4-hydroxybenzaldehyde

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

May 2, 2018 - June 28, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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)

Version / remarks:

July 29, 2016

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No. 440/2008 laying down test methods pursuant to Regulation (EC) No. 1907/2006. B.40.bis. In vitro skin corrosion: human skin model test

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: INVITTOX Protocol SkinEthic Skin Corrosivity Test, April 2012.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-hydroxybenzaldehyde

EC Number:

204-599-1

EC Name:

4-hydroxybenzaldehyde

Cas Number:

123-08-0

Molecular formula:

C7H6O2

IUPAC Name:

4-hydroxybenzaldehyde

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

foreskin from a single donor

Justification for test system used:

The reconstituted human epidermis (RHE)-model is a three-dimensional human skin model and is histologically comparable to the in vivo human tissue. It consists of a main basal, supra basal, spinous and granular layer and a functional stratum corneum. The RHE-model is an accepted in vitro alternative model for skin corrosion testing. It involves topical application of test item to the surface of the epidermis and the subsequent assessment of the effects on cell viability. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT.

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiSkin/SkinEthic Laboratories, Lyon, France
- Tissue batch number(s): 18-RHE-060

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: At the end of the exposure periods, the test item, positive and negative control was removed immediately by gently rinsing with a minimum volume of 20 mL DPBS using a pipette. Excess DPBS was removed by gently shaking the tissue inserts and blotting the bottom of the tissue inserts with blotting paper.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 1 mg/mL
- Incubation time: 3 hours ± 15 min
- Spectrophotometer: ELx800, BioTek Instruments GmbH, Bad Friedenshall, Germany
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- Viability: OD 1.2 (CV =7.5%)
- Barrier function: 4.9 h
- Morphology: 5 cell layers, absence of significant histological abnormalities, well differentiated epidermis consisting of basal, spinous, granular layers and a stratum corneum
- Contamination: no

NUMBER OF REPLICATE TISSUES: 2

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

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be corrosive to skin if 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%.
- The test substance is considered to be non-corrosive to skin if the viability after 3 minutes exposure is greater than or equal to 50% and the viability after 1 hour exposure is greater than or equal to 15%.

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:
The negative control OD values for the SkinEthic RHE-model have to be in the range of ≥ 0.8 and ≤ 3.0 at 570 nm as given in OECD Guideline 431.

Acceptance Criteria stated by Episkin/SkinEthic Laboratories:
Negative control acceptance criteria: The negative control data meet the acceptance criteria if the mean OD value of the 2 tissues is≥ 0.8 and ≤ 3.0 at 570 nm.
Positive control acceptance criteria: The positive control data meet the acceptance criteria if it classified as corrosive (% viability < 15% after 1 hour exposure).
Test substance data acceptance criteria: The range between identically treated tissues has to be less than 30%, with the exception of cases with OD ≤ 0.3 and for viabilities out the range 20 – 100%.

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:
The positive control data meet the acceptance criteria if the mean viability value, expressed as % of the negative control, is lower than or equal to a historically established boundary. The boundary is three standard deviations above the current historical mean (0.94%). The negative control data meet the acceptance criteria if the mean OD value is higher or equal than a historically established boundary at 570 nm. The boundary is two standard deviations below the current historical mean (3 minutes exposure: 1.596; 1 hour exposure: 1.467).

Negative Control, Positive Control and Test Substance Data Acceptance Criteria stated by the Testing Laboratory:
The range between identically treated tissues has to be less than 30%, with the exception of cases with OD ≤ 0.3 and for viabilities out the range 20 – 100%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 20 mg ± 3 mg of solid test material
Before adding 20 ± 3 mg of the test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve further contact between the test item and the epidermis.

NEGATIVE CONTROL
- Amount(s) applied: 40 µL ± 3 µL (deionised water )

POSITIVE CONTROL
- Amount(s) applied: 40 µL ± 3 µL
- Concentration: An 8N potassium hydroxide solution dissolved deionised water pure

Duration of treatment / exposure:

3 min & 1 hour

Number of replicates:

2

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / 3 min

Value:

100.4

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

Experiment 1 / 1 hour

Value:

23.5

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: none
- Direct-MTT reduction: none
- Colour interference with MTT: none

ACCEPTANCE OF RESULTS:

Acceptability of the Quality Control Data of the Skin Model with Reference to Historical Batch Data:

Acceptance Criterion Result
Negative control OD ≥ 0.8 and ≤ 3.0 1.563 to 1.703

Acceptability of the Positive and Negative Control stated by Episkin/SkinEthic Laboratories:

Acceptance Criterion Result
Mean OD negative control ≥ 0.8 and ≤ 3.0 1.694 (3 min)
1.608 (1 hour)

Mean viability positive control < 15% after 1-hour exposure 0.9%

Range between identically treated < 30% 1.4% (3 min)
tissues with test item 17.6% (1 hour)

Acceptability of the Positive and Negative Control based on Historical Data of the Testing Laboratory:

Acceptance Criterion Result
Mean OD negative control ≥ 1.596 (3 min) 1.694 (3 min)
≥ 1.467 (1 hour) 1.608 (1 hour)

Mean viability positive control ≤ 0.94% 0.9%

Negative Control, Positive Control and Test Substance Data Acceptance Criteria stated by the Testing Laboratory:

Group Acceptance Criterion Result
Range between identically Negative control < 30% 1.0% (3 min)
treated tissues 5.8% (1 hour)
Positive control < 30% 83.3% (1 hour)
Test substance < 30% 1.4% (3 min)
17.6% (1 hour)

The range between identically treated tissues with the positive control was higher than 30% after 1 hour exposure (83.3%), but the optical densities measured were <0.3.
The study met all acceptance criteria.

Any other information on results incl. tables

Table 1: Results

Group	Time / [min]	Mean OD	Mean Relative viability / [%]
Negative Control	3	1.694	100.0
Negative Control	60	1.608	100.0
Positive Control	60	0.014	0.9
Test Material	3	1.701	100.4
Test Material	60	0.379	23.5

Table 2: Historical Data

Positive Control		Negative Control   3min		Negative Control 1 hours
Mean Viability [%]	0.58	Mean Absorption [OD570]	1.898	Mean Absorption [OD570]	1.963
Standard Deviation [%]	0.12	Standard Deviation	0.151	Standard Deviation	0.248

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

This study was performed according to GLP and the methods applied are fully compliant with OECD TG 431. Under the conditions of the present study, the test item is not considered to possess a corrosive potential to skin.

Executive summary:

A study according OECD TG 431 was conducted to investigate the potential of the test item to induce skin corrosion in an in vitro human skin model. The test item was applied topically to a human reconstructed skin model followed by determination of the cell viability. Cell viability was determined by enzymatic conversion of vital dye MTT into a blue formazan salt and measurement of the formazan salt after extraction from tissues. The percent reduction of cell viability in comparison to untreated negative controls was used to predict the skin corrosion potential. Duplicates of the human skin RHE-model were treated with the test item or the negative control for 3 minutes and additional 1 hour. Duplicates with the positive control were only treated for 1 hour. 40 ± 3 µL of either the negative control (deionised water) or the positive control (potassium hydroxide, 8N) were applied to the tissues. Before application of 20 ± 3 mg of the solid test item, 20 ± 2 µL of deionised water was spread to the epidermis surface to improve the contact between the test item and the epidermis. After treatment with the positive control (potassium hydroxide, 8N) the mean viability value was 0.9% and, thus, lower than the historically established threshold of 0.94%. After treatment with the negative control (deionised water) the mean ODs were 1.694 (3 minutes exposure) and 1.608 (1 hour exposure) and, thus, higher than the historically established thresholds of 1.596 and 1.467, respectively. Thus, the acceptance criteria were met. Following treatment with the test item, the tissue viability was ≥50% after 3 minutes exposure (mean viability: 100.4%) and ≥15% after 1 hour exposure (mean viability: 23.5%), i.e. according to OECD 431 the test item is not considered as corrosive to skin.