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EC number: 949-050-3 | CAS number: -
- 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
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- 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
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- Additional physico-chemical information
- Additional physico-chemical properties of nanomaterials
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- Endpoint summary
- Stability
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- 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
Key value for chemical safety assessment
Genetic toxicity in vitro
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- other: experimental data on similar substance
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study well documented, test procedure in accordance with OECD 471 methods, meets generally accepted scientific principles, acceptable for assessment and GLP compliant. The test has been performed on only four Salmonella strains.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- no
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Salmonella typhimurium
- Species / strain / cell type:
- S. typhimurium TA 98
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 100
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1535
- Additional strain / cell type characteristics:
- not applicable
- Species / strain / cell type:
- S. typhimurium TA 1537
- Additional strain / cell type characteristics:
- not applicable
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 mix from rats
- Test concentrations with justification for top dose:
- For both mutagenicity test the treatment-levels are:
Strain TA98/TA100/TA1535/TA1537 with and without S9 mix: 20, 80, 320, 1280 and 5120 µg/plate - Vehicle / solvent:
- distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: 2-anthramine
- Remarks:
- all strains with S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- TA1537 wihtout S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: MNNG (N-methyl-N-nitro-N-nitrosoguanidine
- Remarks:
- TA100 and TA1535 without S9 mix
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: daunomycine
- Remarks:
- TA98 without S9 mix
- Details on test system and experimental conditions:
- BACTERIAL STRAINS INFORMATION:
The bacterial strains of Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 were kept as frozen broth cultures, in aliquots of 0.5 ml at -70°C with 0.8 % of DMSO.
Fresh cultures were prepared by adding 0.5 ml of a thawed stock culture to 25 ml of nutrient broth (Normal Difco nutrient broth for strains TA1535 abd TA100, and a double strenght Difco Nutrient broth for TA1537 and Ta98, in 0.5 % NaCl.
A nutrient agar (0.8 % Difco nutient broth, 1.5 % Difco Agar, 0.5 % NaCl) was also streaked.
The broth was incubated in the dark in a shaking water bath at 37°C for 16 hours, while the plate was incubated at 37°C overnight. Plates were then transfereed to the refrigerator for up to one week.
CHECKING OUT TESTER STRAINS
All strains were tested for the presence of thei mutations.
a)The mutation in the histidine operon, basic to the test syste, was tested by checking for growth in the presence and absence of histidine on a minimal medium-agar base.
Bacteria of the nutrient agar were streaked on a minimal medium plate supplemented with 0.1 ml of 0.1 M L-histidine and 0.1 ml of 0.5 mM biotin as a growth requirement.
The plates were incubated at 37°C overnight. Growth, for all trains, was seen only where histidine and biotin were present.
b)Sensitivity to crystal violet is a check for the presence of deep rough (rfa) mutation which causes partial loss of the lipopolysaccharide barrier that coats the surface of the bacteria and increases permeability to large molecules that do not penetrate the normal bacteria cell wall.
Three drops of the broth culture were spread on the surface of a nutrient agar plate. A sterile filter paper disc containing crystal violet (10 µl of a 1 mg/ml solution) was placed on this usrfac. A zore of inhibition around the disc, after 24 hours incubation, shows the presence of the rfa mutation.
c) Two of the tested strains (TA98 and TA100) contain a plasmid expressing resistance to ampicillin (R factor). To check for the presence of the lasmid, a sterile filter paper disc conatinign ampicillin (10 µl og 8 mg/ml in 0.02 N NaOH) was placed on a nutrient agar plate spread with the broth.
In strains TA1535 and TA 1537 a zone of inhibition occurs around the disc but for TA98 and TA100 where the R factor is present, no zone of inhibition is seen.
d) The uvr (uvrB for Salmonella typhimurium) the loss of teh excision repair system, makes bacteria sensitive to UV irradiation.
One drop of the broth was cross-streaked in a utrient agar plate. One half of the plate was iradiated for 20 seconds under a15 watt UV lamp at a distance of approx. 30 cm.
