Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
08 May 2018 - 22 June 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

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 KeratinoSens(TM) 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
Test material form:
liquid
Details on test material:
Identification: C12 – 16 alkyletherdiol
Appearance: Opaque white to light yellow liquid (determined by Charles River Den Bosch)
CAS: Not available
Lot/Batch: D7801-171116001
Date of Production: 17.11.2017
Best before Date: Within 2 years
UVCB 100%

In vitro test system

Details on study design:
Based on a solubility test, a concentration of 400 µg/mL in DMSO was selected as highest concentration for the main assay (highest dose required in the current guideline).

In the main experiments the test item was suspended and dissolved in dimethyl sulfoxide (DMSO) at 40 and 5.0 mg/mL in the first and second experiment, respectively (translucent suspension and colorless solution in the first and second experiment, respectively). The stock of the first experiment (40 mg/mL) was sonicated (Time: 15 min; Temp.: 20.0 - 27.5 °C). 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 first experiment 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%) and in the second experiment resulting in final test concentrations of 50, 25, 13, 6.3, 3.1, 1.6, 0.78, 0.39, 0.20, 0.10, 0.049 and 0.024 µg/mL (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate.

Test item concentrations were used within 2.5 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 blank 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.
- Plating of cells: For testing, cells were 80-90% confluent. The passage number used was P+8 in experiment 1 and P+6 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. The treated plates were covered with foil and then incubated for about 48±1 hour at 37±1.0°C in the presence of 5% CO2. Initially, experiment 2 did not pass all the acceptability criteria and therefore this experiment was repeated. In total 2 valid experiments were performed.
- Luciferase activity measurement: Plates with 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 - 4 hours at 37°C in the presence of 5% CO2. Cells were lysed and subsequently the absorption was measured at 570 nm with the TECAN Infinite® M200 Pro Plate Reader.

DATA ANALYSIS: according to guideline.

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 KeratinoSensTM prediction is considered negative:
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.

Acceptance 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 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.

Results and discussion

Positive control results:
See other effects.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
1.54
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: 5.7 µg/mL
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
2.63
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: EC1.5: 0.06 µg/mL
Other effects / acceptance of results:
Both tests passed the acceptance criteria:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant 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 (75 µM and 32 µM in experiment 1 and 2, respectively). A dose response was observed in both experiments and the induction at 250 µM was higher than 2-fold in the second experiment only (1.91- and 3.82-fold in experiment 1 and 2, respectively).
- Finally, the average coefficient of variation of the luminescence reading for the vehicle (negative) control DMSO was below 20% (7.0% and 8.7% in experiment 1 and 2, respectively).


CYTOTOXICITY:
The test item showed toxicity in both experiments.

Experiment 1
IC30: 6.6 µg/mL
IC50: 8.3 µg/mL

Experiment 2
IC30: 7.6 µg/mL
IC50: 9.0 µg/mL

PRECIPITATION:
Precipitate was observed at the start of the incubation period at 50 µg/mL and upwards but was not observed at the end of the incubation period in both experiments.

Applicant's summary and conclusion

Interpretation of results:
other: Study cannot be used for classification independently, but in a WoE for the end point Skin Sensitisation.
Conclusions:
The test substance is classified in the Keratinosens assay as positive since positive results (>1.5-fold induction) were observed at test concentrations < 200 µg/mL cell viability of >70% compared to the vehicle control.
Executive summary:

A KeratinoSens(TM) assay was performed with C12-16 alkyletherdiol according to OECD 442D and GLP principles. The test substance was dissolved in dimethyl sulfoxide (DMSO) 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 - 400 µg/mL (2-fold dilution series) in the first experiment. The highest test concentration was the highest dose required in the current guideline.

In the second experiment, the top concentration was 5 mg/mL, to investigate the induction at 6.3 µg/mL in experiment 1 in more detail. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.024 - 50µg/mL (2-fold dilution series) .Two independent experiments were performed.

 

C12-16 alkyletherdiol showed toxicity (IC30 values of 6.6 µg/mL and 7.6 µg/mL and IC50 values of 8.3 µg/mL and 9.0 µg/mL in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 5.7 µg/mL and 0.06 µg/mL in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 1.54 -fold and 2.63-fold in experiment 1 and 2 respectively. The test item is 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.