2,2'-[6-(4-methoxyphenyl)-1,3,5-triazine-2,4-diyl]bis{5-[(2-ethylhexyl)oxy]phenol}

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

In vitro androgen competitive binding assay

GLP compliance:

no

Type of method:

in vitro

Test material

Test material information

Constituent 1

Administration / exposure

Details on study design:

Rat prostates were used as a source of cytosolic androgen receptors (AR).
Ten 8 week-old male Alpk: APfSD rats were each given a single subcutaneous injection of the GnRH antagonist Antarelix dissolved in hydroxypropyl methoxycellulose (0.3mg/kg). The animals were killed 24h later by an overdose of Fluothane followed by cervical dislocation, and ventral prostates were removed.
Finely minced prostates were homogenised in "low salt" buffer (1 gm of tissue per 10ml buffer) consisting of Tris (10 mM), glycerol (10% v/v), sodium molybdate (1 mM), EDTA (1.5 mM), dithiothreitol (1 mM) and phenylmethylsulphonyl fluoride (1mM), pH 7.4. The homogenate was centrifuged at 30,000g for 0.5h at 4° to yield the cytosol fraction.
The assay entailed the incubation of prostate cytosol (200 µl) with 3H-R1881 (0.19μCi; = 5x l0^-9M) and the required dose level of the putative competitor in DMSO (10 μl), all in a final volume of 500 μl, for 17h at 4°C. Serial, ten-fold dilutions of each compound (positive controls and test agents) were made in DMSO. These concentrations were so arranged as to give the specified molarity when 10 µl of each dosing preparation was diluted to 500 μl.
Two separate experiments were conducted. Both experiments contained a range of ten-fold dilutions of each test agent (5x10^-10 to 5x10^-4 M) and of R1881 (5x10^-10 to 5x10^-6 M) and testosterone (5x10^-10 to 5x10^-6 M in Experiment 1 and 5x10^-10 to 5x10^-7M in Experiment 2). Each experiment consisted of duplicate incubations of all the putative androgen receptor competitors and also of incubations with solvent alone (DMSO) to ascertain the 100% binding value. Duplicate incubations o 3H-R1881 in low salt buffer (i.e. in the absence of cytosol) gave a measure of non-specific binding.
The receptor-ligand complex was isolated at the termination of each incubation by the addition of hydroxylapatite (250μl of 60% v/v in Tris buffer [50mM]). Each precipitate was collected by centrifugation and washed three times with Tris buffer (50mM), collecting by centrifugation after each wash. The washed precipitates were suspended and the radioactivity determined in a Liquid Scintillation Analyser.

Results and discussion

Observed effects

The positive control compounds (R1881 [methyl trienolone] and testosterone) both gave the expected responses, competing with 3H-R1881 at concentrations observed previously in this laboratory.
In contrast, the test substance gave no indication of activity at any of the dose level tested.

Any other information on results incl. tables

Experiment 1:

CGF-C1607 binding % compared to solvent control (DMSO)

5x10^-10M:     96.8%

5x10^-9M:       97.3%

5x10^-8M:       100.2%

5x10^-7M:       98.8%

5x10^-6M:       99.8%

5x10^-5M:       98.2%

5x10^-4M:       93.6%

Testosterone (5 x10^-6):       9.8%

R1881 (5 x10^-6):        9.9%

Experiment 2:

CGF-C1607 binding % compared to solvent control (DMSO)

5x10^-10M:     101.7%

5x10^-9M:       99.9%

5x10^-8M:       102.6%

5x10^-7M:       102.6%

5x10^-6M:       100.8%

5x10^-5M:       102.0%

5x10^-4M:       100.7%

Testosterone (5 x10^-7):       13.4%

R1881 (5 x10^-6):        12.0%