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Administrative data

Description of key information

Skin sensitization (OECD 442D): not sensitizing

Skin sensitization (OECD 442C): not sensitizing

Skin sensitization (OECD 442E): sensitizing

Skin sensitization (Open epicutaneous test, 1985): not sensitizing

Skin sensitization (Open epicutaneous test, 1970): not sensitizing

Based on the available in vitro and in vivo tests (DPRA, KeratinoSens, h-CLAT, 2 Open epicutaneous tests) for the read-across substance, the target substance is not considered to be a skin sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January - April 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD guideline 442E h-CLAT
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Details on study design:
TEST SYSTEM
- Cell line: THP-1 cells; human monocytic leukemia cell line
- Source: American Type Culture Collection Manassas, USA (ATCC, TIB-202)

TEST-SUBSTANCE PREPARATION
- Concentrations: Dose range finder: 7.81, 15.63, 31.25, 62.50, 125, 250, 500, 1000 µg/mL; Main experiment: 97.71, 81.42, 67.85, 56.54, 47.12, 39.27, 32.72, 27.27 µg/mL
- Stock: 2x concentration of the highest concentration stock solution
- Solvent: DMSO

CONTROLS
- Negative control (NC):Medium
- Solvent control (SC): 0.2% DMSO
- Isotype control: In order to help distinguish non-specific staining from specific antibody staining each test-substance concentration and control is additionally incubated with mouse IgG1
- Positive control: 1-chloro-2-4-dinitrobenzene (DNCB); 4.0 µg/mL

MEDIUM
- Culture medium: RPMI 1640: with L-glutamine, 25 mM HEPES (Gibco) + 10% FBS inactivated + 1% Penicillin/Streptomycin + 0.05 mM 2-Mercaptoethanol (Gibco)
- FACS Buffer: Phosphate Buffered Saline (DPBS) without Ca2+ / Mg2+ (Gibco) + 0.1% BSA (Sigma A7030)
- Blocking Solution: 0.01% Globulins Cohn fraction II,III with DPBS
- Reagent for cytotoxicity test: Propidium iodide (Sigma)

EXPERIMENTAL PROCEDURE
- Replicates: 1
- Experiments: 3
- Exposure period: 24 hours
- Preparation of cells: THP-1 cells were thawed and cultured in complete RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/mL penicillin, 100 µg/mL streptomycin and 0.05 mM 2 -mercaptoethanol (30 > passage >= 5)

ANALYSIS
- FACS: cell staining and flow cytometric analysis 24 hours after exposure

DATA EVALUATION
- CV75 calculation: Relative survival rate is calculated by linear extrapolation. This value is the substance concentration at which cell viability is 75% compared to the vehicle control.
- Relative cell viability: % relative cell viability = (number of living cells / total number of aquired cells) * 100
- Relative fluorescence intensity: RFI (%) = (MFI of chemical-treated cells - MFI of chemical-treated isotype control cells) / (MFI of solvent control cells - MFI of solvent isotype control cells) * 100
- Calculation of EC 150% and EC 200%: If applicable, the concentration resulting in a positive response (RFI of 150% (CD86) or 200% (CD54) and viability >50%) was calculated for each cell surface marker from each experiment. The calculation is performed by linear regression from the two concentrations directly above and below the EC 150% / EC 200% concentration.

ACCEPTANCE CRITERIA
- A tested concentration is not to be further evaluated when viability is less than 50%
- Cell viability of vehicle control cells must yield at least 90%
- In the positive control (DNCB), RFI (relative fluorescence intensity) values of both CD86 and CD54 should be exceed the positive criteria (RFI CD86 =150% and CD54 =200%) and cell viability should be =50%.
- For all vehicle control, the MFI (mean fluoresence intensity) ratio of both CD86 and CD54 to isotype controls should be =105%.
- A study is considered to be acceptable if the positive, negative and vehicle control data lies within the range of the historic data.

