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EC number: 305-170-2 | CAS number: 94349-52-7
- Life Cycle description
- Uses advised against
- Endpoint summary
- Appearance / physical state / colour
- Melting point / freezing point
- Boiling point
- Density
- Particle size distribution (Granulometry)
- Vapour pressure
- Partition coefficient
- Water solubility
- Solubility in organic solvents / fat solubility
- Surface tension
- Flash point
- Auto flammability
- Flammability
- Explosiveness
- Oxidising properties
- Oxidation reduction potential
- Stability in organic solvents and identity of relevant degradation products
- Storage stability and reactivity towards container material
- Stability: thermal, sunlight, metals
- pH
- Dissociation constant
- Viscosity
- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
- Nanomaterial agglomeration / aggregation
- Nanomaterial crystalline phase
- Nanomaterial crystallite and grain size
- Nanomaterial aspect ratio / shape
- Nanomaterial specific surface area
- Nanomaterial Zeta potential
- Nanomaterial surface chemistry
- Nanomaterial dustiness
- Nanomaterial porosity
- Nanomaterial pour density
- Nanomaterial photocatalytic activity
- Nanomaterial radical formation potential
- Nanomaterial catalytic activity
- Endpoint summary
- Stability
- Biodegradation
- Bioaccumulation
- Transport and distribution
- Environmental data
- Additional information on environmental fate and behaviour
- Ecotoxicological Summary
- Aquatic toxicity
- Endpoint summary
- Short-term toxicity to fish
- Long-term toxicity to fish
- Short-term toxicity to aquatic invertebrates
- Long-term toxicity to aquatic invertebrates
- Toxicity to aquatic algae and cyanobacteria
- Toxicity to aquatic plants other than algae
- Toxicity to microorganisms
- Endocrine disrupter testing in aquatic vertebrates – in vivo
- Toxicity to other aquatic organisms
- Sediment toxicity
- Terrestrial toxicity
- Biological effects monitoring
- Biotransformation and kinetics
- Additional ecotoxological information
- Toxicological Summary
- Toxicokinetics, metabolism and distribution
- Acute Toxicity
- Irritation / corrosion
- Sensitisation
- Repeated dose toxicity
- Genetic toxicity
- Carcinogenicity
- Toxicity to reproduction
- Specific investigations
- Exposure related observations in humans
- Toxic effects on livestock and pets
- Additional toxicological data
Genetic toxicity: in vitro
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:
- 1 995
- 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
- 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
Constituent 1
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
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.