U. S. Food and Drug Administration
Center for Food Safety and Applied Nutrition
Office of Food Additive Safety
February 19, 2002

Alan Richards, Ph.D.
Hayashibara International Inc.
8670 Wolff Court, Suite 200
Westminster, CO 80030

Re: GRAS Notice No. GRN 000085

Dear Dr. Richards:

The Food and Drug Administration (FDA) is responding to the notice, dated June 20, 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 September 21, 2001, and designated it as GRAS Notice No. GRN 000085.

The subject of the notice is isoamylase enzyme preparation from Pseudomonas amyloderamosa (P. amyloderamosa). The notice informs FDA of the view of Hayashibara International Inc. (Hayashibara) that isoamylase enzyme preparation from P. amyloderamosa is GRAS, through scientific procedures, for use in foods containing starch or to hydrolyze starch in the preparation of food ingredients. The isoamylase enzyme preparation from P. amyloderamosa is used at minimum levels necessary to achieve the desired technical effects. Hayashibara estimates that the mean exposure to this enzyme preparation from its intended use would be approximately 71 milligrams of total organic solids per person per day.

The notice includes the report of a panel of individuals (Hayashibara’s GRAS panel) who evaluated the data and information that are the basis for Hayashibara’s GRAS determination. Hayashibara considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Based on the data and information reviewed, Hayashibara’s GRAS panel concludes that isoamylase enzyme preparation from P. amyloderamosa is GRAS when produced and used in accordance with current good manufacturing practice at specified levels and when meeting Hayashibara’s specifications.

Commercial enzyme preparations, including those 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 as well as substances used as stabilizers, preservatives or diluents. Enzyme preparations may also contain constituents that derive from the source organism and constituents that derive from the manufacturing process, e.g., components of the fermentation media or the residues of processing aids. Hayashibara provides information about each of these components of the isoamylase enzyme preparation.

In assessing the safety of the enzyme itself, Hayashibara discusses the history of safe use of isoamylases in food processing. The notice cites published articles reporting the characterization of microbial isoamylases since the 1960's, and use in food production since the 1990's. Hyashibara also describes published studies characterizing the isoamylase secreted from P. amyloderamosa. Isoamylase from three genera of bacteria, including Pseudomonas, is approved for use as a food additive in Japan (Japanese Food Additive Regulations, 1998). In addition, isoamylase enzyme preparation from P. amyloderamosa has been used in the food industry in countries such as Japan for more than 20 years. FDA has recently approved the use of isoamylase to modify food starch (66 FR 17508; April 2, 2001), which amends 21 CFR 172.892.

The source of Hayashibara’s isoamylase enzyme preparation is P. amyloderamosa strain MU 1174. P. amyloderamosa is a naturally occurring soil microorganism, is not a human pathogen, and is nontoxigenic. In the notice, Hayashibara describes information from published studies about the parental P. amyloderamosa strain American Type Culture Collection (ATCC) 21262, from which the production strain was derived. The production strain, P. amyloderamosa MU 1174, was derived from the parental strain by chemical mutation using N-methyl-N’-nitro-N-nitrosoguanadine (NTG), a well-known and accepted method for generating random mutations and enhancing productivity of microbial sources of food ingredients. Hayashibara assesses the genetic stability of the production strain through ongoing testing of enzyme production during the manufacturing process. In the notice, Hayashibara describes unpublished studies to corroborate its view that the production strain, like the parental strain, is nontoxigenic and nonpathogenic.

Hayashibara describes the manufacturing process used to produce the isoamylase enzyme preparation. The production strain secretes the enzyme into the medium. Hayashibara monitors fermentation conditions. Hayashibara separates the enzyme from the production microorganism by membrane filtration, followed by ultrafiltration, purification with a precoat filter, and membrane filtration. The enzyme is stabilized and preserved by addition of glucose (20 percent), maltose (20 percent), and glycerol esters of fatty acids (0.1 percent). Water is added to normalize enzyme activity. Hayashibara’s isoamylase enzyme preparation is manufactured in accordance with current good manufacturing practice (cGMP) using standard methods used by the enzyme industry. Components of the fermentation media comply with Hayashibara’s raw material specifications and cGMP. The isoamylase enzyme preparation complies with the general and additional requirements for enzyme preparations set forth in the Food Chemicals Codex (4th edition, 1996). In addition, when the isoamylase enzyme preparation was tested for the presence of mycotoxins and for antimicrobial (antibiotic) activity, no mycotoxins or antibiotics were detected.

Hayashibara describes an unpublished 90-day oral gavage study in male and female Wistar rats conducted with isoamylase enzyme preparation from P. amyloderamosa. Daily treatment with the isoamylase enzyme preparation for 13 weeks caused no adverse effects at the highest dose tested. Hayashibara describes additional unpublished toxicology studies conducted with isoamylase enzyme preparation from P. amyloderamosa, including a bacterial reverse mutation assay, and an in vitro mammalian chromosome aberration study. These studies showed no treatment-related induction of gene mutation or chromosomal aberrations.

Based on the information provided by Hayashibara, as well as other information available to FDA, the agency has no questions at this time regarding Hayashibara’s conclusion that isoamylase enzyme preparation from P. amyloderamosa 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 isoamylase enzyme preparation. As always, it is the continuing responsibility of Hayashibara to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.

Under section 403(a) of the Federal Food, Drug, and Cosmetic Act (FFDCA), a food is misbranded if its labeling is false or misleading in any particular. Section 403(r) of the FFDCA lays out the statutory framework for a health claim. In describing the intended use of the isoamylase enzyme preparation and in describing the information that Hayashibara relies on to conclude that isoamylase enzyme preparation P. amyloderamosa is GRAS under the conditions of its intended use, Hayashibara raises potential labeling issues under these provisions of the FFDCA. These labeling issues consist of purported physiological effects of isoamylase enzyme preparation from P. amyloderamosa that Hayashibara views as "beneficial" - e.g., whether use of the isoamylase enzyme preparation results in food products that are hypocariogenic or lower in caloric value. If products that contain isoamylase bear any claims about such benefits on the label or in labeling, such claims are the purview of the Office of Nutritional Products, Labeling, and Dietary Supplements (ONPLDS) in the Center for Food Safety and Applied Nutrition (CFSAN). The Office of Food Additive Safety (OFAS) neither consulted with ONPLDS on these labeling issues nor evaluated the information in your notice to determine whether it would support any claims made about isoamylase enzyme preparation from P. amyloderamosa on the label or in labeling.

In the notice, Hayashibara states its intention to use isoamylase enzyme preparation from P. amyloderamosa in several food categories, including foods for which standards of identity exist, located in Title 21 of the Code of Federal Regulations. We note that an ingredient that is lawfully added to food products may be used in a standardized food only if it is permitted by the applicable standard of identity. If you have any questions about the use of this isoamylase enzyme preparation in standardized foods that would be marketed in the United States, you should contact the staff in ONPLDS, Division of Standards and Labeling Regulations, HFS-820, 5100 Paint Branch Parkway, College Park, MD 20740. You can reach this division by telephone at (301) 436-2371.

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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