[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

6 Jan - 12 Feb 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Version / remarks:

adopted in 2018

Deviations:

yes

Remarks:

no historical control data range provided

GLP compliance:

yes

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics

Molecular formula:

not applicable due to UVCB substance

IUPAC Name:

Fatty acids, C6-18 (branched and linear) alkyl and hydrocarbons, C10-18, n-alkanes, branched alkanes, cycloalkanes, aromatics

Test material form:

liquid

In vitro test system

Details of test system:

Keratinoses transgenic cell line [442D]

Details on the study design:

TEST METHOD:
The in vitro KeratinoSens™ assay enables the detection of the skin sensitising potential of a test item by analysing the activation of keratinocytes. This activation step represents the second molecular key event of the adverse outcome pathway, which is the induction of cyto-protective signaling pathways in keratinocytes in response to electrophilic test chemicals. The KeratinoSens™ assay addresses the effect on the antioxidant response element (ARE) Nrf2-dependent pathway in the transgenic KeratinoSens™ cell line, which is an immortalised adherent cell line derived from HaCaT human keratinocytes, stably transfected with a selectable plasmid containing the luciferase gene under the transcriptional control of the Anti-oxidant Response Element (ARE) from a gene that is known to be up-regulated by contact sensitisers. The Nrf2-dependent induction of this reporter gene is analysed following exposure to test chemicals. Luminescence detection in the cell lysate after 48 ± 2 h of exposure at 37.0 ± 1.0 °C indicates luciferase induction and allows the discrimination between skin sensitisers and non-sensitisers. The method was adapted to animal product-free conditions by the testing laboratory. The Foetal Calf Serum was replaced by Human Serum in the protocol following adaptation of the cells, and porcine trypsin was replaced by non-animal recombinant trypsin (Trypzean).

PREPARATION OF TEST SOLUTIONS
- Preparation of the positive controls : The positive control was 8, 16, 32, 64 and 128 µM cinnamic aldehyde, prepared in the solvent 1% ethanol in cell culture medium.
- Preparation of the solvent, vehicle and negative controls: The solvent control was 1% ethanol in cell culture medium. No justification was given for the choice of solvent control.

DOSE RANGE FINDING ASSAY:
The solubility of the test item in ethanol was confirmed up to 40 mg/mL. Subsequent dilution in cell culture medium gave a top concentration of 400 µg/mL, which was selected as the highest concentration in the main test.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Number of replicates : 3
- Number of repetitions: 4 runs were performed. Each run consisted of 3 x 96-well plates for luminescence (n = 9 overall) and 2 x 96-well plates for MTT (n = 6 overall).
- Test chemical concentrations : 0.195, 0.391, 0.781,1.563, 3.125, 6.25, 12.5, 25, 50, 100, 200 and 400 µg/mL
- Application procedure : Per plate, for the test substance a single application of 12 concentrations of the test item was applied in cell culture medium (dilution factor of 2) with a final concentration of ethanol of 1%. For the positive and solvent control, per plate a single application of 5 concentrations of the positive control was applied in cell culture medium (dilution factor of 2) with a final concentration of ethanol of 1% and a single application of culture medium with 1% ethanol was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).
- Exposure time : 48 ± 2 h

- Study evaluation and decision criteria used: A test item is considered positive using the KeratinoSens prediction model if the following conditions are met in 2 of 3 repetitions:
1) The IMAX is ≥ 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 ≥ 1.5-fold (i.e. at the EC1.5 determining concentration). Test items that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.
3) The EC1.5 value is < 1000 µM or < 200 µg/mL for test items with no defined MW.
4) There is an apparent overall dose-response for luciferase induction (or a biphasic response).

- Description on study acceptance criteria : Test results are acceptable if:
1) The positive control (cinnamic aldehyde) produces positive results, i.e. the luciferase gene induction produced by this control is above the threshold of 1.5 in at least one of the tested concentrations and this induction is statistically significant compared to the solvent (negative) control (p < 0.05).
2) The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet either or both of the following targets:
a) Average induction in the three replicates for cinnamic aldehyde at 32 µM is within the test facility's historical range (currently 1.6 and 3)
b) EC1.5 value for cinnamic aldehyde is within the test facility's historical range (currently 6 µM and 39 µM).
Note: At least one of these criteria must be met, otherwise the run is discarded unless there is sufficient reason not to do this as determined by the Study Director. If only one criterion is met, it is recommended to check the dose-response curve of cinnamic aldehyde in order to decide on acceptability.
3) CV% of blank values < 20%.

SEEDING AND INCUBATION
One day prior to testing cells were harvested, and seeded at a density of 10000 cells/well in basic medium in conventional 96-well plates. One plate was used for the luciferase activity measurements, and one parallel replicate was used for the MTT cell viability assay. The cells were incubated overnight in the incubator.
- Seeding conditions: passage number 17, seeding density 10,000 cells per well.
- Incubation conditions : 24 h after seeding, the test and control items were applied and plates were incubated at 37 °C, 5% CO2, ≥ 95% relative humidity for 48 ± 2 h.

LUCIFERASE ACTIVITY MEASUREMENTS
The luciferase activity induction was measured. No further details were given.

DATA EVALUATION
- Cytotoxicity assessment: An MTT assay was performed to measure the cellular viability. No further details were given.
- Other: Test facility's Form F0056: “KeratinoSens data processing” v.3 was used to analyse data. This form is a Microsoft Excel workbook, validated in-house (Mar 2019) containing formulae to process the raw data as described in SOP L0057.

Vehicle / solvent control:

other: 1% ethanol in cell culture medium

Positive control:

cinnamic aldehyde [442D]

Results and discussion

Positive control results:

Cinnamic aldehyde was tested in a concentration range of 8 - 128 µM. The induction at 128 µM was ≥ 2-fold in 4 of 5 runs and showed a dose-dependent increase.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

EC 1.5 [442D]

Value:

12.04 µg/mL

Cell viability:

IC30 = 154.04 µg/mL, IC50 = 167.45 µg/mL
Imax = 4.719 at 100 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Results 2

Key result

Group:

test chemical

Run / experiment:

other: run/experiment 4

Parameter:

EC 1.5 [442D]

Value:

8.16 µg/mL

Cell viability:

IC30 = 253.91 µg/mL, IC50 = 295.65 µg/mL
Imax = 4.927 at 100 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The testing laboratory showed technical proficiency by validation in-house (studies 14XC004, 15XC001, 16XCO04) using the 10 proficiency chemicals and 11 reference chemicals described in TG 442D.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes, CV% < 20% for experiment 3 and 4 (mean CV%: 11.26%).
- Acceptance criteria met for positive control: 4 of 5 replicates showed > 1.5-fold induction in a dose-dependent manner. The average induction was 3.269 and therefore did not meet the acceptance criterium (1.6 - 3.0 at 32 µM). As the other acceptance criteria were met, the results were considered to be valid overall.
- Acceptance criteria met for variability between replicate measurements: Four experiments were performed because the variability (CV%) across the negative control wells in experiment 1 and 2 were greater than 20%. Therefore, experiment 1 (CV%: 52.69) and 2 (CV%: 35.71) were considered invalid and not used to determine the result for the test item. For experiment 3 and 4 the acceptance criteria were met (mean CV: 11.26%).
- Range of historical values if different from the ones specified in the test guideline: No historical control values were given in the study report. However, the results from the validation study were included.

Any other information on results incl. tables

Experiment 1

The test substance showed cytotoxicity, with an IC30 = 203.10 µg/mL and IC50 = 254.22 µg/mL. The Imax was 4.47 at 100 µg/mL and the EC1.5 was 15.06 µg/mL. However, the experiment was not considered to be valid as the variability across the negative control wells (CV%) was 52.69.

Experiment 2

The test substance showed cytotoxicity, with an IC30 = 287.63 µg/mL and IC50 = 317.19 µg/mL. The Imax was 2.83 at 100 µg/mL and the EC1.5 was 28.53 µg/mL. However, the experiment was not considered to be valid as the variability across the negative control wells (CV%) was 35.71.

Experiment 3

The Imax was 4.72 at 100 µg/mL and lead to an EC1.5 = 12.04 µg/mL. The test substance showed cytotoxicity, with an IC30 = 154.04 µg/mL and IC50 = 167.49 µg/mL. In this run the negative and positive control results met the acceptance criteria. As the results were considered to be valid, the overall outcome of the experiment was positive.

Experiment 4

The Imax was 4.72 at 100 µg/mL and the EC1.5 was 12.04 µg/mL. The test substance showed cytotoxicity, with an IC30 = 253.91 µg/mL and IC50 = 295.65 µg/mL. In this run, the negative and positive control results met the acceptance criteria. As the results were considered to be valid, the overall outcome of the experiment was positive.

Tables 1 - 4 with results are included in Attachment 1 under "Overall remarks, attachments".

Applicant's summary and conclusion

Interpretation of results:

other: skin sensitising potential based on the key event “inflammatory response in keratinocytes”

Conclusions:

There is regulatory acceptance in the EU for the application of the KeratinoSens™ test method to address key event 2, keratinocyte activation, in the skin sensitisation Adverse Outcome Pathway. Under the conditions of the test, the test substance induces keratinocyte activation. The result is not conclusive with respect to the non-classification or classification as skin sensitiser of the test substance and therefore further evaluation and/or data generation is required.