Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Description of key information

Initially, skin sensitisation was studied in vitro using the three key events, namely the DPRA, the Keratinosens and the hClat Assay.

In the DPRA assay, no indication for peptide depletion was shown. However, due to the observed precipitation of the test material, the prediction model does not apply. In the Keratinosens assay, an increase of the luciferase activity was shown in the transgenic cells, indicating potential for skin sensitisation.

Therefore, the third key event, the hClat assay, was started to verify this result. In this OECD 442E study, there was no upregulation of the expression of the relevant cell surface markers CD86 and CD54 observed, however, the logP of the test material exceeds the threshold of 3.5. Thus, according to OECD 442E, negative results must be considered as inconclusive.

Given the fact that no firm conclusion based on on vitro data for skin sensitisation could be drawn, a further in vivo test was conducted.

In this assay the OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 429. In this local lymph node assay test item concentrations of 5, 10, and 25% (w/w) were applied. The highest concentration tested was the highest concentration that could technically be achieved. The analysis of the SI values showed that the test item is a skin sensitiser. An EC3 value could not be derived since all SI values obtained were above the threshold index of 3.

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:
2019-02-06 to 2019-02-14
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Qualifier:
according to guideline
Guideline:
other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
other: (in chemico) reactivity against synthetic peptides with a thiol or amino group
Details on the study design:
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.
Positive control results:
The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic cysteine peptide. The mean depletion of the cysteine peptide was 69.94%.
Key result
Run / experiment:
other: cysteine run
Parameter:
other: mean peptide depletion [%]
Value:
5.07
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: lysine run
Parameter:
other: mean peptide depletion [%]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
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.

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

Cysteine Values of the Calibration Curve

Sample

Cysteine Peptide

Peak Area at 220 nm

Peptide Concentration [mM]

STD1

17.4940

0.5340

STD2

8.7090

0.2670

STD3

4.3730

0.1335

STD4

2.1750

0.0667

STD5

1.0820

0.0334

STD6

0.5670

0.0167

STD7

0.0000

0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide

Sample

Peak Area at 220 nm

Peptide Concentration [mM]

Peptide Depletion [%]

Mean Peptide Depletion [%]

SD of Peptide Depletion [%]

CV of Peptide Depletion [%]

Positive Control

4.8780

0.1491

70.09

69.94

0.43

0.62

4.8480

0.1482

70.27

4.9820

0.1523

69.45

Test Item

15.6390

0.4778

4.26

5.07

0.74

14.53

15.4770

0.4728

5.25

15.4040

0.4706

5.70

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model 1

Mean Cysteine andLysine PPD

Reactivity Class

DPRA Prediction²

0.00% ≤ PPD ≤ 6.38%

 No or Minimal Reactivity

Negative

6.38% < PPD ≤ 22.62%

Low Reactivity

Positive

22.62% < PPD ≤ 42.47%

Moderate Reactivity

42.47% < PPD ≤ 100%

High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD

ReactivityClass

DPRA Prediction²

0.00% ≤ PPD ≤ 13.89%

No or Minimal Reactivity

Negative

13.89% < PPD ≤ 23.09%

Low Reactivity

Positive

23.09% < PPD ≤ 98.24%

Moderate Reactivity

98.24% < PPD ≤ 100%

High Reactivity

Categorization of the Test Item

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

-

 -

 -

5.07*

Minimal reactivity*

negative*

Positive Control

-

-

-

69.94

Moderate Reactivity

positive

* only for the sake of completeness

Conclusions:
The samples containing lysine peptide could not be analysed due to strong precipitation. In this study under the given conditions the test item showed minimal reactivity towards the cysteine peptide. Due to the observed precipitation in the cysteine experiment a test item concentration of 100 mM cannot be guaranteed and a prediction cannot be made.

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:
2019-02-15 to 2019-03-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Qualifier:
according to guideline
Guideline:
other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of keratinocytes
Details on the study design:
The in vitro KeratinoSens™ assay 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.

