13.12.2012-Stigma Sterol-EFSA

EFSA Journal 2012;10(5):2659
SCIENTIFIC OPINION
Scientific Opinion on the safety of stigmasterol-rich plant sterols as food

additive1
EFSA Panel on Food additives and Nutrient Sources added to Food (ANS)2, 3
European Food Safety Authority (EFSA), Parma, Italy

This scientific output published on 11 June 2012, replaces the earlier version published on 14 May
20124
ABSTRACT
The Panel on Food Additives and Nutrient Sources added to Food (ANS) delivers a scientific opinion evaluating
the safety of use of stigmasterol-rich plant sterols in ready-to-freeze alcoholic cocktails as a stabiliser. Biological
and toxicological data on stigmasterol-rich plant sterols were not provided, but the available data on phytosterols
and their esters can be used to evaluate the safety of stigmasterol-rich plant sterols provided that the studies are
performed with phytosterol preparations containing also a well-defined percentage of stigmasterol. Less than 5%
of dietary phytosterols, phytostanols, and their esters are absorbed by the gastrointestinal tract of rats and
human. The metabolic fate of phytosterols and their esters is similar between rats and humans. No evidence of
mutagenicity or genotoxicity of phytosterols or phytosterol esters was observed. The Panel concluded that
stigmasterol-rich plant sterols are not of concern with respect to genotoxicity. Toxicity studies on phytosterols
and phytosterol esters were limited to 90-day subchronic toxicity studies and a 2-generation reproductive
toxicity study in rats. No chronic toxicity, carcinogenicity or developmental toxicity studies conducted with
phytosterols, phytostanols, and their esters were identified. For adults, mean and 95th percentile exposures to
stigmasterol-rich plant sterols from the proposed uses and use levels were 0.01-0.2 mg/kg bw/day and
0.4-7.4 mg/kg bw/day, respectively. Using the lowest NOAEL values of 1.54 g phytosterols/kg bw/day (335 mg
stigmasterol/kg bw/day) the calculated Margin of Safety (MOS) values amount to 7700-154 000 at the mean and
208-3850 at the 95th percentile for the phytosterols and to 1675-33 500 at the mean and 45-838 at the 95th
percentile for stigmasterol. The Panel considered these MOS values adequate and concluded that the proposed
use and use levels of stigmasterol-rich plant sterols in ready-to-freeze alcoholic cocktails as a stabiliser would
not be of safety concern.
© European Food Safety Authority, 2012
1
On request from the European Commission, Question No EFSA-Q-2011-00428, adopted on 19 April 2012.
2
Panel members: F. Aguilar, R. Crebelli, B. Dusemund, P. Galtier, J. Gilbert, D.M. Gott, U. Gundert-Remy, J. König, C.
Lambré, J-C. Leblanc, A. Mortensen, P. Mosesso, D. Parent-Massin, I. Stankovic, P. Tobback, I. Waalkens-Berendsen,
R.A. Woutersen, M.C. Wright. Correspondence: [email protected]
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: N. Bemrah, P. Galtier, U. Gundert-Remy, R. Guertler, C.
Lambré, J.C. Larsen, J-C. Leblanc, P. Mosesso, D. Parent-Massin, Ch. Tlustos, I. Stankovic, P. Tobback , M.C. Wright as
well as I.M.C.M. Rietjens (former member of the Working Group A on Food Additives and Nutrient Sources of the ANS
Panel).
4
Editorial change: “I.M.C.M. Rietjens” has been deleted in the footnotes 2 and 3 because when the opinion was
adopted this expert was not any more member neither of the ANS Panel nor of the Working Group A on Food
Additives and Nutrient Sources of the ANS Panel. Editorial change: “as well as I.M.C.M. Rietjens (former
member of the Working Group A on Food Additives and Nutrient Sources of the ANS Panel). “ has been added
in the foodnote 3. The changes do not affect the overall conclusion of the opinion. To avoid confusion the
original version has been removed from the website.
Suggested citation: EFSA Panel on Food additives and Nutrient Sources added to Food (ANS); Scientific Opinion on the
safety of stigmasterol-rich plant sterols as food additive. EFSA Journal 2012;10(5):2659. [39 pp.]
doi:10.2903/j.efsa.2012.2659. Available online: www.efsa.europa.eu/efsajournal
© European Food Safety Authority, 2012
Safety of stigmasterol-rich plant sterols as food additive
KEY WORDS
Stigmasterol-rich plant sterols, food additive, phytosterols, stigmasterol, CAS Registry Number 83-48-7, β-
sitosterol, campesterol, brassicasterol.
EFSA Journal 2012;10(5):2659 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 the use of
stigmasterol-rich plant sterols in ready-to-freeze alcoholic cocktails as a stabiliser. The preparation is
requested to be used as a stabiliser, ice nucleating agent, to generate and maintain the presence of ice
dispersions within a frozen alcoholic cocktail.
Biological and toxicological data on stigmasterol-rich plant sterols were not provided by the applicant.
Instead the applicant refers to data on phytosterols, phytostanols, and their esters which have
previously been evaluated by several scientific authorities, including Scientific Committee for Food
(SCF), the Joint FAO/ WHO Expert Committee on Food Additives (JECFA) and the European Food
Safety Authority (EFSA).
The Panel considered that biological and toxicity data on phytosterols and their esters can be used to
evaluate the safety of stigmasterol-rich plant sterols, provided that the studies are performed with
phytosterol preparations containing also a well-defined percentage of stigmasterol. The Panel
considered that these studies with phytosterol preparations with a well-defined percentage of
stigmasterol can also be used to judge the safety of the other sterols and stanols present in stigmasterol
rich plant sterols, because these phytosterol preparations were recognised to contain also other sterols
and stanols and likely at levels even higher than present in the stigmasterol-rich plant sterols.
The SCF also considered that plant sterols and stanols interfere with the absorption of carotenoids as
deduced from the reduction of carotenoid blood level and that the level of other fat-soluble vitamins,
such as vitamin E and tocopherols, may also be affected, although to a lesser extent. The decreases in
blood carotenoids appear to plateau when doses of sterols or stanols reached 2.2 g/day and amounted
to a reduction of 33% after one-year consumption of an enriched margarine providing 3 g/day. The
SCF indicated that the consequences of such a persistent decrease of blood concentrations of carotene
on human health are largely unknown.
In accordance with the scientific opinions of the SCF and EFSA, phytosterols, phytostanols and their
esters are approved for use in various foods within the EU at levels resulting in intake of up to
3 g/day.
The safety of phytosterols, phytostanols, and their esters were also evaluated by JECFA at their 69th
meeting. Based on the similar metabolic profiles among free phytosterols and phytostanols and their
respective esters, JECFA established a group acceptable daily intake (ADI) of 0-40 mg free
phytosterols/kg bw/day, based on an overall no observed adverse effect level (NOAEL) of
4200 mg/kg bw/day from the combined evidence from several short-term (90-day) toxicity studies
and an uncertainty factor of 100 to account for inter and intra-species differences. Less than 5% of
dietary phytosterols, phytostanols, and their esters are absorbed by the gastrointestinal tract of rats and
human. Studies examining the absorption of individual phytosterols/phytostanols have demonstrated
differences in the percentage absorbed.
Following absorption, phytosterols/phytostanols are transported in the serum via HDLs in rats and
LDLs in humans to various organs and tissues. In the liver, phytosterols may be converted to bile
acids. As the majority (>95%) of phytosterols are not absorbed in the gastrointestinal tract,
phytosterols enter the colon intact and are rapidly excreted in the faeces. Absorbed phytosterols and
phytostanols are predominantly excreted as such or as bile acids via the biliary route into the faeces.
The metabolic fate of phytosterols, phytostanols, and their esters is similar between rats and humans,
thereby making the rat an appropriate laboratory model to characterise the safety of phytosterols in
humans. Furthermore, as the individual plant sterols are handled in a similar manner, data conducted
with a mixture of phytosterols, phytostanols, and their esters are applicable to the safety of an
individual phytosterol, like stigmasterol.
EFSA Journal 2012;10(5):2659 3

The Panel noted that stigmasterol-rich plant sterols are to be used in alcohol rich cocktails but that no
data are available on the effects of alcohol on the absorption, distribution, metabolism or excretion of
stigmasterol-rich plant sterols.
The Panel noted that genotoxicity assay on stigmasterol-rich plant sterols were not available, but that
genotoxicity assays performed with phytosterols containing up to 18% stigmasterol are available and
can be used to evaluate the genotoxicity of stigmasterol-rich plant sterols. No evidence of
mutagenicity or genotoxicity of these phytosterols or phytosterol esters was observed following a
battery of in vitro and in vivo assays. Based on these data the Panel concluded that stigmasterol-rich
plant sterols are not of concern with respect to genotoxicity.
Toxicity studies on stigmasterol-rich plant sterols were not provided and studies on phytosterols and
phytosterol esters were limited to 90-day subchronic toxicity studies in rats and a 2-generation
reproductive toxicity study in rats.
In one of the 90-day studies in rats, dietary administration of up to 8.1% phytosterol esters (equivalent
to 5% phytosterols, 4.1 g phytosterols/kg bw/day, or 875 mg stigmasterol/kg bw/day) did not result in
any toxicologically relevant findings and represented the NOAEL.
In another 90-day study reported by Kim et al. in 2002, due to the suppression of body weight gains in
both sexes and the observed infiltration of mononuclear cells in the heart in males at a dose level of
9 g phytosterol esters/kg bw/day, the NOAEL for both sexes was considered to be 3 g phytosterol
esters/kg bw/day, equivalent to 1.88 g phytosterols/kg bw/day or approximately 350 mg
stigmasterol/kg bw/day.
In a 2-generation reproductive toxicity study in rats the authors determined the NOAEL for
phytosterol esters to be 8.1% in the diet for phytosterol esters, the highest dose tested. This
concentration is equivalent to a dose of 2.5-9.1 g phytosterol esters/kg bw/day or 1.54- 5.62 g
phytosterols/kg bw/day (approximately 335-1219 mg stigmasterol/kg bw/day) dependent on the phase
of the study.
No chronic toxicity, carcinogenicity or developmental toxicity studies conducted with phytosterols,

phytostanols, and their esters were identified.
In a study with human volunteers receiving statin medication it was investigated whether plant sterol
enriched novel foods might adversely affect vascular function (Kelly et al., 2011). In this study the
effects of long-term plant sterol and –stanol consumption on changes in terminal vessel diameter were
characterised. This parameter was indicated to reflect alterations in the microcirculation. The authors
indicated that their results may constitute an explanation for the suggested effects of plant sterols on
vascular function, although they also indicated that this novel finding needs confirmation and further
study. The Panel noted that the study does not provide sufficient evidence to allow a final assessment
of the atherogenic potential of increased phytosterol serum concentrations due to the intake of plant
sterol enriched food. Measured effects were marginally and it has to be taken into account that
volunteers of the studies were undergoing statin treatment and might be at higher risk for venular
changes due to vascular predamage.
The Panel considered that the overall NOAEL, derived from the 90-day subchronic toxicity studies in
rats and the 2-generation reproductive toxicity study in rats, amounts to be 2.5-6.6 g phytosterol
esters/kg bw/day, 1.54-4.1 g phytosterols/kg bw/day and 335-900 mg stigmasterol/kg bw/day.
Given the absence of chronic toxicity, carcinogenicity or prenatal developmental toxicity studies the
Panel could not establish an ADI for stigmasterol-rich plant sterols.
EFSA Journal 2012;10(5):2659 4

As the intended use of stigmasterol-rich plant sterols is for alcoholic beverages only, the Panel
performed long-term (chronic) exposure assessment for the following population groups only: Adults
and the elderly.
The estimated mean stigmasterol-rich plant sterol exposure from the proposed food additive use
ranges from 0.6-11.4 mg/day (0.01-0.2 mg/kg bw/day) for adults and 0.6-8.4 mg/day (0.01-0.1 mg/kg
bw/day) for the elderly. The ranges for the 95th percentile exposures are 24-443.3 mg/day
(0.4-7.4 mg/kg bw/day) for adults and 28.2-192.6 mg/day (0.5-3.2 mg/kg bw/day) for the elderly.
Taking into account the estimated exposure to plant sterols from all sources (i.e. from new
applications, from natural sources and added as novel food ingredient) the average daily intake was
estimated to be approximately 2770 mg/day (46 mg/kg bw/day) for both adults and the elderly.
The overall mean and 95th percentile exposures to stigmasterol-rich plant sterols resulting from the
proposed uses and use levels were 0.01-0.2 mg/kg bw/day and 0.4-7.4 mg/kg bw/day, respectively.
Using the lowest NOAEL values from the available toxicity studies of 1.54 g phytosterols/kg bw/day
and 335 mg stigmasterol/kg bw/day the Panel calculated that the Margin of Safety (MOS) values
amount to 7700-154 000 at the mean and 208-38500 at the 95th percentile for the phytosterols and to
1675-33 500 at the mean and 45-838 at the 95th percentile for stigmasterol.
Taking into account:
- that the mean estimated exposure resulting from the proposed use and use levels of
stigmasterol-rich plant sterols (0.6-11.4 mg/day) is 132 to 4000-fold below the estimated
mean daily plant sterol intake from food and food supplements (as added nutrient) (1510-
2450 mg/day).
- that the estimated exposure resulting from the proposed use and use levels of stigmasterol-
rich plant sterols amounts to < 0.5 % at the mean and <15% at the 97th percentile exposure of
the estimated exposure of people using phytosterols to lower their cholesterol levels at dose
levels up to 3 g/day,
- that the bioavailability of the phytosterols and phytostanols in rat is somewhat higher than that
in human
- that the predicted exposures to stigmasterol-rich plant sterols are arguably large over-
estimations for frozen cocktail products, and provide a worst-case scenario for potential
exposure, and
- the available long-term human data,
the Panel considered these MOS values adequate.
The Panel concluded that the proposed use and use levels of stigmasterol-rich plant sterols in ready-
to-freeze alcoholic cocktails as a stabiliser would not be of safety concern.
EFSA Journal 2012;10(5):2659 5

TABLE OF CONTENTS
Abstract ................................................................................................................................................... 1
Summary ................................................................................................................................................. 3
Table of contents..................................................................................................................................... 6
Background as provided by the European Commission ..................................................................... 7
Terms of reference as provided by the European Commission ......................................................... 7
Assessment .............................................................................................................................................. 8
1. Introduction ................................................................................................................................... 8
2. Technical data ................................................................................................................................ 8
2.1. Identity of the substances ..................................................................................................... 8
2.2. Specifications ......................................................................................................................... 9
2.3. Manufacturing process ....................................................................................................... 11
2.4. Methods of analysis in food ................................................................................................ 12
2.5. Reaction and fate in food .................................................................................................... 12
2.6. Case of need and proposed uses ......................................................................................... 12
2.6.1. Proposed use levels of stigmasterol-rich plant sterols ................................................. 13
2.7. Information on existing authorisations and evaluations ................................................. 13
2.8. Food consumption data used for exposure assessment .................................................... 15
2.8.1. EFSA Comprehensive European Food Consumption Database ................................ 15
2.8.2. Food consumption data for different age and consumer groups................................ 15
2.9. Exposure assessment ........................................................................................................... 16
2.9.1. Contributing food groups .............................................................................................. 16
2.9.2. Exposure via other sources ............................................................................................ 17
2.9.3. Total estimated intake of plant sterols from all sources (food additive, added
nutrient source and natural sources) ......................................................................................... 18
3. Biological and toxicological data ................................................................................................ 19
3.1. Absorption, distribution, metabolism and excretion ....................................................... 19
3.2. Toxicological data ............................................................................................................... 21
3.2.1. Acute oral toxicity ........................................................................................................... 21
3.2.2. Short-term and subchronic toxicity .............................................................................. 21
3.2.3. Genotoxicity .................................................................................................................... 23
3.2.4. Chronic toxicity and carcinogenicity ............................................................................ 24
3.2.5. Reproductive and developmental toxicity .................................................................... 24
3.2.6. Other studies ................................................................................................................... 26
4. Discussion ..................................................................................................................................... 28
Documentation provided to EFSA ...................................................................................................... 31
References ............................................................................................................................................. 31
Glossary [and/or] abbreviations.......................................................................................................... 39
EFSA Journal 2012;10(5):2659 6

BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION

The use of food additives is regulated under the European Parliament and Council Regulation (EC) No
1333/2008 on Food additives. Only food additives that will be included in the Community list in
Annex II of that regulation may be placed on the market and used in foods under the conditions of use
specified therein.
An application has been introduced for the authorisation of the use of stigmasterol-rich plant sterols in
ready-to-freeze alcoholic cocktails. The additive is requested to be used as a stabiliser, ice nucleating
agent, to generate and maintain the presence of ice dispersions within a frozen alcoholic cocktail.
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 about the safety of the use of
stigmasterol-rich plant sterols in ready-to-freeze alcoholic cocktails as a stabiliser.
EFSA Journal 2012;10(5):2659 7

ASSESSMENT
1. INTRODUCTION
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 the use of
stigmasterol-rich plant sterols in ready-to-freeze alcoholic cocktails as a stabiliser. The preparation is
requested to be used as a stabiliser, ice nucleating agent, to generate and maintain the presence of ice
dispersions within a frozen alcoholic cocktail. The Panel noted that plant sterols and –stanols are used
as novel food ingredients to reduce elevated levels serum LDL cholesterol concentrations resulting in
already relatively high daily intakes of up to about 3 g/day (SCF, 2002a).
2. TECHNICAL DATA
2.1. Identity of the substances

Stigmasterol-rich plant sterols are derived from soybeans and are a chemically defined simple mixture
that comprises ≥95% of plant sterols (stigmasterol, β-sitosterol, campesterol, and brassicasterol), with
stigmasterol representing > 85% of the stigmasterol-rich plant sterols. The identity of the major sterols
present in stigmasterol-rich plant sterols are showed in Table 1.
Table 1: Identity of the major sterols present in stigmasterol-rich plant sterols
% in
CAS
stigmasterol Molecular
Common Chemical Name Registry Molecular
-rich plant weight
Name Number formula
sterols (g/mol)
(3S,8S,9S,10R,13R,14S,17R)-17-
(5-ethyl-6- methyl-hept-3-en-2-
yl)-10,13-dimethyl-
Stigmasterol > 85 83-48-7 C29H48O 412.6
2,3,4,7,8,9,11,12,14,15,16,17-
dodecahydro-1H-
cyclopenta[a]phenanthren-3-ol
(3S,8S,9S,10R,13R,14S,17R)-17-
[(E,2R,5R)-5,6- dimethylhept-3-
en-2-yl]-10,13-dimethyl-
1.7 474-67-9 C28H46O 398.6
Brassicasterol 2,3,4,7,8,9,11,12,14,15,16,17-
dodecahydro-1H-
(3S,8S,9S,10R,13R,14S,17R)-17-
[(2S,5S)-5-ethyl- 6-methyl-
heptan-2-yl]-10,13-dimethyl-
3 83-46-5 C29H50O 414.7
β-Sitosterol 2,3,4,7,8,9,11,12,14,15,16,17-
dodecahydro-1H-
(3S,8S,9S,10R,13R,14S,17R)-17-
(5,6- dimethylheptan-2-yl)-10,13-
dimethyl-
1.7 474-62-4 C28H48O 400.6
Campesterol 2,3,4,7,8,9,11,12,14,15,16,17-
dodecahydro-1H-
The structural formulae of stigmasterol, brassicasterol, β-sitosterol and campesterol are presented in
Figure 1.
Stigmasterol-rich plant sterols are available as a white to off-white crystalline powder that is insoluble
in water, but soluble in most organic solvents. The melting point of stigmasterol-rich plant sterols is
EFSA Journal 2012;10(5):2659 8

between 162 and 170 °C.
Figure 1: Structural formulae of major sterols in stigmasterol-rich plant sterols
The applicant indicated that consistent with the specifications for phytosterol, phytostanols and their
esters established by the Joint FAO/WHO Expert Committee on Food Additives (JECFA), the identity
of the stigmasterol-rich plant sterols powder is confirmed using gas chromatography (GC).
2.2. Specifications
The stigmasterol-rich plant sterols contain > 85% stigmasterol and lesser amounts of β-sitosterol,
campesterol, and brassicasterol to provide a high-purity plant sterol ingredient.
The specifications proposed by the applicant for stigmasterol-rich plant sterols are presented in Table
2. The applicant indicated that these specifications for stigmasterol-rich plant sterols are based on
JECFA’s specifications for phytosterols, phytostanols, and their esters (JECFA, 2008). Results from
analysis of stigmasterol-rich plant sterols verified that the production process produces a consistent
product that falls within the proposed specifications.
EFSA Journal 2012;10(5):2659 9

Table 2: Chemical specifications as defined by JECFA for phytosterols, phytostanols, and their
esters (JECFA, 2008) and as proposed by the applicant for stigmasterol-rich plant sterols
JECFA’s Specifications as proposed

Parameter Methods of Analysis
Specifications by the applicant
Free-flowing, white to
off-white powders,
Description pills or pastilles; White crystalline powder
colourless to pale
yellow liquids
Identification
Practically insoluble in
Practically insoluble in water.
water. Phytosterols and
Phytosterols and phytostanols
Solubility phytostanols are
are soluble in acetone and
soluble in acetone and
ethyl acetate.
ethyl acetate.
> 85% (w/w)
Stigmasterol content Gas Chromatography
Other plant
sterols/stanols: either
singularly or in
combination including:
Brassicasterol,
campestanol, < 15% (w/w) Gas Chromatography
campesterol, Δ-7-
campesterol,
cholesterol,
chlerosterol, sitostanol
and β-sitosterol.
Purity
Total Ash < 0.1% < 0.1% (JECFA, 2006)
Hexane, 1-propanol,
ethanol or methanol:
Ethanol: < 5000 mg/kg
Residual Solvents <50 mg/kg either
Methanol: < 50 mg/kg
singly or in
combination
Water < 4% (Karl Fischer) < 4% (Karl Fischer) (JECFA, 2006)
Arsenic < 3 mg/kg < 3 mg/kg ICP-MS
Lead < 1 mg/kg < 2 mg/kg ICP-MS
Assay (products > 95% (w/w)
containing only free On a total free Gas Chromatography
sterols and stanols) sterol/stanol basis
The typical impurities present in plant sterol products include plant sterol esters and free fatty acids.
Analyses of 3 non-consecutive batches of stigmasterol-rich plant sterols demonstrated that sterol esters
represent < 6% of the stigmasterol-rich plant sterol powder, and free fatty acids represent < 0.5% of
the sample. These percentages correspond to the whole preparation to be used as food additive. The
applicant also indicated that the sterol esters are regarded as part of the sterol fraction.
The applicant informed that individual contents of other sterols and stanols in 4 batches of
stigmasterol-rich plant sterols were as follows: β-sitosterol (3.0%), brassicasterol (1.7%), campesterol
(1.7%), Δ-7-campesterol (1.6%), sitostanol (0.7%, n=1), cholesterol (0.4%), campestanol (0.2%, n=3),
chlerosterol (0.1%, n=1). The original specifications provided did not indicate a maximum level for
other plant sterols/stanols. Upon request the applicant suggested to modify the proposed specifications
to include; “other sterols” to be ≤ 15%, where other sterols include brassicasterol, campestanol,
campesterol, Δ-7-campesterol, cholesterol, chlerosterol, sitostanol and β-sitosterol. The Panel agreed
EFSA Journal 2012;10(5):2659 10

with this suggestion. The percentage (≤ 15%) of the other sterols corresponds to the whole preparation
to be used as food additive.
The Panel noted the use of methanol in the manufacturing process, whereas the original specifications
provided did not indicate a maximum level for methanol. Upon request the applicant proposed to
modify the specifications to indicate that methanol should be ≤ 50 mg/kg. The Panel agreed that the
specifications should be extended to include limits for methanol.
The Panel noted that the tolerance limits proposed for ethanol and lead are above those established
within JECFA’s specifications for phytosterols, phytostanols, and their esters.
The Panel noted that the product under consideration may contain steroidal hydrocarbons such as
stigmastadienes and that the specifications for virgin olive oil include maximum levels for
stigmastadienes (Commission Regulation (EC) No 61/20115). However, since these requirements
relate to quality issues that are specific to olive oil, the Panel considered that the possible presence of
these compounds in the final product is not of toxicological relevance.
The Panel noted that microorganisms, if present, cannot proliferate in this material due to its low water
activity. However, since stigmasterol-rich plant sterols are produced from a natural source,
microbiological limits have been specified (see Table 3). Analyses for potential microbial
contaminants have been conducted independently using methods based on internationally recognised
standards. The applicant indicated that analyses of 4 non-consecutive lots of stigmasterol-rich plant
sterols verified that the process yields a consistent product within the required microbiological
specifications.
Table 3: Microbiological specifications for stigmasterol-rich plant sterols as proposed by the

applicant.
Test Specification Method of analysis

Based on ISO 4833:2003
Total Plate Count < 1000 CFU/g
Surface inoculation using Rose Bengal
Yeasts < 100 CFU/g Chloramphenicol Agar
surface inoculation using Rose Bengal
Moulds < 100 CFU/g Chloramphenicol Agar
Using surface inoculation of Tryptone Bile X-
Escherichia coli < 10 CFU/g glucuronide agar
Based on ISO 16649-2:2001(E) Part 2
Based on ISO 6579:2002, with confirmation
Salmonella spp. Not detected/25 g using API 20E
CFU - colony forming unit
2.3. Manufacturing process

