Agency Response Letter GRAS Notice No. GRN 000603
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See also Generally Recognized as Safe (GRAS).
CFSAN/Office of Food Additive Safety
July 28, 2016
Rosemonde Mandeville, M.B., Ch.B., Ph.D.
Phagelux (Canada), Inc.
H4P 2R2 Montreal, Quebec
Re: GRAS Notice No. GRN 000603
Dear Dr. Mandeville:
The Food and Drug Administration (FDA) is responding to the notice, dated September 28, 2015, 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 October 7, 2015, filed it on October 29, 2015, and designated it as GRAS Notice No. GRN 000603. In an amendment dated December 21, 2015, Phagelux (Canada) Inc. (Phagelux) limited the intended use of Salmonella phage preparation to poultry products.
The subject of the notice is a preparation containing two bacterial monophages (BP-63 and BP-12 Triumvirate) specific to Salmonella enterica (Salmonella phage preparation). The notice informs FDA of the view of Phagelux that Salmonella phage preparation is GRAS, through scientific procedures, for use as an antimicrobial to control Salmonella on poultry products, when applied to poultry surfaces up to 108 plaque-forming units (PFUs) per gram (g) of poultry.
Phagelux describes Salmonella phage preparation as a clear to opalescent liquid consisting of two monophages (BP-63 and BP-12 Triumvirate) that are produced and purified separately and mixed in equal concentrations. Salmonella phage preparation has a minimal titer of 109 PFU per milliliter (mL). Phagelux states that this suspension is a concentrated form that is diluted with water at the application sites by a factor of 10 to provide a maximum application rate of 1 x 108 PFU/g of food.
Phagelux describes the method of manufacture for Salmonella phage preparation. Both monophages that make up the final preparation are produced using nonpathogenic strains of Escherichia coli (E. coli 8N2 for monophage BP-63 and E. coli BL-21 for monophage BP-12). Phagelux states that both strains of E. coli lack any virulence genes associated with pathotypes of E. coli and do not harbor any plasmids associated with virulence factors. Phagelux reports that endotoxins are removed by ion-exchange chromatography. Phagelux states that all materials used in the manufacture of Salmonella phage preparation are safe for use in food and that the final preparation does not contain any known allergens.
The two monophages are produced separately using aerobic fermentation. Each host bacterium is grown to a desired optical density, and then each monophage is added at a predetermined multiplicity of infection. The culture is incubated under specific fermentation conditions, and after a determined amount of time, the culture is filtered to remove the bacteria. The filtrate is washed with Tris-magnesium chloride-sodium chloride buffer (TMN) and concentrated by tangential flow filtration. The concentrated product is then passed through an ion exchange column to remove proteins. Finally, each monophage solution is filtered and diluted with TMN to the final specified concentration (1x109 PFU/mL). The monophage solutions are blended (1:1) to form the final product, which is sterile-filtered, packaged, and refrigerated. Phagelux notes that the final product is diluted with water by the user to form a working solution at a concentration of 1x108 PFU/mL.
Phagelux describes specifications for Salmonella phage preparation that includes an analysis of the potency (>1 x109 PFU/mL) and identity by testing the preparation against three different Salmonella strains (Typhimurium (DT-1040), Hadar (SHA), and Enteritidis (SE-1)), negative control is Mbandaka (SM)) and analysis by PCR with reference profiles. Specifications also include limits for endotoxins (<25,000 endotoxin units (EU)/mL) and bacterial sterility (no growth after 14 days). Phagelux discusses the results from an analysis of three batches of Salmonella phage preparation that examine the physical and chemical composition. Phagelux states that Salmonella phage preparation contains, on average, 35.3 mg/L total organic carbon, 1 microgram (μg)/L arsenic, 1.7 μg/L lead, 0 μg/L mercury, and 2800 EU/mL endotoxins.
Phagelux estimates the dietary exposure to Salmonella phage preparation based on its intended use on poultry products. Phagelux estimates that the average per capita consumption of chicken and turkey, combined, is 132.4 g/person/day based on food consumption data from the Economic Research Services of the United States Department of Agriculture (USDA) (2015). For the purpose of estimating dietary exposure to Salmonella phage preparation, Phagelux assumes that all chickens and turkeys are treated with Salmonella phage preparation, a maximum surface area to weight ratio of two square centimeters (cm2) per g of poultry, and an application rate of 108 PFU/cm2 of poultry is used.
Phagelux notes that the monophages are eluted from an anion exchange column using a solution that contains sodium chloride. Based on the concentration of sodium chloride in the solution used, subsequent dilution, and the estimated dietary exposure to Salmonella phage preparation, Phagelux calculates the dietary exposure to sodium chloride from the intended use to be 0.008 g/person/day (equivalent to 0.00003 g of sodium/person/day).
Phagelux discusses the safety of use of the Salmonella phage preparation containing two lytic monophages. Phagelux states that phages are ubiquitously present in the environment. Humans consume phages via various foods; hence phages are found in the human gut. Phagelux states that the biology of lytic phages has been extensively studied and documented. Phages have low inherent toxicity and they are susceptible to a variety of environmental factors, including sunlight and UV light. Phagelux states that the exposure to these environmental factors will cause the number of phages to decrease. Phagelux notes that most lytic phages display very limited host range even among specific bacteria and bacteria strains, and lytic phages have a reduced potential for bacterial development of resistance. Citing repeat dose published studies Phagelux states that there were no adverse effects when different phages were administered through drinking water in mice, rats and human volunteers. Phagelux notes that according to its estimate the maximal amount of its Salmonella phage preparation consumed by humans would be 0.044% of that of phages ingested through regular consumption of drinking water. Additionally, Phagelux reports that the proposed exposure of its Salmonella phage preparation is equivalent to similar phage products that are already on the market.
Use in Products under USDA Jurisdiction
During its evaluation of GRN 000603, FDA consulted with the Risk, Innovations, and Management Staff (RIMS) of the Food Safety and Inspection Service (FSIS) of the United States Department of Agriculture (USDA). Under the Federal Meat Inspection Act, Poultry Products Inspection Act, and the Egg Products Inspection Act, FSIS is responsible for determining the efficacy and suitability of food ingredients in meat, poultry, and egg products as well as prescribing safe conditions of use. Suitability relates to the effectiveness of the ingredient in performing the intended purpose of use and the assurance that the conditions of use will not result in an adulterated product, or one that misleads consumers.
FSIS has no objection to the use of Salmonella phage preparation as an antimicrobial to control Salmonella in poultry applied at 1 x 108 PFU/g. No labeling on poultry products is required when used under the accepted condition of use.
FSIS requested that FDA advise Phagelux to seek regulatory guidance from FSIS, Risk, Innovations, and Management Staff, about the use of Salmonella phage preparation in poultry products. Phagelux should direct such an inquiry to Dr. William K. Shaw Jr., Director, Risk, Innovations, and Management Staff, Office of Policy and Program Development, Food Safety and Inspection Service, via email at William.Shaw@fsis.usda.gov.
Section 301(ll) of the Federal Food, Drug, and Cosmetic Act (FD&C Act)
Section 301(ll) of the FD&C Act prohibits the introduction or delivery for introduction into interstate commerce of any food that contains a drug approved under section 505 of the FD&C Act, a biological product licensed under section 351 of the Public Health Service Act, or a drug or a biological product for which substantial clinical investigations have been instituted and their existence made public, unless one of the exemptions in section 301(ll)(1)-(4) applies. In its review of Phagelux’s notice that Salmonella phage preparation is GRAS for the intended uses, FDA did not consider whether section 301(ll) or any of its exemptions apply to foods containing Salmonella phage preparation. Accordingly, this responseshould not be construed to be a statement that foods that contain Salmonella phage preparation, if introduced or delivered for introduction into interstate commerce, would not violate section 301(ll).
Based on the information provided by Phagelux, as well as other information available to FDA, the agency has no questions at this time regarding Phagelux’s conclusion that Salmonella phage preparation 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 Salmonella phage preparation. As always, it is the continuing responsibility of Phagelux 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 000603, as well as a copy of the information in this notice that conforms to the information in the GRAS exemption claim (proposed 21 CFR 170.36(c)(1)), is available for public review and copying at www.fda.gov/grasnoticeinventory.
Dennis M. Keefe, Ph.D. Director
Office of Food Additive Safety
Center for Food Safety and Applied Nutrition
 BP12 Triumvirate consists of BP-12A, BP-12B, and BP-12C BP-12A and BP-12B are Caudovirales from Podoviridae family, whereas BP-12C is Caudovirales from Siphoviridae (Flagella-specific) family.
 Phage to bacteria infection ratio.
 Phagelux notes that endotoxins are known human toxicants that may be present in Salmonella phage preparation. The E. coli strains used in the method of manufacture are nonpathogenic and are gram-negative bacteria. All gram-negative bacteria produce endotoxins or lipopolysaccharide (LPS). Phagelux states that each batch of Salmonella phage preparation is analyzed for LPS content to ensure it meets specifications. FDA notes that oral consumption of LPS does not usually present a problem.