Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin irritation in vitro (OECD TG 439): not classified

Eye irritation in vitro (OECD TG 438): not classified

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:
20-11-2017 ro 27-11-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
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
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
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)
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).

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:
07-11-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)
Version / remarks:
26 July, 2013
GLP compliance:
yes (incl. QA statement)
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
Species:
cattle
Strain:
not specified
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Vitelco slaughterhouse, 's Hertogenbosch, The Netherlands
- Age at study initiation: young cattle
- Other info: the eyes were excised by a slaughterhouse employee as soon as possible after slaughter. Eyes were collected and transported in physiological saline in a suitable container under cooled conditions.
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 of either the negative control, positive control or undiluted test item was introduced onto the epithelium of the cornea.
Duration of treatment / exposure:
Corneas were incubated in a horizontal position for 10 +/- 1 minutes at 32 +/- 1°C. After the incubation the solutions were removed and the epithelium was washed with MEM with phenol red (Earle’s Minimum Essential Medium, Life Technologies) and thereafter with cMEM.
Duration of post- treatment incubation (in vitro):
Corneas were incubated for 120 +/- 10 minutes at 32 +/- 1°C with cMEM.
Number of animals or in vitro replicates:
Triplicate
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 (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)). The isolated corneas were mounted in a corneal holder (one cornea per holder) of BASF (Ludwigshafen, Germany) 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+/-1°C. The corneas were incubated for the minimum of 1 hour at 32+/-1°C.

QUALITY CHECK OF THE ISOLATED CORNEAS
- Opacity determinations were performed on each of the corneas using an opacitometer (BASF-OP3.0, BASF, Ludwigshafen, Germany). 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 (Eurovet Animal Health, Bladel, The Netherlands)

POSITIVE CONTROL USED :
- Ethanol, Batch K47177483, Identification number RS532, Purity >99.9% Stable under storage conditions until 31 October 2020

APPLICATION DOSE AND EXPOSURE TIME
- Undiluted, 10+/-1 minutes

TREATMENT METHOD:
- 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 +/- 1°C. The corneas were incubated for the minimum of 1 hour at 32 +/- 1°C.

REMOVAL OF TEST SUBSTANCE
- Number of washing steps after exposure period: 2

POST-EXPOSURE INCUBATION:
- yes; 120+/-10 minutes

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: The opacity of a cornea was measured by the diminution of light passing through the cornea.The light was measured as illuminance (I = luminous flux per area, unit: lux) by a light meter.
- Corneal permeability: passage of sodium fluorescein dye measured with the aid of a microplate reader (TECAN Infinite® M200 Pro Plate Reader).

SCORING SYSTEM: In Vitro Irritancy Score (IVIS)

ACCEPTABILITY CRITERIA
The assay is considered acceptable if:
1) The positive control gives an in vitro irritancy score that falls within two standard deviations of the current historical mean.
2) The negative control responses should result in opacity and permeability values that are less than the upper limits of the laboratory historical range.
All results presented in the tables of the report are calculated using values as per the raw data rounding procedure and may not be exactly reproduced from the individual data presented.

EVALUATION CRITERIA
The mean opacity and mean permeability values (OD490) were used for each treatment group to calculate an in vitro score: In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value) Additionally the opacity and permeability values were evaluated independently to determine whether the test item induced irritation through only one of the two endpoints. 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) are: ≤ 3: No Category, > 3 ≤ 55: No prediction can be made, >55: Category 1
Irritation parameter:
in vitro irritation score
Run / experiment:
Main
Value:
-0.5
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:
OTHER EFFECTS:
- Visible damage on test system: not reported.

DEMONSTRATION OF TECHNICAL PROFICIENCY:
- The negative control responses for opacity and permeability were less than the upper limits of the laboratory historical range indicating that the negative control did not induce irritancy on the corneas. The mean in vitro irritancy score of the positive control (Ethanol) was 48 and within two standard deviations of the current historical positive control mean. It was therefore concluded that the test conditions were adequate and that the test system functioned properly.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: yes
- Acceptance criteria met for positive control: yes
- Range of historical values if different from the ones specified in the test guideline:
Negative control
-opacity -2.9-3.0 (SD 1.06, n=101)
-permeability -0.034 – 0.100 (SD 0.01, n=101)
-IVIS -2.8-3.0 (SD 1.12, n=101)
Positive control
-IVIS 28.0 – 110.9 (SD 15.67, n=76)

Summary of Opacity, Permeability and In Vitro Scores

Treatment Mean
Opacity (1) 
Mean
Permeability (1)
Mean In vitro Irritation
Score (1,2) 
Negative control 1.6  -0.004 1.5
Positive control (Ethanol) 21 1.751 48
Test item  -0.5 0.001  -0.5

1 Calculated using the negative control mean opacity and mean permeability values for the positive control and test item.

2 In vitro irritancy score (IVIS) = mean opacity value + (15 x mean OD490 value).

Interpretation of results:
other: not classified
Remarks:
based on CLP criteria (Annex I of 1272/2008/EC)
Conclusions:
Gurjun balsam oil (Gurjunene) induced an IVIS ≤ 3. Based on these results, the test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).
Executive summary:

The eye hazard potential of Gurjun balsam oil (Gurjunene) was evaluated according to OECD TG 437 (BCOP test). The eye damage was assessed through topical application of 750 μl of the undiluted testing material for 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Gurjun balsam oil (Gurjunene) did not induce ocular irritation (no opacity and no permeability), resulting in a mean in vitro irritancy score of -0.5 after 10 minutes of treatment. In conclusion, Gurjun balsam oil (Gurjunene) induced an IVIS ≤ 3, and therefore test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

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

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Skin irritation

The skin irritation potential of Gurjun balsam oil (Gurjunene) was tested in accordance toOECDTG 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 performedusing 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 tissueviability 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 skinirritation 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).

Eye irritation

The eye hazard potential of Gurjun balsam oil (Gurjunene) was evaluated according to OECD TG437 (BCOP test). The eye damage was assessed through topical application of 750 μl ofthe undiluted testing material for 10 minutes on top of the corneas. Both the negative control and the positive control (Ethanol) were considered valid. It was therefore concluded that the test conditions were adequate and that the test system functioned properly. Gurjun balsam oil (Gurjunene) did not induce ocular irritation (no opacity and no permeability), resulting in a mean in vitro irritancy score of -0.5 after 10 minutes of treatment. In conclusion, Gurjun balsam oil (Gurjunene) induced an IVIS ≤ 3, and therefore test substance does not need to be classified as eye irritant according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).

Justification for classification or non-classification

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