U. S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
Office of Food Additive Safety
August 14, 2001

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

Re: GRAS Notice No. GRN 000075

Dear Ms. Gregg:

The Food and Drug Administration (FDA) is responding to the notice, dated April 11, 2001, that you submitted on behalf of Novozymes North America, Inc. (Novozymes) 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 your notice on April 13, 2001 and designated it as GRAS Notice No. GRN 000075.

The subject of the notice is lipase enzyme preparation obtained from Aspergillus oryzae (A. oryzae) carrying a recombinant gene encoding a lipase from Fusarium oxysporum (F. oxysporum). The notice informs FDA of the view of Novozymes that the lipase preparation is GRAS, through scientific procedures, for use as a processing aid in the modification of fats and oils and in baking applications. The enzyme catalyzes the hydrolysis of ester bonds of triglycerides and diacylphospholipids, and the lipase formulations would be used at the following levels: 1 kilogram (kg) to produce 1 ton of de-gummed oil, 1 kg to produce 400 kg modified lecithin, 1 kg to produce 500 kg modified egg yolk, and 5 grams (g) per 100 kg flour.

The notice describes scientific publications and recommendations issued by international organizations on the safety of enzymes used in food processing, including enzymes produced from bioengineered organisms. As discussed in these documents, the safety of an enzyme preparation depends on the safety of the enzyme itself, the host organism, the inserted genetic material, the production organism, and the manufacturing process used in producing the enzyme preparation. The notice includes a safety evaluation of each of these components in support of Novozymes' GRAS determination.

In assessing the safety of the enzyme itself, the notice discusses the history of safe use of lipases in food processing. The notice cites a published article and monograph reporting the use of microbial lipases in food production since 1952. Novozymes also notes that several lipases (animal and Rhizopus niveus) are affirmed as GRAS, lipase from Mucor miehei (now known as Rhizomucor miehei) is approved for use as a food additive, and a lipase preparation produced by A. oryzae expressing a Thermomyces lanuginosus lipase is the subject of GRAS Notice No. 000043 (GRN 000043). The notice includes published and unpublished structural and sequence information for several of these lipases as well as for the subject lipase (produced from A. oryzae containing the F. oxysporum lipase). Novozymes concludes that the subject lipase preparation is substantially equivalent to other known lipases used in food production.

In assessing the safety of the host organism, A. oryzae, the notice refers to a review article on A. oryzae, describing the organism as having a long history of safe industrial use and as being commonly used in the production of food processing enzymes. Novozymes states that A. oryzae is nontoxigenic and nonpathogenic based on criteria given in a published article and notes that A. oryzae is also considered nonpathogenic by JECFA (The Joint Food and Agriculture Organization/World Health Organization's (FAO/WHO) Expert Committee on Food Additives). The notice also describes the specific strain of A. oryzae used as the host organism: Jal228 is an amylase negative, alkaline protease negative, neutral metalloprotease I negative derivative of the fully-characterized, well-known industrial production strain of A. oryzae (Ahlburg) Cohn.

The notice provides information about the plasmid, pMStr20, used in the construction of the A. oryzae production strain. The plasmid contains defined fungal chromosomal DNA fragments and synthetic DNA linker sequences including a promoter sequence from an A. niger neutral amylase II (NA2) gene, the 5' non-translated leader sequence of an A. nidulans triose phosphate isomerase gene, the DNA sequence encoding the F. oxysporum lipase, a terminator sequence from an A. niger amyloglycosidase gene, an A. nidulans acetamidase selectable marker gene, and the E. coli cloning plasmid vector pUC19. The notice cites published scientific articles to support Novozymes' view that these DNA sequences are well-known, well-characterized, and commonly used.

At the request of OFAS, Novozymes provided additional information (dated June 18, 2001) on the safety of the donor strain for the lipase gene, F. oxysporum. Novozymes notes that this particular fungus is not generally regarded as a primary pathogen. However, specific strains of F. oxysporum produce toxic secondary metabolites, including fusaric acid, monoliformine, and zearalenone and have been associated with eye infections in humans. Novozymes concludes that the pathogenic and toxigenic potential of F. oxysporum is not a safety concern in the lipase preparation because only the coding sequence of the lipase enzyme is introduced into the production organism, A. oryzae. No genetic sequences involved in secondary metabolite production or pathogenic properties are introduced into A. oryzae that would increase its toxic potential.

The notice discusses the safety of the A. oryzae production strain, designated MStr115. This strain is a spontaneous mutant of strain MStr110, constructed by transformation of the host strain Jal228 with the lipase expression fragment, a purified DNA fragment from plasmid pMStr20. The production organism complies with the Organization for Economic Co-operation and Development criteria for Good Industrial Large Scale Practice microorganisms and meets the criteria for a safe production microorganism described in scientific publications and recommendations issued by international organizations. Using the Southern hybridization technique, Novozymes assessed the identity and stability of the introduced DNA 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 notice describes the manufacturing process used to produce the lipase preparation as a two-step process: submerged fed-batch pure culture fermentation of the A. oryzae production strain and recovery (which includes purification and formulation). Novozymes follows standard industry practices and uses a quality management system that complies with the requirements of ISO 9001. In addition, the materials used in the fermentation and recovery processes are standard ingredients used by the enzyme industry; the notice cites several published articles to support this statement. The notice also lists specifications for the lipase enzyme preparation that comply with the specifications for enzyme preparations provided in the Food Chemicals Codex (4th ed., 1996) and the specifications provided by JECFA in Compendium of Food Additive Specifications, volume 2 (JECFA, 1992).

Novozymes' notice includes an unpublished summary of toxicology studies performed with the lipase preparation. These studies include a 13-week subchronic oral toxicity study in rats, an Ames mutagenicity test, and an in vitro cytogenetic test in cultured human lymphocytes. The studies showed no treatment related toxicity, induction of gene mutation, or chromosomal aberrations.

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 lipase enzyme preparation obtained from Aspergillus oryzae carrying a recombinant gene encoding a Fusarium oxysporum lipase 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 enzyme preparation. As always, it is Novozymes' continuing responsibility 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).

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