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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:
19 January 2018 to 23 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
yes
Remarks:
temporary deviations to temperature and humidity with no impact on results or integrity of the study
GLP compliance:
yes
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Appearance / physical state: Clear, colourless, liquid
- Storage conditions: Room temperature

In vitro test system

Details on study design:
VEHICLE
- Dimethyl sulfoxide (DMSO, Merck, Darmstadt, Germany).

PREPARATION OF TEST ITEM STOCK, SPIKING AND WORKING SOLUTIONS
- No correction was made for the composition/purity of the test item.
- A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 40 mg/mL (clear colourless solution). The 100-fold dilution of the 40 mg/mL DMSO stock in DMEM glutamax formed also a clear solution (400 μg/mL). This concentration was selected as highest concentration for the main assay (highest dose required in the current guideline).
- In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 40 mg/mL (clear colourless solution). From this 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 400, 200, 100, 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39 and 0.20 μg/mL (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate.
- All formulations formed a clear solution. No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- Test item concentrations were used within 3 hours after preparation. Any residual volumes were discarded.

PREPARATION OF THE POSITIVE CONTROL
- The positive control used in the case of KeratinoSens is Ethylene dimethacrylate glycol (EDMG, Sigma, Zwijndrecht, The Netherlands), for which a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted as described in paragraph 4.7.1, so that the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%). All concentrations of the positive control were tested in triplicate.

PREPARATION OF THE VEHICLE CONTROL
- The vehicle control was 1% DMSO in exposure medium. Eighteen wells were tested per
plate.

BLANK
- On each plate three blank wells were tested (no cells and no treatment).

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 (DMEM glutamax) supplemented with 9.1% (v/v) heat inactivated (56°C; 30 min) fetal calf serum (Life Technologies, Bleiswijk, The Netherlands).
- Maintenance medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 9.1% (v/v) heat inactivated (56°C; 30 min) fetal calf serum and geneticin (500 μg/mL).
- Exposure medium: Dulbecco’s minimal (DMEM glutamax) supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) fetal 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 71 - 98 %), containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 35.5 - 36.4°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature and humidity occurred due to opening and closing of the incubator door. Based on laboratory historical data (attached) these deviations are considered not to affect the study integrity.

SUBCULTURING
- Cells were subcultured upon reaching 80 to 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).

PLATING OF CELLS
- For testing, cells were 80 to 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+10 in experiment 1, P+12 in experiment 2 and P+15 in experiment 3.

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 chemical and control items were added.
- Three wells per plate were left empty (no cells and no treatment) to assess background values.
- The treated plates were covered with foil and then incubated for about 48 hours at 37 ± 1.0 °C in the presence of 5% CO2.
- A total of 3 valid experiments were performed.

LUCIFERASE ACTIVITY MEASUREMENT
- The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega (Leiden, The Netherlands) were mixed together.
- The assay plates were removed from the incubator and the medium 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; Sigma, Zwijndrecht, The Netherlands) 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 (Sigma, Zwijndrecht, The Netherlands) 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:
a) 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).
b) 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.
c) Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.
- All results presented in the tables of the report were calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

INTERPRETATION
- The following parameters are calculated in the KeratinoSens test method:
i) The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
ii) The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5 fold threshold (i.e. 50% enhanced luciferase activity) was obtained
iii) The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
- Fold luciferase activity induction is calculated using the equation Fold Induction = (Lsample – Lblank) / (Lsolvent – Lblank) where Lsample = luminescence reading in the test chemical well; Lblank = luminescence reading in the blank well containing no cells and no treatment; Lsolvent = average luminescence reading in the wells containing cells and vehicle (negative) control. The overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.
- The EC1.5 is calculated by linear interpolation using the equation EC1.5 = (Cb – Ca) * [(1.5 - Ia) / (Ib – Ia)] + Ca where Ca = the lowest concentration in μM with > 1.5 fold induction; Cb = the highest concentration in μM with < 1.5 fold induction; Ia = the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells); Ib = the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells).
- Viability is calculated using the equation Viability = (Vsample – Vblank) / (Vsolvent – Vblank) * 100 where Vsample = the MTT-absorbance reading in the test chemical well; Vblank = the MTT-absorbance reading in the blank well containing no cells and no treatment; Vsolvent = the average MTT-absorbance reading in the wells containing cells and vehicle
(negative) control.
- Control IC50 and IC30 are calculated by linear interpolation using the equation ICx = (Cb – Ca) * [((100-x) – Va) / (Vb – Va)] + Ca where x = the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30); Ca = the lowest concentration in μM (or μg/mL) with > x% reduction in viability; Cb = the highest concentration in μM (or μg/mL) with < x% reduction in viability; Va = the % viability at the lowest concentration with > x% reduction in viability; Vb = the % viability at the highest concentration with < x% reduction in viability. The overall IC50 and IC30 are calculated as the mean of the individual repetitions.
- 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 vehicle (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 (see Figure 1, attached):
1. The Imax is higher than (>) 1.5 fold and statistically significantly different as compared to the vehicle (negative) control (as determined by a two-tailed, unpaired Student’s t-test).
2. 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).
3. The EC1.5 value is less than (<) 1000 μM (or < 200 μg/mL for test chemicals with no defined MW).
4. 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.

