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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18th February 2019 - 19th February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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)
Version / remarks:
Adopted October 09, 2017
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine
EC Number:
831-167-5
Cas Number:
2126827-44-7
Molecular formula:
C92H106N8O8
IUPAC Name:
2,9,16,23-tetrakis(2,5-di-tert-butyl-4-methoxyphenoxy)phthalocyanine
Test material form:
solid
Specific details on test material used for the study:
Batch (Lot) Number: 2715/18/01
Expiry date: 01 July 2020 (expiry date)
Physical Description: Dark green solid
Purity/Composition: 99%
Storage Conditions: At room temperature protected from light

Additional information.

Test Facility test item number: 210026/A
Purity/Composition correction factor: No correction factor required
Test item handling: Use amber glassware or wrap container in aluminum foil

Test animals / tissue source

Species:
cattle
Strain:
not specified

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
356 to 369 mg
Duration of treatment / exposure:
for 240 +/- 10 minutes at 32 +/- 1 °C.
Duration of post- treatment incubation (in vitro):
Opacity determined directly after the treatment; Permiability determined after post-treatment incubation of 90 +/- 5 minutes at 32 +/- 1 °C.
Number of animals or in vitro replicates:
Triplicate.
Details on study design:
Test System.

Test System: Bovine eyes were used as soon as possible after slaughter.

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.

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 +/- 1 °C. The corneas were incubated for the minimum of 1 hour at 32 +/- 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.

Substance Preparation.
No correction will be made for the purity/composition of the substance.
Since no workable suspension of the substance in physiological saline could be obtained, the substance was used as delivered by the sponsor and added pure on top of the corneas.
To protect the substance from light, amber-colored glassware or tubes wrapped in tin-foil were used for substance preparations.

Treatment of Corneas and Opacity Measurements.

The medium from the anterior compartment was removed and 750 ul of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The substance was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (356 to 369 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 +/- 1°C. After the incubation the solutions and the substance 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 substance 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.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
1
Value:
0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
2
Value:
-2.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
3
Value:
0.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

Opacity Score.

Treatment

Opacity

before treatment

Opacity

after treatment

Final Opacity1

Negative control corrected Final Opacity2

Mean Final Opacity

 

Negative control

4.1

5.4

1.2

 

0.7

3.4

3.5

0.1

4.3

5.3

0.9

 

Positive control

4.6

160.2

155.6

155

137

3.2

113.1

109.9

109

3.1

149.6

146.5

146

 

Substance

3.4

4.3

1.0

0.2

-0.6

3.4

2.3

-1.1

-1.8

4.5

5.0

0.5

-0.3

1  Final Opacity = Opacity after treatment – Opacity before treatment.

2  Negative control corrected Final Opacity = Final opacity – Mean final opacity negative control

In vitro Irritancy Score.

Treatment

Final Opacity2

Final OD4902

In vitro Irritancy Score1

 

Negative control

1.2

0.001

1.2

0.1

0.023

0.4

0.9

0.010

1.1

 

Positive control

155

1.225

173

109

2.577

148

146

1.733

172

 

Substance

-0.2

-0.008

0.1

-1.8

-0.019

-2.1

-0.3

0.072

0.8

1  In vitro irritancy score (IVIS) = opacity value + (15 x OD490 value).

2  Positive control and substance are corrected for the negative control.

Permeability Score (uncorrected).

Treatment

Dilution factor

OD490

1

OD490

2

OD490

3

Average OD

Final OD

Mean final negative control

 

Negative control

1

0.001

0.001

0.001

0.001

0.001

0.011

1

0.026

0.028

0.016

0.023

0.023

1

0.007

0.007

0.016

0.010

0.010

 

 

Positive control

1

1.229

1.240

1.241

1.237

1.237

 

6

0.442

0.440

0.441

0.441

2.646

 

6

0.300

0.300

0.301

0.300

1.802

 

 

 

Substance

1

0.003

0.003

0.004

0.003

0.003

 

1

-0.008

-0.008

-0.007

-0.008

-0.008

 

1

0.086

0.082

0.082

0.083

0.083

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The results of an eye corrosion/irritation study employing isolated bovine cornea exposed to undiluted substance for 240-minutes indicate that it is a non-irritant to the eye.
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 undiluted (356 - 369 mg) onto corneas (n=3) for a period of 240 minutes, followed by rinsing of the substance. The opacity of the corneas was then determined. The permeability measurements were determined after 90 minutes of incubation with fluorescein. The results of an eye corrosion/irritation study show that the mean in vitro irritance score was -0.4, indicating that that the substance is non-irritant to the eye.