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EC number: 944-533-5 | CAS number: -
- Life Cycle description
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- Endpoint summary
- Appearance / physical state / colour
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- Ecotoxicological Summary
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- Short-term toxicity to fish
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- Long-term toxicity to aquatic invertebrates
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- Acute Toxicity
- Irritation / corrosion
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Endpoint summary
Administrative data
Description of key information
LLNA, not a skin sensitizer (OECD 429, GLP, K, Rel. 1)
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 May to 25 August 2016
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Remarks:
- GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
- Principles of method if other than guideline:
- Not applicable
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- UK GLP Compliance Programme (inspected on July 05, 2016/ signed on October 28, 2016)
- Type of study:
- mouse local lymph node assay (LLNA)
- Specific details on test material used for the study:
- - Expiration date of the lot/batch: 30 September 2016
- Purity test date: 22 August 2016 - Species:
- mouse
- Strain:
- other: CBA/Ca (CBA/CaOlaHsd)
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 changes per hour
- Photoperiod: 12 hours continuous light and 12 hours darkness
- IN-LIFE DATES: 10 May to 25 August 2016 - Vehicle:
- acetone/olive oil (4:1 v/v)
- Concentration:
- Main test: 50%, 25%, 10% or 2.5% w/w in acetone/olive oil 4:1
- No. of animals per dose:
- 5
- Details on study design:
- PRE-SCREEN TESTS:
- Test Item preparation: For the purpose of the study, the test item was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. To aid preparation, the test item formulations were warmed in a water bath set at 45 °C for 30 minutes and also a sonic bath for 15 minutes. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
- Preliminary Screening Test: As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using two mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the test item at concentrations of 50% or 25% w/w in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation and clinical signs of toxicity were recorded. The body weight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6. The thickness of each ear was measured using a Mitutoyo 547 300S gauge (Mitutoyo Corporation), pre dose and 1 hour post dose on Day 1, 1 hour post dose on Days 2 and 3 and on Days 4 to 6. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization. Following the Day 6 ear thickness measurements, the mice were killed by carbon dioxide asphyxiation followed by cervical separation.
- Results: Brown colored residual test item on the ears was noted post dose on Day 2 and 3 and on Days 4 and 5 in the animal treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1.
No signs of systemic toxicity or visual local skin irritation were noted. No irritation indicated by an equal to or greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 25% w/w in acetone/olive oil 4:1. A greater than 25% increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1. Although this was considered to be due to the presence of residual test item on the ears, it was not entirely possible to exclude underlying irritation being the cause.
Based on this information, and after consultation with the Sponsor, the test item at concentrations of 50%, 25%, 10% and 2.5% w/w in acetone/olive oil 4:1 were selected for the main test.
MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".
TREATMENT PREPARATION AND ADMINISTRATION:
25 μL of control or test material was applied topically on the dorsal surface of both ears using a micropipette daily for three consecutive days (Days 1-3). Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse. Five hours later animals were killed by carbon dioxide asphyxiation followed by cervical separation. The lymph nodes from each group were pooled and a single cell suspension was prepared. Cells were washed with phosphate buffered saline (PBS) and precipitated with 5% trichloroacetic acid (TCA) for 18 h at ca. 4 °C. The pellets were recovered by centrifugation and resuspended in 1 mL of TCA and transferred to a vial containing scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA). The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). - Positive control substance(s):
- hexyl cinnamic aldehyde (CAS No 101-86-0)
- Statistics:
- Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.
- Positive control results:
- The positive control α Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (4.69) when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.
- Key result
- Parameter:
- SI
- Value:
- 1.04
- Test group / Remarks:
- 2.5% w/w in acetone/olive oil 4:1
- Key result
- Parameter:
- SI
- Value:
- 0.71
- Test group / Remarks:
- 10% w/w in acetone/olive oil 4:1
- Key result
- Parameter:
- SI
- Value:
- 0.74
- Test group / Remarks:
- 25% w/w in acetone/olive oil 4:1
- Key result
- Parameter:
- SI
- Value:
- 1.51
- Test group / Remarks:
- 50% w/w in acetone/olive oil 4:1
- Cellular proliferation data / Observations:
- DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 2.5, 10, 25 and 50% w/w in acetone/olive oil 4:1 were 1.04, 0.71, 0.74 and 1.51, respectively.
