| Summary of the safety assessment (food safety): |
Corn, (Zea mays L.) is a species of the Gramineae family, Maydeae tribe, subfamily Panicoideae, which is separated within the subgenus Zea and has chromosome number 2n = 20,21,22,24(1). The corn closest wild species is teosinte, found in Mexico and some regions of Central America, where it may cross with corn cultivated in production fields.
Corn has a history of over eight thousand years in the Americas and is cultivated since the Pre-Columbian era. Among higher plants, corn is one of the best scientifically characterized and is currently the cultivated species that reached the highest degree of domestication and is unable to survive in nature, except when cultivated by man(2). There are currently over 300 identified races of corn and, within each such race there are thousands of cultivars.
Corn is one of the most important food sources worldwide and used as input for a range of food products, rations and industrial products. Brazil is one of the largest world producers of corn, which is cultivated practically in the whole national territory(3).
Occurrence of insects on Earth is higher in the tropics than in temperate regions, where damages caused by such animals are higher. Among corn pests, importance of the fall armyworm (Spodoptera frugiperda) shall be underlined. Cruz et al.(4) estimated that losses in Brazil caused by infestations of Spodoptera frugiperda reached 400 million Dollars each year. Other species of the Lepidoptera order are important pests in the culture of corn, such as the corn earworm (Helicoverpa zea) and stalk borer (Diatraea saccharalis).
The main insect controlling measure in corn farming in Brazil has been the use of insecticides. In some of Brazilian Center-Western areas, for instance, tenths of insecticide spraying are needed for a single cycle of culture. Another measure to control pests would be the use of resistant cultivars.
Contrasted to conventional corn, TC 1507 x NK603 corn has no increased survival ability as a pest. Presence of genes granting resistance to lepidopteran insects and tolerance to glyphosate herbicide grant corn TC 1507 x NK603 a selective advantage when exposed to the herbicide and when in the presence of target-insects. However, such characteristics are not sufficient to turn it into a pest in corn cultures(5,6).
Use of corn containing stacked events represents a future trend – that meets the producers’ demand – by combining two agronomically important characteristics in a same hybrid. Corns with events combined by classic genetic improvement have been approved in Japan, Korea, Argentina, El Salvador, European Union, South Africa, Taiwan and the Philippines(7).
III. Description of the GMO and Proteins Expressed
TC 1507 corn was developed from incorporating plasmid DNA PHP8999 containing gene cry1F and the gene used as selective marker, pat. Plants of TC 1507 lineage were obtained by microparticle bombardment using a Biolistics PDS-1000He – Bio-Rad accelerator(8). Gene cry1F product of expression is protein Cry1F, derived from Bacillus thuringiensis var. aizawai, which has insecticide activity on target-pests, protecting the plants(8).
Gene pat, derived from Streptomyces viridochromogenes, is responsible for coding enzyme phosphinotrycin N-acetyltransferase (PAT), with the function to chemically inactivate herbicides derived from phosphinotrycin, such as glufosinate ammonium, making resistant the cells containing such herbicides(9,10,11).
NK 603 corn contains gene cp4 epsps, from Agrobacterium tumefaciens, strain CP4, responsible for expressing protein CP4 EPSPS (CP4 -5-enolpyruvylshikimate-3-phosphate synthase) that determines expression of the characteristic of resistance to the glyphosate herbicide. Protein CP4 EPSPS expressed in glyphosate-tolerant genetically modified plants is functionally identical to the EPSPS protein endogenous to plants(12). In conventional plants, the glyphosate bonds to the EPSPS enzyme and blocks biosynthesis of 5-hydroxy shikimate-3-phosphate, preventing formation of aromatic amino acids and secondary metabolites(13). In glyphosate-tolerant genetically modified plants, such as NK 603 corn, aromatic amino acids and other metabolites needed in the development of plants keep being produced by protein CP4-EPSPS activity(14).
The mode of action and biologic activities of proteins CP4 EPSPS and Cry1F expressed in TC 1507 x NK603 corn are separate and do not have known mechanisms of interaction that would cause adverse effects to human and animal health and to the environment. In a certain way, proteins CP4 EPSPS and Cry1F present in TC 1507 x NK603 corn are accumulated in different cell compartments and have different and non-interactive metabolic functions. Thus, protein CP4 EPSPS is targeted to the chloroplast, while Cry1F protein is accumulated in the cytoplasm(5,6). Absence of a fresh adverse effect, resulting from the interaction of exogenous genes cry1F, pat and cp4 epsps in TC 1507 x NK603 combined corn is evidenced by studies conducted with the isolated events TC 1507 and NK 603.
The level of CP4 EPSPS and Cry1F proteins expression in the individual events (TC 1507 and NK 603 corn) and, thus, the likelihood that the proteins interact between them is low, what has been confirmed through microscopic analysis of agronomic and phenotypic characteristics regarding efficacy and selectivity of TC 1507 x NK603 corn in the field(5,6).
IV. Aspects Related to Human and Animal Health
Safety of Cry1F and EPSPS proteins was duly assayed by CTNBio(5,6). Protein Cry1F mode of action is sufficiently elucidated in scientific literature(15,16). In vitro experiments were used to assess the increased digestibility of foodstuffs containing proteins Cry1F and CP4 EPSPS after pre-heating. The study showed that pre-heating increases the proteins digestibility in simulated gastric and intestinal fluids, suggesting that the likelihood of an occasional increased allergenicity of Cry1F protein is extremely low, given the ease of digestion, which is a further important component in assessing the security of corn containing protein Cry(17,18).
Cry1F protein produced by TC 1507 corn is a protein with a specific effect of controlling certain important corn pest lepidopteran, such as the armyworm (Spodoptera frugiperda) and stalk borer (Diatraea saccharalis). Action of such insecticide protein is mediated by specific receptors that determine toxicity just for some pest insects. Additionally, in vitro and in vivo studies confirmed that Cry1F protein expressed in Bacillus thuringiensis and TC 1507 x NK603 corn is highly selective and has no action on mammals(19,20).
Protein CP4 EPSPS is an enzyme that is present in all plants and in a large number of microorganisms(21), while protein Cry1F has no enzymatic activity in plants and, therefore, does not affect the plant metabolism.
The likelihood of biochemical interactions between CP4 EPSPS and Cry1F proteins in the complex matrix of a plant is limited, since the proteins accumulate in different loci of the cells and in a low level of expression. With this, potential exposure to such proteins is extremely low in human and animal feeding.
Given that proteins CP4 EPSPS and Cry1F proteins fail to produce toxicity in the maximum doses tested, it his highly unlikely that an interaction happens between these proteins in the normal doses found in food, that may cause additive or synergic effects. There is numerous information in the literature, at the area of toxicology of chemical mixtures, showing that such interactions are inexistent when the substances are administered in doses substantially below the no-observed adverse effect level (22,23,24,25,26).
Due to the rigorous specificity for substrates, EPSPS enzymes bond just S3P, PEP and glyphosate. The only known resulting metabolic product is 5-enolpyruvylshikimate-3-phosphate acid, corresponding to the penultimate product in the shikimic acid pathway. Shikimic acid is a precursor for biosynthesis of aromatic amino acids (phenylalanine, tyrosin and tryptophan), and a number of secondary metabolites such as tetrahydropholate, ubiquinone and K vitamin(27). Though the pathway of shikimic acid (or shikimate) and EPSPS proteins do not occur in mammals, fish, birds, reptiles and insects, they are important to plants. One estimates that aromatic molecules, all of them derived from shikimic acid, represent not less than 35% of a plant’s dry weight(28,29).
In vitro studies with simulated digestive fluids are widely used tools as a model of animal digestion. This simulated system was used to study digestibility of plant proteins(28,29), animal proteins(35), and food additives(32), as well as to assess protein quality(33) and the allergenicity potential through absorption of proteins by the digestive system(34).
Protein Cry1F biologic activity was studied in a range of pest insects that feed on corn, exposing such insects to artificial diets containing Cry1F protein. The results confirmed the action of Cry1F protein on target-insects (fall armyworm, sugarcane stalk borer, European corn borer, corn earworm, elasmo worm and Southwest corn borer). On the other hand, protein action was not reported on non-target insects, showing its specificity according to publications by Evans(35).
Finally, the knowledge on protein EPSPS mode of action, specificity and history of safe use, the potential toxic and allergenic effects of such proteins in humans and other mammals was analyzed through in vitro digestion studies. Experiments used simulated gastric (pH 1.2) and intestinal (pH 7.5) fluids. The degradation rate of protein CP4 EPSPS (mature protein, without the transit peptide) was assayed through Western blot analysis. The result was that protein CP4 EPSPS and peptides degraded in less than fifteen seconds after exposure to the gastric fluid. In simulated intestinal fluid, degradation of CP4 EPSPS protein took place in a time lower than ten minutes(34).
V. Environmental Aspects
Corn is a monoic, allogamic and annual plant the pollination of which is anemochoric and distances that the pollen may cover depend on wind patterns, humidity and temperature. Corn pollen disperses freely in areas located near the culture, and is able to reach stilly-stigmas of the same or different genotypes and, under adequate conditions, to start germination, originating the pollinic tube, promoting ovule fecundation within an average period of 24 hours.
Studies conducted on pollen dispersion demonstrated that pollen may travel long distances. However, the major part of it is deposited close to the corn field, with a very low translocation rate, where over 95% of pollen may reach distances within sixty meters from its source. Luna et al.(36) examined the isolation distance and control for pollen and demonstrated that crossed pollination takes place within 200 meters, though no crossed pollination was recorded, under conditions of non-detasseling, for distances no lower than 300 meters from pollen sources. Results indicate that pollen viability is maintained for two hours and that crossed pollination was not recorded at a distance of 300 meters from the pollen source.
By comparing concentrations at one meter from the source under low-to-moderate wind, one estimated that about 2% of the pollen is recorded at sixty meters, 1.1% at 200 meters, and 0.75-0.5% at 500 meters from the source. At a distance of ten meters from the source, on average, the number of pollen grains by unit of soil is tenfold lower than the number recorded at one meter from the border. Therefore, if separation distances established for corn seeds are observed, one expects that pollen transfer to adjacent varieties be minimal, being unlikely the presence of glyphosate- or gluphosinate-resistant genetic materials.
From the agronomic viewpoint, coexistence between conventional corn (improved or creole) and transgenic corn is possible(37,38). Older communities and modern farmers have known how to live, free from problems, with different corn cultivars while keeping their genetic identity along time.
Field, nursery and laboratory studies conducted with corn containing event TC 1507 x NK603 demonstrated that this genetic transformation event is comparable to conventional corn regarding its reproductive, agronomic, and nutritional and environmental safety(5,6).
The use of insect resistant and herbicide tolerant TC 1507 x NK603 technology became an option to control the invading species(40). Proteins Cry are extremely selective for insects of the Lepidoptera Order(41, 42, 43, 44, 45), free from harmful effects on beneficial and non-target insects, including predators and pollinizers(46,47,48,49). On the other hand, protein EPSPS is held as safe and widely accepted, given its ubiquity and negative record for toxicity, not to mention its lack of association to pathogenicity(5,6).
Field studies were conducted on the 2008/2009 crop in the Dow AgroSciences Operating Units of Indianápolis (State of Minas Gerais) and Mogi Mirim (State of São Paulo) comparing nutritional composition of plants with isolated events TC 1507 and NK 603 and combined events TC 1507 x NK603. The results demonstrated that the hybrid analyzed are genetically similar, except in what relates to the events that were introduced(50).
Besides these studies, nutritional composition was assayed in samples of corn kernels and corn silage where the results showed that TC 1507 x NK603 stacked corn, TC 1507 corn, NK 603 corn and non-transgenic corn are substantially equivalent. Comparative data showed that the combined TC 1507 x NK603 corn is not different from its components TC 1507 and NK 603 for the different nutritional components studied, indicating absence of interaction when event TC 1507 is combined to event NK 603 by classical genetic improvement(50).
The stacked event corn action on non-target organisms was studied in different species(51). Assays on pest lepidopteran control and interaction with natural enemies present in corn farming were studied in different locations within Brazil(50,52,53). The results showed that corn containing the Cry protein expressing gene promoted an efficient control of Spodoptera frugiperda and Helicoverpa zea. Besides, there was no interference of the treatment on population level of the predators’ set (Coccinellidae, Sirfidea, Chrysopidae, Orius sp. and Geocoris sp.), as well as on the earwig predator (Doru lineare).
The impacts that Cry proteins may in general cause to non-target insects and soil organisms have been widely studied as part of the environmental safety of cultures with isolated event TC 1507 that expresses protein Cry1F. Studies demonstrated that the tested non-target terrestrial and soil organisms were not affected by protein Cry1F, though the protein level was above the maximum levels that could be measured in case of natural exposure(44,54,55). Additionally, a comparison between Cry proteins produced by Bacillus thuringiensis and the Cry1F protein produced by TC 1507 corn demonstrated that they may remain for a longer time in tropical soils given their bonds with clay particles; however no effect on the soil microbiota was recorded(56).
Scientific literature papers reached a conclusion that the presence of Cry1F protein, besides not significantly affecting the microbiota and soil animals, was absorbed by cultures subsequent to the sowing of TC 1507 corn(57,58). Cry proteins from three species of Bacillus thuringiensis failed to show microbicide or microbiostatic activity against a variety of bacteria, fungi and algae(59).
A further issue of environmental interest relates to genetically modified corn gene flow and the effects that this flow could cause on conventional corn. The likelihood of crossed pollination between a genetically modified and a conventional plant, followed by introgression, relates to availability and viability of the genetically modified parental pollen and delivery of such pollen at the stigma or the conventional parent. This availability depends on the sowing time and agronomic conditions. Pollen delivery to the stigma depends on wind, vectors, distance, rain precipitation and natural barriers to pollen movement. Still, crossed pollination efficiency shall depend concurrently of the flowering time of the pollen recipient and donor, and pollen viability and ability to compete. Depending on the heritage pattern of the feature, part of the pollen produced by the donor fails to contain the gene of interest. Besides, corn pollen grains are large and heavy, reducing dispersion distances, the larger deposition occurring near the donor plant(60,36).
Ninety-eight percent of pollen dispersion occurs within 25 meters from the issuing field and close to 100% within 100 meters, being the larger part (99%) of crossed pollination outside the issuing field occurring in the range of 18 to 20 meters from its borders(37). Weather conditions (and wind direction) and physical barriers affect dispersion of pollen and corn cross pollination, the closer, the most efficient are the barriers. Dispersion of TC 1507 x NK603 corn pollen may, however, be controlled so as to make coexistence of conventional, organic and genetically modified tillage possible(37), as this is naturally made whenever genotypes targeted for different uses (human food, creole races, etc.) are produced in adjacent areas.
VI. Restrictions to the Use of the GMO and its Derivatives
Studies submitted by applicant demonstrated that there was no significant difference between corn hybrids derived from non-modified lineages and TC 1507 x NK603 corn regarding agronomic characteristics, reproduction, dissemination and survival ability. All evidence submitted in the proceedings and bibliographic references ratify that the risk level of transgenic variety is equivalent to non-transgenic varieties before soil microbiota, as well as that of other plants and to human and animal health. Thus, farming and consumption of TC 1507 x NK603 corn are not potentially causes of significant environment degradation and fail to pose any risk to human and animal health. For these reasons, there are no restrictions to the use of TC 1507 x NK603 corn and its derivatives, except in locations mentioned by Law nº 11460, of March 21, 2007.
