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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:
2019-04-02 to 2019-04-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 492 (Reconstructed Human Cornea-like Epithelium (RhCE) Test Method for Identifying Chemicals Not Requiring Classification and Labelling for Eye Irritation or Serious Eye Damage)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Propionamide
EC Number:
201-172-1
EC Name:
Propionamide
Cas Number:
79-05-0
Molecular formula:
C3H7NO
IUPAC Name:
propanamide
Test material form:
solid

Test animals / tissue source

Species:
other: reconstructed human cornea-like epithelium
Details on test animals or tissues and environmental conditions:
Justification of the test method and considerations regarding applicability :
The reconstructed human cornea-like epithelium (RhCE) model is an accepted in vitro method to replace animal testing. The human eye EpiOcular™-model closely mimics the biochemical and physiological properties of the human eye, i.e. the cornea.

Characterization of the Test System:
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27098
Keratinocyte strain: 4F118 8
Supplier: MatTek In Vitro Life Science Laboratories

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount applied: 50 mg per tissue
Duration of treatment / exposure:
6 hours (± 15 minutes)
Duration of post- treatment incubation (in vitro):
25 minutes (± 2 minutes)
Number of animals or in vitro replicates:
2
Details on study design:
- Details of the test procedure used :

Preparation:
On day of receipt, the tissues were equilibrated in their 24-well shipping container to room temperature for about 15 minutes. Afterwards the tissues were removed from the shipping container using sterile forceps and transferred to 6-well plates containing 1 mL pre-warmed (37 °C) assay medium. Any agarose adhering to the inserts was removed by gentle blotting on gauze or paper towel. Afterwards, the tissues were incubated at 37 °C and 5% CO2 overnight (16-24 hours) without medium exchange.

Pre-Treatment.
After the overnight incubation, the tissues were pre-wetted with 20 pL DPBS and incubated at 37 °C and 5% C02 for 30 minutes (± 2 minutes).

Treatment:
After the 30-minute DPBS pre-treatment, the negative and the positive control were tested by applying 50 µL topically on the EpiOcular™ tissues. The solid test item was tested by evenly applying 50 mg topically on the EpiOcular™ tissues. The tissues were placed back into the culture medium after dosing and incubated at 37 °C and 5% CO2 for 6 hours (± 15 minutes). At the end of the 6 hours treatment time, the positive control, negative control and the test item were removed by extensively rinsing the tissues with pre-warmed (room temperature) DPBS. Three clean beakers, containing a minimum of 100 mL each of DPBS were used per group. The inserts containing the tissue were lifted out of the medium by grasping the upper edge of the plastic "collar" with fine forceps. To assure throughput, the two tissues per group were rinsed simultaneously by holding the replicate inserts together by their collars using forceps. The test item or control articles were decanted from the tissue surface onto a clean absorbent material and the cultures dipped into the first beaker of DPBS, swirled in a circular motion in the liquid for approximately 2 seconds, lifted out so that the inserts were mostly filled with DPBS, and the liquid was decanted back into the container. This process was performed at least two additional times in the first beaker. The culture was then rinsed in the second and third beakers of DPBS at least three times each in the same fashion. Finally, any remaining liquid was decanted onto the absorbent material. After rinsing, the tissues were immediately transferred in 5 mL of pre-warmed (room temperature) assay medium in a 12-well plate for 25 minutes (± 2 minutes) at room temperature.

Post-Treatment Incubation
After the 25 minutes incubation, each insert was removed from the assay medium, the medium was decanted off the tissue, and the insert were blotted on absorbent material and transferred in 6-well plates filled with 1 mL of pre-warmed (37 °C) assay medium for 18 hours (± 15 minutes) at 37°C and 5% C02.

- RhCE tissue construct used, including batch number :
Designation: EpiOcular™ Tissue (OCL-200, OCL-212)
Lot No.: 27098
Keratinocyte strain: 4F118 8
Supplier: MatTek In Vitro Life Science Laboratories

- Doses of test chemical and control substances used : Test item: 50 mg per tissue; control substances: 50 µL per tissue

- Duration and temperature of exposure, post-exposure:
Exposure: 6 hours (± 15 minutes) at 37 °C
Post-exposure: 25 minutes (± 2 minutes) at room temperature and 18 hours (± 15 minutes) at 37 °C

- Indication of controls used for direct MTT-reducers and/or colouring test chemicals: No. The pre-test for direct MTT-reducing capacity of the test item did not result in blue color, i.e. the test item is not a direct MTT reducer and the test item has no colorant properties.

