2-(6-methoxybenzofuran-2-yl)-1,3-dimethyl-5-(methylsulphonyl)1H-benzimidazolium acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Non genotoxic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April from 02 to 18, 2007

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

histidine auxotrophs and tryptophan auxotrophs

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate
Since no relevant toxic effects were observed with 5000 µg/plate in the pre-experiment/experiment I, it was chosen as maximal concentration.

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Remarks:

DMSO treated

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (4-NOPD) for strains TA 1537, TA98 without S9; 2-aminoanthracene (2-AA) as positive control for all the used strains with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) for experiment I; preincubation for experiment II

Pre-Experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA 1535, TA 1537, TA 98, TA 100, and WP2 uvrA. Eight concentrations were tested for toxicity and mutation induction with three plates each. The experimental conditions in this pre-experiment were the same as for the experiment I (plate incorporation test).
Toxicity of the test item results in a reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn.
The pre-experiment is reported as main experiment I, if the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

Dose Selection
In the pre-experiment the concentration range of the test item was 3 - 5000 µg/plate. The pre-experiment is reported as experiment I. Since no relevant toxic effects were observed 5000 µg/plate were chosen as maximal concentration. The concentration range included two logarithmic decades. The following concentrations of the active ingredient were tested in experiment II: 33; 100; 333; 1000; 2500; and 5000 µg/plate.

Experimental Performance
For each strain and dose level including the controls, three plates were used.
The following materials were mixed in a test tube and poured onto the selective agar plates:
100 µl Test solution at each dose level, solvent (negative control) or reference mutagen solution (positive control),
500 µl S9 mix (for test with metabolic activation) or S9 mix substitution buffer (for test without metabolic activation),
100 µl Bacteria suspension (cf. test system, pre-culture of the strains),
2000 µl Overlay agar
In the pre-incubation assay 100 µl test solution, 500 µl S9 mix/ S9 mix substitution buffer and 100 µL bacterial suspension were mixed in a test tube and shaken at 37 °C for 60 minutes. After pre-incubation 2.0 ml overlay agar (45 °C) was added to each tube. The mixture was poured on selective agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Acceptability of the Assay
The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of historical data
- the positive control substances should produce a significant increase in mutant colony frequencies

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.- A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.- An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.- A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

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

Positive controls validity:

valid

Additional information on results:

The plates incubated with the test item showed normal background growth up to 5000 µg/plate. Only in experiment II in the absence of metabolic activation, reduced background growth was observed at 5000 µg/plate in strains used.

No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. Minor reductions in the number of revertants was observed in the absence of metabolic activation in strain TA 1537 at 3 µg/plate in experiment I and in strain TA 100 at 5000 µg/plate in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase of induced revertant colonies.

Conclusions:

The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Executive summary:

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration and the controls were tested in triplicate. In the Pre-Experiment/Experiment I, the test item was tested at the concentrations of 3, 10, 33, 100, 333, 1000, 2500, and 5000 µg/plate, while in the experiment II it was tested at 33, 100, 333, 1000, 2500, and 5000 µg/plate. The plates incubated with the test item showed normal background growth up to 5000 µg/plate. Only in experiment II in the absence of metabolic activation, reduced background growth was observed at 5000 µg/plate in strains used. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. Minor reductions in the number of revertants was observed in the absence of metabolic activation in strain TA 1537 at 3 µg/plate in experiment I and in strain TA 100 at 5000 µg/plate in experiment II. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Conclusion

During the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November from 07 to 23, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

adopted 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: ECACC (European Collection of Cell Cultures).
- Cells: Sub-line (K1) of Chinese hamster ovary cell line CHO. The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO K1 is a sub-line of CHO cell line.
- Methods for maintenance in cell culture if applicable: the cell stocks are kept in liquid nitrogen. For each experiment the cells were thawed rapidly, the cells diluted in Ham's F12 medium containing 10 % foetal bovine serum and incubated at 37 ± C in a humidified atmosphere of 5 % CO2 in air. Growing cells were subcultured in an appropriate number of flasks.
- Culturing: the CHO K1 cells for thie study were grown in Ham's F12 medium (F12-10) supplemented with 1 % Antibiotic-antimycotic solution (containing 10000 U/ml penicillin, 10 mg/ml streptomycin and 25 µg/ml amphotericin-B) and heat-inactivated bovine serum (final concentration 10 %).

MEDIA USED
- Periodically checked for Mycoplasma contamination: each batch of frozen cells was purged of HPRT mutants and was free for mycoplasma infections, tested by Central Agricultural Office, National Animal Health Institute, Budapest, Hungary.

