[No public or meaningful name is available]

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 May 2021 - 05 July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Test material

Test material information

Constituent 1

Reference substance name:

N1,N3‐bis(3‐methylphenyl)‐5‐[(3‐methylphenyl)sulfamoyl]benzene‐1,3‐dicarboxamide

Molecular formula:

C29H27N3O4S

IUPAC Name:

N1,N3‐bis(3‐methylphenyl)‐5‐[(3‐methylphenyl)sulfamoyl]benzene‐1,3‐dicarboxamide

Test material form:

solid: particulate/powder

Details on test material:

- Appearance: Off-white powder
- Storage condition: At room temperature protected from light

Method

Target gene:

Thymidine kinase locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: L5178Y/TK+/--3.7.2C mouse lymphoma cells. American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: recommended test system in international guidelines

For cell lines:
- Absence of Mycoplasma contamination: Yes
- Methods for maintenance in cell culture: stock cultures of the cells were stored in liquid nitrogen (- 196°C).
- Periodically ‘cleansed’ of spontaneous mutants: yes

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature: Basic medium
RPMI 1640 Hepes buffered medium (Dutch modification) (Life Technologies) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively) (Life Technologies), 1 mM sodium pyruvate (Sigma, Zwijndrecht, The Netherlands) and 2 mM L-glutamin (Life Technologies).
Growth medium
Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum.
Exposure medium
Cells will be exposed to the test item in basic medium supplemented with 5% to 10% (v/v)
heat-inactivated horse serum.
Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum and 5 µg/mL trifluorothymidine (TFT) (Sigma).
Non-selective medium
Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum.
Environmental conditions
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 50 - 98%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.1 - 39.9 °C).

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and was prepared from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight).
- method of preparation of S9 mix: S9-mix was prepared immediately before use and kept refrigerated. S9-mix components contained per mL physiological saline: 1.63 mg MgCl2.6H2O (Merck); 2.46 mg KCl (Merck); 1.7 mg glucose-6-phosphate (Roche, Mannheim, Germany); 3.4 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom); 4 µmol HEPES (Life technologies). The above solution was filter (0.22 µm)-sterilized. To 0.5 mL S9-mix components 0.5 mL.
- concentration or volume of S9 mix and S9 in the final culture medium: The concentration of the S9-fraction in the exposure medium was 4% (v/v).

Test concentrations with justification for top dose:

- Dose-range finding test, with S9 (3h) and without S9 (3h): 1, 5, 10, 25, 75 and 125 μg/mL
- Dose-range finding test, without S9 (24h): 1, 5, 10, 25, 75 and 125 μg/mL
- Experiment 1, with S9 (3h) and without S9 (3h): 0.01, 1.0, 5.0, 10, 25, 50, 75, 125, 150, 200, 250, 300 and 400 μg/mL exposure medium.
The dose levels selected to measure mutant frequencies at the TK-locus were:
Without and with S9-mix: 0.01, 1.0, 5.0, 10, 25, 50, 75 and 125 μg/mL
- Experiment 2, without S9 (24h): 0.5, 1.0, 2.5, 5.0, 10, 25, 35, 45, 55, 65, 75, 100 and 125 µg/mL exposure medium.
The dose levels selected to measure mutant frequencies at the TK-locus were:
0.5, 1.0, 2.5, 5.0, 10, 25 and 35 µg/mL exposure medium.

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of vehicle: A solubility test was performed based on visual assessment. The test item formed a clear (light) yellow solution in dimethyl sulfoxide (DMSO, Merck, armstadt, Germany).
Test item concentrations were used within 1 hour after preparation.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): test concentrations: 1 per test concentration; positive control: 1; solvent control: 2
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Short term treatment: 3 hours (with and without S9-mix)
Prolonged treatment: 24 hours (without S9-mix)

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection): 2 days in which at least 4 x 10^6 cells were subcultured every day.
- Selection time (if incubation with a selective agent): 11 or 12 days
- Method used: microwell plates
- Selective agent is used: 5 μg/mL trifluorothymidine (TFT)
- Number of cells seeded and method to enumerate numbers of viable and mutants cells: For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-wel
l dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective m
edium.
For determination of the mutation frequency (MF) a total number of 9.6 x 105 cells per concentration were plated in five 96-well microtiter plates, each well containing 2000 cells in selective medium (TFT-selection), with the exception of the positive control groups (MMS and CP) where a total number of 9.6 x 105 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection). The microtiter plates for CEday2 and MF were incubated for 11 or 12 days. After the incubation period, the plates for the TFT-selection were stained for 1.5-2 hours, by adding 0.5 mg/mL 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma) to each well. The plates for the CE day2 and MF were scored with the naked eye or with the microscope.
- Criteria for small (slow growing) and large (fast growing) colonies: The small colonies are morphologically dense colonies with a sharp contour and with a diameter less than a quarter of a well. The large colonies are morphologically less dense colonies with a hazy contour and with a diameter larger than a quarter of a well. A well containing more than on e small colony is classified as one small colony. A well containing more than one large colony is classified as one large colony. A well containing one small and one large colony is classified as one large colony.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: - Method: relative total growth (see 'Any other information on materials and methods incl. tables' for details on calculations)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/
solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
A test item is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for
positive or negative result can be made after an additional confirmation study.
A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested
concentrations reaches a mutation frequency of MF(controls) + 126.



ACCEPTABILITY CRITERIA
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth (SG) over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency,
that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Statistics:

Not performed.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH and osmolarity: The pH and osmolarity at a concentration of 75 μg/mL were 7.28 and 0.465 Osm/kg respectively (compared to 7.29 and 0.464 Osm/kg in the solvent control).
- Precipitation and time of the determination: The test item precipitated directly and after 3 hours in the test system at concentrations of 125 μg/mL and above. After 24 hours the test item precipitated in the test system at concentrations of 75 μg/mL and above.

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix (3 hours treatment), the relative suspension growth was 59% at the test item concentration of 125 μg/mL compared to the relative suspension growth of the solvent control.
In the presence of S9-mix (3 hours treatment), the relative suspension growth was 68% at the test item concentration of 125 μg/mL compared to the relative suspension growth of the solvent control.
The relative suspension growth was 58% at the test item concentration of 25 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentration of 75 and 125 μg/mL after the 24 hours subculture period without S9-mix.

STUDY RESULTS
- Concurrent vehicle negative and positive control data:
The mutant frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database.
Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutant frequency. In addition, the mutant frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Gene mutation tests in mammalian cells:
- Results from cytotoxicity measurements:
Experiment 1:
In the absence of S9-mix, the relative total growth of the highest test item concentration was 77% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test item concentration was 39% compared to the total growth of the solvent controls.
Experiment 2:
The relative total growth of the highest test item was 8% compared to the total growth of the solvent controls.


- Genotoxicity results:
No biologically relevant increase in the mutant frequency at the TK locus was observed after treatment with the test item in the absence and presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
Mutant frequency per 10^6 survivors
-S9 Mix +S9 mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 961 776 1154
SD 408 236 648
n 94 89 93
Lower Control Limit (95% Control Limits) 162 313 -116
Upper Control Limit (95% Control Limits) 1761 1239 2425
SD = Standard deviation
n = Number of observations
Distribution historical positive control data from experiments performed between May 2018 and May 2021.

- Negative (solvent/vehicle) historical control data:
Mutant frequency per 10^6 survivors
-S9 Mix +S9 mix
3 hour treatment 24 hour treatment 3 hour treatment
Mean 100 99 100
SD 28 28 28
n 96 87 93
Lower Control Limit (95% Control Limits) 45 44 46
Upper Control Limit (95% Control Limits) 154 153 154
SD = Standard deviation
n = Number of observations
Distribution historical negative control data from experiments performed between May 2018 and May 2021.

Applicant's summary and conclusion

Conclusions:

The substance is not mutagenic in the TK mutation test system.

Executive summary:

A gene mutation test in mammalian cells was performed according to OECD guideline 490 and GLP principles, to assess the potential of the substance to induce forward mutations at the thymidine kinase locus in L5178Y mouse lymphoma cells.
In the first experiment, the test item was tested up to concentrations of 125 μg/mL in the absence and presence S9-mix, respectively. The incubation time was 3 hours. The RTG was reduced to 77% and 39% for the concentration of 125 μg/mL in the absence and presence of S9-mix, respectively. The test item precipitated in the culture medium at this dose level.
In the second experiment, the test item was tested up to concentrations of 35 μg/mL in the absence of S9-mix. The incubation time was 24 hours. The RTG was reduced to 8% and 38% for concentrations of 35 and 25 μg/mL, respectively. Reliable negative and positive controls were included in both experiments.
In the absence and presence of S9 -mix, the substance did not induce a biologically relevant increase in the mutation frequency. In conclusion, the substance is not mutagenic in the mouse lymphoma L5178Y test system.