13-β-ethyl-3-methoxygona-2,5(10)-dien-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

Study period:

October to December 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983

Deviations:

yes

Remarks:

- no E. coli WP2 or S. typhimurium TA102 strain tested

Principles of method if other than guideline:

The current OECD TG 471 requires at least 5 test strains and the use of E. coli WP2 strains or Salmonella typhimurium TA 102 to detect certain oxidizing mutagens, cross-linking agents and hydrazines. However, the substance is not a highly reactive agent and is therefore not expected to be a cross-linking agent, has no oxidizing properties and is no hydrazine. Thus, a GLP test according to former versions of OECD TG 471 without E. coli WP2 strains or Salmonella typhimurium TA 102 is considered as sufficient to evaluate the mutagenic activity of the substance in this bacterial test system.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Gona-2,5(10)-dien-17-one, 13-ethyl-3-methoxy

IUPAC Name:

Gona-2,5(10)-dien-17-one, 13-ethyl-3-methoxy

Constituent 2

Reference substance name:

13-β-ethyl-3-methoxygona-2,5(10)-dien-17-one

EC Number:

219-034-4

EC Name:

13-β-ethyl-3-methoxygona-2,5(10)-dien-17-one

Cas Number:

2322-77-2

Molecular formula:

C20 H28 O2

IUPAC Name:

3-Methoxy-18-methyl-2,5(10)-estradien-17-on

Method

Target gene:

Histidine gene locus

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1538

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

not applicable

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

not applicable

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

not applicable

Species / strain 5

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced male rat liver S9 mix

Test concentrations with justification for top dose:

50, 100, 250, 500, 1000, 2500 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

cyclophosphamide

other: 2-aminoanthracene

Details on test system and experimental conditions:

direct plate incorporation procedure; Each concentration, including the controls, was tested in triplicate.


NUMBER OF REPLICATIONS:
- Number of cultures per concentration (triplicate)
- Number of independent experiments: one

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 1 x E+06 dilution
- Test substance added in plate incorporation


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: A toxic effect of the substance on the background lawn of non-revertant bacteria and precipitates in the agar were examined stereomicroscopically.

Evaluation criteria:

A positive response was considered if at least 5 mg/plate or up to a toxic dose had been tested (or the compound formed precipitates in the agar) and if the number of revertants of the test compound group compared to the number of revertants of the negative control group was reproducibly higher than 2-fold. A dose-dependent inerease in the number of revertants was also considered to indicate a mutagenic effect.

Statistics:

The arithmetic means of the number of mutant colonies of the 3 parallel plates in the negative control groups were compared with those of the
compound groups.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate onwards in strain TA98 without S9 mix and from 1000 µg/plate onwards in TA 98

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA100 at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA 1535 at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA1537 at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1000 µg/plate onwards in TA 1538

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: not reported
- Data on osmolality: not reported
- Precipitation and time of the determination: There were precipitates in the agar found starting from 0.25 mg/plate onwards in all strains used in the tests without and with S9 mix.


STUDY RESULTS
- Concurrent vehicle negative and positive control data
0.05 mL of the solvents were plated as negative controls. In order to check the activity of the metabolizing system and the mutability of the bacteria at least two reference mutagens were tested for each strain. Their mutagenic effect occurred either directly (9-AA, 2-NF, NaN3) or after metabolic activation (2-AA, BP, CP). Sterility controls were performed additionally. Please refer to table 1 under any other information on results incl. tables.

Ames test:
- Signs of toxicity: yes: Growth inhibition of the background lawn was observed at the highest concentration tested (2.5 mg/plate) in the strains TA1535, TA100 and TA1537, from 0.5 mg/plate onwards in strain TA98 without S9 mix and from 1.0 mg/plate onwards in the strains TA 1538 and TA98.
- Individual plate counts:
TA1535: 168, 189, 139
TA1537: 189, 200, 192
TA1538: 100, 117, 144
TA100: 165, 185, 215
TA98: 213, 242, 259

- Mean number of revertant colonies per plate and standard deviation
Please refer to table 2 under any other information on results incl. tables.

