Technical Opinion no. 1679/2008

Proceedings:  01200.007232/2006-07
Applicant:  Dow Agrosciences Industrial Ltda.
CNPJ:   47.180.625/0001-46
Address: Rodovia Anhanguera, km 344, Caixa Postal 47, Jardinópolis, SP.
Applicant:  Du Pont do Brasil S.A. – Divisão Pionner Sementes.
CNPJ:   87.082.814/0001-09
Address: Rodovia DF 250, km 20, Caixa Postal 08283, Planaltina, DF.
Matter: Commercial Release of Genetically Modified Corn.
Previous extract: 814/2006. Published in the Federal Official Gazette of 12.27.2006, Section 03, page 09.
Meeting: 119th Regular Meeting held on 12.11.2008.
Decision: GRANTED.

CTNBio, following examination of an application for Technical Opinion related to commercial release of genetically modified corn, resistant to insect of the Lepidoptera order (Bt Cry1F 1507- Event TC1507 corn) as well as all progenies originated from transformation event TC1507 and derivatives from crossing non-transgenic lineages and populations carriers of Event TC1507, was favorable to its GRANTING under the terms of this conclusive technical opinion.
Dow Agrosciences  Industrial Ltda. and Du Pont do Brasil S.A. – Divisão Pioneer Sementes, requested CTNBio  a Technical Opinion related to free registration, use,  essays, tests, sowing, transportation, storage, marketing, consumption, import, release and discarding of corn (Zea mays L.) resistant  to insects of the Lepidoptera  order and corn pests – event TC1507. TC1507 corn was obtained by genetic transformation through microparticle acceleration, or biobalistics. Immature corn embryo calluses of the hybrid Hi-II lineage corn were bombarded with insert PHI899A, containing genes cry1F and pat, region coming from plasmid PHP8999. Though plasmid PHP8999 has the nptII gene, it was not used in the process. Transformed calluses were cultivated in a selective medium containing glufosinate ammonium, and resistant plants were transferred to a plant nursery. The insecticide protein present in TC1507 corn is a Cry1F truncated protein derived from the PS81I (NRRL B-18484) of Bacillus thuringiensis var. aizawai. B. thuringiensis (Bt) is a gram-positive bacterium that has, at the moment of its sporulation, crystalline protein inclusions. The inclusions contain proteins denominated delta-endotoxins. These proteins are produced in the form of protoxines and transformed into toxic peptides at the insect bowels under the action of alkaline intestinal pH and proteases. The active toxin causes epithelial cell lysis and death of the larvae. Bacterium B. thuringiensis may be considered to be the biological agent with the greater potential to control forest and agricultural insect pests and disease vectors; given the specificity of delta-endotoxins to target insects and invertebrates, coupled with the bacterium innocuousness to vertebrates and the environment, including beneficial insects and natural enemies, make this agent a key component for strategies of plague controlled management. Since the sixties, B. thuringiensis (Bt) has been used in the United States as a pesticide to control butterflies. Different toxicity studies conducted in mammals with Bt clearly show the absence of toxicity and pathogenicity. Due to its use as a microbial pesticide, a long history of safe use has been associated to proteins produced by Bt. Nutritional and toxicological security tests have been reported evidencing innocuousness of the expressed protein. In addition to resistance to insects, corn TC1507 contains gene pat, derived from Streptomyces viridochromogenes strand Tu949, which is responsible for codifying enzyme phosphinothricin acetyltransferase (PAT), the sequence of which has 183 amino acids and is identical to the PAT protein present in hybrids of genetically modified commercially released corns. The recombinant enzyme PAT is able to chemically inactivate herbicides derived from phosphinothricin, like glufosinate ammonium, making cells and plants containing such enzyme resistant. PAT protein is degraded by gastric juices of animals and by artificial gastric juice similar to that of humans, losing its physicochemical characteristics after oral exposure. Therefore, one does not expect the protein to be entirely absorbed, being unlikely that it may bring adverse or toxic effects. Innocuousness of transformation by the pat gene is confirmed by works conducted by different research groups. A study with cows fed on a formulation containing isogenic and transgenic corn for such modification, revealed the similarity of yield and composition in milk produced by both groups of animals. The same work analyzed appearance of transgene fragments in 90 samples of milk along the experiment and failed to show any positive result of transposition of natural barriers and appearance of DNA fragments or fragments of its corresponding protein in the milk. Except for the characteristics of resistance to pest Lepidoptera insects and tolerance to glufosinate ammonium introduced by gens cry1F and pat, TC1507 corn did not undergo any other phenotypical change. Western Blot analysis confirmed that the cry1F and pat proteins expressed in the plant have the same molecular weight and immunoreactiveness of the protein derived from the microbial form expressed from P. fluorescens. Analyses of TC1507 corn regarding quality and quantity standards of metabolites normally found in corn demonstrated that event TC1507 is substantially equivalent to conventional varieties of corn. The assessments were conducted to define individual components that are part of human diet. Centesimal composition  data analyses presented in the process encompass profile analysis of proteins, amino acids, fat acids, lipids, carbohydrates, minerals, vitamins, secondary metabolites,  and composition of fodder and kernel, comparing event TC1507 with corn plants not genetically modified. The results obtained in Brazil and other countries failed to show variations that exceed the standards commonly found in non-genetically modified corn hybrids and lineages. Therefore, one may assume that TC1507 corn is substantially equivalent to non-genetically modified corn plants. Samples taken from leaves, pollen, kernel and the whole plant (vegetative tissues) of both, TC1507 and conventional corn lineages, were used to detect Cry1F and PAT proteins in transformed plants. Western Blot analyses showed that protein Cry1F is expressed in all tissues, in contrast to protein PAT that was detected only in leaves of the TC1507 lineage. In order  to analyze the level of  expression, samples  of corn leaves, pollen, silk, stalk, whole plant, kernel, both normal and senescent  of the TC1507 corn lineage, as well as  samples  from non-transformed corn plants  were collected during the 1998-1999  crop and  tested with the ELISA test. The results regarding total protein (TP) showed higher levels of the Cry1F protein expression in the whole plant (1063.8 pg Cry1F/ug TP), senescent whole plant (714.3 pg Cry1F/ug TP), stalk (550.0 pg Cry1F/ug TP) followed by pollen (135.5 pg Cry1F/ug TP), leaf (110.0 pg Cry1F/ug TP), grain (89.9pg Cry1F/ug TP) and silk (50.3 pg Cry1F/ug TP). Dispersion of corn seeds is easily controlled, since domestication of corn eliminated the ancient seed dispersion mechanisms and pollen movement is the only effective means for gene escaping of corn plants. Horizontal gene flow between TC1507 corn and other species, even those closely related, are practically unlikely to happen, since wile species related to corn do not occur naturally in Brazil. Coexistence between cultivars of conventional corn (either cultivated or Creole) and transgenic cultivars is possible from the agronomic viewpoint, and this is a reason to comply with the provisions of CTNBio Ruling Resolution no. 04. The use of genetically modified insect-tolerant plants has positive repercussions also in aspects related to obtaining, distributing and using chemical insecticides, for the significant reduction in the pollution brought by industrial waste and water used in insecticide sprinkling, in addition to avoiding contamination of man, food, rivers and water sources resulting from the use, transportation and storage of insecticides. For the foregoing, a conclusion is reached that cultivation and consumption of TC1507 corn is not a potential cause of significant degradation of the environment; or of risks to human and animal health. For these reasons, there is no restrictions to the use of this corn or its derivatives. The applicant shall conduct post-commercial release monitoring under the provisions of CTNBio Ruling Instruction no. 03. According to Article 1 of Law no. 11,460, of March 21, 2007, "research and cultivation of genetically modified organisms may not be conducted in Amerindian areas and conservation units”. ”. Regarding the scope of Article 14 of Law no. 11,105/05, CTNBio holds that the request complies with applicable legislation and regulation aimed at securing biosafety of the environment, agriculture, human and animal health.

 CTNBio Technical Opinion
1. GMO Identification
GMO name:  Bt Cry1F 1507 – Event TC1507 corn, Herculex Corn.
Applicant:  Dow Agrosciences Industrial Ltda. and Du Pont do Brasil S.A. – Division Pioneer Sementes.
Species:  Zea mays L.
Inserted Feature: Tolerance to glufosinate ammonium herbicide and resistance to insects.
Insertion method: Biobalistics, with the use of particle acceleration.
Prospective use: Production of grains for human and animal consumption from the GMO and its derivatives.
II. General Information
Zea mays L., corn, is a monoic annual plant with height ranging from 1.0 to 4.0 meters(1). Its main stalk is composed of knots and inter-knots clearly defined. Inter-knots are wide at the basis and gradually diminish until inflorescence at the upper part of the plant. Leaves alternate along the stalk. Corn is the only grassy plant having both male and female flower structures in the same plant, though in different places(2).
Corn has over eight thousand years of history in the Americas and is cultivated since the pre-Colombian period. Corn is one of the higher plants more well scientifically characterized, and today it is the cultivated species that reached the highest degree of domestication and can only survive in nature when cultivated by man(3). Currently, over 300 corn varieties have been identified and, within each variety, thousands of cultivars.
Corn is one of the most important sources of food in the world and is the input for the production of a wide range of food products, fodder and industrial products. Brazil is the third largest world’s corn producer, and has harvested about 35 million tons in 2005, behind the United States of America (282 million tons) and China (139 million tons)(4). Corn is the second most cultivated grain in Brazil and is planted basically in two different crops (summer and safrinha, or  small  crop) and cultivated practically all over the domestic territory, with 75.68% concentrated in the Southern and Central Region and 24.32% in the  Northern and Northeastern Region. In terms of Brazilian production, corn is second only to soybeans(5).
It is a known fact that the insect population in the tropics is larger than in temperate zones, and that damages caused by them are  more  significant in  the tropical zone. Among the most important corn plagues there is the fall armyworm (Spodoptera frugiperda). Cruz et. al.(6) estimated that losses in Brazil caused by infestations of S. frugiperda are about 400 million Dollars annually. Starting in 1999, there has been an increased occurrence of fall armyworm, and, consequently, larger losses have been recorded. Other species of the Lepidoptera order are also important corn plagues, such as corn earworm (Helicoverpa zea)  and sugarcane borer (Diatraea saccharalis). It is estimated that these three  species may damage  up to 34% of corn kernel production.
The main measure to control insects in corn culture has been the use of insecticides. In some areas of the Brazilian Center-Western Region, dozens of insecticide applications are needed in a single cultivation cycle. Another measure for pest control would be the use of resistant cultivars. Obtaining  insect-resistant corn cultivars by classical genetic improvement has not attained the expected level  of success. Considering fall armyworm, several attempts have achieved limited success(7).
