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

<p style="text-align: justify;">Cotton, <i>Gossypium hirsutum</i> L., is grown worldwide primarily as a source of fibre in textile manufacturing. Cottonseed, the by-product of fibre production, is processed into four major products: oil, meal, hulls, and linters. Cottonseed oil is routinely used in human food. Linters are also used for human food, but to a lesser extent than oil. Both have a long history of safe use. Cottonseed meal and hulls are used in animal feed.</p>

<p style="text-align: justify;">Cottonseed oil used for human consumption is highly purified, and contains no detectable proteins. Refined cottonseed oil is used as frying oil, salad and cooking oil and in various processed foods such as mayonnaise, salad dressing, shortening, and margarine. Linters are also highly processed to obtain pure cellulose for use in casings for processed meat products such as bologna, sausages and frankfurters. Such cellulose is also used to thicken ice cream and salad dressings.</p>

<p style="text-align: justify;">Whole cottonseed is used as an energy feed for dairy cows and as a protein supplement in feed for other livestock. Hulls are used as a fibre component in livestock feeds.</p>

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

<p style="text-align: justify;">Compositional analyses were conducted on cottonseed samples from cotton line MON 15985, the parental control DP50B (MON 531 hybrid), the non-transgenic ancestral line DP50 and four commercially available non-transgenic varieties. The samples were collected from eight field trial sites at various locations in cotton producing areas of the United States. The cottonseed was analysed for protein, fat, ash, moisture, carbohydrate, fibre and caloric content, amino acid and fatty acid composition, mineral content, vitamin E and the anti-nutritional elements gossypol and cyclopropenoid fatty acid.</p>

<p style="text-align: justify;">To perform compositional analysis on cottonseed oil, the cottonseed was pooled across the eight field sites creating one composite sample per cotton line. Each pooled sample was then processed into oil and analyzed for fatty acid composition, vitamin E content, and levels of the anti-nutritional factor gossypol and cyclopropenoid fatty acid.<br />
For cottonseed samples, protein, fat, carbohydrate, moisture, caloric content, amino acids and minerals in the MON 15985 line and the parent lines were not significantly different and were within the published ranges and reference ranges for commercial varieties.</p>

<p style="text-align: justify;">During the fatty acid composition analyses, small but statistically significant differences in the mean levels of myristic, stearic and linoleic acids were noted between cotton line MON 15985 and its parent. The values for these fatty acids fell within the commercial cotton ranges and were therefore not considered biologically significant.<br />
Cyclopropenoid fatty acid levels were analysed and found to be slightly higher in cotton line MON 15985 as compared to the parental control. These levels were found to be within the reference ranges for commercial cotton varieties. There was no difference in the levels of gossypol found in the MON 15985 line compared to the parental control or commercial reference varieties. In addition, all lines were analysed for aflatoxins, potent animal toxins and carcinogens, which were not detected in cotton line MON 15985, the parental control, or any of the commercial reference varieties.</p>

<p style="text-align: justify;">The levels of acid detergent fibre (ADF) and neutral detergent fibre (NDF) in cottonseed from line MON 15985, DP50B and DP50 were analysed. NDF was found to be elevated above the range of NDF values published for commercial varieties in all three lines, which were collected in large scale field production plots in Argentina. These NDF values were not significantly different from line to line. The ADF values were found to neither be significantly different from line to line nor were they significantly different from ADF values published in literature.</p>

<p style="text-align: justify;">For oil, there were no differences between the MON 15985 and DP50B lines for fatty acid profile. These fatty acid profiles were also consistent with those determined for the commercial varieties. The level of vitamin E in oil from line MON 15985 was slightly above that determined for the parental varieties and the values obtained from the reference varieties, however it was still within the range reported in literature.</p>

<p style="text-align: justify;">In the analyses of anti-nutritional factors, there was no gossypol detected in the oil of either the MON 15985 line or the parental line. The cyclopropenoid fatty acid levels were found to be similar for the MON 15985 line, its parental line and commercial varieties.</p>

