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Description of key information

For determination of the eye irritating potential a study according to OECD 492 was performed. Based on the results the test substance can be stated as non-irritating to eyes.

For determination of the skin irritating potential a study according to OECD 439 was performed. Based on the results the test substance can be stated as non-irritating to skin.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
Endpoint:
skin corrosion: 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
Qualifier:
according to
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Qualifier:
according to
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Vehicle:
other: Dulbecco’s Phosphate Buffered Saline (DPBS buffer)
Details on test system:
The test system is a commercially available EpiDermTM-Kit, procured by MatTek.
The EpiDermTM tissue consists of human-derived epidermal keratinocytes which have been cultured to form a multi-layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers representing main
lipid classes analogous to those found in vivo. The EpiDermTM tissues are cultured on spe-cially prepared cell culture inserts.
Control samples:
yes, concurrent vehicle
yes, concurrent positive control
Amount / concentration applied:
Tissue Amount
1 24.8 mg
2 25.0 mg
3 24.8 mg
Details on study design:
One plate (3 tissues) was used as negative control; each tissue was treated with 30 µL DPBS buffer, a nylon mesh was added in order to ensure sufficient contact with the tissue surface.
One plate was used as positive control; each tissue was treated with 30 µL 5% SDS-solution, a nylon mesh was added in order to ensure sufficient contact with the tissue sur-face.
One plate was used for treatment with the test item:
The tissues were wetted with 25 µL DPBS buffer before applying the test item and spreading it to match the tissue size.

Tissues were dosed in 1-minute-intervals. After dosing the last tissue, all plates were transferred into the incubator for 35 minutes at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% rel-ative humidity.
1 hour after the first application, the inserts were removed from the plates using sterile forceps and rinsed immediately in 1-minute-intervals.
After rinsing thoroughly with DPBS, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL). The sur-face of the inserts was then carefully dried with a sterile cotton tipped swab.
Then, the tissues were set in the incubator for 25 hours and 1 minute at 37 ± 1°C and 5.0 ± 1% CO2 and ≥ 95% relative humidity.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 1
Value:
1.239
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 2
Value:
1.421
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 3
Value:
1.37
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value
Value:
1.343
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Interpretation of results:
GHS criteria not met
Conclusions:
The test item L-Tyrosine, N-acetyl-3,5-dinitro- is considered as non-irritant to skin.
After the treatment, the mean value of relative tissue viability was reduced to 86.9%. This value is above the threshold for skin irritation (50%).
The optical density of the negative control was well within the required acceptability crite-rion of 0.8 ≤ mean OD ≤ 2.8.
The positive control has met the acceptance criterion too, for thus ensuring the validity of the test system.
Variation within replicates was within the accepted range for negative control, positive control and test item (required: ≤ 18%).
For these reasons, the result of the test is considered valid.
Executive summary:

Three tissues of the human skin model EpiDermTMwere treated with the test item for 60 minutes.

The test item was applied directly to each tissue and spread to match the tissue size (0.63 cm2; as indicated by the supplier).

DPBS-buffer was used as negative control and 5% SDS solution was used as positive control.

After treatment with the negative control, the mean absorbance value was within the required acceptability criterion of 0.8 ≤ mean OD ≤ 2.8, OD was 1.5.

The positive control showed clear irritating effects. The mean value of relative tissue viability was reduced to 3.1% (required: ≤ 20%).

The variation within the tissue replicates of negative control, positive control and test item was acceptable (required: ≤ 18%).

 

After the treatment with the test item, the mean value of relative tissue viability was reduced to 86.9 %. This value is above the threshold for skin irritation potential (50%). Test items that induce values above the threshold of 50% are considered non-irritant to skin.

 

 

Therefore, the test itemL-Tyrosine, N-acetyl-3,5-dinitro-is considered

non-irritant to skin in the Reconstructed human Epidermis (RhE) Test Method.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
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
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)
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.
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.
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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Additional information

Justification for classification or non-classification