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Description of key information

For the determination of the skin irritating/corrosive potential a study according to OECD 439 was conducted. It can be concluded that the test substance is not skin irritating.

For the determination of the eye irritating/corrosive potential two studies were conducted. The study according to OECD 492 was not sufficient for a classification. Thus a further study according to OECD 437 was conducted. Based on the results the test substance is classifiied as eye damaging category 1.

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 26.2 mg
2 27.2 mg
3 26.9 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 spread-ing 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 ± 0.5% CO2.
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, each tissue was blotted with sterile cellulose tissue and then transferred into a new 6-well-plate with fresh assay medium (0.9 mL).
Then, the tissues were set in the incubator for 23 hours and 20 minutes at 37 ± 1°C and 5.0 ± 0.5% CO2.
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 1
Value:
70.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 2
Value:
80.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
tissue 3
Value:
69.8
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
mean value
Value:
73.5
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, 3,5-dinitro- (wet product) is considered as non-irritant to skin.
After the treatment, the mean value of relative tissue viability was reduced to 73.5%. This value is above the threshold for skin irritation (50%).
The optical density of the negative control was well within the required acceptability criteri-on 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 con-trol 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 L-Tyrosine, 3,5-dinitro- (wet product) 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 2.8 % (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 73.5 %. 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

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

Eye irritation

Link to relevant study records

Referenceopen allclose all

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 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Qualifier:
according to
Guideline:
EU method B.47 (Bovine corneal opacity and permeability test method for identifying ocular corrosives and severe irritants)
GLP compliance:
yes (incl. certificate)
Vehicle:
Hank's balanced salt solution
Controls:
yes, concurrent vehicle
yes, concurrent positive control
Details on study design:
Method Description
After the initial incubation, the medium was changed and the baseline opacity for each cornea was recorded. None of the corneas showed tissue damage; therefore, all corneas were used. For each treatment group (negative control solution, test item and positive control), three replicates were used. After removal of the pre-incubation medium (cMEM without phenol red), 750 µL of negative control solution, 750 µL of test item suspension and 750 µL of positive control solution were applied to each replicate.
According to the characteristics of the test item, the following treatment procedure was performed:

Open Chamber Method
In order to apply the test item, the nut was unscrewed to remove the glass disc.
750 µL of the test item were tested as suspension at 20% concentration in HBSS. The test item was given directly on the epithelium in such a manner that the cornea was cov-ered with test item.
Exposure time on the corneas was 4 hours at 32 ± 1 °C. After thorough rinsing with cMEM with phenol red and final rinsing with cMEM without phenol red, both chambers were filled with cMEM without phenol red, and the final opacity value of each cornea was recorded.

Permeability Test
After the recording of the final opacity value, the cMEM without phenol red was removed from the front chamber, and 1 mL sodium fluorescein solution was added to the front chamber for the detection of permeability of the corneas.
For the open chamber method, a sodium fluorescein solution with a concentration of 5 mg/mL was used.
The chambers were then closed again and incubated for 90 minutes at 32 ± 1 °C. After incubation, the content of the posterior chamber was thoroughly mixed. Then, its optical density at 492 nm was measured with the microtiter plate photometer.
Irritation parameter:
in vitro irritation score
Remarks:
IVIS (In Vitro Irritancy Score)
Run / experiment:
1st replicate
Value:
154.65
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
IVIS (In Vitro Irritancy Score)
Run / experiment:
2nd replicate
Value:
135.27
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
IVIS (In Vitro Irritancy Score)
Run / experiment:
3nd replicate
Value:
144.83
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
in vitro irritation score
Remarks:
IVIS (In Vitro Irritancy Score)
Run / experiment:
mean value
Value:
144.92
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
According to the guideline, the test is considered as valid if the positive control causes an IVIS that falls within two standard deviations of the current historical mean.
The mean IVIS of the negative control has to show an IVIS ≤ 3.
Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
According to OECD Guideline no. 437 (Oct. 2017), a substance with an IVIS > 55 induces serious eye damage, that should be classified as UN GHS Category I.
In the negative control, no signs of eye irritation were observed.
The positive control induced serious eye damage, which would be classified as GHS cate-gory I.
The test item L-Tyrosine, 3,5-dinitro- (wet product) induced serious eye damage on the cornea of the bovine eye. The calculated IVIS is 144.92.
The experiment is considered as sufficient for the classification of the test item, because all three replicates of the test item lead to the same assessment for the test item.
Executive summary:

Bovine corneas were used. They were collected from slaughtered cattle that were between 12 and 60 months old.

The test itemL-Tyrosine, 3,5-dinitro- (wet product)was brought onto the cornea of a bovine eye which had been previously incubated with cMEM without phenol red at 32 ± 1 °C for 1  hour and whose opacity had been measured.

The test item was incubated on the cornea for 4 hours at 32 ± 1 °C. After removal of the test item, opacity and permeability values were measured.

Hank’s Balanced Salt Solution (HBSS) was used as negative control. The negative control showed no irritating effect on the cornea and the calculated IVIS (In VitroIrritancy Score) is 1.68.

20% imidazole solution was used as positive control. The positive control induced serious eye damage on the cornea and was within two standard deviations of the current historical mean. The calculated IVIS is 98.54.

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: 53.2 mg
Tissue 2: 53.6 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 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:
0.04
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:
0.038
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:
1.9
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:
1.8
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:
1.9
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:
study cannot be used for classification
Conclusions:
Under the conditions of the test, L-Tyrosine, 3,5-dinitro- (wet product) 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 1.9%.
This value is below 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, 3,5-dinitro- (wet product)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 1.9%.

This value is well below the threshold for eye irritation potential (≤ 60%). Test items that induce values below the threshold are considered either eye irritant or inducing serious eye damage.

 

According to the OECD Guideline 492, the EpiOcularTMEye Irritation Test does not allow discrimination between eye irritation/reversible effects on the eye (Category 2) and serious eye damage/irreversible effects on the eye (Category 1). For these purposes, further testing with other suitable test methods is required.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (irreversible damage)

Additional information

Justification for classification or non-classification