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EC number: 244-289-3
CAS number: 21245-02-3
Table 1: Summary of in vitro and in vivo activity
MCF-7 Cell Proliferation
(% of E2)*
Uterotrophic effect in immature rats
Increase of uterine weight over control**
Maximal weight increase (% of EE2)***
EE2 = ethinyloestradiol
PE = Proliferative effect over control; PE = (maximal cell count of
experimental group) / (cell count of control)
RPE = Maximal proliferative effect (% of E2); RPE = (PE of experimental
group – 1) / (PE of oestradiol – 1) × 100
*Maximal cell count increase; (Cell count of experimental group – cell
count of control) / (cell count of oestradiol – cell count of control) ×
**(Uterine weight of experimental group) / (uterine weight of control)
***Maximal weight increase (% of ethinyloestradiol) = (uterine weight of
experimental group – uterine weight of control) / (uterine weight of
ethinyloestradiol – uterine weight of control) × 100.
The test material was examined for oestrogenicity in vitro in MCF-7
breast cancer cells and in vivo in the immature rat uterotrophic assay.
The in vitro test used MCF-7 cells in an E-SCREEN assay in which the
cells were treated with the test material and the cell proliferation and
the effect on pS2 protein was measured. In the in vivo study, starting
on post-natal day 21, female rats were treated with the test material
orally via the diet for a 4 day period. After dosing the animals were
sacrificed and the uterus was excised and weighed.
In MCF-7 Cells, cell proliferation was dose-dependently increased. The
effective concentration range was 1–50 µM and the maximum effect
concentration was around 10 µM. The in vitro EC50 value was 2.63 µM.
According to the maximum effects on cell proliferation in relation to
the positive control, the test material acted as a partial agonist.
Levels of secretion of the oestrogen-regulated protein pS2 into the
culture medium were above control after incubation with the test
material but the difference was not significant.
The test material was completely inactive in the uterotrophic assay at
the doses tested.
Under the conditions of this study, cell proliferation was
dose-dependently increased in the MCF-7 cells in the in vitro test. The
test material was completely inactive in the uterotrophic assay at the
Review comments from the SCCNFP
In Vitro Study
The potency of the positive control is in the order of picomoles; the in
vitro potency of the test material lies in the range of micromoles,
which means a difference of 1 million units. The in vitro potency of the
test material is thus importantly lower than the one observed for the
positive control. Probably a lot of industrial chemicals would show some
in vitro oestrogenic effects when this type of comparisons is taken
It should be emphasised here that in vitro assays can only demonstrate
whether UV-filters bind on the oestrogen receptor or not, but they do
not provide evidence whether the compounds have oestrogenic activity or
not. In vitro assays are therefore screening tests useful in setting
priorities for further in vivo testing. The CSTEE committee clearly
stated in its report on endocrine disrupters (1999) that utilising in
vitro data for predicting in vivo endocrine disrupter effects may
generate false negative as well as false positive results and that major
emphasis should therefore be put on in vivo assays. Claiming that all 5
UV-filters (including the registered material) have oestrogenic
properties based on an in vitro test is premature.
The in vitro ranking for the UV-filters did not correspond with the in
vivo results. The most active UV-filter in vitro displayed only a weak
activity in vivo. In addition the registered material was found to be
inactive. Only precise toxicokinetic data can link the in vitro and in
vivo data, a conclusion that was also reached by the authors.
In Vivo Study
The OECD draft protocol on the rodent uterotrophic assay was issued on
April 21, 2000. The protocol used by the Swiss group dates from before
that time and therefore shows some important deviations. Moreover, GLP
conditions have not been applied. The deviations from the current OECD
guideline proposal included:
- The choice of the rat strains is unusual and not explained
- The exposure period of the rats runs until the 26th day of life which
is too close to the onset of puberty
- The potency of the positive control, ethinylestradiol, is in the order
of 1 µg/kg/day; the potency of the UV-filters tested lies in the range
of 100 to 1000 mg/kg/day, which means a difference of 100.000 to 1
million units. Thein vivo potency of the UV-filters is thus importantly
lower than the one observed for the control hormone. Furthermore, 3 of
the 6 UV-filters have no measurable potency at all.
- The uterotrophic assay can only serve a limited function as a test for
in vivo identification of chemicals with oestrogenic activity. The
uterotropic assay is a short-term high-dose test.
The SCCNFP came to the conclusion that a number of important technical
and scientific shortcomings are present in the study of Schlumpf et al.
The answer to the question 'More generally, does the SCCNFP consider
that organic UV filters used in cosmetic sunscreen products have any
estrogenic effects which have the potential to affect human health?' was
that based on the actual scientific knowledge, the SCCNFP is of the
opinion that the organic UV-filters used in cosmetic sunscreen products,
allowed in the EU market today, have no oestrogenic effects that could
potentially affect human health.
The SCCNFP was asked to evaluate the possible oestrogenic effects of
organic UV filters used in cosmetic products and to respond to the
following questions in a scientific opinion:
- Could the SCCNFP provide a critical analysis of the article "In vitro
and in vivo estrogenicity of UV screens" by Margret Schlumpf et al.?
- More generally, does the SCCNFP consider that organic UV filters used
in cosmetic sunscreen products have any oestrogenic effects which have
the potential to affect human health?
The article published by Schlumpf et al. (2001) suggested that several
UV screens show oestrogenic activity. They used an in vitro test with
the MCF-7 breast cancer cell line and an in vivo rat uterotrophic assay.
The in vitro and in vivo oestrogenicity of 5 UV B-filters, including the
registered substance, and 1 UVA-filter were studied.
In the in vitro study, a general screening assay (E-screen) with a human
breast cancer cell line, MCF-7 cells, was carried out. A positive test
was based upon the binding of the test compound with the oestrogen
receptor leading to cell proliferation. As a positive control,
17ß-oestradiol was used and was, as expected, positive in the assay. The
5 UV-B filters were found to be positive in the assay and caused cell
proliferation. The UV-A filter gave a negative result. EC50 values for
17ß-oestradiol and the registered material were 1.22 pM and 2.63 µM,
respectively. The results were supported by the expression of the
oestrogen-dependent pS2 protein and by an inhibition of effects with the
anti-oestrogen ICI 182,780.
In the in vivo study, a uterotrophic assay was carried out involving
oral exposure of young Long-Evans rats to the 6 UV-filters from day 21
of life until day 24 of life, with ethinylestradiol serving as a
positive control. A dose-dependent increase of uterine weights was
observed for two of the compounds and a slighter response was seen for a
third, but no maximal effect was seen as was the case for the positive
control. The registered material was found to be inactive.
- In Vitro Study
- In Vivo Study
- The exposure period of the rats runs until the 26thday of
life which is too close to the onset of puberty
In the study by Schlumpf et al., cell proliferation was dose-dependently
increased in the MCF-7 cells in the in vitro test. The test material was
completely inactive in the uterotrophic assay at the doses tested.
In the European Commission review, based on the actual scientific
knowledge, the SCCNFP is of the opinion that the organic UV-filters used
in cosmetic sunscreen products, including the test material, allowed in
the EU market today, have no oestrogenic effects that could potentially
affect human health.
Two reports are included in the endpoint; the first is a publication and
the second is a review article.
In the study by Schlumpf et al., the test material was examined for
oestrogenicity in vitro in MCF-7 breast cancer cells and in vivo in the
immature rat uterotrophic assay.
In the review article, the SCCNFP was asked to evaluate the possible
oestrogenic effects of organic UV filters used in cosmetic products and
to respond to the following questions in a scientific opinion:
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