SCIENTIFIC OPINION. and

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1 The EFSA Journal (2008) 822, 1-31 SCIENTIFIC OPINION Vitamin K 2 added for nutritional purposes in foods for particular nutritional uses, food supplements and foods intended for the general population and Vitamin K 2 as a source of vitamin K added for nutritional purposes to foodstuffs, in the context of Regulation (EC) N 258/97 1 Scientific Opinion of the Panel on Dietetic Products, Nutrition and Allergies (Question No EFSA-Q and EFSA-Q ) Adopted on 02 October 2008 PANEL MEMBERS Jean-Louis Bresson, Albert Flynn, Marina Heinonen, Karin Hulshof, Hannu Korhonen, Pagona Lagiou, Martinus Løvik, Rosangela Marchelli, Ambroise Martin, Bevan Moseley, Andreu Palou, Hildegard Przyrembel, Seppo Salminen, John Sean Strain, Stephan Strobel, Inge Tetens, Henk van den Berg, Hendrik van Loveren, and Hans Verhagen. SUMMARY Following a request from the Commission, the European Food Safety Authority has been asked to carry out the safety assessment for vitamin K 2 as source of vitamin K added for nutritional purposes to foodstuffs, in the context of Regulation (EC) N 258/97, and to provide a scientific opinion, based on its consideration of the safety and bioavailability of vitamin K 2 added for nutritional purposes in foods for particular nutritional uses and foods (including food supplements) intended for the general population. The collective term vitamin K, encompasses several lipid-soluble 2-methyl-1,4- naphthoquinone derivatives. Naturally occurring forms of vitamin K include vitamin K 1 (phylloquinones) and vitamin K 2 (menaquinones). 1 For citation purposes: Scientific Opinion of the Panel on Dietetic Products Nutrition and Allergies on a request from the European Commission on the safety of Vitamin K2. The EFSA Journal (2008) 822, European Food Safety Authority, 2008

2 Compared to vitamin K 1, dietary contribution of vitamin K 2 is much less. Dietary sources of vitamin K 2 include chicken, egg yolk, dairy products, cow liver, and natto. Vitamin K 2 can also be of microbiological origin, found primarily in fermented foods, or can be produced by bacteria of the gastrointestinal tract. The petitioner proposes that menaquinone be marketed in the form of a vitamin K 2 -containing oil, which is produced from the fermentation of soybean protein isolate and corn starch in the presence of Gram-positive bacterium Bacillus subtilis natto. The K 2 -containing oil contains vitamin K 2 occurring principally as menaquinone-7 (MK-7) and to a smaller extent, as menaquinone-6 (MK-6). Vitamin K 2 appears to be absorbed rapidly and unchanged from the gastrointestinal tract, is carried in the lymph in mixed micelles composed of bile salts, and subsequently released into the circulation. In human supplementation studies, higher and more stable plasma levels of vitamin K were reached with supplements containing vitamin K 2 compared to those containing vitamin K 1 The International Agency for Research on Cancer monograph on vitamin K substances states that neither phylloquinone nor menaquinones have been adequately studied for mutagenicity. Positive results were reported in a non-specific DNA repair test in a bacterium following incubation of vitamin K 2 with E. coli; however, when subjected to a mammalian test system, vitamin K 2 produced no significant increase in the incidence of single strand DNA breaks. In 2000, the International Agency for Research on Cancer evaluation classified vitamin K substances as group 3 (i.e., not classifiable as to their carcinogenicity to humans), based on inadequate evidence for carcinogenicity in humans and experimental animals. The Panel notes that vitamin K 2 occurs as an endogenous compound in humans. Furthermore there was no evidence of any putative preneoplastic or hyperplastic lesion in several sub-chronic (13 weeks) and two long term (1 year) toxicity studies with rats and dogs receiving oral doses of MK-4, the analogue of MK-7 (the principal constituent of the vitamin K 2 containing oil formulation of the present opinion). Based on the results from one-year study in rats with daily oral dose of 0, 20, 100 and 500 mg/kg bw, the Panel considers that a significant decrease in prothrombin time (PT) in all treated males, could be an adverse effect, and this implies that the study does not provide a No-Observed-Adverse-Effect Level (NOAEL). The Panel considered 20 mg/kg bw/day as Lowest-Observed-Adverse-Effect Level (LOAEL). Results from human intervention studies did not report adverse effects of MK-7 on blood coagulation of at least 6 microgram/kg bw/day for adults and of at least 1.5 microgram/kg bw/day for children. The Panel notes however that these human intervention studies were not designed to assess the safety of vitamin K 2. Estimated mean intakes of MK-7, based on conservative assumptions, resulting from the uses proposed by the applicant ranged from 36 microgram/day (female adults) to 54 microgram/day (male teenagers). High intake levels ranged from 75.0 microgram/day (children) to 115 microgram/day (male teenagers). The highest 97.5th percentile was in children and amounted to 5.4 microgram/kg bw/day. The margin of safety calculated from the highest 97.5th percentile intake estimate for children (5.4 microgram/kg bw/day) and the LOAEL (20 mg/kg bw) from the rat study amounts to As the intake estimates are highly conservative, it provides an extra margin of safety. The conservative intake estimate of 5.4 microgram/kg bw/day is also 3.6 fold higher than the dose shown in a human study with children not to affect blood clotting parameters and is just below the 6 microgram/kg bw/day not affecting blood clotting in adults. The EFSA Journal (2008) 822, 2-31

3 The Panel concludes that vitamin K 2 (menaquinone) from the K 2 containing oil formulation of the present opinion is bioavailable as a source of vitamin K. The Panel also concludes that the use of menaquinone-rich edible oil meeting the specifications provided, in foods for the general population (including food supplements) and in foods for particular nutritional uses, other than baby foods and infant formula, at the proposed use levels is not of safety concern. Keywords: Food supplements, vitamin K2, CAS Registry Numbers: Menaquinone (generic) CAS no ; Menaquinone-7 CAS no ; Menaquinone-6 CAS no ; Natto, Menaquinone-4 CAS no The EFSA Journal (2008) 822, 3-31

4 TABLE OF CONTENTS Panel Members...1 Summary...1 Table of Contents...4 Background as provided by the Commission...5 Terms of reference as provided by the commission...5 Acknowledgements...5 Assessment...6 Introduction...6 I. Specification of the novel food (NF) (Chemistry)...6 II. Effect of the production process applied to the NF (Manufacturing process)...7 III. History of the organism used as the source of the NF...8 IX. Anticipated intake/extent of use of the NF...8 X. Information from previous human exposure to the NF or its source and Information on existing authorisations and evaluations...11 XI. Exposure...11 Information on existing authorisations and evaluations...12 Nutritional information on the NF (Biological data)...13 Bioavailability of vitamin K from its vitamin K2 source...14 Metabolic fate and biological distribution...16 XII. Microbiological information on the NF...18 XIII. Toxicological information on the NF (Toxicological data)...18 Acute toxicity...18 Sub-chronic toxicity...18 Reproductive and developmental toxicity...19 Genotoxicity...20 Carcinogenicity...20 Long term studies...20 Human studies...22 Discussion...23 Conclusions...24 Documentation provided to EFSA...24 References...24 Glossary / Abbreviations...31 The EFSA Journal (2008) 822, 4-31

5 BACKGROUND AS PROVIDED BY THE COMMISSION The European Community legislation lists nutritional substances that may be used for nutritional purposes in certain categories of foods as sources of certain nutrients. The Commission has received a request for the evaluation of Vitamin K 2 added for nutritional purposes in food for particular nutritional uses, food supplements and foods intended for the general population. The relevant Community legislative measures are: Directive 2002/46/EC of the European Parliament and of the Council on the approximation of the laws of the Member States relating to food supplements 2. Commission Directive 2001/15/EC of 15 February 2001 on substances that may be added for specific nutritional purposes in foods for particular nutritional uses 3 Regulation (EC) No 1925/2006 of the European Parliament and of the Council of 20 December 2006 on the addition of vitamins and minerals and of certain other substances to foods 4 TERMS OF REFERENCE AS PROVIDED BY THE COMMISSION In accordance with Article 29 (1) (a) of Regulation (EC) No 178/2002, the European Commission asks the European Food Safety Authority to: - carry out the additional assessment for vitamin K 2 as source of vitamin K added for nutritional purposes to foodstuffs, in the context of Regulation (EC) N 258/97, and - provide a scientific opinion, based on its consideration of the safety and bioavailability of vitamin K 2 added for nutritional purposes in foods for particular nutritional uses and foods (including food supplements) intended for the general population. ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank the members of the Working Group for the preparation of this opinion: Jean-Louis Bresson, Karl-Heinz Engel, Marina Heinonen, Pagona Lagiou, Bevan Moseley, Andreu Palou, Annette Pöting, Seppo Salminen, Hendrik Van Loveren, Hans Verhagen; and ad hoc expert Ivonne Rietjens. 2 OJ L 183, , p OJ L52, , p OJ L 404, p.26. The EFSA Journal (2008) 822, 5-31

6 ASSESSMENT Introduction In accordance with the Commission Recommendation 97/618/EC, the ingredient concerned by the application belongs to Class 2.1. Complex NF from non-gm sources: the source of the NF has a history of food use in the Community. For this reason the Opinion will be an assessment of the safety data provided by the applicant to comply with the information required for novel foods of Class 2.1, i.e. information requirements I, II, III, IX, X, XI, XII and XIII as detailed in the following text and does not include an assessment of the possible nutritional benefits of vitamin K 2. Table 1 provides an overview allowing cross reading of the opinion with the format for the opinions on food supplement use. Table 1. Overview allowing cross reading of the opinion with the format for the opinions on food supplement use. Novel food section I Specifications II Effects of the Production Process III History of the Organism Used as the Source of the Novel Food IX Anticipated Intake/Extent of Use of the Novel Food X Information from Previous Human Exposure to the novel food or its source XI Nutritional information on the Novel Food XII Microbiological information on the Novel Food XIII Toxicological information on the Novel Food I. Specification of the novel food (NF) (Chemistry) Food supplement section Chemistry Specifications Manufacturing Process History of the Organism Used as the Source of the Novel Food Intended use levels Exposure Information on existing authorisations and Case of need, exposure Biological and Toxicological data Specifications Biological and Toxicological data Cantox Health Sciences International on behalf of NattoPharma (Norway) proposes to market a sunflower oil suspension of an vitamin K2-rich extract obtained from the fermentation of soybean protein isolate and corn starch in the presence of Bacillus subtilis natto. The contained vitamin K 2 is principally menaquinone-7 (MK-7) and to a smaller extent, menaquinone-6 (MK-6). The commercial food-grade formulation of the vitamin K 2 -containing oil consists of not less than 0.15 % (1,500 ppm) total vitamin K 2 (occurring principally as MK-7 and, to a minor extent, MK-6), and not more than % sunflower oil as a formulation aid. Minor constituents resulting from the manufacturing process of the K 2 -containing oil include unsaponifiable matter (0.85 %), phospholipids (0.002 %), sterols (0.404 %) and tocopherols (0.075 %). The specifications, limiting values, and reference test methods for menaquinone, formulated as K 2 -containing oil, proposed by the petitioner, are presented in Table 2. Vitamin K 2 (2-methyl-3-all-trans-polyprenyl-1,4-naphthoquinones), or the menaquinone series, is a group of prenylated naphthoquinone derivatives (Merck, 2001). The number of isoprene residues, where one isoprene unit consists of 5 carbons comprising the side chain, is The EFSA Journal (2008) 822, 6-31

