Propatylnitrate

Endpoint summary

Description of key information

The health hazard evaluation of the skin sensitisation potential of Propatyl Nitrate:

ARE-Nrf2 Luciferase test:

The test was performed according to OECD Test Guideline No. 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, Adopted in February 2015 and is used for supporting the discrimination between skin sensitisers and non-sensitisers.

The results were positive, so the test item Propatylnitrate had sensitisation potential predicted in the ARE-Nrf2 Luciferase Test Method.

 

DPRA test:

The test was performed according to OECD TG 442C, In Chemico Skin Sensitisation: Direct Peptide Reactivity assay (DPRA) (Adopted: 4 February 2015)

Mean of percent Cysteine and percent Lysine depletion values is 33.75. Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used positive (skin sensitizer) in DPRA prediction; it was assigned to reactivity class – moderate reactivity.

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:

22.01.2018 – 26.01.2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Adopted in February 2015

GLP compliance:

yes (incl. QA statement)

Type of study:

other: gene induction of luciferase activity above 1.5 treshold

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

This method specifically addresses second key event of the Adverse Outcome Pathway of the skin sensitisation through induction of cyto-protective pathways in cells in response to electrophiles and oxidative stress.

The 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 involvement of this regulatory pathway in skin sensitisation has been demonstrated in a number of in vivo studies.
KeratinoSensTM cell line was maintained in 96-well plate and exposed to test item over a two-fold range of twelve concentrations for 48 hours. After time of exposure the cells from the first plate were stained by MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) for 3 – 4 hours. After dyeing time, the MTT taken up by the viable cells were then extracted and the absorption was measured by spectrophotometer. The cells from the second and third plate are 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.

Positive control results:

Positive control (EC1.5 in µM) =23.4 µM
Historical range: EC1.5 = 4 - 29 µM

Key result

Run / experiment:

other: Mean (1,2)

Parameter:

other: EC1.5

Remarks:

in µM

Value:

197.7

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The doubling time of KeratinoSensTM cells (supplier Givaudan, Schweiz) was about 20 hours. It is in accordance with cell characterisation in scientific literature.
The Mycoplasma contamination was verified on 11.12.2017. The absence of contamination was confirmed.

ACCEPTANCE OF RESULTS:

All presented results met criteria of the acceptability:
1.The luciferase activity induction obtained with the positive control (Cinnamic aldehyde) should be statistically significant above the threshold 1.5 in at least one of the tested concentration (4 – 64 µM).
In the 1st experiment the value above the threshold 1.5 was 5 (mean value) in concentration 64 µM and in the 2nd experiment was 2 in concentration 64 µM.

2.EC1.5 value of the positive control should be in historical range control (of laboratory dataset: 4 - 29 µM). In addition, the average induction in 3 replicates for cinnamic aldehyde in concentration 64 µM should be between 2 – 8.
EC1.5 in the 1st experiment was 21.3 µM and in the 2nd experiment 29.6 µM, which is in the historical range.

3.The average coefficient of variation of the luminescence reading for the negative control (DMSO) should be below 20% in each repetition which consists of 6 wells tested in triplicate. If the variability is higher, results should be discarded.
In the 1st experiment the coefficient of variation was 20.4 % and in the 2nd experiment the coefficient of variation was 13.2 %.
Overview of historical control values of positive control are stored in testing laboratory.

Interpretation of results:

other: sensitisation potential predicted in the ARE-Nrf2 Luciferase Test Method, another in vitro test is needed

Conclusions:

Under the experimental design described above, the results were positive in both experiments, so the test item, Propatyl nitrate, had sensitisation potential predicted in the ARE-Nrf2 Luciferase Test Method.

Executive summary:

Sensitisation Test In Vitro: ARE-Nrf2 Luciferase Method assayed sensitisation potential of the test item Propatylnitrate. This method specifically addresses second key event of the Adverse Outcome Pathway of the skin sensitisation 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, Adopted in February 2015 and is used for supporting the discrimination between skin sensitisers and non-sensitisers.

