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Diss Factsheets

Administrative data

skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 06 to 19 July 2010
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
Chemical structure
Reference substance name:
EC Number:
Cas Number:
Molecular formula:
Details on test material:
- Purity: 99.7% area (HPLC)
- Batch No.: E010010557

In vivo test system

Test animals

other: CBA/J
Details on test animals and environmental conditions:
- Source: Harlan, Horst, The Netherlands.
- Age at study initiation: approx. 10 weeks old.
- Weight at study initiation: Body weight variation was within ±20% of the sex mean.
- Housing: Individual housing in labeled Macrolon cages containing sterilized sawdust as bedding material.
- 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): 21.0±3.0℃ (actual range: 20.8-23.2℃ )
- Humidity (%): 40-70% (actual range: 43-81%)
- 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

IN-LIFE DATES: From: 2010-07-06 To: 2010-07-19

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
In preliminary study: 25 and 50%
In main study: 0, 10, 25 and 50%
No. of animals per dose:
In preliminary study: 1 female per dose
In main study: 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 50% and 25% 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 (in the range of 8 to 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
1) Allocation
Three groups of five animals were treated with one test substance concentration per group (10, 25 and 50% w/w). One group of five animals was treated with vehicle.
2) 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 mixed thoroughly using a vortex mixer 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.
3) 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) (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.
4) 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) and stored in the refrigerator until the next day.
5) 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).
Positive control substance(s):
not specified
None stated

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Remarks on result:
other: see Remark
The SI value for the vehicle control group was 1.0. The SI value for the experimental group treated with test substance concentrations 10% was 5.9. The SI value for the experimental group treated with test substance concentrations 25% was 7.2. The SI value for the experimental group treated with test substance concentrations 50% was 7.8.
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
The mean DPM/animal value for the vehicle control group was 840 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 10% was 4972 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 25% was 6055 DPM. The mean DPM/animal value for the experimental group treated with test substance concentrations 50% was 6573 DPM.

Any other information on results incl. tables

Skin reactions / Irritation:

The slight irritation of the ears as shown by animals treated at 50% was considered not to have a toxicologically significant effect on the activity of the nodes. No oedema was observed in any of the animals examined.

Brown remants of the test substance were present on the ears of animals treated with the 25 and 50% test substance soncentration, which did not hamper scoring of the skin reactions.

Macroscopy of the auricular lymph nodes and surrounding area:

The auricular lymph nodes of the animals treated at 50% appeared enlarged. The auricular lymph nodes of the animals of the control group and animals treated with 10 and 25% were considered normal in size. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

Body weights:

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.

Toxicity and mortality:

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

Disintegrations Per Minute (DPM) and Stimulation Index (SI)


Test substance   (% w/w)






4972 ± 1033

5.9 ± 1.7



6055 ± 779

7.2 ± 1.7



6573 ± 794

7.8 ± 1.8


0% (vehicle)

840 ± 160

1.0 ± 0.3

Applicant's summary and conclusion

Interpretation of results:
Migrated information Criteria used for interpretation of results: EU
It was concluded that the test substance elicited SI values of >3 and hence is considered as a skin sensitiser under this studies conditions.