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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
13 September 2017 - February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
2016
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian comet assay

Test material

Constituent 1
Chemical structure
Reference substance name:
N-{2-[(phenylcarbamoyl)amino]phenyl}benzenesulfonamide
EC Number:
806-543-7
Cas Number:
215917-77-4
Molecular formula:
C19H17N3O3S
IUPAC Name:
N-{2-[(phenylcarbamoyl)amino]phenyl}benzenesulfonamide
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lozt No.of test material: M-1702

Test animals

Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT., Cserkesz u. 90.,H-1103 Budapest, Hungary
- Age at study initiation: 56 - 61 days at the commencement of the treatment
- Weight at study initiation: 247.6 - 263.2 g
- Assigned to test groups randomly: yes, all animals were sorted according to body weight by computer and grouped according to weight ranges.
- Housing: 3 animals / cage; at the positive control group 2 animals / cages
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice (ssniff Spezialdiäten GmbH, D-59494 Soest Germany), ad libitum.
- Water: tap water, ad libitum.
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 - 23.6
- Humidity (%): 35 - 64
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle used: 1 % Methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was formulated in Methylcellulose (1 % in ultrapure water) at the adequate concentrations. Formulations were prepared in the laboratory of the test facility before each treatment. The test item was formulated in the vehicle in concentrations of 200, 100 and 50 mg/mL.
Duration of treatment / exposure:
Administration on day 0 and 24 hours thereafter
Frequency of treatment:
twice
Post exposure period:
3-4 hours after the second treatment
Doses / concentrationsopen allclose all
Dose / conc.:
2 000 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Dose / conc.:
500 mg/kg bw/day (nominal)
No. of animals per sex per dose:
6 animals in the dose groups and negative control group
4 animals in the positive control group.
Control animals:
yes, concurrent vehicle
Positive control(s):
Ethyl methanesulfonate (EMS) in 1% Methylcellulose
- Route of administration: oral (gavage)
- Concentration: 200 mg/kg bw, dissolved and applied in 1% Methylcellulose at a concentration of 20 mg/mL.

Examinations

Tissues and cell types examined:
liver and glandular stomach
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
For dose selection the guideline proposal and the information obtained from a previously performed acute oral toxicity study of the test item in rats (LD50 >2000 mg/kg bw) and repeated dose toxicity study was taken into consideration. Based on this information the maximum dose will be 2000 mg/kg bw/day, and in addition to the maximum dose two additional doses, i.e. 1000 and 500 mg/kg bw/day will be selected.
For the correct identification of the maximum tolerated dose (MTD) where no death, evidence of pain, suffering or distress occurs a range finding test was performed in the testing laboratory, using the same species, strain, sex, and treatment regimen to be used in the main study.
In the range-finding test (based on the availably information) two animals (male rats, CRL (WI) BR of Wistar origin) were treated with the test item by oral administration at 10 mL/kg body weight treatment volume at the concentration of 2000 mg/kg bw/day. The treatment was performed on two consecutive days with 24h interval. At the chosen concentration level neither mortality nor any clinical sign, any suffering of animals were observed; therefore 2000 mg/kg body weight/day is chosen as the highest dose level in the present study.

SAMPLING TIMES:
In this particular test the sampling was performed 3-4 hours after the second treatment (the animals were euthanized consistent with the effective animal welfare legislation and 3Rs principles and the cells of the target tissues were isolated) and care was taken to necropsy all animal at the same time after the last dose.

DETAILS OF SLIDE PREPARATION:
- Liver Single Cell Preparation:
A portion of the left lateral lobe of the liver was removed and washed in the cold mincing buffer until as much blood as possible was removed; thereafter placed in mincing buffer (ice cold Hank’s Balanced Salt Solution (HBSS) containing 20 mM EDTA and 10 % DMSO), minced with a pair of fine scissors to release the cells. The cell suspension was kept on ice for about 30 seconds to allow the large clumps to settle. The supernatant was pipetted into an Eppendorf tube and used for comet slides.

- Glandular Stomach Single Cell Preparation:
The stomach was open and washed free shortly from food using cold phosphate buffered saline. The forestomach was removed and discarded. The glandular stomach was then placed into cold mincing buffer and incubated on ice for 15-30 minutes. After the incubation the surface epithelia was gently scraped about two times using a scalpel blade. This layer was discarded and the gastric mucosa was rinsed with cold mincing buffer. Thereafter the stomach epithelia was carefully scraped for 4-5 times with scalpel blade. The obtained cell suspension was kept on ice for about 30 seconds to allow the large clumps to settle. The supernatant was pipetted into an Eppendorf tube and used for comet slides.

- Preparation of the Comet Assay Slides:
The slide preparation was done within one hour after single cell preparation. At least three slides were prepared for each animal for each tissue sample, with at least 5 animals per dose group. In summary, at least 15 slides per treatment per tissue for the treatment groups and vehicle control and at least 12 slides per tissue for the positive controls. The slides were adequately coded.

