(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date: 30 November 2017
Experimental completion date: 13 March 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

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

other: Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency. Toxicol In Vitro. 2012 Oct; 26(7):1150-60.

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

other: A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 2010 Aug; 38(4):275-84.

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), July 2016

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Test material

Test material information

Constituent 1

Reference substance name:

(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

EC Number:

807-159-2

Cas Number:

69701-99-1

Molecular formula:

C10H16O4

IUPAC Name:

(2-ethyl-2-methyl-1,3-dioxolan-4-yl)methyl prop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

Identification: MEDOL-10
Chemical Name: (2-Ethyl-2-methyl-1,3-dioxolan-4-yl)methyl acrylate
CAS No.: 69701-99-1
Batch: 09892701
Purity: 99.9%
Partition coefficient (n-octanol/water): log Pow: 1.92
Water solubility: 12.92 g/L
Appearance: Colourless liquid
Expiry Date: 06 March 2019
Storage Conditions: At room temperature, protected from light
Stability in Solvent: Stable in water (not quantified)
Purpose of Use: Industrial chemical

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

Test Item Preparation

The maximum concentration of test item was 5000 μg/mL stable suspended in culture medium), as tested by a solubility test.

For the first XTT test (dose finding assay) eight concentrations of the test item were analysed. For this, dilutions were prepared by 1:2 serial dilutions from 5000 μg/mL culture medium.
Due to strong cytoxicity observed in all test item treated cells, the first XTT test was repeated with adjusted test item concentrations, starting with 50 μg/mL in culture medium as the highest concentration.

On the day of the experiment (immediately prior to start) MEDOL-10 was dissolved in culture medium.

Test Systems and Supporting Information

Reasons for the Choice of THP-1 Cells

THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitisers.

THP-1 Cell Cultures

Stocks of the THP-1 cell line are stored in liquid nitrogen in the cell bank (aliquots of cells in freezing medium at 1 × 10 6 to 2 × 10 6 cells/mL) allowing the repeated use of the same cell culture batch in experiments. Therefore, the parameters of the experiments remain similar, because of the reproducible characteristics of the cells. Thawed stock cultures are propagated at 37 ± 1.5 °C in plastic flasks. The cells are sub-cultured twice weekly. The cell density should not exceed 1 × 10 6 cells/mL. The THP-1 cell suspension is incubated at 37 ± 1.5 °C and 5.0 ± 0.5 % carbon dioxide atmosphere. Cells can be used up to two months after thawing (passage number should not exceed 30).

The passage numbers of the used THP-1 cells were 26 in both XTT assays and 18 and 11 in the h-CLAT for runs 1 and 2, respectively.

Culture Medium

RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) is used to culture the cells during the assay. Medium with supplements has to be stored at 2 – 8 °C and used within one month. The culture medium has to be warmed to room temperature just before use.

Preparation and Seeding of THP-1 Cells

On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 10 6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate.

For the main experiment (h-CLAT) 0.9 - 1 × 10 6 cells/well in a volume of 500 μL were seeded in a 24-well plate before the treatment.

Experimental Design and Procedures of XTT

Dose Finding Assay (XTT Test)

The test item concentrations investigated in the main experiment (h-CLAT) were determined with two XTT tests.

The XTT test is based on the cleavage of the yellow tetrazolium salt XTT [= (sodium 3'-(1-phenylaminocarbonyl) - (3,4 - tetrazolium) – bis - (4 – methoxy – 6 - nitro) - benzenesulfonic acid hydrate)] to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria.

XTT Labelling Mixture

The XTT labelling mixture consists of two components, a XTT buffer solution and the substrate solution. Both components were mixed right before application at a ratio of 1:100.

Treatment

After the cell seeding, 100 μL of the test item dilutions, the medium and solvent controls, respectively, were added to the cells. All dose groups were tested in 7 replicates for each XTT test. At the end of the incubation period of 24 ± 0.5 hours, the cell cultures were microscopically evaluated for morphological alterations.

