Registration Dossier

Administrative data

Description of key information

The test item was not sensitizing in an LLNA screening test.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 31, 2006 - October 11, 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP study with reduced animal numbers (screening)
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
no
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands
- Age at study initiation: 7 - 8 weeks
- Weight at study initiation: mean weight 19.6 g
- Housing: single in Makrolon Type I, with wire mesh top
- Diet: pelleted standard diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: Under test conditions after health examination. Duration not specified.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30-74
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethyl sulphoxide
Concentration:
20%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
In two non-GLP conform pre-tests in two mice each, test item concentrations of 2.5, 5, 10 and 20 % (w/v) were tested on one ear each. No irritation effects were observed at these concentrations after a single application.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay (LLNA)
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser 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 3HTdR 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:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 20% (w/v) in DMSO. The application volume, 25 µl, was spread over the entire dorsal surface 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).
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.
Parameter:
SI
Remarks on result:
other: see below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see below

Calculation and Results of Individual Data

Test item concentration
% (w/v)
Group Measurement DPM Calculation Result
DPM-BGa) number of lymph nodes DPM per lymph nodeb) S.I.
--- BG I 15.77 --- --- --- ---
--- BG II 18.81 --- --- --- ---
--- CG 1 8783.78 8766.5 8 1095.8  
20 TG 1 15107.50 15090.2 8 1886.3 1.72

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

CG = Control Group

TG = Test Group

S.I. = Stimulation Index

a) = The mean value was taken from the figures BG I and BG II

b) = Since the lymph nodes of the animals of a dose group were pooled, DPM/node

was determined by dividing the measured value by the number of lymph nodes pooled

The EC3 Value could not be calculated, since only one concentration was tested.

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Body Weights

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Interpretation of results:
GHS criteria not met
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In order to study a possible contact allergenic potential of the test item, one group of four female mice was treated daily with the test item at a concentration of 20% (w/v) in DMSO by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (DMSO) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a β-scintillation counter. All treated animals survived the scheduled study period. A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study a Stimulation Index of 1.72 were determined with the test item at concentrations of 20% (w/v) in DMSO. The EC3 value could not be calculated, since only one concentration was tested. In conclusion, the test item was not a skin sensitiser under the described conditions.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008 (CLP). As a result the substance is not considered to be classified for sensitization under Regulation (EC) No 1272/2008.