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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 29 apr 2003 to 06 oct 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

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:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France.
- Age at study initiation: approximately 9 weeks old
- Weight at study initiation: 21.7 +/- 1.4 g
- Housing: individually in disposable crystal polystyrene cages (22*8.5*8 cm)
- Diet (e.g. ad libitum): free access to A04 C pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France).
- Water (e.g. ad libitum): free access to tap water (filtered using a 0.22 micron filter) contained in bottles.
- Acclimation period: at least 5 days before the start of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 30 to 70% relative humidity
- Air changes (per hr): 12 cycles per hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12h / 12h


IN-LIFE DATES: From 05 jun 2003 to 10 jun 2003

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
0, 2.5, 5, 10, 25 and 50 %
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the test item was freely soluble in the vehicle, consequently, the concentrations selected for the preliminary test were 5, 10, 25 and 50%.
- Irritation: no cutaneous reactions and no noteworthy increase in ear thickness were observed in the animals of the treated groups.
- Lymph node proliferation response: no. No positive lymphoproliferative response was noted and no dose-related increase in the stimulation index was observed at the tested concentrations.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: animals will be assigned to the treatment groups by hand procedure. Identification is individually by a number on the tail using an indelible marker.
- Criteria used to consider a positive response: The test item should be regarded as a skin sensitizer when the SI for a dose group is >=3. Other relevant criteria such as cellularity, radioactivity levels and ear thickness will be also taken into account to evaluate the data.
When appropriate, the EC3 value (theoretical concentration resulting in a SI value of 3) will be determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold. The equation used for calculation of EC3 will be:
EC3 = c + [(3 - d)/(b - d)] x (a - c)
Where a = the lowest concentration giving stimulation > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.



TREATMENT PREPARATION AND ADMINISTRATION: 28 female mice were allocated to 7 groups: five treated groups (2.5, 5, 10, 25 and 50%), one negative control group (vehicle), one positive control group (HCA).
On days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No

Results and discussion

Positive control results:
SI = 13.5 with 25% of hexyl cinnamic aldehyde

In vivo (LLNA)

Resultsopen allclose all
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Per node: from 135 to 116.
Parameter:
SI
Value:
1.87
Test group / Remarks:
2.5% / 4 animals
Parameter:
SI
Value:
1.71
Test group / Remarks:
5% / 4 animals
Parameter:
SI
Value:
1.44
Test group / Remarks:
10% / 4 animals
Parameter:
SI
Value:
1.42
Test group / Remarks:
25% / 4 animals
Parameter:
SI
Value:
1.61
Test group / Remarks:
50% / 4 animals

Any other information on results incl. tables

Groups 1 2 3 4 5 6 7
Concentrations (%) 0
(Acetone/olive oil)
2,5 5 10 25 50 HCA at 25%
Cell count viable 66 100 142 78 118 102 192
Cell count dead 10 9 14 9 Il 10 27
Viability (%) 86.84 91.74 91.03 89.66 91.47 91.07 87.67
Amount of cells (*106) cells) 3.3 5.00 7.10 3.90 5.90 5.1 19.2
Cellularity index   1.52 2.15 1.18 1.79 1.55 5.82
Number of nodes per group 8 8 8 8 8 8 8
Disintegrations per min per gp (dpm) 579.34 1083.16 989.82 833.98 822.97 930.8 7820.28
Disintegrations per min per node (dpm) 72.42 135.4 123.73 104.25 102.87 116.35 977.54
stimulation index (SI) 1.87 1.71 1.44 1.42 1.61 13.5
Increase in ear thickness (% between day 1 and day 6) 0.00 1.98 6.86 7.84 4.85 -2.91  
EC3 value   NA  

- Positive control results:at the concentration of 25%, a moderate increase in cellularity and a stimulation index exceeding the threshold value of 3 (SI=13.50) was noted. The study was therefore considered valid.

Local irritation:

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

Proliferation assay:

In the treated groups, no positive lymphoproliferative response was noted and no dose-related increase in the stimulation index was observed at the tested concentrations.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Not sensitizing
Executive summary:

In a dermal sensitization study (Klein, 2003) with Ethylvanillin in acetone/olive oil (4/1), female CBA/J mice (4 animals per dose) were tested in LLNA, according to the guideline OECD 429 at doses: 0, 2.5, 5, 10, 25 and 50 %.

During the study, no cutaneous reactions (ears irritation) were observed, nor a positive lymphoproliferative response.

In absence of effect no EC3 value could be calculated.

Based on these results, Ethylvanillin is not sensitizing, according to 67/548/EC directive and CLP 1272/2008/EC criteria.