Registration Dossier

Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
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:
solid
Remarks:
LIGHT YELLOW, SOLID MASS
Details on test material:
Name of test material as cited in study report: Cedrol - Cedarwood Texas oil distilled
Mfg Date 01/26/2016
Exp Date 01/25/2019

- Identification: Cedrol, Cedarwood Texas oil distilled
- Appearance: Light yellow, solid mass
- Batch: B-64530
- Purity/Composition: UVCB
- Test item storage: At room temperature
- Stable under storage conditions until: 25 January 2019 (expiry date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature protected from light

ODOUR DESCRIPTION: CONFORMS TO STANDARD
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and batch No.of test material: Provided by sponsor, B-64530
- Expiration date of the lot/batch: 25 January 2019
- Purity test date: 26 January 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Test item was equilibrated to 100°C for several minutes until completely liquefied to obtain a homogeneous sample.

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
Test System
- Source: Vitelco slaughterhouse, 's Hertogenbosch, The Netherlands
- Age at study initiation: young cattle
- Other info: the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.

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 or undiluted test item was introduced onto the epithelium of the cornea.
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 with cMEM.
Number of animals or in vitro replicates:
Triplicate
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 containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum ). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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:
-Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, 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: 3

NEGATIVE CONTROL USED:
- A negative control, physiological saline (Eurovet Animal Health, Bladel, The Netherlands) was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED:
- Ethanol, Batch K47177483, Identification number RS532, Purity >99.9%
Stable under storage conditions until 31 October 2020

APPLICATION DOSE AND EXPOSURE TIME:
- Undiluted, 10 minutes

TREATMENT METHOD:
- The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2
- POST-EXPOSURE INCUBATION: yes; 120 +/- 10 minutes

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)

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
1) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
2) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

EVALUATION 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) are: ≤ 3: No Category, > 3 ≤ 55: No prediction can be made, >55: Category 1

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
Main
Value:
>= -0.6 - <= 0.1
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- 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 61 and 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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:

Negative control
opacity -2.9-3.0 (SD 1.07, n=72)
permeability -0.016-0.042 (SD 0.01, n=65)
IVIS -2.8-3.0 (SD 1.17, n=66)
Positive control
IVIS 34.7-78.2 (SD 12.64, n=47)

Any other information on results incl. tables

Treatment

Mean

Opacity

Mean

Permeability

MeanIn vitroIrritation Score

Negative control

-0.5

 0.003

-0.4

Positive control

(Ethanol)

19.6

2.737

60.7

Test item

-0.2

0.059

0.7

Applicant's summary and conclusion

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
Cedrol, Cedarwood Texas oil distilled induced an IVIS ≤ 3. Based on these results, the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).
Executive summary:

The eye hazard potential of Cedrol, Cedarwood Texas oil distilled was evaluated according to OECD Guideline 437 (BCOP test). The eye damage was assessed through topical application of 750 µl of the undiluted Cedrol, Cedarwood Texas oil distilled for 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Cedrol, Cedarwood Texas oil distilled did not induce ocular irritation (no opacity and no permeability), resulting in a mean in vitro irritancy score of 0.7 after 10 minutes of treatment. In conclusion, since Cedrol, Cedarwood Texas oil distilled induced an IVIS ≤ 3, and the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).