Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 13th to April 4th, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD 442E- In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
GLP compliance:
yes
Type of study:
other: human Cell Line Activation Test (h-CLAT)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vitro test system

Details on study design:
CELL LINE: THP-1 Monocytes from peripheral blood cultured at 37 °C ± 1 °C, 5 % CO2, 90 % ± 10 % relative humidity. The doubling time of the cell were checked before starting with the test.

CONTROL:
- Positive controls: 2,4-dinitrochlorobenzene (DNCB) and nickel sulfate hexahydrate (NiSO4)
- Negative control: lactic acid (LA).

SOLVENT: DMSO. Since the test substance was soluble only in DMSO the solutions for pre-feasibility and dose-finding assay were prepared using this solvent.

EXPERIMENTAL DESIGN:
The test was divided in 4 phases:
1) Reactivity check of the cells which were thawed for the assay, performed by treating cells for 24 hours with 2 positive controls, DNCB and NiSO4 and the negative control lactic acid. The final concentration in reactivity check was 4 μg/ml for DNCB, 100 μg/ml for NiSO4 6H2O and 1000 μg/ml for LA. The plate was incubated for 24 hours at 37 °C and 5 % CO2. After incubation the cell were centrifuged and treated with FACS buffer (D-PBS with 0.1 % (w/v) Bovine Serum Albumin lyophilized powder -BSA), blocking buffer (0.01% globulin solution in FACS buffer) and finally with Propidium Iodide (PI). Expression of cell surface antigen was analysed by flow cytometry at excitation wavelength of 488 nm. Collected data were analysed by a software and Relative fluorescence Intensity (RFI) was calculated based on the mean of geometric fluorescence intensity (RFI).
2) Pre-feasibility test, to verify that the test substance did not interfere with the Propidium iodide fluorescent signal due to intrinsic fluorescence. Some unstained cells (indicated as “without Pl“) were used for pre-feasibility test to verify that the test substance does not interfere with the Propidium iodide fluorescent signal due to intrinsic fluorescence.
3) Dose finding assay, to calculate the CV75 needed for determining the concentrations of test substance to be used in the CD54/CD86 expression experiment; in this phase cells were exposed for 24 hours to 8 different concentrations of test substance. Viability was measured after propidium iodide (Pl) staining as the ration between number of living cells and total number of acquired cells. The final concentrations of test item used for dose finding were 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.82 μg/ml.
4) CD54/CD86 expression assay, in which cells were incubated for 24 hours with 8 doses of test substance. As it was not possible to calculate the CV75 value, due to insufficient cytotoxicity of the test substance, 1000 µg/ml concentration was used as the first concentration for the CD54/CD86 expression assay, the highest concentration of the test chemical stably dissolved in DMSO. Then 7 serial dilutions with a 1.2-fold dilution factor were used. The concentrations tested were 1000, 833.33, 694.44, 578.790, 482.25, 401.88, 334.90 and 279.08 µg/ml. Expression levels of CD54 and CD86 were analyzed by flow cytometry and the relative fluorescence intensity (RFI) was calculated in order to define the test substance as positive (potential sensitizer) or negative (non-sensitizer). The test substance was assayed in 2 independent runs, performed in two different days. A third run was not necessary. were 1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90 and 279.08 µg/ml)

REPLICATE IN MAIN STUDY: Two replicates

EVALUATION CRITERIA:
When the RFI of CD54 is ≥ 200 at any tested dose contemporary showing cell viability > 50 %, in the two independent runs, AND/OR if the RFI of CD86 is ≥ 150 at any tested dose contemporary showing cell viability > 50%, in the two independent runs, the test chemical is considered POSITIVE (sensitizer), otherwise it is considered NEGATIVE (non-sensitizer). If the two independent runs are not concordant for at least one of the markers (CD54 or CD86), it is necessary to perform and additional run and conclusions are based on the majority result of the three individual runs (i.e., 2 out of 3).