<p style="text-align: justify;">The line MON 15985 was determined to be not materially different in composition from commercial cotton varieties currently under cultivation.</p>

<p style="text-align: justify;">To determine the performance of cottonseed meal derived from cotton event MON 15985 as a livestock feed, feeding trials of channel catfish were conducted. Channel catfish were fed a diet of containing approximately 20% processed cottonseed meal either from line MON 15985, from the parental line or from a non-transformed cotton line. There were no significant differences found between the three groups for survival, weight gain and feed conversion ratios.<br />
As ginned cottonseed is used as a feed supplement for dairy cattle, lactating dairy cows were fed cottonseed from the transgenic parental line (DP50B, line MON 15985, the non-transgenic parental line and a transgenic line expressing CP4 EPSPS (Roundup Ready). Cottonseed comprised about 10% of the total dry matter intake of the cows. There was no significant difference in dry matter intake or cottonseed intake for the groups. Milk yield, milk composition, and body condition scores were also comparable across treatments.</p>

<p style="text-align: justify;">From both these studies, it was concluded that processed cottonseed meal and cottonseed can be safely used as livestock feed for catfish and dairy cattle without adversely affected behaviour, growth, feed conversion, or performance.</p>

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

<p style="text-align: justify;"><i>Cry2Ab2</i><br />
Due to the low levels of Cry2Ab2 produced in cotton tissue, microbially-produced Cry2Ab2 protein was used for acute oral toxicity testing. The microbial form of Cry2Ab2 was determined to be equivalent to plant-expressed Cry2Ab2.<br />
In acute oral toxicity testing, mice were exposed to the Cry2Ab2 protein through oral gavage at 1450, 359 and 67 mg/kg body weight and observed over 14 days. There were no treatment-related observable adverse effects noted at these doses. The dose of 1450 mg/kg body weight exceeded the maximum potential exposure to Cry2Ab2 in oil and linters, which is expected to be below 1 ug per day for human dietary exposure.</p>

<p style="text-align: justify;">The Cry2Ab2 protein was also rapidly digested upon exposure to simulated gastric fluid (SGF). Within 15 seconds of exposure to SGF, there were no fragments greater than 2kDa remaining. During simulated intestinal fluid (SIF) digestion, a 50kDa trypsin fragment was produced after 1 minute. This fragment was consistent with the SIF digestion of other <i>Bt</i> proteins in which stable tryptic cores persist.</p>

<p style="text-align: justify;">In addition, <i>Bt</i> proteins in microbial spray formulations have been used for over 30 years with no apparent toxic effects. As well, Cry2Ab2 showed no amino acid homology with known toxins.</p>

<p style="text-align: justify;">These data contributed to a determination of no likely toxic effect of Cry2Ab2.</p>

<p style="text-align: justify;"><i>GUS</i><br />
GUS protein produced by an <i>E. coli</i> platform was used in an acute oral toxicity study in mice. Mice were subjected to doses of 40, 100 and 400 mg/kg of GUS protein and observed over 8 or 9 days. There were no treatment-related adverse effects observed during the course of the study, nor at the termination of the study. GUS was also found to not be homologous to any known toxins. In SGF and SIF studies, GUS was found to degrade to non-detectable levels in 15 seconds in SGF and lost 91% of its enzymatic activity after 2 hours digestion in SIF. These data contributed to the conclusion that GUS protein is not likely to be toxic when expressed in line MON 15985 cotton.</p>

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

<p style="text-align: justify;">Current scientific knowledge indicates that most food allergens share common characteristics, such as being present in high concentrations in the food product, resistance to proteolytic digestion and heat activation, and they are often glycosylated. The Cry2Ab2 protein was not derived from an allergenic source, was present only at very low levels in event MON 15985 cottonseed (0.004% of protein present in the seed, &lt;0.25 ppm in oil), was rapidly digested in SGF and SIF and did not exhibit significant amino acid sequence homology with known protein allergens.</p>
