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Administrative data

Endpoint:
endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study

Data source

Reference
Reference Type:
publication
Title:
Comparative Study of the Endocrine-Disrupting Activity of Bisphenol A and 19 Related Compounds
Author:
Kitamura et al.
Year:
2005
Bibliographic source:
Toxicol Sci. 84(2):249-259

Materials and methods

Type of method:
in vitro
Endpoint addressed:
other: Endocrine-Disrupting Activity

Test material

Constituent 1
Chemical structure
Reference substance name:
4,4'-isopropylidenedi-o-cresol
EC Number:
201-240-0
EC Name:
4,4'-isopropylidenedi-o-cresol
Cas Number:
79-97-0
Molecular formula:
C17H20O2
IUPAC Name:
4,4'-isopropylidenedi-o-cresol

Administration / exposure

Details on study design:
ESTROGENIC ACTIVITY
- Method: ERE-luciferase reporter assay
- Cell culture conditions: Human breast cancer cell-line MCF-7 cells were maintained in DMEM (Sigma Chemical Co.) containing penicillin and streptomycin with 5% fetal bovine serum (FBS; Life Technologies, Rockville, MD).
- Procedure: Transient transfections in MCF-7 cells were performed using Transfast (Promega Co., Madison, WI), according to the manufacturer’s protocol. Transfections were done in 48-well plates with 0.3 µg of p(ERE)3-SV40-luc and 2 ng of phRL-CMV (Promega Co.) as an internal standard. 24 h after addition of the sample (final concentration, 10E-04 – 10E-09 M), the assay was performed with a Dual Luciferase assay kit (Promega Co.). Firefly luciferase reporter activity was normalized to renilla luciferase activity from phRL-CMV, to control for the cytotoxic effects of compounds, as well as differences in transfection efficiency between culture wells. For the assay of anti-estrogens, the inhibitory effect of the test substance on the estrogenic activity of 17-β-estradiol at the concentration of 10E-10 M was examined.

ANDROGENIC ACTIVITY
- Method: ARE-luciferase assay
- Cell culture conditions: Mouse fibroblast cell-line NIH3T3 cells without expressing androgene receptor were maintained in DMEM (Sigma Chemical Co.) containing penicillin and streptomycin with 5% calf serum (Life Technologies).
- Procedure: Cells were maintained in phenol red-free DMEM (Sigma Chemical Co.) containing penicillin, streptomycin, and dextran-charcoal-treated calf serum for 2–3 days. Transient transfections in NIH3T3 cells were performed using Transfast according to the manufacturer’s protocol. Transfections were done in 48-well plates with 0.3 µg of p(ARE)2-luc, 0.05 µg of pSG5-hAR, and 2 ng of phRL-CMV as an internal standard. 24 h after addition of the sample (final concentration, 10E-04 – 10E-08 M) dissolved in 10 µL of ethanol, the assay was performed with a Dual Luciferase assay kit according to the manufacturer’s protocol. Firefly luciferase reporter activity was normalized to renilla luciferase activity from phRL-CMV. For the assay of anti-androgenic activity, the inhibitory effect on the androgenic activity of 10E-10 or 10E-11 M dihydrotestosterone was examined.

THYROID HORMONAL ACTIVITY
- Method: Induction of growth hormone production
- Cell culture conditions: Rat pituitary cancer cell-line GH3 cells were maintained in DMEM/F12 mixed medium (Sigma Chemical Co.) containing penicillin and streptomycin with 8% horse serum (Life Technologies) and 2% fetal bovine serum.
- Procedure: GH3 cells were seeded in 24-well plates and the test substance was added the next day. Two days later, growth hormone in the culture medium was measured. For the assay of anti-thyroid hormonal activity, the inhibitory effect on the activity of 10E-07 or 10E-08 M T3 was examined.

Results and discussion

Details on results:
ESTROGENIC ACTIVITY
The test substance showed estrogenic activity. The determined EC50 value in MCF-7 Estrogen Luciferase Reporter Assay was 0.42 µM.
The EC50 value of the reference substance 17-β-estradiol was 8.6 x 10E-06 µM. The EC50 value of bisphenol A was 0.63 µM.

ANDROGENIC ACTIVITY
The test substance showed anti-androgenic activity. The determined IC50 value for androgen activity of dihydrotestosterone in NIH3T3 Luciferase Reporter Assay was 2.0 µM. The reference substance dihydrotestosterone exhibited markedly the androgenic activity toward NIH3T3 cells at 1x 10E-11 - 1 x 10E-09 M. The IC50 value of bisphenol A was 4.3 µM.

THYROID HORMONAL ACTIVITY
No antagonistic action towards growth hormone production induced by the thyroid hormone was observed after incubation with the test substance.

Applicant's summary and conclusion

Conclusions:
Minimum structural requirements for estrogenic activity of BPA derivatives seems to be a 4-hydroxyl group on the A-phenyl ring and a hydrophobic moiety at the 2-position of the propane moiety. Moreover, substituents at the 3,5-positions of the A-phenyl ring and at the methylene bridge markedly influence the estrogenic activity and the endocrine-disrupting activity (Kitamura et al., 2005). 4,4'-isopropylidenedi-o-cresol showed estrogenic activity, and the determined EC50 value was slightly higher than for bisphenol A (EC50 = 0.63 µM). In addition, the test substance 4,4'-isopropylidenedi-o-cresol showed a higher anti-androgenic activity with an IC50 value of 2 µM than bisphenol A (IC50 = 4.3 µM).

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