After 24 hours incubtaion, growth was found only on the unirradiated parte of the streak for all strains.
METHOD OF APPLICATION: in agar (plate incorporation)
PREPARATION OF S9 mix: The S9 mix consists of induced enzymatic systems contained in rat liver post-mitochondrial fraction (S9 fraction) and the cofactors necessary for their function.
S9 was prepared with the following procedure: three male Sprague-Dawley rats weighting about 200 gr were given an i.p. injection of Aroclor 1254 (Monsanto) in peanut oil at a dose of 500 mg/kg.
Five dayes after the injection, the rats were anaesthetized with ether, decapitaed and the ral livers removed in a sterile manner. The livers were homogenized in three times their volume of a sterile 0.14 M KCl, pooled together and centrifuged for 15 minutes at 9000xg (Beckman refrigerated ultracentrifuge).
The supernatant (termed S9-fraction) was aliquoted into sterile tubes and frozen at - 70°C.
The composition of 0.5 ml of S9 mix was:
4 µ moles MgCl2
16.5 µ moles KCl
2.5 µ moles G-6-P
2.0 µ moles NADP
50 µ moles Sodium phosphate buffer pH 7.4
150 µl liver homogenate - Evaluation criteria:
- A test article is considered as positive if either a significant dose-related increase in the number of revertants or a significant and reproducible increase for at least one test concentration is induced.
A test article producing neither a significant dose-related increase in the number of revertants nor a significant and reproducible positive response at any one of the test points is considered non-mutagenic in this system. - Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Species / strain:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- Preliminary toxicity test:
The test item was freely soluble in the vehicle (water for injections) at 50 mg/mL.
Consequently, with a treatment volume of 100 pL/plate, the dose-levels were 10, 100, 500, 1000,2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. An orange coloration of agar was observed in all test item treated plates at dose-levels > 100 µg/plate.
No noteworthy toxicity was noted towards the four sfrains used, with and without S9 mix, except a decrease in the number of revertants at
dose-levels > 1000 µg/plate, for the TA 98 sfrain with S9 mix.
Mutagenicity experiments:
The number of revertants for the vehicle and positive confrols was as specified in the acceptance criteria. The study was therefore considered valid.
Since the test item was freely soluble and generally non-toxic, the highest dose-level was 5000 pg/plate, according to the criteria specified in the international guidelines.
The selected freatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate, for both mutagenicity experiments with and without S9 mix, except for the first experiment with the TA98 and TA 1537 strains with S9 mix where the following dose-levels were tested: 156.3, 312.5, 625, 1250, 2500 and 5000 µg/plate.
No precipitate was observed in the Petri plates when scoring flie revertants at all dose-levels.
A moderate to marked toxicity was noted in the second experiment with S9 mix (preincubation method), in tiie TA 1535, TA 1537, TA 98 and TA 100 sfrains, generally at dose-levels > 2500 µg/plate.
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the six sfrains. - Remarks on result:
- other: strain/cell type: TA98
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- Interpretation of results: negative
The test item did not induce any noteworthy increase in the number of revertants, both with and without S9 mix, in any of the strains. - Executive summary:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test article did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore the tested substance is considered to be non-mutagenic in this Bacterial reverse mutation assay.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Remarks:
- Type of genotoxicity: chromosome aberration
- Type of information:
- other: experimental data on similar substance
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- not specified
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian chromosome aberration test
- Target gene:
- Chinese Hamster V79 Cells
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Details on mammalian cell type (if applicable):
- No data
- Additional strain / cell type characteristics:
- other: CHO-WBL
- Metabolic activation:
- with and without
- Metabolic activation system:
- The activation mixture consisted of 15 pl/ml S9, 1.5 mg/ml NADP, and 2.7 mg/ml isocitric acid in serum-free medium. S9 was obtained from the livers of Aroclor 1254-treated male Sprague-Dawley rats.