Any other information on results incl. tables

None of the five tester strains showed increased reversion to prototrophy at any of the concentrations tested between 50 and 2500 µg/plate, either in the absence or presence of S9 mix.

 

Growth inhibition of the background lawn was observed at the highest concentration tested (2500 µg/plate) in the strains TA1535, TA100 and TA1537, from 500 µg/plate onwards in strain TA98 without 59 mix and from 1000 µg/plate onwards in the strains TA 1538 and TA98. There were precipitates in the agar found starting from 250 µg/plate onwards in all strains used in the tests without and with S9 mix.

 

Table 1:

	TA1535		TA100		TA1537		TA1538		TA98
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	28±5	17±1	132±1	115±4	17±3	13±5	8±1	22±3	19±4	45±5
phosphate buffer	26±10	19±7	115±8	116±21	23±2	17±5	8±2	25±4	21±3	39±8
2-AA	29±3	9±1	152±10	1081±65	23±3	82±3	16±5	991±28	14±4	936±67
CP	62±7	393±2
BaP 2.5 µg			132±6	633±40					16±3	203±16
BaP 5µg			140±5	793±34					16±3	155±10
2-NF							1106±69	440±16	1250±63	355±6
NaN3	432±23	103±7	546±27	169±8
9-AA					990±153	623±196

 

Tables 2:

	TA1535		TA100		TA1537		TA1538		TA98
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
DMSO	28±5	17±1	132±1	115±4	17±3	13±5	8±1	22±3	19±4	45±5
phosphate buffer	26±10	19±7	115±8	116±21	23±2	17±5	8±2	25±4	21±3	39±8
0.05 mg	29±7	17±3	135±7	128±19	23±8	13±2	10±2	24±5	19±3	42±3
0.10 mg	28±2	19±7	147±12	128±13	25±4	14±3	8±2	19±6	20±2	39±7
0.25 mg	28±8	20±3	152±11	119±8	18±6	13±4	11±2	24±6	22±2	36±9
0.50 mg	30±8	18±2	149±16	118±1	24±3	13±5	10±1	21±4	16±2	28±7
1.00 mg	27±2	16±2	172±24	134±1	21±2	15±3	6±4	19±2	20±2	28±4
2.50 mg	29±2	9±1	125±22	134±7	16±3	10±3	9±5	16±2	18±4	25±6

 

 

 

Applicant's summary and conclusion

Conclusions:

The mutagenic potential of the test substance was evaluated in a Salmonella/microsome test with the S. typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 in the presence and absence of S9 mix according to OECD TG 471. Evaluation of the data does not indicate that the test substance is a mutagen in the Ames Salmonella/microsome test when tested up to the precipitating and cytotoxic dose levels. Appropriate positive control chemicals induced marked increases in revertant colony numbers with all strains.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD TG 471 (adopted 21 July, 1997), strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 of S. typhimurium were exposed to D-ET-Dienon in DMSO at concentrations of 50, 100, 250, 500, 1000, 2500 µg/plate in the presence and absence of mammalian metabolic activation using the plate incorporation method.

 

The test item was tested limit concentration 2500 µg/plate None of the five tester strains showed increased reversion to prototrophy at any of the concentrations tested between 50 and 2500 µg/plate, either in the absence or presence of S9 mix. The positive controls induced the appropriate responses in the corresponding strains. Growth inhibition of the background lawn was observed at the highest concentration tested (2500 µg/plate) in the strains TA1535, TA100 and TA1537, from 500 µg/plate onwards in strain TA98 without 59 mix and from 1000 µg/plate onwards in the strains TA 1538 and TA98. There were precipitates in the agar found starting from 250 µg/plate onwards in all strains used in the tests without and with S9 mix..

 

This study is classified as acceptable. This study satisfies the requirement for Test OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

The test material is considered non-mutagenic under the conditions of the test.