DD2006-58: Determination of the Safety of Bayer CropScience's Glufosinate Ammonium Tolerant Rice (Oryza sativa) Event LLrice62
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This Decision Document has been prepared to explain the regulatory decision reached under the regulatory directive Dir95-03 Guidelines for the Assessment of Novel Feeds: Plant Sources and regulatory directive Dir94-08 Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits.
The Canadian Food Inspection Agency (CFIA), specifically the Feed Section of the CFIA, with advice from the Plant Biosafety Office of the CFIA, has evaluated information submitted by Bayer CropScience regarding the glufosinate ammonium tolerant rice event LLrice62. The CFIA has determined that feed derived from this modified plant does not present a significant risk to the environment, nor does it present livestock feed safety concerns when compared to currently commercialized rice varieties in Canada.
Livestock feed use of rice event LLrice62 is therefore authorized as of June 1, 2006. LLrice62 and all its progeny and sister lines which have been derived from the original transformation event and their respective progenies, are also authorized for use in livestock feed, provided that: (i) no inter-specific crosses are performed, (ii) the intended uses are similar, (iii) based on characterization, these plants do not display any additional novel traits and are substantially equivalent, in terms of their specific use and safety for the environment and for human and animal health, to plants currently being cultivated and (iv) the novel genes are expressed at a level similar to that of the authorized line.
This rice event is subject to the same phytosanitary import requirements as its unmodified counterparts.
Table of Contents
- Development Method
- Glufosinate ammonium tolerance
- Stable Integration into the Plant's Genome
- Potential of LLrice62 to Become a Weed of Agriculture or be Invasive of Natural Habitats
- Potential for Gene Flow from LLrice62 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
- Altered Plant Pest Potential of LLrice62
- Potential Impact of LLrice62 on Non-Target Organisms
- Potential Impact on Biodiversity of LLrice62
- Potential Impact on Livestock Nutrition
- Potential Impact on Livestock and Workers/By-standers
I. Brief Identification of the Modified Plant
Designation(s) of the Plant: LLrice62, OECD identifier ACS-OS002-5
Applicant: Bayer CropScience
Plant Species: Rice (Oryza sativa)
Novel Traits: Tolerance to the herbicide glufosinate ammonium
Trait Introduction Method: Microprojectile bombardment of plant cells
Proposed Use of Plant: Production of rice for livestock feed, human food and industrial uses. These materials will be grown outside of Canada, in the usual production areas for rice.
II. Background Information
Bayer CropScience has developed a rice line tolerant to glufosinate ammonium. The rice event, designated as LLrice62, was developed to impart novel tolerance to the herbicide glufosinate ammonium. This herbicide tolerance trait allows for the control or suppression of weeds in rice production.
LLrice62 was developed using using recombinant DNA technology, resulting in the introduction of a bar gene isolated from soil bacterium Streptomyces hygroscopicus. The bar gene codes for the production of the enzyme Phosphinothricin Acetyl-Transferase (PAT) which acetylates glufosinate ammonium, thereby detoxifying the herbicide, conferring herbicide resistance to the rice plant. The bar gene, producing an equivalent PAT protein to that found in LLrice62, has been previously approved for livestock feed use in LibertyLink® cotton event LLcotton25.
Bayer CropScience has provided data on the identity of LLrice62, a detailed description of the transformation method, data and information on the gene insertion site, gene copy number and levels of gene expression in the plant and the role of the inserted genes and regulatory sequences and the full nucleotide sequence of the bar gene. The novel protein was identified and the mode of action was described and characterized. Data was provided for the evaluation of the potential toxicity of the novel protein to livestock and non-target organisms and potential allergenicity of the novel protein to humans.
Agronomic characteristics of LLrice62 such as plant morphology, agronomic performance, disease susceptibility, seed germination and reproductive fitness were compared to those of an unmodified rice counterpart.
Nutritional components of rice grain and processed fractions of LLrice62, such as proximates, amino acids, fatty acids, vitamins, minerals and antinutrients were compared with those of an unmodified rice counterpart.
