Amines, C12-16 alkyl dimethyl, reaction products with ethyl chloroacetate, 2-(2-aminoethylamino)ethanol and 2-propenoic acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline Study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

(2-carboxyethyl)({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl})azanium; [({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl}carbamoyl)methyl](dodecyl)dimethylazanium; ethanol

IUPAC Name:

(2-carboxyethyl)({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl})azanium; [({2-[(2-carboxyethyl)(2-hydroxyethyl)amino]ethyl}carbamoyl)methyl](dodecyl)dimethylazanium; ethanol

Method

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham’s F10 medium without thymidine and hypoxanthine, supplemented with 10% (v/v) horse serum, L-glutamine (2 mM)
and penicillin/streptomycin (50 U/ml and 50 µg/ml respectively).

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Dose range finding test: 10 to 5000 µg/ml
experiment 1 without S9 mix: 0.1, 0.5, 1, 5, 10, 15, 20 and 25 µg/ml
experiment 1 with 8 % S9 mix: 10, 50, 100, 200, 400 and 500 µg/ml
experiment 2 without S9 mix: 5, 10, 15, 20, 22.5, 25, 27.5 and 30 µg/ml
experiment 2 with 12 % S9 mix: 100, 200, 300, 350, 400, 450, 500 and 600 µg/ml
experiment 3 without S9 mix; 3 hours treatment: 10, 20, 25 and 27.5 µg/ml
experiment 3 without S9 mix; 24 hours treatment: 45, 60, 75 and 90 µg/ml

Vehicle / solvent:

exposure medium (F10-medium)

Controls

Negative solvent / vehicle controls:

yes

Remarks:

exposure medium

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION: Exposure duration: 3 hours and 24 hours
SELECTION AGENT (mutation assays): trifluorothymidine
DETERMINATION OF CYTOTOXICITY: relative total growth

Evaluation criteria:

A test substance was considered positive (mutagenic) in the mutation assay if:
1. It induced at least a 8-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner; and
2. The results were reproducible in an independently repeated test.
A test substance was considered negative (not mutagenic) in the mutation assay if:
1. None of the tested concentrations showed a mutant frequency of at least three-fold compared to the solvent control.
2. The results were confirmed in an independently repeated test.

Statistics:

The experimental results were not subjected to statistical analysis.

Results and discussion

Test results

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: test substance concentration range of 10 to 5000 ug/ml in the absence of SS-mix with a 3 and 24 hour treatment period and in the presence of S9-mix with a 3 hour treatment period.

In the absence of S9 metabolic activation, REWOTERIC QAM 50 induced a 5.3-fold increase in the mutant frequency in the first experiment. No significant increase in the mutant frequency at the TK-locus was observed in the second experiment. Verification of this result was performed in the additional third experiment with a 3 hour treatment period and a prolonged treatment time of 24 hours, in which REWOTERIC QAM 50 showed no significant increase in the mutant frequency at the TK-locus.
Since the mutagenic effect observed in the first experiment was not confirmed by the additional assay nor in the short or prolonged treatment, this increase was considered to be not biologically relevant and REWOTERIC QAM 50 is considered to be not mutagenic in the absence of S9-mix.

In the presence of S9-mix, REWOTERIC QAM 50 induced no significant increase in the mutant frequency in both independent experiments.

Remarks on result:

other: strain/cell type: L5178Y mouse lymphoma cells

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

REWOTERIC QAM 50 is not mutagenic in the TK mutation test system under the experimental conditions described.

Executive summary:

In a mammalian cell gene mutation assay according to OECD guideline 467, L5178Y mouse lymphoma cells cultured in vitro were exposed to Rewoteric Qam 50 in the following concentrations in the presence and absence of mammalian metabolic activation (S9 mix):

First experiment

Without S9-mix, 3 h treatment: 0.1, 0.5, 1, 5, 10, 15, 20 and 25 μg/mL

With 8% S9-mix, 3 h treatment: 10, 50, 100, 200, 400 and 500 μg/mL

Second experiment

Without S9-mix, 3 h treatment: 5, 10, 15, 20, 22.5, 25, 27.5 and 30 μg/mL

With 12% S9-mix, 3 h treatment: 100, 200, 300, 350, 400, 450, 500 and 600 μg/mL

Third experiment

Without S9 -mix, 3 h treatment: 10, 20, 25 and 27.5 µg/mL

Without S9 -mix, 24 h treatment: 45, 60, 75 and 90 µg/mL

Test substance was tested up to cyctotoxic concentrations.The positive controls induced the appropriate response.There was no evidence of induced mutant colonies over background.

In the absence of S9 metabolic activation, the substance induced a 5.3-fold increase in the mutant frequency in the first experiment. Since this mutagenic effect was not confirmed by the additional assay nor in the short or prolonged treatment, this increase was considered to be not biologically relevant and the substance is considered to be not mutagenic in the absence of SS-mix.

In the presence of S9 -mix, the substance induced no significant increase in the mutant frequency in both independent experiments.

In conclusion, the substance is not mutagenic in the TK mutation test system under the experimental conditions described.