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OECD Unique Identifier details

MON-89Ø34-3xMON-ØØØ6Ø3-6
Commodity: Corn / Maize
Traits: Glyphosate tolerance,Lepidoptera resistance
Argentina
Name of product applicant: Monsanto Argentina S.A.I.C.
Summary of application:
The stacked event MON89034xNK603 of maize confers resistant to certain lepidopteran insects and tolerance to herbicides which active principle is glyphosate. The single events MON89034 and NK603, were stacked by conventional crossing (sexual). The stacked event has two genes, cry1A.105 and cry2Ab2, from MON89034 event and cp4 epsps gene from NK603 event. The transgenes are inherited independently, since they presents mendelian segregation. Moreover, the applicant proved the gene stability and the effective levels of the expressed proteins. The proteins Cry1A.105 and Cry2Ab2 confer resistance to lepidopteran insects, some affected species are Spodoptera frugiperda, Helicoverpa zea and Diatraea saccharalis. The protein CP4 EPSPS has similar structure and is functionally identical to the endogenous EPSPS enzyme of the plants, but with a reduced affinity to glyphosate. The allergenicity and toxicity assessment of proteins of new expression were carried out previously in the singles events. Taking into account the assessment of genetic stability, molecular characterization, products and levels of expression, compositional analyses and morphoagronomic studies, no metabolic interaction is expected that might impact on the food safety when single events are stacked in a conventional way. The MON89034xNK603 event is substantial and nutritionally equivalent to its non transgenic counterpart.
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Date of authorization: 31/05/2012
Scope of authorization: Food and feed
Links to the information on the same product in other databases maintained by relevant international organizations, as appropriate. (We recommend providing links to only those databases to which your country has officially contributed.):
Summary of the safety assessment:
Please see decision document weblinks
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Where detection method protocols and appropriate reference material (non-viable, or in certain circumstances, viable) suitable for low-level situation may be obtained:
Relevant links to documents and information prepared by the competent authority responsible for the safety assessment: Principles for the Assessment of Food and Feed derived from GMO in Argentina - Resolution Nº 412
Decision document of food/feed safety assessment of event MON89034xTC1507xNK603
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Authorization expiration date:
E-mail:
mjunco@senasa.gov.ar
Organization/agency name (Full name):
SENASA (National Service for Agrifood Health and Quality)
Contact person name:
Mariano Junco
Website:
Physical full address:
Paseo Colón Avenue 367, 3° floor, City of Buenos Aires
Phone number:
54 11 4121 5276
Fax number:
54 11 4121 5258
Country introduction:
The food risk assessment process of transformation events, as the result of modern biotechnology, is carried out by the National Service for Agrifood Health and Quality (Senasa), regulatory agency depending on the Ministery of Agriculture, Livestock and Fisheries. The Agrifood Quality Directorate of Senasa, is the area responsible for carrying out this task. It has an specific scientific team and the advise of a Technical Advisory Committee composed of experts from different scientific disciplines representing different sectors involved in the production, industrialization, consumption, research and development of genetically modified organisms.
Useful links
Relevant documents
Stacked events:
Argentina hasn't a specific authorization mechanism for food/feed safety assessment for stacked events. In principle, stacked events are assessed like another single event on a case-by-case basis.
Contact details of the competent authority(s) responsible for the safety assessment and the product applicant:
National Service for Agrifood Health and Quality (Senasa) (http://www.senasa.gov.ar)
Brazil
Name of product applicant: Monsanto do Brasil Ltda.
Summary of application:
commercial release of MON 89034 x NK 603 maize, which confers insect resistance and tolerance herbicide
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Date of authorization: 18/11/2010
Scope of authorization: Food and feed
Links to the information on the same product in other databases maintained by relevant international organizations, as appropriate. (We recommend providing links to only those databases to which your country has officially contributed.): Center for Environmental Risk Assessment
Summary of the safety assessment:
The parental MON 89034 has the expression cassettes of genes Cry1A.105 and cry2Ab 2 (derived from Bacillus thuringiensis), which encode the proteins Cry1A.105 and Cry2Ab2, respectively, responsible for insect resistance. The parental NK 603 maize contains two expression cassettes of the cp4 epsps gene (derived from Agrobacterium sp. CP4 strain), with the respective regulatory sequence. The cp4 epsps gene encodes the protein 5-enolpyruvyl-shikimate-3-phosphate synthesis (CP4 EPSPS), conferring tolerance to the herbicide glyphosate. The pyramided parental maize MON 89034 x NK 603 has been extensively tested, and its biosafety been examined in separate cases, both approved by CTNBio. The proteins present in NK 603 x MON89034 maize have a history of safe use by the consumption of MON 89034 maize since 2008, and the NK603 maize since 2000. The MON 89034 x NK 603 maize is approved in Japan, North Korea, the Philippines, Taiwan, and the USA. Concerning the proteins expressed by MON 89034 x NK 603 maize, it is known that Cry proteins accumulate in the cytoplasm and which have selective toxicity for some species of lepidopteran insects, and their mechanism of action mediated by specific receptors on target organisms. The Cry1A.105 protein and Cry2Ab2 protein bind to these receptors located in the midgut of susceptible insects, leading to form pores that cause insect death. The EPSPS protein, accumulated in the chloroplast, catalyses a step in the shikimic acid pathway for biosynthesis of aromatic amino acids, being so essential to normal growth in plants and micro-organisms. The mechanism for glyphosate action is by forming a complex with the EPSPS enzyme, which regards to the natural substrate binding for the enzyme, blocking the biosynthetic pathway. The CP4 EPSPS enzyme is present in the MON 89034 x NK 603 maize, has low affinity for glyphosate compared to wild EPSPS proteins. Thus, when the MON 89034 x NK603 maize is treated with glyphosate, the CP4 EPSPS enzyme activity causes the plants to continue developing normally. In summary, the proteins expressed by transgene cp4 epsps and cry (and Cry1A.105 cry2Ab2) in MON 89034 x NK603 maize are accumulated in different cellular compartments. They act in different pathways and have different functions and not interactive functions. Studies with the proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS show that these are rapidly digested in simulated gastric and intestinal fluids. This contributes to a low allergenic potential of these proteins, together with the fact they are present in MON 89034 x NK 603 maize in low quantities. Tests for acute oral toxicity and for sub-chronic oral toxicity indicated that the protein in question does not produce adverse effects in mammals. Bioinformatics analysis also demonstrated that the proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS show no similarity in amino acid sequence with known allergenic and toxic proteins. The confirmation of presence and of integrity for DNA sequence introduced into MON 89034 x NK603 maize was carried out by using insert-specific identification by Southern blot analysis. The expression of proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS occurs in all tissues of the plant because the promoters used to promote constitutive expression of these proteins. The expression levels of these proteins were determined in leaves, grain, and fodder, tissues relevant to assess the safety of MON 89034 x NK603 maize as human food and animal feed. The results show comparable levels of expression of proteins in the pyramided event and in their parents, with a low expression of these proteins in the grains and a higher expression in leaves, and were intermediate in fodder. During the analysis of agronomic traits in the phenotypic MON 89034 x NK 603 maize were not identified statistically significant differences concerning control maize for any parameter assessed. It was demonstrated also the effectiveness in controlling target pests and the presence of the trait to tolerate the herbicide glyphosate. Together, their results support the conclusion that, except by the specific characteristics of each introduced gene, the phenotype of GM maize has not changed and therefore the MON 89034 x NK 603 maize has no greater potential than conventional maize to become a weed. Chemical composition analyses were performed in grains and fodder of MON 89034 x NK 603 maize, comparing it to conventional control variety, which has similar genetic base, and seven varieties of conventional maize referenda. The samples used were generated in three locations, representative of the area of maize crop in Brazil, during the 2007/2008 harvest. All values of chemical composition on fodder and MON 89034 x NK 603 maize grains were within the range of values for isogenic control maize, or of commercial values found in the database of the composition of the ILSI-CCD. With this, we can say that the MON 89034 x NK 603 maize is substantially equivalent to conventional maize and therefore as safe, healthy, and nutritious as conventional maize. Besides the data provided by the company, CTNBio consulted independent scientific literature to assess the safety and the occurrence of any unexpected effect from the cross between these events. Given the above, it is concluded that the cultivation and consumption of MON 89034 x NK 603 maize is not potentially causer of significant environmental degradation or risk to human and animal health. For this reason, there are no restrictions on the use of maize and its derivatives. CTNBio determines that the monitoring post-commercial release should be conducted in commercial fields and not in experimental fields. The areas chosen to be monitored should not be isolated from the others, have borders or any situation that is out of business standard. Monitoring should be carried out in model comparison between the conventional system of cultivation and cropping system of GMOs, and the data collection done by sampling. Monitoring should be conducted in representative biomes of the main areas of cultivation of GMOs and, where possible, involve different types of producers. The monitoring should be conducted for at least five years. The reports presented should be detailed information about all activities in the pre-planting and planting on their implementation, with reports of activities conducted in the areas of monitoring during the crop cycle, about the activities of harvest and weather conditions. There should be monitoring of any injuries to human and animal health systems through the official notification of adverse effects, such as the SINEPS System (Adverse Event Reporting Related to Health Products) regulated by ANVISA. The analytical methods, results, and their interpretations must be developed in accordance with the principles of independence and transparency, subject to commercial confidentiality issues previously defined and justified as such. With regard to the gene cp4 epsps, which confers resistance to the herbicide, should be monitored: the nutritional status and health of GM plants, the chemical and physical attributes related to soil fertility and other basic soil characteristics, soil microbial diversity; the soil diaspore bank, the weed community, the development of herbicide resistance in weeds, the herbicide residues in soil, in grain and in aerial parts, and the gene flow. With respect to the genes Cry1A.105 and cry2Ab2, which confer resistance to insects, should be monitored: the impact on the target insects and on non-target insects, the impact on soil invertebrates of indicators, not belonging to the class Insecta, the residues of insecticidal proteins in decomposing organic matter, soil and waterways near the area of monitoring, the development of resistance among target insects and the gene flow of the two inserted genes. TECHNICAL REPORT I. Identification of GMOs Name of GMO: MON89034 x NK603 maize Applicant: Monsanto do Brasil Ltda. Species: Zea mays L. Inserted feature: Tolerance to glyphosate herbicide and insect resistance Feature input method: Classified as Risk Class I, the MON 89034 x NK603 maize was developed through classical genetic improvement by sexual reproduction between GM maize lines, containing NK603 event and MON89034 event. Proposed use: cultivation, animal and human consumption, handling, transport, disposal, import and export, and any other activities related to the maize and its progenies II. General information Zea mays L. maize is a species of the Gramineae, tribe Maydae, Panicoidae subfamily that is separated into the subgenus Zea and has a chromosome number 2n = 20,21,22,24(1). The closest wild species of maize is the teosinte, which is found in Mexico and in parts of Central America, where it can be bred with maize grown in the field of production. Corn has a history of over eight thousand years in the Americas, being cultivated since pre-Columbian times. It is one of the higher plants better characterised scientifically, being today the cultivated species that reached the highest degree of domestication. Such species only survives in nature when cultivated by man(2). There are currently about 300 races of maize identified and, within each race, thousands of cultivars. One of the most important sources of food in the world, maize is the input to produce a wide range of food, feed, and industrial products. Brazil is a leading world producer of maize, and its cultivation is performed practically all over the country(3). The occurrence of insects on Earth is higher in the tropics compared to temperate regions where the damage caused by these animals is more pronounced. Among the most important pests of maize, the fall armyworm, Spodoptera frugiperda, stands out. Cruz et al (4) estimated that losses in Brazil due to the infestation by S. frugiperda were nearly 400 million dollars per year. Other species of Lepidoptera are also important pests of maize, like the corn earworm (Helicoverpa zea) and the Diatraea saccharalis. The main measure of insect control for maize has been the use of insecticides. In some areas of Central Brazil, for example, it takes several spraying with insecticides in a single cycle. Another measure of pest control is the use of resistant cultivars. Compared with conventional maize, MON 89034 x NK603 maize does not present greater capacity to survive with pests. The presence of genes that confer resistance to Lepidopteran insects and tolerance to the herbicide glyphosate confer a selective advantage to MON 89034 x NK603 maize when exposed to the herbicide and the presence of target insects. However, these characteristics are not sufficient for it to become a pest in maize(5,6). The use of maize with pyramided events represents a future trend – which meets the demand of producers – to combine two characteristics of agronomic importance in a single hybrid. Maize with combined events by classical breeding have already been approved in Japan, EU, Brazil, Korea, Mexico, Philippines, South Africa, Taiwan, Argentina, and El Salvador(7). III. Describing GMOs and expressed proteins The MON89034 maize was produced through genetic transformation mediated by Agrobacterium tumefaciens by using the binary plasmid PV-ZMIR245. The T-DNA I contains the genes Cry1A.105 and cry2Ab2, while the T-DNA II contains the nptII gene that confers resistance to the antibiotic kanamycin and was used in the initial selection of transformed cells. Technique of classical genetic improvement was used to isolate plants that contained only the genes of interest Cry1A.105 and cry2Ab2, but would be devoid of npt11 gene, thereby producing plants free of selectable marker and only with the characteristic of resistance to some Lepidoptera pests(8). The gene sequence Cry1A.105 encodes the protein Cry1A.105, which presents insecticidal action on lepidopteran pests of maize. The Cry1A.105 protein is a Cry1A protein (derived from Bacillus thuringiensis) modified amino acid sequence which is equivalent to the proteins Cry1Ab, Cry1Ac, and Cry1F in 90.0%, 93.6%, and 76.7%, respectively. The coding sequence of the gene cry2Ab2 produces the protein Cry2Ab2 that is member of the proteins Cry2Ab with which it has in common more than 95% of the amino acid sequence(9). This is a wild variant protein Cry2Ab2 isolated from B. thuringiensis subsp. kurstaki. The general mechanism of insecticidal activity of Cry proteins is well understood (10,11,12,13). It is known that these proteins are able to form crystals containing endotoxins, proteins with insecticidal action that act before and during the sporulation phase of the life cycle of Bacillus thuringiensis. Commercial formulations of B. thuringiensis containing these proteins were used in Brazil and in other countries to control some agricultural pests for over 46 years. The Cry insecticidal proteins are highly selective to target insects of the order Lepidoptera(15,16,17,18,19), which have in their intestine specific receptors for this protein. Mammals and other non-target organisms (including other arthropods, pollinators, natural enemies of pests target) does not possess such binding sites, therefore, are unaffected by the Bt protein(20,21,22,23). The NK603 maize has been produced by biolistics and contains two cassettes of cp4 epsps gene expression (derived from Agrobacterium sp. CP4 strain), with their respective regulatory sequence. Assessments showed that the nucleotide sequence of one of the copies of the cp4 epsps gene differs from the original sequence used in the transformation process in two nucleotides. One of nucleotide exchanges were silent and one resulted in the substitution of one amino acid at position 214. The nucleotide change at position 214 bp encoding resulted in a leucine in place of a proline. The new sequence then became known as cp4 epsps L214p(24). The cp4 epsps gene encoding the CP4 EPSPS protein expression (CP4-5-enolpyruvyl-shikimate-3-phosphate synthase), which confers the trait of tolerance to the herbicide glyphosate. The CP4 EPSPS protein expressed in transgenic plants tolerant to glyphosate is functionally identical to endogenous plant EPSPS protein(25). In conventional plants, because of the strict specificity for substrates, enzymes bind only EPSPS S3P, PEP, and glyphosate. The only known, resulting metabolic product is the acid 5-enolpyruvyl-shikimate-3-phosphate, which is the penultimate product of the shikimic acid pathway. The shikimic acid is a precursor for the biosynthesis of aromatic amino acids (phenylalanine, tyrosine, tryptophan), and many secondary metabolites, such as tetrahydrofolate, ubiquinone, and vitamin K(26). Although the pathway of shikimic acid (or shikimate) and EPSPS proteins do not occur in mammals, fish, birds, reptiles, and insects, they are important for plants. It is estimated that the aromatic molecules, all derived from shikimic acid, represent 35% or more of the dry weight of a plant(27,28). In the presence of glyphosate, herbicide is to link the enzyme EPSPS, which blocks the biosynthesis of 5-enolpyruvyl-shikimate-3-phosphate, preventing the formation of aromatic amino acids and secondary metabolites in conventional plants(29). In genetically modified plants resistant to glyphosate as NK603 maize, the aromatic amino acids and other metabolites required for plant development continue to be produced by the activity of the protein CP4 EPSPS(30). The MON 89034 x NK 603 maize results from the breeding, through classical genetic improvement, from parents of GM MON 89034 and NK603 maize, so that the expression of proteins Cry1A.105 and Cry2Ab2 can be observed (which confer resistance to insects) to the CP4 EPSPS protein (conferring tolerance to glyphosate herbicide) in the pyramided event. The parents of GM MON 603 and NK 89034 maize, which gave rise to the combined event, were previously assessed by CTNBio and released for sale after having been considered as safe to human, to animal health, and to environment as to conventional maize (Technical Reports 2052/2009 and 1596/2008, respectively). In Australia (2008), Canada (2008), Japan (2007/2008), Korea (2009), Philippines (2009), EU (2009), and USA (2007 ), the MON89034 maize is also approved for human and animal consumption. By contrast, the NK603 maize is already approved for human and animal consumption in Argentina (2004), China (2005), Colombia (2007), El Salvador (2009), Mexico (2002), EU (2004), Japan (2001), Korea (2002/2004), and USA (2000)(7). Until the time we do not know the existence of adverse effects of NK603 maize and MON89034 maize. The MON 89034 x NK603 pyramided event has released for human consumption and/or animal consumption in Japan (2008), Korea (2009/2010), Mexico (2010), Philippines (2009) and Taiwan (2Q09) (7), also with no reports of adverse effects up till now. IV. Aspects related to human and animal health While there is a history of safe use of parental maize events NK603 (Technical Opinion No 1.596/2008), and MON 89034 (Technical Opinion No 2.052/2009) one of the concerns raised about the pyramided events regards to the potential side effects derived from unanticipated interactions between the expressed gene products. However, the action mode and biological activities of proteins CP4 EPSPS and Cry expressed in MON 89034 x NK603 maize are distinct and have no known mechanisms of interaction that could cause adverse effects to human and animal health nor the environment. The proteins CP4 EPSPS and Cry present in MON 89034 x NK603 maize are accumulated in different cellular compartments and have different metabolic and non-interactive functions, and the CP4 EPSPS protein directed to the chloroplast as proteins Cry1A. 105 and Cry2Ab2 are accumulated in the cytoplasm(5,6). Thus, considering the metabolic pathways involved, as well as the similarity of the phenotypic characteristics observed in pyramided maize, compared to their parental generation, is not expected to occur interference of a gene on the phenotype of the other. The Biosafety feed of MON 89034 x NK 603 maize was determined through various studies(31), which evaluated different aspects, and the summary of results presented below. One of the aspects assessed to determine the safety of MON 89034 x NK 603 pyramided maize was its compositional equivalence with respect to a conventional control variety with similar genetic base, and seven varieties of conventional corn referenda. The chemical composition analysis for the variables ash, carbohydrates, fats, moisture, and protein was performed on samples of seeds and fodder from cultivated plants in three locations in Brazil (Rolandia/PR, Não-Me-Toque/RS, Cachoeira Dourada/MG) in the 2007/2008 harvest. The values of the referenda used were ILSI Crop Composition Database(32). Mean individual values of composition for ash, carbohydrates, fats, moisture, and protein obtained from MON 89034 x NK 603 maize fodder mostly remained within the ranges of the isogenic control maize. In cases where the mean values were not within the range for the isogenic control, they were within the range for commercial references in the combined analysis of three locations. All average values for the proximate components in MON 89034 x NK603 fodder maize were within the ranges established in the database ILSI-CCD. Similar results were observed with respect to data of chemical composition in MON 89034 x NK 603 maize. Only the mean value of carbohydrates in Cachoeira Dourada/MG was outside the range of values of the references in the combined analysis of locations, yet this within the range of values found in the ILSI-CCD, so that it can be stated that this component has common value to population of hybrid maize in trade, what is important in terms of food security. All other mean values for the components in centesimal grains of MON 89034 x NK603 maize were also within the ranges established by the values found in the database ILSI-CCD. Based on data on the composition of grain and fodder produced in Brazil, we can concluded that MON 89034 x NK603 maize is substantially equivalent to conventional maize and therefore is as safe and healthy and nutritious as conventional maize. The food security assessments for proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS included the characterisation of each oriented ideas, with trials of simulated digestion in gastric and intestinal fluids, acute oral toxicity studies in mice and assessment of bioinformatics. One of the important parameters to determine whether a protein can become an allergen is its stability in the gastrointestinal system, because an immune response can be initiated if the protein will survive this digestion and reach the small intestinal mucosa(33). In tests of digestion in simulated gastric and intestinal fluids, proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS were rapidly degraded. Additionally, foreign proteins produced in the pyramid are present at concentrations below those recommended to develop an allergic reaction(34,35). Bioinformatics analyses showed that the proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS show no similarity in amino acid sequence with known allergens and toxic proteins (36~44). Additionally, acute oral toxicity tests in mice with the proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS purified and sub-chronic toxicity tests, with these proteins being administered in doses substantially above the magnitude of the doses found in the normal consumption of maize, showed they do not produce adverse effects(45,46,47,48) and therefore are not an issue of food safety for animals and humans. How proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS produced no toxicity at maximum doses tested in these studies mentioned above, is highly unlikely to interact among these proteins in normal doses found in food, which could cause synergistic or additive effects. There are plenty of information in the literature in the area of toxicology of chemical mixtures that demonstrate that such interactions are absent where the substances are administered at doses substantially below the levels of no observed adverse effect level (NOAEL = no observed adverse effect level) (49.50,51,52). Security for the feed and food of the Cry proteins was confirmed by several authors. Xu et al(53) observed that the Cry1Ab/Ac protein was rapidly degraded in gastric and intestinal fluids, and showed no adverse effects in mice subjected to an acute dose of 5g (Cry1Ab/Ac protein)/kg of body weight. Again it was observed that this protein has no sequence homology with known toxins or allergens, or sites of N-linked glycosylation, confirming that no harm will result from inclusion of protein Cry1Ab/Ac in food or animal feed. Another recent twenty-eight-day study in rats conducted by Onose et al(54) showed no adverse effects can be attributed to diet containing Cry1Ab, whereas the administration of diet containing Cry1Ab protein had no significant effect on any physiological or biochemical parameter, except a lower level of AST in the serum of animals fed such maize, when compared with control. However, no changes in organ weights or histopathological changes were observed in organs like heart, liver, and kidneys. Also, is usually observed that serum AST levels are elevated with tissue injury, but the interpretation of relatively small changes in AST in toxicology studies should be done sparingly, since the range of variation of this parameter can be broad in healthy animals. The decrease of AST in this experiment, therefore, is not