Novel food information: Imidazolinone-tolerant grain sorghum (ADV-IMI-R grain sorghum)

Background:

Health Canada has notified Advanta Seeds that it has no objection to the food use of imidazolinone herbicide tolerant ADV-IMI-R grain sorghum. The Department 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 Advanta Seeds and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

Advanta Seeds developed ADV-IMI-R grain sorghum to be imidazolinone herbicide tolerant and for use in breeding programs to develop multiple hybrid sorghum varieties for commercial release. Advanta seeds does not intend to sell this line and its hybrids in Canada or cultivate it in Canada.

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 ADV-IMI-R grain sorghum was developed; how the composition and nutritional quality of ADV-IMI-R grain sorghum compared to non-modified varieties; and the potential for ADV-IMI-R grain sorghum to be toxic or cause allergic reactions. Advanta Seeds provided data that demonstrates that ADV-IMI-R grain 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 ADV-IMI-R grain 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

The imidazolinone-tolerant grain sorghum (ADV-IMI-R grain sorghum) was developed using standard mutagenesis and conventional breeding procedures. Tolerance to imidazolinone herbicides is the result of a mutation in the Als (acetolactate synthase, also referred to as acetohydroxyacid synthase, acronym sbAHAS) gene in sorghum. The mutation responsible for imidazolinone tolerance in ADV-IMI-R grain sorghum is a single nucleotide change of guanine to adenine at position +277, which results in a codon change from GCG to ACG which results in a single amino acid substitution of alanine to threonine at position 93 (A93T) in the sbAHAS protein.

The petitioner has provided information describing the methods used to develop ADV-IMI-R grain sorghum and data that characterize the genetic modification that results in the herbicide tolerance through the expression of the sbAHAS protein. ADV-IMI-R grain sorghum was produced by chemically induced ethyl methanesulfonate (EMS) modification and conventional breeding procedures.

ADV-IMI-R grain sorghum was derived from the proprietary sorghum variety 80237 that was subjected to seed mutagenesis followed by plant growth in a confined nursery field plot. Sorghum line 80237 plants were treated with an aqueous solution of EMS. Treated seeds were planted and left for pollination, with 273 M1 plants selected and 2 seeds taken from each. The seeds were planted in the nursery and 546 M2 plants were produced. Pollen from one plant of each pair of seeds was collected and used for pollinating the other plant of the pair. M3 seeds obtained from each of the 273 pollinated M2 plants were harvested. 273 furrows were planted with the M3 progeny and fifty plants from each furrow were sprayed with imazethapyr. There were 68 herbicide resistant plants. Herbicide-tolerant M7 mutants plants were designated VT11-11331-BK and deposited with the NCIMB collection with the Access No. NCIMB 41870.

The initial mutant line VT11-11331-BK was backcrossed to different elite inbred lines to introgress the mutation into parental lines for the production of hybrids. Following three rounds of backcrossing, the lines were selfed for three further generations with selection using 3x the commercial rate of imazethapyr to identify plants homozygous for the mutation, which was confirmed through analysis of the progeny.

3. Characterization of the modified plant

Genomic DNA was isolated from the VT11-11331-BK mutant line along with the wild type line 80237 (source of material used for the mutagenesis). PCR primers specific to the sorghum AHAS sequence were synthesized to generate 6 overlapping DNA fragments covering the complete AHAS coding region. The PCR products were sequenced and the resulting AHAS gene sequences were aligned to allow for a comprehensive DNA sequence comparison. The alignment showed a point mutation at nucleotide position +277 which distinguishes the original sorghum line 80237 from the ADV-IMI-R mutant gene in VT11-11331-BK.

The stability of the trait was demonstrated using both homozygous and heterozygous plants to confirm its stability across multiple generations. The expected genotype ratios based on Mendelian segregation principles were observed from herbicide application assays 20 days after emergence and 10 days after treatment, which indicates the expected inheritance pattern for a stably integrated trait. The expression of the trait was stably maintained over at least 7 generations.

4. Product information

One modified protein is expressed in ADV-IMI-R grain sorghum, the mutant sbAHAS. A single amino acid substitution (alanine to threonine at position 93) results in the imidazolinone herbicide tolerance.

The expression of the AHAS enzyme in selected tissues of the modified VT11-11331-BK line was measured using a LC-MS/MS spectroscopy technique. The results indicated that the expression of modified AHAS is no different in the modified line VT11-11331-BK compared with the non-modified 80237 wild-type.

The petitioner provided an SDS-PAGE analysis, DNA sequence comparison to the recombinant protein, and tryptic mass digest fingerprint analysis that indicated the equivalency of the plant expressed sbAHAS in Advanta sorghum to the baculovirus over-expression system produced protein for use in the toxicological studies.

5. Dietary exposure

The genetic modification of ADV-IMI-R grain sorghum is not intended to alter any of its nutritional aspects when compared to conventional non-genetically modified varieties. It is expected that ADV-IMI-R grain sorghum will be used in applications similar to conventional grain sorghum varieties and thus no change in the food use of grain sorghum is anticipated.

