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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015
Reference Type:
other: Amendment to the report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD: Draft Proposal for a New Guideline on In Vitro Skin Sensitization: human Cell Line Activation Test (h-CLAT)
Principles of method if other than guideline:
Skin sensitisation (In vitro/in chemico test system) - Details on study design:
Three key events of the adverse outcome pathway (AOP) were part of the in vitro assay:
1) protein reactivity (DPRA): incubation with synthetic peptides, ca. 24 hours at room temperature. The peptides are custom material (Supplier: GenScript, Piscataway, NJ, USA and RS Synthesis, Louisville KY, USA) containing phenylalanine to aid in detection and either cysteine or lysine as the reactive center.
2) activation of keratinocytes (LuSens): incubation for ca. 48 hours at 37 °C with a luciferase reporter cell line and measurement of antioxidant response element dependent luciferase activity in a luminometer. Transgenic keratinocyte cell line derived from HaCaT cells, prepared in collaboration with Christoph J. Wruck, RWTH Aachen
3) activation of dendritic cells (h-CLAT): incubation for ca. 24 hours at 37 °C with human pro-monocytic cell line THP-1. The human monocytic leukemia cell line was obtained from “American Type Culture Collection, Manassas, USA” (ATCC, TIB- 202).
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
- Purity: main peak: 86.4 area-% (CE-fingerprint)
- Content: water: 82.7 %
- Test-substance No.: 14/0384-1
- Batch identification: 0011784570
- Physical state/color: liquid, yellowish, clear

In vitro test system

Details on study design:
3) Dendritic Cell Line Activation Assay (h-CLAT):
- Preparation of cell line: growth until 5 passages, but no longer than passage 30 prior to testing
- Seeding concentration: 500 μL of 2.0 x 10^6 cells/mL cell suspensions
- Number of replications: two independent experiments were performed, per experiment duplicates of each treatment were tested
- Test concentrations: 1st experiment: 20261, 16884, 14070, 11725, 9771, 8143, 6785, 5655 µg/mL; 2nd experiment: 35011, 29176, 24313, 20261, 16884, 14070, 11725, 9771 µg/mL
- Test concentrations were determined on the basis of a pre-test with 10 concentration.
- Cell staining and flow cytometric analysis: antibody solutions: Anti-CD86, Anti-CD54, Mouse IgG1, staining solution: propidium iodide
- Acceptance criteria: A tested concentration is not to be further evaluated when relative viability is less than 50%. Cell viability of vehicle control cells must yield at least 90%. In the positive control (DNCB), RFI values of both CD86 and CD54 should be over the positive criteria (CD86≥ 150% and CD54 ≥ 200%) and cell viability should be ≥ 50%. In the negative control (LA), RFI values of both CD86 and CD54 should not exceed the positive criteria (RFI CD86≥ 150% and RFI CD54 ≥ 200%) and cell viability should be ≥ 50%. For all vehicle controls, the MFI ratio of both CD86 and CD54 to isotype controls should be ≥ 105%. A study is considered acceptable if the positive and negative and vehicle control data lies within the range of the historic data.

Results and discussion

Positive control results:
Control substance DNCB: Experiment 1: relative fluorescence intensity of 213.1% (CD 86) and 240.9 (CD 54) and a relative viability of 89.1%; Experiment 2: relative fluorescence intensity of 248.8 (CD86) and 438.7 (CD 54) and a relative viability of 85.5%

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: relative viability (%)
Run / experiment:
h-CLAT; experiment 1
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: significant at all test concentrations
Key result
Parameter:
other: relative viability (%)
Run / experiment:
h-CLAT; experiment 2
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: significant at concentrations 9771, 11725, 14070, 16884, 24313, 35011 µg/mL
Other effects / acceptance of results:
The test substance was soluble in culture medium (2 x stock solution) and in finally tested concentrations. After 24 hours precipitates were not noticed in any concentration.

Any other information on results incl. tables

The acceptance criteria for all three tests were met. The substance is peptide reactive, does not activate keratinocytes and activates dendritic cells.

Applicant's summary and conclusion