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

<p style="text-align: justify;">Humans consume relatively little whole kernel or processed maize, compared to maize-based food ingredients.&nbsp; Maize is a raw material for the manufacture of starch, the majority of which is converted to a variety of sweetener and fermentation products, including high fructose syrup and ethanol. Maize oil is commercially processed from the germ. These materials are components of many foods including bakery and dairy goods, and the human food uses of grain from MIR604 are not expected to be different from the uses of non-transgenic field maize varieties. As such, the dietary exposure to humans of grain from insect resistant hybrids will not be different from that for other commercially available field maize varieties.</p>

<p style="text-align: justify;"><strong>Equivalence of Bacterial Expressed Proteins</strong></p>

<p style="text-align: justify;">As it is very difficult to extract and purify sufficient quantities of the subject protein from transgenic corn plants for laboratory studies, it has become standard practice to instead use equivalent proteins that have been produced using bacterial expression systems.&nbsp; mCry3A and PMI were produced in recombinant <em>E. coli</em>, purified and used in laboratory toxicity and allergenicity studies.</p>

<p style="text-align: justify;">Prior to use in these laboratory studies, the bacterially produced proteins are compared to the proteins produced&nbsp;<em>in planta</em> in order to establish their equivalence. The molecular identity and biochemical characteristics of the proteins expressed <em>in planta</em> and in the bacterial-expression systems were examined by analysis of biochemical and functional parameters, including molecular weight determination, immunoreactivity, glycosylation analysis and biological activity.&nbsp; SDS-PAGE and Western blotting confirmed the expected molecular weight of mCry3A to be approximately 67,700 Daltons. In the plant derived protein sample, a second band was present and was thought to represent mCry3A that had been degraded in the plant cell.&nbsp; PMI was confirmed to be approximately 45,000 Daltons.&nbsp; No glycosylation was observed for either protein.</p>

<p style="text-align: justify;">The insecticidal activity of plant-derived mCry3A was similar to the bacterially produced protein and the enzyme activity of plant-derived PMI was equivalent to that of the bacterially produced enzyme.&nbsp; The two amino acid changes in the plant expressed PMI do not affect the function of this enzyme.</p>

<p style="text-align: justify;">These studies established that bacterially produced mCry3A and PMI are equivalent to those proteins produced in corn line MIR604, thus support the use of the bacterial proteins in the toxicity testing.</p>

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

<p style="text-align: justify;">The mCry3A protein extracted and purified from the recombinant <em>E. coli</em> was used as the test material for an acute oral toxicity study with mice.&nbsp; The mice received a single dose of 2377 mg/kg bw mCry3A and were observed for two weeks.&nbsp; Parameters evaluated included body weights, food consumption and detailed clinical observations. At the end of the study all animals were killed and examined post mortem.&nbsp; Brain, liver, kidneys and spleen were weighed and selected tissues were taken for histopathological examination.</p>

<p style="text-align: justify;">One female mouse was killed on day 2 of the study due to clinical signs consistent with a dosing injury and not related to the test substance.&nbsp; No test substance-related mortalities occurred.&nbsp; There were no test substance-related effects on body weight, food consumption, organ weights or macroscopic and microscopic pathology.&nbsp; Therefore, under the conditions of this study, the acute oral LD50 of mcry3A in mice is greater than 2377 mg/kg bw, the highest dose tested</p>

<p style="text-align: justify;">For PMI, the mice received a single dose of 3080 mg/kg bw of the bacterially expressed protein and were observed for two weeks.&nbsp; Parameters evaluated included body weights and detailed clinical observations. At the end of the study all animals were killed and examined post mortem.&nbsp; Brain, liver, kidneys and spleen were weighed.</p>