TC1507 corn possesses characteristics that grant resistance  to insects and tolerance to the herbicide glufosinate ammonium. This means that the phenotype enables the corn to resist to the main pests of the Lepidoptera order affecting corn culture in Brazil. These characteristics result from genes that were introduced and codify a truncated form of the insecticide protein Cry1F, derived from strand PS81I (NRRL B-18484) of bacterium B. thuringiensis var. aizawai and  one enzyme (phosphinothricin acetyltransferase, PAT), which grants tolerance to the herbicide glufosinate ammonium, also obtained from a soil bacterium, Streptomyces viridochromogenes. Corn varieties containing Cry proteins have been used in different countries of the world without any record that corn hybrids containing cry genes have caused damage to the environment, human and  animal health. Commercial use of TC1507 corn has taken place in the United States of America since 2001, Argentina (2005), Colombia (2006), China (2004), Mexico (2003), South Africa (2002), Canada (2002), Australia (2003), Japan (2002), Korea (2002), Philippines (2003),  Taiwan (2003), and European Union (2006) without identification of problems related to the agronomic characteristics of the event(8).
Brazil is held as the third largest consumer of agriculture pest controlling substances   in the world, where  there are about 142 registered  corn pesticides, from which 107 are intended for lizards. There are several reports of resistance caused by the constant and indiscriminate use of corn culture insecticides in Brazil. Besides, one of the most important causes of harm to the health of farmers in the country is the use of chemical pesticides, responsible  for  intoxicating one million individuals each year(9).
TC1507 corn was tested in Brazilian fields,  in several important  regions for corn production since 1998. The TC1507 lineage has also been assessed in essays conducted in Argentina, Chile, South Africa, Colombia, United States and Europe since 1997. Plants derived from TC1507 corn lineage attained the expected yield, without evidence of  unexpected changes  in morphological and phenotypic characteristics. There was also no evidence that event TC1507 had acquired characteristics of a plant pest, both in experimental plots and in fields where the event is recorded for cultivation.
III. Description of the GO  and Proteins Expressed
TC1507 corn was developed from a germplasm deemed appropriate for genetic  modification.  In order to transform corn embryos, a linear portion of plasmid PHP8999 DNA was extracted containing gene cry1F  and the gene used as selective marker: pat. The linear DNA portion, namely the insert, was used in the transformation process. Corn plants of lineage TC1507 were obtained by microparticle bombarding, using a Biolistics accelerator PDS-1000He-Bio-Rad(10).
No vector was used in the transformation of corn to generate  event TC1507. From plasmid PHP8999 a linear fragment was extracted, named PHI8999A, containing the coding sequence of genes cry1F and pat, jointly with  their associated  elements  of genic expression.
The insecticide protein present in TC1507 corn is a truncated Cry1F protein derived from strain PS81I (NRRL B-18484) of Bacillus thuringiensis var. aizawai(11). B. thuringiensis (Bt) is a gram positive bacterium  belonging to family Bacillaceae, producing, at the moment of sporulation, crystalline protein inclusions. The inclusions contain proteins named delta-endotoxins that  currently form a  family of 300 members, classified in  49 different groups(12).
The proteins are  produced as protoxines and are transformed  into toxic peptides in the insect bowels under action of the intestinal alkaline pH and proteases. The active  toxin causes  lysis of epithelial cells and death  of larvae(13, 14). B. thuringiensis may be considered the biologic agent of greater power in controlling forest, agricultural insect pests and disease vectors; due to the specificity of delta-endotoxins towards target insects and invertebrates and innocuousness to vertebrates and the environment, including beneficial  insects and natural enemies(15), making this agent a key component in pest integrated management strategies(16).
In addition to  resistance to insects, TC1507 corn contains the pat gene, derived from Streptomyces viridochromogenes strain Tu494, responsible  for codifying enzyme phosphinothricin acetyltransferase (PAT), the sequence of which has 183 amino acids and is identical to the PAT  protein present  in hybrids of genetically modified commercially released corns(17).  The original sequence of  the  pat gene was modified to reduce G/C content and change  the codon of the beginning  of GTG  translation into ATG,  in a way to  enable and  optimize  synthesizing the original protein. The final version of gene pat has 558 pb. Again, a sequence of 551 pb of CaMV 35S promoter (isolated Cabb-s) and the 178 pb IVS2 intron sequence of the corn gene adhS1 were used to  promote and enhance the pat gene transcription. Sequence 3’-nos  of 220 pb was used  as a stop element of  the  transgene. The cassette therefore enables  synthesizing the recombinant protein PAT, able to chemically inactivate herbicides  derived from phosphinotricin, such as glufosinate ammonium, making resistant the cells and plants containing it. The PAT enzyme has  its activity described and well known(18, 19, 20).
Except for its  resistance to Lepidoptera pest insects and tolerance to  the glufosinate ammonium herbicide introduced  by genes cry1F and pat, the TC1507 corn has no  other phenotypic change. Western Blot analyses confirmed that proteins Cry1F and PAT expressed  in the plant  have the same molecular weight and immunoreactivity of the protein derived from the microbian form expressed from P. fluorescens (21). Gao et al.(22) also used similar methods to study the expression of gene cry1F in cotton.
The results of a detailed molecular characterization of TC1507 corn using Southern Blot enabled  a  conclusion that this corn contain an almost complete copy of the DNA insert used in the transformation (i.e., 6186 pb  of the 6235 pb fragment of insert PHI8999A, containing cry1F and  pat genes jointly with the regulatory sequences needed in their  expression).
TC1507 corn is free from  nptII gene and from any other detectable fragment of part of plasmid PHP8999 not intended in transforming the TC1507 corn. The flanking regions of the corn genomic DNA on borders 5’ and 3’ in the TC1507 insert were  arranged in sequence and characterized in detail.
IV. Aspects related to  Human and Animal Health
Security assessment of food derived  from genetically modified  raw-materials is based on risk analysis, a scientific methodology encompassing  the phases of risk assessment, risk management and risk communication. In the risk assessment, one pursues the qualitative and quantitative characterization of potential adverse effects,  based  on the concept of substantial equivalence to identify any differences between the new food and its conventional correspondent. The Principle of Substantial  Equivalence  is a key concept in assessing the innocuousness of food generated from new technologies(23).
