Registration Dossier

Diss Factsheets

Administrative data

Description of key information

The eye hazard potential of N,N´-Di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 was evaluated in a Bovine Corneal Opacity and Permeability Assay (BCOP) according to OECD TG 437. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 after 4 hours of treatment. In conclusion, since N,N´-Di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.

The skin irritation and corrosion potential of N,N´-Di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 was evaluated in two studies using human three dimensional epidermal models according to OECD TG 431 and OECD TG 439. The test item did not induce skin irritation or corrosion. In conclusion no classification is required for skin irritation or skin corrosion.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20.05.2020 - 29.05.2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Vehicle:
unchanged (no vehicle)
Details on test system:
TEST SYSTEM
- EpiDerm Skin Model (EPI-SIT, Lot no.: 30869 Kit Q)
- Source: MatTek In Vitro Life Science Laboratories, Bratislava, Slovakia

TREATMENT
Before the assay was started the tissues were transferred to new 6-well plates containing 0.9 mL Assay medium per well. At least 25 mg solid was added into the 6-well plates on top of the skin tissues. Test items tissues were moistened before application of the test item with DPBS (25 µL), to ensure close contact to the tissue. Three tissues were treated with 30 µL DPBS (negative control) and 3 tissues with 30 µL 5% SDS (positive control) respectively.
After the exposure period with the test item (35 ± 1.0 minutes at 37.0 ± 1.0°C and the remaining period of the 60 ± 1 minutes test item exposure at room temperature ), the tissues were thoroughly rinsed with Dulbecco’s phosphate buffered saline (DPBS) to remove residual test item. If necessary cotton wool swabs were used to remove any remaining test item. After rinsing the cell culture inserts were each dried carefully and moved to a new well on 0.9 mL pre-warmed assay medium until all tissues were dosed and rinsed. Subsequently the skin tissues are incubated for 24 ± 2 hours at 37°C. The tissues were transferred to 0.9 mL fresh Assay medium and placed back for a post-incubation period of 18 ± 2 hours at 37°C.

CELL VIABILITY MEASUREMENT
After incubation, cell culture inserts were dried carefully to remove excess medium. The cell culture inserts were transferred into a 24-wells plate prefilled with 0.3 mL MTT-medium (1.0 mg/mL). The tissues were incubated for 3 hours ± 5 minutes at 37°C. After incubation the tissues were placed on blotting paper to dry the tissues. After incubation the tissues were washed with DPBS and formazan was extracted with 2 mL isopropanol for at least 2 hours at room temperature with gentle shaking. The amount of extracted formazan was determined spectrophotometrically at 570 nm in duplicate with the TECAN Infinite® M200 Pro Plate Reader.
Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues. Skin irritation potential of the test item was classified according to remaining cell viability following exposure of the test item.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- A test item is considered irritant in the skin irritation test if:
The relative mean tissue viability of three individual tissues after 60 minutes of exposure to the test item and 42 hours of post incubation is ≤ 50% of the mean viability of the negative controls.
- A test item is considered non-irritant in the in vitro skin irritation test if:
The relative mean tissue viability of three individual tissues after 60 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
32.8 to 38.7 mg
Duration of treatment / exposure:
60 minutes
Duration of post-treatment incubation (if applicable):
42 hours
Number of replicates:
3
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean
Value:
92
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test item was checked for possible direct MTT reduction and color interference in the Skin corrosion test using EpiDerm as a skin model (Test Facility Study No. 20245940). Because the solutions did not turn blue / purple and/or a blue / purple precipitate was observed and/or the OD for the test item solution was ≤0.08, therefore it was concluded that the test item did not interfere with the MTT endpoint.
The relative mean tissue viability obtained after 60 ± 1 minutes treatment with the test item compared to the negative control tissues was 92%. Since the mean relative tissue viability for the test item was above 50% the test item is considered to be non-irritant.
The positive control had a mean cell viability of 3.9% after 60 ± 1 minutes exposure. The absolute mean OD570 of the negative control tissues was slightly above the laboratory historical control data range, but well within the acceptance limits of OECD439 (lower acceptance limit ≥0.8 and upper acceptance limit ≤2.8). As the laboratory historical control data is limited and the OECD439 range is leading, this is acceptable. The standard deviation value of the percentage viability of three tissues treated identically was less than 8%, indicating that the test system functioned properly.

Interpretation of results:
GHS criteria not met
Conclusions:
N,N´-di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 is non-irritant in the in vitro skin irritation test under the experimental conditions described.
Endpoint:
skin corrosion: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27.03.2020 - 17.04.2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)
Version / remarks:
2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Details on test system:
TEST SYSTEM
- EpiDerm Skin Model (EPI-200, Lot no.: 33018 kit G+F)
- Source: MatTek In Vitro Life Science Laboratories, Bratislava, SlovakiaMatTek Corporation, Ashland MA, USA

TREATMENT
The skin tissues were kept in the refrigerator the day they were received. The next day, at least 1 hour before the assay was started the tissues were transferred to 6-well plates containing 0.9 mL DMEM per well. The level of the DMEM was just beneath the tissue (see figure 1). The plates were incubated for approximately 1 hour at 37.0 ± 1.0ºC. The medium was replaced with fresh DMEM just before the test item was applied. The test was performed on a total of 4 tissues per test item together with a negative control and positive control. Two tissues were used for a 3-minute exposure to the test item and two for a 1-hour exposure. The skin was moistened with 25 µL Milli-Q water (Millipore Corp., Bedford, Mass., USA) to ensure close contact of the test item to the tissue and 25.8 to 28.1 mg of the solid test item was added into the 6-well plates on top of the skin tissues.
For the negative and positive controls, 2 tissues were treated with 50 µL Milli-Q water (negative control) and 2 tissues were treated with 50 µL 8N KOH (positive control) for both the 3-minute and 1-hour time point.
After the exposure period, the tissues were washed with phosphate buffered saline (Invitrogen Corporation, Breda, The Netherlands) to remove residual test item. The skin inserts were carefully dried. Rinsed tissues were kept in 24 well plates on 300 µL DMEM until 6 tissues (= one application time) were dosed and rinsed.

