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Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well conducted study meeting generally accepted scientific principles, acceptable for assessment.
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, six extensively used ultraviolet (UV) filters were assessed for transcriptional activation of estrogen receptors using a sensitive in vitro reporter gene assay.
Briefly, Human embryonal kidney 293 (HEK293) cells were stable transfected with hERalpha and hERbeta. The compounds to be tested were added to the cells and incubated for 24h before luciferase reading. Furthermore, an in vivo assay was used for which zebrafish, in which an estrogen responsive luciferase reporter gene has been stably introduced, were incubated for 96h before they were sacrificed and luciferase activity was read from homogenised fish samples.
GLP compliance:
no
Type of method:
other: in vitro and in vivo
Species:
other: human embryonal kidney cells (HEK 293) and zebrafish
Strain:
not specified
Sex:
not specified
Route of administration:
oral: drinking water
Vehicle:
ethanol
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
In vitro assay: 24 h
In vivo assay: 96 h
Frequency of treatment:
one application
Post exposure period:
not applicable
Dose / conc.:
0.1 other: µmol/L
Remarks:
in vitro assay
Basis: nominal in water
Dose / conc.:
1 other: µmol/L
Remarks:
in vitro assay
Basis: nominal in water
Dose / conc.:
10 other: µmol/L
Remarks:
in vitro assay
Basis: nominal in water
Dose / conc.:
100 other: µmol/L
Remarks:
in vitro assay
Basis: nominal in water
Dose / conc.:
10 other: µmol/L
Remarks:
in vivo assay
Basis: nominal in water
No. of animals per sex per dose:
In vitro assay: no data on cell density was given
In vivo assay: 5 - 6 fish were exposed per concentration
Control animals:
yes, concurrent vehicle
Details on results:
IN VITRO ASSAY
The test substance butyl methoxydibenzoylmethane (BMDBM) was able to significantly induce the human estrogen receptor alpha (hERalpha) in stably transfected HEK293 cells at concentrations of 10 and 100 µmol/L up to approximately 40 % of maximal 17beta-estradiol induction. Induction of human estrogen receptor beta (hERbeta) was only achievable with the highest tested concentration of 100 µmol/L (significant induction to 10 - 15 % of maximal 17beta-estradiol induction).
The anti-estrogenic potential was assessed using a competition assay with a sub-maximal concentration of estradiol. BMDBM showed no dose-dependent anti-estrogenic effect at either hERalpha or hERbeta. Whereas at best a slight reducing effect was observable for hERalpha, either tested concentration of BMDBM reduced hERbeta transcription to about 60 % of maximal estradiol induction.

IN VIVO ASSAY
Neither BMDBM at 10 µmol/L nor one of the other tested UV filters tested could not induce transcriptional activation of the estrogen receptor in zebrafish. The positive control (17beta-estradiol, 10 nmol/L) showed a clear response.
Conclusions:
The observed slight induction of human estrogen receptor-alpha by BMDBM (10 - 100 µM) in the in vitro assay could not be confirmed in the in vivo zebrafish assay.
Executive summary:

In this study, six extensively used ultraviolet (UV) filters were assessed for transcriptional activation of estrogen receptors using an in vitro reporter gene assay and an in vivo zebrafish luciferase assay.

 

IN VITRO ASSAY

The test substance butyl methoxydibenzoylmethane (BMDBM) was able to significantly induce the human estrogen receptor alpha (hERalpha) in stably transfected HEK293 cells at concentrations of 10 and 100 µmol/L up to approximately 40 % of maximal 17beta-estradiol induction. Induction of human estrogen receptor beta (hERbeta) was only achievable with the highest tested concentration of 100 µmol/L (significant induction to 10 – 15 % of maximal 17beta-estradiol induction).

The anti-estrogenic potential was assessed using a competition assay with a sub-maximal concentration of estradiol. BMDBM showed no dose-dependent anti-estrogenic effect at either hERalpha or hERbeta. Whereas at best a slight reducing effect was observable for hERalpha, either tested concentration of BMDBM reduced hERbeta transcription to about 60 % of maximal estradiol induction.

IN VIVO ASSAY

Neither BMDBM at 10 µmol/L nor one of the other tested UV filters tested could induce any transcriptional activation of the estrogen receptor in zebrafish. The positive control (17beta-estradiol, 10 nmol/L) showed a clear response.

 

The observed slight induction of human estrogen receptor-alpha by BMDBM (10-100 µM) in the in vitro assay could not be confirmed in the in vivo zebrafish assay.

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
Well conducted study, meets generally acepted scientific principles, acceptable for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The interaction of five polycyclic musk compounds and seven UV filters with the estrogen receptor (ER), androgen receptor (All), and progesterone (PR) receptor were assessed, using sensitive and specific reporter gene cell lines, namely stably transfected HEK293 cells (for ERa and ERb) and the AR- and PR-CALUX® bioassays.
GLP compliance:
no
Type of method:
in vitro
Species:
other: stably transfected HEK293 (human embryonic kidney cells), stably transfected U2-OS (human osteosarcoma) cells (for the CALUX® assays)
Strain:
not specified
Sex:
not specified
Route of administration:
other: incubation in medium
Vehicle:
ethanol
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
24h incubation period
Frequency of treatment:
once
Post exposure period:
no
Remarks:
Doses / Concentrations: 0.1 - 10 µM (AR repression assay)
Basis: nominal conc.
Remarks:
Doses / Concentrations: 1 nM - 10 µM (PR repression assay)
Basis: nominal conc.
No. of animals per sex per dose:
Three independent experiments with each concentration measured in triplicate.
Details on results:
All UV filters were reported to show agonism toward hERa, and additionally, a number were found to show agonism toward hERb as well (Schreurs et al. 2000). None of the UV filters showed anti-estrogenic effects.
None of the test compounds showed AR transactivation, however, nearly all test compounds were found to be AR antagonists. BMDBM showed no distinct hAR transcription repression inside the chosen concentration range. Extrapolation outside the tested dose range resulted in an IC50 value of 11 µM.
None of the test compounds showed PR transactivation (data not shown); however, several test compounds, but not BMDBM, were found to be PR antagonists.
Conclusions:
Butyl methoxydibenzoylmethane (BMDBM) showed weak ERa agonism and weak androgen receptor antagonism in in vitro reporter gene assays. No influence on progesteron receptor was observed.
Executive summary:

The interaction of five polycyclic musk compounds and seven UV filters with the estrogen receptor (ER), androgen receptor (All), and progesterone (PR) receptor were assessed, using sensitive and specific reporter gene cell lines, namely stably transfected HEK293 cells (for ERa and ERb) and the AR- and PR-CALUX® bioassays.

In short, all UV filters were reported to show agonism toward hERa, and additionally, a number were found to show agonism toward hERb as well (Schreurs et al. 2000). None of the UV filters showed anti-estrogenic effects.

None of the test compounds showed AR transactivation, however, nearly all test compounds were found to be AR antagonists. BMDBM showed no distinct hAR transcription repression inside the chosen concentration range. Extrapolation outside the tested dose range resulted in an IC50 value of 11 µM.

None of the test showed PR transactivation (data not shown); however, several test compounds, but not BMDBM, were found to be PR antagonists.

Taken together, butyl methoxydibenzoylmethane (BMDBM) only showed weak ERa agonism and weak AR antagonism.

Description of key information

Schreurs 2002: The observed slight induction of human estrogen receptor-alpha by BMDBM (10-100 µM) in the in vitro assay could not be confirmed in the in vivo zebrafish assay.

Schreurs 2005: Butyl methoxydibenzoylmethane (BMDBM) showed weak ERa agonism and weak androgen receptor antagonism in in vitro reporter gene assays. These effects were observed by nearly all tested compounds. No influence on the progesterone receptor was observed.

Additional information

Two studies were found that report on endocrine activity of butyl methoxydibenzoylmethane (BMDBM). Two publications of Schreurs et al. (2002 and 2005) stated that slight induction of human estrogen receptor-alpha by BMDBM was observed in vitro. But as all tested substances showed a similar induction of the estrogene receptor which could not be reproduced in an in vivo zebrafish assay, and none of the other publications support this finding, the observed induction of the estrogen receptor remains questionable.

Based on these results it was concluded that BMDBM possesses no endocrine activity.