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

<p style="text-align: justify;">Sugar beet is generally converted directly to refined white sugar (which is composed almost entirely of sucrose) through extensive purification processes. Analysis of highly refined sugar liquor and crystalline sugar from sugar beet showed that total protein within derivative food products ranged from 0.004 to 1.2 &micro;g per g. CP4-EPSPS and GUS proteins were expressed at low levels in the sugar beet root, and none of these proteins, including Protein 34550 were detected in refined sugar or molasses derived from sugar beet GTSB77.</p>

<p style="text-align: justify;">Sugar beet pulp is a by-product that, in recent years, has been purified and used as an additive, at less than 1%, in some specific foods such as food fibre for breakfast cereals etc. While no direct analysis of these proteins was undertaken in soluble fibre derivatives, it was expected that they would be at negligible concentrations due to the processing steps involved.</p>

<p style="text-align: justify;">It was concluded that little if any CP4-EPSPS or GUS protein would be consumed as a consequence eating food containing sugar derived from sugar beet GTSB77. Thus exposure to the novel proteins was likely to be extremely low.</p>

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

<p style="text-align: justify;">Detailed compositional analyses conducted over multiple years and geographic regions (United States and Europe) were carried out to establish the nutritional adequacy of glyphosate-tolerant sugar beet GTSB77. The effect of glyphosate use on the composition of sugar beet was also examined. Analyses included crude ash, crude fibre, crude protein, carbohydrate and dry matter in both tops and roots (both raw and processed into brei powder used in sugar production). Additional quality components were measured in roots including, invert sugar (glucose + fructose) content, polarization (% sucrose), sodium, potassium and amino nitrogen. No biologically meaningful differences in any compositional and quality parameters relevant to food were identified between non-transgenic, control sugar beet and sugar beet GTSB77, both untreated or treated with glyphosate at recommended agronomic application rates. It is concluded that glyphosate-tolerant sugar beet is equivalent to other commercially available sugar beet with respect to composition and nutritional quality. No nutritional risks are posed by consuming food derived from glyphosate-tolerant sugar beet GTSB77.</p>

<p style="text-align: justify;">The presence of naturally occurring toxins and allergens in glyphosate-tolerant sugar beet line GTSB77 was investigated. Saponins are the only known toxicants found in sugar beet and are actively eliminated in sugar processing. It was concluded that saponin levels in sugar beet GTSB77, both treated and untreated with glyphosate, are not considered to pose a risk to human health since: saponins are, in general, at very low levels in sugar beet tissues; sugar beet processing aims to eliminate saponins and other extraneous material from refined sugar products; and saponin levels in both glyphosate-treated and untreated GTSB77 do not differ significantly across seasons and sites, and fall within the range described for traditional sugar beet varieties in the literature.</p>

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

<p style="text-align: justify;">The low potential toxicity and allergenicity of the CP4 EPSPS and GUS proteins were assessed. While no evidence of the presence of Protein 34550 was found in sugar beet GTSB77, toxicity studies of the protein were still carried out.<br />
There was no evidence to indicate that the CP4-EPSPS, GUS or Protein 34550 had the potential to be either toxic or allergenic to humans. Proteins from the EPSPS or GUS family of proteins are naturally present in our food supply or expressed in human intestinal microflora. The GOX like protein (Protein 34550) was not normally present in foods, but does not possess any of the characteristics common to many allergens or toxins or any significant sequence similarity to any known allergens or toxins. Furthermore, none of the proteins were detectable in the principal food components sugar and molasses, and in sugar beet pulp; CP4-EPSPS and GUS were negligible, detected at levels of 50 ppm and 1 ppm respectively. The levels of CP4-EPSPS and GUS proteins in sugar beet GTSB77 are extremely low in the root (0.005% and 0.00006% respectively) and Protein 34550 was undetectable at a limit of detection of 100 ppb.<br />
In order to assess the safety of these proteins, sufficient amounts of each respective protein were obtained by cloning the genes, CP4-EPSPS, gus and chimeric-gox, into <i>E. coli</i>. CP4-EPSPS and GUS proteins derived from <i>E. coli</i> were shown to be equivalent for safety assessment purposes. As no Protein 34550 was identified in sugar beet GTSB77 tissue, no equivalence of the <i>E. coli</i> cloned Protein 34550 could be established. PCR analysis using primers specific to the truncated <i>goxv247</i> gene did, however, showed that the sizes of the genes in both sugar beet GTSB77 and <i>E. coli</i> were equivalent at approximately 1273 bp.</p>

<p style="text-align: justify;">To directly assess the potential toxicity of the CP4-EPSPS, GUS and 34550 proteins acute gavage tests were undertaken in mice using purified forms of each protein. Despite the experimental level of all three proteins being well in excess of the likely human dietary exposure factor, no mortality or morbidity resulted and there were no significant differences in terminal body weights between the treated and control groups. Upon necropsy, body cavities were opened and organs examined <i>in situ</i> and removed. No pathological findings attributable to the treatment with any protein were observed. All three proteins had no toxic effects on mice given acute doses of the equivalent bacterially produced proteins.</p>

<p style="text-align: justify;">It was concluded that the food products derived from sugar beet GTSB77 should pose no greater risk as a source of toxins or allergens than food products derived from conventional sugar beets.</p>

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

<p style="text-align: justify;">The potential for the novel proteins 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.</p>

<p style="text-align: justify;">Amino acid sequence similarity with known allergens was used as an indicator of the allergenic potential of novel proteins. The amino acid sequence of the CP4-EPSPS, GUS and 34550 proteins were compared to the amino acid sequences of 219 known allergens present in public domain databases (<i>e.g.,</i> GenBank, EMBL, Swissprot, PIR). Sequence similarity was defined as a sequence identity of greater than seven contiguous amino acids. No biologically significant similarity was found between any of the novel proteins with any of allergens listed in these databases.<br />
Allergenic proteins in known allergenic foods (such as milk, soybeans and peanuts) exist as major proteins. There was very limited potential for the novel proteins from sugar beet GTSB77 to act as allergens in final food products as: none of the novel proteins could be detected in either sugar or molasses derived from sugar beet GTSB77, and dietary fibre refined from pulp derived from sugar beet GTSB77 would contain extremely low levels of CP4-EPSPS and GUS. Protein 34550 was not detected in pulp samples.</p>

<p style="text-align: justify;">The CP4-EPSPS, GUS and 34550 proteins were degraded rapidly after exposure to simulated gastric fluid. Such rapid digestion would severely limit the amount of protein absorbed via the intestine and thus restrict any potential immunological response. The rate of degradation of these novel proteins was assessed in simulated gastric (SGF) and intestinal fluids (SIF). The degradation of C4 EPSPS, GUS and Protein 34550 were followed, either through Western blot or enzyme activity assays, in both SGF and SIF at 37C. CP4-EPSPS protein was shown to have a half-life of 15 sec in SGF and 10 min in SIF. No detectable GUS protein was present after 15 sec exposure to SGF and over 50% had been degraded in SIF after 60 to 120min. Over 90% of GUS activity had dissipated after 4 hr in SIF. Protein 34550 was degraded within 15 seconds exposure to SGF and within 60 seconds exposure to SIF, moreover no intermediate stable protein fragments larger that 2kD were generated by digestion of Protein 34550 in either SGF or SIF. On the basis of these data all three novel proteins were demonstrated to be readily digestible.</p>

<p style="text-align: justify;">In summary, none of the novel proteins expressed in GTSB77 possess characteristics normally associated with protein allergens, and their potential for eliciting an allergenic response was judge to be extremely remote.</p>
