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EC number: 279-919-6 | CAS number: 82205-20-7
- 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 vivo
Administrative data
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1992-03-17 to 1993-01-11
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 1 993
- Report date:
- 1993
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- no
- GLP compliance:
- yes
Test material
- Reference substance name:
- [2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride
- EC Number:
- 259-033-6
- EC Name:
- [2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride
- Cas Number:
- 54229-13-9
- Molecular formula:
- C21H29ClN5O3.Cl
- IUPAC Name:
- [2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2- hydroxypropyl)dimethylammonium chloride
- Test material form:
- solid
- Details on test material:
- see below
Constituent 1
Test animals
- Species:
- mouse
- Strain:
- NMRI
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: F. Winkelmann, Borchen, Germany
- Age at study initiation: 8 - 12 weeks of age
- Weight at study initiation: 28 - 43 g
- Housing: females: in groups of a maximum of three mice; males: individually in Macrolon type I cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least one week
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 1.5
- Humidity (%): 40 -70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: physiological saline
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved in physiological saline solution, stirred with a magnetic mixer during administration and injected intraperitoneally.
- Duration of treatment / exposure:
- test item dose groups: 16, 24 and 48 hours,
negative/positive control: 24 hours - Frequency of treatment:
- once
- Post exposure period:
- no
Doses / concentrations
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- Remarks:
- three test item related experimental dose groups: 16, 24 and 48 hours
- No. of animals per sex per dose:
- 5
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- - Positive control used: cyclophosphamide
- Route of administration: intraperitoneal
- Dosis: 20 mg/kg bw
Examinations
- Tissues and cell types examined:
- bone marrow derived erythrocytes
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. For the results please box "Additional information on results".
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
see Table 1 in box "Any other information on materials and methods incl. tables".
DETAILS OF SLIDE PREPARATION:
Bone marrow was flushed into a tube containing fetal bovine serum and centrifuged (5 min, 1000 rpm)
Air dried smears were automatically stained with an Ames HemaTek Slide Stainer and then destained with methanol, rinsed with deionized water and dried. After drying, the slides were covered with xylene and a cover glass.
METHOD OF ANALYSIS:
Coded slides were evaluated using a light microscope at a magnification of about 1000. Micronuclei appear as stained chromatin particles in the anucleated erythrocytes. Normally, 1000 polychromatic erythrocytes were counted per animal. The incidence of cells with micronuclei was established by scanning the slides in a meandering pattern. Moreover, the ratio of polychromatic to normochromatic erythrocytes was determined (number of normochromatic erythrocytes per 1000 polychromatic ones). Additionally, the number of normochromatic erythrocytes showing micronuclei was also established. - Evaluation criteria:
- A test was considered positive if at any of the intervals, there was a relevant and significant increase in the number of polychromatic erythrocytes showing micronuclei in comparison to the negative control. A test was considered negative if there was no relevant or significant increase in the rate of micronucleated polychromatic erythrocytes at any time. A test was also considered negative if there was a significant increase in that rate which, according to the laboratory’s experience, was within the range of negative controls. A test was considered equivocal if there was an increase of micronucleated polychromatic erythrocytes above the range of attached historical negative controls, provided the increase was not significant and the result of the negative control was not closely related to the data of the respective treatment group.
- Statistics:
- The test item group(s) with the highest mean and the positive control were checked by Wilcoxon’s non parametric rank sum test with respect to the number of polychromatic erythrocytes having micronuclei and the number of normochromatic erythrocytes. A variation was considered statistically significant if its error probability was below 5% and the treatment group figure was higher than that of the negative control.
The rate of normochromatic erythrocytes containing micronuclei was examined if the micronuclear rate for polychromatic erythrocytes was already relevantly increased. In this case, the group with the highest mean was compared with the negative control using the one-sided chi²-test. A variation was considered statistically significant, if the error probability was below 5% and the treatment group figure was higher than that of the negative control. In addition, standard deviations (1s ranges) were calculated for all the means.
Results and discussion
Test results
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Vehicle controls validity:
- valid
- Negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
The selection of the dose was based on a pilot test, in which groups of five animals, including both males and females, were i.p. administered 10 mg/kg, 50 mg/kg, 55 mg/kg, 60 mg/kg, 75 mg/kg and 100 mg/kg bw. The following symptoms were recorded for up to 48 hours, starting at 10 mg/kg bw: apathy, roughened fur, red discoloration of hairless parts of skin, staggering gait, spasm, leaping coloured urine. In addition, 3 of 5 animals died in the 75 mg/kg group and all animals died in the 100 mg/kg bw group. Based on the results, 50 mg/kg bw was considered as 1 MTD for the main test.
RESULTS OF DEFINITIVE STUDY
- Clinical signs: After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. No symptoms were recorded for the control groups.
- Mortality: One of forty treated animals died during the test period due to acute toxicity. No animals died in the control group.
- Induction of micronuclei (for Micronucleus assay): no clastogenic effect, see Table 2
- Ratio of PCE/NCE (for Micronucleus assay): slightly changed, see Table 2
- Statistical evaluation: see Table 2
Any other information on results incl. tables
Table 2: Summary of results of micronucleus test with the test item | |||||||||
experimental groups | Number of evaluated poly-chromatic erythrocytes (PCE) | Number of normo-chromatic erythrocytes per 1000 PCE | micronucleated cells per 1000 | ||||||
normo-chromatic erythrocytes | poly-chromatic erythrocytes | ||||||||
Negative control | 10000 | 811 +/- 208 | 1.2 +/- 1.6 | 1.5 +/- 1.1 | |||||
Test item_16 hours | 10000 | 1321* +/- 319 | 1.3 +/- 1.0 | 1.6 +/- 1.0 | |||||
Test item_24 hours | 10000 | 1088 +/- 549 | 1.3 +/- 1.6 | 1.5 +/- 1.0 | |||||
Test item_48 hours | 10000 | 775 +/- 199 | 0.4 +/- 0.7 | 1.3 +/- 1.2 | |||||
Positive control | 10000 | 557 +/- 231 | 0.4 +/- 1.3 | 12.6* +/- 6.9 | |||||
Concentration: test item: 50 mg/kg bw; positive control: 20 mg/kg bw
* p< 0.01 (tested by non-parametric Wilcoxon ranking test)
Applicant's summary and conclusion
- Conclusions:
- The test item did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse. Therefore, the test item is considered to be non-mutagenic with respect to clastogenicity and aneugenicity in the mammalian erythrocyte micronucleus test.
- Executive summary:
In a NMRI mouse bone marrow micronucleus test conducted in accordance with OECD Guideline 474, 5 mice/sex/treatment group were treated intraperitoneally once with the test item (95.6% purity) at doses of 0 and 50 mg/kg bw. The vehicle used was physiological saline. The animals were sacrificed and bone marrow cells were harvested after 16 hours (test item), 24 hours (negative/positive control and test item) and after 48 hours (test item).
After application the animals showed the following signs of toxicity: apathy, roughened fur, staggering gait, spasm, twitching, difficulty in breathing and orange discoloured urine. The test item was tested at an adequate dose based on a preliminary dose range finding test. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time (16, 24 and 48 hours).
This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenicity.
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