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Skin sensitisation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report Date:
2019

Materials and methods

Test guideline
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).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid

Results and discussion

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.

In vitro / in chemico

Resultsopen allclose all
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.