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

<p style="text-align: justify;">Event COT102 was field tested at several locations in the United States, from 2000 to 2002, to evaluate agronomic performance, disease and insect resistance, and efficacy of the lepidopteran resistance trait in COT102 compared to its non-modified parental line Coker 312. Parameters measured included plant growth, morphology, reproductive traits, fibre yield and quality. At some of the locations, boll yield was significantly greater, and fibre quality significantly superior in COT102 compared to Coker 312. This was attributed to greater insect pressure at these locations; greater insect damage in Coker 312 compared to COT102 resulted in delayed maturity of the bolls, hence lower yield and decreased fibre quality. However, field trial results generally indicated that plant growth and morphology, agronomic performance and disease and non-lepidopteran susceptibility of COT102 was similar to that of its parental line. Differences that were observed were not consistent among locations, or among years, and were within the range of variability for conventional cotton cultivars. Since there were no biologically meaningful differences in disease and non-lepidopteran insect susceptibilities, and in vegetative and reproductive growth, COT102 is not expected to become a plant pest risk, neither in terms of weediness, nor in increased suspectibility to plant diseases and insects other than Lepidoptera.</p>

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

<p style="text-align: justify;">Cotton (<i>G. hirsutum</i>) is mainly a self-pollinating plant, but pollen is also routinely transferred by insects, particularly bumblebees and honey bees. The pollen is heavy and sticky and the range of natural crossing is limited. Outcrossing rates of up to 28% to other cotton cultivars grown directly adjacent to the pollen source have been observed under field conditions when sufficient insect pollinators have been present. The rate of outcrossing declines rapidly with increased distance from the pollen source. Results of outcrossing research trials in the United States have shown that pollen movement decreases rapidly at 12 metres from the pollen source. Nevertheless, certified seed cotton growers in the United States must maintain an isolation distance of 202 metres between fields. If COT102 were to be grown in proximity with other cotton cultivars, and sufficient insect pollinators were present and active, the <i>vip3A(a)</i> gene could possibly introgress into these cultivars. In the event of the formation of hybrids, there would no competitive advantage conferred on any hybrid progeny in the absence of infestations by Lepidopteran insect species.</p>

<p style="text-align: justify;">In the United States, species genetically compatible with COT102 cotton include <i>G. hirsutum</i> (wild or under cultivation), <i>G. barbadense</i> (cultivated Pima cotton), and <i>G. tomentosum</i>. Outcrossing from <i>G. hirsutum</i> to <i>G. barbadense</i> is possible under suitable conditions, such as the presence of pollinators. <i>G. thurberi</i> is another wild species found in the United States, but it is genetically incompatible with <i>G. hirsutum</i>. While <i>G. hirsutum</i> and <i>G. tomentosum</i> are genetically compatible (both possess the AADD genome), the possibility of gene transfer is unlikely due to the non-synchronous flowering periods (day for <i>G. hirsutum</i> and night for <i>G. tomentosum</i>), and lack of common pollinators. Populations of wild <i>Gossypium species</i> occur in southern Florida (<i>G. hirsutum</i>), Hawaii (<i>G. tomentosum</i>), Puerto Rico and the U.S. Virgin Islands. In other cotton growing areas of the world, species that could intercross with <i>G. hirsutum</i> include <i>G. mustelinum</i> in Brazil, and <i>G. lanceolatum</i> in Mexico.</p>

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

<p style="text-align: justify;">Cultivated <i>Gossypium hirsutum</i> is not typically considered a weed species in the United States or other countries, but it is listed as a potential weed in southern Florida. Although cotton is cultivated as an annual crop, it behaves as a perennial in undisturbed environments and suitable climatic conditions. Cotton does not tolerate cold conditions thereby limiting its overwintering potential to southern Florida, Hawaii, and Puerto Rico.</p>

<p style="text-align: justify;">No competitive advantage was conferred to COT102 other than the production of the insecticidal protein VIP3A, and it is not expected to become weedy or invasive of natural habitats since none of the reproductive or vegetative growth characteristics of this line were substantially modified. Differences in these characteristics that were observed were not consistent among locations, and were therefore due to environment, including variability in insect pressure among locations. Seed dormancy, germination, and seedling establishment, were not significantly altered in COT102, compared to the parental line Coker 312, and observed differences were within the range of variability for conventional cotton cultivars.</p>

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

<p style="text-align: justify;">Event COT102 cotton was developed to resist lepidopteran pests of cotton. Due to the insecticidal nature of the VIP3A protein, the impact of COT102 on non-target organisms, including non-target insect pests, was investigated. Ecological toxicity studies were conducted with representative terrestrial and aquatic species that could be inadvertently affected by the insecticidal protein VIP3A. Non-target organisms included insects other than lepidopterans (honeybees, ladybird beetles, and green lacewings), soil arthropods (Collembola, earthworms), and representative terrestrial and aquatic species such as mice, bobwhite quail, catfish and <i>Daphnia magna</i>.</p>

