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Agency Response Letter GRAS Notice No. GRN 000657

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CFSAN/Office of Food Additive Safety

November 23, 2016

Janet Oesterling
Novozymes North America, Inc.
77 Perry Chapel Church Road
Franklinton, NC 27525

Re: GRAS Notice No. GRN 000657

Dear Ms. Oesterling:

The Food and Drug Administration (FDA, we) completed our evaluation of GRN 000657. We received the notice, dated June 3, 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 June 3, 2016, and filed it on July 26, 2016. We received an amendment containing clarifying information on October 24, 2016.

FDA published the GRAS final rule on August 17, 2016 (81 FR 54960), with an effective date of October 17, 2016. As GRN 000657 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 24, 2016.

The subject of the notice is glucoamylase produced in Aspergillus niger carrying a modified glucoamylase gene from Penicillium oxalicum (glucoamylase enzyme preparation). The notice informs FDA of the view of Novozymes North America, Inc. (Novozymes) that glucoamylase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the processing of starch and hydrolysis products of starch at up to a level of 2223 milligram Total Organic Solids per kilogram (mg TOS/kg) starch raw material.

Commercial enzyme preparations that are used in food processing typically contain an enzyme component that catalyzes the chemical reaction as well as substances used as stabilizers, preservatives, or diluents. Enzyme preparations may also contain components derived from the production organism and components derived from the manufacturing process, e.g., constituents of the fermentation media or the residues of processing aids. Novozymes’ notice provides information about each of these components in the glucoamylase enzyme preparation.

According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, glucoamylase is identified by the Enzyme Commission Number 3.2.1.3. The accepted name for the enzyme is glucan 1,4-a-glucosidase; and the systematic name is 4-α-D-glucan glucohydrolase. This enzyme is also known as glucoamylase; amyloglucosidase; y-amylase; lysosomal α- glucosidase; acid maltase; exo-1,4-α-glucosidase; glucose amylase; γ-1,4-glucan glucohydrolase; acid maltase; 1,4-α-D-glucan glucohydrolase. The CAS Registry Number for glucoamylase is 9032-08-0. Glucoamylase catalyzes the hydrolysis of terminal (1---4)-linked α-D-glucose residues successively from non-reducing ends of the chains with release of β-D-glucose.

Novozymes states that the A. niger production strain was constructed from the recipient A. niger M1371.[1] Novozymes describes A. niger as a non-pathogenic, non-toxigenic, well-characterized production organism with a history of safe use in the food industry. Novozymes also states that the production strain is considered suitable for Good Industrial Large Scale Practice worldwide.

Novozymes modified the recipient strain at several chromosomal loci to inactivate genes encoding for several amylases and proteases and deleted several genes including those encoding fumonisin and oxaloacetate hydrolase. Novozymes used an expression plasmid containing an A. niger promoter, a modified glucoamylase gene from P. oxalicum,[2] an A. niger terminator, and a selectable marker. Novozymes states that the DNA sequences of the inserted expression cassettes and the flanking regions at each of the integration loci were verified in the final production strain. Novozymes also states that it confirmed the absence of any antibiotic resistance genes as well as confirming via Southern hybridization analyses that the transformed DNA is stably integrated.

Novozymes states that the glucoamylase enzyme preparation is manufactured by submerged fermentation of a pure culture of the production strain.Novozymes states that fermentation is carried out under controlled conditions, and that glucoamylase is secreted into the culture medium. Novozymes also states that each batch of fermentation is periodically tested to ensure production strain identity and purity, and enzyme-generating ability. After fermentation, the enzyme is recovered from the culture broth by a pretreatment step that involves pH adjustment and addition of a flocculating agent. This is followed by vacuum filtration or centrifugation of the supernatant containing the enzyme, which is then concentrated by ultrafiltration or evaporation. The concentrated enzyme solution is then filtered to ensure the removal of any production strain. The enzyme concentrate is standardized by the addition of sodium chloride and formulated to an enzyme preparation with dextrin or water, for solid or liquid preparations, respectively. Novozymes states that the entire process is performed in accordance with current good manufacturing practices using food grade raw materials. Novozymes also states that the final enzyme preparation contains no major food allergens from the fermentation medium.

