Methanesulfonamide, N-[2-[(4,6-dimethoxy-1,3,5-triazin-2-yl)carbonyl]-6-fluorophenyl]-1,1-difluoro-N-methyl-

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 March - 23 April 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Arbeit, Gesundheit und Soziales des Landes Nordrhein-Westfalen

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

EC Number:

620-056-5

Cas Number:

874195-61-6

Molecular formula:

C14H13F3N4O5S

IUPAC Name:

N-[2-(4,6-dimethoxy-1,3,5-triazine-2-carbonyl)-6-fluorophenyl]-1,1-difluoro-N-methylmethanesulfonamide

Method

Target gene:

HPRT locus

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
*cell culture medium: PAA Ready Mix (10% FBS): Eagle's minimal essential medium (MEM, Earle) containing 1% L-glutamine, 1% MEM-vitamins, 1% MEM NEAA, 1% Pen/Strep and 10% FBS(foetal bovine serum);
*medium during treatment: PAA Ready Mix (2% FBS): Eagle's minimal essential medium (MEM, Earle) containing 1% L-glutamine, 1% MEM-vitamins, 1% MEM NEAA, 1% Pen/Strep and 2% FBS
*medium for selction of mutants: PAA Ready Mix (10% FBS) containing 10 µg/mL of 6-thioguanine (6-TG)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Experiment I
Without S9 mix: 25, 50, 100, 200, 400, 600, 800 µg/mL
With S9 mix: 25, 50, 100, 200, 400, 600, 800 µg/mL

Experiment II
Without S9 mix: 25, 50, 100, 200, 400, 600, 800 µg/mL
With S9 mix: 25, 50, 100, 200, 400, 600, 800 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was soluble up to 410 mg/mL in DMSO.

Controls

Untreated negative controls:

yes

Remarks:

Medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

ethylmethanesulphonate (EMS), 900 µg/mL with S9; dimethylbenzanthracene, 20 µg/mL in DMSO, without S9

Positive control substance:

ethylmethanesulphonate

other: Dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 5 h with and without metabolic activation
- Expression time (cells in growth medium): 6 days
- Selection time: 6-8 days
- Fixation time (start of exposure up to fixation or harvest of cells): 13-15 days

SELECTION AGENT (mutation assays): 10 µg/mL 6-TG

NUMBER OF REPLICATIONS: duplicates each in two independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative population growth

Evaluation criteria:

Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10% or greater.
A trial will be considered positive if a concentration-related and in parallel cultures reproducible increase in mutant frequencies is observed. To be relevant, the increase in mutant frequencies should be at least two to three times that of the highest negative or negative control value observed in the respective trial. If this result can be reproduced in a second trial, the test substance is considered to be mutagenic.
Despite these criteria, a positive result will only be considered relevant, if no significant change in osmolality compared to the negative control can be observed. Otherwise, unphysiological culture conditions may be the reason for the positive result (Scott et al., 1991, Mutation Res. 257, 147-204).
A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more concentrations induce a reproducible and biologically relevant increase in mutant frequencies in all trials.
An assay will be considered negative if no reproducible and relevant increases of mutant frequencies were observed.

Statistics:

All acceptable groups are included in the weighted analysis of variance followed by pairwise comparisons to the negative control on a nominal significance level of alpha = 0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual concentrations thereby omitting the positive, untreated and negative controls. If there is a significant concentration related increase of the mutant frequency (alpha = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until p > 0.05. In that way eliminated concentrations are flagged correspondingly.

Results and discussion

Test results

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit 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: Concentrations of up to 800 µg/mL test substance did not change the pH in the medium of the pre-test.
- Effects of osmolality: The osmolality in the medium of the pre-test was not changed by concentrations of up to 800 µg/mL test substance.
- Precipitation: Without and with S9 mix substance precipitation occurred in the medium at the concentration 600 µg/mL and above.

COMPARISON WITH HISTORICAL CONTROL DATA: The results of the controls are in the range of historical controls.

ADDITIONAL INFORMATION ON CYTOTOXICITY: No cytotoxic effects above 80% were induced by the used concentrations in a pre-test. However, precipitation of the test substance in the culture medium started at 600 µg/mL. Due to these findings, the test substance was tested in the mutation experiments in concentrations ranging from 25 µg/mL to 800 µg/mL without and with metabolic activation.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

In the absence and in the presence of S9 mix Chinese hamster V79 cells were exposed to the test substance at concentrations of up to and including 800 µg/mL. The means of the absolute cloning efficiency for the negative controls in the mutation experiments were 56.4% and 93.4% in the experiments without activation. In experiments with metabolic activation 90.7% and 93.2% were observed. These results demonstrate good cloning conditions for the experiments.

