| Summary of the safety assessment (food safety): |
Cotton MON 15985 x MON 88913 results from the crossing, through classical genetic improvement of the parents of genetically modified corn MON 15985 and MON 88913. Regarding MON 15985, genes cry1Ac and cry2ab2 introduced in its genome and also present in the genome of MON 15985 x MON 88913 cotton, come from Bacillus thuringiensis subspecies kurstaki and code for proteins Cry1Ac and Cry2ab2. Proteins Cry2ab2 and Cry1Ac are very specific in action, exhibiting toxic effect only by ingestion and act
in specific receptors located in the middle intestine of some insect species of the order Lepidoptera. The proteins have toxic effect on lepidopteran insects that hit the cotton farming in Brazil, such as the fall armyworm, Spodoptera frugiperda, and other species of the genus Spodoptera, in addition to: cotton leafworm (Alabama argillacea); tobacco budworm, Heliothis virescens; corn earworm, Helicoverpa zea, and pink bollworm Pectinophora gossypiella. Regarding cotton MON88913, gene cp4 epsps introduced in its genome and also present in the genome of cotton MON 15985 x MON 88913 originates from Agrobacterium strain CP4 and codes for protein cp4EPSPS (5-enolpyruvilshikimate-3-phosfate synthase),
responsible for granting tolerance to herbicide glyphosate. Protein CP4 EPSPS present in cotton MON 88913 is functionally identical to the endogenous plant proteins EPSPS, including that of cotton, except for the fact that protein CP4 EPSPS has a naturally reduced affinity for glyphosate. CTNBio analyzed the reports submitted by applicant as well as independente scientific literature. Analyzes of results in all tests indicated that cotton MON 15985 x MON 88913 is held to be substantially equivalent to other cotton varieties. Zones where cultivation of genetically modified cotton is restricted, as established in MAPA
Directive nº 21/2005, shall be strictly observed.
TECHNICAL OPINION
Identification of GMO
GMO name: Genetically modified cotton resistant to insects and tolerant to
glyphosate MON 15985 x MON 88913
Applicant: Monsanto do Brasil Ltda.
Species: Gossypium hirsutum L.
Inserted Characteristic: Resistance to insects and tolerance to glyphosate
Method of Introduction: Combined cotton MON 15985 x MON 88913 results from crossing,
through classical genetic improvement, of parental genetically
modified cotton MON 15985 and MON 88913
Intended Use: Production, from the GMO and its derivatives, of fibers for the
textile industry and grain for human and animal consumption
I. General Information
In Brazil, cotton culture is well developed in the Center-Western and Northeastern regions that, on the 2011-2012 crop year tiled about 872 and 462 hectares, respectively, in an area of about 1392 hectares. Mato Grosso is the main state in seed cotton production, with about 2700 tons according to an estimate of the 2011-2012 crop published by CONAB, representing an increase of 5.4% against the previous harvest(1). The first generations of genetically modified cotton resistant to pests were of the MON 531 cotton variety and that of glyphosate tolerant cotton were variety MON 1455. Both varieties have been widely used by cotton farmers in different countries where the technologies are approved, either individually of combined in a single product, as it is the case of Brazil. Resistance to target pests granted by proteins from Bacillus thuringiensis in the plant have shown to be very efficient in controlling insects, not only in cotton, but also in other crops such as soybean and maize. Tolerance to glyphosate is interesting from the agronomic viewpoint, since it is a non-selective insecticide, used in leaf applications, granting more effective control of annual and perennial weeds that may cause problems during the later stages of the plant development.
Cotton MON 15985 and MON 88913 represent the second generation of pest-resistant glyphosate-tolerant cotton since MON 15985 expresses two proteins coming from Bacillus thuringiensis, which increases its effectiveness and helps as a tool in the management of insects; and MON 88913 exhibits increased levels of tolerance to glyphosate, attained by using improved promoter sequences that regulate the expression of gene cp4 epsps, which codes protein CP4 EPSPS and grants the characteristic of tolerance to glyphosate.
Therefore, the use of MON 15985 x MON 88913 cotton shall enable more effective control of target pests in cotton farming and the use of glyphosate until later stages of the plant development with minimum risks of damaging the cotton culture.
II. Description of the GMO in Expressed Proteins
Stacked cotton MON 15985 x MON 88913 results from the crossing, through classical genetic improvement, of parent lines of genetically modified MON 15985 and MON 88913. Regarding cotton MON 15985, genes cry1Ac and cry2ab2 introduced in its genome, and also present in the genome of cotton MON 15985 x MON 88913, originate from Bacillus thuringiensis subspecies kurstaki, respectively, and code for proteins Cry1Ac and Cry2Ab2. The bacterium Bacillus thuringiensis is a gram-positive soil microorganism that has the ability to develop crystals containing endotoxins, proteins with insecticide action, during the sporulation phase of its development cycle(2). Among such toxins, proteins Cry, or δ-endotoxins stand out.
