Novel Food Information - Herbicide Tolerant Inzen™ Sorghum

Health Canada has notified E.I. du Pont Canada Company (DuPont) that it has no objection to the food use of herbicide tolerant Inzen™ sorghum. The Department conducted a comprehensive assessment of this sorghum 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.

Background:

The following provides a summary of the notification from DuPont and the evaluation by Heath Canada and contains no confidential business information.

1. Introduction

DuPont has developed a genetically modified (GM) sorghum (Sorghum bicolor L. Moench) variety using conventional breeding techniques and the successful introgression of a mutant acetolactate synthase (ALS)-encoding gene derived from a wild sorghum variety (shattercane) into the grain sorghum genome.

In sorghum, the first step in the branched chain amino acid (i.e. valine, leucine, and isoleucine) biosynthesis pathway is catalysed by the ALS enzyme.

Compared to the native sorghum ALS enzyme, the mutant ALS enzyme from shattercane possesses an amino acid substitution that allows the enzyme to function in the presence of ALS-inhibiting herbicides (i.e. sulfonylurea [SU] and imidazolinone [IM]). Expression of the mutant ALS enzyme confers upon Inzen sorghum tolerance to SU and IM herbicides.

Herbicide tolerant ALS enzymes have been evaluated by Health Canada for a number of GM plants (including Cibus Canola Line 5715, 2013; soybean BPS-CV127-9, 2012; Clearfield rice varieties CL IMINTA 1 and CL IMINTA 4, 2006; Clearfield wheat variety Teal 11A, 2004; etc.).

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 this sorghum variety was developed; how the composition and nutritional quality of this variety compared to non-modified sorghum varieties; and the potential for this sorghum variety to be toxic or cause allergic reactions. DuPont has provided data demonstrating that herbicide tolerant Inzen 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 28). Herbicide tolerant Inzen 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 petitioner has provided information describing the methods used to develop herbicide tolerant Inzen sorghum and the molecular biology data that characterize the genetic change, which results in the expression of a herbicide tolerant ALS enzyme.

Herbicide tolerant Inzen sorghum was developed through conventional plant breeding and selection techniques to incorporate a naturally occurring mutation within the ALS-encoding gene of a wild sorghum variety (shattercane; Sorghum bicolor L.) into the grain sorghum genome.

In 1996, an isolate of wild sorghum displaying tolerance to ALS-inhibiting herbicides was identified in southwest Kansas and reported to the International Survey of Herbicide Resistant Weeds. Seeds from this ALS-tolerant shattercane plant were collected in 2003 from a field referred to as Tailwind by the local farmer, and the genotype was thereafter referred to as the 'Tailwind' line.

The Tailwind line was crossed into grain sorghum and progeny lines were characterised by Kansas State University (KSU) with respect to herbicide tolerance and the genetic mutation within the ALS-encoding gene. DuPont Pioneer obtained two elite inbred sorghum lines containing the herbicide tolerant trait from KSU. These lines were further crossed into different grain sorghum elite lines with multiple backcrosses to produce the resultant Inzen sorghum line.

3. Characterization of the Modified Plant

DNA sequence analysis performed by KSU identified the presence of two single nucleotide polymorphisms (SNPs) in the ALS-encoding gene of herbicide tolerant Inzen sorghum, resulting in two amino acid substitutions in the ALS enzyme (i.e., V560I and W574L). The presence of these two SNPs within the ALS-encoding gene of herbicide tolerant Inzen sorghum was confirmed by nucleotide sequence analysis performed by DuPont Pioneer. Using primers specific to the ALS-encoding gene and template genomic DNA prepared from herbicide tolerant Inzen sorghum and a near isogenic control sorghum (i.e. 86G08), PCR amplification yielded 914-bp fragments that were subsequently cloned and sequenced.

The PCR fragment derived from herbicide tolerant Inzen sorghum contained two SNPs relative to the control sequence: a G®A (guanine®adenine) SNP at position 509 (numbering relative to the start of the 914-bp amplicon sequence) resulting in a V560I amino acid substitution in the ALS enzyme and a G®T (guanine®thymine) SNP at position 552 resulting in a W574L amino acid substitution in the ALS enzyme. These two SNPs are the same mutations reported by KSU.

The W574L substitution is well documented in the scientific literature as conferring tolerance to both SU and IM herbicides. The V560I substitution is not known to confer herbicide tolerance. To confirm the hypothesis that the V560I substitution does not confer herbicide tolerance, the ALS amino acid sequence of nine crop plant varieties sensitive to SU and IM herbicides, including soybean, potato, cucumber, and oranges contain the V560I substitution in the ALS enzyme, indicating that this substitution is not related to herbicide tolerance. Based on this evidence it can be concluded that the herbicide tolerance trait exhibited by herbicide tolerant Inzen sorghum is a direct result of the W574L substitution present in the ALS enzyme derived from shattercane sorghum.

Segregation analysis was conducted to demonstrate the mode of trait inheritance of herbicide tolerant Inzen sorghum and to confirm stability of the herbicide tolerance trait across multiple generations. Analysis was performed on five different generations of herbicide tolerant Inzen sorghum. Statistical analyses were conducted to evaluate the segregation ratios of the herbicide tolerant Inzen sorghum generations. Chi-Square tests were conducted to compare the observed segregation ratio to the expected ratio for each generation. A significant difference was established if the P-value was below 0.05. All Chi-Square tests were found to be not significant, demonstrating stable Mendelian inheritance of the mutant ALS-encoding gene in herbicide tolerant Inzen sorghum.

4. Product Information

Herbicide tolerant Inzen sorghum differs from its traditional counterpart by the presence of a mutant ALS-encoding gene in the host genome. Expression of this gene results in a mutant ALS enzyme that is capable of functioning in the presence of ALS-inhibiting herbicides, thus permitting herbicide tolerant Inzen sorghum to grow in the presence of such herbicides.

5. Dietary Exposure

According to the petitioner, sorghum is considered the fifth most important cereal crop in the world behind wheat, rice, maize, and barley. Approximately 50% of the world production of sorghum grain is used as human food and approximately 95% of food use is in Africa and Asia. According to the Food and Agriculture Organization (FAO), the primary use for sorghum in North America is as animal feed.

The herbicide tolerance trait of Inzen sorghum is not expected to change the overall consumption of sorghum by the general population.

6. Nutrition

The nutritional evaluation was based on the 2015 Organisation for Economic Co-operation and Development (OECD) document, the International Life Sciences Institute (ILSI) crop composition database as well as a 1995 publication of the Food and Agriculture Organisation (FAO). Sorghum includes several related plant species but the grain sorghum data used for assessing this submission is that for shattercane Sorghum bicolor (L.) Moench.

The petitioner submitted data from a field phase (2013 growing season) conducted at five sites in commercial sorghum-growing regions of the United States (Illinois, Kansas, Nebraska, Oklahoma and Texas). Each site utilized a randomized complete block design with four blocks, and each block included herbicide tolerant Inzen sorghum, control sorghum with no Inzen herbicide tolerance trait, as well as four Pioneer brand commercial reference sorghum lines.

Four samples each of the test, control, and 4 reference lines (for a total of 20 samples for each component) were gathered and analysed. The petitioner used reasonable methods to limit bias in the study by using known methods and provided information on all reagents used, methods used for analysis and sample calculations.

The petitioner provided analytical data on the following constituents: crude protein, crude fat, crude fiber, total dietary fiber (TDF), acid detergent fiber (ADF), neutral detergent fiber (NDF), carbohydrates, starch-resistant, starch non-resistant, calcium, phosphorus, fatty acids [lauric acid (C12:0); myristic acid (C14:0); palmitic acid (C16:0), palmitoleic acid (C16:1); heptadecanoic acid (C17:0); heptadecenoic acid (C17:1); stearic acid (C18:0); oleic acid (C18:1); linoleic acid (C18:2); alpha-linolenic acid (C18:3); arachidic acid (C20:0); eicosenoic acid (C20:1); eicosadienoic acid (C20:2); behenic Acid (C22:0); erucic Acid (C22:1); and lignoceric acid (C24:0); amino acids:  alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine; vitamins: B6 (pyridoxine); and anti-nutrients: tannins, and phytic acid.

Statistically significant differences between herbicide tolerant Inzen sorghum and control sorghum were noted in protein, carbohydrates (calculated by difference), and some minor fatty acids. Levels of amino acids, when expressed on a percentage dry matter were also significantly higher in herbicide tolerant Inzen sorghum than control sorghum, in proportion to the higher protein content of herbicide tolerant Inzen sorghum. However, these values are all within the range of values for the reference lines and/or OECD ranges.

7. Chemistry/Toxicology

Toxicologists evaluated the safety of herbicide tolerant Inzen sorghum by assessing the potential toxicity and allergenicity of the mutant ALS enzyme, any changes to endogenous toxins and any residual shattercane proteins that could be expressed in herbicide tolerant Inzen sorghum.

The Canadian Community Health Survey (CCHS, cycle 2.2; 2004) reported one person to have consumed any type of sorghum in over 30,000 survey respondents. The herbicide tolerant trait of Inzen sorghum is not expected to change the overall consumption of sorghum by the general population (see Dietary Exposure). Based on this data, it can be concluded that consumer exposure to herbicide tolerant Inzen sorghum would be expected to be very low.

Data was provided of an ALS enzyme activity assay as a surrogate to compare the relative amounts of functional ALS enzyme in germinating seeds from herbicide tolerant Inzen and wild-type, grain sorghum. The data suggests that the amount of ALS enzyme is similar between herbicide tolerant Inzen sorghum and conventional grain sorghums. Consumers of herbicide tolerant Inzen sorghum would not be exposed to a greater amount of total ALS enzyme than consumers of conventional grain sorghum.

The petitioner performed a bioinformatics analysis using the amino acid sequence of the herbicide tolerant Inzen sorghum ALS enzyme and compared it with sequences of known toxins from their in-house database (7,579 sequences) and the National Center for Biotechnology Information (NCBI) GenPept database (93,788,790 sequences). No significant matches were found. It was concluded that herbicide tolerant Inzen sorghum ALS enzyme does not share significant sequence similarity to known toxins.

Herbicide tolerant Inzen sorghum retained approximately 4% of the shattercane genome due to extensive backcrossing with grain sorghum varieties. However, wild sorghum subspecies, like shattercane, have a history of safe food use. Although the grain from shattercane is not used as food in Canada or the United States, the petitioner stated that wild sorghums (the predecessor to shattercane and grain sorghum) were used by subsistence farmers for up to ten thousand years on the African continent. Wild sorghums, like shattercane, are not known as sources of food toxins.

It was indicated that consumers may have more recent incidental exposure to wild sorghum like shattercane. Outcrossing rates of up to 16% have been documented when shattercane is found within 10 metres of grain sorghums. Despite the attempts of farmers to reduce outcrossing, Canadian consumers may be exposed to shattercane grain or shattercane´grain sorghum hybrid grain when consuming sorghum. There are no apparent adverse effects associated with the consumption of hybrid grains.

The main toxin associated with sorghum is prussic acid (hydrogen cyanide); however, this toxin is only found in the leaves of young plants, which is not the part of the plant consumed by people. Canadian consumers would not be expected to be exposed to prussic acid from sorghum grain.

The petitioner performed a bioinformatics analysis using the amino acid sequence of the herbicide tolerant Inzen sorghum ALS enzyme and compared it with sequences of known allergens retrieved from the AllergenOnline database (version 16; 1,956 sequences). The ALS enzyme did not share ≥ 35% amino acid identity with any known allergen. Based on the results of the bioinformatics analysis, the petitioner concluded that the herbicide tolerant Inzen sorghum ALS enzyme did not match any known human allergens.

The two amino acid substitutions in the herbicide tolerant Inzen sorghum ALS enzyme are also present in various GM and non-GM crops. These two amino acid substitutions are not associated with an increase in allergenicity in any of these commodities and thus, it is not expected that the mutant ALS enzyme expressed in herbicide tolerant Inzen sorghum would increase its allergenicity.

Allergy or sensitivity to ingested sorghum and/or malted sorghum is rare. The literature reported that allergic individuals may exhibit dermal irritation (e.g., cutaneous erythema) and/or gastrointestinal discomfort following the consumption of beer that contained sorghum. There is no mechanistic reason to suggest that herbicide tolerant Inzen sorghum will be any more allergenic than wild-type sorghum.

Based on the available data regarding the toxicological and allergenic potential of herbicide tolerant Inzen sorghum, this variety is considered to be as safe as conventional sorghum varieties currently available in the Canadian marketplace from the perspective of toxicological and allergenic risk.

Conclusion:

Health Canada's review of the information presented in support of the food use of herbicide tolerant Inzen sorghum does not raise concerns related to food safety. Health Canada is of the opinion that food derived from this GM sorghum variety to be as safe and nutritious as food from current commercial sorghum varieties.

Health Canada's opinion deals only with the food use of herbicide tolerant Inzen sorghum. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency (CFIA). The CFIA evaluated information provided on the environmental, animal, and human health safety of herbicide tolerant Inzen sorghum with the intended use in animal feed. From their assessment, the CFIA concluded that there are no concerns from an environmental and feed safety perspective. This perspective is applicable to the food and feed products derived from herbicide tolerant Inzen sorghum destined for commercial sale.

It is the continuing responsibility of the food manufacturer or importer to ensure that their products are in compliance with all applicable statutory and regulatory requirements. Any new information obtained in relation to these products which have potential health and safety implications should be forwarded to Health Canada for our consideration in order to ensure the continued safety and integrity of all foods available in the Canadian marketplace. The sale of a food which poses a hazard to the health of consumers would contravene the provisions of the Food and Drugs Act.