Agency Response Letter GRAS Notice No. GRN 000602
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See also Generally Recognized as Safe (GRAS).
CFSAN/Office of Food Additive Safety
February 1, 2016
Christoph H. Rohrig, Ph.D.
DK-2800 Kgs. Lyngby
Re: GRAS Notice No. GRN 000602
Dear Dr. Rohrig:
The Food and Drug Administration (FDA) is responding to the notice, dated September 24, 2015, 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). FDA received the notice on September 30, 2015, filed it on October 13, 2015, and designated it as GRAS Notice No. GRN 000602.
The subject of the notice is N-acetyl-D-neuraminic acid (NANA) dihydrate. The notice informs FDA of the view of Glycom A/S (Glycom) that NANA dihydrate is GRAS, through scientific procedures, for use as an ingredient in non-exempt infant formulas for term infants. Glycom intends to use NANA dihydrate at a maximum level of 50 milligrams per liter (mg/L) of reconstituted formula. Glycom also intends to use NANA dihydrate in baked goods and baking mixes, beverages and beverage bases, coffee and tea, dairy product analogs, infant and toddler foods, grain products and pastas, milk (whole and skim), milk products, processed fruits and fruit juices, processed vegetables and vegetable juices, and sugar substitutes at maximum levels ranging from 0.4 to 100 mg/serving.
As part of its notice, Glycom includes the report of a panel of individuals (Glycom’s GRAS panel) that evaluated the data and information that are the basis for Glycom’s GRAS determination. Glycom considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Glycom’s GRAS panel evaluated information describing the identity and composition, manufacturing process and specifications, and estimated dietary exposure to NANA dihydrate, as well as published studies supporting the safety of NANA dihydrate. Based on this review, Glycom’s GRAS panel concludes that NANA dihydrate produced in accordance with good manufacturing practices is GRAS under the conditions of its intended use.
Glycom describes NANA dihydrate as a white to off-white, crystalline powder that contains a minimum of 97% N-acetyl-D-neuraminic acid dihydrate. The chemical name for NANA is 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-non-2-ulosonic acid and the CAS registry number is 131-48-6. Glycom notes that NANA predominantly occurs in the dihydrate form (CAS registry number 50795-27-2). Glycom discusses sialic acids, which are a family of nine-carbon acidic monosaccharides that consist of nitrogen- or oxygen-substituted derivatives of D-neuraminic acid that are found in mammalian milk and brain tissue.
Glycom provides a description of the manufacturing process for NANA dihydrate. The first stage of the process involves the coupling of N-acetylmannosamine and sodium pyruvate. This is achieved through an aldolase enzyme-catalyzed aldol condensation reaction that produces NANA in solution. The product of the enzymatic reaction is subjected to a series of processing steps, including washing with acetone, to remove the enzyme and other impurities (e.g. water and acetic acid), resulting in crude NANA. Residual water is removed from the crude NANA, which is subsequently filtered to remove lipophilic impurities. In addition, Glycom notes that re-crystallization may be used to further purify the anhydrous NANA.
In the second stage of the manufacturing process, the anhydrous NANA is dissolved in water and is treated with activated charcoal to remove any coloration and impurities. The mixture is filtered to obtain a NANA solution that is then microfiltered to remove potential bacterial contaminants. Crystallization of NANA dihydrate is initiated by adding 2-propanol to the solution. The crystals are isolated by centrifugation and then washed with 2-propanol. The NANA dihydrate crystals are dried and then packaged. Glycom states that all materials used in the manufacture of NANA dihydrate are of food grade quality.
Glycom provides specifications for NANA dihydrate. Specifications include the minimum levels of NANA dihydrat (≥ 97 % on a dry matter basis (DM)) and limits on water (≤ 12.5 %), acetic acid and/or sodium acetate (≤ 0.5%), residual solvents (≤ 0.1 % for individual solvents and ≤ 0.3% for a combination of solvents), lead (≤ 0.1 mg per kilogram (mg/kg)), residual proteins (≤ 0.01 %), and no detectable Cronobacter sakazakii in a 10 gram sample. Glycom provides the results of five batch analyses of NANA dihydrate to demonstrate that their product meets specifications.
Glycom discusses stability studies conducted with NANA dihydrate. Glycom concludes that the shelf-life of NANA dihydrate is 36 months. Glycom notes that an analysis revealed three unidentified carbohydrate degradation products that were detected after 36 months of storage, however, NANA dihydrate remained within the specifications described above and data reveals that after 48 months of storage only one of the three degradation products remains detectable (approximately 0.1%). In addition, Glycom states that NANA dihydrate is stable when stored under accelerated conditions (40°C and 75% relative humidity) for periods of up to 24 months. Glycom discusses stability studies conducted with NANA dihydrate in powdered infant formulas and other food applications and concludes that NANA dihydrate is stable under the intended conditions of use.
