Ethyl 3,4-dihydroxybenzoate

Skin irritation / corrosion

Administrative data

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May 2016 to 27 May 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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:

2015

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

B.40 BIS equivalent to OECD 431 (2004)

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl 3,4-dihydroxybenzoate

EC Number:

223-529-0

EC Name:

Ethyl 3,4-dihydroxybenzoate

Cas Number:

3943-89-3

Molecular formula:

C9H10O4

IUPAC Name:

ethyl 3,4-dihydroxybenzoate

Test material form:

solid: particulate/powder

In vitro test system

Test system:

human skin model

Remarks:

EpiDerm Skin Model (EPI-200, Lot no.: 23939 kits L and M

Source species:

other: normal, human-derived epidermal keratinocytes

Cell type:

non-transformed keratinocytes

Cell source:

other: MatTek Corporation, Ashland MA, U.S.A.

Source strain:

other: keratinocyte strain 00267

Justification for test system used:

recommended in EC and OECD guidelines

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EpiDerm Skin Model (EPI-200)
- Tissue batch number(s): 23939 kits L and M (receipt 25-05-2016), 23610kit N (freeze dried receipt 10-02-2016)
- Date of initiation of testing: 23-05-2016

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 36.2 – 36.6°C

REMOVAL OF TEST MATERIAL AND CONTROLS: rinsed with phosphate buffer

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration:5 mg/ml diluted (1:5) with MTT diluent (supplemented DMEM).
- Incubation time: 3 hours at 37°C in air containing 5% CO2
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 2/treatment duration for controls, positive controls, test substance

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
- Killed tissues: 2 for test substance and 2 for negative controls to correct MTT interference
- Procedure used to prepare the killed tissues (if applicable): Living epidermis was transferred to a freezer (≤-15°C), thawed, and then again transferred to (≤-15°C). The freeze-killed epidermis was stored at ≤ -15°C until use. Freeze-killed tissues were thawed by placing them for 1 hour at room temperature in a 6 well plate on 0.9 ml DMEM medium.
- N. of replicates : 2/treatment
- Method of calculation used: The net OD of the treated freeze-killed tissues was subtracted from the ODs of the test item treated viable tissues.

DECISION CRITERIA
A test item is considered corrosive in the skin corrosion test if:
a) The relative mean tissue viability obtained after 3-minute treatment compared to the negative control tissues is decreased below 50%.
b) In addition, a test item considered non-corrosive (viability >= 50%) after the 3-minute treatment is considered corrosive if the relative tissue viability after 1-hour treatment with the test item is decreased below 15%.

A test item is considered non corrosive in the in vitro skin corrosion test if:
a) The relative mean tissue viability obtained after the 3-minute treatment compared to the negative control tissues is not decreased below 50%.
b) In addition, the relative tissue viability after the 1-hour treatment is not decreased below 15%.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

yes, concurrent MTT non-specific colour control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied: 28.5 to 38.1 mg (tissue moistened with 25 µl Milli-Q water)

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 50 µl

Duration of treatment / exposure:

3 min and 1 hour

Duration of post-treatment incubation (if applicable):

3 hours at 37°C in air containing 5% CO2.

Number of replicates:

2 replicates (measured in triplicate)

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 min

Value:

ca. 102

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

21%

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour

Value:

ca. 99

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

7%

Results 3

Irritation / corrosion parameter:

other: OD570

Run / experiment:

3 min

Value:

>= 1.816 - <= 1.88

Negative controls validity:

valid

Remarks:

11.832 +/- 0.022

Positive controls validity:

valid

Remarks:

0.387 +/- 0.010

Remarks on result:

other: corrected for MTT direct interference of 4.39%

Results 4

Irritation / corrosion parameter:

other: OD570

Run / experiment:

1 hour

Value:

>= 1.685 - <= 1.772

Negative controls validity:

valid

Remarks:

1.751 +/- 0.027

Positive controls validity:

valid

Remarks:

0.117+/- 0.006

Remarks on result:

other: corrected for MTT direct interference of 14.90%

Other effects / acceptance of results:

- OTHER EFFECTS:
- Visible damage on test system: none reported
- Direct-MTT reduction: yes (4.39 and 14.90%)
- Colour interference with MTT:none

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: met
- Acceptance criteria met for positive control: met
- Acceptance criteria met for variability between replicate measurements: 0.9-4.9%
- Range of historical values if different from the ones specified in the test guideline: met

Applicant's summary and conclusion

Interpretation of results:

other: non-corrosive

Conclusions:

The substance is considered not corrosive to the skin when tested in an in vitro skin corrosion test

Executive summary:

In the EpiDerm (EPI-200) assay with reconstituated human skin, the possible corrosive potential of the substance was tested through topical application for 3 minutes and 1 hour. The substance was found to interefer with MTT reduction. Therefore additional replicates using killed tissues were run in parallel. After correction for the interference (4.39 and 14.9% at 3 min and 1 hour exposure times), the substance was found to be non-corrosive. All validity criteria were met.