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Specific investigations: other studies

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Administrative data

endocrine system modulation
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference Type:
Endocrine disruptor activity of multiple environmental food chain contaminants
Wielogórska E, Elliott CT, Danaher M, and L. Connolly
Bibliographic source:
Toxicology in Vitro 29 (2015) 211–220

Materials and methods

Principles of method if other than guideline:
Reportergene assay with the MMV-Luc cell line for agonistic and antagonistic activity at the estrogenic receptor
GLP compliance:
Type of method:
in vitro
Endpoint addressed:
other: estrogen receptor activation

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Test material form:
Specific details on test material used for the study:
purchased from Sigma–Aldrich, St Louis, MO, USA
no data on purity
Name in publication: 2-(2H-benzotriazol-2-yl)-4,6-bis(1-methyl-1-phenylethyl)phenol (UV-234)

Test animals

Details on test animals or test system and environmental conditions:
The estrogen responsive RGA cell line was developed as described in Willemsen, P., Scippo, M.L., Kausel, G., Figueroa, J., Maghuin-Rogister, G., Martial,
J.A., Muller, M., 2004. Use of reporter cell lines for detection of endocrine disrupter activity. Anal. Bioanal. Chem. 378, 655–663.
The MMV-Luc cell line was generated from a human mammary gland cell line by stable transfection with the luciferase gene under the control of a steroid hormone inducible promoter and is specific for the detection of estrogens.

Cells were routinely cultured in 75 cm2 tissue culture flasks at 37 °C with 5% CO2 and 95% humidity. The cells were routinely grown in cell culture medium containing DMEM without phenol red, 10% (v/v) foetal bovine serum and 1% (v/v) penicillin–streptomycin. Cells were transferred at least two passages prior to RGA analysis into assay media, which was composed of DMEM without phenol red and 10% (v/v) hormone depleted serum.

Administration / exposure

Analytical verification of doses or concentrations:
Duration of treatment / exposure:
Doses / concentrations
Dose / conc.:
0.01 other: mM
No. of animals per sex per dose:
All experiments were performed in triplicate for each experimental point and repeated in 3 independent exposures.
Control animals:
yes, concurrent vehicle


For the initial agonist test, test compounds were spiked in media to give a final ‘on the plate’ concentration of 0.01 mM
An antagonist test was carried out by co-incubating the test compounds (also at a concentration of 0.01 mM) with 0.01 mM estradiol.
Cytotoxicity of the test material was tested in the MTT assay.
Positive control:
The estradiol calibration curve was prepared in the range of 0.0005–10 nM

Results and discussion

Details on results:
The substance did not activate the estrogen receptor in this reportergene assay. Also, the substance did not interfere with normal estrogen binding in the assay for antagonist activity.

The substance was not among those that were mentioned as cytotoxic to the reporter cell line. It is therefore assumed to have been not cytotoxic.

Applicant's summary and conclusion