[No public or meaningful name is available]

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

no

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

(2R,2'R)-3,3'-disulfanediylbis(2-((S)-2-aminopropanamido)propanoic acid)

Cas Number:

115888-13-6

Molecular formula:

C12H22N4O6S2

IUPAC Name:

(2R,2'R)-3,3'-disulfanediylbis(2-((S)-2-aminopropanamido)propanoic acid)

Test material form:

solid

In vitro test system

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.

Results and discussion

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.

In vitro / in chemico

Results

Results 1

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

Results 2

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.