3,3'-thiodi(propionic acid)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04.02.2022 - 04.03.2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-thiodi(propionic acid)

EC Number:

203-841-3

EC Name:

3,3'-thiodi(propionic acid)

Cas Number:

111-17-1

Molecular formula:

C6H10O4S

IUPAC Name:

3-[(2-carboxyethyl)sulfanyl]propanoic acid

Constituent 2

Reference substance name:

3,3'-thiobipropionic acid

IUPAC Name:

3,3'-thiobipropionic acid

Details on test material:

- Name of test material: 3,3'-thiobipropionic acid
- Molecular formula: C6H10O4S
- Molecular weight: 178.21
- Physical state: crystalline powder
- Analytical purity: 100.1% (100%)
- Lot/batch No.: WAR9711
- Stability under test conditions: yes
- Storage condition of test material: cool, dark conditions

In vitro test system

Details of test system:

THP-1 cell line [442E]

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: The test item will be dissolved in 0.9% NaCl solution in a concentration of 500 mg/L
- Preparation of the test chemical serial dilutions: eight stock solutions (i.e. eight concentrations) will be prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions will be further diluted 50-fold (0.9% NaCl) giving the working solutions
- Preparation of the positive controls: 2,4-dinitrochlorobenzene (DNCB; CAS No.: 97-00-7, purity ≥ 99%) at a final concentration of 4 μg/mL (alternatively at the concentration of the CV75) will be tested concurrently with the test item. DNCB will be dissolved in DMSO and diluted according to the procedure described for the test item, resulting in a final DMSO concentration of 0.2% (v/v)
- Preparation of the solvent, vehicle and negative controls: medium control will serve as the solvent control
- Stable dispersion obtained
- Log Kow of the test chemical
- Other:

DOSE RANGE FINDING ASSAY:
- Highest concentration used: 1000 µg/mL
- Solubility in solvents
- Solubility in incubation medium
- Results of selecting appropriate concentration and determination of cytotoxicity e.g. CV75
- Final concentration range selected on basis of: [describe]

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates
- Number of repetitions
- Test chemical concentrations
- Application procedure: The solvent controls, the positive control and the working solutions will be mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate.
- Exposure time: 24± 0.5 h
- Study evaluation and decision criteria used:
For CD86/CD54 expression measurement, the test item will be tested in at least two independent runs to derive a single prediction. Each independent run will be performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions will be prepared and independently harvested cells will be used.
Sensitising potential of the test item will be predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT is considered positive if any of the three scenarios are met
- the RFI of CD86 is equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs
- the RFI of CD54 is equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs
- the RFIs of both the CD86 and CD54 are equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs.

In case of non-concordant results, a third run should be conducted to make the final prediction. Otherwise the result will be considered as inconclusive.

A negative test result of a test item will be only accepted if the cell viability at a concentration of 1.2 x CV75 is < 90%. In contrast, a positive test outcome will be accepted irrespective of cell viabilities > 90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (i.e. 5000 μg/mL for 0.9% NaCl; 1000 μg/mL for DMSO or another organic solvent; or the maximum solubility) even if the cell viability is > 90%.
A negative result for test items with a Log KOW > 3.5 should be considered as inconclusive.
- Description on study acceptance criteria:
The test meets acceptance criteria if:
• the cell viability of the medium and the solvent controls is > 90%,
• the cell viability of at least four tested doses of the test item in each run is > 50%,
• the RFI values of the positive control (DNCB) is ≥ 150% for CD86 and ≥ 200% for CD54 at a cell viability of > 50%,
• the RFI values of the solvent control is not ≥ 150% for CD86 and not ≥ 200% for CD54, and
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for both, the medium and DMSO / organic solvent control, is > 105%.

SEEDING AND INCUBATION
- Seeding conditions (passage number and seeding density): low passage number (< 30), density of 0.1 – 0.2 x 106 cells/mL
- Incubation conditions: 37 °C ± 1 °C and 5% CO2
- Washing conditions: washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer)

MEASUREMENT OF CELL SURFACE EXPRESSION/LUCIFERASE ACTIVITY
For h-CLAT and USENS
- Flow cytometry used: BD FACSLyric
- Plate used: 96-well V-bottom plate.
- Propidium iodide staining/cytotoxicity measurements: propidium iodide staining
- Preparation for CD54 and/or CD86 expression measurements/cell staining: After 24 h ± 0.5 h of exposure, cells will be transferred into sample tubes and collected by centrifugation (approx. 250 x g). The following steps will be carried out on ice with pre-cooled buffers and solutions. The supernatant will be discarded and the remaining cells will be washed twice with FACS buffer. After washing, cells will be blocked using 600 μL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells will be split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells will be stained with 50 μL of FITC-labelled anti-CD86, FITC-labelled anti-CD54 or FITC-labelled mouse IgG1) antibodies in the dark for 30 min at 4°C. All antibodies will be diluted in FACS buffer at an appropriate manner. After washing with FACS buffer two times, cells will be re-suspended in FACS buffer and PI solution is added. PI staining will be done just prior to the measurement by adding PI solutions to each sample (final concentration of PI will be 0.625 μg/mL)

DATA EVALUATION
- Cytotoxicity assessment: PI uptake
- Prediction model used:
Sensitising potential of the test item will be predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT is considered positive if any of the three scenarios are met
- the RFI of CD86 is equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs
- the RFI of CD54 is equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs
- the RFIs of both the CD86 and CD54 are equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs.

In case of non-concordant results, a third run should be conducted to make the final prediction. Otherwise the result will be considered as inconclusive.

Vehicle / solvent control:

cell culture medium

Negative control:

other: 0.9% NaCL (vehicle)

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Results and discussion

Positive control results:

RFI CD86: 252 (experiment 1) and 284 (experiment 2)
RFI CD54: 399 (experiment 1) and 388 (experiment 2)

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD54>150 [442E]

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

at the highest test concentration RFI did not exceed 150

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

RFI CD86>200 [442E]

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

at the highest test concentration RFI did not exceed 200

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD86>200 [442E]

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

at the highest test concentration RFI did not exceed 200

Results 4

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

RFI CD54>150 [442E]

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Remarks:

at the highest test concentration RFI did not exceed 150

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

According to the defined approach for Skin sensitisation, based on hCLAT and Keratinosens study, TDPA can be considered as non sensitising.

Conclusions:

In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in two independent experiment runs. Therefore, the test item might be considered as non-sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study Thiodipropionic Acid was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

In all experiments no precipitation or turbidity of the test item was observed for all concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was 88.8% (CD86), 92.6% (CD54) and 91.7% (isotype IgG1 control) in the first experiment and 91.6% (CD86), 92.2% (CD54) and 91.8% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item can be considered as non-sensitiser.