Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria (Ames test)

This study was performed to investigate the potential gene mutagenic activity of the test item according to the plate incorporation test of Ames et. al. (1) using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. The test was performed with and without liver microsomal activation. The test material was tested at the following concentrations: 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 micrograms per plate. Each concentration, including the controls, was tested in triplicate. No toxic effect of the test material was observed. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain when compared with the corresponding controls. The presence of microsomal activation did not influence these findings. In conclusion, it can be stated that during this in vitro Salmonella/mammalian-microsome assay, no gene mutagenic activity could be demonstrated under the experimental conditions reported.

In-vitro gene mutation study in mammalian cells

The test item was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in N,N-Dimethylformamide (DMF) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9-mix). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below: Experiment 1, 5-hour treatment period without S9-mix: 10, 20, 30, 40, 50, 60 and 70 µg/mL Experiment 1, 5-hour treatment period with S9-mix: 15, 20, 25, 30, 35, 40 and 45 µg/mL Experiment 2, 20-hour treatment period without S9-mix: 5, 10, 15, 20, 25, 30, 35 and 40 µg/mL Experiment 2, 5-hour treatment period with S9 -mix: 15, 20, 25, 30, 35, 40 and 45 µg/mL. In the performed assays the concentration levels were chosen mainly based on the cytotoxicity and solubility (Experiment 1, 5-hour treatment period without S9-mix) of test item. Phenotypic expression was evaluated up to 8 days following exposure. In Experiment 1, 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 biologically significant differences between treatment and control groups and no dose-response relationships were noted. In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency. As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted. The sensitivity of the tests and the efficacy of the S9-mix were demonstrated by large increases in mutation frequency in the positive control cultures. 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) in this in vitro test in Chinese hamster ovary cells. Thus, the substance was not mutagenic under the conditions of this study.

In-vitro cytogenicity / chromosome aberration study in mammalian cells

The test item was tested in a Chromosome Aberration Assay in V79 cells. Cells were pre-incubated for 24 hours. The test item was dissolved in N,N-Dimethylformamide (DMF) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using rodent S9 mix). In two independent experiments (both run in duplicate with concurrent negative and positive controls) at least 200 well-spread metaphase cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below, ranging from no or little to maximum (< 50% survival) toxicity: Experiment A with 3/20 h treatment/sampling time without S9 mix: 2.5, 5, 10 and 20 μg/mL test item Experiment A with 3/20 h treatment/sampling time with S9 mix: 10, 20, 40 and 50 μg/mL test item Experiment B with 20/20 h treatment/sampling time without S9 mix: 2, 3, 4, 5 and 5.5# μg/mL test item Experiment B with 20/28 h treatment/sampling time without S9 mix: 2, 3, 4 and 5 μg/mLtest item Experiment B with 3/28 h treatment/sampling time with S9 mix: 10, 20, 40 and 50 μg/mL test item. The 5.5 μg/ml concentration was tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations. Following treatment (exposure) and sampling (expression) time cells were exposed to selection agent Colchicine (0.2 μg/mL) 2-2.5 hours prior to harvesting. Following harvesting cells were treated with fixative for ca. 10 min. before being placed on slides and stained. Chromosome aberration frequencies were then scored for at least 200 well-spread metaphase cells. In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations without gap, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between test item treatment and concurrent negative 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 without S9 mix over a prolonged treatment period of 20 hours and 20 and 28 hour sampling times. Further, a three-hour treatment with the test item up to the cytotoxic concentration in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps. In both experiments, no statistically significant differences between test item treatment and concurrent negative (solvent) control groups and no dose-response relationships were noted.

No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item. The validity of the test was shown using Ethyl methanesulphonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) as concurrent positive controls, which induced biologically and statistically significant increases in the number of cells with chromosome aberration(s) over background. In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, confirming the suitability of the cell line used. The test item, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, the substance is considered not clastogenic in this system.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 1983
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
OECD Guidelines for testing of chemicals, Draft No. 419 (status 1983)
Qualifier:
equivalent or similar to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
EEC Directive 79/831, Annex V, Method No. 431 (status 1983)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL:
- Identification: P 5256
- Batch/Lot number: not available
- Purity: not available
- Physical appearance: powder
- Stability: pure: not available / in vehicle: not available
- Storage: the test material was stored at room temperature in the dark
To avoid any light effects on the test compound, all experimentation was performed under yellow light
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Bacterial test organisms: Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537
Source: B.N. Ames , University of California, Berkeley CA, 94720, USA, May 1981

Characterization of strains:
The strains used are mutants derived from Salm. typhimurium LT2 and have following genotype:
- S.typhimurium TA 1535 his G46 r fa- uvrB
- S.typhimurium TA 1537 his C3076 r fa- uvrB
- S.typhimurium TA 98 his D3052 r fa- uvrB- R+
- S.typhimurium TA 100 his G46 r fa- uvrB- R+
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
Bacterial test organisms: Salmonella typhimurium TA 1538
Source: B.N. Ames , University of California, Berkeley CA, 94720, USA, May 1981

Characterization of strains:
The strains used are mutants derived from Salmonella typhimurium LT2 and have following genotype: S.typhimurium TA 1538 his D3052 r fa- uvrB
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver fractions of rats, treated with AROCLOR 1254
Test concentrations with justification for top dose:
1.58 , 5, 15.8, 50, 158, 500, 1580, 5000 micrograms per plate; substance was not toxic up to the highest concentration

Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
methylmethanesulfonate
Details on test system and experimental conditions:
Method of application: in agar (plate incorporation)
Evaluation criteria:
A material is identified as a mutagen in this test system if there is a reproducible demonstration of a dose effect relation with a 2-fold increase in the number of revertants over the controls in a minimum of one strain. With the strain TA 100, a 1.5-fold increase is the criterion for a positive result.
Statistics:
no data
Key result
Species / strain:
S. typhimurium TA 98
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 100
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 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 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
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
Conclusions:
It can be stated that during this in vitro Salmonella/mammalian-microsome assay with P 5256, no gene mutagenic activity could be demonstrated under the experimental conditions reported.
Executive summary:

This study was performed to investigate the potential gene mutagenic activity of the test item according to the plate incorporation test of Ames et. al. (1) using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538. The test was performed with and without liver microsomal activation. The test material was tested at the following concentrations: 1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 micrograms per plate. Each concentration, including the controls, was tested in triplicate. No toxic effect of the test material was observed. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain when compared with the corresponding controls. The presence of microsomal activation did not influence these findings. In conclusion, it can be stated that during this in vitro Salmonella/mammalian-microsome assay, no gene mutagenic activity could be demonstrated under the experimental conditions reported.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October - December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
TEST MATERIAL
- Substance identification/name in the report: LZ 596
- Molecular formula: C21H30N2
- Batch no.: 1229
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25ºC, <70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
The CHO cell line was originally derived from the ovary of a female Chinese hamster (Kao and Puck, 1967). The CHO K1 is a sub-line of CHO cell line. The CHO K1 cell line was purchased from ECACC (European Collection of Cell Cultures). Lot. No.: 09J019
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver fractions
Test concentrations with justification for top dose:
In the performed assays the concentration levels were chosen mainly based on the cytotoxicity and solubility (Experiment 1, 5-hour treatment period without S9-mix) of test item. Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below:
- Experiment 1, 5-hour treatment period without S9-mix: 10, 20, 30, 40, 50*, 60* and 70* µg/mL
- Experiment 1, 5-hour treatment period with S9-mix: 15, 20, 25, 30, 35, 40 and 45 µg/mL
- Experiment 2, 20-hour treatment period without S9-mix: 5, 10, 15, 20, 25, 30, 35 and 40 µg/mL
- Experiment 2, 5-hour treatment period with S9-mix: 15, 20, 25, 30, 35, 40 and 45 µg/mL
* Slight precipitation in the medium was observed.
Vehicle / solvent:
N,N-Dimethylformamide (DMF)
Untreated negative controls:
yes
Remarks:
= solvent control
Negative solvent / vehicle controls:
yes
Remarks:
= DMF
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Rationale for test conditions:
Cytotoxicity and slight precipitation was observed at concentrations above 40 µg/mL
Evaluation criteria:
The test item is considered to show mutagenic activity in this study, if the assay is valid and all of the following criteria are met:
- The mutant frequency at one or more concentrations is significantly greater than that of the relevant control
- The increase of the mutant frequency is reproducible
- There is a dose-response relationship
The test item is considered not mutagenic, if one of the criteria listed above is not met.
Statistics:
The Wilcoxon-Mann-Whitney U-test was used to determine whether or not there are statistically significant increases in mutant frequency.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at higher concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The test item, LZ 596 was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in N,N-Dimethylformamide (DMF) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9-mix). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below: Experiment 1, 5-hour treatment period without S9-mix: 10, 20, 30, 40, 50, 60 and 70 µg/mL Experiment 1, 5-hour treatment period with S9-mix: 15, 20, 25, 30, 35, 40 and 45 µg/mL Experiment 2, 20-hour treatment period without S9-mix: 5, 10, 15, 20, 25, 30, 35 and 40 µg/mL Experiment 2, 5-hour treatment period with S9-mix: 15, 20, 25, 30, 35, 40 and 45 µg/mL In the performed assays the concentration levels were chosen mainly based on the cytotoxicity and solubility (Experiment 1, 5-hour treatment period without S9-mix) of test item. Phenotypic expression was evaluated up to 8 days following exposure. In Experiment 1, 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 biologically significant differences between treatment and control groups and no dose-response relationships were noted. In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency. As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted. The sensitivity of the tests and the efficacy of the S9-mix were demonstrated by large increases in mutation frequency in the positive control cultures. LZ 596 tested both without and with metabolic activation (S9 mix), did not induce increases in mutant frequency over the background (negative solvent control) in this in vitro test in Chinese hamster ovary cells. Thus, LZ 596 was not mutagenic under the conditions of this study.

Conclusions:
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) in this in vitro test in Chinese hamster ovary cells. Thus, the substance was not mutagenic under the conditions of this study.
Executive summary:

The test item was tested in a Mammalian Gene Mutation Test in CHO-K1 cells. The test item was dissolved in N,N-Dimethylformamide (DMF) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using S9-mix). Two independent main experiments (both run in duplicate) were performed at the concentrations and treatment intervals given below: Experiment 1, 5-hour treatment period without S9-mix: 10, 20, 30, 40, 50, 60 and 70 µg/mL Experiment 1, 5-hour treatment period with S9-mix: 15, 20, 25, 30, 35, 40 and 45 µg/mL Experiment 2, 20-hour treatment period without S9-mix: 5, 10, 15, 20, 25, 30, 35 and 40 µg/mL Experiment 2, 5-hour treatment period with S9 -mix: 15, 20, 25, 30, 35, 40 and 45 µg/mL. In the performed assays the concentration levels were chosen mainly based on the cytotoxicity and solubility (Experiment 1, 5-hour treatment period without S9-mix) of test item. Phenotypic expression was evaluated up to 8 days following exposure. In Experiment 1, 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 biologically significant differences between treatment and control groups and no dose-response relationships were noted. In Experiment 2, the mutant frequency of the cells did not show biologically or statistically significant alterations compared to the concurrent control, when the test item was tested without S9 mix over a prolonged treatment period (20 hours). Furthermore, a five-hour treatment in the presence of S9 mix did not cause significant increases in mutant frequency. As in Experiment 1, in Experiment 2 no statistical differences between treatment and solvent control groups and no dose response relationships were noted. The sensitivity of the tests and the efficacy of the S9-mix were demonstrated by large increases in mutation frequency in the positive control cultures. 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) in this in vitro test in Chinese hamster ovary cells. Thus, LZ 596 was not mutagenic under the conditions of this study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October - December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell transformation assay
Specific details on test material used for the study:
TEST MATERIAL
- Substance identification/name in the report: LZ 596
- Molecular formula: C21H30N2
- Batch no.: 1229
- Analysis date: August 12, 2014
- Date of production: August 11, 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage conditions: Controlled room temperature (15-25ºC, <70 RH%), protected from light and humidity.
- Stability under test conditions: stable for min. 3 years from manufacturing date
- Expiry date: August 10, 2017
- Safety precautions: Routine safety precautions (lab coat, gloves, safety glasses, face mask) for unknown materials were applied to assure personnel health and safety.
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 it suitable for gene toxicity assays with low background aberrations. These cells were 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).
- Supplier: ECACC (European Collection of Cells Cultures)
- Lot. No.: 05F013
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver fractions
Test concentrations with justification for top dose:
Concentration selection cytotoxicity assay was performed as part of this study to establish an appropriate concentration range for the Chromosome Aberration Assays (Experiment A and B), both in the absence and in the presence of a metabolic activation system (rodent S9 mix):
- Experiment A with 3/20 h treatment/sampling time without S9 mix: 2.5, 5, 10 and 20 μg/mL test item
- Experiment A with 3/20 h treatment/sampling time with S9 mix: 10, 20, 40 and 50 μg/mL test item
- Experiment B with 20/20 h treatment/sampling time without S9 mix: 2, 3, 4 and 5 μg/mL test item
- Experiment B with 20/28 h treatment/sampling time without S9 mix: 2, 3, 4 and 5 μg/mLtest item
- Experiment B with 3/28 h treatment/sampling time with S9 mix: 10, 20, 40 and 50 μg/mL test item
The 5.5 μg/mL concentration was tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations.
Vehicle / solvent:
N,N-Dimethylformamide (DMF)
Untreated negative controls:
yes
Remarks:
= solvent
Negative solvent / vehicle controls:
yes
Remarks:
= DMF
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
cyclophosphamide
ethylmethanesulphonate
Evaluation criteria:
The test item is regarded as non-clastogenic if:
- the number of metaphases with structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data
- and/or no significant increase in the number of metaphases with structural chromosome aberration is observed
A test item is classified as clastogenic if it meets the following criteria:
- increase in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (above the range of our historical control data)
- the increase is reproducible between replicate cultures and between tests (when the treatment conditions are the same)
- the increase is statistically significant
Both, biological and statistical significance should be considered together.
Statistics:
For statistical analysis, Fisher exact and CHI2 tests were utilized. The parameters evaluated for statistical analysis were as follows: number of aberration and number of cells with aberration (with and without gaps).
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
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
Conclusions:
The test item, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, the substance is considered not clastogenic in this system.
Executive summary:

The test item was tested in a Chromosome Aberration Assay in V79 cells. Cells were pre-incubated for 24 hours. The test item was dissolved in N,N-Dimethylformamide (DMF) and the following concentrations were selected on the basis of cytotoxicity investigations made in a preliminary study (without and with metabolic activation using rodent S9 mix). In two independent experiments (both run in duplicate with concurrent negative and positive controls) at least 200 well-spread metaphase cells were analysed at concentrations and treatment (exposure)/sampling (expression) intervals given below, ranging from no or little to maximum (< 50% survival) toxicity: Experiment A with 3/20 h treatment/sampling time without S9 mix: 2.5, 5, 10 and 20 μg/mL test item Experiment A with 3/20 h treatment/sampling time with S9 mix: 10, 20, 40 and 50 μg/mL test item Experiment B with 20/20 h treatment/sampling time without S9 mix: 2, 3, 4, 5 and 5.5# μg/mL test item Experiment B with 20/28 h treatment/sampling time without S9 mix: 2, 3, 4 and 5 μg/mLtest item Experiment B with 3/28 h treatment/sampling time with S9 mix: 10, 20, 40 and 50 μg/mL test item. The 5.5 μg/ml concentration was tested but not evaluated due to sufficient cytotoxicity at the next lower concentration and sufficient number of concentrations. Following treatment (exposure) and sampling (expression) time cells were exposed to selection agent Colchicine (0.2 μg/mL) 2-2.5 hours prior to harvesting. Following harvesting cells were treated with fixative for ca. 10 min. before being placed on slides and stained. Chromosome aberration frequencies were then scored for at least 200 well-spread metaphase cells. In Experiment A, there were no biologically significant increases in the number of cells showing structural chromosome aberrations without gap, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between test item treatment and concurrent negative 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 without S9 mix over a prolonged treatment period of 20 hours and 20 and 28 hour sampling times. Further, a three-hour treatment with the test item up to the cytotoxic concentration in the presence of S9 mix did not cause an increase in the number of cells with structural chromosome aberrations without gaps. In both experiments, no statistically significant differences between test item treatment and concurrent negative (solvent) control groups and no dose-response relationships were noted.

No increase in the rate of polyploid and endoreduplicated metaphases was found after treatment with the different concentrations of the test item. The validity of the test was shown using Ethyl methanesulphonate (0.4 or 1.0 μL/mL) and Cyclophosphamide (5.0 μg/mL) as concurrent positive controls, which induced biologically and statistically significant increases in the number of cells with chromosome aberration(s) over background. In the concurrent negative control group the percentage of cells with structural aberration(s) without gap was equal or less than 5 %, confirming the suitability of the cell line used. The test item, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, the substance is considered not clastogenic in this system.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

According to the Globally Harmonised System of Classification and Labelling of Chemicals, the test item does not require any classification/labelling.