When assessing the  security of  a genetically modified food raw-material, or its equivalence to conventional food, it is recommended that four elements are analyzed, namely:
(1) Parental variety, i.e. the plant originating the new genetically modified raw-material;
(2) Transformation process, including a characterization of the construct used  and the resulting event;
(3) Product of the inserted gene and potential toxicity and allergenicity and, finally;
(4) Composition of the new variety resulting from genetic transformation.
The data set of such analyses  shall enable identifying and characterizing any potential adverse effect associated with consumption of the new raw-material,  providing information to the risk management and risk communication phases.
Since the sixties, B.  thuringiensis (Bt) has been used in the United States of  America as a  pesticide to control Lepidoptera. Several toxicity studies in mammals conducted  with Bt clearly show an absence of toxicity and pathogenicity(24). Due to its use as microbian pesticide, a long  history of safe use has been associated to proteins produced by Bt(25).
According to an assessment conducted by the United States Environment Protection Agency (EPA), Bt corns, including event TC1507 expressing protein Cry1F are harmless to human health(25).
Grain nutrient analysis (proteins, fats, acid detergent  fiber, neutral detergent fiber, carbohydrates, ashes and  moist content) from the TC1507 corn hybrid, showed comparability with grain of commercial corn hybrids.
Nutritional and toxicologic safety tests have been reported  evidencing the innocuousness of the protein expressed. Highly relevant scientific articles attest the low risk and innocuousness of cultures containing the Bt toxin gene(26, 27). Assessment of substantial equivalence was made by Herman et al.(28). Concurrently with insect resistance, the Bt toxin contributes for reducing the development of  corn ears molds, organisms responsible  by the production of mycotoxins and consequent contamination of corn(29).
A subchronic toxicity study was conducted with event TC1507 corn in male and female Sprague-Dawley rats(30). They received ad libitum food containing  either TC1507 or conventional corn (control) grains, in a maximum concentration of 33%, for about ninety days. No significant toxicologic differences were recorded between the animals consuming genetically modified and non-modified corn regarding nutritional, ophthalmic, clinic and neurobehavioral parameters, as well as differences in weight of their organs. According to the authors of the study, these results verify that event TC1507 is as safe and nutritional than the non-genetically modified corn.
Parameters of health status and  development were assessed in lactating cows fed with fodder and event TC1507 corn grains(31). No difference was reported between the group fed with fodder containing and non containing transgenic corn regarding milk production and composition, as well as regarding parameters of health status (physical  measurements and blood tests). Poultry fed with fodder  containing event TC1507 corn failed to display any difference regarding mortality and growth when compared with poultry fed with non-transgenic corn(32).
Compositional analyses of event  TC1507 corn were also conducted for macronutrients, minerals, vitamins, amino acid and fat  acid profiles, antinutrients and secondary metabolites. Results were similar  to the ones involving conventional corn and were recorded in tiers described  in the literature(33, 34, 30). Additionally, a study of acute oral toxicity was  conducted in male and female  CD1 mice, which received 5.050 mg/kg of protein Cry1F through gastric intubation(35). There was no  mortality, clinic signs of toxicity,   effects on animal growth and macroscopic lesion in organs, and therefore oral LD50 was assumed to be higher than 5.050 mg/kg. The PAT protein was degraded by gastric juice of animals and by artificial human gastric juice, losing its physicochemical characteristics  after oral exposure. Therefore, it is not expected the protein to be fully absorbed, making unlikely that it may have adverse or toxic effects.
Innocuousness of the transformation by pat gene is verified in works developed by different research teams. A  study conducted with cows fed with fodder formulated with isogenic and transgenic corn for this modification, showed  the similarity in yield and composition of milk produced by both groups  of animals(36). The same work also analyzed the appearance of transgene fragments in 90 milk samples along the experimental period, with  no positive result of transposing natural barriers or appearance of DNA fragments or its  corresponding protein in the milk.
A detailed study on  PAT protein innocuousness was conducted approaching structural assessment,  research  of glycosylation sites, thermal stability  and in vitro digestibility. The protein expressed by gene pat was analyzed, and the conclusion was for the safety of its use in plant modifications  under all the investigated aspects(37). These results enable, in addition, an inference that the PAT protein fails to present any characteristics of allergenicity to sensitive individuals, either by direct action  or by crossed reaction with other allergenic molecules.
Allergens originated from food are commonly resistant to heat, acid and proteases, may be  glycosed and  are present  in high concentrations. Proteins essayed are  readily digested by  gastric juice, are not glycosylated and the heating leads  to  bioactivity loss. Experiments conducted with animals failed to indicate any allergenic potential.
No significant similarity was recorded between the Cry1F protein and dermal, respiratory and food allergens. Besides, the protein, coming from a non-allergenic  source, is thermolabile, rapidly hydrolyzed when submitted to in vitro essay of  resistance to pepsin, being not  glycosylated(38, 39). The data, simultaneously analyzed, indicate lack  of Cry1F allergenic potential(39). This conclusion was reached by Hérouet et  al.(37) regarding  PAT, after different analyses conducted with the protein. The authors recorded that the protein sequence did not display homology with known allergens or toxins. Besides, PAT has no N-glycosylation site, is rapidly degraded by gastric and intestinal fluids and has a source  held as innocuous. Similar results have been described in other studies(40, 38).
