DD2007-64: Determination of the Safety of BASF's Imidazolinone-Tolerant Clearfield™ Durum Wheat Events DW2, DW6, and DW12

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Issued: 2007-01

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 (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 events DW2, DW6, and DW12 (please note that DW2 and DW6 are not considered novel feeds, as explained in Section V). The CFIA has determined that these plants with a novel trait (PNT) do not present altered environmental risk nor, as a novel feed, does event DW12 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 of the Clearfield™ durum wheat events DW2, DW6, and DW12 and use as livestock feed of durum wheat event DW12 are authorized by the Plant Biosafety Office (PBO) of the Plant Products Directorate and the Feed Section of the Animal Health and Production Division as of January 4, 2007. Any wheat lines derived from events DW2, DW6, and DW12 may also be released into the environment and any wheat lines derived from event DW12 may be used as livestock feed, provided (i) no inter-specific crosses are performed, (ii) the intended uses 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.

Durum wheat events DW2, DW6, and DW12 are subject to the same phytosanitary import requirements as their 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

I. Brief Identification of the Modified Plant

II. Background Information

III. Description and Assessment of the Novel Trait

  1. Development Method
  2. Imidazolinone Tolerance
  3. Stable Expression

IV. Criteria for the Environmental Assessment

  1. Potential of events DW2, DW6, and DW12 to Become Weeds of Agriculture or Invasive of Natural Habitats
  2. Potential for Gene Flow from events DW2, DW6, and DW12 to Wild Relatives Whose Offspring May Become More Weedy or More Invasive
  3. Altered Plant Pest Potential of events DW2, DW6, and DW12
  4. Potential Impact on Non-Target Organisms of events DW2, DW6, and DW12
  5. Potential Impact on Biodiversity of events DW2, DW6, and DW12

V. Nutritional Criteria Assessment as Livestock Feed

  1. Potential Impact of event DW12 on Livestock Nutrition
  2. Potential Impact of event DW12 on Livestock and Workers/By-standers

VI. New Information Requirements

VII. Regulatory Decision

I. Brief Identification of the Modified Plant

Designation(s) of the Modified Plant: Clearfield™ wheat events DW2, DW6, and DW12 (please note that events DW2 and DW6 are not considered to be novel feeds, as explained in Section V)

Applicant: BASF Canada

Plant Species: Durum Wheat (Triticum turgidum spp. durum 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 durum wheat events tolerant to imidazolinone herbicides. These wheat events were developed to provide an alternative strategy for weed control.

The development of the Clearfield™ durum wheat events DW2, DW6, and DW12 was accomplished using chemically induced seed mutagenesis. The DW2, DW6, and DW12 events are allotetraploid (28 chromosomes n=14), belonging to genus and species Triticum turgidum spp. durum L. Durum wheat carries two complete genomes designated "" and "B". Each genome has an AHAS gene (ALS3 and ALS2, respectively). The herbicide tolerance in events DW2, DW6, and DW12 results from a single point mutation modification in the ALS3 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 durum wheat events, 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.

Durum wheat event DW12 was field tested in the United States in 2003 and 2004. Events DW2 and DW6 were field tested in Canada in 1998, 1999, 2000, 2001, 2004, and 2005. Agronomic characteristics of events DW2, DW6, and DW12 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 wheat counterparts.

Nutritional components of durum wheat event DW12 such as proximates, amino acids and fatty acids were compared with unmodified wheat counterparts. 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 Unit (BERA) of the Science Strategies Directorate, CFIA, have 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 events DW2, DW6, and DW12 to become weeds of agriculture or be invasive of natural habitats;
  • potential for gene flow from events DW2, DW6, and DW12 to wild relatives whose hybrid offspring may become more weedy or more invasive;
  • potential of events DW2, DW6, and DW12 to become a plant pest;
  • potential impact of events DW2, DW6, and DW12 or their gene products on non-target species, including humans; and
  • potential impact of events DW2, DW6, and DW12 on biodiversity.

The Feed Section of the Animal Health and Production Division, CFIA, has also reviewed the above information pertaining to durum wheat event DW12 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" (please note that events DW2 and DW6 are not considered to be novel feeds, as explained in Section V). The Feed Section has considered:

  • potential impact of event DW12 on livestock nutrition; and
  • potential impact of event DW12 on livestock and workers/by-standers.

III. Description and Assessment of the Novel Trait

1. Development Method

The original mutant durum wheat events DW2, DW6, and DW12 were isolated from populations derived by chemical-induced mutagenesis with ethyl methanesulfonate (EMS) and sodium azide of wheat seed. 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 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 durum wheat events DW2, DW6, and DW12 was submitted.

The tolerance to imidazolinone was demonstrated by comparison of the activity of the AHAS enzyme extracted from durum wheat events DW2, DW6, and DW12 to that of conventional 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.

The amino acid sequence of mutated AHAS differs by one amino acid from that of unmodified durum wheat. Unlike known food allergens, AHAS is a minor protein in plant tissue, it is heat sensitive and trypsin susceptible. The AHAS protein from durum wheat events DW2, DW6, and DW12 was shown to be heat sensitive, with no detectable activity of AHAS after 1 minute of heating at 100°C AHAS from events DW2, DW6, and DW12 were 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.

BASF provided evidence to show that the protein components of durum wheat events DW2, DW6, and DW12 are not altered in comparison with an unmodified comparator. HPLC was performed on protein extracts from unmodified and modified durum wheat and indicated that no new major proteins or increased protein expression occurred as a result of the mutagenic event.

BASF has provided a method for the detection and identification of durum wheat containing this novel trait to the CFIA.

3. Stable Expression

Selections for durum wheat events DW6 and DW2 were made based on imidazolinone tolerance across at least 11 and 27 generations, respectively. Additionally, the segregation of herbicide tolerance in events DW2 and DW6 was consistent with the inheritance of a single gene. Durum wheat event DW12 demonstrated consistent herbicide tolerance over multiple breeding generations, confirming the stable inheritance of this trait.

IV. Criteria for the Environmental Assessment

1. Potential of events DW2, DW6, and DW12 to Become Weeds 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 biology of durum wheat events DW2, DW6, and DW12, and determined that vegetative vigour, time to maturity, seed production (yield), lodging resistance, and resistance to insects and 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 durum wheat events DW2, DW6, or DW12, other than that conferred by tolerance to imidazolinone herbicides, as none of the reproductive or growth characteristics of these events 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 plants with events DW2, DW6, or DW12 to become weedier or more invasive in managed habitats than unmodified T. 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 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 these durum events have no altered weed or invasiveness potential in Canada when compared to conventional durum varieties.

The agronomic stewardship plan, which contains a herbicide tolerance management plan, submitted by BASF was evaluated by the CFIA and determined to be satisfactory. This stewardship plan applies to the durum wheat events described in this document as well as durum wheat event DW1 (see Decision Document DD2006-63) and 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 this product to BASF, which will facilitate the ongoing monitoring of imidazolinone tolerant durum. 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 events DW2, DW6, and DW12. 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 events DW2, DW6, and DW12 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). A. 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 is Agropyron repens (quackgrass). A. 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 T. 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 events DW2, DW6, and DW12 to wild or weedy species in Canada is very unlikely.

3. Altered Plant Pest Potential of events DW2, DW6, and DW12

The intended effect of the novel trait is unrelated to plant pest potential. T. turgidum is not a plant pest in Canada. Additionally, the agronomic characteristics of durum events DW2, DW6, and DW12 (including the responses to insect and disease pressures) were shown to be within the normal range of conventional durum varieties.

The CFIA has therefore determined that durum events DW2, DW6, and DW12 do not present a plant pest concern.

4. Potential Impact on Non-Target Organisms of events DW2, DW6, and DW12

A 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 wheat events DW2, DW6, and DW12. 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 mutant AHAS in events DW2, DW6, and DW12 has not significantly affected the biosynthesis of the branched- chain amino acids, valine, leucine and isoleucine. The detailed compositional analysis has led to the conclusion that durum wheat events DW2, DW6, and DW12 are substantially equivalent to their parent variety.

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 durum wheat events DW2, DW6, and DW12 are within the range of expected values displayed by currently commercialized durum varieties. The CFIA concluded that there were not likely to be significant unintended changes to durum wheat events DW2, DW6, and DW12 that could have adverse impacts on non target organisms.

5. Potential Impact on Biodiversity of events DW2, DW6, and DW12

Durum wheat events DW2, DW6, and DW12 are safe to non-target organisms, do not present altered weediness or plant pest potential and are 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 wheat events DW2, DW6, and DW12 is equivalent to that of currently commercialized durum lines.

V. Nutritional Criteria Assessment as Livestock Feed

Note: Based on information provided by BASF, the imidazolinone-tolerance trait observed in durum wheat events DW2 and DW6 is the result of a genetic modification that is equivalent to the genetic modification leading to the imidazolinone-tolerance trait observed in durum wheat event DW12, and durum wheat events DW2 and DW6 are substantially equivalent to event DW12 in terms of livestock feed safety. Therefore, with the authorization of event DW12, events DW2 and DW6 are no longer novel livestock feeds.

1. Potential Impact of event DW12 on Livestock Nutrition

Nutritional Composition and Anti-Nutritional Factors

Nutritional composition data was obtained from durum wheat event DW12 and the parental variety, which were grown in the United States 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 event DW12 and the control for protein, fat, ADF, TDF, branched chain amino-acids and essential amino acids for both years. Crude fibre in event DW12 was significantly higher than parent control in the 2003 study but comparable to conventional durum wheat varieties. Fatty acids measured in event DW12 were statistically significantly different from the control in the 2003 study. All fatty acids were within the range of conventional durum wheat varieties. Potassium was significantly higher in event DW12 compared to the control in 2003. Vitamin B1 and folic acid were significantly different in event DW12 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 durum wheat event DW12 and compared to the parental variety. There were no statistically significant differences in phytic acid between event DW12 and the parental variety for both years. Trypsin inhibitor was not detected in event DW12 or the parental control.

The evidence provided by BASF supports the conclusion that the nutritional composition of durum wheat event DW12 is equivalent to conventional durum wheat varieties.

2. Potential Impact of event DW12 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 durum wheat event DW12 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 durum wheat genome.

The evidence provided by BASF supports the conclusion that the potential impact on livestock and workers/by-standers of durum wheat event DW1 is equivalent to that of currently commercialized durum 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 events DW2, DW6, and DW12, their 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 events DW2, DW6, and DW12 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 events DW2, DW6, and DW12.

VII. Regulatory Decision

Based on the review of data and information and stewardship plan, submitted by BASF, and through comparisons of durum wheat events DW2, DW6, and DW12 with unmodified durum wheat counterparts, the Biotechnology Environmental Release Assessment Unit, CFIA, has concluded that the modified gene and its corresponding trait do not confer to durum wheat events DW2, DW6, and DW12 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 durum wheat event DW12 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 DW12. 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 DW12 has been assessed and found to be as safe and nutritious as traditional durum wheat varieties. Durum wheat event DW12 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 of the Clearfield™ durum wheat events DW2, DW6, and DW12 and use as livestock feed of durum wheat event DW12 are authorized by the Plant Biosafety Office of the Plant Products Directorate and the Feed Section of the Animal Health and Production Division as of January 4, 2007. Any wheat lines derived from events DW2, DW6, and DW12 may also be released into the environment and any wheat lines derived from event DW12 may be used as livestock feed, provided no inter-specific crosses are performed, provided the intended uses are 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 durum wheat, in terms of their potential environmental impact and livestock feed safety.

The Clearfield™ durum wheat events are 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 Clearfield™ durum wheat events.

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