Proceedings: 01200.005322/2006-55 Applicant: Dow Agrosciences Industrial Ltda

Proceedings: 01200.005322/2006-55 Applicant: Dow AgroSciences Industrial Ltda. CNPJ: 47.180.625/0001-46 Address: Rua Alexandre Dumas, 1671, 1º Andar, Ala A, 04717-903 São Paulo, SP. Matter: Commercial release of Genetically Modified Cotton. Previous extract: 749/2006. Published on 09.29.2006. Meeting: 121st CTNBio Regular Meeting, held on 03.19.2009. Decision: GRANTED.

CTNBio, following examination of an application a Technical Opinion related to commercial release of genetically modified insect-resistant, glufosinate ammonium-tolerant cotton, including all progenies originated from transformation event 281-24-236/3006-210-23 and its derivatives obtained from the crossing of cotton non-transgenic lineages and populations with lineages carriers of event 281-24-236/3006-210-23, decided for the GRANTING of the application under the terms of this Technical Opinion. Dow AgroSciences Industrial Ltda. requested a CTNBio Technical Opinion related to biosafety of the insect-resistant glufosinate ammonium-tolerant genetically modified cotton (Gossypium hirsutum), namely WideStrike Cotton, for the purpose of free registration, use in the environment, human and animal consumption, marketing and industrial use and any other use and activity related to this GMO including derivative lineages and cultivars as well as byproducts, all under the remaining regulations and requirements applicable to any use of cultivated species of the genus Gossypium effective in Brazil. WideStrike Cotton was produced by retro-crossing (“gene stacking”, “stacking”), between events 281-24-236/3006-210-23, containing the synthetic gene cry1F that codifies the synthetic protoxin Cry1F and event 3006-210-23 containing the synthetic gene cry1Ac that codifies synthetic Protoxin Cr1Ac. Both are insecticide crystallized proteins also referred as d-endotoxins, obtained from Bacillus thuringiensis var. aizawai strain PS811 and Bacillus thuringiensis var. kurstaki strain HD73. In addition to insect resistance given by the action of genes cry1F and cry1Ac, event WideStrike also displays resistance to the herbicide glufosinate ammonium due to the presence of two copies of gene pat, which codifies enzyme phosphinotricin-acetiltransferase (PAT). Gene pat is a synthetic version based on the natural pat gene of Streptomyces viridochromogenes, a non-pathogenic bacterium found in the soil. Inclusion of the pat gene enables selection of plants 341/2009 3 68 from well-succeeded transforms that express proteins Cry1F and Cr1Ac of Bacillus thuringiensis. The PAT protein fails to grant any pesticide activity, and there is no known adverse effect to the environment or to man, such as toxicity or allergenicity. The agronomic purpose of the event is the control of cotton pests: Heliothis virescens, Helicoperva zea, Spodoptera frugiperda, Alabama argillacea, Pectinophora gossypiella, Spodoptera exigua, Spodoptera eridiania, Pseudoplusia includens, and Trichoplusia ni. Events Cry1F 281-24-236 and Cr1Ac 3006-210-23 were obtained from transformation of Agrobacterium tumefaciens from cotton cultivar ‘Germain’s Acala GC510’ (Gossypium hirsutum L.). Each event was later retro-crossed with genotype PSC355 (Phythogen Seed Company). The first generations (F1) of such crossings in each event was then retro-crossed for three additional generations for PSC355, to create a BC3F1 lineage of each event. The two transgenic lineages BC3F1 were crossed to obtain the stacked cotton lineage Cry1F/Cr1Ac that was used in the final product. The source of the cry1F and cry1Ac is the bacterium Bacillus thuringiensis (Bt subspecies aizawai and kurstaki, respectively). The d-endotoxins, insecticides Bt expressed in the cotton plant, are proteolitically cleavaged in the alkaline intestine of lepidopters, resulting in a form of active insecticide. The active insecticide protein interacts with a receptor molecule that is present only in the epithelial cells of the middle intestine of susceptible insects, generating pores in the cell membranes. The process disturbs the cell equilibrium, causing a dialysis of intestine cells of insects, leading them to death(53). However, recent studies suggested that the cause of the insect death may be the presence of pores in the intestinal epithelium that causes the passage of bacteria present in the middle intestine to the hemolymph, leading the insect to death by generalized infection(24). There are no binding sites for the d-endotoxins of Bacillus thuringiensis at the surface of the intestinal cells of mammals, therefore domestic 341/2009 4 68 animals and humans are not susceptible to such proteins. A number of experiments testing Bt proteins showed the lack of toxicity to man and vertebrates, absence of adverse effects to non-target organisms and to the environment. Gene stacking events of 281-24-236/3006-210-23 cotton were fieldtested in 1999, 2000, 2001 and 2002, and are under assessment until now in the main cotton regions in the United States, Costa Rica, Argentina, Australia, Mexico, Spain and China. An experiment in Brazil was conducted in the 2005/2006 crop. Data and information related to the agronomic characteristics, resistance to pests and diseases were collected during such tests. Reports are that events 281-24-236/3006-210-23 do not exhibit pathogenic properties to plants and it is unlikely that may harm other insects that are beneficial to agriculture. There is no evidence or suggestion that proteins Cry1F and Cr1Ac of Bt increased the potential for the transformed cotton plant to change into an invading plant, since its phenology, morphology, and other agronomic aspects remained unaltered. Therefore, according to the results obtained and mentioned in the Cotton Biosafety report, it is unlikely that events of cotton events 281-24- 236/3006-210-23 change into pest plants for agriculture or become invaders o natural habitats; cross with wild relatives or create hybrid descendants that may change into pests or invading plants, despite genetic compatibility among the species. It is also unlikely that they change into a plant pest; have adverse effects on non-target species, including man, or that they have any effect on biodiversity. Summarizing, the focus of this application are the transformation events 281-24- 236 and 3006-210-23, which are combined by retro-crossing (conventional improvement). Cotton cultivars containing Cry1F and Cr1Ac will be marketed with stacked events 281-24-236/3006-210-23. The release of stacked product 281-24- 236/3006-210-23 together with the practices of insect resistance management shall reduce the selection pressure towards development of insecticide resistance 341/2009 5 68 and help maintaining an effective control of Lepidoptera. For the foregoing, commercial release of WideStrike Cotton is not potentially harmful to either human or animal health, and is not an event of significant degradation for the environment. According to Article 1 of Law nº 11,460, of March 21, 2007, “research and cultivation of genetically modified organisms may not be conducted in indigenous lands and areas of conservation units”. Under Article 14 of Law no. 11,105/2005, CTNBio found that the request complies with the applicable rules and legislation securing the biosafety of environment, agriculture, human and animal health. According to Annex I of Regulating Resolution no. 5, of March 12, 2008, the applicant shall have a term of thirty (30) days from the publication date of this Technical Opinion to adjust its proposal to the post-commercial release monitoring plan. 341/2009 6 68 CTNBio TECHNICAL OPINION I. GMO Identification GMO name: WideStrike Cotton, Event 281-24-236/3006-210- 23 . Applicant: Dow AgroScience Industrial Ltda. Species: Gossypium hirsutum L. Inserted characteristics: Tolerance to insects [genes cry1Ac and cry1F] and to herbicide glufosinate ammonium [gene pat] Method of insertion: Transformation mediated by Agrobacterium Prospective use: Production of fibers for the textile industry and kernels for human and animal consumption of the GMO and derivatives. II. General Information Cotton belongs to genus Gossypium, Tribe Gossipiae, Family Malvaceae, order Malvales (62,136). The genus is divided into four sub-genera (Gossypium, Sturtia, Houzingenia and Karpas), which, in turn, are divided into nine sections and a number of subsections(61). Original centers of G. hirsutum are Mexico and Guatemala, while those of G. barbadiense are in Peru and Bolivia(173). Allelotetraploid species exhibit in their genome a combination of two distinct diploid species(136). 341/2009 7 68 Two types of cotton plants are predominantly cultivated in Brazil: conventional cotton and a caterpillar-resistant genetically modified cotton plant. These plants are responsible for practically all the cotton produced in the country. Besides, three other cotton plants featuring genetic or ecologic special characteristics are cultivated: the naturally colored, the organic and the agroecologic cotton plant. The colored cotton is almost exclusively concentrated in the State of Paraíba, being the area planted, in 2007, of about 300 hectares. Certified organic cotton is planted in the States of Paraná and Paraíba, and the area cultivated in 2007 was 250 hectares. Tillage of agroecologic cotton plants were conducted by 235 farmers in the semiarid bioma of four States of the Northeastern region, producing 42 tons(116). Chains of special, conventional and transgenic cotton have satisfactorily lived together, without problems of coexistence being reported. The area planted with cotton in Brazil in the past 2007/2008 harvest reached about one million and one hundred thousand hectares, of which over 85% concentrated in the Cerrado bioma, especially in the states of Mato Grosso, Bahia, Goiás and Mato Grosso do Sul. Other cultures are present in other states of the country, mainly in the semiarid of the Northeastern region, Paraná, Minas Gerais and São Paulo(94). Cotton plants are unique in their utilitarian aspects, including weaving fibers and oil- and protein-bearing seeds used in human and animal food. Cotton plant species were developed since ancient times both in the old and the new world. Cotton (Gossypium spp.) is a plant domesticated by man since 3000 BC and cultivated in all continents. Its main use is in the production of fibers and food, especially for animals. Cotton plant (Gossypium hirsutum L.) is one or the four species cultivated for cotton fiber in the world(147), economically exploited in a wide tropical strip and in 341/2009 8 68 some subtropical regions. Culture of cotton in Brazil ranks among the ten main crops in Brazil and is sixth in cultivated area. Cotton species commercially cultivated in Brazil are Gossypium hirsutum and, in a lesser area, G. barbadense. G. hirsutum is more adaptable, more productive and is prevalent in the world. Its fiber is used in the production of textiles, other non-textile products and is the source of industrial cellulose for different products. G. barbadiense is important for the quality and length of its fiber and is used in the production of fine fabrics. Among the main cotton pests in Brazil, one may mention cotton leafworm (Alabama argillacea), cotton budworm (Heliothis virescens), pink bollworm (Pectinophora gossypiella), fall armyworm (Spodoptera frugiperda), cotton aphid (Aphis gossypii), cotton bug (Horcias nobilellus), and boll weevil (Anthonomus grandis). Control of such pests has been mainly conducted with the use of insecticides. In Brazil, over 10 tons of insecticide are consumed each year in cotton fields only, causing a US$ 190 million increase in production costs. The excessive use of non-specific insecticides leads to negative environmental impacts, such as severe reduction of beneficial organisms and potential upsurge of pests resistant to conventional insecticides. WideStrike Cotton’s purpose is to obtain insect-pest resistant plants through introduction of two gens from bacterium B. thuringiensis (Bt), namely gene cry1Ac (Bt species kurstaki) and gene cry1F (Bt subspecies aizawa) that synthesize endotoxins, mainly against Lepidoptera. In addition, gene pat was also introduced (enzyme phosphinothricin acetyltransferase) from bacterium Streptomyces viridochromogenes. The enzyme produced is used as a marker and imparts resistance to glufosinate ammonium. 341/2009 9 68 The use of Bt technology in Brazil may contribute towards the reduction of the use of such insecticides and, consequently, diminish the impacts from the use of such pesticides to the environment, and human and animal health. Besides, adoption of technologies that reduce spraying of chemical products in crops may favor the appearance of secondary benefits. Such benefits are reduction in the use of inputs to produce agricultural pesticides, conservation of fuels used to produce, distribute and apply the pesticides and elimination of the need for use and discarding of pesticide packaging. III. Description of GMO and Proteins Expressed WideStrike Cotton was developed by retro-crossing (“stacking”) between events 281-24-236, containing synthetic gene cry1F that codifies protein Cry1F and event 3006-210-23, containing synthetic gene cry1Ac that codifies protein Cr1Ac. Both are crystallized insecticide proteins, also referred as d-endotoxins, obtained from Bacillus thuringiensis var. aizawai strain PS811 and Bacillus thuringiensis var. kurstaki strain HD73, respectively. In addition to insectresistance for the action of genes cry1F and cry1Ac, event WideStrike is also glufosinate ammonium-resistant due to the presence of two copies of the gene pat, which codifies enzyme phosphinothricin acetyltransferase (PAT). Gene pat is a synthetic version based on the natural pat gene of Streptomyces viridochromogenes. Events Cry1F 281-24-236 and Cr1Ac 3006-210-23 were obtained through transformation by Agrobacterium tumefaciens of the cotton cultivar ‘Germain’s Acala GC510’ (Gossypium hirsutum L.). Each event was later retro-crossed with genotype PSC355 (Phytogen Seed Company). The first generations (F1) of such crossings in each event were retro-crossed for three additional generations for CSC355 in order to generate a BC3F1 lineage of each event. The two transgenic 341/2009 10 68 lineages BC3F1 were crossed to obtain the cotton stacked lineage Cry1F/Cr1Ac that was finally used in the commercial product. The transformation was made by Agrobacterium tumefaciens using binary vectors pAGM281 and pMYC3006, the maps and other gene elements of which are described by the applicant in the proceedings. Genetic elements of region T-DNA of plasmids pAGM281 and pMYC3006 are described below. Plasmid pAGM281 Genetic Element Size (kpp)1 Location (bp) Details (40CS)Dmas 2’ 0.61 7028-7636 (complementary) Promoter of manopine synthase of Agrobacterium tumefaciens strain LBA 4404 pTi15955 Cry1F (synpro) 3.45 3571-7017 (complementary) Plant optimized synthetic, full length version of Cry1F of B.t. var. aizawai. ORF25 poliA 0.73 2818-3544 Bidirectional terminator of Agrobacterium tumefaciens strain LBA 4404. pat 0.55 2259-2810 Plant optimized synthetic gene of glufosinate ammonium resistance, based on a sequence of phosphinotricin-acetiltransferase from S. viridochromogenes. Ubi Zm1 1.99 260-2252 Zea mays promoter plus Zea mays exon 1 (enhanced not translated) and intron 1. Plasmid pMYC3006 Genetic Element Size (kpp)1 Location (bp) Details Ubi Zm1 1.99 6080-8072 (complementary) Zea mays promoter plus Zea mays exon 1 (enhanced not translated) and intron 1. Cry1Ac (synpro) 3.47 2587-6057 (complementary) Plant optimized full length synthetic version of Cr1Ac1 of B.t. var. kurstaki. ORF25 poliA 0.73 1835-2561 Bidirectional terminator of Agrobacterium tumefaciens pTi5955. pat 0.55 1276-1827 Plant optimized synthetic gene of glufosinate ammonium resistance, based on a gene sequence of phosphinotricin-acetiltransferase from Streptomyces viridochromogenes. (40CS)Dmas 2’ 0.61 643-1251 Promoter of manopine synthase of pTi15955 (Barker et al., 1983, Plant Mol. Biol. 2, 335-350), including four copies of octopine synthase (OCS) of pTiAch5. 