EVALUATION RESULTS
- Positive result: A test is considered to be positive when the dendritic cells are activated meaning that CD86 expression is increased =150% and/or CD54 expression increased =200% in relation to vehicle control in at least 2 independent experiments.
- Negative result: A test is considered to be negative when the criteria mentioned above are not met up to the maximum concentration (=5000 µg/mL for the vehicle culture medium or 2000 µg/mL for 0.2% DMSO in culture medium).
Positive control results:
Positive controls achieved RFI values of both CD86 and CD54 over the positive criteria (CD86= 264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3).
Key result
Parameter:
other: RFI CD54 mean (%) and RFI CD54 mean (%)
Run / experiment:
Please see 'Any other information on results incl. tables'.
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

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

-

94.7

94.7

94.3

2943

1205

613

2330

592

101

111

480

197

Solvent Control

0.20%

94.8

95.2

94.8

2988

1214

679

2309

535

100

100

440

179

DNCB

4.00

80.9

80.5

80.0

6868

2013

779

6089

1234

264

231

882

258

ISOBUTYL SALICYLATE

97.71

12.4

11.9

12.3

4427

3822

942

3485

2880

151

538

470

406

81.43

57.3

58.4

57.4

3480

1781

726

2754

1055

119

197

479

245

67.85

83.9

83.4

84.1

3116

1540

673

2443

867

106

162

463

229

56.55

88.9

89.3

89.5

3066

1380

658

2408

722

104

135

466

210

47.12

93.2

93.5

93.6

2778

1235

669

2109

566

91

106

415

185

39.27

94.5

94.3

93.9

2801

1230

686

2115

544

92

102

408

179

32.72

94.4

94.8

94.9

2996

1287

660

2336

627

101

117

454

195

27.27

94.7

94.6

94.7

3063

1276

669

2394

607

104

113

458

191

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

-

94.7

94.8

93.9

4619

1554

646

3973

908

102

95

715

241

Solvent Control

0.20%

95.0

95.1

94.9

4571

1620

669

3902

951

100

100

683

242

DNCB

4.0

81.4

80.5

81.2

13735

3370

633

13102

2737

336

288

2170

532

ISOBUTYL SALICYLATE

97.71

13.2

12.8

12.4

7467

5420

935

6532

4485

167

472

799

580

81.43

45.9

45.5

45.7

6028

3615

1213

4815

2402

123

253

497

298

67.85

79.3

79.0

79.7

5291

3011

782

4509

2229

116

234

677

385

56.55

90.0

90.8

89.7

4710

2761

874

3836

1887

98

198

539

316

47.12

93.5

93.5

92.7

4543

2413

735

3808

1678

98

176

618

328

39.27

93.4

92.5

92.8

4587

2304

746

3841

1558

98

164

615

309

32.72

94.6

94.1

94.2

4335

2049

756

3579

1293

92

136

573

271

27.27

95.1

95.0

94.9

4489

2013

790

3699

1223

95

129

568

255

CD54 and CD86 Expression Experiment 3

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

-

95.2

94.5

94.3

3732

1289

640

3092

649

88

87

583

201

Solvent Control

0.20%

94.7

94.5

94.5

4176

1395

649

3527

746

100

100

643

215

DNCB

4.0

81.9

81.1

82.4

11303

2502

583

10720

1919

304

257

1939

429

ISOBUTYL SALICYLATE

97.71

40.5

38.6

39.3

4938

2769

816

4122

1953

117

262

605

339

81.43

64.4

64.9

64.8

4625

2417

806

3819

1611

108

216

574

300

67.85

86.4

85.4

86.3

4465

2304

739

3726

1565

106

210

604

312

56.55

93.0

91.8

93.0

4049

1925

742

3307

1183

94

159

546

259

47.12

94.0

93.8

94.4

3792

1728

740

3052

988

87

132

512

234

39.27

94.5

94.1

93.9

3707

1636

730

2977

906

84

121

508

224

32.72

95.0

94.9

95.2

3538

1580

735

2803

845

79

113

481

215

27.27

95.2

95.6

95.0

4119

1701

811

3308

890

94

119

508

210

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Based on the results, it is concluded that the test substance induces dendritic cell activation (CD54 expression increased =200% in at least one assay).

Executive summary:

The potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

 

In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 3 valid experiments were performed. The expression of the cell surface marker CD54 clearly exceeded the threshold at one concentration with a cell viability ≥50% in at least two independent runs.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3) were clearly exceeded.

