<p style="text-align: justify;">Maize, or corn (<i>Zea mays</i> L.) is grown commercially in over 100 countries with a combined harvest of nearly 700 million metric tonnes in 2006. The top five producers of maize in 2005 were the United States, China, Brazil, Argentina, and Mexico, accounting for 70% of world production. Maize is grown primarily for its kernel (grain), the majority of which is used for animal feed, but with significant amounts refined into products used in a wide range of food, medical, and industrial goods.</p>

<p style="text-align: justify;">Maize is a raw material for the manufacture of starch, the majority of which is converted by a complex refining process into sweeteners, syrups, and fermentation products, including ethanol. Maize oil is extracted from the germ of the maize kernel. Only a small proportion of the whole kernel is consumed by humans, while refined maize products such as sweeteners, starch, and oil are abundant in processed foods (<i>e.g.</i>, breakfast cereals, dairy goods, chewing gum).</p>

<p style="text-align: justify;">In the United States maize is typically used as animal feed, with roughly 70% of the crop fed to livestock, however a growing amount is now being used for the production of ethanol. The entire maize plant, the kernels, and several refined products such as glutens and steep liquor, are used in animal feeds. Silage made from the whole maize plant makes up 10-12% of the annual corn acreage, and is a major ruminant feedstuff. Livestock that feed on maize include cattle, pigs, poultry, sheep, goats, fish and companion animals.</p>

<p style="text-align: justify;">Industrial uses for maize products include recycled paper, paints, cosmetics, car parts. Refined maize products are also used in bioproducts such as antibiotics.</p>

<p style="text-align: justify;">The European corn borer (ECB), <i>Ostrinia nubilalis</i>, is the most damaging insect pest of maize in the United States with losses from ECB damage and control costs exceeding $1 billion each year. An average of one ECB cavity per maize stalk across an entire field can reduce yield by as much as 5% when caused by first generation larvae, and 2.5% when caused by second generation larvae, with annual yield losses estimated at 5 to 10 %. Despite consistent losses to ECB, chemical insecticides are utilized on a relatively small acreage (less than 20%). Historically, this reluctance stems from the difficulties in identifying and managing ECB in maize crops: ECB larval damage is hidden, heavy infestations are unpredictable, insecticides are costly, timing of insecticide application is difficult and multiple applications may be required to guarantee ECB control.</p>

<p style="text-align: justify;">Weeds are also a major production problem in maize cultivation. Even a light infestation of weeds can reduce yields by 10 to 15%; severe infestations can reduce yields by 50% or more. Typically, weeds are managed using a combination of cultural (<i>e.g.</i>, seed bed preparation, clean seed, variety selection) and chemical controls. Depending on the production area and the prevalent weed species, herbicides may be incorporated into the soil before planting (pre-plant), applied after planting but before emergence (pre-emergence), or applied after the maize plants emerge (post-emergence). Ideally, for maize production, weeds should be controlled for the full season. However, the most critical period for weed control is usually about six to eight weeks after crop emergence, during the 4th to 10th leaf stages. This critical period in the life cycle of maize must be kept weed free in order to prevent yield loss.</p>

<p style="text-align: justify;">The transgenic maize line DAS-06275-8 (hereafter referred to as <b>TC-6275</b>) was genetically engineered to resist ECB, Southwestern corn borer (SWCB), fall armyworm (FAW), and black cutworm (BCW, and to a limited extent, corn earworm (CEW), by producing its own insecticide. TC-6275 was also developed to express tolerance to the herbicide glufosinate ammonium. Two novel genes, a truncated cry1F gene and the bar gene were introduced into the maize hybrid line Hi-II using <i>Agrobacterium</i>-mediated transformation.</p>

<p style="text-align: justify;">The cry1F gene, isolated from the common soil bacterium <i>Bacillus thuringiensis</i> (<i>Bt</i>) var. <i>aizawai</i>, produces the insect control protein Cry1F, a delta-endotoxin. Cry proteins, of which Cry1F is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to bacterial sepsis. Cry1F is lethal only when eaten by the larvae of lepidopteran insects (moths and butterflies), and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for the delta-endotoxins of <i>B. thuringiensis</i> on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.</p>

