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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:
25 January 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well conducted and well described study in accordance with GLP and OECD Guideline 437 without any deviation.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report date:
2013

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
UK GLP Compliance Programme (inspected on 10 July 2012/ signed on 30 November 2012)

Test material

Constituent 1
Reference substance name:
Cypress, Cupressus sempervirens, ext.
EC Number:
283-626-9
EC Name:
Cypress, Cupressus sempervirens, ext.
Cas Number:
84696-07-1
IUPAC Name:
Cypress, Cupressus sempervirens, ext.
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): Cypress oil
- Physical state: Extremely pale yellow liquid
- Lot/batch No.: L4220024
- Date received: 29 November 2012
- Expiration date of the lot/batch: 01 July 2014
- Storage condition of test material: Approximately 4 °C in the dark

Test animals / tissue source

Species:
other: bovine eye
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Eyes from adult cattle were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised and placed in Hanks' Balanced Salt Solution (HBSS), supplemented with Penicillin/Streptomycin, and transported to the laboratory on ice packs. The corneas were prepared immediately on arrival.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
other: negative control: 0.9 % w/v sodium chloride solution
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.75 mL was applied on each cornea
- Concentration: Test material: undiluted; negative control: 0.9 % w/v sodium chloride solution; positive control: ethanol
Duration of treatment / exposure:
The undiluted test item was applied for 10 minutes at 32 ± 1 °C followed by an incubation period of 120 ± 10 minutes at 32 ± 1 °C.
Observation period (in vivo):
- Corneal opacity was measured before and after 120 ± 10 minutes of incubation.
- Application of Sodium Fluorescein (4 mg/mL) and corneal permeability was measured after 90 min of incubation at 32 ± 1 °C.
Number of animals or in vitro replicates:
Total: 9 corneas - 3 corneas/group for test item, negative and positive controls
Details on study design:
Details of test procedure: Corneas obtained from freshly slaughtered adult cattle (from a local abattoir) were mounted in corneal holders. Both chambers of each BCOP holder were filled with complete MEM culture media (MEM) and incubated for 60 minutes at 32 ± 1 °C. Before the treatment, opacity measurement was performed using an opacitometer. The test item (0.75 mL) was applied on each cornea for 10 minutes at 32 ± 1 °C. At the end of the exposure period the test item and control items were removed from the anterior chamber and the cornea was rinsed three times with fresh complete MEM containing phenol red before a final rinse with complete MEM. The anterior chamber was refilled with fresh complete MEM. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 °C for 120 ± 10 minutes. After incubation the holders were removed from the incubator and a final opacity reading was taken. Each cornea was visually observed. 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 (4 mg/mL). The dosing holes were plugged and the holders incubated, anterior chamber uppermost, at 32 ± 1 °C for 90 minutes. 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 492 nm (OD492) was measured.

Results and discussion

In vivo

Results
Irritation parameter:
other: In Vitro Irritancy Score (IVIS)
Basis:
mean
Time point:
other: 120 minutes
Score:
5.3
Reversibility:
other: not applicable
Irritant / corrosive response data:
- In Vitro Irritancy Score (IVIS) were 5.3, 0.3 and 50.5 for test item, negative and vehicle controls, respectively.
Other effects:
- The corneas treated with the test item were clear post treatment and post incubation.
- The corneas treated with the negative control item were clear post treatment and post incubation.
- The corneas treated with the positive control item were cloudy post treatment and post incubation.

Any other information on results incl. tables

See the attached document for information on tables of results

Applicant's summary and conclusion

Interpretation of results:
other: not an ocular corrosive or severe irritant
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, Cypress oil, induced an IVIS of 5.3 and is not classified as corrosive or severe irritant for the isolated bovine cornea.
Executive summary:

In an in vitro eye irritation study performed according to the OECD Guideline 437 and in compliance with GLP, 0.75 mL of Cypress oil was applied to isolated bovine corneas mounted in corneal holders, and incubated for 120 ± 10 minutes at 32 ± 1 °C using the closed chamber method. Three corneas were used for each treated series (undiluted test item; negative control; positive control: ethanol). Before the treatment, a first opacity measurement was performed using an opacitometer. The two endpoints, decreased light transmission through the cornea (opacity) and increased passage of sodium fluorescein dye through the cornea (permeability) were combined in an empirically derived formula to generate an In Vitro Irritancy Score (IVIS).

 

The test item, negative and positive control induced an IVIS of 5.3, 0.3 and 50.5, respectively. The corneas treated with the negative control and test item were clear post treatment and post incubation. The corneas treated with the positive control item were cloudy post treatment and post incubation. The positive control In Vitro Irritancy Score was within the range of 30.9 to 67.7, therefore the acceptance criterion was satisfied.

Under the test conditions, Cypress oil induced an IVIS of 5.3. Cypress oil is therefore not classified as as an ocular corrosive or severe irritant for the eyes.

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