U. S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
Office of Food Additive Safety
December 19, 2001

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

Re: GRAS Notice No. GRN 000079

Dear Mr. Carroll:

The Food and Drug Administration (FDA) is responding to the notice, dated June 29, 2001, 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 July 3, 2001, and designated it as GRAS Notice No. GRN 000079.

The subject of the notice is alpha-amylase enzyme preparation from Bacillus licheniformis (B. licheniformis) carrying a gene constructed from a modified B. licheniformis alpha-amylase gene and a portion of the Bacillus amyloliquefaciens (B. amyloliquefaciens) alpha-amylase gene. The notice informs FDA of the view of Novozymes North America, Inc. (Novozymes) that this alpha-amylase enzyme preparation is GRAS, through scientific procedures, for use as a processing aid for the liquefaction of starch in the production of syrups and for the thinning of starch in distilling mashes in the production of alcohol. The alpha-amylase enzyme preparation is used at minimum levels necessary to achieve the intended effects. The recommended use level for the alpha-amylase enzyme preparation is 0.35 kilogram per ton of starch.

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 alpha-amylases in food processing. The notice cites a published article and monograph reporting the use of microbial alpha-amylases in food production since the 1950's. FDA has affirmed the GRAS status of a mixed carbohydrase (predominantly alpha-amylase) and protease enzyme product derived from B. licheniformis (21 CFR 184.1027). Novozymes has previously notified FDA about its determination that two other modified alpha-amylase enzyme preparations derived from B. licheniformis are GRAS (GRAS Notices No. GRN 000022 and GRN 000024). FDA responded to these GRAS notices with letters stating that the agency had no questions at that time regarding the GRAS determinations.

The notice includes published and unpublished structural and sequence information for several alpha-amylases, including the alpha-amylase that is the subject of GRN 000079. The alpha-amylase discussed in GRN 000079 is the same as the alpha-amylase that is the subject of GRN 000022 except that four amino acids have been changed. Both of the alpha-amylases are stable to heat, as are several other commercially available alpha-amylases. However, the alpha-amylase that is the subject of GRN 000079 can operate at lower pH and lower calcium levels than other commercially available thermostable alpha-amylases. Novozymes concludes that the alpha-amylase that is the subject of GRN 000079 is functionally equivalent to other alpha-amylases used in food production, catalyzing hydrolysis of 1,4-alpha-glucosidic linkages in amylose and amylopectin.

In assessing the safety of the host organism, B. licheniformis strain SJ1707, the notice describes the host strain as a sporulation deficient and alkaline protease negative derivative of the fully-characterized, well-known natural isolate of B. licheniformis strain ATCC 9789. Novozymes considers B. licheniformis to be nontoxigenic and nonpathogenic based on published criteria for the assessment of the safety of microorganisms used in the manufacture of food ingredients. Strain SJ1707 was the host strain for the production organisms described in GRAS Notices GRN 000022 and 000024. The parent of strain SJ1707 (i.e., strain ATCC 9789) has been described in the published literature and has been used for industrial production of enzymes marketed by Novozymes since 1972, including the mixed carbohydrase and protease enzyme preparation that FDA affirmed as GRAS (21 CFR 184.1027).

The notice provides information about the components of plasmid vectors introduced into the host strain SJ1707 by sequential transformation and conjugation. The notice cites published scientific articles to support Novozymes' 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.

The final production strain, designated B. licheniformis strain MOL2803, 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 techniques of Southern hybridization, amplification by polymerase chain reaction (PCR), and DNA sequencing, Novozymes assessed the identity, location, and stability of the introduced DNA. Novozymes concludes that the DNA is stably integrated at three distinct sites on the B. licheniformis chromosome as expected and is not prone to genetic transfer to other organisms.

The notice describes the manufacturing process used to produce the alpha-amylase enzyme preparation. The first step is submerged fed-batch pure culture fermentation of the B. licheniformis production strain. Fermentation is followed by the recovery process, which includes purification and formulation. The materials used in the fermentation and recovery processes are standard materials used by the enzyme industry. Novozymes follows standard industry practices and uses a quality management system that complies with the requirements of ISO 9001. Novozymes provides specifications for the alpha-amylase enzyme preparation, which comply with the specifications for enzyme preparations provided 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, Food and Agriculture Organization of the United Nations, Rome, 1992).

Novozymes describes an unpublished 2-week oral toxicity study in rats conducted with a test batch of the alpha-amylase enzyme preparation that was manufactured from the production strain (MOL2803) using the manufacturing process described above, except that final standardization was excluded. The study did not show any treatment related toxicity. Novozymes also provides an unpublished summary of toxicology studies performed with several other test substances produced by strains derived from B. licheniformis strain SJ1707 (i.e., the host strain used to develop the production strain). These studies include a 4-week and a 13-week oral toxicity study in rats, an Ames mutagenicity test, chromosome aberration test, and mouse lymphoma test. These studies showed no treatment related toxicity, induction of gene mutation, or chromosomal aberrations for test articles derived from the host strain.

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 alpha-amylase enzyme preparation from B. licheniformis carrying a gene constructed from a modified B. licheniformis alpha-amylase gene and a portion of the B. amyloliquefaciens alpha-amylase gene 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 alpha-amylase 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).

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