Agency Response Letter GRAS Notice No. GRN 000274
CFSAN/Office of Food Additive Safety
June 25, 2009
Novozymes North America, Incorporated
77 Perry Chapel Church Road
P.O. Box 576
Franklinton, NC 27525
Re: GRAS Notice No. GRN 000274
Dear Mr. Carroll:
The Food and Drug Administration (FDA) is responding to the notice, dated December 22, 2008, 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 December 30, 2008, filed it on December 30, 2008, and designated it as GRAS Notice No. GRN 000274.
The subject of the notice is a branching glycosyltransferase enzyme preparation from Bacillus subtilis expressing a branching glycosyltransferase gene from Rhodothermus obamensis (branching glycosyltransferase enzyme preparation). The notice informs FDA of the view of Novozymes North America, Incorporated (Novozymes), that the branching glycosyltransferase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the starch industry to obtain dextrins with improved physical properties, such as higher solubility, lower viscosity, and reduced retrogradation.
Commercial enzyme preparations that are used in food typically contain an enzyme component, which catalyzes the chemical reaction that is responsible for its technical effect, as well as substances used as stabilizers, preservatives, or diluents. Enzyme preparations may also contain constituents derived from the production organism and manufacturing process. In its notice, Novozymes provides information about all the components of the branching glycosyltransferase enzyme preparation.
Branching glycosyltransferase catalyzes the transfer of a segment of a 1,4-alpha-D-glucan chain to a primary hydroxy group in a similar glucan chain to create 1,6-alpha linkages and increase the number of branched points. According to the classification of enzymes established by the International Union of Biochemistry and Molecular Biology, branching glycosyltransferase is identified by the Enzyme Commission number 18.104.22.168. Its accepted name is 1,4-alpha-glucan branching enzyme and its systematic name is 1,4-alpha-D-glucan:1,4-alpha-D-glucan 6 alpha-D-(1,4-alpha-D-glucano)-transferase. The Chemical Abstract Service Registry number for branching glycosyltransferase is 9001-97-2.
Novozymes states that B. subtilis is nonpathogenic and nontoxigenic, meets the criteria for a safe production microorganism described by several expert groups, and is widely used by enzyme manufacturers for the production of enzymes for use in human food. Novozymes describes the host microorganism, B. subtilis strain JA1343, as derived from the parent strain B. subtilis strain 168. Novozymes states that B. subtilis strain 168 is well characterized and has been used to develop production strains expressing enzymes intended for use in food, such as alpha-acetolactate decarboxylase (21 CFR 173.115) and maltogenic amylase (GRASP 7G0326). To construct the B. subtilis JA1343 host strain, Novozymes deleted several genes encoding enzyme activities (proteases and amylase) and a sporulation gene. To construct the production strain, B. subtilis JA1343 was transformed with a plasmid vector carrying an expression cassette with the synthetic branching glycosyltransferase gene under the control of a promoter and terminator derived from several Bacillus species, as well as a chloramphenicol resistance (cat) gene. Novozymes states that the cat gene was used as a selectable marker and was deleted after integration to make the production strain marker free. Novozymes states that the stability of the branching glycosyltransferase gene introduced into the production strain was tested by Southern blot analysis.
Novozymes describes the manufacture of the branching glycosyltransferase enzyme using submerged fed-batch pure culture fermentation of the genetically modified strain of B. subtilis. Each production batch is initiated from a lyophilized stock culture, which is tested for identity, absence of foreign microorganisms, and enzyme generating ability before use. All fermentation steps are conducted under controlled conditions and monitored for microbial contamination. The branching glycosyltransferase enzyme is secreted to the fermentation broth and is subsequently recovered via several purification and concentration steps. The resulting liquid concentrate is stabilized with sorbitol and glycerol and tested to demonstrate that it meets the intended specifications. The concentrate is mixed with water and methionine and sold as a liquid formulation. The total organic solids (TOS) content of the final branching glycosyltransferase enzyme preparation is approximately 4%.
