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last updated: 25 Feb 2016
General Information
Brazil
Risk Assessment of Commercial Release of genetically modified eucalyptus - Event H421
EN
04 Sep 2015
Risk assessment details
  • BCH-LMO-SCBD-109049-3
    FGN-Ø1A21-5 - Eucalyptus modified for increased growth and yield
    | FuturaGene Brasil Tecnologia Ltda | Changes in physiology and/or production (Yield), Changes in quality and/or metabolite content (Cellulose), Resistance to antibiotics (Kanamycin, Neomycin), Selectable marker genes and reporter genes
Methodology and points to consider
Pursuant to Law 11105/2005, CTNBio is committed to assessing the risks that genetically
modified organisms may present to the environment, biodiversity and human and animal
health. Therefore, the comprehensive protection targets designated by the Brazilian Federal
Constitution are reflected in this law. CTNBio has the duty of identifying what constituents of
the environment may be affected by the organism studied and use the results to develop risk
hypothesis and draw conclusions on the product safety. For this purpose, the guidelines of risk
assessment set by the Biodiversity Convention (http://www.cbd.int/doc/meetings/bs/mop-06-
13-add1-en.pdf) and the Office of the Australian Gene Technology Regulator (OGTR)
(http://www.ogtr.gov.au/internet/ogtr/publishing.nsf/(Content/raffinal5-toc) are valuable and
complete the approach prescribed in CTNBio Ruling Resolution no. 05.
Genetically modified eucalyptus event H421 is being assessed under containment since it was
obtained and in fields in the case of Brazil, in planned releases into the environment passed by
CTNBio since 2006, showing that adequate management and intensive cultivation can yield
wood for both, production of biomass for energy and production of fibers.
Experiments were conducted in four Brazilian regions where this eucalyptus is likely to be
cultivated: Angatuba, São Paulo; Araraquara, São Paulo; Caravelas, Bahia; and Passagem
Franca, Piauí. In these regions, event H421 was compared to conventional eucalyptus SP350.
Experiments were installed with six repetitions in each field and were assessed in cycles of 2 to
3 years when targeted to production of energy biomass and in a range from 5 to 7 years to
production of cellulose and paper. Besides assessments of performance and management,
these experiments were examined for environmental risk in the context of the application for
commercial release.eucalyptus event H421 produces more wood because of gene
cel1 isolated from Arabidopsis thaliana, which codes for expression of enzyme β-1,4
endoglucanase (Cel1), one glycosyl hydrolase that links to non-crystalline cellulose and
xyloglucans promoting relaxation of cell walls during the phases of expansion and elongation
of the plant cell. Protein Cel1 is a polypeptide of 492 amino acids with an estimated molecular
weight of 54 kDa.
Event features in addition gene nptII, which codes for protein NPTII (neomycin
phosphotransferase type II), having resistance action on the action of antibiotics such as
neomycin and kanamycin. Enzyme NPTII is a modified protein of Escherichia coli. Gehe nptII
used in transformation of plants is derived from transposon Tn5 of Escherichia coli (Beck et al.,
1982), an enterobacterium that is present in the intestinal flora of humans. Protein NPTII is
produced by different prokaryotic microorganisms found ubiquitously in the environment,
both in aquatic and terrestrial habitats an in human and animal intestinal microflora. The
action of protein NPTII is well characterized and culminates with inactivation of aminoglycoside
antibiotics, such as neomycin, gentamycin, paromomycin and kanamycin A, B and C. Protein
NPTII is degraded in human and animal gastrointestinal tract. The use of selection marker
genes, such as gene nptII is essential in the selection of cells transformed in the process of
genetic modification (Horsch et al., 1984; DeBlock et al., 1984).
Molecular analysis (Southern blot, sequencing and PCR) depict the insertion of a single copy in
the expression cassettes of genes cel1 and nptII of the eucalyptus genome, confirmed by the
Mendelian segregation pattern in progenies. Sequencing of event H421 showed that constructs
present in the vector were preserved in the transformation and that there was neither gene
disruption nor structure disruption of the parental eucalyptus genome expression. Stability of
integrated DNA was shown by PCR, where the constructs inserted in the DNA, as well as their insertion loci, were maintained in the tested progenies, obtained in controlled crossings with
different species and conventional commercial varieties.
Studies show that there was insertion of a single functional copy of genes cel1 and nptII
expression cassettes in a single locus of the eucalyptus genome.
Proteins expressed in eucalyptus H421 do not display toxic activities on other organisms. In
silico assessment of toxicity and allergenicity with renowned databanks failed to indicate that
proteins Cel1 and NPTII are similar or homologous to known allergens and toxins.
Expression levels of proteins in plant tissues (culture tissues, young and mature leaves,
branches, roots, pollen grains) as well as in products of honey bees (honey, pollen balls)
showed to be quite low. Concentration of endoglucanases in young leaves of event H421 was
27.4μg/g of fresh tissue, as against 22.67μg/g of fresh tissue in conventional clone SP530. In
mature leaves, endoglucanases showed concentration of 18,87μg/g of fresh tissue in
eucalyptus H421 and 22.33μg/g of fresh tissue in the conventional isoline of mature leaves.
Average quantities of protein NPTII expressed (in μg/g of fresh tissue) were 5.3ng/g in pollen,
16.7 in young leaves, 22.2 in mature leaves and 27.2 in roots. Eucalyptus is an arboreal species with no known record of use as food in Brazil and other
countries, neither directly nor processed, is not consumed by humans or animals that serve as
food to man or other domestic animals.
Arabidopsis thaliana, the donor organism of the cel1 gene, is not a species possessing
evidence of being pathogenic or toxic to human and animals and is nor customarily allergenic,
being analytic and toxicological tests unjustifiable. Arabidopsis thaliana became a model plant
for genetic and plant development studies by having its genome entirely sequenced. The organism is a genre of the Brassicaceae family, which includes species cultivated and
consumed in human food such as cabbage, broccolis and radish, all with a safe record of use.
Protein Cel1 can be found, in very similar forms and with strong identity, in a number of raw
(cucumber, tomato, grape, apple) and processed food (cocoa, soybean, maize) consumed by
humans and animals. In addition, some hydrolases, such as β-glucanases of Aspergillus niger
and Bacillus subtilis, have been used for a long time in alimentary processing and are included
in the food category; noxious effects have not been documented (Pariza and Foster, 1983; in
Franck Oberaspach and Keller, 1997).
Gene neo (or nptII) and its product, enzyme NPTII , has been largely used as a selection marker
in transforming human, plant and animal cells and in experiments of gene therapy in humans.
The protein has no toxic effect to living beings, is naturally present in the environment and
found in microorganisms in foodstuff or in food that colonize the human digestive system
(Fuchs et al., 1993). The European Food Safety Authority – EFSA, the Australian Food Safety
Authority – OGTR) and the United States Food and Drug Agency – FDA reached a conclusion
that the use of gene nptII as a selection marker in genetically modified plants – GM (and
foodstuffs or rations derived thereto) fails to represent a risk to human and animal health and
to the environment. CTNBio has already passed commercial releases of events containing this
same selection marker.
Risk assessments were conducted in Piauí, Bahia and São Paulo. Centesimal composition
analyses show bromatological equivalence between H421 eucalyptus tissues and conventional
eucalyptus. Phenotype and ecological interaction assessments conducted in H421 eucalyptus
showed that this genetically modified event has no potential different from its conventional
equivalent to be an invading plant, or even to disseminate or perpetuate its genes or to
impact other plant or animal organisms in the environment. Toxicity analyses conducted by exposure to leaves and pollen to indicator organisms showed
that H421 eucalyptus fails to cause adverse effects on aquatic species, such as fish (Danio rerio)
and micro-crustaceans (Daphnia similis), and land species, such as bee honey (Apis) and
different species of native bees with no sting, granting these species the same conditions of
exposure granted by cultivation of conventional eucalyptus. Toxicity assessment in honeybees
was conducted for distances of 400m, 1km and 85km of the cultivation locus.
Physical and chemical analyses of different honeys produced in areas featuring genetically
modified trees and in area planted with conventional trees showed that there is no significant
difference for all parameters analyzed. Total protein content of honeys analyzed, both coming
from areas with transformed trees (0.43%) and coming from conventional area (0.33%) are
very low, being only part of the protein due to presence of pollen in honeys. Cel1 protein
concentration expressed in event H421 in honey and pollen balls is so low that detection
methods available for its measurement were not able to quantify it. For NPTII , concentrations
were among the lowest levels found when compared to other tissues of the plant, such as
leaves, roots and pollen. A pertinent observation is that man and animals do not absorb
proteins, but amino acids and, therefore, concerns related to toxic effects in the consumption
of the transgene’s proteins in such tiny amount are unfounded.
There is another concept that we may introduce, the TTC – Threshold of Toxicologic Concern.
The concept, found in details in Kroes et al., 2004, is being increasingly used by the European
Community, specifically by the following committees: Scientific Committee on Consumer
Safety (SCCS), Scientific Committee on Health and Environment Risks (SHER), Scientific
Committee on Emerging and Newly Identified Health Risks (SCENIHR). According to the TCC
concept, substances that are present in foodstuffs may be held as safe up to the following
doses:
· 1.5μg/individual: General threshold. Substances with positive Ames test or structural alerts shall be assessed on a case by case basis;
· 1.5μg/individual/d: Threshold for chemical substances with no structural alert to
carcinogenicity or negative results in mutagenicity tests (Ames’ Test);
· 45μg/individual/d: Threshold for chemical substances with no structural alert to
carcinogenicity or negative results in mutagenicity tests (Ames’ Test) and LD50 (median
lethal dose)> 1.000 mg/kg of body weight.