XTT Labelling and Measurement

At the end of the incubation period, 50 μL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).


Calculation of the h-CLAT Test Item Concentrations

Two independent cytotoxicity experiments were performed on different days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).

Eight final concentrations (μg/mL) were used for the test item in the main experiment (h-CLAT). The highest concentration used was 1.2 × CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution.

Acceptability of the Assay

The XTT test is considered to be acceptable if it meets the following criteria:

• mean absorbance of the medium control is ≥ 0.5

• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Experimental Design and Procedures of h-CLAT

The test item was tested in two independent runs.

Treatment of the Cells

For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment.

Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.

Each concentration of the test item, medium control, positive and DMSO control was prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the Cells

The triplicates of each test item-treated and not test item-treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control).

All solutions were kept light protected at 2 - 8 °C or on ice during the staining and analysis procedures.

The cells with the different antibodies or the IgG1 were mixed and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).

Sample Preparation for Measurement

After staining with the antibodies, the cells were washed twice (2 - 8 °C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-AAD solution were added.

Flow Cytometry Acquisition

Before using the flow cytometer (FACSCalibur) the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.

The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

Preparation of the acquisition

The following acquisition plots were prepared:

• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)

• Histogram plot of each channel (FL-1 and FL-3, respectively)

The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells.

The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

Acquisition

Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

For full details on Data Analysis and Interpretation, please refer to entry in "Any other information on materials and methods incl. tables" section.

Test Item Concentrations

The following concentrations of the test item (dissolved in culture medium) were tested in the main experiment (h-CLAT):
13.9, 16.7, 20.0, 24.0, 28.8, 34.6, 41.5 and 49.8 μg/mL

The highest test item concentration for the main experiment (h-CLAT) of MEDOL-10 was previously determined by two XTT tests.
Cytotoxic effects were observed following incubation with the highest tested concentration (50 μg/mL) in both XTT tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 41.5 μg/mL.

Vehicle / solvent control:

DMSO

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

CV75 [442D and 442E]

Remarks:

Value is a mean of both XTT tests. CV75 values were 43.4 and 39.7 ug/mL respectively for the first and second XTT tests

Value:

41.5 µg/mL

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI, CD 54 >/= 200 %

Value:

150

Cell viability:

> 50%

Vehicle controls validity:

valid

Positive controls validity:

valid

Results 3

Key result

Group:

test chemical

Parameter:

other: RFI

Value:

200

Vehicle controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

The test item with a log Pow of 1.92 was tested in 2 independent runs. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both independent runs. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Any other information on results incl. tables

Results of the Dose Finding Assay (XTT Test)

Results of the first XTT test for Test Item MEDOL-10

The mean viability of the solvent control in comparison to the medium control was 107.35%. The CV75 value of the first XTT test: 43.3 μg/mL.

For tabulated results please refer to attachment "Results of the first and second XTT tests for Test Item MEDOL-10".

Results of the second XTT test for Test Item MEDOL-10

The mean viability of the solvent control in comparison to the medium control was 91.17%.

The CV75 value of the second XTT test: 39.7 μg/mL
The mean CV75 value of both XTT tests: 41.5 μg/mL

For tabulated results please refer to attachment "Results of the first and second XTT tests for Test Item MEDOL-10".

Results of the h-CLAT Test

For results of the first and second h-CLAT run for the Test Item MEDOL-10 please refer to attachments "Results of the first and second h-CLAT runs for the Test Item MEDOL-10".

 

Applicant's summary and conclusion

Conclusions:

The test item MEDOL-10 with a log Pow of 1.92 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

Introduction-

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of MEDOL-10 dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of MEDOL-10 was previously determined by two XTT tests.

Results-

Cytotoxic effects were observed following incubation with the highest tested concentration (50 μg/mL) in both XTT tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 41.5 μg/mL.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Conclusion-

In conclusion, the test item MEDOL-10 with a log Pow of 1.92 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).