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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
Skin irritation / corrosion
Administrative data
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 20 January 2016 - 22 January 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Test method according to OECD 439. GLP study.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
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
- Test material form:
- solid: particulate/powder
- Remarks:
- migrated information: powder
- Details on test material:
- - Name of test material (as cited in study report): Theobromine- Physical state: White powder.- Analytical purity: 99.8 %- Purity test date: 12/01/2015- Lot/batch No.: M150115C- Expiration date of the lot/batch: 01/2020- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: M150115C
- Expiration date of the lot/batch: 01/2020
- Purity test date: 12/01/2015
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25°C, below 70 RH%)
In vitro test system
- Test system:
- human skin model
- Remarks:
- three-dimensional human epidermis model
- Source species:
- human
- Cell type:
- other: Adult human-derived epidermal keratinocytes
- Cell source:
- other: EPISKIN-SM (Manufacturer: SkinEthic, France, Batch No.:16-EKIN-003, Expiry Date: 25 January 2016)
- Source strain:
- not specified
- Justification for test system used:
- The EPISKIN-SM model has been validated for irritation testing in an international validation study and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be suitable for this study.
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- SKIN DISC PREPARATION
- Procedure used: A disk of epidermis was cut from each skin unit (this involved the maximum area of the disk) using a biopsy punch (supplied as part of the kit). The epidermis was separated with the aid of forceps and both parts (epidermis and collagen matrix) were placed into a tube containing 500 μL acidified isopropanol (one tube corresponded to one well of the assay plate).The capped tubes were thoroughly mixed by using a vortex mixer to achieve a good contact of all of the material and the acidified isopropanol, and then incubated for about two hours at room temperature protected from light with gentle agitation (~150 rpm) for formazan extraction.
TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature (26.1-26.6°C)
- Temperature of post-treatment incubation (if applicable): room temperature
REMOVAL OF TEST MATERIAL AND CONTROLS:
The EPISKIN-SM units were removed and rinsed thoroughly with PBS. The rest of the PBS was removed from the epidermal surface with a pipette (without touching the epidermis).
DYE BINDING METHOD
- Dye used in the dye-binding assay: MTT
- Wavelength:570 nm.
NUMBER OF INDEPENDENT TESTING RUNS / EXPERIMENTS TO DERIVE FINAL PREDICTION:3PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be irritant to skin (Category 2) if the mean tissue viability % is ≤ 50 %
- The test substance is considered to be non-irritant to skin if the mean tissue viability % is > 50 %
- Justification for the selection of the cut-off point(s) if different than recommended in TG 430: According to UN GHS/ CLP Classification - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- yes, concurrent MTT non-specific colour control
- other: acidified isopropanol solution (200 μL/well) was used as blank during cell viability measurements
- Amount/concentration applied:
- TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 10 mg of the test item was applied evenly to the epidermal surface. The amount was sufficient to cover the epidermal surface. The test item was applied in its original form, no formulation was required (although the test item was ground to a fine powder).
NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 μL of negative control Phosphate Buffered Saline
POSITIVE CONTROL
- Concentration (if solution):5% (w/v) Sodium Dodecyl Sulphate solution - Duration of treatment / exposure:
- 15 minutes (± 0.5 min)
- Duration of post-treatment incubation (if applicable):
- 42 hours (± 1h)
- Number of replicates:
- In this assay, three replicates were used for the test item. As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation.
Results and discussion
In vitro
Resultsopen allclose all
- Irritation / corrosion parameter:
- other: Cell viability (%)
- Run / experiment:
- 1
- Value:
- 82
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Compared to the Negative control, both being blank corrected
- Irritation / corrosion parameter:
- other: Cell viability (%)
- Run / experiment:
- 2
- Value:
- 79.1
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Compared to the Negative control, both being blank corrected
- Irritation / corrosion parameter:
- other: cell viability (%)
- Run / experiment:
- 3
- Value:
- 84.2
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Compared to the Negative control, both being blank corrected
- Irritation / corrosion parameter:
- other: cell viability (%)
- Run / experiment:
- mean
- Value:
- 81.8
- Other effects / acceptance of results:
- - OTHER EFFECTS:
- Direct-MTT reduction: the test items did not interact with MTT according to the check-methods conducted.