7 used to characterize the menaquinone homologues. The nomenclature is based on the number of isoprene units, such that the name menaquinone is followed by a number. Although menaquinones with side chains of up to 15 isoprene units have been identified, menaquinones of only 2 to 13 isoprene units have been encountered in human and animal tissues (PDR, 2001). Demethylmenaquinones, which are menaquinones nonsubstituted at the carbon-2 position, also have been identified (Conly and Stein, 1992). The K 2 -containing oil evaluated in the present opinion primarily contains MK-7, and MK-6 to a smaller extent. The structural formula for the vitamin K 2 (menaquinone) series is presented in Figure 1. O 1 2 CH 3 3 CH 3 2-methyl-1,4-naphthoquinone ( menadione moiety) O CH 3 CH 3 n = 1-12 Figure 1. Structural formula of the vitamin K 2 (menaquinone) series with menaquinone-7 (MK-7)(n=6) being C 46 H 64 O 2, menaquinone-6 (MK-6)(n=5) being C 41 H 56 O 2 and menaquinone-4 (MK-4)(n=3) being C 31 H 40 O 2. The collective term vitamin K, encompasses several lipid-soluble 2-methyl-1,4- naphthoquinone derivatives. Naturally occurring forms of vitamin K include vitamin K 1 and vitamin K 2 (i.e., phylloquinone and the menaquinone series, respectively), which are characterised by lipophilic side chains at the 3-position of the 2-methyl-1,4-naphthoquinone ring structure. Menadione (vitamin K 3 ), which serves as a vitamin K 2 precursor in animals, and acetomenaphthone (vitamin K 4 ) comprise the synthetic forms of vitamin K (Gilman et al., 1990; Sweetman, 2002). Table 2. Chemical Specifications for K2-containing oil Specification Parameter Specification Method (a) Total Vitamin K 2 (ppm) Not less than 1,500 HPLC (b) Acid Value (mg KOH/g fatty Not more than 10 AOCS (c) Official Method Te 1a-64 acids) Peroxide Value (meq H 2 O 2 /1,000 g sample) Not more than 3 (at the time of filling) (a) : See Appendix A for details of the analytical methods. (b) : HPLC - High Performance Liquid Chromatography (c) : American Oil Chemists Society II. Acetic Acid-Isooctane Method (AOCS Official Method Cd 8b-90) Effect of the production process applied to the NF (Manufacturing process) The raw materials used in the production of menaquinone, formulated as K2-containing oil, include soybean protein isolate, corn starch, water, B. subtilis natto, ethanol, and sunflower oil. The manufacturing process is adequately described by the petitioner and includes fermentation, ethanol extraction, filtration and purification steps, degumming and the addition of sunflower oil. The EFSA Journal (2008) 822, 7-31

8 The petitioner indicates that the analytical methods for the determination of menaquinone from foods, such as beverages, vegetables, fruits, and dairy products, are similar to those utilised for phylloquinone (Gijsbers et al., 1996; Schurgers and Vermeer, 2000). Foods are homogenised in a blender, and vitamin K (phylloquinones and menaquinones) is extracted using 2-propanol, hexane, or ethanol as extraction solvents, and MK-6 or 2,3-dihydrophylloquinone as internal standards (Gijsbers et al., 1996; Schurgers and Vermeer, 2000). The determination of vitamin K2 is subsequently carried out by HPLC analysis using a C-18- reversed phase column and fluorometric detection after post-column electrochemical reduction, as described by Gijsbers et al. (1996). In order to determine the stability of vitamin K2 present in K2-containing oil over time, the petitioner conducted analyses of its commercial formulation for total vitamin K2 content. Total vitamin K2 concentrations in unopened K2-containing oil products stored at room temperature for a period of up to 700 days were measured by HPLC. The results of the stability tests demonstrate that the total vitamin K2 content is minimally (less than a few percent) affected by storage times of the final product up to at least 2 years. The petitioner recommends that the commercial formulation of menaquinone, K2-containing oil, be stored at a temperature of not more than 15 C in a cool, dry, and dark place, and away from high heat, humidity, and sunlight. When stored properly, it is reported that K2-containing oil remains stable for at least 12 months. III. History of the organism used as the source of the NF The organism used as the source of the novel food is Bacillus subtilis natto which is defined taxonomically as follows: division Firmicutes, class Bacilli, order Bacillales, family Bacillaceae, genus Bacillus, species subtilis subspecies natto. Bacillus subtilis natto is a subspecies of Bacillus subtilis, a Gram-positive, catalase positive bacterium commonly found in soils. Bacillus subtilis natto is used in the commercial production of the Japanese delicacy natto and has a history of consumption in Japan. Three different strains of Bacillus subtilis natto are used in the fermentation stage of the natto manufacturing process in Japan of which the most widely used is the Mira strain, possessing a current market share of %. The petitioner indicates that the Bacillus subtilis natto Mira strain was first isolated over 50 years ago and has been commonly used as a source of natto ever since. Vitamin K 2 (MK-7) production employs a strain of Bacillus subtilis natto obtained by mutation of the Mira strain which is performed with a conventional breeding method well-known to the fermentation industry, in order to optimise the microorganism for efficient MK-7 production. IX. Anticipated intake/extent of use of the NF The petitioner indicates that under the conditions of intended use, the level of use of menaquinone from K 2 -containing oil would be in accordance with existing EU legislation, and are anticipated to be similar to the levels of phylloquinone currently approved for foods for particular nutritional uses (PARNUTS), food supplements, and fortified foods. For all uses, including food supplement use, the petitioner does not envisage an addition level of higher than 30 % of the Reference Labelling Values (RLV) for vitamin K per serving of 75 microgram for adults and 12 micrograms for children aged 6 months to 4 years (SCF, 2003a). Table 3 presents the uses and use levels for vitamin K 2 proposed by the petitioner. The EFSA Journal (2008) 822, 8-31

9 The petitioner also indicates that for supplements the addition level will typically be in the range of 50 microgram per day for adults depending on the target of the supplementation. The level of consumption of vitamin K 2 resulting from this use level would represent approximately 5 % of the vitamin K 2 consumption from a 100 g portion of natto. The petitioner indicates that the vitamin K 2 -containing ingredient will not be added to infant and follow on formula or processed cereal-based baby foods and baby foods. Table 3. Proposed uses and use levels for vitamin K2. Food Category Proposed Food Use Vitamin K 2 per serving (microgra m) Serving size (g) Use level (microgram/kg) Beverages Fruit and Vegetable juices Soft Drinks (low calorie) Soft Drinks (not low calorie) Cereal and Cereal Breakfast Cereals to to 500 Products Cereal Bars Dairy Products Cottage Cheese and Low Fat to to 500 Cheese Fromage frais Frozen Yoghurt Ice Cream to to 200 Milk Other Milk and Creams to to 667 Yoghurt Yoghurt Drinks Fats and Oils Low Fat Margarine Low Fat Mayonnaise Olive Oil Pasta, Rice and Pasta Other Miscellaneous Grains Pizza Crust The petitioner provided estimated daily intakes of vitamin K 2 in the United Kingdom, based on the proposed uses and use-levels and on food consumption data from the UK National Dietary and Nutrition Survey (NDNS) Programme (weighed dietary records). Available data include a survey in children aged 1.5 to 4.5 (UKDA, 1995) and a survey in young people aged 4 to 18 (UKDA, 2001), adults aged 16 to 64 years (Office of National statistics 2005). Individual body weights were available in all population groups. Intakes were calculated from NDNS food consumption data and typical usage levels for vitamin K 2 as presented in Table 3. The NDNS data food codes were matched to the usage categories in Table 3 (assuming maximum usage levels and presence in all foods where it could be used) and potential dietary exposure was calculated for each individual. Individuals were considered consumers if they consumed one or more food products in which vitamin K 2 is proposed for use on one of the 4 ( years) or 7 survey days (other The EFSA Journal (2008) 822, 9-31

10 subgroups). Calculations for the potential mean and high-level (97.5th percentile) intakes of vitamin K 2 were performed for the different food categories as well as for the overall intake from all proposed food uses combined. As shown in Table 4, overall estimated mean intake of vitamin K 2 among consumers ranged from 36 microgram/day (female adults) to 54 microgram/day (male teenagers). High intake levels ranged from 75.0 microgram/day (children) to 115 microgram/day (male teenagers). Intakes per kg body weight were also provided by the petitioner and are presented in Table 5. The highest 97.5th percentile was in children (5.4 microgram/kg bw/day). Table 4. Potential estimated daily intake of vitamin K2 in microgram per person among consumers according to UK food surveys Population Group Age Group User % Actual # of Total Users All-Users Consumption Mean Percentile (microgram) Children 1 ½ - 4 ½ , Young People Female Teenager Male Teenager Female Adults Male Adults Table 5. Potential estimated daily intake of vitamin K2 in microgram/kg bw among consumers according to UK food surveys Population Group Age Group User % Actual # of Total Users All-Users Consumption Mean Percentile (microgram) Children 1 ½ - 4 ½ , Young People Female Teenager Male Teenager Female Adult Male Adult High level intakes from individual foods ranged from <1 to around microgram/day, and resulted for all age groups mainly from soft drinks and milk. The petitioner indicates that all of these estimates are highly conservative because they assume that vitamin K 2 would be present in all foods for which it is proposed to be used. The Panel agrees with this statement. For individual food categories this might be realistic since consumer loyalty and individual preferences might cause a person always to choose particular brands containing the additive. However, when potential dietary exposures from all foods are combined the exposure scenarios become far less likely. The EFSA Journal (2008) 822, 10-31

11 Additional exposure to vitamin K 2 may also occur from the use of food supplements. Assuming a use-level of 50 microgram vitamin K 2 in food supplements the total daily vitamin K 2 intake of an adult exposed at the average level of dietary exposure from fortified foods and food supplements would lie in the range of 86 to 95 microgram/day, corresponding to 1.4 to 1.6 microgram/kg bw/day for women and men with a standard body weight of 60 kg, respectively. A very conservative estimate for high intakes would be in the range of 131 to 157 microgram /day, corresponding to 2.2 to 2.6 microgram/kg bw/day for women and men, respectively. The Panel notes that intake from vitamin K from fortified foods and/or food supplements will add to dietary intake from food sources, assuming an average phylloquinone intake of approximately microgram/day from food sources only. It is noted that these estimates for the high intakes are based on conservative assumptions. X. Information from previous human exposure to the NF or its source and Information on existing authorisations and evaluations Exposure Vitamin K 1 is regarded as the major dietary source of vitamin K, occurring naturally in green vegetables (Gilman et al., 1990; Merck, 2001). In contrast to vitamin K 1, dietary contribution of vitamin K 2 is much less (PDR, 2001). Dietary sources of vitamin K 2 include foods such as chicken egg yolk, butter, cow liver, certain cheeses and natto (Schurgers and Vermeer, 2000; PDR, 2001). Vitamin K 2 also is of microbiological origin, found primarily in fermented foods (e.g. natto), as well as produced by the bacteria of the gastrointestinal tract (Akiyama et al., 1995). The various natural forms of vitamin K have different sources. Table 6 summarizes the vitamin K content of various foods. Table 6. Vitamin K content (ng/g) of various foods (a) Food Item Phylloquinone Menaquinone-4 Menaquinone-9 Meat, fish 10 to to to 20 Pork liver 2 to 5 3 to 5 10 to 20 (b) Milk, yoghurt 4 to 10 4 to 10 0 to 20 Cheese, curd 20 to to to 700 Green vegetables 2,000 to 8, Fruit 1 to Bread 5 to to 20 Vermeer et al. (1998) (a) (b) Various food items were homogenised in a blender and extracted with hexane. Vitamin K analysis was performed after pre-purification of the hexane fraction. All data are based on fresh food and expressed per g of wet weight. No menaquinone-9 (MK-9) was present; data are for the sum of MK-7 and menaquinone-8 (MK-8). In the UK, the Expert Group on Vitamins and Minerals (EVM) estimated the mean intake of vitamin K to be 68 microgram/person/day (EVM, 2003). Including additional sources such as supplements (up to 200 microgram/day), the maximum intake of vitamin K was estimated to be 270 microgram/person/day (EVM, 2003). In the United States several studies indicate that mean intakes of phylloquinone in young adults range from 60 to 110 microgram/day and The EFSA Journal (2008) 822, 11-31

12 in older adults (>55 years) from 80 to 210 microgram /day (Booth et al., 1996). In Finland average intake was estimated to be 120 microgram/day (Koivu-Tikkanen, 2001). In the Netherlands, mean daily per capita intake was estimated to be up to 250 microgram due to the relatively high intake of green vegetables. For menaquinone intake there are no populationbased data available, except for the Netherlands where menaquinones are estimated to form about 10 % of total vitamin K intake, corresponding to an estimated mean daily per capita intake of about 25 microgram (Schurgers et al., 1999). Information on existing authorisations and evaluations Currently, only vitamin K 1 (phylloquinone) is permitted for use as a source of vitamin K in PARNUTS categories (Commission Directive 2001/15/EC), food supplements (Directive 2002/46/EC of the European Parliament and of the Council), and fortified foods (Commission of the European Communities, 2001; European Parliament & Council of the EU, 2002). In the U.S., vitamin K is listed under 21 CFR (FDA, 2004), with labelling provisions for dietary supplement products containing vitamin K prescribed under 21 CFR (FDA, 2004). Vitamin K 2 is currently listed in the Physician s Desk Reference (PDR) for use as a source of vitamin K in supplements or multivitamin preparations with a vitamin K component at doses typically ranging from 25 to 100 microgram (PDR, 2001). As previously mentioned, in Japan, vitamin K 2 (as MK-4) is registered for use as a therapeutic agent for the treatment of postmenopausal osteoporosis (IARC, 2000; PDR, 2001; Schurgers, 2002). B. subtilis natto, the bacterium used in the preparation of the fermented soybean product natto, is used in Japan as a dietary supplement source of vitamin K 2 (PDR, 2001). Natto is a traditional Japanese food produced from fermented soybeans and is reported to have a history of dietary consumption in Japan dating back at least 1,000 years (Sumi et al., 1990). In Japan, natto is sold in a pack that usually contains 40 g of natto, a quantity considered to be suitable for one meal in Japan. One pack of natto contains approximately 350 µg menaquinone-7 (Sakano et al., 1988). The Japanese Population-Based Osteoporosis Study has investigated the effects on bone mineral density of the habitual natto intake of up to 4 packs per week in 944 pre- and postmenopausal women over 3 years (Ikeda et al., 2006). This intake corresponds to up to 200 µg per day MK-7. The baseline mean intake for premenopausal women was 1.4 packs of natto per week, and for postmenopausal women 2.0 packs per week, corresponding to 70 and 100 µg MK-7 per day (Ikeda et al., 2006). Natto is also manufactured and sold as a food product on the U.S. market (U.S. Soyfoods Directory, 2003). Natto is rich in vitamin K 2, present mainly as MK-7. Vitamin K 2 extracted from natto has been also sold in Europe in food supplement products since before The Scientific Committee on Food (SCF) made no recommendations for a Population Reference Intake for vitamin K but considered that an intake of 1 microgram/kg body weight/day appears to be adequate and would be provided by a normal diet (SCF, 1993). This recommendation was also used in the UK (COMA, 1991). More recently recommended intakes in some countries have been determined based on effects on blood coagulation. A recommended daily dietary intake for vitamin K of microgram/day or 1 microgram/kg body weight/day has been proposed (D-A-CH Referenzwerte, 2000). The Institute of Medicine (IOM) established an Adequate Intake (AI) for vitamin K of 120 and 90 microgram/day for male and female adults, respectively (IOM, 2001). For an average 60 kg adult, these levels would correspond to AI values of 2.0 and 1.5 The EFSA Journal (2008) 822, 12-31

13 microgram/kg body weight/day of vitamin K for males and females, respectively. Because of lack of specific information about vitamin K requirement of children reference values for them are set at about 1 microgram/kg body weight (IOM, 2001; D-A-CH Referenzwerte, 2000). In its opinion on the Tolerable Upper Intake Level of Vitamin K, the SCF (2003b) expressed that In human studies of limited numbers, there is no evidence of adverse effects associated with supplementary intake of vitamin K in the form of phylloquinone of up to 10 mg/day (more than 2 orders of magnitude higher than the recommended dietary intake of vitamin K) for limited periods of time. These limited data are supported by experimental animal studies in which no adverse effects were observed after daily administration of extremely high doses (2,000 mg/kg body weight) for 30 days. Because of the antagonistic interaction of phylloquinone and coumarin anticoagulant drugs, people taking these drugs should not significantly increase their phylloquinone intake by dietary change or by using dietary supplements without medical advice. In this report, no tolerable upper intake level for vitamin K could be set based on the available data (SCF, 2003b) Similar to the SCF, the EVM (2003) concluded that, there are insufficient data from studies in humans or animals to establish a Safe Upper Level for vitamin K, and that, there are clear differences in the toxicity of different forms of vitamin K. However, a guidance level of 1 mg/person/day (or mg/kg body weight in a 60 kg adult) for supplementary intake of vitamin K 1 was established (EVM, 2003). This was based on limited human supplementation studies, which showed that doses of up to 10 mg/person/day of vitamin K 1 for one month were not associated with adverse effects (EVM, 2003). Given the lack of adverse effects in individuals consuming high doses of vitamin K, also the IOM (2001) was unable to determine a tolerable upper intake level for the compound. XI. Nutritional information on the NF (Biological data) Vitamin K activity can be defined as the ability to serve as a cofactor for the enzyme γ-glutamylcarboxylase (Vermeer et al., 2004). In this respect, vitamins K 1 and K 2 have similar activity, although MK-4 has been reported to be the most potent vitamin K form on γ- carboxylation in the blood coagulation system (Graul and Castañer, 1996; Spronk et al., 2003; Geleijnse et al., 2004, Vermeer et al., 2004). In addition to its vitamin K activity for blood coagulation, Vitamin K 2 has been reported to directly promoting bone metabolism and reducing the incidence of fracture in osteoporosis (Orimo et al., 1992, 1998; Hara et al., 1995; Kameda et al., 1996; Shiraki et al., 2000; Vermeer, 2003b; Vermeer et al., 2004). When compared with vitamin K 1, vitamin K 2 was noted to be more efficacious in decreasing bone turnover in vitro (Hara et al., 1995; Kameda et al., 1996) and in vivo in humans (Vermeer, 2003b). In contrast to vitamin K 1, vitamin K 2 has been reported to decrease serum cholesterol and cholesterol-ester deposition in the aorta, contributing to the suppression of atherosclerotic plaque progression (Graul and Castañer, 1996; Spronk et al., 2003). Dietary deprivation of vitamin K does not induce vitamin K deficiency in humans (Hollander et al., 1976). The application of broad-spectrum antibiotics in humans has been associated with a reduction in hepatic vitamin K 2 concentration and the occurrence of vitamin K- responsive hypoprothrombinaemia, indicating the utilisation of bacterially synthesised menaquinones by the host (Conly and Stein, 1992; Conly and Stein, 1994). It should be noted that the present opinion does not include an assessment of the possible benefits of vitamin K2. The EFSA Journal (2008) 822, 13-31

14 The available data suggest that the physiological activities of vitamin K2, other than its role for the blood coagulation, might differ from those of vitamin K1. The Panel however does not expect that the proposed vitamin K2-rich sunflower oil suspension in the intended use would be nutritionally disadvantageous to consumer. Bioavailability of vitamin K from its vitamin K 2 source The absorption of vitamin K 2 was studied in vitro, using radiolabeled menaquinone-9 (MK-9) incubated with everted small and large rat intestines (Hollander and Rim, 1976; Hollander et al., 1977). Vitamin K 2 absorption was reported to occur via passive diffusion, was nonsaturable, and was linearly related to the concentration. Colonic and ileal absorption of vitamin K 2 was further investigated in vivo in male Sprague- Dawley rats (Hollander et al., 1977). The colon and ileum were perfused with solutions containing non-radioactive menaquinone and radiolabeled [2-methyl- 3 H]-MK-9. The rate of absorption was linearly related to the vitamin K 2 concentration. Akiyama et al. (1995) compared the physiological activity of menaquinone homologues, menaquinone-1 (MK-1) through menaquinone-14 (MK-14), administered orally to hypoprothrombinaemic male Sprague-Dawley rats at doses of 0.1 mg/kg body weight. In groups treated with MK-4 to MK-10, the corresponding homologue was detected in the plasma at 3 and 6 hours post-dosing, and at 3 hours in rats receiving MK-11. Menaquinone homologues with more than 12 isoprene units were not readily absorbed. Rapid absorption of vitamin K 2 was reported, as evidenced by a maximum plasma concentration (C max ) of 2.91 to 5.31 microgram/ml obtained within approximately 1 hour, following oral administration of 4 mg/kg body weight of MK-4 to 4 male non-fasted beagle dogs in a test or control formulation, encapsulated in soft gelatine capsules (Amemiya et al., 1999). In humans, Conly et al. (1993, 1994) examined the intestinal absorption of bacterially synthesised vitamin K 2. Four healthy male subjects (22 to 30 years of age) were maintained on a vitamin K 1 -restricted diet and were warfarinised to maintain an International Standardised Ratio (INR) of approximately 2.0 for a period of 15 days. During the experimental phase, subjects received 1.5 mg of menaquinone, which was extracted from batches of harvested Staphylococcus aureus (ATCC 29213), via a nasoileal tube 10 days after warfarin treatment was initiated (i.e., Day 15). Approximately 90 % of the administered final dose consisted of MK-7 and MK-8. The diet was continued for an additional 3 days following vitamin K 2 administration. The parameters monitored throughout the study period included prothrombin time, factor VII and II, and serum vitamin K 1 levels. Evaluated immediately prior to menaquinone administration, a 30 % reduction was observed in mean serum vitamin K 1 levels compared to pre-diet values. Within 24 hours of vitamin K 2 administration a significant increase in factor VII, paralleled by changes in INR, and a decrease in prothrombin time were reported compared to pre-administration levels. Furthermore, while serum menaquinone was not detected prior to ileal administration, serum samples obtained 3 and 5 hours after menaquinone was administered revealed a MK-8 level of 1.4 nmol/l in one individual. The plasma pharmacokinetic profile of MK-4 in 194 infants (5 days old) was reported to be similar to that of phylloquinone (Shinzawa et al., 1989). Several studies were conducted in which circulating levels of MK-7 were monitored following ingestion of natto or natto bacilli powder. In eastern Japanese women identified as The EFSA Journal (2008) 822, 14-31

15 frequent natto consumers, serum MK-7 levels were significantly higher (5.26 ± 6.13 ng/ml) compared to women in western Japan and Britain (1.22 ± 1.85 and 0.37 ± 0.20 ng/ml, respectively), where natto is less frequently consumed (Kaneki et al., 2001). A dosedependent increase in plasma MK-7 was observed in 6 healthy male subjects provided 2 to 100 g of natto (Sumi, 1999). Regardless of dose, the highest plasma levels were noted at 4 hours post-administration. Vermeer (2003a) conducted a crossover design study in order to compare the pharmacokinetics of MK-7 following ingestion of natto capsules versus natto food. Six volunteers were recruited and given either a single dose of natto food (1.18 mg MK-7 per dose) or 10 vitamin K2 (menaquinone from natto) capsules (1.13 mg MK-7 in total). Large inter-individual variations were identified among subjects in MK-7 absorption from either source. Maximum average serum MK-7 concentrations were 16.2 and 16.5 ng/ml at 6 and 4 hours after ingestion of natto food and capsules, respectively, compared to an average pretreatment value of 0.4 ng/ml. The area under the curve (AUC 0-8 ) was 89.5 ± 28.4 ng h/ml with natto food ingestion and 69.6 ± 23.5 ng h/ml with natto capsules, indicating that the absorption from capsules was 78 % of that from natto. Another study to examine the bioavailability of vitamin K from foods was conducted with 6 male healthy volunteers (mean age 33.5 years) who received either a vitamin K-poor meal, supplemented with 3.5 μmol of a detergent-solubilised vitamin K1 product, or a vitamin K- rich meal consisting of 400 g cooked spinach (providing 3.5 μmol vitamin K1), or 200 g natto (providing 3.1 μmol MK-7) (Schurgers and Vermeer, 2000). All of the meals were supplemented with corn oil to ensure a total fat content of 30 g. Post-prandial serum vitamin K concentrations were determined for 72 hours. Peak values for serum vitamin K (both phylloquinone and MK-7) were noted at 6 hours following meal consumption. Serum concentrations of phylloquinone were higher following consumption of the vitamin K1 supplement alone (~45 nmol/l) compared to the subjects receiving cooked spinach (~10 nmol/l), with peak serum phylloquinone levels occurring at 4 and 6 hours, respectively. In contrast, receiving MK-7 from natto showed peak serum concentrations of approximately ~80 nmol/l 6 hours after meal consumption. A rapid decline of both, phylloquinone and MK- 7 concentrations was observed after the serum peak levels with a half life time (t 1/2 ) of ~1.5 hours during the first 8 hours for both. The disappearance of MK-7 was considerably protracted due to its higher bioavailability and remained detectable for at least 72 hours (with t 1/2 of ~50 hours for MK-7 after 8 hours). It is noted that this study did not provide an Area Under the Curve (AUC) value and a quantification of the higher bioavailability of MK-7 compared to vitamin K1 in this study. These results were confirmed by other human supplementation studies, confirming that higher and more stable plasma levels of vitamin K were reached with supplements containing vitamin K 2 (MK-7) compared to those achieved with vitamin K 1 (Vermeer, 2003a). It has been demonstrated that the bioavailability of vitamin K is dependent upon the nature of the food matrix (Schurgers, 2002). While oil-solubilised forms of vitamin K (i.e., phylloquinones in plant oils and menaquinones in dairy products) are absorbed relatively well, the phylloquinones present in green leafy vegetables, which represent approximately 80 % of an individual s total dietary vitamin K intake, are tightly bound to the thylakoid membranes in plant chloroplasts, and are not well solubilised by bile salts (Gijsbers et al., 1996; Schurgers and Vermeer, 2000; Schurgers, 2002; Vermeer et al., 2004). However, the concomitant intake of fat increases phylloquinone absorption from vegetables through the stimulation of bile secretion (Gijsbers et al., 1996; Schurgers and Vermeer, 2000; Schurgers, 2002). In contrast, although less than 20 % of the total vitamin K intake is accounted for by The EFSA Journal (2008) 822, 15-31

16 menaquinones, they have a higher bioavailability than phylloquinones since menaquinones occur in the matrix of such foods as cheese, eggs, and meat (Schurgers, 2002). Among the various forms of menaquinones, the length of the side chain plays an additional role in bioavailability, as menaquinones with medium-length side chains (e.g., MK-7) are better absorbed compared to those with short (MK-4) or long (e.g., MK-8 and MK-9) side chains (Schurgers and Vermeer, 2000; Schurgers, 2002). Altogether vitamin K 2 appears to be absorbed rapidly and unchanged from the gastrointestinal tract, is carried in the lymph in mixed micelles composed of bile salts, and subsequently released into the circulation (IARC, 2000). As with other lipid-soluble compounds, optimal absorption is dependent on the presence of bile acids (IARC, 2000). Based on the available data, the Panel concludes that the proposed menaquinone-rich sunflower oil suspension provides a source for vitamin K in the form of MK-7 which seems to be better bioavailable than vitamin K1. Metabolic fate and biological distribution Following absorption, vitamin K 2 is distributed to the liver, in which MK-6 through MK-13 comprise 90 % of the total vitamin K composition. Only 10 % of the hepatic vitamin K stores consist of vitamin K 1 (IARC, 2000). The distribution of the various menaquinone homologues varies considerably among individuals (IARC, 2000). Additionally, significant amounts of MK-4 have been identified in the salivary glands, kidneys, pancreas, and brain (Thijssen and Drittij-Reijnders, 1994). In female rats receiving daily doses of 4 mg/kg body weight of [ 14 C]-MK-4 for a period of 10 days, the highest amounts of radioactivity were identified in the liver, adipose tissue, spleen, and adrenals (Sano et al., 1995a). The liver is the principal site of vitamin K metabolism, involving oxidative degradation of the side-chain and resulting in subsequent elimination via the bile or urine (IARC, 2000). In order to examine MK-4 metabolism and excretion, groups of 3 male, bile duct-cannulated Wistar rats were treated with single oral or intravenous doses of 4 mg [ 14 C]-MK-4 (Tadano et al., 1989). Urine and faeces samples were collected every 24 hours for a total of 5 days. Following oral or intravenous administration of MK-4 to rats, 5- and 7-carbon side chain carboxylic acids were identified in the urine and bile [2-methyl-3-(5-carboxy-3-methyl-2- pentenyl)-1,4-naphthoquinone (vitamin K acid 1) and 3-(3-carboxybutyl)-2-methyl-1,4- naphthoquinone (vitamin K acid 2), respectively]. An additional metabolite was isolated from the bile [2-methyl-3-(15-carboxy-3,7,11-trimethyl-2,6,10,14-hexadecatetraenyl)-1,4- naphthoquinone (ω-cooh)]. All 3 metabolites were excreted as glucuronide conjugates. Generally, excretion in the faeces via bile secretion was identified as the predominant route of MK-4 elimination, as evidenced by a 63.3 % recovery of the dose in the bile within 24 hours. In comparison, depending on the route of administration, only 6.0 to 10.3 % of the dose was eliminated in the urine over the course of the entire 5-day collection period. In the bile, all 3 metabolites as well as the parent compound were detected, whereas only the metabolites, vitamin K acid 1 and vitamin K acid 2, were identified in the urine samples. In another study conducted in male rats, blood concentrations of radioactivity peaked at 1.5 to 2 hours following administration of single oral doses of 4 mg/kg body weight of [ 14 C]-MK-4 and declined slowly thereafter (Sano et al., 1995b). Although distribution to tissues was generally slow at 4 hours post-dosing, the highest levels of radioactivity were detected in the liver, adrenals, and spleen. Major metabolites of MK-4 identified were ω-cooh, K acid 1, and K acid 2. Evaluated at 1.5 hours after administration, MK-4 was found primarily unmetabolised in plasma, liver, adipose tissue, and cancellous tissue of the thighbone. MK-4 The EFSA Journal (2008) 822, 16-31

17 remained unchanged for up to 168 hours in adrenal tissue, pancreas and cancellous tissue, while in the plasma and liver, the ratio of metabolites to unchanged MK-4 increased by 24 hours. The majority of MK-4 was excreted unchanged in faeces, where 88.2 % of dosed radioactivity was reported, while 7.8 % of radioactivity was identified in the urine, some of which was present as polar metabolites. Overall, it can be noted that MK-4 metabolism resembled that of phylloquinone (Tadano et al., 1989), which also is metabolised to carboxylic acids, with either a 5- or 7-carbon side chain (McBurney et al., 1980). Therefore, similar to vitamin K 1, vitamin K 2 metabolite formation appears to proceed via a series of reactions, beginning with ω-methyl oxidation to form the MK-4 carboxylic acid (ω-cooh), followed by β-oxidative degradation of the alkyl side chain to yield vitamin K acids 1 and 2. The proposed metabolic pathway of MK-4 is presented in Figure 2. O Menatetrenone CH 3 ω-methyl oxidation O ω-cooh O CH 3 CH 3 CH 3 CH 3 OH O β-oxidation CH 3 O Vitamin K acid 1 O CH 3 OH O CH 3 O Vitamin K acid 2 O CH 3 OH Figure 2. Metabolic Pathway of Menaquinone-4 (Menatetrenone) (MK-4) (Tadano et al., 1989) It is suggested that vitamin K undergoes metabolic recycling by the actions of liver microsomal enzymes. Initial reduction of the vitamin K-quinone is carried out by the NADPH-dependent reaction involving quinone reductases to form vitamin hydroquinone (vitamin K-H 2 ) (Thorp et al., 1995). This intermediate functions as the cofactor in carboxylation reactions, and is coupled to an epoxidation reaction yielding the vitamin K-2,3-epoxide. In turn, this metabolite serves as the substrate for an epoxide reductase, regenerating the vitamin K-quinone. The EFSA Journal (2008) 822, 17-31

18 XII. Microbiological information on the NF Safety of Vitamin K2 (Menaquinone) The petitioner indicates that as a result of its production process, which includes a 30-minute sterilization of the fermented broth at 120 C and deodorization by steam distillation at 170 C, no viable microbiological organisms are expected to be present in the final K 2 -containing oil product. XIII. Toxicological information on the NF (Toxicological data) Only a few studies were conducted specifically with the vitamin K 2 homologues (MK-6 and MK-7) present in the K 2 -containing oil formulation of the present opinion. However, several published animal toxicity studies, as well as clinical studies, have been conducted with the vitamin K 2 homologue, MK-4. Considering the similar metabolic fate of the vitamin K derivatives, and the structural similarity of the vitamin K 2 homologues, read across from these studies can be used in support of the safety of the vitamin K 2 -containing oil formulation of the present opinion. Acute toxicity No mortality or side effects were reported following administration of an oral dose of 540 mg/kg body weight of vitamin K 2 to mice (Takehara, 1981). No further details were provided. Sub-chronic toxicity A 13-week oral toxicity study was conducted in groups of Sprague-Dawley rats (10/sex/group) to evaluate the potential toxicity resulting from the concurrent administration of 300 mg/kg body weight/day of ipriflavone (TC-80), a synthetic isoflavone preparation, and 30 mg/kg body weight/day of MK-4 (Doi et al., 1995). The MK4 test material used was a pure synthetic sample. A vehicle group and a group receiving only 30 mg/kg body weight/day of MK-4 also were maintained. All animals receiving MK-4 only were reported to survive the entire duration of the study period and no clinical signs of toxicity were noted. Body weights and food consumption were not affected by the treatment with the exception of a slight increase in calcium concentration in males, blood chemistry, urine analysis and ophthalmoscopic examination were unremarkable in MK-4-treated animals. Compared to controls, a slight, but significant increase in reticulocyte count was reported in females receiving oral administrations of MK-4. The increase, however, was deemed by the authors to be toxicologically insignificant, since values fell within the normal physiological range, and the effect was not accompanied by any histopathological variations of the haematopoietic tissue. In males, haematological variations were limited to a significant elevation in platelet count. At necropsy, absolute and relative organ weights, including liver, kidneys and spleen of MK-4-treated rats were comparable to the control group, and histopathology did not reveal any abnormalities. Groups of male and female beagle dogs (number per group not identified) received oral administrations of encapsulated synthetic MK-4 at dose levels of 0 (control), 20, 200, or 2,000 mg/kg body weight/day for a period of 3 months (Goldsmith et al., 1995). Following the 3-month treatment period, control and high-dose (2,000 mg/kg body weight/day) animals were maintained for an additional month in order to monitor recovery. No significant differences in body weights were observed among the groups. Samples for haematology and blood chemistry were obtained on week 7 and at study termination. Compared to controls, a statistically significant increase in platelet count and decrease in red blood cells were reported The EFSA Journal (2008) 822, 18-31