- Test concentrations with justification for top dose:
- The concentration range of the test article applied had been detrmined in a pre-experiment using the plating efficiency assay as indicator for toxicity response.
The following dose levels were evaluated:
without S9 mix:
7h: 100 µg/ml
18h: 6; 100 and 125 µg/ml
28h: 100 µg/ml
with S9 mix:
7h: 125 µg/ml
18h: 6; 100 and 125 µg/ml
28h: 125 µg/ml - Vehicle / solvent:
- DMSO
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- used in trials without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- used in trials with metabolic activation
- Details on test system and experimental conditions:
- On the day of the experiment, the test article was dissolved in DMSO. The solvent was chosen according to its solubility properties and its relative non-toxicity for the cells. The final concentration of the solvents in the culture medium did not exceed 1 % v/v.
Preparation of S9 mix:
The S9 liver microsomal fraction was obtained from the liver of 8-12 weeks old male Wistar rats, Strain WU and weigth 150-200gr, supplied by SAVC-Ivanovas, med. Versuchstierzuchten Gmbh, Kisslegg FRG (D) which received a single i.p. injecton of 500 mg/kg b.w. Aroclor 1524 (Antechnicka, Karlsruhe FRG) in olive oil 5 days previously.
After cervical dislocation the livers of the animals were removed, washed in 150 mM KCl and homogenised.
The homogenate, diluted 1:3 in KCl was centrifuged cold at 9000 rpm for 10 minutes.
A stock of the supernatant containing the microsomes was frozen in apoules are kept at -20°C for only several weeks before use.
The standardization of the protein content was made using the analysis kit of Bio-Rad Laboratories.
The protein concentration in the S9 preparation is usually between 20 and 45 mg/ml.
Before the experiment an appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.3 mg/ml in the cultures.
The composition of the cofactor solution was concentrated to yield the following concentrations in the S9 mix:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
5 mM NADP
100 mM sodium-ortho-phosphate-buffer pH 7.4
During the experiment the S9 mix was stored in an ice bath. - Evaluation criteria:
- Analysis of data and determination of genotoxic activity have been described by Galloway et al. [ 1987al. A significant increase in the aberration assay was based on a binomial sampling assumption [Margolin et al., 19861; the P values were adjusted according to Dunnett’s method to take into account multiple dose comparisons.
The trend test for both assays used a linear regression analysis.
Trials with two or more significant doses were considered positive (+), and trials with one significant dose and a significant trend were judged as having weak evidence of a positive response (+ W). The evaluation scheme used places emphasis on the number of doses that were positive rather than on the magnitude
of the response or its progression with dose; all calls were based upon this scheme. Trials with a significant response at one dose and no significant trend, and trials with no significant responses but having a significant trend were considered equivocal.
Positive and weak-positive trials were repeated to verify the response for those assays conducted after March 1984.
If a positive or a weak-positive response was observed both with and without activation, generally only the trial without activation was repeated. When only one activation condition was positive or weak-positive, only that condition was repeated.
If a weak-positive response was not repeatable, the chemical was generally concluded to be negative for that activation condition. When a positive response was not repeatable under conditions similar to those used in the original test, the chemical was concluded to be equivocal for that activation condition.
The terms positive and weak-positive were used to indicate the level of evidence for a significant response, and + W is considered positive in all evaluations of test performance. Equivocals were considered negative in all evaluations. - Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: all strains/cell types tested
- Remarks:
- Migrated from field 'Test system'.
- Conclusions:
- negative
The tested items did not induced chromosomal aberrations with or without activation. - Executive summary:
The tested substance was assessed for its potential to induce structural chromosome aberrations in V79 cell of the Chinese hamster in vitro, in the absence and presence of metabolic activation by S9 mix, according to the OECD guideline 473.
In conclusion, it can be stated that in the described study and under the experimental conditions reported, the tested substance did not induce structural chromosome aberrations in the Chinese V79 cells.
