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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:
09 January 2017 - 03 February 2017
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:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
Information from in vitro test method(s)assessing inflammatory response in keratinocytes for examining skin sensitisation potential are recognised according to Article 13(3).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Identification: Soybean oil, epoxidized, Me ester, reaction products with propylene glycol
Test item 207811/B
Appearance: Yellow liquid (determined by Charles River Den Bosch)
Purity/Composition: UVCB
Test item storage: At room temperature
Stable under storage conditions until: 17 May 2018 (expiry date)
Test item handling: No specific handling conditions required
Chemical name (IUPAC), synonym or trade name: Not indicated

In vitro test system

Details on study design:
- Test material preparation: Batch EM97160711 of the test item was a yellow liquid. The test item was dissolved in dimethyl sulfoxide at 20 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 0.098 – 200 μg/ml (2-fold dilution series). The highest concentration of 200 μg/ml was selected based on solubility. The highest concentration of 200μg/ml formed a homogeneous suspension (stable dispersion). All other concentrations formed a clear solution. Two independent experiments were performed in triplicate.

- Positive control: Ethylene dimethacrylate glycol, the final concentration of the positive control ranged from 7.81 to 250 μM (final concentration DMSO of 1%) triplicates
- Negative control: The vehicle of the test substance, dimethyl sulfoxide (DMSO). Eighteen wells were tested per plate.

- 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). 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.

- Cell culture
Basic medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetalcalf serum.
Maintenance medium: Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum and geneticin (500 μg/ml).
Exposure medium: Dulbecco’s minimal supplemented with 1% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum.

- Environmental conditions:
Humidity: 80 - 100% (actual range 65 - 90 %),
CO2: 5.0 ± 0.5% CO2 in air
Temperature: 37.0 ± 1.0°C (actual range 35.8 - 37.1°C).

-Subculturing
Confluency: 80-90% confluency when subcultured.
Maximum number of passages: 25

- Treatment
Exposure: 150 mL exposure medium + 50 μL of the 25-fold diluted test chemical and control substances.
Background measurement: Three wells per plate were left empty (no cells and no treatment) to assess background values.
Incubation time: The treated plates were incubated for about 48 hours at 37±1.0 C in the presence of 5% CO2.
Independent repeat of experiment: In total 2 experiments were performed.

- Analysis
Procedure: The assay plates were removed from the incubator and the medium is removed. Addition of 200 μL of the Steady-Glo Luciferase substrate solution (prior to addition 1:1 mixed with exposure medium) to each well. The plates were shaken for at least 3 minutes at room temperature. Plates with the cell lysates were placed in the luminometer to assess the quantity of luciferase (integration time one second).

- Cytotoxicity assessment
Procedure: MTT viability assay

- Interpretation
The following parameters are calculated in the KeratinoSensTM test method:
1. The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
2. 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
3. The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

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 ttest)
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

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 2.83 and the EC1.5 35.2 μM.
Experiment 2: The positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 3.43 and the EC1.5 14.4 μM.

In vitro / in chemico

Resultsopen allclose all
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 1
Value:
1.51
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC1,5 (µg/mL)
Run / experiment:
Experiment 1
Value:
196.4
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Viability (%)
Run / experiment:
Experiment 1
Value:
> 70
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: at all test concentrations
Key result
Parameter:
other: Imax
Run / experiment:
Experiment 2
Value:
3.62
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: EC1.5 (µg/mL)
Run / experiment:
Experiment 2
Value:
115.3
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Parameter:
other: Viability (%)
Run / experiment:
Experiment 3
Value:
> 70
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: at all test concentrations
Other effects / acceptance of results:
Two independent experiments were performed. Both tests 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 (35.2 μM and 14.4 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (2.83-fold and 3.43-fold in experiment 1 and 2, respectively).
- The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% (8.5% and 6.6% in experiment 1 and 2, 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:
study cannot be used for classification
Conclusions:
Based on the results of this KeratinoSensTM assay (OECD442D) study "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" is identified as a potential sensitising substance. However, as the test method only addresses a specific key event (Key event 2 OECD IATA AOP) of skin sensitisation, currently this result should not be used in isolation to identify a skin sensitiser. Further testing is required for classification.
On the basis that the test substance does not fall into the applicability domain of one or more methods (covering at least 2 of 3 of the key events in the OECD IATA AOP, as explained in the Skin sensitisation: in vitro_Waiver), in accordance with REACH Regulation (EC) 1907/2006: Annex VII section 8.3.2 column 2 the conduct of the OECD TG 429: Local Lymph Node Assay is considered justified.
Executive summary:

Evaluation of in vitro skin sensitization potential of "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" was performed using the KeratinoSens™ assay according to OECD TG 442D. The test item was dissolved in dimethyl sulfoxide at 20 mg/ml. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.098 – 200 μg/ml (2-fold dilution series). Two independent experiments were performed. Both a positive control ( Ethylene dimethacrylate glycol 7.81 to 250 μM ) and vehicle control (DMSO) were applied in the test. A transgenic cell line having a stable insertion of the luciferase reporter gene under the control of the ARE-element, was exposed to 150 mL exposure medium + 50 μL of the 25-fold diluted test chemical and control substances. The treated plates were incubated for about 48 hours at 37±1.0 C in the presence of 5% CO2. Doses were tested in triplicates.

Both the positive and the negative control produced a valid result. The test item showed no toxicity (no IC30 and IC50 value). Statistically significant induction of the luciferase activity was measured in both experiments. The maximum luciferase activity induction (Imax) was 1.51-fold and 3.62-fold in experiment 1 and 2 respectively. The EC1.5 of the test item was 196.4 and 115.3 μg/ml in experiment 1 and 2, respectively. "Soybean oil, epoxidized, Me ester, reaction products with propylene glycol" is classified as positive in the KeratinoSensTM assay.