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EC number: 224-764-1 | CAS number: 4482-25-1
- 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 cell study: DNA damage and/or repair
- Remarks:
- Type of genotoxicity: DNA damage and/or repair
- Type of information:
- other: read across from analogue substance
- Adequacy of study:
- key study
- Study period:
- 15. Jul 1996 - 16. Oct 1996
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- guideline study with acceptable restrictions
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 000
- Report date:
- 2000
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
- GLP compliance:
- yes
- Type of assay:
- unscheduled DNA synthesis
Test material
- Reference substance name:
- refer to read across section
- IUPAC Name:
- refer to read across section
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Karl Thomae GmbH, Biberach an der Riss, Germany.
- Weight at study initiation: 254g (mean)
- Housing: Makrolon cages, type III
- Diet: Standardized pelleted feed (Kliba-Haltungsdiät/Provimi Kliba SA, Kaiseraugst, Switzerland) was available ad libitum.
- Water: drinking water from bottles was available ad libitum.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 °C
- Humidity (%): 30-70%
- Photoperiod (hrs dark / hrs light): 12 hours light from 6.00 - 18.00 hours and 12 hours darkness from 18.00 - 6.00 hours). During the time between treatment and perfusion the day/night rhythm was not followed.
Administration / exposure
- Route of administration:
- oral: unspecified
- Vehicle:
- - Vehicle/solvent used: CMC (carboxymethyl cellulose).
- Justification for choice of solvent/vehicle: Due to the Iimited solubility of the test substance in water, a 0.5% CMC formulation was selected as the vehicle.
- Concentration of test material in vehicle: Test group 3 and 4: 5g/100ml; Test group 5 and 6: 10g/100ml - Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: To achieve homogeneity of the test substance in the vehicle, the test substance formulation was stirred constantly during removal and administration.
- Duration of treatment / exposure:
- 12 and 18h
- Frequency of treatment:
- The treatment consisted of a single oral administration.
Doses / concentrationsopen allclose all
- Remarks:
- Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.
dose group 3 and 4
- Remarks:
- Doses / Concentrations:
1000 mg/kg
Basis:
nominal conc.
dose group 5 and 6
- No. of animals per sex per dose:
- 3
- Control animals:
- yes, historical
- Positive control(s):
- 2-acetylaminofluorene;
- Justification for choice of positive control(s): 2-acetylaminofluorene is a well-established UDS-inducing agent.
- Route of administration: oral
- Doses / concentrations: 50 mg/kg
Examinations
- Tissues and cell types examined:
- Isolated primary rat hepatocytes.
- Details of tissue and slide preparation:
- DETAILS OF SLIDE PREPARATION: Culture conditions (seeding and attachment period):
The isolated hepatocytes were seeded on coverslips on 1.9 cm² well containing 2 ml of attachment medium (WMEC).
- about 400,000 viable cells were seeded per well.
- 6 wells/per animal were used for the UDS assay.
After an attachment period of about 2 hours with 5% CO2 at 37°C and > 90% humidity, the medium (WMEC) was replaced by fresh medium (WMF 1) to remove non-adherent celIs.
Labeling
The medium (WMEI) was replaced by 2 ml labeling medium, and the cells were incubated with 5% CO2 at 37°C and > 90% humidity for 4 hours. After the labeling period, cells were washed with HBSS or WMEI (about 37°C); then fresh medium containing 0.25 mM unlabeled thymidine was added and the cells were incubated for another 14 hours. The cells on the coverslips were then fixed with ethanol/acetic acid (3 :1, v/v) for at least 30 minutes, rinsed 2 - 4 times with aqua dest. and air-dried. The dried coverslips were mounted ceIl side up on glass slides using Corbit-Balsam and dried overnight.
Autoradiography:
The slides were coated with KODAK NTB-2 photographic emulsion (about 37°C) for about 5 - 10 seconds. After drying at room temperature in the dark (Iightproof boxes) for about 16 hours, the coated slides were started in the dark with a desiccant (in the presence of a drying agent) at -20°C for 2 -10 days. Thereafter, the slide boxes were left at room temperature for at least 3 hours. The photographic emulsion was then developed with KODAK D-19 (about 15°C), fixed in KODAK Acidofix for about 5 minutes, washed in water for about 5 -10 minutes and stained with methyl green-pyronine Y. After rinsing with water and ethanol and air-drying, the slides were covered with a second coverslip using Corbit-Balsam.
METHOD OF ANALYSIS:
Quantification of UDS/Microscopic evaluation:
The quantification of UDS was performed microscopically generally using 3 slides per test group. 25 - 50 cells in good morphological conditions were randomly selected per slide and examined to achieve a total number of 100 cells/animal. For each celI, the following counts were performed with an automatic image analyzer (ARTEK):
- the nuclear grain (NG) count (= number of silver grains overlying the nucleus)
- the cytoplasmic grain (GG) count (= number of grains in two or three nucleusequivalent areas adjacent to the nucleus).
The following parameters were calculated:
- the net nuclear grain (NNG) count of each cell (= nuclear grain count minus
cytoplasmic grain count; NG - GG)
- the mean nuclear grain (NG) count
- the mean cytoplasmic grain (GG) count
- the mean net nuclear grain (NNG) count
- the percentage of cells in repair (cells showing net nuclear grain counts of > 0)
- the percentage of cells in repair (cells showing net nuclear grain counts of > 5)
Slides were coded before microscopic evaluation. - Evaluation criteria:
- The test substance was considered positive if a dose-related increase was demonstrated in both of the following:
- The mean number of net nuclear grain (NNG) counts, which must exceed zero at one of the test points.
- The percentage of cells in repair (NNG > 5) when > 20.
A dose-related increase in % cells in repair > 5 outside the values of both the concurrent negative control and the historical control data base (> 5 < 20) and a dose-related increase in the mean number of NNG counts near to but without exceeding zero is considered to be an indication for a marginal response which needs to be confirmed/clarified in a further experiment. A test article producing both NNG counts and % cells in repair in the range of the negative control data is considered to be negative in the in vitro UDS assay. - Statistics:
- Due to very clear negative findings, a statistical evaluation was not carried out.
Results and discussion
Test results
- Sex:
- male
- Genotoxicity:
- negative
- Remarks:
- (500 mg/kg and 1000 mg/kg)
- Toxicity:
- no effects
- Remarks:
- no cytotoxicity noted (trypan blue exclusion technique)
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- After the single oral administration of the test substance preparation, all animals showed symptoms in form of:
-piloerection
-orange discoloration of for- and hindlimbs, tail and ears
-yellow to brownish discoloration of the lymphatic organs, the adiposal tissue and the liver
-anogenital region smeared with feces and test substance
The urine of the animals was discolored orange/darkbrown.
Furthermore, 5 out of 11 animals in the 1,000 mg/kg group died.
Any other information on results incl. tables
DNA repair activity
Perfusion 12 hours after treatment (mean of 3 animals ± standard deviation)
Test Group | Vehicle control | 500 mg/kg | 1000 mg/kg | positive control | |||
NG counts | 6.53± 1.84 | 7.06 ±0.70 | 8.19±1.89 | 18.11 ±4.44 | |||
CG counts | 10.17±2.04 | 10.73±0.77 | 10.41 ±1.26 | 11.23 ±1.60 | |||
NNG counts | -3.64 ±0.47 | -3.67 ±0.62 | -2.21 ±2.45 | 6.88 ±2.88 | |||
% cells in repair NGG > 0 |
9.33 ± 4.51 | 7.00 ± 4.36 | 20.00 ± 18.19 | 78.67 ±7.57 | |||
% cells in repair NGG > 5 |
0.00 ± 0.00 | 0.00 ± 0.00 | 5.67 ± 8.96 | 54.00 ±11.53 | |||
NG = nuclear grains
CG = cytoplasmic grains
NNG = net nuclear grains
Perfusion 18 hours after treatment (mean of 3 animals ± standard deviation)
Test Group | Vehicle control | 500 mg/kg | 1000 mg/kg | positive control | |||
NG counts | 6.35 ± 0.36 | 6.54 ± 0.54 | 7.48±0.88 | 20.43 ± 1.63 | |||
CG counts | 11.08 ±0.30 | 9.90 ± 0.85 | 10.94±1.16 | 11.88 ±0.56 | |||
NNG counts | -4.73 ±0.65 | -3.36 ± 0.59 | -3.45 ±0.53 | 8.56 ±1.34 | |||
% cells in repair NGG > 0 |
7.67 ± 4.04 | 15.00 ± 4.00 | 14.67 ±4.73 | 91.67 ±4.04 | |||
% cells in repair NGG > 5 |
0.00 ± 0.00 | 0.33 ±0.58 | 0.67 ±0.58 | 65.67 ±9.50 | |||
NG = nuclear grains
CG = cytoplasmic grains
NNG = net nuclear grains
mean per group (mean of 3 animals)
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results (migrated information): negative
The analogue substance 2 was tested for in-vivo mutagenicity following OECD 486 (UDS Assay). Under the experimental conditions the substance did not cause a relevant increase in unscheduled DNA synthesis, as measured by an increase in net nuclear grain counts, and it was concluded that the test substance was negative in this in vivo assay using primary rat hepatocytes. - Executive summary:
The analogue substance 3 was tested for in-vivo mutagenicity following OECD 486 (UDS Assay). Under the experimental conditions the substance did not cause a relevant increase in unscheduled DNA synthesis, as measured by an increase in net nuclear grain counts, and it was concluded that the test substance was negative in this in vivo assay using primary rat hepatocytes.
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