Agency Response Letter GRAS Notice No. GRN 000624
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See also Generally Recognized as Safe (GRAS).
CFSAN/Office of Food Additive Safety
August 18, 2016
Mr. Gary Yingling
Morgan, Lewis & Bockius LLP
111 Pennsylvania Avenue, NW
Washington D.C. 20004
Re: GRAS Notice No. GRN 000624
Dear Mr. Yingling:
The Food and Drug Administration (FDA) is responding to the notice, dated January 19, 2016, that you submitted on behalf of Matsutani Chemical industry Co. Ltd. (Matsutani) 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 January 28, 2016, filed it on February 17, 2016, and designated it as GRAS Notice No. GRN 000624.
The subject of the notice is D-psicose 3-epimerase enzyme preparation produced by Escherichia coli K-12 W3110 carrying a gene expressing D-psicose 3-epimerase from Arthrobacter globiformis M30 (D-psicose epimerase enzyme preparation). The notice informs FDA of the view of Matsutani that D-psicose epimerase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the epimerization of fructose to D-psicose , at up to 900 milligrams Total Organic Solids (TOS) of D-psicose 3-epimerase enzyme preparation per kilogram of final food (mg TOS/kg).
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 constituents derived from the production organism and constituents derived from the manufacturing process, e.g., components of the fermentation media or the residues of processing aids. Matsutani's notice provides information about each of these components of D-psicose 3-epimerase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, D-psicose 3-epimerase is identified by the Enzyme Commission Number 18.104.22.168. The accepted name for the enzyme is D-psicose 3-epimerase, and the systematic name is D-psicose 3-epimerase. D-psicose 3-epimerase is an isomerase, and has also been known as DPEase (ambiguous). D-psicose 3-epimerase catalyzes the epimerization of D-fructose to D-psicose. Matsutani states that the technical effect of D-psicose 3-epimerase enzyme is to epimerize D-fructose at the C3 position, to D-psicose. The enzyme also catalyzes the epimerization of D- and L-keto-hexoses, keto- pentoses and keto-tetroses. Matsutani states that D-psicose 3-epimerase is a tetramer with four identical sub-units with a molecular weight of ~128 kiloDaltons. Matsutani also states that the primary sequence of D-psicose 3-epimerase consists of 289 amino acids.
Matsutani states that E. coli K-12 W3110 host strain was derived from the well characterized wild-type parent E. coli K12. Matsutani describes E. coli K-12 as a non-pathogenic, non-toxigenic microorganism, with a history of safe use.
Matsutani describes the construction of the production strain using an expression plasmid that caries the D-psicose 3-epimerase gene from the donor, A. globiformis M30 and a selection marker. Matsutani states that the production strain was deposited in the National Institute of Technology and Evaluation (NITE) in Japan under accession number P-1111. Matsutani confirmed the transformation of the intended genes by DNA sequencing of the expression plasmid. Matsutani also confirmed the stability of the expression plasmid, and that it does not contain any transformable rDNA.
Matsutani states that the D-psicose 3-epimerase enzyme is produced by a controlled submerged aerobic fed-batch fermentation of a pure culture of the production strain. The manufacture of the D-psicose 3- epimerase enzyme preparation includes fermentation, downstream processing, and formulation of the final product. Appropriate measures are set in place to control for identity, purity, and enzyme-generating ability of the production strain during and after fermentation. After fermentation is stopped, the cells are separated from the enzyme product by membrane filtration. Any remaining fine particles are removed by further filtration steps. The enzyme concentrate is stabilized with sugar or sugar alcohol, subjected to ultrafiltration, and freeze-dried (solid product) or standardized with food-grade reagents to desired enzyme activity (liquid product); the final product is stored at 4-10° C. According to Matsutani, the raw materials used in the fermentation, recovery, and formulation processes are food grade. The entire process is performed in accordance with ISO 9000 Good Food Manufacturing Practice. Matsutani confirms there are no allergens from the manufacturing process in the final D-psicose 3-epimerase enzyme product.
Matsutani has established food grade specifications for D-psicose 3-epimerase enzyme preparation, and notes that the enzyme preparation conforms to the specifications established for enzyme preparations in the Japanese Specifications and Standards for Food Additives ( 2007, 8th Edition), Japanese Pharmacopeia (2016, 17th Edition), and Japanese Standard Methods of Analysis in Food Safety Regulation, Microorganisms (2015). Matsutani provides validated analytical data from three batches of D-psicose 3- epimerase enzyme concentrate to demonstrate consistency with set specifications. Matsutani confirms via testing that the production organism is absent in the final product.
