Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 19, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD test Guideline No. 437 without any deviation.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
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. certificate)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): CISTUS CONCRETE
- Physical state: Dark brown solid at room temperature
- CAS No.: 89997-74-0
- EINECS-No.: 289-711-7
- Analytical purity: 100%UVCB
- Lot/batch No.: 14822 (réf. 101412)
- Production date: 13 May 2013
- Date of Receipt: 11 February 2014
- Expiration date of the lot/batch: 13 May 2015
- Composition: Paraffins, labdanolic derivated
- Storage condition of test material: Room temperature 20 ± 5 °C, keep away from light and humidity

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 (typically 12 to 60 months old) were obtained from a local abattoir as a by-product from freshly slaughtered animals. The eyes were excised by an abattoir employee after slaughter, and were placed in Hanks’ Balanced Salt Solution (HBSS) supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 µg/mL). They were transported to the test facility over ice packs on the same day of slaughter. The corneas were prepared immediately on arrival.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Test Item Formulation and Experimental Preparation: The test item was warmed and melted to 70 °C and then allowed to cool to 32 °C before use.
- Amount(s) applied (volume or weight with unit): 0.75 mL was applied on each cornea
Duration of treatment / exposure:
Each holder was incubated, anterior chamber uppermost, at 32 ± 1 ºC for 10 minutes.
Duration of post- treatment incubation (in vitro):
The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.
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:
- Treatment of corneas: 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 Eagle’s minimum essential medium (MEM) and incubated for 60 minutes at 32 ± 1 °C. Before the treatment, opacity measurement was performed using an opacitometer. MEM was removed from anterior chamber and the test item (0.75 mL) was applied on each cornea. The holders were incubated, anterior chamber uppermost, at 32 ± 1 °C for 10 minutes. 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 without phenol red. The anterior chamber was refilled with fresh complete MEM without phenol red. A post-treatment opacity reading was taken and each cornea was visually observed. The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes. After incubation the holders were removed from the incubator and a final opacity reading was taken. 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 posterior and anterior chambers were removed, the posterior chamber is replaced with fresh complete MEM and the anterior chamber is 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.
- Permeability determinations: 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 using the Anthos 2001 microplate reader.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
0.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
- In Vitro Irritancy Score (IVIS) for test item, negative and positive controls were 0.2, 1.9 and 46.2, respectively.
- Corneal Epithelium Condition: 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

Criteria for an Acceptable Test

The positive control In Vitro Irritancy Score was within the range of 27.8 – 51.0. The positive control acceptance criterion was therefore satisfied.

 

The negative control gave opacity of ≤4.7 and permeability ≤0.080. The negative control acceptance criteria were therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the test conditions, test item is not classified as eye irritant according to the Regulation (EC) No. 1272/2008.
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 undiluted test item was applied to isolated bovine corneas for 10 minutes followed by an incubation period of 120 minutes. 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 0.2, 1.9 and 46.2, respectively. 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.

 

The positive control In Vitro Irritancy Score was within the range of 27.8 to 51.0, therefore the acceptance criterion was satisfied. The negative control gave opacity of ≤4.7 and permeability ≤0.080, therefore the acceptance criterion was satisfied.

 

Under the test conditions, test item is not classified as eye irritant according to the Regulation (EC) No. 1272/2008 and the Directive 67/548/EEC.