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Endpoint summary

Description of key information

N,N´-di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 showed negative results in the KeratinoSens™ assay and a U-Sens™ assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27.03.2020 - 29.05.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™

Version / remarks:

2018

Deviations:

no

GLP compliance:

yes

Type of study:

activation of keratinocytes

Details on the study design:

PREPARATION OF TEST ITEM SOLUTIONS
- The test item was suspended in DMSO at 200 mM (white homogenous suspension). The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. From this stock 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solutions were diluted 25-fold with exposure medium. These solutions were diluted 4-fold with exposure medium in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 µM (final concentration DMSO of 1%).
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- Test item concentrations were used within 2.5 hours after preparation.

TEST SYSTEM
- A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switzerland).
- All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 50 – 93 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 – 37.0°C).
- For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator.
- The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test chemical and control items were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were covered with foil and then incubated for about 48 hours ± 1 h at 37±1.0°C in the presence of 5% CO2.

LUCIFERASE ACTIVITY MEASUREMENT
The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 5 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).

CYTOTOXICITY ASSESSMENT
For the KeratinoSensTM cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1; Sigma) and cells were incubated for 3 - 4 hours at 37°C ± 1.0°C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution (Sigma) to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

Positive control results:

Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.14 and the EC1.5 was 46 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.84 and the EC1.5 was 57 µM.

Run / experiment:

other: 1

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.07

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: maximum luciferase activity induction (Imax)

Value:

1.1

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Both tests passed the acceptance criteria:
• The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
• The EC1.5 of the positive control was within two standard deviations of the historical mean (46 µM and 57 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (3.14-fold and 2.84-fold in experiment 1 and 2, respectively).
• Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.6% and 3.8% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

The test item showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.07-fold and 1.10-fold in experiment 1 and 2 respectively. the test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to 2000 µM.

Overview EC_1.5, I_max, IC₃₀and IC₅₀Values

	EC1.5(µM)	Imax	IC30(µM)	IC50(µM)
Test item Experiment 1	NA	1.07	NA	NA
Test item Experiment 2	NA	1.10	NA	NA
Pos Control Experiment 1	46	3.14	NA	NA
Pos Control Experiment 2	57	2.84	NA	NA

NA = Not applicable

Interpretation of results:

GHS criteria not met

Conclusions:

N,N´-di-L-Alanyl-L-Cystine / (L-Ala-L-Cys) 2 is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17.04.2020 - 28.05.2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 442E – Annex II "In Vitro Skin Sensitisation: U937 Cell Line Activation Test (U-SENS™)"

Version / remarks:

2018

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EURL ECVAM DB-ALM Protocol n° 183: U937 Cell Line Activation Test for Skin Sensitization (U-SENS™)

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes

Type of study:

activation of dendritic cells

Details on the study design:

TEST ITEM PREPARATION
- In the main experiments the test item was dissolved (clear solution) in complete medium at 0.4 mg/mL. The stock was diluted to a final test concentrations of 200, 100, 50, 20, 10 and 1 µg/mL in the first experiment and 100, 140, 180 and 200 μg/mL in the second experiment in the 96-well plate.
- No precipitation was observed at the end of the incubation period in the 96-well plates.
- Test item concentrations were used within 4 hours after preparation.

TEST SYSTEM
- U937 human monocytes (Inducible CD86-expressing cells; ATCC (American Type Culture Collection, Virginia, USA)
- Stock and treatment cultures were performed in RPMI-1640 medium supplemented with 10% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (FCS), L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 μg/mL respectively).
- All incubations were carried out in a humid atmosphere of 80 - 100% (actual range 55 - 92%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.9 - 36.8°C).
- Cultures were initiated in 96-well plates using 100 µL/well of a cell suspension adjusted at 5.0 x 105 viable cells/mL. Cell viability was > 90%. All assays were performed using two replicate culture-wells for the test item. One replicate was dedicated to the nonspecific IgG1 binding and the other one to the CD86 binding. Three replicates of untreated control (RPMI), vehicle control (in case of DMSO as vehicle), negative (LA) and positive (TNBS) controls were tested.
- Two valid experiments were conducted per test item.

TREATMENT OF CELLS
- Cells are treated for 45 ± 3 hours with the selected doses or controls (100 µL). The test item was in the first experiment evaluated up to 200 µg/mL using six doses: 1.0, 10, 20, 50, 100 and 200 µg/mL.
- In the second experiment cells were treated with four selected doses of test item. At least 2 concentrations were common with the previous experiment. The concentrations selected in the second experiment were 100, 140, 180 and 200 µg/mL.
- In all experiments, an untreated control (RPMI), vehicle control (in case of DMSO as vehicle) and the positive (TNBS) and negative control (LA) items were included. The final volume in the wells was 200 µL.

CELL ANTIBODIES STAINING FOR IgG1 AND CD86
Cultures were transferred into V-shaped 96-well plates. The cells were separated from the exposure medium by centrifugation (5 min, 200 g). The supernatant was discarded and cells were rinsed once with 100 µL/well Phosphate Buffered Saline (PBS) containing 5% FCS. After a second centrifugation step (5 min, 200 g) 100 µL/well of staining buffer (PBS containing 5% FCS) was applied to the cells.
FITC-conjugated antibodies was used for both IgG1 and CD86 staining:
- Mouse IgG1 of unknown specificity, for isotypic control
- Human CD86 specific mouse IgG1
The cells were transferred into new V-shaped 96-well plates (keeping the same plate template) containing 5 µL/well of the appropriate antibody (1:1 diluted in PBS) and placed refrigerated in the dark for 30 minutes. After this staining period, the cells were rinsed twice with a mixture of PBS/FCS and once in PBS alone and re-suspended in 90 µL of PBS.

FLOW CYTOMETRY METHOD
Just before acquisition, 5 µL of a 0.5 µg/mL propidium iodide (PI) solution was added to each well. The size (FSC) was set linear and the granularity (SSC) parameter was set to logarithmic scale and a R1 region was defined in which approximately 10,000 events were acquired for each culture. The acquisition parameters remained unchanged for the acquisition of all the wells. For the acquisition the BD FACSCanto™ flow cytometer was used and for further analysis BD FACSDiva™ software was used.
All analysis parameters were set on the RPMI wells for IgG1 and remained unchanged, for the analysis of all the other wells.

Positive control results:

Experiment 1: The positive control (TNBS) showed a S.I. ≥ 621% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (Lactic acid) showed a S.I. ≤ 101% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The positive control (TNBS) showed a S.I. ≥ 649% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 115% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Run / experiment:

other: 1

Parameter:

other: % Viability (Mean)

Value:

100

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: % Viability (Mean)

Value:

99

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: CD86-IgG1 S.I.

Value:

91

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: CD86-IgG1 S.I.

Value:

75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 1

Parameter:

other: Colour Interference S.I.

Value:

103

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: 2

Parameter:

other: Colour Interference S.I.

Value:

95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

Two independent experiments were performed. The cell viability before incubation with the test item was > 90% (99% and 94% in experiment 1 and 2, respectively). The cells were in these experiments incubated with the test item in a concentration range of 1.0 – 200 µg/mL. The increase of CD86 cell surface marker expression was assessed by measuring the amount fluorescent cell staining of the CD86 cell surface marker compared to the vehicle control. In addition, the viability was assessed with propidium iodide.

Experiment 1:
- No precipitation was observed at the end of the incubation period in the 96-well plates.
- The test item showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.
- No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. No EC150 could be calculated and is considered to be higher than 200 µg/mL.
- The test item showed no colour interference.
- The positive control (TNBS) showed a S.I. ≥ 621% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 101% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Experiment 2:
- No precipitation was observed at the end of the incubation period in the 96-well plates.
- The test item showed no toxicity, the viability of the cells was higher than 70% at all test concentrations and therefore no CV70 values could be calculated and is considered to be higher than 200 µg/mL.
- No increase in expression levels of CD86 compared to the vehicle control was observed at any of the test concentrations after treatment with the test item. No EC150 could be calculated and is considered to be higher than 200 µg/mL.
- The test item showed no colour interference.
- The positive control (TNBS) showed a S.I. ≥ 649% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%). The negative control (LA) showed a S.I. ≤ 115% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).

Both tests passed the acceptance criteria:
- At the end of the incubation treatment period, the mean viability of the triplicate untreated U937 cells was above the threshold of 90% (100% in experiment 1 and 99% in experiment 2).
- The CD86 basal expression of untreated U937 cells is within the range of ≥ 2% and ≤ 25% in both experiments.
- At least two out of three IgG1 values of untreated U937 cells fell within the range of ≥ 0.6% and < 1.5% in both experiments.
- No drift in CD86 expression was observed in the untreated controls and negative controls.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item showed no toxicity (No CV70 value) and no biologically relevant induction of the CD86 activity (No EC150 value) was measured at any of the test concentrations in both experiments. The test item is classified as negative in the U-Sens™ assay since negative results (< 150% increase) were observed at all test concentrations with a cell viability of >70% compared to the vehicle control. In conclusion, N,N´-di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 is classified as negative (no increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

N,N´-di-L-Alanyl-L-Cystine / (L-Ala-L-Cys)2 showed negative results in the KeratinoSens™ assay and a U-Sens™ assay. Therefore, no classification and labelling according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2017) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendment) is needed.