Agency Response Letter
GRAS Notice No. GRN 000149

John Carroll
Novozymes North America, Inc.
77 Perry Chapel Church Road
P.O. Box 576
Franklinton, NC 27525

Re: GRAS Notice No. GRN 000149

Dear Mr. Carroll:

The Food and Drug Administration (FDA) is responding to the notice, dated March 26, 2004, 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 the notice on March 30, 2004, filed it on March 31, 2004, and designated it as GRAS Notice No. GRN 000149.

The subject of the notice is beta-glucanase enzyme preparation from Trichoderma harzianum.⁽¹⁾ The notice informs FDA of the view of Novozymes that beta-glucanase enzyme preparation from T. harzianum is GRAS, through scientific procedures, for use as an enzyme to improve the clarification and filtration of wines, especially those wines produced by Botrytis-infected grapes. The enzyme preparation is intended for use at minimum levels necessary to achieve the desired effect and is produced following good manufacturing practices (GMPs). Novozymes states that typical use levels of beta-glucanase enzyme preparation (standardized to 200 Botrytis beta-glucan units per gram of enzyme preparation) range from 1 to 3 grams per hectoliter of wine.

Commercial enzyme preparations that are used in food processing typically contain an enzyme component, which catalyzes the chemical reaction that is responsible for the technical effect of the enzyme preparation, and 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. Novozymes' notice provides information about each of these components of the beta-glucanase enzyme preparation.

In the wine industry, beta-glucanase may be used to improve clarification and filterability of wines, particularly wines made from botrytized grapes. The use of beta-glucanase in wine clarification and filtration has been described in the published literature. Novozymes describes the catalytic activity of beta-glucanase enzyme, which selectively degrades the beta-glucan polysaccharide produced in the wine grapes by the fungus Botrytis cinerea or by yeast. Beta-glucanase enzyme catalyzes the successive hydrolysis of beta-D-glucose units from the nonreducing ends of 1,3-beta-D-glucans, releasing D-glucose. Beta-glucanase enzyme is identified by the following classification numbers: EC 3.2.1.58 and CAS Registry No. 9073-49-8

Novozymes cites scientific review articles in support of its view that the safety of the production organism is the prime consideration in assessing the safety of an enzyme preparation intended for use in food. Novozymes' notes that strains of Trichoderma are widely used in research and industrial biotechnology as producers of industrial enzymes. Novozymes states the beta-glucanase enzyme preparation is obtained from a safe strain of T. harzianum that belongs to the Hyphomycetes (imperfect fungi) class. According to Novozymes, beta-glucanase from T. harzianum was developed in the 1980s, and has been approved for use in wine production in the European Union since 1992. Further, beta-glucanase enzyme preparation from T. harzianum has been evaluated by the Joint Expert Committee on Food Additives (JECFA) for use in winemaking. Novozymes has reviewed published information on T. harzianum and notes that only one report has associated T. harzianum as a cause of opportunistic infection in a single patient. Novozymes notes that T. harzianum is not considered to be a pathogenic microorganism and is not closely related to any known pathogenic fungi.

Novozymes acknowledges that T. harzianum is known as a producer of a wide variety of secondary metabolites, but also notes that there have been no published reports connecting isolates of T. harzianum or any other closely related species with production of secondary metabolites with known toxicity against humans or animals. In accordance with published recommendations for mycotoxin testing for enzyme preparations derived from certain fungal species, Novozymes has tested its enzyme preparation for trichothecene T-2 mycotoxin, which has been associated with Trichoderma sp. Novozymes' beta-glucanase enzyme preparation has tested negative for the presence of T-2 mycotoxin. Novozymes concludes that T. harzianum is nonpathogenic and nontoxigenic.

Novozymes' notice describes the manufacturing process for beta-glucanase enzyme preparation, which is produced by a submerged batch-fed pure culture fermentation of T. harzianum. Following fermentation, a multi-step recovery process is performed, which includes pH adjustment, purification, and concentration of the product. The resultant liquid concentrate of the beta-glucanase enzyme preparation is spray dried onto a maltodextrin carrier, passed through a sieve, and standardized according to product specifications. The total organic solids (TOS) content for a typical batch of beta-glucanase enzyme preparation is 0.668 mg TOS per unit of enzyme activity. Novozymes provides specifications for the beta-glucanase enzyme preparation that comply with the requirements for enzyme preparations as set forth in the Food Chemicals Codex (5th edition, 2004) and by JECFA (2001).

Novozymes' notice includes a published summary of toxicological studies performed with a liquid beta-glucanase enzyme concentrate, prepared according to the method of manufacture described in GRN 000149, except that stabilization and standardization were omitted. These toxicological studies include a 13-week subchronic oral toxicity study in rats, an in vitro test for mutagenicity (Ames test), and a chromosome aberration assay in mammalian cells. Novozymes' notice concludes that these tests show that the beta-glucanase enzyme preparation does not exhibit mutagenic activity or toxic effect under the specified testing conditions.

Conclusions

Based on the information provided by Novozymes, as well as other information available to FDA, the agency has no questions at this time regarding Novozymes' conclusion that beta-glucanase 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 enzyme preparation. As always, it is the continuing responsibility of Novozymes 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, 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 homepage of the Office of Food Additive Safety (on the Internet at http://www.cfsan.fda.gov/~lrd/foodadd.html).

Sincerely,    /s/ Laura M. Tarantino, Ph.D. Director Office of Food Additive Safety Center for Food Safety      and Applied Nutrition

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⁽¹⁾Novozymes states that "glucanase enzyme preparation" is the classification or generic name for the subject of the notice. However, OFAS refers to the subject of GRN 000149 as "beta-glucanase enzyme preparation" based on information provided in the notice and Novozymes' description of the mode of action of the enzyme.