1-[2-(allyloxy)ethyl-2-(2,4-dichlorophenyl)-1H-imidazolium hydrogen sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 1986-09-01 to 1986-10-20

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Study performed equivalent/similar to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) and EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test) with several data reporting deficiencies and limited detail on methodology.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1990

Report date:

1990

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Remarks:

however low number of cells scored

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Remarks:

however low number of cells scored

GLP compliance:

no

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole

EC Number:

252-615-0

EC Name:

1-[2-(allyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole

Cas Number:

35554-44-0

Molecular formula:

C14H14Cl2N2O

IUPAC Name:

(±)-1-(β-allyloxy-2,4-dichloro-phenylethyl) imidazole / (±)-allyl 1-(2,4-dichlorophenyl)-2-imidazol-1-ylethyl ether

Test material form:

solid: crystalline

Details on test material:

- Physical state: crystalline solid
- Appearance: Yellow to brown crystalline solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: D8601
- Expiration date of the lot/batch: Not specified
- Purity: 98.3 (GS with IS)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Not specified
- Stability under test conditions: Known
- Solubility and stability of the test substance in the solvent/vehicle: Not required as prepared for immediate use

Method

Target gene:

Not applicable

Species / strain

Species / strain / cell type:

lymphocytes: Human

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: primary culture of human lymphocytes
- Suitability of cells: according to guideline
- Normal cell cycle time (negative control): not specified

For lymphocytes:
- Sex, age and number of blood donors: 22 and 24 years
- Whether whole blood or separated lymphocytes were used: whole blood
- Whether blood from different donors were pooled or not: not specified
- Mitogen used for lymphocytes: used but not specified

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature, if applicable:
0.5 mL of blood was added to 5 mL growht medium, which was composed of 4.35 ml Ham's F-10 medium, 0.54 ml bovine serum, 0.07 ml phytohemagglutinin and 0.04 ml penicillin-streptomycin. Cultures were incubated in UGB culture bottles at 37°C in a closed waterbath.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 rat liver homogenate

Test concentrations with justification for top dose:

According to the solubility of the test article, the following dose levels were initially chosen to determine the concentration which caused inhibition of the mitotic index:
23, 227, 455 and 909 µg of the test substance/ml culture without and with metabolic activation (S9-mix).

Based on the observations of the cytotoxicity study the following concentrations were chosen for the chromosome aberration test: 9, 36, 73 and 145 µg of the test substance/ml culture without and with metabolic activation.

Vehicle / solvent:

- Vehicle used: dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: The test substance was found to be soluble in DMSO at the required concentrations.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation (-S9); at 0.9 and 1.8 µg/ml

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation (+S9 -mix); at 91 and 182 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION: Approximately 48h after initiation, the cultures were exposed to the test and control articles as well in the absence as in the presence of S9-mix. The test and control articles were diluted in the vehicle (DMSO) immediately before use. The total DMSO volume in the cultures amounted to 100 µL (1.8% v.v)

DURATION
- Exposure duration: 48h Without S9-mix: 24h, With S9-mix: 2h
- Expression time (cells in growth medium): not specified no expression time
- Fixation time (start of exposure up to fixation or harvest of cells): Without S9-mix: 24h, With S9-mix: 2h

SPINDLE INHIBITOR : colchicine

STAIN: 1% lacto-orcein

NUMBER OF REPLICATIONS: 2 replicates per culture

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
Fixed cells were dropped onto grease-free glass slides marked with the blind code. Four slides were prepared per culture and allowed to dry for at least 48 hours at room temperature. the slides were stained overnight with 1% lacto-orcein and mounted with a cover-slip.

NUMBER OF CELLS EVALUATED: the mitotic index of a culture was determined by counting the number of metaphases per 1000 cells.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 50 methaphase spreads per randomly coded culture; i.e. a total of 100 metaphases per concentration group. Only metaphases containing a minimum of 45 and a maximum of 47 centromers were scored.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: No
- Determination of endoreplication: NO

Evaluation criteria:

A test article is considered positive (clastogenic) in the chromosome aberration test if it induced in one of the test concentrations a statistically significant (P<= 0.05) increase in the number of cells with chromosome aberrations.
A test article is considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statiscally significant increase in the number of cells with chromosome aberrations.

Statistics:

The Fisher exact probability Test (one tailed P-values)

Results and discussion

Test results

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No data
- Water solubility: No data
- Precipitation: No data

RANGE-FINDING/SCREENING STUDIES:
At the concentration of 227 µg/ml, the test substance induced a slight hemolysis and a 96% and 82% reduction in mitotic index respectively in the absence and in the presence of a metabolic activation system. At the concentration of 23 µg of the test substance/ ml culture, a 4% and 21% reduction in mitotic index was found respectively in the absence and presence of a metabolic activation system.

STUDY RESULTS
- Concurrent vehicle negative and positive control data
The positive control chemicals both produced statistically significant increases in the frequency of aberrant cells; Therefore it was assumed that the test conditions were optimal and the metabolic activation system functioned properly.
The number of cells with chromosome aberrations (gaps exluded and included) found in the negative and solvent controls were considered acceptable. Although older donors were used, the age of the donors had no influence on the number of cells with chromosome aberrations. If one can not excluse quantitative differences between different donors there are no reasons to believe that different healthy donors would react in a qualitative different manner.

Chromosome aberration test (CA) in mammalian cells:
- Results from cytotoxicity measurements:
o For lymphocytes in primary cultures: mitotic index (MI):
At the concentration of 145 µg/ml culture, the test item was strongly cytotoxic as only 3 metaphases were found per 1000 cells in the presence of ral liver S9 -mix.
At the concentration of 73 µg of the test item /ml culture, nearly 60% reduction in the mitotic index was found in the absence of a metabolic activation system, whereas no reduction at all was found in the presence of S9 -mix.

- Genotoxicity results
o At the concentration of 9, 36, and 73 µg/ml culture, the test item did not induce a significant and biological increase in the number of cells with chromosome aberrations (gaps excluded and included), neither the presence nor in the abscence of a rat liver metabolic activation system.
o At the concentration of 145 µg of test substance/ml culture, only 23 metaphases could be analysed in cultures incubated with rat liver S9 -mix (due to the strong cytotoxic effects). No increase in cells with chromosome aberrations was found among these 23 metaphases analysed of the 145 µg/ml group concentration.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data: no data

Applicant's summary and conclusion

Conclusions:

The test was performed up to the limit of toxicity. Decreasing concentration levels from this toxic level provided adequate number of data points to ensure that any possible dose-response would have been detected.
Based on the lack of increase in the number of cells with chromosome aberrations, it is concluded that the test item, in the presence and absence of a rat liver metabolic activation system, has no clastogenic properties in human lymphocytes.