Cyclohexyl(ethyl)amine

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:

22.3. - 20.4.2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see Overall remarks

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Cyclohexyl(ethyl)amine

EC Number:

226-733-8

EC Name:

Cyclohexyl(ethyl)amine

Cas Number:

5459-93-8

Molecular formula:

C8H17N

IUPAC Name:

N-ethylcyclohexanamine

Test material form:

other: liquid

Details on test material:

- Name of test material (as cited in study report): N-ethylcyclohexylamine
- Physical state: clear colourless liquid
- Analytical purity: 99.5% (w/w)
- Impurities (identity and concentrations): cyclohexanol 0.22% (w/w), alifatic amines 0.23% (w/w), water 0.07% (w/w)
- Lot/batch No.: 89002001
- Expiration date of the lot/batch: 12/2012
- Storage condition of test material: in a closed dark glass vial, in refrigerator

Method

Target gene:

gene for synthesis histidine or tryptophan

Species / strain

Species / strain 1

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine dependent strain

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

other: tryptophan dependent strain

Metabolic activation:

with and without

Metabolic activation system:

postmitochondrial fraction (S9)

Test concentrations with justification for top dose:

50, 150, 500, 1500, 5000 µg/plate (first experiment)
15, 50, 150, 500, 1500 µg/plate (second experiment)

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene diamine, 2-aminofluorene, 2-aminoanthracene, N-methyl-N´-nitro-N-nitrosoguanidine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: 2.

DETERMINATION OF CYTOTOXICITY
- Method: total growth

Evaluation criteria:

The main criterion for evaluation of results was modified two-fold increase rule which is compatible with the aplication of statistical methods. After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.

Statistics:

For the evaluation of results the modified two-fold increase rule was used, which using is comparable with application of statistical methods (2, 3).

2. Dunkel V. C., Chu K.C. (1980): Evaluation of methods for analysis of microbial mutagenicity assays, in The Predictive Value of Short-Term Screening Tests in Carcinogenicity Evaluation, Elsevier North-Holland Biomedical Press, 231 - 240, 1980
3. Claxton L. D. et al. (1987): Guide for the Salmonella typhimurium/mammalian microsome tests for bacterial mutagenicity, Mutat. Res. 189, 83 - 91, 1987

Results and discussion

Test results

Test results 1

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: only for TA 100 (without metabolic activation), negative. Decrease of number of revertants and reduction of bacterial background occurred in all experiments/strains in the maximum dose 5000 µg per plate with metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Under the above-described experimental design, the test substance N-ethylcyclohexylamine was nonmutagenic for all the Salmonella typhimurium as well as Escherichia coli strains both in experiments without and with metabolic activation.

Executive summary:

Test substance N-ethylcyclohexylamine was assayed for the mutagenicity by the Bacterial Reverse Mutation Test. The performed test was based on EU method B.13/14 Mutagenicity – Reverse mutation test using bacteria, which is analogous to the OECD Test Guideline No. 471.

Four indicator Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and one indicator Escherichia coli WP2 uvrA strain were used. The test substance was dissolved in dimethylsulfoxide and assayed in doses of 15-5000 µg, which were applied to plates in volume of 0.1 mL.

Experiments were performed without as well as with metabolic activation with a supernatant of rat liver and a mixture of cofactors.

In the arrangement given above, the test substance was nonmutagenic for all the used tester strains without as well as with metabolic activation.