Registration Dossier

Administrative data

Description of key information

- in an in chemico study (DPRA) according to OECD test guideline 442C, GLP, Reliability 1, the test item was found not to react with the peptides and was thus considered as non-sensitiser.

- in an in vitro study conducted according to OECD test guideline 442D using the ARE-Nrf2 Luciferase Test Method, GLP, Reliability 1, the test item induced luciferase activity only at cytotoxic test item concentrations (viability <70 %). The test item was considered as non-sensitiser at non-cytotoxic concentrations.

- In an in vitro study according to the OECD Draft Proposal for a new test guideline, "in vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)", GLP, Reliability 1, the test item did upregulate one cell surface marker (CD54) in at least two independent experiments. Therefore, the test item is considered to be a sensitiser in accordance with UN GHS category 1. Cytotoxicity was observed (CV75: 174.92 ± 24.9 µg/mL).

- in a read-across study based on the mouse local lymphnode assay (LLNA) according to OECD test guideline 429, GLP, Reliability 2, the source substance Sophorolipid C18 unsaturated acid was found to be a non-sensitiser.

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:
2016-03-07 to 2016-04-13
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
Qualifier:
according to
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154
Version / remarks:
January 12, 2013
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
The Direct Peptide Reactivity Assay (DPRA) is accepted as a reliable and relevant replacement test for in vivo skin sensitisation testing.
Specific details on test material used for the study:
Preparation of the Test Item
The test item was freshly prepared immediately prior to use, unless stability data demonstrate the acceptability of storage. The test item was pre-weighted 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.
Details on study design:
The DPRA is supposed 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 value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitisers and non-sensitisers.
The correlation of protein reactivity with skin sensitisation potential of a chemical is well established and represents the first and initial key event in the skin sensitisation process as defined by the AOP. It is therefore a crucial step for the sensitising potential of a chemical.
This test may be used for the hazard identification of sensitising chemicals 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 DPRA will be classified into UN GHS “Category 1”.
For detailed information on experimental procedure, materials, methods, controls, prediction model and acceptance criteria please see section “any other details on materials and methods incl. tables”.
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 62.90 %.
Key result
Parameter:
other: mean peptide depletion [%]
Run / experiment:
Both Cysteine and Lysine
Value:
2.03
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: mean peptide depletion [%]
Run / experiment:
Cysteine
Value:
0
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Values were set to zero due to negative depletion
Parameter:
other: mean peptide depletion [%]
Run / experiment:
Lysine
Value:
4.07
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS: all criteria were fulfilled. For details, please see Tables 10 and 11 in section “any other details on results incl. tables”.

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. All test item solutions were freshly prepared immediately prior to use. The test item was not soluble in acetonitrile but completely soluble in water. No turbidity, precipitation and phase separation were observed for the test item solutions. All test item preparations of the main experiment were prepared using water.

Precipitation and Phase Separation

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

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

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

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, positive control, RC A, RC B, RC C and for STD 1 and 2. Samples were not centrifuged prior to the HPLC analysis.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any test item samples. Slight phase separation was observed for the positive control and the respective co-elution control.

After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, positive control, RC A, RC B, RC C and for STD 1, 2 and 3.

After the HPLC run samples of the lysine peptide run were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for the any test item samples. Slight phase separation was observed for the positive control and the respective co-elution control.

Co-elution with the peptide peak

No co-elution of the test item with any of the peptide peaks was observed.

Results Calibration Curve

Table 6: 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

3495.8242

0.5340

3675.5410

0.5340

STD2

1805.4337

0.2670

1867.7395

0.2670

STD3

944.5201

0.1335

985.2802

0.1335

STD4

466.7887

0.0667

516.4465

0.0667

STD5

237.0339

0.0334

282.1445

0.0334

STD6

116.4341

0.0167

179.8809

0.0167

STD7

0.0000

0.0000

0.0000

0.0000

Based on these results, linear regression was performed and the following calibration curves were determined:

Cysteine Peptide Calibration Curve : y = 6546.17x + 26.29 ; R2= 0.9995

Lysine Peptide Calibration Curve : y = 6805.79x + 50.30 ; R2= 0.9996

 

Results of the Cysteine Peptide Depletion

Table 7: Depletion of the Cysteine Peptide. * Values were set to zero due to negative depletion.

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

906.7542

0.1345

71.54

72.27

0.90

0.01

892.3105

0.1323

71.99

851.7282

0.1261

73.27

Test Item

3217.3611

0.4875

0.00*

0.00

0.00

-

3224.2549

0.4885

0.00*

3206.4368

0.4858

0.00*

 

 Results of the Lysine Peptide Depletion

Table 8: 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

1608.7321

0.2290

53.57

53.53

0.22

0.00

1618.3588

0.2304

53.29

1603.4867

0.2282

53.72

Test Item

3266.0764

0.4725

4.10

4.07

0.10

0.02

3271.2881

0.4733

3.95

3264.7769

0.4723

4.14

Detailed results about the reference controls can be found in Table 15.

Categorization of the Test Item

Based on the results of the peptide depletion, categorization according to the prediction model was performed. In case that no co-elution was detected, the prediction model based on the combination of cysteine and lysine peptide should be used. Since no co-elution was observed the prediction model of cysteine and lysine was used.

