cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-08-29 to 2016-10-11

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mouse lymphoma assay

Test material

Test material information

Constituent 1

Reference substance name:

cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

EC Number:

262-765-9

EC Name:

cis-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolane-4-ylmethyl benzoate

Cas Number:

61397-56-6

Molecular formula:

C18H15BrCl2O4

IUPAC Name:

[(2R,4R)-2-(bromomethyl)-2-(2,4-dichlorophenyl)-1,3-dioxolan-4-yl]methyl benzoate

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study reports): JNJ-16479671-AAA (T001095)
- 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: I15DB1648
- Expiration date of the lot/batch: 2020-04-19
- Purity test date: 2016-04-27

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not available
- Solubility and stability of the test substance in the solvent/vehicle: Not available


TREATMENT OF TEST MATERIAL PRIOR TO TESTING
The test item was suspended or dissolved in dimethyl sulfoxide. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension in the dose range finding test or until the test item had completely dissolved in the mutagenicity tests.

OTHER SPECIFICS: correction factor 1.03

Method

Target gene:

TK locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD)
- Cell cycle length, doubling time or proliferation index: not indicated
- Methods for maintenance in cell culture if applicable: Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was preferably kept below 1 x 10^6 cells/ml.


MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
Basic medium: RPMI 1640 Hepes buffered medium containing penicillin/streptomycin (50U/mL and 50μg/mL, respectively), 1mM sodium pyruvate and 2mM L-glutamin
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium)
Exposure medium 3h: basic medium supplemented with 5% (v/v) heat inactivated horse serum (R5-medium)
Exposure medium 24h: basic medium supplemented with 10% (v/v) heat inactivated horse serum (R10-medium)
Selective medium: basic medium supplemented with 20% (v/v) heat inactivated horse serum (R20-medium) and 5 μg/ml trifluorothymidine (TFT)
Non-selective medium: basic medium supplemented with 20% (v/v) heat inactivated horse serum (R20-medium)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically 'cleansed' against high spontaneous background: yes, prior to dose range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10 medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10 medium containing hypoxanthine and thymidine only. After this period cells were returned to R10 medium for at least 1 day before starting the experiment.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9-mix (rat liver metabolic activation system induced by a combination of phenobarbital and ßnaphthoflavone)

Test concentrations with justification for top dose:

Dose range finding test 3h: 7.82, 15.63, 31.25, 62.5, 125 without and with S9-mix.
Dose range finding test 24h: 7.82, 15.63, 31.25, 62.5, 125, 250 μg/ml without S9-mix.
Mutation experiment 1: Without S9-mix: 1.57, 3.13, 6.25, 12.5, 25, 50, 70, 85, 100, 115, 130 μg/mL; With S9-mix: 0.4, 0.79, 1.57, 3.13, 6.25, 12.5, 25, 30, 40, 50, 70, 85, 100 μg/mL
Mutation experiment 2: 3.13, 6.25, 12.5, 25, 50, 70, 85, 100, 115 and 130 μg/mL
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 125 and 250 μg/mL exposure medium for the 3 hour and 24 hour treatment, respectively.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:
In DMSO, the test item formed a suspension at the concentration of 56 mg/mL and above and was soluble at 28 mg/mL. Upon mixing with exposure medium the test item precipitated directly at the concentration of 6.5 mg/mL (= 65 μg/mL) and above. After 3 hours, precipitation was observed at concentrations of 14 mg/mL (= 140 μg/mL) and above. Based on these solubility findings, DMSO was selected as vehicle and 125 and 250 μg/mL were selected as the maximum final concentration for the dose range finding test for the 3 and 24 hour treatment, respectively.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

Without S9; 15 μg/mL (3h treatment), 5 μg/mL (24h treatment)

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; 7.5 μg/mL (3h treatment)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 8 x 10^6 cells (10^6 cells/mL for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/mL for 24 hour treatment) were used.

DURATION

- Exposure duration: 3 h or 24 h
- Expression time (cells in growth medium): 48h (3h and 24h treatment)
- Selection time (if incubation with a selection agent): 11 or 12 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): selective medium (TFT-selection)

STAIN (for cytogenetic assays): After the incubation period, the plates for the TFT-selection were stained for 2 hours, by adding 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide (MTT) to each well.

NUMBER OF REPLICATIONS:
Determination of cloning efficiency: 2 x 96-well microtiter plates/concentration
Determination of mutation frequency: 5 x 96-well microtiter plates/concentration (solvent controls and treatment groups); 10 x 96-well microtiter plates/concentration (positive controls)

NUMBER OF CELLS EVALUATED: Determination of cloning efficiency (CEday2): One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
Determination of mutation frequency: 9.6 x 10^5 cells/concentration plated (5 x 96-well microtiter plates/concentration, each well containing 2000 cells in selective medium (solvent controls and treatment groups)); 9.6 x 10^5 cells/concentration plated (10 x 96-well microtiter plates/concentration), each well containing 1000 cells in selective medium (positive controls)

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth

Rationale for test conditions:

The highest concentration tested in the mutagenicity tests should give a relative total growth (RTG) of approximately 10-20% or should show a slight to heavy test item precipitation at the end of the treatment period or should correspond to 2 mg/ml or 0.01 M (whichever is the lowest).
Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 125 and 250 μg/ml exposure medium for the 3 hour and 24 hour treatment, respectively.

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of MF(controls) + 126.

Statistics:

No statistical analysis

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not measured
- Effects of osmolality: not measured
- Precipitation: Dose range finding test 3h: 125 μg/mL with and without S9-mix
Dose range finding test 24h: at 250 μg/mL with and without S9-mix
Mutation experiment 1: at 115 and 130 μg/mL
Mutation experiment 2: at 115 μg/mL

RANGE-FINDING/SCREENING STUDIES: In the dose range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 7.82 to 125 μg/mL in the absence of S9-mix with 3 hour treatment periods and in the presence of S9-mix with a 3-hour treatment period. In the 24 hour treatment, the cells were treated with a test item concentration range of 7.82 to 250 μg/mL in the absence of S9-mix.
Both in the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test item concentration of 115 μg/mL compared to the suspension growth of the solvent control.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database. Except in the first experiment, in which the mutation frequencies of one of the solvent control cultures both in the absence and presence of S9-mix were just above the upper control limits. However, the observed mutation frequencies of the solvent control cultures were within the acceptability criteria of this assay and these limits are 95% control limits and a slightly higher response is within the expected response ranges.

OTHER:
The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 21 and 25 (3 hour treatment) and 61 and 66 (24 hour treatment)

Remarks on result:

other: 3 h treatment

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative with and without metabolic activation
In the absence of S9-mix, the test item did not induce a significant increase in the mutation frequency after a 3-hour treatment period. This result was confirmed in an independent experiment with a treatment duration of 24 hours.
In the presence of S9-mix, the test item did not induce a significant increase in the mutation frequency.
In conclusion, the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.