341/2009 11 68 For both lineages, the transformation took place using segments of cotton cotyledons isolated from plants with 7-10 days of in vitro germination. The segments were cultivated with disarmed A. tumefaciens (strain LBA4404) containing the above described plasmids. After the transformation, the segments were transferred to a medium of callus induction with herbicide glufosinate ammonium. In the middle of the callus formation the antibiotic carbenicillin was also used with the purpose to destroy any remaining Agrobacterium. The source of genes cry1F and cry1Ac is bacterium Bacillus thuringiensis (B.t. subspecies aizawai and kurstaki, respectively). Insecticide d-endotoxins expressed in the cotton plant are proteolitically cleavaged in the alkaline intestine of Lepidoptera insects, resulting in an active insecticide form. The active insecticide protein interacts with a receptor molecule, present only in the epithelial cells of the medium intestine of susceptible insects, generating pores in the cell membranes. This process causes a disturbance in the cell equilibrium, promoting dialysis of the intestine cells of insects, leading them to death(53). Recent studies, however, identified that the cause of the insect death is that the presence of pores in their intestinal epithelium promotes the passage of medium intestine bacteria to the hemolymph leading the insect to death by generalized infection(24). There are not binding sites for the Bacillus thuringiensis d-endotoxins at the surface of mammal’s intestinal cells and therefore domestic animals and man are not susceptible to such proteins. A detailed molecular analysis of WideStrike cotton was submitted by applicant, discussing the results of the insertion number characterization by Southern Blot analysis; identification of terminations 5’ and 3’ and border regions of the DNA inserted by cloning and PCR; DNA sequencing; and assessment of whether other transcriptions of potential mRNA resulting from the insertion sequences are or not 341/2009 12 68 present. The initial Southern Blot analysis indicated that the commercial WideStrike contains a single copy of the T-DNA from the binary vector pAGM281 jointly with the T-DNA of the binary vector pMYC3006. Insert pMYC3006 contains an intact copy of insect resistance, cry1Ac, jointly with one intact copy of the marker for selection of molecular weight, gene pat, while insertion of pAGM281 contains one intact copy of the insect resistant gene, cry1F and one intact copy of the pat gene. Besides, one copy of promoter UbiZm1 and a truncated copy (231 bp) of the pat gene were identified adjacent to the 3’ T-DNA terminal border. Southern blot analyses also suggested the genotypic stability of insects in different generations of the stacked event or of lineages, individually, besides verifying the absence of antibiotic-resistant genes or sequences of the vector backbone in cotton 281-24- 236/3006-210-23. The results are in line with the expectations, since the transformation method using A. tumefaciens, even with the unplanned, though acceptable, presence of a truncated copy of the gene pat close to the insert Cry1F / pat(108, 109, 194). IV. Aspects Related to Human and Animal Health Transgenic cotton resistant to lepidopters order insects event 281-24-236/3006- 210-23 contains DNA sequences derived from the following organisms: Bacillus thuringiensis, Agrobacterium tumefaciens, Streptmyces viridochromogenes, and Zea mays. None of such donor organisms in known as a source of toxins for mammals or as being allergenic to man. Proteins Cr1F/Cr1Ac are microbial d-endotoxins produced by Bacillus thuringiensis (Bt). The toxins act at the intestine of larvae of different caterpillars of the order Lepidoptera, where they have receptors. This bind prevents the 341/2009 13 68 insects from feeding. Man and animals do not have such receptors and, therefore, in principle, are not subject to the effects caused by such bind. Protein Cry1F is expressed in all the plant parts, except nectar, bran and oil. Protein Cr1Ac is expressed in all the plant parts, except nectar, husk, bran and oil. Protein PAT is expressed in very low levels for event 281-24-236 and for cotton 281-24-236/3006-210-23, however this protein was seldom detected in tissues of event 3006-210-23. Analyses carried out in ashes, total fat, humidity, protein, carbohydrates, calories, total fiber, fiber in acid detergent, fiber in neutral detergent in the modified cotton showed results similar to those of the control cotton, varying within the values of the literature. An analysis of fatty acids in the oil of products processed from cottonseed showed that there is no difference between conventional and transgenic cottons. Regarding anti-nutritional factors, the contents of gossypol and cyclopropenoid fatty acids were essentially the same between the control and the modified cotton. Protein PAT is degraded by gastric juice of animals and by similar to human artificial gastric juice, losing its physicochemical characteristics. Therefore, it is not expected that the protein may be fully absorbed being therefore unlikely that it may produce adverse toxic effects. As mentioned above, PAT protein is expressed in very low levels in event 281-24-236 and cotton 281-24-236/3006- 210-23; this protein was seldom detected in event 3006-210-23. References to acute toxicity are described in the sites of the United States Environment Protection Agency, (http://www.epa.gov/fedrgstr/EPA-PEST/1997/April/Day-11/p9373.htm), and at the European Commission Health and Consumer Protection Directorate341/ 2009 14 68 General, (http://ec.europa.eu/food/fs/sc/oldcomm7/out02en.htm) indicating lack of toxicity of PAT protein. Regarding proteins Cry1F and Cr1Ac, according to the United States Environment Protection Agency (http://www.epa.gov) one may state with reasonable certainty that there are not risks to humans caused by exposure to such proteins. The above considerations are also in the report of Cotton Biosafety, event 281- 24-236/3006-210-23, submitted by Dow AgroSciences. Toxicity and allergenicity data of this same event were submitted to and analyzed by the United States Environment Protection Agency that considered both proteins Cry1F and Cr1Ac, pesticides, innocuous to Human and Animal Health. It shall be mentioned that recently, in Brazil, EMBRAPA released a larvicide to combat dengue using Bacillus thuringiensis (FSP 04.06.2008) to be added to water for human consumption. The release notice contained the phrase “This product is totally health and environment safe. In case a child drinks a little water containing the larvicide, this is not a problem”. It is worth stressing that the greater concentration of the Cry proteins is in the cotton leaves and that in the cottonseed, the part potentially consumed by man and animals, the concentration is smaller. In cotton bran none of the inserted protein (Cr1Ac, Cry1F and PAT) was detected. In case they are consumed by animals (bran and kernel) and by man (cottonseed oil) the proteins will be rapidly digested by the stomach, before being available for absorption. Cotton bran is preferably destined to ruminants because of the presence of gossypol, which is toxic for non-ruminants: not an impediment for its use provided inclusion in the diet remains at levels lower than 10%. For ruminants, cotton bran is used in larger quantities, but a large part is 341/2009 15 68 degraded at the rumen and the remaining is digested by the true stomach, the abomasum. For humans, the main ingestion is through cottonseed oil, that has extremely low levels of proteins (generally below detection threshold) and in case the inserted proteins (Cry1F, Cr1Ac and PAT) are contained in the oil, their quantities will be even smaller. This fact, coupled with the rapid stomach digestion of these proteins and thermal lability above 75ºC makes any risk to human health extremely unlikely. Another fact contributing towards reducing the likelihood of toxicity, both for man and animals, is the specificity of the Cry proteins that, in order to act, bind to receptors that are present only in target-insects. The PAT protein, which grants resistance to glufosinate ammonium, was obtained from Streptomyces