3,4-dihydroxybenzaldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 June to 30 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

3,4-dihydroxybenzaldehyde

EC Number:

205-377-7

EC Name:

3,4-dihydroxybenzaldehyde

Cas Number:

139-85-5

Molecular formula:

C7H6O3

IUPAC Name:

3,4-dihydroxybenzaldehyde

Test material form:

solid

In vitro test system

Test system:

human skin model

Remarks:

SkinEthic RHE model

Source species:

human

Cell type:

non-transformed keratinocytes

Justification for test system used:

The SkinEthic RHE model has been validated for irritation testing and its use is recommended by the relevant OECD guideline for irritation testing (OECD No. 439); therefore, it was considered to be
suitable for this study.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: SkinEthic RHE
- Tissue batch number(s): 22-RHE-089
- Production date: N/A
- Delivery date: 28.06.2022
- Date of initiation of testing: 28.06.2022

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 42 minutes at room temperature.
- Temperature of post-treatment incubation (if applicable): 37ºC

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: 25 x 1 mL of DPBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 300 μL of a MTT solution at 1.0 mg/mL
- Incubation time: 3 hours at 36,8º and 37ºC, 5% CO2
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE: there is no direct interaction between the test item and MTT.

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDIC
TION: 1

PREDICTION MODEL / DECISION CRITERIA
- The test item is considered as non-irritant to skin if the mean percent viability after 60 minutes exposure and 42 hours of post-treatment incubation is > 50%.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2) if the mean percent tissue viability after 60 minutes exposure and 42 hours of post-treatment incubat ion is ≤ 50% and the result of a skin corrosion test is “non corrosive”.
- The test item is identified as requiring classification and labelling according to UN GHS (Category 2 or Category 1) if the mean percent tissue viability after 60 minutes exposure and 42 hours of posttreatment incubation is ≤ 50% and in absence of information on a skin corrosion test.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 16 μL

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 16 μL
- Concentration (if solution): 5% SDS

Duration of treatment / exposure:

42 at room temperature and

Duration of post-treatment incubation (if applicable):

41 hours and 06 minutes

Number of replicates:

3

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean

Value:

104.7

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

(distilled water)

Positive controls validity:

valid

Remarks:

(5% SDS)

Any other information on results incl. tables

	Well ID	OD	Mean OD / disc	Mean OD / product	Viability %	Mean viability %	SD viability	Conclusion
Negative control	SPL 1	1.063 1.088 1.081	1.063	0.989	107.4	100.0	7.7	No Category
	SPL 2	1.012 0.970 1.000	0.994		100.5
	SPL 3	0.877 0.936 0.921	0.911		92.1
Positive control	SPL 4	0.013 0.015 0.014	0.014	0.014	1.4	1.4	0.2	Category 2 'Irritant'
	SPL 5	0.012 0.013 0.013	0.012		1.2
	SPL 6	0.016 0.016 0.017	0.016		1.6
Test item PH-22/0351	SPL 7	0.971 0.990 1.028	0.996	1.036	100.7	104.7	6.4	No Category
	SPL 8	0.994 1.009 1.003	1.002		101.3
	SPL 9	1.085 1.122 1.122	1.109		112.1

# mean of 3 values (triplicate of the same extract).

Applicant's summary and conclusion

Interpretation of results:

other: Not classified (CLP Regulation EC no. 1272/2008)

Conclusions:

The test substance can be considered as not irritant to skin as the mean percent viability of the treated tissues was found 104.7% in the in vitro RhE test.

Executive summary:

An in vitro skin irritation test was conducted for the test item in a reconstructed human epidermis model (Episkin) according to OECD TG 439 (GLP study).

Three epidermis units, previously moistened with 10 μL of distilled water, were treated with 16 mg test item for 42 minutes at room temperature. Exposure of the test item was terminated by rinsing with 25 x 1mL of DPBS. The epidermis units were then incubated for a 41 hours and 06 minutes post-treatment incubation period in fresh medium at 37ºC, 5% CO2. Then placed into 300 μL of an MTT solution at 1.9 mg/mL for 3 hours at 37ºC, 5% CO2.

The viability of each disk was assessed by incubating the tissues with MTT, extracting the precipitated formazan crystals using isopropanol during 2 hours under gentle agitation in the dark, and measuring the concentration of formazan by determining the OD at 570 nm, just after dilution of the extracts 1:2 in isopropanol. Under the test conditions, the mean percent viability of the treated tissues was 104.7%, versus 1.4% in the positive control (5% Sodium Dodecyl Sulfate). Therefore, the test item is considered as not irritant to the skin.