Table 9:     Categorization of the Test Item

Predicition 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

2.03

Minimal reactivity

No sensitiser

0.00

Minimal reactivity

No sensitiser

Positive Control

62.90

High reactivity

sensitizer

72.27

Moderate reactivity

sensitizer

 

Acceptance Criteria

Table 10: Acceptance Criteria for Cysteine Peptide

Cysteine Peptide Run

Acceptance Criterion

Range

Value

pass/fail

coefficient of determination

R2> 0.99

0.9995

pass

mean peptide concentration of RC A

0.45 ≤ x ≤ 0.55 mM

0.4969

pass

mean peptide concentration of RC C (PC)

0.45 ≤ x ≤ 0.55 mM

0.4827

pass

mean peptide concentration of RC C (TI)

0.45 ≤ x ≤ 0.55 mM

0.4584

pass

CV of the peak area of RC B

< 15 %

2.38

pass

CV of the peak area of RC C (PC)

< 15 %

0.85

pass

CV of the peak area of RC C (TI)

< 15 %

9.07

pass

mean peptide depletion of the PC

60.8 % < x < 100 %

72.27

pass

SD of peptide depletion of the PC replicates

< 14.9 %

0.90

pass

SD of peptide depletion of the TI replicates

< 14.9 %

0.00

pass

Table 11: Acceptance Criteria for Lysine Peptide

Lysine Peptide Run

Acceptance Criterion

Range

Value

pass/fail

coefficient of determination

R² > 0.99

0.9996

pass

mean peptide concentration of RC A

0.45 ≤ x ≤ 0.55 mM

0.5067

pass

mean peptide concentration of RC C (PC)

0.45 ≤ x ≤ 0.55 mM

0.5017

pass

mean peptide concentration of RC C (TI)

0.45 ≤ x ≤ 0.55 mM

0.4930

pass

CV of the peak area of RC B

< 15 %

0.54

pass

CV of the peak area of RC C (PC)

< 15 %

0.61

pass

CV of the peak area of RC C (TI)

< 15 %

0.54

pass

mean peptide depletion of the PC

40.2 % < x < 69.0 %

53.53

pass

SD of peptide depletion of the PC replicates

< 11.6 %

0.22

pass

SD of peptide depletion of the TI replicates

< 11.6 %

0.00

pass

Table 12: Historical Data Cysteine Peptide

Cysteine Peptide

 

mean

SD

N

linearity of the calibration curve

0.9991

0.0006

8

mean peptide concentration of reference A [mM]

0.52

0.00

8

mean peptide concentration of reference C [mM]

0.50

0.00

10

CV of the peak area of control B [%]

2.10

0.34

8

CV of the peak area of control C [%]

1.60

0.85

10

mean peptide depletion of the PC [%]

74.67

2.32

8

SD of peptide depletion of the PC replicates [%]

0.84

0.72

8

SD of peptide depletion of the test items [%]

4.60

14.80

20

Table 13:   Historical Data Lysine Peptide

Lysine Peptide

 

mean

SD

N

linearity of the calibration curve

0.9998

0.0001

7

mean peptide concentration of reference A [mM]

0.49

0.02

7

mean peptide concentration of reference C [mM]

0.49

0.24

9

CV of the peak area of control B [%]

1.26

0.24

7

CV of the peak area of control C [%]

0.81

0.89

9

mean peptide depletion of the PC [%]

59.54

6.09

7

SD of peptide depletion of the PC replicates [%]

2.58

1.90

7

SD of peptide depletion of the test items [%]

1.02

1.08

21

Table 14:   Exemplary Analysis Sequence

Run 1

Run 2

Run 3

Run 4

Run 5

Run 6

Run 7

Run 8

Run 9

Run 10

Run 11

 

STD1

STD2

STD3

STD4

STD5

STD6

SDT7 (DB)

Reference Control A, replicate 1

Reference Control A, replicate 2

Reference Control A, replicate 3

Run 12

Run 13

Co-Elution Control Positive Control

Co-Elution Test Item 1

Run 14

Run 15

Run 16

Reference Control B, replicate 1

Reference Control B, replicate 2

Reference Control B, replicate 3

Run 17

Run 18

Run 19

Reference Control C, replicate 1

Positive Control, replicate 1

Test Item 1, replicate 1

Run 20

Run 21

Run 22

Reference Control C, replicate 2

Positive Control, replicate 2

Test Item 1, replicate 2

Run 23

Run 24

Run 25

Reference Control C, replicate 3

Positive Control, replicate 3

Test Item 1, replicate 3

Run 26

Run 27

Run 28

Reference Control B, replicate 4

Reference Control B, replicate 5

Reference Control B, replicate 6

Table 15: Results of the Reference Controls for the Cysteine Peptide

Cysteine Peptide Run

Sample

Peptide Peak Area

Peptide Concentration [mM]

PA

Mean

SD

CV [%]

Peptide Concentration

Mean

SD

CV [%]

Reference A 1

3295.67

3278.9512

26.0108

0.79

0.4994

0.4969

0.0040

0.80

Reference A 2

3292.20

0.4989

Reference A 3

3248.98

0.4923

Reference B 1

3211.87

3162.2388

74.5299

2.36

0.4866

0.4791

0.0114

2.38

Reference B 2

3253.24

0.4930

Reference B 3

3222.13

0.4882

Reference B 4

3097.92

0.4692

Reference B 5

3090.32

0.4681

Reference B 6

3097.95

0.4692

Reference C 1 (PC solvent)

3208.51

3186.06

26.9566

0.85

0.4861

0.4827

0.0041

0.85

Reference C 2 (PC solvent)

3193.52

0.4838

Reference C 3 (PC solvent)

3156.16

0.4781

Reference C 1 (TI solvent)

3205.63

3027.02

272.2805

8.99

0.4857

0.4584

0.0416

9.07

Reference C 2 (TI solvent)

2713.64

0.4105

Reference C 3 (TI solvent)

3161.80

0.4790

Table 16:   Results of the Reference Controls for the Lysine Peptide

Lysine Peptide Run

Sample

Peptide Peak Area

Peptide Concentration [mM]

PA

Mean

SD

CV [%]

Peptide Concentration

Mean

SD

CV [%]

Reference A 1

3484.17

3498.5079

12.6544

0.36

0.5046

0.5067

0.0019

0.37

Reference A 2

3508.10

0.5081

Reference A 3

3503.26

0.5074

Reference B 1

3472.14

3482.0649

18.3933

0.53

0.5028

0.5042

0.0027

0.54

Reference B 2

3506.05

0.5078

Reference B 3

3497.27

0.5065

Reference B 4

3473.01

0.5029

Reference B 5

3487.77

0.5051

Reference B 6

3456.15

0.5004

Reference C 1 (PC solvent)

3445.89

3465.01

20.6789

0.60

0.4989

0.5017

0.0030

0.61

Reference C 2 (PC solvent)

3462.18

0.5013

Reference C 3 (PC solvent)

3486.96

0.5050

Reference C 1 (TI solvent)

3426.42

3405.85

18.0704

0.53

0.4961

0.4930

0.0027

0.54

Reference C 2 (TI solvent)

3392.54

0.4911

Reference C 3 (TI solvent)

3398.60

0.4920

 

Interpretation of results:
other: Expert judgement: no indication of sensitisation
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards the peptides. The test item can be considered as “non-sensitizer”. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and will be considered in the context of integrated approach.
Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential

of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP),

namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides

containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine

peptide is used to categorize a substance in one of four reactivity classes to support discrimination

between skin sensitiser and non-sensitisers.

