Reaction products of 3-aminomethyl-3,5,5-trimethylcyclohexylamine with 2,2'-[(1-methylethylidene)bis(4,1-phenyleneoxymethylene)]bisoxirane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was tested for genetic toxicity in an Ames test (OECD 471; EU Method B.13/14), an in vitro mammalian cell gene mutation test (OECD 476; EU Method B.17), and an in vitro mammalian chromosome aberration test (OECD 473; EU Method B.10). All tests were carried out up to cytotoxic concentrations/limit concentrations, without and with metabolic activation. All three tests showed no signs of genetic toxicity.

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:

from 2007-10-16 to 2007-12-11

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000/32/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

June 1996/August 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– -> trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 Mix

Test concentrations with justification for top dose:

In the above experimental phases the examined concentration levels were different. The test item was dissolved in Dimethyl sulfoxide (DMSO).
In the Plate Incorporation Tests the investigated concentration ranges (summarised): in case of Salmonella typhimurium strains 100-0.003 μg/plate with and without metabolic activation (± S9 Mix); in Escherichia coli WP2 uvrA: 3419-0.158 μg/plate (± S9 Mix).
In the Pre-Incubation Tests: in the Salmonella typhimurium strains 10-0.003 μg/plate (–S9 Mix) and 100-0.003 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 158.1-0.158 μg/plate (± S9 Mix).

Vehicle / solvent:

Dimethyl sulfoxide DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylenediamine, NPD

Remarks:

TA 98 without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 100, TA 1535 without S9 Migrated to IUCLID6: NaN3

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537 without S9 Migrated to IUCLID6: 9AA

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E. coli WP2uvrA without S9 Migrated to IUCLID6: MMS

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2AA

Remarks:

S. typhimurium TA 100, TA 98, TA 1535, TA 1537; E. coli WP2uvrA with S9

Evaluation criteria:

The colony numbers for control, positive control and the test plates were determined, the mean values and appropriate standard deviations were calculated.
The mutation factor (MF) was calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (exact, not rounded values were used for this calculation).

Statistics:

not applicable

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 3.162 µg/plate without S9; <= 31.62 µg/plate with S9

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of the test item in plate incorporation tests (Initial Mutation Test, Complementary Plate Incorporation Test, Second Complementary Plate Incorporation Test) and in pre-incubation tests (Confirmatory Mutation Test and Complementary Pre-Incubation Test).

In general, the pre-incubation method is more sensitive than the plate incorporation method. Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate (positive and negative controls were run concurrently). In the Complementary Pre-Incubation Test only the activated part (+S9 Mix) of assay was examined. The Complementary Pre-Incubation Test was performed with Salmonella typhimurium strains.
Following concentrations of Reaction Product of Bisphenol A (BADGE) with IPDA were tested in different experiments: in the Plate Incorporation Tests the investigated concentration ranges (summarised): in case of Salmonella typhimurium strains 100-0.003 μg/plate without and with addition of metabolic activation (±S9 Mix), in Escherichia coli WP2 uvrA: 3419-0.158 μg/plate (± S9 Mix).

In the Pre-Incubation Tests: in the Salmonella typhimurium strains 10-0.003 μg/plate (–S9 Mix) and 100-0.003 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 158.1-0.158 μg/plate (± S9 Mix).

In the Initial Mutation Test, in case of Salmonella typhimurium strains inhibition was observed in the examined concentration range (1-0.003 μg/plate) neither in presence, nor in absence of metabolic activation (S9 Mix).

The observed revertant colony numbers were slightly lower than the revertant colony numbers of the solvent control plates at different concentrations in case of Salmonella typhimurium TA 98, TA 1535 and TA 1537; however these variations were of the minor intensity and were considered to reflect the biological variability of the test.

In Escherichia coli WP2 uvrA the examined concentration range was higher (3419–1 μg/plate) than in the Salmonella typhimurium strains. In case of Escherichia coli WP2 uvrA no bacterial growth was observed in the concentration range of 3419-100 μg/plate without and in the range of 3419-341.9 μg/plate with addition of metabolic activation. The revertant colony numbers were reduced compared to the revertant colony numbers of the solvent control plates and the background lawn development was inhibited at the concentration of 34.19 μg/plate without, and at 100 μg/plate with addition of metabolic activation. The slightly lower revertant counts (compared to the solvent control plates) at the concentrations of 10 and 3.419 μg/plate (–S9 Mix), furthermore at 34.19 μg/plate (+S9 Mix) were evaluated as reflecting the biological variability in the test.

In the Complementary Plate Incorporation Test the examined concentration levels were 10, 3.162 and 1 μg/plate in the Salmonella typhimurium strains and 50; 15.81; 5; 1.581; 0.500 and 0.158 μg/plate in case of Escherichia coli WP2 uvrA. Unequivocally inhibitory effect (indicated by reduced revertant colony numbers compared to the solvent control plates and reduced background lawn development) of the test item was observed in all Salmonella typhimurium strains at the concentration of 10 μg/plate, and in the Escherichia coli WP2 uvrA at 50 μg/plate, without metabolic activation. Similarly to the Initial Mutation Test the slightly lower than the revertant colony numbers of the solvent control plates at different concentration levels in the examined strains were considered to reflect the biological variability of the test.

