3R
`
`lllllllilllfllllflll
`
`ORIGINAL SUBMISSION
`
`05000.1
`
`NEPN 2008
`
`NEPN 2008
`
`

`
`I
`y =-4
`
`‘
`-'
`‘
`.":..;..-M--'
`,
`~— GRA$;Af$OClATE$1 -WLLC-i
`
`..~
`
`Generally Recognized As Safe
`Bend, 03397702-3074.
`541 i%‘1,i_t-3522
`__2pgs2 Jackltght Léitg
`Id;-’/—r
`.
`-.
`.
`‘mcquate@gras-ass5ociates.com'
`3
`'www.gras-assoclatesfcom
`
`
`
`‘
`7\~.~,.9,/~
`P
`‘V
`
`Food and Drug Administration
`Center for Food Safety & Applied Nutrition
`Office of Food Additive Safety (HFS-200)
`5100 Paint Branch Parkway
`College Park, MD 20740-3835
`
`Attention: Dr. Robert L. Martin
`
`Dear Dr. Martin:
`
`On behalfof Neptune Technologies & Bioressources of Laval (Quebec), Canada, we are submitting for FDA review a
`GRAS notification for High Phospholipid Krill Oil. The attached documentation contains the specific information that
`addresses the safe human food uses for the subject notified substance as discussed in the GRAS guidance document.
`
`As part of the deliberations by our Expert Panel on the safety ofthe subject krill oil as a human food ingredient, two
`unpublished scientific studies were considered. They have been included as Appendix G in the submission. Even
`though each of the reports is identified as being confidential, we recognize that the content submitted cannot be held as
`confidential, and we hereby waive the designation of confidentiality.
`
`if additional infonnation or clarification is needed as you and your colleagues proceed with the review, please feel free
`to contact me via telephone or email.
`
`We look forward to your feedback.
`
`Sincerely,
`
`Robert S. McQuate, Ph.D.
`CEO & Co-Founder
`
`GRAS Associates, LLC
`20482 Jacklight Lane
`Bend, OR 97702-3074
`541-678-5522
`
`mcguate@gras—associates.com
`www.gras-associatescom
`
`Enclosure: GRAS Notification — High Phospholipid Krill Oil (in triplicate)
`’
`
`Page 1 of 1
`
`-
`
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`0 0 0002
`
`GRAS ASSOCIATES, LLC
`
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`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil“
`Page 1
`
`I. GRAS EXEMPTION CLAIM
`
`A. Claim of Exemption From the Requirement for Premarket Approval Pursuant to
`' Proposed 21 CFR 17o.36(c)(1)‘
`
`High phospholipid krill oil, meeting the specifications described below, has been determined to '
`be Generally Recognized As Safe (GRAS), in accordance with Section 201 (s) of the Federal
`Food, Drug, and Cosmetic Act. This determination was made by experts qualified by scientific
`training and experience; it is based on scientific procedures as described in the following -
`sections; and the evaluation accurately reflects the conditions of the ingredient’s intended use
`infoods.
`
`Signed:
`
`Robert S. McQuate, Ph.D.
`GRAS Associates, LLC
`
`20482 Jacklight Lane
`Bend, OR 97702-3074
`
`Qf/L’/at /5, 0107673’
`
`Date
`
`B. Name and Address of Notifier
`
`Neptune Technologies and Bioressources
`2740 Pierre Pelade_au Avenue, Suite H200
`Laval (Quebec)
`Canada H7T 3B3
`
`As the notifier, Neptune Technologies and Bioressources ("Neptune”) accepts responsibility
`for the GRAS determination that has been made for high phospholipid krill oil as described in
`the subject notification; consequently, high phospholipid krill oil meeting the conditions
`described herein is exempt from pre-market approval requirements for food ingredients.
`
`C. Common Name and Identity of the Notified Substance
`
`High phospholipid krill oil; also see Sections ll.A and ll.B.
`
`1 See 62 FR 18938 (17 April 1997).
`
`GRAS Associates, LLC
`
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`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill OilTM
`Page 2
`
`D. Conditions of intended Use in Food
`
`High phospholipid krill oil is intended to be added as a substitute or alternative to fish oils to
`the following food categories at per serving levels ranging from 150 - 500 mg: non-alcoholic
`beverages; breakfast cereals; cheeses; frozen dairy desserts; milk products; processed
`fruits/fruitjuices; and medical foods. The maximum daily consumption of the subject krill oil
`will provide up to 2.2 g/person/day of EPA and DHA.
`
`E. Basis for the GRAS Determination
`
`"Pursuant to 21 CFR § 170.30, high phospholipid krill oil has been determined to be GRAS on
`the basis of scientific procedures as discussed in the detailed description provided below.
`
`F. Availability of Information
`
`The data and information that serve as the basis for this GRAS Notification will be sent to the
`US Food and Drug Administration (FDA) upon request or will be available for review and
`copying at reasonable times at the offices of GRAS Associates, LLC, located at 20482
`Jacklighti Lane, Bend, OR 97702-3074.
`
`GRAS Associates, LLC
`
`000004
`
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`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil"'l
`Page 3
`
`ll.
`
`CHEMISTRY AND MANUFACTURE OF NKOT”
`
`NKOTM is refined krill oil which is derived from the marine organism, Euphasia superba, which
`is harvested from Antarctic waters. Krill is recognized as a vital part of the marine food chain
`for baleen whales, mantas, whale sharks, crabeater seals and other seals, and a few seabird
`species.
`
`The following description of krill is given on Neptune’s website?
`
`It looks
`"Krill is a generic term used to designate all deepwater marine planktonlc crustacean species.
`like miniature shrimp, ranging between one and six centimeters in length. These 85 species represent
`the planet's most abundant animal biomass found in banks that may extend over several square
`kilometers. Krill fisheries can mainly be found in two ocean regions: the Antarctic Ocean and the North
`Pacific Ocean, along the Canadian and Japanese coasts. According to a World Health Organization
`estimate, the total quantity of krill in these two oceans exceeds 500 million tons. Approximately 110,000
`tons of krill are harvested every year, or about less than one-half of 0.1 % of the existing resources.”
`
`NKOTM is a whole lipid extract and is an opaque reddish oil with a seafood odor. The major
`components of NKOTM are triglycerides and phospholipids which are rich in eicosapentaenoic
`acid (EPA, C20:5 n-3 fatty acid) and docosahexaenoic acid (DHA, C22:6 n-3 fatty acid).
`NKOTM is an oil intended for use as a dietary ingredient as a source of omega-3 fatty acids,
`which as noted are found in NKOTM in their phospholipid form. No processing aids or
`additives are included in the final NKOTM product due to naturally occurring antioxidants that
`aid in NKO“""s preservation. Additionally, no proprietary or coloring ingredients are added to
`the oil from Antarctic krillto produce NKOTM.
`
`A. Common or Usual Name of the Subject Material
`
`High phospholipid krill oil is the common or usual name of the material that is the subject of
`the GRAS evaluation. The specific substance evaluated is Neptune Krill Oil”, which is
`commonly referred to throughout this evaluation as NKOTM. As noted in the preceding
`paragraph, NKOTM is a mixture of triglycerides and phospholipids that contain numerous
`constituent fatty acids. The compositional character of NKOTM is described in detail in this
`Section.
`
`B. Manufacturing and Specifications for NKOTM
`
`1. Krill Source and Handling
`
`NKOTM is extracted from Euphasia superba. The current taxonomic placement of
`Euphasia superba is summarized below:
`
`2 See www.neptunebiotech.com/whatiskrill.html.
`
`GRAS Associates, LLC
`
`O 0
`
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`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 4
`
`KINGDOM
`
`-
`
`Animalia
`
`l
`
`FAMILY
`GENUS
`SPECIES
`
`!
`l__
`
`Eu ausiidae
`,
`I
`Eumausia
`-
`_ Euphausia superba ,
`
`NKOT“ is produced from Antarctic krill, which are native to the Atlantic section of the
`Austral-Antarctic Circumpolar Ocean.
`in terms of their phylogeny, Antarctic krill are
`closely related to shrimp and are consumed as human food in a similar fashion. The
`Antarctic krill used in the production of NKOT" are a non—genetically modified source,
`are fished from the wild, and are not the result of breeding. No toxic constituents are
`known to be present in NKOT".
`
`The Antarctic krill used to extract NKOT” are fished in the Atlantic section of the Austral-
`Antarctic Circumpolar Ocean, originating specifically from Statistical Fishing Area 48, a
`fishing area designated by the Commission for the Conservation of Antarctic Marine
`Living Resources (CCAMLR). The Antarctic krill processed by Neptune are supplied by
`a Japanese firm that has been fishing Antarctic krill for more than two decades. The
`Antarctic krill are drained and placed in plastic molds for quick-freezing at about —30°C
`on board factory ships within a few hours of harvest. The resulting 12 kg blocks of
`Antarctic krill are individually wrapped in plastic film and are packaged two per carton
`prior to export to Canada; the packaged Antarctic krill are maintained at temperatures
`of -30 to -20°C. Upon arrival in Canada, they are inspected and approved by the
`Canadian Food Inspection Agency (CFlA). Source manipulations on board the fishing
`vessels are made in compliance with regulations as they apply to the handling of krill
`intended for human consumption.
`
`NKO” Manufacturing Process
`
`The manufacturing of NKOT” begins with the crushing of deep frozen Antarctic krill at a
`temperature of 4°C until the Antarctic krill particles are <5 mm in size. The lipids and
`proteins are separated and extracted from the cmshed Antarctic krill via the addition of
`. acetone under cold extractions conditions for a period of 2 hours. This step also takes
`place at a temperature of 4°C, and requires the addition of enough acetone to ensure a
`ratio of crushed Antarctic krill to acetone of 1:6 (w/v). Filtration occurs through an
`organic solvent-resistant filter under reduced pressure to enable physical separation of
`the Antarctic krill lipids and proteins. The remaining crushed Antarctic krill is then
`subjected to a second round of extraction to ensure that all possible lipids and proteins
`are extracted. Following extraction, the Antarctic krill proteins and lipids are filtered and
`separated from each other with this process yielding an oil. Excess acetone is then
`evaporated and separated from any water remaining in the product. The oil undergoes
`filtration and purification to remove impurities and is then packaged in a modified N2-
`containing atmosphere and stored as NKOW. Figure 1 summarizing the overall NKOT”
`manufacturing process.
`
`SR5‘-S Associates LLC
`
`oocoos
`
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`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Ollm
`Page 5
`
`Figure 1. Manufacturing Process for the Production of NKOTM
`
`Deep Frozen Raw Krill
`
`C
`
`Crushing of the Frozen Raw Krill
`
`Cold Extraction of Krill
`Lipids and Proteins
`
`Filtration of Krill Lipids
`and Proteins
`
`il0
`
`Krill Proteins
`
`Eiaporation of Acetone
`
`Excess Acetone
`
`Separation of Oil and Water
`
`Filtration/Purification
`
`3. Manufacturing Controls
`
`The process by which Neptune produces NKOTM occurs under Canadian Good
`Manufacturing Practices (GMP) requirements, as certified by the Natural Health
`Products Directorate (NHPD) of Canada. For GMP Certificate of Compliance
`(Certification Number 0001349) and Site License (License Number 300191), see
`Appendix A.
`‘
`
`GRz’».S Associates. L LC
`
`000007
`
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`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oi|_TM
`Page 6
`
`In addition to conforming to Canadian GMP standards, the Neptune production method
`is internally controlled under the Hazard Analysis Critical Control Points (HACCP)
`system. Under this system, Neptune has identified potential points for potential
`contamination or corruption of NKOTM. To ensure the quality of the final NKOTM
`product, production lots are consistently tested to ensure compliance with appropriate
`international standards addressing residual solvents, microorganisms, heavy metals,
`arsenic, and pesticides as discussed further.
`
`Additionally, Certificate of Registration for a Fish Processing Establishment
`(Certification Number 5111) was granted to Neptune on February 17, 2005 and was
`renewed on February 17, 2006 by the CFIA following the implementation of a “Quality
`Management Program - Fish” (QMP) and is attached in Appendix A.
`
`Specifications for NKOTM
`
`The compositional specifications for NKOTM as established by Neptune are presented
`in Table 1.
`
`The compositional specifications established by Neptune address all relevant issues
`concerning marine oils. They address the quality of the oil by providing minimum
`requirements for fatty acids and phospholipids content, as well as markers of stability
`and purity. The specifications provide information concerning the fat, cholesterol, and
`protein contents of NKOTM, as well as common contaminants, such as pesticides,
`dioxins, and heavy metals, which may be present in marine oils. Finally, Neptune's
`compositional specifications are similar to those for other marine-derived oils
`considering phospholipid or omega-3 fatty acid-rich oils and are consistent with the
`Codex Alimentarius standard for Edible Fats And Oils Not Covered By individual
`Standards (CODEX STAN 19-1981 (Rev. 2-1999); see Appendix B).
`
`The analytical procedures employed in the analysis have been validated by a variety of
`sources, the most prominent of which are the American Oil Chemists Society (A.O.C.S)
`and the Association of Official Agricultural Chemists (A.O.A.C) International. Both of
`these organizations produce validated microbiological and chemical analytical methods.
`In addition, several methods were developed and validated by the Canadian
`government, and additional information on these methods appears in Appendix C.
`
`Results from the analyses for the detailed composition of three different batches of
`NKOTM are presented in Table 2. As can be seen, overall and detailed compositions
`are consistent among the three batches.
`
`GRAS Associates, LLC
`
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`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 7
`
`TABLE 1 . SPEC|F|CATlONS AND COMPOSITIONAL PROFILE FOR NKOT“
`
`
`Requirement
`Mett-iod(s)
`specification
`
`Appearance
`
`Odor
`
`Viscosity
`
`Humidity
`
`Wsual - Reddish opaque oil
`
`Olfactive — Light shrimp odor
`
`Complies
`
`Complies
`
`Brookfield 021, 1.5 rpm, 25°C
`
`< 1,500 cP
`
`A.O.AC.984.2O
`Identlflcafion
`
`< 0.9%
`
`Peroxide value
`
`AO.A.C.965.33
`
`< 0.2 mEq peroxidelkg
`
`P—Anisidine index
`
`A.O.C.S Cd 18-90 (modified)
`
`< 1.2
`
`Saponification value
`
`A.O.A.C.920.160
`
`Iodine Value
`
`A.O.C.S Ja 14-91
`
`> 165.0 mg KOH/g
`
`>130.0 g I2/100 g
`
`
`
`
`Total Cholesterol
`
`BCA Colorimetric method
`A.O.AC. 976.26 (modified)
`
`0.5 — 3.5 g/100g
`0.8 —- 1.2 9/1009
`
`Saturated fatty acids
`
`Monounsaturated fatty
`
`
`A.O.A.C.996.06
`'
`> 73.0 9/100 g
`
`
`A'O.A'C.996m
`
`
`
`
`
`
`< 30.0 g/100 g
`A.O.A.C.996.06
`
`> 12.0 gl100 g
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`Polyunsaturated fatty
`acids
`
`A O A C 996 06
`'
`'
`'
`'
`'
`
`> 32 0 /100
`' 9
`
`9
`
`A.O.A.C.991.39, 963.22, 996.06
`
`
`AO.AC.991.39, 963.22, 996.06
`
`
`
`>1.3gI100g
`
`'
`
`AOAC. 996.06
`
`<o.1g/1oog
`
`ri
`
`' Docosapentaenoic acid.