EN
Considering the negligible amount of pollen proteins that are present in honey, we may, in this
case, use TTC in a way where there is not necessary conduct this product test in animals.
Independent analyses conducted in the Project “Criação do CDA Eucalyptus: Centro
Colaborador em Defesa Agropecuária para a Biossegurança Relativa a Plantas GM de Eucalipto
(“Establishment of CDA Eucalyptus: Collaborating Center in Agriculture and Livestock Defense
for Biosafety Related to eucalyptus GM plants”), a document prepared by the Agriculture and
Livestock Defense Secretary (SDA), the Ministry of Agriculture, Livestock and Supply (MAPA);
jointly with the National Scientific and Technologic Development Council (CNPq), the Ministry
of Science and Technology (MCT); between December, 2008 and June 2012 with the purpose
of developing and adjusting protocols of assessment, guidelines for comparison and analyses
services related to biosafety of plants and plant genetically modified derivatives, strengthen
conclusions obtained by applicant in assessing the biosafety of H421 eucalyptus. Among the
results of the Project, comparing genetically modified and non-genetically modified derivatives
the following shall be noticed: (i) equivalence of composition in essential oils extracted from
leaves and their in vitro effect on phytopathogenic fungi; (ii) evidence of equivalence (absence)
of in vitro toxic effects of honey and foliar extracts on human colon cells and on the
development of bees (Apis mellifera) larvae, as well as on the anatomy of adult insects of the
species; (iii) establishment of native bee small colonies in laboratory for testing the toxicity of derivatives (pollen and honey) of genetically modified and non-genetically modified plants; (iv)
verification of equivalence of honey samples produced in genetically modified and nongenetically
modified areas.
The data found in the project turned possible the following conclusions:
· Eucalyptus event H421 was molecularly characterized and maintenance of integrity of
the inherited genic construct; to wit, that it is a single and intact insert, integrated in a
single locus or the eucalyptus genome, where all the gene elements remained intact,
and no locus of plasmid pB1121 present in the transformation event generated;
· The eucalyptus is not used in human or animal food, though the inserted proteins are
easily digested;
· There is no indication that proteins Cel1 and NPTII present in H421 eucalyptus may
cause allergy or intoxication in humans and animals;
· Expression levels of exogenous proteins expressed in event H421 are extremely low in
plant tissues and undetectable and honey and pollen balls of the beehive.
· No pleiotropic (multiple effects of a single gene) or epistatic (presence of one allele
that inhibits the action of another one) effects were identified in eucalyptus H421;
being the expected characteristics of increased production of wood the only
observable effect.
Following the above observations, the conclusion is that from the viewpoint of human and
animal health, eucalyptus event H421 is as safe as its conventional equivalent.
EN
One issue raised during the public hearing held to discuss eucalyptus H421 relates to
production of honey, since eucalyptus, according to a recurrent argument of the audience, is
“the main source of nectar and pollen for Brazilian apiculture”. Two papers present studies on
eucalyptus gene flow, that is to say, on the distance the bees can take the pollen: Pacheco et
al. (1996) and Campinhos et al., (1998). In the first studies were conducted up to a distance of
300m and reached a conclusion that “Apis mellifera L. is effective in promoting crossed
pollination in Eucalyptus saligna, being greater the activity at a distance limited to 100 meters
of distance, gradually decreasing up to the studied distance of 300 meters”. The second study
uses alloenzymes as markers to estimate the degree of natural hybridization between
Eucalyptus grandis and Eucalyptus urophylla. Genotypes were found that were not present in
the seed nursery, to wit 14.4% of the genotypes came from populations localized 400m distant
from the study area. Extrapolation is the use of regression models to estimate the value of the
dependent variable (response) for a given value of the independent variable (explanatory) that
are outside the observed values of the explanatory variable. This leads to unreliable and
sometimes ludicrous values (Larose & Larose, 2014). Even using the extrapolation of the
regression model presented in the study by Pacheco et al. (1996) and by Campinhos et al.
(1998), the distances in which the gene flows equal zero are 756m and 467m, respectively. The
study submitted by applicant assessed the gene flow up to a distance of 1592m, reaching a
conclusion that at a distance of 0 (zero) meter the flow was found to be 30.8%. This value
keeps decreasing with the distance, with observations of 14.6% at 10m, 13.1% at 25m, 11.5%
at 50m, 11.2% at 100m, 5.0% at 200m, 5.4% at 300m 1.5% at 400m, tending to 0,0% at a
distance nearing 600m. This value is close to the ones extrapolated of published papers.
