Registration Dossier

Administrative data

Description of key information

OECD Guideline in vitro/ in chemico studies have been performed to address each of the key events of skin sensitisation (molecular interaction with skin proteins, inflammatory response in keratinocytes and activation of dendritic cells). The results of these tests are considered valid and all tests concluded that balsalazide acid is not a skin 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:
7th June 2018 - 19th July 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))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
This test method is able to detect chemicals that cause skin sensitisation and may be used on its own to classify a chemical into 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 IATA, combining them with other complementary information e.g., derived from in vitro assays addressing other key events of the AOP
Specific details on test material used for the study:
Batch No.: 0400
Purity: 96%
Appearance: Orange solid
Storage conditions: Room temperature
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 64.83%.
Key result
Parameter:
other: Mean peptide depletion (%)
Run / experiment:
Test item
Value:
2.41
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Reference controls
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes: The mean depletion of both peptides was 64.83%.

Pre-Experiments

Solubility of the test item was determined prior to the main experiment. The test item was soluble in dist. water : acetonitrile 1:1 (v/v). No turbidity, precipitation and phase separation was observed for the test item solution. All test item preparations of the main experiment were prepared using dist. water : acetonitrile 1:1 (v/v). All test item solutions were freshly prepared immediately prior to use

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. 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 positive control including the co-elution control. No precipitation, turbidity or phase separation was observed for the samples of the test item. Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria for the depletion range of the positive control were fulfilled, the observed phase separation was regarded as not relevant

Co-elution with the Peptide Peaks

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

Depletion of the cysteine peptide 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

4.7290

4.7400

4.7210

0.1451

0.1454

0.1449

71.19

71.12

71.24

 

71.18

 

0.06

 

0.08

Test item

16.1620

16.0220

15.7820

0.4951

0.4908

0.4835

2.31

3.15

4.60

 

3.35

 

1.16

 

34.65

Depletion of the Lysine peptide

Cysteine peptide

Sample

Peak area at 220 nm

Peptide Conc. [mM]

Peptide depletion [%]

Mean peptide depletion [%]

SD of peptide depletion [%]

CV of peptide depletion [%]

Positive control

6.2940

5.9870

5.6530

0.2177

0.2071

0.1955

56.28

58.41

60.73

 

58.47

 

2.23

 

3.81

Test item

14.2650

14.3900

14.3550

0.4938

0.4981

0.4969

1.95

1.10

1.34

 

1.46

 

0.44

 

30.32

Categorization of the test item:

Based on the results of the peptide depletion, categorization according to the prediction model might be performed.

Since no co-elution was observed, prediction model 1 based on the combination of cysteine and lysine peptide depletion should be considered.

Prediction model

Prediction model 1 (Cysteine peptide and lysine peptide/ratio 1:10 and 1:50)

Prediction model 2 (cysteine peptide/test item ratio: 1: 10)

Test substance

Mean peptide depletion [%]

Reactivity category

Prediction

Mean peptide depletion [%]

Reactivity category

Prediction

Test item

2.41

Minimal reactivity

No sensitiser

3.35

 

Minimal reactivity

No sensitiser

Positive control

64.83

High reactivity

Sensitiser

71.18

Moderate reactivity

sensitiser

Interpretation of results:
GHS criteria not met
Conclusions:
Under the given conditions of the study, the test item showed minimal reactivity towards both peptides. Therefore, the test item, balsalazide acid, might be considered as a non-sensitiser.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
25 June 2018 - 04 September 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 09 October 2017
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
Batch no: 0400
Details on study design:
Skin sensitisation (In vitro test system)
Key result
Parameter:
other: dendritic cell activation
Run / experiment:
CD86 upregulation (%)
Value:
<= 150
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: dendritic cell activation
Run / experiment:
CD54 upregulation (%)
Value:
<= 200
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: No cytotoxic effects were observed for the cells treated with the test item.

ACCEPTANCE OF RESULTS: See Table 6

Reactivity check of the cell stock:

Doubling time of the cells was monitored and found to be 39.9 h and 39.4 h which are within the doubling time range specified by the manufacturer (35 - 50 h).

 

Table 1:  Results of the Cell Batch 1 Activation Test

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

87.5

373

>150

88.1

358

>200

yes

pass

NiSO4

100 µg/mL

83.5

295

>150

82.1

603

>200

yes

pass

LA

1000 µg/mL

95.7

81

£150

95.8

100

£200

no

pass

 

Table 2:  Results of the Cell Batch 21 Activation Test

Sample

Concentration
[µg/mL]

CD86

CD54

Activated

Pass /Fail

Cell Viability [%]

RFI

Threshold OECD TG 442E

Cell Viability [%]

RFI

Threshold OECD TG 442E

yes/no

DNCB

4 µg/mL

85.5

251

>150

84.7

287

>200

yes

pass

NiSO4

100 µg/mL

74.9

249

>150

75.5

518

>200

yes

pass

LA

1000 µg/mL

94.8

67

£150

95.0

108

£200

no

pass

 

The positive controls DNCB and NiSO4led to upregulation of the cell surface markers CD54 and CD86. The negative control LA did not induce an upregulation of CD54 and CD86.

The cell batch was accepted for further testing.

 

Solvent Finding

All test item solutions were freshly prepared immediately prior to use. The test item was soluble in DMSO at a concentration of 250 mg/mL.

 

Dose Finding Assay

The dose finding assay was performed using stock solutions with a concentration of 500 mg/mL (applied concentration 1000 µg/mL).

Table 3:  Results of the Dose Finding Assay

Sample

Experiment 1

Concentration applied [µg/mL]

Cell Viability [%]

Medium Control

--

--

95.90

Solvent Control

DMSO

--

95.30

Balsalazide acid

C8

7.81

95.20

C7

15.63

94.60

C6

31.25

93.90

C5

62.50

95.00

C4

125.00

94.80

C3

250.00

95.30

C2

500.00

95.50

C1

1000.00

95.30

Calculated CV75 [µg/mL]

No CV75

Mean CV75 [µg/mL]

No CV75

SD CV 75 [µg/mL]

No SD

 

The dose finding assay was performed in one independent run. Since there was no cytotoxicity, no CV75 could be derived. The main experiment was performed covering a concentration range from 1000 – 279.08 µg/mL (500 – 139.54 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 10 (first experiment) and 12 (second experiment). For each experiment separately weighted samples and preparations were used.

Table4:  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

-

94.3

95.8

95.8

1679

1010

543

1136

467

95

96

309

186

Solvent Control

0.20%

95.3

95.6

95.5

1737

1030

546

1191

484

100

100

318

189

DNCB

4.00

82.5

82.4

81.4

4206

2308

601

3605

1707

303

353

700

384

Balsalazide acid

500

95.7

95.9

95.4

1611

1219

598

1013

621

85

128

269

204

416.67

95.6

95.9

95.8

1658

1230

602

1056

628

89

130

275

204

347.22

95.1

95.2

95.4

1787

1219

592

1195

627

100

130

302

206

289.35

94.8

95.3

95.7

1793

1211

595

1198

616

101

127

301

204

241.13

95.0

95.6

95.8

1875

1231

587

1288

644

108

133

319

210

200.94

95.8

96.0

96.3

1770

1114

558

1212

556

102

115

317

200

167.45

95.1

95.1

95.2

1803

1046

548

1255

498

105

103

329

191

139.54

95.7

95.6

95.6

1575

1082

547

1028

535

86

111

288

198

 

Table5: 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

-

96.1

96.1

95.5

1375

904

547

828

357

97

89

251

165

Solvent Control

0.20%

96.6

96.7

97.0

1396

945

544

852

401

100

100

257

174

DNCB

4.0

84.0

85.0

85.1

4295

2175

553

3742

1622

439

404

777

393

Balsalazide acid

500.00

97.0

96.6

97.1

1483

1161

612

871

549

102

137

242

190

416.67

96.6

97.1

96.9

1601

1120

606

995

514

117

128

264

185

347.22

96.7

97.4

97.3

1535

1100

596

939

504

110

126

258

185

289.35

97.2

96.9

96.9

1719

1093

570

1149

523

135

130

302

192

241.13

96.6

96.2

96.8

1523

1123

590

933

533

110

133

258

190

200.94

96.7

96.9

96.4

1790

1067

571

1219

496

143

124

313

187

167.45

96.4

96.9

96.9

1574

1055

569

1005

486

118

121

277

185

139.54

96.9

96.8

96.5

1835

1096

592

1243

504

146

126

310

185

 

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitiser and non-sensitisers.

Prior to the main study the cell batch was checked for its reactivity towards known positive and negative controls and was found to be acceptable for further testing.

In the present study Balsalazide acid was dissolved in DMSO. For the dose finding assay stock solutions with concentrations ranging from 500.00 mg/mL to 139.54 mg/mL were prepared by a serial dilution of 1:2. Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained with propidium iodide and cell viability was measured by FACS analysis.

Due to a lack of cytotoxicity, no CV75 could be derived. Therefore, the main experiment was performed covering the following concentration steps:

1000, 833.33, 694.44, 578.70, 482.25, 401.88, 334.90, 279.08 µg/mL

In all experiments no precipitation or turbidity of the test item was observed for all the concentration steps when mixing the test item stock solutions with cell culture medium.

Cells were incubated with the test item for 24 h at 37°C. After exposure cells were stained and cell surface markers CD54 and CD86 were measured by FACS analysis. Cell viability was assessed in parallel using propidium iodide staining.

No cytotoxic effects were observed for the cells treated with the test item. Relative cell viability at the highest test item concentration was 95.7% (CD86), 95.9% (CD54) and 95.4% (isotype IgG1 control) in the first experiment and 97.0% (CD86), 96.6% (CD54) and 97.1% (isotype IgG1 control) in the second experiment.

The expression of the cell surface marker CD86 was not upregulated above the threshold of 150% in any of the experiments. The expression of cell surface marker CD54 was not upregulated above the threshold of 200% in any of the experiments. Therefore, the test item can be considered as non-sensitiser.

The controls confirmed the validity of the study for all experiments as shown in Table 6.

 

Table 6:  Acceptance Criteria

Acceptance Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

cell viability solvent controls [%]

>90

94.3

-

95.8

pass

95.5

-

97.0

pass

number of test dosed with viability >50% CD86

≥4

8

pass

8

pass

number of test dosed with viability >50% CD54

≥4

8

pass

8

pass

number of test dosed with viability >50% IgG1

≥4

8

pass

8

pass

RFI of positive control of CD86

≥150

303

pass

439

pass

RFI of positive control of CD54

≥200

353

pass

404

pass

RFI of solvent control of CD86

<150

105

pass

103

pass

RFI of solvent control of CD54

<200

104

pass

112

pass

MFI ratio CD86/IgG1 for medium control [%]

>105

309

pass

251

pass

MFI ratio CD86/IgG1 for DMSO control [%]

>105

318

pass

257

pass

MFI ratio CD54/IgG1for medium control [%]

>105

186

pass

165

pass

MFI ratio CD54/IgG1for DMSO control [%]

>105

189

pass

174

pass

 

 

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.

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface marker in at least two independent experiment runs. Therefore, the test item might be considered as non-sensitiser.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
5th June 2018 - 30th July 2018
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)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The induction of the Keap1-Nrf2-ARE signalling pathway by small electrophilic substances such as skin sensitizers was reported by several studies and represents the second key event of the skin sensitisation process as described by the AOP. The ARE-Nrf2 luciferase test method makes use of an immortalised adherent cell line (e.g. KeratinoSens™) derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances. Therefore, this test method is considered to be able to detect chemicals that cause skin sensitisation when used within Integrated Approaches to Testing and Assessment (IATA), together with other relevant complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP as well as non-testing methods, including read-across from chemical analogues Depending on the regulatory framework, positive results generated with these methods may be used on their own to classify a chemical into UN GHS category 1.
Specific details on test material used for the study:
Batch No.: 0400
Purity: 96%
Appearance: Orange solid
Storage conditions: Room temperature
Key result
Parameter:
other: Luciferase induction
Run / experiment:
Experiments 1 and 2
Value:
< 1.5
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
Solvent control
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
No dose response for luciferase activity induction was observed.
Other effects / acceptance of results:
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.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: Yes: Number of positive control concentration steps with significant luciferase activity induction >1.5: 2.0 (Expt 1) and 3.0 (Expt 2); EC1.5: 18.06 (Expt 1) and 9.77 (Expt 2); Induction at 64 µM: 5.22 (Expt 1) and 5.60 (Expt 2)
- Acceptance criteria met for solvent control: Yes: CV: 15.1% (Expt 1) and 8.9 (Expt 2)

Table 1: Induction of Luciferase activity experiment 1:

Experiment 1

Concentration

[μM]

 

Fold induction

Significance

Rep 1

Rep 2

Rep 3

Mean

SD

Solvent control

-

1.00

1.00

1.00

1.00

0.00

 

 

Positive control

4.00

1.25

1.27

1.23

1.25

0.02

 

8.00

1.28

1.25

1.58

1.37

0.18

 

16.00

1.33

1.40

1.47

1.40

0.07

 

32.00

2.02

2.03

2.47

2.17

0.25

*

64.00

4.42

5.04

6.19

5.22

0.90

*

 

 

 

 

Test item

0.98

0.98

0.95

1.03

0.99

0.04

 

1.95

1.07

0.86

0.80

0.91

0.14

 

3.91

0.92

0.82

0.78

0.84

0.07

 

7.81

1.09

0.74

0.88

0.91

0.18

 

15.63

1.11

0.88

0.78

.92

0.17

 

31.25

0.85

0.79

0.78

0.81

0.03

 

62.50

0.77

0.65

0.71

0.71

0.06

 

125.00

0.76

0.66

0.70

0.70

0.05

 

250.00

0.90

0.59

0.65

0.72

0.17

 

500.00

0.92

0.56

0.62

0.70

0.19

 

1000.00

0.63

0.53

0.56

0.57

0.05

 

2000.00

0.47

0.48

0.51

0.49

0.02

 

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

Table 2: Induction of Luciferase activity experiment 2:

Experiment 2

Concentration

[μM]

 

Fold induction

Significance

Rep 1

Rep 2

Rep 3

Mean

SD

Solvent control

-

1.00

1.00

1.00

1.00

0.00

 

 

Positive control

4.00

1.26

1.26

1.31

1.28

0.03

 

8.00

1.43

1.34

1.45

1.40

0.06

 

16.00

1.92

1.63

1.95

1.83

0.18

*

32.00

2.37

2.06

2.52

2.32

0.24

*

64.00

6.03

4.59

6.19

5.60

0.88

*

 

 

 

 

Test item

0.98

2.35

0.95

0.98

1.43

0.80

 

1.95

0.98

0.99

1.05

1.01

0.04

 

3.91

1.03

1.08

1.04

1.05

0.03

 

7.81

1.12

0.96

1.16

1.08

0.10

 

15.63

1.06

1.14

1.24

1.15

0.09

 

31.25

1.12

1.04

1.13

1.10

0.05

 

62.50

1.07

1.06

1.11

1.08

0.02

 

125.00

1.02

0.99

0.94

0.98

0.04

 

250.00

0.91

0.84

0.92

0.89

0.04

 

500.00

0.88

0.66

0.88

0.81

0.13

 

1000.00

0.72

0.65

0.67

0.68

0.03

 

2000.00

0.52

0.53

0.65

0.57

0.07

 

Table 3: Induction of luciferase activity - overall induction

Concentration

[μM]

 

Fold induction

Significance

Experiment 1

Experiment 2

Mean

SD

Solvent control

-

1.00

1.00

1.00

0.00

 

 

Positive control

4.00

1.25

1.28

1.26

0.02

 

8.00

1.37

1.40

1.39

0.02

 

16.00

1.40

1.83

1.62

0.31

 

32.00

2.17

2.32

2.24

0.10

*

64.00

5.22

5.60

5.41

0.27

*

 

 

 

 

Test item

0.98

0.99

1.43

1.21

0.31

 

1.95

0.91

1.01

0.96

0.07

 

3.91

0.84

1.05

0.95

0.15

 

7.81

0.91

1.08

0.99

0.12

 

15.63

0.92

1.15

1.03

0.16

 

31.25

0.81

1.10

0.95

0.20

 

62.50

0.71

1.08

0.89

0.26

 

125.00

0.70

0.98

0.84

0.20

 

250.00

0.72

0.89

0.80

0.12

 

500.00

0.70

0.81

0.76

0.08

 

1000.00

0.57

0.68

0.63

0.07

 

2000.00

0.49

0.57

0.53

0.06

 

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

Interpretation of results:
GHS criteria not met
Conclusions:
Under the given conditions the test item, balsalazide acid did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as a non-sensitiser.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Balsalazide acid is not a skin sensitiser based on the negative results of the three recommended in vtro/ in chemico OECD Guideline skin sensitisation studies.