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`
`GRAS Associates LLC
`
`000009
`
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`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Kn'li Oil”
`Page 8
`
`TABLE 2. BATCH ANALYSES OF THREE LOTS OF NKOT"
`
`Assessing the Compositional Guidelines for NKO""
`
`Requirement
`
`Specifications
`
`060116
`
`"°' ""'“"°'
`000519
`
`060224
`
`Appearance
`
`Red ovaqueoi
`
`Viscosity
`
`< 1,500 cP W 476.5
`
`Peroxide vaiue
`
`< 0.2 mEq peroxidelkg
`
`P-Anisidine
`
`< 1.2
`
`Identification
`A o -3
`
`A $3 ._.
`
`Saponificaiion value
`
`2 165.0 mg KOHlg
`
`Iodine Vaiue
`
`>130.0 gizI100
`
`174.6
`
`137.9
`
`A
`
`170.8
`
`. 135.6
`
`.
`
`A P_x
`
`177.6
`
`134.3
`
`Total Protein
`
`Total Choiesteroi
`
`0.5 -3.5 g/100g
`
`0.8- 1.2 gI100g
`
`Total Phospholipids
`
`2 40.0 gl100g
`
`“'3' “PMS 39 F“‘“V
`Acids
`
`2 73.0 gI100 g
`
`3.0 gl100 g
`
`1.1 gl100 g
`
`47.3 gI100 g
`
`88.5 gI100 g
`
`0.7 gl100 g
`
`1.0 91100 g
`
`0.8 gI100 g
`
`0.9 91100 g
`
`44.8 gl100 g
`
`43.8 97100 g
`
`81 2 g/100 g
`'
`
`88.2 gl100 g
`
`Saturated fafly acids
`
`< 30.0 gI100 g
`
`29.8 9/100 9
`
`29.2 911100 9
`
`28.9 97100 g
`
`M°"°""3a“"a‘°‘ W ' 212.0 gl100 g
`acids
`
`22.1 gI100 g
`
`21.6 gnoo g
`
`21.9 g7100 g
`
`
`
`Poiyunsaturated fatty
`inS2
`
`Omega-3 Fatty Acids
`EPA
`DHA
`DPA
`Linoieicacid
`Omega—6 Fatty Acids
`Omega-9 Fatty Acids
`Oleic acid
`Total Trans Fat
`Vitamin A
`Vitamin E
`
`2 32.0 gI100 g
`
`36.7 gI100 g
`
`34.2 gl100 g
`
`37.4 91100 g
`
`=30-°9“°°9
`>150 gI100g
`> 9.0 gl100g
`>0.4 gI100 g
`> 1.39,7100g
`1.5-2.5 gI100g
`2 6.0 g/100g
`2 5.0 g1100 g
`< 0.1 gnoo g
`2 100.0 IUlg
`2 0.5 iUIg
`
`1.6 gI100g
`
`1.4 gnoo g
`
`0.65 Mg
`
`0.67 |Ulg
`
`34-W009
`18.0 g/100 g
`11.4 gi100 g
`0.6 gI100 g
`1.3 g/100g
`1.6 gI100g
`9.4 gI100 g
`3.2 gI100 g
`Not detected
`269.9 lUlg
`0.70 |UIg
`
`Esterified Astaxanthin
`
`
`
`2 150.0 mg(100 g
`
`
`
`162.6 mgl100 g
`
`160.7 mg/100 g
`
`154.0 mgl100 g
`
`
`
`GRAS Associaies LLC
`
`,
`
`NEPN 2008
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`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”’'
`Page 9
`
`5. Purity of NKOTM
`
`a. Chemical Characterization and Batch Analyses of NKOTM
`
`The potential impurities and incidental constituents present in NKOTM arise largely
`from environmental exposure of the Antarctic Krill. Neptune routinely analyzes
`production lots of NKOTM for the presence of 17 pesticides, as well as dioxins,
`PCBS, heavy metals, fluorine, and various microorganisms. The incidental
`constituents for which NKOTM are analyzed, as well as the methods of analysis and
`associated specifications are presented in Table 3. Results from the batch analyses
`of NKOTM for solvent residues and incidental metal and pesticide constituents are
`presented in Tables 4 and 5, respectively.
`
`C. Microbiological Characterization of NKOTM
`
`The Neptune production method is internally controlled under the Hazard Analysis Critical
`Control Points (HACCP) system. Under this system, Neptune has identified potential points
`for contamination or corruption of NKOTM. To ensure the quality of the final NKOT“ product,
`production lots are consistently tested to ensure compliance with international standards
`addressing residual solvents, microorganisms, heavy metals, inorganic arsenic, and
`pesticides. The microorganisms for which NKOTM is analyzed, as well as the methods of
`analysis and associated specifications, are presented in Table 6. Results of the batch
`analyses of NKOTM for the presence of microorganisms are presented in Table 7.
`
`:3.‘ Stability of NKOT“
`
`Three tests have been conducted to ensure the stability of NKOTM over time. The peroxide
`values of NKOTM were measured, and the results indicate the extent of oxidation with oils, the
`Oil Stability index (OSI), and the Oxygen Radical Absorption Capacity (ORAC).
