2-Propenoic acid, 2-chloro-, 3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl ester

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 Mar - 22 Mar 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluorooctyl 2-chloroprop-2-enoate

EC Number:

801-260-5

Cas Number:

96383-55-0

Molecular formula:

C11H6ClF13O2

IUPAC Name:

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

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- 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

ENVIRONMENTAL CONDITIONS
- 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)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

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.
Observation:
- 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)

Results

Results 1

Key result

Parameter:

SI

Value:

0.7

Variability:

+/- 0.4

Test group / Remarks:

5 %

Results 2

Key result

Parameter:

SI

Value:

1.9

Variability:

+/- 0.4

Test group / Remarks:

50 %

Results 3

Key result

Parameter:

SI

Value:

0.9

Variability:

+/- 0.6

Test group / Remarks:

100 %

Results 4

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

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

Results 5

Parameter:

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

Macroscopy:

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.

Toxicity/mortality:

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

Remarks:

Migrated information Criteria used for interpretation of results: EU