[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 January 2002 to 27 April 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 21, 1997

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

GLP compliance:

yes

Type of assay:

other: HPRT forward mutation in mammalian cells

Test material

Test material information

Constituent 1

Reference substance name:

[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

EC Number:

275-604-2

EC Name:

[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Cas Number:

71550-24-8

Molecular formula:

C24H30N3O2.Cl

IUPAC Name:

[2-[[2-cyano-3-[4-[ethylbenzylamino]phenyl]-1-oxoallyl]oxy]ethyl]trimethylammonium chloride

Test material form:

solid: particulate/powder

Details on test material:

appearance: yellow powder
sample: laboratory sample purified

Specific details on test material used for the study:

purified laboratory sample

Method

Target gene:

6-thioguanine

Species / strain

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: University of Ulm, Germany
- Cell cycle length, doubling time or proliferation index: 10-14 hours
- Methods for maintenance in cell culture: stock kept under liquid nitrogen
- Modal number of chromosomes: 22


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: maintained in plastic vessels at 37 °C and 5%CO2, recloned by subculturing twice weekly (checked regularly for karyotype and mycoplasma presence)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability:yes

Metabolic activation:

with and without

Metabolic activation system:

S9 derived from Aroclor 1254 induced rat liver homogenate (concentration in test medium 5%)

Test concentrations with justification for top dose:

exp 1 without metabolic activation: 10, 20, 40, 60, 80 and 100 µg/mL
exp 2 without metabolic activation: 15, 30, 60, 90, 120 and 240 µg/mL
exp 1 with metabolic activation: 60, 80, 100, 120, 160, 200 and 240 µg/mL
exp 2 with metabolic activation: 60, 80, 100, 120, 160, 200 and 240 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: substance soluble up to 250 mg/mL in pre-test (precipitate in culture medium at 240 µg/mL and above)

Controls

Untreated negative controls:

yes

Remarks:

untreated

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: dimethylbenzanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: Exponentially growing cells were plated in culture medium in 250 mL flasks for 16-24 hours to allow attachment. Thereafter the cells were exposed for 5 hours. Monolayers were washed with PBS, trypsinized and replated in 20 mL culture medium (1.5E06 cells/250 mL) in flasks and in 3 petri dishes (5 mL medium with 200 cells/dish).
Petri dishes were incubated for 6 days and used to assess cytotoxicity (survival to treatment).
The cells in the flasks were incubated to permit growth and expression of mutations. After 6 days the cells were re-seeded in 8 petri dishes per culture at 3E05 cells/dish in presence of 6-thioguaninine. Three additional petri dishes with 200 cels/dish were used to calculate cloning efficiency.
After 6-8 days of incubation colonies were fixed, stained with Chiemsa and counted.

NUMBER OF REPLICATIONS: 8/concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency and relative total growth

Evaluation criteria:

1) assessment in at least 5 replicates per concentration with relative survival, relative population growth and absolute cloning efficiency > 10%
2) positive result with dose related increase in mutant frequency (at least 2-3 times of vehicle controls) and this finding can be reproduced in a separate experiment
3) no unphysiological culture conditions (effects on pH and osmolality)
4) scientific judgement

Statistics:

ANOVA and Dunnet test

Results and discussion

Test results

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

maximum concentrations based on cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Evaporation from medium: NA
- Precipitation: at and above 240 µg/mL

RANGE-FINDING/SCREENING STUDIES:without metabolic activation: 100% cell death at 100 µg/mL; with metabolic activation 100% cell death at 240 µg/mL

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: included in the report
- Negative (solvent/vehicle) historical control data: included in the report

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- cloning efficiency, rel growth: no effects up to 240 µg/mL:
- survival to treatment: in all experiments with and without metabolic activation cell death (100%) at 240 µg/mL

Applicant's summary and conclusion

Conclusions:

The substance did not induce mutations in mammalian cells

Executive summary:

In a HPRT assay according to OECD 476 the substance did not induce mutations in V79 cells both in presence and absence of metabolic activation. The substance was tested up to cytotoxic concentrations.

It can be concluded that the substance is negative in this test and does not induce mutations in mammalian cells.