Since the cytotoxicity of the test item altered in presence of metabolic activation system (S9 Mix), a Second Complementary Plate Incorporation Test was carried out using the concentration levels of 100 and 31.62 μg/plate in Salmonella typhimurium strains and 500 and 158.1 μg/plate in Escherichia coli WP2 uvrA.
No bacterial growth was observed in the bacterial strains at the above concentration levels in absence of metabolic activation furthermore at 100 μg/plate (Salmonella typhimurium strains) and 500 and 158.1 μg/plate (Escherichia coli WP2 uvrA) in presence of metabolic activation. Furthermore reduced revertant colony number and background lawn development was observed in Salmonella typhimurium TA 98 and TA 1537 at the concentration of 31.62 μg/plate in presence of metabolic activation.

In the Confirmatory Mutation Test (Pre-Incubation Test) the examined concentration levels were: 10; 3.162; 1; 0.316; 0.1; 0.032; 0.01 and 0.003 μg/plate in the Salmonella typhimurium strains and 158.1; 50; 15.81; 5; 1.581; 0.500 and 0.158 μg/plate in case of Escherichia coli WP2 uvrA.

Strong inhibitory effects of the test item were observed in all examined strains, however in Salmonella typhimurium strains only the non-activated part (–S9 Mix) was inhibited. The signs of the inhibitory effects included reduced revertant colony numbers, compared to the solvent control plates, reduced background lawn development and pinpoint colony appearance. No bacterial growth was observed in the Salmonella typhimurium TA 98, TA 1535, TA 1537 strains at 10 μg/plate (–S9 Mix), in Escherichia coli WP2 uvrA at 158.1 and 50 μg/plate (–S9 Mix). In Escherichia coli WP2 uvrA at 158.1 μg/plate (+S9 Mix) only one revertant colony was counted on the three parallels.
In the case of Salmonella typhimurium TA 1537 at 3.162 g/plate (–S9 Mix) the revertant colony numbers were increased, but reduced background lawn development was observed and the colonies were small, pinpoint colonies. The revertant colony count were lower than the revertant count of the solvent control plates in several cases, however they were in the historical control data range in all cases.

A Complementary Pre-Incubation Test was performed to reach the cytotoxic level in presence of metabolic activation (+S9 Mix) in case of Salmonella typhimurium strains. The examined concentration levels were 100 and 31.62 μg/plate. No bacterial growth was observed in Salmonella typhimurium TA 98, TA 1535, and TA 1537 at the concentration level of 100 μg/plate, and inhibition was observed (reduced revertant colony numbers, compared to the solvent control plates, reduced background lawn development and pinpoint colony appearance) in all strains at 31.62 μg/plate, furthermore in Salmonella typhimurium TA 100 at 100 μg/plate.
In the consecutive mutation tests the inhibitory, cytotoxic effect of the test item independently of the examined strains (Salmonella typhimurium or Escherichia coli) appeared down to and including the concentration level of 3.162 μg/plate in absence and down to and including the concentration level of 31.62 μg/plate in presence of metabolic activation.
Positive and negative controls were run concurrently. The revertant colony numbers of solvent control plates without S9 Mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies.

Remarks on result:

other: all strains/cell types tested

Conclusions:

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

Experimental Phases of the Study

The test item was examined in the following phases: in Plate Incorporation Tests (Initial Mutation Test, Complementary Plate Incorporation Test, and Second Complementary Plate Incorporation Test) and Pre-Incubation Tests (Confirmatory Mutation Test and Complementary Pre-Incubation Test).

Exposure Concentrations

In the above experimental phases the examined concentration levels were different. The test item was dissolved in Dimethyl sulfoxide (DMSO).

In the Plate Incorporation Tests the investigated concentration ranges (summarised): in case of Salmonella typhimurium strains 100 - 0.003 μg/plate without and with addition of metabolic activation (± S9 Mix), in Escherichia coli WP2 uvrA: 3419 - 0.158 μg/plate (± S9 Mix). In the Pre-Incubation Tests: in the Salmonella typhimurium strains 10 - 0.003 μg/plate (–S9 Mix) and 100 - 0.003 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 158.1 - 0.158 μg/plate (± S9 Mix).

Bacteria

Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 strains and Escherichia coli WP2 uvrA were used.

Results and Discussion:

Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of the test item in independent experiments, in plate incorporation tests and in a pre-incubation tests. Each assay (except the Complementary Pre-Incubation Test) was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate.

Inhibitory, cytotoxic effects of the test item were observed in the experiments in all examined bacterial strains and included reductions of spontaneous mutation rates, when compared to the solvent control values, reduced background lawn development and appearances of small pinpoint colonies. Furthermore no bacterial growth was observed for the strains at several concentration levels, with and without metabolic activation. Inhibition was stronger in the non-activated part (–S9 Mix) of the experiments.

No significant increases in revertant colony numbers of any of the five test strains were observed following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Further, there was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance in the performed experiments.

Positive and negative controls were run concurrently. The revertant colony numbers of solvent control plates without S9 Mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies.

