DD2006-63: Determination of the Safety of BASF's Imidazolinone-Tolerant CLEARFIELD™ Durum Wheat Event DW1
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This Decision Document has been prepared to explain the regulatory decision reached under Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants with Novel Traits", its companion biology document Bio2006-07, The Biology of Triticum turgidum ssp. durum L. (Durum Wheat), and Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".
The Canadian Food Inspection Agency (CFIA), specifically the Biotechnology Environmental Release Assessment Unit (BERA) of the Science Strategies Directorate and the Feed Section of the Animal Health and Production Division, have evaluated information submitted by BASF. This information is in regard to the imidazolinone tolerant durum wheat event DW1. The CFIA has determined that this plant with a novel trait (PNT) does not present altered environmental risk nor, as a novel feed, does it present livestock feed safety concerns when compared to currently commercialized durum wheat varieties in Canada.
Taking into account these evaluations, unconfined release into the environment and use as livestock feed of the CLEARFIELD™ durum wheat event DW1 is authorized by the Plant Biosafety Office of the Plant Products Directorate and the Feed Section of the Animal Health and Production Division as of December 20, 2006. Any wheat lines derived from DW1 may also be released into the environment and used as livestock feed, provided (i) no inter-specific crosses are performed, (ii) the intended use(s) are similar, (iii) it is known, following thorough characterization, that these plants do not display any additional novel traits and are substantially equivalent to currently grown durum wheat, in terms of their potential environmental impact and livestock feed safety.
The durum wheat event DW1 is subject to the same phytosanitary import requirements as its unmodified counterpart.
Please note that the assessment of livestock feed safety and environmental safety are critical steps in the potential commercialization of these plant types. Other requirements, such as the evaluation of food safety by Health Canada, have been addressed separately from this review.
Table of Contents
- Development Method
- Imidazolinone Tolerance
- Stable Expression
- Potential of event DW1 to Become a Weed of Agriculture or Invasive of Natural Habitats
- Potential for Gene Flow from event DW1 to Wild Relatives Whose Offspring May Become More Weedy or More Invasive
- Altered Plant Pest Potential of event DW1
- Potential Impact on Non-Target Organisms of event DW1
- Potential Impact on Biodiversity of event DW1
- Potential Impact of event DW1 on Livestock Nutrition
- Potential Impact of event DW1 on Livestock and Workers/By-standers
I. Brief Identification of the Modified Plant
Designation(s) of the Modified Plant: CLEARFIELD™ durum wheat event DW1
Applicant: BASF Canada
Plant Species: Durum Wheat (Triticum turgidum L.)
Novel Traits: Tolerance to imidazolinone herbicides
Trait Introduction Method: Chemically induced seed mutagenesis
Proposed Use of the Modified Plant: Production of wheat for livestock feed and human food
II. Background Information
BASF has developed a durum wheat event tolerant to imidazolinone herbicides. This wheat event was developed to provide an alternative strategy for weed control.
The development of the CLEARFIELD™ durum wheat event DW1 was accomplished using chemically induced seed mutagenesis. The DW1 event is allotetraploid (28 chromosomes, n=14), belonging to genus and species Triticum turgidum ssp. durumL. Durum wheat carries two complete genomes designated "A"; and "B". Each genome has an AHAS gene (ALS3 and ALS2, respectively). The herbicide tolerance in event DW1 results from a single point mutation modification in the ALS2 acetohydroxyacid synthase (AHAS) gene in the durum wheat genome such that the resulting modified enzyme has a single amino acid substitution and is no longer affected by imidazolinone herbicides.
BASF has provided data on the identity of the wheat event, a detailed description of the modification method and breeding history, information on the modified gene, the resulting protein and its mode of action and the stability of trait expression.
Event DW1 was field tested in the United States of America in 2003 and 2004.