viridochromogenes, present in the soil, recognized as nonpathogenic to man and animals. Tests conducted with inclusion of cotton bran in poultry rations failed to show any adverse effect in the weight gain and mortality rate. It is worth stressing that the feed conversion rate of modified bran was better than the rates of controls, that is to say, the birds consumed a smaller amount of ration to gain the same weight. Another important fact is that the test begun with newly-born birds, sensitive to any kind of adversity and, even so, the presence of modified cotton in the diet did not have any negative effect. In an acute toxicity test, conducted with CD1 mice, a dose of 2000 mg/kg of microbially produced Cry1F and Cr1Ac was fed, with no record of severe pathologic lesion and with animals gaining weight. For being proteins, the risks of allergenic effects were also assessed. Allergens originated from food are normally resistant to heat, acid and proteases, may be glycosylated and present in high concentrations. The proteins essayed are promptly digested by gastric juice, are not glycosylated, and heating leads to loss of bioactivity. Experiments conducted in animals fail do suggest any allergenic 341/2009 16 68 potential. Both B. thuringiensis and Streptomyces viridochromogenes did not have records of being allergy-triggering factors, and as both are present in the environment, mainly in the soil, they may be ingested as food contaminants, mainly vegetables, without causing adverse effects (no records in the literature). Genes introduced in modified organisms do not codify known allergens and fail to share immunologically significant sequences. The sequence of amino acids of proteins Cry1F, Cr1Ac and PAT were compared with two data banks using the software provided by Genetics Computer Group and no allergenic sequence was identified. The above facts, together with the small quantity of modified proteins present in the diet, rapid gastric digestion (one minute for proteins Cry1F and Cr1Ac and less than 30 seconds for protein PAT) and their thermolability make the risk of triggering an allergic reaction practically inexistent. Safety assessment of food derived from genetically modified organisms is entirely based on the concept of substantial equivalence. The doctrine emerged and has been basically discussed by the international community in the context of safety assessment of new foods. According to the doctrine, if a genetically modified product maintains the same characteristics, composition, nutritional values and utility of another non-modified product, there is no motive to segregate it from the remaining so-called conventional ones for the reason that Molecular Biology tools were used, since they are in fact the same product, obtained by different production methods. According to the substantial equivalence doctrine, a food may only cease being an equivalent of another when a scientific assessment finds a characteristics such as composition, nutritional value, nutritional effect or utility that differentiates the product from a corresponding already existing food. 341/2009 17 68 In general, the assessment needed for approving the marketing of such products includes analysis of the transformation vectors, molecular structure of the newly inserted gene, intentional and non-intentional effects associated to its expression, chemical composition in macro- and micronutrients and toxic compounds and products secondary to metabolism. Besides, biological essays (both nutritional and toxicologic ones), chemical analyses, in silico essays (bioinformatics) and biological and biochemical essays shall be conducted to check any allergenic potential of the heterolog protein. Consumption of alimentary cotton products by man is very limited. Therefore, there will be an insignificant exposure to Cry1F, Cr1Ac and PAT proteins in the human diet. The predominant food product derived from cotton is the seed oil, where the protein content is null. On the other hand, animals may consume cotton seed, bran, husk and byproducts as part of their feeding. However, an investigation of the respective products of heterolog expression showed that the recombinant proteins displayed the expected molecular and catalytic characteristic. One fact in favor of protein PAT safety is that transacetylases (the category of PAT) are very common in nature (found in microbes, plants and animals) and are recognized as being non-toxic and non-allergenic. Besides, up to the present, there was no record of potential glycosylation sites or peptide, a sign that they could result in transportation to the endoplasmatic reticulum, place where potential glycosylation could occur, were recorded. PAT protein rapidly degrades in high temperatures. Data about its behavior in simulated digestive fluids and acute oral toxicity caused EPA to issue a final notice in 1977 exempting PAT of the need to establish a tolerance level for all unprocessed commodities when used as an inert protection incorporated to the plant. 341/2009 18 68 Regarding Bt proteins, decades of experiments testing the proteins showed their absence of toxicity to man and vertebrate animals and absence of adverse effects to non-target organisms and the environment. Besides, commercial formulations of B. thuringiensis containing such proteins have been used in Brazil and other countries to control some agricultural pests for over 40 years. Since this bacterium is a soil microorganism, the exposure of living organisms and environment to this bacterium or any element extracted thereof is an event that occurs abundantly in nature. Besides, proteins Cry1F and Cr1Ac have very specific action and operate, only through ingestion, in some species of the Lepidoptera order. Further favoring the safety of such recombinant proteins, data were submitted from the complete characterization of each protein. The more significant data are: absence of homology of the proteins with known toxins and allergenics; pronounced thermolability; rapid digestion under simulated gastric conditions; absence of glycosylation; absence of acute toxicity in rodents; and absence of adverse effects in poultry fed with rations containing the recombinant proteins. Besides, all possible interactions among proteins Cry1F, Cr1Ac and PAT were assessed to estimate possible unforeseeable interactions that may cause adverse effects to man and animals. All results supported the initial evidences that the stacked cotton safe from the alimentary viewpoint. Data submitted by applicant proved that the heterolog proteins are present in very low quantity and in different tissues, an excellent sign of the recombinant proteins safety for humans, since they are clearly different from those typically presented as allergenic proteins. In general, allergenic proteins are reserve proteins of plant organs or tissues, pathogeny- or stress-related proteins, and, in addition allergenic proteins are abundant in the plant and present in high levels over the 341/2009 19 68 plant tissues. Experiments additionally proved that genetically modified cotton plants fail to exhibit morphologic, phenologic or architectural alterations and that there was no effect of the gene insertion to the quality of fibers. Except for the tolerance to target-insects along the crop, the genetically modified cotton plants displayed equivalence in all the analyzed phenotypic and agronomic characteristics against the standard displayed by the non-transformed parental lineage and other varieties used in commercial farming. Examination of documents submitted supports the conclusion that cultivation of cotton event 281-24-236/3006-210-23 will not cause changes in soil and its ecologic and functional relations different from the ones caused by conventional cotton varieties. Besides, studies conducted did not show changes in main natural components and antinutrients found in cotton. Safety of alimentary products from the transgenic cotton was determined by equivalence in composition of macro- and micronutrients in salubrity studies with animals. The conclusion was that the product, as a component of animal