Reaction mass of 2-methylbutyl nitrite and 2-methyl-1-butanol and 1-pentanol and pentyl nitrite

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

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

Test guideline 2

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

Test material

Test material information

Constituent 1

Reference substance name:

2-methylbutan-1-ol; 2-methylbutyl nitrite; pentan-1-ol; pentyl nitrite

EC Number:

951-169-0

Molecular formula:

C15H46N2O10

IUPAC Name:

2-methylbutan-1-ol; 2-methylbutyl nitrite; pentan-1-ol; pentyl nitrite

Test material form:

liquid

In vitro test system

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.

Results and discussion

In vitro / in chemico

Results

Parameter:

other: Direct Peptide Reactive Assay

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

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.

Applicant's summary and conclusion

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.