Pre-treatment of slides:
Conventional (superfrost) slides were dipped in hot 0.5 % normal melting point agarose in water. After gently remove the underside of the slides were wiped in order to remove the excess of agarose. The slides were then laid on a flat surface and were let allow drying.

Embedding the cells:
Before the use a volume of 130 μL of 0.5 % normal melting point agarose (NMA) was added on a microscope slide pre-layered with 0.5 % NMA and covered with a glass coverslip. The slides were placed on a tray until the agarose hardens (approx. 5 minutes). After the cell isolations each cell suspension was mixed with 0.5 % or 1.0 % Low Melting Point Agarose (LMPA). Thereafter 75 μL (approx. 3 x 10^4 cells) of this mixture was added on the microscope slide after gentle slide off the coverslip.
The microscope slides were covered with a new coverslip. After the LMPA-cell mixture hardens an additional 70 μL of NMA was dropped on the microscope slide after a gentle slide off the (second) coverslip and an additional new coverslip was laid on the slide. After the repeated NMA layer hardens the coverslip was removed.

- Lysis:
After the top layer of agarose solidifies and the last glass coverslip was removed the slides were immersed in chilled lysing solution for at least 1 hour (but preferably overnight) at 2-8 °C (in refrigerator) in the dark. After the incubation period, the slides were rinsed to remove residual detergents and salts prior to the alkali unwinding step. This rinsing procedure was performed in electrophoresis buffer.

METHOD OF ANALYSIS:
- Unwinding and Electrophoresis:
The slides were removed from the lysing solution and randomly placed on a horizontal gel electrophoresis unit. The unit was filled up with freshly prepared electrophoresis solution until the surfaces of the slides are completely covered with the solution (to about 1-2 mm above the slides).
During the unwinding and electrophoresis a balanced design was used to place slides in the electrophoresis tank to mitigate the effects of any trends or edge effect within the tank and to minimize batch to batch variability.
The slides were left for 30 min. for the DNA to unwind. Thereafter the electrophoresis was conducted for 30 min. by applying a constant voltage of 25 V and an electric current of about 300 mA. The current was recorded at the start and end of the electrophoresis period. All of these steps were sheltered from the daylight to prevent the occurrence of additional DNA damage. The temperature of the electrophoresis solution through unwinding and electrophoresis was maintained at a low temperature, i.e. 2-10 °C using a special cooler, and recorded once during the procedure

- Neutralization and Preservation of Slides:
After electrophoresis, the slides were removed from the electrophoresis unit, were covered with neutralization solution, allowed to stand covered for about 5 minutes, thereafter blotted and covered again with neutralization solution. This procedure was repeated twice. Subsequently the slides were exposed for additional 5 minutes to absolute ethanol in order to preserve all of the slides.

- Staining:
The slides were air dried and then stored at room temperature until they were scored for comets. Just prior the scoring the DNA, the slides were stained using 50 μL of 2 μg/mL Ethidium bromide.

- Evaluation of slides:
Coded slides were stained and blind scored. The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis the Komet 6.0 F (Andor Technology) were used.
For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment, per vehicle control and 600 per positive controls).
DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, tail moment and tail length.
Olive Tail Moment (OTM), expressed in arbitrary units, is calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in μm. The tail length is measured between the center of the comet head and the end of the comet tail.
The tail % DNA is determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity.
In addition, each slide was examined for presence of ghost cells. Ghost cells were excluded from the image analysis data collection, however determining of their frequency is useful for the data interpretation. The ghost cells were recorded for each slide per animal, per type of the treatment and per tissue.
Evaluation criteria:
The test chemical is clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- there is no concentration-related increase when evaluated with an appropriate trend test;
- all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

The test chemical is clearly positive if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- the increase is dose-related when evaluated with an appropriate trend test;
- any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration.
Statistics:
The data were checked for normality and for homogeneity of variance with the adequate statistical test methods.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose: 2000 mg/kg bw/day.
- Clinical signs of toxicity in test animals: At the chosen concentration level neither mortality nor any clinical sign, any suffering of animals were observed.

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: The highest dose (2000 mg/kg bw/day) is selected according to the criteria required by the OECD 489 guideline. Exposure via oral route was chosen as this route was considered to be the most relevant exposure route.
- Statistical evaluation: The statistical significance of % tail DNA values, tail length, OTM values and number of ghost cells was carried out using the appropriate statistical method, using SPSS PC+ software. The heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. In case of a positive analysis, Duncan's Multiple Range test was used to assess the significance of inter-group differences. Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. If the data were not normal distributed, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. In case of a positive analysis result, the inter-group comparisons were performed using Mann-Whitney U-test.

Any other information on results incl. tables

Results of Analytical Measurements:

Analysis of formulations (for checking of each concentration and homogeneity) was performed by a HPLC method with UV detection using a previously validated analytical method. Based on the results of the available validation study the test item proved to be adequately stable in 1 % Methylcellulose formulations at 50 and 200 mg/mL concentration levels at least for 1 day in refrigerator (5 ± 3 °C). The test item in 1 % Methylcellulose formulations was considered to be homogeneous. The measured concentration values remained within the ± 10 % of nominal range at all concentrations.

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions, the test item did not induce statistically significant increases in DNA strand breaks at any of the tested dose levels in stomach, duodenum or liver cells. The investigated test item is negative and did not show genotoxic activity in the examined tissues.
Executive summary:

The purpose of the comet assay (single cell gel electrophoresis assay) was to evaluate the mutagenic potential of the test item by measuring its ability to induce DNA damage in the target organs, tissues as specified and requested by ECHA. Formulations were prepared before each treatment. The test item was formulated in the vehicle in nominal concentrations of 200, 100 and 50 mg/mL (corresponding dose levels: 2000, 1000 and 500 mg/kg body weight) was applied and referred throughout the study. Analysis of formulations (for checking of each concentration and homogeneity) was performed using a previously validated analytical method. The measured concentration values remained within the ±10% of nominal range at all concentration levels examined.

The test substance was administered orally by gavage; twice: once on the day 0 and 24 hours thereafter at the test item doses and negative controls. The positive control animals were treated by oral gavage once during the experiment on the day 1. The target tissues were the stomach, the duodenum and the liver. The Sampling time was 3-4 hours after the second treatment (doses and vehicle control) and 3-4 hours after the treatment (positive control). Animals were euthanized and the cells of the target tissues were isolated. Cytotoxicity was determined on a small sample of each isolated cell suspension following the Trypan blue dye exclusion technique, directly after sampling. Prior the scoring the DNA was stained with 50μL of 20μg/mL Ethidium bromide; The comets were measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis the Komet 6.0 F (Andor Technology) was used. In addition, each slide was examined for presence of ghost cells (possible indicator of toxicity and/or apoptosis). Ghost cells were excluded from the image analysis data collection. The Numbers of treated Animals were 6 animals in the dose groups and negative control group; 4 animals in the positive control group. Cells from 5 animals were analysed in the dose groups and negative control group and cells from 3 animals were analysed in the positive control group. For each tissue sample fifty cells per slide were randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment, per vehicle control and 450 per positive control).

All of the validity criteria regarding the negative and positive control treatments as well as the number of analysed cells, and the investigated dose levels were met. No mortality was observed during the treatments and expression period in any dose group up to the limit dose of 2000 mg/kg bw/day and in the controls. Toxic symptoms or any clinical signs were not observed during the treatments in any dose or control group. At the tissue isolation normal appearance and anatomy of the target organs (stomach, duodenum and liver) was observed in all animals of the dose and control groups. However at the highest examined dose of 2000 mg/kg bw/day at three animals slight tympanites were noticed in the gastrointestinal tract. The average body weights increased in all dose groups as well as in the negative and positive control groups. The body weight increases were in the range of 0.984.09 % when compared the weight values just before the first treatment and just before the sacrifice. At the cytotoxicity screening measurements (using Trypan blue dye exclusion method) no cytotoxicity was noticed in any test item and control item treatments at any target tissue. At the examined test item treated groups the number of ghost cells in the stomach, duodenum and liver samples remained nearly in the same range and did not differ statistically significantly from that of the vehicle control. A statistically significant increase of ghost cells was noticed in all tissues after EMS treatment. The ghost cells are a possible indicator of cytotoxicity and/or apoptosis. According to the literature increased frequency of ghost cells may also indicate cells with severe DNA damage (genotoxicity). To be conscious of the mutagenicity results and laboratory’s earlier experience, the relatively higher number of ghost cells at the positive control, EMS treatment are considered being a possible indicator of genotoxicity. DNA strand breaks in the comet assay were measured by independent endpoints such as % tail DNA, tail length and Olive Tail Moment (OTM). The median values (and mean median values) were calculated at all examined-evaluated parameters. The median % tail DNA values of each dose remained in the vehicle control range at the examined tissues. In the stomach and liver samples the slightly different (higher or lower) values did not differ statistically significantly from that of the vehicle control up to the limit dose of 2000 mg/kg bw/day. In the duodenum sample at 1000 mg/kg bw/day a slightly lower mean median % tail DNA value was observed, which differed statistically significantly from that of the vehicle control.The obtained statistical significance was considered as not relevant for mutagenicity assessment since the significance is linked to a lower value which is within the historical control data range. The tail length values determined in the duodenum and liver samples showed no statistical significant differences. In the stomach samples the tail length values at the test item doses 500 and 1000 mg/kg bw/day were statistically significantly lower than the tail length value of the vehicle control. The obtained statistical significances at the tail length values of the stomach samples were considered as not relevant for mutagenicity assessment since the significance is linked to a lower value which is well within the historical control data range. The Olive Tail Moment values determined in the stomach, duodenum and liver cells of the test item treated groups did not differ statistically significantly from that of the vehicle control

The investigated test item is negative and did not show genotoxic activity in the examined tissues.