Commercial formulations of Bacillus thuringiensis containing such proteins have been used, both in Brazil and in other countries, to control certain agricultural pests for over 50 years. Proteins Cry2Ab2 and Cry1Ac have very specific action, displaying toxic effect only by ingestion and act in specific receptors located at the middle intestine of some species of insects of the Order Lepidoptera. The proteins have a toxic effect on lepidopteran insects that hit cotton culture in Brazil, such as armyworm, Spodoptera frugiperda and other species of the genus Spodoptera, in addition to: cotton leafworm (Alabama argillacea); tobacco budworm (Heliothis virescens); corn earworm, (Helicoverpa zea), and pink bollworm Pectinophora gossypiella.
According to applicant, this second generation or pest-resistant cotton is more effective in controlling such pests because it expresses two Cry proteins, besides being an adequate and efficient tool in the management of resistance to insects. One difference from Bollgard cotton is the armyworm control, being that the cause for cotton MON 15985 wider range of control. The genetic transformation process of MON 15985 cotton was bombarding genetically modified cotton MON 531 (Bollgard cotton transformed with vector PV-GHBK04, containing genes cry1Ac, nptll and aad, already commercially approved by CTNBio in 2005 – EPT 513/2005) with particles coated with the genetic material of interest (vector PV-GHBK11 containing genes cry2ab2 and uidA), generating the lineage containing the two genes of Bacillus thuringiensis, namely cry1Ac and cry2ab2. It is known that the action mechanism of Cry proteins is mediated by specific receptors located at the middle intestine of susceptible insects. Association of Cry proteins to the receptors leads to formation of pores that cause the death of the target insect(2). Cotton MON 15985 is classified as an organism of biosafety risk class I. In order to transform cotton MON 15985, the vector PV-GHBK11 was linearized and the fragment containing genes cry2ab2 and uidA, and their regulating elements (PV-GHBK11L), was inserted in the MON 531 cotton genome. Genes originally inserted in cotton MON 531 were cry1Ac, granting resistance to pests, and selection marking genes nptII and aad. However, gene aad has no modification for expression in plants, being used solely as a selection marker in bacterial cells, transformed with the vector containing the genes of interest. Gene uidA, in turn, codes for expression of protein GUS, used as a selection mechanism for transformed cells (selection colorimetrical marker). Gene uidA, also known as gene gus or gusA, derived from E. coli, strain K12, codes for enzyme β-D-glucoronidase (GUS). The bacterium Escherichia coli is na inhabitant of the digestive tract of vertebrates, including humans. Enzyme GUS catalyzes hydrolysis of several β-glucuronides, among which the p-nitrophenyl-β-D-glucuronide that results in a bluish chromogenic compound, which enables the selection of transformants.
Therefore, cotton MON 15985 expresses in its DNA proteins Cry1Ac, Cry2Ab2, NPTII and GUS. Molecular characterization depicts cotton MON 15985 as containing only one insertion of this linear fragment PV-GHBK11L, in a single copy of each cassette of expression of genes cry2ab2 and uidA. Molecular characterization of cotton MON 15985 also determined the insert composition and structure, as well as its stability in multiple generations. As the sequences of the vector (replication sequences or other stability elements) are not part of the insert, it is held null any actual potential of horizontal genetic transference between the donor bacterium of the vector and the receiving cotton.
Regarding cotton MON 88913, gene CP4 EPSPS introduced in its genome and present in the genome of cotton MON 15985 x MON 88913 originates from Agrobacterium tumefasciens strain CP4 and codes for protein CP4 EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), responsible for granting tolerance to the herbicide glyphosate. This is the same protein CP4 EPSPS produced in the event glyphosate tolerant cotton named MON 1445. The genetic transformation method was the system mediated by Agrobacterium tumefasciens, using the binary vector PV-GHGT35 that contains two expression cassettes of gene cp4 epsps for the production of protein CP4 EPSPS. In plants, protein EPSPS is located within chloroplasts.
Protein CP4 EPSPS present in cotton MON 88913 is functionally identical to EPSPS proteins endogenous in plants (including cotton), except for the fact that CP4 EPSPS has a naturally reduced affinity for glyphosate. In conventional plants, glyphosate links to the plant endogenous protein EPSPS, blocking biosynthesis of 5-enolpyruvylshikimate-3-phosphate and causes deficiencies in the production of essential aromatic amino acids and secondary metabolites in plants. In plants genetically modified with gene cp4 epsps, the amino acids and other metabolites requires to plant growth and development are obtained by continuous action of protein CP4 EPSPS tolerant to glyphosate(3). In cotton MON 88913, the gene construct containing gene cp4 epsps contains also the target sequence of the chloroplast, which enables application of the glyphosate over the genetically modified cotton culture until later stages of the plant development, as against cotton MON1445. The extended time for glyphosate application to cotton MON 88913 and cotton MON 15985 x MON 88913 is posible due to the use of improved promoter sequences that regulate expression of coding sequences of gene cp4 epsps. This will enable a more effective control of pests during cultivation, with minimum risks of damages to the cotton culture. Therefore, cotton MON 88913 is a technology that enables this control through the use of glyphosate until the later stages of
plant development. The glyphosate has environmental and safety characteristics that favor its use in the management of pest plants(4,5,6). Cotton MON 88913, as well as cotton MON 15985 and cotton MON 15985 x MON 88913, is also classified as a biosafety risk class I.
Expression levels of proteins Cry1Ac, Cry2Ab2, NPTII, GUS and CP4 EPSPS were studied in leaves and grains produced in field studies in Brazil, using the ELISA quantification method. The materials were collected in two representative locations of cotton farming in the country, in planned releases in the environment conducted in Cachoeira Grande, State of Minas Gerais, and Sorriso, State of Mato Grosso(7), being the experimental design of chance blocks in four repetitions. Levels of the five proteins were measured in micrograms (μg) per gram (g) of wet weight. Humidity was then measured to generate the values in dry weight. In an identical field experiment, cottons MON 15985 and MON 88913 were cultivated, enabling the levels of proteins Cry1Ac, Cry2Ab2, NPTII, and GUS to be assessed in MON 15985 and protein CP4 EPSPS in MON 88913 with additional information to be submitted to CTNBio, since an adequate comparison should be made against conventional cotton. For protein Cry1Ac, averages in leaf and grain tissue of cotton MON 15985 x MON 88913 collected in the two locations was 23 μg/g
and 1.9 μg/g of dry weight, respectively, and that of cotton MON 15985 were 19 μg/g and 1.6 μg/g of dry weight, respectively. For protein Cry2Ab2, averages in leaf and grain tissues of cotton MON 15985 x MON 88913 collected in the two locations were 630 μg/g and 250 μg/g of dry weight, respectively, and that of cotton MON 15985 were 590 μg/g and 250 μg/g of dry weight, respectively. For protein CP4 EPSPS, averages in leaf and grain tissues of cotton MON 15985 x MON 88913 collected in the two locations were 1900 μg/g and 270 μg/g of dry weight, respectively, that of cotton MON 88913 were 2100 μg/g and 280 μg/g of dry weight, respectively. Regarding protein NPTII, averages in leaf and grain tissues of cotton
MON 15985 x MON 88913 collected in the two locations were 45 μg/g and 3.8 μg/g of dry weight, respectively, that of cotton MON 15985 were 42 μg/g and 4.1 μg/g of dry weight, respectively. For protein GUS, the average in leaf and grain tissue of cotton MON 15985 x MON 88913 collected in the two locations were 2500 μg/g and 120 μg/g of dry weight, respectively, of cotton MON 15985 were 4400 μg/g and 130 μg/g of dry weight, respectively. The levels of proteins Cry1Ac, Cry2Ab2, CP4 EPSPS, NPTII and GUS in samples of conventional cotton were below essay LOQ and LOD for each type of tissue, except for CP4 EPSPS in just one leaf sample of the location in Sorriso, State of Mato Grosso. Finally, studies were conducted by applicant to assess any possible interaction between proteins Cry1Ac and Cry2Ab2 in the management of resistance to insects(8). In cotton MON 15985, an independent interactive and additive effect was evidenced between the proteins in the answer of target pests (H. virescens, H. zea and S. frugiperda) when fed with tissues of plants expressing only one of the two or both proteins. Different types of tissues were tested and it became clear, through ELISA, that the quantity expressed of each protein is
not affected by the presence of the other protein or gene. The conclusion of the study was that the joint action of the proteins is additive and, besides, the greater insecticide effect is granted by Cry2Ab2. Other studies mentioned in the records corroborate this finding, which is an important conclusion to establish that these two proteins in a single plant is a tool for the management of resistance to insects.
III. Aspects Related to Human and Animal Health Applicant notes in its document that for cotton MON 15985 and cotton MON 88913, already approved by CTNBio, results were submitted that ratify the alimentary safety results found for cotton MON 15985 x MON 88913. Safety assessment of MON 15985 x MON 88913 for human and animal health involved: biochemical characterization of heterolog proteins produced in the plant; equivalence of such plant-produced proteins with the same proteins produced in bacterium; toxicity and allergenicity potential of heterolog proteins based on studies and available scientific literature; and the centesimal composition as against conventional cotton. Safety assessment of food derived from genetically modified raw materials is based on risk analysis, a scientific methodology that comprises the phases of risk assessment, management and communication. The risk assessment phase, a qualitative and quantitative characterization of potential adverse effects is sought, based on the idea of substantial equivalence, in order to identify possible differences between the new food and its conventional correspondent. Regarding the receiving organism, Gossypium hirsutum, this is a very well characterized species. In the proceedings under examination, a host of information is submitted, encompassing origin, domestication, identity, taxonomy, morphology, genetics, hybridation and crossing of the species.
Concerning the gene donor organisms, the species are also well characterized and easily found
in nature. Cultures of Bacillus thuringiensis, the donor organism of genes cry, are recorded with Agência Nacional de Vigilância Sanitária – ANVISA, the National Agency for Sanitary Surveillance, under different formulations for application in 40 types of plant cultures for alimentary purposes. They are included in toxicological classification of group IV and there is
not any determined upper boundary of residue nor withdrawal period(9). Gene cp4 epsps was
obtained from a soil bacterium that is ubiquitous in nature, identified as Agrobacterium sp.
