Androst-4-ene-3,17-dione

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21. to 24 Nov. 1995

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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

December 29, 1992

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:

Androst-4-ene-3,17-dione

EC Number:

200-554-5

EC Name:

Androst-4-ene-3,17-dione

Cas Number:

63-05-8

Molecular formula:

C19H26O2

IUPAC Name:

androst-4-ene-3,17-dione

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix from the liver of Aroclor 1254 induced male rats

Test concentrations with justification for top dose:

up to 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide (NaN3; for TA1535 and TA100), 4-nitro-o-phenylene-diamine (4-NOPD; for TA1537 and TA98), methylmethanesulfonate (MMS; for TA102), 2-aminoanthracene (2-AA, for all strains)

Remarks:

The positive controls NaN3, 4-NOPD, and MMS were used without S9 mix; the positive control 2-AA with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) in triplicates; plates were incubated at 37 °C for approx. 48 hours; the frequency of revertant colonies were assessed using the Autocount (Biosys GmbH, Karben, Germany).

DETERMINATION OF CYTOTOXICITY
- Method: The background growth of the bacteria was judged visually on a light bench. Normal range of spontaneous reversion rates: TA1535 10-29, TA1537 5-28, TA98 15-57, TA100 77-189, TA102 121-293.

Evaluation criteria:

A test article is considered positive if either a dose related increase in the number of revertants or a biological relevant increase for at least 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 one of the test points is considered nonmutagenic in this system.
A significant response is described as folIows: A test article is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate in strains TA 98 and TA 100 and TA 102 or thrice on TA 1535 and TA 1537. 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 .

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

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 2

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

Species / strain:

S. typhimurium TA 98

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 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(no cytotoxicity with metabolic activation, but tested up to limit concentration)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

(no cytotoxicity with metabolic activation, but tested up to limit concentration)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the substance at any concentration level, either in the presence or absence of metabolic activation.
Appropriate control mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Toxic effects, evident as a reduction in the number of revertants, occurred in all test groups in the presence or absence of metabolic activation except in strains TA 100 and TA 102 with metabolic activation. These toxic effects started to occur in strain TA 1537 at concentrations of 1000 µg/plate and above with and without metabolic activation and in strain 1535 at 1000 µg/plate in the absence and at 2500 µg/plate in the presence of S9 mix. Toxic effects were restricted to the highest concentration of the test article in strains TA 98 with and without S9 mix and TA 100 and TA 102 in the absence of metabolic activation.
The background growth of the bacteria was reduced slightly at concentrations of 1000 µg/plate and severely at 2500 and 5000 µg/plate in strains TA 1535 and TA 1537.

Applicant's summary and conclusion

Conclusions:

The substance did not show a mutagenic potential in a bacterial reverse mutation assay according to OECD TG 471 (Ames test in S. typhimurium TA98, TA1537, TA100, TA102, TA1535) when tested up to the highest recommended dose level of 5.0 mg/plate in the absence or presence of metabolic activation. Appropriate control mutagens showed the expected increases in induced revertant colonies.

Executive summary:

The mutagenic potential of Androstendione was evaluated in a Salmonella/microsome test with the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of S9 mix according to OECD TG 471.

The assay was performed in one experiment with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test article was tested at the following concentrations: 33.3; 100.0; 333.3; 1000.0; 2500.0; and 5000.0 µg/plate.

Toxic effects, evident as a reduction in the number of revertants, occurred in all test groups with and without metabolic activation except in strains TA 100 and TA 102 with metabolic activation.

No substantial increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item 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 frameshifts in the genome of the strains used.

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