Registration Dossier

Diss Factsheets

Administrative data

Description of key information

WoE: Direct Peptide Reactivity Assay (DPRA, OEDC 442C): negative

WoE: ARE-Nrf2 Luciferase Assay(KeratinoSensTM-Assay, OECD 442D): negative

WoE: Human Cell Line Activation Test (h-CLAT, OECD 442E) : positive

Read-across from p-anisic acid (CAS 100-09-4)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Positive control results:
The positive control 2,4-dinitrochlorobenzene (DNCB) test at final concentrations of 2 and 3% led to upregulation of the cell surface markers CD54 and CD86.
Key result
Run / experiment:
other: First run
Parameter:
other: relative fluorescence intensity of CD86 (%)
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
since the RFI of CD86 is > 150% the prediction for sensitisation is considered positive; source: CAS 100-09-4; Roth, 2018; h-CLAT
Key result
Run / experiment:
other: Second run
Parameter:
other: relative fluorescence intensity of CD86 (%)
Value:
150
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
since the RFI of CD86 is > 150% the prediction for sensitisation is considered positive; source: CAS 100-09-4; Roth, 2018; h-CLAT
Key result
Run / experiment:
other: First run
Parameter:
other: relative fluorescence intensity of CD54 (%)
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
since the RFI of CD54 is > 200% the prediction for sensitisation is considered positive; source: CAS 100-09-4, Roth, 2018, h-CLAT
Key result
Run / experiment:
other: Second run
Parameter:
other: relative fluorescence intensity of CD54 (%)
Value:
200
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Remarks:
since the RFI of CD54 is > 200% the prediction for sensitisation is considered positive; source: CAS 100-09-4, Roth, 2018, h-CLAT
Other effects / acceptance of results:
OTHER EFFECTS:
- No test item-induced cytotoxicity was noted during the two runs of the main experiment.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The laboratory has demonstrated technical proficiency for the h-CLAT by successfully testing the 10 profiency chemicals outlined in the OECD 442E guideline.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for DMSO control:
The RFI values of the negative control of both CD86 and CD54 were 100, respectively, and thus did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%). The cell viability was 100.0 % and thus > 90%.
- Acceptance criteria met for positive control:
The RFI values of the positive control for CD86 and CD54 were > 650 and > 230, respectively, and thus exceeded the positive criteria (CD86 > 150% and CD54 > 200%) and the cell viability was > 60 % and thus > 50%.

Table 1: Results first h-CLAT run

 

Concentration (µg/mL)

Antibody

/ ISO

MFI

GeoMean(FITC)

MFI- ISO

RFI (%)

Cyto(Geo)

GeoMean(7-AAD)

Mean Cyto

Viability

(%)

Medium control

 

-

ISO

2.62

 

 

2.85

 

2.7

 

100.0

CD54

3.26

0.64

100.0

2.83

CD86

5.12

2.50

100.0

2.46

DMSO

control

 

-

ISO

2.19

 

 

3.06

 

2.7

 

100.0

CD54

3.26

1.07

100.0

2.73

CD86

5.01

2.82

100.0

2.42

 

Positive control (DNCB)

 

2

ISO

2.92

 

 

4.82

 

3.7

 

74.9

CD54

5.44

2.52

235.5

4.07

CD86

17.05

14.13

501.1

2.07

 

3

ISO

3.25

 

 

6.26

 

4.6

 

59.9

CD54

6.68

3.43

320.6

4.99

CD86

22.22

18.97

672.7

2.45

 

 

 

 

 

 

 

 

 

 

 

 

Test Item

 

274

ISO

2.41

 

 

3.22

 

2.9

 

93.1

CD54

3.14

0.73

114.1

2.93

CD86

4.57

2.16

86.4

2.59

 

329

ISO

2.40

 

 

3.15

 

2.9

 

93.7

CD54

3.20

0.80

125.0

2.96

CD86

4.98

2.58

103.2

2.58

 

395

ISO

2.43

 

 

3.13

 

2.9

 

94.7

CD54

3.29

0.86

134.4

2.93

CD86

4.99

2.56

102.4

2.54

 

474

ISO

2.57

 

 

3.09

 

2.8

 

98.0

CD54

3.38

0.81

126.6

2.85

CD86

5.32

2.75

110.0

2.37

 

569

ISO

2.59

 

 

3.07

 

2.8

 

96.9

CD54

3.53

0.94

146.9

2.88

CD86

5.57

2.98

119.2

2.45

 

683

ISO

2.64

 

 

3.10

 

2.8

 

98.2

CD54

3.60

0.96

150.0

2.81

CD86

5.76

3.12

124.8

2.38

 

819

ISO

2.88

 

 

3.36

 

2.9

 

94.5

CD54

4.17

1.29

201.6

3.01

CD86

7.14

4.26

170.4

2.24

 

983

ISO

2.79

 

 

3.08

 

2.9

 

92.2

CD54

3.90

1.11

173.4

2.82

CD86

6.84

4.05

162.0

2.93

Table 1: Results second h-CLAT run

 

Concentration (µg/mL)

Antibody

/ ISO

MFI

GeoMean(FITC)

MFI- ISO

RFI (%)

Cyto(Geo)

GeoMean(7-AAD)

Mean Cyto

Viability

(%)

Medium control

 

-

ISO

2.44

 

 

3.85

 

3.7

 

100.0

CD54

3.30

0.86

100.0

3.78

CD86

5.05

2.61

100.0

3.61

DMSO

control

 

-

ISO

2.57

 

 

4.15

 

3.7

 

100.0

CD54

3.40

0.83

100.0

3.71

CD86

5.01

2.44

100.0

3.12

 

Positive control (DNCB)

 

2

ISO

3.63

 

 

5.03

 

4.6

 

79.8

CD54

7.18

3.55

427.7

4.70

CD86

26.79

23.16

949.2

4.03

 

3

ISO

3.92

 

 

6.12

 

5.7

 

63.9

CD54

10.08

6.16

742.2

5.66

CD86

19.97

16.05

657.8

5.39

 

 

 

 

 

 

 

 

 

 

 

 

Test Item

 

274

ISO

2.76

 

 

4.36

 

3.9

 

95.5

CD54

3.65

0.89

103.5

3.98

CD86

5.25

2.49

95.4

3.43

 

329

ISO

2.74

 

 

4.19

 

3.9

 

96.6

CD54

3.74

1.00

116.3

3.94

CD86

5.60

2.86

109.6

3.50

 

395

ISO

2.92

 

 

4.28

 

3.9

 

95.7

CD54

3.86

0.94

109.3

4.05

CD86

6.07

3.15

120.7

3.42

 

474

ISO

2.91

 

 

4.21

 

3.8

 

98.6

CD54

3.79

0.88

102.3

3.90

CD86

5.30

2.39

91.6

3.29

 

569

ISO

3.01

 

 

4.27

 

3.8

 

98.3

CD54

3.96

0.95

110.5

3.91

CD86

5.69

2.68

102.7

3.25

 

683

ISO

3.13

 

 

4.35

 

3.7

 

101.7

CD54

4.29

1.16

134.9

3.94

CD86

7.14

4.01

153.6

2.76

 

819

ISO

3.06

 

 

4.28

 

3.8

 

97.7

CD54

4.12

1.06

123.3

3.98

CD86

6.04

2.98

114.2

3.24

 

983

ISO

3.51

 

 

5.28

 

4.6

 

81.8

CD54

5.32

1.81

210.5

4.78

CD86

9.46

5.95

228.0

3.68

Interpretation of results:
other: skin sensitising potential based on the key event “activation of dendritic cells” of skin sensitisation Adverse Outcome Pathway (AOP)
Conclusions:
The read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their skin sensitisation potential. In an h-CLAT test with the source substance p-anisic acid (CAS 100-09-4) a skin sensitising potential based on the key event “activation of dendritic cells” of skin sensitisation AOP was identified. Therefore, a skin sensitisation potential for this key event is also expected for the target substance sodium anisate.
The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation was performed.
Endpoint:
skin sensitisation: in chemico
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Run / experiment:
other: mean of three runs
Parameter:
other: mean cysteine depletion (%)
Value:
1.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
since the cysteine depletion is ≤ 6.38 the prediction for sensitisation is considered negative; source: CAS 100-09-4, Fleet, 2018, DPRA
Key result
Run / experiment:
other: mean of three runs
Parameter:
other: mean lysine depeltion (%)
Value:
1.01
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
since the lysine depletion is ≤ 6.38 the prediction for sensitisation is considered negative; source: CAS 100-09-4, Fleet, 2018, DPRA
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The laboratory has demonstrated technical proficiency for the DPRA by successfully testing the 10 profiency chemicals outlined in the OECD 442C guideline

ACCEPTANCE CRITERIA:
The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent positve control 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 depletion,
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine depletion,
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is in the range of 0.45 to 0.55 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Table 2. Overall achieved depletion values

Test item

Cysteine peptide depletion (%)

Lysine peptide depletion (%)

Overall mean depletion (%)

Reactivity class

DPRA prediction

 p-anisic acic

1.40

1.01

1.20

No to minimal

Negative

Table 3. Cysteine peptide depletion

Sample

Peak area (µV.sec)

Peptide concentration1
(µg/mL)

Peptide Depletion (%)

Mean Depletion (%)

SD
 (%)

Positive control

236103

105.70

71.52

71.7

0.08

234831

105.13

71.62

235271

105.33

71.62

p-anisic acic

821285

369.43

1.433

1.40

0.43

825232

371.21

0.9533

818128

368.01

1.813

SD Standard Deviation

1  Samples prepared at a concentration of 376 µg/mL (0.5 mM)

2  Calculated against a mean Reference Control B area of 827610 µV.sec(n=6)

3  Calculated against a mean Reference Control C area of 833170 µV.sec(n=3)

Table 4. Lysine peptide depletion

Sample

Peak area (µV.sec)

Peptide concentration1(µg/mL)

Peptide Depletion (%)

Mean Depletion (%)

SD
 (%)

Positive control

317845

158.71

59.12

58.8

0.29

322285

160.93

58.52

319492

159.53

58.82

p-anisic acic

777172

388.56

-0.2093

1.01

1.78

751867

375.89

3.053

774140

387.03

0.1823

SD Standard Deviation

1   Samples prepared at a concentration of 376 µg/mL (0.5 mM)

2   Calculated against a mean Reference Control B area of 776330 µV.sec(n=6)

3   Calculated against a mean Reference Control C area of 775550 µV.sec (n=3)

Interpretation of results:
other: no skin sensitising potential based on the key event "direct peptide binding" of skin sensitisation Adverse Outcome Pathway (AOP)
Conclusions:
The read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their skin sensitisation potential. In an DPRA test with the source substance p-anisic acid (CAS 100-09-4) no skin sensitising potential based on the key event “direct peptide binding” of skin sensitisation AOP was identified. Therefore, no skin sensitisation potential for this key event is expected for the target substance sodium anisate.

The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation was performed.
Endpoint:
skin sensitisation: in vitro
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: maximum luciferase activity induction (Imax)
Value:
1.618
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Imax at 1000 µM; unit is not applicable since this parameter is defined as the maximum average fold induction of luciferase activity (Imax) value observed at any concentration of the test item
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: EC1.5 (µM)
Value:
849.105
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Unit: µM
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: maximum luciferase activity induction (Imax)
Value:
1.318
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Imax at 500 µM; unit is not applicable since this parameter is defined as the maximum average fold induction of luciferase activity (Imax) value observed at any concentration of the test item
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: EC1.5 (µM)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: N/A - threshold of induction not crossed at any test item concentration.
Remarks:
source: CAS 100-09-4, Bailey, 2019, KeratinoSensTM
Key result
Run / experiment:
other: Experiment 3
Parameter:
other: maximum luciferase activity induction (Imax)
Value:
1.711
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Imax at 1.953 µM; unit is not applicable since this parameter is defined as the maximum average fold induction of luciferase activity (Imax) value observed at any concentration of the test item
Key result
Run / experiment:
other: Experiment 3
Parameter:
other: EC1.5 (µM)
Value:
1.514
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Unit: µM
Key result
Run / experiment:
other: Experiment 4
Parameter:
other: maximum luciferase activity induction (Imax)
Value:
0.819
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other:
Remarks:
Imax at 0.977 µM; unit is not applicable since this parameter is defined as the maximum average fold induction of luciferase activity (Imax) value observed at any concentration of the test item
Key result
Run / experiment:
other: Experiment 4
Parameter:
other: EC1.5 (µM)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: N/A - threshold of induction not crossed at any test item concentration.
Remarks:
source: CAS 100-09-4, Bailey, 2019, KeratinoSensTM
Other effects / acceptance of results:
DEMONSTRATION OF TECHNICAL PROFICIENCY:
The laboratory has demonstrated technical proficiency using the 10 proficiency chemicals and 11 reference chemicals described in OECD 442D. Performance data for the adpatation of the test to animal-free culture coditions are appended to the report.
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The average coefficient of variation (CV) of the luminescence reading for the negative (solvent) control DMSO was < 20% (mean value in experiments 1-3: 15.736% and 9.642% in experiment 4). Therefore the negative control confirmed the validity of the study.
- Acceptance criteria met for positive control: The luciferase activity induced by the positive control at a concentration of 32 μM was between 1.6 and 3.0 (mean value in experiments 1-3: 2.938 and 2.509 in experiment 4). The calculated EC1.5 was between 6 and 39 μM (mean value in experiments 1-3: 13.569 and 7.105 in experiment 4). Therefore the positive control confirmed the validity of the study.

Table 1. Determination criteria for the skin sensitisation potential ofthe test item

 

Repetition1

Repetition

2

Repetition

3

Repetition 4

Does at least one concentration ofTest Iteminduce luciferase activity>1.5-fold:

Yes

No

Yes

No

Does the first concentration inducing luciferase activity above 1.5, have a viability above 70%:

Yes

N/A

Yes

N/A

Is p value < 0.05 for at least one concentration that yielded>1.5-fold induction with viability above 70%

Yes

N/A

Yes

N/A

Does EC1.5value occur at a concentration <1000µM (or <200µg/ml)

Yes

N/A

Yes

N/A

Does the test item induce the luciferase in a dose-dependent manner

Yes

N/A

No

N/A

Classification

Positive

Negative

Inconclusive

Negative

N/A = not applicable

Table 2.Sensitisation Potential of the Test Item: Repetition 1

Rep 1

Test item concentration (µM)

 

0.977

1.953

3.906

7.813

15.625

31.250

62.5

125

250

500

1000

2000

Mean fold induction

0.916

0.957

0.990

1.087

1.071

1.061

1.068

1.368

1.328

1.227

1.618

1.602

Viability %

85.904

84.869

92.513

95.195

90.443

90.041

91.774

94.160

87.317

97.494

85.754

93.566

T-test

7.18E-01

5.86E-01

4.87E-01

2.58E-01

2.90E-01

3.10E-01

2.94E-01

2.18E-02

2.89E-02

8.05E-02

1.08E-03

9.81E-04

SD

0.132

0.164

0.137

0.103

0.063

0.094

0.083

0.377

0.062

0.115

0.408

0.121

IMAX

1.618 at 1000 µM

EC1.5

849.105 µM 

IC30

N/A- Cell viability did not drop below 70%

IC50

N/A- Cell viability did not drop below 50%

Bold font indicates statistical significance (p <0.05)

Table 3.Sensitisation Potential of the Test Item: Repetition 2

Rep 2

Test item concentration (µM)

 

0.977

1.953

3.906

7.813

15.625

31.250

62.5

125

250

500

1000

2000

Mean fold induction

1.030

1.050

1.133

1.095

1.157

1.097

1.244

1.168

1.212

1.318

1.149

1.268

Viability %

108.009

79.898

77.861

101.187

93.561

86.116

80.960

86.007

97.281

93.871

94.702

108.997

T-test

3.82E-01

3.36E-01

1.82E-01

2.46E-01

1.49E-01

2.40E-01

7.33E-02

1.37E-01

9.53E-02

3.41E-02

1.61E-01

5.75E-02

SD

0.108

0.127

0.053

0.179

0.059

0.139

0.305

0.133

0.233

0.250

0.161

0.273

IMAX

1.318 at 500 µM

EC1.5

N/A - threshold of induction not crossed at any test item concentration

IC30

N/A- Cell viability did not drop below 70%

IC50

N/A- Cell viability did not drop below 50%

Bold font indicates statistical significance (p <0.05)

Table 4.Sensitisation Potential of the Test Item: Repetition 3

Rep 3

Test item concentration (µM)

 

0.977

1.953

3.906

7.813

15.625

31.250

62.5

125

250

500

1000

2000

Mean fold induction

1.242

1.711

1.292

1.413

1.615

1.277

1.329

1.283

1.424

1.461

1.430

1.500

Viability %

124.413

112.037

114.138

114.771

104.812

103.686

80.301

87.393

89.960

92.293

87.460

87.460

T-test

9.12E-02

5.79E-04

5.00E-02

1.25E-02

2.50E-03

5.47E-02

3.12E-02

5.17E-02

1.13E-02

7.36E-03

1.10E-02

4.93E-03

SD

0.628

0.696

0.438

0.314

0.804

0.352

0.300

0.357

0.344

0.346

0.380

0.415

IMAX

1.711 at 1.953 µM

EC1.5

1.514 µM

IC30

N/A- Cell viability did not drop below 70%

IC50

N/A- Cell viability did not drop below 50%

Bold font indicates statistical significance (p <0.05)

Table 5.Sensitisation Potential of the Test Item: Repetition 4

Rep 4

Test item concentration (µM)

 

0.977

1.953

3.906

7.813

15.625

31.250

62.5

125

250

500

1000

2000

Mean fold induction

0.819

0.768

0.672

0.743

0.716

0.692

0.731

0.657

0.635

0.682

0.670

0.590

Viability %

80.710

89.558

101.960

114.921

138.078

142.825

166.782

168.341

161.226

139.444

125.368

100.910

T-test

3.46E-01

2.46E-01

9.34E-02

1.85E-01

1.43E-01

1.13E-01

1.64E-01

8.22E-02

7.04E-02

1.03E-01

9.32E-02

3.96E-02

SD

0.120

0.230

0.037

0.100

0.030

0.030

0.068

0.093

0.180

0.048

0.085

0.055

IMAX

0.819

EC1.5

N/A - threshold of induction not crossed at any test item concentration

IC30

N/A- Cell viability did not drop below 70%

IC50

N/A- Cell viability did not drop below 50%

Bold font indicates statistical significance (p <0.05)

Interpretation of results:
other: no skin sensitising potential based on the key event "activation of keratinocytes"
Conclusions:
The read across approach is justified in the analogue justification. The target and source substances are considered unlikely to differ in their skin sensitisation potential. In a KeratinoSensTM test with the source substance p-anisic acid (CAS 100-09-4) no skin sensitising potential based on the key event “activation of keratinocytes” of skin sensitisation AOP was identified. Therefore, no skin sensitisation potential for this key event is expected for the target substance sodium anisate.

The result does not allow for the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation was performed.
Executive summary:

The human skin sensitisation potential of the strucutral analogue was assessed using the validated in vitro method: the KeratinoSens test to determine keratinocyte activation (according to OECD 442D).

In this study, because replicates 1 and 2 provided differing results (Repl. 1 - positive, Repl. 2 - negative), and Repl. 3 was inconclusive (no clear dose response), a 4. Repl. was performed. This 4. Repl. was negative and therefore, based on 2 repetitions in agreement, the test item was classified as negative using the KeratinoSens prediction mode

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

Justification for read-across

There are no experimental data available regarding the sensitisation potential of sodium anisate (CAS 536-45-8). Thus, read-across from an appropriate surrogate substance (p-anisic acid, CAS 100-09-4) is conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 in order to fulfil the standard information requirements defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.1. Common functional groups and structural similarities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

The skin sensitization potential of p-anisic acid (CAS 100-09-4) has been evaluated in an WoE approach in the acknowledged, validated test battery of three in vitro studies the so-called sensitization Adverse Outcome Pathway, AOP, as defined and described in the OECD guideline, consisting of a Direct Peptide Reactivity Assay (OECD 442C), an ARE-Nrf2 luciferase test (KeratinoSensTM, OECD 442D) and a Human Cell Line Activation Test (OECD 442E). In addition a human RIPT is available.

In vitro skin sensitisation

A reliable in vitro Human Cell Line Activation Test (h-CLAT) was performed under GLP and according the OECD 442E (Roth, 2018). To assess the dendritic cell activation potential (the third key event of the so-called sensitization Adverse Outcome Pathway, AOP) p-anisic acid was stably suspended in culture medium and administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of p-anisic acid was previously determined by two XTT tests. In these tests cytotoxic effects were observed following incubation with the test item at the highest applied concentration (1250 µg/mL) in the first XTT test and at 625 µg/mL and 1250 µg/mL in the second XTT test (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 819.4 µg/mL. Accordingly, the following concentrations were tested in the main experiment (h-CLAT): 274, 329, 395, 474, 569, 683, 819 and 983 µg/mL. The test item was tested in 2 independent runs. The RFI of CD86 and CD54 was greater than 150% and 200%, respectively, in at least one concentration of both independent runs. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT. In the solvent control (DMSO), RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%). The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%.

Thus under the conditions of the test, it can be concluded, that the test substance is predicted to possess a skin sensitizing potential based on the key event “activation of dendritic cells” of the skin sensitisation in the human Cell Line Activation Test (h-CLAT).

In a second study, the protein reactivity of p-anisic acid (CAS 100-09-4) was investigated in a Direct Peptide Reactivity Assay (DPRA) according to OECD Guideline 442C and in compliance with GLP (Fleet, 2018). The overall mean percent cysteine and lysine depletion was 1.20% and therefore the test substance is allocated to the “no to minimal reactivity” class (mean % depletion 0 - ≤ 6.38%). Accordingly, the DPRA prediction is negative, and p-anisic acid is predicted as a potential non-skin sensitizer in this test.

The activation of keratinocytes of the test item was investigated in an ARE-Nrf2 Luciferase Test in human transgenic keratinocyte cell line according to OECD 442D and in compliance with GLP (Bailey, 2019). Cells were incubated with test item concentrations of 0.977 - 2000 µg/mL in DMSO for 48 h at 37°C. After exposure, cells were lysed and antioxidant response element (ARE) dependent luciferase activity was assessed by luminescence measurement. Additionally, a MTT assay was performed to assess cytotoxicity of the test substance.In this study, because experiments 1 and 2 provided differing results (1 positive and 1 negative), and experiment 3 was inconclusive (no clear dose response), a fourth experiment was performed.The acceptance criteria were met in all experiments. The test item induced statistically significant luciferase induction > 1.5 in experiment 1, which was dose-responsive. For experiments 2 and 4, there was no induction > 1.5. Experiment 3 had statistically significant luciferase induction > 1.5, but there was no clear dose response observed. The EC1.5 values were calculated as 849.105 µM and 1.514 µM for experiments 1 and 3 respectively. Overall, based on 2 repetitions in agreement, under the conditions of this test the test item is predicted as a potential non-skin sensitizer in this test.

Supporting human data

A reliable (Klinisch score 2) human Repeated Insult Patch Test (RIPT) was conducted (Davis, 1994) with p-anisic acid. In this test the skin sensitization potential of p-anisic acid (3% in glycerine) was tested on 56 subjects (19 males and 37 females). Testing was done in accordance with a modified Draize assay employing an 8 mm Finn Chamber (occlusive patch). For the induction phase the product was applied to the scapular back Monday, Wednesday, and Friday of each week for 3 consecutive weeks, with a final patch on Monday of the 4th week, for a total of ten applications. Scoring of the skin sites was made at the end of each 48 hour patch period (72 hours on weekends). The final reading was made on Wednesday of the fourth week. Following a 12-day rest period each subject received a single 48-hour occlusive challenge patch of p-anisic acid on a naive skin site on the scapular back. Scoring of the challenge sites was made after removal of the patch and again two days later. Parameters analysed / observed were erythema, edema, vesicles, bullae, toxicity/adverse effects. No product-related systemic adverse reactions were noted during the study. Slight dermal irritation (erythema) was noted for 3 subjects at 11 reading time points during the induction phase. No subject showed any indication of skin sensitization during the challenge phase. Based on the results of this study the test substance is not considered a skin sensitizer in humans.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

With respect to the read-across substance p-anisic acid (CAS 100-09-4), the results available from the 3 in vitro tests of the sensitisation AOP test battery reveal one positive (h-CLAT) and one two negative results (DPRA and KeratinoSensTM-Assay). Additionally, human RIPT data were considered for support, and support the assumption that p-anisic acid is not expected a skin sensitiser. Based on these data and a Weight-of-Evidence Approach, the test substance does not meet the criteria for classification according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.