D-Glucitol, 1,5-anhydro-1-C-[3-[[5-(4-fluorophenyl)-2-thienyl]methyl]-4-methylphenyl]-, 2,3,4,6-tetraacetate, (1S)-

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:

2007-06-23 to 2007-08-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

Well documented study report, GLP compliant, and according to Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals. No deviations from the study protocol were recorded.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

no

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-(3-{[5-(4-fluorophenyl)thiophen-2-yl]methyl}-4-methylphenyl)oxan-2-yl]methyl acetate

EC Number:

617-901-5

Cas Number:

866607-35-4

Molecular formula:

C32H33FO9S

IUPAC Name:

[(2R,3R,4R,5S,6S)-3,4,5-tris(acetyloxy)-6-(3-{[5-(4-fluorophenyl)thiophen-2-yl]methyl}-4-methylphenyl)oxan-2-yl]methyl acetate

Test material form:

solid: particulate/powder

Details on test material:

- Physical state: solid
- Appearance: white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M0331013A, Tanabe Seiyaku co., Ltd.
- Expiration date of the lot/batch: Not specified
- Purity test date: Not specified
- Purity: 100%
- Appearance at ordinary temperature: white crytalline powder

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability under test conditions: stable at room temperature under light-shielded consitions
- Solubility and stability of the test substance in the solvent/vehicle: DMSO, 50 mg/mL
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: Not specified

Method

Target gene:

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

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/5,6-benzoflavone induced rat liver (S9) from male Sprague-Dawley rats

Test concentrations with justification for top dose:

Dose-finding assay: 0, 1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate
Main assay: 0, 20, 39, 78, 156, 313 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:The solvent was selected based on the following reasons. As the information from sponsor, the test chemical was reported to be hydrolyzed by water, and its solubility in DMSO was found to be 50 mg/mL or more. The preliminary test on solvent selection in the testing facility indicated that the test chemical was dissolved at 5% in DMSO and there were no form or heat production during the dissolution.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

2AA

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix, all strains, 0.5 µg/plate (TA98), 1.0 µg/plate (TA100), 2.0 µg/plate (TA1535, TA1537), 10.0 µg/plate (WP2 uvrA)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

AF-2

Positive control substance:

other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide

Remarks:

Without S9-mix, 0.01 µg/plate (TA100, WP2 uvrA), 0.1 µg/plate (TA98)

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

NaN3

Positive control substance:

sodium azide

Remarks:

Without S9-mix, 0.5 µg/plate (TA1535)

Controls 4

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

ICR-191

Positive control substance:

other: 6-Chloro-9-[3-(2-chloroethylamino)-propylamino]-2-methoxyacridine dihydrochloride

Remarks:

Without S9-mix, 1.0 µg/plate (TA1537)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation method

DURATION
- Preincubation period: 20 minutes (with shaking) - After preincubation, a 2.0 mL of top agar was added to the mixture and the resultant mixture was overlaid onto a minimum glucose agar plate. Agar soluion supplemented with 0.5 mM L-histidine and 0.5 mM D-biotin (S. typhimurium) or with 0.5 mM L-tryptophan (E. coli) at a volume ratio of 10:1 was used as a top agar.
- Exposure duration: 48h incubation - After incubation, the growth inhibition of the tester bacterial strain was examined with a stereoscopic microscope, and the revertant colonies were counted.

SELECTION AGENT (mutation assays): histidine (S. typhimurium); tryptophan (E. coli)

NUMBER OF REPLICATIONS: duplicate at each dose level; triplicate for negative control

NUMBER OF CELLS EVALUATED: Revertant colonies within a circle with about 80 mm diameter on a plate with 86 mm diameter were counted with an automatic colony counted (and corrected based on the uncounted area by using a personal computer). When the number of revertant colonies on a plate was 1500 or more, the accuracy of the automatic colony counter was lowered and the colonies were counted manually at 5 different places on a plate with a stereoscopic microscope and the average number of colonies was applied to the area-based correction.

DETERMINATION OF CYTOTOXICITY
- Method: growth inhibition (dose-related)

OTHER:
- For all plates, the growth inhibition of bacterial strain was observed with a stereoscopic microscope (40-fold magnification) and the precipitation was observed by unaided eyes.

Evaluation criteria:

When the number of revertant colonies on test chemical treatment plates is remarkably increased compared to the spontaneous level (i.e. twice or more of the solvent control value), with a dose-dependency and reproducibility, it is judged to be positive for mutagenicity. Otherwise, it is judged as negative.

Statistics:

No statistical analysis is performed for data analysis.

Results and discussion

Test results

Key result

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: no information on solubility in water of the test substance. As the information from the sponsor, the test chemical was reported to be hydrolyzed by water.
- Precipitation: White precipitates of the test chemical were observed by unaided eyes at 4.9 μg/plate and higher in the absence of S9 and at 313 μg/plate and higher in the presence of S9 (Dose-finding assay); White precipitates of the test chemical were observed by unaided eyes at 20 μg/plate and higher in the absence of S9 and at 156 μg/plate and higher in the presence of S9 (Main assay).

RANGE-FINDING/SCREENING STUDIES: In the dose-finding assay, the highest dose was set at 5000 μg/plate, and a total of 7 doses were set with a common ratio of 4. Based on these results, the highest dose in the main assay was set at 313 μg/plate, and a total of 5 doses with a common ratio of 2, including at lease one dose producing precipitation.

COMPARISON WITH HISTORICAL CONTROL DATA: Results were check with the laboratories historical control data.

Remarks on result:

other: Dose-finding assay

Any other information on results incl. tables

Validity of assays

- Positive control chemicals increased revertant colonies more than twice of the solvent control value of each strain, indicating the assays were performed properly.

- No bacterial growth due to contamination was observed in the sterility test in both the range-finding assay and the main assay.

- The test chemical did not increase the number of revertant colonies 2-fold or more compared to the solvent control value both in the presence and absence of metabolic activation in any of the strains.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Based on the above results, it was concluded that Ac-GTB was not mutagenic under the test conditions of this study.