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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian germ cell study: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2023

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Performed after approval of testing proposal TPE-D-2114588623-38-01/F

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2023

Report date:

2023

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

29 July 2016

GLP compliance:

yes

Type of assay:

mammalian comet 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

Specific details on test material used for the study:

Expiry date: 23 May 2023
Physical Description: White crystalline powder
Purity/Composition: 95-98.9%
Storage Conditions: At room temperature protected from light

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: 6 weeks
- Weight at study initiation: 163.0 - 172.2 g
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing:
Polycarbonate cages (Makrolon MIV type or 2000P Tecniplast) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles.
During treatment in the dose-range finding study, polycarbonate cages (Makrolon type MIII) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles may be used.
Up to 5 animals of the same sex and same dosing group together.
These housing conditions will be maintained unless deemed inappropriate by the Study Director and/or Clinical Veterinarian. The room(s) in which the animals will be kept will be documented in the study records.

- Diet (e.g. ad libitum): Ad libitum, except during designated procedures
- Water (e.g. ad libitum): Freely available to each animal via water bottles
- Acclimation period: The animals were allowed to acclimate to the Test Facility toxicology accommodation for at least \5 days before the commencement of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): Ten or more air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark (except during designated procedures)

IN-LIFE DATES: From: 02/12/2022 To: 11/01/2023

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
No correction was made for the purity/composition of the test material.
The test material was suspended in corn oil (Fagron Farmaceuticals, Capelle a/d IJssel, the Netherlands). The specific gravity of corn oil is 0.92 g/mL. Test material concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension.
This resulted in yellow suspensions for all formulations. Test material concentrations were dosed within 4 hours after preparation.
Any residual volumes were discarded.

Duration of treatment / exposure:

The first day of dosing was designated as Day 1. The dose was given using a plastic feeding tube. The dosing volume was 10 mL/kg body weight.

Frequency of treatment:

Twice

Control animals:

yes, concurrent vehicle

Positive control(s):

Ethyl methanesulfonate (EMS; RS344) at 200 mg/kg body weight dissolved in physiological saline.

Examinations

Tissues and cell types examined:

Liver
The isolation method was based on the publication of Hu et al. A portion of 0.6-0.7 gram from the liver was removed and minced thoroughly on aluminum foil in ice. The minced liver tissue was added to 10 mL of collagenase (20 Units/mL; Sigma Aldrich, Zwijndrecht, The Netherlands) dissolved in HBSS (Ca2+- and Mg2+-free) and incubated in a shaking water bath at 37 °C for 20 minutes. Thereafter, a low centrifugation force was applied two times to remove large undigested liver debris (40 g for 5 min). The supernatant was collected and centrifuged to precipitate the cells (359 g for 10 min). The supernatant was removed and the cell pellet was resuspended in ice cold HBSS (Ca2+ and Mg2+-free).

Glandular Stomach
This isolation method for glandular stomach is based on the JACVAM Comet validation study.
The stomach is cut open and washed free from food using Hank’s Balanced Salt Solution (HBSS; Ca++, Mg++ free). The fore-stomach is removed and discarded. The glandular stomach is stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The surface epithelia of the glandular epithelia is gently scraped softly. This layer is discarded. The stomach is then scraped softly multiple times in 10 ml of mincing buffer. The mincing buffer consists of 20 mM EDTA (disodium) and 10% DMSO in Hank’s Balanced Salt Solution (HBSS) (Ca++, Mg++ free, and phenol red free if available), pH 7.5 (DMSO will be added immediately before use).
The supernatant is collected and filtered through a 100 μm Cell Strainer to purify the cell suspension.

Isolation of duodenum
This isolation method for duodenum is based on the JACVAM Comet validation study.
The duodenum is stored on ice in mincing buffer incomplete (HBSS containing 20 mM EDTA).
The duodenum is cut open, the surface epithelia is gently scraped to remove apoptotic cells in the upper cell layer. This layer is discarded. The duodenum is then scraped softly multiple times in 10 ml of mincing buffer.
The mincing b

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):

DETAILS OF SLIDE PREPARATION:
To the cell suspension, melted low melting point agarose (LMAgarose; Trevigen, Gaithersburg, USA) was added (ratio 10:140). The cells were mixed with the LMAgarose and 50 µL was layered on a pre-coated comet slide (Trevigen) in duplicate. Three slides per tissue per animal were prepared. The slides were marked with the study identification number, animal number and group number. The slides were incubated for 10-37 minutes in the refrigerator in the dark until a clear ring appears at the edge of the comet slide area.

The cells on the slides were overnight (18h) immersed in pre-chilled lysis solution (Trevigen) in the refrigerator. After this period the slides were immersed/rinsed in neutralization buffer (0.4 M Tris-HCl pH 7.4). The slides were then placed in freshly prepared alkaline solution for 20 – 30 minutes at room temperature in the dark. The slides were placed in the electrophoresis unit just beneath the alkaline buffer solution and the voltage was set to 0.7 Volt/cm. The electrophoresis was performed for 20 (glandular stomach and duodenum) or 30 (liver) minutes under constant cooling (actual temperature 4.0-5.0°C). After electrophoresis the slides were immersed/rinsed in neutralization buffer for 5 minutes. The slides were subsequently immersed for 5 minutes in Absolut ethanol (99.6%, Merck) and allowed to dry at room temperature. The slides were stained for approximately 5 minutes with the fluorescent dye SYBR® Gold (Life Technologies, Bleiswijk, The Netherlands) in the refrigerator. Thereafter the slides were washed with Milli-Q water and allowed to dry at room temperature in the dark and fixed with a coverslip.

Part of the liver, glandular stomach and duodenum from the animals (with exception of the positive control) used (after isolation of a part for the comet assay) was collected and fixed and stored in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution). No histotechnology and histopathology was needed.

Evaluation criteria:

To prevent bias, slides were randomly coded (per tissue) before examination of the comets. An adhesive label with study identification number and code were placed over the marked slide. The slides were examined with a fluorescence microscope connected to a comet assay IV image analysis system (Perceptive instruments Ltd, Suffolk, United Kingdom). One hundred fifty comets (50 comets of each replicate LMAgarose circle) were examined per sample.
The following criteria for scoring of comets were used:
• Only horizontal orientated comets were scored, with the head on the left and the tail on the right.
• Cells that showed overlap or were not sharp were not scored.
In addition, the frequency of hedgehogs was determined and documented based on the visual scoring of at least 150 cells per tissue per animal. The occurrence of hedgehogs was scored in all treatment groups and the control.

Statistics:

ToxRat Professional v 3.3.0 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the comet assay data.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Remarks:

Vehicle control served as negative control

Positive controls validity:

valid

Additional information on results:

In the dose-range finding test, three male and three female animals dosed with 2000 mg/kg bw/day showed no treatment related clinical signs or mortality.

A statistically significant increase in the mean Tail Intensity (%) was observed in liver cells at the highest dose-group of test material treated male treated animals compared to the vehicle treated animals. Furthermore, a positive trend was shown. As this increased tail intensity (1.66%) was clearly within the 95% control limits historical control data of the negative control (1.4 - 12.5%) the increase was considered not biologically relevant.
No statistically significant increase in the mean Tail Intensity (%) was observed in duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals. In addition there was no relevant increase in the number of hedgehogs in test material treated groups compared to the vehicle group.

The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was 0.82 ± 0.35% (mean ± SD), 5.1 ± 1.6% (mean ± SD) and 8.3 ± 1.5% (mean ± SD) in male animals, respectively, which is within or even below the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity of 73 ± 3.5% (mean ± SD; p<0.001 Students t test;), 51 ± 10% (mean ± SD; p<0.001 Students t test;) and 56 ± 2.7% (mean ± SD; p<0.001 Students t test;) in male animals in liver, duodenum and glandular stomach cells, respectively. The mean positive control Tail Intensity was within the 95% control limits of the distribution of the historical positive control database.
Adequate numbers of cells (150 cells per animal) and doses were analyzed and the highest test dose was the maximum dose required by the guidelines. Hence, all criteria for an acceptable assay were met.

Applicant's summary and conclusion

Conclusions:

The comet assay is valid and V123109 (CAS 59941-98-9) is not genotoxic in the comet assay in liver, duodenum and glandular stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 2000 mg/kg bw/day (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this study.

Executive summary:

The objective of this study was to obtain information on the potential genotoxicity of V123109 when administered to rats at the maximum recommended dose in accordance with current regulatory guidelines, by measuring the increase in DNA strand breaks in duodenum, liver and glandular stomach.

The Wistar Han rat was the species and strain of choice because it is a readily available rodent which is commonly used for genotoxicity testing, with documented susceptibility to a wide range of toxic materials. Moreover, historical control background data has been generated with this strain.

The study procedures described in this report were based on the most recent OECD guideline.

Batch V123109/EC of the test material was a white crystalline powder. The test material was suspended in corn oil.

Based on the results of the dose-range finding study test a concentration of 2000 mg/kg bw/day for male animals was selected as maximum dose for the main test (the highest dose required in the current guideline. Since there were no substantial differences in toxicity between sexes only males were used in the main study.

In the main study male animals were dosed with vehicle (corn oil), test material (at 500, 1000 and 2000 mg/kg bw) for two consecutive days. A positive control group was dosed twice by oral gavage with 200 mg Ethyl Methane Sulfonate (EMS) /kg bw.

Approximately 3-4 hours after the last dose the animals were sacrificed by abdominal aorta bleeding under isoflurane anesthesia tissues were isolated. Single cell suspensions from were made followed by comet slide preparation. The slides were analyzed and the
Tail Intensity (%) was assessed.

No biologically relevant statistically significant increase in the mean Tail Intensity (%) was observed in liver, duodenum and glandular stomach cells of test material treated male treated animals compared to the vehicle treated animals.

The mean Tail Intensity in liver, duodenum and glandular stomach cells of vehicle-treated rats was within the 95% control limits of the distribution of the historical control data for the vehicle control. The positive control EMS induced a significant increase and showed a mean Tail Intensity which was within the 95% control limits of the distribution of the historical positive control database. Adequate numbers of cells and doses were analysed and the highest test dose was the maximum dose required by the guidelines. Hence, all criteria for an acceptable assay were met.

In conclusion, the test is valid and V123109 is not genotoxic in the comet assay in liver, duodenum and glandular stomach cells when sampled approximately 3-4 hours post dosing, of male rats that were dosed via oral gavage for two consecutive days up to a dose of 2000 mg/kg bw/day (the maximum recommended dose in accordance with current regulatory guidelines) under the experimental conditions described in this report.