`
`in the first test, the peroxide value of NKOTM was determined by the A.O.C.S Cd.8b.90 method
`to be 0.05 mEq peroxide/kg, indicating that it does not have a high rancidity potential.
`
`The second test conducted on NKOTM by Neptune examined the OSI which is a measure of
`the resistance to oxidation and high temperatures over time.
`in this test, air heated to 97.8°C
`was passed through a sample of NKOTM for a period of 50 hours, during which the
`peroxide values of NKOTM were measured at regular intervals. The‘ results indicate that
`NKOTM maintains a peroxide value of <O.1 mEq peroxide/kg for over 50 hours at a
`temperature of 97.8°C, thereby confirming lack of appreciable rancidity potential.
`
`G RAS Associates, '1. LC
`
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`
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`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 10
`
`TABLE 3. INCIDENTAL CONSTIPTUENTS OF NKOT“
`
`
`
`Requirement
`
`Methods
`Solvent Residues
`
` Acetone
`
`< 10 mg/kg
`
`Specification
`
`Arsenic (inorganic)
`
`Cadmium
`
`Mercury
`
`Lead
`
`Copper
`Tin
`
`Antimony
`
`Incidental Metals and Non-metals
`
`Hydride Generation Atomic
`Absorption
`
`A.O.A.C.986.15, 971.21, 999.11‘
`
`A.O.A.C.986.15, 971.21, 999.11
`
`< 0.1 mg/kg
`
`< 0.1 mg/kg
`
`< 0.1 mg/kg
`
`A.O.A.C.986.15, 971.21, 999.11
`
`< 0.1 mg/kg
`
`ICP“
`ICP
`
`< 5 mg/kg
`< 10 mg/kg
`
`
`
`< 1 mg/kg
`lCP
`Pesticide Residues (including agricultural and veterinary substances)
`A.O.A.C.970. 52, US EPA 8081
`< 0.1 mglkg
`
`A 0 A.C.970.52, US EPA 8081
`
`< 0.1 mg/kg
`
`< 0.05 mg/kg
`< 0.5 mg/kg
`
`< 0.05 mg/kg
`
`Chlordane (alpha and gamma) A.O.A.C.970. 52, US EPA 8081
`Sum of DDT
`A.O.A.C.970. 52, US EPA 8081
`
`Endrin
`
`He achlor
`
`Toxaphene
`Hexachlorobenzene
`
`Alpha~HCH
`Beta-HCH
`
`Lindane
`
`A.0.A.C.970.52, US EPA 8081
`
`A.0.A.C.970.52, US EPA 8081
`
`< 0.1 mg/kg
`
`A.0.A.C.970. 52, US EPA 8081
`
`-
`
`< 0.01 mg/kg
`
`A.O.A.C.970. 52, US EPA 8081
`
`A.0.A.C.970.52, US EPA 8081
`
`<0.1 mg/kg
`
`< 0.1 mg/kg
`
`A.0.A.C.970.52, US EPA 8081
`
`< 0.05 mg/kg
`
`A.0.A.C.970.52, Us EPA 8081
`
`< 1 mg/kg
`
`other Organic or Inorganic Impurities or Toxins
`
`Benzo(a)pyrene
`Sum of PCDD/PCDF
`
`_
`
`US EPA 610, 8310, and 8100
`US EPA Method 1613 revision B
`
`Sum of PCDF/F/PCBs
`
`US EPA Method 1613 revision B
`
`Microbial Specifications
`
`Total aerobic bacteria /
`
`Total coliforms and E. coli
`
`Yeasts and Moulds
`
`Staphylococcus aureus
`
`Listeria monocytogenes
`
`Pseudomonas aemginosa
`
`MFHPB-33
`
`MFHPB—34
`
`MFHPB-32
`
`MFLP—21
`
`MFHPB-30
`
`CFC agar“
`
`MFHPB—20-MFO-11
`Salmonella spp.
`' A.O.A.C. Official Methods of Analysis, 18 Ed. Revision 1, 2006.
`" Inductively coupled plasma mass spectrometry.
`° A pseudomonas selective agar.
`
`< 2.0 ppb
`< 2.0 pglg fat WHO-
`PCDDlF-TEQ/ g fat
`
`< 10.0 pglg fat WHO-
`PCDDIF-TEQI g fat
`
`10 CFUig
`
`10 CFU/g
`
`10 CFUlg
`
`10 CFU/g
`
`Not detectedl25 g
`
`Not detected/25 g
`
`Not detected/25 g
`
`GRAS Associates LLC
`
`NEPN 2003
`
`NEPN 2008
`
`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 11
`
`TABLE 4. BATCH ANALYSES OF 4 LOTS OF ,NKO“‘ FOR THE PRESENCE
`OF SOLVENT RESIDUES
`
`.
`
`‘°“’°°'“°°“°" IEEIIIEZSEEEI ososw ososos
`
`jn1:m;:a:j
`
`Solvent Residues
`
`
`
`I
`
`
`
`
`
`TABLE 5. BATCH ANALYSES OF 3 LOTS OF NKO"" FOR THE PRESENCE
`OF INCIDENTAL METAL AND NON-METAL CONSTITUENTS,
`PESTICIDE RESIDUES AND ORGANIC OR NON-ORGANIC IMPURITIES
`
`
`
`
`
`
`
`EEIIIIEIIEH
`
`EI1'ME-
`'3
`
`< 1 mg/kg
`Antimon
`Pesticide Residues mcludi
`.
`Aldnn
`
`_- ricultuml and veterina
`
`
`
`substances
`< 0.1
`mg/kg
`
`
`
`:
`
`«
`
`< 0.1 mg/kg
`
`
`
`
`
`< 0.05 mg/kg
`< 0.5 mg/kg
`< 0.05 mg/kg
`< 0.1 mg/kg
`< 0.01 mg/kg
`< o_1 mg/kg
`< 0.1 mg/kg
`< 0.05 mg/kg
`
`< 0.05
`mg/kg
`< '5
`3 F
`
`'
`
`;;g
`
`S1333
`
`< 1 mg/kg
`
`< 1 mg/kg
`
`.
`.
`< 2.0 I-
`< 2.0 I-TEQ
`pglg
`TEQ pglg
`< 10.01-TEQ
`< 10.0 I-
`
`
`pglg
`
`TEQ pglg
`
`Chlordane (alpha and
`gamma)
`Sum of DDT
`
`Heptachlor
`Toxaphene
`Hexachiorobenzene
`Alpha-HCH
`Beta-HCH
`
`< 0.05 mg/kg
`< 0.5 mglkg
`< o.o5 mg/kg
`< 0.1 mg/kg
`< 0.01 mg/kg
`
`< 0.1 mg/kg
`< 0.05 mg/kg
`
`< 0.05 mg/kg
`< 0.5 mg/kg
`< 0.05 mg/kg
`< o 1 mg/kg
`< o 01 mg/kg
`<0 1 mg/kg
`< 0.1 mg/kg
`< 0.05 mg/kg
`
`Lindane
`
`< 1 mg/kg
`
`< 1 mg/kg
`
`Benzo a - ene
`
`Sum of PCDD/PCDF
`Sum of
`
`.
`< 2.0 l-TEQ
`pglg
`< 10.01-TEQ
`
`< 2.0 1-TEQ
`, , ,
`< 10.0 I-TEQ
`
`PCDF/FIPCBS
`
`pglg
`
`pg/g
`
`
`
`GRAS Associates LLC
`
`‘
`
`I
`
`NEPN 2003
`
`NEPN 2008
`
`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 12
`
`TABLE 6. MICROBIOLOGICAL SPECIFICATIONS FOR NKOT"
`
`
`
`
`
`
`
`
`
`
`
`MFHPB-32
`
`< 100 CFU/g
`
`Listeria monocytogenes
`
`MFHPB-30
`
`NegativeI25 g
`
`Salmonella spp.
`MFHPB-20-MFO-11
`3 United States Pharmacopeia, 27“§t—ion.
`
`NegativeI25 g
`
`
`
`
`
`TABLE 7. BATCH ANALYSIS OF 3 LOTS OF NKO“" FOR COMPLIANCE
`WITH MICROBIOLOGICAL SPECIFICATIONS
`
`R
`°°
`T t
`I
`
`i
`u remen
`
`t
`
`S
`
`6
`Vi
`
`pecif ca on
`
`Lot Number
`
`bl
`
`050729
`10 CFUI
`
`
`
`Total coliforms
`
`< 10 CFU/g
`
`< 10 CFUIg
`
`< 10 CFU/g
`
`< 10 CFU/g
`
`Escherichia coli
`
`NegativeI25 g
`
`Not Detected
`
`Not Detected
`
`Not Detected
`
`olds
`
`_
`
`Negative/25g
`Negative/25g
`
`’;:°‘:l‘:]‘j,%"s‘;”a5
`
`Not Detected
`Not Detected
`
`
`
`Salmonella spp.
`
`
`
`Negative/25 9
`
`
`Not Detected
`Not Detected
`
`Not Detected
`
`The ORAC value for an oil is an indicator of the oil’s antioxidant capacity against the formation
`of peroxyl and hydroxyl radicals. The presence of peroxyl and hydroxyl radicals increases the
`likelihood of lipid oxidation and associated oxidative rancidity. ORAC analyses involve the
`addition of a fluorescent probe, which measures the scavenging capacity of the antioxidants
`contained in the oil. The results reported were compared to those of Trolox®, a water-soluble
`vitamin E analog which serves as a calibration standard. The ORAC of NKOT" was reported
`to be 378 umol Trolox® equivalentlg, an ORAC value that is more than 300-fold greater than
`that of commercially availably vitamin A andlor vitamin E preparations while also exhibiting
`more than 48-fold greater capacity than a commercial omega—3 18:12 fish oil that is available
`in North America. These results indicate that NKOW is a potent free radical scavenger
`indicating the antioxidant potential of the oil.
`
`GRAS Associates. LLC
`
`‘
`
`INEPN 2003
`
`NEPN 2008
`
`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 13
`
`Additionally, long-term stability testing revealed that there is no significant change in the
`composition of NKOT" following storage at 25°C over a period of 28 months, with an ambient
`relative humidity of 60%. The results of this stability test are presented in Table 8.
`
`TABLE 8. STABILITY TESTING CONDUCTED WITH NKO“‘
`
`
`
`
`
`snarurv TESTING
`svemon
`-W
`u
`III
`
`ET
`'°
`I3I_ -
`
`Phosholi ids
`42.4 mmoll 100 -
` Peroxide Value EE-
`
`mow
`
`I
`
`In
`
`'-
`2
`53.5 mmolI100 -
`0.0 mE k
`
`
`
`
`
`
`
`
`E. Fatty Acid Composition of NKOT"
`
`As an oil extracted directly from Antarctic krill, NKOW is composed primarily of triglycerides
`and phospholipids. These lipids contain a wide variety of naturally occurring fatty acids as
`their side chains, the most prominent of which are EPA and DHA. The fatty acid compositions
`of multiple production batches of NKOT“ were examined by Neptune and the mean values,
`with standard deviations, are presented in Table 9.
`
`F. Similarity of Krill Oil to Other Fish Oils
`
`Several forms of fish oil have GRAS status as acknowledged by FDA. Menhaden oil has been
`affirmed as GRAS.’ in addition, FDA has not objected to GRAS notifications submitted on
`tuna oil (FDA, 2002b), salmon oil (FDA, 2004a) and anchovy oil (FDA, 2004b).
`
`In FDA's review of tuna oil, the fatty acid content of tuna oil was compared to menhaden oil
`(FDA, 2002b). Table 10 captures the values used by FDA and compares them to those of krill
`oil.
`-
`
`Additional information from the scientific literature on the distribution of fatty acids in several
`fish oils is shown in Table 11 and is compared to the Neptune data on krill oil. Krill oil contains
`a high level of the desirable n-3 unsaturated fatty acids that is comparable to that of salmon
`oil.
`its ratio of n-3 to n-6 fatty acids is comparable to the other fish oils in this group.
`
`G. Natural Occurrence of Vaccenic Acid in Food Sources
`
`Vaccenic acid is one of the major fatty acids present in krill oil. The vaccenic acid in krill oil is
`in the cis- isomeric fonn as indicated by the low value of trans-fatty acids found in the oil (see
`Table 2). Vaccenic acid constitutes the majority of the monounsaturated fatty acids present in
`krill oil. Vaccenic acid is found in various fish oils in both the cis- and trans- forms at levels of
`
`3 See 21 CFR 184.1472.
`GRAS Associates LLC.‘
`
`1
`
`NEPN 2003
`
`NEPN 2008
`
`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 14
`
`2-4% and can also be found in a number of other fats and oils. Table 12 lists the vaccenic
`acid concentrations in several widely used vegetable oils as found in two references, although
`it is noted that neither author indicated whether the vaccenic acid was in the cis- or trans-
`form. These oils are reported to be used in a wide variety of food applications.
`
`TABLE 9. FATTY ACID CONTENT OF NKO'"‘
`
`
`
`Mean Fatty Acid
`Fatty Acid Content or NKO" Lots
`(ggflfgrtd
`(%of total lipid)
`
`
`
`omww Deviation»
`
`flflfi
`
`
`
`
`
`Fatty Acid
`
`@H
`
`°«22<°~°5>
`
`E
`ED
`
`EEE@
`EE
`
`EM 2.22
`-
`ET
`HE
`E
`
`
`
`
`
`
`
`
`
`
`
`
`
`EE 1
`
`16.43
`
`17.35
`
`6.71 (0.65)
`
`16.03
`
`0.38
`
`
`
`0.
`
`47 (0.10)
`
`0.53
`
`O.
`
`47 (0.06)
`
`
`
`11-97
`
`
`
`9.50 (1.54)
`
`0.24
`
`0.41
`
`39
`
`0.54
`
`0.48
`
`
`
` (eicosapentaenoic acid,
`EPA)
`.
`
`
`
`
`(docosapentaenoic acid,
`
`DPA)
`C22 :6 n-3
`
`(docosahexaenoic acid,
`
`DHA)
`
`
`
`C22 :1 n-9 (erucic)
`C21 :5 n-3
`
`(heneicosapentaenoic)
`C22 :5 n-3
`
`
`
`GRAB Associates LLC
`
`000016
`
`NEPN 2003
`
`NEPN 2008
`
`

`
`GRAS Assessment for Neptune Technoiogies & Bioressources
`Neptune Krill Oil”
`Page 15
`
`TABLE 10. FATTY ACID COMPOSITION OF KRILL OIL“ COMPARED
`TO TUNA OIL AND MENHADEN OIL” (gI100 g)
`
`
`
`
`
` :IEI1m- Tuna Oil
`
`
`
`
`
`TT
`jTT-
`
`31
`:§l_—
`
`Values for krill oil taken from Table 9.
`° Values for tuna and menhaden oils taken from FDA response to GRN 000109
`(FDA, 2002b).
`
`:
`
`
`
`TABLE 11. COMPARISON OF FATTY ACID CONTENTS OF VARIOUS
`
`FISH OILS (PERCENTAGES)
`
`
`
`Acid
`
`
`
`""°"°"”E
`Oil
`
`
`Oil
`
`Oil
`
`c1e:o
`
`17.4
`
`19.0
`
`10.2
`
`E:
`
`111%
`
`TK
`
`KK
`MEI
`
`
`
`-if .
`If
`T
`
`“ From Lucas et al., 2005.
`
`
`
`A
`
`
`
`15.9
`
`
`
`GRAS Assrzclaies LLC
`
`NEPN 2003
`
`NEPN 2008
`
`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 16
`
`TABLE 12. VACCENIC ACID CONCENTRA'I1ON (% FATI'Y ACIDS)
`IN VARIOUS FATS AND OILS
`
`
`
`Acid”@
`
`
`
`Acid‘
`
`T j
`
`
`
`ii
`
`
`
`
`()_3
`Not Determined
`Cam
`jillltjjli
`Z1
`11
`
`
`
`EIIEEEX
`
`
`
`
`From Wasowicz et., al., 1976
`" From Sauer et al., 1997
`
`
`
`
`Vaccenic acid and other isomers of 18:1 fatty acids occur naturally in milk fats and have
`been found to be protective against cancers in animal and in vitro models, with some
`supporting epidemiological evidence in humans (Doyle et al., 2005). The Vaccenic acid
`present in dairy products is in the trans isomeric form. Cori et al. (2003) showed that the
`level of Vaccenic acid and other trans-18:1 isomers in butter can be increased by varying
`the diets of the producing cows. The levels of trans-1 8:1 and other C18 fatty acids in
`butter from cows on a conventional com ration versus a corn ration supplemented with 2%
`sunflower oil and 1% fish oil is shown in Table 13.
`
`Table 13. DISTRIBUTION OF C18 FATTY ACIDS IN BUTTER“
`
`
`
`
`
`0.41
`
`0.46
`
`18:3 cis-9, cis-12, cis-15
`
`° From Cori et al., 2003
`
`GRAS Associates. LLC
`
`000018
`
`NEPN 2003
`
`
`
`
`
`Ration
`Ration
`Acid
`IT
`.E1
`18:1, trans-9
`0.36
`0.84
`18:1 , trans-10
`0.67
`2.68
`
`NEPN 2008
`
`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 17
`
`Ill.
`
`INTENDED DIETARY USE
`
`A. Current Usage of NKOT" in the Diet
`
`Krill oil is not presently added to foods and is not naturally present in foods consumed in the
`US. Neptune reported that krill has been used in human food since the 1970s, principally in
`Japan, Russia, Ukraine, and, more recently, France.
`
`Neptune submitted a notification to FDA in 2002 for use of krill oil in dietary supplements.
`FDA did not object to this use (FDA, 2002a). The supplement is sold in 300 and 500 mg
`capsules with a recommended dose of 1 - 2 capsules per day.
`
`Softgels containing the NKOT“ have been available as dietary supplements in North America
`for several years; in Canada and the US, NKOT" has been commercially available since
`October of 2002, and in Japan, Korea, Singapore, and Hong Kong, NKOT" has been available
`since December of 2004. A total of 120,000 kg of Neptune krill oil, which is equivalent to
`60,000,000 individual 500 mg softgel capsules, has been ingested by consumers. Throughout
`this time no serious adverse effects have been reported.
`
`B. Intended Uses
`
`Neptune intends to incorporate krill oil into the various food categories as listed in Table 14.
`The krill oil would function as a nutrient supplement as defined in 21 CFR 170.3(o)(20).
`Neptune research has indicated that use levels are seIf—limiting due to the strong taste which
`begins to be detected at levels of 300 - 500 mg per serving, depending on the food type.
`it
`should be noted that krill oil is intended to be used as a substitute or altemative to fish oil.
`
`TABLE 14. PROPOSED FOOD USES FOR NKOT”
`
`
`
`
`
`
`Use Levels Per Serving
`
`.
`
`300 mg
`
`150-250 mg
`
`300-500 mg
`
`300-500 mg
`
`
`
`
`
`
`
`
`
`Food Category
`
`Breakfast cereals
`
`Fruit juices
`
`Milk products
`
`Medical foods
`
`
`
`
`
`
`
`"C. Estimated Daily Intake
`
`The estimated daily intake of NKOT" from proposed food uses above was calculated
`according to FDA guidelines (FDA, 1995) using serving size data and the mean (50"‘
`percentile) consumption of each food type of interest from the CSFII 1994-96 Survey (USDA,
`
`GRAS Associates LLC
`
`NEPN 2003
`
`NEPN 2008
`
`

`
`GRAS Assessment for Neptune Technologies & Bioressources
`Neptune Krill Oil”
`Page 18
`
`2005). According to FDA guidelines, a level twice the mean consumption was calculated to
`estimate use by a high consumer, i. e., at the 90"‘ percentile consumption level. This analysis
`is summarized in Table 15.
`
`TABLE 15. DIETARY INTAKE CALCULATIONS
`
`
`
`2
`
`216
`
`Q53at'g*at5,“
`
`597
`
`576
`
` .a. .1 10U1
`
`1152
`
`«« M
`1182-
`1970
`
`591-985
`
`267-445
`
`534-890
`
`mm
`-100
`
`_o8 8
`
`14e24s
`
`120-200
`
`297-496
`
`1
`
`6164-
`3082-
`
`4140
`8280
`
`
`
`Nonalcoholic
`150-250 m o
`
`
`
`Co -+ =
`
`Total Cottage
`Cheese
`
`One-hall cup of cottage cheese
`= 105 -
`
`Frozen Dairy
`Desserts (300 _ 500 mkcream, lee <_;fghard
`m I
`Milk, Whole & Skim
`One cup of fluid
`(aoo -