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EC number: 232-254-5 | CAS number: 7798-23-4
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Eye irritation
Administrative data
- Endpoint:
- eye irritation: in vitro / ex vivo
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 18 Dec 2012
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
Data source
Reference
- Reference Type:
- study report
- Title:
- Unnamed
- Year:
- 2 016
- Report date:
- 2016
Materials and methods
Test guidelineopen allclose all
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
- Version / remarks:
- 2009
- Deviations:
- no
- Qualifier:
- equivalent or similar 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:
- adopted 9 Oct 2017
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- Department of Health of the Government of the United Kingdom, UK
Test material
- Reference substance name:
- Tricopper bis(orthophosphate)
- EC Number:
- 232-254-5
- EC Name:
- Tricopper bis(orthophosphate)
- Cas Number:
- 7798-23-4
- Molecular formula:
- Cu.2/3H3O4P
- IUPAC Name:
- tricopper bis(orthophosphate)
1
Test animals / tissue source
- Species:
- cattle
- Strain:
- not specified
- Details on test animals or tissues and environmental conditions:
- SOURCE OF COLLECTED EYES
- Source: Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Storage: The eyes were excised by an abattoir employee and placed in Hanks’ Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The eyes were refrigerated on arrival and used within 24 hours of receipt.
Test system
- Vehicle:
- other: 0.9% w/v sodium chloride solution
- Controls:
- yes, concurrent positive control
- yes, concurrent negative control
- Amount / concentration applied:
- TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: 20% (w/v) dilution in 0.9% (w/v) sodium chloride
NEGATIVE CONTROL
- Amount applied: 0.75 mL sodium chloride
- Concentration: 0.9% (w/v) sodium chloride
POSITIVE CONTROL
- Amount applied: 0.75 mL Imidazol
- Concentration: 20 % (v/v) in 0.9% (w/v) sodium chloride - Duration of treatment / exposure:
- 240 minutes
- Observation period (in vivo):
- not applicable
- Number of animals or in vitro replicates:
- not applicable
- 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 (epithelial side uppermost) 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 minimum essential medium (MEM) and plugged. The holders were incubated at 32 ± 1ºC for 60 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 MEM. A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. The average opacity for all corneas was calculated.Three corneas were numerically allocated to the test item. Three corneas were also numerically allocated to the negative control item and three corneas to the positive control item.
TREATMENT OF CORNEAS
The MEM was removed from the anterior chamber of the BCOP holder and the test item preparation or control items were applied to the cornea. 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 ± 1ºC for 240 minutes.
REMOVAL OF TEST SUBSTANCE
- Washing: Cornea rinsed three times with fresh minimum essential medium (MEM) containing phenol red before a final rinse with complete MEM
- Time after start of exposure: 240 minutes
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 ±1ºC for 90 minutes.
PERMEABILITY DETERMINATION
After incubation the medium in the posterior chamber of each holder was decanted and retained. 360 μL of medium representing each cornea was applied to a designated well on a 96-well plate and the optical density at 492nm (OD492) was measured. If values greater than 1.500 OD 492 were obtained a 1 in 5 dilution of the medium in complete MEM was performed and the measurement repeated. The modified value was multiplied by 5 to reflect the 1 in 5 dilution.
HISTOPATHOLOGY
The corneas were retained after testing for possible conduct of histopathology. Each cornea was placed into a pre-labelled tissue cassette fitted with a histology sponge to protect the endothelial surface. The cassette was immersed in 10% neutral buffered formalin.
EVALUATION OF RESULTS
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 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 immediately after rinsing and at the final opacity measurement.
DATA INTERPRETATION
A test item that induces an In Vitro Irritancy Score ≥ 55.1 is defined as an ocular corrosive or severe irritant (EU CLP and UN GHS Hazard statement H318 “Causes Serious Eye Damage” Category 1) and EU DSD (67/548/EEC) Irritant requires symbol “Xi” risk phrase R41 “Risk of Serious Damage to Eyes”.
CRITERIA FOR ACCEPTIBILITY
For an acceptable test the following positive control criterion must be achieved: 20% (w/v) Imidazole was used for positive control purposes. The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 55.8 to 126.1.
Results and discussion
In vitro
Results
- Irritation parameter:
- in vitro irritation score
- Remarks:
- mean value of 3 corneas
- Value:
- 70.7
- Vehicle controls validity:
- valid
- Negative controls validity:
- valid
- Positive controls validity:
- valid
- Other effects / acceptance of results:
- OTHER EFFECTS:
The corneas treated with the test item were cloudy post treatment. The corneas treated with the negative control item were clear post treatment. The corneas treated with the positive control item were cloudy post treatment.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control in vitro irritancy score was 4.2. The negative control acceptance criterion was therefore satisfied.
- Acceptance criteria met for positive control: The positive control in vitro irritancy score was within the range of 55.8 to 126.1. The positive control acceptance criterion was therefore satisfied.
Any other information on results incl. tables
Table 1: In Vitro Irritancy Score
Treatment |
In Vitro Irritancy Score |
Test Item |
70.7 |
Negative Control |
4.2 |
Positive Control |
105.5 |
Table 2: Individual and Mean Corneal Opacity and Permeability Measurements
Treatment |
Cornea Number |
Opacity |
Permeability (OD) |
In vitro Irritancy Score |
||||
Pre-Treatment |
Post-Treatment |
Post-Treatment – Pre-Treatment |
Corrected Value |
|
Corrected Values |
|||
Negative Control⊕ |
1 |
3 |
8 |
5 |
|
0.032 |
|
|
2 |
1 |
4 |
3 |
|
0.047 |
|
|
|
3 |
2 |
5 |
3 |
|
0.031 |
|
|
|
|
|
|
3.7* |
|
0.037 ♦ |
|
4.2 |
|
Positive Control⊕ |
4 |
3 |
75 |
72 |
68.3 |
2.495 |
2.458 |
|
5 |
4 |
80 |
76 |
72.3 |
2.260 |
2.223 |
|
|
6 |
4 |
70 |
66 |
62.3 |
2.930 |
2.893 |
|
|
|
|
|
|
67.7 • |
|
2.525 • |
105.5 |
|
Test Item |
16 |
3 |
74 |
71 |
67.3 |
0.008 |
0.000 |
|
17 |
3 |
71 |
68 |
64.3 |
0.027 |
0.000 |
|
|
18 |
6 |
90 |
84 |
80.3 |
0.033 |
0.000 |
|
|
|
|
|
|
70.7 • |
|
0.000 • |
70.7 |
OD = Optical Density
* = Mean of the post-treatment − pre-treatment values
♦ = Mean permeability • = Mean corrected value
⊕ = Control group shared with Project numbers 41205084, 41205116, 41205121, 41205129 and 41205134
Table 3: Corneal Epithelium Condition Post Treatment
Treatment |
Cornea Number |
Observation |
Post Treatment |
||
Negative Control⊕ |
1 |
clear |
2 |
clear |
|
3 |
clear |
|
Positive Control⊕ |
4 |
cloudy |
5 |
cloudy |
|
6 |
cloudy |
|
Test Item |
16 |
cloudy |
17 |
cloudy |
|
18 |
cloudy |
Applicant's summary and conclusion
- Interpretation of results:
- other: CLP/EU GHS Category 1 (H318) according to Regulation (EC) No 1272/2008
- Conclusions:
- Under conditions of the BCOP test method, the test item is considered to cause serious eye damage.
The available data on eye irritation of the test substance meet the criteria for classification as serious eye damage/eye irritation Cat. 1 (H318) according to Regulation (EC) 1272/2008.
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