Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: according GLP and OECD guideline, well documented

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands B.V. Postbus6174 NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 16.8 - 21.5 g
- Housing: groups of 4
- Diet ad libitum
- Water ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
2.5 %, 5 % and 10 % (w/v)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in two mice with concentrations of 1 %, 2.5 %, 5 % and 10 % (w/v) (pretest excluded from Statement of Compliance).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
A test item is regarded as a sensitizer 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 ^HTdR 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.

TREATMENT PREPARATION AND ADMINISTRATION:
The application volume, 25 pl, was spread over the entire dorsal surface ( 0 - 8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was used to dry the ear's surface as quickly as possible to avoid loss of test item applied.
Five days after the first topical application, all mice were administered with 250 ul of 79.8 uCi/ml 3HTdR (equal to 20.0 uCi 3HTdR) by intravenous injection via a tail vein.
Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of VETANARCOL (Veterinaria AG, Zurich).
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 pm mesh size). After washing two times with phosphate buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of 'Ultima Gold' scintillation liquid and thoroughly mixed.
The level of 3HTdR incorporation was then measured on a |3-scintillation counter. Similarly, background ^HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The p-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: please see below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: please see below

Any other information on results incl. tables

   test item concentration [%]  S.I.  DPM / lymph node
 group 2  2.5  2.3  699
 group 3  5 1.1   339
 group 4  10 1.5   426

Applicant's summary and conclusion

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