4,4'-methylenebis[2,6-diethylaniline]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro gene mutation in bacteria (Ames test)

Two studies were performed. the first 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.

An additional study was conducted on request of ECHA using five strains to meet the current requirements, as the originally submitted test was conducted on four strains only. The second study was performed using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. 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. Precipitate / slight precipitate was detected in the main assays in all examined bacterial strains with and without metabolic activation at higher concentrations. Inhibitory, cytotoxic effect (slightly reduced/minimal background lawn development, with no effect on colony numbers) of the test item was observed in Assay 2 in all examined bacterial strains with and/or without metabolic activation at higher concentrations. The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

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

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November - December 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was conducted on request of ECHA using five strains to meet the current requirements, as the originally submitted test was conducted on four strains only.

Qualifier:

equivalent or similar to guideline

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

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: LZ 596
- Batch/Lot number: 1852
- Purity: 99.8%
- Physical appearance: white to brown crystalline powder
- Expiration date: 09 September 2023
- Storage: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (tight closed container)

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

- Strain: S. typhimurium TA98
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Date of supply: 07 December 2017
- Genotype - trp./his mutation: hisD3052
- Genotype - type of mutation: Frameshift
- Mutations - cell wall: rfa
- Mutations - DNA- repair: uvrB
- Main DNA target: GC
- Plasmid: pKM101

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

- Strain: S. typhimurium TA100
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Date of supply: 07 December 2017
- Genotype - trp./his mutation: hisG46
- Genotype - type of mutation: Base pair substitution
- Mutations - cell wall: rfa
- Mutations - DNA- repair: uvrB
- Main DNA target: GC
- Plasmid: pKM101

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

- Strain: S. typhimurium TA1535
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Date of supply: 07 December 2017
- Genotype - trp./his mutation: hisG46
- Genotype - type of mutation: Base pair substitution
- Mutations - cell wall: rfa
- Mutations - DNA- repair: uvrB
- Main DNA target: GC
- Plasmid: no

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

- Strain: S. typhimurium TA1537
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Date of supply: 07 December 2017
- Genotype - trp./his mutation: hisC3076
- Genotype - type of mutation: Frameshift
- Mutations - cell wall: rfa
- Mutations - DNA- repair: uvrB
- Main DNA target: GC
- Plasmid: no

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Strain: E. coli WP2 uvrA
- Source of cells: MOLTOX - Molecular Toxicology Inc., Boone, North Carolina, USA
- Date of supply: 07 December 2017
- Genotype - trp./his mutation: trpE
- Genotype - type of mutation: Base pair subsitution
- Mutations - cell wall: +
- Mutations - DNA- repair: uvrA
- Main DNA target: AT
- Plasmid: no

Metabolic activation:

with and without

Metabolic activation system:

S9 liver fractions of rats, treated with phenobarbital (PB) and β-naphthoflavone (BNF)

Test concentrations with justification for top dose:

PRE-TEST: 10, 31.6, 100, 316, 1000, 2500, 5000 μg/plate
Precipitate/slight precipitate was detected on the plates in the preliminary test in Salmonella typhimurium TA98 and TA100 bacterial strains at 5000, 2500, 1000 and 316 μg/plate concentrations without metabolic activation and at 5000, 2500 and 1000 μg/plate concentrations with metabolic activation. No inhibitory, cytotoxic effect of the test was observed in the preliminary experiment in any examined bacterial strains.
MAIN TEST: 1,581, 5, 15.81, 50, 158.1, 500, 1581, 5000 μg/plate

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

sodium azide

methylmethanesulfonate

other: 4-nitro-1,2-phenylene-diamine (NPD); 2-aminoanthracene

Details on test system and experimental conditions:

Method of application:
In Assay 1 the plate incorporation method was used, in Assay 2 the pre-incubation method was used.

Evaluation criteria:

A test item is considered mutagenic if:
• a concentration-related increase in the number of revertants occurs and/or;
• a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.
An increase is considered biologically relevant if:
• the number of reversions is more than two times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA98, TA100 and Escherichia coli WP2 uvrA bacterial strains;
• the number of reversions is more than three times higher than the reversion rate of the negative (solvent) control in Salmonella typhimurium TA1535 and TA1537 bacterial strains.
According to the guidelines, statistical method may be used as an aid in evaluating the test results. However, statistical significance should not be the only determining factor for a positive response.

Statistics:

no data

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

see comments below

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:

cytotoxicity

Remarks:

see comments below

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:

see comments below

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:

see comments below

Vehicle 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:

see comments below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitate / slight precipitate was detected in the main assays in all examined bacterial strains with and without metabolic activation at higher concentrations.
Inhibitory, cytotoxic effect (slightly reduced/minimal background lawn development, with no effect on colony numbers) of the test item was observed in Assay 2 in all examined bacterial strains with and/or without metabolic activation at higher concentrations.

Conclusions:

It can be stated that during this in vitro Salmonella/mammalian-microsome assay on five strains, no gene mutagenic activity could be demonstrated under the experimental conditions reported.

Executive summary:

This study was conducted on request of ECHA using five strains to meet the current requirements, as the originally submitted test was conducted on four strains only.

The study was performed using the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. 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. Precipitate / slight precipitate was detected in the main assays in all examined bacterial strains with and without metabolic activation at higher concentrations. Inhibitory, cytotoxic effect (slightly reduced/minimal background lawn development, with no effect on colony numbers) of the test item was observed in Assay 2 in all examined bacterial strains with and/or without metabolic activation at higher concentrations. The reported data of this mutagenicity assay show that under the experimental conditions applied the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
In conclusion, the test item had no mutagenic activity on the growth of the bacterial strains under the test conditions used in this study.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

April 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

This study was repeated on request of ECHA using five strains to meet the current requirements, as the originally submitted test was conducted on four strains only.

Qualifier:

equivalent or similar to guideline

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

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. This study had to be repeated on request of ECHA using five strains to meet the current requirements, as the originally submitted test was conducted on four strains only.

Reference 3

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

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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.

Reference 4

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

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

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.