Agency Response Letter GRAS Notice No. GRN 000406
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CFSAN/Office of Food Additive Safety
October 31, 2012
Takashi Kuriki, Ph.D.
Chief Scientific Officer
Ezaki Glico Co., Ltd.
4-6-5, Utajima, Nishioyogogawa
Re: GRAS Notice No. GRN 000406
Dear Dr. Kuriki:
The Food and Drug Administration (FDA) is responding to the notice, dated September 20, 2011, 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 September 28, 2011, filed it on October 11, 2011, and designated it as GRAS Notice No. GRN 000406.
The subject of the notice is 1,4-α-glucan branching enzyme preparation from Bacillus subtilis strain 168 expressing the synthetic glucan branching enzyme gene from Aquifex aeolicus strain VF5 (branching enzyme preparation). The notice informs FDA of the view of Ezaki Glico Co., Ltd. (Ezaki) that branching enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the production of cyclic dextrin and enzymatically-synthesized glycogen. Ezaki states that the typical use level of branching enzyme preparation in the production of cyclic dextrin is 500 Units per gram (g) substrate, which is equivalent to 0.07 milligram (mg) total organic solids (TOS) per g substrate. Ezaki states that the typical use level of branching enzyme preparation in the production of enzymatically-synthesized glycogen is 5000 Units/g substrate, which is equivalent to 0.67 mg TOS/g substrate.
As part of its notice, Ezaki includes the report of a panel of individuals (Ezaki’s GRAS panel) who evaluated the data and information that are the basis for Ezaki’s GRAS determination. Ezaki considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Ezaki’s GRAS panel evaluated information related to the safety of the production strain, method of manufacture and product specifications, as well as the results of published studies. Based on this review, Ezaki’s GRAS panel concluded that branching enzyme preparation that meets its established food grade specifications is GRAS under the conditions of its intended use.
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. The enzyme preparation may contain constituents derived from the production organism and manufacturing process. Ezaki states that water is added to adjust enzyme activity and that no other stabilizers, preservatives, diluents, or carriers are added to branching enzyme preparation. In its notice, Ezaki provides information about all the components of branching enzyme preparation.
The system established by the International Union of Biochemistry and Molecular Biology classifies branching enzyme as Enzyme Commission number 188.8.131.52. The accepted name is 1,4-α-glucan branching enzyme and its systematic name is 1,4-α-D-glucan: 1,4-α-D-glucan 6-α-D-(1,4-α-D-glucano) transferase. The CAS Registry Number of branching enzyme is 9001-97-2. Branching enzyme has a molecular weight between 62,000 and 66,000, is thermostable, and has optimal activity at 50° C and pH 7.5.
Branching enzyme catalyzes the intra- or intermolecular transglycosylation reactions in glucan substrates, resulting in the formation of α-(1,6) glucosidic bonds. The reaction product is either cyclic dextrin or enzymatically-synthesized glycogen.
Ezaki states that branching enzyme is produced by B. subtilis strain NCIMB12378, which is derived from B. subtilis strain 168 using classical mutagenesis techniques. Published information shows that B. subtilis strain 168 is nonpathogenic and nontoxigenic. B. subtilis strain 168 is also described in the literature as a well-known wild-type strain and as the progenitor of many B. subtilis strains that are used as sources of food processing enzymes.
Ezaki states that the production strain was developed by transformation of B. subtilis strain 168 with the pUAQ2 plasmid. The plasmid contains a branching enzyme gene (glgB) that was chemically synthesized based on the amino acid sequence of branching enzyme from A. aeolicus strain VF5. The inserted DNA sequence was limited to that required for branching enzyme expression and no other sequences were incorporated into the plasmid. The expression of the glgB gene was placed under the regulation of a plasmid promoter sequence. The pUAQ2 plasmid contains a kanamycin resistance cassette, which was used for selecting the production strain. The production strain was confirmed by extraction and restriction analysis of the pUAQ2 plasmid from selected colonies.
Ezaki states that branching enzyme is produced by pure culture fermentation of the B. subtilis production strain using a fermentation medium comprised of food grade ingredients. These include casein (a milk protein), which Ezaki states is removed during the branching enzyme purification process. Sodium hydroxide is added to the main fermentation culture to adjust the pH. Egg white lysozyme is added to lyse the bacterial cells and release the intracellular branching enzyme. Ezaki states that the egg white lysozyme is removed during the branching enzyme purification process. Branching enzyme is solubilized using sodium carbonate and then filtered to remove insoluble materials. The solution containing branching enzyme is purified by microfiltration, concentrated by ultrafiltration, and then water is added to adjust the enzymatic activity. Ezaki states that the manufacturing process is carried out in accordance with current good manufacturing practices. The typical activity of branching enzyme preparation is 40,000 Units per milliliter and the content of TOS is approximately 6.9%.
Ezaki states that branching enzyme preparation conforms to: 1) the specifications for enzyme preparations described in the Food Chemicals Codex (7th edition, 2010), and 2) the current (2006) General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives.
Ezaki states that branching enzyme preparation will be used in the production of cyclic dextrin from food-grade waxy corn starch and enzymatically-synthesized glycogen from food-grade dextrin. Ezaki states that branching enzyme preparation is inactivated and removed from the final food ingredients. Based on this information, Ezaki concludes that the carryover of branching enzyme preparation to cyclic dextrin and enzymatically-synthesized glycogen would be negligible. However, to provide a conservative estimate of possible human exposure, Ezaki assumes that all cyclic dextrin and enzymatically-synthesized glycogen are produced using branching enzyme preparation and that all of the branching enzyme preparation remains in the final foods. Based on these assumptions, Ezaki estimates the exposure to branching enzyme preparation to be 0.46 mg TOS per kilogram (kg) body weight (bw) per day at the 90th percentile all-user intake of cyclic dextrin and enzymatically-synthesized glycogen.
To support the safety of branching enzyme preparation, Ezaki discusses published toxicological studies. A repeated-dose study conducted using Crl:CD(SD) rats showed that the administration of branching enzyme preparation did not result in mortality or abnormalities. A bacterial reverse mutation assay conducted using Salmonella typhimurium strains showed that treatment with branching enzyme preparation did not increase the mean number of revertant colonies. A chromosomal aberration test conducted using Chinese hamster ovary cells showed that treatment with branching enzyme preparation did not result in cytotoxicity. Based on these results, Ezaki concludes that branching enzyme preparation does not cause toxicity following repeated oral exposure nor is it mutagenic or genotoxic.
Ezaki provides information regarding the potential food allergenicity of branching enzyme. Ezaki conducted an amino acid sequence homology search for branching enzyme against known allergens in the Food Allergy Research and Resource Program database. No identity matches of greater than 35% were found, nor were any matches of 8 contiguous amino acids identified. Based on this information, Ezaki considers that the intended use of branching enzyme would not be expected to cause allergic reactions.
Based on the data and information summarized above, Ezaki concludes that branching enzyme preparation is GRAS for its intended uses.
The Food Allergen Labeling and Consumer Protection Act of 2004 (FALCPA) amends the Federal Food, Drug, and Cosmetic Act (FD&C Act) to require that the label of a food that is or contains an ingredient that bears or contains a “major food allergen” declare the presence of the allergen (section 403(w)). FALCPA defines a “major food allergen” as one of eight foods or food groups (i.e., milk, eggs, fish, Crustacean shellfish, tree nuts, peanuts, wheat, and soybeans) or a food ingredient that contains protein derived from one of those foods. Branching enzyme preparation may require labeling under FALCPA because it may contain casein (from milk) and lysozyme (from eggs).
FALCPA provides mechanisms by which a manufacturer may request that a food ingredient covered by FALCPA be exempted from FALCPA's labeling requirements. An ingredient is eligible for exemption if it does not cause an allergic response that poses a risk to human health or contain allergenic protein.
In the notice, Ezaki states that casein and lysozyme are removed during the manufacturing process for branching enzyme preparation. If Ezaki has scientific evidence that one or both of the components of the branching enzyme preparation is eligible for exemption from FALCPA’s labeling requirements, we recommend that Ezaki contact the Food Allergen Coordinator in FDA’s Center for Food Safety and Applied Nutrition (Food Allergen Coordinator, Office of Food Additive Safety, HFS-200, 5100 Paint Branch Parkway, College Park, Maryland 20740) regarding procedures for exemption from FALCPA’s labeling requirements for branching enzyme preparation. More information about FALCPA submissions is available at http://www.fda.gov/Food/LabelingNutrition/FoodAllergensLabeling/GuidanceComplianceRegulatoryInformation/default.htm.
Section 301(ll) of the 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 Ezaki’s notice that branching 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 branching enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain branching enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Ezaki, as well as other information available to FDA, the agency has no questions at this time regarding Ezaki’s conclusion that branching 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 branching enzyme preparation. As always, it is the continuing responsibility of Ezaki 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 000406, 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.
Dennis M. Keefe, Ph.D.
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition