1,4,10,13-pentaoxacyclopentadecane

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Test material

Test material information

Constituent 1

Reference substance name:

1,4,10,13-pentaoxacyclopentadecane

EC Number:

251-379-6

EC Name:

1,4,10,13-pentaoxacyclopentadecane

Cas Number:

33100-27-5

Molecular formula:

C10H20O5

IUPAC Name:

1,4,7,10,13-pentaoxacyclopentadecane

Specific details on test material used for the study:

Purity: 96.9%
Description (physical state): clear colorless non-viscous liquid
Lot/batch no: 1909004 (TSN404665)

In chemico test system

Details of test system:

other:

Details on the study design:

Test System
The DPRA is an in chemico assay which measures the depletion of the cysteine and lysine containing peptides using high performance liquid chromatography (HPLC). The peptides were custom materials containing phenylalanine to aid in detection of either cysteine or lysine as the reactive center. The concentrations of peptide within each sample or control were measured by HPLC with UV detection at 220 nm.

Study Design
The experimental design of the study consists of one definitive assay to determine the peptide depletion of the test substance after 24 hours reactivity period. The percent depletion of the cysteine and lysine containing peptides was then measured by HPLC.

Preparation of reaction mixture
Reaction mixtures were prepared in triplicate. If precipitates or phase separation was observed, samples were centrifuged at low speed (100 to 400 x g) to force the precipitate to the bottom of the vial as a precaution, since a large amount of precipitate may clog the HPLC tubing or column. If the total run time for the cysteine and lysine sample sets was greater than 30 hours, then the cysteine and lysine assays were run on separate days. If analysis occurs on separate days, all test substance dilutions were freshly prepared for each day.

After each addition to the vials, the vials were gently shaken to mix the reaction mixtures. Once prepared, the reaction mixtures were incubated for 24 ± 2 hours in the dark at room temperature before separation and analysis on the HPLC.
Preparation of control articles
Reference controls A, B, and C reaction mixtures were prepared with acetonitrile or the appropriate solvent replacing the test substance.
Reference control A: It consisted of 3 replicates prepared with acetonitrile as the test substance and was run in the same run sequence as the assay standards to verify the accuracy of the standard curve used for peptide quantification.
Reference control B: It was the same 3 replicates as reference control A and was run at the beginning and the end of the run sequence of the test substances. The purpose of reference control B was to verify the stability of the peptide over the analysis time.
Reference control C: It was made as 3 replicates for each solvent used in the assay. It was used in the calculation to determine percent peptide depletion of the test substance treated peptides.

Data Analysis and Calculation
The concentration of peptide was determined in each sample from absorbance at 220 nm measuring the peak area of either the cysteine of lysine containing peptide peaks and calculating the concentration of peptide using the linear standard curves generated from the standards. The peak area data and chromatograms were created using the Empower™ software. The percent depletion of peptide was determined in each sample from absorbance at 220 nm.
Criteria for Determination of a Valid Definitive Assay
The assay was accepted when the following criteria were met:
1) Each standard curve must have an r2 >0.990 and the mean peptide concentration of the reference controls A and C must equal 0.50 ± 0.05 mM. The peak area CV for reference controls B and C must be <15%
2) The percent peptide depletion for the cysteine peptide exposed to the positive control (cinnamic aldehyde) must be >60.8% and the SD for the replicates must be <14.9. For the lysine peptide, the percent depletion by the positive control must be >40.2% and <69.0% and the SD for the replicates must be <11.6.
3) The SD criteria for the test substance replicates must be <14.9% for the percent cysteine depletion and <11.6% for the percent lysine depletion.
Evaluation of Test Results
Based on the calculated % depletion, the reactivity potential of the test substance will be assessed as described below.

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Results and discussion

In vitro / in chemico

Results

Results 1

Key result

Group:

other: positive control

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

53.84 %

At concentration:

0.65 mM

Results 2

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean lysine depletion

Value:

0.7 %

At concentration:

0.5 mM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Results 3

Key result

Group:

other: positive control

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

74.66 %

At concentration:

0.12 mM

Results 4

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

mean cystein depletion

Value:

15.85 %

At concentration:

0.4 mM

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Definitive assays
According to the Cysteine 1:10/Lysine 1:50 prediction model, the test substance, 15-CROWN-5, was predicted to be a sensitizer.

Any other information on results incl. tables

Test substance preparation

Prior to the definitive trial, a solubility test was performed to determine appropriate solvents for preparing the test substance at 100 mM. The following observations were determined during the solubility test.

The test substance, 15-CROWN-5, was found to be soluble in acetonitrile with vortexing. The test substance dilution was a clear colorless non-viscous solution.

Test substance preparation

Prior to the definitive trial, a solubility test was performed to determine appropriate solvents for preparing the test substance at 100 mM. The following observations were determined during the solubility test.

The test substance, 15-CROWN-5, was found to be soluble in acetonitrile with vortexing. The test substance dilution was a clear colorless non-viscous solution.

Definitive assays

According to the Cysteine 1:10/Lysine 1:50 prediction model, the test substance, 15-CROWN-5, was predicted to be a sensitizer.

Table 4:                    DPRA Results – Cysteine – Positive Control and 15-CROWN-5

Treatment	Replicate	Peptide depletion (%)	Peptide conc. Mean ± SD (CV)	Peptide depletion Mean ± SD (CV)
Cinnamic aldehyde (positive control)	r1	74.38	0.12 ± 0.00 (0.01)	74.66 ± 0.28 (0.00)
	r2	74.94
	r3	74.67
15-CROWN-5	r1	11.42	0.40 ± 0.02 (0.05)	15.85 ± 3.89 (0.25)
	r2	17.43
	r3	18.70

 

Table 5:                    DPRA Results – Lysine – Positive Control and 15-CROWN-5

Treatment	Replicate	Peptide depletion (%)	Peptide conc. Mean ± SD (CV)	Peptide depletion Mean ± SD (CV)
Cinnamic aldehyde (positive control)	r1	53.35	0.65 ± 0.72 (1.11)	53.84 ± 0.69 (0.01)
	r2	54.33
	r3	-195.93
15-CROWN-5	r1	0.69	0.50 ± 0.00 (0.00)	0.70 ± 0.26 (0.37)
	r2	0.97
	r3	0.45

 

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

According to the Cysteine 1:10/Lysine 1:50 prediction model, the test substance, 15-CROWN-5, was predicted to be a sensitizer.

Executive summary:

The Direct Peptide Reactivity Assay was used to assess the skin sensitization potential of the test substance, 15-CROWN-5. Synthetic peptides containing cysteine or lysine were reacted with each test substance for 24 ± 2 hours.  After the incubation period, the extent of peptide depletion was analyzed using High Performance Liquid Chromatography (HPLC) coupled with ultra-violet (UV) spectrometric detection. According to the Cysteine 1:10/Lysine 1:50 prediction model, the test substance, 15-CROWN-5, was predicted to be a sensitizer.