N,N-bis(1-methylethyl)formamide

Endpoint summary

Description of key information

The study was to determine the skin sensitization potential of the test article through the ARE-Nrf2 (Antioxidant Response Element-Nuclear Factor) Transcription Factor pathway induction. This protocol was designed based on the OECD Guideline 442D.

Test article did not produce a positive sensitization response in the KeratinoSens™ cell line in two valid independent tests. Therefore, the test article is not considered a potential skin sensitizer in the KeratinoSens™ (ARENrf2) Assay.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 Mar 2021 to 30 Apr 2021

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Specific details on test material used for the study:

Batch number: M-O303190723301F
Purity: 99.68%

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: A 200 mM test article in DMSO was prepared.
From the 200mM concecentration, 11 additional stock solutions were prepared in DMSO by serial
two-fold dilutions (1:1) yielding stock solutions at concentrations of 0.098, 0.195, 0.39, 0.78, 1.56,
3.125, 6.25, 12.5, 25, 50, 100, and 200 mM.
- Preparation of the test chemical serial dilutions: The 100x stock solutions were diluted 25-fold.
From each of the 12 stock concentrations, 40 μl of the stock solution (100x), and 960 μl of dosing
medium) were mixed thoroughly to yield working formulations at concentrations of 3.92, 7.8, 15.6,
31.2, 62.4, 125, 250, 500, 1000, 2000, 4000, and 8000 μM.
- Preparation of the positive controls: Initially, a 12.8 mM cinnamic aldehyde in dimethyl
sulfoxide (DMSO) stock solution was prepared. From the 12.8 mM solution, five diluted solutions
were prepared in DMSO by serial two-fold dilutions (1:1) yielding stock solutions at concentrations of 0.4, 0.8, 1.6, 3.2, and 6.4 mM. The 100x stock solutions were diluted 25-fold.
From each of the five stock concentrations, 40 μl of the stock solution (100x) and 960 μl of dosing medium were mixed thoroughly to yield working formulations at concentrations of 16, 32, 64, 128, and 256 μM.
- Preparation of the solvent, vehicle and negative controls: The dimethyl sulfoxide (DMSO), as received, was diluted 25-fold with dosing medium to yield a 4% (v/v) DMSO solution.

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 2000 μM
- Solubility in solvents: An appropriate vehicle was determined by visually assessing the solubility of the test article in various solvents at the maximum allowable concentration (i.e., 200 mM).
The test article was soluble when formulated to 200 mM in dimethyl sulfoxide (DMSO). The 200 mM test article in DMSO formulation was used to prepare a 2000 μM test article concentration in dosing medium. After allowing the 2000 μM test article formulation to settle for at least two hours, it was determined that the test article was soluble in DMSO.
Following solubilization testing, DMSO was selected as the vehicle for test article stock solution
preparations and dosing medium was selected as the vehicle for final dosing solution preparations.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 2
- Test chemical concentrations: 0.98, 1.95, 3.9, 7.8, 15.6, 31.25, 62.5, 125, 250, 500, 1000, and 2000 μM
- Application procedure: A volume of 50 μl of each test article working formulation (4x) was added to one well, containing seeded cells and 150 μl of dosing medium, on each test plate. Plates were covered with sealing tape to minimize evaporation of volatile compounds and avoid cross-contamination between wells and were maintained in a humidified incubator at 37 (± 1) °C with 5 (± 1) % CO2 for 48 (± 4) hours.
- Exposure time: 48-hour exposure
- Study evaluation and decision criteria used:
The test article was considered positive for skin sensitization potential if the below conditions are true. If any of the criteria were not met, the test article was considered negative for skin sensitization potential.
• At least a 1.5-fold increase in luciferase activity induction was measured and was statistically
significant above the 1.5 threshold (e.g., using two-tailed unpaired Student’s t-test).
• The lowest test article concentration that yielded at least a 1.5-fold increase resulted in relative
cellular viability at least 70% or greater.
• The EC1.5 was less than 1000 μM.
• There was an overall dose-response increase in luciferase induction (or a biphasic response).
- Description on study acceptance criteria:
Positive Control (cinnamic aldehyde):
• At least a 1.5-fold increase in luciferase activity induction was measured at least one concentration and was statistically significant above the 1.5 threshold (e.g., using two-tailed unpaired Student’s ttest).
• The EC1.5 value was within two standard deviations of the historical mean (i.e., between -9.71 μM and 38.52 μM).
• The average induction of 64 μM cinnamic aldehyde (positive control) was between 2 and 8.
Alternatively, there was a clear dose-response with increasing luciferase activity induction
associated with the increasing concentration of cinnamic aldehyde.
Solvent/Vehicle Control (1% DMSO):
• The average coefficient of variation of the luminescence values of all 18 wells (six wells per plate, three total plates) was less than 20%.
Test Article:
• At least two consecutive test article concentrations resulted in relative cellular viability at least
70% or greater.

SEEDING AND INCUBATION
Following the 48-hour exposure, cellular viability was assessed using the MTT assay on the clear 96-well plate.
A 0.5 mg/ml solution of Thiazolyl Blue Tetrazolium Bromide (MTT) solution in dosing medium was prepared prior to each use. The MTT solution was warmed in a water bath at 37°C for at least
10 minutes. Undissolved MTT powder was pelleted by centrifugation at 1000 rpm for five minutes. The MTT solution was decanted (without disturbing any undissolved MTT) into a new container and protected from light until use.
The supernatant from each well of the clear 96-well plate was removed by aspiration and replaced with 200 μl of MTT solution. The plate was maintained in a humidified incubator at 37 (± 1) °C with 5 (± 1) % CO2 for four hours (± 15 minutes).
Following the four-hour incubation, MTT solution was removed by aspiration. Cells were immediately lysed following MTT solution aspiration.
In each well, cells were lysed with 200 μl of lysing agent 10% (w/v) sodium dodecyl sulfate (SDS) in distilled water. The plate was covered with sealing tape and maintained protected from light in a humidified incubator at 37 (± 1) °C with 5 (± 1) % CO2 overnight.
Prior to the collection of absorbance values, the μQuant™ microplate reader (BioTek® Instruments, Inc., model μQuant™ 1010-1) was verified to meet factory specifications at a wavelength of 620 nm with an absorbance test plate (provided by the manufacturer).
Following overnight incubation, the plate was agitated on an orbital shaker for at least 10 minutes. The absorbance of each well was determined at a wavelength of 600 nm.

LUCIFERASE ACTIVITY MEASUREMENTS
Following the 48-hour exposure, luciferase activity was assessed using the Steady-Glo® Luciferase Assay System (Promega™) on the three white 96-well plates.
Steady-Glo®Reagent (luciferase substrate stock solution) was prepared by transferring the contents of one bottle of Steady-Glo®Buffer to one bottle of Steady-Glo®Substrate (components were equilibrated at room temperature) and mixing by inversion. Steady-Glo®Reagent was stored frozen at -18 to -30°C for up to two weeks. Prior to use, Steady-Glo®Reagent was equilibrated at room temperature.
A luciferase substrate working solution was prepared fresh daily prior to use by diluting Steady-
Glo® Reagent two-fold (1:1) with Dulbecco's Modified Eagle Medium (DMEM).
Prior to collecting luminescence values, the GloMax® 96 Microplate Luminometer (Promega™,
model 9101-002) was verified to conform to the specified requirements of the manufacturer with a luminescence light plate (provided by the manufacturer).
The supernatant from each well of the three white 96-well plates was removed by aspiration and replaced with 100 μl of luciferase substrate working solution. The plates were protected from light and agitated on an orbital shaker for at least five minutes. The luminescence of each well was determined using the Steady-Glo® program on the luminometer.

DATA EVALUATION
- Cytotoxicity assessment: For each independent experiment, the absorbance readings from the MTT assay were used to determine cellular viability of each test article-treated well and positive control-treated well.

Vehicle / solvent control:

DMSO

Negative control:

not applicable

Positive control:

cinnamic aldehyde [442D]

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

Imax [442D]

Value:

1.23 µg/mL

Cell viability:

10,000 cells per well

Vehicle controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

GHS criteria not met

Conclusions:

Test article did not produce a positive sensitization response in the KeratinoSens™ cell line in two valid independent tests. Therefore, the test article is not considered a potential skin sensitizer in the KeratinoSens™ (ARENrf2) Assay.

Executive summary:

The purpose of this study was to determine the skin sensitization potential of the test article through the ARE-Nrf2 (Antioxidant Response Element-Nuclear Factor) Transcription Factor pathway induction. This protocol was designed based on the OECD Guideline 442D.

Test article did not produce a positive sensitization response in the KeratinoSens™ cell line in two valid independent tests. Therefore, the test article is not considered a potential skin sensitizer in the KeratinoSens™ (ARENrf2) Assay.

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

Skin sensitisation - in vitro study give negative result.

According to Regulation (EC) No 1272/2008, table 3.4.2, this substance should not be classified for this endpoint.