t-Butyl (2S,2aS,5aR)-1-azabicyclo[3.3.0] oct-2-carboxylate monooxalate salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

no data to 2007-04-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD Guideline 429 (Skin Sensitization: Local Lymph Node Assay) and EU Method B.42 (Skin Sensitization: Local Lymph Node Assay) without deviations.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Remarks:

The testing facility indicated that the protocol was followed without deviation.

Test guideline 2

Qualifier:

according to guideline

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. QA statement)

Remarks:

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-126028, P2, VRT-126028 oxalate salt
- Molecular formula (if other than submission substance): C14H23NO6
- Molecular weight (if other than submission substance): 301.34
- Smiles notation (if other than submission substance): not applicable
- InChl (if other than submission substance): not applicable
- Structural formula attached as image file (if other than submission substance): not applicable
- Substance type: no data
- Physical state: white powder
- Analytical purity: 99.3% area by GC
- Impurities (identity and concentrations): no data
- Composition of test material, percentage of components: no data
- Isomers composition: no data
- Purity test date: no data
- Lot/batch No.: Lot WYJ1141040533/Batch No. 25414
- Expiration date of the lot/batch: May 2008
- Stability under test conditions: no data
- Storage condition of test material: room temperature
- Other: Odor: slight odor
Rotation: [a]29D=+37.2 deg C (C=5515 CH3OH)
Analytical method: GC, chiral GC, NMR, Polarity, MS
Water content: 0.10% by Karl Fischer Titration
LOD: 0.03%
Residue on ignition: 0.06%

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 to 12 weeks
- Weight at study initiation: 14.6 to 20.8 g
- Housing: individual
- Diet (e.g. ad libitum): standard laboratory rodent diet (Special Diet Services RMI (E) SQC), ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days prior to the start of the study

ENVIRONMENTAL CONDITIONS
- Temperature (deg C): 21 +/- 2 deg C
- Humidity (%): 40 to 70%
- Air changes (per hr): no data
- 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: no data To: no data

Study design: in vivo (LLNA)

Vehicle:

other: acetone:olive oil (4:1 v/v)

Concentration:

Preliminary and main study: 5, 10 and 25% w/v

No. of animals per dose:

Preliminary study: one female per dose
Main study: four females per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A vehicle trial conducted with the test substance at 50% w/v showed that it formed a thick paste in acetone:olive oil (4:1 v/v), dimethylformamide, ethyl methyl ketone, propylene glycol and dimethylsulphoxide, which were all too viscous for dose administration. In a further trial at 25% w/v in acetone:olive oil (4:1 v/v), dimethylformamide, ethyl methyl ketone, propylene glycol and dimethylsulphoxide all formed liquid suspensions which were all satisfactory for dose administration.
Acetone:olive oil (4:1 v/v) was chosen as this was the preferred vehicle from those recommended in the protocol.
- Irritation: The ears were examined for signs of irritation.
- Lymph node proliferation response: not applicable

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The mice were allocated without conscious bias to cages within the treatment groups.
- Criteria used to consider a positive response: The test substance is regarded as a sensitizer if at least one concentration of the test substance results in a three-fold greater increase in 3HTdR incorporation compared to control values.

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.
In the main study, five days following the first topical application of test substance (Day 6) all mice were injected via the tail vein with 250 uL of phosphate buffered saline containing 3H-methyl Thymidine (Specific activity 2.0 Ci/mmo, concentation 1.0 mCi/mL) (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 gauge) after the mouse had been heated in a
warming chamber.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

no data

Results and discussion

Positive control results:

Disintegrations per minute
Solvent control: 2034.7
10% v/v: 16123.9
25% v/v: 27440.8
50% v/v: 40300.6

Stimulation index (test/control ratio, a stimulation index greater than 3 indicates a positive result)
Solvent control:
Not applicable
10%:
7.9
25% v/v
13.5
50% v/v
19.8

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks on result:

other: Vehicle control: not applicable 5% w/v: 1.1 10% w/v: 1.7 25% w/v: 0.6

Results 2

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: Vehicle control: 1288.95 5% w/v: 1442.35 10% w/v: 2170.05 25% w/v: 729.25

Any other information on results incl. tables

PRELIMINARY STUDY

Mortality and clinical signs:

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 1 and was still present at termination on Day 4. In addition, particles on the ears were seen post dose from Day 2; this reaction had resolved by 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. In addition, dose residue or particles on the ears were seen post dose from Day 1; this reaction had resolved by Day 4.

Group 3 (25% w/v): Particles and dose residue were seen post dose from Day 1 and test substance staining (white) was observed post dose from Day 2. In addition, greasy fur around the cranial region was noted on Day 3 only. Additional signs had resolved by termination on Day 4.

Bodyweight:

A loss in bodyweight was recorded for the female in Group 2 and no bodyweight gain was seen in the female from Group1 during the preliminary study. The remaining animals from Group 3 gained weight during the study. On the basis of the results of the preliminary study, 25% w/v was considered a suitable high dose level for the main study.

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 by Day 5. Wet fur-cranial region was noted for one animal post dose from the control group on Day 1 only. Particles on the ears were seen in some animals from each of Groups 2, 3 and 4; this sign had resolved by day 4. Dose residue was seen in all animals in Group 4 from either Day 1 or 2 after dosing, and had resolved by Day 4. Test substance staining (white) over the dosed area was noted for one animal on Day 1 only. In addition, yellow staining on bedding was observed from Day 3 for all animals in Groups 2, 3 and 4.

Bodyweight:

A loss of bodyweights was recorded for one female in Group 2, one female in Group 3 and three females from Group 4 during the study. In addition, there was no bodyweight gain for one animal from Group 1. All remaining animals gained weight during the study.

Applicant's summary and conclusion

Conclusions:

The test substance is not regarded as a potential skin sensitizer.

Executive summary:

Not applicable