Document 08
Adviesraad voor Bioveiligheid
Conseil consultatif de Biosécurité
Compilation of comments of the experts in charge of evaluating
notification B/BE/19/V1
Ref. SC/1510/BAC/19_0178
Coordinator:
(VUB)
Experts:
(VIVES),
(ULg),
(Sciensano,
GMOLAB) en
(Sciensano, GMOLAB)
SBB:
INTRODUCTION
Dossier B/BE/19/V1 concerns a notification of the VIB, for deliberate release in the environment of
genetically modified higher plants (GMHP) according to Chapter II of the Royal Decree of 21 February
2005.
The notification has been officially acknowledged on 09 January 2019 and concerns a field trial with a
maize with an impaired DNA-repair mechanism.
Experts were invited to evaluate the genetical y modified organisms considered in the notification as
regards their potential impacts on the environment, including human and animal health, and
information relating to pre- and post-release treatment of the site.
The comments of the experts are roughly structured as in
- Annex II (principles for the risk assessment) of the Royal Decree of 21 February 2005
- Annex III (information required in notifications) of the Royal Decree of 21 February 2005
- Commission Decision 2002/623/EC of 24 July 2002 establishing guidance notes supplementing
Annex II to Directive 2001/18/EC.
Comments sent to the VIB are indicated in grey. It should be noted that all the comments received
from the experts are considered in the evaluation of this dossier and in formulating the final advice of
the Biosafety Advisory Council.
Biosafety Advisory Council -
Service Biosafety and biotechnology (SBB)
Rue Juliette Wytsmanstraat 14 B-1050 Brussels Belgium
T + 32 2 642 52 93 xxx@xxxxxxxxx.xx www.bio-council.be
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LIST OF COMMENTS RECEIVED
The comments below have served as a basis for a list of questions that the competent authority
forwarded to the notifier with a request to provide additional information. The comments highlighted in
grey correspond to the questions/comments selected and sent to the notifier.
1.
INFORMATION RELATED TO THE RECIPIENT OR (WHERE APPROPRIATE) PARENTAL PLANTS
(e.g. reproduction, survivability, dissemination, geographic distribution,...)
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
Page 4: it is mentioned that the maize plants will be detasseled; this is in contradiction with p 15
and 17 where it is said that some plants wil not be detasseled (G1b and G4). See further
2.
INFORMATION RELATED TO THE GENETIC MODIFICATION
(e.g. methods used for the modification, description of the vector,...)
Comment 1
C1 Page 7: off target analysis; provide info about in silico analysis to guarantee specificity. Are
there ATR and/or ATM homologues?
C2 Page 9: plasmid; provide a legend and info on the guides
Where are the guides situated? Provide scheme of the gene (exons/introns) and position
guides.
The deletion in ATRA is unusual big (1200 bp): did VIB use two guides?
Comment 2
As pointed out by the SBB/BAC, this is the first dossier with CRISPR/Cas9 modified plants and,
to the best of my knowledge, no European guidelines are available for the molecular
characterization and risk assessment of such genetical y-modified plants. However, taking into
account the principles and requirements of transgenic plants, the following information might be
relevant for the risk assessment of such plants:
1) The risk assessment needs to consider both intended and unintended effect of the
genetic modification. Regarding CRISPR/Cas9 edited plants, the design of the guide-
RNAs is a critical step to minimize off-target mutations. Little information is provided at
this level. The applicant claims that the guide-RNAs were designed in such a way to
minimize possible matches with other sites of the maize genome, but which stringency
criteria were applied and with which results are worth describing in the dossier. For
instance, whether and which possible secondary targets were identified by the
bioinformatic analysis would be interesting to know. In case such sites are identified,
targeted sequencing in the final GM events could be performed. Discussing possible off
targets is of relevance from the point of view of the aim and justification of the trial
(probably more than for identifying possible hazards and risks).
2) Out-segregation of the effector molecules Cas9 and guide-RNAs needs to be confirmed.
In this dossier both phenotypic characterization via linked herbicide resistance marker
Biosafety Advisory Council -
Service Biosafety and biotechnology (SBB)
Rue Juliette Wytsmanstraat 14 B-1050 Brussels Belgium
T + 32 2 642 52 93 xxx@xxxxxxxxx.xx www.bio-council.be
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(glufosinate in this dossier) and molecular characterization by PCR allowed to rule out the
persistence of these sequences in the final GM plants conducted to the field trial ing.
