N,N'-bis(2,3-dihydroxypropyl)-5-nitrobenzene-1,3 dicarboxamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

27.1. - 16.3.2020

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Test material

Test material information

Constituent 1

Reference substance name:

N,N'-bis(2,3-dihydroxypropyl)-5-nitrobenzene-1,3 dicarboxamide

EC Number:

616-392-7

Cas Number:

76820-34-3

Molecular formula:

C14H19N3O8

IUPAC Name:

N,N'-bis(2,3-dihydroxypropyl)-5-nitrobenzene-1,3 dicarboxamide

impurity 1

Reference substance name:

Unknown impurities

Molecular formula:

Not available

IUPAC Name:

Unknown impurities

Test material form:

solid

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 00601016
- Expiration date of the lot/batch: 03/2022

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.
- Stability under test conditions: Stable

In vitro test system

Details on the study design:

PRINCIPLE OF ASSAY
This in vitro assay quantifies luciferase gene induction as a measure of the activation of the Keap1-Nrf2-antioxidant/electrophile response element (ARE)-dependent pathway in an immortalized adherent cell line derived from HaCaT human keratinocytes transfected with a selectable plasmid (KeratinoSensTM).
The KeratinoSensTM cell line was maintained in duplicate 96-well plates and exposed to the test item over a two-fold range of twelve concentrations for 48 hours. After exposure, the cells for viability measurement from the first plate were incubated with MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 3 – 4 hours. After incubation, the MTT taken up by viable cells was then extracted and the absorption was measured on a spectrophotometer. The cells from the second plate were mixed with luciferase substrate and the luminiscence was measured. The viability of the cells and the gene induction of the luciferase activity were then calculated.
The amount of the viable cells in the presence of the test item concentrations was compared to the amount of the viable cells in the negative control cultures. The percentage of the viability was calculated and the IC50 was determined.
The maximal gene induction of the luciferase activity (Imax) in the presence of the test item concentrations was calculated and compared to the gene induction in the blank and solvent (negative) control cultures. The EC1.5 was then calculated.

TEST SYSTEM
The immortalized adherent cell line derived from HaCaT human keratinocytes transfected with a selectable plasmid (KeratinoSensTM) is used for testing. It is supplied by acCELLerate GmbH, Germany (Lot. No. 92 180810HB01 in the 1st, 2nd and 3rd experiment and Lot. No. 92 191122JP04 in the 4th and 5th experiment). Cells are stored in liquid nitrogen. For the test, the cells are seeded in a 96-well plate.
In the 1st, 2nd and 3rd experiment were used cells which can be kept in culture for a maximum passage number of 25. For these experiments, the absence of mycoplasma contamination is verified. The cell cycle of this cells is about 24 hours and it is evaluated with every new lot of the cells or after one year.
In the 4th and 5th experiment were used instant cells, which do not need to be kept in culture. Passage lower than 25, absence of mycoplasma and properties of cells were declarated by certificate of analysis of cells.

STUDY DESIGN
Test item preparation:
The test item was dissolved in DMSO, which is the first of choice solvent according OECD TG 442D. The test item was ultrasounded for better dissolution.
Maximal concentration of stock solution was 200 mM. A fresh stock solution of the test item was prepared before each experiment.
The test item was diluted by DMSO and the final concentration of DMSO was 1 % in each well.

Preparation of cells:
In the 1st, 2nd and 3rd experiment the KerationoSens cells were cultivated and prepared for testing according to internal SOP.
In the 4th and 5th experiment were used cells which do not need to be cultivated before experiments. The cells were thawed just before the start of experiment. Preparation of the cell suspension before application on 96 well plates was done according to internal SOP.

Sensitisation test:
Five independent experiments were performed.
In the 1st, 2nd and 3rd experiment cells were contamined by mycoplasma. So the 4th and 5th experiment were proceeded with new cells, which were without contamination of mycoplasma. The 4th and 5th experiment, which fulfilled the acceptability criteria, were used for further evaluation.
In each experiment 12 test item concentrations in range 0.98 2000 µM were tested.
No precipitation was observed and the highest test concentration was 2000 µM.
Each experiment included corresponding positive controls (PC = Cinnamic aldehyde), negative controls (NC = untreated wells with cells and 1% DMSO) and blank of the negative controls (BL = untreated wells without cells with 1% DMSO).
Two 96-well plates were used in the 1st, 2nd and 3rd experiment (one plate for luminescence measurement and one plate for viability measurement). In the 4th and 5th experiment three 96 well plates were used (two plates for luminescence measurement and one plate for viability measurement).
In the 1st, 2nd and 3rd experiment every concentration, PC and BL was tested in tetraplicates and NC in 24 replicates for luminescence measurement. For viability measurement NC was tested in 12 replicates, PC and BL in duplicates and every concentration in triplicates. In the 4th and 5th experiment every concentration was tested in triplicates, NC was tested in 24 replicates, PC and BL was tested in tetraplicates for luminescence measurement. For viability measurement NC was tested in 12 replicates, PC and BL in duplicates and every concentration in triplicates.

Results and discussion

In vitro / in chemico

Results

Results 1

Parameter:

other: Imax

Value:

1.5

Positive controls validity:

valid

Results 2

Parameter:

other: cellular viability

Value:

70

Positive controls validity:

valid

Results 3

Parameter:

other: EC1.5

Value:

1 000

Positive controls validity:

valid

Results 4

Parameter:

other: apparent overall dose-response

Value:

0

Any other information on results incl. tables

PREDICTION OF SENSITISATION POTENTIAL

1. the Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control

The Imax at the highest concentration 2000 µM was 0.9 in the 4th experiment and 0.8 in the 5th experiment which is not increased compared to the negative control.

 

2. the cellular viability is higher than (>) 70 % at the lowest concentration with induction of luciferase activity above 1.5 fold

The viability of cells at the highest concentration 2000 µM was 108.8 % in the 4th experiment and 93.0 % in the 5th experiment. Both values were higher than 70%.

 

3.the EC1.5 value is less than (<) 1000 µM

The EC1.5 was >2000 µM in both experiments.

 

4.there is an apparent overall dose-response for luciferase induction

There was not dose-response for luciferase induction in both experiments (see Fig. 1).

 

The test item was not cytotoxic so all concentrations (0.98 – 2000 µM) were used for evaluation of sensitisation potential.

The results of the positive control were evaluated by statistical software Statgraphics Centurion – version XV, USA (GLP software).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the experimental design described above, the luciferase induction at the highest concentration (2000 µM) is lower than 1.5 threshold in both experiments. The test item, NBA-A, had not sensitisation potential predicted by the ARE-Nrf2 Luciferase KeratinoSens™ Test Method.

Executive summary:

An in vitro skin sensitisation test (ARE-Nrf2 Luciferase KeratinoSens™ Method) was performed to assess the sensitisation potential of NBA A. This method specifically addresses the second key event of the Adverse Outcome Pathway (AOP) of skin sensitisation (keratinocyte activation) through induction of cyto-protective pathways in cells in response to electrophiles and oxidative stress. The test was performed according to OECD Test Guideline No. 442D: In Vitro Skin Sensitisation: ARE Nrf2 Luciferase Test Method (2018) and it is used for supporting the discrimination between skin sensitisers and non-sensitisers.

 

The cell line KeratinoSensTM was seeded into 96-well plates and incubated overnight. The test item was dissolved in DMSO to prepare stock concentration 200 mM. The stock solution was diluted into 12 final concentrations, ranging from 0.98 – 2000 µM. Overall five independent experiments were performed, however only two experiments were acceptable. Cells were assayed for viability and luciferase activity in each experiment. The positive control was cinnamic aldehyde and the negative control was untreated wells with cells and 1% DMSO. There was also 1% DMSO in the test item wells.

Under the experimental design described above, the luciferase induction at maximal tested soluble concentration (2000 µM) is less than 1.5 threshold in both acceptable experiments. The test item does not lead to cytotoxicity in the highest test concentration 2000 µM.

 Therefore, the test item NBA A had not sensitisation potential predicted by the ARE Nrf2 Luciferase Test Method.