Novozymes states that branching glycosyltransferase enzyme preparation conforms to the general and additional requirements for enzyme preparations as described in the Food Chemicals Codex (6th edition) and the current (2006) General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the Joint FAO/WHO Expert Committee on Food Additives.
Novozymes states that the branching glycosyltransferase is intended for use in the starch industry to obtain dextrins with improved physical properties, such as higher solubility, lower viscosity, and reduced retrogradation. The recommended use levels range from 0.4 to 40 kilograms (kg) per ton of starch dry substance. The enzyme is expected to be inactivated and removed during starch processing. Novozymes estimates a maximum daily intake of the branching glycosyltransferase enzyme to be 1.8 mg TOS per kg of body weight per day (mg TOS/kg bw/d) under the assumption that 100% of the enzyme would remain in the final foods.
Novozymes summarizes unpublished toxicological studies conducted with the branching glycosyltransferase enzyme which include a subchronic (13-week) oral toxicity study in rats, a bacterial reverse mutation assay (Ames test), an in vitro cytotoxicity test, and an in vitro human lymphocyte micronucleus test. Novozymes states that no systemic toxicity was observed in the subchronic study at the highest dose tested of 769 mg TOS/kg bw/d. Novozymes also states that the genotoxicity studies demonstrated no mutagenic, clastogenic, or aneugenic effects.
Novozymes provides general information regarding potential allergenicity of enzymes used in food processing. Novozymes states that only a small percentage of all dietary proteins are allergens, and enzymes do not generally raise safety concerns. Novozymes also discusses an amino acid sequence comparison and published literature to provide a rationale for their conclusion that the allergenic potential of branching glycosyltransferase enzyme is low. Novozymes compared the amino acid sequence of the branching glycosyltransferase enzyme to the sequences of known allergens according to the FAO/WHO (2001) recommendations. Novozymes states that the results of this analysis revealed a 35% amino acid match within a window of 80 amino acids between the branching glycosyltransferase enzyme and the Aspergillus oryzae TAKA amylase A (Asp o 21), which is an occupational allergen. Novozymes states that because both enzymes belong to the family of 13 glycosyl hydrolases, it is not unexpected that some areas of homology can be found. However, Novozymes states that there are large differences in the loop regions between branching glycosyltransferase and TAKA amylase A, and the overall identity between the enzymes is only about 32%. Novozymes further states that published reports describe only four cases of potential food allergy to Asp o 21; of these, three were linked to occupational sensitization, and none of these reported cases have been clinically confirmed. Novozymes cites the results of a published allergenicity study in which exaggerated intakes of 19 commercial enzymes, including bacterial alpha-amylases, did not result in diagnosed food allergy. Novozymes also states that there have been no reports of sensitization towards a protein of bacterial origin, and they are not aware of any allergic reactions caused by the ingestion of branching glycosyltransferase. Novozymes concludes that based on all available evidence, the risk of allergy due to ingestion of branching glycosyltransferase used in starch processing is negligible.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FFDCA)
Section 301(ll) of the FFDCA prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FFDCA, 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 Novozymes’ notice that the branching glycosyltransferase enzyme preparation is GRAS for use in the starch industry to obtain dextrins with improved physical properties, such as higher solubility, lower viscosity, and reduced retrogradation, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing the branching glycosyltransferase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the branching glycosyltransferase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Novozymes, as well as other information available to FDA, the agency has no questions at this time regarding Novozymes’ conclusion that branching glycosyltransferase enzyme preparation 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 the branching glycosyltransferase enzyme preparation. As always, it is the continuing responsibility of Novozymes 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 000274 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 via the FDA home page at http://www.fda.gov. To view or obtain an electronic copy of the text of the letter, follow the hyperlinks from the “Food” topic to the “Food Ingredients and Packaging” section to the “Generally Recognized as Safe (GRAS)” page where the GRAS Inventory is listed.
Laura M. Tarantino, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition