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Description of key information

Keratinosens Assay: 2-propanone, reaction products with diphenylamine, was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.

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:
05 October 2017 to 27 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
OECD guideline 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method, adopted on February 2015.
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The test is a part of a tiered strategy for the evaluation of skin sensitisation 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.
Specific details on test material used for the study:
No further details specified in the study report.
Details on the study design:
Vehicle and negative control
Based on solubility results, the retained vehicle was DMSO.
This vehicle was used as the negative control, and was applied to cells at a concentration of 1% in culture medium.
Since several test items were assayed concurrently, the results of the negative control were shared.

Positive control
Name: Cinnamic Aldehyde (CA)
Synonym: trans-Cinnamaldehyde
CAS No.: 14371-10-9
Storage conditions: At +4 °C and under nitrogen gas
Since several test items were assayed concurrently, the results of the positive control were shared.
For each run, the positive control item was dissolved in DMSO to a final concentration of 200 mM. This solution was then further diluted to a final concentration of 6.4 mM. It was diluted in DMSO by serial dilutions in the Master plate 100x, using a dilution factor of 2, to obtain a total of 5 concentrations.
Subsequently, each formulation of the Master plate 100x was diluted 25-fold in treatment medium in another 96-well plate called "Master plate 4x". The final tested concentrations ranged from 4 to 64 μM.
All these formulations were prepared within 4 hours before use, then kept at room temperature and protected from light until use.

Test item formulations
On the basis of solubility results, the test item dissolved in DMSO at 40 mg/mL.
One formulation was prepared for each run. It was then diluted in DMSO by serial dilutions, using a dilution factor of 2, to obtain a total of 12 concentrations in a 96-well plate; this 96-well plate was called "Master plate 100x". Subsequently, each formulation of the Master plate 100x was 25-fold diluted in treatment medium in another 96-well plate called "Master plate 4x" taking care to adjust all wells to the same DMSO level.
All formulations were prepared within 4 hours before use, and kept at room temperature and protected from light until use.

TEST SYSTEM
KeratinoSens cells: the cell line KeratinoSens is stably transfected with a modified plasmid. This plasmid contains an ARE sequence from the AKR1C2 gene and a SV40 promotor which are inserted upstream of a luciferase gene. The resulting plasmid was transfected into HaCaT keratinocytes and clones with a stable insertion selected in the presence of Geneticin / G-418. Induction of luciferase gene is the endpoint evaluated and reflects the activation by the test item of the Nrf2 transcription factor in this test.
Supplier: this cell line was provided by Givaudan.
Batch: D1.
Storage condition: at -80°C.
Mycoplasm: absence of mycoplasm was confirmed.

STUDY DESIGN
The test item was tested in two independent runs using cells from a different passage number.

Solubility assay
A solubility assay was performed prior the first treatment in order to select the vehicle (among DMSO, water for injections or treatment culture medium).
Since the test item was found soluble in DMSO at 40 mg/mL, this stock formulation was diluted in treatment culture medium to the final concentration of 400 μg/mL. Then, a visual inspection of the sample was performed to evaluate the presence or absence of precipitate/emulsion.

Method for a run of KeratinoSens assay
Cell seeding for testing
Cells were grown using general culture procedures up to 80-90% confluence,
the day prior to treatment, cells were washed twice with D-PBS containing 0.05% EDTA, harvested, re-suspended in Maintenance medium No. 2 and counted using Trypan Blue dye. Cell concentration was adjusted to a density of 8 x 104 cells/mL,
cells were then distributed into four 96-well plates (three white plates and one transparent plate), by adding 125 μL (representing 1 x 104 cells) per well taking care to avoid sedimentation of the cells during seeding,
after seeding, the cells were grown for 24 (± 1) hours in the 96-well microtiter plates prior to test item addition.

Treatment
After the 24-hour growing period, the medium was removed by aspiration and replaced by 150 μL of treatment medium,
from the Master plate 4x, a volume of 50 μL was added to each well of the three white assay plates and 50 μL to the transparent plate for the cytotoxicity evaluation,
all plates were covered by a sealing membrane to avoid evaporation of volatile test items and to avoid cross-contamination between wells,
the plates were then incubated for 48 (± 2) hours at 37 °C, 5% CO2, 90% humidity.

Endpoint measurements
Microscopic observation to evaluate the presence or absence of precipitate - transparent plate
After the 48 (± 2) hours incubation period, the presence or absence of precipitate/emulsion was determined in each well by microscopic inspection.

Luminescence flash signal to evaluate induction signal - white plates
After incubation, the supernatants from the white assay plates were discarded,
the cells were washed once with D-PBS,
a volume of 20 μL of passive lysis buffer was added to each well and the cells were incubated for 20 (± 2) minutes at room temperature and under orbital shaking,
the plates containing the passive lysis buffer were then placed in the luminometer for reading using the following program:
− 50 μL of the luciferase substrate was added to each well,
− 1 second after this addition, the luciferase signal was integrated for 2 seconds.

Absorbance signal to evaluate the cytotoxicity - transparent plate
For the cell viability assay plate, the medium was replaced by 200 μL of treatment medium,
a volume of 27 μL of a MTT solution at 5 mg/mL in D-PBS was then added to each well of the transparent 96-well plate,
the plates were covered with a sealing membrane and returned at 37 °C in the incubator in humidified atmosphere for 4 hours (± 10 minutes),
at the end of the incubation period, the medium was removed and a volume of 200 μL of a 10% SDS solution was added to each well,
the plates were covered with a sealing membrane and placed at 37°C in the incubator in humidified atmosphere for an overnight period to extract the formazan from cells,
after the overnight incubation, the absorption of each well was determined at 600 nm using the plate reader.
Positive control results:
Detailed in table form - see "Any other information"
Key result
Run / experiment:
other: Geometric mean of 2 runs
Parameter:
other: IC30
Value:
149.17
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Run / experiment:
other: Geometric mean of 2 runs
Parameter:
other: IC50
Value:
230.3
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Other effects / acceptance of results:
The geometric means IC30 and IC50 of both runs were calculated to be 149.17 and 230.30 μg/mL, respectively.
The evaluation criteria for a positive response are met in both runs. The final outcome is therefore positive.
This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment. It cannot be used on its own to conclude on a skin sensitisation potential.

Evaluation of the viability (%) of cultures treated with the test item for each run

 

Concentrations (μg/mL)

2-PROPANONE, REACTION PRODUCTS WITH DIPHENYLAMINE

0.20

0.39

0.78

1.56

3.13

6.25

12.5

25

50

100

200

400

Viability (%) in Run 1

108

112

113

114

118

135

138

137

109

77

17

8

Viability (%) in Run 2

104

106

111

122

121

145

168

192

153

112

70

46

Mean viability (%)

106

109

112

118

120

140

153

165

131

95

43

27

Geometric Mean (%)

106

109

112

118

119

140

152

162

130

93

34

19

SD

3

4

1

6

2

7

21

39

31

25

37

27

 

Gene induction values, Imax, IC30, IC50and EC1.5values, mean and SD values obtained after treatment with the test item in each run

 

Concentrations (μg/mL)

2-PROPANONE, REACTION PRODUCTS WITH DIPHENYLAMINE

0.20

0.39

0.78

1.56

3.13

6.25

12.5

25

50

100

200

400

Induction values in Run 1

0.9

0.8

1.0

1.1

1.3

1.4

1.4

1.6

1.6

5.3

12.8

0.4

Induction values in Run 2

1.0

1.1

1.2

1.3

1.3

1.3

1.3

1.6

1.8

4.3

12.7

19.1

Mean induction

1.0

1.0

1.1

1.2

1.3

1.3

1.4

1.6

1.7

4.8

12.8

9.8

SD

0.1

0.2

0.1

0.1

0.0

0.1

0.1

0.0

0.41

0.7

0.1

13.2

 

2-PROPANONE, REACTION PRODUCTS WITH DIPHENYLAMINE

Imax

EC1.5

(μg/mL)

IC50

(μg/mL)

IC30

(μg/mL)

Run 1

12.84

17.64

144.82

111.57

Run 2

19.10

19.73

366.22

199.45

Mean

15.97

n.r.

n.r.

n.r.

Geometric Mean

n.r.

18.66

230.30

149.17

SD

4.42

1.48

156.55

62.15

-: no data available

n.r.: not requested by the OECD Guideline

 

Evaluation of the viability (%) of cultures treated with the positive control for each run

 

Concentrations (μM)

Cinnamic aldehyde

4

8

16

32

64

Viability (%) in Run 1

103

112

122

138

136

Viability (%) in Run 2

106

108

108

121

127

Mean viability (%)

104

110

115

130

131

Geometric Mean (%)

104

110

115

130

131

SD

3

3

10

12

7

 

Gene induction values, Imax, IC30, IC50 and EC1.5 values obtained with the positive control for each run

 

Concentrations (μM)

Cinnamic aldehyde

4

8

16

32

64

Imax

EC1.5(μM)

IC50(μM)

IC30(μM)

Run 1

1.3

1.6

1.8

2.7

5.8

5.76

2.33

-

-

Run 2

1.2

1.3

1.7

2.3

4.4

4.40

11.34

-

-

Mean

1.2

1.5

1.7

2.5

5.1

5.08

n.r.

n.r.

n.r.

Geometric Mean

n.r.

n.r.

n.r.

n.r.

n.r.

n.r.

5.14

-

-

SD

0.1

0.2

0.0

0.3

1.0

0.96

6.37

-

-

-: no data available

n.r.: not requested by the OECD Guideline

 

Luminescence values for the negative control wells and the %CV between these values for each run

Negative control

Luminescence reading

Mean

%CV

Run 1

Replicate 1

686335

551958

645832

678502

733562

6095636

678887

19.36

Replicate 2

532141

564294

522337

556253

607948

642271

Replicate 3

770156

835439

785694

710659

953357

914608

Run 2

Replicate 1

465025

437700

449591

475976

456380

410325

442915

14.33

Replicate 2

413840

302893

496341

390090

415075

349945

Replicate 3

540682

383437

474913

473977

475055

561227

 

Historical Data

From 13 October 2015 to 13 October 2016

Control Items

Negative control

Positive control

Parameter

Mean RLU

%CV

EC1.5

Imax

n

28

28

28

28

Mean

469622.2

15.3

13.9

4.7

SD

302495.1

4.2

6.6

3.3

Lower CL 95%

352326.9

13.6

11.4

3.4

Upper CL 95%

586917.6

16.9

15.5

6.0

5thPercentile

181303.3

8.3

4.1

2.8

Median

326775.0

15.0

12.1

3.7

95thPercentile

1045640.0

22.5

22.6

15.1

Min

180496.0

8.3

2.8

2.6

Max

1093648.0

25.7

29.0

15.9

Mean – 2SD

/

6.8

0.8

/

Mean +2SD

1074612.4

23.7

27.1

11.2

CL: Confidence limit

CV: Coefficient of Variation

EC1.5: Extrapolated concentration for a 1.5 fod luciferase gene induction

Imax: Maximal induction factor of luciferase activity compared to the negative control over the complete dose-response range measured

Min: Minimal value

Max: Maximal value

n: Number of values

RLU: Relative Luminescence Unit

SD: Standard deviation

/: not applicable, negative calculated value

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the experimental conditions of the study, the test item, 2-propanone, reaction products with diphenylamine, was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.
Executive summary:

The objective of the study was to evaluate the potential of the test item, 2-propanone, reaction products with diphenylamine, to activate the Nrf2 transcription factor. This test is a part of a tiered strategy for the evaluation of skin sensitisation 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.

 

Principle

This in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

 

Methods

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37 °C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37 °C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed as part of this study.

 

Results

All acceptance criteria were met for the positive and negative controls in each run, both runs were therefore considered to be valid.

 

Both runs were performed using the following concentrations 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL in culture medium containing 1% DMSO.

 

At these tested concentrations and for both runs:

-slight to strong precipitates were observed in treated wells at concentrations ≥ 50 μg/mL,

-a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 200 μg/mL,

-the corresponding IC30 were calculated to be 111.57 and 199.45 μg/mL and IC50 were calculated to be 144.82 and 366.22 μg/mL in the first and the second run, respectively,

-statistically significant gene-fold inductions above the threshold of 1.5 compared to the negative control were noted at non-cytotoxic concentrations ≥ 25 μg/mL with an apparent overall dose-response relationship,

-the Imax were 12.84 or 19.10 and the calculated EC1.5 were 17.64 or 19.73 μg/mL in the first and the second runs, respectively.

 

The geometric means IC30 and IC50 of the two runs were calculated to be 149.17 and 230.30 μg/mL, respectively.

 

The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive.

This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

Conclusion

Under the experimental conditions of this study, the test item, 2-propanone, reaction products with diphenylamine, was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.

Endpoint:
skin sensitisation: in vitro
Data waiving:
other justification
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Keratinosens Test

The in vitro test uses the KeratinoSens cell line, an immortalized and genetically modified Human adherent HaCaT keratinocyte cell line. The KeratinoSens cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 origin of replication promoter. This promoter is fused with an ARE sequence. Sensitizers with electrophilic properties provoke the dissociation of Keap-1 from the transcription factor Nrf2. The free Nrf2 binds to the ARE sequence contained in the plasmid and therefore induces transcription of firefly luciferase.

The KeratinoSens cells were first plated on 96-well plates and grown for 24 hours at 37 °C. Then the medium was removed and the cells were exposed to the vehicle control or to different concentrations of test item and of positive controls. The treated plates were then incubated for 48 hours at 37 °C. At the end of the treatment, cells were washed and the luciferase production was measured by flash luminescence. In parallel, the cytotoxicity was measured by a MTT reduction test and was taken into consideration in the interpretation of the sensitisation results. Two independent runs were performed as part of this study.

 

Results

Both runs were performed using the following concentrations 0.20, 0.39, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL in culture medium containing 1% DMSO.

At these tested concentrations and for both runs:

-slight to strong precipitates were observed in treated wells at concentrations ≥ 50 μg/mL,

-a decrease in cell viability (i.e. cell viability < 70%) was noted at concentrations ≥ 200 μg/mL,

-the corresponding IC30 were calculated to be 111.57 and 199.45 μg/mL and IC50 were calculated to be 144.82 and 366.22 μg/mL in the first and the second run, respectively,

-statistically significant gene-fold inductions above the threshold of 1.5 compared to the negative control were noted at non-cytotoxic concentrations ≥ 25 μg/mL with an apparent overall dose-response relationship,

-the Imax were 12.84 or 19.10 and the calculated EC1.5 were 17.64 or 19.73 μg/mL in the first and the second runs, respectively.

 

The geometric means IC30 and IC50 of the two runs were calculated to be 149.17 and 230.30 μg/mL, respectively.

The evaluation criteria for a positive response are met in both runs, the final outcome is therefore positive.

This positive result can be used to support the discrimination between skin sensitizers and non-sensitizers in the context of an integrated approach to testing and assessment.

 

Conclusion

Under the experimental conditions of this study, the test item, 2-propanone, reaction products with diphenylamine, was positive in the KeratinoSens assay and therefore was considered to activate the Nrf2 transcription factor.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the positive result obtained in an in vitro KeratinoSens assay, the substance is determined to be classified as a Skin Sensitiser Category 1B in accordance with Regulation 1272/2008.