Hexa-1,5-diene

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

BCOP Test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 09 January 2018. Experimental completion date: 09 January 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

Guideline:

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

Version / remarks:

Adopted July 26, 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Hexa-1,5-diene

EC Number:

209-754-7

EC Name:

Hexa-1,5-diene

Cas Number:

592-42-7

Molecular formula:

C6H10

IUPAC Name:

hexa-1,5-diene

Specific details on test material used for the study:

1,5-hexadiene. 99.4% purity.

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BSC-433-4-0488-5
- Expiration date of the lot/batch: 01 June 2018
- Purity test date: 26 June 2017

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

Analyses conducted to support the information cited in the certificate of analysis for the test item were not conducted in compliance with the GLP or GMP regulations. The characterization of the test item was conducted under a sponsor or sponsor subcontractor quality system.

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), where 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.

SOURCE OF COLLECTED EYES
- Source: Slaughterhouse (Vitelco, 's Hertogenbosch, The Netherlands)
- Indication of any existing defects or lesions in ocular tissue samples: 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:

750 µL

Duration of treatment / exposure:

10 +/- minutes at 32ºC

Duration of post- treatment incubation (in vitro):

120 +/- 10 minutes at 32ºC

Number of animals or in vitro replicates:

Three corneas for each treatment group

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 (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 +/- 1ºC. The corneas were incubated for the minimum of 1 hour at 32 +/- 1ºC.

QUALITY CHECK OF THE ISOLATED CORNEAS
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.

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

NEGATIVE CONTROL USED
Physiological saline (Eurovet Animal Health, Bladel, The Netherlands).

POSITIVE CONTROL USED
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. 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 +/- 1ºC.

POST-INCUBATION PERIOD: yes
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 +/- 1ºC. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of [UV/VIS spectrophotometry / microtiter plate reader] (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS) = mean opacity value + (15 x mean OD490 value)

DECISION CRITERIA:
IVIS <= 3: No category.
3 < IVIS <= 55: No prediction can be made
IVIS > 55: Category 1

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Value:

3.7

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

fluorescein leakage

Remarks:

Mean permeability

Value:

0.143

Negative controls validity:

valid

Positive controls validity:

valid

Results 3

Irritation parameter:

in vitro irritation score

Value:

5.8

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

The corneas were clear, however two corneas had a spot after the 10 minutes of treatment with 1,5-hexadiene. No pH effect of the test item was observed on the rinsing medium.

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 64 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.

The corneas were clear following exposure, however two corneas had a spot after the 10 minutes of treatment with 1,5-hexadiene. No pH effect of the test item was observed on the rinsing medium.

Any other information on results incl. tables

Summary of opacity, permeability and in vitro scores:

Treatment	Mean opacity	Mean permeability	Mean in vitro irritation score
Negative control	-0.1	0.007	0.0
Positive control	17	3.092	64
1,5 -hexadiene	3.7	0.143	5.8

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Since 1,5-hexadiene induced an IVIS > 3 ¿ 55, no prediction on the classification can be made.

Executive summary:

The objective of this study was to evaluate the eye hazard potential of 1,5-hexadiene as measured by its ability to induce opacity and increase permeability in an isolated bovine cornea 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 1,5-hexadiene was tested through topical application for 10 minutes.

The study procedures described in this report were based on OECD guideline 437. Batch BSC-433-4-0488-5 of 1,5-hexadiene was a clear colourless liquid with a purity of 99.4%. The test item was applied as it is (750 ¿L) 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 64 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. 1,5-hexadiene induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 5.8 after 10 minutes of treatment. In conclusion, since 1,5-hexadiene induced an IVIS > 3 ¿ 55, no prediction on the classification can be made.