Food

Agency Response Letter GRAS Notice No. GRN 000659

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CFSAN/Office of Food Additive Safety

Christoph H. Rohrig, Ph.D.
Glycom A/S
Diplomvej 373
DK-2800 Kgs. Lyngby
Denmark

Re: GRAS Notice No. GRN 000659

Dear Dr. Rohrig:

The Food and Drug Administration (FDA, we) completed our evaluation of GRN 000659. We received the notice, dated July 5, 2016, that you submitted in accordance with the agency’s proposed regulation, proposed 21 Code of Federal Regulations (CFR) 170.36 (62 FR 18938; April 17, 1997; Substances Generally Recognized as Safe (GRAS); the GRAS proposal) on July 12, 2016, and filed it on August 10, 2016. We received an amendment containing additional safety information on September 27, 2016.

FDA published the GRAS final rule on August 17, 2016 (81 FR 54960), with an effective date of October 17, 2016. As GRN 000659 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 lacto-N-neotetraose (LNnT). The notice informs us of Glycom A/S’ (Glycom) view that LNnT is GRAS, through scientific procedures, for use as an ingredient in nonexempt infant formulas for term infants at a maximum use level of 600 milligrams (mg)/liter (L) and in beverages and beverage bases, dairy product analogs, milk (whole and skim), milk products, processed fruits and juices, grain products and pastas, infant foods, and toddler foods (including follow-on formulas) at use levels ranging from 0.02 to 3 grams (g)/serving.

Glycom provides information on the identity and composition of LNnT. Glycom describes LNnT as a white to off-white powder containing ≥ 92% LNnT. Glycom reports that the final LNnT product may also contain other carbohydrates, including lactose, lactose-N-triose II, and para-lacto-N-neohexaose. LNnT is a tetrasaccharide composed of D-galactose, N-acetyl-D-glucosamine, D-galactose, and D-glucose. The chemical name is β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→3)-β-D-galactopyranosyl-(1→4)-D-glucopyranose. Glycom concludes that the LNnT described in this notice is chemically and structurally identical to the LNnT present in human milk.

Glycom describes the two-stage manufacturing process for LNnT. The first stage involves the production of LNnT by fermentation usingthe production strain, which secretes LNnT into the fermentation broth. The production strain Escherichia coli K-12 MP572[1] is transformed with two exogenous genes[2] that are well characterized and not associated with pathogenicity or toxicity.

After fermentation is complete, the microbial biomass is removed by filtration. The second stage includes several purification steps. The filtration permeate from the first stage is concentrated by filtration and treated with ion exchange resin to remove water, minerals, and other small molecules. The solution is subsequently decolored with activated charcoal, filtered, and pH maintained with potassium or sodium hydroxide. The solution is concentrated by distillation or filtration and subjected to chromatography to remove carbohydrate impurities. The resulting LNnT solution is concentrated by vacuum distillation or filtration. Methanol and LNnT seed crystals are added under controlled conditions to initiate crystallization of LNnT. LNnT crystals are separated, washed with methanol, and dried to obtain the final LNnT product. Glycom states that all processing aids used in the manufacture of LNnT are food-grade and used in accordance with U.S. regulations or are GRAS for their respective uses. Glycom states that there are no allergenic proteins in the final product.

Glycom provides specifications for LNnT that include minimum levels of LNnT (≥ 92% on a dry matter basis (DM)) and total oligosaccharides[3] (≥ 95% DM), and limits on D-lactose (≤ 3%), lacto-N-triose II (≤ 3%), para-lacto-N-neohexaose (≤ 3%), LNnT fructose isomer (≤ 1%), moisture (≤ 9%), lead (≤ 0.1 mg/kilogram (kg)), residual proteins (≤ 0.01%), methanol (≤ 100 mg/kg), and microbial contaminants, including Cronobacter sakazakii (absent in 10 g). Glycom provides the results of four nonconsecutive batch analyses of LNnT to demonstrate that its product meets specifications. Glycom incorporates by reference the results described in GRN 000547[4] for a 5-year stability study conducted under ambient conditions and a 2-year stability study conducted under accelerated conditions (increased temperature and humidity) and concludes that LNnT is stable under the conditions tested.

