Agency Response Letter
GRAS Notice No. GRN 000130

Philip C. Olsson
Olsson, Frank and Weeda, P.C.
1400 Sixteenth Street, N.W.
Suite 400
Washington, D.C. 20036-2220

Re: GRAS Notice No. GRN 000130

Dear Mr. Olsson:

The Food and Drug Administration (FDA) is responding to the notice, dated May 23, 2003, that you submitted on behalf of aLF Ventures, LLC (aLF Ventures) 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 May 28, 2003, filed it on May 28, 2003, and designated it as GRAS Notice No. GRN 000130. aLF Ventures is a joint venture of Farmland National Beef Packaging Company, L.P. (National Beef) and DMV International.

The subject of the notice is bovine milk-derived lactoferrin. The notice informs FDA of the view of aLF Ventures that bovine milk-derived lactoferrin is GRAS, through scientific procedures, for use as a component of an antimicrobial spray that would deliver one gram (g) of lactoferrin per dressed beef carcass, followed by a wash with tempered water and rinse with lactic acid, resulting in less than 800 micrograms (µg) lactoferrin per kilogram (kg) of edible beef.

In a previous GRAS notice (GRN 000077) dated May 2, 2001, DMV International informed FDA that it had determined that bovine milk-derived lactoferrin is GRAS, through scientific procedures, for use as an ingredient in sports foods and functional foods at a level of 100 milligrams (mg) per product serving, provided that the ingredient statement of food products that contain this ingredient includes its source (e.g., milk or cow's milk). Estimated daily intake of lactoferrin from that use is 1.0 gram per person per day (g/p/d) at the mean and 2.1 g/p/d at the 90^th percentile. In GRN 000077, DMV International provided a report of a specially convened panel of individuals (DMV International's allergy and immunology panel) who evaluated the data and information that are the basis for DMV International's GRAS determination. DMV International considers the members of its panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. DMV Internationa's allergy and immunology panel concluded that bovine milk-derived lactoferrin at the proposed levels and for the uses specified by DMV International is GRAS, provided that food products containing milk-derived lactoferrin are clearly labeled as containing a milk protein so that milk-allergic consumers can avoid these products. In a letter dated August 14, 2001, FDA responded to DMV International that the agency had no questions at that time regarding DMV International's conclusion that bovine milk-derived lactoferrin is GRAS under the intended conditions of use.

In a previous GRAS notice (GRN 000067) dated January 4, 2001, National Beef informed FDA that it had determined that bovine milk-derived lactoferrin is GRAS, through scientific procedures, for use as a component of an antimicrobial spray containing up to 2 percent lactoferrin on uncooked beef, provided that the ingredient statement of food products that contain milk-derived lactoferrin identifies the source of the protein. The uncooked beef included carcasses, subprimals (i.e., wholesale cuts), and finished cuts of beef. Estimated daily intake of lactoferrin from that use is 4.1 mg/p/d at the mean and 9.1 mg/p/d at the 90^th percentile. During its evaluation of GRN 000067, FDA consulted with the Food Safety and Inspection Service (FSIS), U.S. Department of Agriculture (USDA), Labeling and Consumer Protection Staff. As part of the consultation, FDA, FSIS and representatives of National Beef discussed practical aspects associated with tracking the presence of lactoferrin from the point of application to the consumer, so that consumers who are allergic to milk would be aware of the presence of this milk-derived ingredient. In addition, National Beef provided to FDA a description of the controls that would be put in place to ensure that all lactoferrin-treated beef, from slaughter to retail, would be labeled as to the presence of a milk protein. In a letter dated October 23, 2001, FDA responded to National Beef that the agency had no questions at that time regarding National Beef's conclusion that milk-derived lactoferrin is GRAS under the intended conditions of use.

In GRN 000130, aLF Ventures incorporates by reference data and information contained in GRN 000067 and informs FDA that it has now concluded that the use of milk-derived lactoferrin as a component of an antimicrobial spray that would be applied to dressed beef carcasses and subsequently washed to reduce the residues of milk-derived lactoferrin is GRAS, for the general population, including individuals who are allergic to milk, without declaring the presence of the milk-derived ingredient on the label of beef products that derive from the treated carcasses. aLF Ventures does not extend this conclusion to the use of milk-derived lactoferrin on subprimal cuts of beef or finished cuts of beef. In GRN 000130, aLF Ventures includes the report of a panel of individuals (aLF Ventures' GRAS panel) who evaluated the data and information that are the basis for aLF Ventures' GRAS determination. The members of this panel also served as the allergy and immunology panel for GRN 000077. aLF Ventures considers the members of its GRAS panel to be qualified by scientific training and experience to evaluate the safety of substances added to food. aLF Ventures' GRAS panel discusses the safety of bovine milk-derived lactoferrin for milk-allergic individuals and concludes that bovine milk-derived lactoferrin is safe for milk-allergic individuals when applied to dressed beef carcasses as an antimicrobial spray and subsequently washed to leave a residual concentration of no more than 800 parts per billion (ppb).⁽¹⁾

aLF Ventures describes generally available information about the identity and characteristic properties of bovine milk-derived lactoferrin.⁽²⁾ Lactoferrin is an iron-binding glycoprotein found in mammalian milk and in the secondary granules of neutrophils (a type of white blood cell). In bovine milk, lactoferrin constitutes 0.3 percent of total protein.⁽³⁾ In beef products, aLF Ventures estimates that neutrophil-derived lactoferrin is present at a level of approximately 200 µg lactoferrin per kg of beef due to the neutrophil content of residual blood.

A single gene encodes the lactoferrin that is present in both bovine milk and bovine neutrophils. Bovine lactoferrin is expressed as a single glycosylated polypeptide chain of 689 amino acids.⁽⁴⁾ Information provided by aLF Ventures and otherwise available to FDA shows that there are five potential N-linked glycosylation sites on bovine lactoferrin and that both milk-derived and neutrophil-derived lactoferrin are glycosylated with a mixture of high-mannose and complex glycans at four or more of these potential sites.

aLF Ventures provides data and information on the susceptibility of bovine milk-derived lactoferrin, with or without complexed iron, to digestion by proteases and to denaturation by heat. Bovine milk-derived lactoferrin containing iron is more resistant to the activity of proteases in vitro than lactoferrin without iron; in vivo observations in infants and adults are consistent with these findings. Bovine milk-derived lactoferrin is relatively stable to the heat treatment that occurs during pasteurization, regardless of whether it contains iron. However, although bovine milk-derived lactoferrin that contains iron is more resistant to denaturation by heat than unsaturated lactoferrin, both forms are denatured by brief treatments at high temperatures or longer treatments at lower temperatures (e.g., above approximately 68 degrees C). According to aLF Ventures, denatured lactoferrin is rapidly and completely broken down by hydrolytic enzymes.

aLF Ventures acknowledges that both cow's milk and beef are allergenic foods. Information in GRN 000130 and otherwise available to FDA shows that the incidence of cow's milk protein allergy is generally reported as 2-3 percent in infants under one year of age, with remission to an asymptomatic state reaching 80-90 percent by the third year. aLF Ventures cites published reports to support its view that casein, beta-lactoglobulin and alpha-lactalbumin are generally regarded as the major allergens in Immunoglobulin E-mediated (IgE-mediated) cow's milk allergy. Persistent milk allergy may be linked to casein sensitization. The prevalence of beef allergy is less than that of milk allergy, and most individuals with beef allergy have previously been sensitized to milk. According to a recent literature review, approximately 13 to 20 percent of children who are allergic to cow's milk are allergic to beef, while approximately 80 to 90 percent of children who are allergic to beef are also allergic to cow's milk. aLF Ventures reports that bovine serum albumin (present at higher concentrations than lactoferrin in bovine milk) is most often associated with allergic reactions to beef, but acknowledges that there is less information available about the allergenic components of beef than about the allergenic components of milk.

Based on published information, aLF Ventures compares the characteristics of lactoferrin from milk with those of lactoferrin from beef, with a focus on structural information about the proteins that may be relevant to allergenicity. Because both proteins are encoded by the same gene and are expected to share the same amino acid sequence, any structural differences between milk-derived lactoferrin and endogenous lactoferrin in beef should be limited to differential glycosylation of the protein. aLF Ventures discusses published reports and information regarding the glycosylation of milk-derived and neutrophil-derived lactoferrin as well as general information about the clinical significance of protein glycosylation for food allergy. aLF Ventures concludes that there is no evidence to suggest that the differences in glycosylation reported for lactoferrin derived from either milk or neutrophils would result in clinically significant differences in immunogenicity or allergenicity.⁽⁵⁾

aLF Ventures describes its manufacturing process for milk-derived bovine lactoferrin, including the potential that its lactoferrin product is contaminated by other milk proteins (alpha-lactalbumin, beta-lactoglobulin, caseinmacropeptide,⁽⁶⁾ and bovine serum albumin) that are abundant and allergenic. Because the purification process for lactoferrin includes separation based on the ionic charge of extracted proteins, and because these allergenic proteins are opposite in ionic charge to that of lactoferrin, aLF Ventures did not expect that its lactoferrin product would contain these proteins. To verify that assumption, aLF Ventures used chromatographic techniques to demonstrate that the four proteins of concern were not detectable at a detection limit of approximately 10 parts per million (ppm) in a one percent lactoferrin solution.⁽⁷⁾

aLF Ventures describes experiments that it conducted to determine the residual level of exogenously applied lactoferrin on beef carcasses, assumptions used to estimate the amount of that lactoferrin present in edible beef products derived from the treated carcasses, and two scenarios that it considered regarding how the residual lactoferrin would be distributed in those edible beef products.⁽⁸⁾ One scenario assumed equal distribution of applied lactoferrin by weight across the edible carcass, while the other scenario assumed equal distribution by area. Distribution by area yielded higher lactoferrin concentrations in portions of the edible beef and was considered more conservative. aLF Ventures also describes experiments it conducted, and published data it used, to estimate the level of endogenous neutrophil-derived bovine lactoferrin in beef. aLF Ventures developed exposure estimates for exogenous milk-derived lactoferrin based on each distribution scenario, as well as an exposure estimate for neutrophil-derived lactoferrin endogenous to beef. aLF Ventures calculates the single-occasion eaters-only intake of both exogenous milk-derived bovine lactoferrin (applied as a component of an antimicrobial spray that delivers 1 g of lactoferrin per dressed beef carcass) and endogenous neutrophil-derived lactoferrin (normally present in beef) as described in Table 1 and concludes that intake of neutrophil-derived lactoferrin and milk-derived lactoferrin by consumers of beef would be comparable.

Table 1

	Neutrophil-derived Lactoferrin Intake (µg/occasion)		Milk-derived Lactoferrin Intake (µg/occasion)
Age Group			Weight-based Distribution		Area-based Distribution
	50th Percentile	90th Percentile	50th Percentile	90th Percentile	50th Percentile	90th Percentile
Infants < 1y	3	8	5	14	2	20
Infants 1 y	3	12	5	20	6	35
Children 2-5 y	4	14	7	24	10	44
Children 6-12 y	6	19	9	31	12	62
Teenagers 13-19 y	7	26	11	44	15	76
Adults 20+ y	9	30	16	50	16	76

In GRN 000130, aLF Ventures considers published data and information to evaluate whether lactoferrin is an allergenic component of bovine milk. aLF Ventures discusses published studies in which some infants with reported milk allergy carry IgE antibodies directed against lactoferrin. Some of these studies describe quantitative estimates of the level of anti-lactoferrin IgE in the serum of infants with reported milk allergy compared to the level of IgE to other milk proteins in the serum of the same patients. The remaining studies describe the prevalence of anti-lactoferrin IgE in the populations tested but do not provide quantitative information about the level of anti-lactoferrin IgE. Based on the quantitative studies, aLF Ventures reports that lactoferrin-specific IgE was usually found in lower quantities than IgE directed against the major milk allergens casein, beta-lactoglobulin and alpha-lactalbumin. In a study conducted with 11 infants, one infant appeared to have IgE only to lactoferrin but not to other milk proteins (i.e., the infant appeared to be monosensitized to lactoferrin). In another study conducted with 92 infants, two infants appeared to be monosensitized to lactoferrin. In all but one case, anti-lactoferrin IgE was found in less than 50 percent of the milk-allergic population; one study reported anti-lactoferrin IgE in 7 of 11 infants (64 percent). There have been no reports of allergic reactions induced by direct challenge of lactoferrin alone. aLF Ventures considers that the studies and information discussed above are consistent with the hypothesis that lactoferrin is a minor allergenic component of bovine milk (i.e., that less than half of milk-allergic individuals are sensitized).

Given the possibility that lactoferrin may be an allergenic component of bovine milk, aLF Ventures first considers published data and information and the opinions of its GRAS panel regarding those data and information in order to address the question of whether lactoferrin plays a clinically significant role in milk allergy. Based on the very low level of lactoferrin in bovine milk and the frequency of anti-lactoferrin IgE in the milk-allergic population, aLF Ventures' GRAS panel considers that lactoferrin would be, at most, a minor allergen (i.e., of potential clinical significance in only a minority of milk allergic individuals). aLF Ventures' GRAS panel notes that the studies identifying individuals monosensitized to lactoferrin do not make clear whether blinded oral challenges were done to document and confirm milk allergy in the study participants or whether some other predictive test was used (such as the RadioAllergoSorbent Test or skin prick test, which test for the presence of food-specific IgE). Thus, the clinical significance of in vitro lactoferrin-specific IgE in these monosensitized individuals remains uncertain. During a meeting between FDA and aLF Ventures' GRAS panel on July 10, 2003, panel members emphasized that positive IgE reactivity in vitro is of limited use in conclusively establishing that a patient is in fact allergic to a particular food or component of food, and that only blinded food challenge could establish the clinical significance of the anti-lactoferrin IgE in individuals who are reported to be allergic to milk. At that meeting, panel members also emphasized that lactoferrin is present at very low levels in bovine milk. They expressed the view that such low protein exposure levels are not consistent with the high protein exposures characteristic of most food allergens and would make clinically significant allergy to lactoferrin a rare event that would be difficult to detect and study in the sensitive population. In light of these considerations, the panel members stated that a clinical trial involving lactoferrin food challenge would contribute little additional information to the panel's safety evaluation of lactoferrin for its proposed use.

Given the possibility that lactoferrin may be an allergenic component of bovine milk, aLF Ventures also considers published data and information regarding the concept of a threshold-provoking dose of an allergenic food. For example, they discuss a published report of a roundtable conference between twelve clinical allergists regarding the establishment of a threshold-provoking dose of allergenic foods. In this report, the panel of allergists examined pooled data from an array of oral challenge studies using commonly allergenic foods and attempted to determine the lowest dose or quantity of each food that provoked an allergic response. The report concludes, in part, that non-zero threshold doses of allergenic foods do exist, but are not yet well-defined by currently available data. During the meeting between FDA and aLF Ventures' GRAS panel on July 10, 2003, panel members discussed this concept. In that discussion, the panel members emphasized that the situation that they had evaluated (i.e., the addition of a small amount of milk-derived bovine lactoferrin to beef that already contains the same lactoferrin gene product) is, strictly speaking, different from most of the common scenarios in which the threshold concept has been discussed.

Based on the totality of available data and information, aLF Ventures considers that the intended use of bovine milk-derived lactoferrin as a component of an antimicrobial spray that will be applied to dressed beef carcasses, and subsequently washed to leave a residual concentration of no more than 800 ppb, presents a situation in which a small amount of a protein that may be a minor allergenic component of milk is added as a processing aid to a food (i.e., beef) that already contains the protein in a comparable amount. aLF Ventures concludes that its intended use of bovine milk-derived lactoferrin will be safe for the milk-allergic population.

During its evaluation of GRN 000130, FDA consulted with the Labeling and Consumer Protection Staff of USDA-FSIS regarding the intended use of bovine milk-derived lactoferrin as a component of an antimicrobial spray applied to dressed beef carcasses. Under the Federal Meat Inspection Act and the Poultry Products Inspection Act, FSIS is responsible for determining the efficacy and suitability of food ingredients in meat and poultry 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 had previously evaluated the use of bovine milk-derived lactoferrin as a component of an antimicrobial spray on uncooked beef as described in GRN 000067. FSIS advised FDA that it considers its earlier suitability determination regarding the use of bovine milk-derived lactoferrin described in GRN 000067 to be applicable to the more restricted use described in GRN 000130. In a teleconference on July 31, 2003 between representatives of FDA and representatives of FSIS, FSIS also advised FDA that the assumptions used by aLF Ventures to estimate the distribution of residual lactoferrin on treated beef carcasses are reasonable assumptions that are supported by available data and information.

FDA's authority under the Federal Food, Drug, and Cosmetic Act does not extend to the labeling of meat and poultry products, except with regard to label information that may be related to the conditions of safe use of an ingredient. Therefore, FDA defers to USDA-FSIS on labeling of the beef. Under the Federal Meat Inspection Act, FSIS has the authority to ensure that meat products are accurately labeled under the adulteration and misbranding provisions of that Act, and the ingredient labeling provisions in the Federal meat inspection regulations. Thus, FSIS has the responsibility to address labeling issues associated with lactoferrin-treated beef, particularly as they relate to suitability and the assurance that the conditions of use will not result in an adulterated product, or one that misleads consumers.

Based on the information provided by aLF Ventures, as well as other information available to FDA, the agency has no questions at this time regarding the conclusion of aLF Ventures that bovine milk-derived lactoferrin 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 bovine milk-derived lactoferrin. As always, it is the continuing responsibility of aLF Ventures to ensure that food ingredients that the firm markets are safe, and are otherwise in compliance with all applicable legal and regulatory requirements.

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

cc: Dr. Robert Post, Director
Labeling and Consumer Protection Staff
Office of Policy, Program Development and Evaluation
Food Safety and Inspection Service
1400 Independence Ave., SW, Suite 602, Annex
Washington, DC 20250-3700

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⁽¹⁾Equivalent to 800 mg lactoferrin per kg edible beef.

⁽²⁾Because aLF Ventures incorporates GRN 000067 by reference, in this letter FDA does not distinguish between information presented by aLF Ventures in GRN 000130 and information presented by National Beef in GRN 000067.

⁽³⁾FDA notes that bovine milk contains 3.6 percent protein.

⁽⁴⁾The reported molecular weight of the glycosylated lactoferrin protein ranges from 82-91 kDa

⁽⁵⁾FDA notes that the analysis provided by aLF Ventures is largely theoretical and does not provide a direct experimental demonstration of immunological comparability for these lactoferrin isoforms.

⁽⁶⁾Caseinmacropeptide is a soluble casein fragment.

⁽⁷⁾FDA notes that if any of the four proteins were present in the lactoferrin spray at the level of the limit of detection, approximately one nanogram of that protein would be present per microgram of lactoferrin.

⁽⁸⁾Edible beef from the carcass falls into two categories: "boxed beef" and "trim and grind." Boxed beef consists of subprimal cuts such as rounds, loins, ribs, chucks, and briskets. Trim and grind consists of lean trimmings and some fat removed during subprimal production and is used for ground beef and other processed meats.