<p style="text-align: justify;"><b>Field Testing</b></p>

<p style="text-align: justify;">TC-6275 was field tested at several locations, from 1999 to 2003, in the United States and Puerto Rico. These field tests were conducted to evaluate the agronomic performance of TC-6275 (in 2002 only), the efficacy of the insecticidal protein against the targeted lepidopteran pests, and susceptibility to plant diseases (<i>e.g. </i>, Northern Corn Leaf Blight, smut) and non-lepidopteran insect pests (<i>e.g. </i>, thrips, aphids, spider mites).</p>

<p style="text-align: justify;">Agronomic parameters that were evaluated included seed germination, emergence vigour and stand density, plant height, accumulated growing degree days to 50% pollen shed and silking, percent stalk and root lodging, percent moisture at harvest, grain yield and bushel weight (grain density). Grain yield in TC-6275 was significantly higher, compared to the near-isogenic control hybrid, at locations where the infestations of ECB were higher. However, there were generally no statistically significant differences between TC-6275 and the control hybrid for most of the agronomic parameters. While there were some statistically significant differences observed for characteristics, such as emergence vigour, stand counts, and maturity, these were not consistent and could be attributed to the effect of location rather than genotype. Seed germination in TC-6275 was not significantly different than the control hybrid and was comparable to other maize hybrids.</p>

<p style="text-align: justify;">Results of field monitoring for susceptibility to plant diseases and non-lepidopteran insect pests revealed no differences between TC-6275 and the control hybrid. Specifically, there were no observed differences in the severity of disease symptoms and, in non-lepidopteran insect damage.</p>

<p style="text-align: justify;"><b>Outcrossing</b></p>

<p style="text-align: justify;">Since pollen production and viability were unchanged by the genetic modification resulting in TC-6275, pollen dispersal by wind and outcropping frequency should be no different than for other maize varieties. Gene exchange between TC-6275 maize and other cultivated maize varieties will be similar to that which occurs naturally between cultivated maize varieties at the present time. In the United States, where there are no plant species closely-related to maize in the wild, the risk of gene flow to other species appears remote. Feral species related to corn, as found within the United States, cannot be pollinated due to differences in chromosome number, phenology (periodicity or timing of events within an organism&rsquo;s life cycle as related to temperature and photoperiod, <i>e.g. </i>, flowering time) and habitat.<br />
Maize (<i>Zea mays</i> ssp. <i>mays</i>) freely hybridizes with annual teosinte (<i>Zea mays</i> ssp. <i>mexicana</i>) when in close proximity. These wild maize relatives are native to Central America and are not present in the United States, except for special plantings. <i>Tripsacum</i>, another genus related to <i>Zea</i>, contains sixteen species, of which twelve are native to Mexico and Guatemala. Three species of <i>Tripsacum</i> have been reported in the continental United States: <i>T. dactyloides</i>, <i>T. floridanum</i> and <i>T. lanceolatum</i>. Of these, <i>T. dactyloides</i>, Eastern Gama Grass, is the only species of widespread occurrence and of any agricultural importance. It is commonly grown as a forage grass and has been the subject of some agronomic improvement (<i>i.e</i>., selection and classical breeding). <i>T. floridanum</i> is known from southern Florida and <i>T. lanceolatum</i> is present in the Mule Mountains of Arizona and possibly southern New Mexico. Even though some <i>Tripsacum</i> species occur in areas where maize is cultivated, gene introgression from maize under natural conditions is highly unlikely, if not impossible. Hybrids of <i>Tripsacum</i> species with <i>Zea mays</i> are difficult to obtain outside of the controlled conditions of laboratory and greenhouse. Seed obtained from such crosses are often sterile or progeny have greatly reduced fertility.</p>

<p style="text-align: justify;"><b>Weediness Potential</b><br />
No competitive advantage was conferred to TC-6275 that would render maize weedy or invasive of natural habitats, since none of the reproductive or growth characteristics were modified. Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. <i>Zea mays</i> is not invasive and is a weak competitor with very limited seed dispersal.</p>

<p style="text-align: justify;">Volunteers can occur in fields, the year following cultivation, when maize is grown in rotation with other crops. Although maize volunteers are a minor weed problem, these can cause harvesting problems. TC-6275 maize volunteers will be tolerant to glufosinate ammonium, thereby compromising the use of this herbicide in glufosinate-tolerant crops (<i>e.g.</i>, glufosinate-tolerant soybeans). However, volunteer plants may be controlled by mechanical means or by the use of other herbicides registered for use on maize volunteers.</p>

<p style="text-align: justify;"><b>Secondary and Non-Target Adverse Effects</b></p>

<p style="text-align: justify;">The history of use and literature suggest that bacterial <i>Bt</i> proteins are not toxic to humans, other vertebrates, and beneficial insects. The insecticidal active core of the <i>Bt</i> protein (Cry1F) expressed in TC-6275 was shown to be nearly equivalent to the bacterial protein, except for one amino acid substitution.</p>

