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Toxicological information

Specific investigations: other studies

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Administrative data

Endpoint:
mechanistic studies
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study meets basic scientific principles. The principles of the method are similar to OECD guideline 455. Instead of transfected hERα-HeLa-9903 cells, stably transfected MCF-7 ERE-Luc cells were used in the current study. At the highest positive control concentration the induction of luciferase activity was not maximal. Some discrepancies between study protocol and raw data were obvious (e.g. concentration range of 17-beta-estradiol from 0-300 pM was stated in the study protocol. Raw data however, suggested a dose range from 0 to 150 pM). Data on cytotoxicity are not well documented. Although some deviations to OECD guideline 455 occurred, the study is considered to be acceptable with restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report Date:
2000

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
other: OECD guideline 455 (Stably Transfected Human Estrogen Receptor-α Transcriptional Activation Assay for Detection of Estrogenic Agonist-Activity of Chemicals) (2009)
Deviations:
yes
Remarks:
Instead of transfected hERα-HeLa-9903 cells, stably transfected MCF-7 ERE-Luc cells were used. The luciferase activity after treatment with positive control was not maximal.
Principles of method if other than guideline:
The method based on principles similar to OECD guideline 455. However, instead of transfected hERα-HeLa-9903 cells, stably transfected MCF-7 ERE-Luc cells were used. Therefore, no acceptable range values as given in the OECD guideline 455 for hERα-HeLa-9903 cells are available. It is however mentioned in the guideline, that despite the assay using the hERα-HeLa-9903 cell line, further ER transcriptional activation assays are currently being developed and validated.
GLP compliance:
no
Type of method:
in vitro
Endpoint addressed:
not applicable

Test material

Reference
Name:
Unnamed
Type:
Constituent

Administration / exposure

Route of administration:
other: test substance is added to the growth medium
Vehicle:
ethanol
Details on exposure:
The MCF-7 luciferase induction assay is an in vitro technique to identify estrogenic and anti-estrogenic compounds. Human breast carcinoma MCF-7 cells stably transfected with an estrogen receptor-controlled luciferase reporter gene construct were used in this study (MCF-7 ERE-Luc; Pons et al., Biotechniques 9; 450-457, 1990). These cells express firefly luciferase in response to estrogen agonists. Luciferase activity is measured using a plate-scanning luminometer. The technique has been modified from the published procedure to be conducted in 96-well plates. The potential to induce luciferase activity was assessed by comparing the response after treatment with the response after treatment wit positive control (E2).
Cell Culture
Human breast carcinoma cells (MCF-7 ERE-Luc) were seeded into 96 well culture plates and cultured in medium supplemented with either defined fetal bovine serum (FBS for routine culturing) or charcoal-stripped FBS (for treatment and exposure period).
Cells were routinely cultured at 37° C and 5% CO2 in a humidified atmosphere.
Treatment of Cells and Luciferase Assay
24 hr after seeding, medium was changed to a medium containing reduced estrogen (dextran-coated charcoal-stripped FBS) and cells were dosed with either no treatment (blanks), solvent only, Estrogen (E2), or test substances.
Cells were dosed in triplicate with E2 in ethanol (0-150 pM final concentration) or test compounds dissolved in ethanol at doses of 0 - 6500 µM. Luciferase activity was measured by incubating cells with LucLiteTM reagent (Packard Instruments) for twenty minutes at room temperature. Light production, a measure of luciferase activity, was determined with a luminometer at 30° C. Confluence of wells was verified microscopically. A normalization to protein was stated to be not necessary. Therefore, luciferase activity is reported as either relative light units (RLU) or percent of solvent control. According to the standard operation protocol (included in the study report) viability of cells was assessed prior the measurement of luminescence to exclude cytotoxicity.

Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
cells were exposed to test substance in medium for 3 days
Frequency of treatment:
continous fore 3 days
Post exposure period:
none
Doses / concentrations
Remarks:
Doses / Concentrations:
0.065, 0.65, 6.5, 65, 650, 6500 µM
Basis:
nominal conc.

Examinations

Examinations:
Increase in luciferase activity was measured as marker for ER-alpha induction.
Positive control:
17-beta-estradiol (E2) in concentrations of 0.5, 1.5, 5, 15, 50, 150 pM was used as positive control.
Nonylphenol (a substance known for estrogenicity) was used in concentrations of 0.5, 5, 50, 500, 5000 and 50000 nM.

Results and discussion

Details on results:
A slight increase of luciferase activity was seen at concentrations close to the solubility limit of the test substance (at and above 65 µM the luciferase activity was 115 to 122 % of control). Since the luciferase activity at the highest positive control concentration was not maximal, relative potencies compared to 17-beta-estradiol were only estimated. Relative potency of this sample was calculated to be approximately E+7-fold less potent than E2. In comparison, nonylphenol (a substance known for its estrogenicity) was about 1E+4 to3E+4 less active then 17-beta-estradiol.

Any other information on results incl. tables

Table1: ER-alpha dependent increase in luciferase activity (RLU and % of control)

 

17-beta-estradiol (E2)

Controls

Concentration[pM]

0.5

1.5

5

15

50

150

Blank

solvent

Mean

Mean RLU (xE-2)

1.96

2.50

3.31

4.76

5.80

6.24

2.14

2.29

2.22

% of control

88

113

149

215

262

282

97

104

100

 

TEST SUBSTANCE

Controls

Concentration [µM]

0.065

0.65

6.5

65

650

6500

Blank

solvent

Mean

Mean RLU (xE-2)

1.88

2.08

2.25

2.54

2.70

2.71

2.14

2.29

2.22

% of control

85

94

102

115

122

123

97

104

100

The luciferase activities seen after treatment with test substance (RLU) corresponded to -8.5, -3.5, 0.8, 8.0, 11.9 and 12.2 % of maximal luciferase activity at 6.5E-8, 6.5E-7, 6.5E-6, 6.5E-5, 6.5E-4 and 6.5E-3 M. The calculation based on luciferase activities (RLU) after subtraction of background activity (control = 0.222 RLU).Data regarding cytotoxicity given in the study protocol were not well documented.

Applicant's summary and conclusion