Agronomic characteristics of event DW1 such as seed germination, seedling emergence, seeding vigor, foliar disease susceptibility, insect damage, plant height, days to heading, days to maturity, harvest moisture, test weight and grain yield were compared to those of unmodified durum wheat counterparts.
Nutritional components of event DW1 such as proximates, amino acids and fatty acids were compared with unmodified wheat counterparts. Levels and/or absences of anti-nutritional factors were also determined.
BASF has provided an agronomic stewardship plan for imidazolinone tolerant durum wheat in the Canadian environment. This plan includes information regarding a safe and sustainable deployment of imidazolinone-tolerant durum wheat and an efficient mechanism for growers to report agronomic problems with this product to BASF.
The Biotechnology Environmental Release Assessment (BERA) Unit of the Science Strategies Division, CFIA, has reviewed the above information, in light of the assessment criteria for determining environmental safety of PNTs, as described in the Directive 94-08 (Dir94-08), entitled "Assessment Criteria for Determining Environmental Safety of Plants With Novel Traits". BERA has considered the:
- potential of event DW1 to become a weed of agriculture or be invasive of natural habitats;
- potential for gene flow from event DW1 to wild relatives whose hybrid offspring may become more weedy or more invasive;
- potential of event DW1 to become a plant pest;
- potential impact of event DW1 or its' gene products on non-target species, including humans; and
- potential impact of event DW1 on biodiversity.
The Feed Section of the Animal Health and Production Division, CFIA, has also reviewed the above information with respect to the assessment criteria for determining the safety and efficacy of livestock feed, as described in Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources". The Feed Section has considered the:
- potential impact of event DW1 on livestock nutrition; and
- potential impact of event DW1 on livestock and workers/by-standers.
III. Description and Assessment of the Novel Trait
1. Development Method
The original mutant event DW1 was isolated from populations derived by chemical-induced mutagenesis with ethyl methanesulfonate (EMS) and sodium azide of seed of a parental durum wheat variety. Whole plant selection procedures for herbicide tolerance were used (seedlings were sprayed with imidazolinone herbicides; only tolerant plants lived and were used in the following breeding generations).
2. Imidazolinone Tolerance
Imidazolinone herbicides are active against the enzyme acetohydroxyacid synthase (AHAS), also known as acetolactate synthase (ALS). AHAS is an enzyme found in bacteria, certain other micro-organisms and plants. This enzyme catalyses the first step in the biosynthesis of the essential branched chain amino acids isoleucine, leucine and valine. Herbicide-induced AHAS inhibition results in a lethal decrease in protein synthesis. Unmodified durum wheat is not tolerant to imidazolinone herbicides.
A single amino acid substitution in the AHAS enzyme was sufficient to alter the binding site for imidazolinone herbicides, resulting in the tolerant phenotype.
The novel imidazolinone tolerance is under the control of the native AHAS promoter and is believed to be constitutively expressed. Sequence information for the modified AHAS gene in DW1 was submitted.
The tolerance to imidazolinone was demonstrated by comparison of the activity of the AHAS enzyme extracted from DW1 durum wheat plants to that of conventional type durum wheat plants. In the absence of the herbicide, the AHAS enzyme activity was similar, but when imidazolinone herbicide was added to the assay, the enzyme activity of the susceptible plants was inhibited much more than enzyme from the tolerant plants.
The levels of valine, leucine and isoleucine produced in wheat are regulated by feedback inhibition of AHAS. BASF provided data to demonstrate that the modified AHAS shows similar feedback inhibition by valine and leucine as compared to unmodified AHAS. The modification of the AHAS does not affect feedback inhibition and hence, the regulation and levels of these amino acids.
Unlike known food allergens, AHAS is a minor protein in plant tissue, it is heat sensitive and trypsin susceptible. The AHAS protein from DW1 was shown to be heat sensitive, with no detectable activity of AHAS after 1 minute of heating at 100°C. AHAS from DW1 was shown to be equivalent to parental controls with respect to trypsin degradation. The unmodified form of the AHAS protein shows no amino acid similarity to known allergens. The amino acid sequence of mutated AHAS differs by one amino acid from that of unmodified durum wheat.
BASF provided evidence to show that the protein components of DW1 is not altered in comparison with an unmodified comparator. HPLC performed using protein extracts from unmodified and modified durum wheat indicated that no new major proteins or increased protein expression occurred as a result of the mutagenic event.
BASF has provided to the CFIA a method for the detection and identification of durum wheat containing this novel trait.
3. Stable Expression
Durum wheat event DW1 demonstrated consistent herbicide tolerance over multiple breeding generations, confirming the stable inheritance of this trait.
IV. Criteria for the Environmental Assessment
1. Potential of event DW1 to Become a Weed of Agriculture or Invasive of Natural Habitats
Durum wheat possesses few of the characteristics that are common to weeds and invasive plants. It is an annual crop that does not persist in unmanaged ecosystems without human intervention. Durum plants can grow as volunteers in cultivated fields in the seasons following a durum crop, but they are usually eliminated by soil cultivation or the use of herbicides. There have been no reports of durum wheat becoming an unmanageable weed of agriculture or an invasive pest, in North America or the world.
The CFIA evaluated data submitted by BASF on the agronomic characteristics and biology of durum event DW1, and determined that vegetative vigour, time to maturity, seed production, and resistance to disease were within the normal range of expression of these traits currently displayed by the parent variety.
No competitive advantage was conferred on plants with event DW1, other than that conferred by tolerance to imidazolinone herbicides, as none of the reproductive or growth characteristics of DW1 were different from those of the parent variety. Tolerance to imidazolinone herbicides provides a competitive advantage only when these herbicides are used, and will not, in and of itself, make a plant weedier or more invasive of natural habitats. Imidazolinone tolerance will not cause DW1 to become weedier or more invasive in managed habitats than unmodified Triticum turgidum spp. durum. Imidazolinone-tolerant durum volunteers will not be controlled in subsequent crops if imidazolinone is used as the only weed control tool. However, control of imidazolinone- tolerant durum wheat as a volunteer weed in other crops or in fallow ground can readily be achieved by the use of classes of herbicides other than imidazolinones, or by mechanical means.
The novel trait has no intended or observed effects on weediness or invasiveness. The CFIA has therefore concluded that this durum event have no altered weed or invasiveness potential in Canada when compared to conventional durum wheat varieties.
The agronomic stewardship plan, which contains a herbicide tolerance management plan, was submitted by BASF and determined to be satisfactory when evaluated by the CFIA. This stewardship plan also applies to events DW2, DW6, and DW12 (see Decision Document 64) as well as bread wheat events BW255-2 and BW238-3 (see Decision Document DD2006-60). The herbicide tolerant stewardship plan includes recommendations on agricultural practices concerning imidazolinone tolerant durum and provides an efficient mechanism for growers to report agronomic problems with DW1 to BASF, which will facilitate the ongoing monitoring of imidazolinone tolerant durum wheat. In addition, BASF is required to monitor grower compliance to determine the effectiveness of the stewardship plan and make any changes to the plan as appropriate.
In the longer term, widespread adoption of several different crop species with specific associated weed management systems (i.e. numerous crop species each with one or more tolerances to different herbicides) may lead to the development of crop volunteers with a combination of novel tolerances to different herbicides. This could result in the loss of the use of these herbicides and any of their potential benefits. Therefore, BASF will make their stewardship plan readily available to growers and agriculture extension personnel, in both private and public sectors, to promote careful management practices for durum wheat DW1. These practices include the use of alternate control tools as appropriate to achieve complete weed and volunteer control (which is also recommended to help minimize the development of resistant weed populations).
