reaction product of diphenylmethanediisocyanate, toluenediisocyanate ( reaction mass of isomers: 65 % 2,4- and 35 % 2,6-diisocyanate), octylamine, oleylamine and 4-ethoxyaniline (molar ratio 3.88:1:6.38:0.47:2.91)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item was tested for in vitro mutagenic/ genotoxic activity in a bacterial reverse mutation test (Ames test), in an in vitro mammalian chromosome aberration test according to OECD Guideline 471 and 473, respectively, and in an in vitro mammalian cell gene mutation test (HPRT) according to OECD 476.
The test item did not induce point or frameshift mutations in the bacterial reverse mutation test performed with five strains of S. typhimurium with and without metabolic activation. Therefore, the test item was considered non-mutagenic in the Ames test performed. The test item did not induce structural chromosome aberrations in the in vitro chromosome aberration test performed with Chinese hamster V79 cells in the presence and absence of metabolic activation. Therefore, the test item is not considered clastogenic in this chromosome aberration test. The test item, tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control and historical controls) in this in vitro test in Chinese hamster ovary cells. Thus, the test item was not mutagenic under the conditions of this study.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-04-14 to 1999-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

adopted 1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 1992-12-29

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

EPA712-C-96-223, June 1996

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):

- Type and identity of media: Minimum Essential Medium (MEM), supplemented with 10% foetal calf serum (FCS)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (S9 liver microsomal fraction induced with beta-Naphtoflavone and Phenobarbital)

Test concentrations with justification for top dose:

Experiment I: 4 h treatment, 20 h preparation interval
with and without metabolic activation: 25, 250, 500 µg/mL

Experiment II: 20 h treatment and preparation interval:
without metabolic activation: 25, 100, 250 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (final concentration 1 %)
- Justification for choice of solvent/vehicle: The utilised final concentration of DMSO is compatible with the survival of the cells and the S9 activity.

Untreated negative controls:

yes

Remarks:

, culture medium

Negative solvent / vehicle controls:

yes

Remarks:

, DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

TWO INDEPENDENT EXPERIMENTS
-Experiment I: Exposure period (with and without metabolic activation): 4 hours
-Experiment II: Exposure period (without metabolic activation): 20 hours

Fixation time:
With and without S9 mix: 20 h

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: (with and without metabolic activation): 4 hours in experiment I and 20 hours in experiment II
- Fixation time (start of exposure up to fixation or harvest of cells): approx. 20 hours after start of the exposure
- SPINDLE INHIBITOR (cytogenetic assays): colcemide, 17.5 hours after the start of the treatment.
- STAIN (for cytogenetic assays): After fixation the cells were stained with Giemsa

NUMBER OF REPLICATIONS: 2 replications

NUMBER OF CELLS EVALUATED: At least 200 well spread metaphases per concentration and control were scored for cytogenetic damage.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploid: the number of polyploid cells was scored. Polyploid means a near tetraploid karyotype in the case of this aneuploid cell line.

Evaluation criteria:

There are several criteria for determining a positive result:
- dose-related increase in the number of cells with aberration
- biologically relevant positive response for at least one of the test points

Statistics:

The chi-square test was used as an aid for the interpretation. A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose level.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight cytotoxic effects at concentrations >= 250 µg/mL, only in the absence of metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

slight cytotoxic effects at concentrations >= 100 µg/mL

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the two highest concentrations the precipitate of the test item began to stick to the cells.

RANGE-FINDING/SCREENING STUDIES: Not performed

Remarks on result:

other: all strains/cell types tested

In experiment I, with metabolic activation, a reduction of the mitotic index accompanied by a slight reduction of the cell density was observed at the 250 and 500 µg/mL concentrations. In exp. I, with metabolic activation, as well as in exp. II, without S9, a clear reduction of the relative cell density was seen at the two highest concentrations, but no reduction of the mitotic index. These findings, suggesting slight toxic properties of the test item, may be explained by the solubility properties of the test item. At the two highest concentrations the precipitate of the test item began to stick to the cells.

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Under the conditions of the study, the test substance did not induce structural chromosomal in the in vitro mammalian chromosome aberration test with and without metabolic activation using V79 Chinese hamster cells. Based on the study results, the test item was considered to be non-mutagenic.

