4-hydroxycoumarin

Skin sensitisation

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxycoumarin

EC Number:

214-060-2

EC Name:

4-hydroxycoumarin

Cas Number:

1076-38-6

Molecular formula:

C9H6O3

IUPAC Name:

4-hydroxycoumarin

Test material form:

solid: particulate/powder

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%

In vitro test system

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.

Results and discussion

In vitro / in chemico

Results

Results 1

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

Results 2

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

Results 3

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

Results 4

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.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.