Long-chain alkenyl amide

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2016 - 12 July 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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)

Version / remarks:

Adopted 26 July 2013

Deviations:

no

GLP compliance:

yes

Test material

Test material information

1

Reference substance name:

Reaction products of fatty acids, C16-18, C18 unsatd. with Amines, polyethylenepoly-, triethylenetetramine fraction and 3-(C9–C15, C12 rich, alk-1-enyl)dihydro-2,5-furandione

EC Number:

947-263-6

Cas Number:

68478-81-9

Molecular formula:

C36H68N4 - C76H136N4O6

IUPAC Name:

Reaction products of fatty acids, C16-18, C18 unsatd. with Amines, polyethylenepoly-, triethylenetetramine fraction and 3-(C9–C15, C12 rich, alk-1-enyl)dihydro-2,5-furandione

Test material form:

solid

Details on test material:

Appearance: Dark red-brown solid
Storage conditions: At room temperature

Test animals / tissue source

Species:

cattle

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Source: slaughterhouse (Vitelco, 's-Hertogenbosch, The Netherlands)
- Storage, temperature and transport conditions of ocular tissue: Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- indication of any existing defects or lesions in ocular tissue samples: Eyes exhibiting defects were discarded.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: 751 to 1186 mg per cornea (complete coverage)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight with unit): 750 µl of physiological saline per cornea

POSITIVE CONTROL
Amount(s) applied (volume or weight with unit): 750 µl per cornea
Concentration (if solution): 20% (w/v) Imidazole

Duration of treatment / exposure:

240 +/- 10 minutes

Number of animals or in vitro replicates:

3

Details on study design:

TEST SITE
- Isolated bovine cornea
- Preparation: after being checked for unacceptable defects, the isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF 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

REMOVAL OF TEST SUBSTANCE
- Washing: yes (with MEM with phenol red (Eagle’s Minimum Essential Medium, Invitrogen Corporation) and thereafter with cMEM)
- Time after start of exposure: 240 minutes

ADDITIONAL MEASUREMENTS:
- The opacity of a cornea was measured by the diminution of light passing through the cornea. Following the final opacity measurement, permeability of the cornea to Na-fluorescein (Sigma-Aldrich, Germany) was evaluated by incubating corneas in sodium-fluorescein solution for 90 ± 5 minutes (both incubations at 32 ± 1°C). After the incubation period, 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).

SCORING SYSTEM:
- The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score:

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

TOOL USED TO ASSESS SCORE:
- opacitymeter and microplate reader

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.

NUMBER OF REPLICATES
3

NEGATIVE CONTROL USED
yes, 3 replicates

POSITIVE CONTROL USED
yes, 3 replicates

APPLICATION DOSE AND EXPOSURE TIME
Test item was applied directly on the corneas in such a way that the cornea was completely covered. 751-1186 mg of test item was used. Corneas were incubated in a horizaontal position for 240 +/- 10 minutes.

TREATMENT METHOD: holders with corneas were placed in a horizontal position to ensure uniform distribution the solutions over the entire cornea.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: at least 3 times
- POST-EXPOSURE INCUBATION: following opacity measurments, corneas were incubated with sodium-fluorescein solution in a horizontal position for 90 +/- 5 minutes to determine permeability.

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 by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490)

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
A test substance that induces an IVIS ≤ 3 is not classified for eye irritancy (UN GHS: no category);
A test substance that induces IVIS >55 is defined as a corrosive or severe irritant (UN GHS: category 1);
A test substance that induces an IVIS >3 and ≤55, no prediction on irritant potency can be made.

Results and discussion

In vitro

Results

Irritation parameter:

in vitro irritation score

Run / experiment:

Single run

Value:

3.8

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: corneas treated with the positive control were turbid after 240 minutes of treatment and corneas treated with the test item were slightly translucent after 240 minutes of treatment.

DEMONSTRATION OF TECHNICAL PROFICIENCY: Imidazole was used as a positive control and indicated that the test system was accurate and reliable.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, results were within historical range.
- Acceptance criteria met for positive control: yes, results were within historical range.

Applicant's summary and conclusion

Interpretation of results:

other: No prediction on the classification can be made.

Conclusions:

Based on the outcome of a Bovine Corneal Opacity and Permeability test (BCOP) performed according to OECD guideline and GLP principles, it is concluded that no prediction for the classification of X-19555 for irritancy can be made, under the experimental conditions of this study, based on an IVIS of 3.8 after 240 minutes of treatment.