Executive summary:

An in vitro mammalian chromosome aberration test was performed with the test item according to EU Method B.10, OECD Guideline 473 and EPA OPPTS 870.5375. The test item potential to induce structural chromosome aberrations in V79 Chinese hamster cells was investigated in two independent experiments. In the two independent experiments the chromosomes were prepared 20 h after start of treatment with the test item. Experiment I was performed with and without S9 mix using a treatment interval of 4 h and a preparation interval of 20 h. Experiment II was performed only without metabolic activation with a treatment and preparation interval of 20 h. Two parallel cultures were set up per test group. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated (higher concentrations could not be evaluated due to the solubility characteristics of the test item):

Experiment I: 4 h treatment, 20 h preparation interval:

with and without metabolic activation (S9 mix) and test item concentrations: 25, 250, 500 µg/mL

Experiment II: 20 h treatment and preparation interval:

without metabolic activation (S9 mix) and test item concentrations: 25, 100, 250 µg/mL

Reference mutagens as positive controls were tested in parallel to the test item and showed an expected distinct increase in cells with structural chromosome aberrations.

Mild cytotoxic effects of the test item, indicated by a reduced relative cell density value, were only observed in experiments performed in the absence of metabolic activation starting at a concentration of 250 µg/ml in experiment II and at a concentration of 100 µg/m in experiment II.

Treatment of the cells with the test item in both experiments did not lead to a relevant decrease of the relative mitotic index or of the cell density.

When compared to the corresponding solvent control no biologically relevant increase in aberrant cells was obtained with and without metabolic activation in both independent experiments. No biologically relevant increase in the frequencies of polyploidy metaphases was found after treatment with the test item if compared to the frequencies of the controls.

It was concluded that the test item did not induce structural chromosome aberrations with and without metabolic activation. Thus, the test substance was considered non-mutagenic in this chromosome aberration test.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-03-24 to 1999-05-18

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:

adopted 1997-07-21

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

dated 1992-12-29

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

EPA712-C96-219, June 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

bacteria, other: TA 1535, TA 1537, TA 98, TA 100, TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (S9 liver microsomal fraction induced with beta-Naphtoflavone and Phenobarbital)

Test concentrations with justification for top dose:

Concentration range in the two main tests (with and without metabolic activation): 31.6, 100, 316.2, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

yes

Remarks:

Water

Negative solvent / vehicle controls:

yes

Remarks:

diluted DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

For TA 1535, TA 100 strains without metabolic activation

Positive control substance:

other: 4-nitro-o-phenylene-diamine, 4-NOPD

Remarks:

For TA 1537, TA 98 strains without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

For TA 102 strains without metabolic activation

Positive control substance:

other: 2-aminoanthracene, 2-AA

Remarks:

For TA 1535, TA 1537, TA 98, TA 100 and TA 102 strains with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- experiment I: plate incorporation test
- experiment II: pre-incubation test

DURATION
- Pre-incubation period: 60 min at 37°C (pre-incubation test)
- Expression time (cells in growth medium): 48 hours
- Number of independent experiments: 2 main experiments
- Number of replicates: 3 plates per concentration

Evaluation criteria:

A test item is considered as mutagenic if:
- a dose-related increase in the number of revertants occurs and/or
- a reproducible biologically relevant positive response for at least one of the test points occurs in at least one strain with or without metabolic activation.

A biologically relevant increase is described as follows:
- the number of reversions produced in strains TA 100 and TA 102 is at least twice as high as compared to the spontaneous reversion rate.
- the number of reversions produced in strains TA 1535, TA 1537 and TA 98 is at least three times higher as compared to the spontaneous reversion rate

Statistics:

no statistics required

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation of the test item was observed at a concentrations of ≥ 316.2 µg/plate


RANGE-FINDING/SCREENING STUDIES: The toxicity of the test item was determined with strains TA 98 and TA 100 in a pre-experiment. In total, 8 concentrations of the test item (3.16, 10.0, 31.6, 100.0, 316.2, 1000.0, 2500.0 and 5000.0 µg/plate) were evaluated for toxicity and induction of mutations with 3 plates each. The experimental conditions in this pre-experiment were the same as in experiment I (plate incorporation test).

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects of the test item were not observed.

- No substantial increases in the revertant colony numbers of any of the five test strains were detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments.