CRITICAL COMPUTERISED SYSTEMS
- REES Centron version SQL 2.0: Temperature and humidity (laboratory facilities) data collection.
- Magellan Tracker version 7.0: Optical Density and Luminescence Measurement.

Results and discussion

Positive control results:
- See Table 2 and Figures 5 to 7 (attached)

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: fold induction
Run / experiment:
1 to 3
Value:
> 1.5
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Parameter:
other: percentage cell viability relative to vehicle control
Run / experiment:
1 to 3
Value:
> 70
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Other effects / acceptance of results:
RESULTS
- The test item was evaluated for the ability to activate the antioxidant/electrophile responsive
element (ARE)-dependent pathway.
- An overview of the viability and luciferase activity induction is summarised in Table 1 and Figures 2 to 4 (attached).
- The results of the positive control are summarised in Table 2 and Figures 5 to 7 (attached).
- An overview of EC1.5, Imax, IC30 and IC50 values is given in Table 3 (attached).
- Three independent experiments were performed. The cells were in these experiments
incubated with test item in a concentration range of 0.20 to 400 μg/mL (2-fold dilution steps) for 48 hours. The activation of the ARE-dependent pathway was assessed by measuring the luminescence induction compared to the vehicle control. In addition, the viability was assessed with an MTT assay.

EXPERIMENT 1
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test item showed toxicity. The calculated IC30 was 7.0 μg/mL and the calculated IC50 was 8.6 μg/mL.
- No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with test item. The Imax was 1.47 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 2.83 and the EC1.5 48 μM.

EXPERIMENT 2
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test item showed toxicity. The calculated IC30 was 14 μg/mL and the calculated IC50 was 17 μg/mL.
- A dose related luminescence activity induction was observed after treatment with test item. The Imax was 1.83 and the EC1.5 9.1 μg/mL.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.62 and the EC1.5 85 μM.
- Since the results of the first two experiments were not concordant (no biologically relevant induction in the first experiment and positive result in the second experiment) a third experiment was performed.

EXPERIMENT 3
- No precipitation was observed at the start and end of the incubation period in the 96-well plates.
- The test item showed toxicity. The calculated IC30 was 13 μg/mL and the calculated IC50 was 16 μg/mL. At 6.3 μg/mL a decrease in the viability was observed (viability of 61.1%), however, since at 13 μg/mL the viability was higher than 70% it was considered that this decrease was caused by variation in the data and the decrease was considered not relevant for the cytotoxicity endpoint.
- A dose related luminescence activity induction was observed after treatment with test item. The Imax was 1.62 and the EC1.5 10 μg/mL.
- The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.92 and the EC1.5 36 μM.

ACCEPTANCE CRITERIA
- All 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 (48 μM, 85 μM and 36 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.83-fold, 2.62-fold and 2.92-fold in experiment 1, 2 and 3 respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.4%, 7.8% and 6.4% in experiment 1, 2, and 3 respectively).
- Overall it is concluded that the test conditions were adequate and that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:
other: positive
Conclusions:
The test item was classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

GUIDELINE

The study was conducted in accordance with OECD Guideline TG 442D: In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method (adopted February, 2015).

 

METHODS

The objective of this study was to evaluate the ability of test item to activate the

antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The clear, colourless, test material was dissolved in dimethyl sulfoxide at 40 mg/mL. 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 0.20 to 400 μg/mL (2-fold dilution series). The highest test concentration was the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Three independent experiments were performed since the results of the test item in the first two experiments were not concordant.

 

RESULTS

All 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 (48 μM, 85 μM and 36 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.83-fold, 2.62-fold and 2.92-fold in experiment 1, 2 and 3 respectively). Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.4%, 7.8% and 6.4% in experiment 1, 2, and 3 respectively). Overall it was concluded that the test conditions were adequate and that the test system functioned properly.

 

The test item showed toxicity (IC30 values of 7.0 μg/mL, 14 μg/mL and 13 μg/mL and IC50 values of 8.6 μg/mL, 17 μg/mL and 16 μg/mL in experiment 1, 2 and 3, respectively). In the first experiment no biologically relevant induction of the luciferase activity was observed. In the second and third experiment, a biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 9.1 μg/mL and 10 μg/mL in experiment 2 and 3, respectively) was measured. The maximum luciferase activity induction (Imax) was 1.47-fold, 1.83-fold and 1.62-fold in experiment 1, 2 and 3 respectively. The test material was classified as positive in the KeratinoSens assay since positive results (>1.5- fold induction) were observed at test concentrations < 200 μg/mL with a cell viability of >70% compared to the vehicle control in 2 out of 3 experiments.

 

CONCLUSION

The test item was classified as positive (activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.