6. Nutrition

Compositional data for IMI-R-2 (test), IMI-S-2 (control) and three reference varieties collected from four field trials in Argentina during the 2016-2017 growing season was provided. In each trial, four replicates were planted in a randomized complete block design. Typical commercial agriculture production practices were used for the field trials. The IMI-R-2 hybrid was also treated with imidazolinone herbicide.

Grain samples were harvested and analyzed for ash, crude fat, crude protein, moisture, acid detergent fiber, neutral detergent fiber, total dietary fiber, calcium, phosphorus, vitamin B6, phytic acid, starch, tannins, amino acids, and fatty acids. The data provided was for all key nutrients and anti-nutrients as described in the Organization for Economic Co-Operation and Development (OECD) “Consensus Document on Compositional Considerations for New Varieties of Grain Sorghum (Sorghum bicolor (L.) Moench): Key Food and Feed Nutrients and Anti-nutrients” (2010).

Statistically significant differences between the control and the hybrid were noted when P-values were < 0.05. When a statistical difference was identified, the nutritional relevance of these differences where further examined by comparing the results to expected ranges for conventional sorghum as described in the OECD consensus document (2010) and International Life Sciences Institute Crop Composition Database.

In the data set provided six statistically significant differences were observed in linolenic acid, lysine, glycine, total dietary fibre, neutral detergent fibre, and ash; all of which were higher in the test compared to the control. In all cases the differences were small (<11%), and the analyte levels were within the references ranges provided by the petitioner and the expected range for conventional sorghum. These differences were not considered nutritionally relevant.

Based on the information provided on the composition of ADV-IMI-R grain sorghum, there are no nutritional safety concerns with the sale of foods derived from ADV-IMI-R grain sorghum.

7. Chemistry/toxicology

The safety of the modified acetohydroxyacid synthase (AHAS) protein was based on both the lack of systemic exposure and the lack of similarity to known toxins, as demonstrated by in vitro assays and in silico analyses.

Sorghum is not a common ingredient in western diets, but is a staple in India and Africa. The Canadian Community Health Survey (2015) reported one person to have consumed any type of sorghum in over 19,000 survey respondents. The herbicide-tolerant trait of ADV-IMI-R grain sorghum is not expected to change the overall consumption of sorghum by the general population. General consumption of ADV-IMI-R grain sorghum would be expected to be very low.

Levels of anti-nutrients (i.e., tannins and phytic acid) in the grains of the modified sorghum were not different from those of conventional sorghum. Levels of AHAS in the modified sorghum and the conventional sorghum, were both below the limit of detection (LOD) in the grain (LOD not specified, limit of quantitation was 10 µg/g). Consumers of Advanta Seeds sorghum would not be exposed to a greater amount of total AHAS protein than consumers of conventional sorghum.

Processing of sorghum for human consumption involves cooking at high temperatures, commonly baking or boiling. The modified AHAS protein lost all activity after 1 minute or more at 100 degrees Celsius. The modified protein is not expected to be found in the food, as consumed, due to its inactivation and degradation during cooking.

The modified AHAS protein was digested after 0.5 minutes of exposure in a simulated gastric fluid assay. If the modified AHAS protein were found in the food (e.g., undercooked food), it would likely be digested in the stomach and would not be available to cause any toxicological effects.

A bioinformatics analysis was provided using the amino acid sequence of the ADV-IMI-R grain sorghum AHAS protein and comparing it with sequences of known toxins using the NCBI entrez protein database. No significant matches were identified. The modified AHAS does not share sequence similarity with known toxins.

The allergenic safety of the ADV-IMI-R grain sorghum was based on both the lack of systemic exposure and the lack of similarity to known allergens as demonstrated by in vitro assays and in silico analyses.

As noted above, the modified protein would be expected to be denatured by heat and digested by enzymes in the stomach, thus it would not be available to cause allergenic effects.

A bioinformatics analysis was provided using the amino acid sequence of the ADV-IMI-R grain sorghum AHAS protein and comparing it with sequences of known allergens using the AllergenOnline database (2035 sequences). No significant matches were identified. The modified AHAS does not share sequence similarity with known allergens.

AllergenOnline does not include any identified allergens for sorghum (Sorghum bicolor). One record of a small number of people with positive skin prick tests to sorghum and other components of beer was identified in PubMed. Overall, sorghum is not a common food allergen despite being eaten at high levels in some parts of the world.

ADV-IMI-R grain sorghum would not be expected to pose a toxicological safety concern and is not expected to pose an allergenic safety concern.

Conclusion:

Health Canada’s review of the information presented in support of the food use of ADV-IMI-R grain sorghum does not raise concerns related to food safety. Health Canada is of the opinion that food derived from ADV-IMI-R grain sorghum is as safe and nutritious as food from current commercial sorghum varieties.

Health Canada's opinion deals only with the food use of ADV-IMI-R grain sorghum. Issues related to its environmental release and use as animal feed have been addressed separately through existing regulatory processes in the CFIA.

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

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Date modified:: 2022-03-15

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