In the present study, the test item was dissolved in water and 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 analysis.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1 and 2 of the cysteine run. No centrifugation was necessary to perform the HPLC analysis. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.

After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1, 2 and 3 of the cysteine runs. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.

Since the turbidity noted for the test item samples was also observed for reference controls, positive controls and standard solutions it can be considered that it is related to the peptide and that it is not a precipitation of the test substance. Additionally, the turbidity did not change during the HPLC analysis period. Since stability of the cysteine peptide in the used acetonitrile batch was demonstrated successfully, the reactivity of the positive control towards the cysteine peptide and peptide depletion were identified correctly and the validity of the cysteine run was acceptable the precipitation was considered as not relevant.

No co-elution of test item with the peptide peaks was observed. Sensitizing 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 C).

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 % (2.03 %). 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 62.90 %.

The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was >0.99 (0.9995). The mean peptide depletion of the cysteine peptide by the positive control was between 60.8 % and 100 % (72.27 %).

The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and <0.55 mM (RC A: 0.4969 mM, RC C (acetonitrile): 0.4827 mM, RC C (water): 0.4584 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 2.38 %, RC C (acetonitrile): 0.85 %, RC C (water): 9.07 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <14.9 % (PC: 0.90 %; test item 0.00 %).

For the lysine run the coefficient of determination for the calibration curve was >0.99 (0.9996). The mean peptide depletion of the lysine peptide by the positive control was between 40.2 % and 69.0 % (53.53 %). The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and < 0.55 mM (RC A 0.5067 mM, RC C (acetonitrile): 0.5017 mM, RC C water): 0.4930 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 0.54 %, RC C (acetonitrile: 0.61 %, RC C water): 0.54 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <11.6 % (PC: 0.22 %; test item: 0.10 %).

The test item can be considered as non-sensitiser. The data generated with this test will be considered in the context of an integrated approached such as IATA, combining this result with other complementary information.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-06-13 to 2016-07-21
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: February 04, 2015
Deviations:
no
Qualifier:
according to
Guideline:
other: KeratinoSensTM, EURL ECVAM DB-ALM Protocol No. 155
Version / remarks:
July 1st, 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The ARE-Nrf2 Luciferase Test Method is accepted as a reliable and relevant replacement test for in vivo skin sensitisation testing. This test method is able to detect chemicals that cause skin sensitisation and allows for hazard identification in accordance with UN GHS “Category 1”. 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 an integrated approach such as an IATA, combining them with other complementary information e.g., derived from in chemico or in vitro assays addressing other key events of the AOP.
Details on study design:
Skin sensitisation (In vitro test system) ARE-Nrf-2 Luciferase Test Method (KeratinoSens™): The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers represents the second key event of the skin sensitisation process. This in vitro method is designed to predict and classify the skin sensitising potential of a chemical by assessment of its potential to induce the Keap1-Nrf2-ARE signalling pathway by quantifying the luciferase gene expression using luminescence detection.
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 KeratinoSens™ will be classified into UN GHS “Category 1”.
For details on study design, experimental procedure and material and methods please see section “any other information on materials and methods incl. tables”.
Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (2.98 (experiment 1), 5.07 (experiment 2); 4.36 (experiment 3)). The calculated EC 1.5 was between 7 and 30 µM (21.66 µM (experiment 1), 14.38 µM (experiment 2); 15.30 µM (experiment 3)). The positive control result is therefore considered valid.
Parameter:
other: Imax, maximal induction factor of luciferase activity
Run / experiment:
mean of 3 experiments at a test item concentration of 125 µM
Value:
6.79
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: induction of luciferase was observed only at cytotoxic test item concentrations (viability < 70 %) of 125 µM
Parameter:
other: EC1.5 [µM], concentration for a 1.5 fold luciferase induction
Run / experiment:
mean of 3 experiments
Value:
70.07
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: induction of luciferase was observed only at cytotoxic test item concentrations (viability < 70 %) of 125 µM
Parameter:
other: IC30, concentration effecting a reduction of cellular viability by 30 %.
Run / experiment:
mean of 3 experiments
Value:
32.27
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Parameter:
other: IC50, concentration effecting a reduction of cellular viability by 50 %.
Run / experiment:
mean of 3 experiments
Value:
65.12
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS: all criteria were fulfilled. For details, please see Table 7 in section “any other details on results incl. tables”.

In the first experiment, a max luciferase activity (Imax) induction of 7.96 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 21.8 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC 1.5 was < 1000 µM (67.67 µM).

In the second experiment, a max luciferase activity (Imax) induction of 4.63 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 24.0 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of I max . The calculated EC 1.5 was < 1000 µM (73.70 µM).

In the third experiment, a max luciferase activity (Imax) induction of 7.78 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 18.0%. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC 1.5 was < 1000 µM (68.85 µM).

Since the induction of the luciferase was observed only at cytotoxic test item concentrations (viability < 70 %), the effect cannot be considered for sensitization evaluation. Additionally, no clear dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Controls

The controls confirmed the validity of the study. The luciferase activity induced by the positive control at a concentration of 64µM was between 2 and 8 (2.98 (experiment 1), 5.07 (experiment 2); 4.36 (experiment 3)). The calculated EC1.5 was between 7 and 30 µM (21.66 µM (experiment 1), 14.38 µM (experiment 2); 15.30 µM (experiment 3)). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (13.9 % (experiment 1), (9.4 % (experiment 2), (14.0 % (experiment 3)).

Cytotoxicity

Table 1: Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell viability [%]

 

 

Experiment 1

Experiment 2

Mean

SD

Solvent control

-

100

100

100

0.0

Positive control

4.00

98.1

115.2

102.3

11.3

8.00

192.2

130.5

137.7

51.3

16.00

98.9

136.9

112.2

21.4

32.00

103.0

141.3

120.7

19.3

64.00

95.8

141.8

115.8

23.6

Test item

0.98

72.3

61.0

69.6

7.7

1.95

103.1

55.1

71.5

27.4

3.91

100.2

58.7

70.5

25.9

7.81

102.9

56.4

70.0

28.6

15.63

91.5

54.9

65.6

22.5

31.25

90.5

57.1

63.7

24.3

62.50

70.6

70.3

64.9

9.6

125.00

21.8

24.0

21.3

3.0

250.00

0.2

1.7

0.7

0.9

500.00

0.4

0.6

0.4

0.2

1000.00

0.1

0.2

0.1

0.1

2000.00

0.1

0.4

0.3

0.1

Luciferase induction

Table 2: Induction of Luciferase Activity Experiment 1. * = significant induction according to Student’s t-test, p < 0.05.

Experiment 1

Concentration [µM]

Fold induction

 

 

Rep. 1

Rep. 2

Rep. 3

Mean

SD

 

Solvent control

-

1.00

1.00

1.00

1.00

0.0

 

Positive control

4.00

1.23

1.19

1.00

1.14

0.12

 

8.00

1.26

1.16

1.21

1.21

0.05

 

16.00

1.48

1.40

1.36

1.36

0.14

 

32.00

1.88

1.73

1.75

1.75 *

0.12

 

64.00

3.38

2.52

2.98

2.98 *

0.43

 

Test item

0.98

0.83

0.94

0.90

0.90

0.06

 

1.95

1.14

1.10

1.09

1.09

0.05

 

3.91

1.10

1.07

1.10

1.10

0.03

 

7.81

1.23

1.06

1.10

1.10

0.11

 

15.63

1.06

1.41

1.16

1.16

0.22

 

31.25

0.99

1.13

1.08

1.08

0.08

 

62.50

0.98

0.69

0.92

0.92

0.20

 

125.00

4.95

6.85

12.08

7.96 *

3.96

 

250.00

0

0

0

0

0

 

500.00

0

0

0

0

0

 

1000.00

0

0

0

0

0

 

2000.00

0

0

0

0

0

 

Table 3: Induction of Luciferase Activity Experiment 2. * = significant induction according to Student’s t-test, p < 0.05.

Experiment 1

Concentration [µM]

Fold induction

 

 

Rep. 1

Rep. 2

Rep. 3

Mean

SD

 

Solvent control

-

1.00

1.00

1.00

1.00

0.0

 

Positive control

4.00

1.09

1.27

1.11

1.15

0.10

 

8.00

1.34

1.37

1.20

1.30

0.09

 

16.00

1.59

1.62

1.44

1.55 *

0.09

 

32.00

2.50

2.31

2.05

2.29 *

0.22

 

64.00

5.44

5.49

4.28

5.07 *

0.68

 

Test item

0.98

0.90

1.15

1.02

1.02

0.13

 

1.95

1.02

1.11

0.98

1.04

0.07

 

3.91

0.89

1.02

1.00

0.97

0.07

 

7.81

0.91

1.06

1.07

1.01

0.09

 

15.63

0.95

1.09

0.99

1.01

0.08

 

31.25

0.85

0.99

0.92

0.92

0.07

 

62.50

0.75

0.94

0.77

0.82

0.10

 

125.00

4.33

5.41

4.14

4.63 *

0.68

 

250.00

0

0

0

0

0

 

500.00

0

0

0

0

0

 

1000.00

0

0

0

0

0

 

2000.00

0

0

0

0

0

 

Table 4: Induction of Luciferase Activity Experiment 3. * = significant induction according to Student’s t-test, p < 0.05.

Experiment 1

Concentration [µM]

Fold induction

 

 

Rep. 1

Rep. 2

Rep. 3

Mean

SD

 

Solvent control

-

1.00

1.00

1.00

1.00

0.0

 

Positive control

4.00

1.07

1.17

1.15

1.13

0.05

 

8.00

1.58

1.13

1.06

1.26

0.28

 

16.00

1.57

1.70

1.30

1.52 *

0.20

 

32.00

1.98

2.29

1.82

2.03 *

0.24

 

64.00

4.45

4.95

3.67

4.36 *

0.64

 

Test item

0.98

0.79

1.06

1.17

1.01

0.19

 

1.95

0.82

0.94

0.33

0.70

0.32

 

3.91

0.92

1.12

0.82

0.95

0.15

 

7.81

0.82

1.09

1.03

0.98

0.14

 

15.63

0.80

1.99

1.30

1.36

0.60

 

31.25

0.67

0.97

0.93

0.86

0.17

 

62.50

0.70

0.83

0.84

0.79

0.08

 

125.00

10.47

8.16

4.70

7.78 *

2.90

 

250.00

0

0

0

0

0

 

500.00

0

0

0

0

0

 

1000.00

0

0

0

0

0

 

2000.00

0

0

0

0

0

 

Table 5: Induction of Luciferase Activity - Overall Induction. * = significant induction according to Student’s t-test, p < 0.05.

Overall induction

Concentration [µM]

Fold induction

 

 

 

Experiment 1

Experiment 2

Experiment 3

Mean

SD

Solvent control

-

1.00

1.00

1.00

1.00

0.00

Positive control

4.00

1.14

1.15

1.13

1.14

0.01

8.00

1.21

1.30

1.26

1.26

0.05

16.00

1.36

1.55

1.52

1.48

0.10

32.00

1.75

2.29

2.03

2.02

0.27

64.00

2.98

5.07

4.36

4.14

1.06

Test item

0.98

0.90

1.02

1.01

0.98

0.07

1.95

1.09

1.04

0.70

0.94

0.21

3.91

1.10

0.97

0.95

1.01

0.08

7.81

1.10

1.01

0.98

1.03

0.06

15.63

1.16

1.01

1.36

1.18

0.18

31.25

1.08

0.92

0.86

0.95

0.11

62.50

0.92

0.82

0.79

0.84

0.07

125.00

7.96

4.63

7.78

6.79

1.87

250.00

0

0

0

0

0

500.00

0

0

0

0

0

1000.00

0

0

0

0

0

2000.00

0

0

0

0

0

Table 6: Additional Parameters; n.a. = not applicable

Parameter

Experiment 1

Experiment 2

Experiment 3

Mean

SD

EC1.5

67.67

73.70

68.85

70.07

3.19

Imax

7.96

4.63

7.78

6.79

1.87

IC30

63.29

n.a.