The cells of the line KeratinoSensTM were seeded into 96-well plates and incubated overnight. The test item was dissolved in DMSO to prepare stock concentration 200mM. Then the stock concentration was dissolved into final concentration two-fold range 0.98 – 2000 µM in medium for exposure. The plates with cells were incubated for next 48 hours. After time of exposure the cells from the first plate were stained by vital dye MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and incubated next 4 hours. After dyeing time, the MTT was desorbed by fixative solution and the absorbance was measured. The cells from the second and third plate were mixed with luciferase substrate (supplemented by Promega) and the luminescence was measured. The gene induction of sensitisation pathways is compared to amount of luminescence.

Under the experimental design described above, the results were positive, so the test item Propatylnitrate had sensitisation potential predicted in the ARE-Nrf2 Luciferase Test Method.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15.02.-16.03.2018

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))

Version / remarks:

Adopted: 4 February 2015

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:
The DPRA is a chemistry-based assay. Nucleophile-containing synthetic peptides (cysteine peptide­Ac-RFAACAA-COOH; lysine peptide- Ac-RFAAKAA-COOH) are used to screen for skin sensitisation potential by measuring peptide depletion following incubation with allergens and non-allergens.
Synthetic heptapeptides containing either cysteine or lysine are incubated with the test substance for 24 hours. Depletion of the peptide in the reaction mixture is measured by high pressure liquid chromatography (HPLC) using UV detection. Average peptide depletion data for cysteine and lysine are then calculated.

According to DB-ALM (INVITTOX) Protocol No.154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing 2012, acetonitrile is the preferred solvent for test chemicals.
An approximately 100 mM solution of Propatyl Nitrate in acetonitrile was prepared. Test item was dissolved completely, therefore acetonitrile was used for further tests. The weighed amount of Propatyl Nitrate (approximately 82.0 mg) was dissolved in 3 ml of acetonitrile.

Positive control: The weighed amount of Cinnamic aldehyd (approximately 39.7 mg) was dissolved in 3 ml of acetonitrile.
Negative control: The weighed amount of 1-butanol (approximately 22.7 mg) was dissolved in 3 ml of acetonitrile.

Reference Control is a peptide solution where the test chemical is replaced by the solvent used to dissolve it.
• Reference Control A was made with acetonitrile and used to verify the accuracy of the calibration curve for peptide quantification.
• Reference Control B was made with acetonitrile and its replicates are injected in the beginning and in the end of the experimental run to verify the stability of the peptide over the analysis time.
• Reference Controls C was made with acetonitrile (solvent used to solubilise the test chemicals). They are used to verify that the solvent does not impact the Percent Peptide Depletion. The appropriate Reference Controls C for each chemical are used to calculate Percent Peptide Depletion.
• Co-elution control was constituted by the test chemical alone without the addition of peptides. Instead of the addition of peptides, the appropriate amount of buffer was added. The co-elution test serves to verify that the test substance does not have the same retention time and does not absorb at 220 nm as the Cysteine and Lysine peptides.

Preparation of peptide stock solution (0.667 mM)
The necessary amount of peptide was estimated. For each analysis with addition of peptide, 800 µl of stock solution is needed. 30 ml of the stock solution of Cysteine or Lysine peptide (0.667 mM) was prepared. Based on the amount of peptide stock solution needed, was weighed an appropriate amount of peptide into a volumetric flask. The appropriate amount of peptide was calculated.
The appropriate amount of buffer to individual peptides was added just before testing itself.
Approximately 15.0 mg of the Cysteine peptide was added to 30 ml of phosphate buffer, pH 7.5. Approximately 15.5 mg of the Lysine peptide was added to 30 ml of ammonium acetate buffer, pH 10.2.

Standard preparation for the determination of the calibration curve
Using serial dilution was prepared standards of the peptide stock solution covering the range from 0.534 – 0.0167 mM.
The vials were closed, mixed and placed in the HPLC autosampler (dark) at 25 °C for 24 hours.

Preparation of samples for analysis:
1:10 ratio, Cysteine peptide (0.5 mM Peptide, 5 mM test chemical)
750 µL Cysteine peptide solution (or 750 µL Phosphate buffer, pH= 7.5 for co-elution controls)
200 µL Acetonitrile
50 µL test chemical solution
or 50 µL solvent (ACN) for reference controls
or 50 µL positive (negative) control solution for positive (negative) controls

1:50 ratio, Lysine peptide (0.5 mM Peptide, 25 mM test chemical)
750 µL Lysine peptide solution (or 750 µL Ammonium acetate buffer, pH= 10.2 for co-elution controls)
250 µL test chemical solution
or 250 µL solvent (ACN) for reference controls
or 250 µL positive (negative) control solution for positive (negative) controls

The vials were closed, mixed and placed in the HPLC autosampler (dark) at 25 °C for 24 hours. Then, samples were visually inspected prior to HPLC analysis. Each test chemical was analysed in triplicate for both peptides.

The validated HPLC method - conditions:
Column: Agilent Zorbax SB-C18, 100x2.1 mm, 3.5µm
Precolumn: Phenomex security guard C18, 4.0 x 2.0 mm
Mobile phase A: 0.1% Trifluoracetic acid in water
Mobile phase B: 0.085% Trifluoracetic acid in acetonitrile
Time programmer:
0 min 10 % B
10 min 25 % B
11 min 90 % B
13 min 90 % B
13.5-20 min 10 % B
Column temperature: 30 °C
Sample temperature: 25°C
Flow rate: 0.35 mL/min
Injection volume: 10 μL
Detection: 220 nm

Positive control results:

Cinnamic aldehyd was used as positive control at a concentration of 100 mM in acetonitrile.
The mean Percent peptide depletion value of three replicates for Cinnamic aldehyd was calculated.

Percent Cystein depletion 71.9 %
Percent Lysine depletion 64.7 %

Key result

Run / experiment:

other: 1

Parameter:

other: mean of percent Cysteine and percent Lysine depletion values

Value:

33.75

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

All acceptance criteria have been successfully met.
The following criteria should be met for a run to be considered valid:
a) the standard calibration curve should have an r2˃0.99
b) the mean percent peptide depletion value of the three replicates for the positive control Cinnamic aldehyde should be between 60.8% and 100% for the Cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be <14.9% for the percent Cysteine depletion and <11.6% for the percent lysine depletion
c) the mean peptide concentration of reference controls A should be 0.50±0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%. If one or more of these criteria is not met the run should be repeated.
The following criteria should be met for a test item results to be considered valid:
a) the maximum standard deviation for the test item replicates should be <14.9% for the percent Cysteine depletion and <11.6% for the percent lysine depletion
b) the mean peptide concentration of the free Reference controls C in the appropriate solvent should be 0.50±0.05 mM.

Interpretation of results:

other: positive (skin sensitizer) in DPRA prediction; it was assigned to reactivity class – moderate reactivity

Conclusions:

Mean of percent Cysteine and percent Lysine depletion values is 33.75. Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used.
Test item, Propatyl Nitrate, was classified as positive (skin sensitizer) in DPRA prediction; it was assigned to reactivity class – moderate reactivity.

Executive summary:

The purpose of the test is to contribute to the evaluation of the skin sensitisation potential of Propatyl Nitrate. The study is a part of test item health hazard evaluation.

The test was performed according to OECD TG 442C, In Chemico Skin Sensitisation: Direct Peptide Reactivity assay (DPRA) (Adopted: 4 February 2015)

All acceptance criteria have been successfully met.

Mean of percent Cysteine and percent Lysine depletion values is 33.75. Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used.

Test item, Propatyl Nitrate, was classified as positive (skin sensitizer) in DPRA prediction; it was assigned to reactivity class – moderate reactivity.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available