4-hydroxycoumarin

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date - 03 November 2020; Experimental completion date - 05 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Reconstructed Human Epidermis (RHE) Test Method)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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:

Identification: 4-Hydroxycoumarin
CAS Number: 1076-38-6
Batch: HCN20002
Purity: >99%
Molecular Weight: 162.14
Physical state/Appearance: Beige powder
Expiry Date: 26 September 2022
Storage Conditions: Room temperature in the dark

In vitro test system

Test system:

human skin model

Source species:

human

Vehicle:

water

Remarks:

Distilled

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Supplier: MatTek In Vitro Life Sciences Laboratories
Date received: 03 November 2020
EpiDermTM Tissues (0.63cm2) lot number: 34102
Assay Medium lot number: 102920LHB
Upon receipt of the EpidermTM tissues, the sealed 24-well plate was stored in a refrigerator until use.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37 °C.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1.0 mg/mL
- Incubation time: 60 minutes
- Spectrophotometer: Labtech LT-4500 microplate reader
- Wavelength: 570 nm
- Filter band pass: 10 nm

NUMBER OF REPLICATE TISSUES: Two

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: One

PREDICTION MODEL / DECISION CRITERIA (choose relevant statement)
- The test substance is considered to be corrosive to skin if [complete, e.g. the viability after 3 minutes exposure is less than 50%, or if the viability after 3 minutes exposure is greater than or equal to 50 % and the viability after 1 hour exposure is less than 15%.]

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 25 mg

VEHICLE
- Amount(s) applied: 25 μL Distilled water

NEGATIVE CONTROL
- Amount(s) applied: 50 μL Distilled water

POSITIVE CONTROL
- Amount(s) applied: 50 μL of 8.0 N Potassium Hydroxide

Duration of treatment / exposure:

3 minutes and 60 minutes.

Number of replicates:

Two

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment I

Value:

100.3

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: None
- Direct-MTT reduction: No
- Colour interference with MTT: The solution containing the test item was a white color. This color was attributed to the intrinsic color of the test item itself. It was therefore unnecessary to run color correction tissues.

DEMONSTRATION OF TECHNICAL PROFICIENCY: The mean OD570 for the negative control treated tissues was 2.029 for the 3-Minute exposure period and 1.911 for the 60-Minute exposure period. The negative control acceptance criteria were therefore satisfied.
The relative mean tissue viability for the positive control treated tissues was 0.070% relative to the negative control following the 60-Minute exposure period. The positive control acceptance criterion was therefore satisfied.
In the range 20 to 100% viability the Coefficient of Variation between the two tissue replicates of each treatment group did not exceed 30%. The acceptance criterion was therefore satisfied.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be non-corrosive to the skin.

Executive summary:

Introduction

The purpose of this test was to evaluate the corrosivity potential of the test item using the EpiDerm™ Human Skin Model after treatment periods of 3 and 60 minutes. This test was conducted in accordance with OECD test guideline 431 and Method B.40 in a GLP certified laboratory.

Methods

Duplicate tissues were treated with the negative control, positive control and test item for exposure periods of 3 and 60 minutes. At the end of the exposure period the control items and test item were rinsed from the tissues before each tissue was taken for MTT-loading.

After MTT-loading the tissues were placed into 2 mL of isopropanol for formazan extraction. At the end of the formazan extraction period each well was mixed thoroughly and triplicate 200 µL samples were transferred to the appropriate wells of a pre-labeled 96-well plate. The optical density (OD) was measured at 570 nm (OD570).

Data are presented in the form of percentage viability (MTT reduction in the test item treated tissues relative to negative control tissues).

Results

The relative mean viabilities for each treatment group were as follows:

Exposure Period	Percentage Viability
	Negative Control	Positive Control	Test Item
3 minute	100*	3.6	100.3
60 minute	100*	3.6	109.3

*The mean viability of the negative control tissues is set at 100%

Acceptance criteria: The criteria required for acceptance of results in the test were satisfied.

Conclusion: In this study and under the experimental conditions reported, the test item was considered to be non-corrosive to the skin.