Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
TNO Quality of Life, Zeist, The Netherlands
Type of study:
mouse local lymphnode assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Perfluoro-3,6-dioxaoctanoic acid ammonium salt (EEA-NH4)
- Physical state: white powder
- Analytical purity: 99%
- Lot/batch No.: #070921
- Storage condition of test material: room temperature
- Specific activity (if radiolabelling): 185 GBq/mmol
- Locations of the label (if radiolabelling): lymph nodes

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan, The Netherlands
- Age at study initiation: 6-10 weeks
- Weight at study initiation: unknown
- Housing: From arrival until allocation, group housing in macrolon cages (Type VI, maximally 5/cage). After allocation the animals were housed individually (macrolon Type II cages).
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 40-70
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12 (7:00 to 19:00 light)

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
10, 25 and 50% w/v
No. of animals per dose:
five
Details on study design:
Three groups of 5 animals each were treated with increasing concentrations of EEA-NH4 (10%, 25% and 50% in vehicle (w/v), respectively) applied on the dorsum of both ears (25 µL per ear) for three consecutive days. Three days after the last treatment, all animals received an intravenous injection of 3H thymidine. Five hours later, the 3H-thymidine incorporation in the draining auricular lymph nodes was determined. The results were compared with those of a control group which was treated with the vehicle (acetone/olive oil, 4:1 v/v). A positive control group treated with Hexyl Cinnamic Aldehyde (a 25% HCA solution in vehicle) was included in the study as a reliability check.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical evaluation of the data (body weights and 3H-thymidine incorporation) was performed by analysis of variance followed by a Dunnett’s multiple comparison tests. 3H-thymidine incorporation of treatment groups 2, 3 and 4 were compared to vehicle-treated animals. The validity of the model was tested by comparing the positive control and the vehicle-treated group. Probability values of p<0.05 were considered significant.

Results and discussion

Positive control results:
No signs of irritation were observed after three consecutive applications of a 25% concentration of HCA in all animals. 3H-thymidine incorporation in the auricular lymph nodes was statistically significantly enhanced in comparison with the vehicle controls. The calculated stimulation index of 4.0 supported this observation and demonstrated the sensitivity of the test system and validity of the model.

Disintegrations per minute: 16475.5
Stimulation index: 4.0
Result: positive

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Concentration (% w/v) Stimulation index Result 0 1.0 - 10 1.1 negative 25 2.2 negative 50 1.9 negative
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Concentration (% w/v) Disintegrations per minute 0 4137.1 10 4562.3 25 9113.3 50 7877.1

Any other information on results incl. tables

No signs of irritation were observed after application of a 10%, 25% and 50% concentration of EEA-NH4.

3H-thymidine incorporation in the auricular lymph nodes in animals treated with a 10% concentration EEA-NH4 was similar to that in vehicle control animals, whereas3H‑thymidine incorporation was statistically significantly enhanced at 25% and 50% concentrations. Stimulation indices (SI’s) of 1.1, 2.2 and 1.9 were calculated in response to a 10%, 25% and 50% EEA-NH4 concentration respectively. Since the SI’s were lower than 3 at all concentrations the results indicated that EEA-NH4 should not be considered a skin sensitiser (a SI of 3 is the limiting value required for classification as a skin sensitiser).

Applicant's summary and conclusion