[Federal Register Volume 71, Number 160 (Friday, August 18, 2006)]
[Rules and Regulations]
[Pages 47729-47732]
From the Federal Register Online via the Government Publishing Office [www.gpo.gov]
[FR Doc No: E6-13621]


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DEPARTMENT OF HEALTH AND HUMAN SERVICES

Food and Drug Administration

21 CFR Part 172

[Docket No. 2002F-0316 (formerly 02F-0316)]


Food Additives Permitted for Direct Addition to Food for Human 
Consumption; Bacteriophage Preparation

AGENCY: Food and Drug Administration, HHS.

ACTION: Final rule.

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SUMMARY: The Food and Drug Administration (FDA) is amending the food 
additive regulations to provide for the safe use of a bacteriophage 
preparation on ready-to-eat meat and poultry products as an 
antimicrobial agent against Listeria monocytogenes. This action is in 
response to a petition filed by Intralytix, Inc.

DATES: This rule is effective August 18, 2006. Submit written or 
electronic objections and requests for a hearing by September 18, 2006. 
See section VII of this document for information on the filing of 
objections. The Director of the Office of the Federal Register approves 
the incorporation by reference in accordance with 5 U.S.C. 552(a) and 1 
CFR part 51 of certain publications in new 21 CFR 172.785 as of August 
18, 2006.

ADDRESSES: You may submit objections and requests for a hearing, 
identified by Docket No. 2002F-0316 (formerly 02F-0316), by any of the 
following methods:
Electronic Submissions
    Submit electronic objections in the following ways:
     Federal eRulemaking Portal: http://www.regulations.gov. 
Follow the instructions for submitting comments.
     Agency Web site: http://www.fda.gov/dockets/ecomments. 
Follow the instructions for submitting comments on the agency Web site.
Written Submissions
    Submit written objections in the following ways:
     FAX: 301-827-6870.
     Mail/Hand delivery/Courier [For paper, disk, or CD-ROM 
submissions]: Division of Dockets Management (HFA-305), Food and Drug 
Administration, 5630 Fishers Lane, rm. 1061, Rockville, MD 20852.
    To ensure more timely processing of objections, FDA is no longer 
accepting objections submitted to the agency by e-mail. FDA encourages 
you to continue to submit electronic objections by using the Federal 
eRulemaking Portal or the agency Web site, as described in the 
Electronic Submissions portion of this paragraph.
    Instructions: All submissions received must include the agency name 
and Docket No(s). and Regulatory Information Number (RIN) (if a RIN 
number has been assigned) for this rulemaking. All objections received 
will be posted without change to http://www.fda.gov/ohrms/dockets/default.htm, including any personal information provided. For detailed 
instructions on submitting objections, see the ``Objections'' heading 
of the SUPPLEMENTARY INFORMATION section of this document.
    Docket: For access to the docket to read background documents or 
objections received, go to http://www.fda.gov/ohrms/dockets/default.htm 
and insert the docket number, found in brackets in the heading of this 
document, into the ``Search'' box and follow the prompts and/or go to 
the Division of Dockets Management, 5630 Fishers Lane, rm. 1061, 
Rockville, MD 20852.

FOR FURTHER INFORMATION CONTACT: Raphael A. Davy, Center for Food 
Safety and Applied Nutrition (HFS-265), Food and Drug Administration, 
5100 Paint Branch Pkwy., College Park, MD 20740, 301-436-1272.

SUPPLEMENTARY INFORMATION:

I. Background

    In a notice published in the Federal Register of July 22, 2002 (67 
FR 47823), FDA announced that a food additive petition (FAP 2A4738) had 
been filed by Intralytix, Inc., c/o Lewis & Harrison, 122 C St. NW., 
suite 740, Washington, DC 20001, now represented by Keller & Heckman 
LLP, 1001 G St., NW., suite 500 West, Washington, DC 20001. The 
petition proposed to amend the food additive regulations to provide for 
the safe use of a mixture of bacteriophages\1\ (phages) as an 
antimicrobial agent against Listeria monocytogenes (L. monocytogenes) 
on foods, including fresh meat, meat products, fresh poultry, and 
poultry products. On December 18, 2003, the petitioner amended the 
petition to limit the petitioned use to ready-to-eat (RTE) meat and 
poultry products only.\2\
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    \1\ Bacteriophages are viruses that infect bacteria only.
    \2\ Ready-to-eat products, as used in this final rule, are 
defined in 9 CFR 430.1.
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    The food additive consists of a mixture of equal proportions of six 
individually purified phages. The petitioner's rationale for 
incorporating multiple phages in one formulation is to minimize the 
possibility of L. monocytogenes developing a resistance to the 
additive. Each phage in the additive is specific against various L. 
monocytogenes strains, including those strains known to be associated 
with foodborne illness (e.g., L. monocytogenes strains, serotypes 1/2a, 
4b and 1/2b). The phages are lytic\3\ double-stranded DNA phages. The 
petitioner has characterized each phage with respect to physical 
properties and other appropriate identifying factors (e.g., host range, 
structural protein profile, and DNA sequence of complete genome\4\).
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    \3\ Lytic bacteriophages lyse (destroy) their host bacteria as a 
normal part of their life cycle without integrating into the host 
genome.
    \4\ Genome means the genetic content of a cell or virus.
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    In the manufacturing process, each phage contained in the additive 
is separately produced using a strain of L. monocytogenes that can 
serve as a host to the specific phage. The host L. monocytogenes strain 
is first cultured in microbiological media and the specific phage is 
added to the culture when a specified cell density is achieved. After 
phage multiplication, which results in lysis (destruction) of host 
cells, the phage is purified by use of multiple filtration steps (to 
remove bacteria and their components). The six phages produced by this 
process are then

[[Page 47730]]

blended in phosphate buffered saline solution to formulate the 
additive. The six phages contained in the additive have been deposited 
with the American Type Culture Collection\5\ (ATCC).
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    \5\ ATCC is a nonprofit bioresource center that maintains 
deposits of bacteria and bacteriophages among other biological 
materials. Their primary mission is to acquire, authenticate, 
preserve, develop, and distribute biological material.
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    The phage preparation will be used as an antimicrobial agent to 
control L. monocytogenes in the production of RTE meat and poultry 
products. The phage preparation is directly sprayed on the surface of 
the RTE food articles at a level of approximately 1 milliliter (mL) of 
the preparation per 500 square centimeters (cm\2\) of food surface area 
just prior to packaging.

II. Determination of Safety

    Under the general safety standard in section 409 of the Federal 
Food, Drug, and Cosmetic Act (the act) (21 U.S.C. 348), a food additive 
cannot be approved for a particular use unless a fair evaluation of the 
data available to FDA establishes that the additive is safe for that 
use. FDA's food additive regulations (21 CFR 170.3(i)) define safe as 
``a reasonable certainty in the minds of competent scientists that the 
substance is not harmful under the intended conditions of use.''
    In evaluating the safety of the petitioned substance, FDA 
considered the following factors in determining the safety of the 
proposed food additive use: (1) The safety of the six phages 
constituting the food additive; (2) the safety of potential residues 
from L. monocytogenes used in the manufacture of the food additive and 
the need for limits related to their levels; (3) whether undesirable 
genes are potentially carried by the food additive; and (4) the need 
for additional identity and safety specifications.

A. Safety of the Petitioned Use of the Phage Preparation

    Phages infect only bacteria, rather than mammalian or plant 
cells.\6\ Moreover, phages are ubiquitous and humans are routinely 
exposed to them at high levels through food, water, and the environment 
without adverse effect.\7\ Phages also are a part of the normal 
microbial population of the humangut.\8\ However, the petitioner's 
bacteriophages are specific to L. monocytogenes only. Therefore, FDA 
concludes that the food additive under consideration does not present a 
toxicological concern for use in food as proposed by the petitioner 
based upon the explanations provided in the following sections (Refs. 1 
and 3).
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    \6\ T.D. Brock and M.T. Madigan, 1998, Biology of 
Microorganisms, 5th edition; Prentice-Hall, Inc., Inglewood Cliffs, 
NJ.
    \7\ Bergh, O., K.Y. Borsheim, G. Bratbak, and M. Heldal, 1989, 
High abundance of viruses found in aquatic environments, Nature, 
vol. 340 (10): 467-468.
    \8\ Breitbart et al., 2003; Journal of Bacteriology 185 (20): 
6220-6223.
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B. Safety Evaluation of Potential Residue Components From L. 
monocytogenes

    FDA considered the possibility that the proposed food additive may 
contain L. monocytogenes components as residues from use of the 
organism as host for phage multiplication in the manufacturing process. 
Such residues may include the toxin Listeriolysin O (LLO). Potential 
residues of L. monocytogenes other than LLO do not present a safety 
concern (Ref. 1). Based on our review of scientific literature on the 
pathogenicity of L. monocytogenes (Ref. 1), FDA finds that LLO is the 
only substance known to be toxic that may potentially be present as a 
residue in this food additive after the manufacturing process.
    LLO was not detected in the finished food additive within the assay 
limits of detection of 5 hemolytic units\9\ (HU)/ml, and the petitioner 
provided information on the purification process used in the production 
of the food additive as additional assurance that LLO would not be 
present at detectable levels in the finished food additive. 
Nevertheless, the agency has calculated a worst-case exposure to LLO 
from consumption of food products treated with the phage preparation. 
Assuming LLO is present at a maximum level of 5 HU/ml in the additive, 
the worst-case exposure to LLO for males aged 20 years or more that 
consume RTE foods treated with the additive at the maximum intended use 
level is 52 HU/person/day (HU/p/d) at the mean and 104 HU/p/d at the 
90th percentile. Males aged 20 years or more represent the worst-case 
scenario because this population group consumes the highest amount of 
food intended to be treated with the additive (Ref. 2). In this safety 
evaluation, FDA reviewed all available information on the identity, 
toxicity, and the stability of LLO. Even if LLO were present at the 
level of 5 HU/ml, this level does not present a toxicological concern 
for the following reasons (Ref. 1):
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    \9\ 1 HU of LLO is equal to one nanogram of protein (as reported 
in Geoffroy, C. et al. 1987, Purification, Characterization, and 
Toxicity of the Sulfhydryl-Activated Hemolysin Listeriolysin O from 
Listeria monocytogenes, Infection and Immunity, vol. 55(7): pp. 
1641-1646).
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1. Inactivation of LLO by Cholesterol
    The toxicity of LLO has been shown to be significantly reduced (by 
as much as 200- to 2000-fold) following pre-incubation of LLO with 
added cholesterol in vitro (Ref. 1). Since the phage preparation will 
be used on meat and poultry products and these products normally 
contain significant (milligram) amounts of cholesterol, then any 
residual amounts of LLO at levels no greater than 5 HU/ml that may be 
present in the additive are likely to be inactivated by the 
cholesterol.
2. pH and LLO Activity
    Studies show that LLO activity is lost or significantly decreased 
in acidic (low pH of less than 4) environments (Ref. 1). Residual 
amounts of LLO, if present, are likely to be inactivated by the low pH 
(less than 4) within the human stomach.
3. Inactivation of Orally Consumed LLO by Human Defense Mechanisms
    In vivo studies demonstrate that both normal intestinal microflora 
and cell-mediated immunity reactions in the intestines inhibit LLO 
(Ref. 1). These defense mechanisms provide some protection against low 
incidental oral exposures to LLO (no greater than 5 HU/ml). 
Additionally, at these levels, LLO is expected to be rapidly and 
irreversibly degraded by proteolytic enzymes that may be presented in 
the diet or in the stomach. Thus, LLO at these residual levels would 
not pose a toxic threat to humans.
    Considering all of the above factors, FDA concludes that potential 
residues of LLO that may be found in the food additive are negligible 
(5 HU/ml or less) and do not pose a safety concern for the use of the 
additive as an antimicrobial agent on RTE meat and poultry products.
    Although LLO was not detected in the food additive, the agency 
concludes that a specification is necessary to ensure that LLO is not 
present in detectable amounts to ensure the purity and safe use of the 
petitioned food additive. Thus, the agency is including in this 
regulation a specification of not more than 5 HU/ml for LLO (the limit 
of detection for the method).

C. Undesirable Genes (Bacterial Toxin Genes) Potentially Carried by 
Phages

    Lysogenic phages, as opposed to those that are lytic, have the 
capacity to integrate into the host genome and may facilitate transfer 
of toxin or drug resistance genes between bacterial cells. FDA has 
determined that the phages contained in the petitioned food additive 
are lytic based on the petitioner's information on host lysis

[[Page 47731]]

characteristics and on genomic analysis of each phage (Ref. 4). 
Therefore, FDA concludes that the use of this food additive would not 
result in the spread of toxin or drug genes.

D. The Need for Other Specifications

    We are also including specifications for potency, absence of 
undesirable genes, phage titer\10\, absence of L. monocytogenes and 
other microbiological pathogens, and total organic carbon (Ref. 2). 
These specifications ensure the identity and safe use of the additive.
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    \10\ A term that refers to the number of phage particles per 
milliliter of phage solution.
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III. Other Considerations

    FDA recognizes that while this rule is issued under the authority 
of the Federal Food, Drug, and Cosmetic Act, use of the ingredient must 
also comply with the Federal Meat Inspection Act or the Poultry 
Products Inspection Act, which are administered by the U.S. Department 
of Agriculture (USDA). In particular, those statutes provide that the 
ingredient must be suitable for its intended use. FDA recognizes that 
there may be meat or poultry products considered RTE for which use of 
the additive may not be suitable within the meaning of those statutes. 
This regulation addresses only the safety standard under section 409 of 
the Federal Food, Drug, and Cosmetic Act and does not address 
requirements for suitability administered by the USDA.

IV. Conclusion

    FDA reviewed data in the petition and other available relevant 
material to evaluate the safety of the use of a phage preparation as an 
antimicrobial agent against L. monocytogenes on RTE meat and poultry 
products. Based on this information, the agency concludes that the 
proposed use of the additive is safe. Therefore, the regulations in 
part 172 (21 CFR part 172) should be amended as set forth in this 
document.
    In accordance with Sec.  171.1(h) (21 CFR 171.1(h)), the petition 
and the documents that FDA considered and relied upon in reaching its 
decision to approve the petition are available for inspection at the 
Center for Food Safety and Applied Nutrition by appointment with the 
information contact person (see FOR FURTHER INFORMATION CONTACT). As 
provided in Sec.  171.1(h), the agency will delete from the documents 
any materials that are not available for public disclosure before 
making the documents available for inspection.

V. Environmental Impact

    The agency has previously considered the environmental effects of 
this rule as announced in the notice of filing for FAP 2A4738 (67 FR 
47823). No new information or comments have been received that would 
affect the agency's previous determination that there is no significant 
impact on the human environment and that an environmental impact 
statement is not required.

VI. Paperwork Reduction Act of 1995

    This final rule contains no collection of information. Therefore, 
clearance by the Office of Management and Budget under the Paperwork 
Reduction Act of 1995 is not required.

VII. Objections

    Any person who will be adversely affected by this regulation may 
file with the Division of Dockets Management (see ADDRESSES) written or 
electronic objections. Each objection shall be separately numbered, and 
each numbered objection shall specify with particularity the provisions 
of the regulation to which objection is made and the grounds for the 
objection. Each numbered objection on which a hearing is requested 
shall specifically so state. Failure to request a hearing for any 
particular objection shall constitute a waiver of the right to a 
hearing on that objection. Each numbered objection for which a hearing 
is requested shall include a detailed description and analysis of the 
specific factual information intended to be presented in support of the 
objection in the event that a hearing is held. Failure to include such 
a description and analysis for any particular objection shall 
constitute a waiver of the right to a hearing on the objection. Three 
copies of all documents are to be submitted and are to be identified 
with the docket number found in brackets in the heading of this 
document. Any objections received in response to the regulation may be 
seen in the Division of Dockets Management between 9 a.m. and 4 p.m., 
Monday through Friday.

VIII. References

    The following references have been placed on display in the 
Division of Dockets Management (see ADDRESSES) and may be seen by 
interested persons between 9 a.m. and 4 p.m., Monday through Friday.
    1. Memorandum dated June 3, 2005, from Division of Petition 
Review, Toxicology Group I, Tina Walker, to Raphael Davy, DPR, 
entitled ``Safety Review of LMP-102\TM\ as an antimicrobial agent in 
ready-to-eat foods, fresh meat, meat products, fresh poultry, and 
poultry products.''
    2. Memorandum dated April 11, 2005, from Division of Petition 
Review, Chemistry Review Group, Hyoung Lee, to Regulatory Group II, 
R. Davy, entitled ``FAP 2A4738 (MATS1137 M 2.3), Petition 
for the use of LMP-102\TM\--a mixture of several monoclonal 
bacteriophages as an antimicrobial agent in ready-to-eat meat and 
poultry, Submissions of 10/25/04, 1/18/05, 1/25/05, and 2/18/05.''
    3. Memorandum dated February 1, 2006, from Division of Petition 
Review, Toxicology Group I, Tina Walker, to Raphael Davy, DPR, 
entitled ``Addendum to the June 3, 2005 Final Toxicology Memorandum: 
Additional toxicological evaluation of the potential allergenicity/
immunotoxicity of the Listera bacteriophage, LMP-102\TM\.''
    4. Memorandum dated June 1, 2005, from Division of Biotechnology 
and GRAS Notice Review, Negash Belay, to Raphael A. Davy, Division 
of Petition Review, entitled ``Revised FAP 2A4738.''

List of Subjects in 21 CFR Part 172

    Food additives, Incorporation by reference, Reporting and 
recordkeeping requirements.

0
Therefore, under the Federal Food, Drug, and Cosmetic Act and under 
authority delegated to the Commissioner of Food and Drugs, 21 CFR part 
172 is amended as follows:

PART 172--FOOD ADDITIVES PERMITTED FOR DIRECT ADDITION TO FOOD FOR 
HUMAN CONSUMPTION

0
1. The authority citation for 21 CFR part 172 continues to read as 
follows:

    Authority: 21 U.S.C. 321, 341, 342, 348, 371, 379e.

0
2. Section 172.785 is added to subpart H to read as follows:


Sec.  172.785  Listeria-specific bacteriophage preparation.

    The additive may be safely used as an antimicrobial agent specific 
for Listeria monocytogenes (L. monocytogenes) in accordance with the 
following conditions:
    (a) Identity. (1) The additive consists of a mixture of equal 
proportions of six different individually purified lytic-type (lacking 
lysogenic activity) bacteriophages (phages) specific against L. 
monocytogenes.
    (2) Each phage is deposited at, and assigned an identifying code 
by, a scientifically-recognized culture collection center, and is made 
available to FDA upon request.
    (3) The additive is produced from one or more cell cultures of L. 
monocytogenes in a safe and suitable nutrient medium.
    (b) Specifications.
    (1) The additive achieves a positive lytic result (OD600 
<= 0.06) when tested

