Safety evaluation of certain food additives - ipcs inchem

STEVIOL GLYCOSIDES (addendum) 193
2.2.3 Long-term studies of toxicity and carcinogenicity
The aim of a study with stevia material of unspecified purity was to investigate
potential inhibitory effects on tumour promotion in mice (Yasukawa et al., 2002).
This was not informative for the current evaluation.
In a study to investigate chemoprevention, the effect of rebaudioside A
(purity >99.5%) on azoxymethane (AOM)–induced aberrant crypt foci (ACF) was
studied in groups of male F344 rats. One group of 16 rats was given three weekly
subcutaneous injections of AOM for 2 weeks and a diet containing 200 mg
rebaudioside A/kg for 5 weeks, from 1 week before to 2 weeks after AOM administration,
and was then sacrificed. The dose expressed as steviol was 6.6 μg/kg bw
per day. Other groups received a diet containing rebaudioside A with no AOM
injections (6 rats), a basal diet with no AOM (6 rats) or AOM injections and a basal
diet only (16 rats). At the end of the study, the colons were removed from eight
animals in the test group and from one in each of the control groups and examined
for ACF. The colonic mucosa of the remaining animals from each group were pooled
and examined for ornithine decarboxylase (ODC) activity. Silver-stained nucleolar
organizer region (AgNOR) protein count was also determined for each group. Both
ODC and AgNOR number are biomarkers for cell proliferation. The average body
weights and mean liver weights of the animals receiving the test compound and the
AOM injections were significantly lower than those of the animals receiving AOM
alone. No signs of toxicity were observed, and food consumption was unaffected
by treatment. There was a non-significant trend for rebaudioside A to reduce the
number of AOM-induced ACF, mucosal ODC activity and the number of AgNORs
(Kawamori et al., 1995).
One other study was available on steviol-related material of unspecified
composition, for which the administered dose of steviol could not be identified
(Yamada et al., 1985).
2.2.4 Genotoxicity
A number of genotoxicity studies not previously reviewed were available,
including four reverse mutation assays, all giving negative results up to concentrations
of 10–50 mg/plate. One sister chromatid exchange assay, one chromosome
aberration assay and one rec assay were also available, all giving negative results.
Two micronucleus assays using human cells (lymphocytes and buccal mucosal
cells) gave positive results (Hohn & Zankl, 1990). A forward mutation assay with
Salmonella typhimurium strain TM677 gave positive results. In a review by Brusick
(2008), the author concluded that the strain TM677 is uniquely sensitive to steviol
when it is incubated in the presence of S9 from rats induced by polychlorinated
biphenyls only, and therefore this is not likely to be relevant for the situation in vivo.
A comet assay by Nunes et al. (2007) reviewed by the Committee at its sixtyeighth
meeting, using rat liver, brain, blood and spleen cells, gave positive results.
The validity of this assay has been questioned by other authors (Geuns, 2007;
Williams, 2007), and the Committee previously concluded that it did not provide
convincing evidence of genotoxicity.
The results of the genotoxicity assays for stevioside and rebaudioside A are
summarized in Table 2.