In 2013, Brazil has exported about 16 thousand tons of honey, and about 78% of this total was
exported to the United States of America (ABAMEL, 2014). There is a concern about the loss of
honey market value due to presence of transgenic material in the product. Article 2 of Decree
no. 4680, of April 24, 2003 defines, in the marketing of foodstuff and alimentary ingredient goods targeted to human or animal consumption that either contain or are produced from
genetically transformed organisms, presence of GMO above one percent of the good this
transgenic nature of the product shall be informed to consumers.
However, analyses conducted in honey collected in beehives within the tillage of H421
eucalyptus, Cel1 protein was not detected and the content of protein NPTII was 3.9% of
honey, which is way below the 1% defined in the Decree 4680 for the product to be labeled as
transgenic. Law no. 10831, of December 23 2003, regulated by Decree no. 6323, of December
27, 2007, making provisions on organic agriculture, considers, in its Article 1, elimination in the
use of genetically modified organisms in the agriculture and livestock organic production
system. Regulations on production of organic honey are defined in Ruling Instructions no. 46,
of October 6, 2011, and no. 17, of June 18, 2014. Article 8(IX) of RI 46 it is defined that Plano de
Manejo Orgânico, the Organic Management Plan shall include measures to prevent and
mitigate the risks of external contamination, including GMO and derivatives. However, RI 17, in
its chapter on honey production, particularly in Article 21(V) defines that the organic
production system of honeybees shall use “only non-genetically modified honeybees”. The 20th
Edition of Diretrizes para o Padrão de Qualidade Orgânico IBD – Associação de Certificação
Instituto Biodinâmico, the Directives of IBD – Biodynamic Institute Certification Association
Organic Quality Standard, of August, 2014, an organization responsible for over 100
certifications of organic honey producers, in subparagraphs 4.2 and 4.3 defines that only
products containing not less than 95% of certified ingredients of organic agriculture may use
the seal IBD CERTIFICAÇÕES Orgânico, the Organic IBD CERTIFICATIONS. Special labeling
requirements apply to products that use in their composition at least 95% to 70% of organic
raw materials. The products’ mail labels may display the notice “made with organic
ingredients”. The Animal Health Sector Committee, of the National Organic Standards Board,
the body that recommends to the United States of America Department of Agriculture the
rules to be followed in the production of honey, in a document dated as of December 27, 2010, does not restricts the use of transgenic material. Therefore, the concerns raised in the public
hearing on destination of honey in Brazil are apparently unfounded.
Considering that the a route to possible damages is very unlikely and that the expected
damage is small, one may conclude that the risk for bees and honey production is negligible
EN
In all procedures for release into the environment, the constituents quality of soil, quality of
water (yet no the abundance, which depend on human use), valued organisms in agriculture,
iconic species and native species with which the genetically modified organism may produce a
fertile offspring are regarded as biodiversity representatives. One shall additionally bear in
mind the cross border movement of the living GMO whenever this GMO may affect the
biodiversity of the host country.
In order for these constituents, still very comprehensive, to be translated in final assessment
points, indispensable to the establishment and tests of risk hypothesis, it becomes necessary,
at the outset, to extract from the eucalyptus biology and from observation of phenotypic
changes linked to the transformation of the event, the elements that would enable discarding
certain constituents representative of biodiversity. Eucalyptus biology is conveniently
described by OECD (2014), among other scientific literature of comparable value. Other perils
may be derived from the public perception of risk and are commented in this opinion.
Eucalyptus originated in Australia, with a number of secondary diversity center in Asia. There
are no species sexually compatible with eucalyptus in Brazil. The plant is pollinized by insects
and, though the pollen may be transported by wind, this is not the species reproduction
mechanism. Insects that pollinize eucalyptus in Brazil are mostly bees, the flight range of which
is lower than 1,000m. Each mature plant is able to produce large quantities of seeds, yet
eucalyptuses are commercially propagated through seminal or clonal seeding and not by direct seeding in the fields.
Although eucalyptus exhibits certain characteristics of invading plant, in general it fails to
behave accordingly (Silva et al., 2010b). The hybrid employed in this event was never reported
to be an invader. Only the Eucalyptus grandis species may behave as an invading plant, though
it does not exhibit this behavior in Brazil. According to Comunicado Técnico 83, the Technical
Notice 83, by Embrapa Florestas (Embrapa, 2002), eucalyptus was introduced in Brazil in 1904,
with the initial purpose of meeting the demand for firewood, posts and railway sleepers for the
Brazilian Southeastern region. Later, in the fifties, eucalyptus came to be used also as raw
material in the production of cellulose and paper, charcoal and fiber or particle sheet. It shall
be stressed that after over one hundred years of cultivation in different Brazilian
edaphoclimatic conditions, eucalyptus failed to exhibit invasive behavior.
