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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 4-aminobenzoyl-b-alanine 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:
6th June 2018 to 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
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:
not specified
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
Reduction in animal testing by use of a validated non-vertebrate test, in accordance with the requirements to consider non-vertebrate testing for this endpoint, in accordance with the REACH regulation.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: PharmaZell, batch no. 12/18
- Expiration date of the lot/batch: not specified
- Purity test date: not specfied, but CoA states retest data in March 2019

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: assumed to be stable
- Solubility and stability of the test substance in the solvent/vehicle: assumed to be stable
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: assumed non-reactive

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was pre-weighed into a glass vial and was dissolved in an appropriate solvent previously determined in a pre-experiment. A stock solution with a concentration of 100 mM was prepared. A factor of 1.04 was used to correct for the purity of the test item
- Preliminary purification step (if any): none
- Final dilution of a dissolved solid, stock liquid or gel: None; stock solution was used directly with peptide solutions

FORM AS APPLIED IN THE TEST (if different from that of starting material) : a solution of the solid was used in the test, and mixed at specified ratios with the peptide solutions
Details on study design:
Skin sensitisation (In chemico test system) - Details on study design:

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 sensitisers and non-sensitisers.

Positive control results:
Mean Cysteine peptide depletion = 71.18% (SD= 0.06%, CV = 0.08%)
Mean Lysine peptide depletion = 58.47% (SD = 2.23%, CV = 3.81%)
Prediction model 1, mean of peptides depletions = 64.83%
Key result
Parameter:
other: % Cysteine Peptide depletion
Run / experiment:
1
Value:
0.41
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: % Lysine peptide depletion
Run / experiment:
1
Value:
1.07
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Mean of both % peptide depletions
Run / experiment:
1
Value:
0.74
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Other effects / acceptance of results:
OTHER EFFECTS:
- Visible damage on test system: none stated

DEMONSTRATION OF TECHNICAL PROFICIENCY: not stated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Acceptance criteria met for variability between replicate measurements: Yes

Depletion of the Cysteine 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

4.7290

0.1451 

71.19

71.18

 

0.06 

0.08

4.7400 

0.1454 

71.12

4.7210 

0.1449 

71.24

Test Item

16.6830 

0.5110 

0.00

0.41 

 

0.57 

140.65

16.3680 

0.5014 

1.06

16.5170 

0.5060

0.16

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

6.2940 

0.2177 

56.28

58.47

2.23

3.81

5.9870 

0.2071 

58.41

5.6530 

0.1955 

60.73

Test Item

14.5170 

0.5025 

0.22

1.07

0.80

74.95

14.3800

0.4977 

1.16

14.2860 

0.4945 

1.81

 

 

Interpretation of results:
GHS criteria not met
Conclusions:
In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item is considered as “non-sensitiser”.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
02 July 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 activtation of dendritic cells on the adverse outcome pathway for skin sensitisation
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Specific details on test material used for the study:
Batch no: 12/18

Key result
Parameter:
other: dendritic cell activation
Run / experiment:
CD86 upregulation (%)
Value:
<= 150
Negative controls validity:
valid
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
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

Evaluation of results:

For CD86/CD54 expression measurement, each test item was tested in at least two independent runs to derive a single prediction. Each independent run was performed on a different day of on the same day provided that for each run: independent fresh stock solutoins and working solutions of the test chemicals and antibody solutions were prepared and independently harvested cells were used.

Sensitising potential of the test item was predicted from the mean percentage expression of CD86 and CD54. Any test chemical tested by the h-CLAT was considered positive if the RFI of CD86 was equal or greater than 150% at any tested dose at a cell viability of 50% in at least two independent runs or if the RFI of CD54 was equal to or greater than 200% at any tested dose at a call viability 50% in at least two independent runs or if the RFIs of both the CD86 and CD54 were equal to or are greater than 150% and 200% respectively at any tested dose at a cell viability 50% in at least two independent runs.

