Benzenesulfonamide, N-[2-[[(phenylamino)carbonyl]amino]phenyl]-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial reverse mutation test

Based on an bacterial reverse mutation test using strains of Salmonella typhimurium and Escherichia coli the test substance was considerd to be non mutagenic.

Chromosomal aberration test

In a chromosomal aberration test with cultured mammalian cells a weakly positive result was oberved.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro gene mutation study in mammalian cells does not need to be conducted because adequate data from a reliable in vivo mammalian gene mutation test are available

Justification for type of information:

An in vivo comet assay according to OECD TG 489 is available. 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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Test guideline under Japan chemical Control Law

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

- Type and identity of media: Oxoid Nutrient Broth No. 2 dissolved in purified water and autoclaved. This was stored in cold storage until use.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: Oxoid Nutrient Broth No. 2 dissolved in purified water and autoclaved. This was stored in cold storage until use.

Metabolic activation:

with and without

Metabolic activation system:

Preincubation with S9 mix

Test concentrations with justification for top dose:

Range-finding test: 1.22, 4.88, 19.5, 78.1, 313, 1250 and 5000 µg/plate (with and without S9 mix)
Main test I and II: 39.1, 78.1, 156, 313, 625 and 1250 (with and without S9 mix) for the S.typhimurium strains and 39.1, 78.1, 156, 313, 625, 1250,
2500 and 5000 µg/plate (with and without S9 mix) for the E.coli strain.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The test substance was insoluble in water, and soluble in DMSO with no increases in temperature, foaming or discolouration observed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 9-Aminoacridine hydrochloride monohydrate (9-AA), 2-Aminoanthracene (2-AA), and 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide (AF-2)

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 plates per dose (main tests)

Evaluation criteria:

The test substance was judged to have mutagenicity (positive) when the test substance induced a dose-dependant increase in the number of the
revertant colonies (mean) to a level equal to or greater than 2-fold of the negative (solvent) control value (mean value) in any one of the tester strains with or without S9-mix, and when the dose-dependant increase was reproducible. Other results were judged to be negative.

Statistics:

No statistical analysis was performed with the test in the study.

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

The test substance was negative for mutagenic activity.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Guideline relevant to Japanese CSCL.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

other: CHL/IU (derived from lung fibroblasts of female Chinese hamsters

Details on mammalian cell type (if applicable):

- Type and identity of media: Minimum Essential Medium (MEM) or Eagle's MEM.
- Properly maintained: yes
- Specificity: The specificity of the cells were confirmed to be appropriate; Modal chromosome number (2n): 25, Doubling time: 16.4 hours, Mycoplasm infection: Negative.

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix prepared from the livers of 7-week old Sprague-Dawley rats treated with phenobarbital and 5,6-benzoflavone.

Test concentrations with justification for top dose:

Preliminary toxicity test: 37, 370 and 3700 µg/mL.
Cell growth inhibition test: 2.5, 5, 10 and 20 µg/L.
Chromosomal aberration test: 1, 2, 3, 4, 5, 6, 7, and 8 µg/L.

Vehicle / solvent:

Negative control and test substance vehicle: Dimethyl Sulfoxide (DMSO) was used. In a solvent selection test, DMSO did not show an exothermic reaction, discolouration, or foaming and as such, was selected as an appropriate vehicle for use on study.

Positive control (MMC solution): Material was dissolved in water for injection and serially diluted with saline to obtain test concentrations.
Positive control (BP solution): DMSO was used as the solvent.

All test/vehicle solutions were added to plates containg MEM culture media and S9 mix where applicable.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other:

Remarks:

Without S9: Mitomycin C ; with S9: benzo[a]pyrene; 24 hour assay: MMC

Details on test system and experimental conditions:

Cells were cultured in minimum essential medium (MEM) or Eagle's MEM. Eagle's MEM was prepared by weighing 8.3 g of Eagle's MEM and dissolving in 880 mL of distilled water. This wa sthen autoclaved at 121°C for 20 minutes. This solution was supplemented with L-glutamine solution and sodium bicarbonate solution. Heat inactivated bovin serum was added to MEM.

