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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:
April 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
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)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Cas Number:
28973-99-1
Molecular formula:
C15H24
IUPAC Name:
(3Z,6Z)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Constituent 2
Chemical structure
Reference substance name:
(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene
Cas Number:
25532-79-0
Molecular formula:
C15H24
IUPAC Name:
(E)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)cyclohex-1-ene
Constituent 3
Chemical structure
Reference substance name:
(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)
Cas Number:
29837-07-8
Molecular formula:
C15H24
IUPAC Name:
(Z)-1-methyl-4-(6-methylhepta-2,5-dien-2-yl)
Constituent 4
Chemical structure
Reference substance name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
EC Number:
242-582-0
EC Name:
(E)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Cas Number:
18794-84-8
Molecular formula:
C15H24
IUPAC Name:
7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Constituent 5
Chemical structure
Reference substance name:
(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Cas Number:
53585-13-0
Molecular formula:
C15H24
IUPAC Name:
(E)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Constituent 6
Chemical structure
Reference substance name:
(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Cas Number:
13062-00-5
Molecular formula:
C15H24
IUPAC Name:
(Z)-1-methyl-4-(6-methylhept-5-en-2-ylidene)
Constituent 7
Chemical structure
Reference substance name:
1-methyl-4-(6-methylhepta-1,5-dien-2-yl)
Cas Number:
869843-05-0
Molecular formula:
C15H24
IUPAC Name:
1-methyl-4-(6-methylhepta-1,5-dien-2-yl)
Constituent 8
Chemical structure
Reference substance name:
(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Cas Number:
26560-14-5
Molecular formula:
C15H24
IUPAC Name:
(3Z,6E)-3,7,11-trimethyldodeca-1,3,6,10-tetraene
Constituent 9
Chemical structure
Reference substance name:
(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Cas Number:
28973-97-9
Molecular formula:
C15H24
IUPAC Name:
(Z)-7,11-dimethyl-3-methylenedodeca-1,6,10-triene
Constituent 10
Chemical structure
Reference substance name:
1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene
Cas Number:
72345-84-7
Molecular formula:
C15H24
IUPAC Name:
1-methyl-4-(6-methylhept-5-en-2-yl)cyclohexa-1,4-diene
Constituent 11
Chemical structure
Reference substance name:
2,6,10-trimethyldodeca-2,6,9,11-tetraene
EC Number:
207-948-6
EC Name:
2,6,10-trimethyldodeca-2,6,9,11-tetraene
Cas Number:
502-61-4
Molecular formula:
C15H24
IUPAC Name:
3,7,11-trimethyldodeca-1,3,6,10-tetraene
Constituent 12
Chemical structure
Reference substance name:
Likely sesquiterpene hydrocarbons
Cas Number:
n/a
Molecular formula:
C15H24
IUPAC Name:
Likely sesquiterpene hydrocarbons
Test material form:
liquid
Details on test material:
UVCB substance
Specific details on test material used for the study:
Batch: VE00446520
Purity/Composition: UVCB
Test item storage At room temperature protected from light
Stable under storage conditions until 27 June 2017 (expiry date)

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands). 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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
750ul of either the negative control, positive control (Ethanol) or 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.
Duration of post- treatment incubation (in vitro):
Subsequently the corneas were incubated for 120 +/- 10 minutes at 32 +/- 1'C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.
Details on study design:
Study design
5.5.1. 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-glutamine
(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.
5.5.2. Cornea selection and Opacity reading
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 (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.
5.5.3. Treatment of corneas and opacity measurements
The medium from the anterior compartment was removed and 750 l of either the negative
control, positive control (Ethanol) or test item was introduced onto the epithelium of the
cornea. The holders were slightly rotated, with the corneas maintained in a horizontal
position, to ensure uniform distribution of the control or the test item over the entire cornea.
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. Possible pH effects of the test item on the corneas were recorded.
The medium in the posterior compartment was removed and both compartments were refilled
with fresh cMEM. Subsequently the corneas were incubated for 120  10 minutes at
32  1C. After the completion of the incubation period opacity determination was
performed. Each cornea was inspected visually for dissimilar opacity patterns.
5.5.4. Opacity measurement
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.
The opacity value (measured with the device OP-KIT) was calculated according to:
𝑂𝑝𝑎𝑐𝑖𝑡𝑦 =
𝐼0
𝐼
− 0.9894
0.0251
With I0 the empirically determined illuminance through a cornea holder but with windows
and medium, and I the measured illuminance through a holder with cornea.
The change in opacity for each individual cornea (including the negative control) was
calculated by subtracting the initial opacity reading from the final post-treatment reading. The
corrected opacity for each treated cornea with the test item or positive control was calculated
by subtracting the average change in opacity of the negative control corneas from the change
in opacity of each test item or positive control treated cornea.
The mean opacity value of each treatment group was calculated by averaging the corrected
opacity values of the treated corneas for each treatment group.
5.5.5. Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein
(Sigma-Aldrich, Germany) was evaluated.
The medium of both compartments (anterior compartment first) was removed. The posterior
compartment was refilled with fresh cMEM. The anterior compartment was filled with 1 ml
of 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/ml cMEM solution. The
holders were slightly rotated, with the corneas maintained in a horizontal position, to ensure
uniform distribution of the sodium-fluorescein solution over the entire cornea. Corneas were
incubated in a horizontal position for 90  5 minutes at 32  1C.
5.5.6. Permeability determinations
After the incubation period, the medium in the posterior compartment of each holder was
removed and placed into a sampling tube labelled according to holder number. 360 μl of the
medium from each sampling tube was transferred to a 96-well plate. The optical density at
490 nm (OD490) of each sampling tube was measured in triplicate using a microplate reader
(TECAN Infinite® M200 Pro Plate Reader). Any OD490 that was 1.500 or higher was diluted
to bring the OD490 into the acceptable range (linearity up to OD490 of 1.500 was verified
before the start of the experiment). OD490 values of less than 1.500 were used in the
permeability calculation.
The mean OD490 for each treatment was calculated using cMEM corrected OD490 values. If a
dilution has been performed, the OD490 of each reading of the positive control and the test
item was corrected for the mean negative control OD490 before the dilution factor was applied
to the reading.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Run / experiment:
mean
Value:
-1.2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / 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 47 and 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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Bisabolene did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.2 after 10 minutes of treatment.
Since Bisabolene induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

Evaluation of the eye hazard potential of Bisabolene using the Bovine Corneal Opacity and Permeability test (BCOP test).

This report describes the potency of chemicals to induce serious eye damage using isolated bovine corneas. The eye damage of Bisabolene was tested through topical application for 10 minutes.

The study procedures described in this report were based on the most recent OECD guideline.

Batch VE00446520 of Bisabolene was a colourless to pale yellow liquid. The test item was applied as it is (750 μl) directly on top of the corneas.

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 47 and 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.

Bisabolene did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of -1.2 after 10 minutes of treatment.

Since Bisabolene induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

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