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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Available in vitro GLP studies

• Charles River (2017) Study No. 516995. Evaluation of the Mutagenic Activity of V123109 in the Salmonella typhimurium Reverse Mutation Assay and the Escherichia coli Reverse Mutation Assay (Plate Incorporation and Pre-Incubation Methods). According to OECD 471, GLP-compliant. Outcome: Negative with and without mammalian metabolic activation system (S9).


• Charles River (2017) Study No. 516996. Evaluation of the Ability of V123109 to Induce Chromosome Aberrations in Cultured Peripheral Human Lymphocytes. According to OECD 473, GLP-compliant.
  Outcome: Negative with and without mammalian metabolic activation system (S9).


• Charles River (2017) Study No. 516997. Evaluation of the Mutagenic Activity of V123109 in an in vitro Mammalian Cell Gene Mutation Test with L5178Y Mouse Lymphoma Cells. According to OECD 490, GLP-compliant.
  Outcome: Positive for gene mutations, with and without mammalian metabolic activation system (S9).

Based on the in vitro mammalian cell gene mutation test with mouse lymphoma cells, the substance shows indications for gene mutations in in vitro tests. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 29 July 2016

GLP compliance:

yes

Type of assay:

in vitro mammalian cell transformation assay

Target gene:

thymidine kinase (TK)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Suitability of cells: Recommended test system in international guidelines (e.g. OECD).
- Stock cultures of the cells were stored in liquid nitrogen (-196°C). The cultures were checked for mycoplasma contamination. Cell density was kept below 1 x 106 cells/ml.


MEDIA USED
Horse serum
Horse serum (Life Technologies) was inactivated by incubation at 56°C for at least 30 minutes.
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 (=R10 medium).
Exposure medium
Cells were exposed to the test item in basic medium supplemented with 5% (v/v)
heat-inactivated horse serum (R5-medium).
Selective medium
Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) 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 (total amount of serum = 20%, R20-medium).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate)

Test concentrations with justification for top dose:

In the dose-range finding test, L5178Y mouse lymphoma cells were treated with a test item concentration range of 125 to 2000 µg/ml in the absence and presence ofS9-mix with a 3 hour treatment period.

Based on the results of the dose range finding test, the following dose ranges were selected for the mutagenicity test:
Without S9-mix: 63, 125, 250, 500, 600, 700, 800, 850, 900, 950 and 1000 μg/ml exposure medium.
With S9-mix: 250, 500, 750, 1000, 1200, 1400, 1500, 1600, 1700, 1800 and
1900 μg/ml exposure medium.

Vehicle / solvent:

The vehicle for the test item was dimethyl sulfoxide (SeccoSolv, Merck Darmstadt, Germany).
Rationale: The test substance was solvable in this vehicle.

Untreated negative controls:

yes

Remarks:

The negative control was dimethyl sulfoxide (DMSO), the vehicle of the test item.

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 8 x 10^6 cells (10^6 cells/ml for 3 hour treatment) or 6 x 10^6 cells (1.25 x 10^5 cells/ml for 24 hour treatment)

DURATION
- Preincubation period:
Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine (Sigma), 2 x 10^-7 M aminopterine (Fluka Chemie AG, Buchs, Switzerland) and 1.6 x 10^-5 M thymidine (Sigma) (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 days after the treatment period.
- Fixation time (start of exposure up to fixation or harvest of cells): The microtiter plates were incubated for 11 or 12 days to determine the cloning efficiency and the mutation frequency.

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
After the incubation period, the plates for the TFT-selection were stained for 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.

NUMBER OF CELLS EVALUATED:
For determination of the CEday2 the cell suspensions were diluted and seeded in wells of a 96-well dish. One cell was added per well (2 x 96-well microtiter plates/concentration) in non-selective medium.
For determination of the mutation frequency (MF) a total number of 9.6 x 10^5 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 10^5 cells/concentration were plated in ten 96-well microtiter plates, each well containing 1000 cells in selective medium (TFT-selection).


DETERMINATION OF CYTOTOXICITY
- Method: cell growth and cloning efficiency
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
The colonies were divided into small and large colonies. Mutant cells that have suffered extensive genetic damage have prolonged doubling times and thus form small colonies. Less severely affected mutant cells grow at rates similar to the parental cells and form large colonies. The small colonies can be associated with the induction of chromosomal mutations. The large colonies appear to result from mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene.
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 one 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.

- OTHER:

Rationale for test conditions:

All incubations were carried out in a humid atmosphere (80 - 100%, actual range 65 - 93%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.4 - 37.4°C).

Evaluation criteria:

In addition to the criteria stated below, any increase of the mutation frequency should be evaluated for its biological relevance including comparison of the results with the historical control data range.
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.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the dose levels of 1700, 1800 and 1900 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

the dose levels of 900, 950 and 1000 μg/ml were not used for mutation frequency measurement, since these dose levels were too toxic for further testing.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, the relative suspension growth was 8% at the test item concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. No cell survival was observed at the test item concentration of 2000 μg/ml.
In the presence of S9-mix, the relative suspension growth was 68% at the test item concentration of 1000 μg/ml compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentration of 2000 μg/ml.
Based on the results of the dose range finding test, the following dose ranges were selected for the mutagenicity test:
Without S9-mix: 63, 125, 250, 500, 600, 700, 800, 850, 900, 950 and 1000 μg/ml exposure medium.
With S9-mix: 250, 500, 750, 1000, 1200, 1400, 1500, 1600, 1700, 1800 and
1900 μg/ml exposure medium.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%):
Mutation frequency per 10^6 survivors [95% conficence interval]
- Positive historical control data:
3-hour treatment: 857 [289 - 1425]
24-hour treatment: 1710 [-793 - 4214]
- Negative (solvent/vehicle) historical control data:
3-hour treatment: 86 [36 - 135]
24-hour treatment: 87 [28 - 145]
The mutation 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 mutation frequency. In addition, the mutation 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.


OTHER:
The suspension growth over the two-day expression period for cultures treated with DMSO was 19 and 20.
In the presence of S9-mix, V123109 induced dose related increases in the mutation frequency. The increases were above the 95% control limits of the distribution of the historical negative control database and also above the GEF + MF(controls) (220 per 106 survivors).
Although the increases in the mutation frequency at the TK locus were only observed at toxic dose levels (RTG ≤21%), the mutation frequencies at these concentrations were above the GEF and the results are considered to be biological relevance and the test item is mutagenic at toxic dose levels

Conclusions:

V123109 is mutagenic in the TK mutation test system under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the mutagenic potential of V123109 by testing its ability to induce forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, either in the absence or presence of a metabolic system (S9-mix). The TK mutational system detects base pair mutations, frame shift mutations and small deletions.

The test was performed in the absence and presence of S9-mix with a 3 hour treatment period.  

The study procedures described in this report were based on the most recent OECD guideline (OECD 490, 29 July 2016). 

Batch V123109/SA of V123109 was a white crystalline powder with a purity of >95%. The test item was dissolved in dimethyl sulfoxide

In the mutation experiment, V123109 was tested up to concentrations of 850 and 1500 µg/ml in the absence and presence S9-mix, respectively. The incubation time was 3 hours. Relative total growth (RTG) was reduced to 14 and 15% compared to the negative control in the absence and presence of S9-mix, respectively. Above the dose level of 1500 µg/ml the RTG was below the acceptable limit of 10%. The test item did not precipitate in the culture medium up to the concentration of 1500 µg/ml.

The mutation 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 mutation frequency. In addition, the mutation 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.

In the absence of S9-mix, V123109 induced dose related increases in the mutation frequency. The increases were above the95% control limits of the distribution of the historical negative control databaseand also abovethe GEF + MF_(controls)(225per 10⁶survivors).

In the presence of S9-mix, V123109 induced dose related increases in the mutation frequency. The increases were above the95% control limits of the distribution of the historical negative control databaseand also abovethe GEF + MF_(controls)
(220per 10⁶survivors).

Although the increases in the mutation frequency at the TK locus were only observed at toxic dose levels (RTG ≤21%), the mutation frequencies at these concentrations were above the GEF and the results are therefore considered to be biological relevance and the test item mutagenic at toxic dose levels.

In conclusion, V123109 is mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 March 2017 - 18 June 2017

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)

Version / remarks:

Adopted 29 July 2016

GLP compliance:

yes

Type of assay:

comet assay

Species / strain / cell type:

lymphocytes:

Remarks:

cultured peripheral human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Based on the results of the dose-range finding test an appropriate range of dose levels was chosen for the cytogenetic assays considering the highest dose level was the recommended 2000 µg/ml or had an inhibition of the mitotic index of 50% or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control (24 h and 48 h continuous exposure times).

First Cytogenetic Assay:
Without and with S9-mix: 10, 125, 500, 1000, 1500 and 2000 µg/ml culture medium
(3 h exposure time, 24 h fixation time).

Second Cytogenetic Assay:
Without S9-mix: 10, 100, 200, 400, 500, 600 and 700 µg/ml culture medium
(24 h exposure time, 24 h fixation time).
10, 50, 100, 200, 300, 400 and 500 µg/ml culture medium
(48 h exposure time, 48 h fixation time).

Vehicle / solvent:

A solubility test was performed based on visual assessment.
V123109 was dissolved in dimethyl sulfoxide of spectroscopic quality (SeccoSolv, Merck, Darmstadt, Germany).
The final concentration of the solvent in the culture medium was 1.0% (v/v).

Untreated negative controls:

yes

Remarks:

the solvent control is also the negative control

Negative solvent / vehicle controls:

yes

Remarks:

the solvent control is also the negative control

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

Cultured peripheral human lymphocytes were used as test system. Peripheral human lymphocytes are recommended in international guidelines (e.g. OECD, EC).
Blood was collected from healthy adult, non-smoking volunteers (approximately 18 to 35 years of age). The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined (December 2016) are presented below:
Dose-range finding study: age 22, AGT = 13.7 h
First cytogenetic assay: age 35, AGT = 15.8 h
Second cytogenetic assay: age 28, AGT = 13.4 h


1) Cell Culture
Blood samples:
Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.
Culture medium:
Culture medium consisted of RPMI 1640 medium (Life technologies), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum (Life technologies), L-glutamine (2 mM) (Life technologies), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (Life technologies) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).
Lymphocyte cultures:
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., Dartford, United Kingdom) was added.
Environmental conditions:
All incubations were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 38 - 89%), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.5 - 37.0°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature, humidity and CO2 percentage may occur due to opening and closing of the incubator door. Based on laboratory historical data these deviations are considered not to affect the study integrity.
Metabolic Activation System
Rat S9 homogenate was obtained from Trinova Biochem GmbH, Giessen, Germany and is 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).
Preparation of S9-Mix:
S9-mix was prepared immediately before use and kept on ice. 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 S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

Evaluation criteria:

A chromosome aberration test is considered acceptable if it meets the following criteria:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control item induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analyzed by the Fisher’s exact test (one-sided, p < 0.05).

Statistics:

GraphPad PRISM version 4.03 will be used for statistical analysis of the data.
A test item is considered positive (clastogenic) in the chromosome aberration test if all of the following criteria are met:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with a trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one- sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with a trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

In case the Fisher’s exact test shows that there are statistically significant differences between one or more of the test item groups and the vehicle control group a trend test (p < 0.05) will be performed to test whether there is a significant trend in the induction (ToxRat Professional v 3.2.1).

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Both in the absence and presence of S9-mix, V123109 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.4 in the highest concentration, 7.8 in the solvent control.
- Effects of osmolality: 418 in the highest concentration, 441 in the solvent control.
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
In the dose-range finding test blood cultures were treated with 125, 250, 500, 1000 and 2000 µg V123109/ml culture medium with and without S9-mix.
Table 1 (see attached background material) shows the mitotic index of cultures treated with various V123109 concentrations or with the negative control item.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: See attached background material
- Negative (solvent/vehicle) historical control data: See attached background material

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Other observations when applicable: [complete, e.g. confluency, apoptosis, necrosis, metaphase counting, frequency of binucleated cells]: See attached background material

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. In addition, the number of cells with chromosome aberrations 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.

Both in the absence and presence of S9-mix V123109 did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments.