Eucalyptus is not used as foodstuff in Brazil, and leaves, branches and seeds of the plant are
not consumed by protected animals of the Brazilian fauna. Honey made by bees based on
eucalyptus flowers is collected and marketed in Brazil and also exported. This honey may
contain small amounts of proteins NPTII and Cel1, depending on presence of the transgenic
event’s pollen. The alimentary innocuousness of such honey was assessed by CTNBio. The issue
of impact on pollinating insects imply selecting a final point of assessment. Given its abundance
and importance in pollinating commercial species, the bee Apis mellifera was elected as the
final assessment point.
Areas where eucalyptus is planted are customarily surrounded or bordered by legal
environment conservation areas and, in some cases, with collections of surfacing waters. Part
of the material coming from eucalyptus trees and also exudates may reach the soil, subsoil or
in surrounding collections of water. This is already the case in conventional eucalyptus tillage
and there is no reason to imagine that protein Cel1 if Arabidopsis thaliana, present in tiny
amounts in the eucalyptus plant tissues, maintained within plants, may negatively impact in the quality of soil and waters. Protein NPTII shall be also promptly degraded in the
environment and its antimicrobial action could only be felt in high concentrations on an
environment rich in bacteria. Degradability of the two proteins and their impact on different
indicator organisms have already been studied, especially for protein NPTII, including by the
applicant.
A comparative analysis of biomass degradation was conducted and failed to indicate difference
in speed and amount of residues between the GM eucalyptus and its conventional
counterpart.
Applicant submitted studies of indicator species, such as two aquatic species (fish and
Daphnia), a soil decompositor (earthworm), microorganisms and species of beneficial insects
(such as honeybees and stingless native bees). Honey decomposition studies failed to show
significant differences and also to find any difference in colony decomposition, being therefore
unlikely that the inserted proteins may cause any impact on such insects, as already reported
on expression, quantification and effects of the protein.
It shall be noted that applicant submitted plant material to different independent laboratories,
belonging to the project “CDA Eucalyptus: Collaborating Center for Agriculture and Livestock
Biosafety Related to Eucalyptus Genetically Modified Plants”, sponsored by MAPA, MCTI and
CNPq, which forwarded data on:
· Proof of morphologic equivalence and anatomic of pollen, seeds, flowers and fruits;
germination of pollen and seeds and development of seedlings;
· Composition equivalence in essential oils of leaf extracts and their effects on in vitro
phytopathogenic fungi;
· Proof of equivalence (or absence) of toxic effects in honey and leaf extracts on in vitro
cells of human colon and on development of bee larvae (Apis mellifera) as well as on the anatomy of adult insects of the species;
· Establishment of mini-colonies of native bees for toxicity of derivatives (pollen and
honey) of GM and non-GM plants;
· Proof of bromatologic equivalence of samples of honey produced in areas of GM and
non-GM trees;
· Definition of conditions for detection of GM samples by PCR and qRT-PCR;
· Beginning of comparative studies on transcriptomic and proteotomic with a view to
determine equivalence in transcripts and peptides in leaves and branches de GM and
non-GM Eucalyptus.
No negative effect was recorded. Based on the characteristics of the event, the eucalyptus
biology and the intended use of the GM variety, one may conclude that the only protection
target that could suffer a direct impact from the GMO other than the impact caused by the
conventional variety is the pollinator. As shown, proteins inserted, their degradability and
effects, quantified in honey and plant tissues, evidence that event H421 has no identifiable
effect on Apis mellifera.
EN
Taking into account that only the impact on pollinizers is a real threat, that is directly related
to the protection target recognized by the country Constitution (biodiversity including
protection of insects beneficial to the agro-ecosystem, etc.), this technical opinion considers
the indispensable elements for risk ranking, according to internationally agreed procedures, to
wit: exposure to danger and damage class (or magnitude). This opinion also draws a damage
pathway, from the hypothetical ingestion of transgenic pollen by bees to damages to
individuals, beehives or populations. Apart from this pathway, the results contained in the
dossier make clear that there is no negative impact of genetically modified pollen on bees.
All molecular data indicate that the event in study has only one copy of the insert, which is also
present in pollen. The construct is constituted by:
(1) The coding sequence of gene nptII from Escherichia coli, which is directed by a NOS
promoter and the termination sequence of transcription and polyadenilation derived
from the terminating sequence of 3’ of nopaline synthase (nos) of Agrobacterium
tumefaciens;
(2) The coding sequence of gene cel1 from Arabidopsis thaliana, which is directed by a
promoter of the Strawberry Vein Banding Caulimovirus (SVBV), and the terminating
sequence of transcription and polyadenilation derived from the termination sequence of 3’ nopaline synthase (nos) of Agrobacterium tumefaciens. This T-DNA was inserted
into the eucalyptus genome by transformation with Agrobacterium tumefaciens, to
express genes cel1 and nptII, resulting in the synthesis of proteins Cel1 and NPTII .
