Registration Dossier

Diss Factsheets

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 19 Nov to 15 Dec 2014
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study run to a method comparable with current guidelines and to GLP

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Purity: 96.6%

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were group housed in labeled Makrolon cages containing sterilised sawdust as bedding material. Paper and shelters were supplied as cage-enrichment.
- Diet (e.g. ad libitum): Free access to pelleted rodent diet.
- 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.

- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): a 12-hour light/12-hour dark cycle

IN-LIFE DATES: From 19 Nov to 15 Dec 2014

Study design: in vivo (LLNA)

methyl ethyl ketone
10, 25, 50% w/w
No. of animals per dose:
5 females
Details on study design:
- Compound solubility:
- Irritation: No irritation and no signs of systemic toxicity were observed in any of the animals examined. White staining of test substance remnants on the dorsal surface of the ears of all animals between Days 1 and 5 did not hamper scoring of erythema. Bald spots on the nose were noted for one animal treated at 50% on Day 6. Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Based on these results, the highest test substance concentration selected for the main study was a 50% concentration.
- Lymph node proliferation response:

- Name of test method: Local Lymph Node Assay in the Mouse
- Criteria used to consider a positive response: If the results indicate a SI≥ 3, the test substance may be regarded as a skin sensitizer.

Three groups of five animals were treated with one test substance concentration per group. The highest test substance concentration was selected from the pre-screen test. One group of five animals was treated with vehicle.

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

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 μСi 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 mm). 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) and 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 as the scintillation fluid. Radioactive measurements were performed using a Packard 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).

- Mortality/Viability: Twice daily.
- Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
-Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
-Irritation Once daily on Days 1-6 (on Days 1-3 within 1 hour after dosing) according to the following numerical scoring system. Furthermore, a description of all other (local) effects was recorded.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Test group / Remarks:
Remarks on result:
Key result
Test group / Remarks:
25 %
Key result
Test group / Remarks:
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 632, 797 and 748 DPM, respectively. The mean DPM/animal value for the vehicle control group was 404 DPM.

Any other information on results incl. tables

No irritation of the ears was observed in any of the animals examined. White staining of test substance remnants on the dorsal surface of the ears of all experimental animals between Days 1 and 6 did not hamper scoring for erythema.

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.

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.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU
Since there was no indication that the test substance elicited a SI ≥ 3 when tested up to 50%, the test substance was not considered to be a skin sensitizer.
Based on these results, the test substance would not be regarded as a skin sensitizer according to the recommendations made in the test guidelines. The test substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2011) (including all amendments) and the Regulation (EC) No 1272/2008 on classification, labelling and packaging of substances and mixtures (including all amendments).