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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 July - 27 Oct 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)

Deviations:

yes

Remarks:

males/females of the 16h treatment period were not observed for clinical signs of toxicity at the 2-4h post treatment interval; males of the 16h treatment period were observed for clinical signs ca. 30 min after treatment instead of 1h after treatment

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)

Deviations:

yes

Remarks:

males/females of the 16h treatment period were not observed for clinical signs of toxicity at the 2-4h post treatment interval; males of the 16h treatment period were observed for clinical signs ca. 30 min after treatment instead of 1h after treatment

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

unscheduled DNA synthesis

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

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 7-11 weeks
- Weight at study initiation: males: 271.5±11.1 g; females: 186.4±8.4 g
- Housing: 4 animals/cage; Makrolon Type III/IV, with mesh top (Ehret GmbH, Emmendingen, Germany)
- Diet: pelleted standard diet (Harlan Laboratories B.V., Horst, The Netherlands); ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2
- Humidity (%): 45-70
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: the test item was dissolved in 30% DMSO and 70% PEG 400
- Justification for choice of solvent/vehicle: The vehicle was chosen due to its relative non-toxicity for the animals.
- Amount of vehicle: 10 mL/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
On the day of the experiment, the test item was formulated in 30% DMSO and 70% PEG 400. All animals received a single standard volume orally. A correction factor of 1.07 was applied.

Duration of treatment / exposure:

4 and 16 h

Frequency of treatment:

once orally

Post exposure period:

4 and 16 h after a single oral administration, the animals were anaesthetised and sacrificed by liver perfusion.

Doses / concentrations

Remarks:

Doses / Concentrations:
1000, 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

4

Control animals:

yes, concurrent vehicle

Positive control(s):

N,N´-dimethylhydrazinedihydrochloride (DMH): for the 4 h preparation interval
- Route of administration: oral (gavage)
- Dosing: 80 mg/kg bw (10 mL/kg bw)

2-acetylaminofluorene (2-AFF): for the 16 h preparation interval
- Route of administration: oral (gavage)
- Dosing: 100 mg/kg bw (10 mL/kg bw)

Examinations

Tissues and cell types examined:

primary hepatocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
The maximum tolerated dose is determined to be the dose that causes toxic reactions (e.g. reduced spontaneous activity, eyelid closure, apathy, etc.) without having major effects on survival within 24 hours. If no toxic reactions are observed the highest dose recommended by the OECD guideline to be used should be 2000 mg/kg bw.
In a pre-experiment two males and two females received a single dose of 2000 mg/kg bw test item dissolved in 30% DMSO and 70% PEG 400 once orally. The animals treated with the test item did no show any clinical signs of toxicity.

TREATMENT AND SAMPLING TIMES:
The animals received the test item once orally and were examined for acute toxic symptoms at intervals of approx. 1 h, 2 h and 4 h for the 4 hours treatment, and 1 h, and 16 h for the 16 hours treatment after administration of the test item. Except the females of the 16 h treatment period were examined for acute toxic symptoms approximately 30 minutes after treatment instead of 1 hour after treatment.
4 and 16 h after the single oral administration, the animals were anaesthetised and sacrificed by liver perfusion.

ISOLATION OF THE PRIMARY HEPATOCYTES
After anaesthetising the rats with 46% Ketamin, 23% Xylazin and 31% Midazolam (approx. 2 mL/kg body weight) the liver was perfused through the vena portae with Hanks' balanced salt solution supplemented with collagenase (0.05% (w/v)), adjusted to pH 7.4 and maintained at 37 °C.
The isolated hepatocytes were washed twice with HBSS. The crude cell suspension was filtered through a stainless steel mesh to yield a single cell suspension. The quality of the performed perfusion was determined by the trypan blue dye exclusion method for cell viability. In addition, the number of the cells was determined.

CULTURE CONDITIONS:
The washed hepatocytes were centrifuged and transferred into Williams medium E (WME) supplemented with:
Hepes 2.38 mg/mL, L-Glutamine 0.29 mg/mL, Penicillin 100 units/mL, Insulin 0.50 µg/mL, Streptomycin 0.10 mg/mL, Fetal calf serum (FCS) 100 µL/mL
This complete medium was adjusted to pH 7.6.
At least three cultures were established from each animal. Aliquots with freshly isolated hepatocytes in complete culture medium (2.0 × 105 viable cells/mL) were added to six-well dishes. After an attachment period of ca. 1.5 h in an humidified incubator at 37 °C the culture medium was discarded. Then, the cell layer was rinsed once with PBS to remove non-adherent cells. Subsequently, 3HTdR (5 XCi/mL, specific activity 70-90 Ci/mmol) was added to the cultures. After a labelling time of 4 h the cells were washed twice with WME supplemented with 1% (v/v) FCS and 0.25 mM unlabelled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1% (w/v) sodium citrate for 10 min to swell the nuclei for better grain detection.
The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3+1 v/v) for 20 min each, rinsed with 96% (v/v) ethanol, and air-dried.

METHOD OF ANALYSIS:
Evaluation was performed microscopically on coded slides using NIKON microscopes with oil immersion objectives. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the Sorcerer UDS device version 2.0 DT3152 (Perceptive Instruments). In addition, the number of grains of the most heavily labelled nuclear-sized cytoplasm area adjacent to the nucleus was counted At least two slides per animal and 50 cells per slide were evaluated. Heavily radio-labelled cells undergoing replicative DNA synthesis were excluded from counting.

Evaluation criteria:

Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.
A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.
A group average between 0 and 5 net grains is considered as a marginal response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains.
Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test (4).
A test item producing net grains not greater than 0 or not significantly greater than the concurrent control, at anyone of the test points is considered non-effective in this system.
However, the primary point of consideration is the biological relevance of the results.

Statistics:

A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding controls.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Ruffled fur, reduction in spontaneous activity, abdominal posture, salivation and diarrhoea were signs of systemic toxicity observed in males.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw
- Clinical signs of toxicity in test animals: None

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals:
Ruffled fur, reduction in spontaneous activity, abdominal posture, salivation and diarrhoea were signs of systemic toxicity observed in males.
- Evidence of cytotoxicity in tissue analyzed:
Overall, the viability of the hepatocytes was not substantially affected by the in vivo treatment with the test item. Except the cell viability of the hepatocytes isolated from one male animals of the 1000 mg/kg bw 16 h, one female of the 1000 mg/kg bw 4 h and one female of the 2000 mg/kg bw 4 h treatment group which were below 60% and therefore the results of these animals were scored but not reported.
- There was no biologically relevant UDS induction in the hepatocytes of the treated animals as compared to the current vehicle controls. The net grain counts were not distinctly increased after in-vivo treatment of the animals with the test item at 4 hours or 16 hours, respectively. Net grain counts obtained after treatment with the test item remained consistently negative at all tested doses. In addition, no substantial shift to higher values of percentage of cells in repair was reported.

Any other information on results incl. tables

 Table 1: Group mean nucleus, cytoplasmic area and net grains: males, preparation interval 4 h

Exposure group	Number of animals	Nuclear count	Cytoplasmatic count	Net grain count	Net nuclear grains of cells in repair	Cells in repair [%]
Vehicle control (30% DMSO/ 70% PEG 400)	4	27.64±11.75	39.76±14.46	-12.12±11.24	9.45±4.5	6.5
Positive control (DMH 80 mg/kg bw)	4	77.10±24.20	38.71±13.10	38.39±20.93	40.43±19.28	95.5
Test substance 1000 mg/kg bw	4	21.02±8.9	27.16±10.86	-6.14±10.19	10.62±5.16	13.75
Test substance 2000 mg/kg bw	4	17.91±7.76	26.21±10.01	-8.30±8.84	7.76±2.78	7.25

N,N´-dimethylhydrazinedihydrochloride (DMH)

Table 2: Group mean nucleus, cytoplasmic area and net grains: males, preparation interval 16 h

Exposure group	Number of animals	Nuclear count	Cytoplasmatic count	Net grain count	Net nuclear grains of cells in repair	Cells in repair [%]
Vehicle control (30% DMSO/ 70% PEG 400)	4	23.82±10.51	39.12±15.99	-15.30±11.83	7.29±2.74	3.75
Positive control (2-AAF 100 mg/kg bw)	3*	50.16±11.70	26.23±8.38	23.92±11.09	25.05±9.91	95.33
Test substance 1000 mg/kg bw	3**	28.74±14.57	41.49±18.33	-12.76±11.81	9.86±5.65	5.33
Test substance 2000 mg/kg bw	4	27.54±11.14	42.40±13.68	-14.86±10.70	7.90±1.99	3.25

*For one animal, perfusion failed

**For one animal, data not reported due to low cell viability (<60%)

2-acetylaminofluorene (2-AFF)

Table 3: Group mean nucleus, cytoplasmic area and net grains: females, preparation interval 4 h

Exposure group	Number of animals	Nuclear count	Cytoplasmatic count	Net grain count	Net nuclear grains of cells in repair	Cells in repair [%]
Vehicle control (30% DMSO/ 70% PEG 400)	3*	13.70±6.07	23.49±7.989	-9.79±7.09	7.67±0.0	1.33
Positive control (DMH 80 mg/kg bw)	4	58.35±20.71	22.39±8.32	35.96±20.37	37.04±19.6	97.00
Test substance 1000 mg/kg bw	3**	13.43±6.08	24.68±7.19	-11.25±7.02	4.73±0.37	2.00
Test substance 2000 mg/kg bw	3**	14.11±6.23	27.06±8.94	-12.95±8.66	9.87±0.38	2.33

*For one animal, perfusion failed

**For one animal, data not reported due to low cell viability (<60%)

N,N´-dimethylhydrazinedihydrochloride (DMH)

 

Table 4: Group mean nucleus, cytoplasmic area and net grains: females, preparation interval 16 h

Exposure group	Number of animals	Nuclear count	Cytoplasmatic count	Net grain count	Net nuclear grains of cells in repair	Cells in repair [%]
Vehicle control (30% DMSO/ 70% PEG 400)	3*	13.24±7.66	23.11±9.63	-9.87±9.00	7.04±2.24	5.67
Positive control (2-AAF 100 mg/kg bw)	4	37.86±16.12	25.92±132.01	11.94±13.54	18.48±10.59	68.5
Test substance 1000 mg/kg bw	4	18.42±10.51	29.54±14.34	-11.12±10.95	10.56±6.06	4.5
Test substance 2000 mg/kg bw	4	24.92±12.3	38.73±15.27	-13.81±11.75	9.68±3.89	5.25

*For one animal, perfusion failed

2-acetylaminofluorene (2-AFF)

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative