DD2008-73: Determination of the Safety of BASF Canada Inc.'s Imidazolinone-Tolerant Clearfield® Canola Quality Indian Mustard Event S006
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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 BIO2007-01, The Biology of Brassica juncea (Canola / Mustard), and Directive 95-03 (Dir95-03), entitled "Guidelines for the Assessment of Novel Feeds: Plant Sources".
The Canadian Food Inspection Agency (CFIA) has evaluated information submitted by BASF Canada Inc. regarding the imidazolinone tolerant canola quality Brassica juncea event S006. 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 canola quality B. juncea varieties in Canada.
Taking into account these evaluations, unconfined release into the environment and use as livestock feed of the Clearfield® B. juncea event S006 is therefore authorized by the Plant Biosafety Office of the Plant Products Directorate and the Animal Feed Division of the Animal Health Directorate as of May 2, 2008. Any B. juncea lines derived from S006 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 B. juncea varieties, in terms of their potential environmental impact and livestock feed safety.
The B. juncea event S006 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.
On this page
- Brief Identification of the Modified Plant
- Background Information
- Description and Assessment of the Novel Trait
- Development Method
- Imidazolinone Tolerance
- Stable Expression
- Criteria for the Environmental Assessment
- Potential of Event S006 to Become a Weed of Agriculture or Invasive of Natural Habitats
- Potential for Gene Flow from Event S006 to Wild Relatives Whose Offspring May Become More Weedy or More Invasive
- Altered Plant Pest Potential of Event S006
- Potential Impact on Non-Target Organisms of Event S006
- Potential Impact on Biodiversity of Event S006
- Nutritional Criteria Assessment as Livestock Feed
- Potential Impact of Event S006 on Livestock Nutrition
- Potential Impact of Event S006 on Livestock and Workers/By-standers
- New Information Requirements
- Regulatory Decision
Appendix I: Best Management Practice Program for the Clearfield® Brassica juncea Production System
I. Brief Identification of the Modified Plant
Designation of the Modified Plant: Clearfield® canola quality Brassica juncea event S006
Applicant: BASF Canada Inc.
Plant Species: Indian mustard (Brassica juncea (L.) Czern)
Novel Traits: Tolerance to imidazolinone herbicides
Trait Introduction Method: Chemically induced seed mutagenesis
Proposed Use of the Modified Plant: Production of canola quality B. juncea for livestock feed and human food.
II. Background Information
BASF Canada Inc. has developed a canola quality Brassica juncea line tolerant to imidazolinone herbicides. This B. juncea line was developed to provide an alternative strategy for weed control in B. juncea.
The development of B. juncea event S006 was accomplished using chemically induced seed mutagenesis and interspecific crossing. B. juncea carries two complete genomes designated "A" and "B". Each genome has an Als gene (Als3 and Als1, respectively) coding for an acetohydroxyacid synthase (AHAS) enzyme. A B. juncea line carrying a single point mutation in the Als1 gene was developed by seed mutagenesis. A second B. juncea line was developed by interspecific crossing with Brassica napus to transfer of the modified Als3 gene from commercial Clearfield® Brassica napus. The two modified B. juncea lines were crossed to create B. juncea event S006 containing both mutations. Each mutation results in a modified AHAS enzyme carrying a single amino acid substitution and that is no longer affected by imidazolinone herbicides.
BASF Canada Inc. has provided data on the identity of B. juncea event S006, a detailed description of the modification method and breeding history, information on the modified genes, the resulting proteins and their mode of action and the stability of trait expression.
B. juncea event S006 was field tested in Canada from 2002 to 2006 and agronomic data for 2006 were provided.
Agronomic characteristics of event S006 such as seed germination, pollen viability, seedling emergence, seedling vigour, days to flowering, duration of flowering, days to maturity, plant height, disease damage, insect damage and grain yield were compared to those of unmodified B. juncea counterparts.
Nutritional components of event S006 such as proximates, amino acids and fatty acids were compared with unmodified B. juncea counterparts. Levels of anti-nutritional factors were also determined.
BASF Canada Inc. has provided an agronomic stewardship plan for imidazolinone tolerant B. juncea in the Canadian environment. This plan includes information regarding a safe and sustainable deployment of imidazolinone-tolerant B. juncea and is included as an appendix of this document. BASF Canada Inc. has provided details of efficient communication strategies and mechanisms for growers to report agronomic problems with this product back to BASF Canada Inc. as part of their Clearfield® Commitment program.