<p style="text-align: justify;">Toxicological effects of were determined from acute feeding studies. Leaf tissue and cottonseed from COT102 were not used in these studies to avoid possible confounding effects of the natural toxicants of cotton (<i>e.g.</i>, gossypol). The organisms were fed VIP3A protein derived from either recombinant <i>E. coli</i>, or maize expressing the <i>vip3A(a)</i> gene. The VIP3A protein from both these sources was shown to be equivalent to that expressed by COT102 cotton, as demonstrated by similarity in molecular weight, immunoreactivity, results of mass spectral analysis of the peptides, and similarity in the rank-order of bioactivity against four species of VIP3A-sensitive lepidopteran larvae.</p>

<p style="text-align: justify;">Most of the organisms in the acute feeding studies were fed, at the highest treatments, levels of VIP3A that were several times greater than the estimated environmental concentrations, based on the expression levels in COT102 tissues. No adverse effects were observed in any of the tested organisms at the highest treatment levels. For each of these organisms, the highest treatment levels represented the following exposure to VIP3A from COT102: Bobwhite quail, 21-fold the level in leaves, and &gt;1200-fold the level in cottonseed; honeybee larvae, &gt;77-fold the level in pollen; adult honeybees, &gt;8-fold the level in pollen; ladybird beetles and lacewings, &gt; 133-fold the level in pollen; Collembola and earthworm, &gt;2100- and 180-fold the estimated soil concentration, respectively.</p>

<p style="text-align: justify;">Environmental concentrations of VIP3A from COT102 were not estimated for either <i>Daphnia magma</i> or catfish. Aquatic organisms would most likely be exposed to VIP3A protein from COT102 pollen drifting from nearby fields and depositing on the surface of the water. This represents a very minimal amount of exposure since cotton flowers are mostly self-pollinating, and the pollen is heavy, thereby minimizing dispersal by wind. No adverse effects were observed in either <i>Daphnia magna</i> or catfish when fed at the highest levels of the VIP3A protein.</p>

<p style="text-align: justify;">The effect of VIP3A on mammals was investigated in an acute feeding study conducted with mice. VIP3A expressed in maize tissue was fed, by gavage, at 5000 mg/kg of body weight. VIP3A expressed in <i>E. coli</i> was also fed at 5050 mg/kg of body weight. No adverse effects were observed in the mice, fed either diet, during the 14 days of the acute feeding study. The digestibility of VIP3A in simulated gastric fluids was also investigated. The VIP3A protein expressed in E. coli was degraded, at time zero, to low molecular weight (&lt; 14,000) peptides, and no peptides were detectable after 2 min. The VIP3A protein is therefore rapidly digested in a mammalian gut environment.</p>

<p style="text-align: justify;">VIP3A protein in COT102 cotton was shown to be insecticidal toward several lepidopteran pests of cotton, including cotton bollworm, tobacco budworm, pink bollworm, fall armyworm, beet armyworm, soybean looper, and cabbage looper. However, several lepidopteran pest species were not adversely affected by the protein, including the European Corn Borer (Ostrinia nubilalis), Fall webworm (<i>Hyphantria curea</i>) and Diamondback moth (<i>Plutella zylostella</i>).</p>

<p style="text-align: justify;">The potential for exposure of non-target lepidopterans to the VIP3A protein was investigated. Insect sensitivity studies were conducted on the Monarch butterfly (<i>Danaus plexippus</i>) and demonstrated no adverse effects. The most likely route of exposure to non-target lepidopterans would be through the pollen. Cotton flowers are mostly self-pollinated and therefore very little pollen would be dispersed into the environment. Cotton pollen is also not readily dispersed by wind due to its mass and high moisture content. The exposure to VIP3A via cotton pollen would also be minimal due to the low levels of expression in the pollen.</p>

<p style="text-align: justify;">Based on the results of the ecotoxicological studies, the VIP3A protein in COT102 poses a negligible risk to non-target organisms, including beneficial arthropod species. The cultivation of COT102 therefore poses no greater risk to interacting, non-target organisms, than conventional cotton cultivars.</p>

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

<p style="text-align: justify;">Event COT102 did not exhibit novel phenotypic characteristics that would increase its survival in either unmanaged habitats, or in areas outside of the current geographical range of cotton production. Since the potential of gene transfer to wild relatives in the United States is quite minimal, the risk of transferring the insect resistance trait to species in unmanaged habitats is insignificant. In countries (<i>e.g.</i>, Brazil, Mexico) where hybrids could form with wild relatives of cotton, the introgression of the insect resistance trait into these wild species would confer no other advantage than increased resistance to attack by certain Lepidopteran insects.</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 COT102 cotton, 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>