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9

Test concentrations with justification for top dose:

Without S9 mix: 125, 250, 500, 1000 and 2000 µg/ml
With S9 mix: 250, 500, 1000, 1250, 1500 and 1750 µg/ml

Vehicle / solvent:

- Solvent used: the test item was dissolved in DMSO and diluted prior to treatment.
- Justification for choice of solvent/vehicle: the solvent is compatible with the survival of the CHO cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability was confirmed with the available laboratory’s historical database.
- Preparation: test item was prepared in a concentration of 100 mg/ml in DMSO (stock solution) at the first step. Correction of concentration for active component content was made (correction factor 1.117).The necessary amount of test item was weighed into a calibrated volumetric flask. A partial volume of solvent was added and the formulation was stirred until homogeneity is reached. The formulation was diluted by serial dilutions to obtain the dosing formulations for lower doses. The appropriate amount of these dosing formulations were diluted with Ham's F12 medium or Ham's F12 medium + S9 mix to obtain the test concentrations. All dose formulations were prepared directly prior to the treatment of the cells.

Untreated negative controls:

yes

Remarks:

Ham's F12 medium

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

PROCEDURES
- Preparation of the dishes: on the day of treatment the culture medium of exponentially growing cell cultures were replaced with medium (F12-5) containing the test item.
- Treatment: a 5-hour treatment in the presence and absence of S9-mix was performed.
- Number of cells: 5 x10^6 cells were each placed in sterile dishes.
- Incubation conditions: dishes were incubated for approximately 24 hours before treatment at 37 °C in a humidified atmosphere of 5 % CO2.
- Replicates: duplicate cultures were used at each test item concentration, for negative (solvent) controls and the positive controls for treatment without and with S9-mix.
- Post-exposure incubation: following the exposure period the cells were washed with F12-5 medium and incubated in fresh F12-10 medium for 19 hours.
- Count: after the 19-hour incubation period, cells were washed twice with F12-10 medium and suspended by treatment with trypsin-EDTA solution and counted using a Bürker chamber.
- Precipitation check: solubility of the test item in the cultures was assessed by the naked eye, at the beginning and end of treatment.
- Adjustement of cell number: in samples where sufficient cells survived, cell number was adjusted to 10^5 cells/ml. Throughout the expression period, cells were transferred to dishes for growth or diluted to be plated for survival.

PLATING SURVIVAL
Following adjustment of the cultures to 10^5 cells/ml, samples from these cultures were diluted to 40 cells/ml.
A total of 5 ml (200 cells/dish) of the final concentration of each culture was plated into 3 parallel dishes (diameter is approx. 60 mm).
The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for growing colonies.
Then, colonies were fixed with methanol, stained with Giemsa and counted. Survivals were assessed by comparing the cloning efficiency of the test item treated groups to the negative (solvent) control.

EXPRESSION OF MUTANT PHENOTYPE: during the phenotypic expression period the cultures were subcultured. Aliquots of approximately 2x10^6 cells were taken
on days 1, 3, 6 and evaluated on day 8.

SELECTION OF THE MUTANT PHENOTYPE: at the end of the expression period, cultures from each dose level were adjusted to 2 x 10^5 cells / dish ( 4 x five dishes) in selection medium (hypoxanthine Ham's F12-SEL medium) containing 3.4 µg/ml of thioguanine (6-TG).

PLATING OF VIABILITY: at the end of the expression period cell number in the samples was adjusted to 2 × 10^5 cells/ml. Cells were plated in 3 parallel dishes (diameter is approx. 60 mm) for a viability test as described in “Plating for Survival“ section for the survival test.

FIXATION AND STAINING OF COLONIES: after the selection period, the colonies were fixed with methanol for five minutes, stained with Giemsa and counted for either mutant selection or cloning efficiency determination.

DETERMINATION OF CYTOTOXICITY
A GLP Pre-test on Toxicity was performed; treatment concentrations for the mutation assay were selected on the basis of the result of Pre-test on Toxicity. During the Pre-test on Toxicity, the cultures (more than 50 % confluent) was trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells was seeded into petri dishes at 5x10^6 cells each and incubated with culture medium. After 24 hours the cells were treated with the suitable concentrations of the test item in absence or in presence of S9 mix (50 µL/ml) and incubated at 37 °C for 5 hours. After the treatment cells were washed and incubated in fresh Ham's F12-10 medium for 19 hours. 24 hours after the beginning of treatment, the cultures were washed with Ham's F12-5 medium and the cells were covered with trypsin-EDTA solution, counted and the cell concentration was adjusted to 40 cells/ml with Ham's F12-10 medium. For each concentration of test solution or control solution, 5 ml was plated in parallel into 3 sterile dishes (diameter is approx. 60 mm).
The dishes were incubated at 37 °C in a humidified atmosphere of 5 % CO2 in air for 6 days for colony growing.
Colonies were then fixed with methanol and was stained with Giemsa and the colonies were counted. In order to determine cytotoxicity, survivals were assessed by comparing the colony forming ability of the treated groups to the negative (solvent) control.
Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments. In addition, pH and osmolality was considered for dose level selection.

