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EC number: 600-039-9 | CAS number: 10023-48-0
- 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 gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 09 March - 12 April 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 guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
Test material
- Reference substance name:
- 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-[2-(hydrogen phosphonatooxy)ethyl]-4-methyl-1,3-thiazol-3-ium
- EC Number:
- 600-039-9
- Cas Number:
- 10023-48-0
- Molecular formula:
- C12H17N4O4PS
- IUPAC Name:
- 3-[(4-amino-2-methylpyrimidin-5-yl)methyl]-5-[2-(hydrogen phosphonatooxy)ethyl]-4-methyl-1,3-thiazol-3-ium
- Test material form:
- solid
Constituent 1
Method
- Target gene:
- HPRT
Species / strain
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- - Type and identity of media: MEM
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
- Metabolic activation:
- with and without
- Metabolic activation system:
- co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital and β-naphtoflavone.
- Test concentrations with justification for top dose:
- 4 hours treatment with and without S9 mix: 132.0, 263.0, 526.0, 1053.0, and 2105.0 µg/mL
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
Controlsopen allclose all
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 7,12-dimethylbenzanthracene
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hours with and without metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days
SELECTION AGENT (mutation assays): 6-Thioguanine
NUMBER OF REPLICATIONS: 2
NUMBER OF CELLS EVALUATED: > 1.5 x 10 exp. 6
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency - Evaluation criteria:
- A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concen¬trations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95% confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). - Statistics:
- A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were considered together.
Results and discussion
Test results
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not applicable
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not affected (pH 7.68 in the solvent control versus pH 7.31 at 2105.0 µg/mL).
- Effects of osmolality: No relevant increase (465 mOsm in the solvent control versus 501 mOsm at 2105.0 µg/mL).
- Precipitation: Precipitation did not occur.
RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20% relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation or phase separation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 16.4 µg/mL and 2105 µg/mL were used. The highest concentration was chosen with respect to the current OECD Guideline 476 regarding the purity of the test item (95%).
In the pre-experiment no relevant toxic effects were observed after 4 hours treatment up to the maximum concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. No precipitation or phase separation occurred up to the maximum concentration with and without metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The concentrations used in the main experiment were selected based on the data of the pre-experiment. Again, the maximum concentration was 2105 µg/mL. The individual concentrations were spaced by a factor of 2.
COMPARISON WITH HISTORICAL CONTROL DATA: Complies
ADDITIONAL INFORMATION ON CYTOTOXICITY:
No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation.
Any other information on results incl. tables
Summary Table
conc. µg/mL | S9 mix |
relative CE I |
relative cell density |
rel. adj. CE I | mutant colonies per 106 cells |
95% conf. interval |
|
Culture I |
|
|
|
|
|||
Solvent control (DMSO) | - | 100.0 | 100.0 | 100.0 | 26.0 | 1.7 - 30.2 | |
Positive control (EMS) | 300.0 | - | 112.4 | 89.6 | 100.7 | 293.3 | 1.7 - 30.2 |
Test Item |
65.8 | - | 124.9 | 100.9 | 126.1 | # | 1.7 - 30.2 |
Test Item |
132.0 |
- |
109.3 |
102.5 |
112.1 |
33.5 |
1.7 - 30.2 |
Test Item |
263.0 |
- |
102.6 |
81.0 |
83.1 |
36.4 |
1.7 - 30.2 |
Test Item |
526.0 |
- |
97.6 |
101.8 |
99.4 |
20.6 |
1.7 - 30.2 |
Test Item |
1053.0 |
- |
111.8 |
104.3 |
116.6 |
28.3 |
1.7 - 30.2 |
Test Item |
2105.0 |
- |
117.6 |
111.1 |
130.7 |
21.4 |
1.7 - 30.2 |
|
|
|
|
|
|
|
|
Solvent control (DMSO) |
|
+ |
100.0 |
100.0 |
100.0 |
24.1 |
2.0 - 29.4 |
Positive control (DMBA) | 2.3 | + | 107.3 | 95.9 | 102.9 | 82.4 | 2.0 - 29.4 |
Test Item | 65.8 | + | 107.6 | 86.9 | 93.6 | # | 2.0 - 29.4 |
Test Item | 132.0 | + | 90.0 | 86.4 | 77.8 | 21.8 | 2.0 - 29.4 |
Test Item | 263.0 | + | 98.6 | 127.1 | 125.3 | 21.3 | 2.0 - 29.4 |
Test Item | 526.0 | + | 100.7 | 97.4 | 98.1 | 25.1 | 2.0 - 29.4 |
Test Item | 1053.0 | + | 109.8 | 89.3 | 98.1 | 16.7 | 2.0 - 29.4 |
Test Item | 2105.0 | + | 96.2 | 84.2 | 81.0 | 32.3 | 2.0 - 29.4 |
conc. µg/mL | S9 mix |
relative CE I |
relative cell density |
rel. adj. CE I | mutant colonies per 106 cells |
95% conf. interval |
|
Culture II |
|
|
|
|
|||
Solvent control (DMSO) | - | 100.0 | 100.0 | 100.0 | 23.5 | 1.7 - 30.2 | |
Positive control (EMS) | 300.0 | - | 84.1 | 114.9 | 96.6 | 292.4 | 1.7 - 30.2 |
Test Item |
65.8 | - | 82.3 | 95.5 | 78.6 | # | 1.7 - 30.2 |
Test Item | 132.0 | - | 78.5 | 131.4 | 103.2 | 11.3 | 1.7 - 30.2 |
Test Item | 263.0 | - | 91.4 | 109.1 | 99.8 | 14.1 | 1.7 - 30.2 |
Test Item | 526.0 | - | 57.9 | 75.4 | 43.7 | 8.7 | 1.7 - 30.2 |
Test Item | 1053.0 | - | 66.7 | 89.5 | 59.7 | 11.8 | 1.7 - 30.2 |
Test Item | 2105.0 | - | 93.0 | 119.6 | 111.2 | 8.2 | 1.7 - 30.2 |
Solvent control (DMSO) | + | 100.0 | 100.0 | 100.0 | 20.7 | 2.0 - 29.4 | |
Positive control (DMBA) | 2.3 | + | 106.0 | 77.9 | 82.6 | 58.0 | 2.0 - 29.4 |
Test Item | 65.8 | + | 122.3 | 78.4 | 95.9 | # | 2.0 - 29.4 |
Test Item | 132.0 | + | 97.2 | 87.4 | 84.9 | 10.9 | 2.0 - 29.4 |
Test Item | 263.0 | + | 111.7 | 87.5 | 97.7 | 30.9 | 2.0 - 29.4 |
Test Item | 526.0 | + | 111.2 | 78.6 | 87.4 | 9.5 | 2.0 - 29.4 |
Test Item | 1053.0 | + | 121.6 | 73.9 | 89.8 | 16.6 | 2.0 - 29.4 |
Test Item | 2105.0 | + | 108.0 | 83.4 | 90.1 | 15.4 | 2.0 - 29.4 |
CE = Cloning Efficiency
# culture was not continued as a minimum of only four analysable concentrations is required
DMBA: 7,12-dimethylbenzanthracene
EMS: ethylmethanesulphonate
Applicant's summary and conclusion
- Conclusions:
- Interpretation of results: negative
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