Registration Dossier

Administrative data

Description of key information

An initial evaluation of the potential for the two main constituents of the substance to cause skin sensitisation was carried out using in silico (Quantitative) Structural Activity Relationship ((Q)SAR) approaches. The majority of models predicted CAS 56968-08-2 to be sensitising with however only no, low or moderate confidence in the predictions. For CAS 62568-43-8, an equal number of positive and negative results were obtained with either no or low confidence in the prediction. Overall, there were no arguments for a weight of evidence conclusion on skin sensitisation and therefore in vitro testing was carried out to further investigate the skin sensitisation potential of the substance.

The potential for the substance to cause skin sensitisation has been investigated using a battery of in vitro methods, in accordance with Section 8.3.1 of Annex VII of the REACH Regulation which respectively address the key events of the adverse outcome pathway (AOP) associated with skin sensitisation: molecular interaction with skin proteins (key event 1); inflammatory response in keratinocytes (key event 2) and activation of dendritic cells (key effect 3). Three respective assays: the Direct Peptide Reactivity Assay (DPRA) conducted according to OECD TG 442C, the ARE-Nrf2 Luciferase (KeratinoSensTM) assay conducted according to OECD TG 442D and the Human Cell Line Activation Test (h-CLAT) conducted according to OECD 442E were performed as part of this assessment. While the h-CLAT gave positive results, negative results were obtained in ARE-Nrf2 Luciferase and the DPRA assays respectively. The analysis of the predictive properties of this battery of three tests using known skin sensitisers has indicated that in a weight of evidence approach, a substance giving 2/3 negative results can be regarded as a non-sensitiser. Based on the findings of these tests, the substance is not regarded to be a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date: 11 March 2019 Experimental Completion Date: 28 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Annex VIII Data Requirement
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In Chemico / in vitro tests are required to be performed prior to the performance of in vivo studies (Section 8.3.1 of Annex VII of the REACH Regulation).
Positive control results:
The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in all tests.

The EC1.5 values of the positive control, cinnamic aldehyde, were 8.00 μM, 14.30 μM and 14.61 μM for test 1, 2 and 3, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 4.46, 4.33 and 4.69 for test 1, 2 and 3, respectively, which met the acceptance criterion of between 2 and 8.
Key result
Parameter:
other: EC1.5
Run / experiment:
1
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
The Imax was 1.14 in test 1 which was <1.5 fold compared to the DMSO control and therefore the EC1.5 could not be calculated.
Key result
Parameter:
other: EC1.5
Run / experiment:
2
Value:
>= 7.48
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC1.5
Run / experiment:
3
Value:
7.12
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation

Reaction mass of disodium N,N’-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate – Test 1

Test item conc. (µg/mL)

0.20

0.39

0.78

1.56

3.13

6.25

12.5

25

50

100

200

400

Mean fold induction

0.90

0.93

0.97

1.11

1.14

0.86

0.99

1.10

0.17

0.00

0.00

0.00

Statistically significant

No

No

No

No

No

No

No

No

No

No

No

No

Viability (%)

109.16

99.06

94.61

94.52

78.25

77.05

55.48

44.18

23.63

3.85

0.60

0.77

Imax

1.14

 

EC1.5(µg/mL)

N/A

IC30(µg/mL)

8.29

IC50(µg/mL)

18.56

 

Determination criteria for the skin sensitisation potential of the test item

Result

Is the Imax>1.5 fold and statistically significant

No

Is the cellular viability >70% at the EC1.5determining concentration

N/A

Is the EC1.5value < 200 µg/mL

N/A

Is there an apparent overall dose-response for luciferase induction

No

KeratinoSens™ prediction

Negative

 

 

 

 

Cinnamic aldehyde – Test 1

Positive control conc. (µM)

4

8

16

32

64

Mean fold induction

1.18

1.50

2.17

2.82

4.46

Statistically significant

No

Yes

Yes

Yes

Yes

Viability (%)

96.66

108.56

113.10

114.81

114.73

Imax

4.46

 

EC1.5(µM)

8.00

IC30(µM)

N/A

IC50(µM)

N/A

 

Test Acceptance Criteria

Result

Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations

Yes

Pass

Average induction of positive control at 64 µM between 2 – 8

Yes (4.46)

Pass

EC1.5of positive control within two standard deviations of the historical mean (‑4.70 to 32.10)

Yes (8.00)

Pass

CV% of blank values < 20%

Yes (15.5%)

Pass

 

 

Reaction mass of disodium N,N’-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate – Test 2

Test item conc. (µg/mL)

0.20

0.39

0.78

1.56

3.13

6.25

12.5

25

50

100

200

400

Mean fold induction

0.98

0.97

1.05

1.15

1.16

1.12

3.05

1.04

0.24

0.00

0.00

0.00

Statistically significant

No

No

No

No

No

No

Yes

No

No

No

No

No

Viability (%)

103.92

109.63

102.02

95.70

97.09

89.82

62.82

44.48

28.04

4.07

0.69

1.90

Imax

3.05

 

EC1.5(µg/mL)

7.48

IC30(µg/mL)

10.84

IC50(µg/mL)

21.24

 

Determination criteria for the skin sensitisation potential of the test item

Result

Is the Imax>1.5 fold and statistically significant

Yes

Is the cellular viability >70% at the EC1.5determining concentration

Yes

Is the EC1.5value < 200 µg/mL

Yes

Is there an apparent overall dose-response for luciferase induction

Yes

KeratinoSens™ prediction

Positive

 

 

 

 

 

Cinnamic aldehyde – Test 2

Positive control conc. (µM)

4

8

16

32

64

Mean fold induction

1.26

1.26

1.57

2.28

4.33

Statistically significant

No

No

 

 

 

Viability (%)

104.27

106.43

105.13

121.75

116.12

Imax

4.33

 

EC1.5(µM)

14.30

IC30(µM)

N/A

IC50(µM)

N/A

 

Test Acceptance Criteria

Result

Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations

Yes

Pass

Average induction of positive control at 64 µM between 2 – 8

Yes (4.33)

Pass

EC1.5of positive control within two standard deviations of the historical mean (-4.70 to 32.10)

Yes (14.30)

Pass

CV% of blank values < 20%

Yes (15.4%)

Pass

 

 

Reaction mass of disodium N,N’-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate – Test 3

Test item conc. (µg/mL)

0.20

0.39

0.78

1.56

3.13

6.25

12.5

25

50

100

200

400

Mean fold induction

1.22

1.16

1.24

1.44

1.47

1.14

3.72

0.77

0.01

0.00

0.00

0.00

Statistically significant

No

No

No

No

No

No

Yes

No

No

No

No

No

Viability (%)

90.47

82.76

72.73

66.21

64.62

64.29

49.98

29.34

3.30

3.23

0.99

1.78

Imax

3.72

 

EC1.5(µg/mL)

7.12

IC30(µg/mL)

1.11

IC50(µg/mL)

12.49

 

Determination criteria for the skin sensitisation potential of the test item

Result

Is the Imax>1.5 fold and statistically significant

Yes

Is the cellular viability >70% at the EC1.5determining concentration

No

Is the EC1.5value < 200 µg/mL

Yes

Is there an apparent overall dose-response for luciferase induction

Yes

KeratinoSens™ prediction

Negative

 

 

 

 

 

Cinnamic aldehyde – Test 3

Positive control conc. (µM)

4

8

16

32

64

Mean fold induction

1.14

1.24

1.56

2.20

4.69

Statistically significant

No

No

Yes

Yes

Yes

Viability (%)

99.04

106.82

98.78

109.92

113.95

Imax

4.69

 

EC1.5(µM)

14.61

IC30(µM)

N/A

IC50(µM)

N/A

 

Test Acceptance Criteria

Result

Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations

Yes

Pass

Average induction of positive control at 64 µM between 2 – 8

Yes (4.69)

Pass

EC1.5of positive control within two standard deviations of the historical mean (-4.70 to 32.10)

Yes (14.61)

Pass

CV% of blank values < 20%

Yes (10.1%)

Pass

Interpretation of results:
GHS criteria not met
Conclusions:
It was concluded that the test item gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the test item is not a skin sensitizer.
Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, Reaction mass of disodium N,N’-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The Imaxwas 1.14 in test 1 which was <1.5 fold compared to the DMSO control and therefore the EC1.5could not be calculated. The IC30value was 8.29 µg/mL and the IC50value was 18.56 µg/mL. The graph showed no overall dose-response for luciferase induction.

The Imaxwas 3.05 in test 2 which was >1.5 fold and statistically significant when compared to the DMSO control. The EC1.5was 7.48 µM. The IC30value was 10.84 µg/mL and the IC50value was 21.24 µg/mL. The graph showed an overall dose-response for luciferase induction.

The Imaxwas 3.72 in test 3 which was >1.5 fold and statistically significant when compared to the DMSO control. The EC1.5was 7.12 µM. The IC30value was 1.11 µg/mL and the IC50value was 12.49 µg/mL. The graph showed an overall dose-response for luciferase induction.

The KeratinoSens™ prediction was negative in the first test and positive in the second test. As these results were discordant a third test was conducted. In test 3 the test item gave a positive response but only at a cytotoxic concentration and therefore the prediction was negative.

Endpoint:
skin sensitisation: in chemico
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date: 25 March 2019 Experimental Completion Date: 29 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Annex VIII Data Requirement
Qualifier:
according to
Guideline:
OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))
Version / remarks:
04 February 2015
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
direct peptide binding assay
Justification for non-LLNA method:
In Chemico / in vitro tests are required to be performed prior to the performance of in vivo studies (Section 8.3.1 of Annex VII of the REACH Regulation).
Details on study design:
Solutions of DABCO-TMR-31 were incubated with two synthetic peptides (containing respectively a cysteine and a lysine amino acid) for approximate 24 hours and then the concentration of each peptide was measured relative to a series of peptide control solutions.
Positive control results:
Positive Control Result for Cysteine PeptideDepletion = 71.6% (+/- 0.25%)
Positive Control Result for Lysine PeptideDepletion = 60.4% (+/- 0.79%)
Parameter:
other: Cysteine Peptide Depletion (%)
Run / experiment:
1
Value:
>= 0
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Remarks:
Actual Mean Result = -1.27, but negative results count as zero for predictive purposes.
Parameter:
other: Lysine Peptide Depletion (%)
Run / experiment:
1
Value:
>= 2.69
Positive controls validity:
valid
Remarks on result:
no indication of skin sensitisation
Key result
Parameter:
other: Overall Mean Depletion
Run / experiment:
1
Value:
>= 1.34
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

Cysteine peptide depletion (%)

Lysine peptide depletion (%)

Overall mean depletion (%)

Reactivity class

DPRA prediction

DABCO TMR-31

0.001

2.69

1.34

No to minimal

Negative

1Negative results count as zero for predictive purposes

Interpretation of results:
GHS criteria not met
Conclusions:
With an overall mean depletion of 1.34%, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate (DABCO TMR-31) is therefore categorized into the reactivity class of “no to minimal” and hence it is predicted by DPRA as negative and hence is not a potential skin sensitizer as defined by this assay.
Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals,In chemicoSkin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C, adopted 04 February 2015) was to assess the reactivity and sensitising potential of the test item, Reaction mass of disodium N,N'-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate (DABCO TMR-31). Solutions of DABCO-TMR-31 were incubated with two synthetic peptides (containing respectively a cysteine and a lysine amino acid) for approximate 24 hours and then the concentration of each peptide was measured relative to a series of peptide control solutions.

 

Solutions of DABCO TMR-31 in acetonitrile/water 50/50 v/v were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. For each peptide analysis all acceptance criteria were met (linearity of standard response, reference and stability control concentrations, positive control depletion and test item reproducibility). With an overall mean depletion of 1.34%, DABCO TMR-31 is therefore categorized into the reactivity class of “no to minimal” and hence it is predicted by DPRA as negative and hence does not have the potential to be a skin sensitizer as defined by this assay.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Start Date: 27 March 2019 Experimental Completion Date: 11 April 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Annex VIII Data Requirement
Qualifier:
according to
Guideline:
other: OECD 442E: In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on Activation of Dendritic Cells on the Adverse Outcome Pathway for Skin Sensitisation
Version / remarks:
June 2018
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of dendritic cells
Justification for non-LLNA method:
In Chemico / in vitro tests are required to be performed prior to the performance of in vivo studies (Section 8.3.1 of Annex VII of the REACH Regulation).
Positive control results:
RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
Key result
Parameter:
other: RFI (%) CD54 Antibody
Run / experiment:
1
Value:
> 200
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI (%) CD86 Antibody
Run / experiment:
1
Value:
> 150
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI (%) CD54 Antibody
Run / experiment:
2
Value:
> 200
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: RFI (%) CD86 Antibody
Run / experiment:
2
Value:
> 150
Vehicle controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation

Reaction mass of disodium N,N’-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate – h-CLAT Test 1

 

Concentration
(u
g/mL)

RFI (%)
CD54 Antibody

RFI (%)
CD86 Antibody

Cell Viability
(%)

Medium Control

-

100.0

100.0

96.94

DMSO Control

-

100.0

100.0

96.80

Positive Control (DNCB)

3.0

435.5*

324.1*

88.74

4.0

624.2*

395.0*

85.54

Test Item

21

282.0*

125.1*

95.61

25

392.0*

142.5*

94.93

30

572.0*

188.0*

92.26

36

934.0*

377.5*

80.98

43

1382.0*

580.3*

67.79

51

1848.0*

609.7*

66.37

62

2734.0*

747.6*

65.72

74

3542.0*

844.8*

63.63

*RFI value of CD86 or CD54 fulfilled the positive criteria (CD86150% and CD54200%).

