2-Propenamide, N-[4-[[(4,6-dimethyl-2-pyrimidinyl)amino]sulfonyl]phenyl]-2-methyl-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

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)

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-{4-[(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl}-2-methylprop-2-enamide

EC Number:

611-915-5

Cas Number:

59941-98-9

Molecular formula:

C16 H18 N4 O3 S

IUPAC Name:

N-{4-[(4,6-dimethylpyrimidin-2-yl)sulfamoyl]phenyl}-2-methylprop-2-enamide

Test material form:

solid: particulate/powder

Remarks:

white crystalline

Method

Target gene:

thymidine kinase (TK)

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).
- Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 106 cells/ml.


MEDIA USED
Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium
Cells were exposed to the test item in basic medium supplemented with 5% (v/v)
heat-inactivated horse serum (R5-medium).
Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 µg/ml trifluorothymidine (TFT) (Sigma).
Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v)
heat-inactivated horse serum (total amount of serum = 20%, R20-medium).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 125 to 2000 µg/ml in the absence and presence ofS9-mix with a 3 hour treatment period.

Based on the results of the dose range finding test, the following dose ranges were selected for the mutagenicity test:
Without S9-mix: 63, 125, 250, 500, 600, 700, 800, 850, 900, 950 and 1000 μg/ml exposure medium.
With S9-mix: 250, 500, 750, 1000, 1200, 1400, 1500, 1600, 1700, 1800 and
1900 μg/ml exposure medium.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (SeccoSolv, Merck Darmstadt, Germany).
Rationale: The test substance was solvable in this vehicle.

Controls

Untreated negative controls:

yes

Remarks:

The negative control was dimethyl sulfoxide (DMSO), the vehicle of the test item.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

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)

DURATION
- Preincubation period:
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 (Sigma), 2 x 10^-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10^-5 M thymidine (Sigma) (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.
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period.
- Fixation time (start of exposure up to fixation or harvest of cells): The microtiter plates were incubated for 11 or 12 days to determine the cloning efficiency and the mutation frequency.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
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) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.

NUMBER OF CELLS EVALUATED:
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).


DETERMINATION OF CYTOTOXICITY
- Method: cell growth and cloning efficiency
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than one small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

- OTHER:

Rationale for test conditions:

All incubations were carried out in a humid atmosphere (80 - 100%, actual range 65 - 93%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 37.4°C).

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.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the dose levels of 1700, 1800 and 1900 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the dose levels of 900, 950 and 1000 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, the relative suspension growth was 8% at the test item concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 2000 μg/ml.
In the presence of S9-mix, the relative suspension growth was 68% at the test item concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentration of 2000 μg/ml.
Based on the results of the dose range finding test, the following dose ranges were selected for the mutagenicity test:
Without S9-mix: 63, 125, 250, 500, 600, 700, 800, 850, 900, 950 and 1000 μg/ml exposure medium.
With S9-mix: 250, 500, 750, 1000, 1200, 1400, 1500, 1600, 1700, 1800 and
1900 μg/ml exposure medium.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%):
Mutation frequency per 10^6 survivors [95% conficence interval]
- Positive historical control data:
3-hour treatment: 857 [289 - 1425]
24-hour treatment: 1710 [-793 - 4214]
- Negative (solvent/vehicle) historical control data:
3-hour treatment: 86 [36 - 135]
24-hour treatment: 87 [28 - 145]
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 negative control database.
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.


OTHER:
The suspension growth over the two-day expression period for cultures treated with DMSO was 19 and 20.
In the presence of S9-mix, V123109 induced dose related increases in the mutation frequency. The increases were above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (220 per 106 survivors).
Although the increases in the mutation frequency at the TK locus were only observed at toxic dose levels (RTG ≤21%), the mutation frequencies at these concentrations were above the GEF and the results are considered to be biological relevance and the test item is mutagenic at toxic dose levels

Applicant's summary and conclusion

Conclusions:

V123109 is mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of V123109 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence and presence of S9-mix with a 3 hour treatment period.  

The study procedures described in this report were based on the most recent OECD guideline (OECD 490, 29 July 2016). 

Batch V123109/SA of V123109 was a white crystalline powder with a purity of >95%. The test item was dissolved in dimethyl sulfoxide

In the mutation experiment, V123109 was tested up to concentrations of 850 and 1500 µg/ml in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 14 and 15% compared to the negative control in the absence and presence of S9-mix, respectively. Above the dose level of 1500 µg/ml the RTG was below the acceptable limit of 10%. The test item did not precipitate in the culture medium up to the concentration of 1500 µg/ml.

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 negative control database. 

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.

In the absence of S9-mix, V123109 induced dose related increases in the mutation frequency. The increases were above the95% control limits of the distribution of the historical negative control databaseand also abovethe GEF + MF_(controls)(225per 10⁶survivors).

In the presence of S9-mix, V123109 induced dose related increases in the mutation frequency. The increases were above the95% control limits of the distribution of the historical negative control databaseand also abovethe GEF + MF_(controls)
(220per 10⁶survivors).

Although the increases in the mutation frequency at the TK locus were only observed at toxic dose levels (RTG ≤21%), the mutation frequencies at these concentrations were above the GEF and the results are therefore considered to be biological relevance and the test item mutagenic at toxic dose levels.

In conclusion, V123109 is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.