Referenceopen allclose all
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Link to relevant study records
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- other: experimental data on similar substance
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Study well documented, meets generally accepted scientific principles, acceptable for assessment.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- micronucleus assay
- Species:
- mouse
- Strain:
- other: OF-1 albino mice
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- Weight: male/female 25g
Supplier: from a SPF colny IFFA-CREDO, L'Arbresie France
Quarantine period of 1 week, the animals were allowed a libitum access to food (Aliment Rats-Souris Charles River, produced by U.A.R, Villemoisson/Orge France) and drinking water.
Animals were housed 5 of the same sex per cage in Makrolon type III cages. - Route of administration:
- oral: gavage
- Vehicle:
- deionised water
- Remarks:
- Doses / Concentrations:
0
Basis:
nominal in water - Remarks:
- Doses / Concentrations:
500 mg/kg bw
Basis:
nominal in water - No. of animals per sex per dose:
- 5 mice / sex / Group
- Control animals:
- yes, concurrent vehicle
- Sex:
- male/female
- Genotoxicity:
- negative
- Toxicity:
- no effects
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- At low magnification of the microscope no neticeable differences in bone marros nucleated cells were observed between animals with FAT20261/D and negative control.
In the positive control group (Thio-TEPA) decreased numbers of nucleated bone marrow cells were noted.
There was no statistically significant increase in the number of micronucleated polychromatic erythrocytes in animals exposed to 500 mg/kg of the tested substance compared to negative controls. In animals treated with positive control there was a statistically significant increased number of micronucleated cells.
The ration of polychromatic to normochromatic erythrocytes was markedly decreased in mice treated with positive control. There is no difference between animals treated with the tested substance and the negative control for this ratio. - Conclusions:
- non-mutagenic in micronucleus assay test
- Executive summary:
Under the experimental conditions used the tested substance did not induce a biologically relevant increase in the number of PCEs with micronuclei in bone marrow cells of treated mice and, consequently the tested substance is not genotoxic (clastogenic and/or aneugenic) in bone marrow cells of mice.
Reference
In the pre-experiment on acute toxicity with an exposure of 2000 mg/kg bw D&C Orange 4 exclusively a ruffled fur was found up to 4 h after administration. In the main experiment reduction of spontaneous activity, eyelid closure and ruffled fur were found at 2000 mg/kg
bw up to 6 or 24 h (ruffled fur only).
Treatment with D&C Orange 4 did not result in a decreased PCE/NCE ratios compared to the untreated controls indicating that D&C Orange 4 had no cytotoxic properties in the bone marrow.
However, the quantitative analysis of the test item in the plasma of the treated animals showed significant amounts of D&C Orange 4 after 1 h. This level dropped after 4 h.
Biologically relevant or statistically significant increases in the number of micronucleated PCEs compared to the concurrent vehicle controls were not found at any dose tested, neither 24 nor 48 h after treatment and neither for males nor for females.
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
Both in vitro and in vivo mutagenicity tests are available for similar substance.
Both tests of Bacterial reverse mutation assay, Ames test, showed negative results both with and without metabolic activation.
The results of the in vitro mammalian cell gene mutation assay and the in vitro chromosome aberration test, made on Acid Yellow 194 are both negative.
In addiction also the in vivo micronucleus (bone marrow) test showed negative results.
Based on these tests, the tested substance could be considered as not mutagenic.
Short description of key
information:
Not mutagenic
Endpoint Conclusion: No adverse effect observed (negative)
Justification for classification or non-classification
This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.
For the purpose of classification for germ cell mutagenicity, substances are classified if there is at least positive evidence obtained from experiments in mammals and/or in some cases from in vitro experiments, obtained from:
- somatic cell mutagenicity tests in vivo, in mammals; or
- other in vivo somatic cell genotoxicity tests which are supported by positive results from in vitro mutagenicity assays.
The presented results from in vivo and the in vitro tests are negative, therefore it is concluded that the tested substance is not genotoxic with or without metabolic activation.
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