- Number of tissue replicates used per test chemical and controls: 2

- Wavelength used for quantifying MTT formazan: 570

- Description of the method used to quantify MTT formazan
After the post-treatment incubation period, the treated tissues were transferred in a 24-well plate filled with 300 µL MTT solution (1.0 mg/mL MTT). Once all the tissues were placed into the 24-well plate, the plate was incubated for 180 minutes (± 10 minutes) at 37°C and 5% CO2.
The inserts were removed from the 24-well plate after 180 minutes (± 10 minutes). The bottom of the inserts was blotted on absorbent material, and then transferred to a 6-well plate containing 2 mL isopropanol so that no isopropanol was flowing into the inserts. The plate was sealed with a standard plate sealer. To extract the MTT, the plate was placed on an orbital plate shaker and shaken for 2 to 3 hours at room temperature. The corresponding negative and positive controls were treated identically.
The extract solution was mixed and 2 x 200 µL were transferred into a 96-well plate. The OD was read using a spectrophotometer at 570 nm wavelength. A functional test of the microplate reader was performed using a filter test plate.

- Description of evaluation criteria used including the justification for the selection of the cut-off point for the prediction model :
The test item is identified as not requiring classification and labeling according to UN GHS (No Category) if the mean percent tissue viability is more than 60%. In this case no further testing in other test methods is required.
If the mean percent tissue viability is less than or equal 60%, no prediction can be made. In this case, further testing with other test methods will be required because RhCE test methods show a certain number of false positive results and cannot resolve between UN GHS Categories 1 and 2.

Acceptance Criteria
The results are acceptable if:
1. The negative control OD >0.8 and <2.5
2. The mean relative viability of the positive control is:
a) 30-minute exposure (treatment of liquid test items): below 50% of control viability
b) 6-hour exposure (treatment of solid test items): below 50% of control viability
3. The difference of viability between the two relating tissues of a single chemical is <20% in the same run (for positive and negative control tissues and tissues of single chemicals). This applies also to the killed controls (single chemicals and negative killed control) and the colorant controls which are calculated as percent values related to the viability of the relating negative control.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: Viability (%)
Remarks:
Tissue 1
Run / experiment:
1
Value:
1.6
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability (%)
Remarks:
Tissue 2
Run / experiment:
2
Value:
1.2
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
other: Viability (%)
Run / experiment:
Mean (tissue 1 and 2)
Value:
1.4
Vehicle controls validity:
not examined
Negative controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

The results obtained after treatment of the reconstructed human cornea-like epithelium (RhCE) model with the test item are given in the following table:

Table 1: Results

Group

Tissue 1

Tissue 2

Mean

SD

Difference between tissue replicates

OD

Viability

OD

Viability

OD

Viability

Viability

Negative Control

1.732

97.8%

1.809

102.1%

1.771

100.0%

3.04

4.3%

Positive Control

0.564

31.8%

0.608

34.3%

0.586

33.1%

1.77

2.5%

Test item

0.028

1.6%

0.021

1.2%

0.025

1.4%

0.28

0.4%

Following treatment with the test item, the tissue viability was 1.4% and, thus, lower than 60%. i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Applicant's summary and conclusion

Interpretation of results:
other: Category 1 or 2 based on GHS criteria
Conclusions:
Following treatment with the test item, the tissue viability was 1.4% and, thus, lower than 60%. i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.
Executive summary:

The potential of the test item to induce eye irritation was investigated in an in vitro human cornea model according to OECD guideline 492.

The test item was applied topically to a reconstructed human cornea-like epithelium model (EpiOcular™) followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the eye irritation potential. Duplicates of the EpiOcular™-model were treated with the test item, the negative or the positive control for 6 hours (±15 minutes). 50 mg of the test item and 50 µL of either the negative control (sterile deionized water) or the positive control (methyl acetate) were applied to the tissues. After treatment with the negative control (sterile deionized water) the mean OD was 1.771 (study acceptance criterion: >0.8 and <2.5). Treatment with the positive control (methyl acetate) revealed a mean viability value of 33.1% (study acceptance criterion: <50%). Thus, the acceptance criteria were met.

Following treatment with the test item, the tissue viability was 1.4% and, thus, lower than 60%, i.e. according to OECD 492 no prediction can be made regarding the eye hazard potential of the test item.

Under the conditions of the present study, the eye hazard potential of the test item cannot be predicted.