fodder and the recombinant proteins expressed in the plant tissues proved to be safe and displaying a nutritional value equivalent for human and animal consumption. Quality and composition analyses of seeds from cotton event 281-24-236/3006-210-23 showed that the properties of the genetically modified cotton and its processed fractions were comparable to the properties of conventional cotton. For the foregoing, based on the data submitted by applicant and independent papers consulted in the literature and considering the internationally accepted criteria in the process of risk analyses of genetically modified raw-materials, based on the concept of substantial equivalence, a conclusion emerges that cotton event 281-24-236/3006-210-23 is as safe for human and animal 341/2009 20 68 consumption as its conventional equivalent. V. Environmental Aspects Modern agriculture is an activity responsible for significant negative environmental impacts(9, 42, 79, 155, 193) and, therefore, the risk assessment of any GM event shall be conducted in relation to that impact inherent to conventional agriculture(13, 46, 138). Therefore, the analysis conducted by CTNBio intended to assess whether the impact caused by WideStrike Cotton is significantly higher than the one caused by conventional cotton varieties considering the practices associated to each system. All species of the Gossypium genus posses perfect flowers. Fecundation takes place promptly after anthesis, and either self-fecundation, crossed pollination or both are possible. The cotton plant pollen is relatively large, ranging from 81 to 143 micra, viscous (making the pollen grains to adhere to each other), spherical in format, covered by a large amount of spicules and in practice is not transported by wind(47). In the fields, its viability extends to late afternoon, but may last for up to 24 hours if stored at temperatures from 2ºC to 3ºC(27). In order for crossed fecundation to happen, presence of pollinating insects is necessary, mainly those insects of the Hymenopterae family(29, 149, 150, 164). The crossing rate recorded between cotton plant cultures is relatively low, displaying figures that enable classifying cotton as a partially autogamous species or a species of a mixed reproduction system. Some authors suggest that gene flow from GM plants to wild genotypes may result in biodiversity reduction. However, reduction of genetic variability results from gene introgression, a process far more complex than simple hybridization(46, 49, 80, 175). In order for introgression to happen, hybridization is first necessary and 341/2009 21 68 later a series of retro-crossings so that a gene be incorporated in a permanent way into a new genome(80, 81). Ecotoxicity studies with Cry1F and Cr1Ac were conducted in soil invertebrates and the results showed that protein Cry1F or Cr1Ac derived by microbial way, either pure or in combination with protein Cr1Ac or Cry1F failed to display toxicity to earthworms (Eisenia foetida). A laboratory study was also conducted to determine the chronic effects of the protein Cry1F to survival and reproduction of the collembolan soil invertebrate (Folsimia candida), which plays an important role in soil ecosystems due to the fact that it feeds on decomposed plant material, using Cry1F derived by microbial way added to beer yeast, the staple collembolan diet(192). The tested concentration was 709 mg of Cry1F protein per kg of diet, or 702 mg of Cry1F protein per kg of diet in combination with protein Cr1Ac. There was no noticeable effect with the exposure to protein Cry1F in the diet. A laboratory study was conducted with protein Cr1Ac to determine the chronic effects of this protein by microbial way added to beer yeast, the staple collembolan diet(192). Protein Cr1Ac failed to cause significant effects to survival and reproduction of adults. The effects of proteins Cry were tested in water organisms and no adverse effects were recorded for the water invertebrate Daphnia magna. Acute toxicity from protein Cry1F for rainbow trout (Onchorynchus mykis) was determined for fish exposed for eight days to a commercial type of trout pelletized diet containing 10% of bran prepared with cotton expressing proteins Cry1F and Cr1Ac(128). This produced a diet containing an initial dose of 0.209 gram of Cry1F per gram of food in combination with protein Cr1Ac. The control diet consisted of the same commercial fish diet prepared with non transgenic cotton bran. No mortality of fish of sub-lethal effects were reported for both the fish feeding on control diet and fish 341/2009 22 68 feeding on the GMO diet. Acute toxicity from the diet with protein Cr1Ac to the rainbow trout (Onchorynchus mykiss) was determined for individuals exposed for eight days to a commercial-type trout pelletized diet containing 10% of bran prepared with cotton expressing proteins Cry1F and Cr1Ac(128). This produced a diet containing an initial dose of 0.118 gram of Cr1Ac per gram of food in combination with protein Cr1Ac. The control diet consisted of the same commercial diet prepared with non-transgenic cotton bran. No mortality of fish or sub-lethal effect was recorded for both, the control diet and the diet containing the GMO. Regarding non-target arthropods, no effects were recorded in average survival of bees exposed to 2 mg of pollen of event expressing Cry1F , or 1.98 mg/ml of protein Cry1F in combination with protein Cr1Ac(124). CL50 in a diet for green lacewing (Chryosperia carnea) exposed to protein Cry1F, pure or in combination with protein Cr1Ac, was investigated in a series of studies with the microbial protein administered in a diet of moth eggs(185). There was no effect of Cry1F at 5.2 mg/g in combination with Cr1Ac. The test with Cry1F alone also failed to show any effect. Toxicity for green lacewing is not held as ecologically relevant for risk assessment of cotton event 281-24-236, since the exposure, as case it happens, shall be indirect and results of field censuses failed to reveal any impact of protein Cry1F(125). Parasite hymenoptera (Nasonia vitripennis) were exposed to a single boundary concentration of protein Cry1F, pure and in combination with protein Cr1Ac, in sugared water for 10 days. There were no significant difference in mortality between the two groups of treatment and a control negative for sugared water on day 9, which was the last day of observation before mortality increased beyond the ceiling established in the protocol for negative control(20%). The same result was obtained with protein Cr1Ac, pure and in combination with protein Cry1F. Adult ladybugs (Hipodamia convergens) were not affected when 341/2009 23 68 exposed to protein Cry1F or Cr1Ac expressed by microbial way, pure or in combination with protein Cr1Ac or Cry1F. Non-target organisms may be either directly or indirectly exposed to protein Cry1F expressed in 281-24-236. Exposure estimates for organisms feeding directly on cotton tissues expressing protein Cry1F are based on the upper limit of expression in the specific plant tissue to which the non-target organism may be exposed through direct ingestion. Estimates of upper exposure limit (UEL) represent 90% of the upper limit of expression recorded(200). Indirect exposures represent unadvised exposures to protein Cry1F through the soil, pollen on tissues of the host plant, and multitrophic interactions. Such exposures are known as Estimated Environmental Concentrations (EEC) and are calculated in a conservative way using the higher estimates for the parameters used in the calculation. Direct feeding on plants of parts or plants constitute the primary ways of exposure of organisms to protein Cry1F expressed in event 281-24-236 and to protein Cr1Ac expressed in event 3006-210-23. The plant parts subject to feeding are mainly the leaves, roots, stalks and, possibly, nectar. Organisms feeding directly on cotton as a primary source of food within agroecosystems are characterized as plant pests and are not analyzed here. Organisms incidentally exposed to residues of plants and organisms consuming cotton plants of parts or cotton plants as an occasional or