2-propylvaleronitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Remarks:

Keratinosens

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Report date:

2017

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

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

Alternative in vitro method recommended in first intention by ECHA.

Test material

Test material information

Constituent 1

Reference substance name:

2-propylvaleronitrile

EC Number:

236-338-2

EC Name:

2-propylvaleronitrile

Cas Number:

13310-75-3

Molecular formula:

C8H15N

IUPAC Name:

2-propylpentanenitrile

In vitro test system

Details on the study design:

The test article was dissolved in dimethyl sulfoxide (DMSO) to the final concentration (200 mM). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article (0.098, 0.196, 0.391, 0.781, 1.563, 3.125, 6.25, 12.5, 25, 50, 100 and 200 mM).
The master concentrations were then further diluted 25 fold into culture medium containing serum and finally used for treatment with a further 4 fold dilution factor so that the final concentrations of test article ranged from 0.98 to 2000 μM in 1% DMSO.
Aliquots of 50 μL of each of the final concentrations were transferred to each of three luciferase plates and a single viability plate. Each plate was sealed using a plate sealer and then incubated at 37±1°C, 5% (v/v) CO2 in air, in a humidified environment for 48±1 hours.
After the 48-hour exposure period, the medium in the luciferase plates was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT).The plate was sealed and incubated for 4 hours at 37±1°C, 5% (v/v) CO2. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator at 37±1°C, 5% (v/v) CO2 in air and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm using a SpectraMax M2e. After the 48-hour exposure period, the cells in the viability plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plates were then incubated for 20 minutes at 25±2°C, loaded into the luminescence plate reader and read.

Results and discussion

Positive control results:

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 8 to 64 μM in Experiment 1, at concentrations of 4 to 64 μM in Experiment 2 and at concentrations of 16 to 64 μM in Experiment 3. The EC1.5 values for the positive control were 7.99, 2.45 and 11.53 μM in Experiments 1, 2 and 3, respectively. The average induction in the three replicates for the positive control at 64 μM was 22.07, 6.59 and 17.42 in Experiments 1, 2 and 3, respectively.

In vitro / in chemico

Results

Results 1

Key result

Run / experiment:

other: experiment 1

Parameter:

other: EC 1.5 (ug/mL)

Value:

805

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: I(max) 1.73

Results 2

Key result

Run / experiment:

other: experiment 2

Parameter:

other: EC 1.5 (ug/mL)

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

I (max) 1.15; below thre threshold of 1.5 therefore EC1.5 could not be calculated

Results 3

Key result

Run / experiment:

other: experiment 3

Parameter:

other: EC 1.5 (ug/mL)

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Remarks:

I (max) 1.15; below thre threshold of 1.5 therefore EC1.5 could not be calculated

Other effects / acceptance of results:

Test item: The maximal fold increases (Imax) were 1.73, 1.15 and 1.16 for Experiments 1, 2 and 3, respectively. The EC1.5 value for Experiment 1 was 805 μg/mL. There were no EC1.5 values for Experiments 2 and 3 as there were no statistically significant increases in induction.

Positive control: The EC1.5 value for the positive control in Experiment 2 and the average induction at 64 μM in Experiments 1 and 3 were outside the range specified in the Protocol. These deviations from Protocol did not affect the integrity or outcome as the positive control gave a satisfactory dose response.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test article, 2-propylvaleronitrile (CAS 13310-75-3), was considered to be negative in the ARE-Nrf2 Luciferase Test.