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EC number: 308-020-4 | CAS number: 97808-97-4
- 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
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 19th September 2019 - 10th October 2019
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 019
- Report date:
- 2019
Materials and methods
Test guidelineopen allclose all
- 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)
- Version / remarks:
- Updated 09 October 2017
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
- Version / remarks:
- EC No. 440/2008
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: Guidance Document on ‘The Collection of Tissues for Historical Evaluation and Collection of Data’. Series on Testing and Assessment, No. 160.
- Version / remarks:
- Adopted July 6, 2018 Paris.
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
Test material
- Reference substance name:
- Water
- EC Number:
- 231-791-2
- EC Name:
- Water
- Cas Number:
- 7732-18-5
- Molecular formula:
- H2O
- IUPAC Name:
- Water
- Reference substance name:
- C12-C14 alcohol
- IUPAC Name:
- C12-C14 alcohol
- Reference substance name:
- Mono (C12-C14 alkyl) phosphate ester
- IUPAC Name:
- Mono (C12-C14 alkyl) phosphate ester
- Reference substance name:
- Di (C12-C14 alkyl) diphosphate ester
- IUPAC Name:
- Di (C12-C14 alkyl) diphosphate ester
- Reference substance name:
- Tri (C12-C14 alkyl) triphosphate ester
- IUPAC Name:
- Tri (C12-C14 alkyl) triphosphate ester
- Reference substance name:
- C12-C14 alkyl pyrophosphate esters
- IUPAC Name:
- C12-C14 alkyl pyrophosphate esters
- Reference substance name:
- Ortho-phosphoric acid
- IUPAC Name:
- Ortho-phosphoric acid
- Reference substance name:
- unknown
- Molecular formula:
- unknown
- IUPAC Name:
- unknown
- Test material form:
- solid: bulk
Constituent 1
Constituent 2
Constituent 3
Constituent 4
Constituent 5
Constituent 6
Constituent 7
Constituent 8
- Specific details on test material used for the study:
- CAS Number: 68412-59-9
Batch: 1023S17201
Purity: 100%
Physical state/Appearance: White solid
Expiry Date: 09 July 2020
Storage Conditions: Room temperature in the dark
Test animals / tissue source
- Species:
- cattle
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- Eyes from adult cattle (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.
Test system
- Vehicle:
- physiological saline
- Remarks:
- sodium chloride 0.9% w/v
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- 0.75 mL of Substance 20% w/v solution in sodium chloride 0.9% w/v;
0.75 mL of negative control;
0.75 mL of positive control; - Duration of treatment / exposure:
- 240 min at 32 ± 1 ºC.
- Duration of post- treatment incubation (in vitro):
- Following opacity measurements the corneas were used further for pereability measurement. The corneas were rinsed to remove the test/control substances and further incubated for 90 min with sodum fluorescein at 32 ± 1 ºC.
- Number of animals or in vitro replicates:
- triplicate
- Details on study design:
- Preparation of Corneas
All eyes were macroscopically examined before and after dissection. Only corneas free of damage were used.
The cornea from each selected eye was removed leaving a 2 to 3 mm rim of sclera to facilitate handling. The iris and lens were peeled away from the cornea. The isolated corneas were immersed in a dish containing HBSS until they were mounted in Bovine Corneal Opacity and Permeability (BCOP) holders.
The anterior and posterior chambers of each BCOP holder were filled with complete Eagle’s Minimum Essential Medium (EMEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 75 minutes. At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
Selection of Corneas and Opacity Reading.
The medium from both chambers of each holder was replaced with fresh complete EMEM.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer.
Three corneas were randomly allocated to the test substsance, negative and positive control.
Treatment of Corneas.
The EMEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the substance preparation or control items were applied to the appropriate corneas. The holders were gently tilted back and forth to ensure a uniform application of the item over the entire
cornea. Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 240 minutes.
At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed 3 times with fresh complete EMEM containing phenol red before a final rinse with complete EMEM without phenol red. The anterior chamber was refilled with fresh complete EMEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed.
Application of Sodium Fluorescein.
Following the opacity measurement the permeability of the corneas to sodium fluorescein was evaluated. The medium from the anterior chamber was removed and replaced with 1 mL of sodium fluorescein solution (5 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 ºC for 90 minutes.
Permeability Determinations.
After incubation the medium in the posterior chamber of each holder was decanted and retained.
360 μL of media representing each cornea was dispensed into the appropriate wells of a pre-labeled 96-well plate. The optical density was measured (quantitative viability analysis) at 492 nm (without a reference filter) using the Labtech LT-4500 microplate reader.
Histopathology.
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labeled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
Data Evaluation.
Results from the two test method endpoints, opacity and permeability, were combined in an empirically derived formula to generate an In Vitro Irritancy Score.
Opacity Measurement.
The change in opacity for each cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final opacity reading. These values were then corrected by subtracting the average change in opacity observed for the negative control corneas. The mean opacity value of each treatment group was then calculated by averaging the corrected opacity values of each cornea for that treatment group.
Permeability Measurement.
The corrected OD492 was calculated by subtracting the mean OD492 of the negative control corneas from the OD492 value of each treated cornea. The OD492 value of each treatment group was calculated by averaging the corrected OD492 values of the treated corneas for the treatment group.
In Vitro Irritancy Score.
The following formula was used to determine the In Vitro Irritancy Score:
In Vitro Irritancy Score = mean opacity value + (15 x mean permeability OD492 value)
Additionally, the opacity and permeability values were evaluated independently to determine whether the test item induced a response through only one of the two endpoints.
Visual Observation.
The condition of the cornea was visually assessed post treatment.
Results and discussion
In vitro
Results
- Irritation parameter:
- in vitro irritation score
- Run / experiment:
- mean
- Value:
- ca. 87.4
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Remarks:
- IVIS = 2.1
- Positive controls validity:
- valid
- Remarks:
- IVIS=108.1
- Remarks on result:
- positive indication of irritation
Any other information on results incl. tables
Treatment |
Mean Opacity1 |
Mean Permeability1 |
Mean In vitro Irritation Score1, 2 |
Negative control |
2.0 |
0.003 |
2.1 |
Positive control (20% Imidazole) |
85 |
1.538 |
108.1 |
Substance |
86 |
0.095 |
87.4 |
1 Calculated using the negative control corrected mean opacity and mean permeability values for the positive control and substance.
2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490value).
Corneal epithelium post-treatment condition was visually examined. For positive control and substance treated corneas appeared cloudy; the negative control treated corneas appeared clear.
Applicant's summary and conclusion
- Interpretation of results:
- Category 1 (irreversible effects on the eye) based on GHS criteria
- Conclusions:
- The mean in vitro irritancy score (IVIS) of the test substance is 87.4. Therefore, the substance is classified to cause serious eye damage (Category 1).
- Executive summary:
In a reliable in vitro eye irritation study, conducted according to 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’, the ability of the substance to induce opacity and permeability in an isolated bovine cornea were determined. The substance was applied 20% w/v solution (in sodium chloride 0.9% w/v) at 750 µL onto corneas (n=3) for a period of 240 min at 32 ± 1 ºC, followed by rinsing of the substance. A post-treatment opacity reading was taken and each cornea was visually observed. Following the opacity measurement the permeability of the corneas to sodium fluorescein (following further 90 min incubation) was evaluated. According to opacity and permeability measurements, the substance resulted in a mean in vitro irritancy score (IVIS) of 87.4. In conclusion, since the substance induced an IVIS > 55, the substance is classified to cause serious eye damage.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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