Registration Dossier

Administrative data

Description of key information

- study conducted according to OECD guideline 442 C, reactivity of the test item towards a predefined lysine and cysteine peptide, direct peptide  reactivity  assay (DPRA), high reactivity towards the cysteine peptide

- study conducted according to OECD guideline 442 D, activation of keratinocytes measured via a luciferase assay, induction of luciferase activity more than 1.5 fold in at least two experiments, positive result

- study conducted according to OECD guideline 442E, human Cell Line Activation Test (hCLAT), the test item induced upregulation of the cell surface markers CD86 and CD54 in at least two independent experiment runs. Therefore, the test item is considered to be positive in the hCLAT

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:
2017-09-07 to 2018-02-28
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))
Version / remarks:
adopted February 04, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Details on study design:
Skin sensitisation (In chemico test system)
- Details on study design:
- Pre-experiments: Solubility of the test item was determined prior to the main experiment and was tested at the highest final concentration applied in the study (100 mM). Solubility was investigated in the following
solvents suitable for the test:
- acetonitrile
- dist. water
- dist. water : acetonitrile 1:1 (v/v),
- isopropanol
- methanol
- 1,4-butanediol
- N,N-dimethylformamide (DMF)
- ethanol
- tert. butanol
The test item was completely soluble in DMF, therefore DMF was chosen as suitable vehicle for the main experiments.
- Preparation of the test item: The test item was freshly prepared immediately prior to use. The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. A factor of 1.11 was used to correct for the purity of the test item.
- Peptides: 19.90 mg cysteine peptide with an amino acid sequence of Ac-RFAACAA were pre-weighed in a vial and dissolved in a defined volume (38.69 mL) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM.
22.01 mg lysine peptide with an amino acid sequence of Ac-RFAAKAA were pre-weighed in a vial and dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (41.50 mL) to reach a concentration of 0.667 mM.
- Controls:
Reference controls, co-elution controls and a positive control (PC) were set up in parallel to the test item in order to confirm the validity of the test.
Positive Control
Cinnamic aldehyde ((2E)-3-phenylprop-2-enal) was solved in acetonitrile and was used as positive control. A stock concentration of 100 mM was prepared and was included in every assay run for both peptides.
Co-elution Control
Co-elution controls were set up in parallel to sample preparation but without the respective peptide solution. The controls were used to verify whether a test chemical absorbs at 220 nm and co-elutes with the cysteine or lysine peptide. The co-elution controls were prepared for every test item preparation and the positive control and were included in every assay run for both peptides. Reference Control
Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
Reference control A was prepared using acetonitrile in order to verify the accuracy of the calibration curve for peptide quantification. Its replicates were injected in the beginning of each HPLC run.
Reference control B was prepared using acetonitrile in order to verify the stability of the respective peptide over the analysis time. Its replicates were injected in the beginning and in the end of each HPLC run.
Reference control C was set up for the test item and the positive control. RC C for the positive control was prepared using acetonitrile. RC C for the test item was prepared using the respective solvent used to solubilise the test item. The RC C was used to verify that the solvent does not impact the percent peptide depletion (PPD). Additionally reference control C was used to calculate PPD. The RC C was included in every assay run for both peptides and was injected together with the samples.
The incubation scheme is displayed in tabular form under 'any other information on materials and methods'.
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. 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 the test item was analysed in triplicate for both peptides.
- HPLC: Peptide depletion was monitored by HPLC coupled with an UV detector at λ = 220 nm using a reversed-phase HPLC column (Zorbax SB-C-18 2.1 mm x 100 mm x 3.5 micron) as preferred
column. The entire system was equilibrated at 30 °C with 50% phase A and 50% phase B for at least 2 hours before running the analysis sequence. The HPLC analysis was performed using a flow rate of 0.35 mL/min and a linear gradient from 10% to 25% acetonitrile over 10 minutes, followed by a rapid increase to 90% acetonitrile. The column was re-equilibrated under initial conditions for 7 minutes between injections. Equal volumes of each standard, sample and control were injected.
- Prediction Model: The Prediction Models for both peptides are displayed under 'any other information on material and methods.
- 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.


Positive control results:
The mean peptide depletion by the positive control was 67.81% Cysteine depletion and 58.82 % Lysine depletion. Results are illustrated under 'any other information on results' in a tabular form.
Key result
Parameter:
other: mean percent cysteine depletions
Value:
100
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: highly reactive towards peptide residues
Key result
Parameter:
other: mean percent lysine depletions
Value:
1.79
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Values are only considered as estimation due to co-elution, not used for evaluation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: slight precipitations and phase separation in the positive controls were observed. Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, the mean peptide concentration for the reference control C was 0.4975 ± 0.0028 mM
- Acceptance criteria met for positive control: yes, mean Peptide concentration for the cysteine peptide was > 60.8 % (67.81 %) and the standard deviation of the positive control was < 14.9% (0.00%), mean Peptide concentration for the lysine peptide was > 40.2% (58.82%) and the standard deviation of the positive control was > 11.6% (0.17 %)
- Acceptance criteria met for variability between replicate measurements: yes, the CV[%] of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0% (0.17 and 0.57 %, respectively)

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

1453.8585

0.1612

68.18

67.81

0.64

0.94

1504.0075

0.1667

67.08

1453.6510

      0.1612

      68.18

Test Item

0.0000

0.0013

100.00

100.00

0.00

0.00

0.0000

0.0013

100.00

0.0000

0.0013

100.00

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

3944.2044

0.4895

1.62

1.79*

0.17

9.27

3936.1248

0.4885

1.82

3930.9854

0.4878

1.95

* A minor/major peak in the co-elution control of the test item was observed at the retention time of the lysine peptide. Therefore, co-elution of test item and peptide occurred and the given peak areas do not properly reflect the amount of lysine peptide present in the test item samples. The values can only be considered as an estimation of the peptide depletion and will not be used for evaluation.

Interpretation of results:
other: positive results in the direct peptide reactivity assay (DPRA)
Conclusions:
In this study under the given conditions the test item showed high reactivity towards the cysteine peptide. The test item is thus considered to be “positive" in the direct peptide reactivity assay (DPRA). The data generated with this method may be not sufficient to conclude on the skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-10-10 to 2018-04-12
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)
Version / remarks:
adopted 04 February, 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155
Version / remarks:
01 July, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Details on study design:
Skin sensitisation (In vitro test system) ARE-Nrf-2 Luciferase Test Method (KeratinoSens™)
- Details on study design: A cell suspension of 8E+04 cells/mL in assay medium was prepared. 125 µL of the cell suspension corresponding to 1E+04 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 measurement: 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) or over the weekend (experiment 2 and 3). After the incubation period the plate was shaken for 10 min and the OD was measured at λ = 600 nm.
- 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 (AppliChem; Lot No.: 0000978834, 0001055932) 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%; Alpha Aesar; Lot No.: 10176010) was used as positive control. CA was dissolved in DMSO (AppliChem; Lot No.: 0000978834, 0001055932) 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.

- Prediction model: A KeratinoSens™ prediction is considered positive 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. A negative result for test items with a log KOW > 7 has to be interpreted with care due to the applicability of the test method.


Positive control results:
The positive control (cinnamic aldehyde) values are considered to be valid because they are within the acceptability range demanded by the OECD test guideline 442d and the determined historical data (see also Table 1).
Key result
Parameter:
other: EC1.5 / µM
Run / experiment:
Experiment 1
Value:
65.14
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
IC30 values are > than EC50 values
Key result
Parameter:
other: EC1.5 / µM
Run / experiment:
Experiment 2
Value:
67.33
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
IC30 values are > than EC1.5 values
Key result
Parameter:
other: EC1.5 / µM
Run / experiment:
Experiment 3
Value:
65.21
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
IC30 values are > thatn EC1.5 values
Key result
Parameter:
other: IC30 / µM
Run / experiment:
Experiment 1
Value:
156.08
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: IC30 /µM
Run / experiment:
Experiment 2
Value:
182.51
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: IC30 / µM
Run / experiment:
Experiment 3
Value:
137.81
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No

ACCEPTANCE OF RESULTS:
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.

- Acceptance criteria met for negative control: yes,
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes

Table 2: Results of the Cytotoxicity Measurement

 

Conc. [µM]

Cell Viability [%]

Experiment 3

Experiment 1

Experiment 2

Mean

SD

Conc. [µM]

Cell Viability [%]

Solvent Control

-

100.0

100.0

100.0

0.0

-

100.0

Positive Control

4.00

100.1

99.2

99.7

0.6

4.00

102.9

8.00

103.5

110.9

107.2

5.2

8.00

103.3

16.00

110.4

115.8

113.1

3.8

16.00

105.8

32.00

113.7

117.1

115.4

2.4

32.00

108.1

64.00

117.1

121.3

119.2

3.0

64.00

107.3

Test Item

0.98

87.9

119.5

103.7

22.4

32.24

93.4

1.95

87.7

91.6

89.7

2.8

37.08

95.8

3.91

95.0

104.7

99.8

6.9

42.64

93.2

7.81

94.3

99.8

97.1

3.9

49.04

91.7

15.63

96.2

112.3

104.2

11.4

56.39

91.5

31.25

98.5

108.4

103.5

7.0

64.85

90.3

62.50

97.5

100.0

98.7

1.7

74.58

93.1

125.00

92.1

127.9

110.0

25.3

85.76

92.5

250.00

3.2

2.1

2.7

0.8

98.63

93.2

500.00

3.7

2.1

2.9

1.1

113.42

93.3

1000.00

19.2

8.3

13.8

7.7

130.43

101.5

2000.00

1.0

0.1

0.6

0.6

150.00

18.0

Table 3: 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

1.05

1.18

1.10

1.11

0.06

 

8.00

1.26

1.31

1.41

1.33

0.08

 

16.00

1.43

1.45

1.49

1.46

0.03

 

32.00

1.88

2.04

2.18

2.03

0.15

*

64.00

3.14

3.12

3.47

3.24

0.19

*

Test Item

0.98

0.92

0.98

1.00

0.97

0.04

 

1.95

0.98

1.07

0.98

1.01

0.05

 

3.91

0.96

1.10

1.03

1.03

0.07

 

7.81

1.06

1.10

0.98

1.04

0.06

 

15.63

1.06

1.09

1.08

1.08

0.01

 

31.25

1.27

1.21

1.25

1.24

0.03

 

62.50

1.37

1.43

1.39

1.40

0.03

 

125.00

3.47

4.41

3.66

3.84

0.50

*

250.00

1.99

1.09

0.80

1.29

0.62

 

500.00

0.01

0.00

0.01

0.01

0.01

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

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

Table 4: 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

0.99

1.04

0.04

 

8.00

1.42

1.04

1.07

1.18

0.21

 

16.00

1.49

1.40

1.47

1.46

0.05

 

32.00

1.85

2.54

1.55

1.98

0.51

*

64.00

3.29

2.92

2.76

2.99

0.27

*

Test Item

0.98

1.04

1.01

0.77

0.94

0.15

 

1.95

0.98

0.99

0.92

0.97

0.04

 

3.91

0.96

1.01

0.84

0.94

0.08

 

7.81

0.97

1.01

1.01

1.00

0.02

 

15.63

0.98

1.06

1.03

1.02

0.04

 

31.25

1.06

1.12

0.97

1.05

0.08

 

62.50

1.29

1.21

1.12

1.20

0.09

 

125.00

5.94

4.88

4.28

5.03

0.84

*

250.00

0.10

0.07

0.06

0.08

0.02

 

500.00

0.01

0.01

0.00

0.01

0.00

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

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

Table 5: Induction of Luciferase Activity Experiment 3

Experiment 3

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

1.16

1.21

1.35

1.24

0.10

 

8.00

1.09

1.24

1.16

1.17

0.08

 

16.00

1.57

1.62

1.59

1.59

0.02

*

32.00

1.91

2.23

2.04

2.06

0.16

*

64.00

2.89

3.14

3.48

3.17

0.30

*

Test Item

32.24

1.20

1.58

1.47

1.42

0.20

 

37.08

1.22

1.49

1.31

1.34

0.14

 

42.64

1.40

1.36

1.30

1.35

0.05

 

49.04

1.31

1.47

1.38

1.38

0.08

 

56.39

1.25

1.46

1.42

1.38

0.11

 

64.85

1.45

1.56

1.48

1.50

0.06

 

74.58

1.55

1.56

1.70

1.60

0.09

*

85.76

1.69

1.77

2.12

1.86

0.23

*

98.63

2.52

2.57

2.38

2.49

0.10

*

113.42

4.52

2.50

3.21

3.41

1.03

*

130.43

8.45

7.42

7.17

7.68

0.68

*

150.00

40.82

28.65

75.30

48.26

24.20

*

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

Interpretation of results:
other: positifve results in the KeratinoSens™assay
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 positive in the KeratinoSens™assay.
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.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017-09-07 to 2018-04-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline No. 442E: In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)
Version / remarks:
2017
Deviations:
no
Qualifier:
according to
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Details on study design:
Skin sensitisation (In vitro test system)
- Details on study design:
EXPERIMENTAL PROCEDURE
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 in culture flasks for 72 h or 48 h at a cell density of 0.1 E+06 cells/mL or 0.2 E+06 cells/mL, respectively. 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 106 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1E+06 cells/well). The solvent controls 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, i.e. the concentration showing 75% cell survival, was calculated by log-linear interpolation. The CV75 value was used to calculate the concentration range of the test item for the main experiment.

CD54 and CD86 Expression
The test item was dissolved using DMSO as determined in the pre-experiment. Based on the concentration of the pre-determined CV75 value 8 concentrations of the test item were defined for the measurement of the surface marker expression, corresponding to 1.2*CV75; CV75; CV75/1.2; CV75/1.22; CV75/1.23; CV75/1.24; CV75/1.25; CV75/1.26. If the CV75 could not be determined due to insufficient cytotoxicity of the test item in the dose finding assay, the highest soluble concentration of the test item prepared with each solvent was used as starting dose.
The test item was diluted to the concentration corresponding to 500-fold of the 1.2 × CV75. Then, 1.2-fold serial dilutions were made using the corresponding solvent to obtain the 8 stock solutions to be tested. The stock solutions were further diluted 250-fold into the culture medium (working solutions). These working solutions were finally used for cell treatment with a further final 2-fold dilution factor.
For testing, THP-1 cells were pre-cultured in culture flasks for 72 h or 48 h at a cell density of 0.1 E+06 cells/mL or 0.2 E+06 cells/mL, respectively. Prior to test item application, cells were harvested from the cell culture flask by centrifugation (125 x g) and were re-suspended in fresh culture medium at a density of 2 E+06 cells/mL. Then, 500 µL of the cell suspension were seeded into a 24 well flat-bottom plate (1E+06 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 following steps were carried out on ice with pre-cooled buffers and solutions. The supernatant was discarded and the remaining cells were washed twice with FACS buffer. After washing, cells were blocked using 600 µL of a FcR blocking buffer (FACS buffer containing 0.01% (w/v) Globulin Cohn Fraction) and incubated at 4 °C for 15 min. After blocking, cells were split in three aliquots into a 96-well V-bottom plate. After centrifugation (approx. 250 x g), cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54, or mouse IgG1 (isotype) antibodies in the dark for 30 min at 4°C. All antibodies were diluted in FACS buffer at an appropriate manner (CD86 1:8.3, CD54 1:16.6, IgG 1:16.6). After washing with FACS buffer twice, cells were re-suspended in FACS buffer and PI solution was added. PI staining was done just prior to the measurement by adding PI solutions to each sample (final concentration of PI was 0.625 µg/mL).
The expression levels of CD86 and CD54 as well as cell viability were analysed by flow cytometry using an excitation wavelength of λ = 488 nm and an emission wavelength of λ = 530 nm ± 15 nm for FITC and λ > 650 nm for PI. Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 were calculated. The cell viability was calculated.

Controls
Solvent Control: 0.2% DMSO (v/v) in cell culture medium
Positive Control: 4 µg/mL DNCB

Prediction model
For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day or on the same day provided that for each run: independent fresh stock solutions and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.
Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal to or greater than 150% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a cell viability ≥ 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability ≥ 50% in at least two independent runs. In case of not concordant results a third run should be conducted to make the final prediction. Otherwise the results were considered as inconclusive.
A negative test result of a test item was only accepted if the cell viability at a concentration of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accepted when the test item is tested at the highest soluble concentration (5000 µg/mL for 0.9% NaCl solution; 1000 µg/mL for DMSO or a different organic solvent) even if the cell viability is >90%.

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%.
Positive control results:
The positive controls DNCB and NiSO4 led to upregulation of the cell surface markers CD54 and CD86.

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both experiments. The threshold of 150% for CD86 (413% experiment 1; 295% experiment 2) and 200% for CD54 (259% experiment 1; 339% experiment 2) were clearly exceeded.
Key result
Parameter:
other: RFI of CD86
Remarks:
at 196.19 and 163.49 µg/mL, with cell viability of > 50%
Run / experiment:
#1
Value:
> 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI of CD54
Remarks:
at 196.19 µg/mL, with cell viability of > 50%
Run / experiment:
#1
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI of CD86
Remarks:
at any tested concentration with cell viability ≥ 50%
Run / experiment:
#2
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: RFI of CD54
Remarks:
at 235.43 and 196.19 µg/mL, with cell viability of > 50%
Run / experiment:
#2
Value:
> 200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
Doubling time of the cells was monitored and found to be 48.7 h (Batch 17), 38.6 h (Batch 18) and 44.6 h (Batch 19) which is within the doubling time range specified by the manufacturer (35 - 50 h).
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.


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes, cell viability was > 90% in both experiments (Experiment 1: 96.4 - 97.4; Experiment 2: 96.4 - 96.9), RFI of the solvent control was not > 150 for CD 86 (Experiment 1: 115; Experiment 2: 128) and not > 200 for CD54 (Experiment 1: 137; Experiment 2: 110)
- Acceptance criteria met for positive control: yes, the RFI values of the positive control (DNCB) is ≥150% for CD86 (Exp. 1: 413; Exp. 2: 295) and ≥200% for CD54 (Exp. 1: 259; Exp. 2: 339) at a cell viability of >50%
- Acceptance criteria met for variability between replicate measurements: not applicable
- Range of historical values if different from the ones specified in the test guideline: see table
- test chemicals exhibiting a cell viability of less than 90% at the highest concentration tested
- the cell viability was more than 50% in at least four tested concentrations in each run

Table1:  Results of the Cell Batch Activation Test (Batch 17)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Cell Viability [%]

RFI

Cell Viability [%]

RFI

yes/no

DNCB

4 µg/mL

86.2

282

86.5

256

Yes

NiSO4

100 µg/mL

79.6

229

80.2

573

Yes

LA

1000 µg/mL

96.1

84

96.1

96

No

Table 2: Results of the Cell Batch Activation Test (Batch 18)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Cell Viability [%]

RFI

Cell Viability [%]

RFI

yes/no

DNCB

4 µg/mL

88.8

224

87.6

217

Yes

NiSO4

100 µg/mL

84.4

261

84.3

377

Yes

LA

1000 µg/mL

96.7

68

97.1

86

No

Table 3: Results of the Cell Batch Activation Test (Batch 19)

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Cell Viability [%]

RFI

Cell Viability [%]

RFI

yes/no

DNCB

4 µg/mL

81.1

347

79.7

269

Yes

NiSO4

100 µg/mL

82.1

347

82.5

391

Yes

LA

1000 µg/mL

96.7

89

96.4

109

No

Table 4:  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

95.30

0.00

96.30

Solvent Control

0.00

95.40

0.00

96.10

Tetrahydrofolic acid (THFA)

7.81

96.30

7.81

97.00

15.63

96.20

15.63

95.70

31.25

95.50

31.25

96.70

62.50

95.80

62.50

96.60

125.00

95.30

125.00

96.50

250.00

50.10

250.00

70.50

500.00

34.50

500.00

28.70

1000.00

34.50

1000.00

23.70

Calculated CV75 [µg/mL]

170.65

221.74

Mean CV75 [µg/mL]

196.19

SD CV 75 [µg/mL]

36.12

Table 5: 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

-

97.4

96.6

96.4

2072

1022

578

1494

444

87

73

358

177

Solvent Control

0.20%

97.3

96.6

96.7

2300

1186

577

1723

609

100

100

399

206

DNCB

4.00

84.4

83.7

83.4

7681

2140

560

7121

1580

413

259

1372

382

Tetrahydrofolic acid (THFA)

235.43

27.1

27.4

25.0

5700

3209

2513

3187

696

185

114

227

128

196.19

61.6

61.4

60.5

5836

4391

1018

4818

3373

280

554

573

431

163.49

87.6

86.9

87.6

3888

1605

654

3234

951

188

156

594

245

136.24

94.6

95.1

95.3

2831

1176

598

2233

578

130

95

473

197

113.54

96.3

96.2

95.5

2483

1225

609

1874

616

109

101

408

201

94.61

95.9

95.7

95.6

2482

1138

599

1883

539

109

89

414

190

78.85

96.2

95.5

95.2

2482

1130

581

1901

549

110

90

427

194

65.70

96.2

95.7

95.7

2567

1105

679

1888

426

110

70

378

163

Table 6: 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

IgG Isotype

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

96.9

96.4

96.7

2026

1030

554

1472

476

78

91

366

186

Solvent Control

0.20%

96.9

96.5

96.5

2436

1073

549

1887

524

100

100

444

195

DNCB

4.0

79.9

80.0

81.0

6284

2495

717

5567

1778

295

339

876

348

Tetrahydrofolic acid (THFA)

235.43

38.6

34.5

48.6

5271

3181

1863

3408

1318

181

252

283

171

196.19

74.5

77.3

77.3

3592

2113

846

2746

1267

146

242

425

250

163.49

94.1

94.0

94.2

2775

1066

669

2106

397

112

76

415

159

136.24

96.0

96.1

95.7

1993

1032

653

1340

379

71

72

305

158

113.54

96.2

96.0

95.5

2205

1036

567

1638

469

87

90

389

183

94.61

95.4

95.9

95.7

2340

957

537

1803

420

96

80

436

178

78.85

96.8

96.7

96.8

2017

1058

602

1415

456

75

87

335

176

65.70

96.5

96.3

96.3

2159

986

541

1618

445

86

85

399

182

Table 7: Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

96.4

 

-

97.4

 

pass

 

96.4

 

-

96.9

 

pass

 

number of test dosed with viability >50% CD86

≥4

7

pass

7

pass

number of test dosed with viability >50% CD54

≥4

7

pass

7

pass

number of test dosed with viability >50% IgG1

≥4

7

pass

7

pass

RFI of positive control of CD86

≥150

413

pass

295

pass

RFI of positive control of CD54

≥200

259

pass

339

pass

RFI of solvent control of CD86

<150

115

pass

128

pass

RFI of solvent control of CD54

<200

137

pass

110

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

358

pass

366

pass

MFI ratio IgG1/CD86 for DMSO control [%]