<p style="text-align: justify;">One male in the control group and two in the test group died shortly after dosing or were in distress after dosing and subsequently died.&nbsp; Necropsy revealed perforated oesophagi in these animals, a sign of gavage error and not test-substance related.&nbsp; One replacement animal was available for each group and dosed in the same manner on day 0.&nbsp; There was no test article related mortality during the study.&nbsp; No clinical signs of toxicity were observed in either group.&nbsp; There were no test-substance related effects on body weight, organ weights or gross pathology.&nbsp; Under the conditions of this study, the acute oral LD50 of the PMI protein in mice is greater than 3080 mg /kg bw.</p>

<p style="text-align: justify;">To determine whether mCry3A has any significant homology with known protein toxins, its amino acid sequence was systematically compared to the latest posting of the National Centre for Biotechnology Information Entrez Protein Database containing all the publicly available protein sequences.&nbsp; The appropriate cut-off expectation (E) value was determined to be 0.38 and amino acid sequences with E values lower than this were considered to be significant.</p>

<p style="text-align: justify;">Two hundred and twenty three entries in GenBank returned E values below 0.38.&nbsp; Most of these (216) were identified as known or putative delta-endotoxins&nbsp; with a similar function to mCry3A.&nbsp; None of other entries were identified as known or putative toxins other than delta-endotoxins.</p>

<p style="text-align: justify;">To determine whether the PMI protein sequence has any significant homology with known protein toxins, it was compared to known protein sequences in a similar manner.&nbsp; The sequence used in this study did not take into account the two amino acid changes present in PMI in MIR604.&nbsp; The appropriate cut-off expectation (E) value was determined to be 0.17 and amino acid sequences with E values lower than this were considered to be significant.</p>

<p style="text-align: justify;">One hundred and thirty three protein entries in GenBank returned E values below 0.17. One hundred and fourteen of these were identified as known or putative PMI proteins.&nbsp; Sixteen entries were hypothetical proteins and 3 were unnamed proteins.&nbsp; No association with known or putative toxins was described for these entries.</p>

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

<p style="text-align: justify;">Current scientific knowledge indicates that many food allergens tend to be abundant proteins within the food product, are resistant to proteolytic digestion and heat inactivation and are often glycosylated.&nbsp; The mCry3A and PMI proteins were assessed for allergenicity by examining their digestibility, glycosylation profile, heat stability and amino acid sequence homology to known allergens.</p>

<p style="text-align: justify;">The results of an <em>in vitro</em> digestive fate study using simulated gastric fluid (SGF) indicated mCry3A protein is rapidly degraded by gastric fluid. In a solution of simulated gastric fluid, 1 mg/mL mCry3A test protein mixed with simulated gastric fluid (pH 1.2, containing 2 mg/mL NaCl, 14 mL 6 N HCl, and 2.7 mg/mL pepsin) resulting in 10 pepsin activity units/ mg test protein (complies with 2000 US Pharmacopoeia recommendations), complete degradation of detectable mCry3A protein occurred within 2 minutes, determined by both SDS-PAGE and western blot analysis.</p>

<p style="text-align: justify;">PMI was digested in simulated gastric fluid containing pepsin and in simulated intestinal fluid containing pancreatin.&nbsp; Samples were examined by SDS-PAGE.&nbsp; PMI was degraded rapidly by pepsin: no PMI was detected by SDS-PAGE upon immediate sampling of the reaction mix (0 seconds).&nbsp; When the pepsin was diluted to 0.0001X of the standard concentration, no PMI remained after 10 minutes of incubation.&nbsp; Similarly, no PMI enzymatic activity was detectable after 10 minutes under these conditions.&nbsp; PMI was degraded by pancreatin in simulated intestinal fluid after two minutes.&nbsp; In the unlikely event that PMI survived digestion by pepsin, it would be digested in the mammalian intestinal environment by pancreatin.&nbsp; This indicates that PMI is not stable to digestion and is unlikely to be a food allergen</p>

