4-hydroxycoumarin

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study Initiation Date - 05 November 2020; Experimental Start Date - 07 December 2020; Experimental Completion Date -15 December 2020; Study Completion Date:

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals Method 442E: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), adopted June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of dendritic cells

Specific details on test material used for the study:

4-Hydroxycoumarin CAS number: 1076-38-6
Molecular weight: 162.14 g/mol),
Batch number: HCN20002
Colour: Off-white powder.
Test item received: 25 September 2020
Storage: 15-25°C, protected from light.
Purity: 100.3%

Details on the study design:

Dose Finding Assay
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24 ± 0.5 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, all cells from the 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 µL of phosphate buffered saline containing 0.1% bovine serum albumin (FACS buffer) and re-suspended in 190 µL of FACS buffer. 10 µL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 µg/mL).
PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.
Cell viability was calculated using the following equation:
Cell Viability = number of living cells / total number of acquired cells x100

The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation using the following equation:
Log CV75 = [(75 - c) x Log (b) - (75-a) x Log (d)]/ (a-c)

Where:
a was the minimum value of cell viability over 75% in testing groups
c was the maximum value of cell viability below 75% in testing groups
b and d were the concentrations showing the value of cell viability a and c respectively.


CD86/CD54 Expression Measurement
Two independent runs (experiments) were needed to drive a prediction. Each independent run was performed on the same day provided that for each run:
a) Independent fresh stock solutions and working solutions of the test article and antibody solutions were prepared and
b) Independently harvested cells were used (i.e. cells were collected from different culture flasks). The cells may have come from the same passage.
On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24-well plate (500 µL/1 x 10E6 cells per well).
The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24 ± 0.5 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 µL of blocking solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes.
After blocking, the cells were split into three aliquots of 180 µL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes).
After centrifugation, the cells were stained with 50 µL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.
The stained cells were washed three times with an excess of FACS buffer, re suspended in FACS buffer and 12.5 µg/mL PI solution was added (to give a final PI concentration of 0.625 µg/mL).
The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

Run / experiment:

other: Experiment I

Parameter:

other: RFI of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment I

Parameter:

other: RFI of CD54

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Experiment II

Parameter:

other: RFI of CD86

Value:

150

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Experiment II

Parameter:

other: RFI of CD54

Value:

200

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Assay Acceptance Criteria Results
All assay acceptance criteria were met.
The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run. In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.
For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Dose Finding Assay: No effect on viability was noted, therefore no CV75 value could be calculated.
Main assay: The EC200 value for CD54 calculated by log-linear extrapolation of endpoint assay data was 265.45 μg/mL.

The relative fluorescence intensity (RFI) values for the test article were calculated as follows:

Concentration (µg/mL)	RFI (CD86)		RFI (CD54)
	Exp 1	Exp 2	Exp 1	Exp 2
279.08	66	143	514	336
334.90	68	134	726	877
401.88	73	153	1169	1546
482.25	52	151	2549	1193
578.70	85	156	3823	3198
694.44	56	155	4097	3125
833.33	60	143	4980	3495
1000.00	67	136	4751	2505
Solvent/vehicle control (DMSO)	100	68	88	104
Positive control (DNCB)	459	680	2934	1620

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

4-Hydroxycoumarin, was considered to be a skin sensitizer in the human Cell Line Activation Test.

Executive summary:

The study was conducted to investigate the potential of 4-Hydroxycoumar into activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E in a GLP certified laboratory

.

For the dose finding assay, the test article was formulated in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL giving a maximum test concentration of 1000 µg/mL. No reduction in viability was observed.

For the expression measurements, test concentrations in a range from 279.08 to 1000 μg/mL (after dilution in medium) were used.

Aliquots of 500 µL of each of the working solutionswere mixed 1:1 with cell suspensions at 1 x 10⁶cells per well.

After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes.

The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

In both experiments, the RFI values for CD54 we were >200% (with cell viability >50%) at all concentrations. The RFI values for CD86 were <150% in Experiment 1 and >150% (with cell viability >50%) at multiple concentrations in Experiment 2. The test article therefore gave a positive prediction in the assay.

