Novel Food Information: High Oleic Algae Oil from Prototheca moriformis strain S8695B

On this page

• Background
• 1. Introduction
• 2. Development of the Modified Microorganism
• 3. Characterization of the Modified Microorganism
• 4. Product Information
• 5. Dietary Exposure
• 6. Nutrition
• 7. Microbiology
• 8. Chemistry/Toxicology
• Conclusion

Background:

Health Canada has notified Corbion Biotech, Inc. that it has no objection to the food use of High Oleic Algae Oil (HOAO) derived from genetically modified Prototheca moriformis strain S8695B. The Department conducted a comprehensive assessment of the HOAO 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 Corbion Biotech, Inc. and the evaluation by Health Canada and contains no confidential business information.

1. Introduction

Corbion Biotech, Inc. has developed a HOAO derived from a genetically modified Prototheca moriformis strains, S8695B, using recombinant-DNA techniques. The HOAO contains ≥80% oleic acid and is expected to replace a portion of the dietary fats and oils in the Canadian market for various conventional foods which lack a standard of identity. Genetic modifications were made to P. moriformis strain S8695B to achieve a desired oil composition rich in oleic acid [C18:1(n-9)], and low in saturated fat content. Health Canada has previously approved two HOAOs from Corbion Biotech, Inc. which were produced by genetically modified P. moriformis strains S2532 and S6697 (April 2017). The Department has also previously approved other algal-derived products such as whole algal flour and protein.

The safety assessment performed by Food Directorate scientific 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 approach in this area (e.g., Codex Alimentarius). The safety assessment considered: how the HOAO was developed; how the composition and nutritional quality of the HOAO compared to conventional oils; and what the potential is for the HOAO to be toxic or cause allergic reactions. Corbion Biotech, Inc. has provided data which demonstrates that the HOAO is as safe as other oils 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). Foods which contain HOAO are considered novel foods 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 Microorganism

The production strain P. moriformis S8695B is derived from the parental wild-type strain S376 that was clonally purified from the original wild-type P. moriformis strain. HOAOs from previously assessed strains S2532 and S6697 were also derived from the same parent strain S376. Classical mutagenesis and genetic modifications were made to further obtain the desired oil composition. Information was provided regarding the methods used to develop the HOAO. The evaluation of HOAO from P. moriformis strain S8695B did not raise any safety concerns regarding the method of development.

3. Characterization of the Modified Microorganism

Molecular characterization of strain S8695B indicated that Cassette #1 integrated stably into allele 1 of the targeted locus. Two copies of each transgene were present within the integrated cassette due to gene duplication events. The arrangement of the transgenes was examined using Southern blot analysis and there was no indication of rearrangements. Southern blot analysis also detected one copy of the marker gene, as expected.

Molecular characterization indicated that Cassette #2 integrated stably into allele 1 of the targeted locus of S8695B. An additional off-target integration of Cassette #2 was detected by Southern blot analysis. Southern analysis also demonstrated that there were at least 12 copies of the RNA hairpin present in S8695B, some of which were located at off-target integration sites.

Molecular characterization indicated that Cassette #3 integrated off-target. The transgenes introduced by Cassette #3 were all detected in S8695B by Southern blot analysis. Strain S8695B was shown to be phenotypically stable over 68 generations on non-selective media. The genotypic stability of S8695B transgenes was demonstrated through Southern blot analysis over the same 68 generations.

Given that the final product is a highly refined oil which will be devoid of any protein, there was no reason to expect any new hypothetical protein to be present in the final HOAO product.

4. Product Information

The HOAO is produced via a axenic fermentation under sterile conditions at a facility compliant with current Good Manufacturing Practices (cGMP). Throughout the fermentation process, the pH, temperature, agitation and aeration rates are controlled. Cell banks are prepared to verify and identify the axenic nature of the cultures used in the fermentation. Following fermentation, the broth is inactivated, further processed and purified after which the dried biomass is treated to release the crude algae oil. The crude oil is then highly refined using standard edible oil refining steps including degumming, bleaching, and deodorization. The results of real-time quantitative PCR of the HOAO from S8695B indicated the lack of any endogenous or transgenic P. moriformis DNA in the final oil products.

5. Dietary Exposure

The algal oil is expected to replace a portion of the dietary fats and oils in the Canadian market for various conventional foods which lack a standard of identity. Consumption estimates were provided assuming that the notified algal oil would be used in all foods to which HOAO was proposed to be added (i.e. complete substitution of olive oil, canola oil, corn oil and other vegetable oils); these estimates were deemed to be sufficiently conservative for the purposes of this evaluation. It is not expected to change the overall consumption of these foods by the general population.

The estimated daily intakes (EDIs) of HOAO were provided for individuals consuming the food groups selected for the addition of HOAO using 2004 CCHS data. When CCHS data were used, the mean and 90th percentile of HOAO consumption were estimated to 5.96 and 10.57 g/day, respectively, in all populations (based on age and gender). Among all reference groups, males ages 14-18 were estimated to have the highest usual intakes, with a mean and 90th percentile of 9.41 and 17.37 g/day, respectively. On a body weight basis, children ages 4 to 8 have the highest mean intake with 260.2 mg/kg bw per day, and children ages 1 to 2 have the highest intake at 90th percentile with 449.2 mg/kg bw per day.

Also, a second dietary exposure was provided using NHANES (2011-12) data with modified intake profiles (amount and frequency) and higher use levels of HOAO in targeted food products. In that dietary exposure, the estimated mean and 90th percentile of HOAO consumption was approximately 12.0 and 27.5 g/day, respectively. In any case, a more realistic assumption was considered that only 10% of the products would be used at the maximum level. Therefore, using the dietary exposure with higher levels of addition, a more realistic mean and 90th percentile EDIs would be approximately 1.2 g/day and 2.8 g/day, respectively. Although all food categories were used for the calculation, the notified HOAO would be added only to foods for which a standard of identity does not exist.

6. Nutrition

The nutritional composition of the HOAO derived from strain S8695B was reviewed. The physical and chemical properties as well as the fatty acid composition for 3 lots of HOAO were provided. The fatty acid composition of HOAO was provided by the fatty acid methyl esters (FAMES) analysis using gas chromatography equipped with a flame ionization detector. The methodology was reviewed and deemed acceptable.

The compositional analysis showed that all fatty acids identified in HOAO derived from strain S8695B were also found in HOAO derived from strains S2532 and/or S6697 (previously reviewed and accepted). Oleic acid, a cis n-9 monounsaturated fatty acid, is the predominant fatty acid with a mean level of approximately 93%. Cis n-9 monounsaturated fatty acid can be synthesized by the body and confers no known independent health benefits. No Dietary Reference Intakes have been set for cis-monounsaturated fatty acids.

The second and third most abundant fatty acids are stearic acid and palmitic acid. These saturated fatty acids (SFAs) were found at levels of 1.32% and 1.30%, respectively. There is no Estimated Average Requirement (EAR) or Adequate Intake (AI) set for SFAs because they are not essential and have no known role in preventing chronic disease. Intakes of SFAs should be minimized due to a positive linear trend between SFA intake and total and low-density lipoprotein (LDL) cholesterol levels and an increased risk of coronary heart disease. The levels of palmitic acid and stearic acid in HOAO are similar to or less than the levels found in olive, canola, or soybean oil. Therefore, the level of SFAs found in HOAO does not pose nutritional concerns.

Altogether, oleic, palmitic, and stearic fatty acids account for approximatively 95% of fatty acids in HOAO. Other fatty acids analyzed, including essential fatty acids linoleic (LA) and alpha-linolenic (ALA), were detected at levels of less than 1%. Erucic acid, a potential toxicant, was not detected in the tested samples. As in previously reviewed submissions of HOAO derived from P. moriformis, all fatty acids founds in HOAO are common in many oils and fats, and are expected to be digested through the same normal physiological processes by which other plant-derived oils common to the human diet are digested and utilized.

The levels of tocopherols in HOAO derived from strain S8695B were not provided. Based on published results from Szabo et al. (2014)^{Footnote 1}, HOAO manufactured from P. moriformis strain S2532 does not contain α-tocopherol, and thus will not contribute to the vitamin E intake. Similar results are expected in the current submission, thus HOAO derived from strain S8695B will not contribute to the vitamin E intake.

The information on the levels of antinutrients was not provided. In the previous submissions of HOAO derived from P. moriformis, a search of the scientific literature was conducted and there were no studies reporting that P. moriformis is able to produce any appreciable amounts of the most common antinutrients found in foods, that is phytate, tannins, protease inhibitors, calcium oxalate, and lectins. Also, antinutrients are generally not a concern in purified/refined oil as some processing steps (e.g. heat treatment and degumming) are effective against antinutrients. Therefore, it is expected that HOAO derived from S8695B would also not contain any antinutrients.

