Novel Food Information: High Oleic Algae Oils from Prototheca moriformis strains S2532 and S6697

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. (formerly TerraVia Holdings, Inc.) that it has no objection to the food use of High Oleic Algae Oils (HOAOs) derived from genetically modified Prototheca moriformis strains S2532 and S6697. The Department conducted a comprehensive assessment of these HOAOs 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 two HOAOs derived from two genetically modified Prototheca moriformis strains, S2532 and S6697, using recombinant-DNA techniques. The two HOAOs contain ≥80% oleic acid and are 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. The genetic modifications made to S2532 and S6697 were not identical and lead to oils with slightly different fatty acid profiles, thus two separate novel food data submissions were required. The HOAO from S2532 produces an oil rich in oleic acid (C18:1) and low in linoleic acid (C18:2). The HOAO from S6697 has higher oleic content and lower saturated fat content than the HOAO from S2532. Health Canada has previously approved other algal derived products such as oils and whole algal flour and protein. However, P. moriformis as a production organism has not been evaluated previously. The two submissions were assessed simultaneously.

The safety assessments performed by Food Directorate evaluators were 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 the two HOAOs were developed; how the composition and nutritional quality of the two HOAOs compared to conventional oils; and what the potential is for the two HOAOs to be toxic or cause allergic reactions. Corbion Biotech, Inc. has provided data which demonstrates that the two HOAOs are 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 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.

Both classical mutagenesis and genetic modification techniques were used to produce strains S2532 and S6697. Information was provided regarding the methods used to develop the HOAOs. The production strains P. moriformis S2532 and S6697, which were both derived from the parental wild-type strain S376 that was clonally purified from the original wild-type P. moriformis strain, were modified to overexpress oleic acids. The evaluation of HOAO from P. moriformis strains S2532 and S6697 did not raise any safety concerns regarding the method of development.

3. Characterization of the Modified Microorganism

Molecular characterization of strain S2532 indicated that there were two copies of the gene cassette present at the two targeted insertion sites within both alleles of an endogenous desaturase gene. The arrangement of the transgene was examined using Southern blot analysis and there was no indication of rearrangements. Strain S2532 was shown to be phenotypically stable over 30 generations on non-selective media. The genotypic stability was demonstrated through Southern blot analysis over the same 30 generations.

Molecular characterization of strain S6697 indicated that there were eight copies of the RNA hairpin present at multiple off-target integration sites. Information was provided to explain that this is not uncommon in plant and algal transformations and that the transformed cells do not seem to be affected in a deleterious way given the cells remain viable, have the same morphology as the parent strain S5587, and produce oil normally. The arrangement of the transgene was examined using Southern blot analysis and there was no indication of rearrangements. Strain S6697 was shown to be phenotypically stable over 30 generations on non-selective media. The genotypic stability was demonstrated through Southern blot analysis over the same 30 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 products.

4. Product Information

The HOAOs are produced via axenic fermentation under sterile conditions at a facility compliant with current Good Manufacturing Practices (cGMP). Throughout the fermentation process 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 by heat, further processed and purified, after which the dried biomass is treated to release the crude algae oil. The crude oil is then refined using standard edible oil refining steps including degumming, bleaching, and deodorization. The results of real-time quantitative PCR of the HOAO from S2532 and S6697 indicated the lack of any endogenous P. moriformis DNA in the final oil products.

5. Dietary Exposure

The algal oils are 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. They are not expected to change the overall consumption of these foods by the general population.

Using CCHS 2004 data, the daily intake (EDI) for individuals consuming the food groups selected for the addition of HOAO was estimated. Using these data, the mean and 90th percentile of HOAO consumption were estimated at 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. A more realistic, but still highly conservative, projection was proposed that 10% of the foods in each category would contain HOAO added at the maximum proposed level of use. The mean and 90th percentile of HOAO consumption in all populations in this scenario would be 0.6 and 1.06 g/day, respectively. Although all food categories were used for the calculation, Corbion Biotech, Inc. specified that HOAO would be added only to foods for which a standard of identity does not exist.

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 0.6 and 1.06 g HOAO/day, respectively, for the mean and 90th percentile in all populations.