Protein NPTII is produced by different prokaryotic organisms found in nature different habitats,
including the human and animal microflora(10). Gene nptII is derived from transposon Tn5 of E.
coli, a bacterium of the human digestive system. Protein GUS is also largely found in the
environment, and no adverse effects were reported despite the large exposure to bacteria and
food containing the protein. Its occurrence in different plant and animal species used in human
and animal nutrition is well characterized(12,13,14).
Proteins Cry1Ac and Cry2Ab2 are δ-endotoxins produced by Bacillus thuringiensis exhibiting
specific activity on the digestive tract of some insect families. To remain active, the proteins
must be ingested by the target insects and solubilized by the stomach pH. The proteins, by
action of proteases, are activated and bond to high affinity specific receptors that are present
in insects(2).
Proteins Cry1Ac and Cry2Ab2 are toxic solely for the abovementioned target pests, specifically
lepidopterans (caterpillars) possessing, in their guts, specific receptors for such proteins.
Mammals fail to have such bonding sites and, therefore, human beings, animals and other
non-target organisms are not affected by the Cry proteins of Bacillus thuringiensis, including
other arthropods and also other natural enemies of the target-pests(15,16,17,18,19). As a result, the
proteins display a long history of safe use and the data of studies and literature submitted
enable a conclusion about their alimentary safety. It is worth mentioning that genetically
modified cultures resistant to pests expressing these same proteins, or other of the same
family, have been cultivated in several countries all over the world, including Brazil, with no
record of adverse effects to human and animal health.
Protein CP4 EPSPS, in turn, has a structure homologous to EPSPS proteins naturally found in
plants and other types of food, such as yeasts used to make bread and beverages. There is a
similarity between the amino acid sequence of protein CP4 EPSPS produced in cotton
MON 15985 x MON 88913 and protein CP4 EPSPS expressed in cultures tolerant to glyphosate
already used in several countries. There are no reports that protein CP4 EPSPS may cause
adverse effects on human and animal health, and studies submitted by the applicant of the
commercial release of cotton MON 15985 x MON 88913 corroborate the conclusion for safety
of the protein.
The analysis of the chemical composition of the transgenic variety, mainly of the levels of
nutrients and eventual toxic components naturally present, aims at securing that this new
variety is as nutritious and safe as its commercial equivalent. This way, it is becomes a
confirmation that the intended effects of the modification do not compromise its safety and
fail to result in any unintended effect. Nutritional composition and centesimal component data
shown in the processes of events MON 15985 and MON 88913 are corroborated by
centesimal components shown for MON 15985 x MON 88913 cotton, generated from samples
collected in two occasions and two different places, in planned released to the environment in
Brazil during the 2008/2009(20) crop. Centesimal composition (ashes, fat, humidity, proteins
and carbohydrates by calculation) of grain and forage of cotton MON 15985 x MON 88913 was
contrasted to the conventional control cotton and commercial references. In all places, the
values of centesimal components in forage and cotton grain of MON 15985 x MON 88913
cotton were similar to that of control values. In the analysis by location, average values of
centesimal components were within the intervals of control values within each location or
within the interval of references calculated in the combined analyses of locations. ILSI
databank was used to show that the values found in the study were within the values of the
technical literature (sss.cropcomposition.org). The result enables a conclusion for substantial
equivalence of cotton MON 15985 x MON 88913 as against conventional cotton, a
fundamental component in assessing alimentary safety.
The above analyses made the consideration that introduction of genes cry1Ac, cry2ab2, epsps,
nptII, and uidA failed to result in any substantial nutritional change cotton
MON 15985 x MON 88913, since the profiles of the centesimal composition, coupled with the
previously disclosed results for parental events, were similar to those normally observed in
other varieties or produced under different cultivation conditions.
Besides safety of donor organisms and analyses of centesimal composition, the records
displayed other studies supported by scientific literature that ratify the positive assertion on
alimentary safety of MON 15985 x MON 88913 cotton. One of such assertions is that
alimentary safety of proteins Cry1Ac, Cry2Ab2, NPTII and CP4 EPSPS was assessed in acute
oral toxicity studies with mice, showing that the proteins fail to cause any adverse effect, even
in the highest doses tested, of 4200,1450, 5000 and 572 mg/kg of body weight, respectively.
Essays in simulated gastric and intestinal systems were conducted to assess protein
degradability. Proteins Cry1Ac, Cry2Ab2, and CP4 EPSPS are rapidly digested in simulated, and
results show that 99% and 98% of the two proteins, Cry1Ac and Cry2Ab2 were digested in no
later than 30 seconds and over 95% of protein CP4 EPSPS was digested within 15 seconds of
incubation. It shall be stressed that studies with animals were previously submitted by the
applicant in the records for commercial release of parental events, already passed by CTNBio.
