CFSAN/Office of Food Additive Safety
November 23, 2016
Christoph H. Rohrig, Ph.D.
DK-2800 Kgs. Lyngby
Re: GRAS Notice No. GRN 000650
Dear Dr. Rohrig:
The Food and Drug Administration (FDA, we) completed our evaluation of GRN 000650. We received the notice, dated April 21, 2016, that you submitted in accordance with the agency's proposed regulation, proposed 21 CFR 170.36 (62 FR 18938; April 17, 1997; Substances Generally Recognized as Safe (GRAS); the GRAS proposal) on April 29, 2016, and filed it on May 26, 2016. We received amendments containing clearer figures and identification and discussion of data discrepancies on August 29, 2016, August 30, 2016, and October 3, 2016.
FDA published the GRAS final rule on August 17, 2016 (81 FR 54960), with an effective date of October 17, 2016. As GRN 000650 was pending on the effective date of the GRAS final rule, we requested some additional information consistent with the format and requirements of the final rule. We received an amendment responding to this request on October 27, 2016.
The subject of the notice is 2′-O-fucosyllactose (2′-FL). The notice informs us of the view of Glycom A/S (Glycom) that 2′-FL is GRAS, through scientific procedures, for use as an ingredient in nonexempt infant formulas for term infants at a maximum use level of 2.4 grams per liter (g/L) of reconstituted formula; and, in beverages and beverage bases, dairy product analogs, infant and toddler foods including follow-on formulas, grain products and pastas, milk and milk products, and processed fruits and fruit juices at use levels ranging from 0.084 to 2.04 g/serving.
Glycom describes the identity and composition of 2′-FL. Glycom describes 2′-FL as a white to off-white powder that consists of a minimum of 94% 2′-FL. Glycom states that 2′-FL is a trisaccharide present in human milk; it consists of L-fucose, D-galactose, and D-glucose. The chemical name for 2′-FL is α-D-fucopyranosyl-(1→2)-β-D-galactopyranosyl-(1→4)-D-glucopyranose and the CAS registry number is 41263-94-9. Glycom states that 2′-FL manufactured by the process described in GRN 000650 is chemically and structurally identical to the 2′-FL in human milk.
Glycom describes the production microorganism as a modified strain of Escherichia coli K-12 DH1, referred to as strain SCR6. Glycom states that E. coli K-12 and its derivatives are safe for use in constructing modified strains that are used in food ingredient production, and that strain SCR6 is non-toxicogenic and non-pathogenic. Strain SCR6 was developed by recombinant DNA techniques, including the addition of genes from Klebsiella pneumoniae and Salmonella Typhimurium encoding functions involved in sugar metabolism, and genes from Helicobacter pylori or E. coli K-12 DH1 encoding functions involved in 2′-FL biosynthesis. Glycom states that all genes introduced into strain SCR6 are well characterized.
Glycom describes the two-stage manufacturing process for 2′-FL. Glycom states that 2′-FL is manufactured according to current good manufacturing practices (cGMPs), and that all raw materials and processing aids are food-grade and are used in accordance with U.S. regulations or are GRAS for their respective uses. In the first stage of the manufacturing process, 2′-FL is produced from D-lactose during the fermentation of strain SCR6. Glycom states that isopropyl-β-D-1-thiogalactoside is added to the fermentation medium to induce 2′-FL production. Strain SCR6 secretes 2′-FL into the fermentation medium. After fermentation is complete, the microbial biomass is removed from the fermentation medium by filtration. The second stage of the manufacturing process consists of a series of purification steps. The filtrate is concentrated by additional filtration with optional ion exchange chromatography or electrodialysis to remove water, minerals, and other small molecules. The pH of the resulting solution is maintained by the addition of sodium hydroxide and the solution is decolored using activated charcoal and filtration. The solution is then concentrated by vacuum distillation or filtration. Acetic acid and 2′-FL seed crystals are added under controlled conditions to initiate the crystallization of 2′-FL. The 2′-FL crystals are separated by filtration, washed with acetic acid, and then dried to obtain the final 2′-FL product.
Glycom provides specifications for 2′-FL. Specifications include the minimum content of 2′-FL (≥ 94 % on a dry matter basis), and limits on difucosyllactose (≤ 1 %), 2′-fucosyl-D-lactulose (≤ 1%), D-lactose (≤ 3 %), L-fucose (≤ 1 %), moisture (≤ 5 %), lead (≤ 0.1 milligrams per kilogram (mg/kg)), residual proteins (≤ 0.01 %), and acetic acid (≤ 1 % as acetic acid or sodium acetate), as well as limits for microbial contaminants, including no detectable Cronobacter sakazakii in a 10 g sample. Glycom provides the results of four independent batch analyses of 2′-FL to demonstrate compliance with these specifications.
Glycom incorporates, by reference to GRN 000546, an estimate of the dietary exposure to 2′-FL for infants consuming non-exempt term infant formulas and toddlers consuming follow-on formulas based on the maximum intended use level of 2.4 g/L and body weight data from the U.S. National Center for Health Statistics’ 2009-2010 National Health and Nutrition Examination Surveys (NHANES). Infants aged 0 to 6 months are identified as the largest consumer group, and Glycom estimates the users-only mean and 90th percentile exposures to 2′-FL for infants 0 to 6 months of age to be 2.02 and 2.91 g/person (p)/day (d) (332.8 and 535.6 mg/kg body weight (bw)/d), respectively. The mean and 90th percentile exposures to 2′-FL from infant formulas for infants 7 to 12 months of age are estimated at 1.70 and 2.63 g/p/d (188.9 and 295.8 mg/kg bw/day), respectively. The mean and 90th percentile exposures to 2′-FL from follow-on formulas in toddlers 1 to 3 years of age are estimated at 1.08 and 1.41 g/p/d (89.3 and 117.1 mg/kg bw/day), respectively.
Glycom estimates the dietary exposure to 2′-FL based on the intended uses, and food consumption and body weight data from the 2011-2012 NHANES. The mean and 90th percentile dietary exposures to 2′-FL for the total population are estimated at 0.64 and 1.31 g/p/d (20.0 and 33.1 mg/kg bw/d), respectively. The mean and 90th percentile exposures for toddlers are estimated at 1.12 and 1.97g/p/d (84.9 and 146.0 mg/kg bw/d), respectively.
Glycom discusses published and unpublished studies in animals and human infants to support the safety of its intended use of 2′-FL. Glycom incorporates published data and information by reference from GRN 000546 and GRN 000571 on the safety of 2′-FL for use in nonexempt infant formulas for term infants, and briefly discusses the main findings. Glycom states that 2′-FL does not undergo significant digestion in the upper gastrointestinal tract and only a small fraction of it is absorbed intact. In a published study in juvenile rats, administration of up to 5000 mg 2′-FL/kg bw/d for 90 days by gavage was well tolerated and did not elicit any treatment-related, toxicologically relevant effects. In a published study in neonatal piglets, up to ~292 mg 2′-FL/kg bw/d in males and ~299 mg 2′-FL/kg bw/d in females, administered through diet, did not result in any adverse health effects and did not impact piglet growth. Glycom discusses a new unpublished 90-day oral toxicity study conducted with their 2′-FL. The results of this study corroborate the safety conclusions from the published studies. Glycom notes that 2′-FL is not mutagenic or genotoxic. Glycom discusses human studies demonstrating that infants consuming up to 1.0 g 2′-FL/L of formula in combination with 1.4 g galactooligosaccharide/L or 0.5 g lacto-N- neotetraose /L demonstrated normal growth. Glycom also cites published references stating that most infants are exposed to 2′-FL because it is one of the most common human milk oligosaccharides.
Glycom reports that it conducted a comprehensive literature search on the safety of 2′-FL published since the previous GRAS notices through November 2015 to identify new data and information relevant to the safety of 2′-FL. Glycom states that the new studies did not report any adverse effects of 2′-FL and, therefore, do not contradict the current safety conclusions.
Glycom includes the report of a panel of individuals (Glycom’s GRAS panel). Based on its review, Glycom’s GRAS panel concluded that 2′-FL is safe under the conditions of its intended use.
Based on the data and information summarized above, Glycom concludes that 2′-FL is GRAS for its intended use.
Standards of Identity
In the notice, Glycom states its intention to use 2′-FL in several food categories, including foods for which standards of identity exist, located in Title 21 of the Code of Federal Regulations. We note that an ingredient that is lawfully added to food products may be used in a standardized food only if it is permitted by the applicable standard of identity.
Potential Labeling Issues
Under section 403(a) of the Federal Food, Drug, and Cosmetic Act (FD&C Act), a food is misbranded if its labeling is false or misleading in any way. Section 403(r) of the FD&C Act lays out the statutory framework for labeling claims characterizing a nutrient level in a food or the relationship of a nutrient to a disease or health-related condition (also referred as nutrient content claims and health claims). The notice raises a potential issue under these labeling provisions. In the notice, Glycom cites studies that describe 2′-FL as having certain health benefits. If products containing 2′-FL bear any nutrient content or health claims on the label or in labeling, such claims are the subject to the applicable requirements and are under the purview of the Office of Nutrition and Food Labeling (ONFL) in the Center for Food Safety and Applied Nutrition. The Office of Food Additive Safety (OFAS) did not consult with ONFL on this issue or evaluate any information in terms of labeling claims. Questions related to food labeling should be directed to ONFL.
The FD&C Act requires that the label of a food that is or contains an ingredient that contains a “major food allergen” declare the allergen’s presence (section 403(w)). The FD&C Act defines a “major food allergen” as one of eight foods or food groups (i.e., milk, eggs, fish, Crustacean shellfish, tree nuts, peanuts, wheat, and soybeans) or a food ingredient that contains protein derived from one of those foods. 2′-FL derived from lactose may require labeling under the FD&C Act because it may contain protein derived from milk. Questions about petitions or notifications for exemptions from the food allergen labeling requirements should be directed to the Division of Biotechnology and GRAS Notice Review in OFAS. Questions related to food labeling in general should be directed to ONFL.
Intended Use in Infant Formulas
Under section 412 of the FD&C Act, a manufacturer of a new infant formula must make a submission to FDA providing required assurances about the formula at least 90 days before the formula is marketed. Our response to Glycom’s GRAS notice does not alleviate the responsibility of any infant formula manufacturer that intends to market an infant formula containing 2′-FL to make the submission required by section 412. Infant formulas are the purview of ONFL.
Section 301(ll) of the FD&C Act
Section 301(ll) of the FD&C Act prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FD&C Act, a biological product licensed under section 351 of the Public Health Service Act, or a drug or a biological product for which substantial clinical investigations have been instituted and their existence made public, unless one of the exemptions in section 301(ll)(1)-(4) applies. In our evaluation of Glycom’s notice concluding that 2′-FL is GRAS under its intended conditions of use, we did not consider whether section 301(ll) or any of its exemptions apply to foods containing 2′-FL. Accordingly, our response should not be construed to be a statement that foods containing 2′-FL, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information that Glycom provided, as well as other information available to FDA, we have no questions at this time regarding Glycom’s conclusion that 2′-FL is GRAS under its intended conditions of use. This letter is not our affirmation that 2′-FL is GRAS under 21 CFR 170.35. Unless noted above, our review did not address other provisions of the FD&C Act. Food ingredient manufacturers and food producers are responsible for ensuring that marketed products are safe and compliant with all applicable legal and regulatory requirements.
In accordance with 21 CFR 170.275(b)(2), the text of this letter responding to GRN 000650 is accessible to the public at www.fda.gov/grasnoticeinventory.
Dennis M. Keefe, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
 Serving sizes are based on Reference Amounts Customarily Consumed as described in 21 CFR 101.12.
 Glycom states that food-grade lactose is used as the main substrate of the 2¹-FL fermentation technology and that food-grade lactose may introduce milk proteins into the fermentation medium.
 2′-FL was the subject of GRN 000546, which informed FDA of the view of Glycom that 2'-FL is GRAS, through scientific procedures, for use in term infant formula at a maximum level of 2.4 g/L; and, for use in baked goods and baking mixes, beverages and beverages bases, coffee and tea, dairy product analogs, infant and toddler foods, grain products and pastas, milk (whole and skim), processed fruits and juices, processed vegetables and juices, and sugar substitutes at maximum levels ranging from 0.084 to 2.4 g/serving. FDA responded in a letter dated August 14, 2015, stating that the agency had no questions at that time regarding Glycom’s GRAS determination.
 In GRN 000546, Glycom provides an estimate of the dietary exposure to 2′-FL based on the intended uses in food for the U.S. population (persons aged 4 years and greater), and food consumption data from the 2009-2010 NHANES. Glycom estimates the mean and 90th percentile exposure to 2′-FL for the U.S. population to be 1.61 and 3.23 g/person/day (34.1 and 65.5 mg/kg bw/day), respectively.
 2′-FL was the subject of GRN 000571, which informed FDA of the view of Jennewein Biotechnologie, GmgH (Jennewein) that 2′-FL is GRAS, through scientific procedures, for use as an ingredient in nonexempt, milk-based infant formulas for term infants, and in toddler formulas at a maximum level of 2 g/L of reconstituted formula. FDA responded in a letter dated November 6, 2016, stating that the agency had no questions at that time regarding Jennewein’s GRAS determination.