Decision Document DD2006-61
Determination of the Safety of Monsanto Canada Inc.'s Corn (Zea mays L.) Event LY038

The DHDPS protein is a member of the lyase subfamily of pyruvate-dependent class I aldolases found in a wide range of organisms including bacteria, rodents and humans. The DHDPS enzyme mediates a critical rate-limiting step in the lysine biosynthesis pathway that in corn is controlled by lysine feedback inhibition. The enzyme catalyses the condensation of L-aspartate-4-semialdehyde and pyruvate to form 2,3-dihydrodipicolinate that is converted to lysine through a series of subsequent enzymatic reactions. In contrast to the native corn DHDPS the variant of this enzyme, from Cornybacterium glutamicum (cDHDPS), is less sensitive to lysine feedback inhibition. Since the cDHDPS enzyme is less sensitive to lysine feedback inhibition, its expression in corn event LY038 is expected to result in the elevated levels of free lysine in the plant when compared to conventional corn. The transcription of cordapA is under the control of the corn promoter globulin 1 (Glb1), which directs cDHDPS expression predominantly in the germ resulting in accumulation of free lysine in this portion of the grain.

Cornybacterium glutamicum is a common soil bacterium that is widespread in the environment; therefore, animals and humans are regularly exposed without adverse consequences to this organism and its components, such as the cDHDPS protein. The supplemental lysine that is most commonly provided for supplementing maize-soybean based animal diets is from commercially available lysine sources in the form of lysine monohydrochloride or lysine sulphate which are produced via fermentation by Cornybacterium glutamicum or Brevibacterium lactofermentum. In addition, DHDPS proteins functionally related to the cDHDPS protein found in corn event LY038 are present in plants and microbes that make lysine, many of which are consumed as feed and/or food. Consistent with the fact that all of these DHDPS proteins, including cDHDPS, catalyze the first enzymatic step in lysine biosynthesis in a wide range of organisms it was expected that an algorithm identified (i) high amino acid homology (27-37%) along the entire length of the protein, and (ii) high sequence similarity (36-47%), between cDHDPS and DHDPS proteins from other species.

Expression levels of cDHDPS in corn event LY038

The cordapA gene expressed in corn event LY038 is linked to the Glb1 promoter which directs the cDHDPS expression predominantly in the germ. Samples of grain, forage, whole plant (V2-V4), forage root, root (V2-V4) and pollen tissues were collected from five representative US field trial sites. The average protein expression in the different plant tissues, in micro-grams of protein per gram of dry weight tissue, as evaluated by an ELISA test, is as follows:

These results confirm transcription of the cordapA gene expressing the cDHDPS protein and that cDHDPS expression is predominantly in grain tissue.

Corn event LY038 was tested in 17 locations in two consecutive years in the USA which provided a range of environmental and agronomic conditions representative of major corn growing regions where commercial production of corn event LY038 is intended. A total of 14 phenotypic characteristics were evaluated in 2002 and 2003: seedling vigor, early stand count, days to 50% pollen shed, days to 50% silking, ear height, plant height, staygreen, final stand count, dropped ears, stalk lodging, root lodging, grain test weight, grain moisture and yield. Data generated from these studies represent observations that are typically recorded by plant breeders and agronomists to evaluate the qualities of maize over a broad range of environmental conditions and agronomic practices that corn event LY038 likely would encounter. The measured characteristics provide crop biology data useful in establishing a basis to assess phenotypic equivalence and familiarity of corn event LY038 compared to conventional maize in the context of ecological risk assessment. Detected differences were considered alone, in consideration of other observed differences, and for trends across locations. The phenotypic characteristic data showed no biologically meaningful differences between corn event LY038 and the negative segregant control, or a selection of conventional reference maize hybrids, and support a conclusion of phenotypic equivalence as it relates to familiarity and a lack of increased weed potential. Likewise, assessment of the phenotypic data detected no biologically significant differences between corn event LY038 and negative segregant control, indicative of a selective advantage that would result in increased weed potential for corn event LY038 or other plants if the trait were transferred to a sexually compatible species.

