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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
Endpoint summary
Administrative data
Description of key information
Key study: OECD 429. GLP study. The substance was determined to be not sensitizing to the skin.
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vivo (LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 13 January 2016 - 2 Februaray 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- Test method according to OECD 429. GLP study.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Lot/batch No.of test material:M15015C
- 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: Room temperature - Species:
- mouse
- Strain:
- other: CBA/CaOlaHsd
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: Envigo (formerly: Harlan Laboratories S.r.l.), San Pietro al Natisone (UD), Zona Industriale Azzida, 57, 33049 Italy.
- Age at study initiation: 8-weeks old.
- Weight at study initiation: 18.9 - 20.8 g.
- Housing: Group caged, mice were provided with glass tunnel-tubes, bedding consisted of certified wood chips, nest building material was also provided.
- Diet: Ad libitum.
- Water: Ad libitum.
- Acclimation period: 7 days.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.7 - 24.3 ºC
- Humidity (%): 27 - 77 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours dark. - Vehicle:
- dimethyl sulphoxide
- Concentration:
- 50, 25 and 10 % (w/v)
- No. of animals per dose:
- 4
- Details on study design:
- RANGE FINDING TESTS:
The preliminary toxicity test was conducted under identical conditions to the main test (See any other information on material and methods incl. tables) except that was finalized on day 6 and no assessment of lymph node proliferation was performed. 2 animals per dose were used and 2 doses were tested (50 and 25%). All mice were observed daily for clinical signs of systemic toxicity and local irritation, ear thickness measures were taken at day 1, 3 and 6 and body weights were recorded at the start and at the end of the test.
- Compound solubility: The solubility of the test item was examined in a Preliminary Compatibility Test. The following OECD vehicles were assessed: Acetone:Olive oil 4:1 (v:v), DMF, EMK, Propylene glycol, DMSO and 1% Pluronic. Due to the physical characteristics of the test item (powder), the 100 % (w/v) concentration was not achievable. The formulation at 50 % (w/v) using DMSO as vehicle was suitable for the test. As DMSO is one of the vehicles recommended by the relevant OECD guideline, it was selected for vehicle of the study.- Irritation: No indications of irritancy at the site of application.
MAIN STUDY (See any other information of material and methods inc. tables)
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay.
- Criteria used to consider a positive response: DPM was measured for each pooled group of nodes. The measured DPM values were corrected with the background DPM value (“DPM”). The average of the two measured DPM values of 5 % (w/v) TCA solutions was used as background DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes) following the industry standard for data presentation. Stimulation index (SI = DPN value of a treated group/ DPN value of the negative control group) for each treatment group was calculated. A stimulation index of 3 or greater is an indication of a positive result.
TREATMENT PREPARATION AND ADMINISTRATION (See any other information of material and methods inc. tables) - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Positive control results:
- Stimulation index Positive control (25 % (w/v) HCA in DMSO)= 7DPM Positive control (25 % (w/v) HCA in DMSO)= 26214.0
- Key result
- Parameter:
- SI
- Value:
- 1.7
- Test group / Remarks:
- Thebromine 10%
- Key result
- Parameter:
- SI
- Value:
- 1.2
- Test group / Remarks:
- Theobromine 25%
- Key result
- Parameter:
- SI
- Value:
- 1.5
- Test group / Remarks:
- Theobromine 50%
- Cellular proliferation data / Observations:
- See "Any other information on results" below.
- Interpretation of results:
- not sensitising
- Remarks:
- Based on EU criteria
- Conclusions:
- Theobromine did not show skin sensitisation potential under the tested contitions in the LLNA assay. The stimulation indeces were 1.5, 1.2 and 1.7 at concentrations of 50, 25 and 10% in DMSO.
- Executive summary:
A local lymph node assay was conducted with theobromine according to the OECD guideline 429 under GLP conditions. In the preliminary studies 50 % (w/v) of the test item in DMSO was selected as the top dose for the main experiments. Two animals were used for each concentration in the preliminary studies. On day 1, 2 and 3, 25 µL of each of the theobromine solutions (50, 25 and 10%) and positive and negative controls were applied to the dorsal surface of each ear of the mice. There was no treatment on day 4, 5 or 6. Clinical signs and signs of irritancy were monitored during the 6 days, there were no signs of irritancy or signs of systemic toxicity. Additionally ear thickness measures were performed at day 1, 3 and 6 in the preliminary test; there were no significant changes which could indicate excessive local irritation. Four mice were used per concentration and for each of the controls in the main experiment. On day 6 the weight was determined, the preliminary test finished at this point. In the main tests 250 µL of sterile phosphate buffered saline (PBS) containing approximately 20 µCi of 3HTdR was intravenously injected via tail vein. Five hours (± 30 minutes) after intravenous injection the mice were euthanized by asphyxiation with ascending doses of carbon dioxide. The draining auricular lymph nodes were prepared and pooled. Single cell suspensions (SCS) of pooled lymph node cells (LNCs) were prepared and collected in disposable tubes for each group of pooled lymph nodes. The determination of incorporated3HTdR was determined as DPM for each pooled group. The measured DPM values were corrected with a background blank DPM value. The results were expressed as “DPN” (DPM divided by the number of lymph nodes). The stimulation index was calculated (SI = DPN value of a treated group/ DPN value of the negative control group) for each treatment group. A stimulation index of 3 or greater was considered a positive result. The stimulation indices of theobromine at concentrations of 50, 25 and 10 % were 1.5, 1.2 and 1.7 respectively, no dose-related response was observed, the SI of the positive and negative controls were within the acceptable range. It was concluded that theobromine was not as a skin sensitizer under the conditions of this study.
