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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:
weight of evidence
Study period:
19 January 2018 - 23 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
One of the validated in vitro skin sensitization tests is the KeratinoSensTM assay, which is recommended in international guidelines (e.g. OECD) and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.

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:
February 2015
Deviations:
no
GLP compliance:
yes
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The KeratinoSensTM assay is recommended in international guidelines (e.g. OECD) for substitution of animal testing and mentioned in the ECHA guidance as the in vitro test to be performed as part of weight of evidence.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Appearance: Off-white powder
Test item storage: At room temperature

In vitro test system

Details on study design:
TEST ITEM PREPARATION:
Based on a solubility test, a concentration of 500 μM was selected as highest concentration for the main assay (limit of solubility).

In the main experiments the test item was dissolved in dimethyl sulfoxide (DMSO) at 50 mM (clear colorless 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 500, 250, 125, 63, 31, 16, 7.8, 3.9, 2.0, 0.98, 0.49 and 0.24 μM (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. All formulations formed a clear solution. Precipitation was observed at the start and end of the incubation period in the
96-well plates at 250 and 125 μM and upwards, respectively.

Test item concentrations were used within 3 hours after preparation.

CONTROL ITEMS
- Positive control: ethylene dimethacrylate glycol (tested in triplicate)
Amount used: 0.78 to 25 mM in DMSO, diluted so that the final concentration ranged from 7.8 to 250 μM (final concentration DMSO of 1%)
- Negative control: eighteen wells per plate of a solvent control of 1% DSMO were tested
- Blank control: on each plate three bank wells were tested (no cells and no treatment)

TEST DESIGN
- Test system: The KeratinoSens™ cell line, having a stable insertion of the luciferase reporter gene under the control of the ARE-element is used. 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 (i.e. 25) and are employed for routine testing using the appropriate maintenance medium.

- 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+10 in experiment 1, P+12 in experiment 2 and P+16 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. In total 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 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 a microplate reader to assess the quantity of luciferase (integration time two seconds).

- Cytotoxicity assessment: For the KeratinoSensTM 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 and subsequently 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 a microplate reader.

The KeratinoSensTM 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) The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition whichconsists of 18 wells tested. If the variability is higher, results should be discarded.

A KeratinoSensTM 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 KeratinoSensTM prediction is considered negative
1. 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)
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.

Results and discussion

Positive control results:
Experiment 1: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 1.96 and the EC1.5 120 µM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.47 and the EC1.5 81 µM.
Experiment 3: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.86 and the EC1.5 59 µM.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
1.56
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: 200 μM
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
1.32
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
Parameter:
other: Imax
Run / experiment:
Experiment 3
Value:
1.32
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 showed at 31 μM an induction higher than 1.5 but since at 63 and 125 μM the induction was lower than 1.5, the induction observed at 31 μM was considered to be caused by variation in the data and considered not relevant for the skin sensitization endpoint.

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 (120 μM, 81 μM and 59 μM in experiment 1, 2 and 3, respectively). A dose response was observed and the induction at 250 μM was 1.96-fold, 2.47-fold and 2.86-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%, 7.0% and 7.2% in experiment 1, 2, and 3 respectively).

Cytotoxicity

The test item showed toxicity.

Experiment 1:
The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated.

Experiment 2:
IC30: 122 µM


Experiment 3
IC30: 99 µM

Precipitation:
In all experiments precipitation was observed at the start and end of the incubation period in the 96-well plates at 250 and 125 μM and upwards, resepetively.

Applicant's summary and conclusion

Interpretation of results:
study cannot be used for classification
Remarks:
Study is part of a weight of evidence approach and is not used for classification on its own.
Conclusions:
D6-NORETH is classified as inconclusive in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed in two out of three experiments at test concentrations < 1000 μM.