Agency Response Letter GRAS Notice no. GRN 000479
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CFSAN/Office of Food Additive Safety
March 7, 2014
Gary L. Yingling
Morgan, Lewis & Bockius LLP
1111 Pennsylvania Avenue, NW
Washington, D.C. 20004
Re: GRAS Notice No. GRN 000479
Dear Mr. Yingling:
The Food and Drug Administration (FDA) is responding to the notice, dated April 24, 2013, 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 July 3, 2013, filed it on August 5, 2013, and designated it as GRAS Notice No. GRN 000479.
The subject of the notice is beta-glucanase, cellulase, and xylanase enzyme preparation from Rasamsonia emersonii, (beta-glucanase, cellulase, and xylanase enzyme preparation). The notice informs FDA of the view of DSM that beta-glucanase, cellulase, and xylanase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the production of beer and fermented beverages at levels up to 23 milligrams Total Organic Solids per kilogram (mg TOS/kg) of raw material. DSM intends to use beta-glucanase, cellulase, and xylanase enzyme preparation to improve wort viscosity and reduce haze formation in beer and fermented beverages.
Commercial enzyme preparations that are used in food processing 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 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 beta-glucanase, cellulase, and xylanase enzyme preparation.
According to the classification system of enzymes established by the International Union of Biochemistry and Molecular Biology (IUBMB), beta-glucanase is identified by the Enzyme Commission (EC) Number 220.127.116.11. The accepted name for this enzyme is endo-1, 3(4)-beta-glucanase and the systematic name is 3-(1, 3-1,4)-beta-D-glucan 3(4) glucanohydrolase. The CAS Registry Number for beta-glucanase is 62213-14-3. Beta-glucanase catalyzes the endo-hydrolysis of the 1, 3- or 1, 4-linkages in beta-D-glucans. Cellulase is identified as EC 18.104.22.168. The accepted name for the enzyme is cellulase and the systematic name is 4-(1, 3;1,4)-beta-D-glucan 4-glucanohydrolase. The CAS Registry Number for cellulase is 9012-54-8. Cellulase catalyzes the endo-hydrolysis of 1, 4-beta-D-glucosidic linkages in beta-D-glucans present in celluloses, lichenins, and cereals. Cellulase also catalyzes the hydrolysis of 1, 4-linkages in beta-D-glucans containing 1, 3-linkages. Xylanase is identified as EC 22.214.171.124. The accepted name for the enzyme is 1, 4,-beta-xylanase and the systematic name is 4-β-D-xylan-xylanohydrolase. The CAS Registry Number for xylanase is 9025-57-4. Xylanase catalyzes the hydrolysis of the 1, 4-beta-D-xylosidic linkages in xylans.
DSM describes the development of the production strain, R. emersonii strain FBG1.1,2 Strain FBG1 was derived as a single isolate from the wild-type strain and was treated with ultraviolet light and methyl-nitro-nitrosoguanidine to induce mutagenesis and improve the production of beta-glucanase, cellulase, and xylanase enzymes. DSM cites publicly available information describing R. emersonii as a nonpathogenic fungus as well as published and unpublished information describing the nontoxigenic nature of the organism. DSM reports on tests of the production strain’s ability to produce secondary metabolites, including mycotoxins, under various growth conditions. DSM states that no mycotoxins were found. DSM also states that levels of secondary metabolites found are not toxicologically relevant compared to published literature values.
DSM states that the beta-glucanase, cellulase, and xylanase enzymes are concurrently produced by submerged, aerobic batch fermentation of a pure culture of the production strain. Each fermentation batch is initiated using a fresh culture of the production strain. During fermentation, the dissolved oxygen content, pH, temperature, and viscosity of the culture are controlled and appropriate measures are taken to ensure purity. The beta-glucanase, cellulase, and xylanase enzymes are secreted to the broth during fermentation. After fermentation, the enzymes are recovered using several filtration and concentration steps, during which production strain residues are removed. Glycerol is added to standardize the enzyme concentrate and to ensure that the desired enzyme activities are achieved, followed by the addition of sodium benzoate as a stabilizer. Beta-glucanase, cellulase, and xylanase enzyme preparation is commercialized as a liquid product. DSM states that beta-glucanase, cellulase, and xylanase enzyme preparation is produced in accordance with current good manufacturing practices, and the materials used in fermentation, recovery, and formulation meet predefined quality standards and are food grade. DSM also states that there are no published or unpublished data to suggest that allergenic proteins present in the fermentation media would be present in the final enzyme preparation.
