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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

August 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

• Sponsor’s identification: 4,4'-méthylène bis (2,6-diméthylaniline)
• Synonym: DURCISSEUR DX
• Chemical name: 4-[(4-amino-3,5-dimethylphenyl)methyl]-2,6-dimethylaniline

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: GNJ170717 160 209 0004 (Lot Isochem)
- Expiration date of the lot/batch: 09.09.2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (15°C to 25°C) non hygroscopic
- Stability under test conditions: Stable under normal conditions storage
- Solubility and stability of the test substance in the solvent/vehicle: soluble in DMSO

Target gene:

Histidine and Tryptophane

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats

Test concentrations with justification for top dose:

TA1535, TA1537, TA98, TA100 and E. Coli WP2: 50, 150, 500, 1500, 2500 and 5000 µg/plate, with and without S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Antramine (2µg/plate with S9 metabolic activation), 9-Aminoacridine (50µg/plate without metabolic activation), cis-Platinum (II) Diammine dichloride (1µg/plate without metabolica activation)

Details on test system and experimental conditions:

SOURCE OF THE TEST SYSTEM: Strains were obtained from MOLTOX TM.

METHOD OF APPLICATION: In agar (plate incorporation); preincubation

NUMBER OF REPLICATES: Controls and treatment were performed in triplicate.

DURATION
- Preincubation period: 30 minutes at 37 °C, with shaking
- Incubation period: Plates were inverted and incubated at 37 °C in the dark for 48-72 hour in both direct plate and preincubation methods.

Evaluation criteria:

The following criteria were checked to validate the study:
- the bacteriostatic activity of the highest concentration tested shall be equal to or less than 75 %,
- the spontaneous reversion rate of the absolute negative control shall comply with the historical values of the laboratory,
- the spontaneous reversion rate of the solvent shall not be statistically different from absolute negative control,
- the mean number of revertant colonies obtained for each strain and the corresponding positive control, with and/or without metabolic activation shall comply with the historical values of the laboratory.
- Negative and positive values should not show significant difference with the historical values of the laboratory (± 2 standard deviations).

Statistics:

No data

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

presence of whitish precipitates not hindering the scoring

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:

presence of whitish precipitates not hindering the scoring

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

presence of whitish precipitates not hindering the scoring

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:

presence of whitish precipitates not hindering the scoring

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

presence of whitish precipitates not hindering the scoring

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Results show the absence of any bacterial growth in the presence of the various concentrations of the test item.
Results show the absence of any bacterial growth in the presence of "S9-mix".
Results show that in presence of the highest dose tested 5000 µg/plate a high level of toxicity is measured (compatible with the maximum tolerated). For the first assay, a supplementary dose is studied 2500 µg/plate.
The test item is tested at these doses (5 000, 2 500, 1 500, 500, 150 and 50 µg/plate) for the first assay and (5 000, 1 500, 500, 150 and 50 µg/plate) for the second assay.
One can observe for the highest doses tested (1 500 to 5 000µg/plate) the presence of whitish precipitates not hindering the scoring.

Conclusions:

Under the test conditions, the test item is not considered as mutagenic in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and in Escherichia coli WP2 (uvrA-) (pKM 101) strains without, or with metabolic activation.

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and E.coli WP2) were exposed to test item, 4,4'-méthylène bis (2,6-diméthylaniline) at the following concentrations: 

- TA1535, TA1537, TA98, TA100 and E.coli WP2: 50, 150, 500, 1500, 2500 and 5000 µg/plate, with and without S9-mix

 

Metabolic activation system used in this test 10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats . Vehicle, negative and positive control groups were also included in mutagenicity tests.

 

For assay n° 1, various concentrations of GNJ070817-S1 were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay n° 2, various concentrations of GNJ210817-S1 were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Under the test conditions, test item is not considered as mutagenic in this bacterial system.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Table 7.6/1: Summary of genotoxicity test

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1 Savineau, 2018	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100, WP2 uvrA-	-S9 +S9	Up to cytotoxic or highest recommended concentration	-S9 : non mutagenic +S9 : non mutagenic

 

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, strains ofSalmonella typhimurium(TA1535, TA1537, TA98, TA100 and E.coli WP2) were exposed to test item,4,4'-méthylène bis (2,6-diméthylaniline)at the following concentrations: 

- TA1535, TA1537, TA98, TA100 and E.coli WP2: 50, 150, 500, 1500, 2500 and 5000 µg/plate, with and without S9-mix

 

Metabolic activation system used in this test 10 % S9 mix; S9 fraction prepared from liver homogenates of male Sprague Dawley rats . Vehicle, negative and positive control groups were also included in mutagenicity tests.

 

For assay n° 1, various concentrations of GNJ070817-S1 were put in contact with the strains in the absence and presence of a metabolic activation system (S9-mix 10% (v/v)).

For assay n° 2, various concentrations of GNJ210817-S1 were put in contact with the strains in the absence of metabolic activation and with pre-incubation in the presence of metabolic activation system (S9-mix 10% (v/v)).

For the two assays, negative and positive controls were carried out in parallel. Positive controls induced a significant increase in the number of revertant colonies compared to negative controls. There is no significant difference between the number of spontaneous reversions, the number of reversions obtained in the positive controls (without and with metabolic activation), and the mean of corresponding experimental “historical” values obtained in the laboratory

 

No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, at any dose level either with or without metabolic activation. 

 

Under the test conditions, test item is not considered as mutagenic in this bacterial system.

Justification for classification or non-classification

Harmonized classification:

The registered substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, the registered substance does not require classification for mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP).