Registration Dossier

Administrative data

Description of key information

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In vitro study in accordance with Testing and assessment strategy for evaluating the skin sensitisation potential of substances of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Test material information:
Composition 1
Specific details on test material used for the study:
Name: 9-Octadecenoic acid (9Z)-, propyl ester
Product Description: Propyl oleate
CAS No.: 111-59-1
Physical state: pale yellow to yellow liquid at 20 °C
Batch No.: 37584
Re-certification date of batch: 08 March 2018
Purity: > 99 % (fatty acid propyl esters, max water content 0.3 % (w/w))
Free Fatty Acid, % Oleic: 0.1
Color, Gardner: 3
Iodine Value, cg I2/g 75
Cloud Point, degrees C 0
Saponification Number, mg KOH/g 177
Moisture, % 0,07
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light
Details on study design:
The KeratinoSens assay is supposed to address the second key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the induction of cyto-protective signalling pathways in keratinocytes in response to electrophiles and oxidative stress. The KeratinoSens assay addresses the effect on the antioxidant response element (ARE)-dependent pathway in the KeratinoSens cell line by measuring the induction of an ARE dependent gene product, the luciferase gene. The luciferase gene induction following exposure to test chemicals is measured in cell lysates by luminescence detection, allowing the discrimination between sensitisers and non-sensitisers.

Preparation of the Test Item

All test item solutions were freshly prepared immediately prior to use.
The test item was dissolved in tetrahydrofuran (THF; CAS No.: 109-99-9). A stock solution of 200 mM was prepared by pre-weighing the test material into a glass vial.
These 4x concentrated test item solutions were finally diluted 1:4 when incubated with the cells. Based on this procedure the final concentration of the solvent was 1% (v/v) in all test item concentrations and controls.

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 9 experiment 1; P 3 experiment 2) were used. Cells were cultured in 75 cm2 culture flasks (Greiner) in maintenance medium at 37 +- 1°C and 5% CO2. For test material exposure, cells were cultured in medium.

Composition of Media

Maintenance Medium

Dulbecco’s Modified Eagle Medium with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum
- 1% geneticin (final concentration: 500 µg/mL)

Assay Medium
Dulbecco’s Modified Eagle Medium with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 10% fetal bovine calf serum

Test Item Exposure Medium
Dulbecco’s Modified Eagle Medium with 1.0 g/L D-glucose and Na-Pyruvate. The medium was supplemented with the following components:
- 1% fetal bovine calf serum

Experimental Procedure

A cell suspension of 8 × 10^4 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1 × 10^4 cells were dispensed in each well, except for the blank. To determine the luciferase activity cells were seeded in white 96-well plates (flat bottom). In parallel cells were seeded in a transparent 96-well plate (flat bottom) for the determination of the cell viability. After seeding cells were grown for 24 h ± 1 h in assay medium at 37 °C ± 1 °C and 5% CO2. Thereafter, the assay medium was discarded and replaced by 150 µL test item exposure medium. 50 µL of the shortly before prepared 4x master concentrations were transferred to the luciferase and cell viability plates, resulting in an additional 1:4 dilution of the test item. All plates were sealed using a sealing tape to avoid evaporation of volatile compounds and cross-contamination between wells by the test items. Treated plates were incubated for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.

Luciferase activity

After 48 h ± 1 h of exposure, the supernatant was aspirated from the white assay plates and discarded. Cells were washed once with DPBS. Subsequently 20 µL of passive lysis buffer were added into each well and the plate was incubated for 20 min at room temperature in the absence of light. Plates with the cell lysate were placed in the plate reader for luminescence measurement. Per well 50 µL of the luciferase substrate were injected by the injector of the plate reader. The plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms. This procedure was repeated for each individual well.

Cell viability
For the cell viability plate the medium was replaced with 200 µL test item exposure medium. 27 µL MTT solution were added directly to each individual well. The plate was covered with a sealing tape and incubated for 4 h at 37 °C ± 1 °C and 5% CO2. Afterwards the medium was removed and replaced by 200 µL 10% SDS solution per well. The plate was covered with sealing tape and incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.

Calculation of EC1.5, IC50 and IC30, Cell Viability and Maximal Induction of the Luciferase Activity was performed according to OECD 442 d guideline.


Prediction Model

The test item is considered positive in accordance with UN GHS “Category 1” if the following conditions were met in at least two independently prepared test repetitions:

- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 µM
- an apparent overall dose-response for luciferase induction

If in a given repetition, all of the three first conditions are met but a clear dose-response for the luciferase induction cannot be observed, the result of that repetition is considered as inconclusive and further testing may be required. In addition, a negative result obtained with concentrations <1000 µM is considered as inconclusive.

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.


Positive control results:
Controls
A blank, a negative control and a positive control were set up in parallel in order to confirm the validity of the test.

Blank
A blank well with no seeded cells was included in every plate to determine the background. The well was incubated with the negative control.

Negative Control
DMSO at a final concentration of 1% (v/v) in test item exposure medium was used as negative control. Six wells were included in every testing plate. The preparation of the negative control was carried out analogous to the test item.

Positive Control
Cinnamic aldehyde (CA, (2E)-3-phenylprop-2-enal; CAS 104-55-2; >98%) was used as positive control. CA was dissolved in DMSO at a concentration of 6.4 mM and was further diluted four times with a constant dilution factor of 1:2 resulting in a concentration range of 0.4 mM – 6.4 mM. The following preparation of the positive control was carried out analogous to the preparation of the test item, resulting in a final concentration range of 4 µM – 64 µM. The final concentration of the solvent DMSO was 1% (v/v) for all wells.
Key result
Parameter:
other: EC1.5
Run / experiment:
1
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Key result
Parameter:
other: EC1.5
Run / experiment:
2
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Key result
Parameter:
other: Imax
Run / experiment:
1
Value:
1.3
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Imax
Run / experiment:
2
Value:
2.09
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Imax
Run / experiment:
mean of experiment 1 and 2
Value:
1.69
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
no indication of skin sensitisation
Parameter:
other: IC30
Remarks:
[µM]
Run / experiment:
Experiment 1
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Parameter:
other: IC30
Remarks:
[µM]
Run / experiment:
Experiment 2
Value:
916.17
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Parameter:
other: IC50
Remarks:
[µM]
Run / experiment:
Experiment 1 & 2
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Parameter:
other: EC1.5 PC
Run / experiment:
Positive control 1
Value:
13.46
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation
Parameter:
other: EC1.5 PC
Run / experiment:
Positive control 2
Value:
12.77
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks on result:
positive indication of skin sensitisation

Results

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. In the present study Propyl oleate was dissolved in THF. Based on a molecular weight of 324.5 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, 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, a max luciferase activity (Imax) induction of 2.09 was determined at a test item concentration of 3.91 µM. The corresponding cell viability was 94%. Within the tested concentration range two concentrations with a significant luciferase induction were observed (3.91 µM and 31.25 µM). Since no dose response for luciferase activity induction was observable and no significant induction was calculated at the higher test item concentrations, these two values were regarded as outlier. 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. The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (6.10 in experiment 1; 5.16 in experiment 2). The calculated EC1.5 was between 7 and 34 µM (13.46 µM in experiment 1; 12.77 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (19.4% in experiment 1; 14.5% in experiment 2).

