Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 Sep 2017 - 03 Oct 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guideline
Qualifier:
according to
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:
26 July, 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Remarks:
Pale yellow liquid
Details on test material:
Batch 04455//PGP7216
Appearance: Pale yellow liquid
UVCB

- Expiration date of the lot/batch: 30 March 2019
- Purity test date: 15 dec 2016
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 04455//PGP7216
- Expiration date of the lot/batch: 30 March 2019
- Purity test date: 15 dec 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature
- Stability under test conditions: Not available

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was tested neat.

OTHER SPECIFICS: UVCB

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: Vitelco slaughterhouse, 's Hertogenbosch, The Netherlands
- Characteristics of donor animals (e.g. age, sex, weight): young cattle
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions
- Time interval prior to initiating testing: as soon as possible after slaughter
- Indication of any existing defects or lesions in ocular tissue samples: The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µl of either the negative control, positive control (Ethanol) or test item.
- Concentration (if solution): Undiluted.
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1 °C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.
Duration of post- treatment incubation (in vitro):
Corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.
Number of animals or in vitro replicates:
triplacates
Details on study design:
SELECTION AND 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-gl utamine (Life Technologies) and 1% (v/v) Foetal 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.

QUALITY CHECK OF THE ISOLATED CORNEAS
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 (BASFOP3.0, 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.

NUMBER OF REPLICATES
Three corneas were selected at random for each treatment group. 2 experiments were performed.

NEGATIVE CONTROL USED
Physiological saline (Eurovet Animal Health, Bladel, The Netherlands).

POSITIVE CONTROL USED
Ethanol, purity >99.9%

APPLICATION DOSE AND EXPOSURE TIME
750 µl undiluted test item was introduced onto the epithelium of the cornea. Corneas were incubated in a horizontal position for 10 ± 1 minutes at 32 ± 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM. Possible pH effects of the test item on the corneas were recorded.
POST-EXPOSURE INCUBATION: Corneas were incubated for 120 ± 10 minutes at 32 ± 1 °C with fresh cMEM.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microplate reader (TECAN Infinite® M200 Pro Plate Reader).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value).

DECISION CRITERIA:
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value) Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints. The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN GHS Category 1) and test items not requiring classification for eye irritation or serious eye damage (UN GHS No Category): In vitro score range ≤ 3: No Category, > 3 to ≤ 55: No prediction can be made, >55: Category 1.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
First experiment
Value:
3.9
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
1.4
Positive controls validity:
valid
Remarks:
5
Irritation parameter:
in vitro irritation score
Run / experiment:
Second experiment
Value:
5.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.4
Positive controls validity:
valid
Remarks:
57
Irritation parameter:
cornea opacity score
Run / experiment:
First experiment
Value:
4.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
1.2
Positive controls validity:
valid
Remarks:
18
Irritation parameter:
cornea opacity score
Run / experiment:
Second experiment
Value:
5.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.4
Positive controls validity:
valid
Remarks:
22
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Any other information on results incl. tables

Historical control data

  Negative control Positive control
  Opacity Permeability In vitro Irritancy Score In vitro Irritancy Score
Range -2.9 – 3.0 -0.016 – 0.042 -2.8 – 3.0 34.7 – 78.2
Mean 0.08 0.01 0.17 56.01
SD 1.04 0.01 1.14 12.51
n 84 77 78 55

SD = Standard deviation

n = Number of observations

The above mentioned historical control data range of the controls were obtained by collecting all data over the period of Aug 2014 to Aug 2017.

Applicant's summary and conclusion

Interpretation of results:
other: No prediction on classification can be made
Remarks:
Based on CLP criteria (EC 1272/2008 and its updates)
Conclusions:
Under the conditions of this study, Petitgrain Oil (citrus aurantium) induced an IVIS > 3 ≤ 55, therefore no prediction on the classification can be made in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).
Executive summary:

The eye irritation potential of Petitgrain oil (citrus aurantium) was measured by its ability to induce opacity and increase permeability in an isolated bovine cornea using the Bovine Corneal Opacity and Permeability test (BCOP test). The study procedures described in this report were based on the most recent OECD test guideline 437. Petitgrain oil (citrus aurantium) was applied as received to the test system, and induced a mean in vitro irritancy score of 3.9 (first experiment) and 5.8 (second experiment) after 10 minutes of treatment. The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Under the conditions of this study, Petitgrain Oil (citrus aurantium) induced an IVIS > 355, therefore no prediction on the classification can be made in accordance with the criteria outlined in Annex I of the CLP regulations (1272/2008/EC).