Agency Response Letter
GRAS Notice No. GRN 000198

Edward A. Steele
President, AAC Consulting Group
7361 Calhoun Place
Suite 500
Rockville, MD 20855-2765

Re: GRAS Notice No. GRN 000198

Dear Mr. Steele:

The Food and Drug Administration (FDA) is responding to the notice, dated April 5, 2006, that you submitted on behalf of EBI Food Safety B.V. (EBI) 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 this notice on April 6, 2006, filed it on April 20, 2006, and designated it as GRN No. 000198.

The subject of the notice is bacteriophage P100 preparation from Listeria innocua.⁽¹⁾ For clarity, in the remainder of this letter FDA will use the term bacteriophage P100 preparation when referring to the subject of the notice and use the term bacteriophage when referring to the phage that is a component of the preparation. The notice informs FDA of the view of EBI that bacteriophage P100 preparation is GRAS, through scientific procedures, as an antimicrobial to control L. monocytogenes in brie, cheddar, Swiss, and other cheeses that are normally aged and ripened.

Our use of the term "bacteriophage P100 preparation" in this letter should not be considered an endorsement or recommendation of this term as an appropriate common or usual name for the purpose of declaring this substance in the ingredient statement of foods that contain this ingredient. As stated in 21 CFR 101.4, all ingredients must be declared by their common or usual name. In addition, 21 CFR 102.5 outlines general principles to use when establishing common or usual names for non-standardized foods. Issues associated with labeling and the appropriate common or usual name of a food are the responsibility of the Office of Nutritional Products, Labeling, and Dietary Supplements in the Center for Food Safety and Applied Nutrition.

As part of its notice, EBI includes the report of a panel of individuals (EBI's GRAS panel) who evaluated the data and information that are the basis for EBI's GRAS determination. EBI considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. EBI's GRAS panel discusses host and phage identities, method of manufacture, specifications, an estimated dietary intake for the intended use in cheese, and safety studies for the bacteriophage P100 preparation. EBI's GRAS panel also discusses a published assessment of the gene protein products in bacteriophage P100 preparation to determine its potential pathogenicity, virulence, and allergenicity. EBI's GRAS panel concludes that bacteriophage P100 preparation is GRAS as an antimicrobial at levels up to 1 x 10⁹ pfu/g of cheese.

EBI describes the bacteriophage in the bacteriophage P100 preparation as an isolate from wastewater sources. Bacteriophage P100 preparation targets L. monocytogenes as well as several other species of Listeria. The bacteriophage is cultivated for commercial production in L. innocua. EBI describes a published review of publications demonstrating that L. innocua is a non-pathogenic bacterial species that genetically is similar to L. monocytogenes but lacks functions associated with pathogenicity. EBI notes that virulent factors associated with L. monocytogenes, (listeriolysin O, Phospholipase B & C, Actin polymerization protein, Internalin A & B), are absent from L. innocua. This bacteriophage is strictly lytic ⁽²⁾ and it is one of the few known virulent phages for the genus Listeria. The bacteriophage features a broad host range within the genus Listeria; once an infection has been established it infects and kills more than 95 percent of the different strains belonging to serovars 1/2, 4 (L. monocytogenes) and 5 (L. ivanovii).

EBI describes the method and manufacture for the bacteriophage P100 preparation. EBI notes that the production process is a common fermentation batch process using standard media for bacterial culture and considers substances used in the process to be GRAS. L. innocua cells are cultured to a specified density, then infected with the bacteriophage, which are amplified during further incubation. In a subsequent series of filtrations, host cells and cell debris are removed. EBI provides a table detailing the specifications for the bacteriophage P100 preparation.

In estimating the consumer's intake of bacteriophage P100 preparation, EBI assumes that the maximum concentration of bacteriophage P100 preparation is used (1 x 10⁹ pfu/g of cheese) and is ingested by the consumer. Assuming the 90^th percentile consumption of cheese,⁽³⁾ intake of bacteriophage P100 preparation from all treated cheese would be 5.7 x 10¹⁰ pfu. Based on the total mass of each phage unit (1.2 x 10⁸ daltons), the total weight of phage consumed is 6.8 x 10¹⁸ daltons. The total intake for bacteriophage P100 preparation can then be calculated as approximately 11.35 micrograms per person per day (µg/p/d) or 0.16 micrograms per kilogram body weight per day (µg/kg/d) for a 70 kg person. EBI believes this to be a conservative estimated intake. EBI indicates that cheese manufacturers would use the minimal amount of bacteriophage to achieve the intended technical effect. Further, after the host bacteria L. monocytogenes is depleted in the cheese, the bacteriophage would cease replication and would gradually become inactive.

EBI notes that there are numerous published references attesting to the fact that humans are exposed to huge numbers of phages daily, through food and water, without notable evidence of any harm.

EBI describes their conclusions drawn from a published assessment of the potential pathogenicity, virulence, allergenicity and toxicity of bacteriophage P100 preparation or the bacteriophage, including the complete genome sequence of the bacteriophage, as follows:

• EBI concludes that an analysis of the sequences does not reveal any similarities of the bacteriophage P100 preparation genes or any of its 174 predicted gene products to any genes or proteins or other factors known or supposed to play a direct or indirect role in pathogenicity or virulence of L. monocytogenes, or any other infectious, toxin-producing or otherwise harmful microorganism.
• Based on their analysis of the genetic structure, EBI concludes that there is little likelihood for lysogeny because there is no homology to known lysogenic factors.
• Predicted gene products of the bacteriophage P100 preparation were aligned with proteins known or suspected to be potential food allergens and one protein (gp71) displayed a local similarity in its C terminal domain to a gamma-gliadin protein of wheat. However, through bioinformatic analyses and through sequence comparisons, EBI concludes that such protein has a negligible probability to act as a potential immunoreactive allergen.
• Based on a subacute toxicity study in rats, EBI concludes that the bacteriophage P100 preparation had no gastrointestinal effects and is not toxic.

Standards of Identity

In the notice, EBI states its intention to use bacteriophage P100 preparation in brie, cheddar, Swiss, and other cheeses that are normally aged and ripened, including those 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.

Conclusions

Based on the information provided by the notifier, as well as other information available to FDA, the agency has no questions at this time regarding EBI's conclusion that bacteriophage P100 preparation is GRAS under the intended conditions of use. The agency has not, however, made its own determination regarding bacteriophage P100 preparation. As always, it is the continuing responsibility of EBI 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 responding to GRN 000198, as well as a copy of the information in this notice that conforms to the information in the proposed GRAS exemption claim (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).

Sincerely,

Laura M. Tarantino, Ph.D.
Director
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition

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⁽¹⁾ FDA notes that the bacteriophage preparation listed in 21 CFR 172.785 utilizes L. monocytogenes as the host microorganism.

⁽²⁾ Lytic bacteriophages lyse (destroy) their host bacteria as a normal part of their life cycle without integrating into the host genome.

⁽³⁾ EBI notes that in USDA (1991), the 90^th percentile consumption of cheese, excluding cottage and cream cheese, for the total US population is 57 g/day, with 50^th percentile intake of 28 g/day.