1,4-Benzenedicarboxylic acid, compd. with 1,6-hexanediamine (1:1)

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 05 May to 25 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study OECD 437 compliant

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

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)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

benzene-1,4-dicarboxylic acid; hexane-1,6-diamine

EC Number:

700-230-8

Cas Number:

3160-86-9

Molecular formula:

C8-H6-O4.C6-H16-N2

IUPAC Name:

benzene-1,4-dicarboxylic acid; hexane-1,6-diamine

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Test animals / tissue source

Species:

other: bovine

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.

Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing (1-6). As a consequence a validated and accepted in vitro test for eye irritation should be performed before in vivo tests are conducted. One of the proposed validated in vitro eye irritation tests is the Bovine Corneal Opacity and Permeability (BCOP) test.

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.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: not applicable

Duration of treatment / exposure:

240 minutes

Number of animals or in vitro replicates:

Not applicable

Details on study design:

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 (Eagle’s Minimum Essential Medium (Invitrogen Corporation, Breda, The Netherlands) containing 1% (v/v) L-glutamine (Invitrogen Corporation) and 1% (v/v) Foetal Bovine Serum (Invitrogen 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.

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 (OP-KIT, MC2, Clermont, France). The opacity of each cornea was read against an air 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.

Treatment of corneas and opacity measurements:
The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. Terephthalic acid, hexane-1,6-diamine (1:1) was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (302 to 315 mg).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, Invitrogen 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.

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 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 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

Irritation parameter:

other: IVIS score

Basis:

mean

Time point:

other: 240 minutes

Score:

4.5

Irritant / corrosive response data:

Terephthalic acid, hexane-1,6-diamine (1:1) induced ocular irritation through one endpoint (opacity), resulting in a mean in vitro irritancy score of 4.5 after 240 minutes of treatment.

Other effects:

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 (20% (w/v) Imidazole) was 165 and within the historical positive control data range. 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:

other: Not corrosive

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

IVIS score is 4.5. The substance is therefore not corrosive for the eye. No conclusion can be made between non irritating or irritating for the eye classifications even if IVIS score is only slightly over the limit for the not irritating classification.

Executive summary:

This study (2014) is a screening for the eye irritancy potential of Terephthalic acid, hexane-1,6-diamine (1:1) using the Bovine Corneal Opacity and Permeability test (BCOP test, OECD 437), under GLP conditions. It was tested through topical application for approximately 240 minutes.

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 (20% (w/v) Imidazole) was 165 and within the historical positive control data range. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

Terephthalic acid, hexane-1,6-diamine (1:1) induced ocular irritation through one endpoint (opacity, resulting in a mean in vitro irritancy score of 4.5 after 240 minutes of treatment.

Since Terephthalic acid, hexane-1,6-diamine (1:1) induced an IVIS ≤55, the substance is definitely not corrosive for the eye. As the IVIS is > 3 it is not possible to conclude that the substance is not irritating for the eye and no definitive classification can be made.

However with an IVIS score of 4.5, the substance is very close to the limit for the non irritating classification.