considered to be toxicologically significant. Healy et al(55) presented the results of a study of thirteen weeks of feeding rats with pyramided maize grains, which express CryBb1 and CP4 EPSPS. The responses for rats fed diets containing this variety of maize were comparable to those of rats fed a diet containing grains of its nearly isogenic control variety, confirming that such pyramided maize is as safe and nutritious as grain from existing commercial maize hybrids. Additionally, Paul and colleagues(56) studied the degradation of Cry1Ab protein in GM maize regarding total protein in the digestion of dairy cows. The results indicated that Cry1Ab is increasingly degraded during digestion in these animals in small fragments of 42kDa, 34kDa, and 17kDa. Also, skin tests with Cry1Ab in 27 children with a history of inhalant allergy and 50 patients with asthma/rhinitis(57) demonstrated that the events in genetically modified organisms are safe from potential allergenicity to humans. Another study of Japanese patients with food allergy did not detect significant levels of IgE specific against the proteins CP4 EPSPS or Cry9C in the serum of these patients by ELISA(58). Accordingly, it can also cite a study published by Taylor et al(59) which describes that both chickens fed with pyramided maize as the non-transgenic control showed the same growth characteristics and meat quality, indicating the nutritional equivalence of MON89034 x NK603 maize. It is also important to note that the safety of Cry protein family has been established since when preparations of whole Bacillus thuhngiensis were approved for direct application to crops as a method of biological control of harmful insects, much safer for non-target insects, for environment and for humans and animals than the conventional chemical insecticides. Being completely safe for vertebrates (tomatoes so treated need not be washed before marketing) and allowed its use for direct application in preparations containing up to four different strains of bacilli to the action spectrum be wider on the many existing pests. So we have example of pyramiding Bt proteins by direct application, without considering possible toxic or allergenic potential of other proteins and chemical components of the bacillus. Moreover, there is no personal injury or environmental reported by some users of these formulations organic farmer. Modern biology has allowed technology to simplify this by equipping maize with genes that express the insecticidal proteins endogenously, the combination of distinct proteins broadening the action spectrum of effectiveness and on (insect) pests. Another benefit of technology is the quality of maize obtained since, as a result from its lower insect infestation, it ends up being less affected by fungi, so that shows appreciable reduction in highly toxic mycotoxins, such as fumonisin(60). V. Environmental aspects Molecular characterisation of the MON 89034 x NK 603 pyramided event included the confirmation of the stability of transferred genes by cross-fertilisation for the MON 89034 x NK603 event, demonstrated by identification of insert-specific Southern blot analysis. The presence of hybridisation signals of the expected size in pyramided maize when compared to individual events showed that the structure of genes Cry1A.105, cry2Ab2, and cp4 epsps is preserved in the combined event, which confirms the integrity of the event MON 89034 x NK603. In addition to the molecular characterisation of MON 89034 x NK 603 maize were assessed for expression of heterologous proteins. The expression of proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS occurs in all tissues of the plant because the promoters used in genetic constructs promote its constitutive expression. Data of the proteins Cry1A.105, Cry2Ab2 and CP4 EPSPS were obtained in experiments with leaves, grain, and fodder, made in Brazil (2007/2008) at three different locations (Rolândia/PR; Não-Me-Toque/RS, and Cachoeira Dourada/MG), representative of the maize production in the country. These tissues are relevant for assessing the food security of MON 89034 x NK603 maize as human food and animal feed. The levels of these proteins were also introduced in samples of leaves, seeds, roots, grass, and pollen from maize plants grown in five locations in Argentina. The samples were assessed by ELISA (enzyme-linked immunosorbent assay) using polyclonal antibodies, specific for Cry1A.105 protein and monoclonal antibodies for Cry2Ab2 and CP4 EPSPS. The results of experiments conducted in Brazil showed no substantial variation in levels of protein expression and Cry1A.105 protein and Cry2Ab2 protein between NK 603 x MON 89034 maize and MON 89034 event unique in the tissues studied. Although levels of CP4 EPSPS protein were also determined in experiments conducted in Brazil, the comparison of expression levels between the MON 89034 x NK 603 pyramided event and NK 603 individual event was determined in experiments conducted in five localities of Argentina. Such trials also showed no variation in the expression of CP4 EPSPS protein between the pyramid and parental NK 603 maize event. We can conclude that from this set of analysis that the levels of expression of heterologous proteins in GM MON 89034 x NK 603 maize plants is comparable to that observed in their parents, with a low expression of these proteins in the grains and a higher expression in leaves, and intermediate in the pasture. The results from molecular characterisation of MON 89034 x NK603 maize and expression of proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS presented show that the inserts of the events MON 89034 and NK603 are present and functional in the combined product, the MON 89034 x NK603 maize. Field studies were conducted at four locations in the 2007/2008 season (Rolândia/PR, Cachoeira Dourada/MG, Sorriso/MT, and Não-Me-Toque/RS) in order to compare the MON 89034 x NK603 maize to the conventional control maize and to trade references regarding its vitality, initial and final stand, 50% of plants with tassel exposed, 50% of plants with pollen, plant height, ear height, physiological maturity, stay green, yield grain, 1000 grain weight, and hectolitre weight. In the same location, in the 2008/2009 harvest, we assessed the same agronomic and phenotypic features but now comparing MON 89034 x NK603 maize without the application of glyphosate and the MON 89034 x NK603 maize with glyphosate application to conventional control maize and to trade references. No statistically significant differences were detected between MON 89034 x NK603 maize and control maize in the 2007/2008 harvest and between the tested maize (MON 89034 maize, MON 89034 x NK603 maize [without glyphosate], and MON 89034 x NK603 maize [with glyphosate]) and control maize in the 2008/2009 harvest for any of the assessed phenotypic and agronomic characteristics, indicating that the phenotypic characteristics of MON 89034 x NK603 maize were typical of maize grown in Brazil, and, therefore, the potential for MON 89034 x NK603 maize to behave in a weed and is considered negligible when compared to conventional control maize. The data also show the presence of the characteristic of glyphosate tolerance conferred by expression of CP4 EPSPS protein. It also indicates that the trait of tolerance to glyphosate did not significantly alter plant performance compared to conventional material in both conditions, without and with glyphosate glyphosate to control weeds. The absence of characteristics in discrepant pyramided event indicates that management used in conventional maize is also suitable for pyramided maize, preventing the expression of resistance proteins to represent a drain on the primary metabolism of plants, which would limit their agronomic performance(61). Along with the study of agronomic traits performed in Rolandia/PR, Cachoeira Dourada/MG, Sorriso/MT Não-Me-Toque/RS, in the 2008/2009 harvest and were also assessed the damage caused by lepidopteran pests. The results of harm caused by lepidoptera pests on leaves, ear, and stem in MON 89034 maize, MON 89034 x NK603 maize (without glyphosate), and MON 89034 x NK603 maize (with glyphosate) were significantly lower compared to the results of the control plants (conventional control and commercial references). For other features such as rot stems or ears the results are lower, but not significantly lower. The notes for rot ear and stem denote the smallest lepidopteran pest attack genetically modified plants may be delaying the entry of fungi, but not preventing. This is important in food safety, as previously mentioned, since these fungi are responsible for the production of mycotoxins. Arthropod collections held in the four regions showed no significant differences among the treatments for several organisms belonging to various orders including Orthoptera (Gryllidae), Homoptera (Cicadellidae), Diptera (Cyclorrapha) Dermoptera, Hymenoptera (Formicidae, Apidae), Coleoptera (Elateridae), and Araneae. Based on these results and existing knowledge about the proteins Cry1A.105 protein and Cry2Ab2 protein (62,63), it is confirmed to maintain the selectivity of the insecticidal activity of the pyramided event against target insects of lepidoptera types, and the lack of effect on insects is non-target assessed. The number of volunteers plants did not differ between the treatments was assessed in different locations. Likewise, no statistical differences were found for plant height in yield and in length of pollen formation. Soil samples collected in the field treatment showed average values (pH, Ca, Mg, P, Al, clay, silt, sand) within the ranges of referenda for maize, and there were no outliers among the treatments within each region assessed. Together, the assessments of agronomic data results on control of damage caused by lepidoptera pests demonstrate the presence of the characteristic of glyphosate tolerance and that this did not significantly alter target plant performance for insect control regarding conventional material in the two conditions, without glyphosate and with glyphosate. These data show that there is no interaction between the Cry proteins (Cry1A.105 and Cry2Ab2) and CP4 EPSPS that in any way will interfere in the resistance of MON 89034 x NK603 maize to target lepidopteran. Therefore, the efficacy in controlling target pests and the presence of the glyphosate tolerance trait in MON 89034 x NK603 maize is attested. The assessment of these characteristics enables us to state that MON 89034 maize, MON 89034 x NK603 maize (with or without glyphosate), and control maize are substantially equivalent, except for the specific characteristics of each introduced gene. Another issue of environmental nature referred to gene flow from GM maize and the effects this might have on conventional maize. The possibility that there is cross-pollination between a GM plant and other conventional, followed by introgression, is correlated with the availability and viability of the GM parental pollen and delivery of such pollen on the stigma of conventional parentage. This will depend on availability of planting and agronomic conditions, while the supply of pollen on the stigma depends on wind, vector, distance, precipitation, and natural barriers to movement of pollen. Thus, the efficiency of cross-pollination will depend, in parallel, on the time of flowering of parental receiver and of parental donor, of pollen viability, and of competitive ability of pollen. One must also consider that the pollen grains of maize are large and heavy, and it reduces the distances of dispersal, considering that the highest deposition occurs near the donor plant (64,65). The pollen dispersal is 98% and occurs up to 25 metres from the field emitter and nearly 100% up to 100 feet away, and most (99%) of cross-pollination occurs outside the field emitter up to 18 to 20 meters of its edges(66). The weather conditions (and wind direction) and physical barriers affect the pollen dispersion and the maize cross-pollination rate, and closer barriers are more efficient. The pollen dispersal of MON 89034 x NK603 maize can therefore be controlled so that the coexistence of conventional crops, organic, and GM should be possible(66), as it is naturally done and when genotypes for different uses (seed, food, creole breeds, etc.) are produced in continuous areas. Finally, maize is an exotic species, without wild sexual parentage that are compatible in Brazil. It has a high degree of domestication, without scientific reasons to predict the survival of GM and non-GM plants outside the agricultural environment. Moreover, in the absence of selective pressure (use of herbicides and insecticides), the expression of the inserted genes do not confer adaptive advantage. VI. Restrictions on the use of GMOs and their derivatives As established in art. 1 of Law 11,460, of 21 March 2007, 'the research and cultivation of GM organisms on indigenous lands and areas of conservation is banned'. The studies submitted by the applicant revealed no significant differences between the GM maize and its conventional isoline compared with the agronomic characteristics, mode of reproduction, dissemination, or survivability. All evidence presented in the proceedings and references confirm the risk level of the transgenic variety as equivalent to non-transgenic varieties compared to soil microbes, as well as to other vegetable and to animal and human health. Thus, the cultivation and consumption of MON 89034 x NK 603 maize are not potentially causer of significant environmental degradation or risk to human and animal health. For these reasons, there are no restrictions on the use of maize and its derivatives, except in places covered by Law 11,460, of 21 March 2007. After 15 years of use in several countries, no problem was detected for human and animal health or the environment that may be attributed to the transgenic maize. It is necessary to emphasise that the lack of negative effects resulting from cultivation of transgenic maize does not mean they cannot happen. Zero risk and absolute security does not exist in the biological world, although there is an accumulation of trustworthy scientific information and a history of safe use of GM crops in agriculture. VII. Considerations about the particularities of different regions of the country (subsidies to monitoring agencies) As established in art. 1 of Law 11,460, of 21 March 2007, 'the research and cultivation of GM organisms on indigenous lands and areas of conservation is banned'. VIII. Conclusion Whereas the variety of MON 89034 x NK603 maize (Zea mays) belongs to well characterised species and solid safety record for human consumption and that genes Cry1A.105, cry2Ab2, and cp4 epsps introduced in this variety encode proteins ubiquitous in nature, plants, fungi and micro-organisms that are part of the diet of humans and animals; Whereas the construction of this genotype occurred through classical genetic improvement, which resulted in the inheritance of a stable and functional copies of genes Cry1A.105, cry2Ab2, and cp4 epsps, which provided resistance to insects and tolerance to glyphosate; Whereas composition data showed no significant differences among GM and conventional varieties, suggesting the nutritional equivalence among them; Whereas CTNBio assessed the events separately and gave its assent to its commercial release; Considering also that: 1. The MON 89034 x NK 603 event has been well characterised molecularly, and have been attested to maintain the integrity of the gene constructs inherited from their parents during the process of classical genetic improvement; 2. There is no evidence of interaction among the metabolic pathways that act on proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS; 3. Epistatic or pleiotropic effects on parenting and events together were not identified; 4. The expression of proteins Cry1A.105, Cry2Ab2, and CP4 EPSPS in MON 89034 x NK603 maize is not significantly different from the expression observed in parental events separately; 5. There is no evidence that the expressed proteins to cause allergy or poisoning in humans and animals; 6. Evaluations and agronomic effectiveness of MON 89034 x NK603 maize showed that combining these events by methods of classical genetic improvement (sexual reproduction) did not lead to expression of any other characteristic than that expected, i.e. resistance to certain insects and tolerance to glyphosate; 7. There were no botanical changes in MON 89034 x NK 603 maize that may confer adaptive advantages; 8. Internationally accepted criteria in the process of risk analysis of GM raw materials regarding events pyramided (67); 9. The other risk assessments carried out by countries that have assessed the MON 89034 x NK603 pyramided maize(62.68). And it may be concluded that MON 89034 x NK603 maize is as safe as its conventional equivalent. Within the scope of the powers conferred by art. 14 of Law 11,105/05, CTNBio considered that the application meets the standards and existing laws designed to ensure the biosafety of the environment, agriculture, human, and animal health, and concluded that the MON 89034 x NK 603 maize and substantially equivalent to conventional maize, as being its safe use for human and animal health. Concerning the environment, CTNBio concluded that MON 89034 x NK 603 maize is not a potential cause of significant degradation of the environment, saving with the biota similar relationship to conventional maize. CTNBio considers that this activity is not potentially significant degradation cause of the environment or of harm to human and animal health. Restrictions on the use of GMO in question and its derivatives are subject to the provisions of Law 11,460, of 21 March 2007. Regarding the monitoring plan for post-commercial release, CTNBio determines that instructions should be followed and techniques should be implemented to monitor the actions below: I) – Instructions a) Monitoring should be conducted in commercial fields and not in experimental fields. The areas chosen to be monitored should not be isolated from the others, have borders or any situation that is out of business standard. b) Monitoring should be carried out in model comparison between the conventional system of cultivation and cropping system of GMOs, and the data collection done by sampling. c) Monitoring should be conducted in representative biomes of the main areas of cultivation of GMOs and, where possible, involve different types of producers. d) The monitoring should be conducted for at least five years. e) For all monitoring, the proponent should detail information on all activities performed in the pre-planting and planting, on their implementation, with reports of activities conducted in the areas of monitoring during the crop cycle, about harvest activities, and weather conditions. f) There should also be monitoring of any injuries to human and animal health systems through the official notification of adverse effects, such as the SINEPS System (Adverse Event Reporting Related to Health Products) regulated by ANVISA. g) The analytical methods, results, and their interpretations must be developed in accordance with the principles of independence and transparency, subject to commercial confidentiality issues previously defined and justified as such. h) Based on scientific and technical justifications, CTNBio reserves the right to revise this opinion at any time. II) – Monitoring technique actions to be performed: 1 – Regarding the cp4 epsps gene, which confers resistance to the herbicide must be monitored: a) Nutritional status and health of GM plants. b) Chemical and physical attributes related to soil fertility and other basic soil characteristics. c) Soil microbial diversity. d) Bank of seeds in the soil. e) Community weed. f) Development of herbicide resistance in weeds. g) Glyphosate residues in soil, in grain, and in aerial parts. h) Gene flow. 2 – With respect to genes cry1A. 105 and cry2Ab2, that confer resistance to insects must be monitored: a) Impact on target insects and non-target insects. b) Impact on soil invertebrates that are indicators, belonging to the class Insecta. c) Residues of insecticidal proteins in decomposing organic matter, in soil and in waterways near the monitoring area. d) Development of resistance among target insects. e) Gene flow of the two inserted genes. The assessment of CTNBio considered the opinions expressed by members of the Commission, contributed documents in the Executive Secretariat of CTNBio by the applicant and results of planned releases into the environment. Independent scientific studies and publications regardless of what was required, made by third parties, were also considered and consulted. IX. Bibliographic References 1. Food and Agriculture Organization of the United Nations / World Health Organization. FAO/WHO - 2000a. Grassland Index. Zea mays L. (Source:http://www.fao.org/WAICENT/faoinfo/aaricult/aQp/agpc/doc/qbase/data/pf000342. htm). 2. BAHIA FILHO, A.F.C.; GARCIA, J.C. 2000. Análise e avaliação do mercado brasileiro de sementes de milho. In: UDRY, C.V.; DUARTE, W.F. (Org.) Uma história brasileira do milho: o valor de recursos genéticos. Brasília: Paralelo 15, 167-172. 3. Companhia Nacional de Abastecimento - CONAB. 2007. Milho total (la e 2a safra) Brasil - Série histórica de área plantada: safra 1976-77 a 2009-10. http://www.conab.aov,br/QlalaCMS/uploads/arquivos/10 11 12 14 45 40 milhot otalseriehist.xls. 4. CRUZ, I.; FIGUEIREDO, M.L.C.; OLIVEIRA, A.C.; VASCONCELOS, C.A. 1999. Damage of Spodoptera frugiperda (Smith) in different maize genotypes cultivated in soil under three levels of aluminium saturation. International Journal of Pest Management 45:293-296. 5. Comissão Técnica Nacional de Biossegurança. CTNBio 2009. Parecer Técnico 2052/2009. Publicado no Diário Oficial da União de 16/10/2009, Seção 1, pag. 3. 6. Comissão Técnica Nacional de Biossegurança. CTNBio 2008. Parecer Técnico 1596/2008. Publicado no Diário Oficial da União de 14/10/2008, Seção 1, pag. 3. 7. Center for Enviromental Risk Assessment. GM Crop Database 2010 (http://cera- qmc.orq/index.php?action=qm crop database). 8. Monsanto do Brasil. 2008. Relatório Técnico Liberação Comercial Milho MON 89034. Crickmore, N., D.R. Zeigler, J. Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, e D,H. Dean, 1998. Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol Mol Biol Rev 62:807-13. 9. Gill, S.S., E.A. Cowles, e P.V. Pietrantonio. 1992. The mode of action of Bacillus thuringiensis endotoxins. Ann. Rev. Entomol. 37:615-636. 10. Schnepf, E., N. Crickmore, J. Van Rie, D. Lereclus, J, Baum, J. Feitelson, D.R. Zeigler, e D.H. Dean. 1998. Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Mol. Biol. Rev. 62:775-806. 11. Zhuang, M., e S.S. Gill. 2003. Mode of action of Bacillus thuringiensis toxins., p. Pp 213-236, In G. Voss and G. Ramos, eds. Chemistry of Crop Protection, Progress and Prospects in Science and Regulation. Wiley-VCH, Weinheim, Germany 12. Griffitts and Aroian, 2005 J. Griffitts and R. Aroian, Many roads to resistance: how invertebrates adapt to Bt toxins, BioEssays 27 (2005), pp. 614-624. 13. Shimada, N., Miyamoto, K., Kanda, K., Murata, H., 2006a, Bacillus thuringiensis insecticidal Cry1Ab toxin does not affect the membrane integrity of the mammalian intestinal epithelial cells: an in vitro study. In vitro Cellular and Developmental Biology - Animal, 42: 45-49. 14. DULMAGE, H. T. Microbial control of pests and plant diseases 1970 - 1980. In: BURGES, H. D. (Ed). London: Academic Press, 1981. p. 193-222. 15. KLAUSNER, A. Microbial insect control. Bio/Technology, v. 2, p. 408-419, 1984. 16. ARONSON, A. I.; BACKMAN, W.; DUNN, P. Bacillus thuringiensis and related insect pathogens. Microbiol. Rev., v. 50, p. 1-24, 1986. 17. MACINTOSH, S. C; STONE, T. B,; SIMS, S. R.; HUNST, P.; GREENPLATE, J. T.; MARRONE, P. G.; PERLAK, F. J.; FISCHHOFF, D. A.; FUCHS, R. L. Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects. J, Insect Path., v. 56, p. 258-266, 1990. 18. WHITELEY, H. R.; SCHNEPF, H. E. The molecular biology of parasporal crystal body formation in Bacillus thuringiensis. Ann. Rev. Microbiol., v. 40, p. 549-576, 1986. 19. CANTWELL, G. E.; LEHNERT, T.; FOWLER, J. Are biological insecticides harmful to the honey bee. Am. Bee J., v. 112, p. 294-296, 1972. 20. KRIEG, A.; LANGENBRUCH, G. A. Susceptibility of arthropod species to Bacillus thuringiensis. In: Microbial Control of Pests and Plant Diseases. BURGES, H. D. (Ed). London: Academic Press, 1981. p. 837-896. 21. FLEXNER, J. L; LIGHTHART, B.; CROFT, B. A. The effects of microbial pesticides on non-target beneficial arthropods. Agric. Ecosys. Environ., v. 16, p. 203-254, 1986. 22. UNITED STATES ENVIRONMENTAL PROTECTION AGENCY. Guidance for the re-registration of pesticide products containing Bacillus thuringiensis as the active ingredient. Springfield, VA.: US EPA/National Technical Information Service, 1988. v. 89, p. 164-198. 23. Monsanto do Brasil Ltda. 2004. Avaliação de Biossegurança do Milho NK 603 tolerante ao glifosato. Processo: 01200.002293/2004-16. 24. Padgette, SR., G.F. Barry, D.B. Re, D.A. Eichholtz, M. Weldon, K. Kolacz, e G.M. Kishore. 1993. Purification, cloning and characterization of a highly glyphosate-tolerant 5-enolpyruvylshikimate- 3-phosphate synthase from Agrobacterium sp. strain CP4. Monsanto Technical Report MSL 12738. 25. TAYLOR, M.L.; HARTNELL, G.; NEMETH, M.; KARUNANANDAA, K.; GEORGE, B. 2005. Comparison of broiler performance when fed diets containing corn grain with insect-protected (corn rootworm and European corn borer) and herbicide-tolerant (glyphosate) traits, control corn, or commercial reference corn—revisited. Poult. Sci. 84: 1893-1899. 26. TAN, S.; EVANS, R.; SINGH, B. 2006. Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops. Amino Acids 30: 195-204. 27. SILVA-WERNECK, J.O.; SOUZA, M.T.; DIAS, J.M.C.S.; RIBEIRO, B.M. 1999. Characterization of Bacillus thuringiensis subsp. kurstaki strain S93 effective against the fall armyworm (Spodoptera frugiperda). Canadian Journal of Microbiology 45: 464-471. 28. Haslam, E. 1993. Shikimic acid: metabolism and metabolites. University of Sheffield, UK; 29. Steinrucken, H.C.; Amrhein, N. 1980. The herbicide glyphosate is a potent inhibitor of 5enolpyruvyl- shikimic acid-3-phosphate synthase. Biochem Biophys Res Commun 94:1207-1212. Monsanto do Brasil Ltda. 2009. Relatório de Biossegurança Ambiental e Alimentar do Milho MON 89034 x NK603. Processo: 01200.003952/2009-38. 30. International Life Sciences Institute (ILSI) 2006. Crop Compositions Database, Version 3.0. http://www.cropcomposition.org. Search criteria: corn seed or corn forage, all locations, all years, all proximates dry weight, other than moisture. Acessado em 17 de fevereiro de 2009. 31. Kimber, I., N.I. Kerkvliet, S.L. Taylor, ID. Astwood, K. Sarlo, e R.J. Dearman. 1999. Toxicology of protein allergenicity: prediction and characterization. Toxicol Sci 48:157-62. 32. Fuchs, R.L. 1996a. Allergenicity assessment of foods derived from genetically modified plants. Food technology 50:83-88. 33. Fuchs, R.L. 1996b. Assessment of the allergenic potential of foods derived from genetically engineered plants: glyphosate tolerant soybean as a case study. DFG: Food allergies and intolerances Chapter 17:212-221. 34. Astwood, J.D., e R.L. Fuchs. 1996a. Allergenicity of Foods Derived from Transgenic Crops. Monographs in Allergy: 105-120. 35. Astwood, J.D., R.L. Fuchs, e P.B. Lavrik. 1996b. Food biotechnology and genetic engineering, second edition ed., St Louis 36. Metcalfe, D., J. Astwood, T. R., S. H., T.M. L, e F. R. 1996. Assessment of the allergenic potential of foods derived from genetically engineered crop plants. Critical Reviews in Food Science and Nutrition 36:165-186. 37. Astwood, J.D. 1995a. Bacillus thuringiensis subsp. kurstaki HD-1 insecticidal protein (Btk HD-1 protein) is homologous to proteins of the Bacillus thuringiensis insecticidal crystal protein gene family, but not to protein toxins found in public domain sequence databases. Monsanto Technical Report MSL 14283. 38. Astwood, J.D. 1995b. Bacillus thuringiensis subsp. kurstaki HD-1 insecticidal protein (Btk HD-1 protein) shares no significant sequence similarity with proteins associated with allergy or coeliac disease. Monsanto Technical Report MSL 14172. 