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U.S. Department of Health and Human Services

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Agency Response Letter GRAS Notice No. GRN 000292

CFSAN/Office of Food Additive Safety

September 29, 2009

Richard H. Jundzil
Dyadic International (USA), Inc
140 Intracoastal Pointe Drive
Suite 404
Jupiter FL 33477-5094

Re: GRAS Notice No. GRN 000292

Dear Mr. Jundzil:

The Food and Drug Administration (FDA) is responding to the notice, dated May 27, 2009, 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 May 28, 2009, filed it on June 02, 2009, and designated it as GRAS Notice No. GRN 000292.

The subject of the notice is cellulase enzyme preparation derived from a genetically modified strain of Myceliophthora thermophila (cellulase enzyme preparation). The notice informs FDA of the view of Dyadic International (USA), Inc. (Dyadic) that the cellulase enzyme preparation is GRAS, through scientific procedures, for use as an enzyme in the production of wine, beer and fruit juices.

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 manufacturing process. In its notice, Dyadic provides information about all the components of the cellulase enzyme preparation.

Cellulase is responsible for the endohydrolysis of 1,4-β-D-glucosidic linkages in cellulose, lichenin and cereal β-D-glucans and the hydrolysis of 1,4-linkages in β-D-glucans that contain 1,3-linkages. According to the classification of enzymes established by the International Union of Biochemistry and Molecular Biology, cellulase is identified by the Enzyme Commission number 3.2.1.4. Its accepted name is cellulase and its systematic name is 4-(1,3;1,4)-β-D-glucan 4-glucanohydrolase. The Chemical Abstract Service Registry number of cellulase is 9012-54-8.

Dyadic states that the production organism, M. thermophila, is nonpathogenic, nontoxigenic and meets the criteria for a safe production microorganism as described by expert groups. Dyadic details the development of the cellulase production strain, designated as Eg5#27, from the host strain, UV18pyr5. Dyadic describes the UV18pyr5 strain as a derivative of the wild type strain C1. The C1 strain was initially classified as Chrysosporium lucknowense based on morphological characteristics and was subsequently reclassified as M. thermophila based on genetic tests. Dyadic states that the C1 strain was deposited with the International Depository of the All Russian Collection of Microorganisms of the Russian Academy of Sciences, and was assigned Accession Number VKM F-3500D. Dyadic distinguishes the host strain UV18pyr5 from the C1 strain by its decreased sporulation capability, mycelial fragmentation, high level of protein synthesis and a mutation in the pyr5 gene encoding orotate phosphoribosyltransferase. As a result of this mutation, the UV18pyr5 strain requires uracil or uridine for growth.

The production strain was constructed by introducing into the UV18pyr5 strain two DNA fragments, one carrying the pyr5 gene and the other the eg5 gene, which encodes cellulase. Both genes originated from the ancestral strain C1. One of the resulting transformants, Eg5#27, which lost the requirement for uracil or uridine, was chosen as the production organism based on the elevated production of cellulase. The strain was further evaluated via Southern hybridization to confirm that the integrated DNA is stable and does not contain the sequences of cloning vectors.

Dyadic describes the manufacture of the cellulase using a liquid medium in a submerged fermentation of the genetically modified strain of M. thermophila. Each production batch is initiated from a frozen stock culture, which is tested for strain identity, purity and enzyme-production capability before use in manufacturing. All fermentation steps are conducted under controlled conditions and monitored for contaminating organisms. The cellulase is secreted to the fermentation broth and is subsequently recovered via purification and concentration steps including separation of the production strain by drum filtration followed by ultrafiltration and polish filtration. The resulting liquid concentrate is formulated as a liquid product and standardized according to product specifications using sorbitol, sodium chloride, sodium benzoate, potassium benzoate and caramel coloring. The total organic solids (TOS) content of the final cellulase enzyme preparation is approximately 19.5%. The cellulase preparation contains other active enzymes including β-glucanase and xylanase.

Dyadic states that the cellulase preparation conforms to the specifications for enzyme preparations described in the Food Chemicals Codex (6thedition) and to the current (2006) General Specifications and Considerations for Enzyme Preparations Used in Food Processing established by the FAO/WHO Joint Expert Committee on Food Additives.

Dyadic states that the cellulase is intended for use in production of wine, beer and fruit juices to hydrolyze 1,4-β-D-glucosidic linkages in cellulose. The recommended use levels range from 0.004 to 0.02 percent of the enzyme preparation by weight of food. Dyadic calculates a maximum intake of cellulase to be 0.194 mg TOS/kg body weight/day based on the maximum intended level in food of 0.02%. In its exposure calculation, Dyadic assumes no enzyme removal or inactivation though in reality the cellulase is expected to be removed from the food or inactivated during manufacture.

Dyadic summarizes unpublished toxicological studies conducted with the cellulase enzyme that include 14-day and 13-week oral toxicity studies in rats, a bacterial reverse mutation test (Ames test), an in vitro mammalian chromosomal aberration test, and a gene mutation test at the TK-locus of L5178Y mouse lymphoma cells. Dyadic states that the enzyme was not toxic in the 14-day toxicity study and that no systemic toxicity was observed in the 13-week study at the highest dose tested of 2.8 gram per kilogram body weight per day. Dyadic also concludes that the genotoxicity studies demonstrated no mutagenic or clastogenic effects.

Dyadic provides general and specific information to address potential allergenicity of cellulase. Dyadic states that enzymes have a long history of use in food and there are very few reports in the literature describing allergy symptoms elicited by ingested enzymes. Dyadic also provides the results of bioinformatics studies in which the amino acid sequence of cellulase is compared to the amino acid sequences of known allergens using criteria recommended in the FAO/WHO expert consultation (2001). Dyadic reports that no meaningful homology was found. Dyadic also states that consumer exposure to cellulase will be very low and concludes that the likelihood of allergic reactions to the enzyme would be extremely low.

Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FFDCA)

Section 301(ll) of the FFDCA prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FFDCA, 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 Dyadic’s notice that the cellulase enzyme preparation is GRAS for use in the production of wine, beer, and fruit juices, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing the cellulase enzyme preparation. Accordingly, this response should not be construed to be a statement that foods that contain the cellulase enzyme preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).

Conclusions

Based on the information provided by Dyadic, as well as other information available to FDA, the agency has no questions at this time regarding Dyadic’s conclusion that the cellulase 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 the cellulase enzyme preparation. As always, it is the continuing responsibility of Dyadic 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 000292, 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 via the FDA home page at http://www.fda.gov. To view or obtain an electronic copy of the text of the letter, follow the hyperlinks from the “Food” topic to the “Food Ingredients and Packaging” section to the “Generally Recognized as Safe (GRAS)” page where the GRAS Inventory is listed.

Sincerely,

Mitchell A. Cheeseman, Ph.D.
Acting Director
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition