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Description of key information

Skin irritation (OECD TG 439): not irritating

Eye irritation (OECD TG 437): not irritating

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records
Reference
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)
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)
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
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
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.
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).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records
Reference
Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16-09-2016
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes (incl. QA statement)
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
Species:
other: Bovine
Strain:
not specified
Details on test animals or tissues and environmental conditions:
SOURCE OF COLLECTED EYES
- Source:Vitelco, 's Hertogenbosch, The Netherlands
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Eyes were collected and transported in physiological saline in a suitable container under cooled conditions. The isolated corneas were stored in a petri dish with cMEM (Earle’s Minimum Essential Medium (Life Technologies, Bleiswijk, The Netherlands) containing 1% (v/v) L-glutamine (Life Technologies) and 1% (v/v) Foetal Bovine Serum (Life Technologies)).
- Time interval prior to initiating testing: Bovine eyes were used as soon as possible after slaughter
- indication of any existing defects or lesions in ocular tissue samples: no
Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit):750 μl
- Concentration (if solution): unchanged
Duration of treatment / exposure:
10 minutes
Duration of post- treatment incubation (in vitro):
120 minutes
Number of animals or in vitro replicates:
3
Details on study design:
SELECTION AND PREPARATION OF CORNEAS:
-The eyes were checked for unacceptable defects, such as opacity, scratches, pigmentation and neovascularization by removing them from the physiological saline and holding them in the light. Those exhibiting defects were discarded. The isolated corneas were stored in a petri dish with cMEM containing 1% (v/v) L-glutamine and 1% (v/v) Foetal Bovine Serum. The isolated corneas were mounted in a corneal holder (one cornea per holder) with the endothelial side against the O-ring of the posterior half of the holder. The anterior half of the holder was positioned on top of the cornea and tightened with screws. The compartments of the corneal holder were filled with cMEM of 32°C. The corneas were incubated for the minimum of 1 hour at 32°C.

QUALITY CHECK OF THE ISOLATED CORNEAS:
-After the incubation period, the medium was removed from both compartments and replaced with fresh cMEM. Opacity determinations were performed on each of the corneas using an
opacitometer. The opacity of each cornea was read against a cMEM filled chamber, and the initial opacity reading thus determined was recorded. Corneas that had an initial opacity reading higher than 7 were not used. Three corneas were selected at random for each treatment group.

NUMBER OF REPLICATES: 3

NEGATIVE CONTROL USED: Physiological saline

POSITIVE CONTROL USED: Ethanol

APPLICATION DOSE AND EXPOSURE TIME:
-750 µl of either the positive control, the negative control or the test item, Corneas were incubated in a horizontal position for 10 minutes at 32°C
TREATMENT METHOD: [closed chamber / open chamber]
POST-INCUBATION PERIOD: -

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2
- Post-exposure incubation: Corneas were incubated for 120 minutes at 32°C

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Opacity value is calculated (measured with the device OP-KIT)
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of microplate reader (OD490)
- Others: After the completion of the incubation period opacity determination was performed. Each cornea was inspected visually for dissimilar opacity patterns.

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

DECISION CRITERIA:
-The IVIS cut-off values for identifying the test items as inducing serious eye damage (UN
GHS Category 1) and test items not requiring classification for eye irritation or serious eye
damage (UN GHS No Category)
In vitro score range UN GHS
≤ 3 No Category
> 3; ≤ 55 No prediction can be made
>55 Category 1
-Acceptability of the assay: The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean: The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
Irritation parameter:
cornea opacity score
Run / experiment:
Mean
Value:
1.2
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.5
Positive controls validity:
valid
Remarks:
26.3
Remarks on result:
no indication of irritation
Irritation parameter:
in vitro irritation score
Run / experiment:
Mean
Value:
1.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Remarks:
0.6
Positive controls validity:
valid
Remarks:
69.5
Remarks on result:
no indication of irritation
Interpretation of results:
other: not classified
Remarks:
based on CLP criteria
Conclusions:
The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment. The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage under the conditions of this test.
Executive summary:

In this Bovine Corneal Opacity and Permeability test (BCOP test), the eye hazard potential of Gurjun balsam oil was tested. 750 µl of the testing material was tested through topical application directly on top of the corneas for 10 minutes. The negative and positive controls for opacity and permeability were within the laboratory historical range indicating that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment. The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage under the conditions of this test.  

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

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%.

Eye irritation

In this Bovine Corneal Opacity and Permeability test (BCOP test), the eye hazard potential of Gurjun balsam oil was tested. 750 µl of the testing material was tested through topical application directly on top of the corneas for 10 minutes. The negative and positive controls for opacity and permeability were within the laboratory historical range indicating that the test conditions were adequate and that the test system functioned properly. The test item did not induce ocular irritation through both endpoints, resulting in a mean in vitro irritancy score of 1.4 after 10 minutes of treatment. The test item induced an IVIS ≤ 3, therefore no classification is required for eye irritation or serious eye damage under the conditions of this test.  

Justification for classification or non-classification

Based on the available data, Gurjun Balsam oil - Copaene does not need to be classified for skin and eye irritation in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).