HOAO is almost exclusively in the form of triglycerides (≥ 95%) with small amounts of diglycerides (< 5%) and trace amounts of monoglycerides (< 0.5%). The specification values were reported for free fatty acids, unsaponifiable matter, peroxide value, and p-anisidine for HOAO. Since there is no Codex Standard for microalgal oil, these specification values were compared to other specifications for other types of algal oil. The anisidine value, free fatty acids, peroxide value, and unsaponifiable matter for HOAO meet the specifications of DHA (docosahexaenoic acid) from Algal Schizochytrium sp. oil and DHA from Algal Crypthecodinium sp. oil, as published by the Food Chemical Codex 10, as well as the specifications for anisidine value, peroxide value, and unsaponifiable matter for Crypthecodinium cohnii oil and Schizochytrium sp. oil as published by the United States Pharmacopeia (2016)^{Footnote 2}. Therefore, HOAO is similar to other algal oil with respect to these specifications.

As noted previously, linoleic acid (LA) and alpha-linolenic (ALA) are present at low levels in HOAO. The dietary replacement of vegetable oils containing higher levels of LA and ALA with HOAO could potentially lead to lower intakes of these essential fatty acids in the Canadian population. However, a significant shift of the intakes of LA and ALA is unlikely given the highly conservative EDI of 1.2 and 2.8 g HOAO/day, respectively, for the mean and 90th percentile in all populations.

Overall, no nutritional hazards were identified related to the composition of HOAO that would be any different from other food-grade vegetable oils. Given the information provided, no nutritional safety concerns have been identified in regards to the use of HOAO as food.

7. Microbiology

In April 2017, Health Canada approved two HOAOs produced by genetically modified P. moriformis strains S2532 and S6697.The host strain P. moriformis (current accepted name: Prototheca zopfii W. Krüger) is a eukaryotic microalgae in the same family as the genus Chlorella. Prototheca species are ubiquitous in the environment and have been isolated from soil, decaying plant matter, sewage, water, as well as human and animal mucosal fluids. Microalgae such as Chlorella vulgaris and Chlorella pyrenoidosa are used as sources of food and food ingredients.

The HOAO is produced under a highly rigorous process compliant with cGMP to avoid and prevent microbial contamination. This includes extensive heat treatments during its processing (bleaching, deodorization, etc.). Refined oils do not contain sufficient water/moisture needed for microbial growth. The HOAO has less than 0.1% moisture which is similar to other refined food oils. During the refinement process, samples of the oil product are incubated for 5 days to verify the absence of viable cells.

Wild-type P. moriformis is not known to produce antimicrobial compounds of clinical importance. Whole genome sequencing has not indicated the presence of fully functional genes encoding antimicrobial compounds or antimicrobial-resistance genes.

There are no microbiological concerns with the food use of HOAO from P. moriformis strain S8695B.

8. Chemistry/Toxicology

A safety assessment of the HOAO from P. moriformis strain S8695B was conducted to ensure that the oil does not pose any chemical or toxicological hazards.

The finished oil specifications and analytical results were provided for lead, arsenic, mercury, cadmium, phosphorus and sulfur, which were based on the analysis of three batches of HOAO. The addition of HOAO as an ingredient in the foods specified and at the proposed levels of use is not expected to significantly increase the total dietary exposure of Canadians to these trace elements.

The HOAO was analyzed for a wide array of phycotoxins of potential concern to human health, including those for which Canada has maximum levels (MLs) as well as a number of compounds for which insufficient toxicity data was available to quantitatively assess at the time of this evaluation. None of the toxins tested were detected above their respective limits of detection (LODs), which were deemed to be reasonably sensitive. Furthermore, P. moriformis is not among the types of algae known to produce any of the 9 classes of marine biotoxins which were analyzed. Based on estimates of exposure under the conservative assumption that these phycotoxins were present at concentrations equal to their respective LODs, they would not pose a safety concern for consumers. Furthermore, no adverse effects were observed in the toxicology studies provided that would suggest the presence of phycotoxins in HOAO at concentrations that might pose a safety concern. Therefore, it is considered unlikely that the consumption of HOAO from foods at the indicated levels of use would pose a safety concern to consumers from a phycotoxin perspective.

Certificates of analysis were provided for a number of mycotoxins and polychlorinated biphenyls (PCBs) in the auxiliary materials used to release the oil from the algal biomass. Since none of these contaminants were detected in these auxiliary materials above their respective LODs, the LODs were deemed to be sufficiently low, and since the auxiliary materials are fully removed following their use in the manufacturing process and are not present in the final oil, exposure to these contaminants from the use of the auxiliary materials for isolation of the oil in producing HOAO is not expected to be of concern for human health.

Numerous substances are used during the course of manufacturing HOAO. The acceptability of particular uses of substances for a technological purpose in manufacturing the HOAO is separate from the acceptability of the oil itself. The manufacturer is responsible for using any additives (such as antioxidants) in HOAO in accordance with applicable food additive provisions for such use. More generally, the manufacturer is responsible for ensuring that the use of any substance in the manufacture of HOAO does not result in a violation of Section 4 of the Food and Drugs Act.

The safety of the S8695B HOAO was evaluated by assessing the potential toxicity of the finished product. All toxicity tests were conducted in accordance with GLP and OECD guidelines.

As described previously, the S8695B algal oil was assessed for the presence of several known algal toxins (e.g, including amnesic shellfish poisoning toxins, paralytic shellfish poisoning toxins, diarrhetic shellfish poisoning toxins, neurotoxic shellfish poisoning toxins, cyanobacterial toxins and pheophorbide A). No algal toxins were detected in the algal oil as determined by high performance liquid chromatography with tandem mass spectrometry detection or enzyme-linked immunosorbent assays (LOD range: 0.005-0.15 μg/g oil).

The S8695B strain of P. moriformis differs from those strains previously assessed by Health Canada (S2532 and S6697). The classical mutation-induced and engineered genetic differences in S8695B were not considered to pose a toxicological concern. This was concluded because the final S8695B oil is devoid of proteins and is not expected to contain biologically relevant quantities of small interfering RNAs. On this basis, the toxicological information could be bridged between the algal oils.

Due to the absence of a history of safe food exposure to HOAO, A subchronic (90-day) oral toxicity study was performed in rats. Groups of animals (10 animals/sex/group) were provided the S2532 algal oil at a constant dietary concentration of 25 000, 50 000 or 100 000 ppm in the diet (equivalent to 1366, 2658 and 5200 mg/kg b.w. per day in males and 1624, 3183 and 6419 mg/kg b.w. per day in females, respectively). The control groups were provided the basal diet with similar levels of dietary fat from soybean oil. Based on the absence of treatment-related toxicity, the NOAEL was observed to be 5200 mg/kg b.w. per day, the highest dose tested in males.

The S2532 algal oil was shown not to be mutagenic in a bacterial reverse mutation (Ames) test or clastogenic in an in vivo mammalian bone marrow chromosomal aberration test when administered by injection to NMRI mice. Under the test conditions, the evidence indicates that the algal oils from P. moriformis are not genotoxic.

On a body weight basis, young children (1-3 years) are the highest consumers of the foods that could contain the high oleic algal oils. The 90th percentile of consumers in this subpopulation could be exposed to 449.2 mg/kg b.w. per day which is about 11-fold less than the NOAEL reported in the subchronic (90-day) oral toxicity study.

Because the algal oil is composed of lipids with a composition similar to other edible oils, which are expected to be handled by the body as any other dietary fat, the margin of exposure is considered sufficient from a safety perspective. In addition, the potential allergenicity of the S8695B high oleic algal oil finished product was assessed. S8695B high oleic algal oil is not expected to contain known allergens as it is highly refined and does not contain measurable quantities of protein (nitrogen combustion method; LOD: < 0.1%). Based on the evidence provided, high oleic algal oil S8695B would not be expected to pose an allergenic safety concern.

Based on the evidence provided, the S8695B high oleic algal oil would not be expected to pose a safety concern, from a toxicological perspective.

Conclusion:

Health Canada's review of the information presented in support of the food use of high oleic algae oil S8695B does not raise concerns related to food safety. Health Canada is of the opinion that food containing HOAO derived from P. moriformis strains S8695B is as safe and nutritious as food containing other oils used in Canada.

Health Canada's opinion deals only with the food use of High Oleic Algae Oil from P. moriformis strain S8695B.

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.

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For further information, please contact:

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