Positive control results:
The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (3.48 (experiment 1); 2.39 (experiment 2)).
Key result
Run / experiment:
other: 1
Parameter:
other: luciferase activity
Value:
4.09
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 3.91 µM
Key result
Run / experiment:
other: 1
Parameter:
other: cell viability [%]
Value:
110.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 1
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Key result
Run / experiment:
other: 2
Parameter:
other: luciferase activity
Value:
3.16
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 1.95 µM
Key result
Run / experiment:
other: 2
Parameter:
other: cell viability [%]
Value:
100.9
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: 2
Parameter:
other: EC1.5 [µM]
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
not determinable
Other effects / acceptance of results:
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 µM 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.

The controls fullfilled the validity criteria of the test.

Results of the Cytotoxicity Measurement

 

Concentration [µM]

Cell Viability [%]

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

100

100

100

0.0

Positive Control

4.00

95.7

99.3

97.5

2.5

8.00

93.9

97.3

95.6

2.4

16.00

95.5

99.9

97.7

3.1

32.00

85.0

87.4

86.2

1.7

64.00

73.7

100.1

86.9

18.7

Test Item

0.98

91.4

116.8

104.1

17.9

1.95

107.4

100.9

104.2

4.6

3.91

110.3

101.8

106.0

6.0

7.81

93.9

109.5

101.7

11.1

15.63

85.5

27.0

56.3

41.4

31.25

1.2

0.2

0.7

0.6

62.50

0.5

0.9

0.7

0.3

125.00

0.2

0.6

0.4

0.3

250.00

0.2

1.4

0.8

0.9

500.00

2.3

2.7

2.5

0.3

1000.00

2.6

4.1

3.3

1.1

2000.00

0.3

0.2

0.3

0.1

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

1.13

1.19

1.22

1.18

0.05

 

8.00

1.29

1.57

1.40

1.42

0.14

 

16.00

1.42

1.56

1.40

1.46

0.09

 

32.00

1.32

1.97

2.12

1.81

0.43

*

64.00

3.01

3.42

4.01

3.48

0.51

*

Test Item

0.98

2.38

2.28

2.32

2.33

0.05

*

1.95

2.93

3.21

2.84

2.99

0.19

*

3.91

3.98

4.48

3.80

4.09

0.35

*

7.81

3.75

4.15

3.77

3.89

0.23

*

15.63

2.04

2.32

2.05

2.14

0.16

*

31.25

11.99

11.96

15.08

13.01

1.79

*

62.50

0.00

0.00

0.01

0.00

0.00

 

125.00

0.00

0.00

0.00

0.00

0.00

 

250.00

0.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

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

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

1.03

0.98

0.97

0.99

0.04

 

8.00

1.15

1.08

1.01

1.08

0.07

 

16.00

1.24

1.16

1.20

1.20

0.04

 

32.00

1.64

1.67

1.45

1.59

0.12

*

64.00

2.38

2.47

2.32

2.39

0.08

*

Test Item

0.98

2.46

2.27

2.49

2.41

0.12

*

1.95

3.18

3.14

3.17

3.16

0.02

*

3.91

3.32

3.22

2.82

3.12

0.26

*

7.81

1.74

1.86

1.73

1.78

0.08

*

15.63

6.81

10.06

7.39

8.09

1.73

*

31.25

0.07

0.08

0.05

0.06

0.01

 

62.50

0.00

0.00

0.00

0.00

0.00

 

125.00

0.00

0.00

0.00

0.00

0.00

 

250.00

0.00

0.00

0.00

0.00

0.00

 

500.00

0.00

0.00

0.00

0.00

0.00

 

1000.00

0.00

0.00

0.00

0.00

0.00

 

2000.00

0.00

0.00

0.00

0.00

0.00

 

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

Induction of Luciferase Activity – Overall Induction

 

Concentration [µM]

Fold Induction

Experiment 1

Experiment 2

Mean

SD

Solvent Control

-

1.00

1.00

1.00

0.00

Positive Control

4.00

1.18

0.99

1.09

0.13

8.00

1.42

1.08

1.25

0.24

16.00

1.46

1.20

1.33

0.18

32.00

1.81

1.59

1.70

0.15

64.00

3.48

2.39

2.93

0.77

Test Item

0.98

2.33

2.41

2.37

0.06

1.95

2.99

3.16

3.08

0.12

3.91

4.09

3.12

3.60

0.69

7.81

3.89

1.78

2.83

1.49

15.63

2.14

8.09

5.11

4.21

31.25

13.01

0.06

6.54

9.15

62.50

0.00

0.00

0.00

0.00

125.00

0.00

0.00

0.00

0.00

250.00

0.00

0.00

0.00

0.00

500.00

0.00

0.00

0.00

0.00

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

Additional Parameters

Parameter

Experiment 1

Experiment 2

Mean

SD

EC1.5[µM]

n.a.

n.a.

n.a.

n.a.

Imax

13.01

8.09

10.55

3.48

IC30[µM]

18.50

11.55

15.03

4.91

IC50[µM]

22.20

13.45

17.83

6.19

n.a.: not applicable

Acceptance Criteria

Criterion

Range

Experiment 1

pass/fail

Experiment 2

pass/fail

CV Solvent Control

< 20%

7.9

pass

12.3

pass

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.0

pass

2.0

pass

EC1.5 PC

± 2 x SD of historical mean

17.73

pass

28.31

pass

Induction PC at 64 µM

2 .00 < x < 8.00

3.48

pass

2.39

pass

Historical Data

Acceptance Criterion

Range

Mean

SD

N

CV Solvent Control

< 20%

11.6

3.5

96

No. of positive control concentration steps with significant luciferase activity induction >1.5

≥ 1

2.4

0.6

96

EC1.5 PC

7 < x < 34 µM

18.5

6.0

96

Induction PC at 64 µM

2.00 < x < 8.00

3.8

1.5

96
Conclusions:
In this study under the given conditions the test item did 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 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:
2019-06-27 to 2019-08-01
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018
Qualifier:
according to guideline
Guideline:
other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015
GLP compliance:
yes (incl. QA statement)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of study:
activation of dendritic cells
Details on the study design:
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 sensitisers and
non-sensitisers.
Positive control results:
The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in both
experiments. The threshold of 150% for CD86 (244% experiment 1; 366% experiment 2) and
200% for CD54 (298% experiment 1; 459% experiment 2) were clearly exceeded.
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
120
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 9.78 µg/mL
Key result
Run / experiment:
other: 1
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
126
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 5.66 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD86 [%]
Value:
93
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 9.78 µg/mL
Key result
Run / experiment:
other: 2
Parameter:
other: relative fluorescence intensity CD54 [%]
Value:
128
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Concentration: 6.79 µg/mL
Other effects / acceptance of results:
Acceptance criteria:

The test meets acceptance criteria if:
• the cell viability of the solvent controls is > 90%,
• the cell viability of at least four tested doses of the test item in each run is > 50%,
• the RFI values of the positive control (DNCB) is ≥ 150% for CD86 and ≥ 200% for CD54 at a cell viability of > 50%,
• the RFI values of the solvent control is not ≥ 150% for CD86 and not ≥ 200% for CD54,
• the MFI ratio of CD86 and CD54 to isotype IgG1 control for the medium and DMSO control, is > 105%.

The test mets the acceptance criteria.

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

-

95.1

94.3

94.6

1982

1209

752

1230

457

86

94

264

161

Solvent Control 2 (THF)

0.20%

94.9

95.3

95.3

1786

1281

808

978

473

100

100

221

159

Solvent Control 1 (DMSO)