 

In summary, it has to be concluded that the test substance induces dendritic cell activation.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01.06.2017-08.02.2018
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:
February 04, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany
Type of study:
direct peptide binding assay
Details on study design:
TEST-SUBSTANCE PREPARATION
- Stock solution: 100 mM
- Vehicle: Acetonitrile main experiment 1 (exp. 1) and methanol main experiment 2 (exp. 2).
- Reason for the vehicle: The test item was not soluble in dist. water and dist. water : acetonitrile 1:1 (v/v).

CONTROLS
- Reference controls (RCs) were set up in parallel to sample preparation in order to verify the validity of the test run.
- Co-elution control: buffer and test substance without the peptide
- Positive control: Cinnamic aldehyde in acetonitrile

PEPTIDES
- Synthetic peptides:
-- Cysteine- (C-) containing peptide: 22.61 mg (exp. 1) and 20.15 mg (exp. 2); amino acid sequence: Ac-RFAACAA; dissolved in a defined volume (43.9 mL exp. 1 / 39.18 mL exp. 2) of a phosphate buffer with pH 7.5 to reach a concentration of 0.667 mM
-- Lysine- (K-) containing peptide: 20.94 mg (exp. 1) and 19.32 mg (exp. 2); amino acid sequence: Ac RFAAKAA; dissolved in a defined volume of ammonium acetate buffer with pH 10.2 (36.69 mL exp. 1 / 39.74 mL exp. 2) to reach a concentration of 0.667 mM

EXPERIMENTAL PROCEDURE
- Replicates: 3 for each peptide
- Determination remaining non-depleted peptide concentration: HPLC at 220nm: HPLC analysis started 22 to 26 hours after sample preparation and the analysis time was less than 30 hours.
- Calibration samples: samples of a known peptide concentration are measured in parallel

PREPARATIONS SAMPLES
- Calibration sample was prepared from the peptide stock solution in 20% acetonitrile in the respective buffer using serial concentration: 0.534, 0.267, 0.134, 0.067, 0.033, 0.017 or 0.000 mM peptide
- Test-substance samples: samples were incubated at 25°C +/- 2.5°C in the dark for 24 +/- 2 hours
- Reference controls, co-elution controls as well as the positive control were set up in parallel (see table below)

MEASUREMENT PEPTIDE CONCENTRATIONS
- Method: HPLC Agilent 1200 series
- Wavelength: 220 nm and 258 nm
- Detector: UV detector

DATA EVALUATION
- Concentration of the cysteine and lysine peptide determined from absorbance at λ = 220 nm, measuring the area of the appropriated peaks (peak area (PA)) and calculating the concentration of peptide using the linear calibration curves.
- The percent peptide depletion (PPD) was calculated according to the following formula: PPD = [ 1 – ( Peptide Peak Area in the Replicate Injection / Mean Peptide Peak Area in the Reference Control C)] * 100

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

EVALUATION RESULTS
Cysteine 1:10/ Lysine 1:50 Prediction Model:
- Chemical reactivity was determined by mean peptide depletion [%] and was rated as
-- high: mean peptide depletion > 42.47
-- moderate: mean peptide depletion > 22.62 ≤ 42.47
-- low: mean peptide depletion > 6.38 ≤ 22.62
-- minimal: mean peptide depletion ≤ 6.38
High, moderate and low reactivity are evaluated as positive.
In case of co-elution of the test item with a peptide peak, the peak cannot be integrated correctly and the calculation of the PPD is not possible.
If severe co-elution occurs with both peptides then the analysis was reported as "inconclusive".

- In cases where the co-elution occurs only with the lysine peptide prediction model 2 can be applied (cysteine 1:10 prediction model) :
-- high: mean peptide depletion > 98.24
-- moderate: mean peptide depletion > 23.09 ≤ 98.24
-- low: mean peptide depletion > 13.89 ≤ 23.09
-- minimal: mean peptide depletion ≤ 13.89
High, moderate and low reactivity are evaluated as positive.

DEVIATIONS FROM THE STUDY PLAN
change of supplier for some reagents: the deviation did not influence the quality or integrity of the present study.
Positive control results:
Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control (Positive control values for cystein: 72.27 %, SD 0.27 %; positive control values for lysine: 63.51 %, SD 0.86 %) were fulfilled, the observed precipitations and phase separation were regarded as insignificant.
Parameter:
other: mean depletion % of peptide
Run / experiment:
Experiment 1, both peptides
Value:
0.32
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
no prediction can be made (tested in concentration < 100 mM)
Key result
Parameter:
other: mean depletion % of peptide
Run / experiment:
Experiment 2, cysteine peptide
Value:
0.3
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Based on the prediction model 2
Parameter:
other: mean depletion % of peptide
Run / experiment:
Experiment 2, lysine peptide
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
Phase separation
Other effects / acceptance of results:
Exp. 1: Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

Exp. 2: Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as insignificant.

Results of the Cysteine Peptide Depletion

Depletion of the Cysteine Peptide (Experiment1)

Sample

Peptide concentration
(mM)

Peptide Depletion
(%)

Mean Peptide Depletion
(%)

SD of Peptide Depletion (%)

CV (%)

Positive control

0.1438

71.3

71.38

0.25

0.35

0.1444

71.18

0.1420

71.66

Test item

0.5005

0.30

0.64

0.32

50.31

0.4986

0.68

0.4976

0.93

Depletion of the Cysteine Peptide (Experiment2)

Sample

Peptide concentration
(mM)

Peptide Depletion
(%)

Mean Peptide Depletion
(%)

SD of Peptide Depletion (%)

CV (%)

Positive control

0.1446

72.07

72.27

0.27

0.38

0.1441

72.17

0.1419

72.58

Test item

0.5155

0.00

0.30

0.52

173.21

Results of the Lysine Peptide Depletion

Depletion of the Lysine Peptide (Experiment1)

Sample

Peptide concentration
(mM)

Peptide Depletion
(%)

Mean Peptide Depletion
(%)

SD of Peptide Depletion (%)

CV (%)

Positive control

0.2081

58.68

57.65

1.11

1.93

0.2125

57.80

0.2193

56.47

Test item

0.5085

0.00

0.00

0.00

#DIV/0 !

0.5072

0.00

0.5074

0.00

Depletion of the Cysteine Peptide (Experiment2)

Sample

Peptide concentration
(mM)

Peptide Depletion
(%)

Mean Peptide Depletion
(%)

SD of Peptide Depletion (%)

CV (%)

Positive control

0.1777

64.50

68.51

0.86

1.35

0.1848

63.08

0.1854

62.95

Test item

0.4932

3.47

3.87

0.39

10.08

0.4911

3.89

0.4893

4.25

Interpretation of results:
GHS criteria not met
Conclusions:
In the first experiment using acetonitrile as solvent under the given conditions, the test item showed minimal reactivity towards the peptides. Due to the observed precipitation in both experiments and phase separations in the lysine run, the prediction model does not apply and a prediction cannot be made.

In the second experiment using methanol as solvent under the given conditions, the test item showed minimal reactivity towards the cysteine peptide. The test item can be considered as “non-sensitiser”.
Executive summary:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine. The test was performed according to OECD TG 442C and in compliance to GLP.

Experiment 1

In the present study the test item was dissolved in acetonitrile based on the results of the pre-experiments. Based on a molecular weight of 194.23 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. A slight turbidity was observed for the standard 1 (0.534 mM). Precipitation at the bottom was noted in the samples of the positive control (excluding the co-elution control) as well as for the samples and co-elution control of the test item. Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM stock solution of the test item, turbidity was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the positive controls. However, the turbity noted for the test item sample 24 h before was gone. Instead phase separation was observed for the samples of the test item and their co-elution control. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the linearity of the standard curve as well as for the depletion range of the positive control were fulfilled, the observed precipitations and phase separation were regarded as insignificant.

The stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was < 6.38% (0.32%). However, due to the observed precipitation in both experiments and phase separations in the lysine run prior to the HPLC analysis, a test item concentration of 100 mM as well as the full contact of peptide and test item is not guaranteed. According to the evaluation criteria in the guideline, no firm conclusion on the lack of reactivity should be drawn from a negative result, if a test chemical is tested in concentration < 100 mM.

Therefore, no prediction can be made.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 64.52%.

Experiment 2:

In the present study the test item was dissolved in methanol based on the results of the pre-experiments. Based on a molecular weight of 194.23 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM stock solution of the test item turbidity was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. No precipitation, turbidity or phase separation was observed for any of the samples.

For the 100 mM stock solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation was observed for the samples of the test item (including co-elution control) and the co-elution of the positive control. Samples were not centrifuged prior to the HPLC analysis.

