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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
toxicity to reproduction
Remarks:
other: Androgen receptor binding potential assay
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Neither guideline test nor obey with GLP, but fulfill with basically scientific principles.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2002
Reference Type:
publication
Title:
A rapid, specific protocol for determination of available androgen receptor sites in unfractioned rat ventral prostate cytosol preparations
Author:
Boesel RW, Shain SA
Year:
1974
Bibliographic source:
Biochem. Biophys. Res. Comm. 61: 1004-1011.

Materials and methods

Test guideline
Qualifier:
no guideline available
Principles of method if other than guideline:
The principles of this method is using a rat recombinant fusion protein containing both hinge region and ligand binding domain of the AR as receptor source and radiolabeled methyltrienolone as ligand. Then the test substance was added to the system, which aimed to investigate the affinity of the test substance for the AR by comparing the replacement potential of test substance for "radiolabeled methyltrienolone" with the potential of the reference compounds of dihydrotestosterone and androstenedione.
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
In vitro method with a rat recombinant fusion protein containing both the hinge region and ligand binding domain of the AR as receptor source and methyltrienolone as ligand.

Administration / exposure

Route of administration:
other: not relevant for in vitro method
Vehicle:
DMSO
Remarks:
2 %
Details on exposure:
100 μL assay buffer [50 mM Tris-HCl pH 7.5, 0.8 M sodium chloride, 2 mM DTT, 10% glycerol (V/V)] containing γ-globulin (20 mg/mL) and 2% DMSO (with and without test substance), 50 μL of an 8 nM solution of radiolabeled R1881 in assay buffer containing 1.1% ethanol and 50 μL of an 8 nM AR solution in assay buffer were fully mixed in microtitre plate wells. Final concentrations were 10 mg/mL γ-globulin, 2 nM R1881 and 2 nM AR. Following incubation at 4 ℃ overnight under continuous shaking, 50 μL of a 5 % charcoal suspension in assay buffer was added. After samples mixed at 4 ℃ for 10 min, charcoal was sedimented by centrifugation at 4000 rpm for 5 min. Then 50 μL aliquots of the clear supernatant were analyzed by liquid scintillation counting.
Details on mating procedure:
not applicable
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not applicable
Duration of treatment / exposure:
10 min
Frequency of treatment:
Not applicable
Details on study schedule:
not applicable
Doses / concentrations
Remarks:
Doses / Concentrations:
10 nM to 100000 nM
Basis:
other: Nominal in assay buffer
No. of animals per sex per dose:
Not applicable
Control animals:
other: six fold incubations of vehicle (DMSO) alone
Details on study design:
3H activity as a measure of ligand concentrations was determined by liquid scintillation counting (LSC). Aliquots (50 μl) of assay buffer were taken at least in triplicate and were mixed with 5 mL scintillation cocktail (Ultima Gold). Samples were counted for 5 minutes. Decays per minute were calculated from counts per minutes using a calibration curve established and regularly checked by a set of differentially quenched 3H standards.

Aliquots (50 μL) containing the AR-ligand complex were mixed with 200 μL Ultima Flo AP scintillation cocktail and radioactivity was counted for 10 min in the LSC microplate reader with one hour delay allowing samples to equilibrate.
Positive control:
Dihydrotestosterone (Strong binding reference) and androstenedione (Weak binding reference) in the range of 0.1 nM to 300 nM and 30 nM to 100000 nM, respectively.

Examinations

Parental animals: Observations and examinations:
Not relevant
Oestrous cyclicity (parental animals):
Not relevant
Sperm parameters (parental animals):
Not relevant
Litter observations:
Not relevant
Postmortem examinations (parental animals):
Not relevant
Postmortem examinations (offspring):
Not relevant
Statistics:
Samples were counted for 5 minutes. Decays per minute were calculated from counts per minutes using a calibration curve established and regularly checked by a set of differentially quenched 3H standards.
Reproductive indices:
Not relevant
Offspring viability indices:
Not relevant

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
not specified
Description (incidence and severity):
not applicable
Body weight and weight changes:
not specified
Description (incidence and severity):
not applicable
Food consumption and compound intake (if feeding study):
not specified
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
not specified
Description (incidence and severity):
not applicable
Gross pathological findings:
not specified
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
not specified
Description (incidence and severity):
not applicable
Other effects:
not specified
Description (incidence and severity):
Test substance intake: not applicable

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not specified
Description (incidence and severity):
not applicable
Reproductive function: sperm measures:
not specified
Description (incidence and severity):
not applicable
Reproductive performance:
not specified
Description (incidence and severity):
not applicable

Details on results (P0)

not applicable

Effect levels (P0)

Dose descriptor:
other: in vitro binding to the androgen receptor
Basis for effect level:
other: see 'Remark'
Remarks on result:
not measured/tested
Remarks:
Effect level not specified Generation not specified (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
not specified
Description (incidence and severity):
not applicable
Mortality / viability:
not specified
Description (incidence and severity):
not applicable
Body weight and weight changes:
not specified
Description (incidence and severity):
not applicable
Sexual maturation:
not specified
Description (incidence and severity):
not applicable
Organ weight findings including organ / body weight ratios:
not specified
Description (incidence and severity):
not applicable
Gross pathological findings:
not specified
Description (incidence and severity):
not applicable
Histopathological findings:
not specified
Description (incidence and severity):
not applicable

Details on results (F1)

not applicable

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

The results from the test shows the test substance can moderately displace the radiolabeled ligand methyltrienolone from the androgen receptor (AR) in a concentration dependent manner. However, this displacement was weak, occurred only at high concentrations. The concentration-response relationship - especially at higher concentrations was flat, and a corresponding IC50 -value can not be established. In contrast, the reference substances dihydrotestosterone (DHT) and androstenedione (ANDRO) can displace the radiolabeled ligand methltrienolone from the AR with a significantly steep concentration - response relationship. The concentration-response curve obtained for the test substance was not paralleled with DHT and ANDRO thus suggesting an unspecific interaction with AR binding.

Applicant's summary and conclusion

Conclusions:
The test substance inhibited to some extent the binding of the test ligand methyltrienolone to the androgen receptor, but even at the highest tested concentration of 0.1 mM did not achieve 50% inhibition; from this and the shallow dose-response curve compared to the other ligands dihydrotestosterone and androstendione, it was concluded that homosalate did not bind to the hormone binding site on the androgen receptor.
Executive summary:

Potential of interaction of the registration substance with the androgen receptor (AR) was investigated by means of a classical receptor binding assay using a rat recombinant fusion protein containing both hinge region and ligand binding domain of AR as receptor source and radiolableled methyltrienolone as ligand.

The test substance weakly displaced the radiolabeled ligand methyltrienolone from the AR in a concentration - dependent manner. However, displacement at the maximum concentration of 100 mM can not exceed 32% and 41%, respectively, in two separated tests and accordingly IC50 can not be obtained. In addition, the concentration - response curve was flat and did not parallel those observed for DHT and ANDRO. Thus, it can be concluded that the test substance can not generate the androgen binding concern.