Use of lycopene as a food colour 1. Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food

Transcription

1 The EFSA Journal (2008) 674, 1-66 Use of lycopene as a food colour 1 Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food (Questions No EFSA Q , Q , Q ) Adopted on 30 January 2008 PANEL MEMBERS Fernando Aguilar, Herman Autrup, Sue Barlow, Laurence Castle, Riccardo Crebelli, Wolfgang Dekant, Karl-Heinz Engel, Natalie Gontard, David Gott, Sandro Grilli, Rainer Gürtler, John Chr. Larsen, Catherine Leclercq, Jean-Charles Leblanc, F. Xavier Malcata, Wim Mennes, Maria Rosaria Milana, Iona Pratt, Ivonne Rietjens, Paul Tobback, Fidel Toldrá. SUMMARY Following a request from the Commission, the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food was asked to deliver a scientific opinion on the safety in use of synthetic lycopene as a food colour for use in the food categories specified in the dossier. The Scientific Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food has also been asked to evaluate the safety in use of lycopene from Blakeslea trispora as a food colour in the food categories and use levels as proposed in the dossier. In addition, in view of the re-evaluation of lycopene from tomatoes within the re-evaluation programme of all food colours, the Panel decided to make a global safety assessment of lycopene from all sources. Thus, in the present opinion the Panel evaluates the safety in use of lycopene from different sources as a food colour. Results from animal and human intervention studies with synthetic lycopene or tomato extract as lycopene source indicate that lycopene from these sources is bioavailable. It is expected that lycopene from B. trispora when used in foodstuffs of comparable composition will be bioavailable to a similar extent as lycopene from tomatoes. The toxicity of synthetic lycopene was investigated in subchronic and chronic toxicity studies in rats, a carcinogenicity study and a two-generation study in rats, and developmental toxicity 1 For citation purposes: Scientific Opinion of the Panel on Food Additives, Flavourings, Processing Aids and Materials in Contact with Food on a request from the Commission on the safety in use of lycopene as a food colour. The EFSA Journal (2008) 674, member of the Panel did not participate in the discussion on the subject referred to above because of possible conflict with declared interests. European Food Safety Authority, 2008

2 studies in the rat and rabbit. Mutagenicity has been studied in an extensive program using formulated forms of lycopene. The results did not raise a safety concern. The following No Observed Adverse Effect Levels (NOAELs) for formulated, synthetic lycopene, were established in guideline-conforming toxicity studies: 500 mg/kg bw/day (the highest dose level tested) in a 14-week rat study 500 mg/kg bw/day (the highest dose level tested) in a developmental toxicity study in the rat 500 mg/kg bw/day (the highest dose level tested) in a two-generation study in the rat 400 mg/kg bw/day (the highest dose level tested) in a developmental toxicity study in the rabbit 50 mg/kg bw/day in a one-year rat study, and 50 mg/kg bw/day (the highest dose level tested) in a two-year rat carcinogenicity study. The NOAEL from a 90-day oral toxicity study with lycopene extracted from B. trispora amounted to about 600 mg/kg bw/day. The Panel derives an ADI of 0.5 mg/kg bw/day using a safety factor of 100 based on a NOAEL of 50 mg/kg bw/day from a one year rat study and a non-reversible increase in alanine transaminase (ALT). This ADI refers to lycopene from all sources. The Panel noted that the ADI set by JECFA does not include lycopene from tomatoes. This was probably due to the fact that JECFA was not evaluating lycopene from tomatoes. The Panel concludes however that the ADI defined in the present opinion should include lycopene from tomatoes as well, because the toxicological data base contains several toxicity studies on lycopene from tomatoes showing no adverse effects up to the highest dose levels tested. These studies include a 10-week study in rats, and a 28-week study in mice revealing NOAEL values of respectively 60 mg/kg bw/day and 35 mg/kg bw/day both being the highest dose levels tested. From this it is concluded that the NOAEL values for these studies with lycopene from tomatoes are in the same range as the value of 50 mg/kg bw/day from which the ADI is derived, and that therefore the ADI refers to lycopene from all sources. The Panel noted that total daily exposure to lycopene from B. trispora as a food colour could potentially range from 2 to 6 mg on the average and go up to 11 to 23 mg at the high level. The Panel does not exclude an occasionally combined high exposure from both natural dietary sources and food colours up to 43 mg of lycopene per day. The proposed use of lycopene as a novel food ingredient is not taken into account, because this is not in the remit of the AFC Panel but in the remit of the NDA Panel. The use levels used in these calculations were generally 40-90% lower than the maximum uselevels permitted for food colours, including lycopene extracted from tomatoes, under Directive 94/36/EC. These intake estimates of lycopene used as a food colour are based on conservative assumptions and may therefore overestimate potential intake since lycopene is assumed to be present in all categories where it is authorised. The EFSA Journal (2008) 674, 2-66

3 It is concluded that use of lycopene as a food colour adds significantly to the overall intake of lycopene. The Panel also concludes that with the uses and use levels presented in the present opinion, which are lower than the maximum use-levels permitted for food colours under Directive 94/36/EC, the intake of lycopene from natural sources and as a food colour would be expected to remain within the ADI of 0.5 mg/kg bw/day. However, this does not hold for the high level intakes by pre-school and school children. The Panel notes that non-alcoholic flavoured drinks were found to be by far the largest potential source in all population groups considered, with percentage contribution of the total calculated intake of lycopene ranging from 66% in male adults to more than 90% in pre-school children. The Panel notes that specifications for lycopene from tomatoes may need to be updated taking the actual lycopene content in current colouring preparations into account. Key words: Lycopene, E160d, food colour, synthetic lycopene, lycopene from B. trispora, CAS Registry Number The EFSA Journal (2008) 674, 3-66

4 TABLE OF CONTENTS Panel Members... 1 Summary... 1 Table of Contents... 5 Background as provided by the Commission... 7 Terms of reference as provided by the Commission... 9 Acknowledgements... 9 Assessment Technical data Chemistry Manufacturing Process Specifications Methods of analysis in foods Reaction and fate in foods, stability Case of need and proposed uses Dietary exposure Existing authorisations and evaluations Toxicological data Absorption, Distribution, Metabolism and Excretion Acute Oral Toxicity Short-term and sub-chronic toxicity Reproductive and developmental toxicity Genotoxicity Carcinogenicity and long-term studies Human data Discussion Conclusions Documentation provided to EFSA References Appendices Glossary / Abbreviations The EFSA Journal (2008) 674, 4-66

5 BACKGROUND AS PROVIDED BY THE COMMISSION Directive 94/36/EC on colours for use in foodstuffs authorises the use of Lycopene (E160d) in certain categories of foodstuffs listed in Annex III and Annex V, part 2 of that Directive and at maximum levels of use. Directive 95/45/EC laying down specific purity criteria for food colours specifies that E160d Lycopene is obtained by solvent extraction of the natural strains of red tomatoes (Lycopersicon esculentum L.) with subsequent removal of the solvent. In 1999, following the request from a manufacturer the Commission requested the SCF to assess the safety of synthetic lycopene containing not less than 96% lycopene, when used as a food colour. In its opinion of 2 December 1999, the SCF concluded that on the basis of the available toxicological database the Committee was not able to allocate an ADI and considered its use in food unacceptable at that time. Consequently, the Commission did not propose the authorisation of synthetic lycopene. In 2004 a manufacturer requested the authorisation of a fermentation-produced lycopene from Blakeslea trispora as an alternative source of lycopene for use as a colour in the same food categories for which lycopene (E160d) extracted from tomatoes is permitted. In its opinion of 5 October 2005, EFSA concluded that the range of the potential intake exceeds the range for the mean intake from natural dietary sources and that without further safety data the Panel was unable to conclude that the proposed use levels of lycopene from B. trispora as a food colour would be safe. Consequently, the Commission has not proposed the substance for approval under Directive 94/36/EC on food colours. The applicant has then carried out a refined exposure study in which the estimated intakes were recalculated based on anticipated food categories and actual levels of use rather than those indicated in Directive 94/36/EC on food colours. In practice the study takes into account foods in which the use of lycopene would be technologically necessary and the actual levels of use to achieve the technological effect. It is worth noting that Directive 94/36/EC sets maximum levels of use for lycopene together with a number of other food colours and specifies that these levels need to be respected when the colours are used singly or in combination. These levels may not always represent the actual levels used for each colour, as this would depend on the intensity and nature of the colour used. In addition Article 2(7) of the Directive sets that colours shall be used according to good manufacturing practice at a level not higher than is necessary to achieve the intended purpose. In the meantime another applicant has requested the authorisation of a synthetic lycopene containing a minimum of 96% lycopene for use as a food colour in the same food categories for which lycopene (E160d) extracted from tomatoes is permitted. The EFSA Journal (2008) 674, 5-66

6 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 provide a scientific opinion on the safety in use of: 1. lycopene obtained from a fermentation process with Blakeslea trispora as a food colour in the food categories and use levels as proposed by the applicant and 2. synthetic lycopene as a food colour in the food categories listed in Annex III and Annex V, part 2 of Directive 94/36/EC on colours for use in foodstuffs, 3. taking into account the various requests concerning lycopene currently under consideration, including the re-evaluation of lycopene from tomatoes as part of the systematic re-evaluation of all food colours. ACKNOWLEDGEMENTS The European Food Safety Authority wishes to thank the members of the Working Group for the preparation of this opinion: Fernando Aguilar, Dimitrios Boskou, David Gott, Sandro Grilli, Werner Grunow, Karin Hulshof, John Chr. Larsen, Catherine Leclercq, Jean-Charles Leblanc, Wim Mennes, Alicja Mortensen, Dominique Parent-Massin, Iona Pratt, Ivonne Rietjens, Gerrit Speijers, Paul Tobback, Fidel Toldrá. The EFSA Journal (2008) 674, 6-66

7 ASSESSMENT 1. Technical data 1.1. Chemistry Lycopene is a carotenoid with the formula C 40 H 56. It has a molecular weight of and CAS Registry Number Its structural formula is: H 3 C CH 3 CH 3 CH 3 H 3 C CH 3 CH 3 CH 3 H 3 C CH 3 Lycopene occurs in food predominantly in an all-trans form (Cronin 2000; Boileau et al., 2002). Lycopene can be obtained from various sources: i) Lycopene from tomatoes Lycopene can be obtained by solvent extraction of natural strains of red tomatoes (Lycopersicon esculentum L.) with subsequent removal of the extraction solvent, leaving lycopene and other tomato constituents dissolved and suspended in the tomato s natural lipid phase. Lycopene extracted from tomatoes, is authorised within the EU as food colouring agent (E160d) (EC, 1995). ii) Synthetic lycopene Synthetic lycopene as produced by the petitioner is a highly pure product containing a minimum of 96% lycopene. The main impurity (below 0.15%) is apo-12 -lycopenal. Other impurities that can also be present at very low concentrations are residual solvents (not more than 0.5%) and triphenylphosphine oxide (not more than 0.01%). Various formulations of synthetic lycopene are manufactured by the petitioner. Various beadlet formulations contain typically approximately 5-10% of synthetic lycopene. The other ingredients present in these formulations are common dietary ingredients or approved food additives such as corn starch, gelatine and stabilizers such as ascorbyl palmitate and α- tocopherol. Table 1 gives an overview of the cis-trans isomer ratios of lycopene from various sources including the synthetic lycopene of the present opinion. Synthetic lycopene contains the same cis-isomers as the ones found in tomatoes, tomato products and in human blood plasma. The portion of 9-cis- and 13-cis-isomers lies in the range found in untreated and cooked tomatoes, while the portion of 5-cis-lycopene is about the same as the one found in cooked tomato foodstuffs and in human blood plasma. The petitioner indicates that with respect to the cis/trans The EFSA Journal (2008) 674, 7-66

8 isomer ratio, synthetic lycopene can be regarded as equivalent to lycopene found in natural food sources or to lycopene present in human blood plasma. Table 1. Cis/trans isomer ratio of lycopene in tomatoes, tomato-based foodstuffs, in human blood and in synthetic lycopene (in % of total lycopene) (Schierle et al., 1997) Sample (all-trans)- Lycopene (5-cis)- Lycopene (9-cis)- Lycopene (13-cis)- Lycopene Other cis isomers of Lycopene Raw tomatoes < 1 Cooked tomato-based foodstuffs Blood plasma Relative composition of synthetic lycopene manufactured by the petitioner >70 < 23 < 1 < 1 <5 iii) Lycopene from Blakeslea trispora Lycopene from Blakeslea trispora is biosynthesised by the fungus B. trispora through the same pathway as lycopene produced in the tomato. The predominant isomer is all-trans lycopene. The lycopene is formulated into a 20% or 5% sunflower oil suspension with α-tocopherol at 1% of the lycopene level, and is also available as an α-tocopherol-containing 10% and 20% lycopene cold water dispersible (CWD) product Manufacturing Process i) Lycopene from tomatoes. Lycopene from tomatoes (E160d) is obtained by solvent extraction of the natural strains of red tomatoes (Lycopersicon esculentum L.) with subsequent removal of the solvent, leaving lycopene and other tomato constituents dissolved and suspended in the tomato s natural lipid phase. ii) Synthetic lycopene The manufacturing process for synthetic lycopene is provided by the petitioner. The resulting lycopene meets the specifications defined below. Lycopene is susceptible to chemical changes such as isomerization and degradation when exposed to light, heat and oxygen (Lee and Chen, 2002). The petitioner indicates that each step in the production of synthetic lycopene is well controlled and conducted under inert gas protection to avoid formation of oxidative degradation products. The dry, pure lycopene obtained as a result of the synthesis is directly processed further into commercial lycopene formulations. The EFSA Journal (2008) 674, 8-66

9 iii) Lycopene from B. trispora The manufacturing process is carried out in two phases: the fermentation step and the isolation of the biosynthesised product via extraction. Details of these steps are described in the Opinion of the Scientific Panel on Dietetic Products, Nutrition and Allergies on a request from the Commission related to an application on the use of α-tocopherol-containing oil suspension of lycopene from Blakeslea trispora as a novel food ingredient (EFSA, 2005a). To overcome the stability issues, all the recovery, formulation, and packaging processes of lycopene from B. trispora are carried out in the dark, at controlled temperature and under nitrogen atmosphere conditions. Manufacturing and further processing of lycopene is carried out in a continuous process in which lycopene crystals are not accumulated but immediately suspended in high oleic sunflower oil Specifications i) Lycopene from tomatoes Specifications for lycopene from tomatoes are laid down in the Specification Directive 95/45/EC (EC, 1995). Table 2 presents an overview. The major colouring principle of tomatoes is lycopene, minor amounts of other carotenoid pigments may be present. Beside the other colour pigments the product may contain oils, fats, waxes, and flavour components naturally occurring in tomatoes (EC, 1995). Lycopene in tomatoes consists of 94-96% all-trans, 3-5% 5-cis, 0.1% 9-cis, 1% 13-cis, and <1% other cis isomers (SCF, 1999). Only the following extraction solvents may be used: dichloromethane, carbon dioxide, ethyl acetate, acetone, propan-2-ol, methanol, ethanol, or hexane (EC, 1995). Commission Directive 95/45/EC specifies the purity of lycopene from tomatoes at a content not less than 5% colouring matters. And also according to the SCF, the material prepared from tomatoes has a low lycopene content, namely not less than 5% of total colouring matters (SCF, 1999). Lycopene oleoresin from tomatoes may also be prepared using an additional concentration step to obtain an oleoresin that contains 60-70% lycopene. The remainder of the product may be accounted for by (unquantified) amounts of other carotenoids pigments, oils, fats, waxes, and flavour components naturally occurring in tomatoes. This is however never specifically mentioned. The carotenoid pigments other than lycopene that may be present in the product are not further specified and/or quantified. This also holds for the glycoalkaloids (e.g. tomatine) possibly present. ii) Synthetic lycopene The introduction of specifications for synthetic lycopene would require an amendment of the specifications for lycopene laid down in the Commission Directive 95/45/EC. The petitioner suggests the amendments to the lycopene specifications laid down in Commission Directive 95/45/EC (EC, 1995) that are given in Table 2. The EFSA Journal (2008) 674, 9-66

10 The petitioner indicates that the specifications for lycopene obtained via synthesis are the same as proposed by JECFA for synthetic lycopene (JECFA, 2006b). Table 2. Specification of lycopene from Lycopersicon esculentum L. and proposed specifications for synthetic lycopene as provided by the petitioner. E 160d (i) LYCOPENE Lycopene extracted from Lycopersicon esculentum L. Synonyms Natural Yellow 27 Definition Lycopene is obtained by solvent extraction of the natural strains of red tomatoes (Lycopersicon esculentum L.) with subsequent removal of the solvent. Only the following solvent may be used: dichloromethane, carbon dioxide, ethyl acetate, acetone, propan- 2-ol, methanol, ethanol, hexane. The major colouring principle of tomatoes is lycopene, minor amounts of other carotenoid pigments may be present. Besides the colour pigments the product may contain oil fats, waxes and flavour components naturally occurring in tomatoes. Class Carotenoid Colour index No Chemical names Lycopene, Ψ,Ψ-carotene Chemical formula C 40 H 56 Molecular weight Assay 1% E1cm at nm in hexane (for 100% pure all-translycopene) is Content not less than 5% total colouring matters Description Dark red viscous liquid Identification Spectrometry Purity Solvent residues Ethylaceta te Methanol Ethanol Acetone Hexane Propane 2 ol Dichloromethane Maximum in hexane at ca 472 nm Not more than 50 mg/kg; singly or in combination Not more than 10 mg/kg Sulphated ash Not more than 0.1% Arsenic Not more than 3 mg/kg Lead Not more than 10 mg/kg Mercury Not more than 1 mg/kg Cadmium Not more than 1 mg/kg Heavy metals (as Pb) Not more than 40 mg/kg The EFSA Journal (2008) 674, 10-66

11 E 160d (ii) LYCOPENE Lycopene from synthesis Synonyms Natural Yellow 27 Definition Synthetic lycopene is produced by the Wittig condensation of synthetic intermediates commonly used in the production of other carotenoids used in food. Synthetic lycopene consists predominantly of all-trans-lycopene together with 5-cis-lycopene and minor quantities of other isomers. Commercial lycopene preparations intended for use in food are formulated as suspensions in edible oils or water-dispersible powder and are stabilized with anti-oxidants. Class Carotenoid Colour index No Chemical names Ψ,Ψ-carotene, all-trans-lycopene, (all-e)-lycopene, (all-e)- 2,6,10,14,16,18,20,22,24,26,30-dotriacontatridecaene Chemical formula C 40 H 56 Molecular weight Assay Description Identification Solubility Solution in chloroform Spectrometry Purity Loss on drying Lead Apo-12 -lycopenal Triphenyl phosphine oxide Not less than 96% total lycopenes (not less than 70% all-translycopene) 1% E1cm at nm in hexane (for 100% pure all-translycopene) is Red crystalline powder Insoluble in water, freely soluble in chloroform A 1% solution is clear and has intensive red-orange colour A solution in hexane shows an absorption maximum at approximately 470 nm Not more than 0.5% Not more than 1 mg/kg Not more than 0.15% Not more than 0.01% The degree of purity of synthetic lycopene is specified as 96%. Due to manufacturing process, a carotenoid aldehyde, apo-12 -lycopenal (previously also known as lycopene-c 25 -aldehyde), is found as a by-product. The final apo-12 -lycopenal content is specified as max. 0.15%. In the past, the content of this by-product was higher (up to 0.3%). Due to an improved manufacturing process, this by-product could be reduced to 0.1% (see Table 3). Other impurities likely to be present in lycopene are residual solvents. The following four solvents might be present: acetone, hexane, methanol, iso-propanol. The EFSA Journal (2008) 674, 11-66

12 iii) Lycopene from B. trispora Specifications for lycopene obtained from B. trispora have been described in the opinion of the AFC Panel on the use of α-tocopherol-containing oil suspensions and cold water dispersible forms of lycopene from B. trispora as a food colour (EFSA, 2005b) and the opinion of the NDA Panel on the use of α-tocopherol-containing oil suspension of lycopene from B. trispora as a novel food ingredient (EFSA, 2005a). For comparison Table 3 presents a comparison of the chemical compositions of synthetic lycopene, lycopene from tomatoes and lycopene from B. trispora. Table 3. Comparison of the chemical compositions of synthetic lycopene, lycopene from tomatoes and lycopene from B. trispora Synthetic lycopene (present opinion) Synthetic lycopene* Lycopene from tomatoes* Lycopene from B. trispora** Purity 96% 96% 5% of total colouring matters*** 95% Impurities, other pigments Up to 0.15% Apo- 12 -lycopenal Up to 0.3% of C 25 aldehyde = Apo-12 - lycopenal Other pigments, oils, fats, waxes and natural flavours Other carotenoids All-trans isomer >70% >70% 94-96% 90% 5-cis isomer <23% <25% 3-5% 9-cis isomer <1% <1% 0-1% 13-cis isomer <1% <1% 1% Other cis-isomers <5% <3% <1% Formulation 5-10% lycopene with corn starch, gelatine and stabilizers such as ascorbyl palmitate and α-tocopherol 10% lycopene with ascorbyl palmitate (5%) and α- tocopherol (1.5%) Oleoresin: 2-3% lycopene Powder: 5% lycopene 1-5% 5 or 20% oil suspension with α-tocopherol (1% of lycopene level) 10-20% CWD product * SCF, 1999 ** EFSA, 2005a,b. *** Lycopene oleoresin from tomatoes might also be prepared using an additional concentration step to obtain an oleoresin that contains % lycopene. The EFSA Journal (2008) 674, 12-66

13 1.4. Methods of analysis in foods Methods for analysis of lycopene in food are based on extraction followed by among others liquid chromatography with UV-Vis detection and/or gas chromatography - mass spectrometry. Care has to be taken to avoid isomerization and degradation when exposed to light, heat and oxygen. Cis- and trans-isomers of lycopene can be discriminated and determined by HPLC (Ishida et al., 2001) Reaction and fate in foods, stability Lycopene is sensitive to oxygen and light (SCF, 1999). Aust et al. (2003) demonstrated that in vitro oxidation of lycopene with hydrogen peroxide/osmium tetroxide yielded the dialdehyde 2,7,11-trimethyltetradecahexaene-1,14-dial. In addition, Zhang et al. (2003) demonstrated the formation of (E,E,E)-4-methyl-8-oxo-2,4,6-nonatrienal through the (auto-)oxidative cleavages at the 5, 6- and 13, 14-single bonds of lycopene. i) Lycopene from tomatoes In a publication by Bošković (1979) the possible fate of lycopene in dehydrated tomato products is described as a two-way reaction pathway. In stage one all-trans lycopene oxidizes or isomerizes to less coloured, more oxidizable cis-forms (up to 20%) which either autoxidize or revert to all-trans lycopene. Processing and storage conditions influence distribution between the two routes. In the second stage all forms autoxidize and oxidized degradation products and fragments accumulate. ii) Synthetic lycopene The stability of formulated synthetic lycopene has been determined in different foodstuffs under various temperature and storage conditions. The product forms as manufactured by the petitioner proved to be stable over a period of up to 36 months under various temperature and storage conditions (5, 25, and 35 C, glass, aluminium, PET containers, and different humidity). For technological reasons (obtaining a water or oil dispersible form of the hydrophobic lycopene, easier handling, improved stability and others), lycopene has to be converted using food-approved carriers, stabilizers and other additives into suitable product forms. The petitioner indicates that during the investigations on the stability of lycopene in foodstuffs it has once more been proven essential that care has to be taken that inert conditions during the whole production process and further storage are assured. This is in order to prevent losses of lycopene due to oxidative degradation, a natural process that also occurs during processing of other foodstuffs which contain carotenoids, being naturally present in or added to foodstuff. In addition, stability testing performed with lycopene-containing products indicated that the addition of antioxidants such as ascorbic acid or sodium ascorbate is required to maintain stability. The EFSA Journal (2008) 674, 13-66

14 iii) Lycopene from B. trispora Stability trials with α-tocopherol-containing 5% and 20% oil suspensions and CWD products of lycopene from B. trispora have been described in detail in the previous EFSA opinions (EFSA, 2005a; EFSA, 2005b). Lycopene from B. trispora was shown to be stable for at least 6 months under all conditions tested. It is expected that any breakdown products derived from lycopene from B. trispora in the oil suspension or in the CWD product in the presence of α-tocopherol or in food would be identical to those that would occur with lycopene from other natural sources Case of need and proposed uses Lycopene is a naturally occurring red food colour found in tomatoes and other fruits. With its red colour, lycopene modifies other orange-red carotenoids, broadening the spectrum of carotenoid food colours already available. Lycopene imparts a yellow-orange to red colour at concentrations ranging from 5 to 500 mg/kg food. However, lycopene extracted from tomatoes has a distinct tomato flavour which limits the application as a food colour (Hendry and Houghton 1992). i) Lycopene from tomatoes Table 4 presents the uses and maximum use levels as defined in Annex III and Annex V, Part 2 of Council Directive 94/36/EC (EC,1994) for E160 lycopene derived from tomatoes. The EFSA Journal (2008) 674, 14-66

15 Table 4. Uses and maximum use levels of E160d lycopene as listed in Annex III and Annex V, Part 2 of Council Directive 94/36/EC (EC, 1994). Note that according to the specifications E160d contains only > 5% lycopene and that the maximum levels refer to the quantity of colouring principle contained in the colour preparation. Foodstuffs Maximum level (mg/kg or mg/l) Non-alcoholic flavoured drinks 100 Candied fruits and vegetables, Mostarda di frutta 200 Preserves of red fruits 200 Confectionery 300 Decorations and coatings 500 Fine bakery wares (e.g. viennoiserie, biscuits, cakes and wafers) 200 Edible ices 150 Flavoured processed cheese 100 Desserts including flavoured milk products 150 Sauces, seasonings (for example, curry powder, tandoori), pickles, relishes, 500 chutney and picalilli Jam, jellies and marmalades as mentioned in Directive 79/693/EC and other 100 similar fruit preparations including low calorie products Mustard 300 Fish paste and crustacean paste 100 Pre-cooked crustaceans 250 Salmon substitutes 500 Surimi 500 Fish roe 300 Smoked fish 100 Snacks: extruded or expanded savoury snack products 200 Snacks: other savoury snack products and savoury coated nuts 100 Edible cheese and edible casings Quantum Satis Complete formulae for weight control intended to replace total daily intake or an 50 individual meal Complete formulae and nutritional supplements for use under medical 50 supervision Liquid food supplements / dietary integrators 100 Solid food supplements / dietary integrators 300 Soups 50 Meat and fish analogues based on vegetable proteins 100 Spirituous beverages (including products less than 15% alcohol by volume) 200 except those mentioned in Annex II or III Aromatized wines, aromatized wine-based drinks and aromatized wine products 200 cocktails as mentioned in Regulation (EEC) No 1601/91 except those mentioned in Annex II or III Fruit wines (still or sparkling) 200 Cider (except cidre bouche) and perry 200 Aromatized fruit wines, cider and perry 200 The EFSA Journal (2008) 674, 15-66

16 Actual data on the usage of lycopene from tomatoes as a food colour came from a food additive usage survey conducted in by the Natural Food Colours Association (NATCOL) in a supplementary report (Tennant, 2007). Manufacturers of tomato-extract lycopene were asked to provide information for the colour produced and for each application. Data from the different manufacturers were combined to establish the overall range of usage of the colour in each food category. Typical use levels were collated and where more than one figure was provided an upper range figure (corresponding to approximately the 75 th percentile) was selected. Where typical use levels were not provided the upper limit of the use range was used. The collated results are given in Table 5. The EFSA Journal (2008) 674, 16-66

17 Table 5. Reported uses and use levels for lycopene from tomatoes as a food colour Foodstuffs Range (mg/kg or mg/l) Non-alcoholic flavoured drinks Candied fruits and vegetables, Mostarda di frutta Preserves of red fruits - - Confectionery Decorations and coatings Fine bakery wares (e.g. viennoiserie, biscuits, cakes and wafers) Edible ices Flavoured processed cheese - - Desserts including flavoured milk products Sauces, seasonings (for example, curry powder, tandoori), pickles, relishes, chutney and picalilli Jam, jellies and marmalades as mentioned in Directive 79/693/EC and other similar fruit preparations including low calorie products Mustard - - Fish paste and crustacean paste Pre-cooked crustaceans Salmon substitutes Surimi Fish roe Smoked fish Snacks: extruded or expanded savoury snack products Snacks: other savoury snack products and savoury coated nuts Edible cheese and edible casings Complete formulae for weight control intended to replace total daily intake or an individual meal Complete formulae and nutritional supplements for use under medical supervision Liquid food supplements / dietary integrators Solid food supplements / dietary integrators Soups Meat and fish analogues based on vegetable proteins Spirituous beverages (including products less than 15% alcohol by volume) except those mentioned in Annex II or III Aromatized wines, aromatized wine-based drinks and aromatized wine products cocktails as mentioned in Regulation (EEC) No 1601/91 except those mentioned in Annex II or III Fruit wines (still or sparkling) Cider (except cidre bouche) and perry Aromatized fruit wines, cider and perry Source: Tennant, 2007 Typical (mg/kg or mg/l) The EFSA Journal (2008) 674, 17-66

18 The Panel noted that the typical use levels currently applied by the food industry are generally 40-90% lower than the maximum use-levels permitted for food colours including lycopene extracted from tomatoes under Directive 94/36/EC (EC, 1994). The Panel also noted that these use-levels included use levels for synthetic lycopene and lycopene from B. trispora. ii) Synthetic lycopene A petitioner on synthetic lycopene indicated that synthetic lycopene in appropriate product forms offers an alternative to the food industry for lycopene from tomatoes used in bakery, beverages, convenience food, breakfast cereals, dairy, sauces, and sweets. The intended field of application for lycopene is covered by the list of foodstuffs permitted to be coloured in accordance with Council Directive 94/36/EC (EC, 1994). The petitioner also indicated that the approved maximum levels for lycopene as listed in Annex III and Annex V, Part 2 of Council Directive 94/36/EC are exceeding the levels technologically needed to achieve the desired coloration effect for synthetic lycopene. Table 6 provides the uses and use levels for synthetic lycopene provided by the petitioner. The applicants have conducted enquiries in the food industry to identify current use levels. Since synthetic lycopene is presently very rarely used as a colour little information was available. The petitioner reported that, although a different colour, the mode of use of synthetic lycopene would be similar to the related food colour beta-carotene. Therefore, beta-carotene use levels have been used by the petitioner to establish a range of potential usage levels for lycopene in each food, whether extracted from tomatoes or synthetic. The range reflects typical colour intensities from pink to red corresponding to different food flavours. According to the petitioner the typical use level is based on typical usage of beta-carotene and represents a conservative assumption because a smaller proportion in each food category will be coloured red or pink than orange or yellow as it is the case for beta-carotene usage. The EFSA Journal (2008) 674, 18-66

19 Table 6. Proposed uses and use levels for synthetic lycopene as a food colour Directive 94/36 EC category Range (mg/kg or mg/l) Non-alcoholic flavoured drinks -Carbonates -Dilutables - Juice-based drinks Confectionary Decorations and coatings Fine bakery ware (e.g. viennoiserie, biscuits, cakes and wafers) Edible ices Flavoured processed cheese Desserts including flavoured milk products Sauces, seasonings Fish, paste and crustacean paste Surimi 5 5 Snacks, extruded of expanded savoury snack products Soups Spirituous beverages (excluding products <15% alcohol by volume) except those mentioned in Annex II or III Aromatized wines, aromatized wine based drinks and aromatized wine product cocktails as mentioned in Regulation (EEC) No 160 except those mentioned in Annex II or III Fruit wine (still or sparkling); Cider (except cidre bouche) and perry; Aromatized fruit wines, cider, perry Typical (mg/kg or mg/l) The EFSA Journal (2008) 674, 19-66

20 iii) Lycopene from B. trispora The uses and use levels for lycopene colour in the European Union proposed by the petitioner for lycopene from B. trispora are as given in Table 7. Comparison of the maximum permitted use levels of E160d lycopene as listed in Annex III and Annex V, Part 2 of Council Directive 94/36/EC (EC, 1994) (Table 4) reveals that the proposed use levels are generally 40-90% lower. Table 7. Summary of the individual food uses and use levels for lycopene colour in the EU (presented by the petitioner for B. trispora) Food category Proposed Food-uses Use-levels (mg/kg or mg/l) Beverages Non-alcoholic flavoured drinks 30 Spirituous beverages 30 Cereals and cereal products Viennoiserie, biscuits, cakes and wafers 20 Fish and fish dishes Pre-cooked crustaceans 25 Smoked fish 30 Milk and milk products Desserts including flavoured milk products 15 Edible ices 25 Miscellaneous Sauces, seasonings, pickles and relishes 50 Soups and soup mixes 30 Sugar, preserves and confectionery Confectionery 30 For comparison an overview of all permitted uses and use levels, actual uses and use levels and/or proposed uses and use levels for the different lycopene preparations is presented in Appendix A Dietary exposure Natural lycopene Lycopene is a natural pigment occurring in tomatoes and to a lesser extent in some other foods. An overview of average dietary exposure to lycopene from foods in different populations was presented in previous EFSA evaluations (EFSA, 2005a,b). It was concluded that regular exposure to lycopene from natural dietary sources in different populations are, according to dietary surveys, estimated to be on average between 0.5 and 5 mg/day, with high exposures up to about 8 mg/day. High consumption of fruits and vegetables, especially tomato products, may result in occasional exposure to 20 mg lycopene/day or more. Lycopene from tomatoes as a food colour Exposure estimates were provided by NATCOL (Tennant, 2007) using food consumption data extracted from the most recent UK National Dietary and Nutrition Survey (NDNS) programme (weighed dietary records). Available data include a 4-day survey in pre-school children aged 1½ to 4½ (n=1,675; Gregory et al., 1995), and a 7-day survey in school children aged 4 to 18 (n=1,686; Gregory 2000), adults aged 16 to 64 (n=2,251, Henderson et al., 2002) and elderly aged 65 and over (n=1,632, Finch et al., 1998). Schoolchildren have been sub-divided into preand post- 12 years olds (teenagers). A tiered approach was used to assess the potential exposure to lycopene from tomato-extract. In the first step of the screening maximum use levels set out in The EFSA Journal (2008) 674, 20-66

21 Directive 94/36 (see Table 4) and average consumption levels of the food categories from UK NDNS were used. The two highest 97 th percentiles for those food categories that were the main contributors for exposure plus the mean exposure for other categories for the whole population represented a worst case exposure, where a high level consumer of two foods always chooses foods containing lycopene from tomato-extract. In the second step of the exposure assessment the former calculations were conducted with typical use levels derived from the NATCOL usage survey (Table 5). As shown in Table 8 estimates based on maximum use levels in Directive 94/36/EC (EC, 1994) resulted in average potential dietary exposures ranging from 22 to nearly 75 mg/day, corresponding to less than 1 to around 3 mg/kg bw/day. Worst case estimates for potential high level exposure could reach approximately 120 up to 736 mg/day (corresponding to 2 to 10 mg/kg bw/day) (Table 8). However, based on actual typical usage levels the refined exposure estimates were around 10 times lower (Table 9). Average potential dietary exposures ranged from 3.0 to 6.3 mg/day (or less than 0.1 to nearly 0.3 mg/kg bw/day) whereas potential worst case high level exposures ranged from 10 to nearly 60 mg/day (corresponding to 0.25 to 0.75 mg/kg bw/day). Non alcoholic flavoured drinks were identified as the most important potential source of lycopene for most consumers. However, it was stated by the rapporteur that the physicochemical properties of lycopene oleoresin limit its application in water-based drinks meaning that it can only be used in small proportion of the market and the contribution might be overestimated. Other important contributors were desserts and spirituous beverages, and among adults fruit wines, perry and particularly cider. Since lycopene is unlikely to be used in last application, exposures from this source are probably also overestimated. Therefore, it was concluded that all of the reported calculations of the exposure to lycopene from tomatoes can be regarded as very conservative and the higher intakes reported unlikely to be reached in reality (Tennant, 2007). The Panel also considers these estimates as very conservative. Table 8. Potential estimated daily exposure to lycopene from tomato-extract as a food colour based on maximum permitted usage levels according to UK food surveys Estimated exposure in mg/day Estimated exposure in mg/kg bw/day Mean High level* Mean High level* Pre-school children ( yrs) Schoolchildren (4-11 yrs) Schoolchildren (12-18 yrs) Adults (16-64 yrs) Elderly (65+ yrs) *high level based on the two highest 97.5 th percentiles + mean exposure for other food categories The EFSA Journal (2008) 674, 21-66

22 Table 9. Potential estimated daily exposure to lycopene from tomato-extract as a food colour based on typical usage levels according to UK food surveys Estimated exposure in mg/day Estimated exposure in mg/kg bw/day Mean High level* Mean High level* Pre-school children ( yrs) Schoolchildren (4-11 yrs) Schoolchildren (12-18 yrs) Adults (16-64 yrs) Elderly (65+ yrs) *high level based on the two highest 97.5 th percentiles + mean exposure for other food categories Petitioners for synthetic lycopene and for lycopene from B. trispora also presented exposure estimates. In this opinion only estimates based on individual dietary surveys are included. Synthetic lycopene as a food colour The petitioner provided estimated daily exposures to synthetic lycopene in the United Kingdom, based on the typical use-levels as a food colour and on raw data on food consumption surveys from the UK NDNS Programme. Food codes in the NDNS were matched to food classifications in Directive 94/36 EC. Exposures were calculated from NDNS food consumption data and typical usage levels for synthetic lycopene as presented in Table 6. Where the food descriptions permitted it, foods that would never be coloured red or pink (e.g. clear drinks as lemonade, tonic water, colas and orange flavoured drinks) were eliminated from the exposure estimate. For other categories of food, where the variety of flavours was not provided, all foods in that category were included. Individuals were considered consumers if they consumed one or more food products in which lycopene is proposed for use on at least one of the survey days. Calculations for the potential mean and high-level (97.5 th percentile) exposure to lycopene were performed for the different food categories as well as for the overall exposure from all proposed food uses combined. The Panel noted that the estimates of synthetic lycopene were based on the same dietary records as used by the estimates of lycopene from tomatoes, only the school children aged 4 to 18 were not split up into two age groups. However, since individual data were available for each person who participated in the surveys, the estimates for high consumers of synthetic lycopene can be considered as much more accurate than the exposure estimates to lycopene from tomatoes, based on the consumption of food categories. As shown in Table 10, overall estimated mean exposure to synthetic lycopene as food colour among consumers ranged from 2.0 mg/day (elderly) to 5.6 mg/day (young people). High exposure levels (97.5 th percentile) ranged from 8.4 mg/day to 12.4 mg/day. Exposures per kg bw were provided by the petitioner for pre-school children, school children and adults and mean potential daily exposures were 0.26 mg/kg bw, 0.16 mg/kg bw and 0.05 mg/kg bw, respectively. The highest 97.5 th percentile was in pre-school children (0.61 mg/kg bw/day) (Table 10). The EFSA Journal (2008) 674, 22-66

23 Table 10. Potential estimated daily exposure to synthetic lycopene as a food colour among consumers according to UK food surveys Estimated exposure in mg/day Estimated exposure in mg/kg bw/day Mean 97.5 th percentile Mean 97.5 th percentile Pre-school children ( yrs) School children (4-18 yrs) Adults (16-64 yrs) Elderly (65+ yrs) NA NA NA = not available High level exposures from individual foods ranged from <1 to 7 mg/day, and resulted for all age groups mainly from soft drinks, and for adults and elderly also from soups. The Panel noted that these exposure estimates are based on uses and use levels proposed by the petitioner for use of synthetic lycopene as a food colour (Table 6), whereas for fortified foods the petitioner suggested applications in a range of foods (18 food groups) and typical use levels varying from 9 to 100 mg/kg. These figures were not taken into account in the present exposure estimate for the evaluation of synthetic lycopene as a food colour. Lycopene from B. trispora as a food colour A petitioner provided a refined study on the estimated daily exposure to lycopene from B. trispora. Estimates for the exposure to lycopene colour in the EU were based on the proposed use levels (Table 7) and raw data of food consumption surveys collected as part of the UK NDNS Programme. The exposure estimates were performed for pre-school children, aged 1.5 to 4.5, school children aged 4 to 10, female and male teenagers aged 11 to 18 and male and female adults aged 16 to 64. The Panel noted that the estimates of lycopene from B. trispora were based on the same surveys as used by the estimates of lycopene from tomatoes and synthetic lycopene, except for the exposure for adults. For this group the estimates were now based on data published in 1990 (Gregory et al, 1990). Furthermore, in the calculations for lycopene from B. trispora the total group of school children was split up in groups with smaller age ranges (4-10 and 11-18) and for teenagers and adults exposure estimates are made for males and females separately. As described for synthetic lycopene, food codes in the NDNS were matched to the food use categories as presented in Table 7. Only the food codes in which the addition of lycopene was possible were included. Exposures were calculated from NDNS food consumption data and typical usage levels for lycopene from B. trispora. The Panel noted that compared to the exposure assessment of lycopene from tomatoes and synthetic lycopene the number of food categories for B. trispora was more limited. However, the proposed use-levels for lycopene from B. trispora for non-alcoholic drinks were much higher than the typical use levels applied in the other calculations (30 mg/kg for lycopene from B. trispora versus 4-10 and 5 mg for synthetic lycopene and lycopene from tomatoes, respectively). Calculations for the mean and higher levels (90 th, 95 th and 97.5 th percentile) all-person and alluser exposures were performed for each of the individual proposed food uses. Similar The EFSA Journal (2008) 674, 23-66

24 calculations were used to determine the estimated total exposure to lycopene from B. trispora from all proposed food uses combined. Table 11 and 12 summarise the potential estimated exposure to lycopene colour from B. trispora in respectively mg/day and in mg/kg bw/day from all proposed food uses in the EU by the UK population. Table 11. Population group Pre-school children School children Female Teenager Male Teenager Female Adults Male Adults Table 12. Population group Pre-school children School children Female Teenager Male Teenager Female Adults Male Adults Estimated daily exposure to lycopene from B. trispora (mg/day) from all proposed food categories in the UK by population groups Age Group (Years) % Users Actual # of Total Users All-Person Consumption Mean (mg) Percentile (mg) Mean (mg) All-Users Consumption Percentile (mg) ½-4 ½ , Estimated daily exposure to lycopene from B. trispora (mg/kg bw/day) from all proposed food categories in the UK by population groups Age % Actual All-Person Consumption All-Users Consumption Group Users # of (Years) Total Users Mean (mg/kg) Percentile (mg/kg) Mean (mg/kg) Percentile (mg/kg) ½-4 ½ , As shown in Table 11, overall estimated mean exposure to lycopene as food colour from B. trispora among consumers ranged from 1.9 mg/day (female adults) to 6 mg/day (pre-school children). Highest exposure levels (97.5 th percentile) ranged from approximately 11 mg/day (female adults) to 23 mg/day (pre-school children). Mean potential exposure per kg body weight per day ranged between 0.03 mg/kg bw (adults) and 0.42 mg/kg (pre-school children) The EFSA Journal (2008) 674, 24-66