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EC number: 235-517-2 | CAS number: 12262-25-8 This substance is identified in the Colour Index by Colour Index Constitution Number, C.I. 53430.
- 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
Based on a local lymph node assay according to OECD guidline 429, the test item was detemined not to be skin sensitising.
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:
- 10 January 2017 - 20 March 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Version / remarks:
- 2010
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of study:
- mouse local lymph node assay (LLNA)
- Species:
- mouse
- Strain:
- CBA/Ca
- Remarks:
- CBA/Ca Ola Hsd mice
- Sex:
- female
- Details on test animals and environmental conditions:
- Source: TOXI-COOP ZRT., H-1103, Budapest, Cserkesz u. 90.
Hygienic level at arrival: SPF
Hygienic level during
the study: Good conventional
Justification of strain: On the basis of comparative investigations in other laboratories, mice of the CBA/Ca strain were found to exhibit a more marked response than other strains. Females are used because the existing database is predominantly based on females.
Number of animals: 28 animals/main test (4 animals/treatment group, 12 shared control animals)
Sex: Female, nulliparous, non-pregnant
Age of animals: Young adult mice; 11-12 weeks old (at start of the main test)
Body weight range
at starting: 19.2 – 23.2 g
The weight variation in animals involved in the study did not exceed +/- 20 % of the mean weight.
Acclimatization time: 7 days
Housing during
acclimatization period: Grouped caging in small groups
Housing during the test: Grouped caging (4 animals/cage)
Cage type: Type II. Polypropylene / polycarbonate
Bedding: Laboratory bedding
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 – 70 %
Housing/Enrichment: Mice were group-housed to allow social interaction, and with deep wood sawdust bedding, to allow digging and other
normal rodent activities.
Animals received ssniff® Rat/Souris-Elevage E complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany, ad libitum. Animals received tap water from watering bottles ad libitum. - Vehicle:
- dimethyl sulphoxide
- Concentration:
- 1 %, 0.5 %, 0.25 % or 0.1 % (w/v) formulations in DMSO
- No. of animals per dose:
- 4 animals/treatment group
- Details on study design:
- Based on the preliminary test results the test item was examined in the main test at concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v).
Formulations were prepared with Dimethyl sulfoxide (DMSO). The test item was weighed and the 1 % (w/v) formulation (stock solution) was
prepared with the vehicle in a final volume of 10 mL using ultrasonic dispersion and stirring. Further test concentrations were diluted from the
stock solution. All formulations were freshly prepared just before the daily treatments.
Animals in the treatment groups were treated with the negative (vehicle) controls (DMSO or AOO), appropriate formulations of the test item
or 25 % (w/v) concentration of the positive control substance. The test item was administered at four different concentrations according to
the results of the dose range finding test.
Criteria for Erythema Scores
Observation Score
No erythema 0
Very slight erythema (barely perceptible) 1
Well-defined erythema 2
Moderate to severe erythema 3
Severe erythema (beet redness) to eschar formation preventing grading of erythema 4
Test Concentrations in the Main Test
Groups Test item concentration Positive control concentration No. of animals
(% w/v) (% w/v)
1 Vehicle control for the positive control: AOO - - 4
2 Positive control: HCA in AOO - 25 4
3 Vehicle control for the test item: DMSO - - 4
4 Leuco Sulfur Blue 9 in DMSO 1 - 4
5 Leuco Sulfur Blue 9 in DMSO 0.5 - 4
6 Leuco Sulfur Blue 9 in DMSO 0.25 - 4
7 Leuco Sulfur Blue 9 in DMSO 0.1 - 4
AOO = Acetone: Olive oil 4:1 mixture
DMSO = Dimethyl sulfoxide
In vivo Treatment
Each mouse was topically treated with 25 µL of the appropriate formulations of the test item, the positive control substance or the vehicles using a pipette, on the dorsal surface of each ear. After the treatments animals were returned to their cages. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
Proliferation Assay
No animals showed symptoms of systemic toxicity or excessive skin irritation, and no technical treatment failures were observed during the test: all animals treated were processed. Therefore no treatment group was excluded from the evaluation.
Injection of 3HTdR
On Day 6 each mouse was intravenously injected via the tail vein with 250 µL of sterile PBS (1 x PBS, diluted from 10x concentrate) containing approximately 20 µCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, mice were left for 5 hours (± 30 minutes).
Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the nodes were removed using forceps. Once removed, the nodes of the mice from each test group were pooled and collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs), pooled according to groups, was prepared and collected in disposable tubes by gentle
mechanical disaggregating of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with
PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4 °C. After centrifugation,
the supernatant was removed, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated before making up to 10 mL with PBS
and re-suspending the LNCs. The washing procedure was repeated twice. This procedure was repeated for each group of pooled lymph nodes.
Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently
agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % TCA at 2-8 °C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4 °C and decanting the supernatants, than the pellets were re-suspended in 1 mL of 5 % TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and loaded into the beta-scintillation counter. 3HTdR incorporation was measured for up to 10 minutes per sample. The beta-counter expressed the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were measured in two 1 mL aliquots of 5 % TCA. - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- The randomization was checked by computer software [SPSS/PC+ (4.0.1)] according to the actual body weights verifying the
homogeneity and deviations between the groups.
DPM (disintegration per minute) was measured for each treatment group. The measured DPM values were corrected with the background DPM value:
the average of the two measured DPM values of 5 % (w/v) TCA solutions was used as the background DPM value. The results were expressed
as DPM/mouse. The stimulation index (SI = the DPM/mouse of a treated (positive control or test item) group divided by the DPM/mouse of the respective
negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.
Dose-response relationship was evaluated by linear regression using SI values. All calculations were made by Microsoft Excel Software.
Based on the results an EC3 value (dose calculated to induce a stimulation index of 3) was not calculated for the test item. - Positive control results:
- The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group. A significant lymphoproliferative response (SI >= 3) was noted for HCA (SI = 8.7). The results of the positive control item demonstrated an appropriate performance of the test in accordance with the relevant guidelines and confirmed the validity of the assay.
- Key result
- Parameter:
- SI
- Value:
- 0.9
- Test group / Remarks:
- 1 %
- Key result
- Parameter:
- EC3
- Remarks on result:
- not determinable
- Remarks:
- the SI values were below 3 at all test concentrations
- Parameter:
- SI
- Value:
- 0.3
- Test group / Remarks:
- 0.5 %
- Parameter:
- SI
- Value:
- 1
- Test group / Remarks:
- 0.25 %
- Parameter:
- SI
- Value:
- 1
- Test group / Remarks:
- 0.1 %
- Cellular proliferation data / Observations:
- No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score >= 3) or any other local effect were observed in any treatment group.
A body weight decrease by > 5 % was observed in the positive control group (2 of the 4 animals, 6 % decrease both), in the DMSO group (2 of the 4 animals, 8 % decrease both) and also in the test item treated groups (1 of the 4 animals in each; 7 %, 9 %, 8 % or 7 % decrease in the 1 %, 0.5 %, 0.25 % and 0.1 % (w/v) dose groups, respectively). No body weight decrease by > 5 % was observed in the AOO group. The mean body weights did not decrease significantly in any dose group. The observed effect was not dose-related and was considered not significant or toxicological relevant.
No significant lymphoproliferative response (SI >= 3) compared to the relevant control (DMSO) was noted for Leuco Sulfur Blue 9 at the applied test concentrations. The observed stimulation index values were 0.9, 0.3, 1.0 and 1.0 at test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.80, r = 0.20). - Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item was determined not to have a skin sensitization potential.
- Executive summary:
The aim of this study according to OECD 429 was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. As vehicle DMSO was chosen and the following test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v) were evaluated.
Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control group only. The appearance of the lymph nodes was normal in the negative control groups (AOO or DMSO) and in the test item treated groups. No significant lymphoproliferative response (SI >= 3) compared to the relevant control (DMSO) was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.9, 0.3, 1.0 and 1.0 at test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.80, r = 0.20). No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score >= 3) or any other local effect were observed in any treatment group. A body weight decrease by > 5 % was observed in the positive control group (2 of the 4 animals, 6 % decrease both), in the DMSO group (2 of the 4 animals, 8 % decrease both) and also in the test item treated groups (1 of the 4 animals in each; 7 %, 9 %, 8 % or 7 % decrease in the 1 %, 0.5 %, 0.25 % and 0.1 % (w/v) dose groups, respectively). No body weight decrease by > 5 % was observed in the AOO group. The mean body weights did not decrease significantly in any dose group. The observed effect was not dose-related and was considered not significant or toxicological relevant. According to the evaluation criteria of the relevant guidelines the lack of a significantly increased lymphoproliferation (indicated by an SI >= 3) up to the maximum attainable concentration of 1 % (w/v, based on solubility) and also the lack of a dose-related response are considered as evidence that the test item is not a skin sensitizer.
