Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted according to internationally accepted guidelines and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 437. 7 September 2009, OECD Guideline for the Testing of Chemicals: Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants.
Deviations:
no
Principles of method if other than guideline:
Bovine Corneal Opacity and Permeability Test: An In Vitro Assay of Ocular Irritancy. Fundamental and Applied Toxicology 18: 442-449, 1992.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Diisoamyl phthalate (Diisopentyl phthalate)

- Substance type: organic
- Physical state: colorless to slightly yellow liquid
- Analytical purity: 99.98%

- Lot/batch No.: 7920-1-1
- Expiration date of the lot/batch: August 12, 2012

- Stability under test conditions: not reported
- Storage condition of test material: at room temperature (ca. 20°C), in the dark

Test animals / tissue source

Species:
other: in vitro assay on bovine ocular tissue
Strain:
other: n/a
Details on test animals or tissues and environmental conditions:
Source
The bovine eyes, supplied by a reputable Abattoir, were excised by an abattoir employee and collected as soon after slaughter as possible (excised at 13.00 hours, 15 June 2010).
Instructions were given to avoid damaging the corneas during excision. Excised eyes were maintained and transported to the laboratory, at ambient temperature, in sufficient HBSS, containing 1% (v/v) penicillin/streptomycin solution, to cover all the eyes in the receptacle.
The eyes were used within 4 hours of slaughter (incubation of mounted corneas commenced at 15.22 hours, 15 June 2010).

Preparation of Corneas
All eyes were carefully examined, macroscopically, for defects (opacity, scratches, pigmentation, cuts, etc.) and those exhibiting defects were discarded. The tissue surrounding the eyeball was carefully pulled away and the cornea dissected leaving approximately 2 to 3 mm of sclera present around the cornea. The isolated corneas were stored in a petri dish containing HBSS plus 1% penicillin/streptomycin solution until all the corneas had been dissected. Once all the corneas had been dissected, they were rinsed in fresh HBSS plus 1% penicillin/streptomycin solution prior to mounting.

The corneas were mounted in the cornea holders with the endothelial side against the O-ring of the posterior half of the holder. Each cornea was gently flattened over the O-ring and holder surface with a wetted, gloved finger to expel any air. The anterior half of the holder was then positioned on top of the cornea and secured with screws. Both compartments of the holder were filled with cMEM, using a syringe. The posterior compartment was always filled first to return the cornea to its natural shape. Care was taken to ensure no air bubbles were present within the holders.
The holders were then plugged and incubated, in an upright position, for 60 minutes ± 5 minutes at 32°C ± 1°C in a waterbath. The waterbath temperature remained within the limits of 32°C ± 1°C throughout the experiment.

At the end of the 60 minute incubation period, the medium was removed from both the anterior and posterior compartments using a pipette tip attached to a vacuum pump. The compartments were refilled with fresh cMEM. Again care was taken to ensure no air bubbles were present within the holders. The posterior compartment was then plugged and the basal opacity measurements performed.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: excised corneas employed for testing
Amount / concentration applied:
TEST MATERIAL
- Amount applied (volume) 0.75 mL
- Concentration : undiluted

Duration of treatment / exposure:
Applied once, allowed to remain in contact with the cornea for 10 mins, then washed off with MEM seven times to ensure complete removal of test material.
Observation period (in vivo):
Following washing to remove the test material the anterior compartment holding the cornea was then filled with cMEM taking care to ensure no air bubbleswere present in the compartment. Once all the air bubbles had been removed, the anterior compartment was re-plugged and the holders returned to the waterbath and incubated, in an upright position, for two hours ± 10 minutes at 32°C ± 1°C.
Following completion of the two hour incubation period, the medium was removed from both compartments and replaced with fresh cMEM. The posterior compartment was re-plugged and the opacity of each cornea measured and recorded. The opacity values obtained at this stage were used in calculating the final In Vitro Irritancy Score.
Throughout the assay the corneas were examined for opaque spots or other irregularities.
Number of animals or in vitro replicates:
Not applicable as excised corneas only employed for testing.
Details on study design:
Control corneas were treated with the positive control (ethanol) in exactly the same manner as for the test corneas.

Results and discussion

In vivo

Irritant / corrosive response data:
Throughout the assay the corneas were examined for opaque spots or other irregularities.
Following treatment with test substance, Diisoamyl Phthalate, the corneas were noted as clear.
The corneas treated with the positive control, ethanol, were opaque and the corneas treated with the negative control, 0.9% saline, were clear.
Other effects:
pH determination: The pH of the test substance, measured using pH sticks, was approximately 7.0.

Any other information on results incl. tables

Diisoamyl Phthalate

-0.333 ± 0.577

-0.003± 0.016

-0.4 ± 0.3

Non Corrosive/
Non Severe

Ethanol

21.000 ± 3.000

1.625 ± 0.074

45.4 ± 4.0

Non Corrosive/ Non Severe

09% saline

0.000 ± 1.000

0.021 ± 0.011

N/A

N/A

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: as specified in the study design
Conclusions:
Based upon the referred classification criteris the test article Diisopentyl phthalate is classified as non corrosive/non severe eye irritant
Executive summary:

The Bovine Corneal Opacity and Permeability Assay (BCOP) was performed to assess the ocular irritancy potential in vitro of the test substance. Ethanol was tested in parallel as a positive control.

The assay uses isolated bovine corneas as a means of assessing the ocular corrosivity or severe irritancy potential of test substances in vitro. The isolated corneas were obtained as a by-product of the meat production industry. Two endpoints, corneal opacity and permeability, were measured and combined to give an In Vitro Irritancy Score which can be used to classify and rank test substances as potential eye irritants according to OECD guideline 437 (OECD 437).

The test substance, Diisoamyl Phthalate, elicited an In Vitro Irritancy Score of -0.4 ± 0.3 and was predicted to be a non corrosive/non severe eye irritant.