<p style="text-align: justify;">mCry3A was assessed for glycosylation by DIG Glycan analysis.&nbsp; The limit of detection was 2.5 ng. mCry3A samples of 1000 ng were tested and neither the E. coli-expressed mCry3A nor the corn-expressed mCry3A were found to be glycosylated.&nbsp; The PMI amino acid sequence contains no consensus sequences for N-glycosylation, although O-glycosylation could theoretically occur.&nbsp; Mass spectrometric analysis of human PMI indicates that this protein is not post-translationally modified</p>

<p style="text-align: justify;">The effect of temperature on mCry3A was determined by incubation for 30 minutes at a range of temperatures (4&ordm;C, 25&ordm;C, 37&ordm;C, 65&ordm;C, and 95&ordm;C) followed by a bioassay against Western corn rootworm larvae.&nbsp; At 95&ordm;C mcry3A was completely inactivated.&nbsp; Some reduction in activity was observed after incubation at 65&ordm;C and temperatures of 4&ordm;C, 25&ordm;C and 37&ordm;C had no effect on mCry3A bioactivity.</p>

<p style="text-align: justify;">The heat stability of PMI was evaluated in a similar manner.&nbsp; Loss of enzyme activity was used to determine the instability of the protein after exposure to various temperatures (25, 37, 55, 65 and 95&ordm;C) for 30 minutes.&nbsp; Incubation at ambient temperature (25&ordm;C), 37&ordm;C or 55&ordm;C for 30 minutes had little effect on enzyme activity.&nbsp; Incubation at 65&ordm;C and 95&ordm;C essentially inactivated the protein.</p>

<p style="text-align: justify;">To determine whether mCry3A has any significant homology with allergenic proteins, the protein sequence was systematically compared to a database of allergen sequences.&nbsp; This database was compiled from entries identified as allergens or putative allergens in public protein databases, and was supplemented with additional amino acid sequences identified from the scientific literature.&nbsp; Overall similarity was examined by comparing sequential 80-amino acid sequences covering the entire mcry3A protein sequence (such that each 80-amino acid window was offset from the previous one by one residue and overlapped by 79 residues) to the allergen sequences using the FASTA search algorithm. Any 80-amino acid peptide having greater than 35% amino acid identity was defined as having significant similarity to the allergen sequence.&nbsp; The mCry3A sequence was also screened for matches of eight or more contiguous amino acids.&nbsp; The purpose of this is to identify any short local regions of identity that might indicate the presence of common IgE binding epitopes.</p>

<p style="text-align: justify;">No significant sequence homology was found between any of the sequential mCry3A 80-amino acid peptides and any entries in the database.&nbsp; No alignments of eight or more contiguous identical amino acids between mcry3A and any of the proteins in the database were identified.</p>

<p style="text-align: justify;">The PMI protein sequence was compared to allergenic proteins in a similar manner and there was no significant similarity between any of the sequential PMI 80-amino acid peptides and any entries in the database.&nbsp; There was one region of eight identical amino acids between PMI and the known allergen &Alpha;-parvalbumin from <em>Rana species</em> CH2001.&nbsp; One case of severe food-induced anaphylaxis in a single individual who consumed Indonesian frogs legs has been shown to be due to the protein &Alpha;-parvalbumin from <em>Rana species</em>.&nbsp; To determine if the IgE antibodies in this patient&rsquo;s serum recognized PMI, a PMI protein sample was tested for cross-reactivity. &nbsp;</p>

<p style="text-align: justify;">The PMI sample was bacterially derived and did not contain the two amino acid changes present in MIR604 PMI, however, the region of identity between PMI and &Alpha;-parvalbumin was not affected by this change and therefore the use of bacterially derived PMI would not affect the outcome of this test.&nbsp; No cross reactivity between the human serum IgE and PMI occurred.&nbsp; This indicates that the allergic patient&rsquo;s serum IgE does not recognise any portion of the PMI protein as an allergenic epitope.&nbsp; Therefore, the similarity between PMI and <em>Rana species </em>&Alpha;-parvalbumin is not considered to be biologically relevant.</p>

<p style="text-align: justify;">In summary, no significant homology was found between the novel proteins and known protein allergens.&nbsp; <em>In vitro</em> digestibility studies indicate that the novel proteins are quickly digested.&nbsp; Glycosylation and thermolability studies did not indicate a cause for concern.&nbsp; It is unlikely that either mCry3A or PMI have any allergenic potential.</p>

<p style="text-align: justify;"><strong>Nutritional and Compositional Data</strong><br />
Grain and forage from transgenic event MIR604 derived plants and their near-isogenic, non-transgenic control were grown at 13 different locations over a two year period, with three replicate plots of each genotype planted in randomized complete blocks. Compositional data were statistically analyzed using a randomized block design with locations serving as the blocks. Statistical significance was assigned at p&amp;lt;0.05 indicating that the difference between the treatments was statistically different at the 5% customary level. The treatment-location interaction was also assessed.&nbsp; Values for each analyte from published literature&nbsp; were provided to assess whether statistically significant differences in the composition of the test and control maize are biologically meaningful.</p>

<p style="text-align: justify;">Forage samples were analyzed for proximates and the minerals calcium and phosphorus.&nbsp; Of all the analytes measured, only the moisture level was different in MIR604 when compared with its near-isogenic non-transgenic control. However, moisture is a function of maturity and conditions at harvest and this difference was not considered meaningful. All other values measured for the analytes fell within published literature ranges.</p>

<p style="text-align: justify;">The levels of various nutritional components were determined from mature grain, including: proximates (crude protein, crude fat, moisture, ash, and carbohydrate, acid detergent fibre, neutral detergent fibre, total dietary fibre); minerals (calcium, phosphorus, magnesium, copper, iron, manganese, potassium, sodium, selelnium, chromium and zinc); amino acids; fatty acids; vitamins (beta carotene, cryptoxanthin, folic acid, B1, B2, B3, B5, B6, C and tocopherols); anti-nutrients (phytic acid, trypsin inhibitor); and secondary metabolites (raffinose, furfural, ferulic acid, p-coumaric acid and inositol). &nbsp;</p>

<p style="text-align: justify;">The MIR604 hybrids had statistically significantly higher levels of protein and total fat, and statistically significantly lower levels of starch and total dietary fibre than the near-isogenic non-transgenic control corn. MIR604 hybrids also had statistically significantly higher levels of the amino acids asparagine, threonine, serine, glutamate, alanine, valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine than did the near-isogenic non-transgenic control corn, whereas, the control corn showed statistically higher cystine levels than did the MIR604 corn. However, mean values for all proximates and amino acids, measured for grain from MIR604 and non-isogenic non-transgenic controls were within published literature ranges and it was concluded that the differences in composition between MIR604 and control lines are not biologically significant.</p>

<p style="text-align: justify;">Statistically significantly higher levels of palmitic, oleic, and linoleic acids and statistically significantly lower levels of linolenic acid in MIR604 compared to the near-isogenic non-transgenic control were noted. MIR604 also had statistically significantly higher levels of calcium and phosphorus than did their near-isogenic non-transgenic control corn. Once again, mean values for these were within published ranges and the differences in composition between MIR604 and control lines are not considered to be biologically significant.</p>

<p style="text-align: justify;">Vitamin levels in both MIR604 hybrids and the near-isogenic non-transgenic controls were found to be within published literature ranges.&nbsp; The values for phytic acid were at or below literature values for both MIR604 and the near-isogenic non-trangenic control corn. The values for the other anti-nutrient, trypsin-inhibitor, and all of the secondary metabolites for both the MIR604 hybrids and the near-isogenic non-trangenic control corn were within published literature ranges.</p>

<p style="text-align: justify;">The results of these compositional analyses led to the conclusion that MIR604 forage and grain is not different in nutritional and anti-nutritional composition compared to maize hybrids currently marketed, grown and consumed.&nbsp;</p>
