Agency Response Letter GRAS Notice No. GRN 000428
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CFSAN/Office of Food Additive Safety
November 26, 2012
Gary L. Yingling
Morgan, Lewis & Bockius LLP
1111 Pennsylvania Avenue, NW
Washington, D.C. 20004
Re: GRAS Notice No. GRN 000428
Dear Mr. Yingling:
The Food and Drug Administration (FDA) is responding to the notice, dated March 6, 2012, that you submitted on behalf of DSM Food Specialties (DSM) 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 March 27, 2012, filed it on March 28, 2012, and designated it as GRAS Notice No. GRN 000428.
The subject of the notice is asparaginase enzyme preparation from genetically modified Aspergillus niger (asparaginase enzyme preparation). The notice informs FDA of the view of DSM that asparaginase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme at specified levels to reduce the levels of free L-asparagine,(1) specifically in (1) bread (such as tin bread, buns, rolls, French sticks or batards, variety breads like multigrain types of bread, raisin bread, biscuits, crackers); (2) other cereal-based products (such as corn crackers, cakes, Swiss rolls, Dutch honey cake, breakfast cereals); and (3) potato-based products (such as French fries, potato chips).
Commercial enzyme preparations that are used in food typically contain an enzyme component which catalyzes the chemical reaction 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, DSM provides information about all the components of asparaginase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, asparaginase is identified by the Enzyme Commission number 126.96.36.199. The accepted name for the enzyme is asparaginase and the systematic name is L-asparagine amidohydrolase. The CAS Registry Number for asparaginase is 9015-68-3 and its calculated molecular weight is 39.58 kiloDaltons. Asparaginase catalyzes the hydrolysis of L-asparagine to L-aspartic acid and ammonia. Asparaginase also acts on glutamine, with a lower preference, and it has no activity on other amino acids, or on asparagine residues in peptides or proteins.
DSM states that A. niger strain ISO 528 is the host strain used to develop the production strain. The host strain is derived from A. niger strain NRRL 3122, purchased from the United States Department of Agriculture. The host strain was developed using several classical mutagenesis steps that inactivated genes encoding glucoamylase, amylase, and protease and increased enzyme secretion capacity. The host strain was derived from a well-characterized lineage that has been used to develop production strains for other enzymes used in food. A. niger is a nonpathogenic, nontoxigenic microbe with a long history of safe industrial use.
DSM describes the development of the production strain, A. niger strain AGN7-41. The host strain is transformed with a DNA cassette containing a synthetic gene encoding A. niger asparaginase enzyme with an altered pH profile compared to wild type asparaginase enzyme. The host strain is also transformed with a cassette containing the selectable marker gene amdS from A. nidulans. The amdS gene is subsequently removed to create a marker-free production strain. DSM states that the production microbe contains multiple copies of the gene encoding the modified asparaginase enzyme and is genetically stable. Tests confirm that the production strain neither contains antibiotic resistance markers nor does it produce mycotoxins under the controlled fermentation conditions.
DSM states that the asparaginase enzyme is produced by controlled submerged fermentation of a pure culture of the production strain. During fermentation, the asparaginase enzyme is secreted to the fermentation broth. After fermentation is stopped to effectively kill the production organism, the enzyme is purified and concentrated to obtain a liquid product during downstream processing. The liquid product is then standardized with ~50% glycerol to the desired enzyme activity at pH 3.5-5.0. DSM states that the asparaginase enzyme preparation is produced in accordance with Good Manufacturing Practice (GMP). The raw materials used in the fermentation, recovery, and formulation meet predefined quality standards and are food grade. Tests confirm the absence of both transformable recombinant DNA and the production microbe in the final asparaginase enzyme preparation.
DSM notes that asparaginase enzyme preparation conforms to the specifications for enzyme preparations described in the Food Chemicals Codex (7th edition, 2010), and to the current General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives (2006).
DSM intends to use asparaginase enzyme preparation at levels of 1.6-33.6 milligram per kilogram (mg/kg) total organic solids (TOS) in wheat-based products, 1.7-35.1 mg/kg TOS in cereal-based products, and 4.1-73.8 mg/kg TOS in potato-based products, respectively. Since the asparaginase enzyme is denatured during baking and frying, no enzyme activity is expected to remain in the final food. DSM states that the intended use of the asparaginase is substitutional with other asparaginases in commerce. To estimate possible human exposure, DSM assumes that 100% of the enzyme TOS used in the intended food applications would remain in the finished foods. Based on this assumption, DSM estimates the maximum daily intake of TOS from all of the intended food applications to be 0.01-0.25 mg TOS per kg body weight (bw) per day. DSM anticipates that the action of asparaginase enzyme will not result in the formation of any reaction products that are not already part of the human diet.
DSM notes that FDA had no questions in response to GRN 000214(2) (wild type asparaginase enzyme preparation). DSM considers that the following support the substantial equivalence of the notified asparaginase enzyme, the subject of GRN 000428, to naturally occurring asparaginase and wild type asparaginase enzyme: 1) the enzymes catalyze the same hydrolysis reaction; 2) the notified enzyme has 77.5% sequence identity to the wild-type asparaginase, and 3) the notified enzyme and the wild-type asparaginase are produced from A. niger under GMP using food grade ingredients. Based on the weight of evidence for these enzymes, DSM concludes that safety studies conducted using wild type asparaginase enzyme preparation support the safety of the notified asparaginase enzyme preparation.
DSM summarizes unpublished toxicological studies conducted with wild type asparaginase enzyme preparation and an unpublished genotoxicity study conducted using the notified asparaginase enzyme preparation. The results of chromosomal aberration tests show that the wild type asparaginase enzyme concentrate is not clastogenic to cultured human lymphocytes. Studies conducted using mouse lymphoma cells showed that wild type asparaginase enzyme preparation did not increase the induction of mutation frequency. Tests conducted using bacterial cells showed that neither the wild type nor the notified asparaginase enzyme preparation is mutagenic. A 90-day oral toxicity study conducted in rats showed that consumption of wild type asparaginase enzyme preparation did not cause adverse effects at 271 mg TOS/kg bw/day, the highest dose tested. A developmental toxicity study conducted in rats showed that consumption of wild type asparaginase enzyme preparation did not cause adverse effects at 1081 mg TOS/kg bw/day, the highest dose tested. These levels were identified as no observed adverse effect levels (NOAELs). Based on the NOAEL from the 90-day study and the highest intended use level of the notified asparaginase enzyme preparation, DSM calculates margins of safety to be 1111.
DSM discusses potential food allergenicity of asparaginase enzyme. DSM conducted amino acid sequence homology searches for the notified and wild type asparaginase enzymes against known allergens in two publicly available allergen databases (Allermatch and the Structural Database of Allergenic Proteins). No identity matches of greater than 35% were found, nor were any matches of 8 contiguous amino acids identified. DSM therefore considers it unlikely that the enzyme will produce an allergenic or sensitization response upon oral consumption. In addition, in silico analysis of pepsin digestibility of the notified asparaginase protein sequence reveals easy degradability. Based on the available information, DSM concludes that the notified asparaginase enzyme is not a food allergen.
Based on the data and information summarized above, DSM concludes that asparaginase enzyme preparation is GRAS for its intended uses.
Standards of Identity
In the notice, DSM states its intention to use asparaginase enzyme preparation in several food categories, including foods for which standards of identity exist, located in Title 21 of the Code of Federal Regulations. We note that an ingredient that is lawfully added to food products may be used in a standardized food only if it is permitted by the applicable standard of identity.
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 DSM’s notice that asparaginase 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 asparaginase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain asparaginase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by DSM, as well as other information available to FDA, the agency has no questions at this time regarding DSM’s conclusion that asparaginase 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 asparaginase enzyme preparation. As always, it is the continuing responsibility of DSM 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 000428, 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
(1)DSM describes that the intended effect of the asparaginase is to convert L-asparagine to aspartic acid. L-asparagine is a precursor in the formation of acrylamide, a food contaminant. Acrylamide forms when foods containing asparagine and reducing sugars are baked or fried at temperatures above 120°C. FDA has not evaluated the efficacy of acrylamide reduction by asparaginase in the specified foods.
(2)The wild-type asparaginase enzyme preparation is the subject of GRN 000214, asparaginase enzyme preparation from A. niger.