Gene flow to local varieties (so-called creole corns) of open pollination is possible and poses the same risk caused by commercial genotypes available in the market (80% of conventional corn planted in Brazil comes from commercial seeds that underwent genetic improvement). Coexistence between conventional corn (either improved or creole) and transgenic corn cultivars is possible from the agronomic viewpoint(37,38) and shall comply with the provisions of CTNBio Ruling Resolution nº 4.
After ten years of use in different countries, there is no record of problems for human and animal health that could be attributed to transgenic corns. It shall be stressed that the lack of negative effects from cultivation of transgenic corn plants do not entail that such effects are free from occurring. Zero risk and absolute safety do not exist in a biological world, although there is a mass of reliable scientific information and a safe history of ten years of use that enable us to assert that TC 1507 x NK603 corn is as safe as conventional corn versions. Therefore, applicant shall conduct post commercial-release monitoring under CTNBio Ruling Resolution nº 3.
VII. Consideration on the Particulars of Different Regions of the Country (Information to supervisory agencies)
As established by 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.”
VIII. Conclusion
Considering that TC 1507 x NK603 corn (Zea mays) variety belongs to a well characterized species with a solid safety record for human consumption and that cr1F, pat and cp4 epsps genes introduced in this variety codify proteins ubiquitous in nature and present in plants, fungi and microorganisms that are part of the alimentary diet of humans and animals;
Considering that the insertion of the genotype took place though classical genetic improvement, resulting in insertion of one stable and functional copy of genes cr1F, pat and cp4 epsps that granted the plants tolerance to glyphosate herbicide, glufosinate ammonium, and resistance to insects;
Considering that data on centesimal composition fail to show significant differences between genetically modified varieties and conventional varieties, suggesting nutritional equivalence between them;
Considering that CTNBio assayed the events individually and was favorable to their commercial release;
Whereas:
1. TC 1507 x NK603 corn is a genetically modified product that features resistance to several lepidopteran held as pests and tolerance to the herbicide glyphosate, developed through classic genetic improvement by sexual crossing between lineages containing events TC 1507 and NK 603, both previously approved for commercial release;
2. comparative molecular analysis of TC 1507 x NK603 corn evidenced that integrity of inserts was maintained during the process of classic genetic improvement carried out with the purpose of combining the events;
3. segregation and generic inheritance analysis of corn TC 1507 x NK603 demonstrated that genes of events TC 1507 and NK 603 are independent and segregate in a stable way along successive generations;
4. agronomic and efficacy assessments of TC 1507 x NK603 corn indicated that the combination of the two events through classic genetic improvement methods (sexual crossings) failed to lead to expression of any other trait beyond expected, that is to say, resistance to certain insects and tolerance to glyphosate herbicide;
5. expression of proteins Cry1F, PAT and EPSPS in corn TC 1507 x NK603 is not significantly different from the proteins expressed in corn containing each event separately.
Therefore, considering internationally accepted criteria in the process of analyzing risks in genetically modified raw-material it is possible to conclude that TC 1507 x NK603 corn is as safe as its conventional equivalent.
CTNBio considers the activity free from being a potential cause of significant degradation to the environment, or harm to human and animal health. Restrictions to the use of this GMO and its derivatives are conditioned to the provisions of Law nº 11460, of March 21, 2007, CTNBio Ruling Resolution nº 03, and CTNBio Ruling Resolution nº 04.
CTNBio analysis took into consideration opinions of the Commission members; ad hoc consultants; documents delivered by applicant to CTNBio Executive Secretary; results of planned releases into the environment; and lectures, texts and debates of the public hearing held on 03.20.2007. Also considered and consulted were applicant’s independent studies and scientific literature conducted by third parties.
According to Annex I to Ruling Resolution nº 5, of March 12, 2008, the applicant shall have a term of thirty (30) days from publication of this Technical Opinion to adjust its proposal to the post-commercial release monitoring plan.
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