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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:
8 Mar - 22 Mar 2004
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guidelineopen allclose all
according to guideline
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
according to guideline
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
according to guideline
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-chloroprop-2-enoate
EC Number:
Cas Number:
Molecular formula:
3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-chloroprop-2-enoate
Details on test material:
- Name of test material (as cited in study report): Alpha-Chloro C-6 Monomer
- Physical state: colourless liquid
- Expiration date of the lot/batch: 2004-08-31
- Stability under storage conditions: stable
- Storage condition of test material: in refrigerator in the dark

In vivo test system

Test animals

Details on test animals and environmental conditions:
- Source: Charles River France, L'Arbesle Cedex, France
- Age at study initiation: approx. 10 weeks (main study); approx. 17 weeks (preliminary study)
- Weight at study initiation: 19-23 g
- Housing: individual housing in labelled Macrolon cages (type I, height 12.5 cm) containing purified sawdust as bedding material (Woody-Clean type 3/4; Tecnilab-BMI BV, Someren, The Netherlands); during the acclimatisation period the animals were housed in polycarbonate cages (Macrolon II type, height 15 cm)
- Diet: standard animal laboratory diet (Altromin, code VRF 1, Lage, Germany), ad libitum
- Water: tap-water, ad libitum
- Acclimation period: at least 5 days before the start of the treatment

- Temperature (°C): 21.0 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

acetone/olive oil (4:1 v/v)
Preliminary screening test: 10, 25, 50 and 100% (v/v)
Main test: 5, 50 and 100% (v/v)
No. of animals per dose:
Preliminary screening test: 1
Main test: 5
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 at the highest.
A series of 4 test substance concentrations were tested. The test system, procedures and techniques were identical to those used during days 1 to 3 of the main study unless otherwise specified.
4 young adult animals were selected, Each animal was treated with one concentration on two consecutive days. Approx. 4 hours after the last exposure, the skin was cleaned off residual test substance with water and the irritation was assessed. No necropsy was performed and no bodyweights were determined after termination.

Main study:
Three groups of 5 animals were treated with the test substance concentrations, one group was treated with the vehicle.
Induction (Days 1, 2 and 3):
The dorsum surface of both ears was epidermally treated (25 µL/ear) with the test substance concentration, at approx. the same time each day. The control animals were treated in the same way except that instead of the test substance the vehicle alone was administered.
Treatment (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 (Amersham Pharmacia Biotech). After approx. 5 h all animals were killed by peritoneal injection with an overdose of pentobarbital. The draining (auricular) lymph node of each ear was excised. The relative size of the nodes (as compared to the normal) was estimated by visual examination and abnormalities of the nodes were recorded. The nodes were pooled for each animal in 3 mL PBS.
Tissue processing:
A single cell suspension of lymph node cells (LNC) was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). The LNC were washed twice and the DNA was precipitated. Precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold (Packard) as the scintillation fluid.
Radioactivity measurements:
Radioactive measurements were performed using a Packard scintillation counter (1900TR). Counting time was to a statistical precision of +/- 0.2% or a maximum of 5 min whichever comes first. The scintillation counter was programmed to automatically subtract background and convert CPM to DPM.
- Mortality/Viability: Twice daily
- Toxicity: At least once daily
- Body weights: On days 1 (pre-treatment) and 6. Body weight of the animal found dead was determined at the day of death
- Necropsy: The animal found dead was subjected to necropsy for gross macroscopic examination.
- Irritation: On day 3 (3-4 h after treatment), the skin reactions were assessed
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
Treatment with the current positive control substance α-Hexylcinnamaldehyde, 25% in acetone:olive oil (4:1 v/v) in a reliability check resulted in a stimulation index (SI) of 16.8, thus meeting the reliability criteria for the LLNA (SI > 3).

In vivo (LLNA)

Resultsopen allclose all
Key result
+/- 0.4
Test group / Remarks:
5 %
Key result
+/- 0.4
Test group / Remarks:
50 %
Key result
+/- 0.6
Test group / Remarks:
100 %
Remarks on result:
other: see Remark
The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group were as follows: 5%: 0.70+/- 0.4 50%: 1.9 +/- 0.4 100%: 0.9 +/- 0.6 0% (vehicle control): 1.0
other: disintegrations per minute (DPM)
Remarks on result:
other: Treatment with 0 (vehicle), 5, 50 and 100% test item resulted in DPM/node values of 195 +/-44 , 130+/- 49, 375 +/- 117, and 181 +/- 105 respectively.

Any other information on results incl. tables


No macroscopic abnormalities of the nodes were noted.

Body weights:

Body weight loss was noted in all animals treated with the 100% test substance concentration. The slight body weight loss noted in some animals treated with the vehicle, 5% or 50% test substance concentration was considered not toxicologically significant. Body weights and body weight gain in the other animals remained in the same range as controls over the study period.


One animal treated with 100% test substance concentration was found dead on day 5. Post mortem examination of this animal revealed no abnormalities. No symptoms of systemic toxicity were observed in the animals of the main study.

Table 1: Body weights

test substance concentration (%) animal no. bw (g) on day 1 bw (g) on day 6
5 1 19 19
5 2 19 18
5 3 19 20
5 4 19 19
5 5 18 19
50 6 20 20
50 7 21 21
50 8 23 23
50 9 21 20
50 10 23 23
100 11 22 21
100 12 20 18
100 13 21 19
100 14 19 14
100 15 24 22
0 16 23 23
0 17 20 19
0 18 22 23
0 19 20 21
0 20 21 21

Table 2: Calculation of the Stimulation Index (SI)

group test substance concentration (%) mean DPM +/- SD SI +/- SD
1 5 130 +/- 49 0.7 +/- 0.4
2 50 375 +/- 117 1.9 +/- 0.4
3 100 181 +/- 105 0.9 +/- 0.6
4 0 195 +/- 44 1.0

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Migrated information Criteria used for interpretation of results: EU