Dietary Exposure
As GA21 maize was intended primarily for use in livestock feed, the level of dietary exposure to the mEPSPS protein was predicted to be very low. 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. Overall, the dietary exposure of consumers in Canada and the United States to grain from GA21 maize was anticipated to be the same as for other lines of commercially available field corn.
Nutritional Data
Maize line GA21 and non-transformed control lines were grown at five different locations in 1996. Several samples of both forage material and grains were collected and analyzed. Forage was analyzed for proximate composition: ash, calcium, carbohydrates, fibre [acid detergent fibre (ADF) and neutral detergent fibre (NDF)] moisture, and phosphorus, protein, and fat. Grains were analyzed for protein, fat, ash, carbohydrate (calculated), fibre (ADF and NDF), amino acid and fatty acid composition, calcium and phosphorus. Complementary analyses were conducted on trace elements, trypsin inhibitors, phytic acid and Vitamin E. There were no significant differences in nutrient composition between GA21 and commercial maize varieties in either the grain or forage material.
The data assessed from animal feeding studies clearly demonstrated the nutritional equivalence of grain from GA21 to isogenic material, on the basis of growth performance and body composition of broiler chickens receiving GA21 maize compared to isogenic grain for 40 days.
Toxicity
Because the amino acid sequence of the mEPSPS was 99.3% identical with the sequence of the wild-type enzyme the amino acid substitutions were not predicted to result in an increased potential for toxicity. Furthermore, the amino acid sequence of the inserted mEPSPS enzyme was compared to that of known protein toxins listed in the PIR, SwissProt, EMBL and GenBank genetic databases. Based on these computer searches the mEPSPS protein did not show homologies with known toxins and was judged not to have any potential for human toxicity.
The two putative open reading frames (ORFs) identified proximal to the mEPSPS insert were both derived from maize sequences and assessed for potential toxicity. Northern blot analysis of poly(A+) RNA from GA21 and control lines demonstrated that transcription of these ORFs did not occur in maize line GA21. Sequence analysis indicated no homologies with known toxins or allergens.
Bacterial-expressed mEPSPS was further evaluated for acute oral toxicity in a laboratory animal feeding trial. The test protein was administered by a single oral gavage to ten male and ten female CD-1 mice at doses corresponding to 3.7, 11.8 and 45.6 mg/kg. The highest dose of 45.6 mg mEPSPS/kg body weight was claimed to be at least 500-fold higher than the likely human exposure. A control group of ten mice/sex was administered only the carrier substance, at the same delivery volume as the test substance. An additional control group of ten mice/sex was administered bovine serum albumin (BSA) in the same carrier substance at the highest target dose (45.6 mg/kg). At defined stages throughout the duration of the study, clinical observations were performed for mortality and signs of toxicity, and body weights and food consumption measured. At the termination of the study (day 13-14), animals were sacrificed, examined for gross pathology and numerous tissues were collected and saved.
It was concluded that there was no evidence of toxicity in mice following a single oral dose of 45.6 mg/kg mEPSPS protein. The results of the study showed no statistically significant differences in group mean body weights, cumulative weight gains or food consumption in either males or females at any level of either the BSA control or test material, when compared with the respective carrier control group. All animals survived to the end of the study, and there were no clinical signs observed that could be related to the test material.
Allergenicity
The wild-type maize EPSPS enzyme has not been associated with any allergic effects, nor is its amino acid sequence homologous with any known protein allergens. The near complete identity (99.3%) between the amino acid sequences of the mEPSPS and wild-type EPSPS enzymes was expected to ensure a lack of allergenicity of the novel protein. The amino acid sequence of the inserted mEPSPS enzyme was compared to a database of 219 known allergens and gliadin sequences constructed from public domain databases. There were no significant sequence similarities discovered between mEPSPS and known allergens and gliadins. Data presented also showed that the protein was not glycosylated, a property common to many allergens. Also, unlike common allergens the mEPSPS protein was present at very low levels (Unlike known protein allergens, the mEPSPS was rapidly degraded by acid and/or enzymatic hydrolysis when exposed to simulated gastric or intestinal fluids. EPSPS isolated from leaves of GA21 maize, including mEPSPS (70% of activity), was rapidly degraded in vitro in artificial human gastric and intestinal fluids. The results of these experiments demonstrated that the mEPSPS protein was no longer detectable after 15 seconds in the gastric system and within one minute in the intestinal system.
Together these results strongly supported the conclusion that maize line GA21 expressing mEPSPS did not pose any greater risk than conventional maize, with respect to potential allergenicity.