>105

399

pass

444

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

177

pass

186

pass

MFI ratio IgG1/CD54 for DMSO control [%]

>105

206

pass

195

pass

Table 8: 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: positive results in the h-CLAT
Conclusions:
In this study under the given conditions the test item induced upregulation of the cell surface markers CD86 and CD54 in at least two independent experiment runs. Therefore, the test item is considered to be positive in the hCLAT. 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.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

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 for skin sensitisation testing to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. The DPRA is proposed to address the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic model peptides containing either lysine or cysteine. The percentage depletion values of the cysteine and lysine peptide are used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers. This test method can be used, in combination with other complementary information, to support the discrimination between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers in the context of IATA. This method cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by UN GHS nor to predict potency for safety assessment decisions. All substances yielding a mean peptide depletion value above the threshold for “No or minimal reactivity” of the respective prediction model will be considered “positive” in the DPRA. Depending on the regulatory framework, a positive result with the DPRA may be used on its own to classify a chemical into UN GHS category 1.

The in vitro KeratinoSens™ assay is proposed to address the second molecular key event of the skin sensitisation 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.This test method can be used to support the discrimination between skin sensitisers (i.e. UN GHS Category 1) and non-sensitisers in the context of IATA. It cannot be used on its own, neither to sub-categorise skin sensitisers into subcategories 1A and 1B as defined by the UN GHS, for authorities implementing these two optional subcategories, nor to predict potency for safety assessment decisions. However, depending on the regulatory framework, a positive result may be used on its own to classify a chemical into UN GHS category 1. Therefore, all substances inducing a statistically significant dose-dependent induction of the luciferase activity above a given threshold (i.e. > 1.5 fold or 50% increase), below a defined concentration which does not significantly affect cell viability (i.e. below 1000 µM and at a concentration at which the cellular viability is above 70% are considered positive in the KeratinoSens™.

The h-CLAT is supposed to address the third key event of the skin sensitisation process as defined by the adverse outcome pathway (AOP), the activation of dendritic cells (DC) typically accompanied by expression of specific cell surface markers, chemokines and cytokines. The h-CLAT quantifies the expression of the two surface markers CD86 and CD54 which are considered to be associated with the process of DC activation by using the human monocytic leukemia cell line THP-1 as a surrogate. The expression level of CD86 and CD54 following exposure to test chemicals are used for supporting the discrimination between sensitisers and non-sensitisers. This test may be used for supporting the discrimination between skin sensitisers and non-sensitisers in accordance with UN GHS “Category 1”. It does not allow the classification of chemicals to the subcategories 1A and 1B as defined by UN GHS nor predict potency for safety assessment decisions. Therefore, all substances giving a positive result in the h-CLAT will be classified into UN GHS “Category 1”.

In a study conducted according to OECD guideline 442 C, the test item was tested for reactivity towards a predefined lysine and cysteine peptide. The test item is thus considered to be “positive" in the direct peptide reactivity assay (DPRA).

As a second step in context of IATA, the KeratinoSens™ assay was performed according to OECD guideline 442 D. 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 positive in the KeratinoSens™ assay.

In the third step of the IATA, a h-CLAT assay was performed according to OECD guideline 442 E. In this study under the given conditions the test item induced upregulation of the cell surface markers CD86 and CD54 in at least two independent experiment runs. Therefore, the test item is considered to be positive in the hCLAT.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available, reliable and relevant data generated by the testing strategy as defined by IATA Tetrahydrofolic acid needs to be classified as skin sensitiser according to Regulation (EC) No 1272/2008 (CLP) and the Globally Harmonized System for Classification and Labelling of Chemicals (GHS).