0.20%

93.6

94

92.8

2206

1268

780

1426

488

100

100

283

163

DNCB

4.00

85.7

86

84.3

4288

2264

808

3480

1456

244

298

531

280

Test item

11.74

33.6

30.7

31.9

2102

1238

941

1161

297

119

63

223

132

9.78

38.1

36.9

38.5

2200

1407

1031

1169

376

120

79

213

136

8.15

66.4

66

64.1

1586

1495

977

609

518

62

110

162

153

6.79

84.6

85.3

85.1

1473

1517

947

526

570

54

121

156

160

5.66

90.6

90

90.1

1594

1446

849

745

597

76

126

188

170

4.72

92.2

91.8

91.2

1572

1365

890

682

475

70

100

177

153

3.93

92.6

92.6

92.6

1466

1305

878

588

427

60

90

167

149

3.28

93.6

93.4

94.3

1552

1341

892

660

449

67

95

174

150

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

-

96.8

96.2

96

1140

882

682

458

200

112

102

167

129

Solvent Control 2 (THF)

0.20%

96

95.9

95.9

1153

905

704

449

201

100

100

164

129

Solvent Control 1 (DMSO)

0.20%

95.5

95.7

95.9

1105

892

696

409

196

100

100

159

128

DNCB

4.00

85.7

85.3

83.8

2287

1691

792

1495

899

366

459

289

214

Test item

11.74

55.1

54.9

55.8

1241

1057

869

372

188

83

94

143

122

9.78

47.5

46.1

47.1

1239

946

821

418

125

93

62

151

115

8.15

75.7

75.5

75.8

1095

1107

861

234

246

52

122

127

129

6.79

91.7

91.4

90

1010

1122

865

145

257

32

128

117

130

5.66

92.6

93.9

93.9

848

947

781

67

166

15

83

109

121

4.72

92.2

93.7

90.5

943

989

821

122

168

27

84

115

120

3.93

93.3

93.2

93.5

926

935

730

196

205

44

102

127

128

3.28

95.3

94.2

94.4

949

967

763

186

204

41

101

124

127
Conclusions:
In this study under the given conditions the test item did not upregulate the expression of the cell surface markers in at least two independent experiment runs.
Therefore, the test item might 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 such as IATA.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 15, 2019 - Jan 23, 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: Pre-test test: 12 - 13 weeks
Main Test: 9-9 weeks
- Weight at study initiation: Pre-test and Main test: 16.9 - 21.2 g
- Housing: grouped per dose
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 22-65%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 6
Vehicle:
propylene glycol
Concentration:
5, 10, and 25 (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 25% in MEK
- Irritation: No
- Lymph node proliferation response: -

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: Stimulation index > 3

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Standard statistical methods have been applied for data processing.
Positive control results:
Conc. SI
0%: 1.0
5% 7.7
10% 7.5
25% 11.7
Key result
Parameter:
SI
Value:
7.7
Test group / Remarks:
Test Group: 5% in MEK
Key result
Parameter:
SI
Value:
7.5
Test group / Remarks:
Test Group: 10% in MEK
Key result
Parameter:
SI
Value:
11.7
Test group / Remarks:
Test Group: 25% in MEK
Cellular proliferation data / Observations:

EC3 CALCULATION : EC3 = (a-c) [(3-d)/(b-d)] + c; a,b,c,d = co-ordinates of the two pairs of data lying immediately above and below the S.I. value of 3 on the LLNA dose response plot

CLINICAL OBSERVATIONS: No deaths occurred during the study period. No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

BODY WEIGHTS: The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Calculation of Stimulation Indices per Dose Group:

Test item concentration

Group Calculation

Mean DPM per
animal (2 lymph nodes) a)

SD

S.I.

Vehicle Control Group (MEK)

1214.4

351.3

1.0

5% Test Item

9299.6

4525.9

7.7S

10% Test Item

9048.8

2631.7

7.5S

25% Test Item

14237.6

6932.5

11.7 S

a)      Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)

S      statistically significant (p<0.05) vs. concurrent vehicle control


Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item was found to be a skin sensitiser. An EC3 value could not be derived since all S.I. values obtained were above the threshold index of 3.
Executive summary:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 429.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could technically be achieved.

The test item was found to be a skin sensitiser. An EC3 value could not be derived since all S.I. values obtained were above the threshold index of 3.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the provided information there is need to classified according to the EU Regulation (EC) No 1272/2008 on Classification, Labelling and Packaging of Substances and Mixtures. As the EC3 value could not be determined and may be below 2 % Category 1B is justified.