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Description of key information

- DPRA: not a potential skin sensitizer (OECD TG 442C)

- H-CLAT: not a potential skin sensitizer (OECD TG 442E)

- KeratinoSens: inconclusive (OECD TG 442D)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

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
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)
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%
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

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

 

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.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 13-Dec-2016 to 30-Aug-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD guideline 442E (h-CLAT method)
Version / remarks:
2016
GLP compliance:
yes
Type of study:
activation of dendritic cells
Details on the study design:
Test system and culture:
THP-1 (monocytic leukeamia cell line) cells provided from American Type Culture Collection (batch 61077351).
Cryopreserved cells have been thawed. Cells were cultured, at 37°C under 5% CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10% Foetal Calf Serum, 100 units/mL penicillin and 100 μg/mL streptomycin. THP-1 were routinely seeded every 3-4 days at the density of 0.15 to 0.2 x 106
For testing, THP-1 cells were seeded at a density of 0.2 x 10 cells/mL.

Vehicle: DMSO

Preliminary study: Cytotoxicity assays:
The cytotoxicity of the test item was evaluated in order to select at least 4-5 concentrations able to induce cytotoxicity, around 50%, for the highest one. Assessment of cell toxicity was performed by determining cell viability on THP-1 cells, using the 7-AAD inclusion methods.

Main study: Activation test:
Based on the cytotoxicity assay eight final test item concentrations were selected. The doses range for activation test was from 7.81 to 1 000 μg/mL.
THP-1 cells were plated at 1*10E6 cells/mL/well in 24 well plates and treated for 24±0.5 hours with selected test item concentrations. After treatment cells were washed twice with FACS buffer. Then cells were stained for 30 min at 4°C with the following fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAbs): anti-human CD54, anti-human CD86; FITC labelled-mouse IgG1. Cells were incubated with above mAbs at 6 μL/3*10E5 cells /50μL for the anti-human CD86 mAb, and 3 μL/3*10E5 cells /50μL for the anti-human CD54 mAb. FITC labelled-mouse IgG1 was used as an isotype control at a dilution of 3 μL/3*10E5 cells /50μL. Then, the cells were stained also with 7-AAD for 30 min at 4°C. After washing and resuspension with FACS buffer, the fluorescence intensities of the THP-1 cell surface markers were then analysed by flow cytometry using GUAVA (Merck Millipore, France) and InCyte software.

Data analysis and interpretation:
The Relative Fluorescence Intensity (RFI) is used as an indicator of CD86 and CD54 expression. RFI is calculated with the following formula:
RFI = [MFI of test item-treated cells – MFI of test item-treated isotype control cells] / [MFI of vehicle control cells – MFI of vehicle isotype control cells]

Activation test positive criteria:
The positive criteria are described as following: RFI of CD54 ≥ 200% and RFI of CD86 ≥ 150% of their respective control.
The chemicals must be tested in two independents experiments at least. If two of three independent experiments at any dose exceed 150% of RFI for CD86, or exceed 200% of RFI for CD54, the chemical could be identified as a sensitizer. Otherwise it is identified as a non-sensitizer.
Key result
Run / experiment:
other: Experiments 1 and 2 (8 different concentrations)
Parameter:
other: CD54 RFI
Value:
200
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Run / experiment:
other: Experiments 1 and 2 (8 different concentrations)
Parameter:
other: CD86 RFI
Value:
150
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Cytotoxicity profile of 2,5-DIMETHOXYTETRAHYDROFURAN was assessed with the 7-AAD dye. % cell viability was 98% at concentrations from 7.81 to 1000 µg/mL.

ACCEPTANCE OF RESULTS:
Test system was validated as all criteria were fully accepted.
- Acceptance criteria met for negative control: Negative controls (DMSO) showed cell viability values acceptable regarding the acceptance criteria.
- Acceptance criteria met for positive control: Positive controls showed an increase of CD54/86 expression (RFI ≥ 200/150 respectively) compared to the negative control.
Experiments show eight concentrations that can be analysed in the two experiments.

Table 1 - RFI of CD54/86 expressions

Sample

Dose level

Experiment

1

 

Experiment

2

 

 

µg/mL

CD54

CD86

CV (%)

CD54

CD86

CV (%)

RPMI

 

100

100

98

100

100

98

 

-

100

100

97

100

100

98

DNCB

4

857

440

78

967

563

79

 

7.81

107

123

97

109

105

98

 

15.6

107

107

97

192

122

97

18937/01

31.3

107

96

97

121

114

98

(vehicle:

62.5

113

96

97

114

103

97

DMSO

125

128

95

97

132

127

97

0.2%)

250

119

89

98

139

114

98

 

500

119

82

97

139

126

97

 

1000

147

96

97

146

96

97

 

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these results, 2,5-DIMETHOXYTETRAHYDROFURAN did not demonstrate an in vitro sensitizing potential in condition of the experimental human Cell Line Activation Test, during this study.
Executive summary:

The objective of this study was to evaluate the in vitro intrinsic sensitizing potential of 2,5-DIMETHOXYTETRAHYDROFURAN. The method used for this study was the human cell line activation test (h-CLAT), tested in DMSO at eight concentrations up to 1000 µg/mL.

No cytotoxicity was induced on THP-1 cells by 2,5-DIMETHOXYTETRAHYDROFURAN.

Negative controls (DMSO) showed cell viability values acceptable regarding the acceptance criteria. Positive controls showed an increase of CD54/86 expression (RFI ≥ 200/150 respectively) compared to the negative control.

In the same conditions, no reproducible "increase" of the CD54 and CD86 expression compared with the negative control for all dose-level of 2,5-DIMETHOXYTETRAHYDROFURAN was noticed.

An absence of any dose-response increase of the CD54 and CD86 expression was noticed with the test item. None of the tested doses induced a 1.5 or 2 fold increase of CD86 and CD54 expression respectively compared to the negative control.

Based on these results, 2,5-DIMETHOXYTETRAHYDROFURAN did not demonstrate an in vitro sensitizing potential in condition of the experimental human Cell Line Activation Test, during this study.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 19/09/2016 to 08/03/2018
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:
Feb, 4th, 2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Details on the study design:
Test system:
- Cells: KeratinoSens (Givaudan), cultured in maintenance medium at 37°C, 5% CO² and exempt of mycoplama

Controls:
- Positive control: Cinnamaldehyde in DMSO
- Negative controls: treatment culture medium, 1% DMSO and 1% non-heat inactivated foetal calf serum

Series definition:
- Test item: tested at 12 concentrations according to a geometric progression of ratio 2 from 0.2 µg/mL to 400 µg/mL
- Negative control: 6 wells of solvent control (1% DMSO in treatment medium) with cells and 1 well of solvent control without cell by culture plate
Positive control: 5 concentrations of cinnamaldehyde on each culture plate (4 to 64 µM)

Replications:
The study was composed of 3 independent repetitions. for each repetition the test item and the reference items were replicated on 3 independent plates for the measurement of induction and 2plates for the measurement of cytotoxicity.

Test protocol:
- Cells seeding: Cell suspensions were adjusted to a density of 8.10E4 cells/mL in seeding medium. 125 µL of the cell suspension at 8.10E4 cells/mL (10E4 cells per well) were distributed in 3 white plates for the induction measurement and 2 transparent plates to assess the cytotoxicity. The seeded plates were incubated 24 hours at 37°C, 5% CO².
- Test item stock solution: 40 mg/mL in sterile water
- Plate preparation: 100 µL sterile water + 200 µL of the stock solution. Then a the series dilutions were prepared.
- Contact between the cells and the test and reference items: in the seeded plates, the medium was aspirated and replaced with 150 µL of treatment medium, and incubated for 48 hours (37°C, 5% CO²)
- Luciferase activity: after 48 hours, the medium was aspirated and each well was gently washed with 200 µL PBS. Then 100 µL of luciferase substrate were added in each well. The plates were incubated at least 15 minutes at room temperature. The plates were placed in the luminometer the the lucifarase activity was measured.
- Cell viability assessment: MTT method
Positive control results:
EC1.5 = 16.67 µM (validity criteria met)
Key result
Run / experiment:
other: Repetition 1
Parameter:
other: EC1.5
Remarks:
mean value
Value:
0.2
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: Repetition 2
Parameter:
other: EC1.5
Remarks:
mean value
Value:
2.51
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: Repetition 3
Parameter:
other: EC1.5
Remarks:
mean value
Value:
30.07
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

   Viability  Induction    
   IC70 µg/mL  I max  Linear EC1.5 µg/mL  EC1.5 Lin/Log µG/mL
 Rep 1  > 400  3.42  < 0.20  < 0.20
 Rep 2  > 400  1.67  2.51  2.38
 Rep 3  > 400  2.46  30.07  28.78
 Mean  -  2.52  -  -
 Geometric mean  > 400  -  -  -

Repetition 1: Induction higher than 1.5 are observed on the whole concentration range without any dose effect. The repetition can not be taken into consideration.

Repetition 2: Induction is significantly higher only at 3.13 µg/mL without dose effect. The repetition should be considered inconclusive and further testing is required.

Repetition 3: dose-response with increasing luciferase activity induction at increasing concentrations. Inductions are significantly higher than 1.5 from 50 to 400 µg/mL. The EC1.5 is lower than 200 µg/mL and the viability at the EC1.5 is higher than 70%. The repetition is considered as positive.

Interpretation of results:
other: inconclusive
Conclusions:
Further analysis would be necessary, however, under the retained experimental conditions and in the absence of confirmation, the test item 2,5-dimethoxytetrafuran may be considered as a potential sensitizer
Executive summary:

2,5-dimethoxytetrafuran was tested for skin sensitisation according to OECD TG 442D (KeratinoSens) at concentrations from 0.2 to 400 µg/mL.

3 Repetitions were performed as the two first ones were inconclusive due to a lack of dose-response. The last repetition was conclusive and would show a potential for skin sensitisation.

Further analysis would be necessary, however, under the retained experimental conditions and in the absence of confirmation, the test item 2,5-dimethoxytetrafuran may be considered as a potential sensitizer.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The following in-vitro studies are available on 2,5-Dimethoxytetrahydrofuran (OECD guidelines and GLP):

HPRT assay:

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

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.

H-CLAT assay:

The in vitro intrinsic sensitizing potential of 2,5-DIMETHOXYTETRAHYDROFURAN was tested according to the human cell line activation test (h-CLAT), in DMSO at eight concentrations up to 1000 µg/mL.

No cytotoxicity was induced on THP-1 cells by 2,5-DIMETHOXYTETRAHYDROFURAN.

Negative controls (DMSO) showed cell viability values acceptable regarding the acceptance criteria. Positive controls showed an increase of CD54/86 expression (RFI ≥ 200/150 respectively) compared to the negative control.

In the same conditions, no reproducible "increase" of the CD54 and CD86 expression compared with the negative control for all dose-level of 2,5-DIMETHOXYTETRAHYDROFURAN was noticed.

An absence of any dose-response increase of the CD54 and CD86 expression was noticed with the test item. None of the tested doses induced a 1.5 or 2 fold increase of CD86 and CD54 expression respectively compared to the negative control.

Based on these results, 2,5-DIMETHOXYTETRAHYDROFURAN did not demonstrate an in vitro sensitizing potential in condition of the experimental human Cell Line Activation Test, during this study.

KeratinoSens Assay:

2,5-dimethoxytetrafuran was tested for skin sensitisation according to OECD TG 442D (KeratinoSens) at concentrations from 0.2 to 400 µg/mL.

3 Repetitions were performed as the two first ones were inconclusive due to a lack of dose-response. The last repetition was conclusive and would show a potential for skin sensitisation.

Further analysis would be necessary, however, under the retained experimental conditions and in the absence of confirmation, the test item 2,5-dimethoxytetrafuran may be considered as a potential sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the classification criteria of UN/EU GHS, and given the absence of confirmed positive reactions in the in-vitro assays, 2,5-Dimethoxytetrahydrofuran is not classified as a skin sensitizer.

No data are available for respiratory sensitisation; therefore no conclusion can be made on the classification of this endpoint.