Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 December 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
in accordance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: waxy solid
Details on test material:
- Name of test material (as cited in study report): Guaiacwood oil
- Appearance: Yellow-brown waxy solid
- Storage condition of test material: at room temperature in the dark

Test animals / tissue source

Species:
cattle
Strain:
other: Bovine
Details on test animals or tissues and environmental conditions:
TEST SYSTEM
- Source: Local abattoir
- Age at study initiation: 12-60 months
From freshly slaughtered animals, eyes were transported to the test facility at day of slaughter and used within 24 hours.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent no treatment
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 0.75 mL
- Concentration: 100%
Duration of treatment / exposure:
10 minutes (incubation at 32+/-1 °C)
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
Test substance: 3 corneas
Negative control: 3 corneas
Positive control: 3 corneas
Details on study design:
SELECTION AND 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 Eagle’s Minimum Essential Medium (MEM) without phenol red and plugged. The holders were incubated at 32 ± 1 ºC for 60 minutes.

QUALITY CHECK OF THE ISOLATED CORNEAS
At the end of the incubation period each cornea was examined for defects. Only corneas free of damage were used.
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.

NUMBER OF REPLICATES
Three corneas with opacity values close to the median value of all corneas were allocated to the negative control. Three corneas were also allocated to the test item and three corneas to the positive control item.

NEGATIVE CONTROL USED
Identification: 0.9% w/v sodium chloride solution
Batch: 301038501
Purity: 0.9%
Expiry Date: 01 September 2015
Storage Conditions: Room temperature

POSITIVE CONTROL USED
Identification: Ethanol
Batch: STBD7546V
Purity: >99.8%
Expiry Date: 11 August 2015
Storage Conditions: Room temperature

APPLICATION DOSE AND EXPOSURE TIME
The MEM was removed from the anterior chamber of the BCOP holder and 0.75 mL of the test item or control items were applied to the appropriate corneas. 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 ± 1 ºC for 10 minutes.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 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.
- POST-EXPOSURE INCUBATION: The holders were incubated, anterior chamber facing forward, at 32 ± 1 ºC for 120 minutes.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity:
Light transmission measured quantitatively with the aid of an opacitometer.
A pre-treatment opacity reading was taken for each cornea using a calibrated opacitometer. After exposure and removal of the test substance, a post-treatment opacity reading was taken. After the post-exposure incubation, a final opacity reading was taken.
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.
- Corneal permeability:
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 ºC 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 using the Anthos 2001 microplate reader.
- Others (e.g, pertinent visual observations, histopathology):
Each cornea was observed visually before treatment, after the exposure and removal of the test substance, and after the post-exposure incubation.
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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) was calculated as follows:
Mean opacity value + (15*mean OD492 value)
- The change in opacity for each cornea was calculated by subtracting the initial opacity reading from the final opacity reading. This value was corrected by subtracting the average change in opacity for the negative control. The average is used to calculate the IVIS.
- The corrected OD492 was calculated by subtracting the OD492 of the negative control from the OD492 value of each treated cornea. The average is used to calculate the IVIS.

DECISION CRITERIA:
IVIS≤3 = "No category. Not requiring classification to UN GHS or EU CLP",
IVIS >3; ≤55 = "No prediction of eye irritation can be made",
IVIS >55 = "Category 1. UN GHS or EU CLP Causes serious eye damage".

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
IVIS: 2.2
Positive controls validity:
valid
Remarks:
IVIS: 41.2
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system:
The corneas treated with the test item were clear post treatment and clear 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The test was acceptable if the negative control produced an In Vitro Irritancy Score which is less than or equal to the upper limit for background opacity and permeability values during 2013 for bovine corneas treated with the respective negative control. When testing liquids the negative control range for opacity should be ≤4.7 and for permeability ≤0.080.
The negative control gave a mean opacity of 1.0 and mean permeability of 0.078. The negative control acceptance criteria were therefore satisfied.
- Acceptance criteria met for positive control:
The test was acceptable if the positive control produced an In Vitro Irritancy Score which fell within two standard deviations of the historical mean collated during 2013 for this testing facility. Therefore the In Vitro Irritancy Score should fall within the range of 27.8 to 51.0.
The positive control In Vitro Irritancy Score was 41.2. The positive control acceptance criterion was therefore satisfied.

Any other information on results incl. tables

Individual and Mean Corneal Opacity and Permeability Measurements 

Treatment

Cornea number

Opacity

Permeability (OD)

In Vitro Irritancy Score

Pre-Treatment

Post-Treatment

Post-Incubation

Post-Incubation – Pre-Treatment

Corrected Value

 

Corrected Value

 

Negative Control

9

1

1

2

1

 

0.094

 

 

18

1

1

3

2

 

0.089

 

 

22

1

0

0

0

 

0.051

 

 

 

 

 

 

1.0a

 

0.078c

 

2.2

 Positive Control

1

0

25

29

29

28.0

0.820

0.742

 

3

0

23

28

28

27.0

0.944

0.866

 

16

2

28

30

28

27.0

1.244

1.166

 

 

 

 

 

 

27.3b

 

0.925b

41.2

Test Item

17

2

4

4

2

1.0

0.147

0.069

 

19

2

2

1

-1

0.0

0.052

0.000

 

20

0

1

1

1

0.0

0.040

0.000

 

 

 

 

 

 

0.3b

 

0.023b

0.7

OD = Optical density

a= Mean of the post-incubation – pre-treatment values

b= Mean corrected value

c= Mean permeability

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of this study, the IVIS score for Guaiacwood oil was determined to be 0.7, which is below the limit for classification (IVIS ≤3). Therefore, the test item does not need to be classified in accordance with the classification criteria outlined in Annex I of 1272/2008/EC (CLP).
Executive summary:

An in vitro Bovine Corneal Opacity and Permeability (BCOP) Assay was performed with Guaiacwood oil according to OECD guideline 437 and under GLP conditions. Bovine cornea were acquired from a local abattoir, prepared and treated with the test substance, positive control or negative control. In Vitro Irritancy Scores (IVIS) were calculated based on the measured opacity and permeability of the cornea after exposure.

Under the conditions of this study, the IVIS score for Guaiacwood oil was determined to be 0.7, which is below the limit for classification (IVIS ≤3). Therefore, the test item does not need to be classified in accordance with the classification criteria outlined in Annex I of 1272/2008/EC (CLP).