Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2006-09-20 to 2007-04-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study performed according to OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay) and EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay) without deviations.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Remarks:
The testing facility indicated that the protocol was followed without deviation.
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Remarks:
The testing facility indicated that the protocol was followed without deviation.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate from "The Department of Health of the Government of the United Kingdom"
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: VRT-126016
- Molecular weight: not applicable
- Smiles notation: not applicable
- InChl: not applicable
- Structural formula attached as image file: not applicable
- Substance type: no data
- Physical state: white powder
- Analytical purity: 99.8 % (area)
- Impurities: <0.5 % Z-D-cyclohexylglycine and <0.1 % chloride
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: 2006-02-02
- Lot/batch No.: batch 25719, lot 1-4107-6-02-02
- Expiration date of the lot/batch: 2008-02
- Stability under test conditions: no data
- Storage condition of test material: room temperature
- Other: received on 2006-09-15

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan UK Ltd., Bicester, Oxon, England
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15.7-21.4 g
- Diet: A standard laboratory rodent diet (Special Diet Services RM1 (E) SQC) was provided ad libitum.
- Water: Drinking water was provided ad libitum.
- Acclimation period: 6 days
- Other: The mice were allocated without conscious bias to cages within the treatment groups.They were housed individually in polycarbonate cages with woodflake bedding. The mice were also given Nestlets for environmental enrichment.

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 21 +/- 2
- Humidity (%): 40-70
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES
- From: no data to no data

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
Not applicable
Challengeopen allclose all
Route:
other: not applicable
Vehicle:
other: not applicable
Concentration / amount:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Challenge controls:
Not applicable

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25 and 50 % w/v
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS
- Compound solubility: Vehicle trials conducted with the test substance at 50 % w/v formed a thick paste in acetone:olive oil (4:1 v/v) which was too viscous for dose administration. At 50 % w/v in dimethylformamide, a clear solution was obtained which was satisfactory for dose administration. As dimethylformamide was the next preferred vehicle in the protocol, this was used for the study. The dosages were chosen based on the physical properties of the test substance (i.e. solubility, viscosity). The maximum practical concentration for pinna dosing was 50 % w/v in dimethylformamide. Based on this information, the dose levels of 10, 25 and 50 % w/v were used in the main study. Prior to dose administration, the Study Director authorized the choice of vehicle and dose levels in the raw data.
- Irritation: no data
- Lymph node proliferation response: no data

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: incorporation of 3H-methyl Thymidine by B-scintillation counting of lymph node cell (LNC) suspensions
- Criteria used to consider a positive response: The test substance was regarded as a sensitizer if at least one concentration of the test substance resulted in a three-fold greater increase in 3HTdR incorporation compared to the control. Results were expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio).

TREATMENT PREPARATION AND ADMINISTRATION
The test substance formulations were prepared in the vehicle at the required concentrations freshly on each day of dosing. The absorption of the test substance was not determined. Chemical analysis of the homogeneity, stability and purity of the test substance was not undertaken as part of the study.

The mice were treated by daily application of 25 uL of the appropriate concentration of the test substance, to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was spread over the dorsal surface of each ear using the tip of an automatic micropipette. Another group of four mice received the vehicle alone in the same manner and further group received the positive control substance. Five days following the first topical application of the test substance (Day 6) all mice were injected via the tail vein with 250 uL of phosphate buffered saline containing 3H-methyl Thymidine (3HTdR: 80 uCi/mL) giving a nominal 20 uCi to each mouse. The injection into the tail vein was carried out using a plastic syringe and needle (26 guage) after the mouse had been heated in a warming changer.

All animals were observed daily for signs of ill health or toxicity. The ears were also examined for signs of irritation. The weight of each mouse was recorded on arrival (these data were not reported), Day 1 (first day of dosing) and prior to termination (Day 6).
Five hours following the administration of 3HTdR on Day 6, all mice were killed by carbon dioxide asphyxiation and the draining auricular lymph nodes excised and pooled for each experimental group. 1.0 mL of phosphate buffered saline was added to the pooled lymph nodes for each group. The animals were then discarded and no further investigations were carried out. The following procedures were carried out with the lymph nodes from these animals only.
1. Preparation of single cell suspensions: A single cell suspension of lymph node cells (LNC) was prepared by gentle mechanical disaggregation through a stainless steel gauze (200 mesh size). The pooled LNC were then washed by adding 10 mL phosphate buffered saline, pelleted at 190 x g for 10 minutes and resuspended. The cells were washed twice again and resuspended in 3 mL trichloroacetic acid (TCA: 5 %) following the final wash.
2. Determination of incorporated 3H-methyl Thymidine: after overnight incubation with 5 % TCA at 4 degrees C, the precipitate was recovered by centrifugation and resuspended in 1 mL 5 % TCA and transferred to 10 mL Ultima gold scintillation fluid on Day 7. 3HTdR incorporation was measured by B-scintillation counting.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not applicable

Results and discussion

Positive control results:
-dpm=17870.40
-dpm/node (8)=2233.80
-positive control/control ratio=6.9
The positive control induced a positive result.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Test substance/control ratio- 10%= 1.8, 25 %=1.9 and 50%=2.9 As a SI of 3 or more was not recorded for any of the test substance groups, the test substance was considered not to have the potential to cause skin sensitization (delayed contact hypersensitivity).
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM-10 %=4072.90, 25 %=4928.60 and 50 %=7573.90 DPM/node- 10 %=581.84, 25 %=616.08 and 50 %=946.74

Any other information on results incl. tables

Results:

There were no deaths and no signs of ill health or toxicity observed during the study. Wet fur, cranial area, was noted for all vehicle control, positive control and test animals post dose from Day 1. This sign had resolved by Day 2 in the positive control group and by Day 4 in the vehicle and low and mid dose groups and by Day 5 in the high dose group. Greasy fur, cranial region, was seen in all animals in the positive control group post dose from Day 1 resolving by Day 5. In addition white dose residue was seen on the ears and the fur around the ears, in all animals in the high dose group post dose from Day 1 and Day 3 only for 2 females from the mid dose group. This sign had resolved by Day 4 in the two animals from the mid dose group and was still present on Day 6 at study termination in all animals from the high dose group. One female in the vehicle control group lost weight at study termination. All remaining animals had satisfactory weight gains during the study.

Applicant's summary and conclusion

Conclusions:
This study was performed to assess the skin sensitization potential of the test substance using the murine local lymph node assay. Based on the results, the test substance was not regarded as a potential skin sensitizer.
Executive summary:

Not applicable