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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:
weight of evidence
Study period:
From 2017-11-24 to 2017-12-15
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)
Version / remarks:
2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Specific details on test material used for the study:
Batch: 17019FS4B4
Purity: 94.4%

In vitro test system

Details on study design:
VEHICLE
-Vehicle: dimethyl sulfoxide (DMSO)
-Justification for choice of vehicle: A solubility test was performed. The test item was suspended in DMSO to a final concentration of 200 mM (brown suspension). The suspension was sonicated (25 min; 24 min with a maximum temperature of 24°C) to get a homogeneous suspension. The 100-fold dilution in DMEM-glutamax of the 200 mM stock showed slight precipitation (2000 μM). A 100 mM stock in DMSO (brown solution) was prepared by diluting the 200 mM stock. The 100-fold dilution of the 100 mM DMSO stock showed no precipitation (1000 μM).

Dose Formulation and Analysis
-Preparation of Working Solutions: In the main experiments the test item was suspended in DMSO at 200 mM (brown to slight brown). The stock was sonicated (11 minutes with a maximum temperature of 23.0°C in experiment 1 and for 16 minutes with a maximum temperature of 31.5°C in experiment 2) to get a homogeneous suspension. 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 2000, 1000, 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0 and 0.98 μM (final concentration DMSO of 1%). All formulations formed a clear solution.
-Preparation of the Positive Control: Ethylene dimethacrylate glycol, a 2-fold dilution series ranging from 0.78 to 25 mM were prepared in DMSO and diluted, the final concentration of the positive control ranges from 7.8 to 250 μM (final concentration DMSO of 1%).
-Preparation of the Solvent Control: 1% DMSO in exposure medium

Test System
-Test System: A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used.
-Rationale: In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD).
-Source: Givaudan (Duebendorf, Switserland).

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+8 in experiment 1 and P+10 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 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 then incubated for about 48 hours at 37±1.0°C in the presence of 5% CO2. In total 2 experiments were performed.
-Luciferase Activity Measurement: The Steady-Glo Luciferase Assay Buffer (10 mL) and Steady-Glo Luciferase Assay Substrate (lyophilized) from Promega 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: medium was replaced after the 48 hour exposure time with fresh medium containing MTT 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.

Results and discussion

Positive control results:
- Experiment 1:
Caused a dose related induction of the luciferase activity. The Imax was 2.36 and the EC1.5 100 μM.

- Experiment 2:
Caused a dose related induction of the luciferase activity. The Imax was 2.51 and the EC1.5 55 μM.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: IC30
Run / experiment:
experiment 1
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Remarks:
The viability of the cells was higher than 70% at all test concentrations
Key result
Parameter:
other: IC50
Run / experiment:
experiment 1
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Remarks:
The viability of the cells was higher than 70% at all test concentrations
Key result
Parameter:
other: Imax
Run / experiment:
experiment 1
Value:
2.06
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
other: at 2000 μM
Key result
Parameter:
other: EC1.5 (μM)
Run / experiment:
experiment 1
Value:
1 413
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Parameter:
other: EC1.5 (μM)
Run / experiment:
experiment 2
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Key result
Parameter:
other: Imax
Run / experiment:
experiment 2
Value:
1.35
Vehicle controls valid:
yes
Positive controls valid:
yes
Key result
Parameter:
other: IC30
Run / experiment:
experiment 2
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Remarks:
The viability of the cells was higher than 70% at all test concentrations
Key result
Parameter:
other: IC50
Run / experiment:
experiment 2
Vehicle controls valid:
yes
Positive controls valid:
yes
Remarks on result:
not determinable
Remarks:
The viability of the cells was higher than 70% at all test concentrations
Other effects / acceptance of results:
ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.5% and 7.5% in experiment 1 and 2, respectively).

Any other information on results incl. tables

The test item showed no toxicity (no IC30 and IC50 value). In experiment 1, no biologically relevant induction of luciferase activity was observed. The maximum induction was with 2.06-fold > 1.5-fold and the EC1.5 was 1413 μM. Since, the EC1.5 was 1000 μM the induction was not relevant for the skin sensitization endpoint. In experiment 2, no induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.35-fold. The test item is classified as negative in the KeratinoSensTM assay since no relevant induction was observed up to test concentrations of 2000 μM.

Applicant's summary and conclusion

Interpretation of results:
other: negative
Conclusions:
The test item is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.
Executive summary:

To evaluate the ability of test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensTM assay according to OECD guideline 442D.

Two independent experiments were performed. No precipitate was observed at any dose level tested.

The test item showed no toxicity (no IC30 and IC50 value). In experiment 1, no biologically relevant induction of luciferase activity was observed. The maximum induction was with 2.06-fold > 1.5-fold and the EC1.5 was 1413μM. Since, the EC1.5 was1000μM the induction was not relevant for the skin sensitization endpoint. In experiment 2, no induction of the luciferase activity (no EC1.5 value) was measured at any of the test concentrations. The maximum luciferase activity induction (Imax) was 1.35-fold. The test item is classified as negative in the KeratinoSensTM assay since no relevant induction was observed up to test concentrations of 2000 μM.

 

In conclusion, the test item is classified as negative (no activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.