CELL VIABILITY MEASUREMENT
The DMEM was replaced by 300 µL MTT-medium and tissues were incubated for 3 hours at 37°C in air containing 5% CO2. After incubation the tissues were washed with PBS and formazan was extracted with 2 mL isopropanol (MatTek corporation) over night at room temperature. The amount of extracted formazan was determined spectrophotometrically at 570 nm in triplicate with the TECAN Infinite® M200 Pro Plate Reader.

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
A test item is considered corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability ≥50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.
A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
25.8 to 28.1 mg
Duration of treatment / exposure:
3 minutes / 1 hour
Number of replicates:
3 for the 3-minute application, 3 for the 1-hour application
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean 3-minute application
Value:
93
Negative controls validity:
valid
Positive controls validity:
valid
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Mean 1-hour application
Value:
92
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The test item was checked for color interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because the solutions did not turn blue / purple nor a blue / purple precipitate was observed it was concluded that the test item did not interfere with the MTT endpoint.
The relative mean tissue viability obtained after the 3-minute and 1-hour treatments with the test item compared to the negative control tissues was 93% and 92% respectively. Because the mean relative tissue viability for the test item was not below 50% after 3 minutes treatment and not below 15% after 1 hour treatment the test item is considered to be not corrosive.
The absolute mean OD570 (optical density at 570 nm) of the negative control tissues was within the acceptance limits of OECD 431 (lower acceptance limit ≥0.8 and upper acceptance limit ≥2.8) and the laboratory historical control data range. The mean relative tissue viability following the 1-hour exposure to the positive control was 7.2%.
In the range of 20 - 100% viability the Coefficient of Variation between tissue replicates was ≤13%, indicating that the test system functioned properly.
Interpretation of results:
GHS criteria not met
Conclusions:
N,N´-di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 is not corrosive in the in vitro skin corrosion test under the experimental conditions described.
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
Study period:
27.03.2020 - 07.04.2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
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)
Version / remarks:
2017
Deviations:
no
GLP compliance:
yes
Species:
other: bovine eyes
Details on test animals or tissues and environmental conditions:
TISSUES
- Source: Slaughterhouse (Vitelco, -'s Hertogenbosch, The Netherlands)
- Age at study initiation: young cattle
- Transport: in physiological saline under cooled conditions
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was applied directly on the corneas in such a way that the cornea was completely covered (308.4 to 339.3 mg).
Duration of treatment / exposure:
240 +/- 10 minutes at 32 +/- 1°C
Duration of post- treatment incubation (in vitro):
90 +/- 5 minutes at 32 +/- 1°C
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS :
The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded.
The isolated corneas were stored in a petri dish with cMEM (Eagle’s Minimum Essential Medium containing 1% (v/v) L-glutamine and 1% (v/v) Fetal Bovine Serum). The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS :
After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NEGATIVE CONTROL USED : Physiological saline

POSITIVE CONTROL USED : 20% (w/v) Imidazole solution prepared in physiological saline

TREATMENT METHOD:
The medium from the anterior compartment was removed and 750 µL of the negative control and 20% (w/v) Imidazole solution (positive control) were introduced onto the epithelium of the cornea. The test item was weighed in a bottle and applied directly on the corneas in such a way that the cornea was completely covered. The holder was slightly rotated, with the corneas maintained in a horizontal position, to ensure uniform distribution of the solutions over the entire cornea. Corneas were incubated in a horizontal position for 240 +/- 10 minutes at 32 +/- 1°C.

REMOVAL OF TEST SUBSTANCE
After the incubation the solutions and the test item were removed and the epithelium was washed at least three times with MEM with phenol red. Possible pH effects of the test item on the corneas were recorded. Each cornea was inspected visually for dissimilar opacity patterns. The medium in the posterior compartment was removed and both compartments were refilled with fresh cMEM and the opacity determinations were performed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea. The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter (OP-KIT). The change in opacity for each individual cornea (including the negative control) was calculated by subtracting the initial opacity reading from the final post-treatment reading. The corrected opacity for each treated cornea with the test item or positive control was calculated by subtracting the average change in opacity of the negative control corneas from the change in opacity of each test item or positive control treated cornea. The mean opacity value of each treatment group was calculated by averaging the corrected opacity values of the treated corneas for each treatment group.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microtiter plate reader (OD490) .

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA: Decision criteria as indicated in the TG was used.
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
0.2
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
-0.3
Negative controls validity:
valid
Positive controls validity:
valid
Irritation parameter:
fluorescein retention score
Run / experiment:
Mean
Value:
0.031
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (20% (w/v) Imidazole) was 133 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 0.2 after 240 minutes of treatment.
Interpretation of results:
GHS criteria not met
Conclusions:
In conclusion, since N,N´-Di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 induced an IVIS ≤ 3, no classification is required for eye irritation or serious eye damage.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

N,N´-Di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 induced an IVIS ≤ 3. Therefore, no classification is required for eye irritation or serious eye damage.

N,N´-Di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 did not induce skin irritation or skin corrosion in studies using EpiDerm as skin model. Therefore, no classification is required for skin irritation or skin corrosion.