3-methoxyoestra-1,3,5(10)-trien-17-one

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-methoxyoestra-1,3,5(10)-trien-17-one

EC Number:

216-613-3

EC Name:

3-methoxyoestra-1,3,5(10)-trien-17-one

Cas Number:

1624-62-0

Molecular formula:

C19 H24 O2

IUPAC Name:

3-methoxyoestra-1,3,5(10)-trien-17-one

Specific details on test material used for the study:

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
On the day of the experiment, the test article was dissolved in the vehicle. The test article formed a suspension in the stem solution.

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 102 and TA 100

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 microsomal fraction

Test concentrations with justification for top dose:

10.0, 33.3, 100.0, 333.3, 1000.0, 2500.0, and 5000.0 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: - S9 Mix: Sodium azide: TA100, TA 1535; 4-NOPD: TA 1537, TA 98; MMS: TA 102; + S9 Mix: 2-AA all strains

Details on test system and experimental conditions:

plate incorporation assay

For each strain and dose level, including controls three plates were used as a minimum.

The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl: Test solution at each dose level, solvent (negative control) or control mutagen solution (positive control)
500 µl: S9 mix (metabolic activation) or S9 mix substitution buffer (without metabolic activation)
100 µl: Bacteria suspension
2000 µl: Overlay agar

After solidification the plates were incubated upside down for at least 48 hours at 37°C in the dark.

Data recording:
The colonies were counted using the AUTOCOUNT (Artek Systems Corporation, Biosys GmbH, 51184 Karben)

The background growth of the bacteria was judged visually on a light bench.

Evaluation criteria:

Generally accepted conditions for the evaluation of the results are:
- corresponding background growth on both negative control and test plates
- normal range of spontaneous reversion rates

Evaluation of results:

A test article is considered positive if either a dose related increase in the number of revertants or a biological relevant increase for at lest one test concentration is induced.
A test article producing neither a dose related increase in the number of revertants nor a biological relevant positive response at any of the test points is considered non-mutagenic in this system.
A significant response is described as follows:
A test article is considered mutagenic if the number of reversions is at least twice the spontaneous rate in strains TA 100 and TA 102 or thrice TA 1535, TA 1537, and TA 98.
Also, a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test article regardless whether the highest dose induced the criteria described above or not.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Minor toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 1537 without metabolic activation at concentrations of 1000 µg/plate and above.

Applicant's summary and conclusion

Conclusions:

negative
Estrone methyether did not show a mutagenic potential in the bacterial reverse mutation assay.

Executive summary:

The mutagenic potential of Estrone methylether was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 in the presence and absence of S9 mix according to OECD TG 471.

The assay was performed in one experiment with and without Iiver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations:

10.0; 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate.

Minor toxic effects, evident as a reduction in the number of revertants, occurred in strain TA 1537 without metabolic activation at concentrations of 1000 µg/plate and above.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with ZK 5512 at any dose level, either in the presence or absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate control mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base-pair changes or frame-shifts in the genome of the strains used.

Therefore, the substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.