supplementary source of food are held as non-target organisms that deserve consideration in this exposure assessment. Secondary exposure to protein residues through tritrophic interactions may occur for predators or parasites of organisms that feed on plants. Residues occurring in the soil or water matrices may be a way of additional secondary exposure to protein Cry1F and Cr1Ac. Absence of effects in ecotoxicity tests for non-target organisms 341/2009 24 68 show large safety margins related to environment concentrations of foreseen exposures, in a conservative manner, and such observations are supported by field monitoring of the species abundance(125). Exposure ways postulated in here are therefore relevant for exposure and risk characterization for the rate of Lepidoptera potentially susceptible. Conditions favorable to degradation of proteins Cry1F and Cr1Ac in the soil were described in studies where proteins Cry1F and Cr1Ac, either derived from plants or derived by microbial way were mixed to the soil, incubated under standard laboratory conditions, and then sampled for bioassay in different time intervals(84). Bioessays were performed in insects to measure protein degradation, as loss of biological activity, by applying mixtures of water-agar in soil samples put in artificial diets and leaving the neonate caterpillars of tobacco budworm (Heliothis virescens) to feed on this prepared medium. Based on the bio-essay results (GI50) for soil added of Cry1F, derived by microbial way, the half-life was 1.3 days under laboratory conditions, suggesting a high rate of decomposition in the soil. This fast decomposition was verified in liofilized cotton tissue containing protein Cry1F in combination with protein Cr1Ac, where the half life of bioactivity was below one day. Bioessays with truncated protein Cry1F also displayed a half live in soil of less than one day(85). Microbial protein Cr1Ac failed to decompose when applied to microorganisms in non viable soil(84), but the rapid decomposition took place when the leaf tissue of the liofilized cotton 281-24-236/3006-210-23 containing proteins Cry1F and Cr1Ac was applied in viable soil(84). These results are consistent with degradation of protein Cr1Ac in soil mediated by microorganisms. Based on results of the bioassay (GI50) in soil to which liofilized cotton tissue was added, the half life of crystallized insecticides Cry1F/Cr1Ac was 1.3 day under laboratory conditions, suggesting a high decomposition rate in soil. 341/2009 25 68 A soil representative of cotton agroecosystem was examined in a laboratory study. The protein decomposed very fast and was consistent with what has been seen for other Bt proteins in a lineage of soils(86, 87). The study submitted demonstrates the ability of soil microbes to rapidly degrade such Bt proteins. In addition, field studies including multiple locations and a lineage of soils were planned to focus the potential accumulation of proteins Cry1F and Cr1Ac expressed in cotton 281-24-236/3006-210-23. A report published in one of such multiple field studies found non accumulation(83) and served as a model for the study conducted with cotton 3006-210-23/281-24-236. Bt cotton, when compared with conventional cotton with insecticides, failed to affect arthropod populations collected in soil pitfalls, yellow tray and yellow adhesive card. The main arthropods collected in such pitfalls were the phytophagous Euxesta sp., Chaetopsis sp., Liriomyza sp., Aphis sp. and thrips of the Thripidae family; predators Dolichopodidae sp., parasitoids Aphididae, Aphidius sp. and Encyrtidae; and decomposers Phora sp and Sarcophagidae. Bt cotton failed to affect occurrence of predator arthropods in plants, among which one may mention bugs Orius sp., Nabis sp. Geocoris sp. and Zelus sp; ladybugs Cycloneda sanguine, Scymnus sp., Hyppodamia convergens and Stetorus sp.; spiders Chiracantum sp., Oxyopes sp., Phidippus sp., Misumenopsis sp., and Latrodectus, sp.; crysopid Ceraeochrysa sp., Doru luteipes and the sylphid Toxomerus dispar. Hymenoptera parasitoids captured in yellow trays were not abundant in any of the treatments, totaling 3% of collected insects. The conclusions were that: - Bt cotton failed to cause a negative impact on arthropod populations in the soil; - Bt cotton, when compared with conventional cotton with insecticides, 341/2009 26 68 failed to affect the arthropod population collected in soil pitfalls, yellow tray and adhesive card; - Bt cotton failed to harm the occurrence of predator arthropods in plants. Cotton is basically a culture of self-fecundation, with some crossed pollination by bumble bees, Melisode bees and honeybees; Lepidoptera insects are not pollinators of cultivated cotton(129). Consequently, environmental exposure of a lepidopter sensitive to cotton pollen would be indirect, through contamination of their source of food by noxious species. Indirect exposure to cotton pollen was considered to be non significant. Incidental exposure of a non-target butterfly or moth, in the larval stage sensitive to Cry1F may occur if the pollen of 281-24-236 cotton is present in host plants and is consumed. In a study conducted in the United States, indirect exposure of a larva of a hypothetical non-target lepidopter sensitive to cotton pollen was insignificant, as shown in the case of the Monarch butterfly feeding on Sapium Glandulatum, as an example of a non-target lepidopter occurring in host plants inside or close to cotton fields. The likelihood of exposure is remote due to the non-significant pollen flow of cotton; there is, therefore, an insignificant risk for non-target butterflies and moths present in cotton fields of event 281-24-236. The Monarch butterfly was simply used as a model species for the purpose of this assessment, since it would be minimally exposed to pollen of event 281-24-236, since its migration in spring through cotton cultivation regions takes place before flowering time. Incidental exposure of a non-target butterfly or moth, during the larval stage sensitive to Cr1Ac may occur if the pollen of cotton 3006-210-23 is present in host plants and is consumed. Indirect exposure of a non-target lepidopter larva sensitive to cotton pollen is insignificant. The likelihood of exposure is remote due to the insignificant dispersion of cotton pollen. The 341/2009 27 68 Monarch butterfly was used as a model species for the purpose of this assessment. The consequences of cultivating cotton expressing proteins Cry1F and Cr1Ac on beneficial insects was studied by Wolt (2002)(206). Over 300 species of beneficial insects are known inhabitants of cotton fields. Common arthropods, predators and parasites in cotton fields represent orders that are not sensitive to Cry1 proteins. Besides, such organisms are predominantly predators and parasites, and only in few examples they consume plant products (pollen and nectar) and, in these cases, consumption is made by adults, in a stage of life when the species is no longer sensitive. Direct risks to beneficial insects, therefore, of exposure to protein Cry1F expressed in cotton event 281-24-236 and to protein Cr1Ac expressed in cotton event 3006-210-23 are negligible for practical purposes. Indirect risk to proteins Cry1F and Cr1Ac through tritrophic feeding with host/prey insects is also non-significant, due to the low levels of exposure foreseen in comparison with levels of effects shown in the tests conducted. Security margins shown in risk assessments are very conservative, since they are based on a level of expression of the proteins in plants, while secondary exposure would be significantly reduced in tritrophic feeding. Based on the above analysis, there are no ecologically relevant concerns with cultivation of cotton 281-24-236/3006-210-23 expressing proteins Cry1F and Cr1Ac. Selectivity, exposure ways and concentrations restrict any potential risk posed to lepidopter insects directly exposed to residues of event 281-24- 236/3006-210-23. For this rate, the likelihood of an adverse environmental consequence is insignificant, as shown by the toxicity test of non-target organisms, which indicate concentrations with no effect in doses well above the concentrations of the environmental exposure foreseen in a conservative manner. 