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EC number: 201-494-2 | CAS number: 83-67-0
- 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 vivo
Administrative data
- Endpoint:
- in vivo mammalian cell study: DNA damage and/or repair
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- significant methodological deficiencies
- Remarks:
- Test method similar to EPA OPPTS 870.5915 In vivo Sister Chromatid Exchange. No data on GLP
Data source
Reference
- Reference Type:
- publication
- Title:
- Unnamed
- Year:
- 1 999
Materials and methods
Test guideline
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- other: EPA OPPTS 870.5915 In Vivo Sister Chromatid Exchange Assay
- Deviations:
- yes
- Remarks:
- animals are exposed to BdrU and then exposed to the chemical. 5 males are used.
- Principles of method if other than guideline:
- Paraffin-coated (approximately 80% of the surface) BrdU tables (50mg each) were implanted subcutaneously following the methodology of McFee et al.,and Sharief et al. for in vivo SCE study and cell replication kinetic analysis. Theobromine was injected as single intraperitoneal injection 1 hour after the tablet implantation. Colchicine (4mg/kg) was injected i.p. 22 hours after BdrU implantation. Two hours after, bone marrow cells were obtained,fixed and prepared with fluorescence-plus Giemsa to score SCE frequencies.
- GLP compliance:
- not specified
- Type of assay:
- sister chromatid exchange assay
Test material
- Reference substance name:
- Theobromine
- EC Number:
- 201-494-2
- EC Name:
- Theobromine
- Cas Number:
- 83-67-0
- Molecular formula:
- C7H8N4O2
- IUPAC Name:
- theobromine
- Details on test material:
- - Name of test material (as cited in study report): Theobromine
Constituent 1
Test animals
- Species:
- mouse
- Strain:
- other: Swiss albino
- Sex:
- male
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Age at study initiation: 10-12 weeks old
- Weight at study initiation: 25-30 g- Housing: They were kept five per cage with husk bedding.
- Diet (e.g. ad libitum): standard rodent pellet diet Gold Mohar, Lipton India, Chandigarh, India.
- Water (e.g. ad libitum): yes
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 28±2ºC
- Humidity (%): 60±5%
- Photoperiod (hrs dark / hrs light): Light cycle was 12 h light and 12 h dark.
Administration / exposure
- Route of administration:
- intraperitoneal
- Vehicle:
- - Vehicle(s)/solvent(s) used: DMSO
- Details on exposure:
- TEST MATERIAL
- Concentration: 12.5 mg/kg, 25 and 50 mg/kg - Duration of treatment / exposure:
- 23 hours.
- Frequency of treatment:
- Once per animal.
Doses / concentrationsopen allclose all
- Dose / conc.:
- 12.5 mg/kg bw/day (nominal)
- Dose / conc.:
- 25 mg/kg bw/day (nominal)
- Dose / conc.:
- 50 mg/kg bw/day (nominal)
- No. of animals per sex per dose:
- 5 males per dose.
- Control animals:
- yes, concurrent vehicle
- not specified
- Positive control(s):
- Mitomycin-C (1.5 mg/kg)- Route of administration: injection- Doses / concentrations: 1.5 mg/kg
Examinations
- Tissues and cell types examined:
- In vivo sister chromatid exchanges (SCEs) in bone marrow cells of mice.
- Details of tissue and slide preparation:
- TREATMENT AND SAMPLING TIMES:For SCE analysis, colchicine (4 mg/kg) was injected i.p.. 22 h after BrdU tablet implantation. Two hours later, bone marrow was expelled with 0.075 M KCl solution. After hypotonic treatment (0.075 M KCl at 37ºC) for 20 min, cells were fixed three times with methanol:acetic acid (3:1).
DETAILS OF SLIDE PREPARATION:The slides were prepared and chromosomes were differentially stained with fluorescence-plus-Giemsa technique. All the slides were coded and 30 division metaphase cells (40±2 chromosomes) per animal were scored for SCE frequencies, i.e., a total of 150 cells were scored from five animals per dose tested. - Statistics:
- Results of the in vivo sister chromatid exchanges assay were analysed using an analysis of variance test statistics with Dunnett’s multiple comparisontest with control. Trend test for the evidence of dose response effects was carried out following the method of Margolin et al..
Results and discussion
Test results
- Key result
- Sex:
- male
- Genotoxicity:
- positive
- Toxicity:
- not examined
- Negative controls validity:
- valid
- Positive controls validity:
- valid
Any other information on results incl. tables
A significant increase in sister chromatid exchange was observed in all the three doses tested. Trend test for the evidence of dose response effects was also significant. No significant changes in replicative indices were observed for any of the three doses tested for any of the drugs. Positive control compound mitomycin-C induced very high frequencies of sister chromatid exchange over those of solvent control and treated groups.
Table 1. Frequencies of sister chromatid exchanges induced by theobromine in vivo in bone marrow cells of mice
Chemicals (mg/kg) |
Sister chromatid exchange/cell of five animals |
Sister chromatid exchange/cell (mean ± S.D.)a |
Regression coefficient ± S.E. |
Replicative indices (mean± S.D.)a |
Solvent control(75µl of DMSO) |
3.4, 3.5, 4.9, 4.7, 4.2 |
4.1± 0.6 |
|
1.91± 0.07 |
Theobromine (mg/kg) |
||||
12.5 |
4.7, 5.9, 5.4, 6.1, 5.3 |
5.5± 0.5* |
|
1.90± 0.06 |
25 |
5.8, 6.3, 6.4, 6.9, 6.7 |
6.4± 0.4* |
0.057± 0.008* |
1.93± 0.05 |
50 |
6.9, 7.3, 7.6, 6.5, 7.4 |
7.2± 0.4* |
|
1.95± 0.08 |
Positive control |
||||
Mitomycin-C (1.5 mg/kg) |
18.3, 19.8, 23.0, 25.6, 21.3 |
21.6± 2.7 |
|
1.89± 0.10 |
aMean ± S.D. of five animals (30 cells per animal). Results of each concentration were compared with the solvent control by Dunnett’s multiple comparison with control.
*p<0.01.
Applicant's summary and conclusion
- Conclusions:
- Theobromine is able to increase sister chromatid exchanges in a dose related manner in bone marrow cells after an in vivo exposure from concentrations of 12.5 mg/kg b.w to 50 mg/kg b.w.
- Executive summary:
An in vivo sister chromatid exchanges assay in bone marrow cells of mice was conducted with theobromine. 5 Swiss albino male mice were used for the test per dose group. Paraffin-coated BdrU tablets (approximately 50mg) were implanted subcutaneously in the flank of mice under anaesthesia. Theobromine was administered by intraperitoneal injection at three doses (12.5, 25, 50 mg/kg b.w.) with DMSO as vehicle to different groups of mice 1 hour after the tablet implantation. Negative control mice were injected with 75 µL of DMSO while mitomycin- C was used as a positive control at a dose of 1.5 mg/kg of body weight. Colchicine (4 mg/kg.) was injected i.p. 22 h after BrdU tablet implantation. Two hours later, slides of bone marrow cells were prepared and chromosomes were differentially stained with fluorescence-plus-Giemsa technique. All the slides were coded and 30 division metaphase cells (40±2 chromosomes) per animal were scored for sister chromatid exchange frequencies, i.e., a total of 150 cells were scored from five animals per dose tested. Randomly selected metaphase cells (100/animal) were scored for replicative indices (RI) analysis by their staining pattern as first, second and third division metaphases. Results indicate that theobromine can induce significant increases in sister chromatid exchanges in bone marrow cells of mice after an in vivo exposure from concentrations of 12.5 mg/kg b.w to 50 mg/kg b.w. No significant changes in replicative index were observed for any of the three tested doses.
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