2-ethylhexyl 4-(dimethylamino)benzoate

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test material was examined for oestrogenicity in vitro in MCF-7 breast cancer cells and in vivo in the immature rat uterotrophic assay.

GLP compliance:

not specified

Type of method:

other: in vitro and in vivo

Endpoint addressed:

other: oestrogenicity

Species:

rat

Strain:

Long-Evans

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Bred in house
- Age at study initiation: Post-natal day 21
- Mean weight at study initiation: 38.0 ± 4.5 g
- Housing: Sperm-positive females were housed in groups of two to three and separated 1 day before parturition. Immature pups were housed in groups of 4 to 6 to avoid stress.
- Diet: powdered chow ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 1 °C
- Photoperiod: Lights on from 02.00 to 16.00

Route of administration:

other: in vivo study; oral: feed

Vehicle:

other: acetone or ethanol

Details on exposure:

PERORAL ADMINISTRATION (IN VIVO)
PREPARATION OF DOSING SOLUTIONS:
The test material was dissolved either in acetone or in 99 % ethanol and added to powdered diet. The mixture was prepared at least 48 hr before the experiment to allow for complete evaporation of the solvent. Evaporation was assisted by continuous stirring.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

Beginning on PN 21, female pups received diet containing one of several concentrations of test material for 4 days, until 1200 hr on PN 25.

Frequency of treatment:

Continuous in the diet

Dose / conc.:

0 other: M

Remarks:

in vitro

Dose / conc.:

0 other: M

Remarks:

in vitro

Dose / conc.:

0 other: M

Remarks:

in vitro

Dose / conc.:

0 other: M

Remarks:

in vitro

Dose / conc.:

0 other: M

Remarks:

in vitro

Dose / conc.:

596 mg/kg bw/day (nominal)

Remarks:

Uterotrophic assay

Dose / conc.:

761 mg/kg bw/day (nominal)

Remarks:

Uterotrophic assay

Dose / conc.:

1 419 mg/kg bw/day (nominal)

Remarks:

Uterotrophic assay

No. of animals per sex per dose:

In the in vivo assay, 4, 6 and 6 animals were dosed at 596, 761 and 1419 mg/kg/day, respectively.

Control animals:

yes, concurrent vehicle

yes, plain diet

Details on study design:

IN VITRO STUDY
- Cell line
MCF-7 human breast cancer cells were frozen every 10 passages. In this experiment, samples from frozen stock were used for a maximum of 6 - 13 passages. Mycoplasma status, which was regularly checked, was negative. Cells were cultured in Dulbecco’s modified Eagle Medium (DME) supplemented with 5 % foetal bovine serum (FBS) in 5 % CO2/95 % air at 37 °C under saturated humidity in 50 mL Falcon flasks. Sex steroids were removed from the serum by charcoal dextran treatment (steroid hormone-free FBS, CD-FBS).

- E-SCREEN
MCF-7 cells were trypsinised, mechanically dissociated, and plated into 24-well plates at an initial concentration of 40,000 cells/well. Cells were allowed to attach in the seeding medium (DME supplemented with 5 % FBS) for 24 hours. The seeding medium was then aspirated and replaced by the experimental medium containing phenol red-free DME with 10 % CD-FBS. Cells were incubated with the test material (final concentrations 5 x 10^-5 to 1 x 10^-7 M), E2 as positive control (10^-8 to 10^-13 M), or chemical-free medium. For each concentration, 4 wells per plate were used.
Concentrations of stock solutions in absolute ethanol were 2 × 10^-3 M for E2 and 10^-2 M for test material; final concentrations of ethanol in culture medium were between 1.0 and 0.001 % (v/v) with test material and were = 0.0005 % (v/v) with E2. No difference in the cell proliferation rate was observed in control experiments with chemical- free medium or medium with 1.0 % ethanol. Therefore, chemical-free medium was used as a control.
Antagonism was tested by the pure antioestrogen ICI 182,780 in MCF-7 cells exposed for 6 days to E2 (10 pM ) in the presence or absence of 1, 10 or 100 nM ICI 182,780.

- Measurement of cell proliferation
Experiments were terminated after 6 days of incubation by removing the media from the wells. Cells were fixed with 10 % trichloroacetic acid, washed with phosphate-buffered saline, and stained with 1 % sulforhodamine blue (0.4 % in 1 % acetic acid, 1 mL/well) for 15 min at room temperature. Stained cells were dissolved in TRIS buffer (pH 10.6), and optical density (OD) was measured in an Anthos Labtec 2000 spectrophotometer at 492 nm. OD values were converted into cell numbers by a standard curve. Experimental readings were in the linear range of the standard curve.

- pS2 protein assay
MCF-7 cells were incubated for 72 hours with the test material at 5, 10 and 50 µM; E2 (10 pM) served as a positive control. Culture media were harvested and centrifuged to avoid floating of detached cells. Samples were kept at -80 °C until the radioimmunoassay for pS2 protein was performed according to the protocol of the manufacturer (ELSA - PS2; CIS Bio International, Gif-sur-Yvette, France). Media were analysed in duplicate.

IN VIVO STUDY
The experiment was performed on offspring of time-pregnant rats. Receptive females were mated with a male between 1600 and 1900 hr. The stage 24 hours after onset of the 3-hour mating period was defined as gestational day (GD) 1 and the day of birth as postnatal day (PN) 1 (GD 23).

- Peroral administration
From PN 16, the pups and their dam were habituated to powdered chow which continued after weaning at PN 20. Beginning on PN 21, female pups received chow containing the test material. To limit the number of experimental animals, the size of the treatment groups was adjusted according to statistical needs.
Body weight was recorded at the beginning and at the end of the treatment period. Animals from different litters were randomly assigned to the various treatment groups to give similar mean body weights at the onset of treatment, with no more than 15 % deviation of individual animals from the mean. Mean body weights of experimental groups were in the same range as that of the controls (initial weight 38.0 ± 4.5 g, final weight 48.8 ± 3.8 g). The mean body weight of the 4-day treatment period was calculated for each animal.
Food consumption of the group of four to six animals was measured for the 4-day period. The mean daily dose was calculated from the average amount of test material consumed per animal (ingested powdered chow per animal × concentration of test material in chow per mean body weight of a given animal). The advantage of using the average values of consumption is that the animals were not disturbed. The consistency of the condition is indicated by an SD/mean ratio of uterine weights of 5.6 % in controls and 9.8 % in treated groups.
At the end of the treatment period, pups were decapitated under light ether anaesthesia. The uterus was excised, trimmed free of fat and connective tissues, and blotted with sterile gauze to remove adherent fluid. The uterus was cut just above the junction between the cervix and vagina and at the top of the uterine horns. It was then weighed (wet weight) and either frozen in liquid nitrogen or fixed in buffered 4 % formaldehyde for further analysis.

Positive control:

In the in vitro test, 17ß-oestradiol was used as positivecontrol. In the uterotrophic assay 17ß-ethinylestradiol (0.3–10 µg/kg) was used as a positive control.

Details on results:

IN VITRO
- 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.
- pS2 Protein: 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.

IN VIVO
After administration in powdered feed for 4 days, the positive control material ethinylestradiol elicited dose-dependent increases in uterine weight of immature Long-Evans rats.
The test material was inactive in vivo at the doses tested. Mean body weights were similar and in the range of the vehicle control group (mean ± SD of 38.0 ± 4.5 g at PN 21 and 48.8 ± 3.8 g at PN 25).
In the in vivo assay, the uterine weights at 596, 761 and 1419 mg/kg/day were 26.05 ± 0.95, 24.75 ± 1.29 and 26.13 ± 3.10 mg, respectively.

Table 1: Summary of in vitro and in vivo activity

Parameter		Result
MCF-7 Cell Proliferation	PE	9.13
	RPE	51.77
	(% of E2)*	55.54
	EC50 (µM)	2.63
Uterotrophic effect in immature rats	Increase of uterine weight over control**	1.04
	Maximal weight increase (% of EE2)***	1.15
	ED50	Inactive

 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) × 100

**(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.

Conclusions:

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 doses tested.

Executive summary:

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 doses tested.

Reference 2

Endpoint:

endocrine system modulation

Type of information:

other: Review of experimental publication

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Reliable review of an existing publication

Qualifier:

no guideline followed

Principles of method if other than guideline:

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?

GLP compliance:

no

Type of method:

other: in vitro and in vivo

Endpoint addressed:

other: oestrogenicity

Details on study design:

An 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 investigations of this Swiss group on the safety of UV-filters have alerted the popular press and the public. Controversial interpretations of the results initiated vigorous debates about the safety of sunscreens. More particularly, oestrogenic properties were attributed to a number of UV-filters including the registered substance; even suggestions towards carcinogenic effects were made.
On 27/4/2001, an ad hoc working group of the SCCNFP discussed the issue and published a preliminary report on the scientific review of the investigations of Schlumpf et al. (2001). Serious concerns were expressed as to the validity of the results published. As a follow-up, a final expert opinion of the SCCNFP is given below, consisting of:
(1) A scientific review of the investigations of Schlumpf et al. (2001);
(2) A scientific review of the investigations carried out by the industry concerned;
(3) A risk assessment and margin of safety according to the EU-procedure for UV-filters;
(4) Data on human exposure to environmental and dietary oestrogens; and
(5) Conclusions.

SCIENTIFIC REVIEW OF THE RESULTS OF SCHLUMPF ET AL. (2001)
- Study protocol and results
The in vitro and in vivo oestrogenicity of 5 UV B-filters, including the registered substance, and 1 UVA-filter were studied.

- 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.

- 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.

Details on results:

- In vitro study
As positive control, 17ß-oestradiol 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 vivo study
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.

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 seriously.

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.

Conclusions:

The SCCNFP came to the conclusion that a number of important technical and scientific shortcomings are present in the study of Schlumpf et al.
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.

Executive summary:

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.

 

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 seriously.

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 26^thday 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.

Description of key information

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.

Additional information

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.

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 doses tested.

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:

- 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.

 

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 seriously.

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 26^thday 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.