Registration Dossier

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:
2016
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

1
Chemical structure
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(2+) diphosphate

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.