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

<p style="text-align: justify;">Field testing of Event 176 demonstrated that there were no significant differences observed between hybrids derived using original elite lines and Event 176 for the agronomic traits of yield, moisture at harvest, root lodging rating, ear height, plant height, heat units to silking or pollen shed. Other than resistance to ECB and tolerance to glufosinate ammonium herbicide, the disease, pest and other agronomic characteristics of Event 176 maize were comparable to non-transgenic lines of maize did not demonstrate any altered plant pest potential.</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 Event 176, pollen dispersal by wind and outcrossing frequency should be no different than for other maize varieties. Gene exchange between Event 176 and other cultivated maize varieties will be similar to that which occurs naturally between cultivated maize varieties at the present time. In North America, where there are few plant species closely-related to maize in the wild, the risk of gene flow to other species appears remote. Cultivated maize, or maize, Zea mays L. subsp. mays, is sexually compatible with other members of the genus Zea, and to a much lesser degree with members of the genus Tripsacum.</p>

<p style="text-align: justify;">Wild diploid and tetraploid members of Zea collectively referred to as teosinte are normally confined to Mexico, Guatemala, and Nicaragua; however, a fairly rare, sparsely dispersed feral population of teosinte has been reported in Florida. All teosinte members can be crossed with cultivated maize to produce fertile F1 hybrids. Nonetheless, in the wild, introgressive hybridization from maize to teosinte is currently limited, in part, by several factors including distribution, genetic incompatibility, differences in flowering time, developmental morphology, dissemination, and dormancy. First-generation hybrids are generally less fit for survival and dissemination in the wild, and show substantially reduced reproductive capacity which acts as a significant constraint on introgression.</p>

<p style="text-align: justify;">In Central America, many of these species occur where maize might be cultivated, however, gene introgression from Event 176 under natural conditions is highly unlikely. It is further unlikely that potential introgression of European corn borer resistance or glufosinate tolerance traits from Event 176 would cause teosinte to become more weedy in the absence of glufosinate herbicide selection.</p>

<p style="text-align: justify;">The genus Tripsacum contains up to 16 recognized species, most of which are native to Mexico, Central and South America, but three of which exist as wild and/or cultivated species in the U.S. Hybrids of Tripsacum species with Zea are difficult to obtain outside of a laboratory and are often sterile or have greatly reduced fertility, and none are able to withstand even the mildest winters. Furthermore, none of the sexually compatible relatives of maize in the U.S. are considered to be weeds in the U.S. therefore it is unlikely that introgression of the bar gene would provide a selective advantage to these populations as they would not be routinely subject to herbicide treatments.</p>

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

<p style="text-align: justify;">No competitive advantage was conferred to Event 176, other than that conferred by resistance to European corn borer and herbicide tolerance to glufosinate. Maize is not a weed, and resistance to European corn borer or herbicide tolerance will not render maize weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified. Potential introgression from maize Event 176 into wild relatives is not likely to increase the weediness potential of any resulting progeny nor adversely effect genetic diversity of related plants any more than would introgression from traditional maize hybrids.</p>

<p style="text-align: justify;">Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. Maize volunteers are not uncommon but are easily controlled by mechanical or by using herbicides that are not based on glufosinate as appropriate. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.</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 the bacterial Bt protein is not toxic to humans, other vertebrates, and beneficial insects. This protein is active only against specific lepidopteran insects and no lepidopteran species are listed as threatened or endangered species in North America.</p>

<p style="text-align: justify;">Studies were conducted on several nontarget organisms to determine the potential toxic effects of Cry1Ab protein on test organisms including adult honeybees (Apis melifera,), a predator lady beetle (Coleomegilla maculata), juveniles of the soil-dwelling invertebrate Collembola (springtails,Folsomia candida), earthworms (Eisenia foetida), juveniles of the freshwater invertebrate Daphnia magna, fall armyworm and black cutworm, Northern bobwhite chicks, and mice. Additional studies assessed the impact of Cry1Ab protein on the relative abundance of beneficial arthropods.</p>

<p style="text-align: justify;">Results demonstrated that the larval development of honeybees and lady beetles were not affected when reared on pollen collected from Event 176 maize plants as compared to pollen from nontransgenic plants. Similarly, there was no effect on survival, immobilization, or sublethal toxicity reported for the small aquatic insect, Daphnia magna when exposed to pollen collected from Event 176. Survival rates, signs of toxicity, or loss of weight were not observed in earthworms exposed to leaf tissue from Event 176 maize as compared to the control treatments. Two lepidopteran insects (fall armyworm and black cutworm) that are not susceptible to native Cry1Ab were likewise not affected when feed Cry1Ab derived from leaf tissues. Three insects (ECB, corn earworm, and cabbage looper) that are susceptible to native Cry1Ab were also susceptible to plant-produced Cry1Ab. Results from high dose feeding studies of bobwhite quail fed a protein extract enriched in Cry1Ab isolated from Event 176 maize demonstrated no adverse effects on the bird.</p>

<p style="text-align: justify;">Negative effects were observed in Collembola fed the Event 176 leaf protein (5 % mortality at 0.088 mg Cry1Ab /kg soil) while Collembolans fed with non-Bt maize protein were not adversely affected. The level of Cry1Ab was approximately 10 times greater than the maximum soil concentration that would occur if Event 176 plants were to be incorporated into the soil at anthesis. Under normal conditions, maize would be plowed under in the fall when plants have senesced, and the Cry1Ab protein in Event 176 would be present at very low levels at this time.</p>

<p style="text-align: justify;">Acute oral toxicity studies were conducted with northern bobwhite quail and mice. These animals were fed Event 176-produced protein, and in the case of mice, were also fed bacterial-expressed protein. No mortality was observed and was not expected since Event 176-produced protein was shown to degrade very rapidly under simulated mammalian gastric conditions.</p>

<p style="text-align: justify;">Small scale field studies demonstrated that the number of insects and insect diversity observed on plots planted to either Event 176 maize or a non-transformed counterpart were not significantly different. However, when compared to insect populations on plants treated with a common chemical insecticide (permethrin) versus Event 176 maize plants, the total numbers of beneficial insects (especially lady beetles) associated with Event 176 maize plants were higher.<br />
Results from a study using an enriched leaf extract from Event 176 maize on the soil arthropod Collembola, Folsomia candida, showed that there was a reduction in adult survival and the number of offspring when fed concentrations of Cry1Ab protein at concentrations 200-fold higher than normal. This insect is one of a number of organisms that recycle plant debris in the field. The result was not totally unexpected since a related B. thuringiensis subsp. galleriae has been reported to kill Collembolla. Nonetheless, postharvest monitoring of field test with Event 176 maize plants showed no increase in visible amount of maize debris when compared to non-transgenic plants concluding that there should be no significant adverse effect on Collembola and no increase in maize plant debris as result of the cultivation of Event 176 maize.</p>

<p style="text-align: justify;">In summary, it was concluded that cultivation of Event 176 maize should not have a significant potential to harm nontarget and beneficial organisms common to agricultural ecosystems, nor should it significantly impact species recognized as threatened or endangered.</p>

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

<p style="text-align: justify;">Event 176 had 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 North America is remote, it was determined that the risk of transferring genetic traits from Event 176 to species in unmanaged environments was insignificant.</p>

<p style="text-align: justify;">Event 176 maize plants are not likely to eliminate the use of chemical insecticides which are traditionally applied to about 25 to 35% of the total maize acreage planted, since the primary target for most of these applications has been the coleopteran, corn rootworm. Event 176 maize may positively impact current agricultural practices used for insect control by 1) offering an alternative method for control of European corn borer (and potentially other Cry1Ab-susceptible pests of maize); 2) reducing the use of insecticides to control European corn borer and the resulting potential adverse effects of such insecticides on beneficial insects, farm worker safety, and ground water contamination; and 3) offering a new tool for managing insects that have become resistant to other insecticides currently used or expressed in maize, including other Bt-based insecticides.</p>

<p style="text-align: justify;">Event 176 maize, along with glufosinate ammonium herbicides, is expected to positively impact current agricultural practices used for weed control by 1) offering growers a broad spectrum, post-emergent weed control system; 2) providing the opportunity to continue to move away from pre-emergent and residually active herbicides; 3) providing a new herbicidal mode of action in maize that allows for improved management of weeds which have developed resistance to herbicides with different modes of action; and 4) decreasing cultivation needs and increasing the amount of no-till acres. Volunteers of Event 176 can be easily controlled by selective mechanical or manual weed removal or by the use of herbicides with active ingredients other than glufosinate ammonium.</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 Bt toxins, and the microbial spray formulations of these same toxins, regulatory authorities in Canada and U.S. have required developers to implement specific Insect Resistant Management (IRM) Programs. These programs are mandatory for all transgenic Bt-expressing plants, including Event 176, and require that growers plant a certain percentage of their acreage to non-transgenic varieties in order to reduce the potential for selecting Bt-resistant insect populations. Details on the specific design and requirements of individual IRM programs are published by the relevant regulatory authority.</p>