Two independent experiments were performed with and without S9 mix (Tables 1-4). The mutant frequencies of the untreated controls and of the solvent controls were all within the normal range. The positive controls induced clear mutagenic and statistically significant effects in all trials. The test substance did not induce cytotoxic effects of 80% to 90% both with and without metabolic activation. However, the test substance was tested up to at least its limits of solubility in the medium. The test substance induced no relevant increases in mutant frequencies with and without metabolic activation. In addition, the overall statistical analysis reveals no statistically significant increase. Therefore, neither with nor without S9 mix, the test substance was evaluated as non-mutagenic.

 

Table 1: Experiment I - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Population Growth [%]	Mutant Frequency x 10^6
Negative control	71.7±3.8	118.2	1.2
	60.0±3.5	151.3	2.1
DMSO	60.7±3.3	100	2.1
	52.0±5.6	100	0.8
25	51.5±3.6	97.0	3.2
	62.5±3.5	146.8	0.0
50	69.7±4.6	75.3	4.8
	60.2±1.3	137.9	4.2
100	59.8±0.8	87.9	0.7
	60.0±2.2	147.4	1.4
200	57.2±2.8	110.5	2.2
	58.0±2.8	130.4	0.7
400	58.3±4.5	84.9	1.4
	64.2±2.5	129.2	0.6
600 P	52.8±5.5	122.3	2.4
	68.0±4.4	125.0	0.6
800 P	55.3±2.1	107.2	1.5
	61.5±9.0	158.7	0.0
EMS, 900	46.3±3.2	19.3	624.1
	38.7±9.0	30.3	574.4

EMS: Ethylmethanesulphonate

P: Precipitate

 

 

Table 2: Experiment I - With Metabolic Activation 

Concentration [µg/mL]	Cloning efficiency [%]	Relative Population Growth [%]	Mutant Frequency x 10^6
Negative control	91.2±8.1	128.9	3.2
	84.8±2.8	75.1	4.9
DMSO	96.3±4.0	100	6.9
	90.0±3.8	100	2.8
25	77.3±7.8	120.6	7.5
	88.2±4.9	82.1	4.7
50	81.2±2.8	129.3	13.3
	88.0±5.9	92.5	8.5
100	81.8±0.3	117.0	7.6
	91.3±3.2	81.7	7.3
200	87.5±3.0	94.5	4.8
	76.3±2.8	108.5	2.7
400	93.7±3.1	115.9	5.8
	93.3±0.8	63.8	11.6
600 P	86.2±8.8	106.5	8.7
	85.0±5.6	85.6	4.9
800 P	90.3±1.5	80.0	10.6
	96.7±7.5	77.1	3.9
DMBA	83.5±3.3	61.7	141.7
	83.7±3.3	25.1	151.4

DMAM: dimethylbenzanthracene

P: Precipitate

 

Table 3: Experiment II - Without Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Population Growth [%]	Mutant Frequency x 10^6
Negative control	66.8±5.3	64.1	4.4
	75.5±3.9	107.2	3.9
DMSO	92.7±4.9	100	0.4
	94.0±5.3	100	6.2
25	89.5±4.4	151.4	3.3
	93.0±6.5	102.6	1.8
50	90.2±7.0	130.7	1.8
	82.7±6.3	108.3	3.5
100	96.7±6.8	155.7	2.2
	88.8±2.0	123.0	1.9
200	65.2±8.1	37.8	3.8
	93.7±8.5	83.7	3.1
400	88.7±5.8	86.5	0.5
	92.5±6.5	108.9	0.5
600 P	101.7±3.2	100.6	2.5
	97.5±6.8	117.1	1.3
800 P	95.5±6.8	97.7	3.5
	85.2±5.0	114.9	4.9
EMS, 900	56.7±4.8	51.6	1212.5
	68.0±2.3	27.5	890.3

EMS: Ethylmethanesulphonate

P: Precipitate

 

Table 4: Experiment II - With Metabolic Activation

Concentration [µg/mL]	Cloning efficiency [%]	Relative Population Growth [%]	Mutant Frequency x 10^6
Negative control	87.8±1.3	108.9	3.3
	90.0±2.3	97.9	1.4
DMSO	88.5±11.8	100	6.1
	92.8±5.1	100	3.1
25	87.0±0.7	83.9	5.7
	79.2±9.0	100.4	5.3
50	85.2±3.4	118.9	3.4
	81.0±6.1	106.0	2.1
100	78.2±3.5	166.1	6.4
	94.0±7.5	79.3	11.5
200	83.2±2.9	111.7	10.0
	84.7±4.4	92.7	5.4
400	84.7±1.9	116.5	4.4
	77.8±6.8	87.0	4.8
600 P	81.7±4.2	123.2	10.2
	76.7±5.0	100.8	8.7
800 P	86.5±6.9	106.5	4.3
	78.2±5.3	89.2	13.3
DMBA 20	89.0±6.6	16.5	126.9
	83.3±3.0	14.6	100.5

DMAM: dimethylbenzanthracene

P: Precipitate

 

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative