Food

Agency Response Letter GRAS Notice No. GRN 000645

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CFSAN/Office of Food Additive Safety

November 1, 2016

Janet Oesterling
Novozymes North America, Inc.
77 Perry Chapel Church Road, P.O. Box 576
Franklinton, North Carolina 27525

Re: GRAS Notice No. GRN 000645

Dear Ms. Oesterling:

The Food and Drug Administration (FDA, we) completed our evaluation of GRN 000645. We received the notice, dated April 1, 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 1, 2016, and filed it on May 5, 2016. We received an amendment containing additional safety information on September 30, 2016.

FDA published the GRAS final rule on August 17, 2016 (81 FR 54960), with an effective date of October 17, 2016. As GRN 000645 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 25, 2016.

The subject of the notice is the pullulanase enzyme preparation produced by Bacillus licheniformis carrying a modified synthetic pullulanase gene from B. deramificans and B. acidopullulyticus (pullulanase enzyme preparation). The notice informs us of the view of Novozymes North America, Inc. (Novozymes) that the pullulanase enzyme preparation is GRAS through scientific procedures, for use as an enzyme in the manufacture of fermentable sugars for use in brewing and starch processing at a maximum level of 129 milligrams Total Organic Solids (mg TOS) per kilogram (kg) starch raw material.

Commercial enzyme preparations that are used in food processing typically contain an enzyme component that catalyzes a 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 pullulanase enzyme preparation.

According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, pullulanase is identified by the Enzyme Commission Number 3.2.1.41. The accepted name for the enzyme is pullulanase, and the systematic name is pullulan 6-α-glucanohydrolase. Pullulanase is also known as amylopectin 6-glucanohydrolase; bacterial debranching enzyme; debranching enzyme; α-dextrin endo-1,6-α-glucosidase; R-enzyme; and, pullulan α-1,6-glucanohydrolase.[1] The CAS Registry Number for pullulanase is 9075-68-7. Pullulanase catalyzes the hydrolysis of (1→6)-α-D-glucosidic linkages in pullulan, amylopectin, and glycogen, and in the α - and β-limit dextrins of amylopectin and glycogen.

Novozymes states that the B. licheniformis HyGe486 production strain is constructed from the host strain B. licheniformis AEB1763. Novozymes describes B. licheniformis as a non-pathogenic, non-toxigenic, well-characterized production organism with a history of safe use in the food industry. Novozymes also states that B. licheniformis AEB1763 is considered suitable for use according to Good Industrial Large Scale Practice worldwide.

Novozymes modified the host strain by deleting several genes encoding proteases, a sporulation gene, and three additional genes encoding proteins. Novozymes used an expression plasmid integrated into five specific loci in the host strain. The expression plasmid contained promoter elements from B. licheniformis, B. amyloliquefaciens, and B. thuringiensis, a modified synthetic gene based on pullulanase coding sequences from B. deramificans and B. acidopullulyticus obtained from public databases, and a B. licheniformis terminator. Novozymes states that all extraneous DNA was excised from the genome leaving only the expression cassette of the pul256 gene containing the promoter and the terminator at each target locus. Novozymes states that the sequence of the inserted expression cassettes and the flanking regions at each of the integration loci was determined in the final production strain, as was the absence of any antibiotic resistance genes. Novozymes also states that it confirmed that the transformed DNA is stably integrated via Southern hybridization analyses.

Novozymes states that the pullulanase enzyme preparation is manufactured by submerged fermentation of a pure culture prepared from a stock culture of the production strain. Novozymes states that fermentation is carried out under controlled conditions and the culture 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 primary separation step following pH adjustment and the addition of a flocculating agent. The supernatant containing the enzyme is concentrated by ultrafiltration or evaporation. The concentrated enzyme solution is then filtered to ensure the removal of any production strain. The liquid enzyme is then formulated to an enzyme preparation by the addition of sucrose as a stabilizer; potassium sorbate and sodium benzoate are added as preservatives, and water is added to standardize the enzyme preparation. Novozymes states that the entire process is performed in accordance with current good manufacturing practices using raw materials of food grade quality. Novozymes also states that the final enzyme preparation contains no major food allergens from the manufacturing process.

Novozymes has established food grade specifications and notes that the pullulanase 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 pullulanase enzyme to demonstrate consistency with the specifications.

Novozymes proposes to use the pullulanase enzyme preparation in the manufacture of fermentable sugars for use in brewing and starch processing. Novozymes states that pullulanase enzyme is largely heat inactivated during starch processing and that the majority of pullulanase enzyme would be removed during various processing steps. Novozymes also states that pullulanase enzyme, if present in the final food, will be broken down like all other proteins in the human digestive system. However, Novozymes estimates dietary exposure to pullulanase enzyme preparation based on the maximum intended use levels, and the assumption that all of the enzyme preparation will remain in the final food to be 0.34 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 toxicity assessment of the pullulanase enzyme based on a bioinformatics analysis. Novozymes reports < 20% sequence homology when comparing the sequence of pullulanase against known toxin sequences on the publicly available UNIPROT database. [2] Novozymes also summarizes corroborative unpublished toxicological studies of the pullulanase enzyme concentrate. Novozymes demonstrates that the pullulanase enzyme is not cytotoxic, mutagenic, or clastogenic based on results from an in vitro cytotoxicity assay, a bacterial reverse mutation assay, and an in vitro mouse micronucleus assay, respectively. Novozymes reports results from a 90-day oral toxicity study conducted in rats to show that consumption of pullulanase enzyme did not cause any treatment-related adverse effects at up to the highest dose tested (1000 mg TOS/kg bw/d).

Novozymes discusses the potential food allergenicity of the pullulanase enzyme. Novozymes conducted an 80-amino acid sequence homology search for pullulanase enzyme against known allergens stored in the FARRP allergen protein database, and found no sequence identity matches over 35%, to known allergens. Novozymes did not find any matches of contiguous stretches of eight amino acids in the pullulanase sequence that would be cross reactive with an allergenic protein. Novozymes further discusses the conclusions of several organizations and working groups about the low risk of allergenicity posed by enzymes due to their low use levels and the extensive processing of enzyme-containing foods during manufacturing. Based on the totality of the information available, Novozymes concludes that it is unlikely that oral consumption of pullulanase enzyme will result in allergenic responses.

Based on the data and information summarized above, Novozymes concludes that pullulanase 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’s notice concluding that pullulanase 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 pullulanase enzyme preparation. Accordingly, our response should not be construed to be a statement that foods containing pullulanase 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’s conclusion that the pullulanase enzyme preparation produced in B. licheniformis carrying a modified synthetic gene from B. deramificans and B. acidopullulyticus is GRAS under its intended conditions of use. This letter is not an affirmation that pullulanase enzyme preparation produced in B. licheniformis carrying a modified synthetic gene from B. deramificans and B. acidopullulyticus 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 170.175(b)(2), the text of this letter responding to GRN 000645 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] Pullulanase is also known as limit dextrinase (erroneous). However, while limit dextrinase catalyzes the same reaction in the same sugars, it has the Enzyme Commission Number 3.2.1.142.

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

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