Conclusion:

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2007-10-08 to 2007-11-07

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2000/32/EEC

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

August 1988

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line is well established in toxicology studies. Stability of karyotype and morphology makes them suitable for gene toxicity assays with low background aberrations. These cells are chosen because of their small number of chromosomes (diploid number, 2n=22) and because of the high proliferation rates (doubling time 12 - 14 h). The V79 cell line was established after spontaneous transformation of cells isolated from the lung of a normal Chinese hamster (male). This cell line was purchased from ECACC (European Collection of Cells Cultures). The cell stocks are kept in a freezer at -80 ± 10 °C. Checking of mycoplasma infections is carried out before freezing. Trypsin-EDTA (0.25 % Trypsin, 1mM EDTA x 4 Na) solution is used for cell detachment to subculture. The laboratory cultures are maintained in 75 cm² plastic flasks at 37 ± 0.5 °C in a humidified atmosphere containing 5 ± 0.3 % CO2. The V79 cells for this study are grown in DME (Dulbecco’s Modified Eagle’s) medium supplemented with L-glutamine (2mM) and 1 % of Antibiotic-antimycotic solution (containing 10000 NE/mL penicillin, 10 mg/mL streptomycin and 25 µg/mL amphoptericin-B) and heat-inactivated foetal bovine serum (final concentration 10 %). During the 3 and 20 hours treatments with test item, solvent (negative control) and positive controls, the serum content was reduced to 5 %.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with

Metabolic activation system:

S9 fraction is derived from male WISTAR rats induced with Phenobarbitone (PB) and ß-naphthoflavone (BNF) at a dose level of 80 mg/kg bw/day.

Test concentrations with justification for top dose:

0.61, 1.22, 2.44, 3.66, 4.88, 7.32, 9.76, 14.64, 17.08 µg/mL

Vehicle / solvent:

Dimethyl sulfoxide DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

Remarks:

with S9 mix

Details on test system and experimental conditions:

The test item was prepared in a concentration of 10 mg/mL with DMSO (stock solution) at the first step. The necessary amount of test item was weighed into a calibrated volumetric flask. A partial volume of DMSO was added and the solution was stirred until homogeneity is reached. Thereafter, DMSO was added up to final volume. The appropriate amount of this stock solution was diluted with medium to obtain the examination concentrations. Test solutions were prepared just before the treatment of the cells. For examined test item concentrations, no precipitation in the medium was noted.

Evaluation criteria:

At least 200 metaphase cells containing 2 N ± 2 centromeres were evaluated for structural aberrations from each experimental group. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally the number of polyploid and endoreduplicated cells were scored. The nomenclature and classification of chromosome aberrations were given based upon ISCN, 1985, and Savage, 1976, 1983.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: Tested up to cytotoxic concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility and Dose Selection
The test item was dissolved in DMSO. A clear solution was obtained up to a concentration of 10 mg/mL. There was no precipitation in the medium at any concentration tested.
The dose selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays, both in the absence and in the presence of a metabolic activation system (rodent S9-mix). Toxicity was determined by cell counting and results noted as % cells in relation to the solvent control. Detailed results of the cytotoxicity assay with the test item are presented in Tables 1 and 2 in the Appendix. These results were used to select concentrations of the test item for the Chromosome Aberration Assays.

The following concentrations were selected ranging from little to maximum (< 50 % survival) toxicity and evaluated in the main studies (Experiment A and B). All concentrations were run in duplicate (incl. negative and positive controls) and at least 200 well-spread metaphases were assessed:

Experiment A with 3/20 h treatment/sampling time
without S9 mix: 0.61, 1.22, 2.44, 4.88 and 7.32 µg/mL test item.
with S9 mix: 2.44, 4.88, 9.76 and 14.64 µg/mL test item.

Experiment B with 20/28 h treatment/sampling time
without S9 mix: 0.61, 1.22, 2.44 and 3.66 µg/mL test item.

Experiment B with 3/28 h treatment/sampling time
with S9 mix: 4.88, 9.76, 14.64 and 17.08 µg/mL test item.

Chromosome Aberration Assay
The cytotoxicity at the highest concentrations was adequate in the studies (experiment A and experiment B) as indicated by a reduction of % cell survival of at least 50 % .
In Experiment A, the test item did not induce an increase in the number of cells with aberrations without gaps at any examined concentration, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationship was noted.

In Experiment B, the test item was examined (0.61, 1.22, 2.44 and 3.66 µg/mL) without S9 mix, over a long treatment period (20 hours). Again, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent controls. A three-hour treatment with the test item in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps at 4.88, 9.76, 14.64 and 17.08 µg/mL, further indicating that the findings in Experiment A were within the normal biological variation. As in experiment A, in Experiment B no statistical differences between treatment and control groups and no dose-response relationships were noted.
The occurrence of polyploid and endoreduplicated metaphases is shown. No biologically relevant increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item.

In the control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, proving the suitability of the cell line used.

The positive controls Ethylmethane sulphonate (0.4 and 1.0 µg/mL) and N-Nitrosodimethylamine (1.0 µl/mL) caused the expected biologically relevant increases of cells with structural chromosome aberrations. The studies are, therefore, considered valid.

Conclusions:

The test item tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster ovary cells. Therefore, the test item is considered not clastogenic in this system.

Executive summary:

The test item was tested in a Chromosome Aberration Assay in V79 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).

In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 0.61, 1.22, 2.44, 4.88 and 7.32 µg/mL test item.

with S9 mix: 2.44, 4.88, 9.76 and 14.64 µg/mL test item.

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 0.61,1.22, 2.44 and 3.66 µg/mL test item.

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 4.88, 9.76, 14.64 and 17.08 µg/mL test item.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control, when the test item was examined up to cytotoxic concentrations (0.61, 1.22, 2.44 and 3.66 g/mL) without S9 mix over a prolonged treatment period (20 hours). Further, a three-hour treatment with the test item up to cytotoxic concentrations (4.88, 9.76, 14.64 and 17.08 g/mL) in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps, further indicating that the findings in Experiment A were within the normal biological variation. As in experiment A, in Experiment B no statistical differences between treatment and control groups and no dose-response relationships were noted.

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

The validity of the test was shown using Ethylmethane sulphonate (0.4 and 1.0 L/mL) and N-Nitrosodimethylamine (1.0 L/mL) as positive controls.

In summary, the test item tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster ovary cells.

Therefore, the test item is considered not clastogenic in this system.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2008-10-31 to 2009-05-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

EC Regulation 440/2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: ICH Tripartite Harmonised Guideline on Genotoxicity S2A: “Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals” (1996) and S2B: “Guidance on Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals “(1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

Thymidine kinase (tk) locus L5178Y 3.7.2 C mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Thymidine kinase (tk) locus L5178Y 3.7.2 C mouse lymphoma cells; the tk locus is autosomal and the L5178Y cell line is heterozygous (tk+/-), producing the enzyme thymidine kinase.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction derived from male WISTAR rats induced with Phenobarbitone (PB) and beta-Naphthoflavone (BNF) at a dose level of 80 mg/kg bw/day

Test concentrations with justification for top dose:

Assay 1
3-hour treatment with S9-mix:
5.0, 10.0, 12.5, 15.0, 17.5, 20.0, 22.5 µg/mL
3-hour treatment without S9-mix:
1.75, 3.5, 3.75, 4.0, 4.25, 4.5, 4.75, 5 µg/mL

Assay 2
3-hour treatment withS9-mix:
4, 6, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20 µg/mL
3-hour treatment without S9-mix:
1.2, 1.4, 1.6, 1.8, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8 µg/mL

24-hour treatment without S9-mix:
1.0, 1.2, 1.4, 1.6, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.6, 2.8, 3.0 µg/mL

Vehicle / solvent:

Dimethyl sulfoxide DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9-mix

Details on test system and experimental conditions:

Dose Selection / Preliminary Toxicity Test
The treatment concentrations for the mutation assay were selected on the basis of the results of a short preliminary toxicity test (LAB Study Code: 07/523-033ELE) to cover the range from cytotoxic to non-cytotoxic. In this preliminary experiment, a 3-hour treatment in the presence and absence of S9-mix and a 24-hour treatment in the absence of S9-mix were performed with a range of test concentrations to determine toxicity immediately after the treatments. Based on information available from other in vitro study performed at LAB Research Ltd. the following concentrations were selected for the three and 24 hour range-finding experiment: 100 µg/mL, 50 µg/mL, 25 µg/mL, 12.5 µg/mL, 6.25 µg/mL, 3.12 µg/mL and 1.56 µg/mL.
Single cultures were tested and positive controls were not included. Following treatments, cell concentrations were determined using a haemocytometer and cells were plated for survival in duplicates. Precipitation of the test item in the final culture medium was visually examined at beginning and end of the treatments.

Main Mutation Assays / Treatment of the cells
In the main assays, 3-hour treatments were performed with and without metabolic activation (in the presence and absence of S9-mix). The 24-hour treatment was performed without metabolic activation (in the absence of S9-mix).
For the 3-hour treatment incubations, at least 1E07 cells were placed in each of a series of sterile flasks (culturing surface 75 cm²).
For the 24-hour treatment, at least 4E06 cells were placed in each of a series of sterile flasks (culturing surface 25 cm²). Treatment medium contained a reduced serum level of 5 % (v/v) (RPMI 5). A suitable volume (0.2 mL) of solvent, test compound or positive control solution, and 1.0 mL of S9-mix or of 150 mM KCl (except 24-hour treatment) was added to a final volume of 20 mL per culture per experiment (at least 1E07 cells or 4E06 cells, respectively). Duplicate cultures were used for each treatment. Solubility of the test item in the cultures was visually examined at the beginning and end of each treatment.
Flasks were incubated at 37 °C ± 1 °C (approximately 5 % CO2) with (3-hour treatments) or without gentle shaking (24-hour treatment). After the incubation period, cells were centrifuged at 2000 rpm (approximately 836g) for 5 minutes, washed with tissue culture medium and resuspended in 20 mL RPMI 10/tube. Cell density, where sufficient cells survived, was adjusted to a concentration of 2E05/mL. Cells were transferred to flasks for growth through the expression period (maximum 25 mL of suspension) or diluted to be plated for survival.

Evaluation criteria:

The test item is considered to be mutagenic in this assay if all the following criteria are met (based on Moore 2006 (Ref. 6)):
1. The assay is valid;
2. statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding negative control values at one or more concentrations;
3. the increases are reproducible between replicate cultures and between tests (when treatment conditions were the same);
4. there is a significant dose-relationship as indicated by the linear trend analysis (p < 0.05);
5. the mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 1E06 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.