The EC200 value for CD54 was calculated to be 265.45 μg/mL. All acceptance criteria of the h-CLAT assay parameters were met in each experiment. The test article, 4-Hydroxycoumarin, was considered to be positive in the human Cell Line Activation Test.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 05 November 2020; Experiment start date - 23 November 2020; Experiment end date - 26 November 2020; Study completion date - 26 January 2021.

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))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Specific details on test material used for the study:

4-Hydroxycoumarin CAS number: 1076-38-6
Molecular weight: 162.14 g/mol),
Batch number: HCN20002
Colour: Off-white powder.
Test item received: 25 September 2020
Storage: 15-25°C, protected from light.
Purity: 100.3%

Details on the study design:

Test Article Formulation
The test article was dissolved in 1:1 mixture water:acetonitrile. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 100 mM.
The positive control was dissolved in acetonitrile at a concentration of 100 mM.
A stock solution containing cysteine at approximately 0.667 mM was prepared in 100 mM Phosphate Buffer pH 7.5 and a stock solution containing lysine at approximately 0.667 mM was prepared in 100 mM ammonium acetate buffer pH 10.2. Formulations were prepared shortly before testing.

Test Article Incubation
Each test solution was prepared at ratios of 1:10 and 1:50 (based on molecular weight) with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25 °C for 24 ± 2 hours. Prior to, and at the end of the incubation period the samples were visually inspected for precipitate formation. No precipitation was observed pre-incubation. Precipitation was observed post-incubation in the lysine positive control samples. Precipitate was removed from all affected post incubation samples by centrifugation at 400 g for 5 minutes; and the remaining sample aspirated to a clean autosampler vial.

Analytical Method
The following HPLC conditions were applied:
Column: Agilent Zorbax SB-C18 2.1 mm x 100 mm, 3.5 µm
Wavelength: 220 nm
Guard column: Phenomenex Security Guard C18 4 mm x 2 mm
Flow rate: 0.35 mL/min
Oven temperature: 30°C
Sample temperature: 25°C
Injection volume: 5 µL

Mobile Phase:
Phase A: 0.1% (v/v) of trifluoroacetic acid in MilliQ water
Phase B: 0.085% (v/v) of trifluoroacetic acid in acetonitrile
Gradient: Time (min) Phase A Phase B
0 90 10
10 75 25
11 10 90
13 10 90
13.5 90 10
20 90 10


Reference and Co-elution Controls
Reference controls were prepared for each peptide.
Reference Control A and B for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile.
Reference Control C (positive control vehicle) for each peptide were prepared by adding 750 µL of peptide stock solution to 250 µL of acetonitrile.
Reference Control C (test article vehicle) for cysteine was prepared by adding 750 µL of peptide stock solution to 200 µL of acetonitrile and 50 µL of 1:1 mixture water:acetonitrile.
Reference Control C (test article vehicle) for lysine was prepared by adding 750 µL of peptide stock solution to 250 µL of 1:1 mixture water:acetonitrile.

Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the stability of the reference controls over time and Reference Control C (in triplicate) was used to verify that the vehicle did not impact the percent peptide depletion.
Co-elution controls were prepared to detect possible co-elution of the test article with the peptides. A mixture of 750 µL of 100 mM Phosphate Buffer pH 7.5, 200 µL of acetonitrile and 50 µL of test article solution was used to detect possible co-elution of the test article with cysteine. A mixture of 750 µL of 100 mM ammonium acetate buffer pH 10.2 and 250 µL of test article solution was used to detect possible co elution of the test article with lysine.

Calibration Curves for Peptides
Calibration curves were prepared for each peptide using a range of concentrations from approximately 0.534 mM to 0.0167 mM (Standards 1 to 6).
Standard 1 was prepared at approximatively 0.534 mM by dilution of 1600 µL of the peptide stock solution (0.667 mM) with 400 µL of acetonitrile.
Standards 2 to 6 for cysteine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM Phosphate Buffer pH 7.5).
Standards 2 to 6 for lysine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM ammonium acetate buffer pH 10.2).
Samples of dilution buffer alone were also prepared.

Parameter:

other: Mean cysteine and lysine depletion value

Value:

2.15

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Cysteine Depletion

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD	CV
Test Article	16.63	16.63	0.00	3.19	5.52	173.21
	16.68		0.00
	15.04		9.56
Positive Control	5.78	17.29	66.57	67.01	0.92	1.38
	5.52		68.07
	5.81		66.40

The r²value for the standard calibration curve was 0.99850.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)				SD	CV
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.51	0.51	0.52	0.51	0.01	1.30
C (Positive Control)	0.48	0.52	0.45	0.48	0.03	6.89
C (Test Article)	0.49	0.43	0.48	0.47	0.03	7.01

 

The peak area results for Reference Controls B and C (acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	18.15
	2	18.74
	3	19.97
	4	17.40
	5	16.70
	6	16.43
C (Positive Control)	1	17.27
	2	18.54
	3	16.07
Mean		17.70
SD		1.26
CV		7.13

The peak area results for Reference Controls B and C (1:1 mixture water:acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	18.15
	2	18.74
	3	19.97
	4	17.40
	5	16.70
	6	16.43
C (Test Article)	1	17.59
	2	15.27
	3	17.03
Mean		17.48
SD		1.37
CV		7.86

 

Lysine Depletion

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD	CV
Test Article	25.84	26.22	1.45	1.11	0.34	31.03
	26.02		0.76
	25.93		1.11
Positive Control	10.07	26.38	61.83	61.36	0.43	0.69
	10.29		60.99
	10.22		61.26

The r²value for the standard calibration curve was 0.99987.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM)				SD	CV
	Replicate 1	Replicate 2	Replicate 3	Mean
A	0.49	0.50	0.50	0.50	0.00	0.38
C (Positive Control)	0.50	0.50	0.50	0.50	0.00	0.11
C (Test Article)	0.49	0.49	0.49	0.49	0.00	0.33

 

The peak area results for Reference Controls B and C (acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	26.37
	2	26.39
	3	26.43
	4	26.35
	5	26.38
	6	26.71
C (Positive Control)	1	26.41
	2	26.37
	3	26.35
Mean		26.42
SD		0.11
CV		0.42

The peak area results for Reference Controls B and C (1:1 mixture water:acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1	26.37
	2	26.39
	3	26.43
	4	26.35
	5	26.38
	6	26.71
C (Test Article)	1	26.31
	2	23.23
	3	26.13
Mean		26.37
SD		0.16
CV		0.60

 

Interpretation of results:

GHS criteria not met

Conclusions:

4‑Hydroxycoumarin was considered to be negative with no or minimal reactivity in the Direct Peptide Reactivity Assay. The test item was found to be a "non-sensitizer"

Executive summary:

The study was conducted to quantify the reactivity of 4‑Hydroxycoumarin towards model synthetic peptides containing either lysine or cysteine. This study was conducted in accordance with OECD test guideline 442C in a GLP certified laboratory.

The test article was dissolved in 1:1 mixture water:acetonitrile at a concentration of 100 mM. The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24 ± 2 hours in glass autosampler vials, protected from light, in an incubator set to 25°C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The cysteine depletion value was 3.19%, the lysine depletion value was 1.11% and the mean of the cysteine and lysine depletion values was 2.15%.

Based on the findings of this study, 4‑Hydroxycoumarin was considered to be negative with no or minimal reactivity in the Direct Peptide Reactivity Assay. The test item was found to be a "non-sensitizer"

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 05 November 2020; Experiment start date - 24 November 2020; Experiment end date - 19 February 2021.

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

other: In vitro skin sensitisation

Specific details on test material used for the study:

Identification: 4-Hydroxycoumarin
CAS Number: 1076-38-6
Appearance/Physical State: Beige powder
Batch Number: HCN20002
Purity: 100.3 % w/w
Expiry Date: 26 September 2022
Storage Conditions: Room temperature, in the dark

Details on the study design:

METHODS
Treatment Plate Preparation
The cells (passages 12 and 13) were 80 - 90% confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated for 24±1 hours an incubator set to 37°C, 5% CO2.
For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.