Bees transport the pollen to the beehives in different manners. The pollen may also be
transported to beehives by the wind. However, pollen is not food for bees, and is ingested
together with nectar or honey. The mass of pollen to which a bee may be exposed is very
small. Therefore, the amount of proteins Cel1 and NPTII that may be present in the bee diet is
many orders of magnitude lower. Protein Cel1 itself is widely distributed in plants and hence,
with a record of contacts with humans and animals, mainly through consumption of in natura
plants. Thus, in the first step of the damage pathway, we may assume that the likelihood of the
bee being exposed to significant amounts of the protein is very low. Protein NPTII is of
microbial origin and determines resistance to antibiotics, and this is a reason for the emphasis
in the protein risk analysis.
In the second step of the pathway it is worth creating a plausible hypothesis on how the
protein behaves in the digestive tract of a bee. Same as with pollen itself, the most probable is
that the protein is promptly digested, which actually occurs with the majority of proteins
ingested by the insect, the digestive tract of which is rich in proteases. Thus, the likelihood that
this protein comes to be distributed, in an active form, over the greater part of the digestive
tract is very remote.
In the third step we shall question through which mechanism enzyme NPTII could cause a
negative impact on the bee. There is no record in the literature that the enzyme would impact
the epithelium of the digestive tract of any organism, but it may inhibit the action of some
antibiotics that would otherwise be important in the management of bees in an apiary. It shall
be kept in mind that the enzyme has no antibiotic action and fails to kill bacteria. The amount
of protein remaining after proteolytic digestion within the digestive tract should not be sufficient to inhibit the action of antibiotics purposely added to the bee diet in apiaries. Then,
the likelihood that antibiotic concentrations capable of inhibiting NPTII are present in the
digestive tract of bees that come to graze on flowers of genetically modified eucalyptus is also
very small.
In the fourth step, it becomes intuitive asking why the apiarist would use the antibiotic that,
even in a very low likelihood, could be inhibited by the presence of NPTII. Therefore, the
probability that another antibiotic could be inhibited by NPTII is clearly very small. It shall be
kept in mind that both neomycin and kanamycin are not used in the treatment of bacterioses
in apiaries, being oxytetracycline the preferred drug and, less frequently, some
aminoglicosydes, anphenicols and sulfonamides (Monteiro et al.,2010). The probability that
the whole pathway comes to be realized is therefore very remote.
We may now assess the extension of the damage. In case all paths are realized and the bee
master could not control an eventual bacterial infection in his or her bees, there would be a
loss of the beehives. The same would not take place in the remaining beehives. The, the
damage would be restricted to some few beehives previously affected by a bacterial infection.
A damage that does not affect a whole population (in this case damage would be restricted to
some boxes of the apiary) can be held to be a minor damage.
Damage pathways similar to this one may be established for non-target arthropods, soil, water,
beneficial microorganisms or other protection targets, in case a suspicion based on science
might come to happen and the threat materialize itself in damage. Once more, the data shown
in the dossier strongly suggest that there are no impacts to soil, waters, microbiota and biota
other than those that could be caused by conventional eucalyptus.
A risk hypothesis that has been raised considers the horizontal transfer of gene nptII to
bacteria. In this case, the transfer would be to the bacteria of the bee alimentary canal microflora. This hypothesis, however, contradicts what is known of plant and microorganism
biology and, above all, what is known of gene organization and expression in these groups of
living beings. The probability of transfer of functional genes from plants to microorganisms is
extremely low, as shown more than twenty years ago by Fuchs et al. (1998). It is not surprise
that the transfer of a certain antibiotic resistance marker from genetically modified plants to
bacteria was never recorded in natural conditions (EFSA, 2007). Identical conclusions were
reached by OGTR (OGTR, 2012), Petersen et al. (2014) and many others.
EN
During the public hearing, several other threats were brought by the perception of actors in
the Brazilian and international scenario. Most hazards bear no relation to the direct GMO
impact, yet supposedly they do bear this relation to the monoculture or the technology
associated to the event.
Among the issues raised in explanations, the possibility of a gene that determines increase in
wood productivity causing an effect on plant metabolism was examined. It must be emphasized that after assessment of the event for several years, it became possible to say that
changes are insignificant and, moreover, that no change in characteristics linked to behavior of
the plant was observed, being this an important factor that emerged during the selection
phase of elite events for commercial release, similar to what happens with elite events
obtained by classical genetic improvement.
Another point discussed was the fact that essays displayed an absence of competitive
differential (and consequently invasiveness). This way, concerns were voiced around ability to
germinate and establishment of spontaneous, voluntary plants, coming from H421 and its
isogenic plant. Questions were raised on possible contempt of ability for development higher
than that of H421, after established (characteristics directly related to competitiveness) and on
existence of data accounting for H421 being no longer competitive. Answering to this question,
it was recognized that invasiveness depends on a series of plant characteristics, but rapid
growth is not included among these traits. Though a plant featuring faster growth may be
more competitive, it needs invading potential to start with, which is not the case of the hybrid
eucalyptus in study. It shall be noticed that no botanical characteristics of eucalyptus was
changed, therefore, its reproduction mode and phonological behavior remain unchanged. Two
essays on eucalyptus invasiveness through seeds produced in commercial tillage were
published (Silva et al., 2011a and Silva et al., 2011b). The first was conducted in 18 different
regions where eucalyptus is planted (Minas Gerais, São Paulo, Rio Grande do Sul and Espírito
Santo). The results show that inside the plot and on the adjacent area, that is to say to 10m
distant from the plot, the number of plantlets found ranged from 37 to 157, respectively.
Authors concluded that, though the plants have germinated, eucalyptus plants apparently fail
to establish in conditions found in commercial cultivars. Besides, the management system
used, keeping grazed the inner part of the plot and the surrounding area, as well as the
rotation used, prevent this establishment. Another published paper studies invasiveness of
commercial eucalyptus seeds in fragments of native forests, previously artificially seeded with Eucalyptus grandis or a hybrid of Eucalyptus grandis and Eucalyptus urophylla. On the 270th
day after sowing, no eucalyptus plantlet was recorded in the three loci studied. Applicant also
conducted a study reported in the dossier submitted, on two areas under Planned Release into
the Environment, located in Angatuba, São Paulo; and Caravelas, Bahia. The study show that
there was no significant difference between the percentage of seed germination for eucalyptus
H421 and the percentage of germination for the conventional eucalyptus, in field conditions.
Essays shown by applicant were conducted in important culture regions. Given the lack of
changes on the genetically modified eucalyptus phenological behavior, the impact on Brazilian
ecosystems is not likely to be different from the conventional eucalyptus.
Transformation of wood quality for greater content of cellulose versus productivity for
cellulose production was assessed. The question was whether the generated data actually
support the request for commercial release of H421. Besides, it was questioned whether there
are important experiments to be completed and, in the affirmative, whether they could
compromise the decision taken. Applicant clarified that the data brought by it and the
previously available information on eucalyptus, genes employed and their safety are sufficient
to reach a scientifically sound decision. Results extracted from planned releases are among the
date brought by applicant, though some results were not completed by the date of submission
of the dossier. Consistency of data was, nevertheless, assessed, with special emphasis on data
related to the impact on bees, which are important to support the assessment of the risks to a
protection target that may actually be impacted. Other data came to support the hypothesis,
fully satisfied, that there are no unanticipated effects derived from the transformation
operated in this event.
It is worth stressing that at the Conference of Parties to the Convention on Biological
Diversity - COP-9 (Decision IX-5), an agreement was reached between the parties that the use
of transgenic trees should be approved after studies based on science, under domestic legislations. Brazil, in this regard, complied with the terms of the agreement, through field and
laboratory tests considering the proposed use of the eucalyptus.
The effect of nptII was studied in bacteria present both in the intestinal tract of bees and of
human beings (in the consumption of honey). It was noted that in other countries antibiotics
are regulated for use in controlling diseases in bees. In Brazil, this use is not yet allowed.
However, presence of these substances in beehives could select resistant diseases, which
could lead to a future tragedy in case a severe disease turns epidemic, a case in which control
would be more difficult. As shown, antibiotics for which protein NPTII is effective are not in
clinic use. Besides, the protein is easily degradable and expressed in tiny amounts in plant
tissues.
Another theme discussed in the hearing relates to the ideal time to reach a conclusion on
whether there are not negative or unwanted interactions. Even before experiments start and
time length are inferred, performing a risk assessment is mandatory. Risk assessments
preceded all planned releases and in part supplied data for the commercial release risk
assessment. Considering that the only concrete harm to a protection target that represents
biodiversity is represented by consumption of GM pollen by pollinators, the time for
conclusion on risks may be small and indeed it was extrapolated in this case. However, the
safety of inserted proteins, the use context of eucalyptus and the data analyzed up to now
enable a conclusion on the use of this plant.
Another issue analyzed was whether the tests were conducted in an adequate form to enable a
conclusion that there are no negative interactions in bees and in the presence of pollen
containing GMO material. The information is relevant, taking into consideration that three
times more protein Cel1 was detected in pollen than in other tissues. It shall be stressed that
the amount of protein by gram was calculated with tissues of different humidity degrees.
Besides, the actual amount of the protein to which a bee may be exposed through pollen is minute, since the total mass of ingested pollen is very small compared with the mass of nectar
or of the bee itself. Besides, protein Cel1 is ubiquitous and one of its isoforms is undoubtedly
present in pollen of plants that are part of the bee diet. Summarizing, the most important is
that this protein is present in so minute amounts in the adult plant that it is undetectable.
Besides, the point was raised that no studies were conducted in animals (mice and rats) for the
purpose of assessing stability on digestion and possible (cito)toxic, mutagenic and teratogenic
effects. It shall be noted that this is a commercial release of a novel transgenic organism. It
shall be emphasized that eucalyptus is not used as food by protected and iconic sylvan animals
and therefore the issue of alimentary innocuousness lacks sense in nutritional assessment,
remaining the impact on bees being the main issue. For being highly unlikely, the impact on
animals that consume honey in non-managed both inside and outside the planted area was
also excluded. Protein NPTII was already extensively assessed and there are no robust reasons
to discard any non-negligible risk posed by this protein to human and animal health in
concentrations common in typical diets. The amount of protein Cel1, as mentioned above, is
undetectable in the adult plant and in a number of other plants consumed. Moreover, the
proteins are already distributed in nature, with no record of adverse effects caused by their
consumption.
Another important fact discussed in the public hearing was the possible increase on the use of
water and agrochemicals given the two years reduction in harvest cycle. It was noted that the
cutting time is an issue related to technology and management of the tillage, and has no
relation with the GMO direct impact in soil. About the faster water consumption by the plants,
if it proves to be the case, there is no reason to expect that this increase may imply a
considerable amount in consumption.
According to Lima et al. (2012), there is evidence that the increase in forest culture growth rate
affects positively the consumption of water. The magnitude of this possible impact is strongly affected by climate and soil in the planting locations. To ease this impact, the paper
recommends using forest management strategies. Among these strategies, according to the
authors, encouraging effects come from adequate spacing and percentage of occupation of the
hydrographic microbasin, and deliberate inclusion of critical areas as protection. Among the
results shown in defense of their conclusions, the authors divide the yearly amount of water of
atmospheric precipitation in a microbasin reforested with eucalyptus in two parts, one named
yearly production of water (Q) and the other, evapotranspiration in the scale of the
hydrographic microbasin. Therefore, atmospheric precipitation water has two different
destinations: watercourses and atmosphere. The same as in any other agricultural or forestry
ecosystem, water does not vanish and shall be always put to use.
Regarding herbicides, a reduction in their use shall take place, since the plants grow faster, cast
shadows on the soil and may produce more rapidly substances that can inhibit the growth of
competitive plants (allelopathy), reducing the period in which controlling practices are
necessary. These aspects relate directly with culture management rather than with GMO
safety. It shall be stressed that this technology is not turned to tolerance to herbicides.
The conventional genetic improvement program of eucalyptus has already produced clones
with properties that are similar to the ones of the genetically modified plant. This is due to the
fact that the clone used in the transformation has about 10 years, being more modern clones
significantly more productive. Therefore, water consumption in this genetically modified
culture is not different from consumption in any other garden clonal cultivation.
Some participants argued that the studies also lack scientific trustworthiness when indicating a
20% volume increase in wood with the H421 event, since samples average was leveled up, but
the change was in the range of 6% to 20% in different locations and also whether there may
be clone varieties currently studied that show the same increment without the transgenes.
Effective gain in productivity shall be assessed after the commercial release and has no bearing in risk aspects. It shall be stressed that this technology may be used in the future to elite
clones, with similar or even higher productivity when compared to the growth rates found in
this event.
However, this aspect relates to forest management of the culture and not with safety of the
GMO currently assessed by CTNBio.
EN
microflora. This hypothesis, however, contradicts what is known of plant and microorganism
biology and, above all, what is known of gene organization and expression in these groups of
living beings. The probability of transfer of functional genes from plants to microorganisms is
extremely low, as shown more than twenty years ago by Fuchs et al. (1998). It is not surprise
that the transfer of a certain antibiotic resistance marker from genetically modified plants to
bacteria was never recorded in natural conditions (EFSA, 2007). Identical conclusions were
reached by OGTR (OGTR, 2012), Petersen et al. (2014) and many others. The genetic modification introduced fails to change the basic characteristics of the plant, and
therefore the eucalyptus behaves the same as its conventional counterpart in cultivation
conditions, except for the characteristics that has been inserted.
EN
traditional molecular methods
EN
Additional information
EN
Records referencing this document Show in search
Record type Field Record(s)
Country's Decision or any other Communication Risk assessment 1

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