Preliminary toxicity test:
A preliminary toxicity test was conducted on cell cultures over a 6-hour exposure time with and without metabolic activation, followed by a 18-hour recovery period and a continuous exposure of 24 hours without metabolic activation. The test material dose range used was 37 - 3700 µg/mL. One plate was used per concentration for each treatment and the cell density (observed by an inverted phase-contrast microscope) was presented as the relative percentage of the negative control plate.

Test and control materials:
Test material was weighed and dissolved into DMSO by vortex mixing and sonication. The final volume of the solution was adjusted with DMSO to prepare the highest test substane solution of 100-fold concentration of the final concentration. A portion of this solution was diluted with DMSO to prepare each test substance solution of 100- fold concentration of the final concentration. All procedures were conducted under yellow light and UV-cut light at room temperature. The test substance solutions were used immediately after preparation.

The positive controls were prepared as follows:
- In the absence of S9: mitomycin C was dissolved in water for injection. This was serially diluted with saline to prepare 100-fold concentrations of the prescribed concentrations for each treatment condition (short term assays: 10 ug/mL, continuous treatment assays: 5 ug/mL. These stocks were frozen until required for use.
- In the presence of S9: Benzo[a]pyrene was dissolved in dimethyl sulphoxide to prepare 100-fold concentration of the prescribed concentration (1.5 mg/mL). This solution was divided into tubes and frozen until use.

Cell growth inhibition test conditions:
Cells were seeded in 5 mL of culture medium (4x10^3 cells/mL) using plastic plates (6cm diameter) and incubated for 3 days. After this the MEM culture medium was removed and the test substance solution, MEM culture medium and S9 mix added (where appropriate). In the negative control group, DMSO was used instead of the test substance solution. With solutions in the presence and absence of S9 mix, treatment was undertaken for 6 hours (then washed with MEM and fresh MEM added), followed by an 18-hour recovery period. For the 24-hour assay (absence of S9 mix) cells were continuously treated for 24 hours. Two plates per concentration were used in each treatment (tables provided below showing treatment details). At the beginning and end of each treatment, all plates were examined macroscopically for precipitation of the test substance. All Cultures were incubated at 37 degrees C in 5% CO2 in humidified air.

Chromosome abberation test conditions:
Cells were seeded in 5 mL of culture medium (4x10^3 cells/mL) using plastic plates (6cm diameter) and incubated for 3 days. After this the MEM culture medium was removed and the test substance solution, MEM culture medium and S9 mix added (where appropriate). In the negative control group, DMSO was used instead of the test substance solution. With solutions in the presence and absence of S9 mix, treatment was undertaken for 6 hours (then washed with MEM and fresh MEM added), followed by an 18-hour recovery period. For the 24-hour assay (absence of S9 mix) cells were continuously treated for 24 hours. Two plates per concentration were used in each treatment (tables provided below showing treatment details). At the beginning and end of each treatment, all plates were examined macroscopically for precipitation of the test substance. All Cultures were incubated at 37 degrees C in 5% CO2 in humidified air. The positive control groups were included.

Cell harvest:
Cell growth inhibition test:
The surface of the cells were washed with phosphate buffer saline (PBS(-)). The cells were then treated with 0.25% trypsin-EDTA at 37°C for 5 minutes. The MEM culture medium was added to the plate and the cells were dissociated by pipetting. The number of cells were counted with a cell counter and the cell growth index calculated for the cell growth inhibition test (relative cell number compared to the negative control). The IC50 value was calculated from a linear equation derived from the two data points showing cell growth indices higher and lower than 50%, but closest to 50%.

Chromosome abberation test:
Two hours before the end of the respective culture periods, colcemid was added to the medium in each tube at a final concentration of 0.1 µg/mL to accumulate the metaphase cells. At the end of the respective culture periods, the cell surface were washed with PBS(-) and the cells were treated with 0.25% trypsin-EDTA (37°C for 5 minutes). MEM culture medium was added and the cells were dissociated by pipetting.
The cell suspension was collected in a centrifuge tube and centrifuged to collect the cells. The resulting supernatant was removed and the cells subjected to hypotonic treatment with potassium chloride solution at 37°C for 15 minutes. The cells and 0.5 mL of the ice-cold fixative mixture (ethanol - acetic acid mix (3:1)) were mixed and then centrifuged. The supernatant was removed and then re-centrifuged twice, and the cells suspended in a small amount of the fixative. Drops of the cell suspension were then added onto two sites on a glass slide and dried. Two slides were prepared for each plate. The cells were stained with 3% v/v Giemsa'ssolution and the slides washed with water and dried. The slides were then mounted with a cover glass and mounting medium.

