Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well performed GLP and OECD guidline study - reliability resticted because of testing only a single concentration

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

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
Details on test material:
- Name of test material (as cited in study report): Anilo Acid

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Strain: CBA/CaOlaHsd
- Source: Harlan Netherlands
- Age at study initiation: 7-8 weeks (beginning of acclimatization)
- Weight at study initiation: 17.6 g to 20.5 g
- Housing: single caging, Makrolon(R) Type I, with wire mesh top, granulated soft wood bedding
- Diet (e.g. ad libitum): Pelleted standard diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: yes

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 30 - 72 %
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
0 (control), 25 % (w/v)
No. of animals per dose:
4 animals per treatment group, 4 animals in the control group
Details on study design:
Topical Application

Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with a test item concentrations of 25% (w/v) in propylene glycol. The application volume, 25 μl, was spread over the entire dorsal surface (∅ ∼ 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).


Administration of 3H-Methyl Thymidine

Five days after the first topical application, all mice were administered with 250 μl of 80.4 μCi/ml 3HTdR (corresponds to 20.1 μCi 3HTdR per mouse) by intravenous injection via a tail vein.


Determination of Incorporated 3HTdR

Approximately five hours after treatment with 3HTdR all mice were euthanised by intraperitoneal injection of Na-thiopental
.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions (in 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 scintillation liquid and thoroughly mixed.

The level of 3HTdR incorporation was then measured on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid. The β-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)

Results and discussion

Positive control results:
EC3: 6.1% (w/v)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 25 % (w/v): 2.77
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Measurement DPM Control Group: 3852.11 25 % (w/v): 10624.50

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item (Anilo Acid) was found to be not a skin sensitiser in the LLNA when tested at 25% in propyleneglycol (w/v).
Executive summary:

In the study the test item suspended in propylenglycol was assessed for its possible contact allergenic potential. For this purpose a local lymph node assay was performed using a test item concentration of 25 %. The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. In this study a stimulation index (S.I) of 2.77 was determined with a test item concentration of 25 % (w/v) in propyleneglycol. The test item was found to be not a skin sensitiser in this assay.