L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester

Eye irritation

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:

2020

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)

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

ethyl (2S)-3-[3,5-diamino-4-(4-methoxyphenoxy)phenyl]-2-acetamidopropanoate

Cas Number:

440667-78-7

Molecular formula:

C20H25N3O5

IUPAC Name:

ethyl (2S)-3-[3,5-diamino-4-(4-methoxyphenoxy)phenyl]-2-acetamidopropanoate

Test material form:

solid: particulate/powder

Test system

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

two replicates were tested with the following concentration
Tissue 1 48.7 mg
Tissue 2 50.0 mg

Duration of treatment / exposure:

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 ≥ 95% relative humidity for 30 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 ≥ 95% relative humidity.

Duration of post- treatment incubation (in vitro):

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 ≥ 95% relative humidity.
After the post-treatment incubation, the MTT assay was performed.

Number of animals or in vitro replicates:

two replicates

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

mean percent tissue viability 

Run / experiment:

2

Value:

109.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation parameter:

mean percent tissue viability 

Run / experiment:

1

Value:

102.9

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

1       Findings and Results

1.1      Measured Values

As blank, the optical density of isopropanol was measured in eight wells of the 96-well-plate. The measured values and their mean are given in the following table:

Table 9.1‑a      Absorbance Values Blank Isopropanol (OD at 570 nm)

Replicate	1	2	3	4	5	6	7	8	Mean
Absorbance	0.032	0.034	0.033	0.034	0.033	0.034	0.034	0.035	0.034

 

The absorbance values of negative control, test item and positive control are given in the following table:

Table 9.1‑b      Absorbance Values Negative Control, Positive Control and Test Item (OD at 570 nm)

Designation	Measurement	Negative Control	Positive Control	L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester
Tissue 1	1	1.666	0.592	1.718
	2	1.671	0.594	1.732
Tissue 2	1	1.694	0.564	1.816
	2	1.682	0.568	1.840

 

From the measured absorbances, the mean of each tissue was calculated, subtracting the mean absorbance of isopropanol as given in table 9.1-a (= corrected values).

Table 9.1‑c      Mean Absorbance Negative Control, Positive Control and Test Item

Designation	Negative Control	Positive Control	L-Tyrosine, N-acetyl-3,5 -diamino-O-(4-methoxyphenyl)-, ethyl ester
Mean – blank (Tissue 1)	1.635	0.559	1.691
Mean – blank (Tissue 2)	1.654	0.532	1.794

 

 

 

1.2      Comparison of Tissue Viability

For the test item and the positive control, the following percentage values of tissue viability were calculated in comparison to the negative control:

Table 9.2‑a      % Viability Positive Control and Test Item

Designation	Positive Control	L-Tyrosine, N-acetyl-3,5 -diamino-O- (4-methoxyphenyl)-, ethyl ester
% Viability (Tissue 1)	34.0%	102.9%
% Viability (Tissue 2)	32.4%	109.1%
% Viability Mean	33.2%	106.0%

1.3      Assessment

Eye hazard potential is assessed using the criteria given in the following table:

Table 9.3‑a      Assessment of Eye Hazard Potential

% Viability	Assessment	UN GHS classification
> 60 %	Non eye irritant	No Category
≤ 60 %	At least eye irritant	No prediction can be made (category 1 or 2)

1.1      Validity

Validity criteria and results are stated in the following table:

Table 9.4‑a      Validity

Criterion	Demanded	Found
Mean OD of negative control	> 0.8 and < 2.8	1.6
% mean relative viability of positive control	< 50% of negative control	33.2%
Variation within replicates	< 20%	1.2% (negative control) 1.6% (positive control) 6.3% (test item)

 

The values for negative control and for positive control were within the range of historical data of the test facility (see page 20).

 

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-diamino-O-(4-methoxyphenyl)-, ethyl ester 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 increased to 106.0%. 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 1.6 (> 0.8 and < 2.8).
The positive control induced a decrease in tissue viability as compared to the negative con-trol to 33.2%. Variation within the replicates of the controls and the test item was accepta-ble (< 20%).
For these reasons, the result of the test is considered valid.

Executive summary:

Under the conditions of the test, L-Tyrosine, N-acetyl-3,5-diamino-O-(4-methoxyphenyl)-, ethyl ester is considered non-eye irritant in the EpiOcular^TM Eye Irritation Test.