Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 14 to 26 January 2009
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)
Details on test animals and environmental conditions:
- Source: Charles River France, L'Arbresle Cedex, France.
- Age at study initiation: approx. 9 weeks old
- Weight at study initiation: with +/- 20% of the sex mean
- Housing: Individual housing in labeled Macrolon cages containing sterilized sawdust as bedding material. Paper 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.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: at least 5 days

- Temperature (°C): 18.0 - 23.9 ℃
- Humidity (%): 35 - 64%
- Air changes (per hr): 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day

No additional data
acetone/olive oil (4:1 v/v)
0%, 10%, 25%, 50%
No. of animals per dose:
5 females per dose
Details on study design:
Preliminary Irritation study
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 25% and 50% 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.

Main study
Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the preliminary irritation study. One group of five animals was treated with vehicle.

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 throughly 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
All animals:
Each animal was injected via the tail vein with 0.25 mL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine.
After approximately five hours, all animals were killed by intraperitoneal injection with pentobarbital Euthesate. 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 200 g for 10 minutes at 4 ℃. To precipitate the DNA, the LNC were 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 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 comes first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
not specified
None stated
Remarks on result:
other: The Stimulation index/animal values calculated for the test substance concentrations 10, 25 and 50% were 1.3, 1.7 and 1.3 respectively. The Stimulation index/animal value for the vehicle control group was 1.0.
other: disintegrations per minute (DPM)
Remarks on result:
other: Mean DPM/animal values for the experimental groups treated with test substance concentrations 10, 25 and 50% were 468, 607 and 456 respectively. The mean DPM/animal value for the vehicle control group was 364.

Preliminary irritation study

No irritation was observed in any of the animals examined.

Base on the results, the highest test substance concentration selected for the main study was a 50% concentration.

Main study

Skin reactions /Irritation:

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

Macroscopy of the auricular lymph nodes and surrounding area:

All nodes of the experimental and control groups were considered normal in size.

No macroscopic abnormalities of the surrounding area were noted.

Body weights:

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Toxicity and mortality:

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

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU
The SI values calculated for the substance concentrations 10, 25 and 50% were 1.3, 1.7 and 1.3 respectively.
There was no indication that the test substance could elicit an SI ≥ 3 when tested up to 50%.
Based on these results and according to the recommendations made in the test guidelines, the test substance would not be regarded as skin sensitizer.
The six monthly reliability check with Hexylcinnamaldehyde, indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing contact hypersensitivity.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A LLNA study was conducted according to OECD 429 using mouse (van den Bogaard, 2009). Key study. This study indicate that the test substance could not elicit an SI ≥ 3, hence the test substance should be classfied as a non skin sensitizer.

Migrated from Short description of key information:
A LLNA (van den Bogaard, 2009) study was available which is key study. This study showed that the test substance is not skin sensitizing.

Justification for selection of skin sensitisation endpoint:
This study was conducted according to OECD 429 under GLP.

Justification for classification or non-classification

Skin sensitisation: Animal tests gave negative results (LLNA SI < 3 (actual value 1.7)).

Therefore in accordance with Regulation (EC) No. 1272/2008 Table 3.4.2 the substance is classified as a non skin sensitizer.