ACCEPTANCE CRITERIA:
- Dose range finding: Viability of cells treated with culture medium with DMSO > 90 %
- Main test:
Viability of cells treated with culture medium > 90 %
Viability of cells treated with culture medium with physiological solution > 90 %
Viability of cells treated with culture medium with DMSO >90 %
CD54 RFI of cells treated with culture medium with physiological solution < 200 %
CD86 RFI of cells treated with culture medium with physiological solution < 150 %
CD54 RFI cells treated with culture medium with DMSO < 200 %
CD86 RFI cells treated with culture medium with DMSO < 150 %
CD54 MFI / lgG1 MFI for cells treated with culture medium with physiological solution > 105 %
CD86 MFl / lgG1 MFI for cells treated with culture medium with physiological solution > 105 %
CD54 MFI / lgG1 MFl for cells treated with culture medium with DMSO > 105%
CD86 MFl / lgG1 MFI for cells treated with culture medium with DMSO >105 %
CD54 RFI DNCB ≥ 200 %
CD86 RFI DNCB ≥ 150 %
Viability of cells treated with DNCB > 50 %
Cell viability of at least 4 doses of test substance in each assay > 50% : > 4

Results and discussion

Positive control results:
The data related to 2,4-dinitrochlorobenzene (DNCB) satisfied the acceptance criteria:
- RFI CD54: Run 1= 731.77 % ; Run 2= 717.85 % (CD54 RFI DNCB ≥ 200 %)
- RFI CD86: Run 1= 323.10 % ; Run 2= 347.44 % (CD86 RFI DNCB ≥ 150 %)
- Viability of cell treated: Run 1= 82.08% ; Run 2= 90.89% (Viability for DNCB> 50 %)

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: RFI CD86
Run / experiment:
1st and 2nd run
Value:
< 150
Vehicle controls validity:
valid
Remarks:
cultur medium+DMSO
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: See remarks
Remarks:
For all concentrations of test item: 1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90 and 279.08 µg/ml.
Key result
Parameter:
other: RFI CD54
Run / experiment:
1st and 2nd run
Value:
< 200
Vehicle controls validity:
valid
Remarks:
cultur medium+DMSO
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: See remarks
Remarks:
For all concentrations of test item: 1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90 and 279.08 µg/ml.
Other effects / acceptance of results:
MAIN TEST: In the CD54/CD86 expression assay, RFI of CD54 is < 200 and RFI CD86 is < 150 at any tested dose, therefore it was not possible to calculate the EC200 and EC150 values. The test item is to be considered NEGATIVE (non-sensitizer) up to the concentration 1000 µg/ml.

MONITORING THE DOUBLING TIME: Before starting with the test the doubling time of the cell batch was monitoring: the final doubling time for the cell culture is the average value of the three calculated consecutive doubling times (47 hours) and it should fall in the range 30 - 55 hours.

REACTIVITY CHECK:
Both DNCB and NiSO4 produced a positive response for both CD54 and CD86 (i.e. RFI CD54 ≥ 200 and RFI CD86 ≥ 150) and contemporary showing cell viability > 50%.
LA should produced a negative response for both CD54 and CD86 (i.e. RFI CD54 < 200 and RFI CD86 < 150) and contemporary showing cell viability > 50%.
All acceptance criteria of reactivity check were fulfilled.

DOSE FINDING ASSAY: in the dose finding assay, cell viability was more than 75 % at the highest dose, so it was not possible to calculate the CV75 value. The highest stably dispersed concentration of the test substance in DMSO (1000 µg/ml) was used as first concentration for the CD54/CD86 expression assay.

ACCEPTANCE CRITERIA:
- Dose range finding: fulfilled: Viability of cells treated with culture medium with DMSO = 96.72 (> 90 %)
- Main test: all fulfilled
Viability of cells treated with culture medium > 90 %: Run 1= 97.91 % ; Run 2= 98.75 %
Viability of cells treated with culture medium with physiological solution > 90 % : Run 1= 97.84 % ; Run 2= 98.62 %
Viability of cells treated with culture medium with DMSO > 90 %: Run 1= 97.68 % ; Run 2= 98.79%
CD54 RFI of cells treated with culture medium with physiological solution < 200 % : Run 1= 82.59 % ; Run 2= 75.79 %
CD86 RFI of cells treated with culture medium with physiological solution < 150 % : Run 1= 94.15 % ; Run 2= 110.75 %
CD54 RFI cells treated with culture medium with DMSO < 200 %: Run 1= 79.76 % ; Run 2= 80.47 %
CD86 RFI cells treated with culture medium with DMSO < 150 %: Run 1= 103.05 % ; Run 2= 112.25 %
CD54 MFI / lgG1 MFI for cells treated with culture medium with physiological solution > 105 % : Run 1= 132.44 % ; Run 2= 119.09 %
CD86 MFl / lgG1 MFI for cells treated with culture medium with physiological solution > 105 %: Run 1= 164.45 % ; Run 2= 154.98 %
CD54 MFI / lgG1 MFl for cells treated with culture medium with DMSO > 105%: Run 1= 131.79 % ; Run 2= 120.50 %
CD86 MFl / lgG1 MFI for cells treated with culture medium with DMSO > 105 % : Run 1= 171.56 % ; Run 2= 156.36 %
CD54 RFI DNCB ≥ 200 %: Run 1= 731.77 % ; Run 2= 717.85 %
CD86 RFI DNCB ≥ 150 %: Run 1= 323.10 % ; Run 2= 347.44 %
Viability of cells treated with DNCB > 50 %: Run 1= 82.08 % ; Run 2= 90.89 %
Cell viability of at least 4 doses of test substance in each assay > 50% > 4: Run 1=8; Run 2= 8

Any other information on results incl. tables

Dose range finding
Test dose in well (µg/ml) Mean cell viability (%)
C1 1000 87.3
C2 500 95.73
C3 250 98.24
C4 125 99.08
C5 62.5 97.46
C6 31.25 96.12
C7 15.63 96.15
C8 7.82 96.19
NC+DMSO - 96.72

Main test RFI CD54 (%) RFI CD86 (%) Viability%
Test dose in well (µg/ml) Run 1 Run 2 Run 1 Run 2 Run 1 Run 2
C1 1000 100.07 172.83 40.50 44.03 96.31 98.06
C2 833.33 103.30 155.00 39.46 42.63 96.32 98.38
C3 694.44 91.42 125.04 38.97 39.11 96.67 98.65
C4 578.7 83.10 120.50 46.56 41.45 97.13 98.86
C5 482.25 85.51 103.87 52.78 44.80 97.57 99.04
C6 401.88 85.71 95.78 63.79 51.90 98.14 99.13
C7 334.9 83.15 89.35 59.93 51.15 98.55 99.29
C8 279.08 87.82 92.41 60.37 59.94 98.53 99.28
DNCB 4 731.77 717.85 323.10 347.44 82.08 90.89
NC / / / / / 97.91 98.75
NC+PS / 82.59 75.79 94.15 110.75 97.84 98.62
NC+DMSO / 79.76 80.47 103.05 112.25 97.68 98.79

Applicant's summary and conclusion

Interpretation of results:
other: not classified as skin sensitizer according to the CLP Regulation (EC n.1272/2008)
Conclusions:
The test item resulted to be negative (non-sensitizer) in h-CLAT test.
Executive summary:

The solid substance was tested by h-CLAT assay conducted according to OECD 442E:2016. The test was carried out on the human cell line THP-1, in two independent runs. Cells were exposed to 8 different concentrations of the test chemical for 24 hours. CD54/CD86 over-expression, strictly related to the sensitizing potential of the test substance, was assessed byow cytometry. Under the test conditions above described, the test substance resulted to be NEGATIVE (NON-SENSITIZER) up to the maximal tested concentration of 1000 μg/ml.