Reference
DPM, DPN and Stimulation Index Values for all Groups
Test Group Name |
Measured DPM / group |
DPM |
Number |
DPN |
Stimulation Index |
Background (5 % (w/v) TCA) |
30 / 26 |
- |
- |
- |
- |
Negative control (DMSO) |
3747 |
3719.0 |
8 |
464.9 |
1.0 |
Theobromine in DMSO |
5672 |
5644.0 |
8 |
705.5 |
1.5 |
Theobromine 25 % (w/v) inDMSO |
4605 |
4577.0 |
8 |
572.1 |
1.2 |
Theobromine 10 % (w/v) inDMSO |
6262 |
6234.0 |
8 |
779.3 |
1.7 |
Positive control (25 % (w/v) HCA |
26242 |
26214.0 |
8 |
3276.8 |
7.0 |
The stimulation index values were 1.5, 1.2 and 1.7 at concentrations of 50%, 25 % and 10% (w/v), respectively.
No mortality or signs of systemic toxicity were observed during the study. Test item precipitate or minimal amount of test item precipitate was observed on the ears of the experimental animals in the 50% and 25 % (w/v) dose groups on Days 1‑4 and in the 10%.(w/v) dose group on Days 1-3. There were no indications of any irritancy at the site of application.
No treatment related effects were observed on the mean body weight changes of experimental animals. Individual and mean bodyweights are given in the following table:
Individual Body Weights for all Animals with Group Means
Animal Number |
Identity Number |
Test Group Name |
Initial Body Weight (g) |
Terminal Body Weight* (g) |
Change# (%) |
915 |
1 |
Negative (vehicle) control DMSO
|
20.7 |
20.4 |
-1.4 |
928 |
2 |
19.3 |
20.4 |
5.7 |
|
910 |
3 |
20.1 |
20.5 |
2.0 |
|
936 |
4 |
19.2 |
19.3 |
0.5 |
|
|
|
Mean |
19.8 |
20.2 |
1.7 |
941 |
5 |
Theobromine 50 (w/v) % in DMSO |
20.8 |
21.2 |
1.9 |
912 |
6 |
18.9 |
20.3 |
7.4 |
|
933 |
7 |
19.3 |
20.1 |
4.1 |
|
943 |
8 |
19.4 |
20.6 |
6.2 |
|
|
|
Mean |
19.6 |
20.6 |
4.9 |
934 |
9 |
Theobromine 25 (w/v) % in DMSO |
20.1 |
21.7 |
8.0 |
921 |
10 |
20.2 |
20.5 |
1.5 |
|
938 |
11 |
19.1 |
19.7 |
3.1 |
|
944 |
12 |
19.6 |
19.8 |
1.0 |
|
|
|
Mean |
19.8 |
20.4 |
3.4 |
939 |
13 |
Theobromine 10 (w/v) % in DMSO |
20.5 |
20.8 |
1.5 |
924 |
14 |
19.3 |
19.3 |
0.0 |
|
942 |
15 |
19.9 |
20.0 |
0.5 |
|
923 |
16 |
20.0 |
19.5 |
-2.5 |
|
|
|
Mean |
19.9 |
19.9 |
-0.1 |
950 |
17 |
Positive control 25 (w/v) % HCA in DMSO
|
20.7 |
20.6 |
-0.5 |
961 |
18 |
19.0 |
19.1 |
0.5 |
|
946 |
19 |
19.5 |
20.3 |
4.1 |
|
948 |
20 |
19.5 |
19.2 |
-1.5 |
|
|
|
Mean |
19.7 |
19.8 |
0.7 |
*: Terminal body weights were measured on Day 6.
#: = (Terminal Body Weight – Initial Body Weight) / Initial Body Weight x 100
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
- Key
study: A local lymph node assay was conducted according to the OECD
guideline 429 under GLP conditions with the test substance theobromine. The
stimulation indexes of theobromine at concentrations of 50, 25 and 10 %
were 1.5, 1.2 and 1.7 respectively, no dose-related response was observed,
the SI of the positive and negative controls were within the acceptable
and historical range. It was concluded that theobromine had no skin
sensitisation potential in the LLNA study.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Skin sensitisation: Based on the available experimental results, the test substance is not classified for skin sensitisation in accordance with CLP Regulation (EC) No. 1272/2008.
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