Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

Specific investigations: other studies

Administrative data

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Meets generally accepted scientific standards, well documented and acceptable for assessment. No data on purity were available.

Data source

Reference

Reference Type:

publication

Title:

Estrogenic activities of chemicals related to food contact plastics and rubbers tested by the yeast two-hybrid assay.

Author:

Ogawa Y et al.

Year:

2006

Bibliographic source:

Food Additives and Contaminants (23) 4: 422-430.

Materials and methods

Principles of method if other than guideline:

A yeast two-hybrid assay was performed. The results were evaluated on the basis of the relative activity, expressed as 10% relative effective concentration (REC10), which is the concentration of the test chemical showing 10% of the agonist activity of 10xE-6 mol/L 17-beta-Estradiol, the highest activity level of 17-beta-Estradiol.

GLP compliance:

no

Type of method:

in vitro

Endpoint addressed:

other: estrogenic activity

Test material

Test material information

Constituent 1

Reference substance name:

Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

EC Number:

265-512-0

EC Name:

Calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

Cas Number:

65140-91-2

Molecular formula:

C17 H29 O4 P. 1/2Ca

IUPAC Name:

calcium diethyl bis[[[3,5-bis(1,1-dimethylethyl)-4-hydroxyphenyl]methyl]phosphonate]

Details on test material:

Purity: No data

Administration / exposure

Details on study design:

- The yeast two-hybrid cells were preincubated overnight at 30°C with vigorous shaking in a SD medium which was free from tryptophan and leucine. The culture was diluted with 4 volumes of the fresh SD medium and 250 µl of this solution put into a small test tube. The test chemical solution (2.5 µl) was added and incubated for 4 h at 30° C. After incubation, 150 µl 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 supernatant was removed. The cells were enzymatically digested by incubation with 4mg/mL Zymolyase 20T (200 µl) at 30°C for 15 min. The cell lysate was mixed with 4 mg/mLI ONPG (40 µl) and incubated at 30°C for exactly 30 min. The reaction was stopped by the addition of 1 mol/L Na2CO3. After centrifugation at 10000 rpm for 5 min, the supernatant (150 µL) was placed into each well of a microplate. The absorbances at 420 and 570 nm were read using a microplate reader. The ß-galactosidase activity was calculated using the following equation: U=1000 x ([OD420] - [1.75 x OD570])/([t] x [v] x [OD595]), where t = time of reaction (min), v = volume of culture used in the assay (mL), OD595 =cell density at the start of the assay, OD420 = absorbance by o-nitrophenol at the end of the reaction, and OD570 = light scattering at the end of the reaction. The ß-galactosidase activity was expressed as the mean and standard deviation of the results from three separate test tubes.
- Preparation of metabolites and their measurement of estrogenic activity:
To a tube containing 990 µl of the S9-mix, 10 µL of the test chemical solution (mainly 10xE-1 to 10xE-5 mol/L) 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 µL of the cell solution and 125 µL of the metabolite solution were mixed and then incubated at 30°C for 4 h. Thereafter, the same procedure as described in the first part was carried out.
- Data analysis
The results were evaluated on the basis of the relative activity, expressed as 10% relative effective concentration (REC10), which is the concentration of the test chemical showing 10% of the agonist activity of 10xE-6 mol/L 17-beta-Estradiol, the highest activity level of 17-beta-Estradiol. When the activity of the test chemical was higher than the REC10 within the concentration range tested, the chemical was judged to be positive. When it was judged to be negative, more than the highest dose tested was indicated.

Results and discussion

Details on results:

The test substance was negative in this test system. REC10 values of >1.0 x 10E-4 (parent compound) and >5.0 x 10E-5 (metabolite) were obtained.