Novel Food Information: Herbicide Tolerant DT Sorghum

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• Background
1. Introduction
2. Development of the Modified Plant
3. Characterization of the Modified Plant
4. Dietary Exposure
5. Nutrition
6. Chemistry/Toxicology
• Conclusion

Background:

Health Canada has notified S&W Seed Company that it has no objection to the food use of herbicide tolerant DT Sorghum. Health Canada conducted a comprehensive assessment of this variety according to its Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based upon internationally accepted principles for establishing the safety of foods with novel traits.

The following provides a summary of the notification from S&W Seed Company and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

S&W Seed Company developed DT Sorghum to tolerate quizalofop-p-ethyl herbicide application.

The safety assessment performed by Food Directorate evaluators was conducted according to Health Canada's Guidelines for the Safety Assessment of Novel Foods. These Guidelines are based on harmonization efforts with other regulatory authorities and reflect international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how DT Sorghum was developed; how the composition and nutritional quality of DT Sorghum compared to non-modified sorghum varieties; and the potential for DT Sorghum to be toxic or cause allergic reactions. S&W Seed Company provided data that demonstrates that DT Sorghum is as safe and of the same nutritional quality as traditional sorghum varieties used as food in Canada.

The Food Directorate has a legislated responsibility for pre-market assessment of novel foods and novel food ingredients as detailed in the Food and Drug Regulations (Division B.28). Food use of DT Sorghum is considered a novel food under the following part of the definition of novel foods:

• "c) a food that is derived from a plant, animal or microorganism that has been genetically modified such that
  • (i) the plant, animal or microorganism exhibits characteristics that were not previously observed in that plant, animal or microorganism."

2. Development of the Modified Plant

DT Sorghum was developed through ethyl-methanesulfonate mutagenesis followed by conventional breeding. Calli of sorghum line R.TX430 were treated with aqueous ethyl-methanesulfonate and sodium azide and transferred to selection media containing quizalofop-p-ethyl herbicide. Calli that continued to survive after 21-24 weeks were considered herbicide tolerant.

Herbicide tolerant calli were transferred to shoot regeneration medium with or without quizalofop-p-ethyl herbicide. Resultant shoots were transferred to hormone free rooting medium containing quizalofop-p-ethyl herbicide. Rooted plants were transferred to a greenhouse in potting soil, and challenged with quizalofop-p-ethyl herbicide. One plant, designated HT-F14 was selected for advancement.

HT-F14 was determined to be tolerant to leaf application of quizalofop-p-ethyl herbicide. Molecular analysis of HT-F14 determined that this it possessed a W1999C mutation in the carboxyl transferase (CT) domain of the Sorghum bicolor acetyl-CoA carboxylase (sbACCase) gene.

HT-F14 was male sterile, meaning it does not generate viable pollen. The plant was therefore propagated by fertilizing with pollen from sorghum variety R.TX430. Resulting plants were selected on the basis of male fertility, phenotypic and genotypic similarity to R.TX430 and homozygosity for the sbACCase gene W1999C mutation. One plant was identified with the full complement of the desired characteristics and named R.9000.

To generate the commercial line, R.9000 was crossed and backcrossed with commercial, elite sorghum variety R.M42. The progeny of this cross were self-pollinated for 3 generations and crossed with a commercial male sterile line to produce to the commercial hybrid that has been characterized through regulatory trials.

3. Characterization of the Modified Plant

Given that other crops where herbicide tolerance was conferred by mutations in the carboxyl transferase (CT) domain of the ACCase genes, characterization focused on this region. The coding region of the CT domain is made up of exons 32 and 33 of the sbACCase gene. Therefore, to characterize the sbACCase gene sequence, 4 PCR primer pairs were designed in the CT domain to amplify these regions in order to identify mutations through direct sequencing. The resulting herbicide tolerant sbACCase gene sequence was compared against the sbACCase gene sequence from wildtype sorghum (R.TX430). A pairwise sequence alignment revealed two nucleotide mutations: a silent mutation that resulted in no predicted change to the resulting sbACCase amino acid sequence, and one mutation that is predicted to result in a W1999C amino acid substitution.

To evaluate the trait stability and mode of inheritance of the DT Sorghum mutant sbACCase gene and herbicide tolerance characteristic, a plant that was homozygous for the W1999C mutant allele was crossed with a plant that was homozygous for the wildtype (R.TX430) allele. F1 plants from this cross were self-pollinated to produce F2 seeds that were harvested. F2 seed were planted, grown in a greenhouse and sprayed with quizalofop-p-ethyl herbicide. Surviving plants were genotyped using Kompetitive allele specific PCR (KASP), and F3 seed was harvested from a subset of these plants. F3 seed were planted, sprayed and segregation ratios observed, and results presented. The supplied information showed that the KASP study could effectively differentiate between the two sbACCase alleles despite them differing by a single nucleotide. The results of the genotyping and phenotyping analysis support inheritance of a single trait conferring locus according to Mendelian principles.