2. Potential for Gene Flow from event DW1 to Wild Relatives Whose Offspring May Become More Weedy or More Invasive
No known wild Triticum species exist in North America. The closest known relatives in North America are members of the Aegilops genus. Aegilops cylindrica, jointed goat grass, is present in the United States where it can be found as a weed in winter wheat fields, but is not reported in Canada (nor are any other Aegilops species). Additionally, Aegilops cylindrica does not readily produce fertile progeny when hybridized with durum (research studies indicate that Aegilops cylindrica x durum hybrids are sterile). Aegilops cylindrica is included in the provincial Noxious Weed List in British Columbia to prevent and/or delay the spread of this weed into Canada.
The only weed species found in Canada that is closely related to durum wheat is Agropyron repens (quackgrass). Agropyron repens is a common and troublesome weedy grass of agricultural areas throughout Canada. However, no known naturally-occurring hybrids between durum and Agropyron species have been reported, and published literature on the genetics of Triticum and Agropyron indicate that natural crossing between these two genera is unlikely.
Triticale is a crop that has been developed by humans by crossing wheat (Triticum aestivum or Triticum turgidum) with rye (Secale cereale). There have been no reports of triticale serving as a hybridization bridge between wheat and rye.
The CFIA has therefore determined that gene flow from durum wheat DW1 to wild or weedy species in Canada is very unlikely.
3. Altered Plant Pest Potential of event DW1
The intended effect of the novel trait is unrelated to plant pest potential. Triticum turgidum is not a plant pest in Canada. Additionally, the agronomic characteristics of durum event DW1 (including the responses to diseases) were shown to be within the normal range of conventional durum varieties.
The CFIA has therefore determined that durum event DW1 does not present a plant pest concern.
4. Potential Impact on Non-Target Organisms of event DW1
Single amino acid modification of the AHAS enzyme, which alters the herbicide binding site on the enzyme, is the molecular basis for imidazolinone tolerance in durum event DW1. BASF has submitted data indicating that the modified AHAS is substantially equivalent to its unmodified counterparts with respect to enzyme activity (biosynthesis of the precursor molecule of valine, leucine, and isoleucine) and feedback inhibition (from valine and leucine). The modified AHAS enzyme only differs from the unmodified enzyme by being less inhibited by imidazolinone herbicides. The detailed compositional analysis has led to the conclusion that durum event DW1 is substantially equivalent to its parent line.
The AHAS enzyme is not a known toxin, does not confer resistance to agricultural pests and is commonly found in a wide variety of plants and micro-organisms with a history of safe use. No novel toxins were introduced into this variety. Therefore, no negative interactions with non-target symbiotic or consumer organisms are anticipated.
In addition, agronomic characteristics, pathogen interactions, and insect pest susceptibility of event DW1 is within the expected range of values displayed by currently commercialized durum wheat varieties. The CFIA concluded that there were not likely to be significant unintended changes to durum event DW1 that could have adverse impacts on non target organisms.
5. Potential Impact on Biodiversity of event DW1
Durum event DW1 is safe to non-target organisms, does not present altered weediness or plant pest potential and is not intended to be grown in Canada. In addition the novel trait has not altered the ability of this line to persist in the Canadian environment.
The CFIA has therefore concluded that the potential impact on biodiversity of durum event DW1 is equivalent to that of currently commercialized durum wheat lines.
V. Nutritional Criteria Assessment as Livestock Feed
1. Potential Impact on Livestock Nutrition
Nutritional Composition and Anti-Nutritional Factors
Nutritional composition data was obtained from event DW1 and the parental variety, which were grown in the U.S.A. in 2003 and 2004. Grain samples were analyzed for protein, fat, fibre, ADF, TDF, amino acids, fatty acids, minerals and vitamins. There were no statistically significant differences between DW1 and control for protein, fat, ADF, TDF, branched chain amino-acids and essential amino acids for both years. Fatty acids measured in DW1 were statistically significantly different from the control in the 2003 study while oleic acid was statistically significant different from the parental variety in the 2004 study. All fatty acids were within the range of conventional durum wheat varieties. Potassium was significantly lower in DW1 compared to the control in 2003 and 2004, respectively. Vitamin B1 and folic acid were significantly different in DW1 compared to the control in the 2003 study. However vitamin and mineral levels were within ranges of commercial durum wheat varieties for both years.
Phytic and trypsin inhibitor were analyzed in DW1 and compared to parental variety. There were no statistically significant differences in phytic acid between DW1 and the parental variety for both years. Trypsin inhibitor was not detected for both DW1 and the parental control.
The evidence provided by BASF supports the conclusion that the nutritional composition of durum wheat event DW1 is equivalent to conventional durum wheat varieties.
2. Potential Impact on Livestock and Workers/By-standers
The AHAS enzyme is found in a wide variety of plants and micro-organisms. AHAS is not a known toxin or allergen and a single base pair change would not be expected to change this. AHAS from event DW1 is feedback inhibited as is unmodified AHAS, it is present in small amounts in the feed, it is heat labile and it is rapidly degraded under conditions in the gastrointestinal tract. The expression of AHAS is not changed by the modification. Based on the information provided by BASF, the modified AHAS is unlikely to be a novel toxin or allergen.
Based on the detailed characterization provided (nutritional composition, agronomic data and HPLC protein profiles of the modified plant compared to the unmodified comparator) it is unlikely that secondary mutations causing unintended effects have occurred in the genome of durum wheat event DW1.
The evidence provided by BASF supports the conclusion that the potential impact on livestock and workers/by-standers of durum event DW1 is equivalent to that of currently commercialized durum wheat lines.
VI. New Information Requirements
If at any time, BASF becomes aware of any information regarding risk to the environment, including risk to human or animal health, that could result from release, in Canada or elsewhere, of durum wheat event DW1, its descendants, or products derived there from, BASF must immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of event DW1 on the environment, livestock and human health, and may re-evaluate its decision with respect to the livestock feed use and environmental release authorizations of event DW1.
VII. Regulatory Decision
Based on the review of data and information and stewardship plan, submitted by BASF, and through comparisons of DW1 with unmodified durum wheat counterparts, the Biotechnology Environmental Release Assessment Unit, CFIA, has concluded that the modified gene and its corresponding traits does not confer to durum wheat event DW1 any characteristic that would result in intended or unintended significant environmental effects following unconfined release.
Based on the review of data and information submitted by BASF, including comparisons of event DW1 with unmodified durum wheat counterparts, the Feed Section, CFIA, has concluded that the modified gene and its' corresponding novel trait will not confer to these plants any characteristic that would raise any concerns regarding the safety or nutritional composition of durum wheat event DW1. Wheat grain, its byproducts and wheat germ oil, are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. Durum wheat event DW1 has been assessed and found to be as safe and nutritious as traditional wheat varieties. DW1 and its' products are considered to meet the present ingredient definitions and are approved for use as livestock feed ingredients in Canada.
Taking into account these evaluations, unconfined release into the environment and livestock feed use of the CLEARFIELD™ durum wheat event DW1 is therefore authorized by the Plant Biosafety Office of the Plant Products Directorate and the Feed Section of the Animal Health and Production Division as of December 20, 2006. Any other wheat lines derived from this event may be imported and/or released, provided no inter-specific crosses are performed, provided the intended use is similar, and provided it is known, following thorough characterization that these plants do not display any additional novel traits and are substantially equivalent to currently grown wheat, in terms of their potential environmental impact and livestock feed safety.
The CLEARFIELD™ durum wheat event DW1 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 CLEARFIEL™ durum wheat event DW1.
This bulletin is published by the Canadian Food Inspection Agency. For further information, please contact the Feed Section or Plant Biosafety Office at:
Animal Health and Production Division
Animal Products Directorate
59 Camelot Drive
Ottawa, Ontario K1A 0Y9
Plant Biosafety Office
Plant Products Directorate
59 Camelot Drive
Ottawa, Ontario K1A 0Y9
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