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EC number: 947-359-8 | 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
- 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
Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Remarks:
- The Bovine Corneal Opacity and Permeability Assay (BCOP)
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From October 09, 2017 to October 10, 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 017
- Report date:
- 2018
Materials and methods
Test guideline
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
- Deviations:
- yes
- Remarks:
- Deviations does not impact the outcome of the study
- GLP compliance:
- yes
Test material
- Reference substance name:
- Phosphoric acid, C6-12-alkyl esters, potassium salts
- EC Number:
- 291-900-4
- EC Name:
- Phosphoric acid, C6-12-alkyl esters, potassium salts
- Cas Number:
- 90506-39-1
- Molecular formula:
- UVCB
- IUPAC Name:
- potassium decyl octyl phosphate
- Test material form:
- solid
- Details on test material:
- Appearance: White solid
Batch: RE 14-6
Constituent 1
- Specific details on test material used for the study:
- Appearance: White solid
Batch: RE 14-6
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 27 July 2018 (expiry date)
Test animals / tissue source
- Species:
- other: Bovine
- Details on test animals or tissues and environmental conditions:
- Test System: Bovine eyes were used as soon as possible after slaughter.
Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
Test system
- Vehicle:
- unchanged (no vehicle)
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- 303.7 to 363.6 mg
- Duration of treatment / exposure:
- 4 h
- Observation period (in vivo):
- -
- Duration of post- treatment incubation (in vitro):
- 90 minutes
- Number of animals or in vitro replicates:
- 3
- Details on study design:
- Preparation of Corneas:
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Fetal Bovine Serum (Life Technologies)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32°C ± 1°C. The corneas were incubated for the minimum of 1 hour at 32°C ± 1°C.
Cornea Selection and Opacity Reading:
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.
Test Item Preparation:
No correction will be made for the purity/composition of the test compound. Since no workable suspension of the test item in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.
Treatment of Corneas and Opacity Measurements:
The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (303.7 to 363.6 mg). The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ± 10 minutes at 32°C ± 1°C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Earle’s Minimum Essential Medium Life Technologies). Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.
Opacity Measurement :
The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
The opacity value (measured with the device OP-KIT) was calculated according to:
Opacity=(I0/I-0.9894)/0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
Application of Sodium Fluorescein:
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml of 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany).
The holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were incubated in a horizontal position for 90 ± 5 minutes at 32°C ±1°C.
Permeability Determinations:
After the incubation period, the medium in the posterior compartment of each holder was removed and placed into a sampling tube labelled according to holder number. 360 µL of the medium from each sampling tube was transferred to a 96-well plate. The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader (TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified before the start of the experiment). OD490 values of less than 1.500 were used in the permeability calculation. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a dilution has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.
Results and discussion
In vitro
Results
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- 4 h exposure
- Value:
- 126
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other: Positive indication of corrosive
- Other effects / acceptance of results:
- The individual in vitro irritancy scores for the negative controls ranged from 0.8 to 4.7. One of the negative control eyes was excluded from the analysis since the permeability was above the historical data base which resulted in an IVIS outside the historical data base. Since the other two eyes completely met the criteria and the test substance results were not influenced by this result, this does not affect the study outcome. The individual positive control in vitro irritancy scores ranged from 112 to 199. The corneas treated with the positive control were turbid after the 240 minutes of treatment. The corneas treated with the test item showed opacity values ranging from 25 to 42 and permeability values ranging from 5.680 to 6.980. The corneas were turbid after the 240 minutes of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 110 to 134 after 240 minutes of treatment with the test substance.
Any other information on results incl. tables
Table1: Summary of opacity, permeability and in vitro scores
Treatment |
Mean Opacity |
Mean Permeability |
Mean In vitro Irritation Score1, 2 |
Negative control |
2.5 |
0.021 |
2.8 |
Positive control |
123 |
1.758 |
150 |
Test item |
32 |
6.253 |
126 |
1 Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.
2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).
Table 2: Individual Opacity, Permeability and in vitro Scores
Treatment |
Opacity before treatment |
Opacity after treatment |
Final Opacity1 |
Negative control corrected Final Opacity2 |
Mean Final Opacity |
|
|
||||||
Negative control |
4.8 |
5.5 |
0.7 |
|
2.5 |
|
4.54 |
8.64 |
4.04 |
||||
3.9 |
8.1 |
4.2 |
||||
|
||||||
Positive control |
3.5 |
183.0 |
179.5 |
177 |
123 |
|
3.3 |
93.2 |
89.9 |
87 |
|||
3.4 |
111.0 |
107.6 |
105 |
|||
|
||||||
Test item |
5.0 |
49.6 |
44.6 |
42 |
32 |
|
4.4 |
36.6 |
32.1 |
30 |
|||
6.5 |
34.2 |
27.7 |
25 |
1 Final Opacity = Opacity after treatment – Opacity before treatment.
2 Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control
3 Calculations are made without rounding off.
4 Excluded from analysis (see study plan deviation).
Table 3: Permeability Score Individual Values (Corrected)
Treatment |
Dilution factor |
Negative control corrected OD490 11 |
Negative control corrected OD490 21 |
Negative control corrected OD490 31 |
Negative control corrected OD490 Average |
Negative control corrected final OD490 |
Average OD |
|
|||||||
Positive control |
1 |
1.423 |
1.424 |
1.453 |
1.433 |
1.433 |
1.758 |
6 |
0.281 |
0.277 |
0.277 |
0.278 |
1.670 |
||
6 |
0.363 |
0.365 |
0.358 |
0.362 |
2.172 |
||
|
|||||||
Test item |
6 |
1.023 |
1.006 |
1.020 |
1.016 |
6.098 |
6.253 |
6 |
1.172 |
1.160 |
1.158 |
1.163 |
6.980 |
||
6 |
0.955 |
0.944 |
0.941 |
0.947 |
5.680 |
1 OD490 values corrected for the mean final negative control permeability (0.021).
2 Calculations are made without rounding off.
Table 4: In VitroIrritancy Score
Treatment |
Final Opacity2 |
Final OD4902 |
In vitroIrritancy Score1 |
|
|||
Negative control |
0.7 |
0.007 |
0.8 |
4.03 |
0.5583 |
123 |
|
4.2 |
0.035 |
4.7 |
|
|
|||
Positive control |
177 |
1.433 |
199 |
87 |
1.670 |
112 |
|
105 |
2.172 |
138 |
|
|
|||
Test item |
42 |
6.098 |
134 |
30 |
6.980 |
134 |
|
25 |
5.680 |
110 |
1 In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).
2 Positive control and test item are corrected for the negative control.
3 Excluded from analysis (see study plan deviation).
Applicant's summary and conclusion
- Interpretation of results:
- other: Category 1 (irreversible effects on the eye) based on CLP criteria
- Conclusions:
- Under the study conditions, the test substance was considered to be causing serious eye damage (category 1) to Bovine Corneal Opacity and Permeability Test (BCOP Test) (IVIS-126).
- Executive summary:
A study was conducted to determine the in vitro eye damage potential of the test substance, according to OECD Guideline 437 measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test), in compliance with GLP. The eye damage of the test substance was tested through topical application for 4 h. The study procedures described in this report were based on the most recent OECD guideline. The test substance was a white solid. The test substance was applied as it is (303.7 to 363.6 mg) directly on top of the corneas. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The individual positive control in vitro irritancy scores ranged from 112 to 199. The corneas treated with the positive control were turbid after the 4 h of treatment. The corneas treated with the test substance showed opacity values ranging from 25 to 42 and permeability values ranging from 5.680 to 6.980. The corneas were turbid after the 240 minutes of treatment with the test substance. No pH effect of the test substance was observed on the rinsing medium. Hence, the in vitro irritancy scores ranged from 110 to 134 after 240 minutes of treatment with the test substance. Under the study conditions, the test substance was considered to be causing serious eye damage (category 1) to Bovine Corneal Opacity and Permeability Test (BCOP Test) (IVIS-126) (Groot, 2017).
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