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Skin sensitisation

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Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 June - 01 July 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
/ cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry, no demonstration of technical proficiency in the report

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test)
Version / remarks:
29 July 2016
Deviations:
yes
Remarks:
/ cytotoxicity measurement and estimation of the CV75 value of the dose finding assay was performed by XTT test instead of flow cytometry, no demonstration of technical proficiency in the report
GLP compliance:
yes (incl. certificate)
Remarks:
Hess. Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany
Type of study:
activation of dendritic cells

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vitro test system

Details on study design:
TEST CELL LINE
- Source: American Type Culture Collection (ATCC)
- Passage number: 4 (XTT test); 10 + 11 (h-CLAT)

CELL CULTURE CONDITIONS
- Type and identity of media: RPMI-1640 supplemented with 10% (v/v) FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and 1% (v/v) L-glutamine
- Temperature (°C): 37 ± 1.5
- CO2 (%): 5.0 ± 0.5

INFORMATION FOLLOWING IN THE NEXT 9 PARAGRAPHS RELATE TO DETAILS ON XTT:

The XTT test is a method to investigate the cytotoxicity of a test substance. The test is based on the cleavage of the yellow tetrazolium salt, sodium 3'-(1-phenylaminocarbonyl)-(3,4 - tetrazolium)-bis-(4–methoxy-6-nitro)-benzenesulfonic acid hydrate (XTT), to form an orange water soluble formazan dye by dehydrogenase activity in active mitochondria. Two independent cytotoxicity experiments were performed with different cell cultures and on different days to obtain a reliable concentration showing 75% cell viability (CV75).

CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: dimethyl sulfoxide (DMSO)
- Concentration: 0.2 - 0.5%

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h

TEST CONCENTRATIONS
- Justification for top dose: The test substance was soluble in DMSO up to and including 125 µg/mL.
0.975, 1.95, 3.91, 7.81, 15.63, 31.25, 62.5 and 125 µg/mL

NUMBER OF REPLICATIONS: 4 samples per dose group were tested in two independent experiments

MEASUREMENT
- Device: microplate reader (Versamax, Molecular Devices)
- Wavelength: 450 nm
- Reference wavelength: 690 nm

EVALUATION CRITERIA
A decrease in number of living cells results in a decrease in the overall activity of mitochondrial dehydrogenases in the sample. This decrease directly correlates to the amount of orange formazan formed, as monitored by the absorbance. The relative absorbance as compared to the solvent control is calculated using the formula:
Relative absorbance = 100 x [(mean absorbance test substance) - (absorbance blank)] / [(mean absorbance vehicle control) - (absorbance blank)]

To calculate the concentration of test substance needed to reduce the relative absorbance to 75% of the vehicle control (CV75) the following formula is used:
CV75 = Conc. > 75 - ([(Conc. > 75) - (Conc. < 75)] x [(% > 75) - 75] / [(% > 75) - (% < 75)])
a) Conc. > 75: max. measured concentration with the % of vehicle control > 75%
b) Conc. < 75: min. measured concentration with the % of vehicle control < 75%
c) % > 75: relative absorbance at a) in %
d) % < 75: relative absorbance at b) in %

ACCEPTANCE CRITERIA
The XTT test is considered to be acceptable if it meets the following criteria:
- mean absorbance of the negative control is ≥ 0.5
- mean viability of the vehicle control is ≥ 70%


INFORMATION FOLLOWING IN THE NEXT PARAGRAPHS RELATE TO DETAILS ON H-CLAT:

The h-CLAT method is an in vitro method which quantifies changes of cell surface marker expression (CD86 and CD54) on a human cell line, THP-1 cells, following 24 h exposure to the test substance. The changes of surface marker expression are measured by flow cytometry following cell staining with fluorescein isothiocyanate (FITC)-labelled antibodies. The relative fluorescence intensity of surface markers compared to solvent control are calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

CONTROLS
Negative Control
- Substance: culture medium
Vehicle control
- Substance: DMSO
- Concentration: 0.2%
Positive control
- Substance: 2,4-dinitrochlorobenzene in DMSO
- Concentration: 2 and 3 µg/mL

EXPOSURE CONDITIONS
- Exposure duration: 24 ± 1 h

TEST CONCENTRATIONS
- Justification for top dose: The highest concentration should have been 1.2 × highest soluble test item concentration (125 μg/mL), i.e. 150 μg/mL. However, due to a calculation error, the highest test substance concentration used was 300 μg/mL instead of 150 μg/mL.
83.7, 100.5, 120.6, 144.7, 173.6, 208.3, 250 and 300 μg/mL

NUMBER OF REPLICATIONS: at least in triplicates for the different stainings in two independent experiments

STAINING
- Antibodies: FITC anti-CD54 (Clone: Fun-1); FITC anti-CD86 (Clone: 6.5B5); FITC mouse IgG1 (isotype control)
- Temperature: 2 - 8 °C (on ice)
- Duration: 30 ± 5 min

MEASUREMENT
- Device: FACSCalibur (Becton Dickinson)

EVALUATION CRITERIA
The relative fluorescence intensity (RFI) is used as an indicator of CD86 and CD54 expression, and is calculated for each concentration as follows:
RFI (%) = [(MFI of test substance treated cells) - (MFI of test substance isotype control cells) / (MFI of vehicle control cells) - (MFI of vehicle isotype control cells)] x 100
MFI: geometric mean fluorescence intensity (GeoMean)

The cell viability is calculated as follows for each concentration:
Cell viability (%) = (mean cytotox of vehicle control cells) / (mean cytotox of test substance treated cells) x 100
Where the mean cytotox is the mean of GeoMean (7-AAD) isotype control, GeoMean (7-AAD) CD54 and GeoMean (7-AAD) CD86.

The test substance is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test substance is considered to be a sensitizer. Otherwise it is considered to be a non-sensitizer.

ACCEPTANCE CRITERIA
The study is considered as valid, if the following criteria are met:
- Cell viability of negative control is adjusted to 100% and the cell viability of the vehicle control should be more than 90% in comparison to the negative control.
- In the positive control, RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
- In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- For negative and vehicle controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
- For the test substance resulting in negative outcome, the cell viability at the 1.2 × CV75 value should be less than 90%. If the cell viability at the 1.2 × CV75 value is more than 90% for a positive tested test substance, the data will be acceptable. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test substance are accepted independent by the cell viability.
- The cell viability of at least 4 doses in each experiment should be ≥50%.

Results and discussion

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: relative fluorescence intensity (RFI) of CD54 (%)
Run / experiment:
24 h incubation
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: exceeding positive criteria, except of at 100.5 μg/mL in exp.1 and from 83.7 to 144.7 μg/mL in exp.2 (< 200%)
Key result
Parameter:
other: relative fluorescence intensity (RFI) of CD86 (%)
Run / experiment:
24 h incubation
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: only in exp.1 at 300 μg/mL an RFI of > 150% (exceeding positive criteria) was observed
Other effects / acceptance of results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
XTT test
No cytotoxic effects (i.e. cell viability less than 50%) were observed following incubation with the tested concentrations.

DEMONSTRATION OF TECHNICAL PROFICIENCY: no information provided

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for vehicle control: In the vehicle control, RFI values compared to the negative control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥150% and CD54 ≥ 200%).
- Acceptance criteria met for positive control: The RFI values of the positive control for CD86 and CD54 exceeded the positive criteria (CD86 >150% and CD54 >200%) and the cell viability was >50%.

Any other information on results incl. tables

Table 1: Results of the first XTT test

 

Concentration [µg/mL]

Mean absorbance*

Blank

Absorbance in % of vehicle control**

Negative control

-

0.806 ± 0.019

0.223

97.03

Vehicle control

-

0.870 ± 0.018

0.268

100.0

Test item

0.98

0.798 ± 0.033

0.265

88.72

1.95

0.785 ± 0.021

0.271

85.33

3.91

0.774 ± 0.021

0.267

84.19

7.81

0.728 ± 0.036

0.274

75.43

15.63

0.782 ± 0.034

0.273

84.59

31.25

0.809 ± 0.028

0.278

88.41

62.5

0.810 ± 0.040

0.280

87.95

125

0.828 ± 0.052

0.293

88.85

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

 

Table 2: Results of the second XTT test

 

Concentration [µg/mL]

Mean absorbance*

Blank

Absorbance in % of vehicle control**

Negative control

-

0.733 ± 0.006

0.261

96.29

Vehicle control

-

0.758 ± 0.027

0.268

100.0

Test item

0.98

0.728 ± 0.026

0.254

96.65

1.95

0.738 ± 0.010

0.253

98.88

3.91

0.741 ± 0.018

0.255

98.88

7.81

0.735 ± 0.012

0.253

98.35

15.63

0.742 ± 0.019

0.264

97.33

31.25

0.767 ± 0.006

0.262

102.91

62.5

0.814 ± 0.035

0.262

112.48

125

0.823 ± 0.040

0.268

113.05

*: mean absorbance (absolute) of 4 wells

**: relative absorbance

 

Table 3. Results of the first h-CLAT run

 

Concentration [µg/mL]

Antibody / ISO

MFI

GeoMean (FITC)

MFI - ISO

Cyto(Geo) GeoMean

(7-AAD)

Mean Cyto

RFI (%)

Viability (%)

Negative control

-

ISO

2.51

-

2.35

2.1

-

100.0

CD54

3.16

0.65

2.00

100.0

CD86

4.37

1.86

1.94

100.0

Vehicle control

-

ISO

2.35

-

2.26

2.0

-

100.0

CD54

3.17

0.82

2.06

100.0

CD86

4.32

1.97

1.82

100.0

Positive control

(DNCB)

2

ISO

2.73

-

2.61

2.0

-

104.6

CD54

9.45

6.72

1.74

819.5*

CD86

12.57

9.84

1.52

499.5*

3

ISO

2.67

-

2.88

2.1

-

98.7

CD54

11.36

8.69

1.77

1059.8*

CD86

13.66

10.99

1.57

557.9*

Test item

83.7

ISO

2.74

-

1.38

2.1

-

97.9

CD54

4.70

1.96

1.93

239.0*

CD86

4.75

2.01

1.96

102.0

100.5

ISO

2.71

-

2.34

2.1

-

99.8

CD54

4.16

1.45

2.00

176.8

CD86

5.21

2.50

1.81

126.9

120.6

ISO

2.75

-

2.35

2.1

-

98.6

CD54

4.43

1.68

1.99

204.9*

CD86

4.85

2.10

1.89

106.6

144.7

ISO

2.90

-

2.53

2.2

-

92.3

CD54

5.09

2.19

2.10

267.1*

CD86

5.52

2.62

2.02

133.0

173.6

ISO

2.92

-

2.83

2.4

-

84.8

CD54

4.90

1.98

2.26

241.5*

CD86

5.35

2.43

2.15

123.4

208.3

ISO

3.01

-

2.76

2.4

-

85.5

CD54

5.07

2.06

2.30

251.2*

CD86

5.79

2.78

2.12

141.1

250

ISO

3.03

-

2.95

2.5

-

81.9

CD54

5.33

2.30

2.34

280.5*

CD86

5.77

2.74

2.21

139.1

300

ISO

3.41

-

4.20

3.6

-

57.5

CD54

6.50

3.09

3.35

376.8*

CD86

7.57

4.16

3.13

211.2*

  *: RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥ 150% and CD54 ≥ 200%).

DNCB = 2,4-dinitrochlorobenzene

Table 4. Results of the second h-CLAT run

 

Concentration [µg/mL]

Antibody / ISO

MFI GeoMean (FITC)

MFI - ISO

Csto(Geo) GeoMean (7-AAD)

Mean Cyto

RFI (%)

Viability (%)

Negative control

-

ISO

2.28

-

2.35

2.2

-

100

CD54

2.94

0.66

2.19

100

CD86

3.86

1.58

1.98

100

Vehicle control

-

ISO

2.22

-

2.39

2.1

-

100

CD54

2.96

0.74

2.24

100

CD86

4.09

1.87

1.52

100

Positive control

2

ISO

2.42

-

2.78

2.4

-

87.2

CD54

7.23

4.81

2.29

650.0*

CD86

6.42

4.00

1.98

213.9*

3

ISO

2.61

-

3.25

2.3

-

89.8

CD54

8.83

6.22

1.83

840.5*

CD86

9.39

6.78

1.77

362.6*

Test substance

83.7

ISO

2.55

-

2.71

2.3

-

88.6

CD54

3.68

1.13

2.23

152.7

CD86

4.31

1.76

2.00

94.1

100.5

ISO

2.54

-

2.48

2.2

-

93.5

CD54

3.82

1.28

2.16

173.0

CD86

4.54

2.00

1.94

107.0

120.6

ISO

2.60

-

2.52

2.2

-

94.8

CD54

3.97

1.37

2.18

185.1

CD86

5.10

2.50

1.79

133.7

144.7

ISO

2.60

-

2.67

2.3

-

87.7

CD54

3.99

1.39

2.25

187.8

CD86

4.20

1.60

2.09

85.6

173.6

ISO

2.61

-

2.76

2.4

-

84.6

CD54

4.29

1.68

2.33

227.0*

CD86

4.56

1.95

2.18

104.3

208.3

ISO

2.72

-

2.95

2.5

-

80.5

CD54

4.87

2.15

2.42

290.5*

CD86

5.00

2.28

2.27

121.9

250

ISO

2.81

-

2.99

2.6

-

78.6

CD54

4.98

2.17

2.53

293.2*

CD86

5.48

2.67

2.30

142.8

300

ISO

3.01

-

3.23

2.8

-

74.1

CD54

5.18

2.17

2.63

293.2*

CD86

5.80

2.79

2.44

149.2

  *: RFI value of CD86 or CD54 exceeded the criteria for a positive response (CD86 ≥ 150% and CD54 ≥ 200%).

DNCB =2,4-dinitrochlorobenzene

In the first run the relative fluorescence intensity of CD86 exceeded the threshold of 150% in the cells treated with test item concentration of 300 μg/mL. In the second run the relative fluorescence intensity of CD86 did not exceed the threshold at the test item concentrations tested.

In the first run the relative fluorescence intensity of CD54 exceeded the threshold of 200% in the cells treated with test item concentration of 83.7 μg/mL and between 120.6 and 300 μg/mL. In the second run the relative fluorescence intensity of CD54 exceeded the threshold in cells treated with the test item concentration between 173.6 and 300 μg/mL. Even if higher test item concentrations were used in the main experiment as recommended according to the guideline, the result is considered to be valid and acceptable since the cell viability at this test substance concentration did not fall below 50% cell viability.

Applicant's summary and conclusion

Interpretation of results:
other: skin sensitising potential based on the key event “activation of dendritic cells”
Conclusions:
Under the conditions of the test, the test substance induces dendritic cell activation. The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.
Executive summary:

In the Human Cell Line Activation Test (h-CLAT) (OECD 442E) (2016),THP-1cells were treated for 24 h with concentrations ranging from83.7 to 300 μg/mL, resulting in a relative fluorescence intensity (RFI) of CD54 >200% (at 83.7 μg/mL and between 120.6 and 300 μg/mL in exp. 1 and at 173.6 and 300 μg/mL in exp. 2) and of CD86 < 150% (at all concentrations in both exp., except at 300 μg/mL in exp. 1).

Based on this result, the h-CLAT prediction is considered positive for skin sensitisation.