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Description of key information

From the conducted study in accordance with OECD 429 (LLNA study) there was no indication that the substance elicit an SI ≥ 3 when tested up to 100%. The median SI values calculated for the substance concentrations 25, 50 and 100% were 1.7, 2.8 and 2.8 respectively.

Any significant inhalation of the substance is unlikely as the vapour pressure of the substance is extremely low.

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:
08 February 2010 - 22 March 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River France, L’Arbresle Cedex, France.
- Age at study initiation:Young adult animals (approx. 9 weeks old) were selected
- Weight at study initiation:Body weight variation was within +/- 20% of the sex mean.
- Housing: Individual housing in labeled Macrolon cages (MI type; height 12.5 cm) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France). Paper (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd), Surrey, United Kingdom) was supplied as cage-enrichment. The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on Day 3. During the acclimatization period the accommodation was as described above except that the animals were group housed in Macrolon cages (MIII type; height 18 cm).
- Diet (e.g. ad libitum): Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Health inspection: A health inspection was performed prior to treatment, to ensure that the animals are in a good state of health. Special attention was paid to the ears, which were intact and free from any abnormality.

Results of analysis for diet (nutrients and contaminants), sawdust, paper and water were assessed and did not reveal any findings that were considered to have affected the study integrity. All certificates and results of analysis are retained in the NOTOX archives.

ENVIRONMENTAL CONDITIONS
- Temperature (°C):20.1 – 24.1ºC
- Humidity (%):39 - 65%
- Air changes (per hr):approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light):12 hours artificial fluorescent light and 12 hours darkness per day.
Temporary deviations from the maximum level of temperature and minimum level of relative humidity occurred. Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 08 February 2010 To: 22 March 2010
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0-25-50-100%
No. of animals per dose:
5
Details on study design:
The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

RANGE FINDING TESTS:
- Irritation: A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (maximally grade 2) at the highest concentration.
Two test substance concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines. (undiluted for liquids, 50% for solids)
The test system, procedures and techniques were identical to those used during Days 1 to 3 of the main study unless otherwise specified. Two young adult animals were selected (8-14 weeks old. Each animal was treated with one concentration on three consecutive days. Approximately 3-4 hours after the last exposure, the irritation of the ears was assessed. Bodyweights were determined on Day 3. The animals were sacrificed after the final observation and no necropsy was performed.
- Lymph node proliferation response: Not determined in the preliminary irritation study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group.
If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer, based on the test guideline and recommendations done by ICCVAM (Reference 1).
The results were evaluated according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008 on classification, labeling and packaging of substances and mixtures.

Ref 1. National Institute of Environmental Health Sciences, The Murine Lymph Node Assay: A test method for assessing the allergic contact dermatitis potential of chemicals/compounds. Independent peer review by the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Program Center for the Evaluation of Alternative Toxicological Methods (NICEATM), NIH publication; No 99-4494, February 1999.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3:
The dorsal surface of both ears was epidermally treated (25 μL/ear) with the test substance concentration, at approximately the same time per day. The concentrations were mixed thoroughly using a vortex mixer immediately prior to dosing. The control animals were treated the same as the experimental animals, except that, instead of the test substance, the vehicle alone was administered.

Excision of the nodes - Day 6:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) (Merck, Darmstadt, Germany)
containing 20 μCi of 3H-methyl thymidine (PerkinElmer Life and Analytical Sciences, Boston, MA, US). After approximately five
hours, all animals were killed by intraperitoneal injection with Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).
The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to normal) was
estimated by visual examination and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled
for each animal in approximately 3 mL PBS.

Tissue processing for radioactivity - Day 6:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze
(diameter 125 μm). LNC were washed twice with an excess of PBS by centrifugation at 200g for 10 minutes at 4ºC. To
precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) stored in the
refrigerator until the next day.

Radioactivity measurements - Day 7
Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail
(PerkinElmer Life and Analytical Sciences, Boston, MA, US) as the scintillation fluid. Radioactive measurements were performed
using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes
whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per
Minute (CPM) to Disintegrations Per Minute (DPM).

Observations:
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On Days 1 (pre-treatment) and 6.
Necropsy: No necropsy was performed according to protocol.
Irritation: On Day 3 (3-4 hours after treatment), the skin reactions were assessed. Skin reactions were graded according to the following numerical scoring system. Furthermore descriptions of all other (local) effects were recorded.

Grading Irritation Reactions:
Erythema and eschar formation:
0: No erythema
1: Slight erythema (barely perceptible)
2: Well-defined erythema
3: Severe erythema (beef redness) to slight eschar formation (injuries in depth)

Oedema formation:
0: No oedema
1: Slight oedema (barely perceptible)
2: Moderate oedema
3: Severe oedema
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.
Positive control results:
The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

See attached document "493024 Reliability check".
Parameter:
SI
Value:
1.7
Test group / Remarks:
25%
Remarks on result:
other:
Remarks:
calculated median value
Parameter:
SI
Value:
2.8
Test group / Remarks:
50%
Remarks on result:
other:
Remarks:
calculated median value
Key result
Parameter:
SI
Value:
2.8
Test group / Remarks:
100%
Remarks on result:
other:
Remarks:
calculated median value
Cellular proliferation data / Observations:
Since outliers are present in this study, median values were used to interpret the data. Median DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 530, 879 and 865 DPM respectively. The median DPM/animal value for the vehicle control group was 312 DPM.

Tables and figures have been included in the attached document "LLNA tables and figures".

Other results - main study:

No irritation of the ears was observed in any of the animals examined.

The majority of auricular lymph nodes were considered normal in size, except for the left node of animal no. 8 (25% concentration). No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. The slight body weight loss, noted in some animals, was considered not toxicologically significant.

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

 

Preliminary irritation study:

At a 50 and 100% test substance concentration no signs of systemic toxicity were noted and no irritation was observed in any of the animals examined. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration. See also table 1 in the attached document LLNA tables and figures.

Interpretation of results:
other:
Remarks:
not sensitising, CLP criteria not met.
Conclusions:
Since there was no indication that the test substance elicit an SI ≥ 3 when tested up to 100%, Blown rapeseed oil was considered to be a non skin sensitizer.

Based on these results Blown rapeseed oil would not be regarded as skin sensitizer according to the recommendations made in the test guidelines and does not have to be classified and has no obligatory labeling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007) and the Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Skin sensitisation:

Based on a conducted LLNA study, the substance is considered to be not classified.

Respiratory sensitisation:

Not classified due to the lack of data. Any significant inhalation of the substance is unlikely as the vapour pressure of the substance is extremely low.