Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vitro
Remarks:
KeratinoSens assay
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 October 2017 to 03 November 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin sensitisation tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).

Test material

Constituent 1
Chemical structure
Reference substance name:
Tungsten Oxide (WO3), caesium and tin-doped
EC Number:
945-942-1
Molecular formula:
Cs0.29Sn0.04WO3
IUPAC Name:
Tungsten Oxide (WO3), caesium and tin-doped
Test material form:
solid: particulate/powder
Details on test material:
- Appearance: Dark blue powder
Specific details on test material used for the study:
- No correction was made for the composition/purity of the test material.
- A solubility test was performed. No homogenous suspension could be obtained in DMSO at 200 mM. Next, the test material was suspended in DMSO to a final concentration of 100 mM and 50 mM (both homogeneous blue suspensions). The 100-fold dilution of the 100 mM DMSO stock in DMEM formed slight precipitation (1000 µM) and the 100-fold dilution of the 50 mM stock showed no precipitate (500 µM). A concentration of 1000 µM was selected as highest concentration for the main (limit based on solubility).
- In the main experiments the test material was suspended in dimethyl sulfoxide (DMSO) at 100 mM (blue suspension). The suspensions were vortexed and sonicated for 11 minutes with a maximum temperature of 35 °C in experiment 1 and for 17 minutes with a maximum temperature of 22.0 °C in experiment 2. From the stock, 11 spike solutions in DMSO were prepared (2-fold dilution series). The stock and spike solution were diluted 25-fold with exposure medium. These solutions were diluted 4-fold in the assay resulting in final test concentrations of 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98 and 0.49 µM (final concentration DMSO of 1 %). All concentrations of the test material were tested in triplicate.
- At concentrations of 6.3 mM and higher the test material was suspended in DMSO, at concentrations of 3.1 mM and below the test material was fully soluble.
- No precipitation was observed at the start and end of the incubation period in the 96-well plates. Test material concentrations were used within 2 hours after preparation. Any residual volumes were discarded.

In vitro test system

Details on the study design:
TEST SYSTEM
- A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used (e.g. the KeratinoSens™ cell line). The KeratinoSens™ cell line was generated by and obtained from Givaudan (Duebendorf, Switserland). Upon receipt, cells are propagated (e.g. 2 to 4 passages) and stored frozen as a homogeneous stock. Cells from this original stock can be propagated up to a maximum passage number from the frozen stock (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

CELL CULTURE
- Basic medium: Dulbecco’s minimal supplemented with 9.1 % (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum.
- Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum and geneticin (500 µg/ml).
- Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56 °C; 30 min) foetal calf serum. 
- Environmental conditions: All incubations, were carried out in a controlled environment, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 28 – 99 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0 °C (actual range 35.6 – 37.4 °C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day.
- Sub-culturing: Cells were sub-cultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages from the frozen stock (p+25).

EXPERIMENTAL DESIGN
- Plating of Cells: For testing, cells were 80-90% confluent. One day prior to testing cells were harvested, and distributed into 96-well plates (10,000 cells/well) in basic medium. For each repetition, three replicates were used for the luciferase activity measurements, and one parallel replicate used for the MTT cell viability assay. The cells were incubated overnight in the incubator. The passage number used was p+17 in experiment 1 and p+3 in experiment 2.
- Treatment of Cells: The medium was removed and replaced with fresh culture medium (150 μL culture medium containing serum but without Geneticin) to which 50 μL of the 25-fold diluted test material and controls were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours at 37±1.0 °C in the presence of 5% CO2. Initially, experiment 1 and 2 did not pass all the acceptability criteria (not reported) and therefore these parts of the study were repeated.
- Luciferase Activity Measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilised) were mixed together. The assay plates were removed from the incubator and the medium is removed. Then 200 µL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) was added to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two seconds).
- Cytotoxicity Assessment: For the KeratinoSens cell viability assay, medium was replaced after the 48 hour exposure time with fresh medium containing MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue tetrazolium bromide; CAS No. 298-93-1) and cells were incubated for 3 hours at 37 °C in the presence of 5% CO2. The MTT medium was then removed and cells were lysed overnight by adding 10% SDS solution to each well. After shaking, the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

ACCEPTABILITY CRITERIA
The KeratinoSens test is considered acceptable if it meets the following criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, should be above the threshold of 1.5 in at least one of the tested concentrations (from 7.8 to 250 µM).
- The EC1.5 should be between 5 and 125 µM. Moreover, the induction for Ethylene dimethacrylate glycol at 250 μM should be higher than 2-fold. If the latter criterion is not fulfilled, the dose-response of Ethylene dimethacrylate glycol should be carefully checked, and tests may be accepted only if there is a clear dose-response with increasing luciferase activity induction at increasing concentrations for the positive control.
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

INTERPRETATION
- Data analysis: The following parameters are calculated in the KeratinoSens test method:

- Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
Equation 1: Fold induction = (Lsample - Lblank) / (Lsolvent - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control

- The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual reepetitions.
Equation 2: EC1.5 = (Cb - Ca) x [ (1.5 - Ia) / (Ib - Ia) ] + Ca
Where:
Ca is the lowest concentration in μM with > 1.5 fold induction
Cb is the highest concentration in μM with < 1.5 fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

- Viability is calculated by Equation 3
Equation 3: Viability = (Vsample - Vblank) / (Vsolvent - Vblank) × 100
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.
Equation 4: ICx = (Cb – Ca) x [ ((100−x)- Va) / (Vb−Va) ] + Ca
x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
Ca is the lowest concentration in μM (or µg/mL) with > x% reduction in viability
Cb is the highest concentration in μM (or µg/mL) with < x% reduction in viability
Va is the % viability at the lowest concentration with > x% reduction in viability
Vb is the % viability at the highest concentration with < x% reduction in viability
In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using a two-tailed Student’s t-test, comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p <0.05). ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used for statistical analysis of the data. The lowest concentration with > 1.5 fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.

- Data interpretation:
A KeratinoSens prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens prediction is considered negative:
- The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s t-test)
- The cellular viability is higher than (>) 70% at the lowest concentration with induction of luciferase activity above 1.5 fold (i.e. at the EC1.5 determining concentration)
- The EC1.5 value is less than (<) 1000 μM (or < 200 µg/mL for test chemicals with no defined MW)
- There is an apparent overall dose-response for luciferase induction
- Negative results obtained with concentrations <1000 µM or 200 µg/mL should be considered as inconclusive

Results and discussion

Positive control results:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 µM (66 µM and 78 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.24-fold and 2.13-fold in experiment 1 and 2, respectively).

In vitro / in chemico

Resultsopen allclose all
Key result
Run / experiment:
other: Experiment 1
Parameter:
other: Imax
Value:
1.12
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5 could not be calculated
Key result
Run / experiment:
other: Experiment 2
Parameter:
other: Imax
Value:
1.14
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5 could not be calculated
Other effects / acceptance of results:
EXPERIMENT 1
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test material showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. The Imax was 1.12 and therefore no EC1.5 could be calculated.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.24 and the EC1.5 66 µM.

EXPERIMENT 2
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test material showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test material. The Imax was 1.14 and therefore no EC1.5 could be calculated.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.13 and the EC1.5 78 µM.

Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was between 5 and 125 µM (66 µM and 78 µM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 µM was higher than 2-fold (2.24-fold and 2.13-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (4.0% and 4.5% in experiment 1 and 2, respectively).
Overall it is concluded that the test conditions were adequate and that the test system functioned properly.


DISCUSSION
The test material showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.12-fold and 1.14-fold in experiment 1 and 2 respectively. The test material is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations ≤ 1000 µM.

Any other information on results incl. tables

Table 1: Overview of Luminescence Induction and Cell Viability of Test Material in Experiment 1 and 2

Concentration (µM)

0.49

0.98

2.0

3.9

7.8

16

31

63

125

250

500

1000

Exp 1 luminescence

0.90

0.99

0.93

1.01

1.00

1.02

1.05

1.10

1.12

1.01

0.87

0.90

Exp 1 viability (%)

97.8

95.9

98.7

99.8

109.4

94.7

91.5

98.3

91.6

85.9

83.8

76.4

Exp 2 luminescence

1.07

1.05

1.07

1.07

1.12

1.11

1.13

1.07

1.01

0.81

0.90

1.14

Exp 2 viability (%)

104.8

100.6

98.5

98.6

100.7

102.6

99.8

97.6

93.4

168.4

84.5

87.8

Table 2: Overview of Luminescence Induction and Cell Viability of Positive Control EDMG in Experiment 1 and 2

Concentration (µM)

7.8

16

31

63

125

250

Exp 1 luminescence

1.01

1.15

1.27

1.49

1.72***

2.24***

Exp 1 viability (%)

90.4

95.1

101.6

109.6

105.0

118.9

Exp 2 luminescence

0.99

1.16

1.25

1.37

1.89***

2.13***

Exp 2 viability (%)

102.4

104.0

104.9

118.7

115.4

132.7

***p<0.001 Student’s t test

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU Criteria
Conclusions:
Under the conditions of this study, the test material is classified as negative as it showed no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes.
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD TG 442D, under GLP conditions.

The objective of this study was to evaluate the ability of the test material to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay.

Since no workable suspension of the test material could be obtained at 200 mM, it was suspended in dimethyl sulfoxide at 100 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.49 – 1000 µM (2-fold dilution series). No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria; the luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was above the threshold of 1.5-fold in at least one concentration, the EC1.5 of the positive control was between 5 and 125 µM (66 µM and 78 µM in experiment 1 and 2, respectively), a dose response was observed and the induction at 250 µM was higher than 2-fold (2.24-fold and 2.13-fold in experiment 1 and 2, respectively) and finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (4.0% and 4.6% in experiment 1 and 2, respectively). Overall it is concluded that the test conditions were adequate and that the test system functioned properly. 

The test material showed no toxicity (no IC30 and IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.12-fold and 1.14-fold in experiment 1 and 2 respectively. The test material is classified as negative in the KeratinoSens assay since negative results (<1.5-fold induction) were observed at test concentrations of ≤1000 µM.

Under the conditions of this study, the test material is classified as negative as it showed no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes.