Summary of the safety assessment (food safety): |
Monsanto do Brasil Ltda. requested a CTNBio Technical Opinion related to
biosafety of the insect-resistant genetically modified cotton (Gossypium hirsutum),
namely Bollgard II Cotton, Event MON15895, for the purpose of free registration,
use in the environment, human and animal consumption, marketing and industrial
use and any other use and activity related to this GMO including derivative
lineages and cultivars as well as byproducts, all under the remaining regulations
and requirements applicable to any use of cultivated species of the genus
Gossypium effective in Brazil. Bollgard II Cotton was produced by introducing,
through biobalistics, genes cry2Ab2 and uidA in the Bollgard cotton genome, as
approved by CTNBio in 2005. Plasmid PV-GHBK11 was used to insert genes
cry2Ab2 and uidA to Bollgard cotton genome, generating MON 15895 15985
cotton. Therefore, Bollgard II cotton Event 15985 contains the exogenous genes
340/2009
3 49
cry1Ac, cry2Ab2, nptII, aad and uidA, expresses proteins Cry1Ac, Cry2Ab2,
NPTII and GUS, differing from its Bollgard parental in proteins Cry2Ab2 and GUS.
Combination of proteins Cry2Ab2 and Cry1Ac represents an additional tool to
fight plague resistance to protein Cry1Ac, since Cry2A is a class of proteins
coming from Bacillus thuringiensis, different from Cry1Ac. The uidA gene, also
known as gus or gusA gene, derived from the K12 strain of Escherichia coli,
codifies the GUS enzyme, which was used as a selection mechanism of
transformed cells. The cry2Ab2 gene that codifies the Cry2Ab2 protein is derived
from bacterium B. thuringiensis, a gram-positive soil micro-organism. Commercial
formulations of B. thuringiensis have been used in Brazil and other countries to
control some agricultural plagues for over forty years. Cry2Ab2 and Cry1Ac are
proteins that feature very specific action, showing toxic effect through ingestion
only and acting in specific receptors located in the middle intestine of some
insects of the Lepidoptera Order. Stability and segregation analyses, in ELISA
essays for protein Cry2Ab2 in four generations, support the conclusion that event
MON 15985 is a single copy event of stable insertion. Chi-square analysis
indicates that the insert segregates according to Mendelian genetics, with a
segregation pattern of a single gene, against detection of protein Cry2Ab2.
Southern Blot analyses in generations R1, R2, R3 and R4 and two second
generation retro-crossing lineages (BC2F3), digested with enzyme SphI and
hybridized with a probe of the coding region of gene cry2Ab2, evidenced that the
transgene is stable across different generations, since no difference was noticed
in the band patterns obtained. From the molecular analyses showed, it becomes
340/2009
4 49
evident that Bollgard II cotton event MON 15985 possesses a copy of genes
cry2Ab2, uidA, cry1Ac, nptII and aad, in which the latter is not expressed in
plants. As the vector sequences are not part of the insert, the real potential
horizontal genetic transfer from the bacterium donor of the plasmid to the
receiving cotton can be considered null. Agronomic characteristics of MON 15985
cotton are equally comparable to, or better than, those of conventional cotton.
The control of A. Argillacea, H. virescens and P. gossypiella was efficient,
especially under conditions of high infestation by the pests. In artificial infestations
of Spodoptera frugiperda there was a significant reduction in the number of
caterpillars and defoliation during Bollgard II treatment, yet efficacy in controlling
the pest was decreased when compared with other target pests. Apparently,
insertion of segment PV-GHBI11L was not harmful for the plant development.
Assessment of agronomic performance of MON 15985 lineages and cultivars
against conventional cultivars in Brazilian agricultural conditions revealed normal
variability between genetically modified and conventional plants regarding their
agronomic features (plant height, cycle up to flowering, precocity of maturation,
cycle up to harvest and boll weight) productivity and fiber quality. Though the
combination Cry1Ac and Cry2Ab2 has higher efficacy than Bollgard, Bollgard II is
currently susceptible to damages caused by Spodoptera ssp. and Helicoverpa
zea in conditions of high infestation, especially flowering times. Practices of pest
management associated to cotton Bt have caused a dramatic reduction in the use
of insecticides, which leads to a significant increase in the population of beneficial
insects and, consequently, contributes towards the natural control of some pests.
340/2009
5 49
Studies were conducted with non-target organisms, such as birds, fish and
beneficial invertebrate species. The results evidenced that protein Cry2Ab2 in
MON 15985 cotton fails to impose premature risks for non-target organisms.
Adverse effects were not observed in concentrations significantly higher than the
ones foreseen by exposure to the environment. In all cases, the concentration of
the non-observed effect exceeds the top environmental concentration, indicating
minimum risk of protein Cry2Ab2 to non-target organisms. Results of several
studies indicated that protein Cry2Ab2 poses minimum risk for non-target
beneficial organisms. Studies with populations of predator species, such as
Geocoris spp., Orius Insidiosus, Nabis spp., Slenopsis invicta, spiders,
coccinellidae, chrysopidae and hemerobiidae, evidenced that the populations
were either equal or larger in treatments containing Bollgard and Bollgard II cotton
contrasted with treatments with conventional cotton. In a sample of over 40 field
experiments with cotton and maize expressing proteins Cry it became clear that,
in general, non-target invertebrates are more abundant in cotton and Bt corn
fields than in fields where conventional cultures were treated with insecticides.
However, in insect-resistant genetically modified cotton and corn fields, when
compared to fields with cultures that were not treated with pesticides, show a
statistically significant reduction in the number of some non-target organisms.
Other studies evidenced that, in general, there was no significant difference on
populations of natural enemies between Bollgard cotton and conventional cotton.
Whenever significant differences were apparent, natural enemies were more
abundant in Bollgard cotton fields, probably resulting from the decreased
340/2009
6 49
employment of chemical pesticides. It was additionally observed that, when insect
eggs or larvae were presented as preys, natural populations in Bollgard cotton
fields exhibited predation rates significantly higher. In China, a monitoring of nontarget
organisms was conducted in the northwestern region for Bt cotton fields
and the results suggested an increase in natural predator populations such as
ladybirds, earwigs, spiders and other non-target organisms, in addition to the
reappearance of cotton aphis. In Brazil, it was shown that the Bt cotton is either
harmless or brings positive effects to changes in life cycle, survival, fertility, and
appearance of colonies of Aphys gossypii, under nursery conditions. Results
obtained by the authors and data available in technical literature show the high
specificity of the Bollgard technology in the control of target-organisms, without
causing either positive or negative effects in non-target populations, such as
Aphis gossypii. Regarding the risk of gene flow to wild populations and potential
reduction of biodiversity, it is important to consider that for gene introgression, it is
first necessary hybridization and later a series of retro-crossing to take place for
permanent incorporation of a gene in a genome. Further, the potential of vertical
genetic transfer from genetically modified corn to wild species in non cultivated
ecosystems is low, due to the relatively isolated distribution of Gossypium
species. In Brazil, there are not species sexually compatible with G. hirsutum that
display characteristics of invading plants, and it is highly improbable that cry1AC
and cry2AB2 be transferred to pests, making the latter more invasive. The
likelihood that a Bollgard herbaceous cotton plant becomes a pest is negligible.