Table 1: Results of the cytotoxicity measurement

   Concentration [µM] Cell Viability [%]
     Experiment 1  Experiment 2  Mean  SD
 Solvent control

 -

 100  100  100  -
 Positive control 

4.00

8.00

16.00

32.00

64.00

109.5

114.1

119.1

120.7

111.1

106.5

114.3

119.3

117.4

123.8

108.0

114.2

119.2

119.0

117.5

2.1

0.2

0.2

2.3

9.0

 Test item

0.98

1.95

3.91

7.81

15.63

31.25

62.50

125.00

250.00

500.00

1000.00

2000.00

84.4

92.4

101.9

111.2

120.6

117.0

116.2

116.7

112.0

115.8

116.5

104.9

86.7

73.0

94.0

81.1

84.3

91.2

70.4

76.8

74.3

77.8

69.3

67.9

85.6

82.7

97.9

96.1

102.4

104.1

93.3

96.7

93.2

96.8

92.9

86.4

1.6

13.8

5.5

21.3

25.7

18.3

32.4

28.2

26.7

26.9

33.3

26.2

Table 2: 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

8.00

16.00

32.00

64.00

0.86

1.08

1.64

2.18

6.52

1.13

1.21

1.35

1.68

4.64

1.26

1.27

1.95

2.49

7.13

1.08

1.18

1.65

2.12

6.10

0.20

0.10

0.30

0.41

1.30

*

*

 Test item

0.98

1.95

3.91

7.81

15.63

31.25

62.50

125.00

250.00

500.00

1000.00

2000.00

0.72

0.60

0.95

0.88

0.79

0.93

1.02

0.91

1.08

1.07

1.11

1.25

1.51

1.48

1.43

1.14

1.60

1.24

1.40

1.17

1.25

1.11

1.13

1.41

1.00

1.15

1.05

1.19

1.33

1.15

1.33

1.26

1.09

1.29

1.25

1.24

1.08

1.08

1.14

1.07

1.24

1.11

1.25

1.12

1.14

1.15

1.16

1.30

0.40

0.44

0.25

0.17

0.41

0.16

0.20

0.18

0.09

0.12

0.08

0.10

 

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

Table 3: 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

8.00

16.00

32.00

64.00

1.33

1.28

1.43

2.86

4.39

1.22

1.64

1.67

2.42

6.08

1.10

1.36

1.55

3.95

5.00

1.22

1.43

1.55

3.08

5.16

0.12

0.19

0.12

0.79

0.86

*

*

 Test item

0.98

1.95

3.91

7.81

15.63

31.25

62.50

125.00

250.00

500.00

1000.00

2000.00

1.20

1.24

2.07

1.26

1.39

1.84

1.35

1.24

1.43

1.07

0.97

1.08

1.03

1.21

2.48

1.22

0.97

2.02

1.32

1.43

1.33

1.01

0.74

1.30

0.90

1.19

1.72

1.15

1.06

1.87

1.39

1.36

1.02

1.06

0.79

0.97

1.04

1.21

2.09

1.21

1.14

1.91

1.35

1.34

1.26

1.05

0.83

1.11

0.15

0.03

0.38

0.05

0.22

0.10

0.04

0.10

0.22

0.03

0.12

0.17

*

*

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

Table 4: 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

8.00

16.00

32.00

64.00

1.08

1.18

1.65

2.12

6.10

1.22

1.43

1.55

3.08

5.16

1.15

1.31

1.60

2.60

5.63

0.10

0.17

0.07

0.68

0.66

 

*

*

 Test item

0.98

1.95

3.91

7.81

15.63

31.25

62.50

125.00

250.00

500.00

1000.00

2000.00

1.08

1.08

1.14

1.07

1.24

1.11

1.25

1.12

1.14

1.15

1.16

1.30

1.04

1.21

2.09

1.21

1.14

1.91

1.35

1.34

1.26

1.05

0.83

1.11

1.06

1.15

1.62

1.14

1.19

1.51

1.30

1.23

1.20

1.10

1.00

1.21

0.03

0.10

0.67

0.10

0.07

0.57

0.07

0.16

0.09

0.08

0.23

0.13

 

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.

Interpretation of results:
GHS criteria not met
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 this KeratinoSenss tudy 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.

Conclusion:

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
In vitro study in accordance with testing and assessment strategy for evaluating the skin sensitisation potential of substances of Chapter R.7a: Endpoint specific guidance Version 6.0 – July 2017.
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: OECD 442E guideline: human cell line activation test (h-CLAT)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dentritic cells
Test material information:
Composition 1
Specific details on test material used for the study:
Name: 9-Octadecenoic acid (9Z)-, propyl ester
Product Description: Propyl oleate
CAS No.: 111-59-1
Physical state: pale yellow to yellow liquid at 20 °C
Batch No.: 37584
Re-certification date of batch: 08 March 2018
Purity: > 99 % (fatty acid propyl esters, max water content 0.3 % (w/w))
Free Fatty Acid, % Oleic: 0.1
Color, Gardner: 3
Iodine Value, cg I2/g 75
Cloud Point, degrees C 0
Saponification Number, mg KOH/g 177
Moisture, % 0,07
Stability: stable under test conditions
Storage condition of test material: Room temperature, protected from light
Details on study design:
Test method

This in vitro method is designed to predict and classify the skin sensitising potential of a chemical or multi-constituent substances or mixtures by assessment of its potential to upregulate cell surface markers using fluorescence-activated cell sorting (FACS).

Preparation of the Test Item

The test item was freshly prepared immediately prior to use. The test item was soluble in Tetrahydrofuran (THF) at a concentration of 500 mg/mL. Vortex mixing was used to aid solubilisation. Stock solutions were prepared by diluting the highest soluble concentration seven times with a constant dilution factor of 1:2. The working stock solutions were prepared by diluting each stock solution 250 times with cell culture medium. The working stock solutions were applied to the cells by adding equal volumes of each solution to prepared cells, resulting in a further 1:2 dilution of the working solutions. The solvent was present at a constant volume ratio of 0.2% (v/v) in all cultures, i.e. in all concentrations of the test item and the solvent control.

Controls

A medium control, a solvent control, and a positive control were set up in parallel in order to confirm the validity of the test.

Medium Control

A medium control was included in the test.

Solvent Controls

Solvent controls were included in the test. The solvent controls were set up depending on the appropriate solvent previously determined. Since the test item was solubilized in THF, a THF control was included and served as solvent control for the test item.

Positive Control
2,4-dinitrochlorobenzene (DNCB) at a final concentration of 4 µg/mL (alternatively at the concentration of the CV75) was tested concurrently with the test item. DNCB was dissolved in DMSO and diluted according to the procedure described for the test item, resulting in a final DMSO concentration of 0.2% (v/v).

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 10^6 cells/mL. Cells were cultured in 75 cm2 culture flasks (Greiner) in Roswell Park Memorial Institute medium (RPMI-1640) 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.

Reactivity Check of the Cells Stock

Prior to testing, the quality of freshly thawed cell batch was checked by monitoring the doubling time and checking the reactivity towards positive controls. For the reactivity check of the cell batch additional negative and positive controls were included. DNCB at a final concentration of 4 µg/mL and nickel sulphate at a final concentration of 100 µg/mL served as positive control while lactic acid at a final concentration of 1000 µg/mL served as negative control. Cells were accepted when both, DNCB and nickel sulphate produce a positive response for CD86 and CD54, and lactic acid produces a negative response for CD86 and CD54.

Solvent Finding

Solubility of the test item was determined prior to the dose finding assay. The test item was dissolved in 0.9% NaCl at a final concentration of 100 mg/mL. Test items not soluble in 0.9% NaCl solution were dissolved in DMSO at a concentration of 500 mg/mL. If the test item was not soluble in DMSO, other solvents (e.g. THF) were used. It was taken care that the test chemical is dissolved or stably dispersed in the chosen solvent and that it does not interfere with the test design. If the test item was not soluble in DMSO or a different organic solvent at 500 mg/mL, the highest soluble concentration was tested by diluting the solution from 500 mg/mL with a constant factor of 1:2 up to a minimal concentration of 1 mg/mL.

