1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

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:

2018-10-11 to 2018-11-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

Adopted July 21, 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

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:

1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

EC Number:

813-811-7

Cas Number:

253265-97-3

Molecular formula:

C11H13NO7

IUPAC Name:

1-[([[(3R,3aS,6aR)-hexahydrofuro[2,3-b]furan-3-yl]oxy]carbonyl)oxy]pyrrolidine-2,5-dione

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: powder
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I18IB2522
- Expiration date of the lot/batch: 2019-03-19 (retest date)
- Purity test date: 2018-10-04

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability under test conditions: not indicated
- Solubility in vehicle: Acetone 150 g/L
- Stability in vehicle: Acetone Stable for 6 hours

OTHER SPECIFICS: A correction factor of 1.00 for the purity/composition of the test item was applied in this study. Test item concentrations were used within 1.5 hours of preparation.

Method

Target gene:

Histidine locus (histidine-dependent S. typhimurium strains); Tryptophan locus (tryptophan-dependent E. coli strains)

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):

not applicable

Additional strain / cell type characteristics:

not specified

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

Not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (Aroclor 1254 induced rat liver metabolic activation system)

Test concentrations with justification for top dose:

The maximum final concentration for the dose range finding test was selected based on the solubility of the test item in acetone (highest concentration recommended in OECD test guideline).
Dose-range finding test: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in TA100 and WP2uvrA with and without 5% (v/v) S9-mix

The top doses for the mutation experiments were selected based on the toxicity observed in the dose range finding test.
Mutation experiment 1: 17, 52, 164, 512, 1000 and 2500 μg/plate in TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix
Mutation experiment 2: 154, 275, 492, 1000, 1400 and 2000 µg/plate in all tester strains with and without 10% (v/v) S9-mix

Since in the second mutation experiment in tester strain WP2uvrA no dose level with precipitation and/or cytotoxicity was observed in the presence of S9-mix an additional experiment was performed with this tester strain at the following dose levels:
Mutation experiment 2A: 2000, 3500 and 5000 μg/plate in the presence of 10% (v/v) S9-mix.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in ethanol and THF at 50 and 100 mg/ml, respectively. In acetone, the test item was soluble at 100 mg/ml (= 5000 μg/plate). Based on these solubility findings and the stability findings of Test Facility Study No. 20161739, acetone was selected as vehicle and 5000 μg/plate was selected as the maximum final concentration for the dose range finding test.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

Without S9-mix; 5 μg/plate (TA1535)

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: ICR-191

Remarks:

Without S9-mix; 2.5 μg/plate (TA1537)

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

Without S9-mix; 10 μg/plate (TA98)

Controls 4

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9-mix; 650 μg/plate (TA100)

Controls 5

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9-mix; 10 μg/plate (WP2uvrA)

Controls 6

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

2.5 μg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix), 5 μg/plate (TA1537 with 10% S9-mix), 1μg/plate (TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2 μg/plate (TA100 with 10% S9-mix), 15 μg/ plate (WP2uvrA with 5 and 10% S9-mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Top agar in top agar tubes was melted by heating to 45 ± 2°C.
The following solutions were successively added to 3 ml molten top agar:
- 0.1 ml of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains
- 0.5 ml of a dilution of the test item in acetone and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of nonactivation assays).
The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies were counted.

DURATION
- Exposure duration: 48 ± 4 h
- Selection time (if incubation with a selection agent): 48h (simultaneous with exposure)

SELECTION AGENT (mutation assays): Histidine (S. typhimurium histidine-dependent strains); Tryptophan (E. coli tryptophan-dependent strains)

NUMBER OF REPLICATIONS: all concentrations for all experiments were tested in triplicate

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

Evaluation criteria:

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strain TA1535, TA1537 or TA98 is greater than three (3) times the concurrent vehicle control.
b) A concentration related effect is observed.
c) In case a follow up 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 follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants was evaluated for its biological relevance including a comparison of the results with the historical control data range.

Statistics:

No formal hypothesis testing was done.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium, other: TA1535, TA1537, TA98, TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: The test item was observed to be insoluble in water at 50 mg/ml.
- Precipitation:
Mutation experiment 1: not observed at the start or at the end of the incubation period in any tester strain
Mutation experiment 2: not observed at the start of the incubation period but was observed at the end of the incubation period in tester strains TA1535 and TA1537 at the dose levels of 1000 and 1400 μg/plate in the absence of S9-mix and at 1400 μg/plate in the presence of S9-mix
Mutation experiment 2A: not observed at the start or at the end of the incubation period

RANGE-FINDING/SCREENING STUDIES: In the dose-range finding test, the test item was tested at a concentration range of 1.7 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in the tester strains TA100 and WP2uvrA. The dose range finding test results are reported as a part of mutation experiment I.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: The vehicle 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.

- Cytoxicity:
Mutation experiment 1: Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix at the two highest dose levels tested, except in tester strain TA98 in the presence of S9-mix, where toxicity was only observed at 2500 μg/plate.
Mutation experiment 2: Cytotoxicity was observed in all tester strains in the absence and presence of S9-mix at 1000, 1400 and/or 2000 μg/plate, except in tester strain WP2uvrA in the presence of S9-mix.
Mutation experiment 2A: Cytotoxicity was observed at all concentrations tested.

- Mutagenicity:
Mutation experiment 1: In tester strain TA1537 in the presence of S9-mix, the test item induced a 4.3-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was only observed at the mid dose level tested (512 μg/plate). Furthermore, this increase was within the historical control data range. Therefore, this increase is considered to be not biologically relevant. No increase in the number of revertants was observed upon treatment with the test item under all conditions tested in any of the tester strains.
Mutation experiment 2: In tester strain TA1537 in the absence of S9-mix, the test item induced a 3.2-fold increase in the number of revertant colonies compared to the solvent control. However, this increase was only observed at the mid dose level tested (492 μg/plate). Furthermore, this increase was within the historical control data range. Therefore, this increase is considered to be not biologically relevant. No increase in the number of revertants was observed upon treatment with the test item under all conditions tested in any of the tester strains.
Mutation experiment 2A: No increase in the number of revertants was observed upon treatment with the test item under all conditions tested.

Applicant's summary and conclusion

Conclusions:

Interpretation of the results: negative with and without metabolic activation
All bacterial strains showed negative responses over the entire dose range, i.e. no biologically relevant and/or significant dose-related increase in the number of revertants in any of the experiments.
Based on the results of this study it is concluded that the test item is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.