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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 2017-09-22 to 2017-11-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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

Test material

Constituent 1
Chemical structure
Reference substance name:
2-[4-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)phenyl]butyric acid
EC Number:
303-995-2
EC Name:
2-[4-(1,3-dihydro-1,3-dioxo-2H-isoindol-2-yl)phenyl]butyric acid
Cas Number:
94232-67-4
Molecular formula:
C18H15NO4
IUPAC Name:
2-[4-(1,3-dioxo-2,3-dihydro-1H-isoindol-2-yl)phenyl]butanoic acid
Specific details on test material used for the study:
Batch No.: 17001R73A
Purity: 102%

In vitro test system

Details on the study design:
VEHICLE
- Vehicle: dimethyl sulfoxide (DMSO)
- Justification for choice of vehicle: A solubility test was performed. The test item was dissolved in DMSO to a final concentration of 200 mM.

Dose Formulation and Analysis
-Preparation of Working Solutions: The solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.98 μM (final concentration DMSO of 1%).
-Preparation of the Positive Control: The positive control used is Ethylene dimethacrylate glycol, for which 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 7.81 to 250 μM (final concentration DMSO of 1%).
-Preparation of the Solvent Control: The solvent control was 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 (e.g. the KeratinoSensTM cell line).
-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+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 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 3 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 TECAN Infinite® M200 Pro Plate Reader to assess the quantity of luciferase (integration time two second).
-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:
The luciferase activity induction obtained with the positive control 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 (120 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 (2.53-fold and 2.11-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:
1.13
Vehicle controls validity:
valid
Positive controls validity:
valid
Key result
Run / experiment:
other: experiment 2
Parameter:
other: Imax
Value:
1.14
Vehicle controls validity:
valid
Positive controls validity:
valid
Other effects / acceptance of results:
OTHER EFFECTS:
-Precipitation: No precipitation was observed at any of the test concentrations.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control and negative (solvent) control: yes
-The test conditions were adequate and that the test system functioned properly.

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:

The ability of test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSensassay was evaluated based on the most recent OECD guideline TG 442D.

Test item was dissolved in dimethyl sulfoxide at 200 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 0.98 – 2000 μM (2-fold dilution series). The highest test concentration is the highest dose required in the current guideline. No precipitate was observed at any dose level tested. Two independent experiments were performed.

Both experiments passed the acceptance criteria, the test conditions were adequate and that the test system functioned properly.

Test item showed no toxicity (no IC30and IC50value) and no biologically relevant induction of the luciferase activity (no EC1.5value) was measured at any of the test concentrations in both experiments. The maximum luciferase activity induction (Imax) was 1.13-fold and 1.14-fold in experiment 1 and 2 respectively. Test item is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction)

were observed at test concentrations1000 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.

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