In terms of protein expression levels, DT Sorghum is a result of chemical mutagenesis that introduced a single point mutation in the open reading frame of the sbACCase gene. There is no reason to expect that the new allele would be expressed in a way that differs from the wildtype sbACCase gene and in a way that would negatively impact food safety. Nevertheless, the petitioner also performed western blot analysis to compare the expression levels of sbACCase in wildtype and W1999C mutant sorghum lines. This analysis findings suggested that the sbACCase protein accumulates to similar levels in wildtype and W1999C mutant sorghum lines. The amount of sbACCase in DT Sorghum represents approximately 0.01 % of total protein in grain.

Protein equivalence was established by sequencing the plasmid which encodes the recombinant protein and verifying that the sbACCase gene encoded on the recombinant expression plasmid has the same encoding sequence as DT Sorghum's W1999C sbACCase.

Based on the available data provided, the BMH has no safety concerns regarding DT Sorghum from a molecular biology perspective.

4. Dietary Exposure

It is expected that DT Sorghum will be consumed similarly to sorghum currently sold in the Canadian marketplace.

DT Sorghum is a product of chemical mutagenesis, introducing a single point mutation in the open reading frame of the sbACCase gene. It is not expected that the new allele would be expressed in a way that differs from the sbACCase that is part of sorghum currently on the Canadian market.

The petitioner also performed semi-quantitative western analysis, probing the relative abundance of sbACCase protein in DT Sorghum compared to wildtype sorghum. This analysis suggests that the proteins in wildtype and DT Sorghum lines accumulate to comparable levels.

5. Nutrition

Compositional data for DT Sorghum and its closely related conventional counterpart were obtained from three field trials conducted across three locations in the United States during the 2020 growing season. In each trial, three replicates of each entry were planted in a randomized complete block design.

Grain samples were harvested and analyzed using acceptable methods for proximates, fibres, amino acids, fatty acids, vitamin B6, minerals, and anti-nutrients.

The petitioner conducted a statistical analysis comparing the modified cultivar with its conventional counterpart. If a statistically significant difference (P-value < 0.05) was indicated, the nutritional relevance of the difference was determined through comparison to the reported range for conventional sorghum based on values from the Agriculture & Food Systems Institute Crop Composition Database, the Organisation for Economic Co-operation and Development (OECD) consensus document on compositional considerations for new varieties of sorghum, and the Canadian Nutrient File (CNF #4432).

The analysis indicated statistically significant differences (modified cultivar vs. conventional counterpart, units) in ash (1.53 vs. 1.75, % DW), alanine (1.18 vs. 1.23, % DW), palmitic acid (12.79 vs. 12.51, % total fatty acids), palmitoleic acid (0.56 vs. 0.58, % total fatty acids), oleic acid (35.76 vs. 34.59, % total fatty acids), linoleic acid (47.61 vs. 48.97, % total fatty acids), arachidic acid (0.21 vs. 0.20, % total fatty acids), eicosenoic acid (0.31 vs. 0.28, % total fatty acids), behenic acid (0.11 vs. 0.10, % total fatty acids), and calcium (0.0167 vs. 0.0200, % DW). These observed differences in the modified cultivar were within the reported range for conventional sorghum, therefore, the modified cultivar is not expected to pose any nutritional safety concern.

The Bureau of Nutritional Sciences has not identified any nutritional concerns with the food use of herbicide tolerant DT Sorghum.

6. Chemistry/Toxicology

Data for toxic trace elements were not provided by the petitioner. However, the petitioner provided the results of a compositional assessment comparing samples of DT Sorghum to conventional sorghum hybrids, where samples were analyzed for proximates, minerals (calcium and phosphorous), vitamin B6, phytic acid, trypsin inhibitor, tannins, and amino acids. The petitioner concluded that the levels of these analytes, including the levels of calcium and phosphorous, in grain derived from DT Sorghum were comparable to those of conventional sorghum. This conclusion was supported by the Bureau of Nutritional Sciences and suggests that there would be no indication that the modifications would affect the transport and uptake or result in significant differences in the concentrations of toxic trace elements in DT Sorghum relative to conventional sorghum.