The Feed Section, CFIA, with input from the Plant Biosafety Office, CFIA, has reviewed the above information. The following assessment criteria, as described in regulatory directives Dir95-03 and Dir94-08, were used to determine the safety and efficacy as livestock feed and the environmental safety of this novel feed:
- potential impact of event LLrice62 on livestock and workers/by-standers,
- potential impact of event LLrice62 on livestock nutrition,
- potential of event LLrice62 to become a weed of agriculture or invasive of natural habitats,
- potential for gene flow from event LLrice62 to wild relatives whose hybrid progeny may become more weedy or more invasive,
- potential for event LLrice62 to become a plant pest,
- potential impact of event LLrice62 or its gene products on non-target species, including humans, and
- potential impact of event LLrice62 on biodiversity.
Additionally, the CFIA has reviewed a method submitted by Bayer CropScience for the detection and identification of rice containing the LLrice62 event.
III. Description of the Novel Traits
1. Development Method
Rice callus tissue was transformed with a purified plasmid fragment containing the bar gene using microprojectile bombardment. Transformants were selected for on media containing phosphinothricin. LLrice62 was identified as a successfully transformed rice event and was chosen for further development.
2. Glufosinate ammonium tolerance
Phosphinothricin, the active ingredient of glufosinate ammonium, inhibits glutamine synthetase, which results in the accumulation of lethal levels of ammonia in susceptible plants within hours of application. Plants produce ammonia as a result of normal metabolic processes.
The bar gene engineered into LLrice62 codes for the production of the enzyme Phosphinothricin Acetyl-Transferase (PAT) which acetylates glufosinate ammonium, thereby detoxifying the herbicide, conferring herbicide resistance to the rice plant. PAT has extremely high substrate specificity.
The introduced bar gene was originally isolated from Streptomyces hygroscopicus, a common gram-positive soil-borne bacterium. The PAT enzyme is therefore naturally occurring in the soil. More generally, acetyltransferases are ubiquitous in nature.
The bar gene expressed in event LLrice62 is linked to a constitutive promoter (i.e. results in expression in all rice tissues). PAT protein expression was determined from plants grown in Crowley, Louisiana in 1998. Mean protein expression in event LLrice62 was 12.1 µg/g fresh weight, 75.3 µg/g fresh weight, 1.56 µg/g fresh weight, 12.7 µg/g fresh weight, 30.9 µg/g fresh weight, 84.7 µg/g fresh weight in grain, straw, rice hulls, roots, stems, and leaves respectively.
Protein allergens are normally resistant to digestion unlike the PAT protein, which was shown to degrade readily in simulated gastric fluid within 30 seconds and simulated intestinal fluid within 5 minutes. Unlike many known allergens the PAT protein is not glycosylated and is present at low levels (0.02% in grain, 0.32% in straw). Using sera from patients with known rice allergen sensitivity, it was demonstrated that levels of endogenous rice allergens were comparable between the modified rice and the unmodified parent.
A search for amino acid sequence similarity between the PAT protein and known allergens and toxins, using a database assembled from the public domain databases SwissProt, trEMBL, GeneSeq-Prot, PIR, PDB, DAD and GenPept, revealed no overall homology (based on 35% identity on a window of 80 amino acids). PAT only had high similarity with other acetyltransferase proteins. Additionally, no significant epitope similarities between the PAT protein and known allergens (based on 100% sequence identity of 8 or more contiguous amino acids) were found.
Due to the low levels of PAT protein expressed in the rice plant, it was necessary to produce PAT protein by bacterial fermentation to obtain sufficient quantities to conduct some studies. The bacterially produced protein was compared to the plant produced protein and shown to be of similar molecular weight and immunological reactivity. Neither the plant nor bacterially expressed PAT is glycosylated.
3. Stable Expression
Southern blot analysis of event LLrice62 indicated that there is one site of integration of the introduced DNA which includes a single copy of the bar gene. The data demonstrates that the bar coding region and associated promoter and terminator sequences are intact.
Southern blot analyses conducted over 4 different generations (T2, T3, T5 and T6) and 3 different rice varieties demonstrated the stability of the LLrice62 DNA insert.
IV. Criteria for the Environmental Assessment
Note: Event LLrice62 will not be grown in Canada and will only be imported as human food or livestock feed. The majority of rice imported to Canada does not have an intact hull, which results in the seed being incapable of germination and growth. Due to the unfavorable climatic conditions for rice in Canada, it is unlikely that grain from event LLrice62 would be capable of persisting in the Canadian environment.
1. Potential of LLrice62 to Become a Weed of Agriculture or be Invasive of Natural Habitats
The centre of origin of rice is considered to be in the subtropics of Southeast Asia. Rice is not grown in Canada and is not adapted to the environmental conditions encountered in Canadian agricultural environments.
The CFIA evaluated data submitted by Bayer CropScience on the biology of the event LLrice62 and determined that vegetative vigour, time to maturity and seed production were within the normal range of expression of these traits currently displayed by commercial rice varieties.
No competitive advantage was conferred to these plants, other than that conferred by tolerance to glufosinate ammonium herbicide. Tolerance to glufosinate ammonium will not, in itself, render rice weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified.
The event LLrice62 is not intended for cultivation in Canada and the novel traits have no intended effects on weediness or invasiveness. The CFIA has therefore concluded that this event has no altered weed or invasiveness potential in Canada when compared to conventional rice varieties.
2. Potential for Gene Flow from LLrice62 to Wild Relatives Whose Hybrid Offspring May Become More Weedy or More Invasive
Species sexually compatible with rice do not occur in Canada. The wild "rice" which occurs in Canada (Zizania aquatica) belongs to a species that is not sexually compatible with domesticated rice (Oryza sativa). Event LLrice62 will not be cultivated in the Canada and if released, would not persist.
The CFIA has therefore determined that gene flow to sexually compatible species in Canada is not possible.
3. Altered Plant Pest Potential of LLrice62
Oryza sativa is not a plant pest in Canada. Additionally, the agronomic characteristics of LLrice62 were shown to be within the normal range of conventional rice varieties.
The CFIA has therefore determined that the event LLrice62 does not present a plant pest concern.
4. Potential Impact on Non-Target Organisms of LLrice62
The detailed characterization of the introduced gene and the resulting enzyme, has led to the conclusion that the expression of the novel protein does not result in altered toxic or allergenic properties. The PAT enzyme is not a known toxin, does not confer resistance to agricultural pests and acetyltransferases are commonly found in nature with a history of safe use.
Based on the above, the CFIA has determined that the event LLrice62 will not result in altered impacts on non-target organisms, including humans, compared to current rice varieties.
5. Potential Impact on Biodiversity of LLrice62
This event is safe to non-target organisms, does not present altered weediness or plant pest potential and will not be grown in Canada. In addition, the novel trait has not altered the ability of this event to persist in the Canadian environment.
The CFIA has therefore concluded that LLrice62 will have no impact on biodiversity in Canada.
V. Criteria for the Livestock Feed Assessment
1. Potential Impact on Livestock Nutrition
Nutritional Composition and Anti-Nutritional Factors
Composition of rice grain and straw samples of LLrice62 and its control (Bengal) was determined for samples from replicated trials conducted over two years in Louisiana, Mississippi, and Arkansas. At each site the treatments consisted of LLrice62 (unsprayed) and control Bengal (unsprayed), and at 10 of the 14 trial sites a third treatment, LLrice62 (sprayed with Liberty). Components analysed were as follows:
- Grain: Proximates, acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fibre, amino acids, minerals, vitamins, phytic acid, trypsin inhibitor, lectin;
- Straw: Proximates, ADF, NDF, total dietary fibre.
There were no differences between LLrice62 and control Bengal rice grain in crude protein, fat, ash, total dietary fibre, amino acids except for Tyrosine and Tryptophan (no consistent pattern of differences), most fatty acids (14:0, 16:0, 18:0, 18:1, 18:2, 18:3, 20:0,20:1, 24:0), most minerals and most vitamins. Trypsin inhibitor and lectin (hemagglutinin) were not detected in any sample. Phytic acid levels were equivalent in the three treatments. Some differences were observed in ADF, NDF, and crude fibre, which showed no consistent pattern across locations. Thiamin, Ca, Fe, and pantothenic acid were higher in LLrice62 than control. There were no differences in straw composition between LLrice62 and control.
Additionally, material from a subset of the trials used to generate the rice grain/straw composition data was further processed into various rice fractions. The composition of the following fractions of LLrice62 and control was determined: rice hulls, brown rice, polished rice, rice flour, rice bran, rice bran oil. These studies further supported the equivalence in composition between LLrice62 and control.
Finally, a 42-day broiler chicken study was conducted, in which diets containing 30% rice (LLrice62 vs. control) were compared. There were no differences in weight gains, feed intake, carcass weight, abdominal fat weight, breast muscle weight, or post-mortem macroscopic findings, between birds fed LLrice62 and those fed the control diet.
Overall the applicant has demonstrated the equivalence of LLrice62 to conventional Bengal rice in terms of nutritional composition and feeding value.
2. Potential Impacts on Livestock and Workers/By-standers
PAT is a highly substrate specific enzyme that has been well defined. Exposure to PAT protein is not new. The bar gene is isolated from Streptomyces hygroscopicus, a common soil bacterium. The bar gene is present in the environment with no known adverse effects on humans and animals. In addition, an equivalent PAT protein from the bar gene has been expressed in a previously authorized crop in Canada, LibertyLink® LLcotton25.
While the PAT protein from the bar gene has been shown to have heat stability, unlike typical allergens, the PAT protein was shown to be rapidly digested under simulated gastric and intestinal fluid conditions, is not glycosylated and is present in LLrice62 at very low levels. Also, sera testing showed that the levels of endogenous allergens in LLrice62 did not differ from conventional rice. Homology comparisons between the LLrice62 PAT protein and known toxins, allergens and allergenic epitopes revealed no similarities.
In addition to the above information, an acute intravenous mouse toxicity study with the PAT protein was carried out and revealed no treatment-related adverse effects at 9.5 mg/kg body weight, the highest dose tested. Also, no adverse affects were demonstrated in the broiler chicken feeding study utilizing rice (30% dietary incorporation).
Based on the expected exposure levels and the results of these tests, the introduced gene is unlikely to produce a novel toxin or allergen and no significant risk to livestock and workers/by-standers is expected from exposure to the PAT enzyme.
VI. New Information Requirements
If at any time, Bayer CropScience becomes aware of any information regarding risk to the environment, animal or human health that could result from release of these materials in Canada, or elsewhere, Bayer CropScience must immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of the proposed use and will re-evaluate its decision with respect to the livestock feed authorization of this event.
VII. Regulatory Decision
Based on the review of data and information submitted by Bayer CropScience, including comparisons of rice LLrice62 with unmodified rice counterparts, the Feed Section, CFIA, has concluded that the introduced gene and its corresponding novel trait will not confer to LLrice62 any characteristic that would raise any concerns regarding safety or nutritional composition. Rice grain, groats, hulls, bran and rice bran oil are currently listed in Schedule IV of the Feeds Regulations and are, therefore approved for use in livestock feeds in Canada. LLrice62 has been assessed and found to be as safe as and as nutritious as traditional rice varieties. LLrice62 and its products are considered to meet present ingredient definitions and are approved for use as livestock feed ingredients in Canada. This event will not be grown in Canada nor can the seed overwinter, therefore the release of the feed into the environment would result in neither intended nor unintended environmental effects.
Livestock feed use of LLrice62 is therefore authorized as of June 1, 2006.
All LLrice62 progeny and sister lines which have been derived from the original transformation event and their respective progenies, are also authorized for unconfined release and livestock feed, provided that no inter-specific crosses are performed, provided the intended uses are similar, provided that based on characterization, these plants do not display any additional novel traits and are substantially equivalent, in terms of their specific use and safety for the environment and for human and animal health, to plants currently being cultivated and provided the novel genes are expressed at a level similar to that of the authorized line.
LLrice62 is subject to the same phytosanitary import requirements as its unmodified counterparts.
Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of LLrice62. The food safety decisions are available on the Health Canada web site.
This bulletin is published by the Canadian Food Inspection Agency. For further information, please contact the Feed Section at:
Animal Health and Production Division
Animal Products Directorate
59 Camelot Drive, Ottawa
Ontario K1A 0Y9
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