A further assessment uses bioinformatics tools to contrast amino acid sequences in the
proteins of interest with proteins recognizedly toxic or allergenic. Bioinformatics analyses were
conducted with the sequences of proteins Cry1Ac, Cry2Ab2, NPTII, GUS, and CP4 EPSPS so
assess similarity to allergens and identify immunologically relevant peptides. Comparisons
were conducted using public databanks containing allergen sequences and failed to reveal
significant coincidences between the sequences with that of the proteins analyzed. As far as
toxicity is concerned, similarity of biologically relevant sequence with a known toxin (that is to
say, the sequence apparently derived from a common ancestor gene) may indicate that
additional toxicologic assessments are necessary. Homology is determined by criteria
published to find the degree of similarity of amino acids among proteins. The results obtained
with proteins Cry1Ac, Cry2Ab2, NPTII, GUS and CP4 EPSPS show that there are no sequence
similarities with toxins that may impact the alimentary safety of MON 15985 x MON 88913
corn. Therefore, proteins Cry1Ac, Cry2Ab2, and CP4 EPSPS record no similarity of amino acid
sequences with allergens such as gliadins, glutenins or toxic proteins that may harm mammals,
which is relevant from the viewpoint of alimentary safety.
Cotton may be consumed in natura (seed) by ruminants or in food (cotton seed oil) and
processed ration. The records show that cotton MON 15985 x MON 88913 is held at the same
level of safety than conventional cotton and, therefore, the use in the abovementioned forms
as food or ration would not imply greater risk than that of conventional cotton.
IV. Environmental Aspects
Applicant mentions, in its submission, that cotton MON 15985 e and cotton MON 88913,
already approved by CTNBio, exhibit results that corroborate the results found for
MON 15985 x MON 88913 cotton. The results show that MON 15985 x MON 88913 cotton, as
well as its parental events, fail to display greater potential as an invading plant when compared
with conventional cotton. This conclusion is based on studies carried out in Brazil and the
United States and on experimental evidences involving: phenotypic and agronomic
characteristics, and ecologic interactions that evidence that MON 15985 x MON 88913 cotton
is not a potential cause of negative impacts to the environment nor becoming a weed;
assessment of the impact on non-target organisms showing that MON 15985 x MON 88913
cotton, as well as its parental events, fails to cause adverse effects on such organisms in the
culture conditions of use.
Cotton belongs to genus Gossypium, and the known germplasm may be divided into sylvan and
cultivated species, diploid species (2n= 2X=26) and tetraploid (2n=4x=52) and species able and
unable to produce textile fibers. Four species of agronomic importance possess commercially
valuable fibers, out of which two (Gossypium arboreum and Gossypium herbaceum) are
European diploid species and two (Gossypium barbarensis and Gossypium hirsutum) are New
World allotetraploid species. The cotton variety used as the recipient of gene cp4 epsps to
generate MON 88913 was Coker 312. This is a traditional variety of high ground cotton
(Gossypium hirsutum) that was used to generate MON 1445 cotton, MON 531 cotton and
MON 15985 cotton. Three species of cotton may be found in Brazil: Gossypium hirsutum L.,
Gossypium barbadensis L. and Gossypium mustelinium (Miers & Watt). Gossypium hirsutum is
represented by two exotic races: the first is Gossypium hirsutum r. latifolium Hutch, native of
Mexico and introduced through the United States, widely cultivated in the country and is
present almost exclusively under the form of cultivars. The second race is Gossypium hirsutum
r. marie galante (Watt) Hutch, known as mocó, or arboreal, cotton, originated in the Antilles
and brought to the country by the Dutch or by Africans during colonial times. Species
Gossypium barbadensis, that has its domestication center in the Northern Peru and South of
Ecuador, was introduced by pre-Columbian peoples, and its use as a textile plant widened
before its decadence with the dissemination of the two Gossypium hirsutum races. This species
is not found in natural environments and is maintained basically as a backyard plant. The plant
is largely distributed, present in almost the whole of the country and its in situ conservation is
directly linked to maintenance of use traditions as a medicinal plant. The only active species in
Brazil is Gossypium mustelinum, with natural distribution restricted to the semi-arid Brazilian
Northeast. Just three small populations are known, two in Bahia and one in Rio Grande do
Norte, and the aggregate number of adult plants of all such populations is below two hundred.
All species of such cotton found in Brazil are sexually compatible and crossings are mediated by
pollinating insects. In the absences of complete sexual barriers, geographic segregation among
cultivars and the populations one wishes to protect has been used to reduce the likelihood of
such crossings. The United States and Australia used this strategy to avoid occurrence of gene
flow between sylvan populations and genetically modified cultivars of cotton. The measure
proved to be efficient, since no record of transgene transfer has been, neither its introgression
has been reported up to this moment. In Brazil, “Transgenic Cotton Plants Exclusion Zones for
preservation of species of Gossypium (native and naturalized)” have been set by Embrapa
Algodão Communiqué nº 242 and MAPA Directive nº 21/2005 (Brazil, 2005). These exclusion
zones for cultivation of genetically modified cotton contemplate the locations where they
occur and feral populations of Gossypium hirsutum r. marie galante (mocó cotton) and
Gossypium mustelinum are distributed. Eventual and potential risks to the environment were
considered and analyzed, in addition to issues related to distribution of the sylvan form of
endemic Gossypium mustelinum in the south of Rio Grande do Norte and Northeast of Bahia,
or sub-spontaneous forms of Gossypium barbadense L., in the whole Amazon region,
southeastern Piauí and west of Pernambuco, and in the Atlantic Forest, comprising the
following states: RN, PB, AL, SE, BA, MG and ES, in an area equivalent to the Brazilian cerrado.
As mentioned above, in these areas, farming of MON 15985 x MON 88913 cotton is not
recommended. The environmental consequences of pollen transfer from
MON 15985 x MON 88913 cotton to other cotton plants or to other species related to
Gossypium are held as minimal. This is because the limited movement of cotton pollen, due to
the safety of proteins expressed in the plant and also due to the absence of any competitive
advantage granted by the exogenous genes. Therefore the potential gene flow in sexually
compatible species is unlikely, since the sylvan compatible species are found in few isolated
areas in Brazil.
Modern agriculture is an activity blamed for significant negative impacts(21,22,23) and therefore
risk assessment of any GM event shall be conducted as against the impact already inherent to
conventional agriculture(24,25,26). Thus, the CTNBio analysis sought assessing whether
environmental impact caused by MON 15985 x MON 88913 cotton is significantly more
harmful than the one caused by conventional cotton varieties considering the agriculture
practices associated to each system.
All species of genus Gossypium have perfect flowers. Fecundation takes place right after
anthesis, and self-fecundation or crossed pollination or even both may occur. Cotton plant
pollen is relatively large, ranging from 81 to 143 micra (making the grains to adhere to each
other), spherical in format, covered by a large amount of spicules, practically not prone to be
transported by the wind(27). In the field, its viability extends up to the end of the afternoon,
though it may last for 24 hours if kept at temperatures from 2o to 3oC(28).
Cotton is usually held as a partial crossed pollination culture, although many developers treat
this plant as if it were completely self-fertile and self-pollinating, except for crossed pollination
through pollinating insects. One publication shows that the cotton plant features an
reproductive system that is intermediate between allogamous and autogamous plants, with
pollination rates from 5% to 95%. Self-pollination is the form of hybridation that preferably
occurs in cotton culture, though natural crossing may be the case(30). Production of sees ranges
from 20 to 30 per fruit when crossing and self-pollination are well performed. Cotton plant
flowering time may vary according to environmental conditions and variety, though in general
it starts on the 50th day after emergence and extends up to 120 days or more, with the curve
peak around 70 to 80 days. Procedures of self-pollination and crossing shall be developed at
the most propitious time, 30 to 40 days after flowering.
Genetic improvement requires controlled pollination and maintenance of purity through
physical barriers or isolation by distance. Cotton pollen grains are heavy and viscous, which
makes dispersion by the wind quite unlikely. Pollen transfer is carried out by insects, especially
wild bees, bumblebees (Bombus sp.) and honeybees (Apis mellifera) that reach freshly open
flowers. In Brazil, genetic cotton improvement programs are targeted to aggregate the most
desirable features according to the seeding region, taking into account the components of
agricultural production and adequacy, fiber and thread quality, as well as the product
characteristics for a specific purpose.
Under normal conditions, cotton fails to propagate vegetatively, but does it through bolls or
seeds(29). Natural crossing may take place through pollinating insects, since there is no pollen
dispersion caused by the wind. However, the reach of pollen tends to be limited among very
close cotton flowers, surrounded by bee colonies. The pollen movement is small, just 1.6% of
flowers receive materials from other plants. Pollinating insects are used as a tool in plant
development programs to attain other varieties. One of the most important effects of crossing,
referred as heterosis or hybrid vigor, may be the result of inter-specific, intra-specific and intervarietal
crossings. The use of hybrid vigor in cotton proved to be interesting after the evidence
that excessive introgression (self-pollination) has detrimental effects(31). The rate of natural
crossing recorded in Brazil has ranged from 1% to 100% in the Northeast, and from 0% to 71%
in the Center-West. Different crossing rates in regions close to each other may be explained by
the presence of native forests and pollinator insects, mainly honeybees. It must be emphasized
that crossing rates in the cerrado tillage, reaching around 6%. Yet in the cerrado regions, where
native vegetation has wide occurrence, rates change from 19% to 42% and, in areas cultivated
by small farmers, the rates are higher (45% to 62%) given forest preservation and high
population of bees(26).
Phenotypic and ecological assessments were conducted with MON 15985 x MON 88913 cotton
and included characteristics as dormancy, vigor and germination, emergence, vegetative and
reproductive stages, seed retention and interactions with diseases, insects and abiotic stress.
Characteristics analyzed for samples collected in Brazil show that cotton
MON 15985 x MON 88913 and control cotton are equivalent. Ability of
MON 15985 x MON 88913 cotton to change into a weed when compared to the conventional
control cotton was assessed in the 2008/2009 and 2009/2010 harvests, considering phenotypic
and agronomic characteristics, ecological interactions, pollen morphology and viability,
volunteer plants assessment and germination rates. The results of phenotypic and ecological
assessments conducted in Brazil suggest that MON 15985 x MON 88913 cotton has no
characteristics that may grant higher risk than conventional cotton to change into a weed or
cause ecological impact. Ecological interactions also failed to show higher susceptibility or
tolerance to diseases, abiotic stress and insects in MON 15985 x MON 88913 cotton.
Phenotypic and ecologic data indicate that the characteristics of resistance to insects and
tolerance too glyphosate that are present in cotton MON 15985 x MON 88913 fail to grant it
any selective advantage, being the MON 15985 x MON 88913 cotton as safe as conventional
cotton.
In Brazil, the applicant has conducted assessments of phenotypic and agronomic
characteristics of volunteer plants, of vigor and germination and pollen characteristics in the
2008/2009 and 2009/2010 harvests. Experiments were conducted during the 2008/2009
harvest in Sorriso/MT and Cachoeira Dourada/MG.
The experimental design used was that of randomized blocks with four repetitions. Cotton
MON 15985 x MON 88913 was compared with control cotton and commercial references for
emergence (initial stand), vigor, first flower date, 50% flowering date, first open boll date,
plant height, physiologic maturation, final stand, yield, grain yield and abundance of non-target
organisms. Experiments were also conducted during the 2009/2010 harvest in Sorriso/MT and
Cachoeira Dourada/MG. The experimental design was the same as for the 2008/2009 harvest
and the same parameters were assessed, except for abundance of non-target organisms. The
data were submitted to t test statistical analysis to compare MON 15985 x MON 88913 cotton
to the control cotton at a significance level of 5%. Eight varieties of commercial cotton were
taken as references, four in each seeding location. The results suggest that
MON 15985 x MON 88913 cotton has no characteristics that may grant significant risk of
changing into a pest plant or causing ecological impact different from that of conventional
control cotton. Impact assessment on usual agronomical practices showed that cultivation of
MON 15985 x MON 88913 cotton would not bring impact on cultivation and rotation practices,
and even on insect and diseases management. The only difference would be the control of
pest lepidopterans. Besides, the use of cotton MON 15985 x MON 88913 enables controlling a
large range of grasses and large leaf pest and perennial plants through post-emergence
application of the glyphosate herbicide, similar to what happens with MON 1445 and
MON 88913 cottons.
Regarding environmental safety, phenotypic and agronomic assessments conducted in Brazil
for MON 15985 x MON 88913 cotton ratified the data previously submitted for parental
events, indicating that MON 15985 x MON 88913 cotton has no characteristics that may grant
a significantly changed risk of changing into a pest plant or causing an ecological impact
different from that of conventional cotton. Besides, data submitted from ecologic interactions
indicate that the new features present in MON 15985 x MON 88913 cotton fail to grant higher
susceptibility or tolerance to diseases and abiotic stress and insects. In addition, it was proven
that cotton MON 15985 x MON 88913 has no potential to adversely affect beneficial
organisms, plants or non-target organisms. Safety of Cry1Ac, Cry2Ab2 and CP4 EPSPS proteins
is very well characterized and widely discussed in the proceedings. As a whole, alimentary and
environmental safety data of MON 15985 x MON 88913 cotton evidenced that this plant fails
to impose any risk to human and animal health and to the environment when compared with
conventional cotton.
V. Restriction to use of the GMO and its derivatives
Technical reports related to agronomic performance reached the conclusion that genetically
modified plants are equivalent to conventional ones. Thus, the information suggests that
genetically modified plants are not fundamentally different from the non-modified cotton
genotypes, except for resistance to lepidopteran insects and tolerance to glyphosate. Besides,
there is no evidence of adverse effects in the use of MON 15985 x MON 88913 cotton. Based
on the foregoing, there are no restrictions to the use of such cotton and its derivatives as
either human of animal food.
Aimed at avoiding vertical gene flow to native or naturalized varieties, the seeding of
MON 15985 x MON 88913 cotton shall obey the exclusion zones in sowing genetically modified
cotton according to Embrapa Algodão Communiqué 242(32) and MAPA Directive nº 21/2005
(Brazil, 2005). The exclusion zones for sowing genetically modified cotton refer to locations
where feral populations of G. hirsutum r. marie galante (mocó cotton) and G. mustelinum
cotton occur and are distributed.
