6H-Furo[2',3':4,5]oxazolo[3,2-a]pyrimidin-6-one, 2,3,3a,9a-tetrahydro-3-hydroxy-2-(hydroxymethyl)-7-methyl-, (2S,3S,3aR,9aS)-

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:

06 May - 05 June 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2008

Report date:

2008

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2S,3S,3aR,9aS)-3-hydroxy-2-(hydroxymethyl)-7-methyl-2,3,3a,9a-tertryhydro-6H-furo[2’,3’:4,5][1,3]oxazolo [3,2-a]pyrimidin-6-one

EC Number:

700-330-1

Cas Number:

433733-92-7

Molecular formula:

C10H12N2O5

IUPAC Name:

(2S,3S,3aR,9aS)-3-hydroxy-2-(hydroxymethyl)-7-methyl-2,3,3a,9a-tertryhydro-6H-furo[2’,3’:4,5][1,3]oxazolo [3,2-a]pyrimidin-6-one

Test material form:

solid: bulk

Details on test material:

- Name of test material (as cited in study report): Anhydrothymidine
- Substance type: White powder
- Physical state: powder
- Analytical purity: 99.7%
- Purity test date: not stated
- Lot/batch No.: 073904/05
- Expiration date of the lot/batch: 11 December 2008
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Method

Target gene:

histidine gene in Salmonella typhimurium

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium, other: TA 1535, TA 97, TA 98, 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:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix for TA100
First assay: 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix for TA1535, TA97, TA98 and TA102
Second assay: 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix for TA1535, TA97, TA98, TA100 and TA102

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 5 μg/plate in saline for TA1535

Controls 2

Positive controls:

yes

Positive control substance:

other: ICR-191 1 μg/plate in DMSO for TA97

Remarks:

Without metabolic activation (-S9-mix)

Controls 3

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 10 μg/plate in DMSO for TA98

Controls 4

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 650 μg/plate in DMSO for TA100

Controls 5

Positive controls:

yes

Positive control substance:

cumene hydroperoxide

Remarks:

Without metabolic activation (-S9-mix) Migrated to IUCLID6: 0.1 µg in DMSO for TA102

Controls 6

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO (5 and/or 10%) for all tester strains

Remarks:

With metabolic activation (-S9-mix) for all tested strains

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 hours


NUMBER OF REPLICATIONS: doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.


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


OTHER EXAMINATIONS:
- Determination of precipitation

Rationale for test conditions:

Selection of an adequate range of doses was based on a dose range finding test with tester strain
TA100 with and without S9-mix. Eight concentrations, 3, 10, 33,100,333, 1000, 3330 and
5000 μg/plate were tested in triplicate. This dose range finding test was reported as a part of the
first experiment of the mutation assay. The highest concentration of Anhydrothymidine used in
the subsequent mutation assay was 5 mg/plate.

Evaluation criteria:

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 the tester strain TA100, TA97 and TA102 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, and TA98 is not greater than three (3) times the concurrent 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, TA97 and TA102 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535 and, TA98 is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA 1535, TA 97, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: no increase in the number of revertants was observed upon treatment with Anhydrothymidine under all conditions tested.


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.

Any other information on results incl. tables

Precipitate

Precipitation of Anhydrothymidine on the plates was not observed at the start or at the end of the incubation period.

Toxicity

In both mutation assays, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Mutagenicity

In both mutation assays, no increase in the number of revertants was observed upon treatment with Anhydrothymidine under all conditions tested.

Applicant's summary and conclusion

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
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.
It is concluded that the test substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.