 

Reaction mass of disodium N,N’-[(2-hydroxy-5-nonylphen-1,3-ylene)bis(methylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate – h-CLAT Test 2

 

Concentration
(u
g/mL)

RFI (%)
CD54 Antibody

RFI (%)
CD86 Antibody

Cell Viability
(%)

Medium Control

-

100.0

100.0

95.75

DMSO Control

-

100.0

100.0

96.27

Positive Control (DNCB)

3.0

280.6*

489.4*

87.19

4.0

435.5*

478.2*

83.25

Test Item

21

281.8*

113.9

93.67

25

384.1-

173.4*

93.21

30

529.5*

275.2*

87.19

36

883.0*

420.0*

74.79

43

1106.8*

621.8*

67.33

51

1517.0*

704.1*

66.47

62

1930.7-

655.7*

67.95

74

2377.3*

660.3*

67.782

*RFI value of CD86 or CD54 fulfilled the positive criteria (CD86150% and CD54200%).

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
The test item Reaction mass of disodium N,N-[(2-hydroxy-5-nonylphen-1,3-ylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate, with an estimated log Pow of 2.0129, activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) ofReaction mass of disodium N,N-[(2-hydroxy-5-nonylphen-1,3-ylene)]bis[N-methylaminoacetate] andsodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate which formed a stable suspension/dispersion in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test itemconcentration for the main experiment (h-CLAT)of Reaction mass of disodium N,N-[(2-hydroxy-5-nonylphen-1,3-ylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate was previously determined by two cytotoxicity tests.

 

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 156 µg/mL up to the highest tested concentration (1250 µg/mL) in the first cytotoxicity test and starting with the concentration of 78.1 µg/mL up to the highest tested concentration (1250 µg/mL) in the second cytotoxicity test (threshold of cytotoxicity: < 75%). The mean CV75 value of both cytotoxicity tests was calculated as 61.54 µg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

 

21, 25, 30, 36, 43, 51, 62 and 74 µg/mL

 

The test item with an estimated log Pow of 2.0129 was tested in 2 independent runs.The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs. A dose response could be observed for CD54 and CD86 in both independent runs. Therefore, the h-CLAT prediction is considered positive for the test item in this h-CLAT.

 

In the DMSO control, 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%.

 

In conclusion, the test itemReaction mass of disodium N,N-[(2-hydroxy-5-nonylphen-1,3-ylene)]bis[N-methylaminoacetate] and sodium N-[(2-hydroxy-5-nonylphenyl)methyl]-N-methylaminoacetate with an estimated log Pow of 2.0129 activated THP-1 cells under the test conditions of this study. Therefore, the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

An initial evaluation of the potential for the substance to cause skin sensitisation was carried out using in silico (Quantitative) Structural Activity Relationship ((Q)SAR) approaches. The prediction of the skin sensitising potential of the two main constituents of DABCO TMR-31 (CAS 56968-08-2 and CAS 62568-43-8) was performed with BIOVIA Discovery Studio (TOPKAT) 4 .5, VEGA NIC 1.1.4 (CAESAR), OECD QSAR Toolbox 4.2, Toxtree 3.1.0 and DEREK Nexus 6.0.1. In addition, results from the Danish QSAR Database were also considered in this assessment.

The majority of models predicted CAS 56968-08-2 to be sensitising with however only no, low or moderate confidence in the predictions. For CAS 62568-43-8, an equal number of positive and negative results were obtained with either no or low confidence in the prediction. Overall, there were no arguments for a weight of evidence conclusion on skin sensitisation and therefore in vitro testing was carried out to further investigate the skin sensitisation potential of the substance.

The potential for the substance to cause skin sensitisation has been investigated using a battery of in vitro methods, in accordance with Section 8.3.1 of Annex VII of the REACH Regulation which respectively address the key events of the adverse outcome pathway (AOP) associated with skin sensitisation: molecular interaction with skin proteins (key event 1); inflammatory response in keratinocytes (key event 2) and activation of dendritic cells (key effect 3).

The reactivity and sensitising properties, and the potential for the substance to initiate key event 1 of the skin sensitisation AOP (the molecular initiating event involving the covalent binding of electrophilic substances to nucleophilic centres in skin proteins), were investigated using a Direct Peptide Reactivity Assay (DPRA) in a study conducted according to OECD TG 442C. In the study, solutions of the substance were incubated with two synthetic peptides (containing respectively a cysteine and a lysine amino acid) for approximate 24 hours and the concentration of each peptide relative to a series of peptide control solutions was subsequently measured. All acceptance criteria were met for each peptide assay. A negative result was obtained for the substance in the study, on the basis that the overall mean peptide depletion was 1.34%. Based on these findings, the substance was considered to lack skin sensitisation potential.

The second key event of the skin sensitisation AOP relates to events taking place in keratinocytes including inflammatory responses as well as gene expression associated with specific cell signalling pathways such as antioxidant/electrophilic response (ARE)-dependent pathways. The potential for the substance to initiate key event 2, by inducing genes regulated by the antioxidant response element (ARE) was investigated using the ARE-Nrf2 Luciferase (KeratinoSens) method in a study conducted according to OECD TG 442D. In two initial tests performed, negative and positive results were obtained respectively. A third test was subsequently performed, which gave a positive result at a cytotoxic concentration only and was regarded as negative with respect to gene induction. In accordance with the interpretation of non-concordant results obtained in the assay discussed in OECD TG 442D, where two out of three independent repetitions give negative results, the overall outcome for the assay is determined to be negative.

The potential for the substance to induce the third key event of the skin sensitisation AOP (activation of dendritic cells) was investigated using the Human Cell Line Activation Test (h-CLAT) in a study conducted according to OECD 442E. The assay is designed to evaluate skin sensitisation potential by measuring phenotypic changes, such as the expression of CD86 and CD54 surface markers on dendritic cells, using the human leukemia cell line THP-1 as a surrogate from human myeloid dendritic cells since these cells show enhanced CD86 and/or CD54 expression when treated with sensitisers. Following an assessment of the cytotoxicity of the substance, cultured cells were incubated with concentrations of 21, 25, 30, 36, 43, 51, 62 and 74 µg/mL. Based on an estimated log Pow of 2.0129, two independent test runs were performed in which the relative fluorescence intensity (RFI) was determined at each dose tested in order to provide an indication of the expression of CD86 and CD54, respectively. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of both runs and a dose response relationship could be observed for CD54 and CD86 in both independent runs. Based on these findings, the h-CLAT prediction was considered to be positive for the substance; indicating a potential to initiate the third key event of the skin sensitisation Adverse Outcome Pathway (AOP); activation of dendritic cells.

The evaluation of the skin sensitisation potential of the substance is based on three in vitro tests, each respectively assessing the potential of the substance to initiate a key event in the adverse outcome pathway for skin sensitisation. While negative results were obtained in the peptide-reactivity assay (DPRA) and in the ARE-dependent keratinocyte activation assay, positive results were obtained in the dendritic cell activation assay. Since the findings overall were conflicting, a weight of evidence approach has been used to characterise the potential skin sensitisation properties of the substance. The prediction properties of the three in vitro assays included in the testing battery for the substance have been previously investigated using test substances of known sensitising potential (Bauch et. al. (Reg. Tox. Pharmacol. 63: 489 -504; 2012). On the basis of these studies, is was considered that any two of the three tests rule the overall prediction (any two assays must be positive to rate the substance as a skin sensitiser and any two assays must be negative to rate the substance as a non-sensitiser). On this basis, two of the three in vitro assays are deemed to demonstrate that overall, the substance can be regarded as a non sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

No data are available on the potential of the substance to cause respiratory sensitisation. There are no standard tests available to investigate the respiratory sensitisation hazard of the substance. The substance is not a skin sensitiser and there are no reported cases of occupational asthma related to its use. The substance is unlikey to present a hazard in respect of this endpoint.

Justification for classification or non-classification

The skin sensitisation potential of the substance has been characterised using three in vitro studies that respectively assess the potential induction of three key events in the Adverse Outcomes Pathway (AOP) for skin sensitisation. Based on a weight of evidence analysis of the findings, the substance is not considered to have potential to cause skin sensitisation and classification according to Regulation (EC) 1272/2008 in respect of this endpoint is not required.