Registration Dossier

Administrative data

Description of key information

The test material was not considered to be a skin sensitiser.

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 September 2016 to 12 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Additional details on strain: Inbred, SPF-Quality
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Young adult animals (approximately 10 weeks old)
- Weight at study initiation: 18.6 and 25.5 g. Body weight variation was within ± 20 % of the sex mean.
- Housing: Animals were group housed in labelled cages (height 18 cm) containing sterilised sawdust as bedding material. Paper and shelters (disposable paper corner home) were supplied as cage-enrichment. On Day 6 after acclimatisation, the animals were group housed in cages with a sheet of paper instead of sawdust and cage enrichment.
- Diet: ad libitum access to pelleted rodent diet
- Water: ad libitum
- Acclimation period: At least 5 days before the start of treatment
- Indication of any skin lesions: No. It was ensured that the animals were healthy and that the ears were intact and free from any abnormality.

ENVIRONMENTAL CONDITIONS
- Temperature: 18 to 24 °C
- Humidity: 40 to 70 % (relative)
- Air changes: At least 10 air changes/hour
- Photoperiod: 12-hour light/12-hour dark cycle

IN-LIFE DATES: Not reported
Vehicle:
methyl ethyl ketone
Concentration:
10, 25 and 50 % w/w
No. of animals per dose:
Three groups of five animals were treated with one test material concentration per group.
Details on study design:
PRE-SCREEN TESTS
- Compound solubility: The vehicle was selected on the basis of maximising the solubility using information provided by the sponsor and through trials at the testing facility. A weight of evidence analysis was performed prior to the start of this study and all available information was evaluated. It was concluded that no severe effects were to be expected.
A pre-screen test was conducted in order to select the highest test material concentration to be used in the main study. Two test material concentrations were tested, 25 and 50 %.
Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.
Very slight erythema was noted for the animals treated at 50 % between Days 2 and 4. Yellow test material remnants were present on the dorsal surface of the ears of all animals throughout the observation period, which did not hamper scoring of the skin reactions. Staining by the test material prevented scoring for erythema in the animals treated at 50 % on Day 1 only. Variations in ear thickness during the observation period were less than 25 % from Day 1 pre-dose values.
No signs of systemic toxicity were noted for any of the animals. Yellow discoloration of the urine was noted for all animals between Days 2 and 6. This was considered to be due to the colour of the test material.
Based on these results, the highest test material concentration selected for the main study was a 50 % concentration.

MAIN STUDY
- Induction: Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test material, 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 material.

- Excision of the Nodes: Day 6
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 five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol® 20 %. 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
Following excision of the nodes, 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 x g for 10 minutes at 4 °C. To precipitate the DNA, the LNC were exposed to 5 % trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.

- Radioactivity Measurements: Day 7
Precipitates were recovered by centrifugation, re-suspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as the scintillation fluid. Radioactivity measurements were performed using a scintillation counter. 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 and viability were observed twice daily. Body weights were observed on Day 1 (pre-dose) and Day 6 (prior to necropsy). Clinical signs were observed once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing). Irritation was assessed once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. In addition, a description of all other (local) effects was recorded.
Grading Irritation Reactions (erythema and eschar formation):
0: No erythema
1: Very slight erythema (barely perceptible)
2: Well-defined erythema
3: Moderate to severe erythema (beet redness) to slight eschar formation (injuries in depth)
4: Severe erythema (beet redness) to eschar formation preventing grading of erythema

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- 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 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 an SI ≥ 3, the test material may be regarded as a skin sensitiser. Consideration was given to the EC3 value (the estimated test material concentration that will give an SI =3).

TREATMENT PREPARATION AND ADMINISTRATION:
The test material preparations (w/w) were prepared within 4 hours prior to each dosing. No adjustment was made for specific gravity of the vehicle. Homogeneity was assessed by visual inspection of the solutions.
The dosing formulations were applied to the ear using a spatula for the two higher concentration and a pipette for the control and low dose.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Positive control results:
Concentrations of Alpha- Hexylcinnamaldehyde used for this study were 5, 10 and 25 % in Acetone/Olive oil (4:1 v/v; AcOO).
The SI values calculated for the 5, 10 and 25 % concentrations were 1.4, 1.5 and 4.3 respectively. An EC3 value of 18.0 % was calculated using linear interpolation.
The calculated EC3 value was found to be in the acceptable range of 4.8 and 19.5 %. Based on the results, it was concluded that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
SI
Value:
1.1
Variability:
± 0.2
Test group / Remarks:
10 % concentration
Key result
Parameter:
SI
Value:
1.2
Variability:
± 0.2
Test group / Remarks:
25 % concentration
Key result
Parameter:
SI
Value:
1.1
Variability:
± 0.3
Test group / Remarks:
50 % concentration
Cellular proliferation data / Observations:
SKIN REACTIONS / IRRITATION
No erythema was observed in any of the animals. The scaliness as shown by the control animals and several animals throughout the dosing groups between Days 2 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes.
Yellow test material remnants were present on the dorsal surface of the ears of all test material treated animals throughout the observation period, which did not hamper scoring of the skin reactions.

SYSTEMIC TOXICITY
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. Yellow discoloration of the urine was noted for all test material treated animals between Days 2 and 6. Yellow discoloration of the faeces was noted for the animals treated at 50 % on Days 2 and 3. This was considered to be due to the colour of the test material.

MACROSCOPIC EXAMINATION OF THE AURICULAR LYMPH NODES AND SURROUNDING AREA
All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. No macroscopic abnormalities of the surrounding area were noted for any of the animals.

RADIOACTIVITY MEASUREMENTS AND SI VALUES
Mean DPM/animal values for the experimental groups treated with test material concentrations 10, 25 and 50 % were 566, 614 and 580 DPM, respectively. The mean DPM/animal value for the vehicle control group was 508 DPM. The SI values calculated for the test material concentrations 10, 25 and 50 % were 1.1, 1.2 and 1.1, respectively.
Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material was not considered to be a skin sensitiser.
Executive summary:

A study to investigate the potential of the test material to cause skin sensitisation in the Mouse (Local Lymph Node Assay) was conducted in accordance with the standardised guidelines OECD 429, EU Method B.42 and US EPA OPPTS 870.2600 under GLP conditions.

Test material concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 10, 25 or 50 % w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Methyl ethyl ketone).

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.

No erythema was observed in any of the animals. The scaliness as shown by the control animals and several animals throughout the dosing groups between Days 2 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. 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 material concentrations 10, 25 and 50 % were 566, 614 and 580 DPM, respectively. The mean DPM/animal value for the vehicle control group was 508 DPM. The SI values calculated for the test material concentrations 10, 25 and 50 % were 1.1, 1.2 and 1.1, respectively.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicated that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

Under the conditions of this study, the test material was not considered to be a skin sensitiser.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A study to investigate the potential of the test material to cause skin sensitisation in the Mouse (Local Lymph Node Assay) was conducted in accordance with the standardised guidelines OECD 429, EU Method B.42 and US EPA OPPTS 870.2600 under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Test material concentrations selected for the main study were based on the results of a pre-screen test. In the main study, three experimental groups of five female CBA/J mice were treated with test material concentrations of 10, 25 or 50 % w/w on three consecutive days, by open application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Methyl ethyl ketone).

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.

No erythema was observed in any of the animals. The scaliness as shown by the control animals and several animals throughout the dosing groups between Days 2 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes. All auricular lymph nodes of the animals of the experimental and control groups were considered normal in size. 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 material concentrations 10, 25 and 50 % were 566, 614 and 580 DPM, respectively. The mean DPM/animal value for the vehicle control group was 508 DPM. The SI values calculated for the test material concentrations 10, 25 and 50 % were 1.1, 1.2 and 1.1, respectively.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicated that the Local Lymph Node Assay as performed at the testing facility is an appropriate model for testing for contact hypersensitivity.

Under the conditions of this study, the test material was not considered to be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to skin sensitisation.