No biological relevant effects of V123109 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that V123109 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report. 

Conclusions:

In conclusion, this test is valid and V123109 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate V123109 for its ability to induce structural chromosome aberrations in cultured human lymphocytes, either in the presence or absence of a metabolic activation system (S9-mix).

The possible clastogenicity of V123109 was tested in two independent experiments.

The study procedures described in this report are in compliance with the most recent OECD guidelines.

Batch V123109/SA of V123109 was a white crystalline powder with a purity of >95%. V123109 was dissolved in dimethyl sulfoxide.

In the first cytogenetic assay, V123109 was tested up to 2000 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. This is the highest concentration recommended for testing in the guideline.

In the second cytogenetic assay, V123109 was tested up to 400 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 300 μg/ml for a 48 h continuous exposure time with a 48 h fixation time, both in the absence of S9-mix. Appropriate toxicity was reached at these dose levels (45 ± 5%).

The number of cells with chromosome aberrations found in the solvent control cultures was within the 95% control limits of the distribution of the historical negative control database. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations. In addition, the number of cells with chromosome aberrations 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.

V123109 did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently performed experiments.

No biologically relevant effects of V123109 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that V123109 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.

In conclusion, this test is valid and V123109 is not clastogenic in human lymphocytes under the experimental conditions described in this report.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

20 March - 23 April 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

E. coli WP2uvrA

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose-range finding test: Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Direct plate assay: 52, 164, 512, 1600 and 5000 μg/plate., based on the results of the dose-range finding test.
Pre-Incubation Assay: 17, 52, 164, 512, 1600 and 5000 μg/plate., based on the results of the dose-range finding test.

Vehicle / solvent:

A solubility test was performed based on visual assessment. The test item was dissolved in dimethyl sulfoxide based on this solubility test.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: Acridine Mutagen ICR 191

Details on test system and experimental conditions:

Cell Culture
Preparation of bacterial cultures: Samples of frozen stock cultures of bacteria were transferred into enriched nutrient broth (Oxoid LTD, Hampshire, England) and incubated in a shaking incubator (37 ± 1°C, 150 rpm), until the cultures reached an optical density of 1.0 ± 0.1 at 700 nm (109 cells/ml). Freshly grown cultures of each strain were used for a test.
Agar plates: Agar plates (ø 9 cm) contained 25 ml glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma) and the agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).
Top agar: Milli-Q water containing 0.6% (w/v) bacteriological agar (Oxoid LTD) and 0.5% (w/v) sodium chloride (Merck) was heated to dissolve the agar. Samples of 3 ml top agar were transferred into 10 ml glass tubes with metal caps. Top agar tubes were autoclaved for 20 min at 121 ± 3°C.
Environmental conditions: All incubations were carried out in a controlled environment at a temperature of 37.0 ± 1.0°C (actual range 33.4 - 38.5°C). The temperature was continuously monitored throughout the experiment. Due to addition of plates (which were at room temperature) to the incubator or due to opening and closing the incubator door, temporary deviations from the temperature may occur. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Metabolic Activation System
S9-Fraction: Rat liver microsomal enzymes (S9 homogenate) were obtained from Trinova Biochem GmbH, Giessen, Germany and were prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).
Each S9 batch was characterized with the mutagens benzo-(a)-pyrene (Sigma) and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively.
Preparation of S9-Mix: S9-mix was prepared immediately before use and kept on ice. S9-mix contained per 10 ml: 30 mg NADP (Randox Laboratories Ltd., Crumlin, United Kingdom) and 15.2 mg glucose-6-phosphate (Roche Diagnostics, Mannheim, Germany) in 5.5 ml Milli-Q water (Millipore Corp., Bedford, MA., USA); 2 ml 0.5 M sodium phosphate buffer pH 7.4; 1 ml 0.08 M MgCl2 solution (Merck); 1 ml 0.33 M KCl solution (Merck). The above solution was filter (0.22 μm)-sterilized. To 9.5 ml of S9-mix components 0.5 ml S9-fraction was added (5% (v/v) S9-fraction) to complete the S9-mix.

TEST DESIGN

Dose-range Finding Test
Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and the WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate.
The highest concentration of the test item used in the subsequent mutation assays was 5000 μg/plate. At least five different doses (increasing with approximately half-log steps) of the test item were tested in triplicate in each strain in the absence and presence of S9-mix. The first experiment was a direct plate assay and the second experiment was a pre-incubation assay. Initially, the second experiment was rejected due to technical reasons. This experiment was repeated.
The negative control (vehicle) and relevant positive controls were concurrently tested in each strain in the presence and absence of S9-mix.

First Experiment: Direct Plate Assay
The above mentioned dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested both in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were successively added to 3 ml molten top agar: 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO and either 0.5 ml S9- mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Second Experiment: Pre-Incubation Assay
The test item was tested both in the absence and presence of S9-mix in all tester strains. Top agar in top agar tubes was melted by heating to 45 ± 2°C. The following solutions were pre-incubated for 30 minutes by 70 rpm at 37°C, either 0.5 ml S9-mix (in case of activation assays) or 0.5 ml 0.1 M phosphate buffer (in case of non-activation assays), 0.1 ml of a fresh bacterial culture (109 cells/ml) of one of the tester strains, 0.1 ml of a dilution of the test item in DMSO. After the pre-incubation period the solutions were added to 3 ml molten top agar. The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli) were counted.

Colony Counting
The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

Guideline test conditions.

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Statistics:

No formal hypothesis testing was done.

Species / strain:

S. typhimurium TA 100

Remarks:

Direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

Direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

Direct plate assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Thee number of revertant colonies was below the laboratory historical control range at 512 and 5000 μg/plate in the absence of S9-mix. However, no dose-relationship was observed and these reductions were considered as not biologically relevant.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Remarks:

Pre-incubation assay

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Remarks:

The positive control values were outside the historical laboratory range. Since the value was more than 4 times greater than the concurrent solvent control values, this deviation was considered to have no effect on the results of the study.

Additional information on results:

All bacterial strains showed negative responses over the entire dose-range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative control values were within the laboratory historical control data ranges.
The strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly, except the response for WP2uvrA in the absence of S9-mix in the second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 4 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Conclusions:

Based on the results of this study it is concluded that V123109 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of this study was to determine the potential of V123109 and/or its metabolites to induce reverse mutations at the histidine locus in several strains ofSalmonella typhimurium(S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus ofEscherichia coli(E. coli) strain WP2uvrAin the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay. The study procedures described in this report were based on the most recent OECD 471 and EC 440/2008 guidelines.

Batch V123109/SA of V123109 was a white crystalline powder with a purity of >95%. The test item was dissolved in dimethyl sulfoxide.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrAin the direct plate assay. The test item did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 at the highest dose level tested. No toxicity was observed in tester strain WP2uvrA.

In the first mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the strains TA1535, TA1537 and TA98. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in all three tester strains at the highest dose level tested.

In the second mutation experiment, the test item was tested up to concentrations of 5000 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrAin the pre-incubation assay. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA1535 and TA100 at the highest dose level tested. No toxicity was observed in the tester strains TA1537, TA98 and WP2uvrA.

In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrAboth in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that V123109 is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Available in vitro GLP studies

• Charles River (2013) Study No. 20373456. The Alkaline in vivo Comet Assay with V123109 in Liver/Duodenum/Stomach of Wistar Han Rats. According to OECD 489, GLP-compliant.
  Outcome: Not genotoxic in the comet assay in liver, duodenum and glandular stomach cells.

Following REACH Guidance R.7a, an in vivo mutagenecity test was performed after indications for genetic toxicity in vitro. A comet assay was performed as there were indications for gene mutations. The outcome of this in vivo test was negative and thus the substance is considered not to cause genetic toxicity.

Link to relevant study records

Reference

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

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

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

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

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.

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.

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.

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.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Three in vitro test were performed: OECD 471 (negative with and without metabolic activation), OECD 473 (negative with and without metabolic activation) and OECD 490 (positive with and without metabolic activation). Following REACH Guidance R.7a, an in vivo comet assay (OECD 489) was performed to further investigate. As the in vivo test was negative, it was concluded that the substance does not cause to genetic toxicity.