VI. Considerations on particulars of different regions of the Country (subsidies to
monitoring bodies):
As established by Article 1 of Law 11450, of March 21, 2007, “research and cultivation of
genetically modified organisms are forbidden in indigenous lands and areas of preservation
units”,
VII. Conclusion
Whereas:
Cotton MON 15985 x MON 88913 (Gossypium hirsutum) belongs to a well characterized
species with a solid safety background for human consumption, considering Opinions issued
and individually read by the members in charge of analyzing the records and that genes cry1Ac,
cry2ab2, nptII, uidA and cp4 epsps introduced in this variety code for known and well
characterized proteins, harmless to humans;
Stacked cotton MON 15985 x MON 88913 is the result of crossing, through classical genetic
improvement, of parental of genetically modified cottons MON 15985 and MON 88913,
previously approved by CTNBio (MON 15985 – EPT 1832/2009; MON 88913 – EPT 2956/2011)
and held as safe as conventional cotton;
Centesimal composition data collected from analyses of plants cultivated in Brazil have
ratified the data previously submitted for parental events and failed to show significant
differences between cotton MON 15985 x MON 88913 and conventional cotton, suggesting
nutritional equivalence between them and their alimentary safety;
Data on agronomic and phenotypic characteristics assessed in the Brazilian environment were
submitted in detail and also corroborate the previously submitted data for parental events
MON 15985 and MON 88913, and failed to record any significant differences between cotton
MON 15985 x MON 88913 and conventional cotton, suggesting substantial equivalence
between them and their environmental safety; and
Whereas
1. The levels of proteins Cry1Ac, Cry2Ab2, NPTII, GUS, and CP4 EPSPS in tissues studied
are low, susceptibility to digestion in simulated gastric fluids is large and digestion is fast, there
is no acute toxicity in mammals in higher doses tested for all heterolog proteins, and no
similarity of sequences of such proteins were found with known allergens or toxic proteins
when assessed through bioinformatics tools;
2. The genetic modification introduced in MON 15985 x MON 88913 cotton failed to
result in important differences in chemical composition of nutrients, being the result within the
normal variation range of conventional varieties;
3. The DNA molecule is a natural component of food, with no evidence that the molecule
may have adverse effect for humans when ingested in acceptable amount of food (no direct
toxic effect);
4. The applicant prepared his document based on Article 5 of Ruling Resolution nº 5
(published in the Federal Official Gazette nº 50, Section 1, pages 6 to 8, on 03.13.2008),
providing on rules for commercial release of Genetically Modified Organisms and their
derivatives;
5. The likelihood of a transgenic plant to become a plant pest, as well as the crossing of
MON 15985 x MON 88913 cotton with other cotton species originate a plant pest is negligible;
6. Bacillus thuringiensis is a soil microorganism and exposure of living organisms and the
environment to this bacterium or to any element thereof is an event of abundant occurrence in
nature, with no record of significant risk for soil microbiota;
106/2013
22 29
7. Cultures of Bacillus thuringiensis are registered with Agência Nacional de Vigilância
Sanitária – ANVISA under different formulations for application in thirty types of plant
cultivation for alimentary use(9);
8. Biopesticides based on the toxins of Bacillus thuringiensis are widely used as an
alternative for chemical insecticides, even in organic crops, for its safety for non-target
organisms, providing assistance in cases where development of resistance to chemical
insecticides has been detected;
9. Plants genetically modified with genes cry ov Bacillus thuringiensis and gene
cp4 epsps of Agrobacterium strain CP4 have been approved by regulatory bodies in
different countries, including CTNBio, with no record of adverse effects to the
environment and human and animal health;
10. Enzymes EPSPS are widely distributed in nature and occur in all plants and protein CP4
EPSPS has safe background of safe consumption through its use of other genetically modified
products with gene cp4 epsps;
11. Field studies conducted in Brazil on insect populations present in
MON 15985 x MON 88913 cotton fields showed that abundance of non-target insects was not
affected when compared to conventional cotton, evidencing the specificity of Cry to targetpests;
12. Among the benefits of using cry genes as opposed to other lepidopteran control
methods are the absence of negative effects to non-target insects, mammals and human
beings, the high specificity and efficiency against target-insects, the environmental
degradability and safe manipulation and use;
13. Any insect-control measure that enables reducing the use of chemical pesticides shall
be considered, mainly from the environmental, safety and economic viewpoints;
14. The use history of MON 15985 x MON 88913 cotton in the world is an indication that
the variety is as safe to the environment and human and animal health as the conventional
cotton that have been used.
15. Since 1996 transgenic cultures have been used in different countries, and over 30
million hectares are currently planted with transgenic cultures (soybeans, maize and cotton) in
Brazil, which is the second world producer of GM cultures.
16. The history of transgenic cultures in Brazil during this period of time has no record of
any problem to human and animal health and to the environment which may be attributed to
these cultures, including transgenic cultures. It must be emphasized that the lack of negative
effects from cultivating transgenic cotton plants is not a guarantee that the problems may not
happen. Zero risk and absolute safety do not exist in the biological world, although there is a
host of scientific information and a safe use history that enable us to conclude that transgenic
plants are as safe as conventional versions. Therefore, the applicant shall conduct postcommercial
release monitoring according to CTNBio Ruling Resolution nº 9.
For the foregoing, and taking into account internationally accepted criteria in the process of
analyzing the risk of genetically modified raw materials, it is possible to conclude that cotton
MON 15985 x MON 88913 is as safe as its conventional equivalent.
CTNBio considers this activity is not a potential cause of significant degradation of the
environment nor harmful to human and animal health. Restrictions to the use of the GMO and
its derivatives under analysis are conditioned to the provisions of CTNBio Ruling Resolution nº
09, Embrapa Algodão Communiqué nº 242 and MAPA Directive nº 21/2005 (Brazil, 2005).
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