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Toxicological information

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:
Jan-Feb 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
ARE-Nrf2 luciferase LuSens test method

Test material

Constituent 1
Chemical structure
Reference substance name:
1,6-anhydro-β-D-glucose
EC Number:
207-855-0
EC Name:
1,6-anhydro-β-D-glucose
Cas Number:
498-07-7
Molecular formula:
C6H10O5
IUPAC Name:
(1R,2S,3S,4R,5R)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-triol
Constituent 2
Chemical structure
Reference substance name:
Glycollaldehyde
EC Number:
205-484-9
EC Name:
Glycollaldehyde
Cas Number:
141-46-8
Molecular formula:
C2H4O2
IUPAC Name:
2-hydroxyacetaldehyde
Constituent 3
Reference substance name:
Organic acids
IUPAC Name:
Organic acids
Constituent 4
Reference substance name:
Ketones
IUPAC Name:
Ketones
Constituent 5
Reference substance name:
Phenols
IUPAC Name:
Phenols
Constituent 6
Reference substance name:
Water
EC Number:
231-791-2
EC Name:
Water
Cas Number:
7732-18-5
Molecular formula:
H2O
IUPAC Name:
water
Constituent 7
Reference substance name:
Oligomers of sugars and anhydrosugars
IUPAC Name:
Oligomers of sugars and anhydrosugars
Test material form:
liquid: viscous

In vitro test system

Details of test system:
Lusens transgenic cell line [442D]
Details on the study design:
442D

PREPARATION OF TEST SOLUTIONS
- Preparation of the test chemical stock solution: DMSO was used as solvent. A stock solution containing 200 mg/mL (CRFT) and 40 mg/mL (experiment) test item in 1% DMSO was prepared. The test item was stirred and heated in the water bath at 37 °C for 30 min before use. The stock solution was then shaken at 500 rpm for 10 min. In the experiment the stock solution was afterwards diluted (factor 1:10) to achieve a second stock solution of 4 mg/mL. Subsequent dilution to 1 % finally yielded a maximum concentration of 2000 μg/mL in the CRFT and 40 μg/mL in the experiment.
- Preparation of the test chemical serial dilutions: The stock solution were subsequently diluted toachieve a maximum concentration of 2000 μg/mL in the CRFT and 40 μg/mL. in the experiment. In the end, the total dilution factor was 1:100.
- Positive controls: Positive control used EGDMA (Ethylene glycol dimethylacrylate) in DMSO solvent. EGDMA purity is lower than the one suggested in the OECD Guideline 442D. This difference does not change the results and course of the study and has no impact on the results.
- Negative controls: Negative control used DL-Lactic acid in DMSO solvent. DL-Lactic acid purity is lower than the one suggested in the OECD Guideline 442D. This difference does not change the results and course of the study and has no impact on the results.
-Test system: LuSens cell line was designed using human keratinocyte cell line transfected with the pGL4.20 [luc2/Puro] vector, carrying the regulatory antioxidant response element (ARE) upstream of the luciferase gene (Luc2).

DOSE RANGE FINDING ASSAY:
- Dose range finder study: Cytotoxicity Range Finder Test (CRFT)
- Number of replicates: 3
- Number of repetitions: 1
- CRFT preparation: The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05 % EDTA and then trypsinized until the cells detached. Medium no. 2 was added to stop reaction and after centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2 adjusting the cell count to 83 000 (± 10 %) cells per mL. 120 μL of the cell suspension were seeded in a clear flat bottom 96 well plate. The plate was incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 23 h and 50 min.
- CRFT performance: After incubation medium no.2 was removed and 150 μL medium no. 3 was added to each well. Then 50 μL of the single test item concentrations as well as controls were added to the cells in triplicates (only test item concentrations). Finally the plate was incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
- Viability assay preparation: MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution.
- Viability assay performance: All solutions were removed from the wells of the 96 well plate and 200 μL MTT working solution was added to each well. The plate was incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere and then MTT working solution was removed and 100 μL MTT-lysis buffer was added to each well. The plate was agitated for 5 min before it was measured at a wavelength of 570 nm and of 690 nm at the photometer.
- Concentrations used: 0.98 μg/mL, 1.95 μg/mL, 3.91 μg/mL, 7.81 μg/mL, 15.63 μg/mL, 31.25 μg/mL, 62.5 μg/mL, 125 μg/mL, 250 μg/mL, 500 μg/mL, 1000 μg/mL, 2000 μg/mL
- Final concentration range selected on basis of: A test item concentration inducing a cell viability reduction below 70 % is considered as cytotoxic and is not allowed to be evaluated for luciferase induction. CV75 was also calculated (9 μg/mL)
- Final concentration range selected: 40 μg/mL, 33.33 μg/mL, 27.78 μg/mL, 23.15 μg/mL, 19.29 μg/mL, 16.08 μg/mL, 13.40 μg/mL, 11.16 μg/mL, 9.30 μg/mL, 7.75 μg/mL, 6.46 μg/mL, 5.38 μg/mL

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates: 3
- Number of repetitions: 2
- Luciferase test preparation: The cells were washed twice with PBS (without Ca2+/Mg2+) containing 0.05 % EDTA and then trypsinized until the cells detached. Medium no. 2 was added to stop reaction and after centrifugation (5 min at 380 * g), the supernatant was discarded and the cells were resuspended in medium no. 2 adjusting the cell count to 83 000 (± 10 %) cells per mL. 120 μL of the cell suspension were seeded in two clear flat bottom 96 well plates. The plates were incubated at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere for 23 h 50 min in repetition I and 24 h in repetition II. After incubation medium no.2 was removed and 150 μL medium no. 3 was added to each well. Then 50 μL of the single test item concentrations as well as controls were added to the cells in triplicates (only test item concentrations). Finally the plate was incubated for 48 h at 37 ± 1 °C in a humidified atmosphere containing 5.0 ± 0.5 % CO2.
- Luciferase test performance: After the 48 h exposure period, all solutions were removed from the second 96 wells plate and the cells were washed twice with 300 μL PBS (with Ca2+/Mg2+), then 100 μL per well of a Lysis buffer and 100 μL Steady-Glo® Reagent were added to each well and the plate was shaken slowly for 5 min at room temperature. Finally, 60 μL per well were transferred to a white flat bottom 96 well plate and the luminescence was measured for 2 seconds using a luminometer.
- Viability assay preparation: MTT working solution was prepared by mixing 9 parts of medium no. 3 with 1 part of MTT solution.
- Viability assay performance: All solutions were removed from the wells of the first 96 well plate and 200 μL MTT working solution was added to each well. The plate was incubated for 2 h at 37 ± 1 °C and 5.0 ± 0.5 % CO2 in a humidified atmosphere and then MTT working solution was removed and 100 μL MTT-lysis buffer was added to each well. The plate was agitated for 5 min before it was measured at a wavelength of 570 nm and of 690 nm at the photometer.
- Test chemical concentrations: 40 μg/mL, 33.33 μg/mL, 27.78 μg/mL, 23.15 μg/mL, 19.29 μg/mL, 16.08 μg/mL, 13.40 μg/mL, 11.16 μg/mL, 9.30 μg/mL, 7.75 μg/mL, 6.46 μg/mL, 5.38 μg/mL

DATA EVALUATION
- Study evaluation and prediction model used: A test compound is considered to have the potential to activate the Nrf2 transcription factor if the luciferase induction is ≥ 1.5 fold and statistically significant compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic (relative viability ≥ 70 %) tested concentrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid repetitions. A test compound is considered not to have the potential to activate the Nrf2 transcription factor if the effects mentioned above are not observed. In order to come to a conclusion, a minimum of two valid and independent repetitions need to indicate a positive or negative result according to the criteria described above.


- For the calculation of the luciferase induction as well as the CV75 and relative viability, see section "Any other information on materials and methods incl. tables" below.

Vehicle / solvent control:
DMSO
Negative control:
DL-Lactic acid
Positive control:
EGDMA (120 M) [442D]

Results and discussion

Positive control results:
- Aceptance criteria for positive controls: The average induction for the positive control should be ≥ 2.5 fold and it should have a relative viability of at least 70 %.
- Positive control results in Repetition I: Fold induction 7.7 and Relative Viability 95.9%
- Positive control results in Repetition II: Fold induction 5.8 and Relative Viability 97.5%

In vitro / in chemico

Results
Key result
Remarks on result:
other: details on results are provided in "Any other information on results"
Outcome of the prediction model:
positive [in vitro/in chemico]
Other effects / acceptance of results:
**RESULTS CAN BE SEEN ON THE TABLES UNDER THE SECTION "ANY OTHER INFORMATION ON RESULTS INCL. TABLES" BELOW**


ACCEPTANCE CRITERIA OF RESULTS:

- Acceptance criteria for negative control: The induction triggered by the negative control and growth control should be < 1.5 fold as compared to the induction of the solvent control and the viability should be above 70 %.
- Acceptance criteria for positive control: The average induction for the positive control should be ≥ 2.5 fold and it should have a rela-tive viability of at least 70 %.
- Acceptance criteria for standard deviation of the variability: The average percentage standard deviation (luciferase induction) of the variability in at least 21 solvent control wells should be below 20 %.
- At least 3 test concentrations must be within viability limits ≥70%
- In case a result is to be considered negative, at least one concentration should be cytotoxic (Relative viability < 70 %), or the maximum concentration of 2000 μM (2000 μg/mL) should have been tested

ACCEPTANCE CRITERIA FULFILLMENT:

- Acceptance criteria met for negative control:
Yes. Repetition I - Fold induction is 1.0 (<1.5 fold) and Relative viability is 107.2% (>70%). Repetition II - Fold induction is 0.9 (<1.5 fold) and Relative viability is 107.2% (>70%)
- Acceptance criteria met for growth control: Yes. Repetition I - Fold induction is 1.0 (<1.5 fold) and Relative viability is 122.9% (>70%). Repetition II - Fold induction is 1.1 (<1.5 fold) and Relative viability is 136.5% (>70%)
- Acceptance criteria met for positive control:
Yes. Repetition I - Fold induction is 7.7 (≥ 2.5 fold) and Relative viability is 95.9% (≥70%). Repetition II - Fold induction is 5.8 (≥ 2.5 fold) and Relative viability is 97.5% (≥70%
- Acceptance standard deviation met: Yes. Repetition I - 8.66%. Repetition II - 6.99%
- At least 3 test concentrations within viability limits (≥70%): Yes. Repetition I - 10/12 concentrations are analysable. Repetition II - 9/12 concentrations are analysable.
- Any result considered to be negative: No. Overall result for Repetition I and Repetition II is positive as per parameters described above.

A test compound is considered to have the potential to activate the Nrf2 transcription factor (sensitisation potential) if the luciferase induction is ≥ 1.5 fold and statistically significant compared to the vehicle control in 2 (or more than) consecutive non-cytotoxic (relative viability ≥ 70 %) tested concentrations whereby at least three tested concentrations must be non-cytotoxic in two independent valid repetitions.

- As the results for Repettion I and Repetition II are considered to be positive, the test item BTG Pyrolytic Sugar is considered to have the potential to activate the Nrf2 transcription factor under the conditions of the LuSens test.

Any other information on results incl. tables

Results of Relative Viability [%] in CRFT.

Parameter

Concentration

Relative

Viability

Standard

Deviation

Standard

Deviation

[µg/mL]

[%]

 

[%]

Solvent Control

-

100.0

4.26

4.26

Growth Control

-

138.2

3.06

2.22

Negative Control

5000 µM

104.5

1.92

1.84

Positive Control

120 µM

88.8

0.46

0.52

Test item

0.98

105.2

0.52

0.50

Test item

1.95

101.3

1.39

1.37

Test item

3.91

101.4

5.84

5.76

Test item

7.81

99.0

3.95

3.99

Test item

15.63

90.9

4.77

5.24

Test item

31.25

63.5**

2.42

3.81

Test item

62.5

1.0**

0.41

39.74

Test item

125

0.3**

0.28

86.60

Test item

250

1.8**

0.43

24.05

Test item

500

1.8**

0.16

9.09

Test item

1000*

3.8**

0.34

8.92

Test item

2000*

8.9** 

0.09

1.06

* = At these concentrations, precipitates were visible.

** = Cytotoxic Concentrations

Results of repetition I

 

 

Induction of Luciferase

Viability of the Cells

Parameter

Concentration

Induction

Standard

Deviation

Standard

Deviation

Relative

Viability

Standard

Deviation

Standard

Deviation

[µg/mL]

fold

 

[%]

[%]

 

[%]

Solvent Control

-

1.0

0.07

7.01

100.0

3.03

3.03

Growth Control

-

1.0

0.09

9.37

124.6

4.15

3.33

Negative Control

5000 µM

1.0

0.04

3.59

108.2

1.54

1.43

Positive Control

120 µM

6.6

0.81

12.32

90.8

10.02

11.04

Test item

5.38

1.5

0.03

2.23

104.5

4.24

4.06

Test item

6.46

1.5

0.10

6.91

98.1

4.43

4.51

Test item

7.75

1.6

0.04

2.39

96.6

4.54

4.70

Test item

9.30

1.8

0.10

5.79

98.1

3.31

3.37

Test item

11.16

2.1

0.08

3.74

96.2

5.15

5.35

Test item

13.40

2.1

0.01

0.54

95.3

3.20

3.35

Test item

16.08

2.6

0.16

6.04

93.4

4.33

4.64

Test item

19.29

3.2

0.08

2.51

91.3

3.08

3.38

Test item

23.15

3.8

0.32

8.54

91.5

3.13

3.42

Test item

27.78

4.7

0.19

3.95

83.8

3.25

3.88

Test item

33.33

6.3

0.44

7.00

80.0

3.13

3.91

Test item

40

9.5*

0.45

4.77

58.3*

4.76

8.17

* = Due to cytotoxicity this value was not used for the final evaluation of luciferase induction.

Results of repetition II

 

 

Induction of Luciferase

Viability of the Cells

Parameter

Concentration

Induction

Standard

Deviation

Standard

Deviation

Relative

Viability

Standard

Deviation

Standard

Deviation

[µg/mL]

fold

 

[%]

[%]

 

[%]

Solvent Control

-

1.0

0.06

6.35

100.0

2.97

2.97

Growth Control

-

1.2

0.06

5.05

142.3

3.67

2.58

Negative Control

5000 µM

1.0

0.04

4.19

108.4

1.11

1.02

Positive Control

120 µM

5.7

0.12

2.11

95.6

2.86

2.99

Test item

5.38

1.4

0.02

1.40

98.7

0.52

0.53

Test item

6.46

1.5

0.10

6.51

94.7

4.46

4.71

Test item

7.75

1.6

0.05

3.06

95.2

1.83

1.92

Test item

9.30

1.6

0.08

4.68

97.0

0.55

0.56

Test item

11.16

1.9

0.08

4.11

95.0

2.07

2.18

Test item

13.40

2.0

0.14

6.72

92.6

1.00

1.08

Test item

16.08

2.3

0.08

3.51

90.9

1.12

1.24

Test item

19.29

2.8

0.08

2.78

85.1

2.87

3.38

Test item

23.15

3.4

0.18

5.30

82.3

2.77

3.37

Test item

27.78

4.3

0.11

2.47

73.8

3.04

4.12

Test item

33.33

5.7*

0.18

3.15

69.1*

11.05

15.99

Test item

40

7.4*

0.17

2.28

47.9*

2.33

4.85

* = Due to cytotoxicity these values were not used for the final evaluation of luciferase induction.

Applicant's summary and conclusion

Interpretation of results:
other: The study alone cannot be used for the classification purpose but in a WoE approach of minimu m 2 in vitro assessment representing the key events in AOP
Conclusions:
In conclusion, it can be stated that under the experimental conditions of this study, the test item was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).
Executive summary:

This in vitro study evaluates the potential of the test item to activate the Nrf2 transcription factor by using the LuSens cell line. This test is part of a tiered strategy for the evaluation of skin sensitization potential. Thus, data generated with the present Test Guideline should be used to support the discrimination between skin sensitizers and non- sensitizers in the context of an integrated approach to testing and assessment. The LuSens test is an ARE Reporter Gene Assay performed according to the OECD 442D Guideline with the title “In Vitro Skin Sensitisation assays addressing the AOP Key Event on Keratinocyte activation”. It employs the use of a reporter gene for luciferase placed under the control of the antioxi- dant response element (ARE) and hence monitors Nrf2 transcription factor activity. The measured endpoint is the up-regulation of luciferase activity after 48 h of incubation with the test substance at different concentrations. This up-regulation is an indicator for the activation of the Keap1/Nrf2/ARE signalling pathway. In order to conclude on the Nrf2 transcription factor activity of the test substance, at least two independent and valid repetitions have to be performed.

The assay included a cytotoxicity range finder test (CRFT) using the concentrations 0.98 μg/mL, 1.95 μg/mL, 3.91 μg/mL, 7.81 μg/mL, 15.63 μg/mL, 31.25 μg/mL, 62.5 μg/mL, 125 μg/mL, 250 μg/mL, 500 μg/mL, 1000 μg/mL, 2000 μg/mL and one experiment, consisting of two independent repetitions (repetition I and II at concentrations 40 μg/mL, 33.33 μg/mL, 27.78 μg/mL, 23.15 μg/mL, 19.29 μg/mL, 16.08 μg/mL, 13.40 μg/mL, 11.16 μg/mL, 9.30 μg/mL, 7.75 μg/mL, 6.46 μg/mL, 5.38 μg/mL) with a treatment period of 48 h. The CRFT was performed to detect a potential cytotoxic effect of the test item and based on these results, the concentrations to be tested in the repetitions were determined.

Under the experimental conditions of this study, the test item, BTG Pyrolytic Sugar, was positive in the LuSens assay and is therefore considered to have the potential to activate the Nrf2 transcription factor (sensitizing potential).

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