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

<p style="text-align: justify;">The main purpose of compositional studies is to determine if any unexpected changes in composition have occurred to the food and to establish its nutritional adequacy. In the case of cottonseed, the key components that should be considered in the comparison include protein, fat, carbohydrate, amino acids, fatty acids, vitamins, minerals and the antinutrients, gossypol and cyclopropenoid fatty acids. Compositional analyses were done on fuzzy seed collected from GHB614 cotton and the non-GM counterpart, Coker 312, grown in field trials typical of commercial agricultural production.</p>

<p style="text-align: justify;">Nine field trials were conducted in 2005 at sites representing primary cotton-growing regions of the south-eastern United States. At each test site, six plots of transgenic event GHB614 cotton and three non-transgenic plots of Coker 312 were planted. Three of the six plots containing GHB614 cotton were sprayed three times with glyphosate herbicide (0.75 pounds active ingredient/acre). Compositional analysis of the cottonseed samples included proximates (protein, fat, ash and moisture), acid detergent fibre (ADF), neutral detergent fibre (NDF), minerals (calcium, iron, magnesium, phosphorus, potassium and zinc), amino acids, fatty acids, vitamin E (alpha tocopherol) and carbohydrates by calculation. In addition, three known anti-nutrients found in cotton (gossypol, cyclopropenoid fatty acids and phytic acid) were analysed. Methods of analysis were based on internationally recognised procedures (e.g. AOAC International methods) or other published methods.</p>

<p style="text-align: justify;">No differences of biological significance were observed between GHB614 cotton and its conventional counterpart. Statistically significant differences in some of the key constituents were noted, however the differences observed were minor, and in each case the levels observed were within the range of literature values reported for conventional cotton varieties. The composition of cotton is known to vary significantly with the site, agricultural conditions and season of production, and differences reported here most likely reflect normal biological variability. Food from GHB614 cotton is therefore considered to be compositionally equivalent to food from conventional cotton varieties.</p>

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

<p style="text-align: justify;">In terms of its potential toxicity and allergenicity, it is worth noting that the 2mEPSPS protein has been evaluated previously as the novel protein present in glyphosate-tolerant corn line GA21 which has been approved for food and feed use in numerous countries. It is therefore likely to have been widely distributed in corn based foods. This modified enzyme is derived from the native EPSPS enzyme in maize (99.5% amino acid homology), and is closely related to other EPSPS enzymes from plants and microorganisms which are natural constituents of human diets.<br />
Bioinformatic studies with the 2mEPSPS protein sequence has confirmed the absence of any significant amino acid sequence similarity to known protein toxins or allergens.</p>

<p style="text-align: justify;">Two independent acute oral toxicity studies using purified 2mEPSPS protein have been conducted by different applicants. In the study conducted by Monsanto for GA21 maize, the modified EPSPS (2mEPSPS) protein was administered by a single oral gavage dose to ten male and ten female CD-1 mice, at target doses of 5, 15 and 50 mg/kg bodyweight. In this study, this corresponded to actual doses of 3.7, 11.8 and 45.6 mg/kg respectively. The results of the study showed no statistically significant differences between test and control treatment groups in mean body weights, cumulative weight gains or food consumption in either males or females at any treatment level. The study concluded that there was no evidence of toxicity in mice following a single oral dose of 45.6 mg/kg modified EPSPS (2mEPSPS) protein. In the study conducted by Bayer for GHB614 cotton, the 2mEPSPS protein was administered by a single oral gavage dose of 2000 mg protein/kg bodyweight to 5 female OF1 mice. A second group of female mice received the same dose of bovine serum albumin as a negative control. All animals were observed for clinical signs daily for fifteen days and body weights were measured weekly. At termination, all animals were subjected to necropsy including macroscopic examination. There were no clinical signs, mortalities or treatment related effects on bodyweight in female OF1 mice observed during this study. Based on these findings, it was concluded that no oral toxicity was demonstrated in mice at a very high dose of 2000 mg/kg bodyweight.</p>

<p style="text-align: justify;">One of the criteria for assessing potential allergenicity is to determine the stability of novel proteins in conditions that simulate human digestion. Proteins that are rapidly degraded in such conditions are considered less likely to be involved in eliciting an allergic response. The 2mEPSPS protein was subjected to digestibility studies using simulated human gastric fluid (SGF) containing pepsin and simulated intestinal fluid (SIF) containing porcine pancreatin, which is a mixture of enzymes including amylase, trypsin, lipase, ribonuclease and protease. In SGF (with pepsin), there was no full length or partially degraded 2mEPSPS protein observed at 30 seconds and at subsequent time points. In SIF, the 2mEPSPS protein band was only faintly visible after scanning the gel even at time zero. At all subsequent incubation times, there was no full length or partially degraded 2mEPSPS protein observed.</p>

<p style="text-align: justify;">The weight of evidence shows that the 2mEPSPS protein is not toxic and unlikely to be allergenic in humans.</p>
