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EC number: 288-897-7 | CAS number: 85940-09-6
- 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
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- Nanomaterial surface chemistry
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- 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
Skin irritation:
Based on the results of an OECD 439 study, the test item is considered not to be irritant to skin.
Eye irritation:
Based on the results of an OECD 437 study, the test item is considered not to have an eye damaging potential.
Key value for chemical safety assessment
Skin irritation / corrosion
Link to relevant study records
- Endpoint:
- skin irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 12 June 2017 - 21 July 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
- Version / remarks:
- 2015
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
- Version / remarks:
- 2017
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Test system:
- human skin model
- Source species:
- human
- Cell type:
- non-transformed keratinocytes
- Justification for test system used:
- The EPISKIN model has been validated for irritation testing in an international trial. After a review of scientific reports and peer reviewed publications on the EPISKIN method, it showed evidence of being a reliable and relevant stand-alone test for predicting rabbit skin irritation, when the endpoint is evaluated by MTT reduction and for being used as a replacement for the Draize Skin Irritation test (OECD TG 404 and Method B.4 of Annex V to Directive 67/548/EEC) for the purposes of distinguishing between skin irritating and non-skin irritating test substances (STATEMENT OF VALIDITY OF IN-VITRO TESTS FOR SKIN IRRITATION; ECVAM; Institute for Health & Consumer Protection; Joint Research Centre; European Commission; Ispra; 27 April 2007).
- Vehicle:
- unchanged (no vehicle)
- Details on test system:
- Units: EpiSkinTMSM plate containing up to 12 reconstructed epidermis units (area: 0.38 cm2) each reconstructed epidermis is attached to the base of a tissue culture vessel with an O-ring set and maintained on nutritive agar for transport.
Plate: 12-well assay plate
Punch: EpiSkinTMSM biopsy punch for easy sampling of epidermis
Medium: sterile “Maintenance Medium” for incubations (Batch No.: 17 MAIN3 024; Exp. Date: 21 June 2017)
sterile “Assay Medium” for use in MTT assays (Batch No.: 17 ESSC 023; Exp. Date: 21 June 2017)
The EpiSkinTMSM units were kept in their packaging at room temperature until the pre-incubation was started. The maintenance and assay medium were stored at 2-8°C. - Control samples:
- yes, concurrent negative control
- yes, concurrent positive control
- yes, concurrent MTT non-specific colour control
- Amount/concentration applied:
- The epidermal surface was first moistened with 5 µL deionised water in order to improve further contact between powder and epidermis. Subsequently, 10 mg of the test item was applied evenly to the epidermal surface. A volume of 10 µL positive control (SDS 5 % aq.) or negative control (1x PBS) was applied on the skin surface by using a suitable pipette. Chemicals were gently spread with the pipette tip in order to cover evenly all the epidermal surface if necessary.
- Duration of treatment / exposure:
- The plates with the treated epidermis units were incubated for the exposure time of 15 minutes (± 0.5 min) at room temperature.
- Duration of post-treatment incubation (if applicable):
- After the incubation time the EpiSkinTMSM units were removed and rinsed thoroughly with approximately 25 mL PBS 1x solution to remove all of the test material from the epidermal surface. The rest of the PBS was removed from the epidermal surface with suitable pipette tip linked to a vacuum source (care was taken to avoid the damage of epidermis). After rinsing the units were placed into the plate wells with fresh pre-warmed “maintenance medium” (2 mL/well) below them and then incubated for 42 hours (± 1h) at 37°C in an incubator with 5 % CO2, ≥95% humidified atmosphere. After the 42 hours incubation the EpiSkinTMSM units were transferred into the MTT solution filled wells (2 mL of 0.3 mg/mL MTT per well) and then incubated for 3 hours (± 5 min) at 37 °C in an incubator with 5 % CO2 protected from light, ≥95% humidified atmosphere.
- Number of replicates:
- In this assay 3 replicates per test item and 3 replicates negative controls, 3 replicates positive controls, 2 replicates colour controls and 2 replicates non-specific colour control were used. Furthermore, 3 killed treated tissues and 3 killed negative control tissues are used for the MTT evaluation.
