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Diss Factsheets

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:
20th September 2019 to 17th April 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
other: OECD 442E In Vitro Skin Sensitisation Assays addressing the key event on activation of Dendritic cells on the adverse outcome pathway for Skin Sensitistation Adopted: 25 June 2018
Qualifier:
according to guideline
Guideline:
other: EURL ECVAM DB-ALM Protocol no. 158
GLP compliance:
no
Remarks:
The study has been conducted with a reputable lab for another international submission.
Type of study:
activation of dendritic cells

Test material

Constituent 1
Chemical structure
Reference substance name:
benzyl (3R,4S)-3-{2-[(ethoxycarbonyl)[5-(4-methylbenzenesulfonyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]amino]acetyl}-4-ethylpyrrolidine-1-carboxylate
EC Number:
944-343-2
Cas Number:
2050038-81-6
Molecular formula:
C32H35N5O7S
IUPAC Name:
benzyl (3R,4S)-3-{2-[(ethoxycarbonyl)[5-(4-methylbenzenesulfonyl)-5H-pyrrolo[2,3-b]pyrazin-2-yl]amino]acetyl}-4-ethylpyrrolidine-1-carboxylate
Test material form:
solid: particulate/powder

In vitro test system

Details on the study design:
The h-CLAT is an in vitro assay which measures the changes in expression of cell surface markers CD54 (ICAM-1) and CD86 associated with the process of dendritic cell activation in the human acute monocytic leukemia cell line, THP-1. Dendritic cell activation is considered one of the key biological events in the adverse outcome pathway for skin sensitisation, where CD54 and CD86 are subsequently involved in dendritic cell migration to the lymph nodes and T-cell priming. THP-1 cells, seeded at a density of 2.0 x 10 6 cells/ml in culture medium in 24-well plate format, define the Test System. After treatment of the test item or control articles to the test system, the expression of cell surface markers are measured by flow cytometry following cell staining with flourescein isothiocyanate (FITC) labelled antibodies. Cytometry measurement, using propidium iodide (PI) staining, is conducted concurrently to assess whether upregulation of surface expression occurs at subcytotoxic concentrations.

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 observations 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. 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 calculated for the test article.

Results and discussion

Positive control results:
For the positive control, 2,4-Dinitrochlorobenzene, RFI values of both CD86 and CD54 were predicted to be positive (CD86 RFI ≥150 and CD54, RFI ≥200) and cell viability was >50%.

In vitro / in chemico

Resultsopen allclose all
Run / experiment:
other: Dose Range and Definitive Assay
Parameter:
other: CV75 µg/mL
Value:
1 000
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: µg/mL
Key result
Run / experiment:
other: B1 assay 01 Oct 2019
Parameter:
other: CD54 EC200 µg/mL
Value:
370
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
µg/mL
Key result
Run / experiment:
other: B1 assay 01 Oct 2019
Parameter:
other: CD86 EC150 µg/mL
Value:
1 000
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
µg/mL
Key result
Run / experiment:
other: B2 assay 08 Oct 2019
Parameter:
other: CD54 EC200 µg/mL
Value:
1 000
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
µg/mL
Key result
Run / experiment:
other: B2 assay 8 Oct 2019
Parameter:
other: CD86 EC150 µg/mL
Value:
1 000
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
µg/mL
Key result
Run / experiment:
other: B4 assay 15 Oct 2019
Parameter:
other: CD54 EC200 µg/mL
Value:
1 000
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
µg/mL
Key result
Run / experiment:
other: B4 assay 15 Oct 2019
Parameter:
other: CD86 EC150 µg/mL
Value:
1 000
Vehicle controls validity:
not specified
Negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
µg/mL
Other effects / acceptance of results:
Definitive trial 3 did not meet assay acceptance criteria and therefore, was not considered a valid trial

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 values 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:
The test article A1634356.0 was considered a non-sensitizer according to h-CLAT.
Executive summary:

The Human Cell Line Activation Test (h-CLAT) was used to assess the skin sensitisation potential of the test article by monitoring the upregulation of cell surface markers, CD54 and CD86 on the surface of human acute monocytic leukemia cell line, THP-1. The upregulation of CD54 and CD 86 in response to a skin sensitiser is correlated to dendritic cell actication, which is the third key event of the skin sensitisation pathway.

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.

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 calculated for the test article. 

As the test article elicted an EC200 >1000 for CD54 and an EC150 >1000 for CD86, with cell viability ≥50% in at least two independent runs, A-1634356.0 is considered a non-sensitiser.