Transcript Scientic strategic for risk assessment - YL.ppsx

Committed since 2002
to ensuring that Europe’s food is safe
Scientific Strategy for GMO Risk
Assessment within EU
Yi Liu
Scientific officer
Taiex workshop on biosafety AGR 46779
Kiev, 19-20 April 2012
EFSA Guidance Documents on GMO Plants
PLANTS
• Guidance for risk assessment of food and feed from GM plants (2011)
• Environmental Risk Assessment (ERA) of GM Plants (2010)
Supporting documents
• Application submission guidance (2012)
• Selection of comparators for the risk assessment of GM plants (2011)
• Statistical considerations (2009)
• Use of animal feeding trials for safety assessment of whole GM food/feed (2008)
• Scientific Committee 90-day study on whole food/feed in rodents (2011)
• Allergenicity assessment of GM plants and microorganisms (2010)
• Post market environmental monitoring – PMEM (2011)
• Potential impacts on non-target organisms (2010)
• Guidance for non-food/non-feed use of GM plants (2009)
Committed since 2002
to ensuring that Europe’s food is safe
2
EFSA Guidance Documents
 Documents for:
 applicants: to follow in their risk assessment
 evaluators: to assess the risk assessment
 Living documents: e.g. 2006 GD being updated
with FF GD (2011) and ERA GD (2010), more
prescriptive, but the principles remain
 Detailed list of all the information necessary to
assess the safety of GM plants and derived
food/feed products
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to ensuring that Europe’s food is safe
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GMO RA methodology and principles
Science-based
Comparative approach Weight-of-evidence
Case-by-case principle
Step-by-step principle (tiered approach)
Scopes of GMO applications
Food (human consumption)
 GMO for food use
 Food containing or consisting of GMOs
 Food produced from or containing ingredients produced from GMO
Feed (animal consumption)
 GMO for feed use
 Feed containing or consisting of GMOs
 Feed produced from GMOs
Release into the environment
 Import and processing
 Seeds and plant propagation material for cultivation
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RA principle logic
Comparative approach
• Compare the GMO and derived products to their non-GM
counterparts
•
Assessment of the identified differences regarding environmental,
food/feed safety and nutritional impact
•
Intended effects = those occurring because of the genetic
modification
•
Unintended effects = additional effects which were NOT the
objective of the genetic modification
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Case-by-case approach
Within the frame of the RA approach described in the GD,
the GMO Panel requires a CASE-by-CASE approach
•
•
•
In the context of the intended uses of the GM product
In light of the evaluation of all available scientific information
(application + MS comments + scientific literatures)
In case the Panel needs more specific information: the applicant is
asked for additional information.
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to ensuring that Europe’s food is safe
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Guidance documents for Food Feed RA
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•
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•
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•
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Per section introductory paragraphs to explain why information is required
Per section summary of conclusion
Requirements for molecular characterisation
Assessment of protein expression
Requirements for the RA of GM plants containing stacked events
Criteria for selection of appropriate comparator(s) under different scenarios
(comparator GD 2011)
Design of field trials for compositional / agronomic / phenotypic traits (stats opinion 2010)
Statistical analysis of field trial data: difference / equivalence tests (stats opinion 2010)
Toxicological assessment: reference to internationally agreed protocols
Allergenicity assessment: newly expressed proteins & whole GM food/feed
(allergenicity opinion 2010)
•
Animal feeding studies: when considered necessary (EFSA 2008, 2011)
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Molecular Characterisation (1)
•
Description of methods used for the genetic modification

•
Method for the transformation & relevant bibliographic references
Source and characterisation of nucleic acid used for transformation

The complete sequence of nucleic acid intended to be inserted,
including information on deliberate alteration(s) to the
corresponding donor sequence(s).
•
Nature and source of vector(s) used

Physical map with the indication of the position of e.g. relevant
restriction sites, primers used in PCR, probes for Southern
analysis.
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Molecular Characterisation (2)
•
General description of traits introduced or modified

Description including their mode of action (e.g. novel proteins,
gene silencing traits).
•
Information on the sequences actually inserted/deleted

Sequence information from the 5’ and 3’ flanking regions with the
aim of identifying interruptions of known genes.

Bioinformatic analyses performed using up-to-date databases
and searching for intra- and inter-species similarity.

ORFs present within the insert and spanning the junction sites,
analysed for similarity to known toxins and allergens.
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Molecular Characterisation (3a)
•
Information on the expression of the inserted/modified sequences

Other than protein expression data (RNA, metabolites) are now
foreseen for non-protein based traits (e.g. RNA interference).

Expression data derived from plants grown under conditions
representative of typical cultivation practices (i.e. from field
trials).

The minimum requirement is defined as: 1 site/3 seasons, 3
sites/one season or any permutation of these.

The specificity of the protein analysis method should be
demonstrated.
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Molecular Characterisation (3b)
•
Information on the expression of the inserted/modified sequence

List of comparator(s) that are needed for single and stacked events.

Where appropriate, e.g. HR crops, the effect of specific treatment(s)
linked to the trait should be assessed for single events.

For stacked events, expression data for specific treatment(s) linked
to the trait(s) are only required if data obtained from the single
events indicate a potential safety concern.

For cultivation scope, depending on the trait, information may be
required to assess the impact on target and non-target organisms.
In such cases, data from various parts of the plant are required from
plants grown under conditions representative of typical European
cultivation practices.
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Molecular Characterisation (4)
•
Genetic and phenotypic stability

genetic stability of the insert(s), phenotypic stability, and
inheritance pattern(s) of the introduced trait(s).

For single events, data should usually be provided from five
generations or vegetative cycles.

For stacked events, the structure of the insert should be
compared to their respective single events, using plant materials
representative for commercial production.

Safety implications of the loss-of-function of gene(s) in a multigene cassette should be considered.
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RA of Stacked events
•
•
RA of singles is a pre-requisite
RA of higher stack should address
•
•
•
whenever relevant, sub-combinations from targeted breeding approaches
Focus on:
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all sub-combinations from natural segregation
Stability of inserts
Expression of the introduced genes and their products
Potential interactions (synergistic or antagonistic effects)
Choice of Comparator:
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•
•
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Conventional counterpart or
Negative-segregant or
Any set of GM plants already RA (experimental data)
non-GM reference varieties in the field for the equivalence component of the
comparative assessment
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RA of stacks: RA of singles is a pre-requisite
RA of plant containing stacked events:
requires the RA of plant containing single events
•
If RA of all singles involved is not completed, EFSA will stop the clock
for the stack.
EFSA will re-start the clock for the stack once the RA of all singles is
completed and no outstanding issues remain.
EFSA may require an update of the stack application to include cross
reference to the singles dossiers.
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•
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Allows a comprehensive evaluation of stacks on the basis of
knowledge acquired during the evaluation of all singles involved.
Allows the evaluation of potential interactions: stacked events in
comparison to single events.
Allows flexibility on the choice of comparator (when conventional
counterpart is not available).
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RA of stacks: sub-combinations
Natural segregation
Sub-combinations of events
Targeted breeding programs
Sub-combinations of events for crops with segregating progeny
(e.g. maize):
•
•
All sub-combinations arising by natural segregation should
be addressed (EFSA GD, 2011).
Import & processing applications should cover (scope) all
sub-combinations independently of their origin (either arising
by natural segregation or by targeted breeding programs; EC
request, 2010).
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RA of stacks: sub-combinations (cont)
Sub-combinations of events for crops without
segregating progeny (e.g. cotton):
•
•
Sub-combinations should not be included in the scope
(Panel may require demonstration of lack of segregation).
For each sub-combination intended for marketing, EFSA
requires the presentation of a separate application.
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Comparator – single events
FF risk assessment
• The comparator should be the conventional counterpart ,
• Additional comparators may be included if deemed useful
(e.g. negative segregant ).
Terminology
•
Conventional counterpart:
 non-GM isogenic variety (vegetatively propagated)
•
•
 genotype as close as possible (sexually propagated)
Comparator: all other cases
Non-GM reference varieties: those in the field to establish ranges of natural
variation (equivalence test)
Source: available at http://www.efsa.europa.eu/en/efsajournal/pub/2149.htm
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Comparator – stacked events by
conventional breeding
The comparator of choice is the conventional counterpart
Pre-requisite: the risk assessment of the GM plants containing the
events independently (i.e. GM plants containing single events) is
available (opinions adopted).
When the conventional counterpart is not available
negative segregant(s) derived from crosses between GM plants containing
events which have been risk assessed and which are all stacked in the GM plant
under assessment or
any set of GM plants that have all been risk assessed on the basis of
experimental data and that include between them all of the events stacked in the
GM plant under assessment, and no others.
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Comparator – events stacked by other
methods
The guidance takes into account :
- re-transformation
- co-transformation
- transformation with mutiple gene cassettes
The issue of possible segregation of the new event(s) is discussed and,
depending on the outcomes, the comparator requested are based on the
requirements for the GM plants containing single and stacked events.
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Comparator - Cases where the comparative
approach is not applicable
• GM plants with major modifications in metabolic pathways, possibly
leading to extensive compositional alterations (e.g. nutritionally enhanced
foods)
• GM plants with traits facilitating adaptation to environmental
stresses (e.g. drought/salinity).
Selection of appropriate comparators may be difficult.
FF risk assessment should focus on specific characteristics, should be
based on a comprehensive safety and nutritional assessment of the GM
plant and derived food and feed per se.
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to ensuring that Europe’s food is safe
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Comparator for ERA
When comparative approach is applicable,
ERA: Case-by-case basis depending on the plant’s characteristics and on the
purpose of the study for which the comparator is used (details in EFSA, 2010).
When comparative approach is NOT applicalbe
ERA: focus on the environmental impacts/management of the GM plant compared to
what is currently grown and/or against environmental protection goals. Comparators
should be chosen on a case-by-case basis:
• Non-GM line derived from the breeding scheme used to develop the GM plant
• Non-GM plant with agronomic properties as similar as possible to the GM plant
• Non-GM line having other characteristics as close as possible to those of the
GM plant.
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Design for field trial
GM
CV CV CV
1 2
3
C
CV
4
GM
GM
GM
C
C
CV CV CV
1 7
8
CV CV CV
1 2
3
CV CV CV
1 2
4
C
GM
GM
C
C
GM
C
CV CV CV CV CV
1 5
7
8
9
must be at least
8 sites, over one
or more years
must be the same GM,
non-GM comparator
at each site
may be different
commercial varieties
at each site
CV CV CV
1 5
6
must be at least 6
commercial varieties
over all the sites
CV CV CV
5 6
7
GM
C
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CV CV CV
5 6
7
CV
8
GM, non-GM comparator
& commercial varieties
are all randomised
and replicated at each site
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Design for field trials - herbicide tolerant crops
RA of herbicide-tolerant GM plants, containing single or
stacked events, the experimental design should include a
comparison of:
• GM plants exposed to the intended herbicide
• Comparator treated with conventional herbicide
management regimes
• GM plants treated with the same conventional
herbicide management regimes
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Statistical analysis
Test of Difference:
to verify whether the GMO is different from the non-GM
comparator (identification of possible hazard)
Test of Equivalence:
to verify whether the GMO is equivalent to appropriate
reference varieties (natural variation)
Difference
YES
NO
biologically relevant
biologically NOT relevant
Equivalence
YES
NO
within natural range of variation
outside natural range of variation
Committed since 2002
to ensuring that Europe’s food is safe
Source: available at
http://www.efsa.europa.eu/en/efsajournal/pub/1250.htm
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Toxicological assessment
Focused on
• Presence and levels of newly expressed proteins
• Potential presence of other constituents
• Possible changes in the levels of endogenous constituents (beyond
normal variation)
• Impact of other changes linked to the modification
No. OECD
402
406
407
408
410
415
416
417
421
Title
Acute Dermal Toxicity
Skin Sensitisation
Repeated Dose 28-day Oral Toxicity Study in Rodents
Repeated Dose 90-Day Oral Toxicity Study in Rodents
Repeated Dose Dermal Toxicity:21/28-Day
One-Generation Reproduction Toxicity
Two-Generation Reproduction Toxicity Study
Toxicokinetics
Reproduction/Developmental Toxicity Screening Test
Bacterial reverse mutation test
471
In vitro mammalian chromosome aberration test
473
Mammalian erythrocyte micronucleus test
474
Mammalian bone marrow chromosome aberration test
475
In vitro mammalian cell gene mutation test
476
In vitro sister chromatid exchange (SCE) assay in mammalian cells
479
Committed
2002
DNAsince
damage
and repair, unscheduled DNA synthesis in mammalian cells in vitro
482to ensuring
that Europe’s food is safe
Draft guideline on: In vitro mammalian cell micronucleus test
487
GLP recommended
Non-exhaustive lists
of OECD guidelines
selectively
applicable for
(geno)toxicological
testing for GMO RA
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Animal feeding studies (not mandatory)
Safety on newly-expressed proteins:
• Repeated dose 28-day oral tox-study (unless safety – including mode
of action – is documented).
Safety & nutrition of whole food/feed:
• When: if composition is substantially modified, or if molecular and
comparative analysis indicate possible unintended effects
• When (stacks): if indication for interactions
• What: 90-day toxicity study in rodents
• When being not equivalent, supplementary comparative nutritional
studies on rapidly growing animals.
Committed since 2002
to ensuring that Europe’s food is safe
Source: available at
http://www.efsa.europa.eu/en/efsajournal/pub/1057.htm
http://www.efsa.europa.eu/en/efsajournal/pub/2438.htm
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Allergenicity assessment
• Allergenicity is not an intrinsic fully predictable property of a protein.
• Genetic diversity and variability in atopic humans as well as geographic
and environmental factors are important.
• A cumulative body of evidence is necessary for the risk assessment.
weight-of-evidence approach to assess

