4-[(4-HYDROXYPYRIMIDIN-2-YL)AMINO]BENZONITRILE

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:

2016-04-05 to 2016-05-31

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

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:

4-[(4-Oxo-1,4-dihydropyrimidin-2-yl)amino]benzonitrile

EC Number:

606-195-4

Cas Number:

189956-45-4

Molecular formula:

C11H8N4O

IUPAC Name:

4-[(4-Oxo-1,4-dihydropyrimidin-2-yl)amino]benzonitrile

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-40370226-AAA (T002487)
- Physical state: solid (powder)
- 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: M15GC2731
- Expiration date of the lot/batch: 2016-07-23 (retest date)
- Purity test date: 2015-03-29

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature protected from light
- 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
- 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 1.00

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
- Normal (negative control) cell cycle time: 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: 5.4, 17, 52, 164 and 512 μg/ml without and with S9-mix.
Dose range finding test 24h: 5.4, 17, 52, 164 and 512 μg/ml without S9-mix.
Mutation experiment 1: 0.054, 0.17, 0.54, 1.7, 5.4, 17, 52 and 164 μg/ml without and with S9-mix.
Mutation experiment 2: 1.31, 2.63, 5.25, 10.5, 21, 41, 82 and 164 μg/ml without S9-mix (rejected: no appropriate levels of toxicity were obtained for the determination of the mutation frequency)
Mutation experiment 2A (repeat): 0.75, 1.25, 2.5, 5, 10, 12.5, 15, 17.5, 20, 25, 30, 40 and 50 μg/ml without S9-mix.

Since the test item was poorly soluble in the exposure medium, the highest tested concentration was 512 μg/ml exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In DMSO, the test item formed an off-white suspension at concentrations of 51.2 mg/ml and above and a translucent solution at 5.2 and 16.4 mg/ml. Upon mixing with exposure medium the test item precipitated at concentrations
of 16.4 mg/ml (= 164 μg/ml) and above. Based on these solubility findings, DMSO was selected as vehicle and 512 μg/ml was selected as the maximum final concentration for the dose range finding test.

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-diphenyltetrazolium bromide (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:

Solubility limitations: No solubility test was performed in water/exposure medium based on the information provided by the sponsor. Since the test item was poorly soluble and precipitated upon mixing with exposure medium, the highest tested concentration was 512 μg/ml exposure medium.
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).

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

Test results 1

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

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the second experiment the relative total growth of the highest test item concentration was 6% compared to the total growth of the concurrent solvent controls

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
- Water solubility: No solubility test was performed in water based on the information provided by the sponsor
- Precipitation:
Dose range finding test 3h: at 164 and 512 µg/mL with and without S9-mix
Dose range finding test 24h: at 164 and 512 µg/mL with and without S9-mix
Mutation experiment 1: at 164 µg/mL
Mutation experiment 2A: no precipitation up to the highest concentration evaluated (30 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 5.4 to 512 μg/mL in the absence of S9-mix with 3- and 24-hour treatment periods and in the presence of S9-mix with a 3-hour treatment period.
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 512 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

OTHER: The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 12 and 15 (3 hour treatment) and 143 and 153 (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 in the first experiment. This result was confirmed in a repeat experiment with modification in the duration of treatment.
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 TK mutation test system under the experimental conditions described in this report.