- Irritation / corrosion parameter:
- % tissue viability
- Remarks:
- mean
- Run / experiment:
- 1-3
- Value:
- 72
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Irritation / corrosion parameter:
- % tissue viability
- Run / experiment:
- 1-3
- Value:
- >= 69 - <= 74
- Vehicle controls validity:
- not applicable
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- The mean OD value of the three negative control tissues was 0.720. The mean OD value obtained for the positive control was 0.081 and this result corresponds to 11 % viability when compared to the results obtained from the negative control. Each calculated standard deviation value (SD) for the % viability was below 18. All validity criteria were within acceptable limits and therefore the study can be considered as valid.
Indicator for potential false viability
Possible direct MTT reduction with test item:
As the test item has an intrinsic colour (black), the check-method for possible direct MTT reduction with test item was impossible. The direct interaction with MTT was not defined. However, to avoid the effect of possible interactions with the MTT, an additional control was necessary. The non-specific MTT reduction (NSMTT) was determined (0%)*, the correction of viability percentage was not necessary.
*: The calculated NSMTT was -2.939%. However, for the calculation of non-specific MTT reduction, small negative numbers are counted as zero, because the reason of the small negative number is a slight difference between the used killed epidermis (biological variability).
Colouring potential of test item:
As the test item has an intrinsic colour (black), two additional test item-treated tissues were used for the non-specific OD evaluation. Mean OD (measured at 570 nm) of these tissues was determined as 0.030. The Non Specific Colour % (NSC %) was calculated as 4.2 % (below 5 %). Therefore additional data calculation was not necessary. A false estimation of viability can be precluded. - Interpretation of results:
- GHS criteria not met
- Conclusions:
- The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
- Executive summary:
EpiSkinTMSM test with the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. 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 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item has an intrinsic colour (black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. As the test item is a possible MTT-reducer and has an intrinsic colour (black) to avoid a possible double correction for colour interference, a third control for non-specific colour in killed tissues was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 72 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Reference
OD values and viability percentages of the controls:
Substance |
Optical Density (OD) |
Viability (%) |
|
Negative Control: |
1 |
0.771 |
107 |
2 |
0.602 |
84 |
|
3 |
0.786 |
109 |
|
mean |
0.720 |
100 |
|
standard deviation (SD) |
14.23 |
||
Positive Control: |
1 |
0.056 |
8 |
2 |
0.062 |
9 |
|
3 |
0.124 |
17 |
|
mean |
0.081 |
11 |
|
standard deviation (SD) |
5.27 |
OD values and viability percentages of the test item:
Test Item |
Optical Density (OD) |
Viability (%) |
|
1 |
0.496 |
69 |
|
2 |
0.534 |
74 |
|
3 |
0.514 |
71 |
|
mean |
0.515 |
72 |
|
standard deviation (SD) |
2.69 |
OD values of additional controls for MTT-interacting test item:
Additional controls |
Optical Density (OD) |
|
Negative control killed tissues: |
1 |
0.054 |
2 |
0.044 |
|
3 |
0.066 |
|
mean |
0.055 |
|
Test item treated killed tissues: |
1 |
0.032 |
2 |
0.036 |
|
3 |
0.034 |
|
mean |
0.034 |
OD values and NSC % of additional control:
Additional colour control |
Optical Density (OD) |
Non Specific Colour %(NSC %) |
|
Test item |
1 |
0.034 |
4.2 |
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Eye irritation
Link to relevant study records
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 30 October 2017 - 05 March 2018
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Species:
- cattle
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- - Source: Schlachthof Aschaffenburg, 63739 Aschaffenburg, Germany
- Age at study initiation: at least 9 month old donor cattle - Vehicle:
- physiological saline
- Controls:
- yes, concurrent vehicle
- yes, concurrent positive control
- Amount / concentration applied:
- The test item was tested as a 20% suspension (w/v) in saline using sonication for 10 minutes. The pH value of the suspension prior to application was 5.75.
- Duration of treatment / exposure:
- 240 minutes
- Number of animals or in vitro replicates:
- 3 corneae per group (test item, negative control, positive control)
- Details on study design:
- Three corneas were exposed to each 0.75 mL of a 20% (w/v) suspension of the test item in physiological saline for 240 minutes.
After treatment the test item suspension was rinsed off the corneas and the corneas' opacity was determined. In a second step the permeability of the corneas was determined photometrically after 90 minutes treatment with fluorescein solution.
SCORING SYSTEM:
Opacity measurement
The opacitometer determines changes in the light transmission passing through the corneae, and displays a numerical opacity value. This value was recorded in a table. The opacitometer OP_KiT opacitometer (Electro Design, 63-Riom France) was calibrated as described in the manual and the opacity of each of the corneae was determined by reading each holder placed in the photoreceptor compartment for treated cornea.
For equilibration and prior to application of the test item or controls, the corneae in the holder were incubated in a vertical position for about one hour at 32 ± 1 °C in a water-bath. At the end of the incubation period, the basal opacity was determined (t0). After exposure of the corneae to the test groups, after rinsing and further incubation of the corneae for two hours, the opacity value was determined again (t240).
Permeability Determination
Following to the opacity readings, the permeability was measured as an indication of the integrity of the epithelial cell sheets. After the final opacity measurement was performed, the incubation medium will be removed from the anterior compartment and replaced by 1 mL of a 0.5% (w/v) sodium fluorescein solution in HBSS. Corneae were incubated again in a horizontal position for 90 ± 10 minutes in a water-bath at 32 ± 1 °C. Incubation medium from the posterior compartment were removed, well mixed and transferred into a 96 well plate and the optical density at 490 nm (OD490) was determined with a spectrophotometer (Versamax® Molecular Devices). The absorbance values will be determined using the software SoftMax Pro Enterprise (version 4.7.1).
DATA EVALUATION
Opacity
The change of opacity value of each treated cornea or positive and negative control corneae is calculated by subtracting the initial basal opacity from the post treatment opacity reading (t240 – t0), for each individual cornea.
The average change in opacity of the negative control corneae is calculated and this value is subtracted from the change in opacity of each treated cornea or positive control to obtain a corrected opacity.
Permeability
The corrected OD490 value of each cornea treated with positive control and test item is calculated by subtracting the average negative control cornea value from the original permeability value for each cornea.
IVIS Calculation
The following formula is used to determine the IVIS of the negative control:
IVIS = opacity value + (15 x OD490 value)
The following formula is used to determine the IVIS of the positive control and the test item:
IVIS = (opacity value – opacity value mean negative control) + (15 x corrected OD490 value)
The mean IVIS value of each treated group is calculated from the IVIS values.
Depending on the score obtained, the test item is classified into the following category according to OECD guideline 437:
IVIS: In vitro Irritancy Score (according to OECD 437):
≤ 3 No Category (according to GHS)
> 3; ≤ 55 No prediction can be made
> 55 Serious eye damaging according to CLP/EPA/GHS (Cat 1)
Criteria for Determination of a Valid Test
The test will be acceptable if
• the positive control gives an IVIS that falls within two standard deviations of the current historical mean (updated every three months), and if
• the negative control responses result in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneae treated with the respective negative control. - Irritation parameter:
- in vitro irritation score
- Remarks:
- Replicate 1
- Run / experiment:
- 1
- Value:
- 2.67
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Irritation parameter:
- in vitro irritation score
- Remarks:
- Replicate 2
- Run / experiment:
- 1
- Value:
- 0.77
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Irritation parameter:
- in vitro irritation score
- Remarks:
- Replicate 3
- Run / experiment:
- 1
- Value:
- 1.59
- Vehicle controls validity:
- valid
- Negative controls validity:
- not examined
- Positive controls validity:
- valid
- Interpretation of results:
- GHS criteria not met
- Conclusions:
- In conclusion, according to the current study and under the experimental conditions reported, the test item is not categorised as an eye irritant (GHS).
- Executive summary:
This in vitro study according to OECD guideline 437 was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneas. After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) suspension in saline of the test item, the positive, and the negative controls were applied to the different corneas and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneas and opacity was measured again (t240). After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
For the negative control (saline) neither an increase of opacity nor permeability of the corneas could be observed (mean IVIS = 1.28). The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas (mean IVIS = 126.41) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The IVIS of the positive control fell within two standard deviations of the HCD. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative control. Therefore the acceptability criteria were fulfilled. The test item was tested as suspension. Relative to the negative control, the test item did not cause a relevant increase of the corneal opacity or permeability. The calculated mean IVIS was 1.68 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorised as an eye irritant (GHS).
Reference
Results after 240 Minutes Treatment Time
Test Group |
Opacity value = Difference (t240-t0) of Opacity |
Permeability at 490 nm (OD490) |
IVIS |
Mean IVIS |
Proposedin vitroIrritancy Score |
Standard deviation ofin vitroscore |
||
|
|
Mean |
|
Mean |
|
|
|
|
Negative Control |
0 |
0.33 |
0.072 |
0.063 |
1.08 |
1.28 |
No Category |
0.56 |
1 |
0.061 |
1.92 |
||||||
0 |
0.056 |
0.84 |
||||||
Positive Control |
121.67* |
0.233* |
125.16 |
126.41 |
Category 1 |
1.76 |
||
125.67* |
0.184* |
128.43 |
||||||
122.67* |
0.198* |
125.64 |
||||||
Test item |
2.67* |
0.000* |
2.67 |
1.68 |
No Category |
0.95 |
||
0.67* |
0.007* |
0.77 |
||||||
1.67* |
-0.005* |
1.59 |
*corrected values
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (not irritating)
Respiratory irritation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
Skin irritation:
EpiSkinTMSM test with the test item has been performed to predict its irritation potential by measurement of its cytotoxic effect, as reflected in the MTT assay, according to the OECD Test Guideline No. 439. Disks of EPISKIN (three units) were treated with test item and incubated for 15 minutes at room temperature. Exposure of test material was terminated by rinsing with PBS 1x solution. 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 5% CO2 protected from light. The precipitated formazan was then extracted using acidified isopropanol and quantified spectrophotometrically. SDS (5% aq.) and 1×PBS treated (three units / positive and negative control) epidermis were used as positive and negative controls respectively. For each treated tissue viability was expressed as a percentage relative to negative control. The test item has an intrinsic colour (black), therefore two additional test item treated tissues were used for the non-specific OD evaluation. The test item is a possible MTT-reducer, therefore additional controls (test item treated killed tissues and negative control treated killed tissues) were used to detect and correct for test substance interference with the viability measurement. As the test item is a possible MTT-reducer and has an intrinsic colour (black) to avoid a possible double correction for colour interference, a third control for non-specific colour in killed tissues was performed. Two killed treated tissues were used to avoid a possible double correction for colour interference. The test chemical is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1), if the mean relative viability after 15 minutes exposure and 42 hours post incubation is less or equal (≤) to 50% of the negative control.
In this in vitro skin irritation test using the EPISKIN model, the test item did not show significantly reduced cell viability in comparison to the negative control (mean viability: 72 %). All obtained test item viability results were above 50 % when compared to the viability values obtained from the negative control. Positive and negative controls showed the expected cell viability values within acceptable limits. The experiment was considered to be valid. The test item is considered to be non-irritant to skin and is therefore not classified (UN GHS No Category).
Eye irritation:
An in vitro study according to OECD guidline 437 was performed to assess the corneal damage potential of the test item by means of the BCOP assay using fresh bovine corneas. After a first opacity measurement of the fresh bovine corneas (t0), the 20% (w/v) suspension in saline of the test item, the positive, and the negative controls were applied to the different corneas and incubated for 240 minutes at 32 ± 1 °C. After the incubation phase, the test item as well as the positive and the negative controls were each rinsed from the corneas and opacity was measured again (t240). After the opacity measurements, permeability of the corneas was determined by measuring spectrophotometrically the transfer of sodium fluorescein after incubation in a horizontal position for 90 minutes at 32 ± 1 °C.
For the negative control (saline) neither an increase of opacity nor permeability of the corneas could be observed (mean IVIS = 1.28). The positive control (10% (w/v) benzalkonium chloride in saline) showed clear opacity and distinctive permeability of the corneas (mean IVIS = 126.41) corresponding to a classification as serious eye damaging (CLP/EPA/GHS (Cat 1)). The IVIS of the positive control fell within two standard deviations of the HCD. The negative control responses resulted in opacity and permeability values that are less than the established upper limits for background opacity and permeability values for bovine corneas treated with the respective negative control. Therefore the acceptability criteria were fulfilled. The test item was tested as suspension. Relative to the negative control, the test item did not cause a relevant increase of the corneal opacity or permeability. The calculated mean IVIS was 1.68 (threshold for serious eye damage: IVIS > 55). According to OECD 437, the test item is not categorised as an eye irritant (GHS).
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 does not meet the classification criteria for skin or eye irritation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.
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