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

<p style="text-align: justify;">The cotton line 3006-210-23 was field tested in the United States from 1999 to 2002, and in Puerto Rico in 2000-01 at a total of 57 location-years. These trials were conducted to evaluate agronomic performance and crop quality, and to determine the plant pest risk potential of 3006-210-23. Comparisons were made between the modified line and one of its parental lines, PSC355. While small but significant differences were observed in some of the vegetative growth parameters, these were within the observed range of commercial cultivars. Also observed were small but significant differences in some reproductive parameters (<i>e.g.</i>, percent retention of bolls) and fibre quality. These slight differences would not be expected to confer weediness characteristics to line 3006-210-23. The susceptibility of line 3006-210-23 to cotton diseases, such as seed rot, <i>Fusarium</i> and <i>Verticilium</i> wilts, and boll rot, was evaluated and determined similar to that of the parental line PSC355. Results from these field trials demonstrate that the growth, agronomic performance and disease susceptibility of line 3006-210-23 is similar to that of conventional cotton. This cotton line is therefore not expected to become a plant pest risk, neither in terms of weediness, nor in becoming a more suitable host for plant diseases.</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 the cotton line 3006-210-23 were to be grown in proximity with other cotton cultivars, and sufficient insect pollinators were present and active, the <i>cry</i>1Ac 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.<br />
In the United States, species genetically compatible with 3006-210-23 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 the cotton plant is cultivated as an annual crop, it behaves as a perennial plant 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 the cotton line 3006-210-23 other than the production of the insecticidal protein Cry1Ac. The levels of expression of the PAT protein in this cotton line do not confer tolerance to herbicidal applications of glufosinate ammonium. The purpose of introducing the <i>pat</i> gene was to provide a mechanism for selection of transformants. The cotton line 3006-210-23 was not expected to become weedy or invasive of natural habitats since none of the reproductive or growth characteristics of this line were substantially modified. While there were observed significant differences in some vegetative growth characteristics (number of vegetative branches and vegetative bolts) and reproductive parameters (percent retention of bolls), these were within the ranges observed in conventional cotton varieties. Seed dormancy, germination, and seedling establishment, were not significantly altered in 3006-210-23, compared to the parental line. The reproductive potential, in terms of number of seeds per boll, was also not significantly different in this cotton line.</p>

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

<p style="text-align: justify;">The impact of 3006-210-23 cotton on non-target organisms was investigated. Ecological toxicity studies were conducted with representative terrestrial and aquatic species that could be inadvertently affected by Cry1Ac. Non-target organisms included insects other than lepidopterans (<i>e.g.</i>, honeybees, ladybird beetles, green lacewings, parasitic wasps), soil arthropods (collembola, earthworms), and representative terrestrial and aquatic species such as mice, bobwhite quail, rainbow trout and <i>Daphnia magna</i>. Toxicological effects were determined from feeding studies. The bobwhite quail and rainbow trout were fed Cry1Ac as expressed in cottonseed meal. The remaining studies were conducted with the full-length chimeric Cry1Ac from a microbial source (<i>Pseudomonas fluorescens</i>). Data were provided to substantiate the equivalency between the plant and microbial Cry1Ac, in terms of biochemistry and bioactivity. Results of these feeding studies showed no observable adverse effects of that could be attributed to the Cry1Ac protein.</p>

<p style="text-align: justify;">Cry1Ac protein in cotton line 3006-210-23 was shown to be efficacious against the target lepidopteran pests. Insecticidal effects on non-target lepidopterans, such as the monarch butterfly, although not directly assessed in the field, were not expected given the low expression of the Cry1Ac protein in the pollen. Based on the results from dietary studies of monarch butterfly larvae with Cry1Ac protein, the estimated concentration of Cry1Ac necessary for a 50% reduction in larval growth is 10 times the concentration in pollen from line 3006-210-23. This, combined with the low volume of pollen produced, would result in very little risk to non-target lepidopterans, such as the monarch butterfly. While cotton is mostly self-pollinated, any cross-pollination would be achieved by non-lepidopteran insects, such as bees, which are unaffected by Cry proteins.</p>

<p style="text-align: justify;">Based on this information, it was concluded that, compared to conventional cotton varieties, the cultivation of line 3006-210-23 would pose no greater risk to interacting organisms, with the exception of specific lepidopteran insect pests of cotton.</p>

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

<p style="text-align: justify;">The cotton line 3006-210-23 did not exhibit any 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 3006-210-23 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>
