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

<p style="text-align: justify;">Only refined cottonseed oil and cellulose from processed linters of cottonseed are consumed by humans. Processed linters are essentially pure cellulose (&gt;99%) and are subjected to heat and solvent treatment that would be expected to remove and destroy DNA. CP4 EPSPS was detected in combed lint but not in brown linter stock, the first product in the sequence of processing linters for cellulose. It is generally accepted that the refined oil does not contain protein. The refining process for cottonseed oil includes heat, solvent and alkali treatments that remove and destroy DNA and protein. Although refined cottonseed oil from these lines was not tested for the presence of CP4 EPSPS or NPTII protein, other data were presented confirming that proteins are generally not detectable in such oil at a sensitivity of 1.3 ppm. It was concluded that there would be no dietary exposure to the novel proteins expressed in cotton line 1445.</p>

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

<p style="text-align: justify;">Compositional comparison of cottonseed oil from line 1445 was made to commercial non-transgenic cottonseed oil. The cottonseed used for compositional analyses was taken from six trial sites during two growing seasons. The compositional analyses of cottonseed components measured included proximates (protein, fat, ash, carbohydrates, moisture, crude fibre), amino acid composition, fatty acids profile, and levels of tocopherols.</p>

<p style="text-align: justify;">Some minor differences in proximate analysis between the control, the modified line and the treated modified line (pThe composition of the oil from cotton line 1445 was comparable to that for cottonseed oil from the control line Coker 312 and components were within the normal range reported for cottonseed oil. Based on these data it was determined that refined oil and processed linters derived from glyphosate-tolerant cotton line 1445 were nutritionally and compositionally comparable to that from conventional cotton and were not considered to pose a risk to human health and safety.</p>

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

<p style="text-align: justify;">Toxicity of cotton lines was assessed by analysis of naturally occurring toxins in cotton and an evaluation of the novel proteins CP4 EPSPS and NPTII. Toxicant analyses in cottonseed and refined oil evaluated levels of gossypol and cyclopropenoid fatty acids (sterculic and malvalic acid).</p>

<p style="text-align: justify;">Cottonseed contains a high number of anti-nutritional factors and the raw seed is unsuitable for monogastric animals. Gossypol has numerous toxic effects on mammals, including heart damage and heart failure, such that whole cottonseed or cottonseed meal is not used in human foods and the presence of gossypol limits its use as in animal feed. However, removal or inactivation of gossypol during processing enables the use of some cottonseed meal in feed for fish, poultry and pigs.</p>

<p style="text-align: justify;">The gossypol content was higher in the transformed line compared to the control parent line Coker 312, but still within the range found for field-grown cotton. Gossypol was below the level of detection in cottonseed oil from both the transformed lines and the control.</p>

<p style="text-align: justify;">The cyclopropenoid fatty acids, sterculic acid (C-17), dihydrosterculic acid (C-19) and malvalic acid (C-18), are unique fatty acids common in cotton and produce undesirable biological effects. These include the inhibition of biodesaturation of stearic to oleic acid, affecting phospholipid biosynthesis. There were no statistical differences detected in the content of these fatty acids in cottonseed from the control parent line Coker 312 and from line 1445 with or without glyphosate treatment. In refined oil (single samples processed from cottonseed pooled from the six field sites) the levels of dihydrosterculic and malvalic acids were virtually identical in line 1445 and the control line Coker 312. The level of sterculic acid was slightly higher but within the range for cotton.</p>

<p style="text-align: justify;">Anthocyanin, flavonoid and tannin in the transformed lines were within acceptable levels for cotton and within the range of the controls.</p>

<p style="text-align: justify;">The toxicity of the CP4 EPSPS was previously tested by acute gavage exposure in mice and no deleterious effects were observed. The CP4 EPSPS protein was administered at levels 1000 fold to those anticipated in consumption of food products (high dose: 572 mg/kg body weight). A similar toxicity study on NPTII protein was performed and no deleterious effects were observed. The NPTII protein was administered in three cumulative target dosages of up to 5000 mg/kg body weight. Clinical observations in both studies detected no abnormalities and it was concluded that the toxicity of the two novel proteins was low.</p>

<p style="text-align: justify;">The deduced amino acid sequences of both CP4 EPSPS and NPTII were compared to the amino acid sequence of known protein toxins. No significant similarities were found.</p>

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

<p style="text-align: justify;">The CP4 EPSPS and NPTII proteins are not likely to be toxic or allergenic. The potential for allergenicity was assessed based upon characteristics such as glycosylation, resistance to heat and digestive degradation, and sequence similarity to known allergens.</p>

<p style="text-align: justify;">Refined cottonseed oil and cellulose from linters are devoid of protein and, given that most allergens are proteins, their consumption is unlikely to result in an allergic reaction. Therefore the potential for cottonseed oil or linters from cotton line 1445 to constitute a source of allergens is extremely low. Furthermore EPSPS proteins are naturally present in foods derived from plants and microbes and have no history of being allergenic. Both CP4 EPSPS and NPTII proteins are widespread in nature and no toxic or allergenic effects have ever been reported.</p>

<p style="text-align: justify;">The deduced amino acid sequences of CP4 EPSPS and NPTII showed no homologies with sequences of known toxins or allergens assembled from the Swissprot, Genepept, and PIR protein databases. Unlike known allergens that are resistant to digestion, previous studies demonstrated that the CP4 EPSPS and NPTII proteins were rapidly degraded in a simulated digestive system and inactivated by heat treatment. Data presented showed that the CP4 EPSPS enzyme was rapidly inactivated by heat and by digestion in simulated mammalian gastric fluid. Similarly, NPTII was shown to be rapidly degraded under simulated mammalian digestive conditions, being completely removed after 10 seconds in simulated gastric fluid and with 50% digested after five minutes in simulated intestinal fluid.</p>

<p style="text-align: justify;">The CP4 EPSPS protein sequence does not contain typical glycosylation sequences and comparisons between CP4 EPSPS protein in crude extracts from cottonseed of line 1445 did not differ from purified CP4 EPSPS, supporting the conclusion that the CP4 EPSPS was not glycosylated.</p>

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

<p style="text-align: justify;">The metabolism of glyphosate has been investigated in several varieties of plants, the metabolic pathway in tolerant crops being the same as in non-tolerant crops. In tolerant plants containing the enzyme CP4 EPSPS, like cotton line 1445, glyphosate is only slowly metabolized to AMPA (aminomethylphosphonic acid) as in non-tolerant crops. No detectable residues would occur in cottonseed oil.</p>

<p style="text-align: justify;">Studies with livestock animals also show that glyphosate and AMPA are not metabolized, or are only insignificantly metabolized. Therefore, residues would not be present in meat, milk and eggs of animals fed with tolerant or non-tolerant crops treated with the herbicide glyphosate.</p>
