Registration Dossier

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Administrative data

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20-11-2017 ro 27-11-2017
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)
Version / remarks:
28 July 2015
Qualifier:
according to guideline
Guideline:
EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Essential oil of Gurjun obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Gurjunene quality)
EC Number:
947-839-7
IUPAC Name:
Essential oil of Gurjun obtained from the resin tapped from Dipterocarpus trees (Dipterocarpaceae) by steam distillation (Gurjunene quality)
Test material form:
liquid
Details on test material:
Name in used in report: Gurrjun Balsam oil (Gurjunene)
Test facility number 209144/A


SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: obtained from sponsor, Batch no. 2642704.
- Expiration date of the lot/batch: 28-02-2018 and extended to 28-12-2018 (30-03-2018)
- Purity test date: 20-09-2017


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempersture.

OTHER SPECIFICS: UVCB

Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: obtained from sponsor, Batch no. 2642704. Test facility number 209144/A
- Expiration date of the lot/batch: 28-02-2018 and extended to 28-12-2018 (30-03-2018)
- Purity test date: 20-09-2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room tempersture.

OTHER SPECIFICS: UVCB

In vitro test system

Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 17 EKIN 047)
Source strain:
other: 09-KERA-007 and 11-KERA-010
Justification for test system used:
In the interest of sound science and animal welfare, a sequential testing strategy is recommended to minimize the need of in vivo testing. One of the validated in vitro skin irritation tests is the EPISKIN te st, 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)
- Tissue batch number(s): 17-EKIN-047
- Production date: 21-11-2017

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure: room temperature
- Temperature of post-treatment incubation (if applicable): 36.5 - 37°C

REMOVAL OF TEST MATERIAL AND CONTROLS
- Observable damage in the tissue due to washing: not reported

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
- MTT concentration: MTT concentrate (Sigma Aldrich, Zwijndrecht, The Netherlands; 3 mg/ml in
PBS) diluted (10x) in Assay medium (final concentration 0.3 mg/ml).
- Incubation time: 3 h at 37°C
- Spectrophotometer: TECAN Infinite® M200 Pro Plate Reader
- Wavelength: 570 nm

FUNCTIONAL MODEL CONDITIONS WITH REFERENCE TO HISTORICAL DATA
- The absolute mean OD570 of the negative control tissues was within the laboratory historical
control data range.
-The standard deviation value of the percentage viability of three tissues treated identically was ≤ 13%, indicating that the test system functioned properly.

NUMBER OF REPLICATE TISSUES: triplicates

DECISION CRITERIA
- The test substance is considered to be 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.
- 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
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied: 25μL undiluted

NEGATIVE CONTROL
- Amount(s) applied: 25μL undiluted

POSITIVE CONTROL
- Amount(s) applied: 25μL undiluted
- Concentration (if solution): 5%
Duration of treatment / exposure:
15 ± 0.5 minutes
Duration of post-treatment incubation (if applicable):
42 hours at 37°C
Number of replicates:
three

Results and discussion

In vitro

Results
Irritation / corrosion parameter:
% tissue viability
Run / experiment:
Main experiment in triplicate
Value:
85
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: not reported
- Direct-MTT reduction: no
- Colour interference with MTT: no

DEMONSTRATION OF TECHNICAL PROFICIENCY:
The positive control had a mean cell viability after 15 ± 0.5 minutes exposure of 16%. The absolute mean OD570 of the negative control tissues was within the laboratory historical control data range. The standard deviation value of the percentage viability of three tissues treated identically was < 13%, indicating that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for variability between replicate measurements: yes
- Range of historical values if different from the ones specified in the test guideline:
Negative control: Absorption OD570=0.422 – 1.547, Mean=0.98, SD=0.18, n=174
Positive control: Absorption OD570=0.023 – 0.437, Mean=0.13, SD=0.08, n=173
(SD = Standard deviation, n = Number of observations, test facility data over the period of November 2014 to November 2017)

Applicant's summary and conclusion

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria (Annex I 1272/2008/EC)
Conclusions:
The relative mean tissue viablility after exposure to test item was 85%. Based on the results obtained, it can be concluded that Gurjun balsam oil (Gurjunene) does not need
to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin irritation potential of Gurjun balsam oil (Gurjunene) was tested in accordance to OECDTG 439. Undiluted testing material was topically applied to EPISKIN-SMTM for 15 minutes.

After a 42 hour post-incubation period, determination of the cytotoxic (irritancy) effect was performed using MTT conversion measurements. The relative mean tissue viability obtained after 15 ± 0.5 minutes treatment with Gurjun balsam oil (Gurjunene) compared to the negative control tissues was 82%. Since the mean relative tissue viability for testing material was above 50% after 15 minutes treatment it is not considered to be an irritant. Both the positive and the negative control were within the historical control data range and therefore considered valid. Furthermore, the standard deviation value of the percentage viability of three tissues treated identically was less than 13%, indicating that the test system functioned properly. Gurjun balsam oil (Gurjunene) was determined to non-irritant in the in vitro skin irritation test under the experimental conditions described in this report. Therefore it can be concluded that Gurjun balsam oil (Gurjunene) does not need to be classified for skin irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).