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

Genetic toxicity: in vitro

Administrative data

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.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Test guideline 1

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)

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

237-185-4

EC Name:

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

Cas Number:

13680-35-8

Molecular formula:

C21H30N2

IUPAC Name:

4,4'-methylenebis(2,6-diethylaniline)

Test material form:

solid: crystalline

Details on test material:

- Physical state: see above
- Appearance: see above

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)

Method

Species / strain

Species / strain 1

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 2

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 3

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 4

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 5

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

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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

Test results 3

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

Test results 4

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

Test results 5

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.

Applicant's summary and conclusion

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.