Corn and its derivatives are not considered toxic. The genetic modification of TC507 corn lineage results from the expression of Cry1F and PAT proteins. Protein Cry1F displays specific toxicity against certain Lepidoptera plague insects (target organisms), however there is no evidence  that Cry proteins generated from Bacillus thuringiensis may harm human and animal health(41, 42).
The potential toxicity for humans and animals of protein Cry1F was examined in an acute  oral toxicological study where acute toxicity potential in  rats(35) of the  Cry1F delta-endotoxin of B. thuringiensis var. aizawai was assessed. The highest dose used in the essay was 5050 mg/kg LW, adjusting the purity of the essayed material (11.4%), the dose was 576 mg Cry1F/kg of body weight. In the course of the study, notes were taken on mortality, clinical  pathology and behavioral symptoms, as well as on  body weights, performing full necropsies at  the end of the study. No mortality was recorded in the course of the study.  During the experiment, no adverse clinical signs were present, and no adverse results were recorded in the necropsies.  Changes in the dose used in this study failed to provoke mortality among individuals submitted  to the essay, and therefore it was not possible to determine  the LD50 of the Cry1F protein.
In another oral acute toxicity study, rats were  fed with 6000 mg/kg of an essay material containing about 500 mg of protein PAT/kg LW(43). No clinical  observations were produced related to  the treatment. All rats  gained weight during the two weeks of observation and none  displayed pathologic lesions. In study conditions, and due to absence of any observable  toxicity, determination of PAT protein LD50 was not possible.
PAT protein toxicity safety was determined in detail during  the evaluation of glufosinate ammonium tolerant corn(41, 44, 45, 46, 47). Gene pat was originally obtained from strain Tü494 of bacterium Streptomyces  viridochromogenes that does not have  known  toxic or pathogenic  potential. PAT  protein is enzymatically  active. However, it displays high specificity for a substratum that does not exist in corn plants or in human and animal diets.
A study was conducted  on feeding chicken  through incorporation to the diet of  TC1507 corn lineage grains and non-transgenic control grains of comparable  germplasm(48). Mortality, weight gain and  alimentary conversion of chicken fed on  a diet containing  grain from corn TC1507 lineage were compared  with chicken fed on a standard diet containing common corn.  No significant statistical difference in mortality, weight gain  and alimentary conversion was recorded between chicken feeding on the TC1507 corn and those  on the  control  diet.
When assessing the allergenic potential, the most important issue to consider is  the biologic origin of the  gene introduced and whether it expresses the allergenic product(49). Both, Bacillus thuringiensis (origin of the cry1F gene) and Streptomyces viridochromogenes (origin of the pat gene) have no history  of allergy triggering factors. These donors are common soil bacteria.  In over 30 years of commercial use, there was no verifiable information of Bacillus thuringiensis allergenicity,  including occupational allergies related to the manufacture of products containing it(41).
The biochemical profile of proteins Cry1F and PAT provide the background for an allergenic  assessment by comparing them with allergens of known proteins. Comparison of amino acid sequences of an introduced protein with amino acid sequences of known allergens  may result in a useful  indicator of allergenic potential(50). Meyer(38) conducted a search assisted by the Wisconsin Genetics Computer Group (GCG) sequence analyzer computer program, looking up for “allergenic” in the database of standard DNA and protein sequences. A significant homology is the one recording a sequence identity of 8 or  more contiguous amino acids. A comparison of the 15  sequences of the most homologous data bases  confirmed that protein Cry1F does not share a  significant amino acid sequence  homology with known allergenic proteins.
In a similar way, PAT protein amino acid sequences were compared with allergens of known proteins(38). The  comparison showed that the  PAT protein does not share significant amino acid homology with known allergenic  proteins. The PAT protein had  already  been assessed for previous safety of genetically modified plants(41, 44, 45, 46, 47), including commercial release proceedings passed by CTNBio.
Allergens in food  proteins are generally stable in digestion  by pepsin and trypsin and in acid conditions of the human digestive tract, so  that  they may pass through the  intestinal mucosa to  generate  an allergenic response. Both Cry1F  and PAT proteins are easily degradable in  simulated digestive fluid, minimizing any potential the proteins  may have of  being absorbed by the  intestinal mucosa when  consumed. After one minute, protein Cry1F   is almost completely hydrolyzed in simulated gastric conditions at a 100:1 molar reaction (Cry1F:pepsin)(51). The immunoelectrotransfer blot detection technique also showed  that protein Cry1F is not glycosylated. On the other hand, protein Cry1F loses  immunoreactivity after being processed by heat and has not history of harmful use in microbial pesticides. The PAT protein was degraded to undetectable levels within 5 seconds from introduction of a simulated gastric fluid containing pepsin (40,47).
Therefore, genes cry1F and pat  introduced  in the TC1507 corn lineage do  not code known allergens  and both proteins, Cry1F and PAT,  do not share immunologically significant amino acid sequences with known allergens. These results, coupled with the rapid rupture of the proteins under digestive conditions, confirm that Cry1F and PAT proteins are unlikely to pose any significant allergenic risk.
Corn is extensively cultivated and  has a history of safe use as human and animal food. Corn is not considered  to be harmful to humans, domestic  animals and the wild fauna. With the exception of the  new characteristics  introduced, including resistance to certain Lepidoptera pests granted by gene cry1F  and  tolerance to glufosinate ammonium granted by gene pat, the TC1507 corn lineage is substantially equivalent to other corn lineages commercially found. No other characteristic of the original organism was modified that may be harmful or pose a risk to health. No adverse effects were recorded in  TC1507 corn lineage to  human health and the environment.
V. Environmental and Agronomic Aspects
Corn is an annual plant with low dormancy ability. The corn seed can survive from one cultivation season to another, and may successfully germinate under adequate temperature and moist conditions. These so-called  volunteer plants are easily  identified and controlled by manual, mechanical and chemical means. Corn does not exhibit tendency to proliferate as a plant pest and is not invasive in natural ecosystems(45).  Some species of the Zea genus are sylvan plants developing successfully in Central America without any considerable trend to proliferate as a plant pest.
Event TC1507 was carefully cultivated and monitored in what regards its proliferation ability as plant pest and agronomic behavior in over eighty locations around the world, including Argentina, United States, Chile, Italy, Brazil, France and South Africa. In Brazil, several planned  releases to the environment were presented by applicants and duly passed by CTNBio. In all  cases, TC1507 corn exhibited a behavior similar to the one expected from non-transgenic  corn, without evidencing any development of unforeseen morphologic or phenotypic characteristics.
In experimental and field essays conducted in Brazil by Dow AgroSciences during the 2005-2006 crops to compare TC1507 corn with the conventional material, several agronomic parameters were measured, such as: plant height, ear, stalk breaking, root size and yield, among other agronomic characteristics and resistance to diseases. Results reached in  experiments conducted in domestic soil were comparable to those attained in Argentina and United States, where it was demonstrated that the genetic  modification does not affect  the plant phenotype and field behavior.
Experimental essays conducted  all over  the  world with TC1507 corn lineage since 1997 confirmed that event TC1507 does not  show any unexpected change in plant vigor. Assessment by simple observation of  field essays showed  that the development from an emerging  plantlet to one-leaved plantlet, and from three to  five  leaves plantlet, TC1507 corn lineage is  comparable to the non-genetically modified corn.
Applicants additionally conducted field essays in Brazil, where resistance  to  common rust (Puccinia sorghi), Polysora rust (Puccinia Polysora),  cercosporiosis (Cercospora zea-maydis), Northern corn leaf blight (Exserohilum turcicum), Phaeosphaeria leaf spot (Phaeosphaeria  maydis), and Diplodia leaf spot (Diplodia macrospora) was assessed. The data indicate that in the four hybrid essayed there was no differences in disease severity between the hybrid  with TC1507 event and the correspondent conventional hybrid.
Comparatively, essays were conducted to assess  resistance characteristics  of hybrids derived from TC1507 corn lineage and their corresponding non-genetically modified corn to diseases such as Exserohilum turcicum leaf spot, Bipolaris maydis   leaf spot, Polysora  rust, cercosporiosis, Erwinia stewartii bacterial spot,  Ustilago zeae smut and resistance to pests, such as armyworm Spodoptera frugiperda, corn earworm Helicoverpa zea,  Frankliniella sp. thrips, Aphis sp. aphis, Chaetocnema pulicaria corn flea beetle, red acarus, among others. These essays showed that there was no difference to be recorded on severity of disease symptoms; damage caused by insects, except for organisms identified as susceptible to protein Cry1F among plants of event TC1507 and those of genetically modified corn.
The biological activity of protein Cry1F was studied in a range of pest insects feeding on corn plants. The essays were conducted  by exposing insects to artificial diets  treated with aqueous formulations of Cry1F  protein produced from a microbial source (P. fluorescens). Evans(51) showed  that the biochemical characteristics of a protein produced in either plant or microbial  form are equivalent. Insects studied were: armyworm (Spodoptera Frugiperda), moth borer (Diatraea saccharalis),  European corn borer (Ostrinia nubialis), corn earworm (Helicoverpa zea), black cutworm (Agrotis ipsilon), lesser cornstalk borer (Elasmopalpus lignosellus), Southwest  corn borer (Diatraea grandiosella), Western corn rootworm (Diabrotica virgifera virgifera), corn leaf aphid (Rhopalosiphum maidis) and corn leafhopper (Dalbulus maidis). Huang et al.(55) have already assessed Cry proteins specificity through essays  in connection with cell vesicles, evidencing the high specificity of this protein complex  to insect receptors.
Efficacy essay was conducted in the cities  of Itumbiara, (GO), Toledo (PR), Indianápolis (MG), and Jardinópolis (SP) during the 2005 calendar  year. Experiments were conducted according to cultural practices recommended  for each region.  In that same year, a first assessment was  made, including incidence of initial pests and predators. Incidence of the lesser cornstalk borer, Elasmopalpus lignoselus (Zeller), was not  recorded in any of the localities. Pooled variance analysis of data in the four localities revealed significant difference among the three treatments studied: conventional Pioneer P30F33 corn with application  of  insecticide, conventional Pioneer P30F33 corn without application  of  insecticide, and the same hybrid P30F33 – 1507 (Bt), using F test. Analysis of data related to assessment of herbivory and incidence  of green stink bug,  based on the percentage  of plants  with damage symptoms, revealed that interaction site x treatment was  significant. The result showing the comparison of averages for herbivory is lower for the P30F33 – TC1507 in each site.
Currently there is an indiscriminate use  of insecticides in Brazil, including a mix of chemical products, in an attempt to control insects, especially S frugiperda. With the massive employment of these chemical products an agricultural desert is created  in certain Brazilian regions,  since  the natural enemies  of  such pests  are  the first to be eliminated. Frequent employment of chemical insecticides  contributes to environmental  degradation, pollution and an environmental breakdown in corn culture  and even in other  rotation  crops. By adopting insect  resistant genetically modified  plants, reduction in insecticides has been considerable in countries  where this technology has been adopted for over  ten year. In the United States, for instance, farmers have obtained reductions of over 8,000 tons of active insecticide ingredient in 2001  alone(52, 53, 54). In China, the employment of insecticides were reduced 67% on average, and reduction in volume of active insecticide ingredient reached 80%(55). In South Africa, the  reduction was  around 66%(56). For the foregoing, one may argue that the use of the Bt  technology in Brazil may contribute towards a reduced employment of insecticides  and, consequently, mitigating the  impacts to the  environment and human and animal health resulting from the use  of these pesticides. Furthermore, the use of Bt technologies may positively affect the preservation of non-target populations and beneficial insects, facilitating an integrated management of farm pests(57, 55, 58). In addition, adoption of technologies that minimize the spraying of chemical products in crops  may bring secondary benefits such as reduced use of inputs in the  production of  pesticides, conservation of fuels used  to produce, distribute and  apply the pesticides and elimination of  the  need for use and  discard of pesticide packing.
