Novel food information: Camelina oil derived from thifensulfuron-tolerant camelina line 14CS0851-01-14

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Background:

Health Canada has notified Smart Earth Camelina Corporation that it has no objection to the food use of cold-pressed camelina oil derived from herbicide tolerant camelina line 14CS0851-01-14. The Department conducted a comprehensive assessment of this camelina line 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 Smart Earth Camelina Corp. and the evaluation by Health Canada. This document contains no confidential business information.

1. Introduction

Smart Earth Camelina Corp. has developed a novel camelina line (Camelina sativa (L.) Crantz, Family Brassicaceae), 14CS0851-01-14, that exhibits tolerance to the acetohydroxyacid synthase (AHAS)-inhibiting herbicide thifensulfuron, a sulfonylurea (SU) herbicide.

This novel characteristic was achieved through chemical mutagenesis resulting in two mutant genes, CsAHAS1 and CsAHAS3, which produce AHAS enzymes possessing the same amino acid substitution, P197S (proline to serine at position 197). This point mutation in AHAS confers herbicide tolerance, allowing the enzyme to function in the presence of SU herbicides. 

Smart Earth Camelina Corp. intends to market a cold-pressed camelina oil derived from the SU-tolerant camelina line 14CS0851-01-14. Health Canada previously indicated no objection to the use of cold-pressed oil derived from conventional camelina as a food ingredient (2010).

Health Canada has previously indicated no objection to the sale of SU-tolerant crops due to mutagenized AHAS (ExpressSun™ Sunflower SU7; Cibus Canola Line 5715). In addition, several other AHAS-mutagenized herbicide-tolerant crops have been previously assessed [canola (NS738, NS1471, NS1473); corn (XI-12); lentils (RH44); rice (CL121, CL141, CFX51, PWC16, CL IMINTA 1, CL IMINTA 4, RTC1); soybean BPS-CV127-9; durum wheat (DW1, DW12)].

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 reflects international guidance documents in this area (e.g., Codex Alimentarius). The assessment considered: how herbicide tolerant camelina 14CS0851-01-14 was developed, how the composition and nutritional safety of this variety compared to its unmodified comparator, and what the potential is for this variety to present a toxic or allergenic concern.  Smart Earth Camelina Corp. has provided data to support that this variety is safe for use as food in Canada.

The Food Directorate has a legislated responsibility for the pre-market assessment of novel foods and novel food ingredients, as detailed in Division 28 of Part B of the Food and Drug Regulations (Novel Foods).  SU-tolerant camelina line 14CS0851-01-14 is considered to be 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  

iii. One or more characteristics of the plant, animal, or microorganism no longer fall within the anticipated range for that plant, animal, or microorganism”.

2. Development of the modified plant

SU-tolerant camelina line 14CS0851-01-14 was produced through chemical mutagenesis of the parental variety SRS 934, using ethyl methanesulfonate (EMS). Mutagenized seeds were then grown and tolerant plants were selected using the herbicide thifensulfuron-methyl. Two SU-tolerant plants were crossed and the SU-tolerance trait of the progeny was stabilized through selfing in order to produce a stable homozygous variety. The resulting camelina 14CS0851-01-14 line demonstrated stable SU-tolerance over several generations.

3. Characterization of the modified plant

The gene mutations in the 14CS0851-01-14 camelina line were characterised using Sanger DNA sequencing which confirmed a point mutation of C→T (cytosine→thymine) at position 580 in CsAHAS1 and CsAHAS3. This DNA mutation corresponds to a single amino acid substitution, proline to serine, at position 197.

Segregation analysis was conducted to demonstrate the mode of trait inheritance of 14CS0851-01-14 to confirm stability of the SU-tolerance trait across multiple generations. Results indicated that the SU-tolerance trait in 14CS0851-01-14 was inherited in a Mendelian fashion for 2 independently segregating resistance genes.

A bacterially synthesized AHAS was produced in order to express adequate amounts of mutant AHAS for pepsin digestion and thermolability studies. An Escherichia coli expression system was used to produce a synthetic CsAHAS3 gene encoding the P197S point mutation. The Bureau of Microbial Hazards (BMH) deems the bacterially synthesized mutant CsAHAS3 to be equivalent to the camelina-derived mutant AHAS based on: the verified DNA sequences; the molecular mass of the expressed synthetic protein as determined by SDS-PAGE and Western analysis; the verified amino acid sequence of the recombinant AHAS by mass spectrometry; and the enzyme activity profile of the bacterially synthesized AHAS.

