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  1. Generally Recognized as Safe (GRAS)

Agency Response Letter GRAS Notice No. GRN 000592

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See also Generally Recognized as Safe (GRAS).


CFSAN/Office of Food Additive Safety

October 7, 2015

Dr. Vincent Sewalt
Danisco US Inc.
(operating as DuPont Industrial Biosciences) 925 Page Mill Road
Palo Alto, CA 94304

Re: GRAS Notice No. GRN 000592

Dear Dr. Sewalt:

The Food and Drug Administration (FDA) is responding to the notice, dated July 17, 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 July 20, 2015, filed it on August 6, 2015, and designated it as GRAS Notice No. GRN 000592.

The subject of the notice is beta-glucanase enzyme preparation produced by Bacillus subtilis expressing the gene encoding beta-glucanase from B. subtilis (beta-glucanase enzyme preparation). The notice informs FDA of the view of Danisco US Inc. (operating as DuPont Industrial Biosciences) (DuPont), that beta-glucanase from B. subtilis is GRAS, through scientific procedures, for use as an enzyme in the production of beer and potable alcohol at levels up to 37 milligrams total organic solids per kilogram (mg TOS/kg) of grist.

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 from the manufacturing process, e.g., constituents of the fermentation media or the residues of processing aids. DuPont’s notice provides information about the components in the beta-glucanase enzyme preparation.

According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, beta-glucanase is identified by the Enzyme Commission Number 3.2.1.6. Beta- glucanase catalyzes the endohydrolysis of (1---3)- or (1---4)-linkages in beta-D-glucans when the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is itself substituted at C-3. The systematic name for this enzyme is 1,3-(1→3;1→4)-β-D-glucan3(4)-glucanohydrolase. Other names include endo-1,3-β-D-glucanase; laminarinase; laminaranase; β-1,3-glucanase; β-1,3-1,4-glucanase; endo-1,3-β-glucanase; endo-β-1,3(4)-glucanase; endo-β-1,3-1,4-glucanase; endo-β-(1→3)-D-glucanase; endo- 1,3-1,4-β-D-glucanase; endo-β-(1-3)-D-glucanase; endo-β-1,3-glucanase IV; endo-1,3-β-D-glucanase; 1,3-(1,3;1,4)-β-D-glucan 3(4)-glucanohydrolase. The CAS Registry Number for beta-glucanase is 62213-14-3. DuPont states that the primary sequence of beta-glucanase consists of 214 amino acids.

DuPont states that the production strain, CF 624B-1, is obtained from the recipient strain B. subtilis BG 3934 via a series of modifications that included classical mutagenesis, rDNA and protein engineering.1 DuPont describes B. subtilis as a ubiquitous, gram positive, spore-forming bacterium that is non- pathogenic and non-toxigenic with a history of safe use in the production of industrial enzymes. DuPont confirms that the production strain is stable for at least two years, does not produce any antibiotics, or contain any antibiotic resistance genes.2

DuPont describes the construction of the production strain to introduce multiple copies of the beta- glucanase gene encoding the wild type B. subtilis beta-glucanase, under the regulation of an endogenous aprE promoter, B. amyloliquefaciens apr terminator, and the chloramphenicol resistance marker gene. DuPont verified the copy number of the integrated beta-glucanase cassettes and the absence of bacterial vector DNA by Southern blot analysis.

DuPont states that the beta-glucanase enzyme is produced by submerged fermentation of a pure culture of the production strain. The fermentation is carried out under controlled conditions and the culture is periodically tested until the desired enzyme production is achieved. After fermentation, the enzyme is recovered from the culture broth by a series of filtration or centrifugation steps, followed by a concentration step. The resulting concentrate containing the enzyme is formulated by the addition of glycerol, sodium citrate, citric acid, sodium benzoate, and potassium sorbate. DuPont states that the entire process is performed in accordance with current Good Manufacturing Practices using raw materials of food grade quality. DuPont also states that the final enzyme preparation contains no major food allergens from the fermentation medium.

DuPont has established food grade specifications and notes that the beta-glucanase enzyme preparation conforms to specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 9th edition, 2014) and 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).

DuPont confirms the absence of the production organism in the final enzyme preparation, as established by the set specifications. DuPont also provides analytical data from three batches of beta-glucanase enzyme concentrate to demonstrate consistency with the set specifications.

DuPont intends to use beta-glucanase enzyme preparation in the production of beer and potable alcohol to hydrolyze beta-glucans and thereby reduce wort viscosity and improve mashing efficiency. The maximum intended use level of beta-glucanase enzyme preparation is 37 mg TOS/kg of grist. DuPont estimates the dietary exposure to beta-glucanase enzyme preparation based on the maximum intended use levels and an assumption that all of the enzyme preparation will remain in the final food to be 156 micrograms TOS per kg body weight per day (μg TOS/kg bw/d) from all intended uses. DuPont states that the reaction products resulting from beta-glucanase enzyme activity are already part of the human diet.

DuPont summarizes corroborative toxicological studies using beta-glucanase enzyme liquid concentrate to support the safety of the enzyme for the intended uses. Tests conducted using bacterial cells showed that the beta-glucanase is not mutagenic at the highest dose tested both in the presence and absence of metabolic activation. DuPont also demonstrates that the enzyme is not clastogenic based on results from in vitro chromosomal aberration tests. A 90-day acute oral toxicity study conducted using rats showed that consumption of beta-glucanase enzyme did not cause any treatment-related adverse effects up to the highest dose tested, i.e., 1000 mg TOS/kg bw/d. Based on the highest dose tested in the 90-day study, and the estimated dietary exposure from the intended use of beta-glucanase enzyme preparation, DuPont calculates the margin of safety to be approximately 6400. DuPont states that based on the results of these safety studies and other information in published literature, the beta-glucanase enzyme preparation is considered safe for human consumption.

DuPont examines the potential allergenicity of beta-glucanase enzyme by conducting an amino acid sequence homology search for beta-glucanase against known allergens stored in Food Allergy Research and Resource Program’s FARRP AllergenOnline database for sequence identity matches over 35%.

DuPont reported search results that revealed multiple stretches throughout the peptide sequence with 40.7% identity to Asp f 9 (also called probable glycosidase crf 1) from Aspergillus fumingatus. DuPont states that Asp f 9 has not been identified as a food allergen by the World Health organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Sub-committee. Also, DuPont did not find any matches of contiguous stretches of eight or more amino acids in the beta- glucanase sequence that would be cross reactive with an allergen protein. DuPont 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, DuPont concludes that it is unlikely that oral consumption of beta-glucanase enzyme will result in allergenic responses.

Based on the data and information summarized above, DuPont concludes that beta-glucanase enzyme preparation is GRAS for its intended use.

Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FD&C Act)

The Food and Drug Administration Amendments Act of 2007 which was signed into law on September 27, 2007, amends the FD&C Act to, among other things, add section 301(ll). 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 DuPont’s notice that beta-glucanase enzyme preparation is GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing beta-glucanase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain beta-glucanase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).

Conclusions

Based on the information provided by DuPont, as well as the information in GRAS No. GRN 000592, and other information available to FDA, the agency has no questions at this time regarding DuPont’s conclusion that beta-glucanase enzyme preparation from Bacillus subtilis 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 beta-glucanase from B. subtilis. As always, it is the continuing responsibility of DuPont 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 000592, 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.

Sincerely,

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


(1) B. subtilis strain 1A10 was developed into the recipient strain B. subtilis BG3934 after the deletion of several genes. B. subtilis 1A10 was obtained from the wild type strain B. subtilis 168.

(2) DuPont states that the endogenous chloramphenicol resistance marker would not be readily transferable to other species once integrated into the genome.