3) Description of the mutation and of the likely expressed peptides resulting from the genetic
modification. In two out of the three mutations introduced, single nucleotide additions
have been obtained. The consequences of such additions depend on their location in the
mutated gene (/ORF). New polypeptides are likely to result from frameshift mutations. As
these changes are intentionally introduced into the GM plants, they could be detailed by
the application. The same holds true for the intended deletion caused by the third
mutation. In case new peptides are produced, bioinformatic analysis could provide insight
into their potential al ergenicity and toxicity, as it is done for the newly expressed
polypeptides in transgenic GMOs. However, extensive bioinformatic analysis of the newly
expressed peptides is not required within the scope of field trials, hence this information
should not be demanded for CRISPR/Cas9-edited plants.
Comment 3:
In order to allow detection of the modified lines, it would be relevant to know if the induced
mutations occur naturally in maize.
Note SBB
1. needs to give an estimation of the copy number. In line with this, we think it is worthwhile
to ask for information on the number of ATR and ATM (homologue) genes in maize.
2. does not need to analyse which maize genes have been interrupted due to the insertion
of a new DNA construct. In line with previous field trial evaluations we consider that
information on which off-target mutations have occurred (through targeted sequence
analysis), should not be asked in the early stages of development.
3.
INFORMATION RELATED TO THE GENETICALLY MODIFIED PLANT
3.1. Information related to the traits and characteristics, which have been introduced or
modified
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
We are dealing here with a special case, where the intended modification is to enhance the rate
of stress-induced mutations. In other words, the intended effect of the targeted mutations is to
enhance the rate of non-targeted mutations. However, as described by the applicant, previous in
vitro experiments have indicated that reduced growth is observed upon chemical treatments
diminishing the rate of DNA replication, but not in greenhouse conditions, suggesting that
detrimental effects become evident in specific conditions only. Overall, the available information
suggests a possible reduction of fitness of the GM plant, as compared with the parental
genotype, but no increase that might impact the persistence and invasiveness of the plant,
which is a key issue in the environmental risk assessment.
3.2. Information on the molecular characteristics of the final GMO
(e.g. number of copies of the transgenes,...)
Comment 1
D1 and D2: it is mentioned that no donor material is present in the crispr cas lines.
Biosafety Advisory Council -
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Rue Juliette Wytsmanstraat 14 B-1050 Brussels Belgium
T + 32 2 642 52 93 xxx@xxxxxxxxx.xx www.bio-council.be
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It is mentioned in Bijlage 2 that (q)PCR was performed to demonstrate absence of backbone;
these data are not shown/ Please provide data demonstrating the absence of donor material.
Comment 2
Many of the characteristics of transgenic GM plants subjected to molecular analysis (e.g. copy
number, integrity, expression of the introduced sequences) are not applicable to edited GM
plants.
3.3. Information on the expression (of the insert)
(e.g. parts of plants where the insert is expressed, (expected) expression of the insert during the
lifecycle of the plant,...)
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
Not applicable.
3.4. Information on how the GM plant differs from the recipient plant
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
See comments in 3.1.
3.5. Genetic stability of the insert and phenotypic stability of the GMHP
Comment 1
Page 12 D5
investigate whether
Comment 2
The applicant explains that no genetic instability is expected for the introduced genetic
modification, which should behave like any other mutations. It is also mentioned that the
phenotypic stability will be studied during the field trial. I do not understand which phenotypic
traits wil actually be followed. If we refer to section D4a), no phenotype distinguishing the GM
plant from the reference line B104 is well established, a delay of flowering being the only
expected phenotype based on greenhouse observations. The trial can hardly study at the same
time which phenotypic trait characterizes the GM plant and how stable is this property.
Furthermore, whether the genetic/phenotypic stability can be studied at al in only one
generation of field-trial ed material is questionable. The applicant should clarify these issues.
Note SBB and coordinator: Information on which phenotypic characteristics wil be measured
in the field, is not a risk assessment-related question.