METABOLIC ACTIVATION FRACTION
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH (Rathenau Strasse 2; D-35394 Giessen, Germany; Manufacturer: MOLTOX INC., P.O. BOX 1189; BOONE, NC 28607 USA).

ACCEPTANCE CRITERIA
The assay was considered valid as all the following criteria were met:
- The mutant frequency of concurrent negative controls is within the 95% control limits of the distribution of the laboratory’s historical negative control database.
- The positive control chemicals induced a statistically significant and biologically relevant increase in mutant frequency compared to the concurrent negative control. The increases are compatible with the laboratory historical positive control data base.
- Adequate number of cells and concentrations were analysable.
- Two experimental conditions with and without metabolic activation were tested.
- The highest concentration is adequate.
- The cloning efficiency of the negative controls is between the range of 60 % to 140 % on Day 1 and 70 % to 130 % on Day 8.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- any of the results are outside the distribution of the laboratory historical negative control data (based 95 % control limit),
- the increase of mutant frequency is concentration-related when evaluated with an appropriate trend test.

Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- 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 (based 95 % control limit).

Statistics:

Statistical Analysis was performed with SPSS PC+ software for the following data:
- mutant frequency between the negative (solvent) control group and the test item or positive control item treated groups.
- mutant frequency between the laboratory historical negative (solvent) control group and concurrent negative (solvent) control, the test item or positive control item treated groups.
- The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

on Day 1, in presence of metabolic activation (S9 mix)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

On Day 1, there was very clear evidence of toxicity at the highest tested concentration with the test item in presence of metabolic activation (S9 mix) when compared to the negative (solvent) controls, confirming the response seen in the dose selection cytotoxicity assays. The Day 8 cloning efficiency data indicate that in general the cells had recovered during the expression period.
No biologically or statistically significant increases in mutation frequency at any concentration tested was recorded, either in the absence or in the presence of metabolic activation. There were no significant differences between treatment and control groups and no dose-response relationships were noted.

SOLVENT CONTROL
All values were within the range of the laboratory historical solvent control data and no dose-related increase was observed in any of the cultures.

POSITIVE and NEGATIVE CONTROLS
The sensitivity of the tests and the efficacy of the S9 mix were demonstrated by large and statistically significant (p < 0.01) increases in mutation frequency in the positive control cultures with Ethyl methanesulfonate (1.0 µl/ml) and 7,12-Dimethyl benz[a]anthracene (20 µg/ml). The mutation frequencies of the positive and negative control cultures were consistent with the historical control data from the previous studies performed at this laboratory. Thus, the study is considered valid.

OSMOLALITY and pH
The osmolality and pH values of test item solutions did not show any significant alterations compared to the concurrent control groups in the Pre-test on Toxicity and Main Mutation Assay.

SOLUBILITY
A clear solution was obtained up to a concentration of 100 mg/ml. For examined test item concentrations no precipitation in the medium was noted.

CYTOTOXICITY
In the performed Mutation Assay the concentration levels were chosen mainly based on the cytotoxicity and the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 µg/ml (based on the updated OECD Guideline 476 (2016)).

Conclusions:

The test item is considered as being non-mutagenic in the system.

Executive summary:

The substance was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in DMSO and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study without and with metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver. Mutation Assay was performed at the concentrations and treatment intervals given below: Mutation Assay 5-hour treatment period without S9-mix at 125, 250, 500, 1000 and 2000 µg/ml; Mutation Assay 5-hour treatment period with S9-mix at 250, 500, 1000, 1250, 1500 and 1750 µg/ml.

In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. The maximum recommended concentration for soluble, lower -cytotoxic substances is 2000 µg/ml (based on the updated OECD Guideline 476 (2016)). Phenotypic expression was evaluated up to 8 days following exposure.

In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µl/ml) and 7, 12-Dimethyl benzanthracene (20 µg/ml) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Conclusion

The substance tested up to the maximum recommended concentration with and without metabolic activation system over a 5 hour treatment period did not induce statistically and biologically significant increases in mutant frequency over the background (negative solvent control).

Thus, the test item is considered as being non-mutagenic in the system.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 16th to January 17th, 2018 (pre- and main experiments)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 29th July, 2016

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: from ECACC (European Collection of Cells Cultures).
- Suitability of cells: stability of karyotype and morphology makes it suitable for gene toxicity assays with low background aberrations.
- Cell cycle length: approximately 1.5 normal cell cycles from the beginning of treatment, after 20 hours.
- Storage: the cell stocks were kept in liquid nitrogen.
- Donors: Chinese hamster lung male.
- Grown: the V79 cells for the study were grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 units/ml penicillin, 10 mg/ml streptomycin and 25 µg/ml amphoptericin-B) and heat-inactivated bovine serum (final concentration 10 %).

MEDIA USED
- Type and identity of media: the laboratory cultures were maintained in 75 cm2 plastic flasks at 37 +/- 0.5 °C in a humidified atmosphere in an incubator, set at 5% CO2.
- Periodically checked for Mycoplasma contamination: yes; cells were routinely checked for mycoplasma infections.

Metabolic activation:

with and without

Metabolic activation system:

liver microsome preparations (S9 mix)

Test concentrations with justification for top dose:

Experiment A with 3/20 h treatment/sampling time: without S9 mix 250, 500, 1000 and 2000 µg/ml test item; with S9 mix 500, 1000 and 2000 µg/ml test item
Experiment B with 20/20 h treatment/sampling time: without S9 mix: 31.3, 62.5, 125 and 250 µg/ml test item
Experiment B with 20/28 h treatment/sampling time: without S9 mix: 31.3, 62.5, 125 and 250 µg/ml test item
Experiment B with 3/28 h treatment/sampling time: with S9 mix: 500, 1000 and 2000 µg/ml test item

Vehicle / solvent:

- Solvent: DMSO
- Justification for choice of solvent/vehicle: vehicle is compatible with the survival of the V79 cells and the S9 activity and was chosen based on the results of the preliminary solubility test, and its suitability is confirmed with the available laboratory’s historical database.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

MAIN TEST
The main chromosome aberration assays were conducted in two independent experiments in the presence and in the absence of S9 mix.

Experimental Design Cytogenetic Experiment (Experiment A)
- Replicates: duplicate cultures were used at each test item concentration and the negative control cultures as well as the positive controls for treatment without and with S9 mix.
- Cell number: 5 x 10^5 cells per culture were seeded for each group. The culture medium of exponentially growing cell cultures was replaced with medium containing the test item.
- Exposure period: 3 hours.
- Post-exposure: after test item exposure the cells were washed with DME (Dulbecco’s Modified Eagle’s) medium and then growth medium was added.
- Sampling: sampling was made at 20 hours after start of treatment (approximately 1.5 normal cell cycles from the beginning of treatment).

Experimental Design Cytogenetic Experiment (Experiment B)
- Exposure period without metabolic activation: 20 hours.
- Exposure period with metabolic activation: 3 hours.
- Cell number: for each group 5 x 10^5 cells were seeded per culture.
- Sampling: sampling was made at 1.5 cell cycles (20 hours, without S9 mix only) and at approximately 2 normal cell cycles (28 hours, without and with S9 mix) from the beginning of treatment to cover a potential mitotic delay.

Measurement of pH and Osmolality
The pH value and osmolality of negative (solvent) control and test item treatment solutions (for every treatment concentrations) were measured in the main cytogenetic experiments.

PREPARATION AND ANALYSIS OF CHROMOSOMES
Preparation
Cell cultures were treated with colchicine (0.2 µg/ml) 2.5 hours prior to harvesting. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes free of cytoplasm) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies.

Analysis of Methaphase Cells
300 well-spread metaphase cells containing 22 ± 2 chromosomes were scored per test item concentration, negative and positive controls and were equally divided among the duplicates (150 metaphases/slide). Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally, the number of polyploid and endoreduplicated cells were scored.

PRE-TEST FOR CYTOTOXICITY
The cells were seeded into 92 x 17 mm dishes at 5 x 10^5 cells each and were incubated for 24 hours in 10 ml of DME (Dulbecco’s Modified Eagle’s) medium containing 10 % foetal bovine serum. After 24 hours the cells were treated using increasing concentrations of test item in the absence or presence of S9 mix (50 mg/ml) and were incubated at 37 °C for 3 hours. After treatment the cultures were washed with DME medium and covered with DME (Dulbecco’s Modified Eagle’s) medium containing 10 % foetal bovine serum.
Cell counts were performed after 20 hours (approximately 1.5 normal cell cycles from the beginning of treatment) using a Bürker chamber. Additional groups of cells were treated for 20 hours without metabolic and for 3 hours with metabolic activation, with cell counts conducted after 20 hours (without S9 mix only) and 28 hours (without and with S9 mix). Additionally, 4 cultures were set up for determining the initial cell count. At harvest the cells were trypsinised, collected and cell counts were determined. Based on the cell counts the Relative Increase in Cell Counts (RICC) was calculated, which is an indicator of cytotoxicity. The volume of culture medium was 5 ml/dish for each per group.