39. Croon, K.A., R.S. Sidhu, e C. Deatherage. 2000a. Safety, Compositional and nutritional aspects of Roundup Ready corn line NK603. Conclusions based on studies and information evaluated according to FDA's policy on foods from plant varieties (FDA - Food and Feed Safety). (February/2000). Monsanto Company, St. Louis, MO, USA 40. Croon, K.A., R.S. Sidhu, e C. Deatherage. 2000b. Request for extension of determination of nonregulated status for Roundup Ready corn line NK603 (USDA -Nonregulated Status). (January/2000). Monsanto Company, St. Louis, MO, USA. 41. Croon, K.A., T.G.A. Clemence, R.S. Sidhu, C. Deatherage, L.K. Lahman, E. Jácobs, e J. Costa. 2001. Application for consent to place on the market NK603 Roundup Ready® maize for import and use as any other maize, including the cultivation of varieties, in the European Union. Monsanto Company represented by Monsanto Europe S.A., Brussels, Belgium 42. Rice, E.A., R.E. Goodman, A. Silvanovich, R.E. Hieman, e J.D. Astwood. 2001. Bioinformatic analysis of the CP4 EPSPS protein utilizing ALLERGEN3 and current public domain sequence databases. Monsanto Technical Report MSL 17172. 43. McClintock, J.T., R.D. Sjoblad, e R. Engler. 1992. Toxicological evaluation of genetically engineered plant pesticides. Food Safety Assessment, ACS Symposium Series 484:41-47. 44. Mcclintock, J.T., C.R. Schaffer, e R.D. Sjoblad. 1995a. A Comparative Review of the Mammalian Toxicity of Bacillus Thuringiensis-Based Pesticides. Pesticide Science 45:95-105 45. Pariza, M.W., e E.A. Johnson. 2001. Evaluating the safety of microbial enzyme preparations used in food processing: update for a new century. Regul Toxicol Pharmacol 33:173-86. 46. Harrison, L, M. Bailey, M. Taylor, J. Ream, B.G. Hammond, D. Nida, e B. Burnette. 1996. The expressed protein in glyphosate-tolerant soybean, 5-enolypyryvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice. Journal of Nutrition 126:728-740. 47. Groten, J.P., E.D. Schoen, P.J. Van Bladeren, C.F. Kuiper, J.A. van Zorge, e VJ. Feron. 1997. Subacute toxicity of a mixture of nine chemicals in rats: detecting interactive effects with a fractionated two level factorial design. Fundam. Appl. Toxicol. 36:15-29. 48. Jonker, D., R.A. Woutersen, e V.J. Feron. 1996. Toxicity of mixtures of nephrotoxicants with similar or dissimilar mode of action. Food Chem. Toxicol. 34:1075-1082. 49. Jonker, D., R.A. Woutersen, P.J. van Bladeren, H.P. Til, e V.J. Feron. 1990. 4-week oral toxicity of a combination of eight chemicals in rats: comparison with the toxicity of the individual compounds. Food Chem. Toxicol. 28:623-631, 50. Jonker, D., R.A. Woutersen, P.J. van Bladeren, H.P. Til, e V.J. Feron. 1993. Subacute (4-wk) oral toxicity of a combination of four nephrotoxins in rats: comparison with the toxicity of the individual compounds. Food Chem, Toxicol. 31:125-136. 51. XU, W.J CAO, S.; HE, X.; LUO, Y.; GUO, X.; YUAN, Y.; HUANG, K. Safety assessment of Cry1Ab/Ac fusion protein. Food Chemistry Toxicology, 47, 1459¬1465, 2009 52. ONOSE, J.; IMAI, T.; HASUMURA, M.; UEDA, M.; OZEKI, Y.; HIROSE, M. Evaluation of subchronic toxicity of dietary administered Cry1Ab protein from Bacillus thuringiensis var. Kurustaki HD-1 in F344 male rats with chemically induced gastrointestinal impairment. Food Chemistry Toxicology, 46, 2184-2189, 2008. 53. HEALY, C; HAMMOND, B.; KIRKPATRICK.,J. Results of a 13-week safety assurance study with rats fed grain from corn rootworm-protected, glyphosate-tolerant MON88017 corn. Food Chemistry Toxicology, 46, 2517-2524, 2008 54. PAUL, V.; GUERTLER, P.; WIEDEMANN, S.; MEYER, H.H. Degradation of Cry1Ab protein from genetically modified maize (MON810) in relation to total dietary feed proteins in dairy cow digestion. Transgenic Research, PubMed PMID: 19888668, 2009 55. BATISTA, R.; NUNES, B.; CARMO, M.; CARDOSO, C; JOSE, H.S; ALMEIDA, ABD. MANIQUE, A.; BENTO, L.P., RICARDO, CP. E OLIVEIRA, M.M. Lack of detectable allergenicity of transgenic maize and soya samples. J. Allergy Clin. Immunol. 116:403-410, 2005 56. TAKAGI, K.; TESHIMA, R. e NAKAGIMA, O. Improved ELISA method for screening human antigen-specific IgE and its application for monitoring specific IgE for novel proteins in genetically modified foods. Regulatory Toxicology and Pharmacology, 44: 182-188,2006 57. Taylor, M,; Lucas, D.; Nemeth, M.; Davis, S.; Hartnell, G. 2007. Comparison of Broiler Performance and Carcass Parameters When Fed Diets Containing Combined Trait Insect-Protected and Glyphosate-Tolerant Corn (MON 89034 x NK603), Control, or Conventional Reference Corn. Poultry Science Association Inc. 58. Wu F, Miller JD, Casman, EA (2004) The economic impact of Bt corn resulting from mycotoxin reduction. Journal of Toxicology 23:397-424. 59. Heil, M.; Baldwin, I. T. Fitness costs of induced resistance: emerging experimental support for a slippery concept. Trends in Plant Science. 7(2):61-67. 60. Japanese Biosafety Clearing House, Ministry of Environment. Outline of the biological diversity risk assessment report: Type 1 use approval for MON89034xNK603 61. World Health Organization, 1999. Microbial Pest Control Agent: Bacillus thuringiensis. Environmental Health Criteria 217. 62. SANDERS, P. R.; LEE, T. C; GROTH, M. E.; ASTWOOD, J. D.; FUCHS, R. L. Safety assessment of insect-protected corn. In: THOMAS, J. A. Biotechnology and Safety Assessment. 2 ed. Taylor and Francis, 1998. p. 241-256. 63. LUNA, S.V.; FIGUEROA, J.M.; BALTAZAR, M.B.; GOMEZ, L.R.; TOWNSEND, R. E SCHOPER, J.B. 2001. Maize pollen longevity and distance isolation requirements for effective pollen control. Crop Sci. 41:1551-1557, 64. BROOKES, G.; BARFOOT, P.; MELE, E.; MESSEGUER, J.;BENETRIX, F. BLOC, D.; FOUEILLASSAR, X; FABIE, A.; POEYDOMENGE, C. 2004. Genetically modified maize: pollen movement and crop co-existence. Dorchester, UK: PG Economics, 20pp. 65. EFSA. European Food Safety Authority. Guidance Document of the Scientific Panel on Genetically Modified Organisms for the risk assessment of genetically modified plants containing stacked transformation events. The EFSA Journal (2007) 512, 1¬5. 66. EFSA. Scientific Opinion on application (EFSA-GMO-NL-2007-38) for the placing on the market of insect resistant and herbicide tolerant genetically modified maize MON89034 x NK603 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto. EFSA Journal (2009) 7(9):1320
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Where detection method protocols and appropriate reference material (non-viable, or in certain circumstances, viable) suitable for low-level situation may be obtained:
Molecular traditional methods
Relevant links to documents and information prepared by the competent authority responsible for the safety assessment: National Biosafety Commission
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Authorization expiration date: Not Applicable
E-mail:
gutemberg.sousa@mct.gov.br
Organization/agency name (Full name):
National Biosafety Technical Commission
Contact person name:
Flavio Finardi
Website:
Physical full address:
SPO Area 5 Qd 3 Bl B S 10.1 Brasilia DF
Phone number:
556134115516
Fax number:
556133177475
Country introduction:
The Brazilian National Biosafety Commission – CTNBio , is responsible to the technical decision on biological risk as a response to a request from the proponent. The technical decision is given on a definitive basis. Only the National Biosafety Council (CNBS) can revoke the decision (in case of commercial release), based on social-economical reasons and not on biosafety reasons. Once a decision is taken by CTNBio favorable to the commercial release of a new GMO (being it a plant or any other organism), CNBS has 30 days to issue a revoke. After these steps, the new product must be evaluated for conformity to the Brazilian standards by the registration and enforcement agencies (ANVISA – Ministry of Health, Ministry of Agriculture, Ministry of Environment and Ministry of Fisheries, according to the intended use of the product). If it conforms to the standards, it may be offered to the market. Every institution dealing with GMOs (including universities and public research institutes) has to have an Internal Biosafety Commission (CIBio), which is legally responsible of everything that may happen to be done or caused by the GMO
Useful links
Relevant documents
Stacked events:
At the discretion of, and upon consultation with, CTNBio, a new analysis and issuance of technical opinion may be released on GMOs containing more than one event, combined through classic genetic improvement and which have been previously approved for commercial release by CTNBio
Contact details of the competent authority(s) responsible for the safety assessment and the product applicant:
Dr. Edivaldo Domingues Velini (President of national Biosafety Commission)
Philippines
Name of product applicant: Monsanto Philippines
Summary of application:
Monsanto has developed a biotechnology derived product corn MON 89034 through Agrobacterium mediated transformation to express the Bacillus thuringiensis insecticidal proteins, Cry1A.105 and Cry2Ab2. The introduction of corn MON 89034 is expected to provide enhanced benefits for the control of lepidopteran insects pests such as Ostrinia furnacalis (Asian corn borer, ACB), Spodoptera litura (CCW) and Helicoverpa zea (corn earworm, CEW) compared to existing products.

Corn NK603 contains cp4epsps gene from Agrobacterium sp. Strain CP4. The cp4epsps sequence encodes for the production of the naturally-occurring CP4 EPSPS protein that renders the corn NK 603 tolerant to glyphosate herbicide.

The transgenic corn traits from Event MON89034 and Event NK603 were combined through conventional breeding to produce the Corn MON89034 x NK603. This stacked hybrid produces the three transgenic proteins present in MON89034 x Nk603 corn plants.
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Date of authorization: 22/07/2012
Scope of authorization: Food and feed
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Summary of the safety assessment:
Monsanto Philippines has filed an application with attached technical dossiers to the Bureau of Plant Industry (BPI) for a biosafety notification for direct use as food, feed and for processing under Department of Agriculture (DA)- Administrative Order (AO) No. 8 Part 5 for combined trait corn: corn MON89034 x NK603 which has been genetically modified for insect protection. A safety assessment of combined trait product corn: MON89034 x NK603 was conducted as per Administrative Order No. 8 Series of 2002 and Memorandum Circulars Nos. 6 and 8, Series of 2004. The focus of risk assessment is the gene interactions between the transgenes. Review of results of evaluation by the BPI Biotech Core Team in consultation with DA-Biotechnology Advisory Team (DA-BAT) completed the approval process.
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Where detection method protocols and appropriate reference material (non-viable, or in certain circumstances, viable) suitable for low-level situation may be obtained:
Relevant links to documents and information prepared by the competent authority responsible for the safety assessment:
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Authorization expiration date:
E-mail:
bpibiotechsecretariat@yahoo.com
Organization/agency name (Full name):
Bureau of Plant Industry
Contact person name:
Thelma L. Soriano
Website:
Physical full address:
San Andres St., Malate, Manila
Phone number:
632 521 1080
Fax number:
632 521 1080
Country introduction:
The Philippines is the first ASEAN country to establish a modern regulatory system for modern biotechnology. The country's biosafety regulatory system follows strict scientific standards and has become a model for member-countries of the ASEAN seeking to become producers of agricultural biotechnology crops. Concerns on biosafety in the Philippines started as early as 1987 when scientists from the University of the Philippines Los Banos (UPLB) and International Rice Research Institute (IRRI), the Quarantine Officer of the Bureau of Plant Industry (BPI) and the Director for Crops of the Philippine Council for Agriculture, Forestry and Natural Resources Research and Development (PCARRD) recognized the potential for harm of the introduction of exotic species and genetic engineering. The joint committee formed the biosafety protocols and guidelines for genetic engineering and related research activities for UPLB and IRRI researchers. This proposal was eventually adapted into a Philippine Biosafety policy by virtue of Executive Order No 430, Series of 1990, issued by then President Corazon C. Aquino on October 15, 1990, which created the National Committee on Biosafety of the Philippines (NCBP). The NCBP formulates, reviews and amends national policy on biosafety and formulates guidelines on the conduct of activities on genetic engineering. The NCBP comprised of representative from the Department of Agriculture (DA); Department of Environment and Natural Resources (DENR); Health (DOH); and Department of Science and Technology (DOST), 4 scientists in biology, environmental science, social science and physical science and 2 respected members of the community. The Philippines’ Law, Executive Order No.514 (EO514), Series of 2006 entitled “Establishing the National Biosafety Framework (NBF), Prescribing Guidelines for its Implementation, Strengthening the National Committee on Biosafety of the Philippines, and for Other Purposes was also issued. This order sets the establishment of the departmental biosafety committees in the DA, DENR, DOH and DOST. The mandates jurisdiction and other powers of all departments and agencies in relation to biosafety and biotechnology is guided by the NBF in coordination with the NCBP and each other in exercising its power. The Department of Agriculture (DA) issued Administrative Order No 8, Series of 2002, (DA AO8, 2002), which is part of EO 514, for the implementation of guidelines for the importation and release into the environment of plants and plant products derived from the use of modern biotechnology. The DA authorizes the Bureau of Plant Industry (BPI) as the lead agency responsible for the regulation of agricultural crops developed through modern biotechnology. The BPI has adopted a protocol for risk assessment of GM crops for food and feed or for processing based on the Codex Alimentarius Commission’s Guideline for the Conduct of Food Safety assessment of Foods Derived from Recombinant-DNA plants and a protocol for environmental risk assessment in accordance with the Cartagena Protocol on Biosafety and with the recommendation of the Panel of Experts of the Organization for Economic Cooperation and Development (OECD). DA AO8, 2002 ensures that only genetically food crops that have been well studied and found safe by parallel independent assessments by a team of Filipino scientists and technical personnel from the concerned regulatory agencies of the Department are allowed into our food supply and into our environment. The DA AO 8, 2002 has a step by step introduction of GM plant into the environment. The research and development phase would require testing the genetically modified (GM) crop under controlled conditions subject to regulation by the government agencies. The first stage of evaluation for GM crops is testing under contained facilities such as laboratories, greenhouses and screenhouses. After satisfactory completion of testing under contained facilities, confined environmental release or field trial is done. Confined field trial (CFT) is the first controlled introduction of the GM crop into the environment. The approval for field trial shall be based on the satisfactory completion of safety testing under contained conditions. Unconfined environmental release or commercialization of the product would follow after the safe conduct of the CFT. Approval for propagation shall only be allowed after field trials and risk assessment show no significant risk to human and animal health and the environment.
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Relevant documents
Stacked events:
Gene stacking in plants can be conferred either through genetic engineering or conventional breeding A full risk assessment as to food and feed or for processing shall be conducted to plant products carrying stacked genes conferred through genetic engineering or conventional breeding, where the individual traits have no prior approval for direct use as food and feed or processing from the Bureau of Plant Industry (BPI) A desktop or documentary risk assessment on the possible or expected interactions between the genes shall be conducted for stacked gene products with multiple traits conferred through conventional breeding and individual events granted prior approval by the Bureau of Plant Industry.
Contact details of the competent authority(s) responsible for the safety assessment and the product applicant:
Bureau of Plant Industry 692 San Andres St, Malate, Manila 1004