Pentyl nitrite

Endpoint summary

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

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: 4th February 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM (INVITTOX): Protocol No. 154_ Direct Peptide Reactivity Assay (DPRA) for Skin Sensation Testing

Version / remarks:

2012

Deviations:

no

Principles of method if other than guideline:

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.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: direct peptide reactive assay

Details on the study design:

SKIN SENSITISATION (IN VITRO) - DETAILS ON STUDY DESIGN

PREPARATION OF REAGENTS AND MOBILE PHASE
- Preparation of mobile phase
Mobile Phase A: 1000 ml UPW and 1 ml trifluoracetic acid were mixed together.
Mobile Phase B: 1000 ml ACN and 850 µl trifluoracetic acid were mixed together.
The mobile phases were filtered using the filtering apparatus.
- Preparation of 100mM Sodim phosphate monobasic
13.8 g Sodium phosphate monobasic monohydrate was dissolved in 1000 ml ultra pure water. The solution was stored in the refrigerator.
- Preparation of 100mM Sodim phosphate dibasic
26.8 g Sodium phosphate dibasic heptahydrate was dissolved in 1000 ml ultra pure water. The solution was stored in the refrigerator
- Preparation of 100mM Phosphate buffer, pH=7.5
18 ml 100mM Sodium phosphate monobasic and 82 ml 100 mM Sodium phosphate dibasic were mixed together, pH was measured. The pH was adjusted to 7.5±0.05 with either monobasic (to acidify)
or dibasic (to basify) solution.
- Preparation of 100mM Ammonium acetate buffer, pH=10.2
1.542 g Ammonium acetate was dissolved in 200 ml ultra pure water. The pH was adjusted to 10.2 by dropwise addition of ammonium hydroxide.
- Preparation of Dilution buffer for calibration
8 ml of buffer (pH 7.5 phosphate buffer for Cysteine peptide or pH 10.2 ammonium acetate buffer for Lysine peptide) and 2 ml Acetonitrile were mixed together.

PRE-WORK PREPARATION AND PROCEDURES
- Solvent selection
Solvent selection was performed according to DB-ALM (INVITTOX) Protocol No.154: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitisation Testing, 2012. According to this protocol, acetonitrile
is the preferred solvent for test chemicals.
An approximately 100 mM solution of Pentyl nitrite in acetonitrile was prepared. Test item was dissolved completely, therefore acetonitrile was used for further tests.
- Preparation of test item solution
Test item was pre-weighted into clean, dry 4 ml glass vials. The target weight of test item needed to prepare 3.0 ml of 100 mM solution was calculated according to the formula:
The weighed amount of Pentyl nitrite (approximately 97.63 mg) was dissolved in 3 ml of acetonitrile.
- Preparation of positive and negative control solution
As a positive control, a 100 mM solution of Cinnamaldehyde was used. The amount required to prepare this solution was calculated according to the formula above. The weighed amount of Cinnamic
aldehyd (approximately 40.13 mg) was dissolved in 3 ml of acetonitrile.
As a negative control, a 100 mM solution of 1-butanol was used. The amount required to prepare this solution was calculated according to the formula above. The weighed amount of 1-butanol
(approximately 22.7 mg) was dissolved in 3 ml of acetonitrile.
- Preparation of reference control and co-elution control
Three types of "Reference Controls" were included in this study. 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.
20 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 based on this formula:
The appropriate amount of buffer to individual peptides was added just before testing itself.
Approximately 10.02 mg of the Cysteine peptide was added to 20 ml of phosphate buffer, pH 7.5. Approximately 10.36 mg of the Lysine peptide was added to 20 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.
- Preparation of samples for analysis
Using 1 ml autosampler vials as container, was prepare the sample by adding the reagents in the quantity and order listed in Table No. 1 and Table No. 2 in final report.
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. Test chemical was analysed in triplicate
for both peptides.

Parameter:

other: Direct Peptide Reactive Assay

Remarks on result:

positive indication of skin sensitisation

Mean of percent Cysteine and percent Lysine depletion values is 48.5.Test item Pentyl nitrite was included in the reactivity class – high reactivity and was classified as positive (skin sensitizer) in DPRA prediction.

The criteria of quality are in accordance with theOECD TG 442C,In ChemicoSkin Sensitisation: Direct Peptide Reactivity assay (DPRA) (Adopted: 4 February 2015), chapter 27, 28, page 7.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

All study acceptance criteria have been successfully met.

Test item, Pentyl nitrite, was classified as positive (skin sensitizer) in DPRA prediction; it was assigned to reactivity class – high reactivity.

Executive summary:

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

 

The test was performed according toOECD TG 442C,In ChemicoSkin 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 48.5.Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used (see chapter3.7of final report).

 

Pentyl nitrite was classified as positive (skin sensitizer) in DPRA prediction; it was assigned to reactivity class – high reactivity.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: DB-ALM Protocol No. 155: KeratinoSensTM, ECVAM

Version / remarks:

29th January 2015

Deviations:

no

Principles of method if other than guideline:

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 (1, 2).
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 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.

The test was performed according to the methods listed above. The sensitisation potential is indicated by gene induction of luciferase activity above 1.5 treshold and the results were interpreted according to prediction model in OECD TG 442D (paragraph 39).

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Details on the study design:

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 Givaudan, Schweiz. For the test
the cells are seeded in 96-well plate.
- Maintenance and manipulation with the cell line in this study is documented in laboratory diary No. 259/N in detail.
- Original cell stock can be kept in culture for a maximum passage number of 25. For every study absence of mycoplasma contamination should be verified.
- The cell cycle of this cell line is about 24 hours and it is evaluated with every new lot of the cells or after one year.

STUDY DESIGN:
- Overall 5 experiments were done. Because of contamination of plates the 1st, 2nd and 4th experiment was stopped and they were not used for evaluation. For evaluation of senzibilization it was used
3rd and 5th experiment.
- Every tested concentration, NC and PC was tested in 2 independent experiments. Two 96-well plates were used for each experiment (one plate for luminescence measurement and one plate for viability
measurement).
- Every tested concentration was tested in tetraplicates or pentaplicates, NC was tested at least in 3 x hexaplicates and PC was tested in triplicates in every experiment.
- Each experiment included corresponding positive controls (PC = Cinnamic aldehyde), negative controls (NC = untreated wells with cells and solvent 1% DMSO) and blank of the negative controls (BL =
untreated wells with solvent 1% DMSO).
- The 3rd and 5th experiment, which fullfilleed acceptability criteria were used further for results evaluation. Three experiments were non acceptable (because of contamination of plates). Primary data from each experiment are archived with this study.
- The result of both independent acceptable experiments was positive, so the next experiment did not have to be done.

