Registration Dossier

Administrative data

Description of key information

The potential of the test material to cause skin sensitisation was assessed in vitro by means of AOP related assays:

The OECD 442 C study provided inconclusive results, the OECD 442 D and OECD 442 E experiments were both positive. Two out of three key events for skin sensitisation were thus positive indicating that the test material causes a potential for skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-11-16 to 2017-12-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
other: (in chemico) reactivity against synthetic peptides with a thiol or amino group
Details on study design:
The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.
Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 63.50%.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
cysteine run
Value:
0.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
lysine run
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample

Cysteine Peptide

Lysine Peptide

Peak Area
at 220 nm

Peptide Concentration [mM]

Peak Area
at 220 nm

Peptide Concentration [mM]

STD1

5221.6792

0.5340

4311.3857

0.5340

STD2

2648.5273

0.2670

2143.2170

0.2670

STD3

1327.5009

0.1335

1043.3549

0.1335

STD4

670.7400

0.0667

519.3511

0.0667

STD5

334.8562

0.0334

259.5146

0.0334

STD6

166.3505

0.0167

130.4601

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1609.4324

0.1633

68.11

68.17

0.05

0.08

1604.4860

0.1628

68.21

1605.4248

0.1629

68.19

Test Item

4967.2417

0.5065

0.03

0.30

0.37

122.03

4961.0894

0.5059

0.15

4932.7520

0.5030

0.72

Depletion of the Lysine Peptide

Lysine Peptide

Sample

Peak Area
at 220 nm

Peptide Conc. [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

1589.2585

0.1982

59.95

58.82

0.99

1.68

1662.4030

0.2073

58.11

1650.5492

0.2058

58.41

Test Item*

4055.4836

0.5032

0.00

0.00

0.00

n/a

4028.6870

0.4999

0.00

4043.9385

0.5018

0.00

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model 1

Mean Cysteine andLysine PPD

Reactivity Class

DPRA Prediction²

0.00% PPD 6.38%

 No or Minimal Reactivity

Negative

6.38% < PPD 22.62%

Low Reactivity

Positive

22.62% < PPD 42.47%

Moderate Reactivity

42.47% < PPD 100%

High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD

ReactivityClass

DPRA Predictio

0.00% PPD 13.89%

No or Minimal Reactivity

Negative

13.89% < PPD 23.09%

Low Reactivity

Positive

23.09% < PPD 98.24%

Moderate Reactivity

98.24% < PPD 100%

High Reactivity

Categorization of the Test Item

Prediction Model

Prediction Model 1
(Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)

Prediction Model 2
(Cysteine Peptide / Test Item Ratio: 1:10)

Test Substance

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Mean Peptide Depletion [%]

Reactivity Category

Prediction

Test Item

 -

0.30

Minimal Reactivity

no sensitiser

Positive Control

63.50

High Reactivity

sensitiser

68.17

Moderate Reactivity

sensitiser

Interpretation of results:
other: Prediction cannot be made. The result must be considered in the context of integrated approach.
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. However, due to the observed precipitation, the prediction model does not apply and a prediction cannot be made.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.
Executive summary:

In the present study the test material, was dissolved in DMF, based on the results of the pre-experiments.

Based on a molecular weight of 296 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the

24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the samples of the test item (including the co-elution control of the test item). Samples were centrifuged prior to the HPLC analysis.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive control (including the co-elution control of the positive control). Precipitation was observed for the samples of the test item (including the co-elution control of the test item). Samples of the test item were centrifuged at 400g for 5 minutes prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Co-elution of the test item with the lysine peptide peak was observed. Therefore sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC CDMF). However, precipitation was observed in the cysteine experiment as well.

The 100 mM stock solution of the test item showed minimal reactivity towards the cysteine peptide. The mean depletion of cysteine peptide was  6.38% (0.30%).

According to the evaluation criteria in the guideline, if a precipitation is observed after the incubation period, peptide depletion may be underestimated and a conclusion on the lack of reactivity cannot be drawn with sufficient confidence in case of a negative result. Due to the observed precipitation in the cysteine experiment no prediction can be made.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 63.50%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-11-14 to 2018-02-05
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details on study design:
The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by
addressing the second molecular key event of the adverse outcome pathway (AOP), namely
activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line
KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared
to the respective solvent controls is used to support discrimination between skin sensitisers
and non-sensitisers.

Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.60 (experiment 1) and 3.43 (experiment 2)).
Key result
Parameter:
other: luciferase activity
Run / experiment:
1
Value:
2.64
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 125 µM
Key result
Parameter:
other: cell viability [%]
Run / experiment:
1
Value:
213.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
1
Value:
2.59
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Key result
Parameter:
other: luciferase activity
Run / experiment:
2
Value:
2.79
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 15.63 µM
Key result
Parameter:
other: cell viability [%]
Run / experiment:
2
Value:
146.6
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Parameter:
other: EC1.5 [µM]
Run / experiment:
2
Value:
1.4
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other:
Other effects / acceptance of results:
Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

100

100

100

0.0

Positive Control

4.00

105.2

83.5

94.4

15.3

8.00

108.8

87.0

97.9

15.4

16.00

114.3

96.3

105.3

12.7

32.00

123.0

100.6

111.8

15.9

64.00

167.6

111.7

139.6

39.5

Test Item

0.98

84.6

110.8

97.7

18.6

1.95

84.9

113.3

99.1

20.1

3.91

99.1

136.3

117.7

26.3

7.81

138.9

150.9

144.9

8.5

15.63

195.8

146.6

171.2

34.8

31.25

221.9

124.6

173.3

68.8

62.50

225.5

121.6

173.5

73.5

125.00

213.6

93.2

153.4

85.1

250.00

164.6

123.9

144.2

28.8

500.00

101.4

128.4

114.9

19.1

1000.00

195.0

147.1

171.0

33.9

2000.00

192.3

149.9

171.1

30.0

 

Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.44

1.05

1.43

1.31

0.22

 

8.00

1.26

1.24

1.48

1.33

0.13

 

16.00

1.70

1.25

1.80

1.58

0.29

*

32.00

2.46

1.88

2.36

2.23

0.31

*

64.00

5.70

3.23

4.86

4.60

1.26

*

Test Item

0.98

1.13

1.09

1.19

1.14

0.05

 

1.95

1.44

1.34

1.30

1.36

0.07

 

3.91

2.04

1.83

1.50

1.79

0.27

*

7.81

2.26

2.06

1.68

2.00

0.29

*

15.63

2.68

2.29

2.27

2.41

0.23

*

31.25

2.90

2.50

2.42

2.61

0.26

*

62.50

2.95

2.57

2.37

2.63

0.29

*

125.00

2.99

2.51

2.42

2.64

0.31

*

250.00

2.90

2.12

2.14

2.39

0.45

*

500.00

2.89

2.53

2.18

2.53

0.36

*

1000.00

2.69

2.26

2.06

2.34

0.32

*

2000.00

2.37

2.00

1.78

2.05

0.30

*

* = significant induction according to Student’s t-test, p<0.05

Induction of Luciferase Activity Experiment 2

Experiment 2

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.05

1.07

1.12

1.08

0.04

 

8.00

1.17

1.29

1.25

1.24

0.07

 

16.00

1.31

1.35

1.68

1.45

0.20

 

32.00

1.93

1.85

1.69

1.83

0.12

*

64.00

3.67

2.88

3.74

3.43

0.48

*

Test Item

0.98

1.16

1.27

1.36

1.26

0.10

 

1.95

1.86

1.80

1.75

1.81

0.05

*

3.91

2.07

2.23

2.26

2.19

0.10

*

7.81

2.71

2.57

2.67

2.65

0.07

*

15.63

2.79

2.89

2.68

2.79

0.10

*

31.25

2.42

2.75

2.75

2.64

0.19

*

62.50

2.81

2.55

2.49

2.61

0.17

*

125.00

2.54

2.66

2.35

2.52

0.16

*

250.00

2.62

2.79

2.68

2.69

0.09

*

500.00

2.48

2.49

2.70

2.55

0.13

*

1000.00

2.47

2.27

2.70

2.48

0.22

*

2000.00

2.44

2.14

2.27

2.28

0.15

*

* = significant induction according to Student’s t-test, p<0.05

Induction of Luciferase Activity – Overall Induction

Overall Induction

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.31

1.08

1.19

0.16

 

8.00

1.33

1.24

1.28

0.06

 

16.00

1.58

1.45

1.51

0.10

*

32.00

2.23

1.83

2.03

0.29

*

64.00

4.60

3.43

4.01

0.83

*

Test Item

0.98

1.14

1.26

1.20

0.09

 

1.95

1.36

1.81

1.58

0.32

 

3.91

1.79

2.19

1.99

0.28

*

7.81

2.00

2.65

2.32

0.46

 

15.63

2.41

2.79

2.60

0.26

*

31.25

2.61

2.64

2.62

0.02

*

62.50

2.63

2.61

2.62

0.01

*

125.00

2.64

2.52

2.58

0.09

*

250.00

2.39

2.69

2.54

0.22

*

500.00

2.53

2.55

2.54

0.01

*

1000.00

2.34

2.48

2.41

0.10

*

2000.00

2.05

2.28

2.17

0.17

*

* = significant induction according to Student’s t-test, p<0.05

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

2.59

1.40

2.00

0.84

Imax

2.64

2.79

2.71

0.10

IC30[µM]

n/a

n/a

-

-

IC50[µM]

n/a

n/a

-

-

n/a: not applicable

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

10.1

pass

9.1

pass

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

3.0

pass

2.0

pass

EC1.5 PC

7 < x < 34 µM

13.39

pass

18.27

pass

Induction PC at 64 µM

2.00 < x < 8.00

4.60

pass

3.43

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.3

3.3

41

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.3

0.6

41

EC1.5 PC

7 < x < 34 µM

20.4

6.7

41

Induction PC at 64 µM

2.00 < x < 8.00

3.3

1.1

41

Interpretation of results:
other: The data generated with this test should be considered in the context of integrated approached such as IATA
Conclusions:
In this study under the given conditions the test item did induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as sensitiser.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

In the present study CPG-3 was dissolved in tetrahydrofuran.

Based on a molecular weight of 296 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution

factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by

luminescence measurement.

In both experiment no cytotoxic effect was observable.

In the first experiment, a max luciferase activity (Imax) induction of 2.64 was determined at a test item concentration of 125 µM. The corresponding cell viability was 213.6%. The lowest tested concentration with a significant luciferase induction >1.5 (1.79) was found to be 3.91 µM. The corresponding cell viability was >70% (99.1%). The calculated EC1.5 was <1000 µM (2.59 µM).

In the second experiment, a max luciferase activity (Imax) induction of 2.79 was determined at a test item concentration of 15.63 µM. The corresponding cell viability was 146.6%. The lowest tested concentration with a significant luciferase induction >1.5 (1.81) was found to be 1.95 µM. The corresponding cell viability was >70% (113.3%). The calculated EC1.5 was < 1000 µM (1.40 µM). A dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2018-05-07 to 2018-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: “In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)” adopted 29 July 2016
Qualifier:
according to
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Details on study design:
The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test
item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely
dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in
the human monocytic cell line THP-1. The expression of the cell surface markers compared to the
respective solvent controls is used to support discrimination between skin sensitisers and
non-sensitisers.
Positive control results:
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both
experiments. The threshold of 150% for CD86 (330% experiment 1; 305% experiment 2) and
200% for CD54 (304% experiment 1; 303% experiment 2) were clearly exceeded.
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
1
Value:
210
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 833.33 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
1
Value:
106
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 694.44 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD86 [%]
Run / experiment:
2
Value:
215
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 833.33 µg/mL
Key result
Parameter:
other: relative fluorescence intensity CD54 [%]
Run / experiment:
2
Value:
119
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 694.44 µg/mL
Other effects / acceptance of results:
Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is >90%,
• the cell viability of at least four tested doses of the test item in each run is >50%,
• the RFI values of the positive control (DNCB) is ≥150% for CD86 and ≥200% for CD54 at a cell viability of >50%,
• the RFI values of the solvent control is not ≥150% for CD86 and not ≥200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is >105%.

The test mets the acceptance criteria.

Results of the Cell Batch 20 Activation Test

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

89.2

266

>150

88.4

206

>200

yes

pass

NiSO4

100 µg/mL

82.3

220

>150

80.6

283

>200

yes

pass

LA

1000 µg/mL

96.2

79

150

96.9

101

200

no

pass

 Results of the Cell Batch 21 Activation Test

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

85.5

251

>150

84.7

287

>200

yes

pass

NiSO4

100 µg/mL

74.9

249

>150

75.5

518

>200

yes

pass

LA

1000 µg/mL

94.8

67

150

95.0

108

200

no

pass

Results of the Dose Finding Assay

Sample

Experiment 1

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

92.20

Solvent Control

THF

--

93.00

Test item

C8

7.81

92.50

C7

15.63

93.50

C6

31.25

94.80

C5

62.50

93.90

C4

125.00

94.40

C3

250.00

93.50

C2

500.00

93.40

C1

1000.00

93.90

Calculated CV75 [µg/mL]

No CV75

Mean CV75 [µg/mL]

No CV75

SD CV 75 [µg/mL]

No SD

CD54 and CD86 Expression Experiment 1

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

96.4

96.0

95.8

2089

1122

641

1448

481

94

96

326

175

Solvent Control 2 (THF)

0.20%

96.3

96.0

95.8

1975.0

1166.0

609.0

1366

557

100

100

324

191

Solvent Control 1 (DMSO)

0.20%

96.3

95.4

95.9

2160

1124

621

1539

503

100

100

348

181

DNCB

4.00

84.3

83.6

84.1

5750

2207

677

5073

1530

330

304

849

326

Test item

1000

93.2

92.6

93.0

2937

1146

607

2330

539

171

97

484

189

833.33

91.6

91.2

90.8

3507

1205

641

2866

564

210

101

547

188

694.44

92.3

91.7

92.4

3327

1223

632

2695

591

197

106

526

194

578.70

91.9

92.2

90.4

3369

1177

640

2729

537

200

96

526

184

482.25

91.8

92.0

92.5

3300

1230

679

2621

551

192

99

486

181

401.88

93.1

92.1

92.4

2980

1161

621

2359

540

173

97

480

187

334.90

92.0

92.1

92.5

3158

1122

606

2552

516

187

93

521

185

279.08

93.5

94.6

94.4

2746

1128

626

2120

502

155

90

439

180

 

CD54 and CD86 Expression Experiment 2

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

95.7

95.7

95.3

2063

980

607

1456

373

93

83

340

161

Solvent Control 2 (THF)

0.20%

95.8

96.3

95.8

1801

1005

624

1177

381

100

100

289

161

Solvent Control 1 (DMSO)

0.20%

95.5

95.0

95.5

2153

1035

588

1565

447

100

100

366

176

DNCB

4.0

84.3

83.6

83.7

5409

1994

641

4768

1353

305

303

844

311

Test item

1000.00

92.00

91.30

91.30

3085

1045

675

2410

370

205

97

457

155

833.33

92.30

92.50

91.90

3192

1051

662

2530

389

215

102

482

159

694.44

92.90

93.00

93.70

2893

1065

611

2282

454

194

119

473

174

578.70

93.00

92.60

93.30

2952

1047

637

2315

410

197

108

463

164

482.25

93.10

93.40

93.50

2671

1024

712

1959

312

166

82

375

144

401.88

93.90

93.00

93.80

2730

1007

636

2094

371

178

97

429

158

334.90

92.90

92.40

92.80

2897

1004

594

2303

410

196

108

488

169

279.08

94.40

94.30

94.50

2363

1022

599

1764

423

150

111

394

171

Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

95.4

-

96.4

pass

95.0

-

96.3

pass

number of test dosed with viability >50% CD86

≥4

8

pass

8

pass

number of test dosed with viability >50% CD54

≥4

8

pass

8

pass

number of test dosed with viability >50% IgG1

≥4

8

pass

8

pass

RFI of positive control of CD86

≥150

330

pass

305

pass

RFI of positive control of CD54

≥200

304

pass

303

pass

RFI of DMSO solvent control of CD86

<150

106

pass

107

pass

RFI of DMSO solvent control of CD54

<200

105

pass

120

pass

RFI of THF solvent control of CD86

<150

94

pass

81

pass

RFI of THF solvent control of CD54

<200

116

pass

102

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

326

pass

340

pass

MFI ratio IgG1/CD86 for DMSO control [%]

>105

348

pass

366

pass

MFI ratio IgG1/CD86 for THF control [%]

>105

324

pass

289

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

175

pass

161

pass

MFI ratio IgG1/CD54 for DMSO control [%]

>105

181

pass

176

pass

MFI ratio IgG1/CD54 for THF control [%]

>105

191

pass

161

pass

Historical Data

Criterion

mean

SD

N

cell viability solvent controls [%]

97.0

1.3

672

number of test doses with viability >50%

-

-

1786

RFI of positive control of CD86

401.0

146.8

112

RFI of positive control of CD54

576.6

312.0

112

RFI of solvent control of CD86

115.0

15.1

112

RFI of solvent control of CD54

118.8

25.5

112

MFI ratio IgG1/CD86 for medium control [%]

202.4

50.0

112

MFI ratio IgG1/CD86 for DMSO control [%]

221.6

58.5

112

MFI ratio IgG1/CD54 for medium control [%]

141.0

24.7

112

MFI ratio IgG1/CD54 for DMSO control [%]

147.7

25.6

112

Interpretation of results:
other: The data generated with this test should be considered in the context of integrated approached such as IATA
Conclusions:
In this study under the given conditions the test item did upregulate the cell surface marker in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser in accordance with UN GHS category 1.
The data generated with this method may be not sufficient to conclude on skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Executive summary:

In the present study the test material was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from 500 mg/mL to 3.91 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore the main experiment was performed covering the following concentration steps:

1000; 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

The test item precipitates in all test item concentrations in the dose finding assay and in the main experiments when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 93.2% (CD86), 92.6% (CD54) and 93.0% (isotype IgG1 control) in the first experiment and to 92.0% (CD86), 91.3% (CD54) and 91.3% (isotype IgG1 control)in the second experiment.

The expression of the cell surface marker CD86 was upregulated to a maximum of 210% (experiment 1) and 215% (experiment 2). The upregulation above the threshold of 150% was observed in all concentrations (experiment 1) and at concentrations of 334.90 - 1000.00 µg/mL (experiment 2). In contrast, the expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments

Since one of the cell surface markers clearly exceeded the threshold in two independent experiments the test item considered to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the provided information there is need for classification according to the EU Regulation (EC) No 1272/2008 on Classification,Lab elling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776. The test material should be classified for skin sensitisation class 1.