Bulnesia sarmienti, ext.

Skin irritation / corrosion

Administrative data

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-01-2016 to 25-01-2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

In accordance with GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bulnesia sarmienti, ext.

EC Number:

289-632-8

EC Name:

Bulnesia sarmienti, ext.

Cas Number:

89958-10-1

Molecular formula:

This reference substance is a UVCB of the NCS type. It is a complex mixture of compounds and therefore molecular formula, molecular weight, and structural formula canoot be given.

IUPAC Name:

Essential oil of Guaiacwood obtained from the wood of Bulnesia sarmientoi by steam distillation

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): Guaiacwood oil
- Physical state: viscous liquid
- Storage condition of test material: at room temperature protected from light

Specific details on test material used for the study:

Guaiacwood oil was heated until approximately 55°C to obtain a homogeneous liquid sample.

In vitro test system

Test system:

human skin model

Source species:

human

Cell type:

non-transformed keratinocytes

Cell source:

other: Adult donors

Source strain:

other: Strains no: 09-KERA-009, 08-KERA-008, 08-KERA-001, 10-KERA-005

Details on animal used as source of test system:

Not relevant

Justification for test system used:

In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimise the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN test, which is recommended in international guidelines (e.g. OECD and EC).

Vehicle:

unchanged (no vehicle)

Details on test system:

RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 16-EKIN-003)

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: 37.0 ± 1.0°C (actual range 36.2 - 36.7°C), with the exception of the test item incubation for 15 minutes at room temperature
- Temperature of post-treatment incubation (if applicable): 37.0 ± 1.0°C (actual range 36.2 - 36.7°C)

REMOVAL OF TEST MATERIAL AND CONTROLS
- Volume and number of washing steps: The tissues were washed with phosphate buffered saline to remove residual test item
- Observable damage in the tissue due to washing: The test item clotted during rinsing and was difficult to remove, however since in the end the tissue was clean and no test item was left this did not affect the results.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml
- Incubation time: 3 hours at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

NUMBER OF REPLICATE TISSUES: 3

PREDICTION MODEL / DECISION CRITERIA
- The test substance is considered to be irritant to skin if the relative mean tissue viability of three viability tissues after 15 minutes exposure and 42 hours post incubation is less than or equal to 50% of the mean viability of the negative controls.
- The test substance is considered to be non-irritant to skin if the relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test item and 42 hours of post incubation is > 50% of the mean viability of the negative controls.

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 (volume or weight with unit): 25 µL of the test item was applied (undiluted) to each of triplicate tissues

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL PBS

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25 µL SDS
- Concentration (if solution): 5% in PBS

Duration of treatment / exposure:

15 ± 0.5 min at room temperature

Duration of post-treatment incubation (if applicable):

42 hours at 37°C

Number of replicates:

Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control.

Results and discussion

In vitro

Results

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

mean (percentage of control)

Value:

11

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks:

Mean tissue viability (percentage of control): 17%

Remarks on result:

positive indication of irritation

Other effects / acceptance of results:

- OTHER EFFECTS:
Guaiacwood oil was checked for colour interference in aqueous conditions and possible direct MTT reduction by adding the test item to MTT medium. Because no colour changes were observed it was concluded that Guaiacwood oil did not interact with the MTT endpoint.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 (optical density at 570 nm) of the three tissues of the negative control should reasonably be within the laboratory historical control data range and the Standard Deviation value (SD) of the % viability should be ≤18. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The mean relative tissue viability of the positive control should be ≤50% relative to the negative control and the Standard Deviation value (SD) of the % viability should be ≤18. The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 17%.
- Acceptance criteria met for variability between replicate measurements: The SD calculated from individual % tissue viabilities of the three identically treated replicates should be ≤18. The standard deviation value of the percentage viability of three tissues treated identically was less than 10%, indicating that the test system functioned properly.

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Remarks:

No discrimination between irritation and corrosivity possible (additional test needed).

Conclusions:

Under the conditions of this test, the relative cell viability values decreased to 11% which is below the threshold for irritancy of ≤50%. Therefore, it can be concluded that the test item Guaiacwood oil is at least an irritant to skin and further research is needed to assign a classification in accordance with 1272/2008/EC (CLP).

Executive summary:

This in vitro study was performed according to OECD 439 to assess the irritation potential of Guaiacwood oil by means of the Human Skin Model Test (OECD TG 439). Three tissues of the human skin model EpiSkin™ were treated with the test item, the negative or the positive control for 15 minutes. Tissue viability was determined for the test item and the positive control (as a percentage of the negative control viability) and mean tissue viability was calculated accordingly.

After treatment with Guaiacwood oil, the mean tissue viability was found to be 11%. This value is below the threshold for irritancy of ≤50%. Based on the results obtained, it can be concluded that Guaiacwood oil is at least an irritant to skin and further research is needed to be able to assign a classification in accordance with 1272/2008/EC (CLP).