February 23, 2001

Scott H. Shore, Ph.D.
Novozymes North America, Inc.
77 Perry Chapel Church Road
Box 576
Franklinton, NC 27525-0576

Re: GRAS Notice No. GRN 000054

Dear Dr. Shore:

The Food and Drug Administration (FDA) is responding to the notice, dated July 26, 2000, that Novozymes North America, Inc. (Novozymes; formerly Novo Nordisk BioChem North America, Inc.) 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)). FDA received the notice on July 28, 2000 and designated it as GRAS Notice No. GRN 000054.

The subject of your notice is xylanase enzyme preparation obtained from a strain of Fusarium venenatum. The strain of F. venenatum contains a recombinant gene, derived from Thermomyces lanuginosus, encoding endo 1,4- beta-xylanase. The notice informs FDA of the view of Novozymes that xylanase enzyme preparation is GRAS, through scientific procedures, for use as a processing aid in baked goods in accordance with current good manufacturing practices to improve handling and stability of the dough. The xylanase enzyme preparation would be used at a level of 2-16 grams per 100 kg flour (corresponding to 50-400 Fungal Xylanase Units per kg flour) to degrade the insoluble fraction of arabinoxylans in flour to improve handling and stability of the dough.

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 that derive from the source organism and constituents that derive from the manufacturing process, e.g., components of the fermentation media or the residues of processing aids. Your notice provides information about each of these components of xylanase enzyme preparation.

Your notice describes published information about xylanase enzyme (EC No.3.2.1.8) and the arabinoxylans that are the substrate for xylanase enzyme. Arabinoxylans are highly branched xylans that are found in the outer cell walls and endosperm of various cereals, and they exist in soluble and insoluble form. The soluble fraction is essential for the water retention capacity of flour. The insoluble fraction is coupled to proteins and is believed to reduce the elasticity of the gluten complex. The xylanase enzyme cleaves the beta-(1,4)-D-xylosidic linkages in the arabinoxylan backbone, resulting in depolymerization of the arabinoxylan into smaller oligosaccharides. The practical impact of xylanase activity towards both soluble and insoluble forms of arabinoxylans is increased elasticity of the gluten network.

Your notice describes scientific publications and recommendations issued by international organizations on the safety of enzymes used in food processing, including enzymes derived from genetically modified microorganisms. Consistent with those documents, your notice describes published and unpublished information pertaining to the safety of the host organism (F. venenatum strain MLY3) used in the construction of the xylanase production strain (F. venenatum strain LyMC4.B). F. venenatum strain MLY3 is a derivative of the F. venenatum strain CC1-3, which is a morphological mutant of the wild type F. venenatum strain A3/5 previously known as Fusarium graminearum strain A3/5. The A3/5 strain was deposited in the American Type Culture Collection as F. graminearum Schwabe ATCC 20334. Your notice describes a published scientific study that shows that F. venenatum is capable of producing several types of mycotoxins under specific culture conditions conducive to mycotoxin formation. These mycotoxins include a trichothecene diacetoxyscirpenol (DAS) and related metabolites isotrichodermin and isotrichodermol, the modified trichothecenes sambucinol and apotrichothecene, the sesquiterpenes culmorin, culmorone, and their derivatives, and trace amounts of the cyclic peptide enniatin B. Your notice also describes an internal report that summarizes published information on human and animal infections associated with Fusarium species. Novozymes found no reports connecting F. venenatum, F. sambucinum or any species within the Discolor section with human or animal infections. ⁽¹⁾ Novozymes concludes that F. venenatum is not a human pathogen.

Consistent with scientific publications and recommendations regarding the development of enzyme preparations that would be safe for use in food processing, your notice describes published and unpublished information pertaining to the safety of the various components of the plasmids that were introduced into F. venenatum strain MLY3, including the source of the xylanase gene (T. lanuginosus). The xylanase gene from T. lanuginosus is the same gene that was used in the construction of a recombinant strain of Aspergillus oryzae that is a commercially available source of xylanase enzyme. One of the plasmids used in the construction of the F. venenatum production strain LyMC4.B is used to delete the trichodiene synthase gene (tri5) from the F. venenatum MLY3 host strain by replacing it with the A. nidulans amdS gene. Because the trichodiene synthase is the first enzyme in the trichothecene biosynthetic pathway, deletion of the tri5 gene abolishes the synthesis of trichothecene mycotoxins in the xylanase production strain. The replacement gene amdS serves as a selectable marker by enabling the transformed F. venenatum cells to use acetamide as a sole carbon and nitrogen source. This selection system is commonly used in fungal cloning.

Consistent with scientific publications and recommendations regarding the development of enzyme preparations that would be safe for use in food processing, your notice describes published and unpublished information pertaining to the safety of the production strain (F. venenatum strain LyMC4.B). Using the technique of Southern hybridization, Novozymes assessed the identity and stability of the introduced DNA and demonstrated that the coding sequence of the tri5 gene is absent from the production strain LyMC4.B. Your notice describes a published study that compares the potential of the production strain LyMC4.B to produce mycotoxins to that of the wild type strain and several genetically modified strains of F. venenatum. The production strain LyMC4.B does not produce trichothecenes and modified trichothecenes. However, it does produce several culmorins, albeit at much lower levels that the wild type strain ATCC 20334. It also produces trace levels of enniatin B. Novozymes provides a scientific article, which has been accepted for publication, on the toxicity of culmorins and its own literature review on enniatins. Novozymes concludes that culmorins and enniatins have low toxicity to higher animals and that the low toxicity profile and low levels of culmorins and enniatins that may potentially be produced by the production strain do not present a toxicological concern.

Your notice also discusses the potential that the production strain LyMC4.B may synthesize fusarin C, which can be produced by F. venenatum under inducible conditions. Novozymes describes its own literature survey on fusarin C biosynthesis and concludes that the fermentation conditions used during enzyme production are different from those known to induce fusarin biosynthesis. Novozymes also describes its literature survey of toxicity data for fusarin C and concludes that the acute toxicity of fusarin C to humans is low. Finally, laboratory tests show no evidence that the production strain LyMC4.B produces fusarin C under normal industrial enzyme manufacturing conditions. For all of these reasons, you conclude that fusarin C does not present a toxicological concern.

Your notice includes a published article that describes several studies performed with the xylanase preparation produced from the production strain F. venenatum strain LyMC4.B. These studies include a 13-week oral toxicity study in rats, a test for mutagenicity (Ames test), and a test for cytogenicity (chromosomal aberrations in cultured human lymphocytes). The highest dose level used in the oral toxicity study (10 ml/kg bw /day; equivalent to 1.12 g total organic solids/kg bw/day) revealed no signs of toxic effects.

Your notice describes the manufacturing process for xylanase enzyme preparation, which is produced by a contained system of submerged fed-batch pure culture fermentation of F. venenatum strain LyMC4.B in accordance with current good manufacturing practices and a quality management system that complies with ISO 9001. The xylanase enzyme is recovered from the fermentation broth by a multi-step process that includes filtration, centrifugation, ultrafiltration, evaporation, and pre- and germ filtration. The latter step assures that the concentrated enzyme solution is free of the production strain and insoluble components from the fermentation medium. The enzyme concentrate is subsequently stabilized with sodium chloride, mixed with granulation aids such as dextrin and sorbitol syrup and spray dried. Xylanase enzyme preparation complies with the general and additional requirements for enzyme preparations set forth in the Food Chemical Codex (4th ed., 1996). Xylanase enzyme preparation also conforms to the general specifications for enzyme preparations used in food processing provided in 1992 by the Joint Expert Committee on Food Additives (JECFA; a joint committee of the Food and Agriculture Organization/World Health Organization).

Based on the information provided by Novozymes, as well as other information available to FDA, the agency has no questions at this time regarding your conclusion that 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 xylanase enzyme preparation. As always, it is the continuing responsibility of Novozymes to ensure that food ingredients that you market 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, as well as a copy of the information in your notice that conforms to the information in proposed 21 CFR 170.36(c)(1), is available for public review and copying on the Office of Premarket Approval's homepage on the World Wide Web.

⁽¹⁾The genus Fusarium is divided into sections. Fusarium venenatum belongs to the Discolor section along with several other Fusarium species such as F. graminearum, F. sambucinum. F. sulphureum, and F. cerealis (F. crookwellense).

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