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Description of key information

Skin Sensitization

REACH_not sensitizing | DPRA | OECD 442c | #key study#

REACH_not sensitizing | KeratinoSens | OECD 442d | #key study#

REACH_sensitizing | h-CLAT | OECD 442e | #key study#

REACH_sensitizing | mouse | OECD 429 | #key study##Analogy#

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:
key study
Study period:
2019-01-14 to 2019-01-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
04 February 2015
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
Version / remarks:
January 12, 2013
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
direct peptide reactivity assay (DPRA)
Justification for non-LLNA method:
In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the direct peptide reactivity assay (DPRA) showed evidence of being a reliable and relevant method to test for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use
Details on the 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.

Experimental Procedure
The test item solutions were incubated with the cysteine and lysine peptide solutions in glass vials using defined ratios of peptide to test item (1:10 cysteine peptide, 1:50 lysine peptide). The reaction solutions were left in the dark at 25 ± 2.5 °C for 24 ± 2 h before running the HPLC analysis. Reference controls, co-elution controls as well as the positive control were set up in parallel.
Test item solutions were inspected on a visual basis for the formation of precipitates, turbidity and phase separation prior and after HPLC analysis. If a precipitate or phase separation was observed after the reaction period and prior to the HPLC analysis, samples might have been centrifuged at low speed (100 - 400x g) to force precipitates to the bottom of the vial. After the incubation period of 24 ± 2 h the test item was analysed in triplicate for both peptides using the HPLC procedure.
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 67.86%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion
Value:
1.55
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value in %
Key result
Run / experiment:
other: lysine run
Parameter:
other: mean peptide depletion
Value:
0.76
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value in %
Other effects / acceptance of results:
Acceptance Criteria
Cysteine Peptide Run
- coefficient of determination R² > 0.99: 0.9999 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM: 0.5097 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM: 0.5049 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM: 0.4945 pass
- CV of the peak area of RC B < 15%: 0.81 pass
- CV of the peak area of RC C (PC) < 15%: 0.34 pass
- CV of the peak area of RC C (TI) < 15%: 1.97 pass
- mean peptide depletion of the PC 60.8% < x < 100%: 69.38 pass
- SD of peptide depletion of the PC replicates < 14.9%: 0.58 pass
- SD of peptide depletion of the TI replicates < 14.9%: 1.40 pass

Lysine Peptide Run
- coefficient of determination R² > 0.99: 1.0000 pass
- mean peptide concentration of RC A 0.45 ≤ x ≤ 0.55 mM: 0.5014 pass
- mean peptide concentration of RC C (PC) 0.45 ≤ x ≤ 0.55 mM: 0.5001 pass
- mean peptide concentration of RC C (TI) 0.45 ≤ x ≤ 0.55 mM: 0.5067 pass
- CV of the peak area of RC B < 15%: 0.84 pass
- CV of the peak area of RC C (PC) < 15%: 0.26 pass
- CV of the peak area of RC C (TI) < 15%: 0.43 pass
- mean peptide depletion of the PC 40.2% < x < 69.0%: 66.34 pass
- SD of peptide depletion of the PC replicates < 11.6%: 0.83 pass
- SD of peptide depletion of the TI replicates < 11.6%: 1.20 pass

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

In the present study the test item was dissolved in isopropanol, based on the results of the pre-experiments. Based on a molecular weight of 395.66 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.

All test item solutions were freshly prepared immediately prior to use. For the 100 mM stock solution of the test item turbidity 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. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item turbidity 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. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged 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 not relevant.

No co-elution of the test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cisopropanol).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was <= 6.38% (1.15%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

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 67.86%.

The controls confirmed the validity of the study for both, the cysteine and lysine run.

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

16.4390

0.5340

14.6340

0.5340

STD2

8.2610

0.2670

7.3130

0.2670

STD3

4.0740

0.1335

3.6450

0.1335

STD4

1.9450

0.0667

1.8080

0.0667

STD5

0.9340

0.0334

0.9000

0.0334

STD6

0.4240

0.0167

0.4440

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

4.7360

0.1551

69.56

69.38

0.58

0.84

4.8660

0.1593

68.73

4.6910

0.1537

69.85

Test Item

14.8270

0.4812

2.72

1.55

1.40

90.32

15.4360

0.5008

0.00

14.9480

0.4851

1.92

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

4.4950

0.1644

67.20

66.34

0.83

1.25

4.6220

0.1690

66.27

4.7210

0.1726

65.55

Test Item

13.9810

0.5102

0.00

0.76

1.20

158.59

13.8660

0.5060

0.13

13.5870

0.4959

2.14

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 Prediction²

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

1.15

Minimal Reactivity

negative

1.55

Minimal Reactivity

negative

Positive Control

67.86

High Reactivity

positive

69.38

Moderate Reactivity

positive
Interpretation of results:
GHS criteria not met
Remarks:
DPRA
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.
The data generated with this method may be not sufficient to conclude on the absence of 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 item was dissolved in isopropanol, based on the results of the pre-experiments. Based on a molecular weight of 395.66 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.

All test item solutions were freshly prepared immediately prior to use.

For the 100 mM stock solution of the test item turbidity 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. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item turbidity 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. No precipitation, turbidity or phase separation was observed for the samples of the test item. Phase separation was observed for the samples of the positive control (including the co-elution control). Samples were not centrifuged 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 not relevant.

No co-elution of the test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cisopropanol).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was <= 6.38% (1.15%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

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 67.86%.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-01-23 to 2019-03-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
25 June 2018
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155
Version / remarks:
09 March 2018
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the KeratinoSens™ assay showed evidence of being a reliable and relevant method to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use.
Details on the 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.

CELL LINE
The test was carried out using the transgenic cell line KeratinoSens™ (Givaudan, Switzerland), a cell line derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and were used for routine testing. Only cells at a low passage number <25 (P 02 in experiment 1; P 04 in experiment 2) were used.
Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 ± 1°C and 5% CO2 in a humidified incubator. For test item exposure, cells were cultured in medium for test item exposure.
Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.59 (experiment 1); 4.74 (experiment 2)).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
1.23
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value = fold induction; Concentration: 0.98 µM
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability
Value:
124.5
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value in %; Concentration: 0.98 µM
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
1.18
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value = fold induction; Concentration: 1.95 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability
Value:
92.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: value in %; Concentration: 1.95 µM
Other effects / acceptance of results:
The controls confirmed the validity of the study.

In the first experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

In the second experiment, no significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated.

No 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 non-sensitiser.

Results of the Cytotoxicity Measurement

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Mean

SD

Solvent / Neg. Control

-

100

100

100

0.0

Positive Control

4.00

94.8

116.6

105.7

15.4

8.00

103.7

122.0

112.9

12.9

16.00

101.1

112.0

106.6

7.7

32.00

88.2

129.8

109.0

29.4

64.00

107.5

126.0

116.7

13.1

Test Item

0.98

124.5

75.5

100.0

34.6

1.95

100.3

92.9

96.6

5.2

3.91

106.0

105.1

105.5

0.6

7.81

96.7

105.4

101.1

6.1

15.63

111.1

144.4

127.8

23.6

31.25

105.1

130.5

117.8

18.0

62.50

107.8

125.2

116.5

12.3

125.00

102.7

151.2

126.9

34.3

250.00

97.5

125.7

111.6

19.9

500.00

98.0

106.2

102.1

5.8

1000.00

99.4

97.5

98.5

1.3

2000.00

111.1

101.3

106.2

6.9

Induction of Luciferase Activity Experiment 1

Experiment 1

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent / Neg. Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.14

1.21

1.16

1.17

0.04

 

8.00

1.21

1.22

1.31

1.25

0.05

 

16.00

1.41

1.58

1.71

1.57

0.15

*

32.00

2.29

2.21

2.17

2.23

0.06

*

64.00

4.24

4.60

4.91

4.59

0.34

*

Test Item

0.98

1.18

1.30

1.23

1.23

0.06

 

1.95

1.10

1.16

1.06

1.11

0.05

 

3.91

1.03

1.11

1.13

1.09

0.05

 

7.81

1.08

1.10

1.11

1.10

0.02

 

15.63

1.13

1.28

1.21

1.21

0.07

 

