agreement for the usefulness of chronic toxicity studies, however some disagreement on their length.
901. Finally, the US EPA FIFRA expert panel mentioned earlier recommends multi year non
vertebrate studies for environmental risk assessment. It states that:
"The Panel found that testing of multi-year field effects on non-target vertebrates is
generally essential. It can readily be seen from the numerous publications Brewer et
al. (1989, 1989a, 1990, 1992; Tank et al. 1992, 1992a) that significant annual
differences are apparent with the same pesticide treatments by the same applicators in
the same fields"
902. Although realising the limitations of such studi es, chronic toxicology studies are needed
especially in the screening for unintended effects of GMOs because there are presently no better
methods sufficiently developed.
Question 113

Please provide an assessment of the statements regarding the purpose and use of whole food studies
in paragraphs 142-144 of the US Supplemental Re buttal Submission. What relevance does the
foreseen end product use(s) have in the context of using whole food studies in the evaluation of the
safety of that product?

General comments

903. The Panel sought advice on the relevance of th e US assertion referred to in the question. The
US stated that whole food studies were not warranted as a matter of principle, dismissing both a case
by case approach, and taking into account the final u se of the product, and the target animal with
which such studies may have been requested.
904. Only Dr. Nutti replies to this question, essentia lly repeating the US arguments, that requests
for whole food studies in different species does not have scientific support. The European
Communities notes that her reply to this question cont radicts her reply to question 112, as she stated
here that the request for whole food studies was not supported by Science, on the basis of availability

of 90 days oral studies, and of at least one more feeding study (usually 48 day broiler chicken). This
seems to indicate that if such studies were not available, she would support the request for whole feed
studies, and it is anyhow in contradiction with her reply on question 112, where she dismisses all
chronic or other studies apart from one acute toxicity study in mice.
905. Besides, she does not address the quality and relia bility of the information provided in each
application, and in particular she does not address the molecular characterisation data, nor does she
addresses the end use of the GM crop, where different target animals might need to be tested before
the product can be used safely as feed with these animals .
Detailed comments

906. The European Communities will refer the Pane l to its detailed comments on the previous
question and in section III on general and methodol ogical issues, where, again, there is ample
scientific explanation why these studies may be justified.
907. Whole food studies are necessary to complete th e assessment of the safety of new feeds or
foods for the following reasons:
The determination of the nutrients-toxicants (substantial equivalence) can not detect
all unintended effects (products);
The level of proteins may be increasing significantly ins successive products (See the
comparative levels of CRY IA (b) protein level in the Bt MON 810 compared to that
found in Bt 176 maize;
As is well known, acute gavage with recombinant proteins and in vitro degradation of
purified proteins have limited value;
Alternatively, use of well establishe d protocols for tolerance studies of
pharmaceutical are available but sometimes difficult to follow;
Whole food studies can and must be used to complement other safety testing
approaches.
306
908. There are still a lot of methodological issues that need to be clarified for the conduct of whole
food studies. The duration of tests on unintended effects must be standardised (42 days for poultry and
dairy cows; 100-120 days for pigs and steers). It is not possible to administrate the product at doses
that are multiple of the expected human exposure. Extended clinical and histo-pathological studies
may not possible and not appropriate for such studies.
909. There is the need, in order to achieve a reasona ble level of certainty in the safety assessment,
to use a combination of methods. As indicated in of the text FAO/WHO on Expert consultation
(Geneva 2000), completing the work of the OE CD (1993): " Integration of nutritional and
toxicological expertise was needed for the evaluation and be encouraged and facilitated".
Whole food studies must be performed on specific target speci es: monogastric for
grains, herbivores or ruminants for forages; recombinant proteins are more expressed
in leaves (x10 compared to grain); rodents could be used but are more resistant to
specific toxicants;

306 See König et al., 2004.Food Chem.Tox. 42-1047.

Numerous results have been published in the scientific refereed literature on
nutritional equivalence: as safe as….. no r ecombinant material found in animal
products (milk, eggs, meat…).
Successive approaches of whole food studies ha ve been performed in the US on RR
soybean
307, even as late as 2003:
910. "In vitro degradation and acute toxicity test s were performed on CP4EPSPS protein in order
to support that RR soybean was safe 308. But complementary results from whole food studies have
been provided from experiments performed on pi gs fed whole soybean meal up to slaughter. 309.
Additional toxicological data have been recently p ublished on mice fed soybean meal, on the basis of
data on the testicular development310.
911. This demonstrates clearly that even for a GM crop released already 10 years ago, a sort of
"monitoring" based on whole food studies has been performed for the oldest GM plant placed on the
market.
912. The US claims (3rd written rebuttal submission summary) that the request for chronic toxicity
when acute studies show no effect are not warranted in the case of NK 603 (page 8). However, just to
take one single example, in the case of maize NK603, there where two proteins were expressed
(CP4EPSPS and CP4EPSPS L 214 P) and these acute toxicity tests were notably insufficient (See
Q. 54).
913. Furthermore, it is clear that different parts of the GM crop may have different properties, and
express the individual genes integrated in the gene tic modification at different levels. This warrants
studies on different target animals, with different diet s as regards the plant parts, in order to assess the
unintended effects in the different part consumed.