RAT LIVER FRACTION
The S9 fraction of phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH.
The complete S9 Mix was freshly prepared containing components with the following ratios: S9 fraction 3 ml; N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid 20 mM 2 ml; KCl 330 mM 1 ml; MgCl2 50 mM 1 ml; β-Nicotinamide Adenine Dinucleotide Phosphate 40 mM 1 ml; Glucose-6-phosphate 50 mM 1 ml; DME medium 1 ml.
Before adding to the culture medium the S9 mix was kept in an ice bath.

ACCEPTABILITY OF THE ASSAY
The chromosome aberration assay is considered acceptable because it meets the following criteria:
- the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data,
- concurrent positive controls induce responses that are compatible with the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control,
- cell proliferation in the solvent control is adequate,
- adequate number of cells and concentrations are analyzable,
- all requested experimental conditions were tested unless one resulted in a positive result
- the criteria for the selection of top concentration are fulfilled.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- 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 laboratory historical negative control data.
Providing that all acceptability criteria are fulfilled, the test item is considered clearly negative if, in all experimental conditions examined:
- 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 laboratory historical negative control data.
Both biological and statistical significance should be considered together.

Statistics:

For statistical analysis CHI test was utilized. The parameters evaluated for statistical analysis were the number of aberrations (with and without gaps) and number of cells with aberrations (with and without gaps). The number of aberrations in the treatment and positive control groups were compared to the concurrent negative control.
The concurrent negative and positive controls and the treatment groups were compared to the laboratory historical controls, too. The lower and upper 95 % confidence intervals of historical control were calculated with C-chart.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.
In experiment A in the absence and presence of metabolic activation, one value at the dose of 2000 µg/ml (5 aberrant cells excluding gaps/150 cells) were slightly above the 95 % control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells).
In the experiment B in the presence of metabolic activation one value at the dose of 1000 µg/ml(5 aberrant cells excluding gaps/150 cells) were slightly above the 95% control limits of the historical control data (upper limit approximately 4 aberrant cells excluding gaps/150 cells).
However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant.
No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of test item.

CONTROLS
The number of aberrations found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µl/ml) and Cyclophosphamide (5 µg/ml) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

PRE-TEST FOR CYTOTOXICITY
In both experiments, clear cytotoxicity of about 50% was observed at the highest concentration of 2000 µg/ml after test item 3hours treatment in the absence and presence of metabolic activation. After 20-hours treatment, ca 50 % of cytotoxicity was recorded at 250 µg/ml in plates without S9 mix.

SOLUBILITY and OSMOLALITY
A clear solution of test item was obtained in DMSO up to a concentration of 100 mg/ml. There was no precipitation in the medium at any concentration tested. There were no relevant changes in pH or osmolality after treatment with the test item.

MEAN NUMBER OF CELLS WITH STRUCTURAL - CHROMOSOME ABERRATION(s) EXPERIMENT A

Concentration (µg/ml)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150 cells
				incl. gaps	excl. gaps
Negative (Solvent) control	-	3 h	20 h	8	4
Test item
250 µg/ml	-	3 h	20 h	7	3
500 µg/ml	-	3 h	20 h	8	3
1000 µg/ml	-	3 h	20 h	6	3
2000 µg/ml	-	3 h	20 h	7	5
Pos. Control	-	3 h	20 h	38**	34**
(Ethyl methanesulphonate)
Negative (Solvent) control	+	3 h	20 h	8	4
Test item
500 µg/ml	+	3 h	20 h	8	3
1000 µg/ml	+	3 h	20 h	6	2
2000 µg/ml	+	3 h	20 h	9	5
Pos. Control (Cyclophosphamide)	+	3 h	20 h	49**	43**

Positive control (-S9): Ethyl methanesulphonate (1.0 µl/ml)

Positive control (+S9): Cyclophosphamide (5.0 µg/ml)

** = p < 0.01 to the concurrent negative control and to the historical control

MEAN NUMBER OF CELLS WITH STRUCTURAL - CHROMOSOME ABERRATION(s) EXPERIMENT B

Concentration (µg/ml)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150 cells
				incl. gaps	excl. gaps
Negative (Solvent) control	-	20 h	20 h	7	3
Test item
31.3 µg/ml	-	20 h	20 h	7	4
62.5 µg/ml	-	20 h	20 h	6	3
125 µg/ml	-	20 h	20 h	6	3
250 µg/ml	-	20 h	20 h	7	3
Pos. Control	-	20 h	20 h	43**	37**
(Ethyl methanesulphonate)
Negative (Solvent) control	-	20 h	28 h	7	3
Test item
31.3 µg/ml	-	20 h	28 h	7	3
62.5 µg/ml	-	20 h	20 h	8	4
125 µg/ml	-	20 h	28 h	9	4
250 µg/ml	-	20 h	28 h	8	4
Pos. Control	-	20 h	28 h	43**	35**
(Ethyl methanesulphonate)

Positive control (-S9): Ethyl methanesulphonate (0.4 µl/ml)

** = p < 0.01 to the concurrent negative control and to the historical control

MEAN NUMBER OF CELLS WITH STRUCTURAL - CHROMOSOME ABERRATION(s) EXPERIMENT B

Concentration (µg/ml)	S9 mix	Treatment time	Harvesting time	Mean aberrant cells/150 cells
				incl. gaps	excl. gaps
Negative (Solvent) control	+	3 h	28 h	8	3
Test item
500 µg/ml	+	3 h	28 h	8	3
1000 µg/ml	+	3 h	28 h	9	5
2000 µg/ml	+	3 h	28 h	8	4
Pos. Control (Cyclophosphamide)	+	3 h	28 h	45**	38**

Cyclophosphamide: 5.0 µg/ml

** = p < 0.01 to the concurrent negative control and to the historical control

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS - EXPERIMENT A

Concentration (µg/ml)	S9 mix	Treatment/ Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	-	3/20 h	0	0
Test item
250 µg/ml	-	3/20 h	0	0
500 µg/ml	-	3/20 h	0	0
1000 µg/ml	-	3/20 h	0	0
2000 µg/ml	-	3/20 h	0	0
Pos. Control	-	3/20 h	0	0
(Ethyl methanesulphonate)
Negative (Solvent) control	+	3/20 h	0	0
Test item
500 µg/ml	+	3/20 h	0	0
1000 µg/ml	+	3/20 h	0	0
2000 µg/ml	+	3/20 h	0	0
Pos. Control (Cyclophosphamide)	+	3/20 h	0	0

Ethyl methanesulphonate: 1.0 µl/ml

Cyclophosphamide: 5.0 µg/ml

The number of polyploid and endoreduplicated cells was determined in 300 cells of each test group.

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS - EXPERIMENT B

Concentration (µg/ml)	S9 mix	Treatment/ Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	-	20/20 h	0	0
Test item
31.3 µg/ml	-	20/20 h	0	0
62.5 µg/ml	-	20/20 h	0	0
125 µg/ml	-	20/20 h	0	0
250 µg/ml	-	20/20 h	0	0
Pos. Control	-	20/20 h	0	0
Negative (Solvent) control	-	20/28 h	0	0
Test item
31.3 µg/ml	-	20/28 h	0	0
62.5 µg/ml	-	20/28 h	0	0
125 µg/ml	-	20/28 h	0	0
250 µg/ml	-	20/28 h	0	0
Pos. Control	-	20/28 h	0	0

Positive control (-S9): Ethyl methanesulphonate (0.4 µl/ml)

The number of polyploid and endoreduplicated cells was determined in 300 cells of each test group.

NUMBER OF POLYPLOID CELLS AND ENDOREDUPLICATED CELLS - EXPERIMENT B

Concentration (µg/ml)	S9 mix	Treatment/ Harvesting time	Polyploid Cells (mean)	Endoredup-lication (mean)
Negative (Solvent) control	+	3/28 h	0	0
Test item
500 µg/ml	+	3/28 h	0	0
1000 µg/ml	+	3/28 h	0	0
2000 µg/ml	+	3/28 h	0	0
Pos. Control	+	3/28 h	0	0

Cyclophosphamide: 5.0 µg/ml

The number of polyploid and endoreduplicated cells was determined in 300 cells of each test group.

SUMMARIZED RESULTS OF THE CONCENTRATION SELECTION CYTOTOXICITY ASSAY

3-hour treatment without and with S9 mix / 20-hour sampling time

Test group	Concentration (µg/ml)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	2100000	2100000	2056250	-	-	-
	-	B	–	2050000	1900000
	-	C	–	1950000	2150000
	-	D	–	2100000	2100000
Solvent control (DMSO)	-	A	–	7050000	7100000	7025000	4968750	100	0
	-	B	–	7000000	6950000
Test item	250	A	–	6850000	6900000	6875000	4818750	96.98	3.02
	500	A	–	6000000	6100000	6050000	3993750	80.38	19.62
	1000	A	–	5750000	5650000	5700000	3643750	73.33	26.67
	2000	A	–	4100000	4200000	4150000	2093750	42.14	57.86
	EMS 1 µl/ml	A	–	4550000	4600000	4575000	2518750	50.69	49.31
Solvent control (DMSO)	-	A	+	6300000	6500000	6450000	4393750	100	0
	-	B	+	6550000	6450000
Test item	500	A	+	6400000	6250000	6325000	4268750	97.16	2.84
	750	A	+	6200000	5950000	6075000	4018750	91.47	8.53
	1000	A	+	5300000	5400000	5350000	3293750	74.96	25.04
	1250	A	+	5300000	5450000	5375000	3318750	75.53	24.47
	1500	A	+	5200000	5250000	5225000	3168750	72.12	27.88
	2000	A	+	4000000	3850000	3925000	1868750	42.53	57.47
	Cycl. 5 µg/ml	A	+	4300000	4150000	4225000	2168750	49.36	50.64

RICC= Relative Increase in Cell Counts       

Cytotoxicity= 100-RICC

EMS: Ethyl methanesulfonate (EMS)

Cycl: Cyclophosphamide monohydrate

SUMMARIZED RESULTS OF THE CONCENTRATION SELECTION CYTOTOXICITY ASSAY

20-hour treatment without S9 mix / 20-hour sampling time

Test group	Concentration (µg/ml)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	2100000	2100000	2056250	-	-	-
	-	B	–	2050000	1900000
	-	C	–	1950000	2150000
	-	D	–	2100000	2100000
Solvent control (DMSO)	-	A	–	6000000	5850000	6062500	4006250	100	0
	-	B	–	6300000	6100000
Test item	31.3	A	–	6000000	6000000	6000000	3943750	98.44	1.56
	62.5	A	–	4750000	4550000	4650000	2593750	64.74	35.26
	125	A	–	4200000	4300000	4250000	2193750	54.76	45.24
	250	A	–	3950000	3900000	3925000	1868750	46.65	53.35
	500	A	–	4000000	3750000	3875000	1818750	45.4	54.6
	1000	A	–	500000	700000	600000	-1456250*	-36.35**	136.35***
	1500	A	–	150000	250000	200000	-1856250*	-46.33**	146.33***
	EMS 1 µl/ml	A	–	3950000	4200000	4075000	2018750*	50.39	49.61

RICC= Relative Increase in Cell Counts       

Cytotoxicity= 100-RICC

EMS: Ethyl methanesulfonate (EMS)

*: cell number decrease ; **: zero RICC value; ***:100% cytotoxicity

SUMMARIZED RESULTS OF THE CONCENTRATION SELECTION CYTOTOXICITY ASSAY

20-hour treatment without S9 mix and 3-hour treatment with S9 mix / 28-hour sampling time

Test group	Concentration (µg/ml)	Parallels	S9-mix	Cell counts		Mean cell counts	Increase in cell counts	RICC (%)	Cytotoxicity (%)
				First count	Second count
Initial cell count	-	A	–	2100000	2100000	2056250	-	-	-
	-	B	–	2050000	1900000
	-	C	–	1950000	2150000
	-	D	–	2100000	2100000
Solvent control (DMSO)	-	A	–	8150000	8300000	8175000	6118750	100	0
	-	B	–	8000000	8250000
Test item	31.3	A	–	8000000	8100000	8050000	5993750	97.96	2.04
	62.5	A	–	6950000	6800000	6875000	4818750	78.75	21.25
	125	A	–	5450000	5700000	5575000	3518750	57.51	42.49
	250	A	–	5000000	4900000	4950000	2893750	47.29	52.71
	500			4950000	4700000	4825000	2768750	45.25	54.75
	1000	A	–	1200000	1050000	1125000	-931250*	-15.22**	115.22***
	1500	A	–	300000	250000	275000	-1781250*	-29.11**	129.11***
	EMS 1 µl/ml	A	–	5200000	5400000	5300000	3243750	53.01	46.99
Solvent control (DMSO)	-	A	+	8000000	7850000	7825000	5768750	100	0
	-	B	+	7750000	7700000
Test item	500	A	+	7500000	7450000	7475000	5418750	93.93	6.07
	750	A	+	7100000	7050000	7075000	5018750	87	13
	1000	A	+	6250000	6500000	6375000	4318750	74.86	25.14
	1250	A	+	6250000	6250000	6250000	4193750	72.7	27.3
	1500	A	+	5850000	5900000	5875000	3818750	66.2	33.8
	2000	A	+	4500000	4650000	4575000	2518750	43.66	56.34
	Cycl. 5 µg/ml	A	+	5150000	4950000	5050000	2993750	51.9	48.1

RICC= Relative Increase in Cell Counts       

Cytotoxicity= 100-RICC

EMS: Ethyl methanesulfonate (EMS)

Cycl: Cyclophosphamide monohydrate

*: cell number decrease; **: zero RICC value; ***:100% cytotoxicity

Conclusions:

The test item is considered as being non-clastogenic in this system.

Executive summary:

The test item, dissolved in DMSO, was tested in a chromosome aberration assay in V79 cells in two independent experiments. For the cytogenetic experiments the following concentrations were selected on the basis of a pre-test on (without and with metabolic activation using rodent S9 mix) in accordance with the current OECD Guideline 473:

Experiment A with 3/20 h treatment/sampling time without S9 mix 250, 500, 1000 and 2000 µg/ml test item; with S9 mix 500, 1000 and 2000 µg/ml test item;

Experiment B with 20/20 h treatment/sampling time without S9 mix: 31.3, 62.5, 125 and 250 µg/ml test item;

Experiment B with 20/28 h treatment/sampling time without S9 mix: 31.3, 62.5, 125 and 250 µg/ml test item;

Experiment B with 3/28 h treatment/sampling time with S9 mix: 500, 1000 and 2000 µg/ml test item

Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/ml) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item.

Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts.

No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.

In experiment A in the absence and presence of metabolic activation, one value at the dose of 2000 µg/ml and in the experiment  B one value at the dose of 1000 µg/ml in the presence of metabolic activation was slightly above the 95 % control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µl/ml) and cyclophosphamide (5 µg/ml) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Conclusion

In conclusion, the substance did not induce structural chromosome aberrations in Chinese Hamster lung V79 cells, when tested up to cytotoxic concentrations in the absence and presence of metabolic activation. Thus, the test item is considered as being non-clastogenic in this system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Investigation on the genetic toxicity has been performed with the integrated evaluation of the following studies: in vitro AMES tests, in vitro gene mutation in mammalian cells and in vitro chromosomal aberration assay.

IN VITRO GENE MUTATION ASSAY IN BACTERIA

The test item was assessed for its potential to induce gene mutations in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA98, and TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. Only in experiment II in the absence of metabolic activation, reduced background growth was observed at 5000 µg/plate in strains used. No toxic effects, evident as a reduction in the number of revertants, occurred in the test groups with and without metabolic activation. Minor reductions in the number of revertants was observed in the absence of metabolic activation in strain TA 1537 at 3 µg/plate in experiment I and in strain TA 100 at 5000 µg/plate in experiment II. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore, during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

IN VITRO GENE MUTATION ON MAMMALIAN CELLS

The substance was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. In the performed Mutation Assay the concentration levels were chosen mainly based on the maximum recommended concentration. In both experimental parts, there were no biologically or statistically significant increases in mutation frequency at any concentration tested, either in the absence or in the presence of metabolic activation. There were no statistically and biologically significant differences between treatment groups when was compared to the concurrent and historical control groups and no dose-response relationships were noted.

There was no precipitation of the test item at any dose level tested. No biologically relevant changes in pH or osmolality of the test system were noted at the different dose levels tested.

The mutation frequency found in the solvent controls was in the range of historical laboratory control data. The concurrent positive controls Ethyl methanesulfonate (1.0 µl/ml) and 7, 12-Dimethyl benzanthracene (20 µg/ml) caused the expected biologically relevant increases of cells with mutation frequency as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

IN VITRO CHROMOSOMAL ABERRATION ASSAY

The test item was tested in a chromosome aberration assay in V79 cells in two independent experiments. Following treatment and recovery the cells were exposed to the spindle inhibitor colchicine (0.2 µg/ml) 2.5 hours prior to harvesting. Harvested cells were treated with fixative for ca. 10 minutes before being placed on slides and stained. In each experimental group duplicate cultures were evaluated for cytogenetic damage (150 metaphases per culture).

No precipitation of the test item was observed at any of the applied concentrations. There were no relevant changes in pH or osmolality after treatment with the test item. Clear cytotoxicity of about 50 % was observed after test item treatment in all experimental parts.

No relevant increases in cells carrying structural chromosomal aberrations were observed, neither in the absence nor in the presence of metabolic activation.

In experiment A in the absence and presence of metabolic activation, one value at the dose of 2000 µg/ml and in the experiment  B one value at the dose of 1000 µg/ml in the presence of metabolic activation was slightly above the 95 % control limits of the historical control data. However, no statistical significant differences were observed after test item treatment when compared to the concurrent solvent as well as the historical control groups. In addition, no dose-response relationship was observed and therefore, the findings were not considered as being biologically relevant.

There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases in either experiment in the presence or absence of metabolic activation.

The number of aberrations found in the solvent controls was in the range of the historical laboratory control data. The concurrent positive controls ethyl methanesulphonate (0.4 and 1.0 µl/ml) and cyclophosphamide (5 µg/ml) caused the expected biologically relevant increases of cells with structural chromosome aberrations as compared to solvent controls and were compatible with the historical positive control data. Thus, the study is considered valid.

Justification for classification or non-classification

According to the CLP Regulation (EC) No 1272/2008, for the purpose of the classification for germ cell mutagenicity, substances are allocated in one of two categories in consideration of the fact that they are:

- substances known to induce heritable mutations or to be regarded as if they induce heritable mutations in the germ cells of humans or substances known to induce heritable mutations in the germ cells of humans or

- substances which cause concern for humans owing to the possibility that they may induce heritable mutations in the germ cells of humans.

The available information suggests that test substance did not show any reasons of concern from the genotoxicity point of view.

In conclusion, the substance does not meet the criteria to be classified for genetic toxicity, according to the CLP Regulation (EC) No 1272/2008.