Matsutani proposes to use D-psicose 3-epimerase enzyme preparation in the production of D-psicose at up to 60 g/kg fructose raw material. Matsutani states that D-psicose can be used in a wide range of food applications such as cereals, chewing gum, confections & frostings, dressings for salads, jams & jellies, sugar, sugar substitutes (carrier), and various low-calorie or dietetic foods including low calorie, reduced- calorie, sugar-free beverages (non-alcoholic), cereals, frozen dairy desserts (ice cream, soft serve, sorbet), yogurt and frozen yogurt, gelatins, pudding & fillings, hard candies, soft candies, and sweet sauces and syrups. Matsutani states that no activity from D-psicose 3-epimerase enzyme preparation is expected to be present in the final food. However, in order to estimate dietary exposure to D-psicose 3-epimerase enzyme preparation, Matsutani assumes that all enzyme TOS will remain in the D-psicose used in preparation of final foods. Based on this assumption, Matsutani estimates the maximum daily intake of D-psicose 3- epimerase enzyme from all the intended food applications to be 0.29 mg TOS/kg bodyweight per day (mg TOS/kg bw/d). Matsutani states that D-psicose 3-epimerase enzyme activity will not produce reaction products that are not already part of the human diet.
Matsutani relies on published information for the safety of microbial enzyme preparations used in food processing and summarizes unpublished toxicological studies using the D-psicose 3-epimerase enzyme concentrate to support the safety of the enzyme. A reverse mutation test conducted using bacterial cells (Ames Test) showed that D-psicose 3-epimerase enzyme is not mutagenic. Matsutani also demonstrates that the enzyme is not clastogenic to cultured human lymphocytes under the conditions employed in this study. The results of a 90-day oral toxicity study conducted using rats showed that consumption of D-psicose 3-epimerase enzyme concentrate did not cause any treatment-related adverse effects at the highest dose tested, which corresponds to 1020 mg TOS/kg bw/d of the D-psicose 3-epimerase enzyme.
Matsutani states that based on the results of these safety studies, and from that present in published literature, the D-psicose 3-epimerase enzyme is considered safe for human consumption. Based on the highest dose tested in the 90-day study, and the maximum estimated daily intake from the proposed use levels of D-psicose 3-epimerase enzyme, i. e., 1020 mg TOS/kg bw/d and 0.29 mg TOS/kg bw/d, respectively, Matsutani calculates a margin of safety to be 3504.
Matsutani discusses potential food allergenicity of D-psicose 3-epimerase enzyme. Matsutani conducted an amino acid sequence homology search for D-psicose 3-epimerase enzyme against known allergens using the Allergen Database for Food Safety (ADFS). No amino acid identity matches greater than 35% over 80 amino acids were found and neither were matches of contiguous stretches of eight amino acids shared between the D-psicose 3-epimerase enzyme amino acid sequence and those of known allergens. Based on these results, Matsutani concludes that it is unlikely that oral consumption of D-psicose 3- epimerase enzyme will result in allergic responses. Matsutani further cites the conclusions of several organizations and working groups about the low risk of allergenicity posed by enzymes, and notes the extremely low use levels and extensive processing of the enzyme-containing foods during manufacturing.
Based on the data and information summarized above, Matsutani concludes that D-psicose 3-epimerase 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 its review of Matsutani's notice that D-psicose 3- epimerase 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 D-psicose 3-epimerase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain D-psicose 3- epimerase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Matsutani Chemical industry Co. Ltd., as well as other information available to FDA, the agency has no questions at this time regarding Matsutani's conclusion that D-psicose 3-epimerase enzyme preparation produced by Escherichia coli K-12 W3110 carrying a gene expressing D-psicose 3-epimerase from Arthrobacter globiformis M30 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 D-psicose 3-epimerase enzyme preparation produced by Escherichia coli K-12 W3110 carrying a gene expressing D-psicose 3-epimerase from Arthrobacter globiformis M30. As always, it is the continuing responsibility of Matsutani. 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 00 0624, 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
 D-psicose is also known as allulose or D-ribo-2-hexulose.