3β-hydroxyandrost-5-en-17-one acetate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiated in March 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Prasterone

EC Number:

200-175-5

EC Name:

Prasterone

Cas Number:

53-43-0

Molecular formula:

C19H28O2

IUPAC Name:

(3S,8R,9S,10R,13S,14S)-3-hydroxy-10,13-dimethyl-1,2,3,4,7,8,9,11,12,14,15,16-dodecahydrocyclopenta[a]phenanthren-17-one

Test material form:

solid

Details on test material:

white solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: EM-760-17
- Expiration date of the lot/batch: not indicated

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: not indicated

OTHER SPECIFICS:
- CAS Registry Number: 53-43-0
- Generic Name: Prasterone
- Chemical Name: 3β-hydroxy-5-androsten-17-one or 3β-hydroxyandrost-5-en-17-one
- Molecular Weight: 288.4

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: according to OECD Guideline 473
- Whether whole blood or separated lymphocytes were used if applicable: whole blood

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
RPMI 1640 = culture medium

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Definitive study:
-S9 (4hr exposure): 400, 300, 225, 200, 175,……50 µg/ml - Doses evaluated for chromosome aberrations: 138, 175, 200, 225 µg/ml
-S9 (19 hr exposure): 138, 100, 66, 50……..6.6 µg/ml - Doses evaluated for chromosome aberrations: 25, 50, 66, 100 µg/ml
+S9 (4 hr exposure): 400, 300…………….66 µg/ml - - Doses evaluated for chromosome aberrations: 138, 175, 225; 300 µg/ml

Highest dose was selected based on the solubility limit of the test system

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not indicated

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

1 % ethanol vehicle

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9; 1.00 µg/mL (4h treatment), 0.300 µg/mL (19h treatment)

Controls 2

Untreated negative controls:

yes

Remarks:

1 % ethanol vehicle

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9; 25.0 µg/mL (4h treatment)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (RPMI 1640)
Lymphocytes normally do not divide, but they were stimulated to divide in cultures by exposure to phytohemagglutinin (PHA). At predetermined intervals after exposure to the test article, the lymphocytes were treated with a metaphase arresting substance, Colcemid, harvested, stained, and metaphase cells were analyzed for the presence of chromosomal aberrations.

DURATION
- Exposure duration: 4h or 19h
- Expression time (cells in growth medium): 18h (4h treatment) or 3h (19h treatment); 3h prior to fixation, Colcemid was added
- Fixation time (start of exposure up to fixation or harvest of cells): 22h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN (for cytogenetic assays): yes

NUMBER OF REPLICATIONS: Duplicates in each test.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not specified

NUMBER OF CELLS EVALUATED: 200 cells per dose level scored for aberrations, polyploidy and endoreduplication

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 200

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% mitotic index reduction)

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

according to the OECD Guideline 473.
The negative and vehicle control cultures must contain less than 5% cells with aberrations.

Statistics:

Positive control must be significantly higher (P<0.01) that vehicle controls. Statistical analysis method not specified.

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

mitotic index reduced up to 70% at the highest tested dose levels

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: not specified

RANGE-FINDING/SCREENING STUDIES:
no data

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: all positive control values were significantly higher than the vehicle controls (p<0.01)
- Negative (solvent/vehicle) historical control data: not specified

Any other information on results incl. tables

The study was considered valid

Applicant's summary and conclusion

Conclusions:

In conclusion, the study was negative with and without metabolic activation