Benzoin Sumatra resinoid

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:

November 2018- April 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Resinoid of Styrax tonkinensis (Styracaceae) obtained from exudate by ethanol extraction

EC Number:

946-573-9

IUPAC Name:

Resinoid of Styrax tonkinensis (Styracaceae) obtained from exudate by ethanol extraction

Test material form:

solid

In vitro test system

Test system:

human skin model

Source species:

other: reconstituted epidermis (epiCS®, CellSystems®)

Cell type:

other: reconstituted epidermis (epiCS®, CellSystems®)

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: 0.60 cm² reconstituted epidermis (epiCS®)

EXPOSURE
- The test item was applied as supplied, at the dose of 25 mg, to 2 living Human skin model surfaces (epiCS®, CellSystems®) during 3 minutes and 1 hour.


REMOVAL OF TEST MATERIAL AND CONTROLS
- 3 minutes and 1 hour after the test item application, the human epidermis was washed with 20 mL of PBS.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The cell viability is quantified by measurement of the cellular mitochondrial dehydrogenases activity. These enzymes are responsible for the MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Thiazolyl blue; EINECS number 206-069-5, CAS number 298-93-1)] reduction into blue formazan in the viable cells. The skin sample is placed in MTT solution of appropriate concentration (e.g. 0.3 or 1 mg/mL) for 3 hours at 37°C ± 1°C. The precipitated blue formazan product is then extracted using a solvent (e.g. isopropanol), and the concentration of formazan is measured by determining the Optical Density (OD) at a wavelength between 540 and 600 nm (preferably 570 nm). The measured absorbances are proportional to the number of living cells.
The measurement of OD was performed using the ELx800 absorbance microplate reader supplied by BioTek and the validated software Gens ELISA V1.05.11 supplied by BioTek.

VIABILITY
Viability = (OD test item / OD negative control) x 100
For each tissue, OD values and calculated percentage cell viability data for the test item, positive and negative controls, should be reported in tabular form, including data from replicate repeat experiments as appropriate, mean and individual values.

Control samples:

yes, concurrent negative control

yes, concurrent positive control

Amount/concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25 mg
- Concentration (if solution): Undiluted

Duration of treatment / exposure:

3 minutes and 1 hour

Number of replicates:

duplicate

Results and discussion

In vitro

Results

Results 1

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes replicate 1

Value:

83.41

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3 minutes replicates 2

Value:

74.47

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour replicate 1

Value:

74.6

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1 hour replicate 2

Value:

81.82

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

VIABILITY
- 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with the test item were 78.94 and 78.21%, respectively.

ACCEPTANCE OF RESULTS:
- The mean optical density obtained with the negative control at 3 minutes and 1 hour was 0.727 and 0.790, respectively, which is within the expected range of 0.3 to 0.9.

- Acceptance criteria met for positive control: Yes; 3 minutes and 1 hour after the positive control application, the viability of the human skin model has been 5.51 and 0.13 %, respectively.

Any other information on results incl. tables

Table 7.3.1/1: Skin corrosion assay: Results   Skin OD Mean OD / disc (#) Mean OD / product Viability % Mean viability % Viability difference between replicates % Treatment: 3 min Negative control 1 0.690 0.653 0.727 89.88 100 20.2 0.609 0.672 2 0.743 0.800 110.12 0.909 0.748 Positive control 3 0.039 0.042 0.040 5.78 5.51 0.6 0.046 0.042 4 0.040 0.038 5.23 0.038 0.036 Test item 13 0.550 0.606 0.574 83.41 78.94 8.9 0.641 0.629 14 0.519 0.541 74.47 0.558 0.547

Treatment: 1 hour Negative control 1 0.659 0.790 0.790 98.67 100 2.7 0.865 0.815 2 0.793 0.800 101.33 0.886 0.723 Positive control 3 0.001 0.001 0.001 0.13 0.13 0.0 0.001 0.002 4 0.001 0.001 0.13 0.001 0.002 Test item 13 0.589 0.589 0.618 74.60 78.21 7.2 0.589 0.590 14 0.606 0.646 81.82 0.659 0.674

Applicant's summary and conclusion

Interpretation of results:

other: GHS not met for classification as corrosive

Conclusions:

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that test item does not have to be classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.

Executive summary:

An in vitro skin corrosion study was performed according to OECD Guideline 431 and in compliance with GLP to evaluate the possible corrosive effects of the test item after topical administration on in vitro human reconstituted epidermis (epiCS®, CellSystems®). Test item was applied after being reduced in fine powder, during 3 minutes and 1 hour, at the dose of 25 mg to 2 living Human skin model surfaces (epiCS^®, supplied by CellSystems^®) previously moistened with 25 µL of distilled water. The application wasfollowed by a rinse with 20 mL of DPBS. Cell viability was then measured by enzymatic conversion of the vital dye MTT into a blue formazan salt that was quantitatively measured after extraction from tissues.

The experimental protocol was established in accordance with OECD Test Guideline No. 431 dated 29 July 2016 and method B.40 bis of the Council regulation No. 440/2008 of 30 May 2008.

 

 3 minutes and 1 hour after the test item application, the mean percent viability of the epidermis skins treated with test item were78.94%and78.21%, versus 5.51% and 0.13%, respectively, with the positive control item (potassium hydroxide 8N).

In accordance with Regulation (EC) No. 1272/2008, the results obtained under these experimental conditions enable to conclude that test item does not have to be classified in Category 1 “Corrosive”. The hazard statement “H314: Causes severe skin burns and eye damage” with the signal word “Danger” are not required.