4,4'-(2-ethylhexane-1,1-diyl)diphenol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March to May 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: fully compliant GLP guideline study without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-(2-ethylhexane-1,1-diyl)diphenol

EC Number:

680-046-1

Cas Number:

74462-02-5

Molecular formula:

C20H26O2

IUPAC Name:

4,4'-(2-ethylhexane-1,1-diyl)diphenol

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: BisP-IOTD
Molecular formula: C20H26O2
Molecular weight: 298.43
CAS Number: 74462-02-5
Description: White powder
Batch: 120601
Purity/Composition: 99.6%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 12 March 2014 (allocated by WIL Research Europe B.V., 1 year after receipt of the test substance)

Test animals / tissue source

Species:

other: bovine eyes

Strain:

not specified

Details on test animals or tissues and environmental conditions:

Test System: Bovine eyes were used as soon as possible after slaughter on the same day.
Source: Bovine eyes from young cattle were obtained from the slaughterhouse (Vitelco, ‘s Hertogenbosch, The Netherlands), where the eyes were excised by a slaughterhouse employee as soon as possible after slaughter.
Transport: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
All eyes were carefully examined for defects by holding the eyes submersed in physiological saline. Those exhibiting unacceptable defects, such as opacity, scratches, pigmentation and neovascularization were discarded. The isolated corneas were stored at 32 ±1 °C in a petri dish with cMEM (Eagle's Minimum Essential Medium (lnvitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (lnvitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (lnvitrogen Corporation)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of MC2 (Clermont, France) 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.

Test system

Vehicle:

unchanged (no vehicle)

Remarks:

Since no workable suspension of BisP-IOTD in physiological saline could be obtained, the test substance was used as delivered by the sponsor and added pure on top of the corneas.

Controls:

other: positive and negative controls applied

Amount / concentration applied:

The medium from the anterior compartment was removed and 750 µ l of the negative control and positive control were introduced onto the epithelium of the cornea. BisP-IOTD was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (302 to 312 mg, first experiment and 345 to 355 mg, second experiment).The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 ±10 minutes at 32 ±1 °C. After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle's Minimum Essential Medium, lnvitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Duration of treatment / exposure:

Corneas were incubated in a horizontal position for 240 ±10 minutes at 32 ±1 °C.

Observation period (in vivo):

After the incubation the solutions and the test compound were removed and the epithelium was washed at least three times with MEM with phenol red (Eagle's Minimum Essential Medium, lnvitrogen Corporation). Possible pH effects of the test substance on the corneas were recorded. The anterior and the posterior compartment were refilled with fresh cMEM and an opacity determination was performed without any further incubation. After the completion of the incubation period each cornea were inspected visually for dissimilar opacity patterns and the opacity determination was performed.

Number of animals or in vitro replicates:

In total 18 bovine eyes were used (2 experiments with each 3 for negative control, 3 for positive control and 3 for test substance measurement)

Details on study design:

Opacity measurement
The opacitometer determined the difference in the light transmission between each control or treated cornea and an air filled chamber. The numerical opacity value (arbitrary unit) was displayed and recorded. 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 positive control or test substance treated cornea was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each positive control or test substance 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.
Application of sodium fluorescein
Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Merck) 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 5 mg Na-fluorescein/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.
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 lnfinite® 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 was performed, the OD490 of each reading was corrected for the mean negative control OD490 before the dilution factor was applied to the readings.
Electronic data capture
Observations/measurements in the study were recorded electronically using the following programme: Magellan Tracker 7.0 (TECAN, Austria) for optical density measurement.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

other: opacity

Basis:

mean

Score:

4

Max. score:

135

Reversibility:

other: not applicable

Remarks on result:

other: Experiment 1 (max score is value for positive control)

Results 2

Irritation parameter:

other: opacity

Basis:

mean

Score:

3

Max. score:

87

Reversibility:

other: not applicable

Remarks on result:

other: Experiment 2 (max score is value for positive control)

Results 3

Irritation parameter:

other: permeability

Basis:

mean

Score:

3 790

Max. score:

2 544

Reversibility:

other: not applicable

Remarks on result:

other: Experiment 1 (max score is value for positive control)

Results 4

Irritation parameter:

other: permeability

Basis:

mean

Score:

2 338

Max. score:

3 300

Reversibility:

other: not applicable

Remarks on result:

other: Experiment 2 (max score is value for positive control)

Results 5

Irritation parameter:

other: in vitro irritation score (IVIS)

Basis:

mean

Score:

61

Max. score:

173

Reversibility:

other: not applicabe

Remarks on result:

other: Experiment 1 (max score is value for positive control)

Results 6

Irritation parameter:

other: in vitro irritation score (IVIS)

Basis:

mean

Score:

38

Max. score:

137

Reversibility:

other: not applicable

Remarks on result:

other: Experiment 2 (max score is value for positive control)

Any other information on results incl. tables

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 (lmidazole) were 173 and 137 in the first and second experiment respectively and were within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

BisP-IOTD induced severe ocular irritation through one endpoint (permeability) only, resulting in a mean in vitro irritancy score (IVIS) of 61 after 240 minutes of treatment. In the second experiment, BisP-IOTD induced again ocular irritation through one endpoint (permeability), however the mean in vitro irritancy score was 38 after 240 minutes of treatment with BisP-IOTD. Since in vitro irritancy score for 5 out of 6 corneas treated with BisP-IOTD was below 55.1 after 240 minutes treatment, the IVIS of 84 observed in one cornea is judged as an outlaying value and therefore this increase is considered to be not relevant. Moreover the OD.,9° before the dilution step of this outlaying cornea was comparable with the other two corneas (results not reported), but after the dilution step to bring the OD into the acceptable range for measurement an increase in the OD490 was observed in this cornea.

Since BisP-IOTD induced an IVIS below 55.1 in 5 out of 6 corneas, it is concluded that BisP-IOTD is not corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.

Applicant's summary and conclusion

Interpretation of results:

other: not corrosive or severe irritant

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of this test, BisP-IOTD was found not being corrosive and thus is not classified for eye damage. However, a final decision upon eye irriation is not possible based on the BCOP test model and thus this will have to be assessed in vivo, once the next tonnage level is reached.

Executive summary:

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 (lmidazole) were 173 and 137 in the first and second experiment respectively and were within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

BisP-IOTD induced severe ocular irritation through one endpoint (permeability) only, resulting in a mean in vitro irritancy score (IVIS) of 61 after 240 minutes of treatment. In the second experiment, BisP-IOTD induced again ocular irritation through one endpoint (permeability), however the mean in vitro irritancy score was 38 after 240 minutes of treatment with BisP-IOTD. Since in vitro irritancy score for 5 out of 6 corneas treated with BisP-IOTD was below 55.1 after 240 minutes treatment, the IVIS of 84 observed in one cornea is judged as an outlaying value and therefore this increase is considered to be not relevant. Moreover the OD.,9° before the dilution step of this outlaying cornea was comparable with the other two corneas (results not reported), but after the dilution step to bring the OD into the acceptable range for measurement an increase in the OD490 was observed in this cornea.

Since BisP-IOTD induced an IVIS below 55.1 in 5 out of 6 corneas, it is concluded that BisP-IOTD is not corrosive or severe irritant in the Bovine Corneal Opacity and Permeability test under the experimental conditions described in this report.