31.25

1.21

1.24

1.21

1.22

0.02

 

62.50

1.08

1.17

1.21

1.15

0.07

 

125.00

1.08

1.20

1.08

1.12

0.07

 

250.00

1.11

1.09

1.14

1.11

0.03

 

500.00

1.11

1.13

1.01

1.08

0.06

 

1000.00

1.07

1.14

0.92

1.04

0.11

 

2000.00

1.11

1.19

1.06

1.12

0.07

 

Induction of Luciferase Activity Experiment 2

Experiment 2

Concentration [µM]

Fold Induction

Significance

Rep. 1

Rep. 2

Rep. 3

Mean

SD

Solvent / Neg. Control

-

1.00

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.10

1.27

1.22

1.19

0.09

 

8.00

1.20

1.49

1.07

1.25

0.22

 

16.00

1.60

1.88

1.39

1.62

0.25

*

32.00

2.02

2.76

1.64

2.14

0.57

*

64.00

3.72

6.81

3.68

4.74

1.80

*

Test Item

0.98

1.31

1.15

1.06

1.17

0.13

 

1.95

1.09

1.41

1.04

1.18

0.20

 

3.91

1.12

1.09

1.01

1.08

0.05

 

7.81

1.09

1.05

1.02

1.05

0.04

 

15.63

1.18

1.05

1.14

1.12

0.07

 

31.25

1.05

1.02

0.97

1.02

0.04

 

62.50

1.07

0.95

0.93

0.99

0.07

 

125.00

1.07

0.99

1.05

1.04

0.04

 

250.00

1.06

0.96

1.02

1.02

0.05

 

500.00

1.02

1.02

1.02

1.02

0.00

 

1000.00

1.09

1.10

0.92

1.03

0.10

 

2000.00

1.07

1.21

1.04

1.10

0.09

 

Induction of Luciferase Activity – Overall Induction

Concentration [µM]

Fold Induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent / Neg. Control

-

1.00

1.00

1.00

0.00

 

Positive Control

4.00

1.17

1.19

1.18

0.02

 

8.00

1.25

1.25

1.25

0.00

 

16.00

1.57

1.62

1.60

0.04

*

32.00

2.23

2.14

2.18

0.06

*

64.00

4.59

4.74

4.66

0.11

*

Test Item

0.98

1.23

1.17

1.20

0.04

 

1.95

1.11

1.18

1.14

0.05

 

3.91

1.09

1.08

1.08

0.01

 

7.81

1.10

1.05

1.08

0.03

 

15.63

1.21

1.12

1.16

0.06

 

31.25

1.22

1.02

1.12

0.15

 

62.50

1.15

0.99

1.07

0.12

 

125.00

1.12

1.04

1.08

0.06

 

250.00

1.11

1.02

1.07

0.07

 

500.00

1.08

1.02

1.05

0.04

 

1000.00

1.04

1.03

1.04

0.01

 

2000.00

1.12

1.10

1.11

0.01

 

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n.a.

n.a.

n.a.

n.a.

Imax

1.23

1.18

1.21

0.04

IC30[µM]

n.a.

n.a.

n.a.

n.a.

IC50[µM]

n.a.

n.a.

n.a.

n.a.

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Negative Control

< 20%

8.2

pass

15.5

pass

CV Solvent Control

<20%

10.2

pass

8.3

pass

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

≥ 1

3.0

pass

3.0

pass

EC1.5 PC

7 < x < 34 µM

14.31

pass

13.32

pass

Induction PC at 64 µM

2.00 < x < 8.00

4.59

pass

4.74

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Negative Control

< 20%

11.6

3.5

96

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

≥ 1

2.4

0.6

96

EC1.5 PC

7 < x < 34 µM

18.5

6.0

96

Induction PC at 64 µM

2.00 < x < 8.00

3.8

1.5

96
Interpretation of results:
GHS criteria not met
Remarks:
KeratinoSens
Conclusions:
In this study under the given conditions the test item did not 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 non-sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of 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 item was dissolved in THF. Based on a molecular weight of 395.66 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. 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 the first experiment, nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated.

In the second experiment,nosignificant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5value could be calculated.

No 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 non-sensitiser.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2019-01-23 to 2019-03-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”
Version / remarks:
25 June 2018
Qualifier:
according to guideline
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158
Version / remarks:
01 July 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
In order to replace in vivo experiments validation studies on alternative, mechanistically based in chemico and in vitro test methods on skin sensitisation were conducted under the auspices of ECVAM and have been considered scientifically valid for the evaluation of the skin sensitisation hazard of chemicals. It was concluded that the human cell line activation test (h-CLAT) showed evidence of being a reliable and relevant method to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling for skin sensitisation testing. However, only combinations of several non-animal testing methods within an Integrated Approach to Testing and Assessment (IATA) will be able to fully substitute for the animal test currently in use
Details on the 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.

CELL LINE
The test was carried out using THP-1 cells (ATCC® TIB-202TM), an acute human monocytic leukemic cell line used as a surrogate for DC. Cells from frozen stock cultures, tested routinely for mycoplasma, were seeded in culture medium at an appropriate density and subcultured at least 2 weeks before they were used in the in vitro h-CLAT test. Cells at passage number (<30) were used. Cells are routinely passaged every 2-3 days at a density of 0.1 – 0.2 x 10E6 cells/mL. Cells were cultured in 175 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640, Gibco Life Science; Cat. No.: 31870-025) supplemented with 10% fetal bovine serum, 25 mM HEPES, L-glutamine, 0.05 mM 2-mercaptoethanol and 100 U/ml penicillin/ 100 μg/mL streptomycin at 37 ± 1°C and 5% CO2.
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 (351% experiment 1; 364% experiment 2) and 200% for CD54 (301% experiment 1; 401% experiment 2) were clearly exceeded.
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
160
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value in %; Concentration: 1000 µg/mL Cytotoxicity: 95.9%
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
129
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value in %; Concentration: 279.08 µg/mL Cytotoxicity: 95.3%
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
377
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value in %; Concentration: 401.88 µg/mL Cytotoxicity: 96.10%
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
625
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Value in %; Concentration: 1000 µg/mL Cytotoxicity: 95.40%
Other effects / acceptance of results:
The controls confirmed the validity of the study for all experiments.

In the main experiments precipitation of the test item was observed for all concentration steps 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 95.9% (CD86), 96.1% (CD54) and 96.5% (isotype IgG1 control) in the first experiment and 97.3% (CD86), 95.4% (CD54) and 94.6% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 160% (1000 μM) in the first experiment and up to 377% (401.88 μM) in the second experiment. The expression of the cell surface marker CD54 was upregulated up to 625% (1000 μM) only in the second experiment.

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

Results of the Dose Finding Assay

Sample

Experiment 1

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

96.20

Solvent Control (THF)

THF

--

95.70

dialkyl-methyldihydro-heteropolycycle

C8

7.81

96.30

C7

15.63

96.00

C6

31.25

95.80

C5

62.50

95.90

C4

125.00

95.80

C3

250.00

95.70

C2

500.00

96.20

C1

1000.00

95.50

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 Fluorescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

96.2

95.9

95.6

1456

924

590

866

334

81

80

247

157

Solvent Control 2(THF)

0.20%

96.0

95.7

96.1

1371.0

918.0

637.0

734

281

100

100

215

144

Solvent Control 1 (DMSO)

0.20%

94.1

95.3

94.8

1639

983

565

1074

418

100

100

290

174

DNCB

4.00

89.9

90.1

89.1

4449

1938

680

3769

1258

351

301

654

285

 

dialkyl-methyldihydro-heteropolycycle

1000

95.9

96.1

96.5

1817

902

639

1178

263

160

94

284

141

833.33

95.9

96.2

96.3

1298

812

603

695

209

95

74

215

135

694.44

95.9

96.0

96.0

1443

854

624

819

230

112

82

231

137

578.70

97.1

96.7

96.9

1368

859

613

755

246

103

88

223

140

482.25

96.3

96.0

95.7

1427

832

606

821

226

112

80

235

137

401.88

96.5

96.6

96.5

1283

839

604

679

235

93

84

212

139

334.90

96.3

96.0

95.9

1305

819

602

703

217

96

77

217

136

279.08

95.8

95.3

95.9

1625

1018

655

970

363

132

129

248

155

CD54 and CD86 Expression Experiment 2

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Fluorescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium Control

-

96.5

97.0

97.4

1614

838

619

995

219

109

127

261

135

Solvent Control 2 (THF)

0.20%

96.1

97.0

97.1

1495

816

630

865

186

100

100

237

130

Solvent Control 1 (DMSO)

0.20%

97.6

97.1

97.6

1568

826

654

914

172

100

100

240

126

DNCB

4.0

88.7

88.9

88.1

4012

1377

688

3324

689

364

401

583

200

 

dialkyl-methyldihydro-heteropolycycle

1000.00

97.30

95.40

94.60

2352

1913

751

1601

1162

185

625

313

255

833.33

97.00

97.20

97.10

1725

878

637

1088

241

126

130

271

138

694.44

97.60

97.60

96.90

1570

855

626

944

229

109

123

251

137

578.70

97.10

96.80

97.10

1571

856

613

958

243

111

131

256

140

482.25

97.30

97.40

96.50

1715

848

678

1037

170

120

91

253

125

401.88

96.10

96.90

96.70

3903

1174

645

3258

529

377

284

605

182

334.90

97.10

97.00

96.70

3672

889

633

3039

256

351

138

580

140

279.08

96.70

97.30

96.60

1776

852

619

1157

233

134

125

287

138
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
hCLAT
Conclusions:
In this study under the given conditions the test item did upregulate the cell surface markers in at least two independent experiment runs. Therefore, the test item is considered to be a skin sensitiser.
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 item 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

In the main experiments precipitation of the test item was observed for all concentration steps 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 95.9% (CD86), 96.1% (CD54) and 96.5% (isotype IgG1 control) in the first experiment and 97.3% (CD86), 95.4% (CD54) and 94.6% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was upregulated to 160% (1000 µM) in the first experiment and up to 377% (401.88 µM) in the second experiment. The expression of the cell surface marker CD54 was upregulated up to 625% (1000 µM) only in the second experiment.

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

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
Aug - Sept 2009
Reliability:
1 (reliable without restriction)
Reason / purpose for cross-reference:
read-across source
Key result
Parameter:
EC3
Value:
34.4
Parameter:
SI
Remarks:
25%
Value:
2.11
Parameter:
SI
Remarks:
50%
Value:
4.47
Parameter:
SI
Remarks:
100%
Value:
4.8
Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The analogue test item 2-Undecanal-bi-oxazolidine Sa 190 was found to be a skin sensitiser under the described conditions.
According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures, the test item should be classified as skin sensitiser (Category 1B).
According to the ECETOC classification scheme for potency (ECETOC Technical Report No. 87, Contact sensitization: Classification according to potency, April 2003, Brussels), the test item would be regarded as weak sensitiser.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The three in vitro tests DPRA, KeratinoSens and h-CLAT were performed according to OECD principles (OECD 442c,d,e). Two tests (DPRA, KeratinoSens) resulted that the substance is not a skin sensitiser, the third test (h-CLAT) considered the substance to be a skin sensitiser. With the 2 out of 3 approach and considering the results of the OECD QSAR toolbox (V 4.4), which did not find any alerts for skin sensitization, it could be believed that, the substance might not be a skin sensitizer. But due to its physchem properties (low solubility in water, high log Pow) the test substance is hard to test in vitro. Furthermore, the substance can hydrolize, forming an aldehyde, which is a skin sensitizer. Thus, the results of the three in vitro tests are not considered reliable. That is why a read-across approach was considered using the very similar substance Sa 190. For this substance an LLNA was performed (OECD 429) resulting that Sa 190 was a weak sensitizer. Since both substances are very similar it is considered that dialkyl-methyldihydro-heteropolycycle is a weak skin sensitizer as well.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin Sensitization:

Based on a read accross to the analogue substance Sa 190 the test item is considered to be a weak skin sensitiser.

According to the Regulation (EC) No 1272/2008 on classification, labeling and packaging (CLP) of substances and mixtures, the test item should be classified as a weak skin sensitiser (Category 1B).