Reference
Individual Body Weights of the Animals with Group Means, the Associated Error Termsand Body Weight Changes in the Main Test
Animal |
Dose Group |
Initial |
Terminal |
Body Weight |
Number |
Body Weight |
Body Weight |
Change |
|
(g) |
(g) |
(%) |
||
1 |
Vehicle control for the positive control: |
21.4 |
21.7 |
1 |
2 |
AOO |
19.9 |
19.8 |
-1 |
3 |
|
23.2 |
22.9 |
-1 |
48 |
|
20.8 |
21.0 |
1 |
|
Mean |
21.3 |
21.4 |
0 |
|
SD |
1.4 |
1.3 |
|
4 |
Positive control: |
19.3 |
19.3 |
0 |
5 |
25 % HCA |
21.8 |
20.6 |
-6 |
49 |
in AOO |
22.0 |
20.7 |
-6 |
50 |
|
20.8 |
20.4 |
-2 |
|
Mean |
21.0 |
20.3 |
-3 |
|
SD |
1.2 |
0.6 |
|
6 |
Vehicle control for the test item: |
19.9 |
18.4 |
-8 |
7 |
DMSO |
22.9 |
22.4 |
-2 |
8 |
|
21.2 |
19.6 |
-8 |
51 |
|
20.9 |
20.6 |
-1 |
|
Mean |
21.2 |
20.3 |
-5 |
|
SD |
1.2 |
1.7 |
|
19 |
Leuco Sulfur Blue 9 |
21.9 |
20.3 |
-7 |
20 |
1 % |
19.2 |
18.3 |
-5 |
58 |
in DMSO |
20.4 |
20.8 |
2 |
59 |
|
22.4 |
21.7 |
-3 |
|
Mean |
21.0 |
20.3 |
-3 |
|
SD |
1.5 |
1.4 |
|
21 |
Leuco Sulfur Blue 9 |
20.7 |
20.3 |
-2 |
22 |
0.5 % |
21.7 |
19.8 |
-9 |
60 |
in DMSO |
19.8 |
19.9 |
1 |
61 |
|
22.4 |
22.2 |
-1 |
|
Mean |
21.2 |
20.6 |
-3 |
|
SD |
1.1 |
1.1 |
|
23 |
Leuco Sulfur Blue 9 |
20.5 |
19.9 |
-3 |
24 |
0.25 % |
22.8 |
20.9 |
-8 |
62 |
in DMSO |
19.8 |
20.2 |
2 |
63 |
|
21.0 |
21.7 |
3 |
|
Mean |
21.0 |
20.7 |
-2 |
|
SD |
1.3 |
0.8 |
|
25 |
Leuco Sulfur Blue 9 |
22.3 |
23.2 |
4 |
26 |
0.1 % |
20.8 |
19.4 |
-7 |
64 |
in DMSO |
19.4 |
19.1 |
-2 |
65 |
|
21.9 |
21.8 |
0 |
|
Mean |
21.1 |
20.9 |
-1 |
|
SD |
1.3 |
2.0 |
|
HCA =a-Hexylcinnamaldehyde AOO = Acetone: Olive oil 4:1 (v/v) mixture
DMSO = Dimethyl sulfoxide SD = Standard Deviation
DPM and Stimulation Index Values for all Groups in the Main Test
Dose Group |
Measured |
Group* |
DPM/Mouse# |
Stimulation |
DPM/group |
DPM |
Index Values |
||
Vehicle control for the positive control: |
2998 |
2972.0 |
743.0 |
1.0 |
AOO |
|
|
|
|
Positive control: |
25961 |
25935.0 |
6483.8 |
8.7 |
25 % HCA in AOO |
|
|
|
|
Vehicle control for the test item: |
7728 |
7702.0 |
1925.5 |
1.0 |
DMSO |
|
|
|
|
Leuco Sulfur Blue 9 |
7104 |
7078.0 |
1769.5 |
0.9 |
1 % in DMSO |
|
|
|
|
Leuco Sulfur Blue 9 |
2609 |
2583.0 |
645.8 |
0.3 |
0.5 % in DMSO |
|
|
|
|
Leuco Sulfur Blue 9 |
7487 |
7461.0 |
1865.3 |
1.0 |
0.25 % in DMSO |
|
|
|
|
Leuco Sulfur Blue 9 |
7521 |
7495.0 |
1873.8 |
1.0 |
0.1 % in DMSO |
|
|
|
|
HCA =a-Hexylcinnamaldehyde
AOO = Acetone: Olive oil 4:1 (v/v) mixture
DMSO = Dimethyl sulfoxide
*Group DPM = measured DPMgroup- average DPMbackground
Average DPMbackground= 26
# Number of animals/group = 4
Individual Ear Thickness Values and the Deviations from the Initial Values in the Dose Range Finding Test
Dose Group |
Animal |
Ears |
Day 1* |
Day 3$ |
Day 3 |
Day 6# |
Day 6 |
|
Number |
value (mm) |
value (mm) |
% deviation |
value (mm) |
% deviation |
|
|
981 |
L |
0.19 |
0.21 |
10.5 |
0.22 |
15.8 |
Leuco Sulfur Blue 9 |
R |
0.19 |
0.21 |
10.5 |
0.22 |
15.8 |
|
1 % in DMSO |
982 |
L |
0.20 |
0.20 |
0.0 |
0.22 |
10.0 |
|
R |
0.20 |
0.20 |
0.0 |
0.22 |
10.0 |
|
|
983 |
L |
0.20 |
0.20 |
0.0 |
0.21 |
5.0 |
Leuco Sulfur Blue 9 |
R |
0.20 |
0.20 |
0.0 |
0.21 |
5.0 |
|
0.5 % in DMSO |
34 |
L |
0.20 |
0.20 |
0.0 |
0.22 |
10.0 |
|
R |
0.20 |
0.20 |
0.0 |
0.22 |
10.0 |
|
|
984 |
L |
0.20 |
0.20 |
0.0 |
0.21 |
5.0 |
Leuco Sulfur Blue 9 |
R |
0.20 |
0.20 |
0.0 |
0.21 |
5.0 |
|
0.25 % in DMSO |
35 |
L |
0.19 |
0.20 |
5.3 |
0.20 |
5.3 |
|
R |
0.19 |
0.20 |
5.3 |
0.20 |
5.3 |
L = Left
R = Right
DMSO = Dimethyl sulfoxide
* Ear thickness was measured prior to the first treatment.
$ Ear thickness was measured approximately 48 hours after the first treatment (prior to the third treatment).
# Ear thickness was measured at the end of the test.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
The aim of the study according to OECD 429 was to evaluate the skin sensitization potential of the test item following dermal exposure in the Local Lymph Node Assay. Preliminary tests were performed according to the relevant guidelines to find an appropriate vehicle and the maximum applicable concentration. Solubility of the test item in vehicles preferred in the LLNA was evaluated. As vehicle DMSO was chosen and the following test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v) were evaluated.
Visually larger lymph nodes compared to the relevant vehicle control (AOO) were observed in the positive control group only. The appearance of the lymph nodes was normal in the negative control groups (AOO or DMSO) and in the test item treated groups. No significant lymphoproliferative response (SI >= 3) compared to the relevant control (DMSO) was noted for the test item at the applied test concentrations. The observed stimulation index values were 0.9, 0.3, 1.0 and 1.0 at test item concentrations of 1 %, 0.5 %, 0.25 % and 0.1 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. No statistical significance was observed (p = 0.80, r = 0.20). No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of irritation (indicated by an erythema score >= 3) or any other local effect were observed in any treatment group. A body weight decrease by > 5 % was observed in the positive control group (2 of the 4 animals, 6 % decrease both), in the DMSO group (2 of the 4 animals, 8 % decrease both) and also in the test item treated groups (1 of the 4 animals in each; 7 %, 9 %, 8 % or 7 % decrease in the 1 %, 0.5 %, 0.25 % and 0.1 % (w/v) dose groups, respectively). No body weight decrease by > 5 % was observed in the AOO group. The mean body weights did not decrease significantly in any dose group. The observed effect was not dose-related and was considered not significant or toxicological relevant. According to the evaluation criteria of the relevant guidelines the lack of a significantly increased lymphoproliferation (indicated by an SI >= 3) up to the maximum attainable concentration of 1 % (w/v, based on solubility) and also the lack of a dose-related response are considered as evidence that the test item is not a skin sensitizer.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Classification, Labeling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. Based on this data, the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.
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