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Skin sensitisation

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skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
GLP compliance:
yes (incl. QA statement)
Type of study:
ARE-Nrf2 luciferase LuSens test method
Justification for non-LLNA method:

Test material

Constituent 1
Chemical structure
Reference substance name:
EC Number:
EC Name:
Cas Number:
Molecular formula:
Specific details on test material used for the study:
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: B 2024 v. 23.09.2020
- Purity, including information on contaminants, isomers, etc.: 97.8 area-% (Analysis)

In vitro test system

Details on the study design:
The chemical and biological mechanisms associated with skin sensitisation are summarised in the form of an Adverse Outcome Pathway (AOP). This AOP includes four key events:
1) The molecular initiating event is the covalent binding of electrophilic substances to nucleophilic centres in skin proteins.
2) The inflammatory responses and gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways in the keratinocytes.
3) The activation of dendritic cells, typically assessed by expression of specific cell surfac markers, chemokines and cytokines.
4) The T-cell proliferation. This ARE-Nrf2 luciferase test method is proposed to address the second key event of the skin sensitisation AOP, namely keratinocytes activation. Skin sensitisers have been reported to induce genes that are regulated by the antioxidant response element(ARE). Small electrophilic substances such as skin sensitisers can act on the sensor protein Keap1(Kelch-like ECHassociated protein 1), by e.g. covalent modification of its cysteine residue, resulting in its dissociation from the transcription factor Nrf2 (nuclear factor-erythroid 2-related factor 2). The dissociated Nrf2 can then activate ARE-dependent genes such as those coding for phase II detoxifying enzymes.The test method is technically applicable to the testing of multiconstituent substances and mixtures. The test method is applicable to test items which are soluble or could be formulated as a homogeneous suspension/dispersion either in DMSO, water or culture medium.A LuSens prediction should be considered in the framework of a Defined Approach or of an IATA and in accordance with the provision paragraph 4 and paragraphs 7 and 8 of the OECD 442D general introduction.The ARE-Nrf2 luciferase test (LuSens) can be used as part of a testing battery (including e.g. DPRA (Direct Peptide Reactivity Assay), human cell line activation test method (h-CLAT)) based on the OECD adverse outcome pathway for the assessment of the skin sensitisation potential of chemicals.The technical proficiency of the LuSens with the OECD 442D guideline recommended proficiency substances was demonstrated.
Vehicle / solvent control:
Negative control:
DL-Lactic acid
Positive control:
EGDMA (120 M) [442D]

Results and discussion

In vitro / in chemico

Key result
test chemical
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Remarks on result:
no indication of skin sensitisation
1,5-Pentanediol did not activate the LuSens cells up to a concentration of 2000 µM under the test conditions of this study.

Any other information on results incl. tables

Results and historic control data are listed in the annex under "attached background material"

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
In conclusion, the test item 1,5-Pentanediol did not activate the LuSens cells up to a concentration of 2000 µM under the test conditions of this study. Therefore, the test item is considered negative for the second key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

This in vitro Skin Sensitisation Test ARE-Nrf2 Luciferase Test Method (LuSens) was performed to assess the inflammatory responses in the keratinocytes as changes in gene expression associated with specific cell signalling pathways such as the antioxidant/electrophile response element (ARE)-dependent pathways (second key event of a skin sensitization AOP) of 1,5-Pentanediol. Dose calculation was adjusted to purity to test a final maximal concentration of 2000 µM of the test item. Due to adaptation of the purity of 97.8 area-% instead of 97.96 area-% after experimental completion at request of the sponsor, the adjustment to purity actually carried out resulted in a maximum concentration <2000 µM. Since the deviation occurred in the calculation was in the range <1%, the undercutting of the guideline defined maximum concentration is evaluated as biologically irrelevant. In the cytotoxicity test, cytotoxic effects were not observed following incubation with the test item up to the highest tested concentration (2000 µM). Due to the lack of cytotoxicity, a CV75 value could not be calculated. In this case, the OECD 442D guideline recommend to test a test item concentration of 2000 µM in the main experiments. Five further test item dilutions were prepared by serial dilution with a dilution factor of 1.2. The test item was tested in 4 independent main experiments. The following concentrations of the test item were tested in the main experiments: 804, 965, 1157, 1389, 1667, 2000 µM In the first experiment two concentrations (804 and 1667 µM) showed a luciferase induction ≥ 1.5 fold compared to the solvent control. Since, theses concentrations were not consecutive, the first experiment is considered negative. In the second experiment only the highest tested concentration of 2000 µM was < 1.5 fold. All other concentrations showed a luciferase induction in a range of 5.19 to 5.75 fold. Therefore the second experiment is considered positive. In the third experiment all tested concentrations showed luciferase induction < 1.5 fold and is considered negative. For better interpretation of the date, a fourth experiment was performed. The fourth experiment confirmed the negative outcome of the first and third experiment. After treatment with the test item for 48 ± 1 hours the luciferase induction is < 1.5 fold compared to the solvent control in three of four experiment. Therefore the LuSens prediction is considered negative.
The acceptance criteria were met:
The average luciferase activity induction obtained with the positive control, 120 μM EGDMA was ≥ 2.5 (ME 1: 7.33; ME 2: 6.50; ME 3: 6.25; ME 4: 4.31) and statistically significant. The positive control had a relative cell viability ≥ 70% as compared to the solvent control (ME 1: 111.53%; ME 2: 137.01%; ME 3: 126.21; ME 4: 92.19). The average luciferase activity induction obtained with the negative control, 5000 μM Lactic acid, as well as the basal expression of untreated cells was < 1.5 fold as compared to the average solvent control (ME 1: 1.11; ME 2: 1.21; ME 3: 1.12; ME 4: 1.01). The average coefficient of variation (CV%) of the luminescence reading for the solvent controls (DMSO) was below 20% in each main experiment (ME 1: 7.5%; ME 2: 10.2%; ME 3: 6.5; ME 4: 7.3). At least three test item concentrations had a cell viability of at least 70% relative to the solvent controls. The maximum concentration of 2000 μM has been tested.