Plant height tended to be greater in corn event LY038 than in the negative segregant control, although the height of both was within the range of values seen in reference hybrids grown in the same locations. Seedlings of corn event LY038 were consistently and significantly less vigorous than the negative segregant control in two years of agronomic testing, although the values for both were each within the range of values seen in reference hybrids grown in the same locations. No increase or decrease outside of the reference range was observed in any insect, disease, or abiotic stressor in corn event LY038 (abiotic stressors include chemical stress, chlorosis, cold, compaction, crusting, drought, flood, heat, leaf curl, poor emergence, wind, and wrapped whorl; biotic stressors are listed under section IV-3. altered plant pest potential). No significant differences in percent viable pollen or average pollen diameter were detected between corn event LY038 and the negative segregant control corn line.

The seed dormancy and germination of corn event LY038 was compared with its' negative segregant control. No significant differences were detected in percent normal germinated seed, percent viable hard seed, percent dead seed, and percent viable firm swollen seed. Additionally, no viable hard seeds were detected in corn event LY038 (viable hard seed is associated with seed dormancy).

In some locations, approximately 4 to 7% of the corn event LY038 crop was comprised of plants with a white leaf phenotype (described as completely white plants). The white leaf phenotype is only transiently expressed from germination up to the second leaf stage, and only under certain planting conditions.

The white leaf phenotype observed in a small percentage of the plants was determined to be associated with the LY038 trait of increased lysine in the seed. The literature has shown that similar altered phenotypes (which include but are not limited to decreased chlorophyll content, loss of apical dominance, and degeneration of floral organs) have been observed in other plant species that accumulate high levels of lysine. The effects of the white leaf characteristic did not result in significant changes in other growth and development characteristics on a whole-plot basis and it would not contribute to increased pest potential. On the basis of these data, the CFIA concluded that the corn event LY038 has no increased weediness or invasiveness potential compared to currently commercialized corn hybrids.

The CFIA has, therefore, determined that the corn event LY038 does not display any altered pest potential.

Based on the above, the information cited below in Section V. Criteria for the Livestock Feed Safety Assessment, and the agronomic properties of corn event LY038, the CFIA has determined that the unconfined release of corn event LY038 will not result in altered impacts on interacting organisms, including humans, compared to current commercial corn hybrids.

The CFIA has therefore concluded that the potential impact on biodiversity of corn event LY038 is equivalent to that of currently commercialized corn hybrids.

Compositional equivalence of corn event LY038 to the negative segregant control and 20 commercial corn varieties was assessed from five replicated sites in the US in the 2002 growing season. Forage and grain samples were analyzed for proximate, fibre, amino acids, fatty acids, minerals, vitamins and secondary metabolites. With the exception of phosphorus, all forage nutrients were not statistically significantly different between corn event LY038 and negative segregant control. Specifically, the intended change in total and free lysine in corn event LY038 grain was statistically significantly different from the negative segregant control at all sites. The range of lysine content in corn event LY038 was outside the tolerance interval of the reference corn varieties, but was within the literature range reported for field corn.

Statistically significant differences were observed between corn event LY038 and the control for glutamic acid, histidine, isoleucine, and phenylalanine (% total aa), alanine, aspartic acid, cysteine, glutamic acid, leucine, methionine, phenylamine, serine and valine (%DW). These amino acids were within the range of commercial corn varieties. Statistically significant differences were observed between corn event LY038 and the negative segregant control for CP, NDF, TDF, Ca, Mn, Cu, Zn, folic acid, Vitamin E, oleic, linoleic, linolenic, arachidic and eicosenoic acid. No consistent difference was observed within sites for these nutrients, which were also within the range of commercial corn varieties and literature values.

Lysine-related catabolites, L-pipecolic acid, saccharopine and - aminoadipic acid in corn event LY038 grain were statistically significantly higher than negative segregant control at all sites. L-pepecolic acid in corn event LY038 was within the tolerance interval of the commercial corn varieties, while saccharopine and a-aminoadipic acid were outside the tolerance interval. Monsanto Canada Inc. provided scientific evidence to support the safety of the high levels of saccharopine and a-aminoadipic acid in corn event LY038 grain. They noted that both catabolites are intermediary metabolites of normal endogenous lysine catabolism in plants and animals and are rapidly degraded in the animal's liver ultimately entering the tricarboxylic acid cycle as acetoacetyl-CoA. The applicants also calculated the levels of these catabolites from a broiler study to show that the amounts consumed by birds on corn event LY038 diets were degraded, with no negative effects on bird performance or health.

Anti-Nutrients

According to OECD, "in considering the anti-nutrients and natural toxins in maize, only phytic acid is significant to the animal feed" (OECD, 2002). Levels of anti-nutrients (phytic acid and raffinose) and secondary metabolites (ferulic acid and p-coumaric acid) were measured in grain of LY038 and compared to conventional maize. No statistically significant differences were observed. Therefore, it is concluded that the levels of these antinutrients and secondary metabolites in corn event LY038 are comparable to those found in conventional maize.

Bioefficacy

1400 male broilers were used in a 42-day broiler study to establish the bioefficacy and safety of corn event LY038 grain. Starter and finisher diets with corn event LY038 grain were compared to LY038 (-) and five commercial reference corn varieties (with or without supplemental lysine). 14 treatment diets were randomly assigned to 140 pens (10 pens/treatment) with 10 birds per pen in a randomized complete block design. Diet formulations targeted similar lysine content in corn event LY038 diets and treatments with supplemental lysine. Broilers on corn event LY038 diets had statistically significantly higher intakes, weight gains and gain:feed than birds on the negative segregant control and reference corn diets with no supplemental lysine. No statistically significant differences in intakes, weight gains and gain:feed were observed between birds on corn event LY038 diets and birds on LY038 (-) and the commercial corn diets with supplemental lysine. Carcass characteristics and meat composition of broilers on corn event LY038 diets were similar to dietary treatments with supplemental lysine. Statistically significant improvement in carcass characteristics and meat composition was observed in broilers on corn event LY038 diets when compared to those on diets without supplemental lysine. Chick mortality throughout the trial was low and not related to dietary treatments. No mortality was reported on birds on corn event LY038 diets during the 42 day trial. No unintended effects occurred when broilers were fed diets formulated with corn event LY038 grain.

The evidence provided by Monsanto Canada Inc. supports the conclusion that the nutritional composition of corn event LY038 corn is equivalent to conventional corn except for the intended increase in lysine and the associate increase in the levels of the lysine-related catabolites, saccharopine and a-aminoadipic acid. No detrimental effect of these catabolites was seen on the performance and health of broilers on diets containing corn event LY038 grain. The bioefficacy of corn event LY038 grain was similar to corn grain with supplemental lysine.

Due to the low levels of the cDHDPS protein expressed in corn event LY038 it was necessary to express the cordapA gene in an E. coli production system in order to obtain sufficient quantities of the test article for conducting some safety studies (e.g., acute oral mouse toxicity study and simulated gastric fluid digestion study). The bacterial-produced protein was compared to the plant-produced protein and shown to be of similar molecular weight, immunological reactivity and, to have similar functional activity as the plant produced protein. Matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) and N-terminal sequencing demonstrated amino acid sequence equivalency of the plant and bacterial produced proteins. Based on the results, both proteins were found to be physiochemically and functionally equivalent.

Proteins in the lyase subfamily, such as cDHDPS, are present in many foods with a long history of safe use in Canada, and, therefore, would not be expected to be toxic or allergenic. Unlike known food allergens, cDHDPS is heat sensitive and susceptible to digestion. Data demonstrated rapid degradation of the E. coli-produced cDHDPS protein in simulated gastric fluid. Greater than 96% of the cDHDPS protein was degraded within 30 seconds. Unlike many known allergens, the cDHDPS protein is not glycosylated, and is present at low levels.

Using a database assembled from the public domain databases such as SwissProt, GenBank, EMBL, NRL3D, bioinformatic analyses revealed no biologically relevant structural or immunological similarities of the cDHDPS protein sequence to known allergens, toxins or pharmacologically active proteins. Furthermore, no short (eight amino acids) polypeptide matches were shared between the cDHDPS protein sequence and known protein allergens. These analyses establish the lack of both structurally and immunologically relevant similarities between allergens and the cDHDPS protein sequence. Also, a mouse acute oral toxicity study using bacterially expressed cDHDPS protein indicated there were no adverse effects at 780 mg/kg body weight.

The cDHDPS protein shares no significant homology with known toxins or allergens, it is present in small amounts in the feed, and it is rapidly degraded under the conditions present in the gastrointestinal tract. An acute oral toxicity study performed in mice using bacterially expressed cDHDPS protein indicated there were no adverse effects at 780 mg/kg body weight. From the information provided by Monsanto Canada Inc., the cDHDPS protein is unlikely to be a novel toxin or allergen. The cDHDPS protein has a history of safe use, as it is found in other livestock feed ingredients, without evidence of toxicological effects or allergenicity.

Based on the predicted exposure levels and the results of the above tests, no significant risk to livestock and workers/by-standers is expected from exposure to the cDHDPS protein.

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