- Colour interference with MTT: As the test item was coloured, two additional test item-treated tissues were used for the non specific OD evaluation. The optical density (measured at 570 nm) of tissues were 0.017, Non Specific Colour % was calculated as 2.1% . This value was below 5%, therefore additional data calculation was not necessary.
DEMONSTRATION OF TECHNICAL PROFICIENCY:
This proficiency verification with 10 reference chemicals, in the in vitro skin irritation test, in the EPISKIN model, used in OECD 439 (2010) demonstrated that the laboratory is fully proficient to perform this study. The 5 non-irritant chemicals all gave a clearly non-irritant response and the 5 irritant chemicals all gave a clearly irritant response.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:The mean OD value of the three negative control tissues should be between 0.6 and 1.5, and the standard deviation value (SD) of the % viability values should be ≤ 18.
- Acceptance criteria met for positive control:The acceptable mean percentage viability range for positive controls is 0-40% and the standard deviation value (SD) of the % viability values should be ≤ 18.
- Acceptance criteria met for variability between replicate measurements:The SD calculated from individual % tissue viability values of the three test item treated replicates should be <18.
Any other information on results incl. tables
Table 1: Optical Density (OD) and the calculated Non
Specific Colour % (NSC%)
of the Additional Control Tissues
Additional control |
Optical density (OD) |
Non Specific Colour (%) |
||
Measured |
Blank corrected |
|||
Treated with Theobromine |
1 |
0.056 |
0.010 |
2.1 |
2 |
0.071 |
0.024 |
||
Mean |
|
0.017 |
Mean blank value was 0.047.
Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).
Table 2: Optical Density (OD) and the calculated relative viability %
Substance |
Optical Density (OD) |
Viability (% RV) |
||
|
Measured |
Blank corrected |
||
Negative control |
1 |
0.868 |
0.821 |
103.5 |
2 |
0.842 |
0.796 |
100.3 |
|
3 |
0.811 |
0.764 |
96.3 |
|
Mean |
|
0.794 |
100.0 |
|
Positive control |
1 |
0.112 |
0.065 |
8.2 |
2 |
0.086 |
0.039 |
4.9 |
|
3 |
0.090 |
0.044 |
5.5 |
|
Mean |
|
0.049 |
6.2 |
|
Test item Theobromine |
1 |
0.698 |
0.651 |
82.0 |
2 |
0.675 |
0.628 |
79.1 |
|
3 |
0.715 |
0.669 |
84.2 |
|
Mean |
|
0.649 |
81.8 |
Mean blank value was 0.047.
Optical density means the mean value of the duplicate wells for each sample (rounded to three decimal places).
Applicant's summary and conclusion
- Interpretation of results:
- not classified
- Remarks:
- Based on EU criteria.
- Conclusions:
- Following exposure to theobromine, the mean cell viability of reconstructed human epidermis was 81.8% compared to the negative control. This is above the threshold of 50% , therefore, theobromine was considered as being non-irritant to skin.
- Executive summary:
An in vitro skin irritation test of Theobromine was performed in a reconstructed human epidermis model according to the OECD guideline 439 under GLP conditions. Disks of EPISKIN (three units) were treated with the test item and incubated for 15 minutes at room temperature. Exposure of the test item was terminated by rinsing with Phosphate Buffered Saline (PBS). The epidermis units were then incubated at 37°C for 42 hours in an incubator with 5% CO2. The viability of each disk was assessed by incubating the tissues for 3 hours with MTT solution at 37°C in an incubator with 5% CO2 protected from light. The precipitated formazan crystals were then extracted using acidified isopropanol and quantified spectrophotometrically. PBS and 5% (w/v) Sodium Dodecyl Sulphate (SDS) solution treated epidermis were used as negative and positive controls, respectively (three units/control). For each treated tissue, the viability was expressed as a % relative to the negative control. If the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control, the test item is considered to be irritant to skin. Following exposure with Theobromine, the mean cell viability was 81.8 %compared to the negative control. This is above the threshold of 50%, therefore the test item was considered as being non-irritant to skin. The experiment met the validity criteria, therefore the study was considered to be valid.
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