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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:
May 30th to July 28th, 2017
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
other: OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome pathway for Skin Sensitisation)
GLP compliance:
Type of study:
activation of dendritic cells

Test material


In vitro test system

Details on study design:
Vehicle control: RPMI
Positive controls: 2,4-dinitrochlorobenzene (DNCB), nickel sulfate (NiSO4)
Negative control: lactic acid (LA)

- Cells used: THP-1 (monocytic leukeamia cell line) cells.
- Supplier: American Type Culture Collection (batch 61077351).

PREPARATION OF CELLS: cells were stored in liquid nitrogen and the assays were performed thanks to a master bank (batch THP101008) supplied by Biopredic International (Saint Grégoire – France). Cryopreserved cells have been thawed. Cells were cultured, at 37 °C under 5 % CO2 and humidified atmosphere, in RPMI-1640 medium supplemented with 10 % Foetal Calf Serum, 100 units/ml penicillin and 100 µg/ml streptomycin. THP-1 were routinely seeded every 3-4 days at the density of 0.15 to 0.2 x 10^6 cells/ml. For testing, THP-1 cells were seeded at a density of 0.2 x 106 and pre-cultured in culture flasks for 72 hours. The quality of each batch of THP-1 cells should be checked. Viability of the cells must be above 90 %.

The cytotoxicity of the test item was evaluated in order to select at least 4-5 concentrations able to induce cytotoxicity, around 50 %, for the highest one. Assessment of cell toxicity was performed by determining cell viability on THP-1 cells, using the 7-AAD inclusion methods. Eight concentrations of test item have been prepared by a two-fold serial dilution and a final maximum concentration of 1000 µg/ml, obtaining a final range of concentrations in the plate of 7.81 to 1000 µg/ml. In the day of testing, cells harvested from culture flask were suspended with fresh culture medium at 2 x 10^6 cells/ml. Then, THP-1 cells were distributed into a 24 well flat-bottom plate with 500 µl (1 x 10^6 cells/well) with various concentrations of test item (1:1 ratio) for 24 ± 0.5 hours at 37 °C under 5 % CO2. After treatment cells were washed twice with phosphate-buffered containing 0.1 % (w/v) bovine serum albumin identified as FACS buffer (Fb). The cells were stained with 7-AAD (5 µg/ml final concentration). Then cells were analysed with flow cytometry using GUAVA (Merck Millipore, France) and InCyte software to measure cell viability. The living cells (7-AAD) gate was set in the 7-AAD negative area. 10^4 7-AAD cells were counted as the living population. According to the results the dose levels for the main study were selected.

Based on the cytotoxicity assay the eight final test item concentrations were selected. The test material was soluble in RPMI, the highest concentration that has been reached was 5 mg/ml. The highest dose used in this study was 5000 µg/ml after dilution in medium.
In case of non-toxic concentration for the top dose used in preliminary experiment, the maximum concentration selected for activation test reaches 5000 µg/ml when the test item could be dissolved or stably dispersed in medium. The doses range for activation test was therefore the following (two fold dilution factor): from 39.1 to 5000 µg/ml. Each experiment of activation test was performed on eight concentrations.
THP-1 cells were plated at 1*10^6 cells/ml/well in 24 well plates and treated for 24 ± 0.5 hours with selected test item concentrations. After treatment cells were washed twice with Fb. Then cells were stained for 30 min at 4 °C with the following fluorescein isothiocyanate (FITC) conjugated monoclonal antibodies (mAbs): anti-human CD54, anti-human CD86; FITC labelled-mouse IgG1. Using the manufacturer’s recommended dilutions, cells were incubated with above mAbs at 6 µl/3*105 cells /50µl for the anti-human CD86 mAb, and 3 µl/3*105 cells /50µl for the anti-human CD54 mAb. FITC labelled-mouse IgG1 was used as an isotype control at a dilution of 3 µl/3*10^5 cells /50µl. Then, the cells were stained also with 7-AAD for 30 min at 4 °C. After washing and resuspension with Fb, the fluorescence intensities of the THP-1 cell surface markers were then analysed by flow cytometry using GUAVA (Merck Millipore, France) and InCyte software, on 10000 living cells.

The positive criteria are described as following:
RFI of CD54 ≥ 200 % and RFI of CD86 ≥ 150 % of their respective control at any tested concentration.
The chemicals must be tested in two independents experiments at least. If two of three independent experiments at any dose exceed 150 % of RFI for CD86, or exceed 200 % of RFI for CD54, the chemical could be identified as a sensitizer. Otherwise it is identified as a non-sensitizer.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
other: RFI of CD54
Run / experiment:
Experiment 1
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
other: RFI of CD54
Run / experiment:
Experiment 2
Vehicle controls validity:
Negative controls validity:
Positive controls validity:
Other effects / acceptance of results:
No contamination was noticed during the study.
Based on linear regression and according to the guideline, the concentrations inducing 200 % of CD54 RFI (EC200, EC: Estimated Concentration) is calculated: EC200 = 2617 µg/ml.
EC150 is not calculated because the mean CD86 of the two experiments for the higher concentration is less than 150.
A dose-response increase was noticed for the CD54 expression with the test item. From 2.3 to 3.8 fold increase of CD54 expression compared to the negative control were induced in the first experiment and the fold increase was lower for CD86 marker (under threshold). For the second experiment a 2.8 fold increase of CD54 expression and a 1.6 fold increase of CD 86 compared to the negative control was noted.
No cytotoxicity was induced on THP-1 cells by the substance.

QUALITY CONTROL OF THE SYSTEM: according to the RFI values obtained for vehicles and controls, the test system was validated in the test facility.

- Acceptance criteria met for negative control: negative controls (RPMI) showed cell viability values acceptable regarding the acceptance criteria.
- Acceptance criteria met for positive control: positive controls showed an increase of CD54/86 expression (RFI ≥ 200/150 respectively) compared to the negative control.

Any other information on results incl. tables

The test system was checked after thawing.

Table: CV and ratio of both markers CD54 and CD86 to isotype control obtained for vehicles and controls

Sample CV (%) Ratio CD54/IgG Ratio CD86/IgG
Criteria  Results Criteria  Results Criteria  Results
RPMI > 90 % 98 > 105 130 > 105 162
NaCl > 90 % 98 > 105 127 > 105 158
DMSO > 90 % 98 > 105 132 > 105 161
NiSO4 > 50 % 85
DNCB > 50 % 85
LA > 50 % 98

Table: RFI values obtained for vehicles and controls

Sample  RFI
Criteria  Results CD54 Results CD86
NaCl NaCl ≤200 (CD54) and ≤150 (CD86) 88 91 88 91
DMSO DMSO ≤200 (CD54) and ≤150 (CD86) 102 92 102 92
NiSO4 ≥200 (CD54) and ≥150 (CD86) 3895 355
DNCB ≥200 (CD54) and ≥150 (CD86) 636 674
LA ≤200 (CD54) and ≤150 (CD86) 118 97

The cytotoxic profile of the test item on THP-1 after a 24-hour exposure period is presented below.

Dose level μg/ml Cell viability %
1000 98
500 98
250 98
125 98
62.5 98
31.3 98
15.6 98
7.81 97

CD54/86 expression of the test item after a 24-hour exposure period using the RFI is presented below.

Sample Dose level (μg/ml) Experiment 1 Experiment 2
CD54 CD86 CV (%) CD54 CD86 CV (%)
RPMI 100 100 98 100 100 98
DMSO 100 100 98 100 100 98
DNCB 4 636 674 85 1040 630 81
(vehicle RPMI)
39.1 64 59 98 86 130 98
78.1 71 56 98 81 102 98
156 76 72 98 76 102 98
313 90 63 98 87 108 98
625 97 56 98 102 109 98
1250 125 58 98 118 93 98
2500 232 80 98 156 90 97
5000 382 102 95 276 159


Applicant's summary and conclusion

Interpretation of results:
other: classified as a Skin Sensitiser Cat.1, according to the CLP Regulation (EC) No.1272/2008
Skin sensitiser
Executive summary:

The in vitro intrinsic sensitizing potential of the test item was evaluated according to the human cell line activation test (h-CLAT) following the OECD Guideline 442E. THP-1 cell cultures were exposed to eight concentrations of the test item, ranging from 39.1 to 5000 μg/ml, for 24 hours. After exposure, the expression of two cell surface antigens, CD86 and CD54, was measured by flow cytometry method. Vehicle control (RPMI), negative control (LA) and positive controls (DNCB and NiSO4) run in parallel. The chemicals were tested in two independents experiments.

RFI of CD54 was higher than 200 in two experiments at 5000 μg/ml concentration.

The substance is considered to be a skin sensitiser.