Treatment
At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added.
One well per plate was left empty (no cells and no treatment) to assess background values.
Aliquots of 50 µL of each of the final concentrations were transferred to give three luciferase replicates on a white walled plate and a single viability replicate on a clear walled plate. Each plate was sealed and incubated for 48 ± 1 hours in a humidified incubator set to 37°C, 5% CO2.
Two assays were needed to derive a prediction (positive or negative), comprising three independent repetitions (experiments), each consisting of three replicates of each concentration. Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different passages.
Discordant results were obtained between the two repetitions, Experiments 1 and 2, therefore additional experimentation was performed in order to derive a majority prediction. As however, the test chemical induced luciferase activity close to the cytotoxic levels in Experiments 1 and 2, two additional treatments were performed with more narrow dose-response analysis using a lower dilution factor (1.33-fold dilution between wells), to determine if induction occurred at cytotoxic levels or not.
Discordant results were obtained between the two repetitions, Experiments 3 and 4, therefore a third repetition (Experiment 5) containing three replicates was performed.
Due to a technical error, Experiment 5 treatments were performed using the incorrect fold dilution between wells, therefore a further repetition (Experiment 6) was conducted.
It may be noted that initial Experiment 4 treatments were invalidated as the negative control failed to meet the acceptance criteria and the data are not reported. These treatments were repeated in order to provide the Experiment 4 data which are presented in this report.

Cytotoxicity Assessment
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The plate was sealed and placed in an incubator set to 37°C, 5% CO2 for 4 hours. The MTT medium was removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight.
After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.

Luciferase Activity Measurements
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25 °C, loaded into the luminescence plate reader and read using the following parameters: 100 µL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

Positive control results:

See "Any other information on results incl. tables"

Run / experiment:

other: Experiment 1

Parameter:

other: EC 1.5

Remarks:

(µM)

Value:

1 025.79

Negative controls validity:

valid

Remarks:

% CV = 12.85

Positive controls validity:

valid

Remarks:

EC1.5 = 7.11

Remarks on result:

other: Cell viability = 119.25%; Imax = 2.36

Remarks:

Prediction: Negative

Run / experiment:

other: Experiment 2

Parameter:

other: EC 1.5

Remarks:

(µM)

Value:

982.71

Negative controls validity:

valid

Remarks:

% CV = 5.62

Positive controls validity:

valid

Remarks:

EC1.5 = 9.52

Remarks on result:

other: Cell viability = 71.12%; Imax = 1.54

Remarks:

Prediction: Positive

Run / experiment:

other: Experiment 3

Parameter:

other: EC 1.5

Remarks:

(µM)

Value:

727.42

Negative controls validity:

valid

Remarks:

% CV = 12.56

Positive controls validity:

valid

Remarks:

EC1.5 = 6.02

Remarks on result:

other: Cell viability = 87.36%; Imax = 1.71

Remarks:

Prediction: Positive

Run / experiment:

other: Experiment 4

Parameter:

other: EC 1.5

Remarks:

(µM)

Value:

1 126.68

Negative controls validity:

valid

Remarks:

% CV = 9.38

Positive controls validity:

valid

Remarks:

EC1.5 = 15.58

Remarks on result:

other: Cell viability = 97.43%; Imax = 2.0

Remarks:

Prediction: Negative

Run / experiment:

other: Experiment 5

Parameter:

other: EC 1.5

Remarks:

(µM)

Value:

1 171.18

Negative controls validity:

valid

Remarks:

% CV = 8.95

Positive controls validity:

valid

Remarks:

EC1.5 = 6.95

Remarks on result:

other: Cell viability = 67.13%; Imax = 1.78

Remarks:

Prediction: Negative

Results

These data are summarised as follows:

Experiment	Tested Chemical Range	Imax	EC1.5	Cell Viability at EC1.5 Determining Concentration	Dose Response for Luciferase Induction	Prediction
1	0.98 – 2000 μM	2.36#	1025.79 µM	119.25%	No	Negative
2	0.98 – 2000 μM	1.54#	982.17 µM	71.12%	Yes	Positive
3	86.83 – 2000 μM	1.71#	727.42 µM	87.36%	Yes	Positive
4	86.83 – 2000 μM	2.00#	1126.68 µM	97.43%	Yes	Negative
5	0.98 – 2000 μM	1.78#	1171.18 µM	67.13%	Yes	Negative
6	86.83 – 2000 μM	1.35	NA	NA	No	Negative

# - Luciferase activity induction statistically significant above the threshold of 1.5

NA - Not applicable as no concentrations with luciferase activity induction above 1.5.

