4,4'-methylenebis(cyclohexylamine)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02.04.1982- 31.08.1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study performed according to standard Ames testing guidelines with no major deviations (minor deviations: the test substance was tested up to very slight toxicity, no more than 27% toxicity in strain TA98 (without S9-mix) and 2-aminoanthracene alone is insufficient as a positive control with S9).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1982

Report date:

1982

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Directive 79/831/EEC

Deviations:

yes

Remarks:

: 2-aminoanthracene alone is insufficient as a positive control with S9

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-methylenebis(cyclohexylamine)

EC Number:

217-168-8

EC Name:

4,4'-methylenebis(cyclohexylamine)

Cas Number:

1761-71-3

Molecular formula:

C13H26N2

IUPAC Name:

4,4'-methylenedicyclohexanamine

Details on test material:

- Name of test material (as cited in study report): cyclohexyl,4,4'-methylenebis-
- Analytical purity: no data ("rein")

Method

Target gene:

histidine locus

Species / strain

Species / strain / cell type:

other: S.typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Clophen A 50 induced rat liver

Test concentrations with justification for top dose:

- Dosing: 4, 12.5, 40, 125, 400 ug/plate
- Additional experiment: 125, 400, 800 ug/plate

Vehicle / solvent:

- Vehicle/solvent used: ethanol (70%)

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: 70% ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine for TA1537, sodium azide for TA100 and TA1535, 2-nitrofluorene for TA98 and TA1538

Remarks:

without S9

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

: 70% ethanol

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene for all strains

Remarks:

with S9

Details on test system and experimental conditions:

PLATE INCORPORATION TEST
The following components were mixed in a sterile tube:
-2 mL top-agar
-0,1 mL of the bacterial overnight culture (0,8- 1,5x 10^8 bacteria/ mL)
-0,01 mL of the test item or solvent
-0,5 mL S9 mix
The mixing was done in triplicate for each bacterial strain and for each concentration of the test material and the mixture was then poured onto the surface of minimal agar plates.
-Incubation: 72 hours, 37 °C and then the number of revertant colonies was counted

Rationale for test conditions:

The toxicity of the test item was tested with and without metabolic activation. Doses from 62,5 µg - 2000 µg/ plate were tested. As the highest test dose 800 µg/ plate were chosen (survival of 10-15 % without and 70 % with metabolic activation)

Evaluation criteria:

CRITERIA FOR EVALUATING RESULTS:
- Dose related increase in the number of revertant colonies

Statistics:

not applicable

Results and discussion

Test results

Species / strain:

other: S. typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 800 ug/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

GENOTOXIC EFFECTS:
- With metabolic activation: negative
- Without metabolic activation: negative,
TA1538 additional experiment; 3-fold increase. However, in one experiment only.

PRECIPITATION CONCENTRATION:
- no precipitate

CYTOTOXIC CONCENTRATION:
- With metabolic activation: very slight toxicity at 800 ug/plate
- Without metabolic activation: very slight toxicity at 800 ug/plate

STATISTICAL RESULTS:
- not applicable

Applicant's summary and conclusion

Conclusions:

An Ames-test was performed to assess the mutagenis properties of the test item. The test item was proven to be non mutagenic in the absence and presence of S9 mix.

Executive summary:

The test item was tested for its ability to induce reverse mutations in an in vitro bacterial system. Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 1538 were treated with the test compound by the Ames test plate incorporation. Dose levels up to 800 µg/ plate, in triplicate both with and without the addition of metabolising system were employed.

All bacterial strains exhibited mutagenis responses to the appropriate positive control substances. Solvent controls were also tested with each strain and the mean numbers of spontaneous revertants were in an acceptable range. 

A reproducible mutagenic activity of the test compound to any of the tester strains was not observed with and without metabolic activation.

It is therefore concluded, that the test item is not a bacterial mutagen.