Safety evaluation of certain food additives - ipcs inchem

128 PHYTOSTEROLS, PHYTOSTANOLS AND THEIR ESTERS
However, no differences in pup mortality were observable when analysed on a litter
basis (Waalkens-Berendsen & Wolterbeek, 1998; Waalkens-Berendsen et al.,
1999a). A NOEL of 8.1% phytosterol esters in the diet can be derived from this study.
This concentration in the diet equals a range of 3.3–6.5 g phytosterol esters/kg bw
per day for F0 and F1 females during premating (10 weeks) and 2.3–3.8 g phytosterol
esters/kg bw per day during gestation (3 weeks). A time-weighted average for these
exposure periods is 4.4 g phytosterol esters/kg bw per day, which corresponds to
2.7 g phytosterols/kg bw per day.
(b) Minks
In a limited, non-guideline study, the reproductive toxicity of -sitosterol at
low doses was investigated in the American mink. Sixty minks (7 weeks old) per
group and sex received -sitosterol at 0, 5, 10 or 50 mg/kg bw per day for 10 months.
After 3 months of exposure, 15 males per group were killed and investigated for
organ weights and haematological and clinical chemistry parameters. Selection was
non-randomized: smaller animals with low fur quality were preferred. The authors
reported differences in body fat masses (omental, mesenteric, retroperitoneal, intraabdominal
fat), but increases in fat masses were not dose dependent, and no
experimental details on the determination of fat masses were given. In the second
part of the study, after 7 months of exposure, male (“top-rated”) animals (10–11 per
group) were mated with 4–5 females each. Reproductive parameters (number of
pregnant females, litter and kit numbers, postnatal mortality and development)
measured did not reveal any treatment-related changes (Nieminen et al., 2008).
2.2.4 Special studies on estrogenic activity
In older studies with parenteral application, some estrogenic activity of
phytosterols was observed (Scientific Committee on Food, 2000). This raised
concerns that estrogenic activity may also be present after oral uptake and that
uptake from food may exert effects on the hormonal system in humans.
The possible estrogenicity of phytosterols and phytosterol esters was
investigated in a series of in vitro (competitive estrogen receptor binding assay,
recombinant yeast assay) and in vivo test systems (immature rat uterotrophic
assay). The competitive estrogen receptor binding assay uses a preparation of
estrogen receptors isolated from rat uteri and measures the concentrationdependent
substitution of [2,4,5,6- 3 H]estradiol at the estrogen receptor. In this
assay, a mixture of phytosterols failed to compete for binding at the estrogen
receptor when tested in concentrations up to 1 × 10 4 mol/l. The recombinant yeast
assay is an assay to measure estrogen-induced expression of reporter genes in
yeast. The assay is therefore able to detect the stimulation by test compounds of
the transcription of the human estrogen receptor. When tested in concentrations up
to 2 × 10 4 mol/l, phytosterols did not stimulate the transcriptional activation of the
human estrogen receptor. In the uterotrophic assay, phytosterols were administered
by gavage on 3 consecutive days to 22- to 23-day-old rats followed by determination
of absolute uterus weights on day 4. Groups of 10 animals received a single dose
of phytosterols or phytosterol esters of 0, 5, 50 or 500 mg/kg bw. Treatment with