2-ethyl-3-hydroxy-4-pyrone

Endpoint summary

Description of key information

1. Direct Peptide Reactivity Assay (DPRA): Negative (OECD 442C/GLP)

2.ARE-Nrf2 luciferase test assay ( KeratinoSens): Negative (OECD 442D/GLP)

3. Human Cell Line Activation Test (h-CLAT) assay: Positive (OECD442E/GLP)

The 3 tests were used as part of a tiered strategy for skin sensitization assessment. Out of three in vitro/in chemico tests, two were negative (DPRA and KeratinoSens), while the h-CLAT test gave an indication of a positive result. Based on the negative results in 2 out of 3 tests, the overall conclusion is that Ethyl Maltol is not a skin sensitiser.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 Mar 2017 - 11 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Details on the study design:

Peptides and positive control
-Synthetic peptide containing cysteine: Ac-RFAACAA-COOH, lot number 1556171, purity 95% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
-Synthetic peptide containing lysine: AC-RFAAKAA-COOH, lot number 1556172, purity 94% (by HPLC), supplied by AnaSpec, stored frozen (-10°C to -30°C).
-Positive control: Cinnamic aldehyde, Batch number MKBR2427V purity > 95%, supplied by SAFC, stored at ambient temperature.

Preparation of peptide stock solutions: Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (for cysteine, 100 mM phosphate buffer pH 7.5, for lysine 100 mM ammonium acetate buffer pH 10.2).
Preparation of peptide calibration standards: Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Reference (Stability) Controls and Precision Controls: Reference (stability) controls and precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile. These were injected throughout the analytical run to confirm consistency of peptide response throughout each analytical run.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls: A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Cysteine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in more acetonitrile and cysteine peptide stock solution. The final sample concentration was 5 mM of the test substance, 0.5 mM cysteine. In place of test substance, the positive control solution contained cinnamic aldehyde at a concentration of 5 mM with 0.5 mM cysteine. The co-elution control sample contained 5 mM of the test substance in phosphate buffer solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co-elution Controls: A 100 mM solution in acetonitrile of the test substance was prepared and further diluted in HPLC vials. Lysine peptide depletion samples (in triplicate) were prepared by dilution of the 100 mM test substance solution in lysine peptide stock solution. The final sample concentration was 25 mM of the test substance, 0.5 mM lysine. In place of the test substance, the positive control solution contained cinnamic aldehyde at a concentration of 25 mM with 0.5 mM lysine. The co-elution control sample contained 25 mM of the test substance in ammonium acetate buffer solution.

Incubation: The appearance of the Ethyl Maltol, positive control samples and co-elution controls in the HPLC vials was documented following preparation with the vials then placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to initiation of the analysis run.

Analysis: The concentration of both the cysteine and lysine peptides in the presence of Ethyl Maltol and the associated positive controls were quantified by HPLC using UV detection. Equipment: HPLC Waters Alliance 2695 separation module and 2487 dual wavelength detector (Column: Agilent Zorbax SB C18, 3.5 μm, 100 × 2.1 mm, Guard column: Phenomenex AJO4286)

Calculations: The peak area response for each peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation: % peptide depletion = 100 - [(Peptide peak area in replicate depletion samples x 100) / (Mean peptide peak area of reference (stability) control samples)]

Positive control results:

69.9% depletion (SD 0.21%, n = 3) and 57.9% depletion (SD 1.26%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.

Key result

Run / experiment:

other: mean n=3

Parameter:

other: cysteine depletion, %

Value:

0.026

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: mean n=3

Parameter:

other: lysine depletion, %

Value:

1.28

Vehicle controls validity:

valid

Remarks:

stability and precision controls

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

mean

Parameter:

other: mean cysteine and lysine depletion, %

Value:

0.652

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Reference (stability) controls and precision controls of both peptides were met (CV 0.97%, n = 6 and CV 0.26%, n = 6, for cysteine and lysine, respectively, at 0.50 mM and 0.51 mM).
- Acceptance criteria met for positive control: yes, 69.9% depletion (SD 0.21%, n = 3) and 57.9% depletion (SD 1.26%, n = 3) of cysteine and lysine, respectively, was observed with the positive control cinnamic aldehyde.
- Acceptance criteria met for variability between replicate measurements: yes, SD 0.25% and SD 0.56%, respectively, for cysteine and lysine depletion by the test item.
 