CLINICAL OBSERVATIONS: Brown colored residual test item on the ears and fur loss were noted in animals treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1.
There were no deaths. No signs of systemic toxicity or local skin irritation (visual and ear thickness measurements) were noted in the test or control animals during the test.
BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period. - Interpretation of results:
- GHS criteria not met
- Conclusions:
- Under the test conditions, test material is classified as a non-sensitizer according to Regulation (EC) No. 1272/2008.
- Executive summary:
A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP.
Following a preliminary screening test in which no clinical signs of toxicity or excessive local irritation were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Four groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25%, 10% or 2.5% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v inacetone/olive oil 4:1.The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).
Stimulation index for 2.5, 10, 25 and 50% w/w in acetone/olive oil 4:1 were 1.04, 0.71, 0.74 and 1.51, respectively. Brown colored residual test item on the ears and fur loss were noted in animals treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1. There were no deaths. No signs of systemic toxicity or local skin irritation (visual and ear thickness measurements) were noted in the test or control animals during the test.
The positive control α‑Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (4.69) when tested at a concentration of 25% v/v inacetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.
Under the test conditions, test material is classified as a non-sensitizer according to Regulation (EC) No. 1272/2008.
This study is considered as acceptable and satisfies the requirement for sensitisation endpoint.
Reference
Table 7.4.1/1: Individual Disintegrations per Minute and Stimulation Index
Treatment Group |
Animal Number |
dpm/ |
Mean dpm/Animal |
Stimulation Indexb |
Result |
Vehicle |
1-1 |
2356.30 |
2333.28# |
na |
na |
1-2 |
1086.70 |
||||
1-3 |
3284.15 |
||||
1-4 |
2605.96 |
||||
1-5 |
974.52Ä |
||||
Test Item |
2-1 |
1930.08 |
2424.62 |
1.04 |
Negative |
2-2 |
3305.14 |
||||
2-3 |
2441.30 |
||||
2-4 |
2646.11 |
||||
2-5 |
1800.49 |
||||
Test Item |
3-1 |
1817.78 |
1651.68 |
0.71 |
Negative |
3-2 |
957.73 |
||||
3-3 |
2003.08 |
||||
3-4 |
1488.72 |
||||
3-5 |
1991.10 |
||||
Test Item |
4-1 |
1694.82 |
1730.69 |
0.74 |
Negative |
4-2 |
2485.82 |
||||
4-3 |
843.58 |
||||
4-4 |
1906.19 |
||||
4-5 |
1723.06 |
||||
Test Item |
5-1 |
4397.82 |
3522.31 |
1.51 |
Negative |
5-2 |
1303.85 |
||||
5-3 |
4812.06 |
||||
5-4 |
4630.69 |
||||
5-5 |
2467.15 |
||||
Positive Control Item |
6-1 |
10786.47 |
10933.49*** |
4.69 [5.30] |
Positive |
6-2 |
12704.13 |
||||
6-3 |
13812.96 |
||||
6-4 |
9099.31 |
||||
6-5 |
8264.60 |
Theresults of the statistical analysis of the data indicated there was no significant difference between the control group and the test item test groups
dpm = Disintegrations per minute
a = Total number of lymph nodes per animal is 2
b = Stimulation Index of 3.0 or greater indicates a positive result
Ä = Identified as an outlier by the Grubbs test
# = Value based on four animals due to exclusion of outlier value
[ ] = Result with inclusion of outlier value
*** = Significantly different from control group p<0.001
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not sensitising)
- Additional information:
A GPMT study was available on the reistered substance. In this study, 2 out of 10 animals were sensitised after 3 challenges. However, in the absence of positive control, the sensitivity and validity of the experiment was confirmed. Additionally the substance contains bisabolene alpha, an ingredient known to have sensitising properties.
The testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (December 2016), was used to evaluate the skin sensitisation potential of the registered substance:
Element
Information
Conclusion
Comments
Existing data on physico-chemical properties
1
Is the substance a strong acid (pH≤ 2.0) or base (pH≥ 11.5), corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature?
NO
Existing human data
2
Are there adequate existing human data, which provide evidence that the substance is a skin sensitiser?
NO
Existing animal data from sensitisation studies
3
Are there data from existing studies on skin sensitisation in laboratory animals (LLNA, GPMT, or Buehler test, EU B.42, B.50, B.51 and B.6/OECD TGs 429, 442A, 442B and 406), which provide sound conclusive evidence that the substance is a sensitiser, or non-sensitiser?
NO
In a GPMT assay, 2 out of 10 animals were sensitised. However, in the absence of positive control, the sensitivity and validity of the experiment was not confirmed.
Additionally, the substance contains bisabolene alpha, an ingredient known to have sensitising properties.Existing/new (Q)SAR data and read-across
4
Do “read-across” from structurally and mechanistically related substances and/or do suitable (Q)SAR predictions reliably indicate skin sensitisation potential or the absence thereof of the substance?
NO
Not applicable - UVCB substance
Existing in chemico and in vitro data
5a
Is there evidence/hypothesis of dermal bioavailability based on physico-chemical, in silico, in vitro or in vivo data?
NO
Not applicable - UVCB substance
5b
Has the substance demonstrated peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. OECD TG 442c)? (Key event 1 of the AOP), and/or
Has the substance demonstrated activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. OECD TG 442d)?(Key event 2 of the AOP), and/or
Has the substance demonstrated induction of the cell surface markers (CD54 and/or CD86) on monocytic cells in a validated in vitro test, e.g. h-CLAT? (Key event 3 of the AOP).
Data from in chemico/in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.NO
(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)
5c
Are there data from (a) non-validated in vitro test(s), which provide evidence that the substance may be a skin sensitiser?
NO
Weight-of- Evidence analysis
6
The “elements” described above may be arranged as appropriate.Taking all existing and relevant data (elements 1-5) into account, is there sufficient information to meet the information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?
NO
Generation of new non-animal data
7a
Does the substance demonstrate peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. B. 59/OECD TG 442c)? (Key event 1 of the AOP)
In chemico test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.NO
(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)
7b
Does the substance demonstrate activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. B.60/OECD TG 442d)? (Key event 2 of the AOP)
In vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.NO
(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)
7c
Does the substance demonstrate induction of the cell surface markers (CD54 and/or CD86) of monocytic cells in a validated in vitro test (e.g. h-CLAT)? (Key event 3 of the AOP)
In vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.NO
(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)
7d
Is any additional testing/generation of data considered necessary in order to conclude on classification, or e.g. to explain the inconsistent data obtained in previous elements or to address the Key event 4 of the AOP (T-cell proliferation) with an in vitro test?
NO
(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)
Weight-of-Evidence analysis
8
The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-7) into account, is there sufficient information to meet the respective information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?
NO
Generation of new in vivo data for sensitisation as a last resort (Annex VII to the REACH Regulation)
8b
Does the substance demonstrate sensitising properties in an EU/OECD adopted in vivo test, the LLNA (EU B.42/OECD TG 429)?
YES
Since it was not possible to get a conclusion based on available data, a LLNA was initiated: Not a skin sensitizer
Therefore, a new LLNA study was performed according to the OECD test guideline No. 429 and in compliance with GLP (Envigo, 2017, rel.1) [NB: at the time of test performance, i.e. before 11 October 2016, in vitro testing was not the REACH default requirement].
Following a preliminary screening test in which no clinical signs of toxicity or excessive local irritation were noted at a concentration of 50% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Four groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 50%, 25%, 10% or 2.5% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v inacetone/olive oil 4:1.The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).
Stimulation index for 2.5, 10, 25 and 50% w/w in acetone/olive oil 4:1 were 1.04, 0.71, 0.74 and 1.51, respectively. Brown colored residual test item on the ears and fur loss were noted in animals treated with the test item at a concentration of 50% w/w in acetone/olive oil 4:1. There were no deaths. No signs of systemic toxicity or local skin irritation (visual and ear thickness measurements) were noted in the test or control animals during the test.
The positive control α‑Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (4.69) when tested at a concentration of 25% v/v inacetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.
Under the test conditions, test material is classified as a non-sensitizer according to Regulation (EC) No. 1272/2008.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
Harmonized classification:
The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008.
Self-classification:
Based on the available data, no additional classification is proposed regarding skin sensitisation according to the Regulation (EC) No. 1272/2008 (CLP).
No data was available for respiratory sensitisation. However, this substance is not a skin sensitizer, therefore according to Figure R.7.3 -2 of the Chapter R.7 (V 4.1 - October 2015) the chemical is not considered as a respiratory sensitizer.
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