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against any of the following L. monocytogenes isolates available from 
American Type Culture Collection (ATCC): ATCC 35152 (serogroup 1/2a), 
ATCC 19118 (serogroup 4b), and ATCC 15313 (serogroup 1/2b). The 
analytical method for determining the potency of the additive entitled 
``Determination of Potency of LMP-102\TM\,'' dated October 9, 2003, and 
printed by Intralytix, Inc., is incorporated by reference. The Director 
of the Office of the Federal Register approves this incorporation by 
reference in accordance with 5 U.S.C. 552(a) and 1 CFR part 51. You may 
obtain a copy from the Office of Food Additive Safety (HFS-200), Center 
for Food Safety and Applied Nutrition, Food and Drug Administration, 
5100 Paint Branch Pkwy., College Park, MD 20740, or you may examine a 
copy at the Center for Food Safety and Applied Nutrition's Library, 
5100 Paint Branch Pkwy., College Park, MD 20740, or at the National 
Archives and Records Administration (NARA). For information on the 
availability of this material at NARA, call 202-741-6030, or go to: 
http://www.archives.gov/federal_register/code_of_federal_regulations/ibr--locations.html.
    (2) The mean phage titer of each monophage in the additive is 1 x 
10\9\ plaque forming units (PFU)/ml. The analytical method for 
determining phage titer entitled ``Method to Determine Lytic Activity/
Phage Titer,'' dated November 6, 2001, and printed by Intralytix, Inc., 
is incorporated by reference. Copies are available at locations cited 
in paragraph (b)(1) of this section.
    (3) The phages present in the preparation must not contain a 
functional portion of any of the toxin-encoding sequences described in 
40 CFR 725.421(d). No sequences derived from genes encoding bacterial 
16S ribosomal RNA are present in the complete genomic sequence of the 
phages.
    (4) L. monocytogenes toxin, listeriolysin O (LLO), is not greater 
than 5 hemolytic units (HU)/ml. The analytical method for determining 
LLO entitled ``Quantitation of Listeriolysin O Levels in LMP-102\TM\,'' 
dated September 27, 2004, and printed by Intralytix, Inc., is 
incorporated by reference. Copies are available at locations cited in 
paragraph (b)(1) of this section.
    (5) The additive is negative for L. monocytogenes. The modified 
version of the U.S. Department of Agriculture's method for determining 
L. monocytogenes entitled ``LMP-102\TM\ Listeria monocytogenes 
Sterility Testing,'' dated May 24, 2004, and printed by Intralytix, 
Inc., is incorporated by reference. Copies are available at locations 
cited in paragraph (b)(1) of this section.
    (6) The additive is negative for gram-positive and gram-negative 
bacteria capable of growing in commonly used microbiological media 
(e.g., Luria-Bertani (LB) medium), including Escherichia coli, 
Salmonella species and coagulase-positive Staphylococci, as determined 
by the ``Method to Determine Microbial Contamination,'' dated July 11, 
2003, and printed by Intralytix, Inc., is incorporated by reference. 
Copies are available at locations cited in paragraph (b)(1) of this 
section.
    (7) Total organic carbon (TOC) is less than or equal to 36 mg/kg. 
The analytical method for determining TOC entitled ``Determination of 
Total Organic Carbon by Automated Analyzer,'' dated March 30, 2001, and 
printed by Intralytix, Inc., is incorporated by reference. Copies are 
available at locations cited in paragraph (b)(1) of this section.
    (c) Conditions of use. The additive is used in accordance with 
current good manufacturing practice to control L. monocytogenes by 
direct application to meat and poultry products that comply with the 
ready-to-eat definition in 9 CFR 430.1. Current good manufacturing 
practice is consistent with direct spray application of the additive at 
a rate of approximately 1 mL of the additive per 500 cm\2\ product 
surface area.

    Dated: August 3, 2006.
Jeffrey Shuren,
Assistant Commissioner for Policy.
[FR Doc. E6-13621 Filed 8-17-06; 8:45 am]
BILLING CODE 4160-01-S