Androstan-17-one, 3-[[(4-methylphenyl)sulfonyl]oxy]-, (3.beta.,5.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:

2013-02-07 to 2013-03-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP, OECD guideline 471 and EU Method B.13/14 with only one minor deviation in temperature which did not adversely affect the study integrity.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The temperature was recorded to be outside the range of 37.0 ± 1.0°C as specified in the protocol for approximately 2 hours in the dose range finding study (with a maximum of 39.8°C) and second mutation experiment (with a maximum of 39.4°C).

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The temperature was recorded to be outside the range of 37.0 ± 1.0°C as specified in the protocol for approximately 2 hours in the dose range finding study (with a maximum of 39.8°C) and second mutation experiment (with a maximum of 39.4°C).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Androsterone tosylate

EC Number:

600-553-3

Cas Number:

10429-07-9

Molecular formula:

C26H3604S

IUPAC Name:

Androsterone tosylate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Trantos
- Molecular formula (if other than submission substance): C26H36O4S
- Molecular weight (if other than submission substance): 444.63
- Substance type: Pure active substance
- Physical state: solid, white powder
- Analytical purity: 99.9%
- Lot/batch No.: CQE299K1DL2
- Expiration date of the lot/batch: 15 January 2014
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Method

Species / strain

Species / strain 1

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

Test concentrations with justification for top dose:

3, 10, 33, 100, 333, 1000, 3330, 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Controls

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

yes

Positive controls:

yes

Remarks:

See table below

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); Preparation of bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth and incubated in a shaking incubator (37°C, 150 spm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test. Agar plates: Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar in Vogel-Bonner Medium E, 20 g glucose. The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin and 15 μg/plate histidine and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan. Top agar: Milli-Q water containing 0.6% (w/v) bacteriological agar and 0.5% (w/v) sodium chloride was heated to dissolve the agar. Samples of 3 mL top agar were transferred into 10 mL glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.

DURATION
- Preincubation period: 48-72 hours
- Exposure duration: Not reported
- Expression time (cells in growth medium): 48 ± 4 h


NUMBER OF REPLICATIONS: 3 replicate plates

NUMBER OF CELLS EVALUATED: The revertant colonies (histidine independent or tryptophan independent) were counted manually if less than 40 colonies per plate were present. If more than 40 colonies were present, these were counted automatically with a Biocount 4000 Pro-S-colony counter.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

Acceptability of the assay: A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain.
b) The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.

Data evaluation and statistical procedures: No formal hypothesis testing was done.
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: (Dose range finding study) Precipitation of TRANTOS on the plates was observed at the start and at the end of the incubation period at concentrations of 1000 μg/plate and upwards. (Mutation study) Precipitation of TRANTOS on the plates was observed at the start and at the end of the incubation period at the concentration of 1000 μg/plate.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES:
- TRANTOS was dissolved in dimethyl sulfoxide (DMSO) at concentrations of 1 mg/mL and below. At concentrations of 3.3 mg/mL and higher TRANTOS was suspended in dimethyl sulfoxide. In both mutation experiments, TRANTOS was dissolved in dimethyl sulfoxide up to the highest dose level of 10 mg/mL. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension (dose range finding) or until the test substance had completely dissolved (mutation experiments). Test substance concentrations were used within 2.5 hours after preparation.
-In the dose range finding test, TRANTOS was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. TRANTOS precipitated on the plates at dose levels of 1000 μg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values were within the laboratory 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'.

Any other information on results incl. tables

Summary Table of Experiment 1: Mutagenic response of TRANTOS in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-Mix
Positive control	829 ± 11	532 ± 27	956 ± 32	903 ± 36	1151 ± 41
Solvent control	8 ± 1	4 ± 1	18 ± 2	93 ± 6	27 ± 5
3	N/A	N/A	N/A	93 ± 12	21 ± 2
10	9 ± 4	5 ± 2	22 ± 3	108 ± 6	26 ± 8
33	7 ± 2	5 ± 2	16 ± 2	88 ± 10	23 ± 9
100	8 ± 1	4 ± 3	26 ± 4	102 ± 20	18 ± 1
333	6 ± 3	4 ± 1	15 ± 4	98 ± 10	29 ± 8
1000 SP	5 ± 3	4 ± 2	18 ± 2	97 ± 6	20 ± 1
3330 MP	N/A	N/A	N/A	97 ± 11	24 ± 2
5000 MP	N/A	N/A	N/A	93 ± 12	27 ± 3
	TA1535	TA1537	TA98	TA100	WP2uvrA
With S9-Mix^1
Positive control	422 ± 41	416 ± 29	927 ± 49	1198 ± 91	342 ± 20
Solvent control	8 ± 2	5 ± 3	22 ± 4	92 ± 7	24 ± 4
3	N/A	N/A	N/A	93 ± 13	29 ± 3
10	11 ± 1	4 ± 1	28 ± 4	109 ± 9	35 ± 9
33	9 ± 3	5 ± 1	26 ± 4	109 ± 4	32 ± 2
100	6 ± 2	5 ± 3	26 ± 4	99 ± 3	38 ± 4
333	6 ± 1	6 ± 1	17 ± 4	113 ± 12	30 ± 8
1000 SP	7 ± 2	7 ± 2	21 ± 4	117 ± 7	30 ± 9
3330 MP	N/A	N/A	N/A	103 ± 5	36 ± 2
5000 MP	N/A	N/A	N/A	108 ± 14	32 ± 2
Summary Table of Experiment 2: Mutagenic response of TRANTOS in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay
	TA1535	TA1537	TA98	TA100	WP2uvrA
Without S9-Mix
Positive control	979 ± 33	629 ± 22	1141 ± 42	1028 ± 25	1263 ± 25
Solvent control	10 ± 3	9 ± 3	23 ± 4	127 ± 11	24 ± 2
10	9 ± 3	10 ± 4	20 ± 4	140 ± 4	22 ± 4
33	9 ± 2	10 ± 3	21 ± 2	138 ± 38	19 ± 4
100	10 ± 2	6 ± 4	26 ± 3	127 ± 18	21 ± 5
333	11 ± 3	9 ± 2	21 ± 4	142 ± 7	19 ± 2
1000 SP	9 ± 1	6 ± 3	16 ± 2	124 ± 11	28 ± 4
	TA1535	TA1537	TA98	TA100	WP2uvrA
With S9-Mix^1
Positive control	260 ± 16	516 ± 26	485 ± 63	1262 ± 56	305 ± 23
Solvent control	12 ± 4	14 ± 3	22 ± 2	101 ± 30	26 ± 9
10	10 ± 3	9 ± 3	30 ± 4	132 ± 9	26 ± 4
33	13 ± 4	9 ± 2	24 ± 3	145 ± 11	23 ± 4
100	11 ± 4	13 ± 3	28 ± 3	127 ± 14	25 ± 7
333	10 ± 5	7 ± 3	31 ± 7	133 ± 14	23 ± 1
1000 SP	7 ± 1	9 ± 3	29 ± 6	117 ± 19	28 ± 3
Notes: 1: The S9-mix contained 10% (v/v) S9 fraction SP: Slight precipitation MP: Moderate Precipitation

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Based on the results of this study it is concluded that TRANTOS is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.