Calium lignosulfonate work as carrier of vatamine

EFSA Journal 2010; 8(3):1525
1© European Food Safety Authority, 2010
SCIENTIFIC OPINION
Scientific Opinion on the use of calcium lignosulphonate (40-65) as a carrier
for vitamins and carotenoids1
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. 8061-52-7, lignosulphonic acid calcium salt, calcium lignosulphate,
lignin calcium sulphonate, carrier, vitamins and carotenoids
1
On request from the European Commission, Question No EFSA-Q-2009-00374, adopted on 26 February 2010
2 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:10.2903/j.efsa.2010.1525. Available online: www.efsa.europa.eu

The use of calcium lignosulphonate as a carrier for vitamins and carotenoids
2EFSA Journal 2010; 8(3):1525
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 40000 and 65000 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 11-17 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 224.3 mg/day for children under

3EFSA Journal 2010; 8(3):1525
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 366.9 to 410.4 mg/day for children aged 11 to 17
years, ranges from 278.7 to 318.7 mg/day for children aged 3-10 years, is 265.3 mg/day for children
under 3 years old, and is 439.3 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.

4EFSA Journal 2010; 8(3):1525
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............................................................................................................................................... 6
1. Introduction ..................................................................................................................................... 6
2. Technical data.................................................................................................................................. 6
2.1. Identity of the substance ......................................................................................................... 6
2.2. Specifications.......................................................................................................................... 7
2.3. Manufacturing process............................................................................................................ 8
2.4. Methods of analysis in foods .................................................................................................. 8
2.5. Stability, reaction and fate in food.......................................................................................... 8
2.6. Case of need and proposed uses.............................................................................................. 9
2.7. Information on existing authorisations and evaluations ......................................................... 9
2.8. Exposure ............................................................................................................................... 10
3. Biological and toxicological data .................................................................................................. 12
3.1. Absorption, distribution, metabolism and excretion............................................................. 12
3.2. Toxicological data................................................................................................................. 14
3.2.1. Acute oral toxicity ............................................................................................................ 14
3.2.2. Short-term and subchronic toxicity .................................................................................. 14
3.2.3. Genotoxicity ..................................................................................................................... 17
3.2.4. Chronic toxicity and carcinogenicity................................................................................ 17
3.2.5. Reproductive and developmental toxicity ........................................................................ 17
3.2.6. Other studies..................................................................................................................... 18
4. Discussion...................................................................................................................................... 19
Conclusions............................................................................................................................................ 20
Documentation provided to EFSA ......................................................................................................... 21
References .............................................................................................................................................. 21
Glossary /Abbreviations......................................................................................................................... 24

5EFSA Journal 2010; 8(3):1525
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 40000 and 65000 g/mol.
The additive was evaluated by the 69th
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.

6EFSA Journal 2010; 8(3):1525
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 8061-52-7. However, the Panel notes that the
proposed CAS Registry Number (8061-52-7) 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 69th
meeting were sufficiently different as to require

7EFSA Journal 2010; 8(3):1525
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.
2.2. Specifications
Calcium lignosulphonate (40-65) consists of molecules with a molecular weight in the range of 1000
to 250000 g/mol (> 90% of the sample ranges), with an average molecular weight between 40000 to
65000 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.
Calcium
lignosulphonate
(FCC VI, 2008)
Calcium lignosulphonate
(40-65)
(JECFA, 2008)
Calcium lignosulphonate
(Specifications proposed by
the petitioner)
Definition 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) 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 40000 to
65000 g/mol.
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 40000 to
65000 g/mol.
Assay ≥ 5.0% sulphonate
sulphur.
- no specific assay is proposed
pH 3 – 11 (1:100 aqueous
solution)
2.7 – 3.3 (10% solution) 2.7 - 3.5 (10%)
Degree of
sulphonation
- 0.3 – 0.7 (on the dried basis) 0.3- 0.7

8EFSA Journal 2010; 8(3):1525
Calcium ≤ 7.0% ≤ 5.0% ≤ 5.0% (on the dried basis)
Loss on drying ≤ 10.0% ≤ 8.0% (105o
C, 24 h) ≤ 8.0%
Reducing sugars ≤ 30.0% ≤ 5.0% (on the dried basis) ≤ 5.0% (a)
Residue on
ignition
≤ 20.0% - -
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
50% solution
≤ 3000 centipoises - -
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.
2.3. Manufacturing process
The manufacturing process has been adequately described by the petitioner. In summary, calcium
lignosulphonate (40-65) is obtained from the sulphite pulping4
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.
2.4. 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.
2.5. 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.

9EFSA Journal 2010; 8(3):1525
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 40000 - 65000 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).

10EFSA Journal 2010; 8(3):1525
In the United States lignosulphonates are approved as indirect food additives in feed up to a
concentration of 4% (21 CFR 573.600), as adhesive for use in food packaging (21CFR 175.105) and
as dispersion agents and stabilizers in pesticides for specific applications (21 CFR 172. 715).
2.8. 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 (90th
,
97.5th
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 (95th
or 97.5th
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 11-14 and 15-17 years where these amounts of calcium
lignosulphonate (40-65) vary from approximately 500-700 µg/day and from 2600-3700 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
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
E ranges from 88.8 to 224.3 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”.

11EFSA Journal 2010; 8(3):1525
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 97.5th
percentile intake (adults or children) and on the other hand, a
ratio of 0.5 between 97.5th
percentile intake for adults and the 97.5th
percentile for children under 18
years old.
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
1- 3 800 930.4a
- 1:3.5 to 1:5 -
4 - 6 1100 1707b
- 1:3.5 to 1:5
7- 10 1500 1707 b
- 1:3.5 to 1:5
11-14 2000 1860b
140 1:3.5 to 1:5 490 to 700
15- 17 2600 1860 b
740 1:3.5 to 1:5 2590 to 3700
Adults* 3000 6564c
- 1:3.5 to 1:5
Vitamin D
years μg/day μg /day μg /day μg /day
0-2 25 14.4a
10.6 1:200 2114
3-10 25 5.9d
19.1 1:200 3820
11-17 50 9.7 d
40.3 1:200 8060
Adults 50 17.3 d
32.7 1:200 6540
Vitamin E
years mg/day mg/day mg/day mg/day
1-3 100 11.2a
88.8 1:1 88.8
4-6 120 19.5 d
100.5 1:1 100.5
7-10 160 19.5 d
140.5 1:1 140.5
11-14 220 35.7 d
184.3 1:1 184.3
15-17 260 35.7 d
224.3 1:1 224.3
Adults 300 36 d
264 1:1 264
Vitamin K
years mg/day mg/day mg/day mg/day
<18 10 0.313 9.7 1:18 174.4
Adults 10 0.625 9.4 1:18 168.8
* including women of child-bearing age
a
Fantino, 2005.
b
Lioret et al., 2009.
c
SCF (2002a)
d
Flynn et al., 2009.

12EFSA Journal 2010; 8(3):1525
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 11-17 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 366.9 to
410.4 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 265.3 mg/day for children under 3
years old, varies from 278.7 to 318.7 mg/day for 3-10 year old children and is 439.3 mg/day for
adults.
Carotenoids
mg/day for β-carotene and zeaxanthin, from less than 10 to 95 mg/day for lutein and from less than 10
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

13EFSA Journal 2010; 8(3):1525
affected. The calculated 3
H-labelled calcium lignosulphonate (molecular weight > 200000 g/mol)
permeability coefficients were not significantly different (0.002 x 10-6
cm/s and 0.005 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 (< 200000
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 24-48
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 1000 – 25000 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 ± 1.0 74.1 ± 1.3 70.7 ± 0.3 71.4 ± 2.4
Gastrointestinal tract (a)
1.98 ± 0.07 2.22 ± 0.37 0.09 ± 0.01 0.22 ± 0.15
Skin/fur (b)
4.61 ± 0.14 4.24 ± 0.68 0.44 ± 0.05 0.43 ± 0.03
Urine 2.81 ± 0.28 3.12 ± 0.11 0.11 ± 0.05 0.05(c)
Blood 0.56 ± 0.05 0.66 ± 0.12 0.01 ± 0.001 0.012 ± 0.002
Liver 0.76 ± 0.03 0.71 ± 0.07 0.079 ±0.005 0.074 ± 0.006
Remaining carcass 12.88 ± 0.87 13.23 ± 0.6 0.68 ± 0.01 0.64 ± 0.05

14EFSA Journal 2010; 8(3):1525
Total 98.35 98.28 72.04 72.83
(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
H2O) 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 > 200000 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 gastro-
intestinal tract of mice. Overall, the Panel considers that calcium lignosulphonate (40-65) is poorly
transported and absorbed in vivo.
3.2. Toxicological data
3.2.1. Acute oral toxicity
An acute oral LD50 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 LD50 was
estimated to lie between 10 and 20 g/kg bw (Keller, 1978).
3.2.2. 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.

15EFSA Journal 2010; 8(3):1525
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.

16EFSA Journal 2010; 8(3):1525
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).

17EFSA Journal 2010; 8(3):1525
3.2.3. Genotoxicity
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.
3.2.4. 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.
3.2.5. 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

18EFSA Journal 2010; 8(3):1525
foetuses, abnormal and embryo or foetal resorptions) did not differ from the control group. Fetal data
reported showed that the mean fetal weight development (statistically significant differences were
considered minimal and incidental), sex ratios and external, visceral and skeletal examinations were
unaffected by the treatment. Local thinning of the diaphragm and rudimentary cervical ribs were found
in fetuses of treatment groups but these effects were considered either not significant compared to
controls, or of low incidence or within the historical data. According to the authors a NOAEL of 1000
mg/kg bw/day (the highest dose tested) was derived from this study.
3.2.6. Other studies
A mouse Local Lymph Node Assay (LLNA) was conducted with increasing doses of calcium
lignosulphonate (not described if identical to calcium lignosulphonate (40-65)) administered to CBA
female mice (SPF-bred) according to OECD Guideline 429 and GLP (van Huygevoort, 2004). Four
groups of 5 animals were exposed through the epidermis to calcium lignosulphonate at dose levels of
0, 2.5%, 10% and 25% during three consecutive days. Control group received the vehicle alone
(propylene glycol). Calculated Stimulation Index (SI) values for tested animals groups were 1.0
(control), 0.8 (2.5%), 0.5 (10%) and 0.9 (25%). All values were below a SI value of 3 and it was thus
considered that calcium lignosulphonate is not a sensitising substance by skin contact. The Panel
considers that this assay is not relevant for the assessment of calcium lignosulphonate (40-65) as a
food additive.
The petitioner mentions that calcium lignosulphonate (40-65) can be considered as a non-digestible
dietary fiber and that, therefore, could lead to similar nutritional effects attributed to other fibers of the
same type appearing at high levels of exposure. The influence of calcium lignosulphonates on nutrient
digestibility and hindgut fermentation was investigated in three groups of 5 adult male Sprague-
Dawley rats (Flickinger et al., 1998). Apparent digestibility was measured from dry matter and
organic matter contents in feed and faecal samples. After 14 days administration of approximately 40,
55 and 61% calcium lignosulphonate in the diet apparent digestibility was lower in all exposed groups
as compared to controls. Faecal samples from treatment groups were all brown coloured but no
diarrhoea was reported. Body weight gain was unaffected by the treatments but cecal and colonic
organ weights were found to be statistically significantly higher than controls. Colonic pH values were
lower than control and these changes were reported to affect slightly colonic microflora composition.
Calcium lignosulphonate was shown to induce colonic ulceration in four out of seven (57%) adult
Dutch rabbits fed an aqueous solution of 40 g/L of calcium lignosulphonate over a period of 8 weeks
(Marcus and Watt, 1977). Fresh solutions were prepared every day and daily fluid intake of each
animal was measured. No effects were reported on average weight gain as compared to controls and
the mean daily intake of calcium lignosulphonate per animal was reported to be 2320 mg/kg bw. The
same effects, with the same incidence, were reported in rabbits fed sodium lignosulphonate but not in
those animals fed magnesium lignosulphonate. Similar effects have been reported previously in rabbits
and in guinea-pigs (Marcus and Watt, 1974).
In female Wistar rats (7 per group) fed diets containing 2.5, 5, 10 and 20% (w/w) lignosulphonate
supplemented with 1% cholesterol, serum cholesterol concentrations were shown to slightly decrease
with increasing intake of lignosulphonate (Meijer and Beynen, 1991). This effect was reported to be
more marked at the highest dose tested (20%), similarly to liver cholesterol which was also shown to
decrease in the same experiments particularly at the highest dose tested of lignosulphonate.
A single case-report of contact allergy attributed to calcium lignosulphonate was reported in a 22-year-
old man developing eczema of the face, hands and forearms (Andersson and Göransson, 1980).

19EFSA Journal 2010; 8(3):1525
4. Discussion
The present opinion deals with 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.
Lignosulphonates belong to a family of complex phenolic polymers derived from lignin. According to
the petitioner calcium lignosulphonate (40-65) is characterised by an average molecular weight
between 40000 and 65000 g/mol and the proposed name reflects the considerations of JECFA about
the need to distinguish the product from other food-grade calcium lignosulphonates.
In vivo assays showed that upon oral intake calcium lignosulphonate (40-65) is poorly absorbed. After
48 hours of administration more than 98% of a radiolabelled oral dose of calcium lignosulphonate (40-
65) was detected in faeces. Some remaining radioactivity was found in urine and in some organs but it
was shown that the majority of the radioactivity was composed of low molecular weight radiolysis
products. In vitro assays showed that per hour less than 1 - 2% radiolabelled calcium lignosulphonate
(40-65) crossed Caco-2 cell layers. However the majority of the transported radioactivity is composed
of low molecular weight compounds arising from radiolysis in the stock solution. The Panel considers
that these data indicate that calcium lignosulphonate (40-65) is poorly absorbed following oral
administration.
Calcium lignosulphonate (40-65) has undergone in vitro genotoxicity, short-term, subchronic and
developmental toxicity testing in accordance with recognised guidelines. No chronic toxicity or
carcinogenicity studies were presented for 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).
In a short-term 28-day toxicity study, a NOAEL of 1500 mg/kg bw/day has been 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 to be 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.
The developmental toxicity study reported no treatment-related effects in dams or fetuses. A NOAEL
of 1000 mg/kg bw/day (the highest dose tested) can be derived from this 21-day toxicity study.
The lack of chronic toxicity and carcinogenicity studies on calcium lignosulphonate (40-65) was
justified by the petitioner on the basis that two other substances (the sodium salt of polypentosan
sulphate (PPS) and polyvinylpyrrolidon copolymer), considered as similar to calcium lignosulphonate
(40-65), also showed histiocytosis in mesenteric lymph nodes of animals upon oral administration.
However, in light of the view of the Panel the 90-day toxicity study with a 4-week recovery period is
inadequate for the safety evaluation of calcium lignosulphonate (40-65), the Panel considers that long-
term toxicity/carcinogenicity studies are needed to elucidate whether the histiocytosis observed in the
inadequate 90-day study is indeed a treatment-related histopathological change which may or may not
progress to a more adverse lesion with time.
Exposure estimates were based on the reported European high percentile intakes of vitamins from food
and the 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 11-17 years. The maximum
intake of calcium lignosulphonate (40-65) resulting from its use as a carrier for vitamin D ranges from

20EFSA Journal 2010; 8(3):1525
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 224.3 mg/day for children under 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 366.9 to 410.4 mg/day for children aged 11 to 17
years, ranges from 278.7 to 318.7 mg/day for children aged 3-10 years, is 265.3 mg/day for children
under 3 years old, and is 439.3 mg/day for adults.
mg/day for -carotene and zeaxanthin, from less than 10 to 95 mg/day for lutein and from less than 10
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) when used as carrier for these colours since these colours are still under
evaluation in Europe and their intakes have not yet been evaluated.
CONCLUSIONS
The Panel considers that the available data on calcium lignosulphonate (40-65) were insufficient to
establish an ADI.
Furthermore, the Panel considers the 90-day study with a 4-week recovery period to be inadequate for
evaluating the safety of calcium lignosulphonate (40-65). Therefore, the Panel considers that long-
term toxicity studies are needed to elucidate whether the histiocytosis observed in the mesenteric
lymph nodes of the rats of the inadequate 90-day toxicity study may progress into a more adverse
status 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.

21EFSA Journal 2010; 8(3):1525
DOCUMENTATION PROVIDED TO EFSA
1. Calcium lignosulphonate (40-65). Application for the authorisation of calcium lignosulphonate
(40-45) as a new food additive under the provision of Commission Directive 89/107/EEC. October
2008. Submitted by DSM Nutritional Products France, France. Additional data submitted in
October 2009.
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Preformed Vitamin A (retinol and retinyl esters). Available at:
http://ec.europa.eu/food/fs/sc/scf/out145_en.pdf
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Preformed Vitamin E. Available at: http://ec.europa.eu/food/fs/sc/scf/out195_en.pdf
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mutation assay with Ultrazine FG-R. Report No. 2500196. DSM Nutritional Products Ltd.
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hamster V79 cells with Ultrazine FG-R. Report No. 2500268. DSM Nutritional Products Ltd.
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24EFSA Journal 2010; 8(3):1525
GLOSSARY /ABBREVIATIONS
ADI Acceptable Daily Intake
ANS Scientific Panel on Food Additives and Nutrient Sources added to Food
CAS Chemical Abstracts Service
EC European Commission
EFSA European Food Safety Authority
EU European Union
FCC Food Chemicals Codex
FDA US Food and Drug Administration
FAO/WHO Food and Agriculture Organization/World Health Organization
FEEDAP Scientific Panel on Additives and Products or Substances used in Animal Feed
GLP Good Laboratory Practice
JECFA Joint FAO/WHO Expert Committee on Food Additives
LLNA Local Lymph Node Assay
LSC Laser Scanning Cytometer
LD50 Lethal Dose, 50 % i.e. dose that causes death among 50 % of treated animals
NOAEL No Observed Adverse Effect Level (NOAEL
OECD Organisation for Economic Co-operation and Development
PPS Polypentosan Sulphate
SAR Structure Activity Relationship
SCF Scientific Committee on Food
SEC Size Exclusion Chromatography
SI Stimulation Index
UL Tolerable Upper Intake Levels
UV Ultra Violet

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