A negative test result of a test item is only accepted if the cell viability of 1.2 x CV75 is <90%. In contrast, a positive test outcome was accepted irrespective of cell viabilities >90% at a concentration of 1.2 x CV75. If no CV75 could be derived negative test results can be accpeted when the test item is tested at the highest soluble concentration (5000 µg/ml for 0.9% NaCl solution; 1000 µg/ml for DMSO or a different organic solvent) even if cell viability is >90%.

Table 1: Results for CD54 and CD86 expression experiment 1

Sample

Conc (µg/ml)

Cell viability (%)

Mean fluorescence intensity

Corrected mean fluorescence intensity

Relative fluorescence intensity (RFI)

Ratio isotype IgG1 to (%)

 

-

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium control

 

96.6

95.7

95.3

1873

978

521

1352

457

97

91

360

188

Solvent control

0.20%

95.4

96.0

95.6

1899

1016

512

1387

504

100

100

371

198

DNCB

4.00

84.2

84.5

83.9

5529

2516

543

4986

1973

359

391

1018

463

 

 

4-Aminobenzoyl b-alanine

1000

95.7

95.2

95.1

1769

984

525

1244

459

90

91

337

187

833.33

95.4

95.0

95.0

1864

984

553

1311

431

95

86

337

178

694.44

95.3

95.4

94.6

1912

995

540

1372

455

99

90

354

184

578.70

95.4

95.3

95.1

1998

994

582

1416

412

102

82

343

171

482.25

95.8

95.7

95.3

1944

1011

554

1390

457

100

91

351

182

401.88

95.0

95.2

95.2

2012

988

555

1457

433

105

86

363

178

334.90

95.7

95.5

95.2

1969

1019

555

1414

464

102

92

355

184

279.08

95.3

95.5

94.6

1803

991

568

1235

423

89

84

317

174

Table 2: Results for CD54 and CD86 expression experiment 2

Sample

Conc (µg/ml)

Cell viability (%)

Mean fluorescence intensity

Corrected mean fluorescence intensity

Relative fluorescence intensity (RFI)

Ratio isotype IgG1 to (%)

 

-

CD86

CD54

Isotype IgG1

CD86

CD54

Isotype IgG1

CD86

CD54

CD86

CD54

CD86

CD54

Medium control

 

96.7

96.1

96.5

1515

1005

559

956

446

84

94

271

180

Solvent control

0.20%

97.0

96.9

96.8

1668

1002

528

1140

474

100

100

316

190

DNCB

4.00

85.1

85.3

84.1

4510

2139

580

3930

1559

345

329

778

369

 

 

4-Aminobenzoyl b-alanine

1000

95.6

95.8

95.3

1675

1076

673

1002

403

88

85

249

160

833.33

95.1

95.8

96.0

1796

1027

569

1227

458

108

97

316

180

694.44

96.2

96.2

96.3

1638

1040

567

1071

473

94

100

289

183

578.70

96.7

96.6

96.8

1735

1072

582

1153

490

101

103

298

184

482.25

97.3

97.2

96.9

1612

975

663

949

312

83

66

243

147

401.88

96.6

96.9

96.9

1631

1056

567

1064

489

93

103

288

186

334.90

96.0

96.3

95.7

1651

998

667

984

331

86

70

248

150

279.08

95.8

96.5

96.5

1592

972

572

1020

400

89

84

278

170

Interpretation of results:
GHS criteria not met
Conclusions:
In the study under the given conditions the test substance did not upregulate the expression of the cell surface markers in at least two independent cell runs. Therefore it is conisdered as a non-sensitiser.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8th June 2018 - 29th June 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)
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Reduction in animal testing by use of a validated non-vertebrate test, in accordance with the requirements to consider non-vertebrate testing for this endpoint, in accordance with the REACH regulation.
Specific details on test material used for the study:
Batch no: 12/18
Key result
Parameter:
other: Luciferase induction
Run / experiment:
Experiments 1 and 2
Value:
< 1.5
Negative controls validity:
valid
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:
ACCEPTANCE OF RESULTS:
The test meets the acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the thresehold 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 64µM is between 2 and 8
-the EC1.5 value of the positive control is within two standard devations 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.

Table 1: Results of the Cytotoxicity Measuerment

 

Concentration

[µM]

Cell Viability [%}

 

-

Experiment 1

Experiment 2

Mean

SD

Solvent control

 

100.0

100.0

100.0

0.0

 

Positive control

4.00

107.0

99.8

103.4

5.1

8.00

111.9

102.8

107.3

6.5

16.00

114.0

107.5

110.7

4.6

32.00

116.6

106.9

111.8

6.8

64.00

112.8

96.9

104.9

11.2

 

 

 

 

 

Test item

0.98

106.9

101.9

104.4

3.5

1.95

105.5

96.3

100.9

6.5

3.91

103.7

102.3

103.0

1.0

7.81

98.8

99.2

99.0

0.3

15.63

93.6

102.7

98.1

6.5

31.25

95.1

93.6

94.4

1.1

62.50

97.3

93.1

95.2

3.0

125.00

88.4

89.5

89.0

0.8

250.00

81.9

85.7

83.8

2.7

500.00

76.7

69.2

72.9

5.3

1000.00

66.4

52.3

59.4

10.0

2000.00

66.3

56.8

61.6

6.8

Table 2: Induction of Luciferase activity experiment 1

Experiment 1

Concentration [µm]

Fold induction

Significance

Rep 1

Rep 2

Rep 3

Mean

SD

Solvent control

-

1.00

1.00

1.00

1.00

0.00

 

 

 

Positive control

4.00

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

0.74

0.93

0.88

0.13

 

1.95

1.08

0.69

0.80

0.86

0.20

 

3.91

0.87

0.77

0.92

0.85

0.08

 

7.81

0.89

0.71

0.81

0.80

0.09

 

15.63

1.07

0.86

0.86

0.93

0.12

 

31.25

0.89

0.70

0.75

0.78

0.10

 

62.50

0.95

0.77

0.77

0.83

0.10

 

125.00

0.92

0.76

0.86

0.85

0.08

 

250.00

1.01

0.81

0.82

0.88

0.11

 

500.00

1.00

0.83

0.91

0.91

0.09

 

1000.00

0.83

0.71

0.76

0.77

0.06

 

2000.00

0.97

0.81

0.99

0.92

0.10

 

*= significant induction according to Students t-test p<0.05

Table 3: Induction of Luciferase Activity experiment 2

Experiment 2

Concentration [µm]

Fold induction

Significance

Rep 1

Rep 2

Rep 3

Mean

SD

Solvent control

-

1.00

1.00

1.00

1.00

0.00

 

 

 

Positive control

4.00

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

1.47

0.93

0.84

1.08

0.34

 

1.95

0.99

0.91

0.83

0.91

0.08

 

3.91

1.00

1.01

0.96

0.99

0.02

 

7.81

1.10

0.93

1.19

1.07

0.13

 

15.63

0.99

0.95

0.96

0.97

0.02

 

31.25

1.10

1.03

1.01

1.05

0.04

 

62.50

1.05

1.02

1.06

1.04

0.02

 

125.00

1.10

1.09

1.09

1.09

0.01

 

250.00

1.17

0.96

1.00

1.04

0.11

 

500.00

1.09

1.08

1.07

1.08

0.01

 

1000.00

1.06

1.02

1.07

1.05

0.03

 

2000.00

0.97

0.93

0.99

0.96

0.03

 

*= significant induction according to Students t-test p<0.05

Interpretation of results:
GHS criteria not met
Conclusions:
Under the conditions of the study the test item is not considered to be a skin 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

4-aminobenzoyl-b-alanine is not a skin sensitiser based on the negative results of the three recommended in vtro/ in chemico OECD Guideline skin sensitisation studies.