Stigmasterol-rich plant sterols are manufactured using a process that is commonly used within
industry for other commercially available phytosterol products. Soybean oil deodorizer distillate
(SODD), a by-product in the soybean oil refining process, is used as the source material for the plant
sterols, which undergoes subsequent esterification and trans-esterification steps (conducted using
methanol) to convert the free fatty acids and triglycerides, respectively, to fatty acid methyl ester
(FAME). The phytosterol- and FAME-containing liquor is subsequently filtered and re-crystallized
5
Commission Regulation (EU) No 61/2011 of 24 January 2011 amending Regulation (EEC) No 2568/91 on the
characteristics of olive oil and olive-residue oil and on the relevant methods of analyis. OJ L 23, 27.1.2011, 1-14.
EFSA Journal 2012;10(5):2659 11

using ethanol to obtain an intermediate phytosterol product, containing 95% phytosterols. The
intermediate product is subsequently re-crystallized and purified several times to produce a final
product that contains at least 85% stigmasterol, based on the whole preparation (stigmasterol-rich
plant sterols).
2.4. Methods of analysis in food

Stigmasterol-rich plant sterols can be detected in ready-to-freeze alcoholic beverages using a GC
method developed by the applicant based on ISO 6799 (ISO, 1991) using an internal standard for
quantification and also based on Commission Regulation (EEC) No 2568/916 (Annex V). Prior to
injection, stigmasterol-rich plant sterols plus internal standard are extracted from the beverage sample
using diethyl ether, dried under nitrogen, and converted to trimethylsilyl derivatives.
2.5. Reaction and fate in food

Studies were conducted to assess the bulk stability of the stigmasterol-rich plant sterols powder under
various storage conditions, as well as to assess their stability within the proposed beverage uses.
Under accelerated conditions (40 °C, 70 ± 5% relative humidity), stigmasterol-rich plant sterols were
stable, with > 99% of the sample remaining after 2 years of storage in an open beaker or aluminium
bag. Likewise > 99% of a sample of the stigmasterol-rich plant sterols remained following 3 years of
storage at approximately 20 °C in a sealed plastic bag, with minimal light exposure.
When added to a ready-to-freeze vodka and lemonade cocktail with a pH of 2.8, stigmasterol-rich
plant sterols also were highly stable (> 99% of sample remained) when stored under accelerated
condition (52 °C) for up to 14 days. The applicant indicated that these accelerated conditions represent
a worst-case scenario of the conditions experienced within distribution channels and trade storage over
1 year. The applicant also informed that the stability of the stigmasterol-rich plant sterol powder in the
vodka and lemonade cocktail is expected to be applicable to the other proposed beverage formulations
(piña colada, strawberry daiquiri, mudslide). Although the pH values for these products range between
acidic and neutral (2.8 to 7.5), with the vodka and lemonade, and mudslide formulations having the
lowest and highest pH value, respectively, studies conducted with phytosterol esters added to various
food products over a range of pH values demonstrated high stability over long-term storage (up to 1
year) (Cantrill, 2008).
The applicant also provided data from accelerated stability tests at approximately neutral pH (pH 7.5),
which demonstrated stigmasterol-rich plant sterols to be stable over 14 days at 52 °C.
2.6. Case of need and proposed uses

The applicant has developed a range of ready-to-freeze alcoholic cocktails containing stigmasterol-
rich plant sterols, designed to be purchased by the consumer in liquid form and placed in a domestic
freezer to produce a semi-frozen beverage. Stigmasterol-rich plant sterols are added to the cocktails as
an ice-nucleating agent (stabiliser) to ensure that the cocktail will freeze and produce a satisfactory
semi-frozen beverage in the consumer’s freezer. Without the use of stigmasterol-rich plant sterols,
supercooling of the beverage can occur and ice formation cannot be guaranteed resulting in product
failure.
An ice-nucleator induces heterogeneous ice nucleation once the temperature of an aqueous solution is
lowered below its melting point, but is still at a temperature above the homogeneous ice nucleation
point. The use of water-stable sterols, such as stigmasterol-rich plant sterols, provides predictable
nucleation temperatures that remain stable over long periods of time, especially when the beverage is
frozen and thawed repeatedly. The ready-to-freeze cocktail products require such ingredients in order
to obtain a commercially viable product, by ensuring that ice formation occurs consistently within
6
Commission Regulation (EEC) No 2568/91 of 11 July 1991 on the characteristics of olive oil and olive-residue oil and on
the relevant methods of analysis. OJ L 248, 5.9.1991, 1-83.
EFSA Journal 2012;10(5):2659 12

domestic freezers.
2.6.1. Proposed use levels of stigmasterol-rich plant sterols

Investigation by the applicant into the minimum dosing required for the ready-to-freeze cocktails has
identified a preferred dosing of 60 mg of stigmasterol-rich plant sterols per 750 ml product in water-
based products and up to 600 mg per 750 ml in cream-based products to achieve the desired effect.
Table 4 provides information on the proposed use levels and food categories in which stigmasterol-
rich plant sterols are intended to be used.
Table 4: Proposed use levels of stigmasterol-rich plant sterols by the applicant.
Proposed use level

Foods
(mg/l)
Water based alcoholic drinks/cocktails 80
Cream based alcoholic drinks/cocktails 800
2.7. Information on existing authorisations and evaluations

The SCF has evaluated the long-term safety of phytosterol consumption in foods (SCF, 2000, 2002a).
Subsequent safety evaluations conducted by the SCF (SCF, 2002b, 2002c, 2003) and the European
Food Safety Authority (EFSA, 2003, 2005a,b, 2006, 2007a,b,c) have corroborated the SCF’s
conclusions.
The SCF (SCF, 2002a) evaluated the benefits of using phytosterol-enriched foods with the purpose of
helping hypercholesterolaemic individuals reduce their LDL-cholesterol blood levels and concluded
that a daily intake in the range of 1-3 g plant sterols lowers LDL-cholesterol levels by about 5-15%, in
different populations, ages and conditions and that no additional effect on cholesterol levels is derived
from an intake of phytosterols above the range of 1-3 g/day.
The SCF also considered that plant sterols and stanols interfere with the absorption of carotenoids as
deduced from the reduction of carotenoid blood level and that the level of other fat-soluble vitamins,
such as vitamin E and tocopherols, may also be affected, although to a lesser extent. The decreases in
blood carotenoids appear to plateau when doses of sterols or stanols reached 2.2 g/day and amounted
to a reduction of 33% after one-year consumption of an enriched margarine providing 3 g/day. The
SCF indicated that the consequences of such a persistent decrease of blood concentrations of carotene
on human health are largely unknown (SCF, 2002a).
The SCF concluded that the available data do not provide a basis for setting a numerical upper level of
total daily intake of phytosterols. In consideration of the dosages found to be effective for cholesterol
lowering, without evidence of additional benefits at higher intakes and the possibility that high intakes
might induce undesirable effects, the SCF concluded that it is prudent to avoid plant sterol intakes
exceeding a range of 1-3 g/day (SCF, 2002a). In 2005, the EFSA Panel on Dietetic Products, Nutrition
and Allergies (NDA) evaluated the studies reported by Miettinen et al. (2000) concerning the
combined intake of simvastatin (lipid-lowering medication) and plant stanols and by Sudhop et al.
(2002a) on elevated serum levels of plant sterols as potential risks factors for coronary heart disease.
The NDA Panel concluded that the principles raised by these publications had been dealt with
adequately in the SCF opinion (SCF, 2002a) and that it was unnecessary at the present time to update
the advice or to modify the SCF’s conclusions (EFSA, 2005b).
EFSA Journal 2012;10(5):2659 13

Regulation (EU) Nº 1169/20117 ammending Commission Regulation (EC) Nº 608/20048 concerning

the labelling of foods and food ingredients with added phytosterols, phytosterol esters, phytostanols
and/or phytostanol esters indicates that the consumption of more than 3 g/day of added plant
sterols/plant stanols should be avoided. In accordance with the opinion of the SCF (2002a), the NDA
Panel evaluated the safety of plant sterols (present as free sterols or sterol esters) in the context of
safety assessment of Novel Foods under Regulation (EC) No 258/979; this resulted in Commission
Decisions on authorising the placing on the market of products with added plant sterols10.
Specifications of phytosterol and phytostanols for the addition to foods and food ingredients are
established in each Commission Decision authorising its addition in a given food and food ingredient
(e.g Commission Decision 2008/36/EC11 authorising the placing on the market of rice drinks with
added phytosterols/phytostanols as novel food). The Panel noted that in all Commission Decisions,
published until now, the maximum permitted stigmasterol content of phytosterol/phytostanol
ingredients is 30%; therefore, the maximum stigmasterol intake in foods containing phytosterols
would be equivalent to 900 mg/day from approved maximum food uses.
From 2008 onwards the NDA Panel focussed on scientific substantiation of health claims (efficacy
aspects only) related to plant sterols and plant stanols based mainly on the EC authorised
uses/proposed condition of use pursuant to Regulation (EC) No 1924/200612 (EFSA, 2008a, 2009,
2010).
The safety of phytosterols, phytostanols, and their esters also were evaluated by JECFA at their 69th
meeting (JECFA, 2009). Based on the similar metabolic profiles among free phytosterols and
phytostanols and their respective esters, JECFA established a group acceptable daily intake (ADI) of
0-40 mg free phytosterols/kg bw/day, using the combined evidence from several short-term (90-day)
toxicity studies to identify an overall no observed adverse effect level (NOAEL) of 4200 mg/kg
bw/day and an uncertainty factor of 100. Based on a 60 kg individual, the maximum daily intake is
equivalent to 2.4 g phytosterols/day.
A variety of different vegetable oil and tall oil derived phytosterol ingredients are considered
Generally Recognized as Safe (GRAS) for use in a large variety of different food products in the U.S.
Typical use levels range from 0.6-1.1 g/serving (GRN, 39, 48, 53, 61, 112, 176, 177, 181, 206, 250,
335).
Health Canada approved the use of phytosterols, phytostanols, and their esters in a variety of food
products including unstandardised spreads, mayonnaise, margarine, calorie-reduced margarine, salad
dressing and unstandardised salad dressings, yogurt and yogurt drinks, and vegetable and fruit juices at
a maximum level of 1 g (free phytosterols) per serving and per reference amount (Health Canada,
2010).
Vegetable oil phytosterol esters and non-esterified tall oil derived phytosterols are permitted for
addition to edible oil spreads, breakfast cereals, milk, and yogurt in Australia and New Zealand. Using
worst-case scenario intake modelling, Food Standards Australia New Zealand (FSANZ) estimated that
7
Regulation (EU) No 1169/2011 of the European Parliament and of the council of 25 October 2011 on the providsion of food
information to consumers, amending Regulations (EC) No 1924/2006 and (EC) No 1925/2006 of the European Parliament
of the Council and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive
199/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and
2008/5/EC and Commission Regulation (EC) No 608/2004. OJ L 304, 22.11.2011, pp 18-62.
8
Commission Regulation (EC) No 608/2004 of 31 March 2004 concerning the labeling of foods and food ingredients with
added phytosterols, phytosterol esters, phytostanols and/or phytostanol esters. OJ L 97, 1.4.2004, pp 44-45.
9
Regulation (EC) No 258/97 of the European Parliament and of the Council of 27 January 1997 concerning novel foods and
novel food ingredients. OJ L 43, 14.2.1997
10
http://ec.europa.eu/food/food/biotechnology/novelfood/authorisations_en.htm
11
Commission Decision, of 10 January 2008, authorising the placing on the market of rice drinks with added
phytosterols/phytostanols as novel food under Regulation (EC) No 258/97 of the European Parliament and of the Council.
OJ L 8, 11.1.2008
12
Regulation (EC) No 1924/2006 of the European Parliament and of the Council of 20 December 2006 on nutrition and
health claims on foods. OJ L 404, 30.12.2006.
EFSA Journal 2012;10(5):2659 14

the intake of phytosterols from these uses would result in mean and 90th percentile intakes of 1.9 and
4.7 g/person among the highest users (users aged 40 to 64) of phytosterol containing foods. Whole
population (aged 15+) exposures were 1.8 and 4.6 g/person per day in mean and 90th percentile users
(FSANZ, 2004).
2.8. Food consumption data used for exposure assessment
2.8.1. EFSA Comprehensive European Food Consumption Database

In 2010, the EFSA Comprehensive European Food Consumption Database (Comprehensive Database)
has been built from existing national information on food consumption at a detailed level. Competent
authorities in the European countries provided EFSA with data from the most recent national dietary
survey in their country at the level of consumption by the individual consumer. This included food
consumption data concerning infants (2 surveys from 2 countries), toddlers (8 surveys from 8
countries), children (17 surveys from 14 countries), adolescents (14 surveys from 12 countries), adults
(21 surveys from 20 countries), elderly (9 surveys from 9 countries) and very elderly (8 surveys from
8 countries) for a total of 32 different dietary surveys carried out in 22 different European countries
(EFSA, 2011a).
Overall, the food consumption data gathered at EFSA in the Comprehensive Database are the most
complete and detailed data currently available in the EU. However, consumption data were collected
by using different methodologies and thus they are not suitable for direct country-to-country
comparison.
2.8.2. Food consumption data for different age and consumer groups
The ANS Panel considered that only chronic dietary exposure to stigmasterol-rich plant sterols needs
to be assessed. Therefore, as suggested by the EFSA Working Group on Food Consumption and
Exposure (EFSA, 2011a) dietary surveys with only one day per subject were not considered for the
calculation of stigmasterol-rich plant sterols exposure, as they are not adequate to assess repeated
dietary exposure. Similarly, subjects who participated only one day in the dietary studies where the
protocol prescribed more reporting days per individual were excluded.
performed long-term (chronic) exposure assessment for the following population groups only: Adults
and the elderly. Data on adolescents was not considered in this evaluation due to the very low number
of consumers recorded in all available consumption surveys, which does not allow for robust exposure
estimation. However, estimates derived from this very limited dataset (not included) indicated a
considerably lower exposure than that calculated for adults and therefore it can be assumed that
adolescents are sufficiently covered by the exposure assessment performed for adults.
Thus, for the present assessment, food consumption data were available from 23 different dietary
surveys carried out in 13 different European countries as mentioned in Table 5.
Table 5: Population groups considered for the exposure estimates of stigmasterol-rich plant sterols
Population Age range Countries with chronic food consumption

statistics
Belgium, Czech Republic, Denmark, Finland,
from 18 up to and including 64
Adults France, Germany, Hungary, Ireland, Italy, Latvia,
years of age
Netherlands, Spain, UK
Belgium, Germany, Denmark, Finland, France,
Elderly* Older than 65 years
Hungary, Italy
* includes elderly and very elderly populations listed in the comprehensive database as published
EFSA Journal 2012;10(5):2659 15

Data from the Comprehensive Database were used by the Panel to calculate both mean and high level
exposure to stigmasterol-rich plant sterols using the highest proposed use level listed in Table 4. High
level exposure (95th percentile of consumers only) was calculated by adding the 95th percentile of
exposure from one food group (i.e. the one having the highest value) to the mean exposure resulting
from the consumption of all other food groups.
This is based on the assumption that an individual might be a high level consumer of one food
category and would be an average consumer of the others. This approach has been tested several times
by the Panel in re-evaluation of food colours and has shown reasonable correlation with high level
total intakes when using the raw food individual consumption data. Therefore, this approach was
preferred for the calculations based on the maximum reported use levels in order to avoid excessively
conservative estimates.
2.9. Exposure assessment

Exposure estimates have been performed using the maximum proposed use levels (Table 4) combined
with national consumption data for two population groups: adults and the elderly.
Table 6 presents a summary of anticipated exposure to stigmasterol-rich plant sterols in these 2

subpopulations.
The Panel noted that its estimates should be considered as being conservative as it is assumed that all
alcoholic beverages considered contain the stigmasterol-rich plant sterols added at the maximum
proposed use level of 800 mg/l.
Table 6: Summary of anticipated exposure to stigmasterol-rich plant sterols in 2 population groups
Adults Elderly
(18-64 years old) (65 years old and older)
mg/kg bw/day mg/day mg/kg bw/day mg/day
Mean exposure 0.01-0.2 0.6-11.4 0.01-0.1 0.6-8.4
Exposure 95th percentile 0.4-7.4 24.0-443.3(1) 0.5-3.2 28.2-192.6(2)
(1)
95th percentile calculated based on 8 surveys (of 15 available) as remaining surveys did not have sufficient subjects to
calculate a robust 95th percentile.
(2)
95th percentile calculated on a low number of subjects (only 1 survey had sufficient subjects to calculate a robust 95th
percentile).
The estimated average stigmasterol-rich plant sterols intake is 0.6-11.4 mg/day (0.01-0.2 mg/kg
bw/day) for adults and 0.6-8.4 mg/day (0.01-0.1 mg/kg bw/day) for the elderly. The ranges of high
percentile intakes are 24-443.3 mg/day (0.4-7.4 mg/kg bw/day) for adults and 28.2-192.6 mg/day
(0.5-3.2 mg/kg bw/day) for the elderly.
2.9.1. Contributing food groups

Contributing food groups to the exposure estimates are given in Table 7. It presents the main
contributing food groups (> 10%) to total exposure and the number of surveys in which each food
groups contribute.
Table 7: Contributing food groups to the exposure estimates of stigmasterol-rich plant sterols.
% contribution to total exposure (number of surveys)

Food Category
Adults Elderly
Alcoholic beverages (unspecified) 13.5-24.7 (2) 35.5-41.8 (2)
Liqueur 12.5-49.5 (7) 11-38.2 (6)
Spirits 21.2-100 (all) 20.6-100 (13)
Alcoholic mixed drinks 28.3-73.9 (6) 21.7-71.4 (5)
EFSA Journal 2012;10(5):2659 16

2.9.2. Exposure via other sources
2.9.2.1. Via regular diet

Plant-based foods contain a large number of plant sterols as minor lipid components (Jimenez-Escrig
et al., 2006). Phytosterols are not endogenously synthesized in the body, and therefore they are derived
solely from the diet. The most important natural sources of plant sterols are oils and margarines. Nuts,
seeds and cereal products are also significant sources of phytosterols, whereas the phytosterol content
of vegetables, given on a fresh weight basis, is considerably lower (Hearty et al., 2008).
Dietary intake estimates for naturally occurring plant sterols have been reported in the literature and
are available for a number of European countries (see Table 8).
Table 8: Dietary intake estimates of naturally occurring plant sterols reported in the literature
Men Women Adults Children

Country 97.5th 97.5th References
Mean Mean Mean Mean
percentile percentile
mg/day 301 605 229 427 331
(Sioen et
Belgium mg/kg
5.0 10.1 3.8 7.1 22.1 al., 2011a)
bw/day*
mg/day 305 237
(Valsta et
Finland mg/kg
5.1 4.0 al., 2004)
bw/day*
mg/day 283 512 228 387
(Hearty et
Ireland mg/kg
4.7 8.5 3.8 6.5 al., 2008)
bw/day*
mg/day 307 263
(Normèn et
Netherlands mg/kg
5.1 4.4 al., 2001)
bw/day*
mg/day 276 (Jimenez-
Spain mg/kg Escrig et
4.6
bw/d* al., 2006)
mg/day 252 212 (Klingberg
Sweden mg/kg et al.,
4.2 3.5
bw/day* 2008a)
mg/day 300 293 (Klingberg
UK mg/kg et al.,
5.0 4.9 2008b)
bw/day*
* reported data expressed as mg/kg bw/day based on a 60 kg adult and 15 kg child
Intake estimates for adults are available for seven European countries and are of comparable
magnitude, ranging on average from 212-307 mg/day (3.5–5.1 mg/kg bw/day), estimates for children
(2.5-6.2 years old) are available for Belgium at a mean of 331 mg/day (22.1 mg/kg bw/day).
2.9.2.2. Via addition as novel food ingredient

esters are approved for use in various foods within the EU at levels resulting in intake of up to 3 g/day
(Commission Decision 2008/36/EC).
Consumption of food and beverages with added plant sterols in the European Union was reviewed by
EFSA in 2008 (EFSA, 2008b). Whilst it was stressed that estimation of the actual intake was difficult
due to a lack of information, the report concluded that there seemed to be little over-consumption of
food products with added plant sterols.
EFSA Journal 2012;10(5):2659 17

More detailed information is available from two European studies designed to quantify intake of plant
sterols from enriched sources:
Sioen et al. (2011b) derived regional intake estimates of foods enriched with plant sterols and
supplements containing plant sterols available on the Belgian market and reported an average intake of
0.70 g/day for pre-school children and 1.51 g/day for adults. The 95th percentile intake in adults was
estimated at 4.2 g/day and 16.4% of the adult population were found to have a plant sterol intake
above 3 g/day (Sioen et al., 2011b).
Hearty et al. (2009) estimated a mean phytosterol intake from enriched sources of 2.45 g/day for Irish
adults. In total, 23% had phytosterol intakes >3.0 g/day, the mean intake for respondents with
phytosterol intakes >3.0 g/day was 4.1 g/day.
These two studies indicate that the mean intake of plant sterols from consumption of enriched foods in
adults ranges from 1.5-2.5 g/day, whereas high intake resulted in intakes above the recommended
maximum of 3 g/day, ranging from 4.1-5.5 g/day.
2.9.3. Total estimated intake of plant sterols from all sources (food additive, added nutrient
source and natural sources)
Table 9 presents the total anticipated exposure to plant sterols from all sources, including natural
occurrence and use as added nutrient reported in the literature (see Tables 6 and 8) and proposed use
as food additive estimated in this opinion (see Table 6).
Table 9: Total estimated intake of plant sterols from all sources (food additive, added nutrient
source and natural sources)
Adults Elderly
95th 95th
Mean Mean
percentile percentile
Intake range from food mg/day 0.6-11.4 24-443.3 0.6-8.4 28.2-192.6
in Europe (proposed (3)
mg/kg bw/day 0.01-0.2 0.4-7.4 0.01-0.1 0.5-3.2
food additive use)
(1) (2)
Intake range of plant mg/day 212-307 387-605 212-307 387-605(1,2)
sterols from food in
mg/kg bw/day(4) 3.5-5.1 6.5-10.1(1) 3.5-5.1(2) 6.5-10.1(1,2)
Europe (natural sources)
Estimated daily plant mg/day 1510-2450 4200-5500 1510-2450(2) 4200-5500(2)
sterol intake from food
and food supplements mg/kg bw/day(4) 25.2-40.8 70-91.6 25.2-40.8(2) 70-91.6(2)
(as added nutrient)
Total anticipated mg/day 2768.4 2765.4
exposure to plant sterols
mg/kg bw/day 46.1 46.0
from all sources
(1)
97.5th percentile
(2)
Adult data
(3)
Based on body weight of individual consumers
(4)
Calculated based on a standard 60 kg adult
Based on data reported in the literature, total exposure to plant sterols from all sources was estimated
at an average daily intake of approximately 2770 mg/day (46 mg/kg bw/day) for both adults and the
elderly.
Estimated mean exposure to stigmasterol-rich plant sterols from the proposed use as food additive
ranges from 0.6-11.4 mg/day (0.01-0.2 mg/kg bw/day) for adults and 0.6-8.4 mg/day (0.01-0.1 mg/kg
EFSA Journal 2012;10(5):2659 18

bw/day) for the elderly, respectively, contributing between 0.02-0.4% in adults and 0.02-0.3% in the
elderly to the estimated mean total anticipated exposure to plant sterols, respectively.
Two surveys conducted in Europe (Belgium and Ireland) indicated that high consumers of products
with added plant sterols already exceed the recommended intake of 3 g/day, with intakes ranging from
4.2-5.5 g/day at the 95th percentile (see section 2.9.2.2). Assuming a worst case scenario, namely that
the same consumers are also high consumers of alcoholic beverages containing stigmasterol-rich plant
sterols from the proposed use as food additive, this intake could potentially rise to 4.6-5.9 g/day.
The Panel noted that the mean estimated exposure resulting from the proposed use and use levels of
stigmasterol-rich plant sterols (0.6-11.4 mg/day) is well below the estimated mean daily plant sterol
intake from food and food supplements (as added nutrient) (1510-2450 mg/day) namely between 132
to 4000-fold lower on a total plant sterol basis.
3. BIOLOGICAL AND TOXICOLOGICAL DATA

previously been evaluated by several scientific authorities, including JECFA (2009), EFSA (2003,
2005a,b, 2006, 2007a,b,c) and the SCF, (2000, 2002a,b,c, 2003). The test articles used in the majority
of these studies were high-purity phytosterols and phytostanols (≥ 95%) and could be classified into 3
test articles as follows:
1. Phytosterol esters: Derived from vegetable oil distillates (soya bean) esterified with fatty acids from
sunflower oil. Main constituents of the oil were β-sitosterol (45-51%), stigmasterol (17-23%),
campesterol (6-15%);
2. Phytostanol-phytosterol mixtures: Derived from tall oil. Main constituents were β-sitosterol (40-
65%), β-sitostanol (16-31%), campesterol (6-5%), and campestanol (2-11%);
3. Phytostanol esters: Wood-derived mixtures consisting of 94% β-sitostanol and approximately 6%

campestanol and vegetable oil-derived mixtures consisting of 68% β-sitostanol and 32% campestanol.
The Panel considered that biological and toxicity data on phytosterols and their esters can be used to
evaluate the safety of stigmasterol-rich plant sterols, provided that the studies are performed with
phytosterol preparations containing also a well defined percentage of stigmasterol. The Panel
stigmasterol can also be used to judge the safety of the other sterols and stanols present in
stigmasterol-rich plant sterols, because these phytosterol preparations were recognised to contain also
other sterols and stanols and likely at levels even higher than present in the stigmasterol-rich plant
sterols.
Summarised in the sections below, are studies previously reviewed by JECFA at their 69th meeting
(JECFA, 2009) that employed a test material containing stigmasterol.
3.1. Absorption, distribution, metabolism and excretion

Given that phytosterols are plant sterols structurally similar to cholesterol, phytosterols may influence
the absorption, distribution, metabolism and excretion (ADME) of cholesterol. For this reason studies
on ADME characteristics of phytosterols often also quantify bioavailability of cholesterol and/or
compare the bioavailability of the phytosterols to that of cholesterol.
Animals
In rats administered phytosterols and phytostanols via gavage, cholesterol absorption was greater than
that of the individual phytosterols or phytostanols (Sanders et al., 2000; Hamada et al., 2006). At
24 hours after administration of 14C- and 3H-radiolabelled sterols or stanols to male and female CD
EFSA Journal 2012;10(5):2659 19

rats, cholesterol absorption was between 24 and 27% (Sanders et al., 2000). The absorption of
campesterol (13%) was greater than that of stigmasterol (4.3%) or β-sitosterol (4.0%) as well as β-
sitostanol and campestanol (1 to 2%). However, as biliary excretion was not taken into consideration,
the reported absorption figures are expected to be slight underestimates of total absorption. Among
thoracic-duct-cannulated male rats administered cholesterol and phytosterol emulsions, 59.2, 15.4, 3.3,
2.4, 0.5, and 0.3% of cholesterol, campesterol, brassicasterol, sitosterol, stigmasterol, and sitostanol
respectively, were recovered within the lymph (Hamada et al., 2006).
In rats, once absorbed, phytosterols (esterified or non-esterified) are transported in the blood by high-
density lipoproteins (HDL) and distributed to various organs and tissues (Moghadasian, 2000).
In animals given phytosterols, the highest concentrations of β-sitosterol and campesterol were detected
in the abdominal organs, small intestine, kidney, adrenal cortex, ovary and testis, and liver
(Bhattacharyya and Lopez, 1979; Moghadasian, 2000; Sanders et al., 2000). Campesterol was only
detected in the peripheral tissues, subcutaneous adipose tissue, muscle, skin, tendon, and aorta
following the dietary administration to rabbits for 10 weeks of a mixture of β-sitosterol, campesterol,
and stigmasterol providing approximately 600 mg plant sterols/kg bw/day (Bhattacharyya and Lopez,
1979). No stigmasterol was detected in any of the sampled organs or tissues or in the blood.
The plant sterols including stigmasterol and stanols can cross the blood-brain barrier as indicated
by their increased levels in the brain following 90-day feeding of Watanabe heritable hyperlipidemic
rabbits with these compounds (Fricke et al., 2007).
In rats absorbed phytosterols are transformed to metabolites identified as polar C21 bile acids and these
C21 bile acids were reported to be major metabolites of sitosterol (Boberg et al., 1990). Formation of
bile acids from sitosterol in bile-fistulated female Wistar rats was studied with use of [4-14C]-labelled
sitosterol and sitosterol labelled with 3H in specific positions. After intravenous or intraperitoneal
administration of [4-14C]- labelled sitosterol to female bile fistula rats, 7.2 +/- 1.1% (mean +/- S.E., n =
4) of the injected dose was recovered in bile after 24 hours. During the next 24 hours, an additional 6.2
+/- 1.2% was recovered. The authors indicated that this degree of excretion is in accordance with
previous investigations (Subbiah and Kuksis, 1973). 85 +/- 2% of the recovered radioactivity has
extraction properties similar to those of bile acids. The study also reported that considerably less C21
bile acids were formed from [4-14C]sitosterol in male than in female Wistar rats. Experiments with
isolated perfused liver gave direct evidence that the overall conversion of sitosterol into C21 bile acids
occurs in this organ (Boberg et al., 1990). The authors also indicated that the formation of C21-bile
acids may not be restricted to sitosterol as substrate. They showed that campesterol was also converted
into polar compounds with chromatographic properties identical to those of the metabolites of
sitosterol.
Another study (Sanders et al., 2000) in rats reported absorbed phytosterols and phytostanols are
predominantly excreted via the biliary route into the faece, in which 75-96% of phytosterols were
recovered within a 24-hour period following dosing.
Humans
Among healthy humans, approximately 55 to 60% of cholesterol is absorbed from the gastrointestinal
tract (Patel and Thompson, 2006), whereas less than 5% of total dietary phytosterols are absorbed
(Ling and Jones, 1995; Pegel, 1997; Ostlund et al., 2002; Rozner and Garti, 2006; Ellegård et al.,
2007). In 10 healthy men, the most extensively absorbed phytosterol over a 50 cm segment of the
upper jejunum was campesterol (9.6%) followed by stigmasterol (4.8%) and sitosterol (4.2%), while
cholesterol absorption averaged 33% in these subjects (Heinemann et al., 1993). In a crossover study,
the systemic absorption of 600 mg of lecithin-emulsified deuterated phytosterols or phytostanols
derived from soya beans following administration in 2 consecutive single-meal absorption tests
2 weeks apart, was investigated in 10 healthy humans (7 females, 3 males) (Ostlund et al., 2002).
Measurements made by a mass spectroscopic technique reported lower absorption values in
EFSA Journal 2012;10(5):2659 20

comparison to estimates from previous studies that utilized radioactive counting methods. The percent
absorption for campesterol, β-sitosterol, campestanol, and β-sitostanol was 1.9, 0.5, 0.2, and 0.04%,
respectively.
In humans, in contrast to animals, phytosterols are transported in the blood by low-density lipoproteins
(LDL); however, < 1% of the total plasma sterol concentration consists of phytosterols in healthy
adults (Moghadasian, 2000). According to Ling and Jones (1995), there is little information available
regarding the distribution of phytosterols in various tissues of the body in humans. Nguyen et al.
(1990) noted only trace levels of phytosterols in whole liver and liver microsomes of normal subjects.
Phytosterols cannot be endogenously synthesized in humans; thus, phytosterols within the body
originate solely from dietary sources (Ling and Jones, 1995; Calpe-Berdiel et al., 2009). In humans,
metabolism of β-sitosterol to pregnolenone, following the same pathways as cholesterol, was reported
to occur in the adrenal glands, testes, and placenta (Katan et al., 2003). Phytosterols are converted to
bile acids in the liver and the mechanism is speculated to involve initial hydroxylation at C21 and
subsequent beta-oxidation by peroxisomes or mitochondria (Lund et al., 1991).
It has been speculated that in the lower gastrointestinal tract, unabsorbed phytosterols may be
converted to coprostanol and coprostanone by the intestinal microflora (Moghadasian, 2000).
In a two-period, parallel dosing, randomised, placebo-controlled dietary study, the ingestion of control
margarine or test margarine product providing 8.6 g phytosterols/day (46% β-sitosterol, 26%
campesterol, 20% stigmastanol by w/w) in 24 healthy subjects (12/sex) for up to 28 days had no
significant effect on the faecal bacterial profile (Ayesh et al., 1999; Weststrate et al., 1999).
In humans, ingested phytosterols (~95%) are generally not absorbed and enter the colon (Ling and
Jones, 1995). They are eliminated, primarily unchanged, in the faeces, similar to rats (Weststrate et al.,
1999). In male volunteers, the biliary secretion rate of β-sitosterol was quicker (1.23 mg/hour) than
that of campesterol (0.76 mg/hour) (Sudhop et al., 2002b). In a female patient, trace amounts of
phytosterols absorbed into the plasma from the diet were reported to appear in the skin surface lipids
(Bhattacharyya et al., 1983).
Overall the Panel noted that despite the similarities in structure between phytosterols and cholesterol,
in comparison to cholesterol phytosterols are not readily absorbed from the gastrointestinal tract in
both rats and humans. Among healthy humans, less than 5% of total dietary phytosterols are absorbed.
Absorbed phytosterols and phytostanols are predominantly excreted as such or as bile acids via the
biliary route into the faeces. The Panel noted that stigmasterol-rich plant sterols are to be used in
alcohol rich cocktails but that no data are available on the effects of alcohol on the absorption,
distribution, metabolism or excretion of stigmasterol-rich plant sterols.
3.2. Toxicological data
3.2.1. Acute oral toxicity

No data on acute toxicity of stigmasterol-rich plant sterols or of related phytosterols were available for
evaluation.
3.2.2. Short-term and subchronic toxicity

In a subchronic toxicity study, Alpk:APfSD (Wistar-derived) rats (20/sex/group) were fed diets
containing phytosterol esters at levels of 0, 0.16, 1.6, 3.2 and 8.1% (w/w) in the diet (equivalent to
phytosterol concentrations of 0, 0.1, 1, 2 or 5%, respectively) for 90 days (Horner, 1997; Hepburn et
al., 1999). The study was performed under good laboratory practice (GLP). The phytosterols were
derived from a variety of common edible vegetable oil distillates (mainly soya bean) and re-esterified
with fatty acids from sunflower oil to increase their oil solubility. The phytosterol profile in the
mixture was mainly β-sitosterol (48.7%), stigmasterol (21.6%), and campesterol (25.8%), with minor
EFSA Journal 2012;10(5):2659 21

amounts of β-sitostanol (1.4%), brassicasterol (1.1%), unknowns (0.8%), and cholesterol (0.4%). The
consumption of phytosterol esters, phytosterols, and stigmasterol, on a body weight basis,
administered to male and female rats are summarised in Table 10.
Table 10: Dietary consumption of phytosterol esters, phytosterol, and stigmasterol (Hepburn et al.,
1999)
Dietary Mean consumption of Dietary Mean consumption (as Mean consumption as

percentage phytosterol esters1 percentage phytosterol) stigmasterol
of (g/kg bw/day) of (g/kg bw/day) (mg/kg bw/day)3
phytosterol phytosterols2
Males Females Males Females Males Females
esters (%) (%)1
0 (control) 0 0 0 (control) 0 0 0 0
0.16 0.1 0.1 0.1 0.1 0.1 17 19
1.6 1.3 1.5 1.0 0.8 0.9 174 195
3.2 2.6 2.9 2.0 1.6 1.8 347 390
8.1 6.6 7.4 5.0 4.1 4.6 879 987
1
Dietary intake was only reported for the high-dose group; therefore, intakes at lower doses were estimated based on the
reported values for the dietary concentration and intakes reported for the high-dose group.
2
Calculated as g phytosterol esters/kg bw/day x 62% (reported phytosterol content)
3
Calculated as g phytosterols/kg bw/day x 21.6% (reported stigmasterol content)
During the study there were no effects on mortality, clinical signs of toxicity, food consumption or
utilization, body weights or organ weights that were attributed to the consumption of phytosterol
esters. Minor statistically significant changes were noted in some haematological and clinical
chemistry parameters (reduced platelet counts in females at all doses and in males at dietary levels of
1.6 and 3.2%; marginally reduced prothrombin time in females of all dose groups and activated partial
thromboplastin time in males at the 3.2% and 8.1% dietary level; minor increases in plasma albumin,
phosphorous, and magnesium levels and slight increases in several serum and plasma enzymes in
males and/or females at dietary phytosterol ester levels of 1.6, 3.2, or 8.1%). Due to the absence of
organ weight changes or macroscopic or histopathological findings attributed to the consumption of
phytosterol esters, the changes in haematological and clinical chemistry parameters were considered
by the authors to be of no toxicological significance. Based on the lack of compound-related changes,
the NOAEL was determined to be 8.1% phytosterol esters in the diet, the highest dose tested
(equivalent to approximately 6.6 g phytosterol esters/kg bw/day or 4.1 g phytosterols/kg bw/day).
Based on the content of stigmasterol in the phytosterol ester mixture (21.6%), the authors derived a
NOAEL expressed as stigmasterol to be equivalent to approximately 879 mg/kg bw/day, the highest
dose level tested.
Kim et al. (2002) likewise evaluated the subchronic toxicity of plant sterol esters following daily oral
gavage administration to male and female Sprague-Dawley rats (10 to 16/sex/group) at dose levels of
0, 1, 3 and 9 g phytosterol esters/kg bw/day for 90 days. The study was performed according to OECD
guidelines and under GLP. The plant sterols were isolated from soy bean and subsequently esterified
with unsaturated fatty acids (oleic acid ≥ 70%) from olive oil to increase their oil solubility. The plant
sterol esters were composed of 49.4% β-sitosterol, 27.9% campesterol, 18.5% stigmasterol, and others.
The doses administered to animals, calculated as phytosterols and stigmasterol, are presented in Table
11. At the end of the 13-week treatment period 10 rats/sex/group were necropsied while 6 rats/sex in
the negative control and highest dose groups were necropsied following a 4-week recovery period.
Changes noted in haematology or clinical chemistry parameters were not considered of toxicological
significance as they were either within normal levels established by historical controls, not dose-
dependent, or occurred at a very low incidence. Thus it was concluded by the authors that there were
no adverse effects on mortality, clinical signs of toxicity, food and water consumption,
ophthalmoscopy, urinalysis, haematology, serum biochemistry, necropsy findings and organ weights
in any treatment group. Significant body weight gain reductions, independent of food consumption,
were noted in both sexes at the high-dose when compared to the negative controls. These differences
EFSA Journal 2012;10(5):2659 22

were no longer present at the end of the recovery period. An increased incidence of mononuclear cell
infiltration in the heart was observed in males of the 9 g/kg bw/day group. Due to the suppression of
body weight gains in both sexes and the observed infiltration of mononuclear cells in the heart in
males at a dose level of 9 g phytosterol esters/kg bw/day, the NOAEL for both sexes was considered
to be 3 g phytosterol esters/kg bw/day. As demonstrated in Table 11, the NOAEL is equivalent to
1.88 g phytosterols/kg bw/day or approximately 350 mg stigmasterol/kg bw/day.
Table 11: Dose levels of phytosterol esters, free phytosterols, and stigmasterol in a 90-day
subchronic study in rats (Kim et al., 2002)
Dose as phytosterol esters Dose as phytosterols Dose as stigmasterol

(g/kg bw/day) (g/kg bw/day)1 (g/kg bw/day)2
0 (control) 0 0
1 0.63 0.12
3 1.88 0.35
9 5.63 1.04
1
Free phytosterols calculated assuming the conversion factor for phytosterol esters to phytosterols is 1.6 (Hepburn et al.,
1999).
2
Calculated as g phytosterols/kg bw/day x 18.5% (reported stigmasterol content).
3.2.3. Genotoxicity
Phytosterol mixtures, phytosterol esters, and phytosterol oxide demonstrated a lack of mutagenic or
genotoxic activity in a battery of in vitro and in vivo assays (Table 12). Additional in vitro and in vivo
assays on the potential mutagenicity and genotoxicity of phytosterol-phytostanol mixtures and
phytostanol esters were evaluated by JECFA at their 69th meeting, which further corroborated the lack
of mutagenic or genotoxic activity of these compounds (JECFA, 2009). The Panel noted that
genotoxicity assays on stigmasterol-rich plant sterols were not available, but that genotoxicity assays
performed with phytosterols containing up to 18% stigmasterol have been performed (Table 12) and
can be used to evaluate the genotoxicity of stigmasterol-rich plant sterols. Based on these data the
Panel concluded that stigmasterol-rich plant sterols are not of concern with respect to genotoxicity.
Table 12: Overview of genotoxicity studies conducted with phytosterols (adapted from JECFA,
2009)
Test
End-point Test system Concentration/dose Result Reference
material
In vitro
Salmonella
typhimurium
Reverse Wolfreys and
TA98, TA100, Phytosterol 5 to 5000 μg/plate Negativeb
mutation Hepburn (2002)
TA1535 and mixturea
TA1537
S. typhimurium
TA98, TA100,
TA1535 and
Reverse Phytosterol Wolfreys and
TA1537;  50 to 5000 μg/plate Negativeb
mutation estersa Hepburn (2002)
Escherichia coli
WP2 uvrA
(pKM101)
S. typhimurium
Reverse TA98, TA100, Phytosterol
1.6 to 5000 μg/plate Negativeb Lea et al. (2004)
mutation TA102, TA1535 oxide
and TA1537 concentrate
Human peripheral
Chromosom Phytosterol Wolfreys and
blood 40 to 160 μg/ml Negativeb
al aberration mixturea Hepburn (2002)
lymphocytes
EFSA Journal 2012;10(5):2659 23

Test
End-point Test system Concentration/dose Result Reference
material
Human peripheral
Chromosom Wolfreys and
blood Phytosterol 25 to 200 μg/ml Negativeb
al aberration Hepburn (2002)
lymphocytes estersa
Human peripheral Phytosterol
Chromosom Wolfreys and
blood Oxide 131.1 to 500 μg/ml Negativeb
al aberration Hepburn (2002)
lymphocytes concentrate
Mouse lymphoma
Gene L5178Y cells, Phytosterol 5 to 80 μg/ml Negativeb
Lea et al. (2004)
mutation Tk+/- locus estersa
Clastogenicit
Human peripheral Phytosterol
y
blood oxide Up to 625 μg/ml Negativeb Lea et al. (2004)
(micronucle
lymphocytes concentrate
us induction)
In vivo
Micronucleu Male rats, bone 500 to 2000 mg/kg Wolfreys and

Phytosterol Negative
s induction marrow bw/day for 2 days Hepburn (2002)
estersc
Unscheduled
Phytosterol 800 or 2000 mg/kg Wolfreys and
DNA Male rats, liver Negative
estersc bw Hepburn (2002)
synthesis
Triols and
epoxides of Epoxides: up to
Abramsson-
Micronucleu a mixture of 67 mg/kg bw;
Male mice, blood Negative Zetterberg et al.
s induction β- sitosterol triols: up to
(2007)
and 9.4 mg/kg bw
campesterol
Sister
Male NIH mice of 200, 400, 600, or Paniagua- Pérez et
chromatid Negative
8 weeks of age β- sitosterol 1000 mg/kg bw al. (2005)
exchange
Cellular
proliferation Negative
8 weeks of age β- sitosterol 1000 mg/kg bw al. (2005)
kinetics
Mitotic Male NIH mice of 200, 400, 600, or Paniagua- Pérez et
Negative
index 8 weeks of age β- sitosterol 1000 mg/kg bw al. (2005)
Micro-
nucleated
polychromat β- sitosterol Negative
8 weeks of age 1000 mg/kg bw al. (2005)
ic
erythrocytes
a
Phytosterol composition: 26.7% campesterol, 17.7% stigmasterol, 51% β-sitosterol.
b
With and without metabolic activation (S9 mix from Aroclor 1254–induced rat liver).
c
Phytosterol composition: 28.1% campesterol, 18.7% stigmasterol, 45.5% β-sitosterol.
3.2.4. Chronic toxicity and carcinogenicity

No chronic toxicity or carcinogenicity studies were evaluated by JECFA during their 69th meeting
(JECFA, 2009). Additionally, a search of the available public literature did not identify any chronic
toxicity or carcinogenicity studies conducted with phytosterol or phytostanol esters or free
phytosterols or phytostanols published subsequent to JECFA’s evaluation.
3.2.5. Reproductive and developmental toxicity

The reproductive toxicity of plant phytosterol esters derived from vegetable oils was evaluated in a
2-generation reproductive toxicity study in Wistar rats (Waalkens-Berendsen et al., 1999). Rats aged
4-5 weeks (117 males and 116 females) comprised the F0 generation and were fed diets containing 0
(control), 1.6, 3.2, or 8.1% phytosterol esters (corresponding to 1.0, 2.0, or 5.0% phytosterols,
EFSA Journal 2012;10(5):2659 24

respectively) for a 10-week premating period. The phytosterol esters were composed mainly of β-
sitosterol (48.7%), campesterol (25.8%), and stigmasterol (21.6%). The intake of phytosterols esters,
phytosterols, and stigmasterol, on a body weight basis, for the F0 and F1 generations are presented in
Table 13.
Table 13: Dose levels of phytosterol esters, phytosterols, and stigmasterol in a 2-generation
reproductive toxicity study in rats (Waalkens-Berendsen et al., 1999)
Concentration of phytosterol esters in diet (%)

1.6 % (1%)a 3.2 % (2%)a 8.1 % (5%)a
Phyto Phyto Stigma Phyto Phyto Stigma Phyto Phyto Stigma
sterol sterolsb sterolc sterol sterolsb sterolc sterol sterolsb sterolc
esters esters esters
(g/kg bw/day)
F0 generation
Premating
(1-10 wk) 0.9-0.5 0.6-0.3 0.1-0.06 1.8-1.0 1.1-0.6 0.2-0.1 4.4-2.7 2.8-1.7 0.6-0.4
males
Premating
(1-10 wk) 1.1-0.6 0.7-0.4 0.1-0.08 2.1-1.3 1.3-0.8 0.3-0.2 5.6-3.4 3.5-2.1 0.8-0.5
females
Gestation
0.8 0.5 0.1 1.5 0.9 0.2 3.8 2.4 0.5
(wk 1)
Gestation
0.8 0.5 0.1 1.5 0.9 0.2 3.9 2.4 0.5
(wk 2)
Gestation
0.5 0.3 0.07 1.0 0.6 0.1 2.5 1.6 0.3
(wk 3)
Lactation
1.1 0.7 0.1 2.1 1.3 0.3 5.7 3.6 0.8
(wk 1)
Lactation
1.8 1.1 0.2 3.5 2.2 0.5 9.1 5.7 1.2
(wk 2)
Lactation
2.3 1.4 0.3 4.5 2.8 0.6 12.6 7.9 1.7
(wk 3)d
F1 generation
Premating
(1-10wk) 1.3-0.6 0.8-0.4 0.2-0.1 2.6-1.1 1.6-0.7 0.4-0.1 6.2-2.8 3.9-1.8 0.8-0.4
males
Premating
(1-10wk) 1.3-0.6 0.8-0.4 0.2-0.1 2.6-1.2 1.6-0.8 0.4-0.2 6.5-3.3 4.1-2.1 0.9-0.4
females
Gestation
0.7 0.4 0.1 1.4 0.9 0.2 3.6 2.3 0.5
(wk 1)
Gestation
0.7 0.4 0.1 1.4 0.9 0.2 3.6 2.3 0.5
(wk 2)
Gestation
0.5 0.3 0.1 0.9 0.6 0.1 2.3 1.4 0.3
(wk 3)
Lactation
1.0 0.6 0.1 2.1 1.3 0.3 4.8 3.0 0.6
(wk 1)
Lactation
1.7 1.1 0.2 3.4 2.1 0.5 8.5 5.3 1.1
(wk 2)
Lactation
2.1 1.3 0.3 4.4 2.8 0.6 10.9 6.8 1.5
(wk 3)d
a
Number in parentheses represents equivalent concentrations of phytosterols (%) in the diet.
b
Calculated as g phytosterol esters/kg bw/day x 62% (reported phytosterol content).
c
Calculated as g phytosterols/kg bw/day x 21.6% (reported stigmasterol content).
d
As pups started to eat around postnatal Day 14, test substance intake in lactation week 3 is an unrealistic figure.
EFSA Journal 2012;10(5):2659 25

At the end of the pre-mating period, the rats were pair-mated, and the females were checked every
morning for 3 weeks for evidence of mating via the presence of sperm cells in a vaginal smear. Day 0
of gestation was considered to be the day that copulation was confirmed. The morning after parturition
was defined as postnatal day (PND) 1. The dams from the F0 generation and their litters (F1
generation) were held within the same cages during lactation. On PND 21, F1 pups were weaned and
randomly selected (28/sex) from each diet group to be fed the same pre-mating diet at the same dose
levels as their parents. The same procedures for pre-mating, mating, gestation, and lactation as in the
F0 generation were followed for the F1 generation. F1 generation litters (F2 generation) were reared in
an identical manner to those of the F0 generation.
There were no compound-related mortalities or differences in the appearance, general behaviour, and
general condition of the rats consuming phytosterol esters in comparison to the control rats for all
generations. Slight, but statistically significant increases in food consumption were observed among F0
males fed 1.6% phytosterol esters, F1 males fed 8.1% phytosterol esters, and F0 females fed 8.1%
phytosterol esters during the pre-mating phase of the study. Although significant decreases in body
weights were observed among F0 and F1 males at various points during the pre-mating phase in the
groups fed 1.6 or 8.1% phytosterol esters, the changes in food consumption and body weights were not
consistent throughout this phase of the study and therefore were not attributed to the consumption of
phytosterol esters. Similarly, food consumption was significantly decreased among F0 and F1 females
at various points during gestation, but that was not associated with any changes in body weights. No
changes in food consumption or body weights were observed in either generation during the lactation
phase of the study. Moreover, no differences were observed between the treatment or control groups in
organ weight, macroscopic observations at necropsy, and histopathological findings. There were no
effects on fertility and reproductive parameters including sexual maturity, oestrous cycle length, pre-
coital time, and the histopathology of reproductive tissues. There were no developmental or
reproductive effects and no estrogenic activity reported in the study. Due to the lack of significant
changes in any of the other parameters tested, the observed changes in food consumption patterns and
body weights were not considered toxicologically relevant. The authors determined the NOAEL for
phytosterol esters to be 8.1% in the diet for phytosterol esters, the highest dose tested. This
concentration is equivalent to 3.3-6.5 g phytosterol esters/kg bw/day during pre-mating
(approximately 2.1-4.1 g phytosterols/kg bw/day or 400-900 mg stigmasterol/kg bw/day) or 2.5-9.1 g
phytosterol esters/kg bw/day during gestation (approximately 1.4-5.7 g phytosterols/kg bw/day or
300-1200 mg stigmasterol/kg bw/day). The authors concluded that a dose of 2.5-9.1 g phytosterol
esters/kg bw/day or 1.54-5.62 g phytosterols/kg bw/day (approximately 335-1219 mg stigmasterol/kg
bw/day) (dependent on the phase of the study) was considered to be the NOAEL following daily oral
administration of phytosterol esters for two successive generations. The Panel agreed with this
NOAEL and noted that it was the highest dose levels tested.
3.2.6. Other studies

In humans ingestion of phytosterols can lead to reduced serum levels of carotenoids (Plat and
Mensink, 2005; JECFA, 2009).
Plat and Mensink (2005) indicated that because plant stanol esters lower the solubility of cholesterol in
mixed micelles, it is reasonable to suggest that the incorporation, and consequently absorption, of
other lipophilic dietary substances (such as fat-soluble antioxidants) in humans would also be reduced.
In a plant stanol esters human intervention trial, a reduction in serum β-carotene concentrations was
found of approximately 25% after the consumption of 3.8 to 4.0 g/day plant stanols as fatty acid esters
for 8 weeks (Plat and Mensink, 2001). Concentrations of the other lipophilic hydrocarbon carotenoids
(α-carotene and lycopene) were also lowered, whereas concentrations of the more polar oxygenated
carotenoids (lutein/zeaxanthin and β-cryptoxanthin) and tocopherols were unchanged.
Also, in another study in which volunteers were given once a day 2.5 g stanols or 2.5 g stanols divided
across 3 meals, it was found that serum hydrocarbon carotenoid concentrations in particular were
lowered during plant stanol esters consumption (Plat et al., 2000).
EFSA Journal 2012;10(5):2659 26

The SCF (SCF 2002a) also considered that plant sterols and stanols interfere with the absorption of
carotenoids as deduced from the reduction of carotenoid blood level and that the level of other fat-
soluble vitamins, such as vitamin E and tocopherols, may also be affected, although to a lesser extent.
The decreases in blood carotenoids appear to plateau when doses of sterols or stanols reached 2.2
g/day and amounted to a reduction of 33% after one-year consumption of an enriched margarine
providing 3 g/day. The SCF indicated that the consequences of such a persistent decrease of blood
concentrations of carotene on human health are largely unknown.
As consumption of phytosterols results in decreased cholesterol absorption, reduction of the

absorption of carotenoids and lipid-soluble vitamins also may occur. Several clinical trials were
conducted with endpoints related to the effects of phytosterols or phytosterol esters on the circulating
lipid soluble vitamin and carotenoid levels (Saito et al., 2006; Colgan et al., 2004; Ntanios et al., 2002
Judd et al., 2002; Noakes et al., 2005; Plana et al., 2008; Hansel et al., 2007; Polagruto et al., 2006;
Devaraj et al., 2006; Quílez et al., 2003; Hendriks et al., 2003; Raeini- Sarjaz et al., 2002; Richelle et
al., 2004; Gonçalves et al., 2007; Thomsen et al., 2004; Korpela et al., 2006). These studies generally
applied phytosterols or phytosterol esters at dose levels of 1.2 up to 4 g/day for 3-8 weeks up to one
year.
These investigations did not reveal any clinical study in which a significant decrease in circulating
levels of fat-soluble vitamins was reported following consumption of up to 3.6 g/day of phytosterols
for periods of up to 52 weeks in healthy subjects or hypercholesterolaemic subjects (Judd et al., 2002;
Ntanios et al., 2002; Raeini-Sarjaz et al., 2002; Hendriks et al., 2003; Quílez et al., 2003; Colgan et al.,
2004; Thomsen et al., 2004; Korpela et al., 2006; Saito et al., 2006; Polagruto et al., 2006; Devaraj et
al., 2006). Likewise, there were no significant reductions in absolute or lipid-adjusted plasma/serum
carotenoid concentrations (Judd et al., 2002; Raeini-Sarjaz et al., 2002; Quílez et al., 2003; Colgan et
al., 2004; Thomsen et al., 2004; Saito et al., 2006; Polagruto et al., 2006; Devaraj et al., 2006; Hansel
et al., 2007; Gonçalves et al., 2007; Plana et al., 2008). Of the reviewed studies, a single study reported
decreased bioavailability of β-carotene and α-tocopherol (Richelle et al., 2004), while 5 studies
reported decreased circulating levels of carotenoids (Judd et al., 2002; Ntanios et al., 2002; Hendriks
et al., 2003; Noakes et al., 2005; Polagruto et al., 2006). It should be noted that there were no adverse
biological or physiological effects observed in the studies that reported decreases in plasma carotenoid
levels or bioavailability. Furthermore, in hypercholesterolaemic males consuming 1.92 g
phytosterols/day for 21 days and provided with diets formulated to meet the Canadian Recommended
Nutrient Intake, there was no effect on plasma carotenoid levels (Raeini- Sarjaz et al., 2002).
The Panel noted however that the studies described above were not designed to test the safety of the
phytosterol or phytosterol esters.
In another study, it was investigated whether plant sterol enriched novel foods might adversely affect
vascular function in human volunteers receiving statin medication (Kelly et al., 2011). In this study the
characterised. This parameter was indicated to reflect alterations in the microcirculation. The studies
included three randomized groups that were studied at baseline and after 85 weeks of intervention.
Group one (n = 11) consumed plant sterol enriched margarine (30 g margarine containing 2.5 g plant
sterols/day), the second (n = 8) plant stanol enriched margarine (30 g margarine containing 2.5 g plant
stanols/day), and the control group (n = 11) consumed non enriched margarine (30 g/day). Plant
sterols and –stanols were provided as fatty acid esters. The composition of the plant sterol and –stanol
samples was not provided. Serum cholesterol-standardized campesterol and sitosterol concentrations
increased (p< 0.001) in the sterol group, while decreasing non-significantly in the stanol group. Serum
LDL-cholesterol concentrations decreased significantly in both the plant sterol (p = 0.016) and –stanol
groups (p = 0.018) but showed an increase in the controls. The mean change in venular diameters for
the plant sterol group (2.3 ± 3.1 μm), plant stanol groups (−0.8 ± 3.4 μm) and control group
(−0.8 ± 5.1 μm) did not reach significance but the change in cholesterol-standardized campesterol
concentrations correlated positively with the change in venular diameter independent of changes in
serum LDL-cholesterol concentrations (r = 0.39, N = 30, p = 0.033). Increased serum campesterol
EFSA Journal 2012;10(5):2659 27

concentration correlated positively with increased retinal venular diameter, independent from changes
in serum LDL-cholesterol concentrations. The authors indicated that their results may constitute an
explanation for the suggested effects of plant sterols on vascular function, although they also indicated
that this novel finding needs confirmation and further study. The Panel noted that the study does not
provide sufficient evidence to allow a final assessment of the atherogenic potential of increased
phytosterol serum concentrations due to the intake of plant sterol enriched food. Measured effects
were marginally and it has to be taken into account that volunteers of the studies were undergoing
statin treatment and might be at higher risk for venular changes due to vascular predamage.
4. DISCUSSION
The present opinion evaluates the safety of the use of stigmasterol-rich plant sterols in ready-to-freeze
alcoholic cocktails as a stabiliser. The preparation is requested to be used as a stabiliser, ice nucleating
agent, to generate and maintain the presence of ice dispersions within a frozen alcoholic cocktail.
previously been evaluated by several scientific authorities, including the SCF (2000, 2002a,b,c, 2003),
JECFA (2009) and EFSA (EFSA, 2003, 2005a,b, 2006, 2007a,b,c).
The Panel considered that biological and toxicological data on phytosterols and their esters can be
used to evaluate the safety of stigmasterol-rich plant sterols provided that the studies are performed
with phytosterol preparations containing also a well defined percentage of stigmasterol. The Panel
stigmasterol can also be used to judge the safety of the other sterols and stanols present in
stigmasterol-rich plant sterols, because these phytosterol preparations were recognised to contain also
other sterols and stanols and likely at levels even higher than present in the stigmasterol-rich plant
sterols.
The SCF (SCF, 2002a) considered that plant sterols and stanols interfere with the absorption of
carotenoids as deduced from the reduction of carotenoid blood levels and moreover that the levels of
other fat-soluble vitamins, such as vitamin E and tocopherols, may also be affected, although to a
lesser extent. The decreases in blood carotenoids appear to plateau when doses of sterols or stanols
reached 2.2 g/day and amounted to a reduction of 33% after one-year consumption of an enriched
margarine providing 3 g/day. The SCF indicated that the consequences of such a persistent decrease of
blood concentrations of carotene on human health are largely unknown.
esters are approved for use in various foods within the EU at levels resulting in intake of up to 3 g/day.
Specifications of phytosterol and phytostanols for the addition to foods and food ingredients are
established in each Commission Decision authorising its addition in a given food and food ingredient.
The Panel noted that in all Commission Decisions, published until now the maximum permitted
stigmasterol content of phytosterol/phytostanol ingredients is 30%; therefore, based on a level of
3 g/day the maximum stigmasterol intake in foods containing phytosterols should not exceed
900 mg/day equivalent to 15 mg/kg bw/day for a 60 kg person.
The safety of phytosterols, phytostanols, and their esters was also evaluated by JECFA at their 69th
meeting (JECFA, 2009). Based on the similar metabolic profiles among phytosterols and phytostanols
and their respective esters, JECFA established a group ADI of 0-40 mg phytosterols/kg bw/day, based
on an overall NOAEL of 4200 mg/kg bw/day from the combined evidence from several short-term
(90-day) toxicity studies and an uncertainty factor of 100 to account for inter and intra-species
differences. Based on a 60 kg individual, the maximum daily intake would be equivalent to 2.4 g
phytosterols/day. Assuming that phytosterol-containing soya bean extracts contain up to 23%
stigmasterol (JECFA, 2009), the ADI for stigmasterol derived from this group ADI for phytosterols
would be 0-9.2 mg/kg bw/day.
EFSA Journal 2012;10(5):2659 28

Less than 5% of dietary phytosterols, phytostanols, and their esters are absorbed by the gastrointestinal
tract of rats and human. Studies examining the absorption of individual phytosterols/phytostanols have
demonstrated differences in the percentage absorbed, with the absorption of campesterol being greater
than that of stigmasterol or β-sitosterol, β-sitostanol, or campesterol. These differences in absorption
among phytosterols as observed in the animal and human studies may be attributed to structural
differences in the side chains (i.e., absorption of campesterol > stigmasterol > sitosterol) (Heinemann
et al., 1993; Ling and Jones, 1995; Igel et al., 2003).
Following absorption, phytosterols/phytostanols are transported in the serum via HDLs in rats and
LDLs in humans to various organs and tissues; however < 1% of the total plasma sterol concentration
consists of phytosterols in healthy humans. In the liver, phytosterols may be converted to bile acids.
As the majority (>95%) of phytosterols are not absorbed in the gastrointestinal tract, phytosterols enter
the colon intact and are rapidly excreted in the faeces. Absorbed phytosterols and phytostanols are
predominantly excreted as such or as bile acids via the biliary route into the faeces. The metabolic fate
of phytosterols, phytostanols, and their esters is similar between rats and humans, thereby making the
rat an appropriate laboratory model to characterise the safety of phytosterols in humans. Furthermore,
as the individual plant sterols are handled in a similar manner, data conducted with a mixture of
phytosterols, phytostanols, and their esters are applicable to the safety of an individual phytosterol,
like stigmasterol.
The Panel noted that stigmasterol-rich plant sterols are to be used in alcohol rich cocktails but that no
data are available on the effects of alcohol on the absorption, distribution, metabolism or excretion of
stigmasterol-rich plant sterols.
No evidence of mutagenicity or genotoxicity of phytosterols or phytosterol esters was observed in a

battery of in vitro and in vivo assays performed with phytosterol mixtures containing 18% stigmasterol
or with mixtures containing structurally related phytosterols. Based on these data the Panel concluded
that stigmasterol-rich plant sterols are not of concern with respect to genotoxicity.
Toxicity studies on stigmasterol-rich plant sterols were not provided and studies on phytosterols and
phytosterol esters were limited to 90-day subchronic toxicity studies in rats (Horner, 1997; Hepburn et
al., 1999; Kim et al., 2002) and a 2-generation reproductive toxicity study in rats (Waalkens-
Berendsen et al., 1999). In one of the 90-day studies in rats dietary administration of up to 8.1%
phytosterol esters (equivalent to 5% phytosterols, 4.1 g phytosterols/kg bw/day, or 879 mg
stigmasterol/kg bw/day) did not result in any toxicologically relevant findings and represented the
NOAEL (Horner, 1997; Hepburn et al., 1999).
In the 90-day study reported by Kim et al. (2002) due to reduced body weight gains in both sexes and
the observed infiltration of mononuclear cells in the heart in males at a dose level of 9 g phytosterol
esters/kg bw/day, the NOAEL for both sexes was considered to be 3 g phytosterol esters/kg bw/day,
equivalent to 1.88 g phytosterols/kg bw/day or approximately 350 mg stigmasterol/kg bw/day. In the
2-generation reproductive toxicity study in rats the authors determined the NOAEL for phytosterol
esters to be 8.1% in the diet for phytosterol esters, the highest dose tested (Waalkens-Berendsen et al.,
1999). This concentration is equivalent to a dose of 2.5-9.1 g phytosterol esters/kg bw/day or 1.54-
5.62 g phytosterols/kg bw/day (approximately 335-1219 mg stigmasterol/kg bw/day) dependent on the
phase of the study.
No chronic toxicity, carcinogenicity or prenatal developmental toxicity studies conducted with

phytosterols, phytostanols, and their esters were identified.
In a study with human volunteers receiving statin medication it was investigated whether plant sterol
enriched novel foods might adversely affect vascular function (Kelly et al., 2011). In this study the
characterised. This parameter was indicated to reflect alterations in the microcirculation. The authors
indicated that their results may constitute an explanation for the suggested effects of plant sterols on
EFSA Journal 2012;10(5):2659 29

vascular function, although they also indicated that this novel finding needs confirmation and further
study. The Panel noted that the study does not provide sufficient evidence to allow a final assessment
of the atherogenic potential of increased phytosterol serum concentrations due to the intake of plant
sterol enriched food. Measured effects were marginally and it has to be taken into account that
volunteers of the studies were undergoing statin treatment and might be at higher risk for venular
changes due to vascular predamage.
The Panel considered that the overall NOAEL derived from the 90-day subchronic toxicity studies in
rats (Horner, 1997; Hepburn et al., 1999; Kim et al., 2002) and the 2-generation reproductive toxicity
study in rats (Waalkens-Berendsen et al., 1999) amounts to 2.5-6.6 g phytosterol esters/kg bw/day,
1.54-4.1 g phytosterols/kg bw/day and 335-900 mg stigmasterol/kg bw/day.
Given the absence of chronic toxicity, carcinogenicity or prenatal developmental toxicity studies the
Panel could not establish an ADI for stigmasterol-rich plant sterols.
performed long-term (chronic) intake assessment for the following population groups only: adults and
the elderly.
The estimated mean stigmasterol-rich plant sterol exposure from the proposed use as stabiliser ranges
from 0.6-11.4 mg/day (0.01-0.2 mg/kg bw/day) for adults and 0.6-8.4 mg/day (0.01-0.1 mg/kg
bw/day) for the elderly. The ranges for the 95th percentile exposures are 24-443.3 mg/day
(0.4-7.4 mg/kg bw/day) for adults and 28.2-192.6 mg/day (0.5-3.2 mg/kg bw/day) for the elderly. The
Panel noted that total exposure to plant sterols from all sources (i.e. from new applications, from
natural sources and added as novel food ingredient) was estimated at an average daily intake of
approximately 2770 mg/day (46 mg/kg bw/day) for both adults and the elderly.
The overall mean and 95th percentile exposures to stigmasterol-rich plant sterols resulting from the
proposed uses and use levels were 0.01-0.2 mg/kg bw/day and 0.4-7.4 mg/kg bw/day, respectively.
Using the lowest NOAEL values from the available toxicity studies of 1.54 g phytosterols/kg bw/day
and 335 mg stigmasterol/kg bw/day the Panel calculated that the Margin of Safety (MOS) values
amount to 7700-154 000 at the mean and 208-38500 at the 95th percentile for the phytosterols and to
1675-33 500 at the mean and 45-838 at the 95th percentile for stigmasterol.
Taking into account:
- that the mean estimated exposure resulting from the proposed use and use levels of
stigmasterol-rich plant sterols (0.6-11.4 mg/day) is 132 to 4000-fold below the estimated mean
daily plant sterol intake from food and food supplements (as added nutrient) (1510-2450
mg/day).
- that the estimated exposure resulting from the proposed use and use levels of stigmasterol-
rich plant sterols amounts to < 0.5 % at the mean and <15% at the 97th percentile exposure of
the estimated exposure of people using phytosterols to lower their cholesterol levels at dose
levels up to 3 g/day,
- that the bioavailability of the phytosterols and phytostanols in rat is somewhat higher than
that in human
- that the predicted exposures to stigmasterol-rich plant sterols are arguably large over-
estimations for frozen cocktail products, and provide a worst-case scenario for potential
exposure, and
- the available long-term human data,
EFSA Journal 2012;10(5):2659 30

the Panel considered these MOS values adequate. Thus, the Panel concluded that the proposed use and
use levels of stigmasterol-rich plant sterols would not be of safety concern.
CONCLUSIONS
The Panel concluded that the MOS between the lowest NOAEL values from the available toxicity
studies and the exposure resulting from intake of stigmasterol-rich plant sterols resulting from the
proposed uses and use levels is adequate.
The Panel concluded that the proposed use and use levels of stigmasterol-rich plant sterols in ready-to-
freeze alcoholic cocktails as a stabiliser would not be of safety concern.
DOCUMENTATION PROVIDED TO EFSA

1. Application for the approval of Stigmasterol-Rich Plant Sterols. Regulation (EC) No. 1333/2008
of the European Parliament and Council of 16 December 2008 on food Additives. February 2011.
Submitted by Diageo plc. Additional information on 6th January 2012. 9th March 2012, 13th April
2012 and 27th April 2012.
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EFSA Journal 2012;10(5):2659 38

GLOSSARY [AND/OR] ABBREVIATIONS

ADI Acceptable Daily Intake
ADME Absorption, distribution, metabolism and excretion
ANS Scientific Panel on Food Additives and Nutrient Sources added to Food
CFU Colony forming unit
EFSA European Food Safety Authority
EC European Commission
FAME Fatty acid methyl esters
FSANZ Food Standards Australia New Zealand
GC Gas chromatography
GLP Good laboratory practice
GRAS Generally recognized as safe
GRN GRAS Registry Number
HDL High density lipoproteins
ICP-MS Inductively coupled plasma mass spectrometry
ISO International Organization for Standardization
JECFA Joint FAO/WHO Expert Committee on Food Additives
LDL Low-density lipoproteins
MOS Margin of safety
NDA EFSA Panel on Dietetic Products, Nutrition and Allergies
NOAEL No-Observed-Adverse-Effect Level
PND Postnatal day
SCF Scientific Committee for Food
SODD Soybean oil deodorizer distillate
EFSA Journal 2012;10(5):2659 39

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EFSA Journal 2012;10(5):2659

Scientific Opinion on the safety of stigmasterol-rich plant sterols as food

European Food Safety Authority (EFSA), Parma, Italy

© European Food Safety Authority, 2012

EFSA Journal 2012;10(5):2659 2

EFSA Journal 2012;10(5):2659 3

No chronic toxicity, carcinogenicity or developmental toxicity studies conducted with phytosterols,

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Taking into account:

- the available long-term human data,

the Panel considered these MOS values adequate.

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EFSA Journal 2012;10(5):2659 6

BACKGROUND AS PROVIDED BY THE EUROPEAN COMMISSION

TERMS OF REFERENCE AS PROVIDED BY THE EUROPEAN COMMISSION

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2.1. Identity of the substances

Table 1: Identity of the major sterols present in stigmasterol-rich plant sterols

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between 162 and 170 °C.

Figure 1: Structural formulae of major sterols in stigmasterol-rich plant sterols

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JECFA’s Specifications as proposed

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Table 3: Microbiological specifications for stigmasterol-rich plant sterols as proposed by the

Test Specification Method of analysis

CFU - colony forming unit

2.3. Manufacturing process

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2.4. Methods of analysis in food

2.5. Reaction and fate in food

2.6. Case of need and proposed uses

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2.6.1. Proposed use levels of stigmasterol-rich plant sterols

Table 4: Proposed use levels of stigmasterol-rich plant sterols by the applicant.

Proposed use level

2.7. Information on existing authorisations and evaluations

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Regulation (EU) Nº 1169/20117 ammending Commission Regulation (EC) Nº 608/20048 concerning

EFSA Journal 2012;10(5):2659 14

2.8. Food consumption data used for exposure assessment

2.8.1. EFSA Comprehensive European Food Consumption Database

Population Age range Countries with chronic food consumption

EFSA Journal 2012;10(5):2659 15

2.9. Exposure assessment

Table 6 presents a summary of anticipated exposure to stigmasterol-rich plant sterols in these 2

Table 6: Summary of anticipated exposure to stigmasterol-rich plant sterols in 2 population groups

2.9.1. Contributing food groups

% contribution to total exposure (number of surveys)

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2.9.2. Exposure via other sources

2.9.2.1. Via regular diet

Men Women Adults Children

2.9.2.2. Via addition as novel food ingredient

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3. BIOLOGICAL AND TOXICOLOGICAL DATA

3. Phytostanol esters: Wood-derived mixtures consisting of 94% β-sitostanol and approximately 6%

3.1. Absorption, distribution, metabolism and excretion

EFSA Journal 2012;10(5):2659 19