4,4'-Methylene-bis-(3-chloro-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:

April - June 1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1985

Report date:

1985

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

402-130-7

EC Name:

-

Cas Number:

106246-33-7

Molecular formula:

C21 H28 Cl2 N2

IUPAC Name:

4-[(4-amino-2-chloro-3,5-diethylphenyl)methyl]-3-chloro-2,6-diethylaniline

Test material form:

solid: crystalline

Details on test material:

- Name: LONZACURE® M-CDEA
- Internal substance code: P5367
- Storage: Keep container tightly closed. Keep in a dry, cool and well-ventilated place.
- Aggregate state/appearance: Crystalline solid
- Colour: white to off-white
- Odour: Odourless

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Dr. Bruce Ames, University of California, USA
- Cultures of all organisms were prepared by overnight incubation of freshly inoculated nutrient broth (Oxoid No. 2).

TA 1535:
contains a histidine missense mutation but is also deficient in a DNA repair system (uvr B) and has a defective lipopolysaccharide coat on the cell wall. It is reverted by many agents causing base-pair substitutions, but is not sensitive to
frameshift mutagens.

TA 1537:
bears a histidine frameshift mutation. Like TA 1535, it is defective in DNA repair and in the lipopolysaccharide coat. It is sensitive to agents causing frameshift mutations involving insertion or deletion of a single base-pair.

TA 100
is the same as TA 1535 but contains a resistance transfer factor conferring ampicillin resistance (plasmid pKM 101). In addition to base-pair substitutions, it is also able to detect certain frameshift mutagens.

TA 98
is TA 1538 with the addition of the pKM 101 plasmid

Additional strain / cell type characteristics:

other: see above

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells: Dr. Bruce Ames, University of California, USA
- Cultures of all organisms were prepared by overnight incubation of freshly inoculated nutrient broth (Oxoid No. 2).

TA 1538
contains another histidine frameshift mutation. Again it has a defective DNA repair system and lipopolysaccharide coat. It is reverted by agents causing deletion of two adjacent basepairs (double frameshift mutations).

Additional strain / cell type characteristics:

other: see above

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

was tested in a separate study (see reference LR2718)

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

- Concentration range in the main test (with metabolic activation): 8 - 5000 µg/plate
- Concentration range in the main test (without metabolic activation): 8 - 5000 µg/plate

Vehicle / solvent:

Solvent: Dimethylsulfoxide

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

other: 2-Aminoanthracene: with and without S9-mix

Details on test system and experimental conditions:

A preliminary toxicity test was conducted at eight concentrations from 2.5 µg up to 5000 µg.

Rationale for test conditions:

A preliminary toxicity test was conducted at eight concentrations from 2.5 µg up to 5000 µg. In the main study, concentrations of 8 up to 5000 µg were tested.

Evaluation criteria:

no data

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Observations:
No increase in revertants in any strain following exposure to test substance. Positive controls gave the expected response.

Remarks on result:

no mutagenic potential (based on QSAR/QSPR prediction)

Applicant's summary and conclusion

Conclusions:

The test item was found to be non-mutagenic in this Ames test, in presence and absence of S9-mix.

Executive summary:

The study was performed 1985 as GLP-test following EC-test method B.14 on Salmonella typhimurium strains TA 98, 100, 1535, 1537 and 1538. P5367 was dissolved in DMSO and tested at concentrations of 8 - 5000 ug/plate, with and without metabolic activation (Aroclor 1254 induced liver S9 -mix). Cytoxicity was observed at concentrations above 5000 ug/plate, however, there was no increase in revertants in any strain following exposure to test substance.

In conclusion, the substance is not mutagenic with and without metabolic activation.