Agency Response Letter GRAS Notice No. GRN 000068

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CFSAN/Office of Premarket Approval*

May 29, 2001

Mr. David R. Joy
Keller and Heckman, LLP
Name of Company
1001 G Street, N.W.
Washington, DC 20001

Re: GRAS Notice No. GRN 000068

Dear Mr. Joy:

The Food and Drug Administration (FDA) is responding to the notice, dated January 31, 2001, that you submitted on behalf of Amano Enzyme, Inc., (Amano) 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 the notice on January 31, 2001 and designated it as GRAS Notice No. GRN 000068.

The subject of the notice is lipase enzyme preparation derived from Penicillium camembertii. The notice informs FDA of the view of Amano that this lipase enzyme preparation is GRAS, through scientific procedures, for use in the production of ingredients derived from fats and oils. The lipase enzyme preparation would be added at up to one percent of the weight of the oil or fat that is the intended substrate for the enzyme. Amano estimates that dietary exposure to this enzyme preparation from its intended use would be approximately one milligram (mg) per person per day for a 60 kilogram adult, equivalent to 0.017 mg per kilogram body weight per day (mg/kg bw/day).

Commercial enzyme preparations that are used in food processing 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 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. Amano provides information about each of these components of the lipase enzyme preparation.

Consistent with scientific publications and recommendations regarding the development of enzyme preparations that would be safe for use in food processing, Amano describes published and unpublished information pertaining to the safety of the production strain, P. camembertii U-150. In general, this information falls into two categories - i.e., human consumption of food containing P. camembertii and a scientific study of the pathogenic potential of P. camembertii. P. camembertii is the organism that traditionally was used in the production of Camembert cheese, although another species (P. candidum) has largely supplanted this use of P. camembertii in recent years. In addition, Amano describes a published study in which mice were inoculated with up to one million viable spores of P. camembertii. Because fungi were not recovered from the mice, Amano concludes that the mouse immunological system rapidly inactivated P. camembertii spores.

Amano describes the manufacturing process for lipase enzyme preparation, which is produced by a controlled fermentation of the P. camembertii strain with conditions specified for time, temperature, and rate of rotation. The production strain secretes the enzyme into the medium. Amano separates enzyme from the fungal cells by a sequence of filtration steps (using press filters and ultrafilters for a 6000 molecular weight cut), followed by heat treatment and pH adjustment (to pH 6.4 to 6.6), then additional filtration (press filter and ceramic filter). The resulting fluid is then precipitated with the addition of cold ethanol and acidification to pH 4.9 to 5.1. The resulting precipitate is centrifuged, dried, crushed, and blended with a diluent to the desired enzyme activity for the finished product. To dilute the enzyme preparation to its final activity, Amano uses dextrin, which FDA affirmed as GRAS in 21 CFR 184.1277. The final enzyme preparation is sieved through 42 mesh screen. The total organic solids (TOS) content of the commercial enzyme is approximately 35 percent. Amano describes lipase enzyme preparation as a powder, pale yellowish brown to pale brown in color, with an activity of at least 50,000 U/g.(1) Lipase enzyme preparation complies with the general and additional requirements for enzyme preparations set forth in the Food Chemicals Codex, fourth edition (National Academy of Sciences, 1996). In addition, when lipase enzyme preparation was tested for the presence of mycotoxins (including aflatoxin B1, cyclopiazonic acid, ochratoxin A, penicillic acid, roridin A, zearalenone, roquefortine, and penitrem A) and for antibiotic production, no mycotoxins or antibiotics were detected (all analytical results were below detection limits, ranging from 0.5 to 250 parts per billion).

Amano's notice includes a published article that describes studies performed with lipase enzyme preparation produced from the production strain P. camembertii U-150. For these studies, the investigators utilized a crude preparation of lipase enzyme preparation (referred to as LGK). A 90-day gavage study conducted in rats using LGK suspended in distilled water at 0, 500, 1000, and 2000 mg/kg bw/day resulted in no observable adverse effects. Amano compares these exposures to its estimated daily intake for humans (i.e., 0.017 mg/kg bw/day) and concludes that the exposure to humans is within a safe range. Amano also discusses a bacterial mutagenicity study conducted in Salmonella typhimurium with and without metabolic activation. Because no increase in revertants was reported, with or without metabolic activation, Amano concludes that the lipase enzyme preparation does not show mutagenic potential.

Amano describes a published report of a single adverse event involving P. camembertii: a case of allergic alveolitis in a subject employed by a salami factory that used the organism in its fermentation process. Amano considers that the reaction was caused by inhalation of the organism. Because the lipase enzyme preparation does not contain the production microorganism, Amano concludes that this published report does not raise a safety concern.

Based on the information provided by Amano Enzyme, Inc., as well as other information available to FDA, the agency has no questions at this time regarding Amano's conclusion that lipase enzyme preparation derived from P. camembertii 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 lipase enzyme preparation. As always, it is the continuing responsibility of Amano 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 Amano's notice that conforms to the information in proposed 21 CFR 170.36(c)(1), is available for public review and copying on the Office of Premarket Approval's homepage on the Internet (at


Alan M. Rulis, Ph.D.
Office of Premarket Approval
Center for Food Safety and Applied Nutrition

(1)The enzyme is assayed by the vinyl laurate emulsion method, in which a unit of activity is defined as the quantity of enzyme that liberates one micromole of fatty acid per minute under the conditions of the assay.

* The Office of Premarket Approval became the Office of Food Additive Safety on June 18, 2001.

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