Agency Response Letter GRAS Notice No. GRN 000628
CFSAN/Office of Food Additive Safety
September 14, 2016
Ms. Candice Cryne
AB Enzymes GmbH
Regulatory Affairs Specialist (The Americas)
Ontario, CANADA M6J3L9
Re: GRAS Notice No. GRN 000628
Dear Ms. Cryne:
The Food and Drug Administration (FDA) has received the notice, dated December 30, 2015, 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 this notice on February 5, 2016, filed it on March 2, 2016, and designated it as GRN No. 000628.
The subject of the notice is endo-1,4-β-xylanase enzyme preparation produced in Trichoderma reesei carrying a gene that expresses endo-1,4-β-xylanase from Thermopolyspora flexuosa (endo-1,4-β-xylanase enzyme preparation). The notice informs FDA of the view of AB Enzymes GmbH (hereafter, AB Enzymes) that the endo-1,4-β-xylanase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in grain processing, manufacture of cereals, cereal-based beverages, potable alcohol, and baked goods at up to a level of 10 milligrams of Total Organic Solids per kilogram (mg TOS/kg) raw material.
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 components derived from the production organism and components derived from the manufacturing process, e.g., constituents of the fermentation media or the residues of processing aids. AB Enzymes’ notice provides information about each of these components in the endo-1,4-β-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 126.96.36.199. The accepted name for the enzyme is endo-1,4-β-xylanase, 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-β-D-xylanase; 1,4-β-xylan xylanohydrolase; β-xylanase; β-1,4-xylan xylanohydrolase; endo-1,4-β-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 arabinoxylans resulting in depolymerization of the arabinoxylan into smaller oligosaccharides.
AB Enzymes describes the construction of the production strain, T. reesei RF5427, from the host strain T. reesei F4847. AB states that the endo-1,4-β-xylanase gene used in this construction was isolated from the genome of T. flexuosa DSM43186, and contained in the expression cassette that was inserted into RF4847. The expression cassette was generated using standard molecular biology methods, and included a T. reesei signal sequence, the endo-1,4-β-xylanase gene, and the Aspergillus nidulans amdS gene as a selectable marker. AB Enzymes states that T. reeseiis a nonpathogenic, nontoxigenic microbe, and that it has a long history of safe use for the production of enzymes used in food. AB Enzymes confirms that the transformed DNA is well-characterized, stably integrated, and does not contain any harmful genes.
AB Enzymes states that the endo-1,4-β-xylanase enzyme preparation is manufactured by the submerged fermentation of a pure culture preparation of T. reesei RF5427. AB Enzymes states that fermentation is carried out under well-defined process conditions (pH, temperature, mixing, etc.) and that endo-1,4-ß-xylanase is excreted into the culture media. After fermentation, the enzyme is separated from the cell mass by the addition of a flocculating agent to facilitate the primary solid/liquid separation. This is followed by filtration or centrifugation. The supernatant containing the enzyme is concentrated at controlled pH and temperature, and filtered to remove insoluble material and the production organism. The final enzyme product is either spray dried, or standardized as a liquid product with appropriate food- grade ingredients. AB Enzymes states that the entire process is performed in accordance with current good manufacturing practices using raw materials of food-grade quality. AB Enzymes also states that the final enzyme preparation contains no major food allergens from the fermentation medium.
AB Enzymes notes that the endo-1,4-β-xylanase enzyme preparation conforms to 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). AB Enzymes provides analytical data from three batches of endo-1,4-β-xylanase enzyme preparation to demonstrate consistency with the specifications.
AB Enzymes proposes to use the endo-1,4-β-xylanase enzyme preparations as an enzyme in grain processing, production of cereal-based beverages, and brewing, potable alcohol production, and manufacture of baked goods at up to 10 mg TOS/kg of raw material. AB Enzymes states that the endo- 1,4-β-xylanase enzyme is expected to be inactivated and/or removed during manufacturing in all the proposed uses. AB Enzymes also states that the hydrolysis products of endo-1,4-β-xylanase are oligosaccharides of variable lengths, and natural constituents of cereals. However, AB Enzymes estimates dietary exposure to endo-1,4-β-xylanase enzyme preparation, based on the maximum intended use levels and the assumption that all of the enzyme preparation will remain in the final food, to be 0.4 mg TOS/kg bodyweight per day (mg TOS/kg bw/d). AB Enzymes also states that endo-1,4-β-xylanase enzyme, if present in the final food, will be broken down like all other proteins in the human digestive system.
AB Enzymes relies on published information for the safety of microbial enzyme preparations used in food processing and summarizes unpublished toxicological studies using the endo-1,4-β-xylanase enzyme concentrate. Tests conducted with bacterial cells showed that the endo-1,4-β-xylanase enzyme is not mutagenic at the highest dose tested both in the presence and absence of metabolic activation. AB Enzymes also demonstrates that the endo-1,4-β-xylanase enzyme concentrate is not clastogenic based on results from in vitro chromosomal aberration tests. A ninety day oral toxicity study (90-day) in rats using the endo-1,4-β-xylanase enzyme concentrate at the highest dose tested (1000 mg/kg bw/d), corresponding to 940 mg TOS/kg bw/d, showed slight effects that were not considered toxicologically significant by the notifier. Based on the highest dose tested in the 90-day study, and the estimated dietary exposure from the intended uses of the endo-1,4-β-xylanase enzyme preparation, i.e. 940 mg/kg bw/d and 0.4 mg TOS/kg bw/d, respectively, AB Enzymes calculates the margins of safety to be approximately 2300.
AB Enzymes discusses potential food allergenicity of endo-1,4-β-xylanase enzyme. AB Enzymes conducted an 80-amino acid sequence homology search for endo-1,4-β-xylanase enzyme against known allergens stored in a variety of allergen databases, and found no sequence identity matches over 35% to known allergens. Additionally, AB Enzymes did not find any matches of contiguous stretches of six amino acids in the endo-1,4-β-xylanase enzyme sequence that would be cross reactive with an allergenic protein. AB Enzymes further cites the conclusions of several organizations and working groups about the low risk of allergenicity posed by enzymes due to their low use levels and the extensive processing of enzyme-containing foods during manufacturing. Based on the totality of the information available, AB Enzymes concludes that it is unlikely that oral consumption of endo-1,4-β-xylanase enzyme will result in allergenic responses.
Based on the data and information summarized above, AB Enzymes concludes that endo-1,4-β-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 AB Enzymes’s notice that endo-1,4-β-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 endo-1,4-β-xylanase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain endo-1,4-β-xylanase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by AB Enzymes, as well as other information available to FDA, the agency has no questions at this time regarding AB Enzymes’ conclusion that the endo-1,4-β-xylanase enzyme preparation produced in T. reesei carrying a gene that expresses endo-1,4-β-xylanase enzyme in T. flexuosa is GRAS under the intended conditions of use. The agency has not, however, made its own determination regarding the GRAS status of the use of endo-1,4-β-xylanase enzyme preparation produced in T. reesei carrying a gene that expresses endo-1,4-β-xylanase enzyme in T. flexuosa. As always, it is the continuing responsibility of AB Enzymes 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 000628, 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
 Previously named as Actinomadura flexuosa, Microtetraspora flexuos, or Nonomuria flexuosa.
 RF5427 has been deposited in the Centraalbureau voor Schimmelcultures (CBS) in the Netherlands with the deposit number CBS114044.
 RF4847 is a classical mutant strain originating from T. reesei QM6a, and its identity has been confirmed by CBS, based on DNA sequencing.