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EC number: 238-914-9 | CAS number: 14852-17-6
- 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
- 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
Repeated dose toxicity: oral
Administrative data
- Endpoint:
- short-term repeated dose toxicity: oral
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Justification for type of information:
- Data is from an experimental study report.
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
- Principles of method if other than guideline:
- According to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
- GLP compliance:
- yes
- Limit test:
- no
Test material
- Reference substance name:
- Ethylenediamine, salt with phosphoric acid
- EC Number:
- 238-914-9
- EC Name:
- Ethylenediamine, salt with phosphoric acid
- Cas Number:
- 14852-17-6
- Molecular formula:
- C2H8N2.xH3O4P
- IUPAC Name:
- ethane-1,2-diamine; phosphoric acid
- Test material form:
- solid: crystalline
- Remarks:
- White crystals
- Details on test material:
- Name (as per report): Ethylenediamine, salt with phosphoric acid
CAS No.: 14852-17-6
Molecular Weight: 158.093 g/mol
Molecular Formula: C2-H8-N2.x-H3-O4-P
SMILES: C(CN)N.P(=O)(O)(O)O
Purity: 98.04%
Constituent 1
Test animals
- Species:
- rat
- Strain:
- Wistar
- Details on species / strain selection:
- The rat is a commonly used species for toxicity studies and is recommended by the stated regulatory guideline.
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: In-bred animals of animal house facility at testing facility.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 6 to 7 weeks at receipt
- Weight at study initiation:
Males: 240.00 g to 250.40 g
Females: 183.80g to 185.80 g
- Fasting period before study: No Data Available
- Housing: Two to three rats were housed in each polycarbonate cage (length 37 cm X 21 cm breadth X height 20 cm). Rotation of the cage positions in each rack was carried once a week to nullify any possible effects arising from the cage placement. Sterilized corn-cob produced from pure corn, dried and free from dust, was used as bedding material.
- Diet (e.g. ad libitum): A conventional laboratory pellet diet was offered ad libitum.
- Water (e.g. ad libitum): Aquaguard™ filtered drinking water was offered ad libitum in bottles.
- Acclimation period: Male and female animals were acclimatised to the test conditions prior to test Item administration for a period of 12 and 13 days respectively.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.00 and 24.00 °C
- Humidity (%): 43.00 and 64.60 %.
- Air changes (per hr): at least 12 changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark.
IN-LIFE DATES:
From: May 22, 2019
To: July 12, 2019
Administration / exposure
- Route of administration:
- oral: gavage
- Details on route of administration:
- The oral route was used, since it is a commonly used route of administration
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5%
- Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
Required quantitity of test item was weighed and triturated in a mortar-pestle, first without vehicle and then in small volume of the vehicle. After thorough trituration, the test-item and vehicle were transferred quanititavely into a calibrated measuring cylinder and diluted using vehicle upto the required volume of the formulation, before being transferred to the final container.
DIET PREPARATION
- Rate of preparation of diet (frequency): No Data Available
- Mixing appropriate amounts with (Type of food): No Data Available
- Storage temperature of food: No Data Available
VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was found to be insoluble in water and also did not form a homogenous suspension in water, whereas 0.5% CMC provided adequate suspensibility and the formulation was found stable in this vehicle. Further, 0.5% CMC is well tolerated by rats at the dose volumes administered in this study.
- Concentration in vehicle:
Control and Control Recovery: 0 mg/ml
Low Dose: 20 mg/ml
Mid Dose: 40 mg/ml
High Dose and High Dose Recovery: 60 mg/ml
- Amount of vehicle (if gavage): 10 ml/kg bw
- Lot/batch no. (if required): SLBX2402
- Purity: No Data Available - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Samples from the dose formulations of G1/G1R, G2, G3 and G4/G4R were analyzed for homogeneity and concentration. The dose formulation analysis was carried out once on Day 1 of dose administration to males and once on Day 22. Results were calculated and reported.
- Duration of treatment / exposure:
- 28 Days with 2 weeks recovery period (For Control and High Dose Recovery Groups)
- Frequency of treatment:
- Daily
Doses / concentrationsopen allclose all
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Control and Control Recovery Group
- Dose / conc.:
- 200 mg/kg bw/day (actual dose received)
- Remarks:
- Low Dose Group
- Dose / conc.:
- 400 mg/kg bw/day (actual dose received)
- Remarks:
- Mid Dose Group
- Dose / conc.:
- 600 mg/kg bw/day (actual dose received)
- Remarks:
- High Dose and High Dose Recovery Group
- No. of animals per sex per dose:
- 5 animals per sex per group
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale:
The above-stated dose levels were set based on suggestion by the Sponsor and the available literature (Ethylenediamine, Salt with Phosphoric Acid, End Point Summary, ECHA and OECD SIDS Initial Assessment Report, Ethylenediamine, Bern, Switzerland November 6-9, 2001).
- Rationale for animal assignment (if not random): During the acclimatisation phase, male and female animals were separately randomized into six different experimental groups, based on the most recently recorded body weights, using the “Daniel’s XL Toolbar” for MS Excel. After randomisation, it was ensured that individual body weights were within ± 20% of the respective group means. Details of the randomization were documented in the study raw data. The mean body weights were analyzed by One-way ANOVA and it was found that the group means were statistically comparable to each other within each sex.
- Fasting period before blood sampling for clinical biochemistry: Yes, the animals were fasted before the collection of blood
- Rationale for selecting satellite groups: The satellite or recovery group was selected to observe an revertions of the effects caused due to the administration of the test chemical, if any.
- Post-exposure recovery period in satellite groups: 2 weeks (14 days)
- Section schedule rationale (if not random): No Data Available
- Other: No Data Available - Positive control:
- No Data Available
Examinations
- Observations and examinations performed and frequency:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily (once before and once after the day’s activities) for any morbidity and/or mortality, throughout the acclimatization and study periods. Also, All animals were observed for clinical signs and symptoms before and after dose administration on the treatment Day 1 and once daily thereafter. These observations were made approximately 1 hour after completion of dose administration, since onset of clinical signs after oral gavage are anticipated in that time frame.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals were subject to a detailed clinical examination on treatment Day 1 and weekly thereafter throughout the treatment and recovery periods.
BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed once on receipt, once during randomization, once at the start of treatment and once every week thereafter, till the end of the experimental period.
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No Data Available
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified
FOOD EFFICIENCY: No Data Available
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified
- Time schedule for examinations: No Data Available
OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Ophthalmological examination was conducted in all animals once prior to exposure (during acclimatization period) to test for treatment-realted changes, the examination was repeated in the high-dose (G4) and control group (G1) animals during the final week of dosing.
- Dose groups that were examined: High-dose (G4) and Control group (G1)
HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just before sacrifice.
- Anaesthetic used for blood collection: Not Specified
- Animals fasted: Yes
- How many animals:
- Parameters checked in table [No.?] were examined.
CLINICAL CHEMISTRY: Yes / No / Not specified
- Time schedule for collection of blood:
- Animals fasted: Yes / No / Not specified
- How many animals: All animals from all dose grroups.
URINALYSIS: Yes
- Time schedule for collection of urine: Prior to euthanasia
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4 for main group animals and week 6 for recovery group animals.
- Dose groups that were examined: All Dose Groups
- Battery of functions tested: Home-cage observations (for posture and convulsions), handling observations (for ease of removal from cage, handling reactivity, palpebral closure, lacrimation, eye examination, piloerection, skin examination, salivation), open-field observations (gait, mobility, arousal, vocalizations, rears, respiration, clonic movements, tonic movements, urination, defecation, stereotypy, bizarre behaviour), sensory reactivity (responses to approach, touch, click, tail pinch and reflexes to air-righting and pupil-constriction), grip strength (forelimb and hindlimb) and foot splay (landing hindlimb).
IMMUNOLOGY: Not specified
- Time schedule for examinations: No Data Available
- How many animals: No Data Available
- Dose groups that were examined: No Data Available
OTHER: No Data Available - Sacrifice and pathology:
- GROSS PATHOLOGY: Yes, All animals were subjected to Gross Pathology.
HISTOPATHOLOGY: Yes, Full histopathology was carried out on the preserved organs and tissues of all animals in the control and high-dose groups. - Other examinations:
- Oestrus Cycle: The stages of oestrus cycle were determined for females by microscopic examination of the vaginal smears. This observation was carried out daily during week 4 for main group females and daily during week 6 for recovery group females.
Bone Marrow Smear Examination: Bone marrow smear (from femur) were prepared at the time of necropsy. - Statistics:
- Raw data was analysed using statistical software Sigma Plot v14.0. All continuous data (body weight, percent change in body weight with respect to day 1, feed consumption, functional observation battery/neurobehavioral observation, foot splay record, grip strength, motor activity, haematology, clinical chemistry, absolute and relative organ weights, etc.) were checked first for their normality and homogeneity, and then analysed using one-way ANOVA (or unpaired t-test) for comparison of means. Heterogneous data was analysed using ANOVA on ranks (Kruskal-Wallis test) or Mann Whitney test on ranks. For means found significantly different by ANOVA, Dunnett’s post-hoc test was applied to identify the group pairs with significant differences. P values of less than or equal to 0.05 were accepted as being statistically significant.
Results and discussion
Results of examinations
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment-related clinical signs or symptoms were observed in any of the animals of either sex through the study period.
- Mortality:
- no mortality observed
- Description (incidence):
- No morbidities or mortality were found in animals of any of the experimental groups throughout the duration of the experiment.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Mean body weights remained comparable among the main groups and also between the recovery groups for every time point recorded in both the sexes during and after the exposure period. The percent change in body weights with respect to the first day of exposure was also statistically comparable among all the groups of males. However, G2 females showed a statistically significant increase in the percent change in body weight as compared to G1 females. This difference was only seen on day 08 and not for any other time points recorded, due to which, it can be safely treated as an inconsequential result. It was noted that females of vehicle recovery group (G1R) showed a statistically significant increase in the percent change in body weight as compared to the high-dose group (G4R) at days 8, 22, 28 and 42. This observation was not found consistent with the trend in means of percent change in body weight for the main groups G1 and G4. To gain clarity and to check the significance of this result, an additional statistical analysis (t-test) was run by collating the data for G1 and G1R into one group and G4 and G4R into another group. The treatment received and all experimental procedures for main and recovery groups were identical. The t-test returned a result showing no statictically significant difference between the control and high-dose groups (of ten animals each) for Days 8, 22 and 28. Hence the difference observed between the recovery groups could have arisen out of chance.
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- The feed consumed per animal per day remained statistically comparable among the experimental groups within each sex at every point of observation
- Food efficiency:
- not specified
- Water consumption and compound intake (if drinking water study):
- not specified
- Ophthalmological findings:
- no effects observed
- Description (incidence and severity):
- No treatment related opthalmological changes were observed in the tested group.
- Haematological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Data for main groups and males of recovery groups did not return any statistical variation in the means of hematology parameters. While an increase in haemoglobin content was noted for females of G4R as compared to the respective controls, there was no change in any other hematology parameter which could be attributed to test-item administration. The stated change in haemoglobin content, although statistically significant, can be regarded as irrelevant for the current study.
- Clinical biochemistry findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- While ALT was significantly higher in males of G3 (mid-dose) and G4 (high-dose) group sera as compared to G1 (control) sera, the levels of ALP were found significantly higher in males and females of G3, but not in G4, as compared to controls. Another observation of significance was that of cholesterol levels in females, which was found significantly higher in all treatment groups, viz., G2, G3 and G4 as compared to controls. Serum triglycerides were also significantly higher in sera of G3 males as compared to G1 males. Additionally, serum glucose was found significantly high in G4 males, as compared to controls. However, of the above-stated changes seen in high-dose groups (ALT and glucose levels in males and cholesterol in females), none were persistent after the 14 days recovery period, since G4R data remained comparable with that of G1R for these parameters. Further, ALP levels in males of G4R did show a significant hike as compared to G1R. Bile acids were found increased in G4R animals of both sexes as compared to G1R after the treatment-free recovery period. Mean glucose level in females was found reduced in G4R sera, when compared to G1R at the end of recovery period. These findings were not substantiated by any observations at the level of histology. Increased ALP levels did not show up as any tissue damage in the G4R males and serum ALP levels often show variablility and can have multiple origins. Therefore, this result was inconclusive, with the current data.
- Urinalysis findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Urine parameters were largely unaffected in the treatment groups of this study. pH values, however, were found significantly high in G4 females, as compared to G1 females. Urine pH variation is not uncommon in mammals and the currently reported change in pH could not be corroborated by any data from other clinical chemistry or histopathological parameters. This result is therefore treated as one with no major significance from the toxicology perspective.
- Behaviour (functional findings):
- effects observed, non-treatment-related
- Description (incidence and severity):
- The battery of functional and behavioural tests carried out during the last week of observations for the respective groups revealed no treatment-related abnormalities or deviation from the normal. Among the females, however, an increased grip strength of the forelimb was recorded in G3 and G4 animals, as compared to the controls. Although these increases were statistically significant, they could not be correlated with any other functional observation or histopathology or clinical pathology observations. With the current data, it does not seem possible to definitively attribute this result to test-item treatment and hence a negative effect of dose administration on muscle function can safely be ruled out, in view of the histopathological and clinical pathological observations herein.
- Immunological findings:
- not specified
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Among the main groups, the weights of thyroid and parathyroid relative to body weights were significantly higher in G2 males as compared to controls. The mean liver weight relative to body weight of G4 males was significantly higher than that of G1 males. The mean absolute weight of spleens in the high-dose recovery (G4R) females was significantly lower than those in G1R females and this also resulted in statistically significantly lower weights of G4R spleens with respect to both body weight as well as brain weight. Further, a significant increase in absolute weights of pituitary and pituitary weights in relation to brain weight was observed for G4R males as compared to G1R males.
- Gross pathological findings:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Apart from mild reduction in size of testes in two animals of G4 males, which is a spontaneous occurrence unrelated to test-item effects, no notable observations were reported from the necropsy examination.
- Neuropathological findings:
- not specified
- Histopathological findings: non-neoplastic:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No treatment-related histopathological findings are reported in this study that could arise out of test-item administration. All observed tissues were normal at the level of histology. Minimal and focal lymphocytic infiltrates were observed in some slides of liver, kidney, heart and epididymis, which were distributed randomly across the experimental groups and did not show any pattern of either dose-dependency or sex-based selectivity.
- Histopathological findings: neoplastic:
- not specified
- Other effects:
- no effects observed
- Description (incidence and severity):
- At the end of treatment period (for main groups) and at the end of the recovery period (for recovery groups), the females of all groups were found to have a normal pattern of ovarian cycle as reflected from the vaginal smears studied over a period of one week.
Bone marrow smears were tested from 3 animals each of G1 and G4 from each sex. There were no pathologies noted from the differential cell counts made on these smears. The low- and mid-dose slides were therefore not evaluated.
Effect levels
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 600 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- behaviour (functional findings)
- body weight and weight gain
- clinical biochemistry
- clinical signs
- food consumption and compound intake
- gross pathology
- haematology
- histopathology: non-neoplastic
- mortality
- ophthalmological examination
- organ weights and organ / body weight ratios
- urinalysis
- other: Oestrous Cycle and Bone Marrow Smear Examinations
- Remarks on result:
- not determinable due to absence of adverse toxic effects
Target system / organ toxicity
- Critical effects observed:
- no
Applicant's summary and conclusion
- Conclusions:
- Based on all the observations and results, it was concluded that the NOAEL for the test chemical was observed to be 600 mg/kg bw after the administration of the test chemical to Wistar rats, for 28 days and with 2 weeks recovery period.
- Executive summary:
A repeated dose 28 day study with 2 week recovery period, according to OECD TG 407 was performed using the test chemical in the male and female Wistar rats. A total of 60 Wistar rats (30 males and 30 females) were randomized into 6 experimental groups each of 5 animals per sex per group. The groups, viz., G1, G2, G3 and G4 received 0, 200, 400 and 600 mg/kg body weight of test-item respectively and were referred to as the main groups. The groups G1R and G4R received 0 and 600 mg/kg body weight of test-item respectively and were referred to as the recovery groups. Dose administration was through oral gavage and the vehicle used in this study was 0.5% solution of Carboxymethyl cellulose, sodium salt. All groups were administered with dose formulation for a period of 28-days, following which all animals of main groups were euthanized while the recovery groups animals were retained for another 14 days for observation before being euthanized. To ensure accurate administration of test-item, the concentrations and homogeneity of the dose formulation were verified through formulation analyses on two occasions – Day 1 and Day 22 of treatment. The results of these were found within acceptable limits on both occasions. Observations on the animals encompassed mortality/morbidity, onset of any clinical signs/symptoms, detailed clinical examination, body weight, body weight changes, feed consumption, ophthalmological examination, functional observational battery/neurobehavioral observation, hematology, clinical biochemistry, urinalysis, gross pathology and histopathology. The results of this study reveal that the test chemical acid does not cause mortality up to a dose of 600 mg/kg body weight when administered orally for 28 days. Further, no clinical signs or symptoms were observed in any animals of the study. While feed consumption remained comparable across groups through the course of the study, a difference in the percent change in body weights was noted between females of G1R and G4R groups, which, nevertheless, was found unrelated to test-item administration. A battery of observations carried out for functional and behavioral assessment of animals revealed no effect of test-item administration at any of the tested doses. These observations included in-cage observations, handling response, open-field behavior, hind limb foot splay, sensory response, motor activity measurement and grip strength measurements. Ophthalmological profile was also found normal. Clinical chemistry parameters tested herein were inclusive of serum biochemistry, electrolytes, hematology and urine analysis. Whereas the values largely remained unperturbed due to test-item administration, the serum levels of Alanine aminotransferase (ALT) was elevated in G3 and G4 males, in comparison to the controls. Although this data prima facie indicates stress on the liver, no change in the liver histology profiles of any animals were reported. The rise in ALT is therefore likely a result of inter-animal variations commonly cited for this enzyme, which itself is sensitive to various non-treatment related physiologic and metabolic factors. Examination of external features and gross pathology during necropsy revealed no noteworthy observations attributable to test-item administration. Histopathological observations were made for G1 (vehicle control) and G4 (high-dose) animals and no treatment related changes showed up in any of the observed tissues at the tested dose (600 mg/kg body weight) in either sex. Based on all the observations and results, it was concluded that the NOAEL for the test chemical was observed to be 600 mg/kg bw after the administration of the test chemical to Wistar rats, for 28 days and with 2 weeks recovery period.
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