1.26

32.27

43.86

IC50

88.88

89.91

16.58

65.12

42.04

Table 7: Acceptance criteria

Criterion

Range

Experiment 1

Pass/fail

Experiment 2

Pass/fail

Experiment 3

Pass/fail

CV Solvent Control

< 20 %

13.9

 pass

9.4

 pass

14.0

 pass

No. of positive control concentration steps with significant luciferase

activity induction > 1.5

1

2.0

 pass

3.0

 pass

3.0

 pass

EC1.5 PC [µM]

7 < x < 30µM

21.66

 pass

14.38

 pass

15.30

 pass

Induction PC at 64 µM

2 < x < 8

2.98

 pass

5.07

 pass

4.36

 pass

Table 8: Historical data

Acceptance criterion

Range

Mean

SD

n

CV Solvent Control

< 20 %

11.5

2.4

15

No. of positive control concentration steps with significant luciferase

activity induction > 1.5

1

2.5

0.5

15

EC1.5 PC [µM]

7 < x < 30µM

16.96

5.56

15

Induction PC at 64 µM

2 < x < 8

3.98

1.02

15

 

 

  

Interpretation of results:
other: expert statement: no indication of sensitisation
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 at non-cytotoxic concentrations. 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 will be considered in the context of integrated approach.
Executive summary:

In this study conducted according to OECD test guideline 442D (adopted February 04, 2015), transgenic keratinocytes constitutively expressing an ARE-reporter gene were incubated with Sophorolipids, fermentation products of glucose and fatty acids, at concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0 µM (solvent control) for 48 h at 37 °C. Afterwards the test substance containing medium was removed and the cells lysed and luminescence subsequently measured. Beside the luminescence the cell viability was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method.

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

- EC 1.5 value is < 1000 µM

- an apparent overall dose-response for luciferase induction

In the first experiment, a max luciferase activity (Imax) induction of 7.96 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 21.8 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC1.5 was < 1000 µM (67.67 µM).

In the second experiment, a max luciferase activity (Imax) induction of 4.63 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 24.0 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of I max . The calculated EC1.5 was < 1000 µM (73.70 µM).

In the third experiment, a max luciferase activity (Imax) induction of 7.78 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 18.0 %. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC1.5 was < 1000 µM (68.85 µM).

Since the induction of the luciferase was observed only at cytotoxic test item concentrations (viability < 70 %), the effect cannot be considered for sensitization evaluation. Additionally, no clear dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

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 (2.98 (experiment 1), 5.07 (experiment 2); 4.36 (experiment 3)). The calculated EC1.5 was between 7 and 30 µM (21.66 µM (experiment 1), 14.38 µM (experiment 2); 15.30 µM (experiment 3)). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (13.9 % (experiment 1), (9.4 % (experiment 2), (14.0 % (experiment 3)).

Under the conditions of this study the test item is therefore considered as non-sensitiser at non-cytotoxic concentrations. The data generated with this method may be not sufficient to conclude definitely on the absence of skin sensitisation potential of the test item and will thus be considered in the context of integrated approach.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016-03-07 to 2016-04-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
OECD draft proposal for a new test guideline for "h-CLAT"
Qualifier:
according to
Guideline:
other: OECD guidelines for Testing of Chemicals, Draft Proposal for a new test guideline, "in vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)"
Version / remarks:
July 2014
Qualifier:
according to
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol Nr. 158
Version / remarks:
July 1st, 2015
Principles of method if other than guideline:
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”.
For detailed information on experimental procedure, materials, methods, controls, prediction model and acceptance criteria please see section “any other details on materials and methods incl. tables”.
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
The correlation of upregulation of immunological relevant cell surface markers with the skin sensitising potential of a chemical has been reported and represents the third key event in the skin sensitisation process as described by the AOP. This method that measures the markers of DC activation, based on DC-like cell line THP-1 is considered relevant for the assessment of the skin sensitisation potential of chemicals.
This test method is able to detect chemicals that cause skin sensitisation and allows hazard identification in accordance with UN GHS “Category 1”. 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 IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP.
Positive control results:
The positive control (DCNB) led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (252 % in experiment 1 and 351 % in experiment 2) and 200 % for CD54 (431 % in experiment 1 and 772 % in experiment 2) were clearly exceeded.
Key result
Parameter:
other: upregulation of CD54 [% control]
Run / experiment:
174.93 µg/mL (1)
Value:
ca. 348
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
Corresponding CV was 90.1 %
Key result
Parameter:
other: upregulation of CD54 [% control]
Run / experiment:
174.93 µg/mL (2)
Value:
ca. 446
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
Corresponding CV was 84.1 %
Key result
Parameter:
other: upregulation of CD86 [% control]
Run / experiment:
both experiments
Value:
< 150
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
no dose response observed.
Parameter:
other: upregulation of CD54 [% control]
Run / experiment:
209.91 µg/mL (1)
Value:
ca. 1 476
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Corresponding CV was 52.5 %
Remarks:
cytotoxic effects observed
Parameter:
other: upregulation of CD54 [% control]
Run / experiment:
209.91 µg/mL (2)
Value:
ca. 2 198
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: Corresponding CV was 48.0 %
Remarks:
cytotoxic effects observed
Parameter:
other: CV75 [µg/mL]
Run / experiment:
mean of two experiments (dose range finding]
Value:
ca. 174.92
Vehicle controls validity:
valid
Negative controls validity:
valid
Remarks:
Lactic acid
Positive controls validity:
valid
Remarks on result:
other: cytotoxicity was observed
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: not reported
ACCEPTANCE OF RESULTS: all criteria were fulfilled. For details, please see section “any other details on results incl. tables”.

Reactivity Check of the Cell Stock

Doubling time of the cells was monitored and found to be 40.07 h which is within the doubling time range specified by the manufacturer (35 - 50 h).

Table 1: Results of the Cell Batch Activation Test

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Cell Viability [%]

RFI

Cell Viability [%]

RFI

yes/no

DNCB

µg/mL

85.1

298

86.3

385

Yes

NiSO4

100 µg/mL

90.5

219

91.2

260

Yes

LA

1000 µg/mL

98.2

68

98.1

65

No

Solvent finding

All test item solutions were freshly prepared immediately prior to use. The test item was soluble in 0.9 % NaCl solution (Braun, Lot No.: 140696, 140698, 152418002) at a concentration of 100 mg/mL.

Dose finding assay

The dose finding assay was performed using stock solutions with a concentration of 100 mg/mL.

Table 2: Results of the Dose Finding Assay

 

Experiment 1

Experiment 2

Sample

Concentration applied [µg/ml]

Cell Viability [%]

Concentration applied [µg/ml]

Cell Viability [%]

Medium Control

(= solvent control)

0.00

98.20

0.00

97.30

Test item

7.81

98.20

7.81

96.50

15.63

97.40

15.63

96.60

31.25

97.20

31.25

96.90

62.50

97.10

62.50

96.80

125.00

96.50

125.00

95.60

250.00

62.00

250.00

33.50

500.00

0.60

500.00

0.80

1000.00

11.00

1000.00

7.20

Calculated CV75 [µg/mL]

192.53

157.31

Mean CV75 [µg/mL]

174.92

SD CV75 [µg/mL]

24.90

The mean CV75 was derived from two single runs and was found to be 174.92±24.9µg/mL. Based on the mean CV75, the main experiment was performed covering a concentration range from 209.91 – 58.58µg/mL (20.99 – 5.86 mg/mL stock solution).

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 18 (first experiment) and passage 19 (second experiment). For each experiment separately weighted samples and preparations were used.

Table 3: 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

-

97.8

97.8

97.7

1817

809

546

1271

263

100

100

333

148

DMSO Control

0.20 %

97.6

97.9

97.8

2041

829

484

1557

345

123

131

422

171

DNCB

4.00

84.7

84.6

83.4

4555

2114

627

3928

1487

252

431

726

337

Test item

209.91

52.3

52.5

53.1

1803

4546

665

1138

3881

90

1476

271

684

174.93

90.4

90.1

92.3

1856

1462

548

1308

914

103

348

339

267

145.77

95.0

95.0

94.9

1616

948

523

1093

425

86

162

309

181

121.48

96.6

96.7

96.8

1534

833

526

1008

307

79

117

292

158

101.23

96.8

96.1

96.8

1370

763

514

856

249

67

95

267

148

84.36

97.4

97.3

97.4

1400

777

521

879

256

69

97

269

149

70.30

97.3

97.2

96.9

1429

820

525

904

295

71

112

272

156

58.58

97.7

97.2

97.5

1444

795

537

907

258

71

98

269

148

Table 4: CD54 and CD86 Expression Experiment 2

Sample

Conc.
[μg/mL]

Cell Viability [%]

Mean Fluorescence Intensity

corrected Mean Fluorescence Intensity

Relative Flourescence Intensity (RFI)

Ratio Isotype IgG1 to [%]

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

C86

CD54

Medium Control

-

98.1

98.1

98.4

1117

617

498

619

119

100

100

224

124

DMSO Control

0.20 %

98.1

98.3

98.3

1190

633

483

707

150

114

126

246

131

DNCB

4.0

73.4

72.3

72.9

3097

1770

612

2485

1158

351

772

506

289

Test item

209.91

47.9

48.0

46.2

1183

3316

700

483

2616

78

2198

169

474

174.93

84.1

84.1

83.9

1140

1064

533

607

531

98

446

214

200

145.77

94.1

94.3

94.4

1013

689

478

535

211

86

177

212

144

121.48

95.4

95.4

96.0

875

634

476

399

158

64

133

184

133

101.23

97.6

97.2

97.2

960

617

491

469

126

76

106

196

126

84.36

97.4

97.4

97.8

930

620

484

446

135

72

114

192

128

70.30

97.7

97.9

97.8

932

615

489

443

126

72

106

191

126

58.58

98.0

98.0

97.9

953

622

487

466

135

75

113

196

128

Table 5: Acceptance criteria

Acceptance criterion

range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

97.6 – 97.9

 

 

pass

 

98.1 – 98.4

 

pass

number of test dosed with viability >50% CD86

≥4

8

pass

7

pass

number of test dosed with viability >50% CD54

≥4

8

pass

7

pass

number of test dosed with viability >50% IgG1

≥4

8

pass

7

pass

RFI of positive control of CD86

≥150

252

pass

351

pass

RFI of positive control of CD54

≥200

431

pass

772

pass

RFI of solvent control of CD86

<150

123

pass

114

pass

RFI of solvent control of CD54

<200

131

pass

126

pass

MFI ratio IgG1/CD86 for medium control [%]

>105

333

pass

224

pass

MFI ratio IgG1/CD86 for solvent control [%]

>105

422

pass

246

pass

MFI ratio IgG1/CD54 for medium control [%]

>105

148

pass

124

pass

MFI ratio IgG1/CD54 for solvent control [%]

>105

171

pass

 

pass

Table 6: Historical data

Criterion

mean

SD

N

cell viability solvent controls [%]

94.7

3.2

70

number of test doses with viability >50 %

-

-

242

RFI of positive control of CD86

292.8

136.2

11

RFI of positive control of CD54

386.0

75.4

11

RFI of solvent control of CD86

109.3

16.2

10

RFI of solvent control of CD54

121.8

15.2

10

MFI ratio IgG1/CD86 for medium control [%]

224.0

77.9

12

MFI ratio IgG1/CD86 for DMSO control [%]

146.3

12.9

12

MFI ratio IgG1/CD54 for medium control [%]

242.5

89.6

12

MFI ratio IgG1/CD54 for DMSO control [%]

156.4

14.2

12

 

 

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Remarks:
the result must be considered in the context of an integrated approach
Conclusions:
The test item did upregulate one cell surface marker in at least two independent experiments. Therefore, the test item is considered to be a sensitiser in accordance with UN GHS category 1. The data generated in this test should be considered in the context of an integrated approach combining the result with other complementary information derived from other in vitro studies addressing the key events of the skin sensitisation AOP.
Executive summary:

In an in vitro study according to the OECD Draft Proposal for a new test guideline, "in vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)", the test item was dissolved in 0.9 % NaCl. A CV75 of 174.92 ± 24.9 µg/mL was derived in a dose range finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 209.91, 174.93, 145.77, 121.48, 101.23, 84.36, 70.30 and 58.58 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.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 52.3 % (CD86), 52.5 % (CD54) and 53.1 (isotype IgG1 control) in the first experiment and to 47.9 % (CD86), 48.0 % (CD54) and 46.2 % (isotype IgG1 control) in the second experiment.

The expression of cell surface marker CD86 was not upregulated above the threshold of 150 % in both independent experiments. No dose response was observed for the induction of CD86.

The expression of the cell surface marker CD54 was upregulated at two concentration steps in the first experiment. The relative expression of CD54 was found to be 1476 % at a concentration of 209.91 µg/mL and 348 % at a concentration of 174.93 µg/mL. The corresponding cell viability was 52.5 % and 90.1 %, respectively, indicating cytotoxic effects triggered by the test item at the highest concentration. Therefore, the upregulation of CD54 at the highest test item concentration should be considered with care.

For the second experiment, upregulated expression of CD54 was observed at a concentration of 209.91 µg/mL (2198 %) and at a concentration of 174.93 µg/mL (446 %). The corresponding cell viability was 48.0 % and 84.1 %, respectively. For the second experiment, the cell viability of the highest tested dose (209.91 µg/mL) should not be considered for evaluation with respect to a sensitising potential since cytotoxicity was below the threshold of 50 %. Irrespective of that, the expression of CD54 exceeded the threshold of 200 % in both independent experiments at a concentration of 174.93µg/mL.

The positive control DNCB led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (252 % experiment 1; 351 % experiment 2) and 200 % for CD54 (431 % experiment 1; 772 % experiment 2) were clearly exceeded.

In this study, the test item did upregulate one cell surface marker in at least two independent experiments. Therefore, the test item is considered to be a sensitiser in accordance with UN GHS category 1.

The data generated in this test should be considered in the context of an integrated approach combining the result with other complementary information derived from other in vitro studies addressing the key events of the skin sensitisation AOP.

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

DPRA

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by addressing the molecular initiating event of the adverse outcome pathway (AOP), namely protein reactivity, by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The percentage depletion value of the cysteine and lysine peptide is used to categorize a substance in one of four reactivity classes to support discrimination between skin sensitiser and non-sensitisers.

In the present study, the test item was dissolved in water and 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 analysis.

After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples of the cysteine and lysine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1 and 2 of the cysteine run. No centrifugation was necessary to perform the HPLC analysis. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.

After the HPLC run samples of the cysteine peptide run were inspected for precipitation, turbidity or phase separation. Precipitation was observed for the test item samples, RC A, RC B, RC C, positive control and for STD 1, 2 and 3 of the cysteine runs. Slight phase separation was observed for the positive control and the respective co-elution control of the lysine run.

Since the turbidity noted for the test item samples was also observed for reference controls, positive controls and standard solutions it can be considered that it is related to the peptide and that it is not a precipitation of the test substance. Additionally, the turbidity did not change during the HPLC analysis period. Since stability of the cysteine peptide in the used acetonitrile batch was demonstrated successfully, the reactivity of the positive control towards the cysteine peptide and peptide depletion were identified correctly and the validity of the cysteine run was acceptable the precipitation was considered as not relevant.

No co-elution of test item with the peptide peaks was observed. Sensitizing 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 C).

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 % (2.03 %). 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 62.90 %.

The controls confirmed the validity of the study for both, the cysteine and lysine run. For the cysteine run the coefficient of determination for the calibration curve was >0.99 (0.9995). The mean peptide depletion of the cysteine peptide by the positive control was between 60.8 % and 100 % (72.27 %).

The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and <0.55 mM (RC A: 0.4969 mM, RC C (acetonitrile): 0.4827 mM, RC C (water): 0.4584 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 2.38 %, RC C (acetonitrile): 0.85 %, RC C (water): 9.07 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <14.9 % (PC: 0.90 %; test item 0.00 %).

For the lysine run the coefficient of determination for the calibration curve was >0.99 (0.9996). The mean peptide depletion of the lysine peptide by the positive control was between 40.2 % and 69.0 % (53.53 %). The mean peptide concentration of reference control A and reference control C (acetonitrile and water) was between >0.45 and < 0.55 mM (RC A 0.5067 mM, RC C (acetonitrile): 0.5017 mM, RC C water): 0.4930 mM). The coefficient of variation (CV) of the peak area (PA) of reference control B and reference control C (acetonitrile and water) was <15 %. (RC B: 0.54 %, RC C (acetonitrile: 0.61 %, RC C water): 0.54 %). The SD of the peptide depletion for the replicates of the positive control as well as for the tested test item samples was <11.6 % (PC: 0.22 %; test item: 0.10 %).

The test item can be considered as non-sensitiser in the DPRA.

 

KeratinoSens assay

In a study conducted according to OECD test guideline 442D (adopted February 04, 2015), transgenic keratinocytes constitutively expressing an ARE-reporter gene were incubated with Sophorolipids, fermentation products of glucose and fatty acids, at concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 and 0 µM (solvent control) for 48 h at 37 °C. Afterwards the test substance containing medium was removed and the cells lysed and luminescence subsequently measured. Beside the luminescence the cell viability was measured using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay method.

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

- EC 1.5 value is < 1000 µM

- an apparent overall dose-response for luciferase induction

In the first experiment, a max luciferase activity (Imax) induction of 7.96 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 21.8 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC1.5 was < 1000 µM (67.67 µM).

In the second experiment, a max luciferase activity (Imax) induction of 4.63 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 24.0 % indicating severe cytotoxicity. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of I max. The calculated EC1.5 was < 1000 µM (73.70 µM).

In the third experiment, a max luciferase activity (Imax) induction of 7.78 was determined at a test item concentration of 125.00 µM. The corresponding cell viability was 18.0 %. The lowest tested concentration with a significant luciferase induction >1.5 was identical to that of Imax. The calculated EC1.5 was < 1000 µM (68.85 µM).

Since the induction of the luciferase was observed only at cytotoxic test item concentrations (viability < 70 %), the effect cannot be considered for sensitization evaluation. Additionally, no clear dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

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 (2.98 (experiment 1), 5.07 (experiment 2); 4.36 (experiment 3)). The calculated EC1.5 was between 7 and 30 µM (21.66 µM (experiment 1), 14.38 µM (experiment 2); 15.30 µM (experiment 3)). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20 % (13.9 % (experiment 1), (9.4 % (experiment 2), (14.0 % (experiment 3)).

Under the conditions of this study the test item is therefore considered as non-sensitiser at non-cytotoxic concentrations.

 

h-CLAT

In an in vitro study according to the OECD Draft Proposal for a new test guideline, "in vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)", the test item was dissolved in 0.9 % NaCl. A CV75 of 174.92 ± 24.9 µg/mL was derived in a dose range finding assay. Based on the CV75, the main experiment was performed covering the following concentration steps: 209.91, 174.93, 145.77, 121.48, 101.23, 84.36, 70.30 and 58.58 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.

Cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was reduced to 52.3 % (CD86), 52.5 % (CD54) and 53.1 (isotype IgG1 control) in the first experiment and to 47.9 % (CD86), 48.0 % (CD54) and 46.2 % (isotype IgG1 control) in the second experiment.

The expression of cell surface marker CD86 was not upregulated above the threshold of 150 % in both independent experiments. No dose response was observed for the induction of CD86.

The expression of the cell surface marker CD54 was upregulated at two concentration steps in the first experiment. The relative expression of CD54 was found to be 1476 % at a concentration of 209.91 µg/mL and 348 % at a concentration of 174.93 µg/mL. The corresponding cell viability was 52.5 % and 90.1 %, respectively, indicating cytotoxic effects triggered by the test item at the highest concentration. Therefore, the upregulation of CD54 at the highest test item concentration should be considered with care.

For the second experiment, upregulated expression of CD54 was observed at a concentration of 209.91 µg/mL (2198 %) and at a concentration of 174.93 µg/mL (446 %). The corresponding cell viability was 48.0 % and 84.1 %, respectively. For the second experiment, the cell viability of the highest tested dose (209.91 µg/mL) should not be considered for evaluation with respect to a sensitising potential since cytotoxicity was below the threshold of ≤50 %. Irrespective of that, the expression of CD54 exceeded the threshold of 200 % in both independent experiments at a concentration of 174.93 µg/mL.

The positive control DNCB led to an upregulation of CD54 and CD86 in both experiments. The threshold of 150 % for CD86 (252 % experiment 1; 351 % experiment 2) and 200 % for CD54 (431 % experiment 1; 772 % experiment 2) were clearly exceeded.

In this study, the test item did upregulate one cell surface marker in at least two independent experiments. Therefore, the test item is considered to be a sensitiser in accordance with UN GHS category 1.

 

Further supporting data are available. In a dermal sensitisation study with a source substance, groups of 4 female CBA mice were tested using the LLNA method according to OECD Guideline 429, 24 April 2002. The source substance was considered to be a non-sensitiser under the conditions of the test.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfiled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.

Justification for classification or non-classification

The test item “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed”, is considered to be a non-sensitiser. This decision is based on the following experimental approach:

Two out of three reliable in chemico and in vitro studies gave no indication of a sensitising potential of “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed”. Additionally, in a read-across study based on the mouse local lymphnode assay (LLNA), the source substance Sophorolipid C18 unsaturated acid was found to be a non-sensitiser.

The DPRA as well as the ARE-Nrf2 Luciferase Test Method both clearly lead to the result that the test item is a non-sensitiser. In the third study (h-CLAT), the test item did upregulate one cell surface marker in at least two independent experiments and was considered to be a sensitiser in accordance with UN GHS category 1. Cytotoxicity of the test item was observed (CV75: 174.92 ± 24.9 µg/mL).

The Direct Peptide Reactivity Assay (DPRA), the ARE-Nrf2 Luciferase Test Method and the in vitro human cell line activation test (h-CLAT) are validated test methods for the assessment of skin sensitisation which have not been developed as stand-alone test methods, but to be used in a Weight-of-Evidence approach. When used in an AOP-based IATA, the outcome of these studies targets key events along the defined toxicity pathway and the results enable a regulatory decision. The AOP “2 out of 3” approach predicts skin sensitization hazard by sequential testing in up to three internationally accepted non-animal methods that map to key events 1-3 of the AOP (ENV/JM/MONO(2016)67, ENV/JM/MONO(2016)29). The overall result is based on the two concordant findings.

In this AOP-based IATA, no indication of skin sensitisation was found regarding key event 1 and 2 tested with the DPRA and the ARE-Nrf2 Luciferase Method. Even though the third assay (h-CLAT) lead to a positive result, the criteria of the “2 out of 3” approach are fulfilled, and a regulatory decision can be made. Besides, it must be taken into consideration that the test item showed pronounced cytotoxicity in the h-CLAT assay which may render these results less reliable. Furthermore, related scientific literature supports the higher relevance of DPRA results- which were clearly negative here- for exploration of the AOP as compared to other tests (Asturiol et al. 2016; Benigni et al. 2016).

Taken together, the test item “Sophorolipids: fermentation products of glucose and fatty acids, C18-unsatd., esters with glycerol with yeast Candida Bombicola, partially hydrolysed” is considered to be a non-sensitiser according to the AOP “2 out of 3” approach and a supporting read-across study.