Dose Finding Assay

Starting from 500 mg/mL solutions of the test chemicals, eight stock solutions (eight concentrations) were prepared, by 2-fold serial dilutions using the corresponding solvent. These stock solutions were further diluted 250-fold into culture medium (working solutions). The working solutions were finally used for treatment by adding an equal volume of working solution to the volume of THP-1 cell suspension in a 96-well plate to achieve a further 2-fold dilution. For testing, THP-1 cells were pre-cultured for at least 48 h in culture flasks at a cell density of 0.1 – 0.2 x 10^6 cells/mL. Prior to test item application, cells were harvested from the cell culture flask by centrifugation and were re-suspended in fresh culture medium at a density of 2 x 10^6 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1 x 10^6 cells/well).
The solvent controls, the positive control and the working solutions were mixed 1:1 (v/v) with the cell suspensions prepared in the 24-well plate. Treated plates were incubated for 24 h ± 0.5 h at 37 °C ± 1 °C and 5% CO2. After 24 h ± 0.5 h of exposure, cells were transferred into sample tubes and collected by centrifugation (approx. 250 x g). The supernatant was discarded and the remaining cells were washed twice with Dulbecco’s phosphate buffered saline (DPBS) containing 0.1% bovine serum albumin (BSA; i.e. FACS buffer). After washing, cells were re-suspended in 600 µL FACS buffer. 200 µL of the cell suspension were transferred into a FACS tube and stained by using propidium iodide (PI) solution at a final concentration of 0.625 µg/mL. The PI uptake of the cells and therefore cytotoxicity was analysed immediately after the staining procedure by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ > 650 nm. A total of 10,000 living (PI negative) cells were acquired and cell viability was calculated for each test concentration. The CV75 value was used to calculate the concentration range of the test item for the main experiment.




Positive control results:
The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86. The cell batches were accepted for further testing.
Key result
Parameter:
other: Relative Flourescence Intensity (RFI)
Run / experiment:
Experiment 1 - CD86 - 1000 µg/ml
Value:
101
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
DNCB
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Relative Flourescence Intensity (RFI)
Run / experiment:
Experiment 1 - CD54 - 1000 µg/ml
Value:
120
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
DNCB
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Relative Flourescence Intensity (RFI)
Run / experiment:
Experiment 2 - CD86 - 1000 µg/ml
Value:
82
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
DNCB
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Relative Flourescence Intensity (RFI)
Run / experiment:
Experiment 2 - CD54 - 1000 µg/ml
Value:
116
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Remarks:
DNCB
Remarks on result:
no indication of skin sensitisation
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%.

Results

Dose Finding Assay

The dose finding assay was performed using stock solutions with a concentration of 1000 µg/mL.

Table 1:  Results of the Dose Finding Assay

 Sample  Experiment 1     Experiment 2   
   Concentration applied [µg/ml] Cell viability [%] Concentration applied [µg/ml]  Cell viability [%]
 Medium control  0.00 96.50 0.00 93.80
 Solvent control  0.00 95.70 0.00 93.40
 Test item

 7.81

15.63

31.25

62.50

125.00

250.00

500.00

1000.00

92.70

81.60

56.30

31.60

96.30

96.40

96.50

95.60

 7.81

15.63

31.25

62.50

125.00

250.00

500.00

1000.00

94.90

94.50

94.60

94.50

94.80

94.70

95.30

94.80

 Calculated CV75 [µg/mL] No CV75       No CV75       
 Mean CV75 [µg/mL] No CV75 
 SD CV75 [µg/mL] No CV75 

Results CD54 and CD86 Expression

For determination of the cell surface markers CD54 and CD86 two independent experiments were performed using separate cultivated cells at passage 14 (first experiment) and 17 (second experiment). For each experiment separately weighted samples and preparations were used.

Discussion

n 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 sensitiser and non-sensitisers. Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing. In the present study Propyl oleate was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from (500.00 mg/mL to 139.54 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

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 95.5% (CD86), 94.7% (CD54) and 94.5% (isotype IgG1 control) in the first experiment and to 95.6% (CD86), 95.9% (CD54) and 96.3% (isotype IgG1 control) in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is considered to be no skin sensitiser.The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (277% experiment 1; 322% experiment 2) and 200% for CD54 (309% experiment 1; 372% experiment 2) were clearly exceeded. The controls confirmed the validity of the study. The viability of the solvent control was > 90% (90.9-94.9% experiment 2; 93.4-96.3% experiment 2). The number of tested test item concentrations with cell viability > 50% was ≥ 4 (8 experiments 1 and 2). The RFI for CD86 and CD54 of cells treated with the solvent DMSO was ≤ 150% (125% experiment 1; 97% experiment 2) and ≤ 200% (122% experiment 1; 109% experiment 2). The MFI ratio of the medium control and isotype IgG1control was ≥ 105% for CD86 (228% experiment 1; 227% experiment 2) and CD54 (152% experiment 1; 163% experiment 2). The MFI ratio of the solvent control (DMSO) and isotype IgG1 control was ≥ 105% for CD86 (275% experiment 1; 224% experiment 2) and CD54 (169% experiment 1; 169% experiment 2). 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.

Table 2: 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    93.1  90.9  91.7  1634  1087  717  917  370  80  82  228

 152

Solvent Control

 0.2

 94.9

 94.0

 93.6

 1663

 1022

 711

 952

 311

 100

 100

 234

 144

 DNCB (positive control)

 4.0

 74.2

 72.8

 71.2

 3803

 2083

 636

 3167

 1402

 277

 309

 598

 320

 Test item

1000

833.33

694.44

578.70

482.25

401.88

334.90

279.08

95.5

93.8

94.4

93.5

93.1

90.9

91.4

86.8

94.7

92.6

93.8

93.1

93.7

91.0

91.4

88.4

94.5

92.9

94.1

92.4

93.2

89.8

89.6

88.2

1526

1559

1571

1556

1523

1368

1579

1576

939

927

932

1007

934

932

985

1093

567

546

600

576

567

587

582

769

959

1013

971

980

956

781

997

807

372

381

332

431

367

345

403

324

101

106

102

103

100

82

105

85

120

123

107

139

118

111

130

104

269

286

262

270

269

233

271

205

166

170

155

175

165

159

169

142

Table 3: CD54 and CD86 Expression Experiment 2

 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

 

 95.9

 95.6

 95.2

 1544

 1108

 680

 864

 428

 103

 92

 227

 163

Solvent Control

 0.2

 95.5

 93.4

 95.2

 1567

 1096

 722

 854

 374

 100

 100

 218

 152

 DNCB (positive control)

 4.0

 81.7

 80.8

 81.1

 3356

 2404

 671

 2685

 1733

 322

 372

 500

 358

 Test item

1000.00

833.33

694.44

578.70

482.25

401.88

334.90

279.08

95.6

95.2

95.6

95.2

96.4

96.5

96.5

96.1

95.9

95.3

95.6

95.6

96.0

95.5

95.8

95.7

96.3

95.1

95.5

95.6

96.4

95.7

95.8

95.5

1285

1356

1387

1453

1220

1277

1208

1365

1019

999

991

1045

991

962

989

1091

585

592

594

682

593

611

608

646

700

764

793

771

627

666

600

719

434

407

397

363

398

351

381

445

82

89

93

90

73

78

70

84

116

109

106

97

106

94

102

119

220

229

234

213

206

209

199

211

174

169

167

153

167

157

163

169

Table 4: Acceptance Criteria

Acceptance Criterion 

 Range

 Experiment 1

 pass/fail

 Experiment 2

 pass/fail

cell viability medium and solvent controls [%]

number of test dosed with viability >50% CD86

number of test dosed with viability >50% CD54

number of test dosed with viability >50% IgG1

RFI of positive control of CD86

RFI of positive control  of CD54

RFI of solvent control of CD86

RFI of  solvent control  of CD54

MFI ratio IgG1/CD86 for medium control [%]

MFI ratio IgG1/CD86 for DMSO control [%]

MFI ratio IgG1/CD54 for medium control [%]

MFI ratio IgG1/CD54 for DMSO control [%]

>90

≥4

≥4

≥4

≥150

≥200

<150

<200

>105

>105

>105

>105

90.9 - 94.9

8

8

8

277

309

125

122

228

275

152

169

pass

pass

pass

pass

pass

pass

pass

pass

pass

pass

pass

pass

93.4 - 96.3

8

8

8

322

372

97

109

227

224

163

169

pass

pass

pass

pass

pass

pass

pass

pass

pass

pass

pass

pass

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore the test item considered to be no skin 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:

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. In the present study Propyl oleate was dissolved in THF. For the dose finding assay stock solutions with concentrations ranging from (500.00 mg/mL to 139.54 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.

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 95.5% (CD86), 94.7% (CD54) and 94.5% (isotype IgG1 control) in the first experiment and to 95.6% (CD86), 95.9% (CD54) and 96.3% (isotype IgG1 control) in the second experiment. The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item is considered to be no skin sensitiser. The positive control (DNCB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (277% experiment 1; 322% experiment 2) and 200% for CD54 (309% experiment 1; 372% experiment 2) were clearly exceeded.

Conclusion:

Based on the OECD GUIDANCE DOCUMENT ON THE REPORTING OF DEFINED APPROACHES AND INDIVIDUAL INFORMATION SOURCES TO BE USED WITHIN INTEGRATED APPROACHES TO TESTING AND ASSESSMENT (IATA) FOR SKIN SENSITISATION Series on Testing & Assessment No. 256 (ENV/JM/MONO(2016)29 and ENV/JM/MONO(2016)29/ANN1) an Adverse Outcome Pathway-based "2 out of 3" integrated testing strategy approach to skin hazard identification (BASF) was chosen. This defined approach describes an integrated testing strategy (ITS) for the identification of the skin sensitisation hazard of a substance primarily for the purposes of classification and labelling without the use of animal testing. The combination of test methods used covers the first three key events (KEs) of the adverse outcome pathway (AOP) leading to skin sensitisation as formally described by the OECD: KE 1: protein binding (e.g. via the direct peptide reactivity assay (DPRA); OECD TG 442C); KE 2: keratinocyte activation (e.g. via the KeratinoSensTM or LuSens assay; OECD TG 442D); and dendritic cell activation [e.g. via the human cell line activation test (h-CLAT); OECD TG 442E. The prediction model entails that two concordant results obtained from methods addressing different steps of first three KEs of the AOP, determine the final classification. Performance and classifications derived from the “2 out of 3 - Sens ITS” of 213 substances were compared to both high quality animal and human data. Depending on the combination of tests used, the “2 out of 3 - Sens ITS” prediction model generally achieved accuracies slightly exceeding those of the murine local lymph node assay (LLNA) when compared to human data. These results compellingly verify the applicability of this easy to understand integrated testing approach (ITS) for a wide range of chemicals.[Ref. CASE STUDY I of ENV/JM/MONO(2016)29/ANN1, Dr. Robert Landsiedel, BASF SE] The test item does neither activate the Nrf2-Keap1-ARE toxicity pathway (key event 2 of skin sensitization AOP) nor induce incresed number of cell surface markers CD54 and CD86 (key 3 of the skin sensitization AOP). In line with the "2 out of 3" integrated testing strategy approach to skin hazard identification presented classification of the test item as skin sensitizer is not justified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification