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Toxicological information

Genetic toxicity: in vitro

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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:
22. May to 22. Aug. 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
(1997)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Androstendion
- Lot/batch No.: 72009028
- Purity: 99.86 %
- Stability under test conditions: The stability of the test substance in the vehicle was analytically verified for at least 4 hours.

Method

Target gene:
HPRT locus
Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM with supplements; for the selection of mutant cells the medium was supplemented with 11 µg/mL 6-thioguanine.
- Properly maintained: yes
- Periodically "cleansed" against high spontaneous background: yes
- Periodically checked for karyotype stability: yes
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix from the liver of phenobarbital/ß-naphthoflavone induced rats.
Test concentrations with justification for top dose:
Experiment I: treatment time 4 hours, with and without S9 mix 7.5, 15.0, 30.0, 60.0, 120.0, and 240.0 µg/mL
Experiment II: treatment time 4 hours, with S9 mix 7.5, 15.0, 30.0, 60.0, 120.0, 180.0, and 240.0 µg/mL; treatment time 24 hours, without S9 mix 1.89, 3.75, 7.5, 15.0, 30.0, 60.0, 90.0, and 120.0 µg/mL.
Vehicle / solvent:
DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: ethylmethanesulfonate (EMS; 1.2 mM), 7,12-dimethylbenzanthracene (DMBA; 4.3 µM)
Remarks:
EMS was used without and DMBA with metabolic activation
Details on test system and experimental conditions:
PRE-TEST: Test item concentrations ranged between 15.0 ¿g/mL and 1920 ¿g/mL in the pre-experiment. As result no relevant toxic effect occurred up to the maximum concentration tested with and without metabolic activation following 4 hour treatment. A transient reduction of the cloning efficiency to 44.1 % at 480 ¿g/mL without metabolic activation was judged as precipitation artefact rather than true cytotoxicity as the cloning efficiency was back to normal at higher concentrations. Following 24 hours treatment without metabolic activation cytotoxic effects occurred at 120 ¿g/mL and above.

METHOD OF APPLICATION: in medium
Approximately 1.5×10exp6 (single culture) and 5×102 cells (in duplicate) were seeded in plastic culture flasks. The cells were grown for 24 hours prior to treatment. After 24 hours the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 ¿l/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 hours this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 hours in complete medium, supplemented with 10 % FBS, in the absence of metabolic activation.
Three or four days after treatment 1.5×10exp6 cells per experimental point were sub-cultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×10exp5 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution.
The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 8 days

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points. A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows: A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment. The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed. However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory¿s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolarity: There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
- Water solubility: The maximum concentration of the pre-experiments (1920 ¿g/mL) was based on the solubility properties of the test item in DMSO and aqueous medium.
- Precipitation: No precipitation of the test item was observed up to the maximum analyzable concentration in both main experiments. In the pre-experiments precipitation occurred at 120 ¿g/mL and above after 4 hours treatment with and without metabolic activation, and at 480 ¿g/mL and above following 24 hours treatment without metabolic activation.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Cytotoxic effects indicated by a relative cloning efficiency I or relative cell density below 50 % of the corresponding solvent control were noted at 120 ¿g/mL in the first main experiment without metabolic activation. In the presence of metabolic activation no cytotoxicity was observed at the maximum analyzable concentration of 120 ¿g/mL. At the next higher concentration of 240 ¿g/mL exceedingly severe cytotoxic effects precluded analysis of any data on mutagenicity. In the second experiment cytotoxicity as described above occurred at 180 ¿g/mL with and at 120 ¿g/mL without metabolic activation. The recommended cytotoxic range of approximately 10-20 % relative cloning efficiency I or relative cell density was covered with and without metabolic activation.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
negative
Executive summary:

The test item did not induce substantial and reproducible dose dependent increase of the mutation frequency in a mammalian cell gene mutation assay (HPRT) according to OECD TG 476. In this assay two independent experiments were conducted with V79 cells being treated for either 4 hours with and without metabolic activation or 24 hours without metabolic activation. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. Therefore, the substance was considered to be non-mutagenic in the specified test.