<p style="text-align: justify;">The impact of TC-6275 maize on non-lepidopteran pests, and on non-target organisms was investigated. In field trials conducted to evaluate the efficacy of TC-6275 against the targeted lepidopteran pests of maize, plots were also monitored for damage caused by non-lepidopteran pests, such as thrips, aphids and red spider mites. There were no observed differences between TC-6275 and the non-transgenic control line in the extent of damage caused by non-lepidopteran pests.</p>

<p style="text-align: justify;">The Cry1F protein expressed in TC-6275 is similar to that expressed by the maize line <a href="dbase.php?action=ShowProd&amp;data=TC1507&amp;frmat=LONG"><b>TC1507</b></a>, which received regulatory approval for environmental release in the U.S. (2001), Canada (2002), and Japan (2002). This was substantiated by data, provided by Dow Agro Sciences, to demonstrate the equivalency in terms of biochemical properties and biological activity, of Cry1F in TC-6275 to that in TC1507. The following data and information on the effects of Cry1F on non-target organisms are those that were provided in support of the environmental safety of TC1507.</p>

<p style="text-align: justify;">Maize inbreds and hybrids expressing the Cry1F protein were compared to their non-transformed counterpart for relative abundance of beneficial arthropods, including: lady beetles (<i>Cycloneda munda</i> and <i>Coleomegilla maculata</i>), predacious Carabids, brown lacewings (<i>Hemerobiidae</i>), green lacewings (<i>Chrysoperla plorabunda</i>), minute pirate bugs (<i>Orius insidiosus</i>), assassins bugs (<i>Reduviidae</i>), damsel bugs (Nabidae), Ichneumonid and Braconids (parasitic wasps), damselflies, dragonflies, and spiders. Visual counts showed no significant differences between the number of arthropods collected in the <i>Bt</i> maize plots and those cultivated to the non-transgenic isolines with two exceptions. There was a significantly greater number of lady beetles in the <i>Bt</i> maize plots (1.2 per test plant <i>vs</i> 0.6 per control plant), as well as significantly more <i>Orius</i> than in the non-transgenic control plots on two of the three sample dates. These field studies demonstrated that Cry1F had neither a direct, nor an indirect effect on beneficial arthropod populations.</p>

<p style="text-align: justify;">Species representative of non-target and beneficial organisms in an agricultural environment were selected for acute dietary toxicity studies. The species were: honey bee larvae (<i>Apis melifera</i>), predatory ladybird beetle (<i>Hippodamia convergens</i>), green lacewing (<i>Chysoperla carnea</i>), parasitic Hymenoptera (<i>Nasonia vitripennis</i>), as well as the soil arthropod Collembola (<i>Folsomia candida</i>). Acute toxicity studies were also conducted with earthworms, the freshwater invertebrate <i>Daphnia magna</i>, Northern bobwhite quail, and mice. In all cases there were no observable adverse effects attributed to the Cry1F insecticidal protein.</p>

<p style="text-align: justify;">An additional study was conducted on the effect of Cry1F on neonate monarch butterfly larvae when fed a 10000 ng/mL diet dose. First instar larval weight and mortality were recorded after seven days of feeding. Although there was some growth inhibition, there was no mortality to monarchs fed the 10000 ng/mL diet, the highest rate tested. Since pollen doses equivalent to 10000 ng/mL are not likely to occur on milkweed leaves in nature, it can be concluded that Cry1F will not pose a risk to monarchs.</p>

<p style="text-align: justify;">The levels of Cry1F in TC-6275 pollen and grain were substantially lower than in TC1507 (83% less in pollen and 50% less in grain). Non-target organisms consuming TC-6275 pollen and grain would be exposed to considerably less Cry1F than from TC1507. However, soil exposure to Cry1F from TC-6275 was determined to be approximately 1.5-fold greater that from TC1507. The exposure to Cry1F in soil would nevertheless be minimal since the LC50 for Cry1F for Collembola and earthworms was at least 40-fold the estimated soil concentration from ploughed-under TC-6275 senescent plants.</p>

<p style="text-align: justify;"><b>Impact on Biodiversity</b></p>

<p style="text-align: justify;">TC-6275 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of maize production. Since the risk of outcrossing with wild relatives in the United States is remote, it was determined that risk of transferring genetic traits from TC-6275 maize to species in unmanaged environments was insignificant.</p>

<p style="text-align: justify;"><b>Other Considerations</b></p>

<p style="text-align: justify;">In order to prolong the effectiveness of plant-expressed <i>Bt</i> toxins, and the microbial spray formulations of these same toxins, regulatory authorities in the United States have required developers to implement specific Insect Resistant Management (IRM) Programs. These programs are mandatory for all transgenic <i>Bt</i>-expressing plants, including TC-6275 maize, and require that growers plant a certain percentage of their acreage to non-transgenic varieties in order to reduce the potential for selecting <i>Bt</i>-resistant insect populations. Details on the specific design and requirements of individual IRM programs are published by the relevant regulatory authority.</p>
