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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 September, 2019 - 13 March, 2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
other: OECD Guideline 442E
GLP compliance:
no
Remarks:
The study was performed in accordance with UN GHS standards, OECD Test Guideline 442E
Type of study:
other: Activation of human acute monocytic leukemia cells
Justification for non-LLNA method:
The Human Cell Line Activation Test was used to assess the skin sensitization potential of the test articles by monitoring the upregulation of cell surface markers, C054 and CD86, on the surface of human acute monocytic leukemia cells (THP-1). The upregulation of CD54 and CD86 in response to a skin sensitizer is correlated to dendritic cell activation, which is the third key event of the skin sensitization pathway.

Test material

Constituent 1
Chemical structure
Reference substance name:
ethyl {5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamate
EC Number:
943-940-5
Cas Number:
1869118-24-0
Molecular formula:
C16H16N4O4S
IUPAC Name:
ethyl {5-[(4-methylphenyl)sulfonyl]-5H-pyrrolo[2,3-b]pyrazin-2-yl}carbamate
Test material form:
solid: crystalline

In vitro test system

Details on the study design:
Solubility Determination:

Prior to the preliminary dose range finding assay, the test article was tested in a solubility test to determine an appropriate solvent. The following ovservations were determined during the test. The test article was found to be soluble at 500 mg/mL in DMSO with approximately 1 minute of vortexing. The test article dilution appeared to be a cloudy white non-viscous suspension. During all of the valid trials, the media tubes 1-8 used in the test substance dilution scheme appeared to contain small white particles. The tubes were vortexed immediately prior to dosing and the cloudiness remained.

Dose Range Finding Assay:

A preliminary dose range finding assay was performed to determine the viability of the THP-1 cells after 24 +/- 0.5 hour exposure to 8 test article concentrations. The CV75, which is the calculated test article concentration leading to 75% cell viability was calculated for the test article.

Definitive assays:

Based on the results from the dose range finding assay, the doses were chosen for the test article for the definitive assay. At least 2 valid definitive trials were performed.

The positive control, 2,4-Dinitrochlorobenzene, was tested in the dose rang and definitive trials.

Evaluation of test results:

The relative fluorescence intensity was calculated for the test article and control treated cell population. The EC200 and EC150 values, which are the calculated test article concentrations leading to an RFI of 200 or 150 respectively, were calcued for the test article.

Results and discussion

Positive control results:
The positive control results were considered valid on the days that the test material assays were run. On October 1, 2019 the positive control had a cell viability (%) of 83.19 and passed the test. On October 8, 2019 the positive control had a cell viability of 77.60 and passed the test.

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: B1 Assay Date: 1 October 2019
Parameter:
other: CD54 (μg/mL), CD86 (μg/mL)
Value:
1 000
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
μg/mL
Key result
Run / experiment:
other: B2 Assay Date: 8 October 2019
Parameter:
other: CD54 (μg/mL), CD86 (μg/mL)
Value:
1 000
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
μg/mL
Key result
Run / experiment:
other: Dose Range and Definitive Assays
Parameter:
other: CV75 (μg/mL)
Value:
1 000
Remarks on result:
other: μg/mL
Other effects / acceptance of results:
The assay met acceptance criteria when:

The cell viability values of the solvent control was > 90%.

For the solvent control, RFI values of both CD86 and CD54 were less than the positive criteria (CD86 RFI < 150 and CD54 RFI < 200).

For the positive control (DNCB), RFI valus of both CD86 and CD54 were predicted to be positive (CD86 RFI >= 150 and CD54 RFI >= 200), and cell viability was > 50%.

For the medium and solvent controls, the MFI ration of both CD86 and CD54 to isotype control was >105%.

The cell viability of the test article-treated cultures was > 50% in at least four doses.

All acceptance criteria for a valid assay were met for the definitive trials.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of the h-CLAT definitive assays, the test material was considered to be a non-sensitizer.