Novel Food Information: 2′-fucosyllactose (2′-FL) from Escherichia coli BL21 (DE3) Strain #1242
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Background
Health Canada has notified Chr. Hansen A/S that it has no objection to the food use of 2′-fucosyllactose (2′-FL) derived from a genetically modified strain of E. coli BL21 (DE3), “Strain #1242”, as a food ingredient in infant and toddler formula. The Department conducted a comprehensive assessment of this 2′-FL 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 Chr. Hansen A/S and the evaluation by Health Canada. This document contains no confidential business information.
1. Introduction
The Food Directorate received a submission from Chr. Hansen A/S (formerly Jennewein Biotechnologie) regarding their 2′-FL product which is derived from E. coli BL21 (DE3) strain #1242. This strain is the direct progenitor (parent or precursor strain) of the previously-assessed E. coli BL21 (DE3) strain #1540, “Strain #1540”. The intended use of the 2′-FL product derived from Strain #1242 is as an ingredient in infant and toddler formula. The Food Directorate previously reviewed and did not object to the use of Strain #1540 for this purpose.
The intended use level of 2′-FL produced by Strain #1242 is the same as that which was authorized for 2′-FL produced by Strain #1540, i.e., up to 1.2 g/L in infant and toddler formula, as consumed and inclusive of overage.
With respect to the original submission for 2′-FL produced from Strain #1540, it was considered a novel food by virtue of the source organism being a genetically modified bacterium, under the following part of the definition of novel foods: “a food that is derived from a plant, animal, or microorganism that has been genetically modified such that one or more characteristics of the plant, animal, or microorganism no longer fall within the anticipated range for that plant, animal, or microorganism". 2′-FL produced from Strain #1242 formally meets the same definition of novel food as did 2′-FL from Strain #1540.
The 2′-FL produced by Strain #1540 was authorized as a novel food on the basis of the full safety review, which considered the characterization and safety of the strain, the manufacturing process, and the final product specifications with respect to contaminants. Given that (1) the final purified ingredient is the same due to identical downstream manufacturing steps, and (2) the production strains were fully characterized under the original submission for 2′-FL from Strain #1540, the approach taken for this request was to evaluate the equivalency of 2′-FL from Strain #1242 to the previously authorized 2′-FL from Strain #1540. This equivalency determination considered both the safety of the strain and the manufacturing of the 2′-FL (i.e., fermentation, purification, and final product testing). Further, the safety and nutritional quality end-points considered for a novel food were assessed.
2. Development of the Modified Microorganism
The method of development and characterization of genetic modifications in Strain #1242 were previously assessed during the evaluation of Strain #1540. There were no additional genetic modifications introduced into Strain #1242 since the previous assessment. The downstream manufacturing and purification steps for 2′-FL produced by Strain #1242 are identical or highly similar to those described in the previous notification.
Strain #1242 was confirmed to be equivalent from a safety perspective to the authorized Strain #1540 as a production organism for 2′-FL, based on the following rationale: a) Strain #1242 is the direct progenitor (parent strain) of the previously-assessed strain #1540 and no further genetic modifications were made to Strain #1242; b) genetic modifications and stability for both strains were fully characterized with equal robustness using whole genome sequencing; and c) the downstream purification steps and microbiological specifications are identical for 2′-FL made by Strains #1242 and #1540, as was demonstrated by the non-consecutive batch test results provided for both. Since the fermentation conditions are the same for both strains, and Strain #1242 is a direct progenitor of #1540 with no further genetic modifications introduced, then no novel proteins are expected to be expressed by Strain #1242.
Due to the equivalence of Strain #1242 to the authorized Strain #1540 as a production organism for 2′-FL, a new molecular biological assessment was not required. There are no safety concerns regarding the method of development or the molecular characterization of 2′-FL produced by Strain #1242.
3. Nutrition
Chr. Hansen’s 2′-FL produced with Strain #1242 is structurally identical to 2′-FL in human milk, and the ingredient meets identical specification parameters as 2′-FL produced with Strain #1540 (which was previously approved for use in both term infant formula and toddler formulas). The finished material continues to be a spray-dried powder containing ≥90% 2′-FL dry weight, with the remaining components comprising small amounts of inorganic material, moisture, and residual carbohydrate by-products. The 2′-FL produced with Strain #1242 continues to meet the same specification limits for carbohydrate by-products.
2′-FL is a non-digestible oligosaccharide. Intestinal microbiota in infants and toddlers metabolize human milk oligosaccharides into short chain fatty acids that are used for energy by colonocytes (Rodricks et al. 2007; Engfer et al. 2000), or are excreted in the feces.
Chr. Hansen provided data comparing nutritional, chemical and microbiological parameters between the 2′-FL products using data from five batches for each product. From a nutritional point of view, the two products are considered similar, and the minor differences in the percentages of 2′-FL and the residual sugars (e.g., lactose, fucose, 3-fucosyllactose, difucosyllactose, fucosyl-galactose, glucose, galactose) are unlikely to significantly affect nutritional safety such as absorption of nutrients. Therefore, there are no differences with respect to the nutritional considerations for 2′-FL produced with Strain #1242 or Strain #1540. Moreover, there are no differences expected with respect to bioavailability, gastrointestinal and other physiological effects based on the similarity of 2′-FL produced with Strain #1242 with the 2′-FL produced using the previously approved Strain #1540.
There are no nutritional safety concerns with the use of Chr. Hansen’s 2′-FL made using Strain #1242 in term infant formula and toddler formulas intended for children 1-3 years of age at a maximum level of 1.2 g/L of formula or the liquid product as fed (as consumed and inclusive of overage). Further assessment of the proposed new infant formula with 2′-FL will be conducted when the premarket notification is submitted, as per Division 25 of the Food and Drug Regulations.
4. Chemistry
The product specifications and manufacturing process of 2′-fucosyllactose (2′-FL) produced by Strain #1242, including all substances used and their levels, are identical or highly similar to 2′-FL produced by Strain #1540. The few minor differences in the fermentation media components are not expected to have any impact on the composition or safety profile of the final 2′-FL product.
The specifications for the 2′-FL (CAS RN 41263-94 9) product produced by Strain #1242, including limits on trace elements, are identical to the specifications for the approved 2′-FL produced by Strain #1540. The specifications for arsenic, cadmium, lead and mercury can be consistently met based on analytical results for 2′-FL provided by Chr. Hansen. The estimated concentrations of these trace elements in the foods proposed would be well below the background levels typically seen in similar foods on the Canadian Market. Furthermore, potential exposure to these trace elements from the consumption of 2′-FL in foods proposed at the maximum requested levels of use was determined to contribute negligible amounts of arsenic and lead to overall dietary exposure and result in exposure to mercury and cadmium that are orders of magnitude lower than their respective toxicological reference values. Overall, the potential exposure to arsenic, cadmium, lead and mercury from the proposed uses of 2′-FL are not expected to pose a chemical safety concern.
Chr. Hansen provided proton (1H) and carbon (13C) Nuclear Magnetic Resonance (NMR) spectra data demonstrating that the molecular structure of 2′-FL produced by fermentation of Strain #1242 is chemically equivalent to 2′-FL isolated from human milk and 2′-FL produced by Strain #1540. There are no chemical food safety concerns with the proposed uses of 2′-FL produced by Strain #1242.
5. Microbiology
The downstream purification and manufacturing steps for 2′-FL produced by Strain #1242 are the same as for Strain #1540. In addition, the microbiological specifications are identical for 2′-FL made by Strains #1242 and #1540, and compliance with those specifications was demonstrated by the non-consecutive batch test results provided for both. Consequently, a new microbiological assessment was not required for 2′-FL derived from Strain #1242. There are no microbiological safety concerns with the use of 2′-FL produced by Strain #1242 in infant and toddler formula.
6. Toxicology
The requested level of use of 2′-FL(i.e., 1.2 g/L) for infant formula, as well as toddler formula, falls within the range of 2′-FL concentrations found in human milk (ranges from 0.06 to 4.65 g/L). Chr. Hansen provided supporting evidence that their 2′-FL from Strain #1242 is chemically and nutritionally highly similar to the 2′-FL already approved for use in Canada (i.e., from the related Strain #1540), for addition to infant and toddler formula, up to 1.2 g/L (including overages). Chr. Hansen is requesting the same level of use that has already been approved.
Strain #1242 is the direct progenitor of Strain #1540, and the manufacturing processes were confirmed to be highly similar. Therefore, the toxicological data for 2′-FL produced by Strain #1540 can be used to support the safety of 2′-FL produced by Strain #1242. The studies reviewed for 2′-FL produced by Strain #1540 (from Jennewein, which was acquired by Chr. Hansen) included in vitro and in vivo genotoxicity testing, a 13-week oral toxicity study in rats, and a 3-week tolerability study in neonatal piglets. No toxicological safety concerns were identified in that assessment.
Based on the results of these studies, and the ability to bridge the safety data (given the highly similar nature of Strain #1242 and Strain #1540), there are no toxicological concerns with the addition of Chr. Hansen’s 2′-FL produced by Strain #1242 to infant and toddler formula, up to 1.2 g/L, including overage.
7. Allergenicity
2′-FL is a human milk oligosaccharide and would not be expected to elicit an allergic response in consumers. Strain #1242 contains fewer genetic modifications than Strain #1540, as it is the direct progenitor of this previously authorized production strain. Therefore, no additional proteins will be expressed in the production of 2′-FL from Strain #1242.
Chr. Hansen submitted analytical data for protein levels. Based on the five certificates of analysis provided (five different batches tested), the protein content of the 2′-FL from Strain #1242, continues to be < 10 μg/g (using a modified version of the Bradford assay). This is below the specified limit of ≤ 100 μg/g.
There are no allergenicity concerns for the addition of Chr. Hansen’s 2′-FL to infant and toddler formula.
Conclusion
Health Canada’s review of the information presented in support of the use of 2′-FL product derived from E. coli BL21 (DE3) strain #1242 does not raise concerns related to food safety.
Health Canada’s opinion refers only to the use of 2′-FL as an ingredient in infant and toddler formula.
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 Sir Frederick Banting Driveway
Ottawa, Ontario K1A 0K9
bmh-bdm@hc-sc.gc.ca
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