Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 2003 to 12 February 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was carried out in compliance with principles of GLP and according to OECD TG 429. The test is well described and raw test results are available.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
The study was based on a design adopted by ICCVAM and ECETOC (technical report No. 78 Skin sensitization testing: methodological considerations, Brussels, Dec 1999. OECD guideline 429.
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Name: Vératrole
CAS number: 91-16-7
Description: colorless liquid
Container: one smoked glass flask
Date of receipt: 7 Nov. 2003
Storage conditions: RT

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: 9 weeks old
- Weight at study initiation: 21.6 ± 1.3 g
- Housing: individually in disposable crystal polystyrene cages (22x8.5x8 cm)
- Diet (e.g. ad libitum): free access to A04 C pelleted dieet
- Water (e.g. ad libitum):free access to tap water (contained in bottles)
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22± 2∙C
- Humidity (%): 30-70%
- Air changes (per hr): 12 cycles/hours of filtered, non-recylced air
- Photoperiod (hrs dark / hrs light):12/12 h

IN-LIFE DATES: From: To: from 12 to 25 November 2003

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
batch N°I049312232 (Merck, Schelles, France) and olive oil, batch N°102K6001 (Sigma Saint-Quentin-Fallavier, France)
Concentration:
Preliminary test: 10-25-50-100% Vératrole
Main test: 5-10-25-50-100% Vératrole
No. of animals per dose:
8 females for the preliminary test (2 per concentration); 28 females for the main test (4 per concentration).
Details on study design:
RANGE FINDING TESTS:
- compound solubility: the test item was soluble in acetone/olive oil (4/1 v/v) (recommended vehicle). A homogeneous dosage form preparation was obtained at all concentrations.
- Irritation: 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 group 1 to 6 was measured using a micrometer. No measurement of ear thickness was performed for the animals of positive control group. Any irritation reaction (erythema and oedema) was recorded in parallel. Any other observation (coloration, presence of residual test item,...) was noted. Non irritant effect observed.
- Lymph node proliferation response: no increased lymph node proliferation

MAIN STUDY
ANIMAL ASSIGNEMENT AND TREATMENT
- Name of test method: Murine local lymph node assay (LLNA)
- Criteria used to consider a positive response: The test item was considererd as a skin sensitizer when the stimulation index (SI) for a dose group is > or equal to 3.

TREATMENT PREPARATION AND ADMINISTRATION: On days 1, 2 and 3 a volume of 25 uL using an adjustable pipette fitted with a plastic tip of the control or dosage was applied to the dorsal surface of both ears. Animals were slightly anaesthesized with isoflurane during application to avoid licking and ensure optimal application of the test material. No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.


On day 6, all animals recieved a single intravenous injection of 250 uL of 0.9% NaCl containing 20 uCi of 3H-TDR (specific acitivity of 25 Ci/mmol) via the tail vein. Approx. 5 hours later, the animals were killed by cervical dislocation and the auricular lymph nodes were excised. The lymph nodes were pooled for each experimental group.
For each experimental group, a single cell suspension of auricular lymph node cells (ALNC) was prepared by mechanical dissagregation in Petri dishes with the plunger of a syringe. Cell suspensions were washed with 15 mL of 0.9% NaCl and pellets obtained were re-suspended in 0.9% NaCl for numeration of lymphocytes (cellularity) and determination of their viability by exclusion of trypan blue. Each cell suspension was then centrifuged and pellets were precipitated with 3 mL of 5% (w/v) trichloroacetic acid (TCA) in purified water at +4°C overnight. After a last centrifugation, the pellets were precipitated with 1 mL of 5% TCA. Three mL of Ultima GoldxR scintillation fluid (Packard) were added in order to measure incorporation of 3H-TdR using β-scintillation counting.
The results were expressed as disintegration's/mn (dpm) per group.
Stimulation Indices (SI) were calculated according to the following formula:
SI =dpm of treated group /dpm of control group
The stimulation index (SI) is expressed as the disintegrations per minute (dpm) in the treated groups divided by the disintegrations per minute (dpm) in the control group.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
not performed

Results and discussion

Positive control results:
In the positive control group given HCA at the concentration of 25%, a moderate increase in cellularity and a stimulation index (SI) exceeding the threshold value of 3 (SI=10.78) were noted. The study was therefore considered as valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: Control: / Vératrole 5%: 2.03 Vératrole 10%: 1.18 Vératrole 25%: 1.31 Vératrole 50%: 1.00 Vératrole 100%: 1.70 HCA 25%: 10.78
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPI per group: Control: 562.51 Vératrole 5%: 1140.05 Vératrole 10%: 660.97 Vératrole 25%: 738.05 Vératrole 50%: 564.84 Vératrole 100%: 718.34 HCA 25%: 6061.20

Any other information on results incl. tables

Table 7.4.1/2: Results of the main study

Groups

Treatment and concentrations

Cell count

viability (%)

Number of nodes per gp

dpm per group

dpm per node

Stimulation index (SI)

increase in ear thickness (% between day 1 and 6)

Irritation classe

EC3 value

1

Acetone/olive oil

0%

106

10

91,38

8

562,51

70,31

 

7,29

 

NA

2

Veratrole

5%

228

16

93,44

8

1140,05

142,51

2,03

4,17

I : non-irritant

3

Veratrole

10%

133

17

88,67

8

660,97

82,62

1,18

3,06

4

Veratrole

25%

113

19

85,61

8

738,05

92,26

1,31

7,45

5

Veratrole

50%

126

16

88,73

8

564,84

70,61

1.00

10,64

6

Veratrole

100%

123

21

85,42

6

718,34

119,72

1.70

4.30

7

HCA

25%

199

28

87,67

8

6.061

757,65

10,78

 

 

dpm = disintegrations per minute                                                                                                   

viability = (viable cells)/(viable cells + dead cells)

cellularity index= amount of cells (*10^6 cells) in the treated group/amount of cells (*10^6 cells) in the vehicle group

stimulation index = dpm of treated group/dpm of control group                                                           

EC3 value = theoretical concentration resulting in a SI value of 3

NA= not applicable

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions, the test item Vératrole does not induce delayed contact hypersensitivity in the murine local lymph node assay.
Executive summary:

The aim of the study (2004) was to evaluate the potential of the test item Veratrole to induce delayed contact hypersensitivity using the murine local lymph node assay (LLNA), according to the OECD guideline 429 and under GLP conditions.

A preliminary test was first performed in order to define the concentrations of test item to be used in the main test.

In the main test, twenty-eight female CBA/J mice were allocated to seven groups:

• five treated groups of four animals receiving the test item Vératrole at the concentration of 5, 10, 25, 50 or 100%,

• one negative control group of four animals receiving the vehicle (mixture acetone/olive oil (4/1)),

• one positive control group of four animals receiving the reference item, α-hexylcinnamaldehyde (HCA), a moderate sensitizer, at the concentration of 25%.

During the induction phase, the test item, vehicle or reference item was applied over the ears (25 μL per ear) for three consecutive days (days 1, 2 and 3). After 2 days of resting, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of tritiated methyl thymidine (day 6). The obtained values were used to calculate stimulation indices (SI).

The irritant potential of the test item was assessed in parallel by measurement of ear thickness on days 1, 2, 3 and 6.

The test item was soluble in the first recommended vehicle, acetone/olive oil (4/1, v/v). A homogeneous dosage form preparation was obtained whatever the proportion. Consequently, the concentrations selected for the main test were 5, 10, 25, 50 and 100%.

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

No cutaneous reactions were observed during the study.

Except for a very slight increase in ear thickness recorded at the concentration of 50%, no noteworthy increase in ear thickness was observed in the treated groups.

No noteworthy lymphoproliferation was noted at any tested concentration, while significant lymphoproliferation was observed with HCA at 25%.

Under these experimental conditions, the test item Veratrole does not induce delayed contact hypersensitivity in the murine Local Lymph Node Assay and it is not classified as a skin sensitizer according to the regulation (EC) 1272/2008 (CLP) and the directive 67/548/EEC.