Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007-01-31 to 2007-05-22
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.
GLP compliance:
yes (incl. certificate)
Remarks:
Certificate issued by 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 (as cited in study report): VRT-753136 hcl
- Molecular formula: not applicable
- 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 to off white powder
- Analytical purity: 100 area %
- Impurities: 0.03 area% of unknowns
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: 2006-09-15
- Lot/batch No.: Batch number 25515/Lot No. AF6-001
- Expiration date of the lot/batch: 2008-05
- Stability under test conditions: no data
- Storage condition of test material: room temperature
- Other: no data

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Obtained from Harlan UK Ltd., Bicester, Oxon, England
- Age at study initiation: approximately eight to twelve weeks of age prior to dosing on Day 1
- Weight at study initiation: 18.5 and 21.8 g
- Housing: Animals were housed individually in polycarbonate cages with woodflake bedding.
- Diet: Standard laboratory rodent diet (Special Diet Services RM1 (E) SQC)
- Water: Drinking water was provided ad libitum.
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 21+/- 2 deg C
- Humidity (%): 40 to 70%
- Air changes (per hr): Not applicable
- Photoperiod (hrs dark / hrs light): Lighting was controlled by means of a time switch to give 12 hours of artificial light (0600 - 1800 hours GMT) in each 24-hour period.

IN-LIFE DATES:
From: 2007-02-01 To: 2007-02-27

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
Positive control substance(s):
not required

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
4:1 v/v
Concentration:
5, 10 and 25% w/v VRT-753136 hcl
No. of animals per dose:
Four mice were dosed at three dosage levels, as well as a vehicle control.
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Groups of one female were treated at one of three concentrations of the test substance. The mice were treated by daily application of 25 uL of appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days (Days 1-3). The test substance was applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette.
- There were no deaths during the study.

Clinical signs of reaction to treatment comprised:
- Group 1 (5% w/v): Greasy fur around the cranial region was seen post dose from Day 2 and was still present at termination on Day 4.
- Group 2 (10% w/v): Greasy fur around the cranial region was seen post dose from Day 1 and was still present at termination on Day 4. Wet fur cranial region was noted post dose on Day 1 only. In addition, particles on the ears were seen post dose from Day 1 and had resolved by Day 3.
- Group 3 (25% w/v): Greasy fur around the cranial region was seen post dose from Day 1 and was still present at termination on Day 4. In addition, wet fur around the cranial region was noted post dose on Days 1 and 2 only.
- No signs of irritation were seen over the dosed area during the study.
- Bodyweight increases were recorded for all mice over the period of the preliminary study.
- Results indicated that 25% w/v was a suitable high dose level for the main study.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: In the main study, the proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine by beta-scintillation counting of LNC suspensions. The response was 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:
- Groups of four mice were treated at one of three concentrations of the test substance. 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 applied to the dorsal surface of each ear using an automatic micropipette. The test substance was spread over the entire dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.
- A vehicle trial was conducted and identified acetone olive oil (4:1 v/v) as the appropriate vehicle for the main study. This is also the preferred vehicle recommended in the protocol.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
no data

Results and discussion

Positive control results:
Responses to the positive control substance hexyl cinnamic aldehyde (HCA), in contemporaneous studies demonstrate the reliability and sensitivity of this assay to detect skin sensitization potential in this laboratory.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
In this study, the stimulation indexes (test/control ratio) obtained for 10, 25 or 50% v/v HCA were 7.9, 13.5 and 19.8, respectively. This indicates that HCA showed the potential to induce skin sensitization (delayed contact hypersensitivity) and confirms the sensitivity of the technique to detect skin sensitization.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Group 1 (vehicle control): 4283.65 dpm/2.0 lymph nodes per animal (535.46 dpm/node). Test control ratio: not applicable Result: not applicable Group 2 (5% w/v): 2676.95 dpm/2.0 lymph nodes per animal (334.62 dpm/node) Test control ratio: 0.6 Result: negative Group 3 (10% w/v): 2090.05 dpm/2.0 lymph nodes per animal (261.26 dpm/node) Test control ratio: 0.5 Result: negative Group 4(25% w/v): 2751.85 dpm/2.0 lymph nodes per animal (343.98 dpm/node) Test control ratio: 0.6 Result: negative

Any other information on results incl. tables

Estimation of the Lymph node proliferation response:

- The test/control ratios obtained for 5, 10 and 25% w/v VRT-753136 hcl were 0.6, 0.5 and 0.6, respectively.

- As a test/control ratio of 3 or more was not recorded for any of the concentrations tested, VRT-753136 hcl is not considered to have the potential to cause skin sensitization (delayed contact hypersensitivity).

Main Study

Mortality and clinical signs:

- There were no deaths and no signs of ill health or toxicity observed during this study.

- No signs of irritation were seen over the dosed area during the study.

- Greasy fur was noted for all control and test animals post-dose from Day 1. This sign had resolved completely in all animals in Groups 1, 2 and 3 by Day 5 and was still present in all animals in Group 4 on Day 6 at study termination. Dose residue was seen post dose in one animal in Group 2 from Day 2, and in the remaining animals in Group 2 on Day 3. In addition, dose residue was also seen post dose from Day 1 in some animals from Groups 3 and 4. This sign had resolved by Day 4 in all animals.

Bodyweight:

- Bodyweight increases were recorded for all mice over the period of the main study.

Applicant's summary and conclusion

Conclusions:
VRT-753136 hcl is not regarded as a potential skin sensitizer.
Executive summary:

Not applicable