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

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:

2016-05-18 to 2016-10-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chrommosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

283-585-7

EC Name:

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

Cas Number:

84682-23-5

Molecular formula:

C14H14Cl2N2O3

IUPAC Name:

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

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-16479489-AAA (T001159)
- Physical state: solid
- Appearance: off-white powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: M15LB5093
- Expiration date of the lot/batch: 2016-11-30 (retest date)
- Purity test date: 2016-03-08

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: In DMSO, the test item was dissolved at concentrations of 65 mg/ml and below but formed a suspension at concentrations of 160 mg/ml and above.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The stock solutions used for the dose range finding study and first cytogenetic assays were treated with ultrasonic waves until the test item had completely dissolved or to obtain a homogeneous suspension.

OTHER SPECIFICS: correction factor: 1.00

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age):
Dose range finding study: age 30, AGT = 12.9 h
First cytogenetic assay: age 33, AGT = 12.7 h
Cytogenetic assay 1A: age 30, AGT = 13.8 h
Second cytogenetic assay: age 35, AGT = 12.9 h
Cytogenetic assay 2A: age 33, AGT = 14.2 h
Cytogenetic assay 2B: age 26, AGT = 12.8 h
Cytogenetic assay 2C: age 30, AGT = 12.9 h
AGT = Average Generation Time of the cells
- Suitability of cells: Peripheral human lymphocytes are recommended in international guidelines (OECD, EC).
- Sex, age and number of blood donors if applicable: see in "Source of cells"
- Whether whole blood or separated lymphocytes were used if applicable: whole blood
- Methods for maintenance in cell culture if applicable: Immediately after blood collection lymphocyte cultures were started. Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: no
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: no

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/ß-naphthoflavone induced rat liver homogenate (S9-mix)

Test concentrations with justification for top dose:

Dose range finding test (3h exposure): 52, 164, 512, 1600 and 2000 µg/mL with and without 1.8% S9-mix
Dose range finding test (24h/48h continuous exposure): 52, 164, 512, 1600 and 2000 µg/mL without S9-mix

Cytogenetic assay 1 (3 h exposure time, 24 h fixation time): 50, 150, 500, 600, 700, 800, 900 and 1000 µg/mL without S9-mix; 145, 579, 772, 965, 1061, 1254 and 1447 µg/mL with 1.8% S9-mix (rejected since no appropriate dose levels could be selected for scoring)
Cytogenetic assay 1A (3 h exposure time, 24 h fixation time): 150, 500, 525, 550, 575, 600 and 650 µg/mL without S9-mix; 150, 500, 525, 550, 575, 600, 625 and 650 µg/mL with 1.8% S9-mix
The following dose levels were selected for scoring of chromosome aberrations, with and without S9-mix: 150, 575 and 650 µg/ml culture medium

Cytogenetic assay 2 (24 h continuous exposure, 24 h fixation time): 10, 25, 50, 75, 100, 125 and 150 µg/mL without S9-mix (rejected since no appropriate dose levels could be selected for scoring)
Cytogenetic assay 2A (24 h continuous exposure, 24 h fixation time): 1, 5, 10, 15, 20, 30, 40, 50 and 75 µg/mL without S9-mix (rejected since no appropriate dose levels could be selected for scoring)
Cytogenetic assay 2B (24 h continuous exposure, 24 h fixation time): 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL without S9-mix (rejected since no appropriate dose levels could be selected for scoring)
Cytogenetic assay 2C (24 h continuous exposure, 24 h fixation time): 5, 10, 20, 40, 60, 80, 100, 120, 140, 160, 180 and 200 µg/mL without S9-mix;
The following doses were selected for scoring of chromosome aberrations: 40, 160 and 180 µg/ml culture medium.

Since the test item precipitated in culture medium at 160 mg/mL (= 1600 µg/mL) and above in the solubility test, 2000 µg/mL was selected as maximum concentration for the dose range finding test.
The highest concentration analysed was selected based on toxicity, inhibition of the mitotic index of about 50% or greater.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test item was observed to be insoluble in culture medium and ethanol. In DMSO, the test item was soluble up to the concentration of 65 mg/mL, and after treatment with ultrasonic waves up to the concentration of 160 mg/mL. Based on the 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:

mitomycin C

Remarks:

without S9-mix; 0.5 and 0.75 µg/mL (3h), 0.2 and 0.3 µg/mL (24h)

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix; 10 µg/mL (3h)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Cytogenetic assay 1A: 3h; Cytogenetic assay 2C: 24h continuous exposure
- Expression time (cells in growth medium): Cytogenetic assay 1A: 20-22h; Cytogenetic assay 2C: 0h.
- Fixation time (start of exposure up to fixation or harvest of cells): 24h

SPINDLE INHIBITOR (cytogenetic assays): During the last 2.5 - 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/ml medium). Thereafter the cell cultures were centrifuged for 5 min at 365 g and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56% (w/v) potassium chloride solution at 37°C. After hypotonic treatment, cells were fixed with 3 changes of methanol (Merck):acetic acid (Merck) fixative (3:1 v/v).

STAIN (for cytogenetic assays): 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8

NUMBER OF REPLICATIONS: duplicate

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the Test Facility Study number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex.

NUMBER OF CELLS EVALUATED: The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells. At least three analysable concentrations were used for scoring of the cytogenetic assay.

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): 150 metaphase chromosome spreads per culture were examind by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined.
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Rationale for test conditions:

The highest concentration examined for chromosome aberrations should be cultures that produce an inhibition of the mitotic index of 55 ± 5 %, whereas the mitotic index of the lowest concentration should have little or no cytotoxicity (approximately the same as vehicle control). Also cultures treated with an intermediate concentration should be examined.
For poorly soluble test items, the highest concentration analysed should be the lowest insoluble concentration (determined at the end of treatment) irrespective of toxicity. The extent of precipitation may not interfere with the scoring of chromosome aberrations.
If no precipitate or limiting toxicity is observed, the highest concentration examined should correspond to 2 mg/ml or 0.01 M, whichever is the lowest.

Evaluation criteria:

A test item is considered positive (clastogenic) in the chromosome aberration test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical vehicle control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent vehicle control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the historical vehicle control data range.

Statistics:

Graphpad Prism version 4.03 and ToxRat Professional v 3.2.1 were used for statistical analysis of the data.
Since the Fisher’s exact test shows that there are statistically significant differences between one or more of the test item groups and the concurrent vehicle control group a Cochran Armitage Trend test (p < 0.05) was performed to test whether there is a significant trend in the induction.

Results and discussion

Test results

Test results 1

Species / strain:

lymphocytes: human

Remarks:

Cytogenetic Assay 1A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

appropriate toxicity was reached at 650 µg/mL (mitotic index of 44%)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

lymphocytes: human

Remarks:

Cytogenetic Assay 2C

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Appropriate toxicity was reached at 180 µg/mL (mitotic index of 39%)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No marked changes in pH and osmolality were observed in culture medium containing the highest, non-precipitating test item concentration of 512 μg/ml (pH 7.827) compared to the concurrent vehicle control (pH 7.866)
- Effects of osmolality: No marked changes in osmolality were observed in culture medium containing the highest, non-precipitating test item concentration of 512 μg/ml (424 mOsm/kg) compared to the concurrent vehicle control (432 mOsm/kg)
- Water solubility: no data
- Precipitation:
Dose range finding test (3h and 24h exposure): at 1600 µg/mL and above
Cytogenetic Assay 1A: No test item precipitation up to the highest tested concentration.
Cytogenetic Assay 2C: No test item precipitation up to the highest tested concentration.
- Definition of acceptable cells for analysis: Only metaphases containing 46 ± 2 centromeres (chromosomes) were analysed. The number of cells with structural aberrations and the number of structural aberrations were recorded. Gaps were also recorded and reported separately, but not included in the total aberration frequency.

RANGE-FINDING/SCREENING STUDIES: In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. Lymphocytes were cultured for 48 ± 2 h and thereafter exposed to selected doses of the test item for 3h, 24h and 48h in the absence of S9-mix or for 3h in the presence of 1.8% (v/v) S9-mix. For the 3 hour exposure time, blood cultures were treated with 52, 164, 512, 1600 and 2000 μg test item/ml culture medium with and without S9-mix. For the 24 and 48 hour exposure time, blood cultures were treated with 52, 164, 512, 1600 and 2000 μg test item/ml culture medium without S9-mix. A complete cell lysis was observed at 1600 μg/ml and above for all the exposed groups. Based on the results of the dose range finding test, the following dose levels were selected for the Cytogenetic Assay 1A: 50, 150, 500, 600, 700, 800, 900 and 1000 μg/ml culture medium without S9-mix and 145, 579, 772, 965, 1061, 1254 and 1447 μg/ml culture medium with S9-mix.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. The number of cells with chromosome aberrations found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
- Negative (solvent/vehicle) historical control data: The number of cells with chromosome aberrations found in the vehicle control cultures was within the 95% control limits of the distribution of the historical vehicle control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the vehicle control cultures was within the 95% control limits of the distribution of the historical vehicle control database.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: mitotic index

Any other information on results incl. tables

Cytogenetic Assay 1A:

In the absence of S9-mix, the test item induced a statistically significant increase in the number of cells with chromosome aberrations at the highest concentration only, both when gaps were included and excluded. The increase was considered not biologically relevant since the sum of the number of cells with chromosome aberrations in the duplicate cultures was within the historical vehicle control data range.

In the presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations.

Cytogenetic Assay 2C:

At the 24 h continuous exposure time, the test item induced a statistically significant increase in the number of cells with chromosome aberrations at the highest concentration only, both when gaps were included and excluded. The increase was considered not biologically relevant since the increase could not be confirmed by 2 independent scorers.

It was noted that the test item increased the number of polyploid cells and cells with endoreduplicated chromosomes both in the absence and presence of S9-mix. This may indicate that the test item has the potential to disturb mitotic processes and cell cycle progression.

Applicant's summary and conclusion

Conclusions:

It is concluded that this test is valid and that the test item is not clastogenic in human lymphocytes under the experimental conditions described in this report.