EXPERIMENTAL PROCEDURE:
- Preparation of the test item:
- The test item was dissolved in DMSO in stock concentration 200mM (concentration 69.65 mg/mL) and next diluted into 12 concentrations in medium for exposure (DMEM with low glucose and
supplemented with glutamax + 1% FBS) in two-fold range (0.98 – 2000 µM) according procedure in OECD 442D. Fresh stock solutions were prepared before each experiment in glass vials
and 96-well plates.
- Controls:
- The final concentrations of Cinnamic aldehyde (= PC) in wells were 4, 8, 16, 32 and 64 µM. Stock solution 200 mM of Cinnamic aldehyde was prepared fresh before each experiment or
stored in the freezer in glass vials (no older than 1 month).
- Cell cycle length determination:
- The cell cycle length of KeratinoSensTM cells was evaluated by doubling time experiment: first day the cells in twelve Petri dishes (60 mm) was seeded in three concentration 2000, 4200 and 9000 cells/mL. Every following day in similar time the number of cells was counted always in three Petri dishes.
- Then the proliferation curves were constructed for each concentration (average number of counted cells in three Petri dishes vs. time in hours) and cell cycle length was determined. Results of the experiment are archived in the laboratory.
- Manipulation with cell line:
- Passage number of the cells used for seeding in experiments was 20 in the 3rd experiment and 22 in the 5th experiment. The level of confluence was 80 – 90%.
- The density of the cells for calculation of cell concentration for experiments was evaluated microscopically by Cellometer Auto T4.
- Demonstration of the proficiency of the luminescence measurement:
- For the luminescence measurement the luminometer Synergy HTX Biotek, made by Biotek, USA, was used. The luminescence is measure without any filter.
- The demonstration of the proficiency of the laboratory was done according OECD TG 442D, Annex 2 and Annex 3. Detailed report about results of this demonstration is available on request.
- For the measurements, the Luciferase glow substrate made by Promega was used (ONE Glo Ex Cat. No. E6110).
- Treatment and cultivation:
- The KeratinoSensTM cells were seeded into three 96-well plates in density 80 000 cells per well (125 µL/well) and incubated for 24 ± 2 hours. After that, the cultivation medium was
aspirated and 150 µL of exposure medium and 50 µL of prepared test item solutions were added to each well (according procedure in OECD 442D) of the plates. It was carried out in a
laminar box at room temperature in aseptic conditions. Cultures were treated with the test item for 48 hours ± 30 minutes in incubator (5% CO2, 37 ± 1°C, humidity). After time of exposure the medium was aspirated from the second plate, cells were mixed with luciferase substrate (50 µL per well substrate + 50 µL per well medium for exposure) and after 3 - 5 minutes shaking the luminescence was measured (luminometer Synergy HTX Biotek). The medium from the first plate was aspirated, fresh medium (200 µL/well) was added and the cells were dyed by MTT
solution (concentration 5 mg/mL – 27 µL/well) and incubated next 4 hours. After dyeing time, the MTT was extracted by isopropanol (200 µL/well) and absorbance was measured (600 nm)
by spectrophotometer.

Positive control results:

Positive control (EC1.5 in µM): Mean - 5.6, SD 1.8
Historical range: EC1.5 = 2 – 39.8 µM

Key result

Parameter:

other: Sensitional potential

Vehicle controls validity:

not specified

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

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 20/11/2018 and 03/12/2018. The absence of contamination was confirmed (see Annex 3).

ACCEPTABILITY OF EXPERIMENTAL RESULTS:
All presented results met criteria of the acceptability mentioned in chapter 3.8, in detail:
- The luciferase activity induction obtained with the postitive 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 2.17 (mean value) in concentration 8 µM and in the 5th experiment was 4.32 in concentration 16 µM.
- EC1.5 value of the postitive control should be in historical range control (of laboratory dataset: 2 – 39.8 µM).
EC1.5 in the 1st experiment was 4.4 µM and in the 2nd experiment 7 µM, which is in the historical range.
- The average coefficient of variation of the luminescence reading for the negative control (DMSO) should be below 20%. If the variability is higher, results should be discarded.
In the 1st experiment the coefficient of variation was 19 % and in the 2nd experiment the coefficient of variation was 19 %.
- Overview of historical control values of positive control are stored in testing laboratory.

RESULTS OVERVIEW

Results of experiments are summarized in the Annex 1 (Raw data) and Annex 2 (Summary tables) of final report. The tables and curves contain thedose applied per culture in µM, calculated luciferase induction values, OD₅₇₀values, % of viability and statistical evaluations.

Overall 2 independent experiments, which fullfiled acceptability criteria, were done. Every experimental plate was controlled visually under the inverted microscope to check right cell confluence in wells.

The measured values from experiments were evaluated according equations in chapter 3.7 of ifnal report and the acceptability criteria were controlled. Further, dose-response curve was constructed (in MS Excel).

The 4 criteria for the prediction of sensitisation potential were met, so the prediction is considered as positive. The positive result of the test item (and positive control) was evaluated by statistical software Statgraphics Centurion – version XV, USA (GLP software) and these data are stored in Archive of CETA.

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

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

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Justification for classification or non-classification

Mean of percent Cysteine and percent Lysine depletion values is 48.5. Based on the results obtained, the Cysteine 1:10/Lysine 1:50 prediction model was used.

Pentyl nitrite was classified as positive (skin sensitizer) in DPRA prediction; it was assigned to reactivity class – high reactivity.