Evaluation criteria:

Cell growth index was also measured in the chromosome abberation test (same conditions as per the cell growth inhibition test).
The mitotic and relative mitotic index were calculated as follows:
- The number of metaphase cells were counted by examining 500 cells per plate. Mitotic index (%) = (number of metaphase cells)/(Number of cells examined) x 100. From the mitotic index, the relative mitotic index (%) = (mitotic index in cells treated with the negative control) x100. This was not conducted for the positive controls. Because of cytotoxicity, the mitotic index could not be measured at 80 or 100 ug/mL in the 24-hour assay.

Scoring chromosomal damage:
The first 100 consecutive well-spread metaphases from each plate was counted.
Structural aberration - Number of chromosomes was 25 +/- 2
Numerical aberration - Number of chromosomes was 25 +/- 2, or 38 or more
Structural aberration - Chromatid break/exchange, chromosome break/exchange and fragmentation. Gaps were also analysed and numerical aberrations (polyploidal cells with 38 or more centromeres and endoreduplicated cells.

Statistics:

No statistical analysis was performed

Key result

Species / strain:

other: CHL/IU (derived from lung fibroblasts of female Chinese hamsters

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cell growth index <50%; 70, 100 or 60 in the -S9, +S9 and 24 hour assay respectively.

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Preliminary phase: At 370 µg/mL the cell density (relative percentage to the negative control plate) was 0% in all tests.

Cell growth inhibition test: The IC50 values were 56.9, 59.6 and 50.2 ug/mL in the -S9, +S9 and 24-hour assay respectively.
Concentration for metaphase analysis: Cell growth index <50% (measured as part of the chromosome abberation assay); 70, 100 or 60 in the -S9, +S9 and 24 hour assay respectively.

Chromosome aberration assay: No precipitation was observed at any of the concentrations. From the results of the cell growth indices, the preliminary microscopic observation was conducted at 50, 60 or 70 ug/mL (-S9), 70, 80 or 100 µg/mL (+S9) and at 40, 50 or 60 (24-hour assay).
The incidence of structural aberrant cells was 19% at 70 µg/mL (-S9) and 10.5 % at 100 µg/mL (+S9). The incidences of numerical aberrant cells in the -S9/+S9 and those of both structural aberrant cells and numerical aberrant cells in the 24 hour assay were <5% in the treatment groups.
The D20 values were 0.074 mg/mL in the -S9 and 0.13 mg/mL in the +S9.

In the vehicle control group, the incidence of structural and numerical aberrant cells was <5% and in the positive control groups, the incidence of structural aberrant cells were >10% indicting the test system was satisfactory.

Conclusions:

The test substance was considered to have the potential to induce chromosomal aberrations in CHL/IU cells under the conditions employed in the study, although the results obtained were only slightly above the threshold for a positive result.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Comet assay

In an vivo comet assay on isolated stomach, duodenum and liver cells under alkaline conditions in the male WISTAR rats 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.

Link to relevant study records

Reference

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

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

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

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

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.

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.

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.

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.

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 0.98‑4.09 % 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Genetic toxicity in-vitro

Bacterial reverse mutation test

From the results described, it was concluded that the test material was not mutagenic under the conditions employed in the bacterial reverse mutation test.

 

Chromosomal aberration test

In the chromosome aberration assay, the test result for clastogenicity was positive (incidence of structural aberrant cells was 19% at 70 µg/mL (-S9) and 10.5% at 100 µg/mL (+S9), both being over the 10% threshold for the number of structurally aberrant cells, indicating a positive response (although only slightly over).

 

Genetic toxicity in vivo

Comet-Assay study

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 0.98‑4.09 % 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.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. It was concluded that the test material was not mutagenic based on the conditions employed in the bacterial reverse mutation test and would not be classified based on the negative result obtained. A weak positive result available in the in vitro chromosome aberration assay, was evaluated further in vivo. In an in vivo mammalian alkaline comet assay on isolated stomach, duodenum and liver cells no genotoxic activity was observed. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the seventeenth time in Regulation (EU) 2021/849