Androstane-3,17-diol, 2-(4-morpholinyl)-16-(1-pyrrolidinyl)-, (2.beta.,3.alpha.,5.alpha.,16.beta.,17.beta.)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2 August to 8 November 1999

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(3aS,3bR,9aS,9bR,11aS)-9a,11a-dimethyl-8-(morpholin-4-yl)-2-(pyrrolidin-1-yl)-hexadecahydro-1H-cyclopenta[a]phenanthrene-1,7-diol

EC Number:

601-595-5

Cas Number:

119302-20-4

Molecular formula:

C27H46N2O3

IUPAC Name:

(3aS,3bR,9aS,9bR,11aS)-9a,11a-dimethyl-8-(morpholin-4-yl)-2-(pyrrolidin-1-yl)-hexadecahydro-1H-cyclopenta[a]phenanthrene-1,7-diol

Test material form:

other: Solid

Details on test material:

- Name of test material (as cited in study report): Pymordiol
- Physical state: Cream white solid
- Purity of test material: 96 wt%
- Lot/batch No.: DGM096K1A
- Expiration date of the lot/batch: 1 January 2001
- Stability under test conditions: Stable throughout study period
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

Thymidine kinase (TK)

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Mouse lymphoma cells were cultured in F10 complete culture medium. Cell density was preferably kept below 7 x 10^5 cells/mL. F10 complete culture medium consisted of Ham's F10 medium without thymidine and hypoxanthine (Gibco), supplemented with 10% (v/v) horse serum, L-glutamine (2 mM) and penicillin/streptomycin (50 U/mL and 50 µg/mL respectively).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically "cleansed" against high spontaneous background: Yes, Prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in F10 complete culture medium containing 10^-4 M hypoxanthine, 2x10^-7 M aminopterin and 1.6 x10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on medium containing hypoxanthine and thymidine only. After this period cells were returned to normal medium at least for 1 day before starting the experiment.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-fraction

Test concentrations with justification for top dose:

Within and without S9-mix: 0.1, 0.3, 0.56, 1.0, 1.8, 3.3, 5.6, and 10 µg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulphoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: Dimethylnitrosamine (DMN)

Details on test system and experimental conditions:

METHOD OF APPLICATION: In medium, F10 complete culture medium.

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 5 days
- Selection time (if incubation with a selection agent): 9 days
- Fixation time (start of exposure up to fixation or harvest of cells): 3 days

SELECTION AGENT (mutation assays): F10 complete culture medium, supplemented with 10% (v/v) horse serum and 5µg/mL TFT-Selection
SPINDLE INHIBITOR (cytogenetic assays):
STAIN (for cytogenetic assays): 0.5 mg/mL MTT (3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Sigma)

NUMBER OF REPLICATIONS: 9.6 x 10^5 cells/concentrations, each well containing 2500 cells in selective medium (TFT-selection)

NUMBER OF CELLS EVALUATED: 8 x 10^6 cells (10^6/mL), if test substance concentration was expected to be toxic, 16 x 10^6 cells (10^6/mL) were used per culture.

DETERMINATION OF CYTOTOXICITY
- Method: Cloning efficiency (CE)

Evaluation criteria:

Colonies were divided in small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severe affected mutant cells have grown at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appeared to result from mutants with single gene mutation (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphological dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphological dense colonies with a hazy contour and with a diameter larger than a quarter of a well.

Statistics:

Calculation of the cloning efficiency (CE) was determined by dividing the number of empty wells by the total number of wells. This value was called P(0), the zero term of the Poisson distribution (equation below).
The mutation frequency (MF) was expressed as the number of mutants per 10^5 surviving cells (equation below).

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in the exposition medium at a concentration of 10 µg/mL.

RANGE-FINDING/SCREENING STUDIES: In the absence of S9-mix after 3 hours of treatment, the toxicity in the suspension growth was 32% at the test substance concentration of 10 µg/mL compared to the suspension growth of the solvent control.
In the absence of S9-mix after 24 hours treatment, the toxicity in the suspension growth was 34% at the test substance concentration of 10 µg/mL compared to the suspension growth of the solvent control.
In the presence of S9-mix after 3 hours treatment, no toxicity in the suspension growth was observed in all concentrations tested compared to the solvent control.

COMPARISON WITH HISTORICAL CONTROL DATA: Cloning efficiency of the remaining cells was comparable to the solvent controls even at the highest tested dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Both in the absence and presence of S9 -mix, after 3 hours treatment no significant reduction in the cell count was observed even in the highest dose level tested.

The test substance did not induce the mutant frequency at the TK locus either in the absence or in the presence of S9 -mix.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the current test conditions the test substance was concluded to be non-mutagenic in mouse lymphoma L5178Y.