- The positive controls showed a distinct increase of induced revertant colonies.

Conclusions:

Under the experimental conditions reported, the test substance did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test item was considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Executive summary:

The test item was investigated for its mutagenic potential in a bacterial reverse mutation test according to EU Method B.13/14, OECD Guideline 471 and EPA OPPTS 870.5265. Five S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were tested with and without metabolic activation. A plate incorporation test and a pre-incubation test were performed independently and the following concentrations of the test item, tested in triplicate, were used in both experiments: 31.6; 100.0; 316.2; 1000.0; 2500.0 and 5000.0 µg/plate.

Precipitation of the test item was observed on the agar plates at concentrations >= 316.2 µg/plate. Cytotoxic effects of the test item were not observed. Reference mutagens showed a distinct increase of induced revertant colonies.

No substantial increase in the revertant colony numbers of any of the five test strains was detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments. Thus, the test substance was considered non-mutagenic in this bacterial reverse mutation assay.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-05-21 to 2019-06-06

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 using the Hprt and xprt genes)

Version / remarks:

2016-07-29

Deviations:

yes

Remarks:

Negative results were not confirmed as the confirmation of negative results is not required by the most current Guideline (OECD 476, 29 Jul 2016)

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-31

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Target gene:

The objective of this study was to determine whether the test item or its metabolites can induce forward mutation at the hypoxanthine-guanine phosphoribosyl transferase enzyme locus (hprt) in cultured Chinese hamster ovary cells.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data

Metabolic activation:

with and without

Metabolic activation system:

S9 mix of phenobarbital and β-naphthoflavone induced rat liver

Test concentrations with justification for top dose:

5-hour treatment period without S9-mix: 15.7, 31.3, 62.5, 125 and 250 μg/mL

5-hour treatment period with S9-mix: 15.7, 31.3, 62.5, 125 and 250 μg/mL

In the performed assay the concentration levels were chosen mainly based on the solubility of test item. At concentration of 250 μg/mL slight precipitation in the medium was observed.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: N,N-Dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: The vehicle was chosen based on results of the preliminary Solubility Test. Its suitability is confirmed by the available laboratory’s historical database.

Untreated negative controls:

yes

Remarks:

N,N-Dimethylformamide (DMF)

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

no

Remarks:

Solvent control was run concurrently with treatment groups.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide (DMSO)

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 24 hours
- Exposure duration: 5 hours
- Expression time (cells in growth medium): 1, 3 and 6 days
- Selection time: 6 days

SELECTION AGENT: Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 2000000

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

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.
Test item is then considered able to induce gene mutations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical 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).
• The test item is then considered unable to induce gene mutations in cultured mammalian cells in this test system.

Statistics:

Statistical analysis was done 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 heterogeneity of variance between groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.

Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a none-normal distribution, the non-parametric method of Kruskal-Wallis one-way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

strain/cell type: Sub-line (KI) of Chinese hamster ovary cell line CHO

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no
- Effects of osmolality: no
- Precipitation: at a concentration of 250 µg/mL slight precipitation was observed in the medium


RANGE-FINDING/SCREENING STUDIES:
Treatment concentrations for the mutation assay were selected on the basis of the result of a pre-test on toxicity. For the pre-test on toxicity (cytotoxicity assay), the cultures (more than 50 % confluent) were trypsinised and cell suspensions were prepared in Ham's F12-10 medium. Cells were seeded into petri dishes (tissue cultures quality: TC sterile) at 5x106 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 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. Five concentrations were selected for the treatment in the main mutation assay without and with metabolic activation.


COMPARISON WITH HISTORICAL CONTROL DATA:
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.

Conclusions:

The test substance tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control and historical controls) in this in vitro test in Chinese hamster ovary cells. Thus, the test substance was not mutagenic under the conditions of this study.

Executive summary:

The test item, dissolved in N,N-Dimethylformamide, was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix: 15.7, 31.3, 62.5, 125 and 250 μg/mL

5-hour treatment period with S9-mix: 15.7, 31.3, 62.5, 125 and 250 μg/mL

At a concentration of 250 μg/mL precipitation in the medium was observed. In the performed assay the concentration levels were chosen mainly based on the solubility of test item. There were no relevant changes in pH or osmolality after treatment with the test item. 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 observed between treatment groups when compared to the concurrent and historical control groups and no dose-response relationships were noted.

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.

The test substance tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control and historical controls) in this in vitro test in Chinese hamster ovary cells.

Thus, the test substance was not mutagenic under the conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation test:

The test item was investigated for its mutagenic potential in a bacterial reverse mutation test according to EU Method B.13/14, OECD Guideline 471 and EPA OPPTS 870.5265. Five S. typhimurium strains (TA 1535, TA 1537, TA 98, TA 100 and TA 102) were tested with and without metabolic activation. A plate incorporation test and a pre-incubation test were performed independently and the following concentrations of the test item, tested in triplicate, were used in both experiments: 31.6; 100.0; 316.2; 1000.0; 2500.0 and 5000.0 µg/plate.

Precipitation of the test item was observed on the agar plates at concentrations >= 316.2 µg/plate. Cytotoxic effects of the test item were not observed. Reference mutagens showed a distinct increase of induced revertant colonies.

No substantial increase in the revertant colony numbers of any of the five test strains was detected at any dose level of the test item either with or without metabolic activation in both independently performed experiments. Thus, the test item was considered non-mutagenic in this bacterial reverse mutation assay.

 

In vitro mammalian cell chromosome aberration test:

An in vitro mammalian chromosome aberration test was performed with the test item according to EU Method B.10, OECD Guideline 473 and EPA OPPTS 870.5375. The test item potential to induce structural chromosome aberrations in V79 Chinese hamster cells was investigated in two independent experiments. In the two independent experiments the chromosomes were prepared 20 h after start of treatment with the test item. Experiment I was performed with and without S9 mix using a treatment interval of 4 h and a preparation interval of 20 h. Experiment II was performed only without metabolic activation with a treatment and preparation interval of 20 h. Two parallel cultures were set up per test group. Per culture 100 metaphases were scored for structural chromosomal aberrations. The following concentrations were evaluated (higher concentrations could not be evaluated due to the solubility characteristics of the test item):

Experiment I: 4 h treatment, 20 h preparation interval:

with and without metabolic activation (S9 mix) and test item concentrations: 25, 250, 500 µg/mL

Experiment II: 20 h treatment and preparation interval:

without metabolic activation (S9 mix) and test item concentrations: 25, 100, 250 µg/mL

Reference mutagens as positive controls were tested in parallel to the test item and showed an expected distinct increase in cells with structural chromosome aberrations.

Mild cytotoxic effects of the test item, indicated by a reduced relative cell density value, were only observed in experiments performed in the absence of metabolic activation starting at a concentration of 250 µg/ml in experiment II and at a concentration of 100 µg/m in experiment II.

Treatment of the cells with the test item in both experiments did not lead to a relevant decrease of the relative mitotic index or of the cell density.

When compared to the corresponding solvent control no biologically relevant increase in aberrant cells was obtained with and without metabolic activation in both independent experiments. No biologically relevant increase in the frequencies of polyploidy metaphases was found after treatment with the test item if compared to the frequencies of the controls.

It was concluded that the test item did not induce structural chromosome aberrations with and without metabolic activation. Thus, the test item was considered non-mutagenic in this chromosome aberration test.

 

In vitro mammalian cell gene mutation test

The test item, dissolved in N,N-Dimethylformamide was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The following concentrations were selected on the basis of a pre-test on cytotoxicity with and without metabolic activation using S9 mix of phenobarbital and β-naphthoflavone induced rat liver and solubility of test item.

5-hour treatment period without S9-mix: 15.7, 31.3, 62.5, 125 and 250 μg/mL

5-hour treatment period with S9-mix: 15.7, 31.3, 62.5, 125 and 250 μg/mL

At a concentration of 250 μg/mL precipitation in the medium was observed. In the performed assay the concentration levels were chosen mainly based on the solubility of test item. There were no relevant changes in pH or osmolality after treatment with the test item. 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 observed between treatment groups when compared to the concurrent and historical control groups and no dose-response relationships were noted.

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.

The test item tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control and historical controls) in this in vitro test in Chinese hamster ovary cells. Thus, the test item was not mutagenic under the conditions of this study.

Justification for classification or non-classification

Based on the available in vitro studies, the test item was not classified and labelled as mutagenic according to Regulation (EC) No 1272/2008 (CLP).