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Description of key information

Skin sensitisation (OECD TG 429): sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 Jun 2017 - 06 Jul 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Qualifier:
according to guideline
Guideline:
other: EC, No 440/2008, part B: "Skin Sensitization: Local Lymph Node Assay"
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: L4285828, obtained from sponsor
- Expiration date of the lot/batch: 30 April 2018 (retest date)
- Purity test date: 04 July 2016

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

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. No adjustment was made for specific gravity of the vehicle and no correction was made for the purity/composition of the test item, since the test method requires a logical concentration range rather than specific dose levels.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in acetone/olive oil (4:1 v/v)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF-Quality
- Age at study initiation: Approx. 9 weeks old
- Weight at study initiation: 20.1 to 25.1 g
- Housing:
Arrival/Assignment phase: up to 5 animals of the same sex and same dosing group
Cahes: polycarbonate, labeled cages containing sterilized sawdust as bedding material equipped with water bottles.
Procedures/activities: animals were separated only during designated procedures/activities.
- Diet (e.g. ad libitum): Pelleted rodent diet available ad libitum
- Water (e.g. ad libitum): Municipal tap-water available ad libitum
- Acclimation period: 5 days before the commencement of dosing
- Indication of any skin lesions: before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 01 Jun 2017 (dosing intitation) To: 03 Jul 2017
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Pre-screen test: 50% and 100% (initial pre-test), 10% and 25% (additional pre-test)
Main study: 5, 10 and 25%
No. of animals per dose:
Pre-screen: 2 animals per dose
Main study: 5 animals per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: no solubility effects reported
- Irritation: very slight erythema, scaliness and/or scabs were noted for the animals treated at 50% and 100% between Days 2 and 6. No irritation was observed in any of the pre-screen animals treated at 10% and 25%.
- Systemic toxicity: Piloerection, hunched posture and/or labored respiration were noted for the animals treated at 50% and 100% on Day 3. No i signs of systemic toxicity were observed in any of the pre-screen animals treated at 10% and 25%.
- Ear thickness measurements: > 25% increase for animals treated at 100% and <25% increase for animals treated at 50%. For the 10 and 25% dosed animals variations in ear thickness were <25%.
- Erythema scores: maximum level 1 (Very slight erythema,barely perceptible) with scaliness at day 2 and 3 in the 100 adn 50% dosed groups

MAIN STUDY
In the main study, three experimental groups of five female CBA/J mice were treated with test item concentrations of 5, 10 or 25% w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Aceton:olive oil 4:1 (v:v)). Three days after the last exposure, all animals were injected with 3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. After precipitating the DNA of the lymph node cells, radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: A Stimulation Index (SI) is calculated for each group using the individual SI values. The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.

Classification of results
UN-GHS 2015; EC-CLP 2008 EC Hazard statement
SI < 3 No sensitizer
SI ≥ 3 Cat 1 Skin sensitizer
EC3 value ≤ 2%: sub-category 1A
EC3 value > 2%: sub-category 1B H317: May cause an allergic skin reaction

Consideration was given to the EC3 value (the estimated test item concentration that will give a SI =3)

TREATMENT PREPARATION AND ADMINISTRATION:
Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily and dosed within 4 hours after adding the vehicle to the test item. The dosing formulations were kept at room temperature until dosing. The dorsal surface of both ears was topically treated (25 μL/ear) with the test item, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing. The control animals were treated in the same way as the experimental animals, except that the vehicle was administered instead of the test item.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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.
The individual SI is calculated as the ratio of the DPM/animal compared to the DPM/vehicle control group mean.
The EC3 value (the estimated item concentration that will give a SI=3) may be determined if possible, based on the dose response relationship or calculated using linear interpolation.
Positive control results:
A reliability check is carried out at regular intervals to check the sensitivity of the test system and the reliability of the experimental techniques as used by Charles River Den Bosch. In June 2017 the reliability check was performed with Alpha- Hexylcinnamaldehyde in CBA/J mice. Based on the valid results, it was concluded that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
EC3
Remarks:
(%) Gurjun Balsam Oil
Value:
4.9
Test group / Remarks:
An EC3 value of 4.9% was calculated using a log-linear extrapolation
Parameter:
SI
Value:
46
Variability:
9.5
Test group / Remarks:
25% Gurjun Balsam Oil
Parameter:
SI
Value:
25.9
Variability:
7.0
Test group / Remarks:
10% Gurjun Balsam Oil
Parameter:
SI
Value:
3.8
Variability:
0.8
Test group / Remarks:
5% Gurjun Balsam Oil
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
The auricular lymph nodes in the animals treated with the vehicle and at 5% were considered normal in size, the nodes in the animals treated at a concentration of 10% and 25% which were considered enlarged.
The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted for any of the animals.
Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 1940, 13360 and 23689 DPM, respectively. The mean DPM/animal value for the vehicle control group was 515 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 3.8, 25.9 and 46.0, respectively.

DETAILS ON STIMULATION INDEX CALCULATION:
The individual SI was calculated as the ratio of the DPM/animal compared to the DPM/vehicle control group mean

EC3 CALCULATION:
The EC3 value (the estimated item concentration that will give a SI=3) was determined based on the dose response relationship or calculated using log-linear extrapolation. according to the method of Ryan CA,et al. (Extrapolating local lymph node assay EC3 values to estimate relative sensitizing potency. Cutan Ocul Toxicol 2007;26:135-145.)

CLINICAL OBSERVATIONS:
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.

BODY WEIGHTS:
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Interpretation of results:
other: Category 1B
Remarks:
based on the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC)
Conclusions:
Based on the results of this study Gurjun balsam oil needs to be classified for skin sensitisation, Category 1B in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

The skin sensitisation potential of Gurjun Balsam Oil was examined in a Local Lymph Node Assay according to OECD TG 429. In a pre-screen study it was determined that concentrations >50% induced irritant effects. Therefore concentrations of 0, 5, 10 and 25% in Aceton:olive oil 4:1 (v:v) were used for the main study. The test items were applied for 3 consequtive days on the ears of 5 female mice per dose, by open application. Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. Radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The auricular lymph nodes in the animals treated with the vehicle and at 5% were considered normal in size, the nodes in the animals treated at a concentration of 10% and 25% were considered enlarged. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 1940, 13360 and 23689 DPM, respectively. The mean DPM/animal value for the vehicle control group was 515 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 3.8, 25.9 and 46.0, respectively. These results show that the test item elicits a SI ≥ 3. An EC3 value (the estimated test item concentration that will give a SI =3) of 4.9% was calculated using a log-linear extrapolation according to Ryan et al . (2007). Based on the results of this study Gurjun balsam oil needs to be classified as skin sensitizer (Category 1B) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The skin sensitisation potential of Gurjun Balsam Oil was examined in a Local Lymph Node Assay according to OECD TG 429. In a pre-screen study it was determined that concentrations >50% induced irritant effects. Therefore concentrations of 0, 5, 10 and 25% in Aceton:olive oil 4:1 (v:v) were used for the main study. The test items were applied for 3 consequtive days on the ears of 5 female mice per dose, by open application. Three days after the last exposure, all animals were injected with3H-methyl thymidine and after five hours the draining (auricular) lymph nodes were excised and pooled for each animal. Radioactivity measurements were performed. The activity was expressed as the number of disintegrations per minute (DPM) and a stimulation index (SI) was subsequently calculated for each group.

The auricular lymph nodes in the animals treated with the vehicle and at 5% were considered normal in size, the nodes in the animals treated at a concentration of 10% and 25% were considered enlarged. The largest auricular lymph nodes were found in the higher dose groups. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

Mean DPM/animal values for the experimental groups treated with test item concentrations 5, 10 and 25% were 1940, 13360 and 23689 DPM, respectively. The mean DPM/animal value for the vehicle control group was 515 DPM. The SI values calculated for the test item concentrations 5, 10 and 25% were 3.8, 25.9 and 46.0, respectively. These results show that the test item elicits a SI ≥ 3. An EC3 value (the estimated test item concentration that will give a SI =3) of 4.9% was calculated using a log-linear extrapolation according to Ryan et al. (2007). Based on the results of this study Gurjun balsam oil needs to be classified as skin sensitizer (Category 1B) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).

Justification for classification or non-classification

Based on the results of this study Gurjun balsam oil needs to be classified for skin sensitisation (Skin Sens. 1B / H317) in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).