Registration Dossier

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