Agency Response Letter GRAS Notice No. GRN 000589
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See also Generally Recognized as Safe (GRAS).
CFSAN/Office of Food Additive Safety
September 17, 2015
Mr. Gary Yingling
Morgan, Lewis & Bockius LLP
1111 Pennsylvania Avenue, NW
Washington, DC 20004
Re: GRAS Notice No. GRN 000589
Dear Mr. Yingling:
The Food and Drug Administration (FDA) is responding to the notice, dated July 2, 2015, that you submitted on behalf of DSM Food Specialties 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 10, 2015, filed it on August 3, 2015, and designated it as GRAS Notice No. GRN 000589.
The subject of the notice is endo-1,4-β-xylanase produced by Aspergillus niger carrying a endo-1,4-β-xylanase gene synthesized in vitro from a cDNA coding sequence obtained from Rasamsonia emersonii (xylanase enzyme preparation). The notice informs FDA of the view of DSM Food Specialties (DSM) that xylanase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in baking applications and during brewing of beer at levels up to 27.6 milligrams total organic solids (mg TOS) per kilogram flour and 6.9 mg TOS per liter of malt, respectively.
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. DSM’s notice provides information about each of these components of xylanase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology, endo-1,4-β-xylanase is identified by the Enzyme Commission Number 220.127.116.11. The accepted name for the enzyme is endo-1,4-β-, and the systematic name is 4-β-D-xylan xylanohydrolase. Endo-1,4-β-xylanase is also known as endo-(1→4)-β-xylan 4-xylanohydrolase; endo-1,4-xylanase; xylanase; β-1,4-xylanase; endo-1,4-xylanase; endo-β-1,4-xylanase; endo-1,4-B-D-xylanase; 1,4-β-xylan xylanohydrolase; B-xylanase; B-1,4-xylan xylanohydrolase; endo-1,4-B-xylanase; β-D- xylanase. The CAS Registry Number for endo-1,4-β-xylanase is 9025-57-4. Endo-1,4-β-xylanase catalyzes the endohydrolysis of (1→4)-β-D-xylosidic linkages in xylans.
DSM states that A. niger ISO-502 host strain was derived from the fully characterized strain, A. niger GAM-53,(1) which has been taxonomically identified by the Dutch culture collection, the Centraalbureau voor Schimmelcultures, CBS. A. niger ISO-502 was modified at several chromosomal loci to delete genes encoding glucoamylase, protease and amylase.(2) DSM also describes A. niger as a non-pathogenic, non- toxigenic, and well-characterized microbe, with a history of safe use.(3)
DSM describes that the construction of the production strain used two plasmids, one carrying the expression cassette for the xylanase gene, and the other, a selectable marker. DSM states that the gene coding for xylanase was a codon optimized, in vitro synthesized, cDNA sequence from R. emersonii. DSM chose the transformant with the highest copy numbers for xylanase expression and one that did not contain any selectable marker. DSM confirmed the absence of the selectable marker via Southern analysis, and the transformation by gel electrophoresis. DSM states that the strain lineage is stable for over 30 years, and that the expression cassettes do not contain any transformable rDNA.(4)
DSM states that the xylanase enzyme preparation is produced by a controlled submerged aerobic fed- batch fermentation of a pure culture of the production strain. The manufacture of xylanase enzyme preparation includes pre-culture fermentation, 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 production organism cells are killed by incubating with sodium benzoate. The cells are then separated from the enzyme product by membrane filtration. Any remaining fine particles are removed by further filtration steps, and the enzyme is concentrated by ultrafiltration. The enzyme concentrate is spray dried and standardized with flour to desired enzyme activity. Alternatively, a liquid xylanase enzyme preparation is obtained by standardizing the xylanase enzyme concentrate with glycerol, and adjusting it to desired activity. According to DSM, the raw materials used in the fermentation, recovery, and formulation processes are food grade. The entire process is performed in accordance with Good Food Manufacturing Practice. DSM states that tests show the production strain does not generate any known toxins under the fermentation conditions described. DSM also states that the final enzyme preparation contains no major food allergens from the fermentation media.
DSM demonstrates that the product is tested to ensure compliance with specifications prior to release of the final enzyme preparation. DSM has established food grade specifications for xylanase enzyme preparation, and notes that the enzyme preparation conforms to the specifications established for enzyme preparations in the Food Chemicals Codex (FCC, 9th edition, 2014), and to 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). DSM provides analytical data from three batches of xylanase enzyme preparation to demonstrate consistency with set specifications.
DSM proposes to use xylanase enzyme preparation in baking applications for improved dough handling, at up to 27.6 mg TOS per kilogram flour. DSM also proposes to use xylanase enzyme preparation in beer production to decrease viscosity, at up to 6.9 mg TOS per liter of malt. DSM states that no enzyme activity is present in the final food product. However, in order to estimate dietary exposure to xylanase enzyme preparation, DSM assumes that all the enzyme TOS will remain in the final food. Based on this assumption, DSM estimates the maximum daily intake of xylanase enzyme TOS from all the intended food applications to be 0.097 mg TOS/kilogram bodyweight per day (mg TOS/kg bw/d). DSM states that xylanase enzyme activity will not produce reaction products that are not already part of the human diet.
DSM summarizes corroborative toxicological studies, using the xylanase enzyme concentrate, to support the safety of the enzyme. Tests conducted using bacterial cells showed that xylanase enzyme is not mutagenic. DSM 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 xylanase enzyme concentrate did not cause any treatment-related adverse effects at up to 6400 mg/kg bw/d, the highest dose tested; this corresponds to 1850 mg TOS/kg bw/d of the xylanase enzyme. DSM states that based on the results of these safety studies, and from that in the published literature, the xylanase 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 xylanase enzyme, i. e., 1850 mg TOS/kg bw/d and 0.097 mg TOS/kg bw/d, respectively, DSM calculates a margin of safety to be 19072.
DSM discusses potential food allergenicity of xylanase enzyme. DSM conducted an amino acid sequence homology search for xylanase enzyme against known allergens using the AllergenOnline database. 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 xylanase enzyme amino acid sequence and those of known allergens. Based on these results, DSM concludes that it is unlikely that oral consumption of xylanase enzyme will result in allergic responses. DSM 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, DSM concludes that xylanase 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 DSM’s notice that xylanase 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 the xylanase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain xylanase 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 information available to FDA, the agency has no questions at this time regarding DSM’s conclusion that the xylanase enzyme preparation is GRAS under the intended conditions use. The agency has not, however, made its own determination regarding the GRAS status of the subject use of xylanase 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 000589, 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) A. niger GAM 53 was modified from A. niger NRRL 3122 via classical mutagenesis. A. niger NRRL 3122 has been deposited in the Culture Collection Unit of the Northern Utilization Research and Development Division, U.S. Department of Agriculture, Peoria, IL, USA.
(2) GAM-53 contains 7 loci for the glucoamylase gene that were removed to create ‘plug-sites’ for integration of expression units containing the xylanase gene in the construction of the production strain.
(3) A. niger GAM-53 has been used for the large-scale production of glucoamylase, phytase and xylanase.
(4) DSM states that the production strain has been classified by Dutch and French authorities as a Group I safe microorganism.