The cry genes were isolated from a soil bacterium, B. thuringiensis and, therefore
340/2009
7 49
the exposure of living and environment organisms to this bacterium or to any
element derived from it is an event that occurs abundantly in nature. It was
verified that adoption of Bt cotton in different countries caused significant
reduction in the use of pesticides, with benefits to the environment and field
workers. Available information suggests that transgenic plants are not
fundamentally different from genotypes of non-transformed cotton, safe for
resistance to some insects of the order Lepidoptera. There are no restrictions to
the use of this cotton or derivatives, either for human or animal feeding. According
to Article 1 of Law nº 11,460, of March 21, 2007, “research and cultivation of
genetically modified organisms may not be conducted in indigenous lands and
areas of conservation units”. The Bollgard technology proved to be useful under
all agricultural practices commonly used in different regions and conditions, either
for the availability of inputs, labor, among others, used in the cotton culture. There
are no creole varieties of cotton plants and the chains of special, conventional
and transgenic cottons have lived together in a satisfactory fashion, without
records of coexistence problems. According to Annex I of Regulating Resolution
no. 5, of March 12, 2008, the applicant shall have a term of thirty (30) days from
the publication date of this Technical Opinion to adjust its proposal to the postcommercial
release monitoring plan. Under Article 14 of Law no. 11,105/2005,
CTNBio found that the request complies with the applicable rules and legislation
securing the biosafety of environment, agriculture, human and animal health.
340/2009
8 49
TECHNICAL OPINION
I. GMO Identification
GMO name: Bollgard II Cotton, Event MON 15985.
Species: Gossypium hirsutum
Inserted characteristics: Tolerance to certain pest insects
Method of insertion: Plant transformation by particle acceleration
Prospective use: Release into the environment, marketing,
consumption and any other activities related to
this GMO and its derivatives.
II. General Information
Herbaceous cotton (Gossypium hirsutum L.) of the Malvaceae family is a
allelotetraploid plant, native of Mexico and sexually compatible with all the
remaining allelotretraploid species of the same genus. It is one of the most
cultivated plants used by humankind(12) and is cultivated in Brazil in small and
large properties in regions featuring distinct ecological conditions(18).
Cotton plant is one of the main cultivated plants, represented by commercial
species, such as G. hirsutum, G. barbadense, G. arboretum, and G. herbaceum.
G. hirsutum is the main such plants, with a production of about 90% of the total
cotton fibers produced worldwide, being such fibers responsible for 40% of
340/2009
9 49
human clothes(7). Cotton is held to be one of the prime agricultural products and is
very important in Brazil, for its complex production/industry process and high use
of manpower.
Two types of cotton plants are predominantly cultivated in Brazil: conventional
cotton and genetically modified caterpillar-resistant cotton. These plants are
responsible for practically all the cotton produced in the country. In addition, other
three types of cotton featuring special genetic or ecologic features are cultivated:
the naturally colored fiber cotton and the agro-ecological cotton. Colored cotton is
almost exclusively concentrated in the State of Paraíba, with a crop area in 2007
of about 300 hectares. Crops of agro-ecological were sown by 235 farmers in the
semiarid bioma in four states of the Brazilian Northeastern region and produced
42 tons(37). Chains of special, conventional and transgenic cotton have
satisfactorily lived together, without problems of coexistence being reported. The
area planted with cotton in Brazil in the past 2007/2008 harvest reached about
one million and one hundred thousand hectares, of which over 85% concentrated
in the Cerrado bioma, especially in the states of Mato Grosso, Bahia, Goiás and
Mato Grosso do Sul. Other cultures are present in other states of the country,
mainly in the semiarid of the Northeastern region, Paraná, Minas Gerais and São
Paulo(28).
Besides the herbaceous one, other three cotton plants grow in Brazil, all of them
allelootetraploids and, therefore, sexually compatible with the cultivars. None of
such species is considered to be a pest in agricultural or natural environments.
340/2009
10 49
The species G. barbadense was domesticated mainly in the Northern Peru and
Southern Ecuador(9). It was introduced by pre-Columbian peoples and its fibers
were used in the production of textile craftsmanship by some indigenous ethnic
groups before the arrival of Portuguese colonizers(42). Its use as a textile plant
was disseminated among colonizers but suffered a decline driven by the
dissemination of two exotic races of G. hirsutum races. G. barbadense cannot be
found in natural environments and is basically kept as a backyard plant. It is
widely distributed, present in almost the whole country and the in situ
conservation is directly linked to the maintenance of traditional use as a
medicine plant(4).
The only species indigenous to Brazil is G. mustelinum, being its natural
distribution restricted to the Northeastern semiarid(19,32). Populations are known
only in the States of Bahia and Rio Grande do Norte, in places that do not
produce herbaceous cotton. Two problems affect the in situ maintenance of G.
mustelinum. The first and most severe is the destruction of non-perennial rivers
and rivulets gallery forests, the natural habitat of the species. The second is the
extensive cattle raising conducted in the region, especially caprines. The animals
feed on buds, leaves, fruits, seeds and bark, harming the development and, in
some cases, killing adult plants. Renewal of populations is also jeopardized, since
the grazing on young individuals destroys part of the plants(10). The distance
between known populations of G. mustelinum and cotton producing regions
prevents the crossing between G. mustelinum and herbaceous cotton present in
cultivars.
340/2009
11 49
The third cotton plant is known as mocó cotton and belongs to a race different
from the same species of herbaceous cotton (G. hirsutum var. marie galante
(Watt) Hutch.). This cotton originated in the Antilles and its introduction to Brazil is
uncertain. One conjectures that it may have been brought by the Dutch or
Africans during colonial times(42). Mocó cotton was widely cultivated in the
Northeastern semiarid until the end of the eighties, when a series of problems
caused an abrupt interruption in planting(6). A small amount of arboreal cotton
plants, mainly inter-racial hybrids of colored and white fiber cotton produced by
the Embrapa improvement program are still cultivated. However, the planting of
this material is in decline; 5,692 hectares were harvested during the 2004/2005
crop and just 1,326 hectares during the 2005/2006 crop(28). Tillage is cultivated
with a minimum of external inputs and the most important one is the insecticide
to control insect pests. Control of weed is almost exclusively conducted through
manual clearing. Transient populations of this race with high biologic importance,
derived from forsaken farming, may be found in the high ridges of some
municipalities of the Seridó area in the States of Paraíba and Rio Grande do
Norte(4). These populations are geographically isolated from herbaceous cotton
farms and well represented in the Embrapa germplasm banks.
The cotton leafworm (Alabama argillacea), cotton budworm (Helotes virescens),
pink bollworm (Pectinophora gossypiella), fall armyworm (Spodoptera frugiperda),
cotton aphid (Aphis gossypii), cotton bug (Horcias nobilellus), and boll weevil
(Anthonomus grandis) are the main cotton pests in Brazil. Control of such pests
has mainly been conducted with the use of insecticides. In Brazil, over 10 tons of
340/2009
12 49
insecticides are consumed each year in cotton fields only, causing a US$ 190
million increase in production costs. The excessive use of non-specific
insecticides leads to negative environmental impacts, such as severe reduction of
beneficial organisms and potential upsurge of pests resistant to conventional
insecticides.
Bollgard II cotton (Event MON 15985) was developed from Bollgard cotton, Event
531, through introduction of the gene cry2Ab2 of Bacillus thuringiensis, of the
variety kurstaki. Therefore, Bollgard II expresses d-endotoxins Cry1Ac and
Cry2Ab2 that are highly specific and toxic to caterpillars and some Lepidoptera,
including Spodoptera frugiperda (J.D. Smith) (Lepidoptera: Noctuidae) and other
species of the Spodoptera genus(13, 39, 54, 53), in addition to Bollgard cotton
target-pests, Alabama argillacea (Hüeb.) (Lepidoptera: Noctuidae), cotton
budworm (Heliothis virescens), (Fabr.) (Lepidoptera: Noctuidae), and pink
bollworm (Pectinophora gossypiella) (Saund.) (Lepidoptera: Gelechiidae).
Expression of two toxic proteins gives large scope for action to control pest
Lepidoptera and makes possible, in some cases, to delay the evolution of
resistance.
Taking into account that Bollgard II cotton was developed from Bollgard cotton,
which has been approved for commercial use by CTNBio in 2005, the biosafety
analysis in this technical opinion shall be focused in additional proteins expressed
in Bollgard II: GUS and Cry2Ab2 and possible interactions.
340/2009
13 49
After ten years from the first commercial release of a genetically modified
organism, the genetically modified cotton – GM took 20% of all worldwide planted
area in 2005, corresponding to 1/9 of the whole area sowed with GM plants in the
world. However, China and USA were responsible for the most part of this
increase in planted area, where GM crops exceeded 2/3 and 4/5, respectively.
Other countries featuring high rates of transgenic cotton adoption in the world in
2005 were Australia and South Africa, both featuring about 4/5 of their respective
cultivated areas planted with cotton. In 2008, out of the 15.8 million hectares
covered with transgenic cultures in Brazil, 14 million are soybeans, 1.4 million
corn, and 0.4 million cotton.
Bollgard II cotton Event MON 15985 is marketed in different countries, such as:
United States of America (2002), Australia (2002), Japan (2002), South Africa
(2003), Philippines (2003), Mexico (2003), Korea (2003), Canada (2003),
European Union (2005), China (2006), India (2006), and Burkina Faso (2008)(2).
Up to this moment, no severe damage to human and animal health and to the
environment was recorded by such commercial use in the above countries. In
Brazil, field experiments were conducted in different Brazilian states.
III. Description of GMO and Proteins Expressed
Bollgard II cotton (Event MON 15985) was developed from Bollgard cotton by
introducing another gene cry1Ab2 from B. thuringiensis var. kurstaki. Therefore,
Bollgard II expresses d-endotoxins Cry1Ac and Cry2Ab2, which are highly
340/2009
14 49
specific and toxic to caterpillars of some Lepidoptera, including Spodoptera
frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) and other species of the genus
Spodoptera(13, 53), in addition to target-pests of Bollgard cotton, Alabama
argillacea (Hüeb) (Lepidoptera: Noctuidae), Heliothis virescens (Fabr.)
(Lepidoptera: Noctuidae) and Pectinophora gossypiella (Saund.) (Lepidoptera:
Gelechiidae). Expression of two toxic proteins increases the scope of action for
controlling pest Lepidoptera and makes possible, in some cases, delay the
evolution of resistance. CTNBio approves the release of Bollgard cotton Event
531 in 2005.
Commercial event MON 15985 (Bollgard II) was obtained by genetic
transformation of Bollgard cotton (variety CP50B) using the methodology of
microparticle acceleration or biobalistics(36). Bollgard cotton, already approved for
marketing in Brazil(14) contains genes cry1Ac, nptII, aad, introduced using the
transformation technique via Agrobacterium tumefaciens, in the conventional
variety Coker 312, by using plasmid PV-GHBK04. Despite the presence of gene
aad, the Bollgard cotton expresses only proteins Cry1Ac and NPTII. The gene
aad has no modifications for expression in plants, and is used only as a marker
for selection in bacterial cells, transformed with the vector containing the genes
of interest.
Bollgard II cotton, in turn, was generated through introducing, by biobalistics
(which results in direct entry of the DNA of interest to the plant cell) of genes
cry2Ab2 and uidA in the Bollgard cotton genome, which was approved by CTNBio
340/2009
15 49
in 2005. Plasmid PV-GHBK11 was used to insert genes cry2Ab2 and uidA in the
Bollgard cotton genome to generate MON 15985 cotton. The plasmid was
propagated in Escherichia coli, purified of bacterial suspensions and used for
transformation. The exogenous DNA was introduced in cotton meristems using
the method of particle acceleration and the DNA integration was detected by
histochemical coloring for GUS (b-glucoronidase) in the vascular tissue. Selected
plants were then tested for expression of the protein of interest Cry2Ab2.
Therefore, cotton MON15985 contains exogenous genes cry1Ac, cry2Ab2, nptII,
aad and uidA, and expresses proteins Cry1Ac, Cry2Ab2, NPTII and GUS,
differing from its parental Bollgard in proteins Cry2Ab2 and GUS. The
combination of proteins Cry2Ab2 and Cry1Ac represents one additional tool for
resistance of pests to protein Cr1Ac, since Cry2A is a class of proteins coming
from B. thuringiensis that is different from protein Cry1Ac.
Protein GUS is a product of expression of gene uidA, and was used as a
selection mechanism for transformed cells (calorimetric selection marker). Gene
uidA, also known as gene gus and gusA, derived from E. coli strain K12, codifies
enzyme b-D-glucoronidase (GUS).The enzyme catalyzes the hydrolysis of
different b-glucoronides, among them the p-nitrophenyl-b-D-glucoronide, resulting
in a chromogemic bluish compound. Bacterium E. coli is an inhabitant of the
digestive system of vertebrates, including humans.
Gene cry2Ab2, which codifies protein Cry2Ab2, is derived from bacterium B.
thuringiensis, a gram-positive soil microorganism, first isolated in Japan by
340/2009
16 49
Ishiwata and formally described by Berliner in 1915. This pathogen displays the
ability to form crystals containing endotoxins, which are proteins featuring
insecticide action, during the sporulation phase of its development cycle. Among
toxins, are the well known proteins Cry, or d-endotoxins. Commercial formulations
of B. thuringiensis containing such proteins have been used in Brazil and other
countries in controlling some farm pests for over forty years. Cry2Ab2 and Cry1Ac
are proteins featuring very specific action, displaying toxic effects in case of
ingestion and acting in specific receptors located at the middle intestine of some
species of insects of the Order Lepidoptera.
Stability and segregation analyses, in ELISA essays for protein Cry2Ab2 in four
generations, support the conclusion that event MON 15985 is a single copy event
of stable insertion. Chi-square analysis showed that the insert segregates
according to Mendelian genetics, with a single gene segregation pattern in
relation to detection of protein Cry2Ab2. Southern Blot analysis of generations
R1, R2, R3 and R4 and two lineages of the second retrocrossed generation
(BC2F3), digested with enzyme SphI and hybridized with a probe of the codifying
region of gene cry2Ab2, shows that the transgene is stable across different
generations, since no difference was apparent in the pattern of bands obtained.
The Mendelian segregation and stability of the transgene across tested
generation of the MON 15985 cotton progeny was submitted by applicant.
As evidenced by molecular analyses shown, Bollgard II Event MON 15985
possesses a copy of genes cry2Ab2, uidA, cry1Ac, nptII and aad, the latter not
340/2009
17 49
expressed in plants. Since the sequences of the vector (replication sequences or
other elements of plasmid stability) are not part of the insert, any actual potential
of horizontal genetic transfer from the bacterium donor of the plasmid to the
receiving cotton is deemed null.
IV. Aspects Related to Human and Animal Health
Assessment and alimentary and nutritional safety studies for MON 15985 cotton
were conducted based on the principle of Substantial Equivalence adopted by
international organizations and regulatory bodies, such as WHO, FAO, OECD
and ILSI. Under such approach, in case a new ration or a new food derived from
a genetically modified culture is substantially equivalent to its conventional
counterpart and the new proteins produced are held as safe, this genetically
modified culture is held to be “as safe as” the conventional culture.
Protein GUS is an enzyme (b-glucoronidase), codified by gene uidA of E. coli
and catalyzes the hydrolysis of b-d-glucoronides. By adding the artificial substract
p-nitrophenyl-b-glucoronide, it is hydrolyzed imparting a bluish color that acts as a
visible marker of the selection, being this the reason for its introduction in MON
15895 cotton. The protein is normally existent in the human organism due to the
presence of E. coli and also for its presence in several foods derived from
conventional plants such as potato, apple, oat, beet and others. Besides, it is
degraded in the intestinal tract of humans and animals.
Gene uidA was not obtained from any clearly allergenic source, since bacterium
340/2009
18 49
E. coli(31) is prevalent in the gastrointestinal tract of humans and animals. A data
bank containing sequences of proteins associated to allergy and celiac disease
was assembled from public domain data banks (GenBank, EMBL, PIR and
SwissProt). A search for the sequence of enzyme GUS in such data banks shows
that this protein has no similarity with allergenic sequences.
Protein Cry2Ab2, similarly to Cry1Ac, is a microbial d-endotoxin produced by B.
thuringiensis (Bt.). The toxin acts in the intestine of larvae of different caterpillars
of the Order Lepidoptera that have the related receptor. This bond causes the
opening of pores for cations and prompts an osmotic imbalance between the
digestive system and the hemolymph, causing the death of such insects.
Humans and animals cannot be under the effects resulting from this bond
because they lack the related receptors.
According to the United States of America Environmental Protection Agency(58),
no effects caused by this transgene were detected and, even with high doses, the
Cry2Ab2 d-endotoxin was not considered to be toxic. Since the protein is promptly
digested upon ingestion, effects of a chronic exposure to this protein are not
expected.
For being proteins, the risks of allergenic effects were also assessed. Allergens
originated from food are normally resistant to heat, acids and proteases, may be
glycosylated and present in high concentrations. The proteins tested were
promptly digested by gastric juices, are not glycosylated and their heating leads to
340/2009
19 49
loss of bioactivity. The amino acid sequences of this protein were searched
against a data bank containing about 600 sequences of allergenic proteins and no
similarity became apparent. Experiments conducted in animals failed to suggest
any allergenic potential.
According to data submitted by applicant, oral acute toxicity of proteins Cry1Ac
and Cry2Ab2 is low and are held to be non toxic to mammals. Besides, food
products derived from cotton are highly processed, with in general degrades the
proteins expressed by the Bolgard II cotton. However, in case the proteins are
ingested, they will be immediately broken into their respective amino acids, which
disable any chronic exposure. Protein Cry2Ab2 was searched against a data
bank containing 4667 proteins held to be toxic and no similarity was found. A test
with rats was also conducted with the supply of high doses, without evidence of
any toxic effect.
In vitro digestion studies showed that when exposed to gastric juice, 98% of the
protein was digested in just 15 seconds. In the intestinal fluid, it resisted for a
quite longer period, but as almost all of the protein is digested in the stomach, the
importance of intestinal digestion is low.
Results from 14 tests conducted in the United States evidenced that there was no
bromatologic change in the composition of MON 15895 cotton against its
conventional counterpart considering elements such as ashes, calories,
carbohydrates, total fat, total fiber, fiber in acid detergent, fiber in neutral
detergent and protein. An analysis of eighteen essential amino acids also failed to
340/2009
20 49
find differences between the two varieties.
Though the composition of fat acids in MON 15895 cotton seeds is similar to that
of conventional cotton, some acids were found in higher amounts in the new
variety (myristic, stearic, linolenic, arachidonic and dehydro-sterculius). It is worth
stressing that linolenic and arachidonic are essential fatty acids for man and
animals. Despite the differences, the averages found were within the 95%
confidence interval of conventional references. The composition of fatty acids in
processed cottonseed oil was also similar between MON 15895 cotton and
conventional cotton. Regarding minerals (Ca, Cu, Fe, Mg, P, K, Na and Zn), the
contents were quite similar between the two varieties, with MON 15895 displaying
lower values for copper, iron and phosphor, though all values were also within the
expected variation range of conventional cotton. For gossypol, which is the toxic
factor of the cotton kernel, the contents found were practically the same in the two
varieties
Considering the analyses conducted in Brazil, MON 15895 cotton showed results
similar to those of conventional cotton for ashes, carbohydrates, fat, protein and
gossypol. The average of the latter was 12% lower in MON 15895 cotton.
Nutritional composition also varied within the limits for conventional cotton
adopted by ILSI(30). The conclusion was that Bollgard II Event MON 15895 has a
composition similar to that of conventional cotton.
Cotton is primarily cultivated for the value of its fiber and, secondly, for the use of
its kernel in the production of cottonseed oil and animal fodder(29). Cottonseed oil
340/2009
21 49
and cellulose from processed fibers are the only products derived from cotton
used in human food(45). Short fibers are the main source of cellulose used in the
chemical and food industry (their cellulose content reaches 99%). Cotton kernel
produces high quality oil that is used in a variety of foods as frying oil, salad and
cooking oil, mayonnaise, salad dressing and margarine, among other uses. It is
the oldest oil industrially produced and has been largely consumed in Brazil
before the increased use of soybean oil. The high quality of refined cottonseed oil
is due to the presence of essential fatty acids (such as linoleic acid) and high
content of E vitamin and a-tocopherol (a natural antioxidant) that increases its
value for consumption compared with corn and soybean oils(16).
Kernel quality and composition analyses of Bollgard II event MON 15895 cotton
showed that this genetically modified insect-resistant cotton and its processed
fractions are comparable to those of conventional cotton, taking into consideration
the natural variability between market cotton varieties. Studies performed with
animas (dairy cows, catfish, quails and rats) assessed the nutritional quality of
MON 15895 cotton and the effects of diets containing modified cotton kernels on
the development of animals(26). MON 15895 cotton, as a component of animal
fodder, and proteins Cry2Ab2, Cry1Ac, NPTII and GUS in plant tissues proved to
be safe and had similar nutritional value for human and animal consumption.
Field experiments with MON 15895 cotton were conducted in three locations in
Brazil (Santa Cruz das Palmeiras, SP; Santa Helena de Goiás, GO and Sorriso,
MT) during the 2005/2006 harvest, with the purpose of generating samples to
340/2009
22 49
quantify proteins CryAb2, Cry1Ac, NPTII and GUS in tissues of leaves and
kernels. Samples were analyzed regarding the content of such proteins by the
ELISA method. Average levels or proteins in leaves and kernels in the three
locations for MON 15895 cotton were: Cry2Ab2, 660 and 250 mg/g of dry weight,
respectively; Cry1Ac, 53 and 1.9 mg/g of dry weight, respectively; NPTII, 35 and
2.7 mg/g of dry weight, respectively; and GUS, 2600 and 140 mg/g of dry weight,
respectively.
Proteins Cry2Ab2, Cry1Ac, NPTII and GUS have higher levels of expression in
leaves, but they were also detected in samples from other tissues. After
processing the fibers and kernels, such proteins are undetected. Since oil and
processed fibers are the only products derived from cotton used in human food,
consumption of exogenous bioactive proteins or any product of their degradation
is not expected(33, 51, 52).
Nutritional equivalence of MON 15895 cotton with conventional varieties of cotton
was assessed in dairy cows, catfish, quails and poultry, and the results showed
that the MON 15895 cotton is as healthy and nutritious as conventional corn when
used as fodder for those animals.
The studies in animals were conducted by comparing MON 15895 cotton with
conventional cotton. A survey with dairy cows consuming an average of 2.250 kg
of raw cotton kernel per day failed to detect any difference in milk production and
composition. Among ruminants, a productive dairy cow is an animal very sensitive
340/2009
23 49
to external factors: a change in temperature can affect milk production. Fodder
may change not only milk production but mainly its composition, which did not
happen with MON 15895. As an effect of ruminal microorganisms, gossypol is
inactivated and about 60% of cotton meal protein is degraded, remaining 40% to
be digested by the true stomach (abomasus), in addition to intestinal digestion.
Studies were also conducted with catfish, quails and poultry (very sensitive to
proteic quality), without any record of adverse effect. Given the presence of
gossypol and the high content of fibers, cotton meal is either seldom used or used
in small quantities (no more than 5%) as fodder for monogastric animals. As
gossypol impairs the use of lysine (an amino acid of great importance to animals),
its use for monogastric animals is not recommended, and it is only used to reduce
the cost of industrial fodder.
Despite the absence of exogenous proteins in food products, the way of action,
specificity and exposure history, the absence of similarity with allergenic and toxic
proteins, the rapid digestion in simulated gastric and intestinal fluids and the lack
of acute oral toxicity in mice demonstrate the safety of these proteins for human
and animal consumption.
As the proteins are internal to cells, field workers are not exposed to them.
Besides, fibers are mainly cellulose and practically devoid of proteins. Cotton
plant is highly self-pollinating and the pollen is large and sticky, which makes
dispersion by wind difficult, reducing human exposure.
340/2009
24 49
For people who use short fiber products and cottonseed oil, besides the
minimum(sometimes undetectable) level of protein in such products, thermal and
chemical treatment generally inactivates or removes the residual protein, making
the risk practically inexistent. As already said, in case of ingestion of some
amount of protein, which will be minimal, its digestion in the stomach is very rapid.
Considering that protein Cry1Ac represents no more than 0.002% of cotton kernel
total proteins, and protein Cry2Ab2, 0.02%, a possible effect to man is unlikely.
V. Environmental and Agronomic Aspects
Modern agriculture is an activity responsible for significant negative
environmental impacts(3, 11, 57) and, therefore, the risk assessment of any GM
event shall be conducted in relation to that impact inherent to conventional
agriculture(5, 15, 43). Therefore, the analysis conducted by CTNBio intended to
assess whether the impact caused by Bollgard II Cotton Event MON 15895 is
significantly higher than the one caused by conventional cotton varieties
considering the practices associated to each system.
All species of the Gossypium genus posses perfect flowers. Fecundation takes
place promptly after anthesis, and either self-fecundation, crossed pollination or
both are possible. The cotton plant pollen is relatively large, ranging from 81 to
143 micra, viscous (making the pollen grains to adhere to each other), spherical
in format, covered by a large amount of spicules and in practice is not transported
by wind(47). In the fields, its viability extends to late afternoon, but may last for up
to 24 hours if stored at temperatures from 2ºC to 3ºC(10).
340/2009
25 49
Cotton is often described as a partially crossed pollinating culture, though a large
number of improvers treat the plant as fully auto-fertile and self-pollinating, except
for crossed pollination through pollinating insects. Freire (2000) argues that the
cotton plant has a reproductive system intermediating between that of allogamic
and autogamic plants, with crossed pollinating rates between 5% and 95%(19).
Self-pollination is the preferred form of hybridization in cotton culture, though
natural crossing may also occur(46). Seeds are produced at a rate of 20 to 30 per
fruit when crossing and self-pollination are well performed(21). The cotton plant
flowering time may vary according to environmental conditions and cotton variety,
but in general starts about 50 days after emergence and lasts 120 or more days,
with the peak of the curve situated around 70 or 80 days. Self-pollinating and
crossing procedures shall take place at the most opportune time, 30 to 40 days
from flowering.
Genetic improvement requires controlled pollination and maintenance of purity by
physical barriers or isolation by distance. Cotton pollen grains are heavy and
viscous, which makes dispersion unlikely. Pollen transfer is made by insects,
especially by wild bees, bumble bees (Bombus sp.) and honeybees (Apis
mellifera) that reach semi-open flowers. In Brazil, cotton genetic improvement
programs focus on gathering the most desirable features, according to the region
of culture, taking into consideration production components and agricultural
adequacy, fiber and thread quality, as well as characteristics of the product for
special purposes(21).
340/2009
26 49
Cotton commonly does not propagates by vegetation, but through apples or
seeds(19). Natural crossing may occur thorough pollinating insects, since there is
not pollen dispersion by wind. However, pollen reach tend to be limited to very
close cotton flowers, surrounded by bee colonies. Pollen movement is small, just
1.6% of flowers receive material from other plants. Pollinating insects are used as
a tool in improvement programs in order to obtain fresh cotton varieties. One of
the most important effects of crossing, known as heterosis or hybrid vigor, may
result from interspecific, intraspecific and intervarietal crossing. The use of hybrid
vigor in cotton became interesting after it was noticed that excessive introgression
(self-pollinating) causes detrimental effects(50). The natural crossing rate detected
in Brazil has ranged from 1% to 100% in the Northeastern region, and from 0% to
71% in the Central-Western region. Different crossing rates in boundary regions
are explained by the presence of native forests and pollinating insects, mainly
honeybees. It shall be emphasized that crossing rates in the Cerrado crops have
always been low, about 6%. However, in Cerrado regions with significant
occurrence of native vegetation, rates range from 19% to 42% and, in areas
cultivated by small farmers, rates are even higher (45% to 69%), because of
preserved forests and high population of bees(20).
The application for commercial release of Bollgard II cotton is based on three field
experiments conducted in the 2005/2006 crop by the Monsanto do Brasil Ltda.
Experimental Stations, located in Santa Cruz da Palmeira, SP; Sorriso, MT and
Santa Helena de Goiás, GO. According to data submitted, the agronomic features
of MON 15895 cotton (including phenotype, fiber quality, productivity) are
340/2009
27 49
comparable or better than those of conventional cotton (DP50). According to the
results, control of A. argillacea was excellent in the three experimental areas,
mainly in conditions of high infestation of the pest in Sorriso, MT and Santa
Helena de Goiás, GO. The high efficacy of Bollgard II cotton was also observed
against H. virescens in these two locations. Apparently, there was not infestation
by H. virescens in Santa Cruz das Palmeiras, SP. Infestation by P. gossypiella
took place only in Sorriso, MT, and the performance of MON 15895 cotton was
also excellent in controlling such species. Efficacy of Bollgard II cotton in
controlling H. virescens and P. gossypiella was already recognized from studies
conducted in other countries, especially the United States. Due to the low
infestation by S. frugiperda it was not possible to assess efficacy of Bollgard II in
natural infestations in the three experimental areas. Therefore, the results shown
were based in artificial infestations by the pest, conducted only in Santa Helena
de Goiás, GO. Leaves and flower buds were infested with two caterpillars by
structure, comprising ten blocks (10 repetitions), each with five structures. The
structures were separately infested with large and small caterpillars. Each
repetition was screen protected to avoid any interference from the environment
and escaping by the caterpillars. Assessments were conducted three days after
infestations. Significant reduction was recorded in the number of caterpillars and
defoliation in the treatment with Bolgard II, however the efficacy in controlling S.
frugiperda was lower when compared with other target pests (A. argillacea, H.
virescens and P. gossypiella).
Regarding agronomic characteristics, insertion of the segment PV-GHBK11L
340/2009
28 49
apparently has not harmed the plant development. Since 1998, assessments of
field essays have been conducted in the United States, Porto Rico, Argentina,
South Africa, Costa Rica and Australia.
Comparison studies between event MON 15895 and conventional corn DP50,
conducted in the United States, show that features such as yield, morphology and
fiber maturity and quality are within a normal range of variability, with significant
variation. Assessment of agronomic performance of MON 15895 cotton lineages
and cultivars against conventional cotton cultivars under Brazilian conditions also
show normal variability between genetically modified plants and conventional
ones regarding agronomic characteristics (plant height, cycle up to flowering,
precocity of maturation, cycle up to harvest and boll weight), productivity and fiber
quality.
Results obtained abroad evidenced an additional and independent activity of
Cry1Ac and Cry2Ab2, due to the absence of crossed resistance between the
proteins(22, 34, 38), enabling greater biologic activity and enhancing the scope for
action against species of genus Spodoptera(54). However, though the combination
of Cry1Ac and Cry2Ab2 has proved to be more efficient than Bollgard, Bolgard II
is still susceptible to damages caused by Spodoptera ssp. and H. zea under
conditions of high infestation, especially when flowering(1, 13, 54). Under such
conditions, insecticides are still needed to control the pests. These fields
observations are in line with toxicological data reported by Sivasupramanian et al.
(2008) verifying the greater tolerance of S. frugiperda (CL50=82ppm) to protein
340/2009
29 49
Cry2Ab2 regarding H. virescens (CL50=0.549 ppm) and P. gossypiella
(CL50=0.036 ppm)(53).
With the worldwide increase in area cultivated with insect-resistant genetically
modified cultures, concerns about the impact of the technology in non-target
organisms, including important ones in biologic control, have been frequently
raised. However, management practices of pests associated with cotton Bt have
resulted in dramatic reduction in the use of insecticides, leading to a significant
increase in populations of beneficial insects and, consequently, contributing to
natural control of some pests(59).
Studies were conducted with non-target indicator organisms such as birds, fish
and beneficial invertebrate species. Non-target organisms were exposed to
leaves or seeds of MON 15895 cotton or to purified protein Cry2Ab2 incorporated
in a diet during five to eight weeks, depending of the study. Doses were chosen in
so to exceed envisaged environmental exposure, therefore increasing the safety
margin of conclusions generated by the studies. Results showed that protein
Cry2Ab2 in MON 15895 cotton does not impose previous risks to non-target
organisms. Adverse effects were not recorded in concentrations significantly
higher than the ones foreseen for exposure to the environment. In all cases, the
no observable effect concentration (NOEC) largely exceeds the maximum
environmental concentration, indicating minimum risk posed by protein Cry2Ab2
to non-target organisms. Besides, results obtained in different international
research centers showed that the populations of A. mellifera (honeybee),
340/2009
30 49
Folsomia candida (collembolan), Chrysomera carnea (green lacewing),
Hippodamia convergens (ladybug), Nasonia vitripennis (jewel wasp), Eisenia
foetida (redworm) fail do display any significant adverse effect in concentrations
exceeding the one forecasted by exposure in the environment. Study results
indicate that protein Cry2Ab2 poses minimum risk to such non-target beneficial
organisms. Adverse effects were not recorded at the maximum concentration
foreseen in the environment to which such organisms may be exposed.
Additionally, Hagerty et al. (2005) conducted studies with populations of predator
species, such as Geocoris ssp., Orius insidiosus, Nabis ssp., Slenopsis invicta,
spiders, coccinellidae, chrysopidae and hemerobiidae, evidenced that the
populations were either equal or larger in treatments containing Bollgard cotton
and Bolgard II cotton contrasted with treatments with conventional cotton(23).
Marvier et al. (2007) analyzed over forty field experiments with cotton and corn
expressing proteins Cry, including Cry1Ac, and found that, in general, non-target
invertebrates are more abundant in fields of Bt cotton and corn than in
conventional ones treated with insecticides(40). On the other hand, fields of insectresistant
genetically modified cotton and corn, when compared to fields of
cultures untreated with pesticides, display a reduction statistically significant in the
number of some non-target organisms(41, 49). Such differences are expected: in
general, insecticides are little selective, which explains the fact that fields with Bt
plants (and, consequently, with reduced applications of insecticides) show more
non-target organisms; however, tillage of conventional plants without pest control
(and, consequently, without application of insecticides) will not display reduction
340/2009
31 49
in the population of pests and non-target organisms.
Head et al. (2005) conducted field studies comparing populations of natural
enemies in fields of Bollgard and conventional cotton, in the period from 2000 to
2002 in the United States. Results show that, in general, there were no significant
differences in populations of natural enemies between Bollgard and conventional
cotton. Whenever significant differences were recorded, there was greater
abundance of natural enemies in the fields of Bollgard cotton, probably due to the
lower use of chemical pesticides. The study also observed that, when insect eggs
or larvae were offered as preys, populations of natural enemies in the field of
Bollgard cotton exhibited predation rates significantly higher(27).
In China, a monitoring or non-target organisms was conducted in the northeast of
the country in Bt cotton fields(60). The results indicate an increase in populations of
natural predators, such as ladybug (Coccinella septempunctata), lacewing
(Chrysopa sinica), spider and other non-target organisms, in addition to the
reappearance of cotton aphis.
In Brazil, Sujii et al. (2008) verified that the Bt cotton plant, the expressing of
protein Cry1Ac has no harmful action and fails to positively favor changes in life
cycle, survival, fecundity and colony formation of Aphis gossypii in nursery
conditions. Results obtained by the authors and data available in the scientific
literature show the high specificity of Bollgard technology for controlling target
organisms, without positive or negative effects to non-target populations, such as
cotton aphis(56).
340/2009
32 49
Regarding the risk of gene flow to wild populations and potential reduction of
biodiversity, it is worth stressing that in order for gene introgression to occur, first,
hybridization and then a series or retro-crossing are necessary for a gene to be
permanently incorporated into the genome(24,25). Additionally, the potential of
vertical gene transfer from genetically modified cotton to wild species in noncultivated
ecosystems is low, due to the relatively isolated distribution of the
species of Gossypium. Some conditions are necessary for vertical gene transfer
and gene introgression: physical proximity of the plants (less than 30 meters),
simultaneous fecundity times, sexual compatibility of parents, production of viable
seeds, generation of fertile progeny ecologically adapted to the environment and
occurrence of gene transfer in the following generation, at least(55).
There are not in Brazil species sexually compatible with G. hirsutum displaying
characteristics of invading plants, and it is extremely unlikely that genes cry1Ac
and cry2Ab2 be transferred to pests making them more invasive. The cotton plant
has not any characteristic associated to potential invasiveness, such as seed
dormancy, persistence in soil, germination under adverse environmental
conditions, rapid vegetative growth, short life cycle, high production of seeds and
dispersion of seeds at long distance. Therefore, it is deemed unlikely that
herbaceous Bollgard II cotton may change into a pest plant.
It is worth noticing that genes cry were isolated from a soil bacterium B.
thuringiensis. Therefore, exposure of living organisms and environment to this
bacterium or to any element thereof is an event that occurs abundantly in nature.
340/2009
33 49
The fear of adverse effects to the environment and concerns of alimentary safety
were not justified during the first decade of adoption of the Bollgard technology.
On the contrary, data suggest that water and soil quality improved, due to the less
use of pesticides in GM cotton cultivars. King (2003) concluded that there is no
evidence that GM plants are harmful to the environment, human and animal
health(35).
An important characteristics regarding adoption of Bt cotton in different countries
is that the use of pesticides has been significantly reduced(44). An ensuing benefit
favors the environment and field workers due to the reduced use of pesticides.
According to FAO – Food and Agriculture Organization of the United Nations, the
use of Bt cotton has caused a strong positive environmental impact, resulting in
significant reduction in contamination of water sources and less impact to
beneficial insects(17).
VI. Restrictions to the use of GMO and GMO derivatives
Technical opinions related to agronomic performance concluded that there is
equivalence between transgenic and conventional plants. Therefore, the data
suggest that transgenic cotton plants are not fundamentally different from the
genotypes of non-transformed cotton plants, except for the resistance to certain
insects of the order Lepidoptera. In addition, there is no evidence of adverse
reactions to the use of Bollgard II cotton. For the foregoing, there are no
restrictions to the use of this cotton or its derivatives, either as human or animal
food.
340/2009
34 49
As established by Article 11 of Law no. 11,460, of March 21, 2007 “research and
cultivation of genetically modified organisms may not be conducted in indigenous
lands and areas of conservation units.”
VII. Considerations on particulars of different regions of the country
(contribution to supervision agencies)
Bollgard technology was shown to be usable under all agricultural practices
commonly used in different regions in different conditions, considering availability
of inputs and labor, among other inputs used in the culture of cotton.
There are not creole varieties of cotton plants and the chains of special cotton
plants, both conventional and transgenic, have lived together in a satisfactory
fashion, without any record of coexistence problems.
VIII. Conclusion
Long experience with traditional plant improvement techniques, over three
decades of experience in research and over one decade of marketing transgenic
varieties over the world, in addition to knowledge advancements in the structure
and dynamics of genomes, indicating whether a certain gene or characteristic is
safe, indicate that the genetic engineering process, by its own, leaves little room
for appearance of unexpected consequences that would not be identified or
eliminated during the process of development of commercial genetically modified
varieties(8).
340/2009
35 49
Considering that Bollgard II cotton belongs to a well characterized species
(Gossypium hirsutum) with a solid background of safety for human use and that
the cry1Ac and cry2Ab2 genes introduced in this variety do not codify any toxic
protein, and is harmless to humans;
Considering that Bollgard II cotton was developed from Bollgard cotton, which
was approved for commercial use by CTNBio in 2005 and, up to this moment,
there is no evidence of risk to human and animal health and to the environment;
Considering that Event MON 15895 is a single-copy event of stable insertion and
that Chi-square analysis evidenced that the insert segregates according to
Mendelian genetics, with a segregation pattern of a single gene regarding
detection of protein Cry2Ab2;
Considering that composition data failed to point significant differences between
the genetically modified and conventional varieties, suggesting an equivalence
between such varieties; and
Whereas:
1. Cotton plant is one of the most used plants among those cultivated by the
human being;
2. Proteins Cry1Ac and Cry2Ab2 are promptly digested after ingestion and
that effects of chronic exposure to such proteins are not expected;
3. Acute oral toxicity of proteins Cr1Ac and Cr1Ac is low, and they are
340/2009
36 49
considered to be non toxic for mammals;
4. The DNA molecule is a natural component of food and there is no
evidence that it may have any adverse effect to humans when ingested in
food within acceptable amounts (no direct toxic effect);
5. There is no evidence that intact genes of plants may be transferred and
functionally integrated to the human or other mammals genome exposed
to the DNA or to food produced with such elements.
6. The likelihood that the herbaceous Bollgard II cotton plant becomes a
pest plant is negligible;
7. Exposure of living organisms and environment to B. thuringiensis or to
any element extracted from this bacterium is an event that occurs
abundantly in nature;
8. Insertion of segment PV-GHBK11L apparently failed to harm the
development of the plant regarding agronomic characteristics;
9. There are no reports in change of agronomic performance observed in
the commercial cultivation of this event in other countries;
10. Analysis of biochemical composition showed that event MON 15895
displays substantial equivalence with non-genetically modified varieties, a
robust suggestion that such event has no undesirable pleiotropic effects;
340/2009
37 49
11. Literature and field experiment data suggest that event MON 15895 has
no impact against non-target organisms except those already inherent to
the culture of cotton;
12. Field data indicate that water and soil quality improved due to the less
use of pesticides in cultivars of GM cotton;
13. Adoption of Bt cotton in different countries has significantly reduced
applications of pesticides;
Summarizing, considering the criteria internationally accepted in the process of
analyzing the risk of genetically modified raw materials, it is possible to reach a
conclusion that Bollgard II Cotton Event MON 15895 is as safe as its conventional
equivalent.
For the foregoing, commercial release of Bollgard Cotton Event MON 15895 is
not potentially harmful to human and animal health and does not cause significant
environment degradation.
The CTNBio analysis considered the opinions issued by the Commission
members; ad hoc consultants; documents delivered by the applicant to the
CTNBio Executive Secretariat; results of planned releases into the environment;
lectures, texts and discussions in a public hearing held on 08.17.2007.
Independent third party scientific studies and publications submitted by the
applicant were also considered.
340/2009
38 49
According to Annex I of Ruling Resolution 5, of March 12, 2008, applicant shall
make adjustments to its proposed post-commercial release monitoring plan within
thirty (30) days from publication of this Technical Opinion.
IX. Bibliography
1. ADAMKZYK, J.J.; SUMERFORD, D.V.2001. Increased tolerance of fall
armyworms (Lepidoptera: Noctuidae) to Cry1Ac d-endotoxins when fed
transgenic B. thuringiensis cotton: impact on the development of subsequent
generations. Florida Entomologist 84: p.1-6.
2. AGBIOS 2008. AGBIOS Database Product Description:
http://www.agbios.com.
3. AMMANN, K. 2005. Effects of biotechnology on biodiversity: herbicidetolerant
and insect-resistant GM crops. Trends Biotech. 23:388-394.
4. BARROSO P.A.V.; FREIRE E.C.; AMARAL J. A. B. do; SILVA M.T.
2005. Zonas de exclusão de algodoeiros transgênicos para preservação
de espécies de Gossypium nativas ou naturalizadas. Campina Grande:
Embrapa Algodão, 7 p. ( Comunicado Técnico, 242).
5. BATSCH, D.; SCHUPHAN, I. 2002. Lessons we can learn from ecological
biosafety research. J. Biotech. 98: 71-77.
340/2009
39 49
6. BELTRÃO, N.E. de M. 1999. O Agronegócio do Algodão no Brasil.
Brasília: EMBRAPA-CTT, 1.023P.
7. BELTRÃO N. E. de M. 2003. Documento 117: Breve História do Algodão
no Nordeste do Brasil. Campina Grande: Empresa Brasileira de
Agropecuária, Centro Nacional de Pesquisa de Algodão, 20p.
8. BRADFORD K. J.; DEYNZE A.V.; GUTTERSON N.; PARROTT W.;
STRAUSS S.H. 2005. Regulating transgenic crops sensibly: lessons from
plant breeding, biotechnology and genomics. Nat. Biotechnol. 23: 439-
444.
9. BRUBAKER C.; BOURLAND E.M.; WENDEL J.E. 1999. The origin and
domestication of cotton. In: SMITH C.W.; COTHREN J.T. Cotton: Origin,
history, and production. New York: John Wiley & Sons, p. 3-31.
10. CALHOUN, D.S.; BOWMAN, D.T.1999. Techniques for development of
new cultures. In: SMITH, C.W.; COTHREN, J.T. Cotton, origin, history
technology and production, p.361-414. New York: John Willey & Sons, p.
361-414.
11. CHAPIN FRS.; AZALEA E.G.; EVENER VAT; NAYLOR R.; VIRTUOSIC
P.M.; REYNOLDS H.L.; HOOPER .U.; LABORED S.; SALE I.E.;
HOBBIES S.E.; MACK MUCH; DIAZ S. 2000. Consequences of changing
340/2009
40 49
biodiversity. Nature 405: 234-242.
12. CHERRY, J.P.; LIFER, H.R. 1984. Seed. In: LEWIS, C.F.; KOHL, R.J.
Cotton. Madison: American School of Agronomy, 512-570.
13. CHITOWSKI, R.L.; TURNIPSEED, S.G.; SULLIVAN, M.J.; BRIDGES JR,
W.C. Field and laboratory evaluations of transgenic cottons expressing
one or two Bacillus thuringiensis var. kurstaki Berliner proteins for
management of Noctuid (Lepidoptera) pests. J. Econ. Entomol. 96(3):
755-762.
14. Comissão Técnica Nacional de Biossegurança – CTNBio. Parecer nº
513/2005 – Liberação Comercial de Algodão Geneticamente Modificado
resistente a insetos Evento 531 – Processo 01200.001471/2003-01:
Parecer Técnico Prévio Conclusivo nº 513/2005.
http://www.ctnbioi.gov.br/upd_blog/0000/615.doc.
15. CONNER A.J.; GLARE T.E.; NAP J-P. 2003. The release of genetically
modified crops into the environment. Plant J. 33: 19-46.
16. EMBRAPA. 2003. Cultura do Algodão Herbáceo na Agricultura Familiar –
subprodutos do Algodão. Sistemas de Produção. Janeiro/2003. 7p.
17. FAO/WHO – Food and Agriculture Organization of the United Nations,
2004. The State of Food and Agriculture. Agricultural Biotechnology:
340/2009
41 49
Meeting the Needs of the Poor? Food and Agriculture Organization.
Rome [s.n.], 2004. Available at
http:www.fao.org/DOCREP/006/Y5160E/Y160E00.htm.
18. FONTES, E.M.G.; RAMALHO, F. de S.; UNDERWOOD, E.; BARROSO,
P.A.V.; SIMON, M.F.; SUJII, E.R.; PIRES, C.S.S.; BELTRÃO, N.;
LUCENA, W.A.; FREIRE, F.C.2006. The cotton agricultural context in
Brazil. In: HILBECK, A.;ANDOW, D.A.; FONTES, E.M.G.; KAPUSCINSKI,
A.R.;SCHEI, P.J. (Ed). Environmental risk assessment of genetically
modified organisms: methodologies for assessing Bt cotton in Brazil.
Wallingford, UK: CABI Publishing, v.2. p.21-66.
19. FREIRE E.C. 2000. Distribuição, coleta, uso e preservação das espécies
silvestres de algodão no Brasil. Embrapa: Campina Grande, 22pp.
20. FREIRE, C.C.2000.Viabilidade de cruzamentos entre algodoeiros
transgênicos e comerciais e silvestres no Brasil. O. Fibras, 6: 465-470.
21. FUZATTO M.G. 1999. Melhoramento genético do algodão. In: Cultura do
algodeiro. FREIRE E.C.; SANTOS W.J. (eds.) Piracicaba: Potrafós, p. 15-
32.
22. GREENPLATE, J.T.; MULLINS, J.W.; PENN, S.R.; DAHAM, A.; REICH,
B.J.; OSBORN, J.A.; RAHN, P.R.; RUSCHKE, L.; SHAPPLEY, Z.W.
340/2009
42 49
2003. Partial characterization of cotton plants expressing two toxin
proteins from Bacillus thuringiensis: relative contribution, toxin interaction
and resistance management. J. Appl. Entomol. 127, 340-347.
23. HAGERTY, A.M.; KILPATRICK, A.L.; TURNIPSEED, S.G.; SULLIVAN,
M.J.; BRIDGES, W.C. 2005. Predaceous arthropods and Lepitopteran
pests on conventional, Bollgard and Bollgard II cotton under untreated
and disrupted conditions. Environ. Entomol. 34(1): 105-114.
24. HAILS R.S.; MORLEY K. 2005. Genes invading new populations: a risk
assessment perspective. Trends in Ecol. Evol. 20: 245-252
25. HANSEN, L.B.; SIEGISMUND, H.R.; JØRGENSEN, R.B. 2001.
Introgression between oilseed rape (Brassica napus L.) and its weedy
relative B. rapa in a natural population. Gen. Res. and Crop. Evol. 48:
621-627.
26. HAMILTON, K.A.; PYLA, P.D.; BREEZE, M.; OLSON, T.; LI, E.;
ROBINSON, E.; GALLAGHER, S.SP; SORBET, R.; CHEN, Y. 2004.
Bollgard II Cotton: Compositional analysis and feeding studies of
cottonseed from insect protected cotton (Gossypium hirsutum L.)
producing the Cry1Ac and Cry2Ab2 proteins. J. Agric. Food Chem.
52:6969-6976.
340/2009
43 49
27. HEAD, G.; MOAR, W.; EUBANKS, M.; FREEMAN, B.; RUBERSON, J.;
HAGERTY, A.; TURNIPSEED, S. 2005. A multiyear, large-scale
comparison of arthropod populations on commercially managed Bt and
non-Bt cotton fields. Environmental Entomology 34: 1257-1266.
28. IBGE – Instituto Brasileiro de Geografia e Estatística. 2008.
http://www.ibge.gov.br.
29. ICAC. International Cotton Advisory Committee – Technical Information
Section. 2000. Economics of growing transgenic cotton. Vol XVIII nº 1
March 2000, p. 7-11.
30. International Life Science Institute – Crop Composition Database
http://www.cropcomposition.org/cgi-perl/search_ora_cgi.
31. JEFFERSON, R.A.; KAVANAGH, T.A.; BEVAN, M.W. 1986. b-
Glucoronidase from Escherichia coli as a genefusion marker. PNAS USA.
83:8447-8451.
32. JOHNSTON J.A.; MALLORY-SMITH C.; BRUBAKER C.L.; GANDARA F.;
ARAGÃO F.J.L.; BARROSO P.A.V.; QUANG V.D.; CARVALHO L.P. de;
KAGEYAMA P.; CIAMPI, A.Y.; FUZATTO, M.; CIRINO, V.; FREIRE, E.
2006. Assessing cotton flow from Br cotton in Brazil and its possible
consequences. 2006. In: HILBECK A.; ANDOW D.; FONTES E.M.G.
340/2009
44 49
Environmental ri |