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

Phase separation in the lysine experiment was observed. Therefore, the given peak areas and corresponding lysine peptide values can only be considered as an estimation of the peptide depletion and cannot be used for evaluation.

Sensitising potential of the test item was predicted from the mean peptide depletion of the cysteine peptide by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC Cmethanol). The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptide. The mean depletion of the cysteine peptide was ≤ 13.89% (0.30%). Based on the prediction model 2 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 67.89%.

Conclusions:

In the first experiment using acetonitrile as solvent under the given conditions, the test item showed minimal reactivity towards the peptides. Due to the observed precipitation in both experiments and phase separations in the lysine run, the prediction model does not apply and a prediction cannot be made.

In the second experiment using methanol as solvent under the given conditions, the test item showed minimal reactivity towards the cysteine peptide in the second experiment. The test item can be considered as “non-sensitiser”.

The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01.06.-22.09.2017
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:
February 04, 2015
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Rathausgasse 4, 91126 Schwabach, Germany
Type of study:
activation of keratinocytes
Details on study design:
Vehicle:
dimethyl sulfoxide (DMSO)

Concentrations:
the following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 pM

Controls:
- negative control: DMSO: 1%
- positive control: Cinnamic aldehyde 4 pM, 8 pM, 16 pM; 32 pM; 64 pM

Cell line:
- transgenic cell line KeratinoSensTM (Givaudan, Switzerland), derived from human keratinocytes (HaCaT) transfected with a stable insertion of the Luciferase construct

Replicates:
- three replicates in every independent run (test item and positive control)
- The negative control was assessed using six replicates per plate in every independent run.

Experimental Procedure:
- After seeding cells were grown for 24 h ± 1 h at 37 °C ± 1 °C and 5% CO2
- Cells were incubated with the test item for 48 h ± 1 h at 37 °C ± 1 °C and 5% CO2.
- Luciferase activity:
-- Cells were washed once with DPBS, ubsequently 20 pL 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
-- luminescence measurement. in the plate reader waited for 1.000 ms before assessing the luciferase activity for 2.000 ms
- Cell viability:
-- incubated for 4 h at 37 °C ± 1 °C and 5% CO2 with MTT solution
-- after removal of the MTT incubated in the incubator at 37 °C ± 1 °C and 5% CO2 overnight (experiment 1 and 2)
-- OD was measured at 600 nm.

Prediction Model:
- The test item is considered positive in accordance with UN GHS "Category 1" if the following conditions were met in at least two independently prepared test repetitions:
- Imax is >1.5 fold increased and statistically significant (p <0.05) compared to the negative control
- cell viability is >70% at the lowest concentration with an induction of luciferase activity >1.5
- EC1.5 value is <1000 pM
- 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 pM is considered as inconclusive.

Acceptance Criteria:
The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of 64 pM 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.

Deviations from the Study Plan:
There were no deviations from the study plan.
Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 pM was between 2 and 8 (3.39 in experiment 1; 2.27 in experiment 2).
Parameter:
other: max luciferase activity (Imax) induction
Run / experiment:
Exp. 1
Value:
1.12
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Parameter:
other: max luciferase activity (Imax) induction
Run / experiment:
Exp. 2
Value:
1.13
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
The controls confirmed the validity of the study. The calculated EC1.5 was between 7 and 34 µM (24.15 µM in experiment 1; 21.59 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (14 % in experiment 1; 6.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 3.39 and thus the criteria was fulfilled. In the second experiment the activity was 2.27. Thus the acceptance criteria were met.

Induction of Luciferase Activity - Overall Induction

  Concentration (microM) Fold induction
Experiment 1 Experiment 2 Mean SD
Solvent Control   1.00 1.00 1.00 0.00
Positive Control 4.0 1.09 1.09 1.09 0.00
8.0 1.12 1.24 1.18 0.09
16.0 1.28 1.33 1.31 0.04
32.0 1.71 1.81 1.76* 0.07
64.0 3.39 2.27 2.83 0.08
Test Item 0.98 1.07 1.13 1.10 0.04
1.95 1.11 1.10 1.10 0.01
3.91 1.07 1.04 1.05 0.02
7.81 1.00 1.09 1.05 0.07
15.63 0.98 1.03 1.01 0.04
31.25 1.03 1.08 1.05 0.03
62.50 1.12 1.00 1.06 0.09
125.00 0.00 1.05 0.53 0.74
250.00 0.00 0.00 0.00 0.00
500.00 0.00 0.01 0.01 0.01
1000.00 0.00 0.00 0.00 0.00
2000.00 0.00 0.00 0.00 0.00

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

Interpretation of results:
GHS criteria not met
Conclusions:
In this study according to OECD 442D under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSensTM cell line in at least two independant runs.
Therefore, the test item can be considered as non-sensitiser. The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach.
Executive summary:

The in vitro KeratinoSensTM assay according to OECD 442D and GLP enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSensTM. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and

non-sensitisers.