 

Discordant results were obtained between the two repetitions, Experiments 1 and 2, therefore additional experimentation was performed in order to derive a majority prediction. As however, the test chemical induced luciferase activity close to the cytotoxic levels in Experiments 1 and 2 (EC_1.5 value (representing the lowest concentration for which induction of luciferase activity was above the 1.5-fold threshold) was 1025.79 and 982.17µM in Experiments 1 and 2 respectively, and viability at the EC_1.5 determining concentration in Experiment 2 was 71.12%), two additional treatments (Experiments 3 and 4) were performed with more narrow dose-response analysis using a lower dilution factor of 1.33-fold between wells, to determine if induction occurred at cytotoxic levels or not.

Discordant results were again obtained from these two experiments (Experiments 3 and 4), therefore a third repetition (Experiment 5) containing three replicates was performed. Due to a technical error, Experiment 5 treatments were performed using the incorrect fold dilution between wells (2-fold rather than 1.33-fold), therefore a further repetition with 1.33-fold dilution between wells was conducted (Experiment 6).

For Experiments 1, 2 and 5, which were performed with tested chemical range 0.98 to 2000 µM, the mean I_max was1.89 and the geometric mean EC_1.5 was 1056.71 µM.

In Experiments 3, 4 and 6, which were performed using narrow dose response analysis and a tested chemical range of 86.83 to 2000 µM, the mean I_max was 1.69 and the geometric mean EC_1.5 was 905.30 µM. The geometric mean EC_1.5 value was calculated using the EC_1.5 values from Experiments 3 and 4 only, as there were no statistically significant increases in induction in Experiment 6.

There was no dose response for luciferase induction in Experiment 1 as >1.5-fold luciferase induction was observed only at the highest test concentration of 2000µM in this experiment. There was no dose response for luciferase induction in Experiment 6 as there were no test concentrations with >1.5-fold luciferase induction.

The cell viability was above 70% at all concentrations in Experiments 1 to 4, at concentrations of 0.98 to 1000 μM in Experiment 5 and at concentrations of 86.83 to 1504 μM in Experiment 6.

The IC₃₀ values could not be calculated for Experiments 1 to 4 as all viability results were above 70%. In Experiments 5 and 6 the IC₃₀ values were 1705.77 μM and 1902.42 μM,respectively. The IC₅₀ values could not be calculated for Experiments 1 to 6 as the minimum viability did not drop below 50% in any experiment.

Assay Acceptance

Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 8 to 64 µM in Experiments 1, 3 and 5 and at concentrations of 16 to 64 µM in Experiments 2, 4 and 6.

The EC_1.5 values and average induction at 64 µM for the positive control, and the average coefficient of variation of the luminescence reading for the negative control (DMSO) were as follows:

	Positive Control		Negative Control
Experiment	EC1.5	Average Induction at 64 µM	%CV
1	7.11	7.37	12.85
2	9.52	6.26	5.62
3	6.02	21.99	12.56
4	15.58	3.87	9.38
5	6.95	10.36	8.95
6	15.29	6.21	9.08

The EC_1.5 for the positive control in all experiments was not within two standard deviations of the historical mean and in Experiments 3 and 5 the average induction for the positive control at 64 μM was above the range specified in the protocol. As however the EC_1.5 was within the range seen at these laboratories and a clear positive dose response was observed for luciferase induction, these data were considered to be acceptable.

Interpretation of results:

GHS criteria not met

Conclusions:

The test article 4-Hydroxycoumarin was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Executive summary:

The study was conducted according to OECD test guideline 442D and to investigate the potential of 4-Hydroxycoumarin to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

4-Hydroxycoumarin was assessed for induction of luciferase activity in three repetitions using a 2-fold dilution scheme and test concentrations of 0.98 to 2000 mM, and in three repetitions using a more narrowed dose-response analysis with a lower dilution factor of 1.33-fold and test concentrations of 86.83 to 2000 mM, to determine if induction occurred at cytotoxic concentrations or not.

Aliquots of 50 µL of each of the final concentrations were transferred to give three luciferase replicates on a white‑walled plate and a single viability replicate on a clear‑walled plate. Each plate was sealed using a plate sealer and then incubated for 48±1 hours in a humidified incubator set to 37°C, 5% CO₂.

After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours in an incubator set to 37°C, 5% CO₂. The MTT medium was then removed and SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO₂, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.

After the 48-hour exposure period, the cells in the luciferase plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read.

The results are summarised as follows:

Criteria	Experiment
	1	2	3	4	5	6
Imax	2.36	1.54	1.71	2.00	1.78	1.35
Cell Viability	119.25%	71.12%	87.36%	97.43%	67.13%	-
EC1.5	1025.79 µM	982.17 µM	727.42 µM	1126.68 µM	1171.18 µM	-
Dose Response	No	Yes	Yes	Yes	Yes	-
Prediction	Negative	Positive	Positive	Negative	Negative	Negative

- Concentration range of 0.98 to 2000 µM was tested in Experiments 1, 2 and 5

- Concentration range of 86.83 to 2000 µM was tested in Experiments 3, 4 and 6

All acceptance criteria were met, with the exception that in all experiments the EC_1.5 for the positive control was not within two standard deviations of the historical mean and in Experiments 3 and 5 the average induction for the positive control at 64 μM was above the range specified in the protocol. As however the EC_1.5 was within the range seen at these laboratories and a clear positive dose response was observed for luciferase induction, these data were considered to be acceptable.

The test article4-Hydroxycoumarin did not meet the 4 conditions required for a positive prediction in the same 2 out of 3 repetitions when assessed over a treatment range of 0.98 – 2000 μM in Experiments 1, 2 and 5. The KeratinoSens prediction from these experiments was therefore considered to be negative.

A narrow dose-response analysis of 86.83 – 2000 μM in Experiments 3, 4 and 6, which were performed due to cytotoxicity observations in the initial experiments, confirmed this negative prediction, as the test article 4-Hydroxycoumarin did not meet the 4 conditions required for a positive prediction in the same 2 out of 3 repetitions in these experiments either.

The test article4-Hydroxycoumarin was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

InVitro OECD TG 442E

The study was conducted to investigate the potential of4-Hydroxycoumarin to activate monocytes and dendritic cells in the human monocytic leukemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54).The human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E in a GLP certified laboratory.

In both experiments, the RFI values for CD54 we were >200% (with cell viability >50%) at all concentrations. The RFI values for CD86 were <150% in Experiment 1 and >150% (with cell viability >50%) at multiple concentrations in Experiment 2. The test article therefore gave a positive prediction in the assay. The EC200 value for CD54 was calculated to be 265.45 μg/mL. All acceptance criteria of the h-CLAT assay parameters were met in each experiment. The test article,4-Hydroxycoumarin, was considered to be positive in the human Cell Line Activation Test.

InVitro OECD TG 442C

The study was conducted to quantify the reactivity of 4‑Hydroxycoumarin towards model synthetic peptides containing either lysine or cysteine. This study was conducted in accordance with OECD test guideline 442C in a GLP certified laboratory.

Based on the findings of this study, 4‑Hydroxycoumarin was considered to be negative with no or minimal reactivity in the Direct Peptide Reactivity Assay. The test item was found to be a "non-sensitizer"

InVitro 442D

The study was conducted according to OECD test guideline 442D and to investigate the potential of 4-Hydroxycoumarin to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Based on the findings of this study, 4-Hydroxycoumarin was therefore considered to be negative in the ARE-Nrf2 Luciferase Test.

Justification for classification or non-classification

Skin sensitization (DPRA: OECD TG 442C): Negative

Skin sensitisation (Keratinosens): OECD TG 442D): Negative

Skin sensitization (h-CLAT: OECD TG 442E): Positive

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 negative results in 2 out of 3 tests, the overall conclusion of the in vitro/in chemico tests is that the test substance has no skin sensitizing potential in accordance with Bauch et al., 2012 (Reference: Bauch, C., Kolle, S.N., Ramirez, T., Eltze, T., Fabian E., Mehling, A., Teubner, W., van Ravenzwaay, B., Landsiedel, R., 2012, Putting the parts together: Combining in vitro methods to test for skin sensitizing potentials, Regul. Toxicol. Pharmacol., 63, 489–504.)

 

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