2,2'-azobis[2,4-dimethylvaleronitrile]

Endpoint summary

Description of key information

one Study equivalent to OECD Guideline 442D and one study according to OECD Guideline 442C available.

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:

09. Oct. 2017 - 08.Mar. 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Principles of method if other than guideline:

The LuSens cell line was specially designed for this test system by the BASF SE (Ludwigshafen,
Germany). It employs the use of a reporter gene for luciferase placed under the
control of the antioxidant response element (ARE) and hence monitors Nrf-2 transcription
factor activity. For designing this cell line, a human keratinocyte cell line (provided by
RWTH, Aachen, Germany) was transfected with the pGL4.20 [luc2/Puro] vector (Promega,
Germany) carrying the regulatory antioxidant response element (ARE) upstream of the
luciferase gene (Luc2, Promega, Germany) at the Institute of Anatomy and Cell Biology of
the RWTH, Aachen (laboratory of PD Dr. Wruck).

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: CF1001
- Expiration date of the lot/batch: Jul. 2018
- Purity test date: not stated

RADIOLABELLING INFORMATION (if applicable)
not applicable

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Freezer: (-15 - -25 °C)
- Stability under test conditions: assumed stable under given conditions.
- Solubility and stability of the test substance in the solvent/vehicle: a Solubility Test
was performed to verify whether the test item is sufficiently soluble in DMSO. The
solubility test was performed under non-GLP conditions. The test item was soluble at the
required concentration of 200 mM.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: assumed stable under given conditions.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
none

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Details on the study design:

Cell Cultures
For mycoplasma contamination screened stocks of LuSens cells are stored in liquid nitrogen
in the cell bank of LAUS GmbH to allow a continuous stock of cells, which guarantees
similar parameters of the experiment and reproducible characteristics of the cells.
For the Cytotoxicity Range Finder Assay cells of passage 8 were used. For the main experiments
cells of passage 10 were used. After thawing the cells were cultivated in DMEM
(9 % FCS) in cell culture flasks at 37 ± 1 °C in a humidified atmosphere with 5.0 ± 0.5 %
CO2.

Negative Control
Name DL-Lactic acid
CAS no. 50-21-5
Solvent DMSO
Supplier: Sigma-Aldrich
Purity: 90.5 %
Lot no.: BCBP5043V
Expiry Date: 26. Jan. 2021
Final concentration: 5000 μM
The solution was freshly prepared on the day of the experiment.
Positive Control
Name EGDMA (Ethylene glycol dimethylacrylate)
CAS no. 97-90-5
Solvent DMSO
Supplier: Sigma-Aldrich
Purity: 98 %
Lot no.: SHBG0572V
Expiry Date: 27. Jan. 2021
Final concentration: 120 μM
The solution was freshly prepared on the day of the experiment.
Solvent Control
Name DMSO
CAS no. 67-68-5
Supplier: Carl Roth
Purity: > 99.5 %
Lot no.: 187256959
Expiry Date: 04. Apr. 2020
Final concentration: 1 %

Experimental Performance
Experiment I and II were performed in the same way. Experiment II serves only to confirm
the results of experiment I. The exposure dates were 24. Oct. 2017 and 25. Oct. 2017.
At the time of seeding the cells were 80 % confluent. The cells were washed twice with
PBS (without Ca2+/Mg2+) containing 0.05% EDTA. Afterwards the cells were trypsinized
until the cells detached. To stop this reaction, medium no. 2 was added. After centrifugation
(5 min at 380 * g), the supernatant was discarded and the cells were resuspended in
medium no. 2. After quantification, the cell suspension was adjusted to 83 000 (±10 %)
cells per mL. 120 μL of the cell suspension (≙ 10 000 cells) were seeded in two clear flat
bottom 96 well plates (one for viability and one for luciferase induction measurement)9.
Both plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere
for 24 h in Experiment I and 23 h and 30 min in Experiment II.
The treatment procedure was performed on both 96 well plates identically: After the incubation
time the medium was removed from the cells and 150 μL medium no. 3 were added
to each well. Afterwards 50 μL of each single test item concentration and the controls were
added to the cells in triplicates (test item concentrations). 24 wells were used for solvent
control, 12 wells were used for growth control (cells + medium no. 3), 6 wells were used for
negative control, 5 wells for positive control and 1 well for blank. The plates were sealed
with breathable tape to avoid evaporation of volatile compounds and to avoid cross contamination
between wells. Afterwards the plates were incubated for 48 h at 37 ± 1 °C in a
humidified atmosphere containing 5.0 ± 0.5 % CO2.
For the evaluation of the viability, one of the plates was used:
The MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of
MTT solution. All solutions were removed from the wells of the 96 well plate and 200 μL
MTT working solution were added to each well. The plate was incubated for 2 h at 37 ±
1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere. Afterwards the solution was removed
and 100 μL of lysis buffer were added to each well. The plate was agitated for
5 min before it was measured at 570 nm and at 690 nm (reference) at the photometer. The
cell viability is measured by the reduction of the tetrazolium dye MTT (3-(4,5-
Dimethyl thiazole 2-yl)-2,5-diphenyltetrazolium-bromide) (yellow color) to its insoluble
formazan (purple color) in living cells and therefore indicates the amount of living cells.
After the measurement of the color change, the values were transferred in a validated
spreadsheet for the calculation of the viability (see chapter 7.3.2, page 19).
For the evaluation of the Luciferase induction, the second plate was used:
For the evaluation of the Luciferase expression all solutions were removed from the wells
and the cells were washed twice with 300 μL PBS (with Ca2+/Mg2+). Afterwards 100 μL per
well of a Lysis buffer were added to the cells and incubated for 5 min at room temperature.
During this process, the plate was slightly moved. Afterwards 100 μL Steady-Glo® Reagent
were added to each well and the plate was shaken again slowly for 5 min at room temperature.
Then, 160 μL per well were transferred to a white flat bottom 96 well plate and the
luminescence was measured for 2 seconds using a luminometer.

Positive control results:

First Experiment:
the positive control induced a clear effect with an induction value of 5.1 fold in comparison to the solvent control.
second experiment
the positive control induced a clear effect with an induction value of 4.0 fold in comparison to the solvent control.

Key result

Run / experiment:

other: Eperiment I

Parameter:

other: Induction of Luciferase, expressed as "fold" compared to Solvent control

Value:

2

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Experiment II

Parameter:

other: Induction of Luciferase, expressed as "fold" compared to Solvent control

Value:

1.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the experimental conditions of this study, the test item, 2,2'-Azobis(2,4-dimethylvaleronitrile), was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

Executive summary:

This in vitro study evaluates the potential of the test item 2,2'-Azobis(2,4-dimethylvaleronitrile) to activate the Nrf2 transcription factor (sensitizing potential) by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non-sensitizers in the context of

an integrated approach to testing and assessment.

The LuSens test is an ARE Reporter Gene Assay that was developed by the BASF SE (Ludwigshafen, Germany) and is based on the OECD 442D Guideline (KeratinoSens Assay).

The assay differs in some points from the OECD guideline. All differences are marked with footnotes. In addition all deviations are indicated in chapter 11 page 27.

The assay included a cytotoxicity range finder test (CRFT) and two independent experiments (experiment I and II) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item. Based on the results of this test the concentrations for the two experiments were determined. In the experiments, the highest nominal applied concentration (2000 μM) was chosen based on the results obtained in the CRFT. A geometric series (factor 1.21) of eleven dilutions thereof was prepared. Precipitation of the test item was not visible in any of the experiments.

DMSO (final concentration: 1 %) was used as solvent control and medium no. 3 as growth control. Lactic acid (5000 μM) was used as negative control and EGDMA2 (120 μM) as positive control.

The evaluated experimental points and the results are summarised in chapter 8. A substantial and reproducible dose-dependent increase in luciferase induction above 1.5 fold in more than two non-cytotoxic test item concentrations was observed in both experiments.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13. April - 28. April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

HPLC system
Designation: HPLC_4
Components: Degasser G1322A
Quaternary pump G1311A
Autosampler G1313A
Column compartment G1316A
UV/VIS-Detector DAD G1315A
Manufacturer: Agilent Technologies
Software: CHROMELEON 6.80 SR15b Build 4981

Test solutions
Dilution buffers
• 2 mL Acetonitrile were mixed with 8 mL phosphate buffer, pH 7.5 (Peptide dilution buffer C)
• 2 mL Acetonitrile were mixed with 8 mL ammonium acetate buffer, pH 10.2 (Peptide dilution buffer K)
Peptide stock solutions
The peptide stock solutions were freshly prepared for each assay.
• 0.667 mM Cys-Peptide solution was prepared by dissolving 22.5 mg of the peptide in 45.0 mL phosphate buffer, pH 7.5. (batch no. 20180425)
• 0.667 mM Lys-Peptide solution was prepared by dissolving 23.3 mg of the peptide in 45.0 mL ammonium acetate buffer, pH 10.2. (batch no. 20180426)
Peptide calibration standards
From each peptide stock solution the following calibration standards were prepared in the appropriate dilution buffer (see chapter 7.2.1): 0.534 / 0.267 / 0.134 / 0.067 / 0.033 / 0.017 mM Peptide.
Calibration samples were analysed before the samples containing the test item. Blank dilution buffer was also measured.
Test item samples
Samples were prepared in triplicate for each peptide. The Cys-peptide samples were pre-pared in 1:10 molar ratio (0.5 mM peptide: 5 mM test item), the Lys-peptide samples in 1:50 molar ratio (0.5 mM peptide and 25 mM test item) using the stock solutions described in chapters 7.2.2 and 7.1. A final volume of 1 mL per sample was prepared for each sample.

Incubation
The positive control, solvent control sets C, and test item samples were incubated in closed amber glass HPLC vials in an incubation chamber at 25.0 ± 2.5 °C for 22.08 h for Cys-peptide and 24 h for Lys-peptide.
All three replicates of test item incubated with Cys-peptide and all three replicates of Posi-tive Control Cinnamaldehyde were turbid after incubation.

Positive control results:

a) The mean peptide depletion and standard deviation of the three replicates of the positive control Cinnamaldehyde were in the acceptable range of 60.8 – 100.0 % and ≤ 14.9 %, respectively, for the Cys-peptide.
b) The mean peptide depletion and standard deviation of the three replicates of the positive control 2,3-Butanedione were in the acceptable range of 10.0 – 45.0 % and ≤ 11.6 %, respectively, for the Lys-peptide.

Key result

Run / experiment:

other: Lys-Peptide, mean

Parameter:

other: Depletion %

Value:

20.35

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Cys-Peptide, mean

Parameter:

other: Depletion %

Value:

99.44

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Conclusions:

The DPRA predicition is “positive” with high reactivity according to the Cysteine 1:10/Lysine 1:50 prediction model.

Executive summary:

The study was performed in order to evaluate the reactivity of the test item2,2'-Azobis(2,4-dimethylvaleronitrile)towards Cysteine (Cys-) and Lysine (Lys-) containing peptides. A test item solution in Acetonitrile was incubated 24 ± 2 h at 25 °C together with Cysteine and Lysine peptides, respectively, and the peptide concentration after the incubation was measured using HPLC-UV.

Three replicates were prepared using 1:10 and 1:50 molar ratio of the test item with the Cys- and Lys-peptide, respectively. Triplicate samples of the solvent without test item were incubated and measured in parallel.

The peptide depletion values after incubation are shown in Table

Results

	Cys-peptide depletion [%]	Lys-Peptide depletion [%]	Mean peptide depletion [%]
Experiment	99.44	20.35	59.89

 

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The Adverse Outcome Pathway (AOP) set up for the endpoint skin sensitisation includes three major steps for skin sensitisation. The available studies provide results on two of the steps and no contradictory results are obtained. Therefore, the information is sufficient for assessment of the hazard.

Both studies are evaluated to show that the substance is a skin sensitiser. Based on the fact that the substances shows a high activity in the OECD 442C study (mean Protein Depletion ca. 60 %, Depletion of Cystein-proteine nearly 100 %), it can be presumed that the substance has the potential to produce significant sensitisation in humans.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The available information is conclusive and sufficient for classification as skin sensitising cat. 1A