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EC number: 433-460-1 | CAS number: 210880-92-5
- 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:
- 25 Jul - 30 Aug 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2017
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- adopted 2016
- Deviations:
- yes
- Remarks:
- Animals did not receive enriched environments, only males were used (justification was provided)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Certified by Food safety and Consumer Affairs Bureau, Ministry of Agriculture, Forestry and Fisheries, Japan
- Type of assay:
- mammalian erythrocyte micronucleus test
Test material
- Reference substance name:
- -
- EC Number:
- 433-460-1
- EC Name:
- -
- Cas Number:
- 210880-92-5
- Molecular formula:
- C6H8ClN5O2S
- IUPAC Name:
- (E)-N'-[(2-chloro-1,3-thiazol-5-yl)methyl]-N-methyl-N''-nitroguanidine
Constituent 1
Test animals
- Species:
- rat
- Strain:
- other: Crl:CD(SD) (SPF)
- Details on species / strain selection:
- commonly used for micronucleus tests, historical data is available
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Hino Breeding Center, Charles River Laboratories Japan, Inc, Shiga, Japan
- Age at study initiation: 8 weeks
- Weight at study initiation: 302.7-338.4 g
- Housing: plastic cages (W290xD340xH170 mm, Crea Japan Inc., Tokyo, Japan) with shavings (Whiteflake, Charles River Laboratories Japan, Inc.), in groups of maximum three animals
- Diet: Laboratory animal chows (CRF-1, Oriental Yeat Co., Ltd., Tokyo, Japan), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.0 - 24.1
- Humidity (%): 49.6 - 61.0
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- oral: gavage
- Vehicle:
- 0.5% aqueous cremophor (Kolliphor EL) was used as a vehicle due its relative non-mutagenicicty and non-toxicity
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS: test substance was weighed to the approriate amount, ground with pestle and mortar and mixed with the cremophor solution. Constant stirring was achieved using a magnetic stirrer at room temperature. Stability of the test substance in the vehicle was proven for 2 h at room temperature for concentrations of 1 and 250 mg/mL. The dose volume was 10 mL/kg bw. The individual dose volumes were calculated based on body weights taken just before administration.
The positive control cyclophosphamide (CP) was dissolved in purified water (12 mg/mL) and administered once at 5 mL/kg bw (orally via gavage). - Duration of treatment / exposure:
- 2 days
- Frequency of treatment:
- Rats were dosed twice, once per day (24 h in between)
rats that received the positive control were only dosed once, together with the second dosing in animals receiving the test item and solvent control - Post exposure period:
- 24 h
Doses / concentrationsopen allclose all
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Dose / conc.:
- 1 000 mg/kg bw/day (nominal)
- Dose / conc.:
- 2 000 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5 (for 0, 500 and 1000 mg/kg bw/day, and for the positive control), 7 (for 2000 mg/kg bw group)
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- Cyclophosphamide
- Justification for choice of positive control: known mutagen
- Route of administration: orally via gavage
- Doses / concentrations: as described above, CP was solubilized at 12 mg/mL and administered at 5 mL/kg bw
Examinations
- Tissues and cell types examined:
- Bone marrow cells
- Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
No range finding study was conducted but doses were based on an alkaline comet assay in rats. The rats were of the same strain, age and were treated in the way described for this study (including dosing volume, route, frequency). No toxicity differences between the sexes were found and the rats tolerated all doses, up to the highest of 2000 mg/kg bw/day. As this is the limit dose recommended by the guideline, the doses in this experiment were chosen accordingly.
TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Groups of 5 or 7 male rats were dosed twice, approximately 24 h apart, with the vehicle alone (negative control) or the test item (500, 1000 or 2000 mg/kg bw/day). At the end of the observation period (approximately 24 h after the second test item administration), rats were euthanized by carbon dioxide inhalation.
OBSERVATIONS
Clinical signs and mortality were observed immediately, 3 h and 1 day after dosing. Body weights were recorded immediately before each dosing and 1 day after second dosing.
DETAILS OF SLIDE PREPARATION:
Bone marrow cells were washed out from the femurs with 1.0 mL of fetal bovine serum with 25 mM EDTA. After that, the cell suspension was centrifuged and the pellet was resuspended in residual serum and diluted with fetal bovine serum (including 25 mM EDTA). Small portions of this solution were smeared on a glass slide and the slides were fixed with methanol for 5 min within one day after preparation.
METHOD OF ANALYSIS:
The cells were analysed using acridine orange (40 µg/mL) using a fluorescent microscope (B excitation) using a 40-fold objective lens and 15-fold eye lens in a dry system. Erythrocytes with yellowish green fluorescence emitting particles (nuclei) and sharp outlines were identified as micronucleated erythrocytes. Due to the staining, polychromatic erythrocytes (PCEs) appeared bluish-grey and normochromatic erythrocytes (NCEs) appeared red. The incidences of micronucleated cells were scored in 4000 PCEs (per animal) and the incidence of PCEs in 1000 erythrocytes (including PCEs and NCEs) was examined. - Evaluation criteria:
- Acceptability
The test was considered acceptable if
- incidence of PCEs (micronucleated) in the vehicle control group was within the acceptable negative control range from the historical control data
- the positive control induced a significant increase in micronucleated PCEs when compared with the concurrent vehicle control group.
Evaluation
An assay was considered positive if
- there was a statistically significant increase in the incidence of micronucleated PCEs when compared to the solvent or vehicle control
- the increase was dose-related
- the incidence of micronucleated PCEs in the dose group showing the increase exceeds the historical control range. - Statistics:
- To statistically evaluate the incidences of PCEs, Kastenbaum-Bowman tables were used. Levels of significance were p=0.05 and 0.01. In case of an increased incidence, the dose-response relationship was tested with the Cochran-Armitage trend test (one-tailed, levels of significance were p=0.025 and 0.005).
The ratio of PCEs to total erythrocytes and animal body weight changes were evaluated using the t-test. The Student's t-test was used if p >= 0.05 and the F-test and the Welch's t-test was used if p<0.05 in the F-test (all two-tailed, levels of significance p=0.05 and 0.01).
Results and discussion
Test results
- Key result
- Sex:
- male
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- - Clinical signs: no clinical signs were observed in any of the treatment or control groups
- Body weight: Body weight and body weight gain was reduced in treatment groups before second dosing (500 and 1000 mg/kg bw/day) and one day after second dosing (all treatment groups)
- ratio of PCEs: the ratio of PCEs to total erythrocytes was decreased significantly (at all dose levels), a sign for bone marrow toxicity
- Induction of micronuclei: incidences of micronucleated PCEs were similar to vehicle controls for all dose levels and within the range of historical control data. Occassional incidences of slightly elevated increases were considered incidental and not of biological importance.
- Positive control: the positive control induced a statistically significant increase in the number of PCEs with micronuclei.
Historical control data
The historical control data was provided by the testing facility and can be found in Attachment 2 in the attached background material
Any other information on results incl. tables
The study is considered acceptable since
1) the vehicle controls fell within the historical control range,
2) a sufficient number of animals were evaluable and
3) the positive control induced the expected result, confirming the
sensitivity of the assay.
ADME data had already been generated and decrease in % PCEs confirmed target organ exposure.
Applicant's summary and conclusion
- Conclusions:
- The study was performed according to OECD guideline 474 and in compliance with GLP. According to the bone marrow micronucleus assay, there was no evidence of clastogenicity or aneugenicity following oral (gavage) administration of the test substance up to the limit dose of 2000 mg/kg bw/day in male rats.
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