2-ethoxyethyl ethyl carbonic acid ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 November 2018 to 16 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The KeratinoSens™ test is a method for which validation studies have been completed followed by an independent peer review conducted by the European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM). The KeratinoSens™ test method was considered scientifically valid to be used as part of an IATA (Integrated Approach to Testing and Assessment), to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxyethyl ethyl carbonic acid ester

EC Number:

809-934-0

Cas Number:

627034-93-9

Molecular formula:

C7H14O4

IUPAC Name:

2-ethoxyethyl ethyl carbonic acid ester

Test material form:

liquid

Details on test material:

- Appearance: clear, colourless liquid
- Storage conditions: room temperature, in the dark

In vitro test system

Details on the study design:

CONTROLS
- Negative Control: DMSO
- Positive Control: Cinnamic Aldehyde

TEST SYSTEM
- The KeratinoSens™ cell line (test system) 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 luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test substances.
- Method of administration of test material: Per plate, a single application of 12 concentrations of the test material was applied in cell culture medium (dilution factor of 2) with a final concentration of DMSO of 1%. The top concentration was previously determined by solubility testing.
- Method of administration of reference materials: 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 DMSO of 1% and a single application of culture medium with 1% DMSO was applied as the negative control (6 wells per plate). One well per plate was left empty (no cells).
- Exposure times of test materials and reference materials: Cells were incubated with the test or reference material for 48 ± 2h prior to endpoint measurements.
- Number of repetitions: Three repetitions (runs) were performed. Each repetition consisted of 3 x 96-well plates for luminescence (n=9 overall) and 2 x 96-well plates for MTT (n=6 overall). The validity of each repetition was assessed following acceptance criteria.

OVERVIEW
- Preliminary testing: Determination of the top concentration by solubility testing
- Day 1: Cell seeding (3 x 96-well plates for Luminescence; 2 x 96-well plate for MTT); 10,000 cells per well, passage number 17.
- Day 2: 24 hours after seeding, the test and control materials were applied and plates were incubated at 37 °C, 5 % CO2, ≥ 95 % relative humidity for 48 ± 2 hours.
- Day 4: Evaluation of luciferase activity by luminescence (3 plates) and cell viability by MTT testing (2 plates)

DATA ANALYSIS
- XCellR8 Form F0056: “KeratinoSens data processing” v.2 was used to analyse data. This form is a Microsoft Excel workbook, validated in-house (24JUL18) containing formulae to process the raw data as described in SOP L0057.
- The following parameters were calculated using the KeratinoSens™ test method:
• The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration of the test material and positive control.
• The EC1.5 value representing the concentration at which the induction of luciferase activity was above the 1.5-fold threshold (i.e. 50% enhanced luciferase activity).
• For each concentration showing > 1.5-fold luciferase activity induction, statistical significance is calculated (e.g. by a two-tailed Student’s t-test), comparing the luminescence values for the three replicate samples with the luminescence values in the solvent (negative) control wells to determine whether the luciferase activity induction is statistically significant (p < 0.05). The lowest concentration with > 1.5-fold luciferase activity induction is the value determining the EC1.5 value. It is checked in each case whether this value is below the IC30 value, indicating that there is less than 30% reduction in cellular viability at the EC1.5 determining concentration.
• The percentage of viability as compared to the Negative control.

ACCEPTANCE OF THE ASSAY
Test results are acceptable if:
• 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).
• The lMAX and the EC1.5 for cinnamic aldehyde is calculated and meet either or both of the following targets: Average induction in the three replicates for cinnamic aldehyde at 32 µM is within the XCellR8 historical range (currently 1.6 and 3) or EC1.5 value for cinnamic aldehyde is within the XCellR8 historical range (currently 6 µM and 39 µM). 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.
• CV % of blank values < 20 %

INTERPRETATION OF RESULTS
A test material is considered positive using the KeratinoSens prediction model if the following conditions are met in 2 of 3 repetitions:
• 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).
• 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). Test materials that only induce the gene activity at cytotoxic levels are not rated positive, as in the case for some non-sensitising skin irritants.
• The EC1.5 value is < 1000 µM or < 200 µg/mL for test materials with no defined MW.
• There is an apparent overall dose-response for luciferase induction (or a biphasic response).

Results and discussion

Positive control results:

The acceptance criteria stated that the Positive Control (PC) (Cinnamic aldehyde) must have induction >1.5-fold in at least one concentration, it showed this at 4/5 concentrations and therefore met the acceptance criteria.

In vitro / in chemico

Results

Results 1

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

1.226

At concentration:

500 mM

Cell viability:

103.327 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration.

Results 2

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

0.854

At concentration:

1 000 mM

Cell viability:

94.869 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration.

Results 3

Key result

Group:

test chemical

Run / experiment:

run/experiment 3

Parameter:

Imax [442D]

Value:

1.443

At concentration:

1 000 mM

Cell viability:

108.383 %

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: The maximal average fold induction of luciferase activity (IMAX) value observed at any concentration.

Results 4

Key result

Group:

test chemical

Run / experiment:

mean

Parameter:

EC 1.5 [442D]

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: N/A - threshold of induction not crossed at any test material concentration in any repetition.

Other effects / acceptance of results:

SOLUBILITY ASSESSMENT
- The test concentrations of the test material used in the KeratinoSens™ method were selected on the basis of a solubility test carried out during the study: Solubility of the test material was confirmed up to 200 mM in DMSO. Subsequent dilution in cell culture medium gave a top concentration of 2000 µM.

SKIN SENSITISATION
- In this study, the test material was classified as a Negative using the KeratinoSens prediction model.
- The sensitisation potential of the test material was quantified by calculating 2 parameters known as the EC1.5 and the IMAX value:
• The EC1.5 value is the Effective Concentration (EC) of test material that yielded an induction of luciferase activity greater than 1.5-fold over untreated controls. If at least one concentration induces statistically significant luciferase activity > 1.5, then the product is classified as positive provided the cell viability measured by MTT is greater than 70%. The test material did not induce statistically significant luciferase induction > 1.5 in any of the 3 repetitions. The statistical significance, viability, dose response and dose acceptance criteria were all met and therefore: The test material was classified as negative using the KeratinoSens prediction model.
• The IMAX value is the maximum induction observed within the concentration range tested. Although the KeratinoSens™ test is not validated to predict potency, the IMAX value can provide a useful tool for preliminary comparison of sensitisation potential between test materials. The maximum induction for rep 1 was 1.226 at 500.000 µM, 0.854 for rep 2 at 1000.000 µM and for rep 3 the maximum induction was 1.443 at 1000.000 µM. For reference, during test validation, sensitising proficiency chemicals produced IMAX values of up to 36-fold over untreated controls.
- All of the formal acceptance criteria of the tests were met.

Any other information on results incl. tables

Table 1: Results of Repetition 1

	Test Material Concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.500	125.000	250.000	500.000	1000.000	2000.000
Mean fold induction	0.716	1.043	1.043	1.094	1.128	1.061	1.187	1.165	1.158	1.226	1.059	0.818
Viability %	104.281	85.624	89.579	93.600	88.757	86.403	98.080	93.171	102.439	103.327	102.210	107.861
T-test	3.48E-03	6.54E-01	6.37E-01	3.24E-01	1.78E-01	5.18E-01	4.94E-02	7.79E-02	9.33E-02	1.67E-02	5.25E-01	5.80E-02
SD	0.122	0.209	0.054	0.166	0.155	0.145	0.124	0.073	0.111	0.043	0.024	0.160
IMAX	1.226 at 500.000 µM
EC1.5	N/A - threshold of induction not crossed at any test material concentration
IC30	N/A- Cell viability did not fall below 70%
IC50	N/A- Cell viability did not fall below 50%

 

Table 2: Results of Repetition 2

	Test Material Concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.500	125.000	250.000	500.000	1000.000	2000.000
Mean fold induction	0.519	0.689	0.667	0.699	0.673	0.731	0.804	0.752	0.770	0.831	0.854	0.526
Viability %	88.937	72.463	86.221	97.222	96.247	101.615	109.586	92.829	105.862	90.815	94.869	93.426
T-test	2.59E-06	1.73E-03	6.08E-04	2.09E-03	8.45E-04	5.86E-03	3.81E-02	9.41E-03	1.53E-02	7.48E-02	1.19E-01	3.51E-06
SD	0.060	0.170	0.046	0.128	0.107	0.148	0.101	0.076	0.062	0.118	0.089	0.065
IMAX	0.854 at 1000.000 µM
EC1.5	N/A - threshold of induction not crossed at any test material concentration
IC30	N/A- Cell viability did not fall below 70%
IC50	N/A- Cell viability did not fall below 50%

 

Table 3: Results of Repetition 3

	Test Material Concentration (µM)
	0.977	1.953	3.906	7.813	15.625	31.250	62.500	125.000	250.000	500.000	1000.000	2000.000
Mean fold induction	0.793	0.825	0.853	1.006	0.958	0.975	1.031	1.099	1.122	1.203	1.443	1.271
Viability %	141.968	97.441	105.926	96.755	102.259	102.534	98.897	94.869	107.917	109.373	108.383	136.691
T-test	2.87E-02	6.22E-02	1.16E-01	9.53E-01	6.52E-01	7.87E-01	7.39E-01	2.96E-01	1.93E-01	3.25E-02	1.38E-05	4.65E-03
SD	0.086	0.069	0.062	0.133	0.037	0.006	0.061	0.160	0.087	0.111	0.113	0.069
IMAX	1.443 at 1000.000 µM
EC1.5	N/A - threshold of induction not crossed at any test material concentration
IC30	N/A- Cell viability did not fall below 70%
IC50	N/A- Cell viability did not fall below 50%

 

Applicant's summary and conclusion

Interpretation of results:

other: Negative according to the KeratinoSens prediction model.

Conclusions:

Under the conditions of this study, the test material is negative according to the KeratinoSens prediction model.

Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guideline OECD 442D, under GLP conditions.

The human skin sensitisation potential of the test material was assessed using the validated in vitro method, the KeratinoSens™ assay, adapted to animal product-free conditions by the Test Facility, and validated in-house to determine keratinocyte activation.

After 48h exposure of cells with 12 concentrations of the test material, Luciferase measurements and MTT viability testing were performed.

The EC1.5 value is the Effective Concentration (EC) of test material that yielded an induction of luciferase activity greater than 1.5-fold over untreated controls. If at least one concentration induces statistically significant luciferase activity >1.5, then the product is classified as positive provided the cell viability measured by MTT is greater than 70%. The test material did not induce statistically significant luciferase induction >1.5 in any of the 3 repetitions. The statistical significance, viability, dose response and dose acceptance criteria were all met and therefore: The test material was classified as negative using the KeratinoSens prediction model.

The IMAX value is the maximum induction observed within the concentration range tested. Although the KeratinoSens™ test is not validated to predict potency, the IMAX value can provide a useful tool for preliminary comparison of sensitisation potential between test materials. The maximum induction for rep 1 was 1.226 at 500.000 µM, 0.854 for rep 2 at 1000.000 µM and for rep 3 the maximum induction was 1.443 at 1000.000 µM. For reference, during test validation, sensitising proficiency chemicals produced IMAX values of up to 36-fold over untreated controls. All of the formal acceptance criteria of the tests were met.

Under the conditions of this study, the test material is negative according to the KeratinoSens prediction model.