Estr-4-ene-3,17-dione, 11-hydroxy-, (11.alpha.)-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: According to guideline; under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

11a-Hydroxy-estr-4-ene-3,17-dione

EC Number:

613-901-4

Cas Number:

6615-00-5

Molecular formula:

C18H24O3

IUPAC Name:

11a-Hydroxy-estr-4-ene-3,17-dione

Details on test material:

- Name of test material (as cited in study report): 11-HN (11 alpha-hydronorandrostenedione)
- Physical state: solid
- Storage condition of test material: room temperature
- Other: Safety precautions - the routine hygienic procedures are sufficient to assure personnel health and safety.

Method

Target gene:

histidine

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: Stored as stock cultures in ampoules with nutrient broth (OXOID) supplemented with DMSO (8% w/w) )over liquid nitrogen.
- All strains contain mutations in the histidine operon. They contain the deep rough (rfa) mutation and deletion of the uvrB gene. Strains TA98, TA100 and TA102 contain the R-factor plasmid, pkM101.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction from rats treated with phenobarbital/beta-napthoflavone

Test concentrations with justification for top dose:

Preliminary test concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 ug/plate
Main test: 31.6, 100, 316, 1000, 2500 and 5000 ug/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the vehicle based on solubility of the test material and compatibility with the target cells.

Controls

Untreated negative controls:

yes

Remarks:

distilled water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Without metabolic activation: sodium azide (NaN3); 4-nitro-o-phenylene-diamine (4-NOPD); methylmethanesulfonate (MMS). With metabolic activation: 2-aminoanthracene (2-AA).

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment 1 - in agar (plate incorporation); Experiment 2 - preincubation.

DURATION
- Preincubation period: 60 minutes (experiment 2 only).
- Expression time (cells in growth medium): Plates were incubated at 37 degrees C for at least 48 hours

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used as a minimum.

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was determined by a clearing or diminuation of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.

OTHER: PRE-EXPERIMENT FOR TOXICITY: To evaluate the toxicity of the test article, a pre-experiment was performed with strains TA98 and TA100. Eight concentrations (3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 ug/plate) were tested for toxicity and mutation induction with each 3 plates.

Evaluation criteria:

Criteria of validity: A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical response to ampicillin (TA98, TA100, TA102)
- the control plates with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range): TA98: without S9: 16-46, with S9: 18-56; TA100: without S9: 77-174, with S9: 80-165; TA1535: without S9: 5-29, with S9: 5-27; TA1537: without S9: 5-28, with S9: 5-34; TA102: without S9: 164-399, with S9: 163-458.
- Corresponding background growth on negative control, solvent control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate.

Evaluation of mutagenicity: The mutation factor is calculated by dividing the mean value of the revertant counts through the mean values of the solvent control (the exact and not the rounded values are used for calculation). A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA98, TA100 and TA102 the number of reversions is at least twice as high as the reversion rate of the solvent control.
- If in tester strains TA1535 and TA1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Results and discussion

Test results

Species / strain:

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

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

Additional information on results:

In a preliminary toxicity assay, the maximum dose tested was 5000 ug/plate. Based on the findings of the preliminary toxicity assay, the maximum doses tested in the mutagenicity assay 5000 ug/plate in the presence and absence of S9 with all tester strains.

No precipitation of the test material was observed in any tester strains evaluated in experiments 1 and 2.

Toxic effects of the test material were noted in several tester strains evaluated in experiments 1 and 2. In experiment 1, toxic effects of the test item were observed in strain TA1535 at a concnetration of 5000 ug/plate (without metabolic activation) and at concentrations of 2500 ug/plate and higher (with metabolic activation). In experiment 2, toxic effects of the test material were noted in strains TA100 and TA1537 at a concentration of 5000 ug/plate (without metabolic activation). In strain TA1535, toxic effects of the test material were noted at concentrations of 2500 ug/plate and higher (without metabolic activation). The reduction in the number of revertants down to a mutation factor of 0.4 found in experiment 2 in strain TA1537 at a concentration of 100 ug/plate (without metabolic activation) was regarded as not biologically relevant due to the lack of a dose-response relationship.

No biologically relevant increases in revertant colony numbers of any of the 5 tester strains were observed following treatment with the test material at any concentration level, neither in the presence nor absence of metabolic activation in experiments 1 and 2.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the conditions of this study, the test material did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore the test material is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The study was performed to evaluate the potential mutagenicity of the test material in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA102, in both the presence and absence of S9 mix. The study was performed to GLP and was designed to the requirements of OECD 471, EU Method B.13/14, EPA OPPTS 870.5100. In a preliminary toxicity assay, the maximum dose tested was 5000 ug/plate. Based on the findings of the preliminary toxicity assay, the maximum doses tested in the mutagenicity assay 5000 ug/plate in the presence and absence of S9 with all tester strains. Positive controls appropriate for each strain, in the presence and absence of S9 mix, were included.

Two independent experiments were performed with and without metabolic activation. The concentrations, including the controls were tested in triplicate. The test concentrations used in the main study were: 31.6, 100, 316, 1000, 2500 and 5000 ug/plate. The test material did not induce any significant, reproducible increases in the observed number of revertant colonies in any of the strains tested, either in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix were clearly demonstrated by the increases in the numbers of reverant colonies induced by the positive control substances. It was concluded that, under the conditions of this assay, the test substance gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and TA102 in both the presence and absence of S9 mix.