VI. Restrictions to the use of the GMO and its derivatives.
Studies submitted by applicant showed  that there was no  significant difference between corn hybrids derived  from non-modified lineages and TC1507 corn regarding  agronomic characteristics such as  productivity,  moist at harvest, root bedding and plant height, among others. Besides, there was no significant differences in the method of reproduction, dissemination and survival ability of  the genetically modified corn compared with non-modified lineages. All evidences submitted in the proceedings  and bibliographic references(60, 61, 62, 63, 64, 65, 66) confirm the risk level of the transgenic variety as equivalent to those of non-transgenic in what regards soil microbiota,  non-target vertebrate and invertebrate animals, other plants and human and  animal health. Therefore, cultivation of TC1507 corn is not potentially a cause of  significant degradation of the environment and of risks  to  human and animal health. For the foregoing, there is no restrictions to the  use of this  corn and its derivatives.
 After being used for ten years in different countries, there was no record of problem to human and animal health or to the  environment that may be attributable to transgenic corns. It is worth emphasizing that absence of negative effects  resulting from cultivation of transgenic corn plants does not imply that they may not happen in the future. Zero  risk coupled with absolute safety does not exist in the biologic world, although there  is a host of trustworthy scientific information and a safe use history of ten years that  enables us to  say  that TC1507 corn is as safe as conventional  corn versions.
This way, applicant shall  conduct  a post-commercial release monitoring  according  to  the provisions of CTNBio Ruling Resolution no. 03.
The vertical genic flow to local varieties, the so-called Creole corns, of open pollination is possible  and displays the same risk caused by  commercial genotypes available in the  market  (80% of conventional corn planted in Brazil comes from commercial seed that underwent a process of genetic improvement. Coexistence between cultivars of conventional  corns (improved or Creole) and transgenic  corn cultivars is possible from the  agronomic viewpoint(43, 67) and shall  follow the provisions of CTNBio Ruling Resolution no. 4.
VII. Considerations about Particulars of Different Regions in the Country (subsidy to monitoring agencies):
According to Article 1 of Law no. 11,460, of March 21, 2007 ”research and  cultivation of  genetically modified organisms may not be conducted in  Amerindian areas and conservation units”.
VIII. Conclusion
Considering that TC1507 corn is derived from a transformation of common Zea mays corn, a fully characterized species with a  solid history of safety  for  human and animal  consumption; that the transformation process caused insertion of a  single  copy of a DNA fragment containing genetic constructs with pat and cry1f  genes.
Considering that  safety  of  corn  containing the pat gene was exhaustively assessed by  CTNBio in proceedings 01200.005154/1998-36, and  analyzed  corn containing genes cry1A(b) and  pat in proceedings 01200.002109/2000-04; and that all  aspects  regarding corn biosafety were studied  for corns Liberty Link and Bt11  in Technical Opinions no. 987/2007 and 1255/2008.
Whereas:
1. Corn is the species that reached the highest domestication level among cultivated plants, and is  unable to survive  in nature with no human intervention.
2. In Brazil, there are no wild species with which corn may intercross, since  the closest wild corn species is teosinte, found only in Mexico and in some Central America locations, where it may cross  with corn cultivated in production fields.
3. Protein Cry1F was detected in low levels  in tissues analyzed and displayed  high  susceptibility to digestion in simulated gastric fluids, failing  to show acute toxicity in mammals and similarity with known  allergens(38).
4. The DNA molecule is  a natural  component of food and there is no  evidence that this molecule may have adverse effect  to man when ingested  in food  in acceptable amounts.
5. There is no evidence that intact  genes of plants may be  transferred and functionally integrated to the human genome or genome of  other mammals exposed to such DNA or to foods manufactured with such elements(50).
6. Applicant answered to all questions mentioned in CTNBio Ruling Instruction  no. 05 and there is no issue indicating that this corn may present  adverse  effects to human and animal health.
7. There  is no likelihood  that TC1507 corn may perform  or cause invasion  of uncultivated areas.
8. Proteins Cry1F and PAT  are  rapidly degraded in gastric conditions, thus  minimizing any absorption potential in an intestinal system(51, 40).
9. Bacterium B. thuringiensis may be considered the most potent biological agent to control forest and agricultural pest insects and disease vectors for  the specificity of delta-endotoxins to insects and target-invertebrates, and its innocuousness to  vertebrates and the environment, including  beneficial insects and natural enemies,  making this agent a key component in integrated management of pests.
10. Cultures of B. thuringiensis are filed with Agência Nacional de Vigilância Sanitária – ANVISA, the National  Sanitary Surveillance Agency, under  different formulations for application in thirty types of plant  cultures  for food  use(68).
11. Biopesticides  based on such toxin are widely used as an alternative to chemical insecticides in terms  of safety to non-target organisms and when  development of resistance to chemical insecticides is the  case(69).
12. The use of Bt technology in Brazil may contribute to  reduce  the use of insecticides and, consequently,  mitigate  the impact resulting from the  use of  such pesticides  to the environment, human and  animal  health, and to positively affect the preservation of non-target  organisms and beneficial  insects, facilitating the integrated management of farm pests.
13. This corn variety shows low risk to human health, animal health  and is no likely to change into a  plant pest.
14. The gene insertion did  not change the composition and  nutritive value and the presence of protein Cry1F in proportion to the total corn protein does not imply significant contribution to the amount or proteins in human diet.
15. Nutritional, equivalence and toxicological tests have  been reported showing the expressed protein to be innocuous(26, 27, 28).
16. Concurrently to resistance to insects, the Bt toxin contributes to reduce mold  development in corn ears, which are responsible for production and  contamination of corn  with mycotoxins(29).
17. No  other characteristic  of the  original organisms that represents risks to human health was modified and there  was no record  of adverse effects resulting from  TC1507 corn in studies related to human health and the environment.
18. Commercial use of TC1507 corn lineage is occurring  in the United States since 2001, Argentina (2005), Colombia (2006), China (2004), Mexico (2003), South Africa (2002), Canada (2002), Australia (2003), Japan (2002), Korea (2002), Philippines (2003), Taiwan (2003)  and European Union (2006)  without  any record of problems linked to the agronomic characteristics of the event.
19. Comments, opinions, suggestions and documents resulting from a Public Hearing related to  TC1507 corn held on March 20, 2007, failed to register any relevant scientific fact, corroborated by scientific evidence, that may compromise the environmental safety and  human and animal  health.
20. Coexistence of  conventional  corn cultivars (improved or Creole) and transgenic cultivars is  possible from  the agronomic viewpoint, and the provisions  of CTNBio Ruling Instruction no. 04 shall be complied  with.
For the foregoing, and  considering internationally accepted criteria in the process of  risk analysis  for  genetically modified raw-materials, a conclusion  emerges that TC1507 corn is as safe as  its conventional  equivalent.
CTNBio holds that commercial cultivation and consumption of TC1507 corn are not potential causes of significant degradation to the environment or of harm to human and animal health. Restrictions to the use of the GMO analyzed and its derivatives are conditioned to the provisions of CTNBio Ruling Resolutions no. 03 and 04. Additionally, this risk analysis took into consideration and consulted third party  independent studies and scientific publication submitted by applicant.
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Walter Colli
President of CTNBio

 Dissenting vote:
CTNBio  members Doctor Kenny Bonfim (Ms.), Doctor Rodrigo Roubach, Doctor Graziele Almeida da Silva (Ms.) and Doctor José Maria Ferraz (Permanent Sector Subcommission: Human and Animal Health) opposed  approval of commercial release of TC1507 corn.
Author of the  Technical Opinion, Doctor Paulo Brack (Environmental Permanent Sector Subcommission) issued an opinion opposed to approval of this product on the following grounds:
1. The large amount  of unknowns resulting  from technology shall be considered.
2. Corn is a strategic food culture for the economy and small farmers.
3. There are no efficient mechanisms of segregation and coexistence.
4. There is no efficient follow-up by monitoring government  agencies.
Authors of a Re-examination Request, under  Article 22, Paragraph 26, of  MCT  Directive no. 146, Doctor Paulo Kageyama  and Doctor Leonardo Melgarejo (Environmental Permanent Sector Subcommission) issued an opinion contrary to approval of TC1507 corn  on the following grounds:
1. An analysis of the proceedings enabled a  conclusion that the study lacked scientific basis to maintain that the  toxins extracted  from plants are identical, or similar, to  the ones extracted  from Pseudomonas fluorescens.
2. No study on the intact additional copy of transgene Cry1F was  mentioned in the dossier.
3. There is no description of the technology used to determine the  degree of genotypic stability of Herculex corn and, in addition,   it  shall be  emphasized that  there are no  data on stability  of the genotypes the cultivation of which is  intended in Brazil.
4. Results of nutrition  assessment in experimental animals  for two generations”, and lack of information on “time length of experiments”.
5. There is no information on the dossier on “possible poisonous effect of the GMO in  pregnant animals and its teratogenic potential” and on  immunologic analyses” of animals tested, as well as its teratogenic potential.
6. “potential pollinating agents” were not enumerated, and likewise not mentioned the corn seed disseminating agents, and “geographical distribution in Brazil” of each of them.
7. The dossier fails  to  consider the  risks for the Brazilian avifauna and  there is no record of studies with species included in the list  of endangered species.
8. There were no  studies on environmental impacts of  Herculex corn tillage on the microorganism communities of Brazilian soils, such as mycorrhizal fungi, bacteria involved in  symbiotic fixation  of atmospheric nitrogen, lignin decomposing fungi and bacterial antagonists (Pseudomonades  type).
10. A good part of studies submitted in the dossier relate to bacterium-synthesized proteins that fail to carry the whole information related to the set of interactions  associated to the proteins incorporated in TC1507 corn and,  therefore, fail to  support the intended conclusions.
11. Some animals used in tests are unjustifiably  not  described or are not representative  in our biomes, and therefore result in insufficient grounds the purposes of the dossier.
12. The time length of assessments involving animals is no longer  than one generation  and fails to consider the likelihood of teratogenic  impacts, as required by CTNBio Ruling Regulation no. 05.
13. The risk of contaminating Creole corn cultures, fundamental in maintaining production systems linked to the culture and living standards of  familiar, indigenous and quilombola farmers, are significant and tending to inevitability, threatening the majority of Brazilians that live in rural  areas.
14.  Several issues mentioned in CTNBio Ruling  Resolution no. 05 are not complied with, while other issues are insufficiently addressed.