Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2003-01-15 to 2003-01-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline and GLP-compliant study

Data source

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

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
RCC ltd.
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
444-370-5
EC Name:
-
Cas Number:
669005-94-1
Molecular formula:
C28 H23 F3 N6
IUPAC Name:
4-methyl-2,6-bis[(4-methylphenyl)amino]-5-{2-[2-(trifluoromethyl)phenyl]diazen-1-yl}pyridine-3-carbonitrile
Details on test material:
- Description: orange solid
- Stability of test item: Stable under storage conditions
- Expiry date: 01-OCT-2005

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.; Postbus 6174; NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 17.3 - 21.8 g
- Housing: in groups of 4 mice, in Macolon type-3 cages
- Diet: Pelleted standard Kliba 3433, ad libitum
- Water: Community tap water from Itingen, available ad libitum.


ENVIRONMENTAL CONDITIONS
- Temperature: 22 + 3 °C
- Humidity: 30 - 70 %
- Air changes: 10 - 15 air changes per hour
- Photoperiod: 12 hour fluorescent light / 12 hour dark cycle

In-Life phase: no data

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
1 %, 2.5 % and 5 % (w/v)
No. of animals per dose:
3
Details on study design:
- Topical application:
Test group of mice were treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with test item concentrations of 1 %, 2.5 % or 5 % (w/v) in acetone:olive oil, 4:1 (v/v). The application volume, 25 µL, 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 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.
- ADMINISTRATION OF 3H-METHYL THYMIDINE:
Five days after the first topical application, all mice were administered with 250 µL of 79.8 µCi/ml 3HTdR (equal to 20.0 pCi 3HTdR) by intravenous injection via a tail vein.
- DETERMINATION OF INCORPORATED 3HTDR
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 µm 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 beta-scintillation counter. Similarly,
background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid.
The beta-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:
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 3 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.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 1 %) 1.3 2.5 %: 0.8 5 %: 1.0
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: 0% :3050 dpm (measurement; 2969 dpm (-Background) 1 %: 3867 dpm (measurement); 3786 dpm (- Background) 2.5 %: 2450 dpm (measurement); 2369 dpm (- Background) 5 %: 3122 dpm (measurement); 3041 dpm (- Background)

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information