<p style="text-align: justify;">TC-6275 maize also was developed to allow for the use of glufosinate ammonium as a weed control option, and as a breeding tool for selecting plants containing the <i>cry</i>1F gene. The herbicidal mode of action of glufosinate ammonium is related to the activity of glutamine synthetase (GS), the enzyme required for the synthesis of the amino acid glutamine. L-phosphinothricin, the active ingredient of glufosinate ammonium, is a structural analog of glutamate, and acts as a competitive inhibitor. After application of the herbicide, L-phosphinothricin competes with glutamine for its active sites on GS. The results of the inhibition of GS are an accumulation of ammonia in the plant, a reduction in the synthesis of glutamine, and an inhibition of photosynthesis. This causes the death of plant cells, and eventually, the entire plant. TC-6275 maize contains the bar gene, which codes for the production of the enzyme phosphinothricin acetyl-transferase (PAT). This enzyme acetylates L-phosphinothricin rendering it inactive in the plant. The PAT enzyme is not known to have any toxic properties. The <i>bar</i> gene was isolated from the soil bacterium <i>Streptomyces hygroscopius</i>, the same organism from which L-phosphinothricin was originally isolated.</p>

<p style="text-align: justify;">TC-6275 was tested in field trials in the United States and Puerto Rico from 1999 to 2003. Data collected from these trials demonstrated that TC-6275 was not different from conventional maize varieties. TC-6275 grew normally and exhibited the expected morphology, reproductive and physiological characteristics of maize. TC-6275 was also shown not to have unexpected pest or disease susceptibility compared to conventional maize.</p>

<p style="text-align: justify;">Maize does not have any closely related species growing in the wild in continental United States. Cultivated maize can naturally cross with annual teosinte (<i>Zea mays</i> ssp. <i>mexicana</i>) when grown in close proximity, however, these wild maize relatives are native to Central America and are not naturalized in the United States. Gene exchange between TC-6275 and maize relatives was determined to be negligible in managed ecosystems, with no potential for transfer to wild species in the United States.</p>

<p style="text-align: justify;">TC-6275 maize was compared to its non-transgenic counterpart, a near-isogenic maize line, for the relative abundance of beneficial arthropods, such as ladybird beetles, minute pirate bugs and ichneumonids. Field studies demonstrated that Cry1F expressed in TC-6275 had neither a direct nor an indirect effect on the beneficial arthropod populations. In summary, it was determined that when compared with currently commercialized maize varieties, TC-6275 maize did not present an increased risk to or impact on interacting organisms, including humans, with the exception of specific lepidopteran insect species.</p>

<p style="text-align: justify;">Regulatory authorities in the United States have mandatory requirements for developers of <i>Bt</i> maize to implement specific Insect Resistant Management (IRM) Programs. The potential exists for <i>Bt</i>-resistant ECB populations to develop as acreages planted with transgenic <i>Bt</i> hybrids expand. Hence, these IRM programs are designed to reduce this potential and prolong the effectiveness of plant-expressed <i>Bt </i>toxins, and the microbial <i>Bt</i> spray formulations that contain these same toxins.</p>

<p style="text-align: justify;">The food and livestock feed safety of TC-6275 maize was established based on several standard criteria. As part of the safety assessment, the nutritional composition of TC-6275 grain was found to be equivalent to conventional maize as shown by the analyses of key nutrients including proximates (<i>e.g. </i>, protein, fat, fibre, ash and carbohydrate), amino acid composition, fatty acid profiles, minerals, and vitamins. Similar compositional analyses were conducted on TC-6275 forage, which was also found to be compositionally equivalent to forage from conventional maize.</p>

<p style="text-align: justify;">The low potential for toxicity of plant-expressed Cry1F protein was demonstrated by the lack of amino acid sequence homology with known protein toxins, by <i>in vitro</i> studies showing that the protein was rapidly degraded in simulated gastric fluids, and from results of acute oral toxicity studies demonstrating no acute toxicity when Cry1F protein was fed to laboratory mice. In the latter study, mice were fed high doses of Cry1F protein with no negative consequences. The doses of Cry1F were 26,000-fold greater than levels to which humans would be exposed, based on the typical consumption of foods derived from TC-6275 maize grain.</p>

<p style="text-align: justify;">The potential allergenicity of Cry1F was assessed by examining: physiochemical characteristics; amino acid sequence homology to known protein allergens; and digestibility in simulated gastric fluids. The Cry1F protein has a history of safe use, demonstrated by its use in microbial <i>Bt</i> spray formulations in agriculture and forestry for more than 30 years with no evidence of adverse effects. This fact, combined with the lack of amino acid sequence homology between Cry1F protein and known allergens, and the rapid degradation of Cry1F protein in simulated gastric fluids, were sufficient to provide with reasonable certainty that Cry1F has no allergenic potential.</p>

<p style="text-align: justify;">The PAT protein in TC-6275 was also evaluated for its potential for toxicity and allergenicity. A sequence homology search revealed no similarity to known toxins and allergens. Results of previous in vitro digestibility studies on PAT protein reveal that it is rapidly digested in simulated gastric fluid. These results, and those of other studies previously submitted to regulatory authorities in support of the safety of PAT, demonstrate its low potential for toxicity and allergenicity.</p>
