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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 29-Jun-2018 to 03-Sept-2018
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
GLP compliance:
yes (incl. QA statement)
Type of study:
direct peptide reactivity assay (DPRA)

Test material

Constituent 1
Chemical structure
Reference substance name:
Tetrahydro-2,5-dimethoxyfuran
EC Number:
211-797-1
EC Name:
Tetrahydro-2,5-dimethoxyfuran
Cas Number:
696-59-3
Molecular formula:
C6H12O3
IUPAC Name:
tetrahydro-2,5-dimethoxyfuran
Test material form:
liquid

In chemico test system

Details on the study design:
Formulation of the Test Item: 100 mM in acetonitrile (a homogenous and clear solution was formed) (prepared just before use)

Positive Control: Cinnamaldehyde

Peptide solutions: Cysteine and lysine peptide stock solutions were prepared at the concentrations of 0.501 mg/mL and 0.518 mg/mL with sodium phosphate buffer (pH=7.5) and ammonium acetate buffer (pH=10.2) respectively. Calibration standards were made by serial dilutions from these peptide stock solutions. The test chemical stock solutions were combined with the peptide stock solutions (1:10 and 1:50 ratio with cysteine and lysine peptides respectively) as reaction samples.

HPLC System Conditions:
- HPLC system: SHIMADZU LC2030 (Prominence-i LC-2030C)
- Serial number: L21445402951AE
- Column: Zorbax SB-C18 (2.1 x 100 mm, 3.5 μm)
- Serial number: USRY003976
- Column temperature: 30°C
- Sample temperature: 25°C
- Detector: 220 nm (258 nm)
- Injection volume: 7μL
- System equilibration: 50% phase A (0.1 % v/v trifluoroacetic acid in ultra-pure water) and 50% phase B (0.085 % v/v trifluoroacetic acid in acetonitrile) for 2 hours at 30°C and running the gradient twice before injecting the first sample
- Run time: 20 min
- Flow conditions: gradient flow

Percent peptide depletion:
The concentration of the peptide was determined in each reaction sample from absorbance at 220 nm, measuring the peak area of the appropriate peaks and calculating the concentration of the peptide using the linear calibration curves derived from the standards.
The percent peptide depletion was determined in each reaction sample and positive control sample measuring the quotient of the peak area and the mean reference control peak area.

Assigning the test chemical to a reactivity class and category:
The average percent peptide depletion was calculated for the test item. By using the cysteine 1:10 / lysine 1:50 prediction model (see table below), the threshold of 6.38% average peptide depletion was used to support the discrimination between a skin sensitizer and a non-sensitizer. On the basis of the prediction model, chemicals assigned to the minimal reactivity class should be classified as non-sensitisers whereas chemicals assigned to the low, moderate or high reactivity class should be classified as sensitisers:
- No or minimal reactivity: Mean % depletion < 6.38%
- Low reactivity: 6.38% < Mean % depletion < 22.62%
- Moderate reactivity: 22.62% < Mean % depletion < 42.47%
- High reactivity: 42.47% < Mean % depletion < 100%

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: mean (3 replicates)
Parameter:
other: % cysteine peptide depletion
Value:
5.64
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: mean (3 replicates)
Parameter:
other: % lysine peptide depletion
Value:
1.05
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

Table 1. Cysteine peptide depletion values for the positive control and the test item

Name, replicate number

Peptide

peak area

at 220 nm

Peptide conc. calculated (mM)

Peptide depletion

%

SD

(%)

ref ControlAcetonitril, rep I

1709414

0.48

-

-

ref ControlAcetonitril, rep II

1826152

0.51

-

ref ControlAcetonitril, rep III

1602686

0.45

-

2,5-Dimethoxytetrahydrofuran, rep I

1702104

0.48

0.43

5.11

2,5-Dimethoxytetrahydrofuran, rep II

1631700

0.46

10.65

2,5-Dimethoxytetrahydrofuran, rep III

1509173

0.42

5.83

CINNAMALDEHYDE, rep I

550299

0.15

67.81

2.03

CINNAMALDEHYDE, rep II

532919

0.15

70.82

CINNAMALDEHYDE, rep III

529501

0.15

66.96

 

Table 2. Lysine peptide depletion values for the positive control and the test item

Name, replicate number

Peptide

peak area

at 220 nm

Peptide conc. calculated (mM)

Peptide depletion

%

SD

(%)

ref ControlAcetonitril, rep I

2423559

0.50

-

-

ref ControlAcetonitril, rep II

2423810

0.50

-

ref ControlAcetonitril, rep III

2427200

0.50

-

2,5-Dimethoxytetrahydrofuran, rep I

2379022

0.49

1.84

0.89

2,5-Dimethoxytetrahydrofuran, rep II

2421761

0.50

0.08

2,5-Dimethoxytetrahydrofuran, rep III

2397130

0.50

1.24

CINNAMALDEHYDE, rep I

1118469

0.23

53.85

2.35

CINNAMALDEHYDE, rep II

1166689

0.24

51.87

CINNAMALDEHYDE, rep III

1233686

0.26

49.17


 

Table 3. Mean peptide depletion values for the positive control and the test chemical

Name, replicate number

Obtained mean % cysteine peptide depletion

Obtained mean % lysine peptide depletion

Mean % obtained peptide depletion

2,5-Dimethoxytetrahydrofuran

5.64

1.05

3.35

CINNAMALDEHYDE

68.53

51.63

60.08

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
In this assay, 2,5 Dimethoxytetrahydrofuran has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitizer.
Executive summary:

This study was undertaken to evaluate the skin sensitization potential of the test item 2,5-Dimethoxytetrahydrofuran in chemico. The Direct Peptide Reactivity Assay (DPRA) according to OECD TG 442C was used.

The positive control replicates showed the expected percent peptide depletion values within acceptable limits (65.53% with cysteine peptide and 51.63 % mean percent lysine peptide depletion). The back-calculated values of the reference control replicates were within the expected molarity concentration range of 0.50 ± 0.05 mM. The experiment was considered to be valid.

The percent cysteine peptide depletion value of 2,5-Dimethoxytetrahydrofuran was 5.64 % while the percent lysine peptide depletion was 1.05 %. The Cysteine 1:10 / Lysine 1:50 prediction model was used for the discrimination between sensitisers and non-sensitisers based on the threshold of 6.38 %. The average percent peptide depletion value of the test item was 3.35 % which does not exceed the threshold.

Results obtained from this in chemico Direct Peptide Reactivity Assay with the test item 2,5 Dimethoxytetrahydrofuran indicated that the test item has no or minimal reactivity towards the synthetic peptides, thus is not a potential skin sensitizer.