Androstan-17-one, 2,3-epoxy-16-(1-pyrrolidinyl)-, (2.alpha.,3.alpha.,5.alpha.,16.beta.)-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two Ames assays, both in S. typhimurium and E. coli, and two chromosomal aberration assays in peripheral human lymphocytes were negative.

Link to relevant study records

Reference

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:

8 October 1997 to 10 November 1997

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

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

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine-requiring S. typhimurium, and
Tryptophan-requiring E. coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Properly maintained: Yes
- Periodically "cleansed" against high spontaneous background: Yes, E. coli

Metabolic activation:

with and without

Metabolic activation system:

S9-fraction

Test concentrations with justification for top dose:

Up to 3330 µg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: No data

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

methylmethanesulfonate

other: daunomycine, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: In agar

DURATION
- Preincubation period: No data
- Exposure duration: 48 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): 48 hours

SELECTION AGENT (mutation assays): Incubation time

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - number of revertnants

Evaluation criteria:

The test substance is considered negative (not mutagenic) in the test if:
- The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
- The negative response should be reproducible in at least one independently repeated experiment.

The test substances is considered positive (mutagenic) in the test if:
- It induces at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 is considered to be not significant.
- The positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance precipitated in top agar at concentration of 1000 and 3330 µg/plate. Precipitation of the test substance on the plates was observed at the concentration of 3330 µg/plate at the start and at the end of the incubation period in all tester strains.

RANGE-FINDING/SCREENING STUDIES: The test substance was tested in the tester strains TA100 and WP2uvrA with concentration of 3, 10, 33, 100, 333, 1000, 3330, and 5000 µg/plate in the absence and presence of S9-mix.

The test substance precipitated in the top agar at concentrations of 1000 µg/plate and upwards.
Precipitate of the test substance on the plates was observed at concentrations of 3330 and 5000 µg/plate at the start and at the end of the incubation period in tester strain TA100 and WP2uvrA.

To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.
In strain TA100, in the absence of S9-mix, a moderate reduction of the bacterial background lawn and in the number of His+ revertants was observed at the concentration of 3330 µg/plate. An extreme reduction of the bacterial background lawn and an increase in the size of microcolonies was observed at the concentration of 5000 µg/plate.
In the presence of S9-mix, a slight reduction of the bacterial background lawn and in the number of His+ revertants was observed at the concentration of 3330 µg/plate. A moderate reduction of the bacterial background lawn and of the His+ revertants was observed at the concentration of 5000 µg/plate.
In tester strain WP2uvrA, no reduction of the bacterial background lawn and no decrease in the number of revertant colonies, which was less than the minimal value of the historical control data range was observed at all concentration tested.

COMPARISON WITH HISTORICAL CONTROL DATA: All other concentrations, not present in tables 1 and 2 below, and strain Wp2uvrA showed no reduction in the bacterial background lawn or showed no reduction in the number of revertants, which was less than the minimal value of the historical control data range.

The negative and strain-specific positive control values were within our laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Reduction in the Bacterial Background Lawn
Strain	Dose (µg/plate)	Without S9-Mix	With S9-Mix
TA1535	3330	-	-
TA1537	3330	Slight	-
TA98	3330	-	-
TA100^1	3330 5000	Moderate Extreme	Slight Moderate
- No reduction in the number of revertant colonies, which was less than the minimal value of the historical control data range. ^1The first experiment was tested in the dose range finding test.

Table 2: Reduction in the Number of Revertants
Strain	Dose (µg/plate)	Without S9-Mix	With S9-Mix
TA1537	3330	-	-
TA98	3330	-	-
TA100^1	3330 5000	Moderate Extreme^2	Slight Moderate
- No reduction in the number of revertant colonies, which was less than the minimal value of the historical control data range. ^1The first experiment was tested in the dose range finding test. ^2Microcolonies

Number of Revertants: All bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two-fold, increase in the number of revertants in two independently repeated experiments.

Conclusions:

Interpretation of results (migrated information):
negative

Under test conditions, the test substance is considered non-mutagenic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Two Ames assays, both in S. typhimurium and E. coli, and two chromosomal aberration assays in peripheral human lymphocytes were negative.

Justification for selection of genetic toxicity endpoint
Reliable test (Guideline and GLP) test data indicate that epyrron is not genotoxic.

Justification for classification or non-classification

Taking into account all data, it is not expected that epyrron exhibits any genotoxic/mutagenic potential. Based on the results there is no classification required according to Directive 67/548/EEC and Regulation (EC) 1272/2008 (CLP).