likelihood of the novel protein to be allergenic

likelihood of the GM plant to be more allergenic than the
comparator (endogenous allergens).
Committed since 2002
to ensuring that Europe’s food is safe
Source: available at
http://www.efsa.europa.eu/en/efsajournal/pub/1700.htm
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Allergenicity assessment
Assessment of the newly expressed protein (1)
• Amino acid sequence homology comparison:
i) aligment-based criterion: involving 35% sequence identity over
a window of at least 80 amino acids is a minimal requirement.
ii) for short peptidic fragments (e.g. ORFs) a search for sequences
of continuous identical amino acids.
• Specific serum screening: if there is indication of sequence homology
and/or the source of the gene is allergenic:
i) individual (not pooled) sera from well-characterised allergic
individuals .
ii) IgE-binding assays
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Allergenicity assessment
Assessment of the newly expressed protein (2)
• Pepsin resistance and other in vitro digestibility tests:
i) more physiological conditions as well as specific sub-population
(e.g. infants) to be considered.
ii) interactions between the protein and matrix, and effect of
processing to be taken into account.
• Other tests might provide additional information:
i) in vitro cell based assays
ii) in vivo tests on animal models
•
When evidence shows potential adjuvant activity, the possible role of
these proteins as adjuvants should be considered.
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Allergenicity assessment
Assessment of the whole GM plant
If recipient is known to be allergenic, allergen repertoire should be
compared between GM plant and its comparator.
- Performed by analytical methodologies, e.g. proteomics, together with
allergic human sera as probes.
- To minimise the use of human sera, preliminary information from the
inclusion of allergens in the compositional analysis and using sera from
animals experimentally sensitised.
- The integrated process applies to edible components and the pollen of
GM plants (i.e. covers both food and respiratory allergy risk).
- Available information on the prevalence of occupational allergy in
workers or farmers should be provided.
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ERA Guidance Document
Environmental Risk Assessment (ERA)
(1) Problem formulation (including hazard
identification)
Systematic approach
(2) Hazard
characterisation
6 steps as described in
Feedback
7 area of risks
(3) Exposure
characterisation
(4) Risk characterisation
(5) Risk management strategies
Directive 2001/18/EC
(6) Overall risk evaluation and conclusions
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to ensuring that Europe’s food is safe
Overall Risk Management, including
Post Market Environmental
Monitoring (PMEM)
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ERA Guidance Document
Strategies for ERA of GM plants
Cross-cutting issues
Specific areas of risk
1.
Choice of comparators
1.
Persistence and invasiveness
2.
Statistical considerations
2.
Horizontal gene transfer
3.
Long-term effects
3.
Target organisms (TO)
4.
Receiving environments
4.
Non-target organisms (NTO)
5.
Farming practices
6.
Biogeochemical processes
7.
Human and animal health
5.
Stacked events
Source: available at
http://www.efsa.europa.eu/en/efsajournal/pub/1879.htm
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ERA Guidance Document
Cross-cutting considerations
Comparators
Receiving environments
Long-term effects
Step 1
Step 2
Step 3
Step 4
Step 5
1
2
Stacks
3
4
5
NonTarget
Organisms
Persistence &
invasiveness
Statistics
Horizontal
Gene
Transfer
Target
Organisms
6
7
Human and
animal
health
Impact of
cultivation
practices
Impact on
biogeochemical
processes
Step 6
Conclusions ERA & PMEM
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Interplay RA-RM-PMEM
ERA as starting point


Overall conclusions of ERA, including
identified risk, if any, and critical
uncertainty
(e.g., knowledge gap)

CSM to confirm assumptions of the ERA
(case-by-case) &
GS to monitor unanticipated adverse
effects (Mandatory!)
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Source: available at http://www.efsa.europa.eu/en/efsajournal/pub/2316.htm
Methodology for general surveillance
GS is not hypothesis-driven (as targeting unanticipated adverse
effects) but focused on protection goals.
When setting up the GS plan, applicants should consider the
complementary tools for GS.
At the desk: comprehensive literature review
At field scale: farmers survey for first-hand info
At landscape level: existing networks
 surveying the environment
(e.g., fauna)
 surveying production and land use
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(e.g., fertilisers, pesticides)
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GS reporting
Annually & periodically (e.g., every 3 years)
Request for raw data
e.g., re-analysis of farmer questionnaires for MON810
Applicant remains sole responsible for PMEM but… GS goes beyond
monitoring of GMOs
 Monitoring of agro-ecosystems necessitating close cooperation between
≠ parties
 Common platform for PMEM data collection & storage
Close collaboration amongst applicants, RA and RM
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Risk assessment of GMOs at EFSA
All guidance documents are available at
http://www.efsa.europa.eu/en/gmo/gmoguidance.htm
Further questions on guidance documents to:
[email protected]
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Yi Liu
EFSA GMO Unit
[email protected]
Thank you for your attention!
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