Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
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)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid

Test system

Vehicle:
water
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Tissue 1: 53.5 mg
Tissue 2: 52.3 mg
Duration of treatment / exposure:
6-well-plates were labelled with test item, negative control and positive control and filled with 1 mL assay medium in the appropriate wells. All inserts were inspected for viability and the presence of air bubbles between agarose gel and insert. Viable tissues were transferred in the prepared 6-well-plate and incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 1 hour.
After the pre-incubation, the medium was replaced and the wells were filled with 1 mL fresh assay medium. All 6-well-plates were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 16 hours.
Duration of post- treatment incubation (in vitro):
After overnight incubation, the tissues were pre-wetted with 20 µL DPBS buffer and the tissues were incubated at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity for 29 minutes. After that, 50 µL of the controls and a defined amount of the test item (see table 7.2-a) were applied in duplicate in one- minute- intervals.
At the beginning of each experiment (application of negative controls), a stop watch was started. After dosing the last tissue, all plates were transferred into the incubator for 6 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
At the end of exposure time, the inserts were removed from the plates in one-minute-intervals using sterile forceps and rinsed immediately. The inserts were thoroughly rinsed with DPBS. Then, the tissues were immediately transferred into 5 mL of assay medium in pre-labelled 12-well plate for 25 minutes post soak at room temperature.
After that, each insert was blotted on absorbent material and transferred into a pre-labelled 6-well plate, containing 1 mL assay medium. For post-treatment incubation, the tissues were incubated for 18 hours at 37 ± 1 °C, 5 ± 1 % CO2 and 80 – 100 % relative humidity.
After the post-treatment incubation, the MTT assay was performed.

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
in vitro irritation score
Remarks:
absorbance
Run / experiment:
mean value tissue 1
Value:
1.948
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
absorbance
Run / experiment:
mean value tissue 2
Value:
1.998
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
% viability
Run / experiment:
mean value tissue 1
Value:
93.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
% viability
Run / experiment:
mean value tissue 2
Value:
96.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
% viability
Run / experiment:
mean value all tissues
Value:
95.1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Values for negative control and for positive control were within the range of historical data of the test facility.
Therefore, the experiment is considered valid.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the test, L-Tyrosine, N-acetyl-3,5-dinitro- is considered non-eye irritant in the EpiOcularTM Eye Irritation Test.
After treatment with the test item, the mean value of relative tissue viability was reduced to 95.1%.
This value is above the threshold for eye irritation potential (≤ 60%).
All validity criteria were met. The criterion for optical density of the negative control was fulfilled: The OD value was 2.1(> 0.8 and < 2.5).
The positive control induced a decrease in tissue viability as compared to the negative control to 38.3%. Variation within the replicates was acceptable (< 20%).
For these reasons, the result of the test is considered valid.
Executive summary:

The test item L-Tyrosine, N-acetyl-3,5-dinitro-was applied to a three-dimensional human cornea tissue model in duplicate for an exposure time of 6 hours.

The solid test item was applied to two tissue replicates.

After treatment, the respective substance was rinsed from the tissue; then, cell viability of the tissues was evaluated by addition of MTT, which can be reduced to formazan. The formazan production was evaluated by measuring the optical density (OD) of the resulting solution.

Demineralised water was used as negative control and methyl acetate was used as positive control.

The controls showed the following results: After treatment with the negative control, the absorbance values were within the required acceptability criterion of mean OD>0.8 and < 2.5, OD was 2.1. The positive control showed clear eye irritating effects, mean value of the relative tissue viability was 38.3% (< 50%).

Variation within tissue replicates was acceptable (< 20%).

After treatment with the test item, the mean value of relative tissue viability was 95.1%.

This value is well above the threshold for eye irritation potential (≤ 60%). Test items that induce values above the threshold are considered non-eye irritant

 

Under the conditions of the test,L-Tyrosine, N-acetyl-3,5-dinitro-is considered

non- eye irritant in the EpiOcularTMEye Irritation Test.