Canola (Brassica napus) line GT73 (synonym RT73) was developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option in canola crops. Two novel genes were introduced into canola line GT73, which in combination provide field level tolerance to glyphosate. The novel plants express a glyphosate tolerant version of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene isolated from Agrobacterium tumefaciens strain CP4 (CP4 EPSPS). Glyphosate specifically binds to and inactivates EPSPS, which is involved in the biosynthesis of the aromatic amino acids tyrosine, phenylalanine and tryptophan. The EPSPS enzyme is present in all plants, bacteria and fungi, but not in animals, which do not synthesize their own aromatic amino acids. The modified enzyme (CP4 EPSPS) has a reduced binding affinity for glyphosate and allows the plant to function normally in the presence of the herbicide.

The second gene codes for a modified version of glyphosate oxidase, a bacterial enzyme from Ochrobactrum anthropi strain LBAA. The gene was modified to improve the affinity of the enzyme for glyphosate and is referred to as the gox variant (goxv247). Glyphosate oxidase (GOX) accelerates the normal degradation of the herbicide glyphosate into aminomethylphosphonic acid (AMPA) and glyoxylate. AMPA is the principal metabolite of glyphosate and is degraded by several microorganisms while glyoxylate is commonly found in plant cells and is broken down by the glyoxylic pathway for lipid metabolism.

These two enzymes, CP4 EPSPS and glyphosate oxidase, allow GT73 canola to be protected against herbicide damage when glyphosate is used for weed control in the cultivation of canola.

Canola line GT73 was produced by Agrobacterium-mediated transformation of the B. napus cultivar 'Westar'. The T-DNA region of the Ti plasmid was "disarmed" by removal of virA genes, normally associated with the pathogenicity and disease-causing properties of A. tumefaciens, and replacement with the CP4 EPSPS encoding and goxv247 genes. During transformation, the T-DNA portion of the plasmid was transferred into the plant cells and stably integrated into the plant's genome.

GT73 was produced using plasmid PV-BNGT04 and contained the CP4 EPSPS gene and the goxv247 gene. Constitutive expression of the CP4 EPSPS gene was regulated using the figwort mosaic virus 35S promoter and the 3' end of the pea rbcS E9 gene (E9 3'). The CP4 EPSPS gene was fused to a chloroplast transit peptide (CTP 2) sequence derived from the Arabidopsis thaliana to allow targeting of the newly translated EPSPS enzyme into the chloroplast, the site of aromatic amino acid biosynthesis and endogenous EPSPS and GOX activity.

Similarly expression of goxv247 gene was under control of the 35S promoter from a modified figwort mosaic virus and the 3' end of the pea rbcS E9 gene (E9 3'). Expression of the goxv247 gene was targeted to the chloroplast by the action of the N-terminal of the small subunit 1A (SSU1A) of the ribulose 1,5-bisphosphate carboxylase chloroplast transit peptide of A. thaliana (CTP1).

There was no incorporation of translatable plasmid DNA sequences outside of the T-DNA region. No antibiotic resistance marker genes were introduced into GT73.

The Introduced DNA
Southern blot and PCR analysis were used to demonstrate that there was a single DNA insertion in line GT73 consisting of the T-DNA containing one complete copy of the CP4 EPSPS gene and a complete copy of the gox gene and their respective regulatory sequences.

The CP4 EPSPS gene encodes a protein of 47.6 KDa consisting of a single polypeptide of 455 amino acids. It was determined that once translocated to the chloroplast, the chloroplast transit signal sequence was cleaved and rapidly degraded in vivo leaving only the only newly expressed CP4 EPSPS.

The gox gene encodes a single polypeptide of 431 amino acids with a molecular mass of 46.1 KDa. Nucleotide sequencing comparing the gox variant protein with normal version determined that goxv247 differs by three amino acid substitutions and the two proteins were greater than 99% identical.

Genetic Stability of the Introduced Trait
Southern blot analysis of inserted DNA from the third generation and fifth generation demonstrated that the CP4 EPSPS and gox genes were stably transferred into the plant genome and stably inherited from parent to offspring over several generations.

The insertion site was characterized by backcrossing studies. B. napus is an amphidiploid composed of two genomes B. rapa/B. oleracea. Backcrossing studies with B. rapa were successful and suggested that the genetic insert was integrated within the B. rapa portion of the genome.

Expressed Material
The expression of both CP4 EPSPS and glyphosate oxidoreductase was detected in the leaves and seeds of transgenic GT73 canola. Enzyme linked immunosorbent assay (ELISA) of seed and leaf tissue demonstrated that the introduced proteins were expressed in GT73 at very low levels, constituting less than 0.02% of the seed on a fresh weight basis. No detectable CP4 EPSPS or GOX protein was measured in non-transgenic control 'Westar' seed or leaf tissue from any year. In line GT73, expression of both CP4 EPSPS and GOX proteins in the seed was stable when observed in different trials. Levels of CP4 EPSPS ranged from 18 - 34 µg/g fresh weight and levels of GOX protein ranged from 108 - 193 µg/g fresh weight.

Field Testing
The canola line GT73 was field tested in Canada starting in 1992, in the United States since 1995 and in Europe. Agronomic and adaptation characteristics such as vegetative vigour, overwintering capacity, days to flowering, time to maturity, seed production or yield, germination and dormancy were within the normal range of expression of characteristics in unmodified counterparts. Stress adaptation was evaluated, including resistance to major B. napus pests such as the fungal pathogen Leptosphaeria maculans (blackleg) and other pests (e.g., sclerotinia, flea beetles, diamondback moth larvae) and determined to fall within the ranges currently displayed by commercial varieties. Similarly no differences were observed for abiotic stress factors such as heat, drought and frost. The only significant difference between GT73 canola and the parental non-transformed variety was the increase in the CP4 EPSPS enzyme and GOX protein that confer tolerance to glyphosate. Overall the field data reports demonstrated that canola GT73 has no potential to pose a plant pest risk.

Outcrossing
Transgenic canola line GT73 displayed normal reproductive characteristics. B. napus plants are known to outcross up to 30% with other plants of the same species, and potentially with plants of related species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum. Previous studies have demonstrated that gene flow is most likely to occur with B. rapa.

The genes coding for glyphosate tolerance, CP4 EPSPS and goxv247 are not expected to confer an ecological advantage to potential hybrid offspring. If glyphosate tolerant individuals arose through interspecific or intergeneric hybridization, the novel trait would confer no competitive advantage to these plants unless these populations were routinely subject to herbicide treatments. This may occur in managed ecosystems where glyphosate containing herbicides are applied for broad spectrum weed control, or when used to control weeds in plant varieties developed for tolerance to glyphosate. In the event that a glyphosate tolerant plant survived, the herbicide-tolerant individual would be easily controlled using mechanical and other available chemical means.

Weediness Potential
Field studies on weediness potential, such invasiveness and survival characteristics, comparing GT73 canola to non-transgenic counterparts determined that the transgenic lines were not different from their counterparts in this respect. Studies on fitness characteristics such as germination, seed production, pest and disease resistance, response to abiotic factors and sensitivity to herbicides other than glyphosate, demonstrated that there was no increase in weediness potential for canola line GT73. It was determined that glyphosate tolerance did not confer a competitive advantage to GT73 canola over non-transgenic varieties since the herbicide resistance trait did not confer any pest resistance, alter reproductive biology or change any physiology related to survival. It was determined that weed management control would not be affected, since glyphosate tolerant B. napus volunteer plants were shown to be easily managed using alternative herbicides with different modes of action. It was concluded that GT73 canola had no altered weed or invasiveness potential compared to currently commercialized B. napus varieties.

Secondary and Non-Target Adverse Effects
It was determined that genetically modified canola line GT73 did not have a significant adverse impact on organisms beneficial to plants or agriculture, nontarget organisms, and was not expected to impact on threatened or endangered species. The novel proteins CP4 EPSPS and GOX expressed in GT73 did not result in altered toxicity or allergenicity properties as previously determined from studies using simulated digestive fluids, acute toxicity studies in mice, and amino acid sequence homology studies.

Plant residue studies were conducted to determine the effect of residues from GT73 on the agronomic performance of succeeding crops of barley and peas. No significant differences in either plant counts or grain yield were identified between field plots where GT73 and the non-modified counterpart 'Westar' had been previously grown.

Impact on Biodiversity
The transgenic canola line GT73 has no novel phenotypic characteristics which would extend the use beyond the current geographic range of canola/rapeseed production. Since outcross species are only found in disturbed habitats, transfer of novel traits would not have an impact on unmanaged environments. Studies have demonstrated that GT73 was not invasive of natural habitats, and that it was not more competitive than its counterparts, both in natural and managed ecosystems. It was determined that the relative impact on biodiversity of GT73 was equivalent to that of currently commercialized canola lines.

Dietary Exposure
The human consumption of canola products is limited to the refined oil. Typically, canola oil is used by itself as a salad oil or cooking oil, or blended with other vegetable oils in the manufacture of margarine, shortening, salad oil and cooking oils. Refined edible canola oil consists of purified triglycerides (96-97%) and does not contain any detectable protein, hence no amounts of CP4 EPSPS or GOX proteins were detected in the refined oil of GT73 canola. The genetic modification of GT73 canola will not result in any change in the consumption pattern for this product. As the introduced gene products were not detectable in the refined oil produced from transgenic canola, there is little or no anticipated human exposure to these proteins

Nutritional Data
The analysis of nutrients from transgenic GT73 canola and non-transgenic canola did not reveal any significant differences in the levels of crude protein, crude fat, crude fibre, ash and gross energy in either whole seed or processed meal. The fatty acid composition of oils and levels of the shikimate pathway aromatic amino acids phenylalanine, tyrosine and tryptophan extracted from both transgenic GT73 and non-transgenic canola was statistically the same and, in the case of fatty acids, within the normal range for canola oil. These results collectively demonstrated that the introduction of the novel genes in GT73 should not result in any secondary effects impacting on composition or nutritional quality. Furthermore, the transgenic line GT73 meets the standards for canola oil in Canada of containing less than 2% erucic acid and less than 30 micromoles/g glucosinolates in the oil-free meal. It was determined that the consumption of refined oil from GT73 would have no significant impact on the nutritional quality of the food supply in the United States and Canada.

Several animal feeding studies were conducted to demonstrate the wholesomeness of canola meal. Canola meal is used only for animal feed and is not considered a human food fraction. The feeding studies were conducted to establish the nutritional adequacy of canola meal for animal feeds and represented a worst case scenario in terms of canola consumption by humans. These studies included a four-week rat study on processed and unprocessed meal, a ten-week trout study on processed meal and a five-day quail (Northern Bobwhite) study on unprocessed meal. The studies all supported the safety of meal from glyphosate-tolerant canola as a component in animal feed.

The rat feeding study examined six week-old Sprague-Dewley rats (10/sex/group) fed either 0, 5 or 15% w/w ground (unprocessed) and processed (toasted and defatted) glyphosate-tolerant canola (GT73 and GT200) and 'Westar' canola meal and a diet control (commercial rodent chow with no added canola meal). The study determined that rats fed on diets with unprocessed canola seed had relative liver and kidney weights 5 - 20% greater compared to the diet controls. However, there were no differences in absolute or relative organ weights between the glyphosate-tolerant canola and parental line groups. When fed processed canola meal, there were no meaningful differences observed in body weights and body weight gains between groups fed processed glyphosate-tolerant canola meal, parental line canola meal and a control diet. Liver weights, however, increased approximately 12-16% for both sexes fed 15% GT73 meal. The increase in liver weight was attributed to a higher level of alkyl glucosinolate toxicants in the glyphosate-tolerant canola line GT73 which was 4 g/kg compared to 1.8 g/kg for the parental line. The higher level of glucosinolates present in glyphosate-tolerant canola was not attributed to the genetic modification.

The quail feeding study examined thirty northern bobwhite (Colinus virginianus) chicks fed diets for five days and observed for a further three days. Treatment groups were fed a control diet, glyphosate-tolerant canola (both line GT73 and GT200) or the parental line 'Westar', where canola was incorporated at a rate of 20% of the total weight of the diet. The study did not find any significant effects on body weight or feed consumption between birds in the control or treatment groups. There were no mortalities or overt signs of toxicity in either treatment or control groups.

Similarly a trout feeding study examined triplicate groups of 15 fish rainbow trout (Oncorhynchus mykiss) fed canola meal at 0, 5, 10, 15 or 20% weight of the dry diet for 10 weeks (i.e., 45 fish/treatment).There was statistical overlap in weight gain of fish fed each dietary treatment and no differences were detected between glyphosate-tolerant canola (both line GT73 and GT200) diets and controls. Fish fed the glyphosate-tolerant canola did not exhibit any adverse effects.

Toxicity
It was determined that there were no toxicity or allergenicity concerns with GT73, since refined canola oil is the only product for human consumption and does not contain any detectable amount of protein. The absence of toxicity was further demonstrated by examining the amino aid sequence homology and the physiochemical characteristics of the CP4 EPSPS and GOX proteins, and acute toxicity study in mice.

The deduced amino acid sequences of both the CP4 EPSPS and GOX proteins were compared to the amino acid sequences of 1935 known protein toxins. No significant similarities were found other than would be expected given that certain functional domains are generally conserved between proteins.

The CP4 EPSPS enzyme belongs to the same family of EPSPS enzymes that occur in plants, fungi and bacteria. The EPSPS proteins are naturally present in foods derived from plants and microbes and have no history of being toxic or allergenic. Similarly, the gox gene was sourced from a common soil bacterium, which has no history of pathogenicity. The GOX enzyme degrades glyphosate and experiments demonstrated that the enzyme displayed a narrow substrate specificity, and appeared not to have any secondary effects on other plant specific pathways.

The acute oral toxicity of CP4 EPSPS, GOX and GOXv247 proteins, was studied in groups of ten CD-1 mice/sex in order to directly assess the potential for toxicity associated with these proteins. Based on previously assessed studies, there were no adverse effects or mortalities noted in mice administered CP4 EPSPS protein by gavage at doses up to 572 mg/kg. Similarly, no adverse effects were observed in mice administered the GOX protein by gavage at doses up to 100 and 104 mg/kg for GOX and GOXv247 respectively. The doses were well above the level of expression of the proteins found in glyphosate-tolerant canola plants, estimated to be a 1300-fold increase of CP4 EPSPS and 5000-fold increase of GOX. Clinical observations observed up to 9 days after dosing showed no signs of toxicity (i.e., no adverse effects for either protein on body weight, food consumption, survival, or gross pathology).

Allergenicity
The possibility of exposure to CP4 EPSPS and GOX is significantly limited by the combined facts that these proteins are present at low levels in leaf tissue and are absent in refined oil. The low potential for allergenicity was further demonstrated by examining the amino aid sequence homology and a study on the digestibility of the CP4 EPSPS and GOX proteins.

The amino acid sequence of the CP4 EPSPS and GOX proteins were compared to the amino acid sequences of 219 known allergens present in public domain databases (e.g., GenBank, EMBL, Swissprot, PIR). No significant similarities (i.e. a sequence of more than 8 consecutive amino acids) were found with any of these known allergens.

The study of the digestibility of both proteins in model digestion systems was done in vitro using simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) as mammalian digestion models. The exposure of CP4 EPSPS and GOX proteins to SGF and SIF was conducted over a series of timed incubations at 37°C. The products of the digestion were analyzed using gel electrophoresis, Western blot analysis and enzymatic activity assays. From the simulated digestion experiments and Western blot analyses, the CP4 EPSPS protein had a half-life of less than 15 seconds in the gastric system and 10 minutes in the intestinal system. The GOX protein had a half-life of less than 30 seconds in the intestinal system as determined by Western blot analyses. In summary, both the CP4 EPSPS and GOX proteins were digested by proteases present in the mammalian digestive system, suggesting that they would not survive peptic and tryptic digestion or the acidic conditions of the human digestive system.

Draft Risk Analysis Report: Application A363, food produced from glyphosate tolerant canola line GT73

Decision Document DD95-02: Determination of Environmental Safety of Monsanto Canada Inc.'s Roundup® Herbicide-Tolerant Brassica napus Canola Line GT73

COMMISSION DECISION of 31 August 2005 concerning the placing on the market, in accordance with Directive 2001/18/EC of the European Parliament and of the Council, of an oilseed rape product (Brassica napus L., GT73 line) genetically modified for tolerance to the herbicide glyphosate

Notification of the placing on the Community Register of MON-ØØØ73-7.

Opinion of the Scientific Panel on Genetically Modified Organisms on a request from the Commission related to the Notification (Reference C/NL/98/11) for the placing on the market of herbicide-tolerant oilseed rape GT73, for import and processing, under Part C of Directive 2001/18/EC from Monsanto (Question N° EFSA-Q-2003-078). Opinion adopted on 11 February 2004

Outline of the biological diversity risk assessment report: Type 1 use approval for RT73

Product safety description

NOVEL FOOD INFORMATION - FOOD BIOTECHNOLOGY GLYPHOSATE TOLERANT CANOLA, GT73

General release of Roundup Ready canola (Brassica napus) in Australia.

Determination of the Safety of Monsanto‘s Canola RT73 (Glyphosate- Herbicide Tolerant Canola) for Direct Use as Food, Feed and Processing

Petition for Determination of Nonregulated Status for Roundup Ready Canola RT73 (CBI-deleted)

Monsanto Co.; Availability of determination of nonregulated status for canola genetically engineered for glyphosate herbicide tolerance.

Response to Monsanto Petition 98-216-01p for Determination of Nonregulated Status for Glyphosate-Tolerant Canola Line RT73

Giesy, J.P., Dodson, S. & Solomon, K.R. (2000). Ecotoxocological risk assessment for Roundup herbicide. Reviews of Environmental Contamination and Toxicology 167, 35-120.

Williams, G.M., Kroes, R. & Munro, I.C. (2000). Safety evaluation and risk assessment of the herbicide Roundup and its active ingredient, glyphosate, for humans. Regulatory Toxicology and Pharmacology 31, 117-165.