3.6. Any change to the ability of the GMHP to transfer genetic material to other organisms
Have evaluated this section and have no comments/questions: 2 experts
Biosafety Advisory Council -
Service Biosafety and biotechnology (SBB)
Rue Juliette Wytsmanstraat 14 B-1050 Brussels Belgium
T + 32 2 642 52 93 xxx@xxxxxxxxx.xx www.bio-council.be
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3.7. Information on any toxic, allergenic or other harmful effects on human health arising
from the genetic modification
Comment 1
Page 13 D7: effect of environmental stress at DNA level; see further
Comment 2
The increased rate of mutations expected in the GM plants potential y leads to new peptides
and the accumulation of intermediate metabolites, both raising potential safety issues. However,
this corresponds to an increased frequency of events which spontaneously happen in the maize
genome and have not raised any safety issues so far. Furthermore, the field trial is a
fundamental research aiming at quantifying the impact of environmental stress on the frequency
of mutations and the role of the knocked-out genes at that level. In conclusion, no further
information on the potential toxic or al ergenic constituents of the GM plant seems necessary.
3.8. Information on the safety of the GMHP to animal health, particularly regarding any toxic,
allergenic or other harmful effects from the genetic modification, where the GMHP is intended
to be used in animal feedstuffs
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
See comments under 3.7 above.
3.9. Mechanism of interaction between the genetically modified plant and target organisms (if
applicable)
Have evaluated this section and have no comments/questions: 2 experts
3.10. Potential changes in the interactions of the GMHP with non-target organisms resulting
from the genetic modification
Have evaluated this section and have no comments/questions: 1 expert
3.11. Potential interactions with the abiotic environment
Have evaluated this section and have no comments/questions: 1 expert
3.12. Description of detection and identification techniques for the GM plant
Have evaluated this section and have no comments/questions: 1 expert
Comment 1:
We want to note that the provided protocol only al ows detection of nucleotide modifications in
(planted) homozygote material. With this protocol one is unable to check heterozygous material
for nucleotide modifications, if requested. For example, one wil not be able to verify whether
GM pollen have contaminated maize occurring in the surroundings. For the latter, more
appropriate methods that can distinguish with sufficient sensitivity single nucleotide differences
need to be applied (e.g. NGS or digital PCR). We welcome any feedback on this observation.
Biosafety Advisory Council -
Service Biosafety and biotechnology (SBB)
Rue Juliette Wytsmanstraat 14 B-1050 Brussels Belgium
T + 32 2 642 52 93 xxx@xxxxxxxxx.xx www.bio-council.be
SC/1510/BAC/19_0178_
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Annex 9 gives the sequences of the oligonucleotide primers proposed for the PCR detection of
the modified ATR and ATM genes, but the exact map locations of these primers are not
indicated. The position of the primers and their targets have to be il ustrated by a figure. Further,
the amplicon sequence should be provided to be able to analyse the sequence. Final y, data
should be provided to demonstrate that the method is working (e.g. PCR with gel and
visualisation of the PCR fragment, sequencing results and analysis, such as a BLAST or other
type of sequence alignment).
3.13. Information about previous releases of the GM plant, if applicable
Have evaluated this section and have no comments/questions: 1 expert
4.
INFORMATION RELATING TO THE SITE OF RELEASE
(e.g. description of the site ecosystem, presence sexually compatible species, proximity of protected
areas, ...)
Have evaluated this section and have no comments/questions: 2 experts
5.
INFORMATION RELATING TO THE RELEASE
(e.g. purpose of release, dates and duration of the release, methods for preparing and managing the
release site, number of plants, ...)
Comment 1
Purpose of the release:
It is mentioned that the aim of the trial is to monitor the GMO plants in the field.
It is advisabl
methods that wil be used to analyze the plants from the trial (NGS for mutations?). It is not clear
modified phenotype
while it was stated several times that the lines do not show a phenotype as compared to WT
plants.
F1)
Environmental pol ution: the GMO maize wil be planted in a classic non-polluted field. What
does VIB mean by environmental pollution? This needs more explanation. Also VIB is hoping for
not be control ed in the field, while in the greenhouse,
conditions such as light and drought and other stresses can be well regulated.
Note SBB and coordinator: The specific analyses and methodologies that will be used during
the trial and the information the applicants want to obtain from this trial, are not relevant for risk
assessment; consequently, there is no need to ask for further clarification.
Comment 2
The applicant proposes to cultivate the plot with commercial maize the year after the trial, based
on the absence of any regrowth of GM maize following previous releases since 2012. However,
I have no access to the information about the post-release operations in the previous trials and
on how the monitoring of the possible regrowth was performed, hence it is difficult to validate the
claims of the applicant.
Biosafety Advisory Council -
Service Biosafety and biotechnology (SBB)
Rue Juliette Wytsmanstraat 14 B-1050 Brussels Belgium
T + 32 2 642 52 93 xxx@xxxxxxxxx.xx www.bio-council.be
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Note SBB: The monitoring reports of B/BE/11/V4 and B/BE/14/V2 state that no volunteers were
found in years fol owing the trial.
6.
INFORMATION RELATED TO THE RISKS FOR THE ENVIRONMENT
6.1. Information on the likelihood for the GMHP to become more persistent than the recipient
or parental plants or more invasive
Have evaluated this section and have no comments/questions: 2 experts
6.2. Information on the selective advantage or disadvantage conferred to the GMHP
Have evaluated this section and have no comments/questions: 2 experts
6.3. Information on potential of gene transfer to other sexually compatible plant species
under conditions of planting and its consequences
Have evaluated this section and have no comments/questions: 2 experts
6.4. Information on the environmental impact resulting from direct and indirect interactions of
the GMHP with target organisms
Have evaluated this section and have no comments/questions: 2 experts
6.5. Information on the environmental impact resulting from direct and indirect interactions of
the GMHP with non-target organisms, including herbivores, parasites, symbionts...
Have evaluated this section and have no comments/questions: 2 experts
6.6. Information on possible effects on human health resulting from potential direct and
indirect interactions of the GMHP and persons working with, coming into contact with or
living in the vicinity of the GMHP release
Have evaluated this section and have no comments/questions: 2 experts
6.7. Information on possible effects on animal health and consequences for the food/feed
chain resulting from consumption of the GMO and any product derived from it, if it is
intended to be used as animal feed
Have evaluated this section and have no comments/questions: 2 experts
6.8. Possible immediate and/or delayed effects on biogeochemical processes resulting from
potential direct and indirect interactions of the GMO and target and non-target organisms
in the vicinity of the GMO release(s)
Have evaluated this section and have no comments/questions: 2 experts
Biosafety Advisory Council -
Service Biosafety and biotechnology (SBB)
Rue Juliette Wytsmanstraat 14 B-1050 Brussels Belgium
T + 32 2 642 52 93 xxx@xxxxxxxxx.xx www.bio-council.be
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6.9. Information on environmental impact of the specific cultivation, management and
harvesting techniques used for the GMHP where these are different from those used for
non-GMHPs
Have evaluated this section and have no comments/questions: 2 experts
7.
INFORMATION RELATED TO CONTROL, MONITORING, POSTRELEASE AND WASTE TREATMENT
7.1. Precautions taken
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
G1b page 16 and G4 page 17: why wil some plants not be detasseld?
Is
there a reason to assume that the lines wil not produce pol en?
Referee suggests to detassel al plants (in order to avoid dispersal of pollen)
7.2. Information on methods for post release treatment of site
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
See previous comment under section 5.
7.3. Information on post release treatment methods for the GM plant material, including
wastes
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
nd leaves are considered as GMO (due to the
method used to generate the plants) but it can be said that these plant residues do not pose
harm for the environment since these plant residues wil break down.
7.4 Information related to monitoring plans and the detection techniques
Have evaluated this section and have no comments/questions: 2 experts
7.5. Information on the emergency plan(s) proposed by the notifier
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
Biosafety Advisory Council -
Service Biosafety and biotechnology (SBB)
Rue Juliette Wytsmanstraat 14 B-1050 Brussels Belgium
T + 32 2 642 52 93 xxx@xxxxxxxxx.xx www.bio-council.be
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7.6. Information on methods and procedures to protect the site
Have evaluated this section and have no comments/questions: 1 expert
Comment 1
I could not find this information in the dossier.
Note SBB: information can be found in G.6
8.
OTHER INFORMATION
Comment 1
guidelines for the risk assessment of CRISPR-edited GM plants are lacking. It might be useful to
see whether such information is available from other competent authorities in EU (and/or from
EFSA or ISPRA JRC?).
Biosafety Advisory Council -
Service Biosafety and biotechnology (SBB)
Rue Juliette Wytsmanstraat 14 B-1050 Brussels Belgium
T + 32 2 642 52 93 xxx@xxxxxxxxx.xx www.bio-council.be
SC/1510/BAC/19_0178_
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