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". The BERA Unit has considered the:
- potential of event S006 to become a weed of agriculture or be invasive of natural habitats;
- potential for gene flow from event S006 to wild relatives whose hybrid offspring may become more weedy or more invasive;
- potential of event S006 to become a plant pest;
- potential impact of event S006 or its' gene products on non-target species, including humans; and
- potential impact of event S006 on biodiversity.
The Animal Feed Division of the Animal Health Directorate, 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 Animal Feed Division has considered the:
- potential impact of event S006 on livestock nutrition; and
- potential impact of event S006 on livestock and workers/by-standers.
III. Description of the Novel Traits
1. Development Method
A mutation in the Als1 gene was produced by chemical mutagenesis of seed from the conventional canola quality B. juncea variety Arid. 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). Separately, the modified Als3 gene was introduced into B. juncea from the commercial imidazolinone tolerant Clearfield® B. napus variety 45A71 by interspecific crossing followed by backcrossing to conventional B. juncea. Event S006 is derived from a cross between the two modified lines to combine the two mutations into a single line.
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 B. juncea is not tolerant to imidazolinone herbicides.
The introduced single nucleotide mutations in the Als1 and Als3 genes result in single amino acid substitutions in the AHAS1 and AHAS3 enzymes respectively. These single amino acid substitutions alter the binding site for imidazolinone herbicides, resulting in the imidazolinone tolerant phenotype of B. juncea lines containing either or both mutations. One of the introduced single nucleotide substitutions will also confer some tolerance to sulfonylurea herbicides, although this tolerance is not strong enough to confer protection in B. juncea to commercial rates of non-imidazolinone group 2 herbicides.
The novel imidazolinone tolerance is under the control of the native Als1 and Als3 promoters and is believed to be constitutively expressed. Sequence information for the modified Als genes in event S006 was submitted.
The tolerance to imidazolinone was demonstrated by comparison of the activity of the AHAS enzyme extracted from S006 B. juncea plants to that of conventional type B. juncea 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 conventional plants was inhibited much more than enzyme from the S006 plants.
The levels of valine, leucine and isoleucine produced in B. juncea are regulated by feedback inhibition of AHAS. BASF Canada Inc. 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 are unaffected in event S006.
Unlike known food allergens, AHAS is a minor protein in plant tissue, and it is heat-sensitive and trypsin-susceptible. The AHAS protein from event S006 was shown to be heat sensitive, with no detectable activity of AHAS after 1 to 3 min of heating at 100 °C. AHAS protein from event S006 was shown to be equivalent to the control line with respect to trypsin degradation. In addition, the unmodified and modified forms of the AHAS1 and AHAS3 proteins show no amino acid similarity to known allergens.
BASF Canada Inc. provided evidence to show that the protein components of S006 grain are not altered in comparison with an unmodified comparator. HPLC performed using protein extracts from unmodified and modified B. juncea indicated that no new proteins or major increase in protein expression occurred in grain as a result of the introduction of the novel trait.
BASF Canada Inc. has provided to the CFIA a method for the detection and identification of B. juncea containing this novel trait.
3. Stable Expression
The two mutations in Als genes that are responsible for the imidazolinone herbicide tolerance trait are inherited in a Mendelian fashion across multiple generations. In addition, plants from an advanced breeding generation of B. juncea event S006 demonstrated consistent imidazolinone herbicide tolerance, confirming the stable inheritance of this trait.
IV. Criteria for the Environmental Assessment
1. Potential of event S006 to Become a Weed of Agriculture or Invasive of Natural Habitats
The Indian mustard (B. juncea) possesses some of the characteristics that are common to weeds and invasive plants. It is an annual crop that may persist in unmanaged ecosystems without human intervention. It has some tolerance to drought, heat, frost, and disease. There have been reports of B. juncea becoming a weed of agriculture or an invasive pest in North America and other parts of the world, however it has not become an abundant or problematic weed in Canada, despite being cultivated in Canada for many years. The seeds do not shatter as readily as those of B. napus or B. rapa, and its' smaller seed size and thinner seed coat may decrease the ability of the seed to persist in the environment relative to other canola-quality Brassica species in Canada. B. juncea plants can grow as volunteers in cultivated fields in the seasons following a B. juncea crop, but they are usually eliminated by soil cultivation or the use of herbicides.
The CFIA evaluated data submitted by BASF Canada Inc. on the agronomic characteristics and biology of B. juncea event S006. These characteristics were: seed germination, seedling emergence, seedling vigour, days to flowering, duration of flowering, pollen viability, flea beetle damage, disease incidence, plant height, days to maturity and grain yield. S006 was not significantly different from the parent variety "Arid" and/or was within the range of expression of these traits in other conventional B. juncea varieties for all measured characteristics. As in the case of control varieties, no noticeable disease pressure was observed on event S006 at any of the sites, for Albugo candida (white rust), Leptosphaeria maculans or L. biglobosa (blackleg), or Alternaria spp. (Alternaria blackspot). Monitoring of trial locations did not indicate any changes in seed dormancy of S006 in comparison to other Brassica juncea varieties, nor is the imidazolinone tolerance trait expected to contribute to increased seed dormancy.
No competitive advantage was conferred to plants of event S006, other than that conferred by tolerance to imidazolinone herbicides, as the reproductive or growth characteristics of event S006 were comparable to those of unmodified B. juncea varieties. Tolerance to imidazolinone herbicides provides a competitive advantage only when these herbicides are used, and will not, in and of itself, make an imidazolinone tolerant plant weedier or more invasive of natural habitats. Imidazolinone tolerance will not cause B. juncea event S006 to become weedier or more invasive in unmanaged habitats than unmodified B. juncea. Imidazolinone-tolerant volunteers will not be controlled in subsequent crops if imidazolinones are used as the only weed control tool, and may not be controlled if other group 2 herbicides are used as the sole weed management tool. However, control of imidazolinone- tolerant B. juncea 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 B. juncea event S006 has no altered weed or invasiveness potential in Canada when compared to conventional B. juncea varieties.
BASF Canada Inc. has submitted a herbicide tolerance stewardship plan (Best Management Practice Program for the Clearfield® Brassica juncea Production System) to the CFIA which was determined to be satisfactory when evaluated by the BERA Unit.
BASF Canada Inc. will make this stewardship plan readily available to growers and agriculture extension personnel, in both private and public sectors, to promote careful management practices for event S006 and other imidazolinone tolerant Brassica juncea. These practices include the use of alternate control tools as appropriate to achieve complete volunteer control of imidazolinone-tolerant B. juncea, as well as additional recommendations on agricultural practices concerning imidazolinone-tolerant B. juncea. BASF Canada Inc. will use its' Clearfield® Commitment Program to provide an efficient mechanism for growers to report agronomic problems to BASF Canada Inc., which will facilitate the ongoing monitoring of imidazolinone tolerant B. juncea. BASF Canada Inc. will monitor grower compliance to determine the effectiveness of the stewardship plan and make any changes to the plan as appropriate.
2. Potential for Gene Flow from event S006 to Wild Relatives Whose Offspring May Become More Weedy or More Invasive
Successful interspecific and intergeneric crosses between B. juncea and some related species have been reported in the scientific literature (see BIO2007-01, The Biology of Brassica juncea (Canola / Mustard)). However, many of these crosses have required extensive human intervention and the rates of natural hybridization between B. juncea and weedy relatives resulting in fertile offspring appear to be very low. Sinapsis arvensis is considered the worst of the weedy relatives of B. juncea in Western Canada. Hybrids between both species can be produced under field conditions, however at very low frequency. Additionally, backcrossing of the hybrids to S. arvensis failed to produce viable progeny. Therefore, the likelihood of introgression of traits from B. juncea to S. arvensis appears to be very low. In crosses with other wild related species (i.e. Raphanus raphanistrum and Erucastrum gallicum), no viable hybrid seed was produced.
Stable gene transfer from B. juncea is most likely with Brassicasp. crops such as B. napus and B. rapa. The introgression of the Als mutations from B. juncea event S006 into B. napus is of minimal concern as both mutations are already present in commercial Clearfield® B. napus varieties (the Als1 mutation is identical to the corresponding Als mutation present in Clearfield® B. napus varieties, and the mutated Als3 gene of B. juncea event S006 was transferred from Clearfield® B. napus by conventional breeding). Any hybrids resulting from outcrossing between B. rapa and B. juncea event S006 could be controlled by herbicides other than imidazolinones or by cultivation. Any unintended presence of imidazolinone tolerance in non-imidazolinone tolerant varieties of B. juncea can be controlled by typical crop management practices.
If imidazolinone-tolerant individuals arose through interspecific or intergeneric hybridization, the novel trait would confer no competitive advantage to these plants unless challenged by imidazolinones. This would only occur in managed ecosystems where imidazolinones are used for weed control. As with imidazolinone-tolerant B. juncea event S006, these herbicide tolerant individuals, should they arise, could be controlled using mechanical means or herbicides other than imidazolinones . Hybrids, if they developed, could potentially result in the loss of imidazolinones as a tool to control these species. This, however, can be avoided by the use of sound crop management practices.
The CFIA has therefore determined that the likelihood gene flow from B. juncea event S006 to wild relatives in Canada is very low. Gene flow from B. juncea event S006 to Brassica relatives is possible, but would not result in increased weediness or invasiveness of the resulting progeny.
Additionally, BASF Canada Inc.'s herbicide tolerance stewardship plan for Clearfield® Brassica juncea contains recommendations to minimize and manage outcrossing with related species.
3. Altered Plant Pest Potential of event S006
B. juncea is not a plant pest in Canada, and the intended effect of the novel trait (tolerance to imidazolinone herbicides) is unrelated to plant pest potential. Although no noticeable disease pressure was observed for any of the varieties tested at any of the sites, for Albugo candida (white rust), Leptosphaeria maculans or L. biglobosa (blackleg), or Alternaria spp. (Alternaria blackspot), the agronomic characteristics of event S006 were shown to be within the normal range of conventional canola quality B. juncea varieties. This indicates that it is unlikely that there are any major unintended effects in S006 that could alter its plant pest potential relative to that of other B. juncea.
The CFIA has therefore determined that B. juncea event S006 does not present a plant pest concern.
4. Potential Impact on Non-Target Organisms of event S006
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. Single amino acid modification of the AHAS enzyme, which alters the imidazolinone herbicide binding site on the enzyme, is the molecular basis for imidazolinone tolerance in B. juncea event S006. The mutations in B. juncea event S006 are identical to the corresponding mutations in commercial Clearfield® B. napus canola varieties which have a history of safe use in Canada and other countries. BASF Canada Inc. has submitted data indicating that event S006 is substantially equivalent to its unmodified counterpart with respect to AHAS enzyme activity in leaf and seed tissues and feedback inhibition of AHAS enzyme from valine and leucine. The AHAS enzyme activity of event S006 only differs from the AHAS enzyme activity of its unmodified counterpart by being less inhibited by imidazolinone herbicides. No novel toxins were introduced into event S006. Therefore, no negative interactions with non-target symbiotic or consumer organisms are anticipated.
The CFIA has therefore concluded that B. juncea event S006 is unlikely to cause adverse impacts on non target organisms.
5. Potential Impact on Biodiversity of event S006
B. juncea event S006 is safe to non-target organisms and does not present altered weediness, invasiveness or plant pest potential. The novel trait is not expected to expand the range of cultivation of B. juncea in Canada. No changes in current agronomic practices for B. juncea are expected, with the exception of the use of imidazolinone herbicides for weed control. BASF Canada Inc.'s herbicide tolerance stewardship plan for Clearfield® Brassica juncea contains recommendations for delaying the development of imidazolinone resistance in weeds.
Imidazolinone herbicides are somewhat persistent in soil, and this persistence increases when soil moisture is low (as is commonly found in the dark brown and brown soil zones of Western Canada). Some crops are sensitive to the carry-over of persistent herbicides from previous growing seasons. The introduction of imidazolinone-tolerant Brassica juncea is not likely to affect typical crop rotation practices in these soil zones, as most of the crops that are expected to follow Brassica juncea in rotation are not subject to lengthy re-cropping restrictions when imidazolinone herbicides are used.
The CFIA has therefore concluded that the potential impact on biodiversity of B. juncea event S006 is equivalent to that of currently commercialized B. juncea lines.
V. Criteria for the Livestock Feed Assessment
1. Potential Impact on Livestock Nutrition
Nutritional Composition and Anti-Nutritional Factors
The compositional equivalence of Canola Quality Brassica juncea event S006 to its parental control (Arid) and three conventional Brassica juncea varieties was assessed from three sites in Western Canada during the 2006 growing season. Grain samples were collected from replicated plots and analyzed for protein crude fat, crude fibre, ash, acid detergent fibre (ADF), neutral detergent fibre (NDF), total dietary fibre (TDF), amino acids, fatty acids, minerals and vitamins. No statistically significant differences were observed between B. juncea event S006 and the control for crude fat, crude fibre, crude protein, ADF and TDF. There were statistically significant differences between B. juncea event S006 and its control for ash and NDF, but the means were with the range of conventional varieties. There were no statistically significant differences between B. juncea S006 and Arid for the branched-chain amino acids (valine, isoleucine and leucine), lysine, methionine, cystine and threonine. All means were within the range of commercial varieties and literature values. Apart from eicosenoic (gadoleic) acid, there were no statistically significant differences between B. juncea event S006 and Arid for palmitic, palmitoleic, stearic, oleic, linoleic, linolenic, arachidic, lignoceric and nervonic acids. Eicosenoic acid for both B. juncea event S006 and its control were within literature values. Erucic acid was below the level of detection for B. juncea event S006, Arid and the conventional varieties. No statistically significant differences were observed between B. juncea event S006 and Arid for zinc, iron, manganese, calcium, sodium and potassium, vitamin B1, pantothenic acid, vitamin E, vitamin B2, and vitamin A. There were statistically significant differences for phosphorus, magnesium, copper, niacin, vitamin B6 and folic acid, but the means were within the range for conventional varieties and literature values.
Phytic acid, trypsin inhibitor and glucosinolates were analyzed in grain samples from Canola Quality Brassica juncea event S006 and compared to its parental control (Arid) and three conventional B. juncea varieties. There were no statistically significant differences between B. juncea event S006 and its control for phytic acid. Significantly lower levels of trypsin inhibitor and glucosinolates were observed for B. juncea event S006 than Arid as expected for Canola quality B. juncea. Means values were within the range for commercial B. juncea varieties.
The evidence provided by BASF Canada Inc. supports the conclusion that the nutritional composition of Clearfield Canola Quality Brassica juncea event S006 is substantially equivalent to conventional Brassica juncea 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 in each of the AHAS enzymes in B. juncea event S006 would not be expected to change this. Crude extracts from B. juncea event S006 displayed similar AHAS enzyme activity and were feedback inhibited in a similar manner to crude extracts from an appropriate control cultivar. AHAS activity of B. juncea event S006 is heat labile, and the modified AHAS enzymes do not have homology with any known allergens or toxins. Based on the information provided by BASF Canada Inc., the modified AHAS enzymes are unlikely to be a novel toxin or allergen.
Based on the detailed characterization provided (nutritional composition and agronomic data of the modified plant compared to an unmodified comparator) it is unlikely that secondary mutations causing unintended effects have occurred in the B. juncea genome.
The evidence provided by BASF Canada Inc. supports the conclusion that the potential impact on livestock and workers/by-standers of B. juncea event S006 is equivalent to that of currently commercialized B. juncea lines.
VI. New Information Requirements
If at any time, BASF Canada Inc. 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 B. juncea event S006, its descendants, or products derived there from, BASF Canada Inc. must immediately provide such information to the CFIA. On the basis of such new information, the CFIA will re-evaluate the potential impact of B. juncea event S006 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 B. juncea event S006.
VII. Regulatory Decision
Based on the review of data and information and the stewardship plan submitted by BASF Canada Inc., and through comparisons of B. juncea event S006 with unmodified B. juncea counterparts, the Biotechnology Environmental Release Assessment Unit, CFIA, has concluded that the modified genes and their corresponding trait do not confer to B. juncea event S006 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 Canada Inc., including comparisons of B. juncea event S006 with unmodified B. juncea counterparts, the Animal Feed Division, CFIA, has concluded that the modified genes and their corresponding novel trait will not confer to these plants any characteristic that would raise any concerns regarding the safety or nutritional composition of B. juncea event S006. Oil and meal prepared from canola-quality B. juncea are currently listed in Schedule IV of the Feeds Regulations and are, therefore, approved for use in livestock feeds in Canada. B. juncea event S006 has been assessed and found to be as safe and nutritious as traditional canola-quality B. juncea varieties. B. juncea event S006 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 use as livestock feed of the Clearfield® B. juncea event S006 is therefore authorized by the Plant Biosafety Office of the Plant Health and Biosecurity Directorate and the Animal Feed Division of the Animal Health Directorate as of May 2, 2008. Any B. juncea lines derived from S006 may also be released into the environment and used as livestock feed, provided no inter-specific crosses are performed, provided the intended use(s) 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 B. juncea varieties, in terms of their potential environmental impact and livestock feed safety.
The B. juncea event S006 is subject to the same phytosanitary import requirements as its unmodified counterpart.
Please refer to Health Canada's Decisions on Novel Foods for a description of the food safety assessment of Clearfield® B. juncea event S006.
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