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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
January 1995 to March 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP and guideline compliant study report. Read across was performed with ethyl red violet chloride.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
N-ethyl-4-[[4-(ethylamino)-m-tolylphenyl][4-(ethylimino)-3-methylcyclohexa-2,5-dien-1-ylidene]methyl]-m-toluidine monoacetate
EC Number:
305-170-2
EC Name:
N-ethyl-4-[[4-(ethylamino)-m-tolylphenyl][4-(ethylimino)-3-methylcyclohexa-2,5-dien-1-ylidene]methyl]-m-toluidine monoacetate
Cas Number:
94349-52-7
Molecular formula:
C28H38N3.C2H3O2
IUPAC Name:
[4-[bis[4-(ethylamino)-3-methyl-phenyl]methylene]-2-methyl-cyclohexa-2,5-dien-1-ylidene]-ethyl-ammonium;acetate
Details on test material:
- Name of test material (as cited in study report): Ethylrotviolett-Chlorid
- Physical state: powder
- Analytical purity: 83.7 %
- Lot/batch No.: Vers. M 94-153
- Stability under test conditions: The stability of the test substance in water and DMSO has been verified analytically.
- Storage condition of test material: at room temperature

Method

Species / strain
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM (Minimal Essential Medium incl. glutamine), supplemented with 10 % FCS (Fetal Calf Serum) and antibiotics.
- Properly maintained: yes, Stocks of the V79 cell line (1 mL portions) were maintained at -196 °C in liquid nitrogen using 7 % DMSO in culture medium as a cryoprotectant.
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (Aroclor 1254 induced rat liver)
Test concentrations with justification for top dose:
1st experiment:
0.1, 0.2, 0.4, 0.6, 0.8 µg/mL without S9 mix (harvest time 18 hours)
2.0, 3.0, 4.0, 5.0, 6.0 µg/mL with S9 mix (harvest time 18 hours)

2nd experiment:
0.4, 0.6, 0.8 µg/mL without S9 mix (harvest time 18 hours)
0.8 µg/mL without S9 mix (harvest time 28 hours)
3.0, 4.0, 5.0 µg/mL with S9 mix (harvest time 18 hours)
5.0 µg/mL with S9 mix (harvest time 18 hours)
Vehicle / solvent:
- Vehicle used: aqueous culture medium (MEM)
- Justification for choice of vehicle: Good solubility of the test substance in water.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
CPP
Positive control substance:
cyclophosphamide
Remarks:
with S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
EMS
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9 mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
CV
Positive control substance:
other: Basic violet 3
Remarks:
with and without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Treatment of the test cultures
About 24 - 30 hours after seeding and incubating the cells the medium was replaced by fresh medium. The test article, dissolved in 1 mL medium with FCS, was added to the culture medium with or without 1 mL S9 mix. Concurrent negative and positive controls were tested in parallel. After incubation < 37 °C, 5 % CO2, >90 % humidity) for 4 hours the medium was replaced after rinsing twice with Hanks balanced salt solution (HBSS). Subsequently, the Quadriperm dishes were incubated again for another 14 hours or 24 hours until the cells were harvested.

Cell harvest and preparation of metaphase spreads
2 - 3 hours prior to harvesting the cells, 0.2 µg Colcemid/mL culture medium (= 1 µg Colcemid dissolved in 0.1 mL PBS/culture) was added in each chamber in order to arrest mitosis in the metaphase. After incubation at 37 °C the culture medium was completely removed. For hypotonic treatment 5 mL of a 0.4 % KCl solution which was at 37 °C was added for about 20 minutes. Subsequently 5 mL of fixative (methanol : glacial acetic acid/3 : 1) which was at 4 °C was added and kept for at least 15 minutes and then replaced. After about another 10 minutes fixative was replaced again and kept for at least 5 minutes at room temperature for complete fixation. The slides were taken out of the Quadriperm chambers, briefly dripped off and then rapidly passed through a Bunsen burner flame. The preparations were dried in the air and subsequently stained in a solution of Giemsa and Titrisol (15 mL Giemsa, 185 mL Titrisol pH 7.2) for 10 minutes. After being rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: the first 100 consecutive well-spread metaphases of each culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
Evaluation criteria:
As a rule, the first 100 consecutive well-spread metaphases of each culture were counted for all test groups and if cells had 20 - 22 chromosomes, they were analyzed for chromosome aberrations, i.e. structural chromosome aberrations and numerical chromosome aberrations (so-called heteroploidies).
Statistics:
The statistical evaluation of the data was carried out using the MUCHAN program System (BASF AG). For each group the proportion of metaphases with aberrations was calculated. A comparison of each dose group with the vehicle control group was carried out using Fisher`s exact test for the hypothesis of equal proportions. This test was Bonferroni-Holm corrected over the dose groups separately for each time point and was performed one-sided.

Results and discussion

Test results
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The doses for the main experiments (18 hours sampling time) were determined from appropriate pretests with cultures exposed for the duration of 4 hours to a wide dose range of the test article, i.e. 0.16 µg/mL - 8.0 µg/mL culture medium without S9 mix and 0.16 µg/mL - 10.0 µg/mL culture medium after adding a metabolizing system. In the course of this, various parameters were checked for all or at least for some selected doses. As a rule the highest dose/concentration for non-toxic test substances should not exceed a limit of 5 mg/mL as recommended by the EEC Directive 92/69 or 10 mM as recommended by the OECD and by an ICPEMC Task Group. According to the findings of the pretests, 0.6 µg/mL without S9 mix and 5.0 µg/mL with metabolic activation were selected as top doses. This selection was based on a reduction of the mitotic index, a decrease in the cell count, on the quality of chromosomes and partly on the observed cell attachment.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
For determination of cytotoxicity additional cell cultures (using 25 cm2 plastic flasks) were treated in the same way as in the main experiment. Growth inhibition was estimated by counting the number of cells in the dose groups in comparison to the concurrent vehicle control at the end of the culture period using a counting chamber. According to the results, a growth inhibition was observed under most of the experimental conditions. Please refer to Table 2 under "any other results incl. tables"
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1 Results

Experiment

Exposure period (Sampling time) [h]

Dose (µg/mL)

S9 mix

Prec.

Aberrations (%)

1

4 (18)

Negative control

-

-

0.5

 

 

0.4

-

-

0.5

 

 

0.6

-

-

1.5

 

 

0.8

-

-

2.0

 

 

Positive control (EMS)

-

-

16.0

 

 

Positive control (CV)

-

-

14.0

 

 

 

 

 

 

2

4 (18)

Negative control

-

-

2.0

 

 

0.4

-

-

2.5

 

 

0.6

-

-

3.0

 

 

0.8

-

-

1.0

 

 

Positive control (EMS)

-

-

12.0

 

4 (28)

Negative control

-

-

3.0

 

 

0.8

-

-

3.0

 

 

 

 

 

 

1

4 (18)

Negative control

+

-

1.5

 

 

3

+

-

1.0

 

 

4

+

-

1.0

 

 

5

+

-

1.5

 

 

Positive control (CPP)

+

-

11.0

 

 

Positive control (CV)

+

-

30.0

 

 

 

 

 

 

2

4 (18)

Negative control

+

-

4.5

 

 

3

+

-

2.0

 

 

4

+

-

0.0

 

 

5

+

-

0.5

 

 

Positive control (CPP)

+

-

10.0

 

4 (28)

Negative control

+

-

2.5

 

 

5

+

-

4.5

Table 2 Cytoxicity

Experiment 1 (18 h)

 

S9 mix

Cytotoxicity

[%]

Negative control

-

100.0

0.4

-

30.6

0.6

-

47.2

0.8

-

36.1

 

 

 

Negative control

+

100.0

3

+

70.5

4

+

50.0

5

+

72.7

Experiment 2 (18h)

 

S9 mix

Cytotoxicity

[%]

Negative control

-

100.0

0.4

-

80.0

0.6

-

68.6

0.8

-

47.1

 

 

 

Negative control

+

100.0

3

+

54.4

4

+

58.8

5

+

61.8

Experiment 2 (28 h)

 

S9 mix

Cytotoxicity

[%]

Negative control

-

100.0

0.8

-

41.5

 

 

 

Negative control

+

100.0

5

+

45.3

Applicant's summary and conclusion