DSM states that beta-glucanase, cellulase, and xylanase enzyme preparation conforms to the specifications for enzyme preparations described in the Food Chemicals Codex (8th edition, 2012) 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 intends to use beta-glucanase, cellulase, and xylanase enzyme preparation in the production of beer and fermented beverages at levels up to 23 mg TOS/kg of raw material. DSM states that beta-glucanase, cellulase, and xylanase enzyme preparation is applied during the mashing phases of the manufacturing processes for beer and fermented beverages, followed by boiling steps. DSM states that boiling denatures the beta-glucanase, cellulase, and xylanase enzymes, and thus no activities from these enzymes remain in the final foods. However, to estimate dietary exposure to beta-glucanase, cellulase, and xylanase enzyme preparation, DSM assumes that all beers and fermented beverages are produced using this enzyme preparation and that the entire enzyme TOS would remain in the final foods. Based on these assumptions and the proposed maximum use level of beta-glucanase, cellulase, and xylanase enzyme preparation TOS in these foods, DSM estimates the maximum daily intake of the enzyme preparation from all of the intended food applications to be 4.56 mg TOS per liter of beer or fermented beverages, which corresponds to 0.027 mg TOS per kg bodyweight per day (TOS/kg bw/d).
In addition to published and unpublished information describing the safety of the production strain, DSM describes the natural occurrence of beta-glucanase, cellulase, and xylanase enzymes in nature and the long history of the uses of these enzymes in food processing. DSM cites previous GRAS notices (GRN 000054, GRN 000149, and GRN 000195) for enzyme preparations containing beta-glucanase, cellulase, and xylanase enzymes individually and in combination, and notes that FDA had no questions in response to these GRAS notices. DSM further states that JECFA assigned acceptable daily intakes of “not specified” for enzyme preparations containing beta-glucanase, cellulase, or xylanase enzymes.
DSM summarizes unpublished toxicological studies supporting the safety of beta-glucanase, cellulase, and xylanase enzyme concentrate in the absence of standardization and stabilization ingredients. Tests conducted using bacterial cells showed that beta-glucanase, cellulase, and xylanase enzyme concentrate is not mutagenic. Tests conducted using human lymphocytes showed that beta-glucanase, cellulase, and xylanase enzyme concentrate is not clastogenic. The results of a 90-day oral toxicity study conducted using rats showed that consumption of beta-glucanase, cellulase, and xylanase enzyme concentrate did not cause any treatment-related adverse effects at 1600 mg/kg bw/d, the highest dose tested, which corresponds to 84.8 mg TOS/kg bw/d of the beta-glucanase, cellulase and xylanase enzyme concentrate. DSM designated this level as the no observed adverse effect level (NOAEL). Based on this NOAEL and the highest intended consumption level (0.027 mg TOS/kg bw/d) for beta-glucanase, cellulase, and xylanase enzyme preparation, DSM calculates a maximum margin of safety of 3140.
DSM discusses the potential food allergenicity of beta-glucanase, cellulase, and xylanase enzyme preparation. DSM conducted an amino acid sequence homology search for the beta-glucanase, cellulase, and xylanase enzymes against known allergens using the AllergenOnline database. No amino acid identity matches greater than 35% over 80 amino acids were found nor were matches of 8 amino acids or more observed for the amino acid sequences of any of the enzymes. Based on these results, DSM concludes that it is unlikely that oral consumption of beta-glucanase, cellulase, and xylanase enzymes will result in allergic responses.
Based on the data and information summarized above, DSM concludes that beta-glucanase, cellulase, and xylanase enzyme preparation is GRAS for the intended uses.
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 beta-glucanase, cellulase, and 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 beta-glucanase, cellulase, and xylanase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain beta-glucanase, cellulase, and 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 the information available to FDA, the agency has no questions at this time regarding DSM’s conclusion that beta-glucanase, cellulase, and xylanase 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 beta-glucanase, cellulase, and 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
000479, 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
1The host strain is a filamentous eukaryotic thermophilic fungus isolated from compost material.
2The published literature shows that Rasamsonia emersonii, formerly known as Talatomyces emersonii, has also been cited in literature as Pencillium emersonii or Geosmithia emersonii, and most recently been renamed as Rasamsonia emersonii.