Data for mycotoxins were not provided. However, the petitioner provided data related to agronomic characteristics of DT Sorghum relative to its near-iso-hybrids and a leading commercial hybrid. DT Sorghum was found to be similar to its near-iso hybrids with respect to most of the assessed characteristics, many of which related to plant growth and yield. For the characteristics where a difference was observed between DT Sorghum and its near-iso-hybrids, there was no sustained pattern to suggest any impact on plant morphology or agronomics due to the mutation of W1999C allele. These findings suggest that DT Sorghum is not expected to exhibit an increased uptake of toxic trace elements or susceptibility to infection by mycotoxin-producing fungi compared to other conventional sorghum varieties.

The molecular characterization assessment by BMH did not identify any risk hypothesis that would suggest that the W1999C mutation in DT Sorghum would cause an increase in uptake of chemical contaminants or greater susceptibility to mycotoxin producing fungi relative to non-GM sorghum. Therefore, DT Sorghum is comparable to conventional sorghum.

The only modification in DT Sorghum is the introduction of a point mutation, which results in a single amino acid substitution in the ACCase enzyme. The ACCase enzyme is well-characterized, ubiquitously present in all plant species, and has a long history of safe consumption with no intrinsic toxic activity. Herbicide-tolerant novel foods carrying a similar trait (i.e., ACCase with a single point mutation) are already approved in Canada and have not been associated with any adverse effects. ¹

ACCase is primarily found in actively growing portions of the plant (e.g., leaf, shoots, roots), with minimal expression in the grains. As such, dietary exposures to this protein from sorghum grains is expected to be low. A semi-quantitative western blot assay conducted by the petitioner demonstrated that ACCase was significantly more pronounced in the shoot when compared to the grain.

The same semi-quantitative western blot assay, as well as an enzyme activity assay conducted by the petitioner, further demonstrated that the expression level of the modified ACCase in DT Sorghum is not increased relative to the parental line expressing the native ACCase. Thus, there is no expectation that dietary exposures to this enzyme will be increased compared to the non-modified form from conventional sorghum.

Given that there are no major alterations in the sequence of the modified ACCase, and the structure (and resultant expression and activity) are similar to that of the native enzyme, it is unlikely that the single amino acid substitution will result in the development of a novel toxic mode of action.

The petitioner conducted an in silico search of the National Center for Biotechnology Information (NCBI) protein database to compare the amino acid sequence of the modified ACCase to known protein toxins. The search did not identify any similarities to protein sequences of known toxins.

In considering the above, the overall weight-of-evidence suggests that DT Sorghum is as safe as conventional sorghum from a toxicological perspective.

Sorghum (Sorghum bicolor) is not a common allergenic food, and is not listed as a priority food allergen in Canada.

ACCase enzymes are naturally present in sorghum, and this class of enzymes is not known to be an allergen.

The petitioner conducted an amino acid sequence homology search using the AllergenOnline database to identify potential cross-reactivity of the modified ACCase against known allergens. Searches included a full FASTA alignment, a sliding window of 80 amino acids stretches with greater than 35 % identity, and a search for 8 contiguous amino acids to assess for presence of epitopes (i.e., parts of an allergenic molecule that bind to an antibody). The search did not identify any matches against known allergens.

Furthermore, in an in vitro digestion assay conducted by the petitioner, the modified ACCase was rapidly degraded in simulated gastric fluid (SGF). Thus, even if the (low levels of) protein were to remain stable throughout the processing and cooking conditions for sorghum, the protein would be susceptible to rapid digestion in conditions representative of the human stomach, and would not be absorbed systemically.

These findings suggest that DT Sorghum does not pose any additional allergenic risk in comparison to conventional sorghum.

Taken together, the data presented supports the conclusion that DT Sorghum is just as safe as the conventional corn in terms of potential chemical contaminants, toxicants and allergens.

Conclusion:

Health Canada's review of the information presented in support of the food use of DT Sorghum does not raise concerns related to food safety. Health Canada is of the opinion that food derived from DT Sorghum is as safe and nutritious as food from current commercial sorghum varieties.

Health Canada's opinion deals only with the food use of DT Sorghum.

This Novel Food Information document has been prepared to summarize the opinion regarding the subject product provided by the Food Directorate, Health Products and Food Branch, Health Canada. This opinion is based upon the comprehensive review of information submitted by the petitioner according to the Guidelines for the Safety Assessment of Novel Foods.

(Également disponible en français)

For further information, please contact:

Novel Foods Section
Food Directorate
Health Products and Food Branch
Health Canada, PL2204A1
251 Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca