Scientific Opinion on the use of calcium lignosulphonate (40-65) as a carrier for vitamins and carotenoids 1

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1 SCIENTIFIC OPINION Scientific Opinion on the use of calcium lignosulphonate (40-65) as a carrier for vitamins and carotenoids 1 EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS) 2, 3 European Food Safety Authority (EFSA), Parma, Italy ABSTRACT The Panel on Food Additives and Nutrient Sources added to Food provides a scientific opinion on the safety of calcium lignosulphonate (40-65) when used as a carrier for vitamins and carotenoids intended to be added to foods for colouring and nutrient purposes. Calcium lignosulphonate (40-65) has been evaluated by JECFA and an ADI of 20 mg/kg bw/day was established. Calcium lignosulphonate (40-65) is poorly absorbed following oral administration. From the results obtained in vitro from one bacterial reverse mutation assay and one mammalian chromosomal aberration assay it can be concluded that there is no indication for a genotoxic potential of calcium lignosulphonate (40-65). In a short-term 28-day toxicity study, a NOAEL of 1500 mg/kg bw/day was identified for calcium lignosulphonate (40-65) based on minimal focal/multifocal chronic inflammation in the rectum of male rats. In a 90-day subchronic toxicity study the petitioner identified a NOAEL of 2000 mg/kg bw/day for calcium lignosulphonate (40-65), the highest dose tested. The Panel, however, considered this study inadequate for evaluating the safety of calcium lignosulphonate (40-65) due to the high incidence of lymphoid hyperplasia and lymphoid infiltration in the mandibular and mesenteric lymph nodes, in the Peyer s patches and in the liver in all animals, including controls. Therefore, the Panel considers that available data on calcium lignosulphonate (40-65) are insufficient to establish an ADI. Furthermore, the Panel considers that long-term toxicity studies are needed to elucidate whether the histiocytosis in the mesenteric lymph nodes of the rats observed in the inadequate 90-day toxicity study may progress into a more adverse state with time. Overall, based on the available information, the Panel concludes that the safety of use of calcium lignosulphonate (40-65), as a carrier for vitamins and carotenoids intended to be added to foods for colouring and nutrient purposes, cannot be assessed. KEY WORDS Calcium lignosulphonate (40-65), CAS No , lignosulphonic acid calcium salt, calcium lignosulphate, lignin calcium sulphonate, carrier, vitamins and carotenoids 1 On request from the European Commission, Question No EFSA-Q , adopted on 26 February Panel members: F. Aguilar, U.R. Charrondiere, B. Dusemund, P. Galtier, J. Gilbert, D.M. Gott, S. Grilli, R. Gürtler, J. Koenig, C. Lambré, J-C. Larsen, J-C. Leblanc, A. Mortensen, D. Parent-Massin, I. Pratt, I.M.C.M. Rietjens, I. Stankovic, P. Tobback, T. Verguieva, R.A. Woutersen. Correspondence: ans@efsa.europa.eu 3 Acknowledgement: The Panel wishes to thank the members of the Working Group A on Food Additives and Nutrient Sources of the ANS Panel for the preparation of this opinion: F. Aguilar, N. Bemrah, P. Galtier, J. Gilbert, S. Grilli, R. Gürtler, NG. Ilback, C. Lambré, J.C. Larsen, J-C. Leblanc, A. Mortensen, I. Pratt, Ch. Tlustos, I. Stankovic. Suggested citation: EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS); Scientific Opinion on the use of calcium lignosulphonate (40-65) as a carrier for vitamins and carotenoids on request of the European Commission. EFSA Journal 2010;8(3):1525. [24 pp.]. doi: /j.efsa Available online: European Food Safety Authority,

2 SUMMARY Following a request from the European Commission, the Panel on Food Additives and Nutrient Sources added to Food (ANS) was asked to deliver a scientific opinion on the safety of calcium lignosulphonate (40-65) when used as a carrier for vitamins and carotenoids intended to be added to foods for colouring and nutrient purposes. According to the petitioner, the proposed name of calcium lignosulphonate (40-65) distinguishes the product from other available calcium lignosulphonates presenting lower degrees of lignin polymerisation and higher content of sugars. Lignosulphonates are used in a variety of food manufacturing applications including dispensing, binding, complexing and emulsifying. Calcium lignosulphonate (40-65) is an amorphous yellow-brown to brown polymer derived from lignin, not having a well defined structural or molecular formula, with an average molecular weight between and g/mol. In vitro and in vivo assays have shown that calcium lignosulphonate (40-65) is poorly absorbed by the oral route. Calcium lignosulphonate (40-65) has been tested in in vitro genotoxicity, short-term, subchronic and developmental toxicity studies in accordance with recognised guidelines. No long-term or carcinogenicity studies were conducted with calcium lignosulphonate (40-65). From the results obtained in vitro from one bacterial reverse mutation assay and one mammalian chromosomal aberration assay it can be concluded that there is no indication for a genotoxic potential of calcium lignosulphonate (40-65). The Panel noted that a test for induction of gene mutations in mammalian cells in vitro, as recommended by the Guidance on submissions for food additive evaluations (SCF, 2001), has not been performed. The petitioner considered that such an assay was unnecessary since, given its high molecular weight, calcium lignosulphonate (40-65) is unlikely to enter the cells. The Panel agreed with this argument. In a short-term 28-day toxicity study a No Observed Adverse Effect Level (NOAEL) of 1500 mg/kg bw/day was identified for calcium lignosulphonate (40-65) based on minimal focal/multifocal chronic inflammation in the rectum of male rats. In a 90-day subchronic toxicity study, the petitioner identified a NOAEL of 2000 mg/kg bw/day for calcium lignosulphonate (40-65), the highest dose tested. The Panel, however, considers this study inadequate for evaluating the safety of calcium lignosulphonate (40-65) due to the high incidence of lymphoid hyperplasia and lymphoid infiltration in the mandibular and mesenteric lymph nodes, in the Peyer s patches and in the liver in all animals, including controls. In a developmental toxicity study (21 days) in the rat, no treatment-related effects in dams or fetuses were reported up to the highest dose tested and a NOAEL of 1000 mg/kg bw/day can be identified for calcium lignosulphonate (40-65) from this study. Exposure estimates were based on the reported European high percentile intakes of vitamins from food and Tolerable Upper Intake Levels (ULs) of vitamins for children and adults, and on the percentage of calcium lignosulphonate (40-65) proposed by the petitioner to be used as a carrier. The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin A cannot be estimated for children under 10 years old and adults, as the food intake of this vitamin is higher than the UL. The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin A varies between approximately 500 and 3700 µg/day for children aged years. The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin D ranges from approximately 2100 to 8000 µg/day for children under 18 years old and it is approximately 6500 µg/day for adults. The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin E ranges from 88.8 to mg/day for children under 2

3 18 years old and is 264 mg/day for adults. The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin K is less than 200 mg/day. The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for multivitamins (vitamins A, D, E and K) ranges from to mg/day for children aged 11 to 17 years, ranges from to mg/day for children aged 3-10 years, is mg/day for children under 3 years old, and is mg/day for adults. Exposure estimates of calcium lignosulphonate (40-65), resulting from its use as carrier for carotenoids as proposed by the petitioner, were based on the estimated intake of carotenoids from natural sources, food additives and food supplements. The estimated intakes of calcium lignosulphonate (40-65), resulting from its use as a carrier, ranges from less than 10 to over 100 mg/day for -carotene and zeaxanthin, from less than 10 to 95 mg/day for lutein and from less than 10 to 125 mg/day for lycopene. No estimates have been made for intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for canthaxanthin (food colour limited to saucisses de Strasbourg) and -apo-8 carotenal (no intake data available and no uses as food supplement) as their intakes are likely to be low. The Panel cannot provide a more refined exposure assessment for calcium lignosulphonate (40-65) resulting from its use as carrier for these colours since these colours are still under evaluation in Europe and their intakes have not yet been evaluated. The Panel considers that the available data on calcium lignosulphonate (40-65) were insufficient to establish an ADI. The Panel further considers that the 90-day study with a 4-week recovery period is inadequate for the evaluation of the safety of calcium lignosulphonate (40-65). Therefore, the Panel considers that long-term toxicity studies are needed to elucidate whether the histiocytosis in the mesenteric lymph nodes of the rats observed in the inadequate 90-day toxicity study may progress into a more adverse state with time. Overall, based on the available information, the Panel concludes that the safety of use of calcium lignosulphonate (40-65), as a carrier for vitamins and carotenoids intended to be added to foods for colouring and nutrient purposes, cannot be assessed. 3

4 TABLE OF CONTENTS Abstract... 1 Key words... 1 Summary... 2 Table of contents... 4 Background as provided by the European Commission... 5 Terms of reference as provided by the European Commission... 5 Assessment Introduction Technical data Identity of the substance Specifications Manufacturing process Methods of analysis in foods Stability, reaction and fate in food Case of need and proposed uses Information on existing authorisations and evaluations Exposure Biological and toxicological data Absorption, distribution, metabolism and excretion Toxicological data Acute oral toxicity Short-term and subchronic toxicity Genotoxicity Chronic toxicity and carcinogenicity Reproductive and developmental toxicity Other studies Discussion Conclusions Documentation provided to EFSA References Glossary /Abbreviations

5 BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION Carriers for food additives are a functional class of food additive which are regulated under Directive 95/2/EC of the European Parliament and the Council on food additives other than colours and sweeteners. In addition Regulation (EC) No 1333/2008 on Food additives will also regulate the use of carriers for nutrients and other substances added to food for nutritional and/or for other physiological purposes. A manufacturer has requested the authorisation of calcium lignosulphonate (40-65) under Directive 95/2/EC as a carrier for vitamins and carotenoids. According to the applicant the calcium lignosulphonate is derived from lignin and the substance requested for authorisation has an average molecular weight between and g/mol. The additive was evaluated by the 69 th meeting of the Joint FAO/WHO Expert Committee on Food additives (JECFA) in 2008, at which time JECFA established an ADI of 0-20 mg/kg bw. Lignosulphonates (E565) are also approved as feed additives in the European Community under Council Directive 70/524/EEC of 23 November 1970 concerning additives in feeding stuffs. TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN 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 of calcium lignosulphonate (40-65) as a carrier for vitamins and carotenoids intended to be added to foods for colouring and nutrient purposes. 5

6 ASSESSMENT 1. Introduction The present opinion deals with the safety of calcium lignosulphonate (40-65) when used as a food additive. According to the petitioner the proposed name of calcium lignosulphonate (40-65) distinguishes the product from other commercial available calcium lignosulphonates presenting lower degrees of lignin polymerisation and higher content of sugars. Lignosulphonates are used in a variety of food manufacturing applications including dispensing, binding, complexing and emulsifying. 2. Technical data 2.1. Identity of the substance Calcium lignosulphonate (40-65) is identified as an amorphous yellow-brown to brown polymer derived from soft wood lignin which is a naturally occurring polymer of highly irregular structure consisting of randomly polymerized coniferyl alcohol. Calcium lignosulphonate (40-65) does not have a well defined structural formula and a tentative structure proposed by the petitioner is presented in Figure 1. Figure 1. Structural formula of calcium lignosulphonate (40-65) as proposed by the petitioner. According to the petitioner the CAS Registry Number is However, the Panel notes that the proposed CAS Registry Number ( ) corresponds generally to the calcium salt of lignosulphonic acid, rather than specifically to calcium lignosulphonate (40-65). The specifications for calcium lignosulphonate available in Food Chemicals Codex (FCC VI, 2008) define the limits for reducing sugars ( 30.0%), calcium ( 7.0%), and loss on drying ( 10%). Due to these differences and the proposed usages, the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 2008 decided that calcium lignosulphonate according to FCC specifications and the calcium lignosulphonate (40-65) evaluated during its 69 th meeting were sufficiently different as to require 6

7 different chemical names, hence the substance was named calcium lignosulphonate (40-65). The JECFA specifications do not contain a CAS Registry number, nor a defined molecular formula or chemical name (JECFA, 2008). More than 22 synonyms for calcium lignosulphonate, not specifically the 40-65, can be found in the open literature, the most common being lignosulphonic acid calcium salt, calcium lignosulphate, lignin calcium sulphonate Specifications Calcium lignosulphonate (40-65) consists of molecules with a molecular weight in the range of 1000 to g/mol (> 90% of the sample ranges), with an average molecular weight between to g/mol. The degree of sulphonation, expressed as the ratio between organically bound sulphur and methoxyl groups, is in the range of 0.3 and 0.7. It is described as soluble in water and practically insoluble in organic solvents like methanol, ethanol and hexane. The ph of a 10% solution is reported to vary from 2.7 to 3.5, the loss on drying not more than 8.0%, and the total ash not more than 14% on the dried basis (reported as composed of calcium sulphate 65%, calcium oxide 30% and calcium carbonate < 5%). Chemical specifications proposed by the petitioner are the same as existing JECFA specifications (JECFA, 2008) and are summarised in Table 1. Table 1: Comparative presentation of specifications for calcium lignosulphonate according to Food Chemicals Codex (FCC VI, 2008), calcium lignosulphonate (40-65) according to JECFA (2008) and the specifications proposed by the petitioner. Definition Calcium lignosulphonate (FCC VI, 2008) Calcium lignosulphonate occurs as a brown, amorphous polymer. It is obtained from the spent sulphite and sulphate pulping liquor of wood or from the sulphate (kraft) pulping process. It may contain up to 30% reducing sugars. It is soluble in water, but not in any of the common organic solvents. The ph of a 1:100 aqueous solution is between approximately 3 and 11. Calcium lignosulphonate (40-65) (JECFA, 2008) Calcium lignosulphonate (40-65) is an amorphous material obtained from the sulphite pulping of softwood. The lignin framework is a sulphonated random polymer of three aromatic alcohols: coniferyl alcohol, p-coumaryl alcohol, and sinapyl alcohol, of which coniferyl alcohol is the principal unit. After completion of the pulping, the water-soluble calcium lignosulphonate is separated from the cellulose, purified (ultrafiltration), and acidified. The recovered material is evaporated and spray dried. The commercial product has a weight average molecular weight range of to g/mol. Calcium lignosulphonate (Specifications proposed by the petitioner) Calcium lignosulphonate (40-65) is an amorphous material obtained from the sulphite pulping of softwood. The lignin framework is a sulphonated random polymer of coniferyl alcohol. After completion of the pulping, the water-soluble calcium lignosulphonate is separated from the cellulose, purified (ultrafiltration), and acidified. The recovered material is evaporated and spray dried. The commercial product has a weight-average molecular weight range of to g/mol. Assay 5.0% sulphonate - no specific assay is proposed sulphur. ph 3 11 (1:100 aqueous (10% solution) (10%) solution) Degree of (on the dried basis) sulphonation 7

8 Calcium 7.0% 5.0% 5.0% (on the dried basis) Loss on drying 10.0% 8.0% (105 o C, 24 h) 8.0% Reducing sugars 30.0% 5.0% (on the dried basis) 5.0% (a) Residue on 20.0% - - ignition Total ash - 14% (on the dried basis) 14% (on the dried basis) Sulphite - 0.5% (on the dried basis) 0.5% (on the dried basis) Viscosity of a 3000 centipoises % solution Arsenic - 1 mg/kg 1 mg/kg Lead - 2 mg/kg 2 mg/kg (a) It can contain mannose, xylose, galactose, glucose, arabinose and rhamnose The petitioner proposes the JECFA specifications with exclusion of p-coumaryl alcohol and sinapyl alcohol from the definition. The Panel notes that, according to the literature data, soft wood lignins consist of 85-90% of coniferyl alcohol monomers (Glasser, 1980) and the petitioner did not submit analytical data supporting the claim that lignin framework of calcium lignosulphonate (40-65) consists of coniferyl alcohol units only Manufacturing process The manufacturing process has been adequately described by the petitioner. In summary, calcium lignosulphonate (40-65) is obtained from the sulphite pulping 4 of spruce (Picea sp.) soft wood, in which wood chips are treated with an acidic calcium bisulphite solution rendering the lignin molecules soluble in water. After completion of the pulping, calcium lignosulphonate is separated from insoluble cellulose by filtration. The filtrate is further purified to remove depolymerisation products, evaporated and spray dried. According to the petitioner calcium lignosulphonate (40-65) is manufactured under current good manufacturing practices and meets the proposed specifications. Additionally, according to the petitioner all raw materials used and isolated intermediate products are checked for compliance with specifications. Upon request from the Panel, information provided by the petitioner states that the wood used for production of calcium lignosulphonate (40-65) has not been treated with pesticides and those single lot analytical results from wood harvested in two different seasons show that nine pesticides residues measured are below of the limit of detection of 0.03 mg/kg for lignosulphonate Methods of analysis in foods According to the petitioner no analytical method could be identified or developed to quantify calcium lignosulphonate (40-65) in food. The petitioner developed an analytical method for determination of calcium lignosulphonate (40-65) in nutrient formulations Stability, reaction and fate in food No studies were presented on the reaction and fate in food of calcium lignosulphonate (40-65). Data on the stability by itself and in carotenoid preparations show that the powder is stable at least for 24 months and during 48 weeks in lots of supplements containing 10% of β-carotene, stored under normal and accelerated conditions. 4 The process used to extract lignin from the wood for the production of pulp. 8

9 2.6. Case of need and proposed uses The case of need to use calcium lignosulphonate (40-65) is attributed by the petitioner to its protective properties mediated by the encapsulation of vitamins and carotenoids, leading to increased stability in foods. The product is intended to be used as a carrier for vitamins and carotenoids in food. It is indicated by the petitioner that the amount of calcium lignosulphonate (40-65) used in food could vary depending on specific technological needs, as well as on authorised levels of use of vitamins and carotenoids in food (Table 2). Vitamins and carotenoids intended to be carried by calcium lignosulphonate are water dispersible forms of β-carotene, zeaxanthin, lutein, lycopene, canthaxanthin, β-apo-8 -carotenal, vitamin A, vitamin D, Vitamin E and vitamin K. The proposed percentage of the carrier and the vitamins/carotenoids in the final formulations is indicated in Table 2. It is indicated that as the calcium lignosulphonate (40-65) has not yet been introduced commercially, this list is compiled using the best of the current knowledge and corresponds to a good overview of the technical possibilities. Table 2: Percentages proposed by the petitioner of the carrier (calcium lignosulphonate (40-65)) and the vitamins/carotenoids in the final formulations. Caroteoid/vitamin Active carotenoid/vitamin Lignosulphonate Beta-carotene 1%-10% 50% Zeaxanthin 5%-10% 50% Lutein 10% 50% Lycopene 10% 50% Canthaxanthin 10% 50% Beta-apo-8'-carotenal 10% 50% Vitamin A 10%-15% 50% Vitamin E 50% 50% Vitamin D3 0.25% 50% Vitamin K 5% 90% 2.7. Information on existing authorisations and evaluations Calcium lignosulphonate (40-65) (INS No. 1522) was evaluated by JECFA (JECFA, 2009). The committee established an Acceptable Daily Intake (ADI) of 20 mg/kg bw/day and prepared new specifications. The Scientific Panel on Additives and Products or Substances used in Animal Feed (FEEDAP) evaluated a feed additive formulation containing, among other components, 4.0% calcium lignosulphonate. It was not defined if specifications for calcium lignosulphonate in this feed formulation matched the average g/mol molecular weight range ascribed to the substance in the present opinion. The FEEDAP Panel concluded that the new formulation would not be expected to introduce any additional risks or concerns for the safety of the target species, consumers of animal products or for those handling the product (EFSA, 2005). Lignosulphonates (E 565) are approved as feed additives in the European Community and may be used in all animal species and animal categories without maximum levels specified (EC, 2004). 9

10 In the United States lignosulphonates are approved as indirect food additives in feed up to a concentration of 4% (21 CFR ), as adhesive for use in food packaging (21CFR ) and as dispersion agents and stabilizers in pesticides for specific applications (21 CFR ) Exposure The petitioner proposed an exposure assessment for calcium lignosulphonate (40-65) based on its use as a carrier for some vitamins and carotenoids. The petitioner concluded that the key findings from their assessment were that intakes of lignosulphonate resulting from its use as a carrier for carotenoids are unlikely to exceed 100 mg/day or 4 mg/kg bw/day for the highest intake groups (90 th, 97.5 th percentiles). Intakes of lignosulphonate resulting from its use as a carrier for vitamins would generally result in maximum intakes of lignosulphonate of less than 20 mg/day except for vitamin E, where intakes could exceed 300 mg/day. In multi-vitamin supplements the maximum lignosulphonate intake would not exceed 400 mg/day. The carotenoids are not likely to be used together because they share similar technological and nutritional properties and therefore substitute for each other. A consumer who consumed foods fortified or coloured with carotenoids would have a maximal intake of lignosulphonate from this source of 100 mg/day. If he also took a multivitamin supplement he could obtain up to 370 mg from that source. However, the total intake is unlikely to reach 470 mg/day because of limited market share and the fact that lignosulphonate cannot be used in oil-based products, which make up approximately 50 % of the total market. For the use of calcium lignosulphonate (40-65) as a carrier for vitamins, the Panel made its own exposure estimates based on the reported European high percentile intakes of vitamins (95 th or 97.5 th intake) from food and the Tolerable Upper Intake Levels (ULs) of vitamins for children and adults (SCF, 2002b; 2003a), and on the percentage of calcium lignosulphonate (40-65) proposed by the petitioner to be used as a carrier. The Panel has evaluated the maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier in a food supplement by assuming that vitamins are added to the carrier at a maximum level not exceeding established UL for each particular vitamin. Vitamin A The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin A has only been calculated for children aged and years where these amounts of calcium lignosulphonate (40-65) vary from approximately µg/day and from µg/day, respectively. The high percentile of vitamin A intake from food is higher than the UL for children under 10 years old and adults. Vitamin D The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin D ranges approximately from 2100 to 8000 µg/day for children under 18 years old and is approximately 6500 µg/day in adults. Vitamin E The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin E ranges from 88.8 to mg/day for children under 18 years old and is 264 mg/day in adults. Vitamin K In 2003, the SCF was unable to determine a UL for vitamin K. The SCF opinion indicated that In human studies of limited numbers, there is no evidence of adverse effects associated with supplementary intakes of vitamin K in the form of phylloquinone of up to 10 mg/day (more than two 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 (2000 mg/kg body weight) for 30 days. 10

11 Available data on European intakes of vitamin K from food only provide average values ranging from 57 to 250 µg/day for adults (SCF, 2003b). No available data have been published on high percentile intakes or on the intake of vitamin K from food for children. Nevertheless the available data on the intake of vitamins and nutrients from food indicate, on the one hand, a ratio of 2.5 between the mean intake (adults or children) and the 97.5 th percentile intake (adults or children) and on the other hand, a ratio of 0.5 between 97.5 th percentile intake for adults and the 97.5 th percentile for children under 18 years old. The maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin K is less than 200 mg/day for both adults and children under 18 years old. Table 3: The estimated maximum intakes of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin A, vitamin D, vitamin E or vitamin K or multivitamin supplements. Age Tolerable Upper Intake Level (UL) Food Intake (high percentile) UL minus Food Intake Ratio vitamin: calcium lignosulphonate (40-65) Maximum intake of calcium lignosulphonate (40-65) resulting from its use as a carrier in food supplements Vitamin A (preformed vitamin A = retinol and retinyl esters) years μg /day μg /day μg /day μg /day a - 1:3.5 to 1: b - 1:3.5 to 1: b - 1:3.5 to 1: b 140 1:3.5 to 1:5 490 to b 740 1:3.5 to 1: to 3700 Adults* c - 1:3.5 to 1:5 Vitamin D years μg/day μg /day μg /day μg /day a : d : d : Adults d : Vitamin E years mg/day mg/day mg/day mg/day a : d : d : d : d : Adults d 264 1:1 264 Vitamin K years mg/day mg/day mg/day mg/day < : Adults : * including women of child-bearing age a Fantino, b Lioret et al., c SCF (2002a) d Flynn et al.,

12 Multivitamin supplements The intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for multivitamin supplements (vitamins A, D, E and K) is difficult to be estimated as intakes of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin A cannot be calculated for all population groups. For the group of children aged years, the intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for multivitamin supplements (vitamins A, D, E and K) ranges from to mg/day. For the other population groups the intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for multivitamin supplements (vitamins A, D, E and K) is mg/day for children under 3 years old, varies from to mg/day for 3-10 year old children and is mg/day for adults. Carotenoids Exposure estimates of calcium lignosulphonate (40-65), resulting from its use as carrier for carotenoids as proposed by the petitioner, were based on the estimated intake of carotenoids from natural sources, food additives and food supplements. The estimated intakes of calcium lignosulphonate (40-65), resulting from its use as a carrier, ranges from less than 10 to over 100 mg/day for β-carotene and zeaxanthin, from less than 10 to 95 mg/day for lutein and from less than 10 to 125 mg/day for lycopene. No estimates have been made for intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for canthaxanthin (food colour limited to saucisses de Strasbourg) and β apo-8 carotenal (no intake data available and no uses as food supplement) as their intakes are likely to be low. The Panel cannot provide a more refined exposure assessment for calcium lignosulphonate (40-65) when used as carrier for these colours since these colours are still under evaluation in Europe and their intakes have not yet been evaluated. 3. Biological and toxicological data 3.1. Absorption, distribution, metabolism and excretion In vitro studies To study transepithelial transport of calcium lignosulphonate, Caco-2 cells (HTB37) cultured as monolayer on a collagen coated filter were exposed to uniformely 3 H-labelled calcium lignosulphonate (40-65) at concentrations of 1, 3, 10 and 30 mg/ml (Beck et al., 2008). Radioactivity in the cells was determined after 30 minutes, 1, 1.5, 2 and 3 hours. The flux of radioactivity on the receiver side of the culture monolayer was calculated from the slope of the regression line obtained from the curve of radioactivity plotted versus the time. The radioactivity of 3 H-labelled calcium lignosulphonate was analyzed by Laser Scanning Cytometer (LSC) of eluting fractions. Calcium lignosulphonate in solution was quantified spectrophotometrically under UV light and Size Exclusion Chromatography (SEC) was used to determine the molecular weight distribution of calcium lignosulphonate. In the absence of any cytotoxicity, the relative transepithelial transport of radioactivity was approximately the same for the three lower concentrations tested. It was slightly lower for the highest concentration tested (1.31% of the total radioactivity per hour) for which cellular integrity was slightly 12

13 affected. The calculated 3 H-labelled calcium lignosulphonate (molecular weight > g/mol) permeability coefficients were not significantly different (0.002 x 10-6 cm/s and x 10-6 cm/s) after incubation with 3 and 30 mg/ml, respectively. The apparent permeability coefficient calculated from these data was 2.1 ± 0.3 x 10-6 cm/s and appeared relatively high. However, SEC analysis showed that 99.5% of this radioactivity came from small molecular weight molecules (< g/mol) formed by radiolysis of 3 H-labelled calcium lignosulphonate. In vivo studies 3 H-labelled calcium lignosulphonate was administered by oral gavage to three male Wistar albino rats (SPF) as a single dose of 10 mg/kg bw (200 µci/kg bw) in a pilot oral dose study (Beck and Rossi, 2005). In this study multiple samples were taken from cannulated vena jugularis at 1, 2, 4, 6 and 24 hours post dosing. Urine and faeces were collected during the first 24 hours and during the hours period. Rats were sacrificed at 48 hours post dosing. Total radioactivity was determined in blood, urine (radioactivity measured in several molecular weight fractions), faeces and in gastrointestinal tracts (stomach, small intestine, colon), in liver, skin/fur, and in the rest of the carcass. Aliquots of biological samples, tissues and samples combusted in an oxidizer (drying) were analyzed for total radioactivity by Liquid Scintillation Counting (LSC). The SEC fractionated urine samples were analyzed by the same procedure. In the main study three female and three male Wistar albino rats were administered by oral gavage with a single oral dose of 10 mg/kg bw (250 µci/kg bw) by oral gavage and samples were treated and measured as in the pilot study (Beck and Rossi, 2005). The molecular weight distribution of calcium lignosulphonate in both studies showed a broad distribution within a molecular weight range between g/mol. As mentioned before a significant portion (> 25%) of the radioactivity was found to elute as low molecular weight molecules, which was attributed to radiolysis of 3 H-labelled calcium lignosulphonate stock solution. Therefore an ultrafiltration procedure, the same as used in the in vitro study mentioned below, was used to reduce as much as possible small sized molecules from the tritiated calcium lignosulphonate (40-65) application solutions. Radioactivity recovery after 48 hours from the main study was 98.4 ± 0.7%, the majority coming from faeces (74.6 ± 1.1%) and less from urine (3.12% in females and 2.56% in males) (Table 4). These findings were reported to be similar to those found in the pilot study. The Panel observes that around 20% of the radioactivity was found in the carcass. The Panel further notes the differences reported between wet and dry sample results for urine and tissue radioactivity. Table 4: Radioactivity recovered in Wistar albino rats from biological fluids and organs after 48 hours exposure to a single dose of 3 H-labelled calcium lignosulphonate (40-65). Radioactivity, mean % of initial dose ± SD Biological samples Liquid/wet samples Dried samples Males Females Males Females Faeces 75.0 ± ± ± ± 2.4 Gastrointestinal tract (a) 1.98 ± ± ± ± 0.15 Skin/fur (b) 4.61 ± ± ± ± 0.03 Urine 2.81 ± ± ± (c) Blood 0.56 ± ± ± ± Liver 0.76 ± ± ± ± Remaining carcass ± ± ± ±

14 Total (a): stomach, small intestine and colon including contents; (b) polled samples from neck, belly and back, (c) no standard deviation (SD) reported since samples were pooled in this case Radioactivity levels measured in dried samples from the pilot and the main study were lower than in liquid/wet samples, with no significant interindividual differences observed. According to the authors this discrepancy can be attributed to radioactivity coming from tritiated water ( 3 H 2 O) present as a radiolysis by-product from 3 H-labelled calcium lignosulphonate (40-65) in stock solutions. Data presented showed that upon storage (3 weeks, -20 C) tritiated water forms in stock solutions of 3 H- labelled calcium lignosulphonate (40-65) and that a large fraction of the tritiated molecules in urine elutes with spiked tritiated water. Furthermore, SEC data presented showed that less than 1% of the radioactivity is found in blood and urine samples within the high molecular weight fractions (molecular weight > g/mol). The Panel considers that the findings would be consistent with lability of the radiolabel (tritium exchange) which was also seen in the in vitro studies. The in vitro studies indicate that the low transepithelial transport measured in Caco2 cell layers could be explained by the high molecular weight of calcium lignosulphonate (40-65). Furthermore, the in vivo studies demonstrated that calcium lignosulphonate (40-65) is poorly absorbed from the gastrointestinal tract of mice. Overall, the Panel considers that calcium lignosulphonate (40-65) is poorly transported and absorbed in vivo Toxicological data Acute oral toxicity An acute oral LD 50 of calcium lignosulphonate (molecular weight not specified) was established as greater than 31.6 g/kg bw in young albino Sprague-Dawley rats (sex not specified) (Bio-Test, 1962). Other sparse information in the dossier mentions a study undertaken on three groups of 6 male rats (strain not identified) administered between 5 and 20 g/kg bw of calcium lignosulphonate and observed for 14 days post-dosing. All animals in the 20 g/kg bw group died and the LD 50 was estimated to lie between 10 and 20 g/kg bw (Keller, 1978) Short-term and subchronic toxicity A 28-day oral toxicity study was conducted with increasing doses of calcium lignosulphonate (40-65) administered to in-house random bred Wistar rats (HsdCpb) according to OECD Guideline 407 and Good Laboratory Practice (GLP) (Weber and Ramesh, 2005). Four groups of 6 animals per sex were administered ad libitum diet containing calcium lignosulphonate (40-65) at target dose levels of 0, 500, 1500 and 4000 mg/kg bw/day. Clinical observations revealed that most of the animals were healthy. Food consumption and mean body weights or body weight gains of any treated group did not differ significantly from the controls. Opthalmological findings did not reveal abnormalities related to the administration of calcium lignosulphonate (40-65). Regarding haematology, any findings were considered as incidental by the authors based on the lack of dose-response. Regarding clinical chemistry, males at the intermediate dose group were reported having significant higher cholesterol levels but no other changes were reported. No significant changes were reported in females. 14

15 Statistically significant increases in the absolute weight of ovaries and thymus at the low dose and of heart and brain at the intermediate dose groups were reported in females. No significant changes were reported in males. At gross examination an increased incidence of unilateral flabby testes was reported at the low dose in males which upon microscopic examination was diagnosed as unilateral atrophy of seminiferous tubules. Furthermore, a few cases of dilated kidney-pelvis were reported and confirmed microscopically. No other significant findings were reported. Histopathologic examination revealed higher incidence of chronic inflammation in the rectum of males at the high dose group (4000 mg/kg bw/day) as compared to controls. This inflammation was classified as being of minimal severity, with a focal/multifocal distribution comprising fibrosis with a minimal inflammatory cell infiltration. This finding was considered as treatment-related by the authors, who derived a No Observed Adverse Effect Level (NOAEL) of 1500 mg/kg bw/day. No other significant histopathologic findings were reported (Weber and Ramesh, 2005). A 90-day subchronic oral toxicity study, including additional neurotoxicity testing, was conducted with increasing doses of calcium lignosulphonate (40-65) administered to Wistar (SPF-bred) rats according to OECD Guideline 408, FDA Redbook 2000 Principles and GLP (Thiel et al., 2007). Four groups of 20 animals per sex were administered ad libitum diet containing calcium lignosulphonate (40-65) at target dose levels of 0, 500, 1000 and 2000 mg/kg bw/day (Allocation A). A further 6 animals per sex and group were used to assess possible changes in the primary immunological response (Allocation C). Additionally, 10 rats per sex and group were treated for 13 weeks with 0 and 2000 mg/kg bw/day and then allowed a 28-day treatment-free recovery period (Allocation B). General parameters evaluated included recording of body weight and food consumption as well as ophthalmological examinations. Functional observational battery, locomotor activity and grip strength were performed during 13 and 17 weeks. Sperm count, motility and morphology were examined at necropsy in all treatment and control males after 13 weeks (Allocation A) and 17 weeks (Allocation B). Oestrus cycle was determined over a two-week period in females starting at week 10 (Allocation A and B) and at week 15 (Allocation B). Haematology and plasma chemistry were performed after 2 and 6 weeks of treatment and at the end of the study in all allocation groups. Urine and faecal samples were collected in all allocation groups. All animals were necropsied and examined. Histopathological examinations were performed on organs and tissues from all animals of Allocations A and B. Mesenteric lymph nodes and kidneys were particularly examined in all animals from Allocation A and B. Immunotoxicity testing consisted of analysis of primary immune response to immunogene (Type 2 B testing) and of leukocyte populations in blood (Level 1 extended). No general adverse clinical signs related to the compound were found. Food consumption and mean body weights or body weight gains of any treated group did not differ significantly from the controls. Opthalmological findings were not related to the administration of calcium lignosulphonate (40-65) at any dose and period tested. Functional observational battery recorded after 13 weeks and after 4 weeks of recovery did not reveal neurotoxicological effects. No dose-related effects were reported in the mean fore- and hind limb grip strength or in the mean locomotor activity. Although reduced (p<0.05) force limb grip strength was observed in males administered 2000 mg/kg bw/day and in females administered 1000 mg/kg bw/day as compared to controls, they were considered to be not treatment-related as consistent reductions were not observed in other extremities. No differences were noted in animals after a 4-week recovery period. No significant dose-related differences in sperm motility, sperm morphology or sperm head count were noted at any dose during a 13-week period, nor during a 4-week recovery period. No differences were noted in the duration of oestrus, dioestrus, proestrus or metoestrus phases after 13 weeks nor after a 4-week recovery period. 15

16 At 2 or 6 weeks after the start of the treatment, statistically significant haematological changes were reported in males and/or female rats, however according to the authors, reported values were within the historical control data. At 13 weeks after the start of treatment at the highest dose tested a significant increase in the mean number of basophils and platelets in females was reported. At the intermediate dose significant reductions in white blood cell count, and in mean absolute neutrophils and eosinophils counts were reported in males. No differences were noted in females. After the recovery period, reduced white blood cell counts (reduced neutrophils, eosinophils, lymphocytes, monocytes) were reported in male rats at the highest dose tested. In females, a reduced mean corpuscular hemoglobin concentration and elevated platelet count were observed at the highest dose tested. According to the authors all these changes remained within the ranges of historical controls and were considered as not treatment-related. The Panel agreed with this conclusion. Regarding clinical biochemistry, differences to the controls were observed at all doses tested during the treatment period. According to the authors, all differences remained within the ranges of historical control data. After the recovery period nearly all parameters were reported to return to control levels, excluding persistent reductions in aspartate aminotranferase and alkaline phosphatase activities, as well as decreased phosphorus and protein levels and globulin fraction. No treatment-related effects were reported on urinalysis parameters or faecal ph, other than a statistically significant increase in urinary erythrocytes, reported in females at 2000 mg/kg bw/day, but this was considered as unrelated to the treatment by the authors since the value was within upper limits of historical control data. Regarding immunotoxicity no treatment-related differences were reported in primary immune response or in leukocyte populations after 13-week or 4-week of recovery period. Statistically significant increases in mean thyroid-to-body weight ratio were reported in males at 2000 mg/kg bw/day. At intermediate dose a significant increase in mean thyroid-to-body weight and a decrease in mean thyroid-to-brain ratio were reported in males. No-treatment related changes in other organ weights or ratios were reported in females at any dose level and treatment period. Upon microscopical examination, signs of lymphoid hyperplasia or lymphoid infiltration in different organs were reported. These histopathological changes were particularly seen in mesenteric and mandibular lymph nodes, in the Peyer s patches and in the liver of male and female animals of all groups, including controls. Furthermore, proliferation of histiocytes with foamy cytoplasm, characterised as histiocytosis, was seen in the mesenteric lymph nodes of several males and females of the low dose group and almost all animals of the mid- and top-dose groups, but not in controls. Kidneys in females were affected by tubular vacuolation at the two highest doses tested. No such changes were reported in male rats. All these findings persisted after the recovery period in animals treated with 2000 mg/kg bw/day. The pathologists involved in this study discarded these effects as adverse on the basis of absence of co-existing tissue damage or reaction or on the basis of lack of dose-dependent severity grade, absence of tubular damage or other sign of renal toxicity or impairment. However, the Panel considers that the high incidence of lymphoid hyperplasia and lymphoid infiltration observed in the mandibular and mesenteric lymph nodes, in the Peyer s patches and in the liver of most animals, including controls, may point to a poor health status of the rats, which could have influenced the integrity of the study. Therefore, the Panel considered that this study is inadequate and cannot be used for the safety evaluation of calcium lignosulphonate (40-65) (Thiel et al., 2007). 16

17 Genotoxicity The use of calcium lignosulphonate as a carrier for vitamins and carotenoids A bacterial reverse mutation assay, conducted according to OECD guideline 471 and GLP, was performed on calcium lignosulphonate (40-65) (Thiel et al., 2005). No substantial increase in revertant colony numbers on any of the five tested strains (TA 1535, 1537, 98, 100, WP2 uvra) was reported at any dose level tested (from 3 to 5000 µg/plate), in the presence or absence of metabolic activation (S9- mix). No cytotoxicity was reported in the test groups. An in vitro mammalian chromosome aberration assay, conducted according to OECD guideline 473 and GLP, was conducted on calcium lignosulphonate (40-65) (Thiel et al., 2006a). No relevant increases of structural or numerical chromosomal aberrations in V79 cells were reported at any dose level tested (up to 5000 µg/ml), in the presence or absence of metabolic activation (S9-mix) in two independent experiments. A test for induction of gene mutations in mammalian cells in vitro as recommended by the Guidance on submissions for food additive evaluations (SCF, 2001) has not been performed. The petitioner considered that such an assay was unnecessary since, given its high molecular weight, calcium lignosulphonate (40-65) is unlikely to enter the cells. The Panel agreed with this argumentation Chronic toxicity and carcinogenicity No chronic toxicity and carcinogenicity studies were performed on calcium lignosulphonate (40-65). The petitioner justified the lack of long-term studies on the arguments that: a) calcium lignosulphonate (40-65) did not show potential genotoxic effects in in vitro assays, b) given the low bioavailability of the compound it would be difficult to achieve significant systemic exposure to obtain meaningful results, c) a consideration of Structure Activity Relationship (SAR) performed on lignosulphonates (not described if identical to calcium lignosulphonate (40-65)) did not identify concerns for genotoxicity or carcinogenicity, d) results from short term and subchronic toxicity studies did not produce lesions indicative of neoplastic activity, and e) foamy hystiocytosis observed in a 90-day toxicity study performed with calcium lignosulphonate (40-65) is not of concern since similar histopathological findings were reported with other two compounds (polypentosan sulphate sodium salt and polyvinylpyrrolidon copolymer) (Thiel, 2008). However, taking into account that the 90-day study was considered inadequate for evaluating the safety of calcium lignosulphonate (40-65), the Panel estimated that long-term toxicity/carcinogenicity studies are needed to elucidate whether the histiocytosis observed may or may not progress to a more adverse lesion with time Reproductive and developmental toxicity A developmental toxicity study was conducted with increasing doses of calcium lignosulphonate (40-65) administered to HanRco Wistar rats (SPF) according to OECD Guideline 414, FDA Redbook 2000 Principles and GLP practices (Thiel et al., 2006b). Four groups of 22 mated female rats were administered ad libitum diet containing calcium lignosulphonate (40-65) at target dose levels of 100, 300 and 1000 mg/kg bw/day. The diets containing calcium lignosulphonate (40-65) were made available at the end of day 5 post coitum (to assure exposure at day 6) and until the end of day 15 (to assure exposure at day 16). All animals were sacrificed on day 21 post coitum and the foetuses analyzed. Due to a technical failure leading to the loss of fetuses for visceral investigations from mid (7 foetuses lost from one litter) and high dosing groups (25 foetuses lost from 5 litters) an additional control group consisting of 22 animals and an additional high dose treated group (1000 mg/kg bw/day) of the same number of animals were started to remain in compliance with the guidelines. The full set of analysis was performed in these two additional groups. During the study no deaths occurred and the compound was reported to be well tolerated. Food consumption of dams in general and mean body weights or body weight gains of treated groups did not differ significantly from the controls. At necropsy no treatment-related findings were reported. Reproduction data (number of implantation sites, pre- and post-implantation losses, number of live 17