Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

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:
Estrogenic activities of chemieals related to food contact plastics and rubbers tested by the yeast two-hybrid assay
Ogawa et al
Bibliographic source:
Food Additives and Contaminants, April, 2006; (23)4: 422-430
Report date:

Materials and methods

Test guideline
no guideline followed
GLP compliance:
Type of method:
in vitro
Endpoint addressed:
other: estrogenic activity

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Details on test material:
no further data provided
Specific details on test material used for the study:
purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan), Wako Pure Chemical Industries, Ltd. (Osaka, Japan), Sigma-Aldrich Japan Co. (Tokyo, Japan), or obtained from the manufacturers

Test animals

other: yeast, expressing the estrogen receptor alpha and the coactivator TIF-2

Administration / exposure

Route of administration:
other: in the culture medium
Analytical verification of doses or concentrations:
Doses / concentrations
Doses / Concentrations:
final concentrations: 10-3 to 10-7 mol/l
nominal conc.
Details on study design:
Test chemicals were dissolved in dimethylsulfoxide (DMSO) at 10-1 to 10-5 mol/l (final concentrations: 10-3 to 10-7 mol/l). When the chemical could not be dissolved at 10-1 mol/l, the concentration was changed to 10-2 to 10-5 mol/l (final concentrarions: 10-4 to 10-7 mol/l).
The concentrarion of DMSO was 1% in the assay, which did not inhibit the yeast growth. Each experiment was accompanied by 17 ß-estradiol (E2) as the positive control and DMSO as the negative control.
The yeast two-hybrid cells were preincubated overnight at 30°C with vigorous shaking in a SD mediumwhich was free from tryptophan and leucine. The
culture was diluted with 4 volumes of the fresh SD medium and 250 ul of this solution put into a small test tube. The test chemical solution (2.5 ul) was added and incubated for 4h at 30°C. After incubation, 150 ul of the culture solution was placed into each of the 96 wells of a microplate and the absorbancy measured at 595 nm. The rest of the culture was centrifuged at 10,000 rpm for 7 min, after which the supematant was removed. The cells were enzymatically digested by incubation with 1 mg/ml Zymolyase 20T (200 ul) at 30°C for 15 min. The celllysate was mixed with 4mg/ml ONPG (40 ul) and incubated at 30°C for exactly 30 min. The reaction was stopped by the addition of 1 mol/l Na2C03 (100 uI). After centrifugation at 10,000 rpm for 5 min, the supernatant (150 ul) was placed into each well of a micrcplate. The absorbances at 420 and 570nm were read using a microplate reader.
Preparation of metabolites and their measurement of estrogenic activity:
To a tube containing 990 Jll of the S9-mix, 10 ul of the test chemical solution (mainly 10-1 to I0-5 mol/1 which corresponds to 5 x 10-4 to 5 x I0-8 mol/l of final concentration) was added, incubated at 37°C for 4h and then stored at -80°C until the yeast two-hybrid test was run as metabolite solution. Each experiment was accompanied by trans-stylbene to confirm the metabolic activity. The yeast two-hybrid cells were pre-incubated overnight at 30° C with vigorous shaking in a SD medium free from tryptophan and leucine, then diluted with 1. 5 volumes of fresh 2 x SD medium. In a small test tube, 125 ul ofthe cell solution and 125 ul of the metabolite solution were mixed and then incubated at 30°C for 4 h. Thereafter, the same procedure as the Measurement of estrogenic activity
by yeast two-hybrid assay was carried out.

Results and discussion

Details on results:
The results were evaluated based on relative activity, expressed as 10% relative effective concentration of the test chemical showing 10% agonist activity of 10-6 M β-estradiol. The test item did not show estrogenic activity in this yeast two hybride system

Applicant's summary and conclusion