cis-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-ylmethyl methanesulphonate monohydrochloride

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:

2016-05-23 to 2016-06-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

cis-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-ylmethyl methanesulphonate monohydrochloride

EC Number:

282-252-3

EC Name:

cis-2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolane-4-ylmethyl methanesulphonate monohydrochloride

Cas Number:

84145-27-7

Molecular formula:

C15H16Cl2N2O5S.HCl

IUPAC Name:

[2-(2,4-dichlorophenyl)-2-(1H-imidazol-1-ylmethyl)-1,3-dioxolan-4-yl]methyl methanesulfonate hydrochloride

Test material form:

solid: particulate/powder

Details on test material:

- Name of the test material (as cited in study reports): JNJ-17336007-AAC (T001202)
- 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: I14KB4717
- Expiration date of the lot/batch: 2016-11-25 (retest date)
- Purity test date: 2015-03-04

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

OTHER SPECIFICS: A correction factor of 1.09 for the purity/composition of the test item was applied in this study.

Method

Target gene:

The histidine locus in S. typhimurium strains (TA98, TA100, TA1535 and TA1537) and the tryptophan locus in E. coli strains (WP2uvrA)

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: 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate with and without 5% S9-mix (TA100 and WP2uvrA) (top dose selected based on the solubility findings)
Mutation experiment I: 17, 52, 164, 512, 1600 and 5000 μg/plate with and without 5% S9-mix (TA98, TA1535 and TA1537) (top dose selected based on the dose-range finding test results)
Mutation experiment II: 154, 275, 492, 878, 1568 and 2800 μg/plate with and without 10% S9-mix (TA1535, TA1537, TA98 and TA100); 492, 878, 1568, 2800 and 5000 μg/plate with and without 10% S9-mix (WP2uvrA)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in water and ethanol at 50 mg/ml, the test item formed suspensions in both vehicles. In DMSO, the test item was soluble at 50 mg/ml after treatment with ultrasonic waves. 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 metabolic activation; 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 metabolic activation; 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 metabolic activation; 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 metabolic activation; 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 metabolic activation; 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 ± 4 h
- Selection time: 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 of the bacterial background lawn; increase in the size of the microcolonies; reduction of the 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.
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

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at highest dose levels. See details below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

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
- Water solubility: The test item was observed to be insoluble in water
- Precipitation: DRF test: at 1600 and 5000 µg/plate at the start of the incubation period, at 5000 µg/plate at the end of the incubation period; experiment I: at 5000 µg/plate at the start and at the end of the incubation period; experiment II: at 2800 and 5000 at the start of the incubation period, at 2800 and 5000 µg/plate at the end of the incubation period (WP2uvrA) and at 5000 µg/plate at the end of the incubation period (TA98, TA100, TA1535 and TA1537).

RANGE-FINDING/SCREENING STUDIES: The test item was tested in the tester strains TA100 and WP2uvrA at concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix. Based on the results, the results of the dose range finding test are reported as a part of the mutation experiment.

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:
Mutation experiment 1: Cytotoxicity was observed in the tester strains TA100, TA1535, TA1537 and TA98 in the absence and presence of S9-mix at the highest dose level(s) tested. Microcolonies were also observed in the absence and presence of S9-mix in the tester strain TA1535 at 5000 μg/plate. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.
Mutation experiment 2: Cytotoxicity was observed in the tester strains TA100, TA1535, TA1537 and TA98 in the absence and presence of S9-mix at the highest dose level(s) tested. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

Any other information on results incl. tables

Mutation experiment 2

Toxicity: In strain TA1537 (absence of S9-mix; experiment II), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid dose of 492 μg/plate. However, since no dose-relationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without 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.