(3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl[(methylsulfonyl)oxy]acetate

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:

04-nov-2003 to 13-nov-2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

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 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

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:

(3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl[(methylsulfonyl)oxy]acetate

EC Number:

700-396-1

Cas Number:

60924-38-1

Molecular formula:

C23 H36 O7 S

IUPAC Name:

(3aS,4R,5S,6S,8R,9R,9aR,10R)-6-ethenyl-5-hydroxy-4,6,9,10-tetramethyl-1-oxodecahydro-3a,9-propanocyclopenta[8]annulen-8-yl[(methylsulfonyl)oxy]acetate

Details on test material:

- Name of test material (as cited in study report): SB-322069
- Substance type: White solid
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

- S. typhimurium: Histidine gene

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 102

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Preliminary test (without and with S9-mix) TA100: 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate
Main study:
Without and with S9-mix: 1.5, 5, 15, 50, 150, 500 and 1500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO;
- Justification for choice of solvent/vehicle:
The test material was insoluble in water but was fully soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines .

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Migrated to IUCLID6: at 3 µg/plate for TA100 and 5 µg/plate for TA1535

Controls 2

Positive control substance:

mitomycin C

Remarks:

Without S9-mix

Migrated to IUCLID6: at 0.5 µg/plate for TA102

Controls 3

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9-mix

Migrated to IUCLID6: at 0.2 µg/plate for TA98

Controls 4

Positive control substance:

9-aminoacridine

Remarks:

Without S9-mix

Migrated to IUCLID6: at 80 µg/plate for TA1537

Controls 5

Positive control substance:

benzo(a)pyrene

Remarks:

With S9-mix

Migrated to IUCLID6: at 5 µg/plate for TA98

Controls 6

Positive control substance:

other: 2-aminoanthracene at 1 µg/plate for TA100

Remarks:

With S9-mix

Controls 7

Positive control substance:

other: 2-aminoanthracene at 2 µg/plate for TA1535 and TA1537

Remarks:

With S9-mix

Controls 8

Positive control substance:

other: DAN at 10 µg/plate for TA102

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

The test material may be considered positive in this test system if the following criteria are met: The test material should have induced a dose-related and statistically (Dunett's method of linear regression) significant increase in the revertant count in at least one strain of bacteria.

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium 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:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was observed at dose level of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- In tester strain TA100, toxicity was observed at dose levels of 50 μg/plate and above in the absence and presence of S9-mix.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: without S9: 500 µg/plate and above and with S9: 500 µg/plate and above
TA1537: without S9: 500 µg/plate and above and with S9: 500 µg/plate and above
TA98: without S9: 500 µg/plate and above and with S9: 500 µg/plate and above
TA100: without S9: 50 µg/plate and above and with S9: 150 µg/plate and above
TA102: without S9: 500 µg/plate and above and with S9: 500 µg/plate and above

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

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.

It is concluded that this test is valid and that SB-322069 is not mutagenic in the Salmonella typhimurium reverse mutation assay.