Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
June 2018 to September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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:
2017
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Specific details on test material used for the study:
Clear colourless liquid
Purity: 97.2%

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, 's Hertogenbosch,The Netherlands). Eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
- Age at study initiation: 6 – 60 months of age

ENVIRONMENTAL CONDITIONS
- Eyes were collected and transported in physiological saline without antibiotics 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, Postive Control, Negative Control
- Amount applied: 750 µL
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. Subsequently the corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.
Duration of post- treatment incubation (in vitro):
The corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.
Number of animals or in vitro replicates:
3 replicates/treatment
Details on study design:
SELECTION AND PREPARATION OF CORNEAS
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.

Cornea selection & 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.

QUALITY CHECK OF THE ISOLATED 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.

NUMBER OF REPLICATES: Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED: Yes, physiological saline

POSITIVE CONTROL USED: Yes, ethanol

APPLICATION DOSE AND EXPOSURE TIME: The medium from the anterior compartment was removed and 750 µl of either the negative control, positive control (Ethanol) or 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.

POST-INCUBATION PERIOD: yes

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. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM.
- POST-EXPOSURE INCUBATION: The corneas were incubated for 120 ± 10 minutes at 32 ± 1°C.

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. 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 mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated with the aid of a microplate reader (TECAN Infinite® M200 Pro Plate Reader). The optical density at 490 nm (OD490) of each sampling tube was measured in triplicate. The mean OD490 for each treatment was calculated using cMEM corrected OD490 values.

SCORING SYSTEM:
- In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

DECISION CRITERIA: Yes, the IVIS cut-off values for identifying the test item as inducing serious eye damage were based on the UN GHS criteria: GHS Category 1 for serious eye damage; and the GHS No Category for not requiring classification for eye irritation/serious eye damage.

If: IVIS Range ≤ 3; UN GHS “No category”
If: IVIS Range > 3, ≤ 55: UN GHS “No prediction can be made”
If: IVIS Range >55; UN GHS “Category 1”

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
-0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
mean
Value:
-0.7
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The corneas treated with test item showed opacity values ranging from -1.3 to -0.3 and permeability values ranging from 0.000 to 0.006. The corneas were clear after the 10 minutes of treatment with the test item. No pH effect of the test item was observed on the rinsing medium.

The individual in vitro irritancy scores for the negative controls ranged from 2.0 to 2.6. The corneas treated with the negative control item were clear after the 10 minutes of treatment. The individual positive control in vitro irritancy scores ranged from 29 to 31. The corneas treated with the positive control item were turbid after the 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 30 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.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of -0.7 after 10 minutes of treatment. In conclusion, since 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Executive summary:

1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) did not induce ocular irritation through both endpoints (opacity and permeability), resulting in a mean in vitro irritancy score of -0.7 after 10 minutes of treatment. In conclusion, since 1,1’(1,1,3-trimethylpropane-1,3-diyl)bis(cyclohexane) induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.