Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 21 July, 2017 to 13 September, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP/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)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source: eyeballs were obtained from a local abattoir as a by-product from freshly slaughtered animals.
- Characteristics of donor animals (e.g. age, sex, weight): Eyeballs were obtained from adult healthy cattle (typically 12 to 60 months old).
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): The eyeballs collected were immersed in Hanks’ Balanced Salt Solution (HBSS), and stored and transported at less than 20 ºC until use in the test operation (temperature range during transportation: 14ºC to 17ºC).
- indication of any existing defects or lesions in ocular tissue samples: All eyeballs were macroscopically examined. Only corneas free of damage were used.
- Indication of any antibiotics used: Prior to transportation the eyeballs were placed in
HBSS supplemented with antibiotics (penicillin at 100 IU/mL and streptomycin at 100 μg/mL).

Test system

Vehicle:
other: not applicable
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 0.75 mL
- Concentration: undiluted test substance
Duration of treatment / exposure:
Exposure period of 10 minutes (at 32 ± 1°C), followed by rinsing
Duration of post- treatment incubation (in vitro):
120 minutes (at 32 ± 1°C)
Number of animals or in vitro replicates:
Three corneas were assigned to the negative control, test substance and the positive control groups.
Details on study design:
COLLECTION OF CORNEAS
The eyeballs were macroscopically observed and examined foe defects including corneal opacity, scratches, and neovascularization, and the corneas free of these abnormalities were used in the study. The corneas were dissected with a 2 to 3 mm rim of the sclera remaining, and were immersed in HBSS.

MOUNTING OF CORNEAS TO CORNEAL HOLDERRS AND EQUILIBRATION
After assigning the corneal numbers (Corneal Nos.1 to 12) for identification, the isolated corneas were mounted in corresponding corneas holders consisting of anterior and posterior chambers, which interfaced with the epithelial and endothelial sides of the cornea, respectively. Both chambers were filled with phenol red-free MEM, and then the corneas were immersed in the medium and equilibrated in an incubator set at 32 ± 1°C.

MEASUREMENT OF BASELINE CORNEAL OPACITY VALUES
Following the equilibration, the medium was collected and removed from both chambers of the corneal holders, and the chambers were filled with fresh phenol red-free MEM. The baseline opacity value of each cornea was measured using the opacitometer (BASF-OP3.0, BASF).
The cornea was then macroscopically observed again for defects including opacity, scratches, and neovascularization. As the results of measurement and observation of the corneas, one cornea (corneal No. 4) had scratches. This cornea was not used in subsequent operation and excluded from the study.

STUDY DESIGN
Among the corneas for which measurement of the baseline corneal opacity value was completed, 3 each were assigned to the negative control, test substance, and positive control groups in ascending order of the corneal Nos. The corneas not assigned to test groups were not used in subsequent operation and excluded from the study.

APPLICATION OF TEST SUBSTANCE AND CONTROLS
The medium in the anterior chamber of the corneal holder was removed, and 750 µL of the test substance or the control was directly applied onto the surface of the corneal epithelium. The cornea was exposed to the test substance or the control in the incubator set at 32°C ± 1°C for 10 min, keeping the holder in a horizontal position. After completion of exposure, the test substance or the control was removed from the anterior chamber of the corneal holder, and the epithelial layer was rinsed with phenol red-free MEM, and the chambers were filled with phenol red-free MEM and incubated at 32°C ± 1°C.for 2 hours.

MEASUREMENT OF CORNEAL OPACITY VALUES FOLLOWING TREATMENT WITH TEST SUBSTANCE AND CONTROL
After-2-hr incubation, phenol red-free MEM in both of the anterior and posterior chambers was replaced with fresh MEM, and the opacity value of each cornea following treatment with the test substance or the control was measured using the opacitometer.

MEASUREMENT OF PERMEABILITY
After measurement of corneal opacity values, the medium was removed from the anterior chamber of the corneal holder and 1 mL of sodium fluorescein solution was added to anterior chamber. The corneal holder was then incubated in a horizontal position in an incubator set at 32°C ± 1°C for 90 min. After completion of incubation, the total volume of the medium in the posterior chamber of the corneal holder was collected in a test tube, and the amount of the sodium fluorescein dye that passed across the cornea was measured as absorbance at 490 nm wavelength with an ultraviolet visible spectrophotometer (UV-2600, shimadzu Corporation).

CALCULATION OF OPACITY VALUE
The difference (“Final opacity”) between the opacity value following treatment with the test substance or control and the baseline opacity value was calculated for each cornea, and then group was subtracted to serve as the “Mean corrected final opacity.” The mean corrected final opacity value was used for calculated of the In Vitro Irritancy Score (IVIS).
Mean corrected final opacity
= mean final opacity of test substance or positive control – mean final opacity of negative control
For the negative control group, the mean final opacity value of each cornea was used for calculation of IVIS.
Mean opacity of negative control = mean final opacity of negative control

CALCULATION OF PERMEABILITY
For the test substance and positive control group, the blank OD450 value was subtracted from each measured OD450 value, and subsequently the mean OD490 value of the negative control was subtracted to calculate the “Corrected OD490 change” of each cornea. The corrected OD490 changes were averaged for each test group (“Mean corrected OD490 change”) and used for calculation of IVIS.
Corrected OD490 change of each cornea
= measured OD490 – blank OD490 – mean OD490 of negative control
Mean corrected OD490 change
= total OD490 of each cornea treated with test substance or positive control / number of corneas treated with test substance of positive control
For the negative control group, the blank OD490 value was subtracted from each measured OD490 value (“OD490 change”), which was averaged and used for calculation of IVIS.
OD490 change of each cornea treated with negative control
= measured OD490 of negative control – blank OD490
Mean OD490 change of negative control
=total OD490 of each cornea treated with negative control / number of corneas treated with negative control

IN VITRO IRRITANCY SCORE
In Vitro Irritancy Score (IVIS) was calculated from the opacity and permeability values.
IVIS = (mean opacity) + 15 [mean permeability (OD490)]

EVALUATION
According to the evaluation criteria described in OECD TG 437, IVIS ≤ 3 was classified as UN GHS No Category, IVIS > 3; ≤ 55 as No prediction can be made for the degree of irritancy, and IVIS > 55 as UN GHS Category 1, a severe irritant or a corrosive.

STUDY ACCEPTANCE CRITERIA
The following study acceptance criteria were adopted according to those described in OECD TG 437:
A test is considered acceptable if the IVIS of the positive control is within 2 standard deviations of the latest historical control mean. A test is considered acceptable if the opacity and permeability values of the negative control are less than the upper limit of opacity and permeability values of the historical control data obtained from the bovine corneas treated with the negative control.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Remarks:
(IVIS)
Value:
-0.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of irritation

Applicant's summary and conclusion

Interpretation of results:
other: UN GHS No Category
Conclusions:
As the results, the IVIS of the test substance was -0.4 (IVIS ≤ 3), and the test substance was classified as UN GHS No category and identified as non-irritants under the present study conditions.
Executive summary:

The potential ocular irritancy of(3S,6E)-3,7,11-trimethyldodeca-6,10-dien-1-ol was evaluated by the BCOP test using indicators of effects on the opacity and permeability to fluorescein dye in the isolated bovine corneas.

To each of the negative control (distilled water), test substance ((3S,6E)-3,7,11-trimethyldodeca-6,10-dien-1-ol), and positive control (dimethylformamide) groups, 3 corneas were assigned and exposed to the test substance or control for 10 min. From the measured opacity and permeability values, the In Vitro Irritancy Scores (IVIS) were calculated and used for evaluation of the potential ocular irritancy.

As the results, the IVIS of the negative control was 0.7 and that of the test substance was -0.4 (IVIS ≤ 3), and the negative control and the test substance were classified as UN GHS No category and identified as non-irritants. While the IVIS of the positive control was 86.1 (IVIS > 55), and the positive control was classified as UN GHS Category 1 and identified as a severe irritant or a corrosive.

In conclusion, (3S,6E)-3,7,11-trimethyldodeca-6,10-dien-1-ol was classified as UN GHS No category and was not identified as an ocular corrosive or severe irritant under the present study conditions.