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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:
Oct 07 - 26, 2011
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD TG 429.
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)
Details on test animals and environmental conditions:
- Source: Harlan Laboratories B.V. Postbus 6174, 5960 AD Horst / The Netherlands
- Age at study initiation: 9- 10 weeks
- Weight at study initiation: 20 g
- Housing: group
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period: at least 5 days prior start of dosing

- Temperature (°C): 22 +/- 2°C
- Humidity (%): 31-65%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 6 am - 6 pm

IN-LIFE DATES: From: day 1 To: day 6
acetone/olive oil (4:1 v/v)
5, 10, 25 %
No. of animals per dose:
Control group: 5
each test goup: 5
Details on study design:

- Name of test method: LLNA

- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
First, that exposure to at least one concentration of the test item resulted in an
incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as
indicated by the Stimulation Index.
Second, that the data are compatible with a conventional dose response, although
allowance must be made (especially at high topical concentrations) for either local
toxicity or immunological suppression.

Mortality / Viability:
At least once daily from experimental start to necropsy.

Body weights: In the pre-test: prior to the first application and prior to sacrifice. In the main experiment: prior to the first application and prior to treatment with 3HTdR.

Ear thickness: In the pre-test prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6).

Ear weights: In the pre-test after sacrifice; biopsy punches were taken from each ear.

Clinical signs (local / systemic): Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully.

The test item was placed into a volumetric flask on a tared balance and acetone:olive oil (4+1 (v/v)) was quantitatively added. The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer. The preparations were made freshly before each dosing occasion. Concentrations were in terms of material as supplied.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with different test item concentrations of 5, 10, and 25% (w/v) in acetone:olive oil (4+1 (v/v)). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface ( 8 mm) of each ear once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Standard Statistics
Remarks on result:
other: Group 5%: 1.12 Group 10%: 0.91 Group 25%: 1.03
other: disintegrations per minute (DPM)
Remarks on result:
other: see Table 1
Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU
The test item was not a skin sensitiser under the test conditions of this study and thus no classification for skin sensitisation is needed according to CLP-Regulation (EC) No 1272/2008.
Executive summary:

Study Design

In order to study a possible skin sensitising potential of the test material, three groups each of five female mice were treated once daily with the test item at concentrations of 5, 10, and 25% (w/v) in acetone:olive oil (4+1 (v/v)) by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by a pre-experiment. A control group of five mice was treated with the vehicle (acetone:olive oil (4+1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per animal. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a beta-scintillation counter.


All treated animals survived the scheduled study period. No systemic findings were observed during the study period. Animals treated with 5 and 10% test item concentration did not show any signs of local skin irritation. The animals treated with a test item concentration of 25% showed an erythema of ear skin (Score 1) on day 4 and 5 only.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 1.12, 0.91; and 1.03 were determined with the test item at concentrations of 5, 10, and 25% in acetone:olive oil (4+1 (v/v)). The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.


The test item was not a skin sensitiser under the test conditions of this study and thus no classification for skin sensitisation is needed according to CLP-Regulation (EC) No 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:
Migrated from Short description of key information:
No activation of the lymph nodes of mice were observed in the LLNA performed with the test material

Justification for selection of skin sensitisation endpoint:
Only one study available

Justification for classification or non-classification

Fluorphlogopite is not classified with regard to skin sensitisation.