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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:
weight of evidence
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
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

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Remarks:
light yellow
Details on test material:
CH03216 / E1A

Test animals / tissue source

Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- 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.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions):
Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
- Time interval prior to initiating testing:
Bovine eyes were used as soon as possible after slaughter
- 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.
- Indication of any antibiotics used: no

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
Since no workable suspension of 3,4,5-TRIMETHOXYCINNAMIC ACID in physiological saline could be obtained, the test item was used as delivered by the sponsor and added pure on top of the corneas.
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):
3,4,5-TRIMETHOXYCINNAMIC ACID was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (315.6 to 384.3 mg).
- Concentration (if solution):
The medium from the anterior compartment was removed and 750 l of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea.

VEHICLE
No vehicle used
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 240  10 minutes at 32  1C.
Number of animals or in vitro replicates:
3 replicates per treatment (negative control, positive control and test item)
Details on study design:
SELECTION AND PREPARATION OF 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. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES
3 replicates per treatment (negative control, positive control and test item)

NEGATIVE CONTROL USED
A negative control, physiological saline was included to detect non-specific changes in the test system and to provide a baseline for the assay endpoints.

POSITIVE CONTROL USED
20% (w/v) Imidazole (Merck Schuchardt OHG, Germany) [CAS Number 288-32-4] solution prepared in physiological saline.

APPLICATION DOSE AND EXPOSURE TIME
The medium from the anterior compartment was removed and 750 l of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. 3,4,5-TRIMETHOXYCINNAMIC ACID was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered (315.6 to 384.3 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  1C.

POST-INCUBATION PERIOD: no

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 3

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 (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: 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 5 mg Na-fluorescein/ml cMEM solution (Sigma-Aldrich Chemie GmbH, Germany). 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  1C.
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 l 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.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Value:
20
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Value:
19
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Value:
0.111
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: none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control:
The individual in vitro irritancy scores for the negative controls ranged from 0.1 to 1.6.
- Acceptance criteria met for positive control:
The individual positive control in vitro irritancy scores ranged from 110 to 135 .
- Range of historical values if different from the ones specified in the test guideline: NA

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Conclusions:
In conclusion, since 3,4,5-TRIMETHOXYCINNAMIC ACID induced an IVIS between 3 and 55, no prediction on the classification can be made.
Executive summary:

The objective of this study was to evaluate the eye hazard potential of 3,4,5-TRIMETHOXYCINNAMIC ACID 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 3,4,5-TRIMETHOXYCINNAMIC ACID was tested through topical application for approximately 240 minutes. 

The study procedures described in this report were based on the most recent OECD guideline (OECD 437, adopted July 26, 2013).

Batch CH03216 / E1A of 3,4,5-TRIMETHOXYCINNAMIC ACID was a light yellow crystallinesolid. Since no workable suspension in physiological saline could be obtained, the test item was used as delivered and added pure on top of the corneas.

The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical rangeindicating that the negative control did not induce irritancy on the corneas. The meanin vitroirritancy score of the positive control (20% (w/v) Imidazole) was 123 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. 

3,4,5-TRIMETHOXYCINNAMIC ACID induced ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 20 after 4 hours of treatment.

In conclusion, since 3,4,5-TRIMETHOXYCINNAMIC ACID induced an IVIS between 3 and 55, no prediction on the classification can be made.