In the present study the test item was dissolved in DMSO. Based on a molecular weight of 194.23 g/mol a stock solution of 200 mM was prepared. Based on the stock solution, a set of twelve master solutions in 100% solvent was prepared by serial

dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay: 2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 pM.

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

In the first experiment, a max luciferase activity (Imax) induction of 1.12 was determined at a test item concentration of 62.50 pM. The corresponding cell viability was 67.7%. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. Cytotoxicity was observed in the concentration range 62.50 pM — 2000 pM.

In the second experiment, a max luciferase activity (Imax) induction of 1.13 was determined at a test item concentration of 0.98 pM. The corresponding cell viability was 95.1%. No significant luciferase induction > 1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. Cytotoxicity was observed in the concentration range 125 pM — 2000 pM.

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSensTM cell line in at least two independent experiment runs. Therefore, the test item can be considered as non-sensitiser.

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

Skin sensitisation in vitro (key studies)

In vitro studies covering the three key steps of the adverse outcome pathway for skin sensitization as identified by the OECD (OECD Publication No.168; ENV/JM/MONO(2012)10) were performed. These study types have initially undergone in-house validation in the test facility using 54 substances (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504). Recently, a validation performed with 213 substances has been published (Urbisch et al. 2015 Regul Toxicol Pharmacol. 71: 337-351).

 

Based on the results of the in-house validation (Bauch et al., 2012 Regul Toxicol Pharmacol. 63: 489-504) the predictive capacity of the evaluation scheme using DPRA (peptide reactivity), LuSens/KeratinoSens™ (keratinocyte activation) and (m)MUSST/h-CLAT (dendritic cell activation) is comparable to that of the local lymph node assay. This was confirmed in the validation study with more substances (Urbisch 2015): When compared to the sensitization potential of the substances in humans, the classification based on an evaluation scheme using results obtained from the DPRA, LuSens and mMUSST had a sensitivity of 90%, a specificity of 90 % and an accuracy of 90 %.

 

In the evaluation scheme used here any two of the three test results determine the overall classification, i.e. any two positive test results drive the prediction of a sensitizer, while any two negative test results drive the prediction of a test substance to be a non-sensitizer (Bauchet al. 2012; Table 1). If two assays (DPRA ,LuSens or KeratinoSens, MUSST or h-CLAT) yield concordant results, the result of the third assay is not necessarily required to determine the overall outcome of the evaluation scheme.

 

Table 1: Decision matrix for combinations of DPRA, LuSens/KeratinoSens and MUSST/ h-CLAT assays.

DPRA

LuSens/

KeratinoSensTM

MUSST/

h-CLAT

Test battery evaluation

positive

positive

positive

sensitizer

positive

positive

negative

sensitizer

positive

negative

positive

sensitizer

positive

negative

negative

non-sensitizer

negative

positive

positive

sensitizer

negative

positive

negative

non-sensitizer

negative

negative

positive

non-sensitizer

negative

negative

negative

non-sensitizer

Each individual assay was performed under GLP-conditions. All three assays follow the procedures of the respective OECD testing guidelines (OECD 442C, D and E).

 

The test battery applicability is limited when testing substances insoluble in the commonly used vehicles and highly volatile substances. Substances susceptible to base-catalyzed hydrolysis cannot be evaluated reliably for binding to lysine as the incubation is performed at pH 10.2. Also substances that interfere with the experimental measurements (e.g., co-elution with the peptide in the DPRA-assay) are not or only partially suitable for testing using in vitro methods.

In accordance with the published evaluation scheme (Bauchet al., 2012 Regul Toxicol Pharmacol. 63: 489-504) and Sections 1.2 and 1.4 of Annex XI of EC regulation 1907/2006, the test substance is judged not to be a skin sensitizer.

Details on the assessed tests:

OECD 442C:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of the test item. All analytical acceptance criteria for each peptide run were met. Solutions of the test item were successfully analysed by the DPRA analytical method in both Cysteine and Lysine containing synthetic peptides.

In the first experiment using acetonitrile as solventunder the given conditions, the test item showed minimal reactivity towards the peptides. Due to the observed precipitation in both experiments and phase separations in the lysine run,the prediction model does not apply and a prediction cannot be made.In the second experiment using methanol as solvent under the given conditions, the test item showed minimal reactivity (maxium depletion of 0.30%) towards the cysteine peptide in the second experiment. The test item can be considered as “non-sensitiser”.The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

OECD 442D:

The in vitro KeratinoSens™ assay according to OECD 442D enables detection of the sensitising potential of a test item by addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers. In the present study the test substance was dissolved in DMSO. Based on a molecular weight of 194.23 g/mol a stock solution of 200 mM was prepared. Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using a constant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium. The following concentration range was tested in the assay: 2000,1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement. In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. In the second experiment, no significant luciferase induction >1.5 was found in the tested concentration range. Therefore, no EC1.5 value could be calculated. No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction. Under the condition of this study the test item is therefore considered as non sensitiser. The controls confirmed the validity of the study. The calculated EC1.5 was between 7 and 34 µM (24.15 µM in experiment 1; 21.59 µM in experiment 2). The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (14 % in experiment 1; 6.4% in experiment 2). The luciferase activity induced by the positive control at a concentration of 64 µM should be between 2 and 8. In the first experiment the luciferase activity was 3.39, in the second experiment 2.27 and thus the criteria was fulfilled.

OECD 442E

In the third key study the potential of test substance to induce the cell membrane markers CD86 and CD54 expression was evaluated in the Human Cell Line Activation Test (h-CLAT). For this purpose the test substance was incubated with human pro-monocytic cell line THP-1 for ca. 24 hours at 37°C and membrane markers expression measured by flow cytometry. In order to determine the concentrations suitable for the main experiment a pre-test was performed. Cells were exposed to 10 concentrations of the test substance and cytotoxicity was determined thereafter by propidium iodide (PI) intercalation into the DNA. The CV75 value (=estimated concentration that affords 75% cell viability) was determined by linear regression from the concentration response curve.

In the main test after 24 hour exposure THP-1 cells were stained with FITC labelled anti-human-CD86/ anti-human-CD54 antibody and propidium iodide and the fluorescence intensity was analysed using flow cytometry. A total of 3 valid experiments were performed. The expression of the cell surface marker CD54 clearly exceeded the threshold at oneconcentration with acellviability ≥50%inatleasttwoindependentruns.

The positive control (DNCB) led to an upregulation of CD54 and CD86 in all three experiments. The threshold of 150% for CD86 (264% experiment 1; 336% experiment 2; 304% experiment 3) and 200% for CD54 (231% experiment 1; 288% experiment 2; 257% experiment 3) were clearly exceeded.

Skin sensitisation in vivo (supporting studies)

In the Open Epicutaneous Test (OET, 1985) the skin sensitizing effect of the test item was evaluated.

The study was not according to GLP or OECD, however, it is similar to OECD 406. Various test concentrations were used and per group at least 6 guinea pigs were included.

The maximum applied doses for the induction phase and the challenge are unknown. The test item was considered not to be a sensitiser as no reactions occured.

In the second study (1970) study, the sensitisation propreties of the test item has been determined probing white male guinea pigs. The test item was first dissolved in alcohol and then suspended in distilled water so that a 0.1% suspension was in 5% ethyl alcohol in distilled water.

All injection were intradermal. The first and the Challenge injections were 0.05 mL. The second and the consecutive tenth injections were 0.1 mL. The induction injections was performed on alternative days during 3.5 weeks. The callenge injection was performed 10 days to two weeks after the last induction injection. The reading was conducted 24 hours after the challenge injection. The observation demonstrated that the reaction after the challenge injection did not go over the average reaction of the induction injection.

In conclusion, the results show that the test item does not have sensitising propreties under that test conditions.

Overall conclusion

Considering all studies of the source substance, the target substance is not considered to be a skin sensitizer.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on skin sensitization for the read-across substance, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.