cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolane-4-methanol

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:

2000-12-01 to 2004-12-21

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Remarks:

No information was provided on the test substance including purity.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

No information was provided on the test substance including purity.

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-1,2,4-triazol-1-ylmethyl)-1,3-dioxolane-4-methanol

EC Number:

267-747-4

EC Name:

cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolane-4-methanol

Cas Number:

67914-85-6

Molecular formula:

C13H13Cl2N3O3

IUPAC Name:

(+-)-cis-2-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolane-4-methanol

Details on test material:

- Name of test material (as cited in study report): T001207
- Physical state: solid
- Analytical purity: no data
- Lot/batch No.:ZT001207PUA441
- Storage condition of test material: The test substance was stored in closed containers.

Method

Target gene:

Histidine locus

Species / strain

Species / strain / cell type:

S. typhimurium, other: TA1535, TA1537, TA98, TA100 and TA102

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9 (40 µL S9/mL-initial study and 100 µl S9/mL-repeat study)

Test concentrations with justification for top dose:

First and repeat experiments: 50, 100, 250, 500, 1000, 2500, and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: A solubility test was performed. The test substance was found to be insoluble in water, but soluble in DMSO up to the highest concentration.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene

Remarks:

without S9; at 5 µg/plate for TA98

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9; at 1 µg/plate for TA100 and TA1535

Controls 3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9; at 50 µg/plate for TA1537

Controls 4

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; at 5 µg/plate for TA102

Controls 5

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Amino-anthracene

Remarks:

with S9; at 1 µg/plate for TA98, TA100 and TA1535 and at 2.5 µg/plate for TA1537 and TA102

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

EXPERIMENTAL PERFORMANCE
The following solutions were successively added to 2 ml histidine-biotine supplemented top agar: 0.1 ml of an overnight bacterial culture of the tester
strain, 0.1 ml of a dilution of T001207 and either 0.5 ml S9-mix containing 100 μl S9/ml for the activation portion, or 0.5 ml phosphate buffer for the
non-activation portion. The content of the tube was then mixed and poured onto minimal glucose agar petri dishes. The plates were incubated in the
dark at 37°C for 48 to 72 h.

DURATION
Exposure duration: 48 hours
Selection simultaneous wiht exposure

SELECTION AGENT: histidine

NUMBER OF REPLICATIONS: All concentration levels of T001207, vehicle controls and positive controls were plated in triplicate

DETERMINATION OF CYTOTOXICITY
- Method: reduction in bacterial background lawn

OTHER EXAMINATIONS:
The genotypes and viability of the bacteria were checked when a new set of frozen permanent cultures was prepared. Overnight cultures for the plate incorporation tests were checked for their genotypes and viability as follows: 1) Histidine requirement (his- character) of all the strains were confirmed by demonstrating the histidine requirement for growth on selective agar. 2) The rfa character of the strains was confirmed by testing their sensitivity to the lethal effect of crystal violet. 3) The strains carrying the uvrB mutation were checked for their sensitivity to UV irradiation. 4) The strains carrying the plasmid pKM101 were tested for their resistance to ampicillin. 5) The strain carrying the plasmid pAQ1 was tested for its resistance to tetracyclin. 6) Spontaneous reversion frequencies were determined by plating the strains on three minimal glucose agar plates. 7) Positive control substances were used to determine the reversion properties of the strains. 8) Viability of the bacteria was assessed by cell counting on nutrient agar plates.

Sterility check: Agar, top agar, S9 mix and the highest concentration of the test substance were checked for sterility and were incubated at the same time and for the same length of time as the test plates.

Density of cultures: The number of viable cells per mL overnight culture were determined for each strain used in the study by plating an appropriate dilution of the overnight culture onto nutrient broth.

Evaluation criteria:

The mean number of the revertant colonies on the vehicle control plates will be taken as the background level of mutation of the test. Mean reversion values will be considered as positive if:
- the test is valid;
- the test substance produces at least a twofold increase in the mean number of revertants with one of the strains TA98, TA102, or TA100, or a threefold increase in the mean number of revertants with one of the strains TA1535 and TA1537 at one or more concentration levels;
- a dose effect relationship is observed;
- these effects can be reproduced in an additional study.
The above mentioned criteria will be applied in most cases. When equivocal results are obtained, more tests may be required, in order to evaluate the mutagenic potential of the test substance. If the test substance produces a positive response in a single test that cannot be reproduced in additional runs, the initial positive test data will be discounted.

Statistics:

No statistical analysis necessary.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA98, TA100 TA102, TA1535 and TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Evaporation from medium: no data
- Water solubility: not soluble
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES:
A range finding study was performed with the strain TA100, with and without activation using ten concentrations ranging from 25-5000 µg/plate. All concentration levels of the test substance, vehicle controls and positive controls were plated in triplicate. Plates were incubated for 48 to 72 hours.
Thinning of the background lawn was observed at 5000 µg/plate in the presence and in the absence of metabolic activation. No biologically significant increase in the number of revertant colonies was found either in the presence or in the absence of metabolic activation. Based on these results, 5000 µg/plate was selected as the highest concentration for the initial main mutation study described above.

COMPARISON WITH HISTORICAL CONTROL DATA:
In both tests, the number of spontaneous and vehicle control revertant colonies for the strains in the absence and in the presence of metabolic activation fell within the range of the laboratory historical control data. The positive controls showed a significant increase in the number of revertant colonies indicating their mutagenic activity. On basis of these findings, this study was considered acceptable for the evaluation of the mutation potential of the test substance.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
-Initial assay- In the absence of S9, thinning of the bacterial background lawn was observed at 5000 µg/plate for TA98, TA100 and TA102 and from 1000 µg/plate onwards for TA1537, evolving into pinpoint colonies at higher concentrations. In the presence of S9 mix, thinning of the bacterial background lawn was observed at 5000 µg/plate for TA98, TA100 and TA1535 and pinpoints were observed at 5000 µg/plate for TA1537.
-Repeat assay- In the absence of S9, thinning of the bacterial background lawn was observed at 5000 µg/plate for all strains. In the present of S9, thinning of the bacterial background lawn was observed at 5000 µg/plate for TA102.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Concentration analysis revealed the maximum concentration of 50 mg test substance/mL DMSO was indeed present in the highest sock solution and that no test substance was present in the 0 mg test substance/mL DMSO concentration.

Sterility checks, genotypes of bacterial stains and bacterial titer were according to the criteria.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The test substance was evaluated for mutagenic potential in the Ames test using S. typhimurium strains TA98, TA100, TA102, TA1535 and TA1537. The test substance was not found to be mutagenic to any strain tested, with or without metabolic activation, at concentrations up to 5000 µg/plate.