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

<p style="text-align: justify;">BT11 maize was intended mainly for use in animal feed. The major human food uses for maize are extensively processed starch and oil fractions prepared by wet or dry milling procedures and products include corn syrup and corn oil, neither containing protein. Human exposure to the modified protein from whole grain corn in the diet was considered to be very low due both to its low abundance in the protein fraction of the grain and to the proportionately low percentage of protein in the kernel, compared with the major starch component. The level of the Cry1Ab protein in maize kernels, the only part of the plant used for human food, was very low - less than 5 ng/g fresh weight or 5 parts per billion. The dietary exposure will be lower than that experienced through eating products sprayed with <i>Bt</i>-based insecticides. Furthermore, the processing steps for maize would be expected to remove and/or destroy the Cry1Ab protein. Thus the level of Cry1Ab protein present in processed products derived from BT11 maize would be extremely low. Overall, the dietary exposure of people to insect resistant hybrids of BT11 maize was anticipated to be the same, or lower, as for other lines of commercially available field maize.</p>

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

<p style="text-align: justify;">Detailed compositional analyses were carried out to establish the nutritional adequacy of BT11 maize, and to look for any unintended effects by comparing it to non-modified control lines. The nutritional composition of the ECB resistant maize hybrids was analysed at six locations for grain and silage. For the grain analysis, protein, oil, starch and fibre content of the ECB resistant maize lines were shown to be substantially equivalent to the untransformed controls. Silage samples from the ECB resistant maize hybrids had significantly higher levels of ADF and NDF than samples from the control lines, however the values were well within the normal range for corn silage. All other nutrients tested (protein, calcium, magnesium, phosphorus and potassium) were shown to be equivalent to their respective controls.<br />
Proximate analysis (protein, fat, fibre) was performed using Near Infrared Reflectance (NIR) technology. The method was conducted with appropriate calibration and statistical analysis, to show equivalence between the NIR technique and traditional chemistry. Sample falling outside the normal range using NIR were analysed using AOAC approved, traditional chemical methods. The proximate analysis of the ECB resistant maize hybrids gave values well within the published range for traditional maize cultivars.</p>

<p style="text-align: justify;">Animal feeding studies demonstrated that were few significant differences in results between BT11 maize and non-transformed maize. Four separate studies were made with cattle. The first, a short-term experiment with dairy cattle was designed to study any carry over of the Cry1Ab and PAT proteins into milk. Neither protein could be detected in the milk of any animals fed whole-crop BT11 maize. The second longer-term trial with dairy cattle compared performance of animals fed BT11 maize or its conventional counterpart. There was no effect on dry matter intake, milk production, milk composition or a number of rumen parameters relating to feed utilization.<br />
Laying hens were also fed a diet for 14 days incorporating 64% maize meal from BT11 maize. The diet with transgenic maize contained 240 - 263 ng/g Cry1Ab and 59 - 74 ng/g PAT. The control diet was slightly contaminated with transgenic kernels, accounting for 18 ng/g Cry1Ab. No significant differences were observed for feed intake, bodyweight, egg production, and egg weight. Egg whites, yolks and tissue samples (breast, thigh, liver) were analysed for Cry1Ab and PAT at the end of the experimental period. Neither protein was found in these tissues above the detection limits of 6 ng/g for CRY1Ab and 30 ng/g for PAT.</p>

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

<p style="text-align: justify;">The potential for acute toxicity of the Cry1Ab and PAT proteins was assessed by evaluating physical and chemical characteristics of the proteins and also by acute oral toxicity in mice. Data were generated that demonstrated that the active Cry1Ab protein product of the inserted <i>cry1Ab</i> gene was equivalent to the protein contained in microbial <i>Bt</i> spray formulations that have been safely used in agriculture for more than 40 years. Studies on the Cry1Ab protein expressed in plants and the resulting proteolytic fragments were compared to the bacterial proteins and shown to be of similar molecular weight, amino acid sequence, immunological reactivity and susceptibility to trypsin digestion. The protein was not glycosylated and showed similar bioactivity and host range specificity to the native protein. The biological activity of the trypsinized Cry1Ab protein from the modified maize was the same as that of the native Cry1Ab on ECB, with a LC50 of 0.46-0.51 &micro;g/ml (f.w.) and an EC50 of 0.060-0.067 &micro;g/g (f.w.). After one week incubation in soil, 99% of the bioactivity of Cry1Ab in transgenic leaf tissue was lost due to aerobic degradation.<br />
The potential acute oral toxicity of Cry1Ab was assessed in mice. High oral dose feeding studies were conducted on mice fed Cry1Ab protein, or its truncated form, at up to 4000 mg/kg bodyweight without toxic effects. These results were consistent with other studies on the acute toxicity of Cry1Ab in mice and in rabbits and did not demonstrate any potential mammalian toxicity from Cry1Ab protein.</p>

<p style="text-align: justify;">The toxicity of PAT protein was assessed using similar animal studies. Results from acute oral toxicity testing in mice did not indicate any toxic effects. A comparison of the amino acid sequence of the PAT protein to a database of known toxins demonstrated that it does not share any significant similarity with any known protein toxins. The sequences were obtained from the GenBank, EMBL, Swissprot and PIR databases. Furthermore, the PAT enzyme has very specific enzymatic activity and does not possess proteolytic or heat stability, and does not affect plant metabolism. Acetyltransferases are common enzymes in bacteria, plants and animals and no toxic or allergic effects were expected.</p>

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

<p style="text-align: justify;">The potential for the novel proteins Cry1Ab and PAT to be allergenic was investigated using a number of criteria, including amino acid sequence homology with known allergens, history of use and common physicochemical properties of allergens, including the sensitivity to digestion by digestive enzymes.<br />
The deduced amino acid sequences of the introduced proteins were compared to 219 known allergens present in public domain databases (GenBank, EMBL, Swissprot, PIR) and no homologies were detected. Unlike known protein allergens, studies have shown that Cry1Ab proteins were rapidly inactivated when subjected to simulated mammalian gastric fluids. Similarly, the PAT protein was found to be rapidly digested in conditions that mimic human digestion. Also, PAT was found to be present at very low levels, if at all, in maize kernels.</p>

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

<p style="text-align: justify;">The principal residue identified in transgenic BT11 maize plants after post-emergence use of glufosinate ammonium was N-acetyl-glufosinate with lesser quantities of glufosinate and 3-[hydroxy(methyl)phosphinoyl]propionic acid (MPP) which was also found in non-transgenic plants. In maize grain, which exhibits much lower residues than the other plant parts, the principal residue identified was MPP with lesser amounts of N-acetyl-glufosinate. About 80 field trials in Europe were conducted with different application rates and residues in harvested grain for each metabolite was below 0.05 mg/kg. In green maize, forage and fodder, higher residues can occur. The glufosinate-derived residues do not concentrate in any maize processed fraction, which are relevant food or feed items. These include flour, starch, grits and oil. Residues are not detectable in crude and refined oil. In ruminant and poultry feeding studies no detectable residues were found in meat, milk or eggs at the dose calculated to represent the highest residues in livestock feed under Good Agricultural Practices and taking into account the potential use of glufosinate herbicide in several tolerant crops. It was concluded, on the basis of the available data, that residues of glufosinate ammonium and its metabolites, N-acetyl-glufosinate and 3-[hydroxy(methyl)phosphinoyl]propionic acid (MPP) expressed as glufosinate free acid equivalents, were below 0.1 mg/kg maize grain. In food of animal origin from livestock animal fed with feedstuffs after application of glufosinate herbicide in tolerant maize no residues above the limit of determination are to be expected.</p>
