Agency Response Letter
GRAS Notice No. GRN 000122

Denise Bernstein
Novozymes North America, Inc.
77 Perry Chapel Church Road
P.O. Box 576
Franklinton, North Carolina 27525

Re: GRAS Notice No. GRN 000122

Dear Ms. Bernstein:

The Food and Drug Administration (FDA) is responding to the notice, dated January 9, 2003, 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 January 17, 2003, filed it on January 22, 2003, and designated it as GRAS Notice No. GRN 000122.

The subject of the notice is laccase enzyme preparation from Aspergillus oryzae expressing the gene encoding a laccase from Myceliophthora thermophila. The notice informs FDA of the view of Novozymes North America, Inc. (Novozymes) that the laccase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in breath-freshening products (such as breath mints and chewing gum) and other food products at minimum levels necessary in accordance with good manufacturing practice. Novozymes estimates that this use of laccase enzyme preparation as a direct food ingredient would result in the consumption of up to approximately 14 milligrams per person per day of the total organic solids present in the laccase enzyme preparation.

Commercial enzyme preparations that are used in food 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. Novozymes' notice provides information about each of these components of the laccase enzyme preparation from A. oryzae.

Novozymes describes generally available information about the identity and technical effect of laccase. The subject laccase is an 85 kDa protein, with three internal disulfide bonds and four copper atoms. Novozymes identifies the laccase enzyme by the following classification numbers: EC 1.10.3.2 and CAS Registry No. 80498-15-3. Laccase catalyzes the oxygen-dependent oxidation of phenolic substrates such as o-diphenols and p-diphenols to form their corresponding quinones and water. In the applications described in the notice, these quinone reaction products react non-enzymatically with odor causing compounds such as sulfides, thiols, and amines to eliminate odors in the oral cavity. Novozymes describes a published article that discusses the characterization of the gene encoding the laccase of M. thermophila and the subsequent analysis of the recombinant laccase as expressed in A. oryzae.

Novozymes cites published articles reporting the presence of laccase enzymes and phenolic compounds as naturally occurring components commonly found in human food sources such as fruits, vegetables, fungi, herbs, spices, teas, and coffee. Novozymes also notes that interactions involving laccase and phenolic substrates are responsible for the browning of freshly peeled or bruised fruit and vegetables. The browning reaction plays a role in the curing of tea and coffee. Novozymes describes the use of laccases in beverage production. In Denmark, the laccase enzyme is approved for use in brewing beer, where it is applied during the mashing process to prevent the formation of an off-flavor compound, trans-2-nonenal. Laccase has also been immobilized on a copper-chelate carrier and used to remove phenols from white grape must during clarification of wine.

Novozymes describes the host microorganism, A. oryzae strain How B711, as a derivative of a well-known industrial production strain of A. oryzae (Ahlburg) Cohn, which Novozymes obtained from the Institute for Fermentation, Osaka, Japan.⁽¹⁾ Novozymes considers A. oryzae to be nontoxigenic and nonpathogenic based on published criteria for the assessment of the safe use of microorganisms used in the manufacture of food ingredients.

Novozymes describes the development of its bioengineered production strain as a three-phase process: construction of a host strain with the necessary genetic background for subsequent genetic manipulations, transformation of the host strain with the laccase gene, and mutagenesis of the transformed host strain to obtain a high-yielding laccase production strain (designated Mt-3). Novozymes cites published scientific articles to support its view that all of the DNA sequences that were used in the construction of the production strain are well-known, well-characterized, and commonly used. Novozymes assessed the identity and stability of the introduced DNA using the technique of genomic DNA hybridization and concluded that the DNA is integrated into the A. oryzae chromosome as expected and is not prone to genetic transfer to other organisms. The resulting production strain meets the criteria for Good Industrial Large-Scale Practice published in the Organization for Economic Co-operation and Development's 1992 report entitled "Safety Considerations for Biotechnology."

Novozymes describes the manufacturing process for the laccase enzyme preparation, which is produced by submerged, fed-batch pure culture fermentation of the A. oryzae production strain. The enzyme is secreted into the fermentation broth and separated from the cells using filtration. The enzyme preparation is concentrated by ultrafiltration and evaporation. The enzyme preparation is then preserved and stabilized with the addition of sodium chloride. Novozymes follows standard industry practices and uses a quality management system that complies with the requirements of ISO 9001. Novozymes cites several published sources to support the conclusion that the production and control methods used are generally accepted methods that are commonly used for the production of microbial enzyme preparations. Novozymes states that the three mycotoxins reported to be made by some strains of A. oryzae (i.e., kojic acid, cyclopiazonic acid, and beta-nitropropionic acid) were not detected in four different batches of laccase preparation. Novozymes states that the enzyme preparation complies with the general and additional requirements for enzyme preparations set forth in the Food Chemicals Codex (4th ed., 1996) and the specifications established by the Joint Food and Agriculture Organization/World Health Organization's (FAO/WHO) Expert Committee on Food Additives (Compendium of Food Additive Specifications, Volume 2, Annex 1, Food and Agriculture Organization of the United Nations, 1992 as supplemented in Appendix B to Annex 1, FAO Food and Nutrition Paper 52, Addendum 6, 1998).

Novozymes includes in the notice a published summary of toxicological studies performed on the laccase preparation produced as described in the notice, except that stabilization and standardization were omitted. Novozymes also includes in the notice an unpublished summary of the same studies. These studies include a two-week and 13-week oral gavage study in rats, acute inhalation and acute dermal toxicity studies in rats, acute skin and acute eye irritation studies in rats, human skin sensitization test, and tests for genetic toxicity, including an Ames test and a chromosome aberration test with human lymphocytes. Novozymes concludes that these toxicity studies showed no treatment related toxicity and no induction of gene mutation in bacteria or chromosomal aberrations in cultured human blood lymphocytes.

Non-food uses of laccase enzyme preparation

In its notice, Novozymes provides use levels for non-food items such as toothpaste and mouthwash. Under proposed 21 CFR 170.36, FDA evaluates the view of a notifier that a particular use or uses of a substance in food is safe and that this safety is generally recognized by experts qualified by scientific training and experience to evaluate the safety of substances added to food. In its evaluation of GRN 000122, FDA did not evaluate the use of laccase enzyme in non-food items.

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 laccase enzyme preparation produced by A. oryzae expressing the gene encoding laccase from M. thermophila 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 this laccase 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 the 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. Acting Director Office of Food Additive Safety Center for Food Safety      and Applied Nutrition

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⁽¹⁾The Institute for Fermentation, Osaka designates A. oryzae (Ahlburg) Cohn as IFO 4177 or A1560.