Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 14 March 2008 to 7 May 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: study performed according to OECD guideline and GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

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): Vegeflux soy
- Substance type: UVCB
- Physical state: Clear yellowish liquid
- Stability under test conditions: data not available
- Storage condition of test material: at room temperature

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelman GmbH, D-33176 Borchen
- Age at study initiation: About 8 weeks at the first administration
- Weight at study initiation: 15.8 - 20.7 g
- Housing: Single caging. Makrolon cages type II (22 cm x 16.5 cm ground area, 15 cm high)
- Diet: Maintenance diet for rats and mice R/M-H (item V1534-3), autoclavable, ad libitum
- Water: Tap water from Makrolon-bottles with staineless steel canules, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: Average of 21.0°C (continuous monitoring and recording)
- Humidity: Average of 45.1 % (continuous monitoring and recording)
- Air changes (per hr): data not available
- Photoperiod: Only artificial light from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: from 1 APRIL 2008 to 8 APRIL 2008

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
(AOO)
Concentration:
10%, 25% and 50% Vegeflux soy in AOO
No. of animals per dose:
5 animals/group (including spare animals):
- 15 animals for 3 test substance groups,
- 5 animals for the negative control group,
- 5 animals for the positive control group.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: the test substance is well soluble in AOO (min. 50%, v/v)
- Irritation:
A range finding study was performed with two animals/concentration with following concentrations: 100% and 50%. All animals of both groups had slight erythema at the application sites on Days 3 and 4. An important increase of ear thickness was measured in animals of the high dosed group (mean: +0.13 mm), whereas in the animals of the 50% test substance group only a slight increase of ear thickness (mean: +0.05 mm) was measured. There is evidence of systemic toxicity and excessive skin irritation at a concentration of 100%, due to body mass loss and an important increase of ear thickness. Therefore, 50% was chosen as highest test substance concentration.


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The individual animals were allocated to their groups by random numbers
- Criteria used to consider a positive response: A substance is regarded as a sensitiser in the LLNA if the test substance induces a 3-fold or greater increase in 3HTdR incorporation into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes, as indicated by the SI, together with the consideration of dose response.

TREATMENT PREPARATION AND ADMINISTRATION:
Administration was performed epicutaneously to the dorsal surface of both ears, once a day on three consecutive days. The volume administered was 25 μL per ear.
5 days after the first topical application, 3H-methyl thymidine was intravenously administered to all mice via a tail vein. Approximately 5 hours later all animals were sacrificed, the draining auricular lymph nodes were excised, pooled for each group, and single cell suspensions were prepared.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
not applicable

Results and discussion

Positive control results:
The positive control substance led to a stimulation index of 7.2, thus demonstrating the validity of the experiment.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: The SIs of the tested test substance concentrations were 1.0 (low dose), 1.0 (mid dose) and 2.5 (high dose).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see table 1

Any other information on results incl. tables

General:

All animals survived till the end of the study.

No adverse effects were noted in any animal.

Body masses and body mass gains were in the range to be expected from animals of the same strain, sex and age.

At the application sites no skin irritating effects were observed in all animals of the test substance groups A and B and the negative control group throughout the whole study. Slight erythema was noted in all animals of the test substance group C and the positive control group on Days 3 and 4, indicating slight local skin irritation.

Table 1: dpm results and calculated SIs:

Group

dpm

SI

group K (negative control)

5741

1

group A (low dose)

5557

1.0

group B (mid dose)

5898

1.0

group C (high dose)

14371

2.5

group P (positive control)

41407

7.2

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information according to EU criteria
Conclusions:
Vegeflux soy is regarded as a non sensitiser in the LLNA assay and needs not to be labelled with "R43 May cause sensitisation by skin contact".
Executive summary:

The Local Lymph Node Assay was performed to evaluate a possible skin sensitising potential of "VEGEFLUX SOY" according to the OECD-Guideline 429 and GLP.

The test substance was administrated epicutaneously on three consecutive days to three groups of 5 mices at the concentration of 10, 25 and 50% in acetone:olive oil (AOO). Two groups with 5 animals each served as positive and negative controls.

5 days after the first topical application, 3H-methyl thymidine was intravenously administered to all mice via a tail vein. Approximately 5 hours later all animals were sacrificed, the draining auricular lymph nodes were excised, pooled for each group, and single cell suspensions were prepared. The desintegration per minut (dpm) and the stimulation index (SI) were measured and compared with the negative control.

No adverse effect was observed, except a slight erythema in all animals of the high dose group and the positive control.

The SIs of the tested test substance concentrations were 1.0 (low dose), 1.0 (mid dose) and 2.5 (high dose).

The positive control substance led to a stimulation index of 7.2, thus demonstrating the validity of the experiment.

According to this test, Vegeflux soy is regarded as a non sensitiser in the LLNA assay and needs not to be labelled with "R43 May cause sensitisation by skin contact".