TEST SUBSTANCE RESULTS:
Mean depletion of 0.0260% and 1.28% was observed for the test substance with cysteine and lysine peptides, respectively. The mean of results depletion by Ethyl Maltol is 0.652% With the test substance not reacting with the cysteine nor lysine peptide it is classed as “no to minimal”, hence the DPRA prediction is negative.

Interpretation of results:

other: DPRA was negative

Conclusions:

It can be concluded that this DPRA test is valid, and that the test substance was negative in the DPRA and is classified in the “no or minimal reactivity class” when using the Cysteine 1:10 / Lysine 1:50 prediction model.

Executive summary:

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA; FD95MW), Ethyl Maltol (>99%) in acetonitrile was evaluated by monitoring peptide depletion between the test item and synthetic cysteine and lysine peptides (24 hrs at 25°C). Subsequently samples were analysed by HPLC. Reference (stability) controls and precision controls, co-elution controls and a positive control (cinnamic aldehyde in acetonitrile) were set up in parallel to the test item in order to confirm the validity of the test.

The acceptance criteria for the calibration curve samples, the reference (stability) controls and precision controls and co-elution controls, as well as for the study samples were satisfied. The acceptance criteria for positive control (cinnamic aldehyde) were met; 69.9% depletion (SD 0.21%, n = 3) and 57.9% depletion (SD 1.26%, n = 3) of cysteine and lysine, respectively. The study was therefore considered to be valid.

The test substance caused 0.0260% cysteine peptide depletion and 1.28% lysine peptide depletion. The mean of results depletion by Ethyl Maltol is 0.652% and the test substance was therefore classified as “no to minimal reactivity” based on the Cysteine 1:10 / Lysine 1:50 prediction model and was thus considered to be negative in the DPRA.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442D and OECD 442E were also performed. The data generated with this test will be considered in the context of an integrated approaches such as IATA, combining the result with other complementary information from the other 2 tests.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Mar 2017 - 24 Mar 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E; In vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT)

Version / remarks:

July 2016

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: human Cell Line Activation Test (h-CLAT)

Details on the study design:

Details on study design
Skin sensitisation (In vitro test system) - Details on study design:

Solvent: DMSO (final concentration 0.2% in culture medium, also used as a solvent for positive control)
Positive control: DNCB in DMSO diluted with culture medium (2 and 3 μg DNCB/mL)
Cell line: THP-1 cells (Human monocytic leukemia cell line) were purchased from ATCC, #TIB-202. THP-1 cells are used as surrogate for human myeloid dendritic cells and show enhanced CD86 and/or CD54 expression when treated with sensitizers. The cell density did not exceed 1 × 10^6 cells/mL. The passage numbers of the used THP-1 cells was 16 in both XTT assays and 21 and 22 in the h-CLAT for runs 1 and 2, respectively.

Culture medium: RPMI-1640 supplemented with 10 % FBS (v/v), 0.05 mM 2-mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate, 1% (v/v) L-glutamine and appropriate antibiotics (100 U/mL of penicillin and 100 μg/mL of streptomycin) was used to culture the cells during the assay.

Preparation and seeding of THP-1 cells: On the day of the cytotoxicity experiment (XTT) directly before the application of the test item, solvent and medium control, a volume of 100 μL with a cell density of 0.9 - 1 × 10^6 THP-1 cells/mL was seeded in each well of a 96-well flat bottom plate. For the main experiment (h-CLAT) 0.9- 1 × 10^6 cells/well in a volume of 500 μL was seeded in a 24-well plate before the treatment.
Dose finding assay: The doses investigated in the main experiment (h-CLAT) were determined with two XTT tests, instead of flow cytometry recommended by the guideline. Two independent cytotoxicity experiments were performed with different cell cultures days to obtain a reliable CV75. The mean of two CV75 values was used to determine the dose-range for the main experiment (h-CLAT). CV75 is defined as the concentration of toxicant required to reduce the relative absorbance to 75% of the solvent control and is calculated as:
CV75 = Conc.>75 - [(Conc.>75 - Conc.<75) x (%>75 - 75)]/(%>75 - %<75), where:
a) Conc.>75 = maximal measured concentration with the % of solvent control > 75%
b) Conc.<75 = minimal measured concentration with the % of solvent control < 75%
c) %>75 = relative absorbance at a) in %
d) %<75 = relative absorbance at b) in %

Test item preparation: immediately prior to start the substance was dissolved in culture medium. The maximum concentration of test item was 2500 μg/mL in culture medium, as tested by a solubility test. For the XTT test (dose finding assay) eight concentrations of the test item were analysed. Therefore, dilutions were prepared by 1:2 serial dilutions from 2500 μg/mL in culture medium.

XTT Labelling and Measurement: At the end of the incubation period, 50 μL of the XTT labelling mixture were added to each well. The cells were incubated and subsequently transferred to a microplate reader (Versamax® Molecular Devices). The absorbance was measured at 450 nm (reference wavelength 690 nm). The absorbance values were determined using the software SoftMax Pro Enterprise (version 4.7.1).
Acceptability criteria of XTT assay: The XTT test is considered to be acceptable if it meets the following criteria:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

Main test: The test item was tested in two independent runs.
Test item preparation: For the test item exposure the highest dose solution calculated from the XTT assay was prepared corresponding to 1.2 × CV75. Further 7 dilutions were prepared by serial 1:1.2 dilution. The dilutions were prepared freshly before each experiment. The following concentrations of the test item (solved in culture medium) were tested in the main experiment (h-CLAT): 81, 98, 117, 141, 169, 203, 243 and 292 μg/mL.
Treatment of the cells: Each volume (500 μL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. Each concentration of the test item, medium control, positive and DMSO control was tested in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

Staining of the cells: The triplicates of each test item-treated and not test item treated cells were pooled and equally distributed into three sample tubes, collected by centrifugation (approx. 250 × g, 5 min) and then washed twice with approx. 2 mL of FACS buffer (PBS with 0.1% (w/v) BSA). Thereafter, the cells were centrifuged, re-suspended and blocked with 600 μL of blocking solution at 2 - 8 °C (on ice) for approx. 15 min. After blocking, the cells were centrifuged and the cell pellets were re-suspended in 100 μL FACS buffer. The cells were stained with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1 (isotype control). All solutions were kept light protected at 2-8 °C or on ice during the staining and analysis procedure. The cells were gently mixed by hand and incubated light protected for 30 ± 5 min. at 2 - 8 °C (on ice).
Sample preparation for measurement: After staining with the antibodies, the cells were washed twice (2-8°C) with 2 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 μL of a 7-aminoactinomycin D (7-AAD) solution were added.
Flow cytometry acquisition: The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNAbound 7-AAD fluorescence signal.

Acquisition: A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was not calculated, if the cell viability was less than 50 % (due to diffuse labelling of cytoplasmic structures that are generated due to cell membrane destruction).

Data analysis and interpretation: The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical: RFI (%) = 100 x (MFI of test item treated cells - MFI of test item treated isotope control cells) / (MFI of solvent control cells - MFI of solvent isotope control cells), where MFI is geometric mean fluorescent intensity. The cell viability is calculated as follows: Cell viability (%) = 100 x (Mean cytotoxicity of solvent control cells) / (Mean cytotoxicity of the test item treated cells), where Mean cytotoxicity is the mean of geometric mean (7-AAD) isotype control, geometric mean (7-AAD) CD54 and geometric mean (7-AAD) CD86.

Acceptability criteria of the h-CLAT assay:
The study is considered as valid, if the following criteria are met:
• Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50%.
• In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For medium and DMSO controls, the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• For the test item resulting in negative outcome, the cell viability at the 1.2 × CV75 should be less than 90%. (If the cell viability at the 1.2 × CV75 is more than 90% for a positive tested test item, the negative result should be discarded. If 5 mg/mL in saline, 1 mg/mL in DMSO or the highest soluble dose will be used as the maximal test concentration instead of CV75-based dose, the data for test item are accepted independent by the cell viability.)
• The cell viability of at least 4 doses in each experiment should be ≥50%.

Evaluation of results: The test item is tested in 2 independent runs. If the RFI of CD86 is ≥ 150% or if the RFI of CD54 is ≥ 200% in both independent run data, the test item is considered to be a sensitizer. Otherwise, it is considered to be a non-sensitizer. In case of different results in both runs, a third run has to be performed. If the RFI of CD86 is ≥ 150% at any dose in at least 2 of 3 independent run data, or if the RFI of CD54 is ≥ 200% in at least 2 of 3 independent run data, the test item is considered to be a sensitizer. Otherwise it is considered to be a non-sensitiser.

Positive control results:

The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%.

Key result

Run / experiment:

other: 1, CD86

Parameter:

other: % RFI

Value:

442

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(>150.0%)

Key result

Run / experiment:

other: 2, CD86

Parameter:

other: % RFI

Value:

355.6

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(>150.0%)

Key result

Run / experiment:

other: 1, CD54

Parameter:

other: % RFI

Value:

521.4

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(>200.0%)

Key result

Run / experiment:

other: 2, CD54

Parameter:

other: % RFI

Value:

300.8

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Remarks:

(>200.0%)

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: In the DMSO solvent control, RFI values compared to the medium control of both CD86 and CD54 did not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
- Acceptance criteria met for positive control: The RFI values of the positive controls (DNCB) for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%.
- The cell viability of the two highest tested test item concentrations in the first h-CLAT run were below 50% and therefore excluded from the evaluation

Results of the first XTT test for Test Item Ethyl Maltol

		Microscopic Evaluation
Test Group	Concentration [μg/mL]	Cytotoxicity	Mean Absorbance*	Standard-Deviation	Chem Blank	Mean Absorbance - Chemical Blank	Absorbance in % of Solvent Control**
Medium control	/	no	0.831	0.049	0.203	0.628	106.07
Solvent Control	/	no	0.791	0.045	0.199	0.592	100.00
Test Item	19.5	no	0.757	0.045	0.196	0.561	94.82
	39.1	no	0.763	0.054	0.193	0.570	96.40
	78.1	no	0.744	0.065	0.193	0.551	93.14
	156.3	no	0.694	0.046	0.200	0.494	83.53
	312.5	yes	0.563	0.020	0.204	0.359	60.74
	625	yes	0.491	0.010	0.210	0.281	47.43
	1250	yes	0.469	0.013	0.238	0.231	39.06
	2500	yes	0.330	0.008	0.290	0.041	6.86

Bottom four test concentrations: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

* mean absorbance (absolute) of 7 wells

** relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 94.3%.

Results of the second XTT test for Test Item Ethyl Maltol

		Microscopic Evaluation
Test Group	Concentration [μg/mL]	Cytotoxicity	Mean Absorbance*	Standard-Deviation	Chem Blank	Mean Absorbance - Chemical Blank	Absorbance in % of Solvent Control**
Medium control	/	no	0.871	0.033	0.202	0.669	107.87
Solvent Control	/	no	0.818	0.031	0.198	0.620	100.00
Test Item	19.5	no	0.812	0.028	0.198	0.614	99.04
	39.1	no	0.788	0.019	0.199	0.589	94.94
	78.1	no	0.809	0.026	0.195	0.615	99.09
	156.3	no	0.806	0.015	0.200	0.605	97.62
	312.5	yes	0.612	0.019	0.196	0.416	67.11
	625	yes	0.539	0.017	0.213	0.326	52.59
	1250	yes	0.503	0.017	0.241	0.262	42.29
	2500	yes	0.344	0.007	0.277	0.067	10.74

Bottom four test concentrations: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

* mean absorbance (absolute) of 7 wells

** relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 92.7%.

The CV75 value of the second XTT test: 272.1 μg/mL / The mean CV75 value of both XTT tests: 243.4 μg/mL

Results of the first h-CLAT run for the Test Item Ethyl Maltol

	Concentration (μg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	/	100.0	100.0	100.0
DMSO Control	/	100.0	100.0	100.0
Positive Control (DNCB)	2.0	292.1*	458.2*	64.0
	3.0	440.6*	618.8*	58.4
Test Item	81	154.8	129.9	85.2
	98	194.0	148.4	83.7
	117	222.6*	157.3*	83.8
	141	333.3*	208.9*	81.7
	169	469.0*	414.0*	58.9
	203	521.4*	442.0*	54.3
	243	661.9*	589.2*	47.4
	292	688.1*	565.0*	42.6

Bottom two test groups: cell viability below 50%, are excluded from the evaluation

* RFI value of CD86 or CD54 fulfilled the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Results of the second h-CLAT run for the Test Item Ethyl Maltol

	Concentration (μg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)
Medium Control	/	100.0	100.0	100.0
DMSO Control	/	100.0	100.0	100.0
Positive Control (DNCB)	2.0	210.4*	417.2*	69.5
	3.0	303.2*	745.5*	63.6
Test Item	81	93.4	93.7	92.4
	98	96.7	73.2	90.9
	117	111.6	112.0	91.0
	141	124.8	114.1	88.7
	169	153.7	132.4	87.5
	203	183.5	168.3*	84.1
	243	233.1*	259.2*	76.3
	292	300.8*	355.6*	67.6

* RFI value of CD86 or CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).

Interpretation of results:

other: h-CLAT was positive

Conclusions:

In a GLP-compliant guideline study, the test substance was found to be positive in the in vitro h-CLAT test, indicating a possible skin sensitizing potential of the substance.

Executive summary:

In an in vitro skin sensitisation: human Cell Line Activation Test (h-CLAT; 1820900), Ethyl Maltol (>99%) was evaluated by the ability to induce an increase in cell surface marker expression (CD54, CD86) in THP-1 cells. Ethyl Maltol (in 0.2% DMSO in RPMI culture medium) was applied to THP-1 cells at concentrations from 81, 98, 117, 141, 169, 203, 243 and 292 μg/mL for 24 hrs in 2 independent runs. Flow cytometry analysis was carried out on IgG1-FITC, CD86-FITC and CD54-FITC antibody labelled cells. The % Relative Fluorescence Index (%RFI) for CD86 and CD54 expression for each tested concentration was then calculated. 7-aminoactinomycin D (7-AAD) was used for viability discrimination. The positive control was 2,4-Dinitrochlorobenzene (DNCB).

All acceptance criteria for controls and test item were reached in each run. The study was therefore considered to be valid. The RFI values DNCB for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%. The cell viability of the two highest tested test item concentrations in the first h-CLAT run were below 50% and therefore excluded from the evaluation. The RFI of CD86 and CD54 was greater than 150% and 200%; (442.0% (CD86 run 1) 355.6% (CD86 run 2) and 521.4% (CD54 run 1) 300.8% (CD54 run 2). Both runs were therefore considered positive for CD54 and CD86. Therefore, the test item is considered to have an indication of a skin sensitizing potential.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C and OECD 442D were also performed. The data generated with this test will be considered in the context of integrated approached such as IATA, combining the result with other complementary information from the other 2 tests.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 May 2017 - 12 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

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

Details on the study design:

Controls:
Positive control: ethylene dimethacrylate glycol, 7.81-250 µM, tested in triplicate, final concentration DMSO of 1%
Negative control: DMSO (vehicle) (1% in exposure medium)
Blank: on each plate three blank wells were tested (no cells and no treatment) to assess background values

Number of replicates: three independent experiments, each concentration tested in triplicate for the luciferase activity measurements, one parallel replicate for MTT cell viability assay.

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 (the passage number used was 21 in experiment 1 and 23 in experiment 2) and are employed for routine testing using the appropriate maintenance medium. Cells were subcultured upon reaching 80-90% confluency. To maintain the integrity of the response, the cells were grown for more than one passage from the frozen stock, and were not cultured for more than 25 passages. 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.

Test item preparation:
No correction was made for the composition/purity of the test item.
The test item was dissolved in DMSO to a final concentration of 200 mM (clear colourless solution). From this stock 11 spike solutions in DMSO were prepared. A 2-fold dilution series was prepared in experiment 1 and a 1.25-fold dilution series was prepared in experiment 2.
The stock and spike solution were diluted 25-fold (final concentration DMSO of 4%). These solutions were diluted 4-fold in the assay resulting in final test concentrations of 2000, 1000, 500, 250, 125, 62.5, 31.3, 15.6, 7.81, 3.91, 1.95 and 0.977 μM (final concentration DMSO of 1%) in experiment 1 and of 2000, 1600, 1280, 1024, 819, 655, 524, 419, 336, 268, 215 and 172 μM in experiment 2 (final concentration DMSO of 1%). All concentrations of the test item were tested in triplicate. No precipitate was observed at the start or end of the treatment period. Test item concentrations were used within 3 hours after preparation.

Media:
Basic medium
Dulbecco’s minimal supplemented with 9.1% (v/v) heat-inactivated (56°C; 30 min) foetal calf 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.

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 substances were added. Three wells per plate were left empty (no cells and no treatment) to assess background values. The treated plates were then incubated for about 48 hours in a humid atmosphere of 80 - 100% (actual range 59 – 86 %) at 37.0 ± 1.0°C (actual range 36.5 – 37.5°C), in the presence of 5% ± 0.5%CO2.

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 the luminometer to assess the quantity of luciferase (integration time one second).

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; CAS No. 298-93-1) and 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 (e.g. by adding 10% SDS solution to each well) overnight. After shaking, the absorption is measured with the TECAN Infinite® M200 Pro Plate Reader.

Data analysis:
The following parameters are calculated in the KeratinoSensTM test method:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the tested chemical and positive control
• 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
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.

Fold luciferase activity induction is calculated by Equation 1, and the overall maximal fold induction (Imax) is calculated as the average of the individual repetitions.

Equation 1: Fold induction= (Lsample - Lblank)/(Lsolvent - Lblank)
Where:
Lsample is the luminescence reading in the test chemical well
Lblank is the luminescence reading in the blank well containing no cells and no treatment
Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative) control

The EC1.5 is calculated by linear interpolation according to Equation 2, and the overall EC1.5 is calculated as the mean of the individual repetitions.

Equation 2: EC1.5 = (Cb - Ca) x [(1.5 - Ia) / (Ib - Ia)] + Ca
Where:
Ca is the lowest concentration in μM with > 1.5 fold induction
Cb is the highest concentration in μM with < 1.5 fold induction
Ia is the fold induction measured at the lowest concentration with > 1.5 fold induction (mean of three replicate wells)
Ib is the fold induction at the highest concentration with < 1.5 fold induction (mean of three replicate wells)

Viability is calculated by Equation 3:
Equation 3: Viability = 100 x (Vsample - Vblank) / (Vsolvent - V blank)
Where:
Vsample is the MTT-absorbance reading in the test chemical well
Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
Vsolvent is the average MTT-absorbance reading in the wells containing cells and solvent (negative) control
Control IC50 and IC30 are calculated by linear interpolation, and the overall IC50 and IC30 are calculated as the mean of the individual repetitions.

In case the luciferase activity induction is larger than 1.5 fold, statistical significance is shown by using 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.

Data interpretation:
A KeratinoSensTM prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in 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

Acceptance criteria:
• 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 7.81 to 250 µM).
• 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.
• Finally, the average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO should be below 20% in each repetition which consists of 18 wells tested. If the variability is higher, results should be discarded.

Positive control results:

Experiment 1: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 18.6 and the EC1.5 14.6 µM.
Experiment 2: the positive control Ethylene dimethacrylate glycol caused a dose related induction of the luciferase activity. The Imax was 2.68 and the EC1.5 83.5 µM.

Key result

Run / experiment:

other: Experiment 1

Parameter:

other: Imax

Value:

1.46

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Experiment 2

Parameter:

other: Imax

Value:

0.93

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

In experiment 1, the test substance showed slight toxicity. The calculated IC30 was 1741 μM. Fifty percent toxicity was not reached and thus no IC50 could be calculated. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test substance. The Imax was 1.46 and therefore no EC1.5 could be calculated. Since a slight increase of luciferase activity was observed at a concentration that was just toxic a more narrow dose-response analysis using a lower dilution factor of 1.25 was used in experiment 2, to investigate this effect in more detail.
In experiment 2, the test substance showed no toxicity. The viability of the cells was higher than 70% at all test concentrations and therefore no IC30 and IC50 values could be calculated. No luminescence activity induction compared to the vehicle control was observed at any of the test concentrations after treatment with the test substance. The Imax was 0.93 and therefore no EC1.5 could be calculated.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for positive control: yes. 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 (14.6 μM and 83.5 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold (18.6-fold and 2.68-fold in experiment 1 and 2, respectively).
- Acceptance criteria met for variability between replicate measurements: yes. The average coefficient of variation of the luminescence reading for the negative (solvent) control DMSO was below 20% in each repetition (respectively 9.2% and 19.9% for experiment 1 and 2).

Summary tables

Table 1. Overview luminescence induction and cell viability of the test substance in Experiments 1 and 2

Concentration (µM)	0.98	1.95	3.91	7.81	15.6	31.3	62.5	125	250	500	1000	2000
Exp 1 luminescence	0.79	0.73	0.74	0.72	0.79	0.84	0.93	1.07	1.28	0.89	0.77	1.46
Exp 1 viability (%)	103.8	102.8	110.1	100.6	92.3	102.1	103.0	110.7	112.8	103.6	93.7	61.7

Concentration (µM)	172	215	268	336	419	524	655	819	1024	1280	1600	2000
Exp 2 luminescence	0.81	0.87	0.84	0.87	0.87	0.90	0.80	0.93	0.80	0.83	0.91	0.88
Exp 2 viability (%)	110.6	101.1	100.8	91.7	92.7	95.5	94.6	96.4	107.6	102.4	86.7	103.1

Table 2. Overview of luminescence induction and cell viability positive control Ethylene dimethacrylate glycol in Experiments 1 and 2

Concentration (µM)	7.81	15.6	31.3	62.5	125	250
Exp 1 luminescence	1.16	1.55	2.18*	2.49**	4.27***	18.63***
Exp 1 viability (%)	118.3	125.6	135.5	150.1	151.2	142.2
Exp 2 luminescence	0.88	1.09	1.15	1.34	1.82***	2.68***
Exp 2 viability (%)	135.1	12.8.7	138.3	139.4	140.8	152.1

* p<0.05; ** p<0.01; *** p<0.001 Students t-test

Table 3. Overview of EC1.5, IC30 and IC50 values

	EC1.5 (µM)	Imax	IC30 (µM)	IC50 (µM)
Test substance Experiment 1	NA	1.46	1741	NA
Test substance  Experiment 2	NA	0.93	NA	NA
Pos Control Experiment 1	14.6	18.6	NA	NA
Pos Control Experiment 2	83.5	2.68	NA	NA

NA = Not applicable

Interpretation of results:

other: KeratinoSens assay was negative

Conclusions:

In a GLP-compliant guideline study, the test substance did not cause a biologically relevant induction in luciferase activity in Keratinosens assay. Based on this, the test substance is considered to give a negative result under the experimental conditions in this assay.

Executive summary:

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay (KeratinoSens; 517938), Ethyl Maltol (>99%) was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. Ethyl Maltol (in 1% DMSO in DMEM culture medium) was applied to KeratinoSens cells at concentrations from 0.98 to 2000 µM (first experiment) and 172 – 2000 μM (second experiment) for 48 h at 37°C. Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. The positive control was ethylene dimethacrylate glycol.

The controls confirmed the validity of the study. The test substance showed slight toxicity (IC30 value of 1741 μM and no IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured in the first experiment. Since a slight increase of luciferase activity was observed at a concentration that was just toxic a more narrow dose-response analysis using a lower dilution factor of 1.25 was used in experiment 2, to investigate this effect in more detail. The test substance showed no toxicity (cell viability>70%; no IC30 value and no IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured in the second experiment.

The maximum luciferase activity induction (Imax) was 1.46-fold and 0.93-fold in experiment 1 and 2 respectively. The test substance is therefore found to be negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 μM with a cell viability of >70% compared to the vehicle control.

This test is part of a tiered strategy for skin sensitization assessment. OECD 442C and OECD 442E were also performed. The data generated with this test will be considered in the context of integrated approached such as IATA, combining the result with other complementary information from the other 2 tests.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation (in vitro)

There are 3 in vitro/in chemico studies from Ethyl Maltol available (DPRA, KeratinoSens, h-CLAT). The 3 tests were used as part of a tiered strategy for skin sensitization assessment.

1. Direct Peptide Reactivity Assay (DPRA).

In an in chemico skin sensitization: direct peptide reactivity assay (DPRA; OECD 442C/GLP), Ethyl Maltol (>99%) in acetonitrile was evaluated by monitoring peptide depletion between the test item and synthetic cysteine and lysine peptides (24 hrs at 25°C). Subsequently samples were analysed by HPLC. Reference (stability) controls and precision controls, co-elution controls and a positive control (cinnamic aldehyde in acetonitrile) were set up in parallel to the test item in order to confirm the validity of the test.

The acceptance criteria for the calibration curve samples, the reference (stability) controls and precision controls and co-elution controls, as well as for the study samples were satisfied. The acceptance criteria for positive control (cinnamic aldehyde) were met; 69.9% depletion (SD 0.21%, n = 3) and 57.9% depletion (SD 1.26%, n = 3) of cysteine and lysine, respectively. The study was therefore considered to be valid. The test substance caused 0.0260% cysteine peptide depletion and 1.28% lysine peptide depletion. The mean of results depletion by Ethyl Maltol is 0.652% and the test substance was therefore classified as “no to minimal reactivity” based on the Cysteine 1:10 / Lysine 1:50 prediction model and was thus considered to be negative in the DPRA.

2. ARE-Nrf2 luciferase test assay (KeratinoSens)

In an in vitro skin sensitisation: ARE-Nrf2 luciferase test method assay (KeratinoSens; OECD 442D/GLP), Ethyl Maltol (>99%) was evaluated for its potential to activate the Nrf2 transcription factor in KeratinoSens cells. Ethyl Maltol (in 1% DMSO in DMEM culture medium) was applied to KeratinoSens cells at concentrations from 0.98 to 2000 µM (first experiment) and 172 – 2000 μM (second experiment) for 48 h at 37°C. Luciferase production was measured by flash luminescence. Cytotoxicity was measured using an MTT assay. The positive control was ethylene dimethacrylate glycol. The controls confirmed the validity of the study. The test substance showed slight toxicity (IC30 value of 1741 μM and no IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured in the first experiment. Since a slight increase of luciferase activity was observed at a concentration that was just toxic a more narrow dose-response analysis using a lower dilution factor of 1.25 was used in experiment 2, to investigate this effect in more detail. The test substance showed no toxicity (cell viability>70%; no IC30 value and no IC50 value) and no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured in the second experiment. The maximum luciferase activity induction (Imax) was 1.46-fold and 0.93-fold in experiment 1 and 2 respectively. The test substance is therefore found to be negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations of ≥1000 μM with a cell viability of >70% compared to the vehicle control.

3. Human Cell Line Activation Test (h-CLAT) assay

In an in vitro skin sensitisation: human Cell Line Activation Test (h-CLAT; 1820900), Ethyl Maltol (>99%) was evaluated by the ability to induce an increase in cell surface marker expression (CD54, CD86) in THP-1 cells. Ethyl Maltol (in 0.2% DMSO in RPMI culture medium) was applied to THP-1 cells at concentrations from 81, 98, 117, 141, 169, 203, 243 and 292 μg/mL for 24 hrs in 2 independent runs. Flow cytometry analysis was carried out on IgG1-FITC, CD86-FITC and CD54-FITC antibody labelled cells. The % Relative Fluorescence Index (%RFI) for CD86 and CD54 expression for each tested concentration was then calculated. 7-aminoactinomycin D (7-AAD) was used for viability discrimination. The positive control was 2,4-Dinitrochlorobenzene (DNCB).

All acceptance criteria for controls and test item were reached in each run. The study was therefore considered to be valid. The RFI values DNCB for CD86 and CD54 exceeded the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability was >50%. The cell viability of the two highest tested test item concentrations in the first h-CLAT run were below 50% and therefore excluded from the evaluation. The RFI of CD86 and CD54 was greater than 150% and 200%; (442.0% (CD86 run 1) 355.6% (CD86 run 2) and 521.4% (CD54 run 1) 300.8% (CD54 run 2). Both runs were therefore considered positive for CD54 and CD86. Therefore, the test item is considered to have an indication of a skin sensitizing potential, based on the results of this test.

Overall conclusion

The 3 tests are part of a tiered strategy and address specific events of the skin sensitisation AOP. Out of three in vitro/in chemico tests, two were negative (DPRA and KeratinoSens), while h-CLAT test gave an indication of a positive result. Based on the ‘2 out of 3’ prediction model in the OECD guidance (OECD 2016), it is concluded that Ethyl Maltol is not a skin sensitiser.

OECD (2016) Guidance Document On The Reporting Of Defined Approaches And Individual Information Sources To Be Used Within Integrated Approaches To Testing And Assessment (IATA) For Skin Sensitisation (ENV/JM/MONO(2016)29).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the available information in the dossier, the substance Ethyl Maltol (CAS No. 4940-11-8) does not need to classified for skin sensitisation when the criteria outlined in Annex I of 1272/2008/EC and Annex I of 286/2011/EC are applied.