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Description of key information

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:
2003-08-19 - 2004-01-16
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD TG 429) performed under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted on 24th April 2002
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: approximately 8 weeks old
- Body weight at study initiation: 20.5 ± 1.4 g
- Housing: individually in disposable crystal polystyrene cages (22 cm x 8.5 cm x 8 cm), each cage contained autoclaved sawdust (SICSA, Alfortville, France)
- Diet: A04 C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water: tap water (filtered using a 0.22 micron filter), ad libitum
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 30 - 70 %
- Air changes: approximately 12 air changes/hour (filtered, non-recycled air)
- Photoperiod: 12 h / 12 h

IN-LIFE DATES: From: 2003-09-17 To: 2003-09-22
Vehicle:
propylene glycol
Concentration:
0 % (control), 0.5 %, 1 %, 2.5 %, 5 %, 10 % in propylene glycol (w/v)
No. of animals per dose:
4 animals per group
Details on study design:
MAIN STUDY
- Criteria used to consider a (lymphoproliferative) positive response: Stimulation Index (SI) > 3

TREATMENT PREPARATION AND ADMINISTRATION:
- The test item was prepared in the vehicle at the chosen concentrations and sonicated during approximately 10 minutes for the highest tested concentration (10 %). All dose formulations were made freshly on the morning of administration.

TOPICAL APPLICATION (each test group of mice):
- Topical (epidermal) application of different test item concentrations to the dorsal surface of both ears
- Application volume: 25 µL, spread over the entire dorsal surface of each ear lobe once daily for three consecutive days (on days 1, 2 and 3)
- Control animals: Application of equivalent volume of the vehicle alone
- No rinsing was performed between each application

ADMINISTRATION OF 3H-METHYL THYMIDINE (³HTdR):
- Three days before the injections, the required quantity of ³HTdR was diluted in 0.9% NaCl (20 µCi in 250 µL of 0.9% NaCl per animal). The obtained solution was stored at +4°C and protected from light before use.

OBSERVATIONS:
- Clinical signs, morbidity and mortality: once a day during the study
- Body weight: on the first day of the study (day 1) and on the day of sacrifice (day 6).
- Ear thickness measurements and recording of local reactions: On days 1, 2 and 3 (before application) as well as on day 6 (after sacrifice), the thickness of the left ear of each animal of the vehicle and treated groups was measured using a micrometer. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (e.g. coloration, presence of residual test item) was noted.

PROLIFERATION ASSAY / DETERMINATION OF INCORPORATED ³HTdR:
- On day 6, all animals of all groups received a single intravenous injection of 250 µL of 0.9% NaCl containing 20 µCi of ³HTdR (specific activity of 25 Ci/mmol), via the tail vein.
- Approximately 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised and pooled for each experimental group.
- For each experimental group (and the positive control group), a single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation and measured using ß-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated for the body weight.
Positive control results:
In the positive control group given HCA at the concentration of 25 %, an increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI = 21.96) were noted. The study was therefore considered valid.
Parameter:
SI
Remarks on result:
other: Control Group: - Test Group (0.5 %): 3.98 Test Group (1 %): 0.86 Test Group (2.5 %): 1.65 Test Group (5 %): 1.17 Test Group (10 %): 1.34 Positive control group (25 % HCA): 21.96
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: dpm per node: Control Group: 61.48 dpm Test Group (0.5 %): 244.43 dpm Test Group (1 %): 53.00 dpm Test Group (2.5 %): 101.15 dpm Test Group (5 %): 71.93 dpm Test Group (10 %): 82.67 dpm Positive control group (25 % HCA): 1350.3 dpm

Systemic clinical signs and mortality:

No clinical signs and no mortality were observed during the study.

 

Body weight:

The body weight change of the treated animals was similar to that of controls.

 

Local irritation:

No cutaneous reactions and no increase in ear thickness were observed at any of the tested concentrations

LLNA:

The quantity of cells obtained in each group was satisfactory and the cellularity correlated with incorporation of H-TdR. The cell viability was higher than 80 % in each group. In the treated groups, a positive lymphoproliferative response (SI = 3.98) was noted at the concentration of 0.5 %. However, as no positive response was present and no dose-response relationship was observed at the highest tested concentrations, this positive response could not be considered as biologically relevant. Therefore, no lymphoproliferative responses attributed to delayed contact hypersensitivity were observed during the study.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In a skin sensitization study (CIT 26399, 2004, OECD 429, GLP) the potential of non-micronized ETH50 to induce delayed contact hypersensitivity was evaluated using the murine Local Lymph Node Assay (LLNA). Twenty-eight female CB A/J mice were allocated to seven groups of four animals each:

- five treated groups receiving the test item at the concentration of 0.5, 1, 2.5, 5 or 10 % in propylene glycol,

- one negative control group receiving the vehicle (propylene glycol),

- one positive control group receiving the reference item, alpha-hexylcinnamaldehyde (HCA), at the concentration of 25 %.

The test item, vehicle or reference item was applied over the ears (25 uL per ear) for 3 consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of the lymph node cells in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6 and by assessment of local skin effects.

No mortality and no clinical signs were observed during the study. No cutaneous reactions and no increase in ear thickness were observed in the animals of the treated groups. A lymphoproliferative response with an SI > 3 was noted at the concentration of 0.5 % only. The SI values were 3.98, 0.86, 1.65, 1.17, 1.34 for the 0.5%, 1%, 2.5%, 5% and 10% concentration group, respectively. However, as no response and no dose-response relationship were observed up to the highest concentration tested, this response could not be considered as biologically relevant. Therefore, no lymphoproliferative responses attributed to delayed contact hypersensitivity were observed during the study. The reference item induced the appropriate response (SI: 21.96), thus demonstrating sensitivity of the test system.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The present data on dermal sensitization do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.