Glycom calculates the dietary exposure to NANA dihydrate based on a maximum use level of 50 mg/L of NANA dihydrate in term infant formulas and the intended use levels in various food categories using food consumption data from the 2009-2010 National Health and Nutrition Examination Surveys (NHANES) (CDC, 2011; USDA, 2012). The estimated mean and 90th percentile exposures for infants aged 0 to 6 months are 46.5 and 67.1 mg/person/day (mg/p/d) (7.3 and 11.8 mg/kg body weight (bw)/d), respectively. For infants aged 7 to 11 months, the estimated mean and 90th percentile exposures to NANA dihydrate are 55.5 and 79.2 mg/p/d (6.0 and 9.2 mg/kg bw/d), respectively. For toddlers aged 1 to 3 years, the estimated mean and 90th percentile exposures to NANA dihydrate are 52.3 and 87.9 mg/p/d (4.0 and 6.6 mg/kg bw/d), respectively. For individuals in the U.S. population aged 4 years and older, the estimated mean and 90th percentile exposures to NANA dihydrate are 65.3 and 130.9 mg/p/d (1.2 and 2.6 mg/kg bw/d), respectively.
Glycom discusses publicly available information as well as published studies supporting the safety of NANA. Glycom discusses the chemical and physical properties of NANA, as well as the fact that it is found ubiquitously throughout the human body and is present in human breast milk. Glycom reviews published metabolic studies which show that ingested NANA is eliminated unchanged in urine or is metabolized and utilized as a carbon source for cell metabolism, ultimately leading to expiration as carbon dioxide. Glycom evaluates published animal studies, including a published 13-week oral toxicity study in rats with an in utero exposure phase, to evaluate the effects of NANA on female reproduction and on general growth and development. Glycom states that there were no compound-related adverse effects at the highest levels tested (974 mg/kg bw/d and 1246 mg/kg bw/d, in males and females, respectively). In addition, Glycom assesses published genetic toxicology studies, including a bacterial reverse mutation assay and an in vitro mammalian cell micronucleus test, and concludes that NANA is not mutagenic or genotoxic.
Based on the totality of the data and information described in the notice, Glycom concludes that NANA dihydrate is GRAS for its intended uses in food.
Standards of Identity
In the notice, Glycom states its intention to use NANA dihydrate in several food categories, including foods for which standards of identity exist, located in Title 21 of the Code of Federal Regulations. FDA notes 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
In describing the intended uses of NANA dihydrate and in describing the information that Glycom relies on to conclude that NANA dihydrate is GRAS under the conditions of its intended use, Glycom raises a potential issue under the labeling provisions of the Federal Food, Drug, and Cosmetic Act (FD&C Act). Under section 403(a) of the FD&C Act, a food is misbranded if its labeling is false or misleading in any particular. Section 403(r) of the FD&C Act lays out the statutory framework for the use of labeling claims that characterize the level of a nutrient in a food or that characterize the relationship of a nutrient to a disease or health-related condition. If products that contain NANA dihydrate bear any claims on the label or in labeling, such claims are 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 neither consulted with ONFL on this labeling issue nor evaluated the information in your notice to determine whether it would support any claims made about NANA dihydrate on the label or in labeling.
Intended Use in Infant Formula
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. FDA’s response to Glycom’s GRAS notice does not alleviate the responsibility of any infant formula manufacturer who intends to market an infant formula that contains NANA dihydrate to make the submission required by section 412.
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 its review of Glycom’s notice that NANA dihydrate is GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing NANA dihydrate. Accordingly, this response should not be construed to be a statement that foods that contain NANA dihydrate, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Glycom, as well as other information available to FDA, the agency has no questions at this time regarding Glycom’s conclusion that NANA dihydrate is GRAS under the intended conditions of use. The agency has not, however, made its own determination regarding the GRAS status of the subject use of NANA dihydrate. As always, it is the continuing responsibility of Glycom to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.
In accordance with proposed 21 CFR 170.36(f), a copy of the text of this letter responding to GRN 000602, as well as a copy of the information in this notice that conforms to the information in the GRAS exemption claim (proposed 21 CFR 170.36(c)(1)), is available for public review and copying at www.fda.gov/grasnoticeinventory.
Dennis M. Keefe, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
 In this letter, FDA uses the terms NANA and NANA dihydrate to refer to N-acetyl-D-neuraminic acid and its dihydrate form, respectively. The method of manufacture described in GRN 000602 results in a crystalline product that binds two molecules of water per molecule of NANA (i.e., NANA dihydrate).
 Glycom notes that the aldolase enzyme preparation (i.e. N-acetylneuraminate lyase; CAS: 9027-60-5; EC: 126.96.36.199) is obtained from a modified strain of Escherichia coli K12 and meets the general purity specifications for enzyme preparations as described in the Food Chemicals Codex (FCC, 2014).
 Glycom states that degradation products were found at a maximum level of 0.4% for individual products and at a maximum level of 1.0% for the total of all degradation products.
 Glycom concludes that the two other degradation products found were either artifacts or transient intermediates to a final degradation product. Glycom notes that the remaining impurity increases over stressed temperature conditions, and based on additional analytical data, Glycom concludes that it is likely formed by the elimination of water from NANA. Glycom states that the level of this impurity (~ 0.1 %) at the intended use level of NANA dehydrate in reconstituted infant formula would result in dietary exposures below 8 micrograms/kg body weight/day.