Benzonitrile, 4-[(1,6-dihydro-4-hydroxy-6-oxo-2-pyrimidinyl)amino]-

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:

2016-05-23 to 2016-08-02

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)

Version / remarks:

A deviation to the study plan is reported in the results section, but does not consist in a deviation to the guideline.

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-[(4,6-dihydroxypyrimidin-2-yl)amino]benzonitrile

EC Number:

609-425-1

Cas Number:

374067-80-8

Molecular formula:

C11H8N4O2

IUPAC Name:

4-[(4,6-dihydroxypyrimidin-2-yl)amino]benzonitrile

Test material form:

solid: particulate/powder

Details on test material:

- Name of test material (as cited in study report): JNJ-4370561-AAA (T002326)
- Physical state: solid (powder)
- Appearance: slight beige powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: I15CB0987
- Expiration date of the lot/batch: 2016-07-29 (retest date)
- Purity test date: 2015-08-17

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: not indicated
- Solubility and stability of the test substance in the solvent/vehicle: not indicated
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not indicated

Method

Target gene:

TK+/- locus in L5178Y mouse lymphoma cells

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)
- 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). Cell density was kept below 1 x 10^6 cells/ml.
- Normal (negative control) cell cycle time: not indicated

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 exposure: basic medium supplemented with 5% (v/v) heat-inactivated horse serum (R5-medium)
Exposure medium 24h exposure: 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 x 10-5 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 ß-naphthoflavone)

Test concentrations with justification for top dose:

Dose-range finding test (3h treatment): 0, 5.4, 17, 52, 164, 512 µg/mL with and without S9
Dose-range finding test (24h treatment): 0, 4.95, 15.6, 47.7, 150, 512 µg/mL without S9

Mutation experiment I (3h treatment): 0, 0.18, 0.55, 1.7, 5.4, 17, 52, 164, 512 µg/mL with and without S9
Mutation experiment II (24h treatment): 0, 4, 8, 16, 32, 64, 128, 256, 512 µg/mL without S9

Justification for top dose: In all experiments, the highest concentration was determined by the solubility of the test item in the culture medium since the test item was not toxic and difficult to dissolve in aqueous solutions.

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 at 228 mg/ml after a few seconds of treatment with ultrasonic waves. Upon mixing with exposure medium the test item precipitated at concentrations of 51.2 mg/ml (= 512 μg/ml) and above.

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; 15µg/mL (3h treatment), 5µg/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) per culture

DURATION
- Exposure duration: 3h or 24h
- 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: 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 con
taining 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).
Solubility limitation: the test item was poorly soluble in the exposure medium and precipitated at the dose of 512 µg/mL, which was the highest test concentration.
Toxicity: no severe toxicity was observed and all dose levels were evaluated in the absence and presence of S9-mix

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 on pH and osmolality: not reported
- Precipitation: The test item precipitated in the exposure medium at the highest concentration tested (512 μg/ml), in all experiments.

RANGE-FINDING/SCREENING STUDIES: L5178Y mouse lymphoma cells were treated with a test item concentration range of 5.4 to 512 μg/ml in the absence and presence of S9-mix with a 3-hour treatment period and with a test item concentration range of 4.95 to 512 μg/ml in the absence of S9-mix with a 24-hour treatment period. Cytotoxicity of the test item was calculated by the suspension growth and the relative suspension growth.
No toxicity in the 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 both in the absence of S9-mix and presence of S9-mix, for the 3-hour and the 24-hour treatments.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: 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. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
- Negative (solvent/vehicle) historical control data: The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical solvent control database, except in the second experiment, in which the mutation frequency of one of the solvent control cultures was just below the acceptability criteria range of this assay. Since the other solvent control culture was within the range, this deviation has no (adverse) effect on the results of the second mutation assay.

OTHER:
The suspension growth (SG) over the two-day expression period for cultures treated with DMSO was between 19 and 21 (3 hour treatment) and 110 and 128 (24 hour 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.
It is concluded that the test item is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.