Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05-09-2016 to 12-09-2016
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
2015
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Balsams, gurjun
EC Number:
232-444-8
EC Name:
Balsams, gurjun
Cas Number:
8030-55-5
IUPAC Name:
Essential oil of Gurjun obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Copaene quality)
Test material form:
liquid
Remarks:
Clear colourless to pale yellow liquid
Details on test material:
- Name of test material (as cited in study report): Gurjun Balsam oil
- Substance type: pure substance
- Physical state: liquid
- Analytical purity: no data
- Lot/batch No.: confidential information
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Test item: 207786/A
- Source and lot/batch No.of test material:L4285828
- Expiration date of the lot/batch:30 April 2018 (retest date)

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:At room temperature protected from light

OTHER SPECIFICS: UVCB

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
other: epidermal keratinocytes
Cell source:
other: SkinEthic Laboratories, Lyon, France
Source strain:
other: Not applicable
Vehicle:
unchanged (no vehicle)
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
- Model used: EPISKIN Small ModelTM
- Tissue batch number: 16-EKIN-036
- Production / shipping / delivery date: No data
- Date of initiation of testing: 5 September 2016

PRE-TEST PROCEDURE:
- Pre-incubation: On day of receipt the tissues were transferred to 12-well plates and preincubated with prewarmed maintenance medium for approximately 22 hours at 37°C.
- Test item colour interference: To assess colour interference, 10 μL of testing material was added to 90 μL Milli-Q water. The mixture was mixed for approximately 15 minutes. A negative control, 10 μL Milli-Q water was tested concurrently. At the end of the shaking period a colour check was performed.
- Test item MTT reduction: To assess the ability of the test item to reduce MTT, 25 μL of the test item was added to 2 mL MTT solution (0.3 mg/mL in PBS). The mixture was incubated for 3 hours at 37°C. A negative control, sterile Milli-Q water was tested concurrently. At the end of the incubation period a colour
check was performed.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during exposure: Room temperature
- Temperature used during treatment / post-treatment incubation: 36.1 - 37.1 °C

RREMOVAL OF TEST MATERIAL AND CONTROLS
Tissues were washed with phosphate buffered saline to remove residual test item.

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: 0.3 mg/ml in PBS
- Incubation time: 3 hours at 37 °C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm
- Filter: No data
- Linear OD range of spectrophotometer: No data

DECISION CRITERIA
- A test substance is considered irritant in the skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance and 42 hours of post incubat ion is ≤ 50% of the mean viability of the negative controls.
- A test substance is considered non-irritant in the in vitro skin irritation test if: The relative mean tissue viability of three individual tissues after 15 minutes of exposure to the test substance 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
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 25μl

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): 25μl (aq)Phosphate buffered saline (PBS, Merck KGaA, Darms tadt, Germany).

POSITIVE CONTROL
- Amount(s) applied (volume or weight): 25μl Sodium dodecyl sulphate (SDS, Sigma, Steinheim, Germany) [CAS Number 151-21-3] in PBS
- Concentration (if solution): 5% (aq)
Duration of treatment / exposure:
15 ± 0.5 minutes (the positive control was re-spread after 7 minutes contact time).
Duration of post-treatment incubation (if applicable):
After rinsing with PBS tissues were moved to a new well with 2 ml pre-warmed maintenance medium and incubated for 42 hours at 37°C. After exposure and incubation, tissues were dried and transferred to 2 ml MTT-solution (0.3 mg/ml in P BS). The tissues were incubated for 3 h at 37°C. Epidermis was separated from the collagen matrix a nd both parts were placed in prelabeled microtubes and extracted with 500 μl isopropanol (Merck, Da rmstadt, Germany). Tubes were stored refrigerated and protected from light for 71 hours.
Number of replicates:
3

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Relative mean tissue viability compared to the negative control tissues (100%)
Value:
97
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
100
Positive controls validity:
valid
Remarks:
5.3
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
- OTHER EFFECTS:
- Direct-MTT reduction: No colour changes observed
- Colour interference with MTT: Not colour changes observed

DEMONSTRATION OF TECHNICAL PROFICIENCY: No data

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range.
- Acceptance criteria met for positive control: The positive control had a mean cell viability of 5.3% after 15 ± 0.5 minutes exposure.
- Acceptance criteria met for variability between replicate measurements: The standard deviation value of the percentage viability of three tissues treated identically was 11% or less, indicating that the test system functioned properly. The standard deviation value of the percentage viability of the three tissues treated with GURJUN BALSAM OIL was 30%. The individual results of the percentage viability of these tissues were 107, 120 and 63%. Since these were all above 50% and therefore clearly negative, this does not influence the study outcome.

Applicant's summary and conclusion

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria
Conclusions:
Under the conditons of this test, the relative mean tissue viability for the test item determined to be 97%. Based on this result, the substance is considered to be non-irritant and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).
Executive summary:

The possible skin irritation potential of Gurjun balsam oil was tested in vitro using a human skin model through topical application for 15 minutes. The study procedures described in this report were according to OECD TG 439 guideline and GLP principles. Skin tissue was treated by topical application of 25 μL undiluted test substance. After 42 hours incubation period, determination of the cytotoxic (irritancy) effect was performed. Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) MTT at the end of treatment.

Skin irritation is expressed as the remaining cell viability after exposure to the test substance. Reliable negative and positive controls were included. The positive control had a mean cell viability of 53% after 15

minutes exposure. The standard deviation value of the percentage viability of three tissues treated identically was less than 11%, except for The standard deviation of the viability of the test item treated tissues was 30% which is above acceptance criteria. Since all individual viabilities were >50% the outcome was clearly negative, this does not influence the outcome of the test.

The relative mean tissue viability obtained after 15 minutes treatment with the substance compared to the negative control tissue was 97%. Based on this result, the substance is considered to be non-irritant and does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of CLP (1272/2008/EC).