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Skin sensitisation

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skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September 2016 - 19 October 2016
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Information used for read across to Veilex #3.

Data source

Reference Type:
study report
Report Date:

Materials and methods

Test guidelineopen allclose all
according to
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
July 2010
according to
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
May 2008
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Test material form:
other: liquid
Details on test material:
- Storage condition of test material: Store in cool dark place at room temperature

In vivo test system

Test animals

Details on test animals and environmental conditions:
Female CBA/J strain mice were supplied by Janvier, Le Genest-Saint-Isle, France. On receipt the animals were randomly allocated to cages. The animals were nulliparous and non pregnant and the acclimatisation period was at least five days. At the start of the study the animals were in the weight range of 18.2 to 25.2 g, and were approximately ten weeks old.

Animals were group housed in labeled Makrolon cages containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment. Free access to tap water and food (pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany)) was allowed throughout the study.
The temperature and relative humidity were controlled to remain within target ranges of 18 to 24°C a nd 40 to 70 %, respectively.

The animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
Undiluted test item or the test item at concentrations of 5%, 10% or 25% v/v in vehicle.
No. of animals per dose:
Groups of five mice were treated per dose.
Details on study design:
Preliminary Screening Test:
A pre-screen test was conducted in order to select the highest test item concentration to be used in the main study. In principle, this highest concentration should cause no systemic toxicity, may give well-defined irritation as the most pronounced response (maximum grade 2) and/or an increase in ear thickness < 25%) and/or is the highest possible concentration that can be technically applied.
Initially, two test item concentrations were tested; a 50% and 100% concentration. The highest concentration was the maximum concentration as required in the test guidelines.
Based on the results of the initially treated animals, four additional animals were treated in a similar manner with two lower concentrations (10% and 25%) at a later stage.
The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 10-11 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed. Two young adult animals per concentration were selected. Each animal was treated with one concentration on three consecutive days. Animals were group housed in labeled Makrolon cages (MII type, height 14 cm). 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.

Main Test
Test Item Administration
Groups of five mice were treated with the undiluted test item or the test item at concentrations of 5%, 10 % or 25 % v/v in acetone/olive oil 4:1. The vehicle was selected on the basis of the test item data provided by the Sponsor and trial preparation results performed at Charles River Den Bosch. The vehicle was chosen from the vehicles specified in the test guideline: Acetone/Olive oil (4:1 v/v), N,N-dimethylformamide, methylethylketone, propylene glycol, dimethylsulfoxide and 1% Pluronic© L92 in Elix water (in case an aqueous vehicle is suitable). There was no information available regarding the solubility or stability in vehicle. No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation. This is an exception with regard to GLP and has been reflected in the GLP compliance statement. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local irritation at the highest suitable concentration. 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. A further group of five mice received the vehicle alone in the same manner.

The positive control animals were similarly treated to the test animals except that 25 μL of the positive control item, α Hexylcinnamaldehyde, techical grade, at a concentration of 5, 10 and 25 % v/v in acetone/olive oil 4:1 was applied to the dorsal surface of each ear. The positive control group served as common control with another study according to the summary of positive control data for the local lymph node assay.

Induction - Days 1, 2 and 3
The dorsal surface of both ears was topically treated (25 μL/ear) with the test substance, at approximately the same time on each day. The concentrations were stirred with a magnetic stirrer immediately prior to dosing.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item, 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).

Terminal Procedures - Day 6
Termination: After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of 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.

Preparation of Single Cell Suspension: 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 200g for 10 minutes at 4ºC. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) (Merck, Darmstadt, Germany) and then stored in the refrigerator until the next day.

Determination of 3HTdR Incorporation: 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. Radioactivity 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 back ground and convert Counts Per Minute (CPM) to Disintegrations Per Minute(DPM).

Mortality/Viability: Twice daily.
Clinical Observations: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing). Any signs of toxicity or signs of ill health during the test were recorded.
Bodyweights: The bodyweight of each mouse was recorded on Day 1 (prior to dosing) and on Day 6 (prior to termination).
Irritation: Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the numerical scoring system (see any other information on materials and methods incl. tables). In addition, a description of all other (local) effects was recorded.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Not performed.

Results and discussion

Positive control results:
The positive control item, α-Hexylcinnamaldehyde, techical grade, gave a Stimulation Index of greater than 3 when tested at a concentration of 25 % v/v in acetone/olive oil 4:1.

In vivo (LLNA)

Resultsopen allclose all
Key result
Remarks on result:
other: EC3 was not reached
Remarks on result:
other: 5% test group
Remarks on result:
other: 10% test group
Remarks on result:
other: 25% test group
Cellular proliferation data / Observations:
The SI values calculated for the test item concentrations 5, 10 and 25% were 0.8, 0.7 and 0.9, respectively.
There was no indication that the test item elicits a SI ≥ 3 when tested up to 25%, Veilex 4 was not considered to be a skin sensitizer. It was established that the EC3 value (the estimated test item concentration that will give a SI =3) (if any) exceeds 25%.
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.

Any other information on results incl. tables

Pre-screen Test:

At a concentration of 50%, hunched posture was noted on Day 3 and slight erythema was noted on Days 2 and 3. At a concentration of 100%, slight erythema was noted on Day 3 and scaliness was noted between Days 2 and 6. At concentrations of 25% and 10%, no signs of systemic toxicity or erythema were noted. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values for all animals.

Based on these results, the highest test item concentration selected for the main study was a 25% concentration.

Applicant's summary and conclusion

Interpretation of results:
other: Not sensitising.
According to Regulation (EC) No 1272/2008 and its amendments.
The SI values calculated for the test item concentrations 5, 10 and 25% were 0.8, 0.7 and 0.9, respectively. These results show that the test substance does not elicit a SI ≥ 3. An EC3 is derived of >25%. A NOEC of >25% is derived. The test substance was considered not to be a sensitiser under the conditions of the test.
Executive summary:

The skin sensitisation potential of the test substance has been tested according to OECD TG 429: Local Lymph Node Assay" method and GLP principles. The test substance was tested at 5, 10 and 25%.

No higher dose was tested in the main study, as in the preliminary study systemic effects were observed- at the concentration of 50% hunched posture was noted (dose at 100% is ca 2400 mg/kg bw (50 ul = 50 mg/0.021 kg mouse). At the concentration of 100%, scaliness was noted. The substance showed SI values of 0.8, 0.7 and 0.9, respectively. Reliable negative and positive controls were included. The test substance did not elicit an SI ≥ 3 when tested up to and including the maximum concentration of 25% and therefore the substance was not considered to be a skin sensitizer.