April 20, 2001

Mr. Robert H. Sindt
Attorney at Law
1850 M Street, N. W.
Suite 400
Washington, DC 20036

Re: GRAS Notice No. GRN 000065

Dear Mr. Sindt:

The Food and Drug Administration (FDA) is responding to the notice, dated December 19, 2000, that you submitted on behalf of Rhodia, Inc. (Rhodia), 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 December 20, 2000, and designated it as GRAS Notice No. GRN 000065.

The subject of the notice is nisin derived from Lactococcus lactis subsp. lactis. The notice informs FDA of the view of Rhodia that nisin is GRAS, through scientific procedures, for use on casings for frankfurters and on cooked meat and poultry products as an antimicrobial agent. Nisin would be used in casings for frankfurters at a level of 3.15 milligrams (mg) per pound (lb) (equivalent to approximately 6.9 mg per kilogram (kg)) in the finished frankfurter and in cooked meats and poultry products sold ready-to-eat at a level of 2.5 mg/ lb (equivalent to 5.5 mg/kg).

Rhodia describes generally available information about the identity, characteristic properties, and functionality of nisin. Nisin is a small antimicrobial peptide produced by certain strains of Lactococcus lactis subsp. lactis, a common lactic acid-producing bacterium. Nisin is composed of 34 amino acids and has a molecular weight of approximately 3,510 Da. The Chemical Abstract Service (CAS) registry number for nisin is 1414-45-5. FDA has affirmed that nisin derived from certain strains of Lactococcus lactis subsp. lactis⁽¹⁾ is GRAS for use as an antimicrobial agent in various cheese products when used at a level that delivers a maximum of 250 parts per million of nisin in the finished product (21 CFR 184.1538).

Rhodia describes the manufacturing process for nisin. A fermentation medium, including yeast extract, corn steep liquor, soy protein peptone and starch is sterilized in high-grade stainless steel fermentation tanks. The medium is cooled to an appropriate incubation temperature and inoculated with a pure culture of nisin-producing Lactococcus lactis subsp. lactis. Nisin production is allowed to proceed under controlled time and temperature conditions until optimum production is achieved. The fermentation is then stopped by chilling and/or heat treatment. The nisin produced is concentrated by processes which may include foam precipitation, sodium chloride precipitation, precipitation under acidic conditions, or ultrafiltration. The resulting nisin concentrate is centrifuged and dried either at 140 degrees Fahrenheit (140F) for 4 hours in an oven, or at 180 F in a spray dryer. The dried concentrate is standardized with sodium chloride to achieve an activity of not less than 10⁶ IU nisin/gram as measured by the British Standards Institution Methods, "Methods for the Estimation and Differentiation of Nisin in Processed Cheese," BS4020 (1974). This method is currently cited in 21 CFR 184.1538(d) for use in determining nisin in cheese. The final product complies with the specifications for nisin preparation in 21 CFR 184.1538(b).

Rhodia provides intake estimates for nisin from the proposed use based on the 1994-1996 Continuing Survey of Food Intakes by Individuals (CSFII) 2-day survey. Rhodia estimates the eaters-only intake of nisin from the intended use to be approximately 0.6 milligrams per person per day (mg/p/d) at the mean consumption level and approximately 1.1 mg/p/d at the 90th percentile consumption level.

Rhodia's notice describes an unpublished 3-generation rat reproduction study, and published and unpublished studies on antibiotic resistance potential. Rhodia's notice also describes a published rat reproduction/chronic toxicity study by Frazer et al. (Ref. 3). The authors of the study reported no adverse effects of nisin on survival or reproductive performance in animals of the parental generation. No alterations of gastrointestinal, kidney, or liver function were observed in the F1 rats. The authors also concluded organ weights and gross or microscopic pathological findings were normal in either the parental or F1 generations. This same publication also examined the potential of sensitization to nisin by testing guinea pigs via both oral and intraperitoneal (i.p.) injection routes. The authors reported that nisin can be sensitizing via i.p. injection but not by oral treatment.

Based on its review of the published rat reproduction/chronic toxicity study by Frazer et al. (Ref. 3), FDA accepted the highest dose tested in that study (3,330,000 units of nisin per kg of diet, equivalent to 83.25 mg per kg of diet) as the no-observed-effect-level (NOEL) and affirmed the GRAS status of nisin derived from Lactococcus lactis subsp. lactis for use in processed cheese ¬53 FR 11247 (April 6, 1988)|. The Joint Food and Agriculture Organization/World Health Organization's (FAO/WHO) Expert Committee on Food Additives (JECFA) also accepted the highest dose tested in that study as the NOEL (Ref. 4).

The notice includes the report of a panel of individuals (Rhodia's GRAS panel) who evaluated the data and information that are the basis for Rhodia's GRAS determination. Rhodia considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. Rhodia's GRAS panel concludes that nisin, produced in accordance with current GMP and meeting appropriate food grade specifications, is GRAS based on scientific procedures for use as an anti-microbial on cooked meat and poultry products as specified in the notice. Rhodia considers this conclusion to be consistent with FDA's decision to affirm the GRAS status of nisin derived from Lactococcus lactis subsp. lactis for use as an antimicrobial in processed cheese.

Based on the information provided by Rhodia, as well as other information available to FDA, the agency has no questions at this time regarding Rhodia's conclusion that nisin derived from Lactococcus lactis subsp. lactis 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 nisin. As always, it is the continuing responsibility of Rhodia to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.

During its evaluation of GRN 000065, FDA consulted with the Labeling and Consumer Protection Staff of the Food Safety and Inspection Service of the United States Department of Agriculture (FSIS). The conclusions from FSIS are on Rhodia's antimicrobial formulations containing nisin rather than on nisin itself. Based on the information submitted by Rhodia, FSIS concludes that Rhodia's nisin-containing antimicrobial formulations, when used under the conditions specified in the notice, would be acceptable to control L. monocytogenes in various non-standardized meat and poultry products. FSIS requests that Rhodia be advised that it may be necessary to modify regulatory standards where a standard of identity prohibits or limits the use of an ingredient. At this time, FSIS is not initiating any substance-specific rulemaking activities to amend food standards until its ongoing standards modernization activities are completed. However, FSIS will be able to address requests for the use of any antimicrobial formulations containing nisin on meat and poultry products with prevailing standards of identity provided they are descriptively labeled to clearly distinguish them from the traditional standardized products. It is our understanding that Rhodia will seek regulatory guidance from FSIS concerning the use of these antimicrobial formulations in standardized meat and poultry products. Rhodia should direct its inquiry to Dr. Robert Post, Director, Labeling and Consumer Protection Staff, Office of Policy, Program Development and Evaluation, Food Safety and Inspection Service, 300 12th Street, SW, Room 602, Washington, DC 20250-3700. The telephone number of his office is (202) 205-0279 and the FAX number is (202)205-3625.

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 Rhodia'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 http://www.cfsan.fda.gov/~lrd/foodadd.html).

References

1. Schleifer, K. H., Kraus, J., Dvorak, C., Kilpper-Bälz, R., Collins, M. D., Fischer, W.: Transfer of Streptococcus lactis and related streptococci to the genus Lactococcus gen. nov. System. Appl. Microbiol. 6:183-195 (1985).

2. W. A. Volk et al., Essentials of Medical Microbiology, 4th ed., 1991; M. E. Stiles and W. H. Holzapfel, Int. J. Food Microbiol 36:1-29,1997.

3. Frazer, A.C., Sharratt, M., and Hickman, J.R., The biological effects of food additives. I. Nisin. J. Sci. Food Agric. 13: 32-42, 1962.

4. World Health Organization Technical Report Series No. 430, Specifications for the Identity and Purity of Food Additives and Their Toxicological Evaluation: Some Antibiotics, Twelfth Report of the Joint FAO/WHO Expert Committee on Food Additives, Geneva, 1969.

⁽¹⁾Under 21 CFR 184.1538, the source of nisin is listed as Streptococcus lactis Lancefield Group N. Streptococcus lactis has been reclassified as Lactococcus lactis subsp. lactis (Ref. 1). The Lancefield grouping is a serological classification for streptococci. Streptococci that are found in dairy products, the so-called lactic streptococci, are of antigen group N. (Ref. 2).

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