6. Nutrition

The nutritional composition of HOAO derived from strains S2532 and S6697 was reviewed. The physical and chemical properties as well as fatty acid composition for 3 lots of HOAO from each strain was provided. The fatty acid composition of HOAO by 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 oleic acid is the predominant fatty acid in HOAO derived from strains S2532 and S6697 with mean levels of 86.61% and 88.27%, respectively. Oleic acid is a cis n-9 monounsaturated fatty acid; it can be synthesized by the body and confers no known independent health benefits. No Dietary Reference Intake values have been set for cis-monounsaturated fatty acids.

The next two most abundant fatty acids in HOAO are palmitic acid and stearic acid. Palmitic acid is present at mean levels of 8.11% and 3.54%, while stearic acid is present at 1.55% and 3.67% in HOAO from strains S2532 and S6697, respectively. Palmitic acid and stearic acid are saturated fatty acids (SFAs). There is no EAR or 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. HOAO is low in both of the essential fatty acids, linoleic (at less than 2%) and alpha-linolenic (at less than 1%). Other fatty acids (e.g., heneicosanoic, erucic, tricosanoic acids) were detected at levels of less than 1%. All fatty acids 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.

Szabo et al. (2014)^{Footnote 1} published results of a compositional analysis of HOAO from P. moriformis (lot # RDB540, strain S2532) that included levels of sterols. The levels of total and individual sterols found in HOAO are within the ranges of other vegetable oils listed in the FAO's Codex Standards for Fats and Oils from Vegetable Sources and within the range of other vegetable oils available commercially^{Footnote 2}. Based on analysis published by Szabo et al. (2014), HOAO contains no α-tocopherol, thus will not contribute to the vitamin E intake.

Erucic fatty acid, a potential toxicant, was present in one HOAO sample derived from strain S2532 at a level of 0.01%. The level of erucic acid in HOAO is well below the maximum of 5% permitted by B.09.022 of the Food and Drug Regulations in cooking oil, margarine, salad oil, simulated dairy product, shortening or food that resembles margarine or shortening. The level of erucic acid in HOAO does not pose nutritional concerns.

HOAO is almost exclusively in the form of triglycerides (≥ 95%) with small amounts of diglycerides (< 5%) and trace amounts of monoglycerides (< 0.5%). Corbion Biotech, Inc. reported specification values 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 oil and DHA from Algal Crypthecodinium oil, as published in the Food Chemicals Codex 10, as well as the specifications for anisidine value, peroxide value, and unsaponifiable matter for Crypthecodinium cohnii oil and Schizochytrium oil as published in the United States Pharmacopeia 39 (2016).

A scientific literature search was conducted and there were no studies reporting that P. moriformis is able to produce any appreciable amounts of the most common anti-nutrients found in foods, such as phytate, tannins, protease inhibitors, calcium oxalate, and lectins.

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 to the use of HOAO as food.

7. Microbiology

A microbiological safety assessment of HOAOs derived from strains S2532 and S6697 was conducted. 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.). Highly refined oils do not contain sufficient water/moisture needed for microbial growth. The HOAOs have less than 0.1% moisture which is similar to other highly refined food oils. During the refinement process, samples of the oil product are incubated for 5 days to verify the absence of viable cells. Whole genome sequencing of P. moriformis has not indicated the presence of fully functional genes encoding antimicrobial compounds. In addition, the absence of proteins and DNA in the final oil product would make it very unlikely that antimicrobial compounds would be present. There are no microbiological concerns with the food use of HOAO from P. moriformis strains S2532 and S6697.

8. Chemistry/Toxicology

A safety assessment of the HOAO from P. moriformis strains S2532 and S6697 was conducted to ensure that the oil does not pose any chemical or toxicological hazards.

Finished oil specifications were provided for lead, arsenic, mercury and cadmium, which was based on a batch analysis of the 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 phycotoxins of potential concern to human health are not known to be produced by the Prototheca genus of algae; nevertheless, a study was referenced showing test results for phycotoxins in the HOAO. None of the phycotoxins were detected above their respective limits of detection (LOD). With respect to phycotoxins, it is considered unlikely that consumption of HOAO at the indicated levels of use reported in the submission would pose a safety concern to consumers.

Certificates of analysis were provided for a number of mycotoxins, pesticides and polychlorinated biphenyls in the auxiliary materials used for the treatment of the biomass to isolate the oil and in the final oil. Since none of these contaminants were detected above their respective LOD, and the auxiliary materials are fully removed in the manufacturing process and are not present in the final oil, exposure to these contaminants from the consumption of HOAO is not expected to be of concern for human health.

Numerous substances are used during the course of manufacturing the 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 mixed tocopherols, in the oil in accordance with food additive provisions for such use. More generally, the manufacturer is responsible for ensuring that the use of any substance in the manufacture of the oil does not result in a violation of section 4 of the Food and Drugs Act.

The safety of the S2532 and S6697 HOAOs was evaluated by assessing the potential toxicity of the finished product.

The S6697 strain of P. moriformis differs from the S2532 strain through genetic modification. The genetic differences between S6697 and S2532 were not considered to pose a toxicological concern, since the final S6697 HOAO will be devoid of proteins and is not expected to contain biologically relevant quantities of small interfering RNAs. Therefore, toxicological information could be bridged between these HOAOs.

The HOAOs are composed of a mixture of lipids that are produced from highly processed algae. The body's ability to digest the HOAOs is expected to be normal, like that of lipids from other food sources derived from plant materials.

The algae are not known to be toxigenic or pathogenic. Nevertheless, the algal oil of the parent strain of S2532 and S6697 was assessed for the presence of several known algal toxins, 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 fluorescence or mass spectrometry detection. On this basis, the S2532 and S6697 HOAOs are not expected to contain toxins that are present in other toxigenic algae.

Due to the absence of a history of safe food exposure to HOAO, a standard 90-day subchronic oral toxicity study was performed in rats (10 animals/sex/group) that were provided the S2532 HOAO 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 bw/day in males and 1624, 3183 and 6419 mg/kg bw/day in females, respectively). The control groups were provided the basal diet with similar levels of dietary fat from soybean oil. The results showed no mortalities, clinical signs of toxicity, toxicologically significant differences in body weight, body weight gain, food efficiency, clinical chemistry, hematology, urinalysis or blood coagulation associated with S2532 HOAO treatment. No treatment-related toxicities were observed in the gross or histopathology examinations. The ophthalmoscopic examinations were normal in all HOAO-treated animals. Based on the absence of toxicity, the NOAEL was estimated to be 5200 mg HOAO/kg bw/day (based on the highest dose tested in males).

On a body weight basis, young children (3-5 years) are the highest consumers of the foods that could contain the HOAOs. The 90th percentile of consumers in this subpopulation could be exposed to 552.44 mg HOAO/kg bw/day. The HOAO was calculated to have a margin of exposure (MOE) that is 9-fold less than the NOAEL reported in the 90-day subchronic oral toxicity study (the highest dose tested in male rats).

The S2532 HOAO was shown not to be mutagenic in a standard Ames test or clastogenic in a standard, in vivo mammalian bone marrow chromosomal aberration test when administered by injection to NMRI mice. Under standard test conditions, the evidence suggests that the HOAO is not genotoxic.

This margin of exposure is considered sufficient from a safety perspective because the HOAOs are composed of lipids that have similar composition to other foods, specifically plant derived-oils. They are expected to be handled by the body as any other dietary fat. As well, the HOAO was demonstrated not to be toxic in a subchronic oral toxicity study and not genotoxic in standard in vitro and in vitro assays. In addition, the potential allergenicity of the HOAO finished product was assessed. HOAOs from strains S2532 and S6697 are not expected to contain known allergens as the oils are highly refined and do not contain measurable quantities of protein (AOAC method 990.03, combustion; LOD: < 0.1%). Based on the evidence provided, high oleic algal oil derived from strains S2532 and S6697 would not be expected to pose an allergenic safety concern.

Based on the evidence provided, the HOAOs from strains S2532 and S6697 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 S2532 and S6697 does not raise concerns related to food safety. Health Canada is of the opinion that food containing HOAOs derived from P. moriformis strains S2532 and S6697 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 Oils S2532 and S6697.

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