341/2009 28 68 Abundance in fields corroborates the absence of risk at the typical rates of cotton agroecosystems. Experience with commercial farming in other countries corroborates such experimental results. No adverse environmental effect associated with this product was detected. VI. Restrictions to the use of GMO and derivatives Technical opinions related to agronomic performance concluded that there is equivalence between transgenic and conventional plants. Therefore, the data suggest that transgenic cotton plants are not fundamentally different from the genotypes of non-transformed cotton plants, except for the resistance to certain insects. In addition, there is no evidence of adverse reactions to the use of WideStrike Cotton event 281-24-236/3006-210-23. For the foregoing, there are no restrictions to the use of this cotton or its derivatives, either as human or animal food. As established by Article 11 of Law no. 11,460, of March 21, 2007 “research and cultivation of genetically modified organisms may not be conducted in indigenous lands and areas of conservation units.” VII. Considerations on particulars of different regions of the country (contribution to supervision agencies) WideStrike cotton event 281-24-236/3006-210-23 technology was shown to be usable under all agricultural practices commonly used in different regions under different conditions, considering availability of inputs and labor, among other inputs used in the culture of cotton. There are not creole varieties of cotton plants and the chains of special cotton plants, both conventional and transgenic, have lived together in a satisfactory 341/2009 29 68 fashion, without any record of coexistence problems. VIII. Conclusion Whereas:*** 1. Event 281-24-236/3006-210-23 Cotton is as safe for human and animal consumption as the conventional cotton; 2. All possible interactions among proteins Cry1F, Cr1Ac and PAT were assessed in order to estimate any possible unforeseen interactions that my cause adverse effects to man and animals, and all results supported the initial evidences that stacked cotton is safe from the alimentary point of view; 3. Event 281-24-236/3006-210-23 Cotton contributes to reduce the use of insecticides and, consequently, reduces the impacts of such pesticides in the environment, human and animal health; 4. The use of technology such as insect-resistant Bt cotton may have a positive impact to population preservation of non-target organisms and beneficial insects, making integrated management of crop pests easier; 5. Cultivation of event 281-24-236/3006-210-23 cotton will not cause changes in the soil and its functional and ecologic relations different from the changes caused by conventional corn varieties; 6. Proteins Cry1F and Cr1Ac have very specific action and act, through ingestion only, in some species of the Lepidoptera order; 7. Protein PAT was extensively discussed by CTNBio during analysis 341/2009 30 68 related to commercial release of Liberty Link corn, Bt corn and Liberty Link cotton, where the conclusion was that such protein has no known toxic and allergenic potential and that is rapidly digested by enzymes (proteases) in the digestive system of animals, eliminating most of this protein potential to be allergenic when consumed; 8. Assessment of proteins Cry1F and PAT expressed in event 281-24-236 and proteins Cr1Ac and PAT expressed in event 3006-210-23 failed to identify any allergenicity potential in food products; 9. The event produces a protein that is toxic to some cotton pest insects only, and does not have properties allergenic to mammals; 10. The source organism of proteins Cry1F and Cr1Ac, Bacillus thuringiensis, and PAT, Streptomyces viridochromogenes are not recognized as allergenic; 11. There are no ecologically relevant concerns with cultivation of 281-24- 236/3006-210-23 cotton expressing proteins Cry1F and Cr1Ac, since no adverse environmental effect associated to this product was recorded; 12. Event 281-24-236/3006-210-23 Cotton is nutritionally equivalent to conventional cotton; 13. Nutritional and productive equivalence of WideStrike Cotton was evidenced in different research works conducted in the United States and herein enclosed; 14. Nutritional quality of cotton remained unchanged related to ashes, total fat, humidity, protein, carbohydrate, calorie, total fiber, fiber in acid 341/2009 31 68 detergent, fiber in neutral detergent, amino acids in seeds and bran, fatty acids in seed and seed oil, tocopherols in seed oil fraction, antinutrients in leaf and floral bud and seed, oil and bran; 15. Crossing of WideStrike Cotton event 281-24-236/3006-210-23 with wild kindred cotton and generation of hybrid offspring that may change into pest plants harmful to agriculture or plants invaders of natural habitats is unlikely; 16. Finally, given the detailed data submitted by applicant, the results obtained in control and safety essays on this genetically modified organism, the elements credited to authors of scientific works mentioned and inexistence of facts contrary to nutritional, toxicological and allergenic safety, no matter how much they have been scrutinized; CTNBio finds that this activity is not a potential cause of significant degradation to the environment nor harmful to human and animal health. Restrictions to the use of such GMO and its derivatives are conditions to the provisions of CTNBio Ruling Resolution nº 03 and Ruling Resolution nº 04. According to Annex I of Ruling Resolution 5, of March 12, 2008, applicant shall make adjustments to its proposed post-commercial release monitoring plan within thirty (30) days from publication of this Technical Opinion. IX. Bibliography 1. AGBIOS. Agbios. Database product Description. www.agbios.com. 2008. 2. ABEL, C.A.; ADAMCZYK, J. J. JR. Relative concentration of Cry1A in maize leaves and cotton bolls with diverse chlorophyll content and 341/2009 32 68 corresponding larval development of fall armyworm (Lepidoptera: Noctuidae) and southwestern corn borer (Lepidoptera: Crambidae) on maize whorl leaf profiles. J. Econ. Entomol. 2004 Oct; 97 (5): 1737-44. 3. ADAMCZYK, J. J., JR; ADAMS, L. C; HARDEE, D. D. Field efficacy and seasonal expression profiles for terminal leaves of single and double Bacillus thuringiensis toxin cotton genotypes. J. Econ. Entomol. 2001 Dec; 94(6) 1589-93. 4. ADAMCZYK, J. J., J.R.; SUMERFORD, D. V. Potential factors impacting season-long expression of Cry1Ac in 13 commercial varieties of Bollgard cotton. J. Insect Sci. 2001; 1:13. Epub 2001 Nov 9. 5. ADAMCZYK, J. J.; HARDEE, D. D.; ADAMS L. C.; SUMERFORD, D. V. Correlating differences in larval survival and development of bollworm (Lepidoptera: Noctuidae) and fall armyworm (Lepidoptera: Noctuidae) to differential expression of Cry1A(c) d-endotoxin in various plant parts among commercial cultivars of transgenic Bacillus thuringiensis cotton. J. Econ Entomol. 2001 Feb; 94(1): 284-90. 6. AKHURST, R. J.; JAMES, W.; BIRD L. J.; BEARD, C. Resistance to the Cry1Ac delta-endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). J. Econ Entomol. 2003 Aug; 96(4): 1290-9 7. AKIAMA, H.; WATANABEAT, T.; KIKUCHI, H.; SAKATA, K.; TOKISHITA, S.; HAYASHI, Y.; HINO, A.; TESHIMA, R.; SAWADA, J.; MAITANI, T. A detection method of Cry1Ac protein for identifying genetically modified rice using the lateral flow strip assay. Shokuhin Eiseigaku Zasshi. 2006 Jun; 47(3): 111-4. 341/2009 33 68 8. ALI, M.I.; LUTTREL, R. G.; YOUNG, S.Y. 3 rd. Susceptibilities of Helicoverpa zea and Heliothis virescens (Lepidoptera: Noctuidae) populations to Cry1Ac insecticidal protein. J. Econ Entomol. 2006 Feb; 99(1):164-75. 9. AMMANN, K. 2005. Effects of biotechnology on biodiversity: herbicidetolerant and insect-resistant G. M. crops. Trends Biotech. 23:388-394. 10. A., M., SHELTON; J., -Z., ZHAO; AND, R. T., ROUSH (2002) Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annual Review of Entomology Vol. 47: 845-881 11. ANILKUMAR, K. J.; RODRIGO-SIMÓN, A.; FERRÉ, J.; PUSZTAICAREY, M.; SIVASUPRAMANIAM, S.; MOAR, W. J. Production and characterization of Bacillus thuringiensis Cry1Ac-resistant cotton bollworm Helicoverpa zea (Boddie). Appl. Environ. Microbiol. 2008 Jan; 74(2): 462-9. Epub 2007 Nov 16. 12. ASHFAQ, M.; YOUNG, S. Y.; MCNEW, R., W. Larval mortality and development of Pseudoplusia includens (Lepidoptera: Noctuidae) reared on a transgenic Bacillus thuringiensis-cotton cultivar expressing Cry1Ac insecticidal protein. J. Econ Entomol. 2001 Oct;94(5): 1053-8. 13. BARTSCH, D.; SCHUPHAN; I. 2002. Lesson we can learn from ecological biosafety research. J. Biotech. 98: 71-11. 14. BATES, S. L.; ZHAO, J. Z.; ROUSH R. T.; SHELTON, A. M. Insect resistance management in G. M. crops: past, present and future. Nat Biotechnol. 2005 Jan; 23(1): 57-62. 341/2009 34 68 15. BENEDICT, J.; & ALTMAN; D, 2001. Commercialization of transgenic cotton expressing insecticidal crystal protein. In J. Jenkins & S. Saha (Eds), Genetic improvement of cotton: Emerging technologies (pp. 137- 201).Enfield, NH: Science Publishers Inc. 16. BERTRAND, J. A.; SUDDUTH, T. Q.; CONDON, A.; JENKINS, T. C.; CALHOUN, M. C. Nutrient content of whole cottonseed. J. Dairy Sci. 2005 Apr; 88 (4): 1470-7. 17. BETZ, F. S.; HAMMOND, B. G.; FUCHS S. L. Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regul Toxicol Pharmacol. 2000 Oct; 32(2): 156-73. Review. 18. BIRD, L. J.; AKHURST, R. J. Fitness of Cry1Ac-resistant and susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on transgenic cotton with reduced levels of Cry1Ac. J. Econ. Entomol.2005 Aug; 98 (4):1311-9. 19. BIRD, L. J.; AKHURST, R. J. Relative fitness of Cry1Ac-resistant and susceptible Helicoverpa armigera (Lepidoptera: Noctuidae) on conventional and transgenic cotton. J. Econ. Entomol. 2004 Oct; 97 (5): 1699-709. 20. BIRD, L. J.; AKHURST, R. J. Variation in susceptibility of Helicoverpa armigera (Hübner) and Helicover papunctigera (Wallengren) (Lepidoptera: Noctuidae) in Australia to two Bacillus thuringiensis toxins. J Invertebr Pathol. 2007 Feb; 94(2): 84-94.Epub 2006 Oct 17. 21. BLANCO, C. A.; PERERA, O. P.; BOYKIN, D.; ABEL, C.; GORE, J.; MATTEN, S. R.; RAMIREZ-SAGAHON, J. C.; TERÁN-VARGAS, A. P. Monitoring Bacillus thuringiensis-susceptibility in insect pests that occur in 341/2009 35 68 large geographies: how to get best information when two countries are involved. J Invertebr Pathol. 2007 Jul; 95(3):201-7. Epub 2007 Mar. 22. BLANCO, C. A.; STORER, N. P.;ABEL, C. A.; JACKSON, R; LEONARDO, R.; LOPES, J. D. J. R.; PAYNE, G.; SIEGFRIED, B. D.; SPENCER, T.; TERÁN-VARGAS, A. P. Baseline susceptibility of tobacco budworm (Lepidoptera: Noctuidae) to Cry1Ftoxin from B bacillus thuringiensis. J. Econ. Entomol. 2008 Feb; 101 (1): 168-73. 23. BRADFORD, K. J.; DEYNZE, A. V.; GUTTERSON, N.; PARROT, W.; STRAUSS, S. H. Regulating transgenic crops sensibly: lessons from plant breeding, biotechnology and genomics. Nature Biotechnology, 23(4): 439-444, 2005. 24. BRODERICK, A. N.; RAFFA, K. F.; HANDELSMAN, J. Midgut bacteria required for Bacillus thuringensis insecticidal activity. Proc. Natl. Acad. Sci,103:15196-151999. 25. BUCCHINI, L., GOLDMAN, L. R. STARLINK corn: a risk analysis. Environ Health Perspect. 2002 Jan;110 (1):5-13. Review. 26. BUNDY, C.S., MCPHERSON, R. M. Dynamics and seasonal abundance of stink bugs (Heteroptera: Pentatomidae) in a cotton-soybean ecosystem.J. Econ. Entomol. 2000 Jun; 93(3):697-706. 27. CALHOUN, D. S.; BOWMAN, D. T. 1999 Techniques for development of new cultivars. In: SMITH, C. W.; COTHREN, J. T. Cotton: origin, history, technology and production, p. 361-414. New York : John Wiley e Sons, p. 361-414. 341/2009 36 68 28. CAPRIO, M. A.; FAVER, M. K.; HANKINS, G. Evaluating the impacts of refuge width on source-sink dynamics between transgenic and nontransgenic cotton. J Insect Sci. 2004; 4:3. Epub 2004 Feb 9. 29. CARDOSO, C. F.; SILVEIRA, F. A.; OLIVEIRA, G.; CAVÉCHIA, L. A.; ALMEIDA, J. P. S.; SUJII, E. R.; FONTES, E. M. G.; PIRES, C. 2007.Principais polonizadores de Gossypium hirsutum var. latifolium Malvaceae, cv. Delta Opal, em uma localidade do Distrito Federal, Brasil. In: VI Congresso Brasileiro do Algodão, Uberlandia. Anais do VI Congresso Brasileiro do Algodão. 30. CARPENTER, J.;FELSOT, A.; GOODE, T.; HAMMIG, M. ONSTAD, D.; & SANKULA, S. 2002. Comparative environmental impacts of biotechnology-derived and traditional soybean, corn, and cotton crops (CAST: I-189). Ames, IA: Council for Agricultural Science and Technology. 31. CARRIERE, Y; DENNEHY, T. J.; PEDERASEN, B.; HALLER S.; ELLERS-KIRK, C. ANTILLA, L; LIU, Y. B.; WILLOTT, E.; TABASHNIK, B. E. Large-scale management of insect resistance to transgenic cotton in Arizona: can transgenic insecticidal crops be sustained? J. Econ. Entomol. 2001 Apr; 94(2): 315-25. 32. CARRIERE, Y; ELLERS-KIRK, C.; BIGGS, R.; DEGAIN, B.; HOLLEY D.;YAFUSO C.; EVANS, P.; DENNEHY, T. J.;TABASHNIK, B. E. Effects of cotton cultivar on fitness costs associated with resistance of pink bollworm (Lepidoptera: Gelevhiidae) to Bt cotton. J. Econ. Entomol.2005 Jun; 98(3):947-54. 341/2009 37 68 33. CARRIERE, Y.; ELLERS-KIRK, C.; BIGGS, HIGGINSON, D. M.; DENNEHY, T. J.;TABASHNIK;B.E.; Effects of gossypol on fitness costs associated with resistance to Bt cotton in pink boll worm. J. Econ. Entomol. 2004 Oct; 97(5):1710-8. 34. CARRIERE, Y.; ELLERS-KIRK, C; BIGGS, R. W., NIBOER, M. E.; UNNITHAN, G. C.; DENNEHY, T. J.; TABASHNIK, B. E. Cadeherinbased resistance to Bacillus thringiensis cotton in hybrid strains of pink boll worm: fitness costs and incomplete resistance. J. Econ. Entomol. 2006 Dec; 99(6): 1925-35. 35. CARRIERE, Y.; ELLERS-KIRK, C; KUMAR, K.; HEUBERGER, S.;WHITLOW, M.; ANTILLA, L.; DENNEHY, T. J.;TABASHNIK, B. E. Long-term evaluation of compliance with refuge requirement for Bt cotton. Pest Manag. Sci. 2005 Apr; 61(4): 327-30. 36. CARRIERE, Y.; ELLERS-KIRK, C; LIU, Y. B.; SIMS, M. A.; PATIN, A. L.; DENNEHY, T. J.; TABASHNIK, B. E. Fitness costs and maternal effects associated with resistance to transgenic cotton in the pink bollworm (Lepidoptera: Gelevhiidae). J. Econ. Entomol. 2001 Dec; 94(6): 1571-6. 37. 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Walter Colli President of CTNBio

341/2009 67 68 Dissenting Vote: Rapporteurs, Doctor Paulo Brack and Doctor Paulo Kageyama (Permanent Sector Subcommission for the Environment) voted against approval of commercial release genetically modified insect-resistant WideStrike Cotton event 281-24-236/3006-210-23 arguing that: 1. Scattering of seeds through roads is a risk regarding contamination and gene flow between GM and non-GM varieties; 2. Although apparently maintenance of polycultures and use of natural control mechanisms – with no toxic insecticides and no GMOs – are unable to secure immediate return in modern agricultural systems, in the middle term they may be a solution for restoration of equilibrium in agroindustrial systems; 3. A generalized introduction of genetically modified plants may cause an undesirable impact even greater than the one promoted by conventional agriculture, affecting also flora and fauna; 4. The single industrial productivistic pattern of modern agriculture with GMO fails to consider the necessary complexity of natural systems and the inherent sustainability of such different systems that have several million years of evolutionary history; 5. By planting the GM cotton with Bt gen, in one decade the soil would be impoverished in life, negatively affecting food production; 6. There are no studies regarding the main groups of non-target invertebrates, especially those endangered ones mentioned in IBAMA 341/2009 68 68 lists, such as Lepidoptera, and Hymenoptera of the Meliponidae bee group; 7. There is a small number of studies regarding molecular changes in sequences inserted and assessment or such risks in the environment is based on generic assumptions resulting from properties of the species, gene inserted and laboratory studies; 8. Studies on gene stability in multiple situations of several Brazilian biomes should also be conducted prior to commercial release; 9. There are doubts, r of a highly invasive technology that gains space in an indiscriminate way.