Based on the information provided, there are no concerns regarding the food use of the SU-tolerant camelina line 14CS0851-01-14 from a molecular perspective.

4. Product information

Herbicide tolerant camelina line 14CS0851-01-14 differs from its conventional counterpart by the presence of mutant AHAS-encoding genes in the host genome. Expression of these genes results in two mutant AHAS enzymes that are capable of functioning in the presence of AHAS-inhibiting herbicides, thus permitting herbicide tolerant camelina line 14CS0851-01-14 to grow in the presence of such herbicides, namely thifensulfuron.

Smart Earth Camelina Corp. provided a scientific rationale to explain that the point mutations in the SU-tolerant AHAS in camelina are highly unlikely to affect the expression levels of AHAS compared to wild-type. The SU-tolerant AHAS gene is regulated by the endogenous AHAS promoter and is expected to produce the same amount of enzyme as in wild-type camelina. The BMH agrees with the petitioner’s rationale and has no safety concerns regarding the expression levels of SU-tolerant AHAS in camelina line 14CS0851-01-14.

5. Dietary exposure

It is expected that herbicide tolerant camelina 14CS0851-01-14 will be used in applications similar to conventional camelina varieties. Smart Earth Camelina Corp. does not anticipate a significant change in the food use of camelina with the introduction of herbicide tolerant camelina 14CS0851-01-14.

6. Nutrition

The Bureau of Nutritional Sciences (BNS) assessed the nutritional composition of camelina 14CS0851-01-14 compared with its parental line control SRS 934 and an oil-rich reference commercial variety MIDASTM, grown in field trials conducted at 3 sites (Taber, AB; Saskatoon, SK; Morris, MN) in 2016, in a randomized complete block design with 4 replicates of each line.

The compositional analytes measured in 14CS0851-01-14, the control line, and MIDASTM included, in whole grain: crude fat, crude protein, amino acids, glucosinolates, sinapine, phytic acid, trypsin inhibitor; and in extracted oil: fatty acids, vitamin E.

There were no differences in fat, protein, amino acids, or the antinutrients sinapine, phytic acid, and trypsin inhibitor, in grain from 14CS0851-01-14 compared with its parental line control, SRS 934.  There was a significant difference in total glucosinolates, with 14CS0851-01-14 containing significantly lower total glucosinolates than SRS 934.

In the analysis of the oil, no differences were observed in linolenic, linoleic, arachidic, or behenic acids, while small but significant differences were reported for eicosenoic (lower in 14CS0851-01-14), palmitic (higher in 14CS0851-01-14 ), stearic (lower in 14CS0851-01-14), oleic (lower in 14CS0851-01-14), and  erucic (higher in 14CS0851-01-14).  Erucic acid levels in all three lines were well below the maximum level (5%) permitted in cooking oils, salad oils, margarines, and shortening or foods that resemble margarine or shortening, as per B.09.022 of the Food and Drug Regulations. The levels of all fatty acids were within the range for camelina oil reported in Vollman and Eynck (2015)Footnote 1.  There was no difference in vitamin E content between 14CS0851-01-14 and the parental line control.

Smart Earth Camelina Corp. has demonstrated that 14CS0851-01-14 has similar nutritional composition to its control and to conventional varieties of camelina, including MIDASTM.  Therefore, the BNS has no safety concerns with the food use of thifensulfuron-tolerant camelina line 14CS0851-01-14, from a nutritional perspective.

7.  Chemistry

Smart Earth Camelina Corp. did not provide data on the concentrations of any chemical contaminants in the novel camelina oil, such as trace elements of most concern on human health (e.g. lead, arsenic) or mycotoxins. However, a compositional assessment was conducted comparing the mutagenized camelina seed (camelina line 14CS0851-01-14) with 2 concurrently grown control seed varieties (a commercial variety and the parent line). Samples were assessed for various minerals of nutritional importance (e.g. calcium, copper). As the BNS is of the opinion that nutritive trace elements in the camelina line 14CS0851-01-14 and the control lines are comparable, it is not expected that camelina line 14CS0851-01-14 would uptake more toxic trace elements relative to conventional varieties.

Smart Earth Camelina Corp. also provided analytical results from the analysis of samples from 3 non‑consecutive batches, for arsenic, cadmium, lead, mercury, and 2 non-consecutive batches for aflatoxins (B1, B2, G1 and G2) and ochratoxin in their conventional camelina oil that is sold in Canada. The vast majority of results were either very low or below their analytical limits of detection, which were deemed by the Food Contaminants Section to be suitably low. The reported concentrations were within the range of background levels of these contaminants normally seen in cooking fats and oils sold on the Canadian market. This information may suggest that camelina oil derived from thifensulfuron-tolerant camelina line 14CS0851-01-14 would not be expected to have higher concentrations of toxic trace elements relative to the conventional oil.

The BMH is of the opinion that camelina oil derived from thifensulfuron-tolerant camelina line 14CS0851-01-14 is highly comparable to its non-mutagenized counterpart, which also suggests that it is unlikely that the mutations in the AHAS gene would cause an increase in chemical uptake or susceptibility to mycotoxins relative to conventional camelina oil.

The use of nitrogen gas and diatomaceous earth during the manufacturing process of this camelina oil are not considered food additives and are suitable for such use from a safety perspective. The seller is responsible for ensuring that any ingredient used in foods is of food grade and does not result in a violation of Section 4 of the Food and Drugs Act.

Based on the information provided, the Food Additives Section and Food Contaminants Section of CHHAD is of the opinion that camelina oil derived from thifensulfuron-tolerant camelina line 14CS0851-01-14 would not be expected to pose a concern to consumers from a chemical safety perspective.

8. Toxicology

Safe use of oil derived from the camelina line 14CS0851-01-14, containing a mutant AHAS enzyme, is supported by the results of submitted toxicity data. This information includes bioinformatics analyses, equivalence studies, heat denaturation, and in vitro digestion.

AHAS protein is a natural constituent from plants in the diet and is not considered a toxin. The mutation is a minor change, consisting of a single amino acid substitution. A bioinformatics analysis of the mutant AHAS protein was performed, using the Toxin and Toxin Target Database (Wishart, 2015) and a Basic Local Alignment Tool (BLAST) similarity search. No matches with toxins were detected.

No unintended effects were detected in the mutant line, as demonstrated by normal functioning of the altered enzyme in AHAS enzyme activity assays and no significant changes in seed composition.

Potential toxicity to the novel AHAS protein is unlikely, due to heat denaturation during oil processing and rapid digestion in the stomach following ingestion. Exposure to the intact modified AHAS protein is considered negligible.

There has been exposure to modified AHAS proteins, which Health Canada has approved in the past and have been present in commercial plant varieties for years, with no association of adverse effects.

Based on the available toxicity data, no toxicological food safety concerns were identified with use of oil derived from camelina line 14CS0851-01-14.

9. Allergenicity

The mutant AHAS protein was sensitive to denaturation from heat exposure and digestion. Substantial loss of enzyme activity occurred with incubation at 50°C for 15 minutes. Complete digestion occurred within 0.5 minutes of incubation in the simulated gastric fluid assay. Heat treatment during oil extraction and digestion in the stomach will likely leave no protein to elicit an allergic reaction.

The bioinformatics analysis of the amino acid sequence of the mutant AHAS protein did not detect any sequence homology matches with known allergens, using the Allergenicity database Version 17 http://www.allergenonline.com (January 18, 2017). An additional search, with the database AllerBase_BLAST, did not detect any hits.

Although Camelina sativa is related to mustard, a known food allergen, no reports of allergic reactions were located in the literature.

Based on the information provided, there are no allergenic concerns identified for the use of oil derived from camelina line 14CS0851-01-14.

Conclusion:

Health Canada’s review of the information presented in support of the use of thifensulfuron-tolerant camelina line 14CS0851-01-14 does not raise concerns related to food safety. 

Health Canada's opinion refers only to the food use of thifensulfuron-tolerant camelina line 14CS0851-01-14. Issues related to its use as animal feed have been addressed separately through existing regulatory processes in the Canadian Food Inspection Agency.

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 

Footnote 1

Vollmann, J., & Eynck, C. (2015). Camelina as a sustainable oilseed crop: Contributions of plant breeding and genetic engineering. Biotechnology journal, 10(4), 525-535.

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