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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 October 2017 to 01 February 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 i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Deviations:
no
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
3-{2-[2-(3-azaniumylpropoxy)ethoxy]ethoxy}propan-1-aminium di[(2Z)-3-carboxyprop-2-enoate]
Cas Number:
1629579-82-3
Molecular formula:
C18H32N2O11
IUPAC Name:
3-{2-[2-(3-azaniumylpropoxy)ethoxy]ethoxy}propan-1-aminium di[(2Z)-3-carboxyprop-2-enoate]
Test material form:
liquid
Details on test material:
CAS Number: 1629579-82-3
EC Number: 818-033-1
Molecular formula: C18H32N2O11
Specific details on test material used for the study:
Identification: Bis-Aminopropyl Diglycol Dimaleate
Appearance: Clear colourless to pale yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: Not indicated
Test item storage: At room temperature
Stable under storage conditions until: 31 November 2019 (expiry date)

Test animals / tissue source

Species:
cattle
Details on test animals or tissues and environmental conditions:
- Test System: Bovine eyes were used as soon as possible after slaughter.
- Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize 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:
yes
Amount / concentration applied:
750 µl
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 10 ± 1 minutes
Duration of post- treatment incubation (in vitro):
120 ± 10 minutes
Number of animals or in vitro replicates:
3
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 (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 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 (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. Three corneas were selected at random for each treatment group.

Test Item Preparation
No correction was made for the purity/composition of the test item. The test item was tested neat.

Treatment of Corneas and Opacity Measurements
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 ± 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. 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 ± 1 °C. After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns. Initially an experiment was performed in which the acceptability criteria were not met. The experiment was rejected and a repeat experiment was performed.

Opacity Measurement
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 opacity value (measured with the device OP-KIT) was calculated according to:

𝑂𝑝𝑎𝑐𝑖𝑡𝑦 = 𝐼0 / 𝐼 − 0.9894 / 0.0251

With I0 the empirically determined illuminance through a cornea holder but with windows and medium, and I the measured illuminance through a holder with cornea. 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 treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control 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 (Sigma-Aldrich, Germany) 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 4 mg Na-fluorescein (Sigma-Aldrich Chemie GmbH, Germany)/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 has been performed, the OD490 of each reading of the positive control and the test item was corrected for the mean negative control OD490 before the dilution factor was applied to the reading.

Results and discussion

In vitro

Results
Irritation parameter:
in vitro irritation score
Value:
4.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation

Any other information on results incl. tables

The corneas treated with Bis-Aminopropyl Diglycol Dimaleate showed opacity values ranging from 1.6 to 4.1 and permeability values ranging from 0.129 to 0.140.

The corneas were translucent after the 10 minutes of treatment with Bis-Aminopropyl Diglycol Dimaleate.

A pH effect of the test item was observed on the rinsing medium, the corneas were rinsed until no color change of the medium was observed. Hence, the in vitro irritancy scores ranged from 3.7 to 6.2 after 10 minutes of treatment with Bis-Aminopropyl Diglycol Dimaleate.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
no prediction on the classification can be made.
Conclusions:
Bis-Aminopropyl Diglycol Dimaleate induced ocular irritation through one endpoint (permeability), resulting in a mean in vitro irritancy score of 4.8 after 10 minutes of treatment.
In conclusion, since Bis-Aminopropyl Diglycol Dimaleate 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 Bis-Aminopropyl Diglycol Dimaleate 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 Bis-Aminopropyl Diglycol Dimaleate was tested through topical application for 10 minutes. The study procedures described in this report were based on the most recent OECD guideline. Batch 11_13_17_1 of Bis-Aminopropyl Diglycol Dimaleate was a clear colourless to pale yellow liquid liquid.

The test item was applied as it is (750 µl) 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 51 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.

Bis-Aminopropyl Diglycol Dimaleate induced ocular irritation through one endpoint (permeability), resulting in a mean in vitro irritancy score of 4.8 after 10 minutes of treatment. In conclusion, since Bis-Aminopropyl Diglycol Dimaleate induced an IVIS > 3 ≤ 55, no prediction on the classification can be made.