2-phenyl-1H-benzimidazole-5-sulphonic acid

Toxicity to reproduction: other studies

Administrative data

Endpoint:

toxicity to reproduction: other studies

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 2002

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: scientifically valid method was used and study is adequately reported

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Qualifier:

no guideline available

Principles of method if other than guideline:

The aim of this in vitro investigation was to evaluate a potential affinity of test article sodium salt for the androgen receptor (AR). The experimental system employed was capable of detecting strongly and weakly binding compounds such as dihydrotestosterone and androstenedione.
ln principle, the method described by Boesel and Shain (A rapid, specific protocol for determination of available androgen receptor sites in unfractioned rat ventral prostate cytosol preparations. Biochem Biophys Res Comm 61: 1004-1011, 1974) was applied, however, a rat recombinant fusion protein containing both the hinge region and ligand binding domain of the AR served as receptor source, whereas radiolabeled methyltrienolone was used as receptor ligand according to Kelce et al. (Kelce WR, Monosson E, Gamcsik MP, Laws SC, Gray LE jr., Environmental hormone disruptors: Evidence that vinclozolin developmental toxicity is mediated by antiandrogenic metabolites. Toxicol Appl Pharmacol 126: 276-285, 1994).

GLP compliance:

yes (incl. QA statement)

Type of method:

in vitro

Test material

Test material information

Constituent 1

Reference substance name:

2-phenyl-1H-benzimidazole-5-sulphonic acid

EC Number:

248-502-0

EC Name:

2-phenyl-1H-benzimidazole-5-sulphonic acid

Cas Number:

27503-81-7

Molecular formula:

C13H10N2O3S

IUPAC Name:

2-phenyl-1H-benzimidazole-5-sulphonic acid

Test animals

Details on test animals or test system and environmental conditions:

Biological material:
Androgen receptor (AR): rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain of the AR (PanVera, Madison, Wl, USA).
Equipment:
Labofuge A (Heraeus, Osterode, FRG)
Liquid scintillation counter 1900 TR (Canberra-Packard, Frankfurt, FRG)
Liquid scintillation counter 1450 Microbeta™ Trilux (Wallac, Freiburg, FRG)
Chemicals:
Tris(hydroxymethyl)-aminomethane (Tris) p.a. (E. Merck, Darmstadt, FRG)
Sodium chloride (KMF, St. Augustin, FRG)
Dithiothreitol (DTT ) (Sigma, Deisenhofen, FRG)
Glycerol anhydrous pure (E. Merck, Darmstadt, FRG)
Human y-globulin 96 - 99 % (Sigma, Deisenhofen, FRG)
Dimethylsulfoxid p.a. (DMSO) (E. Merck, Darmstadt, FRG)
Ethanol p.a. (E. Merck, Darmstadt, FRG)
Ultima Gold™ scintillation cocktail (Canberra-Packard, Frankfurt, FRG)
Ultima Flo AP™ scintillation cocktail (Canberra-Packard, Frankfurt, FRG)
Radiochemicals:
Methyltrienolone, [ 17a-Methyl -3 H] (R 1881) 3089.5 GBq/mmol (NEN Du Pont, Dreieich, FRG)
Fine chemicals, reference:
Dextrane coated charcoal (Sigma, Deisenhofen, FRG)
Methyltrienolone (R 1881) (NEN Du Pont, Dreieich, FRG)
4-Androstene-3,17-dione 98 % (Sigma, Steinheim, FRG)
Dihydrotestosterone (DHT) >99 % (Sigma, Steinheim, FRG)

Administration / exposure

Details on exposure:

Test compound was incubated overnight with 2 nM androgen receptor and 2 nM radiolabeled methyltrienolone. After removal of unbound ligand by adsorption to charcoal and centrifugation, receptor-bound methyltrienolone was determined by liquid scintillation counting. Incubations were performed in triplicate (control: n = 6 / coefficient of variation =3 %). Receptor binding in the presence of excess (5 µM) unlabeled R1881, i.e., unspecific binding was substracted. Ligand bound in the presence of test compound was related to ligand bound in the absence of compound and expressed as percentage of control.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

overnight

Doses / concentrations

Dose / conc.:

1 other: mmol

Remarks:

Basis:
analytical conc.
test article sodium salt

Control animals:

other: dihydrotestosterone (DHT) and androstenedione (ANDRO)

Details on study design:

In order to screen for a potential affinity of test article sodium salt for the androgen receptor (AR), potential AR binding of test article sodium salt was investigated by means of a classical receptor binding assay using a rat recombinant fusion protein containing both the hinge region and ligand binding domain of the AR as receptor source and radiolabeled methyltrienolone as ligand. The physiological androgens dihydrotestosterone and androstenedione served as reference compounds with strong and weak affinity for the AR, respectively.

Statistics:

no data

Results and discussion

Effect levels

Key result

Dose descriptor:

other:

Basis for effect level:

other: No binding to the androgen receptor at the maximally employable concentration of 1 mM.

Remarks on result:

not measured/tested

Any other information on results incl. tables

An affinity of test article sodium salt for the androgen receptor (AR) at the maximally employable concentration of one millimolar could not be demonstrated in two independent experiments. Within the experimental error, the quantity of radiolabeled ligand methyltrienolone bound in the presence of the test compound was comparable to that of the control. The reference compounds dihydrotestosterone (DHT) and androstenedione (ANDRO) reproducibly displaced radiolabeled methyltrienolone from the AR with IC50-values of 4.7 nM and 4.6 nM for DHT and 2.4 pM and 2.0 pM for ANDRO, respectively. In the presence of test article sodium salt generally no displacement of ligand was observed. A slight drop in ligand binding at the maximally testable concentration of 1 mM in one experiment representing the edge of the experimental error, was indistinguishable from that observed in the presence of 1 µM in the same experiment, and thus was not confirmed in the other experiment.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this assay system, test article sodium salt was found to have no affinity for the androgen receptor (AR).

Executive summary:

This study was performed to investigate the potential affinity of test article for the androgen receptor (AR) by means of a classical receptor binding assay using a rat recombinant fusion protein containing both the hinge region and ligand binding domain of the AR as receptor source and radiolabeled methyltrienolone as ligand.

Whereas reference compounds dihydrotestosterone and androstenedione reproducibly displaced radiolabeled methyltrienolone from the AR, an affinity of test article sodium salt for the AR at the maximally employable concentration of one mmol could not be demonstrated.