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EC number: 226-408-0 | CAS number: 5395-50-6
- 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:
- key study
- Study period:
- 2001 - 2002
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 002
- Report date:
- 2002
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Version / remarks:
- 1997
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- other: Micronucleus assay in bone marrow cells
Test material
- Reference substance name:
- Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione
- EC Number:
- 226-408-0
- EC Name:
- Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione
- Cas Number:
- 5395-50-6
- Molecular formula:
- C8H14N4O6
- IUPAC Name:
- 1,3,4,6-tetrakis(hydroxymethyl)-octahydro-[1,3]diazolo[4,5-d]imidazole-2,5-dione
- Test material form:
- liquid
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source and batch No.of test material: BASF AG and THOR GmbH and batch no.: 0767
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature
- Stability of the test substance in the solvent: The stability of the test substance in water was determined analytically and may be requested from the sponsor.
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: On the day of the experiment, the test substance was formulated in deionised water.
- Purity: 49.5 g / 100 g (aqueous solution)
Test animals
- Species:
- mouse
- Strain:
- NMRI
- Details on species / strain selection:
- The mouse has been used for many years as suitable experimental animal. Thus, many data are available which may be helpful in the interpretation of the results from the micronucleus test.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: RCC Ltd., Biotechnology and Animal Breeding Division; CH-4414 Füllingsdorf
- Age at study initiation: 8 -10 weeks
- Weight at study initiation: mean weight males: 39.6 +/- 3.3 g and females: 31.7 +/- 2.0 g
- Assigned to test groups randomly: yes
- Housing: individually
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: minimum five days
- number of animals: 84 (42 males and 42 females)
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 4
- Relative humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12
Administration / exposure
- Route of administration:
- intraperitoneal
- Vehicle:
- deioised water
The vehicle was chosen to its relative non-toxicity for the animals. - Details on exposure:
- - Single injection intraperitoneally
- Dose volume: 10 mg/kg bw - Duration of treatment / exposure:
- - single expsoure
- 24 h and additionally 48 h for the vehicle control and highest dose
Doses / concentrationsopen allclose all
- Dose / conc.:
- 375 mg/kg bw/day (nominal)
- Remarks:
- male animal
- Dose / conc.:
- 437.5 mg/kg bw/day (nominal)
- Remarks:
- female animal
- Dose / conc.:
- 750 mg/kg bw/day (nominal)
- Remarks:
- male animal
- Dose / conc.:
- 875 mg/kg bw/day (nominal)
- Remarks:
- female animal
- Dose / conc.:
- 1 500 mg/kg bw/day (nominal)
- Remarks:
- male animal
- Dose / conc.:
- 1 750 mg/kg bw/day (nominal)
- Remarks:
- female animal
- No. of animals per sex per dose:
- six males and six females were assigned to each test group and additionally six males and six females for control group and highest dose for the 48 h period. Animals were identified by their cage number as shown in table 1 (any other information).
- Control animals:
- yes, concurrent vehicle
- Positive control(s):
- cyclophosphamide disolved in deionised water
- Route of administration: intraperitoneally, once
- Doses: 40 mg/kg bw
- Dose volume: 10 ml/kg bw
Examinations
- Tissues and cell types examined:
- bone marrow
polychromatic erythrocytes (PCE)
normochromatic erythrocytes (NCE) - Details of tissue and slide preparation:
- CRITERIA FOR DOSE SELECTION:
The active ingredient content of the test substance was originally given by the sponsor as approximately 47%. Therefore, the doses used in this study and reported presently are expressed in terms of this indicated content.
lt is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test substances. The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours. The volume to be administered should be compatible with physiological space available. Three adequate spaced dose leveis spaced by a factor of 2 were applied at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.
TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
At the beginning of the treatment the animais (including the controls) were weighed and the individual volume to be administered was adjusted to the animais body weight. The animais received the test substance, the vehicle or the positive control substance once i.p.. Six males were treated per dose group and sampling time. The animais of the highest dose group were examined for acute toxic symptoms at intervals of around 1 h, 2-4h, 6 h and 24 h after administration of the test substance. Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively.
DETAILS OF SLIDE PREPARATION:
The animais were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf Serum, using a syringe. The ceII Suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supenatant was discarded. A small drop of the resuspended celI pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.
METHOD OF ANALYSIS:
Evaluation of the sildes was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To desoribe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 2000 PCEs. The analysis was performed with coded slides. Five males per test group were evaluated as described. - Evaluation criteria:
- The study was considered valid as the following criteria are met:
- the negative controls are in the range of our historical control data (0.20 - 1.50 %; mean = 0.86 ± 0.32 % PCEs with micronuclei).
- the positive controls are in the range of our historical control data (10.0 - 27.1 %;mean = 16.78±4.79 % PCEs with micronuclei).
- at least 80 % of animals are evaluable
The test substance was classified as mutagenic if it induced either a dose-related increase in the number of micronucleated polychromatic erythrocytes (which clearly exceeds the negative control range) or a relevant positive response for at least one of the test points. Statistical methods can be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test substance producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a positive response at any of the test points is considered non-mutagenic in this system. - Statistics:
- Statistical methods (nonparametric Mann-Whitney test) can be used as an aid in evaluating the results. Statistical significance at the five percent level (p < 0.05) was evaluated. However, the primary point of consideration is the biological relevance of the results.
Results and discussion
Test results
- Key result
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- yes
- Remarks:
- In animals treated with the highest dose
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RESULTS OF RANGE-FINDING STUDY
- Dose range: 1,000 - 2,000 mg/kg
- Clinical signs of toxicity in test animals: clear signs of toxicity in the highest test groups. Reduction of spontaneous activity, abdominal position, eyelid closure, apathy, ruffled fur
RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): affected by treatment (see tables 1 and 2 in any other information on results)
- Statistical evaluation: Mann-Whitney Test
- Appropriateness of dose: yes
Any other information on results incl. tables
In comparison to the corresponding vehicle control there was no substantial enhancement in the frequency of the detected micronuclei at the 24 h preparation interval. The value obtained for the males treated with 1500 mg/kg at the 48 h preparation interval (1 .6 %) was statistically significant. However, this increase is within the historical range for the negative control males (0.0 - 2.1%) and significance was obtained due to the low vehicle control value. Therefore, it is considered as biologically irrelevant.
Historical controls (1999 - 2000)
|
Micronucleated PCE’s per thousand (‰) |
|||
|
Negative controls |
Positive controls |
||
|
Males |
Females |
Males |
Females |
Mean* |
0.76 |
0.62 |
18.70 |
14.54 |
SD** |
0.42 |
0.34 |
5.79 |
5.32 |
range |
0.0 – 2.1 |
0.0 – 1.4 |
9.30 – 34.10 |
7.00 – 35.50 |
*= mean of 62 experiments
Table 1: Summary of micronucleus test results for males
Test group |
Dose [mg/kg bw] |
Sampling time [h] |
PCEs with micronuclei [‰] |
Range |
PCE/NCE |
significance |
P |
Vehicle |
0 |
24 |
0.3 |
0 – 1 |
2000/1587 |
|
|
Test substance |
375 |
24 |
0.2 |
0 – 1 |
2000/1778 |
n.t. |
- |
“ |
750 |
24 |
1.4 |
0 – 6 |
2000/1612 |
- |
0.0873 |
“ |
1500 |
24 |
1.2 |
0 – 4 |
2000/2176 |
- |
0.0873 |
Positive control |
40 |
24 |
15.5 |
12 – 46 |
2000/1419 |
+ |
0.0040 |
vehicle |
0 |
48 |
0.0 |
0 – 0 |
2000/1706 |
- |
- |
Test substance |
1500 |
48 |
1.6 |
2 – 6 |
2000/2564 |
+ |
0.0040 |
- = not significant
+ = significant (p < 0.05)
n.t. = not tested, as the mean micronucleus frequency was not above the vehicle control value
Table 2: Summary of micronucleus test results for females
Test group |
Dose [mg/kg bw] |
Sampling time [h] |
PCEs with micronuclei [‰] |
Range |
PCE/NCE |
significance |
P |
Vehicle |
0.0 |
24 |
0.8 |
0 – 3 |
2000/1288 |
|
|
Test substance |
437.5 |
24 |
0.4 |
0 – 2 |
2000/1615 |
n.t. |
- |
“ |
875.0 |
24 |
0.5 |
0 – 3 |
2000/1854 |
n.t. |
- |
“ |
1750.0 |
24 |
0.9 |
1 – 2 |
2000/2335 |
- |
0.5000 |
Positive control |
40.0 |
24 |
16.5 |
27 – 39 |
2000/1894 |
+ |
0.0040 |
vehicle |
0.0 |
48 |
0.4 |
0 – 2 |
2000/1572 |
- |
- |
Test substance |
1750.0 |
48 |
0.6 |
1 – 2 |
2000/2191 |
- |
0.3571 |
- = not significant
+ = significant (p < 0.05)
n.t. = not tested, as the mean micronucleus frequency was not above the vehicle control value
Applicant's summary and conclusion
- Conclusions:
- In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow celIs of the mouse. Therefore, the test substance is considered to be non-clastogenic and non-aneugenic in this micronucleus assay.
- Executive summary:
This study was performed to investigate the potential of the test substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.
The test substance was formulated in deionised water, which was also used as vehicle control. The volume administered intraperitoneally (i.p.) was 10 mI/kg b.w.. 24 h and 48 h after a single i.p. administration of the test substance the bone marrow cells were collected for micronuclei analysis. Five males or five females per test group were evaluated for the occurrence of micronuclei. At least 2000 polychromatic erythrocytes (PCEs) per animal were scored for micronuclei.
To describe a cytotoxic effect due to the treatment with the test substance the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCEs per 2000 PCEs. The test substance is an aqueous solution. The active ingredient content of the test substance was originally given by the sponsor as approximately 47%. Therefore, the doses used in
this study are expressed in terms of this indicated content. The exact value of the test substance purity is, however, 49.5 g / 100 g as indicated later by the sponsor.
The following dose levels of the test substance were investigated:
males:
24 h preparation interval: 375, 750, and 1500 mg/kg b.w..
48 h preparation interval: 1500 mg/kg b.w..
females:
24 h preparation interval: 437.5, 875, and 1750 mg/kg b.w..
48 h preparation interval: 1750 mg/kg b.w..
The highest dose (1500 mg/kg b.w. for males and 1750 m g/kg b.w. for females) was estimated by pre-experiments to be suitable.
After treatment with the highest dose of the test substance the number of NCEs was increased as compared to the mean value of NCEs of the vehiole controls thus indicating that the test substance had cytotoxic effectiveness in the bone marrow.
In comparison to the corresponding vehicle controls there was no substantial enhancement in the frequency of the detected micronuclei at any preparation interval after administration of the test substance and with any dose level used. 40 mg/kg b.w. cyclophosphamide administered intraperitoneally (i.p.) was used as positive control which showed a substantial increase of induced micronucleus frequency.
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