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

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:

2016-05-30 to 2016-06-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Well-documented GLP study report in accordance with OECD guideline 471 and EU Method B. 13/14. No deviations were recorded.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

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

EC Number:

695-878-0

Cas Number:

1283129-18-9

Molecular formula:

C44H57FO9S

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-42808415-AAA (T003422)
- Physical state: solid (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: I15AB0305
- Expiration date of the lot/batch: 2017-01-23 (retest date)
- Purity test date: 2015-09-22

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Stock solutions were treated with ultrasonic waves to obtain homogenous suspensions or until the test item had completely dissolved

Method

Target gene:

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

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:

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

Test concentrations with justification for top dose:

Dose-range finding test: 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in absence and presence of S9
Mutation experiment I: 0, 17, 52, 164, 512 and 1600 μg/plate in absence and presence of S9
Mutation experiment II: 0, 154, 275, 492, 878, 1568 and 2800 μg/plate in absence and presence of S9

Justification for top dose: at 1600 µg/plate the test item exhibited limited solubility and was therefore used as the highest concentration of the test item in the subsequent mutation assay

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: in Test Facility Study no. 513615 (Micronucleus test study with test item T003422), the test item was suspended at 51.2 mg/mL in DMSO. Based on these solubility findings, DMSO was selected as vehicle.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

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:

yes

Negative solvent / vehicle controls:

yes

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:

yes

Negative solvent / vehicle controls:

yes

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:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9-mix; 650 µg/plate (TA100)

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

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:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With S9-mix; 2.5µg/plate (TA1535 with 5 and 10% S9-mix, TA1537 with 5% S9-mix, 5µg/plate (TA1537 with 10% S9-mix), 1µg/plate (TA98 with 5 and 10% S9-mix, TA100 with 5% S9-mix), 2µg/plate (TA100 with 10% S9-mix), 15µg/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,
- 0.1 ml of a dilution of the test item in DMSO and
- either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation 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 ± 4h
- Selection time: 48h (simultaneous with exposure)

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

Statistics:

No formal hypothesis testing was done.
In addition to the criteria stated below, 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

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

Key result

Species / strain:

S. typhimurium 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 3

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

decrease in number of revertants at 1600 µg/plate (mutation experiment I)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1535

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 5

Key result

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 test: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 512 μg/plate and upwards. In strain WP2uvrA, precipitation at the end of the incubation period was already observed at 164 μg/plate.
First mutation experiment: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 512 and 1600 μg/plate and at 1600 μg/plate at the end of the incubation period in all tester strains.
Second mutation experiment: Precipitation of the test item on the plates was observed at the start and at the end of the incubation period at concentrations of 878 μg/plate and upwards, except in tester strain TA100 in the absence of S9-mix, where precipitation was only observed at 1568 and 2800 μg/plate 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.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: the strain-specific positive control values were within the laboratory historical control data ranges
- Negative (solvent/vehicle) historical control data: the vehicle control values were within the laboratory historical control data ranges

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: Reduction in bacterial background lawn; increase in size of the microcolonies; reduction of revertant colonies
- Other observations when applicable: cytotoxicity (decrease in number of revertant colonies) was only observed in tester strain TA1537 at 1600 µg/plate (top dose) in the first mutation experiment in the presence and absence of S9

Any other information on results incl. tables

Mutation experiment I:

Toxicity: In strain TA98 (absence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the lowest dose of 17 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. This reduction is more likely caused by incidental fluctuations in the number of revertant colonies.

Mutation experiment II:

Toxicity: In strain TA100 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed at 878 and 2800 μg/plate. However, since no dose-relationship was observed and the reductions in the mean number of revertant colonies were only minor when compared against relevant historical control data, these reductions are caused by incidental fluctuations in the number of revertant colonies and are considered as not biologically relevant.

In strain TA100, a fluctuation in the number of revertant colonies above the laboratory historical control data range was observed in the presence of S9-mix at the mid-dose level of 878 μg/plate. However, since the increase was not two-fold (a maximum of 1.5-fold was reached), this increase was not considered to be relevant.

Applicant's summary and conclusion

Conclusions:

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

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.