Novozymes states that the glucoamylase enzyme preparation conforms to specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 9th edition, 2014), and to the General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives (JECFA, 2006). Novozymes provides analytical data from three batches of glucoamylase enzyme to demonstrate consistency with the specifications.

Novozymes intends to use glucoamylase enzyme preparation as an enzyme in the processing of starch for baking, brewing, and other cereal-based products at up to 2223 mg TOS/kg starch raw material. Novozymes states that glucoamylase enzyme is expected to be inactivated or removed during the production of the food categories intended for use. Novozymes also states that glucoamylase, if present in the final food, will be susceptible to breakdown in the human digestive system like other proteins. However, Novozymes estimates maximum daily intake to glucoamylase enzyme preparations based on the highest intended use levels and the assumption that all of the enzyme preparation will remain in the final food, to be 12.15 mg TOS/kg bodyweight per day (mg TOS/kg bw/d).

Novozymes relies on published information for the safety of microbial enzyme preparations used in food processing, and reports a toxicity assessment of the glucoamylase enzyme based on bioinformatics analysis. Novozymes reports the absence of sequence homology between glucoamylase and known toxins on the publicly available database, UNIPROT.[3] Novozymes also summarizes unpublished toxicological studies using the glucoamylase enzyme concentrate. Tests conducted using the glucose enzyme concentrates with bacterial cells showed that the glucoamylase is not mutagenic at the highest dose tested both in the presence and absence of metabolic activation. Novozymes demonstrates that the glucoamylase enzyme is neither clastogenic nor aneugenic based on results from in vitro chromosomal aberration tests. Novozymes reports results of a 90-day oral toxicity study conducted using rats showing that consumption of glucoamylase enzyme concentrate did not cause any treatment-related adverse effects at 10 milliliter/kg bw/d, the highest dose tested, which corresponds to 1360 mg TOS/kg bw/d of the glucoamylase enzyme. Based on the highest dose tested in the 90-day study, and the estimated maximum daily intake from the proposed use levels of glucoamylase enzyme preparation, i.e., 1360 mg TOS/kg bw/d and 12.15 mg TOS/kg bw/d, respectively, Novozymes calculates the margin of safety to be approximately 112.

Novozymes discusses the potential for food allergenicity of glucoamylase enzyme. Novozymes conducted an 80-amino acid sequence homology search for glucoamylase against known allergens stored in the FARRP allergen protein database, and the WHO/IUIS Allergen Nomenclature Sub-committee. Novozymes summarizes that the modified glucoamylase sequence had > 35% identity to the sequence of a respiratory allergen, 1,4-alpha-D-glucan glucohydrolase from Sch c 1 from Schizophyllum commune. Novozymes relies on published studies that demonstrate gluco-amylases from fungal sources have been reported to trigger occupational respiratory sensitization, but with no resulting oral allergenic responses. Novozymes also performed amino acid sequence identity for matches of contiguous stretches of eight or more amino acids in the modified glucoamylase sequence that would be cross reactive with an allergen protein, and did not report any match. Novozymes further cites the conclusions of several organizations and working groups about the low risk of allergenicity posed by enzymes due to their low use levels and extensive processing of the enzyme-containing foods during manufacturing. Based on the totality of information available, Novozymes concludes that it is unlikely that oral consumption of glucoamylase enzyme will result in allergenic responses.

Based on the data and information summarized above, Novozymes concludes that glucoamylase enzyme preparation is GRAS for its intended use.

Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (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 Novozymes’ notice concluding that glucoamylase enzyme preparation 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 glucoamylase enzyme preparation. Accordingly, our response should not be construed to be a statement that foods containing glucoamylase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).

Conclusions

Based on the information that Novozymes provided, as well as other information available to FDA, we have no questions at this time regarding Novozymes’ conclusion that glucoamylase produced in A.nigercarrying a modified glucoamylase gene from P. oxalicum is GRAS under its intended conditions of use. This letter is not an affirmation that glucoamylase produced in A.niger carrying a modified glucoamylase gene from P. oxalicum 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 000657 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] A. niger M1371 is a natural isolate of A. niger strain BO-1.

[2] Novozymes states that the glucoamylase gene from P. oxalicum varies from its wild-type sequence by one amino acid residue.

[3] The UNIPROT database contains entries from SWISSPROT and TREMBL PIR-PSD databases.

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