Statistics:

Assessment of statistical significance of mutant frequency
Statistical significance of mutant frequencies (total wells with clones) was carried out using Microsoft Excel 2000 software. The control log mutant frequency (LMF) was compared to the LMF from each treatment dose, using Dunnett's test for multiple comparisons and the data were checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend is one-tailed, therefore negative trend was not considered significant. These tests required the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Mutation Assays
In the mutation assays cells were exposed to the test item for 3 or 24 hours, with or without metabolic activation (S9-mix), then the cells were plated for determination of survival data and in parallel sub-cultured without test item for approximately 3 days to allow the expression of the genetic changes (if any occurred). At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

Assay 1
No precipitation of the test item was observed visually either in the presence or absence of S9-mix at the start of treatment or at the end of the treatment period.
Data of Assay 1 are presented for survival, viability and mutagenicity.
In the presence of S9-mix (3h treatment), excessive cytotoxicity was observed at 22.5 µg/mL concentration. Cells were not plated for survival after treatment or transferred for expression period due to the insufficient number of surviving cells. Due to the marked cytotoxicity observed, the dose of 20 µg/mL (relative survival value of 4 %) was excluded from the further evaluation. The observed cytotoxicity at the next concentration of 17.5 µg/mL resulted in 17 % relative survival value, which met the acceptance criteria of 10 - 20 % of relative survival. An evaluation was made at concentration of 17.5 µg/mL and below (a total of five doses).
No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis.

In the absence of S9-mix (3h treatment), excessive cytotoxicity was observed at concentrations of 5, 4.75, 4.5, 4.25 and 4 µg/mL. Cells were not plated for survival after treatment or transferred for expression period due to the insufficient number of surviving cells after treatment. The number of the remaining doses did not meet the acceptance criteria (minimal number of four doses), thus this experiment was considered invalid and was not evaluated. The experiment was repeated in the Assay 2 with modified test concentrations.

Assay 2
No precipitation of the test item was observed visually (either in the presence or absence of S9-mix) at treatment. However, precipitation was observed at the end of the incubation period in some test item treated samples both in the experiments with and without metabolic activation (the amount and homogeneity of precipitation was dose-dependent). The appearance of the controls was normal.
Data of Assay 2 are presented for survival, viability and mutagenicity.

In the presence of S9-mix (3h treatment), excessive cytotoxicity was observed at concentrations of 20 and 18 µg/mL. Cells were not plated for survival or transferred for expression period in these cases due to the insufficient number of surviving cells after treatment. Marked cytotoxicity was observed at 16 µg/mL concentration (with 4 % relative survival value), thus this dose was excluded from further evaluation. The observed cytotoxicity at the next concentration of 15 µg/mL resulted in 12 % relative survival value, which met the acceptance criteria of 10 - 20 % of relative survival. An evaluation was made at a concentration of 15 µg/mL and the next six concentrations (a total of 7 doses). No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by linear trend analysis.

In the absence of S9-mix (3h treatment), excessive cytotoxicity was observed at a concentration of 3.8 µg/mL. Cells were not plated for survival or transferred for expression period due to the insufficient number of surviving cells after treatment. Marked cytotoxicity was observed at 3.6, 3.4 and 3.2 µg/mL concentrations (relative survival values of 5, 6 and 6 %, respectively) and these samples were excluded from further evaluation. The next concentration of 3 µg/mL resulted in 15 % relative survival value, which met the acceptance criteria regarding cytotoxicity (see Table 2). An evaluation was made at concentration of 3 µg/mL and the next four concentrations (a total of 5 doses). No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by linear trend analysis.

In the absence of S9-mix (24h treatment), excessive cytotoxicity was observed at concentrations of 3, 2.8 and 2.6 µg/mL. Cells were not plated for survival or viability and mutation due to the insufficient number of surviving cells after treatment or in the expression period. The observed cytotoxicity at the next concentration of 2.4 µg/mL resulted in 21 % relative survival value, which is very close to the acceptance criteria of 10 - 20 % relative survival. However, the heterogeneity in the samples of this dose (2.4 µg/mL) was above the acceptance criteria, so it was excluded from further evaluation. (Note: according to the relevant SOP for each dose of the viability plates and mutation plates a heterogeneity factor is calculated using the data of all plates. If the heterogeneity factor of a given dose is higher than the 10.8 times of the historical heterogeneity factor, that dose will be excluded from further evaluation). An evaluation was made at the remaining highest seven doses (2.3 µg/mL and the next six concentrations) as indicated in the Study plan.
No significant increase of the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis.

Validity of the Mutation Assays
The spontaneous mutation frequency of the negative control was within the expected range in line with historical data in all assays.

The positive controls (Cyclophosphamide in the presence of metabolic activation and 4-Nitroquinoline-N-oxide in the absence of metabolic activation) gave the anticipated increases in mutation frequency over the negative controls and were in accordance with historical data in all assays.

The plating efficiencies for the negative control at the end of the expression period (PEviability) were within the acceptable range in all assays.

The number of test concentrations evaluated for each treatment (3h or 24h, with or without S9-mix) was at least four, which met the acceptance criteria.

The defined acceptance criteria regarding cytotoxicity produced by the highest evaluated concentrations (80 - 90 % toxicity, i.e. 10 - 20 % relative survival) was attained in this study. The one exception was the 24-hour treatment without metabolic activation in Assay 2. In this experiment, the relative survival value observed in case of the highest dose used for evaluation (2.3 µg/mL) was 26 %, which is out of the default range of 10 - 20 %. However, this value is close to the acceptance criteria and cells in the samples with an acceptable level of cytotoxicity died during the expression period. Thus the overall study was considered to be valid.

Conclusions:

The Mouse Lymphoma Assay with the test item on L5178Y TK+/- 3.7.2 C cells was considered valid. Treatment with the test item did not result in a statistically or biologically significant dose-dependent increase in mutation frequencies either in the presence or absence of a rat metabolic activation system (S9). In conclusion, no mutagenic effect of the test item was observed either in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of the test item to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (S9-mix) and for 24 hours without metabolic activation.

Dimethyl sulfoxide (DMSO) was used as the solvent of the test item in this study. Treatment concentrations for the mutation assay were selected based on the results of a preliminary cytotoxicity test. The following concentrations of the test item were selected for the main assays:

Assay 1

3-hour treatment in the presence of S9-mix: 22.5; 20; 17.5; 15; 12.5; 10 and 5 µg/mL.

3-hour treatment in the absence of S9-mix: 5; 4.75; 4.5; 4.25; 4; 3.75; 3.5 and 1.75 µg/mL.

Assay 2

3-hour treatment in the presence of S9-mix: 20; 18; 16; 15; 14; 13; 12; 11; 10; 9; 8; 6 and 4 µg/mL.

3-hour treatment in the absence of S9-mix: 3.8; 3.6; 3.4; 3.2; 3; 2.8; 2.6; 2.4; 2.2; 2; 1.8; 1.6; 1.4 and 1.2 µg/mL.

24-hour treatment in the absence of S9-mix: 3; 2.8; 2.6; 2.4; 2.3; 2.2; 2.1; 2; 1.9; 1.8; 1.6; 1.4; 1.2 and 1 µg/mL.

In the main assays, a measurement of the cytotoxicity survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of an approximately 3 day expression period following the treatment) of the cells was determined.

In Assay 1, in the presence of S9-mix (3h treatment), excessive cytotoxicity was observed at 22.5 µg/mL concentration. Cells were not plated for survival after treatment or transferred for expression period due to the insufficient number of surviving cells. Due to the marked cytotoxicity observed, the dose of 20 µg/mL was excluded from further evaluation. The observed cytotoxicity at the next concentration of 17.5 µg/mL resulted in 17 % relative survival value, which met the acceptance criteria of 10 - 20 % of relative survival. An evaluation was made at concentration of 17.5 µg/mL and below (a total of five doses). No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis.

In Assay 1, in the absence of S9-mix (3h treatment), excessive cytotoxicity was observed at concentrations of 5, 4.75, 4.5, 4.25 and 4 µg/mL. Cells were not plated for survival after treatment or transferred for expression period due to the insufficient number of surviving cells after treatment. The number of the remaining doses did not meet the acceptance criteria (minimal number of four doses), thus this experiment was considered invalid and was not evaluated. The experiment was repeated in the Assay 2 with modified test concentrations.

In Assay 2, in the presence of S9-mix (3h treatment), excessive cytotoxicity was observed at concentrations of 20 and 18 µg/mL. Cells were not plated for survival or transferred for expression period in these cases due to the insufficient number of surviving cells after treatment. Marked cytotoxicity was observed at 16 µg/mL concentration, thus this dose was excluded from further evaluation. The observed cytotoxicity at the next concentration of 15 µg/mL met the acceptance criteria of 10 - 20 % of relative survival. An evaluation was made at concentration of 15 µg/mL and the next six concentrations (a total of 7 doses). No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by linear trend analysis.

In Assay 2, in the absence of S9-mix (3h treatment), excessive cytotoxicity was observed at concentration of 3.8 µg/mL. Cells were not plated for survival or transferred for expression period due to the insufficient number of surviving cells after treatment. Marked cytotoxicity was observed at 3.6, 3.4 and 3.2 µg/mL concentrations, these samples were excluded from further evaluation. The next concentration of 3 µg/mL resulted in 15 % relative survival value, which met the acceptance criteria regarding cytotoxicity. An evaluation was made at concentration of 3 µg/mL and the next four concentrations (a total of 5 doses). No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by linear trend analysis.

In Assay 2, in the absence of S9-mix (24h treatment), excessive cytotoxicity was observed at concentrations of 3, 2.8 and 2.6 µg/mL. Cells were not plated for survival or viability and mutation due to the insufficient number of surviving cells. The observed cytotoxicity at the next concentration of 2.4 µg/mL resulted in 21 % relative survival value, which is very close to the acceptance criteria of 10 - 20 % relative survival. However, the heterogeneity in the samples of this dose (2.4 µg/mL) was above the acceptance criteria, so it was excluded from further evaluation. An evaluation was made at the remaining highest seven doses (2.3 µg/mL and the next six concentrations) as indicated in the Study plan. No significant increase of the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis.

In all assays, the mutation frequencies of negative and positive controls (Cyclophosphamide in experiments with metabolic activation system and 4-Nitroquinoline-N-oxide in experiments without metabolic activation system) were acceptable and were in accordance with historical data. The plating efficiencies of the negative control at the end of the expression period were within the acceptable range in each assay.

The defined acceptance criteria regarding cytotoxicity produced by the highest evaluated concentrations (80 - 90 % toxicity, i.e. 10 - 20 % relative survival) was attained in this study. The one exception was the 24-hour treatment without metabolic activation in Assay 2. In this experiment, the relative survival value observed in case of the highest dose used for evaluation (2.3 µg/mL) was 26 %, which is out of the default range of 10 - 20 %. However, this value is close to the acceptance criteria; and cells in the samples with an acceptable level of cytotoxicity died during the expression period. Thus the overall study was considered to be valid.

Conclusion

In conclusion, no mutagenic effect of the test item was observed either in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

The test item was examined in the following phases: in Plate Incorporation Tests (Initial Mutation Test, Complementary Plate Incorporation Test, and Second Complementary Plate Incorporation Test) and Pre-Incubation Tests (Confirmatory Mutation Test and Complementary Pre-Incubation Test).

In the above experimental phases the examined concentration levels were different. The test item was dissolved in Dimethyl sulfoxide (DMSO).

In the Plate Incorporation Tests the investigated concentration ranges (summarised): in case of Salmonella typhimurium strains 100 - 0.003 μg/plate without and with addition of metabolic activation (±S9 Mix), in Escherichia coli WP2 uvrA: 3419 - 0.158 μg/plate (± S9 Mix).

In the Pre-Incubation Tests: in the Salmonella typhimurium strains 10 - 0.003 μg/plate (–S9 Mix) and 100 - 0.003 μg/plate (+S9 Mix); in case of Escherichia coli WP2 uvrA 158.1 - 0.158 μg/plate (± S9 Mix).

Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 strains and Escherichia coli WP2 uvrA were used.

Results and Discussion:

Five bacterial strains, Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA were used to investigate the mutagenic potential of the test item in independent experiments, in plate incorporation tests and in a pre-incubation tests. Each assay (except the Complementary Pre-Incubation Test) was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate.

Inhibitory, cytotoxic effects of the test item were observed in the experiments in all examined bacterial strains and included reductions of spontaneous mutation rates, when compared to the solvent control values, reduced background lawn development and appearances of small pinpoint colonies. Furthermore no bacterial growth was observed for the strains at several concentration levels, with and without metabolic activation. Inhibition was stronger in the non-activated part (–S9 Mix) of the experiments.

No significant increases in revertant colony numbers of any of the five test strains were observed following treatment with the test item at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Further, there was no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance in the performed experiments.

Positive and negative controls were run concurrently. The revertant colony numbers of solvent control plates without S9 Mix were within the historical control data range. The reference mutagens showed a distinct increase of induced revertant colonies.

Conclusion:

The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the strains used. Therefore, the test item is considered non-mutagenic in this bacterial reverse mutation assay.

Chromosome aberration test

The test item was tested in a Chromosome Aberration Assay in V79 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).

In two independent experiments (both run in duplicate) at least 200 well-spread metaphase cells were analysed at concentrations and incubation/expression intervals given below, ranging from little to maximum (< 50 % survival) toxicity:

Experiment A with 3/20 h treatment/sampling time

without S9 mix: 0.61, 1.22, 2.44, 4.88 and 7.32 µg/mL test item.

with S9 mix: 2.44, 4.88, 9.76 and 14.64 µg/mL test item.

Experiment B with 20/28 h treatment/sampling time

without S9 mix: 0.61,1.22, 2.44 and 3.66 µg/mL test item.

Experiment B with 3/28 h treatment/sampling time

with S9 mix: 4.88, 9.76, 14.64 and 17.08 µg/mL test item.

In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted.

In Experiment B, the frequency of the cells with structural chromosome aberrations without gaps did not show significant alterations compared to the concurrent control, when the test item was examined up to cytotoxic concentrations (0.61, 1.22, 2.44 and 3.66 g/mL) without S9 mix over a prolonged treatment period (20 hours). Further, a three-hour treatment with the test item up to cytotoxic concentrations (4.88, 9.76, 14.64 and 17.08g/mL) in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps, further indicating that the findings in Experiment A were within the normal biological variation. As in experiment A, in Experiment B no statistical differences between treatment and control groups and no dose-response relationships were noted.

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

The validity of the test was shown using Ethylmethane sulphonate (0.4 and 1.0 L/mL) and N-Nitrosodimethylamine (1.0 L/mL) as positive controls.

In summary, the test item tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster ovary cells.

Therefore, the test item is considered not clastogenic in this system.

In vitro mammalian cell assay

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK ± 3.7.2 C cells at the tk locus to test the potential of the test item to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (S9-mix) and for 24 hours without metabolic activation.

Dimethyl sulfoxide (DMSO) was used as the solvent of the test item in this study. Treatment concentrations for the mutation assay were selected based on the results of a preliminary cytotoxicity test. The following concentrations of the test item were selected for the main assays:

Assay 1

3-hour treatment in the presence of S9-mix: 22.5; 20; 17.5; 15; 12.5; 10 and 5 µg/mL.

3-hour treatment in the absence of S9-mix: 5; 4.75; 4.5; 4.25; 4; 3.75; 3.5 and 1.75 µg/mL.

Assay 2

3-hour treatment in the presence of S9-mix: 20; 18; 16; 15; 14; 13; 12; 11; 10; 9; 8; 6 and 4 µg/mL.

3-hour treatment in the absence of S9-mix: 3.8; 3.6; 3.4; 3.2; 3; 2.8; 2.6; 2.4; 2.2; 2; 1.8; 1.6; 1.4 and 1.2 µg/mL.

24-hour treatment in the absence of S9-mix: 3; 2.8; 2.6; 2.4; 2.3; 2.2; 2.1; 2; 1.9; 1.8; 1.6; 1.4; 1.2 and 1 µg/mL.

In the main assays, a measurement of the cytotoxicity survival (colony-forming ability at the end of the treatment period) and viability (colony-forming ability at the end of an approximately 3 day expression period following the treatment) of the cells was determined.

In Assay 1, in the presence of S9-mix (3h treatment), excessive cytotoxicity was observed at 22.5 µg/mL concentration. Cells were not plated for survival after treatment or transferred for expression period due to the insufficient number of surviving cells. Due to the marked cytotoxicity observed, the dose of 20 µg/mL was excluded from further evaluation. The observed cytotoxicity at the next concentration of 17.5 µg/mL resulted in 17 % relative survival value, which met the acceptance criteria of 10 - 20 % of relative survival. An evaluation was made at concentration of 17.5 µg/mL and below (a total of five doses). No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis.

In Assay 1, in the absence of S9-mix (3h treatment), excessive cytotoxicity was observed at concentrations of 5, 4.75, 4.5, 4.25 and 4 µg/mL. Cells were not plated for survival after treatment or transferred for expression period due to the insufficient number of surviving cells after treatment. The number of the remaining doses did not meet the acceptance criteria (minimal number of four doses), thus this experiment was considered invalid and was not evaluated. The experiment was repeated in the Assay 2 with modified test concentrations.

In Assay 2, in the presence of S9-mix (3h treatment), excessive cytotoxicity was observed at concentrations of 20 and 18 µg/mL. Cells were not plated for survival or transferred for expression period in these cases due to the insufficient number of surviving cells after treatment. Marked cytotoxicity was observed at 16 µg/mL concentration, thus this dose was excluded from further evaluation. The observed cytotoxicity at the next concentration of 15 µg/mL met the acceptance criteria of 10 - 20 % of relative survival. An evaluation was made at concentration of 15 µg/mL and the next six concentrations (a total of 7 doses). No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by linear trend analysis.

In Assay 2, in the absence of S9-mix (3h treatment), excessive cytotoxicity was observed at concentration of 3.8 µg/mL. Cells were not plated for survival or transferred for expression period due to the insufficient number of surviving cells after treatment. Marked cytotoxicity was observed at 3.6, 3.4 and 3.2 µg/mL concentrations, these samples were excluded from further evaluation. The next concentration of 3 µg/mL resulted in 15 % relative survival value, which met the acceptance criteria regarding cytotoxicity. An evaluation was made at concentration of 3 µg/mL and the next four concentrations (a total of 5 doses). No significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by linear trend analysis.

In Assay 2, in the absence of S9-mix (24h treatment), excessive cytotoxicity was observed at concentrations of 3, 2.8 and 2.6 µg/mL. Cells were not plated for survival or viability and mutation due to the insufficient number of surviving cells. The observed cytotoxicity at the next concentration of 2.4 µg/mL resulted in 21 % relative survival value, which is very close to the acceptance criteria of 10 - 20 % relative survival. However, the heterogeneity in the samples of this dose (2.4 µg/mL) was above the acceptance criteria, so it was excluded from further evaluation. An evaluation was made at the remaining highest seven doses (2.3 µg/mL and the next six concentrations) as indicated in the Study plan. No significant increase of the mutation frequency was observed at the evaluated concentrations. No significant dose response to the treatment was indicated by the linear trend analysis.

In all assays, the mutation frequencies of negative and positive controls (Cyclophosphamide in experiments with metabolic activation system and 4-Nitroquinoline-N-oxide in experiments without metabolic activation system) were acceptable and were in accordance with historical data. The plating efficiencies of the negative control at the end of the expression period were within the acceptable range in each assay.

The defined acceptance criteria regarding cytotoxicity produced by the highest evaluated concentrations (80 - 90 % toxicity, i.e. 10 - 20 % relative survival) was attained in this study. The one exception was the 24-hour treatment without metabolic activation in Assay 2. In this experiment, the relative survival value observed in case of the highest dose used for evaluation (2.3 µg/mL) was 26 %, which is out of the default range of 10 - 20 %. However, this value is close to the acceptance criteria; and cells in the samples with an acceptable level of cytotoxicity died during the expression period. Thus the overall study was considered to be valid.

Conclusion

In conclusion, no mutagenic effect of the test item was observed either in the presence or absence of metabolic activation system under the conditions of this Mouse Lymphoma Assay.

Justification for classification or non-classification

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

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.