Glycom estimates the dietary exposure to LNnT based on the intended uses and food consumption and body weight data from the U.S. National Center for Health Statistics’ 2011-2012 National Health and Nutrition Examination Surveys (NHANES). Glycom also discusses the estimates of dietary exposure to LNnT presented in GRN 000547 for infants and toddlers based on the maximum intended use level of 600 mg/L in nonexempt term infant formulas and follow-on formulas and consumption and body weight data from the 2009-2010 NHANES. Glycom reports the mean and 90th percentile exposures to LNnT from infant formulas for infants 0 to 6 months of age to be 0.51 and 0.73 g/person (p)/day (d) (83.2 and 133.9 mg/kg body weight (bw)/d), respectively. The mean and 90th percentile exposures to LNnT from infant formulas for infants 7 to 12 months of age are reported to be 0.42 and 0.66 g/p/d (48.5 and 79.5 mg/kg bw/d), respectively. The mean and 90th percentile exposures to LNnT from follow-on formulas in toddlers 1 to 3 years of age are reported to be 1.08 and 1.41 g/p/d (89.3 and 117.1 mg/kg bw/d), respectively. The mean and 90th percentile exposures from other toddler foods are reported to be 0.5 and 0.9 g/p/d (38.4 and 67.7 mg/kg bw/d), respectively. The mean and 90th percentile dietary exposures to LNnT for the total population are reported to be 0.3 and 0.6 g/p/d (8.1 and 16.8 mg/kg bw/d), respectively.

Glycom incorporates by reference all published information included in GRN 000547 and discusses pivotal published data cited therein for its safety assessment. Glycom also discusses publicly-available information, including information published since our evaluation of GRN 000547 through March 2016. Glycom states that LNnT synthesized chemically (GRN 000547) or enzymatically (GRN 000659) is identical to the LNnT that is a component in human milk. Glycom notes that the exposure to LNnT from its intended uses, including infant formula use, is not expected to present safety concerns because of the safe consumption of human milk. Glycom states that all available data suggest that the majority of LNnT reaches the large intestine undigested, and serves as a substrate for gut microflora or is excreted intact in the feces. Glycom states that LNnT is neither mutagenic nor genotoxic, and published subchronic repeat dose studies in rats showed no toxicologically-relevant adverse effects at the highest dose tested (5000 mg/kg bw/d). Glycom also cites a published study showing that LNnT produced using a yeast fermentation method was well-tolerated and showed no adverse effects on growth in infants 6-24 months of age at a use level of 200 mg/L. In addition, Glycom provides a detailed discussion of the unpublished studies conducted with LNnT, including LNnT produced from bacterial fermentation, to support its safety conclusion.

Glycom includes the report of a panel of individuals (Glycom’s GRAS panel). Based on its review, Glycom’s GRAS panel concluded that LNnT is safe under the conditions of its intended use.

Based on the totality of information discussed above, Glycom concludes that LNnT is GRAS for its intended use.

Standards of Identity

In the notice, Glycom states its intention to use LNnT in several food categories, including foods for which standards of identity exist, located in Title 21 of the CFR. 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 (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 to 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 LNnT as having certain health benefits. If products containing LNnT 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 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.

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 LNnT 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 LNnT 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 LNnT. Accordingly, our response should not be construed to be a statement that foods containing LNnT, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).

Conclusions

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 LNnTis GRAS under its intended conditions of use. This letter is not an affirmation that LNnTis 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 170.175(b)(2), the text of this letter responding to GRN 000659 is accessible to the public at www.fda.gov/grasnoticeinventory.

Sincerely,

Dennis M. Keefe, Ph.D.
Director
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition


[1] We extensively reviewed and determined that E. coli K-12 is a non-pathogenic, non-toxigenic, and safe production strain when used in accordance with good manufacturing practices (55 FR 10932 at 10934; March 23, 1990). E. coli K-12 MDO (the parent strain of E. coli K-12 MP572) is derived from E. coli K-12 DH1, a nalidixic acid-resistant strain altered by seven genetic modifications.

[2] These two genes encode the final two enzymes in the biosynthetic pathway for LNnT: β-1,3-N-acetylglucosaminyltransferase from Neisseria meningitidis and β-1,4-galactosyltransferase from Helicobacter pylori.

[3] Glycom states that the specified minimum of total oligosaccharides includes the total of LNnT, lactose, lacto-N-triose II, and para-lacto-N-hexaose.

[4] GRN 000547 describes the use of LNnT as an ingredient 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 0.02 to 1.2 g per serving and for use as an ingredient in term infant formulas at a maximum level of 600 mg/L of reconstituted formula. We evaluated this notice and responded in a letter dated October 2, 2015, stating that we had no questions at that time regarding Glycom’s GRAS determination.

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